Brassinosteroid deficiency due to truncated steroid 5alpha-reductase causes dwarfism in the lk mutant of pea.

The endogenous brassinosteroids in the dwarf mutant lk of pea (Pisum sativum) were quantified by gas chromatography-selected ion monitoring. The levels of castasterone, 6-deoxocastasterone, and 6-deoxotyphasterol in lk shoots were reduced 4-, 70-, and 6-fold, respectively, compared with those of the wild type. The fact that the application of brassinolide restored the growth of the mutant indicated that the dwarf mutant lk is brassinosteroid deficient. Gas chromatography-selected ion monitoring analysis of the endogenous sterols in lk shoots revealed that the levels of campestanol and sitostanol were reduced 160- and 10-fold, respectively, compared with those of wild-type plants. These data, along with metabolic studies, showed that the lk mutant has a defect in the conversion of campest-4-en-3-one to 5alpha-campestan-3-one, which is a key hydrogenation step in the synthesis of campestanol from campesterol. This defect is the same as that found in the Arabidopsis det2 mutant and the Ipomoea nil kbt mutant. The pea gene homologous to the DET2 gene, PsDET2, was cloned, and it was found that the lk mutation would result in a putative truncated PsDET2 protein. Thus it was concluded that the short stature of the lk mutant is due to a defect in the steroidal 5alpha-reductase gene. This defect was also observed in the callus induced from the lk mutant. Biosynthetic pathways involved in the conversion of campesterol to campestanol are discussed in detail.

A dominant mutation in the pea PHYA gene confers enhanced responses to light and impairs the light-dependent degradation of phytochrome A.

Phytochrome A (phyA) is an important photoreceptor controlling many processes throughout the plant life cycle. It is unique within the phytochrome family for its ability to mediate photomorphogenic responses to continuous far-red light and for the strong photocontrol of its transcript level and protein stability. Here we describe a dominant mutant of garden pea (Pisum sativum) that displays dramatically enhanced responses to light, early photoperiod-independent flowering, and impaired photodestruction of phyA. The mutant carries a single base substitution in the PHYA gene that is genetically inseparable from the mutant phenotype. This substitution is predicted to direct the replacement of a conserved Ala in an N-terminal region of PHYA that is highly divergent between phyA and other phytochromes. This result identifies a region of the phyA photoreceptor molecule that may play an important role in its fate after photoconversion.

Uncoupling brassinosteroid levels and de-etiolation in pea.

The suggestion that brassinosteroids (BRs) have a negative regulatory role in de-etiolation is based largely on correlative evidence, which includes the de-etiolated phenotypes of, and increased expression of light-regulated genes in, dark-grown mutants defective in BR biosynthesis or response. However, we have obtained the first direct evidence which shows that endogenous BR levels in light-grown pea seedlings are increased, not decreased, in comparison with those grown in the dark. Similarly, we found no evidence of a decrease in castasterone (CS) levels in seedlings that were transferred from the dark to the light for 24 h. Furthermore, CS levels in the constitutively de-etiolated lip1 mutant are similar to those in wild-type plants, and are not reduced as is the case in the BR-deficient lkb plants. Unlike lip1, the pea BR-deficient mutants lk and lkb are not de-etiolated at the morphological or molecular level, as they exhibit neither a de-etiolated phenotype or altered expression of light-regulated genes when grown in the dark. Similarly, dark-grown WT plants treated with the BR biosynthesis inhibitor, Brz, do not exhibit a de-etiolated phenotype. In addition, analysis of the lip1lkb double mutant revealed an additive phenotype indicative of the two genes acting in independent pathways. Together these results strongly suggest that BR levels do not play a negative-regulatory role in de-etiolation in pea.

Control of gibberellin levels and gene expression during de-etiolation in pea.

Gibberellin A(1) (GA(1)) levels drop significantly in wild-type pea (Pisum sativum) plants within 4 h of exposure to red, blue, or far-red light. This response is controlled by phytochrome A (phyA) (and not phyB) and a blue light receptor. GA(8) levels are increased in response to 4 h of red light, whereas the levels of GA(19), GA(20), and GA(29) do not vary substantially. Red light appears to control GA(1) levels by down-regulating the expression of Mendel's LE (PsGA3ox1) gene that controls the conversion of GA(20) to GA(1), and by up-regulating PsGA2ox2, which codes for a GA 2-oxidase that converts GA(1) to GA(8). This occurs within 0.5 to 1 h of exposure to red light. Similar responses occur in blue light. The major GA 20-oxidase gene expressed in shoots, PsGA20ox1, does not show substantial light regulation, but does show up-regulation after 4 h of red light, probably as a result of feedback regulation. Expression of PsGA3ox1 shows a similar feedback response, whereas PsGA2ox2 shows a feed-forward response. These results add to our understanding of how light reduces shoot elongation during de-etiolation.