Overexpression of cinnamyl alcohol dehydrogenase gene from sweetpotato enhances oxidative stress tolerance in transgenic Arabidopsis

Cinnamyl alcohol dehydrogenase (CAD) is the enzyme in the last step of lignin biosynthetic pathway and is involved in the generation of lignin monomers. IbCAD1 gene in sweetpotato (Ipomoea batatas) was identified, and its expression was induced by abiotic stresses based on promoter analysis. In this study, transgenic Arabidopsis plants overexpressing IbCAD1 directed by CaMV 35S promoter were developed to determine the physiological function of IbCAD1. IbCAD1-overexpressing transgenic plants exhibited better plant growth and higher biomass compared to wild type (WT), under normal growth conditions. CAD activity was increased in leaves and roots of transgenic plants. Sinapyl alcohol dehydrogenase activity was induced to a high level in roots, which suggests that IbCAD1 may regulate biosynthesis of syringyl-type (S) lignin. Lignin content was increased in stems and roots of transgenic plants; this increase was in S lignin rather than guaiacyl (G) lignin. Overexpression of IbCAD1 in Arabidopsis resulted in enhanced seed germination rates and tolerance to reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂). Taken together, our results show that IbCAD1 controls lignin content by biosynthesizing S units and plays an important role in plant responses to oxidative stress.

     

Localization and expression of serine racemase in Arabidopsis thaliana

Arabidopsis plants transformed by promoter of A. thaliana serine racemase fused with β-glucuronidase (GUS) reporter gene showed strong GUS staining in elongating and developing cells such as tip regions of primary and lateral roots, developing leaves, and shoot meristems. RT-PCR and digital northern hybridization showed that expression of the serine racemase gene was not induced by l- and d-serine, light irradiation, biotic and abiotic stresses.     

BWMK1 Responds to Multiple Environmental Stresses and Plant Hormones

Many plant mltogen-actlvated protein klnases （MAPKs） play an important role In regulating responses to both ablotlc and biotic stresses. The first reported rice MAPK gene BWMK1 Is Induced by both rice blast （Magnaporthe grisea） Infection and mechanical wounding. For further analysis of Its response to other environmental cues and plant hormones, such as jasmonlc acid （JA）, salicylic acid （SA）, and benzothladlazole （BTH）, the promoter of BWMKf was fused with the coding region of the β-glucuronldase （GUS） reporter gene. Two promoter-GUS constructs with a 1.0- and 2.5-kb promoter fragment, respectively, were generated and transformed into the Japonica rice cultIvars TP309 and Zhonghua 11. Expression of GUS was Induced in the transgenic lines by cold, drought, dark, and JA. However, light, SA, and BTH treatments suppressed GUS expression. These results demonstrate that BWMK1 Is responsive to multiple ablotlc stresses and plant hormones and may play a role In cross-talk between different signaling pathways.     

Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweetpotato and its expression in response to stress

A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1 , was isolated from cell cultures of sweetpotato (Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated. swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32. The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform. RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweetpotato. The level of swAPX1 RNA progressively increased during cell growth in suspension cultures. In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis. swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 M), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 M) or exposure to high temperature (37°C). In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen (Pectobacterium chrysanthemi). These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses.Communicated by G. Jürgens     

Differential expression of a senescence-enhanced metallothionein gene in Arabidopsis in response to isolates of Peronospora parasitica and Pseudomonas syringae

