摘要
土壤有效磷供给不足和磷资源匮乏将引起未来的农业危机。在我国玉米主栽区,土壤有效磷不足已成为限制作物产量和增加生产成本的重要因素之一。同时,磷肥的大量使用也带来了水域富营养化等生态问题。因此,利用作物固有的生物学特性,挖掘作物自身对磷素高效吸收利用的潜力来解决上述问题已成为植物生物学研究中的热点领域。由于土壤磷素移动性差,根系的生长发育和形态结构对磷素吸收尤为重要,同时根系还可通过合成生长调节物质(如CTK、ABA等)来调控整个植株的状态包括叶片衰老、光合速率、气孔开度等。玉米是最重要的农作物之一,其根系特点与拟南芥明显不同,其发育调控机理也可能有差异,研究磷营养影响玉米根系发育的机制对于培育磷高效玉米新材料有重要意义。
本研究以玉米骨干自交系齐319(Q319)和其细胞工程突变体99038去胚乳幼苗为材料,比较了它们在不同供磷水平下的转录组差异；分析了低磷诱导的转录因子基因ZmPTF1的表达强度变化对玉米植株生长发育的影响及其作用机制；克隆了与生长素浓度梯度形成与维持相关的包含Zea mays auxin transporter protein 3(ZmAUX1)在内的4个生长素极性转运流入载体基因和包含ZmPIN1a和ZmPIN1b在内的13个生长素极性转运流出载体基因,并进行了表达谱分析和启动子序列分析；利用农杆菌介导法将正反向的ZmAUX1、ZmPIN1a和ZmPIN1b基因导入到了玉米骨干自交系DH4866中,获得了纯合的转基因植株,对转基因植株的生长发育状况、产量性状、根系构型和对低磷环境的反应进行了分析。在本工作中,获得了具有很好应用前景的转ZmPTF1基因和转ZmPIN1a基因的玉米育种材料。
玉米自交系Q319和99038的根系转录组比较分析
利用玉米全基因组芯片(microarray)检测了低磷处理0、2、8d的玉米自交系Q319和99038的根尖和侧根原基发生区的转录组变化。该芯片含47K的玉米寡核苷酸探针,后者代表3万多个基因。设在3个生物学重复中符合ratio≤0.66 or ratio≥1.5且p≤0.1 (t-test)的基因为有意义的差异基因。低磷处理前,在根尖区中99038比Q319中表达上调的基因有470个,表达下调的基因有542个；而在侧根原基发生区99038比Q319中表达上调的基因为621个,表达下调的有572个。在低磷处理2d后,与Q319的相比,99038根尖表达上调的基因有343个下调的有245个；在侧根原基发生区99038比Q319上调表达的基因有272个下调表达的有270个。在低磷处理8d后,在根尖区,99038比Q319表达上调的基因为984个,表达下调的为812个；在侧根原基发生区99038比Q319表达上调的基因为601个,表达下调的为457个。这些差异表达基因的功能分类表明,Q319和99038根系在低磷处理的不同时间点上存在着不同的应答反应,根的不同部位对低磷胁迫的响应也存在差异。在这些差异表达基因中,约有50%的差异基因功能不清楚,其余大多数与代谢相关,其次与细胞信号转导、转录、细胞增殖关联等。这些结果表明,玉米根系对低磷胁迫的应答是一个复杂的过程,存在多个适应或调节机制。
在低磷处理前,一些参与乙烯和生长素代谢及信号途径的基因,其表达强度在两基因型之间存在显著差异。1-aminocyclopropane-1-carboxylic acid(ACC) oxidase编码基因(对应探针号：TM00025945、TM00042027)和acc synthase编码基因(TM00018872)的表达在99038的侧根原基发生区显著低于Q319的,而在根尖区却高于Q319的,提示由腺苷甲硫氨酸生成乙烯的强度可能在99038和Q319的不同部位根区段中存在差异。在乙烯信号途径中位于CTR1下游的膜结合蛋白EIN2(ethylene insensitive 2, TM00057348)和乙烯应答转录因子(ethylene-responsive factor-like protein 1,ERF1, TM00030445)和ethylene-responsive small GTP-binding protein(TM00014304)在99038侧根原基发生区的表达量也显著低于Q319的,乙烯响应元件结合蛋白(ethylene responsive element binding factor 3,TM00018574和TM00042351)等基因的表达在99038的根尖高于Q319的。这些结果表明乙烯信号转导途径参与了99038更发达根系的形成。
