Search

In Arabidopsis thaliana, the epidermis is the outermost cell layer composed of many specialized types of epidermal cells, such as pavement cells, trichomes, and guard cells. The homeodomain-leucine zipper (HD-ZIP) class Ⅳ transcription factors (TFs), which are unique to the plant kingdom, have been recognized as key regulators of epidermis development. Unlike animal HD proteins, which can bind to DNA as monomers, plant HD-ZIP class Ⅳ TFs bind to DNA as dimers, although little is known about the regulation of their dimerization process. Here, we show that the homodimerization of ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) - HD-ZIP class Ⅳ TF that is required for protoderm development - is regulated by the lipid-binding steroidogenic acute regulatory protein-related lipid transfer (START) domain. We found that ATML1 forms homodimer through interaction via its ZIP motif in yeast and plant cells, although the interaction is abolished by generating a mutation into the lipid-binding START domain to disrupt the lipid-binding ability. These results suggest that lipidic ligands function as key regulators of protoderm development via modulating the dimerization of ATML1.

Summary The Arabidopsis thaliana PROTODERMAL FACTOR1 (PDF1) gene encoding a putative extracellular proline-rich protein is exclusively expressed in the L1 layer of shoot apices and the protoderm of organ primordia. In order to identify essential cis-regulatory sequences required for the L1 layer-specific expression, a series of 5′ deletions of the PDF1 promoter were fused to the β-glucronidase (GUS) gene and introduced into Arabidopsis plants. Our analysis revealed that the minimum region necessary to confer L1-specific expression of PDF1 is confined within a 260-bp fragment upstream of the transcription start site. We identified an 8-bp motif in this region that is conserved between promoter regions of all the L1-specific genes so far cloned, and we designated it the L1 box. Electrophoretic mobility shift assays demonstrated that the L1-specific homeodomain protein ATML1 can bind to the L1 box sequence in vitro. The GUS expression in transgenic plants disappeared when a mutation that abolishes binding of ATML1 was introduced into the PDF1 l1 box sequence of the construct. These results suggest that the L1 box plays a crucial role in the regulation of PDF1 expression in L1 cells and that ATML1 could cooperate to drive L1-specific expression.

Summary 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.

Summary Development of the epidermis involves members of the class–IV homeodomain-leucine zipper (HD-ZIP IV) transcription factors. The Arabidopsis HD-ZIP IV family consists of 16 members, among which PROTODERMAL FACTOR 2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) play an indispensable role in the differentiation of shoot epidermal cells; however, the functions of other HD-ZIP IV genes that are also expressed specifically in the shoot epidermis remain to be fully elucidated. We constructed double mutant combinations of these HD-ZIP IV mutant alleles and found that the double mutants of pdf2-1 with homeodomain glabrous1-1 (hdg1-1), hdg2-3, hdg5-1 and hdg12-2 produced abnormal flowers with sepaloid petals and carpelloid stamens in association with the reduced expression of the petal and stamen identity gene APETALA 3 (AP3). Expression of another petal and stamen identity gene PISTILATA (PI) was less affected in these mutants. We confirmed that AP3 expression in pdf2-1 hdg2-3 was normally induced at the initial stages of flower development, but was attenuated both in the epidermis and internal cell layers of developing flowers. As the expression of PDF2 and these HD-ZIP IV genes during floral organ formation is exclusively limited to the epidermal cell layer, these double mutations may have non-cell-autonomous effects on AP3 expression in the internal cell layers. Our results suggest that cooperative functions of PDF2 and other members of the HD-ZIP IV family in the epidermis are crucial for normal development of floral organs in Arabidopsis.

In a number of land plants, Golden2-like (GLK) genes encode a pair of partially redundant nuclear transcription factors that are required for the expression of nuclear photosynthetic genes and for chloroplast development. As chloroplast biogenesis depends on close co-operation between the nuclear and plastid compartments, GLK gene function must be dependent on tight intracellular control. However, the extent to which GLK-mediated chloroplast development depends on intercellular communication is not known. Here we used sector analysis to show that GLK proteins operate cell-autonomously in leaf mesophyll cells. To establish whether GLK proteins are able to influence adjacent cell layers, we used tissue-specific promoters to restrict GLK gene expression to the epidermis and to the phloem. GLK genes driven by the Arabidopsis epidermal FIDDLEHEAD (FDH) and MERISTEM LAYER1 (AtML1) promoters failed to rescue the pale-green Atglk1 Atglk2 mutant phenotype, confirming the suggestion that GLK proteins can only influence chloroplast development intracellularly. An exception to this rule was seen in lines in which GLK genes were expressed in the phloem. However, we believe that the partial complementation of the mutant phenotype that was observed resulted from phloem unloading, as opposed to inherent properties of GLK proteins. We conclude that GLK proteins act in a cell-autonomous manner to coordinate and maintain the photosynthetic apparatus within individual cells. Significantly, this suggests that GLK proteins provide a means to fine-tune photosynthesis according to the differential requirements of cells within the leaf.

