Epidermal injury-induced derepression of key regulator ATML1 in newly exposed cells elicits epidermis regeneration.

Plant cell fate determination depends on the relative positions of the cells in developing organisms. The shoot epidermis, the outermost cell layer of the above-ground organs in land plants, protects plants from environmental stresses. How the shoot epidermis is formed only from the outermost cells has remained unknown. Here we show that when inner leaf mesophyll cells are exposed to the surface, these cells show up-regulation of ATML1, a master regulator for epidermal cell identity in Arabidopsis thaliana. Epidermal cell types such as stomatal guard cells regenerate from young inner-lineage tissues that have a potential to accumulate ATML1 protein after epidermal injury. Surgical analyses indicate that application of pressure to the exposed site was sufficient to inhibit ATML1 derepression in the outermost mesophyll cells, suggesting this process requires pressure release. Furthermore, pharmacological analyses suggest that ATML1 derepression in the outermost mesophyll cells require cortical microtubule formation, MAPK signaling and proteasome activity. Our results suggest that surface-positional cues involving mechanical signaling are used to restrict ATML1 activity to the outermost cells and facilitate epidermal differentiation.

N-terminal domain of ARF-GEF GNOM prevents heterodimerization with functionally divergent GNL1 in Arabidopsis.

Evolutionary change following gene duplication can lead to functionally divergent paralogous proteins. If comprising identical subunits their random assortment would also form potentially detrimental heteromeric proteins. In Arabidopsis, the ARF GTPase guanine-nucleotide exchange factor GNOM is essential for polar recycling of auxin-efflux transporter PIN1 from endosomes to the basal plasma membrane whereas its paralog GNL1 mediates retrograde Golgi-endoplasmic reticulum traffic. Here we show that both GNOM and GNL1 form homodimers but no heterodimers. To assess the biological significance of this, we generated transgenic plants expressing engineered heterodimer-compatible GNOM variants. Those plants showed developmental defects such as the failure to produce lateral roots. To identify mechanisms underlying heterodimer prevention, we analyzed interactions of the N-terminal dimerization and cyclophilin-binding (DCB) domain. Each DCB domain interacted with the complementary fragment (ΔDCB) both of their own and of the paralogous protein. However, only DCBGNOM interacted with itself whereas DCBGNL1 failed to interact with itself and with DCBGNOM . GNOM variants in which the DCB domain was removed or replaced by DCBGNL1 revealed a role for DCB-DCB interaction in the prevention of GNOM-GNL1 heterodimers whereas DCB-ΔDCB interaction was essential for dimer formation and GNOM function. Our data suggest a model of early DCB-DCB interaction that facilitates GNOM homodimer formation, indirectly precluding formation of detrimental heterodimers.

A Quarter Century History of ATML1 Gene Research.

The cloning of the ATML1 gene, encoding an HD-ZIP class IV transcription factor, was first reported in 1996. Because ATML1 mRNA was preferentially detected in the shoot epidermis, cis-regulatory sequences of ATML1 have been used to drive gene expression in the outermost cells of the shoot apical meristem and leaves, even before the function of ATML1 was understood. Later studies revealed that ATML1 is required for developmental processes related to shoot epidermal specification and differentiation. Consistent with its central role in epidermal development, ATML1 activity has been revealed to be restricted to the outermost cells via several regulatory mechanisms. In this review, we look back on the history of ATML1 research and provide a perspective for future studies.

ATML1 activity is restricted to the outermost cells of the embryo through post-transcriptional repressions.

Cell fate determination in plants relies on positional cues. To investigate the position-dependent gene regulation in plants, we focused on shoot epidermal cell specification, which occurs only in the outermost cells. ATML1, which encodes an HD-ZIP class IV transcription factor, is a positive regulator of shoot epidermal cell identity. Despite the presence of a weak ATML1 promoter activity in the inner cells, ATML1 protein was detected mostly in the outermost cells, which suggests that ATML1 accumulation is inhibited in the inner cells. ATML1 nuclear localization was reduced in the epidermis and there was a positive, albeit weak, correlation between the amount of ATML1 in the nuclei and the expression of a direct target of ATML1. Nuclear accumulation of ATML1 was more strongly inhibited in the inner cells than in the outermost cells. Domain deletion analyses revealed that the ZLZ-coding sequence was necessary and partially sufficient for the post-transcriptional repression of ATML1 Our results suggest that post-transcriptional repressions contribute to the restriction of master transcriptional regulator activity in specific cells to enable position-dependent cell differentiation.

Specification of epidermal cell fate in plant shoots.

