Two distinct blue-light responses regulate epicotyl elongation in pea

Blue light induces a long-term suppression of epicotyl elongation in red-light-grown pea (Pisum sativum L.) seedlings. The fluence-response characteristics are bell-shaped, indicating the possibility of two different blue-light responses: a lower fluence response causing suppression and a higher fluence response alleviating the suppression. To determine if two responses are in effect, we have grown pea seedlings under dark conditions hoping to eliminate one or the other response. Under these growth conditions, only the lower fluence portion of the response (suppression of elongation) is apparent. The kinetics of suppression are similar to those observed for the lower fluence response of red-light-grown seedlings. The response to blue light in the dark-grown seedlings is not due to the excitation of phytochrome because a pulse of far-red light large enough to negate phytochrome-induced suppression has no effect on the blue-light-induced suppression. Furthermore, treatment of the dark-grown seedlings with red light immediately prior to treatment with high fluence blue light does not elicit the higher fluence response, indicating that the role of red light in the blue high fluence response is to allow the plant to achieve a specific developmental state in which it is competent to respond to the higher fluences of blue light.     

Evidence for the nuclear location of the gene for chloroplast elongation factor G.

The chloroplast protein synthesis factor responsible for the translocation step of polypeptide synthesis on chloroplast ribosomes (chloroplast elongation factor G [EF-G]) has been detected in whole cell extracts and in isolated chloroplasts from Euglena gracilis. This factor can be detected by its ability to catalyze translocation on 70 S prokaryotic ribosomes such as those from E. coli. Chloroplast EF-G is present in low levels when Euglena is grown in the dark and can be induced more than 20-fold when the organism is grown in the light. The induction of this factor by light is inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. However, inhibitors of chloroplast protein synthesis such as streptomycin or spectinomycin have no effect on the induction of this factor by light. Furthermore, chloroplast EF-G can be partially induced by light in an aplastidic mutant (strain W₃BUL) which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-G resides in the nuclear genome, and that this protein is synthesized on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts.     

Development of cytosol and chloroplast aldolases during germination of spinach seeds

The total activity of aldolase (EC 4.1.2.13) and the activities of cytosol and chloroplast aldolase were determined in seeds, cotyledons, primary leaves and secondary leaves of spinach (Spinacia oleracea L., cv. Monopa) during germination. Total aldolase activity in cotyledons increased from low levels to a low maximum in the dark after one week and to a high maximum in white light after three to four weeks and declined thereafter. The activity in primary and secondary leaves started to rise strongly from the 18th and 26th days, respectively, up to the 42nd day of germination. The levels of aldolase activity paralleled the development of leaf area, chlorophyll content and protein content per leaf except that the leaf area of cotyledons continued to increase steadily up to the 42nd day after the maximum of aldolase activity was reached. Resolution of cytosol- and chloroplast-specific isoenzymes by chromatography on diethylaminoethylcellulose indicated that in the light the cytosol enzyme represented approx. 8% of the total activity in cotyledons, primary and secondary leaves throughout germination, and the chloroplast enzyme represented the remaining 92%. Only in cotyledons of dark-grown seedlings was the cytosol aldolase between 25 and 50% of the total activity. Seeds contained almost exclusively a cytosol aldolase. In cotyledons the increase of total activity in the light was specifically the consequence of an increase in chloroplast aldolase while the cytosol aldolase was little affected by light. The light effect was mediated by phytochrome as demonstrated by classical induction and reversion experiments with red and far-red light and by continuous far-red light treatment.Abbreviation DEAE-cellulose diethylaminoethylcellulose     

Hormone levels and response during de-etiolation in pea

The objective of this study was to increase our understanding of the hormonal regulation of de-etiolation by investigating endogenous hormone levels and response in etiolated pea ( Pisum sativum L.) seedlings after exposure to continuous white light. Recent reports suggest that de-etiolation may result from the down-regulation of an enzyme in the brassinosteroid (BR) biosynthesis pathway in pea. A subsequent review highlighted the need for direct measurements of BR levels to support this hypothesis. We have shown that endogenous castasterone and 6-deoxocastasterone levels are not greatly reduced after exposure to light; indeed, 6-deoxocastasterone levels were actually increased. Similarly, the elongation response to exogenous brassinolide was greater in plants grown in continuous light, or in dark-grown plants that had been transferred into the light, than in plants that were grown in continuous darkness. These results provide further evidence to suggest that BRs do not negatively regulate de-etiolation in pea. However, changes in the levels of several other hormones have also been implicated in light-regulated development. We have simultaneously quantified indole-3-acetic acid (IAA), gibberellin (GA), and abscisic acid levels in whole seedlings, which revealed a complex pattern of changes in the levels of these substances after exposure to light. The first and most dramatic of these changes was a significant reduction in GA(1) levels, which reached a minimum 8 h after exposure to light. Whilst GA(1) levels rapidly decreased, IAA levels remained unchanged in the short term after exposure to light, suggesting that GA(1) levels may be the primary factor regulating the reduction in elongation growth during de-etiolation. In the long term after exposure to light, IAA levels underwent a transitory increase, which peaked at 48 h, and had abated by 96 h. However, abscisic acid levels remained unchanged in the first 1 h after exposure to light before undergoing a steady decline over time. The relative importance of these changes in mediating light-induced changes in plant morphology is discussed.     

