Salt-induced changes in cell wall composition precede stress responses in the xylem
A remarkable result of this study was the clear distinction of salt responses related to xylem modification and stress adaptation. Transporters putatively involved in salt exclusion were constituively up-regulated in P. euphratica compared with salt-sensitive poplars (Ding et al., 2010; Janz et al., 2010). Here we show that this trait led to exclusion of salt from the above-ground transport path, in particular from the developing xylem. By contrast, highest Na accumulation occurred in the developing xylem of P. × canescens, consequently requiring strong activation of defenses to prevent injury. In the overrepresented GO term categories for genes with increased transcript abundance typical representatives for osmotic stress as well as for antioxidative defenses were found, for example, genes coding for proteins involved in osmotic adjustment (LEA protein, osmotin precurser, myo-inositol-1-phosphate synthase, trehalose phosphatase), detoxification (glutathione-S-transferases, superoxide dismutase, alcohol dehydrogenase) and in signal perception and transduction (ABA-induced protein phosphatase 2C (PP2C), WRKY and MYB transcription factors, the salt responsive homeobox leucin zipper transcription factor HB-7, various serine/threonine protein kinases, 9-cis-epoxycarotenoid dioxygenase involved in ABA biosynthesis) (Table S2). The activation of these genes has also been reported in previous salt screens in roots or leaves of poplar as well as in other plant species (Broschéet al., 2005; Gong et al., 2005; Ottow et al., 2005; Ma et al., 2006; Teichmann et al., 2008; Brinker et al., 2010; Ding et al., 2010; Qiu et al., 2011). Since our plants were acclimated to high salinity and still showed wood formation, this suite of genes must be important to sustain a new degree of cellular homeostasis under stress.
Two GO categories enriched under salt stress in the developing xylem of P. × canescens deserve specific attention: ‘multicellular process’ and ‘secondary metabolism’. The first category contains a collection of transcription factors involved in cell division and vascular development (e.g. homologs to the Arabidopsis genes KNAT-3 (Truernit et al., 2006) recently identified in Juglans nigra during heartwood formation (Huang et al., 2009), MONOPTEROS (Ohashi-Ito & Fukuda, 2010), ANAC087 and ANAC082 (Guan & Nothnagel, 2004), and ARF16 (Ding et al., 2010) required for meristem differentiation), thus supporting our cytological and morphometric analyses, which revealed ongoing growth. Significant accumulation of carbohydrates along the transport route (this study) as well as in roots of osmotically stressed poplars (Luo et al., 2009a,b; Galvez et al., 2011) may, however, point to disturbed phloem unloading. Because massive carbohydrate depletion in the developing xylem was not observed, the reasons for the growth reductions remain unclear. Decreased carbon flux to fuel cell wall formation and inhibited vessel expansion by hydraulic stress may have diminished radial growth. It is possible that decreased potassium availability plays a role in this respect (Langer et al., 2002; Wind et al., 2004), but this requires further analyses because it is not clear if elevated concentrations of Na can replace K.
The second category ‘secondary metabolism’ was not identified in P. euphratica. However, this species exhibits constitutive activation of secondary metabolism, even in the absence of salt stress (Janz et al., 2010). Quantitative trait locus (QTL) analyses in poplar progenies revealed that genes for secondary metabolites correlate with drought tolerance (Street et al., 2006). Increased concentrations of flavonoids and phenolics may protect plants against environmental cues because they act as antioxidants preventing cellular injury (Rice-Evans et al., 1997). The pathways for flavonoid production and precursors for lignin (flavonoid 3 hydroxylase, flavonol synthase, flavonol-O-methyl transferase, cytochrome P 450 family protein, cinnamoyl CoA reductase, peroxidases) were stimulated in response to salt stress in the developing xylem of P. × canescens. Substantial increases in lignin relative to cellulose were not detected, suggesting that the production of phenol-based compounds served to increase the antioxidant capacity. It should also be recalled that the FTIR analyses were conducted on the surface of young, not fully differentiated xylem, where lignification was not yet accomplished and therefore the question of whether salt stress led to increased lignin concentrations cannot be conclusively answered yet. However, both species showed changes in cell wall carbohydrates. P. euphratica displayed only moderate salt accumulation, which resulted neither in detectable osmotic stress nor in anatomical changes. Our results indicate that processes involved in cell wall modification are responsive to subtle alterations in hydraulic signals or the ion balance and can clearly be distinguished from cellular defense reactions.
Changes in xylem anatomy and cell wall composition adapt poplar to hydraulic stress through generation of ‘pressure wood’
Osmotic stress causes strong negative xylem pressures, which can lead to cavitation and subsequently to conduit collapse in the xylem (Hacke & Sperry, 2001). Because the integrity of the water transport system is essential for survival under stressful conditions, plants across a vast range of biomes increase their wall strength by decreasing the ratio of vessel lumen to cell wall thickness in response to water limitation (Hacke et al., 2001). This phenomenon is also known as the hydraulic safety principle because single cavitation incidents pose a lower threat when a high number of conduits are available (Zimmermann, 1983; Tyree & Ewers, 1991). Notably, this hydraulic adaptation strategy can also be found when comparing salt-adapted mangrove species (many small vessels) with nonmangrove species (fewer, larger vessels) of the same genus (Janssonius, 1950). Flexible adjustment of vessel lumina to changes in osmotic conditions by salt or drought has also been reported for poplars (Junghans et al., 2006; Arend & Fromm, 2007; Beniwal et al., 2010; Schreiber et al., 2011; this study) and results in formation of false year rings under field conditions (Liphschitz & Waisel, 1970).
