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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.

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.

The shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration. Advances have been made in our understanding of stomatal patterning and differentiation, and a set of ‘master regulatory’ transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis. Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages. Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata. A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2. Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta. Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.

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 β-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 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 beta-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.

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.

Background: Dryopteris fragrans, which is densely covered with glandular trichomes, is considered to be one of the ferns with the most medicinal potential. The transcriptomes from selected tissues of D. fragrans were collected and analyzed for functional and comparative genomic studies. The aim of this study was to determine the transcriptomic characteristics of wild D. fragrans sporangium in tissues from the SR (root), SL (sporophyll), and TRL (sporophyll with glandular trichomes removed). Results: Cluster analysis identified genes that were highly expressed in an organ-specific manner according to read mapping, feature counting, and normalization. The functional map identified gene clusters that can uniquely describe the function of each tissue. We identified a group of three tissue-specific transcription factors targeting the SL, SR, and TRL. In addition, highly expressed transcription factors (TFs) were found in each tissue-specific gene cluster, where ERF and bHLH transcription factors were the two types showing the most distinct expression patterns between the three different tissues. The specific expression of transcription factor genes varied between the different types of tissues. The numbers of transcription factors specifically expressed in the roots and sporophylls were 60 and 30, respectively, while only seven were found for the sporophylls with glandular trichomes removed. The expression of genes known to be associated with the development of glandular trichomes in flowering plants, including MIXTA, ATML1, and MYB106, were also validated and are discussed. In particular, a unigene encoding MIXTA was identified and exhibited the highest expression level in SL in D. fragrans. Conclusions: This study is the first report of global transcriptomic analysis in different tissues of D. fragrans, and the first to discuss these findings in the context of the development of homologous glandular trichomes. These results set the stage for further research on the development, stress resistance, and secondary metabolism of D. fragrans glandular trichomes.

Homeobox genes are master regulatory genes that specify the body plan and control development of many eukaryotic organisms, including plants. We isolated and characterized a cDNA designated ATML1 (for Arabidopsis thaliana meristem L1 layer) that encodes a novel homeodomain protein. The ATML1 protein shares high sequence homology inside and outside of the homeodomain with both the Phalaenopsis O39 and the Arabidopsis GLABRA2 (GL2) homeodomain proteins, which together define a new class of plant homeodomain-containing proteins, designated HD-GL2. The ATML1 gene was first expressed in the apical cell after the first asymmetric division of the zygote and continued to be expressed in all proembryo cells until the eight-cell stage. In the 16-cell proembryo, the ATML1 gene showed a distinct pattern of expression, with its mRNA becoming restricted to the protoderm. In the torpedo stage of embryo development, ATML1 mRNA disappeared altogether but reappeared later only in the L1 layer of the shoot apical meristem in the mature embryo. After germination, this L1 layer-specific pattern of expression was maintained in the vegetative shoot apical meristem, inflorescence, and floral meristems, as well as in the young floral organ primordia. Finally, ATML1 mRNA accumulated in the protoderm of the ovule primordia and integuments and gradually became restricted in its expression to the endothelium surrounding the embryo sac. We propose that ATML1 may be involved in setting up morphogenetic boundaries of positional information necessary for controlling cell specification and pattern formation. In addition, ATML1 provides an early molecular marker for the establishment of both apical-basal and radial patterns during plant embryogenesis.

Background: Genome- wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. We have developed a sensitive approach based on ultra-low RNA sequencing coupled to laser assisted microdissection for analyzing different tissues of the small Arabidopsis embryo. Methods and results: We first characterized the number of genes detected according to the quantity of tissue yield and total RNA extracted. Our results revealed that as low as 0.02 mm(2) of tissue and 50 pg of total RNA can be used without compromising the number of genes detected. The optimised protocol was used to compare the epidermal versus mesophyll cell transcriptomes of cotyledons at the torpedo- shaped stage of embryo development. The approach was validated by the recovery of well- known epidermal genes such AtML1 or AtPDF2 and genes involved in flavonoid and cuticular waxes pathways. Moreover, the interest and sensitivity of this approach were highlighted by the characterization of several transcription factors preferentially expressed in epidermal cells. Conclusion: This technical advance unlocks some current limitations of transcriptomic analyses and allows to investigate further and efficiently new biological questions for which only a very small amounts of cells need to be isolated. For instance, it paves the way to increasing the spatial accuracy of regulatory networks in developing small embryo of Arabidopsis or other plant tissues.

