Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H2O2 elevation.

In this report a full-length cDNA, SPCAT1, was isolated from ethephon-treated mature L3 leaves of sweet potato. SPCAT1 contained 1479 nucleotides (492 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 71.2-80.9%) with several plant catalases, including Arabidopsis, eggplant, grey mangrove, pea, potato, tobacco and tomato. Gene structural analysis showed that SPCAT1 encoded a catalase and contained a putative conserved internal peroxisomal targeting signal PTS1 motif and calmodulin binding domain around its C-terminus. RT-PCR showed that SPCAT1 gene expression was enhanced significantly in mature L3 and early senescent L4 leaves and was much reduced in immature L1, L2 and completely yellowing senescent L5 leaves. In dark- and ethephon-treated L3 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24h, then decreased gradually until 72h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H(2)O(2) amounts. Effector treatment showed that ethephon-enhanced SPCAT1 expression was repressed by antioxidant reduced glutathione, NADPH oxidase inhibitor diphenylene iodonium (DPI), calcium ion chelator EGTA and de novo protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H(2)O(2), NADPH oxidase, external calcium influx and de novo synthesized proteins are required and associated with ethephon-mediated enhancement of sweet potato catalase SPCAT1 expression. Exogenous application of expressed catalase SPCAT1 fusion protein delayed or alleviated ethephon-mediated leaf senescence and H(2)O(2) elevation. Based on these data we conclude that sweet potato SPCAT1 is an ethephon-inducible peroxisomal catalase, and its expression is regulated by reduced glutathione, DPI, EGTA and cycloheximide. Sweet potato catalase SPCAT1 may play a physiological role or function in cope with H(2)O(2) homeostasis in leaves caused by developmental cues and environmental stimuli.

Antimicrobial, dehydroascorbate reductase, and monodehydroascorbate reductase activities of defensin from sweet potato [Ipomoea batatas (L.) Lam. 'Tainong 57'] storage roots.

A cDNA encoding a small cysteine-rich protein designated defensin (SPD1) was isolated from sweet potato storage roots. On the basis of the amino acid sequence similarity and conserved residues, it is suggested that SPD1 is a member of the plant defensin family. Recombinant SPD1 protein overproduced in Escherichia coli was purified by Ni (2+)-chelated affinity chromatography. A recombinant protein from the storage root cDNA clone effectively inhibited the trypsin activity in a dose-dependent manner. Both the corresponding mRNA and protein level were found to be highest in the storage roots, followed by sprout. SPD1 reduced dehydroascorbate (DHA) in the presence of glutathione to regenerate l-ascorbic acid (AsA). However, without glutathione, SPD1 has very low DHA reductase activity, and AsA was oxidized by AsA oxidase to generate monodehydroascorbate (MDA) free radical. MDA was also reduced by SPD1 to AsA in the presence of NADH, mimicking the MDA reductase catalyzed reaction. These data suggest that SPD1 has both DHA reductase and MDA reductase activities. SPD1 was also shown to inhibit the growth of both fungi and bacteria. SPD1 is apparently the first reported plant defensin exhibiting DHA and MDA activities in vitro.

Inhibitory activities of semicarbazide-sensitive amine oxidase and angiotensin converting enzyme of pectin hydroxamic acid.

Solutions of 100 mL of 1% commercial pectin each with a different degree of esterification (DE), DE94, DE65, and DE25, were reacted with 100 mL of 2 M alkaline hydroxylamine (pH 12.0) at room temperature for 4 or 18 h. These pectin hydroxamic acids (PHAs; DE94T4, DE94T18, DE65T4, and DE25T4) were used to test the inhibitory activities against semicarbazide-sensitive amine oxidase (SSAO) and angiotensin-converting enzyme (ACE). Compared to different DE pectins (DE94, DE65, and DE25), the PHAs of DE94T4, DE94T18, DE65T4, and DE25T4 showed different inhibition activities against SSAO or ACE. Commercial pectins with different DE values showed negligible SSAO or ACE inhibitions. The order of SSAO inhibition was DE65T4 > DE94T18 approximately DE25T4 >> DE94T4. However, the order of ACE inhibition was DE94T4 > DE94T18 >> DE65T4 > DE25T4. The SSAO activity staining or ACE-hydrolyzed products on TLC chromatogram also confirmed the inhibitory activities of PHAs against SSAO or ACE.