Direct conversion of root primordium into shoot meristem relies on timing of stem cell niche development.

To understand how the identity of an organ can be switched, we studied the transformation of lateral root primordia (LRP) into shoot meristems in Arabidopsis root segments. In this system, the cytokinin-induced conversion does not involve the formation of callus-like structures. Detailed analysis showed that the conversion sequence starts with a mitotic pause and is concomitant with the differential expression of regulators of root and shoot development. The conversion requires the presence of apical stem cells, and only LRP at stages VI or VII can be switched. It is engaged as soon as cell divisions resume because their position and orientation differ in the converting organ compared with the undisturbed emerging LRP. By alternating auxin and cytokinin treatments, we showed that the root and shoot organogenetic programs are remarkably plastic, as the status of the same plant stem cell niche can be reversed repeatedly within a set developmental window. Thus, the networks at play in the meristem of a root can morph in the span of a couple of cell division cycles into those of a shoot, and back, through transdifferentiation.

Mitochondrial Defects Confer Tolerance against Cellulose Deficiency.

Because the plant cell wall provides the first line of defense against biotic and abiotic assaults, its functional integrity needs to be maintained under stress conditions. Through a phenotype-based compound screening approach, we identified a novel cellulose synthase inhibitor, designated C17. C17 administration depletes cellulose synthase complexes from the plasma membrane in Arabidopsis thaliana, resulting in anisotropic cell elongation and a weak cell wall. Surprisingly, in addition to mutations in CELLULOSE SYNTHASE1 (CESA1) and CESA3, a forward genetic screen identified two independent defective genes encoding pentatricopeptide repeat (PPR)-like proteins (CELL WALL MAINTAINER1 [CWM1] and CWM2) as conferring tolerance to C17. Functional analysis revealed that mutations in these PPR proteins resulted in defective cytochrome c maturation and activation of mitochondrial retrograde signaling, as evidenced by the induction of an alternative oxidase. These mitochondrial perturbations increased tolerance to cell wall damage induced by cellulose deficiency. Likewise, administration of antimycin A, an inhibitor of mitochondrial complex III, resulted in tolerance toward C17. The C17 tolerance of cwm2 was partially lost upon depletion of the mitochondrial retrograde regulator ANAC017, demonstrating that ANAC017 links mitochondrial dysfunction with the cell wall. In view of mitochondria being a major target of a variety of stresses, our data indicate that plant cells might modulate mitochondrial activity to maintain a functional cell wall when subjected to stresses.

The SBT6.1 subtilase processes the GOLVEN1 peptide controlling cell elongation.

The GOLVEN (GLV) gene family encode small secreted peptides involved in important plant developmental programs. Little is known about the factors required for the production of the mature bioactive GLV peptides. Through a genetic suppressor screen in Arabidopsis thaliana, two related subtilase genes, AtSBT6.1 and AtSBT6.2, were identified that are necessary for GLV1 activity. Root and hypocotyl GLV1 overexpression phenotypes were suppressed by mutations in either of the subtilase genes. Synthetic GLV-derived peptides were cleaved in vitro by the affinity-purified SBT6.1 catalytic enzyme, confirming that the GLV1 precursor is a direct subtilase substrate, and the elimination of the in vitro subtilase recognition sites through alanine substitution suppressed the GLV1 gain-of-function phenotype in vivo Furthermore, the protease inhibitor Serpin1 bound to SBT6.1 and inhibited the cleavage of GLV1 precursors by the protease. GLV1 and its homolog GLV2 were expressed in the outer cell layers of the hypocotyl, preferentially in regions of rapid cell elongation. In agreement with the SBT6 role in GLV precursor processing, both null mutants for sbt6.1 and sbt6.2 and the Serpin1 overexpression plants had shorter hypocotyls. The biosynthesis of the GLV signaling peptides required subtilase activity and might be regulated by specific protease inhibitors. The data fit with a model in which the GLV1 signaling pathway participates in the regulation of hypocotyl cell elongation, is controlled by SBT6 subtilases, and is modulated locally by the Serpin1 protease inhibitor.

