Joachim Forner

Transcriptional Control of a Plant Stem Cell Niche

Despite the independent evolution of multicellularity in plants and animals, the basic organization of their stem cell niches is remarkably similar. Here, we report the genome-wide regulatory potential of WUSCHEL, the key transcription factor for stem cell maintenance in the shoot apical meristem of the reference plant Arabidopsis thaliana. WUSCHEL acts by directly binding to at least two distinct DNA motifs in more than 100 target promoters and preferentially affects the expression of genes with roles in hormone signaling, metabolism, and development. Striking examples are the direct transcriptional repression of CLAVATA1, which is part of a negative feedback regulation of WUSCHEL, and the immediate regulation of transcriptional repressors of the TOPLESS family, which are involved in auxin signaling. Our results shed light on the complex transcriptional programs required for the maintenance of a dynamic and essential stem cell niche.

RNA PROCESSING FACTOR2 Is Required for 5′ End Processing of nad9 and cox3 mRNAs in Mitochondria of Arabidopsis thaliana

Posttranscriptional processes play a crucial role in plant mitochondrial gene expression. This work describes the identification and characterization of a pentatricopeptide repeat protein required for 5′ end processing of distinct mitochondrial transcripts. This protein is related to RESTORERS OF FERTILITY, which can suppress cytoplasmic male sterility, a mitochondrially inherited trait. In mitochondria of higher plants, the majority of 5′ termini of mature mRNAs are generated posttranscriptionally. To gain insight into this process, we analyzed a natural 5′ end polymorphism in the species Arabidopsis thaliana. This genetic approach identified the nuclear gene At1g62670, encoding a pentatricopeptide repeat protein. The functional importance of this mitochondrial restorer of fertility-like protein, designated RNA PROCESSING FACTOR2 (RPF2), is confirmed by the analysis of a respective T-DNA knockout mutant and its functional restoration by in vivo complementation. RPF2 fulfills two functions: it is required for the generation of a distinct 5′ terminus of transcripts of subunit 9 of the NADH DEHYDROGENASE complex (nad9) and it determines the efficiency of 5′ end formation of the mRNAs for subunit 3 of the CYTOCHROME C OXIDASE (cox3), the latter also being influenced by mitochondrial DNA sequences. Accordingly, recombinant RPF2 protein directly binds to a nad9 mRNA fragment in vitro. Two-dimensional gel electrophoresis and immunodetection analyses reveal that altered 5′ processing does not influence accumulation of the nad9 and cox3 polypeptides. In accessions C24, Oystese-1, and Yosemite-0, different inactive RPF2 alleles exist, demonstrating the variability of this gene in Arabidopsis. The identification of RPF2 is a major step toward the characterization of 5′ mRNA processing in mitochondria of higher plants.

GreenGate---a novel, versatile, and efficient cloning system for plant transgenesis.

Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.

Two DEAD-Box Proteins May Be Part of RNA-Dependent High-Molecular-Mass Protein Complexes in Arabidopsis Mitochondria

Posttranscriptional processes are important for regulation of gene expression in plant mitochondria. DEAD-box proteins, which form a huge protein family with members from all kingdoms, are fundamental components in virtually all types of processes in RNA metabolism. Two members of this protein family, designated PMH1 and PMH2 (for PUTATIVE MITOCHONDRIAL RNA HELICASE), were analyzed and characterized in mitochondria of Arabidopsis (Arabidopsis thaliana). Green fluorescent protein tagging with N-terminal PMH1 and PMH2 sequences supports the mitochondrial localization of these proteins. Northern experiments, as well as histochemical β-glucuronidase staining of transgenic plants carrying respective promoter:β-glucuronidase fusion constructs, revealed differing transcription patterns for the two genes. In response to cold, however, transcript levels of both genes increased. Immunodetection analyses of mitochondrial protein complexes after two-dimensional blue native/urea SDS-PAGE and after fractionation on sucrose gradients strongly suggest that one or both proteins are part of RNA-dependent complexes. Cold treatment of cell cultures or solubilization of mitochondria in the presence of MgCl2 favored the detection of high-molecular-mass complexes. This study paves the way for detailed analysis of high-molecular-mass complexes in mitochondria of higher plants.

