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.     

Different Mechanisms of Four Aluminum (Al)-Resistant Transgenes for Al Toxicity in Arabidopsis

We have characterized the mechanism of action of four transgenes (AtBCB [Arabidopsis blue copper-binding protein], parB [tobacco (Nicotiana tabacum) glutathione S-transferase], NtPox [tobacco peroxidase], and NtGDI1 [tobacco GDP dissociation inhibitor]) that independently Al resistance on transgenic Arabidopsis. All four transgenic lines showed lower deposition of callose after Al treatment than the Landsberg erecta ecotype of Arabidopsis, confirming that the four genes function to ameliorate Al toxicity. Influx and efflux experiments of Al ions suggested that the AtBCB gene may suppress Al absorption, whereas expression of the NtGDI1 gene promotes a release of Al in the root tip region of Arabidopsis. The total enzyme activities of glutathione S-transferases or peroxidases in transgenic lines carrying either the parB or NtPox genes were significantly higher than in the Landsberg erecta ecotype of Arabidopsis, and these enzyme activities were maintained at higher levels during Al stress. Furthermore, lipid peroxidation caused by Al stress was repressed in these two transgenic lines, suggesting that overexpression of these two genes diminishes oxidative damage caused by Al stress. Al-treated roots of transgenic plants were also stained by 4',6-diamino-2-phenylindole to monitor cell death caused by Al toxicity. The result suggested that cell death is repressed in the NtPox line. Analysis of F(1) hybrids between the four transgenic lines suggests that more resistant transgenic plants can be constructed by combinations of these four genes.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyl-transferase (FTase) gene was examined using transgenic expression of the β-glucuronidase (GUS) gene fused to a 3.2 kb 5′ upstream sequence of the gene encoding the pea FTase β subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase β subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

Functional characterization of NtCDPK1 in tobacco

We previously showed that NtCDPK1, a tobacco cal-cium-dependent protein kinase, interacts with and phosphorylates the Rpn3 regulatory subunit of the 26S proteasome, and that both NtCDPK1 and Rpn3 are mainly expressed in rapidly proliferating tissues, in-cluding shoot and root meristem. In this study, we ex-amined NtCDPK1 expression in roots using GUS ex-pression in transgenic Arabidopsis plants, and investi-gated its function in root development by generating transgenic tobacco plants carrying a sense NtCDPK1 transgene. GUS activity was first detected in roots two days after sowing. In later stages, strong GUS expres-sion was detected in the root meristem and elongation zone, as well as the initiation sites and branch points of lateral roots. Transgenic tobacco plants in which NtCDPK1 expression was suppressed were smaller, and their root development was abnormal, with reduced lateral root formation and less elongation. These re-sults suggest that NtCDPK1 plays a role in a signaling pathway regulating root development in tobacco.     

An Arabidopsis thaliana root-specific kinase homolog is induced by dehydration, ABA, and NaCl

Genomic and cDNA clones have been isolated for an Arabidopsis thaliana gene, ARSK1, that encodes a protein with structural similarities to serine/threonine kinases. Expression of ARSK1 is root specific and is induced by exposing roots to air during growth or by treatment of roots with ABA or NaCl. ARSK1 gene expression in transgenic plants is confined to cells in the tissues of the root as measured by β-glucuronidase (GUS) expression from an ARSK1 gene promoter—GUS gene construct. Transverse sections of the stained roots further defined the tissue-specificity; high levels of expression in the epidermal, endoepidermal and cortex regions, but no or very little expression in the vascular system. Another feature of the expression pattern of the ARSK1 gene was a gradual increase in the expression level along the root with the highest level of expression in the region closest to the root meristem. These studies suggest that ARSK1 may have a role in the signal transduction pathway of osmotic stress.     

The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana, indicating the conservation of signalling pathways between angiosperms and gymnosperms

A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.     