Predicting gene function from uncontrolled expression variation among individual wild-type Arabidopsis plants.

Gene expression profiling studies are usually performed on pooled samples grown under tightly controlled experimental conditions to suppress variability among individuals and increase experimental reproducibility. In addition, to mask unwanted residual effects, the samples are often subjected to relatively harsh treatments that are unrealistic in a natural context. Here, we show that expression variations among individual wild-type Arabidopsis thaliana plants grown under the same macroscopic growth conditions contain as much information on the underlying gene network structure as expression profiles of pooled plant samples under controlled experimental perturbations. We advocate the use of subtle uncontrolled variations in gene expression between individuals to uncover functional links between genes and unravel regulatory influences. As a case study, we use this approach to identify ILL6 as a new regulatory component of the jasmonate response pathway.

The GLV6/RGF8/CLEL2 peptide regulates early pericycle divisions during lateral root initiation.

Small peptides of the Arabidopsis GLV/RGF/CLEL family are involved in different developmental programmes, including meristem maintenance and gravitropic responses. In addition, our previous report suggested that they also participate in the formation of lateral roots. Specifically, GLV6 is transcribed during the first stages of primordium development and GLV6 overexpression results in a strong reduction of emerged lateral roots. To investigate the cause of this phenotype we analysed primordium development in gain-of-function (gof) mutants and found that GLV6 induces supernumerary pericycle divisions, hindering the formation of a dome-shaped primordium, a prerequisite for successful emergence. The GLV6 phenotype could be reproduced by ectopic expression of the gene only in xylem-pole pericycle cells. Furthermore, GLV6 seems to function at the very beginning of lateral root initiation because GLV6 excess-either gene overexpression or peptide treatment-disrupts the first asymmetric cell divisions required for proper primordium formation. Our results suggest that GLV6 acts during lateral root initiation controlling the patterning of the first pericycle divisions.

Expanding the repertoire of secretory peptides controlling root development with comparative genome analysis and functional assays.

Plant genomes encode numerous small secretory peptides (SSPs) whose functions have yet to be explored. Based on structural features that characterize SSP families known to take part in postembryonic development, this comparative genome analysis resulted in the identification of genes coding for oligopeptides potentially involved in cell-to-cell communication. Because genome annotation based on short sequence homology is difficult, the criteria for the de novo identification and aggregation of conserved SSP sequences were first benchmarked across five reference plant species. The resulting gene families were then extended to 32 genome sequences, including major crops. The global phylogenetic pattern common to the functionally characterized SSP families suggests that their apparition and expansion coincide with that of the land plants. The SSP families can be searched online for members, sequences and consensus (http://bioinformatics.psb.ugent.be/webtools/PlantSSP/). Looking for putative regulators of root development, Arabidopsis thaliana SSP genes were further selected through transcriptome meta-analysis based on their expression at specific stages and in specific cell types in the course of the lateral root formation. As an additional indication that formerly uncharacterized SSPs may control development, this study showed that root growth and branching were altered by the application of synthetic peptides matching conserved SSP motifs, sometimes in very specific ways. The strategy used in the study, combining comparative genomics, transcriptome meta-analysis and peptide functional assays in planta, pinpoints factors potentially involved in non-cell-autonomous regulatory mechanisms. A similar approach can be implemented in different species for the study of a wide range of developmental programmes.

Transcriptional and functional classification of the GOLVEN/ROOT GROWTH FACTOR/CLE-like signaling peptides reveals their role in lateral root and hair formation.

