The expression of early light-inducible proteins (ELIPs) under high-light stress as defense marker in Northern- and Southern European cultivars of barley (Hordeum vulgare)

Clones coding for the two small early light-inducible proteins (ELIP)-gene families of 13.5 and 17 kDa have been used as markers to study the effect of high-light intensities on gene expression in cultivars of barley which were bred for growth in Southern and Northern Europe. The mRNA levels of the light-harvesting chlorophyll a / b protein (LHC-II) and the small subunit of ribulose-1,5-bisphosphate carboxylase (SSU) were determined in addition. These data were correlated to the decay of PSII activity during high-light stress and its recovery. In all cultivars, the induction of ELIP mRNAs by high light was accompanied by a correspondent reduction of the LHC-II mRNA level. Furthermore, the LHC-II mRNA levels observed under low-light conditions used for the growth of the plants, were in all cases found to be inversely related to the amount of the ELIP which could be induced by a high-light treatment. In contrast, the amount of the SSU mRNA was reduced only at the highest investigated light intensity of 2 000 μmol m⁻² s⁻¹. During recovery from light-stress, the activity of PSII was quickly restored in all European cultivars. Of these cultivars, however, the cv. Otis which expressed the highest ELIP levels recovered considerably faster than the cultivar p4266N which accumulated the lowest amounts of ELIP under high light. Thus, it appears likely that ELIPs contribute to the restoration of PSII activity during and after photoinhibition.     

Coordinate Expression of Rubisco Activase and Rubisco during Barley Leaf Cell Development

We have utilized the cellular differentiation gradient and photomorphogenic responses of the first leaf of 7-day-old barley (Hordeum vulgare L.) to examine the accumulation of mRNA and protein encoded by the ribulose-1,5-biphosphate carboxylase holoenzyme (rubisco) activase gene (rca). Previous studies have revealed a pattern of coordinate expression of rubisco subunit polypeptides during development. We compared the expression of rubisco polypeptides and mRNAs with those encoded by rca. The mRNAs encoding both rubisco activase and rubisco are expressed exclusively in leaf tissue of 7-day-old barley seedlings; mRNAs and polypeptides of rca accumulate progressively from the leaf base in a pattern that is qualitatively similar to that of rubisco subunit mRNAs and polypeptides. The parallel pattern of rca protein and mRNA accumulation indicate that a primary control of rca gene expression in this system lies at the level of mRNA production. Light-induced expression of rca in etiolated barley follows a different pattern from that of the acropetal barley leaf gradient, however. Etiolated, 7-day-old barley seedlings contain levels of rca mRNA near the limit of detection in Northern blot hybridization assays. White light induces a 50- to 100-fold accumulation of rca mRNA, which is detectable within 30 min after the onset of illumination. In contrast, steady state levels of mRNAs encoding the small rubisco subunit are affected little by light, and mRNAs encoding the large subunit accumulate about 5-fold in response to illumination. While rca mRNA levels are low in etiolated barley leaves, levels of the protein are approximately 50 to 75% of those found in fully green leaves.     

Expression of Early Light-Inducible Proteins in Flag Leaves of Field-Grown Barley

Early light-inducible protein (ELIP) mRNA and protein levels were analyzed during maturation and senescence of barley (Hordeum vulgare L.) flag leaves under field conditions. The data clearly demonstrate that ELIP mRNA levels are related to the sunlight intensity before sample collection. Levels of mRNAs encoding both low and high molecular mass ELIPs fluctuate in parallel. Changes in mRNA levels are accompanied by corresponding changes in protein levels except for days when average temperatures are high. Comparison of flag leaves at different stages of development in spring and winter barley varieties suggests that light-stress-regulated ELIP gene expression is independent of the developmental stage of the leaves. Although chlorophyll content, photosystem II (PSII) efficiency, and 32-kD herbicide-binding protein of PSII levels decrease drastically after the onset of senescence, ELIP mRNA and protein still accumulate to high levels on bright days.     

