Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 2014 Apr 4;165(2):648–657. doi: 10.1104/pp.114.237008

The Pea Photoperiod Response Gene STERILE NODES Is an Ortholog of LUX ARRHYTHMO1,[W],[OPEN]

Lim Chee Liew 1,2,3, Valérie Hecht 1,2, Frances C Sussmilch 1, James L Weller 1,*
PMCID: PMC4044833  PMID: 24706549

Early flowering and insensitivity to daylength in garden pea is caused by disruption of a gene important for circadian rhythms.

Abstract

The STERILE NODES (SN) locus in pea (Pisum sativum) was one of the first photoperiod response genes to be described and provided early evidence for the genetic control of long-distance signaling in flowering-time regulation. Lines homozygous for recessive sn mutations are early flowering and photoperiod insensitive, with an increased ability to promote flowering across a graft union in short-day conditions. Here, we show that SN controls developmental regulation of genes in the FT family and rhythmic regulation of genes related to circadian clock function. Using a positional and functional candidate approach, we identify SN as the pea ortholog of LUX ARRHYTHMO, a GARP transcription factor from Arabidopsis (Arabidopsis thaliana) with an important role in circadian clock function. In addition to induced mutants, sequence analysis demonstrates the presence of at least three other independent, naturally occurring loss-of-function mutations among known sn cultivars. Examination of genetic and regulatory interactions between SN and two other circadian clock genes, HIGH RESPONSE TO PHOTOPERIOD (HR) and DIE NEUTRALIS (DNE), suggests a complex relationship in which HR regulates expression of SN and the role of DNE and HR in control of flowering is dependent on SN. These results extend previous work to show that pea orthologs of all three Arabidopsis evening complex genes regulate clock function and photoperiod-responsive flowering and suggest that the function of these genes may be widely conserved.

Flowering time is an important determinant of crop adaptation and yield, and in recent years, much effort has been devoted to understanding molecular basis for flowering-time control across a range of model species and taxonomic groups (Andrés and Coupland, 2012). Legumes are a major group of crop plants and globally provide an important complement to cereals, both as a protein source for human and animal nutrition and in crop rotations, where they supplement soil nitrogen and help disrupt disease cycles (Smykal et al., 2012). Major crop legumes fall into two major groups with respect to their flowering-time control: warm season crops such as soybean (Glycine max) and common bean (Phaseolus vulgaris), which require short days for flowering, and the temperate, cool-season crops such as pea (Pisum sativum), lentil (Lens culinaris), and chickpea (Cicer arietinum), which are long-day plants (Nelson et al., 2010). While ancestors of many legume crops are geographically constrained by their photoperiod requirements, the isolation of variants with relaxed requirements has allowed cultivation across a much wider range and provided adaptation to a range of agronomic practices (Watanabe et al., 2012; Weller et al., 2012).

Among temperate legumes, the molecular and genetic control of flowering time is best understood in garden pea (Hecht et al., 2007, 2011; Liew et al., 2009a; Weller et al., 2009, 2012), although other model legumes such as Medicago truncatula and Lotus japonicus are beginning to provide a useful complement (Laurie et al., 2011; Yamashino et al., 2013). Early studies on flowering in pea distinguished several major loci responsible for existing variation in flowering time and provided one of the first examples of the use of controlled photoperiod conditions to resolve photoperiod-dependent phenotypes in genetic analysis (Barber, 1959; Murfet, 1971, 1973). These early investigations identified a major locus controlling photoperiod sensitivity, which was named STERILE NODES (SN), reflecting the increased number of vegetative (sterile) nodes produced before flowering in photoperiod-responsive (SN) lines relative to less-sensitive (sn) lines (Wellensiek, 1925; Barber, 1959).

Emerging evidence from several species shows that a reduction in photoperiod sensitivity can result from altered function of genes that have a primary role in the maintenance of circadian rhythms (Doyle et al., 2002; Watanabe et al., 2011; Faure et al., 2012; Campoli et al., 2013). In pea, we previously identified photoperiod response loci DIE NEUTRALIS (DNE), LATE BLOOMER1 (LATE1), and HIGH RESPONSE TO PHOTOPERIOD (HR) as orthologs of Arabidopsis (Arabidopsis thaliana) circadian clock genes EARLY FLOWERING4 (ELF4), GIGANTEA (GI), and ELF3, respectively (Hecht et al., 2007; Liew et al., 2009a; Weller et al., 2012), suggesting that other photoperiod response loci in pea might also affect circadian clock components. The molecular nature of the SN locus has not yet been determined, and the aim of this study was to examine in more detail the nature of the SN locus and identify the underlying gene. We report here that SN is the pea ortholog of Arabidopsis circadian clock gene LUX ARRHYTHMO (LUX) and investigate its roles and interactions with the DNE and HR genes in control of flowering time and rhythmic gene expression.

RESULTS

SN Contributes to Photoperiodic Flowering and the Maintenance of Circadian Rhythms

Initial genetic and physiological characterization of the SN locus involved the use of two pea lines with contrasting flowering behavior, cv Telephone (SN) and Massey (sn; Barber, 1959). Subsequent studies showed that many of the earliest pea cultivars, including cv Alaska, Progress #9, Meteor, Sparkle, Vinco, and Kleine Rheinländerin, all carry recessive alleles at the SN locus (Murfet, 1971, 1977; Weeden et al., 1988). An induced mutant allele (sn-2) was recovered from γ-irradiation of cv Borek (Arumingtyas and Murfet, 1994), and we identified two further mutant alleles (sn-3 and sn-4) in ethyl methanesulfonate mutagenesis of line NGB5839 (Hecht et al., 2007) that also confer early flowering under short-day (SD) conditions (Fig. 1A). As previously reported for naturally occurring sn mutants (Murfet, 1977), the sn-4 mutation also confers a complete insensitivity to photoperiod (Fig. 1A), similar to the previously described dne mutant (King and Murfet, 1985; Liew et al., 2009a). As seen for dne, the early flowering of the sn-4 mutant is also associated with elevated expression of several FT genes in leaf and shoot apex tissue (Fig. 1B).

