HDACs MADS-domain protein interaction: a case study of HDA15 and XAL1 in Arabidopsis thaliana.

Cellular behavior, cell differentiation and ontogenetic development in eukaryotes result from complex interactions between epigenetic and classic molecular genetic mechanisms, with many of these interactions still to be elucidated. Histone deacetylase enzymes (HDACs) promote the interaction of histones with DNA by compacting the nucleosome, thus causing transcriptional repression. MADS-domain transcription factors are highly conserved in eukaryotes and participate in controlling diverse developmental processes in animals and plants, as well as regulating stress responses in plants. In this work, we focused on finding out putative interactions of Arabidopsis thaliana HDACs and MADS-domain proteins using an evolutionary perspective combined with bioinformatics analyses and testing the more promising predicted interactions through classic molecular biology tools. Through bioinformatic analyses, we found similarities between HDACs proteins from different organisms, which allowed us to predict a putative protein-protein interaction between the Arabidopsis thaliana deacetylase HDA15 and the MADS-domain protein XAANTAL1 (XAL1). The results of two-hybrid and Bimolecular Fluorescence Complementation analysis demonstrated in vitro and in vivo HDA15-XAL1 interaction in the nucleus. Likely, this interaction might regulate developmental processes in plants as is the case for this type of interaction in animals.

The MADS-box genes SOC1 and AGL24 antagonize XAL2 functions in Arabidopsis thaliana root development.

MADS-domain transcription factors play pivotal roles in numerous developmental processes in Arabidopsis thaliana. While their involvement in flowering transition and floral development has been extensively examined, their functions in root development remain relatively unexplored. Here, we explored the function and genetic interaction of three MADS-box genes (XAL2, SOC1 and AGL24) in primary root development. By analyzing loss-of-function and overexpression lines, we found that SOC1 and AGL24, both critical components in flowering transition, redundantly act as repressors of primary root growth as the loss of function of either SOC1 or AGL24 partially recovers the primary root growth, meristem cell number, cell production rate, and the length of fully elongated cells of the short-root mutant xal2-2. Furthermore, we observed that the simultaneous overexpression of AGL24 and SOC1 leads to short-root phenotypes, affecting meristem cell number and fully elongated cell size, whereas SOC1 overexpression is sufficient to affect columella stem cell differentiation. Additionally, qPCR analyses revealed that these genes exhibit distinct modes of transcriptional regulation in roots compared to what has been previously reported for aerial tissues. We identified 100 differentially expressed genes in xal2-2 roots by RNA-seq. Moreover, our findings revealed that the expression of certain genes involved in cell differentiation, as well as stress responses, which are either upregulated or downregulated in the xal2-2 mutant, reverted to WT levels in the absence of SOC1 or AGL24.

ULTRAPETALAs in action: Unraveling their role in root development.

The epigenetic complex Trithorax (TrxG) regulates gene transcription through post-translational histone modifications and is involved in a wide range of developmental processes. ULTRAPETALA1 (ULT1) is a SAND domain plant-exclusive TrxG protein that regulates the H3K4me3 active mark to counteract PcG repression. ULT1 has been identified to be involved in multiple tissue-specific processes. In the Arabidopsis root, ULT1 is required to maintain the stem cell niche, a role that is independent of the histone methyltransferase ATX1. Here we show the contribution of ULT2 in the maintenance of root stem cell niche. We also analyzed the gene expression in the ult1, ult2, and ult1ult2 mutants, evidencing three ways in which ULT1 and ULT2 regulate gene expression, one of them, where ULT1 or ULT2 regulate specific genes each, another where ULT1 and ULT2 act redundantly, as well as a regulation that requires of ULT1 and ULT2 together, supporting a coregulation, never reported. Furthermore, we also evidenced the participation of ULT1 in transcriptional repression synergically with CLF, a key histone methyltransferase of PcG.

XAANTAL1 Reveals an Additional Level of Flowering Regulation in the Shoot Apical Meristem in Response to Light and Increased Temperature in Arabidopsis.

Light and photoperiod are environmental signals that regulate flowering transition. In plants like Arabidopsis thaliana, this regulation relies on CONSTANS, a transcription factor that is negatively posttranslational regulated by phytochrome B during the morning, while it is stabilized by PHYA and cryptochromes 1/2 at the end of daylight hours. CO induces the expression of FT, whose protein travels from the leaves to the apical meristem, where it binds to FD to regulate some flowering genes. Although PHYB delays flowering, we show that light and PHYB positively regulate XAANTAL1 and other flowering genes in the shoot apices. Also, the genetic data indicate that XAL1 and FD participate in the same signaling pathway in flowering promotion when plants are grown under a long-day photoperiod at 22 °C. By contrast, XAL1 functions independently of FD or PIF4 to induce flowering at higher temperatures (27 °C), even under long days. Furthermore, XAL1 directly binds to FD, SOC1, LFY, and AP1 promoters. Our findings lead us to propose that light and temperature influence the floral network at the meristem level in a partially independent way of the signaling generated from the leaves.

