Primary Root Meristem Research Articles

Gallic acid regulates primary root elongation via modulating auxin transport and signal transduction.

Gallic acid is an important secondary metabolite in plants, with great value in medicine, food, and chemical industry. However, whether and how this widely existing natural polyphenolic compound affects the growth and development of plants themselves remains elusive. In this study, we revealed that exogenous application of gallic acid has a dual effect on the elongation of primary root in Arabidopsis. While lower concentrations of gallic acid slightly stimulate primary root growth, excessive gallic acid profoundly reduces primary root length and root meristem size in a dose-dependent manner, probably via suppressing cell division in root meristem as indicated by CYCB1;1::GUS. Moreover, as suggested by the DR5::GFP line analysis and confirmed by the LC-MS assay, auxin contents in root tips were dramatically decreased upon excessive gallic acid treatment. Additional application of IAA partially rescued the shortened primary root and root meristem upon excessive gallic acid treatment, suggesting that auxin is required for excessive gallic acid-caused root growth inhibition. Then, we further revealed that excessive gallic acid down-regulated the expression of auxin transporters PIN1, PIN2, PIN3, and PIN7, and triple mutant pin1 pin3 pin7 exhibited a reduced sensitivity to gallic acid treatment. Meanwhile, excessive gallic acid decreased the degradation of AXR3/IAA17 protein as revealed by HS::AXR3NT-GUS reporter line. Auxin signaling mutant tir1 afb2 afb3 and axr3-3 were also less sensitive to excessive gallic acid treatment in terms of primary root length and root meristem size. Taken together, these findings suggested that excessive gallic acid inhibits primary root growth by modulating auxin transport and signaling in Arabidopsis.

ABSCISIC ACID INSENSITIVE 3 promotes auxin signalling by regulating SHY2 expression to control primary root growth in response to dehydration stress.

Plants combat dehydration stress through different strategies including root architectural changes. Here we show that when exposed to varying levels of dehydration stress, primary root growth in Arabidopsis is modulated by regulating root meristem activity. Abscisic acid (ABA) in concert with auxin signalling adjust primary root growth according to stress levels. ABSCISIC ACID INSENSITIVE 3 (ABI3), an ABA-responsive transcription factor, stands at the intersection of ABA and auxin signalling and fine-tunes primary root growth in response to dehydration stress. Under low ABA or dehydration stress, induction of ABI3 expression promotes auxin signalling by decreasing expression of SHY2, a negative regulator of auxin response. This further enhances the expression of auxin transporter gene PIN1 and cell cycle gene CYCB1;1, resulting in an increase in primary root meristem size and root length. Higher levels of dehydration stress or ABA repress ABI3 expression and promote ABSCISIC ACID INSENSITIVE 5 (ABI5) expression. This elevates SHY2 expression, thereby impairing primary root meristem activity and retarding root growth. Notably, ABI5 can promote SHY2 expression only in the absence of ABI3. Such ABA concentration-dependent expression of ABI3 therefore functions as a regulatory sensor of dehydration stress levels and orchestrates primary root growth by coordinating its downstream regulation.

Control of lateral root formation by rapamycin-induced dimerization of engineered RGI/BAK1 and by BIR3 chimera.

Leucine-rich repeat receptor kinases (LRR-RKs) play a pivotal role in diverse aspects of growth, development, and immunity in plants by sensing extracellular signals. Typically, LRR-RKs are activated through the ligand-induced interaction with a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) coreceptor, triggering downstream signaling. ROOT MERISTEM GROWTH FACTOR1 (RGF1) INSENSITIVEs (RGIs) LRR-RLK receptors promote primary root meristem activity while inhibiting lateral root (LR) development in response to RGF peptide. In this study, we employed rapamycin-induced dimerization (RiD) and BAK1-INTERACTING RECEPTOR-LIKE KINASE3 (BIR3) chimera approaches to explore the gain-of-function of RGI1, RGI4, and RGI5. Rapamycin induced the association of cytosolic kinase domains (CKDs) of RGI1 and the BAK1 coreceptor, activating both mitogen-activated protein kinase 3 (MPK3) and MPK6. Rapamycin significantly inhibited LR formation in RiD-RGI1/RGI4/RGI5-BAK1 plants. Using transgenic Arabidopsis expressing RGI1CKD fused to the BIR3-LRR chimera under estradiol control, we observed a substantial reduction in LR density upon β-estradiol treatment. Additionally, we identified a decrease in root gravitropism in BIR3 chimera plants. In contrast, RiD-RGI/BAK1 plants did not exhibit defects in root gravitropism, implying the importance of combinatorial interactions between RGIs and SERK coreceptors in the inhibition of root gravitropism. Constitutive activation of RGIs with BAK1 in RiD-RGI/BAK1 plants by rapamycin treatment resulted in the inhibition of primary root growth, resembling the inhibitory effects observed with high concentrations of phytohormones on primary root elongation. Our findings highlight that the interactions between CKDs of RGIs and BAK1, constitutively induced by rapamycin or BIR3 chimera, efficiently control LR development.

