,1,*, 杨新笋,2,*Identification and expression analysis of cell wall invertase IbCWIN gene family members in sweet potato
SONG Tian-Xiao1,2,**, LIU Yi2,3,**, RAO Li-Ping2,3, Soviguidi Deka Reine Judesse2,3, ZHU Guo-Peng,1,*, YANG Xin-Sun,2,*通讯作者:
收稿日期:2020-08-6接受日期:2020-11-13网络出版日期:2020-12-24
| 基金资助: |
Received:2020-08-6Accepted:2020-11-13Online:2020-12-24
| Fund supported: |
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宋天晓, E-mail:
刘意, E-mail:
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宋天晓, 刘意, 饶莉萍, Soviguidi Deka Reine Judesse, 朱国鹏, 杨新笋. 甘薯细胞壁蔗糖转化酶基因IbCWIN家族成员鉴定及表达分析[J]. 作物学报, 2020, 47(7): 1297-1308. doi:10.3724/SP.J.1006.2021.04180
SONG Tian-Xiao, LIU Yi, RAO Li-Ping, Soviguidi Deka Reine Judesse, ZHU Guo-Peng, YANG Xin-Sun.
甘薯[Ipomoea batatas (L.) Lam.]具有极高的营养保健价值, 是重要的粮食、饲料、工业原料作物和新型的生物能源作物, 在世界粮食生产中总产排列第7位[1,2,3]。蔗糖转化酶是植物蔗糖代谢中的一个关键酶, 不可逆的催化蔗糖裂解形成葡萄糖和果糖, 在授粉、果实成熟和纤维素生物合成过程中, 所有的蔗糖转化酶在植物生长中都起着关键的作用[4,5,6]。蔗糖转化酶是β呋喃果糖苷酶, 根据其溶解性、亚细胞定位以及最适pH值, 可分为细胞质、液泡和细胞壁转化酶(cell wall invertase, CWIN)。细胞质形式(中性/碱性转化酶)在pH 7.0~8.0范围内表现出最佳的活性, 而液泡(可溶性酸性转化酶)和细胞壁相关(胞外)在酸性pH值下表现出最佳活性。因此, 细胞壁蔗糖转化酶是酸性转化酶, 而酸性蔗糖转化酶可以催化水解含有β-呋喃果糖结构的其他低聚糖, N端存在糖基化, 属于糖基水解酶家族(glycoside hydrolase family 32, GH32)[7]。在功能上, 细胞壁转化酶是植物源、库组织蔗糖代谢的关键酶, 包括蔗糖在源、库组织之间的分配、细胞分化、植物发育和对多种生物或非生物胁迫信号的响应[8,9,10,11]。
前人研究发现, 在胡萝卜中表达细胞壁转化酶反义mRNA的植株叶片积累了较高含量的蔗糖和淀粉, 碳水化合物含量升高, 而主根发育明显减少, 由此产生的较小器官含有较低水平的碳水化合物[12]。提高番茄细胞壁蔗糖转化酶活性可以延缓叶片衰老, 增加种子重量和果实己糖含量[13]; 通过RNAi抑制番茄细胞壁蔗糖转化酶合成基因LeLin5的表达可以导致番茄幼果的败育[14]。在烟草中, 花药组织特异性表达CWIN基因NtNin88表达受到抑制后, 花粉早期发育受阻, 引发植株雄性不育[15]。在马铃薯中, 细胞壁转化酶参与了抗寒马铃薯植株低温耐受性的形成[16]。在烟草叶片过表达SoCIN1, 能提高烟草的CIN活性和可溶性糖含量, 促进植株生长[17]。
在木薯[18]、水稻[19]、马铃薯[20]、杨树[21]、番木瓜[22]等物种中已有关于细胞壁蔗糖转化酶基因家族的研究。对甘薯而言, 吴立军等[23]在甘薯悬浮细胞中进行了可溶性酸性转化酶的部分纯化与鉴定。Wang等[24]在甘薯中克隆了2个液泡转化酶基因并对其理化性质及表达模式进行分析, 但目前在甘薯上关于细胞壁转化酶的功能研究较少, 细胞壁转化酶的调节作用机制尚不清楚。本研究以生物信息学方法鉴定甘薯细胞壁蔗糖转化酶IbCWIN基因家族, 对其理化性质、保守结构域、系统进化关系、启动子作用元件、组织特异性表达模式进行分析, 并选取食用型甘薯鄂薯11号和菜用型甘薯福菜薯18两个品种为材料, 分析甘薯收获期的蔗糖、淀粉含量, 旨在为进一步研究甘薯IbCWIN基因家族的功能及调控甘薯源库关系机制提供理论指导。
1 材料与方法
1.1 试验材料与处理
选取食用型甘薯鄂薯11以及菜用型甘薯福菜薯18两个品种为试验材料。于2019年7月22日采用盆栽方式定植, 每盆3株, 每个品种4盆, 一般田间水肥管理。在块根膨大后期(120 d)时分别取块根、须根、茎、源叶(第3~5片功能叶), 用于蔗糖、淀粉含量测定及qRT-PCR分析。1.2 甘薯IbCWIN基因家族生物信息学分析
根据已报道的文献在NCBI (利用expasy (
1.3 甘薯IbCWIN基因家族组织特异性表达分析
利用何照范[27]、汤章城[28]方法测定蔗糖和淀粉含量。利用通用植物总RNA提取试剂盒(北京全式金生物技术有限公司)提取鄂薯11和福18不同部位的总RNA, 置于-80℃超低温冰箱保存备用, 使用全式金公司的反转录试剂盒反转录成cDNA, -20℃保存备用。利用NCBI primer blast网站和Primer 5软件设计IbCWIN基因家族的荧光定量特异性引物(表1), 以甘薯肌动蛋白β-Actin基因为内参基因, 参照TransStart Tip Green qPCR SuperMix试剂盒说明书(北京全式金生物技术有限公司), 在荧光定量PCR仪(Bio-Rad CFX96)上进行扩增, 每个反应3次重复。荧光定量PCR扩增条件为94℃预变性30 s; 94℃变性5 s, 56℃退火30 s, 40个循环; 溶解曲线设置为65℃到95℃, 增量为0.5℃持续5 s。检测IbCWIN1~ IbCWIN10基因在不同品种、不同组织间的表达模式, 利用2-ΔΔCt法计算出基因的相对表达量。Table 1