Combined Approach of GWAS and Phylogenetic Analyses to Identify New Candidate Genes That Participate in Arabidopsis thaliana Primary Root Development Using Cellular Measurements and Primary Root Length.

Genome-wide association studies (GWAS) have allowed the identification of different loci associated with primary root (PR) growth, and Arabidopsis is an excellent model for these studies. The PR length is controlled by cell proliferation, elongation, and differentiation; however, the specific contribution of proliferation and differentiation in the control of PR growth is still poorly studied. To this end, we analyzed 124 accessions and used a GWAS approach to identify potential causal genomic regions related to four traits: PR length, growth rate, cell proliferation and cell differentiation. Twenty-three genes and five statistically significant SNPs were identified. The SNP with the highest score mapped to the fifth exon of NAC048 and this change makes a missense variant in only 33.3% of the accessions with a large PR, compared with the accessions with a short PR length. Moreover, we detected five more SNPs in this gene and in NAC3 that allow us to discover closely related accessions according to the phylogenetic tree analysis. We also found that the association between genetic variants among the 18 genes with the highest scores in our GWAS and the phenotypic classes into which we divided our accessions are not straightforward and likely follow historical patterns.

Unraveling the role of epigenetic regulation in asymmetric cell division during plant development.

Asymmetric cell divisions are essential to generate different cellular lineages. In plants, asymmetric cell divisions regulate the correct formation of the embryo, stomatal cells, apical and root meristems, and lateral roots. Current knowledge of regulation of asymmetric cell divisions suggests that, in addition to the function of key transcription factor networks, epigenetic mechanisms play crucial roles. Therefore, we highlight the importance of epigenetic regulation and chromatin dynamics for integration of signals and specification of cells that undergo asymmetric cell divisions, as well as for cell maintenance and cell fate establishment of both progenitor and daughter cells. We also discuss the polarization and segregation of cell components to ensure correct epigenetic memory or resetting of epigenetic marks during asymmetric cell divisions.

Live Plant Cell Tracking: Fiji plugin to analyze cell proliferation dynamics and understand morphogenesis.

Arabidopsis (Arabidopsis thaliana) primary and lateral roots (LRs) are well suited for 3D and 4D microscopy, and their development provides an ideal system for studying morphogenesis and cell proliferation dynamics. With fast-advancing microscopy techniques used for live-imaging, whole tissue data are increasingly available, yet present the great challenge of analyzing complex interactions within cell populations. We developed a plugin "Live Plant Cell Tracking" (LiPlaCeT) coupled to the publicly available ImageJ image analysis program and generated a pipeline that allows, with the aid of LiPlaCeT, 4D cell tracking and lineage analysis of populations of dividing and growing cells. The LiPlaCeT plugin contains ad hoc ergonomic curating tools, making it very simple to use for manual cell tracking, especially when the signal-to-noise ratio of images is low or variable in time or 3D space and when automated methods may fail. Performing time-lapse experiments and using cell-tracking data extracted with the assistance of LiPlaCeT, we accomplished deep analyses of cell proliferation and clonal relations in the whole developing LR primordia and constructed genealogical trees. We also used cell-tracking data for endodermis cells of the root apical meristem (RAM) and performed automated analyses of cell population dynamics using ParaView software (also publicly available). Using the RAM as an example, we also showed how LiPlaCeT can be used to generate information at the whole-tissue level regarding cell length, cell position, cell growth rate, cell displacement rate, and proliferation activity. The pipeline will be useful in live-imaging studies of roots and other plant organs to understand complex interactions within proliferating and growing cell populations. The plugin includes a step-by-step user manual and a dataset example that are available at https://www.ibt.unam.mx/documentos/diversos/LiPlaCeT.zip.

A Green Light to Switch on Genes: Revisiting Trithorax on Plants.

