Brassinosteroids Mitigate Cadmium Effects in Arabidopsis Root System without Any Cooperation with Nitric Oxide

The heavy metal cadmium (Cd) affects root system development and quiescent center (QC)-definition in Arabidopsis root-apices. The brassinosteroids-(BRs)-mediated tolerance to heavy metals has been reported to occur by a modulation of nitric oxide (NO) and root auxin-localization. However, how BRs counteract Cd-action in different root types is unknown. This research aimed to find correlations between BRs and NO in response to Cd in Arabidopsis’s root system, monitoring their effects on QC-definition and auxin localization in root-apices. To this aim, root system developmental changes induced by low levels of 24-epibrassinolide (eBL) or by the BR-biosynthesis inhibitor brassinazole (Brz), combined or not with CdSO4, and/or with the NO-donor nitroprusside (SNP), were investigated using morpho-anatomical and NO-epifluorescence analyses, and monitoring auxin-localization by the DR5::GUS system. Results show that eBL, alone or combined with Cd, enhances lateral (LR) and adventitious (AR) root formation and counteracts QC-disruption and auxin-delocalization caused by Cd in primary root/LR/AR apices. Exogenous NO enhances LR and AR formation in Cd-presence, without synergism with eBL. The NO-signal is positively affected by eBL, but not in Cd-presence, and BR-biosynthesis inhibition does not change the low NO-signal caused by Cd. Collectively, results show that BRs ameliorate Cd-effects on all root types acting independently from NO.

New insights on the role of HCN in root hair elongation through single cell proteomics

Root hairs are specialized structures involved in water and nutrient uptake by plants. They elongate from epidermal cells following a complex developmental program. β-cyanoalanine synthase (CAS), which is mainly involved in hydrogen cyanide (HCN) detoxification in Arabidopsis thaliana, plays a role in root hair elongation, as evidenced by the fact that cas-c1 mutants show a severe defect in root hair shape. In addition to root hairs, CAS C1 is expressed in the quiescent center and meristem. However, the cas-c1 mutation has no visible effect on either tissue, in both control and nutrient-deprivation conditions. To identify its role in root hair formation, we conducted single cell proteomics analysis by isolating root hair cells using Fluorescence-Activated Cell Sorting (FACS) from wild type and cas-c1 mutants. We also analyzed the presence of S-cyanylation, a protein post-translational modification (PTM) mediated by HCN and affecting cysteine residues and protein activity, in proteins of wild type and cas-c1 mutants. We found that several proteins involved in root hair development, related to the receptor kinase FERONIA signaling and to DNA methylation, are modified by this new post-translational modification.

Whole-Tissue Three-Dimensional Imaging of Rice at Single-Cell Resolution

The three-dimensional (3D) arrangement of cells in tissues provides an anatomical basis for analyzing physiological and biochemical aspects of plant and animal cellular development and function. In this study, we established a protocol for tissue clearing and 3D imaging in rice. Our protocol is based on three improvements: clearing with iTOMEI (clearing solution suitable for plants), developing microscopic conditions in which the Z step is optimized for 3D reconstruction, and optimizing cell-wall staining. Our protocol successfully 3D imaged rice shoot apical meristems, florets, and root apical meristems at cellular resolution throughout whole tissues. Using fluorescent reporters of auxin signaling in rice root tips, we also revealed the 3D distribution of auxin signaling events that are activated in the columella, quiescent center, and multiple rows of cells in the stele of the root apical meristem. Examination of cells with higher levels of auxin signaling revealed that only the central row of cells was connected to the quiescent center. Our method provides opportunities to observe the 3D arrangement of cells in rice tissues.

