• Journal of Plant Biotechnology
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• v.41 no.2
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• pp.81-88
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• 2014

Salt cress (Thellungiella halophila or Thellungiella parvula), species closely related to Arabidopsis thaliana, represents an extremophile adapted to harsh saline environments. To isolate salt-tolerance genes from this species, we constructed a cDNA library from roots and leaves of salt cress plants treated with 200 mM NaCl. This cDNA library was subsequently shuttled into the destination binary vector [driven by the cauliflower mosaic virus (CaMV) 35S promoter] designed for plant transformation and expression via recombination- assisted cloning. In total, 305,400 pools of transgenic BASTA-resistant lines were generated in Arabidopsis using either T. halophila or T. parvula cDNA libraries. These were used for functional screening of genes involved in salt tolerance. Among these pools, 168,500 pools were used for primary screening to date from which 7,157 lines showed apparent salt tolerant-phenotypes in the initial screen. A secondary screen has now identified 165 salt tolerant transgenic lines using 1,551 (10.6%) lines that emerged in the first screen. The prevalent phenotype in these lines includes accelerated seed germination often accompanied by faster root growth compared to WT Arabidopsis under salt stress condition. In addition, other lines showed non-typical development of stems and flowers compared to WT Arabidopsis. Based on the close relationship of the tolerant species to the target species we suggest this approach as an appropriate method for the large-scale identification of salt tolerance genes from salt cress.

• Plant Biotechnology Reports
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• v.5 no.1
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• pp.71-77
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• 2011

Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Glycine betaine is one of the most important osmolytes present in higher plants that enable them to cope with environmental stresses through osmotic adjustment. In this study, a betaine aldehyde dehydrogenase (BADH) gene from spinach under the control of the stress-induced promoter rd29A from Arabidopsis thaliana was introduced into potato cultivar Gannongshu 2 by the Agrobacterium tumefaciens system. Putative transgenic plants were confirmed by Southern blot analysis. Northern hybridization analysis demonstrated that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants. The BADH activity in the transgenic potato plants was between 10.8 and 11.7 U. There was a negative relationship (y = -2.2083x + 43.329, r = 0.9495) between BADH activity and the relative electrical conductivity of the transgenic potato plant leaves. Plant height increased by 0.4-0.9 cm and fresh weight per plant increased by 17-29% for the transgenic potato plants under NaCl and polyethylene glycol stresses compared with the control potato plants. These results indicated that the ability of transgenic plants to tolerate drought and salt was increased when their BADH activity was increased.

• Molecules and Cells
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• v.38 no.6
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• pp.506-517
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• 2015

Arabidopsis Shaggy-like protein kinases (ASKs) are Arabidopsis thaliana homologs of glycogen synthase kinase 3/SHAGGY-like kinases (GSK3/SGG), which are comprised of 10 genes with diverse functions. To dissect the function of $ASK{\beta}$ (AtSK32), $ASK{\beta}$ antisense transgenic plants were generated, revealing the effects of $ASK{\beta}$ down-regulation in Arabidopsis. Suppression of $ASK{\beta}$ expression specifically interfered with pollen development and fertility without altering the plants' vegetative phenotypes, which differed from the phenotypes reported for Arabidopsis plants defective in other ASK members. The strength of these phenotypes showed an inverse correlation with the expression levels of $ASK{\beta}$ and its co-expressed genes. In the aborted pollen of $ASK{\beta}$ antisense plants, loss of nuclei and shrunken cytoplasm began to appear at the bicellular stage of microgametogenesis. The in silico analysis of promoter and the expression characteristics implicate $ASK{\beta}$ is associated with the expression of genes known to be involved in sperm cell differentiation. We speculate that $ASK{\beta}$ indirectly affects the transcription of its co-expressed genes through the phosphorylation of its target proteins during late pollen development.

• Molecules and Cells
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• v.27 no.1
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• pp.75-81
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• 2009

The Arabidopsis gene AtLEC (At3g15356) gene encodes a putative 30-kDa protein with a legume lectin-like domain. Likely to classic legume lectin family of genes, AtLEC is expressed in rosette leaves, primary inflorescences, and roots, as observed in Northern blot analysis. The accumulation of AtLEC transcript is induced very rapidly, within 30 min, by chitin, a fungal wall-derived oligosaccharide elictor of the plant defense response. Transgenic Arabidopsis carrying an AtLEC promoter-driven ${\beta}$-glucuronidase (GUS) construct exhibited GUS activity in the leaf veins, secondary inflorescences, carpel heads, and silique receptacles, in which no expression could be seen in Northern blot analysis. This observation suggests that AtLEC expression is induced transiently and locally during developmental processes in the absence of an external signal such as chitin. In addition, mechanically wounded sites showed strong GUS activity, indicating that the AtLEC promoter responds to jasmonate. Indeed, methyl jasmonate and ethylene exposure induced AtLEC expression within 3-6 h. Thus, the gene appears to play a role in the jasmonate-/ethylene-responsive, in addition to the chitin-elicited, defense responses. However, chitin-induced AtLEC expression was also observed in jasmonate-insensitive (coi1) and ethylene-insensitive (etr1-1) Arabidopsis mutants. Thus, it appears that chitin promotes AtLEC expression via a jasmonate- and/or ethylene-independent pathway.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.130-137
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• 2007

