光皮桦miR164及其靶基因NAC1在低氮胁迫中的表达分析

氮是植物生长发育所必需的大量营养元素,植物缺氮后严重影响地上部分生物量的积累,因此,揭示植物如何抵抗或适应低氮胁迫的分子机制具有重要意义。杨树(Populus tremula × P. alba)NAC1(NAM, ATAF, CUC 1)基因位于调控网络上游,在低氮环境下调控下游关键基因的表达,进而调控根系生长以抵抗低氮胁迫。本文以光皮桦(Betula luminifera)G49-3无性系组培苗为材料,探讨了miR164及其靶基因NAC1对低氮胁迫的响应。通过RACE技术克隆了光皮桦NAC1基因(GenBank登录号：KT900889),全长1497 bp,编码358个氨基酸,N端具有高度保守的NAM结构域;运用5′-RACE验证了NAC1为miR164靶基因,切割位点在第10和11位碱基之间;采用qRT-PCR分析miR164与靶基因NAC1在低氮胁迫时的表达模式,发现miR164表达在根中的低氮处理前期(4 d)受到抑制,而后升高,而茎叶中表达模式与根不同;靶基因NAC1与miR164表达水平呈负相关,且在恢复实验组(重新添加全营养液)中,根中miR164表达上升,NAC1显示出相应的表达变化,暗示miR164及其靶基因NAC1可能在低氮胁迫响应中发挥调控功能。本研究结果有助于揭示miR164对NAC1在低氮胁迫响应中转录后水平的分子调控机制,为进一步研究miR164-NAC1在低氮胁迫响应中的功能提供有价值的信息。     

磷和外源生长素对白羽扇豆(Lupinus albus L.)根形态和生理特性的影响

白羽扇豆(Lupinus albus L.)对磷和生长素表现出高度的根系形态和生理可塑性反应, 然而磷和生长素如何调节根形态和生理特性以及它们对根形态和生理的交互效应尚不清楚. 本研究通过水培实验旨在评价磷(0或250 μmol/L)和生长素(10^-8 mol/L NAA)对白羽扇豆根特性的影响及其交互效应. 结果表明, 缺少磷和生长素施用均明显改变了白羽扇豆的根形态(主根长度减少、一级侧根数目增加和排根大量形成)和生理特性(质子释放、柠檬酸分泌和酸性磷酸酶活性增加). 外源生长素的施用增加了缺磷白羽扇豆根系的响应度和敏感性. 磷和生长素对白羽扇豆根系形态和生理具有明显的交互作用. 主成分分析表明, 磷解释了根特性64.8%的信息, 而生长素解释了21.3%的信息, 表明磷供应对白羽扇豆根特性的影响比外源生长素施用更为重要. 白羽扇豆能够通过协调根系形态和生理对外部刺激(如缺磷和施用生长素)作出应答, 以及优化根形态和生理之间的关系从而最大化获取磷素资源.     

6-BA对缺磷白羽扇豆排根形成和有机酸分泌的影响

缺磷条件下白羽扇豆能够形成排根,并增加有机酸分泌.但上述过程的调节机制尚不清楚.该文的结果表明,使用外源6-BA不影响缺磷白羽扇豆的生长和磷在体内的分配,但明显抑制了根簇的形成和有机酸分泌.经低浓度6-BA(10-8 mol/L)处理后转移至不含6-BA的缺磷营养液中继续培养的植株,其根簇形成和有机酸分泌得到恢复,甚至超过未经6-BA处理的缺磷植株;但高浓度6-BA(10-7 mol/L)对根簇形成和有机酸分泌的抑制作用不可恢复.对6-BA影响缺磷的白羽扇豆排根形成和有机酸分泌的可能机制进行了讨论.     

排根的形成及其所分泌的有机酸的调节

排根是在磷、铁、氮等养分缺乏条件下形成的一种特殊根系结构,能够形成排根的植物种类有限.不同植物排根分泌的有机酸种类和数量可能不同,如在缺磷条件下白羽扇豆的排根所释放的柠檬酸可达植物总干重的23%,使根簇周围的柠檬酸浓度达50～90 μmol*g-1土壤.这是一种防止胞质过度酸化以及柠檬酸过度积累而超过液泡储存能力的解毒机制.其分泌可能受阴离子通道的调控.     

