Dynamics of MONOPTEROS and PIN-FORMED1 expression during leaf vein pattern formation in Arabidopsis thaliana

Genetic evidence links the Arabidopsis MONOPTEROS (MP) and PIN-FORMED1 (PIN1) genes to the patterning of leaf veins. To elucidate their potential functions and interactions in this process, we have assessed the dynamics of MP and PIN1 expression during vascular patterning in Arabidopsis leaf primordia. Both genes undergo a dynamic process of gradual refinement of expression into files one to two cells wide before overt vascular differentiation. The subcellular distribution of PIN1 is also gradually refined from a non-polar distribution in isodiametric cells to strongly polarized in elongated procambial cells and provides an indication of overall directions of auxin flow. We found evidence that MP expression can be activated by auxin exposure and that PIN1 as well as DR5::GUS expression is defective in mp mutant leaves. Taken together the results suggest a feedback regulatory loop that involves auxin, MP and PIN1 and provide novel experimental support for the canalization-of-auxin-flow hypothesis.     

The Arabidopsis TALE homeobox gene ATH1 controls floral competency through positive regulation of FLC

Floral induction is controlled by a plethora of genes acting in different pathways that either repress or promote floral transition at the shoot apical meristem (SAM). During vegetative development high levels of floral repressors maintain the Arabidopsis SAM as incompetent to respond to promoting factors. Among these repressors, FLOWERING LOCUS C (FLC) is the most prominent. The processes underlying downregulation of FLC in response to environmental and developmental signals have been elucidated in considerable detail. However, the basal induction of FLC and its upregulation by FRIGIDA (FRI) are still poorly understood. Here we report the functional characterization of the ARABIDOPSIS THALIANA HOMEOBOX 1 (ATH1) gene. A function of ATH1 in floral repression is suggested by a gradual downregulation of ATH1 in the SAM prior to floral transition. Further evidence for such a function of ATH1 is provided by the vernalization-sensitive late flowering of plants that constitutively express ATH1. Analysis of lines that differ in FRI and/or FLC allele strength show that this late flowering is caused by upregulation of FLC as a result of synergism between ATH1 overexpression and FRI. Lack of ATH1, however, results in attenuated FLC levels independently of FRI, suggesting that ATH1 acts as a general activator of FLC expression. This is further corroborated by a reduction of FLC-mediated late flowering in fca-1 and fve-1 autonomous pathway backgrounds when combined with ath1. Since other floral repressors of the FLC clade are not significantly affected by ATH1, we conclude that ATH1 controls floral competency as a specific activator of FLC expression.     

Reviewing models of auxin canalization in the context of leaf vein pattern formation in Arabidopsis

In both plants and animals vein networks play an essential role in transporting nutrients. In plants veins may also provide mechanical support. The mechanism by which vein patterns are formed in a developing leaf remains largely unresolved. According to the canalization hypothesis, a signal inducing vein differentiation is transported in a polar manner and is channeled into narrow strands. Since inhibition of auxin transport affects venation patterns, auxin is likely to be part of the signal involved. However, it is not clear whether the canalization hypothesis, initially formulated over 25 years ago, is compatible with recent experimental data. In this paper we focus on three aspects of this question, and show that: (i) canalization models can account for an acropetal development of the midvein if vein formation is sink-driven; (ii) canalization models are in agreement with venation patterns resulting from inhibited auxin transport and (iii) loops and discontinuous venation patterns can be obtained assuming proper spacing of discrete auxin sources.     

The ADAXIALIZED LEAF1 gene functions in leaf and embryonic pattern formation in rice

The adaxial-abaxial axis in leaf primordia is thought to be established first and is necessary for the expansion of the leaf lamina along the mediolateral axis. To understand axis information in leaf development, we isolated the adaxialized leaf1 (adl1) mutant in rice, which forms abaxially rolled leaves. adl1 leaves are covered with bulliform-like cells, which are normally distributed only on the adaxial surface. An adl1 double mutant with the adaxially snowy leaf mutant, which has albino cells that specifically appear in the abaxial mesophyll tissue, indicated that adl1 leaves show adaxialization in both epidermal and mesophyll tissues. The expression of HD-ZIPIII genes in adl1 mutant increased in mature leaves, but not in the young primordia or the SAM. This indicated that ADL1 may not be directly involved in determining initial leaf polarity, but rather is associated with the maintenance of axis information. ADL1 encodes a plant-specific calpain-like cysteine proteinase orthologous to maize DEFECTIVE KERNEL1. Furthermore, we identified intermediate and strong alleles of the adl1 mutant that generate shootless embryos and globular-arrested embryos with aleurone layer loss, respectively. We propose that ADL1 plays an important role in pattern formation of the leaf and embryo by promoting proper epidermal development.     

