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.     

Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice

Summary We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family.     

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.     

A novel alkaline α-galactosidase gene is involved in rice leaf senescence

We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves. Here we characterized the structure and function of an SAG-Osh69 encoding alkaline α-galactosidase that belongs to a novel family of glycosyl hydrolases. Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8. The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid, salicylic acid), and stresses (H₂O₂ and wounding). The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal α-galactosidase activity at pH 8.0. The enzyme showed good hydrolytic activities towards α-1,6-galactosyl oligosaccharides and galactolipid digalactosyl diacylglycerol. Immunoelectron microscopic analysis demonstrates that Osh69 is specifically localized in the chloroplasts of senescing leaves. These findings strongly suggest an important role for Osh69 in the degradation of chloroplast galactolipids during leaf senescence. The nucleotide sequence data reported will appear in the GenBank Nucleotide Sequence Database under the accession number AF251068.     

马铃薯POTHR-1 cDNA的克隆及晚疫病菌和其他非生物因子诱导表达分析

利用RACE和重叠延伸相结合的方法,从经晚疫病菌接种诱导的马铃薯水平抗性材料叶片中克隆了一个POTHR-I基因(potato Phytophthora infestans-induced hypersensitive response related protein gene)的全长cDNA。序列分析表明,该基因编码225个氨基酸,与烟草harpin诱导蛋白基因hinI有很高的同源性(编码区核苷酸和氨基酸序列分别为83％和81％)。Southern杂交结果显示在马铃薯基因组中有2、3个拷贝。对其诱导表达模式研究表明：晚疫病病原菌接种36h后,该基因表达迅速增加;机械伤害及茉莉酸(JA)处理能够诱导表达;渗透胁迫(NaCI浸泡)能够诱导其微弱表达;但水杨酸(SA)不能诱导表达。该基因可能和病原与寄主互作时寄主产生过敏反应及细胞生理性死亡有关。     

Abscisic acid and jasmonic acid activate wound-inducible genes in potato through separate, organ-specific signal transduction pathways

Mechanical damage to leaf tissue causes an increase in abscisic acid (ABA) which in turn activates the biosynthesis of jasmonic acid (JA). The resulting higher endogenous JA levels subsequently activate the expression of wound-inducible genes. This study shows that JA induces the expression of different sets of genes in roots and leaves of potato plants. When roots of intact plants were treated with JA, high levels of proteinase inhibitor II (pin2), cathepsin D inhibitor, leucine aminopeptidase and threonine deaminase mRNAs accumulated in the systemic leaves. However, in the treated roots, very low, if any, expression of these genes could be detected. In contrast, a novel, root-specific pin2 homologue accumulated in the JA-treated root tissue which could not be detected in leaves, either systemic or those directly treated with JA. Application of okadaic acid and staurosporine revealed that a protein phosphorylation step is involved in the regulation of this differential response. In leaves, a protein phosphatase is required for the JA-induced expression of pin2 and the other genes analysed. This phosphatase activity is not necessary for the JA-induced expression of a pin2 homologue in roots, suggesting the existence of different transduction pathways for the JA signal in these organs. The requirement of a protein phosphatase activity for JA-mediated gene induction has enabled identification of a JA-independent pathway for ABA induction of pin2 and the other wound-inducible genes. This alternative pathway involves a protein kinase, and appears to be selective for wound-inducible genes. Our data suggest the presence of a complex, organ-specific transduction network for regulating the effects of the plant hormones ABA and JA on gene expression upon wounding.