表1
表1试验所用引物
Table 1
| 基因名称 Gene name | 正向引物序列 Forward primer sequence (5'-3') | 反向引物序列 Reverse primer sequence (5'-3') |
|---|---|---|
| IbCWIN1 | GGGTGTTGCATGCTGTTCC | ACCCAACTGCACCGATTGTC |
| IbCWIN2 | ACTTGGATGGGTGTGG | TTGATACCCGGCCCGTTTG |
| IbCWIN3 | GTATGTGGGAGTGCGTGGAT | CGTCGTCGTAACTTCCCAGG |
| IbCWIN4 | ACCCTCATTTGCTGGATTTG | TGCCTCTGTGCCATTGTTG |
| IbCWIN5 | GGTTAGTTACAATAGGGTCCAAGG | TCCACGCATTCCCACATA |
| IbCWIN6 | GTTCTTGCATTCGGTTCCGC | ATCGAGTCTCAACCCGTGTC |
| IbCWIN7 | TAGGGAGCAAAGTTGAGCGG | AGAGGGTGTTCGGCTTTGAC |
| IbCWIN8 | GGATCACAACACTGGTCGGT | ATTAAGTTCGTGCATGCGGC |
| IbCWIN9 | GACACCGAGGACTTCAAGCA | GGTGGTCGAAACCGGGTAAA |
| IbCWIN10 | TGGGGAACGAGAGAAAGCAC | AAAAGTCAGGGCACTCCCAC |
| β-Actin | AGCAGCATGAAGATTAAGGTTGTAGCAC | TGGAAAATTAGAAGCACTTCCTGTGAAC |
新窗口打开|下载CSV
1.4 数据处理
利用Microsoft Excel和SAS 8.1软件分析数据, 进行单因素方差分析(ANOVA), 多重比较采用Duncan法分析。2 结果与分析
2.1 甘薯IbCWIN基因家族成员鉴定及系统进化分析
利用木薯细胞壁蔗糖转化酶基因作为种子序列在甘薯基因组数据库进行Blast序列比对得到10条同源序列。通过SMART、Pfam、NCBI-CDD三个蛋白保守域数据库对甘薯(IbCWIN1~IbCWIN10)、拟南芥(AtCWINV1~AtCWINV4)、马铃薯(StCWIN1~StCWIN4)、木薯(MeCWINV1~MeCWINV6)细胞壁蔗糖转化酶基因进行蛋白保守结构域分析发现, 它们都含有共同的Glyco_32保守结构域, 属于糖基水解酶基因家族GH32; 保守基序分析发现, 它们含有高度相似的motif, 这也进一步证明了我们得到的是甘薯IbCWIN基因家族(图1)。根据它们在染色体上的位置分别命名为IbCWIN1~IbCWIN10 (图2), IbCWIN基因家族编码氨基酸442~1115个, 分子量范围为49.56~124.44 kD, 等电点为5.0~9.1。亚细胞定位预测显示, IbCWIN1、IbCWIN2、IbCWIN3、IbCWIN5位于液泡内, IbCWIN4、IbCWIN6、IbCWIN7、IbCWIN8、IbCWIN9、IbCWIN10位于细胞壁上(表2)。将甘薯与拟南芥、马铃薯、木薯细胞壁蔗糖转化酶基因家族蛋白质序列构建系统进化树(图1)发现, IbCWIN1、IbCWIN2、IbCWIN3、IbCWIN5、 IbCWIN6、IbCWIN9为单独的一类, 但与木薯MeCWINV1和拟南芥AtCWINV6较近; IbCWIN7、IbCWIN8、IbCWIN10与木薯MeCWINV3、MeCWINV4分为一类; IbCWIN4单独为一类但与木薯MeCWINV6较为接近。进化树中, 聚类关系越近, 其功能类似的可能性越大[29], 推测IbCWIN基因家族与这些基因可能具有相似的功能。图1
新窗口打开|下载原图ZIP|生成PPT图1甘薯与其他物种CWIN基因家族比较
MeCWINV1~MeCWINV6 GenBank登录号为JQ339929、JX291160、JN801147、JQ792172、JX291159、JQ339930; AtCWINV1~AtCWINV6登录号为At3g13790、At3g52600、At1g55120、At2g36190、At3g13784、At5g11920; StCWIN1~StCWIN4登录号为Z21486、Z22645、AJ133765、AJ133765。
Fig. 1Comparison of CWIN gene family between sweet potato and other species
Accession numbers of MeCWINV1-MeCWINV6 in GenBank are JQ339929, JX291160, JN801147, JQ792172, JX291159, JQ339930; Accession numbers of AtCWINV1-AtCWINV6 are At3g13790, At3g52600, At1g55120, At2g36190, At3g13784, At5g11920; and accession numbers of StCWIN1-StCWIN4 are Z21486, Z22645, AJ133765, and AJ133765, respectively.