SHORT-ROOT regulates vascular patterning,but not apical meristematic activity in the Arabidopsis root through cytokinin homeostasis

SHORT-ROOT (SHR) is a key regulator of radial patterning and stem-cell renewal in the Arabidopsis root. Although SHR is expressed in the stele, its function in the vascular tissue was not recognized until recently. In shr, the protoxylem is missing due to the loss of expression of microRNA165A (miR165A) and microRNA166B (miR165B). shr is also defective in lateral root formation, but the mechanism remains unclear. To dissect the SHR developmental pathway, we recently have identified its direct targets at the genome scale by chromatin immunoprecipitation followed by microarray analysis (ChIP-chip). In further studies, we have shown that SHR regulates cytokinin homeostasis through cytokinin oxidase 3 and that this role of SHR is critical to vascular patterning in the root. In this communication we report that SHR also regulates miR165A and miR166B indirectly through its effect on cytokinin homeostasis. Although cytokinin is inhibitory to root growth, the root-apical-meristem defect in shr was not alleviated by reduction of endogenous cytokinin. These results together suggest that SHR regulates vascular patterning, but not root apical meristematic activity, through cytokinin homeostasis.     

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.     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

A molecular mechanism that confines the activity pattern of miR165 in Arabidopsis leaf primordia

In Arabidopsis leaf primordia, the expression of HD‐Zip III, which promotes tissue differentiation on the adaxial side of the leaf primordia, is repressed by miRNA165/166 (miR165/166). Small RNAs, including miRNAs, can move from cell to cell. In this study, HD‐Zip III expression was strikingly repressed by miR165/166 in the epidermis and parenchyma cells on the abaxial side of the leaf primordia compared with those on the adaxial side. We also found that the MIR165A locus, which was expressed in the abaxial epidermis, was sufficient to establish the rigid repression pattern of HD‐Zip III expression in the leaf primordia. Ectopic expression analyses of MIR165A showed that the abaxial‐biased miR165 activity in the leaf primordia was formed neither by a polarized distribution of factors affecting miR165 activity nor by a physical boundary inhibiting the cell‐to‐cell movement of miRNA between the adaxial and abaxial sides. We revealed that cis‐acting factors, including the promoter, backbone, and mature miRNA sequence of MIR165A, are necessary for the abaxial‐biased activity of miR165 in the leaf primordia. We also found that the abaxial‐determining genes YABBYs are trans‐acting factors that are necessary for the miR165 activity pattern, resulting in the rigid determination of the adaxial–abaxial boundary in leaf primordia. Thus, we proposed a molecular mechanism in which the abaxial‐biased patterning of miR165 activity is confined.     

BLADE-ON-PETIOLE 1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes

We report a novel function for BLADE-ON-PETIOLE1 (BOP1) and BOP2 in regulating Arabidopsis thaliana lateral organ cell fate and polarity, through the analysis of loss-of-function mutants and transgenic plants that ectopically express BOP1 or BOP2. 35S:BOP1 and 35S:BOP2 plants exhibit a very short and compact stature, hyponastic leaves, and downward-orienting siliques. We show that the LATERAL ORGAN BOUNDARIES (LOB) domain genes ASYMMETRIC LEAVES2 (AS2) and LOB are upregulated in 35S:BOP and downregulated in bop mutant plants. Ectopic expression of BOP1 or BOP2 also results in repression of class I knox gene expression. We further demonstrate a role for BOP1 and BOP2 in establishing the adaxial-abaxial polarity axis in the leaf petiole, where they regulate PHB and FIL expression and overlap in function with AS1 and AS2. Interestingly, during this study, we found that KANADI1 (KAN1) and KAN2 act to promote adaxial organ identity in addition to their well-known role in promoting abaxial organ identity. Our data indicate that BOP1 and BOP2 act in cells adjacent to the lateral organ boundary to repress genes that confer meristem cell fate and induce genes that promote lateral organ fate and polarity, thereby restricting the developmental potential of the organ-forming cells and facilitating their differentiation.     

The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana

The CURLY LEAF (CLF ) gene in Arabidopsis thaliana (L.) Heynh. is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems To clarify the function of CLF in organ development, we characterized clf mutants using an anatomical and genetic approach. The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions. A decrease both in the extent of cell elongation and in the number of cells was evident in the clf mutant leaves, suggesting that the CLF gene might be involved in the division and elongation of cells during leaf morphogenesis. An analysis of the development of clf mutant leaves revealed that the period during which cell division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type. Two phases in the elongation of cells were also recognized from this analysis. From analysis of an angustifolia clf double mutant, we found that the two phases of elongation of leaf cells were regulated independently by each gene. Thus, the CLF gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. Received: 19 February 1998 / Accepted: 24 March 1998     

Mutations in the FASS gene uncouple pattern formation and morphogenesis in Arabidopsis development.

The pattern of cell division is very regular in Arabidopsis embryogenesis, enabling seedling structures to be traced back to groups of cells in the early embryo. Recessive mutations in the FASS gene alter the pattern of cell division from the zygote, without interfering with embryonic pattern formation: although no primordia of seedling structures can be recognised by morphological criteria at the early-heart stage, all elements of the body pattern are differentiated in the seedling. fass seedlings are strongly compressed in the apical-basal axis and enlarged circumferentially, notably in the hypocotyl. Depending on the width of the hypocotyl, fass seedlings may have up to three supernumerary cotyledons. fass mutants can develop into tiny adult plants with all parts, including floral organs, strongly compressed in their longitudinal axis. At the cellular level, fass mutations affect cell elongation and orientation of cell walls but do not interfere with cell polarity as evidenced by the unequal division of the zygote. The results suggest that the FASS gene is required for morphogenesis, i.e., oriented cell divisions and position-dependent cell shape changes generating body shape, but not for cell polarity which seems essential for pattern formation.