Isolation and characterization of a <Emphasis Type="Italic">GAI/RGA</Emphasis>-like gene from <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The aim of the investigation reported here was to assess the role of gibberellin in cotton fiber development. The results of experiments in which the gibberellin (GA) biosynthesis inhibitor paclobutrazol (PAC) was tested on in vitro cultured cotton ovules revealed that GA is critical in promoting cotton fiber development. Plant responses to GA are mediated by DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified from the GenBank database and analyzed with the BLAST program. The full-length cDNA was cloned from upland cotton (Gossypium hirsutum, Gh) and sequenced. A comparison of the putative protein sequence of this cDNA with all Arabidopsis DELLA proteins indicated that GhRGL is a putative ortholog of AtRGL. Over-expression of this cDNA in Arabidopsis plants resulted in the dwarfed phenotype, and the degrees of dwarfism were related to the expression levels of GhRGL. The deletion of 17 amino acids, including the DELLA domain, resulted in the dominant dwarf phenotype, demonstrating that GhRGL is a functional protein that affects plant growth. Real-time quantitative PCR results showed that GhRGL mRNA is highly expressed in the cotton ovule at the elongation stage, suggesting that GhRGL may play a regulatory role in cotton fiber elongation.     

Functional characterization of <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> profilin 1 gene (<Emphasis Type="Italic">GhPFN1</Emphasis>) in tobacco suspension cells

Cotton fiber is an extremely long plant cell. Fiber elongation is a complex process and the genes that are crucial for elongation are largely unknown. We previously cloned a cDNA encoding an isoform of cotton profilin and found that the gene (designated GhPFN1) was preferentially expressed in cotton fibers. In the present study, we have further analyzed the expression pattern of GhPFN1 during fiber development and studied its cellular function using tobacco suspension cells as an experimental system. We report that expression of GhPFN1 is tightly associated with fast elongation of cotton fibers whose growth requires an intact actin cytoskeleton. Overexpression of GhPFN1 in the transgenic tobacco cells was correlated with the formation of elongated cells that contained thicker and longer microfilament cables. Quantitative analyses revealed a 2.5–3.6 fold increase in total profilin levels and a 1.6–2.6 fold increase in the F-actin levels in six independent transgenic lines. In addition to the effect on cell elongation, we also observed delayed cell cycle progression and a slightly lower mitotic index in the transgenic cells. Based on these data, we propose that GhPFN1 may play a critical role in the rapid elongation of cotton fibers by promoting actin polymerization. Hai-Yun Wang and Yi Yu contributed equally to this work.     

Production and characterization of somatic hybrids between upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) and wild cotton (<Emphasis Type="Italic">G. klotzschianum</Emphasis> Anderss) via electrofusion

Symmetric somatic hybrid plants between Gossypium hirsutum Coker 201 and G. klotzschianum were obtained through electrofusion. The fusion products were cultured in KM₈P medium supplemented with 2.685 M -naphthaleneacetic acid and 0.465 M kinetin, and the regenerated plants were morphologically, genetically, and cytologically characterized. Nuclear-DNA flow cytometric analysis revealed that the plants tested (31 of 40) had a relative DNA content close to the total DNA contents of the two parents. Subsequent genome DNA analysis using random amplified polymorphic DNA markers revealed 16 of 18 plants were true somatic hybrids. Cytological investigation of the metaphase root-tip cells of seven hybrids revealed there were 72–81 chromosomes in the hybrids, a value close to the expected 78 chromosomes. The morphology of the hybrids was distinct from that of the parents and from that of the regenerants from protoplasts of Coker 201. Somatic hybridization represents a potent and novel tool for transferring genomes of wild cottons containing economically important traits to cultivars in breeding programs. This is the first report on the regeneration of somatic hybrids via protoplast fusion in cotton.     

