Hongjie Feng

Analysis of Flavonoids and the Flavonoid Structural Genes in Brown Fiber of Upland Cotton

Backgroud As a result of changing consumer preferences, cotton (Gossypium Hirsutum L.) from varieties with naturally colored fibers is becoming increasingly sought after in the textile industry. The molecular mechanisms leading to colored fiber development are still largely unknown, although it is expected that the color is derived from flavanoids. Experimental Design Firstly, four key genes of the flavonoid biosynthetic pathway in cotton (GhC4H, GhCHS, GhF3′H, and GhF3′5′H) were cloned and studied their expression profiles during the development of brown- and white cotton fibers by QRT-PCR. And then, the concentrations of four components of the flavonoid biosynthetic pathway, naringenin, quercetin, kaempferol and myricetin in brown- and white fibers were analyzed at different developmental stages by HPLC. Result The predicted proteins of the four flavonoid structural genes corresponding to these genes exhibit strong sequence similarity to their counterparts in various plant species. Transcript levels for all four genes were considerably higher in developing brown fibers than in white fibers from a near isogenic line (NIL). The contents of four flavonoids (naringenin, quercetin, kaempferol and myricetin) were significantly higher in brown than in white fibers and corresponding to the biosynthetic gene expression levels. Conclusions Flavonoid structural gene expression and flavonoid metabolism are important in the development of pigmentation in brown cotton fibers.

Potential of Endophytic Fungi Isolated from Cotton Roots for Biological Control against Verticillium Wilt Disease

Verticillium wilt is a soil-borne disease, and severely limits the development of cotton production. To investigate the role of endophytic fungi on Verticillium wilt, CEF-818 (Penicillium simplicissimum), CEF-714 (Leptosphaeria sp.), CEF-642 (Talaromyces flavus.) and CEF-193 (Acremonium sp.) isolated from cotton roots were used to assess their effects against cotton wilt disease caused by a defoliating V. dahliae strain Vd080. In the greenhouse, all treatments significantly reduced disease incidence and disease index, with the control efficacy ranging from 26% (CEF-642) to 67% (CEF-818) at 25 days (d) after inoculation. In the disease nursery, compared to controls (with disease incidence of 33.8% and disease index of 31), CEF-818, CEF-193, CEF-714 and CEF-642 provided a protection effect of 69.5%, 69.2%, 54.6% and 45.7%, respectively. Especially, CEF-818 and CEF-714 still provided well protection against Verticillium wilt with 46.9% and 56.6% or 14.3% and 33.7% at the first peak of the disease in heavily infected field, respectively (in early July). These results indicated that these endophytes not only delayed but also reduced wilt symptoms on cotton. In the harvest, the available cotton bolls of plant treated with CEF-818 and CEF-714 increased to 13.1, and 12.2, respectively. And the seed cotton yield significantly increased after seed bacterization with CEF-818 (3442.04 kg/ha) compared to untreated control (3207.51 kg/ha) by 7.3%. Furtherly, CEF-818 and CET-714 treatment increased transcript levels for PAL, PPO, POD, which leads to the increase of cotton defense reactions. Our results indicate that seed treatment of cotton plants with CEF-818 and CET-714 can help in the biocontrol of V. dahliae and improve seed cotton yield in cotton fields. This study provided a better understanding of cotton-endophyte interactions which will aid in developing effective biocontrol agents for Verticillium wilt of cotton in futhre.

Isolation and functional analysis of the pathogenicity-related gene VdPR3 from Verticillium dahliae on cotton.

The fungal plant pathogen Verticillium dahliae is the causal agent of vascular wilt, a disease that can seriously diminish cotton fiber yield. The pathogenicity mechanism and the identity of the genes that interact with cotton during the infection process still remain unclear. In this study, we investigated the low-pathogenic, non-microsclerotium-producing mutant vdpr3 obtained in a previous study from the screening of a T-DNA insertional library of the highly virulent isolate Vd080; the pathogenicity-related gene (VdPR3) in wild-type strain Vd080 was cloned. Knockout mutants (ΔVdPR3) showed lower mycelium growth and obvious reduction in sporulation ability without microsclerotium formation. An evaluation of carbon utilization in mutants and wild-type isolate Vd080 demonstrated that mutants-lacking VdPR3 exhibited decreased cellulase and amylase activities, which was restored in the complementary mutants (ΔVdPR3-C) to levels similar to those of Vd080. ΔVdPR3 postponed infectious events in cotton and showed a significant reduction in pathogenicity. Reintroduction of a functional VdPR3 copy into ΔVdPR3-C restored the ability to infect cotton plants. These results suggest that VdPR3 is a multifunctional gene involved in growth development, extracellular enzyme activity, and virulence of V. dahliae on cotton.

Molecular analysis of proanthocyanidins related to pigmentation in brown cotton fibre (Gossypium hirsutum L.)

