Chloroplast Heat Shock Protein Research Articles

Molecular and Biological Characteristics of a Peach Latent Mosaic Viroid PC Isolate in Peach from China: Base Mutations in Hairpin Stems and Implications for Symptomatology.

Peach latent mosaic viroid (PLMVd) infects peach trees in China and induces a conspicuous albino phenotype (peach calico, PC) that is closely associated with variants containing a 12-to-14 nucleotide hairpin insertion capped by a U-rich loop. Initially, PC disease distribution was limited to parts of Italy, and it was first detected in the field in China in 2019. To explore the molecular and biological characteristics of PLMVd PC isolates in peach in China, we conducted a comprehensive analysis of disease phenotype development and investigated the data-associated pathogenicity and in vivo dynamics of the Chinese isolate PC-A2 using slash-inoculation into GF-305 peach seedlings. Inoculated seedlings displayed PC symptoms much earlier following topping treatment, and PLMVd infectivity was further assessed using bioassay and semiquantitative RT-PCR experiments. Evolutionary analysis showed that the PC isolate and its progeny variants clustered into a single phylogroup distinct from reference PC-C40 isolates from Italy and PC-K1 and PC-K2 from South Korea. Some PC-A2 progeny variants from green leaves of PC-expressing seedlings showed unbalanced point mutations in hairpin stems compared with the PC-C40 reference sequence and constituted a new stem insertion type. The results reveal associations between the recessive phenotypes of peach albino symptoms and base variation in hairpin stem insertions relative to the PC-C40/chloroplastic heat shock protein 90 reference sequence.

Characterization of cellular toxicity induced by sub-lethal inorganic mercury in the marine microalgae Chlorococcum dorsiventrale isolated from a metal-polluted coastal site

Mercury (Hg) is a global pollutant that affects numerous marine aquatic ecosystems. We isolated Chlorococcum dorsiventrale Ch-UB5 microalga from coastal areas of Tunisia suffering from metal pollution and analyzed its tolerance to Hg. This strain accumulated substantial amounts of Hg and was able to remove up to 95% of added metal after 24 and 72 h in axenic cultures. Mercury led to lesser biomass growth, higher cell aggregation, significant inhibition of photochemical activity, and appearance of oxidative stress and altered redox enzymatic activities, with proliferation of starch granules and neutral lipids vesicles. Such changes matched the biomolecular profile observed using Fourier Transformed Infrared spectroscopy, with remarkable spectral changes corresponding to lipids, proteins and carbohydrates. C. dorsiventrale accumulated the chloroplastic heat shock protein HSP70B and the autophagy-related ATG8 protein, probably to counteract the toxic effects of Hg. However, long-term treatments (72 h) usually resulted in poorer physiological and metabolic responses, associated with acute stress. C. dorsiventrale has potential use for Hg phycoremediation in marine ecosystems, with the ability to accumulating energetic reserves that could be used for biofuel production, supporting the notion of using of C. dorsiventrale for sustainable green chemistry in parallel to metal removal.

DJC78 is a cochaperone that interacts with cpHsc70-1 in the chloroplasts

Heat shock proteins 70 (HSP70s) could cooperate with structurally diverse HSP40s (J proteins) to generate diverse chaperone networks in various cellular compartments, performing multiple housekeeping and stress-related functions in the organisms. There are two kinds of chloroplast heat shock protein 70 (cpHsc70-1, cpHsc70-2) and multiple J proteins in the Arabidopsis chloroplasts, while the interaction between cpHsc70s and J proteins and the function of most J proteins are largely unknown. In the present study, we found that AtDJC78 interacts with cpHsc70-1 through its C terminal, according to the results of yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). Bioinformatics analysis showed that DJC78 is one of the widespread and highly conserved J proteins in plants, AtDJC78 could be transported into chloroplasts, and the expression of AtDJC78 was significantly up-regulated under heat stress. Furthermore, we found that AtDJC78 may be associated with regulating hydrogen peroxide levels under heat stress in plants. These findings suggest that AtDJC78 is a new cochaperone interacting with cpHsc70-1 in the chloroplasts.

Melatonin Regulates Chloroplast Protein Quality Control via a Mitogen-Activated Protein Kinase Signaling Pathway.