Gene expression of chloroplast translation elongation factor Tu during maize chloroplast biogenesis

We have examined the expression of a maize nucleartuf gene(tufA) coding for the chloroplast translation elongation factor EF-Tu. Southern analysis revealed that the maize chloroplast EF-Tu was encoded by at least two distinct genes in the nuclear genome. In order to know the effect of light on the expression of thetufA gene during maize chloroplast biogenesis, we have analyzed the steady-state level of thetufA mRNAs by Northern analysis. The steady-state level of thetufA mRNAs was similar in both continuous light- and dark-grown seedlings. The level of thetufA mRNAs also maintained at relatively same level during light-induced greening of etiolated seedlings and all examined developmental stages. These results indicate that the gene expression of the maize chloroplast EF-Tu is rarely light-regulated at it’s mRNA level during chloroplast biogenesis.     

Auxin Transport Is Required for Hypocotyl Elongation in Light-Grown but Not Dark-Grown Arabidopsis

Many auxin responses are dependent on redistribution and/or polar transport of indoleacetic acid. Polar transport of auxin can be inhibited through the application of phytotropins such as 1-naphthylphthalamic acid (NPA). When Arabidopsis thaliana seedlings were grown in the light on medium containing 1.0 μm NPA, hypocotyl and root elongation and gravitropism were strongly inhibited. When grown in darkness, however, NPA disrupted the gravity response but did not affect elongation. The extent of inhibition of hypocotyl elongation by NPA increased in a fluence-rate-dependent manner to a maximum of about 75% inhibition at 50 μmol m⁻² s⁻¹ of white light. Plants grown under continuous blue or far-red light showed NPA-induced hypocotyl inhibition similar to that of white-light-grown plants. Plants grown under continuous red light showed less NPA-induced inhibition. Analysis of photoreceptor mutants indicates the involvement of phytochrome and cryptochrome in mediating this NPA response. Hypocotyls of some auxin-resistant mutants had decreased sensitivity to NPA in the light, but etiolated seedlings of these mutants were similar in length to the wild type. These results indicate that light has a significant effect on NPA-induced inhibition in Arabidopsis, and suggest that auxin has a more important role in elongation responses in light-grown than in dark-grown seedlings.     

Changes in Nucleic Acids in Phytochrome-dependent Elongation of the Alaska Pea Epicotyl

Red light, which produces the physiologically active form of phytochrome (Pfr), inhibited epicotyl elongation in intact dark-grown Alaska pea seedlings. This red light response was detectable 3 hours after the light treatment and became pronounced after 5 hours. The growth inhibition was completely reversed by far red light applied immediately after the red or by pretreatment of the seedlings with the plant hormone gibberellin A₃.     

Loss or retention of chloroplast DNA in maize seedlings is affected by both light and genotype

We examined the chloroplast DNA (cpDNA) from plastids obtained from wild type maize (Zea mays L.) seedlings grown under different light conditions and from photosynthetic mutants grown under white light. The cpDNA was evaluated by real-time quantitative PCR, quantitative DNA fluorescence, and blot-hybridization following pulsed-field gel electrophoresis. The amount of DNA per plastid in light-grown seedlings declines greatly from stalk to leaf blade during proplastid-to-chloroplast development, and this decline is due to cpDNA degradation. In contrast, during proplastid-to-etioplast development in the dark, the cpDNA levels increase from the stalk to the blade. Our results suggest that DNA replication continues in the etioplasts of the upper regions of the stalk and in the leaves. The cpDNA level decreases rapidly, however, after dark-grown seedlings are transferred to light and the etioplasts develop into photosynthetically active chloroplasts. Light, therefore, triggers the degradation of DNA in maize chloroplasts. The cpDNA is retained in the leaf blade of seedlings grown under red, but not blue light. We suggest that light signaling pathways are involved in mediating cpDNA levels, and that red light promotes replication and inhibits degradation and blue light promotes degradation. For five of nine photosynthetic mutants, cpDNA levels in expanded leaves are higher than in wild type, indicating that nuclear genotype can affect the loss or retention of cpDNA.     