The structural characteristics and component differences of proanthocyanidins in brown and white cotton fibres were identified by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analyses. Proanthocyanidins in brown and white cotton fibres were found to contain mainly procyanidin (PC) and prodelphidin (PD) units with 2, 3-cis form (epigallocatechin and epicatechin). However, part of the proanthocyanidins in the white cotton fibres were modified by acylation and were constitutively different from the proanthocyanidins in brown cotton fibres. The relative amount of PD was similar to that of PC in white cotton fibres, while proanthocyanidins in brown cotton fibres consisted mainly of PD units with a relative ratio of 9:1. In brown cotton fibres, the proanthocyanidin monomeric composition was consistent with the expression profiles of proanthocyanidin synthase genes, suggesting that anthocyanidin reductase represented the major flow of the proanthocyanidin biosynthesis pathway. In addition, the structural characteristics and component differences of proanthocanidins in brown and white cotton fibres suggested that quinones, the oxidation products of proanthocyanidins, were the direct contributors to colour development in brown cotton fibre. This was demonstrated by vanillin-HCl staining and Borntragers test. Collectively, these data demonstrated that the biosynthesis of proanthocyanidins is a crucial pigmentation process in brown cotton fibre, and that quinones may represent the main pigments contributing to formation of the the brown colour. This study revealed the molecular basis of pigmentation in brown cotton fibres, and provided important insights for genetic manipulation of pigment production in cotton fibres.

VdCYC8, Encoding CYC8 Glucose Repression Mediator Protein, Is Required for Microsclerotia Formation and Full Virulence in Verticillium dahliae.

Verticillium dahliae is the primary causal agent for Verticillium wilt disease on a diverse array of economically important crops, including cotton. In previous research, we obtained the low-pathogenicity mutant T286 from the T-DNA insertional mutant library of the highly virulent isolate Vd080 derived from cotton. In this study, the target disrupted gene VdCYC8 was identified by TAIL-PCR, encoding a homolog of CYC8 proteins involved in glucose repression. The deletion mutant ΔCYC8 exhibited several developmental deficiencies, including reduced microsclerotia formation, reduced sporulation, and slower growth. Moreover, compared with the wild type strain Vd080, the pathogenicity of strain ΔCYC8 was significantly decreased on cotton seedlings. However, the complementary mutants ΔCYC8-C led to restoration of the wild type phenotype or near wild type levels of virulence on cotton. Interestingly, pathogenicity of the strains was correlated with VdCYC8 gene expression levels in complemented mutants. Gene expression analyses in the wild type strain Vd080, the ΔCYC8-45 strain, and complemented strain ΔCYC8-C26 indicated that VdCYC8 regulates the transcription levels of several genes in V. dahliae that have roles in melanin and production.

The endochitinase VDECH from Verticillium dahliae inhibits spore germination and activates plant defense responses

Chitinases function in the digestion of chitin molecules, which are present principally in insects and fungi. In plants, chitinase genes play important roles in defense, and their expression can be triggered in response to both biotic and abiotic stresses. In this study, we cloned and characterized an endochitinase (VDECH) from Verticillium dahliae, strain Vd080. The VDECH coding region consists of 1845bp with two exons and one 54bp intron, encoding a 615 amino acid protein with the predicted molecular weight (MW) of 63.9kDa. The VDECH cDNA without signal peptide-encoding region was introduced into pCold-TF vector and the recombinant protein HIS-VDECH with a predicted MW of ∼114kDa was expressed. HIS-VDECH showed high tolerance to extreme temperature, exhibiting efficient chitinolytic activity at 50°C. In addition, VDECH triggered typical plant defense responses, including a hypersensitive response, oxidative burst, and elicited increased expression of defense-related genes in both Arabidopsis and cotton. VDECH-treatment of the conidial spores of V. dahliae and Fusarium oxysporum resulted in marked reductions in the germination of these spores in both fungi. After 36h of incubation with VDECH, the inhibition rate of germination was recorded at 99.57% for V. dahliae, and 96.89% for F. oxysporum. These results provide evidence that VDECH is recognized by the plant to elicit defense responses, and also that VDECH is an effective inhibitor of conidia germination, both of which may be exploited for disease control.

The Negative Correlation between Fiber Color and Quality Traits Revealed by QTL Analysis.