The proteome landscape of the root cap reveals a role for the jacalin-associated lectin JAL10 in the salt-induced endoplasmic reticulum stress pathway

Rapid climate change has led to enhanced soil salinity, one of the major determinants of land degradation, resulting in low agricultural productivity. This has a strong negative impact on food security and environmental sustainability. Plants display various physiological, developmental, and cellular responses to deal with salt stress. Recent studies have highlighted the root cap as the primary stress sensor and revealed its crucial role in halotropism. The root cap covers the primary root meristem and is the first cell type to sense and respond to soil salinity, relaying the signal to neighboring cell types. However, it remains unclear how root-cap cells perceive salt stress and contribute to the salt-stress response. Here, we performed a root-cap cell-specific proteomics study to identify changes in the proteome caused by salt stress. The study revealed a very specific salt-stress response pattern in root-cap cells compared with non-root-cap cells and identified several novel proteins unique to the root cap. Root-cap-specific protein–protein interaction (PPI) networks derived by superimposing proteomics data onto known global PPI networks revealed that the endoplasmic reticulum (ER) stress pathway is specifically activated in root-cap cells upon salt stress. Importantly, we identified root-cap-specific jacalin-associated lectins (JALs) expressed in response to salt stress. A JAL10-GFP fusion protein was shown to be localized to the ER. Analysis of jal10 mutants indicated a role for JAL10 in regulating the ER stress pathway in response to salt. Taken together, our findings highlight the participation of specific root-cap proteins in salt-stress response pathways. Furthermore, root-cap-specific JAL proteins and their role in the salt-mediated ER stress pathway open a new avenue for exploring tolerance mechanisms and devising better strategies to increase plant salinity tolerance and enhance agricultural productivity.

On salt stress, PLETHORA signaling maintains root meristems.

Salt stress is one of the unfavorable environmental factors to affect plants. Salinity represses root growth, resulting in reduced biomass of agricultural plants. Little is known about how plants maintain root growth to counteract salt stress. The AP2-domain transcription factors PLETHORA1/2 (PLT1/2) act as master regulators in root meristem maintenance in Arabidopsis. In this study, we report that the salt overly sensitive (SOS) pathway component SOS2 regulates PLT1/2 at the post-transcriptional level. Salt-activated SOS2 interacts and phosphorylates PLT1/2 through their conserved C-terminal motifs to stabilize PLT1/2, critical for root apical meristem maintenance under salt stress. The phospho-mimetic version of PLT1/2 restored meristem and primary root length reduction of sos2-2 and plt1-4 plt2-2 mutants on salt treatment. Moreover, SOS2-mediated PLT1/2 phosphorylation improves root growth recovery after salt stress alleviation. We identify a SOS2-PLT1/2 core protein module that is required for protecting primary root growth and meristem maintenance from salt stress.

The RGI1–BAK1 module acts as the main receptor–coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis

ABSTRACT ROOT MERISTEM GROWTH FACTOR1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs), a group of leucine-rich repeat receptor kinases, promote primary root meristem activity via a mitogen-activated protein kinase (MPK) signaling cascade and control root gravitropism in Arabidopsis. Genetic analyses and in vitro binding assays have indicated that among five RGIs identified in Arabidopsis, RGI1, RGI2, and RGI3 recognize RGF1 peptides. However, it remains unclear whether the RGF1 peptide is redundantly recognized by these RGIs or mainly by a single RGI in the regulation of primary root meristem activity. In the present study, we analyzed root meristem growth of the rgi1, rgi2, and rgi3 single mutants in response to RGF1 treatment and observed a significantly decreased sensitivity in meristem growth of rgi1 and complete insensitivity in rgi1 rgi2 rgi3 triple mutant compared with the wild type but not in the rgi1 and rgi2 single mutants. We also observed that both root gravitropism and meristem growth in the BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (bak1) single mutant were insensitive to RGF1 peptide treatment, whereas other serk mutants, such as serk1, serk2, and serk4, were fully sensitive to RGF1 peptide like the wild type. These mutant analyses suggest that RGI1–BAK1 pair acts as the main receptor–coreceptor pair for regulating primary root gravitropism and meristem activity in response to RGF1 peptide in Arabidopsis.

