小麦TaSSP1基因和其拟南芥同源基因AT3G14067的克隆及其功能分析
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摘要
类枯草杆菌蛋白酶(Subtilases)在植物体内广泛分布,且数目众多,广泛参与植物的生长发育,以及响应外界环境变化参与代谢调控等,但目前该类蛋白酶在植物体内的具体功能和作用机制等方面还不完全清楚。本课题组在前期研究中,从小麦暗诱导衰老的叶片中分离纯化了一种与叶片衰老相关的类枯草杆菌丝氨酸蛋白酶,命名为TaSSP1(Triticum aestivum Subtilisin-like Serine Protease1),并通过质谱鉴定所得数据在NCBI数据库中比对,获得了该蛋白酶氨基酸序列的部分信息,但该蛋白酶在小麦生长发育中到底具有哪些功能不完全清楚。本研究根据已知的该蛋白酶基酸部分序列,采用RACE的方法得到了小麦的该蛋白酶基因TaSSP1全长序列。之后,通过将TaSSP1基酸序列在国际蛋白酶数据库(MEROPS)中进行比对,发现了在拟南芥中与该蛋白酶相似度达77.8%的同源蛋白AT3G14067,而AT3G14067蛋白在拟南芥中的具体功能也尚不清楚。因此,本文在上述研究结果的基础上,进一步通过RT-PCR、荧光定量PCR、Western blot、SDS-PAGE、天然梯度凝胶活性电泳和转基因技术等方法手段,主要从基因的转录和蛋白表达及酶活性表现的水平上研究分析了TaSSP1和AT3G14067基因在小麦和拟南芥生长发育及在响应环境变化中的具体功能,得到的主要研究结果如下：
1)根据已知TaSSP1的氨基酸部分序列,采用RACE的方法获得了小麦蛋白酶基因TaSSP1全长序列,并在GenBank进行了登录,登记号为：JX962746。
TaSSP1基因CDS区全长2361bp,编码786个氨基酸,在丝氨酸蛋白酶家族中属于S8A家族。对TaSSP1蛋白结构进行生物信息学分析,该蛋白酶含有五个结构域,分别为N端信号肽、19Inhibitor区域、PA (Protease-associated)结构域、催化结构域和C端延长区域。
通过荧光定量PCR对jTaSSP1基因的小麦组织特异性表达情况进行分析,发现TaSSP1基因在不同衰老程度的叶片中,衰老程度更严重的叶片中TaSSP1的mRNA表达量更高；在叶片的不同部位中,叶尖部位的TaSSP1表达量较高；暗处理叶片衰老中,发现随着暗处理时间延长,叶片衰老程度加深,叶片TaSSP1的mRNA表达水平和蛋白酶活力均逐渐升高。叶片TaSSPl基因的表达亦受植物激素ABA、6-BA和乙烯的影响,其1nRNA表达和酶活力水平随着ABA处理时间(0-48h,或0-72h)的延长逐渐不断升高；而在6-BA处理下,其mRNA表达和酶活力水平都是呈现先升高后降低并降低到对照(0h)时的以下水平。结果还发现,TnSSP1基因的表达还能明显地被外界渗透胁迫所诱导,如小麦在200mMNaCl、蔗糖和甘露醇以及16%PEG6000的胁迫处理下,叶片中TnSSP1基因的mRNA表达量均明显上升。Western blot与SDS-PAGE实验结果显示,在ABA和16%PEG6000处理的72h过程中,随着TaSSP1蛋白表达水平的不断提高,叶片Rubisco大亚基的含量逐渐减低,表明随着TaSSP1蛋白酶含量的不断升高,Rubisco大亚基的降解程度逐渐加剧。而在6-BA处理下,其蛋白表达水平也是呈现先升后降并降低到低于对照(0h)时以下水平,Rubisco大亚基的含量几乎没有发生明显的变化。另外,实验结果还发现：Cu2+和Ca2+能显著促进TaSSP1酶活力的提高,Zn2+和Mn2+对该酶也有一定的促进作用,但Mg2+和Co2+则对酶活力没有促进作用。以上结果表明：TaSSP1可能是小麦生长发育中的一种重要的Ca2+依赖性的类枯草杆菌丝氨酸蛋白酶,该蛋白酶不仅密切与小麦的衰老进程相关,而且能通过上调基因表达及加快蛋白质周转去积极应对如渗透胁迫等的环境胁迫。
2)对AT3G14067蛋白酶进行生物信息学的结构分析,发现AT3G14067蛋白酶与TaSSP1蛋白酶的结构非常相似,同样属于S8A蛋白家族,亦含有N端信号肽、19Inhibitor区域、PA结构域、催化结构域和C端延长区域等五个结构域。
通过半定量RT-PCR对AT3G14067基因在拟南芥的组织特异性表达进行分析,结果显示：AT3G14067基因在茎、莲座叶和茎生叶中的mRNA表达量最高,这与在植物膜蛋白数据库中比对分析的结果相吻合。研究结果还显示,AT3G14067基因能明显受植物激素ABA、6-BA、乙烯和水杨酸的诱导而上调表达,生长素类似物NAA和赤霉素可轻微提高AT3G14067基因的表达水平,但在茉莉酸甲酯处理下,AT3G14067的转录表达水平没变化。此外,AT3G14067同样能明显受NaCl、甘露醇、PEG6000和蔗糖等渗透胁迫的诱导而上调表达。
暗诱导处理拟南芥莲座叶叶片后,发现处理12h后AT3G14067基因的转录表达水平开始上调,48h时的表达量达到最高；在不同衰老程度的莲座叶叶片中,发现衰老程度严重的叶片中AT3G14067基因的转录表达水平更高,表明AT3G14067基因也与叶片的衰老相关。
在野生型拟南芥种子的萌发实验中,发现在黑暗、蓝光、远红光的培养条件下,AT3G14067基因的mRNA表达量会显著上升,其中以黑暗和远红光处理的最为明显。这表明AT3G14067基因对不同光质也有响应。
上述结果初步表明,AT3G14067蛋白酶也不仅与拟南芥的衰老有关,而且可能与拟南芥的生长发育密切相关。
3)通过转基因获得了AT3G14067基因过表达拟南芥株系OX-3、OX-4和OX-6,经荧光定量PCR鉴定突变体ssp-1为纯合体。对拟南芥的野生型、突变体和过表达株系的表型进行比较发现,在黑暗萌发条件下,AT3G14067基因过表达株系下胚轴显著伸长,突变体株系轻微伸长,突变体与野生型的差异不明显；过表达株系抽苔时间与野生型的相比要提前5天左右,但过表达植株和突变体植株的种子均会提前成熟；在自然生长条件下,过表达植株还表现出主茎顶端生长点丢失,株高变矮,而突变体则株高变高,表型相反；过表达株系植株的分枝数、果荚数显著高于突变体和野生型植株,但过表达株系果荚长度变短、单果荚种子数变少、单果荚重量减轻、单株种子总数提高,而突变体则表现为果荚变长、单果荚种子数、重量均提高；同一时期莲座叶叶片过表达植株叶片的衰老程度与野生型和突变体相比为更严重。
对上述拟南芥不同株系进行生理生化分析,结果表明：当植株生长至50d时,突变体中的莲座叶叶片的叶绿素含量要显著高于野生型和过表达株系；在植株生长至60d、70d时,过表达株系中莲座叶片中的可溶性蛋白含量要显著高于野生型,而突变体与野生型的相比差异不明显；在40d以后,过表达株系中TBARS含量逐渐升高,在60d时株系中的TBARS含量显著高于野生型和突变体植株的含量；在植株生长至50d时,过表达株系中的POD、SOD、CAT酶活力都会明显下降,而脯氨酸含量在40d以后不断升高,与野生型和突变体植株的相比,差异极为显著。
选取拟南芥中与叶片衰老相关的基因,采用荧光定量RT-PCR方法对拟南芥不同株系进一步分析了他们的表达情况,发现SAG12基因的转录水平在过表达株系中明显上调,而在突变体中明显下调；SAG113基因的表达在突变体中轻微下调,在过表达中轻微上调；NAP、NAC2基因的转录水平在过表达株系中上升了2倍左右,而突变体与野生型的相比差异不明显。另外,ATMYB2基因在突变体中的表达有所上调,但在过表达株系中其表达水平比野生型的高出了近5倍；WRKY6和WRKY53两个基因在突变体中的转录表达也轻微上调,但这二者的转录水平在过表达株系中都上调2倍左右。只有SAG29基因的表达在突变体和过表达株系中都呈上调的趋势。以上结果进一步表明：AT3G14067基因不仅直接与拟南芥的衰老相关,而且还可能通过调控其他衰老相关基因的表达促进拟南芥的衰老。
选取拟南芥中与下胚轴发育调控相关基因,同样采用荧光定量RT-PCR方法,对拟南芥不同株系进行其转录表达量分析,发现ABCB19、SOB7和DFL1基因的表达水平在突变体中均显著上升,而三者在野生型和过表达株系中的表达水平差异均不显著；FHY3基因在突变体中有轻微上调趋势,而在野生型和过表达株系中的差异也不明显；而RLP21基因的表达水平在野生型和突变体中的差异不显著,在过表达株系中却呈现上调趋势；DWARF基因的表达在突变体中上调显著,但在过表达株系中却呈明显下调趋势；1EII、RAFL23、CUL1、RPT1、NPL1、HY3、LCL5、MAX2基因的表达在突变体和过表达株系中均呈现上调趋势,但在突变体中的上调程度都更高。
选取与拟南芥花发育调控相关基因的荧光定量RT-PCR分析结果显示,AP2、ATlG01460基因在突变体中的表达水平有上调趋势,但在过表达株系中几乎与野生型的无差异；AP1、AG、AGL2和LFY3基因的表达水平在突变体和过表达株系中均显著下调；AGL9基因的表达在突变体和过表达株系中也均呈明显下调趋势,但突变体中的下调程度更显著；而FLC基因的表达水平在野生型和突变体中几乎无差异,但在过表达株系中的表达水平呈现下调趋势。
以上结果表明：拟南芥AT3G14067基因还可能拟南芥的生长发育,如与下胚轴和开花调控等过程密切相关。
Subtilisin-like proteases (subtilases) belong to the serine protease family(S8) which are widely distributed in all three kingdoms of life. This family of serine proteases is involved in multiple processes in plants, such as plants growth and development, response to environment changes and metabolic regulation. But their specific functions and regulation mechanism et al. are not entirely understanded. In the previous study of our group, a senescence-associated subtilisin-like serine protease was separated and purified from the dark-induced senescence wheat leaves, which was named TaSSPl (Triticum aestivum Subtilisin-like Serine Proteasel). The mass spectrometry data were used to alignment at NCBI protein database and only part of the protease amino acid sequence information was obtained for the database. However, the identified functions of this protease in wheat growth and development were not entirely clear. In this study, according to the known partial amino acid sequence of the protease, the RACE method was used to amplify the full sequence of TaSSP1protease gene. After the full-length sequence of TaSSPl gene was obtained, the CDS region of TaSSPl was used to compare in the MEROPS database and a protease of arabidopsis AT3G14067was found which with a homology was77.8percent compared to TaSSP1. The special function of AT3G14067protease was also not unclear in arabidopsis. Therefore, the methods of RT-PCR, real time quantitative PCR, western blotting, gradient gelatin-PAGE in gel proteolysis assay and transgenic et al. were used to furthermore understand the TaSSPl and AT3G14067function during plants development and response to environmental changes based on the results of the previous study. The main results obtained in this study were showed as follows:
1) According to the known partial amino acid sequence of the TaSSP1, The RACE method was used to obtain the full-length sequence of wheat TaSSP1protease gene. The GenBank database accession No. was JX962746.
This cDNA had a2361bp coding region, and was predicted to encode a protein of786amino acid residues, and that it contained a signal peptide, a19Inhibitor domain, a peptidase S8family domain, a protease-associated domain and a C-terminus domain as analyzed by NCBI's Conserved Domain Database (CDD).
