,1,*, 王润豪1, 陈煜2, 付丽娜1, 刘庆庆1, 王清连1Cloning and functional analysis of promoter of potassium transporter gene GhHAK5 in upland cotton (Gossypium hirsutum L.)
CHAO Mao-Ni1, HU Hai-Yan,1,*, WANG Run-Hao1, CHEN Yu2, FU Li-Na1, LIU Qing-Qing1, WANG Qing-Lian1通讯作者:
收稿日期:2019-04-25接受日期:2019-08-9网络出版日期:2019-09-12
| 基金资助: |
Received:2019-04-25Accepted:2019-08-9Online:2019-09-12
| Fund supported: |
作者简介 About authors
E-mail:chaomaoni@126.com。
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晁毛妮, 胡海燕, 王润豪, 陈煜, 付丽娜, 刘庆庆, 王清连. 陆地棉钾转运体基因GhHAK5启动子的克隆与功能分析[J]. 作物学报, 2020, 46(1): 40-51. doi:10.3724/SP.J.1006.2019.94066
CHAO Mao-Ni, HU Hai-Yan, WANG Run-Hao, CHEN Yu, FU Li-Na, LIU Qing-Qing, WANG Qing-Lian.
棉花是我国重要的经济作物, 也是钾敏感作物[1]。据估计, 我国耕地有1/4~1/3的土壤缺钾或严重缺钾[2], 土壤缺钾不仅会影响棉花的产量和纤维品质的形成, 也是引起棉花早衰的重要原因之一[3,4,5,6,7,8]。研究表明, 植物可以通过提高自身的钾吸收效率或调整内源钾的再分配与利用来适应外界的低钾环境[9], 该过程主要通过钾转运体和钾离子通道蛋白两大钾转运系统来完成[10,11]。近年来, 对钾转运系统基因的研究已成为提高作物钾吸收特性和培育钾高效作物品种的一条新途径。
KUP/HAK/KT钾转运体是植物钾转运系统较早发现的一个基因家族, 其成员在介导植物对K+的高亲和性吸收、转运与分配等方面都起着关键作用[12,13]。AtHAK5是拟南芥KUP/HAK/KT家族成员之一, 其表达受低钾胁迫诱导, 也被认为是拟南芥响应钾缺乏的标志性基因[12]。作为拟南芥KUP/HAK/ KT家族中唯一的能在低于10 μmol L-1钾浓度环境下参与钾吸收的高亲和性钾转运体基因[13], 目前关于AtHAK5表达调控的分子机制已开展许多研究。研究者们发现, AtHAK5启动子区特定的顺式作用元件会与相应的转录因子结合来调控该基因的表达, 使植物更好地适应外界低钾环境[14,15]。例如, 转录因子ARF2通过与AtHAK5启动子区生长素响应元件结合来抑制AtHAK5的表达, 并可通过低钾引起的ARF2磷酸化来解除其对AtHAK5表达的抑制, 使其在低钾条件下被诱导并大量表达[14]; 同样地, 转录因子RAP2.11可通过与AtHAK5启动子区ERE结构域和GCC-box位点结合来正向调控AtHAK5在低钾条件下的表达[15]。AtHAK5的表达除受转录水平调控外, 近年来研究发现激酶CIPK23能磷酸化AtHAK5蛋白的N端, 并在转录后水平调控AtHAK5蛋白的活性, 增强其对环境中钾的吸收能力[16]。这些研究表明, 钾转运体基因的表达调控在植物适应外界低钾胁迫方面发挥着重要作用。
启动子对于基因的表达调控至关重要, 其包含的顺式作用元件种类和数量会影响基因的表达模式和强度[17]。因此, 克隆和分析基因的启动子, 有助于了解其表达模式及表达调控机制。陆地棉钾转运体基因GhHAK5是拟南芥AtHAK5的同源基因, 其CDS序列已被克隆, 关于其序列特征和表达特性已进行了初步研究[18], 然而, 关于该基因表达调控机制的研究尚很少开展。本研究以陆地棉品种百棉1号为材料, 对GhHAK5基因启动子2000 bp片段pGhHAK5进行克隆和顺式作用元件分析。为了研究pGhHAK5启动子的功能, 构建了融合表达载体pGhHAK5::GUS并转化拟南芥, 通过对转pGhHAK5拟南芥植株进行GUS组织定位和低钾诱导表达特性分析来探索GhHAK5基因的表达调控模式。研究结果不仅有助于阐明钾转运体基因表达调控的分子机制, 也可为棉花的钾营养性状的改良提供理论依据。
1 材料与方法
1.1 供试材料与植株培养
试验材料陆地棉品种百棉1号由河南科技学院棉花课题组提供, 用于GhHAK5基因启动子2000 bp片段pGhHAK5的克隆和表达特性的分析。将百棉1号种植于河南科技学院光照培养室, 光照强度为450 μmol m-2 s-1, 光周期为(30~33)℃/ 14 h光照, (23~26)℃/10 h黑暗。挑选整齐一致且饱满的棉花脱绒种子于湿沙中萌发和出苗, 待子叶展开后转移至1/2 Hoagland’s营养液[19]中培养。在棉花幼苗生长24、48、72、96、120 h及子叶期和四片真叶期时, 分别取棉花植株的根和叶, 速冻于液氮中并保存于-80℃冰箱, 以供用于GhHAK5基因不同发育时期的表达特性分析。
1.2 DNA的提取及pGhHAK5扩增
以幼嫩的百棉1号根系为材料, 按照DNA提取试剂盒(TIANGEN, DP321)说明书提取基因组DNA。根据pGhHAK5的序列信息, 利用Primer5.0设计引物, 引物序列为pGhHAK5-F: 5°-TTCACGCCCATCTT TATCTC-3°; pGhHAK5-R: 5°-TCAACGTCCTTATCC AATCC-3°。PCR体系50 μL, 包括10 μL 5× Prime STAR GXL buffer (Mg2+ plus)、4.0 μL 2.5 mmol L-1 dNTPs、10 mmol L-1正反向引物各1.5 μL、1 μL 1.2 U μL-1 PrimeSTAR GXL DNA Polymerase (TaKaRa, R050A)、5.0 μL DNA模板和27.0 μL ddH2O。PCR程序为94°C 5 min; 94°C 30 s, 65°C 2.5 min, 32个循环; 72°C 10 min。PCR产物经1%琼脂糖凝胶电泳检测, 确定为目的基因的经胶回收试剂盒(Axygen, AP-GX-4)回收后送华大基因公司测序。1.3 序列分析
利用植物顺式作用元件数据库PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)在线分析启动子的顺式作用元件。从Phyzotome网站(https://phytozome.jgi.doe.gov/pz/ portal.html)下载拟南芥AtHAK5 (AT4G13420.1)、水稻OsHAK1 (LOC_Os04g3292 0.1)、毛果杨PtHAK5.1 (POPTR_0001s03680)ATG上游2000 bp的启动子序列; 从Cottongen网站(https://www.cottongen.org/)下载海岛棉GbHAK5 (GB_D01G 2145)、雷蒙德氏棉GrHAK5 (Gorai.002G213000.1)和亚洲棉GaHAK5 (Ga02G1270)ATG上游2000 bp的启动子序列。利用Clustal软件进行序列比对后, 使用Bioedit 7.0软件分析序列间的相似性。1.4 基因表达特性分析
不同生长发育时间点(幼苗生长24、48、72、96、120 h及4片真叶期)棉花组织(根和叶)的转录组数据下载于NCBI网站SRA (Sequence read archive)数据库(SRA: PRJNA248163[20])。使用软件cufflinks (Version 2.1.1)的FPKM (Fragments per kilobase of exon model per million mapped reads)方法来计算基因的表达量[21]。采用荧光定量PCR (qRT-PCR)的方法进一步分析GhHAK5的表达特性, 内参基因为棉花组成型表达基因Actin (GenBank登录号为AY907703.1)。内参基因和GhHAK5基因的荧光定量PCR引物序列为GhHAK5-F: 5'-GTAAGGACGGGTGGATA-3'; GhHA K5-R: 5'-AGTAAAGCAGGCAAGGTA-3'; Actin-F: 5'-GACCGCATGAGCAAGGAGAT-3'; Actin-R: 5'- GCTGGAAGGTGCTGAGTGAT-3'。以反转录得到的cDNA为模板, 于BIOER荧光定量PCR仪上进行qRT- PCR。反应体系包括2× SYBR Premix Ex Taq (TaKaRa, RR820L) 10 μL、10 mmol L-1正反向引物各0.8 μL、1 μL cDNA模板和7.4 μL ddH2O, 共计20 μL。反应程序为95°C 30 s; 95°C 5 s, 60°C 20 s, 40个循环后增加熔解曲线。每个样品设3次重复, 采用2-ΔΔCt法[22]计算基因的相对表达量。
1.5 载体的构建及农杆菌介导的转化
选用pCAMBIA1381Z(含有GUS基因, 不含启动子)作为表达载体。首先, 根据GhHAK5启动子2000 bp的序列设计特异性引物, 引物序列为pGhHAK5- GUS-F: 5°-CATGCCATGGATTGATTTCAAAAAAA AAATGATATG-3' (下画线为内切酶Nco I的识别序列); pGhHAK5-GUS-R: 5'-CATGCCATGGAAGTT GCGATGACGGTGAG-3' (下画线为内切酶Nco I的识别序列)。接着, 通过PCR扩增将酶切位点Nco I引入pGhHAK5的上下游。对PCR回收产物和载体pCAMBIA1381Z分别用内切酶Nco I进行酶切并胶回收, 载体胶回收产物再用去磷酸化酶CIAP (2250A, Takara)进行去磷酸化并胶回收, 载体酶切并去磷酸化后的胶回收产物和PCR酶切后的胶回收产物用T4连接酶进行连接反应, 将连接产物转化大肠杆菌感受态DH5α, 涂布含有卡纳霉素的LB抗性平板, 37℃培养12 h后, 挑取单克隆, 并进行菌液PCR检测。检测为阳性的菌液提取质粒后进行进一步的酶切验证, 将含有目的基因片段的菌液送华大基因公司测序。最后, 将测序结果与目标序列比对, 序列一致且目的片段连入方向正确的菌液提取质粒后转化根癌农杆菌EHA105。采用农杆菌介导的蘸花法[23]将上述重组表达载体转化野生型拟南芥(Cloumbia-0), 获得T0代转基因种子。1.6 转基因拟南芥植株的筛选和鉴定
将收获的T0代转基因种子种在含有潮霉素抗性的MS培养基上筛选, 经抗性筛选长出的植株为T1代植株, 待T1代植株长出两片真叶后移栽到蛭石中, 在光照培养箱(22℃, 光16 h/暗8 h)中培养。于幼苗期取T1代转基因拟南芥植株的叶片, 用于进一步PCR检测, 检测为阳性的植株于成熟期收获T1代种子。PCR检测方法如下, 首先, 通过DNA提取试剂盒(Tiangen, DP321)提取转基因拟南芥植株和野生型拟南芥(阴性对照)叶片DNA; 然后, 利用目的基因特异性引物F: 5'-ATTGATTTCAAAAAAAAAATG ATATG-3', R: 5'-AAGTTGCGATGACGGTGAGAAA TG-3', 以叶片DNA和表达载体pCAMBIA1381Z- pGhHAK5质粒(阳性对照)为模板进行PCR扩增。最后, 扩增产物经1%脂糖凝胶电泳检测后, 根据检测结果鉴定目标启动子片段是否插入拟南芥基因组DNA中。以同样方法继续对T1代种子进行潮霉素抗性筛选和PCR检测, 收获的T2代种子用于后续的pGhHAK5功能分析。1.7 拟南芥幼苗的低钾处理
