The Arabidopsis-mei2-like genes play a role in meiosis and vegetative growth in Arabidopsis

The Arabidopsis-mei2-Like (AML) genes comprise a five-member gene family related to the mei2 gene, which is a master regulator of meiosis in Schizosaccharomyces pombe and encodes an RNA binding protein. We have analyzed the AML genes to assess their role in plant meiosis and development. All five AML genes were expressed in both vegetative and reproductive tissues. Analysis of AML1-AML5 expression at the cellular level indicated a closely similar expression pattern. In the inflorescence, expression was concentrated in the shoot apical meristem, young buds, and reproductive organ primordia. Within the reproductive organs, strong expression was observed in meiocytes and developing gametes. Functional analysis using RNA interference (RNAi) and combinations of insertion alleles revealed a role for the AML genes in meiosis, with RNAi lines and specific multiple mutant combinations displaying sterility and a range of defects in meiotic chromosome behavior. Defects in seedling growth were also observed at low penetrance. These results indicate that the AML genes play a role in meiosis as well as in vegetative growth and reveal conservation in the genetic mechanisms controlling meiosis in yeast and plants.     

Gene duplication within the Green Lineage: the case of TEL genes

Recent years have witnessed a breathtaking increase in the availability of genome sequence data, providing evidence of the highly duplicate nature of eukaryotic genomes. Plants are exceptional among eukaryotic organisms in that duplicate loci compose a large fraction of their genomes, partly because of the frequent occurrence of polyploidy (or whole-genome duplication) events. Tandem gene duplication and transposition have also contributed to the large number of duplicated genes in plant genomes. Evolutionary analyses allowed the dynamics of duplicate gene evolution to be studied and several models were proposed. It seems that, over time, many duplicated genes were lost and some of those that were retained gained new functions and/or expression patterns (neofunctionalization) or subdivided their functions and/or expression patterns between them (subfunctionalization). Recent studies have provided examples of genes that originated by duplication with successive diversification within plants. In this review, we focused on the TEL (TERMINAL EAR1-like) genes to illustrate such mechanisms. Emerged from the mei2 gene family, these TEL genes are likely to be land plant-specific. Phylogenetic analyses revealed one or two TEL copies per diploid genome. TEL gene degeneration and loss in several Angiosperm species such as in poplar and maize seem to have occurred. In Arabidopsis thaliana, whose genome experienced at least three polyploidy events followed by massive gene loss and genomic reorganization, two TEL genes were retained and two new shorter TEL-like (MCT) genes emerged. Molecular and expression analyses suggest for these genes sub- and neofunctionalization events, but confirmation will come from their functional characterization.     

The interaction of two homeobox genes,BREVIPEDICELLUS and PENNYWISE,regulates internode patterning in the Arabidopsis inflorescence

Plant architecture results from the activity of the shoot apical meristem, which initiates leaves, internodes, and axillary meristems. KNOTTED1-like homeobox (KNOX) genes are expressed in specific patterns in the shoot apical meristem and play important roles in plant architecture. KNOX proteins interact with BEL1-like (BELL) homeodomain proteins and together bind a target sequence with high affinity. We have obtained a mutation in one of the Arabidopsis BELL genes, PENNYWISE (PNY), that appears phenotypically similar to the KNOX mutant brevipedicellus (bp). Both bp and pny have randomly shorter internodes and display a slight increase in the number of axillary branches. The double mutant shows a synergistic phenotype of extremely short internodes interspersed with long internodes and increased branching. PNY is expressed in inflorescence and floral meristems and overlaps with BP in a discrete domain of the inflorescence meristem where we propose the internode is patterned. The physical association of the PNY and BP proteins suggests that they participate in a complex that regulates early patterning events in the inflorescence meristem.     

CHB2, a member of the SWI3 gene family,is a global regulator in Arabidopsis

The SWI/SNF complex is an ATP-dependent chromatin remodeling complex that plays an important role in the regulation of eukaryotic gene expression. Very little is known about the function of SWI/SNF complex in plants compared with animals and yeast. SWI3 is one of the core components of the SWI/SNF chromatin remodeling complexes in yeast. We have identified a putative SWI3-like cDNA clone, CHB2 (AtSWI3B), from Arabidopsis thaliana by screening the expressed sequence tag database. CHB2 encodes a putative protein of 469 amino acids and shares 23% amino acid sequence identity and 64% similarity with the yeast SWI3. The Arabidopsis genome contains four SWI3-like genes, namely CHB1 (AtSWI3A), CHB2 (AtSWI3B), CHB3 (AtSWI3C) and CHB4 (AtSWI3D). The expression of CHB2, CHB3 and CHB4 mRNA was detected in all tissues analyzed by RT-PCR. The expression of CHB1 mRNA, however, could not be detected in the siliques, suggesting that there is differential expression among CHB genes in different Arabidopsis tissues. To investigate the role of CHB2 in plants, Arabidopsis plants were transformed with a gene construct comprising a CHB2 cDNA in the antisense orientation driven by the CaMV 35S promoter. Repression of CHB2 expression resulted in pleiotropic developmental abnormalities including abnormal seedling and leaf phenotypes, dwarfism, delayed flowering and no apical dominance, suggesting a global role for CHB2 in the regulation of gene expression. Our results indicate that CHB2 plays an essential role in plant growth and development.     