In order to gain a better understanding of the functions of TaSSPl, wheat were cultured and treated with100μM ABA,6-BA, ethephon, and200mM NaCl, D-Mannitol, sucrose or16%PEG6000respectively, whereafter the mRNA, protein and activity levels of TaSSP1in wheat at stated different hours after treatment were analyzed by RT-PCR, fluorescent quantitative RT-PCR, western blotting and gradient gelatin-PAGE in gel proteolysis assay. The results showed that the transcriptional and translational expression activities of TaSSP1in ABA treated wheat were gradually up-regulated with the extension of whole treatment time, but were obviously down-regulated in6-BA treated wheat from the middle to late stages of treatment. The increasing expression levels of TaSSPl were also identified in dark-induced senescence and older wheat leaves. In addition, the TaSSPl expression activities could be obviously up-regulated in wheats treated with200mM NaCl, D-Mannitol and sucrose or16%PEG6000. Furthermore, TaSSP1was showed to be a metal-dependent protease because its activity could be activated by some divalent metal cations, such as Cu2+, Ca2+, Zn2+and Mn2+. The results above indicated the function of TaSSPl was not only related with leaf senescence, but also related with the development of wheat under environment stress, especially to osmosis stress. And suggested that TaSSPl might be important on the response of wheat to environment stresses through its regulation of both gene expression and protein turnover.
2) As AT3G14067protein sequence structure was analyzed through bioinformatics method, we found that AT3G14067has very similar structure compared to TaSSPl, and that it also contained a signal peptide, a19Inhibitor domain, a peptidase S8family domain, a protease-associated domain and a C-terminus domain as analyzed by NCBI's Conserved Domain Database (CDD).
Combined with semi-quantitative RT-PCR for analysis tissue-specific expression of AT3G14067, The result showed that the highest expression sites were in the stem, rosette leaves and cauline leaves which was consistent with the results of the plant membrane protein database. The expression of AT3G14067in response to various plant hormones,200mM NaCl,200mM D-Mannitol,200mM sucrose and16%PEG6000was further analyzed by Real-time quantitative PCR, the results show that it is apparently induced by ABA,6-BA, ETH,200mM NaCl,200mM D-Mannitol,200mM sucrose and16%PEG6000. These results indicated that AT3G14067might also be important in arabidopsis on the response to hormones and abiotic stress.
In wild type arabidopsis, we also found that after the rosette leaves were treated with dark, the expression of AT3G14067was up-regulated after12hours treatment and came to the highest level at48hours treatment. In addition, The increasing expression levels of AT3G14067was also identified in older rosette leaves. This may indicated that AT3G14067gene was also a senescence-associated gene.
In germination experiments of arabidopsis seeds, the results were showed that the expression level of AT3G14067was significantly up-regulated when seeds were cultured in dark, blue light and far-red light situations. Among them, the phenomena above in dark and far-red light treatments were most obviously. This indicated that the responses of AT3G14067gene were different in the seed germination treated with different light quality.
The results preliminary showed that the AT3G14067protease was not only associated with arabidopsis leaf senescence, but may also closely related to the growth and development of Arabidopsis.
3) Over-expression transgenic arabidopsis AT3G14067OX-lines3,4,6were obtained and identified as homozygous by real time quantitative PCR method. Compared with wild type, mutant and over-expression lines phenotype were found that when the seeds germinated in the dark condition, the hypocotyl of AT3G14067gene over-expression lines elongation significantly while the mutant and wild type plants had no significant difference between them. Moreover, the bolting time was5days in advance compared to the over-expression lines and the wild-type. However, the seeds of plants will mature early between the over-expression and the mutant plants. Under natural growth conditions, the plants height of the over-expression lines were lower. In contrast, AT3G14067mutants plants show the opposite phenotype. During the same periods, the rosette leaves of over-expressing plants had deeper leaf senescence degree compared to wild-type and mutant plants.
The physiological and biochemical analysis showed that when the Arabidopsis thaliana grow to the50d stage, the content of chlorophyll in rosette leaves of mutant was significantly higher than that in wild-type and over-expression lines. At the60d and70d stage, the content of soluble protein in rosette leaves of over-expression lines was obvious higher than that in wild-type, while it in mutant is similar to wild-type plants. In over-expression lines, the TBARS content was beginning to increase after40d stage, and clearly higher than that in wild-type and mutant in the60d stage. In the50d stage, the enzyme activity of POD, SOD and CAT was significantly decrease, and the content of proline was rising significantly compared with the wild-type and mutant.
The expression quantity of senescence-related genes in Arabidopsis thaliana leaves were analyzed by qRT-PCR. The expression levels of SAG12in over-expressing lines were significantly increased and in mutant significantly reduced, while SAG13in over-expressing lines were slightly increased and in mutant slightly reduced. NAP and NAC2in over-expressing lines were highly expressed, but in mutant and wild-type ware not significantly difference. The expression profiles for AtMYB2in over-expressing lines was5times higher than in wild-type. The expression patterns of WRKY6and WRKY53in mutant have a slight increase and in over-expressing lines have raised two times. Only SAG29appeared upward trend in both over-expressing lines and mutant. These results further indicate that AT3G14067gene not only directly related to aging, but also possibly promoting aging through regulation of other aging-related genes in Arabidopsis.
Using the qRT-PCR method to analysis the genes associated with the hypocotyls development. The expression profiles for BCB19, SOB7and DFL1in the mutant were all significantly increased and in over-expressing lines and wild-type have slightly different. FHY3only in mutant has a slight upward trend. The expression level of RLP21in wild-type and mutant were similar while in over-expressing lines was up-regulated. The expression patterns of DWARF6in the mutant was significantly up-regulated but in over-expressing lines had an obvious downward trend. FE11, RAFL23, CUL1, RPT1, NPL1, HY3, LCL5, MAX2were up-regulated in both over-expressing lines and mutant, but in the mutant have a higher degree.
The genes related to flowering were analyzed by the qRT-PCR method. Both AP2and AT1G01460in mutant had increased tendency, but had no change in over-expressing lines and in wild-type. The expression level of AP1, AG, AGL2and LFY3in mutant and over-expressing lines were significantly reduced. The expression profiles for AGL9in the mutant and over-expressing lines were all significantly up-regulated. The expression pattern of FLC in mutant and wild-type had no changed but in over-expressing lines had a downward trend.
These results above suggest that Arabidopsis thaliana AT3G14067gene may also have close relationship to the plant growth and development, such as the development of hypocotyl and flowering in Arabidopsis thaliana..
1)根据已知TaSSP1的氨基酸部分序列,采用RACE的方法获得了小麦蛋白酶基因TaSSP1全长序列,并在GenBank进行了登录,登记号为：JX962746。
TaSSP1基因CDS区全长2361bp,编码786个氨基酸,在丝氨酸蛋白酶家族中属于S8A家族。对TaSSP1蛋白结构进行生物信息学分析,该蛋白酶含有五个结构域,分别为N端信号肽、19Inhibitor区域、PA (Protease-associated)结构域、催化结构域和C端延长区域。
通过荧光定量PCR对jTaSSP1基因的小麦组织特异性表达情况进行分析,发现TaSSP1基因在不同衰老程度的叶片中,衰老程度更严重的叶片中TaSSP1的mRNA表达量更高；在叶片的不同部位中,叶尖部位的TaSSP1表达量较高；暗处理叶片衰老中,发现随着暗处理时间延长,叶片衰老程度加深,叶片TaSSP1的mRNA表达水平和蛋白酶活力均逐渐升高。叶片TaSSPl基因的表达亦受植物激素ABA、6-BA和乙烯的影响,其1nRNA表达和酶活力水平随着ABA处理时间(0-48h,或0-72h)的延长逐渐不断升高；而在6-BA处理下,其mRNA表达和酶活力水平都是呈现先升高后降低并降低到对照(0h)时的以下水平。结果还发现,TnSSP1基因的表达还能明显地被外界渗透胁迫所诱导,如小麦在200mMNaCl、蔗糖和甘露醇以及16%PEG6000的胁迫处理下,叶片中TnSSP1基因的mRNA表达量均明显上升。Western blot与SDS-PAGE实验结果显示,在ABA和16%PEG6000处理的72h过程中,随着TaSSP1蛋白表达水平的不断提高,叶片Rubisco大亚基的含量逐渐减低,表明随着TaSSP1蛋白酶含量的不断升高,Rubisco大亚基的降解程度逐渐加剧。而在6-BA处理下,其蛋白表达水平也是呈现先升后降并降低到低于对照(0h)时以下水平,Rubisco大亚基的含量几乎没有发生明显的变化。另外,实验结果还发现：Cu2+和Ca2+能显著促进TaSSP1酶活力的提高,Zn2+和Mn2+对该酶也有一定的促进作用,但Mg2+和Co2+则对酶活力没有促进作用。以上结果表明：TaSSP1可能是小麦生长发育中的一种重要的Ca2+依赖性的类枯草杆菌丝氨酸蛋白酶,该蛋白酶不仅密切与小麦的衰老进程相关,而且能通过上调基因表达及加快蛋白质周转去积极应对如渗透胁迫等的环境胁迫。
2)对AT3G14067蛋白酶进行生物信息学的结构分析,发现AT3G14067蛋白酶与TaSSP1蛋白酶的结构非常相似,同样属于S8A蛋白家族,亦含有N端信号肽、19Inhibitor区域、PA结构域、催化结构域和C端延长区域等五个结构域。
通过半定量RT-PCR对AT3G14067基因在拟南芥的组织特异性表达进行分析,结果显示：AT3G14067基因在茎、莲座叶和茎生叶中的mRNA表达量最高,这与在植物膜蛋白数据库中比对分析的结果相吻合。研究结果还显示,AT3G14067基因能明显受植物激素ABA、6-BA、乙烯和水杨酸的诱导而上调表达,生长素类似物NAA和赤霉素可轻微提高AT3G14067基因的表达水平,但在茉莉酸甲酯处理下,AT3G14067的转录表达水平没变化。此外,AT3G14067同样能明显受NaCl、甘露醇、PEG6000和蔗糖等渗透胁迫的诱导而上调表达。
暗诱导处理拟南芥莲座叶叶片后,发现处理12h后AT3G14067基因的转录表达水平开始上调,48h时的表达量达到最高；在不同衰老程度的莲座叶叶片中,发现衰老程度严重的叶片中AT3G14067基因的转录表达水平更高,表明AT3G14067基因也与叶片的衰老相关。
在野生型拟南芥种子的萌发实验中,发现在黑暗、蓝光、远红光的培养条件下,AT3G14067基因的mRNA表达量会显著上升,其中以黑暗和远红光处理的最为明显。这表明AT3G14067基因对不同光质也有响应。
上述结果初步表明,AT3G14067蛋白酶也不仅与拟南芥的衰老有关,而且可能与拟南芥的生长发育密切相关。
3)通过转基因获得了AT3G14067基因过表达拟南芥株系OX-3、OX-4和OX-6,经荧光定量PCR鉴定突变体ssp-1为纯合体。对拟南芥的野生型、突变体和过表达株系的表型进行比较发现,在黑暗萌发条件下,AT3G14067基因过表达株系下胚轴显著伸长,突变体株系轻微伸长,突变体与野生型的差异不明显；过表达株系抽苔时间与野生型的相比要提前5天左右,但过表达植株和突变体植株的种子均会提前成熟；在自然生长条件下,过表达植株还表现出主茎顶端生长点丢失,株高变矮,而突变体则株高变高,表型相反；过表达株系植株的分枝数、果荚数显著高于突变体和野生型植株,但过表达株系果荚长度变短、单果荚种子数变少、单果荚重量减轻、单株种子总数提高,而突变体则表现为果荚变长、单果荚种子数、重量均提高；同一时期莲座叶叶片过表达植株叶片的衰老程度与野生型和突变体相比为更严重。
对上述拟南芥不同株系进行生理生化分析,结果表明：当植株生长至50d时,突变体中的莲座叶叶片的叶绿素含量要显著高于野生型和过表达株系；在植株生长至60d、70d时,过表达株系中莲座叶片中的可溶性蛋白含量要显著高于野生型,而突变体与野生型的相比差异不明显；在40d以后,过表达株系中TBARS含量逐渐升高,在60d时株系中的TBARS含量显著高于野生型和突变体植株的含量；在植株生长至50d时,过表达株系中的POD、SOD、CAT酶活力都会明显下降,而脯氨酸含量在40d以后不断升高,与野生型和突变体植株的相比,差异极为显著。
选取拟南芥中与叶片衰老相关的基因,采用荧光定量RT-PCR方法对拟南芥不同株系进一步分析了他们的表达情况,发现SAG12基因的转录水平在过表达株系中明显上调,而在突变体中明显下调；SAG113基因的表达在突变体中轻微下调,在过表达中轻微上调；NAP、NAC2基因的转录水平在过表达株系中上升了2倍左右,而突变体与野生型的相比差异不明显。另外,ATMYB2基因在突变体中的表达有所上调,但在过表达株系中其表达水平比野生型的高出了近5倍；WRKY6和WRKY53两个基因在突变体中的转录表达也轻微上调,但这二者的转录水平在过表达株系中都上调2倍左右。只有SAG29基因的表达在突变体和过表达株系中都呈上调的趋势。以上结果进一步表明：AT3G14067基因不仅直接与拟南芥的衰老相关,而且还可能通过调控其他衰老相关基因的表达促进拟南芥的衰老。
选取拟南芥中与下胚轴发育调控相关基因,同样采用荧光定量RT-PCR方法,对拟南芥不同株系进行其转录表达量分析,发现ABCB19、SOB7和DFL1基因的表达水平在突变体中均显著上升,而三者在野生型和过表达株系中的表达水平差异均不显著；FHY3基因在突变体中有轻微上调趋势,而在野生型和过表达株系中的差异也不明显；而RLP21基因的表达水平在野生型和突变体中的差异不显著,在过表达株系中却呈现上调趋势；DWARF基因的表达在突变体中上调显著,但在过表达株系中却呈明显下调趋势；1EII、RAFL23、CUL1、RPT1、NPL1、HY3、LCL5、MAX2基因的表达在突变体和过表达株系中均呈现上调趋势,但在突变体中的上调程度都更高。
选取与拟南芥花发育调控相关基因的荧光定量RT-PCR分析结果显示,AP2、ATlG01460基因在突变体中的表达水平有上调趋势,但在过表达株系中几乎与野生型的无差异；AP1、AG、AGL2和LFY3基因的表达水平在突变体和过表达株系中均显著下调；AGL9基因的表达在突变体和过表达株系中也均呈明显下调趋势,但突变体中的下调程度更显著；而FLC基因的表达水平在野生型和突变体中几乎无差异,但在过表达株系中的表达水平呈现下调趋势。
以上结果表明：拟南芥AT3G14067基因还可能拟南芥的生长发育,如与下胚轴和开花调控等过程密切相关。
Subtilisin-like proteases (subtilases) belong to the serine protease family(S8) which are widely distributed in all three kingdoms of life. This family of serine proteases is involved in multiple processes in plants, such as plants growth and development, response to environment changes and metabolic regulation. But their specific functions and regulation mechanism et al. are not entirely understanded. In the previous study of our group, a senescence-associated subtilisin-like serine protease was separated and purified from the dark-induced senescence wheat leaves, which was named TaSSPl (Triticum aestivum Subtilisin-like Serine Proteasel). The mass spectrometry data were used to alignment at NCBI protein database and only part of the protease amino acid sequence information was obtained for the database. However, the identified functions of this protease in wheat growth and development were not entirely clear. In this study, according to the known partial amino acid sequence of the protease, the RACE method was used to amplify the full sequence of TaSSP1protease gene. After the full-length sequence of TaSSPl gene was obtained, the CDS region of TaSSPl was used to compare in the MEROPS database and a protease of arabidopsis AT3G14067was found which with a homology was77.8percent compared to TaSSP1. The special function of AT3G14067protease was also not unclear in arabidopsis. Therefore, the methods of RT-PCR, real time quantitative PCR, western blotting, gradient gelatin-PAGE in gel proteolysis assay and transgenic et al. were used to furthermore understand the TaSSPl and AT3G14067function during plants development and response to environmental changes based on the results of the previous study. The main results obtained in this study were showed as follows:
1) According to the known partial amino acid sequence of the TaSSP1, The RACE method was used to obtain the full-length sequence of wheat TaSSP1protease gene. The GenBank database accession No. was JX962746.
