Leaf adaxial-abaxial polarity specification and lamina outgrowth: evolution and development

A key innovation in leaf evolution is the acquisition of a flat lamina with adaxial-abaxial polarity, which optimizes the primary function of photosynthesis. The developmental mechanism behind leaf adaxial-abaxial polarity specification and flat lamina formation has long been of interest to biologists. Surgical and genetic studies proposed a conceptual model wherein a signal derived from the shoot apical meristem is necessary for adaxial-abaxial polarity specification, and subsequent lamina outgrowth is promoted at the juxtaposition of adaxial and abaxial identities. Several distinct regulators involved in leaf adaxial-abaxial polarity specification and lamina outgrowth have been identified. Analyses of these genes demonstrated that the mutual antagonistic interactions between adaxial and abaxial determinants establish polarity and define the boundary between two domains, along which lamina outgrowth regulators function. Evolutionary developmental studies on diverse leaf forms of angiosperms proposed that alteration to the adaxial-abaxial patterning system can be a major driving force in the generation of diverse leaf forms, as represented by 'unifacial leaves', in which leaf blades have only the abaxial identity. Interestingly, unifacial leaf blades become flattened, in spite of the lack of adaxial-abaxial juxtaposition. Modification of the adaxial-abaxial patterning system is also utilized to generate complex organ morphologies, such as stamens. In this review, we summarize recent advances in the genetic mechanisms underlying leaf adaxial-abaxial polarity specification and lamina outgrowth, with emphasis on the genetic basis of the evolution and diversification of leaves.     

Characterization of the AE7 gene in Arabidopsis suggests that normal cell proliferation is essential for leaf polarity establishment

The formation of leaf polarity is critical for leaf morphogenesis. In this study, we characterized and cloned an Arabidopsis gene, AS1/2 ENHANCER7 (AE7), which is required for both leaf adaxial-abaxial polarity formation and normal cell proliferation. The ae7 mutant exhibited leaf adaxial-abaxial polarity defects and double mutants combining ae7 with the leaf polarity mutants as1 (asymmetric leaves1), as2, rdr6 (RNA-dependent RNA polymerase6) or ago7/zip (argonaute7/zippy) all resulted in plants with an apparently enhanced loss of adaxial leaf identity. In addition, ae7 also showed decreased cell proliferation in both leaves and roots, compensated by increased cell sizes in leaves. AE7 encodes a protein conserved in many eukaryotic organisms, ranging from unicellular yeasts to humans; however, the functions of AE7 family members from other species have not been reported. In situ hybridization revealed that AE7 is expressed in a spotted pattern in plant tissues, similar to cell-cycle marker genes such as HISTONE4. Moreover, the ae7 endoploidy and expression analysis of several cell-cycle marker genes in ae7 suggest that the AE7 gene is required for cell cycle progression. As the previously characterized 26S proteasome and ribosome mutants also affect both leaf adaxial-abaxial polarity and cell proliferation, similar to the defects in ae7, we propose that normal cell proliferation may be essential for leaf polarity establishment. Possible models for how cell proliferation influences leaf adaxial-abaxial polarity establishment are discussed.     

Novel as1 and as2 defects in leaf adaxial-abaxial polarity reveal the requirement for ASYMMETRIC LEAVES1 and 2 and ERECTA functions in specifying leaf adaxial identity