生长素是调控植物根系发育的核心激素之一,参与了植物对低磷胁迫的反应。生长素的极性转运对生长素浓度梯度的形成、生长素信号的启动、生长素调控生长发育过程至关重要。生长素极性转运流入载体AUX1-like permease (TM00029483)的表达在99038根尖和侧根原基发生区显著高于Q319的,可能与99038侧根数量有关。TM00003447编码phosphatase 2A的调节亚单位,在99038的侧根发生区表达强度远低于Q319的。该酶通过磷酸化与去磷酸化作用可逆调节生长素流出载体PIN蛋白的分布,这暗示着在该区段生长素流出速率大幅度降低,有利于生长素在此处的积累。PGP1 (TM00020888)是参与生长素极性运输的另一类载体,也在两个基因型中的表达强度差异表达。TM00047995编码IAA-Ala hydrolase,该酶水解IAA-Ala复合物产生有活性的IAA,在侧根发生区99038的转录丰度显著高于Q319的,而在根尖区却低于Q319的,这与99038的根系形态相对应。Tryptophan synthase (TM00016868)、putative tyrosine/dopa decarboxylase (TM00005060)、indole-3-glycerol phosphate lyase(TM00005958)和.cinnamic acid 4-hydroxylase (YUCCA,TM00025513)等IAA生物合成中的关键酶基因也在这两个基因型间差异表达,可能导致局部IAA的合成或活性IAA的浓度在99038和Q319中出现差异。另外,玉米中的生长素结合蛋白auxin binding protein1 (ABP1, TM00041857)、auxin-induced in root cultures protein 12(TM00055925)和GH3家族成员(TM00048104)等生长素信号途径或应答基因在两个基因型之间的表达也出现差异。以上这些差异暗示着根系不同区段的IAA含量及活性的变化、以及信号途径及下游基因的表达差异很可能是造成两个基因型根系形态差异的主要因素之一
生长素和乙烯的合成、代谢和信号转导及调控参与了玉米对低磷胁迫的应答。在低磷处理2d和8d的植株中,生长素极性运输和分布调节相关基因的表达在99038和Q139之间存在明显差异,可能低磷胁迫通过影响生长素极性转运而影响整个植株的生长状态。与足磷供给条件下相比,MAP3K、MAPK4、MAPK5、MAPK6等基因在根尖区呈下调表达,MAPK信号系统可能参与了低磷胁迫信号的转导及代谢变化。乙烯信号途径中的EIN3、ERF1、赤霉素信号途径中的Gibberellin-regulated protein 2等基因的差异表达可能与低磷胁迫下侧根发生和两个基因型在低磷胁迫下的根系差异有关。99038和Q319对生长素、乙烯信号反应的差异很可能是造成它们对低磷胁迫响应差异的重要原因。
一些转录因子和在信号转导及生长发育中起重要作用的基因,如14-3-3、ABP1、AUX1、RHD3、ROP6、SGR2、BRI1等,在两个基因型间或／和在低磷处理前后出现表达丰度的差异,提示它们参与了玉米根系发育的调控和对低磷胁迫耐的响应,可作为玉米根系改良和磷高效育种的操控靶标。
过表达ZmPTF1改进了玉米根系发育和耐低磷特性
前期工作中,实验室通过RACE的方法克隆到ZmPTF1基因,该基因编码具有bHLH(basic helix-loop-helix domain)结构域的转录因子,与水稻的OsPTF1有高的相似性。该基因在根中受低磷胁迫诱导表达。ZmPTF1过表达株系在不同介质培养条件下根系发达,根系明显大于对照和转反义基因植株的。当生长于低磷土壤中,ZmPTF1过表达株系具有较多的雄穗分支数和较饱满的籽粒,受低磷胁迫影响较小。过表达ZmPTF1导致了一些低磷胁迫响应基因如RNases. vacuolar H+ pyrophosphatase(H+-PPase]、PEP carboxykinase等在正常磷供给下高量表达。糖含量测定表明,ZmPTF1过表达株系叶片中可溶性糖浓度(Glu+Fru+Suc)低于对照和转反义基因植株的,而根中的可溶性糖浓度高于对照和转反义基因植株的。对蔗糖合成和分解代谢相关基因的表达分析发现,过表达ZmPTF1导致了蔗糖合成关键基因fructose-1,6-bisphosphatase和sucrose phosphate synthase 1的表达在叶片中显著提高,在根中明显低于对照植株的。同时,参与蔗糖代谢的酶基因在过表达株系根中的表达量也低于对照的。ZmPTF1过表达导致了玉米代谢和根系形态发生变化,当遭受低磷胁迫时,这些特征能促进过表达株系较快适应低磷环境,减少了低磷对玉米生长发育和产量的影响。该工作为深入了解ZmPTF1-耐低磷-根系形态-糖之间的关系积累了重要资料,提供了一个通过基因工程育种来提高作物对低磷环境的耐性的成功实例。获得的转基因材料己通过转基因生物安全性中间试验,并提供给多家育种单位用于育种。
生长素极性运输相关基因在玉米根系发育中的表达变化
生长素极性运输使生长素在植株体内形成以器官顶端为中心的浓度梯度,并维持植物不同组织中的生长素浓度差,以调控植物的生长发育。介导生长素极性运输的蛋白有AUX1/LAX家族、PIN-formed家族,ABCB家族蛋白。拟南芥和水稻中分别含有4个可能的生长素流入载体,8个和12个PIN蛋白家族成员。通过生物信息学预测和RT-PCR从玉米克隆出4个可能的生长素流入载体,13个可能的生长素流出载体。与拟南芥和水稻中生长素输入／出载体相比较,发现早期报道的玉米ZmAUX1(rename=Zm auxin transporter protein 3)与拟南芥auxin transporter 2和3、水稻Os auxin transporter protein 3的序列相近,在氨基酸组成、长度、等电点和分子量上差异不大。