图2
新窗口打开|下载原图ZIP|生成PPT图2甘薯IbCWIN基因家族在染色体上的分布
Fig. 2Distribution of IbCWIN gene family on chromosomes in sweet potato
Table 2
表2
表2甘薯IbCWIN基因家族
Table 2
| 基因名称 Gene name | 基因序列号 Gene ID | 氨基酸 Amino acid | 分子量 Molecular weight (kD) | 等电点 Isoelectric point | 预测亚细胞定位 Predicting subcellular localization |
|---|---|---|---|---|---|
| IbCWIN1 | g1732 | 657 | 72.01 | 5.19 | 液泡Vacuole |
| IbCWIN2 | g1764 | 680 | 74.64 | 5.00 | 液泡Vacuole |
| IbCWIN3 | g15600 | 1115 | 124.44 | 5.96 | 液泡Vacuole |
| IbCWIN4 | g25102 | 467 | 52.68 | 6.47 | 细胞壁Cell wall |
| IbCWIN5 | g25369 | 792 | 86.78 | 5.58 | 液泡Vacuole |
| IbCWIN6 | g49879 | 552 | 61.99 | 8.30 | 细胞壁Cell wall |
| IbCWIN7 | g55209 | 442 | 49.56 | 9.01 | 细胞壁Cell wall |
| IbCWIN8 | g55220 | 913 | 100.07 | 8.61 | 细胞壁Cell wall |
| IbCWIN9 | g55625 | 591 | 66.10 | 5.53 | 细胞壁Cell wall |
| IbCWIN10 | g60985 | 577 | 64.50 | 8.78 | 细胞壁Cell wall |
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2.2 甘薯IbCWIN家族结构和保守基序分析
由图3可知, IbCWIN基因家族包含5~14个外显子, 4~13个内含子。IbCWIN1与IbCWIN2基因结构与大小相似; IbCWIN3与IbCWIN9基因结构相近, 但长度差异较大; IbCWIN7与IbCWIN8基因结构与大小差异最大, 推测基因在进化过程中发生了较大的变异。对IbCWIN基因家族保守基序进行分析发现, IbCWIN1~IbCWIN10均含有motif1、motif2。其中IbCWIN1、IbCWIN2、IbCWIN3、IbCWIN5、IbCWIN10均含有10个motif, IbCWIN4、IbCWIN7所含motif最少, 只有6个。IbCWIN基因家族除了共同含有Glyco_32蛋白保守结构域外, IbCWIN1、IbCWIN2、IbCWIN3、IbCWIN5含有一种保守结构域Pfam:DUF3357, IbCWIN3含有Pfam:zf-RVT结构域, 可能是逆转录酶的锌结合区和存在于植物反转录转座子的一部分的Pfam:RVT_3结构域。IbCWIN6、IbCWIN8含有Low complexity普遍存在的区域, IbCWIN8、IbCWIN10含有signal peptide信号肽结构域, IbCWIN8还含有RNA结合蛋白RRM保守域。利用MCScanX软件分析发现, IbCWIN1与IbCWIN2基因存在共线性关系(图4)。图3
新窗口打开|下载原图ZIP|生成PPT图3甘薯IbCWIN基因家族进化树与基因结构
Fig. 3Phylogenetic tree and gene structure of IbCWIN gene family in sweet potato
图4
新窗口打开|下载原图ZIP|生成PPT图4甘薯IbCWIN基因家族共线性分析
Fig. 4Collinearity analysis of IbCWIN gene family in sweet potato
2.3 甘薯IbCWIN基因家族启动子顺式作用元件分析
利用Plantcare预测IbCWIN基因家族启动子上游1500 bp的顺式作用元件(图5)发现, 甘薯IbCWIN基因家族主要含有基本顺式作用元件(TATA-box、CAAT-box)、逆境响应元件(W-box、STRE)、光响应元件(ACE、GT1-motif)、氧调节元件(ARE、GC-motif)、激素调节元件(ERE、CGTCA-motif)、机械伤害防御元件(WUN-motif、WRE3)、发育调节元件(CAT-box、MSA-like)等7大类型元件。其中IbCWIN1~IbCWIN10均含有干旱和ABA应答的顺式作用元件MYC、基本顺式作用元件TATA-box和CAAT-box元件、逆境响应元件和光响应元件。除IbCWIN1外均含有干旱、盐、低温胁迫及外源ABA诱导等多种逆境胁迫反应调控元件MYB; 除IbCWIN9外均含有激素与氧相关的调节元件。伤害防御元件主要集中在IbCWIN3~IbCWIN10基因上, 发育调节元件主要集中在IbCWIN4~IbCWIN10基因上。由此推测甘薯IbCWIN基因家族可能参与多种逆境反应, 并以多种方式调节甘薯生长发育。图 5