Phylogeny and domain evolution in the APETALA2-like gene family

The combined processes of gene duplication, nucleotide substitution, domain duplication, and intron/exon shuffling can generate a complex set of related genes that may differ substantially in their expression patterns and functions. The APETALA2-like (AP2-like) gene family exhibits patterns of both gene and domain duplication, coupled with changes in sequence, exon arrangement, and expression. In angiosperms, these genes perform an array of functions including the establishment of the floral meristem, the specification of floral organ identity, the regulation of floral homeotic gene expression, the regulation of ovule development, and the growth of floral organs. To determine patterns of gene diversification, we conducted a series of broad phylogenetic analyses of AP2-like sequences from green plants. These studies indicate that the AP2 domain was duplicated prior to the divergence of the two major lineages of AP2-like genes, euAP2 and AINTEGUMENTA (ANT). Structural features of the AP2-like genes as well as phylogenetic analyses of nucleotide and amino acid (aa) sequences of the AP2-like gene family support the presence of the two major lineages. The ANT lineage is supported by a 10-aa insertion in the AP2-R1 domain and a 1-aa insertion in the AP2-R2 domain, relative to all other members of the AP2-like family. MicroRNA172-binding sequences, the function of which has been studied in some of the AP2-like genes in Arabidopsis, are restricted to the euAP2 lineage. Within the ANT lineage, the euANT lineage is characterized by four conserved motifs: one in the 10-aa insertion in the AP2-R1 domain (euANT1) and three in the predomain region (euANT2, euANT3, and euANT4). Our expression studies show that the euAP2 homologue from Amborella trichopoda, the putative sister to all other angiosperms, is expressed in all floral organs as well as leaves.     

A description of the Mei2-like protein family; structure,phylogenetic distribution and biological context

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a "mei2-dot" in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.     

KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis.

Plant development depends on the activity of apical meristems, which are groups of indeterminate cells whose derivatives elaborate the organs of the mature plant. Studies of knotted1 (kn1) and related gene family members have determined potential roles for homeobox genes in the function of shoot meristems. The Arabidopsis kn1-like gene, KNAT1, is expressed in the shoot apical meristem and not in determinate organs. Here, we show that ectopic expression of KNAT1 in Arabidopsis transforms simple leaves into lobed leaves. The lobes initiate in the position of serrations yet have features of leaves, such as stipules, which form in the sinus, the region at the base of two lobes. Ectopic meristems also arise in the sinus region close to veins. Identity of the meristem, that is, vegetative or floral, depends on whether the meristem develops on a rosette or cauline leaf, respectively. Using in situ hybridization, we analyzed the expression of KNAT1 and another kn1-like homeobox gene, SHOOT MERISTEMLESS, in cauliflower mosaic virus 35S::KNAT1 transformants. KNAT1 expression is strong in vasculature, possibly explaining the proximity of the ectopic meristems to veins. After leaf cells have formed a layered meristem, SHOOT MERISTEMLESS expression begins in only a subset of these cells, demonstrating that KNAT1 is sufficient to induce meristems in the leaf. The shootlike features of the lobed leaves are consistent with the normal domain of KNAT1's expression and further suggest that kn1-related genes may have played a role in the evolution of leaf diversity.     

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.     

Analysis of the competence to respond to KNOTTED1 activity in Arabidopsis leaves using a steroid induction system

Expression of KNOX (KNOTTED1-like homeobox) genes in the shoot apical meristem of Arabidopsis is required for maintenance of a functional meristem, whereas exclusion of KNOX gene expression from leaf primordia is required for the elaboration of normal leaf morphology. We have constructed a steroid-inducible system to regulate both the amount and timing of KN1 (KNOTTED1) misexpression in Arabidopsis leaves. We demonstrate that lobed leaf morphology is produced in a dose-dependent manner, indicating that the amount of KN1 quantitatively affects the severity of lobing. The KN1-glucocorticoid receptor fusion protein is not detected in leaves in the absence of steroid induction, suggesting that it is only stable when associated with steroid in an active state. By using a second inducible fusion protein to mark exposure of leaf primordia to the steroid, we determined the stage of leaf development that produces lobed leaves in response to KN1. Primordia as old as plastochron 7 and as young as plastochron 2 were competent to respond to KN1.     

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.