The balanced polymorphism in arsenate tolerance previously characterized in H. lanatus (Meharg & Macnair, 1992c; Meharg et al., 1993; Naylor et al., 1996) was also clearly exhibited in the CBG population, as expected. Here we report the first phosphate-specific response for this polymorphism, as previous investigations tried to link tolerance gene frequency to soil phosphate status (Naylor et al., 1996), rather than studying phenotype response to P fertilization. A differential allocation to root and shoot biomass in response to P fertilization was observed, T maintaining a relatively constant shoot : root ratio while N showed the classic plant response to P addition in that shoot : root ratio increased (Gojon et al., 2009). This differential response in shoot : root ratio to P availability is likely to be the reason why this polymorphism is maintained, and this will be outlined in more detail in subsequent publications. Shoot P (root P was not measured as it is impossible to remove all adhering soil, which greatly confounds interpretation) does not differ between phenotypes, and this shoot P status is not P fertilizer responsive, all indicating tight homeostasis, a known characteristic with respect to plant P nutrition (Hill et al., 2006; Gojon et al., 2009).
Transcriptome assembly and expression patterns
With respect to annotation of the transcript data, the highest homologous matches against plant-refseq were invariably identified against protein/transcript sequences of B. distachyon, Sorghum bicolor and O. sativa, of these B. distachyon being the most frequent hit. Holcus lanatus is most closely related to B. distachyon of currently genome-sequenced grasses (Aliscioni et al., 2012). The only previously reported H. lanatus gene sequence, AY704470, a CDC25 (Cell division cycle 25) phosphatase homologue (Bleeker et al., 2006), was identified in all genotypes here, with isotig19077 showing 100% identity to this published sequence (tBLASTn; Fig. S7), giving further verification of the transcriptome assembly.
A key finding of this study was the independence of transcript expression between N/T and P+/P− nutrition; in particular, transcript expression of genes involved directly in P nutrition (i.e. those encoding enzymes involved in P transport and P compound synthesis/degradation) was not the basis of tolerance as would have been hypothesized from a priori knowledge, as these phenotypes results in suppressed phosphate/arsenate transport (Macnair et al., 1992; Meharg & Macnair, 1992a), including T phenotypes screened out of populations established on uncontaminated soils (Meharg & Macnair, 1992c). Given the large differential expression of classes of transcripts involved in post-translational and post-transcriptional regulation, probably regulating phosphate transporter enzyme production/degradation/activation, it is argued here that tolerance is attributable to these post-translational and post-transcriptional regulatory genes.
Many of the transcripts responsive to P nutrition are regulated as expected, with many obviously involved in P nutrition and generally strongly up-regulated in response to P stress. For example, the four phosphate transport-related transcripts and the two SPX transcripts identified in H. lanatus in this study were induced by P stress, consistent with what has been observed generically in studies from the literature, as exemplified by the phosphate-responsive transcriptome of white lupin (Lupin albus) (O'Rourke et al., 2013). Other transcripts coding for proteins involved in P regulation or transport identified (Table S3) include glycerophosphodiester/phosphodiesterase, sucrose phosphate synthase, glucose pyrophosphorylase, purple acid phosphatase, phosphatase, and glucose-6-phosphate phosphate translocator, also differentially regulated when comparing P-stressed and P-repleat lupin plants (O'Rourke et al., 2013). Also, it is now well established that P-responsive genes (1) give rise to transcriptome signal cascades and/or (2) are involved in these signal cascades (Chiou & Lin, 2011; Wu et al., 2013) .
Although we know that arsenate tolerance is under the control of a single gene (Macnair et al., 1992), the differential expression of transcripts between the T and N phenotypes represents a suite of genes, for which there are two potential explanations. The first is that there is an as yet unidentified gene that is itself controlling the transcript production of a host of genes differentially expressed in the T/N comparison. The second, which is not exclusive of the first, is that differences in metabolism resulting from differential function of a gene(s) may lead to feedback regulating transcripts of interrelated functions, such as the obvious impact of P starvation on P metabolism observed here. P stress perception in plants is known to induce a host of differential responses, such as tillering, root biomass production, arbuscular mycorrhizal regulation, and rhizosphere excretion of dicarboxylic acids to mobilize phosphate from iron minerals, and this will lead to differential regulation of a network of genes (Hill et al., 2006; Gojon et al., 2009; Chiou & Lin, 2011). Again, the arsenate tolerance gene has a range of pleiotropic consequences (shoot/root biomass allocation, HAPT suppression and arsenate tolerance itself), consistent with such a feedback and/or upstream regulator model.
