Cloning and functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers

Abstract

Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulated during early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA reductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1 and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues, respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed during the cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2 showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAs were cloned and expressed in yeast haploid ybr159w∆ mutant that was deficient in 3-ketoacyl-CoA reductase activity. Wild-type growth rate was restored in ybr159w∆ cells that expressed either GhKCR1 or 2. Further analysis showed that GhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to the yeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductase activity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest that GhKCR1 and 2 are functional orthologues of ScYbr159p.

Transcriptome Profiling, Molecular Biological, and Physiological Studies Reveal a Major Role for Ethylene in Cotton Fiber Cell Elongation

“…Recombinant ACO1, ACO2, and ACO3 proteins were detected with monoclonal antipolyhistidine antibody (RandD Systems) and visualized after incubating with goat anti-mouse IgG conjugated with horseradish peroxidase (Promega) as previously described (Qin et al, 2005).…”

“…Parallel reactions using cotton UBQ7 primers were performed to normalize the amount of template cDNA added in each reaction. QRT-PCR was performed using the SYBR Green PCR kit (Applied Biosystems) in a DNA Engine Opticon continuous fluorescence detection system (MJ Research) as previously described (Qin et al, 2005). The QRT-PCR cycles were as follows: initiation with a 10-min denaturation at 958C, followed by 42 cycles of amplification with 10 s of denaturation at 948C, 20 s of annealing according to the melting temperatures provided in Supplemental Table 3 online, 20 to 30 s of extension at 728C, and reading the plate for fluorescence data collection at 78 to 808C.…”

Saturated Very-Long-Chain Fatty Acids Promote Cotton Fiber and Arabidopsis Cell Elongation by Activating Ethylene Biosynthesis

“…Interestingly, many cotton genes encoding nonspecific lipid transfer proteins and enzymes involved in various steps during fatty acid chain elongation are highly upregulated during early fiber development (Ji et al, 2003;Qin et al, 2005Qin et al, , 2007Shi et al, 2006;Gou et al, 2007), indicating that the biosynthesis of very-long-chain fatty acids (VLCFAs) or their transport may be required for fiber cell elongation processes. A comprehensive lipid analysis has indicated that linolenic (18:3) and palmitic (16:0) acids are the most abundant fatty acids in developing cotton fibers (Wanjie et al, 2005).…”

Comparative Proteomics Indicates That Biosynthesis of Pectic Precursors Is Important for Cotton Fiber and Arabidopsis Root Hair Elongation

“…Among them, 162 fiber-preferential genes were mapped to 102 metabolic events with ethylene biosynthesis and fatty acid biosynthesis/chain elongation being the most significantly upregulated processes. Systematic studies showed that a large number of genes encoding nonspecific lipid transfer proteins and enzymes that are involved in various steps of fatty acid chain elongation are highly up-regulated during early fiber development, indicating that biosynthesis of saturated verylong-chain fatty acids and/or their transport may also be required for fiber cell growth (3,(7)(8)(9)(10)(11). Exogenously applied lignoceric acid (C24:0) in the ovule culture medium promotes significant fiber cell growth, possibly by activating the transcription of several 1-aminocyclopropane-1-carboxylic acid oxidases involved in ethylene biosynthesis (12).…”