Minkyun Kim

Root-specific expression of OsNAC10 improves drought tolerance and grain yield in rice under field drought conditions.

Drought poses a serious threat to the sustainability of rice (Oryza sativa) yields in rain-fed agriculture. Here, we report the results of a functional genomics approach that identified a rice NAC (an acronym for NAM [No Apical Meristem], ATAF1-2, and CUC2 [Cup-Shaped Cotyledon]) domain gene, OsNAC10, which improved performance of transgenic rice plants under field drought conditions. Of the 140 OsNAC genes predicted in rice, 18 were identified to be induced by stress conditions. Phylogenic analysis of the 18 OsNAC genes revealed the presence of three subgroups with distinct signature motifs. A group of OsNAC genes were prescreened for enhanced stress tolerance when overexpressed in rice. OsNAC10, one of the effective members selected from prescreening, is expressed predominantly in roots and panicles and induced by drought, high salinity, and abscisic acid. Overexpression of OsNAC10 in rice under the control of the constitutive promoter GOS2 and the root-specific promoter RCc3 increased the plant tolerance to drought, high salinity, and low temperature at the vegetative stage. More importantly, the RCc3:OsNAC10 plants showed significantly enhanced drought tolerance at the reproductive stage, increasing grain yield by 25% to 42% and by 5% to 14% over controls in the field under drought and normal conditions, respectively. Grain yield of GOS2:OsNAC10 plants in the field, in contrast, remained similar to that of controls under both normal and drought conditions. These differences in performance under field drought conditions reflect the differences in expression of OsNAC10-dependent target genes in roots as well as in leaves of the two transgenic plants, as revealed by microarray analyses. Root diameter of the RCc3:OsNAC10 plants was thicker by 1.25-fold than that of the GOS2:OsNAC10 and nontransgenic plants due to the enlarged stele, cortex, and epidermis. Overall, our results demonstrated that root-specific overexpression of OsNAC10 enlarges roots, enhancing drought tolerance of transgenic plants, which increases grain yield significantly under field drought conditions.

Direct evidence for selective modulation of psbA, rpoA, rbcL and 16S RNA stability during barley chloroplast development

The turnover of RNAs encoded by seven different barley chloroplast genes was analyzed after treatment of barley shoots with tagetitoxin, a selective inhibitor of chloroplast transcription. Changes in RNA stability were examined during chloroplast development using basal and apical leaf sections of 4.5-day-old dark-grown seedlings and apical leaf sections of 4.0-day-old dark-grown seedlings which had been illuminated for 12 h. Of the RNAs examined, a 2.6 kb unspliced precursor of tRNA(lys) exhibited the shortest half-life, which was estimated to be 3 h. The 16S rRNA and psbA mRNA had the longest estimated half-lives, which were greater than 40 h. Among mRNAs, half-lives were estimated to range from 6 h for psaA mRNA, to over 40 h for psbA mRNA. Therefore, barley chloroplast mRNAs have long half-lives relative to bacterial mRNAs. The stability of atpB mRNA and the unspliced precursor of tRNA-lys was not altered during chloroplast development, while the stability of psaA mRNA decreased 2-fold. In contrast, the stability of the 16S rRNA and mRNAs for rpoA, psbA and rbcL increased during chloroplast development. The stability of 16S rRNA increased markedly during chloroplast development in the dark and this increase was maintained in illuminated seedlings. The stability of rbcL mRNA increased 2.5-fold during chloroplast development in the dark, and then decreased 2-fold in chloroplasts of light-grown plants. The initial increase in rpoA and psbA mRNA stability was also light-independent, with total increases in stability of at least 5-fold. In the case of rpoA, the stability of 2 of the 13 polycistronic rpoA transcripts that were detected in dark-grown plants was selectively increased during chloroplast development. In conclusion, the stability of some transcripts is selectively increased and further modulated during chloroplast development in barley. We propose that the selective stabilization of chloroplast mRNAs, which occurred independent of light, is an indication that non-light regulated developmental signals are involved in barley chloroplast mRNA stability.

Identification of a sequence-specific DNA binding factor required for transcription of the barley chloroplast blue light-responsive psbD-psbC promoter.