将转基因拟南芥种子春化、消毒后分别播种在正常(MS)和低钾(LK, 100 μmol L-1 K+)培养基上[24], 然后于相同条件的光照培养箱(22℃, 光16 h/暗8 h)中培养, 生长2周后, 取整株拟南芥幼苗进行GUS染色。1.8 GUS组织化学染色
参照Jefferson等[25]的方法略加修改, 取拟南芥幼苗和成熟期拟南芥植株的根、叶片、茎、花和荚等组织, 加入GUS染色液, 于37℃温育过夜24 h, 用70%乙醇脱色, 直至底色完全消失, 在体视显微镜(AXIO Zoom.V16, 蔡司)下观察染色结果并照相。2 结果与分析
2.1 GhHAK5基因启动子的克隆
以先前克隆的位于陆地棉D亚组的钾转运体基因GhHAK5[18]ID号Gh_D01G1760检索陆地棉基因组数据库[20], 得到其起始密码子ATG上游2000 bp的启动子序列。在该目标序列的上下游约500 bp范围内设计1对特异性引物, 以百棉1号DNA为模板, 通过PCR方法对GhHAK5启动子2000 bp片段pGhHAK5进行扩增。扩增片段的理论大小为2600 bp, 由图1可以看出, PCR产物电泳检测结果与预期的片段大小相符。PCR产物经测序后与陆地棉TM-1基因组数据库pGhHAK5参考序列比对分析表明, 克隆的2600 bp序列包含完整的目标pGhHAK5片段, 且与TM-1基因组参考序列一致, 表明已成功克隆GhHAK5基因上游2000 bp的启动子序列。图1
新窗口打开|下载原图ZIP|生成PPT图1陆地棉GhHAK5启动子序列的扩增
1: GhHAK5启动子PCR扩增产物; 2: DNA分子量标准(DL2000)。
Fig. 1Amplification of GhHAK5 promoter sequence in upland cotton (Gossypium hirsutum L.)
1: PCR products of GhHAK5 promoter; 2: DNA marker (DL2000).
2.2 pGhHAK5顺式作用元件预测与分析
利用在线启动子元件预测工具PlantCARE对pGhHAK5进行顺式作用元件分析, 发现该启动子除具有CAAT-box和TATA-box等启动子基本核心元件外, 还包括1个提高转录水平的5' UTR Py-rich stretch元件、5个参与光响应的元件(Box I、I-box、G-Box、AT1-motif和Box II 2)、1个生物钟调控元件(circadian)、4个参与植物激素响应的元件(包括1个水杨酸响应元件TCA-element, 1个茉莉酸响应元件CGTCA-motif, 1个生长素响应元件TGA-element和1个乙烯响应元件ERE)、5个逆境胁迫响应元件(包括4个热胁迫响应元件HSE和1个真菌诱导子元件Box-W1); 另外, 还包括4个胚乳表达相关顺式作用调控元件(Skn-1_motif)、1个AT-rich DNA结合蛋白的结合位点(AT-rich element)和5个根特异性表达响应元件[26]等其他类顺式作用元件(表1和图2)。这些结果表明GhHAK5基因的表达可能受光、植物激素、逆境胁迫和生物钟等外界环境条件的调控。Table 1
表1
表1GhHAK5启动子区包含的顺式作用元件
Table 1
| 元件类型 Element type | 名称 Name | 拷贝数 Copy number | 基序 Motif sequence | 功能 Function |
|---|---|---|---|---|
| 基础元件 Basal element | 5UTR Py-rich stretch | 1 | TTTCTTCTCT | 高转录水平顺式作用元件 cis-acting element conferring high transcription levels |
| TATA-box | 61 | TATA/ATATAT/TTTTA | 转录起始位点-30核心启动子元件 Core promoter element around -30 of transcription start | |
| CAAT-box | 18 | CAATT/CAAT/CCAAT | 启动子和增强子区的一般顺式作用元件 Common cis-acting element in promoter and enhancer regions | |
| 光 Light | Box I | 1 | TTTCAAA | 光响应元件 Light responsive element |
| I-box | 1 | ATGATATGA | 部分光响应元件 Part of a light responsive element | |
| G-Box | 1 | CACGTT | 光响应顺式作用调控元件 cis-acting regulatory element involved in light responsiveness | |
| AT1-motif | 1 | ATTAATTTTACA | 部分光响应模块 Part of a light responsive module | |
| Box II | 1 | GTGGATATTATAT | 部分光响应元件 Part of a light responsive element | |
| 生物钟 Circadian | Circadian | 1 | CAANNNNATC | 昼夜节律顺式作用调控元件 cis-acting regulatory element involved in circadian control |
| 植物激素 Phytohormone | CGTCA-motif | 1 | CGTCA | 茉莉酸响应顺式作用元件 cis-acting regulatory element involved in the MeJA-responsiveness |
| TCA-element | 1 | GAGAAGAATA | 水杨酸响应顺式作用元件 cis-acting element involved in salicylic acid responsiveness | |
| TGA-element | 1 | AACGAC | 生长素响应元件 Auxin-responsive element | |
| ERE | 1 | ATTTCAAA | 乙烯响应元件 Ethylene-responsive element | |
| 逆境胁迫 Stress | Box-W1 | 1 | TTGACC | 真菌诱导子响应元件 Fungal elicitor responsive element |
| HSE | 4 | AAAAAATTTC | 热胁迫响应顺式作用元件 cis-acting element involved in heat stress responsiveness | |
| 其他 Other | Skn-1_motif | 4 | GTCAT | 胚乳表达相关顺式调控元件 cis-acting regulatory element required for endosperm expression |
| AT-rich element | 1 | ATAGAAATCAA | AT-rich DNA结合蛋白的结合位点(ATBP-1) Binding site of AT-rich DNA binding protein | |
| Root-specific motif | 5 | ATAAAAT | 根特异性表达响应元件 Root-specific responsive element |
新窗口打开|下载CSV
图2
新窗口打开|下载原图ZIP|生成PPT图2GhHAK5启动子的序列分析
核苷酸的+1位置指的是起始密码子ATG中的A的位置, ATG用红色表示。部分预测的顺式作用元件在图中用灰色阴影表示。1个ARF转录因子结合位点(TGTCNN)在图中用红色加下画线表示。5个根特异性表达元件(ATAAAAT)在图中用红色加粗表示。
Fig. 2Sequence analysis of GhHAK5 promoter
The nucleotide at position +1 is the ATG start codon, the ATG is indicated in red. Parts of the putative cis-regulatory elements are noted under the sequences in shadow. The one ARF binding site (TGTCNN) is underlined in red, and the five root-specific motifs are bold in red.
2.3 GhHAK5与其同源基因启动子序列的比较分析
为了深入了解GhHAK5的表达调控特性, 对GhHAK5及其同源基因包括拟南芥AtHAK5、水稻OsHAK1、毛果杨PtHAK5.1、四倍体棉种海岛棉GbHAK5以及二倍体祖先棉种雷蒙德氏棉(D5)GrHAK5和亚洲棉(A2)GaHAK5基因ATG上游2000 bp的启动子序列比较分析。表明pGhHAK5具有5个根特异性表达调控的元件(ATAAAAT)[26](图3), 暗示着GhHAK5可能是一个在根中特异性高表达的基因。PtHAK5.1是一个在根中特异性高表达的基因(数据来源于Phyzotome网站), 与pGhHAK5一样, pPtHAK5.1也具有5个根特异性表达调控元件(图3)。另外, pGhHAK5含有1个参与低钾条件下转录调控的ARF转录因子结合位点[27](TGTCNN), 但是不含有参与低钾条件下AtHAK5表达调控的RAP2.11转录因子结合位点GCC-box[15](图3)。说明pGhHAK5的表达可能受低钾胁迫调控, 但是可能具有和其他植物不一样的转录调控机制。进一步对棉属4个种HAK5基因启动子序列比较分析发现, pGhHAK5与雷蒙德氏棉pGrHAK5在重要调控元件数量和位置分布上具有较高的一致性, 均含有5个根特异性表达调控元件和1个ARF转录因子结合位点(图3), 且两者启动子间的序列相似性达87.7%; 与海岛棉pGbHAK5在元件数量和分布上差异次之, 且两者启动子间的序列相似性达68.9%, 与亚洲棉pGaHAK5在元件数量和分布上差异最大, 且两者启动子间序列相似性达40.5% (图3)。陆地棉是一个位于D亚组的基因, 陆地棉pGhHAK5与二倍体祖先种雷蒙德氏棉(D5)pGrHAK5在核心元件数量和位置上具较高的一致性, 表明棉花启动子区重要的调控元件或位点在物种进化过程中可能是保守的。图3
新窗口打开|下载原图ZIP|生成PPT图3GhHAK5同源基因启动序列的比较分析
加粗的为棉属4个种钾转运体基因HAK5的启动子。
Fig. 3Comparative analysis of the promoter sequence of GhHAK5 and its homologous gene
The promoter of potassium transporter gene HAK5 of four species in Gossypium are indicated in bold.
