Analysis of gene expression in cotton fiber initials

Abstract

Background: Cotton (Gossypium hirsutum L.) fibers are trichomes that initiate from the ovule epidermis. Little is known about the developmental pathway causing fiber to differentiate from ovular epidermal cells even though limits on the number of cells that differentiate into fiber will limit yield.

Results: A method was developed to isolate RNA from fiber initials 1 day post anthesis (dpa). Complementary DNA libraries representing 1 dpa fibers and other cotton tissues were sequenced and analyzed. Assembly of G. hirsutum Expressed Sequenced Tags (ESTs) identified over 11,000 sequences not previously represented in GenBank. New genes identified among these ESTs were represented on microarrays. The microarrays were used to identify genes enriched in fiber initials (1 dpa fibers) and elongating fibers. Analyses of Gene Ontologies (GO) of differentially expressed genes determined that terms associated with the "membranes" were statistically over represented among genes increased in expression in fiber initials and 10 dpa fibers. Staining ovules with a fluorescent dye confirmed an increase in Endoplasmic Reticulum (ER) occurred in fiber initials on the day of anthesis, persisted through 3 dpa and was absent in a fiberless mutant. Two genes similar to the CAPRICE/TRIPTYCHON (CPC) gene that inhibits differentiation of leaf trichomes in Arabidopsis were also characterized. Genes associated with novel regulation of brassinosterols, GTP mediated signal transduction and cell cycle control and components of a Ca+2 mediated signaling pathway were identified. Staining of cellular Ca+2 indicated that fiber initials had more Ca+2 than other ovule cells supporting a role for Ca+2 in fiber development.

Conclusion: Analysis of genes expressed in fiber initials identified a unique stage in fiber development characterized by an increase in ER and Ca+2 levels that occurred between 0 and 1 dpa. The gene similar to CPC has a MYB domain but appears to lack a transcription activating domain similar to the Arabisopsis gene. The method used to stain the ER also can be used to count fiber initials and showed fiber cells develop from adjacent cells unlike leaf trichomes.

Figure 1

RNA isolated from frozen ovules. One sample was mixed with glass beads and vortexed and a picture was taken. A picture was taken of the other frozen ovules without addition of glass beads or vortexing (A). Polyribosomal RNA was isolated from vortexed 1 dpa, 3 dpa, 5 dpa or 7 dpa ovules and shoots. Free-polyribosomal (F) or membrane bound polyribosomal (Mb) RNA was isolated from vortexed 3 dpa ovules. The RNA was separated on a 1.2% gel and visualized by staining with ethidium-bromide.

Figure 2

Validation of expression of selected genes. Lanes 1, 2 and 3 represent RNA from 0 dpa ovules, 1 dpa fibers and 10 dpa fiber, respectively. Panel A shows RNA blot analysis of 2 ug of total RNA hybridized as indicated. Ethidium bromide stained rRNA bands of the RNA used in these experiments are also shown. Panel B Shows semiquantitative PCR of the indicated transcript. UCE was used a loading control.

Figure 3

Ovules and leaves were stained with DiOC. Panel A. The wild type ST4793R was used unless otherwise noted. Water stained controls showed no auto fluorescence (data not shown). The fiberless mutant SL1-7-1 was used where indicated. The magnification is indicated in parentheses under the picture. Panel B. An image of a 20× magnification was digitalized and stained features identified, highlighted in red and counted using ImageJ.

Figure 4

Alignment of CPC and other MYBs: GhCPC1(contig16590), GhCPC2(Contig17149), AtCPC(NP_182164), AtGL1(NP_189430), GhMYB2(translation of MYB2). Regions of identity shared by all clones lightly shaded. Regions shared by majority of clones darkly shaded. MYB motifs underlined.

Figure 5

Ovules of the indicated age were stained with RhodFF. Water stained controls showed no auto fluorescence (data not shown). Arrow head indicate stained guard cells. The -1 dpa ovule is shown at 20× magnification to visualize a larger area. The 0 dpa ovule is shown at 40× magnification to allow easier visualization of stained cells.