,2Cloning and Functional Analysis of a Root Development Related Gene OsKSR7 in Rice (Oryza sativa L.)
ZHOU JiaQin1, ZHU JunZhao2, YANG SiXue2, ZHU ZhouJie2, YAO Jie2, ZHENG WenJuan2, ZHU ShiHua2, DING WoNa,2通讯作者:
收稿日期:2018-07-18接受日期:2018-08-26网络出版日期:2019-03-01
| 基金资助: |
Received:2018-07-18Accepted:2018-08-26Online:2019-03-01
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周佳琴,E-mail：
朱俊兆,E-mail：
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周佳琴, 朱俊兆, 杨思学, 诸周洁, 姚婕, 郑文娟, 朱世华, 丁沃娜. 水稻根系发育基因OsKSR7 的克隆与功能分析[J]. 中国农业科学, 2019, 52(5): 777-785 doi:10.3864/j.issn.0578-1752.2019.05.001
ZHOU JiaQin, ZHU JunZhao, YANG SiXue, ZHU ZhouJie, YAO Jie, ZHENG WenJuan, ZHU ShiHua, DING WoNa.
0 引言
【研究意义】根系是植物在长期进化过程中形成的重要营养器官,发达的根系为水稻生长发育提供充足的养分和水分,根系也是感知逆境信号的重要器官,与水稻抗逆性密切相关 [1,2,3]。根系形态与产量的关系一直是水稻根系研究的热点,地下部根系性状与地上部性状具有联动关系,根长、根重、根直径等与地上部农艺性状密切相关[4,5,6]。但长期以来由于研究技术的局限性,水稻根系功能基因的研究相对落后。【前人研究进展】水稻是须根系单子叶作物,其根系由主根、不定根、侧根和根毛组成。目前,已克隆了40多个水稻根系发育调控基因[7,8,9],其中部分和产量相关。如,编码K+/Na+/Cl-协同转运蛋白的OsCCC1 通过调控K+/Na+/Cl-平衡参与细胞伸长进程,突变体整个生育期的主根、侧根和冠根均变短,大田下产量下降,穗数、千粒重、每穗粒数均显著降低[10]。编码β-酮脂酰-酰基载体蛋白合酶的OsKASI 参与脂肪酸合成,突变体的主根、侧根和不定根都明显变短,分蘖数、花粉育性和千粒重均降低[11]。编码线粒体蛋白的OsSPR1 与铁的动态平衡有关,突变体胚后根变短,成熟期植株较野生型矮化,分蘖数显著减少[12]。编码中性/碱性转化酶的OsCYT-INV1 催化蔗糖水解成葡萄糖和果糖,在根系发育早期供碳源和能量,突变体的主根、不定根和侧根均变短,花期延迟,花粉部分不育,结实率显著降低[13]。此外,编码WUSCHEL相关的同源异型框基因的WOX11 可能是整合生长素和细胞分裂素信号传导的因子,突变体主根变小,发芽后冠状根数目减少甚至没有冠状根,成熟期株高变矮,分蘖数减少,结实率大幅下降[14]。参与调控根生长的基因编码蛋白的功能差异很大,表明多因素多信号途径调控水稻根生长发育的复杂性。【本研究切入点】通过水稻根系基因的功能鉴定,对水稻根系发育的分子调控模式有了一定程度的认识,但相对于其他器官的研究程度来说仍远远不够,需要分离和鉴定更多的根系调控基因。【拟解决的关键问题】本研究从EMS诱变的籼稻Kasalath突变体库中筛选获得1个短根突变体Osksr7 ,通过对该突变体进行表型鉴定、突变基因的图位克隆和转基因互补验证,为深入解析该基因的功能和在水稻根系育种上的应用奠定基础。1 材料与方法
1.1 试验材料
短根突变体Osksr7 来自浙江大学植物生理学与生物化学国家重点实验室构建的籼稻（Kasalath, indica）EMS诱变突变体库。2011—2015年,在宁波大学科学技术学院试验田连续自交5代,突变性状稳定且不分离。将突变体Osksr7 分别与Kasalath和粳稻Nipponbare杂交,观察F1的根系表型,F1自交获得的F2群体用于遗传分析和基因定位。1.2 突变体的表型鉴定
采用溶液培养法培养水稻幼苗[15],观察生长7、14和21 d的野生型Kasalath和突变体Osksr7 表型并拍摄,对植株的重要性状（株高、种子根长、不定根长、不定根数和侧根长）进行测量,绘制根系生长曲线。样本统计数为20株,3次重复。随机选取大田种植的野生型Kasalath和突变体Osksr7 各10株,成熟期测定主要农艺性状,包括株高、穗数、茎秆粗细、剑叶长宽、穗长、结实率和千粒重等。
1.3 分子标记的选择与设计
基因定位用的SSR（simple sequence repeat）分子标记来自Gramene网站（http://www.gramene.org/）,在12条水稻染色体上按照遗传距离均匀分布的原则选择SSR标记进行初定位。精细定位中新的分子标记发展是根据已公布的Nipponbare全基因组序列和Kasalath全基因组重测序数据,在初定位区间内分析2个亲本间有差异的序列并设计引物,选取有多态性的InDel标记用于进一步定位。1.4 突变基因的图位克隆
采用TPS法提取2个亲本、F1和F2分离群体的DNA[16]。在Osksr7 ×Nipponbare的F2分离群体中随机挑选30个短根株系提取DNA后等量混合形成突变体基因池。PCR扩增挑选的有多态性的SSR标记,用非变性聚丙烯酰胺凝胶电泳进行检测,观察银染显色后的带型判断连锁关系,对疑似连锁的标记进行解包检测。在初定位区间内设计新的分子标记和扩大定位群体,逐渐缩小定位区间。通过水稻基因组数据库网站（http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/ rice/）分析定位区间内的候选基因,测序验证确定突变位点。1.5 转基因互补验证
根据OsKSR7 的mRNA序列和带有35S启动子的pCAMBIA1300载体上的多克隆位点信息,设计带有酶切位点的引物OsKSR7-F（5′ -AAAGGTACCTCCC ATCCCCACCACCTCTC-3′ ）和OsKSR7-R（5′ - AAA GGATCCAATCTCCCACAACTCAGACCT-3′ ）扩增OsKSR7 的CDS序列,上下游引物分别引入Kpn Ⅰ和Bam HⅠ酶切位点（下划线表示）。以野生型Kasalash叶片的cDNA为模板进行PCR扩增,PCR产物和pCAMBIA1300载体用Kpn Ⅰ和Bam HⅠ双酶切后连接,用热击法转化大肠杆菌DH5α感受态细胞。阳性克隆抽提质粒后用Kpn Ⅰ和Bam HⅠ双酶切验证再测序确定。鉴定后的质粒用电击法转化农杆菌感受态EHA105,经农杆菌介导的水稻遗传转化体系转入突变体Osksr7 成熟胚诱导的愈伤组织[17]。用Trizol法分别提取T0代转基因植株、野生型和突变体叶片的总RNA,逆转录成cDNA后用引物RT-F（5′ -TAGGTTCTTGCTCCCGGTGTC-3′ ）和RT-R（5′ -ATGGCCTTGACTGACCAATTGTT-3′ ）进行RT-PCR鉴定,选取基因表达量高的转基因植株繁种获得纯合株系后进行表型分析。2 结果
2.1 突变体表型鉴定
对溶液培养7 d的幼苗进行观察,与野生型相比,突变体Osksr7 植株整体矮小,地上部高度为野生型的75.9%（图1-A,表1）,主根、不定根和侧根的长度分别只有野生型的33%、38.9%和35.3%,但不定根数有显著增加（图1-B,表1）。在体式镜下观察根部表型,发现根毛的发生和伸长也都受抑制（图1-C）。根系生长曲线表明突变体Osksr7 根系的伸长明显比野生型缓慢（图2）。Table 1
表1
表1野生型（WT）和突变体Osksr7 生长7 d的植株表型比较（平均值±标准差）
Table 1
| 性状Trait | WT | Osksr7 |
|---|---|---|
| 主根长Primary root length (cm) | 9.4±0.9 | 3.1±0.3** |
| 苗高Plant height (cm) | 11.2±0.8 | 8.5±0.5* |
| 不定根长Adventitious root length (cm)a | 3.6±0.7 | 1.4±0.2** |