Eukaryotic development and stem cell control depend on the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly link these events are known. The DEFECTIVE KERNEL1 (DEK1) gene encoding the unique plant calpain protein is fundamental for development and growth, being essential to confer and maintain epidermal cell identity that allows development beyond the globular embryo stage. We show that DEK1 expression is highest in the actively dividing cells of seeds, meristems and vasculature. We further show that eliminating Arabidopsis DEK1 function leads to changes in developmental cues from the first zygotic division onward, altered microtubule patterns and misshapen cells, resulting in early embryo abortion. Expression of the embryonic marker genes WOX2, ATML1, PIN4, WUS and STM, related to axis organization, cell identity and meristem functions, is also altered in dek1 embryos. By monitoring cell layer-specific DEK1 down-regulation, we show that L1- and 35S-induced down-regulation mainly affects stem cell functions, causing severe shoot apical meristem phenotypes. These results are consistent with a requirement for DEK1 to direct layer-specific cellular activities and set downstream developmental cues. Our data suggest that DEK1 may anchor cell wall positions and control cell division and differentiation, thereby balancing the plant's requirement to maintain totipotent stem cell reservoirs while simultaneously directing growth and organ formation. A role for DEK1 in regulating microtubule-orchestrated cell wall orientation during cell division can explain its effects on embryonic development, and suggests a more general function for calpains in microtubule organization in eukaryotic cells.