Figure 1.

Figure 1.

Open in a new tab

Mutations at the SN locus confer photoperiod-insensitive early flowering and elevated expression of FT genes. A, Node of flower initiation. Data are mean ± se for n = six to eight plants. B, Relative transcript levels were determined for FT genes and the floral marker PROLIFERATING INFLORESCENCE MERISTEM (PIM) in wild-type (WT; NGB5839) and sn-4 plants at 2-d intervals following seedling emergence into SD conditions (8-h light/16-h dark), in either the uppermost expanded leaf (leaf) or shoot apices dissected to 2 to 3 mm in length (apex). Values are normalized to the transcript level of the ACTIN gene and represent mean ± se for n = two to three biological replicates, each consisting of pooled material from two plants.

In view of the similarity of the sn and dne mutant phenotypes (Fig. 1; Liew et al., 2009a), we considered that sn might also have a primary defect in maintaining normal circadian rhythms. In initial support of this conclusion, we previously showed that rhythmic expression of the pea GI ortholog LATE1 under long-day (LD) conditions was significantly disturbed by the sn-4 mutation (Hecht et al., 2007). We therefore examined the effect of sn-4 on rhythmic gene expression in more detail. Figure 2 shows that under SD conditions, where its flowering phenotype is most clearly expressed, the LATE1 rhythm in the sn-4 mutant also exhibits a clear loss of repression during the night. Under these conditions, expression of DNE also showed a clear advance in peak expression in the mutant of around 4 h compared with the wild type. No consistent statistically significant effects on expression of other genes were detected, although there was a suggestion that the peak expression of the pea LATE ELONGATED HYPOCOTYL (LHY) gene was lower and slightly broader and that expression of TIMING OF CAB2 EXPRESSION1 (TOC1) and PSEUDO RESPONSE REGULATOR59 (PRR59) was slightly elevated during the night (Fig. 2).

Figure 2.

Figure 2.

Open in a new tab

SN affects rhythmic expression of clock gene homologs under SD cycles. Transcript levels were determined in the uppermost fully expanded leaf of 3-week-old wild-type (NGB5839) and sn-4 plants grown under an 8-h photoperiod at 20°C. Data are mean ± se for n = two to three biological replicates, each consisting of pooled material from two plants. Day and night periods are respectively indicated by white and black bars above the graph. Note that both genotypes carry the hr mutation (Weller et al., 2012).

To confirm the photoperiod- and light-independent nature of the rhythmic defects in the sn mutant, we also examined the effect of sn-4 on rhythms of gene expression after transfer to continuous darkness from an entraining photoperiod. Figure 3 shows that rhythmic expression of LHY was maintained in the sn mutant without light input for at least two cycles, but with an apparently shorter period, with peaks at approximately zeitgeber time (ZT) 45 and ZT 60 instead of ZT 48 and ZT 69 as seen in the wild type. As previously described, the amplitude of the DNE expression rhythm was reduced around 10-fold after transfer to continuous darkness (Liew et al., 2009a), and the sn-4 mutation also affected the ELF4 expression rhythm under the entraining photoperiod and in subsequent darkness, with lower peak expression and a clear phase advance. No clear effects of sn on rhythms of TOC1 or LATE1 expression were evident, although the expression level of TOC1 was generally lower in sn, and LATE1 was higher during the subjective night (Fig. 3).

Figure 3.

Figure 3.

Open in a new tab

SN affects circadian rhythms of gene expression in continuous darkness. Transcript levels were determined in the uppermost fully expanded leaf of 3-week-old wild-type (NGB5839) and sn-4 plants grown under a light/dark cycle (12L:12D) at 20°C for 21 d before transfer to continuous darkness at ZT 24. Values are normalized to the transcript level of the ACTIN gene and represent ± se for n = two biological replicates, each consisting of pooled material from two plants. ZT refers to the time since lights-on of the last full entraining cycle. Bars above the graph refer to periods of light (white bars) or darkness (black or gray bars). The gray bars indicate the periods of subjective day during the period of continuous darkness. Note that both genotypes carry the hr mutation (Weller et al., 2012).

SN Is the Pea Ortholog of Arabidopsis LUX

The SN gene was previously mapped between the isozyme loci Aldolase (Aldo) and Galactosidase2 on the lower half of pea linkage group VII (Murfet and Sherriff, 1996), which is syntenic with M. truncatula chromosome 4 (Aubert et al., 2006). To refine this position, we generated an F2 progeny from a cross between the sn-4 mutant and cv Térèse. Initial mapping confirmed the position of SN in linkage group VII between markers Aldo and Pip2 (Aubert et al., 2006), and we then scanned the corresponding region of M. truncatula chromosome 4 for genes potentially related to flowering. This identified homologs of Arabidopsis HUA2, TARGET OF EAT1 (TOE1), FPA, VERNALIZATION1, PRR3/PRR7, PHOTOTROPIN1, and LUX. Scoring of additional genes in this region narrowed the position of SN to an interval between TOE1 and the Clpser marker (Aubert et al., 2006) and excluded PRR37 as a candidate (Liew et al., 2009b). The only other circadian-related gene predicted to lie in this interval, LUX, did not contain an intron in the coding sequence and was identical in sequence in the two parental backgrounds cv Torsdag and Térèse, precluding its mapping in this cross. However, the positions of SN in the pea genetic map and LUX in the M. truncatula physical map between anchor markers Clpser and TOE1 were very similar, and together with the evidence linking SN to circadian function, we therefore considered LUX as a strong candidate for SN.

Like LUX orthologs in other species, the pea LUX gene consists of a central, highly conserved myb-like GARP domain flanked by regions of low conservation and a short, moderately conserved N-terminal domain of no known function (Supplemental Fig. S1). In Arabidopsis, LUX is part of a small gene family that includes its closest homolog NOX/BROTHER OF LUX ARRHYTHMO (Dai et al., 2011; Helfer et al., 2011) and three other genes (Hazen et al., 2005) that fall into a separate subclade (Fig. 4A). By contrast, M. truncatula and L. japonicus each have only a single representative of LUX/NOX and only one gene in the other subclade. Each of these genes is represented in soybean by a pair of homeologs. A third soybean sequence was identified as belonging to this second clade but had neither a soybean homeolog nor a counterpart in the other legumes.