| 不定根数Adventitious root number | 3.5±0.6 | 5.2±0.8* |
| 侧根长Lateral root length (cm)b | 1.7±0.3 | 0.6±0.1** |
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图1
新窗口打开|下载原图ZIP|生成PPT图1野生型（WT）和突变体Osksr7 生长7 d的表型
A：野生型（WT）和突变体Osksr7 全株照,bar=2 cm;B：野生型（WT）和突变体Osksr7 根部照,bar= 2 cm;C：野生型（WT）和突变体Osksr7 主根的体视镜照片,bar=1 mm
Fig. 1Phenotypic characterization of 7-day-old seedlings of wild type (WT) and Osksr7 mutant
A: Seedlings of the WT and Osksr7 mutant, bar=2 cm; B: The root of WT and Osksr7 mutant, bar=2 cm; C: The primary root of WT and Osksr7 mutant under stereoscope, bar=1 mm
图2
新窗口打开|下载原图ZIP|生成PPT图2突变体Osksr7 与野生型（WT）的主根（A）和不定根（B）的生长曲线
Fig. 2Growth curve of primary root (A) and adventitious root (B) of Oskrs7 and wide type (WT)
在成熟期考察重要的农艺性状,与野生型相比,突变体Osksr7 主要表现为穗数减少,为野生型的56.3%;茎秆变细,为野生型的70.8%;结实率明显降低,仅为野生型的37.3%。此外株高变矮,叶片变窄,叶长变短,千粒重显著降低（表2）。说明OsKSR7 呈“一因多效”,不仅影响根系发育,而且影响水稻的产量。
Table 2
表2
表2野生型（WT）和突变体Osksr7 的农艺性状比较（平均值±标准差）
Table 2
| 农艺性状Agronomic trait | WT | Osksr7 |
|---|---|---|
| 株高Plant height (cm) | 167.3±3.3 | 138.7±4.0* |
| 穗数Panicle number | 20.8±1.8 | 11.7±2.5** |
| 剑叶长Flag leaf length (cm) | 63.8±2.9 | 52.2.2±3.0* |
| 剑叶宽Flag leaf width (cm) | 1.9±0.1 | 1.5±0.1* |
| 穗长Panicle length (cm) | 33.1±0.5 | 32.4±0.6 |
| 茎杆周长Stem circumference (cm) | 2.4±0.1 | 1.7±0.1* |
| 千粒重1000-grain weight (g) | 17.5±1.3 | 13.7±1.5* |
| 结实率Seed setting rate (%) | 95.1±1.2 | 35.5±2.1** |
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2.2 突变体的遗传分析
将短根突变体Osksr7 与籼稻Kasalath杂交获得F1,F1个体根系正常,溶液培养7 d的338个F2植株中,野生型表型的有257株,突变体表型的有81株（图1-B）,经卡方检验符合孟德尔的单基因控制的遗传规律（χ2=0.19<χ20.05=3.84）（表3）,表明Osksr7 的突变表型由1对隐性核基因控制,命名突变基因为OsKSR7 。Table 3
表3
表3突变体Osksr7 的遗传学分析
Table 3
| 杂交组合 Cross | F1表型 F1 phenotype | F2群体 F2 population | χ2(3﹕1) | ||
|---|---|---|---|---|---|
| 正常型株数Normal plants | 短根株数Short root plants | 总株数Total plants | |||
| Osksr7 /Kasalath | 正常型 Normal type | 257 | 81 | 338 | 0.19 |
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2.3 OsKSR7 的图位克隆
利用混合分离分析法（bulked segregant analysis,BSA）[18]进行OsKSR7 的初定位。用均匀分布于水稻12条染色体的115对SSR引物进行初步定位,发现突变基因与第11染色体上的RM21标记连锁,再用30株F2单株对RM21进行连锁分析,确定了该分子标记与突变位点连锁。之后,扩大定位群体至1 587株,并在RM21的上下游设计新的InDel标记（表4）,最终将基因锁定在分子标记为InD1和InD2之间的143 kb区间内,覆盖有2个BAC克隆：OSJNBb0013M05和OSJNBa0041K10（图3-A）。根据水稻基因组注释信息,该区间有25个预测基因,其中4个为功能基因,对这4个候选基因PCR扩增后测序,发现基因号为LOC_Os11g24560的第一个外显子上ATG后73 bp处的T突变为A,导致编码的氨基酸序列的25位色氨酸（Trp）突变为精氨酸（Arg）。生物信息学分析结果表明,该基因预测编码一个转运蛋白,是负责内质网到高尔基体蛋白运输的COPII有被小泡的SEC23亚基。LOC_Os11g24560基因全长为7 681 bp,编码区为2 382 bp,有12个外显子和11个内含子（图3-B）,蛋白序列为793个氨基酸。
Table 4
表4
表4用于Osksr7 定位的分子标记序列
Table 4
| 标记 Marker | 引物序列 Primer sequence (5′-3′) | 产物大小 Product size (bp) |
|---|---|---|
| RM21 | F：ACAGTATTCCGTAGGCACGG R：GCTCCATGAGGGTGGTAGAG | 157 |
| RM4862 | F：CAACTTTCTGGCATAAACTA R：TGGTGAAAGATATTTCAGAC | 159 |
| InD1 | F：AGAACATAAGAGTAAAAACCA R：AGTAGGTTTCACCATTTTGGA | 99 |
| InD2 | F：AGTGGCTACATTTAGTTTGCT R：ACTGGGGATTGTATGGAGCAG | 123 |
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图3
新窗口打开|下载原图ZIP|生成PPT图3OsKSR7 的图位克隆
A：OsKSR7 在第11染色体上的精细定位;B：OsKSR7 的结构,黑框代表外显子,白框代表非翻译区
Fig. 3Map-based cloning of OsKSR7 gene
A: Fine mapping of OsKSR7 on rice chromosome 11; B: Gene structure of OsKSR7 , Black boxes represent exons, white boxes indicate the untranslated regions
2.4 OsKSR7 的互补验证分析
为了确认Osksr7 突变体表型是由OsKSR7 的突变引起的,构建35S启动子驱动的OsKSR7 的回复载体,以Osksr7 突变体成熟胚愈伤组织为受体,通过农杆菌EHA105介导,成功获得8个根系变长的T0转基因株系。T2代纯合株系溶液培养鉴定,发现Osksr7 突变体的表型回复至野生型状态（图4-A和图4-B）,经RT-PCR验证OsKSR7 在转基因株系中超表达,且该基因在野生型和突变体中的表达水平没有明显差异（图4-C）。说明Osksr7 突变体表型确实是由OsKSR7 突变引起。图4
新窗口打开|下载原图ZIP|生成PPT图4突变体Osksr7 的转基因互补验证
A：野生型（WT）、突变体Osksr7 和2个超表达转基因回复株系（OV1、OV2）的主根体视镜照片,bar=1 mm;B：野生型（WT）、突变体Osksr7 和2个超表达转基因回复株系（OV1、OV2）的全株表型,bar=2 cm;C：RT-PCR检测OsKSR7 的表达
