Characterization of abiotic stress-responsive <Emphasis Type="Italic">Arabidopsis thaliana RD29A</Emphasis> and <Emphasis Type="Italic">RD29B</Emphasis> genes and evaluation of transgenes

Abiotic stresses have adverse effects on plant growth and productivity. The homologous RD29A and RD29B genes are exquisitely sensitive to various abiotic stressors. Therefore, RD29A and RD29B gene sequences have potential to confer abiotic stress resistance in crop species grown in arid and semi-arid regions. To our knowledge, no information on the physiological roles of the proteins encoded by RD29A and RD29B are available in the literature. To understand how these proteins function, we used reverse genetic approaches, including identifying rd29a and rd29b T-DNA knockout mutants, and examining the effects of complementing transgenes with the genes under control of their native promoters and chimeric genes with the native promoters swapped. Four binary vectors with the RD29A and RD29B promoters upstream of the cognate RD29A and RD29B cDNAs and as chimeric genes with noncognate promoters were used to transform rd29a and rd29b plants. Cold, drought, and salt induced both genes; the promoter of RD29A was found to be more responsive to drought and cold stresses, whereas the promoter of RD29B was highly responsive to salt stress. Morphological and physiological responses of rd29a and rd29b plants to salt stress were further investigated. Root growth, and photosynthetic properties declined significantly, while solute concentration (Ψπ), water use efficiency (WUE) and δ¹³C ratio increased under salt stress. Unexpectedly, the rd29a and rd29b knockout mutant lines maintained greater root growth, photosynthesis, and WUE under salt stress relative to control. We conclude that the RD29A and RD29B proteins are unlikely to serve directly as protective molecules.     

Construction of Stress Responsive Synthetic Promoters and Analysis of Their Activity in Transgenic Arabidopsis thaliana

To obtain strong inducible promoters to drive abiotic stress-inducible transgene expression with minimal negative effects, we constructed three artificial synthetic promoters (EKCM, EKCRM, and ECCRM) comprising multiple cis-acting stress-response elements. Each promoter was fused independently to the β-glucuronidase (GUS) reporter gene, and GUS expression was analyzed in stable expression systems in Arabidopsis thaliana. T2 transgenic progenies showed integration of the promoter-GUS construct in their genome. RT-PCR assays and histochemical staining analysis showed that GUS expression driven by each promoter increased under desiccation, cold, and high salt conditions. The activity of synthetic promoters, assessed by fluorometric quantitative analysis of GUS enzyme activity, was significantly higher than that of the rd29A promoter under various stress treatments. The most powerful promoter, EKCM, allowed about 1.29-fold in GUS activity relative to the rd29A promoter, on average, under dehydration conditions. All three synthetic promoters could drive stress-inducible GUS expression in different organs of transgenic Arabidopsis. These synthetic promoters represent valuable tools for improving the stress tolerance of crops.     

Isolation and characterization of the dehydration stress-inducible GhRDL1 promoter from the cultivated upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Cotton fiber is the basic raw material used in the textile industry. The fiber yield is severely affected by a number of biotic and abiotic factors, such as insects, viruses, drought and salinity. Drought is a major factor that negatively impacts the yields and quality of cotton fiber. Promoters that respond to stress conditions and up-regulate transgenes are of great significance in crop improvement using genetic engineering approach. Although dehydration-responsive gene promoters, such as RD22 and RD29 from Arabidopsis, have been characterized, not much information is available regarding stress-responsive promoters from Gossypium hirsutum, which accounts for approximately 90 % of cultivated cotton. In this study, we isolated and characterized the promoter of a dehydration-responsive gene (GhRDL1) from G. hirsutum using Agrobacterium-mediated transformation in tobacco and cotton. Transgenic tobacco plants expressing uidA under the GhRDL1 promoter showed GUS activity in the trichomes. Also, GUS expression was observed to some extent in leaf, stem and floral tissues. Similar results were observed when GhRDL1 promoter was tested in transgenic cotton. Most importantly, our study showed that the GhRDL1 promoter is up-regulated in the presence of polyethylene glycol that creates water stress under invitro conditions. Thus, the GhRDL1 promoter may find its usefulness in the development of stress-tolerant cotton and other crop species in the near future.     

Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.     

Activation of the NaCl- and drought-induced RD29A and RD29B promoters by constitutively active Arabidopsis MAPKK or MAPK proteins

Mitogen-activated protein (MAP) kinases mediate cellular responses to a wide variety of stimuli. Activation of a MAP kinase (MAPK) occurs after phosphorylation by an upstream MAP kinase kinase (MAPKK). The Arabidopsis thaliana genome encodes 10 MKKs, but few of these have been shown directly to activate any of the 20 Arabidopsis MAPKs (AtMPKs) and NaCl-, drought- or abscisic acid (ABA)-induced genes RD29A or RD29B. We have constructed the constitutively activated form for nine of the 10 AtMKK proteins, and tested their ability to activate the RD29A and RD29B promoters and also checked the ability of the nine activated AtMKK proteins to phosphorylate 11 of the AtMPK proteins in transient assays. The results show that three proteins, AtMKK1, AtMKK2 and AtMKK3, could activate the RD29A promoter, while these three and two additional AtMKK6/8 proteins could activate the RD29B promoter. Four other proteins, AtMKK7/AtMKK9 and AtMKK4/AtMKK5, can cause hypersensitive response (HR) in tobacco leaves using transient analysis. The activation of the RD29A promoter correlated with four uniquely activated AtMPK proteins. A novel method of activating AtMPK proteins by fusion to a cis-acting mutant of a human MAPK kinase MEK1 was used to confirm that specific members of the AtMPK gene family can activate the RD29A stress pathway.     

