• To minimize interspecific pollination, it has been suggested that pollen is placed on different parts of a pollinator's body corresponding to the conspecific location of pollen pickup by the stigma.
• Although Pedicularis is regarded as a classic example of pollinator-mediated floral isolation, such reciprocal pollen placement has not been demonstrated experimentally. This leads us to question previous observations of pollen release in Pedicularis species.
• Here, we show that pollen grains are released from the tip, rather than the basal opening, of the galea (the hoodlike upper lip of the corolla) in eight nectarless Pedicularis species, mimicking pollen release from poricidal anthers. We used safranin-stained pollen within anthers to track pollen placement in three Pedicularis species, and showed that pollen was deposited on numerous parts of the bumblebee's body. However, fluorescent powder placed on the stigmas to detect the contact location on the bumblebee's body was deposited mainly on the major position of pollen placement in each of the three species.
• Such segregation of pollen placement and pickup between species sharing the same pollinator probably helps to reduce reproductive interference, but the positions of pollen placement and stigma contact on the bumblebee's body were not as precise as previously thought.

• How plants tailor their defense responses to attack from different insects remains largely unknown. Here, we studied the role of a mitogen-activated protein kinase (MAPK), MPK4, in the resistance of a wild tobacco Nicotiana attenuata to two herbivores, the specialist Manduca sexta and the generalist Spodoptera littoralis.
• Stably transformed N. attenuata plants silenced in MPK4 (irMPK4) were generated and characterized for traits important for defense against herbivores.
• Only the oral secretions (OS) from M. sexta, but not the OS from S. littoralis or mechanical wounding, induced elevated levels of jasmonic acid (JA) in irMPK4 plants relative to the wild-type plants. Moreover, silencing of MPK4 strongly increased the resistance of N. attenuata to M. sexta in a fashion that was independent of COI1 (CORONATINE INSENSITIVE1)-mediated JA signaling. Untargeted metabolomic screening identified several new MPK4-dependent putative defensive compounds against M. sexta. By contrast, silencing of MPK4 did not affect the growth of the generalist insect S. littoralis, and we propose that this was because of the very low levels of fatty acid–amino acid conjugates (FACs) in S. littoralis OS.
• Thus, MPK4 is likely to be a key signaling element that enables plants to tailor defense responses to different attackers.

• Oaks (Quercus spp.), which are major forest trees in the northern hemisphere, host many biotic interactions, but molecular investigation of these interactions is limited by fragmentary genome data. To date, only 75 oak expressed sequence tags (ESTs) have been characterized in ectomycorrhizal (EM) symbioses.
• We synthesized seven beneficial and detrimental biotic interactions between microorganisms and animals and a clone (DF159) of Quercus robur. Sixteen 454 and eight Illumina cDNA libraries from leaves and roots were prepared and merged to establish a reference for RNA-Seq transcriptomic analysis of oak EMs with Piloderma croceum.
• Using the Mimicking Intelligent Read Assembly (MIRA) and Trinity assembler, the OakContigDF159.1 hybrid assembly, containing 65 712 contigs with a mean length of 1003 bp, was constructed, giving broad coverage of metabolic pathways. This allowed us to identify 3018 oak contigs that were differentially expressed in EMs, with genes encoding proline-rich cell wall proteins and ethylene signalling-related transcription factors showing up-regulation while auxin and defence-related genes were down-regulated.
• In addition to the first report of remorin expression in EMs, the extensive coverage provided by the study permitted detection of differential regulation within large gene families (nitrogen, phosphorus and sugar transporters, aquaporins). This might indicate specific mechanisms of genome regulation in oak EMs compared with other trees.

• Shoot elongation is one of the main plastic responses of plants to burial, a ubiquitous stress factor in dry ecosystems. Yet, intraspecific variation in this response to burial and the extent to which this variation is functionally coordinated with variation in other trait responses are largely unknown.
• We subjected seedlings of the shrub Caragana intermedia from 18 maternal parents (i.e. different half-sib families) to repeated partial burial to investigate how burial affects shoot growth, stem mechanical traits and associated plasticity.
• Burial increased both stem elongation and diameter growth of plants, but decreased biomass production. Half-sib families had different rates of shoot elongation, and differed in their response to burial with respect to biomechanical stem properties. Across half-sib families, the magnitude of these responses in mechanical traits was positively correlated with the magnitude of the stem elongation response.
• These results indicate that plasticity in different stem traits in response to sand burial and intraspecific variation therein are functionally coordinated with respect to mechanical stability. The results emphasize the importance of considering functionally coordinated traits when analyzing phenotypic plasticity in plants.

• Shoot branching is a major determinant of plant architecture. Genetic variants for reduced stem branching in the axils of cauline leaves of Arabidopsis were found in some natural accessions and also at low frequency in the progeny of multiparent crosses.
• Detailed genetic analysis using segregating populations derived from backcrosses with the parental lines and bulked segregant analysis was used to identify the allelic variation controlling reduced stem branching.
• Eight quantitative trait loci (QTLs) contributing to natural variation for reduced stem branching were identified (REDUCED STEM BRANCHING 1–8 (RSB1–8)). Genetic analysis showed that RSB6 and RSB7, corresponding to flowering time genes FLOWERING LOCUS C (FLC) and FRIGIDA (FRI), epistatically regulate stem branching. Furthermore, FLOWERING LOCUS T (FT), which corresponds to RSB8 as demonstrated by fine-mapping, transgenic complementation and expression analysis, caused pleiotropic effects not only on flowering time, but, in the specific background of active FRI and FLC alleles, also on the RSB trait.
• The consequence of allelic variation only expressed in late-flowering genotypes revealed novel and thus far unsuspected roles of several genes well characterized for their roles in flowering time control.

Terpenoids constitute a large and diverse class of natural products that serve many functions in nature. Most of the tens of thousands of the discovered terpenoids are synthesized by plants, where they function as primary metabolites involved in growth and development, or as secondary metabolites that optimize the interaction between the plant and its environment. Several plant terpenoids are economically important molecules that serve many applications as pharmaceuticals, pesticides, etc. Major challenges for the commercialization of plant-derived terpenoids include their low production levels in planta and the continuous demand of industry for novel molecules with new or superior biological activities. Here, we highlight several synthetic biology methods to enhance and diversify the production of plant terpenoids, with a foresight towards triterpenoid engineering, the least engineered class of bioactive terpenoids. Increased or cheaper production of valuable triterpenoids may be obtained by ‘classic’ metabolic engineering of plants or by heterologous production of the compounds in other plants or microbes. Novel triterpenoid structures can be generated through combinatorial biosynthesis or directed enzyme evolution approaches. In its ultimate form, synthetic biology may lead to the production of large amounts of plant triterpenoids in in vitro systems or custom-designed artificial biological systems.

• Changes in atmospheric carbon dioxide concentration ([CO₂]) affect plant carbon/water tradeoffs, with implications for drought tolerance. Leaf-level studies often indicate that drought tolerance may increase with rising [CO₂], but integrated leaf and xylem responses are not well understood in this respect. In addition, the influence of the low [CO₂] of the last glacial period on drought tolerance and xylem properties is not well understood.
• We investigated the interactive effects of a broad range of [CO₂] and plant water potentials on leaf function, xylem structure and function and the integration of leaf and xylem function in Phaseolus vulgaris.
• Elevated [CO₂] decreased vessel implosion strength, reduced conduit-specific hydraulic conductance, and compromised leaf-specific xylem hydraulic conductance under moderate drought. By contrast, at glacial [CO₂], transpiration was maintained under moderate drought via greater conduit-specific and leaf-specific hydraulic conductance in association with increased vessel implosion strength.
• Our study involving the integration of leaf and xylem responses suggests that increasing [CO₂] does not improve drought tolerance. We show that, under glacial conditions, changes in leaf and xylem properties could increase drought tolerance, while under future conditions, greater productivity may only occur when higher water use can be accommodated.

• High vein density (D_V) evolution in angiosperms represented a key functional transition. Yet, a mechanistic account on how this hydraulic transformation evolved remains lacking. We demonstrate that a consequence of producing high D_V is that veins must become very small to fit inside the leaf, and that angiosperms are the only clade that evolved the specific type of vessel required to yield sufficiently conductive miniature leaf veins.
• From 111 species spanning key divergences in vascular plant evolution, we show, using analyses of vein conduit evolution in relation to vein packing, that a key xylem innovation associated with high D_V evolution is a strong reduction in vein thickness and simplification of the perforation plates of primary xylem vessels.
• Simple perforation plates in the leaf xylem occurred only in derived angiosperm clades exhibiting high D_V (> 12 mm mm⁻²). Perforation plates in the vessels of other species, including extant basal angiosperms, consisted of resistive scalariform types that were associated with thicker veins and much lower D_V.
• We conclude that a reduction in within-vein conduit resistance allowed vein size to decrease. We suggest that this adaptation may have been a critical evolutionary step that enabled dramatic D_V elaboration in angiosperms.

• Nodulation and arbuscular mycorrhization require the activation of plant host symbiotic programs by Nod factors, and Myc-LCOs and COs, respectively. The pathways involved in the perception and downstream signaling of these signals include common and distinct components. Among the distinct components, NSP1, a GRAS transcription factor, has been considered for years to be specifically involved in nodulation.
• Here, we analyzed the degree of conservation of the NSP1 sequence in arbuscular mycorrhizal (AM) host and non-AM host plants and carefully examined the ability of Medicago truncatula nsp1 mutants to respond to Myc-LCOs and to be colonized by an arbuscular mycorrhizal fungus.
• In AM-host plants, the selection pressure on NSP1 is stronger than in non-AM host ones. The response to Myc-LCOs and the frequency of mycorrhizal colonization are significantly reduced in the nsp1 mutants.
• Our results reveal that NSP1, previously described for its involvement in the Nod factor signaling pathway, is also involved in the Myc-LCO signaling pathway. They bring additional evidence on the evolutionary relatedness between nodulation and mycorrhization.

• Aquaporins are thought to be associated with water transport and play important roles in cotton (Gossypium hirsutum) fibre elongation. Among aquaporins, plasma membrane intrinsic proteins (PIPs) constitute a plasma-membrane-specific subfamily and are further subdivided into PIP1 and PIP2 groups.
• In this study, four fibre-preferential GhPIP2 genes were functionally characterized. The selective interactions among GhPIP2s and their interaction proteins were studied in detail to elucidate the molecular mechanism of cotton fibre development.
• GhPIP2;3 interacted with GhPIP2;4 and GhPIP2;6, but GhPIP2;6 did not interact with GhPIP2;4. Coexpression of GhPIP2;3/2;4 or GhPIP2;3/2;6 resulted in a positive cooperative effect which increased the permeability coefficient of oocytes, while GhPIP2;4/2;6 did not. GhBCP2 (a blue copper-binding protein) inhibited GhPIP2;6 water channel activity through their interaction. Overexpression of GhPIP2 genes in yeast induced longitudinal growth of the host cells. By contrast, knockdown of expression of GhPIP2 genes in cotton by RNA interference markedly hindered fibre elongation.
• In conclusion, GhPIP2 proteins are the primary aquaporin isoforms in fibres. They selectively form hetero-oligomers in order to regulate their activities to meet the requirements for rapid fibre elongation.

• In gynodioecious plants, females are predicted to produce more and/or better offspring than hermaphrodites in order to be maintained in the same population. In the field, the roots of both sexes are usually colonized by arbuscular mycorrhizal (AM) fungi. Transgenerational effects of mycorrhizal symbiosis are largely unknown, although theoretically expected.
• We examined the maternal and paternal effects of AM fungal symbiosis and host sex on seed production and posterior seedling performance in Geranium sylvaticum, a gynodioecious plant. We hand-pollinated cloned females and hermaphrodites in symbiosis with AM fungi or in nonmycorrhizal conditions and measured seed number and mass, and seedling survival and growth in a glasshouse experiment.
• Females produced more seeds than hermaphrodites, but the seeds did not germinate, survive or grow better. Mycorrhizal plants were larger, but did not produce more seeds than nonmycorrhizal plants. Transgenerational parental effects of AM fungi were verified in seedling performance.
• This is the first study to show transgenerational mycorrhiza-mediated parental effects in a gynodioecious species. Mycorrhizal symbiosis affects plant fitness mainly through female functions with enduring effects on the next generation.