The Anaphase Promoting Complex/Cyclosome Subunit 11 and Its Role in Organ Size and Plant Development

The anaphase promoting complex/cyclosome (APC/C), a member of the E3 ubiquitin ligase family, plays an important role in recognizing the substrates to be ubiquitylated. Progression of anaphase, and therefore, of the cell cycle, is coordinated through cyclin degradation cycles dependent on proteolysis triggered by APC/C. The APC/C activity depends on the formation of a pocket comprising the catalytic subunits, APC2, APC11, and APC10. Among these, the role of APC11 outside the cell division cycle is poorly understood. Therefore, the goal of this work was to analyze the function of APC11 during plant development by characterizing apc11 knock-down mutant lines. Accordingly, we observed decreased apc11 expression in the mutant lines, followed by a reduction in meristem root size based on the cortical cell length, and an overall size diminishment throughout the development. Additionally, crosses of apc11-1 and amiR-apc11 with plants carrying a WUSCHEL-RELATED HOMEOBOX5 (WOX5) fluorescent marker showed a weakening of the green fluorescent protein-positive cells in the Quiescent Center. Moreover, plants with apc11-1 show a decreased leaf area, together with a decrease in the cell area when the shoot development was observed by kinematics analysis. Finally, we observed a decreased APC/C activity in the root and shoot meristems in crosses of pCYCB1;1:D-box-GUS with apc11-1 plants. Our results indicate that APC11 is important in the early stages of development, mediating meristematic architecture through APC/C activity affecting the overall plant growth.

Altered Root Growth, Auxin Metabolism and Distribution in Arabidopsis thaliana Exposed to Salt and Osmotic Stress

Salt and osmotic stress are the main abiotic stress factors affecting plant root growth and architecture. We investigated the effect of salt (100 mM NaCl) and osmotic (200 mM mannitol) stress on the auxin metabolome by UHPLC-MS/MS, auxin distribution by confocal microscopy, and transcript levels of selected genes by qRT-PCR in Arabidopsis thaliana ecotype Columbia-0 (Col-0) and DR5rev::GFP (DR5) line. During long-term stress (13 days), a stability of the auxin metabolome and a tendency to increase indole-3-acetic acid (IAA) were observed, especially during salt stress. Short-term stress (3 h) caused significant changes in the auxin metabolome, especially NaCl treatment resulted in a significant reduction of IAA. The data derived from auxin profiling were consistent with gene expressions showing the most striking changes in the transcripts of YUC, GH3, and UGT transcripts, suggesting disruption of auxin biosynthesis, but especially in the processes of amide and ester conjugation. These data were consistent with the auxin distribution observed in the DR5 line. Moreover, NaCl treatment caused a redistribution of auxin signals from the quiescent center and the inner layers of the root cap to the epidermal and cortical cells of the root elongation zone. The distribution of PIN proteins was also disrupted by salt stress; in particular, PIN2 was suppressed, even after 5 min of treatment. Based on our results, the DR5 line was more sensitive to the applied stresses than Col-0, although both lines showed similar trends in root morphology, as well as transcriptome and metabolome parameters under stress conditions.

Cell Type-Specific Differentiation Between Indica and Japonica Rice Root Tip Responses to Different Environments Based on Single-Cell RNA Sequencing

Background: As Oryza sativa ssp. indica and Oryza sativa ssp. japonica are the two major subspecies of Asian cultivated rice, the adaptative evolution of these varieties in divergent environments is an important topic in both theoretical and practical studies. However, the cell type-specific differentiation between indica and japonica rice varieties in response to divergent habitat environments, which facilitates an understanding of the genetic basis underlying differentiation and environmental adaptation between rice subspecies at the cellular level, is little known.Methods: We analyzed a published single-cell RNA sequencing dataset to explore the differentially expressed genes between indica and japonica rice varieties in each cell type. To estimate the relationship between cell type-specific differentiation and environmental adaptation, we focused on genes in the WRKY, NAC, and BZIP transcription factor families, which are closely related to abiotic stress responses. In addition, we integrated five bulk RNA sequencing datasets obtained under conditions of abiotic stress, including cold, drought and salinity, in this study. Furthermore, we analyzed quiescent center cells in rice root tips based on orthologous markers in Arabidopsis.Results: We found differentially expressed genes between indica and japonica rice varieties with cell type-specific patterns, which were enriched in the pathways related to root development and stress reposes. Some of these genes were members of the WRKY, NAC, and BZIP transcription factor families and were differentially expressed under cold, drought or salinity stress. In addition, LOC_Os01g16810, LOC_Os01g18670, LOC_Os04g52960, and LOC_Os08g09350 may be potential markers of quiescent center cells in rice root tips.Conclusion: These results identified cell type-specific differentially expressed genes between indica-japonica rice varieties that were related to various environmental stresses and provided putative markers of quiescent center cells. This study provides new clues for understanding the development and physiology of plants during the process of adaptative divergence, in addition to identifying potential target genes for the improvement of stress tolerance in rice breeding applications.