Azospirillum brasilense activates peroxidase-mediated cell wall modification to inhibit root cell elongation

The molecular mechanism of beneficial bacterium Azospirillum brasilense-mediated root developmental remain elusive. A.brasilense elicited extensively transcriptional changes but inhibited primary root elongation in Arabidopsis. By analyzing root cell type-specific developmental markers, we demonstrated that A.brasilense affected neither overall organization nor cell division of primary root meristem. The cessation of primary root resulted from reduction of cell elongation, which is probably because of bacterially activated peroxidase that will lead to cell wall cross-linking at consuming of H2O2. The activated peroxidase combined with downregulated cell wall loosening enzymes consequently led to cell wall thickness, whereas inhibiting peroxidase restored root growth under A.brasilense inoculation. We further showed that peroxidase activity was probably promoted by cadaverine secreted by A.brasilense. These results suggest that A.brasilense inhibits root elongation by activating peroxidase and inducing cell wall modification in Arabidopsis, in which cadaverine released by A.brasilense is a potential signal compound.

A histochemical reporter system to study extracellular ATP response in plants.

When cells experience acute mechanical distress, they release ATP from their cellular compartment into the surrounding microenvironment. This extracellular ATP (eATP) can then act as a danger signal-signaling cellular damage. In plants, cells adjacent to damage detect rising eATP concentrations through the cell-surface receptor kinase, P2K1. Following eATP perception, P2K1 initiates a signaling cascade mobilizing plant defense. Recent transcriptome analysis revealed a profile of eATP-induced genes sharing pathogen- and wound-response hallmarks-consistent with a working model for eATP as a defense-mobilizing danger signal. To build on the transcriptional footprint and broaden our understanding of dynamic eATP signaling responses in plants, we aimed to i) generate a visual toolkit for eATP-inducible marker genes using a β-glucuronidase (GUS) reporter system and ii) evaluate the spatiotemporal response of these genes to eATP in plant tissues. Here, we demonstrate that the promoter activities of five genes, ATPR1, ATPR2, TAT3, WRKY46, and CNGC19, were highly sensitive to eATP in the primary root meristem and elongation zones with maximal responses at 2h after treatment. These results suggest the primary root tip as a hub to study eATP-signaling activity and provide a proof-of-concept toward using these reporters to further dissect eATP and damage signaling in plants.

The F-box protein SHORT PRIMARY ROOT modulates primary root meristem activity by targeting SEUSS-LIKE protein for degradation in rice.

Root meristem activity is essential for root morphogenesis and adaptation, but the molecular mechanism regulating root meristem activity is not fully understood. Here, we identify an F-box family E3 ubiquitin ligase named SHORT PRIMARY ROOT (SHPR) that regulates primary root (PR) meristem activity and cell proliferation in rice. SHPR loss-of-function mutations impair PR elongation in rice. SHPR is involved in the formation of an SCF complex with the Oryza sativa SKP1-like protein OSK1/20. We show that SHPR interacts with Oryza sativa SEUSS-LIKE (OsSLK) in the nucleus and is required for OsSLK polyubiquitination and degradation by the ubiquitin 26S-proteasome system (UPS). Transgenic plants overexpressing OsSLK display a shorter PR phenotype, which is similar to the SHPR loss-of-function mutants. Genetic analysis suggests that SHPR promotes PR elongation in an OsSLK-dependent manner. Collectively, our study establishes SHPR as an E3 ubiquitin ligase that targets OsSLK for degradation, and uncovers a protein ubiquitination pathway as a mechanism for modulating root meristem activity in rice.

The ROP2 GTPase Participates in Nitric Oxide (NO)-Induced Root Shortening in Arabidopsis.