在克隆的13个可能的玉米生长素流出PIN-like基因中,与AtPIN1同源的基因有4个,分别为ZmPIN1a、ZmPIN1b、ZmPIN1c和ZmPIN1d。ZmPIN1b和ZmPIN1c在玉米基因组上对应于同一段序列,推测认为它们是同—mRNA前体可变剪接的产物,与OsPIN1a有较高的相似性。ZmPIN1a是水稻OsPIN1c的同源基因,玉米ZmPIN1d与水稻OsPIN1b和OsPIN1d亲缘关系近。与AtPIN5同源的玉米基因有5个,分别定位于玉米的3、4、8、2、1染色体上,编码产物相似性较高。与AtPIN8同源的玉米基因只1个,命名为ZmPIN8。在玉米中还有一个与水稻OsPIN9同源的基因,命名为ZmPIN9。未找到AtPIN2、AtPIN4、AtPIN6和AtPIN7在玉米中的同源基因,在水稻基因组中也未发现AtPIN4、AtPIN6和AtPIN7的同源基因。
启动子元件分析发现Zm auxin transporter protein 3可能更多的参与干旱胁迫诱导的反应,而Zm auxin transporter protein 2和Zm auxin transporter protein 4则在伤害反应中起作用。PIN1家族对冷、热胁迫的响应可能主要是通过ZmPIN1b/c完成的,而ZmPIN1a在干旱、ABA的应答反应中起主要作用。ZmPIN1a表达可能是受干旱、内源ABA、GA、乙烯等调控,在植株形态建成和生长发育中起重要作用。ZmPIN3b基因可能受MYBHvl的调控。ZmPIN3a和ZmPIN3b可能参与了损伤反应及形态发生,并对环境胁迫发生反应,受玉米素、赤霉素和生长素浓度梯度或信号途径的调控。
ZmAUX1在地上部分的表达丰度远高于根中,如在萌发4d的小苗中幼叶的表达丰度是幼根的5倍多。推测该基因在生长素从地上部分向地下部的转运中起关键作用。生长素流出载体ZmPIN1b,ZmPIN1a表达强度相对高,ZmPIN3a、ZmPIN3b、ZmPIN5a、ZmPIN5b、ZmPIN5c、ZmPIN8、ZmPIN9的表达在5叶期玉米中表达强度很低。这些基因在种子萌发期活跃表达暗示着它们参与了植株早期生长与发育。生长素极性转运相关基因在5叶期玉米的叶片、侧根发生区和根尖区具有不同的表达丰度。各基因对低磷胁迫的响应也与器官部位有关。在侧根发生区ZmAUX1、ZmPIN1a、ZmPIN1b受低磷胁迫的诱导,而ZmPIN1c表达却被低磷胁迫抑制。在根尖和叶片中ZmPIN1a的表达受低磷胁迫的诱导,而其它基因的表达受到抑制。
以上结果表明,生长素极性运输相关基因参与了玉米形态建成和生长发育的调控,参与了对低磷胁迫的应答。
转ZmPIN1a/1b基因和ZmAUX1基因对玉米生长发育和磷胁迫抗性的影响
在对生长素极性转运相关基因分析的基础之上,构建了ZmPIN1a、ZmPIN1b和ZmAUX1的正、反向植物表达载体,通过农杆菌介导的转化法将它们分别导入到玉米优良自交系DH4866中,获得了纯合的转基因植株,在此基础上研究了ZmPIN1a、ZmPIN1b和ZmAUX1的表达强度变化对玉米生长发育、根系构型和对低磷环境反应的影响。
转正义ZmPIN1b或ZmPIN1a基因植株的根系比WT和转反义基因植株的发达,其中以转正义ZmPIN1a基因的更为明显,表现为种子根长、侧根数目增多,根体积变大。生物量测定表明,转正义ZmPIN1a基因的根系生物量显著高于WT的,而茎叶生物量明显低于WT的。在SP营养液培育下,转正义ZmPIN1a株系玉米根系较发达,虽然种子根数和冠根数与WT植株基本无差异,但侧根是对照植株的121～173%。转反义ZmPIN1a株系的侧根数目则为WT植株的82%～106%。根长度分析揭示,转正义ZmPIN1a植株的初生根长度显著高于WT及反义株系的,但平均根长却显著低于WT和转反义基因株系的,形成了种子根较长侧根密集的表型。当在LP营养液中生长时,各株系均表现出适应性变化,主要表现在初生根增加,侧根数目减少,而ZmPIN1a基因过表达的效果更加明显。比较低磷条件下不同株系的性状差异,得出转正义ZmPIN1a植株的根数目是WT的141%～261%,但总根长是WT的98%～132%,即过表达ZmPIN1a促进侧根发生这与低磷胁迫下生长素浓度梯度的局部变化相对应。分析成株期植株的形态,发现转正义ZmPIN1a基因植株下部茎节间变短、穗位和株高均降低、侧根数目增多、根系涉猎面积显著增大,从而提高了植株对水分和养分的吸收能力,有利于产量提高。所创造的种质材料可能在玉米耐密植育种中有很好的应用价值。
ZmPIN1b基因转正义基因植株的茎叶生物量高于WT的,但未达到差异显著程度,根系生物量则显著高于WT的。其反义株系的表型和生物量与WT无明显差别。在SP营养液培养下转正义ZmPIN1b基因的植株根数目比WT增加,为WT的129%～146%,根总长是WT的114%～138%。而转反义基因株系的根数目和根总长比WT略有下降。在LP营养液中培养时,转正义基因植株和转反义基因植株都表现出对低磷胁迫的敏感性降低,侧根数、总根数和SP培养液中的植株相差不大,与WT对低磷胁迫的应答有显著差异。可能ZmPIN1b影响了玉米在低磷胁迫条件下根系的适应性变化。