Figure 4.

Figure 4.

Open in a new tab

SN is the pea ortholog of Arabidopsis LUX. A, Phylogram of legume LUX-like protein sequences. Arabidopsis RESPONSE REGULATOR1 (AtARR1), a dissimilar GARP transcription factor (Sakai et al., 1998), is included as an outgroup. All branches have at least 58% bootstrap support. Ps, Pea; At, Arabidopsis; Mt, M. truncatula; Lj, L. japonicus; Gm, soybean. Details of all sequences and the corresponding alignment are presented in Supplemental Figure S1. B, Diagram of the PsLUX gene showing details of functionally significant mutations in sn mutants and cultivars. C, Median-joining network representing relatedness of 23 SN haplotypes, each represented by a numbered circle. Details of haplotypes and their effects on the SN protein sequence are given in Supplemental Table S1. Gray and red circles represent P. sativum var sativum with functional or mutant forms of SN, respectively. Other colors represent P. sativum var humile (purple) or var elatius (green), or Pisum fulvum (yellow). Changes to the protein sequence are represented by circles (substitutions) or triangles (indels), either black (functionally significant changes) or white (likely to be inconsequential).

We found distinct mutations in PsLUX in each of the three induced sn mutants (Fig. 4B). The sn-2 irradiation mutant carries an 8-bp deletion leading to a frameshift in codon 107 with truncation after 13 missense amino acids, and the sn-3 and sn-4 mutants both carry G-to-A mutations, respectively replacing the start codon and W15 with stop codons. We therefore conclude that the SN locus is equivalent to PsLUX. As all three of these mutations are predicted to truncate the LUX protein before the GARP DNA-binding domain, it is likely that they all represent complete loss-of-function alleles.

Naturally Occurring Variation in SN

We also found clear mutations in naturally occurring sn variants. The best known sn line, cv Alaska, carried a 10-bp deletion (sn-1) just prior to the GARP domain, which was also present in several other sn cultivars including cv Massey and Cennia. This mutation introduced a frameshift at residue 121 and truncation after one additional missense amino acid. Two other sn cultivars, cv Vinco and Sparkle, exhibited several differences from the NGB5839 sequence that in each case included a single distinct functionally significant mutation (Fig. 4B). cv Vinco carried a deletion of a single G in codon 3 (sn-5) directing a frameshift and truncation after nine missense amino acids, whereas cv Sparkle carried a single-nucleotide polymorphism (SNP) directing substitution of a residue within the GARP domain (K184R; sn-6) that is highly conserved across the wider clade of LUX-like proteins (Supplemental Fig. S1). Interestingly, this substitution was not present in the SN revertant line E54, which was derived from ethyl methanesulfonate mutagenesis of cv Sparkle (Murfet and LaRue, 1994), further supporting the case for the K184R polymorphism as the causal mutation in the cv Sparkle sn allele.

Sequence analysis of the SN coding sequence from a small selection of diverse Pisum spp. lines revealed highly similar sequences among a number of cultivars and other domesticated lines (Fig. 4C). All lines carrying the sn-1 mutation shared the same haplotype (haplotype 2), and had LUX coding sequences that were otherwise identical to NGB5839 and the majority of SN cultivars examined. Apart from the putative causal mutations in cv Vinco and Sparkle, only five SNPs were identified among other domesticated lines, including three silent changes and two that directed substitutions of highly variable residues (Supplemental Table S1; Supplemental Fig. S1). Three other SNPs in the data set also specified amino acid substitutions, but these also affected poorly conserved residues, while a 6-bp expansion of a CCT microsatellite introduced two additional residues to a series of six prolines within a highly variable region. LUX sequences within the domesticated lines examined showed greatest affinity with a group of wild P. sativum var elatius accessions from Israel and Turkey (haplotypes 1 and 9). A larger group of accessions with more divergent LUX sequences included other var elatius and Pisum fulvum lines, and landraces from Afghanistan and China.

Interactions of SN, DNE, and HR

In view of the fact that SN, DNE, and HR genes all influence the circadian clock, we next examined how they might interact in control of flowering time and rhythmic gene expression. The three induced sn mutants and most commonly available cultivars carrying the sn-1 mutation also carry the recessive loss-of-function hr mutation (Weller et al., 2012), and these mutants are thus double loss-of-function mutants. This is also true for the previously described dne mutant that was isolated in the cv Torsdag (hr) background (King and Murfet, 1985; Liew et al., 2009a). To examine the genetic interactions among these loci, we crossed sn and dne into a functional HR genetic background and compared their effects. Figure 5 shows that the sn-4 mutation is epistatic to HR in the control of flowering time, as plants carrying sn-4 were equivalently early flowering in SD regardless of their HR genotype. By contrast, although dne also conferred slightly earlier flowering under SD in an HR background, dne HR plants were still markedly later than dne hr plants. This suggests that DNE and HR may interact functionally and may both require SN for their action. We also generated an sn dne hr triple mutant, which was indistinguishable from the sn single mutant and the sn hr double mutant.

Figure 5.

Figure 5.

Open in a new tab

Interaction of SN, HR, and DNE in the control of SD flowering. Node of flower initiation for plants grown in the phytotron under an 8-h photoperiod of natural light. Data represent mean ± se for n = six to 12 plants.

To test how genetic interactions might be reflected in effects on circadian gene expression, we first examined SN/LUX and DNE/ELF4 expression rhythms using a NGB5839 near-isoline, into which functional HR alleles had been introgressed (Weller et al., 2012). In Arabidopsis, LUX shows circadian regulation with a peak in the evening, similar to TOC1 and GI (Hazen et al., 2005; Onai and Ishiura, 2005). Figure 6 shows that under a 12-h-light (12L):12-h-dark (12D) entraining photoperiod, SN expression showed a strong rhythm with an afternoon peak at around ZT 8, similar to LATE1 and TOC1 and slightly earlier than DNE (Liew et al., 2009a). In plants with functional HR, this rhythm was maintained under constant light, with peaks at ZT 32 and ZT 56. Loss of HR function had minimal effect on SN rhythmic expression under the entraining photoperiod but led to a clear damping of the rhythm to an intermediate level after transfer to continuous light, as seen for other clock genes LHY, TOC1, and LATE1 (Weller et al., 2012). By contrast, the DNE expression rhythm remained very similar in HR and hr lines under continuous light.

