Wound-inducible and organ-specific expression of ORF13 from Agrobacterium rhizogenes 8196 T-DNA in transgenic tobacco plants

Tissue-specific expression of the ORF13 promoter from Agrobacterium rhizogenes 8196 was assessed throughout the development of transgenic tobacco plants using a GUS reporter gene. ORF13 exhibited high activity in roots but with different patterns of expression. The activity of the ORF13 promoter in vascular tissues increased from the base to the tip of the stem. The ORF13 promoter is wound inducible in a limited area adjacent to the wound site. The time course of wound induction of ORF13 in transgenic tobacco containing an ORF13 promoter-GUS translational fusion was similar to that previously described for genes involved in plant defense responses. A series of 5′ deletions of the ORF13 promoter fused to the β-glucuronidase gene was examined for expression in roots and leaves of transgenic plants. Cis-acting elements that modulate quantitative expression of the transgene after wounding were detected. Received: 11 July 1996 / Accepted: 19 November 1996     

Wounding changes the spatial expression pattern of the arabidopsis plastid omega-3 fatty acid desaturase gene (FAD7) through different signal transduction pathways.

The Arabidopsis FAD7 gene encodes a plastid omega-3 fatty acid desaturase that catalyzes the desaturation of dienoic fatty acids in membrane lipids. The mRNA levels of the Arabidopsis FAD7 gene in rosette leaves rose rapidly after local wounding treatments. Wounding also induced the expression of the FAD7 gene in roots. To study wound-responsive expression of the FAD7 gene in further detail, we analyzed transgenic tobacco plants carrying the -825 Arabidopsis FAD7 promoter-beta-glucuronidase fusion gene. In unwounded transformants, FAD7 promoter activity was restricted to the tissues whose cells contained chloroplasts. Activation of the FAD7 promoter by local wounding treatments was more substantial in stems (29-fold) and roots (10-fold) of transgenic plants than it was in leaves (approximately two-fold). Significant induction by wounding was observed in the overall tissues of stems and included trichomes, the epidermis, cortex, vascular system, and the pith of the parenchyma. Strong promoter activity was found preferentially in the vascular tissues of wounded roots. These results indicate that wounding changes the spatial expression pattern of the FAD7 gene. Inhibitors of the octadecanoid pathway, salicylic acid and n-propyl gallate, strongly suppressed the wound activation of the FAD7 promoter in roots but not in leaves or stems. In unwounded plants, exogenously applied methyl jasmonate activated the FAD7 promoter in roots, whereas it repressed FAD7 promoter activity in leaves. Taken together, wound-responsive expression of the FAD7 gene in roots is thought to be mediated via the octadecanoid pathway, whereas in leaves, jasmonate-independent wound signals may induce the activation of the FAD7 gene. These observations indicate that wound-responsive expression of the FAD7 gene in aerial and subterranean parts of plants is brought about by way of different signal transduction pathways.     

Wound-response regulation of the sweet potato sporamin gene promoter region

Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation. In an effort to demonstrate the regulatory mechanism of wound response on the sporamin gene, a 1.25 kb sporamin promoter was isolated for studying the wound-induced signal transduction. Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter. A construct containing the sporamin promoter fused to a -glucuronidase (GUS) gene was transferred into tobacco plants by Agrobacterium-mediated transformation. The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots. This expression pattern was similar to that of the sporamin gene in sweet potatoes. Exogenous application of methyl jasmonate (MeJA) activated the sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants. A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on sporamin gene expression. In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the sporamin promoter function that was stimulated by wound and MeJA treatments. In conclusion, wound-response expression of the sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.     

Functional Characterization of the Tau Class Glutathione-S-Transferases Gene (SbGSTU) Promoter of Salicornia brachiata under Salinity and Osmotic Stress

