Youn Shic Kim

OsbZIP23 and OsbZIP45, members of the rice basic leucine zipper transcription factor family, are involved in drought tolerance

Many stress-inducible genes including the transcription factor basic leucine zipper (bZIP) are involved in the response of plants to environmental stresses. bZIPs are composed of two domains, a basic region for DNA binding and a leucine zipper region for dimerization. In this study, two drought-induced bZIP genes OsbZIP23 and OsbZIP45 were identified in rice. The transcription factors are orthologs of Arabidopsis bZIPs belonging to groups A and G, respectively, and are known to be involved in drought tolerance. To investigate the regulation of OsbZIP23 and OsbZIP45 expression in rice, quantitative RT-PCR was performed using RNAs from plants grown at drought stress conditions and different developmental stages. Expression of OsbZIP23 and OsbZIP45 showed positive correlation with drought tolerance. To further understand the functions of OsbZIP23 and OsbZIP45, we overexpressed OsbZIP23 and OsbZIP45 in rice using PGD1 promoter. Results of phenotypic and chlorophyll fluorescence analysis on PGD1:OsbZIP23 and PGD1:OsbZIP45 plants showed enhanced tolerance to drought stress. These results suggest that OsbZIP23 and OsbZIP45 are involved in drought stress response in rice and have a great potential for engineering drought-tolerant crops.

The NF-YA transcription factor OsNF-YA7 confers drought stress tolerance of rice in an abscisic acid independent manner.

The mechanisms of plant response and adaptation to drought stress require the regulation of transcriptional networks via the induction of drought-responsive transcription factors. Nuclear Factor Y (NF-Y) transcription factors have aroused interest in roles of plant drought stress responses. However, the molecular mechanism of the NF-Y-induced drought tolerance is not well understood. Here, we functionally analyzed two rice NF-YA genes, OsNF-YA7 and OsNF-YA4. Expression of OsNF-YA7 was induced by drought stress and its overexpression in transgenic rice plants improved their drought tolerance. In contrast, OsNF-YA4 expression was not increased by drought stress and its overexpression in transgenic rice plants did not affect their sensitivity to drought stress. OsNF-YA4 expression was highly induced by the stress-related hormone abscisic acid (ABA), while OsNF-YA7 was not, indicating that OsNF-YA7 mediates drought tolerance in an ABA-independent manner. Analysis of the OsNF-YA7 promoter revealed three ABA-independent DRE/CTR elements and RNA-seq analysis identified 48 genes downstream of OsNFYA7 action putatively involved in the OsNF-YA7-mediated drought tolerance pathway. Taken together, our results suggest an important role for OsNF-YA7 in rice drought stress tolerance.

The rice OsNAC6 transcription factor orchestrates multiple molecular mechanisms involving root structural adaptions and nicotianamine biosynthesis for drought tolerance

Summary Drought has a serious impact on agriculture worldwide. A plants ability to adapt to rhizosphere drought stress requires reprogramming of root growth and development. Although physiological studies have documented the root adaption for tolerance to the drought stress, underlying molecular mechanisms is still incomplete, which is essential for crop engineering. Here, we identified OsNAC6‐mediated root structural adaptations, including increased root number and root diameter, which enhanced drought tolerance. Multiyear drought field tests demonstrated that the grain yield of OsNAC6 root‐specific overexpressing transgenic rice lines was less affected by drought stress than were nontransgenic controls. Genome‐wide analyses of loss‐ and gain‐of‐function mutants revealed that OsNAC6 up‐regulates the expression of direct target genes involved in membrane modification, nicotianamine (NA) biosynthesis, glutathione relocation, 3′‐phophoadenosine 5′‐phosphosulphate accumulation and glycosylation, which represent multiple drought tolerance pathways. Moreover, overexpression of NICOTIANAMINE SYNTHASE genes, direct targets of OsNAC6, promoted the accumulation of the metal chelator NA and, consequently, drought tolerance. Collectively, OsNAC6 orchestrates novel molecular drought tolerance mechanisms and has potential for the biotechnological development of high‐yielding crops under water‐limiting conditions.

Overexpression of OsERF48 causes regulation of OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance.