2.4 转基因拟南芥阳性植株的鉴定
将2000 bp的启动子片段pGhHAK5通过单酶切法(Nco I)构建到含有GUS基因的表达载体pCAMBIA1381Z (有GUS, 无启动子)上, 并通过农杆菌介导法转化野生型拟南芥(Cloumbia-0), 成熟后收获T0代种子, 经含有潮霉素MS培养基筛选后, 进一步对T1代拟南芥幼苗进行PCR检测。由图4可知, T1代拟南芥幼苗和表达载体pCAMBIA1381Z-pGhH AK5质粒均能扩增到2000 bp的目标条带pGhHAK5, 而野生型拟南芥没有扩增到目标条带, 表明本研究检测的T1代拟南芥幼苗为阳性植株, 成熟后收获T1代种子, 经含有潮霉素MS培养基筛选和PCR检测, 成熟后收获的T2代种子用于后续的功能分析。图4
新窗口打开|下载原图ZIP|生成PPT图4T1代转基因拟南芥植株的PCR检测
M: marker; 1: 野生型拟南芥(阴性对照); 2: 表达载体pCAMBIA1381Z-pGhHAK5质粒(阳性对照); 3~8转基因拟南芥。
Fig. 4PCR analysis of T1 generation transgenic Arabidopsis plants
M: marker; 1: wild Arabidopsis thaliana (negative control); 2: expression vector pCAMBIA1381Z-pGhHAK5 plasmid (positive control); 3-8: transgenic Arabidopsis thaliana.
2.5 pGhHAK5的功能分析
对转pGhHAK5拟南芥植株进行GUS组织化学染色, 结果pGhHAK5驱动的GUS主要在转基因拟南芥幼苗的叶脉(图5-B1, B2)和胚轴维管束组织(图5-B1, B3)中表达, 在根(图5-B1, B4)中表达量较低, 而野生型拟南芥幼苗的各个组织均无染色(图5-A1~A4)。先前研究发现GhHAK5是一个在根中特异性高表达的基因[18], 为了探究pGhHAK5驱动GUS在根中的表达是否受发育时期影响, 本研究进一步对成熟期拟南芥植株的根、叶片、茎、花和荚等组织进行GUS染色。结果转基因拟南芥的叶脉(图5-B5)、根(图5-B11, B12)和花萼维管束组织(图5-B7, B8)均染色较深, 茎(图5-B6)和荚皮(图5-B9, B10)染色较浅; 野生型拟南芥的叶片(图5-A5)、茎(图5-A6)、花(图5-A7, A8)、荚(图5-A9, A10)和根(图5-A11, A12)均没染色。说明pGhHAK5驱动GUS主要在转基因拟南芥成熟的根和地上部维管束组织中表达, 且在拟南芥根中的表达受发育时期的影响。图5
新窗口打开|下载原图ZIP|生成PPT图5转pGhHAK5拟南芥植株的GUS组织化学染色
A: 野生型拟南芥; B: 转pGhHAK5拟南芥; 1~4: 拟南芥幼苗(1)及其叶片(2)、胚轴(3)和根(4)对应位置放大图; 5~12: 成熟期拟南芥植株的叶片(5)、茎(6)、花(7)、花对应位置放大图(8)、荚(9)、荚对应位置放大图(10)、根(11)和根对应位置放大图(12)。
Fig. 5GUS histochemical staining in pGhHAK5 transgenic Arabidopsis thaliana
A: wild-type Arabidopsis thaliana seedlings; B: pGhHAK5 transgenic Arabidopsis thaliana; 1-4: Arabidopsis seedlings (1) and enlarged view of its leaf (2), hypocotyl (3) and roots (4); 5-12: the leaf (5), stem (6), flower (7), enlarged view of flower (8), pod (9), enlarged view of pod (10), roots (11), and enlarged view of roots (12) in mature period Arabidopsis thaliana.
2.6 低钾胁迫对转pGhHAK5拟南芥幼苗GUS表达的影响
为了探究pGhHAK5驱动的GUS在转基因拟南芥幼苗根中微弱的表达是否会受低钾胁迫诱导而增强, 对转pGhHAK5拟南芥幼苗进行低钾胁迫处理。结果与正常供钾(HK)水平相比, 低钾(LK)处理后pGhHAK5驱动GUS在拟南芥幼苗根中的微弱表达并未显著增强(图6), 表明PGhHAK5驱动的GUS在拟南芥幼苗的幼嫩根中的表达不受低钾胁迫诱导, 这可能是植株处于幼苗期时, 植物本身对外界环境中钾元素需求量比较低的缘故。这些研究结果表明本研究克隆的pGhHAK5可能是一个主要在成熟根中具有功能的低钾诱导型启动子。图6
新窗口打开|下载原图ZIP|生成PPT图6转pGhHAK5拟南芥幼苗对低钾胁迫的响应
WT: 野生型拟南芥; HK: 正常钾; LK: 低钾。a: 拟南芥幼苗; b: 叶对应位置放大图; c: 根对应位置放大图。
Fig. 6Low potassium stress response analysis in pGhHAK5 transgenic Arabidopsis thaliana
WT: wild type Arabidopsis thaliana seedlings; HK: high potassium; LK: low potassium. a: Arabidopsis thaliana seedlings; b: enlarged view of leaf; c: enlarged view of roots.
2.7 GhHAK5基因的时空表达特性分析
为了验证pGhHAK5的功能, 对GhHAK5的时空表达特性研究表明, 在棉花幼苗发育前5 d (生长24~ 120 h), GhHAK5在根中表达量很低; 在棉花四片真叶期, 与发育前5 d相比, GhHAK5在根中的表达量迅速上升, 在叶片中的表达量一直很低(图7-A)。进一步荧光定量PCR分析表明, 在棉花四片真叶期, GhHAK5在根中的表达量迅速升高, 而在叶片中的表达量一直处于较低水平(图7-B), 该结果与转录组分析结果相一致。另外, 与子叶期幼嫩的根相比(图7-C), GhHAK5在四叶期根中表达要显著高于其在子叶期幼嫩根中的表达(图7-B)。这些结果表明GhHAK5在根中的表达受发育时期的影响, 该结果与转pGhHAK5拟南芥植株根系GUS染色结果相一致; 另外, 本研究观察到的GhHAK5在叶片中较低的表达也与HAK5基因是一个主要在根中负责外界环境中钾离子吸收的功能相一致。图7
新窗口打开|下载原图ZIP|生成PPT图7陆地棉GhHAK5基因的时空表达特性分析
A: 陆地棉GhHAK5基因时空表达特性的转录组分析; B: 陆地棉GhHAK5基因时空表达特性的荧光定量PCR分析; C: 不同发育时期棉花根系形态的比较; **表示在0.01水平上差异显著。
Fig. 7Spatio-temporal expression analysis of GhHAK5 in upland cotton (Gossypium hirsutum L.)
A: transcriptome analysis of spatio-temporal expression of GhHAK5 in upland cotton; B: quantitative real-time PCR analysis of spatio-temporal expression of GhHAK5 in upland cotton; C: the comparison of roots morphological change at different development stages in cotton; ** Significant at the 0.01 probability level.
