Isolation and Characterization of Three New Promoters from Gossypium hirsutum that Show High Activity in Reproductive Tissues

Engineering of plant protection requires well-characterized tissue-specific promoters for the targeted expression of insecticidal resistance genes. Herein, we describe the isolation of five different fragments of promoters of three distinct flower-specific cotton (Gossypium hirsutum) genes. Expression analyses of the three genes GhPME-like1, GhβGal-like1 and GhPL-like1 revealed that they are expressed highly in flowers buds ranging from 4 to 12 mm in size. Several putative regulatory cis-elements were identified in the promoter regions, including elements involved in the control of tissue-specific gene expression in pollen grains and fruits. In vivo analyses of these promoters were performed using the heterologous plant system Arabidopsis thaliana by fusing them with the gene uidA (GUS). GUS staining in Arabidopsis tissues revealed that their expression was restricted to anthers, with the majority of expression in pollen grains and in the upper portion of the carpels and siliques. A comparison between a CaMV35S::GUS constitutive promoter and the promoters isolated in this study revealed that the cotton promoters were more active and were specific to flowers and fruits, which are organs that are preferentially attacked by important pest insects such as the boll weevil (Anthonomus grandis). The activity of the promoters was also confirmed using transient expression assays in flower buds of G. hirsutum. The promoters of GhPME-like1, GhβGal-like1 and GhPL-like1 are specific to reproductive tissues and could represent important biotechnological tools for controlling insect pests, in particular the cotton boll weevil, which attacks floral and fruit tissues.     

<Emphasis Type="Italic">SnRK2</Emphasis> Homologs in <Emphasis Type="Italic">Gossypium</Emphasis> and <Emphasis Type="Italic">GhSnRK2.6</Emphasis> Improved Salt Tolerance in Transgenic Upland Cotton and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SnRK2s are a large family of plant-specific protein kinases, which play important roles in multiple abiotic stress responses in various plant species. But the family in Gossypium has not been well studied. Here, we identified 13, 10, and 13 members of the SnRK2 family from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively, and analyzed the locations of SnRK2 homologs in chromosomes based on genome data of cotton species. Phylogenetic tree analysis of SnRK2 proteins showed that these families were classified into three groups. All SnRK2 genes were comprised of nine exons and eight introns, and the exon distributions and the intron phase of homolog genes among different cotton species were analogous. Moreover, GhSnRK2.6 was overexpressed in Arabidopsis and upland cotton, respectively. Under salt treatment, overexpressed Arabidopsis could maintain higher biomass accumulation than wild-type plants, and GhSnRK2.6 overexpression in cotton exhibited higher germination rate than the control. So, the gene GhSnRK2.6 could be utilized in cotton breeding for salt tolerance.     

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.     

Cloning and Expression Studies of Novel Small RNAs in Tetraploid Cotton

Small RNAs are a group of non-coding RNAs that downregulate gene expression in a sequence-specific manner to control plant growth and development. The objective of the present study was to clone and characterize several small RNAs in cotton. To identify small RNAs that are involved in the development of cotton bolls and fibers, we generated cDNA libraries from cotton bolls at 13?days post-anthesis from two cotton cultivars, Pima Phy 76 (Gossypium bardadense) and Acala 1517?C99 (Gossypium hirsutum). Screening of these libraries identified eight small RNAs, seven of which have not been reported in other plant species and appear to be absent in the known sequences of other plant species. Their predicted target genes are known to be involved in cotton fiber development. The cloned small RNAs displayed lower and differential expression in the examined boll developmental stages using RT-PCR and quantitative RT-PCR. The genetic polymorphism of the small RNAs at the DNA level was evaluated by miRNA-amplified fragment length polymorphism (AFLP) analysis using primers designed from the small miRNA genes in combination with AFLP primers. Homologous small RNA gene sequences were further isolated using this homology-based genotyping approach, and potential hairpin structures were identified. The results represent a novel method to isolate small including miRNA genes at the RNA and DNA levels in many plant species where genome sequences are not available or expressed sequence tags are limited.     

Comparative Genome-Wide Analysis of the Malate Dehydrogenase Gene Families in Cotton

Malate dehydrogenases (MDHs) play crucial roles in the physiological processes of plant growth and development. In this study, 13 and 25 MDH genes were identified from Gossypium raimondii and Gossypium hirsutum, respectively. Using these and 13 previously reported Gossypium arboretum MDH genes, a comparative molecular analysis between identified MDH genes from G. raimondii, G. hirsutum, and G. arboretum was performed. Based on multiple sequence alignments, cotton MDHs were divided into five subgroups: mitochondrial MDH, peroxisomal MDH, plastidial MDH, chloroplastic MDH and cytoplasmic MDH. Almost all of the MDHs within the same subgroup shared similar gene structure, amino acid sequence, and conserved motifs in their functional domains. An analysis of chromosomal localization suggested that segmental duplication played a major role in the expansion of cotton MDH gene families. Additionally, a selective pressure analysis indicated that purifying selection acted as a vital force in the evolution of MDH gene families in cotton. Meanwhile, an expression analysis showed the distinct expression profiles of GhMDHs in different vegetative tissues and at different fiber developmental stages, suggesting the functional diversification of these genes in cotton growth and fiber development. Finally, a promoter analysis indicated redundant but typical cis-regulatory elements for the potential functions and stress activity of many MDH genes. This study provides fundamental information for a better understanding of cotton MDH gene families and aids in functional analyses of the MDH genes in cotton fiber development.     