转ZmAUX1正、反义株系的分析表明,过表达ZmAUX1的植株在低磷环境中能维持较高的生物量和较好长势。并且ZmAUX1的过表达调节其它生长素极性运输相关基因的表达。
该实验结果表明,通过调节生长素极性运输强度来改变玉米植株构型及根系构型是可行的,为玉米抗逆育种和高产育种提供了新思路。ZmPIN1a基因过表达对植株转录组影响
采用Real-time RT-PCR方法分析了过表达和抑制表达ZmPIN1a对生长素极性转运相关基因表达的影响,发现过表达ZmPIN1a引起ZmAUX、ZmPIN1b、ZmPIN1c、ZmPIN3b、ZmPIN3b在幼叶和幼根中上调表达,在多数转反义基因株系中这些基因则不同程度下调表达。ZmAUX1表达强度在过表达株系叶中是对照的3-6倍,在根中是对照的1.4-2.2倍;ZmPIN1b表达强度变化在根中和ZmAUX1相近,在叶中则低于ZmAUX1的,是对照植株的2-5倍；ZmPIN1c也表现出同样的变化趋势,但变化幅度较小。PIN3的2个成员的表达也受到ZmPIN1a过表达的诱导,且变化幅度大,但与ZmPIN1a的表达强度对应关系不明显。ZmPIN5、ZmPIN8、ZmPIN9这几个短loop的PIN基因表达变化不明显。
采用数字表达谱分析了不同供磷状态下ZmPIN1a正、反义株系和对照的叶片和根中的差异表达基因,发现过表达ZmPIN1a对转录组有较大影响,抑制ZmPIN1a表达对转录组影响相对较小,尤其在根中。过表达ZmPIN1a植株的根系形态和株型发生了显著变化,转录组分析表明这些变化是大量基因表达变化引起的体内代谢反应及生长发育调整的结果。生长素和乙烯的代谢及信号转导途径明显受到ZmPIN1a表达水平的影响,光合作用在过表达ZmPIN1a植株中增强。另外,在过表达ZmPIN1a基因植株中一些中间代谢产物出现积累,昼夜节律相关因子出现不同变化趋势。这些资料为深入了解生长素极性转运与玉米株型之间的关系提供了大量信息。
本工作利用两个对低磷胁迫响应有明显差异的玉米自交系为材料进行了转录组比较,鉴别出一些可能在玉米低磷胁迫反应和根系发育中起重要作用的信号途径和相关基因；克隆并系统分析了玉米生长素极性转运相关基因,初步确定了它们的表达与玉米耐低磷特性的关系；通过转基因植株的鉴定和分析确定了过表达或抑制表达ZmPTF1、ZmPIN1a、ZmPIN1b、ZmAUX1基因对玉米生长发育和耐低磷特性的影响,选出了在玉米育种上有很好应用价值的转基因新种质。
Phosphate deficiency in soil and natural resources will be a potential agriculture crisis in the future. Low phosphate concentration is frequently a supply constraint in maize yields and increased the cost for production. At the same time, application of phosphate (Pi) fertilizer in large quantities produces the water pollution by organism in aquatic systems and degradation of the environment. It has been one of hotspots in plant biology to solve the above problems by exploring the potential ability of phosphate absorption of crops. Root postembryonic growth and development are critical for Pi uptake since for the poor mobility of soil phosphate, and root system could regulate the whole plant growth include the leaf aging, photosynthesis and stomata opening by synthesizing plant hormone such as cytokinins and abscisic acid. Maize is an important crop with unique features root system compared to the dicotyledonous model root system from Arabidopsis, and maybe the regulation mechanism was different. It is important to explore the development mechanisms of maize root in low phosphate environment for the improvement of the Pi efficiency by breeding.
