Characterization of the class IV homeodomain-Leucine Zipper gene family in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome contains 16 genes belonging to the class IV homeodomain-Leucine zipper gene family. These include GLABRA2, ANTHOCYANINLESS2, FWA, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), and PROTODERMAL FACTOR2 (PDF2). Our previous study revealed that atml1 pdf2 double mutants have severe defects in the shoot epidermal cell differentiation. Here, we have characterized additional members of this gene family, which we designated HOMEODOMAIN GLABROUS1 (HDG1) through HDG12. Analyses of transgenic Arabidopsis plants carrying the gene-specific promoter fused to the bacterial beta-glucuronidase reporter gene revealed that some of the promoters have high activities in the epidermal layer of the shoot apical meristem and developing shoot organs, while others are temporarily active during reproductive organ development. Expression profiles of highly conserved paralogous gene pairs within the family were found to be not necessarily overlapping. Analyses of T-DNA insertion mutants of these HDG genes revealed that all mutants except hdg11 alleles exhibit no abnormal phenotypes. hdg11 mutants show excess branching of the trichome. This phenotype is enhanced in hdg11 hdg12 double mutants. Double mutants were constructed for other paralogous gene pairs and genes within the same subfamily. However, novel phenotypes were observed only for hdg3 atml1 and hdg3 pdf2 mutants that both exhibited defects in cotyledon development. These observations suggest that some of the class IV homeodomain-Leucine zipper members act redundantly with other members of the family during various aspects of cell differentiation. DNA-binding sites were determined for two of the family members using polymerase chain reaction-assisted DNA selection from random oligonucleotides with their recombinant proteins. The binding sites were found to be similar to those previously identified for ATML1 and PDF2, which correspond to the pseudopalindromic sequence 5'-GCATTAAATGC-3' as the preferential binding site.     

The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia

The Arabidopsis thaliana MERISTEM LAYER 1 (ATML1) gene is expressed in the epidermis of developing embryos and shoot meristems. To identify regulatory sequences necessary for epidermis-specific expression, three fusions of overlapping ATML1 genomic sequences to the GUS reporter gene were introduced into Arabidopsis plants. All fusion genes conferred epidermis-specific expression of both GUS mRNA and protein activity but varied in both the timing and relative levels of expression, suggesting partial redundancy of ATML1 regulatory elements. This study defines L1-specific regulatory sequences that are sufficient to direct foreign gene expression in a layer-specific manner.     

Induction of epidermal cell fate in Arabidopsis shoots

Land plants have evolved a cuticle-bearing epidermis to protect themselves from environmental stress and pathogen attack. Despite its important role, little is known about the molecular mechanisms regulating shoot epidermal cell identity. In a recent study, we found that the Arabidopsis thaliana ATML1 gene is possibly a master regulator of shoot epidermal cell fate. We revealed that ATML1 has the ability to confer shoot epidermis-related traits to non-epidermal cells of the seedlings. These data are consistent with the previous loss-of-function mutant analyses, which implied a positive role of ATML1 in epidermal cell differentiation. Importantly, ectopic epidermal cells induced in ATML1-overexpressing lines provide a novel tool to assess the intrinsic properties of epidermal cells and to study epistatic interactions among genes involved in epidermal/mesophyll differentiation. Using this system, we obtained data revealing that ATML1 negatively influenced mesophyll cell fate. In addition, we provided a working model of how division planes in epidermal cells are determined.     

Cloning and characterization of an L1 layer-specific gene in Arabidopsis thaliana.

The shoot apical meristem (SAM) of Arabidopsis thaliana constitutes the tunica of L1 and L2 and the corpus represented by L3 cells. Regulatory networks involved in establishing and maintaining this structure of shoot meristems remain largely unknown. In order to identify the genes that function in the SAM, we performed cDNA subtraction experiments between wild-type and terminal flower1 shoot apices. Here, we describe the cloning of a gene designated PDF1 (PROTODERMAL FACTOR1). In situ hybridization revealed that the expression of PDF1 is exclusively limited to the L1 layer of vegetative, inflorescence and floral meristems and to the protoderm of organ primordia. By contrast, PDF1 shows no detectable level of expression in the epidermis of mature organs. Specific expression of the PDF1 gene in protodermal cells is also observed during embryogenesis. The deduced amino acid sequence of PDF1 shares no significant homology with that of other known proteins but contains a putative signal peptide and novel proline-rich repeat motifs, suggesting a cell-wall protein. Possible roles of the PDF1 gene in the SAM are discussed.     

