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.     

Employing in vitro directed molecular evolution for the selection of α-amylase variant inhibitors with activity toward cotton boll weevil enzyme

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 10⁸α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control.     

Promoters of nuclear‐encoded respiratory chain Complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen‐specific expression

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.     

Cotton (Gossypium hirsutum) 14‐3‐3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling

Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14‐3‐3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14‐3‐3L, Gh14‐3‐3e and Gh14‐3‐3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14‐3‐3 RNAi transgenic plants were significantly shorter than those of wild type. This ‘short fibre’ phenotype of the 14‐3‐3 RNAi cotton could be partially rescued by application of 2,4‐epibrassinolide (BL). Expression levels of the BR‐related and fibre‐related genes were altered in the Gh14‐3‐3 transgenic fibres. Furthermore, we identified Gh14‐3‐3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14‐3‐3L/e/h were required for Gh14‐3‐3‐GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14‐3‐3 proteins. Additionally, 14‐3‐3‐regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14‐3‐3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation.     

Influence of water deficits on the abscisic Acid and indole-3-acetic Acid contents of cotton flower buds and flowers

A field experiment was conducted during the summer of 1988 to test the hypothesis that water deficit affects the abscisic acid (ABA) and indole acetic acid (IAA) concentrations in cotton (Gossypium hirsutum L.) flower buds in ways that predispose young fruits (bolls) that subsequently develop from them to increased abscission rates. Water deficit had little effect on the ABA content of flower buds but increased the ABA content of flowers as much as 66%. Water deficit decreased the concentrations of free and conjugated IAA in flower buds during the first irrigation cycle but increased them during the second cycle. Flowers contained much less IAA than buds. Water deficit slightly increased the conjugated IAA content of flowers but had no effect on the concentration of free IAA in flowers. Because water deficit slightly increased the ABA content but did not decrease the IAA content of flowers, any carry-over effect of water deficit on young boll shedding might have been caused by changes in ABA but not from changes in IAA.     

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.     

Identification and Characterisation of Cotton Boll Wall-Specific Gene Promoters for Future Transgenic Cotton Varieties

Current transgenic cotton varieties constitutively express transgenes encoding anti-pest proteins to protect against plant damage caused by insect attack. However, restricting the spatial expression of transgenes to the tissues in which their products are required is likely to improve crop performance and reduce environmental impacts. Therefore, we sought to identify native gene promoters that would restrict transgene expression to the boll wall of the cotton plant. Six abundant mRNAs that accumulated preferentially in the boll wall were identified, and the gene promoters of two of these mRNAs were identified, isolated and characterised. The promoters of a proline-rich protein gene (GhPRP3) and a chalcone synthase gene (GhCHS1) were demonstrated to drive boll wall-preferential expression of a reporter gene in a transient transformation system. In silico analyses of the GhPRP3 and GhCHS1 promoters identified numerous previously identified cis-acting regulatory elements (CAREs) as well as the presence of three novel shared CAREs. The identification and characterisation of these promoters provides an important step in the development of the next generation of transgenic plants.     

Global functional analyses of rice promoters by genomics approaches

Promoters play key roles in conferring temporal, spatial, chemical, developmental, or environmental regulation of gene expression. Promoters that are subject to specific regulations are useful for manipulating foreign gene expression in plant cells, tissues, or organs with desirable patterns and under controlled conditions, and have been important for both basic research and applications in agriculture biotechnology. Recent advances in genomics technologies have greatly facilitated identification and study of promoters in a genome scale with high efficiency. Previously we have generated a large T-DNA tagged rice mutant library (TRIM), in which the T-DNA was designed with a gene/promoter trap system, by placing a promoter-less GUS gene next to the right border of T-DNA. GUS activity screens of this library offer in situ and in planta identifications and analyses of promoter activities in their native configurations in the rice genome. In the present study, we systematically performed GUS activity screens of the rice mutant library for genes/promoters constitutively, differentially, or specifically active in vegetative and reproductive tissues. More than 8,200 lines have been screened, and 11% and 22% of them displayed GUS staining in vegetative tissues and in flowers, respectively. Among the vegetative tissue active promoters, the ratio of leaf active versus root active is about 1.6. Interestingly, all the flower active promoters are anther active, but with varied activities in different flower tissues. To identify tissue specific ABA/stress up-regulated promoters, we compared microarray data of ABA/stress induced genes with those of tissue-specific expression determined by promoter trap GUS staining. Following this approach, we showed that the peroxidase 1 gene promoter was ABA up-regulated by 4 fold within 1 day of exposure to ABA and its expression is lateral root specific. We suggest that this be an easy bioinformatics approach in identifying tissue/cell type specific promoters that are up-regulated by hormones or other factors. Su-May Yu and Swee-Suak Ko equally contributed to this work.     

