Evolution and diverse roles of the CUP-SHAPED COTYLEDON genes in Arabidopsis leaf development

CUP-SHAPED COTYLEDON2 (CUC2) and the interacting microRNA miR164 regulate leaf margin dissection. Here, we further investigate the evolution and the specific roles of the CUC1 to CUC3 genes during Arabidopsis thaliana leaf serration. We show that CUC2 is essential for dissecting the leaves of a wide range of lobed/serrated Arabidopsis lines. Inactivation of CUC3 leads to a partial suppression of the serrations, indicating a role for this gene in leaf shaping. Morphometric analysis of leaf development and genetic analysis provide evidence for different temporal contributions of CUC2 and CUC3. Chimeric constructs mixing CUC regulatory sequences with different coding sequences reveal both redundant and specific roles for the three CUC genes that could be traced back to changes in their expression pattern or protein activity. In particular, we show that CUC1 triggers the formation of leaflets when ectopically expressed instead of CUC2 in the developing leaves. These divergent fates of the CUC1 and CUC2 genes after their formation by the duplication of a common ancestor is consistent with the signature of positive selection detected on the ancestral branch to CUC1. Combining experimental observations with the retraced origin of the CUC genes in the Brassicales, we propose an evolutionary scenario for the CUC genes.     

The Arabidopsis BEL1-LIKE HOMEODOMAIN proteins SAW1 and SAW2 act redundantly to regulate KNOX expression spatially in leaf margins

In Arabidopsis thaliana, the BEL1-like TALE homeodomain protein family consists of 13 members that form heterodimeric complexes with the Class 1 KNOX TALE homeodomain proteins, including SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). The BEL1-like protein BELLRINGER (BLR) functions together with STM and BP in the shoot apex to regulate meristem identity and function and to promote correct shoot architecture. We have characterized two additional BEL1-LIKE HOMEODOMAIN (BLH) proteins, SAWTOOTH1 (BLH2/SAW1) and SAWTOOTH2 (BLH4/SAW2) that, in contrast with BLR, are expressed in lateral organs and negatively regulate BP expression. saw1 and saw2 single mutants have no obvious phenotype, but the saw1 saw2 double mutant has increased leaf serrations and revolute margins, indicating that SAW1 and SAW2 act redundantly to limit leaf margin growth. Consistent with this hypothesis, overexpression of SAW1 suppresses overall growth of the plant shoot. BP is ectopically expressed in the leaf serrations of saw1 saw2 double mutants. Ectopic expression of Class 1 KNOX genes in leaves has been observed previously in loss-of-function mutants of ASYMMETRIC LEAVES (AS1). Overexpression of SAW1 in an as1 mutant suppresses the as1 leaf phenotype and reduces ectopic BP leaf expression. Taken together, our data suggest that BLH2/SAW1 and BLH4/SAW2 establish leaf shape by repressing growth in specific subdomains of the leaf at least in part by repressing expression of one or more of the KNOX genes.     

Mechanisms of leaf tooth formation in Arabidopsis

Serration found along leaf margins shows species‐specific characters. Whereas compound leaf development is well studied, the process of serration formation is largely unknown. To understand mechanisms of serration development, we investigated distinctive features of cells that could give rise to tooth protrusion in the simple‐leaf plant Arabidopsis. After the emergence of a tooth, marginal cells, except for cells at the sinuses and tips, started to elongate rapidly. Localized cell division seemed to keep cells at the sinus smaller, rather than halt cell elongation. As leaves matured, the marginal cell number between teeth became similar in any given tooth. These results suggest that teeth are formed by repetition of an unknown mechanism that spatially monitors cell number and regulates cell division. We then examined the role of CUP‐SHAPED COTYLEDON 2 (CUC2) in serration development. cuc2‐3 forms fewer hydathodes and auxin maxima, visualized by DR5rev::GFP, at the leaf margin, suggesting that CUC2 patterns serration through the regulation of auxin. In contrast to a previous interpretation, comparison of leaf outlines revealed that CUC2 promotes outgrowth of teeth rather than suppression of growth at the sinuses. We found that mutants with increased CUC2 expression form ectopic tissues and mis‐express SHOOT MERISTEMLESS (STM) at the sinus between the enhanced teeth. Similar but infrequent STM expression was found in the wild type, indicating STM involvement in the serration of simple leaves. Our study provides insights into the morphological and molecular mechanisms for leaf development and tooth formation, and highlights similarities between serration and compound leaf development.     

