Effects of the Na and Le loci on gibberellin (GA) content and transport in pea ( Pisum sativum L.) shoots were studied. GA 1 , GA 8 , GA 17 , GA 19 , GA 20 , GA 29 , GA 44 , GA 8 catabolite, and GA 29 catabolite were identified by full-scan gas chromatography-mass spectrometry in extracts of expanding and fully expanded tissues of line C79-338 ( Na Le ). Quantification of GAs by gas chromatography-single-ion monitoring using deuterated internal standards in lines differing at the Na and Le alleles showed that na reduced the contents of GA 19 , GA 20 , and GA 29 on average to 1 and GA 8 to Na lines. In expanding tissues from Na le lines, GA 1 and GA 8 concentrations were reduced to approximately 10 and 2%, respectively, and GA 29 content increased 2- to 3-fold compared with those in Na Le plants. There was a close correlation between stem length and the concentrations of GA 1 or GA 8 in shoot apices in all six genotypes investigated. In na/Na grafts, internode length and GA 1 concentration of nana scions were normalized, the GA 20 content increased slightly, but GA 19 levels were unaffected. Movement of labeled GAs applied to leaves on Na rootstocks indicated that GA 19 was transported poorly to apices of na scions compared with GA 20 and GA 1 . Our evidence suggests that GA 20 is the major transported GA in peas.

http://www.plantphysiol.org/content/plantphysiol/100/3/1354.full.pdf

Gibberellin concentration and transport in genetic lines of pea : effects of grafting.

Gibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases ent-kaurene synthase A (KSA) and ent-kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA-responsive ls-1 mutant of pea suggested that GA levels are reduced in a developmental- and organ-specific manner due to reduced GA biosynthesis. Analysis of cellfree enzyme preparations from WT and ls-1 embryos at contact point reveals that ls-1 reduces the activity of KSA but not KSB. To characterize the ls-1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls-1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls-1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to ent-kaurene in vitro. Based on the sequence of RT-PCR products, three different truncated KSA proteins are predicted to exist in ls-1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls-1 allele is not null and is able to encode at least a partially functional KSA since a more severe is allele has been identified. The ls-1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1997.11030443.x

The LS locus of pea encodes the gibberellin biosynthesis enzyme ent-kaurene synthase A

Molecular biology of gibberellin synthesis

The metabolism and growth-promoting activity of gibberellin A20 (GA20) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A29 and GA29-catabolite were the major metabolites of GA20 in the genotype na le. However, low levels of GA1, GA8 and GA8-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a ‘leaky’ mutation. Gibberellin A20 was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3β-hydroxylated metabolite of GA20, GA8 (2β-hydroxy GA1), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA8 levels. A similar relationship was found on comparing GA20 metabolism in the three genotypes le
d, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3β-hydroxylation of GA20 to GA1). These results indicate that GA20 has negligible intrinsic activity and support the contention that GA1 is the only GA active per se in promoting stem growth in pea.

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The gibberellin (GA)-biosynthesis mutations, lh

 i
 , ls and Ie

 5839
 have been used to investigate the role(s) of the GAs in seed development of the garden pea (Pisum sativum L.). Seeds homozygous for lh

 i
 possess reduced GA levels, are more likely to abort during development, and weigh less at harvest, compared with wild-type seeds due to expression of the lh

 i
 mutation in the embryo and/ or endosperm. Compared with wild-type seeds, the lh

 i
 mutation reduces endogenous GA1 and gibberellic acid (GA3) levels in the embryo/endosperm a few days after anthesis and fertilizing lh

 i
 plants with wild-type pollen dramatically increases GA1 and GA3 levels in the embryo/ endosperm and restores normal seed development. By contrast, the ls and le

 5839
 mutations do not appear to reduce GA levels in the embryo/endosperm of seeds a few days after anthesis, and do not affect embryo or endosperm development. However, both the ls and lh

 i
 mutations substantially reduce endogenous GA levels in embryos at contact point (the first day the liquid endosperm disappears). Levels of GAs in seeds from crosses involving the ls and lh

 i
 mutations suggest that GAs are synthesised in both the embryo/endosperm and testa and that the expression of ls depends on the tissue and developmental stage examined. These results suggest that GAs (possibly GA1 and/or GA3) play an important role early in pea seed development by regulating the development of the embryo and/or endosperm. By contrast, the high GA levels found in wild-type seeds at contact point (and beyond) do not appear to have a physiological role in seed development.

Gibberellins and pea seed development

Gibberellins (GAs) A17, A19, A20, A29, A44, 2βOH-GA44 (tentative) and GA29-catabolite were identified in 21-day-old seeds of Pisum sativum cv. Alaska (tall). These GAs are qualitatively similar to those in the dwarf cultivar Progress No. 9 with the exception of GA19 which does not accumulate in Progress seeds. There was no evidence for the presence of 3-hydroxylated GAs in 21 day-old Alaska seeds. Dark-grown shoots of the cultivar Alaska contein GA1, GA8, GA20, GA29, GA8-catabolite and GA29-catabolite. Dark-grown shoots of the cultivar Progress No.9 contain GA8, GA20, GA29 and GA29-catabolite, and the presence of GA1 was strongly indicated. Quantitation using GAs labelled with stable isotope showed the level of GA1 in dark-grown shoots of the two cultivars to be almost identical, whilst the levels of GA20, GA29 and GA29-catabolite were significantly lower in Alaska than in Progress No. 9. The levels of these GAs in dark-grown shoots were 102- to 103-fold less than the levels in developing seeds. The 2-epimer of GA29 is present in dark-grown-shoot extracts of both cultivars and is not thought to be an artefact.

Gibberellins in immature seeds and dark-grown shoots of Pisum sativum : Gibberellins identified in the tall cultivar Alaska in comparison with those in the dwarf Progress No. 9.

Jake MacMillan

Papers

Gibberellins in immature seeds and dark-grown shoots of Pisum sativum : Gibberellins identified in the tall cultivar Alaska in comparison with those in the dwarf Progress No. 9.