Summary The AP2/ERF family is a plant‐specific transcription factor family whose members have been associated with various developmental processes and stress tolerance. Here, we functionally characterized the drought‐inducible OsERF48, a group Ib member of the rice ERF family with four conserved motifs, CMI‐1, ‐2, ‐3 and ‐4. A transactivation assay in yeast revealed that the C‐terminal CMI‐1 motif was essential for OsERF48 transcriptional activity. When OsERF48 was overexpressed in an either a root‐specific (ROXO s ERF 48) or whole‐body (OXO s ERF 48) manner, transgenic plants showed a longer and denser root phenotype compared to the nontransgenic (NT) controls. When plants were grown on a 40% polyethylene glycol‐infused medium under in vitro drought conditions, ROXO s ERF 48 plants showed a more vigorous root growth than OXO s ERF 48 and NT plants. In addition, the ROXO s ERF 48 plants exhibited higher grain yield than OXO s ERF 48 and NT plants under field‐drought conditions. We constructed a putative OsERF48 regulatory network by cross‐referencing ROXO s ERF 48 root‐specific RNA‐seq data with a co‐expression network database, from which we inferred the involvement of 20 drought‐related genes in OsERF48‐mediated responses. These included genes annotated as being involved in stress signalling, carbohydrate metabolism, cell‐wall proteins and drought responses. They included, OsCML16, a key gene in calcium signalling during abiotic stress, which was shown to be a direct target of OsERF48 by chromatin immunoprecipitation‐qPCR analysis and a transient protoplast expression assay. Our results demonstrated that OsERF48 regulates OsCML16, a calmodulin‐like protein gene that enhances root growth and drought tolerance.

Rice OsERF71-mediated root modification affects shoot drought tolerance

ABSTRACT Drought is the most serious problem that impedes crop development and productivity worldwide. Although several studies have documented the root architecture adaption for drought tolerance, little is known about the underlying molecular mechanisms. Our latest study demonstrated that overexpression of the OsERF71 in rice roots under drought conditions modifies root structure including larger aerenchyma and radial root growth, and thereby, protects the rice plants from drought stresses. The OsERF71-mediated root modifications are caused by combinatory overexpression of general stress-inducible, cell wall-associated and lignin biosynthesis genes that contribute to drought tolerance. Here we addressed that the OsERF71-mediated root modifications alter physiological capacity in shoots without evidence of developmental changes for drought tolerance. Thus, the OsERF71-mediated root modifications provide novel molecular insights into drought tolerance mechanisms.

Production of insect-resistant transgenic rice plants for use in practical agriculture

Plant biotechnology provides a powerful solution to boost agricultural productivity and nutritional quality. The development process of a transgenic crop includes multiple steps that consist of gene isolation for a target trait, generation of T0 transgenic crops, characterization of the transgene, evaluation of agronomic performance of transgenic crops, selection of elite transgenic lines and assessment of target trait efficacy. Here, we developed elite insect-resistant transgenic rice plants that may satisfy the standards of biosafety assessments. We made a construct with the insecticide cry1Ac gene for a target trait. A total of 310 T0 transgenic lines were generated and underwent extensive analysis. We selected four T3 lines that contain a single-copy transgene inserted into intergenic regions of the rice genome. During this process, we critically analyzed the transgenic lines with five checkpoints that include single copy of transgene, its integration into intergenic region, clean T-DNA arrangement, stability of transgene through generations and substantial equivalence of transgenic plants in agronomic traits other than insect resistance. Consequently, we obtained insect-resistant transgenic rice plants that can be used in practical agriculture.

Overexpression of the OsbZIP66 transcription factor enhances drought tolerance of rice plants

Drought stress is a major constraint of crop development and productivity. Plants have evolutionally developed several mechanisms at the molecular, cellular, and physiological levels to overcome drought stress. The basic leucine zipper (bZIP) transcription factor (TF) family members are starting to be concerned about their roles in drought stress responses. In this study, we functionally characterized OsbZIP66, a rice group-E bZIP TF, to be associated with rice drought tolerance mechanisms. Expression of OsbZIP66 was significantly induced upon treatments of rice plants with drought, high salinity, and ABA. These observations and the fact that the OsbZIP66 promoter contains ten ABA-responsive cis-elements suggest that OsbZIP66 is up-regulated by drought stress in an ABA-dependent manner. Overexpression of OsbZIP66 both in a whole plant body and specifically in roots enhanced drought tolerance of rice plants, indicating that the rice drought tolerance positively correlates with the expression levels of OsbZIP66. Thus, our results demonstrated that OsbZIP66 has a potential for use in biotechnological development of high-yielding rice plants under drought conditions.