Fig. 4Complementation of the Osksr7 mutant
A: The primary root the WT, Osksr7 and two lines of over-expression transgenic plants (OV1 and OV2) under stereoscope, bar=1 mm; B: Seedlings of the WT, Osksr7 and two lines of over-expression transgenic plants (OV1 and OV2) in the Osksr7 mutant background, bar=2 cm; C: RT-PCR analysis of OsKSR7 expression
3 讨论
3.1 短根突变体Osksr7 突变基因的确定
水稻根系的发育是一个复杂的生物学过程,参与该过程的基因很多。目前报道的根系发育调控基因,多具有“一因多效”性[7]。本研究报道的突变体Osksr7 的主根、不定根、侧根和根毛的生长发育均受到抑制,且成熟期穗数、茎秆粗细和结实率等重要农艺性状都受到显著影响,说明OsKSR7 是一个与水稻生长和单株产量密切相关的重要功能基因。通过基因定位将OsKSR7 定位在水稻第11染色体上,迄今为止,第11染色体上仅报道了一个编码谷氨酰tRNA酰胺基转移酶B亚基的基因OsGatB （LOC_Os11g34210）参与根系发育,Osgatb 突变体主根生长迟缓,但是不定根、侧根以及根毛的生长与野生型相似,苗高也与野生型相似[19]。候选基因测序发现,Osksr7 突变体在LOC_Os11g24560第1个外显子上发生了一个T-A碱基替换,导致编码的Trp突变为Arg。RT-PCR表明突变基因的表达水平在野生型和Osksr7 突变体中无显著差异,35S启动子驱动的OsKSR7 的回复载体能够使Osksr7 突变体的表型回复成野生型,证实LOC_Os11g24560 的单碱基突变是造成Osksr7 突变表型的原因。3.2 突变基因的功能分析
生物信息学分析表明LOC_Os11g24560是COPII有被小泡的SEC23亚基在水稻中的同源蛋白。COPII小泡是真核生物蛋白质分泌途径中的重要组分,介导蛋白质从ER运向Golgi,在保持细胞内各个细胞器动态平衡中发挥重要功能[20]。COPII小泡由5种蛋白亚基组成,分别是小G蛋白Sar1、内衣被组分SEC23和SEC24,外衣被组分SEC13和SEC31,这5个细胞质蛋白在所有真核生物中都高度保守[21]。SEC23作为COPII小泡的内衣被蛋白,折叠形成5个明显的结构域：α螺旋、β折叠、锌指结构、主干区域和C端区域[22]。研究发现不同的物种中存在不同数目的SEC23亚型,酵母中只有1个SEC23亚型（SEC23p）[23],哺乳动物有2个SEC23亚型（SEC23A和SEC23B）[24],拟南芥SEC23有7个亚型（AtSEC23A-G）[25]。SEC23将COPII小泡的各个亚基连接起来,同时也是SAR1的GTP酶激活蛋白（GAP）。研究还发现SEC23能够和其他蛋白相互作用,调控COPII小泡形成的多种过程[26,27,28]。已有研究表明SEC23的部分保守氨基酸残基具有重要作用。酵母SEC23蛋白的第722位精氨酸是其GAP活性所必需的[22]。人SEC23A第382位苯丙氨酸发生突变会降低其对SEC31的亲和力并导致颅缝异型增生病[29,30]。斑马鱼Sec23A第402位亮氨酸突变后表现出和人颅缝异型增生病类似的颅面发育缺陷[31]。研究发现拟南芥AtSEC23A蛋白中保守天冬氨酸残基是其与AtSAR1特异性结合所必需的[32]。本研究OsKSR7 的第25位色氨酸突变为精氨酸后即导致严重的根系发育和农艺性状缺陷,说明该氨基酸残基具有潜在的重要功能。
目前,关于SEC23参与调控植物生长发育的研究较少,主要在拟南芥中进行。TANAKA等[33]研究发现AtSEC24B和AtSEC24C参与雌雄胚子发育,且功能冗余。AtSEC24A对雄性育性[34]、内质网-高尔基体完整性[35]和维持萼片细胞大小分化[36]都是不可或缺的。ZENG等[32]研究表明AtSEC23A和AtSAR1发生特异性结合才能发挥输出内质网蛋白功能。最近研究发现AtSEC23A和AtSEC23D在调控花粉壁形成、花粉粒外壁分化和绒毡层发育中起重要作用[25]。水稻中还没有SEC23 的相关研究报道。OsKSR7 的克隆为揭示SEC23调控水稻根系性状的遗传机理奠定基础,也为水稻根系分子育种提供了基因资源。
4 结论
EMS诱变获得一个水稻短根突变体Osksr7 ,其结实率等重要农艺性状受到显著影响。突变表型受1对隐性核基因控制,为COPII有被小泡的SEC23亚基突变所致。SEC23在调控水稻根系发育及单株产量中起重要作用。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
DOI:10.1093/jxb/ert421URLPMID:24353245 [本文引用: 1]
Abstract Root growth and development are of outstanding importance for the plant's ability to acquire water and nutrients from different soil horizons. To cope with fluctuating nutrient availabilities, plants integrate systemic signals pertaining to their nutritional status into developmental pathways that regulate the spatial arrangement of roots. Changes in the plant nutritional status and external nutrient supply modulate root system architecture (RSA) over time and determine the degree of root plasticity which is based on variations in the number, extension, placement, and growth direction of individual components of the root system. Roots also sense the local availability of some nutrients, thereby leading to nutrient-specific modifications in RSA, that result from the integration of systemic and local signals into the root developmental programme at specific steps. An in silico analysis of nutrient-responsive genes involved in root development showed that the majority of these specifically responded to the deficiency of individual nutrients while a minority responded to more than one nutrient deficiency. Such an analysis provides an interesting starting point for the identification of the molecular players underlying the sensing and transduction of the nutrient signals that mediate changes in the development and architecture of root systems.
