Dool-Yi Kim

Ectopic expression of a hot pepper bZIP-like transcription factor in potato enhances drought tolerance without decreasing tuber yield

Over-expression of group A bZIP transcription factor genes in plants improves abiotic stress tolerance but usually reduces yields. Thus, there have been several efforts to overcome yield penalty in transgenic plants. In this study, we characterized that expression of the hot pepper (Capsicum annuum) gene CaBZ1, which encodes a group S bZIP transcription factor, was induced by salt and osmotic stress as well as abscisic acid (ABA). Transgenic potato (Solanum tuberosum) plants over-expressing CaBZ1 exhibited reduced rates of water loss and faster stomatal closure than non transgenic potato plants under drought and ABA treatment conditions. CaBZ1 over-expression in transgenic potato increased the expression of ABA- and stress-related genes (such as CYP707A1, CBF and NAC-like genes) and improved drought stress tolerance. Interestingly, over-expression of CaBZ1 in potato did not produce undesirable growth phenotypes in major agricultural traits such as plant height, leaf size and tuber formation under normal growth conditions. The transgenic potato plants also had higher tuber yields than non transgenic potato plants under drought stress conditions. Thus, CaBZ1 may be useful for improving drought tolerance in tuber crops. This might be the first report of the production of transgenic potato with improved tuber yields under drought conditions.

N-glycan containing a core α1,3-fucose residue is required for basipetal auxin transport and gravitropic response in rice (Oryza sativa).

In plants, α1,3-fucosyltransferase (FucT) catalyzes the transfer of fucose from GDP-fucose to asparagine-linked GlcNAc of the N-glycan core in the medial Golgi. To explore the physiological significance of this processing, we isolated two Oryza sativa (rice) mutants (fuct-1 and fuct-2) with loss of FucT function. Biochemical analyses of the N-glycan structure confirmed that α1,3-fucose is missing from the N-glycans of allelic fuct-1 and fuct-2. Compared with the wild-type cv Kitaake, fuct-1 displayed a larger tiller angle, shorter internode and panicle lengths, and decreased grain filling as well as an increase in chalky grains with abnormal shape. The mutant allele fuct-2 gave rise to similar developmental abnormalities, although they were milder than those of fuct-1. Restoration of a normal tiller angle in fuct-1 by complementation demonstrated that the phenotype is caused by the loss of FucT function. Both fuct-1 and fuct-2 plants exhibited reduced gravitropic responses. Expression of the genes involved in tiller and leaf angle control was also affected in the mutants. We demonstrate that reduced basipetal auxin transport and low auxin accumulation at the base of the shoot in fuct-1 account for both the reduced gravitropic response and the increased tiller angle.

Expression of potato RNA-binding proteins StUBA2a/b and StUBA2c induces hypersensitive-like cell death and early leaf senescence in Arabidopsis

Highlight Potato StUBA2 RNA-binding proteins promote hypersensitive-like cell death (dependent on the first StUBA2 RNA recognition motif) and premature leaf senescence, and increase transcripts of select pathogen-associated, senescence-associated, and autophagy-associated genes.

Ectopic expression of CaWRKY1 , a pepper transcription factor, enhances drought tolerance in transgenic potato plants

WRKY transcription factors play a major role in plant biotic and abiotic stress responses. In this study, WRKY transcription factor, CaWRKY1, whose expression was strongly induced by cold and abscisic acid treatment in pepper, was cloned into potato via Agrobacterium-mediated transformation of vector expressing CaWRKY1 under control of the CaMV35S promoter. The overexpression of CaWRKY1 in potato plants increased tolerance to drought stress without noticeably affecting other agricultural important factors, such as plant height, leaf size and tuber formation. In addition, CaWRKY1-overexpressing transgenic potato plants showed increased expression of abiotic stress-related genes, such as C-REPEAT BINDING FACTOR 3 (CBF3), ZINC FINGER PROTEIN ZAT10 (ZAT10), TREHALOSE-6-PHOSPHATE SYNTHASE (TPS) and LATE EMBRYOGENESIS ABUNDANT (LEA). These results suggest that CaWRKY1 can regulate drought stress tolerance in potato plants via the activation of putative stress-responsive genes.