• The relative use of new photosynthate compared to stored carbon (C) for the production and maintenance of fine roots, and the rate of C turnover in heterogeneous fine-root populations, are poorly understood.
• We followed the relaxation of a ¹³C tracer in fine roots in a Liquidambar styraciflua plantation at the conclusion of a free-air CO₂ enrichment experiment. Goals included quantifying the relative fractions of new photosynthate vs stored C used in root growth and root respiration, as well as the turnover rate of fine-root C fixed during [CO₂] fumigation.
• New fine-root growth was largely from recent photosynthate, while nearly one-quarter of respired C was from a storage pool. Changes in the isotopic composition of the fine-root population over two full growing seasons indicated heterogeneous C pools; < 10% of root C had a residence time < 3 months, while a majority of root C had a residence time > 2 yr.
• Compared to a one-pool model, a two-pool model for C turnover in fine roots (with 5 and 0.37 yr⁻¹ turnover times) doubles the fine-root contribution to forest NPP (9–13%) and supports the 50% root-to-soil transfer rate often used in models.

• The coding regions of many mitochondrial genes in plants are interrupted by intervening sequences that are classified as group II introns. Their splicing is essential for the expression of the genes they interrupt and hence for respiratory function, and is facilitated by various protein cofactors. Despite the importance of these cofactors, only a few of them have been characterized.
• CRS1-YhbY domain (CRM) is a recently recognized RNA-binding domain that is present in several characterized splicing factors in plant chloroplasts. The Arabidopsis genome encodes 16 CRM proteins, but these are largely uncharacterized.
• Here, we analyzed the intracellular location of one of these hypothetical proteins in Arabidopsis, mitochondrial CAF-like splicing factor 1 (mCSF1; At4 g31010), and analyzed the growth phenotypes and organellar activities associated with mcsf1 mutants in plants.
• Our data indicated that mCSF1 resides within mitochondria and its functions are essential during embryogenesis. Mutant plants with reduced mCSF1 displayed inhibited germination and retarded growth phenotypes that were tightly associated with reduced complex I and IV activities. Analogously to the functions of plastid-localized CRM proteins, analysis of the RNA profiles in wildtype and mcsf1 plants showed that mCSF1 acts in the splicing of many of the group II intron RNAs in Arabidopsis mitochondria.

• Spores of homosporous ferns are small, wind-borne and thus have the potential for long-distance dispersal. This common perception has led to a prediction of near-random spatial genetic structure within fern populations. Spore dispersal and spore bank studies, however, indicate that most spores fall close to the maternal plant (< 5 m), supporting a prediction of significant fine-scale genetic structure (FSGS) within populations.
• To determine which of these two hypotheses is more likely to occur in nature, we measured inbreeding and quantified the spatial distribution of individuals and allozyme-based genotypes using spatial autocorrelation methods within four populations of the fern Cyrtomium falcatum in southern South Korea.
• Inbreeding levels were low, and all populations exhibited significant aggregation of individuals and strong FSGS.
• The present results support the second hypothesis, and the substantial FSGS in C. falcatum could reflect the unique features of most homosporous ferns (outcrossing mating systems that lead a majority of spores to occur at short distances and a very limited dispersal distance of male gametes). Although fern spores are physically analogous to orchid seeds, the intensity of FSGS exhibited in C. falcatum is four times stronger than that in 16 terrestrial orchid species.

• The processes by which the functions of interdependent tissues are coordinated as lineages diversify are poorly understood.
• Here, we examine evolutionary coordination of vascular, epidermal and cortical leaf tissues in the anatomically, ecologically and morphologically diverse woody plant family Proteaceae.
• We found that, across the phylogenetic range of Proteaceae, the sizes of guard, epidermal, palisade and xylem cells were positively correlated with each other but negatively associated with vein and stomatal densities. The link between venation and stomata resulted in a highly efficient match between potential maximum water loss (determined by stomatal conductance) and the leaf vascular system's capacity to replace that water. This important linkage is likely to be driven by stomatal size, because spatial limits in the packing of stomata onto the leaf surface apparently constrain the maximum size and density of stomata.
• We conclude that unified evolutionary changes in cell sizes of independent tissues, possibly mediated by changes in genome size, provide a means of substantially modifying leaf function while maintaining important functional links between leaf tissues. Our data also imply the presence of alternative evolutionary strategies involving cellular miniaturization during radiation into closed forest, and cell size increase in open habitats.

• The restoration fertility complex (RFC) was previously identified in Honglian (HL)-cytoplasmic male sterility (CMS) rice (Oryza sativa), and glycine-rich protein 162 (GRP162) is responsible for binding to the CMS-associated transcript atp6-orfH79.
• Here, we engineered a recombinant GRP162 containing the mitochondrial transit peptide, termed Mt-GRP162, as an artificial restorer of fertility (Rf) gene. Mt-GRP162 was confirmed to bind to CMS-associated RNA and to localize to the mitochondria. The transgenic plants showed restored fertility with partially functional pollen.
• We found that the expression of ORFH79 decreased in transgenic plants, while the expression of atp6-orfH79 was not changed. These findings indicate that Mt-GRP162 restores fertility by suppressing the expression of the cytotoxic protein ORFH79 at the post-transcriptional level rather than via the cleavage of atp6-orfH79 in the presence of RFC.
• These findings contribute to our understanding of the mechanisms of restoration through diverse pathways.

• Autophagy is present at a basal level in all plant tissues and is induced during leaf ageing and in response to nitrogen (N) starvation. Nitrogen remobilization from the rosette to the seeds is impaired in autophagy mutants. This report focuses on the role of autophagy in leaf N management and proteolysis during plant ageing.
• Metabolites, enzyme activities and protein contents were monitored in several autophagy-defective (atg) Arabidopsis mutants grown under low and high nitrate conditions.
• Results showed that carbon (C) and N statuses were affected in atg mutants before any senescence symptoms appeared. atg mutants accumulated larger amounts of ammonium, amino acids and proteins than wild type, and were depleted in sugars. Over-accumulation of proteins in atg mutants was selective and occurred despite higher endopeptidase and carboxypeptidase activities. Specific over-accumulation of the ribosomal proteins S6 and L13 subunits, and of catalase and glutamate dehydrogenase proteins was observed. atg mutants also accumulated peptides putatively identified as degradation products of the Rubisco large subunit and glutamine synthetase 2 (GS2). Incomplete chloroplast protein degradation resulting from autophagy defects could explain the higher N concentrations measured in atg rosettes and defects in N remobilization.
• It is concluded that autophagy controls C : N status and protein content in leaves of Arabidopsis.

• Transcription factors belonging to the APETALA2/Ethylene Responsive Factor (AP2/ERF) family are conservatively widespread in the plant kingdom. These regulatory proteins are involved in the control of primary and secondary metabolism, growth and developmental programs, as well as responses to environmental stimuli. Due to their plasticity and to the specificity of individual members of this family, AP2/ERF transcription factors represent valuable targets for genetic engineering and breeding of crops. In this review, we integrate the evidence collected from functional and structural studies to describe their different mechanisms of action and the regulatory pathways that affect their activity.

• Chinese fir (Cunninghamia lanceolata), a commercially important tree for the timber and pulp industry, is widely distributed in southern China and northern Vietnam, but its large and complex genome has hindered the development of genomic resources. Few efforts have focused on analysis of the modulation of transcriptional networks in vascular cambium during the transition from active growth to dormancy in conifers.
• Here, we used Illumina sequencing to analyze the global transcriptome alterations at the different stages of vascular cambium development in Chinese fir.
• By analyzing dynamic changes in the transcriptome of vascular cambium based on our RNA sequencing (RNA-Seq) data at the dormant, reactivating and active stages, many potentially interesting genes were identified that encoded putative regulators of cambial activity, cell division, cell expansion and cell wall biosynthesis and modification. In particular, the genes involved in transcriptional regulation and hormone signaling were highlighted to reveal their biological importance in the cambium development and wood formation.
• Our results reveal the dynamics of transcriptional networks and identify potential key components in the regulation of vascular cambium development in Chinese fir, which will contribute to the in-depth study of cambial differentiation and wood-forming candidate genes in conifers.

• Artemisia annua, which produces the anti-malaria compound artemisinin, occurs as high-artemisinin production (HAP) and low-artemisinin production (LAP) chemotypes. Understanding the basis of the difference between these chemotypes would assist breeding and optimising artemisinin biosynthesis.
• Here we present a systematic comparison of artemisinin biosynthesis genes that may be involved in determining the chemotype (CYP71AV1, DBR2 and ALDH1). These genes were isolated from the two chemotypes and characterized using transient expression in planta. The enzyme activity of DBR2 and ALDH1 from the two chemotypes did not differ, but structural differences in CYP71AV1 from LAP and HAP chemotypes (AMOLAP and AMOHAP, respectively) resulted in altered enzyme activity.
• AMOLAP displays a seven amino acids N-terminal extension compared with AMOHAP. The GFP fusion of both proteins show equal localization to the ER but AMOHAP may have reduced stability.
• Upon transient expression in Nicotiana benthamiana, AMOLAP displayed a higher enzyme activity than AMOHAP. However, expression in combination with the other pathway genes also resulted in a qualitatively different product profile (‘chemotype’); that is, in a shift in the ratio between the unsaturated and saturated (dihydro) branch of the pathway.

• Increasing atmospheric CO₂ concentrations alter leaf physiology, with effects that cascade to communities and ecosystems. Yet, responses over cycles of disturbance and recovery are not well known, because most experiments span limited ecological time. We examined the effects of CO₂ on root growth, herbivory and arthropod biodiversity in a woodland from 1996 to 2006, and the legacy of CO₂ enrichment on these processes during the year after the CO₂ treatment ceased.
• We used minirhizotrons to study root growth, leaf censuses to study herbivory and pitfall traps to determine the effects of elevated CO₂ on arthropod biodiversity.
• Elevated CO₂ increased fine root biomass, but decreased foliar nitrogen and herbivory on all plant species. Insect biodiversity was unchanged in elevated CO₂. Legacy effects of elevated CO₂ disappeared quickly as fine root growth, foliar nitrogen and herbivory levels recovered in the next growing season following the cessation of elevated CO₂.
• Although the effects of elevated CO₂ cascade through plants to herbivores, they do not reach other trophic levels, and biodiversity remains unchanged. The legacy of 10 yr of elevated CO₂ on plant–herbivore interactions in this system appear to be minimal, indicating that the effects of elevated CO₂ may not accumulate over cycles of disturbance and recovery.

• Ralstonia solanacearum is a major soilborne pathogen that attacks > 200 plant species, including major crops. To characterize MtQRRS1, a major quantitative trait locus (QTL) for resistance towards this bacterium in the model legume Medicago truncatula, genetic and functional approaches were combined.
• QTL analyses together with disease scoring of heterogeneous inbred families were used to define the locus. The candidate region was studied by physical mapping using a bacterial artificial chromosome (BAC) library of the resistant line, and sequencing. In planta bacterial growth measurements, grafting experiments and gene expression analysis were performed to investigate the mechanisms by which this locus confers resistance to R. solanacearum.
• The MtQRRS1 locus was localized to the same position in two recombinant inbred line populations and was narrowed down to a 64 kb region. Comparison of parental line sequences revealed 15 candidate genes with sequence polymorphisms, but no evidence of differential gene expression upon infection. A role for the hypocotyl in resistance establishment was shown.
• These data indicate that the quantitative resistance to bacterial wilt conferred by MtQRRS1, which contains a cluster of seven R genes, is shared by different accessions and may act through intralocus interactions to promote resistance.