Serotonin N-acetyltransferase 1 (SNAT1), the penultimate enzyme for melatonin biosynthesis has shown N-acetyltransferase activity toward multiple substrates, including histones, serotonin, and plastid proteins. Under two different light conditions such as 50 or 100 μmol m−2 s−1, a SNAT1-knockout (snat1) mutant of Arabidopsis thaliana ecotype Columbia (Col-0) exhibited small size phenotypes relative over wild-type (WT) Arabidopsis Col-0. Of note, the small phenotype is stronger when growing at the 50 μmol m−2 s−1, exhibiting a dwarfism phenotype and delayed flowering. The snat1 Arabidopsis Col-0 accumulated less starch than the WT Col-0. Moreover, snat1 exhibited lower Lhcb1, Lhcb4, and RBCL protein levels, compared with the WT Col-0, but no changes in the corresponding transcripts, suggesting the involvement of melatonin in chloroplast protein quality control (CPQC). Accordingly, caseinolytic protease (Clp) and chloroplast heat shock proteins (CpHSPs), two key proteins involved in CPQC, as well as ROS defense were suppressed in snat1. In contrast, exogenous melatonin treatment induced expression of Clp, CpHSP, APX1, and GST, but not other growth-related genes such as DWF4, KS, and IAA1. Finally, the induction of ClpR1, APX1, and GST1 in response to melatonin was inhibited in the mitogen-activated protein kinase (MAPK) knockdown Arabidopsis (mpk3/6), suggesting that melatonin-mediated CPQC was mediated, in part, by the MAPK signaling cascade. These results suggest that melatonin is involved in CPQC, which plays a pivotal role in starch synthesis in plants.

A chloroplast heat shock protein modulates growth and abiotic stress response in creeping bentgrass.

Small heat shock proteins (sHSPs), a family of the ubiquitous stress proteins in plants acting as molecular chaperones to protect other proteins from stress-induced damage, have been implicated in plant growth and development as well as plant response to environmental stress, especially heat stress. In this study, a chloroplast-localized sHSP, AsHSP26.8, was overexpressed in creeping bentgrass (Agrostis stolonifera L.) to study its role in regulating plant growth and stress response. Transgenic (TG) creeping bentgrass plants displayed arrested root development, slow growth rate, twisted leaf blades and are more susceptible to heat and salt but less sensitive to drought stress compared to wild-type (WT) controls. RNA-seq analysis revealed that AsHSP26.8 modulated the expression of genes in auxin signalling and stress-related genes such as those encoding HSPs, heat shock factors and other transcription factors. Our results provide new evidence demonstrating that AsHSP26.8 negatively regulates plant growth and development and plays differential roles in plant response to a plethora of diverse abiotic stresses.

The Chloroplastic Small Heat Shock Protein Gene KvHSP26 Is Induced by Various Abiotic Stresses in Kosteletzkya virginica.

Small heat shock proteins (sHSPs) are a group of chaperone proteins existed in all organisms. The functions of sHSPs in heat and abiotic stress responses in many glycophyte plants have been studied. However, their possible roles in halophyte plants are still largely known. In this work, a putative sHSP gene KvHSP26 was cloned from K. virginica. Bioinformatics analyses revealed that KvHSP26 encoded a chloroplastic protein with the typical features of sHSPs. Amino acid sequence alignment and phylogenetic analysis demonstrated that KvHSP26 shared 30%-77% homology with other sHSPs from Arabidopsis, cotton, durian, salvia, and soybean. Quantitative real-time PCR (qPCR) assays exhibited that KvHSP26 was constitutively expressed in different tissues such as leaves, stems, and roots, with a relatively higher expression in leaves. Furthermore, expression of KvHSP26 was strongly induced by salt, heat, osmotic stress, and ABA in K. virginica. All these results suggest that KvHSP26 encodes a new sHSP, which is involved in multiple abiotic stress responses in K. virginica, and it has a great potential to be used as a candidate gene for the breeding of plants with improved tolerances to various abiotic stresses.

