Modification of phytosterol composition influences cotton fiber cell elongation and secondary cell wall deposition

Abstract

Background: Cotton fiber is a single cell that arises from the epidermis of ovule. It is not only a main economic product of cotton, but an ideal material for studying on the growth and development of plant cell. Our previous study indicated that phytosterol content and the ratio of campesterol to sitosterol fluctuated regularly in cotton fiber development. However, what effects of modified phytosterol content and composition on the growth and development of cotton fiber cell is unknown. In this study, we overexpressed the GhSMT2-1, a cotton homologue of sterol C-24 methyltransferase 2 gene in transgenic upland cotton plants to modify phytosterol content and composition in fiber cells and investigated the changes on fiber elongation and secondary cell wall deposition.

Results: GhSMT2-1 overexpression led to changes of phytosterol content and the ratio of campesterol to sitosterol in fiber cell. At the rapid elongation stage of fiber cell, total phytosterol and sitosterol contents were increased while campesterol content was decreased in transgenic fibers when compared to control fibers. Accordingly, the ratio of campesterol to sitosterol declined strikingly. Simultaneously, the transgenic fibers were shorter and thicker than control fibers. Exogenous application of sitosterol or campesterol separately inhibited control fiber cell elongation in cotton ovule culture system in vitro. In addition, campesterol treatment partially rescued transgenic fiber elongation.

Conclusion: These results elucidated that modification of phytosterol content and composition influenced fiber cell elongation and secondary cell wall formation. High sitosterol or low ratio of campesterol to sitosterol suppresses fiber elongation and/or promote secondary cell wall deposition. The roles of sitosterol and campesterol were discussed in fiber cell development. There might be a specific ratio of campesterol to sitosterol in different developmental stage of cotton fibers, in which GhSMT2-1 play an important role. Our study, at a certain degree, provides novel insights into the regulatory mechanisms of fiber cell development.

Keywords: Cotton fiber; Fiber elongation; GhSMT2–1; Phytosterol; Secondary cell wall.

Fig. 1

Sketch of expression vector and characterization of transgenic cotton plants overexpressing GhSMT2–1. a Sketch of pBI121-GN-OS. RB and LB is right border and left border of T-DNA region, respectively. NOS-P: NOS promoter; NOS-T: NOS terminator; 35S: CaMV 35S promoter. b GUS staining in cotton leaf discs. c Genotyping of transgenic cotton plants by PCR. A fragment of GhSMT2–1 cDNA was amplified from genomic DNA. M, DNA marker; P, positive control, amplified from a plasmid containing the 35S::GhSMT2–1::NOS-T cassette; N, negative control, amplified from the genomic DNA of a non-transgenic cotton plant; W, blank control, amplified from a sample containing only water. d Relative expression level of the GhSMT2–1 gene in the 10-DPA fibers and leaves of control and transgenic cotton plants. Total RNA was isolated from the 10-DPA fibers and leaves of control plants and transgenic lines. The first strand cDNA (from total RNA) was used as the template for RT-qPCR, using cotton HISTONE3 for normalization of the data. OS-1 to OS-10 represents transgenic plant no. 1 to 10

Fig. 2

Change of phytosterols content and composition in transgenic 10-DPA fibers. 10-DPA cotton bolls were harvested and fibers derived from the ovule were rapidly dried at 70 °C. The dried material was ground to powder and quantified. 2.0 g 10-DPA fiber powder was used for phytosterol extraction. a Contents of campesterol, sitosterol, and stigmasterol in 10-DPA fibers from control, OS-6, and OS-9 plants. Error bars represent the SD for three independent experiments. b Total phytosterols in 10-DPA fibers from control and transgenic plants. c The ratio of campesterol to sitosterol in 10-DPA fibers from control, OS-6, and OS-9 plants. Asterisks indicate statistically significant differences between transgenic lines and the control, as determined by Student’s t-test (**, P < 0.01)

Fig. 3

Fiber length in transgenic lines and the control. a and c is the photo and fiber length data from cotton ovule culture in vitro, respectively. The photo was taken before combing and showed the original shape of fiber (with ovule) after culture. b and d is the photo and fiber length data of mature fiber from plant, respectively. The photo was taken after combing. a The fibers (with ovules) of the control and transgenic lines after 10-day culture. b Mature and dried fibers (combed by hand before photographing) from the control plant and transgenic plant OS-6. c Fiber lengths of control and transgenic cotton lines after 10-day culture; error bars represent the SD for at least 15 seeds. d Lengths of mature fiber from control and transgenic lines. OS-6 and OS-9: transgenic lines 6 and 9 overexpressing GhSMT2–1. Error bars represent SD for at least 20 seeds. Statistically significant differences based on paired Student’s t-test at P < 0.01 are denoted by double asterisks

Fig. 4

Micronaire values and secondary cell wall thickness of mature fiber. a-c Microscopic images of cross-sections of mature fibers of the control (a), transgenic OS-6 (b), and OS-9 (c). The thickness of secondary cell wall is indicated by a red demarcation. d Micronaire values of control and transgenic mature fibers. Error bars represent the SD of three biological replicates. Statistically significant differences based on paired Student’s t-test at P < 0.01 are denoted by double asterisks

Fig. 5

Effects of sitosterol or campesterol on fiber growth in ovule culture system in vitro. a and b Mocks for the ovules from control plants (a) and transgenic cotton lines (b). BT medium adjusted with the amount of ethanol equivalent to that used to dissolve sitosterol or campesterol was used as the mock. c and d Sitosterol treatment of fibers from control plants (c) and OS-6 line(d). e and f Campesterol treatment of fibers from control plants(e) and OS-6 line (f). All ovules derived from control and transgenic plants were cultured on liquid BT medium for 10 days. g Fiber length of mock and sitosterol-treated ovules of the control after 10-day culture in BT medium. h Fiber length of mock and campesterol-treated ovules of the control after 10-day culture in BT medium. i Fiber lengths of mock and campesterol-treated ovules of the OS-6 transgenic plant after 10-day culture in BT medium. Error bars represent SD for at least 10 seeds. Asterisks indicate statistically significant differences between the mock and treatment, as determined by Student’s t-test (*, P < 0.05)