• Applied Biological Chemistry
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• v.39 no.2
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• pp.104-111
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• 1996

We have isolated several proteinase inhibitor II genes pin2 from a Russet Burbank potato DNA library. One of these, pin2T was subcloned and a 1.8 kb Xbal/Nsil insert was sequenced. This fragment contained the complete Inhibitor II gene including 965 Up of flanking DNA upstream from the gene and 200 bp of flanking DNA downstream from the gene. The open reading frame encodes a protein that is similar to other reported proteinase Inhibitor II proteins. The DNA sequence of the 5' flanking region of pin2T from -714 to +1 is highly homologous (91% identity) with that of the previously isolated wound-inducible pin2K. There are, however, four small deletions in the pin2T promoter which are located at -221 to -200, -263 to -254, -523 to -426 and -759 to -708 relative to the transcription start site of the wound-inducible pin2K. Three of these deletions map to a portion of the promoter that controls the wound-inducibility of the proteinase inhibitor genes. Chimeric genes containing the promoter of the pin2T gene linked with the both CAT and GUS were constructed and transfered into tobacco plants. Analysis of these plants indicated that pin2T is not a wound-inducible gene but is expressed at low levels. Thus, wound-inducibility is lost with the concomitant natural deletion of three small regions of the promoter. Comparision of the sequences deleted in pin2T relative to the pin2K with Genebank sequences indicates that the deleted sequences contain a motif (consensus 5'-AGTAAA-3') that is found in many other wound-inducible genes but not easily found in the published promoter sequences of other plant genes. Nuclear proteins from unwounded and wounded potato leaves were bound to the proximal promoter region, downstream of the 5'-AGTAAA-3', of pin2T. The comparison of the pin2T gone with the pin2K gene indicates that the natural internal promoter deletions are likely responsible for loss of the wound-inducible phenotype in the pin2T gene.

• Journal of Plant Biotechnology
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• v.2 no.1
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• pp.43-49
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• 2000

An efficient and reliable Agrobacterium transformation procedure based on TDZ (thidiazuron)-induced organogenesis was established and applied to six Brazilian eggp1ant varieties. Optimum transgenic plants recovery was achieved upon the study of the following parameters affecting transformation efficiency, using F-100 variety as a model: i) explant source; ii) pre-culture period; iii) physical state of the pre-culture medium and iv) coculture conditions. The highest frequency of kanamycin-resistant calli derived from leaf explants (5%) was obtained without a pre-culture period and co-cultivation for 24 h in liquid medium followed by five days on solid RM (regeneration medium). For cotyledon explants, best results were achieved upon a pre-culture of 24 h in liquid RM and a co-cultivation period of 24 h in liquid RM followed by three days in solid RM, resulting in a transformation Sequency of 22.7%. Kanamycin-resistant organogenic calli were also obtained from cultivars Emb, Preta Comprida, Round nose Shaded, Campineira and Florida Market. The expression pattern of an epidermis-specific promoter was studied using transformants expressing a chimaeric construct comprised by the promoter Atgrp-5 transcriptionally fused to the coding region of the gus gene. The expression pattern was similar to that previously observed in tobacco and Arabidopsis thaliana, with preferential expression at the epidermis and the stem phloem. These results support the idea that the Atgrp-5 promoter can be used to drive defense genes in these tissues, which are sites of pathogen interaction and spread, in programs for the genetic improvement of eggplant.

• Journal of Microbiology
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• v.44 no.2
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• pp.206-216
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• 2006

In this study, we have attempted to characterize the functions of YlaC and YlaD encoded by ylaC and ylaD genes in Bacillus subtilis. The GUS reporter gene, driven by the yla operon promoter, was expressed primarily during the late exponential and early stationary phase, and its expression increased as the result of hydrogen peroxide treatment. Northern and Western blot analyses revealed that the level of ylaC transcripts and YlaC increased as the result of challenge with hydrogen peroxide. A YlaC-overexpressing strain evidenced hydrogen peroxide resistance and a three-fold higher peroxidase activity as compared with a deletion mutant. YlaC-overexpressing and YlaD-disrupted strains evidenced higher sporulation rates than were observed in the YlaC-disrupted and YlaD-overexpressing strains. Analyses of the results of native polyacrylamide gel electrophoresis of recombinant YlaC and YlaD indicated that interaction between YlaC and YlaD was regulated by the redox state of YlaD in vitro. Collectively, the results of this study appear to suggest that YlaC regulated by the YlaD redox state, contribute to oxidative stress resistance in B. subtilis.