This cDNA had a2361bp coding region, and was predicted to encode a protein of786amino acid residues, and that it contained a signal peptide, a19Inhibitor domain, a peptidase S8family domain, a protease-associated domain and a C-terminus domain as analyzed by NCBI's Conserved Domain Database (CDD).
In order to gain a better understanding of the functions of TaSSPl, wheat were cultured and treated with100μM ABA,6-BA, ethephon, and200mM NaCl, D-Mannitol, sucrose or16%PEG6000respectively, whereafter the mRNA, protein and activity levels of TaSSP1in wheat at stated different hours after treatment were analyzed by RT-PCR, fluorescent quantitative RT-PCR, western blotting and gradient gelatin-PAGE in gel proteolysis assay. The results showed that the transcriptional and translational expression activities of TaSSP1in ABA treated wheat were gradually up-regulated with the extension of whole treatment time, but were obviously down-regulated in6-BA treated wheat from the middle to late stages of treatment. The increasing expression levels of TaSSPl were also identified in dark-induced senescence and older wheat leaves. In addition, the TaSSPl expression activities could be obviously up-regulated in wheats treated with200mM NaCl, D-Mannitol and sucrose or16%PEG6000. Furthermore, TaSSP1was showed to be a metal-dependent protease because its activity could be activated by some divalent metal cations, such as Cu2+, Ca2+, Zn2+and Mn2+. The results above indicated the function of TaSSPl was not only related with leaf senescence, but also related with the development of wheat under environment stress, especially to osmosis stress. And suggested that TaSSPl might be important on the response of wheat to environment stresses through its regulation of both gene expression and protein turnover.
2) As AT3G14067protein sequence structure was analyzed through bioinformatics method, we found that AT3G14067has very similar structure compared to TaSSPl, and that it also contained a signal peptide, a19Inhibitor domain, a peptidase S8family domain, a protease-associated domain and a C-terminus domain as analyzed by NCBI's Conserved Domain Database (CDD).
Combined with semi-quantitative RT-PCR for analysis tissue-specific expression of AT3G14067, The result showed that the highest expression sites were in the stem, rosette leaves and cauline leaves which was consistent with the results of the plant membrane protein database. The expression of AT3G14067in response to various plant hormones,200mM NaCl,200mM D-Mannitol,200mM sucrose and16%PEG6000was further analyzed by Real-time quantitative PCR, the results show that it is apparently induced by ABA,6-BA, ETH,200mM NaCl,200mM D-Mannitol,200mM sucrose and16%PEG6000. These results indicated that AT3G14067might also be important in arabidopsis on the response to hormones and abiotic stress.
In wild type arabidopsis, we also found that after the rosette leaves were treated with dark, the expression of AT3G14067was up-regulated after12hours treatment and came to the highest level at48hours treatment. In addition, The increasing expression levels of AT3G14067was also identified in older rosette leaves. This may indicated that AT3G14067gene was also a senescence-associated gene.
In germination experiments of arabidopsis seeds, the results were showed that the expression level of AT3G14067was significantly up-regulated when seeds were cultured in dark, blue light and far-red light situations. Among them, the phenomena above in dark and far-red light treatments were most obviously. This indicated that the responses of AT3G14067gene were different in the seed germination treated with different light quality.
The results preliminary showed that the AT3G14067protease was not only associated with arabidopsis leaf senescence, but may also closely related to the growth and development of Arabidopsis.
3) Over-expression transgenic arabidopsis AT3G14067OX-lines3,4,6were obtained and identified as homozygous by real time quantitative PCR method. Compared with wild type, mutant and over-expression lines phenotype were found that when the seeds germinated in the dark condition, the hypocotyl of AT3G14067gene over-expression lines elongation significantly while the mutant and wild type plants had no significant difference between them. Moreover, the bolting time was5days in advance compared to the over-expression lines and the wild-type. However, the seeds of plants will mature early between the over-expression and the mutant plants. Under natural growth conditions, the plants height of the over-expression lines were lower. In contrast, AT3G14067mutants plants show the opposite phenotype. During the same periods, the rosette leaves of over-expressing plants had deeper leaf senescence degree compared to wild-type and mutant plants.
The physiological and biochemical analysis showed that when the Arabidopsis thaliana grow to the50d stage, the content of chlorophyll in rosette leaves of mutant was significantly higher than that in wild-type and over-expression lines. At the60d and70d stage, the content of soluble protein in rosette leaves of over-expression lines was obvious higher than that in wild-type, while it in mutant is similar to wild-type plants. In over-expression lines, the TBARS content was beginning to increase after40d stage, and clearly higher than that in wild-type and mutant in the60d stage. In the50d stage, the enzyme activity of POD, SOD and CAT was significantly decrease, and the content of proline was rising significantly compared with the wild-type and mutant.
The expression quantity of senescence-related genes in Arabidopsis thaliana leaves were analyzed by qRT-PCR. The expression levels of SAG12in over-expressing lines were significantly increased and in mutant significantly reduced, while SAG13in over-expressing lines were slightly increased and in mutant slightly reduced. NAP and NAC2in over-expressing lines were highly expressed, but in mutant and wild-type ware not significantly difference. The expression profiles for AtMYB2in over-expressing lines was5times higher than in wild-type. The expression patterns of WRKY6and WRKY53in mutant have a slight increase and in over-expressing lines have raised two times. Only SAG29appeared upward trend in both over-expressing lines and mutant. These results further indicate that AT3G14067gene not only directly related to aging, but also possibly promoting aging through regulation of other aging-related genes in Arabidopsis.
Using the qRT-PCR method to analysis the genes associated with the hypocotyls development. The expression profiles for BCB19, SOB7and DFL1in the mutant were all significantly increased and in over-expressing lines and wild-type have slightly different. FHY3only in mutant has a slight upward trend. The expression level of RLP21in wild-type and mutant were similar while in over-expressing lines was up-regulated. The expression patterns of DWARF6in the mutant was significantly up-regulated but in over-expressing lines had an obvious downward trend. FE11, RAFL23, CUL1, RPT1, NPL1, HY3, LCL5, MAX2were up-regulated in both over-expressing lines and mutant, but in the mutant have a higher degree.
The genes related to flowering were analyzed by the qRT-PCR method. Both AP2and AT1G01460in mutant had increased tendency, but had no change in over-expressing lines and in wild-type. The expression level of AP1, AG, AGL2and LFY3in mutant and over-expressing lines were significantly reduced. The expression profiles for AGL9in the mutant and over-expressing lines were all significantly up-regulated. The expression pattern of FLC in mutant and wild-type had no changed but in over-expressing lines had a downward trend.
These results above suggest that Arabidopsis thaliana AT3G14067gene may also have close relationship to the plant growth and development, such as the development of hypocotyl and flowering in Arabidopsis thaliana..
引文
卜友泉,杨正梅,宋方洲,2006.新基因功能研究的策略与方法.生命科学研究10(2)：095-098.
陈善福,舒庆尧,1999.植物耐干旱胁迫的生物学机理及其基因工程研究进展.植物学通报16：555-560.
董志强,舒文华,翟学军,张保明,刘芳等,2005.棉株不同器官中几种内源激素的变化及相关关系.核农学报19：62-67.
范建平,惠玉琴,2002.光,渗透胁迫和激素对离体小麦叶片衰老的影响.青海师范大学学报(自然科学版)1：015.
郭晓瑞,唐中华,孙艳斐,李晓微,祖元刚,2008.自然干旱胁迫下长春花叶片和根部的激素和可溶性糖代谢变化.植物研究28：422-425.
胡秀丽,杨海荣,李潮海,2009.ABA对玉米响应干旱胁迫的调控机制.西北植物学报29：2345-2351.
卡恩,1990.种子萌发的生理生化,pp.北京：农业出版社.
李德全,邹琦,程炳嵩,1994.植物在水分胁迫下的渗透调节作用.作物抗旱生理生态研究.济南：山东科学技术出版社12.
李彦,张英鹏,孙明,高弼模,2008.盐分胁迫对植物的影响及植物耐盐机理研究进展.中国农学通报24.
梁蕊芳,徐龙,岳明强,2013.干旱胁迫下植物响应机制研究进展.现代农业18：066.
林莺,范海,2006.NaCl对海滨锦葵光合作用特性的效应[D],pp.山东师范大学.
刘楠梅,2000.基因功能研究方法浅介.生物技术通讯11：231-233.
彭立新,李德全,束怀瑞,2002.植物在渗透胁迫下的渗透调节作用.天津农业科学8：40-43.
芮琪,2004.小麦叶片衰老过程中内肽酶同工酶变化和特性及Rubisco降解的研究,pp.南京农业大学.
沈成国,张福锁,1998.植物叶片衰老过程中基因的表达与调控.植物生理学通讯34：304-312.
沈成国,张福锁,毛达如,1998.植物叶片衰老过程中基因的表达与调控.34：304-312.
孙小芳,郑青松,刘友良,2000NaCl胁迫对棉花种子萌发和幼苗生长的伤害.植物资源与环境学报9：22-25.
谭冬梅,许雪峰,李天忠,韩振海,2007.干旱胁迫诱导苹果属植物细胞程序性死亡的研究.园艺学报34：275-278.
汤日圣,梅传生,1997.4PU—30对水稻叶片衰老与内源激素的调控.植物生理学报(ISSN0257-4829)23：169-174.