*Summary The specification of epidermal (L1) identity occurs early during plant embryogenesis. Here we show that, in Arabidopsis, AtDEK1 encodes a key component of the embryonic L1 cell-layer specification pathway. Loss of AtDEK1 function leads to early embryo lethality characterized by a severe loss of cell organization in the embryo proper and abnormal cell divisions within the suspensor. Markers for L1 identity, ACR4 and ATML1, are not expressed in homozygous mutant embryos. In order to clarify the function of AtDEK1 further, an RNAi knockdown approach was used. This allowed embryos to partially complete embryogenesis before losing AtDEK1 activity. Resulting seedlings showed a specific loss of epidermal cell identity within large portions of the cotyledons. In addition, meristem structure and function was systematically either reduced or entirely lost. AtDEK1 expression is not restricted to the L1 epidermal cell layer at any stage in development. This is consistent with AtDEK1 playing an upstream role in the continuous generation or interpretation of positional information required for epidermal specification. Our results not only identify a specific role for AtDEK1 during embryogenesis, but underline the potential key importance of L1 specification at the globular stage for subsequent progression through embryogenesis.

The series of unidirectional events that constitute the cell division cycle renders two cells with an identical genomic content, for example, a 2C diploid nucleus. One of the crucial events is genome replication that occurs faithfully during each S phase of the cell cycle. Later, the two identical genomic copies are transferred to each daughter cell during mitosis. This paradigmatic process is at the base of the necessary increase in cell number required for organogenesis, cell and tissue repair and development. There are, however, alternative cycles where genome replication is not followed by cell division. Instead, mitosis is actively prevented in such a way that nuclear ploidy increases, yielding nuclei (or cells) with a DNA content of 4C, 8C, 16C, 32C, 64C, and so on. The major mechanism whereby ploidy is increased in this manner is endoreplication (also termed as endoreduplication), although other pathways leading to polyploidy also exist in nature. It is worth noting that endoreplication consists of multiple rounds of full genome replication. Consequently, it is a process different from selective amplification of certain genomic regions or from re-replication, triggered in response to various DNA replication stress conditions and by altering the mechanisms controlling genome replication. Endoreplication occurs across the evolutionary scale in many multicellular organisms, but it is far more frequent in plants. In fact, it is normally associated to the plant response to hormonal signals, developmental cues or environmental challenges. The increase in nuclear ploidy by endoreplication requires a very precise arrest of the cell cycle, the entry into the new endoreplication cycle (initiation) and also active mechanisms sustaining several endoreplication cycles (endocycles). A plethora of processes involved in controlling initiation of the endocycle programme have been identified, including cell patterning and differentiation, DNA replication, DNA damage, chromatin status, the retinoblastoma/E2F pathway, and hormonal signals, among others (Gutierrez, 2009; De Veylder et al, 2011). Progression through various endocycles has been linked to cell differentiation and growth. Nuclear ploidy level and size of a cell seem to be correlated in Arabidopsis epidermal cells, although multiple experiments have revealed that cell growth is controlled by different elements, being endoreplication only one of the numerous components that determine the final size of a cell. This key issue has been addressed in a recent work published in The EMBO Journal by Breuer et al (2012). Breuer et al focussed their study on development of Arabidopsis trichomes, unicellular epidermal hairs that increase their cell volume >100-fold. After cell fate specification, trichomes enter a highly complex differentiation programme that proceeds through two major growth phases (Figure 1): first, protodermal cells that have taken the trichome fate undergo several rounds of endocycles and substantial increase in size and branching events, and second, occurrence of a rapid cell expansion driven mainly by vacuolization (Ishida et al, 2008). The first stage involves initiation of the endoreplication programme but crucial questions that still remain elusive include whether there are mechanisms that control termination of the endocycle programme and, as a consequence, arrest of cell differentiation-associated growth, and what are the mechanisms that couple endoreplication and cell growth. A previous study from the same laboratory identified a mutation in the GTL1 gene, encoding a member of the GT-2 family of trihelix transcription factors, that results in extended number of endocycles and increased trichome cell growth (Breuer et al, 2009). Here the authors combined cellular, molecular, genetic and genomic analysis that, together, establish GTL1 as a master regulator of a cohort of genes that control termination of the endoreplication programme and its coupling to cell growth. Figure 1 Coordination between the endoreplication programme and cell growth during trichome development. Trichome development starts with cell fate