Salt stress induced effects on biochemical parameters in etiolated maize leaf segments during greening

Treatment of etiolated maize leaf segments with 0-300 mM NaCl during greening decreased the osmotic potential of leaf sap significantly. Na+ content of the leaf tissue increased significantly on supplying different concentrations of salt. Relative water content of the leaf tissue was marginally affected by the supply of NaCl. Total protein and RNA content of the maize leaf segments gradually increased when treated with lower concentrations of NaCl and subsequently decreased at higher concentrations. Decrease in DNA content was noted with increasing salt treatment in a concentration dependent manner. SDS-PAGE analysis of salt treated maize leaf tissue revealed appearance of one protein band (approx. 73 kD) in samples incubated with ≥50 mM NaCl compared to control. Protein identification performed with the Mascot search engine in NCBI database indicated extensive homology of this protein with chloroplast heat shock protein 70 of Cenchrus americanus (Protein score: 295), hypothetical proteins SELMODRAFT_267815 of Selaginella moellendorffii (Protein score: 125), VITISV_000728 of Vitis vinifera (Protein score: 118) and SELMODRAFT_230659 of Selaginella moellendorffii (Protein score: 51). The results revealed that the dark grown maize leaf segments exhibit high degree of stress due to NaCl treatment, which affects the biochemical parameters governing the metabolic activities of the leaf tissue. Further, the stress induced proteins are being synthesized in leaf tissue in response to salt stress.

Identification of HSP90C as a substrate of E3 ligase TaSAP5 through ubiquitylome profiling

Protein ubiquitination is a major post-translational modification important for diverse biological processes. In wheat (Triticum aestivum) and Arabidopsis thaliana, STRESS-ASSOCIATED PROTEIN 5 (SAP5) is involved in drought tolerance, acting as an E3 ubiquitin ligase to target DRIP and MBP-1 for degradation. To identify further target proteins of SAP5, we implemented two independent approaches in this work. We used ubiquitylome capture with a di-Gly-Lys antibody-based peptide enrichment and affinity purification with a polyubiquitin antibody coupled with mass spectrometry to elucidate the SAP5-dependent ubiquitylation of its target proteins in response to osmotic stress. Wild type or TaSAP5-overexpressing Arabidopsis line, which was more tolerant to osmotic stress according to our previous study, were used here. We identified HSP90C (chloroplast heat shock protein 90) as a substrate of TaSAP5. Further biochemical experiments indicated that TaSAP5 interacts with HSP90C and mediates its degradation by the 26S proteasome. Our work also demonstrates that ubiquitylome profiling is an effective approach to search for substrates of the TaSAP5 E3 ubiquitin ligase when heterologously expressed in Arabidopsis.

Novel DnaJ Protein Facilitates Thermotolerance of Transgenic Tomatoes.

DnaJ proteins, which are molecular chaperones that are widely present in plants, can respond to various environmental stresses. At present, the function of DnaJ proteins was studied in many plant species, but only a few studies were conducted in tomato. Here, we examined the functions of a novel tomato (Solanum lycopersicum) DnaJ protein (SlDnaJ20) in heat tolerance using sense and antisense transgenic tomatoes. Transient conversion assays of Arabidopsis protoplasts showed that SlDnaJ20 was targeted to chloroplasts. Expression analysis showed that SlDnaJ20 expression was induced by chilling, NaCl, polyethylene glycol, and H2O2, especially via heat stress. Under heat stress, sense plants showed higher fresh weights, chlorophyll content, fluorescence (Fv/Fm), and D1 protein levels, and a lower accumulation of reactive oxygen species (ROS) than antisense plants. These results suggest that SlDnaJ20 overexpression can reduce the photoinhibition of photosystem II (PSII) by relieving ROS accumulation. Moreover, higher expression levels of HsfA1 and HsfB1 were observed under heat stress in sense plants, indicating that SlDnaJ20 overexpression contributes to HSF expression. The yeast two-hybrid system proved that SlDnaJ20 can interact with the chloroplast heat-shock protein 70. Our results indicate that SlDnaJ20 overexpression enhances the thermotolerance of transgenic tomatoes, whereas suppression of SlDnaJ20 increases the heat sensitivity of transgenic tomatoes.

Inducible transgenic tobacco system to study the mechanisms underlying chlorosis mediated by the silencing of chloroplast heat shock protein 90.