DOI:10.1016/j.tplants.2014.08.005URLPMID:25239776 [本文引用: 1]
Roots play an essential role in the acquisition of water and minerals from soils. Measuring crop root architecture and assaying for changes in function can be challenging, but examples have emerged showing that modifications to roots result in higher yield and increased stress tolerance. In this review, we focus mainly on the molecular genetic advances that have been made in altering root system architecture and function in crop plants, as well as phenotyping methods. The future for the modification of crop plant roots looks promising based on recent advances, but there are also important challenges ahead.
DOI:10.1016/j.tplants.2014.02.002URLPMID:24630073 [本文引用: 1]
It is becoming increasingly evident that optimization of root architecture for resource capture is vital for enabling the next green revolution. Although cereals provide half of the calories consumed by humans, root and tuber crops are the second major source of carbohydrates globally. Yet, knowledge of root architecture in root and tuber species is limited. In this opinion article, we highlight what is known about the root system in root and tuber crops, and mark new research directions towards a better understanding of the relation between root architecture and yield. We believe that unraveling the role of root architecture in root and tuber crop productivity will improve global food security, especially in regions with marginal soil fertility and low-input agricultural systems.
URL [本文引用: 1]
为开展水稻根系性状的育种提供理论依据和选择指标，本文对１２个不同穗型水稻品种的根系形态特征与地上部关系及根系性状的遗传进行了研究。结果表明：根数、根重和根直径分别与地上部有关性状相关显著，可作为水稻育种中的根系选择指标。遗传分析显示，上述３个根系性状均为数量遗传，与细胞质基因无关。３个根系性状的遗传变异中，加性、显性和上位性都很重要。
URL [本文引用: 1]
为开展水稻根系性状的育种提供理论依据和选择指标，本文对１２个不同穗型水稻品种的根系形态特征与地上部关系及根系性状的遗传进行了研究。结果表明：根数、根重和根直径分别与地上部有关性状相关显著，可作为水稻育种中的根系选择指标。遗传分析显示，上述３个根系性状均为数量遗传，与细胞质基因无关。３个根系性状的遗传变异中，加性、显性和上位性都很重要。
URLMagsci [本文引用: 1]
为了探讨寒地水稻根系性状与地上部性状在不同生育阶段的关系，以黑龙江省的3个超级稻品种和3个普通品种为试验材料进行田间试验。结果表明，根长在不同的生育时期与结实率、各节间长、粗和粒数、一次支梗数呈正相关；根系体积与粒数、株高、与一次枝梗数和二次枝梗数在分蘖盛期为负相关，拔节孕穗期以后为正相关；根系干重在分蘖盛期与穗数呈极显著的负相关（r=-0.92**），而与穗粒数、一次枝梗数和一次枝梗粒数均呈极显著的正相关的关系。根冠比在齐穗后与产量呈显著或极显著正相关（r=0.84*～0.96**）。
URLMagsci [本文引用: 1]
为了探讨寒地水稻根系性状与地上部性状在不同生育阶段的关系，以黑龙江省的3个超级稻品种和3个普通品种为试验材料进行田间试验。结果表明，根长在不同的生育时期与结实率、各节间长、粗和粒数、一次支梗数呈正相关；根系体积与粒数、株高、与一次枝梗数和二次枝梗数在分蘖盛期为负相关，拔节孕穗期以后为正相关；根系干重在分蘖盛期与穗数呈极显著的负相关（r=-0.92**），而与穗粒数、一次枝梗数和一次枝梗粒数均呈极显著的正相关的关系。根冠比在齐穗后与产量呈显著或极显著正相关（r=0.84*～0.96**）。
DOI:10.1016/j.copbio.2014.11.015URLPMID:25448235 [本文引用: 1]
http://linkinghub.elsevier.com/retrieve/pii/S0958166914002031
DOI:10.1007/s12284-008-9016-5URL [本文引用: 2]
Plant roots have a large range of functions, including acquisition of water and nutrients, as well as structural support. Dissecting the genetic and molecular mechanisms controlling rice root development is critical for the development of new rice ideotypes that are better adapted to adverse conditions and for the production of sustainably achieved rice yield potential. Most knowledge regarding the gene networks involved in root development has been accumulated in the model dicotyledon plant species Arabidopsis thaliana . Rice, the model monocotyledon species, presents several singularities compared to A. thaliana , including a root architecture characterized by a fibrous root system comprising five types of embryonic and postembryonic roots. The anatomy and morphology of the rice root system, which is typical for a cereal, differs from that of A. thaliana , for instance, by the presence of a lysigenous cortex and additional cell layers compared to the dicotyledon model. Moreover, the structure and functions of the root apical meristem (RAM) of rice are distinct from those of A. thaliana . Recently, several rice root mutants have been identified via forward or reverse genetics, and these will aid in forming hypothesis to characterize either the divergence or conservation of genetic pathways relative to A. thaliana . Furthermore, these mutants will help to identify key genes in rice roots that may be missing in A. thaliana . This review summarizes both classical and recent data concerning the molecular genetics of rice root development, including root anatomy and morphology, RAM structure, RAM patterning, and root mutants.
DOI:10.1007/s10725-012-9721-3URL [本文引用: 1]
AbstractAdventitious roots account for the majority of the rice root system and play an irreplaceable role in rice growth and development. Rice adventitious roots are formed by division of the innermost ground meristem cells in the central cylinder, and some lateral roots are observable in the adventitious root system. Multiple hormones have been implicated in the regulation of root development. Auxin is involved in the initiation of adventitious roots, whereas cytokinin inhibits adventitious root initiation, but promotes adventitious root elongation. Other phytohormones such as nitric oxide, ethylene, brassinosteroid, jasmonic acid and gibberellin may be also involved in regulating adventitious root initiation and development. Additionally, more than 600 root development related quantitative trait loci (QTLs) have been located by QTL analysis of root traits.
DOI:10.1016/j.tplants.2016.07.011URLPMID:27524642 [本文引用: 1]
Lateral root formation in cereals is unique with respect to the involved cell types, their position relative to the vascular elements, their stochastic pattern of emergence and their root-type specificity. Genetic analyses demonstrated that auxin signal transduction, polar auxin transport, auxin transport regulation and cell cycle regulation are key elements of lateral root formation in cereals. High resolution tissue- and cell-type-specific transcriptome studies identified candidate genes and metabolic pathways associated with lateral root initiation in cereals. Architectural remodeling of lateral root branching contributes largely to the adaptive plasticity of the root system in response to extrinsic abiotic and biotic factors such as water availability, nutrients status and interaction with arbuscular mycorrhizal fungi.