Figure 6.

Figure 6.

Open in a new tab

Effect of HR on circadian expression of SN and DNE under continuous light. Transcript levels were determined in the uppermost fully expanded leaf of 3-week-old plants of NGB5839 (hr) and an HR near-isoline grown under a light/dark cycle (12L:12D) at 20°C for 21 d before transfer to continuous light at ZT 24. Values are normalized to the transcript level of the ACTIN gene and represent mean ± se for n = two biological replicates, each consisting of pooled material from two plants. ZT refers to the time since lights-on of the last full entraining cycle. Bars above the graph refer to periods of light (white or gray bars) or darkness (black bars). The gray bars indicate the periods of subjective night during the period of continuous light.

Finally, we compared SN and sn genotypes on an HR background in plants similarly released to continuous light (LL) following photoperiod entrainment. The data presented in Figure 7 show that rhythmic expression of LHY had slightly lower peak expression in sn and appeared to damp more rapidly to a low level in continuous light following the first subjective dusk at ZT 36. A similar pattern was seen for TOC1 and LATE1, except that these genes damped to an intermediate expression level, and showed a clear phase advance. Under the entraining SD, the sn mutation conferred a phase advance in DNE expression (Fig. 7) that was similar to the effect observed on an hr background (Fig. 2) but had no clear effect in constant light. HR expression was apparently unaffected by the sn mutation, remaining low and essentially arrhythmic under entraining or constant conditions. Interestingly, a distinct difference was observed in the effects of sn on expression of two PRR genes, PRR37 and PRR59 (Liew et al., 2009a). Whereas PRR37 expression was completely unaffected by sn, PRR59 showed 3- to 4-fold higher expression over the latter half of the night phase and an associated earlier shift in the daytime peak (Fig. 7).

Figure 7.

Figure 7.

Open in a new tab

Effect of SN on circadian expression of clock genes under continuous light. Transcript levels were determined in the uppermost fully expanded leaf of 3-week-old plants of SN HR and sn-4 HR near-isolines grown under a light/dark cycle (12L:12D) at 20°C for 21 d before transfer to continuous light at ZT 24. Values are normalized to the transcript level of the ACTIN gene and represent mean ± se for n = two biological replicates, each consisting of pooled material from two plants. ZT refers to the time since lights-on of the last full entraining cycle. Bars above the graph refer to periods of light (white or gray bars) or darkness (black bars). The gray bars indicate the periods of subjective night during the period of continuous light.

DISCUSSION

SN is one of four loci in pea that influence the response to photoperiod by delaying flowering under SD conditions. Like the dne, hr, and photoperiod response (ppd) mutations, sn mutations accelerate flowering, shorten the reproductive phase, and inhibit basal branching under SD (King and Murfet, 1985; Murfet, 1985; Arumingtyas and Murfet, 1994; Weller et al., 2009). We recently demonstrated that DNE and HR are orthologs of Arabidopsis circadian clock genes and presented preliminary evidence that SN might also affect circadian rhythms (Hecht et al., 2007; Liew et al., 2009a; Weller et al., 2012). Here, we have confirmed this conclusion and characterize SN as the pea ortholog of Arabidopsis LUX. This is based on the identification of functionally significant mutations in several independent sn mutants (Fig. 4), backed by observations of tight genetic linkage of sn phenotypes to putative causal polymorphisms, and the general phenotypic similarity of sn mutants to Arabidopsis lux mutants (Hazen et al., 2005). Disruption of LUX homologs has also recently been proposed as the molecular basis for mutations conferring photoperiod-insensitive early flowering in the barley (Hordeum vulgare) early maturity10 (eam10) mutant (Campoli et al., 2013) and in einkorn wheat (Triticum monococcum; Mizuno et al., 2012).

Three of the sn functional variants described here are naturally occurring. One of these, a 10-bp deletion, is the probable basis for the distinctively early flowering in the well-known pea cultivar Alaska. This variety was developed in the United Kingdom by Thomas Laxton and introduced to the United States around 1880, where it achieved wide popularity due to its early maturity and the associated expansion in seasonal and climatic range for cultivation (Shoemaker and Delwiche, 1934). The documented history of pea breeding is too fragmentary to trace the deeper origin of this variant, but cv Alaska was subsequently widely used in the development of other early cultivars, as reflected in our observation that the same mutant allele is present in a number of cultivars previously shown to carry an sn mutation. All of these cultivars also belong to HR haplotype 2, the single widespread haplotype carrying the loss-of-function hr mutation (Weller et al., 2012), consistent with a recent origin for the sn-1 deletion within this group of germplasm. However, sn-dependent early flowering has arisen at least two other times and, in the case of cv Vinco, occurs on a functional HR background. Again, limited pedigree details for cv Sparkle and Vinco unfortunately provide little insight into the origins of these distinct sn functional variants (De Haan, 1955; Gritton and Myers, 2002). Future wider analysis of SN diversity will clearly help clarify these origins and reveal any additional reduced-function alleles not present in the limited selection examined here.