The shoot apical meristem (SAM) of seed plants is the site at which lateral organs are formed. Once organ primordia initiate from the SAM, they establish polarity along the adaxial-abaxial, proximodistal and mediolateral axes. Among these three axes, the adaxial-abaxial polarity is of primary importance in leaf patterning. In leaf development, once the adaxial-abaxial axis is established within leaf primordia, it provides cues for proper lamina growth and asymmetric development. It was reported previously that the Arabidopsis ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) genes are two key regulators of leaf polarity. In this work, we demonstrate a new function of the AS1 and AS2 genes in the establishment of adaxial-abaxial polarity by analyzing as1 and as2 alleles in the Landsberg erecta (Ler) genetic background. We provide genetic evidence that the Arabidopsis ERECTA (ER) gene is involved in the AS1-AS2 pathway to promote leaf adaxial fate. In addition, we show that AS1 and AS2 bind to each other, suggesting that AS1 and AS2 may form a complex that regulates the establishment of leaf polarity. We also report the effects on leaf polarity of overexpression of the AS1 or AS2 genes under the control of the cauliflower mosaic virus (CAMV) 35S promoter. Although plants with as1 and as2 mutations have very similar phenotypes, 35S::AS1/Ler and 35S::AS2/Ler transgenic plants showed dramatically different morphologies. A possible model of the AS1, AS2 and ER action in leaf polarity formation is discussed.     

Distinct Regulation of Adaxial-Abaxial Polarity in Anther Patterning in Rice

Establishment of adaxial-abaxial polarity is essential for lateral organ development. The mechanisms underlying the polarity establishment in the stamen remain unclear, whereas those in the leaf are well understood. Here, we investigated a rod-like lemma (rol) mutant of rice (Oryza sativa), in which the development of the stamen and lemma is severely compromised. We found that the rod-like structure of the lemma and disturbed anther patterning resulted from defects in the regulation of adaxial-abaxial polarity. Gene isolation indicated that the rol phenotype was caused by a weak mutation in SHOOTLESS2 (SHL2), which encodes an RNA-dependent RNA polymerase and functions in trans-acting small interfering RNA (ta-siRNA) production. Thus, ta-siRNA likely plays an important role in regulating the adaxial-abaxial polarity of floral organs in rice. Furthermore, we found that the spatial expression patterns of marker genes for adaxial-abaxial polarity are rearranged during anther development in the wild type. After this rearrangement, a newly formed polarity is likely to be established in a new developmental unit, the theca primordium. This idea is supported by observations of abnormal stamen development in the shl2-rol mutant. By contrast, the stamen filament is likely formed by abaxialization. Thus, a unique regulatory mechanism may be involved in regulating adaxial-abaxial polarity in stamen development.     

miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis

In plants, cell proliferation and polarized cell differentiation along the adaxial-abaxial axis in the primordium is critical for leaf morphogenesis, while the temporal-spatial relationships between these two processes remain largely unexplored. Here, it is reported that microRNA396 (miR396)-targeted Arabidopsis growth-regulating factors (AtGRFs) are required for leaf adaxial-abaxial polarity in Arabidopsis. Reduction of the expression of AtGRF genes by transgenic miR396 overexpression in leaf polarity mutants asymmetric leaves1 (as1) and as2 resulted in plants with enhanced leaf adaxial-abaxial defects, as a consequence of reduced cell proliferation. Moreover, transgenic miR396 overexpression markedly decreased the cell division activity and the expression of cell cycle-related genes, but resulted in an increased percentage of leaf cells with a higher ploidy level, indicating that miR396 negatively regulates cell proliferation by controlling entry into the mitotic cell cycle. miR396 is mainly expressed in the leaf cells arrested for cell division, coinciding with its roles in cell cycle regulation. These results together suggest that cell division activity mediated by miR396-targeted AtGRFs is important for polarized cell differentiation along the adaxial-abaxial axis during leaf morphogenesis in Arabidopsis.     

生长素对拟南芥叶片发育调控的研究进展

叶片（包括子叶）是茎端分生组织产生的第一类侧生器官,在植物发育中具有重要地位。早期叶片发育包括三个主要过程：叶原基的起始,叶片腹背性的建立和叶片的延展。大量证据表明叶片发育受到体内遗传机制和体外环境因子的双重调节。植物激素,尤其是生长素在协调体内外调节机制中起着不可或缺的作用。生长素的稳态调控、极性运输和信号转导影响叶片发育的全过程。本文着重介绍生长素在叶片生长发育和形态建成中的调控作用,试图了解复杂叶片发育调控网络。     