The molecular characterization of PaHB2, a homeobox gene of the HD-GL2 family expressed during embryo development in Norway spruce

PaHB1 (for Picea abies Homeobox1), an evolutionarily conserved HD-GL2 homeobox gene, specifically expressed in the protoderm during somatic embryogenesis in the gymnosperm Norway spruce has been reported previously. An additional HD-GL2 gene designated PaHB2 is reported here. During somatic embryogenesis, the PaHB2 gene is uniformly ex pressed in proembryogenic masses and in early somatic embryos, but it is not detectably transcribed at the beginning of maturation. In mature embryos, PaHB2 expression was essentially detected in the outermost layer of the cortex and the root cap. A similar PaHB2 expression is detected post-embryonically in both the primary root and the hypocotyl. Phylogenetic reconstructions and intron pattern analyses revealed that the PAHB proteins fall within two distinct subclasses comprising highly similar angiosperm homologues. The PAHB1 subclass consists of protoderm/epiderm-specific members. By contrast, the PAHB2 subclass gathers homologues with a subepidermal and protodermal/epidermal activity. This study suggests that at least two distinct HD-GL2 genes with a layer-specific expression already existed in the last common ancestor of angiosperms and gymnosperms. The conserved protodermal/epidermal and subepidermal expression of HD-GL2 genes could be used to study embryo radial pattern formation across seed plants.     

Pattern formation during de novo assembly of the Arabidopsis shoot meristem

Most multicellular organisms have a capacity to regenerate tissue after wounding. Few, however, have the ability to regenerate an entire new body from adult tissue. Induction of new shoot meristems from cultured root explants is a widely used, but poorly understood, process in which apical plant tissues are regenerated from adult somatic tissue through the de novo formation of shoot meristems. We characterize early patterning during de novo development of the Arabidopsis shoot meristem using fluorescent reporters of known gene and protein activities required for shoot meristem development and maintenance. We find that a small number of progenitor cells initiate development of new shoot meristems through stereotypical stages of reporter expression and activity of CUP-SHAPED COTYLEDON 2 (CUC2), WUSCHEL (WUS), PIN-FORMED 1 (PIN1), SHOOT-MERISTEMLESS (STM), FILAMENTOUS FLOWER (FIL, also known as AFO), REVOLUTA (REV), ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) and CLAVATA 3 (CLV3). Furthermore, we demonstrate a functional requirement for WUS activity during de novo shoot meristem initiation. We propose that de novo shoot meristem induction is an easily accessible system for the study of patterning and self-organization in the well-studied model organism Arabidopsis.     

Altered expression of auxin-related genes in the fatty acid elongase mutant oni1 of rice

VLCFAs are the main components of cuticular wax, which covers and protects plants from physical and biological stresses. However, the effect of fatty acid composition or the physiological role of VLCFAs on plant development under normal growth conditions is not well understood. We analyzed loss-of-function mutants of ONION1 (ONI1) which encodes fatty acid elongase (β-ketoacyl CoA synthase) catalyzing an elongation reaction of a carbon chain of VLCFAs. We showed that oni1 shoot contained a reduced amount of VLCFAs, and differentiation and functionality of an outermost cell layer (L1) were highly perturbed in oni1 shoot. In spite of the L1-specific expression of ONI1, the effects of the oni1 mutation were not restricted to L1, but expanded to inner cells, so that the entire shoot development was impaired including failure of the maintenance of the SAM and ectopic expression of SAM-specific KNOX genes in leaf. Thus, ONI1 function is cell non-autonomous, and signaling from L1 to inner cells may support proper development of inner cells. Here we report that expression of auxin-related genes was affected in oni1 shoot, and we speculate the existence of improper auxin distribution due to a lack of normal L1 in oni1 shoot.Key words: fatty acid elongase, very-long-chain fatty acid, auxin, shoot development, rice     

