Subcellular localizations of AS1 and AS2 suggest their common and distinct roles in plant development

During leaf organogenesis, a critical step for normal leaf primordium initiation is the repression of the class 1 KNOTTED1-like homeobox (KNOX) genes. After leaf primordia are formed, they must establish polarity for normal leaf morphogenesis.Recent studies have led to the identification of a number of genes that participate in the class 1 KNOX gene repression and/or the leaf polarity establishment. ASTMMETRIC LEAVES1 and 2 (AS1 and AS2) are two of these genes, which are critical for both of these two processes. As a first step towards understanding the molecular genetic basis of the AS1-AS2 action, we determined the subcellular Iocalizations of the two proteins in both tobacco BY2 cells and Arabidopsis plants,by fusing them to yellow/cyan fluorescent protein (YFPICFP). Our data showed that AS1 and AS2 alone were predominantly localized in the nucleolus and the nucleoplasm, respectively. The presence of both AS1 and AS2 proteins in the same interphase cell demonstrated their co-localization in both nucleolus and nucleoplasm. In addition, AS1 alone was able to associate with the condensed chromosome in the metaphase cell. Our data suggest that AS1, AS2 and the AS1-AS2 protein complex may have distinct functions, which are all required for normal plant development.     

Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 ( as1 ) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED -like homeobox ( KNOX ) genes ( BP , KNAT2 and KNAT6 ) and ETTIN / ARF3 , is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2 . Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.     

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.     

PHANTASTICA regulates development of the adaxial mesophyll in Nicotiana leaves

Initiation and growth of leaf blades is oriented by an adaxial/abaxial axis aligned with the original axis of polarity in the leaf primordium. To investigate mechanisms regulating this process, we cloned the Nicotiana tabacum ortholog of PHANTASTICA (NTPHAN) and generated a series of antisense transgenics in N. sylvestris. We show that NSPHAN is expressed throughout emerging blade primordia in the wild type and becomes localized to the middle mesophyll in the expanding lamina. Antisense NSPHAN leaves show ectopic expression of NTH20, a class I KNOX gene. Juvenile transgenic leaves have normal adaxial/abaxial polarity and generate leaf blades in the normal position, but the adaxial mesophyll shows disorganized patterns of cell division, delayed maturation of palisade, and ectopic reinitiation of blade primordia along the midrib. Reversal of the phenotype with exogenous gibberellic acid suggests that NSPHAN, acting via KNOX repression, maintains determinacy in the expanding lamina and sustains the patterns of cell proliferation critical to palisade development.     

Histone deacetylases and ASYMMETRIC LEAVES2 are involved in the establishment of polarity in leaves of Arabidopsis

We show that two Arabidopsis thaliana genes for histone deacetylases (HDACs), HDT1/HD2A and HDT2/HD2B, are required to establish leaf polarity in the presence of mutant ASYMMETRIC LEAVES2 (AS2) or AS1. Treatment of as1 or as2 plants with inhibitors of HDACs resulted in abaxialized filamentous leaves and aberrant distribution of microRNA165 and/or microRNA166 (miR165/166) in leaves. Knockdown mutations of these two HDACs by RNA interference resulted in phenotypes like those observed in the as2 background. Nuclear localization of overproduced AS2 resulted in decreased levels of mature miR165/166 in leaves. This abnormality was abolished by HDAC inhibitors, suggesting that HDACs are required for AS2 action. A loss-of-function mutation in HASTY, encoding a positive regulator of miRNA levels, and a gain-of-function mutation in PHABULOSA, encoding a determinant of adaxialization, suppressed the generation of abaxialized filamentous leaves by inhibition of HDACs in the as1 or as2 background. AS2 and AS1 were colocalized in subnuclear bodies adjacent to the nucleolus where HDT1/HD2A and HDT2/HD2B were also found. Our results suggest that these HDACs and both AS2 and AS1 act independently to control levels and/or patterns of miR165/166 distribution and the development of adaxial-abaxial leaf polarity and that there may be interactions between HDACs and AS2 (AS1) in the generation of those miRNAs.     