Biolistic transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with the <Emphasis Type="Italic">phyA</Emphasis> gene from <Emphasis Type="Italic">Aspergillus ficuum</Emphasis>

Transgenic cotton with an increased level of phytase activity was generated from cotton (Gossypium hirsutum L.) cv. ND94-7 by subjecting shoot-apex explants to particle bombardment. These tissues were transformed with plasmid pC-KSA2300 carrying a selectable marker (for kanamycin) and a target gene (phytase, or phyA, from Aspergillus ficuum). Primary plants were regenerated in a medium containing 75 mg l⁻¹ kanamycin. Of 1,534 shoot apices, 52 (3.4%) survived on this selection medium. Southern and Northern blot analyses confirmed that phyA was stably integrated and expressed in those primary transgenics. The progenies of the primary transgenic plants were found to have a 3.1- to 3.2-fold increase in root extracellular phytase activity, resulting in improved phosphorus (P) nutrition. Growth also was enhanced when they were supplied with phytate, and their P content was equivalent to that of wildtype plants supplied with inorganic phosphate. These results demonstrate that the expression of phyA in cotton plants improves their ability to utilize organic P in response to a deficiency.     

Fine-mapping qFS07.1 controlling fiber strength in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Key message

qFS07.1 controlling fiber strength was fine-mapped to a 62.6-kb region containing four annotated genes. RT-qPCR and sequence of candidate genes identified an LRR RLK gene as the most likely candidate.

Abstract

Fiber strength is an important component of cotton fiber quality and is associated with other properties, such as fiber maturity, fineness, and length. Stable QTL qFS07.1, controlling fiber strength, had been identified on chromosome 7 in an upland cotton recombinant inbred line (RIL) population from a cross (CCRI35?×?Yumian1) described in our previous studies. To fine-map qFS07.1, an F₂ population with 2484 individual plants from a cross between recombinant line RIL014 and CCRI35 was established. A total of 1518 SSR primer pairs, including 1062, designed from chromosome 1 of the Gossypium raimondii genome and 456 from chromosome 1 of the G. arboreum genome (corresponding to the QTL region) were used to fine-map qFS07.1, and qFS07.1 was mapped into a 62.6-kb genome region which contained four annotated genes on chromosome A07 of G. hirsutum. RT-qPCR and comparative analysis of candidate genes revealed a leucine-rich repeat protein kinase (LRR RLK) family protein to be a promising candidate gene for qFS07.1. Fine mapping and identification of the candidate gene for qFS07.1 will play a vital role in marker-assisted selection (MAS) and the study of mechanism of cotton fiber development.

     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> -mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) using a heterologous bean chitinase gene

Cotton (Gossypium hirsutum L., var. Coker 312) hypocotyl explants were transformed with three strains of Agrobacterium tumefaciens, LBA4404, EHA101 and C58, each harboring the recombinant binary vector pBI121 containing the chi gene insert and neomycin phosphotransferase (nptII) gene, as selectable marker. Inoculated tissue sections were placed onto cotton co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ cepotaxime. Putative calli were subsequently regenerated into cotton plantlets expressing both the kanamycin resistance gene and βglucuronidase (gus) as a reporter gene. Polymerase chain reaction was used to confirm the integration of chi and nptII transgenes in the T₁ plants genome. Integration of chi gene into the genome of putative transgenic was further confirmed by Southern blot analysis. ‘Western’ immunoblot analysis of leaves isolated from T₀ transformants and progeny plants (T₁) revealed the presence of an immunoreactive band with MW of approximately 31 kDa in transgenic cotton lines using anti-chitinase-I polyclonal anti-serum. Untransformed control and one transgenic line did not show such an immunoreactive band. Chitinase specific activity in leaf tissues of transgenic lines was several folds greater than that of untransformed cotton. Crude leaf extracts from transgenic lines showed in vitro inhibitory activity against Verticillium dahliae.Transgenic plants currently growing in a greenhouse and will be bioassayed for improved resistance against V. dahlia the causal against of verticilliosis in cotton.     

Next generation genetic mapping of the Ligon-lintless-2 (<Emphasis Type="Italic">Li</Emphasis><Subscript>2</Subscript>) locus in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Key message

Mapping-by-sequencing and novel subgenome-specific SNP markers were used to fine map the Ligon-lintless 2 ( Li ₂ ) short-fiber gene in tetraploid cotton. These methodologies will accelerate gene identification in polyploid species.