A disease resistance related gene, MbR7, was identified in the wild apple species, Malus baccata. The MbR7 gene has a single open reading frame (ORF) of 3,288 nucleotides potentially encoding a 1,095-amino acid protein. Its deduced amino acid sequence resembles the N protein of tobacco and the NL27 gene of potato and has several motifs characteristic of a TIR-NBS-LRR R gene subclass. Ectopic expression of MbR7 in Arabidopsis enhanced the resistance against a virulent pathogen, Pseudomonas syringae pv. tomato DC3000. Microarray analysis confirmed the induction of defense-related gene expression in 35S::MbR7 heterologous Arabidopsis plants, indicating that the MbR7 gene likely activates a downstream resistance pathway without interaction with pathogens. Our results suggest that MbR7 can be a potential target gene in developing a new disease-resistant apple variety.

• Molecules and Cells
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• v.25 no.3
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• pp.438-445
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• 2008

Leaf senescence is a highly regulated genetic process that constitutes the last stage of plant development and provides adaptive fitness by relocating metabolites from senescing leaves to reproducing seeds. Characterization of various senescence mutants, mostly in Arabidopsis, and genome-wide analyses of gene expression, have identified a wide array of regulatory components, including transcription factors and enzymes as well as signaling molecules mediating growth hormones and environmental stress responses. In this work we demonstrate that a membrane-associated NAC transcription factor, NTL9, mediates osmotic stress signaling in leaf senescence. The NTL9 gene is induced by osmotic stress. Furthermore, activation of the dormant, membrane-associated NTL9 is elevated under the same conditions. A series of senescence-associated genes (SAGs) were upregulated in transgenic plants overexpressing an activated form of NTL9, and some of them were slightly but reproducibly downregulated in a T-DNA insertional NTL9 knockout mutant. These observations indicate that NTL9 mediates osmotic stress responses that affect leaf senescence, providing a genetic link between intrinsic genetic programs and external signals in the control of leaf senescence.

• Journal of Plant Biology
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• v.39 no.4
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• pp.243-247
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• 1996

Phosphoribosylanthranilate transferase (PAT) catalyzes the second step of the tryptophan biosynthetic pathway and is encoded by a single-copy gene that complements all the visible phenotypes of the tryptophan mutant (trp1-100) of Arabidopsis. The trp1-100 is blue fluorescent under UV light becuase it accumulates anthranilate. To obtain a plant with reduced PAT activity, PAT1 genes with several internal deletions in different promoter regions (pHS 101, pHS102, pHS104, pHS105, and pHS107) were induced into trp1-100 via Agrobacterium. Then, homozygous T3 plants were isolated and examined for blue fluorescence. Introduction of the PAT1 gene fusants results in the reversion of fluorescence phenotype except in the case of pHS105. These results prompted us to perform a parallel analysis of anthranilate synthase and PAT interms of the genetic complementation. A plant line carrying pHS105 gene fusant does not completely complement the blue fluorescence but it accumulates less anthranilate than trp1-100. The activity of PAT was reduced in the transgenic mutant as well. The plant carrying these constructs will add to the growing collection of molecular tools for the study of the indolic secondary metabolism.

• Korean Journal of Plant Resources
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• v.21 no.5
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• pp.388-394
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• 2008

This study was carried out to develop new cultivars using the $T_5$ generation of transformed rice by PCR analysis with DNA marker in each generation $(from\;T_3\;to\;T_5)$. In the previous study, we successfully developed the transgenic rice plants over-expressing the Arabidopsis $H^+/Ca^{2+}$ antiporter CAX 1 (accession no. U57411) gene. The calcium concentration in brown rice of transgenic plants was higher than that of donor plants, Iipum, and was selected 3 lines out of 25 lines at cultured GMO field. The major agronomic traits such as culm length, panicle length and panicle number of 3 lines at transgenic plants $(T_5)$ were similar to wild type. Also these lines appeared to have disease resistance to rice blast, cold resistance as compared with donor types. The grain shape was similar to donor plant, however, the 1000 grain weight of brown rice was different from transgenic plants. These finding would be used for basic data of new variety registration.

• Journal of Plant Biotechnology
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• v.5 no.3
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• pp.143-148
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• 2003

Bax, a mammalian pro-apoptotic member of the Bcl-2 family induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MSP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to playa novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2003.04a
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• pp.65-71
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• 2003

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorho-damine 123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MBP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.