The pea SN/LUX gene shows strongly rhythmic expression under light/dark cycles and is coexpressed with several other evening genes including TOC1, LATE1, and PRR59 (Figs. 6 and 7). Like Arabidopsis lux mutants, pea sn mutants entrained to light/dark cycles rapidly lose normal rhythmic expression of these genes following transfer to constant conditions, indicating the importance of SN for maintenance of circadian rhythms (Figs. 3 and 7; Hazen et al., 2005; Onai and Ishiura, 2005). Peak expression of LHY is significantly lower in the sn mutant than in the wild type (Fig. 7), consistent with the role of Arabidopsis LUX as a positive regulator of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LHY (Hazen et al., 2005). The damping of TOC1 and LATE1 to high/intermediate levels observed in sn may also result from the low level of LHY expression, similar to Arabidopsis, where CCA1 and LHY are known to negatively regulate TOC1 and GI expression (Alabadí et al., 2001; Mizoguchi et al., 2002; Nagel and Kay, 2012). Recent analyses of LUX binding specificity have shown that the positive effect of LUX on CCA1/LHY is an indirect effect, involving direct repression by LUX of PRR9, which is itself a direct repressor of CCA1 (Helfer et al., 2011). This is reflected in the fact that lux mutants show elevated PRR9 expression, particularly in the night phase (Helfer et al., 2011; Nusinow et al., 2011; Herrero et al., 2012). We also found significant misregulation of the PRR59 gene in the sn mutant, suggesting that this sequence of regulatory interactions might also be conserved in pea. Interestingly, in barley, Campoli et al. (2013) found no difference between the wild type and the eam10 mutant in rhythmic expression of TOC1 or genes in the PRR5/PRR9 subclade but instead demonstrated elevated expression of PRR37/Ppd-H1 and PRR73 genes.

In Arabidopsis, lux mutants share circadian and flowering phenotypes with two other mutants, elf3 and elf4 (Nagel and Kay, 2012). This similarity is also seen for the corresponding mutants in pea and mutants for LUX and ELF3 orthologs in barley (Liew et al., 2009a; Faure et al., 2012; Weller et al., 2012; Campoli et al., 2013) and is likely to stem from an intimate functional relationship among these three genes. In Arabidopsis, LUX was recently shown to act together with ELF3 and ELF4 proteins in a complex termed the evening complex (EC), in which ELF3 is thought to serve as a adaptor, physically linking ELF4 to LUX (Nusinow et al., 2011; Herrero et al., 2012). Consistent with a role of ELF3 and ELF4 in the EC and in LUX activity, elf3 and elf4 mutants also show elevated expression of PRR9 (Thines and Harmon, 2010; Dixon et al., 2011; Kolmos et al., 2011). Likewise, the pea dne mutant also shows altered regulation of PRR59 with an apparent loss of repression during the latter part of the night phase but had no effect on PRR37 expression (Liew et al., 2009a), similar to sn (Figs. 2 and 7).

The three Arabidopsis EC genes are rhythmically coexpressed with peaks around dusk (Dixon et al., 2011; Nusinow et al., 2011), and the same is true for Eam8 and Eam10 genes in barley (Faure et al., 2012; Campoli et al., 2013). In pea, whereas both SN and DNE exhibit strongly rhythmic expression under light/dark cycles (Fig. 6; Hecht et al., 2007; Liew et al., 2009a), there is no discernable rhythm in HR expression (Fig. 7). However, this may not be particularly consequential for HR function given that the level of ELF3 expression in Arabidopsis is reported to be relatively unimportant for its function (Dixon et al., 2011). Two other aspects of HR and SN action and interaction are potentially more significant. Although HR and SN have generally similar effects on rhythmic expression of LHY, TOC1, and LATE1 under LL conditions, HR does not affect DNE expression, whereas SN clearly does (Figs. 6 and 7). Also, loss of SN function has clear effects on rhythmic expression of several clock genes, including its putative direct target PRR59, even in the presence of the hr mutation (Figs. 2 and 3). These findings would not be expected if SN were only active as part of a complex in which HR was an essential component. One possible explanation is that there may be redundancy for HR action that is exposed differently in SN-dependent regulation of different sets of target genes. Alternatively, the action of SN may not be entirely dependent on HR or on an HR-containing complex. More generally, it appears unlikely that the roles of Arabidopsis EC components are restricted to functions of the EC, as both ELF3 and ELF4 have been shown to interact physically with other proteins. For example, ELF3 interacts with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and GI to facilitate COP1-dependent degradation of GI (Yu et al., 2008), while ELF4 also interacts with GI to control its subnuclear localization (Kim et al., 2013).

Comparison of near-isogenic single null mutants for hr, dne, and sn (Fig. 5) shows that only sn mutants are fully insensitive to photoperiod, while the hr mutation significantly reduces but does not eliminate the photoperiod response, and the dne mutation has only a weak effect. This is in apparent contrast to Arabidopsis and barley, where the equivalent mutants are all reported to be essentially insensitive to photoperiod (Doyle et al., 2002; Hazen et al., 2005; Faure et al., 2012; Campoli et al., 2013). The reason for this is not clear, but one possibility could be that DNE and HR overlap in function such that they have some capacity for mutual compensation. An alternative possibility is that there is redundancy in the function of HR and DNE individually due to the presence of additional homologs (Liew et al., 2009a). The observation that loss of SN function alone is sufficient to eliminate any response to photoperiod is consistent with the fact that legume LUX genes appear to be single copy (Fig. 4A) and shows that the next most closely related genes to SN have no significant role in this process. Flowering-time comparisons also confirm the previously reported epistasis of sn over HR (Murfet, 1973). The genotype HR dne sn was not available for this study, and we therefore could not test directly the interaction of sn and dne on an HR background. Nevertheless, in view of the fact that the sn single mutant was as early flowering as the hr sn double and the hr dne sn triple (Fig. 5), it seems highly probable that sn is also epistatic to dne. These genetic relationships imply that HR and DNE may depend on SN for their function and act by positively modifying SN activity, a conclusion that is consistent with the central role of LUX in recruitment of the EC to target promoters (Nusinow et al., 2011). Epistatic interactions between the Arabidopsis EC genes have also been examined in the control of rhythmic gene expression and suggest that ELF4 may act upstream of a complex that requires both ELF3 and LUX (Herrero et al., 2012). While these interactions have some similarity to the ones we observe, they also differ with respect to the epistasis of SN/LUX over HR/ELF3. It should also be noted that these interactions have been assessed on different traits, and the interactions among the pea genes on clock properties has not yet been examined in detail. The flowering-time interactions described here could thus reflect interaction at the level of FT gene expression rather than in clock function (Hecht et al., 2011). Discrimination between these two possibilities will depend on a better understanding of how clock and light signals are integrated for FT regulation in legumes (Weller et al., 2009).

