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.     

Functional divergence of GLP genes between G. barbadense and G. hirsutum in response to Verticillium dahliae infection

Germin-like proteins (GLPs) play important roles in plant disease resistance but are rarely reported in cotton. We compared the expression of GLPs in Verticillium dahliae inoculate G. hirsutum (susceptible) and G. barbadense (resistant) and enriched 11 differentially expressed GLPs. 2741 GLP proteins identified from 53 species determined that GLP probably originated from algae and could be classified into 7 clades according to phylogenetic analysis, among which Clade I is likely the most ancient. Cotton GLP (two allopolyploids and two diploids) genes within a shared clade were highly conserved. Intriguingly, clade VII genes were mainly located in gene clusters that derived from the expansion of LTR transposons. Clade VII members expressed mainly in root which is the first battle against Verticillium dahlia and could be induced more intensely in G. barbadense than G. hirsutum. The GLP genes are resistant to Verticillium dahliae, which can be further investigated against Verticillium wilt.     

Cotton annexin proteins participate in the establishment of fiber cell elongation scaffold

Cotton plant is one of the most important economic crops in the world which supplies natural fiber for textile industry. The crucial traits of cotton fiber quality are fiber length and strength, which are mostly determined by the fiber elongation stage. Annexins are assumed to be involved in regulating fiber elongation, but direct evidences remain elusive. Recently, we have investigated the activities of fiber-specific expressed annexins AnGb5/6 and their interacted proteins in cotton. AnGb5 and 6 can interact reciprocally to generate a protein macro-raft in cell membrane. This macro-raft is probably a stabilized scaffold for Actin1 organization. The actin assembling direction and density are correlated with AnGb6 gene expression and fiber expanding rate among three fiber length genotypes. These results suggest that annexins may act as the adaptor that linked fiber cell membrane to actin assembling. Due to the strong Ca²⁺ and lipid binding ability of annexins, these results also indicate that annexins complex may function as an intermediate to receive Ca²⁺ or lipid signals during 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.     

Isolation, characterization and mapping of genes differentially expressed during fibre development between Gossypium hirsutum and G. barbadense by cDNA-SRAP

Gossypium hirsutum and G. barbadense are two cultivated tetraploid cotton species with differences in fibre quality. The fibre of G. barbadense is longer, stronger and finer than that of G. hirsutum. To isolate genes expressed differently between the two species during fibre development, cDNA-SRAP (sequence-related amplified polymorphism) was applied. This technique was used to analyse genes at different stages of fibre development in G. hirsutum cv. Emian22 and G. barbadense acc. 3-79, the parents of our interspecific mapping population. A total of 4096 SRAP primer combinations were used to screen polymorphism between the DNA of the parents, and 275 highly polymorphic primers were picked out to analyse DNA and RNA from leaves and fibres at different developmental stages of the parents. A total of 168 DNA fragments were isolated from gels and sequenced: 54, 30, 38 and 41 from fibres of 5, 10, 15 and 20 days post-anthesis, respectively, and five from multi stages. To genetically map these sequences, 104 sequence-specific primers were developed and were used to screened polymorphism between the mapping parents. Finally, six markers were mapped on six chromosomes of our backbone interspecific genetic map. This work can give us a primary knowledge of differences in mechanism of fibre development between G. hirsutum and G. barbadense.     

Cotton Fiber Cell Walls of Gossypium hirsutum and Gossypium barbadense Have Differences Related to Loosely-Bound Xyloglucan