specification of some protodermal cells in the epidermis by expression of the homeobox gene GL2. These cells arrest ... GTL1 is normally expressed in the late phases of trichome development when they have terminated their branching process (Breuer et al, 2009). To test its possible contribution to the temporal control of the endocycle programme, GTL1 was expressed at early stages of trichome development using cell type-specific promoters that are active either early in establishing the trichome fate of protodermal cells (GL2) or active in all leaf epidermal cells (ATML1). These experiments demonstrated that GTL1 is sufficient to inhibit endoreplication and ploidy-dependent cell growth and that its correct spatio-temporal expression is responsible for the termination of the endoreplication programme in trichomes (Figure 1). The fact that ectopic expression of GTL1 in pavement epidermal also inhibits growth suggests that other members of the trihelix transcription factor family may be involved in controlling cell growth arrest. Interestingly, expression of GTL1 in trichome cell precursors also inhibits the expression of the homeobox gene GL2, indicating that it may have an impact on cell fate establishment or maintenance. To identify GTL1 target(s), the authors used a combination of ChIP/chip analysis and trichome-specific microarrays of the gtl1 mutant. Within the set of potential direct GTL1 targets identified, it was striking to find the CCS52A1 gene (Figure 1), encoding a Cdh1/Fzr-type of activators of the APC. This gene possesses a GTL1 binding GT3 box in its promoter and is upregulated in the gtl1 mutant. CCS52A genes were previously implicated in the onset of endoreplication during trichome development (Larson-Rabin et al, 2009). In fact, CCS52A1 cooperates with SIAMESE (SIM), a plant-specific CDK inhibitor, in preventing the accumulation of mitotic cyclins and triggering the endocycle in trichomes (Kasili et al, 2010) and is modulated by the retinoblastoma (RBR) pathway (Magyar et al, 2012). Using a reporter construct driving the expression of H2B-GFP under the control of the CCS52A1 promoter, the authors demonstrate that expression of GTL1 at late stages of trichome development represses CCS52A1, leading to endoreplication arrest. Furthermore, CCS52A1 and GTL1 interact genetically since the ccs52a1-2 mutation completely suppresses the typical increased ploidy phenotype of gtl1-1 trichomes, which in the double mutant are under-branched and under-endoreplicated. This is strong genetic evidence that CCS52A1 is acting directly downstream of GTL1 regulating endocycle and branching, the first phase of trichome growth. It has been postulated that endoreplication could play a predominant role in cell size control setting up the approximate area of cell growth (that might vary between different cell types) and within this range, a DNA-independent growth mechanism is responsible for the final cell size (Schnittger et al, 2003; Harashima and Schnittger, 2010). In this context, Breuer et al (2012) found that trichome cells in the double gtl1-1 ccs52a1-2 mutant are significantly larger than those in the single ccs52a1-2 mutant, suggesting that the ccs52a1-2 mutation does not completely suppress the cell growth phenotype of gtl1-1. This indicates that ploidy and cell growth can be readily uncoupled and points to GTL1 as a transcription factor that controls endocycle-dependent cell growth by repressing CCS52A1 expression and endocycle-independent cell growth through additional targets (Figure 1). Thus, a challenging endeavour would be the identification of GTL1-regulated genes controlling cell growth in an endoreplication-independent manner. Finally, these observations suggest that the extent of the endoreplication programme is established in a way that the cell is able to “count” the number of endocycles. The mechanism is not known but it is very remarkable that gtl1 mutants, as well as other endoreplication mutants (Gutierrez, 2009), do not normally loose completely the control of how many endoreplication cycles occur during trichome development since they extend their programme with one extra endocycle, but only one in most of the cases. This limitation in the number of endocycles is suggestive of one or more factors being synthesized de novo and necessary to initiate new endocycles (or made available in each endocycle), even after altering the counting mechanism, whatever it is in molecular terms. Once certain factors, for example, GTL1, come to play they may inhibit their synthesis, accumulation and/or accessibility thus limiting the number of extra endocycles. It is obvious that a number of questions are now open, for example, what are the windows where the various pathways activating or terminating the endocycle operate or how the various pathways impinging on the endocycle control redundantly or coordinately affect endoreplication progress (Gutierrez, 2009; Roodbarkelari et al, 2010; De Veylder et al, 2011). Also, what is the role of other GTL1-related members and their targets in controlling termination of cell growth and whether there are cell type-specific mechanisms involved. These and other unanswered questions remain to be addressed in the future before we can understand fully the mechanisms controlling the increase in ploidy through the endoreplication programme, the pathways impinging on the activation and termination and its role in cell growth and expansion during cell differentiation.