Chlorosis is one of the most common symptoms of plant diseases, including those caused by viruses and viroids. Recently, a study has shown that Peach latent mosaic viroid (PLMVd) exploits host RNA silencing machinery to modulate the virus disease symptoms through the silencing of chloroplast-targeted heat shock protein 90 (Hsp90C). To understand the molecular mechanisms of chlorosis in this viroid disease, we established an experimental system suitable for studying the mechanism underlying the chlorosis induced by the RNA silencing of Hsp90C in transgenic tobacco. Hairpin RNA of the Hsp90C-specific region was expressed under the control of a dexamethasone-inducible promoter, resulted in the silencing of Hsp90C gene in 2days and the chlorosis along with growth suppression phenotypes. Time course study suggests that a sign of chlorosis can be monitored as early as 2days, suggesting that this experimental model is suitable for studying the molecular events taken place before and after the onset of chlorosis. During the early phase of chlorosis development, the chloroplast- and photosynthesis-related genes were downregulated. It should be noted that some pathogenesis related genes were upregulated during the early phase of chlorosis in spite of the absence of any pathogen-derived molecules in this system.

Callose Deposition in Plasmodesmata and Viroid Invasion of the Shoot Apical Meristem.

Callose Deposition in Plasmodesmata and Viroid Invasion of the Shoot Apical Meristem.

A tomato chloroplast-targeted DnaJ protein protects Rubisco activity under heat stress.

Photosynthesis is one of the biological processes most sensitive to heat stress in plants. Carbon assimilation, which depends on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is one of the major sites sensitive to heat stress in photosynthesis. In this study, the roles of a tomato (Solanum lycopersicum) chloroplast-targeted DnaJ protein (SlCDJ2) in resisting heat using sense and antisense transgenic tomatoes were examined. SlCDJ2 was found to be uniformly distributed in the thylakoids and stroma of the chloroplasts. Under heat stress, sense plants exhibited higher chlorophyll contents and fresh weights, and lower accumulation of reactive oxygen species (ROS) and membrane damage. Moreover, Rubisco activity, Rubisco large subunit (RbcL) content, and CO2 assimilation capacity were all higher in sense plants and lower in antisense plants compared with wild-type plants. Thus, SlCDJ2 contributes to maintenance of CO2 assimilation capacity mainly by protecting Rubisco activity under heat stress. SlCDJ2 probably achieves this by keeping the levels of proteolytic enzymes low, which prevents accelerated degradation of Rubisco under heat stress. Furthermore, a chloroplast heat-shock protein 70 was identified as a binding partner of SlCDJ2 in yeast two-hybrid assays. Taken together, these findings establish a role for SlCDJ2 in maintaining Rubisco activity in plants under heat stress.

A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress

DnaJ proteins act as essential molecular chaperones in protein homeostasis and protein complex stabilization under stress conditions. The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1), whose expression was upregulated by treatment at 4 and 42 °C, and with high light, NaCl, polyethylene glycol, and H2O2, were investigated here using sense and antisense transgenic tomatoes. The sense plants exhibited not only higher chlorophyll content, fresh weight and net photosynthetic rate, but also lower accumulation of reactive oxygen species and membrane damage under chilling stress. Moreover, the maximal photochemistry efficiency of photosystem II (PSII) (F v/F m) and D1 protein content were higher in the sense plants and lower in the antisense plants, and the photoinhibitory quenching was lower in the sense plants and higher in the antisense plants, suggesting that the inhibition of PSII was less severe in the sense plants and more severe in the antisense plants compared with the wild type. Furthermore, the PSII protein complexes were also more stable in the sense plants. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher F v/F m, D1 protein content and PSII stability than the SM-untreated antisense plants. This finding suggested that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, chloroplast heat-shock protein 70 was identified as the partner of LeCDJ1. These results indicate that LeCDJ1 has essential functions in maintaining PSII under chilling stress.

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts.

Chloroplast heat shock protein 90 (Hsp90C) represents a highly conserved subfamily of the Hsp90 family of molecular chaperones whose function has not been defined. We identified Hsp90C as a component that interacts with import intermediates of nuclear-encoded preproteins during posttranslational import into isolated chloroplasts. Hsp90C was specifically coprecipitated with a complex of protein import components, including Tic110, Tic40, Toc75, Tic22, and the stromal chaperones, Hsp93 and Hsp70. Radicicol, an inhibitor of Hsp90 ATPase activity, reversibly inhibited the import of a variety of preproteins during translocation across the inner envelope membrane, indicating that Hsp90C functions in membrane translocation into the organelle. Hsp90C is encoded by a single gene in Arabidopsis thaliana, and insertion mutations in the Hsp90C gene are embryo lethal, indicating an essential function for the chaperone in plant viability. On the basis of these results, we propose that Hsp90C functions within a chaperone complex in the chloroplast stroma to facilitate membrane translocation during protein import into the organelle.

