In recent years the biosynthesis of auxin has been clarified with the aid of mutations in auxin biosynthesis genes. However, we know little about the effects of these mutations on the seed-filling stage of seed development. Here we investigate a key auxin biosynthesis mutation of the garden pea, which results in auxin deficiency in developing seeds. We exploit the large seed size of this model species, which facilitates the measurement of compounds in individual seeds. The mutation results in small seeds with reduced starch content and a wrinkled phenotype at the dry stage. The phenotypic effects of the mutation were fully reversed by introduction of the wild-type gene as a transgene, and partially reversed by auxin application. The results indicate that auxin is required for normal seed size and starch accumulation in pea, an important grain legume crop.

Traditionally, schemes depicting auxin biosynthesis in plants have been notoriously complex. They have involved up to four possible pathways by which the amino acid tryptophan might be converted to the main active auxin, indole-3-acetic acid (IAA), while another pathway was suggested to bypass tryptophan altogether. It was also postulated that different plants use different pathways, further adding to the complexity. In 2011, however, it was suggested that one of the four tryptophan-dependent pathways, via indole-3-pyruvic acid (IPyA), is the main pathway in Arabidopsis thaliana, although concurrent operation of one or more other pathways has not been excluded. We recently showed that, for seeds of Pisum sativum (pea), it is possible to go one step further. Our new evidence indicates that the IPyA pathway is the only tryptophan-dependent IAA synthesis pathway operating in pea seeds. We also demonstrated that the main auxin in developing pea seeds, 4-chloroindole-3-acetic acid (4-Cl-IAA), which accumulates to levels far exceeding those of IAA, is synthesized via a chlorinated version of the IPyA pathway.

Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea.

Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA1, play critical roles in the control of elongation,1-3 along with environmental and endogenous factors, including other hormones such as the brassinosteroids.4-5 The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism,6 since auxin up-regulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and down regulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA1.7 In our recent paper,1 we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action,8-9 since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA1 synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level.10 However, our recent results,1 in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined.

Models describing plant hormone interactions are often complex and web-like. Here we assess several suggested interactions within one experimental system, elongating pea internodes. Results from this system indicate that at least some suggested interactions between auxin, gibberellins (GAs), brassinosteroids (BRs), abscisic acid (ABA) and ethylene do not occur in this system or occur in the reverse direction to that suggested. Furthermore, some of the interactions are relatively weak and may be of little physiological relevance. This is especially true if plant hormones are assumed to show a log-linear response curve as many empirical results suggest. Although there is strong evidence to support some interactions between hormones (e.g. auxin stimulating ethylene and bioactive GA levels), at least some of the web-like complexities do not appear to be justified or are overstated. Simpler and more targeted models may be developed by dissecting out key interactions with major physiological effects.

It is remarkable that although auxin was the first growth-promoting plant hormone to be discovered, and although more researchers work on this hormone than on any other, we cannot be definitive about the pathways of auxin synthesis in plants. In 2001, there appeared to be a dramatic development in this field, with the announcement of a new gene,i and a new intermediate, purportedly from the tryptamine pathway for converting tryptophan to the main endogenous auxin, indole-3-acetic acid (IAA). Recently, however, we presented evidence challenging the original and subsequent identifications of the intermediate concerned.2

The synthesis and deactivation of bioactive gibberellins (GA) are regulated by auxin and by GA signalling. The effect of GA on its own pathway is mediated by DELLA proteins. Like auxin, the DELLAs promote GA synthesis and inhibit its deactivation. Here, we investigate the relationships between auxin and DELLA regulation of the GA pathway in stems, using a pea double mutant that is deficient in DELLA proteins. In general terms our results demonstrate that auxin and DELLAs independently regulate the GA pathway, contrary to some previous suggestions. The extent to which DELLA regulation was able to counteract the effects of auxin regulation varied from gene to gene. For Mendel's LE gene (PsGA3ox1) no counteraction was observed. However, for another synthesis gene, a GA 20-oxidase, the effect of auxin was weak and in WT plants appeared to be completely over-ridden by DELLA regulation. For a key GA deactivation (2-oxidase) gene, PsGA2ox1, the up-regulation induced by auxin deficiency was reduced to some extent by DELLA regulation. A second pea 2-oxidase gene, PsGA2ox2, was up-regulated by auxin, in a DELLA-independent manner. In Arabidopsis also, one 2-oxidase gene was down-regulated by auxin while another was up-regulated. Monitoring the metabolism pattern of GA(20) showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA.