• Plants turn on induced defenses upon insect herbivory. In the current study, we evaluated the role of European corn borer (ECB) elicitors (molecules secreted by herbivores) that either induce/suppress defenses in Solanum lycopersicum (tomato) and Zea mays (maize), two very important crop plants that are grown for food and/or fuel throughout the world.
• We used a combination of molecular, biochemical, confocal and scanning electron microscopy, caterpillar spinneret ablation/cauterization, and conventional insect bioassay methods to determine the role of ECB elicitors in modulating defenses in both tomato and maize crop plants.
• Our results clearly demonstrate that the components present in the ECB saliva induce defense-related proteinase inhibitors in both tomato (PIN2) and maize (MPI). Presence of glucose oxidase in the ECB saliva induced defenses in tomato, but not in maize. However, ECB saliva induced genes present in the jasmonic acid biosynthesis pathway in both tomato and maize.
• Although ECB saliva can induce defenses in both tomato and maize, our results suggest that host-specific salivary components are responsible for inducing host plant defenses. Proteomic analysis of ECB salivary elicitors and plant receptors/signaling mechanisms involved in recognizing different ECB elicitors remains to be determined.

• C₄ plants display higher cyclic electron transport activity than C₃ plants. This activity is suggested to be important for the production of ATPs required for C₄ metabolism.
• To understand the process by which photosystem I (PSI) cyclic electron transport was promoted during C₄ evolution, we conducted comparative analyses of the functionality of PSI cyclic electron transport among members of the genus Flaveria, which contains several C₃, C₃–C₄ intermediate, C₄-like and C₄ species.
• The abundance of NDH-H, a subunit of NADH dehydrogenase-like complex, increased markedly in bundle sheath cells with the activity of the C₄ cycle. By contrast, PROTON GRADIENT REGULATION5 (PGR5) and PGR5-LIKE1 increased in both mesophyll and bundle sheath cells in C₄-like Flaveria palmeri and C₄ species. Grana stacks were drastically reduced in bundle sheath chloroplasts of C₄-like F. palmeri and C₄ species; these species showed a marked increase in PSI cyclic electron transport activity.
• These results suggest that both the expression of proteins involved in PSI cyclic electron transport and changes in thylakoid structure contribute to the high activity of cyclic electron flow in NADP–malic enzyme-type C₄ photosynthesis. We propose that these changes were important for the establishment of C₄ photosynthesis from C₃–C₄ intermediate photosynthesis in Flaveria.

• Deficiency of abscisic acid (ABA) in the sitiens mutant of tomato (Solanum lycopersicum) culminates in increased resistance to Botrytis cinerea through a rapid epidermal hypersensitive response (HR) and associated phenylpropanoid pathway-derived cell wall fortifications. This study focused on understanding the role of primary carbon : nitrogen (C : N) metabolism in the resistance response of sitiens to B. cinerea. How alterations in C : N metabolism are linked with the HR-mediated epidermal arrest of the pathogen has been also investigated.
• Temporal alterations in the γ-aminobutyric acid (GABA) shunt, glutamine synthetase/glutamate synthase (GS/GOGAT) cycle and phenylpropanoid pathway were transcriptionally, enzymatically and metabolically monitored in both wild-type and sitiens plants. Virus-induced gene silencing, microscopic analyses and pharmacological assays were used to further confirm the data.
• Our results on the sitiens–B. cinerea interaction favor a model in which cell viability in the cells surrounding the invaded tissue is maintained by a constant replenishment of the tricarboxylic acid (TCA) cycle through overactivation of the GS/GOGAT cycle and the GABA shunt, resulting in resistance through both tightly controlling the defense-associated HR and slowing down the pathogen-induced senescence.
• Collectively, this study shows that maintaining cell viability via alterations in host C : N metabolism plays a vital role in the resistance response against necrotrophic pathogens.

• Plants have evolved efficient defence mechanisms to defend themselves from pathogen attack. Although many studies have focused on the transcriptional regulation of defence responses, less is known about the involvement of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in plant immunity. This work investigates miRNAs that are regulated by elicitors from the blast fungus Magnaporthe oryzae in rice (Oryza sativa).
• Small RNA libraries were constructed from rice tissues and subjected to high-throughput sequencing for the identification of elicitor-responsive miRNAs. Target gene expression was examined by microarray analysis. Transgenic lines were used for the analysis of miRNA functioning in disease resistance.
• Elicitor treatment is accompanied by dynamic alterations in the expression of a significant number of miRNAs, including new members of annotated miRNAs. Novel miRNAs from rice are proposed. We report a new rice miRNA, osa-miR7695, which negatively regulates an alternatively spliced transcript of OsNramp6 (Natural resistance-associated macrophage protein 6). This novel miRNA experienced natural and domestication selection events during evolution, and its overexpression in rice confers pathogen resistance.
• This study highlights an miRNA-mediated regulation of OsNramp6 in disease resistance, whilst illustrating the existence of a novel regulatory network that integrates miRNA function and mRNA processing in plant immunity.

• ALTERED MERISTEM PROGRAM1 (AMP1) encodes a glutamate carboxypeptidase that plays an important role in shoot apical meristem development and phytohormone homeostasis.
• We isolated a new mutant allele of AMP1, amp1-20, from a screen for abscisic acid (ABA) hypersensitive mutants and characterized the function of AMP1 in plant stress responses.
• amp1 mutants displayed ABA hypersensitivity, while overexpression of AMP1 caused ABA insensitivity. Moreover, endogenous ABA concentration was increased in amp1-20- and decreased in AMP1-overexpressing plants under stress conditions. Application of ABA reduced the AMP1 protein level in plants. Interestingly, amp1 mutants accumulated excess superoxide and displayed hypersensitivity to oxidative stress. The hypersensitivity of amp1 to ABA and oxidative stress was partially rescued by reactive oxygen species (ROS) scavenging agent. Furthermore, amp1 was tolerant to freezing and drought stress. The ABA hypersensitivity and freezing tolerance of amp1 was dependent on ABA signaling. Moreover, amp1 had elevated soluble sugar content and showed hypersensitivity to high concentrations of sugar. By contrast, the contents of amino acids were changed in amp1 mutant compared to the wild-type.
• This study suggests that AMP1 modulates ABA, oxidative and abotic stress responses, and is involved in carbon and amino acid metabolism in Arabidopsis.

• B chromosomes (Bs) are supernumerary chromosomes that vary in number among individuals of the same species. Because of their dispensable nature, their non-Mendelian inheritance and their origin from A chromosomes (As), one might assume that Bs followed a different evolutionary pathway from As, this being reflected in differences in their high-copy DNA constitution. We provide detailed insight into the composition and distribution of rye (Secale cereale) B-located high-copy sequences.

• A- and B-specific high-copy sequences were identified in silico. Mobile elements and satellite sequences were verified by fluorescence in situ hybridization (FISH). Replication was analyzed via EdU incorporation.

• Although most repeats are similarly distributed along As and Bs, several transposons are either amplified or depleted on the B. An accumulation of B-enriched satellites was found mostly in the nondisjunction control region of the B, which is transcriptionally active and late-replicating. All B-enriched sequences are not unique to the B but are also present in other Secale species, suggesting the origin of the B from As of the same genus.

• Our findings highlight the differences between As and Bs. Although Bs originated from As, they have since taken a separate evolutionary pathway.

• Cytosine methylation is an epigenetic mark that promotes gene silencing and plays an important role in genome defence against transposons and invading DNA viruses. Previous data showed that the largest family of single-stranded DNA viruses, Geminiviridae, prevents methylation-mediated transcriptional gene silencing (TGS) by interfering with the proper functioning of the plant methylation cycle.
• Here, we describe a novel counter-defence strategy used by geminiviruses, which reduces the expression of the plant maintenance DNA methyltransferases, METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE 3 (CMT3), in both locally and systemically infected tissues.
• We demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widespread among geminivirus species. Additionally, we identified Rep (Replication associated protein) as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 down-regulation. The presence of Rep suppressed TGS of an Arabidopsis thaliana transgene and of host loci whose expression was strongly controlled by CG methylation. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites.
• Our findings suggest that Rep, the only viral protein essential for replication, displays TGS suppressor activity through a mechanism distinct from that thus far described for geminiviruses.

• The Arctic is already experiencing changes in plant community composition, so understanding the contribution of different vegetation components to carbon (C) cycling is essential in order to accurately quantify ecosystem C balance. Mosses contribute substantially to biomass, but their impact on carbon use efficiency (CUE) – the proportion of gross primary productivity (GPP) incorporated into growth – and aboveground versus belowground C partitioning is poorly known.
• We used ¹³C pulse-labelling to trace assimilated C in mosses (Sphagnum sect. Acutifolia and Pleurozium schreberi) and in dwarf shrub–P. schreberi vegetation in sub-Arctic Finland. Based on ¹³C pools and fluxes, we quantified the contribution of mosses to GPP, CUE and partitioning.
• Mosses incorporated 20 ± 9% of total ecosystem GPP into biomass. CUE of Sphagnum was 68–71%, that of P. schreberi was 62–81% and that of dwarf shrub–P. schreberi vegetation was 58–74%. Incorporation of C belowground was 10 ± 2% of GPP, while vascular plants alone incorporated 15 ± 4% of their fixed C belowground.
• We have demonstrated that mosses strongly influence C uptake and retention in Arctic dwarf shrub vegetation. They increase CUE, and the fraction of GPP partitioned aboveground. Arctic C models must include mosses to accurately represent ecosystem C dynamics.

• Species with extensive ranges experience highly variable environments with respect to temperature, light and soil moisture. Synchronizing the transition from vegetative to floral growth is important to employ favorable conditions for reproduction. Optimal timing of this transition might be different for semelparous annual plants and iteroparous perennial plants.
• We studied variation in the critical photoperiod necessary for floral induction and the requirement for a period of cold-chilling (vernalization) in 46 populations of annuals and perennials in the Mimulus guttatus species complex. We then examined critical photoperiod and vernalization QTLs in growth chambers using F₂ progeny from annual and perennial parents that differed in their requirements for flowering.
• We identify extensive variation in critical photoperiod, with most annual populations requiring substantially shorter day lengths to initiate flowering than perennial populations. We discover a novel type of vernalization requirement in perennial populations that is contingent on plants experiencing short days first. QTL analyses identify two large-effect QTLs which influence critical photoperiod. In two separate vernalization experiments we discover each set of crosses contain different large-effect QTLs for vernalization.
• Mimulus guttatus harbors extensive variation in critical photoperiod and vernalization that may be a consequence of local adaptation.

• The fractionation of stable copper (Cu) isotopes during uptake into plant roots and translocation to shoots can provide information on Cu acquisition mechanisms.
• Isotope fractionation (⁶⁵Cu/⁶³Cu) and intact tissue speciation techniques (X-ray absorption spectroscopy, XAS) were used to examine the uptake, translocation and speciation of Cu in strategy I (tomato–Solanum lycopersicum) and strategy II (oat–Avena sativa) plant species. Plants were grown in controlled solution cultures, under varied iron (Fe) conditions, to test whether the stimulation of Fe-acquiring mechanisms can affect Cu uptake in plants.
• Isotopically light Cu was preferentially incorporated into tomatoes (Δ⁶⁵Cu_{whole plant-solution} = c. −1‰), whereas oats showed minimal isotopic fractionation, with no effect of Fe supply in either species. The heavier isotope was preferentially translocated to shoots in tomato, whereas oat plants showed no significant fractionation during translocation. The majority of Cu in the roots and leaves of both species existed as sulfur-coordinated Cu(I) species resembling glutathione/cysteine-rich proteins.
• The presence of isotopically light Cu in tomatoes is attributed to a reductive uptake mechanism, and the isotopic shifts within various tissues are attributed to redox cycling during translocation. The lack of isotopic discrimination in oat plants suggests that Cu uptake and translocation are not redox selective.

• The spatiotemporal dynamics of, and interspecific differences in, the acquisition of litter-derived nitrogen (N) by natural assemblages of ectomycorrhizal root tips are poorly understood.
• Small cylindrical mesh bags containing ¹⁵N-labelled beech (Fagus sylvatica) leaf litter that permit hyphal but not root ingrowth were inserted vertically into the top soil layer of an old-growth beech forest. The lateral transfer of ¹⁵N into the circumjacent soil, roots, microbes and ectomycorrhizas was measured during an 18-month exposure period.
• Ectomycorrhial fungi (EMF) showed large interspecific variation in the temporal pattern and extent of ¹⁵N accumulation. Initially, when N was mainly available from the leachate, microbes were more efficient at N immobilization than the majority of EMF, but distinct fungal species also showed significant ¹⁵N accumulation. During later phases, the enrichment of ¹⁵N in Tomentella badia was higher than in microbes and other EMF species. Roots and soil accumulated ¹⁵N with a large delay compared with microbes and EMF.
• Because approximately half of the studied fungal species had direct access to N from leaf litter and the remainder to N from leached compounds, we suggest that EMF diversity facilitates the N utilization of the host by capturing N originating from early-released solutes and late degradation products from a recalcitrant source.