The organisation and expression of the genes encoding the mitochondrial glycine decarboxylase complex and serine hydroxymethyltransferase in pea (Pisum sativum)

Summary Restriction fragment length polymorphisms have been used to determine the chromosomal location of the genes encoding the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) of pea leaf mitochondria. The genes encoding the H subunit of GDC and the genes encoding SHMT both show linkage to the classical group I marker i. In addition, the genes for the P protein of GDC show linkage to the classic group I marker a. The genes for the L and T proteins of GDC are linked to one another and are probably situated on the satellite of chromosome 7. The mRNAs encoding the five polypeptides that make up GDC and SHMT are strongly induced when dark-grown etiolated pea seedlings are placed in the light. Similarly, when mature plants are placed in the dark for 48 h, the levels of both GDC protein and SHMT mRNAs decline dramatically and then are induced strongly when these plants are returned to the light. During both treatments a similar pattern of mRNA induction is observed, with the mRNA encoding the P protein of GDC being the most rapidly induced and the mRNA for the H protein the slowest. Whereas during the greening of etiolated seedlings the polypeptides of GDC and SHMT show patterns of accumulation similar to those of the corresponding mRNAs, very little change in the level of the polypeptides is seen when mature plants are placed in the dark and then re-exposed to the light.     

Diurnal and circadian rhythmicity in the expression of light-induced plant nuclear messenger RNAs

The levels of nuclear mRNAs for three light-inducible proteins (light-harvesting chlorophyll a/b protein, small subunit of ribulose-1,5-bisphosphate carboxylase and early light-induced protein) have been analyzed under light-dark and constant light conditions. The levels of all three mRNAs have been found to vary considerably during the day, both under ligh-dark and under constant light conditions, demonstrating the existence of diurnal and circadian rhythmicity in the expressionoof these nuclear-coded plant proteins. The levels of two of these mRNAs have been found to be enhanced 2 h before the beginning of illumination when active phytochrome levels are still low.Abbreviations ELIP early light-inducible protein - LHCP light-harvesting chlorophyll alb protein; poly(A)RNA=polyadenylated RNA - (ss)RuBPCase (small subunit) ribulose-1,5-bisphosphate carboxylase     

High-intensity-light-dependent and transient expression of new genes encoding distant relatives of light-harvesting chlorophyll-a/b proteins in Chlamydomonas reinhardtii

We identified four Lhc-like genes (Lhl) encoding proteins that are distant relatives of light-harvesting chlorophyll a/b-binding (LHC) proteins in the green alga Chlamydomonas reinhardtii. Their mRNA levels after transfer from low-intensity light to high-intensity light (HL) were examined and compared with those of Lhc genes encoding LHC proteins of PSII. The transfer caused a decrease in the mRNA level of Lhl3, a homolog of Arabidopsis thaliana Lil3, within 2 h, followed by gradual restoration depending on the intensity of HL. The response was similar to that of Lhc genes. In contrast, the mRNA levels of Lhl1, Lhl2 and Lhl4 significantly increased, reached a maximum within 1 h after the transfer and then rapidly returned to a low level. The intensity of HL little influenced the response of these genes. While the Lhl1 and Lhl2 proteins were homologs of early light-inducible protein (ELIP) and high-light-inducible protein (HLIP), respectively, Lhl4 encoded a novel protein. The HL-induced expression of Lhl4 was most prominent among the genes tested. Studies using various inhibitors indicate that the HL response is not mediated by the redox state of plastoquinone pool or reactive oxygen species, but required de novo protein synthesis in the cytosol.     

Control of photosynthesis in barley plants with reduced activities of glycine decarboxylase

