<Emphasis Type="Italic">Arabidopsis</Emphasis> <Emphasis Type="Italic">thaliana</Emphasis> expressing a thermostable chimeric Rubisco activase exhibits enhanced growth and higher rates of photosynthesis at moderately high temperatures

Temperature is one of the most important factors controlling growth, development, and reproduction in plants. The rate of photosynthesis declines at moderately high temperatures in plants and particularly in temperate species like Arabidopsis thaliana. This can be attributed to a reduced ability of Rubisco activase to achieve optimum activation of Rubisco, leading to reduced Rubisco activity. In order to overcome this problem, we transformed the Arabidopsis rca mutant with a more thermostable, chimeric activase where a Rubisco recognition domain in the more thermostable tobacco activase was replaced with that from Arabidopsis. Transgenic lines expressing this activase showed higher rates of photosynthesis than the wild type after a short exposure to higher temperatures and they also recovered better, when they were returned to the normal temperature. Moreover, under extended exposure to moderately elevated temperature, the transgenic lines had higher biomass and seed yield when compared with the wild type plants.     

Enhanced Thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress

Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Deltarca) line. In a long-term growth test at either constant 26 degrees C or daily 4-h 30 degrees C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions.     

Introduction of the carrot HSP17.7 into potato (Solanum tuberosum L.) enhances cellular membrane stability and tuberization in vitro

We have examined the ability of a carrot (Daucus carota L.) heat shock protein gene encoding HSP17.7 (DcHSP17.7) to confer enhanced heat tolerance to potato (Solanum tuberosum L.), a cool-season crop. The DcHSP17.7 gene was fused to a 6XHistidine (His) tag to distinguish the engineered protein from endogenous potato proteins and was introduced into the potato cultivar 'Désirée' under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Western analysis showed that engineered DcHSP17.7 was constitutively, but not abundantly, expressed in transgenic potato lines before heat stress. Leaves from multiple regenerated potato lines that contain the transgene exhibited significantly improved cellular membrane stability at high temperatures, compared with wild-type and vector control plants. Transgenic potato lines also exhibited enhanced tuberization in vitro: under a condition of constant heat stress, at 29 degrees C, nodal sections of the transgenic lines produced larger and heavier microtubers at higher rates, compared to the wild type and vector controls. The dry weight and percentages of microtubers that were longer than 5 mm were up to three times higher in the transgenic lines. Our results suggest that constitutive expression of carrot HSP17.7 can enhance thermotolerance in transgenic potato plants. To our knowledge, this is the first study that shows that the thermotolerance of potato can be enhanced through gene transfer.     

Heat causes oligomeric disassembly and increases the chaperone activity of small heat shock proteins from sugarcane

Small heat shock proteins (sHsp) constitute an important chaperone family linked to conformational diseases. In plants, sHsps prevent protein aggregation by acting as thermosensors and to enhance cell stress tolerance. SsHsp17.2 and SsHsp17.9 are the most highly expressed class I sHsps in sugarcane. They exist as dodecamers at 20 °C and have distinct substrate specificities. Therefore, they are useful models to study how class I SHsps work. Here we present data on the effects of heat on the oligomerization and chaperone activity of SsHsp17.2 and SsHsp17.9. Using several biophysical and biochemical probes, we show that the effects of heat are completely reversible, an important property for proteins that act at heat shock temperatures. SsHsp17.2 and SsHsp17.9 dodecamers dissociated to dimers at temperatures ranging from 40 to 45 °C and this dissociation was followed by enhanced chaperone activity. We conclude that high temperature affects the oligomeric state of these chaperones, resulting in enhanced chaperone activity.     