The Effect of Red Irradiation on Plastid Ribosomal RNA Synthesis in Dark-grown Pea Seedlings

Dark-grown pea seedlings (Pisum sativum L.) were irradiated for a short period each day with low intensity red light (662 nm), red light immediately followed by far red light (730 nm), or far red light alone. Other plants were transferred to a white light regime (14 hours light/10 hours dark). There was no change in the amount of RNA in the tissue on a fresh weight basis after the various treatments. However, compared with dark-grown seedlings, those plants irradiated with red light showed an increase in the net RNA content per stem apex. In addition there was a two- to three-fold increase in ribosomal RNA of the etioplasts relative to the total ribosomal RNA. These increases were comparable to those found in plants grown in the white light regime. The changes were much smaller if the dark-grown plants were irradiated either with red light followed by far red light, or with far red light alone. Thus continuous light is not essential for the production of ribosomal RNA in plastids, and the levels of ribosomal RNA found in chloroplasts can also be attained in etioplasts of pea leaves in the dark provided the leaf phytochrome is maintained in its active form.     

Formation of isopentenyl diphosphate via mevalonate does not occur within etioplasts and etiochloroplasts of mustard (Sinapis alba L.) seedlings

Seedlings from the white mustard, Sinapis alba, grown under continuous far-red light exhibit enhanced plastid enzyme activities when compared with dark-grown seedlings (for review, see Mohr 1981). These activities are even more pronounced upon illumination with white light during the etioplast/chloroplast transformation. Etioplasts and etiochloroplasts from the cotyledons of such seedlings show high prenyl-lipid-synthesizing activities when [1-¹⁴C]isopentenyl diphosphate is used as the precursor. They lack, however, any enzymatic activities for the formation of isopentenyl diphosphate via the mevalonate pathway, i.e. hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase and diphosphomevalonate decarboxylase, which are present and easily detectable within the endoplasmic reticulum and cytoplasm. These results corroborate the view that the cytoplasm of the plant cell is the only site of isopentenyl-diphosphate formation via the mevalonate pathway.     

Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.)

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.     

Diurnal periodicity of chalcone-synthase activity during the development of oat primary leaves

Chalcone-synthase (CHS) activity was followed during the development of primary leaves of oat (Avena sativa L.) seedlings grown under different illumination conditions. Continuous darkness and continuous light resulted in similar time courses of enzyme activity. The maximum of CHS activity in etiolated leaves was delayed by 1 d and reached about half the level of that of light-grown leaves. In seedlings grown under defined light-dark cycles a diurnal rhythm of CHS activity and its protein level was observed which followed the rhythm of CHS-mRNA translational activity (Knogge et al. 1986). This rhythm persisted in continuous light after a short-term pre-exposure to the light-dark cycle but not in continuous darkness.Abbreviations CHS chalcone synthase - PAL phenylalanine ammonio lyase Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged (G.W., We 630/9-7; We 630/10-1). Thanks are given to Dr. St. Kellam (Department of Plant Microbiological Sciences, University of Canterbury, New Zealand) for correcting the English.     

The involvement of indole-3-acetic acid in the control of stem elongation in dark- and light-grown pea (Pisum sativum) seedlings

We investigated the role of auxin on stem elongation in pea (Pisum sativum L.) grown for 10d in continuous darkness or under low-irradiance blue, red, far red and white light. The third internode of treated seedlings was peeled and the tissues (epidermis and cortex+central cylinder) were separately analyzed for the concentration of free and conjugated indole-3-acetic acid (IAA). Under red, far red and white light internode elongation was linearly related with the free IAA content of all internode tissues, suggesting that phytochrome-dependent inhibition of stem growth may be mediated by a decrease of free IAA levels in pea seedlings. The correlation between IAA and internode elongation, however, did not hold for blue light-grown seedlings. The hypothesis that the growth response under low-irradiance blue light might be correlated with the lack of phytochrome B signalling and changes in gibberellin metabolism is discussed in view of current knowledge on hormonal control of stem growth.     

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     

Enhanced expression of a distinct plastid DNA region in mustard seedlings by continuous far-red light

Fragments of chloroplast DNA from mustard (Sinapis alba L.) seedlings, generated by the restriction endonuclease Eco RI, were used to assess the frequency of complementary sequences in mustard RNA by DNA/RNA hybridization. A pronounced increase in hybridization to a single DNA fragment was found with RNA from seedlings irradiated with continuous far-red light, compared to RNA from dark-grown seedlings.     

Light-controlled Stem Elongation in Pea Seedlings Grown under Varied Light Conditions

There appears to be an orderly transition from one photosensitive state to another in light-controlled stem elongation in the garden pea, Pisum sativum L. cv. Alaska. Stem elongation in dark-grown plants is known to be phytochrome-controlled. However, seedlings are insensitive to phytochrome after a red light pretreatment. An action spectrum for inhibition in these seedlings has peaks at 440 and 620 nm. Protochlorophyll is suggested as the photoreceptor. If these red pretreated seedlings are further exposed to white light, sensitivity to 440 to 620 nm light is lost. Promotion by blue-green light is the only photoresponse shown by these seedlings. Inhibition of completely white light-grown seedlings required simultaneous exposure to high intensity blue light and 600 nm light.