Pflanzen durchlaufen während ihres Lebenszyklus verschiedene Entwicklungsphasen, die durch Ausprägung neuer morphologischer Merkmale und/oder Bildung neuer Organe gekennzeichnet sind. Die Größe und die Anzahl der in der vegetativen Phase gebildeten Blätter werden durch komplexe Netzwerke reguliert, die durch Umwelteinflüsse, genetische Faktoren und Phytohormone beeinflusst werden. Das zellteilungsfördernde Pflanzenhormon Cytokinin (CK) hat einen wesentlichen Einfluss auf die Wachstumsprozesse eines Organs. Ob CK dabei als nicht-zellautonomer Faktor zwischen verschiedenen Zellschichten eines sich entwickelnden Organs vermittelt, ist bisher nicht geklärt. Das Phytohormon ist zudem ein positiver Regulator der Blühinduktion. Da Pflanzen erst mit dem Übergang zur adulten vegetativen Phase Blühkompetenz erlangen, hat die Dauer der juvenilen Phase einen maßgeblichen Einfluss auf den Blühbeginn. Die Juvenil-adult-Transition wird durch die hochkonservierten microRNAs miR156 und miR172 sowie ihre jeweiligen Zielgene reguliert, wobei miR156 Juvenilität und miR172 den Übergang zur adulten und reproduktiven Phase fördert. Über einen Einfluss von CK auf die Juvenil-adult-Transition ist bislang nur sehr wenig bekannt und auch die molekularen Mechanismen, mit denen das Phytohormon Einfluss auf den Blühbeginn nimmt, sind weitestgehend ungeklärt. Im ersten Teilprojekt dieser Arbeit wurde der Einfluss des epidermalen Cytokininstatus auf das Spross-wachstum untersucht. Dafür wurden mehrere Promotor-Gen-Konstrukte kloniert und in Arabidopsis exprimiert. Der in der Epidermis aktive Promotor des ATML1-Gens wurde mit Genen kombiniert, welche die Cytokininsignaltransduktion hemmen (ARR1-SRDX) bzw. den Katabolismus (CKX1) oder die Biosynthese (LOG4) von CK fördern. Alle Transgene verursachten Veränderungen des Cytokininstatus, wobei ARR1-SRDX lokal in der Epidermis wirkte und CKX1 und LOG4 auch Effekte über die Expressionsdomäne hinaus bewirkten. Es konnte gezeigt werden, dass die Expression von pATML1:LOG4 und pATML1:CKX1-4xMyc die Größe des vegetativen Meristems beeinflusst, möglicherweise durch die Veränderung des von der L1 ausgehenden Cytokiningradienten. pATML1:LOG4-Pflanzen bildeten größere, pATML1:ARR1-SRDX- und pATML1:CKX1-Pflanzen kleinere Rosettenblätter, die durch Veränderungen der Zellteilungsaktivität bewirkt wurden. Die durch lokale Hemmung der Cytokininsignaltransduktion hervorgerufene Reduktion der epidermalen Zellteilung in pATML1:ARR1-SRDX beschränkte das Wachstum der subepidermalen Gewebe. Die Cytokininrezeptoren AHK2 und AHK3 sowie der Typ-B-ARR ARR1 konnten als vermittelnde Faktoren bei der Blattgrößendetermination in den pATML1-Linien identifiziert werden. Darüber hinaus konnten Effekte der Transgenexpression auf die Plazenta-Aktivität, die Apikaldominanz, den Stengeldurchmesser und das Wurzelwachstum beobachtet werden. Die Analyse der pATML1-Linien und weiterer Arabidopsis-Linien mit verändertem Cytokininstatus im zweiten Teilprojekt förderte eine von der Fotoperiode unabhängige, negative Korrelation zwischen dem Cytokininstatus und der Länge der juvenilen vegetativen Phase zutage, wobei der epidermale Cytokininstatus keinen wesentlichen Einfluss auf die Juvenil-adult-Transition zeigte. AHK2, AHK3 und ARR1 konnten als vermittelnde Faktoren der Cytokininsignalkaskade in diesem Prozess identifiziert werden. Neben der Länge der juvenilen Phase wurde der Blühzeitpunkt zudem durch den Einfluss von CK auf die Blattbildungsrate beeinflusst, wobei dem Rezeptor CRE1/AHK4 hierbei eine wesentliche Rolle zukommt. Expressionsanalysen zeigten, dass der positive Einfluss von CK auf Entwicklungsübergänge durch die transkriptionelle Aktivierung von MIR172-Genen zu erklären ist. In einem Yeast-one-hybrid-Matrix-ansatz konnte eine Bindung von ARR1, ARR2 und ARR12 an MIR172-Promotoren gezeigt werden, was eine direkte Aktivierung der MIR172-Transkription durch Typ-B-ARRs vermuten lässt. Expressionsanalysen und genetische Untersuchungen zeigten zudem, dass CK u. a. eine Hemmung der Expression des miR172-Zielgens SMZ bewirkt, welches für einen Blührepressor kodiert, der u. a. die Expression der Florigene FT und TSF reguliert. Da CK TSF in positiver Weise reguliert, kann die Hypothese aufge¬stellt werden, dass CK die TSF-Aktivierung möglicherweise u. a. über die Hemmung von SMZ erreicht., Plants progress through a number of developmental transitions during their life cycle that are characterized by the development of new morphological traits and/or organs. The number and size of leaves formed during the vegetative phase is regulated by complex regulatory networks which are influenced by environmental cues and genetic factors as well as phytohormones. Cytokinins (CKs), a class of phytohormones which promote cell division, are known to contribute considerably to organ growth. However, it remains unclear whether CKs act as non-cell autonomous factors between different cell layers in developing organs. CKs are also positive regulators of flowering time. As plants acquire the competence to flower upon transition to the adult vegetative phase, the length of the juvenile phase considerably influences flowering time. The juvenile-to-adult phase transition is regulated by the highly conserved microRNAs miR156 and miR172 and their respective target genes. MiR156 promotes juvenility, while miR172 stimulates the transition to adult and reproductive growth. The impact of CKs on the juvenile-to-adult phase transition as well as the CK-related molecular mechanisms involved in flowering time determination are still poorly understood. In the first part of this study, the importance of the epidermal CK status for shoot growth was investigated by using promoter-gene constructs which were stably expressed in Arabidospis thaliana. The epidermis-specific ATML1 promoter was fused to the ORFs of genes inhibiting CK signal transduction (ARR1-SRDX), or promoting the catabolism (CKX1) or biosynthesis (LOG4) of CKs. The CK status was altered by all transgenes. ARR1-SRDX acted locally in the epidermis whereas CKX1 and LOG4 also caused effects outside of their expression domain. It was shown that expression of pATML1:LOG4 and pATML1:CKX1-4xMyc influences the size of the vegetative meristem, probably by altering the CK gradient originating from the L1 layer. Compared to the wildtype, rosette leaves of pATML1:LOG4 plants were bigger and those of pATML1:ARR1-SRDX and pATML1:CKX1 plants were smaller due to changes in cell proliferation activity. In pATML1:ARR1-SRDX plants, the reduction of epidermal cell division due to local inhibition of CK signal transduction restricted subepidermal tissue growth. The CK receptors AHK2 and AHK3 and type-B-ARR ARR1 were identified as factors controlling leaf size in pATML1 lines. Moreover, effects of transgene expression on placenta activity, apical dominance, stem diameter and root growth were observed. The analysis of pATML1 lines and additional Arabidopsis lines with altered CK status presented in the second part of this study allowed the identification of a photoperiod- independent negative correlation between CK status and length of the juvenile vegetative phase. However, no substantial influence of the epidermal CK status on juvenile-to-adult transition was observed. AHK2, AHK3 and ARR1 were identified as components of the CK signaling cascade involved in this process. Beside the length of the juvenile phase, flowering time was also affected by the impact of CK on the leaf formation rate, CRE1/AHK4 being the crucial player involved. Expression analyses showed that the positive effect of CK on developmental transitions relies on the transcriptional activation of MIR172. Using a yeast one-hybrid assay, ARR1, ARR2 and ARR12 were shown to bind to MIR172 promoters, suggesting direct transcriptional activation of MIR172 genes by type-B ARRs. Expression analyses and genetic investigations additionally showed that CK inhibits the expression of the miR172 target gene SMZ which encodes a flowering repressor regulating the expression of the florigenes FT and TSF. As CKs positively regulate TSF, it can be hypothezised that this activation is achieved by inhibition of SMZ.

