Accumulation of plastid HSP 23 of Chenopodium rubrum is controlled post-translationally by light

The expression of the 23 kDa plastid heat-shock protein (HSP) of Chenopodium rubrum has been studied at various light intensities at a temperature of 38°C where the 23 kDa protein accumulates to its highest levels. It was observed that the level of mRNA which is induced at this heat-shock temperature is independent of the light intensity between 0 and 1000 W m⁻². Labelling in vivo of all investigated HSP is also not dependent on the light fluxes applied. In clear contrast the accumulation of the mature chloroplast HSP 23 is light dependent: while almost no protein is detectable in the dark the level of the accumulated protein reaches a maximum at a light intensity of 300 W m⁻². The accumulated levels of HSP 23 correlate well with resistance against photoinhibition; photoinhibitory effects are observed at a light intensity of 300 W m⁻² or above as measured by the decline of PS II activity. When high light intensities are applied during recovery from heat shock the amounts of HSP 23 stay elevated for a longer time and at a higher level than at the standard light intensity of 10 W m⁻². This appears to be a peculiar property of the plastid HSP 23 as the accumulation of HSP 17 and 70, as analysed by Western blot, is not influenced by light. When under particular stress conditions the levels of HSP 23 remain low a protein of 31 kDa accumulates that reacts with the antibody to HSP 23 and might represent the precursor of HSP 23.     

Biogenesis of chloroplast membranes. I. Plastid dedifferentiation in a dark-grown algal mutant (Chlamydomonas reinhardi)

This paper describes the morphology and photosynthetic activity of a mutant of Chlamydomonas reinhardi (y-1) which is unable to synthesize chlorophyll in the dark. When grown heterotrophically in the light, the mutant is indistinguishable from the wild type Chlamydomonas. When grown in the dark, chlorophyll is diluted through cell division and the photosynthetic activity (oxygen evolution, Hill reaction, and photoreduction of NADP) decays at a rate equal to or faster than that of chlorophyll dilution. However, soluble enzymes associated with the photosynthetic process (alkaline FDPase, NADP-linked G-3-P dehydrogenase, RuDP carboxylase), as well as cytochrome f and ferredoxin, continue to be present in relatively high concentrations. The enzymes involved in the synthesis of the characteristic lipids of the chloroplast (including mono- and digalactoside glycerides, phosphatidyl glycerol, and sulfolipid) are still detectable in dark-grown cells. Such cells accumulate large amounts of starch granules in their plastids. On onset of illumination, dark-grown cells synthesize chlorophyll rapidly, utilizing their starch reserve in the process. At the morphological level, it was observed that during growth in the dark the chloroplast lamellar system is gradually disorganized and drastically decreased in extent, while other subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) or much less affected (eyespot, ribosomes). It is concluded that the dark-grown mutant possesses a partially differentiated plastid and the enzymic apparatus necessary for the synthesis of the chloroplast membranes (discs). The advantage provided by such a system for the study of the biogenesis of the chloroplast photosynthetic membranes is discussed.     

Comparative analysis of the biogenesis of photosystem II in the wild- type and Y-1 mutant of Chlamydomonas reinhardtii

Expression of the genes of the photosystem II (PSII) core polypeptides D1 and D2, of three proteins of the oxygen evolving complex of PSII and of the light harvesting chlorophyll a/b binding proteins (LHCP) has been compared in wild-type (wt) and in the y-1 mutant of Chlamydomonas reinhardtii. Since wt, but not y-1 cells produce a fully developed photosynthetic system in the dark, comparison of the two has allowed us to distinguish the direct effect of light from the influence of plastid development on gene expression. The PSII core polypeptides and LHCP are nearly undetectable in dark-grown y-1 cells but they accumulate progressively during light induced greening. The levels of these proteins in wt are the same in the light and the dark. The amounts of the proteins of the oxygen evolving complex do not change appreciably in the light or in the dark for both wt and y-1. Steady state levels of chloroplast mRNA encoding the core PSII polypeptides remain nearly constant in the light or the dark and are not affected by the developmental stage of the plastid. Levels of nuclear encoded mRNAs for the oxygen evolving proteins and of LHCP increase during light growth in wt and y-1. In contrast to wt, synthesis of LHCP proteins is not detectable in y-1 cells in the dark but starts immediately after transfer to light, indicating that LHCP synthesis is controlled by a light-induced factor or process. While the rates of synthesis of D1 and D2 are immediately enhanced by light in wt, this increase occurs only after a lag in y-1 and thus must be dependent on an early light-induced event in the plastid. These results show that the biosynthesis of PSII is affected by light directly, by the stage of plastid development, and by the interaction of light and events associated with plastid development.     

Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation

The tomato (Lycopersicon esculentum) chloroplast small heat shock protein (sHSP), HSP21, is induced by heat treatment in leaves, but also under normal growth conditions in developing fruits during the transition of chloroplasts to chromoplasts. We used transgenic tomato plants constitutively expressing HSP21 to study the role of the protein under stress conditions and during fruit maturation. Although we did not find any effect for the transgene on photosystem II (PSII) thermotolerance, our results show that the protein protects PSII from temperature-dependent oxidative stress. In addition, we found direct evidence of the protein's role in fruit reddening and the conversion of chloroplasts to chromoplasts. When plants were grown under normal growth temperature, transgenic fruits accumulated carotenoids earlier than controls. Furthermore, when detached mature green fruits were stored for 2 weeks at 2 degrees C and then transferred to room temperature, the natural accumulation of carotenoids was blocked. In a previous study, we showed that preheat treatment, which induces HSP21, allowed fruit color change at room temperature, after a cold treatment. Here, we show that mature green transgenic fruits constitutively expressing HSP21 do not require the heat treatment to maintain the ability to accumulate carotenoids after cold storage. This study demonstrates that a sHSP plays a role in plant development under normal growth conditions, in addition to its protective effect under stress conditions.     

Quantitative and structural characteristics of lipids in Chlorobium and Chloroflexus

Exposure of dark grown resting Euglena to light induced the synthesis of chloroplast valyl-tRNA synthetase. Ethanol, a specific inhibitor of Euglena chloroplast development had little effect on chloroplast valyl-tRNA synthetase induction during the first 12 h of light exposure. Ethanol, however, completely inhibited enzyme synthesis between 12–72 h of light exposure. Malate, an alternative carbon source, had little effect on the photoinduction of valyl-tRNA synthetase. When dark grown resting cells were exposed to 2 h of light and returned to the dark, chloroplast valyl-tRNA synthetase continued to accumulate for 8–12 h at a rate which was less than the rate in cells maintained continuously in the light. The mutant strain W₃BUL lacks detectable chloroplast DNA and phototransformable protochlorophyllide, but retains a plastid remnant. Exposure of strain W₃BUL to light induced the synthesis of chloroplast valyl-tRNA synthetase and enzyme induction was not inhibited by ethanol. After 72 h of light exposure in the presence or absence of ethanol, enzyme levels in strain W₃BUL were comparable to the levels found in the wildtype strain after 8–14 h of light exposure. These results suggest that the nonchloroplast photoreceptor regulates the initial phase of enzyme synthesis. Mutant strain W₁₀BSmL differs from strain W₃BUL in that the plastid remnant if present, is greatly reduced. Chloroplast valyl-tRNA synthetase was undetectable in the strain W₁₀BSmL suggesting that the levels of active, cytoplasmically synthesized, chloroplast localized enzymes may be related to the developmental status of the chloroplast through the extent to which the enzyme precursor can be accumulated and or posttranslationally processed into an active enzyme within the chloroplast or chloroplast remnant.This research was supported by National Institutes of Health Grant GM26994, Biomedical support grant RR-0755 and funds from the Research Council, University of Nebraska     

TSC1 enables plastid development under dark conditions,contributing to rice adaptation to transplantation shock

