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

Abstract

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.

Figure 1

Phenotypes, experimental procedures for and results of PCR-select cDNA subtraction. (A) Seeds of wild-type upland cotton Xuzhou 142 (left) and fl (fuzzless-lintless) mutant (right). (B) Strategy for isolating and identifying cotton fiber development-related genes via PCR-select cDNA subtraction and differential screening. (C) Products of PCR-select cDNA subtraction. M, Φ174/HaeIII digested molecular marker; UD, unsubtracted driver cDNA; UT, unsubtracted tester cDNA; S1, PCR products obtained after one round of subtraction; S2, PCR products obtained after two rounds of subtraction.

Figure 2

Functional classification of genes included in the subtractive cDNA library. Cotton cDNAs were classified by referring to the SRB embryonic EST project (http://www.mcdb.ucla.edu/Research/Goldberg), in which ∼20 000 ESTs representing messages active 6–7 days after fertilization in embryos of scarlet runner bean were identified and were assigned to 15 different functional categories.

Figure 3

Differential screening of the 280 cDNAs by macroarray, using fiber cDNA (left) and fl ovule cDNA (right) as probes. A cotton ubiquitin (P2D01) gene was used as the positive control. Negative controls include PCR primers (P1D09), ddH₂O (P1B07) and vector DNA (P1D10).

Figure 4

RT–PCR analysis of cotton fiber cDNAs. Total RNA (5 µg) isolated from fl mutant ovules and from fiber cells at different developmental stages was used to synthesize first-strand cDNA for RT–PCR using gene-specific primers. A cotton ubiquitin cDNA was used to normalize the amount of templates added in PCR reactions. M, fl mutant ovule; UF, upland cotton fiber. Days post anthesis (d.p.a., days 0–20) are shown at the top. Capitalized gene names indicate previously reported fiber genes that were not recovered from our subtractive library. These genes did not show significant differences in expression between 10 d.p.a. fiber and fl mutant ovules. KCBP, kinesin-like calmodulin binding protein; VLCFA, very-long chain fatty acid; CAP, adenyl cyclase associated protein; RGP, reversibly glycosylated polypeptide.

Figure 5

Analysis of cotton fiber preferentially expressed genes during early fiber development. Macroarrays were prepared as for Figure 3. ³³P-labeled cDNA probes were prepared from RNA isolated from 0, 5, 10 and 20 d.p.a. cotton fibers.

Figure 6

In situ determination of early fiber-specific expression of three important genes. In situ hybridization: (A–C) a putative MAPK (P3D03); (E–G) a putative ABP (P1F01); (I–K) a profilin (P1E05). (A), (E) and (I) show antisense hybridization to cross-sections of 2 d.p.a. wild-type upland cotton. (B), (F) and (J) show antisense hybridization to 2 d.p.a. fl mutant ovules. (C), (G) and (K) show negative controls of 2 d.p.a. wild-type cotton with the sense strand of each cDNA. (D, H and L) Positive controls of 2 d.p.a. wild-type cotton with the antisense strand of a cotton ubiquitin. Blue signals represent mRNA of the tested genes. f, fiber cells; oi, outer integument; ii, inner integument.