A number of recent studies have found upstream regulators of low P adaption responses, with low P adaption having been postulated as the driver in maintaining the H. lanatus balanced polymorphism under study here (Meharg & Macnair, 1992c; Naylor et al., 1996). ALFIN-LIKE proteins have been implicated in regulating root hair growth in Arabidopsis under P stress (Chandrika et al., 2013). ALFIN-LIKE proteins are a small family of plant homeo domain (PHD)-containing putative transcription factors with a methylated histone residue-binding component, and ALFIN-LIKE 6 was shown to control the transcription of a range of genes involved in growth, in particular root hair growth (Chandrika et al., 2013). A homologue of this gene was identified in our H. lanatus transcriptome (isotig02053) and the translated isotig showed high similarity to Arabidopsis and even higher similarity to B. distachyon ALFIN-LIKE protein (Fig. S8). This transcript was equally highly expressed in all N and T plants and not differentially regulated between either P treatments or phenotypes, suggesting that it has a limited role in P nutrition and arsenate tolerance in H. lanatus.
Kinases were strongly differentially expressed in T/N transcript comparisons, with some kinases having recently been identified as being central in phenotypic differences in plant root response to P status (Gamuyao et al., 2012) as well as shown to be up-regulated in response to arsenate stress (Huang et al., 2012). A kinase that confers adaption to soil P stress in rice, PSTOL1, has been characterized (Gamuyao et al., 2012). This gene is an enhancer of early root growth and its over-expression leads to increased grain yields, hypothesized to be attributable to more efficient P capture as a result of larger root systems, with larger root systems characterizing the H. lanatus T phenotype here (Fig. 2). PSTOL1 homologues, although detected in the transcriptome, had no role in differential tolerance or P nutrition response in H. lanatus. However, Cbl-interacting kinases were differentially expressed between T and N phenotypes here; these are serine/threonine protein kinases, as is PSTOL1 (Gamuyao et al., 2012).
Auxins, and associated expansins, were also differentially expressed between phenotypes and these have a key role in root growth (Cosgrove, 1999), with differential shoot/root allocation in response to P fertilization being identified here as another pleiotropic effect of arsenate tolerance. Ubiquitins, also differentially expressed in T/N comparisons, which mark proteins for proteasome-mediated degradation, are thought to have a key role in regulation of plant SPX domains in response to P stress (Wu et al., 2013).
Transposons and retrotransposons, the class of isotigs most frequently and most strongly differentially expressed between phenotypes, are thought to play a role in post-transcriptional regulation, with silencing of TEs involving both transcriptional and post-transcriptional mechanisms (Okamoto & Hirochika, 2001; Mirouze & Paszkowski, 2011). In plants, retrotransposons are commonly known to be expressed under conditions of stress (Grandbastien, 1998). It is also thought that retrotransposon activation is sensitive to environment (Grandbastien, 1998; Mirouze & Paszkowski, 2011), further enhancing their candidacy for regulating stress responses, such as nutritional deficiencies. TEs can generate gene variation and functional changes (Gao et al., 2012) and are a source of small RNAs, and they have been implicated in gene regulation in both animals and plants (McCue & Slotkin, 2012). A role for small RNAs in regulation of P starvation in plants is emerging (Fang et al., 2009; Hsieh et al., 2009; Chiou & Lin, 2011; O'Rourke et al., 2013). Further to this, a potential role of a small RNA targeting transcripts involved in post-transcriptional/post-translational regulation leading to T and N phenotypes in H. lanatus is worth further investigation.