Reactive oxygen or nitrogen species are generated in the plant cell during the extreme stress condition, which produces toxic compounds after reacting with the organic molecules. The glutathione-S-transferase (GST) enzymes play a significant role to detoxify these toxins and help in excretion or sequestration of them. In the present study, we have cloned 1023 bp long promoter region of tau class GST from an extreme halophyte Salicornia brachiata and functionally characterized using the transgenic approach in tobacco. Computational analysis revealed the presence of abiotic stress responsive cis-elements like ABRE, MYB, MYC, GATA, GT1 etc., phytohormones, pathogen and wound responsive motifs. Three 5’-deletion constructs of 730 (GP2), 509 (GP3) and 348 bp (GP4) were made from 1023 (GP1) promoter fragment and used for tobacco transformation. The single event transgenic plants showed notable GUS reporter protein expression in the leaf tissues of control as well as treated plants. The expression level of the GUS gradually decreases from GP1 to GP4 in leaf tissues, whereas the highest level of expression was detected with the GP2 construct in root and stem under control condition. The GUS expression was found higher in leaves and stems of salinity or osmotic stress treated transgenic plants than that of the control plants, but, lower in roots. An efficient expression level of GUS in transgenic plants suggests that this promoter can be used for both constitutive as well as stress inducible expression of gene(s). And this property, make it as a potential candidate to be used as an alternative promoter for crop genetic engineering.     

Differential expression of antisense in regenerated tobacco plants transformed with an antisense version of a tomato ACC oxidase gene

Ethylene production was measured during vegetative and reproductive development in normal tobacco plants and in transgenic tobacco plants carrying antisense genes for tomato ACC oxidase driven by the 35S CaMV promoter (Hamilton et al., 1990). When expressed in three independently derived transgenic plants, the antisense ethylene gene failed to affect ethylene production in young/mature leaves or in stems but it did inhibit ethylene production in roots by 37–58%. Ethylene production in developing flowers (i.e. from small unopened flower buds up until open flowers at anthesis) was not affected in transgenic plants but ethylene production in fruits was inhibited by 35%. The most dramatic effect on ethylene production in transgenic plants was seen immediately after wounding leaf tissue, in which case the antisense gene inhibited wound ethylene production by 72%. Thus, the antisense gene composed of a 35S CaMV promoter driving a heterologous ACC oxidase sequence had differential effects on ethylene production in tobacco plants.     

Analysis of a maize α-tubulin gene promoter by transient expression and in transgenic tobacco plants

The pattern of expression directed by the promoter of the maize Tub α 1 gene was investigated by analysis of chloramphenicol acetyl transferase (CAT) and β-glucuronidase (GUS) activities in transient expression experiments of maize and tobacco protoplasts. The same promoter was also investigated by histochemical GUS analysis in transgenic tobacco plants containing promoter gene fusions. As determined by histochemical tests, the Tub α 1 promoter gene preferentially directs GUS expression in regenerating root tip meristems and pollen. This pattern corresponds to the distinctive features of natural expression of the gene in maize as determined by Northern analysis. However, no expression is observed in other meristematic tissues of the transgenic tobacco plants, as in shoot apex or in coleoptiles, which is weakly detected in maize. Analysis of the regulatory properties of 5' promoter deletions showed that the proximal region of the promoter, from positions −1410 or −449 to 15 bp upstream of the ATG, is sufficient to establish the qualitative pattern of expression in transgenic tobacco plants. Deletions to positions −352 or −117 abolished the expression in roots, but not in pollen, suggesting that upstream of these positions there are elements responsible for the pattern in root. Further deletions abolished all the promoter activity, suggesting that this promoter region contains the elements essential for expression in pollen. The different patterns and levels of transient and stable expression are discussed.     

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.     

Effect of localized reduction of gibberellins in different tobacco organs on drought stress tolerance and recovery

Drought resistance is increased in plants by the absence of the hormone gibberellic acid (GA) or by a lack of GA sensitivity. We studied the effects of tissue-specific reduction in GA levels on drought tolerance, on recovery from drought stress, and on primary and secondary growth using transgenic tobacco plants expressing the GA-inactivating gene PtGA2ox ₁ (GA 2-oxidase) specifically in leaves, stems, or roots. Localized reduction of bioactive GA₁ levels was achieved by tissue-specific expression of the PtGA2ox ₁ gene in leaves using the rbcs promoter (LD plants), in roots using the TobRB7 promoter (RD plants), and in stems using the LMX5 promoter (SD plants). In response to drought stress, all transgenic tobacco plants exhibited reduced primary and secondary growth and increased drought tolerance with a corresponding reduction in malondialdehyde levels, higher relative water content, increased proline and sugar content, and elevated peroxidase, superoxide dismutase, and catalase activities relative to wild-type plants. The highest level of drought tolerance and the most rapid recovery from stress was achieved by localized reduction of GA₁ in the roots of the RD transgenic plants. In addition, although the total bioactive GA₁ content in RD and LD plants was essentially identical, the heights of LD plants were significantly greater and drought tolerance was significantly less than in RD plants. It is possible that the site of gibberellin-related gene expression plays an important role in the balance between growth and drought tolerance.     