Light at the end of the tunnel: FRAP assay reveals that plant vacuoles start as a tubular network

Plant vacuoles play key roles in cellular homeostasis performing catabolic and storage functions, regulating pH and ion balance 1,2. The essential role of vacuoles for plant cell viability makes them a notoriously difficult subject to study. As a consequence, there is still no consensus on the mechanism of vacuolar establishment and the source of membrane material for it. Our previous suggestion of endoplasmic reticulum (ER) being the main contributor of membrane for growing young vacuoles3 was recently challenged in a study proposing that plant vacuoles are formed de novo via homotypic fusion of multivesicular bodies (MVBs)4. Authors of this work pointed out issues that might explain our seemingly contradictory observations and we have thus carefully revaluated our hypothesis. Using the Arabidopsis thaliana root as a model, we provide a systematic overview of successive vacuolar biogenesis stages, starting from the youngest cells proximate to the quiescent center. We validate our previous conclusions by demonstrating that the vacuolar dye BCECF is fully suitable for studying the organelle morphology and provide 3D models from vacuoles of all developmental stages. We established a customized FRAP assay and proved that even at the earliest stages of biogenesis, vacuoles comprise a connected network. Together, this adds to a growing body of evidence indicating that vacuolar structures cannot originate solely from MVBs.

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.

Molecular Bases for the Regulation of Adventitious Root Generation in Plants

The formation of adventitious roots (ARs) is an ecologically and economically important developmental process in plants. The evolution of AR systems is an important way for plants to cope with various environmental stresses. This review focuses on identified genes that have known to regulate the induction and initiation of ARs and offers an analysis of this process at the molecular level. The critical genes involved in adventitious rooting are the auxin signaling-responsive genes, including the AUXIN RESPONSE FACTOR (ARF) and the LATERAL ORGAN BOUNDARIES-DOMAIN (LOB) gene families, and genes associated with auxin transport and homeostasis, the quiescent center (QC) maintenance, and the root apical meristem (RAM) initiation. Several genes involved in cell wall modulation are also known to be involved in the regulation of adventitious rooting. Furthermore, the molecular processes that play roles in the ethylene, cytokinin, and jasmonic acid signaling pathways and their crosstalk modulate the generation of ARs. The crosstalk and interaction among many molecular processes generates complex networks that regulate AR generation.

A hybrid model connecting regulatory interactions with stem cell divisions in the root

Stem cells give rise to the entirety of cells within an organ. Maintaining stem cell identity and coordinately regulating stem cell divisions is crucial for proper development. In plants, mobile proteins, such as WUSCHEL-RELATED HOMEOBOX 5 (WOX5) and SHORTROOT (SHR), regulate divisions in the root stem cell niche. However, how these proteins coordinately function to establish systemic behaviour is not well understood. We propose a non-cell autonomous role for WOX5 in the cortex endodermis initial (CEI) and identify a regulator, ANGUSTIFOLIA (AN3)/GRF-INTERACTING FACTOR 1, that coordinates CEI divisions. Here, we show with a multi-scale hybrid model integrating ordinary differential equations (ODEs) and agent-based modeling that quiescent center (QC) and CEI divisions have different dynamics. Specifically, by combining continuous models to describe regulatory networks and agent-based rules, we model systemic behaviour, which led us to predict cell-type-specific expression dynamics of SHR, SCARECROW, WOX5, AN3 and CYCLIND6;1, and experimentally validate CEI cell divisions. Conclusively, our results show an interdependency between CEI and QC divisions.