A surprising result of our study was that developing xylem undergoing hydraulic adaptation exhibited a coordinated reduction of gene transcripts identified in tension wood formation of poplar (Andersson-Gunnerås et al., 2006). Tension wood is formed on the upper side of the stem in response to gravitational stimuli and is characterized by low lignin content and strong accumulation of cellulose in the fiber lumina (Timell, 1986). Our results show that hydraulic adaptation apparently involves the opposite regulation of a suite of genes required for tension wood formation, namely FLAs, COBRA-like (homolog to Arabidopsis COBL4), and genes encoding xyloglucan endo transglycolyase, pectin methylesterase, pectin lyase, expansin, xylosidase, and amylase. Most of these genes are members of large gene families in poplars. With the exception of the xylosidase gene, members of these gene families were up-regulated in tension wood (Andersson-Gunnerås et al., 2006) and down-regulated here. Conversely, gene expression of the phenylpropanoid pathway was increased here and significantly decreased in tension wood (Andersson-Gunnerås et al., 2006). In tension wood, cellulose biosynthetic genes (CES) were increased but suppression of cellulose synthase genes was not found here, although cellulose formation was impaired. Genetic analysis showed that cellulose deposition is under the control of AtCOB4 in Arabidopsis and the ortholog BC1 in rice (Brown et al., 2005; Sato et al., 2010). The ortholog of these genes, COBRA-like, was suppressed in salt-stressed poplar, pointing to suppression of cellulose biosynthesis by transcriptional regulation. Furthermore, the β-amylase 3 gene identified here is a homolog of ArabidopsisCYT1, whose failure in knockout mutants resulted in significant decreases in cellulose and accumulation of defense compounds (Lukowitz et al., 2001). Therefore, transcriptional repression of poplar COBRA-like and β-amylase 3 may have caused alterations in cell wall carbohydrate composition.
The only gene with increased transcript abundance in the young xylem of salt-exposed P. euphratica encoded a 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), a gene required for ethylene production. In poplar, ethylene affects the fiber : vessel ratio (Junghans et al., 2004) and fiber extensibility (Qin et al., 2007). Recently, it was shown that ethylene controls tension wood formation (Love et al., 2009). In compression wood of conifers, a strong correlation of increasing lignin : cellulose ratios and ACC oxidase was reported (Plomion et al., 2000), which might point to a role of ethylene in wood formation under pressure stress.
Fasciclin-like arabinogalactan protein genes (or FLA genes or FLAs) are an extremely expanded gene family with several hundred members in poplar (Johnson et al., 2003; Lafarguette et al., 2004; Andersson-Gunnerås et al., 2006). The FLAs identified as down-regulated in this study form a group of paralogs which have no true orthologs in Arabidopsis; this observation has also been made for the FLAs massively up-regulated during the formation of tension wood (Andersson-Gunnerås et al., 2006). FLAs, which form a distinct subgroup of arabinogalactan-proteins (AGPs), are thought to be involved in various processes of xylem differentiation such as cell–cell signaling, cell division, adhesion, and microfibril orientation and may function as wall integrity sensors affecting downstream signal transduction (Humphrey et al., 2007; Seifert & Blaukopf, 2010). In compression wood of loblolly pine, an AGP with GPI anchor (AGP5) showed decreased expression (Zhang et al., 2000). The Arabidopsis Salt Overly Sensitive 5 (SOS5) protein, a synonym for FLA4 (Swiss-Prot:Q9SNC3), is required for controlled cell expansion (Shi et al., 2003). Phylogenetic comparisons showed that the group A of FLA genes was conserved in monocots and dicots and was specific to stems (MacMillan et al., 2010). Double mutants of Atfla12/fla11 showed reduced tensile strength and stiffness, thus rendering cell walls more brittle (MacMillan et al., 2010). Atfla11 and Atfla12/fla11 mutants also contained lower amounts of cellulose and higher lignin concentrations (Persson et al., 2005; MacMillan et al., 2010). In this study, P. × canescens and P. euphratica showed only a few common reactions towards salt stress; one striking commonality was the suppression of five specific FLAs and changes in chemical wood composition resembling those of Arabidopsis fla mutants.
We conclude that salinity caused formation of a novel type of ‘pressure’ wood, presumably as a result of transcriptional coregulation of the same set of genes involved in tension wood biosynthesis, but in the opposite direction. At present, it is mostly unknown how trees sense and signal environmental cues to modify cell walls (Seifert & Blaukopf, 2010). The identification of a putative master relay leading to coordinated stimulation or inhibition of genes for reaction wood formation will be an important research target for future studies.
With regard to practical implications, higher wood density as the result of hydraulic adaptation affects wood biomechanical properties and its technological utilization (Irvine & Grace, 1997; Holtta et al., 2002). Increased lignin : cellulose ratios, as found in compression wood of conifers, have negative effects on pulpability and render such trees less suitable for papermaking (Yeh et al., 2006). However, this wood may be useful as fuelwood for local populations and, therefore, knowledge of the mechanistic basis of wood formed under environmental constraints will help to accelerate the selection of trees that can grow on sodic soils.