The GOLVEN (GLV)/ROOT GROWTH FACTORS/CLE-Like small signaling peptide family is encoded by 11 genes in Arabidopsis (Arabidopsis thaliana). Some of them have already been shown to control root meristem maintenance, auxin fluxes, and gravitropic responses. As a basis for the detailed analysis of their function, we determined the expression domains for each of the 11 GLV genes with promoter-reporter lines. Although they are collectively active in all examined plant parts, GLV genes have highly specific transcription patterns, generally restricted to very few cells or cell types in the root and shoot and in vegetative and reproductive tissues. GLV functions were further investigated with the comparative analysis of root phenotypes induced by gain- and loss-of-function mutants or in treatments with GLV-derived synthetic peptides. We identified functional classes that relate to the gene expression domains in the primary root and suggest that different GLV signals trigger distinct downstream pathways. Interestingly, GLV genes transcribed at the early stages of lateral root development strongly inhibited root branching when overexpressed. Furthermore, transcription patterns together with mutant phenotypes pointed to the involvement of GLV4 and GLV8 in root hair formation. Overall, our data suggest that nine GLV genes form three subgroups according to their expression and function within the root and offer a comprehensive framework to study the role of the GLV signaling peptides in plant development.

Feeding cells induced by phytoparasitic nematodes require γ-tubulin ring complex for microtubule reorganization.

Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound γ-tubulin ring complexes. Here we investigate the requirement of γ-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that γ-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis γ-tubulin (TUBG1 and TUBG2) and two γ-tubulin complex proteins genes (GCP3 and GCP4) are upregulated in galls. Electron microscopy demonstrates association of GCP3 and γ-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both γ-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the γ-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts.

Systems-based analysis of Arabidopsis leaf growth reveals adaptation to water deficit.

Leaves have a central role in plant energy capture and carbon conversion and therefore must continuously adapt their development to prevailing environmental conditions. To reveal the dynamic systems behaviour of leaf development, we profiled Arabidopsis leaf number six in depth at four different growth stages, at both the end-of-day and end-of-night, in plants growing in two controlled experimental conditions: short-day conditions with optimal soil water content and constant reduced soil water conditions. We found that the lower soil water potential led to reduced, but prolonged, growth and an adaptation at the molecular level without a drought stress response. Clustering of the protein and transcript data using a decision tree revealed different patterns in abundance changes across the growth stages and between end-of-day and end-of-night that are linked to specific biological functions. Correlations between protein and transcript levels depend on the time-of-day and also on protein localisation and function. Surprisingly, only very few of >1700 quantified proteins showed diurnal abundance fluctuations, despite strong fluctuations at the transcript level.

GOLVEN peptides as important regulatory signalling molecules of plant development.

The contribution of signalling peptides to plant development is increasingly evident as more new peptide families become identified. The recently discovered GLV/RGF/CLEL secreted peptide family comprises 11 members in Arabidopsis and has been shown by independent research groups to be involved in different plant developmental programmes such as root meristem maintenance, root hair development, and root and hypocotyl gravitropism. This short review summarizes our current knowledge on GLV/RGF/CLEL peptides and highlights future challenges to decipher their function.

Functional modules in the Arabidopsis core cell cycle binary protein-protein interaction network.

As in other eukaryotes, cell division in plants is highly conserved and regulated by cyclin-dependent kinases (CDKs) that are themselves predominantly regulated at the posttranscriptional level by their association with proteins such as cyclins. Although over the last years the knowledge of the plant cell cycle has considerably increased, little is known on the assembly and regulation of the different CDK complexes. To map protein-protein interactions between core cell cycle proteins of Arabidopsis thaliana, a binary protein-protein interactome network was generated using two complementary high-throughput interaction assays, yeast two-hybrid and bimolecular fluorescence complementation. Pairwise interactions among 58 core cell cycle proteins were tested, resulting in 357 interactions, of which 293 have not been reported before. Integration of the binary interaction results with cell cycle phase-dependent expression information and localization data allowed the construction of a dynamic interaction network. The obtained interaction map constitutes a framework for further in-depth analysis of the cell cycle machinery.

Fast-track transformation and genome editing in Brachypodium distachyon.

BACKGROUND: Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd) transgenics span at least 14 weeks from the start of tissue culture to the recovery of regenerated plantlets. RESULTS: We have previously shown that embryogenic somatic tissues grow in the scutellum of immature zygotic Bd embryos within 3 days of in vitro induction with exogenous auxin and that the development of secondary embryos can be initiated immediately thereafter. Here, we further demonstrate that such pluripotent reactive tissues can be genetically transformed with Agrobacterium tumefaciens right after the onset of somatic embryogenesis. In brief, immature zygotic embryos are induced for callogenesis for one week, co-cultured with Agrobacterium for three days, then incubated on callogenesis selective medium for three weeks, and finally transferred on selective regeneration medium for up to three weeks to obtain plantlets ready for rooting. This 7-to-8-week procedure requires only three subcultures. Its validation includes the molecular and phenotype characterization of Bd lines carrying transgenic cassettes and novel CRISPR/Cas9-generated mutations in two independent loci coding for nitrate reductase enzymes (BdNR1 and BdNR2). CONCLUSIONS: With a short callogenesis stage and streamlined in vitro regeneration following co-cultivation with Agrobacterium, transgenic and edited T0 Bd plantlets can be produced in about 8 weeks, a gain of one to two months compared to previously published methods, with no reduction in transformation efficiency and at lower costs.

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis.

Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

Variation in HMA4 gene copy number and expression among Noccaea caerulescens populations presenting different levels of Cd tolerance and accumulation.

There is huge variability among populations of the hyperaccumulator Noccaea caerulescens (formerly Thlaspi caerulescens) in their capacity to tolerate and accumulate cadmium. To gain new insights into the mechanisms underlying this variability, we estimated cadmium fluxes and further characterized the N. caerulescens heavy metal ATPase 4 (NcHMA4) gene in three populations (two calamine, Saint-Félix-de-Pallières, France and Prayon, Belgium; one serpentine, Puente Basadre, Spain) presenting contrasting levels of tolerance and accumulation. Cadmium uptake and translocation varied among populations in the same way as accumulation; the population with the highest cadmium concentration in shoots (Saint Félix-de-Pallières) presented the highest capacity for uptake and translocation. We demonstrated that the four NcHMA4 copies identified in a previous study are not fixed at the species level, and that the copy truncated in the C-terminal part encodes a functional protein. NcHMA4 expression and gene copy number was lower in the serpentine population, which was the least efficient in cadmium translocation compared to the calamine populations. NcHMA4 expression was associated with the vascular tissue in all organs, with a maximum at the crown. Overall, our results indicate that differences in cadmium translocation ability of the studied populations appear to be controlled, at least partially, by NcHMA4, while the overexpression of NcHMA4 in the two calamine populations may result from convergent evolution.

One-Week Scutellar Somatic Embryogenesis in the Monocot Brachypodium distachyon.

Plant somatic embryogenesis (SE) is a natural process of vegetative propagation. It can be induced in tissue cultures to investigate developmental transitions, to create transgenic or edited lines, or to multiply valuable crops. We studied the induction of SE in the scutellum of monocots with Brachypodium distachyon as a model system. Towards the in-depth analysis of SE initiation, we determined the earliest stages at which somatic scutellar cells acquired an embryogenic fate, then switched to a morphogenetic mode in a regeneration sequence involving treatments with exogenous hormones: first an auxin (2,4-D) then a cytokinin (kinetin). Our observations indicated that secondary somatic embryos could already develop in the proliferative calli derived from immature zygotic embryo tissues within one week from the start of in vitro culture. Cell states and tissue identity were deduced from detailed histological examination, and in situ hybridization was performed to map the expression of key developmental genes. The fast SE induction method we describe here facilitates the mechanistic study of the processes involved and may significantly shorten the production of transgenic or gene-edited plants.

Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana.

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)-cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK-cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.

Systematic localization of the Arabidopsis core cell cycle proteins reveals novel cell division complexes.

Cell division depends on the correct localization of the cyclin-dependent kinases that are regulated by phosphorylation, cyclin proteolysis, and protein-protein interactions. Although immunological assays can define cell cycle protein abundance and localization, they are not suitable for detecting the dynamic rearrangements of molecular components during cell division. Here, we applied an in vivo approach to trace the subcellular localization of 60 Arabidopsis (Arabidopsis thaliana) core cell cycle proteins fused to green fluorescent proteins during cell division in tobacco (Nicotiana tabacum) and Arabidopsis. Several cell cycle proteins showed a dynamic association with mitotic structures, such as condensed chromosomes and the preprophase band in both species, suggesting a strong conservation of targeting mechanisms. Furthermore, colocalized proteins were shown to bind in vivo, strengthening their localization-function connection. Thus, we identified unknown spatiotemporal territories where functional cell cycle protein interactions are most likely to occur.

AGRONOMICS1: a new resource for Arabidopsis transcriptome profiling.

Transcriptome profiling has become a routine tool in biology. For Arabidopsis (Arabidopsis thaliana), the Affymetrix ATH1 expression array is most commonly used, but it lacks about one-third of all annotated genes present in the reference strain. An alternative are tiling arrays, but previous designs have not allowed the simultaneous analysis of both strands on a single array. We introduce AGRONOMICS1, a new Affymetrix Arabidopsis microarray that contains the complete paths of both genome strands, with on average one 25mer probe per 35-bp genome sequence window. In addition, the new AGRONOMICS1 array contains all perfect match probes from the original ATH1 array, allowing for seamless integration of the very large existing ATH1 knowledge base. The AGRONOMICS1 array can be used for diverse functional genomics applications such as reliable expression profiling of more than 30,000 genes, detection of alternative splicing, and chromatin immunoprecipitation coupled to microarrays (ChIP-chip). Here, we describe the design of the array and compare its performance with that of the ATH1 array. We find results from both microarrays to be of similar quality, but AGRONOMICS1 arrays yield robust expression information for many more genes, as expected. Analysis of the ATH1 probes on AGRONOMICS1 arrays produces results that closely mirror those of ATH1 arrays. Finally, the AGRONOMICS1 array is shown to be useful for ChIP-chip experiments. We show that heterochromatic H3K9me2 is strongly confined to the gene body of target genes in euchromatic chromosome regions, suggesting that spreading of heterochromatin is limited outside of pericentromeric regions.

Probing the reproducibility of leaf growth and molecular phenotypes: a comparison of three Arabidopsis accessions cultivated in ten laboratories.

A major goal of the life sciences is to understand how molecular processes control phenotypes. Because understanding biological systems relies on the work of multiple laboratories, biologists implicitly assume that organisms with the same genotype will display similar phenotypes when grown in comparable conditions. We investigated to what extent this holds true for leaf growth variables and metabolite and transcriptome profiles of three Arabidopsis (Arabidopsis thaliana) genotypes grown in 10 laboratories using a standardized and detailed protocol. A core group of four laboratories generated similar leaf growth phenotypes, demonstrating that standardization is possible. But some laboratories presented significant differences in some leaf growth variables, sometimes changing the genotype ranking. Metabolite profiles derived from the same leaf displayed a strong genotype x environment (laboratory) component. Genotypes could be separated on the basis of their metabolic signature, but only when the analysis was limited to samples derived from one laboratory. Transcriptome data revealed considerable plant-to-plant variation, but the standardization ensured that interlaboratory variation was not considerably larger than intralaboratory variation. The different impacts of the standardization on phenotypes and molecular profiles could result from differences of temporal scale between processes involved at these organizational levels. Our findings underscore the challenge of describing, monitoring, and precisely controlling environmental conditions but also demonstrate that dedicated efforts can result in reproducible data across multiple laboratories. Finally, our comparative analysis revealed that small variations in growing conditions (light quality principally) and handling of plants can account for significant differences in phenotypes and molecular profiles obtained in independent laboratories.

Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells.

The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3-4 weeks by using Gateway cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.