• Plants form ubiquitous associations with diverse microbes. These interactions range from parasitism to mutualism, depending partly on resource supplies that are being altered by global change. While many studies have considered the separate effects of pathogens and mutualists on their hosts, few studies have investigated interactions among microbial mutualists and pathogens in the context of global change.
• Using two wild grass species as model hosts, we grew individual plants under ambient or elevated CO₂, and ambient or increased soil phosphorus (P) supply. Additionally, individuals were grown with or without arbuscular mycorrhizal inoculum, and after 2 wk, plants were inoculated or mock-inoculated with a phloem-restricted virus.
• Under elevated CO₂, mycorrhizal association increased the titer of virus infections, and virus infection reciprocally increased the colonization of roots by mycorrhizal fungi. Additionally, virus infection decreased plant allocation to root biomass, increased leaf P, and modulated effects of CO₂ and P addition on mycorrhizal root colonization.
• These results indicate that plant mutualists and pathogens can alter each other's success, and predict that these interactions will respond to increased resource availability and elevated CO_2. Together, our findings highlight the importance of interactions among multiple microorganisms for plant performance under global change.

• Plant water status and hydraulics were measured in six woody angiosperms growing in a karstic woodland, during an extreme summer drought. Our aim was to take advantage of an unusual climatic event to identify key traits related to species-specific drought damage.
• The damage suffered by different species was assessed in terms of percentage of individuals showing extensive crown desiccation. Stem water potential (Ψ_stem) and percent loss of hydraulic conductivity (PLC) were measured in healthy and desiccated individuals. Vulnerability to cavitation was assessed in terms of stem water potential inducing 50% PLC (Ψ₅₀). Stem density (ρ_stem) was also measured.
• Species-specific percentage of desiccated individuals was correlated to Ψ₅₀ and ρ_stem. Crown desiccation was more widespread in species with less negative Ψ₅₀ and lower ρ_stem. Desiccated individuals had lower Ψ_stem and higher PLC than healthy ones, suggesting that hydraulic failure was an important mechanism driving shoot dieback. Drought-vulnerable species showed lower safety margins (Ψ_stem − Ψ₅₀) than resistant ones.
• The Ψ₅₀, safety margins and ρ_stem values emerge as convenient traits to be used for tentative predictions of differential species-specific impact of extreme drought events on a local scale. The possibility that carbohydrate depletion was also involved in induction of desiccation symptoms is discussed.

Understanding the context dependence of ecosystem responses to global changes requires the development of new conceptual frameworks. Here we propose a framework for considering how tree species and their mycorrhizal associates differentially couple carbon (C) and nutrient cycles in temperate forests. Given that tree species predominantly associate with a single type of mycorrhizal fungi (arbuscular mycorrhizal (AM) fungi or ectomycorrhizal (ECM) fungi), and that the two types of fungi differ in their modes of nutrient acquisition, we hypothesize that the abundance of AM and ECM trees in a plot, stand, or region may provide an integrated index of biogeochemical transformations relevant to C cycling and nutrient retention. First, we describe how forest plots dominated by AM tree species have nutrient economies that differ in their C–nutrient couplings from those in plots dominated by ECM trees. Secondly, we demonstrate how the relative abundance of AM and ECM trees can be used to estimate nutrient dynamics across the landscape. Finally, we describe how our framework can be used to generate testable hypotheses about forest responses to global change factors, and how these dynamics can be used to develop better representations of plant–soil feedbacks and nutrient constraints on productivity in ecosystem and earth system models.

• Pathogenic oomycetes have evolved RXLR effectors to thwart plant defense mechanisms and invade host tissues. We analysed the function of one of these effectors (Penetration-Specific Effector 1 (PSE1)) whose transcript is transiently accumulated during penetration of host roots by the oomycete Phytophthora parasitica.
• Expression of PSE1 protein in tobacco (Nicotiana tabacum and Nicotiana benthamiana) leaves and in Arabidopsis thaliana plants was used to assess the role of this effector in plant physiology and in interactions with pathogens. A pharmacological approach and marker lines were used to charcterize the A. thaliana phenotypes.
• Expression of PSE1 in A. thaliana led to developmental perturbations associated with low concentrations of auxin at the root apex. This modification of auxin content was associated with an altered distribution of the PIN4 and PIN7 auxin efflux carriers. The PSE1 protein facilitated plant infection: it suppressed plant cell death activated by Pseudomonas syringae avirulence gene AvrPto and Phytophthora cryptogea elicitin cryptogein in tobacco and exacerbated disease symptoms upon inoculation of transgenic A. thaliana plantlets with P. parasitica in an auxin-dependant manner.
• We propose that P. parasitica secretes the PSE1 protein during the penetration process to favour the infection by locally modulating the auxin content. These results support the hypothesis that effectors from plant pathogens may act on a limited set of targets, including hormones.

• Drought-induced defoliation has recently been associated with the depletion of carbon reserves and increased mortality risk in Scots pine (Pinus sylvestris). We hypothesize that defoliated individuals are more sensitive to drought, implying that potentially higher gas exchange (per unit of leaf area) during wet periods may not compensate for their reduced photosynthetic area.
• We measured sap flow, needle water potentials and whole-tree hydraulic conductance to analyse the drought responses of co-occurring defoliated and nondefoliated Scots pines in northeast Spain during typical (2010) and extreme (2011) drought conditions.
• Defoliated Scots pines showed higher sap flow per unit leaf area during spring, but were more sensitive to summer drought, relative to nondefoliated pines. This pattern was associated with a steeper decline in soil-to-leaf hydraulic conductance with drought and an enhanced sensitivity of canopy conductance to soil water availability. Near-homeostasis in midday water potentials was observed across years and defoliation classes, with minimum values of −2.5 MPa. Enhanced sensitivity to drought and prolonged periods of near-zero gas exchange were consistent with low levels of carbohydrate reserves in defoliated trees.
• Our results support the critical links between defoliation, water and carbon availability, and their key roles in determining tree survival and recovery under drought.

Recent advances in the partitioning of autotrophic from heterotrophic respiration processes in soils in conjunction with new high temporal resolution soil respiration data sets offer insights into biotic and environmental controls of respiration. Besides temperature, many emerging controlling factors have not yet been incorporated into ecosystem-scale models. We synthesize recent research that has partitioned soil respiration into its process components to evaluate effects of nitrogen, temperature and photosynthesis on autotrophic flux from soils at the ecosystem level. Despite the widely used temperature dependence of root respiration, gross primary productivity (GPP) can explain most patterns of ecosystem root respiration (and to some extent heterotrophic respiration) at within-season time-scales. Specifically, heterotrophic respiration is influenced by a seasonally variable supply of recent photosynthetic products in the rhizosphere. The contribution of stored root carbon (C) to root respiratory fluxes also varied seasonally, partially decoupling the proportion of photosynthetic C driving root respiration. In order to reflect recent insights, new hierarchical models, which incorporate root respiration as a primary function of GPP and which respond to environmental variables by modifying C allocation belowground, are needed for better prediction of future ecosystem C sequestration.

• We present an intra-annual stable carbon isotope (δ¹³C) study based on a labeling experiment to illustrate differences in temporal patterns of recent carbon allocation to wood structures of two functional types of trees, Podocarpus falcatus (a late-successional evergreen conifer) and Croton macrostachyus (a deciduous broadleaved pioneer tree), in a tropical mountain forest in Ethiopia.
• Dendrometer data, wood anatomical thin sections, and intra-annual δ¹³C analyses were applied.
• Isotope data revealed a clear annual growth pattern in both studied species. For P. falcatus, it was possible to synchronize annual δ¹³C peaks, wood anatomical structures and monthly precipitation patterns. The labeling signature was evident for three consecutive years. For C. macrostachyus, isotope data illustrate a rapid decline of the labeling signal within half a year.
• Our δ¹³C labeling study indicates a distinct difference in carryover effects between trees of different functional types. A proportion of the labeled δ¹³C is stored in reserves of wood parenchyma for up to 3 yr in P. falcatus. By contrast, C. macrostachyus shows a high turnover of assimilates and a carbon carryover effect is only detectable in the subsequent year.

• Introduced plants may quickly evolve new adaptive traits upon their introduction. Canada thistle (Cirsium arvense – Cardueae, Asteraceae) is one of the worst invasive weeds worldwide. The goal of this study is to compare gene expression profiles of native (European) and introduced (North American) populations of this species, to elucidate the genetic mechanisms that may underlie such rapid adaptation.
• We explored the transcriptome of ten populations (five per range) of C. arvense in response to three treatments (control, nutrient deficiency and shading) using a customized microarray chip containing 63 690 expressed sequence tags (ESTs), and verified the expression level of 13 loci through real-time quantitative PCR.
• Only 2116 ESTs (3.5%) were found to be differentially expressed between the ranges, and 4458 ESTs (7.1%) exhibited a significant treatment-by-range effect. Among them was an overrepresentation of loci involved in stimulus and stress responses.
• Cirsium arvense has evolved different life history strategies on each continent. The two ranges notably differ with regard to R-protein mediated defence, sensitivity to abiotic stresses, and developmental timing. The fact that genotypes from the Midwest exhibit different expression kinetics than remaining North American samples further corroborates the hypothesis that the New World has been colonized twice, independently.

• Pathogen infection of higher plants often induces rapid production of phosphatidic acid (PA) and changes in lipid profiles, but the enzymatic basis and the function of the lipid change in pathogen–plant interactions are not well understood.
• Infection of phospholipase D β1 (PLDβ1)-deficient plants by Pseudomonas syringae tomato pv DC3000 (Pst DC30000) resulted in less bacterial growth than in wild-type plants, and the effect was more profound in virulent Pst DC3000 than avirulent Pst DC3000 (carrying the avirulence gene avrRpt2) infection. The expression levels of salicylic acid (SA)-inducible genes were higher, but those inducible by jasmonic acid (JA) showed lower expression in PLDβ1 mutants than in wild-type plants.
• However, PLDβ1-deficient plants were more susceptible than wild-type plants to the fungus Botrytis cinerea. The PLDβ1-deficient plants had lower levels of PA, JA and JA-related defense gene expression after B. cinerea inoculation.
• PLDβ1 plays a positive role in pathogen-induced JA production and plant resistance to the necrotrophic fungal pathogen B. cinerea, but a negative role in the SA-dependent signaling pathway and plant tolerance to infection with biotrophic Pst DC3000. PLDβ1 is responsible for most of the increase in PA production in response to necrotrophic B. cinerea and virulent Pst DC3000 infection, but contributes less to avirulent Pst DC3000 (avrRpt2)-induced PA production.

• The genetic structure of ectomycorrhizal (ECM) fungal populations results from both vegetative and sexual propagation. In this study, we have analysed the spatial genetic structure of Tuber melanosporum populations, a heterothallic ascomycete that produces edible fruit bodies.
• Ectomycorrhizas from oaks and hazels from two orchards were mapped and genotyped using simple sequence repeat markers and the mating type locus. The distribution of the two T. melanosporum mating types was also monitored in the soil. In one orchard, the genetic profiles of the ascocarps were compared with those of the underlying mycorrhizas.
• A pronounced spatial genetic structure was found. The maximum genet sizes were 2.35 and 4.70 m in the two orchards, with most manifesting a size < 1 m. Few genets persisted throughout two seasons. A nonrandom distribution pattern of the T. melanosporum was observed, resulting in field patches colonized by genets that shared the same mating types.
• Our findings suggest that competition occurs between genets and provide basic information on T. melanosporum propagation patterns that are relevant for the management of productive truffle orchards.