Integration of light and metabolic signals for stem cell activation at the shoot apical meristem

A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling. Metabolic signals, on the other hand, are transduced to the meristem through activation of the TARGET OF RAPAMYCIN (TOR) kinase. Surprisingly, TOR is also required for light signal dependent stem cell activation. Thus, the TOR kinase acts as a central integrator of light and metabolic signals and a key regulator of stem cell activation at the shoot apex. DOI: http://dx.doi.org/10.7554/eLife.17023.001

Germline-transmitted genome editing in Arabidopsis thaliana Using TAL-effector-nucleases.

Transcription activator–like effector nucleases (TALENs) are custom-made bi-partite endonucleases that have recently been developed and applied for genome engineering in a wide variety of organisms. However, they have been only scarcely used in plants, especially for germline-modification. Here we report the efficient creation of small, germline-transmitted deletions in Arabidopsis thaliana via TALENs that were delivered by stably integrated transgenes. Using meristem specific promoters to drive expression of two TALEN arms directed at the CLV3 coding sequence, we observed very high phenotype frequencies in the T2 generation. In some instances, full CLV3 loss-of-function was already observed in the T1 generation, suggesting that transgenic delivery of TALENs can cause highly efficient genome modification. In contrast, constitutive TALEN expression in the shoot apical meristem (SAM) did not cause additional phenotypes and genome re-sequencing confirmed little off-target effects, demonstrating exquisite target specificity.

RETINOBLASTOMA RELATED1 mediates germline entry in Arabidopsis

Germ cells on demand Unlike animals, plants do not set aside a germline. Instead, germ cells are developed on demand from somatic lineages. Zhao et al. examined the regulatory pathways that manage the transition from somatic to germ cell development in the small plant Arabidopsis (see the Perspective by Vielle-Calzada). The transcription factor WUSCHEL (WUS) was needed early on for development of ovules. Soon after, a trio of inhibitors that work through a cyclin-dependent kinase allowed a transcriptional repressor to down-regulate WUS. This opened the door to meiosis, while restricting the number of reproductive units per seed to one. Science, this issue p. eaaf6532; see also p. 378 Cell cycle and transcription factors regulate the pathway that converts plant somatic cells into a limited number of germ cells. INTRODUCTION Seeds of flowering plants have evolved to typically carry only a single embryo next to a nourishing tissue, the endosperm that functions analogously to the human placenta. Both structures are formed from a single female gametophyte (embryo sac) that develops from one meiotic product, the functional megaspore, in most sexually reproducing plants. As in many other organisms, including humans, all but one female meiotic product die to assure the development of only one reproductive unit per future seed. RATIONALE In contrast to humans and animals, plants do not set aside a specialized cell lineage (germline) that produces meiocytes in early embryogenesis. Instead, the germline of plants is established de novo from somatic cells in floral reproductive organs. Several genes have been identified that control the formation of ovules, which harbor the meiocytes [megaspore mother cells (MMCs)]. These include the homeodomain transcription factor WUSCHEL (WUS), a key regulator of stem cell fate in plants that is essential for the formation of the integuments from which the seed coat is derived. Moreover, WUS is also involved in the specification of MMCs. However, it is not clear how somatic cells that divide mitotically switch to a meiotic cell division program. RESULTS Following up expression data of young ovules primordia and MMCs, we reveal a regulatory cascade that controls the entry into meiosis, starting with a group of redundantly acting cyclin-dependent kinase (CDK) inhibitors of the KIP-RELATED PROTEIN (KRP) class. KRPs function by restricting CDKA;1–dependent inactivation of the Arabidopsis Retinoblastoma homolog RBR1. In rbr1 and krp triple mutants, designated meiocytes undergo several mitotic divisions, resulting in the formation of supernumerary meiocytes that give rise to multiple reproductive units per future seed. Live observation revealed that these multiple units can successfully attract a pollen tube and can be fertilized. However, subsequent seed development is blocked, resulting in semisterility of the mutant plants. One of the functions of RBR1 is the direct repression of the stem cell factor WUSCHEL (WUS), which ectopically accumulates in meiocytes of triple krp and rbr1 mutants. Depleting WUS in rbr1 mutants restored the formation of only a single meiocyte. However, ectopic expression of WUS by itself is not sufficient to induce mitotic divisions instead of meiosis, revealing that RBR1 is a central hub of meiocyte differentiation. CONCLUSION There is a delicate balance between WUS activation important for ovule primordia formation—including the development of the integuments, as well as a role in specifying the MMC itself—and its inactivation by RBR1 soon afterward to allow entry into meiosis. Different components of the Rb control pathway have been associated with germ cell fate initiation in animals; for example, mutants in the CDK inhibitor dacapo in Drosophila fail to enter meiosis. Similarly, down-regulation of Cdk2-cyclin E, a well-known regulator of Rb, is important for Caenorhabditis elegans germline development. This raises the intriguing question of whether Rb family proteins play a conserved role in germline entry in multicellular organisms. Formation of multiple MMCs in Arabidopsis. The left column shows the wild type, the middle column shows krp4 krp6 krp7 triple mutants, and the right column shows rbr1-2. A designated MMC undergoes a mitotic instead of a meiotic division, leading to the production of multiple MMCs in triple krp and rbr1 mutants (top row) instead of a single MMC, as found in the wild type. MMC fate is highlighted by means of KNU-YFP expression (second row). Multiple MMCs give rise to multiple gametophytes in ovules (third row and highlighted in different shades of blue in the bottom row). To produce seeds, flowering plants need to specify somatic cells to undergo meiosis. Here, we reveal a regulatory cascade that controls the entry into meiosis starting with a group of redundantly acting cyclin-dependent kinase (CDK) inhibitors of the KIP-RELATED PROTEIN (KRP) class. KRPs function by restricting CDKA;1–dependent inactivation of the Arabidopsis Retinoblastoma homolog RBR1. In rbr1 and krp triple mutants, designated meiocytes undergo several mitotic divisions, resulting in the formation of supernumerary meiocytes that give rise to multiple reproductive units per future seed. One function of RBR1 is the direct repression of the stem cell factor WUSCHEL (WUS), which ectopically accumulates in meiocytes of triple krp and rbr1 mutants. Depleting WUS in rbr1 mutants restored the formation of only a single meiocyte.