Land plants have evolved a single layer of epidermal cells, which are characterized by mostly anticlinal cell division patterns, formation of a waterproof coat called cuticle, and unique cell types such as stomatal guard cells and trichomes. The shoot epidermis plays important roles not only to protect plants from dehydration and pathogens but also to ensure their proper organogenesis and growth control. Extensive molecular genetic studies in Arabidopsis and maize have identified a number of genes that are required for epidermal cell differentiation. However, the mechanism that specifies shoot epidermal cell fate during plant organogenesis remains largely unknown. Particularly, little is known regarding positional information that should restrict epidermal cell fate to the outermost cell layer of the developing organs. Recent studies suggested that certain members of the HD-ZIP class IV homeobox genes are possible master regulators of shoot epidermal cell fate. Here, we summarize the roles of the regulatory genes that are involved in epidermal cell fate specification and discuss the possible mechanisms that limit the expression and/or activity of the master transcriptional regulators to the outermost cell layer in plant shoots.

Post-embryonic induction of ATML1-SRDX alters the morphology of seedlings.

Arabidopsis thaliana MERISTEM LAYER 1 (ATML1), an HD-ZIP class IV homeobox gene, is one of the key regulators promoting epidermal cell differentiation in Arabidopsis thaliana. We recently showed that ATML1 was able to confer an ectopic shoot epidermis cell fate to non-epidermal tissues of seedlings, suggesting that ATML1 is a master regulator of epidermal cell fate. To further assess the roles of ATML1 and its homologs in epidermal cell differentiation, I generated transgenic plants expressing ATML1 fused with a transcriptional repressor sequence (ATML1-SRDX). Estradiol-induced expression of ATML1-SRDX in the seedlings decreased transcript levels of several epidermis-related genes. Moreover, these transgenic plants exhibited phenotypes such as increased permeability to a hydrophilic dye and fusion of leaves and cotyledons, which are reminiscent of epidermis and/or cuticle-deficient mutants. Epidermal cell morphology was severely affected in the strong lines: filamentous protrusions were formed on the surface of the cotyledons. Marker gene analyses showed that these protrusions did not have epidermis, mesophyll, root hair, or trichome cell identity, suggesting that post-embryonic expression of ATML1-SRDX was sufficient to alter cell identity in pre-existing protodermal cells of the cotyledons. Taken together, these results suggest that ATML1 and/or its target genes are not only necessary for the initial specification of epidermal cell fate but also may be necessary for the maintenance of epidermal cells in later stages.

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.

ATML1 promotes epidermal cell differentiation in Arabidopsis shoots.

Molecular mechanisms that generate distinct tissue layers in plant shoots are not well understood. ATML1, an Arabidopsis homeobox gene, is expressed in the outermost cell layer, beginning at an early stage of development. The promoters of many epidermis-specific genes, including ATML1, contain an ATML1-binding site called an L1 box, suggesting that ATML1 regulates epidermal cell fate. Here, we show that overexpression of ATML1 was sufficient to activate the expression of epidermal genes and to induce epidermis-related traits such as the formation of stomatal guard cells and trichome-like cells in non-epidermal seedling tissues. Detailed observation of the division planes of these ectopic stomatal cells suggested that a near-surface position, as well as epidermal cell identity, were required for regular anticlinal cell division, as seen in wild-type epidermis. Moreover, analyses of a loss-of-function mutant and overexpressors implied that differentiation of epidermal cells was associated with repression of mesophyll cell fate. Collectively, our studies contribute new information about the molecular basis of cell fate determination in different layers of plant aerial organs.

Efficient yeast one-/two-hybrid screening using a library composed only of transcription factors in Arabidopsis thaliana.

Yeast one-hybrid screening is widely used for the identification of transcription factors (TFs) that interact with specific DNA sequences. However, screening a whole cDNA library is not efficient for the identification of TFs because TF genes represent only a small percentage of clones in a cDNA library. Here, we present the development of an efficient yeast one-hybrid screening system using a prey library composed only of approximately 1,500 TF cDNAs of Arabidopsis thaliana. This library enabled us to isolate a TF that binds to a specific promoter sequence with high efficiency, even when the promoter region of the gene of interest was directly employed as bait. Furthermore, this library was also successfully applied as a yeast two-hybrid library to find TFs that interact with specific proteins. This efficient system will contribute to the elucidation of gene regulatory networks in plants.

Stomatal density is controlled by a mesophyll-derived signaling molecule.

Stomata are composed of a pair of guard cells and a pore between them, and their density and positions are regulated by developmental and environmental signals. In a screen in which we overexpressed many genes coding for putative secretory proteins one by one in Arabidopsis, we identified a gene named STOMAGEN, which increases stomatal density when overexpressed. The STOMAGEN gene encodes a small peptide with a putative secretory signal sequence at its N-terminus and is expressed preferentially in mesophyll cells. This peptide belongs to the EPIDERMAL PATTERNING FACTOR (EPF) family of the cysteine-rich peptides superfamily. The mature form was a 45-amino-acid peptide (stomagen) with three intramolecular disulfide bonds. Stomagen treatment at very low concentrations, as low as 10 nM, increased the stomatal density of wild-type Arabidopsis plants. We propose that stomagen is a mesophyll-to-epidermis signaling molecule that positively regulates stomatal density. We also suggest that stomagen increases stomatal density by competing with negative regulators EPF1 and EPF2 for the receptor-like protein TOO MANY MOUTHS.