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.

The formation of cellular patterns during development requires the coordination of cell division with cell identity specification. This coordination is essential in patterning the highly elongated giant cells, which are interspersed between small cells, in the outer epidermis of the Arabidopsis thaliana sepal. Giant cells undergo endocycles, replicating their DNA without dividing, whereas small cells divide mitotically. We show that distinct enhancers are expressed in giant cells and small cells, indicating that these cell types have different identities as well as different sizes. We find that members of the epidermal specification pathway, DEFECTIVE KERNEL1 (DEK1), MERISTEM LAYER1 (ATML1), Arabidopsis CRINKLY4 (ACR4) and HOMEODOMAIN GLABROUS11 (HDG11), control the identity of giant cells. Giant cell identity is established upstream of cell cycle regulation. Conversely, endoreduplication represses small cell identity. These results show not only that cell type affects cell cycle regulation, but also that changes in the cell cycle can regulate cell type.

Growth and developmental processes are occasionally accompanied by multiple rounds of DNA replication, known as endoreduplication. Coordination between endoreduplication and cell size regulation often plays a crucial role in proper organogenesis and cell differentiation. Here, we report that the level of correlation between ploidy and cell volume is different in the outer and inner cell layers of leaves of Arabidopsis thaliana using a novel imaging technique. Although there is a well-known, strong correlation between ploidy and cell volume in pavement cells of the epidermis, this correlation was extremely weak in palisade mesophyll cells. Induction of epidermis cell identity based on the expression of the homeobox gene ATML1 in mesophyll cells enhanced the level of correlation between ploidy and cell volume to near that of wild-type epidermal cells. We therefore propose that the correlation between ploidy and cell volume is regulated by cell identity.

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.

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.

Cotton fibres are a subset of single epidermal cells that elongate from the seed coat to produce the long cellulose strands or lint used for spinning into yarn. To identify genes that might regulate lint fibre initiation, expression profiles of 0 days post-anthesis (dpa) whole ovules from six reduced fibre or fibreless mutants were compared with wild-type linted cotton using cDNA microarrays. Numerous clones were differentially expressed, but when only those genes that are normally expressed in the ovule outer integument (where fibres develop) were considered, just 13 different cDNA clones were down-regulated in some or all of the mutants. These included: a Myb transcription factor (GhMyb25) similar to the Antirrhinum Myb AmMIXTA, a putative homeodomain protein (related to Arabidopsis ATML1), a cyclin D gene, some previously identified fibre-expressed structural and metabolic genes, such as lipid transfer protein, alpha-expansin and sucrose synthase, as well as some unknown genes. Laser capture microdissection and reverse transcription-PCR were used to show that both the GhMyb25 and the homeodomain gene were predominantly ovule specific and were up-regulated on the day of anthesis in fibre initials relative to adjacent non-fibre ovule epidermal cells. Their spatial and temporal expression pattern therefore coincided with the time and location of fibre initiation. Constitutive overexpression of GhMyb25 in transgenic tobacco resulted in an increase in branched long-stalked leaf trichomes. The involvement of cell cycle genes prompted DNA content measurements that indicated that fibre initials, like leaf trichomes, undergo DNA endoreduplication. Cotton fibre initiation therefore has some parallels with leaf trichome development, although the detailed molecular mechanisms are clearly different.

