Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato

Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings [12]. The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression. Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes. However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters. Chimeric gene fusions of each tomlox promoter with the -glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation. GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment. No GUS activity was detected in tomloxB-gus seedlings. Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter. During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA. In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA. In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella. These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness.     

Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants

Polygalacturonase (PG, EC 3.2.1.15), an enzyme commonly found in ripening fruit, has also been shown to be associated with abscission. A zone-specific rise in PG activity accompanies the abscission of both leaves and flowers of tomato (Lycopersicon esculentum Mill.) plants. Studies of transgenic plants expressing an antisense RNA for fruit PG indicate that although the enzyme activity in transgenic fruit is < 1 % of that in untransformed fruit, the PG activity in the leaf abscission zone increases during separation to a similar value to that in untransformed plants. The timing and rate of leaf abscission in transgenic plants are unaffected by the introduction of the antisense gene. A polyclonal antibody raised against tomato fruit PG does not recognise the leaf abscission protein. Furthermore a complementary DNA (cDNA) clone (pTOM6), which has been demonstrated to code for fruit PG, does not hybridise to mRNA isolated from the abscission-zone region of tomato leaves. These results indicate that the PG protein in abscission zones of tomato is different from that in the fruit, and that the gene coding for this protein may also be different.Abbreviation PG polygalacturonase The authors of this paper are grateful to David Jackson of the John Innes Institute, Norwich, UK for his assistance with the in-situ hybridisation work. This research was supported by an Agricultural and Food Research Council Post-Doctoral award to J.E.T., and by a grant to D.G. from the Science and Engineering Research Council Biotechnology Directorate in association with ICI seeds. The work was carried out under Ministry of Agriculture, Food and Fisheries licences.     

Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants

GUS (uidA) reporter gene expression for two sugarcane polyubiquitin promoters, ubi4 and ubi9, was compared to expression from the maize Ubi-1 promoter in stable transgenic rice (only ubi9) and sugarcane (ubi4 and ubi9). Ubi9 drove high-level GUS expression, comparable to the maize Ubi-1 promoter, in both callus and regenerated plants of rice transformed by Agrobacterium. This high level expression was inherited in R1 plants. Expression from ubi4 and ubi9 was quite high in sugarcane callus transformed via particle bombardment. Expression dropped to very low or undetectable levels in the resulting plants; this drop in expression resulted from PTGS. PTGS in regenerated sugarcane plants also occurred with the maize Ubi-1 promoter. In sugarcane callus, ubi4 was HS inducible, but ubi9 was not. This physiological difference corresponds to a MITE insertion that is present in the putative HSEs of ubi9 but not present in ubi4.     

Developmental regulation of expression of the malate synthase gene in transgenic plants

The cucumber malate synthase (MS) gene, including 1856 bp of 5 non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5 sequence was linked to the -glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of -glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed.     

Regulation of expression of a wound-inducible tomato inhibitor I gene in transgenic nightshade plants

A wound-inducible proteinase Inhibitor I gene from tomato containing 725 bp of the 5 region and 2.5 kbp of the 3 region was stably incorporated into the genome of black nightshade plants (Solanum nigrum) using an Agrobacterium Ti plasmid-derived vector. Transgenic nightshade plants were selected that expressed the tomato Inhibitor I protein in leaf tissue. The leaves of the plants contained constitutive levels of the inhibitor protein of up to 60 g/g tissue. These levels increased by a factor of about two in response to severe wounding. Only leaves and petioles exhibited the presence of the inhibitor, indicating that the gene exhibited the same tissue specificity of expression found in situ in wounded tomato leaves. Inhibitor I was extracted from leaves of wounded transformed nightshade plants and was partially purified by affinity chromatography on a chymotrypsin-Sepharose column. The affinity-purified protein was identical to the native tomato Inhibitor I in its immunological reactivity and in its inhibitory activity against chymotrypsin. The protein exhibited the same M _r of 8 kDa as the native tomato Inhibitor I and its N-terminal amino acid sequence was identical to that of the native tomato inhibitor I, indicating that the protein was properly processed in nightshade plants. These expriments are the first report of the expression of a member of the wound-inducible tomato Inhibitor I gene family in transgenic plants. The results demonstrate that the gene contains elements that can be regulated in a wound-inducible, tissuespecific manner in nightshade plants.     

Local and systemic induction of two defense-related subtilisin-like protease promoters in transgenic Arabidopsis plants. Luciferin induction of PR gene expression

Following a pathogenic attack, plants are able to mount a defense response with the coordinated activation of a battery of defense-related genes. In this study we have characterized the mode of expression of the P69B and P69C genes from tomato (Lycopersicon esculentum Mill.), which encodes two closely related subtilisin-like proteases associated with the defense response. We have compared the mode of gene regulation in heterologous transgenic Arabidopsis plants harboring promoter-beta-glucuronidase (GUS) and promoter-luciferase (LUC) gene fusions for these two genes. These studies revealed that the P69B and P69C promoters are induced by salicylic acid as well as during the course of both a compatible and an incompatible interaction with Pseudomonas syringae. Furthermore, P69B and P69C expression takes place in both the local and the distal (noninoculated) leaves upon inoculation with bacteria but following different and unique tissue-specific patterns of expression that are also different to that described for most other classical PR genes. Also, we report that luciferin, the substrate for the reporter luciferase (LUC) gene, is able to activate expression of PR genes, and this may pose a problem when using this gene reporter system in studies related to plant defense.     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.     