Cotton fiber is an important natural textile fiber due to its exceptional length and thickness. These properties arise largely through primary and secondary cell wall synthesis. The cotton fiber of commerce is a cellulosic secondary wall surrounded by a thin cuticulated primary wall, but there were only sparse details available about the polysaccharides in the fiber cell wall of any cotton species. In addition, Gossypium hirsutum (Gh) fiber was known to have an adhesive cotton fiber middle lamella (CFML) that joins adjacent fibers into tissue-like bundles, but it was unknown whether a CFML existed in other commercially important cotton fibers. We compared the cell wall chemistry over the time course of fiber development in Gh and Gossypium barbadense (Gb), the two most important commercial cotton species, when plants were grown in parallel in a highly controlled greenhouse. Under these growing conditions, the rate of early fiber elongation and the time of onset of secondary wall deposition were similar in fibers of the two species, but as expected the Gb fiber had a prolonged elongation period and developed higher quality compared to Gh fiber. The Gb fibers had a CFML, but it was not directly required for fiber elongation because Gb fiber continued to elongate rapidly after CFML hydrolysis. For both species, fiber at seven ages was extracted with four increasingly strong solvents, followed by analysis of cell wall matrix polysaccharide epitopes using antibody-based Glycome Profiling. Together with immunohistochemistry of fiber cross-sections, the data show that the CFML of Gb fiber contained lower levels of xyloglucan compared to Gh fiber. Xyloglucan endo-hydrolase activity was also higher in Gb fiber. In general, the data provide a rich picture of the similarities and differences in the cell wall structure of the two most important commercial cotton species.     

Genetic dissection of lint yield and fiber quality traits of <Emphasis Type="Italic">G. hirsutum</Emphasis> in <Emphasis Type="Italic">G. barbadense</Emphasis> background

Gossypium hirsutum L. is a widely cultivated species characterized by its high yield and wide environmental adaptability, while Gossypium barbadense is well known for its superior fiber quality. In the present report, we, for the first time, developed G. hirsutum chromosome segment introgression lines (ILs) in a G. barbadense background (GhILs_Gb) and genetically dissected the inheritance of lint yield and fiber quality of G. hirsutum in G. barbadense background. The GhILs_Gb contains introgressed segments spanning 4121.20 cM, which represents 82.20% of the tetraploid cotton genome, with an average length of 18.65 cM. A total of 39 quantitative trait loci (QTLs) for six traits are identified in this IL population planted in Xinjiang. Four QTL clusters are detected. Of them, however, three clusters have deleterious effects on fiber length and strength and boll weight, and only one cluster on Chr. D9 can be used in marker-assisted selection (MAS) to increase lint percentage and decrease micronaire value in G. barbadense. QTL mapping showed that most of yield-related QTLs detected have positive effects and increase lint yield in G. barbadense, while most of fiber quality-related QTLs have deleterious effects except for micronaire. It suggested that G. hirsutum evolved to have a high lint yield. Several lines improved in lint percentage and boll size in G. barbadense by introgressed one fragment of G. hirsutum have been developed from the GhILs_Gb. The ILs developed, and the analyses presented here will enhance the understanding of the genetics of lint yield and fiber quality in G. hirsutum and facilitate further molecular breeding to improve lint yield in G. barbadense.     

The evolution of spinnable cotton fiber entailed prolonged development and a novel metabolism

A central question in evolutionary biology concerns the developmental processes by which new phenotypes arise. An exceptional example of evolutionary innovation is the single-celled seed trichome in Gossypium (“cotton fiber”). We have used fiber development in Gossypium as a system to understand how morphology can rapidly evolve. Fiber has undergone considerable morphological changes between the short, tightly adherent fibers of G. longicalyx and the derived long, spinnable fibers of its closest relative, G. herbaceum, which facilitated cotton domestication. We conducted comparative gene expression profiling across a developmental time-course of fibers from G. longicalyx and G. herbaceum using microarrays with ~22,000 genes. Expression changes between stages were temporally protracted in G. herbaceum relative to G. longicalyx, reflecting a prolongation of the ancestral developmental program. Gene expression and GO analyses showed that many genes involved with stress responses were upregulated early in G. longicalyx fiber development. Several candidate genes upregulated in G. herbaceum have been implicated in regulating redox levels and cell elongation processes. Three genes previously shown to modulate hydrogen peroxide levels were consistently expressed in domesticated and wild cotton species with long fibers, but expression was not detected by quantitative real time-PCR in wild species with short fibers. Hydrogen peroxide is important for cell elongation, but at high concentrations it becomes toxic, activating stress processes that may lead to early onset of secondary cell wall synthesis and the end of cell elongation. These observations suggest that the evolution of long spinnable fibers in cotton was accompanied by novel expression of genes assisting in the regulation of reactive oxygen species levels. Our data suggest a model for the evolutionary origin of a novel morphology through differential gene regulation causing prolongation of an ancestral developmental program.