Abstract

Next generation sequencing offers new ways to identify the genetic mechanisms that underlie mutant phenotypes. The release of a reference diploid Gossypium raimondii (D₅) genome and bioinformatics tools to sort tetraploid reads into subgenomes has brought cotton genetic mapping into the genomics era. We used multiple high-throughput sequencing approaches to identify the relevant region of reference sequence and identify single nucleotide polymorphisms (SNPs) near the short-fiber mutant Ligon-lintless 2 (Li ₂) gene locus. First, we performed RNAseq on 8-day post-anthesis (DPA) fiber cells from the Li ₂ mutant and its wild type near isogenic line (NIL) Gossypium hirsutum cv. DP5690. We aligned sequence reads to the D₅ genome, sorted the reads into A and D subgenomes with PolyCat and called SNPs with InterSNP. We then identified SNPs that would result in non-synonymous substitutions to amino acid sequences of annotated genes. This step allowed us to identify a 1-Mb region with 24 non-synonymous SNPs, representing the introgressed region that differentiates Li ₂ from its NIL. Next, we sequenced total DNA from pools of F₂ plants, using a super bulked segregant analysis sequencing (sBSAseq) approach. The sBSAseq predicted 82 non-synonymous SNPs among 3,494 SNPs in a 3-Mb region that includes the region identified by RNAseq. We designed subgenome-specific SNP markers and tested them in an F₂ population of 1,733 individuals to construct a genetic map. Our resulting genetic interval contains only one gene, an aquaporin, which is highly expressed in wild-type fibers and is significantly under-expressed in elongating Li ₂ fiber cells.

     

Phenolic compounds and somatic embryogenesis in cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Studies of phenolic compounds were performed during cell suspension cultures in relation with the induction of embryogenic structures in two cultivars of cotton. Coker 312 produced embryogenic structures, unlike R405-2000 which was found to be a non-embryogenic cultivar. Embryogenesis induction in Coker 312 was strongly linked to a higher content of caffeic, ferulic and salicylic acids and to the appearance of p-coumaric acid, benzoic acid, trans-resveratrol, catechin and naringenin.     

Molecular cloning and characterization of the promoter for the multiple stress-inducible gene <Emphasis Type="Italic">BjCHI1</Emphasis> from <Emphasis Type="Italic">Brassica juncea</Emphasis>

We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009–1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing the BjCHI1 promoter or its derivatives to β-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from −695 to −620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at −353 to −348 of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter. Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA.     

Isolation and characterization of 14 microsatellite markers in <Emphasis Type="Italic">Macrodasyceras</Emphasis><Emphasis Type="Italic">hirsutum</Emphasis> (Hymenoptera: Torymidae)

Macrodasyceras hirsutum Kamijo is the seed parasitoid wasp of the bird-dispersed, dioecious tree, Ilex integra Thunb. The wasp reduces the level of dispersal mutualism between the Ilex tree and its frugivorous birds by manipulating the color of mature berries. The female trees do not blossom every year and sometimes change sex. Thus, the reproduction biology of I. integra affects the population size and structure of M. hirsutum in a forest and consequently influences the seed dispersal mutualism between the tree and birds, because of limited ability of adult locomotion. To investigate the wasp population structure with reference to the dispersal mutualism between trees and birds, we isolated 14 microsatellite loci of M. hirsutum wasps. Every locus was polymorphic among 20 females, with 3–13 alleles per locus, without linkage disequilibrium. The observed and expected heterozygosities ranged from 0.100 to 0.900 and 0.099 to 0.818, respectively, indicating their utility in molecular analyses of the wasp population.     

<Emphasis Type="Italic">GhDRIN</Emphasis>1, a novel drought-induced gene of upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) confers abiotic and biotic stress tolerance in transgenic tobacco

A novel stress tolerance cDNA fragment encoding GhDRIN1 protein was identified and its regulation was studied in cotton boll tissues and seedlings subjected to various biotic and abiotic stresses. Phylogenetic and conserved domain prediction indicated that GhDRIN1 was annotated with a hypothetical protein of unknown function. Subcellular localization showed that GhDRIN1 is localized in the chloroplasts. The promoter sequence was isolated and subjected to in silico study. Various cis-acting elements responsive to biotic and abiotic stresses and hormones were found. Transgenic tobacco seedlings exhibited better growth on amended MS medium and showed minimal leaf damage in insect bioassays carried out with Helicoverpa armigera larvae. Transgenic tobacco showed better tolerance to water-deficit and fast recovered upon rewatering. Present work demonstrated that GhDRIN1, a novel stress tolerance gene of cotton, positively regulates the response to biotic and abiotic stresses in transgenic tobacco.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Identification of the family of aquaporin genes and their expression in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>L.)