A mutant (LaPr 87/30) of barley (Hordeum vulgare L.) deficient in glycine decarboxylase (GDC; EC 2.1.2.10) was crossed with wild-type plants to generate heterozygous plants with reduced GDC activities. Plants of the F₂ generation were grown in air and analysed for reductions in GDC proteins and GDC activity. The leaves of heterozygous plants contained reduced amounts of H-protein, and when the content of H-protein was lower than 60% of the wild-type, the P-protein was also reduced. The contents of the other two proteins of the GDC complex, T-protein and L-protein were not affected. Glycine decarboxylase activities, measured as the decarboxylation of [1-¹⁴C]glycine by intact mitochondria released from protoplasts, were between 47% and 63% of the wild-type activity in heterozygous plants and between 86% and 100% in plants with normal contents of H-protein. The enzyme activity was linearly correlated with the relative content of H-protein. Plants with reduced GDC activities developed normally and did not show major pleiotropic effects. In air, the reduction in GDC activity had no effect on the leaf metabolite content or photosynthesis, but under conditions of enhanced photorespiration (low CO₂ and high light), glycine accumulated and the rates of photosynthesis decreased compared to the wild-type. The accumulation of glycine did not lead to a depletion of amino donors or to the accumulation of glyoxylate. The lower rates of photosynthesis were probably caused by an impaired recycling of carbon in the photorespiratory pathway. It is concluded that GDC has no control over CO₂ assimilation under normal growth conditions, but appreciable control by GDC becomes apparent under conditions leading to higher rates of photorespiration. Received: 24 November 1996 / Accepted: 23 January 1997     

Developmental and circadian control of the capacity for δ-aminolevulinic acid synthesis in green barley

The synthesis of δ-aminolevulinic acid (δ-ALA) is a key step in the regulation of tetrapyrrole synthesis. To study the developmentally and circadian-clock controlled mechanism that co-ordinates synthesis of chlorophylls and chlorophyll-binding proteins, δ-ALA-synthesising capacity was analysed in barley (Hordeum vulgare L.) primary leaves grown under dark/light or constant light conditions. The δ-ALA-forming activity oscillated within 24 h with a maximum at the transition of dark to light and a minimum 12 h later, indicating the involvement of the circadian oscillator during development. The capacity for δ-ALA synthesis increased transiently in the middle of barley primary leaves. The δ-ALA-forming-activity correlated well with the previously published steady-state level of mRNA for light-harvesting chlorophyll-binding proteins in space and time; this supports the view of a co-ordinate synthesis of chlorophyll and pigment-binding proteins. Steady-state levels of mRNAs encoding the three enzymes of the δ-ALA-synthesising pathway and of proteins for glutamyl-tRNA reductase (GluTR) and glutamate 1-semialdehyde aminotransferase (GSA AT; EC 5.4.3.8) were analysed for their developmental and circadian expression in barley leaves. The contents of GluTR mRNA and protein cycled parallel to the changes in δ-ALA-forming activity. The levels of GSA AT mRNA oscillated in an opposite phase, but the protein content did not show substantial oscillation under diurnal and circadian growth conditions. No circadian oscillation was detected for glutamyl tRNA synthase (GluRS; EC 6.1.1.17). Maximal GluTR mRNA content and protein was observed in the middle (segments 3 and 4) of the barley primary leaves. The developmentally controlled expression of GluTR therefore differs from that of GSA AT and GluRS, but resembles the capacity for δ-ALA synthesis in a barley leaf gradient. These data indicate that the oscillating, light-dependent and spatial expression of GluTR mRNA might contribute to the regulated formation of the chlorophyll precursor δ-ALA. Received: 29 April 1996 / Accepted 11 December 1996     

Glycine decarboxylase controls photosynthesis and plant growth

Photorespiration makes oxygenic photosynthesis possible by scavenging 2-phosphoglycolate. Hence, compromising photorespiration impairs photosynthesis. We examined whether facilitating photorespiratory carbon flow in turn accelerates photosynthesis and found that overexpression of the H-protein of glycine decarboxylase indeed considerably enhanced net-photosynthesis and growth of Arabidopsis thaliana. At the molecular level, lower glycine levels confirmed elevated GDC activity in vivo, and lower levels of the CO₂ acceptor ribulose 1,5-bisphosphate indicated higher drain from CO₂ fixation. Thus, the photorespiratory enzyme glycine decarboxylase appears as an important feed-back signaller that contributes to the control of the Calvin-Benson cycle and hence carbon flow through both photosynthesis and photorespiration.     