Naturally existing colored cotton was far from perfection due to having genetic factors for lower yield, poor fiber quality and monotonous color. These factors posed a challenge to colored cotton breeding and innovation. To identify novel quantitative trait loci (QTL) for fiber color along with understanding of correlation between fiber color and quality in colored cotton, a RIL and two F2 populations were generated from crosses among Zong128 (Brown fiber cotton) and two white fiber cotton lines which were then analyzed in four environments. Two stable and major QTLs (qLC-7-1, qFC-7-1) for fiber lint and fuzz color were detected accounting for 16.01%-59.85% of the phenotypic variation across multiple generations and environments. Meanwhile, some minor QTLs were also identified on chromosomes 5, 14, 21 and 24 providing low phenotypic variation (<5%) from only F2 populations, not from the RILs population. Especially, a multiple-effect locus for fiber color and quality has been detected between flanking markers NAU1043 and NAU3654 on chromosome 7 (A genome) over multiple environments. Of which, qLC-7-1, qFC-7-1 were responsible for positive effects and improved fiber color in offsprings. Meanwhile, the QTLs (qFL-7-1, qFU-7-1, qFF-7-1, qFE-7-1, and qFS-7-1) for fiber quality had negative effects and explained 2.19%-8.78% of the phenotypic variation. This multiple-effect locus for fiber color and quality may reveal the negative correlation between the two types of above traits, so paving the way towards cotton genetic improvement.

Functional Analysis of the Pathogenicity-Related Gene VdPR1 in the Vascular Wilt Fungus Verticillium dahliae

Verticillium dahliae Kleb., the causal agent of vascular wilt, can seriously diminish the yield and quality of many crops, including cotton. The pathogenic mechanism to cotton is complicated and unclear now. To screen pathogencity related genes and identify their function is the reliable way to explain the mechanism. In this study, we obtained a low-pathogenicity mutant vdpr1 from a T-DNA insertional library of the highly virulent isolate of V. dahliae Vd080, isolated from cotton. The tagged gene was named pathogenicity-related gene (VdPR1). The deletion mutant ΔVdPR1 did not form microsclerotia and showed a drastic reduction in spore yield and mycelial growth, compared to wild type. Also, ΔVdPR1 showed significantly lower protease and cellulase activities than those of wild type. Complementation of the mutant strain with VdPR1 (strain ΔVdPR1-C) almost completely rescued the attributes described above to wild-type levels. The knockout mutant ΔVdPR1 showed delayed infection, caused mild disease symptoms, formed a smaller biomass in roots of the host, and showed compromised systemic invasive growth in the xylem. These results suggest that VdPR1 is a multifaceted gene involved in regulating the growth development, early infection and pathogenicity of V. dahliae.

Induction of cotton ovule culture fibre branching by co-expression of cotton BTL, cotton SIM, and Arabidopsis STI genes

The highly elongated single-celled cotton fibre consists of lint and fuzz, similar to the Arabidopsis trichome. Endoreduplication is an important determinant in Arabidopsis trichome initiation and morphogenesis. Fibre development is also controlled by functional homologues of Arabidopsis trichome patterning genes, although fibre cells do not have a branched shape like trichomes. The identification and characterization of the homologues of 10 key Arabidopsis trichome branching genes in Gossypium arboreum are reported here. Nuclear ploidy of fibres was determined, and gene function in cotton callus and fibre cells was investigated. The results revealed that the nuclear DNA content was constant in fuzz, whereas a limited and reversible change occurred in lint after initiation. Gossypeum arboreum BRANCHLESS TRICHOMES (GaBLT) was not transcribed in fibres. The homologue of STICHEL (STI), which is essential for trichome branching, was a pseudogene in Gossypium. Targeted expression of GaBLT, Arabidopsis STI, and the cytokinesis-repressing GaSIAMESE in G. hirsutum fibre cells cultured in vitro resulted in branching. The findings suggest that the distinctive developmental mechanism of cotton fibres does not depend on endoreduplication. This important component may be a relic function that can be activated in fibre cells.

Molecular analysis of caffeoyl residues related to pigmentation in green cotton fibers

The pigment components in green cotton fibers were isolated and identified as 22-O-caffeoyl-22-hydroxymonodocosanoin and 22-O-caffeoyl-22-hydroxydocosanoic acid. The concentration of 22-O-caffeoyl-22-hydroxymonodocosanoin correlated positively with the degree of colour in the green fibers, indicating a role for caffeoyl derivatives in the pigmentation of green cotton fibers. Upland cotton (Gossypium hirsutum L.) contains four genes, Gh4CL1-Gh4CL4, encoding 4-coumarate:CoA ligases (4CLs), key enzymes in the phenylpropanoid biosynthesis pathway. In 15-24-day post-anthesis fibers, the expression level of Gh4CL1 was very low, Gh4CL3 had a similar expression level in both white and green cottons, Gh4CL2 had a significantly higher expression level in green fibers than in white fibers, while Gh4CL4 had a higher expression level in white fibers than in green fibers. According to enzyme kinetics analysis, Gh4CL1 displayed a preference for 4-coumarate, Gh4CL3 and Gh4CL4 exhibited a somewhat low but still prominent activity towards ferulate, while Gh4CL2 had a strong preference for caffeate and ferulate. These results suggest that Gh4CL2 might be involved in the metabolism of caffeoyl residues and related to pigment biosynthesis in green cotton fibers. Our findings provide insights for understanding the biochemical and molecular mechanisms of pigmentation in green cotton fibers.