Background  

Cotton (Gossypium spp.) is produced in over 30 countries and represents the most important natural fiber in the world. One of the primary factors affecting both the quantity and quality of cotton production is water. A major facilitator of water movement through cell membranes of cotton and other plants are the aquaporin proteins. Aquaporin proteins are present as diverse forms in plants, where they function as transport systems for water and other small molecules. The plant aquaporins belong to the large major intrinsic protein (MIP) family. In higher plants, they consist of five subfamilies including plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), NOD26-like intrinsic proteins (NIP), small basic intrinsic proteins (SIP), and the recently discovered X intrinsic proteins (XIP). Although a great deal is known about aquaporins in plants, very little is known in cotton.     

Molecular cloning and characterization of an inducible RNA-dependent RNA polymerase gene, <Emphasis Type="Italic">GhRdRP</Emphasis>, from cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

The RNA-dependent RNA polymerase (RdRP) cDNA, designated as Gossypium hirsutum RdRP (GhRdRP) was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 3,672 bp in size and encoded an open reading frame (ORF) of 1,110 amino acids which contained the RdRP conserved functional domain and the signature motif DbDGD. Amino acid sequence alignment indicated that GhRdRP shared the highest identity (66.37%) with AtRdRP1 and had homology with other plant, fungal, yeast and nematode RdRPs. The corresponding genomic DNA containing five exons and four introns, was isolated and analyzed. Also a 5′-flanking region was cloned, and a group of putative cis-acting elements were identified. Southern blot analysis revealed a single copy of the GhRdRP gene in cotton genome. The expression analysis by semi-quantitative RT-PCR showed that GhRdRP was induced by salicylic acid (SA), 5-chloroSA (5-CSA) and fungal infection of Rhizoctonia solani Kuhn. The cloning and characterization of the GhRdRP gene will be useful for further studies of biological roles of GhRdRP in plants.     

Isolation and characterization of the rice <Emphasis Type="Italic">NPR1</Emphasis> promoter

NPR1 is a positive regulator of systemic acquired resistance in Arabidopsis and rice. Expression of the rice gene OsNPR1 is induced by salicylic acid (SA). To identify the region of the OsNPR1 promoter involved in response to SA, we carried out deletion mutagenesis of the region 1005 bp upstream of the OsNPR1 start codon. Cis-element analysis revealed that the OsNPR1 promoter contains W-boxes and ASF1 motifs, both of which are known to be functional cis-elements of the WRKY and bZIP proteins, respectively. The deletion constructs 1005:LUC and 752:LUC, were induced by up to 4.3- and 3.8-fold, respectively, following SA treatment, suggesting that W-boxes and ASF1 motifs may play an important role in the strong induction of these constructs by SA. Using mutation analysis, we also showed that both the W-box and ASF1 motif are necessary for SA-induced expression of OsNPR1.     

Isolation and characterization of <Emphasis Type="Italic">Brittle2</Emphasis> promoter from <Emphasis Type="Italic">Zea Mays</Emphasis> and its comparison with <Emphasis Type="Italic">Ze19</Emphasis> promoter in transgenic tobacco plants

ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression in embryos, which is different from similar promoters tested in maize.     

Isolation and characterization of melanin pigment from <Emphasis Type="Italic">Pleurotus cystidiosus</Emphasis> (telomorph of <Emphasis Type="Italic">Antromycopsis</Emphasis><Emphasis Type="Italic">macrocarpa</Emphasis>)

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of bacteria and fungi. For more than 40 years, fungi have been known to produce pigments called melanins. Melanin pigment production by mushrooms was not intensively studied. The present study was carried out on isolation and characterization of melanin from an edible mushroom Pleurotus cystidiosus var. formosensis. The mushroom produced dark mucous mass of hyaline arthrospores on mycelium. The coremia exclusively produced dikaryotic arthrospores with the remnant of a clamp connection. Continuous cell extension and division in the coremium stipe supplied cells for arthroconidiation at the coremium apex, which is surrounded by a liquid droplet (coremioliquid). The black coloured coremea (conidia) were produced by Antromycopsis macrocarpa (anamorph of P. cystidiosus) when cultured on potato dextrose agar medium. The agar plate was incubated at continuous light illumination for high amount of pigment (coremea) production. The slimy layer of the coremea was extracted and partially purified by alkaline and acid treatment. The black pigment was confirmed as melanin based on UV, IR and EPR spectra apart from chemical analysis. This is the first report on characterization of melanin obtained from Pleurotus cystidiosus var. formosensis.