Responses of sweet potato peroxidases to sodium nitroprusside-mediated nitric oxide

To ascertain the response of sweetpotato peroxidases (PODs) to nitric oxide (NO), we treated the leaves of sweet potato with the NO generator sodium nitroprusside (SNP) and the NO scavenger carboxyl-PTIO (cPTIO). Exogenous application of more than 5 mM SNP caused damage to sweetpotato leaves at 24 h after treatment. The accumulation of NO in leaves was positively correlated with the SNP dose. The specific activity of PODs in sweet potato leaves was markedly increased by treatment with greater than 1 mM SNP for 24 h, whereas POD activity and accumulated NO content decreased to low levels by treatment with cPTIO. Expression analysis of POD genes in response to treatment with SNP and cPTIO revealed that major stress-inducible acidic genes, such as swpa1, swpa2, swpa3, and swpa4, were specifically regulated. These results indicate that increased NO levels in sweet potato leaves are closely linked to an improved defense capability mediated by stress-inducible PODs.     

Differential expression of 10 sweetpotato peroxidases in response to sulfur dioxide, ozone, and ultraviolet radiation

Secretory class III plant peroxidase (POD, EC 1.11.1.7) is believed to function in diverse physiological processes, including responses to various environmental stresses. To understand the function of each POD in terms of air pollutants and UV radiation, changes in POD activity and expression of 10 POD genes isolated from cell cultures of sweetpotato (Ipomoea batatas) were investigated in the leaves of sweetpotato after treatment with sulfur dioxide (SO(2) 500ppb, 8h/day for 5 days), ozone (O(3) 200ppb, 8h/day for 6 days), and ultraviolet radiation (UV-B 0.6mWm(-2) for 24h, UV-C 0.16mWm(-2) for 24h). All treatments significantly reduced the PSII photosynthetic efficiency (F(v)/F(m)). POD-specific activities (units/mg protein) were increased in leaves treated with SO(2) and O(3) by 5.2- and 7.1-fold, respectively, compared to control leaves. UV-B and UV-C also increased POD activities by 3.0- and 2.4-fold, respectively. As determined by RT-PCR analysis, 10 POD genes showed differential expression patterns upon treatment with air pollutants and UV radiation. Among the POD genes, swpa1, swpa2, and swpa4 were strongly induced following each of the treatments. Interestingly, basic POD genes (swpb1, swpb2, and swpb3) were highly expressed following SO(2) treatment only, whereas neutral swpn1 was highly induced following O(3) treatment only. These results indicated that some specific POD isoenzymes might be specifically involved in the defense mechanism against oxidative stress induced by air pollutants and UV radiation in sweetpotato plants.     

甘薯根腐病菌侵染对甘薯内源激素水平的影响

甘薯根腐病病原菌[Fusarium solani(Mart.)Sacc.f.sp.batatas McClure,简称FSB]侵染及其培养液滤液处理高敏感性甘薯品种‘胜利百号’后,引起甘薯叶片、茎尖和根部组织内源ABA含量大幅升高。其中在根部出现最早,但茎尖中积累浓度最高。侵染后甘薯叶片、茎尖和根部组织内源GA1/3含量显著低于对照。甘薯组培苗经FSB培养滤液处理9h后,ABA含量显著上升,处理15h,ABA含量呈下降趋势,而GA1/3含量在101和102稀释液处理15h(103稀释液处理12h)时出现显著上升。这些结果有助于解释甘薯根腐病株矮小不产生藤蔓,并在秋季大量现蕾开花的生理现象。     

Differential expression of 10 sweetpotato peroxidase genes in response to bacterial pathogen, Pectobacterium chrysanthemi.

To understand the function of each peroxidase (POD, EC 1.11.1.7) in terms of biotic stress, changes in POD specific activity and expression of 10 POD genes were investigated in four cultivars of sweetpotato (Ipomoea batatas) after infection with Pectobacterium chrysanthemi. POD specific activity (units mg(-1) protein) increased from 16 h after inoculation (HAI) in three varieties. POD activities of two cultivars, Shinwhangmi and White Star, reached a maximum level at 24 HAI by about three times compared to mock treatment (MT), and then decreased, whereas those of Zami and Yulmi continuously increased until 36 HAI. Native gel analysis revealed that one POD isoenzyme with a high electrophoretic mobility significantly increased in response to pathogen infection in all cultivars. Additionally, 10 POD genes displayed differential expression patterns upon bacterial infection by northern analysis. Several POD genes such as swpa2, swpa3, swpa4, swpa5, swpb1 were induced upon bacterial infection, but other genes were not. Particularly, swpa4 gene was markedly expressed in response to bacterial infection in four different cultivars, suggesting that this gene has a stress-inducible promoter. These results indicate that some specific POD isoenzymes are involved in defense in relation to pathogenesis of P. chrysanthemi in sweetpotato plants.     