王凡强,马美荣,王正祥,诸葛健,2000a.枯草杆菌蛋白酶的基因工程改性.微生物学通报3：018.
王凡强,马美荣,王正祥,诸葛健,2000b.枯草杆菌蛋白酶基因工程的研究进展.生物工程进展20：41-44.
王清泉,陈云,谢虹,梁建生,2004.干旱和氮素交互作用对玉米叶片水势,气孔导度及根部ABA与CTK合成的影响.中国农学通报3.
徐萌,刘清岱,朱晔荣,王勇,2008.植物蛋白酶研究进展.生物学通报43：7-9.
阳韶昆,蔡璇,邹晶晶,王彩云,2012.桂花开放与衰老过程中花瓣蛋白酶活性与种类的变化.园艺学报39：1967-1974.
杨东清,王振林,尹燕枰,倪英丽,杨卫兵等,2013.外源ABA和6-BA对不同持绿型小麦旗叶衰老的影响及其生理机制.作物学报39：1096-1104.
杨,2004.植物成花的分子调控研究进展!CHINESE JOURNAL OF TROPICAL AGRICULTURE 24.
殷奎德,胡文玉1994.离体小麦叶片衰老过程中细胞分裂素对活性氧的作用.黑龙江八一农垦大学学报4
袁政,张大兵,2002.植物叶片衰老的分子机制.植物生理学通讯38：417-422.
张华,熊运海,2000.切花衰老与保鲜技术研究综述.江西农业大学学报22(3)：455-460
张洁,方少忠,蔡宣梅,郭文杰,林真,2009.赤霉素预处理对'Sorbonne'百合切花衰老的影响[J].江西农业学报21：48-50.
张鹏,2006.黄瓜叶片衰老过程中内肽酶变化及其生化特性的研究[D],pp.南京农业大学.
赵福庚,刘友良,1999.胁迫条件下高等植物体内脯氨酸代谢及调节的研究进展.植物学通报16：540-546.
周相娟,姜微波,胡小松,周立刚,2003.赤霉素和乙烯对香菜叶片衰老的影响.北方园艺3：54-56.
朱诚,曾广文,2000.桂花花衰老过程中的某些生理生化变化.园艺学报27：356-360.
M. Aida, T. Ishida, H. Fukaki, H. Fujisawa, M. Tasaka,1997. Genes involved in organ separation in Arabidopsis:an analysis of the cup-shaped cotyledon mutant. The Plant Cell Online 9: 841-857.
C. M. Antao, F. X. Malcata,2005. Plant serine proteases:biochemical, physiological and molecular features. Plant Physiology and Biochemistry 43:637-650.
K. Arima, T. Uchikoba, H. Yonezawa, M. Shimada, M. Kaneda,2000. Isolation and characterization of a serine protease from the sprouts of Pleioblastus hindsii Nakai. Phytochemistry 54(6):559-565.
H. Attia, N. Karray, M. Rabhi, M. Lachaal,2008. Salt-imposed restrictions on the uptake of macroelements by roots of Arabidopsis thaliana. Acta Physiologiae Plantarum 30:723-727.
A. Azeez, A. P. Sane, D. Bhatnagar, P. Nath,2007. Enhanced expression of serine proteases during floral senescence in Gladiolus. Phytochemistry 68:1352-1357.
S. Balazadeh, M. Kwasniewski, C. Caldana, M. Mehrnia, M. I. Zanor, et al,2011. ORS1, an H2O2-responsive NAC transcription factor, controls senescence in Arabidopsis thaliana. Molecular plant 4(2):346-360.
A. J. Barrett, N. D. Rawlings,1995. Families and clans of serine peptidases. Archives of biochemistry and biophysics 318:247-250.
A. K. Batchelor, K. Boutilier, S. S. Miller, H. Labbe, L. Bowman et al,2000. The seed coat-specific expression of a subtilisin-like gene, SCS1, from soybean. Planta 211:484-492.
E. P. Beers, B. J. Woffenden, C. Zhao,2000. Plant proteolytic enzymes:possible roles during programmed cell death, pp.155-171 in Programmed Cell Death in Higher Plants. Springer.
V. Beilinson, O. V. Moskalenko, D. S. Livingstone, S. V. Reverdatto, et al,2002. Two subtilisin-like proteases from soybean. Physiologia Plantarum 115(4):585-597.
D. Berger, T. Altmann,2000. A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana. Genes & development 14:1119-1131.
C. A. Beveridge,2000. Long-distance signalling and a mutational analysis of branching in pea. Plant Growth Regulation 32:193-203.
G Beyene, C. H. Foyer, K. J. Kunert,2006 Two new cysteine proteinases with specific expression patterns in mature and senescent tobacco (Nicotiana tabacum L.) leaves. Journal of Experimental Botany 57(6):1431-1443
J. Booker, T. Sieberer, W. Wright, L. Williamson, B. Willett et al,2005. MAX1 Encodes a Cytochrome P450 Family Member that Acts Downstream of MAX3/4 to Produce a Carotenoid-Derived Branch-Inhibiting Hormone. Developmental cell 8:443-449.
P. F. Brode, C. R. Erwin, D. S. Rauch, B. L. Barnett, J. M. Armpriester et al.,1996. Subtilisin BPN'variants:increased hydrolytic activity on surface-bound substrates via decreased surface activity. Biochemistry 35:3162-3169.
V. Buchanan-Wollaston, C. Ainsworth,1997. Leaf senescence in Brassica napus:cloning of senescence related genes by subtractive hybridisation. Plant molecular biology 33:821-834.
M. Budic, J. Sabotic, V. Meglic, J. Kos, M. Kidric,2012. Characterization of two novel subtilases from common bean (Phaseolus vulgaris L.) and their responses to drought. Plant Physiology and Biochemistry 62:79-87
W. N. Burnette,1981. "Western blotting":electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical biochemistry 112:195-203.
W.-H. Cao, J. Liu, X.-J. He, R.-L. Mu, H.-L. Zhou et al.,2007. Modulation of ethylene responses affects plant salt-stress responses. Plant Physiology 143:707-719.
M. Chaves, J. Flexas, C. Pinheiro,2009. Photosynthesis under drought and salt stress:regulation mechanisms from whole plant to cell. Annals of Botany 103:551-560.
M. M. Chaves, J. P. Maroco, J. S. Pereira,2003. Understanding plant responses to drought-from genes to the whole plant. Functional Plant Biology 30:239-264.
H. J. Chen, C. T. Su, C. H. Lin, G. J. Huang, Y. H. Lin,2010. Expression of sweet potato cysteine protease SPCP2 altered developmental characteristics and stress responses in transgenic Arabidopsis plants. Journal of plant physiology 167(10):838-847.
T.E. Chen, D. J. Huang, Y. H. Lin YH,2004. Isolation and characterization of a serine protease from the storage roots of sweet potato (Ipomoea batatas [L.] Lam). Plant Science 166(4):1019-1026.
N. V. Chichkova, J. Shaw, R. A. Galiullina, G. E. Drury, A. I. Tuzhikov et al.,2010. Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity. The EMBO journal 29: 1149-1161.
Y.-H. Cho, S.-D. Yoo, J. Sheen,2006. Regulatory functions of nuclear hexokinase1 complex in glucose signaling. Cell 127:579-589.
E. Curotto, G. Gonzalez, S. O'reilly, G. Tapia,1989. Isolation and partial characterization of a protease from Cucurbita ficifolia. FEBS letters 243:363-365.
S. De Folter, R. G. Immink, M. Kieffer, L. Pafenicova, S. R. Henz et al.,2005. Comprehensive interaction map of the Arabidopsis MADS box transcription factors. The Plant Cell Online 17: 1424-1433.
R. Drake, I. John, A. Farrell, W. Cooper, W. Schuch, D. Grierson,1996. Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence. Plant molecular biology 30(4):755-767.
M. De Torres-Zabala, M.H. Bennett, G. Lafforgue, J.W. Mansfield, P.R. Egea et al.,2007 Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease. The EMBO Journal 26(5):1434-1443
R. S. Dhindsa, P. L. Plumb-Dhindsa, D. M. Reid,1982. Leaf senescence and lipid peroxidation: Effects of some phytohormones, and scavengers of free radicals and singlet oxygen. Physiologia Plantarum 56:453-457.
S. Distefano, J. Palma, M. Gomez, L. D. Rio,1997. Characterization of endoproteases from plant peroxisomes. Biochemical Journal 327:399.
T. Eulgem, I. E. Somssich,2007. Networks of WRKY transcription factors in defense signaling. Current opinion in plant biology 10(4):366-371.
F. Dominguez, F. J. Cejudo,1996. Characterization of the endoproteases appearing during wheat grain development. Plant physiology 112:1211-1217.
J. Flexas, J. Bota, J. Galmes, H. Medrano, M. Ribas-Carbo,2006. Keeping a positive carbon balance under adverse conditions:responses of photosynthesis and respiration to water stress. Physiologia Plantarum 127:343-352.
M. A. Frohman,1990. RACE:rapid amplification of cDNA ends. PCR protocols:A guide to methods and applications:28-38.
L. Galli-Taliadoros, J. Sedgwick, S. Wood, H. Korner,1995. Gene knock-out technology:a methodological overview for the interested novice. Journal of immunological methods 181:1-15.
S. Gan, R. M. Amasino,1995. Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270:1986-1988.
S. Gan, R. M. Amasino,1996. Cytokinins in plant senescence:from spray and pray to clone and play. Bioessays 18:557-565.
S. Gan, R. M. Amasino,1997. Making Sense of Senescence (Molecular Genetic Regulation and Manipulation of Leaf Senescence). Plant Physiology 113:313.
E. Gasteiger, A. Gattiker, C. Hoogland, I. Ivanyi, R. D. Appel et al,2003. ExPASy:the proteomics server for in-depth protein knowledge and analysis. Nucleic acids research 31: 3784-3788.
S. Gepstein, K. V. Thimann,1981. The role of ethylene in the senescence of oat leaves. Plant physiology 68:349-354.
U. Gibson, C. A. Heid, P. M. Williams,1996. A novel method for real time quantitative RT-PCR. Genome research 6:995-1001.
I. A. Graham, C. J. Leaver, S. M. Smith,1992. Induction of malate synthase gene expression in senescent and detached organs of cucumber. The Plant Cell Online 4:349-357.
N. Guex, M. C. Peitsch,1997. SWISS-MODEL and the Swiss-Pdb Viewer:An environment for comparative protein modeling. Electrophoresis 18:2714-2723.
J. M. Hamilton, D. J. Simpson, S. C. Hyman, B. K. Ndimba, A. R. Slabas,2003. Ara12 subtilisin-like protease from Arabidopsis thaliana:purification, substrate specificity and tissue localization. Biochemical Journal 370:57.
P. Hare, W. Cress,1997. Metabolic implications of stress-induced proline accumulation in plants. Plant Growth Regulation 21:79-102.
P. M. Hasegawa, R. A. Bressan, J.-K, Zhu, H. J. Bohnert,2000. Plant cellular and molecular responses to high salinity. Annual review of plant biology 51:463-499.
H. Hemmi, O. Takeuchi, T. Kawai, T. Kaisho, S. Sato et al.,2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-745.
K. HoIst, T. Schmulling, T. Werner,2011. Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation. Journal of plant physiology 168:1328-1334.
B. K. Hwang,2011. Proteomics and functional analyses of pepper abscisic acid-responsive 1 (ABR1), which is involved in cell death and defense signaling. The Plant Cell Online 23: 823-842.
H. Ikemura, H. Takagi, M. Inouye,1987. Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. Journal of Biological Chemistry 262(16):7859-7864.
L. Jean, F. Hackett, S. R. Martin, M. J. Blackman,2003. Functional characterization of the propeptide of Plasmodium falciparum subtilisin-like protease-1. Journal of Biological Chemistry 278:28572-28579.
D. Jiang, J. Li, P. Wu, Z. Liu, C. Zhuang,2009. Isolation and Characterization of a Microsporocyte-Specific Gene, OsMSP, in Rice. Plant Molecular Biology Reporter 27:469-475.
L. Jiang, J. C. Rogers,1999. Functional analysis of a Golgi-localized Kex2p-like protease in tobacco suspension culture cells. The Plant Journal 18:23-32.
W. B. Jiang, A. Lers, E. Lomaniec, N. Aharoni,1999. Senescence-related serine protease in parsley. Phytochemistry 50(3):377-382.
R. Jibran, D. A. Hunter, P. P. Dijkwel,2013. Hormonal regulation of leaf senescence through integration of developmental and stress signals. Plant molecular biology:1-15.