新窗口打开|下载原图ZIP|生成PPT图 5甘薯IbCWIN基因家族启动子预测
Fig. 5Promoter prediction of IbCWIN gene family in sweet potato
2.4 甘薯IbCWIN基因家族表达特性分析
在收获期测定甘薯不同部位蔗糖和淀粉含量。由图6可知, 在鄂11中块根蔗糖含量极显著低于须根、叶和茎, 其他部位之间不存在显著性差异。在福18中茎的蔗糖含量极显著高于叶、须根和块根。鄂11和福18相比, 福18茎和块根中的蔗糖极显著高于鄂11, 其余部位无显著差异。鄂11块根淀粉含量极显著高于须根、茎和叶, 茎中淀粉含量显著高于叶; 福18块根淀粉也极显著高于须根、茎、叶, 茎叶间无显著差异。2个品种间淀粉比较发现, 鄂11须根中淀粉含量显著高于福18, 其余部位无显著差异。2个品种不同组织中蔗糖含量从高到低均为茎、叶、须根、块根, 淀粉含量则是块根、须根、茎、叶。块根和须根蔗糖与淀粉含量呈现相反趋势, 茎和叶无这种趋势。可能是因为根部作为库器官不断地将运输来的蔗糖转化为淀粉贮藏起来, 而茎和叶作为运输和光合产物合成的源器官, 与蔗糖转运、分配、消耗速率有关。对甘薯不同组织中IbCWIN家族基因表达进行荧光定量分析(图7)发现, 在地下部位具有较高表达的有IbCWIN2、IbCWIN3、IbCWIN9, 与淀粉含量变化趋势一致的是IbCWIN2和IbCWIN9, 在块根中表达量显著高于其他部位, 推测它们在甘薯块根蔗糖水解与淀粉合成中起到主要作用。与2个品种不同组织部位间蔗糖含量变化趋势一致的是IbCWIN4, 在茎中表达量极显著高于叶、须根和块根; 在地上部位有较高表达的是IbCWIN5、IbCWIN6, 推测它们在甘薯源组织蔗糖合成和运输中起主要作用; IbCWIN8在不同组织部位间表达相对均衡, 推测其在甘薯源、库组织间蔗糖淀粉转化合成的整个过程中起作用; 在须根和茎或叶中同时高表达的有IbCWIN1、IbCWIN3、IbCWIN7、IbCWIN10。进化关系较近的IbCWIN1在鄂11叶和须根中极显著高于福18, 而IbCWIN2在福18块根、须根和叶中极显著高于鄂11; IbCWIN3在福18须根中显著高于鄂11, IbCWIN9在鄂11块根中极显著高于福18。可能IbCWIN基因家族的表达与甘薯基因型有关。总体而言, IbCWIN基因家族在甘薯不同组织中均有表达且有多种表达模式, 推测不同组织中高表达基因在该组织蔗糖淀粉的转化合成中发挥主要作用。
图6
新窗口打开|下载原图ZIP|生成PPT图6鄂11、福18蔗糖和淀粉含量
图中以不同字母表示数据间的显著性差异, 大写字母表示在0.01水平差异显著, 小写字母表示在0.05水平差异显著。
Fig. 6Contents of sucrose and starch in E11 and Fu18
Different letters in the figure indicate significant differences among the treatments. Uppercase letter indicates significant differences at the 0.01 probability level, and lowercase letter indicates significant differences at the 0.05 probability level.
图7
新窗口打开|下载原图ZIP|生成PPT图7IbCWIN基因家族的组织特异性表达分析
图中以不同字母表示数据间的显著性差异, 大写字母表示在0.01水平差异显著, 小写字母表示在0.05水平差异显著。
Fig. 7Tissue specific expression analysis of IbCWIN gene family
Different letters in the figure indicate significant differences among the treatments. Uppercase letter indicates significant differences at the 0.01 probability level, and lowercase letter indicates significant differences at the 0.05 probability level.