Light-induced increases in the glycine decarboxylase multienzyme complex from pea leaf mitochondria

The rates of mitochondrial glycine oxidation estimated by CO2-release and glycine-bicarbonate exchange activities in fully greened tissues are approximately 10 times greater than those of etiolated pea leaves and potato tuber mitochondria. The release of CO2 from glycine in intact mitochondria isolated from dark-grown and nonphotosynthetic tissues was sensitive to inhibitors of mitochondrial electron transport, glycine transport, and glycine decarboxylase activities. The CO2-release and glycine-bicarbonate exchange activities in crude mitochondrial protein extracts from light-grown versus dark-grown tissues exhibited light/dark ratios of 12 and 21, respectively. This suggests that the differences in capacity to oxidize glycine reside with the glycine decarboxylase enzyme complex itself. The complex is composed of four subunit enzymes, the P, H, T, and L proteins, which can be isolated individually and reconstituted into the active enzyme. The activities of P and T proteins were at least 10 times higher in fully greened pea leaves than in the etiolated tissue, while the H and L protein activities were four times higher in these same tissues. The levels of P and T proteins detected immunochemically were substantially lower in total mitochondrial extracts prepared from leaves of dark-grown pea seedlings. Labeling of whole pea seedlings and in vitro protein synthesis with isolated mitochondria indicated that the entire glycine decarboxylase enzyme complex is cytoplasmically synthesized and therefore encoded by the nucleus. Polypeptides synthesized from total leaf polyadenylated mRNA isolated from leaves of both the dark-grown and light-treated peas indicated the presence of P protein. This implies that translatable messages for this enzyme are present at some level throughout leaf development.     

A double SORLIP1 element is required for high light induction of ELIP genes in Arabidopsis thaliana

Promoter elements that contribute to high light (HL) induction of the Arabidopsis ELIP1 gene were defined using a transgenic promoter-reporter system. Two adjacent SORLIP1 elements (double SORLIP1, dSL) were found to be essential for HL induction of a GUS reporter gene. The dSL element was also found to be essential for HL induction conferred by the ELIP2 promoter. SORLIP1 elements were enriched in ELIP promoters throughout the plant kingdom, and showed a clade-specific pattern of gain or loss that suggested functionality. In addition, two G-box elements were found to redundantly contribute to HL induction conferred by the ELIP1 promoter.     

Interaction between photorespiration and respiration in transgenic potato plants with antisense reduction in glycine decarboxylase

Potato (Solanum tuberosum L. cv. Désirée) plants with an antisense reduction in the P-protein of the glycine decarboxylase complex (GDC) were used to study the interaction between respiration and photorespiration. Mitochondria isolated from transgenic plants had a decreased capacity for glycine oxidation and glycine accumulated in the leaves. Malate consumption increased in leaves of GDC deficient plants and the capacity for malate and NADH oxidation increased in isolated mitochondria. A lower level of alternative oxidase protein and decreased partitioning of electrons to the alternative pathway was found in these plants. The adenylate status was altered in protoplasts from transgenic plants, most notably the chloroplastic ATP/ADP ratio increased. The lower capacity for photorespiration in leaves of GDC deficient plants was compensated for by increased respiratory decarboxylations in the light. This is interpreted as a decreased light suppression of the tricarboxylic acid cycle in GDC deficient plants in comparison to wild-type plants. The results support the view that respiratory decarboxylations in the light are restricted at the level of the pyruvate dehydrogenase complex and/or isocitrate dehydrogenase and that this effect is likely to be mediated by mitochondrial photorespiratory products.     

An Arabidopsis thaliana protein homologous to cyanobacterial high-light-inducible proteins

An Arabidopsis thaliana cDNA clone encoding a novel 110 amino acid thylakoid protein has been sequenced. The in vitro synthesized protein is taken up by intact chloroplasts, inserted into the thylakoid membrane and the transit peptide is cleaved off during this process. The mature protein is predicted to contain 69 amino acids, to form one membrane-spanning -helix and to have its N-terminus at the stromal side of the thylakoid membrane. The protein showed similarity to the LHC, ELIP and PsbS proteins of higher plants, but more pronounced to the high-light-inducible proteins (HLIPs) of cyanobacteria and red algae, to which no homologue previously has been detected in higher plants. As for HLIP and ELIP, high light increases the mRNA levels of the corresponding gene. Sequence comparisons indicate that the protein may bind chlorophyll and form dimers in the thylakoid membrane. The level of expression of the protein seems to be far lower than that of normal PSI and PSII subunits.