Harin Jung

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.

The overexpression of OsNAC9 alters the root architecture of rice plants enhancing drought resistance and grain yield under field conditions

Drought conditions limit agricultural production by preventing crops from reaching their genetically predetermined maximum yields. Here, we present the results of field evaluations of rice overexpressing OsNAC9, a member of the rice NAC domain family. Root-specific (RCc3) and constitutive (GOS2) promoters were used to overexpress OsNAC9 and produced the transgenic RCc3:OsNAC9 and GOS2:OsNAC9 plants. Field evaluations over two cultivating seasons showed that grain yields of the RCc3:OsNAC9 and the GOS2:OsNAC9 plants were increased by 13%-18% and 13%-32% under normal conditions, respectively. Under drought conditions, RCc3:OsNAC9 plants showed an increased grain yield of 28%-72%, whilst the GOS2:OsNAC9 plants remained unchanged. Both transgenic lines exhibited altered root architecture involving an enlarged stele and aerenchyma. The aerenchyma of RCc3:OsNAC9 roots was enlarged to a greater extent than those of GOS2:OsNAC9 and non-transgenic (NT) roots, suggesting the importance of this phenotype for enhanced drought resistance. Microarray experiments identified 40 up-regulated genes by more than threefold (P < 0.01) in the roots of both transgenic lines. These included 9-cis-epoxycarotenoid dioxygenase, an ABA biosynthesis gene, calcium-transporting ATPase, a component of the Ca(2+) signalling pathway involved in cortical cell death and aerenchyma formation, cinnamoyl CoA reductase 1, a gene involved in lignin biosynthesis, and wall-associated kinases¸ genes involved in cell elongation and morphogenesis. Interestingly, O-methyltransferase, a gene necessary for barrier formation, was specifically up-regulated only in the RCc3:OsNAC9 roots. Such up-regulated genes that are commonly and specifically up-regulated in OsNAC9 transgenic roots may account for the altered root architecture conferring increased drought resistance phenotype.

Application of two bicistronic systems involving 2A and IRES sequences to the biosynthesis of carotenoids in rice endosperm.

Coordination of multiple transgenes is essential for metabolic engineering of biosynthetic pathways. Here, we report the utilization of two bicistronic systems involving the 2A sequence from the foot-and-mouth disease virus and the internal ribosome entry site (IRES) sequence from the crucifer-infecting tobamovirus to the biosynthesis of carotenoids in rice endosperm. Two carotenoid biosynthetic genes, phytoene synthase (Psy) from Capsicum and carotene desaturase (CrtI) from Pantoea, were linked via either the synthetic 2A sequence that was optimized for rice codons or the IRES sequence under control of the rice globulin promoter, generating PAC (Psy-2A-CrtI) and PIC (Psy-IRES-CrtI) constructs, respectively. The transgenic endosperm of PAC rice had a more intense golden color than did PIC rice, demonstrating that 2A was more efficient than IRES in coordinating gene expression. The 2A and IRES constructs were equally effective in driving transgene transcription. However, immunoblot analysis of CRTI, a protein encoded by the downstream open reading frame of the bicistronic constructs, revealed that 2A was ninefold more effective than IRES in driving translation. The PAC endosperms accumulated an average of 1.3 μg/g of total carotenoids, which was ninefold higher than was observed for PIC endosperms. In particular, accumulation of β-carotene was much higher in PAC endosperms than in PIC endosperms. Collectively, these results demonstrate that both 2A and IRES systems can coordinate the expression of two biosynthetic genes, with the 2A system exhibiting greater efficiency. Thus, the 2A expression system described herein is an effective new tool for multigene stacking in crop biotechnology.

Posttranscriptional Control of Photosynthetic mRNA Decay under Stress Conditions Requires 3′ and 5′ Untranslated Regions and Correlates with Differential Polysome Association in Rice

Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3′ and 5′ untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3′ and 5′ untranslated regions and correlates with differential polysome association.

Oryza sativa COI homologues restore jasmonate signal transduction in Arabidopsis coi1-1 mutants.