3 讨论
KUP/HAK/KT钾转运体家族, 对植物钾营养的吸收具有重要作用。由于土壤中钾离子的浓度往往低于植物生长发育所需的浓度, 植物常常处于低钾环境[28]。已有研究发现, 许多高亲和性的钾转运体基因, 例如拟南芥AtHAK5[29]以及其同源基因大麦HvHAK1[30]、番茄LeHAK5[31]、水稻OsHAK1[32]和陆地棉GhHAK5[18]等, 它们的表达均受低钾条件诱导。研究这些基因表达调控的分子机制, 并通过基因工程手段提高这些基因的表达水平, 对于提高植物的钾吸收效率具有重要意义。本研究对陆地棉钾转运体基因GhHAK5启动子序列分析发现, pGhHAK5除了含有基础元件外, 还含有多个参与光、植物激素、逆境胁迫和生物钟等顺式作用元件(表1和图2), 这些元件的存在, 暗示着GhHAK5基因的表达可能受多种外界条件的影响。先前研究表明, 陆地棉GhHAK5基因的表达受低钾胁迫诱导[18], 但其他逆境胁迫或者养分胁迫是否也能引起GhHAK5基因表达的变化目前还不清楚。在拟南芥中, AtHAK5的表达除受低钾诱导外, 还受其他的一些逆境条件如低钙、盐胁迫和ABA胁迫等不同程度影响[33]。另外, 氮(NO3-)和磷(P)也可诱导拟南芥和番茄HAK5基因表达水平的升高[34]。因此, 在今后的研究中, 有必要在多种逆境胁迫或养分胁迫条件下来进一步研究GhHAK5的表达特性。在许多植物中, 钾转运体基因HAK5的表达具有组织特异性, 其主要在根中表达[12,27,30,32]。本研究对pGhHAK5序列分析发现, pGhHAK5与同源基因的启动子一样, 具有参与根特异性表达的调控元件(ATAAAAT)(图3), 暗示着GhHAK5可能是一个在根中特异性高表达的基因。但是, 本研究对转pGhHAK5拟南芥植株的GUS组织定位分析发现, pGhHAK5驱动的GUS主要在拟南芥成熟的根、叶脉、花萼维管束和胚轴维管束组织中表达(图5), 表明本研究克隆的pGhHAK5片段在拟南芥中不能驱动根特异性的表达调控模式, 尽管先前研究表明GhHAK5是一个在根中特异性高表达的基因[18]。同样地, EgHAK5也是一个在根中特异性高表达的基因, 然而将1.5 kb的EgHAK5启动子片段连上GUS并进行烟草转化后发现, pEgHKK5驱动的GUS除在烟草根中高表达外, 在叶脉和胚轴的维管束组织中也大量地表达[27]。在拟南芥中, AtHAK5基因启动子驱动的GUS仅在拟南芥的根中表达, 本研究观察到的pGhHAK5驱动的非特异性组织表达模式, 可能是由于本研究克隆的启动子片段缺乏一些其他的重要组织特异性调控元件, 也或者是由于pGhHAK5可能需要与棉花中特异的转录因子结合才能激活其组织特异性的表达调控模式。因此, 在今后的研究中, 有必要将pGhHAK5通过转化棉花植株来进一步研究其功能。
钾转运体基因的转录调控被认为是植物响应外界低钾胁迫的一项重要机制[35,36,37]。在拟南芥中, 钾转运体基因AtHAK5是维持植物在低钾条件下高效吸收环境中钾离子的重要基因, 其表达调控除受ARF2和RAP2.11两个转录因子影响外, 可能还有其他多个转录因子参与调控[38]。转录因子ARF2作为低钾条件下调控拟南芥AtHAK5表达的重要参与者, 该基因缺失的突变体与过表达植株具有相同的表型, 它们的高亲和性钾吸收能力均显著提升[14]。本研究克隆的陆地棉GhHAK5启动子具有1个在低钾条件下参与转录调控的与ARF转录因子结合位点(TGTCNN), 说明转录因子ARF可能参与陆地棉GhHAK5的表达调控, 但仍需进一步的实验研究。GCC-box位点是拟南芥AtHAK5启动子序列中可与转录因子RAP2.11结合的重要基序[39], 但是陆地棉GhHAK5启动子序列中不含有GCC-box位点, 表明陆地棉可能具有与拟南芥不一样的表达调控机制。总的来说, 基因的表达调控是一个非常复杂的过程, 一方面单个钾转运体基因表达可能受多个转录因子的调控, 另外多个激酶参与的转录后调控在植物响应钾缺乏和增强钾吸收方面也起着重要作用[40]。因此, 在以后的研究中, 如何将反向遗传学, 如表达数量性状定位方法(eQTL)[41]与正向遗传学的方法相结合来鉴定调控GhHAK5表达的关键因子, 对于我们深入认识陆地棉响应低钾胁迫的分子机理和调控网络至关重要。
4 结论
从陆地棉品种百棉1号中克隆了钾转运体基因GhHAK5上游2000 bp的启动子片段pGhHAK5, 并构建融合表达载体pGhHAK5::GUS。通过转化拟南芥表明pGhHAK5能在拟南芥中驱动GUS的表达, 且其驱动的GUS主要在拟南芥成熟的根和地上部维管束组织中表达, 在幼嫩的根中表达很弱且不受低钾胁迫诱导, 表明本研究克隆的pGhHAK5可能是一个主要在成熟根中具有功能的低钾诱导型启动子。参考文献 原文顺序
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被引期刊影响因子
DOI:10.3724/SP.J.1006.2009.00718URL [本文引用: 1]
缺钾及因缺钾而导致的早衰已成为当前我国棉花生产的主要限制因素, 而根系的生长发育与钾素营养互相影响、关系密切。本试验在生长室内营养液培养条件下，调查缺钾对棉花幼苗根系性状指标的影响, 测定根系游离吲哚乙酸(IAA)和乙烯释放量。结果显示, 与适钾处理(0.50 mmol L-1)相比, 缺钾处理(0.05 mmol L-1)显著抑制了根系伸长和侧根发生, 而且侧根的减少主要由侧根发生区的缩短所致, 侧根发生密度并无变化, 似乎缺钾减慢了侧根发育的进程, 但不改变可以发育为侧根的中柱鞘细胞的发育状况。此外, 细根(0.05~0.20 mm)生长受缺钾的影响最大, 绝对根长、根表面积、根体积及其占总根系的比例均显著降低；中等根(0.25~0.45 mm)受到影响最小, 粗根(>0.45 mm)居中。由于细根的吸收活性强于中等根和粗根, 因而缺钾幼苗的钾营养状况较根系生长更为恶化, 处理4 d和10 d的整株钾积累量仅分别为适钾处理的25%左右和16%左右, 而其总根长和根系总表面积分别相当于适钾处理的35.7%~38.0%(处理4 d)和47.7%~50.6%(处理10 d)。与适钾条件相比，缺钾使根系的游离吲哚乙酸(IAA)含量降低约50%, 而乙烯释放量提高将近6倍, 这可能是缺钾抑制棉花幼苗根系生长的重要原因之一。
DOI:10.3724/SP.J.1006.2009.00718URL [本文引用: 1]
缺钾及因缺钾而导致的早衰已成为当前我国棉花生产的主要限制因素, 而根系的生长发育与钾素营养互相影响、关系密切。本试验在生长室内营养液培养条件下，调查缺钾对棉花幼苗根系性状指标的影响, 测定根系游离吲哚乙酸(IAA)和乙烯释放量。结果显示, 与适钾处理(0.50 mmol L-1)相比, 缺钾处理(0.05 mmol L-1)显著抑制了根系伸长和侧根发生, 而且侧根的减少主要由侧根发生区的缩短所致, 侧根发生密度并无变化, 似乎缺钾减慢了侧根发育的进程, 但不改变可以发育为侧根的中柱鞘细胞的发育状况。此外, 细根(0.05~0.20 mm)生长受缺钾的影响最大, 绝对根长、根表面积、根体积及其占总根系的比例均显著降低；中等根(0.25~0.45 mm)受到影响最小, 粗根(>0.45 mm)居中。由于细根的吸收活性强于中等根和粗根, 因而缺钾幼苗的钾营养状况较根系生长更为恶化, 处理4 d和10 d的整株钾积累量仅分别为适钾处理的25%左右和16%左右, 而其总根长和根系总表面积分别相当于适钾处理的35.7%~38.0%(处理4 d)和47.7%~50.6%(处理10 d)。与适钾条件相比，缺钾使根系的游离吲哚乙酸(IAA)含量降低约50%, 而乙烯释放量提高将近6倍, 这可能是缺钾抑制棉花幼苗根系生长的重要原因之一。
[本文引用: 1]
[本文引用: 1]
DOI:Y2015/V27/I1/71URL [本文引用: 1]
早衰是棉花生长发育的一种异常现象,是大量衰老相关基因差异表达的结果。早衰棉花光合作用、碳水化合物和其他生物大分子合成相关基因大多下调表达,而蛋白、核苷酸、脂类降解和氨基酸、糖类、嘌呤、嘧啶和离子转运体等养分循环利用相关基因大多上调表达；脱落酸（ABA）、乙烯、生长素、茉莉酸（JA）和赤霉素（GA）相关基因大多上调表达,而细胞分裂素合成基因IPT下调表达；NAC和WRKY等转录因子基因也大多上调表达。结合作者在该领域的研究,总结评述了光合作用及大分子降解、养分循环利用、激素和转录因子相关基因在早衰棉花中的表达模式及作用机理。
DOI:Y2015/V27/I1/71URL [本文引用: 1]
早衰是棉花生长发育的一种异常现象,是大量衰老相关基因差异表达的结果。早衰棉花光合作用、碳水化合物和其他生物大分子合成相关基因大多下调表达,而蛋白、核苷酸、脂类降解和氨基酸、糖类、嘌呤、嘧啶和离子转运体等养分循环利用相关基因大多上调表达；脱落酸（ABA）、乙烯、生长素、茉莉酸（JA）和赤霉素（GA）相关基因大多上调表达,而细胞分裂素合成基因IPT下调表达；NAC和WRKY等转录因子基因也大多上调表达。结合作者在该领域的研究,总结评述了光合作用及大分子降解、养分循环利用、激素和转录因子相关基因在早衰棉花中的表达模式及作用机理。
URL [本文引用: 1]
通过分析典型的重衰、轻衰、未衰三类转基因抗虫棉田的土壤养分含量和皮棉产量的相关性,结果表明:土壤有机质、全氮、速效钾含量与棉花早衰有明显的相关性.速效钾含量(x)与皮棉产量(y)呈直线正相关.其回归方程为y=630.77+5.0864x,r=0.9346.
URL [本文引用: 1]
通过分析典型的重衰、轻衰、未衰三类转基因抗虫棉田的土壤养分含量和皮棉产量的相关性,结果表明:土壤有机质、全氮、速效钾含量与棉花早衰有明显的相关性.速效钾含量(x)与皮棉产量(y)呈直线正相关.其回归方程为y=630.77+5.0864x,r=0.9346.