The Degree of Susceptibility of Nectary-Inoculated Cotton Flowers and Bolls to Subsequent Seed Infection by Aspergillus flavus Is Determined at or before Anthesis

In greenhouse and field studies, cotton (Gossypium hirsutum L.) flowers were inoculated with Aspergillus flavus at the involucral nectaries. Bolls developing from early-season flowers had significantly higher percentages of A. flavus-infected seed than did bolls from flowers formed later in the season. Seeds from bolls inoculated 2 weeks after anthesis had the same infection levels as those from flowers inoculated at anthesis. These results indicate that early-season flowers are predisposed to A. flavus infection and that the degree of susceptibility at anthesis is retained through early boll development.     

A pFBP6::Barnase Construct Resulted in Stigma and Style Ablation and Floral Abscission in Transgenic Tobacco

The potential for transgene dispersal through pollen, fruit, and seed is an important argument against the release of genetically modified plants. One approach toward addressing the concerns of gene flow from transgenic crops into closely related wild species involves in the use of tissue-specific promoters to engineer male and/or female sterility. In this study, we investigated the potential of Barnase ectopic expression for engineering floral sterility. A 2.6?kb promoter region of floral binding protein 6 (FBP6) from Petunia hybrida was isolated and fused to a reporter gene encoding ??-glucuronidase (GUS). The construct was introduced into tobacco plants where GUS staining was detected ubiquitously throughout the various tissues. The expression pattern of FBP6 resembled AG promoters, i.e., weak promoter activity was found in vegetative tissues, and strong activity was found in the various floral organs including the carpels and stigma. Meanwhile,The pFBP6::Barnase construct was then cotransformed into tobacco along with the Barstar gene, encoding an enzymatic inhibitor of Barnase, which was expressed at low but ubiquitous levels. Although cotransformed tobacco plants showed near normal vegetative growth, 74% of transgenic plants exhibited stigma and style ablation, and 98% of flower buds abscised before opening. Further analyses confirmed that stigma and style ablation prevented fertilization of the flower, and abscission of the bud followed rapidly. Thus, this approach has advantages for those ornamental/landscaping species where the pollen and fruit represent pollutants of the urban environment (e.g., platanus and poplar).     

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.     

Expression analysis of plant intracellular Ras-group related leucine-rich repeat proteins (PIRLs) in Arabidopsis thaliana

Arabidopsis thaliana contains a family of nine genes known as plant intracellular Ras-group related leucine-rich repeat (LRR) proteins (PIRLs). These are structurally similar to animals and fungal LRR proteins and play important roles in developmental pathways. However, to date, no detailed tissue-specific expression analysis of these PIRLs has been performed. Therefore, in this study, we generated promoter:GUS transgenic plants for the nine A. thaliana PIRL genes and identified their expression patterns in seedlings and floral organs at different developmental stages. Most PIRL members showed expression in the root apical region and in the vascular tissue of primary and lateral roots. Shoot apex-specific expression was recorded for PIRL1 and PIRL8. Furthermore, PIRL1, PIRL3, PIRL5, PIRL6, and PIRL7 showed distinct expression patterns in flowers, especially in pollen and anthers. In addition, co-expression network analysis identified cases where PIRLs were co-expressed with other genes known to have specific functions related to growth and development. Taken together, the tissue-specific expression patterns of PIRL genes improve our understanding of the functions of this gene family in plant growth and development.