Nitric oxide (NO) is a versatile signal molecule that mediates environmental and hormonal signals orchestrating plant development. NO may act via reversible S-nitrosation of proteins during which an NO moiety is added to a cysteine thiol to form an S-nitrosothiol. In plants, several proteins implicated in hormonal signaling have been reported to undergo S-nitrosation. Here, we report that the Arabidopsis ROP2 GTPase is a further potential target of NO-mediated regulation. The ROP2 GTPase was found to be required for the root shortening effect of NO. NO inhibits primary root growth by altering the abundance and distribution of the PIN1 auxin efflux carrier protein and lowering the accumulation of auxin in the root meristem. In rop2-1 insertion mutants, however, wild-type-like root size of the NO-treated roots were maintained in agreement with wild-type-like PIN1 abundance in the meristem. The ROP2 GTPase was shown to be S-nitrosated in vitro, suggesting that NO might directly regulate the GTPase. The potential mechanisms of NO-mediated ROP2 GTPase regulation and ROP2-mediated NO signaling in the primary root meristem are discussed.

ROOT MERISTEM GROWTH FACTOR1 (RGF1)-RGF1 INSENSITIVE 1 peptide-receptor pair inhibits lateral root development via the MPK6-PUCHI module in Arabidopsis.

ROOT MERISTEM GROWTH FACTOR1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs) regulate primary root meristem activity via a mitogen-activated protein kinase (MPK) signaling cascade in Arabidopsis. However, it is unknown how RGF1 regulates lateral root (LR) development. Here, we show that the RGF1-RGI1 peptide-receptor pair negatively regulates LR development via activation of PUCHI encoding AP2/EREBP. Exogenous RGF1 peptides inhibited LR development of the wild type. However, the rgi1 mutants were partially or fully insensitive to RGF1 during LR development, whereas four other rgi single mutants, namely rgi2, rgi3, rgi4, and rgi5, were sensitive to RGF1 in inhibiting LR formation. Consistent with this, the red fluorescent protein (RFP) signals driven by the RGF1 promoter were detected at stage I and the following stages, overlapping with RGI1 expression. PUCHI expression was significantly up-regulated by RGF1 but completely inhibited in rgi1. LR development of puchi1-1 was insensitive to RGF1. PUCHI expression driven by the RGI1 promoter reduced LR density in both the wild type and rgi1,2,3. Further, mpk6, but not mpk3, displayed significantly down-regulated PUCHI expression and insensitive LR development in response to RGF1. Collectively, these results suggest that the RGF1-RGI1 module negatively regulates LR development by activating PUCHI expression via MPK6.

RAV1 mediates cytokinin signaling for regulating primary root growth in Arabidopsis.

Root growth dynamics is an outcome of complex hormonal crosstalk. The primary root meristem size, for example, is determined by antagonizing actions of cytokinin and auxin. Here we show that RAV1, a member of the AP2/ERF family of transcription factors, mediates cytokinin signaling in roots to regulate meristem size. The rav1 mutants have prominently longer primary roots, with a meristem that is significantly enlarged and contains higher cell numbers, compared with wild-type. The mutant phenotype could be restored on exogenous cytokinin application or by inhibiting auxin transport. At the transcript level, primary cytokinin-responsive genes like ARR1, ARR12 were significantly downregulated in the mutant root, indicating impaired cytokinin signaling. In concurrence, cytokinin induced regulation of SHY2, an Aux/IAA gene, and auxin efflux carrier PIN1 was hindered in rav1, leading to altered auxin transport and distribution. This effectively altered root meristem size in the mutant. Notably, CRF1, another member of the AP2/ERF family implicated in cytokinin signaling, is transcriptionally repressed by RAV1 to promote cytokinin response in roots. Further associating RAV1 with cytokinin signaling, our results demonstrate that cytokinin upregulates RAV1 expression through ARR1, during post-embryonic root development. Regulation of RAV1 expression is a part of secondary cytokinin response that eventually represses CRF1 to augment cytokinin signaling. To conclude, RAV1 functions in a branch pathway downstream to ARR1 that regulates CRF1 expression to enhance cytokinin action during primary root development in Arabidopsis.

MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE 1 mediates root sensing of serotonin through jasmonic acid signaling and modulating reactive oxygen species

Serotonin (5-hydroxytryptamine) acts as a neurotransmitter in mammals and is widely distributed in the plant kingdom, where it influences root growth and defense. Mitogen-Activated Protein Kinases (MAPKs) and MAPK phosphatases (MKPs) play critical functions in decoding hormonal signalling, but their possible roles in mediating serotonin responses await investigation. In this report, we unveiled positive roles for the MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE1 (MKP1) in the inhibition of the primary root growth, cell division, meristem structure, and differentiation events in Arabidopsis seedlings. mkp1 mutants were less sensitive to jasmonic acid applications that halted primary root growth in wild-type (WT) plants, and consistently, the neurotransmitter activated the expression of the JASMONATE ZIM-domain (JAZ) proteins JAZ1 and JAZ10, two critical proteins orchestrating jasmonic acid signalling. This effect correlated with exacerbated production of endogenous reactive oxygen species (ROS) in the WT, a process constitutively manifested in mkp1 mutants. These data help to clarify the relationship between serotonin and growth/defense trade-offs, and reveal the importance of the MAPK pathway in root development through ROS production.