Cell-cell communication in Arabidopsis early embryogenesis.

The basic body plan of the adult plant is established during embryogenesis, resulting in the juvenile form of the seedling. Arabidopsis embryogenesis is distinguished by a highly regular pattern of cell divisions. Some of these divisions are asymmetric, generating daughter cells with different fates. However, their subsequent differentiation might still depend on cell-cell communication to be fully accomplished or maintained. In some cases, cell fate specification solely depends on cell-cell communication that in general plays an important role in the generation of positional information within the embryo. Although auxin-dependent signalling has received much attention, other ways of cell-cell communication have also been demonstrated or suggested. This review focuses on aspects of pattern formation and cell-cell communication during Arabidopsis embryogenesis up to the mid-globular stage of development.

Vascular signalling mediated by ZWILLE potentiates WUSCHEL function during shoot meristem stem cell development in the Arabidopsis embryo.

Stem cells are maintained in an undifferentiated state by signals from their microenvironment, the stem cell niche. Despite its central role for organogenesis throughout the plant's life, little is known about how niche development is regulated in the Arabidopsis embryo. Here we show that, in the absence of functional ZWILLE (ZLL), which is a member of the ARGONAUTE (AGO) family, stem cell-specific expression of the signal peptide gene CLAVATA3 (CLV3) is not maintained despite increased levels of the homeodomain transcription factor WUSCHEL (WUS), which is expressed in the organising centre (OC) of the niche and normally promotes stem cell identity. Tissue-specific expression indicates that ZLL acts to maintain the stem cells from the neighbouring vascular primordium, providing direct evidence for a non-cell-autonomous mechanism. Furthermore, mutant and marker gene analyses suggest that during shoot meristem formation, ZLL functions in a similar manner but in a sequential order with its close homologue AGO1, which mediates RNA interference. Thus, WUS-dependent OC signalling to the stem cells is promoted by AGO1 and subsequently maintained by a provascular ZLL-dependent signalling pathway.

Transcriptional regulation of epidermal cell fate in the Arabidopsis embryo.

How distinct cell fates are specified at correct positions within the plant embryo is unknown. In Arabidopsis, different cell fates are generated early on, starting with the two daughter cells of the zygote. To address mechanisms of position-dependent gene activation and cell fate specification, we analyzed the regulatory region of the Arabidopsis thaliana MERISTEM LAYER 1 (ATML1) gene, which is already expressed at the one-cell stage and whose expression is later restricted to the outermost, epidermal cell layer from its inception. A sensitive, multiple GFP reporter revealed a modular organization to the ATML1 promoter. Each region contributes positively to specific spatial and temporal aspects of the overall expression pattern, including position-dependent but auxin-independent regulation along the apical-basal axis of the embryo. A 101 bp fragment that conferred all aspects of ATML1 expression contained known binding sites for homeodomain transcription factors and other regulatory sequences. Our results suggest that expression patterns associated with cell fate determination in the plant embryo result from positional signals targeting different regulatory sequences in complex promoters.

Arabidopsis CUP-SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation.

Overall shoot architecture in higher plants is highly dependent on the activity of embryonic and axillary shoot meristems, which are produced from the basal adaxial boundaries of cotyledons and leaves, respectively. In Arabidopsis thaliana, redundant functions of the CUP-SHAPED COTYLEDON genes CUC1, CUC2, and CUC3 regulate embryonic shoot meristem formation and cotyledon boundary specification. Their functional importance and relationship in postembryonic development, however, is poorly understood. Here, we performed extensive analyses of the embryonic and postembryonic functions of the three CUC genes using multiple combinations of newly isolated mutant alleles. We found significant roles of CUC2 and CUC3, but not CUC1, in axillary meristem formation and boundary specification of various postembryonic shoot organs, such as leaves, stems, and pedicels. In embryogenesis, all three genes make significant contributions, although CUC3 appears to possess, at least partially, a distinct function from that of CUC1 and CUC2. The function of CUC3 and CUC2 overlaps that of LATERAL SUPPRESSOR, which was previously shown to be required for axillary meristem formation. Our results reveal that redundant but partially distinct functions of CUC1, CUC2, and CUC3 are responsible for shoot organ boundary and meristem formation throughout the life cycle in Arabidopsis.

CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation.

