Light-induced changes in the amounts of the 36000-Mr polypeptide of NADPH-protochlorophyllide oxidoreductase and its mRNA in barley plants grown under a diurnal light/dark cycle

Seedlings of barley were grown either in continuous darkness or under a diurnal 12 h light/12 h dark cycle and the effects on NADPH-protochlorophyllide oxidoreductase were followed at two different levels. Firstly, the relative content of the mRNA encoding the NADPH-protochlorophyllide oxidoreductase was measured by dot-blot hybridization. Secondly, changes in the enzyme polypeptide were monitored either by the method of immunoblotting or by immunogold labelling of ultrathin sections of Lowicryl-embedded leaf tissue. Our results demonstrate that drastic diurnal changes in the level of mRNA sequences and the enzyme protein are unlikely to occur in plants which have been grown under natural light/dark conditions. In the dark, protein and mRNA accumulation occurs at an early developmental stage. These results are difficult to reconcile with the suggestion that the massive accumulation of mRNA and enzyme protein in dark-grown seedlings is primarily the consequence of an artificially extended darkperiod. In addition to the plastid-specific NADPH-protochlorophyllide oxidoreductase a closely related polypeptide has been detected outside the plastid in the surrounding cytoplasm (Dehseh et al. 1986b, Planta 169, 172–183). During the diurnal light/dark treatment of seedlings the concentrations of the two protein populations did not show any variation indicative of an exchange between the two protein populations across the plastid envelope.Abbreviation poly(A)⁺RNA polyadenylated RNA     

Two barley catalase genes respond differentially to light

Cloned catalase probes from barley ( Hordeum vulgare L.) and maize ( Zea mays L.) were used to examine catalase gene expression in greened and etiolated leaves of several barley lines. Etiolated leaves had greater levels of an mRNA detected by barley Cat1 , compared with greened leaves, in all lines. In contrast, a Cat2 -like mRNA (homologous to Cat2 of maize) was induced by light and accumulated to high levels in greened leaves, compared to the negligible levels detected in etiolated leaves. This suggests that barley contains light-inducible and light-repressible catalase genes. In the catalase-deficient barley mutant RPr 79/4, no hybridization signal was detected when RNA from greened or etiolated leaves was probed with maize Cat2 , indicating that this mutant is deficient for the light-induced Cat mRNA. In etiolated seedlings of both RPr 79/4 and its motherline, the level of the Cat1 mRNA increased coordinately with a steady increase in catalase activity. Even though the mutant RPr 79/4 was able to grow to maturity in normal air, it sustained chlorosis and significant head sterility, probably due to the lack of a light-inducible catalase. Although the mutant RPr 79/4 is not completely lacking catalase (EC 1.11.1.6), the loss of the CAT-1 isozyme is evidently harmful. This observation underscores the protective role of catalases in plants.     

Regulation by light and metabolites of ferredoxin-dependent glutamate synthase in maize

The regulation of Fd-glutamate synthase (Fd-GOGAT, EC 1.4.1.7) and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) was investigated in maize ( Zea mays L. cv. DEA) (1) during development starting from 7- to 11-day-old seedlings, (2) by treatment of 7-day-old etiolated leaves with intermittent light pulses to activate (red) and inactivate (far-red) phytochromes and (3) in 7-day-old green leaves grown under 16-h light/8-h dark cycles. Fd-GOGAT mRNA accumulated 4-fold, and the enzyme polypeptide (3-fold) and activity (3-fold) also increased in leaf cells, while NADH-GOGAT activity remained constantly low. Leaf-specific induction of Fd-GOGAT mRNA (3-fold) occurred in etiolated leaves by low fluence red light, and far-red light reversibly repressed the mRNA accumulation. Red/far-red reversible induction also occurred for Fd-GOGAT polypeptide (2-fold) and activity (2-fold), implicating the phytochrome-dependent induction of Fd-GOGAT. In contrast, NADH-GOGAT activity remained constant, irrespective of red/far-red light treatments. Fd-GOGAT showed diurnal changes under light/dark cycles with the maximum early in the morning and the minimum in the afternoon at the levels of mRNA, enzyme polypeptide and activity. Gln diurnally changed in parallel with Fd-GOGAT mRNA. The induction of Fd-GOGAT provides evidence that light and metabolites are the major signal for the Gln and Glu formation in maize leaf cells.     

Environmental and genetic effects on circadian clock-regulated gene expression in Arabidopsis.