Since its domestication from wild rice thousands of years ago, rice has been cultivated largely through transplantation. During transplantation from the nursery to the paddy field, rice seedlings experience transplantation shock which affects their physiology and production.However, the mechanisms underlying transplantation shock and rice adaptation to this shock are largely unknown. Here,we isolated a transplant-sensitive chloroplast-deficient(tsc_1)rice mutant that produces albino leaves after transplantation.Blocking light from reaching the juvenile leaves and leaf primordia caused chloroplast deficiencies in transplanted tsc_1 seedlings. TSC_1 encodes a noncanonical adenosine triphosphate-binding cassette(ABC) transporter homologous to At NAP_(14) and is of cyanobacterial origin. We demonstrate that TSC_1 controls plastid development in rice under dark conditions, and functions independently of light signaling.However, light rescued the tsc_1 mutant phenotype in a spectrum-independent manner. TSC_1 was upregulated following transplantation, and modulated the iron and copper levels, thereby regulating prolamellar body formation during the early P_4 stage of leaf development. Therefore, TSC_1 is indispensable for plastid development in the absence of light,and contributes to adaptation to transplantation shock.Our study provides insight into the regulation of plastid development and establishes a framework for improving recovery from transplantation shock in rice.     

Regulation of accumulation of the major thylakoid polypeptides in Chlamydomonas reinhardtii y-1 at 25 degrees C and 38 degrees C

The amount of messenger RNA (mRNA) for polypeptides of the chlorophyll a/b-protein complex of thylakoid membranes in etiolated and greening cells of Chlamydomonas reinhardtii y-1 was examined by immunoprecipitation and electrophoresis of products of in vitro translation to determine at which stage production of these polypeptides is regulated. Cells grown 4 d in the dark at 25 degrees C contained small amounts of translatable mRNA for the major membrane polypeptides. Exposure of these etiolated cells to light, under conditions in which the membrane polypeptides accumulated, resulted in a significant increase in the quantity of the mRNA. In contrast, when etiolated cells were incubated for 1-2 h in the dark at 38 degrees C, translation assays indicated that mRNA for the membrane polypeptides became abundant. Moreover, the quantity of the mRNA did not increase when these cells subsequently were exposed to light. Therefore, at 38 degrees C the cellular level of the polypeptides is not regulated by synthesis of mRNA. The in vitro synthesized polypeptides, which were precipitated with antibodies prepared against the purified thylakoid polypeptides, had apparent molecular weights of 31,500 and 30,000. The corresponding immunoprecipitated polypeptides made in vivo had apparent molecular weights of 29,500 and 26,000. Thus, the membrane polypeptides are made as precursors. No net accumulation of the polypeptides occurred in cells in the dark at 38 degrees C, but immunoreactive polypeptides the size of the mature membrane components were labeled during incubation of cells with [14C]acetate in the dark. These results indicated that the mRNA was translated in the dark, but since the polypeptides did not accumulate, the products of translation were probably degraded. We conclude from our experiments that at 25 degrees C production of the polypeptides is regulated by the level of translatable mRNA in the cells. At 38 degrees C, however, the accumulation of the polypeptides is controlled by posttranslational processes.     

Immunological evidence for accumulation of two high-molecular-weight (104 and 90 kDa) HSPs in response to different stresses in rice and in response to high temperature stress in diverse plant genera

Rice seedlings accumulate stainable amounts of the 104 and 90 kDa polypeptides in response to high temperature stress. We have purified and raised highly specific polyclonal antisera against both of these polypeptides. In western blotting experiments, we find that these proteins are accumulated to different extents in rice seedlings subjected to salinity (NaCl), water stress, low-temperature stress and exogenous abscisic acid application. These proteins also accumulated when rice seedlings grown in pots under natural conditions were subjected to water stress by withholding watering. Seedlings of Triticum aestivum, Sorghum bicolor, Pisum sativum, Zea mays, Brassica juncea and mycelium of Neurospora crassa showed accumulation of the immunological homologues of both the 104 and the 90 kDa polypeptides, in response to high-temperature stress. We have earlier shown that shoots of rice seedlings exposed to heat shock accumulate a 110 kDa polypeptide which is an immunological homologue of the yeast HSP 104 (Singla and Grover, Plant Mol Biol 22: 1177–1180, 1993). Employing anti-rice HSP 104 antibodies and anti-yeast HSP 104 antibodies together, we provide evidence that rice HSP 104 is different from the earlier characterized rice HSP 110.     