*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.

International audience; Transcriptional regulation is an important mechanism underlying gene expression and has played a crucial role in evolution. The number, position and interactions between cis-elements and transcription factors (TFs) determine the expression pattern of a gene. To identify functionally relevant cis-elements in gene promoters, a phylogenetic shadowing approach with a lipase gene (LIP1) was used. As a proof of concept, in silico analyses of several Brassicaceae LIP1 promoters identified a highly conserved sequence (LIP1 element) that is sufficient to drive strong expression of a reporter gene in planta. A collection of ca. 1,200 Arabidopsis thaliana TF open reading frames (ORFs) was arrayed in a 96-well format (RR library) and a convenient mating based yeast one hybrid (Y1H) screening procedure was established. We constructed an episomal plasmid (pTUY1H) to clone the LIP1 element and used it as bait for Y1H screenings. A novel interaction with an HD-ZIP (AtML1) TF was identified and abolished by a 2 bp mutation in the LIP1 element. A role of this interaction in transcriptional regulation was confirmed in planta. In addition, we validated our strategy by reproducing the previously reported interaction between a MYB-CC (PHR1) TF, a central regulator of phosphate starvation responses, with a conserved promoter fragment (IPS1 element) containing its cognate binding sequence. Finally, we established that the LIP1 and IPS1 elements were differentially bound by HD-ZIP and MYB-CC family members in agreement with their genetic redundancy in planta. In conclusion, combining in silico analyses of orthologous gene promoters with Y1H screening of the RR library represents a powerful approach to decipher cis- and trans-regulatory codes.

Elaboration of size and shape in multicellular organisms involves coordinated cell division and cell growth. In higher plants, continuity of cell layer structures exists from the shoot apical meristem (SAM), where organ primordia arise, to mature aboveground organs. To unravel the extent of inter-cell layer coordination during SAM and aboveground organ development, cell division in the epidermis was selectively restricted by expressing two cyclin-dependent kinase inhibitor genes, KRP1/ICK1 and KRP4, driven by the L1 layer-specific AtML1 promoter. The transgenes conferred reduced plant size with striking, distorted lateral organ shape. While epidermal cell division was severely inhibited with compensatory cell size enlargement, the underlying mesophyll/cortex layer kept normal cell numbers and resulted in small, packed cells with disrupted cell files. Our results demonstrate the autonomy of cell number checkpoint in the underlying tissues when epidermal cell division is restricted. Finally, the L1 layer-specific expression of both KRP1/ICK1 and KRP4 showed no effects on the structure and function of the SAM, suggesting that the effects of these cyclin-dependent kinase inhibitors are context dependent.

Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. We have developed a sensitive approach based on ultra-low RNA sequencing coupled to laser assisted microdissection for analyzing different tissues of the small Arabidopsis embryo. We first characterized the number of genes detected according to the quantity of tissue yield and total RNA extracted. Our results revealed that as low as 0.02 mm2 of tissue and 50 pg of total RNA can be used without compromising the number of genes detected. The optimised protocol was used to compare the epidermal versus mesophyll cell transcriptomes of cotyledons at the torpedo-shaped stage of embryo development. The approach was validated by the recovery of well-known epidermal genes such AtML1 or AtPDF2 and genes involved in flavonoid and cuticular waxes pathways. Moreover, the interest and sensitivity of this approach were highlighted by the characterization of several transcription factors preferentially expressed in epidermal cells. This technical advance unlocks some current limitations of transcriptomic analyses and allows to investigate further and efficiently new biological questions for which only a very small amounts of cells need to be isolated. For instance, it paves the way to increasing the spatial accuracy of regulatory networks in developing small embryo of Arabidopsis or other plant tissues.