甜蛋白Brazzein基因在番茄果实中的特异表达

西瓜(Citrullus vulgaris S.)来源的AGPL1启动子在番茄(Lycopersicon esculentum L.)果实中具有较强的特异性驱动功能。将该启动子与甜味蛋白基因Brazzein融合构建植物表达载体, 通过根癌农杆菌(Agrobacterium tumefaciens)介导法成功地进行了对番茄的遗传转化, 获得转化植株。组织化学法、PCR特异扩增、Southern杂交分析及RT-PCR检测, 表明Brazzein基因已整合到转基因番茄植株基因组中并且稳定表达。通过AGPL1果实特异启动子的调控, 在不改变果实其他性状的前提下提高了番茄果实甜味品质, 并为甜蛋白的生产提供经验。     

Sugar regulation of gene expression in plants

The molecular details of sugar sensing and sugar-mediated signal transduction pathways are unclear but recent results suggest that hexokinase functions as an important plant sugar sensor in a way that is similar to that found in yeast. The use of mutants in Arabidopsis defective in specific signaling steps is of particular importance because these give access to the genes encoding components in the signaling pathways. In addition, the physiological analysis of such mutants may reveal the interaction of sugar-induced signaling pathways and those induced by other stimuli such as environmental or biotic stress.     

MicroRNA regulation of gene expression in plants

It has only been a few years since we began to appreciate that microRNAs provide an unanticipated level of gene regulation in both plants and metazoans. The high level of complementarity between plant microRNAs and their target mRNAs has allowed rapid progress towards the elucidation of their varied biological functions. MicroRNAs have been shown to regulate diverse developmental processes, including organ separation, polarity, and identity, and to modulate their own biogenesis and function. Recently, they have also been implicated in some processes outside of plant development.     

The light-dependent regulation of gene expression during plastid development in higher plants

In recent years there has been a considerable increase in our understanding of the manner by which light affects gene expression during chloroplast development. In most systems that have been studied, light acts through sensitive photoreceptor molecules and quantitatively increases or represses the level of expression of specific nuclear-and plastid-encoded genes. Although the mechanisms are obscure, a picture is beginning to emerge in which the coordination of nuclear and plastid gene expression is controlled by regulatory mechanisms originating within their respective subcellular compartments. This review summarizes some of our current knowledge concerning the nature of light-regulated gene expression in higher plants and provides a prospectus for future research in this area.     

Porcine growth hormone gene expression from viral promoters in transgenic swine

Abstract

The production of porcine growth hormone (pGH) from novel expression vectors containing the promoter/enhancer elements of the Moloney murine leukemia virus (MLV) LTR or the human cytomegalovirus (CMV) immediate early gene was examined in transgenic swine. Both fusion genes resulted in elevated levels of serum pGH, elevation of insulin‐like growth factor 1 (IGF‐1), and a pronounced decrease in carcass fat deposition. The two viral promoter/enhancer elements were constitutively active in the transgenic swine throughout the life of the animals. In individual swine, the CMV‐pGH transgene was expressed predominantly in the pancreas while the MLV‐pGH transgene was expressed in a wide variety of tissues. These swine were infertile, had insulin resistance, and demonstrated an accelerated form of osteochondritis dissicans. Our results show that excess pGH produces a phenotype identical to that seen in swine expressing heterologous growth hormones, and provides a baseline for assessing the overall efficiency of producing transgenic swine. Furthermore, our data suggests that unregulated pGH production, even at 15 times normal levels and independent of the tissue source, has adverse effects that outweigh the desired reduction in carcass fat deposition in transgenic swine.     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

Kanamycin resistance during in vitro development of pollen from transgenic tomato plants

Effects of kanamycin on pollen germination and tube growth of pollen from non-transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin, however, severely affected tube growth of pollen from non-transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200–400 mgl-1. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development.Abbreviations ATW Agrobacterium Tomato Wageningen - KAN Kanamycin - KANr Kanamycin resistant - KANs Kanamycin sensitive - mRNA messenger RNA - NPT Neomycin phosphotransferase     

Developmental regulation and tissue-specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plants

The heat shock (hs) response during plant growth and development was analyzed in tobacco and Arabidopsis using chimaeric -glucuronidase reporter genes (hs-Gus) driven by a soybean hs promoter. Fluorimetric measurements and histochemical staining revealed high Gus activities in leaves, roots, and flowers exclusively after heat stress. The highest levels of heat-inducible expression were found in the vascular tissues. Without heat stress, a developmental induction of hs-Gus was indicated by the accumulation of high levels of Gus in transgenic tobacco seeds. There was no developmental induction of hs-Gus in Arabidopsis seeds. In situ hybridization to the RNA of the small heat shock protein gene Athsp17.6 in tissue sections revealed an expression in heat-shocked leaves but no expression in control leaves of Arabidopsis. However, a high level of constitutive expression of hs gene was detected in meristematic and provascular tissues of the Arabidopsis embryo. The developmental and tissue-specific regulation of the hs response is discussed.Abbreviations hs heat shock - Hsp heat shock protein(s) - hs Gus: heat-inducible Gus gene(s) - HSE heat shock element(s) - HSF heat shock factor - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide - Gus -glucuronidase - DAF days after flowering - SAR scaffold attachment region