CORONATINE INSENSITIVE 1 (COI1) encodes an E3 ubiquitin ligase complex component that interacts with JAZ proteins and targets them for degradation in response to JA signaling. The Arabidopsis genome has a single copy of COI1, but the Oryza sativa genome has three closely related COI homologs. To examine the functions of the three OsCOIs, we used yeast two-hybrid assays to examine their interactions with JAZ proteins and found that OsCOIs interacted with OsJAZs and with JAZs, in a coronatine dependent manner. We also tested whether OsCOI1a and OsCOI1b could complement Arabidopsis coi1-1 mutants and found that overexpression of either gene in the coi1-1 mutant resulted in restoration of JA signal transduction and production of seeds, indicating successful complementation. Although OsCOI2 interacted with a few OsJAZs, we were not able to successfully complement the coi1-1 mutant with OsCOI2. Molecular modeling revealed that the three OsCOIs adopt 3D structures similar to COI1. Structural differences resulting from amino acid variations, especially among amino acid residues involved in the interaction with coronatine and JAZ proteins, were tested by mutation analysis. When His-391 in OsCOI2 was substituted with Tyr-391, OsCOI2 interacted with a wider range of JAZ proteins, including OsJAZ1, 2, 5∼9 and 11, and complemented coi1-1 mutants at a higher frequency than the other OsCOIs and COI1. These results indicate that the three OsCOIs are orthologues of COI1 and play key roles in JA signaling.

Rice NAC proteins act as homodimers and heterodimers

Members of the NAM-ATAF-CUC (NAC) protein family are plant-specific transcription factors that contain a highly conserved N-terminal NAC-domain and diverse C-terminal regions. They have been implicated in plant development and abiotic stress responses. To identify interacters of rice NAC-domain proteins (OsNACs), we performed yeast two-hybrid screening of rice cDNA library using OsNAC5 as a bait, and the results showed that OsNAC5 interacts with other OsNACs including itself. To delineate an interacting domain, a series of deletion constructs of four OsNACs were made and transformed into yeast in various combinations. The results revealed that the conserved NAC domain of OsNACs plays a primary role in homodimer and heterodimer formation, and a part of C-terminal sequence is also necessary for the interaction. In vitro pull-down assays using recombinant OsNAC proteins verified the dimer formations, together suggesting that OsNACs may act by forming homodimers and/or heterodimers in plants.

Characterization of the stress-inducible OsNCED3 promoter in different transgenic rice organs and over three homozygous generations

To be effective in crop biotechnology applications, gene promoters need to be stably active over sequential generations in a population of single-copy transgenic lines. Most of the stress-inducible promoters characterized in plants thus far have been analyzed at early (T0, T1 or T2) generations and/or by testing only a small number of transgenic lines. In our current study, we report our analysis of OsNCED3, a stress-inducible rice promoter involved in ABA biosynthesis, in various organs and tissues of transgenic rice plants over the T2–4 homozygous generations. The transgene copy numbers in the lines harboring the OsNCED3:gfp construct were determined and six single- and two double-copy transgenic lines were analyzed for promoter activity in comparison with the Wsi18, a stress-inducible promoter previously characterized. The exogenous promoter activities were found to be significantly enhanced in the roots and leaves, whereas zero or low levels of activity were evident in grains and flowers, under drought and high-salinity conditions. The highest induction levels of gfp transcripts in the OsNCED3:gfp plants upon drought treatments were 161- and 93-fold in leaves and roots, respectively, and these levels were comparable with those of gfp transcripts in the Wsi18:gfp plants. A comparison of the promoter activities between the T2–T4 plants revealed that comparable activity levels were maintained over these three homozygous generations with no evidence of silencing. Thus, our results provide the OsNCED3 promoter that is stress-inducible in a whole rice plant except for in the aleurones and endosperm and stably active over three generations.