M. Kaneda, N. Tominaga,1975. Isolation and characterization of a proteinase from the sarcocarp of melon fruit. Journal of Biochemistry 78:1287-1296.
J. H. Kim, H. R. Woo, J. Kim, P. O. Lim, I. C. Lee et al.,2009. Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science 323(5917):1053-1057.
H. Kano, S. Taguchi, H. Momose,1997. Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis. Applied microbiology and biotechnology 47:46-51.
W. J. Kent,2002. BLAT—the BLAST-like alignment tool. Genome research 12:656-664.
M. A. Khan, R. Ansari, B. Gul, W. Li,2009. Dormancy and germination responses of halophyte seeds to the application of ethylene. Comptes Rendus Biologies 332:806-815.
H. Kinal, C.-M. Park, J. O. Berry, Y. Koltin, J. A. Bruenn,1995. Processing and secretion of a virally encoded antifungal toxin in transgenic tobacco plants:evidence for a Kex2p pathway in plants. The Plant Cell Online 7:677-688.
Z. Kong, M. Li, W. Yang, W. Xu, Y. Xue,2006. A novel nuclear-localized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice. Plant physiology 141: 1376-1388.
M. Koornneef, C. Alonso-Blanco, H. Blankestijn-De Vries, C. Hanhart, A. Peeters,1998a. Genetic interactions among late-flowering mutants of Arabidopsis. Genetics 148:885-892.
M. Koornneef, C. Alonso-Blanco, A. J. Peeters, W. Soppe,1998b. Genetic control of flowering time in Arabidopsis. Annual review of plant biology 49:345-370.
J. Kraut,1977. Serine proteases:structure and mechanism of catalysis. Annual review of biochemistry 46:331-358.
N. Kudryakova, E. Burkhanova, V. Y. Rakitin, L. Yakovleva, A. Smith et al,2001. Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants. Russian Journal of Plant Physiology 48:624-627.
E. Lam, N. Kato, M. Lawton,2001. Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411:848-853.
R. H. Lee, C. H. Wang, L. T. Huang, S. C. G Chen,2001. Leaf senescence in rice plants:cloning and characterization of senescence up-regulated genes. Journal of experimental botany 52(358):1117-1121.
A. Lers, A. Khalchitski, E. Lomaniec, S. Burd, P. J. Green,1998. Senescence-induced RNases in tomato. Plant molecular biology 36(3):439-449.
D. R. Lewis, N. D. Miller, B. L. Splitt, G Wu, E. P. Spalding,2007. Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes. The Plant Cell Online 19:1838-1850.
X.-R. Liao, P.-C. He, X.-C. Zhu,1997. Effect of Zeatin on H～ 2O～ 2 Scavenging System of Vitis vulpina Leaf Disks Under Salt Stress. ACTA BOTANICA SINICA-CHINESE EDITION- 39: 641-646.
P. O. Lim, H. J. Kim, H. Gil Nam,2007. Leaf senescence. Annu. Rev. Plant Biol.58:115-136.
J. X. Liu, R. Srivastava, P. Che, S. H. Howell,2007. Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling. The Plant Journal 51:897-909.
X. Liu, Z. Zhang, N. Barnaby, K. A. Wilson, A. Tan-Wilson,2001. Soybean subtilisin-like protease involved in initiating storage protein degradation. Seed Science Research 11:55-68.
K. J. Livak, T. D. Schmittgen,2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408.
K. N. Lohman, S. Gan, M. C. John, R. M. Amasino,1994. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiologia Plantarum 92(2):322-328.
K. N. Lohman, S. Gan, M. C. John, R. M. Amasino,1994. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiologia Plantarum 92:322-328.
R. Muller, E. C. Sisler, M. Serek,2000. Stress induced ethylene production, ethylene binding, and the response to the ethylene action inhibitor 1-MCP in miniature roses. Scientia horticulturae 83: 51-59.
S. Mahajan, N. Tuteja,2005. Cold, salinity and drought stresses:an overview. Archives of biochemistry and biophysics 444(2):139-158
Y. Matsubayashi, Y. Sakagami,2006. Peptide hormones in plants. Annu. Rev. Plant Biol.57: 649-674.
P. Mcsteen, Y. Zhao,2008. Plant hormones and signaling:common themes and new developments. Developmental cell 14:467-473.
P. Meimoun, G. Vidal, A.-S. Bohrer, A. Lehner, D. Tran et al.,2009. Intracellular ca2+ stores could participate to abscisic acid-induced depolarization and stomatal closure in Arabidopsis thaliana. Plant signaling & behavior 4:830-835.
S. D. Mishra, B. K. Gaur,1977. Role of Petiole in Protein Metabolism of Senescing Betel (Piper betle L.) Leaves. Plant Physiology 59:961-964.
I. A. Mohamed Ahmed, I. Morishima, E. E. Babiker, N. Mori,2009. Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen. Phytochemistry 70(4):483-491.
A. Mouradov, F. Cremer, G. Coupland,2002. Control of flowering time interacting pathways as a basis for diversity. The Plant Cell Online 14:S111-S130.
M. Nakazawa, N. Yabe, T. Ichikawa, Y. Y. Yamamoto, T. Yoshizumi et al.,2001. DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light response of hypocotyl length. The Plant Journal 25: 213-221.
E. Narinx, E. Baise, C. Gerday,1997. Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. Protein engineering 10:1271-1279.
R. Narsai, C. Wang, J. Chen, J. Wu, H. Shou et aL,2013. Antagonistic, overlapping and distinct responses to biotic stress in rice (Oryza sativa) and interactions with abiotic stress. BMC genomics 14:93.
L. D. Nooden, J. J. Guiamet, I. John,1997. Senescence mechanisms. Physiologia plantarum 101: 746-753.
S. A. Oh, S. Y. Lee, I. K. Chung, C. H. Lee, H. G. Nam,1996. A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence. Plant molecular biology 30(4):739-754.
T. Panavas, A. Pikula, P. D. Reid, B. Rubinstein, E. L. Walker,1999. Identification of senescence-associated genes from daylily petals. Plant molecular biology 40(2):237-248.
F. Parcy,2005. Flowering:a time for integration. International Journal of Developmental Biology 49: 585.
A. K. Parida, A. B. Das,2005. Salt tolerance and salinity effects on plants:a review. Ecotoxicology and environmental safety 60:324-349.
C.-M. Park, J. O. Berry, J. A. Bruenn,1996. High-level secretion of a virally encoded anti-fungal toxin in transgenic tobacco plants. Plant molecular biology 30:359-366.
D. L. Parrott, J. M. Martin, A. M. Fischer,2010. Analysis of barley (Hordeum vulgare) leaf senescence and protease gene expression:a family CIA cysteine protease is specifically induced under conditions characterized by high1 carbohydrate, but low to moderate nitrogen levels. New Phytologist 187(2):313-331.
D. L. Parrott, K. Mclnnerney, U. Feller, A. M. Fischer,2007. Steam-girdling of barley (Hordeum vulgare) leaves leads to carbohydrate accumulation and accelerated leaf senescence, facilitating transcriptomic analysis of senescence-associated genes. New Phytologist 176(l):56-69
J. J. Perona, C. S. Craik,1995. Structural basis of substrate specificity in the serine proteases. Protein Science 4:337-360.
T. Popovic, V. Puizdar, J. Brzin,2002. A novel subtilase from common bean leaves. FEBS letters 530:163-168.
H. R. Powell,1999. The Rossmann Fourier autoindexing algorithm in MOSFLM. Acta Crystallogr D Biol Crystallogr 55:1690-1695.
A. Prins, P.D. Van Heerden, E. Olmos, K. J. Kunert, C. H. Foyer,2008. Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves:a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies. Journal of experimental botany 59(7):1935-1950.
C. Rautengarten, D. Steinhauser, D. Biissis, A. Stintzi, A. Schaller et al.,2005. Inferring hypotheses on functional relationships of genes:analysis of the Arabidopsis thaliana subtilase gene family. PLoS computational biology 1:e40.
N. D. Rawlings, A. J. Barrett,1994. [2] Families of serine peptidases. Methods in enzymology 244: 19-61.
I. N. Roberts, C. Caputo, M. Kade, M. V. Criado, A. J. Barneix,2011. Subtilisin-like serine proteases involved in N remobilization during grain filling in wheat. Acta Physiologiae Plantarum33:1997-2001.
I. N. Roberts, P. F. Murray, C. P. Caputo, S. Passeron, A. J. Barneix,2003. Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leaves. Physiologia Plantarum 118:483-490.
I. N. Roberts, S. Passeron, A. J. Barneix,2006. The two main endoproteases present in dark-induced senescent wheat leaves are distinct subtilisin-like proteases. Planta 224:1437-1447.
I. Romero, A. Fuertes, M. Benito, J. Malpica, A. Leyva, J. Paz-Ares,1998. More than 80R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana. Plant journal 14(3):273-284.
S.B. Ryu, X. Wang,1995. Expression of phospholipase D during castor bean leaf senescence. Plant physiology 108(2):713-719.
M. Sajid, C. Withers-Martinez, M. J. Blackman,2000. Maturation and specificity of Plasmodium falciparum subtilisin-like protease-1, a malaria merozoite subtilisin-like serine protease. Journal of Biological Chemistry 275:631-641.
A. Santner, L. I. A. Calderon-Villalobos, M. Estelle,2009. Plant hormones are versatile chemical regulators of plant growth. Nature chemical biology 5:301-307.
A. Schaller,2004. A cut above the rest:the regulatory function of plant proteases. Planta 220: 183-197.
A. Schaller, C. A. Ryan,1994. Identification of a 50-kDa systemin-binding protein in tomato plasma membranes having Kex2p-like properties. Proceedings of the National Academy of Sciences 91: 11802-11806.
A. Schaller, A. Stintzi, L. Graff,2012. Subtilases-versatile tools for protein turnover, plant development, and interactions with the environment. Physiologia Plantarum 145:52-66.
G.-Z. Shen, X.-Y. Liu, S.-K. Shen, Y.-R. Wu, Y.-H. Wang,2008. Effect of 6-BA and NAA on Germination Characteristics of Anneslea fragrans Wall Seed [J]. Seed 3:024.
K. I. Shennan, N. A. Taylor, J. L. Jermany, G Matthews, K. Docherty,1995. Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events. Journal of Biological Chemistry 270: 1402-1407.
U. Shinde, M. Inouye,1995. Folding Mediated by an Intramolecular Chaperone:Autoprocessing Pathway of the Precursor Resolved viaa Substrate Assisted Catalysis Mechanism. Journal of molecular biology 247(3):390-395.
U. Shinde, Y. Li, S. Chatterjee, M. Inouye,1993. Folding pathway mediated by an intramolecular chaperone. Proceedings of the National Academy of Sciences 90:6924-6928.
R. J. Siezen, J. A. Leunissen,1997. Subtilases:the superfamily of subtilisin-like serine proteases. Protein Science 6:501-523.
G G Simpson, C. Dean,2002. Arabidopsis, the Rosetta stone of flowering time? Science 296: 285-289.
C. M. Smart,1994. Tansley review no.64. Gene expression during leaf senescence. New Phytologist: 419-448.
Y. Song, C. Zhang, W. Ge, Y. Zhang, A. L. Burlingame et al,2011. Identification of NaCl stress-responsive apoplastic proteins in rice shoot stems by 2D-DIGE. Journal of proteomics 74: 1045-1067.
C. M. Spickett, N. Smirnoff, R. G Ratcliffe,1992. Metabolic Response of Maize Roots to Hyperosmotic Shock An in Vivo 31P Nuclear Magnetic Resonance Study. Plant physiology 99: 856-863.
R. Srivastava, J. X. Liu, S. H. Howell,2008. Proteolytic processing of a precursor protein for a growth-promoting peptide by a subtilisin serine protease in Arabidopsis. The Plant Journal 56: 219-227.
S. Taguchi, A. Ozaki, H. Momose,1998. Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system. Applied and environmental microbiology 64: 492-495.
T. Tajima, A. Yamaguchi, S. Matsushima, M. Satoh, S. Hayasaka, K. Yoshimatsu, Y. Shioi,2011. Biochemical and molecular characterization of senescence-related cysteine protease-cystatin complex from spinach leaf. Physiologia plantarum 141(2):97-116.
H. Tanaka, H. Onouchi, M. Kondo, I. Hara-Nishimura, M. Nishimura et al,2001. A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants. Development 128:4681-4689.
J. Tao, I. Ginsberg, N. Banerjee, W. Held, Y. Koltin et al.,1990. Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins. Molecular and cellular biology 10:1373-1381.
