Regulation of synthesis of the photosystem I reaction center.

The in vivo biosynthesis of the P700 chlorophyll a-apoprotein was examined to determine whether this process is light regulated and to determine its relationship to chlorophyll accumulation during light-induced chloroplast development in barley (Hordeum vulgare L.). Rabbit antibodies to the 58,000-62,000-mol-wt apoprotein were used to measure relative synthesis rates by immunoprecipitation of in vivo labeled leaf proteins and to detect apoprotein accumulation on nitrocellulose protein blots. 5-d-old, dark-grown barley seedlings did not contain, or show net synthesis of, the 58,000-62,000-mol-wt polypeptide. When dark-grown barley seedlings were illuminated, net synthesis of the apoprotein was observed within the first 15 min of illumination and accumulated apoprotein was measurable after 1 h. After 4 h, P700 chlorophyll a-apoprotein biosynthesis accounted for up to 10% of the total cellular membrane protein synthesis. Changes in the rate of synthesis during chloroplast development suggest coordination between production of the 58,000-62,000-mol-wt polypeptide and the accumulation of chlorophyll. However, when plants were returned to darkness after a period of illumination (4 h) P700 chlorophyll a-apoprotein synthesis continued for a period of hours though at a reduced rate. Thus we found that neither illumination nor the rate of chlorophyll synthesis directly control the rate of apoprotein synthesis. The rapidity of the light-induced change in net synthesis of the apoprotein indicates that this response is tightly coupled to the primary events of light-induced chloroplast development. The data also demonstrate that de novo synthesis of the apoprotein is required for the onset of photosystem I activity in greening seedlings.

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.

Heat shock protein 101 effects in A. thaliana: genetic variation, fitness and pleiotropy in controlled temperature conditions.

The Hsp100/ClpB heat shock protein family is ancient and required for high temperature survival, but natural variation in expression and its phenotypic effects is unexplored in plants. In controlled environment experiments, we examined the effects of variation in the Arabidopsis cytosolic AtHsp101 (hereafter Hsp101). Ten wild-collected ecotypes differed in Hsp101 expression responses across a 22 to 40 degrees C gradient. Genotypes from low latitudes expressed the least Hsp101. We tested fitness and pleiotropic consequences of varying Hsp101 expression in 'control' vs. mild thermal stress treatments (15/25 degrees C D/N vs. 15/25 degrees D/N plus 3 h at 35 degrees C 3 days/week). Comparing wild type and null mutants, wt Columbia (Col) produced approximately 33% more fruits compared to its Hsp101 homozygous null mutant. There was no difference between Landsberg erecta null mutant NIL (Ler) and wt Ler; wt Ler showed very low Hsp101 expression. In an assay of six genotypes, fecundity was a saturating function of Hsp101 content, in both experimental treatments. Thus, in addition to its essential role in acquired thermal tolerance, Hsp101 provides a substantial fitness benefit under normal growth conditions. Knocking out Hsp101 decreased fruit production, days to germination and days to bolting, total dry mass, and number of inflorescences; it increased transpiration rate and allocation to root mass. Root : total mass ratio decayed exponentially with Hsp101 content. This study shows that Hsp101 expression is evolvable in natural populations. Our results further suggest that Hsp101 is primarily an emergency high-temperature tolerance mechanism, since expression levels are lower in low-latitude populations from warmer climates. Hsp101 expression appears to carry an important trade-off in reduced root growth. This trade-off may select for suppressed expression under chronically high temperatures.

The major low-molecular-weight heat shock protein in chloroplasts shows antigenic conservation among diverse higher plant species.