The Trithorax Group (TrxG) is a highly conserved multiprotein activation complex, initially defined by its antagonistic activity with the PcG repressor complex. TrxG regulates transcriptional activation by the deposition of H3K4me3 and H3K36me3 marks. According to the function and evolutionary origin, several proteins have been defined as TrxG in plants; nevertheless, little is known about their interactions and if they can form TrxG complexes. Recent evidence suggests the existence of new TrxG components as well as new interactions of some TrxG complexes that may be acting in specific tissues in plants. In this review, we bring together the latest research on the topic, exploring the interactions and roles of TrxG proteins at different developmental stages, required for the fine-tuned transcriptional activation of genes at the right time and place. Shedding light on the molecular mechanism by which TrxG is recruited and regulates transcription.

Beyond the Genetic Pathways, Flowering Regulation Complexity in Arabidopsis thaliana.

Flowering is one of the most critical developmental transitions in plants' life. The irreversible change from the vegetative to the reproductive stage is strictly controlled to ensure the progeny's success. In Arabidopsis thaliana, seven flowering genetic pathways have been described under specific growth conditions. However, the evidence condensed here suggest that these pathways are tightly interconnected in a complex multilevel regulatory network. In this review, we pursue an integrative approach emphasizing the molecular interactions among the flowering regulatory network components. We also consider that the same regulatory network prevents or induces flowering phase change in response to internal cues modulated by environmental signals. In this sense, we describe how during the vegetative phase of development it is essential to prevent the expression of flowering promoting genes until they are required. Then, we mention flowering regulation under suboptimal growing temperatures, such as those in autumn and winter. We next expose the requirement of endogenous signals in flowering, and finally, the acceleration of this transition by long-day photoperiod and temperature rise signals allowing A. thaliana to bloom in spring and summer seasons. With this approach, we aim to provide an initial systemic view to help the reader integrate this complex developmental process.

Integrative Roles of Phytohormones on Cell Proliferation, Elongation and Differentiation in the Arabidopsis thaliana Primary Root.

The growth of multicellular organisms relies on cell proliferation, elongation and differentiation that are tightly regulated throughout development by internal and external stimuli. The plasticity of a growth response largely depends on the capacity of the organism to adjust the ratio between cell proliferation and cell differentiation. The primary root of Arabidopsis thaliana offers many advantages toward understanding growth homeostasis as root cells are continuously produced and move from cell proliferation to elongation and differentiation that are processes spatially separated and could be studied along the longitudinal axis. Hormones fine tune plant growth responses and a huge amount of information has been recently generated on the role of these compounds in Arabidopsis primary root development. In this review, we summarized the participation of nine hormones in the regulation of the different zones and domains of the Arabidopsis primary root. In some cases, we found synergism between hormones that function either positively or negatively in proliferation, elongation or differentiation. Intriguingly, there are other cases where the interaction between hormones exhibits unexpected results. Future analysis on the molecular mechanisms underlying crosstalk hormone action in specific zones and domains will unravel their coordination over PR development.

Hormonal Regulation of Stem Cell Proliferation at the Arabidopsis thaliana Root Stem Cell Niche.

The root stem cell niche (SCN) of Arabidopsis thaliana consists of the quiescent center (QC) cells and the surrounding initial stem cells that produce progeny to replenish all the tissues of the root. The QC cells divide rather slowly relative to the initials, yet most root tissues can be formed from these cells, depending on the requirements of the plant. Hormones are fundamental cues that link such needs with the cell proliferation and differentiation dynamics at the root SCN. Nonetheless, the crosstalk between hormone signaling and the mechanisms that regulate developmental adjustments is still not fully understood. Developmental transcriptional regulatory networks modulate hormone biosynthesis, metabolism, and signaling, and conversely, hormonal responses can affect the expression of transcription factors involved in the spatiotemporal patterning at the root SCN. Hence, a complex genetic-hormonal regulatory network underlies root patterning, growth, and plasticity in response to changing environmental conditions. In this review, we summarize the scientific literature regarding the role of hormones in the regulation of QC cell proliferation and discuss how hormonal signaling pathways may be integrated with the gene regulatory network that underlies cell fate in the root SCN. The conceptual framework we present aims to contribute to the understanding of the mechanisms by which hormonal pathways act as integrators of environmental cues to impact on SCN activity.

The Epigenetic Faces of ULTRAPETALA1.