Control of plant cell fate transitions by transcriptional and hormonal signals

Plant meristems carry pools of continuously active stem cells, whose activity is controlled by developmental and environmental signals. After stem cell division, daughter cells that exit the stem cell domain acquire transit amplifying cell identity before they are incorporated into organs and differentiate. In this study, we used an integrated approach to elucidate the role of HECATE (HEC) genes in regulating developmental trajectories of shoot stem cells in Arabidopsis thaliana. Our work reveals that HEC function stabilizes cell fate in distinct zones of the shoot meristem thereby controlling the spatio-temporal dynamics of stem cell differentiation. Importantly, this activity is concomitant with the local modulation of cellular responses to cytokinin and auxin, two key phytohormones regulating cell behaviour. Mechanistically, we show that HEC factors transcriptionally control and physically interact with MONOPTEROS (MP), a key regulator of auxin signalling, and modulate the autocatalytic stabilization of auxin signalling output.

Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis

Shoot regeneration can be achieved in vitro through a two‐step process involving the acquisition of pluripotency on callus‐induction media (CIM) and the formation of shoots on shoot‐induction media. Although the induction of root‐meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in de novo shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for de novo shoot regeneration. In developing callus, it catalyzes histone acetylation at several root‐meristem gene loci including WOX5, WOX14, SCR, PLT1, and PLT2, providing an epigenetic platform for their transcriptional activation. In turn, we demonstrate that the transcription factors encoded by these loci act as key potency factors conferring regeneration potential to callus and establishing competence for de novo shoot regeneration. Thus, our study uncovers key epigenetic and potency factors regulating plant‐cell pluripotency. These factors might be useful in reprogramming lineage‐specified plant cells to pluripotency.

A Comprehensive Toolkit for Inducible, Cell Type-Specific Gene Expression in Arabidopsis

A set of transgenic lines enables spatiotemporal control of gene expression in Arabidopsis. Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis (Arabidopsis thaliana) based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic pOp promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. When we combined these transgenic lines with effectors under the control of the pOp promoter, we observed tight temporal and spatial control of gene expression. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows for rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible method is suitable for overcoming the limitations of ubiquitous genetic approaches, the outputs of which often are difficult to interpret due to the widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.