Several plant species are known to synthesize low-molecular-weight nucleus-encoded heat shock proteins (HSPs) which localize to chloroplasts. DNA sequence analysis of chloroplast HSP cDNAs from pea (Pisum sativum) and soybean (Glycine max) has shown that the carboxyl-terminal halves of these proteins are homologous to low-molecular-weight HSPs from a wide range of eucaryotes (E. Vierling, R. T. Nagao, A. E. DeRocher, and L. M. Harris, EMBO J. 7:575-581, 1988). We used a pea cDNA to construct fusion proteins containing either the carboxyl-terminal heat shock domain or the amino-terminal domain of the chloroplast HSP. The fusion proteins were overexpressed in Escherichia coli and used to produce choloroplast HSP-specific polyclonal antibodies. The carboxyl-terminal antibodies recognized chloroplast HSP precursor proteins from pea and from three divergent plant species, Arabidopsis thaliana, petunia (Petunia hybrida), and maize (Zea mays). The amino-terminal antibodies recognized effectively only the pea precursor. When intact plants of each species were subjected to a heat stress regime mimicking field growth conditions, significant levels of the mature forms of the chloroplast HSPs accumulated in pea, A. thaliana, and maize. The levels of accumulated HSPs remained unchanged for 12 h following the stress treatment. We conclude that the synthesis of chloroplast-localized HSPs is an important component of the stree response in all higher plants and that chloroplast HSPs from dicotyledonous and monocotyledonous plants have a conserved carboxyl-terminal domain.

Localization of small heat shock proteins to the higher plant endomembrane system.

Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We describe a fourth LMW HSP family, represented by PsHSP22.7 from Pisum sativum and GmHSP22.0 from Glycine max, and demonstrate that this family of proteins is endomembrane localized. PsHSP22.7 and GmHSP22.0 are 76.7% identical at the amino acid level. Both proteins have amino-terminal signal peptides and carboxyl-terminal sequences characteristic of endoplasmic reticulum (ER) retention signals. The two proteins closely resemble class I cytoplasmic LMW HSPs, suggesting that they evolved from the cytoplasmic proteins through the addition of the signal peptide and ER retention motif. The endomembrane localization of these proteins was confirmed by cell fractionation. The polypeptide product of PsHSP22.7 mRNA was processed to a smaller-M(r) form by canine pancreatic microsomes; in vivo, GmHSP22.0 polysomal mRNA was found to be predominantly membrane bound. In vitro-processed PsHSP22.7 corresponded in mass and pI to one of two proteins detected in ER fractions from heat-stressed plants by using anti-PsHSP22.7 antibodies. Like other LMW HSPs, PsHSP22.7 was observed in higher-molecular-weight structures with apparent masses of between 80 and 240 kDa. The results reported here indicate that members of this new class of LMW HSPs are most likely resident ER proteins and may be similar in function to related LMW HSPs in the cytoplasm. Along with the HSP90 and HSP70 classes of HSPs, this is the third category of HSPs localized to the ER.

Expression of Low Molecular Weight Heat-Shock Proteins under Field Conditions.

Heat-shock proteins (HSPs) are known to be expressed in plants experiencing high-temperature stress. We have examined the expression of class I cytoplasmic low molecular weight (LMW) HSPs and find that these HSPs also frequently accumulate in seeds, seed pods, and flowers during a normal growing season. We first examined the expression of class I cytoplasmic LMW HSPs by western blot analysis in a range of seed samples from both commercially grown and wild legumes. LMW HSPs were present in all seed samples, indicating that these HSPs are regularly expressed in these tissues. To examine more specifically conditions under which LMW HSPs were produced during an average growing season, additional studies of Medicago sativa were carried out during the fall season in Tucson, AZ. Plants were irrigated to avoid conditions of water stress, and canopy temperature was monitored throughout the study period. LMW HSP expression in leaves, flowers, and developing seed pods was analyzed by western blotting. Results show that in the field HSPs are frequently produced in flowers and seed pods, even in plants that show no HSP expression in leaves. Parallel greenhouse studies indicate that HSP expression in seeds is in part developmentally regulated. In total our data suggest a more widespread occurrence of HSPs in optimal growth environments and emphasize their potential role during reproduction.

