The Function Of Campesterol And Sitosterol For Cotton Fiber Development

Cotton is a leading crop to produce fibers.Cotton fibers,as a main production of cotton plants,are unicellular,linear structures derived from the epidermis of the ovule.Cotton fiber development consists of four different and overlapping developmental stages:fiber initiation,cell elongation,secondary wall deposition,and maturation.Fiber cells from upland cotton generally grow up to 20-30 mm in length and about 15μm in thickness at full maturity.Therefore,their lengths are 1,000 to 3,000 times longer than their diameter.In addition to being among the longest plant cells ever characterized,a single cell wall biopolymer,cellulose,accounts for over 95%of the dry weight of the mature cotton fiber.The highly elongated structure and exceptional chemical composition of fiber cell establishes itself as an ideal experimental model for studying plant cell elongation,synthesis of cellulose,and cell wall accumulation.However,the mechanism of fiber development is hitherto largely unknown.The sterols are sort of important intermediates in BR(Brassinosteroids) biosynthetic up-stream pathways.They are not only the precursor of BL(Brassinolide,the most active BR) but also the structure components of membrane.Recent research of the sterol Arabidopsis thaliana mutant proved that the sterols perform different bioactivities and physiological functions from the BL in the complicated network of plant developmental regulations.They play crucial role in the function of cell elongation, cellulose biosynthesis and cell wall formation.The pathway of sterol biosynthesis was branched by SMT2 into two biosynthestic segments:one produces 24-methyl sterols such as campesterol,and the other produces 24-ethyl sterols such as sitosterol.The campesterol was the precursor of BL and the sitosterol is the primer for cellulose biosynthesis.The two kinds of phytosterols,not only their contents but also the ratio of sitosterol to campesterol,play an important role in cell growth and development,and the cotton fibers were characterized to be longest plant cells and with high content of cellulose,so the study of the campesterol and sitosterol was meaningful for understand the molecular mechanism of the cotton fiber development.To understand the function of campesterol and sitosterol for the cotton fiber development,we tested the the content of campesterol and sitosterol in different developmental stages of cotton fibers, Furthermore,the phytosterol biosynthesis inhibitor was used in ovule culture system and field test to study the the function of sterols for the cotton fiber structure formation. Finally,we cloned the GhSMT2 gene,which is a rate-limited enzyme for sitosterol biosynthesis.The transgenic cotton plants of overexpressing GhSMT2 gene were obtained.Phytosterol analysis and the variation analysis of fiber growth were finished. The results are as follows.1 The content of phytosterols in various developmental stages of cotton fibers The phytosterols were extracted and tested to know the variational pattern of the phytosterols content during various development stages of cotton fibers,the result showed that the content of campesterol and sitosterol are the highest in 8DPA (sitoterol:0.435±0.081mg/g,campesterol:0.086±0.016mg/g)of the fiber elongation, however,the content decreased to a lower level in the secondary wall deposition,at the day of 15DPA,the content of sitosterol falled to 0.306±0.036mg/g and campesterol decreased to 0.062±0.09mg/g.The ratio of the campesterol to sitosterol was accounted in different stage of the fiber development.Furthermore,the contents of the two sterols in ovules were held in a low level during all fiber elongation and secondary wall deposition.These results showed that campesterol and sitosterol is important in the fiber elongation stage.2 The phytosterol biosynthesis inhibitor suppressed the fiber cell elongation in in ovule culture system and plants testWe used the cotton ovule in vitro culture system which was added with tridemorph,a kind of morpholinium compound,to study the function of sterols on the cotton fiber development.For quantitative analysis,average fiber lengths of different concentration treatments are measured.The result displayed that these fibers treated with inhibitor of sterols tridemorph were restrained in a dose-dependent manner,that is 6.04mm-9.51mm, and the ovules were also inhibited in the higher concentration of tridemorph.The inhibitor also suppressed the initiation of cotton fibers,the initial protuberances of fiber cells on inhibited plant ovule were smaller than the fiber cells on control plant ovule; The mature fibers treated with tridemorph were measured,the length of the mature fibers which were treated with tridemorph was 27.17±1.56mm while the control fibers length was 30±1.64mm,the mature fibers treated with tridemorph were shorter than the control plant,these results showed that phytosterol play important role in the fiber elongation.3 Overexpressing GhSMT2 Gene results in transgenic cotton fiber length decreaseTo understand the variation of cotton fiber when the content and ratio of campesterol and sitosterol were changed,we constructed p5-CaMV35S::GhSMT2 vector and transformed it into cotton plants by means of Agrobacterium-mediated transformation. The result is that the overexpression of GhSMT2 lead to sitosterol increased and campesterol decreased,the ratio of campesterol to sitosterol was also decreased.At the same time,the fibers from the overexpress GhSMT2 plants were shorter than wildtype, after 10 days fostered in the in ovule culture system,the wildtype fiber length was 11.84±1.30mm while the transgenic fiber length was 4.29±1.25mm,the mature fiber of transgenic plants length was also shorter than the wildtype.The cotton balls of the transgenic plants were smaller than wildtype and the aborted seeds of overexpressing GhSMT2 were more than the WT.Furthermore,circularity of GhSMT2 was higher than wildtype.