Ching-Hui Yeh

Characterization of the genomic structures and selective expression profiles of nine class I small heat shock protein genes clustered on two chromosomes in rice (Oryza sativa L.)

The cytosolic class I small heat shock proteins (sHSP-CI) represent the most abundant sHSP in plants. Here, we report the characterization and the expression profile of nine members of the sHSP-CI gene family in rice (Oryza sativa Tainung No.67), of which Oshsp16.9A, Oshsp16.9B, Oshsp16.9C, Oshsp16.9D and Oshsp17.9B are clustered on chromosome 1, and Oshsp17.3, Oshsp17.7, Oshsp17.9A and Oshsp18.0 are clustered on chromosome 3. Oshsp17.3 and Oshsp18.0 are linked by a 356-bp putative bi-directional promoter. Individual gene products were identified from the protein subunits of a heat shock complex (HSC) and from in vitro transcription/ translation products by two-dimensional gel electrophoreses (2-DE). All sHSP-CI genes except Oshsp17.9B were induced strongly after a 2-h heat shock treatment. The genes on chromosome 3 were induced rapidly at 32  and 41 °C, whereas those on chromosome 1 were induced slowly by similar conditions. Seven of these genes, except Oshsp16.9D and Oshsp17.9B, were induced by arsenite (As), but only genes on chromosome 3 were strongly induced by azetidine-2-carboxylic acid (Aze, a proline analog) and cadmium (Cd). A similar expression profile of all sHSP-CI genes at a lower level was evoked by ethanol, H2O2 and CuCl2 treatments. Transient expression assays of the promoter activity by linking to GUS reporter gene also supported the invivo selective expression of the sHSP-CI genes by Aze treatment indicating the differential induction of rice sHSP-CI genes is most likely regulated at the transcriptional level. Only Oshsp16.9A abundantly accumulated in mature dry seed also suggested additionally prominent roles played by this HSP in development.

A DEAD-Box Protein, AtRH36, is Essential for Female Gametophyte Development and is Involved in rRNA Biogenesis in Arabidopsis

DEAD-box RNA helicases are involved in RNA metabolism, including pre-mRNA splicing, ribosome biogenesis, RNA decay and gene expression. In this study, we identified a homolog of the RH36 gene, AtRH36, which encodes a DEAD-box protein in Arabidopsis thaliana. The gene was expressed ubiquitously throughout the plant. The AtRH36 fused to green fluorescent protein was localized in the nucleus. Homozygosity for the Arabidopsis atrh36 mutants, atrh36-1 and atrh36-2, could not be obtained. Progeny of selfed Arabidopsis atrh36 heterozygote plants were obtained at a heterozygote to wild-type ratio of 1 : 1, which suggested that the AtRH36 gene was involved in gametogenesis. Therefore, we performed a reciprocal cross to determine whether AtRH36 was involved in female gametophyte development. Female gametogenesis was delayed in atrh36-1, and asynchronous development of the female gametophytes was found within a single pistil. Knock-down of AtRH36 gave a pleiotropic phenotype and led to the accumulation of unprocessed 18S pre-rRNA. These results suggest that AtRH36 is essential for mitotic division during female gametogenesis and plays an important role in rRNA biogenesis in Arabidopsis.

Mutation in a homolog of yeast Vps53p accounts for the heat and osmotic hypersensitive phenotypes in Arabidopsis hit1-1 mutant

Previously, the growth of Arabidopsis hit1-1 (heat-intolerant) mutant was found to be inhibited by both heat and water stress (Wu et al. in J Plant Physiol 157:543–547, 2000). In order to determine the genetic mutation underlying the hit1-1 phenotype, map-based cloning of HIT1 gene was conducted. Transformation of the hit1-1 mutant with a HIT1 cDNA clone reverts the mutant to the heat tolerance phenotype, confirming the identity of HIT1. Sequence analysis revealed the HIT1 gene encodes a protein of 829 amino acid residues and is homologous to yeast (Saccharomyces cerevisiae) Vps53p protein. The yeast Vps53p protein has been shown to be a tethering factor that associates with Vps52p and Vps54p in a complex formation involved in the retrograde trafficking of vesicles to the late Golgi. An Arabidopsis homolog of yeast Vps52p has previously been identified and mutation of either the homolog or HIT1 by T-DNA insertion resulted in a male-specific transmission defect. The growth of yeast vps53Δ null mutant also shows reduced thermotolerance, and expression of HIT1 in this mutant can partially complement the defect, supporting the possibility of a conserved biological function for Vps53p and HIT1. Collectively, the hit1-1 is the first mutant in higher plant linking a homolog of the vesicle tethering factor to both heat and osmotic stress tolerance.