A Major Jasmonate-Inducible Protein of Sweet Potato, Ipomoelin, is an ABA-Independent Wound-Inducible Protein

Treatment of sweet potato plants cultured in vitro with a vaporof methyl jasmonate (MeJA) induced an accumulation in leavesof a large amount of protein with an apparent molecular massof 18 kDa. This protein, designated ipomoelin, was purified,and the amino acid sequences of proteolytic fragments were determined.Screening a cDNA library of MeJA-treated leaves by oligonucleotideprobes designed from the peptide sequences identified a clonethat could code for a polypeptide with 154 amino acids. Thededuced amino acid sequence of ipomoelin showed an overall aminoacid identity of 25% with the salt-inducible SalT protein ofrice. In addition, the C-terminal 70 amino acid sequence ofipomoelin showed about 50% identity with the C-terminal aminoacid sequences of seed lectins from Moraceae. The gene for ipomoelinwas present in a few copies in the genome of sweet potato. ThemRNA for ipomoelin was detected in leaves and petioles, butnot in stems and tuberous roots, of sweet potato plants grownin the field. Mechanical wounding of leaves induced ipomoelinmRNA both locally and systemically, while treatment of leaveswith ABA, salt, or a high level of sucrose did not induce ipomoelinmRNA. By contrast, ABA-inducible mRNA for sporamin was not inducedby MeJA. These results suggest that ipomoelin is involved indefensive reactions of leaves in response to wounding and thatJA-mediated wound-induction of ipomoelin occurs independentlyof ABA. (Received January 6, 1997; Accepted March 13, 1997)     

Abscisic acid and osmotic induction of synchronous somatic embryo development of sweet potato

Summary Somatic embryos of sweet potato have potential as synthetic seeds. The effects of abscisic acid (ABA) (0,0,0.1, 1.0, 10.0 and 50.0 μM) were examined to improve synchrony and proliferation of somatic embryos. Transferring embryos compared to those cultures transferred at day 0. The development of embryos in suspension culture supplemented with ABA was poor. However, when calli proliferation cultures were in gelled medium and pulsed with 0.1 μM ABA for 14 d, the number of somatic embryos increased. Proembryonic masses cultured in mannitol-containing medium (Y=−1.5 MPa) increased embryo development and synchrony of embryo development. Thus, in this work ABA and mannitol have been shown to improve both the total number and the synchrony of sweet potato somatic embryos.     

Characterization and expression of Rubisco activase genes in Ipomoea batatas

Two-dimensional electrophoresis, coupled with MALDI-TOF-MS, was used to identify differentially expressed proteins between young and mature leaves of sweet potato [Ipomoea batatas (L.) Lam]. The results showed that there were 25 differential proteins between young and mature leaves. The Rubisco activase (RCA) that catalyzes the activation of Rubisco in vivo and plays a crucial role in photosynthesis was among these 25 proteins. So far, little was known about the molecular biology of RCA in sweet potato. Here, this research reports the cloning and characterization of two genes encoding the short isoform and the long isoform of sweet potato RCAs. Analysis of DNA sequences of RCA suggested that the corresponding mRNAs were transcribed from two different genes. To study the roles of these two RCA isoforms in photosynthesis, we investigated the expression patterns of these RCA genes at the mRNA and protein levels every 2 h in a photoperiod and under different temperatures conditions. The results indicated that these two RCA isoforms may play different roles in regulating photosynthesis and they may be regulated by light, heat or both. In addition, there were interactions between Rubisco large subunit (RBCl) and short isoform RCA (RCAs) as well as RCAs and long isoform RCA (RCAl), but no interaction between RBCl and RCAl, implying they might form a sandwich-like structure (RBCl–RCAs–RCAl), at least in yeast cells. These results provided new information on the modulation of RCA genes in sweet potato, which could be useful in improving photosynthesis and plant growth in sweet potato.     