In conclusion, the fact that very similar patterns of misregulation of CCA1/LHY, GI, TOC1, and other PRR genes are observed in mutants for LUX orthologs in three different species points to a broad conservation not only of LUX function, but also of wider regulatory interactions within the circadian clock. While formation of a functional complex equivalent to the Arabidopsis EC has not yet been demonstrated, a comparison of mutant phenotypes for pea and barley orthologs shows that these genes have a widely conserved role in circadian and photoperiodic regulation and suggests that their mutational inactivation may be a simple and common route to early flowering and a reduction in the LD requirement in many temperate species.

MATERIALS AND METHODS

Plant Material

Origins of the sn-2, sn-3, sn-4, and dne-1 mutations are described previously (King and Murfet, 1985; Arumingtyas and Murfet, 1994; Hecht et al., 2007). The sn-1 mutant used in Figure 1 is a near-isoline in the NGB5839 background obtained by introgression of the sn allele from cv Alaska. Parental lines cv Borek (sn-2) and NGB5839 (sn-3 and sn-4) both carry the hr mutation (Weller et al., 2012), and single mutants were generated by crossing the original sn-4 and dne-1 mutants to a near-isogenic line of NGB5839 carrying a functional HR allele (Weller et al., 2012). Identity of desired allelic combinations was verified in advanced generations by molecular genotyping using high resolution melt-quantitative PCR and cleaved amplified polymorphic sequence markers (Liew et al., 2009a; Weller et al., 2012). Triple sn dne hr mutants were similarly obtained from a cross between the original sn-4 and dne-1 mutants. Details of lines used for diversity analysis are described in Weller et al. (2012) and shown in Supplemental Table S2.

General Growth Conditions

All plants were grown in a 1:1 mixture of dolerite chips and vermiculite topped with potting mix and received nutrient solution weekly. Plants for gene expression experiments (Figs. 1–3, 6, and 7) were grown in growth cabinets at 20°C under 150 µmol m–2 s–1 white light from cool-white fluorescent tubes. Segregating progenies and plants for flowering experiments (Figs. 1 and 5) were grown in the phytotron and received an 8-h photoperiod of natural daylight either with (LD) or without (SD) an 8-h extension with white light (10 µmol m–2 s–1) from cool-white fluorescent tubes (Hecht et al., 2007).

Mapping and Linkage Analysis

Several markers used for linkage analysis were modified from gene-based markers described by Aubert et al. (2006) and Bordat et al. (2011). These markers were supplemented by newly designed markers targeted to introns of appropriate genes identified in the relevant interval of the Medicago truncatula genome (v3.5; http://www.jcvi.org/medicago/) and also present in pea (Pisum sativum) sequence databases in GenBank (http://www.ncbi.nlm.nih.gov). Details of these markers and their method of detection are provided in Supplemental Table S3. Markers used to detect the dne-1 and hr mutations have been described previously (Liew et al., 2009a; Weller et al., 2012).

Sequence and Expression Analysis

The pea LUX gene was initially isolated with specific primers designed on the M. truncatula sequence (Medtr4g064730; MtLUX-1F 5′-TTGATCCCACCGGAGCTCGC-3′ and MtLUX-1R 5′-ACCCTTCAACATTCATCAAC-3′), and full-length complementary DNA was subsequently obtained using RACE-PCR (BD-SMART RACE cDNA Amplification Kit; Clontech) and gene-specific primers (for the 5′ region: LUX-4R 5′-CAGCGTCGTTTCTGTTCTAACC-3′ and LUX-5R 5′-GGTTGACGTCGATGAGTGTACG-3′; for the 3′ region: LUX-4F 5′-GAACAGAAACGACGCTGAAACG-3′ and LUX-5F 5′-AAGAGATTCGTCGACGTTGTGG-3′). Full genomic sequence was obtained by genome walking (GenomeWalker Universal Kit; Clontech) using the same primers. All PCR products were cloned in pGEM-T Easy (Promega) and sequenced at Macrogen.

Sequences for phylogenetic analysis were retrieved by reciprocal BLAST searches in legume databases (http://www.jcvi.org/medicago/; http://www.phytozome.net/soybean; http://www.kazusa.or.jp/lotus/). For the phylogenetic tree shown in Figure 4A, amino acid sequences were aligned using ClustalX (Thompson et al., 1997). Distance and parsimony-based methods were used for phylogenetic analyses in PAUP*4.0b10 (http://paup.csit.fsu.edu/) using the alignment shown in Supplemental Figure S1. For Figure 4C, a median-joining haplotype network was constructed in SplitsTree 4 (http://www.splitstree.org) using the default settings.

For all circadian and diurnal expression experiments (Figs. 2, 3, 6, and 7), plants were 3 weeks old at harvest, and harvested tissue consisted of both leaflets from the uppermost expanded leaf. Tissue harvests for the FT expression experiment presented in Figure 1B were performed as described in Hecht et al. (2011). RNA extraction, reverse transcription, and real-time PCR analysis were performed as described previously (Liew et al., 2009a). Primers for expression analysis are presented in Supplemental Table S4.

Sequence data from this article can be found in the GenBank/EMBL data libraries under the accession number KJ801796.

Supplemental Data

The following materials are available in the online version of this article.

Supplementary Material

Supplemental Data
supp_165_2_648__index.html (887B, html)

Acknowledgments

We thank Ian Cummings, Tracey Winterbottom, and Michelle Lang for help with plant husbandry and Jackie Vander Schoor, Stephen Ridge, and Ray Ali for technical assistance.

Glossary

SD

short-day

LD

long-day

ZT

zeitgeber time

SNP

single-nucleotide polymorphism

12L

12-hour-light

12D

12-hour-dark

EC

evening complex

Footnotes

1

This work was supported by the Australian Research Council through a Discovery Project grant to J.L.W.

[W]

The online version of this article contains Web-only data.