Functional Regions of Rice Heat Shock Protein, Oshsp16.9, Required for Conferring Thermotolerance in Escherichia coli

Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli. To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E. coliXL1-blue cells after isopropyl β-d-thioglactopyranoside induction. The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E. coli incapable of surviving at 47.5°C. To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase. It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase. Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9. Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity.

Involvement of the Arabidopsis HIT1/AtVPS53 tethering protein homologue in the acclimation of the plasma membrane to heat stress

Arabidopsis thaliana hit1-1 is a heat-intolerant mutant. The HIT1 gene encodes a protein that is homologous to yeast Vps53p, which is a subunit of the Golgi-associated retrograde protein (GARP) complex that is involved in retrograde membrane trafficking to the Golgi. To investigate the correlation between the cellular role of HIT1 and its protective function in heat tolerance in plants, it was verified that HIT1 was co-localized with AtVPS52 and AtVPS54, the other putative subunits of GARP, in the Golgi and post-Golgi compartments in Arabidopsis protoplasts. A bimolecular fluorescence complementation assay showed that HIT1 interacted with AtVPS52 and AtVPS54, which indicated their assembly into a protein complex in vivo. Under heat stress conditions, the plasma membrane of hit1-1 was less stable than that of the wild type, as determined by an electrolyte leakage assay, and enhanced leakage occurred before peroxidation injury to the membrane. In addition, the ability of hit1-1 to survive heat stress was not influenced by exposure to light, which suggested that the heat intolerance of hit-1 was a direct outcome of reduced membrane thermostability rather than heat-induced oxidative stress. Furthermore, hit1-1 was sensitive to the duration (sustained high temperature stress at 37 °C for 3 d) but not the intensity (heat shock at 44 °C for 30 min) of exposure to heat. Collectively, these results imply that HIT1 functions in the membrane trafficking that is involved in the thermal adaptation of the plasma membrane for tolerance to long-term heat stress in plants.

Isolation and characterization of the Arabidopsis heat- intolerant 2 (hit2) mutant reveal the essential role of the nuclear export receptor EXPORTIN1A (XPO1A) in plant heat tolerance

*The Arabidopsis heat-intolerant 2 (hit2) mutant was isolated on the basis of its impaired ability to withstand moderate heat stress (37 degrees C). Determination of the genetic mutation that underlies the hit2 thermosensitive phenotype allowed better understanding of the mechanisms by which plants cope with heat stress. *Genetic analysis revealed that hit2 is a single recessive mutation. Map-based cloning was used to identify the hit2 locus. The response of hit2 to other types of heat stress was also investigated to characterize the protective role of HIT2. *hit2 was defective in basal but not in acquired thermotolerance. hit2 was sensitive to methyl viologen-induced oxidative stress, and the survival of hit2 seedlings in response to heat stress was affected by light conditions. The mutated locus was located at the EXPORTIN1A (XPO1A) gene, which encodes a nuclear transport receptor. Two T-DNA insertion lines, xpo1a-1 and xpo1a-3, exhibited the same phenotypes as hit2. *The results provide evidence that Arabidopsis XPO1A is dispensable for normal plant growth and development but is essential for thermotolerance, in part by mediating the protection of plants against heat-induced oxidative stress.

Two rice (Oryza sativa) full-length cDNA clones encoding low-molecular-weight heat-shock proteins

Two rice cDNA clones pTS1 and pTS3 were screened at reduced stringency from a cDNA library generated from rice seedling poly(A) RNA by a partial cDNA fragment of soybean 17.5E (pCE53). The rice seedlings were induced at 41 ° C for 2 h before harvest for RNA extraction. Both clones were identified by a hybrid-selected in vitro translation assay and proved to belong to the low-molecular-mass heat-shock protein group (16-20 kDa). The rice pTS1 clone has an open reading frame encoding a 150 amino acid residue 16.9kDa protein which is 72.3~o, 75.3~o and 83.7~o identical to soybean HSP 17.5E [1], pea HSP 179a [2] and wheat C5-8 [3], respectively. On the other hand, the pTS3 cDNA clone encodes a 155 amino acid residue 17.3 kDa protein which is 70.9~o, 73.1~o, 67.5~o identical to soybean HSP 17.5E, pea HSP 179a and wheat C58, respectively. A series of pairwise sequence comparisons show that the two eDNA clones belong to the class I low-molecular-weight heatshock protein family. Among the members of this family, they share a higher homology at the carboxyl terminal than at the amino terminal, as all HSPs do. Both the cDNA and deduced amino acid sequences of the two clones are shown in Fig. 1 and Fig. 2.