Expression patterns of the cold-circadian rhythm-RNA binding (CCR) and chlorophyll a/b binding (CAB) protein genes have circadian rhythms with phases that are different from each other and are affected differently by cold (4 degrees C) treatment. Cycling of CCR and CAB RNA levels was observed in Arabidopsis seedlings grown for 5 days at 4 degrees C under a light/ dark photoperiod, although the cycling had reduced amplitude compared with normal growth conditions (20 degrees C). CCR RNA levels were elevated in the cold, whereas CAB RNA levels were reduced in the cold relative to levels in control seedlings. Cold pulses (4 degrees C for 12 or 20 hr) under continuous light affected the rhythms of CCR and CAB RNA levels in similar ways. The 12-hr cold pulse caused a 4-hr phase delay in both rhythms, whereas the 20-hr cold pulse resulted in a 12-hr phase delay in both rhythms. The timing of CAB expression 1 (toc1) mutation shortened the period of the CCR rhythm, matching previous results for the regulation of the CAB-luciferase (CAB-luc) transgene in this mutant. The results suggest that CCR and CAB share clock machinery but are regulated by downstream components that are affected differently by the cold. Also, the circadian clock regulating these genes in Arabidopsis has a cold-sensitive phase under continuous light conditions.     

Regulation of Maize Leaf Nitrate Reductase Activity Involves Both Gene Expression and Protein Phosphorylation

Nitrate reductase (NR; EC 1.6.6.1) activity increased at the beginning of the photoperiod in mature green maize (Zea mays L.) leaves as a result of increased enzyme protein level and protein dephosphorylation. In vitro experiments suggested that phosphorylation of maize leaf NR affected sensitivity to Mg2+ inhibition, as shown previously in spinach. When excised leaves were fed 32P-labeled inorganic phosphate, NR was phosphorylated on seryl residues in both the light and dark. Tryptic peptide mapping of NR labeled in vivo indicated three major 32P-phosphopeptide fragments, and labeling of all three was reduced when leaves were illuminated. Maize leaf NR mRNA levels that were low at the end of the dark period peaked within 2 h in the light and decreased thereafter, and NR activity generally remained high. It appears that light signals, rather than an endogenous rhythm, account primarily for diurnal variations in NR mRNA levels. Overall, regulation of NR activity in mature maize leaves in response to light signals appears to involve control of gene expression, enzyme protein synthesis, and reversible protein phosphorylation.     

Effect of tagetitoxin on the levels of ribulose 1,5-bisphosphate carboxylase, ribosomes, and RNA in plastids of wheat leaves

Growth of wheat seedlings in the presence of the phytotoxin tagetitoxin produces pigment-deficient leaves of normal size and morphology whose cells contain only rudimentary plastids. We could not detect the accumulation of either the plastid-encoded large subunit or the nuclear-encoded small subunit of the chloroplast stromal enzyme ribulose 1,5-bisphosphate carboxylase (RuBPCase) in western blots of protein extracted from leaves of such seedlings. Sucrose gradient centrifugation profiles showed that plastid ribosomes were essentially absent in toxin-treated leaf tissue while cytoplasmic ribosomes were relatively unaffected. Northern blot analysis of RNA in toxin-treated leaves showed a deficiency of plastid ribosomal RNA (16S and 23S) as well as reduced levels of plastid mRNAs for the large subunit of RuBPCase and for the 32 kilodalton thylakoid Q_B polypeptide. Northern analysis also showed that the nuclear-encoded rbcS mRNA for the small subunit of RuBPCase is present in only trace amounts in toxin-treated leaves.     

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.     

Effect of light and glucose on the induction of nitrate reductase and on the distribution of nitrate in etiolated barley leaves

Barley seedlings grown in the dark with 10 mm KNO(3) have low levels of nitrate reductase activity even though large amounts of No(3) (-) accumulate in the leaves. When the leaves are excised and transferred to the light, there is an increase in nitrate reductase activity both in the presence and absence of exogenous NO(3) (-). When the leaves are transferred to a glucose solution (0.05 m) but kept in the dark, induction of nitrate reductase activity occurs only when fresh NO(3) (-) is added to the system.In dark-grown leaves, there are small traces of NO(3) (-) in a "metabolic pool." Addition of glucose does not alter this distribution. Light, on the other hand, results in an appreciable accumulation of NO(3) (-) in the metabolic pool. There is a linear correlation between nitrate reductase activity and the size of the metabolic NO(3) (-) pool. Our results thus suggest that NO(3) (-) accumulates in a storage pool when seedlings are grown in continuous darkness. The transfer of this NO(3) (-) to an active metabolic pool is mediated by light but not by glucose. We believe that this transfer of NO(3) (-) leads to the induction of nitrate reductase. When NO(3) (-) is included in the medium, both light and glucose increase its incorporation into the metabolic pool. The results suggest two mechanisms for regulating the metabolic NO(3) (-) pool: (a) a transfer from the storage pool which requires light; and (b) a transfer from the external medium which requires either glucose or light.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Maize w3 disrupts homogentisate solanesyl transferase (ZmHst) and reveals a plastoquinone‐9 independent path for phytoene desaturation and tocopherol accumulation in kernels