[OPEN]

Articles can be viewed online without a subscription.

References

  1. Alabadí D, Oyama T, Yanovsky MJ, Harmon FG, Más P, Kay SA. (2001) Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science 293: 880–883 [DOI] [PubMed] [Google Scholar]
  2. Andrés F, Coupland G. (2012) The genetic basis of flowering responses to seasonal cues. Nat Rev Genet 13: 627–639 [DOI] [PubMed] [Google Scholar]
  3. Arumingtyas EL, Murfet IC. (1994) Flowering in Pisum: a further gene controlling response to photoperiod. J Hered 85: 12–17 [Google Scholar]
  4. Aubert G, Morin J, Jacquin F, Loridon K, Quillet MC, Petit A, Rameau C, Lejeune-Hénaut I, Huguet T, Burstin J. (2006) Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula. Theor Appl Genet 112: 1024–1041 [DOI] [PubMed] [Google Scholar]
  5. Barber HN. (1959) Physiological genetics of Pisum II. The genetics of photoperiodism and vernalisation. Heredity 13: 33–60 [Google Scholar]
  6. Bordat A, Savois V, Nicolas M, Salse J., Chauveau A, Bourgeois M, Potier J, Houtin H, Rond C, Murat F, et al. (2011) Translational genomics in legumes allowed placing in silico 5460 unigenes on the pea functional map and identified candidate genes in Pisum sativum. G3 1: 93-103 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Campoli C, Pankin A, Drosse B, Casao CM, Davis SJ, von Korff M. (2013) HvLUX1 is a candidate gene underlying the early maturity 10 locus in barley: phylogeny, diversity, and interactions with the circadian clock and photoperiodic pathways. New Phytol 199: 1045–1059 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dai S, Wei X, Pei L, Thompson RL, Liu Y, Heard JE, Ruff TG, Beachy RN. (2011) BROTHER OF LUX ARRHYTHMO is a component of the Arabidopsis circadian clock. Plant Cell 23: 961–972 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. De Haan H. (1955) The breeding of peas in the Netherlands. Euphytica 4: 67–75 [Google Scholar]
  10. Dixon LE, Knox K, Kozma-Bognar L, Southern MM, Pokhilko A, Millar AJ. (2011) Temporal repression of core circadian genes is mediated through EARLY FLOWERING 3 in Arabidopsis. Curr Biol 21: 120–125 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Doyle MR, Davis SJ, Bastow RM, McWatters HG, Kozma-Bognár L, Nagy F, Millar AJ, Amasino RM. (2002) The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana. Nature 419: 74–77 [DOI] [PubMed] [Google Scholar]
  12. Faure S, Turner AS, Gruszka D, Christodoulou V, Davis SJ, von Korff M, Laurie DA. (2012) Mutation at the circadian clock gene EARLY MATURITY 8 adapts domesticated barley (Hordeum vulgare) to short growing seasons. Proc Natl Acad Sci USA 109: 8328–8333 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gritton ET, Myers JR (2002) Pea-green. In TC Wehner, ed, Vegetable Cultivar Descriptions for North America, Lists 1–26 Combined. (http://cuke.hort.ncsu.edu/cucurbit/wehner/vegcult/peagreenmz.html) [Google Scholar]
  14. Hazen SP, Schultz TF, Pruneda-Paz JL, Borevitz JO, Ecker JR, Kay SA. (2005) LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms. Proc Natl Acad Sci USA 102: 10387–10392 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hecht V, Knowles CL, Vander Schoor JK, Liew LC, Jones SE, Lambert MJM, Weller JL. (2007) Pea LATE BLOOMER1 is a GIGANTEA ortholog with roles in photoperiodic flowering, deetiolation, and transcriptional regulation of circadian clock gene homologs. Plant Physiol 144: 648–661 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hecht V, Laurie RE, Vander Schoor JK, Ridge S, Knowles CL, Liew LC, Sussmilch FC, Murfet IC, Macknight RC, Weller JL. (2011) The pea GIGAS gene is a FLOWERING LOCUS T homolog necessary for graft-transmissible specification of flowering but not for responsiveness to photoperiod. Plant Cell 23: 147–161 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Helfer A, Nusinow DA, Chow BY, Gehrke AR, Bulyk ML, Kay SA. (2011) LUX ARRHYTHMO encodes a nighttime repressor of circadian gene expression in the Arabidopsis core clock. Curr Biol 21: 126–133 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Herrero E, Kolmos E, Bujdoso N, Yuan Y, Wang M, Berns MC, Uhlworm H, Coupland G, Saini R, Jaskolski M, et al. (2012) EARLY FLOWERING4 recruitment of EARLY FLOWERING3 in the nucleus sustains the Arabidopsis circadian clock. Plant Cell 24: 428–443 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim Y, Lim J, Yeom M, Kim H, Kim J, Wang L, Kim WY, Somers DE, Nam HG. (2013) ELF4 regulates GIGANTEA chromatin access through subnuclear sequestration. Cell Rep 3: 671–677 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. King WM, Murfet IC. (1985) Flowering in Pisum: a sixth locus, Dne. Ann Bot (Lond) 56: 835–846 [Google Scholar]
  21. Kolmos E, Herrero E, Bujdoso N, Millar AJ, Tóth R, Gyula P, Nagy F, Davis SJ. (2011) A reduced-function allele reveals that EARLY FLOWERING3 repressive action on the circadian clock is modulated by phytochrome signals in Arabidopsis. Plant Cell 23: 3230–3246 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laurie RE, Diwadkar P, Jaudal M, Zhang L, Hecht V, Wen J, Tadege M, Mysore KS, Putterill J, Weller JL, et al. (2011) The Medicago FLOWERING LOCUS T homolog, MtFTa1, is a key regulator of flowering time. Plant Physiol 156: 2207–2224 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liew LC, Hecht V, Laurie RE, Knowles CL, Vander Schoor JK, Macknight RC, Weller JL. (2009a) DIE NEUTRALIS and LATE BLOOMER 1 contribute to regulation of the pea circadian clock. Plant Cell 21: 3198–3211 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liew LC, Hecht V, Weeden NF, Weller JL. (2009b) Isolation of Pseudo Response Regulator genes and evaluation as candidate genes for photoperiod response loci. Pisum Genet 41: 21–25 [Google Scholar]