3 讨论
栽培种甘薯为六倍体, 基因组复杂且庞大(2n=6x=90), 具有自交不亲和性, 导致甘薯的遗传研究落后于其他农作物[30]。甘薯的90条染色体中, 30条是来源于二倍体祖先种, 另外60条来源于四倍 体祖先种。在50万年前, 二倍体祖先种和四倍体祖先种之间发生了一次自然杂交后染色体加倍形成今天的栽培种[31]。本研究利用生物信息学方法从甘薯基因组数据库鉴定得到了10个IbCWIN基因, 比拟南芥6条、马铃薯4条、木薯6条CWIN基因数目多, 产生了基因扩增现象, 这可能是由于甘薯在进化过程中发生了全基因组复制和串联复制所导致的, 以便增强适应环境的能力[32]。基因可以通过全基因组复制、串连复制、片段复制和逆转座复制等多种方式进行扩增, 也是基因组进化产生具有新功能的基因和物种的主要分子机制之一[33]。甘薯IbCWIN基因家族编码氨基酸442~1115个, 分子量范围为49.56~124.44 kD, 等电点为5.0~9.1, 包含6~14个外显子, 5~13个内含子。由于其内含子外显子的数目和位置大小差别较大, 导致成员在基因结构以及编码氨基酸数目上存在较大的差异[34]。7个外显子和6个内含子的基因结构为植物酸性转化酶基因的原始结构[22], 本研究中只有IbCWIN7是6个外显子和5个内含子, 其余成员均未有丢失现象。植物转化酶基因和转化酶的表达活性水平似乎受到植物组织中己糖库的调节, 受到反应产物葡萄糖和果糖所抑制[35]。不同的CWINV可以根据组织特异性和酶学特性实现可变的功能[18]。玉米Incw2基因为组织特异性基因, 灌浆期在玉米胚乳中特异表达, 过表达Incw2调控蔗糖卸载, 促进籽粒灌浆, 进而增加籽粒产量[36]。辣椒CaCWIN03在花和果实发育中高表达, CaCWIN01在种子和叶片中较高表达, 表明这2个基因在花发育和生殖过程中及在种子和叶片发育中起重要作用[37]。木薯中细胞壁酸性转化酶和蔗糖合酶mRNA表达量分别在须根和块根中的相对高表达, 初步证实了须根的质外体和块根的共质体卸载途径的主体地位[38]。qRT-PCR和蛋白系统进化树表明, 甘薯IbCWIN基因家族在不同组织部位间具有多种表达模式。IbCWIN1、IbCWIN2、IbCWIN3、IbCWIN5、IbCWIN6、IbCWIN9可以分为为单独的一类, 但它们与木薯MeCWINV1和拟南芥AtCWINV6关系较近; 在木薯叶片和块茎中MeCWINV1和MeCWINV3的表达量较高于其他MeCWINV[39], IbCWIN1、IbCWIN3、IbCWIN6在叶片和须根中表达量也较高。MeCWINV1参与木薯高低温、光照和激素处理中的响应表达, 局部活性提高具有一定的普遍性[40]。IbCWIN2和IbCWIN9在块根中表达量显著高于其他部位, 而在库器官中提高细胞壁酸性转化酶的活性有利于蔗糖在库器官中的积累[8], 因此, 在下一步功能研究中可以重点关注这2个基因。IbCWIN7、IbCWIN8、IbCWIN10与木薯MeCWINV3、MeCWINV4分为一类; 木薯MeCWINV3在成熟叶片中高表达, 响应昼夜节律, 并通过调节糖从源到库的分配来影响木薯产量[39]。IbCWIN7、IbCWIN8, IbCWIN10在甘薯叶片部位表达量也较高, 推测它们具有相似的功能。另一方面, 在木薯中过表达MeCWINV3时, 蔗糖从叶到贮藏根的出口被大大抑制, 叶中的蔗糖水解被加速, 促进了木薯叶片衰老进程, 衰老相关基因表达增加。因叶片中的糖分配急剧减少, 贮藏根系的发育也被推迟[41]。IbCWIN4与MeCWINV6进化关系较近, MeCWINV6 基因表达受到外界环境的调节, 在正常生长条件下表达量极低, 但是经低温胁迫、赤霉素胁迫、水杨酸胁迫和脱落酸胁迫处理后它的表达量发生了显著变化[42], 推测IbCWIN4在甘薯中也可能具有相似的功能。
4 结论
通过对甘薯IbCWIN基因家族进行鉴定, 共获得10个基因, 分布在8条染色体上, 均含有Glyco_32蛋白保守结构域和多种类型顺式作用元件, 基因表达具有多种表达模式, IbCWIN2和IbCWIN9在块根中表达量显著高于其他组织部位, 其可能参与块根中光合产物蔗糖的水解, 进而促进淀粉合成。对于IbCWIN基因家族功能的研究, 还有待进一步探索发现。参考文献 原文顺序
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被引期刊影响因子
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DOI:10.1104/pp.16.01269URLPMID:27694342 [本文引用: 1]
Fruit ripening is a complex process that involves a series of physiological and biochemical changes that ultimately influence fruit quality traits, such as color and flavor. Sugar metabolism is an important factor in ripening, and there is evidence that it influences various aspects of ripening, although the associated mechanism is not well understood. In this study, we identified and analyzed the expression of 36 genes involved in Suc metabolism in ripening tomato (Solanum lycopersicum) fruit. Chromatin immunoprecipitation and gel mobility shift assays indicated that SlVIF, which encodes a vacuolar invertase inhibitor, and SlVI, encoding a vacuolar invertase, are directly regulated by the global fruit ripening regulator RIPENING INHIBITOR (RIN). Moreover, we showed that SlVIF physically interacts with SlVI to control Suc metabolism. Repression of SlVIF by RNA interference delayed tomato fruit ripening, while overexpression of SlVIF accelerated ripening, with concomitant changes in lycopene production and ethylene biosynthesis. An isobaric tags for relative and absolute quantification-based quantitative proteomic analysis further indicated that the abundance of a set of proteins involved in fruit ripening was altered by suppressing SlVIF expression, including proteins associated with lycopene generation and ethylene synthesis. These findings provide evidence for the role of Suc in promoting fruit ripening and establish that SlVIF contributes to fruit quality and the RIN-mediated ripening regulatory mechanisms, which are of significant agricultural value.