Involvement of rice Polycomb protein OsFIE2 in plant growth and seed size

Seed development is a complex but orchestrated process that requires the fine-tuning of parentally governed gene expression, which is regulated by Polycomb proteins. Over the last decade, various Polycomb proteins have been identified and functionally characterized in plants, and it has been found that they form the PRC2 suppressor complex, which is involved in various developmental programs, including seed development. In this study, the function of the rice fertilization-independent endosperm gene OsFIE2, which expresses a protein homologous to the Arabidopsis Polycomb protein FIE, was characterized. We also characterized OsEZ1/OsiEZ1, another key component of the PRC2 complex. Both the OsFIE2 and OsEZ11 genes are strongly expressed in leaf and stem compared to other tissues, including root, anther, ovary, and ovule. We further examined whether OsFIE2 interacted with OsEZ1 using a yeast two-hybrid system. Interaction analysis showed that OsFIE2 interacted with OsEZ1 but not with Arabidopsis MEA protein. To examine the physiological roles of OsFIE2, 35S:OsFIE2Arabidopsis lines were generated. Transgenic plants with 35S:OsFIE2 grew faster than wild-type plants during early development. Importantly, they produced bigger seed than the wild type, indicating that OsFIE2 may play an important role in seed size. In addition, we generated pOsFIE2:GUS plants to examine the spatial expression pattern of OsFIE2. GUS expression was detected in cotyledon but not in any other tissues, suggesting that OsFIE2 expression may be required to suppress homeotic genes in cotyledon.

Rice LIM protein OsPLIM2a is involved in rice seed and tiller development

Yield of major monocotyledonous crops including wheat, rice, barley, and sorghum is greatly influenced by tillering. However, deciphering the underlying mechanisms of the tillering has long been hindered because many changeable factors are involved in the process. Plant two LIM-domain-containing proteins bind to and stabilize actin filaments that are major constituents in the formation of higher-order actin cytoskeleton. Here, we report that rice LIM-domain protein, OsPLIM2a, is involved in rice tillering likely through actin cytoskeleton organization. OsPLIM2 genes (OsPLIM2a, OsPLIM2b, and OsPLIM2c) expressed in reproductive organs including anthers, but not in other tissues. Analysis of both OsPLIM2a and OsPLIM2c promoter fused to GUS reporter revealed that both promoters directed strong and specific GUS expression in pollens. Transient expression of OsPLIM2a-GFP and OsPLIM2c-GFP in tobacco leaves showed that OsPLIM2a and OsPLIM2c could bind to actin filaments, which is consistent with other plant LIM proteins with actin-binding property. To examine further physiological roles of rice OsPLIM2a and OsPLIM2c, transgenic rice plants with 35S:OsPLIM2a or 35S:OsPLIM2c were examined for any phenotypic changes. Transgenic plants overexpressing OsPLIM2a produced bigger seeds than wild type, whereas they exhibited reduction in tiller numbers. These results suggest that OsPLIM2a may participate positively in seed development but negatively in tiller differentiation. Protein interaction assays using OsPLIM2c proteins revealed that OsPLIM2c interacted with at least three proteins including rice Fimbrin, of which homologs in Arabidopsis play crucial roles in pollen tube growth, implying that rice OsPLIM2c and Fimbrin may exert roles together in pollen tube growth.

Overexpression of Nuclear Factor Y subunit StNF-YA7 enhances drought tolerance in potato

Drought, the most devastating abiotic stress, has detrimental effects on crop production. To minimize such adverse effects, it is crucial to improve drought tolerance in crops. Several members of the plant Nuclear Factor Y (NF-Y) gene family are involved in drought tolerance. In this study, we isolated the potato NF-Y subunit A 7 (StNF-YA7) gene from Solanum tuberosum ‘Superior’ and analyzed its relationship to abiotic stress tolerance. StNF-YA7 was induced by drought, salt stress, and abscisic acid (ABA), and a transient expression assay using 35S:StNF-YA7-smGFP showed that StNF-YA7 is localized to the nucleus, as reported for other plant NF-Ys. To examine the involvement of StNF-YA7 in drought tolerance, we generated 35S:StNF-YA7 transgenic potato plants. The transgenic potato plants exhibited enhanced drought tolerance, likely due to reduced leaf water loss. Analysis of the StNF-YA7 promoter (pStNF-YA7) fused to GUS revealed that StNF-YA7 is strongly expressed in vascular tissue. Taken together, these results suggest that enhancing drought tolerance via StNF-YA7 overexpression is associated with reduced leaf water loss, likely through mechanism(s) involving the vascular system.