In this study, the gene expression profiles of root segments of inbred Q319 and 99038 (from a cell mutant) cultured in nutrient solution with different phosphate concentrations were examined using an Arizona Maize 47K Oligonucleotide Array. The expression pattern of transcription factor ZmPTF1 that was induced by low phosphate stress was determined, and the effects of ZmPTF1 expression levels on the maize root morphology and the function of ZmPTF1was carefully examined by transgenic strategy. Four auxin influx transporters genes and 13 auxin efflux transporters genes were cloned and the expression patterns of these genes and their promoter sequences were analyzed. Using Agrobacterium-mediated maize shoot transformation, the sense and antisense Zea mays auxin transporter protein 3 (ZmAUX1), ZmPIN1a and ZmPIN1b were introduced into inbred line DH4866. The morphology, development, yield, root architecture and their response to low phosphate stress of the transgenic homozygous lines were determined. The ZmPTFl and ZmPIN1a overexpressing lines which showed high prospects in maize breeding were obtained.
The main results of this work are follows:
Comparative transcriptome analysis of the inbred line Q319 and 99038 roots
In this study, the gene expression profiles of root segments of inbred Q319 and 99038 were examined using an Arizona Maize 47K Oligonucleotide Array, which representing more than 30,000 identifiable unique maize genes. The samples were the root segments of the plants cultured in nutrient solution. And one kind of the segments were about 0.5cm of seminal root tips, and another were 1.0cm segment of seminal roots from 0.5cm to 1.5cm behind the root tip in which lateral root will take place. The genes of ratio≥1.5 or ratio<0.66 (99038/Q319) and p value less than 0.1 by t-test in the three independent biological repeats were defined as differentially expressed genes between the two genotypes. Before low phosphate treated, there are 470 up-regulated (99038/Q319) and 542 genes down-regulated in the root tips in 99038 compared with Q319, whereas in the segments where lateral root take place,621 genes were up-regulated and 572 genes were down-regulated. After 2d low phosphate culture, compare with Q319 grown in the same solution, there were 343 genes up-regulated in the root tips of 99038 and 245 genes down-regulated, while 272 genes up-regulated and 270 genes down-regulated in the segments where lateral root take place. After 8d low phosphate culture, in the root tip of 99038 there were 984 genes up-regulated and 812 genes down-regulated compare with the Q319, with 601 genes up-regulated and 457 genes down-regulated in the segments where lateral root take place. The catalog of GO indicated that Q319 and 99038 have their different low phosphate response profiles in the some point of treatment, also in the two segments. Among these genes, more than 50% have no hit in the database, others were belong to metabolism, cell signaling, transcription and cell proliferation and so on. The results indicated that it was a complex process of maize root response to low phosphate stress, and the intricate regulations and adaptation were existed.
Before low phosphate treatment, genes involved in ethylene synthesis and signaling were differentially expressed in the two lines. 1-aminocyclopropane-1-carboxylic acid(ACC) oxidase (TM00025945、TM00042027) and acc synthase (TM00018872) which coding for the two enzymes in the synthesis of ethylene had lower expression in the segments where lateral root take place of 99038, but higher in the root tips. These suggested that the synthesis of ethylene via the adenosylmethionine may be different in the root segments of the two lines. The membrane binding protein EIN2 (ethylene insensitive 2, TM00057348) and the ethylene responsive transcription factor (ethylene-responsive factor-like protein 1, ERF1,TM00030445)and ethylene- responsive small GTP-binding protein (TM000 14304) which involved in the ethylene signaling showed a lower expression in the segments where lateral root take place of 99038 compare to Q319. The expression of ethylene responsive element binding factor 3 (TM00018574 and TM00042351) were high in the 99038 root tips. These indicated that ethylene might participate in the formation of more robust root system of 99038.