• The family Geraniaceae is characterized by a beak-like fruit, consisting of five seeds appended by a tapering awn. The awns exhibit coiling or bending hygroscopic movement as part of the seed dispersal strategy. Here we explain the variation in the hygroscopic reaction based on structural principles.
• We examined five representative species from three genera: Erodium, Geranium, and Pelargonium. Using X-ray diffraction, and electron and polarized light microscopy, we measured the cellulose microfibril angles in relation to the cell and cellulose helix axes. The behavior of separated single cells during dehydration was also examined.
• A bi-layered structure characterizes all the representative genera studied, with a hygroscopically contracting inner layer, and a stiff outer layer. We found that the cellulose arrangement in the inner layer is responsible for the type of awn deformation (coiling or bending). In three of the five awns examined, we identified an additional coiling outer sublayer, which adds coiling deformation to the awn.
• We divide the movements into three types: bending, coiling, and coiled-bending. All movement types are found in the Geranium genus. These characteristics are of importance for understanding the evolution of seed dispersal mechanisms in the Geraniaceae family.

• Allotetraploid speciation, that is, the generation of a hybrid tetraploid species from two diploid species, and the long-term evolution of tetraploid populations and species are important in plants. We developed a population genetic model to infer population genetic parameters of tetraploid populations from data of the progenitor and descendant species.
• Two yarrow species, Achillea alpina-4x and A. wilsoniana-4x, arose by allotetraploidization from the diploid progenitors, A. acuminata-2x and A. asiatica-2x. Yet, the population genetic process has not been studied in detail. We applied the model to sequences of three nuclear genes in populations of the four yarrow species and compared their pattern of variability with that in four plastid regions.
• The plastid data indicated that the two tetraploid species probably originated from multiple independent allopolyploidization events and have accumulated many mutations since. With the nuclear data, we found a low rate of homeologous recombination or gene conversion and a reduction in diversity relative to the level of both diploid species combined.
• The present analysis with a novel probabilistic model suggests a genetic bottleneck during tetraploid speciation, that the two tetraploid species have a long evolutionary history, and that they have a small amount of genetic exchange between the homeologous genomes.

• The development of nematode feeding sites induced by root-knot nematodes involves the synchronized activation of cell cycle processes such as acytokinetic mitoses and DNA amplification. A number of key cell cycle genes are reported to be critical for nematode feeding site development. However, it remains unknown whether plant cyclin-dependent kinase (CDK) inhibitors such as the Arabidopsis interactor/inhibitor of CDK (ICK)/Kip-related protein (KRP) family are involved in nematode feeding site development. This study demonstrates the involvement of Arabidopsis ICK2/KRP2 and ICK1/KRP1 in the control of mitosis to endoreduplication in galls induced by the root-knot nematode Meloidogyne incognita.
• Using ICK/KRP promoter-GUS fusions and mRNA in situ hybridizations, we showed that ICK2/KRP2, ICK3/KRP5 and ICK4/KRP6 are expressed in galls after nematode infection. Loss-of-function mutants have minor effects on gall development and nematode reproduction. Conversely, overexpression of both ICK1/KRP1 and ICK2/KRP2 impaired mitosis in giant cells and blocked neighboring cell proliferation, resulting in a drastic reduction of gall size.
• Studying the dynamics of protein expression demonstrated that protein levels of ICK2/KRP2 are tightly regulated during giant cell development and reliant on the presence of the nematode.
• This work demonstrates that impeding cell cycle progression by means of ICK1/KRP1 and ICK2/KRP2 overexpression severely restricts gall development, leading to a marked limitation of root-knot nematode development and reduced numbers of offspring.

• Hemlock woolly adelgid (HWA) is an exotic insect pest causing severe decimation of native hemlock trees. Extensive research has been conducted on the ecological impacts of HWA, but the exact physiological mechanisms that cause mortality are not known.
• Water relations, anatomy and gas exchange measurements were assessed on healthy and infested eastern (Tsuga canadensis) and Carolina (Tsuga caroliniana) hemlock trees. These data were then used in a mechanistic model to test whether the physiological responses to HWA infestation were sufficiently significant to induce changes in whole-plant water use and carbon uptake.
• The results indicated coordinated responses of functional traits governing water relations in infested relative to healthy trees. In response to HWA, leaf water potential, carbon isotope ratios, plant hydraulic properties and stomatal conductance were affected, inducing a reduction in tree water use by > 40% and gross primary productivity by 25%. Anatomical changes also appeared, including the activation of traumatic cells.
• HWA infestation had a direct effect on plant water relations. Despite some leaf compensatory mechanisms, such as an increase in leaf hydraulic conductance and nitrogen content, tree water use and carbon assimilation were diminished significantly in infested trees, which could contribute to tree mortality.

• Previous studies have suggested that the mesophyll contributes to stomatal CO₂ responses.
• The effects of changes in CO₂ concentration (100 or 700 ppm) on stomatal responses in red or white light were examined microscopically in a leaf segment, an epidermal strip and an epidermal strip placed on a mesophyll segment of Commelina communis, all mounted on a buffer-containing gel.
• In both red and white light, stomata of the leaf segment opened/closed rapidly at low/high CO₂. In red light, epidermal strip stomata barely responded to CO₂. In white light, they opened at low CO₂, but hardly closed at high CO₂. Stomata of the epidermal strip placed on the mesophyll responded in the same manner as those on the leaf segment. Insertion of a doughnut-shaped cellophane spacer (but not polyethylene spacer) between the epidermal strip and the mesophyll hardly altered these responses. Stomata in leaf segments treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthesis inhibitor, did not open in red light, but opened/closed at low/high CO₂ in white light.
• These results indicate that the apoplast transfer of ‘mesophyll signals’ and the stomatal opening at low CO₂ are dependent on photosynthesis, whereas the stomatal closure at high CO₂ is independent of photosynthesis.

• Population genetic and phylogenetic studies have shown that Phaeosphaeria nodorum is a member of a species complex that probably shares its center of origin with wheat (Triticum aestivum and Triticum durum). We examined the evolutionary histories of three known necrotrophic effectors (NEs) produced by P. nodorum and compared them with neutral loci.
• We screened over 1000 individuals for the presence/absence of each effector and assigned each individual to a multi-effector genotype. Diversity at each NE locus was assessed by sequencing c. 200 individuals for each locus.
• We found significant differences in effector frequency among populations. We propose that these differences reflect the presence/absence of the corresponding susceptibility gene in wheat cultivars. The population harboring the highest sequence diversity was different for each effector locus and never coincided with populations harboring the highest diversity at neutral loci. Coalescent and phylogenetic analyses showed a discontinuous presence of all three NEs among nine closely related Phaeosphaeria species. Only two of the nine species were found to harbor NEs.
• We present evidence that the three described NEs of P. nodorum were transmitted to its sister species, Phaeosphaeria avenaria tritici 1, via interspecific hybridization.

• Pleistocene climate change has had an important effect in shaping intraspecific genetic variation in many species; however, its role in driving speciation is less clear. We examined the possibility of a Pleistocene origin of the only two representatives of the genus Pugionium (Brassicaceae), Pugionium cornutum and Pugionium dolabratum, which occupy different desert habitats in northwest China.
• We surveyed sequence variation for internal transcribed spacer (ITS), three chloroplast (cp) DNA fragments, and eight low-copy nuclear genes among individuals sampled from 11 populations of each species across their geographic ranges.
• One ITS mutation distinguished the two species, whereas mutations in cpDNA and the eight low-copy nuclear gene sequences were not species-specific. Although interspecific divergence varied greatly among nuclear gene sequences, in each case divergence was estimated to have occurred within the Pleistocene when deserts expanded in northwest China.
• Our findings point to the importance of Pleistocene climate change, in this case an increase in aridity, as a cause of speciation in Pugionium as a result of divergence in different habitats that formed in association with the expansion of deserts in China.

• Global warming may have profound effects on terrestrial ecosystems. However, a comprehensive evaluation of the effects of warming on ecosystem nitrogen (N) pools and dynamics is not available.
• Here, we compiled data of 528 observations from 51 papers and carried out a meta-analysis of experimental warming effects on 13 variables related to terrestrial N pools and dynamics.
• We found that, on average, net N mineralization and net nitrification rate were increased by 52.2 and 32.2%, respectively, under experimental warming treatment. N pools were also increased by warming, although the magnitude of this increase was less than that of N fluxes. Soil microbial N and N immobilization were not changed by warming, probably because microbes are limited by carbon sources. Grassland and shrubland/heathland were less responsive to warming than forest, probably because the reduction of soil moisture by warming offset the temperature effect in these areas. Soil heating cable and all-day treatment appeared to be the most effective method on N cycling among all treatment methods.
• Results of this meta-analysis are useful for better understanding the response of N cycling to global warming and the underlying mechanism of warming effects on plants and ecosystem functions.

• The phytohormone ABA and the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3)/VIVIPAROUS 1 (VP1) function in protecting embryos during the desiccation stage of seed development. In a similar signaling pathway, vegetative tissue of the moss Physcomitrella patens survives desiccation by activating downstream genes (e.g. LEA1) in response to ABA and ABI3.
• We show that the PpLEA1 promoter responds to PpABI3 primarily through the ACTT-core element (5′-TCCACTTGTC-3′), while the ACGT-core ABA-responsive element (ABRE) appears to respond to ABA alone. We also found by yeast-two-hybrid screening that PpABI3A interacts with PpNF-YC1, a subunit of CCAAT box binding factor (CBF)/nuclear factor Y (NF-Y). PpNF-YC1 increased the activation of the PpLEA1 promoter when incubated with PpABI3A, as did NF-YB, NF-YC, and ABI3 from Arabidopsis.
• This new response element (ACTT) is responsible for activating the ABI3-dependent ABA response pathway cooperatively with the nuclear factor Y (NF-Y) complex. These results further define the regulatory interactions at the transcriptional level for the expression of this network of genes required for drought/desiccation tolerance.
• This gene regulatory set is in large part conserved between vegetative tissue of bryophytes and seeds of angiosperms and will shed light on the evolution of this pathway in the green plant lineage.

• It is still an open question as to whether genome size (GS) variation is shaped by natural selection. One approach to address this question is a population-level survey that assesses both the variation in GS and the relationship of GS to ecological variants.
• We assessed GS in Zea mays, a species that includes the cultivated crop, maize, and its closest wild relatives, the teosintes. We measured GS in five plants of each of 22 maize landraces and 21 teosinte populations from Mexico sampled from parallel altitudinal gradients.
• GS was significantly smaller in landraces than in teosintes, but the largest component of GS variation was among landraces and among populations. In maize, GS correlated negatively with altitude; more generally, the best GS predictors were linked to geography. By contrast, GS variation in teosintes was best explained by temperature and precipitation.
• Overall, our results further document the size flexibility of the Zea genome, but also point to a drastic shift in patterns of GS variation since domestication. We argue that such patterns may reflect the indirect action of selection on GS, through a multiplicity of phenotypes and life-history traits.

• Alternative splicing (AS) generates multiple types of mRNA from a single type of pre-mRNA by differential intron splicing. It can result in new protein isoforms or down-regulation of gene expression by transcript decay. The evolutionary conservation of AS events in plants is largely unexplored and only a small number of AS events have been identified as conserved between divergent species.
• We performed a large-scale analysis of cDNA data from Brassica and Arabidopsis to identify and further characterize conserved AS events.
• We identified 537 conserved AS events in 485 genes. Alternative donor and acceptor events are significantly overrepresented among conserved events, whereas intron retention and exon skipping events are underrepresented. Conserved AS events are significantly shorter, less likely to be in the 3′UTR, and they are enriched for genes whose products function in the chloroplast. AS modified a functional domain for about half of the genes with conserved events. We further characterized three genes with conserved AS events.
• This study identifies many AS events that are conserved between Brassica and Arabidopsis, revealing features of conserved AS events. Many of the conserved AS events may have important, but uncharacterized, functions.