拟南芥醛还原酶编码基因At3g04000表达模式分析及过量表达转基因植株获得

植物体内的α,β-不饱和活性醛类化合物对植物细胞具有毒害作用,清除这些α,β-不饱和活性醛类化合物对于植物细胞维持正常的生命活动至关重要。前人研究报道通过体外酶活测定和异源瞬时表达鉴定拟南芥 At3g04000基因编码的蛋白为 NADPH 依赖的叶绿体醛还原酶(Arabidopsis NADPH-dependent chloroplastic aldehyde reductases, AtChlADRs),推测其在清除叶绿体中长链(≥5)α,β-不饱和醛类物质中具有重要的功能。该研究主要构建了拟南芥 At3g04000基因的表达模式分析载体 ProAt3g04000：GUS、亚细胞定位分析载体At3g04000-EGFP 和过量表达载体 At3g04000-OE,并获得了转基因拟南芥,并通过实时定量 PCR 分析了At3g04000基因在拟南芥不同组织中的转录水平。结果表明：拟南芥 At3g04000基因在幼苗中的转录水平最高,在莲座叶、茎生叶、花序和角果中均有较高的转录水平;而在根部和茎秆中的转录水平较低。通过对ProAt3g04000：GUS 转基因植株的 GUS 染色分析可知,At3g04000基因在子叶、莲座叶和萼片的维管组织和保卫细胞中均有较强的表达,在根的维管组织中有较弱的表达。通过共聚焦显微镜对 At3g04000-EGFP 转基因植株的观察和分析发现,At3g04000不是定位于叶绿体中,而是定位在细胞质和细胞核中。该研究结果为深入研究拟南芥醛还原酶编码基因 At3g04000的功能奠定了基础。     

Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana

The plant, Arabidopsis thaliana, contains two S-adenosylmethionine synthetase-encoding genes (sam). Here, we analyze the structure and expression of the sam-2 gene and compare it with the previously described sam-1 gene. Northern-blot analysis using gene-specific probes shows that both sam-1 and sam-2 are highly expressed in stem, root, and callus tissue. This similar expression pattern might be mediated by the presence of three highly conserved sequences in the 5' region of both sam genes. Using a chimeric beta-glucuronidase (GUS)-encoding gene, we show that in transgenic tobacco plants, 748 bp of 5' sam-1 sequences generate high GUS activity in the same type of tissues as previously observed in transgenic A. thaliana plants. A deletion analysis of these 5' sam-1 sequences indicates that 224 bp of 5' sam-1 sequences can still induce higher expression of the gene in stem and root relative to leaf. However, the level of expression is reduced when compared to the expression level obtained with the full-length promoter.     

An RNA-binding protein, AtRBP1, is expressed in actively proliferative regions in Arabidopsis thaliana

A cDNA clone, named XF41, that encodes an RNA-binding protein was isolated from Arabidopsis thaliana. The deduced protein, named AtRBP1, contains two conserved consensus sequence-type RNA-binding domains (CS-RBDs) in the N-terminal half, a putative PY motif (a target of a WW domain) in the center, and uncharacterized C-terminal domain. A binding assay demonstrated that the AtRBP1 can bind to single-stranded nucleic acids in vitro. Analysis of localization of the GUS activity of transgenic Arabidopsis thaliana plants that have the chimeric gene containing the upstream sequence of the AtRBP1 gene and GUS gene demonstrated that the AtRBP1 gene is expressed in meristematic tissues such as the vegetative shoot apex and root tips, developing organs such as floral buds and pistils of young flowers, abscission layers of immature siliques and junctions of pedicels. Considering the specificity of the expression, AtRBP1 may be required in the progress of cell proliferation.     

Engineering a root-specific, repressor-operator gene complex

Strong, tissue-specific and genetically regulated expression systems are essential tools in plant biotechnology. An expression system tool called a 'repressor-operator gene complex' (ROC) has diverse applications in plant biotechnology fields including phytoremediation, disease resistance, plant nutrition, food safety, and hybrid seed production. To test this concept, we assembled a root-specific ROC using a strategy that could be used to construct almost any gene expression pattern. When a modified E. coli lac repressor with a nuclear localization signal was expressed from a rubisco small subunit expression vector, S1pt::lacIn, LacIn protein was localized to the nuclei of leaf and stem cells, but not to root cells. A LacIn repressible Arabidopsis actin expression vector A2pot was assembled containing upstream bacterial lacO operator sequences, and it was tested for organ and tissue specificity using beta-glucuronidase (GUS) and mercuric ion reductase (merA) gene reporters. Strong GUS enzyme expression was restricted to root tissues of A2pot::GUS/S1pt::lacIn ROC plants, while GUS activity was high in all vegetative tissues of plants lacking the repressor. Repression of shoot GUS expression exceeded 99.9% with no evidence of root repression, among a large percentage of doubly transformed plants. Similarly, MerA was strongly expressed in the roots, but not the shoots of A2pot::merA/S1pt::lacIn plants, while MerA levels remained high in both shoots and roots of plants lacking repressor. Plants with MerA expression restricted to roots were approximately as tolerant to ionic mercury as plants constitutively expressing MerA in roots and shoots. The superiority of this ROC over the previously described root-specific tobacco RB7 promoter is demonstrated.     

Arabidopsis Alcohol Dehydrogenase Expression in Both Shoots and Roots Is Conditioned by Root Growth Environment

It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.