DOI:10.1104/pp.16.00017URLPMID:26983995 [本文引用: 1]
Abstract Rice (Oryza sativa) is characterized by having fibrous root systems; however, the molecular mechanisms underlying the root development are not fully understood. Here, we isolated a rice mutant with short roots and found that the mutant had a decreased cell size of the roots and shoots compared with the wild-type rice (WT). A map-based cloning combined with whole genome sequencing revealed that a single nucleotide mutation occurred in a gene, which encodes a putative cation-chloride cotransporter (OsCCC1). Introduction of OsCCC1 cDNA into the mutant rescued the mutant growth, indicating that growth defect of both the roots and shoots are caused by loss of function of OsCCC1. Physiological analysis showed that the mutant had a lower concentration of Cl－ and K＋, and lower osmolality in the root cell sap than WT at all KCl supply conditions tested; however, the mutant only showed a lower Na＋ concentration at high external Na＋. Expression of OsCCC1 in yeast increased accumulation of K＋, Na＋ and Cl－. The expression of OsCCC1 was found in both the roots and shoots although higher expression was found in the root tips. Furthermore, the expression in the roots did not respond to different Na＋, K＋ and Cl－ supply. OsCCC1 was expressed in all cells of the roots, leaf and basal node. Immunoblot analysis revealed that OsCCC1 was mainly localized to the plasma membrane. These results suggest that OsCCC1 is involved in the cell elongation by regulating ion (Cl－, K＋ and Na＋) homeostasis to maintain cellular osmotic potential. 08 2016 American Society of Plant Biologists. All rights reserved.
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DOI:10.1007/s00425-015-2296-2URLPMID:25893869 [本文引用: 1]
The involvement of OsKASI in FA synthesis is found to play a critical role in root development of rice. The root system plays important roles in plant nutrient and water acquisition. However, mechanisms of root development and molecular regulation in rice are still poorly understood. Here, we characterized a rice (Oryza sativa L.) mutant with shortened roots due to a defect in cell elongation. Map-based cloning revealed that the mutation occurred in a putative 3-oxoacyl-synthase, an ortholog of -ketoacyl-[acyl carrier protein] synthase I (KASI) in Arabidopsis, thus designated as OsKASI. OsKASI was found to be ubiquitously expressed in various tissues throughout the plant and OsKASI protein was localized in the plastid. In addition, OsKASI deficiency resulted in reduced fertility and a remarkable change in fatty acid (FA) composition and contents in roots and seeds. Our results demonstrate that involvement of OsKASI in FA synthesis is required for root development in rice.
DOI:10.1111/j.1469-8137.2010.03513.xURLPMID:21039568 [本文引用: 1]
090004A rice mutant, Oryza sativa short postembryonic roots 1 (Osspr1), has been characterized. It has short postembryonic roots, including adventitious and lateral roots, and a lower iron content in its leaves.090004OsSPR1 was identified by map-based cloning. It encodes a novel mitochondrial protein with the Armadillo-like repeat domain.090004Osspr1 mutants exhibited decreased root cell elongation. The iron content of the mutant shoots was significantly altered compared with that of wild-type shoots. A similar pattern of alteration of manganese and zinc concentrations in shoots was also observed. Complementation of the mutant confirmed that OsSPR1 is involved in post-embryonic root elongation and iron homeostasis in rice. OsSPR1 was found to be ubiquitously expressed in various tissues throughout the plant. The transcript abundance of various genes involved in iron uptake and signaling via both strategies I and II was similar in roots of wild-type and mutant plants, but was higher in the leaves of mutant plants.090004Thus, a novel mitochondrial protein that is involved in root elongation and plays a role in metal ion homeostasis has been identified.
DOI:10.1007/s00425-008-0718-0URLPMID:18317796 [本文引用: 1]
A short root mutant was isolated from an EMS-generated rice mutant library. Under normal growth conditions, the mutant exhibited short root, delayed flowering, and partial sterility. Some sections of the roots revealed that the cell length along the longitudinal axis was reduced and the cell shape in the root elongation zone shrank. Genetic analysis indicated that the short root phenotype was controlled by a recessive gene. Map-based cloning revealed that a nucleotide substitution causing an amino acid change from Gly to Arg occurred in the predicted rice gene (Os02g0550600). It coded an alkaline/neutral invertase and was homologous to Arabidopsis gene AtCyt-inv1. This gene was designated as OsCyt-inv1. The results of carbohydrate analysis showed an accumulation of sucrose and reduction of hexose in the Oscyt-inv1 mutant. Exogenously supplying glucose could rescue the root growth defects of the Oscyt-inv1 mutant. These results indicated that OsCyt-inv1 played important roles in root cell development and reproductivity in rice.
DOI:10.1105/tpc.108.061655URLPMID:19258439 [本文引用: 1]
In rice (Oryza sativa), the shoot-borne crown roots are the major root type and are initiated at lower stem nodes as part of normal plant development. However, the regulatory mechanism of crown root development is poorly understood. In this work, we show that a WUSCHEL-related Homeobox (WOX) gene, WOX11, is involved in the activation of crown root emergence and growth. WOX11 was found to be expressed in emerging crown roots and later in cell division regions of the root meristem. The expression could be induced by exogenous auxin or cytokinin. Loss-of-function mutation or downregulation of the gene reduced the number and the growth rate of crown roots, whereas overexpression of the gene induced precocious crown root growth and dramatically increased the root biomass by producing crown roots at the upper stem nodes and the base of florets. The expressions of auxin-and cytokinin-responsive genes were affected in WOX11 overexpression and RNA interference transgenic plants. Further analysis showed that WOX11 directly repressed RR2, a type-A cytokinin-responsive regulator gene that was found to be expressed in crown root primordia. The results suggest that W0X11 may be an integrator of auxin and cytokinin signaling that feeds into RR2 to regulate cell proliferation during crown root development.
DOI:10.3389/fpls.2017.00088URLPMID:5281565 [本文引用: 1]
Cytokinins play important roles in regulating plant development, including shoot and root meristems, leaf longevity, and grain yield. However, thein plantafunctions of rice cytokinin receptors have not been genetically characterized yet. Here we isolated a rice mutant,Osckt1, with enhanced tolerance to cytokinin treatment. Further analysis showed thatOsckt1was insensitive to aromatic cytokinins but responded normally to isoprenoid and phenylurea-type cytokinins. Map-based cloning revealed that the mutation occurred in a putative cytokinin receptor gene, histidine kinase 6 (OsHK6).OsCKT1was found to be expressed in various tissues throughout the plant and the protein was located in the endoplasmic reticulum. In addition, whole-genome gene expression profiling analysis showed thatOsCKT1was involved in cytokinin regulation of a number of biological processes, including secondary metabolism, sucrose and starch metabolism, chlorophyll synthesis, and photosynthesis. Our results demonstrate thatOsCKT1plays important roles in cytokinin perception and control of root development in rice.