DOI:10.1104/pp.16.01601URLPMID:27923989 [本文引用: 1]
Pollination in flowering plants is initiated by germination of pollen grains on stigmas followed by fast growth of pollen tubes representing highly energy-consuming processes. The symplastic isolation of pollen grains and tubes requires import of Suc available in the apoplast. We show that the functional coupling of Suc cleavage by invertases and uptake of the released hexoses by monosaccharide transporters are critical for pollination in tobacco (Nicotiana tabacum). Transcript profiling, in situ hybridization, and immunolocalization of extracellular invertases and two monosaccharide transporters in vitro and in vivo support the functional coupling in supplying carbohydrates for pollen germination and tube growth evidenced by spatiotemporally coordinated expression. Detection of vacuolar invertases in maternal tissues by these approaches revealed metabolic cross talk between male and female tissues and supported the requirement for carbohydrate supply in transmitting tissue during pollination. Tissue-specific expression of an invertase inhibitor and addition of the chemical invertase inhibitor miglitol strongly reduced extracellular invertase activity and impaired pollen germination. Measurements of (competitive) uptake of labeled sugars identified two import pathways for exogenously available Suc into the germinating pollen operating in parallel: direct Suc uptake and via the hexoses after cleavage by extracellular invertase. Reduction of extracellular invertase activity in pollen decreases Suc uptake and severely compromises pollen germination. We further demonstrate that Glc as sole carbon source is sufficient for pollen germination, whereas Suc is supporting tube growth, revealing an important regulatory role of both the invertase substrate and products contributing to a potential metabolic and signaling-based multilayer regulation of pollination by carbohydrates.
DOI:10.1111/nph.14392URLPMID:28032636 [本文引用: 1]
Carbon for cellulose biosynthesis is derived from sucrose. Cellulose is synthesized from uridine 5'-diphosphoglucose (UDP-glucose), but the enzyme(s) responsible for the initial sucrose cleavage and the source of UDP-glucose for cellulose biosynthesis in developing wood have not been defined. We investigated the role of CYTOSOLIC INVERTASEs (CINs) during wood formation in hybrid aspen (Populus tremula x tremuloides) and characterized transgenic lines with reduced CIN activity during secondary cell wall biosynthesis. Suppression of CIN activity by 38-55% led to a 9-13% reduction in crystalline cellulose. The changes in cellulose were reflected in reduced diameter of acid-insoluble cellulose microfibrils and increased glucose release from wood upon enzymatic digestion of cellulose. Reduced CIN activity decreased the amount of the cellulose biosynthesis precursor UDP-glucose in developing wood, pointing to the likely cause of the cellulose phenotype. The findings suggest that CIN activity has an important role in the cellulose biosynthesis of trees, and indicate that cellulose biosynthesis in wood relies on a quantifiable UDP-glucose pool. The results also introduce a concept of altering cellulose microfibril properties by modifying substrate supply to cellulose biosynthesis.
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DOI:10.1104/pp.121.1.1URLPMID:10482654 [本文引用: 1]
DOI:10.1016/j.pbi.2004.03.014URLPMID:15134743 [本文引用: 1]
Sucrose cleavage is vital to multicellular plants, not only for the allocation of crucial carbon resources but also for the initiation of hexose-based sugar signals in importing structures. Only the invertase and reversible sucrose synthase reactions catalyze known paths of sucrose breakdown in vivo. The regulation of these reactions and its consequences has therefore become a central issue in plant carbon metabolism. Primary mechanisms for this regulation involve the capacity of invertases to alter sugar signals by producing glucose rather than UDPglucose, and thus also two-fold more hexoses than are produced by sucrose synthase. In addition, vacuolar sites of cleavage by invertases could allow temporal control via compartmentalization. In addition, members of the gene families encoding either invertases or sucrose synthases respond at transcriptional and posttranscriptional levels to diverse environmental signals, including endogenous changes that reflect their own action (e.g. hexoses and hexose-responsive hormone systems such as abscisic acid [ABA] signaling). At the enzyme level, sucrose synthases can be regulated by rapid changes in sub-cellular localization, phosphorylation, and carefully modulated protein turnover. In addition to transcriptional control, invertase action can also be regulated at the enzyme level by highly localized inhibitor proteins and by a system that has the potential to initiate and terminate invertase activity in vacuoles. The extent, path, and site of sucrose metabolism are thus highly responsive to both internal and external environmental signals and can, in turn, dramatically alter development and stress acclimation.