The activities of four constitutively expressed promoters in single-copy transgenic rice plants for two homozygous generations

AbstractMain conclusionWe have characterized four novel constitutive promotersARP1, H3F3, HSPandH2BF3that are active in all tissues/stages of transgenic plants and stable over two homozygous generations. Gene promoters that are active and stable over several generations in transgenic plants are valuable tools for plant research and biotechnology. In this study, we characterized four putative constitutive promoters (ARP1, H3F3, HSP and H2BF3) in transgenic rice plants. Promoter regions were fused to the green fluorescence protein (GFP) reporter gene and transformed into rice. Single-copy transgenic lines were then selected and promoter activity was analyzed in various organs and tissues of two successive homozygous generations. All four promoters showed a broad expression profile in most tissues and developmental stages, and indeed the expression of the ARP1 and H3F3 promoters was even greater than that of the PGD1 promoter, a previously described constitutive promoter that has been used in transgenic rice. This observation was based on expression levels in leaves, roots, dry seeds and flowers in both the T2 and T3 generations. Each promoter exhibited comparable levels of activity over two homozygous generations with no sign of transgene silencing, which is an important characteristic of promoters to be used in crop biotechnology applications. These promoters therefore have considerable potential value for the stable and constitutive expression of transgenes in monocotyledonous crops.

A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data

BackgroundGenetically modified crops (GM crops) have been developed to improve the agricultural traits of modern crop cultivars. Safety assessments of GM crops are of paramount importance in research at developmental stages and before releasing transgenic plants into the marketplace. Sequencing technology is developing rapidly, with higher output and labor efficiencies, and will eventually replace existing methods for the molecular characterization of genetically modified organisms.MethodsTo detect the transgenic insertion locations in the three GM rice gnomes, Illumina sequencing reads are mapped and classified to the rice genome and plasmid sequence. The both mapped reads are classified to characterize the junction site between plant and transgene sequence by sequence alignment.ResultsHerein, we present a next generation sequencing (NGS)-based molecular characterization method, using transgenic rice plants SNU-Bt9–5, SNU-Bt9–30, and SNU-Bt9–109. Specifically, using bioinformatics tools, we detected the precise insertion locations and copy numbers of transfer DNA, genetic rearrangements, and the absence of backbone sequences, which were equivalent to results obtained from Southern blot analyses.ConclusionNGS methods have been suggested as an effective means of characterizing and detecting transgenic insertion locations in genomes. Our results demonstrate the use of a combination of NGS technology and bioinformatics approaches that offers cost- and time-effective methods for assessing the safety of transgenic plants.

Overexpression of OsTF1L, a rice HD‐Zip transcription factor, promotes lignin biosynthesis and stomatal closure that improves drought tolerance

Summary Drought stress seriously impacts on plant development and productivity. Improvement of drought tolerance without yield penalty is a great challenge in crop biotechnology. Here, we report that the rice (Oryza sativa) homeodomain‐leucine zipper transcription factor gene, OsTF1L (Oryza sativa transcription factor 1‐like), is a key regulator of drought tolerance mechanisms. Overexpression of the OsTF1L in rice significantly increased drought tolerance at the vegetative stages of growth and promoted both effective photosynthesis and a reduction in the water loss rate under drought conditions. Importantly, the OsTF1L overexpressing plants showed a higher drought tolerance at the reproductive stage of growth with a higher grain yield than nontransgenic controls under field‐drought conditions. Genomewide analysis of OsTF1L overexpression plants revealed up‐regulation of drought‐inducible, stomatal movement and lignin biosynthetic genes. Overexpression of OsTF1L promoted accumulation of lignin in shoots, whereas the RNAi lines showed opposite patterns of lignin accumulation. OsTF1L is mainly expressed in outer cell layers including the epidermis, and the vasculature of the shoots, which coincides with areas of lignification. In addition, OsTF1L overexpression enhances stomatal closure under drought conditions resulted in drought tolerance. More importantly, OsTF1L directly bound to the promoters of lignin biosynthesis and drought‐related genes involving poxN/PRX38, Nodulin protein,DHHC4,CASPL5B1 and AAA‐type ATPase. Collectively, our results provide a new insight into the role of OsTF1L in enhancing drought tolerance through lignin biosynthesis and stomatal closure in rice.