• Journal of The Korean Society of Grassland and Forage Science
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• v.41 no.4
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• pp.242-249
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• 2021

Forage crop management is severely challenged by global warming-induced climate changes representing diverse a/biotic stresses. Thus, screening of valuable genetic resources would be applied to develop stress-tolerant forage crops. We isolated two NAC (NAM, ATAF1, ATAF2, CUC2) transcription factors (ANAC032 and ANAC083) transcriptionally activated by multi-abiotic stresses (salt, drought, and cold stresses) from Arabidopsis by microarray analysis. The NAC family is one of the most prominent transcription factor families in plants and functions in various biological processes. The enhanced expressions of two ANACs by multi-abiotic stresses were validated by quantitative RT-PCR analysis. We also confirmed that both ANACs were localized in the nucleus, suggesting that ANAC032 and ANAC083 act as transcription factors to regulate the expression of downstream target genes. Promoter activities of ANAC032 and ANAC083 through histochemical GUS staining again suggested that various abiotic stresses strongly drive both ANACs expressions. Our data suggest that ANAC032 and ANAC083 would be valuable genetic candidates for breeding multi-abiotic stress-tolerant forage crops via the genetic modification of a single gene.

• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.686-696
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• 2011

To deal with pathogens, plants have evolved sophisticated mechanisms including constitutive and induced defense mechanisms. Phytohormones play important roles in plant growth and development, as well as in the systemic response induced by beneficial and pathogen microorganisms. In this work, we identified an Aspergillus ustus isolate that promotes growth and induces developmental changes in Solanum tuberosum and Arabidopsis thaliana. A. ustus inoculation on A. thaliana and S. tuberosum roots induced an increase in shoot and root growth, and lateral root and root hair numbers. Assays performed on Arabidopsis lines to measure reporter gene expression of auxin-induced/ repressed or cell cycle controlled genes (DR5 and CycB1, respectively) showed enhanced GUS activity, when compared with mock-inoculated seedlings. To determine the contribution of phytohormone signaling pathways in the effect elicited by A. ustus, we evaluated the response of a collection of hormone mutants of Arabidopsis defective in auxin, ethylene, cytokinin, or abscisic acid signaling to the inoculation with this fungus. All mutant lines inoculated with A. ustus showed increased biomass production, suggesting that these genes are not required to respond to this fungus. Moreover, we demonstrated that A. ustus synthesizes auxins and gibberellins in liquid cultures. In addition, A. ustus induced systemic resistance against the necrotrophic fungus Botrytis cinerea and the hemibiotrophic bacterium Pseudomonas syringae DC3000, probably through the induction of the expression of salicylic acid, jasmonic acid/ethylene, and camalexin defense-related genes in Arabidopsis.

• Molecules and Cells
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• v.39 no.2
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• pp.111-118
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• 2016

MiR399f plays a crucial role in maintaining phosphate homeostasis in Arabidopsis thaliana. Under phosphate starvation conditions, AtMYB2, which plays a role in plant salt and drought stress responses, directly regulates the expression of miR399f. In this study, we found that miR399f also participates in plant responses to abscisic acid (ABA), and to abiotic stresses including salt and drought. Salt and ABA treatment induced the expression of miR399f, as confirmed by histochemical analysis of promoter-GUS fusions. Transgenic Arabidopsis plants overexpressing miR399f (miR399f-OE) exhibited enhanced tolerance to salt stress and exogenous ABA, but hypersensitivity to drought. Our in silico analysis identified ABF3 and CSP41b as putative target genes of miR399f, and expression analysis revealed that mRNA levels of ABF3 and CSP41b decreased remarkably in miR399f-OE plants under salt stress and in response to treatment with ABA. Moreover, we showed that activation of stress-responsive gene expression in response to salt stress and ABA treatment was impaired in miR399f-OE plants. Thus, these results suggested that in addition to phosphate starvation signaling, miR399f might also modulates plant responses to salt, ABA, and drought, by regulating the expression of newly discovered target genes such as ABF3 and CSP41b.