L. P. Tripathi, R. Sowdhamini,2006. Cross genome comparisons of serine proteases in Arabidopsis and rice. BMC genomics 7:200.
N. Tuteja,2007. Abscisic acid and abiotic stress signaling. Plant signaling & behavior 2:135-138.
C. Uauy, A. Distelfeld, T. Fahima, A. Blechl, J. Dubcovsky,2006. A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314(5803):1298-1301.
R. A. Van Der Hoorn,2008. Plant proteases:from phenotypes to molecular mechanisms. Annu. Rev. Plant Biol.59:191-223.
R. D. Vierstra,1996. Proteolysis in plants:mechanisms and functions. Plant molecular biology 32: 275-302.
R. D. Vierstra,2003. The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends in plant science 8:135-142.
D. Wagner, R. W. Sablowski, E. M. Meyerowitz,1999. Transcriptional activation of APETALA1 by LEAFY. Science 285:582-584.
R. Wang, S. Liu, J. Wang, Q. Dong, L. Xu et al.,2013. Purification, characterization and identification of a senescence related serine protease in dark-induced senescent wheat leaves. Phytochemistry.95:118-26
Y. Wang, K. Li, X. Li,2009. Auxin redistribution modulates plastic development of root system architecture under salt stress in Arabidopsis thaliana. Journal of plant physiology 166: 1637-1645.
Y. T. Wang, C. Y. Yang, Y.-T. Chen, Y. Lin, J.-F. Shaw,2004. Characterization of senescence-associated proteases in postharvest broccoli florets. Plant Physiology and Biochemistry 42:663-670.
L. M. Weaver, R. M. Amasino,2001. Senescence is induced in individually darkened Arabidopsis leaves, but inhibited in whole darkened plants. Plant Physiology 127:876-886.
A.-L. Wei, Y.-Z. Chen,2000. Effect of IAA on soybean seedling's membrance injury and salt resistance. ACTA BOTANICA BOREALI OCCIDENTALIA SINICA 20:410-414.
F.-P. Wen, Z.-H. Zhang, T. Bai, Q. Xu, Y.-H. Pan,2010. Proteomics reveals the effects of gibberellic acid (GA3) on salt-stressed rice (Oryza sativa L.) shoots. Plant Science 178: 170-175.
G C. Whitelam, E. Johnson, J. Peng, P. Carol, M. L. Anderson et al.,1993. Phytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light. The Plant Cell Online 5: 757-768.
K. Xu, K. Griebenow, A. M. Klibanov,1997. Correlation between catalytic activity and secondary structure of subtilisin dissolved in organic solvents. Biotechnology and bioengineering 56: 485-491.
H. Yamagata, T. Masuzawa, Y. Nagaoka, T. Ohnishi, T. Iwasaki,1994. Cucumisin, a serine protease from melon fruits, shares structural homology with subtilisin and is generated from a large precursor. Journal of Biological Chemistry 269:32725-32731.
H. Yamagata, S. Ueno, T. Iwasaki,1989. Isolation and characterization of a possible native cucumisin from developing melon fruits and its limited autolysis to cucumisin. Agricultural and biological chemistry 53:1009-1017.
K. Yamaguchi-Shinozaki, K. Shinozaki,1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. The Plant Cell Online 6(2):251-264.
Y. Yamaoka, Y. Ohba, M. Takeuchi, Y. Morohashi,1993. Isolation and properties of a carboxypeptidase from cotyledons of germinated mung bean seeds. Plant Science 95:1-7.
Y. Yang, Y. Wu, J. Pirrello, F. Regad, M. Bouzayen et al,2010. Silencing Sl-EBF1 and Sl-EBF2 expression causes constitutive ethylene response phenotype, accelerated plant senescence, and fruit ripening in tomato. Journal of experimental botany 61:697-708.
C. Yeh, H. Chang, H. Hsieh, K. Yoda, M. Yamasaki et al,1997. Improved translational efficiency of subtilisin YaB gene with different initiation codons in Bacillus subtilis and alkalophilic Bacillus YaB. Journal of applied microbiology 83:758-763.
K. T. Yoshida, T. Kuboyama,2001. A subtilisin-like serine protease specifically expressed in reproductive organs in rice. Sexual Plant Reproduction 13:193-199.
K. Yu, Y. Wang, J. Wei, Q. Ma, D. Yu et al,2009. Improving rhizome yield and quality of Paris polyphylla through gibberellic acid-induced retardation of senescence of aerial parts. Plant signaling & behavior 4:413-415.
J. Zeevaart, R. Creelman,1988 Metabolism and physiology of abscisic acid. Annual review of plant physiology and plant molecular biology 39(1):439-473
J. Zhang, W. Jia, J. Yang, A. M. Ismail,2006. Role of ABA in integrating plant responses to drought and salt stresses. Field Crops Research 97:111-119.
J.-K. Zhu,2002. Salt and drought stress signal transduction in plants. Annual review of plant biology 53:247.
陈善福,舒庆尧,1999.植物耐干旱胁迫的生物学机理及其基因工程研究进展.植物学通报16：555-560.
董志强,舒文华,翟学军,张保明,刘芳等,2005.棉株不同器官中几种内源激素的变化及相关关系.核农学报19：62-67.
范建平,惠玉琴,2002.光,渗透胁迫和激素对离体小麦叶片衰老的影响.青海师范大学学报(自然科学版)1：015.
郭晓瑞,唐中华,孙艳斐,李晓微,祖元刚,2008.自然干旱胁迫下长春花叶片和根部的激素和可溶性糖代谢变化.植物研究28：422-425.
胡秀丽,杨海荣,李潮海,2009.ABA对玉米响应干旱胁迫的调控机制.西北植物学报29：2345-2351.
卡恩,1990.种子萌发的生理生化,pp.北京：农业出版社.
李德全,邹琦,程炳嵩,1994.植物在水分胁迫下的渗透调节作用.作物抗旱生理生态研究.济南：山东科学技术出版社12.
李彦,张英鹏,孙明,高弼模,2008.盐分胁迫对植物的影响及植物耐盐机理研究进展.中国农学通报24.
梁蕊芳,徐龙,岳明强,2013.干旱胁迫下植物响应机制研究进展.现代农业18：066.
林莺,范海,2006.NaCl对海滨锦葵光合作用特性的效应[D],pp.山东师范大学.
刘楠梅,2000.基因功能研究方法浅介.生物技术通讯11：231-233.
彭立新,李德全,束怀瑞,2002.植物在渗透胁迫下的渗透调节作用.天津农业科学8：40-43.
芮琪,2004.小麦叶片衰老过程中内肽酶同工酶变化和特性及Rubisco降解的研究,pp.南京农业大学.
沈成国,张福锁,1998.植物叶片衰老过程中基因的表达与调控.植物生理学通讯34：304-312.
沈成国,张福锁,毛达如,1998.植物叶片衰老过程中基因的表达与调控.34：304-312.
孙小芳,郑青松,刘友良,2000NaCl胁迫对棉花种子萌发和幼苗生长的伤害.植物资源与环境学报9：22-25.
谭冬梅,许雪峰,李天忠,韩振海,2007.干旱胁迫诱导苹果属植物细胞程序性死亡的研究.园艺学报34：275-278.
汤日圣,梅传生,1997.4PU—30对水稻叶片衰老与内源激素的调控.植物生理学报(ISSN0257-4829)23：169-174.
王凡强,马美荣,王正祥,诸葛健,2000a.枯草杆菌蛋白酶的基因工程改性.微生物学通报3：018.
王凡强,马美荣,王正祥,诸葛健,2000b.枯草杆菌蛋白酶基因工程的研究进展.生物工程进展20：41-44.
王清泉,陈云,谢虹,梁建生,2004.干旱和氮素交互作用对玉米叶片水势,气孔导度及根部ABA与CTK合成的影响.中国农学通报3.
徐萌,刘清岱,朱晔荣,王勇,2008.植物蛋白酶研究进展.生物学通报43：7-9.
阳韶昆,蔡璇,邹晶晶,王彩云,2012.桂花开放与衰老过程中花瓣蛋白酶活性与种类的变化.园艺学报39：1967-1974.
杨东清,王振林,尹燕枰,倪英丽,杨卫兵等,2013.外源ABA和6-BA对不同持绿型小麦旗叶衰老的影响及其生理机制.作物学报39：1096-1104.
杨,2004.植物成花的分子调控研究进展!CHINESE JOURNAL OF TROPICAL AGRICULTURE 24.
殷奎德,胡文玉1994.离体小麦叶片衰老过程中细胞分裂素对活性氧的作用.黑龙江八一农垦大学学报4
袁政,张大兵,2002.植物叶片衰老的分子机制.植物生理学通讯38：417-422.
张华,熊运海,2000.切花衰老与保鲜技术研究综述.江西农业大学学报22(3)：455-460
张洁,方少忠,蔡宣梅,郭文杰,林真,2009.赤霉素预处理对'Sorbonne'百合切花衰老的影响[J].江西农业学报21：48-50.
张鹏,2006.黄瓜叶片衰老过程中内肽酶变化及其生化特性的研究[D],pp.南京农业大学.
赵福庚,刘友良,1999.胁迫条件下高等植物体内脯氨酸代谢及调节的研究进展.植物学通报16：540-546.
周相娟,姜微波,胡小松,周立刚,2003.赤霉素和乙烯对香菜叶片衰老的影响.北方园艺3：54-56.
朱诚,曾广文,2000.桂花花衰老过程中的某些生理生化变化.园艺学报27：356-360.
M. Aida, T. Ishida, H. Fukaki, H. Fujisawa, M. Tasaka,1997. Genes involved in organ separation in Arabidopsis:an analysis of the cup-shaped cotyledon mutant. The Plant Cell Online 9: 841-857.
C. M. Antao, F. X. Malcata,2005. Plant serine proteases:biochemical, physiological and molecular features. Plant Physiology and Biochemistry 43:637-650.
K. Arima, T. Uchikoba, H. Yonezawa, M. Shimada, M. Kaneda,2000. Isolation and characterization of a serine protease from the sprouts of Pleioblastus hindsii Nakai. Phytochemistry 54(6):559-565.
H. Attia, N. Karray, M. Rabhi, M. Lachaal,2008. Salt-imposed restrictions on the uptake of macroelements by roots of Arabidopsis thaliana. Acta Physiologiae Plantarum 30:723-727.
A. Azeez, A. P. Sane, D. Bhatnagar, P. Nath,2007. Enhanced expression of serine proteases during floral senescence in Gladiolus. Phytochemistry 68:1352-1357.
S. Balazadeh, M. Kwasniewski, C. Caldana, M. Mehrnia, M. I. Zanor, et al,2011. ORS1, an H2O2-responsive NAC transcription factor, controls senescence in Arabidopsis thaliana. Molecular plant 4(2):346-360.
A. J. Barrett, N. D. Rawlings,1995. Families and clans of serine peptidases. Archives of biochemistry and biophysics 318:247-250.
A. K. Batchelor, K. Boutilier, S. S. Miller, H. Labbe, L. Bowman et al,2000. The seed coat-specific expression of a subtilisin-like gene, SCS1, from soybean. Planta 211:484-492.
E. P. Beers, B. J. Woffenden, C. Zhao,2000. Plant proteolytic enzymes:possible roles during programmed cell death, pp.155-171 in Programmed Cell Death in Higher Plants. Springer.
V. Beilinson, O. V. Moskalenko, D. S. Livingstone, S. V. Reverdatto, et al,2002. Two subtilisin-like proteases from soybean. Physiologia Plantarum 115(4):585-597.
D. Berger, T. Altmann,2000. A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana. Genes & development 14:1119-1131.
C. A. Beveridge,2000. Long-distance signalling and a mutational analysis of branching in pea. Plant Growth Regulation 32:193-203.
G Beyene, C. H. Foyer, K. J. Kunert,2006 Two new cysteine proteinases with specific expression patterns in mature and senescent tobacco (Nicotiana tabacum L.) leaves. Journal of Experimental Botany 57(6):1431-1443
J. Booker, T. Sieberer, W. Wright, L. Williamson, B. Willett et al,2005. MAX1 Encodes a Cytochrome P450 Family Member that Acts Downstream of MAX3/4 to Produce a Carotenoid-Derived Branch-Inhibiting Hormone. Developmental cell 8:443-449.
P. F. Brode, C. R. Erwin, D. S. Rauch, B. L. Barnett, J. M. Armpriester et al.,1996. Subtilisin BPN'variants:increased hydrolytic activity on surface-bound substrates via decreased surface activity. Biochemistry 35:3162-3169.
