Isolation and Characterization of Hydroxyproline-Rich Glycopeptide Signals in Black Nightshade Leaves

A gene encoding a preprohydroxyproline-rich systemin, SnpreproHypSys, was identified from the leaves of black nightshade (Solanum nigrum), which is a member of a small gene family of at least three genes that have orthologs in tobacco (Nicotiana tabacum; NtpreproHypSys), tomato (Solanum lycopersicum; SlpreproHypSys), petunia (Petunia hybrida; PhpreproHypSys), potato (Solanum tuberosum; PhpreproHypSys), and sweet potato (Ipomoea batatas; IbpreproHypSys). SnpreproHypSys was induced by wounding and by treatment with methyl jasmonate. The encoded precursor protein contained a signal sequence and was posttranslationally modified to produce three hydroxyproline-rich glycopeptide signals (HypSys peptides). The three HypSys peptides isolated from nightshade leaf extracts were called SnHypSys I (19 amino acids with six pentoses), SnHypSys II (20 amino acids with six pentoses), and SnHypSys III (20 amino acids with either six or nine pentoses) by their sequential appearance in SnpreproHypSys. The three SnHypSys peptides were synthesized and tested for their abilities to alkalinize suspension culture medium, with synthetic SnHypSys I demonstrating the highest activity. Synthetic SnHypSys I was capable of inducing alkalinization in other Solanaceae cell types (or species), indicating that structural conformations within the peptides are recognized by the different cells/species to initiate signal transduction pathways, apparently through recognition by homologous receptor(s). To further demonstrate the biological relevance of the SnHypSys peptides, the early defense gene lipoxygenase D was shown to be induced by all three synthetic peptides when supplied to excised nightshade plants.     

Three hydroxyproline-rich glycopeptides derived from a single petunia polyprotein precursor activate defensin I, a pathogen defense response gene

Hydroxyproline-rich glycopeptides (HypSys peptides) are recently discovered 16-20-amino acid defense signals in tobacco and tomato leaves that are derived from cell wall-associated precursors. The peptides are powerful wound signals that activate the expression of defensive genes in tobacco and tomato leaves in response to herbivore attacks. We have isolated a cDNA from petunia (Petunia hybrida) leaves encoding a putative protein of 214 amino acids that is a homolog of tobacco and tomato HypSys peptide precursors and is inducible by wounding and MeJA. The deduced protein contains a leader sequence and four predicted proline-rich peptides of 18-21 amino acids. Three of the four peptides were isolated from leaves, and each peptide contained hydroxylated prolines and glycosyl residues. Each of the peptides has a -GR- motif at its N terminus, indicating that it may be the substrate site for a processing enzyme. The peptides were active in a petunia suspension culture bioassay at nanomolar concentrations, but they did not induce the expression of defense genes that are directed against herbivores, as found in tobacco and tomato leaves. They did, however, activate expression of defensin 1, a gene associated with inducible defense responses against pathogens.     