• Rapidly enhancing oleoresin production in conifer stems through genomic selection and genetic engineering may increase resistance to bark beetles and terpenoid yield for liquid biofuels.
• We integrated association genetic and genomic prediction analyses of oleoresin flow (g 24 h⁻¹) using 4854 single nucleotide polymorphisms (SNPs) in expressed genes within a pedigreed population of loblolly pine (Pinus taeda) that was clonally replicated at three sites in the southeastern United States.
• Additive genetic variation in oleoresin flow (h² ≈ 0.12–0.30) was strongly correlated between years in which precipitation varied (r_a ≈ 0.95), while the genetic correlation between sites declined from 0.8 to 0.37 with increasing differences in soil and climate among sites. A total of 231 SNPs were significantly associated with oleoresin flow, of which 81% were specific to individual sites. SNPs in sequences similar to ethylene signaling proteins, ABC transporters, and diterpenoid hydroxylases were associated with oleoresin flow across sites.
• Despite this complex genetic architecture, we developed a genomic prediction model to accelerate breeding for enhanced oleoresin flow that is robust to environmental variation. Results imply that breeding could increase oleoresin flow 1.5- to 2.4-fold in one generation.

• Dead organic matter (OM) is a major source of nitrogen (N) for plants. The majority of plants support N uptake by symbiosis with arbuscular mycorrhizal (AM) fungi. Mineralization of N is regulated by microfauna, in particular, protozoa grazing on bacteria. We hypothesized that AM fungi and protozoa interactively facilitate plant N nutrition from OM.
• In soil systems consisting of an OM patch and a root compartment, plant N uptake and consequences for plant carbon (C) allocation were investigated using stable isotopes.
• Protozoa mobilized N by consuming bacteria, and the mobilized N was translocated via AM fungi to the host plant. The presence of protozoa in both the OM and root compartment stimulated photosynthesis and the translocation of C from the host plant via AM fungi into the OM patch. This stimulated microbial activity in the OM patch, plant N uptake from OM and doubled plant growth.
• The results indicate that protozoa increase plant growth by both mobilization of N from OM and by protozoa–root interactions, resulting in increased C allocation to roots and into the rhizosphere, thereby increasing plant nutrient exploitation. Hence, mycorrhizal plants need to interact with protozoa to fully exploit N resources from OM.

• Foliar water uptake (FWU) is a common water acquisition mechanism for plants inhabiting temperate fog-affected ecosystems, but the prevalence and consequences of this process for the water and carbon balance of tropical cloud forest species are unknown.
• We performed a series of experiments under field and glasshouse conditions using a combination of methods (sap flow, fluorescent apoplastic tracers and stable isotopes) to trace fog water movement from foliage to belowground components of Drimys brasiliensis. In addition, we measured leaf water potential, leaf gas exchange, leaf water repellency and growth of plants under contrasting soil water availabilities and fog exposure in glasshouse experiments to evaluate FWU effects on the water and carbon balance of D. brasiliensis saplings.
• Fog water diffused directly through leaf cuticles and contributed up to 42% of total foliar water content. FWU caused reversals in sap flow in stems and roots of up to 26% of daily maximum transpiration. Fog water transported through the xylem reached belowground pools and enhanced leaf water potential, photosynthesis, stomatal conductance and growth relative to plants sheltered from fog.
• Foliar uptake of fog water is an important water acquisition mechanism that can mitigate the deleterious effects of soil water deficits for D. brasiliensis.

• Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs.
• Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions.
• Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification.
• Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions.

• Uncertainty surrounds belowground plant responses to rising atmospheric CO₂ because roots are difficult to measure, requiring frequent monitoring as a result of fine root dynamics and long-term monitoring as a result of sensitivity to resource availability.
• We report belowground plant responses of a scrub-oak ecosystem in Florida exposed to 11 yr of elevated atmospheric CO₂ using open-top chambers. We measured fine root production, turnover and biomass using minirhizotrons, coarse root biomass using ground-penetrating radar and total root biomass using soil cores.
• Total root biomass was greater in elevated than in ambient plots, and the absolute difference was larger than the difference aboveground. Fine root biomass fluctuated by more than a factor of two, with no unidirectional temporal trend, whereas leaf biomass accumulated monotonically. Strong increases in fine root biomass with elevated CO₂ occurred after fire and hurricane disturbance. Leaf biomass also exhibited stronger responses following hurricanes.
• Responses after fire and hurricanes suggest that disturbance promotes the growth responses of plants to elevated CO₂. Increased resource availability associated with disturbance (nutrients, water, space) may facilitate greater responses of roots to elevated CO₂. The disappearance of responses in fine roots suggests limits on the capacity of root systems to respond to CO₂ enrichment.

• A new mechanistic model of the photosynthesis–light response is developed based on photosynthetic electron transport via photosystem II (PSII) to specifically describe light-harvesting characteristics and associated biophysical parameters of photosynthetic pigment molecules. This model parameterizes ‘core’ characteristics not only of the light response but also of difficult to measure physical parameters of photosynthetic pigment molecules in plants.
• Application of the model to two C₃ and two C₄ species grown under the same conditions demonstrated that the model reproduced extremely well (r² > 0.992) the light response trends of both electron transport and CO₂ uptake.
• In all cases, the effective absorption cross-section of photosynthetic pigment molecules decreased with increasing light intensity, demonstrating novel operation of a key mechanism for plants to avoid high light damage.
• In parameterizing these previously difficult to measure characteristics of light harvesting in higher plants, the model provides a new means to understand the mechanistic processes underpinning variability of CO₂ uptake, for example, photosynthetic down-regulation or reversible photoinhibition induced by high light and photoprotection. However, an important next step is validating this parameterization, possibly through application to less structurally complex organisms such as single-celled algae.

Bacterial pathogens can cause multiple plant diseases and plants rely on their innate immune system to recognize and actively respond to these microbes. The plant innate immune system comprises extracellular pattern recognition receptors that recognize conserved microbial patterns and intracellular nucleotide binding leucine-rich repeat (NLR) proteins that recognize specific bacterial effectors delivered into host cells. Plants lack the adaptive immune branch present in animals, but still afford flexibility to pathogen attack through systemic and transgenerational resistance. Here, we focus on current research in plant immune responses against bacterial pathogens. Recent studies shed light onto the activation and inactivation of pattern recognition receptors and systemic acquired resistance. New research has also uncovered additional layers of complexity surrounding NLR immune receptor activation, cooperation and sub-cellular localizations. Taken together, these recent advances bring us closer to understanding the web of molecular interactions responsible for coordinating defense responses and ultimately resistance.

• Arbuscular mycorrhizal (AM) symbiosis is stimulated by phosphorus (P) limitation and contributes to P and nitrogen (N) acquisition. However, the effects of combined P and N limitation on AM formation are largely unknown.
• Medicago truncatula plants were cultivated in the presence or absence of Rhizophagus irregularis (formerly Glomus intraradices) in P-limited (LP), N-limited (LN) or combined P- and N-limited (LPN) conditions, and compared with plants grown in sufficient P and N.
• The highest AM formation was observed in LPN, linked to systemic signaling by the plant nutrient status. Plant free phosphate concentrations were higher in LPN than in LP, as a result of cross-talk between P and N. Transcriptome analyses suggest that LPN induces the activation of NADPH oxidases in roots, concomitant with an altered profile of plant defense genes and a coordinate increase in the expression of genes involved in the methylerythritol phosphate and isoprenoid-derived pathways, including strigolactone synthesis genes.
• Taken together, these results suggest that low P and N fertilization systemically induces a physiological state of plants favorable for AM symbiosis despite their higher P status. Our findings highlight the importance of the plant nutrient status in controlling plant–fungus interaction.

• The exocyst is a protein complex that is essential for polarized secretion in mammals and fungi. Although the exocyst is essential for plant development, its precise function has not been elucidated. We studied the role of exocyst subunit SEC3A in plant development and its subcellular localization.
• T-DNA insertional mutants were identified and complemented with a SEC3A-green fluorescent protein (GFP) fusion construct. SEC3A-GFP localization was determined using confocal microscopy.
• sec3a mutants are defective in the globular to heart stage transition in embryogenesis. SEC3A-GFP has similar cell plate localization to the other plant exocyst subunits. In interphase cells, SEC3A-GFP localizes to the cytoplasm and to the plasma membrane, where it forms immobile, punctate structures with discrete lifetimes of 2–40 s. These puncta are equally distributed over the cell surface of root epidermal cells and tip growing root hairs. The density of puncta does not decrease after growth termination of these cells, but decreases strongly when exocytosis is inhibited by treatment with brefeldin A.
• SEC3A does not appear to be involved in polarized secretion for cell expansion in tip growing root hairs. The landmark function performed by SEC3 in mammals and yeast is likely to be conserved in plants.

• Coccolithophores are important calcifying phytoplankton predicted to be impacted by changes in ocean carbonate chemistry caused by the absorption of anthropogenic CO₂. However, it is difficult to disentangle the effects of the simultaneously changing carbonate system parameters (CO₂, bicarbonate, carbonate and protons) on the physiological responses to elevated CO₂.
• Here, we adopted a multifactorial approach at constant pH or CO₂ whilst varying dissolved inorganic carbon (DIC) to determine physiological and transcriptional responses to individual carbonate system parameters.
• We show that Emiliania huxleyi is sensitive to low CO₂ (growth and photosynthesis) and low bicarbonate (calcification) as well as low pH beyond a limited tolerance range, but is much less sensitive to elevated CO₂ and bicarbonate. Multiple up-regulated genes at low DIC bear the hallmarks of a carbon-concentrating mechanism (CCM) that is responsive to CO₂ and bicarbonate but not to pH.
• Emiliania huxleyi appears to have evolved mechanisms to respond to limiting rather than elevated CO₂. Calcification does not function as a CCM, but is inhibited at low DIC to allow the redistribution of DIC from calcification to photosynthesis. The presented data provides a significant step in understanding how E. huxleyi will respond to changing carbonate chemistry at a cellular level.

• Besides photosynthates, dodder (Cuscuta spp.) acquires phloem-mobile proteins from host; however, whether this could mediate inter-species phenotype transfer was not demonstrated. Specifically, we test whether phosphinothricin acetyl transferase (PAT) that confers host plant glufosinate herbicide tolerance traffics and functions inter-specifically.
• Dodder tendrils excised from hosts can grow in vitro for weeks or resume in vivo by parasitizing new hosts. The level of PAT in in vivo and in vitro dodder tendrils was quantified by enzyme-linked immunosorbent assay. The glufosinate sensitivity was examined by dipping the distal end of in vivo and in vitro tendrils, growing on or excised from LibertyLink^® (LL; PAT-transgenic and glufosinate tolerant) and conventional (CN; glufosinate sensitive) soybean hosts, into glufosinate solutions for 5 s. After in vitro tendrils excised from LL hosts reparasitized new CN and LL hosts, the PAT level and the glufosinate sensitivity were also examined.
• When growing on LL host, dodder tolerated glufosinate and contained PAT at a level of 0.3% of that encountered in LL soybean leaf. After PAT was largely degraded in dodders, they became glufosinate sensitive. PAT mRNA was not detected by reverse transcription PCR in dodders.
• In conclusion, the results indicated that PAT inter-species trafficking confers dodder glufosinate tolerance.

• Root hairs are known to be highly important for uptake of sparingly soluble nutrients, particularly in nutrient deficient soils. Development of increasingly sophisticated mathematical models has allowed uptake characteristics to be quantified. However, modelling has been constrained by a lack of methods for imaging live root hairs growing in real soils.
• We developed a plant growth protocol and used Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM) to uncover the three-dimensional (3D) interactions of root hairs in real soil. We developed a model of phosphate uptake by root hairs based directly on the geometry of hairs and associated soil pores as revealed by imaging.
• Previous modelling studies found that root hairs dominate phosphate uptake. By contrast, our study suggests that hairs and roots contribute equally. We show that uptake by hairs is more localized than by roots and strongly dependent on root hair and aggregate orientation.
• The ability to image hair–soil interactions enables a step change in modelling approaches, allowing a more realistic treatment of processes at the scale of individual root hairs in soil pores.

