Light regulates xylem cell differentiation via PIF in Arabidopsis.

The balance between cell proliferation and differentiation in the cambium defines the formation of plant vascular tissues. As cambium cells proliferate, subsets of daughter cells differentiate into xylem or phloem. TDIF-PXY/TDR signaling is central to this process. TDIF, encoded by CLE41 and CLE44, activates PXY/TDR receptors to maintain proliferative cambium. Light and water are necessary for photosynthesis; thus, vascular differentiation must occur upon light perception to facilitate the transport of water and minerals to the photosynthetic tissues. However, the molecular mechanism controlling vascular differentiation in response to light remains elusive. In this study we show that the accumulation of PIF transcription factors in the dark promotes TDIF signaling and inhibits vascular cell differentiation. On the contrary, PIF inactivation by light leads to a decay in TDIF activity, which induces vascular cell differentiation. Our study connects light to vascular differentiation and highlights the importance of this crosstalk to fine-tune water transport.

Reversion of fruit-dependent inhibition of flowering in Citrus requires sprouting of buds with epigenetically silenced CcMADS19.

In Citrus, the response to environmental floral inductive signals is inhibited by the presence of developing fruits. The mechanism involves epigenetic activation of the CcMADS19 locus (FLC orthologue), encoding a floral repressor. To understand how this epigenetic regulation is reverted to allow flowering in the following season, we have forced precocious sprouting of axillary buds in fruit-bearing shoots, and examined the competence to floral inductive signals of old and new leaves derived from them. We have found that CcMADS19 is enriched in repressive H3K27me3 marks in young, but not old leaves, revealing that axillary buds retain a silenced version of the floral repressor that is mitotically transmitted to the newly emerging leaves, which are able to induce flowering. Therefore, we propose that flowering in Citrus is necessarily preceded by vegetative sprouting, so that the competence to respond to floral inductive signals is reset in the new leaves.

Fruit-dependent epigenetic regulation of flowering in Citrus.

In many perennial plants, seasonal flowering is primarily controlled by environmental conditions, but in certain polycarpic plants, environmental signals are locally gated by the presence of developing fruits initiated in the previous season through an unknown mechanism. Polycarpy is defined as the ability of plants to undergo several rounds of reproduction during their lifetime, alternating vegetative and reproductive meristems in the same individual. To understand how fruits regulate flowering in polycarpic plants, we focused on alternate bearing in Citrus trees that had been experimentally established as fully flowering or nonflowering. We found that the presence of the fruit causes epigenetic changes correlating with the induction of the CcMADS19 floral repressor, which prevents the activation of the floral promoter CiFT2 even in the presence of the floral inductive signals. By contrast, newly emerging shoots display an opposite epigenetic scenario associated with CcMADS19 repression, thereby allowing the activation of CiFT2 the following cold season.

PIF4-induced BR synthesis is critical to diurnal and thermomorphogenic growth.

The Arabidopsis PIF4 and BES1/BZR1 transcription factors antagonize light signaling by facilitating co-activated expression of a large number of cell wall-loosening and auxin-related genes. While PIF4 directly activates expression of these targets, BES1 and BZR1 activity switch from a repressive to an activator function, depending on interaction with TOPLESS and other families of regulators including PIFs. However, the complexity of this regulation and its role in diurnal control of plant growth and brassinosteroid (BR) levels is little understood. We show by using a protein array that BES1, PIF4, and the BES1-PIF4 complex recognize different DNA elements, thus revealing a distinctive cis-regulatory code beneath BES1-repressive and PIF4 co-activation function. BES1 homodimers bind to conserved BRRE- and G-box elements in the BR biosynthetic promoters and inhibit their expression during the day, while elevated PIF4 competes for BES1 homodimer formation, resulting in de-repressed BR biosynthesis at dawn and in response to warmth. Our findings demonstrate a central role of PIF4 in BR synthesis activation, increased BR levels being essential to thermomorphogenic hypocotyl growth.

Regulation of Root Angle and Gravitropism.

Regulation of plant root angle is critical for obtaining nutrients and water and is an important trait for plant breeding. A plant's final, long-term root angle is the net result of a complex series of decisions made by a root tip in response to changes in nutrient availability, impediments, the gravity vector and other stimuli. When a root tip is displaced from the gravity vector, the short-term process of gravitropism results in rapid reorientation of the root toward the vertical. Here, we explore both short- and long-term regulation of root growth angle, using natural variation in tomato to identify shared and separate genetic features of the two responses. Mapping of expression quantitative trait loci mapping and leveraging natural variation between and within species including Arabidopsis suggest a role for PURPLE ACID PHOSPHATASE 27 and CELL DIVISION CYCLE 73 in determining root angle.