ULTRAPETALA1 (ULT1) is a versatile plant-exclusive protein, initially described as a trithorax group (TrxG) factor that regulates transcriptional activation and counteracts polycomb group (PcG) repressor function. As part of TrxG, ULT1 interacts with ARABIDOPSIS TRITHORAX1 (ATX1) to regulate H3K4me3 activation mark deposition. However, our recent studies indicate that ULT1 can also act independently of ATX1. Moreover, the ULT1 ability to interact with transcription factors (TFs) and PcG proteins indicates that it is a versatile protein with other roles. Therefore, in this work we revised recent information about the function of Arabidopsis ULT1 to understand the roles of ULT1 in plant development. Furthermore, we discuss the molecular mechanisms of ULT1, highlighting its epigenetic role, in which ULT1 seems to have characteristics of an epigenetic molecular switch that regulates repression and activation processes via TrxG and PcG complexes.

The canonical RdDM pathway mediates the control of seed germination timing under salinity.

Plants respond to adverse environmental cues by adjusting a wide variety of processes through highly regulated mechanisms to maintain plant homeostasis for survival. As a result of the sessile nature of plants, their response, adjustment and adaptation to the changing environment is intimately coordinated with their developmental programs through the crosstalk of regulatory networks. Germination is a critical process in the plant life cycle, and thus plants have evolved various strategies to control the timing of germination according to their local environment. The mechanisms involved in these adjustment responses are largely unknown, however. Here, we report that mutations in core elements of canonical RNA-directed DNA methylation (RdDM) affect the germination and post-germination growth of Arabidopsis seeds grown under salinity stress. Transcriptomic and whole-genome bisulfite sequencing (WGBS) analyses support the involvement of this pathway in the control of germination timing and post-germination growth under salinity stress by preventing the transcriptional activation of genes implicated in these processes. Subsequent transcriptional effects on genes that function in relation to these developmental events support this conclusion.

Interplay between Hormones and Several Abiotic Stress Conditions on Arabidopsis thaliana Primary Root Development.

As sessile organisms, plants must adjust their growth to withstand several environmental conditions. The root is a crucial organ for plant survival as it is responsible for water and nutrient acquisition from the soil and has high phenotypic plasticity in response to a lack or excess of them. How plants sense and transduce their external conditions to achieve development, is still a matter of investigation and hormones play fundamental roles. Hormones are small molecules essential for plant growth and their function is modulated in response to stress environmental conditions and internal cues to adjust plant development. This review was motivated by the need to explore how Arabidopsis thaliana primary root differentially sense and transduce external conditions to modify its development and how hormone-mediated pathways contribute to achieve it. To accomplish this, we discuss available data of primary root growth phenotype under several hormone loss or gain of function mutants or exogenous application of compounds that affect hormone concentration in several abiotic stress conditions. This review shows how different hormones could promote or inhibit primary root development in A. thaliana depending on their growth in several environmental conditions. Interestingly, the only hormone that always acts as a promoter of primary root development is gibberellins.

Beyond What Your Retina Can See: Similarities of Retinoblastoma Function between Plants and Animals, from Developmental Processes to Epigenetic Regulation.

The Retinoblastoma protein (pRb) is a key cell cycle regulator conserved in a wide variety of organisms. Experimental analysis of pRb's functions in animals and plants has revealed that this protein participates in cell proliferation and differentiation processes. In addition, pRb in animals and its orthologs in plants (RBR), are part of highly conserved protein complexes which suggest the possibility that analogies exist not only between functions carried out by pRb orthologs themselves, but also in the structure and roles of the protein networks where these proteins are involved. Here, we present examples of pRb/RBR participation in cell cycle control, cell differentiation, and in the regulation of epigenetic changes and chromatin remodeling machinery, highlighting the similarities that exist between the composition of such networks in plants and animals.

A system-level mechanistic explanation for asymmetric stem cell fates: Arabidopsis thaliana root niche as a study system.

Asymmetric divisions maintain long-term stem cell populations while producing new cells that proliferate and then differentiate. Recent reports in animal systems show that divisions of stem cells can be uncoupled from their progeny differentiation, and the outcome of a division could be influenced by microenvironmental signals. But the underlying system-level mechanisms, and whether this dynamics also occur in plant stem cell niches (SCN), remain elusive. This article presents a cell fate regulatory network model that contributes to understanding such mechanism and identify critical cues for cell fate transitions in the root SCN. Novel computational and experimental results show that the transcriptional regulator SHR is critical for the most frequent asymmetric division previously described for quiescent centre stem cells. A multi-scale model of the root tip that simulated each cell's intracellular regulatory network, and the dynamics of SHR intercellular transport as a cell-cell coupling mechanism, was developed. It revealed that quiescent centre cell divisions produce two identical cells, that may acquire different fates depending on the feedback between SHR's availability and the state of the regulatory network. Novel experimental data presented here validates our model, which in turn, constitutes the first proposed systemic mechanism for uncoupled SCN cell division and differentiation.

