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

Redillas, Mark C.F.R.,Jeong, Jin S.,Kim, Youn S.,Jung, Harin,Bang, Seung W.,Choi, Yang D.,Ha, Sun‐,Hwa,Reuzeau, Christophe,Kim, Ju‐,Kon Blackwell Publishing Ltd 2012 Plant biotechnology journal Vol.10 No.7

<P><B>Summary</B></P><P>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 <I>OsNAC9,</I> a member of the rice NAC domain family. Root‐specific (<I>RCc3</I>) and constitutive (<I>GOS2</I>) promoters were used to overexpress <I>OsNAC9</I> and produced the transgenic <I>RCc3:OsNAC9</I> and <I>GOS2:OsNAC9</I> plants. Field evaluations over two cultivating seasons showed that grain yields of the <I>RCc3:OsNAC9</I> and the <I>GOS2:OsNAC9</I> plants were increased by 13%–18% and 13%–32% under normal conditions, respectively. Under drought conditions, <I>RCc3:OsNAC9</I> plants showed an increased grain yield of 28%–72%, whilst the <I>GOS2:OsNAC9</I> plants remained unchanged. Both transgenic lines exhibited altered root architecture involving an enlarged stele and aerenchyma. The aerenchyma of <I>RCc3:OsNAC9</I> roots was enlarged to a greater extent than those of <I>GOS2:OsNAC9</I> 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 (<I>P </I><<I> </I>0.01) in the roots of both transgenic lines. These included <I>9‐cis‐epoxycarotenoid dioxygenase</I>, an ABA biosynthesis gene, <I>calcium‐transporting ATPase</I>, a component of the Ca<SUP>2+</SUP> signalling pathway involved in cortical cell death and aerenchyma formation, <I>cinnamoyl CoA reductase 1</I>, a gene involved in lignin biosynthesis, and <I>wall‐associated kinases¸</I> genes involved in cell elongation and morphogenesis. Interestingly, <I>O‐methyltransferase</I>, a gene necessary for barrier formation, was specifically up‐regulated only in the <I>RCc3:OsNAC9</I> roots. Such up‐regulated genes that are commonly and specifically up‐regulated in <I>OsNAC9</I> transgenic roots may account for the altered root architecture conferring increased drought resistance phenotype.</P>

The use of JIP test to evaluate drought-tolerance of transgenic rice overexpressing OsNAC10

Redillas, Mark C.F.R.,Strasser, Reto J.,Jeong, Jin-Seo,Kim, Youn-Shic,Kim, Ju-Kon The Korean Society of Plant Biotechnology 2011 Plant biotechnology reports Vol.5 No.2

In this study, the JIP test was exploited to assess drought-tolerance of transgenic rice overexpressing OsNAC10. Two types of promoters, RCc3 (root-specific) and GOS2 (constitutive), were used to drive the transcription factor OsNAC10, a gene involved in diverse functions including stress responses. Three-month-old plants were exposed to drought for 1 week and their fluorescence kinetics was evaluated. Our results showed that drought-treated non-transgenic plants (NT) have higher fluorescence intensity at the J phase (2 ms) compared to transgenic plants, indicating a decline in electron transport beyond the reduced plastoquinone ($Q_A^-$). As manifested by negative L bands, transgenic plants also showed higher energetic connectivity and stability over NT plants under drought conditions. Also, the pool size of the end electron acceptor at the photosystem I was reduced more in NT than in transgenic plants under drought conditions. Furthermore, the transgenic plants had higher $PI_{total}$, a combined parameter that reflects all the driving forces considered in JIP test, than NT plants under drought conditions. In particular, the $PI_{total}$ of the RCc3:OsNAC10 plants was higher than that of NT plants, which was in good agreement with their differences in grain yield. Thus, the JIP test proved to be practical for evaluating drought-tolerance of transgenic plants.

Accumulation of trehalose increases soluble sugar contents in rice plants conferring tolerance to drought and salt stress

Mark C. F. R. Redillas,박수현,Jang Wook Lee,김연식,정진서,Harin Jung,방승운,한태룡,김주곤 한국식물생명공학회 2012 Plant biotechnology reports Vol.6 No.1