The wheat chloroplastic small heat shock protein (sHSP26) is involved in seed maturation and germination and imparts tolerance to heat stress

The nuclear-encoded chloroplast small heat shock proteins (sHSPs) are present in all plant species from algae to angiosperms. Expression analysis shows that the wheat chloroplastic sHSP (HSP26) is highly inducible by heat stress in almost all the vegetative and generative tissues and is also expressed constitutively in certain developmental growth stages. We characterize wheat chloroplastic sHSP 26 through transgenic approach using Arabidopsis and report cloning of the promoter and its characterization. Transgenic Arabidopsis plants were substantially tolerant under continuous high temperature regimen than wild-type plants, as measured by photosystem II (PSII) activity, accumulation of more photosynthetic pigments, higher biomass and seed yield. Transgenic plants produced bold seeds under high temperature, having higher germination potential than the wild-type plants. Further, antisense Arabidopsis plants showed negligible tolerance even for non-lethal heat shock, impaired in basal thermo-tolerance, and accumulated less biomass and seed yield under normal growth conditions. Promoter analysis revealed the presence of several heat and other abiotic stress responsive cis-acting elements along with developmental stage and tissue-specific elements. Analysis of promoter through GUS reporter system in both transgenic rice and Arabidopsis further confirms the role of chloroplastic sHsp26 in heat and other abiotic stresses as well as during seed maturation and germination. Genome-wide expression analysis of overexpression Arabidopsis plants revealed that the transcriptome remained unchanged in the transgenic plants and the tolerance was due to the overexpression of chloroplastic heat shock protein (HSP) only.

Small RNAs containing the pathogenic determinant of a chloroplast‐replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing

How viroids, tiny non-protein-coding RNAs (~250-400 nt), incite disease is unclear. One hypothesis is that viroid-derived small RNAs (vd-sRNAs; 21-24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast-replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12-14-nt hairpin insertion. By dissecting albino and green leaf sectors of Prunus persica (peach) seedlings inoculated with PLMVd natural and artificial variants, and cloning their progeny, we have established that the hairpin insertion sequence is involved in PC. Furthermore, using deep sequencing, semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends (RACE), we have determined that two PLMVd-sRNAs containing the PC-associated insertion (PC-sRNA8a and PC-sRNA8b) target for cleavage the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90), thus implicating RNA silencing in the modulation of host gene expression by a viroid. Chloroplast malformations previously reported in PC-expressing tissues are consistent with the downregulation of cHSP90, which participates in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis. Besides PC-sRNA8a and PC-sRNA8b, both deriving from the less-abundant PLMVd (-) strand, we have identified other PLMVd-sRNAs potentially targeting peach mRNAs. These results also suggest that sRNAs derived from other PLMVd regions may downregulate additional peach genes, ultimately resulting in other symptoms or in a more favorable host environment for viroid infection.

Viroids: How to infect a host and cause disease without encoding proteins

Despite being composed by a single-stranded, circular, non-protein-coding RNA of just 246-401 nucleotides (nt), viroids can incite in their host plants symptoms similar to those caused by DNA and RNA viruses, which have genomes at least 20-fold bigger and encode proteins. On the other hand, certain non-protein-coding plant satellite RNAs display structural similarities with viroids but for replication and transmission they need to parasitize specific helper viruses (modifying concomitantly the symptoms they induce). While phenotypic alterations accompanying infection by viruses may partly result from expressing the proteins they code for, how the non-protein-coding viroids (and satellite RNAs) cause disease remains a conundrum. Initial ideas on viroid pathogenesis focused on a direct interaction of the genomic RNA with host proteins resulting in their malfunction. With the advent of RNA silencing, it was alternatively proposed that symptoms could be produced by viroid-derived small RNAs (vd-sRNAs) -generated by the host defensive machinery- targeting specific host mRNA or DNA sequences for post-transcriptional or transcriptional gene silencing, respectively, a hypothesis that could also explain pathogenesis of non-protein-coding satellite RNAs. Evidence sustaining this view has been circumstantial, but recent data provide support for it in two cases: i) the yellow symptoms associated with a specific satellite RNA result from a 22-nt small RNA (derived from the 24-nt fragment of the satellite genome harboring the pathogenic determinant), which is complementary to a segment of the mRNA of the chlorophyll biosynthetic gene CHLI and targets it for cleavage by the RNA silencing machinery, and ii) two 21-nt vd-sRNAS containing the pathogenic determinant of the albino phenotype induced by a chloroplast-replicating viroid target for cleavage the mRNA coding for the chloroplastic heat-shock protein 90 via RNA silencing too. This evidence, which is compelling for the satellite RNA, does not exclude alternative mechanisms.