Auxin was one of the key hormones in plant root development and the low phosphate response process. Auxin polar transport was essential to the local auxin accumulation, auxin signaling imitation and auxin regulated biological process. AUX1-like permease (TM00029483), which coding for an auxin influx transporter had a higher expression in both the root tip and segments where lateral root take place of line 99038, this might promote auxin to the root, and was a factor to form the root with more lateral roots of 99038. TM00003447, which represents a phosphatase 2A regulate subunit, had a lower expression in the segments where lateral root take place, the protein phosphatase 2A (PP2A) and PID (protein serine/threonine (Ser/Thr) kinase PINOID) act antagonistically on phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. PGP1(TM00020888), which coding for another class of auxin efflux transporter was differentially expressed in the two lines. These differentially expressed auxin polar transporters and their regulation proteins could result in a different local auxin gradient in the root of 99038. TM00047995 which coding for IAA-Ala hydrolase role in the free IAA release from IAA-Ala, this gene was high expressed in the segments where lateral root take place of 99038, while had a lower expressed in the root tips. Tiyptophan synthase (TM00016868)、putative tyrosine/dopa decarboxylase (TM00005060) indole-3-glycerol phosphate lyase (TM00005958) and cinnamic acid 4-hydroxylase (YUCCA, TM00025513) were key enzymes participated in the biological synthesis of IAA. These differentially expressed auxin metabolism related genes may confer to the differential local auxin gradient in root of the two lines. Auxin binding protein1 (ABP1,TM00041857), auxin-induced in root cultures protein 12 (TM00055925) and GH3 family gene (TM00048104) and other auxin response genes and transcription factors showed different expression patterns in the two lines. The different levels of auxin synthesis, metabolism, transport, accumulation, signaling and the auxin regulated development process may be one of the main factors resulted in the differential root system of 99038 and Q319.
The signaling of auxin and ethylene participate the acclimation of maize plants to the low phosphate stress. On the 2d and 8d low phosphate treatment, auxin polar transport and the regulation genes in root had differently expressed to the low phosphate stress between the two lines. It was possible that the auxin polar transport in the root was a key link in the responses to low phosphate stress and resulted in modification of the root morphology, this meant the low phosphate signaling regulated the root architecture by regulating the polar auxin transport and signaling. Compared with plants in SP solution, MAP3K、MAPK4、MAPK5、MAPK6 genes had an relative lower levels in the root tips of 99038, MAPK system usually function in coordination with ethylene signaling pathway, maybe the system participate the low phosphate response and regulate many aspects of plant to low phosphate stress. And the EIN3, ERF1 in the ethylene signaling pathway and Gibberellin-regulated protein 2 might directly involve in the lateral root occurrence and the acclimation of root to low phosphate environment. It was concluded that the different auxin and ethylene signaling between two genotypes was an important factor for their differences in low phosphate circumstance.
Several transcription factors and signaling transduction genes, such as 14-3-3, ABP1, AUX1, RHD3, ROP6, SGR2 and BRIl1were differentially expressed between the two lines or in their response to low phosphate stress. This was inferred that the genes participated in the root morphology and low phosphate acclimation and could be the targets of root breeding and the breeding to tolerate low phosphate stress in maize.
Overexpression of transcription factor ZmPTF1 improves low phosphate tolerance of maize by regulating carbon metabolism and root growth
A bHLH (basic helix-loop-helix domain) transcription factor involved in tolerance to Pi starvation was cloned from Zea mays with an RT-PCR coupled RACE approach and named ZmPTFl in our previous work. ZmPTF1 encoded a putative protein of 481 amino acids that had identity with OsPTFl in basic region. Real-time RT-PCR revealed that ZmPTFl was quickly and significantly up-regulated in the root under phosphate starvation conditions. Overexpression of ZmPTF1 in maize improved root development in different mediums and the roots of the sense lines were significantly development than WT. When cultured in low phosphate soil, the sense plants developed more tassel branches and larger kernels and were less affected by low phosphate stress. Some low phosphate response genes have higher expression levels in the overexpression lines, such as drought induced RNase, vacuolar H+ pyrophosphatase (H+-PPase), PEP carboxykinase. Compared with wild type, overexpressing ZmPTF1 altered the concentrations of soluble sugars in transgenic plants, in which soluble sugars (Glu+Fru+Suc) levels were lower in the leaves and higher in the roots. Overexpression of ZmPTF1 enhanced the expression of fructose-1,6-bisphosphatase and sucrose phosphate synthasel participated in sucrose synthesis in the leaves but decreased them in the root, and reduced the expression of genes involved in sucrose catabolism in the roots. The modifications on the physiology and root morphology of the plants enhanced low phosphate tolerance and increased the yield under low phosphate conditions. This research provides a useful gene for transgenic breeding of maize that is tolerant to phosphate deficiency and is helpful for exploring the relationship between sugar signaling and phosphate concentrations in cells. These transgene lines had confirmed by experts in maize breeding and to be used to breed the variety with trait of tolerating low phosphate stress.