TOPLESS mediates brassinosteroid control of shoot boundaries and root meristem development in Arabidopsis thaliana.

The transcription factor BRI1-EMS-SUPRESSOR 1 (BES1) is a master regulator of brassinosteroid (BR)-regulated gene expression. BES1 together with BRASSINAZOLE-RESISTANT 1 (BZR1) drive activated or repressed expression of several genes, and have a prominent role in negative regulation of BR synthesis. Here, we report that BES1 interaction with TOPLESS (TPL), via its ERF-associated amphiphilic repression (EAR) motif, is essential for BES1-mediated control of organ boundary formation in the shoot apical meristem and the regulation of quiescent center (QC) cell division in roots. We show that TPL binds via BES1 to the promoters of the CUC3 and BRAVO targets and suppresses their expression. Ectopic expression of TPL leads to similar organ boundary defects and alterations in QC cell division rate to the bes1-d mutation, while bes1-d defects are suppressed by the dominant interfering protein encoded by tpl-1, with these effects respectively correlating with changes in CUC3 and BRAVO expression. Together, our data unveil a pivotal role of the co-repressor TPL in the shoot and root meristems, which relies on its interaction with BES1 and regulation of BES1 target gene expression.

Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation.

Spatiotemporal regulation of transcription is fine-tuned at multiple levels, including chromatin compaction. Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of Histone 3 at lysine 27 (H3K27me3), which is the hallmark of a repressive chromatin state. Multiple PRC2 complexes have been reported in Arabidopsis thaliana to control the expression of genes involved in developmental transitions and maintenance of organ identity. Here, we show that PRC2 member genes display complex spatiotemporal gene expression patterns and function in root meristem and vascular cell proliferation and specification. Furthermore, PRC2 gene expression patterns correspond with vascular and nonvascular tissue-specific H3K27me3-marked genes. This tissue-specific repression via H3K27me3 regulates the balance between cell proliferation and differentiation. Using enhanced yeast one-hybrid analysis, upstream regulators of the PRC2 member genes are identified, and genetic analysis demonstrates that transcriptional regulation of some PRC2 genes plays an important role in determining PRC2 spatiotemporal activity within a developing organ.

PRC2 represses dedifferentiation of mature somatic cells in Arabidopsis.

Plant somatic cells are generally acknowledged to retain totipotency, the potential to develop into any cell type within an organism. This astonishing plasticity may contribute to a high regenerative capacity on severe damage, but how plants control this potential during normal post-embryonic development remains largely unknown(1,2). Here we show that POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a chromatin regulator that maintains gene repression through histone modification, prevents dedifferentiation of mature somatic cells in Arabidopsis thaliana roots. Loss-of-function mutants in PRC2 subunits initially develop unicellular root hairs indistinguishable from those in wild type but fail to retain the differentiated state, ultimately resulting in the generation of an unorganized cell mass and somatic embryos from a single root hair. Strikingly, mutant root hairs complete the normal endoreduplication programme, increasing their nuclear ploidy, but subsequently reinitiate mitotic division coupled with successive DNA replication. Our data show that the WOUND INDUCED DEDIFFERENTIATION3 (WIND3) and LEAFY COTYLEDON2 (LEC2) genes are among the PRC2 targets involved in this reprogramming, as their ectopic overexpression partly phenocopies the dedifferentiation phenotype of PRC2 mutants. These findings unveil the pivotal role of PRC2-mediated gene repression in preventing unscheduled reprogramming of fully differentiated plant cells.

BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth.

Signaling by the hormones brassinosteroid (BR) and gibberellin (GA) is critical to normal plant growth and development and is required for hypocotyl elongation in response to dark and elevated temperatures. Active BR signaling is essential for GA promotion of hypocotyl growth and suppresses the dwarf phenotype of GA mutants. Cross-talk between these hormones occurs downstream from the DELLAs, as GA-induced destabilization of these GA signaling repressors is not affected by BRs. Here we show that the light-regulated PIF4 (phytochrome-interacting factor 4) factor is a phosphorylation target of the BR signaling kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2), which marks this transcriptional regulator for proteasome degradation. Expression of a mutated PIF41A protein lacking a conserved BIN2 phosphorylation consensus causes a severe elongated phenotype and strongly up-regulated expression of the gene targets. However, PIF41A is not able to suppress the dwarf phenotype of the bin2-1 mutant with constitutive activation of this kinase. PIFs were shown to be required for the constitutive BR response of bes1-D and bzr1-1D mutants, these factors acting in an interdependent manner to promote cell elongation. Here, we show that bes1-D seedlings are still repressed by the inhibitor BRZ in the light and that expression of the nonphosphorylatable PIF41A protein makes this mutant fully insensitive to brassinazole (BRZ). PIF41A is preferentially stabilized at dawn, coinciding with the diurnal time of maximal growth. These results uncover a main role of BRs in antagonizing light signaling by inhibiting BIN2-mediated destabilization of the PIF4 factor. This regulation plays a prevalent role in timing hypocotyl elongation to late night, before light activation of phytochrome B (PHYB) and accumulation of DELLAs restricts PIF4 transcriptional activity.

