Sandra E. Davidson

The Pea Gene NA Encodes ent-Kaurenoic Acid Oxidase

The gibberellin (GA)-deficient dwarf namutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 hadent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway froment-kaurenoic acid to GA12 when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7α-hydroxykaurenoic acid and GA12-aldehyde, some additional products of the pea KAO activity were detected, includingent-6α,7α-dihydroxykaurenoic acid and 7β-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 andna-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with thena-1 allele cosegregated with the dwarfna phenotype. PsKAO1 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.PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds ofna plants.

The Pea Gene LH Encodes ent -Kaurene Oxidase

The pea (Pisum sativum) homolog, PsKO1, of the Arabidopsis GA3 gene was isolated. It codes for a cytochrome P450 from the CYP701A subfamily and has ent-kaurene oxidase (KO) activity, catalyzing the three step oxidation of ent-kaurene to ent-kaurenoic acid in the gibberellin (GA) biosynthetic pathway when expressed in yeast (Saccharomyces cerevisiae). PsKO1 is encoded by the LH gene because in three independent mutant alleles, lh-1, lh-2, and lh-3, PsKO1 has altered sequence, and the lh-1 allele, when expressed in yeast, failed to metabolize ent-kaurene. The lh mutants of pea are GA deficient and have reduced internode elongation and root growth. One mutant (lh-2) also causes a large increase in seed abortion. PsKO1 (LH) is expressed in all tissues examined, including stems, roots, and seeds, and appears to be a single-copy gene. Differences in sensitivity to the GA synthesis inhibitor, paclobutrazol, between the mutants appear to result from the distinct nature of the genetic lesions. These differences may also explain the tissue-specific differences between the mutants.

Reassessing the Role of N-Hydroxytryptamine in Auxin Biosynthesis

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 biosynthesis in pea is mediated through the COP1/HY5 pathway.

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.

Plant hormone interactions: how complex are they?

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.

Biosynthesis of the Halogenated Auxin, 4-Chloroindole-3-Acetic Acid

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.

Regulation of the gibberellin pathway by auxin and DELLA proteins

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 GA20 showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA.

Developmental regulation of the gibberellin pathway in pea shoots

To investigate gibberellin (GA) biosynthesis in mature tissue of pea (Pisum sativum L.) in the absence of potentially GA-producing meristematic tissue we grafted wild-type scions to rootstocks of the GA-deficient ls-1 mutant and later decapitated the shoot. After 2 d, decapitated shoots contained as much GA19 (a precursor of the bioactive GA1) as comparable tissue from intact plants, even though applied [14C]GA19 was metabolised rapidly during this time. These results show that the pool size of endogenous GA19 was maintained, probably by de novo GA19 synthesis. We also found that the LS gene, which catalyses an early step in GA biosynthesis, is expressed in mature tissue, as are the shoot-expressed GA 20-oxidase and GA 3-oxidase genes. Nevertheless, mature tissue contained very low levels of GA1 and GA20 compared with immature tissue. Levels of GA19, GA29 and GA8 were less affected by tissue age. Metabolism studies using 14C-labelled GAs indicated that mature tissue rapidly converted GA19 to GA20 and GA20 to GA1; the latter step was promoted by IAA. However, the 2-oxidation steps GA1 to GA8, GA20 to GA29 and GA29 to GA29-catabolite appear to proceed very rapidly in mature tissue (regardless of IAA content), and we suggest this is the reason why GA1 and GA20 do not accumulate. This is supported by the high level of expression of a key GA 2-oxidase gene in mature tissue.

Regulation of the early GA biosynthesis pathway in pea

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.

Cytochromes P450 in gibberellin biosynthesis

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.