缺磷或缺铁引起白羽扇豆锰中毒的机理

白羽扇豆在缺磷或缺铁条件下均有排根形成,并且根系还原力显著增加。缺磷、缺铁根系还原力在高峰期分别高于对照。缺磷与缺铁根系还原力高峰不仅出现的时期不同,而且还原力增加部位也不一样。缺磷处理的排根区具有很高的还原力,缺铁处理还原力较高的部位是在主根和侧根的根尖以及排根区。由于Ｍｎ４＋比Ｆｅ３＋更易被还原,致使根系还原力提高促使根际大量锰被还原,这是缺磷和缺铁造成白羽扇豆锰中毒的主要原因之一。     

缺磷白羽扇豆排根与非排根区根尖分泌有机酸的比较

采用根系分泌有机酸原位收集方法及市郊液相色谱技术分析了供磷及缺磷后不同时间白羽扇豆（Ｌｕｐｉｎｕｓ ａｉｂｕｓ Ｌ．）非排根区根尖和排根分泌有机酸的种类和数量,以及相应的根尖、排根组织,茎木质部、韧皮部汁液中有机酸含量的变化。结果表明：⑴缺磷能够诱导白羽扇豆要系产生大量排根,根系的有机酸分泌量也明显增加。⑵无论在供磷或缺磷条件下,排根与非排根区根区根尖组织中的有机酸种类相同,但排根主要分泌柠檬酸和     

缺磷胁迫对小麦根细胞周期蛋白基因cyc1At表达的影响

用液培方法研究了缺磷胁迫对小麦(TriticumaestivumL.)根系生长的影响。结果表明,随着介质磷水平的提高,小麦根轴长度和植株生长素浓度均降低。在低磷条件下用生长素极性运输抑制剂三碘苯甲酸(TIBA)处理后,小麦的根轴长度明显降低,表明生长素参与了缺磷小麦根轴生长的调控。缺磷小麦根部生长素浓度的提高诱导了细胞周期蛋白基因cyclAt的表达,促进了根分生组织细胞的分裂并驱动了根的生长。     

缺磷或缺铁引起白羽扇豆锰中毒的机理

白羽扇豆在缺磷或缺铁条件下均有排根形式,并且根系还原力显著增加。缺磷、缺铁根系还原力在高峰期分别高于对照。缺磷与缺铁根系还原力高峰不仅出现的时期不同,而且还原力增加部位也不一样。缺磷处理的排根区具有很高的还原力,缺铁处理还原力较高的部位是在主根和侧根的根尖以及排根区。由于Ｍｎ＾４＋比Ｆｅ＾３＋更易被还原,致使根系还原力提高促使根际大量锰被还原,这是缺磷和缺铁造成白羽扇豆锰中毒的主要原因之一。     

缺磷白羽扇豆排根与非排根区根尖分泌有机酸的比较

采用根系分泌有机酸原位收集方法及高效液相色谱技术分析了供磷及缺磷后不同时间白羽扇豆(LupinusalbusL .)非排根区根尖和排根分泌有机酸的种类和数量 ,以及相应的根尖、排根组织 ,茎木质部、韧皮部汁液中有机酸含量的变化。结果表明 :(1)缺磷能够诱导白羽扇豆根系产生大量排根 ,根系的有机酸分泌量也明显增加。 (2 )无论在供磷或缺磷条件下 ,排根与非排根区根尖组织中的有机酸种类相同 ,但排根主要分泌柠檬酸和苹果酸 ,而非排根区根尖主要分泌苹果酸和乙酸。 (3)缺磷后非排根区根尖分泌苹果酸的量增加 ,至第 17天达到高峰 ;排根开始分泌柠檬酸的时间相对较晚。缺磷后排根分泌柠檬酸的量随缺磷时间的延长不断增加。 (4 )在缺磷的排根与非排根区根尖组织和茎木质部伤流液中含有大量柠檬酸和苹果酸 ,但在茎韧皮部汁液中则几乎检测不到这两种有机酸。上述结果表明 ,尽管排根和非排根区根尖组织中的有机酸种类相同 ,但它们向外分泌的有机酸种类不同。缺磷后排根及非排根区根尖增加向外分泌的有机酸主要在根中合成     

子叶磷在白羽扇豆缺磷适应性反应中的作用

实验用液体培养的方法,对比分析了在不同供磷条件下,白羽扇豆子叶中的磷对植物生长发育的影响,以及排根和根尖中有机酸积累和分泌的作用,结果表明,子叶中的磷能使白羽扇豆在完全缺磷２３d的环境中,不仅没有使干物质的积累减少,反而使干物质的积累略有增加,相反,如果没有子叶磷的供给,则使白羽扇豆在缺磷环境中产生强烈的抗胁迫反应,表现在干物质的积累明显下降,根系能产生大量的排根,排根能积累和分泌大量的柠檬酸,而根尖能积累和分泌萍果酸,在整个缺磷反应过程中,根尖中苹果酸的分泌要早于排根可柠檬酸的积累和分泌。     

Proteoid Root Development of Phosphorus Deficient Lupin is Mimicked by Auxin and Phosphonate

