Search

In Arabidopsis, the ASYMMETRIC LEAVES2 (AS2) protein plays a key role in the formation of flat symmetric leaves via direct repression of the abaxial gene ETT/ARF3. AS2 encodes a plant-specific nuclear protein that contains the AS2/LOB domain, which includes a zinc-finger (ZF) motif that is conserved in the AS2/LOB family. We have shown that AS2 binds to the coding DNA of ETT/ARF3, which requires the ZF motif. AS2 is co-localized with AS1 in perinucleolar bodies (AS2 bodies). To identify the amino acid signals in AS2 required for formation of AS2 bodies and function(s) in leaf formation, we constructed recombinant DNAs that encoded mutant AS2 proteins fused to yellow fluorescent protein. We examined the subcellular localization of these proteins in cells of cotyledons and leaf primordia of transgenic plants and cultured cells. The amino acid signals essential for formation of AS2 bodies were located within and adjacent to the ZF motif. Mutant AS2 that failed to form AS2 bodies also failed to rescue the as2-1 mutation. Our results suggest the importance of the formation of AS2 bodies and the nature of interactions of AS2 with its target DNA and nucleolar factors including NUCLEOLIN1. The partial overlap of AS2 bodies with perinucleolar chromocenters with condensed ribosomal RNA genes implies a correlation between AS2 bodies and the chromatin state. Patterns of AS2 bodies in cells during interphase and mitosis in leaf primordia were distinct from those in cultured cells, suggesting that the formation and distribution of AS2 bodies are developmentally modulated in plants., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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.

The ASYMMETRIC LEAVES2 (AS2) gene is required for the generation of the flat and symmetrical shape of the leaf lamina in Arabidopsis. AS2 encodes a plant-specific protein with an AS2/LATERAL ORGAN BOUNDARIES (AS2/LOB) domain that includes a cysteine repeat, a conserved single glycine residue and a leucine-zipper-like sequence in its amino-terminal half. The Arabidopsis genome contains 42 genes, including AS2, that encode proteins with an AS2/LOB domain in their amino-terminal halves, and these genes constitute the AS2/LOB gene family. In the present study, we cloned and characterized cDNAs that covered the putative coding regions of all members of this family, and investigated patterns of transcription systematically in Arabidopsis plants. Comparisons among amino acid sequences that had been deduced from the cloned cDNAs revealed eight groups of genes, with two or three members each, and high degrees of identity among entire amino acid sequences, suggesting that some members of the AS2/LOB family might have redundant function(s). Moreover, no member of the family exhibited significant similarity, in terms of the deduced amino acid sequence of the carboxy-terminal half, to AS2. Results of domain swapping between AS2 and other members of the family showed that the AS2/LOB domain of AS2 cannot be functionally replaced by those of other members of the family, and that only a few dissimilarities among respective amino acid residues of the AS2/LOB domain of AS2 and those of other members are important for the specific functions of AS2.

The ASYMMETRIC LEAVES2 (AS2) gene, a member of the AS2/LOB gene family, and the ASYMMETRIC LEAVES1 (AS1) gene of Arabidopsis thaliana participate in the development of a symmetrical, expanded lamina. We report here the patterns of expression of these genes, and the importance of the sites of such expression in leaf development. Transcripts of both genes accumulated in the entire leaf primordia at early stages, but the patterns of accumulation changed as the leaves expanded. AS2 and AS1 transcripts were detected, respectively, in the adaxial domain and in the inner domain between the adaxial and abaxial domains of leaves. The ratios of numbers of adaxial cells to abaxial cells in cotyledons of corresponding mutant lines were greater than the ratios in wild-type cotyledons. The low levels of ectopic expression of AS2 under the control of the AS1 promoter in as2 mutant plants restored an almost normal phenotype in some cases, but also resulted in flatter leaves than those of wild-type plants. Strong expression of the construct in wild-type and as2 plants, but not as1 plants, resulted in the formation of narrow, upwardly curled leaves. Our results indicate that AS2 represses cell proliferation in the adaxial domain in the presence of AS1, and that adaxial expression of AS2 at an appropriate level is critical for the development of a symmetrical, expanded lamina. Real-time RT-PCR analysis revealed that mutation of either AS2 or AS1 resulted in an increase in the levels of transcripts of ETTIN (ETT; also known as AUXIN RESPONSE FACTOR3, ARF3) and KANADI2 (KAN2), which are abaxial determinants, and YABBY5 (YAB5). Thus, AS2 and AS1 might negatively regulate the expression of these genes in the adaxial domain, which might be related to the development of flat and expanded leaves.

