Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization.

In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

Developmental Analysis of Mimulus Seed Transcriptomes Reveals Functional Gene Expression Clusters and Four Imprinted, Endosperm-Expressed Genes.

The double fertilization of the female gametophyte initiates embryogenesis and endosperm development in seeds via the activation of genes involved in cell differentiation, organ patterning, and growth. A subset of genes expressed in endosperm exhibit imprinted expression, and the correct balance of gene expression between parental alleles is critical for proper endosperm and seed development. We use a transcriptional time series analysis to identify genes that are associated with key shifts in seed development, including genes associated with secondary cell wall synthesis, mitotic cell cycle, chromatin organization, auxin synthesis, fatty acid metabolism, and seed maturation. We relate these genes to morphological changes in Mimulus seeds. We also identify four endosperm-expressed transcripts that display imprinted (paternal) expression bias. The imprinted status of these four genes is conserved in other flowering plants, suggesting that they are functionally important in endosperm development. Our study explores gene regulatory dynamics in a species with ab initio cellular endosperm development, broadening the taxonomic focus of the literature on gene expression in seeds. Moreover, it is the first to validate genes with imprinted endosperm expression in Mimulus guttatus, and will inform future studies on the genetic causes of seed failure in this model system.

Characterizing the involvement of FaMADS9 in the regulation of strawberry fruit receptacle development.

FaMADS9 is the strawberry (Fragaria x ananassa) gene that exhibits the highest homology to the tomato (Solanum lycopersicum) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid-1,4-lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10, DIV, ZFN1, ZFN2, GT2, and GT5, or associated with the action of hormones, such as abscisic acid, SHP, ASR, GTE7 and SnRK2.7. The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1, DIV, ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9-specific CArG box was identified in the promoters of these genes.

DOF2.1 Controls Cytokinin-Dependent Vascular Cell Proliferation Downstream of TMO5/LHW.

To create a three-dimensional structure, plants rely on oriented cell divisions and cell elongation. Oriented cell divisions are specifically important in procambium cells of the root to establish the different vascular cell types [1, 2]. These divisions are in part controlled by the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex [3-7]. Loss-of-function of tmo5 or lhw clade members results in strongly reduced vascular cell file numbers, whereas ectopic expression of both TMO5 and LHW can ubiquitously induce periclinal and radial cell divisions in all cell types of the root meristem. TMO5 and LHW interact only in young xylem cells, where they promote expression of two direct target genes involved in the final step of cytokinin (CK) biosynthesis, LONELY GUY3 (LOG3) and LOG4 [8, 9] Therefore, CK was hypothesized to act as a mobile signal from the xylem to trigger divisions in the neighboring procambium cells [3, 6]. To unravel how TMO5/LHW-dependent cytokinin regulates cell proliferation, we analyzed the transcriptional responses upon simultaneous induction of both transcription factors. Using inferred network analysis, we identified AT2G28510/DOF2.1 as a cytokinin-dependent downstream target gene. We further showed that DOF2.1 controls specific procambium cell divisions without inducing other cytokinin-dependent effects such as the inhibition of vascular differentiation. In summary, our results suggest that DOF2.1 and its closest homologs control vascular cell proliferation, thus leading to radial expansion of the root.

Predicting gene regulatory networks by combining spatial and temporal gene expression data in Arabidopsis root stem cells.

Identifying the transcription factors (TFs) and associated networks involved in stem cell regulation is essential for understanding the initiation and growth of plant tissues and organs. Although many TFs have been shown to have a role in the Arabidopsis root stem cells, a comprehensive view of the transcriptional signature of the stem cells is lacking. In this work, we used spatial and temporal transcriptomic data to predict interactions among the genes involved in stem cell regulation. To accomplish this, we transcriptionally profiled several stem cell populations and developed a gene regulatory network inference algorithm that combines clustering with dynamic Bayesian network inference. We leveraged the topology of our networks to infer potential major regulators. Specifically, through mathematical modeling and experimental validation, we identified PERIANTHIA (PAN) as an important molecular regulator of quiescent center function. The results presented in this work show that our combination of molecular biology, computational biology, and mathematical modeling is an efficient approach to identify candidate factors that function in the stem cells.

Inferring Gene Regulatory Networks in the Arabidopsis Root Using a Dynamic Bayesian Network Approach.

Gene regulatory network (GRN) models have been shown to predict and represent interactions among sets of genes. Here, we first show the basic steps to implement a simple but computationally efficient algorithm to infer GRNs based on dynamic Bayesian networks (DBNs), and we then explain how to approximate DBN-based GRN models with continuous models. In addition, we show a MATLAB implementation of the key steps of this method, which we use to infer an Arabidopsis root GRN.

Multi-sample Arabidopsis Growth and Imaging Chamber (MAGIC) for long term imaging in the ZEISS Lightsheet Z.1.

Time-course imaging experiments on live organisms are critical for understanding the dynamics of growth and development. Light-sheet microscopy has advanced the field of long-term imaging of live specimens by significantly reducing photo-toxicity and allowing fast acquisition of three-dimensional data over time. However, current light-sheet technology does not allow the imaging of multiple plant specimens in parallel. To achieve higher throughput, we have developed a Multi-sample Arabidopsis Growth and Imaging Chamber (MAGIC) that provides near-physiological imaging conditions and allows high-throughput time-course imaging experiments in the ZEISS Lightsheet Z.1. Here, we illustrate MAGIC's imaging capabilities by following cell divisions, as an indicator of plant growth and development, over prolonged time periods. To automatically quantify the number of cell divisions in long-term experiments, we present a FIJI-based image processing pipeline. We demonstrate that plants imaged with our chamber undergo cell divisions for >16 times longer than those with the glass capillary system supplied by the ZEISS Z1.