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.     

The cytokinin 2-isopentenyladenine causes partial reversion to skotomorphogenesis and induces formation of prolamellar bodies and protochlorophyllide657 in the lip1 mutant of pea

When grown in darkness the photomorphogenic lip 1 mutant of pea ( Pisum sativum L.) has a slender stem, expanded leaves, prolamellar body (PLB) lacking plastids with the size of chloroplasts and a low level of phytochrome A. The lack of PLBs in a dark-grown material ( lip 1) created a possibility to further study the regulation of their formation in relation to plant development. Inclusion of a cytokinin, 2-isopentenyladenine (2iP), in a medium supporting growth of the pea seedlings in darkness was found to reduce epicotyl length in the wild type. In lip 1 the formation of a slender stem was inhibited and a short epicotyl developed. Furthermore, leaf expansion was inhibited, the plastid size reduced and the formation of PLBs induced. The PLB formation in lip 1 was not accompanied by an increase in the amount of protochlorophyllide (Pchlide) or Pchilde oxidoreductase (POR). In the presence of 2iP the level of phytochrome A protein was increased in lip 1 and the POR mRNA levels decreased in both lip 1 and wild-type plants. The chloroplast characteristic trans -3-hexadecenoate acyl group of phosphatidylglycerol, present in the plastids of dark-grown lip 1, was not influenced by 2iP. Thus, not all photomorphogenic processes reacted similarly in the lip 1 mutant, but leaf expansion and plastid differentiation, including PLB formation, seemed to be regulated by the same signal transduction chain. Exogenously applied brassinolide could rescue neither dark- nor light-grown defects of the lip 1 mutant. Thus, cytokinins but not brassinolides seem to be involved in the regulation of certain characteristic traits of skotomorphogenesis in pea, including plastid development and PLB formation.     

The Casparian strip in pea epicotyls: Effects of light on its development

The Casparian strip, which is specific to roots, was studied in the epicotyls of dark-grown seedlings of pea (Pisum sativum L.) where it was found to have the same morphology and properties as the strip in roots. In dark-grown seedlings, the distance between the upper-most position of the Casparian strip and the bending point of the hook (about 37 mm) did not change during growth of the seedlings. In the uppermost 0.5-mm region of the region in which the Casparian strip could be detected by fluorescence microscopy, the plasma membrane was not firmly attached to the cell wall. The development of the Casparian strip continued for about 42 h after dark-grown seedlings were transferred to the light, indicating that (i) the cells that have been determined to form the Casparian strip in darkness form the strip in the light, and that (ii) it takes about 42 h for the cells to complete formation of the strip. Cells in the hook of dark-grown seedlings did not form a Casparian strip when such seedlings were transferred to the light. The Casparian strip was formed in rapidly elongating internodes of light-grown seedlings when the seedlings were transferred to darkness. Light did not control the formation of the Casparian strip in roots.Abbreviation PBS phosphate-buffered saline     

Altered Phytochrome Regulation of Greening in an aurea Mutant of Tomato

A brief pulse of red light accelerates chlorophyll accumulation upon subsequent transfer of dark-grown tomato (Lycopersicon esculentum) seedlings to continuous white light. Such potentiation of greening was compared in wild type and an aurea mutant W616. This mutant has been the subject of recent studies of phytochrome phototransduction; its dark-grown seedlings are deficient in phytochrome, and light-grown plants have yellow-green leaves. The rate of greening was slower in the mutant, but the extent (relative to the dark control) of potentiation by the red pulse was similar to that in the wild type. In the wild type, the fluence-response curve for potentiation of greening indicates substantial components in the VLF (very low fluence) and LF (low fluence) ranges. Far-red light could only partially reverse the effect of red. In the aurea mutant, only red light in the LF range was effective, and the effect of red was completely reversed by far-red light. When grown in total darkness, aurea seedlings are also deficient in photoconvertible PChl(ide). Upon transfer to white light, the aurea mutant was defective in both the abundance and light regulation of the light-harvesting chlorophyll a/b binding polypeptide(s) [LHC(II)]. The results are consistent with the VLF response in greening being mediated by phytochrome. Furthermore, the data support the hypothesis that light modulates LHC(II) levels through its control of the synthesis of both chlorophyll and its LHC(II) apoproteins. Some, but not all, aspects of the aurea phenotype can be accounted for by the deficiency in photoreception by phytochrome.     

Temporal and light regulation of the expression of three phytochromes in germinating seeds and young seedlings of Avena sativa L.

An oat (Avena sativa L.) plant contains at least three phytochromes, which have monomeric masses of 125, 124, and 123 kilodaltons (kDa) (Wang et al., 1991, Planta 184, 96–104). The 124-kDa phytochrome is most abundant in dark-grown seedlings, while the other two phytochromes predominate in light-grown seedlings. Using three monoclonal antibodies, each specific to one of the three phytochromes, we have monitored by immunoblot assay the expression of these three phytochromes in the 5 d following onset of imbibition of seeds. On a per-organism basis, each of these three phytochromes increased in abundance for the first 3 d in the light, or for the first 4 d in darkness, after which they each began to decrease in quantity. When 3-d-old dark-grown seedlings were transferred to the light, the abundance of each of these three phytochromes decreased both in absolute amount and relative to the phytochrome levels in control seedlings kept in darkness. In contrast, when 3-d-old light-grown seedlings were transferred to darkness, the abundance of the 124-kDa and 125-kDa phytochromes increased while that of 123-kDa phytochrome remained unchanged. In each case, the level of phytochrome was greater than that of control seedlings maintained in the light. Thus, in addition to temporal regulation, all three phytochromes exhibit photoregulated expression at the protein level, although the magnitude of this photoregulation varies substantially. We thank Drs. Elizabeth Williams and Tammy Sage (Botany Department, University of Georgia, USA) for generously permitting us to use their image-analysis system. This research was supported by USDA NRICGP grant 91-37100-6490.     

A Role for Cytokinins in De-Etiolation in Arabidopsis (det Mutants Have an Altered Response to Cytokinins)

When grown in the absence of light, Arabidopsis thaliana deetiolated (det) mutants develop many of the characteristics of light-grown plants, including the development of leaves and chloroplasts, the inhibition of hypocotyl growth elongation, and elevated expression levels of light-regulated genes. We show here that dark-grown wild-type seedlings exhibit similar phenotypic traits if any one of a variety of cytokinins are present in the growth medium. We further show that the striking phenotype of det mutants is unlikely to be caused by different levels of cytokinins in these mutants. The three major Arabidopsis cytokinins, zeatin, zeatin riboside, and isopentenyladenosine, accumulate to similar levels in wild-type seedlings grown in either the light or the dark. There is no consistently different pattern for the levels of these cytokinins in wild-type versus det1 or det2 mutants. However, det1 and det2 have an altered response to cytokinin in a detached leaf senescence assay and in tissue culture experiments. A model is proposed in which light and cytokinins act independently or sequentially through common signal transduction intermediates such as DET1 and DET2 to control the downstream light-regulated responses.