Gain of function assays identify non- rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity

This study tested the morphogenetic potential of 15 open reading frames of the TL-DNA of Agrobacterium rhizogenes strain HRI. These open reading frames were expressed individually under the control of the 35S RNA promoter in transgenic tobacco plants ( Nicotiana tabacum L.). Expression of three T-DNA loci, ORF3n, ORF8 and ORF13, alters plant morphogenesis or the response of transgenic tissues to plant hormones. ORF3n transgenic plants are characterized by retarded flowering, altered internode elongation, altered leaf shape and, in particular, leaf tip necrosis. ORF3n and ORF8 expression reduces the sensitivity to auxin and cytokinin in combination or auxin alone. Tetracycline-dependent expression of ORF13 overcomes a selection of low levels of expression during plant regeneration and reveals a strong inhibitory effect of the ORF13 gene product on cell division and cell elongation. We conclude that the A. rhizogenes TL-DNA harbors genetic information that is important for pathogenicity apart from the well studied rol genes. We propose that these genes play mainly a negative regulatory role during pathogenesis. Moreover, these loci might be relevant to successful infections in specific host plants.     

Cloning and characterization of a cold inducible Pal promoter from Fagopyrum tataricum

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in biosynthesis pathway of flavonoids and plays an important role in plant stress resistance. In this study, the 5’ flanking region of phenylalanine ammonia-lyase gene was isolated from Fagopyrum tataricum by thermal asymmetric interlaced PCR method, named PFtPal (GenBank: KF463139). To investigate the functional properties of PFtPal, we constructed a series of plant expression vectors that contained different promoter fragments resulting from nest deletions and had successfully transformed them into tobacco leaves by Agrobacterium tumefaciens. Histochemical assay of GUS suggested that PFtPal could drive GUS gene expression in leaves and roots, while GUS activity was not detected in the stem. In addition, the region of ?274 bp to ?1 bp was enough to drive normal expression of GUS gene. Low temperature treatment of transgenic tobacco plants demonstrated that PFtPal conferred cold-induced expression. Taken together, our study will help to better understand the Pal promoter, and provides a candidate promoter for molecular breeding in Fagopyrum plants.     

Differential expression of the potato proteinase inhibitor II promoter in transgenic tobacco

We studied temporal and spatial expression patterns of the potato proteinase inhibitor II (PI-II) promoter, using transgenic tobacco (Nkotiana tabacum L cv. Xanthi) plants that carried a fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) gene. Pl-ll promoter activity was low when plants were young, but increased as plants grew. In 8-week-old plants, old leaves showed higher activity than young leaves. At flowering stage (ca. 15 weeks), the overall promoter activity was reduced to a lower level except in the petals. Compared with stems or petioles at the flowering stage, the roots and floral organs showed minimal activity for the Pl-ll promoter. We used several environmental stimuli to examine the induction of the Pl-ll promoter in different organs. Promoter induction was effected by wounding or methyl jasmonate in stems, petioles, sepals, and leaves. The induction was highest in leaves, as was sucrose-enhanced wound induction. These results suggest that the Pl-ll gene is temporally and spatially regulated. We also established a transient assay system in tobacco BY2 suspension cells to elucidate the upstream regulatory region of the Pl-ll promoter. A field strength of 0.75 kV/cm and 400 μF capacitance were optimal electroporation conditions for our transient assay.     

玉米19kD醇溶贮藏蛋白基因启动子种子特异性表达控制区段的分析

为研究玉米（Ｚｅａｍａｙｓ　Ｌ．）１９ｋＤ醇溶贮藏蛋白（ｚｅｉｎ）基因启动子种子特异性表达的控制区段,将全长６９４ｂｐ的启动子进行５’端缺失,共得到６个缺失突变体,长度分别为４８８ｂｐ、３７８ｂｐ、３０２ｂｐ、１５２ｂｐ、１２４ｂｐ和８５ｂｐ。将６个片段分别与报告基因ｇｕｓ连接构建成表达载体ｐＤＧＢ系列,经土壤农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ）介导转化,引入烟草。ＧＵＳ活性检测证明,４８８ｂｐ启动子片段能促使ｇｕｓ基因在种子中特异表达。３７８ｂｐ、３０２ｂｐ、１５２ｂｐ和１２４ｂｐ片段启动子引导的ｇｕｓ基因在烟草根、叶柄、种子中均可表达。