The plastid gene psbD encodes the photosystem II reaction center chlorophyll protein D2. psbD is located in a complex operon that includes psbC, psbK, psbl, orf62, and trnG. The operon is transcribed from at least three different promoters. One of the psbD promoters is differentially activated when plants are exposed to blue light. In this study, the psbD blue light-responsive promoter was accurately transcribed in vitro in high-salt extracts of barley plastids. Transcription required supercoiled templates and was inhibited by tagetitoxin, an inhibitor of plastid transcription. Escherichia coli RNA polymerase did not recognize the psbD light-responsive promoter with the same specificity as plastid RNA polymerase. Deletion analyses demonstrated that sequences between -39 and -68, upstream of the transcription initiation site, were required for transcription of the psbD blue light-responsive promoter. This DNA region is highly conserved among plant species and contains multiple AAG sequences. Gel shift assays and DNase I footprinting experiments demonstrated that the AAG-rich DNA sequence interacts with a sequence-specific DNA binding factor termed AGF. Point mutations in the AAG cis element decreased binding of AGF and inhibited transcription from the psbD light-responsive promoter. We concluded that AGF is an essential factor required for transcription of the psbD light-responsive promoter.

Detailed architecture of the barley chloroplast psbD-psbC blue light-responsive promoter.

The photosystem II reaction center chlorophyll protein D2, is encoded by the chloroplast gene psbD. PsbDis transcribed from at least three different promoters, one which is activated by high fluence blue light. Sequences within 130 base pairs (bp) of the psbD blue light-responsive promoter (BLRP) are highly conserved in higher plants. In this study, the structure of thepsbD BLRP was analyzed in detail using deletion and site-directed mutagenesis and in vitro transcription. Deletion analysis showed that a 53-bp DNA region of thepsbD BLRP, from −57 to −5, was sufficient for transcription in vitro. Mutation of a putative prokaryotic −10 element (TATTCT) located from −7 to −12 inhibited transcription from the psbD BLRP. In contrast, mutation of a putative prokaryotic −35 element, had no influence on transcription. Mutation of a TATATA sequence located between the barley psbA −10 and −35 elements significantly reduced transcription from this promoter. However, site-directed mutation of sequences located between −35 and −10 had no effect on transcription from the psbDBLRP. Transcription from the psbD BLRP was previously shown to require a 22-bp sequence, termed the AAG-box, located between −36 and −57. The AAG-box specifically binds the protein complex AGF. Site-directed mutagenesis identified two different sequence motifs in the AAG-box that are important for transcription in vitro. Based on these results, we propose that positive factors bind to the AAG-box and interact with the chloroplast-encoded RNA polymerase to promote transcription from the psbD BLRP. Transcription from the psbD BLRP is thus similar to type II bacterial promoters that use activating proteins to stimulate transcription. Transcription of the psbD BLRP was ∼6.5-fold greater in plastid extracts from illuminated versus dark-grown plants. This suggests that light-induced activation of this promoter in vivo involves factors interacting with the 53-bp psbDBLRP in vitro.

Involvement of Protein Kinase and Extraplastidic Serine/Threonine Protein Phosphatases in Signaling Pathways Regulating Plastid Transcription and the psbD Blue Light- Responsive Promoter in Barley

We investigated the signaling pathways that control changes in plastid transcription in response to development and light. Plastid gene expression was analyzed in dark-grown barley (Hordeum vulgare L.) seedlings treated in vivo with an inhibitor of protein phosphatases 1 and 2A, okadaic acid (OA), or an inhibitor of protein kinases (K252a), followed by exposure of the seedlings to either red, blue, or white light. OA prevented blue light from activating the plastid psbD blue-light-responsive promoter (BLRP) and prevented red and blue light from activating the expression of the plastid-encoded rbcL and psbA and the nuclear-encoded RbcS and Lhcb genes. OA reduced total plastid transcription activity in dark- and light-grown seedlings by 77 to 80%, indicating that OA prevented light-responsive transcription by reducing total plastid transcription. In contrast, K252a activated the accumulation of mRNAs arising from the BLRP. Blue light in combination with K252a increased psbD mRNA levels in an additive manner. The results indicate that protein phosphatases 1 and/or 2A, which reside external to the organelle, are required for proper function of plastid transcription and chloroplast development, whereas a protein kinase represses the BLRP in plants grown in the dark.