Papers by Hsien-jung Chen
Botanical Bulletin of Academia Sinica, 1999
ABSTRACT
Journal of Experimental Botany, 2004
Journal of Plant Physiology, 2014
Catalase (CAT) functions as one of the key enzymes in the scavenging of reactive oxygen species a... more Catalase (CAT) functions as one of the key enzymes in the scavenging of reactive oxygen species and affects the H2O2 homeostasis in plants. In sweet potato, a major catalase isoform was detected, and total catalase activity showed the highest level in mature leaves (L3) compared to immature (L1) and completely yellow, senescent leaves (L5). The major catalase isoform as well as total enzymatic activity were strongly suppressed by ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). This inhibition could be specifically and significantly mitigated in mature L3 leaves by exogenous CaCl2, but not MgCl2 or CoCl2. EGTA also inhibited the activity of the catalase isoform in vitro. Furthermore, chlorpromazine (CPZ), a calmodulin (CAM) inhibitor, drastically suppressed the major catalase isoform as well as total enzymatic activity, and this suppression was alleviated by exogenous sweet potato calmodulin (SPCAM) fusion protein in L3 leaves. CPZ also inhibited the activity of the catalase isoform in vitro. Protein blot hybridization showed that both anti-catalase SPCAT1 and anti-calmodulin SPCAM antibodies detect a band at the same position, which corresponds to the activity of the major catalase isoform from unboiled, but not boiled crude protein extract of L3 leaves. An inverse correlation between the major catalase isoform/total enzymatic activity and the H2O2 level was also observed. These data suggest that sweet potato CAT activity is modulated by CaCl2 and SPCAM, and plays an important role in H2O2 homeostasis in mature leaves. Association of SPCAM with the major CAT isoform is required and regulates the in-gel CAT activity band.
Plant & cell physiology, Jan 20, 2016
The role of ascorbate (AsA) recycling via dehydroascorbate reductase (DHAR) in the tolerance of C... more The role of ascorbate (AsA) recycling via dehydroascorbate reductase (DHAR) in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress was examined. The activity of DHAR and the abundance of the CrDHAR1 (Cre10.g456750) transcript increased after moderate light (ML; 750 µmol m(-2) s(-1)) or high light (HL; 1,800 µmol m(-2) s(-1)) illumination, accompanied by dehydroascorbate (DHA) accumulation, decreased AsA redox state, photo-inhibition, lipid peroxidation, H2O2 overaccumulation, growth inhibition and cell death. It suggests that DHAR and AsA recycling is limiting under high-intensity light stress. The CrDHAR1 gene was cloned and its recombinant CrDHAR1 protein was a monomer (25 kDa) detected by Western blot that exhibits an enzymatic activity of 965 µmol min(-1) mg(-1) protein. CrDHAR1 was overexpressed driven by a HSP70A:RBCS2 fusion promoter or down-regulated by artificial microRNA (amiRNA) to examine whether DHAR-mediated AsA recycling is critical for the tolerance ...
Botanical Bulletin- Academia Sinica Taipei
Using >-naphthyl myristate (C 14 fatty acid ester) as a screening substrate, we purified fatty ac... more Using >-naphthyl myristate (C 14 fatty acid ester) as a screening substrate, we purified fatty acid esterases (FAEs) from yam (Dioscorea batatas Decne) tuber. Two FAE fractions (named FAE I and FAE II) were obtained after DE-52 ion exchange chromatography and Sephadex G-75 gel filtration purification steps, and then further purified by Con A Sepharose 4B affinity chromatography. FAE I and II fractions contained the same three protein bands about 50-64 kDa corresponding to esterase activity bands on SDS-PAGE gels. Among >-naphthyl esters determined at pH 4.0, 5.0 and 6.0 the best substrate for both FAE fractions was a C 10 -containing one with a maximum pH at 5.0. The K m and V max for >-naphthyl caprate (C 10 fatty acid ester) of FAE I and II at 37°C, pH 5.0 were 0.338 and 0.959 mM; 0.405 and 0.585 nmole >-naphthol/min µg protein, respectively. FAE activity was stable below 50°C and lost completely above 65°C.