Plant cell division from the perspective of polarity.

The orientation of cell division is a major determinant of plant morphogenesis. In spite of considerable efforts over the past decades, the precise mechanism of division plane selection remains elusive. The majority of studies on the topic have addressed division orientation from either a predominantly developmental or a cell biological perspective. Thus, mechanistic insights into the links between developmental and cellular factors affecting division orientation are particularly lacking. Here, I review recent progress in the understanding of cell division orientation in the embryo and primary root meristem of Arabidopsis from both developmental and cell biological standpoints. I offer a view of multilevel polarity as a central aspect of cell division: on the one hand, the division plane is a readout of tissue- and organism-wide polarities; on the other hand, the cortical division zone can be seen as a transient polar subcellular plasma membrane domain. Finally, I argue that a polarity-focused conceptual framework and the integration of developmental and cell biological approaches hold great promise to unravel the mechanistic basis of plant cell division orientation in the near future.

Participation of Auxin Transport in the Early Response of the Arabidopsis Root System to Inoculation with Azospirillum brasilense

The potential of Plant Growth Promoting Rhizobacteria (PGPR) has been demonstrated in the case of plant inoculation with bacteria of the genus Azospirillum which improves yield. A. brasilense produces a wide variety of molecules, including the natural auxin indole-3-acetic acid (IAA), as well as other phytoregulators. However, several studies have suggested that auxin induces changes in plant development during their interaction with the bacteria. The effects of A. brasilense Sp245 on the development of Arabidopsis thaliana root were investigated to help explain the molecular basis of the interaction. The results obtained showed a decrease in primary root length from the first day and remained so throughout the exposure, accompanied by a stimulation of initiation and maturation of lateral root primordia and an increase of lateral roots. An enhanced auxin response was evident in the vascular tissue and lateral root meristems of inoculated plants. However, after five days of bacterization, the response disappeared in the primary root meristems. The role of polar auxin transport (PAT) in auxins relocation involved the PGP1, AXR4-1, and BEN2 proteins, which apparently mediated A. brasilense-induced root branching of Arabidopsis seedlings.

Mutation of MEDIATOR 18 and chromate trigger twinning of the primary root meristem in Arabidopsis.

Plants adapt to soil injury and biotic stress via cell regeneration. In Arabidopsis, root tip damage by genotoxic agents, antibiotics, UV light and cutting induces a program that recovers the missing tissues through activation of stem cells and involves ethylene response factor 115 (ERF115), which triggers cell replenishment. Here, we show that mutation of the gene encoding an MED18 subunit of the transcriptional MEDIATOR complex and chromate [Cr(VI)], an environmental pollutant, synergistically trigger a developmental program that enables the splitting of the meristem in vivo to produce twin roots. Expression of the quiescent centre gene marker WOX5, auxin-inducible DR5:GFP reporter and the ERF115 factor traced the changes in cell identity during the conversion of single primary root meristems into twin roots and were induced in an MED18 and chromate-dependent manner during the root twinning events, which also required auxin redistribution and signalling mediated by IAA14/SOLITARY ROOT (SLR1). Splitting of the root meristem allowed dichotomous root branching in Arabidopsis, a poorly understood process in which stem cells may act to enable whole organ regeneration.

Processes controlling programmed cell death of root velamen radicum in an epiphytic orchid.

Development of the velamen radicum on the outer surface of the root epidermis is an important characteristic for water uptake and retention in some plant families, particularly epiphytic orchids, for survival under water-limited environments. Velamen radicum cells derive from the primary root meristem; however, following this development, velamen radicum cells die by incompletely understood processes of programmed cell death (PCD). We combined the use of transmission electron microscopy, X-ray micro-tomography and transcriptome methods to characterize the major anatomical and molecular changes that occur during the development and death of velamen radicum cells of Cymbidium tracyanum, a typical epiphytic orchid, to determine how PCD occurs. Typical changes of PCD in anatomy and gene expression were observed in the development of velamen radicum cells. During the initiation of PCD, we found that both cell and vacuole size increased, and several genes involved in brassinosteroid and ethylene pathways were upregulated. In the stage of secondary cell wall formation, significant anatomical changes included DNA degradation, cytoplasm thinning, organelle decrease, vacuole rupture and cell wall thickening. Changes were found in the expression of genes related to the biosynthesis of cellulose and lignin, which are instrumental in the formation of secondary cell walls, and are regulated by cytoskeleton-related factors and phenylalanine ammonia-lyase. In the final stage of PCD, cell autolysis was terminated from the outside to the inside of the velamen radicum. The regulation of genes related to autophagy, vacuolar processing enzyme, cysteine proteases and metacaspase was involved in the final execution of cell death and autolysis. Our results showed that the development of the root velamen radicum in an epiphytic orchid was controlled by the process of PCD, which included initiation of PCD, followed by formation of the secondary cell wall, and execution of autolysis following cell death.