Peptide signals for plant defense display a more universal role

Hydroxyproline-rich systemins (HypSys) are small defense signaling glycopeptides found within the Solanaceae family that until recently were thought to only induce defense genes to herbivore attack. The glycopeptides are processed from larger proproteins with up to 3 different glycopeptides being processed out of a single precursor protein. A conserved central hydroxyproline motif within each HypSys is the site of pentose sugar attachment. Recently, it was found that in Petunia hybrida, these defense signaling glycopeptides did not induce protease inhibitor but instead, increased levels of defensin, a gene that is involved in pathogen attack. More recently, a HypSys peptide was isolated from Ipomoea batatas (sweet potato) of the Convolvulaceae family and found to induce sporamin. The proprotein precursor contained six putative peptide signals and had a propeptidase processing region with homology to solanaceous proHypSys. Thus, the HypSys defense peptides are no longer confined to defense against herbivory or exclusivity to the Solanaceae family, redefining both function and dispersion.Key words: systemin, hydroxyproline-rich systemin glycopeptides, HypSys, plant defense, proteinase inhibitorsPlants have evolved an arsenal of defense mechanisms for survival against the wide array of predators and pathogens that they encounter. Each species has evolved within its unique environment and the protective defense mechanisms must evolve and refine over time to allow a plant to compete in its niche.¹ Plant peptide signals have recently been discovered that induce defense genes for protection against both herbivores and pathogens.² This raises the issue of how these peptides, their receptors, signaling pathways, and the downstream regulated defense proteins and compounds have evolved to meet the unique and specific needs of each plant. Our recent papers^3,4 reveal that these defense signaling peptides are not confined to a single family of plants and that the end products of the signaling pathway may be more diverse than expected.Systemin was the first peptide signal discovered in plants.⁵ The 18 amino acid peptide is processed from the C-terminal of a 200 amino acid precursor; prosystemin.⁶ Although lacking a signal sequence, prosystemin reaches the apoplast and the mature peptide is processed upon insect attack, signaling downstream events leading to the production of defense proteins, such as polyphenol oxidase and protease inhibitors.⁷ Systemin has only been found in the Solanaceae family and more specifically, only in the subfamily Solanoideae, which contains tomato, potato, nightshade and pepper.The hydroxyproline-rich systemin glycopeptides are similar to systemin in size (18–20 amino acids in length) and, like systemin, are processed from larger precursors.^2,8 Both systemin and HypSys induce the production of methyl jasmonate and function to amplify the defense response. Each HypSys peptide contains a hydroxyproline-rich inner core that is the site of glycosylation and both the peptide backbone and the carbohydrate moieties are important for receptor recognition (9^,10 Although there is no sequence similarity between prosystemin and hydroxyproline-rich systemins, it has been suggested that because of their size, structure and functional similarities, they should be classified together.¹¹

Table 1

Comparisons of the amino acid sequences of isolated and putative Systemin and HypSys peptides

Molecular cloning and characterization of a cDNA encoding asparaginyl endopeptidase from sweet potato (Ipomoea batatas (L.) Lam) senescent leaves

Asparaginyl endopeptidase is a cysteine endopeptidase that has strict substrate specificity toward the carboxyl side of asparagine residues, and is possibly involved in the post-translational processing of proproteins. In this report one full-length cDNA, SPAE, was isolated from senescent leaves of sweet potato (Ipomoea batatas (L.) Lam). SPAE contained 1479 nucleotides (492 amino acids) in the open reading frame, and exhibited high amino acid sequence homologies (c. 61-68%) with asparaginyl endopeptidases of Vicia sativa, Phaseolus vulgaris, Canavalia ensiformis, and Vigna mungo. SPAE probably encoded a putative precursor protein. Via cleavage of the N- and C-termini, it produced a mature protein containing 325 amino acids (from the 51st to the 375th amino acid residues), the conserved catalytic residues (the 173rd His and 215th Cys amino acid residues), and the putative N-glycosylation site (the 332nd Asn amino acid residue). Semi-quantitative RT-PCR and western blot hybridization showed that SPAE gene expression was enhanced significantly in natural senescent leaves and in dark- and ethephon-induced senescent leaves, but was much less in mature green leaves, stems, and roots. Phylogenic analysis showed that SPAE displayed close association with vacuolar processing enzymes (legumains/asparaginyl endopeptidases), which function via cleavage for proprotein maturation in the protein bodies during seed maturation and germination. In conclusion, sweet potato SPAE is probably a functional, senescence-associated gene and its mRNA and protein levels were significantly enhanced in natural and induced senescent leaves. The possible role and function of SPAE associated with bulk protein degradation and mobilization during leaf senescence were also discussed.     