ULTRAPETALA1 maintains Arabidopsis root stem cell niche independently of ARABIDOPSIS TRITHORAX1.

During plant development, morphogenetic processes rely on the activity of meristems. Meristem homeostasis depends on a complex regulatory network constituted by different factors and hormone signaling that regulate gene expression to coordinate the correct balance between cell proliferation and differentiation. ULTRAPETALA1, a transcriptional regulatory protein described as an Arabidopsis Trithorax group factor, has been characterized as a regulator of the shoot and floral meristems activity. Here, we highlight the role of ULTRAPETALA1 in root stem cell niche maintenance. We found that ULTRAPETALA1 is required to regulate both the quiescent center cell division rate and auxin signaling at the root tip. Furthermore, ULTRAPETALA1 regulates columella stem cell differentiation. These roles are independent of the ARABIDOPSIS TRITHORAX1, suggesting a different mechanism by which ULTRAPETALA1 can act in the root apical meristem of Arabidopsis. This work introduces a new component of the regulatory network needed for the root stem cell niche maintenance.

Natural Root Cellular Variation in Responses to Osmotic Stress in Arabidopsis thaliana Accessions.

Arabidopsis naturally occurring populations have allowed for the identification of considerable genetic variation remodeled by adaptation to different environments and stress conditions. Water is a key resource that limits plant growth, and its availability is initially sensed by root tissues. The root's ability to adjust its physiology and morphology under water deficit makes this organ a useful model to understand how plants respond to water stress. Here, we used hyperosmotic shock stress treatments in different Arabidopsis accessions to analyze the root cell morphological responses. We found that osmotic stress conditions reduced root growth and root apical meristem (RAM) size, promoting premature cell differentiation without affecting the stem cell niche morphology. This phenotype was accompanied by a cluster of small epidermal and cortex cells with radial expansion and root hairs at the transition to the elongation zone. We also found this radial expansion with root hairs when plants are grown under hypoosmotic conditions. Finally, root growth was less affected by osmotic stress in the Sg-2 accession followed by Ws, Cvi-0, and Col-0; however, after a strong osmotic stress, Sg-2 and Cvi-0 were the most resilience accessions. The sensitivity differences among these accessions were not explained by stress-related gene expression. This work provides new cellular insights on the Arabidopsis root phenotypic variability and plasticity to osmotic stress.

MADS-Box Genes Are Key Components of Genetic Regulatory Networks Involved in Abiotic Stress and Plastic Developmental Responses in Plants.

Plants, as sessile organisms, adapt to different stressful conditions, such as drought, salinity, extreme temperatures, and nutrient deficiency, via plastic developmental and growth responses. Depending on the intensity and the developmental phase in which it is imposed, a stress condition may lead to a broad range of responses at the morphological, physiological, biochemical, and molecular levels. Transcription factors are key components of regulatory networks that integrate environmental cues and concert responses at the cellular level, including those that imply a stressful condition. Despite the fact that several studies have started to identify various members of the MADS-box gene family as important molecular components involved in different types of stress responses, we still lack an integrated view of their role in these processes. In this review, we analyze the function and regulation of MADS-box gene family members in response to drought, salt, cold, heat, and oxidative stress conditions in different developmental processes of several plants. In addition, we suggest that MADS-box genes are key components of gene regulatory networks involved in plant responses to stress and plant developmental plasticity in response to seasonal changes in environmental conditions.

MADS-box genes underground becoming mainstream: plant root developmental mechanisms.

Plant growth is largely post-embryonic and depends on meristems that are active throughout the lifespan of an individual. Developmental patterns rely on the coordinated spatio-temporal expression of different genes, and the activity of transcription factors is particularly important during most morphogenetic processes. MADS-box genes constitute a transcription factor family in eukaryotes. In Arabidopsis, their proteins participate in all major aspects of shoot development, but their role in root development is still not well characterized. In this review we synthetize current knowledge pertaining to the function of MADS-box genes highly expressed in roots: XAL1, XAL2, ANR1 and AGL21, as well as available data for other MADS-box genes expressed in this organ. The role of Trithorax group and Polycomb group complexes on MADS-box genes' epigenetic regulation is also discussed. We argue that understanding the role of MADS-box genes in root development of species with contrasting architectures is still a challenge. Finally, we propose that MADS-box genes are key components of the gene regulatory networks that underlie various gene expression patterns, each one associated with the distinct developmental fates observed in the root. In the case of XAL1 and XAL2, their role within these networks could be mediated by regulatory feedbacks with auxin.