Plant responses to environmental stress.

Considerable progress is being made in identifying genes that are important for tolerance to abiotic stress and in defining stress-responsive gene promoters and signal-transduction pathways. Although genetically engineered crop plants with greater resistance to environmental stress have not yet been produced, research is at a turning point where correlative changes can now be tested for effectiveness in conferring stress tolerance.

The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing alpha-crystallin domains (Acd proteins).

Comprehensive analysis of the Arabidopsis genome revealed a total of 13 sHsps belonging to 6 classes defined on the basis of their intracellular localization and sequence relatedness plus 6 ORFs encoding proteins distantly related to the cytosolic class Cl or the plastidial class of sHsps. The complexity of the Arabidopsis sHsp family far exceeds that in any other organism investigated to date. Furthermore, we have identified a new family of ORFs encoding multidomain proteins that contain one or more regions with homology to the ACD (Acd proteins). The functions of the Acd proteins and the role of their ACDs remain to be investigated.

The chloroplast small heat shock protein undergoes oxidation-dependent conformational changes and may protect plants from oxidative stress.

The nuclear-encoded chloroplast-localized Hsp21 is an oligomeric heat shock protein (Hsp), belonging to the protein family of small Hsps and alpha-crystallins. We have investigated the effects of high temperature and oxidation treatments on the structural properties of Hsp21, both in purified recombinant form and in transgenic Arabidopsis thaliana plants engineered to constitutively overexpress Hsp21. A conformational change was observed for the 300 kDa oligomeric Hsp21 protein during moderate heat stress (< or =40 degrees C) of Arabidopsis plants, as judged by a shift to lower mobility in non-denaturing electrophoresis. Similar changes in mobility were observed when purified recombinant Hsp21 protein was subjected to an oxidant. Exposure of Hsp21 protein to temperatures above 70 degrees C led to irreversible aggregation, which was prevented in presence of the reductant dithiothreitol. The transgenic plants that constitutively overexpressed Hsp21 were more resistant to heat stress than were wildtype plants when the heat stress was imposed under high light conditions. These results suggest that the physiological role of Hsp21 involves a response to temperature-dependent oxidative stress.

Analysis of conserved domains identifies a unique structural feature of a chloroplast heat shock protein.

A low molecular weight heat shock protein which localizes to chloroplasts has been identified in several plant species. This protein belongs to a eukaryotic superfamily of small HSPs, all of which contain a conserved carboxyl-terminal domain. To investigate further the structure of this HSP, we isolated and sequenced cDNA clones for the chloroplast LMW HSPs from Petunia hybrida and Arabidopsis thaliana. The cloning of chloroplast HSPs from these two species enabled us to compare the amino acid sequences of this protein from plant species (petunia, Arabidopsis, pea, soybean and maize) that represent evolutionarily divergent taxonomic subclasses. Three conserved regions were identified, which are designated as regions I, II and III. Regions I and II are also shared by cytoplasmic LMW HSPs and therefore are likely to have functional roles common to all eukaryotic LMW HSPs. In contrast, consensus region III is not found in other LMW HSPs. Secondary structure analysis predicts that this region forms an amphipathic alpha-helix with high conservation of methionine residues on the hydrophobic face and 100% conservation of residues on the hydrophilic face. This structure is similar to three helices, termed "methionine bristles", which are found in a methionine-rich domain of a 54 kDa protein component of signal recognition particle (SRP54). The conservation of regions I and II among LMW cytoplasmic and chloroplast HSPs suggests that these HSPs perform related functions in different cellular compartments. However, identification of the methionine bristle domain suggests that chloroplast HSPs also have unique functions or substrates within the special environment of the chloroplast or other plastids.

The expression of small heat shock proteins in seeds responds to discrete developmental signals and suggests a general protective role in desiccation tolerance.