Botanical Bulletin- Academia Sinica Taipei
Journal of Plant Growth Regulation
Journal of Plant Physiology, 2000
Journal of Plant Physiology, 1999
Journal of Cellular Physiology, 1996
Activated Xenopus egg extracts are capable of undergoing cell-free cell cycling. Using these acti... more Activated Xenopus egg extracts are capable of undergoing cell-free cell cycling. Using these activated extracts, we previously showed that purified, bacterially expressed oncogenic human RasH protein arrests cell cycle progression. Because oncogenic Ras activates many serine/threonine protein kinases in Xenopus oocytes and egg extracts, it is possible that induction of cell cycle arrest involves the action of oncogenic Ras-activated kinases. Thus, the identification of the physiological substrates for oncogenic Ras-activated kinases is important for elucidating the molecular mechanism underlying oncogenic Ras-induced cell cycle arrest. We used 32P-orthophosphate as a label to identify the potential substrates. Our results demonstrated that the "P-labeling of both a 32 and a 33 kDa protein were greatly enhanced by oncogenic Ras during the incubation of activated Xenopus egg extracts. The enhanced labeling correlated with the induced cell cycle arrest and was contributed by serine phosphorylation. Moreover, the 33 kDa protein was detected only in the presence of oncogenic Ras and was a serine-hyperphosphorylated form of the 32 kDa protein. Furthermore, new protein synthesis was not required for the enhanced labeling, consistent with the concept that the enhanced serine phosphorylation of the 32 kDa protein is by oncogenic Ras-activated protein kinases. In addition to serine phosphorylation, our results also suggested that an as yet unidentified modification of the 32 kDa protein might also be induced by oncogenic Ras. Our results suggest that the 32 kDa protein is a potential physiological substrate for oncogenic Ras-activated protein kinases. o 1996 WiIey-Liss, Inc.
Botanical Bulletin of …, 2000
Three transgenic tobacco regenerants (lines RP3/2, RP3/7 and RP3/18) harboring rice RP3/GUS chime... more Three transgenic tobacco regenerants (lines RP3/2, RP3/7 and RP3/18) harboring rice RP3/GUS chimeric gene inserts in their genomes were isolated previously and used to establish cell suspension cultures . A differential GUS expression pattern together with morphological, biochemical, and molecular variations were observed among these cell lines . In this report we used pigments such as carotenoids and chlorophylls a and b as markers to study the possible relationship between RP3 promoter activity and cellular pigment contents, and a positive association was found among these cell lines. RP3/2, which contained the highest cellular pigment contents, also exhibited the highest GUS expression level. The RP3 promoter activity in RP3/2 continuously decreased and was parallel with the reduction of pigment contents within a 12-day growth period after subculture. In RP3/18, both also showed a parallel association and remained relatively constant during cell growth. Based on these results, a positive association between RP3 promoter activity and cellular pigment content was found among and within cell lines. The meaning of the association and its possible explanation are discussed.
Botanical Bulletin of Academia …, 2000
Abstract. Dioscorins, the storage proteins of yam tubers, from six cultivars of three Dioscorea s... more Abstract. Dioscorins, the storage proteins of yam tubers, from six cultivars of three Dioscorea species including D. batatas, D. alata, and D. pseudojaponica were detected in crude extracts of yam tubers but not leaves by immuno stainings on PVDF membrane with a polyclonal ...
Journal of plant physiology, Jan 15, 2015
Sweet potato sporamins are trypsin inhibitors and exhibit strong resistance to digestion by pepsi... more Sweet potato sporamins are trypsin inhibitors and exhibit strong resistance to digestion by pepsin, trypsin and chymotrypsin. In addition, they constitute the major storage proteins in the sweet potato and, after degradation, provide nitrogen as a nutrient for seedling regrowth in sprouting storage roots. In this report, four cysteine proteases-one asparaginyl endopeptidase (SPAE), two papain-like cysteine proteases (SPCP1 and SPCP2), and one granulin-containing cysteine protease (SPCP3)-were studied to determine their association with sporamin degradation in sprouting storage roots. Sporamin degradation became significant in the flesh of storage roots starting from week 4 after sprouting and this correlated with expression levels of SPAE and SPCP2, but not of SPCP1 and SPCP3. In the outer flesh near the skin, sporamin degradation was more evident and occurred earlier than in the inner flesh of storage roots. Degradation of sporamins in the outer flesh was inversely correlated with ...
Botanical Studies
In this report a full-length cDNA, SPCP1, was isolated from senescent leaves of sweet potato (Ipo... more In this report a full-length cDNA, SPCP1, was isolated from senescent leaves of sweet potato (Ipomoea batatas (L.) Lam). SPCP1 contained 1020 nucleotides (339 amino acids) in the open reading frame, and exhibited high amino acid sequence homologies (ca. 58% to 74%) with papain-like cysteine proteases of Alnus glutinosa, Arabidopsis thaliana, Astragalus sinicus, Brassica napus, Daucus carota, Gossypium hirsutum, Hordeum vulgare, Iris hollandica, Medicago truncatula, Nicotiana tabacum, Oryza sativa, Ricinus communis, Trifolium repens. Semi-quantitative RT-PCR and Western blot hybridization showed that SPCP1 gene expression was enhanced significantly in natural senescent leaves and in dark-, ethephon-, and ABAinduced senescent leaves, whereas, was almost not detected in mature green leaves, stems, and roots. Initiation of chlorophyll degradation is earlier than the SPCP1 gene expression during leaf senescence. SPCP1 expression was also induced in sweet potato suspension cells treated w...