Analysis of the APX, PGD1 and R1G1B constitutive gene promoters in various organs over three homozygous generations of transgenic rice plants

We have previously characterized the constitutively active promoters of the APX, PGD1 and R1G1B genes in rice (Park et al. 2010 in J Exp Bot 61:2459–2467). To have potential crop biotechnology applications, gene promoters must be stably active over many generations. In our current study, we report our further detailed analysis of the APX, PGD1 and R1G1B gene promoters in various organs and tissues of transgenic rice plants for three (T3–5) homozygous generations. The copy numbers in 37 transgenic lines that harbor promoter:gfp constructs were determined and promoter activities were measured by real-time qPCR. Analysis of the 37 lines revealed that 15 contained a single copy of one of the three promoter:gfp chimeric constructs. The promoter activity levels were generally higher in multi-copy lines, whereas variations in these levels over the T3–5 generations studied were observed to be smaller in single-copy than in multi-copy lines. The three promoters were further found to be highly active in the whole plant body at both the vegetative and reproductive stages of plant growth, with the exception of the APX in the ovary and R1G1B in the pistil and filaments where zero or very low levels of activity were detected. Of note, the spatial activities of the PGD1 promoter were found to be strikingly similar to those of the ZmUbi1, a widely used constitutive promoter. Our comparison of promoter activities between T3, T4 and T5 plants revealed that the APX, PGD1 and R1G1B promoters maintained their activities at comparable levels in leaves and roots over three homozygous generations and are therefore potentially viable alternative promoters for crop biotechnology applications.

Transgenic overexpression of UIP1, an interactor of the 3′ untranslated region of the Rubisco small subunit mRNA, increases rice tolerance to drought

Gene regulation at the post-transcriptional level is a well-organized process to adjust plants in response to environmental changes. Here, we identified a novel RNA-binding protein (RBP) possessing a CBS (cystathionine-β-synthase) domain through yeast three-hybrid screening. This RBP, 3′-UTR-interacting protein 1 (UIP1), interacts with 3′ untranslated region of the Rubisco small subunit mRNA (3′ RbcS)—the major mRNA element that mediates the stress-induced mRNA decay (SMD) under drought and salt stress conditions. Six deletion constructs were made to delineate the binding domain of the UIP1 protein. Co-transformation of yeast with these constructs together with three different hybrid RNAs in various combinations showed that deletion of 51 N-terminal amino acids resulted in a loss of sequence-specific binding affinity. Further deletion at the region between 52 and 212 amino acids revealed that the CBS domain of UIP1 is necessary for binding to 3′ RbcS. Transgenic overexpression of UIP1 in rice resulted in an increase in tolerance to drought stress at the vegetative stage of growth. Under drought, high salt and low temperature conditions, the maximum photochemical efficiency of photosystem II (Fv/Fm) of UIP1 plants was higher than those of the nontransgenic plants. Interestingly, the effect of UIP1 overexpression on tolerance to stress was much more pronounced under drought than under high salt and low temperature conditions. Taken together, our results demonstrate that UIP1 interacts with 3′ untranslated region of RbcS1 mRNA and increases tolerance of transgenic overexpressors to drought stress.

Erratum to: Characterization of the stress-inducible OsNCED3

To be effective in crop biotechnology applications, gene promoters need to be stably active over sequential generations in a population of single-copy transgenic lines. Most of the stress-inducible promoters characterized in plants thus far have been analyzed at early (T0, T1 or T2) generations and/or by testing only a small number of transgenic lines. In our current study, we report our analysis of OsNCED3, a stress-inducible rice promoter involved in ABA biosynthesis, in various organs and tissues of transgenic rice plants over the T2–4 homozygous generations. The transgene copy numbers in the lines harboring the OsNCED3:gfp construct were determined and six singleand two double-copy transgenic lines were analyzed for promoter activity in comparison with the Wsi18, a stress-inducible promoter previously characterized. The exogenous promoter activities were found to be significantly enhanced in the roots and leaves, whereas zero or low levels of activity were evident in grains and flowers, under drought and high-salinity conditions. The highest induction levels of gfp transcripts in the OsNCED3:gfp plants upon drought treatments were 161and 93-fold in leaves and roots, respectively, and these levels were comparable with those of gfp transcripts in the Wsi18:gfp plants. A comparison of the promoter activities between the T2–T4 plants revealed that comparable activity levels were maintained over these three homozygous generations with no evidence of silencing. Thus, our results provide the OsNCED3 promoter that is stress-inducible in a whole rice plant except for in the aleurones and endosperm and stably active over three generations.