DOI:10.1080/01904169709365272URL [本文引用: 1]
DOI:10.11674/zwyf.14565URL [本文引用: 1]
【目的】研究钾肥用量和施用时期对棉花产量、 纤维品质、 钾肥利用率和棉田钾素平衡的影响,确定钾肥正确的用量和合适的施用时期,可为棉花主产区科学施肥提供依据。【方法】连续2年在山东平原县、 河北威县、 新疆昌吉市进行田间试验。钾肥用量试验山东设K2O 0、 30、 60、 120、 180、 240 kg/hm2,河北设K2O 0、 37.5、 75、 150、 225、 300 kg/hm2,新疆设K2O 0、 18.75、 37.5、 75、 112.5、 150 kg/hm2。钾肥施用时期试验3地点均设不施钾 (CK)、 钾肥100%基施、 钾肥50%基施+50%花期追施、 钾肥50%蕾期+50%铃期追施、 钾肥50%蕾期+50%吐絮期追施、 钾肥50%花期+50%吐絮期追施。【结果】山东、 河北、 新疆试验点上棉花开花期后累积的钾占整个生育期钾素累积量的54%~62%、 70%~73%、 49%~67%。施用钾肥增加皮棉产量和收益,钾肥用量对棉花产量的影响2013年比2012年明显,三个地点钾肥用量对产量的影响新疆>山东>河北,其最高产量施钾量分别为142、 240、 174 kg/hm2,经济最佳施钾量分别为136、 212、 150 kg/hm2。钾肥不同用量除影响某些纤维指标外,对品质的影响没有显著差异。秸秆不还田时,山东和河北试验点钾肥用量180 kg/hm2 和150 kg/hm2时可以维持棉田钾素平衡,而在新疆所有钾肥用量下棉田钾素都是负平衡。河北和新疆50%钾肥蕾期+50%钾肥铃期追施、 山东50%钾肥基施+50%钾肥开花期追施的皮棉产量、 钾肥的农学效率和施钾经济效益高于其他施用时期。钾肥施用时期影响纤维长度、 断裂比强度或纤维伸长率,但因地点和试验年份而不同。施钾时期对纤维整齐度和马克隆值没有显著影响。【结论】钾肥用量对棉花纤维品质没有显著影响,主要影响棉花产量和经济效益。山东试验点钾肥的适宜用量为K2O 180~240 kg/hm2,最佳施用时期为钾肥50%基施+50%开花期追施; 河北试验点钾肥的适宜用量为K2O 150~180 kg/hm2,最佳施用时期为钾肥50%蕾期+50%铃期追施; 新疆试验点钾肥的适宜用量为K2O 112.5~150 kg/hm2,最佳施用时期为钾肥50%蕾期+50%铃期追施。
DOI:10.11674/zwyf.14565URL [本文引用: 1]
【目的】研究钾肥用量和施用时期对棉花产量、 纤维品质、 钾肥利用率和棉田钾素平衡的影响,确定钾肥正确的用量和合适的施用时期,可为棉花主产区科学施肥提供依据。【方法】连续2年在山东平原县、 河北威县、 新疆昌吉市进行田间试验。钾肥用量试验山东设K2O 0、 30、 60、 120、 180、 240 kg/hm2,河北设K2O 0、 37.5、 75、 150、 225、 300 kg/hm2,新疆设K2O 0、 18.75、 37.5、 75、 112.5、 150 kg/hm2。钾肥施用时期试验3地点均设不施钾 (CK)、 钾肥100%基施、 钾肥50%基施+50%花期追施、 钾肥50%蕾期+50%铃期追施、 钾肥50%蕾期+50%吐絮期追施、 钾肥50%花期+50%吐絮期追施。【结果】山东、 河北、 新疆试验点上棉花开花期后累积的钾占整个生育期钾素累积量的54%~62%、 70%~73%、 49%~67%。施用钾肥增加皮棉产量和收益,钾肥用量对棉花产量的影响2013年比2012年明显,三个地点钾肥用量对产量的影响新疆>山东>河北,其最高产量施钾量分别为142、 240、 174 kg/hm2,经济最佳施钾量分别为136、 212、 150 kg/hm2。钾肥不同用量除影响某些纤维指标外,对品质的影响没有显著差异。秸秆不还田时,山东和河北试验点钾肥用量180 kg/hm2 和150 kg/hm2时可以维持棉田钾素平衡,而在新疆所有钾肥用量下棉田钾素都是负平衡。河北和新疆50%钾肥蕾期+50%钾肥铃期追施、 山东50%钾肥基施+50%钾肥开花期追施的皮棉产量、 钾肥的农学效率和施钾经济效益高于其他施用时期。钾肥施用时期影响纤维长度、 断裂比强度或纤维伸长率,但因地点和试验年份而不同。施钾时期对纤维整齐度和马克隆值没有显著影响。【结论】钾肥用量对棉花纤维品质没有显著影响,主要影响棉花产量和经济效益。山东试验点钾肥的适宜用量为K2O 180~240 kg/hm2,最佳施用时期为钾肥50%基施+50%开花期追施; 河北试验点钾肥的适宜用量为K2O 150~180 kg/hm2,最佳施用时期为钾肥50%蕾期+50%铃期追施; 新疆试验点钾肥的适宜用量为K2O 112.5~150 kg/hm2,最佳施用时期为钾肥50%蕾期+50%铃期追施。
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DOI:10.11983/CBB16231URL [本文引用: 1]
钾是植物体内含量最大的阳离子, 在植物生长发育过程的诸多生理生化反应中起关键作用。缺钾会抑制植株根系的生长, 使根冠比降低; 同时阻碍光合产物的合成和向韧皮部转运, 导致生物量下降。因此, 提高植物钾营养的吸收转运和利用效率对于作物品种改良和增产具有重要的理论和生产实践意义。该文综述了植物响应低钾的生理机制和提高植物钾利用效率的四大策略, 并对改善钾营养吸收利用以提高作物产量和品质进行了讨论及展望。
DOI:10.11983/CBB16231URL [本文引用: 1]
钾是植物体内含量最大的阳离子, 在植物生长发育过程的诸多生理生化反应中起关键作用。缺钾会抑制植株根系的生长, 使根冠比降低; 同时阻碍光合产物的合成和向韧皮部转运, 导致生物量下降。因此, 提高植物钾营养的吸收转运和利用效率对于作物品种改良和增产具有重要的理论和生产实践意义。该文综述了植物响应低钾的生理机制和提高植物钾利用效率的四大策略, 并对改善钾营养吸收利用以提高作物产量和品质进行了讨论及展望。
DOI:10.1093/jxb/48.Special_Issue.451URLPMID:21245224 [本文引用: 1]
Plant roots accumulate potassium from a wide range of soil concentrations, utilizing at least two distinct plasma membrane uptake systems with different affinities for the cation. Details on the structure and function of these K(+) transporters are accumulating, but many prominent questions remain regarding regulation of these uptake pathways in varying physiological conditions. Efficient use of the K(+) absorption capacity requires that the activity of all membrane K(+) conductances interact. In this paper, it is shown how intrinsic properties of the major K(+) transporters in the root plasma membrane generate sufficient inward K(+) flux at varying levels of external [K(+)]. In the high affinity range, uptake proceeds via K(+):H(+) symport and kinetic control prevents outward K(+) leakage through inward rectifying channels. Leakage through outward rectifying channels is minimized due to a combination of kinetic control and intrinsic open channel rectification as predicted by the constant field theory. At millimolar external K(+), symport activity is down regulated by the K(+) induced membrane depolarization. In these conditions, channel-mediated K(+) uptake can only explain the observed unidirectional fluxes in intact tissue if the cell switches from a state where the K(+) conductance dominates (K(+)-state) to one where the primary pumps dominate the membrane conductance (pump-state).
DOI:10.1073/pnas.91.20.9272URLPMID:7937754 [本文引用: 1]
Potassium is a major nutrient in higher plants, where it plays a role in turgor regulation, charge balance, leaf movement, and protein synthesis. Terrestrial plants are able to sustain growth at micromolar external K+ concentrations, at which K+ uptake across the plasma membrane of root cells must be energized despite the presence of a highly negative membrane potential. However, the mechanism of energization has long remained obscure. Therefore, whole-cell mode patch clamping has been applied to root protoplasts from Arabidopsis thaliana to characterize membrane currents resulting from the application of micromolar K+. Analysis of whole cell current/voltage relationships in the presence and absence of micromolar K+ enabled direct testing of K+ transport for possible energization by cytoplasmic ATP and the respective trans-membrane gradients of Na+, Ca2+, and H+. Subtracted current/voltage relations for K(+)-dependent membrane currents are independent of ATP and reverse at potentials that imply H(+)-coupled K+ transport with a ratio of 1 H+:K+. Furthermore, the reversal potential of the K+ current shifts negative as external H+ activity is decreased. K(+)-dependent currents saturate in the micromolar concentration range with an apparent Km of 30 microM, a value in close agreement with previously reported Km values for high-affinity K+ uptake. We conclude that our results are consistent with the view that high-affinity K+ uptake in higher plants is mediated by a H+:K+ symport mechanism, competent in driving K+ accumulation to equilibrium ratios in excess of 10(6)-fold.
DOI:10.1104/pp.103.034660URLPMID:14988478 [本文引用: 3]
Potassium (K(+)) is the most abundant cation in plants and is required for plant growth. To ensure an adequate supply of K(+), plants have multiple mechanisms for uptake and translocation. However, relatively little is known about the physiological role of proteins encoded by a family of 13 genes, named AtKT/KUP, that are involved in K(+) transport and translocation. To begin to understand where and under what conditions these transporters function, we used reverse transcription-PCR to determine the spatial and temporal expression patterns of each AtKT/KUP gene across a range of organs and tested whether selected AtKT/KUP cDNAs function as K(+) transporters in Escherichia coli. Many AtKT/KUPs were expressed in roots, leaves, siliques, and flowers of plants grown under K(+)-sufficient conditions (1.75 mm KCl) in hydroponic culture. AtHAK5 was the only gene in this family that was up-regulated upon K(+) deprivation and rapidly down-regulated with resupply of K(+). Ten AtKT/KUPs were expressed in root hairs, but only five were expressed in root tip cells. This suggests an important role for root hairs in K(+) uptake. The growth and rubidium (Rb(+)) uptake of two root hair mutants, trh1-1 (tiny root hairs) and rhd6 (root hair defective), were studied to determine the contribution of root hairs to whole-plant K(+) status. Whole-plant biomass decreased in the root hair mutants only when K(+) concentrations were low; Rb(+) (used as a tracer for K(+)) uptake rates were lower in the mutants at all Rb(+) concentrations. Seven genes encoding AtKUP transporters were expressed in E. coli (AtKT3/KUP4, AtKT/KUP5, AtKT/KUP6, AtKT/KUP7, AtKT/KUP10, AtKT/KUP11, and AtHAK5), and their K(+) transport function was demonstrated.
