Analysis of the Protein Constituency of Developing Cotton Fibres

Graves, D. A. and Stewart, J. McD. 1988. Analysis of the proteinconstituency of developing cotton fibres.—J. exp. Bot.39: 59–69. Protein composition of young cotton (Gossypium hirsuium L.)ovules and fibres was examined in order to characterize grosschanges during early development and to determine if changescorrespond to known developmental events. Major emphasis wason the fibre, although ovule development was considered at earlierages. Ovules and fibres from upland cotton (‘Deltapine 61’)were collected at 6, 3, and 1 d pre-anthesis, at anthesis andat 2, 6, 10, 16 and 20 d post-anthesis. Total protein was extractedfrom whole ovules or isolated fibres and subjected to two-dimensionalgel electrophoresis. The gels indicated the following: (1) Theprotein patterns of ovules and fibres 3 d pre-anthesis to 2d post-anthesis were relatively similar, suggesting a plateauin protein expression. The plateau was proposed to reflect thetime during which fibre cells are differentiated from otherepidermal cells. (2) The plateau was delineated by apparentperiods of increased complexity in the population at 3 d pre-anthesisand 2 d post-anthesis. (3) A transition in the protein populationoccurred between 2 and 10 d post-anthesis. (4) By 6 to 10 dpost-anthesis, the fibre began to express proteins putativelyinvolved with secondary wall synthesis. (5) Increases in spotintensity of certain proteins around 16 d post-anthesis maycorrelate with cellulose synthesis reported by others to peakabout this time. These observations suggest that early ovule and fibre developmentis characterized by at least two distinct protein populations.The first occurs around anthesis and the second occurs by 10d and extends to 20 d post-anthesis and beyond. Key words: Gossypium hirsutum, fibres, ovules, protein composition     

Changes in the composition of cotton fibre cell walls during development

Purified cell walls, prepared from cotton fibres (Gossypium arboreum L.) at different growth stages, were subjected to successive extractions to give pectic, hemicellulosic, and -cellulosic fractions. The protein content and sugars obtained after hydrolysis of the total cell walls and of the various fractions were quantitatively estimated. The amount of protein in the fibre cell walls from one ovule reached a maximum value at the end of the elongation growth, decreased, and then reached a second maximum at the end of the secondary wall deposition. The absolute amounts of fucose, galactose, mannose, rhamnose, arabinose, uronic acid, and non-cellulosic glucose residues all reached a maximum at the end of the primary wall formation or at the beginning of the secondary wall formation. Only the absolute amounts of xylose and of the cellulosic glucose residues increased until the end of the fibre development. Most conspicuous was the decrease in the absolute amounts of non-cellulosic glucose and of arabinose residues during the secondary wall formation, possibly indicating a turnover of at least some of the hemicellulosic wall material.Abbreviations DPA days post anthesis - TLC thin layer chromatography - SDS sodium dodecyl sulphate     

GhXB38D represses cotton fibre elongation through ubiquitination of ethylene biosynthesis enzymes GhACS4 and GhACO1

Ethylene plays an essential role in the development of cotton fibres. Ethylene biosynthesis in plants is elaborately regulated by the activities of key enzymes, 1-aminocyclopropane-1-carboxylate oxidase (ACO) and 1-aminocyclopropane-1-carboxylate synthase (ACS); however, the potential mechanism of post-translational modification of ACO and ACS to control ethylene synthesis in cotton fibres remains unclear. Here, we identify an E3 ubiquitin ligase, GhXB38D, that regulates ethylene biosynthesis during fibre elongation in cotton. GhXB38D gene is highly expressed in cotton fibres during the rapid elongation stage. Suppressing GhXB38D expression in cotton significantly enhanced fibre elongation and length, accompanied by the up-regulation of genes associated with ethylene signalling and fibre elongation. We demonstrated that GhXB38D interacts with the ethylene biosynthesis enzymes GhACS4 and GhACO1 in elongating fibres and specifically mediates their ubiquitination and degradation. The inhibition of GhXB38D gene expression increased the stability of GhACS4 and GhACO1 proteins in cotton fibres and ovules, resulting in an elevated concentration of ethylene. Our findings highlight the role of GhXB38D as a regulator of ethylene synthesis by ubiquitinating ACS4 and ACO1 proteins and modulating their stability. GhXB38D acts as a negative regulator of fibre elongation and serves as a potential target for enhancing cotton fibre yield and quality through gene editing strategy.     