A complex case of simple leaves: indeterminate leaves co-express ARP and KNOX1 genes

The mutually exclusive relationship between ARP and KNOX1 genes in the shoot apical meristem and leaf primordia in simple leaved plants such as Arabidopsis has been well characterized. Overlapping expression domains of these genes in leaf primordia have been described for many compound leaved plants such as Solanum lycopersicum and Cardamine hirsuta and are regarded as a characteristic of compound leaved plants. Here, we present several datasets illustrating the co-expression of ARP and KNOX1 genes in the shoot apical meristem, leaf primordia, and developing leaves in plants with simple leaves and simple primordia. Streptocarpus plants produce unequal cotyledons due to the continued activity of a basal meristem and produce foliar leaves termed “phyllomorphs” from the groove meristem in the acaulescent species Streptocarpus rexii and leaves from a shoot apical meristem in the caulescent Streptocarpus glandulosissimus. We demonstrate that the simple leaves in both species possess a greatly extended basal meristematic activity that persists over most of the leaf’s growth. The area of basal meristem activity coincides with the co-expression domain of ARP and KNOX1 genes. We suggest that the co-expression of ARP and KNOX1 genes is not exclusive to compound leaved plants but is associated with foci of meristematic activity in leaves.     

Negative regulation of <Emphasis Type="Italic">KNOX</Emphasis> expression in tomato leaves

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.     

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.     

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.     

Revelation of ancestral roles of KNOX genes by a functional analysis of <Emphasis Type="Italic">Physcomitrella</Emphasis> homologues

KNOX genes are indispensable elements of indeterminate apical growth programmes of vascular plant sporophytes. Since little is known about the roles of such genes in non-vascular plants, functional analysis of moss KNOX homologues (MKN genes) was undertaken using the genetically amenable model plant, Physcomitrella patens. Three MKN genes were inactivated by targeted gene knockout to produce single, double and triple mutants. MKN2 (a class 1 KNOX gene) mutants were characterised by premature sporogenesis, abnormal sporophyte ontogeny and irregular spore development. MKN4 (a second class 1 gene) mutants were phenotypically normal. MKN1-3 (a class 2 KNOX gene) mutants exhibited defects in spore coat morphology. Analysis of double and triple mutants revealed that the abnormal sporophytic phenotype of MKN2 mutants was accentuated by mutating MKN4 and to a lesser degree by mutating MKN1-3. The aberrant spore phenotype of MKN1-3 and MKN2 mutants was exacerbated by mutating MKN4. This study provides the first instance in which an abnormal phenotype has been associated with the disruption of a class 2 KNOX gene as well as the first demonstrated case of functional redundancy between a class 1 and a class 2 KNOX gene. We conclude that KNOX genes play significant roles in programming sporophytic development in moss and we provide evidence that ancestral function(s) of this gene family were instrumental in the successful transition of plants to a terrestrial environment.     

KNOX homeobox genes potentially have similar function in both diploid unicellular and multicellular meristems, but not in haploid meristems

Members of the class 1 knotted-like homeobox (KNOX) gene family are important regulators of shoot apical meristem development in angiosperms. To determine whether they function similarly in seedless plants, three KNOX genes (two class 1 genes and one class 2 gene) from the fern Ceratopteris richardii were characterized. Expression of both class 1 genes was detected in the shoot apical cell, leaf primordia, marginal part of the leaves, and vascular bundles by in situ hybridization, a pattern that closely resembles that of class 1 KNOX genes in angiosperms with compound leaves. The fern class 2 gene was expressed in all sporophyte tissues examined, which is characteristic of class 2 gene expression in angiosperms. All three CRKNOX genes were not detected in gametophyte tissues by RNA gel blot analysis. Arabidopsis plants overexpressing the fern class 1 genes resembled plants that overexpress seed plant class 1 KNOX genes in leaf morphology. Ectopic expression of the class 2 gene in Arabidopsis did not result in any unusual phenotypes. Taken together with phylogenetic analysis, our results suggest that (a) the class 1 and 2 KNOX genes diverged prior to the divergence of fern and seed plant lineages, (b) the class 1 KNOX genes function similarly in seed plant and fern sporophyte meristem development despite their differences in structure, (c) KNOX gene expression is not required for the development of the fern gametophyte, and (d) the sporophyte and gametophyte meristems of ferns are not regulated by the same developmental mechanisms at the molecular level.