  25. Mizoguchi T, Wheatley K, Hanzawa Y, Wright L, Mizoguchi M, Song HR, Carré IA, Coupland G. (2002) LHY and CCA1 are partially redundant genes required to maintain circadian rhythms in Arabidopsis. Dev Cell 2: 629–641 [DOI] [PubMed] [Google Scholar]
  26. Mizuno N, Nitta M, Sato K, Nasuda S. (2012) A wheat homologue of PHYTOCLOCK 1 is a candidate gene conferring the early heading phenotype to einkorn wheat. Genes Genet Syst 87: 357–367 [DOI] [PubMed] [Google Scholar]
  27. Murfet IC. (1971) Flowering in Pisum. Three distinct phenotypic classes determined by the interaction of a dominant early and a dominant late gene. Heredity 26: 243–257 [Google Scholar]
  28. Murfet IC. (1973) Flowering in Pisum. Hr, a gene for high response to photoperiod. Heredity 31: 157–164 [Google Scholar]
  29. Murfet IC (1977) The physiological genetics of flowering. In JF Sutcliffe, JS Pate, eds, The Physiology of the Garden Pea. Academic Press, London, pp 385–430 [Google Scholar]
  30. Murfet IC (1985) Pisum sativum In A Halevy, ed, CRC Handbook of Flowering, Vol 4. CRC Press, Boca Raton, FL, pp 97–126 [Google Scholar]
  31. Murfet IC, LaRue TA. (1994) Late flowering in mutant E54LF results from mutation of sn to Sn. Pisum Genet 26: 21–23 [Google Scholar]
  32. Murfet IC, Sherriff LJ. (1996) Confirmation that the sn locus is between Aldo and Gal2 in linkage group VII of pea. Pisum Genet 28: 13–14 [Google Scholar]
  33. Nagel DH, Kay SA. (2012) Complexity in the wiring and regulation of plant circadian networks. Curr Biol 22: R648–R657 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nelson MN, Berger JD, Erskine W. (2010) Flowering time control in annual legumes: prospects in a changing global climate. CAB Rev: Persp Agric Vet Sci Nut Nat Res 5: 1–14 [Google Scholar]
  35. Nusinow DA, Helfer A, Hamilton EE, King JJ, Imaizumi T, Schultz TF, Farré EM, Kay SA. (2011) The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth. Nature 475: 398–402 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Onai K, Ishiura M. (2005) PHYTOCLOCK 1 encoding a novel GARP protein essential for the Arabidopsis circadian clock. Genes Cells 10: 963–972 [DOI] [PubMed] [Google Scholar]
  37. Sakai H, Aoyama T, Bono H, Oka A. (1998) Two-component response regulators from Arabidopsis thaliana contain a putative DNA-binding motif. Plant Cell Physiol 39: 1232–1239 [DOI] [PubMed] [Google Scholar]
  38. Shoemaker DN, Delwiche EJ (1934) Descriptions of Types of Principal American Varieties of Garden Peas. Miscellaneous Publication No. 170. U.S. Department of Agriculture, Washington, DC, pp 1–38 [Google Scholar]
  39. Smykal P, Aubert G, Burstin J, Coyne CJ, Ellis NTH, Flavell AJ, Ford R, Hybl M, Macas J, Neumann P, et al. (2012) Pea (Pisum sativum L.) in the genomic era. Agronomy 2: 74–115 [Google Scholar]
  40. Thines B, Harmon FG. (2010) Ambient temperature response establishes ELF3 as a required component of the core Arabidopsis circadian clock. Proc Natl Acad Sci USA 107: 3257–3262 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Watanabe S, Harada K, Abe J. (2012) Genetic and molecular bases of photoperiod responses of flowering in soybean. Breed Sci 61: 531–543 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Watanabe S, Xia Z, Hideshima R, Tsubokura Y, Sato S, Yamanaka N, Takahashi R, Anai T, Tabata S, Kitamura K, et al. (2011) A map-based cloning strategy employing a residual heterozygous line reveals that the GIGANTEA gene is involved in soybean maturity and flowering. Genetics 188: 395–407 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weeden NF, Kneen BE, Murfet IC. (1988) Mapping of the SN locus to chromosome 2. Pisum Newsletter 20: 49–51 [Google Scholar]
  45. Wellensiek SJ. (1925) Genetic monograph on Pisum. Bibliogr Genet 2: 343–476 [Google Scholar]
  46. Weller JL, Hecht V, Liew LC, Sussmilch FC, Wenden B, Knowles CL, Vander Schoor JK. (2009) Update on the genetic control of flowering in garden pea. J Exp Bot 60: 2493–2499 [DOI] [PubMed] [Google Scholar]
  47. Weller JL, Liew LC, Hecht VF, Rajandran V, Laurie RE, Ridge S, Wenden B, Vander Schoor JK, Jaminon O, Blassiau C, et al. (2012) A conserved molecular basis for photoperiod adaptation in two temperate legumes. Proc Natl Acad Sci USA 109: 21158–21163 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yamashino T, Yamawaki S, Hagui E, Ueoka-Nakanishi H, Nakamichi N, Ito S, Mizuno T. (2013) Clock-controlled and FLOWERING LOCUS T (FT)-dependent photoperiodic pathway in Lotus japonicus I: verification of the flowering-associated function of an FT homolog. Biosci Biotechnol Biochem 77: 747–753 [DOI] [PubMed] [Google Scholar]
  49. Yu JW, Rubio V, Lee NY, Bai S, Lee SY, Kim SS, Liu L, Zhang Y, Irigoyen ML, Sullivan JA, et al. (2008) COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI stability. Mol Cell 32: 617–630 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data
supp_165_2_648__index.html (887B, html)
supp_pp.114.237008_237008SuppFile_revised_2.pdf (830.3KB, pdf)

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES