miR394 enhances WUSCHEL-induced somatic embryogenesis in Arabidopsis thaliana.

Many plant species can give rise to embryos from somatic cells after a simple hormone treatment, illustrating the remarkable developmental plasticity of differentiated plant cells. However, many species are recalcitrant to somatic embryo formation for unknown reasons, which poses a significant challenge to agriculture, where somatic embryogenesis is an important tool to propagate desired genotypes. The micro-RNA394 (miR394) promotes shoot meristem maintenance in Arabidopsis thaliana, but the underlying mechanisms have remained elusive. We analyzed whether miR394 affects indirect somatic embryogenesis and determined the transcriptome of embryogenic callus upon miR394-enhanced somatic embryogenesis. We show that ectopic miR394 expression enhances somatic embryogenesis in the recalcitrant Ler accession when co-expressed with the transcription factor WUSCHEL (WUS) and that miR394 acts in this process through silencing the target LEAF CURLING RESPONSIVENESS (LCR). Furthermore, we show that higher endogenous miR394 levels are required for the elevated embryogenic potential of the Columbia accession compared with Ler, providing a mechanistic explanation for this natural variation. Our transcriptional analysis provides a framework for miR394 function in regulating pluripotency by expanding WUS-mediated direct transcriptional repression.

B1-type cyclins control microtubule organization during cell division in Arabidopsis.

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.

The role of AUX1 during lateral root development in the domestication of the model C4 grass Setaria italica.

C4 photosynthesis increases the efficiency of carbon fixation by spatially separating high concentrations of molecular oxygen from Rubisco. The specialized leaf anatomy required for this separation evolved independently many times. The morphology of C4 root systems is also distinctive and adapted to support high rates of photosynthesis; however, little is known about the molecular mechanisms that have driven the evolution of C4 root system architecture. Using a mutant screen in the C4 model plant Setaria italica, we identify Siaux1-1 and Siaux1-2 as root system architecture mutants. Unlike in S. viridis, AUX1 promotes lateral root development in S. italica. A cell by cell analysis of the Siaux1-1 root apical meristem revealed changes in the distribution of cell volumes in all cell layers and a dependence of the frequency of protophloem and protoxylem strands on SiAUX1. We explore the molecular basis of the role of SiAUX1 in seedling development using an RNAseq analysis of wild-type and Siaux1-1 plants and present novel targets for SiAUX1-dependent gene regulation. Using a selection sweep and haplotype analysis of SiAUX1, we show that Hap-2412TT in the promoter region of SiAUX1 is an allele which is associated with lateral root number and has been strongly selected for during Setaria domestication.

Regulatory function of homeodomain-leucine zipper (HD-ZIP) family proteins during embryogenesis.

Homeodomain-leucine zipper proteins (HD-ZIPs) form a plant-specific family of transcription factors functioning as homo- or heterodimers. Certain members of all four classes of this family are involved in embryogenesis, the focus of this review. They support auxin biosynthesis, transport and response, which are in turn essential for the apical-basal patterning of the embryo, radicle formation and outgrowth of the cotyledons. They transcriptionally regulate meristem regulators to maintain the shoot apical meristem once it is initiated. Some members are specific to the protoderm, the outermost layer of the embryo, and play a role in shoot apical meristem function. Within classes, homeodomain-leucine zippers tend to act redundantly during embryo development, and there are many examples of regulation within and between classes of homeodomain-leucine zippers. This indicates a complex network of regulation that awaits future experiments to uncover.

The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis.

Upon DNA damage, cyclin-dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology-dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant-specific B1-type CDKs (CDKB1s) and the class of B1-type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1-CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell-cycle activity is blocked after DNA damage, CDKB1-CYCB1 complexes are specifically activated to mediate HR.

ZLL/AGO10 maintains shoot meristem stem cells during Arabidopsis embryogenesis by down-regulating ARF2-mediated auxin response.

BACKGROUND: The shoot meristem gives rise to new organs throughout a plant's life by the activity of pluripotent stem cells in the meristem center. Organ initiation at the periphery of the shoot meristem is triggered by the accumulation of the phytohormone auxin at the initiation site. Loss-of-function mutants of the ZWILLE/ARGONAUTE10/PINHEAD (ZLL/AGO10/PNH) gene terminate shoot meristem stem cells late in embryogenesis and can form a leaf or a leaf-like structure instead, indicating that AGO10 activity is required to maintain shoot meristem stem cells undifferentiated. RESULTS: Here, we addressed whether stem cell maintenance by AGO10 involves regulation of auxin. We found that in zll-1 mutants, auxin accumulation and expression of the response reporter DR5:GFP are elevated, and transcription of the Auxin Response Factor 2 (ARF2) gene is upregulated. Downregulation of ARF2 significantly restores stem cells in zll-1 mutants, whereas increased expression of ARF2 enhances differentiation of stem cells in zll-1 mutants. We further found that upregulation of the AGO10 effector gene REVOLUTA restores ARF2 expression and stem cell maintenance in zll-1 embryos. CONCLUSIONS: Our results indicate that maintenance of shoot meristem stem cells by AGO10 involves negative regulation of auxin signaling and, via REV-mediated downregulation of ARF2 expression, auxin response.

Accession-specific modifiers act with ZWILLE/ARGONAUTE10 to maintain shoot meristem stem cells during embryogenesis in Arabidopsis.

