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Purificatin and Chacterizaion of the 22-kDa Kunitze type Potato Proteinase Inhibitors
Suh, Sang-Gon,Hannapel, David J. 영남대학교 마늘연구소 2001 의성군 ·영남대학교 관 ·학협동, 협약 조인식 및 심포지움 Vol.2001 No.-
Three abundant protein of approximate molecular masses of 22,23, and 24 kilodaltons were purified from potato (Solanum tuberosum L.) tubers by DEAE cellulose and CM-52 cellulose ion exchange culumn chromatography, electroelution, and high-pressure liquid chromatography (HPLC). Antibodies specific to the gel-purified 22-kilodalton protein were prepared. Immunoblot analysis showed that the 22-, 23-, and 24- kilodalton proteins are immunologically related and that these proteins are present in tubers and as higher molecular mass farms in leaves, but not in stems, roots, and stolons. The ratios of amino acid composition were compared among the three purified protiens, and the amino-terminal amino acid sequence were determined for these three proteins. All three proteins have identical amino-terminal sequence that match the deduced amino acid sequence of an abundant tuber protein cDNA. Using a proteinase-inhibition assay, we have demonstrated that the 22-kilodalton (kDa) potato (Solanum tuberosum L.) tuber proteins are strong inhibitors of serine proteinases. Two out of three purified proteins from the 22-kDa family of potato-tuber proteins were effective inhibitors of both trypsin and chymotrypsin, while the third, with a molecular mass (Mr) of apprex.24-kDa, inhibited only trypsin activity. Comparison of the amino-acid sequence of the putative reactive sites of several proteinase inhibitors with the deduced sequence of the 22-kDa protein showed that the 22-kDa protein contained sequences potentially pessessing "double-headed" sites of inhibition, one against trypsin and another against chymotrypsin. The genes coding for the 22-kDa proteins were developmentally regulated in tubers and environmentally regulated in leaves. Wound induction of the genes coding for the 22-kDa potato-tubers proteins was detected at the RNA level. In leaves, transcripts of the 22-kDa protein family were detected 6h after wounding and were highest after 12h in locally wounded leaves. The strongest induction occurred systemically in response to mechanical wounding in non-wounded leaves. Cross-hybridization of a cDNA, p34021, which codes for the 22-kDa tuber protein, with both proteinase-inhibitor I and II cDNAs and with a second family of 20-kDa potato-tuber cDNAs showed no cross-homology. Members of this second group of 20-kDa potato-tuber proteins also exhibited wound-induction in leaves at the RNA level.
Kang, Sang-Gu,Hannapel, David J. 영남대학교 마늘연구소 2001 의성군 ·영남대학교 관 ·학협동, 협약 조인식 및 심포지움 Vol.2001 No.-
Early tuberization involves biological processes such as plastid differentiation, amyloplast development, carbon partitioning, starch accumulation, cell division, and the induction of specific proteins, resulting in morphological changes from undifferentiated unswollen-stolons to differentiated new-tubers. However, genes involved directly in early tuberization are unknown. Here several genes are characterized among genes expressed in early tuber development. A cDNA encoding the potato (Solanum tuberosum L.) chloroplast ribosomal protein S16 (cp rps16) was isolated and characterized. The expression of rps16 is developmentally regulated with transcript levels during tuberization. The rps16 was constitutively expressed in unswollen stolons and axillary buds. A potato MADS-box gene cDNA(POTM1-1) from an early tuber cDNA library has been isolated and characterized. During axillary bud development in a model petiole-leaf cutting system, the levels of POTM1-1 transcripts were abundant in actively growing shoots and the early stages of microtuber development, suggesting that these novel MADS-box genes may be involved in vegetative organ development of potato. The levels of POTM1-1 transcripts were constitutive after treatments of exogenous abscisic acid (ABA), gibberellic acid (GA3), methyl jasmonate (MeJA), and 1-naphthalene acetic acid (NAA). However, when treated with benzyladenine (BA), the level of POTM1-1 transcripts was reduced. Based on these results, it is conceivable that cytokinin is involved in the regulation of POTM1-1 gene expression and vegetative meristem development. Two cDNAs, STGA2 and STGB2, encoding herotrimeric G protein α- and β-subunit proteins were cloned and characterized. Heterotrimeric G proteins on the plasma membrane of eukaryotic cells are involved in the signal transduction pathways. The transcript levels of two genes were abundant in active growing organs. Interestingly, STGA2 and STGB2 gene expression showed synchronous patterns in the examined organs. During the early tuber development, the transcripts of STGA2 and STGB2 were abundant in unswollen stolons, swollen stolons, and new tubers, but were not detected in matured tubers. Results indicate that potato Gα-and β-subunit genes are developmentally regulated and may be involved in signaling pathway during potato tuber development.
Potato lipoxygenase genes associated with development and pathogen defense response
Kolomiets, Michael V.,Chen, Hao,Braun, E.J.,Gladon, Richard J.,Hannapel, David J. 영남대학교 마늘연구소 2001 의성군 ·영남대학교 관 ·학협동, 협약 조인식 및 심포지움 Vol.2001 No.-
Plant lipoxygenases (LOXs) are a functionally diverse class of dioxygenases implicated in physiological processes such as growth, senescence, and stress-related responses. LOXs incorporate oxygen into their fatty acid substrates and produce hydroperoxide fatty acids that are precursors of jasmonic acid and related compounds. Here we report the involvement of a potato class-1 LOX, POTLX-1, in the control of tuber growth and a unique LOX, POTLX-3, in defense responses against pathogens. RNA hybridization analysis showed that accumulation of Lox1-class transcripts was specific to developing tubers and that mRNA accumulation correlated positively with tuber initiation and growth. Suppression mutants produced by expressing antisense coding sequence of the tuber LOX, POTLX-1, exhibited a significant reduction in LOX activity in stolons and tubers. The suppression of LOX activity correlated with reduced tuber yield, decreased average tuber size, and a disruption of tuber formation. POTLX-3 mRNA accumulation was induced specifically in potato leaves infected with either virulent or avirulent strains of Phytophthora infestans, the causal agent of late blight. During the resistance response, POTLX-3 was induced within 6 h, increased steadily through 24 h, and its mRNA continued to accumulate for a week after inoculation. In contrast, when a plant was susceptible to P. infestans, induction of mRNA accumulation in response to inoculation was inconsistent and delayed. LOX activity assayed during an incompatible interaction in leaves peaked three days earlier than during a compatible interaction. POTLX-3 mRNA accumulation also was induced during hypersensitive response development caused by the incompatible pathogen Pseudomonas syringae pv. phaseolicola. Our results show that LOXs of potato function in diverse roles in both development and in defense responses against pathogen infection.