Systemic wound signaling in tomato leaves is cooperatively regulated by systemin and hydroxyproline-rich glycopeptide signals

Hydroxyproline-rich glycopeptides (HypSys peptides) have been isolated recently from tobacco and tomato leaves that are powerful activators of protease inhibitor synthesis. The peptides are processed from polyprotein precursors, two from a single tobacco precursor and three from a single tomato precursor. The precursor genes are expressed in response to wounding and methyl jasmonate, similar to the expression of the systemin precursor prosystemin in tomato leaves. Here we investigate the relationships between systemin and the tomato HypSys peptides in regulating wound signaling in tomato plants. Analysis of transgenic tomato plants over-expressing sense and antisense constructs of the tomato HypSys precursor under the 35S CaMV promoter show that the transgenic plants regulate protease inhibitor gene expression in response to wounding in a manner similar to prosystemin. The evidence indicates that the expression of both the tomato HypSys precursor gene and the prosystemin gene in response to wounding are necessary for strong systemic signaling. The data supports a role for both genes in an amplification loop that up-regulates the octadecanoid pathway and the synthesis of jasmonates to effect strong systemic signaling of defense genes. This report provides the first demonstration of the involvement of two plant peptides derived from two unrelated genes in regulating long distance wound signaling in plants. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors () is Clarence A. Ryan.     

Molecular cloning and characterization of a granulin-containing cysteine protease SPCP3 from sweet potato (Ipomoea batatas) senescent leaves

Granulins are a family of evolutionarily ancient proteins that are involved in regulating cell growth and division in animals. In this report a full-length cDNA, SPCP3, was isolated from senescent leaves of sweet potato (Ipomoea batatas). SPCP3 contains 1389 nucleotides (462 amino acids) in its open reading frame, and exhibits high amino acid sequence homologies (ca. 64-73.6%) with several plant granulin-containing cysteine proteases, including potato, tomato, soybean, kidney bean, pea, maize, rice, cabbage, and Arabidopsis. Gene structural analysis shows that SPCP3 encodes a putative precursor protein. Via cleavage of the N-terminal propeptide, it generates a protein with 324 amino acids (from the 139th to the 462nd amino acid residues), which contains two main domains: the conserved catalytic domain with the putative catalytic residues (the 163rd Cys, 299th His and 319th Asn) and the C-terminal granulin domain (from the 375th to the 462nd amino acid residues). Semi-quantitative RT-PCR and protein gel blot hybridization showed that SPCP3 gene expression was enhanced significantly in natural senescent leaves and in dark- and ethephon-induced senescent leaves, but was almost undetectable in mature green leaves, veins, and roots. Phylogenic analysis showed that SPCP3 displayed close association with a group of plant granulin-containing cysteine proteases which have been implied to be involved in programmed cell death. In conclusion, sweet potato SPCP3 is a functional, senescence-associated gene. Its mRNA and protein levels were significantly enhanced in natural and induced senescing leaves. The physiological role and/or function of SPCP3 associated with programmed cell death during leaf senescence were also discussed.     

Signal transduction and regulation of IbpreproHypSys in sweet potato

Hydroxyproline‐rich glycopeptides (HypSys) are small signalling peptides containing 18–20 amino acids. The expression of IbpreproHypSys, encoding the precursor of IbHypSys, was induced in sweet potato (Ipomoea batatas cv. Tainung 57) through wounding and IbHypSys treatments by using jasmonate and H₂O₂. Transgenic sweet potatoes overexpressing (OE) and silencing [RNA interference (RNAi)] IbpreproHypSys were created. The expression of the wound‐inducible gene for ipomoelin (IPO) in the local and systemic leaves of OE plants was stronger than the expression in wild‐type (WT) and RNAi plants after wounding. Furthermore, grafting experiments indicated that IPO expression was considerably higher in WT stocks receiving wounding signals from OE than from RNAi scions. However, wounding WT scions highly induced IPO expression in OE stocks. These results indicated that IbpreproHypSys expression contributed towards sending and receiving the systemic signals that induced IPO expression. Analysing the genes involved in the phenylpropanoid pathway demonstrated that lignin biosynthesis was activated after synthetic IbHypSys treatment. IbpreproHypSys expression in sweet potato suppressed Spodoptera litura growth. In conclusion, wounding induced the expression of IbpreproHypSys, whose protein product was processed into IbHypSys. IbHypSys stimulated IbpreproHypSys and IPO expression and enhanced lignin biosynthesis, thus protecting plants from insects.     