• We compare the nutrient-dependent photosynthetic efficiencies of the chlorophyte, Dunaliella tertiolecta, with those of the marine diatom, Thalassiosira weissflogii. Despite considerable evolutionary and physiological differences, these two species appear to use nearly identical growth strategies under a wide range of nutrient limitation.
• Using a variety of physiological measurements, we find that, for both species and across all growth rates, 75% of the gross photosynthetic electron flow is invested in carbon fixation and only 30% is retained as net carbon accumulation. A majority of gross photosynthesis (70%) is ultimately used as reductant for biosynthetic pathways and for the generation of ATP.
• In both species, newly formed carbon products exhibit much shorter half-lives at slow growth rates than at fast growth rates. We show that this growth rate dependence is a result of increased polysaccharide storage during the S phase of the cell cycle.
• We present a model of carbon utilization that incorporates this growth rate-dependent carbon allocation and accurately captures (r² = 0.94) the observed time-resolved carbon retention. Together, our findings suggest a common photosynthetic optimization strategy in evolutionarily distinct phytoplankton species and contribute towards a systems-level understanding of carbon flow in photoautotrophs.

• Ion fluxes are ubiquitous processes in the plant and animal kingdoms, controlled by fine-tuned regulations of ion channel activity. Yet the mechanism that cells employ to achieve the modification of ion homeostasis at the molecular level still remains unclear. This is especially true when it comes to the mechanisms that lead to cell death.
• In this study, Arabidopsis thaliana cells were exposed to ozone (O₃). Ion flux variations were analyzed by electrophysiological measurements and their transcriptional regulation by RT-PCR. Reactive oxygen species (ROS) generation was quantified by luminescence techniques and caspase-like activities were investigated by laser confocal microscopy.
• We highlighted the delayed activation of K⁺ outward-rectifying currents after an O₃-induced oxidative stress leading to programmed cell death (PCD). Caspase-like activities are detected under O₃ exposure and could be decreased by K⁺ channel blocker. Molecular experiments revealed that the sustained activation of K⁺ outward current could be the result of an unexpected post-transcriptional regulation of the guard cell outward-rectifying K⁺ (GORK) channels.
• This consists of a likely new mode of regulating the processing of the GORK mRNA, in a ROS-dependent manner, to allow sustained K⁺ effluxes during PCD. These data provide new mechanistic insights into K⁺ channel regulation during an oxidative stress response.

• The dimension of organs depends on the number and the size of their component cells. Formation of polyploid cells by endoreduplication cycles is predominantly associated with increases in the cell size and implicated in organ growth. In plants, the CCS52A proteins play a major role in the switch from mitotic to endoreduplication cycles controlling thus the number of mitotic cells and the endoreduplication events in the differentiating cells.
• Arabidopsis has two CCS52A isoforms; AtCCS52A1 and AtCCS52A2. Here we focused on their roles in endoreduplication and cell size control during plant development.
• We demonstrate their complementary and dose-dependent actions that are dependent on their expression patterns. Moreover, the impact of CCS52A overexpression on organ size in transgenic plants was dependent on the expression level; while enhanced expression of the CCS52A genes positively correlated with the ploidy levels, organ sizes were negatively affected by strong overexpression whereas milder overexpression resulted in a significant increase in the organ sizes.
• Taken together, these finding support both complementary and dose-dependent actions for the Arabidopsis CCS52A isoforms in plant development and demonstrate that elevated ectopic CCS52A expression positively correlates with organ size, opening a route to higher biomass production.

• Submergence 1A (SUB1A), is an ethylene response factor (ERF) that confers submergence tolerance in rice (Oryza sativa) via limiting shoot elongation during the inundation period. SUB1A has been proposed to restrict shoot growth by modulating gibberellic acid (GA) signaling.
• Our transcriptome analysis indicated that SUB1A differentially regulates genes associated with brassinosteroid (BR) synthesis during submergence. Consistent with the gene expression data, the SUB1A genotype had higher brassinosteroid levels after submergence compared to the intolerant genotype. Tolerance to submergence can be activated in the intolerant genotype by pretreatment with exogenous brassinolide, which results in restricted shoot elongation during submergence.
• BR induced a GA catabolic gene, resulting in lower GA levels in SUB1A plants. BR treatment also induced the DELLA protein SLR1, a known repressor of GA responses such as shoot elongation. We propose that BR limits GA levels during submergence in the SUB1A rice through a GA catabolic enzyme as part of an early response and may repress GA responses by inducing SLR1 after several days of submergence.
• Our results suggest that BR biosynthesis is regulated in a SUB1A-dependent manner during submergence and is involved in modulating the GA signaling and homeostasis.

• In order to understand the molecular genetic mechanisms of rice (Oryza sativa) organ development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow-thin grains.
• We found that narrow-curly leaves resulted mainly from reduced lateral-axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow-thin grains.
• Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and NS2 in maize and PRESSED FLOWER in Arabidopsis. OsWOX3A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development-associated genes were altered in nal2/3, and auxin transport-related genes were significantly changed, leading to pin mutant-like phenotypes such as more tillers and fewer lateral roots.
• OsWOX3A is involved in organ development in rice, lateral-axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers and lateral roots.

• Diets rich in broccoli (Brassica oleracea var italica) have been associated with maintenance of cardiovascular health and reduction in risk of cancer. These health benefits have been attributed to glucoraphanin that specifically accumulates in broccoli. The development of broccoli with enhanced concentrations of glucoraphanin may deliver greater health benefits.
• Three high-glucoraphanin F₁ broccoli hybrids were developed in independent programmes through genome introgression from the wild species Brassica villosa. Glucoraphanin and other metabolites were quantified in experimental field trials. Global SNP analyses quantified the differential extent of B. villosa introgression
• The high-glucoraphanin broccoli hybrids contained 2.5–3 times the glucoraphanin content of standard hybrids due to enhanced sulphate assimilation and modifications in sulphur partitioning between sulphur-containing metabolites. All of the high-glucoraphanin hybrids possessed an introgressed B. villosa segment which contained a B. villosa Myb28 allele. Myb28 expression was increased in all of the high-glucoraphanin hybrids. Two high-glucoraphanin hybrids have been commercialised as Beneforté^® broccoli.
• The study illustrates the translation of research on glucosinolate genetics from Arabidopsis to broccoli, the use of wild Brassica species to develop cultivars with potential consumer benefits, and the development of cultivars with contrasting concentrations of glucoraphanin for use in blinded human intervention studies.

• Root responses to lack of iron (Fe) have mainly been studied in nutrient solution experiments devoid of silicon (Si). Here we investigated how Si ameliorates Fe deficiency in cucumber (Cucumis sativus) with focus on the storage and utilization of Fe in the root apoplast.
• A combined approach was performed including analyses of apoplastic Fe, reduction-based Fe acquisition and Fe-mobilizing compounds in roots along with the expression of related genes.
• Si-treated plants accumulated higher concentrations of root apoplastic Fe, which rapidly decreased when Fe was withheld from the nutrient solution. Under Fe-deficient conditions, Si also increased the accumulation of Fe-mobilizing compounds in roots. Si supply stimulated root activity of Fe acquisition at the early stage of Fe deficiency stress through regulation of gene expression levels of proteins involved in Fe acquisition. However, when the period of Fe deprivation was extended, these reactions further decreased as a consequence of Si-induced enhancement of the Fe status of the plants.
• This work provides new evidence for the beneficial role of Si in plant nutrition and clearly indicates that Si-mediated alleviation of Fe deficiency includes an increase of the apoplastic Fe pool in roots and an enhancement of Fe acquisition.

• Strigolactones (SLs) are a class of phytohormones controlling shoot branching. In potato (Solanum tuberosum), tubers develop from underground stolons, diageotropic stems which originate from basal stem nodes. As the degree of stolon branching influences the number and size distribution of tubers, it was considered timely to investigate the effects of SL production on potato development and tuber life cycle.
• Transgenic potato plants were generated in which the CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) gene, key in the SL biosynthetic pathway, was silenced by RNA interference (RNAi).
• The resulting CCD8-RNAi potato plants showed significantly more lateral and main branches than control plants, reduced stolon formation, together with a dwarfing phenotype and a lack of flowering in the most severely affected lines. New tubers were formed from sessile buds of the mother tubers. The apical buds of newly formed transgenic tubers grew out as shoots when exposed to light. In addition, we found that CCD8 transcript levels were rapidly downregulated in tuber buds by the application of sprout-inducing treatments.
• These results suggest that SLs could have an effect, solely or in combination with other phytohormones, in the morphology of potato plants and also in controlling stolon development and maintaining tuber dormancy.

• Eucalypts are one of the most planted tree genera worldwide, and there is increasing interest in marker-assisted selection for tree improvement. Implementation of marker-assisted selection requires a knowledge of the stability of quantitative trait loci (QTLs). This study aims to investigate the stability of QTLs for wood properties and growth across contrasting sites and multiple pedigrees of Eucalyptus globulus.
• Saturated linkage maps were constructed using 663 genotypes from four separate families, grown at three widely separated sites, and were employed to construct a consensus map. This map was used for QTL analysis of growth, wood density and wood chemical traits, including pulp yield.
• Ninety-eight QTLs were identified across families and sites: 87 for wood properties and 11 for growth. These QTLs mapped to 38 discrete regions, some of which co-located with candidate genes. Although 16% of QTLs were verified across different families, 24% of wood property QTLs and 38% of growth QTLs exhibited significant genotype-by-environment interaction.
• This study provides the most detailed assessment of the effect of environment and pedigree on QTL detection in the genus. Despite markedly different environments and pedigrees, many QTLs were stable, providing promising targets for the application of marker-assisted selection.

• We examine the photosynthetic response of submerged plants from streams and lakes with contrasting free-CO₂ and nitrogen (N) availability. We hypothesized that: the photosynthetic capacity of stream plants is higher because of higher N availability; the photosynthetic N-use efficiency (PNUE) is also higher because stream plants are acclimated to higher free-CO₂; and PNUE is lower in aquatic compared to terrestrial plants.
• We tested these hypotheses by measuring tissue-N, photosynthetic capacity and inorganic C extraction capacity in plants collected from streams and lakes and by comparing the PNUE of aquatic plants with previously published PNUE of terrestrial plants.
• We found that the organic N content was consistently higher in stream (3.8–6.3% w/w) than in lake plants (1.2–4.3% w/w). The photosynthetic capacity correlated positively with tissue-N. The relationships were similar for stream and lake plants, indicating that N allocation patterns were similar despite variability in free-CO₂ between the two habitats.
• The slope of the relationship between photosynthetic capacity and tissue-N was lower than found for terrestrial plants, whereas the compensatory N content for photosynthesis was similar. This suggests that PNUE is lower in aquatic plants, perhaps reflecting that the selection pressure for a high C fixation rate per unit N is reduced as a result of low inorganic C availability in the aquatic environment.

• Currently, phloem transport in plants under field conditions is not well understood. This is largely the result of the lack of techniques suitable for the measurement of the physiological properties of phloem.
• We present a model that interprets the changes in xylem diameter and live bark thickness and separates the components responsible for such changes. We test the predictions from this model on data from three mature Scots pine trees in Finland. The model separates the live bark thickness variations caused by bark water capacitance from a residual signal interpreted to indicate the turgor changes in the bark.
• The predictions from the model are consistent with processes related to phloem transport. At the diurnal scale, this signal is related to patterns of photosynthetic activity and phloem loading. At the seasonal scale, bark turgor showed rapid changes during two droughts and after two rainfall events, consistent with physiological predictions. Daily cumulative totals of this turgor term were related to daily cumulative totals of canopy photosynthesis. Finally, the model parameter representing radial hydraulic conductance between phloem and xylem showed a temperature dependence consistent with the temperature-driven changes in water viscosity.
• We propose that this model has potential for the continuous field monitoring of tree phloem function.

• Despite substantial investigations into Oryza phylogeny and evolution, reliable estimates of the divergence times and ancestral effective population sizes of major lineages in Oryza are challenging.
• We sampled sequences of 106 single-copy nuclear genes from all six diploid genomes of Oryza to investigate the divergence times through extensive relaxed molecular clock analyses and estimated the ancestral effective population sizes using maximum likelihood and Bayesian methods.
• We estimated that Oryza originated in the middle Miocene (c. 13–15 million years ago; Ma) and obtained an explicit time frame for two rapid diversifications in this genus. The first diversification involving the extant F-/G-genomes and possibly the extinct H-/J-/K-genomes occurred in the middle Miocene immediately after (within < 1 Myr) the origin of Oryza. The second giving rise to the A-/B-/C-genomes happened c. 5–6 Ma. We found that ancestral effective population sizes were much larger than those of extant species in Oryza.
• We suggest that the climate fluctuations during the period from the middle Miocene to Pliocene may have contributed to the two rapid diversifications of Oryza species. Such information helps better understand the evolutionary history of Oryza and provides further insights into the pattern and mechanism of diversification in plants in general.