The tryptamine pathway is one of five proposed pathways for the biosynthesis of indole-3-acetic acid (IAA), the primary auxin in plants. The enzymes AtYUC1 (Arabidopsis thaliana), FZY (Solanum lycopersicum), and ZmYUC (Zea mays) are reported to catalyze the conversion of tryptamine to N-hydroxytryptamine, putatively a rate-limiting step of the tryptamine pathway for IAA biosynthesis. This conclusion was based on in vitro assays followed by mass spectrometry or HPLC analyses. However, there are major inconsistencies between the mass spectra reported for the reaction products. Here, we present mass spectral data for authentic N-hydroxytryptamine, 5-hydroxytryptamine (serotonin), and tryptamine to demonstrate that at least some of the published mass spectral data for the YUC in vitro product are not consistent with N-hydroxytryptamine. We also show that tryptamine is not metabolized to IAA in pea (Pisum sativum) seeds, even though a PsYUC-like gene is strongly expressed in these organs. Combining these findings, we propose that at present there is insufficient evidence to consider N-hydroxytryptamine an intermediate for IAA biosynthesis.

Light regulation of gibberellin (GA) biosynthesis occurs in several species, but the signaling pathway through which this occurs has not been clearly established. We have isolated a new pea (Pisum sativum) mutant, long1, with a light-dependent elongated phenotype that is particularly pronounced in the epicotyl and first internode. The long1 mutation impairs signaling from phytochrome and cryptochrome photoreceptors and interacts genetically with a mutation in LIP1, the pea ortholog of Arabidopsis thaliana COP1. Mutant long1 seedlings show a dramatic impairment in the light regulation of active GA levels and the expression of several GA biosynthetic genes, most notably the GA catabolism gene GA2ox2. The long1 mutant carries a nonsense mutation in a gene orthologous to the ASTRAY gene from Lotus japonicus, a divergent ortholog of the Arabidopsis bZIP transcription factor gene HY5. Our results show that LONG1 has a central role in mediating the effects of light on GA biosynthesis in pea and demonstrate the importance of this regulation for appropriate photomorphogenic development. By contrast, LONG1 has no effect on GA responsiveness, implying that interactions between LONG1 and GA signaling are not a significant component of the molecular framework for light-GA interactions in pea.

The gibberellins (GAs) are an important class of plant growth regulators that are active in many aspects of plant growth and development. GAs are synthesized by a complex pathway involving three enzyme classes spanning different subcellular compartments. One of these enzyme classes is the cytochrome P450s which catalyze a number of oxidation steps in the middle part of the pathway. Mutants in these cytochrome P450-mediated steps in a number of species have been crucial in isolating the genes encoding these enzymes and have also played an important role in understanding GA physiology. GAs are also synthesized by fungi, in a biosynthesis pathway largely catalyzed by cytochrome P450s. The fungal pathway appears to have evolved independently to that of higher plants.

The early steps in the gibberellin (GA) biosynthetic pathway are controlled by single copy genes or small gene families. In pea (Pisum sativum L.) there are two ent-kaurenoic acid oxidases, one expressed only in the seeds, while ent-copalyl synthesis and ent-kaurene oxidation appear to be controlled by single copy genes. None of these genes appear to show feedback regulation and the only major developmental regulation appears to be during seed development. During shoot maturation, transcript levels do not change markedly with the result that all the three genes examined are expressed in mature tissue, supporting recent findings that these tissues can synthesise GAs. It therefore appears that the regulation of bioactive GA levels are determined by the enzymes encoded by the 2-oxoglutarate-dependent dioxygenase gene families controlling the later steps in GA biosynthesis. However the early steps are nonetheless important as a clear log/linear relationship exists between elongation and the level of GA1 in a range of single and double mutants in genes controlling these steps.