The metallothionein gene, LSC54 , shows increased expression during leaf senescence in Brassica napus and Arabidopsis thaliana . A number of abiotic and biotic stresses have been shown to induce senescence-like symptoms in plants and, to investigate this further, the promoter of the LSC54 gene was cloned and fused to the GUS gene and transformed into Arabidopsis . The promoter was highly induced during leaf senescence and also in response to wounding; histochemical analysis indicated that this induction was localised to a few cells close to the wound site. The transgenic Arabidopsis tissue was infected with compatible and incompatible isolates of both the fungal biotroph, Peronospora parasitica and the bacterial necrotroph, Pseudomonas syringae. Incompatible isolates induced rapid cell death (the hypersensitive response) at the site of infection and, with both pathogens, early, localised expression of the GUS gene was observed. In contrast, relatively slow induction of the GUS gene was seen in the compatible interaction and this was correlated with the appearance of senescence-like symptoms in the biotrophic interaction and cell death by necrosis that occurred in response to the necrotrophic pathogen. These results suggest that there are common steps in the signalling pathways that lead to cell death in the hypersensitive response, pathogen induced necrosis and senescence.     

Isolation of the Chinese rose sHSP gene promoter and its differential regulation analysis in transgenic Arabidopsis plants

In our previous study, we identified a Rosa chinensis heat shock protein (HSP) gene, RcHSP17.8, which was induced by abiotic stresses, such as high temperature and osmotic stress. To analyze the expression of RcHSP17.8 and the function of cis-acting elements in the promoter region, a 1,910 bp fragment of the upstream sequence of the RcHSP17.8 translation initiation codon and five promoter deletion fragments were fused to a β-glucuronidase (GUS) report gene. These plasmids were transferred to Arabidopsis thaliana via Agrobacterium. GUS staining was seen in all the organs, especially in the vascular tissues after heat treatment. In transgenic Arabidopsis, GUS expression driven by the full length promoter was significantly higher under heat shock, but no GUS activity was detected under other abiotic stresses. Deletion analysis indicated that the region from −178 to −771 was essential for the promoter’s response to high temperature.     

Characterization of the Hormone and Stress-Induced Expression of <Emphasis Type="Italic">FaRE</Emphasis>1 Retrotransposon Promoter in Strawberry

Retrotransposons are the most abundant mobile elements in the plant genome and seem to play an important role in genome reorganization induced by environmental challenges. Their success in this function depends on the ability of their promoters to regulate plant adaptation to biotic and abiotic stresses. In this study, the promoter region of FaRE1 was amplified in the strawberry genome, and promoter::GUS fusion was constructed. We produced transgenic strawberry plants carrying FaRE1 promoter::GUS-fusion genes, and monitored GUS reporter activity. Histochemical and fluorimetric GUS analysis these plants showed the characteristics of the FaRE1 promoter were activated by either hormones treatments with ABA, NAA, and 2,4-D or cold stress. In addition, we found the GUS reporter was activated in the leaves of transgenic strawberry plants using 5-azaC. These results suggest that the promoter of FaRE1 may act as different signal transduction pathways, allowing FaRE1 retrotransposon to be activated in response to multiples challenges.     

玉米胚胎发生后期丰富蛋白基因的启动子克隆与功能验证

该研究在生物信息学分析的基础上,克隆玉米胚胎发生后期丰富蛋白基因(MGL3)的启动子序列(pMGL3),进行非生物逆境应答元件分析以及实时定量PCR验证其非生物逆境胁迫响应特性,构建了pMGL3启动子驱动报告基因(GUS)表达载体,基因枪法转化玉米愈伤组织,通过GUS染色验证pMGL3启动子在非生物逆境胁迫下的驱动活性。再根据启动子序列分析结果,去除不同的顺式作用元件,构建不同长度pMGL3启动子驱动报告基因GUS表达载体,农杆菌介导法转化烟草叶盘,以确定pMGL3启动子的最短活性序列。结果显示:pMGL3启动子长1 554bp,存在多种与非生物逆境胁迫应答相关的调控元件,在干旱、高盐、低温胁迫及脱落酸、乙烯诱导下驱动MGL3基因增量表达,用以驱动GUS基因转化玉米愈伤组织,在高渗、高盐、低温胁迫及脱落酸诱导下具有驱动活性,且截短至325bp仍可保持驱动活性。研究表明,pMGL3启动子的确有非生物逆境诱导启动活性,进一步验证其作用机理后可运用于玉米抗逆转基因研究。     