A 9 bp cis-element in the promoters of class I small heat shock protein genes on chromosome 3 in rice mediates L-azetidine-2-carboxylic acid and heat shock responses

In rice, the class I small heat shock protein (sHSP-CI) genes were found to be selectively induced by L-azetidine-2-carboxylic acid (AZC) on chromosome 3 but not chromosome 1. Here it is shown that a novel cis-responsive element contributed to the differential regulation. By serial deletion and computational analysis, a 9 bp putative AZC-responsive element (AZRE), GTCCTGGAC, located between nucleotides –186 and –178 relative to the transcription initiation site of Oshsp17.3 was revealed. Deletion of this putative AZRE from the promoter abolished its ability to be induced by AZC. Moreover, electrophoretic mobility shift assay (EMSA) revealed that the AZRE interacted specifically with nuclear proteins from AZC-treated rice seedlings. Two AZRE–protein complexes were detected by EMSA, one of which could be competed out by a canonical heat shock element (HSE). Deletion of the AZRE also affected the HS response. Furthermore, transient co-expression of the heat shock factor OsHsfA4b with the AZRE in the promoter of Oshsp17.3 was effective. The requirement for the putative AZRE for AZC and HS responses in transgenic Arabidopsis was also shown. Thus, AZRE represents an alternative form of heat HSE, and its interaction with canonical HSEs through heat shock factors may be required to respond to HS and AZC.

Molecular characterization of Oryza sativa 16.9 kDa heat shock protein.

A rice class I low-molecular-mass heat shock protein (LMM HSP) Oshsp 16.9 was overexpressed in Escherichia coli. Oligomerized complexes of Oshsp16.9 were harvested and electron microscopic observations of purified complexes revealed globular structures of 10-20 nm in diameter (with majority of 15-18 nm) and calculated to comprise approx. 12 monomers per complex. In comparison, complexes from native rice class I LMM HSPs were observed as larger ellipsoid- or globular-like random aggregated hetero-oligomers. To characterize the biochemical functions of the hydrophobic N-terminal region of Oshsp16.9, a truncation in the N-terminal region was constructed and introduced into E. coli. Results showed that the N-terminal truncated Oshsp16.9 mutant was capable of forming complexes similar to the full-length Oshsp16.9; however, the deletion protein failed to confer in vitro protein thermostability under elevated temperatures. Protein assays from in vivo treatments at higher temperatures exhibited that non-specific interactions of E. coli cellular proteins only occurred with full-length Oshsp16.9 complexes but not with the mutant complex. In vitro immunoprecipitation of cellular proteins from E. coli overexpressing full-length Oshsp16.9 showed that interactions between plant LMM HSP and E. coli cellular proteins are temperature-dependent. Taken together, the hydrophobic N-terminal region of rice class I LMM HSP is critical in the ability of the protein to interact/bind with its potential substrates.

Expression of a gene encoding a rice RING zinc-finger protein, OsRZFP34, enhances stomata opening.

By oligo microarray expression profiling, we identified a rice RING zinc-finger protein (RZFP), OsRZFP34, whose gene expression increased with high temperature or abscisic acid (ABA) treatment. As compared with the wild type, rice and Arabidopsis with OsRZFP34 overexpression showed increased relative stomata opening even with ABA treatment. Furthermore, loss-of-function mutation of OsRZFP34 and AtRZFP34 (At5g22920), an OsRZFP34 homolog in Arabidopsis, decreased relative stomata aperture under nonstress control conditions. Expressing OsRZFP34 in atrzfp34 reverted the mutant phenotype to normal, which indicates a conserved molecular function between OsRZFP34 and AtRZFP34. Analysis of water loss and leaf temperature under stress conditions revealed a higher evaporation rate and cooling effect in OsRZFP34-overexpressing Arabidopsis and rice than the wild type, atrzfp34 and osrzfp34. Thus, stomata opening, enhanced leaf cooling, and ABA insensitivity was conserved with OsRZFP34 expression. Transcription profiling of transgenic rice overexpressing OsRZFP34 revealed many genes involved in OsRZFP34-mediated stomatal movement. Several genes upregulated or downregulated in OsRZFP34-overexpressing plants were previously implicated in Ca2+ sensing, K+ regulator, and ABA response. We suggest that OsRZFP34 may modulate these genes to control stomata opening.