To learn more about the function and regulation of small heat shock proteins (sHSPs) during seed development, we studied sHSP expression in wild-type and seed maturation mutants of Arabidopsis by western analysis and using an HSP17.4 promoter-driven beta-glucuronidase (GUS) reporter gene in transgenic plants. In the absence of stress, GUS activity increases during development until the entire embryo is stained before desiccation. Heat-stressed embryos stained for GUS at all stages, including early stages that showed no detectable HSP17. 4::GUS activity without heat. Examination of HSP17.4 expression in seeds of the transcriptional activator mutants abi3-6, fus3-3 (AIMS no. CS8014/N8014), and lec1-2 (AIMS no. CS2922/N2922) showed that protein and HSP17.4::GUS activity were highly reduced in fus3-3 and lec1-2 and undetectable in abi3-6 seeds. In contrast, heat-stressed abi3-6, fus3-3, and lec1-2 seeds stained for GUS activity throughout the embryo. These data indicate that there is distinct developmental and stress regulation of HSP17.4, and imply that ABI3 activates HSP17.4 transcription during development. Quantitation of sHSP protein in desiccation-intolerant seeds of abi3-6, fus3-3, lec1-2, and line24 showed that all had <2% of wild-type HSP17.4 levels. In contrast, the desiccation-tolerant but embryo-defective mutants emb266 (AIMS no. CS3049/N3049) and lec2-1 (AIMS no. CS2728/N2728) had wild-type levels of HSP17.4. These data correlate a reduction in sHSPs with desiccation intolerance and suggest that sHSPs have a general protective role throughout the seed.

Cytoplasmic HSP70 homologues of pea: differential expression in vegetative and embryonic organs.

Eukaryotes express several cytoplasmic HSP70 genes, and their encoded proteins participate in diverse cellular processes. Three cDNAs encoding highly expressed cytoplasmic HSP70 homologues from Pisum sativum were cloned and characterized. They were designated PsHSP71.2, PsHSC71.0, and PsHSP70b. These HSP70 genes have different expression profiles in leaves: PsHSP71.2 is observed only in response to heat stress, PsHSC71.0 is present constitutively, and PsHSP70b is weakly constitutively expressed, but induced strongly in response to heat stress. In addition to being heat induced, the PsHSP71.2 mRNA is also expressed in zygotic, but not maternal organs of developing pea seeds, while PsHSC71.0 and PsHSP70b mRNAs are present in maternal and zygotic organs throughout seed development. Immunoblot analysis of parallel protein samples detects a 70 kDa polypeptide in all samples, and a 72 kDa polypeptide that corresponds to the PsHSP71.2 gene product is observed in cotyledons beginning at mid-maturation and in axes beginning between late maturation and desiccation. This polypeptide is not detected in the seed coat. The 72 kDa polypeptide remains abundant in both cotyledons and axes through germination, but declines substantially between 48 and 72 h after the onset of inbibition. Differential control of HSP70 expression during heat stress, seed maturation, and germination is consistent with the hypothesis that there are functional distinctions between cytoplasmic HSP70s.

Ribulose 1,5-Bisphosphate Carboxylase Synthesis during Heat Shock.

Ribulose 1,5-bisphosphate carboxylase (RuBPCase) was chosen as a model protein to study how heat shock (HS) affects both chloroplast protein synthesis and the nuclear-chloroplast interaction in production of chloroplast proteins. Experiments were performed using highly chlorophyllous, soybean (Glycine max L. Merr. var Corsoy) cell suspension cultures active in chloroplast protein synthesis. Synthesis of RuBPCase large (L) and small (S) subunits was followed by in vivo labeling, and corresponding mRNA levels were examined by Northern and dot hybridization analyses. Results demonstrate that L and S synthesis declines with increasing HS temperatures (33-40 degrees C) and reaches minimum levels (20-30% of control) at temperatures of maximum HS protein synthesis (39-40 degrees C). Recovery of L and S synthesis following a 2-hour HS at 38 or 40 degrees C was also studied. The changes in S synthesis during HS and recovery correlate with the steady state levels of S mRNA. In contrast, changes in L synthesis show little relationship to the corresponding mRNA levels; levels of L mRNA remain relatively unchanged by HS. We conclude that chloroplast protein synthesis shows no greater sensitivity to HS than is observed for cytoplasmic protein synthesis and that transport of proteins into the chloroplast (e.g.S subunit) continues during HS. Furthermore, there is no apparent coordination of L and S subunit mRNA levels under the conditions examined.