Development of Marker-free TaGlu-Ax1 Transgenic Rice Harboring a Wheat High-molecular-weight Glutenin Subunit (HMW-GS) Protein

High-molecular-weight glutenin subunits (HMW-GSs) are extremely important determinants of the functional properties of wheat dough. Transgenic rice plants containing a wheat TaGlu-Ax1 gene encoding a HMG-GS were produced from the Korean wheat cultivar ‘Jokyeong’ and used to enhance the bread-making quality of rice dough using the Agrobacterium-mediated co-transformation method. Two expression cassettes with separate DNA fragments containing only TaGlu-Ax1 and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately into the Agrobacterium tumefaciens EHA105 strain for co-infection. Rice calli were infected with each EHA105 strain harboring TaGlu-Ax1 or HPTII at a 3:1 ratio of TaGlu-Ax1 and HPTII. Among 210 hygromycin-resistant T0 plants, 20 transgenic lines harboring both the TaGlu-Ax1 and HPTII genes in the rice genome were obtained. The integration of the TaGlu-Ax1 gene into the rice genome was reconfirmed by Southern blot analysis. The transcripts and proteins of the wheat TaGlu-Ax1 were stably expressed in rice T1 seeds. Finally, the marker-free plants harboring only the TaGlu-Ax1 gene were successfully screened in the T1 generation. There were no morphological differences between the wild-type and marker-free transgenic plants. The quality of only one HMW-GS (TaGlu-Ax1) was unsuitable for bread making using transgenic rice dough. Greater numbers and combinations of HMW and LMW-GSs and gliadins of wheat are required to further improve the processing qualities of rice dough. TaGlu-Ax1 marker-free transgenic plants could provide good materials to make transgenic rice with improved bread-making qualities.

Growth, quality, and yield characteristics of transgenic potato (Solanum tuberosum L.) overexpressing StMyb1R-1 under water deficit

Abstract This study was conducted to evaluate agronomic characteristics such as growth, quality, and yields of StMyb1R-1 transgenic potato and also to obtain the basic data for establishing assessment guidelines of transgenic potato. Three transgenic lines (Myb 1, Myb 2, and Myb 8) were cultivated under conventional irrigation, drought condition, and severe drought condition and were analyzed by com-paring with wild type, non-transgenic cv. Superior. Myb 2 showed a different flower color from wild type and Myb 1 had much bigger secondary leaflets than wild type. Myb 1 and Myb 2 showed higher P 2 O 5 content in both top and root zone and longer shaped tubers than wild type. In yield factors, transgenic lines had more tubers than wild type, however their yield decreases were severe because of the poor enlargement of tuber under water deficit condition. This tendency was noticeable in Myb 1 and Myb 2. In TR ratio, chlorophyll content, dry matter rate, and relative water content, there were no big differences between transgenic lines and wild type. Meanwhile, in phenotype, growth, quality, and yield factors, substantial equivalent was confirmed between Myb 8 and wild type. Then, Myb 8 showed the highest marketable tuber yield under conventional irrigation, while showed lower level than wild type under water deficit. Judged by this result, the enhancing drought-tolerance by

N-terminal region of rice polycomb group protein OsEZ1 is required for OsEZ1–OsFIE2 protein interaction

Polycomb group (PcG) proteins form Polycomb Repressive Complex 2 (PRC2), which regulates seed development by the epigenetic control of gene expression. Interaction assay among Arabidopsis Fertilization-independent-seed2 (FIS) class PcG proteins showed that Fertilization-independent endosperm (FIE) interacts with Medea (MEA), a SET-domain polycomb protein, of which N-terminal region is crucial for the interaction. In this study, rice SET-domain PcG protein OsEZ1, also known as OsiEZ1 in indica rice, was analyzed to identify an interacting domain of OsEZ1 required for OsEZ1–OsFIE2 protein interaction. A series of OsEZ1 deletions were generated and used to determine an interacting domain of OsEZ1 with OsFIE2 using the yeast two-hybrid system. Among OsEZ1 deletions, only OsEZ1∆2 and OsEZ1∆3 interacted with OsFIE2, indicating that the 155K–169R or N-proximal region of OsEZ1 is crucial for OsFIE2–OsEZ1 interaction. To examine the physiological roles of OsEZ1, 35S:OsEZ1Arabidopsis lines were generated. OsEZ1 overexpressors exhibited altered seedling growth and seed size, implying that OsEZ1 may play important roles in seedling and seed development.