V. Buchanan-Wollaston, C. Ainsworth,1997. Leaf senescence in Brassica napus:cloning of senescence related genes by subtractive hybridisation. Plant molecular biology 33:821-834.
M. Budic, J. Sabotic, V. Meglic, J. Kos, M. Kidric,2012. Characterization of two novel subtilases from common bean (Phaseolus vulgaris L.) and their responses to drought. Plant Physiology and Biochemistry 62:79-87
W. N. Burnette,1981. "Western blotting":electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical biochemistry 112:195-203.
W.-H. Cao, J. Liu, X.-J. He, R.-L. Mu, H.-L. Zhou et al.,2007. Modulation of ethylene responses affects plant salt-stress responses. Plant Physiology 143:707-719.
M. Chaves, J. Flexas, C. Pinheiro,2009. Photosynthesis under drought and salt stress:regulation mechanisms from whole plant to cell. Annals of Botany 103:551-560.
M. M. Chaves, J. P. Maroco, J. S. Pereira,2003. Understanding plant responses to drought-from genes to the whole plant. Functional Plant Biology 30:239-264.
H. J. Chen, C. T. Su, C. H. Lin, G. J. Huang, Y. H. Lin,2010. Expression of sweet potato cysteine protease SPCP2 altered developmental characteristics and stress responses in transgenic Arabidopsis plants. Journal of plant physiology 167(10):838-847.
T.E. Chen, D. J. Huang, Y. H. Lin YH,2004. Isolation and characterization of a serine protease from the storage roots of sweet potato (Ipomoea batatas [L.] Lam). Plant Science 166(4):1019-1026.
N. V. Chichkova, J. Shaw, R. A. Galiullina, G. E. Drury, A. I. Tuzhikov et al.,2010. Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity. The EMBO journal 29: 1149-1161.
Y.-H. Cho, S.-D. Yoo, J. Sheen,2006. Regulatory functions of nuclear hexokinase1 complex in glucose signaling. Cell 127:579-589.
E. Curotto, G. Gonzalez, S. O'reilly, G. Tapia,1989. Isolation and partial characterization of a protease from Cucurbita ficifolia. FEBS letters 243:363-365.
S. De Folter, R. G. Immink, M. Kieffer, L. Pafenicova, S. R. Henz et al.,2005. Comprehensive interaction map of the Arabidopsis MADS box transcription factors. The Plant Cell Online 17: 1424-1433.
R. Drake, I. John, A. Farrell, W. Cooper, W. Schuch, D. Grierson,1996. Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence. Plant molecular biology 30(4):755-767.
M. De Torres-Zabala, M.H. Bennett, G. Lafforgue, J.W. Mansfield, P.R. Egea et al.,2007 Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease. The EMBO Journal 26(5):1434-1443
R. S. Dhindsa, P. L. Plumb-Dhindsa, D. M. Reid,1982. Leaf senescence and lipid peroxidation: Effects of some phytohormones, and scavengers of free radicals and singlet oxygen. Physiologia Plantarum 56:453-457.
S. Distefano, J. Palma, M. Gomez, L. D. Rio,1997. Characterization of endoproteases from plant peroxisomes. Biochemical Journal 327:399.
T. Eulgem, I. E. Somssich,2007. Networks of WRKY transcription factors in defense signaling. Current opinion in plant biology 10(4):366-371.
F. Dominguez, F. J. Cejudo,1996. Characterization of the endoproteases appearing during wheat grain development. Plant physiology 112:1211-1217.
J. Flexas, J. Bota, J. Galmes, H. Medrano, M. Ribas-Carbo,2006. Keeping a positive carbon balance under adverse conditions:responses of photosynthesis and respiration to water stress. Physiologia Plantarum 127:343-352.
M. A. Frohman,1990. RACE:rapid amplification of cDNA ends. PCR protocols:A guide to methods and applications:28-38.
L. Galli-Taliadoros, J. Sedgwick, S. Wood, H. Korner,1995. Gene knock-out technology:a methodological overview for the interested novice. Journal of immunological methods 181:1-15.
S. Gan, R. M. Amasino,1995. Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270:1986-1988.
S. Gan, R. M. Amasino,1996. Cytokinins in plant senescence:from spray and pray to clone and play. Bioessays 18:557-565.
S. Gan, R. M. Amasino,1997. Making Sense of Senescence (Molecular Genetic Regulation and Manipulation of Leaf Senescence). Plant Physiology 113:313.
E. Gasteiger, A. Gattiker, C. Hoogland, I. Ivanyi, R. D. Appel et al,2003. ExPASy:the proteomics server for in-depth protein knowledge and analysis. Nucleic acids research 31: 3784-3788.
S. Gepstein, K. V. Thimann,1981. The role of ethylene in the senescence of oat leaves. Plant physiology 68:349-354.
U. Gibson, C. A. Heid, P. M. Williams,1996. A novel method for real time quantitative RT-PCR. Genome research 6:995-1001.
I. A. Graham, C. J. Leaver, S. M. Smith,1992. Induction of malate synthase gene expression in senescent and detached organs of cucumber. The Plant Cell Online 4:349-357.
N. Guex, M. C. Peitsch,1997. SWISS-MODEL and the Swiss-Pdb Viewer:An environment for comparative protein modeling. Electrophoresis 18:2714-2723.
J. M. Hamilton, D. J. Simpson, S. C. Hyman, B. K. Ndimba, A. R. Slabas,2003. Ara12 subtilisin-like protease from Arabidopsis thaliana:purification, substrate specificity and tissue localization. Biochemical Journal 370:57.
P. Hare, W. Cress,1997. Metabolic implications of stress-induced proline accumulation in plants. Plant Growth Regulation 21:79-102.
P. M. Hasegawa, R. A. Bressan, J.-K, Zhu, H. J. Bohnert,2000. Plant cellular and molecular responses to high salinity. Annual review of plant biology 51:463-499.
H. Hemmi, O. Takeuchi, T. Kawai, T. Kaisho, S. Sato et al.,2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-745.
K. HoIst, T. Schmulling, T. Werner,2011. Enhanced cytokinin degradation in leaf primordia of transgenic Arabidopsis plants reduces leaf size and shoot organ primordia formation. Journal of plant physiology 168:1328-1334.
B. K. Hwang,2011. Proteomics and functional analyses of pepper abscisic acid-responsive 1 (ABR1), which is involved in cell death and defense signaling. The Plant Cell Online 23: 823-842.
H. Ikemura, H. Takagi, M. Inouye,1987. Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. Journal of Biological Chemistry 262(16):7859-7864.
L. Jean, F. Hackett, S. R. Martin, M. J. Blackman,2003. Functional characterization of the propeptide of Plasmodium falciparum subtilisin-like protease-1. Journal of Biological Chemistry 278:28572-28579.
D. Jiang, J. Li, P. Wu, Z. Liu, C. Zhuang,2009. Isolation and Characterization of a Microsporocyte-Specific Gene, OsMSP, in Rice. Plant Molecular Biology Reporter 27:469-475.
L. Jiang, J. C. Rogers,1999. Functional analysis of a Golgi-localized Kex2p-like protease in tobacco suspension culture cells. The Plant Journal 18:23-32.
W. B. Jiang, A. Lers, E. Lomaniec, N. Aharoni,1999. Senescence-related serine protease in parsley. Phytochemistry 50(3):377-382.
R. Jibran, D. A. Hunter, P. P. Dijkwel,2013. Hormonal regulation of leaf senescence through integration of developmental and stress signals. Plant molecular biology:1-15.
M. Kaneda, N. Tominaga,1975. Isolation and characterization of a proteinase from the sarcocarp of melon fruit. Journal of Biochemistry 78:1287-1296.
J. H. Kim, H. R. Woo, J. Kim, P. O. Lim, I. C. Lee et al.,2009. Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science 323(5917):1053-1057.
H. Kano, S. Taguchi, H. Momose,1997. Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis. Applied microbiology and biotechnology 47:46-51.
W. J. Kent,2002. BLAT—the BLAST-like alignment tool. Genome research 12:656-664.
M. A. Khan, R. Ansari, B. Gul, W. Li,2009. Dormancy and germination responses of halophyte seeds to the application of ethylene. Comptes Rendus Biologies 332:806-815.
H. Kinal, C.-M. Park, J. O. Berry, Y. Koltin, J. A. Bruenn,1995. Processing and secretion of a virally encoded antifungal toxin in transgenic tobacco plants:evidence for a Kex2p pathway in plants. The Plant Cell Online 7:677-688.
Z. Kong, M. Li, W. Yang, W. Xu, Y. Xue,2006. A novel nuclear-localized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice. Plant physiology 141: 1376-1388.
M. Koornneef, C. Alonso-Blanco, H. Blankestijn-De Vries, C. Hanhart, A. Peeters,1998a. Genetic interactions among late-flowering mutants of Arabidopsis. Genetics 148:885-892.
M. Koornneef, C. Alonso-Blanco, A. J. Peeters, W. Soppe,1998b. Genetic control of flowering time in Arabidopsis. Annual review of plant biology 49:345-370.
J. Kraut,1977. Serine proteases:structure and mechanism of catalysis. Annual review of biochemistry 46:331-358.
N. Kudryakova, E. Burkhanova, V. Y. Rakitin, L. Yakovleva, A. Smith et al,2001. Ethylene and Cytokinin in the Control of Senescence in Detached Leaves of Arabidopsis thaliana eti-5Mutant and Wild-Type Plants. Russian Journal of Plant Physiology 48:624-627.
E. Lam, N. Kato, M. Lawton,2001. Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411:848-853.
R. H. Lee, C. H. Wang, L. T. Huang, S. C. G Chen,2001. Leaf senescence in rice plants:cloning and characterization of senescence up-regulated genes. Journal of experimental botany 52(358):1117-1121.
A. Lers, A. Khalchitski, E. Lomaniec, S. Burd, P. J. Green,1998. Senescence-induced RNases in tomato. Plant molecular biology 36(3):439-449.
D. R. Lewis, N. D. Miller, B. L. Splitt, G Wu, E. P. Spalding,2007. Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes. The Plant Cell Online 19:1838-1850.
X.-R. Liao, P.-C. He, X.-C. Zhu,1997. Effect of Zeatin on H～ 2O～ 2 Scavenging System of Vitis vulpina Leaf Disks Under Salt Stress. ACTA BOTANICA SINICA-CHINESE EDITION- 39: 641-646.
P. O. Lim, H. J. Kim, H. Gil Nam,2007. Leaf senescence. Annu. Rev. Plant Biol.58:115-136.
J. X. Liu, R. Srivastava, P. Che, S. H. Howell,2007. Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling. The Plant Journal 51:897-909.
X. Liu, Z. Zhang, N. Barnaby, K. A. Wilson, A. Tan-Wilson,2001. Soybean subtilisin-like protease involved in initiating storage protein degradation. Seed Science Research 11:55-68.
K. J. Livak, T. D. Schmittgen,2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408.
K. N. Lohman, S. Gan, M. C. John, R. M. Amasino,1994. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiologia Plantarum 92(2):322-328.
K. N. Lohman, S. Gan, M. C. John, R. M. Amasino,1994. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiologia Plantarum 92:322-328.
R. Muller, E. C. Sisler, M. Serek,2000. Stress induced ethylene production, ethylene binding, and the response to the ethylene action inhibitor 1-MCP in miniature roses. Scientia horticulturae 83: 51-59.
S. Mahajan, N. Tuteja,2005. Cold, salinity and drought stresses:an overview. Archives of biochemistry and biophysics 444(2):139-158
Y. Matsubayashi, Y. Sakagami,2006. Peptide hormones in plants. Annu. Rev. Plant Biol.57: 649-674.
P. Mcsteen, Y. Zhao,2008. Plant hormones and signaling:common themes and new developments. Developmental cell 14:467-473.
P. Meimoun, G. Vidal, A.-S. Bohrer, A. Lehner, D. Tran et al.,2009. Intracellular ca2+ stores could participate to abscisic acid-induced depolarization and stomatal closure in Arabidopsis thaliana. Plant signaling & behavior 4:830-835.
S. D. Mishra, B. K. Gaur,1977. Role of Petiole in Protein Metabolism of Senescing Betel (Piper betle L.) Leaves. Plant Physiology 59:961-964.
I. A. Mohamed Ahmed, I. Morishima, E. E. Babiker, N. Mori,2009. Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen. Phytochemistry 70(4):483-491.
A. Mouradov, F. Cremer, G. Coupland,2002. Control of flowering time interacting pathways as a basis for diversity. The Plant Cell Online 14:S111-S130.