DOI:10.3864/j.issn.0578-1752.2011.21.001URLMagsci [本文引用: 1]
【目的】鉴定和克隆水稻根毛突变体新基因，了解水稻根毛发育的分子遗传机理。【方法】通过T-DNA插入获得短根毛突变体。采用溶液培养、形态特征观察、杂交后代的表型分离统计及基于图位克隆技术的基因定位等方法，对突变体Ossrh1的表型、遗传和基因精细定位开展研究。【结果】突变体在苗期表现为根毛长度变短，只有野生型长度的36%左右，遗传分析表明该突变性状受1对隐性基因控制，利用Ossrh1和籼稻品种Kasalath杂交构建的F2群体对OsSRH1进行基因定位, 发现与第6染色体上的SSR（simple sequence repeat）标记RM3183和RM193连锁，OsSRH1距它们的遗传距离分别为0.9 cM和1.0 cM。通过在两标记间发展3个新的STS（sequence-tagged site）标记，将OsSRH1精细定位于标记T1757和T1768之间，物理距离约为115 kb。【结论】水稻短根毛突变体Ossrh1的性状由1对隐性核基因控制，该基因位于第6染色体的STS标记T1757和T1768之间115 kb范围内。
DOI:10.3864/j.issn.0578-1752.2011.21.001URLMagsci [本文引用: 1]
【目的】鉴定和克隆水稻根毛突变体新基因，了解水稻根毛发育的分子遗传机理。【方法】通过T-DNA插入获得短根毛突变体。采用溶液培养、形态特征观察、杂交后代的表型分离统计及基于图位克隆技术的基因定位等方法，对突变体Ossrh1的表型、遗传和基因精细定位开展研究。【结果】突变体在苗期表现为根毛长度变短，只有野生型长度的36%左右，遗传分析表明该突变性状受1对隐性基因控制，利用Ossrh1和籼稻品种Kasalath杂交构建的F2群体对OsSRH1进行基因定位, 发现与第6染色体上的SSR（simple sequence repeat）标记RM3183和RM193连锁，OsSRH1距它们的遗传距离分别为0.9 cM和1.0 cM。通过在两标记间发展3个新的STS（sequence-tagged site）标记，将OsSRH1精细定位于标记T1757和T1768之间，物理距离约为115 kb。【结论】水稻短根毛突变体Ossrh1的性状由1对隐性核基因控制，该基因位于第6染色体的STS标记T1757和T1768之间115 kb范围内。
DOI:10.1046/j.1365-313X.2003.01860.xURLPMID:12974815 [本文引用: 1]
We generated T-DNA insertions throughout the rice genome for saturation mutagenesis. More than 1000 flanking sequences were mapped on 12 rice chromosomes. Our results showed that T-DNA tags were not randomly spread on rice chromosomes and were preferentially inserted in gene-rich regions. Few insertions (2.4%) were found in repetitive regions. T-DNA insertions in genic (58.1%) and intergenic regions (41.9%) showed a good correlation with the predicted size distribution of these sequences in the rice genome. Whereas, obvious biases were found for the insertions in the 5'- and 3'-regulatory regions outside the coding regions both at 500-bp size and in introns rather than in exons. Such distribution patterns and biases for T-DNA integration in rice are similar to that of the previous report in Arabidopsis , which may result from T-DNA integration mechanism itself. Rice will require approximately the same number of T-DNA insertions for saturation mutagenesis as will Arabidopsis . A database of the T-DNA insertion sites in rice is publicly available at our web site ( http://www.genomics.zju.edu.cn/ricetdna ).
DOI:10.1073/pnas.88.21.9828URL [本文引用: 1]
DOI:10.3389/fpls.2016.00599URL [本文引用: 1]
A short-root rice mutant was isolated from an ethyl methane sulfonate-mutagenized library. From map-based cloning strategy, a point mutation, resulting in an amino acid change from proline to leucine, was identified in the fourth exon of a glutamyl-tRNA (Gln) amidotransferase B subunit family protein (OsGatB, LOC_Os11g34210). This gene is an ortholog ofArabidopsis GatBand yeastPET112. GatB is a subunit of tRNA-dependent amidotransferase (AdT), an essential enzyme involved in Gln-tRNAGlnsynthesis in mitochondria. Although previous studies have described that cessation in mitochondrial translation is lethal at very early developmental stages in plants, this point mutation resulted in a non-lethal phenotype of smaller root meristem and shorter root cell length. In the root,OsGatBwas predominantly expressed in the root tip and played an important role in cell division and elongation there. OsGatB was localized in the mitochondria, and mitochondrial structure and function were all affected inOsgatbroot tip cells.
DOI:10.1016/j.tcb.2013.08.005URLPMID:24076263 [本文引用: 1]
The vast majority of proteins that are transported to different cellular compartments and secreted from the cell require coat protein complex II (COPII) for export from the endoplasmic reticulum (ER). Many of the molecular mechanisms underlying COPII assembly are understood in great detail, but it is becoming increasingly evident that this basic machinery is insufficient to account for diverse aspects of protein export from the ER that are observed in vivo. Here we review recent data that have furthered our mechanistic understanding of COPII assembly and, in particular, how genetic diseases associated with the early secretory pathway have added fundamental insights into the regulation of ER-derived carrier formation. We also highlight some unresolved issues that future work should address to better understand the physiology of COPII-mediated transport.
DOI:10.1016/j.ceb.2010.04.003URLPMID:20439155 [本文引用: 1]
The small GTPase Sar1 resides at the core of a regulatory cycle that controls protein export from the ER in COPII vesicles. Recent advances in minimally reconstituted systems indicate continual flux of Sar1 through GTPase cycles facilitates cargo concentration into forming vesicles that ultimately bud from membranes. During export from ER membranes, this GTPase cycle is harnessed through the combinatorial power of multiple coat subunits and cargo adaptors to sort an expanding array of proteins into ER-derived vesicles. The COPII budding machinery is further organized into higher-order structures at transitional zones on the ER surface where the large multi-domain Sec16 protein appears to perform a central function.
DOI:10.1038/nature01040URLPMID:12239560 [本文引用: 2]
COPII-coated vesicles form on the endoplasmic reticulum by the stepwise recruitment of three cytosolic components: Sar1-GTP to initiate coat formation, Sec23/24 heterodimer to select SNARE and cargo molecules, and Sec13/31 to induce coat polymerization and membrane deformation. Crystallographic analysis of the Saccharomyces cerevisiae Sec23/24-Sar1 complex reveals a bow-tie-shaped structure, 15 nm long, with a membrane-proximal surface that is concave and positively charged to conform to the size and acidic-phospholipid composition of the COPII vesicle. Sec23 and Sar1 form a continuous surface stabilized by a non-hydrolysable GTP analogue, and Sar1 has rearranged from the GDP conformation to expose amino-terminal residues that will probably embed in the bilayer. The GTPase-activating protein (GAP) activity of Sec23 involves an arginine side chain inserted into the Sar1 active site. These observations establish the structural basis for GTP-dependent recruitment of a vesicular coat complex, and for uncoating through coat-controlled GTP hydrolysis.