Trehalose is a nonreducing sugar composed of two glucose units linked in an a,a-1,1-glycosidic linkage. Present in a wide variety of organisms, this sugar may serve as a source of energy and carbon and as a protective molecule against abiotic stresses. In this study, trehaloseproducing transgenic rice plants (Oryza sativa) expressing a bifunctional fusion enzyme TPSP (Ubi1:TPSP) were utilized to dissect the enigmatic role of trehalose in conferring stress tolerance to plants. Grown under normal conditions, the Ubi1:TPSP plants produced high amounts of soluble sugars (glucose, fructose and sucrose), ranging from 1.5- to 3.5-fold over NT controls. In the time course of drought treatment, the transcripts for the drought degradable-marker genes (RbcS, FBPase, and PBZ1) persisted for two more days in Ubi1:TPSP plants before being completely degraded relative to those in NT plants, confirming the tolerance of the transgenic plants to drought. This was further supported by a delayed increase in transcript levels of the stress-inducible genes SalT, Dip1, and Wsi18 during drought stress. Similarly, Ubi1:TPSP plants showed tolerance to salt levels of up to 150 mM NaCl, as evidenced by the seedling growth and the delayed decay in RbcS and delayed increase in SalT transcript levels. The growth of NT plants was found to be slightly affected by exogenous trehalose feeding, whereas Ubi:TPSP plants remained resistant, validating the protective role of internally produced trehalose. These results suggest that the elevated production of trehalose in rice, through TPSP overexpression, increases the soluble sugar contents and enhances tolerance to both drought and salt stress.

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

Park, Su-Hyun,Jeong, Jin Seo,Redillas, Mark C.F.R.,Jung, Harin,Bang, Seung Woon,Kim, Youn Shic,Kim, Ju-Kon 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.1

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-${\beta}$-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 ($F_v/F_m$) 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.

Characterization of the root-predominant gene promoter HPX1 in transgenic rice plants

Park, Su-Hyun,Jeong, Jin Seo,Han, Eun Hyang,Redillas, Mark C.F.R.,Bang, Seung Woon,Jung, Harin,Kim, Youn Shic,Kim, Ju-Kon 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.3

Gene promoter(s) specialized in root tissues is an important component for crop biotechnology. In our current study, we report results of promoter analysis of the HPX1, a gene expressed predominantly in roots. The HPX1 promoter regions were predicted, linked to the gfp reporter gene, and transformed into rice. Promoter activities were analyzed in various organs and tissues of six independent transgenic HPX1:gfp plants using the fluorescent microscopy and q-RT-PCR methods. GFP fluorescence levels were high in root elongation regions but not in root apex and cap of the HPX1:gfp plants. Very low levels of GFP fluorescence were observed in anthers and leaves. Levels of promoter activities were 16- to 190-fold higher in roots than in leaves of the HPX1:gfp plants. The HPX1 promoter directs high levels of gene expression in root tissues producing GFP levels up to 0.39 % of the total soluble protein. Thus, the HPX1 promoter is predominantly active in the root elongation region during the vegetative stage of growth.

Transgenic overexpression of UIP1, an interactor of the 30 untranslated region of the Rubisco small subunit mRNA, increases rice tolerance to drought

박수현,Jin Seo Jeong,Mark C. F. R. Redillas,Harin Jung,Seung Woon Bang,Youn Shic Kim,김주곤 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.1

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 RNAbinding protein (RBP) possessing a CBS (cystathionine-bsynthase) domain through yeast three-hybrid screening. This RBP, 30-UTR-interacting protein 1 (UIP1), interacts with 30 untranslated region of the Rubisco small subunit mRNA (30 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. Cotransformation of yeast with these constructs together withthree 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 30 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 30 untranslated region of RbcS1 mRNA and increases tolerance of transgenic overexpressors to drought stress.

Characterization of the root-predominant gene promoter HPX1 in transgenic rice plants

박수현,Jin Seo Jeong,Eun Hyang Han,Mark C. F. R. Redillas,방승운,Harin Jung,Youn Shic Kim,김주곤 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.3

Gene promoter(s) specialized in root tissues isan important component for crop biotechnology. In ourcurrent study, we report results of promoter analysis of theHPX1, a gene expressed predominantly in roots. The HPX1promoter regions were predicted, linked to the gfp reportergene, and transformed into rice. Promoter activities wereanalyzed in various organs and tissues of six independenttransgenic HPX1:gfp plants using the fluorescent microscopyand q-RT-PCR methods. GFP fluorescence levelswere high in root elongation regions but not in root apexand cap of the HPX1:gfp plants. Very low levels of GFPfluorescence were observed in anthers and leaves. Levels ofpromoter activities were 16- to 190-fold higher in rootsthan in leaves of the HPX1:gfp plants. The HPX1 promoterdirects high levels of gene expression in root tissues producingGFP levels up to 0.39 % of the total soluble protein. Thus, the HPX1 promoter is predominantly active in theroot elongation region during the vegetative stage ofgrowth.

Cell Adhesion on Porous and Non-Porous Hydrophobic Membranes

Jennica D. Orata,Grace M. Nisola,Mark C. F. R. Redillas,Gino M. Antioquia,Ye?Kyung Kim,Xiaohong Yang,Eulsaeng Cho,Byung Ro Kim,Wook?Jin Chung 한국대기환경학회 2007 한국대기환경학회 학술대회논문집 Vol.- No.-