White lupin (Lupinus albus L.) develops proteoid (cluster) rootsin response to phosphorus deficiency. Proteoid roots are composedof tight clusters of rootlets that initiate from the pericycleopposite protoxylem poles and emerge from every protoxylem polewithin the proteoid root axis. Auxins are required for lateralroot development, but little is known of their role in proteoidroot formation. Proteoid root numbers were dramatically increasedin P-sufficient (+P) plants by application of the syntheticauxin, naphthalene acetic acid (NAA), to leaves, and were reducedin P-deficient (-P) plants by the presence of auxin transportinhibitors [2,3,5-triiodobenzoic acid (TIBA) and naphthylphthalamicacid (NPA)]. While ethylene concentrations in the root zonewere 1.5-fold higher in -P plants, there was no effect on proteoidroot numbers of the ethylene inhibitors aminoethoxyvinvylglycine(AVG) and silver thiosulphate. Phosphonate, which interfereswith plant perception of internal P concentration, dramaticallyincreased the number of proteoid root segments in +P plants.Activities of phosphoenolpyruvate carboxylase (PEPC), malatedehydrogenase (MDH) and exuded acid phosphatase in proteoidroot segments were not different from +P controls when NAA wasapplied to +P lupin plants, but increased to levels comparableto -P plants in the phosphonate treatment. Addition of TIBAor NPA to -P plants reduced PEPC and MDH activity of -P proteoidroots to levels found in +P or -P normal root tissues, but didnot affect acid phosphatase in root exudates. These resultssuggest that auxin transport from the shoot plays a role inthe formation of proteoid roots during P deficiency. Auxin-stimulatedproteoid root formation is necessary, but not sufficient, tosignal the up-regulation of PEPC and MDH in proteoid root segments.In contrast, phosphonate applied to P-sufficient white lupinelicits the full suite of coordinated responses to P deficiencyCopyright2000 Annals of Botany Company Lupinus albus L., white lupin, proteoid roots, auxin, ethylene, phosphonate, phosphorus deficiency     

Acclimation of white lupin to phosphorus deficiency involves enhanced expression of genes related to organic acid metabolism

White lupin (Lupinus albus L.) acclimates to phosphorus deficiency (–P) by the development of short, densely clustered lateral roots called proteoid (or cluster) roots. These specialized plant organs display increased exudation of citric and malic acid. The enhanced exudation of organic acids from P stressed white lupin roots is accompanied by increased in vitro phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activity. Here we report the cloning of full-length white lupin PEPC and MDH cDNAs. RNA blot analysis indicates enhanced expression of these genes in –P proteoid roots, placing higher gene expression at the site of organic acid exudation. Correspondingly, macroarray analysis of about 1250 ESTs (expressed sequence tags) revealed induced expression of genes involved in organic acid metabolism in –P proteoid roots. In situ hybridization revealed that PEPC and MDH were both expressed in the cortex of emerging and mature proteoid rootlets. A C₃ PEPC protein was partially purified from proteoid roots of P deficient white lupin. Native and subunit Mr were determined to be 440 kD and 110 kD, respectively. Citrate and malate were effective inhibitors of in vitro PEPC activity at pH 7. Addition of ATP partially relieved inhibition of PEPC by malate but had little effect on citrate inhibition. Taken together, the results presented here suggest that acclimation of white lupin to low P involves modified expression of plant genes involved in carbon metabolism.     

miR393 and miR164 influence indeterminate but not determinate nodule development

The roles of auxin in the regulation of symbiotic legume nodule formation are unclear. We recently showed that enhanced sensitivity to auxin resulting from overexpression of miR160 inhibits determinate nodule formation in soybean. We examined the roles of miR393 and miR164 in soybean (that forms determinate nodules) and Medicago truncatula (that forms indeterminate nodules). Our results together with previous studies suggest that indeterminate nodule formation requires a higher, but narrow window of auxin sensitivity and that miR164 regulation is not crucial for determinate nodule formation.     

Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress

White lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to -P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA(i))-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under -P stress. Here, we show that LaMATE had high expression in proteoid roots not only under -P, but also under -Fe, -N, -Mn and +Al stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein. LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA(i)-based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.     

MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for arabidopsis lateral root development

Although several plant microRNAs (miRNAs) have been shown to play a role in plant development, no phenotype has yet been associated with a reduction or loss of expression of any plant miRNA. Arabidopsis thaliana miR164 was predicted to target five NAM/ATAF/CUC (NAC) domain-encoding mRNAs, including NAC1, which transduces auxin signals for lateral root emergence. Here, we show that miR164 guides the cleavage of endogenous and transgenic NAC1 mRNA, producing 3'-specific fragments. Cleavage was blocked by NAC1 mutations that disrupt base pairing with miR164. Compared with wild-type plants, Arabidopsis mir164a and mir164b mutant plants expressed less miR164 and more NAC1 mRNA and produced more lateral roots. These mutant phenotypes can be complemented by expression of the appropriate MIR164a and MIR164b genomic sequences. By contrast, inducible expression of miR164 in wild-type plants led to decreased NAC1 mRNA levels and reduced lateral root emergence. Auxin induction of miR164 was mirrored by an increase in the NAC1 mRNA 3' fragment, which was not observed in the auxin-insensitive mutants auxin resistant1 (axr1-12), axr2-1, and transport inhibitor response1. Moreover, the cleavage-resistant form of NAC1 mRNA was unaffected by auxin treatment. Our results indicate that auxin induction of miR164 provides a homeostatic mechanism to clear NAC1 mRNA to downregulate auxin signals.