DOI:10.1016/j.jplph.2014.01.011URL [本文引用: 2]
Cloning and characterizations of plant K+ transport systems aside from Arabidopsis have been increasing over the past decade, favored by the availability of more and more plant genome sequences. Information now available enables the comparison of some of these systems between species. In this review, we focus on three families of plant K+ transport systems that are active at the plasma membrane: the Shaker K+ channel family, comprised of voltage-gated channels that dominate the plasma membrane conductance to K+ in most environmental conditions, and two families of transporters, the HAK/KUP/KT K+ transporter family, which includes some high-affinity transporters, and the HKT K+ and/or Na+ transporter family, in which K+-permeable members seem to be present in monocots only. The three families are briefly described, giving insights into the structure of their members and on functional properties and their roles in Arabidopsis or rice. The structure of the three families is then compared between plant species through phylogenic analyses. Within clusters of ortologues/paralogues, similarities and differences in terms of expression pattern, functional properties and, when known, regulatory interacting partners, are highlighted. The question of the physiological significance of highlighted differences is also addressed. (C) 2014 Elsevier GmbH.
DOI:10.1105/tpc.16.00684URLPMID:27895227 [本文引用: 3]
Potassium (K+) plays crucial roles in plant growth and development. In natural environments, K+ availability in soils is relatively low and fluctuating. Transcriptional regulation of K+ transporter genes is one of the most important mechanisms in the plant's response to K+ deficiency. In this study, we demonstrated that the transcription factor ARF2 (Auxin Response Factor 2) modulates the expression of the K+ transporter gene HAK5 (High Affinity K+ transporter 5) in Arabidopsis thaliana The arf2 mutant plants showed a tolerant phenotype similar to the HAK5-overexpressing lines on low-K+ medium, whose primary root lengths were longer than those of wild-type plants. High-affinity K+ uptake was significantly increased in these plants. ARF2-overexpressing lines and the hak5 mutant were both sensitive to low-K+ stress. Disruption of HAK5 in the arf2 mutant abolished the low-K+-tolerant phenotype of arf2 As a transcriptional repressor, ARF2 directly bound to the HAK5 promoter and repressed HAK5 expression under K+ sufficient conditions. ARF2 can be phosphorylated after low-K+ treatment, which abolished its DNA binding activity to the HAK5 promoter and relieved the inhibition on HAK5 transcription. Therefore, HAK5 transcript could be induced, and HAK5-mediated high-affinity K+ uptake was enhanced under K+ deficient conditions. The presented results demonstrate that ARF2 plays important roles in the response to external K+ supply in Arabidopsis and regulates HAK5 transcription accordingly.
DOI:10.1093/mp/sss003URL [本文引用: 3]
Plants respond to low-nutrient conditions through metabolic and morphology changes that increase their ability to survive and grow. The transcription factor RAP2.11 was identified as a component in the response to low potassium through regulation of the high-affinity K+ uptake transporter AtHAK5 and other components of the low-potassium signal transduction pathway. RAP2.11 was identified through the activation tagging of Arabidopsis lines that contained a luciferase marker driven by the AtHAK5 promoter that is normally only induced by low potassium. This factor bound to a GCC-box of the AtHAK5 promoter in vitro and in vivo. Transcript profiling revealed that a large number of genes were up-regulated in roots by RAP2.11 overexpression. Many regulated genes were identified to be in functional categories that are important in low-K+ signaling. These categories included ethylene signaling, reactive oxygen species production, and calcium signaling. Promoter regions of the up-regulated genes were enriched in the GCCGGC motif also contained in the AtHAK5 promoter. These results suggest that RAP2.11 regulates AtHAK5 expression under low-K+ conditions and also contributes to a coordinated response to low-potassium conditions through the regulation of other genes in the low-K+ signaling cascade.
DOI:10.1104/pp.15.01401URLPMID:26474642 [本文引用: 1]
Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 μm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.
DOI:10.1105/tpc.010412URLPMID:11971132 [本文引用: 1]
Pathogen-inducible plant promoters contain multiple cis-acting elements, only some of which may contribute to pathogen inducibility. Therefore, we made defined synthetic promoters containing tetramers of only a single type of element and present evidence that a range of cis-acting elements (boxes W1, W2, GCC, JERE, S, Gst1, and D) can mediate local gene expression in planta after pathogen attack. The expression patterns of the promoters were monitored during interactions with a number of pathogens, including compatible, incompatible, and nonhost interactions. Interestingly, there were major differences in the inducibilities of the various promoters with the pathogens tested as well as differences in the speed of induction and in the basal expression levels. We also show that defense signaling is largely conserved across species boundaries at the cis-acting element level. Many of these promoters also direct local wound-induced expression, and this provides evidence for the convergence of resistance gene, nonhost, and wound responses at the level of the promoter elements. We have used these cis-acting elements to construct improved synthetic promoters and show the effects of varying the number, order, and spacing of such elements. These promoters are valuable additions to the study of signaling and transcriptional activation during plant-pathogen interactions.
DOI:10.3724/SP.J.1006.2018.00236URL [本文引用: 6]
KUP/HAK/KT钾转运体基因家族对植物吸收钾离子发挥重要作用, 鉴定和克隆棉花的钾转运体基因, 对于改良棉花的钾吸收特性, 提高棉花的产量和品质具有重要意义。基于已测序的陆地棉基因组序列, 本研究通过同源克隆的方法鉴定到陆地棉钾转运体基因GhHAK5, 并以陆地棉品种百棉1号为材料对其CDS序列进行扩增。结果表明, GhHAK5基因的CDS全长为2451 bp, 编码816个氨基酸, 分子量和等电点分别为91.23 kD和8.15。GhHAK5蛋白具有KUP/HAK/KT家族基因的保守结构域“K-trans”(Pfam02705)和标志性序列GXXXGDXXXSPLY, 并具有11个跨膜区。在进化上, GhHAK5蛋白与拟南芥AtHAK5亲缘关系最近, 其次是与水稻的OsHAK5, 它们同属Cluster I进化簇。亚细胞定位结果显示, GhHAK5是一个定位于质膜的蛋白, 这与其主要作为钾转运子参与K+吸收的功能是一致的。GhHAK5基因在根中表达量最高, 在茎、叶、花瓣、纤维和花萼中表达量很低, 且其表达受外界低钾环境诱导。本研究结果为进一步了解GhHAK5基因的功能及培育钾高效棉花品种奠定了基础。
DOI:10.3724/SP.J.1006.2018.00236URL [本文引用: 6]
KUP/HAK/KT钾转运体基因家族对植物吸收钾离子发挥重要作用, 鉴定和克隆棉花的钾转运体基因, 对于改良棉花的钾吸收特性, 提高棉花的产量和品质具有重要意义。基于已测序的陆地棉基因组序列, 本研究通过同源克隆的方法鉴定到陆地棉钾转运体基因GhHAK5, 并以陆地棉品种百棉1号为材料对其CDS序列进行扩增。结果表明, GhHAK5基因的CDS全长为2451 bp, 编码816个氨基酸, 分子量和等电点分别为91.23 kD和8.15。GhHAK5蛋白具有KUP/HAK/KT家族基因的保守结构域“K-trans”(Pfam02705)和标志性序列GXXXGDXXXSPLY, 并具有11个跨膜区。在进化上, GhHAK5蛋白与拟南芥AtHAK5亲缘关系最近, 其次是与水稻的OsHAK5, 它们同属Cluster I进化簇。亚细胞定位结果显示, GhHAK5是一个定位于质膜的蛋白, 这与其主要作为钾转运子参与K+吸收的功能是一致的。GhHAK5基因在根中表达量最高, 在茎、叶、花瓣、纤维和花萼中表达量很低, 且其表达受外界低钾环境诱导。本研究结果为进一步了解GhHAK5基因的功能及培育钾高效棉花品种奠定了基础。
DOI:10.1038/srep21060URLPMID:26879005 [本文引用: 1]
Proteomics was employed to investigate the molecular mechanisms of apoplastic response to potassium(K)-deficiency in cotton. Low K (LK) treatment significantly decreased the K and protein contents of xylem sap. Totally, 258 peptides were qualitatively identified in the xylem sap of cotton seedlings, of which, 90.31% were secreted proteins. Compared to the normal K (NK), LK significantly decreased the expression of most environmental-stress-related proteins and resulted in a lack of protein isoforms in the characterized proteins. For example, the contents of 21 Class Ш peroxidase isoforms under the LK were 6 to 44% of those under the NK and 11 its isoforms were lacking under the LK treatment; the contents of 3 chitinase isoforms under LK were 11-27% of those under the NK and 2 its isoforms were absent under LK. In addition, stress signaling and recognizing proteins were significantly down-regulated or disappeared under the LK. In contrast, the LK resulted in at least 2-fold increases of only one peroxidase, one protease inhibitor, one non-specific lipid-transfer protein and histone H4 and in the appearance of H2A. Therefore, K deficiency decreased plant tolerance to environmental stresses, probably due to the significant and pronounced decrease or disappearance of a myriad of stress-related proteins.