BACKGROUND: Stem cells located in the centre of the shoot apical meristem are required for the repetitive formation of new organs such as leaves, branches and flowers. In Arabidopsis thaliana, the ZWILLE/PINHEAD/AGO10 (ZLL) gene encodes a member of the ARGONAUTE (AGO) protein family and is required to maintain shoot meristem stem cells during embryogenesis. In the Landsberg erecta (Ler) acession, ZLL is essential for stem cell maintenance, whereas in the Columbia (Col) accession its requirement appears masked by genetic modifiers. The genetic basis for this variation has remained elusive. RESULTS: To understand the impact of natural variation on shoot stem cell maintenance, we analysed 28 wild-type Arabidopsis accessions from around the world and show that ZLL function is essential for stem cell maintenance in accessions mainly originating from Germany, but is dispensable for accessions from other regions. Quantitative Trait Loci (QTL) mapping using Ler/Col recombinant inbred lines indicated that at least five genomic regions, referred to as FLETSCHE (FHE) 1-5, modify ZLL function in stem cell maintenance. Characterisation of Col zll near isogenic lines confirmed that the major QTL, FHE2, is preferentially maintained as a Ler allele in seedlings lacking stem cells, suggesting that this region harbours an important modifier of ZLL function. Comparison of torpedo-stage embryo expression profiles to QTL map data revealed candidate FHE genes, including the Arabidopsis Cyclophilin-40 homologue SQUINT (SQN), and functional studies revealed a previously uncharacterised role for SQN in stem cell regulation. CONCLUSIONS: Multiple genetic modifiers from different Arabidopsis accessions influence the role of ZLL in embryonic stem cell maintenance. Of the five FHE loci modifying stem cell maintenance in Ler-0 and Col-0, FHE2 was the most prominent and was tightly linked to the SQN gene, which encodes a cofactor that supports AGO1 activity. SQN shows variable embryonic expression levels between accessions and altered ZLL-dependency in transgenic assays, confirming a key role in stem cell maintenance. Reduced SQN expression levels in Col-0 correlate with transposon insertions adjoining the transcriptional start site, which may contribute to stem cell maintenance in other ZLL-independent accessions.

BAC-recombineering for studying plant gene regulation: developmental control and cellular localization of SnRK1 kinase subunits.

Recombineering, permitting precise modification of genes within bacterial artificial chromosomes (BACs) through homologous recombination mediated by lambda phage-encoded Red proteins, is a widely used powerful tool in mouse, Caenorhabditis and Drosophila genetics. As Agrobacterium-mediated transfer of large DNA inserts from binary BACs and TACs into plants occurs at low frequency, recombineering is so far seldom exploited in the analysis of plant gene functions. We have constructed binary plant transformation vectors, which are suitable for gap-repair cloning of genes from BACs using recombineering methods previously developed for other organisms. Here we show that recombineering facilitates PCR-based generation of precise translational fusions between coding sequences of fluorescent reporter and plant proteins using galK-based exchange recombination. The modified target genes alone or as part of a larger gene cluster can be transferred by high-frequency gap-repair into plant transformation vectors, stably maintained in Agrobacterium and transformed without alteration into plants. Versatile application of plant BAC-recombineering is illustrated by the analysis of developmental regulation and cellular localization of interacting AKIN10 catalytic and SNF4 activating subunits of Arabidopsis Snf1-related (SnRK1) protein kinase using in vivo imaging. To validate full functionality and in vivo interaction of tagged SnRK1 subunits, it is demonstrated that immunoprecipitated SNF4-YFP is bound to a kinase that phosphorylates SnRK1 candidate substrates, and that the GFP- and YFP-tagged kinase subunits co-immunoprecipitate with endogenous wild type AKIN10 and SNF4.

Cullin 4-ring finger-ligase plays a key role in the control of endoreplication cycles in Arabidopsis trichomes.

One of the predominant cell-cycle programs found in mature tissues is endoreplication, also known as endoreduplication, that leads to cellular polyploidy. A key question for the understanding of endoreplication cycles is how oscillating levels of cyclin-dependent kinase activity are generated that control repeated rounds of DNA replication. The APC/C performs a pivotal function in the mitotic cell cycle by promoting anaphase and paving the road for a new round of DNA replication. However, using marker lines and plants in which APC/C components are knocked down, we show here that outgrowing and endoreplicating Arabidopsis leaf hairs display no or very little APC/C activity. Instead we find that RBX1-containing Cullin-RING E3 ubiquitin-Ligases (CRLs) are of central importance for the progression through endoreplication cycles; in particular, we have identified CULLIN4 as a major regulator of endoreplication in Arabidopsis trichomes. We have incorporated our findings into a bio-mathematical simulation presenting a robust two-step model of endoreplication control with one type of cyclin-dependent kinase inhibitor function for entry and a CRL-dependent oscillation of cyclin-dependent kinase activity via degradation of a second type of CDK inhibitor during endoreplication cycles.

Endoreplication controls cell fate maintenance.

Cell-fate specification is typically thought to precede and determine cell-cycle regulation during differentiation. Here we show that endoreplication, also known as endoreduplication, a specialized cell-cycle variant often associated with cell differentiation but also frequently occurring in malignant cells, plays a role in maintaining cell fate. For our study we have used Arabidopsis trichomes as a model system and have manipulated endoreplication levels via mutants of cell-cycle regulators and overexpression of cell-cycle inhibitors under a trichome-specific promoter. Strikingly, a reduction of endoreplication resulted in reduced trichome numbers and caused trichomes to lose their identity. Live observations of young Arabidopsis leaves revealed that dedifferentiating trichomes re-entered mitosis and were re-integrated into the epidermal pavement-cell layer, acquiring the typical characteristics of the surrounding epidermal cells. Conversely, when we promoted endoreplication in glabrous patterning mutants, trichome fate could be restored, demonstrating that endoreplication is an important determinant of cell identity. Our data lead to a new model of cell-fate control and tissue integrity during development by revealing a cell-fate quality control system at the tissue level.