Botanical Studies
Aspartic proteinases (EC, 3.4.23) cDNA clone (SPAP) of sweet potato (Ipomoea batatas (L.) Lam. &#... more Aspartic proteinases (EC, 3.4.23) cDNA clone (SPAP) of sweet potato (Ipomoea batatas (L.) Lam. 'Tainong 57') storage roots were isolated by differential display. The open reading frame in this cDNA encodes a pre-pro-protein of 508 amino acids with a predicted molecular mass of 55,006 Da (pI 4.91). The SPAP gene shares 81% and 78% homology on the level of nucleotides and amino acids, respectively, with an aspartic proteinase cDNA of sweet potato senescent leaves (SPAPSL). SPAP amino acid sequence was different from other AP sequences in signal and propeptide portions. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant specific insert (PSI) and two active site aspartic acid residues. Examination of the expression patterns in sweet potato by northern blot analyses revealed that the transcripts of SPAP were specifically induced in the storage roots. Recombinant SPAP overproduced in E. coli (M15) was purified by Ni2 ...
Botanical Studies
Trypsin inhibitor (TI), the root storage protein of sweet potato, was shown by spectrophotometric... more Trypsin inhibitor (TI), the root storage protein of sweet potato, was shown by spectrophotometric methods to inhibit angiotensin converting enzyme (ACE) in a dose-dependent manner (50-200 µg/mL, with 31.9-53.2% inhibition) using N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) as a substrate.The 50% inhibition (IC 50 ) of ACE activity required 187.96 μg/mL TI compared to 10 nM (868 ng/ mL)ofCaptopril.TheuseofTLCalsoshowedTIasACEinhibitor.TIactedasamixedtypeinhibitoragainst ACE using FAPGG as a substrate. When 200 μg/mL TI were added, Vmax and Km were, respectively 0.013 ΔA/min and 0.715 mM while without TI they were 0.027 ΔA/min and 0.419 mM. Pepsin was used for TI hydrolysisfordifferenttimes.ACEinhibitoryactivitywasfoundtoincreasefrom34%toabout83%after24 hofhydrolysis.TheresultssuggestedthatwhensmallpeptidesincreasedbypepsinhydrolysisoftheTIACE inhibitorycapacityalsoincreasedupto24handthendecreased,itmaybeduetothedisappearanceofsome conformational requirements. Ten peptides-namely HDHM, LR, SNIP, VRL, TYCQ, GTEKC, RF, VKAGE, AH and KIEL-were synthesized based on the simulated pepsin digestion of trypsin inhibitor and then tested forACE inhibitory activity. IC 50 values of individual peptides were 276.2, 746.4, 228.3, 208.6, 2.3, 275.8, 392.2, 141.56, 523.5 and 849.7 µM, suggesting that TYCQ might represent the main active site for the ACE inhibition.TI and its hydrolysates might be good for control of hypertension and other diseases when people consumesweetpotatotuberousroots.
Botanical Studies
In this report several senescence-associated markers were used to study the ethephon-mediated eff... more In this report several senescence-associated markers were used to study the ethephon-mediated effects on leaf senescence in detached sweet potato leaves. The chlorophyll contents and Fv/Fm values were drastically reduced, however, H2O2 contents detected with diaminobenzidine (DAB) staining and a papain-like cysteine protease SPCP1 expression were significantly enhanced in ethephon-treated leaves compared to untreated dark control. In the presence of reduced glutathione, EGTA or cycloheximide, the reduction of chlorophyll contents and Fv/Fm values were alleviated, however, the induction or enhancement of H2O2 contents and cysteine protease SPCP1 expression were repressed. Both calcium ionophore A23187 and glutathione synthase inhibitor, L-buthionine sulfoximide (BSO), remarkably induced SPCP1 expression in detached leaves, and the induction was also repressed by EGTA and reduced glutathione, respectively. The time effective for cycloheximide repression of SPCP1 expression was ca. 6 t...
Botanical Studies
We have previously isolated an asparaginyl endopeptidase, SPAE, from senescent leaves of sweet po... more We have previously isolated an asparaginyl endopeptidase, SPAE, from senescent leaves of sweet potato (Ipomoea batatas cv. Tainong 57). Gene expression of SPAE was activated and enhanced in natural and induced senescent leaves (Chen et al., 2004). In this report the full-length SPAE cDNA was constructed in the T-DNA portion of recombinant pBI121 vector under the control of CaMV 35S promoter and transferred to Arabidopsis with Agrobacterium-mediated floral dip transformation. Three transgenic Arabidopsis plants were isolated and confirmed by kanamycin-resistance and genomic PCR amplification of SPAE. Protein gel blot also demonstrated sweet potato SPAE expression in these transgenic plants. Phenotypic analysis showed that transgenic plants exhibited earlier floral transition from vegetative growth and leaf senescence than control. Transgenic plants also contained fewer siliques and a higher percentage of incompletely-developed siliques per plant than control. Based on these results w...
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Papers by Hsien-jung Chen