生长素对拟南芥叶片发育调控的研究进展

叶片(包括子叶)是茎端分生组织产生的第一类侧生器官, 在植物发育中具有重要地位。早期叶片发育包括三个主要过程: 叶原基的起始, 叶片腹背性的建立和叶片的延展。大量证据表明叶片发育受到体内遗传机制和体外环境因子的双重调节。植物激素, 尤其是生长素在协调体内外调节机制中起着不可或缺的作用。生长素的稳态调控、极性运输和信号转导影响叶片发育的全过程。本文着重介绍生长素在叶片生长发育和形态建成中的调控作用, 试图了解复杂叶片发育调控网络。     

YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development

During leaf development, the formation of leaf adaxial-abaxial polarity at the primordium stage is crucial for subsequent leaf expansion. However, little is known about the genetic control from polarity establishment to blade outgrowth. The leaf margin, comprising elongated margin cells and hydathodes, is thought to affect leaf expansion. Here, we show that mutants with defective leaf polarity or with loss of function in the multiple auxin-biosynthetic YUCCA (YUC) genes exhibited a similar abnormal leaf margin and less-expanded leaves. Leaf margins of these mutants contained fewer hydathodes and an increased number of cell patches in which the patterns of epidermal cells resembled those of hydathodes. The previously characterized leaf-abaxialized asymmetric leaves2 (as2) revoluta (rev) and leaf-adaxialized kanadi1 (kan1) kan2 double mutants both produce finger-shaped, hydathode-like protrusions on adaxial and abaxial leaf surfaces, respectively. YUCs are required for formation of the protrusions, as those produced by as2 rev and kan1 kan2 were absent in the yuc1 yuc2 yuc4 triple mutant background. Expressions of YUC1, YUC2, and YUC4 were spatially regulated in the leaf, being associated with hydathodes in wild-type leaves and protrusions on as2 rev and kan1 kan2 leaves. In addition, inhibition of auxin transport by treatment of seedlings with N-(1-naphtyl) phtalamic acid or disruption of the auxin gradient by transforming plants with the 35S:YUC1 construct also blocked leaf margin development. Collectively, our data show that expressions of YUCs in the leaf respond to the adaxial-abaxial juxtaposition, and that the activities of auxin mediate leaf margin development, which subsequently promotes blade outgrowth.     

The proteolytic function of the Arabidopsis 26S proteasome is required for specifying leaf adaxial identity

Polarity formation is central to leaf morphogenesis, and several key genes that function in adaxial-abaxial polarity establishment have been identified and characterized extensively. We previously reported that Arabidopsis thaliana ASYMMERTIC LEAVES1 (AS1) and AS2 are important in promoting leaf adaxial fates. We obtained an as2 enhancer mutant, asymmetric leaves enhancer3 (ae3), which demonstrated pleiotropic plant phenotypes, including a defective adaxial identity in some leaves. The ae3 as2 double mutant displayed severely abaxialized leaves, which were accompanied by elevated levels of leaf abaxial promoting genes FILAMENTOUS FLOWER, YABBY3, KANADI1 (KAN1), and KAN2 and a reduced level of the adaxial promoting gene REVOLUTA. We identified AE3, which encodes a putative 26S proteasome subunit RPN8a. Furthermore, double mutant combinations of as2 with other 26S subunit mutations, including rpt2a, rpt4a, rpt5a, rpn1a, rpn9a, pad1, and pbe1, all displayed comparable phenotypes with those of ae3 as2, albeit with varying phenotypic severity. Since these mutated genes encode subunits that are located in different parts of the 26S proteasome, it is possible that the proteolytic function of the 26S holoenzyme is involved in leaf polarity formation. Together, our findings reveal that posttranslational regulation is essential in proper leaf patterning.     