DOI:10.1105/tpc.11.2.177URLPMID:9927637 [本文引用: 1]
To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.
DOI:10.1105/tpc.108.063719URLPMID:19574437 [本文引用: 1]
Invertase plays multiple pivotal roles in plant development. Thus, its activity must be tightly regulated in vivo. Emerging evidence suggests that a group of small proteins that inhibit invertase activity in vitro appears to exist in a wide variety of plants. However, little is known regarding their roles in planta. Here, we examined the function of INVINH1, a putative invertase inhibitor, in tomato (Solanum lycopersicum). Expression of a INVINH1:green fluorescent protein fusion revealed its apoplasmic localization. Ectopic overexpression of INVINH1 in Arabidopsis thaliana specifically reduced cell wall invertase activity. By contrast, silencing its expression in tomato significantly increased the activity of cell wall invertase without altering activities of cytoplasmic and vacuolar invertases. Elevation of cell wall invertase activity in RNA interference transgenic tomato led to (1) a prolonged leaf life span involving in a blockage of abscisic acid-induced senescence and (2) an increase in seed weight and fruit hexose level, which is likely achieved through enhanced sucrose hydrolysis in the apoplasm of the fruit vasculature. This assertion is based on (1) coexpression of INVINH1 and a fruit-specific cell wall invertase Lin5 in phloem parenchyma cells of young fruit, including the placenta regions connecting developing seeds; (2) a physical interaction between INVINH1 and Lin5 in vivo; and (3) a symplasmic discontinuity at the interface between placenta and seeds. Together, the results demonstrate that INVINH1 encodes a protein that specifically inhibits the activity of cell wall invertase and regulates leaf senescence and seed and fruit development in tomato by limiting the invertase activity in planta.
DOI:10.1104/pp.109.136598URLPMID:19439574 [本文引用: 1]
It has been previously demonstrated, utilizing intraspecific introgression lines, that Lycopersicum Invertase5 (LIN5), which encodes a cell wall invertase, controls total soluble solids content in tomato (Solanum lycopersicum). The physiological role of this protein, however, has not yet been directly studied, since evaluation of data obtained from the introgression lines is complicated by the fact that they additionally harbor many other wild species alleles. To allow a more precise comparison, we generated transgenic tomato in which we silenced the expression of LIN5 using the RNA interference approach. The transformants were characterized by an altered flower and fruit morphology, displaying increased numbers of petals and sepals per flower, an increased rate of fruit abortion, and a reduction in fruit size. Evaluation of the mature fruit revealed that the transformants were characterized by a reduction of seed number per plant. Furthermore, detailed physiological analysis revealed that the transformants displayed aberrant pollen morphology and a reduction in the rate of pollen tube elongation. Metabolite profiling of ovaries and green and red fruit revealed that metabolic changes in the transformants were largely confined to sugar metabolism, whereas transcript and hormone profiling revealed broad changes both in the hormones themselves and in transcripts encoding their biosynthetic enzymes and response elements. These results are discussed in the context of current understanding of the role of sugar during the development of tomato fruit, with particular focus given to its impact on hormone levels and organ morphology.
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DOI:10.3390/ijms15057313URLPMID:24786092 [本文引用: 4]
The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a beta-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a beta-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker.
DOI:10.1007/s00299-004-0910-zURLPMID:15759120 [本文引用: 1]
Cell-wall invertase (CIN) catalyzes the hydrolysis of sucrose into glucose and fructose for the supply of carbohydrates to sink organs via an apoplastic pathway. To study the CIN genes in rice (Oryza sativa L.), we isolated cDNA clones showing amino acid similarity to the plant cell wall invertase proteins from a search of rice sequence databases. Profile analyses revealed that the cloned genes are expressed in unique patterns in various organs. For example, transcripts of OsCIN1, OsCIN2, OsCIN4, and OsCIN7 were detected in immature seeds whereas OsCIN3 gene expression was flower-specific. Further transcript analysis of these genes expressed in developing seeds indicated that OsCIN1, OsCIN2, and OsCIN7 might play an important role involving sucrose partitioning to the embryo and endosperm. Sucrose, a substrate of CINs, induced the accumulation of OsCIN1 transcripts in excised leaves and OsCIN2 in immature seeds, while the level of OsCIN5 was significantly down-regulated in excised leaves treated with sucrose. Infecting the tissues with rice blast (Magnaporthe grisea) as a biotic stressor increased the expression of OsCIN1, OsCIN4, and OsCIN5, suggesting that these genes may participate in a switch in metabolism to resist pathogen invasion. These results demonstrate that OsCIN genes play diverse roles involving the regulation of metabolism, growth, development, and stress responses.
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DOI:10.1371/journal.pone.0138540URLPMID:26393355 [本文引用: 1]
Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.
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DOI:10.1021/jf0480851URLPMID:15853418 [本文引用: 1]
Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.