A chloroplast-targeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition.

Dark-grown Chlamydomonas reinhardtii cultures that were illuminated at low fluence rates before exposure to high-light conditions exhibited a faster rate of recovery from photoinhibition than did dark-grown cells that were directly exposed to photoinhibitory conditions. This pretreatment has been shown to induce the expression of several nuclear heat shock protein 70 (HSP70) genes, including HSP70B, encoding a chloroplast-localized chaperone. To investigate a possible role of plastidic HSP70B in photoprotection and repair of photosystem II, which is the major target of photoinhibition, we have constructed strains overexpressing or underexpressing HSP70B. The effect of light stress on photosystem II in nuclear transformants harboring HSP70B in the sense or antisense orientation was monitored by measuring variable fluorescence, flash-induced charge separation, and relative amounts of various photosystem II polypeptides. Underexpression of HSP70B caused an increased light sensitivity of photosystem II, whereas overexpression of HSP70B had a protective effect. Furthermore, the reactivation of photosystem II after photoinhibition was enhanced in the HSP70B-overexpressing strain when compared with the wild type, both in the presence or absence of synthesis of chloroplast-encoded proteins. Therefore, HSP70B may participate in vivo both in the molecular protection of the photosystem II reaction centers during photoinhibition and in the process of photosystem II repair.     

Heme, a plastid-derived regulator of nuclear gene expression in Chlamydomonas

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants, light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX, indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as plastid signals that regulate the expression of nuclear genes.     

Accumulation, stability, and localization of a major chloroplast heat-shock protein

Diverse higher plant species synthesize low molecular weight (LMW) heat shock proteins (HSPs) which localize to chloroplasts. These proteins are homologous to LMW HSPs found in the cytoplasm of all eukaryotes, a class of HSPs whose molecular mode of action is not understood. To obtain basic information concerning the role of chloroplast HSPs, we examined the accumulation, stability, tissue specificity, and intra-chloroplast localization of HSP21, the major LMW chloroplast HSP in pea. Intact pea plants were subjected to heat stress conditions which would be encountered in the natural environment and HSP21 mRNA and protein levels were measured in leaves and roots. HSP21 was not detected in leaves or roots before stress, but the mature, 21-kD protein accumulated in direct proportion to temperature and HSP21 mRNA levels in both tissues. All of the HSP21 in leaves was localized to chloroplasts; there was no evidence for its transport into other organelles. In chloroplast fractionation experiments, greater than 80% of HSP21 was recovered in the soluble chloroplast protein fraction. The half-life of HSP21 at control temperatures was 52 +/- 12 h, suggesting the protein's function is critical during recovery as well as during stress. We hypothesize that HSP21 functions in a catalytic fashion in both photosynthetic and nonphotosynthetic plastids.     

Chromoplast development in ripening tomato fruit: identification of cDNAs for chromoplast-targeted proteins and characterization of a cDNA encoding a plastid-localized low-molecular-weight heat shock protein

During tomato fruit ripening, photosynthetically competent thylakoid membranes are broken down and replaced by membranous deposits of carotenoids. Few of the proteins involved in this transition have been identified. We have used chloroplast protein import assays as a means to identify two cDNAs that encode proteins destined for the developing chromoplast. One of the cDNAs had unexpected properties and its biological function has not been determined. However, the other cDNA encodes a plastid-localized low-MW heat shock protein (hsp). The steady-state level of RNA corresponding to this cDNA increased several-fold during tomato ripening, and the amount of RNA induced by heat stress increased dramatically during this process. These observations suggest a new role for this stress protein in protecting the plastid during the dismantling of the thylakoid membranes or during the buildup of carotenoids.