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.     

Studies on the expression of linalool synthase using a promoter-β-glucuronidase fusion in transgenic Artemisia annua

Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to β-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652 bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that LIS expression will most likely have little or no effect on artemisinin production.     

Isolation and heterologous transformation analysis of a pollen-specific promoter from wheat (Triticum aestivum L.)

The promoter of a pollen-specific gene TaPSG719 was isolated from wheat (Triticum aestivum L.) by inverse-PCR (IPCR). Sequence analysis revealed that the promoter contains two cis-acting elements (AGAAA and GTGA) known to confer anther/pollen-specific gene expression which suggests that the promoter of TaPSG719 gene is a pollen-specific one. To ascertain the regulatory function of TaPSG719 promoter, two deleted fragments (?1,776 to ?1 bp and ?1,019 to ?1 bp) were fused to the β-glucuronidase (GUS) gene and transformed into tobacco plants. Similar GUS expression patterns were observed in all transformed plants and its activity was detected exclusively in pollen. No GUS activity in any other floral or vegetative tissue was observed. The results confirm that TaPSG719 promoter is pollen-specific and active during the middle stages of pollen development till anther matured, and it can drive pollen-specific gene expression across the species.     

DNA addition or deletion is associated with a major karyotype polymorphism in the fungal phytopathogen Colletotrichum gloeosporioides

The bacterial GUS (β-glucuronidase) gene has been used as a reporter gene in plants and bacteria and was recently expressed in filamentous fungi. Here, we report the application of GUS for the establishment of transient and stable gene expression systems in the phytopathogenic fungus Cochliobolus heterostrophus. The utility of the transient expression system is demonstrated in applications involving promoter analysis and in tests of various parameters of a transformation system, for comparing the rates of stable and transient transformation events using GUS as sole screening marker and for comparing different transformation systems using either GUS or a dominant selection marker. For these purposes two plasmids were constructed harbouring the GUS gene and the hph gene of Escherichia coli which confers resistance to the antibiotic hygromycin B (HygB), ligated either to the P1 or GPD1 (glyceraldehyde 3 phosphate dehydrogenase) promoter of C. heterostrophus. In transient expression studies the first appearance of GUS activity was observed within 2 h after transformation and maximal values were obtained after 7 or 10 h, depending on the promoter fused to the GUS gene. At peak activity, the GPD1 promoter was revealed to be five fold stronger than the P1 promoter. The same difference in promoter strenght was observed when the vectors were stably integrated in the fungal genome. Using the GUS gene as a colour selection marker in plate assays, it was possible to detect transformants and monitor the process of transient gene expression visually. Blue transformants obtained by screening for the GUS phenotype were mitotically unstable. Transformants obtained by selecting for HygB resistance were mitotically stable and expressed the β-glucuronidase gene constitutively. GUS activity in fungal colonies was detected fluorometrically in a nondestructive plate assay. The pathogenicity of these strains was unaltered compared with wild type. The GUS phenotype allowed selective blue staining of the colonizing mycelia on maize leaves.     

Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants

Summary We characterized the expression of genes that correspond to a cDNA clone, RD29, which is induced by desiccation, cold and high-salt conditions in Arabidopsis thaliana. Northern analysis of desiccation-induced expression revealed a two-step induction process. Early induction occurs within 20 min and secondary induction occurs 3 h after the start of desiccation. Exogenous abscisic acid (ABA) induces RD29 mRNA within 3 h. Two genes corresponding to RD29, rd29A and rd29B, are located in tandem in an 8 kb region of the Arabidopsis genome and encode hydrophilic proteins. Desiccation induces rd29A mRNA with two-step kinetics, while rd29B is induced only 3 h after the start of desiccation. The expression of both genes is stimulated about 3 h after application of ABA. It appears that rd29A has at least two cis-acting elements, one involved in the ABA-associated response to desiccation and the other induced by changes in osmotic potential. The -glucuronidase (GUS) reporter gene driven by the rd29A promoter was induced at significant levels by desiccation, cold, high-salt conditions and ABA in both transgenic Arabidopsis and tobacco. Histochemical analysis of GUS activity revealed that the rd29A promoter functions in almost all the organs and tissues of vegetative plants during water deficiency.     