Effects of systemic potato response to wounding and jasmonate on the aphid Macrosiphum euphorbiae (Sternorryncha: Aphididae)

Plant induced responses are activated by multiple biotic and abiotic stresses, and may affect the interactions between a plant and phytophagous insects. The objective of this work was to evaluate the effects of different stresses inflicted to potato plants (Solanum tuberosum) on the potato aphid (Macrosiphum euphorbiae). Abiotic wounding, biotic wounding by Leptinotarsa decemlineata and treatment with volatile methyl jasmonate (MeJA) were evaluated with regard to the orientation behaviour, the feeding behaviour and the development of the potato aphids. Dual‐choice olfactometry showed that plants treated with MeJA lost their attractiveness for the potato aphids, while both abiotic and biotic wounding did not alter the orientation of aphids. Electropenetrography revealed that the feeding behaviour of aphids was only slightly disturbed by a previous L. decemlineata wounding, while it was highly disturbed by mechanical wounding and MeJA treatment. Aphid nymph survival was reduced on mechanically wounded plants, the pre‐reproductive period was lengthened and the fecundity reduced on plants treated with MeJA. Our results bring new information about the effects of various stresses inflicted to S. tuberosum on M. euphorbiae. We showed that wounding and MeJA treatment induced an antixenosis resistance in potato plants against M. euphorbiae, which may influence aphid colonization processes.     

Transgenic Analysis Reveals 5′ Abbreviated <Emphasis Type="Italic">Os</Emphasis>RGLP2 Promoter(s) as Responsive to Abiotic Stresses

Germins and germin-like proteins are ubiquitous, expressed at various developmental stages and in response to various abiotic and biotic stresses. In this study, to functionally validate the OsRGLP2 promoter, 5′ deletion analysis of the promoter sequences was performed and the deletion fragments fused with the β-glucuronidase (GUS) and green fluorescent protein reporter genes were used for transient expression in tobacco as well as for generating stable transgenic Arabidopsis plants. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the P-1063 and P-565 when subjected to abiotic stresses. Histochemical analysis of transgenic Arabidopsis plants revealed expression of reporter gene in root, leaf and stem sections of plants harboring P-1063 and P-565. Real-time qPCR analysis of transiently expressed tobacco leaves and transgenic Arabidopsis plants subjected to several abiotic stresses supported histochemical data and showed that P-565 responded to all the stresses to which the full-length promoter was responsive. The data suggest that P-565 may be a good alternative to full-length promoter region that harbors the necessary cis-elements in providing stable and high level of expression in response to wound, salt and temperature stresses.     

Sweetpotato late embryogenesis abundant 14 (<Emphasis Type="Italic">IbLEA14</Emphasis>) gene influences lignification and increases osmotic- and salt stress-tolerance of transgenic calli

Late embryogenesis abundant 14 (LEA14) cDNA was isolated from an EST library prepared from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Quantitative RT-PCR revealed a variety of different IbLEA14 expression patterns under various abiotic stress conditions. IbLEA14 expression was strongly induced by dehydration, NaCl and abscisic acid treatments in sweetpotato plants. Transgenic sweetpotato non-embryogenic calli harboring IbLEA14 overexpression or RNAi vectors under the control of CaMV 35S promoter were generated. Transgenic calli overexpressing IbLEA14 showed enhanced tolerance to drought and salt stress, whereas RNAi calli exhibited increased stress sensitivity. Under normal culture conditions, lignin contents increased in IbLEA14-overexpressing calli because of the increased expression of a variety of monolignol biosynthesis-related genes. Stress treatments elicited higher expression levels of the gene encoding cinnamyl alcohol dehydrogenase in IbLEA14-overexpressing lines than in control or RNAi lines. These results suggest that IbLEA14 might positively regulate the response to various stresses by enhancing lignification.     