Maize white seedling 3 (w3) has been used to study carotenoid deficiency for almost 100 years, although the molecular basis of the mutation has remained unknown. Here we show that the w3 phenotype is caused by disruption of the maize gene for homogentisate solanesyl transferase (HST), which catalyzes the first and committed step in plastoquinone‐9 (PQ‐9) biosynthesis in the plastid. The resulting PQ‐9 deficiency prohibits photosynthetic electron transfer and eliminates PQ‐9 as an oxidant in the enzymatic desaturation of phytoene during carotenoid synthesis. As a result, light‐grown w3 seedlings are albino, deficient in colored carotenoids and accumulate high levels of phytoene. However, despite the absence of PQ‐9 for phytoene desaturation, dark‐grown w3 seedlings can produce abscisic acid (ABA) and homozygous w3 kernels accumulate sufficient carotenoids to generate ABA needed for seed maturation. The presence of ABA and low levels of carotenoids in w3 nulls indicates that phytoene desaturase is able to use an alternate oxidant cofactor, albeit less efficiently than PQ‐9. The observation that tocopherols and tocotrienols are modestly affected in w3 embryos and unaffected in w3 endosperm indicates that, unlike leaves, grain tissues deficient in PQ‐9 are not subject to severe photo‐oxidative stress. In addition to identifying the molecular basis for the maize w3 mutant, we: (1) show that low levels of phytoene desaturation can occur in w3 seedlings in the absence of PQ‐9; and (2) demonstrate that PQ‐9 and carotenoids are not required for vitamin E accumulation.     

Effects of Cycloheximide and Light on Leaf Senescence in Maize and Hydrangea

The senescence of maize and hydrangea leaves after detachmentand darkening was studied in terms of the loss of chlorophylland protein. Chlorophyll contents of the detached leaves decreasedin the dark in both plants. Cycloheximide at 0.1 mM effectivelyinhibited the loss of chlorophyll in maize, but did not do soin hydrangea. Continuous irradiation with white light of 4.6Wm^–2 prevented the loss of chlorophyll in hydrangea leaves,while it caused bleaching of maize leaves. Reducing agents suchas ascorbic acid and glutathione did not prevent the bleachingby light. In maize leaves, the amount of protein decreased inthe dark more slowly than that of chlorophyll, and cycloheximideslightly prevented the protein decrease. Continuous light irradiationof 4.6 Wm^–2 delayed the loss of protein more effectivelythan cycloheximide did. (Received January 31, 1981; Accepted May 21, 1981)     

Comparative proteomic study reveals the involvement of diurnal cycle in cell division, enlargement, and starch accumulation in developing endosperm of Oryza sativa

The development and starch accumulation of cereal endosperms rely on the sugar supply of leaves, which is subject to diurnal cycles, and the endosperm itself also experiences a light/dark switch. However, revealing how the cereal endosperm responds to diurnal input remains a major challenge. We used comparative proteomic approaches to probe diurnally affected processes in rice endosperm (Oryza sativa) 10 days after flowering under 12-h light/12-h dark. Starch granules in rice endosperm showed a growth ring structure under a normal light/dark cycle but not under constant light. Sucrose showed a high level in light and low level in dark. Two-dimensional (2-D) differential in-gel electrophoresis-based proteomic analysis revealed 101 protein spots diurnally changed and 91 identities, which were involved in diverse processes with preferred distribution in stress response, protein synthesis/destination and metabolism. Proteins involved in cell division showed high expression in light and those in cell enlargement and cell wall synthesis high in dark, while starch synthesis proteins were light-downregulated and dark-upregulated. Redox homeostasis-associated proteins showed in-phase peaks under light and dark. These data demonstrate diurnal input-regulated diverse cellular and metabolic processes in rice endosperm, and coordination among these processes is essential for development and starch accumulation with diurnal input.     

A comparative analysis of fructose 2,6-bisphosphate levels and photosynthate partitioning in the leaves of some agronomically important crop species

Starch, sucrose, and fructose 2,6-bisphosphate (F2, 6BP) levels were measured in pea (Pisum sativum L.), maize (Zea mays L.), onion (Allium cepa L.) and soybean (Glycine max L.) leaves throughout a light/dark cycle. Leaf starch accumulated in pea, maize, and soybean but not in onion. Sucrose was a major leaf storage reserve in pea, maize, and onion but was only found at low levels in soybean. In all species examined, the most dramatic changes in F2,6BP concentration coincided with light/dark transitions. During the light period F2,6BP levels were about 0.1 nanomole/milligram chlorophyll in soybean source leaves and there was a small increase in effector concentration in the dark. Levels of F2,6BP were also low in pea and maize leaves during the light period but then increased 10- or 20-fold in the dark. Dark onion leaf F2,6BP levels were about 1.1 to 1.3 nanomole/milligram chlorophyll and these values decreased by 20 to 30% in the light. Thus, three different patterns were identified that describe diurnal F2,6BP levels in source leaves. These results support the suggestion that F2,6BP is involved in the regulation of sucrose biosynthesis. However, it was not possible to demonstrate that high levels of F2,6BP are essential for starch synthesis in the chloroplast.