PIFs get BRright: PHYTOCHROME INTERACTING FACTORs as integrators of light and hormonal signals.

Light and temperature, in coordination with the endogenous clock and the hormones gibberellin (GA) and brassinosteroids (BRs), modulate plant growth and development by affecting the expression of multiple cell wall- and auxin-related genes. PHYTOCHROME INTERACTING FACTORS (PIFs) play a central role in the activation of these genes, the activity of these factors being regulated by the circadian clock and phytochrome-mediated protein destabilization. GA signaling is also integrated at the level of PIFs; the DELLA repressors are found to bind these factors and impair their DNA-binding ability. The recent finding that PIFs are co-activated by BES1 and BZR1 highlights a further role of these regulators in BR signal integration, and reveals that PIFs act in a concerted manner with the BR-related BES1/BZR1 factors to activate auxin synthesis and transport at the gene expression level, and synergistically activate several genes with a role in cell expansion. Auxins feed back into this growth regulatory module by inducing GA biosynthesis and BES1/BZR1 gene expression, in addition to directly regulating several of these growth pathway gene targets. An exciting challenge in the future will be to understand how this growth program is dynamically regulated in time and space to orchestrate differential organ expansion and to provide plants with adaptation flexibility.

Bioinformatic tools in Arabidopsis research.

Bioinformatic tools are an increasingly important resource for Arabidopsis researchers. With them, it is possible to rapidly query the large data sets covering genomes, transcriptomes, proteomes, epigenomes, and other "omes" that have been generated in the past decade. Often these tools can be used to generate quality hypotheses at the click of a mouse. In this chapter, we cover the use of bioinformatic tools for examining gene expression and coexpression patterns, performing promoter analyses, looking for functional classification enrichment for sets of genes, and investigating protein-protein interactions. We also introduce bioinformatic tools that allow integration of data from several sources for improved hypothesis generation.

Identification of novel loci regulating interspecific variation in root morphology and cellular development in tomato.

While the Arabidopsis (Arabidopsis thaliana) root has been elegantly characterized with respect to specification of cell identity, its development is missing a number of cellular features present in other species. We have characterized the root development of a wild and a domesticated tomato species, Solanum pennellii and Solanum lycopersicum 'M82.' We found extensive differences between these species for root morphology and cellular development including root length, a novel gravity set point angle, differences in cortical cell layer patterning, stem cell niche structure, and radial cell division. Using an introgression line population between these two species, we identified numerous loci that regulate these distinct aspects of development. Specifically we comprehensively identified loci that regulate (1) root length by distinct mechanisms including regulation of cell production within the meristem and the balance between cell division and expansion, (2) the gravity set point angle, and (3) radial cell division or expansion either in specific cell types or generally across multiple cell types. Our findings provide a novel perspective on the regulation of root growth and development between species. These loci have exciting implications with respect to regulation of drought resistance or salinity tolerance and regulation of root development in a family that has undergone domestication.

Gene regulatory networks in the Arabidopsis root.

Thanks to the increasing use of high-throughput tools in genetics, genomics, proteomics and metabolomics, a tremendous amount of information has been generated in the recent years. How these genes, transcripts, proteins and metabolites are inter-connected in a spatiotemporal context is one of the most ambitious goals that fundamental biology needs to answer. Owing to high quality data that are available, Arabidopsis thaliana has become an ideal organism for the application of bioinformatics and systems biology studies. The radially symmetrical structure of the Arabidopsis root and the ability to track developmental time in constrained cell files make this organ the perfect model to investigate different types of biological networks at a cell type-specific level. In this review we present the latest findings in this field as well as our perspective on the future of root biological networks.

Transcriptional factor interaction: a central step in DELLA function.

Gibberellins (GAs) affect several growth and developmental responses during the plant life cycle. Components essential for GA perception and GA signaling have been identified in rice and Arabidopsis and are conserved among vascular plants but not in Physcomitrella patens. The recent observation that DELLAs bind in nuclei to different members of the phytochrome interacting factor family, to block their transcriptional activity, is an important breakthrough to the understanding of the functional mechanism of these repressors. Beyond its role in GA-signaling repression, DELLAs were found to regulate GA homeostasis and to represent a convergence point for other hormone-signaling pathways. These repressors impose a growth restraint under environmental adverse conditions, allowing land plants to adapt their life cycle to the changing environment.

A molecular framework for light and gibberellin control of cell elongation.

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments.