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Purification and biosynthesis of cottonseed (Gossypium hirsutum L.) catalase.

ABSTRACT: As part of our research on peroxisome biogenesis, catalase was purified from cotyledons of dark-grown cotton (Gossypium hirsutum L.) seedlings and monospecific antibodies were raised in rabbits. Purified catalase appeared as three distinct electrophoretic forms in non-denaturing gels and as a single protein band (with a subunit Mr of 57,000) on silver-stained SDS/polyacrylamide gels. Western blots of crude extracts and isolated peroxisomes from cotton revealed one immunoreactive polypeptide with the same Mr (57,000) as the purified enzyme, indicating that catalase did not undergo any detectable change in Mr during purification. Synthesis in vitro, directed by polyadenylated RNA isolated from either maturing seeds or cotyledons of dark-grown cotton seedlings, revealed a predominant immunoreactive translation product with a subunit Mr of 57,000 and an additional minor immunoreactive product with a subunit Mr of 64000. Labelling studies in vivo revealed newly synthesized monomers of both the 64000- and 57,000-Mr proteins present in the cytosol and incorporation of both proteins into the peroxisome without proteolytic processing. Within the peroxisome, the 57,000-Mr catalase was found as an 11S tetramer; whereas the 64,000-Mr protein was found as a relatively long-lived 20S aggregate (native Mr approx. 600,000-800,000). The results strongly indicate that the 64,000-Mr protein (catalase?) is not a precursor to the 57,000-Mr catalase and that cotton catalase is translated on cytosolic ribosomes without a cleavable transit or signal sequence.

SUBMITTER: Kunce CM

PROVIDER: S-EPMC1148976 | biostudies-other | 1988 Apr

REPOSITORIES: biostudies-other

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Project description:Naturally-colored brown cotton (NBC) fiber is an environmentally friendly raw source of fiber for textile applications. The fiber of some NBC cultivars exhibits flame-retardant properties, which can be used in textiles that require flame resistance. Proanthocyanidins or their derivatives are responsible for the brown pigment in NBC; however, how flame retardancy is related to pigmentation in NBC is poorly understood. To gain insight into brown pigment biosynthesis, we conducted comparative transcripts and metabolites profiling analysis of developing cotton fibers between the brown (MC-BL) and white (MC-WL) cotton near-isogenic lines (NILs), genetically different only in the Lc1 locus. In this study, mass spectrometry was used to detect metabolites in BL and WL developing fibers at 8, 12, 16, 20, 24, 36, and 40 days post anthesis (DPA) and mature fibers. Transcripts analysis was performed at two critical fiber developmental points, 8 DPA (fiber elongation) and 20 DPA (secondary cell wall deposition). We found 5836 (ESI MS positive mode) and 4541 (ESI MS negative mode) metabolites significantly different accumulated between BL and WL. Among them, 142 were known non-redundant metabolites, including organic acids, amino acids, and derivatives of the phenylpropanoid pathway. Transcript analysis determined 1691 (8 DPA) and 5073 (20 DPA) differentially expressed genes (DEGs) between BL and WL, with the majority of DEGs down-regulated at 20 DPA. Organic acids of the citric acid cycle were induced, while most of the detected amino acids were reduced in the MC-BL line. Both cis- and trans-stereoisomers of flavan-3-ols were detected in developing MC-WL and MC-BL fibers; however, the gallocatechin and catechin accumulated multiple times higher. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acids determined that palmitic acid long-chain alcohols were the main constituents of waxes of mature fibers. Energy-dispersive X-ray spectrometry (EDS) analysis of mature fibers revealed that potassium accumulated three times greater in MC-BL than in MC-WL mature fibers. This study provides novel insights into the biosynthesis of pigments and its association with flame retardancy in NBC fibers.

Project description:BackgroundWe previously reported the development of a set of Gossypium hirsutum-G. australe alien chromosome addition lines. Naturally, however, G. hirsutum-G. australe chromosome exchanges were very limited, impeding the stable transference of useful genes from G. australe (G2G2 genome) into the most cultivated cotton, G. hirsutum (AADD).ResultsIn the present report, the pollen from a pentaploid (2n = AADDG2) of G. hirsutum-G. australe was irradiated with seven different doses ranging from 10 to 40 Grays and used to pollinate emasculated flowers of G. hirsutum over three consecutive years. Irradiation greatly increased the genetic recombination rates of the G. hirsutum and G. australe chromosomes and a total of 107 chromosome introgression individuals in 192 GISH-negative (with no GISH signal on chromosome) survived individuals, 11 chromosome translocation individuals (containing 12 chromosome translocation events) and 67 chromosome addition individuals were obtained in 70 GISH-positive (with GISH signal(s) on chromosome(s)) survived individuals, which are invaluable for mining desirable genes from G. australe. Multicolor genomic in situ hybridization results showed that there were three types of translocation, whole arm translocation, large alien segment translocation and small alien segment translocation, and that all translocations occurred between the G2-genome and the A-subgenome chromosomes in G. hirsutum. We also found that higher doses induced much higher rates of chromosome variation but also greatly lowered the seed viability and seedling survivability.ConclusionsIrradiation has been successfully employed to induce chromosome introgressions and chromosome translocations and promote chromosome exchanges between cultivated and wild species. In addition, by balancing the rates of chromosome introgression and translocation to those of seed set, seed germination, and seedling rates in the M1 generation, we conclude that the dosage of 20 Grays is the most suitable. The established methodology may guide the utilization of the tertiary gene pool of Gossypium species such as G. australe in cotton breeding in the future.

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Purification and biosynthesis of cottonseed (Gossypium hirsutum L.) catalase.

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