Dynamics of small heat shock protein distribution within the chloroplasts of higher plants.

Accumulation of the small heat shock proteins (sHSPs) in response to high temperature stress is thought to contribute to the development of thermotolerance in eukaryotic organisms, but the mechanism of action is unknown. We are investigating the chloroplast-localized sHSP, HSP21, with the goal of defining its contribution to the acquisition of thermotolerance in plants. Following an initial heat stress and period of recovery, HSP21 is localized primarily in the soluble fraction of the chloroplast. During an additional stress, HSP21 undergoes a temperature-dependent redistribution from the soluble to the insoluble chloroplast fraction in both isolated organelles and intact plants. The change in HSP21 partitioning is accompanied by depletion of the 10-11 S HSP21-containing complexes from the soluble chloroplast fraction. HSP21 in the insoluble fraction cannot be solubilized by nonionic detergent under conditions that release essentially all the pigments and proteins from the thylakoid membranes, indicating that HSP21 in its insoluble state is not dependent for its insolubility on attachment to an intact membrane. The temperature-dependent redistribution of HSP21 is affected by light intensity but occurs in both leaf and root plastids, suggesting that the function of this activity is not strictly related to the presence of the photosynthetic apparatus. Our study indicates that the chloroplast sHSP has dynamic properties similar to those of cytoplasmic sHSPs from plants and other organisms and suggests that the ability to partition between a soluble and an insoluble state reflects a functionally important property of all sHSPs.

Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress.

Hsp101 is a molecular chaperone that is required for the development of thermotolerance in plants and other organisms. We report that Arabidopsis thaliana Hsp101 is also regulated during seed development in the absence of stress, in a pattern similar to that seen for LEA proteins and small Hsps; protein accumulates during mid-maturation and is stored in the dry seed. Two new alleles of the locus encoding Hsp101 (HOT1) were isolated from Arabidopsis T-DNA mutant populations. One allele, hot1-3, contains an insertion within the second exon and is null for Hsp101 protein expression. Despite the complete absence of Hsp101 protein, plant growth and development, as well as seed germination, are normal, demonstrating that Hsp101 chaperone activity is not essential in the absence of stress. In thermotolerance assays hot1-3 shows a similar, though somewhat more severe, phenotype to the previously described missense allele hot1-1, revealing that the hot1-1 mutation is also close to null for protein activity. The second new mutant allele, hot1-2, has an insertion in the promoter 101 bp 5' to the putative TATA element. During heat stress the hot1-2 mutant produces normal levels of protein in hypocotyls and 10-day-old seedlings, and it is wild type for thermotolerance at these stages. Thus this mutation has not disrupted the minimal promoter sequence required for heat regulation of Hsp101. The hot1-2 mutant also expresses Hsp101 in seeds, but at a tenfold reduced level, resulting in reduced thermotolerance of germinating seeds and underscoring the importance of Hsp101 to seed stress tolerance.

A 21-kDa chloroplast heat shock protein assembles into high molecular weight complexes in vivo and in Organelle.