DOI:10.1080/07352680701495820URL [本文引用: 1]
All eukaryotes possess a secretory pathway, and the major molecular players involved in secretion are well conserved. However, the morphological manifestation of this pathway at the level of the participant organelles shows great divergences between yeasts, mammals and plants. The unique features of the early secretory pathway in plants-a polydisperse mobile Golgi apparatus and the lack of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus-suggests the participation of many plant-specific molecules in the maintenance and regulation of protein trafficking. The advent of live cell imaging fluorescently-tagged proteins and the increased usage of cryotechniques in electron microscopy has led to dramatic advances in our understanding of the early secretory pathway of plants. In contrast, contradictions have sometimes emerged and interpretations for the same observations have not necessarily reached a consensus. In this review we have attempted to provide the reader with a critical, yet balanced overview of this rapidly expanding research area. Wherever possible we have contrasted a particular event or parameter with the corresponding situation in yeast or mammalian cells. We have also taken the opportunity to suggest suitable experimentation in newly emerging sectors.
DOI:10.1038/ng.405URLPMID:19561605 [本文引用: 1]
Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.
DOI:10.1093/jxb/ery015URLPMID:29390074 [本文引用: 2]
Abstract The specialized multi-layered pollen wall plays multiple functions to ensure normal microspore development. The major components of pollen wall (e.g. sporopollenin and lipidic precursors) are provided from the tapetum. Material export from the endoplasmic reticulum (ER) is mediated by coat protein complex II (COPII) vesicles. The Arabidopsis thaliana genome encodes seven homologs of SEC23, a COPII component. However, the functional importance of this diversity remains elusive. Here, we analyzed knockout and knockdown lines for AtSEC23A and AtSEC23D, two of the A. thaliana SEC23 homologs, respectively. Single atsec23a and atsec23d mutant plants, despite normal fertility, showed an impaired exine pattern. Double atsec23ad mutant plants were semi-sterile and exhibited developmental defects in pollen and tapetal cells. Pollen grains of atsec23ad had defective exine and intine and showed signs of cell degeneration. Moreover, the development of tapetal cells was altered with structural abnormalities in organelles. AtSEC23A and AtSEC23D exhibited the characteristic localization pattern of COPII proteins and were highly expressed in the tapetum. Our work suggests that AtSEC23A and AtSEC23D may organize pollen wall development and exine patterning by regulating ER export of lipids and proteins necessary for pollen wall formation. Also, our results shed light on the functional heterogeneity of SEC23 homologs.
DOI:10.1016/j.tcb.2008.04.006URLPMID:18534853 [本文引用: 1]
Coat protein complex II (COPII) is a multi-subunit protein complex responsible for the formation of membrane vesicles at the endoplasmic reticulum. The assembly of this complex on the endoplasmic reticulum membrane needs to be tightly regulated to ensure efficient and specific incorporation of cargo proteins into nascent vesicles. Recent studies of a genetic disease affecting COPII function, and a structural analysis of COPII subunit interactions emphasize the central role of the Sec23 subunit in COPII coat assembly. Similarly, the demonstration that Sec23 interacts physically and functionally with proteins involved in both vesicle tethering and the transport along microtubules indicates that the Sec23 subunit is crucially important in linking COPII vesicle formation to anterograde transport events.
DOI:10.1038/34438URLPMID:9428766 [本文引用: 1]
Vesicles coated with coat protein complex II (COPII) selectively transport molecules (cargo) and vesicle fusion proteins from the endoplasmic reticulum (ER) to the Golgi complex. We have investigated the role of coat proteins in cargo selection and recruitment. We isolated integral membrane and soluble cargo proteins destined for transport from the ER in complexes formed in the presence of Sar1 and Sec23/24, a subset of the COPII components, and GTP or GMP-PNP. Vesicle fusion proteins of the vSNARE family and Emp24, a member of a putative cargo carrier family, were also found in COPII complexes. The inclusion of amino-acid permease molecules into the complex depended on the presence of Shr3, a protein required for the permease to leave the ER,. Resident ER proteins Sec61, BiP (Kar2) and Shr3 were not included in the complexes, indicating that the COPII components bound specifically to vesicle cargo. COPII-cargo complexes and putative cargo adaptor-cargo complexes were also isolated from COPII vesicles. Our results indicate that cargo packaging signals and soluble cargo adaptors are recognized by a recruitment complex comprising Sar1-GTP and Sec23/24.
DOI:10.1016/j.molcel.2007.03.017URLPMID:17499046 [本文引用: 1]
The mechanism of cargo concentration into ER-derived vesicles involves interactions between the COPII vesicular coat complex and cargo transport signals—peptide sequences of 10–15 residues. The SNARE protein Sec22 contains a signal that binds the COPII subcomplex Sec23/24 and specifies its endoplasmic reticulum (ER) exit as an unassembled SNARE. The 200 kDa crystal structure of Sec22 bound to Sec23/24 reveals that the transport signal is a folded epitope rather than a conventional short peptide sequence. The NIE segment of the SNARE motif folds against the N-terminal longin domain, and this closed form of Sec22 binds at the Sec23/24 interface. Thus, COPII recognizes unassembled Sec22 via a folded epitope, whereas Sec22 assembly into SNARE complexes would mask the NIE segment. The concept of a conformational epitope as a transport signal suggests packaging mechanisms in which a coat is sensitive to the folded state of a cargo protein or the assembled state of a multiprotein complex.
DOI:10.1088/0031-9155/53/6/010URLPMID:16980979 [本文引用: 1]
Abstract Cranio-lenticulo-sutural dysplasia (CLSD) is an autosomal recessive syndrome characterized by late-closing fontanels, sutural cataracts, facial dysmorphisms and skeletal defects mapped to chromosome 14q13-q21 (ref. 1). Here we show, using a positional cloning approach, that an F382L amino acid substitution in SEC23A segregates with this syndrome. SEC23A is an essential component of the COPII-coated vesicles that transport secretory proteins from the endoplasmic reticulum to the Golgi complex. Electron microscopy and immunofluorescence show that there is gross dilatation of the endoplasmic reticulum in fibroblasts from individuals affected with CLSD. These cells also exhibit cytoplasmic mislocalization of SEC31. Cell-free vesicle budding assays show that the F382L substitution results in loss of SEC23A function. A phenotype reminiscent of CLSD is observed in zebrafish embryos injected with sec23a-blocking morpholinos. Our observations suggest that disrupted endoplasmic reticulum export of the secretory proteins required for normal morphogenesis accounts for CLSD.