CUP-SHAPED COTYLEDON (CUC)1 encodes members of the NAC family. These are functionally redundant genes that are involved in shoot apical meristem (SAM) formation and cotyledon separation during embryogenesis in Arabidopsis. We analyzed transgenic plants overexpressing CUC1 (35S::CUC1). The cotyledons of these transgenic seedlings regularly had two basal lobes, small and round epidermal cells between the sinuses, and adventitious SAMs on the adaxial surface of this region. This suggests that CUC1 promotes adventitious SAM formation by maintaining epidermal cells in an undifferentiated state. In 35S::CUC1 cotyledons, the class I knotted-like homeobox (KNOX) genes, including SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS (BP), which are involved in SAM formation and/or maintenance, were ectopically expressed before adventitious SAM formation. In stm mutants, ectopic expression of CUC1 could not induce adventitious SAMs, whereas they continued to be observed in bp mutants. These results suggest that STM, but not BP, is necessary for the formation of adventitious SAMs in 35S::CUC1 cotyledons. Furthermore, we examined the relationship between CUC1 and ASYMMETRIC LEAVES (AS)1 and AS2. The as1 and as2 mutations genetically enhance 35S::CUC1 phenotypes even in the absence of STM function. Interestingly, the as1 mutation can partially rescue the mutant vegetative development phenotypes in the cuc1 cuc2 double mutant. Our results suggest that CUC1 positively regulates SAM formation not only through STM but also through an STM-independent pathway that is negatively regulated by AS1 and AS2.

Terminal flower2, an Arabidopsis homolog of heterochromatin protein1, counteracts the activation of flowering locus T by constans in the vascular tissues of leaves to regulate flowering time.

The flowering time of plants is tightly regulated by both promotive and repressive factors. Molecular genetic studies using Arabidopsis have identified several epigenetic repressors that regulate flowering time. Terminal flower2, (TFL2), which encodes a homolog of heterochromatin protein1 represses flowering locus T (FT) expression, which is induced by the activator constans (CO) in response to the long-day signal. Here, we show that TFL2, CO, and FT are expressed together in leaf vascular tissues and that TFL2 represses FT expression continuously throughout development. Mutations in TFL2 derepress FT expression within the vascular tissues of leaves, resulting in daylength-independent early flowering. TFL2 can reduce FT expression even when CO is overexpressed. However, FT expression reaches a level sufficient for floral induction even in the presence of TFL2, suggesting that TFL2 does not maintain FT in a silent state or inhibit it completely; rather, it counteracts the effect of CO on FT activation.

Arabidopsis TERMINAL FLOWER 2 gene encodes a heterochromatin protein 1 homolog and represses both FLOWERING LOCUS T to regulate flowering time and several floral homeotic genes.

Floral transition should be strictly regulated because it is one of the most critical developmental processes in plants. Arabidopsis terminal flower 2 (tfl2) mutants show an early-flowering phenotype that is relatively insensitive to photoperiod, as well as several other pleiotropic phenotypes. We found that the early flowering of tfl2 is caused mainly by ectopic expression of the FLOWERING LOCUS T (FT) gene, a floral pathway integrator. Molecular cloning of TFL2 showed that it encodes a protein with homology to heterochromatin protein 1 (HP1) of animals and Swi6 of fission yeast. TFL2 protein localizes in subnuclear foci and expression of the TFL2 gene complemented yeast swi6(-) mutants. These results suggested that TFL2 might function as an HP1 in Arabidopsis: Gene expression analyses using DNA microarrays, however, did not show an increase in the expression of heterochromatin genes in tfl2 mutants but instead showed the upregulation of the floral homeotic genes APETALA3, PISTILLATA, AGAMOUS and SEPALLATA3. The pleiotropic phenotype of the tfl2 mutant could reflect the fact that TFL2 represses the expression of multiple genes. Our results demonstrate that despite its homology to HP1, TFL2 is involved in the repression of specific euchromatin genes and not heterochromatin genes in Arabidopsis.

Embryonic shoot apical meristem formation in higher plants.

The shoot apical meristem (SAM) is essential for organ formation in higher plants. How the SAM is formed during plant development is poorly understood, however. In this review, we focus on several recent studies that provide new insights into the mechanism of SAM formation during embryogenesis. Recently, positive and negative regulators of the class I KNOX genes, which are thought to be necessary for SAM formation, have been identified; the Arabidopsis CUP-SHAPED COTYLEDON ( CUC) genes are required for the expression of a class I KNOX gene, SHOOT MERISSTEMLES( STM) during embryogenesis, and the Arabidopsis ASYMMETRIC LEAVES1 ( AS1), AS2, and several other genes negatively regulate KNOX gene expression in cotyledon primordia. Also, several genes that are involved in the formation of the adaxial-abaxial axis of cotyledons seem to regulate embryonic SAM formation.