• Molecules and Cells
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• v.27 no.1
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• pp.67-74
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• 2009

Autophagy degrades toxic materials and old organelles, and recycles nutrients in eukaryotic cells. Whereas the studies on autophagy have been reported in other eukaryotic cells, its functioning in plants has not been well elucidated. We analyzed the roles of OsATG10 genes, which are autophagy-related. Two rice ATG10 genes - OsATG10a and OsATG10b - share significant sequence homology (about 75%), and were ubiquitously expressed in all organs examined here. GUS assay indicated that OsATG10b was highly expressed in the mesophyll cells and vascular tissue of younger leaves, but its level of expression decreased in older leaves. We identified T-DNA insertional mutants in that gene. Those osatg10b mutants were sensitive to treatments with high salt and methyl viologen (MV). Monodansylcadaverine-staining experiments showed that the number of autophagosomes was significantly decreased in the mutants compared with the WT. Furthermore, the amount of oxidized proteins increased in MV-treated mutant seedlings. These results demonstrate that OsATG10b plays an important role in the survival of rice cells against oxidative stresses.

• Journal of Life Science
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• v.33 no.1
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• pp.34-42
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• 2023

In a signal transduction network, from the perception of stress signals to stress-responsive gene ex- pression, binding of various transcription factors to cis-acting elements in stress-responsive promoters coordinate the adaptation of plants to abiotic stresses. Among the AP2/ERF transcription factor family genes, group VII ERF genes, such as RAP2.12, RAP2.2, RAP2.3, AtERF73/HRE1, and AtERF71/ HRE2, are known to be involved in the response to hypoxia stress in Arabidopsis. In this study, we dissected the HRE1 promoter to identify hypoxia-responsive region(s). The 1,000 bp upstream promoter region of HRE1 showed increased promoter activity in Arabidopsis protoplasts and transgenic plants under hypoxia conditions. Analysis of the promoter deletion series of HRE1, including 1,000 bp, 800 bp, 600 bp, 400 bp, 200 bp, 100 bp, and 50 bp upstream promoter regions, using firefly luciferase and GUS as reporter genes indicated that the -200 to -100 region of the HRE1 promoter is responsible for the transcriptional activation of HRE1 in response to hypoxia. In addition, we identified two putative hypoxia-responsive cis-acting elements, the ERF-binding site and DOF-binding site, in the -200 to -100 region of the HRE1 promoter, suggesting that the expression of HRE1 might be regulated via the ERF transcription factor(s) and/or DOF transcription factor(s). Collectively, our results suggest that HRE1 contains hypoxia-responsive cis-acting elements in the -200 to -100 region of its promoter.

• Molecules and Cells
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• v.25 no.4
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• pp.559-565
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• 2008

Members of the TGA family of basic domain/leucine zipper transcription factors regulate defense genes through physical interaction with NON-EXPRESSOR OF PR1 (NPR1). Of the seven TGA family members, TGA4/octopine synthase (ocs)-element-binding factor 4 (OBF4) is the least understood. Here we present evidence for a novel function of OBF4 as a regulator of flowering. We identified CONSTANS (CO), a positive regulator of floral induction, as an OBF4-interacting protein, in a yeast two-hybrid library screen. OBF4 interacts with the B-box region of CO. The abundance of OBF4 mRNA cycles with a 24 h rhythm under both long-day (LD) and short-day (SD) conditions, with significantly higher levels during the night than during the day. Electrophoretic mobility shift assays revealed that OBF4 binds to the promoter of the FLOWERING LOCUS T (FT) gene, a direct target of CO. We also found that, like CO and FT, an OBF4:GUS construct was prominently expressed in the vascular tissues of leaf, indicating that OBF4 can regulate FT expression through the formation of a protein complex with CO. Taken together, our results suggest that OBF4 may act as a link between defense responses and flowering.