M. Nakazawa, N. Yabe, T. Ichikawa, Y. Y. Yamamoto, T. Yoshizumi et al.,2001. DFL1, an auxin-responsive GH3 gene homologue, negatively regulates shoot cell elongation and lateral root formation, and positively regulates the light response of hypocotyl length. The Plant Journal 25: 213-221.
E. Narinx, E. Baise, C. Gerday,1997. Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold. Protein engineering 10:1271-1279.
R. Narsai, C. Wang, J. Chen, J. Wu, H. Shou et aL,2013. Antagonistic, overlapping and distinct responses to biotic stress in rice (Oryza sativa) and interactions with abiotic stress. BMC genomics 14:93.
L. D. Nooden, J. J. Guiamet, I. John,1997. Senescence mechanisms. Physiologia plantarum 101: 746-753.
S. A. Oh, S. Y. Lee, I. K. Chung, C. H. Lee, H. G. Nam,1996. A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence. Plant molecular biology 30(4):739-754.
T. Panavas, A. Pikula, P. D. Reid, B. Rubinstein, E. L. Walker,1999. Identification of senescence-associated genes from daylily petals. Plant molecular biology 40(2):237-248.
F. Parcy,2005. Flowering:a time for integration. International Journal of Developmental Biology 49: 585.
A. K. Parida, A. B. Das,2005. Salt tolerance and salinity effects on plants:a review. Ecotoxicology and environmental safety 60:324-349.
C.-M. Park, J. O. Berry, J. A. Bruenn,1996. High-level secretion of a virally encoded anti-fungal toxin in transgenic tobacco plants. Plant molecular biology 30:359-366.
D. L. Parrott, J. M. Martin, A. M. Fischer,2010. Analysis of barley (Hordeum vulgare) leaf senescence and protease gene expression:a family CIA cysteine protease is specifically induced under conditions characterized by high1 carbohydrate, but low to moderate nitrogen levels. New Phytologist 187(2):313-331.
D. L. Parrott, K. Mclnnerney, U. Feller, A. M. Fischer,2007. Steam-girdling of barley (Hordeum vulgare) leaves leads to carbohydrate accumulation and accelerated leaf senescence, facilitating transcriptomic analysis of senescence-associated genes. New Phytologist 176(l):56-69
J. J. Perona, C. S. Craik,1995. Structural basis of substrate specificity in the serine proteases. Protein Science 4:337-360.
T. Popovic, V. Puizdar, J. Brzin,2002. A novel subtilase from common bean leaves. FEBS letters 530:163-168.
H. R. Powell,1999. The Rossmann Fourier autoindexing algorithm in MOSFLM. Acta Crystallogr D Biol Crystallogr 55:1690-1695.
A. Prins, P.D. Van Heerden, E. Olmos, K. J. Kunert, C. H. Foyer,2008. Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves:a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies. Journal of experimental botany 59(7):1935-1950.
C. Rautengarten, D. Steinhauser, D. Biissis, A. Stintzi, A. Schaller et al.,2005. Inferring hypotheses on functional relationships of genes:analysis of the Arabidopsis thaliana subtilase gene family. PLoS computational biology 1:e40.
N. D. Rawlings, A. J. Barrett,1994. [2] Families of serine peptidases. Methods in enzymology 244: 19-61.
I. N. Roberts, C. Caputo, M. Kade, M. V. Criado, A. J. Barneix,2011. Subtilisin-like serine proteases involved in N remobilization during grain filling in wheat. Acta Physiologiae Plantarum33:1997-2001.
I. N. Roberts, P. F. Murray, C. P. Caputo, S. Passeron, A. J. Barneix,2003. Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leaves. Physiologia Plantarum 118:483-490.
I. N. Roberts, S. Passeron, A. J. Barneix,2006. The two main endoproteases present in dark-induced senescent wheat leaves are distinct subtilisin-like proteases. Planta 224:1437-1447.
I. Romero, A. Fuertes, M. Benito, J. Malpica, A. Leyva, J. Paz-Ares,1998. More than 80R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana. Plant journal 14(3):273-284.
S.B. Ryu, X. Wang,1995. Expression of phospholipase D during castor bean leaf senescence. Plant physiology 108(2):713-719.
M. Sajid, C. Withers-Martinez, M. J. Blackman,2000. Maturation and specificity of Plasmodium falciparum subtilisin-like protease-1, a malaria merozoite subtilisin-like serine protease. Journal of Biological Chemistry 275:631-641.
A. Santner, L. I. A. Calderon-Villalobos, M. Estelle,2009. Plant hormones are versatile chemical regulators of plant growth. Nature chemical biology 5:301-307.
A. Schaller,2004. A cut above the rest:the regulatory function of plant proteases. Planta 220: 183-197.
A. Schaller, C. A. Ryan,1994. Identification of a 50-kDa systemin-binding protein in tomato plasma membranes having Kex2p-like properties. Proceedings of the National Academy of Sciences 91: 11802-11806.
A. Schaller, A. Stintzi, L. Graff,2012. Subtilases-versatile tools for protein turnover, plant development, and interactions with the environment. Physiologia Plantarum 145:52-66.
G.-Z. Shen, X.-Y. Liu, S.-K. Shen, Y.-R. Wu, Y.-H. Wang,2008. Effect of 6-BA and NAA on Germination Characteristics of Anneslea fragrans Wall Seed [J]. Seed 3:024.
K. I. Shennan, N. A. Taylor, J. L. Jermany, G Matthews, K. Docherty,1995. Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events. Journal of Biological Chemistry 270: 1402-1407.
U. Shinde, M. Inouye,1995. Folding Mediated by an Intramolecular Chaperone:Autoprocessing Pathway of the Precursor Resolved viaa Substrate Assisted Catalysis Mechanism. Journal of molecular biology 247(3):390-395.
U. Shinde, Y. Li, S. Chatterjee, M. Inouye,1993. Folding pathway mediated by an intramolecular chaperone. Proceedings of the National Academy of Sciences 90:6924-6928.
R. J. Siezen, J. A. Leunissen,1997. Subtilases:the superfamily of subtilisin-like serine proteases. Protein Science 6:501-523.
G G Simpson, C. Dean,2002. Arabidopsis, the Rosetta stone of flowering time? Science 296: 285-289.
C. M. Smart,1994. Tansley review no.64. Gene expression during leaf senescence. New Phytologist: 419-448.
Y. Song, C. Zhang, W. Ge, Y. Zhang, A. L. Burlingame et al,2011. Identification of NaCl stress-responsive apoplastic proteins in rice shoot stems by 2D-DIGE. Journal of proteomics 74: 1045-1067.
C. M. Spickett, N. Smirnoff, R. G Ratcliffe,1992. Metabolic Response of Maize Roots to Hyperosmotic Shock An in Vivo 31P Nuclear Magnetic Resonance Study. Plant physiology 99: 856-863.
R. Srivastava, J. X. Liu, S. H. Howell,2008. Proteolytic processing of a precursor protein for a growth-promoting peptide by a subtilisin serine protease in Arabidopsis. The Plant Journal 56: 219-227.
S. Taguchi, A. Ozaki, H. Momose,1998. Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system. Applied and environmental microbiology 64: 492-495.
T. Tajima, A. Yamaguchi, S. Matsushima, M. Satoh, S. Hayasaka, K. Yoshimatsu, Y. Shioi,2011. Biochemical and molecular characterization of senescence-related cysteine protease-cystatin complex from spinach leaf. Physiologia plantarum 141(2):97-116.
H. Tanaka, H. Onouchi, M. Kondo, I. Hara-Nishimura, M. Nishimura et al,2001. A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants. Development 128:4681-4689.
J. Tao, I. Ginsberg, N. Banerjee, W. Held, Y. Koltin et al.,1990. Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins. Molecular and cellular biology 10:1373-1381.
L. P. Tripathi, R. Sowdhamini,2006. Cross genome comparisons of serine proteases in Arabidopsis and rice. BMC genomics 7:200.
N. Tuteja,2007. Abscisic acid and abiotic stress signaling. Plant signaling & behavior 2:135-138.
C. Uauy, A. Distelfeld, T. Fahima, A. Blechl, J. Dubcovsky,2006. A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314(5803):1298-1301.
R. A. Van Der Hoorn,2008. Plant proteases:from phenotypes to molecular mechanisms. Annu. Rev. Plant Biol.59:191-223.
R. D. Vierstra,1996. Proteolysis in plants:mechanisms and functions. Plant molecular biology 32: 275-302.
R. D. Vierstra,2003. The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends in plant science 8:135-142.
D. Wagner, R. W. Sablowski, E. M. Meyerowitz,1999. Transcriptional activation of APETALA1 by LEAFY. Science 285:582-584.
R. Wang, S. Liu, J. Wang, Q. Dong, L. Xu et al.,2013. Purification, characterization and identification of a senescence related serine protease in dark-induced senescent wheat leaves. Phytochemistry.95:118-26
Y. Wang, K. Li, X. Li,2009. Auxin redistribution modulates plastic development of root system architecture under salt stress in Arabidopsis thaliana. Journal of plant physiology 166: 1637-1645.
Y. T. Wang, C. Y. Yang, Y.-T. Chen, Y. Lin, J.-F. Shaw,2004. Characterization of senescence-associated proteases in postharvest broccoli florets. Plant Physiology and Biochemistry 42:663-670.
L. M. Weaver, R. M. Amasino,2001. Senescence is induced in individually darkened Arabidopsis leaves, but inhibited in whole darkened plants. Plant Physiology 127:876-886.
A.-L. Wei, Y.-Z. Chen,2000. Effect of IAA on soybean seedling's membrance injury and salt resistance. ACTA BOTANICA BOREALI OCCIDENTALIA SINICA 20:410-414.
F.-P. Wen, Z.-H. Zhang, T. Bai, Q. Xu, Y.-H. Pan,2010. Proteomics reveals the effects of gibberellic acid (GA3) on salt-stressed rice (Oryza sativa L.) shoots. Plant Science 178: 170-175.
G C. Whitelam, E. Johnson, J. Peng, P. Carol, M. L. Anderson et al.,1993. Phytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light. The Plant Cell Online 5: 757-768.
K. Xu, K. Griebenow, A. M. Klibanov,1997. Correlation between catalytic activity and secondary structure of subtilisin dissolved in organic solvents. Biotechnology and bioengineering 56: 485-491.
H. Yamagata, T. Masuzawa, Y. Nagaoka, T. Ohnishi, T. Iwasaki,1994. Cucumisin, a serine protease from melon fruits, shares structural homology with subtilisin and is generated from a large precursor. Journal of Biological Chemistry 269:32725-32731.
H. Yamagata, S. Ueno, T. Iwasaki,1989. Isolation and characterization of a possible native cucumisin from developing melon fruits and its limited autolysis to cucumisin. Agricultural and biological chemistry 53:1009-1017.
K. Yamaguchi-Shinozaki, K. Shinozaki,1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. The Plant Cell Online 6(2):251-264.
Y. Yamaoka, Y. Ohba, M. Takeuchi, Y. Morohashi,1993. Isolation and properties of a carboxypeptidase from cotyledons of germinated mung bean seeds. Plant Science 95:1-7.
Y. Yang, Y. Wu, J. Pirrello, F. Regad, M. Bouzayen et al,2010. Silencing Sl-EBF1 and Sl-EBF2 expression causes constitutive ethylene response phenotype, accelerated plant senescence, and fruit ripening in tomato. Journal of experimental botany 61:697-708.
C. Yeh, H. Chang, H. Hsieh, K. Yoda, M. Yamasaki et al,1997. Improved translational efficiency of subtilisin YaB gene with different initiation codons in Bacillus subtilis and alkalophilic Bacillus YaB. Journal of applied microbiology 83:758-763.
K. T. Yoshida, T. Kuboyama,2001. A subtilisin-like serine protease specifically expressed in reproductive organs in rice. Sexual Plant Reproduction 13:193-199.
K. Yu, Y. Wang, J. Wei, Q. Ma, D. Yu et al,2009. Improving rhizome yield and quality of Paris polyphylla through gibberellic acid-induced retardation of senescence of aerial parts. Plant signaling & behavior 4:413-415.
J. Zeevaart, R. Creelman,1988 Metabolism and physiology of abscisic acid. Annual review of plant physiology and plant molecular biology 39(1):439-473
J. Zhang, W. Jia, J. Yang, A. M. Ismail,2006. Role of ABA in integrating plant responses to drought and salt stresses. Field Crops Research 97:111-119.
J.-K. Zhu,2002. Salt and drought stress signal transduction in plants. Annual review of plant biology 53:247.