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum,a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.     

miR482 Regulation of NBS-LRR Defense Genes during Fungal Pathogen Infection in Cotton

In this study, we characterized the miR482 family in cotton using existing small RNA datasets and the recently released draft genome sequence of Gossypium raimondii, a diploid cotton species whose progenitor is the putative contributor of the D_t (representing the D genome of tetraploid) genome of the cultivated tetraploid cotton species G. hirsutum and G. barbadense. Of the three ghr-miR482 members reported in G. hirsutum, ghr-miR482a has no homolog in G. raimondii, ghr-miR482b and ghr-miR482c each has a single homolog in G. raimondii. Gra-miR482d has five homologous loci (gra-miR482d, f-i) in G. raimondii and also exists in G. hirsutum (ghr-miR482d). A variant, miR482.2 that is a homolog of miR2118 in other species, is produced from several GHR-MIR482 loci in G. hirsutum. Approximately 12% of the G. raimondii NBS-LRR genes were predicted targets of various members of the gra-miR482 family. Based on the rationale that the regulatory relationship between miR482 and NBS-LRR genes will be conserved in G. raimondii and G. hirsutum, we investigated this relationship using G. hirsutum miR482 and G. raimondii NBS-LRR genes, which are not currently available in G. hirsutum. Ghr-miR482/miR482.2-mediated cleavage was confirmed for three of the four NBS-LRR genes analysed. As in tomato, miR482-mediated cleavage of NBS-LRR genes triggered production of phased secondary small RNAs in cotton. In seedlings of the susceptible cultivar Sicot71 (G. hirsutum) infected with the fungal pathogen Verticillium dahliae, the expression levels of ghr-miR482b/miR482b.2, ghr-miR482c and ghr-miR482d.2 were down-regulated, and several NBS-LRR targets of ghr-miR482c and ghr-miR482d were up-regulated. These results imply that, like tomato plants infected with viruses or bacteria, cotton plants are able to induce expression of NBS-LRR defence genes by suppression of the miRNA-mediated gene silencing pathway upon fungal pathogen attack.     

An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense

Cotton fiber is an excellent model system of cellulose biosynthesis; however, it has not been widely studied due to the lack of information about the cellulose synthase (CESA) family of genes in cotton. In this study, we initially identified six full-length CESA genes designated as GhCESA5–GhCESA10. Phylogenetic analysis and gene co-expression profiling revealed that CESA1, CESA2, CESA7, and CESA8 were the major isoforms for secondary cell wall biosynthesis, whereas CESA3, CESA5, CESA6, CESA9, and CESA10 should involve in primary cell wall formation for cotton fiber initiation and elongation. Using integrative analysis of gene expression patterns, CESA protein levels, and cellulose biosynthesis in vivo, we detected that CESA8 could play an enhancing role for rapid and massive cellulose accumulation in Gossypium hirsutum and Gossypium barbadense. We found that CESA2 displayed a major expression in non-fiber tissues and that CESA1, a housekeeping gene like, was predominantly expressed in all tissues. Further, a dynamic alteration was observed in cell wall composition and a significant discrepancy was observed between the cotton species during fiber elongation, suggesting that pectin accumulation and xyloglucan reduction might contribute to cell wall transition. In addition, we discussed that callose synthesis might be regulated in vivo for massive cellulose production during active secondary cell wall biosynthesis in cotton fibers.     

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.     

Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons

MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expression comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression levels in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two cultivated cotton cultivars, Pima Phy 76 (Gossypium barbadense) and Acala 1517-99 (Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA-AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirmation using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies.     

Molecular cloning and characterization of five genes encoding pentatricopeptide repeat proteins from Upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

The pentatricopeptide repeat (PPR) protein family is one of the largest and most complex families in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding RNA. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. In this study, we identified many genes encoding PPR protein in Upland cotton through an extensive survey of the database of Gossypium hirsutum. Furthermore, we isolated five full-length cDNA of PPR genes from G. hirsutum 0-613-2R which were named GhPPR1–GhPPR5. Domain analysis revealed that the deduced amino acid sequences of GhPPR1–5 contained from 5 to 10 PPR motifs and those PPR proteins were divided into two different PPR subfamilies. GhPPR1–2 belonged to the PLS subfamily and GhPPR3–5 belonged to the P subfamily. Phylogenetic analysis of the five GhPPR proteins and 18 other plant PPR proteins also revealed that the same subfamily clustered together. All five GhPPR genes were differentially but constitutively expressed in roots, stems, leaves, pollens, and fibers based on the gene expression analysis by real-time quantitative RT-PCR. This study is the first report and analysis of genes encoding PPR proteins in cotton.