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DOI:10.1038/s41477-017-0002-zURLPMID:28827752 [本文引用: 1]
Here we present the 15 pseudochromosomes of sweet potato, Ipomoea batatas, the seventh most important crop in the world and the fourth most significant in China. By using a novel haplotyping method based on genome assembly, we have produced a half haplotype-resolved genome from ~296 Gb of paired-end sequence reads amounting to roughly 67-fold coverage. By phylogenetic tree analysis of homologous chromosomes, it was possible to estimate the time of two recent whole-genome duplication events as occurring about 0.8 and 0.5 million years ago. This half haplotype-resolved hexaploid genome represents the first successful attempt to investigate the complexity of chromosome sequence composition directly in a polyploid genome, using sequencing of the polyploid organism itself rather than any of its simplified proxy relatives. Adaptation and application of our approach should provide higher resolution in future genomic structure investigations, especially for similarly complex genomes.
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DOI:10.1104/pp.108.1.285URLPMID:7784506 [本文引用: 1]
Photoautotrophic suspension-culture cells of Chenopodium rubrum that were shifted to mixotrophic growth by adding glucose were used as model system to investigate the influence of the source-sink transition in higher plants on the expression and enzyme activities of intracellular and extracellular invertases. The complete cDNA coding for an extracellular invertase was cloned and sequenced from C. rubrum, and its identity has been proven by heterologous expression in Saccharomyces cerevisiae. The higher activity of extracellular invertase after preincubation in the presence of glucose was paralleled by an increased expression of the corresponding gene. The induction by glucose could be mimicked by the nonmetabolizable glucose analog 6-deoxyglucose. Both enzyme activity and mRNA level of extracellular invertase showed a sink-tissue-specific distribution in plants. The activity of neutral and acidic intracellular invertases were not affected by preincubation of autotrophic tissue cultures with sugars, nor did they show a tissue-specific distribution in plants. The data suggest that apoplastic invertase not only has an important function in phloem unloading and carbohydrate partitioning between source and sink tissues but may also have a role in establishing metabolic sinks.
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DOI:10.3389/fpls.2019.00772URLPMID:31316528 [本文引用: 2]
The basic leucine zipper (bZIP) transcription factor family plays crucial roles in multiple biological processes, especially stress responses. Cassava (Manihot esculenta Crantz) is an important tropical crop with a strong tolerance to environmental stresses such as drought, heat, and low-fertility environments. Currently, limited information is available regarding the functional identification of bZIP transcription factors in response to abiotic stress in cassava. Herein, a gene encoding an ABA Insensitive 5 (ABI5)-like transcription factor, designated as MeABL5, was identified in cassava. Sequence and phylogenetic analysis showed that MeABL5 is a cassava bZIP transcription factor that is not included in the previously identified cassava bZIP family members, belongs to subfamily A, and has high sequence similarity to ABI5-like proteins. Subcellular localization and transactivation assays revealed that MeABL5 was a nuclear-localized protein and possessed transactivation activity. Furthermore, MeABL5 was able to specifically interact with the ABRE cis-element in the promoter of the cassava major cell wall invertase gene, MeCWINV3, in vitro and in vivo. MeABL5 and MeCWINV3 exhibited similar expression patterns in various organs or tissues and under abiotic stress in cassava. The expressions of MeABL5 and MeCWINV3 within cassava plantlets were both induced by exogenous abscisic acid (ABA), gibberellic acid (GA3), methyl jasmonate (MeJA), and heat. Overexpression of MeABL5 increased the activity of the MeCWINV3 gene, and the up-regulated expressions of MeCWINV3 were significantly activated under ABA-, salicylic acid (SA)-, and MeJA-induced conditions. Overall, these results suggest that MeABL5 is a positive regulator of MeCWINV3 and might participate in the robust resistance of cassava in response to abiotic stress. This study also provides a foundation for further research on ABA-mediated and stress-related signaling pathways in cassava.
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DOI:10.3389/fpls.2019.00541URLPMID:31114601 [本文引用: 1]
Storage roots are the main sink for photo-assimilate accumulation and reflect cassava yield and productivity. Regulation of sugar partitioning from leaves to storage roots has not been elucidated. Cell wall invertases are involved in the hydrolysis of sugar during phloem unloading of vascular plants to control plant development and sink strength but have rarely been studied in root crops like cassava. MeCWINV3 encodes a typical cell wall invertase in cassava and is mainly expressed in vascular bundles. The gene is highly expressed in leaves, especially mature leaves, in response to diurnal rhythm. When MeCWINV3 was overexpressed in cassava, sugar export from leaves to storage roots was largely inhibited and sucrose hydrolysis in leaves was accelerated, leading to increased transient starch accumulation by blocking starch degradation and reduced overall plant growth. The progress of leaf senescence was promoted in the MeCWINV3 over-expressed cassava plants with increased expression of senescence-related genes. Storage root development was also delayed because of dramatically reduced sugar allocation from leaves. As a result, the transcriptional expression of starch biosynthetic genes such as small subunit ADP-glucose pyrophosphorylase, granule-bound starch synthase I, and starch branching enzyme I was reduced in accordance with insufficient sugar supply in the storage roots of the transgenic plants. These results show that MeCWINV3 regulates sugar allocation from source to sink and maintains sugar balance in cassava, thus affecting yield of cassava storage roots.
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