DOI:10.1016/j.devcel.2007.10.005URLPMID:2262049 [本文引用: 1]
Proteins trafficking through the secretory pathway must first exit the endoplasmic reticulum (ER) through membrane vesicles created and regulated by the COPII coat protein complex. Cranio-lenticulo-sutural dysplasia (CLSD) was recently shown to be caused by a missense mutation in , a gene encoding one of two paralogous COPII coat proteins. We now elucidate the molecular mechanism underlying this disease. In vitro assays reveal that the mutant form of SEC23A poorly recruits the Sec13-Sec31 complex, inhibiting vesicle formation. Surprisingly, this effect is modulated by the Sar1 GTPase paralog used in the reaction, indicating distinct affinities of the two human Sar1 paralogs for the Sec13-Sec31 complex. Patient cells accumulate numerous tubular cargo-containing ER exit sites devoid of observable membrane coat, likely representing an intermediate step in COPII vesicle formation. Our results indicate that the Sar1-Sec23-Sec24 prebudding complex is sufficient to form cargo-containing tubules in vivo, whereas the Sec13-Sec31 complex is required for membrane fission.
DOI:10.1038/ng1880URLPMID:16980978 [本文引用: 1]
Abstract An increasing number of human disorders have been linked to mutations in genes of the secretory pathway. The chemically induced zebrafish crusher variant results in malformed craniofacial skeleton, kinked pectoral fins and a short body length. By positional cloning, we identified a nonsense mutation converting leucine to a stop codon (L402X) in the sec23a gene, an integral component of the COPII complex, which is critical for anterograde protein trafficking between endoplasmic reticulum and Golgi apparatus. Zebrafish crusher mutants develop normally until the onset of craniofacial chondrogenesis. crusher chondrocytes accumulate proteins in a distended endoplasmic reticulum, resulting in severe reduction of cartilage extracellular matrix (ECM) deposits, including type II collagen. We demonstrate that the paralogous gene sec23b is also an essential component of the ECM secretory pathway in chondrocytes. In contrast, knockdown of the COPI complex does not hinder craniofacial morphogenesis. As SEC23A lesions cause the cranio-lenticulo-sutural dysplasia syndrome, crusher provides the first vertebrate model system that links the biology of endoplasmic reticulum to Golgi trafficking with a clinically relevant dysmorphology.
DOI:10.1073/pnas.1519333112URLPMID:26578783 [本文引用: 2]
Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on -amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes.
DOI:10.1007/s00425-013-1913-1URLPMID:23779001 [本文引用: 1]
Anterograde vesicle transport from the endoplasmic reticulum to the Golgi apparatus is the start of protein transport through the secretory pathway, in which the transport is mediated by coat protein complex II (COPII)-coated vesicles. Therefore, most proteins synthesized on the endoplasmic reticulum are loaded as cargo into COPII vesicles. The COPII is composed of the small GTPase Sar1 and two types of protein complexes (Sec23/24 and Sec13/31). Of these five COPII components, Sec24 is thought to recognize cargo that is incorporated into COPII vesicles by directly interacting with the cargo. The Arabidopsis genome encodes three types of Sec24 homologs (AtSec24A, AtSec24B, and AtSec24C). The subcellular dynamics and function of AtSec24A have been characterized. The intracellular distributions and functions of other AtSec24 proteins are not known, and the functional differences among the three AtSec24s remain unclear. Here, we found that all three AtSec24s were expressed in similar parts of the plant body and showed the same subcellular localization pattern. AtSec24B knockout plant, but not AtSec24C knockdown plant, showed mild male sterility with reduction of pollen germination. Significant decrease of AtSec24B and AtSec24C expression affected male and female gametogenesis in Arabidopsis thaliana. Our results suggested that the redundant function of AtSec24B and AtSec24C is crucial for the development of plant reproductive cells. We propose that the COPII transport is involved in male and female gametogenesis in planta.
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DOI:10.1093/jxb/err174URLPMID:21705385 [本文引用: 1]
Eukaryotic cells use COPII-coated carriers for endoplasmic reticulum (ER)-to-Golgi protein transport. Selective cargo capture into ER-derived carriers is largely driven by the SEC24 component of the COPII coat. The Arabidopsis genome encodes three AtSEC24 genes with overlapping expression profiles but it is yet to be established whether the AtSEC24 proteins have overlapping roles in plant growth and development. Taking advantage of Arabidopsis thaliana as a model plant system for studying gene function in vivo, through reciprocal crosses, pollen characterization, and complementation tests, evidence is provided for a role for AtSEC24A in the male gametophyte. It is established that an AtSEC24A loss-of-function mutation is tolerated in the female gametophyte but that it causes defects in pollen leading to failure of male transmission of the AtSEC24A mutation. These data provide a characterization of plant SEC24 family in planta showing incompletely overlapping functions of the AtSEC24 isoforms. The results also attribute a novel role to SEC24 proteins in a multicellular model system, specifically in male fertility.
DOI:10.1105/tpc.109.068270URLPMID:19933201 [本文引用: 1]
The endoplasmic reticulum (ER) is composed of tubules, sheets, and three-way junctions, resulting in a highly conserved polygonal network in all eukaryotes. The molecular mechanisms responsible for the organization of these structures are obscure. To identify novel factors responsible for ER morphology, we employed a forward genetic approach using a transgenic Arabidopsis thaliana plant (GFP-h) with fluorescently labeled ER. We isolated two mutants with defects in ER morphology and designated them endoplasmic reticulum morphology1 (ermo1) and ermo2. The cells of both mutants developed a number of ER-derived spherical bodies, approximately 1 microm in diameter, in addition to the typical polygonal network of ER. The spherical bodies were distributed throughout the ermo1 cells, while they formed a large aggregate in ermo2 cells. We identified the responsible gene for ermo1 to be GNOM-LIKE1 (GNL1) and the gene for ermo2 to be SEC24a. Homologs of both GNL1 and SEC24a are involved in membrane trafficking between the ER and Golgi in yeast and animal cells. Our findings, however, suggest that GNL1/ERMO1 and SEC24a/ERMO2 have a novel function in ER morphology in higher plants.
DOI:10.1104/pp.114.246033URLPMID:25315606 [本文引用: 1]
Size is a critical property of a cell, but how it is determined is still not well understood. The sepal epidermis of Arabidopsis (Arabidopsis thaliana) contains cells with a diversity of sizes ranging from giant cells to small cells. Giant cells have undergone endoreduplication, a specialized cell cycle in which cells replicate their DNA but fail to divide, becoming polyploid and enlarged. Through forward genetics, we have identified a new mutant with ectopic giant cells covering the sepal epidermis. Surprisingly, the mutated gene, SEC24A, encodes a coat protein complex II vesicle coat subunit involved in endoplasmic reticulum-to-Golgi trafficking in the early secretory pathway. We show that the ectopic giant cells of sec24a-2 are highly endoreduplicated and that their formation requires the activity of giant cell pathway genes LOSS OF GIANT CELLS FROM ORGANS, DEFECTIVE KERNEL1, and Arabidopsis CRINKLY4. In contrast to other trafficking mutants, cytokinesis appears to occur normally in sec24a-2. Our study reveals an unexpected yet specific role of SEC24A in endoreduplication and cell size patterning in the Arabidopsis sepal.