• In Arabidopsis thaliana, small peptides (AtPeps) encoded by PROPEP genes act as damage-associated molecular patterns (DAMPs) that are perceived by two leucine-rich repeat receptor kinases, PEPR1 and PEPR2, to amplify defense responses. In particular, expression of PROPEP2 and PROPEP3 is strongly and rapidly induced by AtPeps, in response to bacterial, oomycete, and fungal pathogens, and microbe-associated molecular patterns (MAMPs).
• The cis-regulatory modules (CRMs) within the PROPEP2 and PROPEP3 promoters that mediate MAMP responsiveness were delineated, employing parsley (Petroselinum crispum) protoplasts and transgenic A. thaliana plants harboring promoter-reporter constructs. By chromatin immunoprecipitation in vivo, DNA interactions with a specific transcription factor were detected. Furthermore, the phastCons program was used to identify conserved regions of the PROPEP3 locus in different Brassicaceae species.
• The major MAMP-responsive CRM within the PROPEP2 promoter is composed of several W boxes and an as1/OCS (activation sequence-1/octopine synthase) enhancer element, while in the PROPEP3 promoter the CRM is comprised of six W boxes. The WRKY33 transcription factor binds in vivo to these promoter regions in a MAMP-dependent manner. Both the position and orientation of the six W boxes are conserved within the PROPEP3 promoters of four other Brassicaceae family members.
• WRKY factors are the major regulators of MAMP-induced PROPEP2 and PROPEP3 expression.

• Small RNAs play important roles in resistance to plant viruses and the complex responses against pathogens and leaf-chewing insects. We investigated whether small RNA pathways are involved in Arabidopsis resistance against a phloem-feeding insect, the green peach aphid (Myzus persicae).
• We used a 2-wk fecundity assay to assess aphid performance on Arabidopsis RNA silencing and defence pathway mutants. Quantitative real-time polymerase chain reaction was used to monitor the transcriptional activity of defence-related genes in plants of varying aphid susceptibility. High-performance liquid chromatography-mass spectrometry was employed to measure the accumulation of the antimicrobial compound camalexin. Artificial diet assays allowed the assessment of the effect of camalexin on aphid performance.
• Myzus persicae produces significantly less progeny on Arabidopsis microRNA (miRNA) pathway mutants. Plants unable to process miRNAs respond to aphid infestation with increased induction of PHYTOALEXIN DEFICIENT3 (PAD3) and production of camalexin. Aphids ingest camalexin when feeding on Arabidopsis and are more successful on pad3 and cyp79b2/cyp79b3 mutants defective in camalexin production. Aphids produce less progeny on artificial diets containing camalexin.
• Our data indicate that camalexin functions beyond antimicrobial defence to also include hemipteran insects. This work also highlights the extensive role of the miRNA-mediated regulation of secondary metabolic defence pathways with relevance to resistance against a hemipteran pest.

• Six transcription factors of APETALA2/ethylene-response factor (AP2/ERF) family were cloned and analyzed in Artemisia annua. Real-time quantitative polymerase chain reaction (RT-Q-PCR) showed that AaORA exhibited similar expression patterns to those of amorpha-4,11-diene synthase gene (ADS), cytochrome P450-dependent hydroxylase gene (CYP71AV1) and double bond reductase 2 gene (DBR2) in different tissues of A. annua.
• AaORA is a trichome-specific transcription factor, which is expressed in both glandular secretory trichomes (GSTs) and nonglandular T-shaped trichomes (TSTs) of A. annua. The result of subcellular localization shows that AaORA is targeted to the nuclei and the cytoplasm.
• Overexpression and RNA interference (RNAi) of AaORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71AV1, DBR2 and AaERF1. The up-regulated or down-regulated expression levels of these genes resulted in a significant increase or decrease in artemisinin and dihydroartemisinic acid. The results demonstrate that AaORA is a positive regulator in the biosynthesis of artemisinin.
• Overexpression of AaORA in Arabidopsis thaliana increased greatly the transcript levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2), HEVEIN-LIKE PROTEIN (HEL) and BASIC CHITINASE (B-CHI). After inoculation with Botrytis cinerea, the phenotypes of AaORA overexpression in A. thaliana and AaORA RNAi in A. annua demonstrate that AaORA is a positive regulator of disease resistance to B. cinerea.

• α-Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of tomatinases that degrade α-tomatine into the less fungitoxic compounds β-tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus Cladosporium fulvum is unable to detoxify α-tomatine.
• Here, we present a functional analysis of the glycosyl hydrolase (GH10), CfTom1, which is orthologous to fungal tomatinases.
• We show that C. fulvum hydrolyzes α-tomatine into tomatidine in vitro and during the infection of tomato, which is fully attributed to the activity of CfTom1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, ∆cftom1 mutants of C. fulvum are more sensitive to α-tomatine and are less virulent than the wild-type fungus on tomato. Although α-tomatine is thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by CfTom1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously.
• Altogether, our results show that CfTom1 is responsible for the detoxification of α-tomatine by C. fulvum, and is required for full virulence of this fungus on tomato.

• Fusarium circinatum causes pitch canker disease in a wide range of pine trees, including Pinus radiata, with devastating economic consequences.
• To assess the spatial and temporal dynamics of growth of this pathogen in radiata pine, we examined the process of infection using both real-time PCR to quantify fungal biomass inside the plant host, and confocal microscopy using a green fluorescent protein (GFP)-tagged strain of F. circinatum.
• Pathogen growth exhibited three distinct phases: an initial exponential increase in fungal biomass, concomitant with pathogen colonization of the cortex and phloem; a slowdown in fungal growth coincident with sporulating hyphae deep within the host; and stabilization of the fungal biomass when the first wilting symptoms appeared. The number of resin ducts in the xylem was found to increase in response to infection and the fungus grew inside both constitutive and traumatic resin ducts.
• These results indicate that conidiation may contribute to the spatial or temporal dissemination of the pathogen. Moreover, the present findings raise the intriguing possibility that the generation of traumatic resin ducts may be of more benefit to the fungus than to the plant.

• Global-scale analyses of ectomycorrhizal (ECM) fungi communities emphasize host plant families as the main drivers of diversity. This study aims to test, on Alnus–ECM communities, which fungi are said to be ‘host-specific’, to what extent host species, habitat and distance explain their alpha and beta diversity variations, and their specificity.
• In France, ECM communities associated with two subgenera and five species of Alnus, were sampled on 165 trees from 39 lowland to subalpine sites. In all, 1178 internal transcribed spacer (ITS) sequences of ECM fungi clustered in 86 molecular operational taxonomic units (MOTUs).
• The species richness was low but still variable, and the evenness of communities was lower on organic soils and in Corsica. Similarity between communities was influenced both by host, soil parameters, altitude and longitude, but not by climate and distance. A large majority of ‘specific’ fungi were shared between host species within a subgenus, and showed habitat preferences within the subgenus distribution range.
• Our study confirms that Alnus ECM communities are low in diversity, highly conserved at a regional scale, and partly shared between congeneric host species. A large part of alpha and beta diversity variations remained unexplained, and other processes may shape these communities.

• Much of the macroecological information about microorganisms is confounded by the lack of standardized methodology, paucity of metadata and sampling effect of a particular substrate or interacting host taxa.
• This study aims to disentangle the relative effects of biological, geographical and edaphic variables on the distribution of Alnus-associated ectomycorrhizal (ECM) fungi at the global scale by using comparable sampling and analysis methods.
• Ribosomal DNA sequence analysis revealed 146 taxa of ECM fungi from 22 Alnus species across 96 sites worldwide. Use of spatial and phylogenetic eigenvectors along with environmental variables in model selection indicated that phylogenetic relations among host plants and geographical links explained 43 and 10%, respectively,in ECM fungal community composition, whereas soil calcium concentration positively influenced taxonomic richness.
• Intrageneric phylogenetic relations among host plants and regional processes largely account for the global biogeographic distribution of Alnus-associated ECM fungi. The biogeography of ECM fungi is consistent with ancient host migration patterns from Eurasia to North America and from southern Europe to northern Europe after the last glacial maximum, indicating codispersal of hosts and their mycobionts.

• Cell-to-cell trafficking of transcription factors (TFs) has been shown to play an important role in the regulation of plant developmental events, but the evolutionary relationship between cell-autonomous and noncell-autonomous (NCA) TFs remains elusive.
• AtDof4.1, named INTERCELLULAR TRAFFICKING DOF 1 (ITD1), was chosen as a representative NCA member to explore this evolutionary relationship. Using domain structure–function analyses and swapping studies, we examined the cell-to-cell trafficking of plant-specific Dof TF family members across Arabidopsis and other species.
• We identified a conserved intercellular trafficking motif (ITM) that is necessary and sufficient for selective cell-to-cell trafficking and can impart gain-of-function cell-to-cell movement capacity to an otherwise cell-autonomous TF. The functionality of related motifs from Dof members across the plant kingdom extended, surprisingly, to a unicellular alga that lacked plasmodesmata. By contrast, the algal homeodomain related to the NCA KNOX homeodomain was either inefficient or unable to impart such cell-to-cell movement function.
• The Dof ITM appears to predate the evolution of selective plasmodesmal trafficking in the plant kingdom, which may well have acted as a molecular template for the evolution of Dof proteins as NCA TFs. However, the ability to efficiently traffic for KNOX homeodomain (HD) proteins may have been acquired during the evolution of early nonvascular plants.

• Temperature is considered an important determinant of biodiversity distribution patterns. Grasses (Poaceae) occupy among the warmest and coldest environments on earth but the role of cold tolerance evolution in generating this distribution is understudied.
• We studied cold tolerance of Danthonioideae (c. 280 species), a major constituent of the austral temperate grass flora. We determined differences in cold tolerance among species from different continents grown in a common winter garden and assessed the relationship between measured cold tolerance and that predicted by species ranges. We then used temperatures in current ranges and a phylogeny of 81% of the species to study the timing and mode of cold tolerance evolution across the subfamily.
• Species ranges generally underestimate cold tolerance but are still a meaningful representation of differences in cold tolerance among species. We infer cold tolerance evolution to have commenced at the onset of danthonioid diversification, subsequently increasing in both pace and extent in certain lineages. Interspecific variation in cold tolerance is better accounted for by spatial than phylogenetic distance.
• Contrary to expectations, temperature – low temperature in particular – appears not to limit the distribution of this temperate clade. Competition, time or dispersal limitation could explain its relative absence from northern temperate regions.

• The role of Quaternary climatic shifts in shaping the distribution of Linaria elegans, an Iberian annual plant, was investigated using species distribution modelling and molecular phylogeographical analyses. Three hypotheses are proposed to explain the Quaternary history of its mountain ring range.
• The distribution of L. elegans was modelled using the maximum entropy method and projected to the last interglacial and to the last glacial maximum (LGM) using two different paleoclimatic models: the Community Climate System Model (CCSM) and the Model for Interdisciplinary Research on Climate (MIROC). Two nuclear and three plastid DNA regions were sequenced for 24 populations (119 individuals sampled). Bayesian phylogenetic, phylogeographical, dating and coalescent-based population genetic analyses were conducted.
• Molecular analyses indicated the existence of northern and southern glacial refugia and supported two routes of post-glacial recolonization. These results were consistent with the LGM distribution as inferred under the CCSM paleoclimatic model (but not under the MIROC model). Isolation between two major refugia was dated back to the Riss or Mindel glaciations, > 100 kyr before present (bp).
• The Atlantic distribution of inferred refugia suggests that the oceanic (buffered)–continental (harsh) gradient may have played a key and previously unrecognized role in determining Quaternary distribution shifts of Mediterranean plants.