Down-regulation of GhADF1 gene expression affects cotton fibre properties

Cotton fibre is the most important natural fibres for textile industry. To date, the mechanism that governs the development of fibre traits is largely unknown. In this study, we have characterized the function of a member of the actin depolymerizing factor (ADF) family in Gossypium hirsutum by down-regulation of the gene (designated as GhADF1 ) expression in the transgenic cotton plants. We observed that both the fibre length and strength of the GhADF1 -underexpressing plants increased as compared to the wild-type fibre, and transgenic fibres contained more abundant F-actin filaments in the cortical region of the cells. Moreover, the secondary cell wall of the transgenic fibre appeared thicker and the cellulose content was higher than that of the control fibre. Our results suggest that organization of actin cytoskeleton regulated by actin-associated proteins such as GhADF1 plays a critical role in the processes of elongation and secondary cell wall formation during fibre development. Additionally, our study provided a candidate intrinsic gene for the improvement of fibre traits via genetic engineering.     

A secondary phloic (bast) fibre-shy (<Emphasis Type="Italic">bfs</Emphasis>) mutant of dark jute (<Emphasis Type="Italic">Corchorus olitorius</Emphasis> L.) develops lignified fibre cells but is defective in cambial activity

Bast fibre development in jute (Corchorus spp.) is a complex process that involves the differentiation of secondary phloic fibres (SPF) from the cambium followed by lignification of the fibre wall. We have identified a unique radiation-induced bast fibre-shy mutant of dark jute (C. olitorius L.), which is concurrently defective in the differentiation of SPF and secondary xylem (wood) but develops lignified fibre cells. It displays the most unusual phenotype with stunted growth and abnormal leaf shape, matures earlier, yields significantly less bast fibres and wood, and produces poorer quality fibres than its parental wild-type. Cambial activities in the mutant and the normal type were monitored by estimating the fibre content that entails the total number of fibre cell bundles (FCBs) in an entire transversal section. The results show that a multi-fold reduction of bast fibre yield in the mutant is related to development-specific loss of cambium function along the length of the stem from to top to bottom. Since lignification of the fibre wall in the mutant is not only normal but also developmentally uniform, cambium function may be unrelated to the lignification process during bast fibre development. Lignin does not influence bast fibre strength and fineness. The architecture of the mostly triangular FCB wedges, which is governed by a balanced growth between radially elongating FCBs and tangentially expanding ray cells due to development-specific activation of the fusiform and ray initials of the cambium, conditions fibre fineness. Our study shows that mutation could specifically impair the cambial activity by rendering those initials that differentiate the SPF and secondary xylem nonfunctional.     

Glucan synthesis by intact cotton fibres fed with different precursors at the stages of primary and secondary wall formation

Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 M were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo--1,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight material was found in cellulose and at least 2/3 in -1,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation -1,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.Abbreviations UDPG uridinediphosphoglucose - GDPG guanosinediphosphoglucose - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid - DMSO dimethyl-sulfoxide - DNP 2,4-dinitrophenol - DPA days post anthesis     

Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array

Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule. To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver. We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes. cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases. Twenty-nine cDNAs, including a putative vacuolar (H⁺)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a. fiber cells when compared to that in 0 d.p.a. ovules. Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period. This report constitutes the first systematic analysis of genes involved in cotton fiber development. Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.     

FIBER INITIATION ON THE COTTON OVULE (GOSSYPIUM HIRSUTUM)

Cotton ovules at the stage of fiber initiation were studied with a scanning electron microscope. On the day before anthesis the anatropous ovule had many anomocytic stomata, especially on the chalazal end. At anthesis, fibers first appeared at the crest of the funiculus and then around the lateral circumference of the ovule. Fiber initials were delayed for a few hours at the chalazal cap and for 3 or 4 days at the extreme end of the micropylar region. The morphological differentiation of a fiber began when an epidermal cell rounded up and protruded. After a brief period of diametric expansion, the initial began elongation toward the micropylar end. Between 2 and 3 days of age the fibers segregated into small groups, developed tapered tips, and began spiral growth. Fiber density was about 3,300 fibers per mm², and the ratio of fiber initials to total epidermal cells was 1:3.7 at anthesis.