Cytokinin regulates differentially expression of PAHK-GUS constructs in transgenic Arabidopsis thaliana plants

The expression regulation by cytokinin of genetic constructs P _AHK2 -GUS, P _AHK3 -GUS, and P _AHK4 -GUS in transgenic Arabidopsis thaliana (L.) Heynh plants bearing the gene encoding β-glucuronidase (GUS) under the control of the promoter of one of three genes encoding histidine protein kinases, which are membrane receptors of cytokinin was studied. In 4–5-day-old etiolated A. thaliana seedlings, treatment with cytokinin resulted in the strongest expression activation of the constructs P _AHK2 -GUS and P _AHK3 -GUS. The same constructs were activated by cytokinin also at the seedling transit from scoto- to photomorphogenesis. Long-term seedling growing in darkness on medium containing cytokinin resulted in the substantial promoter activation of the gene encoding the histidine kinase AHK2. In the leaves of three-week-old plants with actively functioning chloroplasts, treatment with cytokinin mainly stimulated expression of the construct P _AHK3 -GUS. In detached senescing leaves, treatment with cytokinin retarded the loss of chlorophyll but did not affect significantly GUS activity under both light and darkness conditions in either of tested lines containing GUS gene under the control of promoters of histidine kinase genes. At the same time, cytokinin activated the promoter of the gene of primary response to cytokinin in the construct P _ARR5 -GUS. Thus, in the studied test-system, treatment with cytokinin of A. thaliana plant grown in darkness or in the light affected differently the expression of histidine kinase genes in dependence of plant age, conditions of plant cultivation, and plant physiological state.     

Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress

The constitutive and drought-induced activities of the Arabidopsis thaliana RD29A and RD29B promoters were monitored in soybean (Glycine max (L.) Merr.] via fusions with the visual marker gene β-glucuronidase (GUS). Physiological responses of soybean plants were monitored over 9 days of water deprivation under greenhouse conditions. Data were used to select appropriate time points to monitor the activities of the respective promoter elements. Qualitative and quantitative assays for GUS expression were conducted in root and leaf tissues, from plants under well-watered and dry-down conditions. Both RD29A and RD29B promoters were significantly activated in soybean plants subjected to dry-down conditions. However, a low level of constitutive promoter activity was also observed in both root and leaves of plants under well-watered conditions. GUS expression was notably higher in roots than in leaves. These observations suggest that the respective drought-responsive regulatory elements present in the RD29X promoters may be useful in controlling targeted transgenes to mitigate abiotic stress in soybean, provided the transgene under control of these promoters does not invoke agronomic penalties with leaky expression when no abiotic stress is imposed.     

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.     

Functional analysis of a cryptic promoter from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> reveals bidirectionality

Cryptic promoter elements play a significant role in evolution of plant gene expression patterns and are prospective tools for creating gene expression systems in plants. In a previous report, a 452 bp promoter fragment designated as cryptic root-specific promoter (AY601849) was identified immediately upstream to T-DNA insertion, in the intergenic region between divergent genes SAHH1 and SHMT4, in T-DNA tagged mutant M57 of Arabidopsis thaliana. In silico analysis of 452 bp promoter revealed typical eukaryotic promoter architecture, presence of root-specific motifs and other cis-regulatory motifs responsible for the spatial and temporal expression. GUS expression driven by 452 bp in M57 was developmentally as well as light-regulated. The AT-rich 452 bp promoter does not show homology to any known sequences. The 452 bp promoter was further proved cryptic and detailed molecular characterization of the promoter carried out through serial 5′ and 3′ deletion analysis, by cloning the promoter fragments upstream to promoter-less GUS vector. A 279 bp fragment obtained by deleting 173 bp from 5′ end of 452 bp was capable of driving root-specific expression, similar to that of full-length promoter. Further, root tip-specific, root-specific and core-regulatory motifs for root-specific expression were identified at positions 173–227, 251–323 and 408–452 bp, respectively, from the 5′ end of 452 bp. The 452 bp promoter was equally functional in inverse orientation, hence bidirectional and symmetric. In heterologous systems, such as Brassica juncea and Oryza sativa, the promoter activity was not significant since GUS was not visually detected in transient assays.     

Seed-specific expression of seven Arabidopsis promoters

Seeds contain storage compounds, from various carbohydrates to proteins and lipids, which are synthesized during seed development. For the purposes of many plant researches or commercial applications, developing promoter systems expressing specifically in seeds or in particular constituents or tissues/compartments of seeds are indispensable. To screen genes dominantly or specifically expressed in seed tissues, we analyzed Arabidopsis ATH1 microarray data open to the public. Thirty-two candidate genes were selected and their expressions in seed tissues were confirmed by RT-PCR. Finally, seven genes were selected for promoter analysis. The promoters of seven genes were cloned into pBI101 vector and transformed into Arabidopsis to assay histochemical β-glucuronidase (GUS) activity. We found that Pro-at3g03230 promoter drove GUS expression in a chalazal endosperm, Pro-at4g27530:GUS expressed in both chalazal endosperm and embryo, Pro-at4g31830 accelerated GUS expression both in radicle and procambium, Pro-at5g10120 and Pro-at5g16460 drove GUS expression uniquely in embryo, Pro-at5g53100:GUS expressed only in endosperm, and Pro-at5g54000 promoted GUS expression in both embryo and inner integument. These promoters can be used for expressing any genes in specific seed tissues for practical application.