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

Overexpression of barley nicotianamine synthase 1 confers tolerance in the sweet potato to iron deficiency in calcareous soil

Background and aims

Iron (Fe) is an essential micronutrient for all higher organisms. Fe is sparingly available in calcareous soils and Fe deficiency is a major agricultural problem worldwide. Nicotianamine (NA) is a metal chelator involved in metal translocation in plants. Sweet potato is an attractive crop that can grow in poor soil and thus is useful for planting in uncultivated soil. In addition, the sweet potato has recently been suggested as a source of bioethanol. Our aim is to increase NA concentration in sweet potato to ameliorate Fe deficiency.

Method

Sweet potato plants expressing the barley NA synthase 1 (HvNAS1) gene under the control of CaMV 35S promoter were produced by Agrobacterium-mediated transformation.

Results

The transgenic sweet potato exhibited tolerance to low Fe availability when grown in calcareous soil. The level of tolerance to low Fe availability was positively correlated with the HvNAS1 expression level. The NA concentration of the transgenic sweet potato leaves was up to 7.9-fold greater than that of the non-transgenic (NT) plant leaves. Furthermore, the Fe and zinc concentrations were 3- and 2.9-fold greater, respectively, in transgenic sweet potato than in NT plant leaves.

Conclusions

Our results suggest that increasing the NA concentration of sweet potato by overexpression of HvNAS1 could significantly improve agricultural productivity and energy source.

     

Influence of Ethephon on Nitrate Reductase, Protein Amino Nitrogen and Nitrate Content of Solanum tuberosum L.

Nitrate reductase activity was stimulated in roots and stems,but suppressed in leaves of potato plants grown in nutrientculture by 30 mg.liter^–1 ethephon applied to the culturesolution. In stems, nitrate reductase activity was stimulatedafter 5 h and by 24 h it was more than two fold that of thecontrol. The magnitude of stimulation by ethephon was less inroots compared to stems. Ethephon treatment enhanced ethyleneproduction by roots, stems and leaves but the level of productionwas not significantly different in these organs. The stimulationof nitrate reductase activity was prevented by cycloheximideand cordycepin suggesting the involvement of new protein synthesis. Ethephon enhanced TCA precipitable protein levels in both rootsand stems while that in leaves was not significantly affected.Amino nitrogen content increased in parallel with protein contentin response to ethephon, with roots exhibiting substantial stimulation.Nitrate accumulation in stem tissues was not affected by ethephontreatment but was increased in roots at 24 and 48 h. Leaf NO³content declined with time in both ethephon-treated and controlplants and after 24 h significantly less NO³ accumulated intreated leaves. These results are explained in terms of ethephonstimulated protein synthesis and increase in cellular metabolismand permeability. (Received August 21, 1984; Accepted January 7, 1985)     

ABA预处理对PEG胁迫下两种草坪草POD同工酶的影响

以假俭草和狗牙根幼苗为材料,分别用PEG(聚乙二醇)处理和ABA预处理24h后再用PEG处理模拟干旱胁迫,研究在0~4d内POD同工酶电泳酶谱的结构及特征。结果表明,随着20%PEG处理时间延长,假俭草POD同工酶Rf 0.12和Rf 0.45区域的主要谱带颜色加深,POD活性提高。ABA预处理24h的假俭草POD同工酶谱带颜色却不同,其POD活性降低;而ABA预处理对狗牙根POD活性影响不明显。PEG处理后假俭草图谱中Rf值为0.56的酶谱随着胁迫时间延长而逐渐消失,但在处理第4d出现了新的Rf 0.324谱带,而ABA预处理24h就出现该谱带并一直存在;在Rf 0.22区域也存在几条类似的谱带。     

Cloning and characterization of the Rubisco activase gene from <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam

A full-length cDNA of Rubisco activase (IBrcaI) was cloned from sweet potato (Ipomoea batatas (L.) Lam) using Rapid-Amplification of cDNA Ends (RACE). IBrcaI contains a 1,347 bp open reading frame encoding a protein of 439 amino acids. The sequence alignment of multiple Rubisco activase genes from sweet potato and other plants showed high homology at two previously described ATP-binding sites. Western blot analysis indicated that there are two Rubisco activase proteins in sweet potato. Expression of IBrcaI was only detected in leaves. In the 14 h light and 10 h dark photoperiods, maximal and minimal IBrcaI mRNA expression levels were detected at 8:00 in the morning and at midnight, respectively.