Identification and expression analysis of AUX1/LAX family and PIN-formed family genes in maize
Auxin polar transport was critical for formation and maintaining of auxin concentration gradient around to the meristem, and this different auxin gradient initiate and regulates many aspects of plant growth and development. The polar auxin transport is mediated by three classes of proteins AUX1/LAX family, PIN-formed family and ABCB subfamily proteins. There are 4 auxin influx transporters and 8 PIN auxin efflux proteins in model plant Arabidopsis, whereas in monocots crop rice there are 4 auxin influx transporters and 12 PIN auxin efflux proteins respectively. Four putative auxin influx transporters and 13 putative PIN auxin efflux proteins were identified from the maize genome by a database search and 10 genes were cloned by RT-PCR. Phytogenetic analysis of auxin influx transporters indicated that the previously reported ZmAUX1 had the highest similarity to Arabidopsis auxin transporter 2 and 3, to the Os auxin transporter protein 3 in rice and renamed the gene Zm auxin transporter protein 3. The 13 auxin efflux transporters genes cloned by us were categorized into branches with Arabidopsis PIN genes and rice. In the PIN1 subfamily, there are four PIN1-like genes in rice and maize, and we named the four PIN1 genes in maize ZmPIN1a, ZmPIN1b, ZmPIN1c and ZmPIN1d sequentially. The mRNA sequences of ZmPIN1b and ZmPIN1c were derived from a identical genomic DNA sequence, we presumed the two mRNA came from one Primary transcript by the alternative splicing. ZmPIN1b and ZmPIN1c have a high similarity to OsPIN1a, while ZmPIN1a was the homologous gene of OsPIN1c, and ZmPIN1d was the homologous gene of OsPIN1b and OsPIN1d. There are two homologous genes of AtPIN3 in maize. There are five homologous genes of AtPIN5 in maize, they locate on the 3,4,8,2,1 chromosome respectively. A homologous gene of AtPINS and a homologous gene of OsPIN9 were found in maize and named them ZmPIN8 and ZmPIN9. The homologous genes of AtPIN2, AtPIN4, AtPIN6 and AtPIN7 did not exist in maize, and also none in rice of OsPIN4,OsPIN6 and OsPIN7.
Promoter analysis of the genes pointed out that Zm auxin transporter protein 3 was more possible involved in drought stress, whereas Zm auxin transporter protein 2 and Zm auxin transporter protein 4 were mainly function in injury and wounding defense. In the PIN1 family, the response to high and low temperature were mainly taken on by ZmPIN1b/c, whereas the ZmPIN1a was function in drought and ABA response and regulated by endogenous or exogenous ABA, GA and ethylene signaling. ZmPIN3b might be the target genes of MYBHvl for there were 4 MYBHv1 binding sites in the promoter. These genes could be regulated by GA, zeatin and auxin.
RT-PCR analysis showed that ZmAUX1 was highly expressed in root and shoot in the 4 days germinated seedlings, especially in the shoot, the expression level in the shoot was more than 5 fold of root. These suggested that ZmAUXl played an important role in the auxin transport from shoot to the root. Auxin influx transporter ZmPIN1b, ZmPIN1a have a relative higher expression to other genes, whereas the expression of ZmPIN3a、ZmPIN3b、ZmPIN5a、ZmPIN5b、ZmPIN5c、ZmPIN8、ZmPIN9 were very low in the 5-leaf stage. The high expression of these genes in the seed germination and organogenesis indicated that the participated the early morphogenesis of plant. They showed different expression patterns in the root tip, the segments where lateral root take place and the leaves of 5-leaf stage maize plant, and the response to low phosphate stress were diverse to the organs. In the segments where lateral root take place, ZmAUX1、ZmPIN1a、ZmPIN1b were up-regulated and ZmPIN1c was down-regulated by low phosphate stress.In the root tip and the leaf, ZmPIN1a was up-regulated by low phosphate stress, whereas ZmAUX1, ZmPIN1b and ZmPIN1c were down-regulated.
The analysis of promoter sequences and expression patterns of these auxin polar transport genes indicated that these genes were involved in the morphogenesis of plant and the response to low phosphate stress.
Introduced ZmPIN1a/1b or ZmAUX1 into maize enhanced the tolerance to low phosphate stress and modified the morphology of plants
Based on the analysis of auxin polar transporter, the sense and antisense constructs of ZmPIN1a, ZmPIN1b and ZmAUX1 were introduced into maize inbred line DH4866. The characteristics, yields, root architecture and their response to low phosphate of the transgenic homozygous lines were determined.