Involvement of hydrogen peroxide and nitric oxide in expression of the ipomoelin gene from sweet potato

The IPO (ipomoelin) gene was isolated from sweet potato (Ipomoea batatas cv Tainung 57) and used as a molecular probe to investigate its regulation by hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) after sweet potato was wounded. The expression of the IPO gene was stimulated by H(2)O(2) whether or not the plant was wounded, but its expression after wounding was totally suppressed by the presence of diphenylene iodonium, an inhibitor of NADPH oxidase, both in the local and systemic leaves of sweet potato. These results imply that a signal transduction resulting from the mechanical wounding of sweet potato may involve NADPH oxidase, which produces endogenous H(2)O(2) to stimulate the expression of the IPO gene. The production of H(2)O(2) was also required for methyl jasmonate to stimulate the IPO gene expression. On the contrary, NO delayed the expression of the IPO gene, whereas N(G)-monomethyl-L-arginine monoacetate, an inhibitor of NO synthase, enhanced the expression of the IPO gene after the plant was wounded. This study also demonstrates that the production of H(2)O(2) stained with 3,3'-diaminobenzidine hydrochloride could be stimulated by wounding but was suppressed in the presence of NO. Meanwhile, the generation of NO was visualized by confocal scanning microscope in the presence of 4,5-diaminofluorescein diacetate after sweet potato was wounded. In conclusion, when sweet potato was wounded, both H(2)O(2) and NO were produced to modulate the plant's defense system. Together, H(2)O(2) and NO regulate the expression of the IPO gene, and their interaction might further stimulate plants to protect themselves from invasions by pathogens and herbivores.     

Catalase activity is modulated by calcium and calmodulin in detached mature leaves of sweet potato

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.     

Primary structure and expression of a 24-kD vacuolar protein (VP24) precursor in anthocyanin-producing cells of sweet potato in suspension culture

A 24-kD vacuolar protein (VP24) accumulates abundantly in intravacuolar pigmented globules in anthocyanin-containing sweet potato (Ipomoea batatas) cells in suspension culture. A cDNA clone encoding VP24 was isolated from a cDNA library constructed from light-irradiated suspension-cultured cells. Sequence analysis revealed that a 2.9-kbp VP24 cDNA encodes a protein of 893 amino acid residues with a molecular mass of 96.3 kD. According to the deduced amino acid sequence of VP24 cDNA, VP24 is probably synthesized as a large precursor protein with an N-terminal extension composed of a signal peptide and a propeptide, plus the polypeptide of the mature VP24 and its C-terminal propeptide, which contains the multiple transmembrane domains. A search in the ProDom database revealed the mature VP24 domain belongs to the zinc metalloprotease family. Northern analysis revealed that the single 2.9-kb VP24 mRNA increases rapidly after light irradiation, whereas VP24 mRNA was undetectable in the dark-cultured cells or in the presence of a high concentration of 2,4-dichlorophenoxyacetic acid. Light-induced VP24 gene expression closely correlated with the accumulation of anthocyanin in the vacuoles. These results suggested that proteins derived from the VP24 precursor protein may be involved in vacuolar transport and/or accumulation of anthocyanin synthesized in the cytosol.     