There were large and systematic differences in SNPs between T and N phenotypes. Ten of these were identified in transcripts annotated as protease, protease subunit and heat-shock proteins. We assume that these SNPs are of genomic origin, but without the genomic sequence of H. lanatus it is not possible to rule out the possibility that targeted mRNA editing may be involved in some of these cases. RNA editing, which was first identified in the cox2 (cytochrome c oxidase subunit) mRNA of Trypanosoma brucei, is thought to play an important role in organelles (plastids and mitochondria) of plants, with those identified typically involving a change of a specific C to U, but other changes can as yet not be ruled out (Grennan, 2011; Jiang et al., 2012). The role of RNA editing in plant plastids as well as nuclear-encoded RNA/mRNA remains to be further investigated by systematic sequencing of the plant genome (DNA) and transcriptome (cDNA), as has been described for identification of RNA editing sites in human studies (Ramaswami et al., 2012). While RNA-binding proteins of the pentatricopeptide repeat family, multiple organellar RNA editing factors and chloroplast ribonucleoproteins are known to be involved in RNA editing, further proteins remain to be identified (Tillich et al., 2009; Grennan, 2011; Takenaka et al., 2012). So it is noteworthy in this context that one of the homozygous SNPs identified between the T and N phenotypes in this study is in isotig05374, which shows 70% homology to an RNA recognition motif-containing protein/predicted ribonucleoprotein. Further to that, an exonuclease (isotig08248), which mediates RNA degradation (Stoppel & Meurer, 2012), was strongly up-regulated in the N phenotype. Thus, both the N/T gene expression findings and the SNPs obtained for the N/T phenotypes suggest that post-translational regulation of proteins via the ubiquitin–proteasome system plays an important role in determining the N and T phenotypes and, furthermore, indicates a potential role of post-transcriptional regulation (RNA degradation and a possible role of RNA editing). A causative upstream master regulatory gene, inducing post-translational and maybe also post-transcriptional events of consequence for arsenate resistance and P uptake efficiency in these plants, remains to be identified, and potential involvement of small RNAs should be investigated in this context.
With respect to arsenate tolerance, the character used to screen the phenotypes under study, one gene with a putative role in arsenic transport/metabolism, in addition to phosphate transporters and their regulators, that was differentially regulated between phenotypes was an arsB-like gene. The alignment of isotig09604 with an arsB-like protein can be seen in Fig. S9. arsB is widely present in arsenic-resistant bacteria, where its role is as an arsenite efflux channel (Yang et al., 2012), although in plants this class of aquaglycerinporins are also involved in silicic acid transport (Zhao et al., 2009). Logoteta et al. (2009) reported that differential efflux of arsenite was not found between H. lanatus T and N phenotypes. A protein, HLASR (H. lanatus arsenate reductase), has been identified in H. lanatus to have a constitutive, but not an adaptive, role in the metabolism of arsenate, as an arsenate reductase (Bleeker et al., 2006). It was thought that the product of this HLASR gene only had a secondary role in arsenic metabolism and that its primary role was homologous to that of CDC25 phosphatases, which activate cycline-dependent kinases in Arabidopsis, which are involved in cell cycle regulation. HLASR is also thought to have a role in GSH (reduced glutathione) oxidation (Bleeker et al., 2006). An exact protein match (isotig19077) to this Cdc25-like H. lanatus ASR, the only gene sequence previously published for this species, was found in all 10 N and 10 T transcriptomes (Fig. S7), and was shown not to be differentially regulated, confirming that it has no adaptive role in tolerance.
We identified 87 transcripts whose expression significantly differed between the T and N phenotypes, and 19 transcripts (17 with functional annotation) with consistent SNPs (36 SNPs in total) in all 10 T and N genotypes (Table S5). It is noteworthy that consistent SNPs and significant gene expression changes between N and T phenotypes are aggregated in transcripts with regulatory functions (Figs 4, 5). Differential post-translational and/or post-transcriptional regulation (involving ubiquitin, proteases, kinases, methylation, transponsons, retrotransposons and RNA-binding proteins), potentially of HAPT proteins or proteins acting on HAPT, therefore, appears to define the N/T phenotypes. Whether the identified SNPs are all genomic SNPs or whether in some cases mRNA editing may be involved remains to be elucidated. A master regulatory gene, potentially to be found within our list of target genes, or possibly in the form of an as yet unidentified small RNA, producing the observed effect on genes involved in post-translational events, including protein degradation via the ubiquitin/proteasome complex and potentially also post-transcriptional events mediated by RNA-binding proteins, is still to be identified. This characterization of the genetic consequences of the P response polymorphism in H. lanatus provides an unparalleled insight into the signal cascades, optimized under natural selection, involved in P nutrition and has major consequences for understanding how plants respond to P nutrition and adapt to arsenate in their environment. We anticipate that this as yet unknown master regulatory gene and its downstream targets, which we have already identified, will be of significant consequence for future study and breeding of P-efficient forage plants and cereal crops.
Furthermore, the transcriptome characterized here will enable future transcriptomic studies on arsenic mine-adapted plants (Macnair et al., 1992) to be more focused, as transcripts identified in this study will be the starting point for looking at further selection that may occur on mine spoil soil. Key to this is the fact that the identification of transcripts involved in arsenate tolerance within a polymorphic population will enable the identification of confounding genes that have no involvement in arsenate tolerance, but appear in comparisons of mine and non-mine population transcriptomes as a result of other selection pressures.