DOI:10.1038/nbt.3207URLPMID:25893781 [本文引用: 2]
Upland cotton is a model for polyploid crop domestication and transgenic improvement. Here we sequenced the allotetraploid Gossypium hirsutum L. acc. TM-1 genome by integrating whole-genome shotgun reads, bacterial artificial chromosome (BAC)-end sequences and genotype-by-sequencing genetic maps. We assembled and annotated 32,032 A-subgenome genes and 34,402 D-subgenome genes. Structural rearrangements, gene loss, disrupted genes and sequence divergence were more common in the A subgenome than in the D subgenome, suggesting asymmetric evolution. However, no genome-wide expression dominance was found between the subgenomes. Genomic signatures of selection and domestication are associated with positively selected genes (PSGs) for fiber improvement in the A subgenome and for stress tolerance in the D subgenome. This draft genome sequence provides a resource for engineering superior cotton lines.
DOI:10.1038/nbt.1621URLPMID:20436464 [本文引用: 1]
High-throughput mRNA sequencing (RNA-Seq) promises simultaneous transcript discovery and abundance estimation. However, this would require algorithms that are not restricted by prior gene annotations and that account for alternative transcription and splicing. Here we introduce such algorithms in an open-source software program called Cufflinks. To test Cufflinks, we sequenced and analyzed >430 million paired 75-bp RNA-Seq reads from a mouse myoblast cell line over a differentiation time series. We detected 13,692 known transcripts and 3,724 previously unannotated ones, 62% of which are supported by independent expression data or by homologous genes in other species. Over the time series, 330 genes showed complete switches in the dominant transcription start site (TSS) or splice isoform, and we observed more subtle shifts in 1,304 other genes. These results suggest that Cufflinks can illuminate the substantial regulatory flexibility and complexity in even this well-studied model of muscle development and that it can improve transcriptome-based genome annotation.
DOI:10.1006/meth.2001.1262URL [本文引用: 1]
DOI:10.1046/j.1365-313x.1998.00343.xURLPMID:10069079 [本文引用: 1]
The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
[本文引用: 1]
[本文引用: 1]
URLPMID:3327686 [本文引用: 1]
We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
DOI:10.1016/j.mod.2012.03.002URL [本文引用: 2]
Marker genes are specifically expressed in a tissue, organ or time of development. Here we used a computational screen to identify marker genes of the root in Arabidopsis thaliana. We mined the existing transcriptome datasets for genes having high expression in roots while being low in all other organs under a wide range of growth conditions. We show that the root-specificity of these genes is conserved in the sister species Arabidopsis lyrata, indicating that their expression pattern is under selective pressure. We delineated the cis-regulatory elements responsible for root-specific expression and validated two third of those in planta as bona fide root-specific regulatory sequences. We identified three motifs over-represented in these sequences, which mutation resulted in alteration of root-specific expression, demonstrating that these motifs are functionally relevant. In addition, the three motifs are also over-represented in the cis-regulatory regions of the A. lyrata orthologs of our root-specific genes, and this despite an overall low degree of sequence conservation of these regions. Our results provide a resource to assess root-identity in the model genus Arabidopsis and shed light on the evolutionary history of gene regulation in plants. (c) 2012 Elsevier Ireland Ltd.
DOI:10.1007/s11240-017-1256-xURL [本文引用: 3]
URL [本文引用: 1]
钾是植物生长发育所必需的矿质营养元素之一。不同种类植物的钾营养效率存在差异, 已有证据表明这种差异是受遗传基因控制的。植物细胞依靠细胞膜上的各种钾转运体和通道蛋白吸收和转运钾离子, 这些膜蛋白的活性调控是植物钾营养效率调控的关键和基础。本文对植物钾营养高效性状分子遗传机制以及相关基因的分子功能和调控机制的研究进展进行了简要评述, 并讨论了改善作物钾营养高效性状的可能途径。
URL [本文引用: 1]
钾是植物生长发育所必需的矿质营养元素之一。不同种类植物的钾营养效率存在差异, 已有证据表明这种差异是受遗传基因控制的。植物细胞依靠细胞膜上的各种钾转运体和通道蛋白吸收和转运钾离子, 这些膜蛋白的活性调控是植物钾营养效率调控的关键和基础。本文对植物钾营养高效性状分子遗传机制以及相关基因的分子功能和调控机制的研究进展进行了简要评述, 并讨论了改善作物钾营养高效性状的可能途径。
DOI:10.1104/pp.104.057216URLPMID:15734909 [本文引用: 1]
Potassium is an important macronutrient and the most abundant cation in plants. Because soil mineral conditions can vary, plants must be able to adjust to different nutrient availabilities. Here, we used Affymetrix Genechip microarrays to identify genes responsive to potassium (K(+)) deprivation in roots of mature Arabidopsis (Arabidopsis thaliana) plants. Unexpectedly, only a few genes were changed in their expression level after 6, 48, and 96 h of K(+) starvation even though root K(+) content was reduced by approximately 60%. AtHAK5, a potassium transporter gene from the KUP/HAK/KT family, was most consistently and strongly up-regulated in its expression level across 48-h, 96-h, and 7-d K(+) deprivation experiments. AtHAK5 promoter-beta-glucuronidase and -green fluorescent protein fusions showed AtHAK5 promoter activity in the epidermis and vasculature of K(+) deprived roots. Rb(+) uptake kinetics in roots of athak5 T-DNA insertion mutants and wild-type plants demonstrated the absence of a major part of an inducible high-affinity Rb(+)/K(+) (K(m) approximately 15-24 microm) transport system in athak5 plants. In comparative analyses, uptake kinetics of the K(+) channel mutant akt1-1 showed that akt1-1 roots are mainly impaired in a major transport mechanism, with an apparent affinity of approximately 0.9 mm K(+)(Rb(+)). Data show adaptation of apparent K(+) affinities of Arabidopsis roots when individual K(+) transporter genes are disrupted. In addition, the limited transcriptome-wide response to K(+) starvation indicates that posttranscriptional mechanisms may play important roles in root adaptation to K(+) availability in Arabidopsis. The results demonstrate an in vivo function for AtHAK5 in the inducible high-affinity K(+) uptake system in Arabidopsis roots.
DOI:10.1105/tpc.9.12.2281URLPMID:9437867 [本文引用: 2]
The high-affinity K+ uptake system of plants plays a crucial role in nutrition and has been the subject of extensive kinetic studies. However, major components of this system remain to be identified. We isolated a cDNA from barley roots, HvHAK1, whose translated sequence shows homology to the Escherichia coli Kup and Schwanniomyces occidentalis HAK1 K+ transporters. HvHAK1 conferred high-affinity K+ uptake to a K(+)-uptake-deficient yeast mutant exhibiting the hallmark characteristics of the high-affinity K+ uptake described for barley roots. HvHAK1 also mediated low-affinity Na+ uptake. Another cDNA (HvHAK2) encoding a polypeptide 42% identical to HvHAK1 was also isolated. Analysis of several genomes of Triticeae indicates that HvHAK1 belongs to a multigene family. Translated sequences from bacterial DNAs and Arabidopsis, rice, and possibly human cDNAs show homology to the Kup-HAK1-HvHAK1 family of K+ transporters.
DOI:10.1104/pp.008854URLPMID:12428001 [本文引用: 1]
Mineral nutrient deficiencies constitute major limitations for plant growth on agricultural soils around the world. To identify genes that possibly play roles in plant mineral nutrition, we recently generated a high-density array consisting of 1,280 genes from tomato (Lycopersicon esculentum) roots for expression profiling in nitrogen (N) nutrition. In the current study, we used the same array to search for genes induced by phosphorus (P), potassium (K(+)), and iron (Fe) deficiencies. RNA gel-blot analysis was conducted to study the time-dependent kinetics for expression of these genes in response to withholding P, K, or Fe. Genes previously not associated with P, K, and Fe nutrition were identified, such as transcription factor, mitogen-activated protein (MAP) kinase, MAP kinase kinase, and 14-3-3 proteins. Many of these genes were induced within 1 h after withholding the specific nutrient from roots of intact plants; thus, RNA gel-blot analysis was repeated for specific genes (transcription factor and MAP kinase) in roots of decapitated plants to investigate the tissue-specific location of the signal triggering gene induction. Both genes were induced just as rapidly in decapitated plants, suggesting that the rapid response to the absence of P, K, or Fe in the root-bathing medium is triggered either by a root-localized signal or because of root sensing of the mineral environment surrounding the roots. We also show that expression of Pi, K, and Fe transporter genes were up-regulated by all three treatments, suggesting coordination and coregulation of the uptake of these three essential mineral nutrients.
DOI:10.1104/pp.007781URLPMID:12376644 [本文引用: 2]
Plants take up large amounts of K(+) from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions. Transport of K(+) from the soil solution to its final destination is mediated by channels and transporters. To better understand K(+) movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativa cv Nipponbare). By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1-17) encoding transporters of this family and obtained evidence of the existence of other two genes. Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions. The three transporters mediate K(+) influxes or effluxes, depending on the conditions of the experiment. A comparative kinetic analysis of HAK-mediated K(+) influx in yeast and in roots of K(+)-starved rice seedlings demonstrated the involvement of HAK transporters in root K(+) uptake. We discuss that all HAK transporters may mediate K(+) transport, but probably not only in the plasma membrane. Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast.