In higher plants, an outer layer of meristematic cells, the protoderm, forms early in embryogenesis and this layer gives rise to the epidermis in differentiating tissues. We proposed previously that an Arabidopsis thaliana homolog of crinkly4 (ACR4), a gene for a receptor-like protein kinase, would be involved in differentiation and/or maintenance of epidermis-related tissues. In the present study, we isolated loss-of-function acr4 mutants by a reverse genetic approach. Our extensive analyses using the transmission electron microscopy and the toluidine blue test -- a method that has recently been developed for the rapid visualization of defects in the leaf cuticle -- showed that the acr4 mutations significantly affected the differentiation of leaf epidermal cells, suggesting similar roles for ACR4 and CR4 in the differentiation of leaf epidermis. Our acr4 mutants also had various abnormalities related to epidermal differentiation, which included disorganized cell layers in the integument and endothelium of ovules. In addition, the green fluorescent protein fused to ACR4 was localized preferentially on the lateral and basal plasma membranes in the epidermis of the leaf primordia, suggesting a role for ACR4 in epidermal differentiation at cell surfaces that make contact with adjacent cells. Furthermore, the loss-of-function mutations in the ACR4 and ABNORMAL LEAF SHAPE1 (ALE1) genes, which encode a putative subtilisin-like serine protease, synergistically affected the function of the epidermis such that most leaves fused. Thus, ACR4 seems to play an essential role in the differentiation of proper epidermal cells in both vegetative and reproductive tissues.

The epidermis of higher plants generates the cuticle layer that covers the outer surface of each plant. The cuticle plays a crucial role in plant development, and some mutants with defective cuticle exhibit morphological abnormalities, such as the fusion of organs. The way in which the cuticle forms and its contribution to morphogenesis are poorly understood. Conventional detection of the cuticle by transmission electron microscopy (TEM) requires laborious procedures, which include fixation, staining with osmium, and preparation of ultra-thin sections. It is also difficult to survey entire surfaces of expanded leaves because of the limited size of specimens that can be examined. Thus, TEM is unsuitable for large-scale screening for mutants with defective cuticle. We describe here a rapid and inexpensive method, designated the toluidine-blue (TB) test, for detection of cuticular defects in whole leaves. We demonstrated the validity of the TB test using mutants of Arabidopsis thaliana, including abnormal leaf shape1 (ale1), fiddlehead (fdh), and five eceriferum (cer) mutants, in which the structure and/or function of the cuticle is abnormal. Genetic screening for mutants using the TB test allowed us to identify seven loci. The cuticle-defective regions of leaves of the mutants revealed five intrinsic patterns of surface defects (classes I through V), suggesting that formation of functional cuticle on leaves involves various spatially regulated factors.

We identified a mutation in Arabidopsis that resulted in defective embryos, and we designated this mutation globular arrest1 (gla1). The predicted amino acid sequence encoded by the GLA1 gene is homologous to the amino acid sequences of folylpolyglutamate synthetase (FPGS) and dihydrofolate synthetase (DHFS), which participate in folate biosynthesis. The defect of gla1 in the formation of calli was rescued by the supplement of 5-formyl tetrahydrofolate. These results indicated that GLA1 is involved in the biosynthesis of tetrahydrofolate. The gla1 embryos developed normally in the early stage of development but did not undergo the transition to the heart stage. Thus, the function of the GLA1 gene in embryogenesis appears to be required after the globular stage. However, when the levels of GLA1 transcripts in transgenic plants were increased by introduction of several copies of a GLA1 transgene (GLA6.8), the gla1 embryos that grew on gla1/gla1 GLA6.8/- plants developed as far as the heart to bent-cotyledon stage. This result suggests that the GLA1 function is provided to embryos by maternal tissues until embryos reach the globular stage.