The CNGCb and CNGCd genes from Physcomitrella patens moss encode for thermosensory calcium channels responding to fluidity changes in the plasma membrane

Land plants need precise thermosensors to timely establish molecular defenses in anticipation of upcoming noxious heat waves. The plasma membrane-embedded cyclic nucleotide-gated Ca²⁺ channels (CNGCs) can translate mild variations of membrane fluidity into an effective heat shock response, leading to the accumulation of heat shock proteins (HSP) that prevent heat damages in labile proteins and membranes. Here, we deleted by targeted mutagenesis the CNGCd gene in two Physcomitrella patens transgenic moss lines containing either the heat-inducible HSP-GUS reporter cassette or the constitutive UBI-Aequorin cassette. The stable CNGCd knockout mutation caused a hyper-thermosensitive moss phenotype, in which the heat-induced entry of apoplastic Ca²⁺ and the cytosolic accumulation of GUS were triggered at lower temperatures than in wild type. The combined effects of an artificial membrane fluidizer and elevated temperatures suggested that the gene products of CNGCd and CNGCb are paralogous subunits of Ca²⁺channels acting as a sensitive proteolipid thermocouple. Depending on the rate of temperature increase, the duration and intensity of the heat priming preconditions, terrestrial plants may thus acquire an array of HSP-based thermotolerance mechanisms against upcoming, otherwise lethal, extreme heat waves.     

Replicon-specific regulation of small heat shock genes in Agrobacterium tumefaciens

Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (sigma32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5' untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive.     

Small heat shock proteins prevent aggregation of citrate synthase and bind to the N-terminal region which is absent in thermostable forms of citrate synthase

Citrate synthase (CS) is often used in chaperone assays since this thermosensitive enzyme aggregates at moderately increased temperatures. Small heat shock proteins (sHsps) are molecular chaperones specialized in preventing the aggregation of other proteins, termed substrate proteins, under conditions of transient heat stress. To investigate the mechanism whereby sHsps bind to and stabilize a substrate protein, we here used peptide array screening covering the sequence of porcine CS (P00889). Strong binding of sHsps was detected to a peptide corresponding to the most N-terminal α-helix in CS (amino acids Leu₁₃ to Gln₂₇). The N-terminal α-helices in the CS dimer intertwine with the C-terminus in the other subunit and together form a stem-like structure which is protruding from the CS dimer. This stem-like structure is absent in thermostable forms of CS from thermophilic archaebacteria like Pyrococcus furiosus and Sulfolobus solfatacarium. These data therefore suggest that thermostabilization of thermosensitive CS by sHsps is achieved by stabilization of the C- and N-terminae in the protruding thermosensitive softspot, which is absent in thermostable forms of the CS dimer.     

The chloroplast‐localized small heat shock protein Hsp21 associates with the thylakoid membranes in heat‐stressed plants

The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity of sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps with a single conserved β‐sandwich fold domain defining the family, the α‐crystallin domain, whereas the N‐terminal and C‐terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non‐metazoan sHsp expressed in the chloroplasts in green plants which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat‐stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic ¹⁵N‐isotope labeling of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat‐stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered.     

Enhancement of Reproductive Heat Tolerance in Plants

Comparison of average crop yields with reported record yields has shown that major crops exhibit annual average yields three- to seven-fold lower than record yields because of unfavorable environments. The current study investigated the enhancement of pollen heat tolerance through expressing an Arabidopsis thaliana heat shock protein 101 (AtHSP101) that is not normally expressed in pollen but reported to play a crucial role in vegetative thermotolerance. The AtHSP101 construct under the control of the constitutive ocs/mas ‘superpromoter’ was transformed into cotton Coker 312 and tobacco SRI lines via Agrobacterium mediated transformation. Thermotolerance of pollen was evaluated by in vitro pollen germination studies. Comparing with those of wild type and transgenic null lines, pollen from AtHSP101 transgenic tobacco and cotton lines exhibited significantly higher germination rate and much greater pollen tube elongation under elevated temperatures or after a heat exposure. In addition, significant increases in boll set and seed numbers were also observed in transgenic cotton lines exposed to elevated day and night temperatures in both greenhouse and field studies. The results of this study suggest that enhancing heat tolerance of reproductive tissues in plant holds promise in the development of crops with improved yield production and yield sustainability in unfavorable environments.     