DOI:10.7668/hbnxb.2014.06.036URL [本文引用: 1]
为研究在不同逆境,如低钾、低钙、NaCl及ABA胁迫下,对拟南芥高亲和性钾转运体基因 AtHAK5 表达的影响,在对含有 promoter AtHAK5::GUS 融合基因的拟南芥转基因植株进行组织化学染色基础上进行Real time RT-PCR检测。结果表明,这些逆境条件可以引起拟南芥 AtHAK5 基因表达量的上调。同时在对拟南芥Col-0和 AtHAK5 缺失突变体 athak5 表型对比分析,发现 AtHAK5 参与植物根对盐胁迫及ABA的反应。
DOI:10.7668/hbnxb.2014.06.036URL [本文引用: 1]
为研究在不同逆境,如低钾、低钙、NaCl及ABA胁迫下,对拟南芥高亲和性钾转运体基因 AtHAK5 表达的影响,在对含有 promoter AtHAK5::GUS 融合基因的拟南芥转基因植株进行组织化学染色基础上进行Real time RT-PCR检测。结果表明,这些逆境条件可以引起拟南芥 AtHAK5 基因表达量的上调。同时在对拟南芥Col-0和 AtHAK5 缺失突变体 athak5 表型对比分析,发现 AtHAK5 参与植物根对盐胁迫及ABA的反应。
DOI:10.1111/ppl.12205URLPMID:24716623 [本文引用: 1]
The high-affinity K(+) transporter HAK5 is a key system for root K(+) uptake and, under very low external K(+), the only one capable of supplying K(+) to the plant. Functional HAK5-mediated K(+) uptake should be tightly regulated for plant adaptation to different environmental conditions. Thus, it has been described that the gene encoding the transporter is transcriptionally regulated, being highly induced under K(+) limitation. Here we show that environmental conditions, such as the lack of K(+), NO(3)(-) or P, that induced a hyperpolarization of the plasma membrane of root cells, induce HAK5 transcription. However, only the deprivation of K(+) produces functional HAK5-mediated K(+) uptake in the root. These results suggest on the one hand the existence of a posttranscriptional regulation of HAK5 elicited by the low K(+) signal and on the other that HAK5 may be involved in yet-unknown functions related to NO(3)(-) and P deficiencies. These results have been obtained here with Solanum lycopersicum (cv. Micro-Tom) as well as Arabidopsis thaliana plants, suggesting that the posttranscriptional regulation of high-affinity HAK transporters take place in all plant species.
DOI:10.1093/jxb/erj034URLPMID:16364949 [本文引用: 1]
The availability of potassium to the plant is highly variable, due to complex soil dynamics, which are strongly influenced by root-soil interactions. A low plant potassium status triggers expression of high affinity K+ transporters, up-regulates some K+ channels, and activates signalling cascades, some of which are similar to those involved in wounding and other stress responses. The molecules that signal low K+ status in plants include reactive oxygen species and phytohormones, such as auxin, ethylene and jasmonic acid. Apart from up-regulation of transport proteins and adjustment of metabolic processes, potassium deprivation triggers developmental responses in roots. All these acclimation strategies enable plants to survive and compete for nutrients in a dynamic environment with a variable availability of potassium.
DOI:10.1146/annurev-arplant-050312-120153URLPMID:23330792 [本文引用: 1]
As one of the most important mineral nutrient elements, potassium (K(+)) participates in many plant physiological processes and determines the yield and quality of crop production. In this review, we summarize K(+) signaling processes and K(+) transport regulation in higher plants, especially in plant responses to K(+)-deficiency stress. Plants perceive external K(+) fluctuations and generate the initial K(+) signal in root cells. This signal is transduced into the cytoplasm and encoded as Ca(2+) and reactive oxygen species signaling. K(+)-deficiency-induced signals are subsequently decoded by cytoplasmic sensors, which regulate the downstream transcriptional and posttranslational responses. Eventually, plants produce a series of adaptive events in both physiological and morphological alterations that help them survive K(+) deficiency.
DOI:10.1093/jxb/ert402URL [本文引用: 1]
Potassium is a major inorganic constituent of the living cell and the most abundant cation in the cytosol. It plays a role in various functions at the cell level, such as electrical neutralization of anionic charges, protein synthesis, long- and short-term control of membrane polarization, and regulation of the osmotic potential. Through the latter function, K is involved at the whole-plant level in osmotically driven functions such as cell movements, regulation of stomatal aperture, or phloem transport. Thus, plant growth and development require that large amounts of K are taken up from the soil and translocated to the various organs. In most ecosystems, however, soil K availability is low and fluctuating, so plants have developed strategies to take up K more efficiently and preserve vital functions and growth when K availability is becoming limited. These strategies include increased capacity for high-affinity K uptake from the soil, K redistribution between the cytosolic and vacuolar pools, ensuring cytosolic homeostasis, and modification of root system development and architecture. Our knowledge about the mechanisms and signalling cascades involved in these different adaptive responses has been rapidly growing during the last decade, revealing a highly complex network of interacting processes. This review is focused on the different physiological responses induced by K deprivation, their underlying molecular events, and the present knowledge and hypotheses regarding the mechanisms responsible for K sensing and signalling.
DOI:10.1093/pcp/pct094URL [本文引用: 1]
Potassium (K) is an essential macronutrient for plant growth and reproduction. HAK5, an Arabidopsis high-affinity K transporter gene, plays an important role in K uptake. Its expression is up-regulated in response to K deprivation and is rapidly down-regulated when sufficient K levels have been re-established. To identify transcription factors regulating HAK5, an Arabidopsis TF FOX (Transcription Factor Full-length cDNA Over-eXpressor) library containing approximately 800 transcription factors was used to transform lines previously transformed with a luciferase reporter gene whose expression was driven by the HAK5 promoter. When grown under sufficient K levels, 87 lines with high luciferase activity were identified, and endogenous HAK5 expression was confirmed in 27 lines. Four lines overexpressing DDF2 (Dwarf and Delayed Flowering 2), JLO (Jagged Lateral Organs), TFII_A (Transcription initiation Factor II_A gamma chain) and bHLH121 (basic Helix-Loop-Helix 121) were chosen for further characterization by luciferase activity, endogenous HAK5 level and root growth in K-deficient conditions. Further analysis showed that the expression of these transcription factors increased in response to low K and salt stress. In comparison with controls, root growth under low K conditions was better in each of these four TF FOX lines. Activation of HAK5 expression by these four transcription factors required at least 310 bp of upstream sequence of the HAK5 promoter. These results indicate that at least these four transcription factors can bind to the HAK5 promoter in response to K limitation and activate HAK5 expression, thus allowing plants to adapt to nutrient stress.
DOI:10.1093/mp/sss003URL [本文引用: 1]
Plants respond to low-nutrient conditions through metabolic and morphology changes that increase their ability to survive and grow. The transcription factor RAP2.11 was identified as a component in the response to low potassium through regulation of the high-affinity K+ uptake transporter AtHAK5 and other components of the low-potassium signal transduction pathway. RAP2.11 was identified through the activation tagging of Arabidopsis lines that contained a luciferase marker driven by the AtHAK5 promoter that is normally only induced by low potassium. This factor bound to a GCC-box of the AtHAK5 promoter in vitro and in vivo. Transcript profiling revealed that a large number of genes were up-regulated in roots by RAP2.11 overexpression. Many regulated genes were identified to be in functional categories that are important in low-K+ signaling. These categories included ethylene signaling, reactive oxygen species production, and calcium signaling. Promoter regions of the up-regulated genes were enriched in the GCCGGC motif also contained in the AtHAK5 promoter. These results suggest that RAP2.11 regulates AtHAK5 expression under low-K+ conditions and also contributes to a coordinated response to low-potassium conditions through the regulation of other genes in the low-K+ signaling cascade.
DOI:10.1016/j.semcdb.2017.07.009URLPMID:28711523 [本文引用: 1]
The HAK/KUP/KT family of potassium (K+) transporters belongs to the amino acid-polyamine-organocation (APC) superfamily of carriers for secondary active transport and has been widely associated with K+ transport across membranes in bacteria, fungi, and plants. The plant genome contains large number of HAK/KUP/KT transporters, and they show the diverse roles in K+ uptake and translocation, salt tolerance and osmotic potential regulation, as well as in controlling root morphology and shoot phenotyping. Recently, significant progress has been achieved towards uncovering the regulatory mechanisms of HAK/KUP/KT transporters at both transcriptional and post-translational levels. Most of the HAK/KUP/KT genes were regulated at transcriptional level, and such regulation may contribute to the alteration of root cell membrane potential by different growth conditions. At least six transcription factors have been identified as positive or negative regulators of HAK/KUP/KT gene expression in responding to external K+ supply. The HAK/KUP/KT transporter proteins can be phosphorylated by CIPK-CBL complexes for activating their function in K+ uptake and probably signaling. Nevertheless, it is still not known if HAK/KUP/KT transporters are involved in K+-sensing and K+-compartmentation in plant cells. Some orthologues of the HAK/KUP/KT transporters from different species show varied physiological functions and some plant species lack an entire sub-clade of HAK/KUT/KT transporters. We are still a long way from unraveling the molecular mechanism of HAK/KUP/KT involved in K+-sensing and signaling pathways in plants.
DOI:10.1111/j.1467-7652.2009.00460.xURLPMID:20055957 [本文引用: 1]
An expression Quantitative Trait Locus or eQTL is a chromosomal region that accounts for a proportion of the variation in abundance of a mRNA transcript observed between individuals in a genetic mapping population. A single gene can have one or multiple eQTLs. Large scale mRNA profiling technologies advanced genome-wide eQTL mapping in a diverse range of organisms allowing thousands of eQTLs to be detected in a single experiment. When combined with classical or trait QTLs, correlation analyses can directly suggest candidates for genes underlying these traits. Furthermore, eQTL mapping data enables genetic regulatory networks to be modelled and potentially provide a better understanding of the underlying phenotypic variation. The mRNA profiling data sets can also be used to infer the chromosomal positions of thousands of genes, an outcome that is particularly valuable for species with unsequenced genomes where the chromosomal location of the majority of genes remains unknown. In this review we focus on eQTL studies in plants, addressing conceptual and technical aspects that include experimental design, genetic polymorphism prediction and candidate gene identification.