The ectopic expression of methionine sulfoxide reductase 4 results in enhanced growth performance in arabidopsis

In response to challenging oxidative environments, most aerobic organisms have developed cellular defense systems that either repair or degrade damaged proteins to prevent them from adversely affecting cellular reactions. For instance, oxidized methionine can be reduced to methionine by the enzyme methionine sulfoxide reductase (MSR). Previously, we reported that the Arabidopsis gene MSR4 (AT4G25130) is induced by salt stress, and during our investigations into this gene, we generated a transgenic Arabidopsis that overexpressed a mutant form of MSR4 ( mMSR4). The transgenic plants exhibited enhanced sensitivity to high salt stress. Consistent with this observation, the ectopic expression of MSR4 enhanced growth performance in normal as well as in stress medium. To determine the targets of MSR4, we performed two-dimensional electrophoresis analyses and observed a significant reduction in the levels of chloroplast heat shock proteins, which may explain the enhanced salt stress sensitivity of mMSR4 transgenic plants.

Chloroplast heat shock protein Cpn60 from Chlamydomonas reinhardtii exhibits a novel function as a group II intron-specific RNA-binding protein

Intron-binding proteins in eukaryotic organelles are mainly encoded by the nuclear genome and are thought to promote the maturation of precursor RNAs. Here, we present a biochemical approach that enable the isolation of a novel nuclear-encoded protein from Chlamydomonas reinhardtii showing specific binding properties to organelle group II intron RNA. Using FPLC chromatography of chloroplast protein extracts, a 61-kDa RNA-binding protein was isolated and then tentatively identified by mass spectrometry as the chloroplast heat shock protein Cpn60. Heterologous Cpn60 protein was used in RNA protein gel mobility shift assays and revealed that the ATPase domains of Cpn60 mediates the specific binding of two group II intron RNAs, derived from the homologous chloroplast psaA gene and the heterologous mitochondrial LSU rRNA gene. The function of Cpn60 as a general organelle splicing factor is discussed.

Heat sensitivity in a bentgrass variant. Failure to accumulate a chloroplast heat shock protein isoform implicated in heat tolerance.

Two variants of creeping bentgrass (Agrostis stolonifera cv palustris), developed using tissue culture, have been used to determine the roles of chloroplast-localized small heat shock proteins (CP-sHSPs) in heat tolerance. Results from previous research indicate that the heat-tolerant variant expressed two additional CP-sHSP isoforms not expressed in the heat-sensitive variant, that accumulation of the additional CP-sHSP isoforms was genetically linked to thermotolerance, and that the presence of the additional isoforms in the heat-tolerant variant provided greater protection to photosystem II during heat stress. To determine the basis of the differential expression, we isolated the genes encoding the CP-sHSPs from both variants and characterized their structure and expression. Two genes, ApHsp26.2 and ApHsp26.7a, were isolated from the heat-tolerant variant, and three genes, ApHsp26.2m, ApHsp26.8, and ApHsp26.7b, were isolated from the heat-sensitive variant. The sequence of ApHsp26.2m from the heat-sensitive variant was identical to ApHsp26.2, except for a point mutation that generated a premature stop codon. Therefore, the protein product of ApHsp26.2m did not accumulate in the heat-sensitive line. Mass spectrometry analysis confirmed that ApHsp26.2 encoded for the CP-sHSP isoforms unique to the heat-tolerant variant. An identical mutation was detected in one of the three parental lines used to develop the creeping bentgrass variants. This suggests that ApHsp26.2m was inherited from this parent and did not arise from a mutation that occurred during tissue culture. The presence of two isoforms encoded by the same gene might be due to differential processing of the N-terminal amino acids during or after import into the chloroplast.