Microbial small heat shock proteins and their use in biotechnology

Small heat shock proteins (sHsps) exist in almost all organisms. Most organisms have more than one sHsp, but their number can be as high as 65, as found in the eukaryote, Vitis vinifera. The function of sHsps is well-known; they confer thermotolerance to cellular cultures and proteins in cellular extracts during prolonged incubations at elevated temperatures. This demonstrates the ability of sHsps to protect cellular proteins, and to maintain cellular viability under conditions of intensive stress, such as heat shock or chemical treatments. sHsps have several properties that distinguish them from heat shock proteins (Hsps): they function as ATP-independent chaperones, require the flexible assembly and reassembly of oligomeric complex structures for their activation, and exhibit a wide range of substrate-binding capacities. Recent studies indicate that sHsps have important biological functions in thermostability, disaggregation, and proteolysis inhibition. These functions can be harnessed for various applications, including nanobiotechnology, proteomics, bioproduction, and bioseparation. In this review, we discuss the properties and diversity of microbial sHsps, as well as their potential uses in the biotechnology industry.     

Two Rubisco activase isoforms may play different roles in photosynthetic heat acclimation in the rice plant

Studies on some plant species have shown that increasing the growth temperature gradually or pretreating with high temperature can lead to obvious photosynthetic acclimation to high temperature. To test whether this acclimation arises from heat adaptation of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activation mediated by Rubisco activase (RCA), gene expression of RCA large isoform (RCA_L) and RCA small isoform (RCA_S) in rice was determined using a 4‐day heat stress treatment [40/30°C (day/night)] followed by a 3‐day recovery under control conditions [30/22°C (day/night)]. The heat stress significantly induced the expression of RCA_L as determined by both mRNA and protein levels. Correlative analysis indicated that RCA_S protein content was extremely significantly related to Rubisco initial activity and net photosynthetic rate (Pn) under both heat stress and normal conditions. Immunoblot analysis of the Rubisco–RCA complex revealed that the ratio of RCA_L to Rubisco increased markedly in heat‐acclimated rice leaves. Furthermore, transgenic rice plants expressing enhanced amounts of RCA_L exhibited higher thermotolerance in Pn and Rubisco initial activity and grew better at high temperature than wild‐type (WT) plants and transgenic rice plants expressing enhanced amounts of RCA_S. Under normal conditions, the transgenic rice plants expressing enhanced amounts of RCA_S showed higher Pn and produced more biomass than transgenic rice plants expressing enhanced amounts of RCA_L and wild‐type plants. Together, these suggest that the heat‐induced RCA_L may play an important role in photosynthetic acclimation to moderate heat stress in vivo, while RCA_S plays a major role in maintaining Rubisco initial activity under normal conditions.     

Overexpression of tomato <Emphasis Type="Italic">tAPX</Emphasis> gene in tobacco improves tolerance to high or low temperature stress

In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants.     

Involvement of small heat shock proteins,trehalose, and lipids in the thermal stress management in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Changes in the levels of three structurally and functionally different important thermoprotectant molecules, namely small heat shock proteins (sHsps), trehalose, and lipids, have been investigated upon heat shock in Schizosaccharomyces pombe. Both α-crystallin-type sHsps (Hsp15.8 and Hsp16) were induced after prolonged high-temperature treatment but with different kinetic profiles. The shsp null mutants display a weak, but significant, heat sensitivity indicating their importance in the thermal stress management. The heat induction of sHsps is different in wild type and in highly heat-sensitive trehalose-deficient (tps1Δ) cells; however, trehalose level did not show significant alteration in shsp mutants. The altered timing of trehalose accumulation and induction of sHsps suggest that the disaccharide might provide protection at the early stage of the heat stress while elevated amount of sHsps are required at the later phase. The cellular lipid compositions of two different temperature-adapted wild-type S. pombe cells are also altered according to the rule of homeoviscous adaptation, indicating their crucial role in adapting to the environmental temperature changes. Both Hsp15.8 and Hsp16 are able to bind to different lipids isolated from S. pombe, whose interaction might provide a powerful protection against heat-induced damages of the membranes. Our data suggest that all the three investigated thermoprotectant macromolecules play a pivotal role during the thermal stress management in the fission yeast.     

The identification and characterization of IbpA,a novel α-crystallin-type heat shock protein from mycoplasma

α-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.