The conservation of the carboxyl-terminal "heat shock" domain among small (sm) cytoplasmic and chloroplast heat shock proteins (HSPs) suggests that these smHSPs perform similar functions. Previous studies have established that cytoplasmic smHSPs are found in higher order structures in vivo (approximately 500 kDa). To determine if the chloroplast smHSP is found in similar complexes, we examined the size of the 21-kDa chloroplast smHSP from Pisum sativum, PsHSP21, under non-denaturing conditions. Following sedimentation of chloroplast stromal extracts on sucrose gradients PsHSP21 is detected in fractions corresponding to 10-11 S. Upon non-denaturing gel electrophoresis, PsHSP21 was detected in two high molecular mass complexes of approximately 230 and 200 kDa, in good agreement with the sucrose gradient data. These PsHSP21-containing particles were stable under different salt and Mg2+ conditions, and their integrity was not affected by 1.0% Triton X-100 or 10 mM ATP. To study assembly of the high molecular weight complexes containing PsHSP21, in vitro translated PsHSP21 was imported into chloroplasts and its size was examined. Following import into chloroplasts isolated from heat-stressed plants, greater than 50% of PsHSP21 was recovered in the higher molecular weight forms. In contrast, following import into chloroplasts isolated from control plants the protein was recovered exclusively in a 5 S (approximately 42-kDa) form. These data suggest that preexisting PsHSP21 or other heat-induced factors may be required for assembly of the higher molecular weight particles. We propose that the 10-11 S particles are the functional form of PsHSP21.

Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress.

The ability of organisms to acquire thermotolerance to normally lethal high temperatures is an ancient and conserved adaptive response. However, knowledge of cellular factors essential to this response is limited. Acquisition of thermotolerance is likely to be of particular importance to plants that experience daily temperature fluctuations and are unable to escape to more favorable environments. We developed a screen, based on hypocotyl elongation, for mutants of Arabidopsis thaliana that are unable to acquire thermotolerance to high-temperature stress and have defined four separate genetic loci, hot1-4, required for this process. hot1 was found to have a mutation in the heat shock protein 101 (Hsp101) gene, converting a conserved Glu residue in the second ATP-binding domain to a Lys residue, a mutation that is predicted to compromise Hsp101 ATPase activity. In addition to exhibiting a thermotolerance defect as assayed by hypocotyl elongation, 10-day-old hot1 seedlings were also unable to acquire thermotolerance, and hot1 seeds had greatly reduced basal thermotolerance. Complementation of hot1 plants by transformation with wild-type Hsp101 genomic DNA restored hot1 plants to the wild-type phenotype. The hot mutants are the first mutants defective in thermotolerance that have been isolated in a higher eukaryote, and hot1 represents the first mutation in an Hsp in any higher plant. The phenotype of hot1 also provides direct evidence that Hsp101, which is required for thermotolerance in bacteria and yeast, is also essential for thermotolerance in a complex eukaryote.

A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein.

Small heat shock proteins (sHsps) are a diverse group of heat-induced proteins that are conserved in prokaryotes and eukaryotes and are especially abundant in plants. Recent in vitro data indicate that sHsps act as molecular chaperones to prevent thermal aggregation of proteins by binding non-native intermediates, which can then be refolded in an ATP-dependent fashion by other chaperones. We used heat-denatured firefly luciferase (Luc) bound to pea (Pisum sativum) Hsp18.1 as a model to define the minimum chaperone system required for refolding of a sHsp-bound substrate. Heat-denatured Luc bound to Hsp18.1 was effectively refolded either with Hsc/Hsp70 from diverse eukaryotes plus the DnaJ homologs Hdj1 and Ydj1 (maximum = 97% Luc reactivation with k(ob) = 1.0 x 10(-2)/min), or with prokaryotic Escherichia coli DnaK plus DnaJ and GrpE (100% Luc reactivation, k(ob) = 11.3 x 10(-2)/min). Furthermore, we show that Hsp18.1 is more effective in preventing Luc thermal aggregation than the Hsc70 or DnaK systems, and that Hsp18.1 enhances the yields of refolded Luc even when other chaperones are present during heat inactivation. These findings integrate the aggregation-preventive activity of sHsps with the protein-folding activity of the Hsp70 system and define an in vitro system for further investigation of the mechanism of sHsp action.