Functional characterization of Arabidopsis†HsfA6a as a heat-shock transcription factor under high salinity and dehydration conditions.

Although heat-shock transcription factors are well characterized in the heat stress-related pathway, they are poorly understood in other stress responses. Here, we functionally characterized AtHsfA6a in the presence of exogenous abscisic acid (ABA) and under high salinity and dehydration conditions. AtHsfA6a expression under normal conditions is very low, but was highly induced by exogenous ABA, NaCl and drought. Unexpectedly, the levels of AtHsfA6a transcript were not significantly altered under heat and cold stresses. Electrophoretic mobility shift assays and transient transactivation assays indicated that AtHsfA6a is transcriptionally regulated by ABA-responsive element binding factor/ABA-responsive element binding protein, which are key regulators of the ABA signalling pathway. Additionally, fractionation and protoplast transient assays showed that AtHsfA6a was in cytoplasm and nucleus simultaneously; however, under conditions of high salinity the majority of AtHsfA6A was in the nucleus. Furthermore, at both seed germination and seedlings stage, plants overexpressing AtHsfA6a were hypersensitive to ABA and exhibited enhanced tolerance against salt and drought stresses. Finally, the microarray and qRT-PCR analyses revealed that many stress-responsive genes were up-regulated in the plants overexpressing AtHsfA6a. Taken together, the data strongly suggest that AtHsfA6a acts as a transcriptional activator of stress-responsive genes via the ABA-dependent signalling pathway.

Rice Rab11 is required for JA-mediated defense signaling.

Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8.

Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants.

The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k(cat) values 0.12 ± 0.007 min(-1) (n = 6) and 0.087 ± 0.002 min(-1) (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm.

OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells.

In yeast and mammals, the Yip/PRA1 family of proteins has been reported to facilitate the delivery of Rab GTPases to the membrane by dissociating the Rab-GDI complex during vesicle trafficking. Recently, we identified OsPRA1, a plant Yip/PRA1 homolog, as an OsRab7-interacting protein that localizes to the prevacuolar compartment, which suggests that it plays a role in vacuolar trafficking of plant cells. Here, we show that OsPRA1 is essential for vacuolar trafficking and that it has molecular properties that are typical of the Yip/PRA1 family of proteins. A trafficking assay using Arabidopsis protoplasts showed that the point mutant OsPRA1((Y94A)) strongly inhibits the vacuolar trafficking of cargo proteins, but has no inhibitory effect on the plasma membrane trafficking of H(+)-ATPase-GFP, suggesting its specific involvement in vacuolar trafficking. Moreover, OsPRA1 was shown to be an integral membrane protein, suggesting that its two hydrophobic domains may mediate membrane integration, and its cytoplasmic N- and C-terminal regions were found to be important for binding to OsRab7. OsPRA1 also interacted with OsVamp3, implying its involvement in vesicle fusion. Finally, we used a yeast expression system to show that OsPRA1 opposes OsGDI2 activity and facilitates the delivery of OsRab7 to the target membrane. Taken together, our results support strongly that OsPRA1 targets OsRab7 to the tonoplast during vacuolar trafficking.

Role of Arabidopsis CHL27 protein for photosynthesis, chloroplast development and gene expression profiling.

In Chl biosynthesis, aerobic Mg-protoporphyrin IX monomethyl ester (MPE) cyclase is a key enzyme involved in the synthesis of protochlorophyllide a, and its membrane-bound component is known to be encoded by homologs of CHL27 in photosynthetic bacteria, green algae and plants. Here, we report that the Arabidopsis chl27-t knock-down mutant exhibits retarded growth and chloroplast developmental defects that are caused by damage to PSII reaction centers. The mutant contains a T-DNA insertion within the CHL27 promoter that dramatically reduces the CHL27 mRNA level. chl27-t mutant plants grew slowly with a pale green appearance, suggesting that they are defective in Chl biosynthesis. Chl fluorescence analysis showed significantly low photosynthetic activity in chl27-t mutants, indicating damage in their PSII reaction centers. The chl27-t mutation also conferred severe defects in chloroplast development, including the unstacking of thylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis, including those encoding components of light-harvesting complex I (LHCI) and LHCII, and PSI and PSII, which accounts for the defects in photosynthetic activity and chloroplast development. In addition, the microarray data also revealed the significant repression of genes such as PORA and AtFRO6 for Chl biosynthesis and iron acquisition, respectively, and, furthermore, implied that there is cross-talk in the Chl biosynthetic pathway among the PORA, AtFRO6 and CHL27 proteins.

AtObgC-AtRSH1 interaction may play a vital role in stress response signal transduction in Arabidopsis.

The interaction of Obg (Spo0B-associated GTP-binding protein) GTPase and SpoT, which is a bifunctional ppGpp (guanosine 3',5'-bispyrophosphate) hydrolase/synthetase, is vital for the modulation of intracellular ppGpp levels during bacterial responses to environmental cues. It has been recently reported that the ppGpp level is also inducible by various stresses in the chloroplasts of plant cells. However, the function of the Obg-SpoT interaction in plants remains elusive. The results from the present and previous studies suggest that AtRSH1 is a putative bacterial SpoT homolog in Arabidopsis and that its transcription levels are responsive to wounding and salt stresses. In this study, we used a yeast two-hybrid analysis to map the regions required for the AtObgC-AtRSH1 interaction. Moreover, protein-protein docking simulations revealed reasonable geometric and electrostatic complementarity in the binding surfaces of the two proteins. The data support our experimental results, which suggest that the conserved domains in AtObgC and the N terminus of AtRSH1 containing the TGS domain contribute to their interaction. In addition, quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AtObgC and AtRSH1 exhibit a similar inhibition pattern under wounding and salt-stress conditions, but the inhibition pattern was not greatly influenced by the presence or absence of light. Based on in vivo analyses, we further confirmed that the AtRSH1 and AtObgC proteins similarly localize in chloroplasts. Based on these results, we propose that the AtObgC-AtRSH1 interaction plays a vital role in ppGpp-mediated stress responses in chloroplasts.

OsRab11 and OsGAP1 are essential for the vesicle trafficking of the vacuolar H(+)-ATPase OsVHA-a1 under high salinity conditions.

To understand the molecular mechanism of the plant vacuolar H(+)-ATPase in endocytic trafficking and adaptation to high salinity, yeast two-hybrid assay, IP-western hybridization, trafficking assay, RT- and qRT-PCR analyses and growth assay were performed here. To confirm the interaction between OsVHA-a1 and OsGAP1, pull-down assay and Co-IP were performed in vitro and in vivo, respectively. qRT-PCR analysis revealed that the transcription of OsVHA-a1, OsGAP1 and OsRab11 was induced under high salinity. Through the protoplast-based trafficking assay, OsVHA-a1 localized predominantly from the TGN to the PVC under stressed conditions. In addition, both OsGAP1 (R385A) and OsRab11 (S28N) mutants did not interact with OsVHA-a1, and blocked the vesicular trafficking of OsVHA-a1 to the PVC. In a seedling growth assay using the dominant negative OsRab11 (S28N), this mutant was much more sensitive to high salinity than the wild-type. Furthermore, the trafficking assay using isolated vacuoles demonstrated directly that OsGAP1 targeted to the tonoplast of the central vacuole under high salinity. Taken together, it is suggested that OsGAP1 and OsRab11 are essential for the vesicle trafficking of OsVHA-a1 to the PVC and/or the central vacuole under high salinity.