Overexpression of ZmPIN1a or ZmPIN1b improved the maize root development, especially for the ZmPIN1a. The ZmPIN1a sense lines have distinctive root architecture with long seminal roots, high density lateral roots, but the length of lateral roots reduced, resulted in the increased root volume and changed root architecture. Biomass determination demonstrated that the root weight of ZmPIN1a sense lines were significantly higher than that of WT, while the shoot were lighter than WT. When cultured in SP solution the root system of ZmPIN1a sense lines were more developed, the number of lateral roots was 121～173% of WT, although the number of seminal roots were not significantly different with WT. At the same time the number of lateral roots of antisense lines was 82%-106% of WT plants. The analysis of root length showed that the length of seminal roots of ZmPIN1a sense lines were more longer than WT, but the average root length were lower than WT, resulted in a root system with long seminal root with more density lateral roots. When cultured in LP solution, all the lines behaved low phosphate acclimation, and showed the increased seminal root length and inhibited lateral root formation. Compare with WT and antisense lines, the ZmPIN1a sense lines were more distinct from others. The root number of sense plant was 141～261% of WT, and the total length of root was 98～132% of WT, this implied the overexpression of ZmPIN1a promoted the occurrence of lateral root, which might be a consequence of local auxin gradient change induced by the low phosphate stress. The sense lines showed the reduced height of plant and ear position, and the increased lateral root number and length of seminal root, and improved yield. These traits offered an excellent prospect in maize breeding.
The dry weights of ZmPIN1b sense line were slightly higher than WT, but the dry weight of root was significantly higher than WT and antisense lines. The latter were similar to WT. When cultured in SP solution, the number of lateral roots was 129～146% of WT and the length of root system was 114%～138% of WT, while the antisense lines were slightly less than WT. When cultured in LP solution, the sense and antisense lines both behaved decreased low phosphate sensitivity and the number and length of lateral roots were not significantly different of plants in SP solution. It was possible that ZmPIN1b expression levels affected the acclimation to low phosphate stress of the plants.
Overexpression of ZmAUX1 in maize could maintain relative higher biomass and better growth development under low phosphate conditions. Moreover, the overexpression of ZmA UX1 regulated the expression of other auxin polar transport related genes.
These transgene plants were very impressed us that it was possible to improve the stress tolerance and produce high yield breeding by changing the root architecture via introducing the auxin polar transport genes into maize.
The transcriptom changes by manipulating the expression level of ZmPINla
The expression of auxin polar transporter genes in ZmPIN1a sense, antisense and WT plants were analysis by Real-time RT-PCR. We found that overexpression of ZmPIN1a enhanced the expression of ZmAUX1, ZmPIN1b, ZmPIN1c, ZmPIN3b and ZmPIN3b in the shoot and root of 4 day germinated seedlings, while in the antisense lines theses genes decreased with various degrees. The expression of ZmAUX1 in the sense lines were 3-6 fold of WT in the shoot and 1.4-2.2 fold in the root. The expression of ZmPIN1b was similar to ZmAUX1 in the root and 2-5 fold of WT in the shoot. The expression of ZmPIN1c was similar to ZmAUX1,but the fold was smaller than others. The expression of two PIN3 genes was induced by ZmPIN1a, but no significant parallelism in expression levels between ZmPIN1a and them.
Used the DGEs, the transcriptions of root and shoot of ZmPIN1a sense, antisense lines and WT cultured in different Pi content solutions were analysis. The overexpression of ZmPIN1a affected largely the transpcripts of the plants compared with the antisense lines, especially in the root. Overexpression of ZmPIN1a led to significant changes of root architecture and plant development. Auxin and ethylene signaling were obviously modified in the ZmPINla sense line, and the remarkable change was the genes involved in photosynthesis were up-regulated on transcribe level. Moreover, many metabolic intermediate might be accumulated in the sense line and the circadian rhythm related factors were differentially expressed in different lines. These provided abundant information to explore the relationships among auxin-root architecture-low phosphate.
In summary, we have examined the gene expression profiles of maize plants from inbred Q319 and its analogical line 99038 using an Oligonucleotide Array; Some important signaling pathway and key genes related to low phosphate response and root morphology were characterized. Thirteen auxin polar transporter genes were cloned and examined for their expression profiles and promoter sequence. Using Agrobacterium mediated maize shoot transformation, the sense and antisense ZmPTF1, Zea mays auxin transporter protein 3 (ZmAUX1), ZmPIN1a and ZmPIN1b were intruded into maize inbred line. The morphology, development, yield, root architecture and their response to low phosphate stress of the transgene lines were determined. We have obtained the overexpression lines of ZmPTFl or ZmPIN1a which have well prospect in maize breeding.
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