Establishing leaf polarity: the role of small RNAs and positional signals in the shoot apex

The flattening of leaves results from the juxtaposition of upper (adaxial) and lower (abaxial) domains in the developing leaf primordium. The adaxial-abaxial axis reflects positional differences in the leaf relative to the meristem and is established by redundant genetic pathways that interpret this asymmetry through instructive, possibly non-cell autonomous, signals. Small RNAs have been found to play a crucial role in this process, and specify mutually antagonistic fates. Here, we review both classical and recently-discovered factors that contribute to leaf polarity, as well as the candidate positional signals that their existence implies.     

叶发育的遗传调控机理研究进展

叶是植物进行光合作用的主要器官。高等植物叶原基起始于顶端分生组织的周边区,在一系列基因精确调控下,叶原基建立近一远轴、基一顶轴和中．侧轴极性,引导原基细胞朝着特定的方向分裂和分化,最终发育戍一定形态和大小的叶片。近年来分子遗传学研究结果表明,数个转录因子家族基因、小分子RNA和细胞增殖相关因子组成一个复杂的遗传控制网络,调节叶片极性建成过程。此外,复叶的形态建成还受到另外一些转录因子的调控。本文对近年来叶发育遗传调控机理研究的新进展做简要介绍。     

Developmental events leading to peltate leaf structure in <Emphasis Type="Italic">Tropaeolum majus</Emphasis> (Tropaeolaceae) are associated with expression domain changes of a YABBY gene

Peltate leaf architecture has evolved from conventional bifacial leaves many times in flowering plant evolution. Characteristics of peltate leaves, such as the differentiation of a cross zone and of a radially symmetric, margin-less petiole, have also been observed in mutants of genes responsible for adaxial-abaxial polarity establishment. This suggests that altered regulation of such genes provided a mechanism for the evolution of peltate leaf structure. Here, we show that evolution of leaf peltation in Tropaeolum majus, a species distantly related to Arabidopsis thaliana, was associated with altered expression of Tropaeolum majus FILAMENTOUS FLOWER (TmFIL), a gene conferring abaxial identity. In situ hybridization indicates that adaxial and abaxial domains are established in early leaf primordia as in species with bifacial leaves. Upon initiation of the cross zone by fusion of the blade margins, localized expansion of TmFIL to the upper leaf side could be seen, indicating a local loss of adaxial leaf identity. The observed changes in expression are consistent with a role of TmFIL in radialization of the petiole and circularization of the leaf blade margin by the cross zone. In addition, expression was observed in segment primordia and during expansion of the bifacial blade, suggesting additional roles for TmFIL in leaf development.     

The ASYMMETRIC LEAVES Complex Employs Multiple Modes of Regulation to Affect Adaxial-Abaxial Patterning and Leaf Complexity

Flattened leaf architecture is not a default state but depends on positional information to precisely coordinate patterns of cell division in the growing primordium. This information is provided, in part, by the boundary between the adaxial (top) and abaxial (bottom) domains of the leaf, which are specified via an intricate gene regulatory network whose precise circuitry remains poorly defined. Here, we examined the contribution of the ASYMMETRIC LEAVES (AS) pathway to adaxial-abaxial patterning in Arabidopsis thaliana and demonstrate that AS1-AS2 affects this process via multiple, distinct regulatory mechanisms. AS1-AS2 uses Polycomb-dependent and -independent mechanisms to directly repress the abaxial determinants MIR166A, YABBY5, and AUXIN RESPONSE FACTOR3 (ARF3), as well as a nonrepressive mechanism in the regulation of the adaxial determinant TAS3A. These regulatory interactions, together with data from prior studies, lead to a model in which the sequential polarization of determinants, including AS1-AS2, explains the establishment and maintenance of adaxial-abaxial leaf polarity. Moreover, our analyses show that the shared repression of ARF3 by the AS and trans-acting small interfering RNA (ta-siRNA) pathways intersects with additional AS1-AS2 targets to affect multiple nodes in leaf development, impacting polarity as well as leaf complexity. These data illustrate the surprisingly multifaceted contribution of AS1-AS2 to leaf development showing that, in conjunction with the ta-siRNA pathway, AS1-AS2 keeps the Arabidopsis leaf both flat and simple.