GhL1L1 affects cell fate specification by regulating GhPIN1‐mediated auxin distribution

Auxin is as an efficient initiator and regulator of cell fate during somatic embryogenesis (SE), but the molecular mechanisms and regulating networks of this process are not well understood. In this report, we analysed SE process induced by Leafy cotyledon1‐like 1 (GhL1L1), a NF‐YB subfamily gene specifically expressed in embryonic tissues in cotton. We also identified the target gene of GhL1L1, and its role in auxin distribution and cell fate specification during embryonic development was analysed. Overexpression of GhL1L1 accelerated embryonic cell formation, associated with an increased concentration of IAA in embryogenic calluses (ECs) and in the shoot apical meristem, corresponding to altered expression of the auxin transport gene GhPIN1. By contrast, GhL1L1‐deficient explants showed retarded embryonic cell formation, and the concentration of IAA was decreased in GhL1L1‐deficient ECs. Disruption of auxin distribution accelerated the specification of embryonic cell fate together with regulation of GhPIN1. Furthermore, we showed that PHOSPHATASE 2AA2 (GhPP2AA2) was activated by GhL1L1 through targeting the G‐box of its promoter, hence regulating the activity of GhPIN1 protein. Our results indicate that GhL1L1 functions as a key regulator in auxin distribution to regulate cell fate specification in cotton and contribute to the understanding of the complex process of SE in plant species.     

PaHB1 is an evolutionary conserved HD-GL2 homeobox gene expressed in the protoderm during Norway spruce embryo development

In angiosperms, the protoderm or outer cell layer is the first tissue to differentiate in the embryo proper. In gymnosperms, it is not known whether a protoderm is defined and similarly differentiated. Here, we report a cDNA designated PaHB1 (for Picea abies Homeobox1), which is expressed during somatic embryogenesis in Norway spruce. PaHB1 exon/intron organization and its corresponding protein are highly similar to those of the HD-GL2 angiosperm counterparts. A phylogenetic analysis reveals that PaHB1 is strongly associated with one subclass consisting of protoderm/epiderm-specific genes. Moreover, PaHB1 expression switches from a ubiquitous expression in proembryogenic masses to an outer cell layer-specific localization during somatic embryo development. Ectopic expression of PaHB1 in somatic embryos leads to an early developmental block. The transformed embryos lack a smooth surface. These findings show that the PaHB1 expression pattern is highly analogous to angiosperm HD-GL2 homologues, suggesting similarities in the definition of the outer cell layer in seed plants.     

Enhancement of the Listeria monocytogenes p60-specific CD4 and CD8 T cell memory by nonpathogenic Listeria innocua

The contact of T cells to cross-reactive antigenic determinants expressed by nonpathogenic environmental micro-organisms may contribute to the induction or maintenance of T cell memory. This hypothesis was evaluated in the model of murine Listeria monocytogenes infection. The influence of nonpathogenic L. innocua on the L. monocytogenes p60-specific T cell response was analyzed. We show that some CD4 T cell clones raised against purified p60 from L. monocytogenes cross-react with p60 purified from L. innocua. The L. monocytogenes p60-specific CD4 T cell clone 1A recognized the corresponding L. innocua p60 peptide QAAKPAPAPSTN, which differs only in the first amino acid residue. In vitro experiments revealed that after L. monocytogenes infection of APCs, MHC class I-restricted presentation of p60 occurs, while MHC class II-restricted p60 presentation is inhibited. L. innocua-infected cells presented p60 more weakly but equally well in the context of both MHC class I and MHC class II. In contrast to these in vitro experiments the infection of mice with L. monocytogenes induced a strong p60-specific CD4 and CD8 T cell response, while L. innocua infection failed to induce p60-specific T cells. L. innocua booster infection, however, expanded p60-specific memory T cells induced by previous L. monocytogenes infection. In conclusion, these findings suggest that infection with a frequently occurring environmental bacterium such as L. innocua, which is nonpathogenic and not adapted to intracellular replication, can contribute to the maintenance of memory T cells specific for a related intracellular pathogen.