The molecular identity of the final two classical gibberellin (GA) biosynthesis mutants of Pisum sativum, na and lh, have been determined. NA and LH encode adjacent cytochrome P450 monooxygenases in the GA biosynthesis pathway and together oxidise the six steps from ent-kaurene to GA l2. The GA deficient dwarf na mutant in pea has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKA01 and PsKA02, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKA01 and PsKA02 had ent-kaurenoic acid oxidase (KAO) activity; catalysing the 3 steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA l2 when expressed in yeast. The NA gene encodes PsKA01, since in two independent mutant alleles, na- 1 and na-2, PsKA01 had altered sequences and the 5 base deletion in PsKA01 associated with the na-1 allele co-segregated with the dwarf na phenotype. PsKA01 was expressed in the stem, apical bud, leaf, pod and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKA02 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants. The pea gene associated with the GA deficient lh dwarf mutant was isolated and found to encode a cytochrome P450 monooxygenase, of the subfamily CYP701A. This gene had high similarity to genes encoding ent-kaurene oxidases in Arabidopsis and pumpkin, AtK01 and CmK01 respectively. The PsK01 gene sequence was altered in all three of the dwarf pea mutant alleles, lh-1, lh-2 and lh-3.This confirmed previous work that showed that the lh-2 mutant blocked the 3 step metabolism of ent-kaurene to ent-kaurenoic acid. The phenotypes of the lh-1 and lh- 3 alleles differ from 1h-2 in a tissue specific manner. In the seed, the lh-1 and lh-3 alleles had little effect while the lh-2 allele phenotype had smaller seeds and decreased seed survival. However, in the vegetative parts of the plants all three alleles are of similar dwarf stature. However, the lh-2 mutant is up to 30 times more sensitive to the KO specific inhibitor paclobutrazol than is the lh-1 or lh-3 alleles. The different phenotypes appear to reflect the different natures of the aberrant proteins produced by the different alleles. With the isolation of genes encoding the two major cytochrome P450 monooxygenases from the GA biosynthesis pathway in pea, control of the early sections of the pathway was investigated. Northern blot analysis was used to measure the mRNA transcript levels of genes encoding enzymes at three steps including the first committed step of GA biosynthesis, cyclization of GGDP to copalyl diphosphate (CPS), and the two monooxygenases which oxidise entkaurene, (KO), and ent-kaurenoic acid, (KA0s). ent-kaurenoic acid oxidase activity is encoded by a 2 membered gene family with tissue specific expression patterns. PsKA02 is only expressed in developing seed and therefore potentially allows differential regulation of GA biosynthesis in seed. In contrast, the single copy ent-kaurene oxidase, PsK01, is expressed at high levels in all tissue and developmental stages tested. Although copalyl diphosphate synthase, PsCPS, was expressed at low levels in all tissues tested, it had higher expression in seed and stem tissue. There is evidence of developmental regulation of the genes in seeds at the transcript level. All four genes examined, PsCPS, PsK01, PsKA01 and PsKA02, have a transcript level peak around the time of contact point coinciding with a peak of GA20accumulation in maturing seeds. PsK01 expression levels were also high in young seeds (11 days after anthesis) coinciding with the tail end of the peak of GA 1 content in young seeds. However, genes for these steps appear unaffected at the transcript level by maturation of the pea shoot. PsCPS, PsK01 and PsKA01 were expressed in mature stem and leaf tissue, even though the mature tissue contained less bioactive GA1and had ceased expanding. Unlike some genes later in the GA biosynthesis pathway, those early in the pathway, PsCPS, PsK01, PsKA01 and PsKA02, are not under feed-back regulation by GA1activity. Expression levels were unaltered in samples from plants with reduced stature and decreased bioactive GA levels as in GA deficient mutants or plants treated with the GA biosynthesis inhibitor, paclobutrazol.