毛竹miR164b前体的克隆及其在叶形态建成中的功能分析

microRNA（miRNA）参与植物多种生理代谢过程,在调控植物形态建成中发挥着重要作用。miR164作为植物特有的miRNA,其主要的靶基因是NAC转录因子,参与调控植物茎、叶顶端分生组织的建立、器官的分化和植株衰老等过程。本研究以毛竹（Phyllostachys edulis（Carr.） Lehaie）为材料,从中分离出miR164b的前体序列（82 bp）,二级结构分析结果发现该前体序列能够形成稳定的茎环结构,其成熟序列（21 bp）产生于茎环结构5'端的臂上,且碱基具有较高的保守性。本研究还构建了由CaMV 35S启动,包含毛竹miR164b前体序列的植物表达载体,并转化野生型拟南芥（Arabidopsis thaliana（L.） Heynh）,获得了转基因植株。结果表明,转基因植株生长瘦弱,莲座叶数量明显减少,叶片变小且叶片边缘锯齿减少,更加光滑。实时定量PCR分析结果显示,转基因拟南芥中毛竹miR164b的表达量极显著上升,而拟南芥内源靶基因CUC1与CUC2的表达量极显著下降。表明毛竹miR164b通过调节CUC1和CUC2的表达来参与植物叶形态建成过程。研究结果可为利用miRNA开展竹子分子育种提供参考。     

植物叶缘和叶脉发育调控的研究进展

通常可通过植物叶片的形态来区分不同植物的种类。叶片由茎顶端分生组织侧翼发育而成,为多种多样大小和形状的扁平结构。叶片的结构看似简单,但调控叶片形态和结构发育的分子机理错综复杂,叶片的发育受植物激素、转录因子、一系列蛋白因子及环境的共同调控。本文回顾了叶片边缘形态和叶脉发育研究的最新进展。在叶边缘形态方面,Aux/IAA生长素响应抑制家族蛋白通过调节生长素浓度最大点的离散分布影响小叶的起始和生长以及叶边缘结构;NAM/CUC转录因子促进叶边缘锯齿的分离以及复叶中小叶的分离和分化,NAM/CUC和Aux/IAA通过不同通路实现对生长素的调控;拟南芥RAX1基因/番茄Potato-leaf基因和拟南芥JAG基因/番茄LYR基因促进叶边缘锯齿发育;RCO调控复叶小叶的发育不通过改变生长素的分布来实现;在番茄中反式小干扰RNA途径中的因子参与叶边缘形态发育;另外,在拟南芥中,mir164A、CUC2、PIN1、DPA4、SVR9-1及SVR9L-1构成复杂的调控网络影响叶边缘锯齿的发育。在叶脉发育方面,PIN1能否正确的定位会影响叶脉发育;AS1和AS2共同参与叶片远近轴极性的分化;另外AXR6、MP、BDL、CVP因子功能的缺失影响叶脉发育;生长素、PIN1、Aux/IAA、MP、ATHB8构成反馈循环调控子叶叶脉的形成。     

Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes

The shoot apical meristem and cotyledons of higher plants are established during embryogenesis in the apex. Redundant CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 as well as SHOOT MERISTEMLESS (STM) of Arabidopsis are required for shoot apical meristem formation and cotyledon separation. To elucidate how the apical region of the embryo is established, we investigated genetic interactions among CUC1, CUC2 and STM, as well as the expression patterns of CUC2 and STM mRNA. Expression of these genes marked the incipient shoot apical meristem as well as the boundaries of cotyledon primordia, consistent with their roles for shoot apical meristem formation and cotyledon separation. Genetic and expression analyses indicate that CUC1 and CUC2 are redundantly required for expression of STM to form the shoot apical meristem, and that STM is required for proper spatial expression of CUC2 to separate cotyledons. A model for pattern formation in the apical region of the Arabidopsis embryo is presented.     

The role of ARGONAUTE1 (AGO1) in meristem formation and identity

The ARGONAUTE gene family is involved in the regulation of gene expression via the RNAi Silencing Complex (RISC). microRNA (miRNA) are 20-22bp RNAs that direct RISC to target genes. Several miRNA have been characterized in plants. Their roles include control of flowering time, floral organ identity, cell division patterns, and leaf polarity. ARGONAUTE1 (AGO1) is required for stem cell function and organ polarity, as is the closely related protein PINHEAD/ZWILLE (PNH/ZLL). Through phenotypic and double mutant analysis, we show that AGO1 regulates stem cell function via SHOOT MERISTEMLESS (STM). CUPSHAPED COTYLEDONS1 and 2 (CUC1 and CUC2) positively regulate STM and are targets of miRNA. The effect of AGO1 on leaf polarity is dependent, in part, on its role in meristem function revealed by interactions with ASYMMETRIC LEAVES1(AS1). AGO1 is required for full expression of LEAFY (LFY), APETALA1 (AP1) and AGAMOUS (AG). Flowering time is unaffected but floral meristem identity is partially restored in a curlyleaf (clf) background and this is not due to clf's affects on AG expression. CLF is over expressed in ago1, showing that the RNAi pathway regulates polycomb-type epigenetic modifiers.