The cysteine-rich receptor-like protein kinase CRK28 modulates Arabidopsis growth and development and influences abscisic acid responses.

CRK28, a cysteine-rich receptor-like kinase, plays a role in root organogenesis and overall growth of plants and antagonizes abscisic acid response in seed germination and primary root growth. Receptor-like kinases (RLK) orchestrate development and adaptation to environmental changes in plants. One of the largest RLK groups comprises cysteine-rich receptor-like kinases (CRKs), for which the function of most members remains unknown. In this report, we show that the loss of function of CRK28 led to the formation of roots that are longer and more branched than the parental (Col-0) plantlets, and this correlates with an enhanced domain of the mitotic reporter CycB1:uidA in primary root meristems, whereas CRK28 overexpressing lines had the opposite phenotype, including slow root growth and reduced lateral root formation. Epidermal cell analyses revealed that crk28 mutants had reduced root hair length and increased trichome number, whereas 35S::CRK28 lines present primary roots with longer root hairs but lesser trichomes in leaves. The overall growth in soil of crk28 mutant and CRK28 overexpressing lines was reduced or enhanced, respectively, when compared to the parental (Col-0) seedlings, while germination, root growth and expression analyses of ABI3 and ABI5 further showed that CRK28 modulates ABA responses, which may be important to fine-tune plant morphogenesis. Our study unravels the participation of RLK signaling in root growth and epidermal cell differentiation.

Mitogen-activated protein kinase 6 integrates phosphate and iron responses for indeterminate root growth in Arabidopsis thaliana.

A MAPK module, of which MPK6 kinase is an important component, is involved in the coordination of the responses to Pi and Fe in the primary root meristem of Arabidopsis thaliana. Phosphate (Pi) deficiency induces determinate primary root growth in Arabidopsis through cessation of cell division in the meristem, which is linked to an increased iron (Fe) accumulation. Here, we show that Mitogen-Activated Protein Kinase6 (MPK6) has a role in Arabidopsis primary root growth under low Pi stress. MPK6 activity is induced in roots in response to low Pi, and such induction is enhanced by Fe supplementation, suggesting an MPK6 role in coordinating Pi/Fe balance in mediating root growth. The differentiation of the root meristem induced by low Pi levels correlates with altered expression of auxin-inducible genes and auxin transporter levels via MPK6. Our results indicate a critical role of the MPK6 kinase in coordinating meristem cell activity to Pi and Fe availability for proper primary root growth.

Optimized Whole-Mount In Situ Immunolocalization for Arabidopsis thaliana Root Meristems and Lateral Root Primordia.

Immunolocalization is a valuable tool for cell biology research that allows to rapidly determine the localization and expression levels of endogenous proteins. In plants, whole-mount in situ immunolocalization remains a challenging method, especially in tissues protected by waxy layers and complex cell wall carbohydrates. Here, we present a robust method for whole-mount in situ immunolocalization in primary root meristems and lateral root primordia in Arabidopsis thaliana. For good epitope preservation, fixation is done in an alkaline paraformaldehyde/glutaraldehyde mixture. This fixative is suitable for detecting a wide range of proteins, including integral transmembrane proteins and proteins peripherally attached to the plasma membrane. From initiation until emergence from the primary root, lateral root primordia are surrounded by several layers of differentiated tissues with a complex cell wall composition that interferes with the efficient penetration of all buffers. Therefore, immunolocalization in early lateral root primordia requires a modified method, including a strong solvent treatment for removal of hydrophobic barriers and a specific cocktail of cell wall-degrading enzymes. The presented method allows for easy, reliable, and high-quality in situ detection of the subcellular localization of endogenous proteins in primary and lateral root meristems without the need of time-consuming crosses or making translational fusions to fluorescent proteins.