甘薯叶片超氧化物歧化酶基因克隆及测序

以甘薯（Ｉｐｏｍｏｅａｂａｔａｔａｓ（Ｌ．）Ｐｏｉｒ）叶为材料提取植物总ＲＮＡ,经反转录后,利用多聚酶链式反应技术,扩增并克隆超氧化物歧化酶基因的ｃＤＮＡ,并进行测序分析。该序列全长４８２ｂｐ,其读码框编码１５２个氨基酸,与国外文献报道的甘薯块根ＳＯＤ基因的ｃＤＮＡ序列相比,具有９９％的同源性。     

Molecular cloning of two metallothionein-like protein genes with differential expression patterns from sweet potato (Ipomoea batatas) leaves

Metallothionein (MT) is a group of proteins with low molecular masses and high cysteine contents, and is classified into different types, which in general contains two domains (domain 1 and domain 2) with typical amino acid sequences (Rauser 1999). In this report two full-length cDNAs (Y459 and G14) encoding MT-like proteins were isolated from leaves of sweet potato (Ipomoea batatas). Their open reading frames contained 249 and 195 nucleotides (82 and 64 amino acids) for Y459 and G14, respectively, and exhibited a relatively low amino acid sequence similarity (ca. 25.8%). Gene structure studies showed that Y459 had the conserved domain 1 region of type 2 MT; however, the domain 2 region was not conserved and contained additional amino acids between the CxC and CxC spacing. G14 had conserved domains 1 and 2 of type 4 MT except that the last CxC of domain 2 was changed to RxC. Semi-quantitative RT-PCR showed that Y459 was expressed in significant quantity in roots and stems, but was much less in green leaves. During natural and induced (with dark and ethephon, an ethylene-releasing compound, treatments) leaf senescence, Y459 gene expression was significantly enhanced. In contrast, relatively constant gene expression levels were found for G14 in all tissues or treatments analyzed. In conclusion, the two MT-like protein genes of sweet potato display differential gene structures and gene expression patterns, which may be associated with the diverse roles and functions they play in plant physiology in order to cope with particular developmental and environmental cues.     

甘薯生物技术研究进展

新兴的生物技术为甘薯这一古老的农作物带来了新的发展契机。细胞大规模培养、体细胞融合、基因转导等技术的研究和应用,可望从根本上改变甘薯传统的生产和育种模式。本文综合近年来国内外甘薯体细胞胚胎发生、原生质体培养和基因工程等方面的研究进展,对影响甘薯体胚发生体系及原生质体再生体系建立的诸多因素进行了详细论述,讨论了甘薯基因工程研究的应用潜力和目前存在的一些问题。     

A pathogen-responsive cDNA from potato encodes a protein with homology to a phosphate starvation-induced phosphatase

Infiltration of potato leaves with the phytopathogenic bacteria Pseudomonas syringae pv. maculicola induces local and systemic defense gene expression as well as increased resistance against subsequent pathogen attacks. By cDNA-AFLP a gene was identified that is activated locally in potato leaves in response to bacterial infiltration and after infection with Phytophthora infestans, the causal agent of late blight disease. The encoded protein has high homology to a phosphate starvation-induced acid phosphatase from tomato. Possibly, decreased phosphate availability after pathogen infection acts as a signal for the activation of the potato phosphatase gene.     

Structural characterization of the dihydroflavonol 4-reductase B (DFR-B) gene in the sweet potato.

A genomic fragment containing the dihydroflavonol 4-reductase B (DFR-B) gene was cloned from the sweet potato (Ipomoea batatas) and its nucleotide sequence was analyzed. The exons and flanking regions were highly homologous to those of previously reported DFR-B genes of the Japanese morning glory, whereas the introns and the intergenic region were less conserved. In addition to the sequences of three miniature inverted-repeat transposable elements (MITEs) and one direct repeat previously reported in the DFR-B gene of Japanese morning glory, two mobile element-like sequences were newly identified in the sweet potato DFR-B gene. At least four allelic sequences were found to exist by amplification of the DFR-B gene from various sweet potato cultivars. One of these allelic sequences had a 2-kb deletion in the intergenic region and was observed in the cultivars with high anthocyanin content in their storage roots.