A Comparison of Constitutive Promoters for Expression of Transgenes in Alfalfa (Medicago Sativa)

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.     

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.     

High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, <Emphasis Type="Italic">Morus indica</Emphasis> cv. K2, by constitutive and inducible expression of tobacco <Emphasis Type="Italic">osmotin</Emphasis>

Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200 mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought.     

Expression of vacuolar H+-pyrophosphatase (OVP3) is under control of an anoxia-inducible promoter in rice

Vacuolar H⁺-pyrophosphatase (V-PPase) expression increases in a number of abiotic stresses and is thought to play a role in adaptation to abiotic stresses. This paper reports on the regulation of six V-PPase genes in rice (Oryza sativa L.) coleoptiles under anoxia, using flood tolerant and intolerant cultivars to test our hypothesis. Quantitative PCR analysis showed that one vacuolar H⁺-pyrophosphatase (OVP3) was induced by anoxia, particularly in flood-tolerant rice. Regulation of OVP3 expression under anoxia was investigated by analysing putative OVP promoters. The putative OVP3 promoter contained more previously identified anoxia-inducible motifs than the putative promoters of the other five OVP genes. GUS activity in transgenic rice plants containing the OVP3 promoter region linked to the GUS reporter gene was induced only by anoxia. Salt and cold treatments had little effect on OVP3 promoter-driven GUS expression when compared to the anoxic treatment.     

Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice

To identify minimal effective promoters for driving abiotic stress-inducible transgene expression in rice, we selected promoter elements of three stress-responsive genes, viz. rab16A coding for dehydrin, OsABA2 coding for zeaxanthin epoxidase, and a gene coding for a hypothetical protein (HP1) based on the presence of ABA-, salt- and drought-responsive cis-acting elements. These were translationally fused to the gusA reporter gene and introduced into rice to study their effect on heterologous gene expression. The OsABA2 promoter was found to be the most effective and desirable promoter among the three in terms of driving a low constitutive transgene expression under normal conditions and high induction in response to ABA, salt and drought stress, the highest being a 12-fold induction in response to ABA. The rab16A and HP1 promoters resulted in high levels of constitutive expression. While induction of GUS activity was generally two- to threefold for all the treatments in roots for both the promoters, induction in leaves was generally insignificant, the exceptions being rab16A in response to continuous salt stress and HP1 in response to water deficit. It was also observed that the three promoters, in general, resulted in lower constitutive expression, but higher induction in roots as compared to leaves. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. R. Wu: Deceased. This paper is humbly dedicated to the memory of Professor Ray Wu.     

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.     

Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana

The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5′ untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.     

Evaluation of heterologous promoters in transgenic Populus tremula × P. alba plants

The pattern and expression level of β-glucuronidase (gus) reporter gene regulated by six heterologous promoters were studied in transgenic Populus tremula × P. alba plants obtained by Agrobacterium-mediated transformation. Binary vector constructs used contained the following promoter sequences: the CaMV35S from cauliflower mosaic virus; its duplicated version fused to the enhancer sequence from alfalfa mosaic virus; CsVMV from cassava vein mosaic virus; ubiquitin 3 from Arabidopsis thaliana (UBQ3); S-adenosyl-L-methionine synthetase (Sam-s) from soybean; and the rolA from Agrobacterium rhizogenes. Histochemical staining of root, stem and leaf tissues showed phloem and xylem-specific gus expression under rolA promoter, and constitutive expression with the other putative constitutive promoters. Quantitative GUS expression of 10 – 15 independently transformed in vitro grown plants, containing each promoter, was determined by fluorimetric GUS assays. The UBQ3-gus fusion induced the highest average expression level, although an extensive variation in expression levels was observed between independent transgenic lines for all the constructs tested.     

Specificity of expression of the GUS reporter gene (uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures

Twelve independent lines were transformed by particle bombardment of soybean embryogenic suspension cultures with the tobacco anthranilate synthase (ASA2) promoter driving the uidA (beta-glucuronidase, GUS) reporter gene. ASA2 appears to be expressed in a tissue culture specific manner in tobacco (Song H-S, Brotherton JE, Gonzales RA, Widholm JM. Tissue culture specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase. Plant Physiol 1998;117:533-43). The transgenic lines also contained the hygromycin phosphotransferase (hpt) gene and were selected using hygromycin. All the selected cultures or the embryos that were induced from these cultures expressed GUS measured histochemically. However, no histochemical GUS expression could be found in leaves, stems, roots, pods and root nodules of the plants formed from the embryos and their progeny. Pollen from some of the plants and immature and mature seeds and embryogenic cultures initiated from immature cotyledons did show GUS activity. Quantitative 4-methylumbelliferyl-glucuronide (MUG) assays of the GUS activity in various tissues showed that all with observable histochemical GUS activity contained easily measurable activities and leaves and stems that showed no observable histochemical GUS staining did contain very low but measurable MUG activity above that of the untransformed control but orders of magnitude lower than the constitutive 35S-uidA controls used. Low but clearly above background levels of boiling sensitive GUS activity could be observed in the untransformed control immature seeds and embryogenic cultures using the MUG assay. Thus in soybean the ASA2 promoter drives readily observable GUS expression in tissue cultures, pollen and seeds, with only extremely low levels seen in vegetative tissues of the plants. The ASA2 driven expression seen in mature seed was, however, much lower than that seen with the constitutive 35S promoter; less than 2% in seed coats and less than 0.13% in cotyledons and embryo axes. The predominate tissue culture specific expression pattern of the ASA2 promoter may be useful for genetic transformation of crops.