Hormonal and stress induction of the gene encoding common bean acetyl-coenzyme A carboxylase

Regulation of the cytosolic acetyl-coenzyme A carboxylase (ACCase) gene promoter from common bean (Phaseolus vulgaris) was studied in transgenic Arabidopsis (Arabidopsis thaliana) plants using a beta-glucuronidase (GUS) reporter gene fusion (PvACCase::GUS). Under normal growth conditions, GUS was expressed in hydathodes, stipules, trichome bases, flowers, pollen, and embryos. In roots, expression was observed in the tip, elongation zone, hypocotyl-root transition zone, and lateral root primordia. The PvACCase promoter was induced by wounding, Pseudomonas syringae infection, hydrogen peroxide, jasmonic acid (JA), ethylene, or auxin treatment. Analysis of PvACCase::GUS expression in JA and ethylene mutants (coronatine insensitive1-1 [coi1-1], ethylene resistant1-1 [etr1-1], coi1-1/etr1-1) suggests that neither JA nor ethylene perception participates in the activation of this gene in response to wounding, although each of these independent signaling pathways is sufficient for pathogen or hydrogen peroxide-induced PvACCase gene expression. We propose a model involving different pathways of PvACCase gene activation in response to stress.     

A comparison of two class 10 pathogenesis-related genes from alfalfa and their activation by multiple stresses and stress-related signaling molecules

A collection of 29 pathogenesis-related 10 (PR10) genes of Medicago sativa and Medicago truncatula showed that they were almost all obtained from cDNA libraries of tissues undergoing abiotic or biotic stresses. The predicted proteins could be divided into two subclasses, PR10.1 and PR10.2, but in silico predicted models of their three-dimensional structures revealed that they could be further divided based on size of the hydrophobic internal cavity and number of β-bulges. A comparison of the expression of two highly similar M. sativa subclass PR10.1 genes, MsPR10.1A and MsPR10.1B, predicted to have similar sized hydrophobic internal cavities, but a different number of β-bulges revealed differences in their expression patterns. MsPR10.1A was induced faster than MsPR10.1B by ABA, ethylene, and X. campestris pv. alfalfae, but slower than MsPR10.1B by harvesting and wounding. Unlike MsPR10.1A, MsPR10.1B expression was induced in non-harvested tissues following harvesting, but was not induced by heat treatment. Histochemical observations of Nicotiana benthamiana transformed with 657 bp of the MsPR10.1A promoter fused to the β-glucuronidase (GUS) gene showed that GUS expression was wound-inducible in leaves, which was consistent with MsPR10.1A expression in alfalfa leaves. GUS expression in stems and leaves was mostly in vascular tissue. The MsPR10.1A promoter may be valuable in controlling the expression in vascular tissues and disease resistance.     

A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells

A strong oxidative stress-inducible peroxidase (POD) promoter was cloned from sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco plants and cultured cells in terms of environmental stress. A POD genomic clone (referred to as SWPA2) consisted of 1824 bp of sequence upstream of the translation start site, two introns (743 bp and 97 bp), and a 1073 bp coding region. SWPA2 had previously been found to encode an anionic POD which was highly expressed in response to oxidative stress. The SWPA2 promoter contained several cis-element sequences implicated in oxidative stress such as GCN-4, AP-1, HSTF, SP-1 reported in animal cells and a plant specific G-box. Employing a transient expression assay in tobacco protoplasts, with five different 5-deletion mutants of the SWPA2 promoter fused to the -glucuronidase (GUS) reporter gene, the 1314 bp mutant deletion mutant showed about 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in transgenic tobacco plants under the control of the –1314 SWPA2 promoter was strongly induced in response to environmental stresses including hydrogen peroxide, wounding and UV treatment. Furthermore, GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the –1314 bp SWPA2 promoter-GUS fusion was strongly expressed after 15 days of subculture compared to other deletion mutants. We anticipate that the –1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.