Glucose inhibits root meristem growth via ABA INSENSITIVE 5, which represses PIN1 accumulation and auxin activity in Arabidopsis

Glucose functions as a hormone‐like signalling molecule that modulates plant growth and development in Arabidopsis thaliana. However, the role of glucose in root elongation remains elusive. Our study demonstrates that high concentrations of glucose reduce the size of the root meristem zone by repressing PIN1 accumulation and thereby reducing auxin levels. In addition, we verified the involvement of ABA INSENSITIVE 5 (ABI5) in this process by showing that abi5‐1 is less sensitive to glucose than the wild type, whereas glucose induces ABI5 expression and the inducible overexpression of ABI5 reduces the size of the root meristem zone. Furthermore, the inducible overexpression of ABI5 in PIN1::PIN1‐GFP plants reduces the level of PIN1‐GFP, but glucose reduces the level of PIN1‐GFP to a lesser extent in abi5‐1 PIN1::PIN1‐GFP plants than in the PIN1::PIN1‐GFP control, suggesting that ABI5 is involved in glucose‐regulated PIN1 accumulation. Taken together, our data suggest that ABI5 functions in the glucose‐mediated inhibition of the root meristem zone by repressing PIN1 accumulation, thus leading to reduced auxin levels in roots.     

WD40‐REPEAT 5a represses root meristem growth by suppressing auxin synthesis through changes of nitric oxide accumulation in Arabidopsis

Although nitric oxide (NO) is known to regulate root growth, the factor(s) modulating NO during this process have not yet been elucidated. Here, we identified Arabidopsis WD40‐REPEAT 5a (WDR5a) as a novel factor that functions in root growth by modulating NO accumulation. The wdr5a‐1 mutant accumulated less NO and produced longer roots than the wild type, whereas the WDR5a overexpression lines had the opposite phenotype. The role of NO was further supported by our observation that the NO donor sodium nitroprusside (SNP) and the NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) rescued the root meristem growth phenotypes of the wdr5a‐1 and WDR5a overexpression lines, respectively. The regulation of root growth by WDR5a was found to involve auxin because the auxin levels were similar in SNP‐treated wdr5a‐1 and wild‐type roots, but higher in untreated wdr5a‐1 roots than in wild‐type roots. In addition, the wdr5a‐1 mutant had higher production and activity levels of the auxin biosynthetic enzyme TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1), in contrast to its reduced expression and activity in the WDR5a overexpression lines, and the increased root meristem growth in wdr5a‐1 was suppressed by treatment with l ‐kynurenine, which inhibits TAA1, as well as by mutating TAA1. WDR5a therefore functions in root meristem growth by maintaining NO homeostasis, and thus TAA1‐mediated auxin biosynthesis.     

RLF,a cytochrome b5‐like heme/steroid binding domain protein,controls lateral root formation independently of ARF7/19‐mediated auxin signaling in Arabidopsis thaliana

Lateral root (LR) formation is important for the establishment of root architecture in higher plants. Recent studies have revealed that LR formation is regulated by an auxin signaling pathway that depends on auxin response factors ARF7 and ARF19, and auxin/indole‐3‐acetic acid (Aux/IAA) proteins including SOLITARY‐ROOT (SLR)/IAA14. To understand the molecular mechanisms of LR formation, we isolated a recessive mutant rlf (reduced lateral root formation) in Arabidopsis thaliana. The rlf‐1 mutant showed reduction of not only emerged LRs but also LR primordia. Analyses using cell‐cycle markers indicated that the rlf‐1 mutation inhibits the first pericycle cell divisions involved in LR initiation. The rlf‐1 mutation did not affect auxin‐induced root growth inhibition but did affect LR formation over a wide range of auxin concentrations. However, the rlf‐1 mutation had almost no effect on auxin‐inducible expression of LATERAL ORGAN BOUNDARIES‐DOMAIN16/ASYMMETRIC LEAVES2‐LIKE18 (LBD16/ASL18) and LBD29/ASL16 genes, which are downstream targets of ARF7/19 for LR formation. These results indicate that ARF7/19‐mediated auxin signaling is not blocked by the rlf‐1 mutation. We found that the RLF gene encodes At5g09680, a protein with a cytochrome b₅‐like heme/steroid binding domain. RLF is ubiquitously expressed in almost all organs, and the protein localizes in the cytosol. These results, together with analysis of the genetic interaction between the rlf‐1 and arf7/19 mutations, indicate that RLF is a cytosolic protein that positively controls the early cell divisions involved in LR initiation, independent of ARF7/19‐mediated auxin signaling.     

A genotypic difference in primary root length is associated with the inhibitory role of transforming growth factor‐beta receptor‐interacting protein‐1 on root meristem size in wheat

Previously we identified a major quantitative trait locus (QTL) qTaLRO‐B1 for primary root length (PRL) in wheat. Here we compare proteomics in the roots of the qTaLRO‐B1 QTL isolines 178A, with short PRL and small meristem size, and 178B, with long PRL and large meristem size. A total of 16 differentially expressed proteins were identified: one, transforming growth factor (TGF)‐beta receptor‐interacting protein‐1 (TaTRIP1), was enriched in 178A, while various peroxidases (PODs) were more abundantly expressed in 178B. The 178A roots showed higher TaTRIP1 expression and lower levels of the unphosphorylated form of the brassinosteroid (BR) signaling component BZR1, lower expression of POD genes and reduced POD activity and accumulation of the superoxide anion O₂^? in the root elongation zone compared with the 178B roots. Low levels of 24‐epibrassinolide increased POD gene expression and root meristem size, and rescued the short PRL phenotype of 178A. TaTRIP1 directly interacted with the BR receptor TaBRI1 of wheat. Moreover, overexpressing TaTRIP1 in Arabidopsis reduced the abundance of unphosphorylated BZR1 protein, altered the expression of BR‐responsive genes, inhibited POD activity and accumulation of the O₂^? in the root tip and inhibited root meristem size. Our data suggested that TaTRIP1 is involved in BR signaling and inhibited root meristem size, possibly by reducing POD activity and accumulation of O₂^? in the root tip. We further demonstrated a negative correlation between the level of TaTRIP1 mRNA and PRL of landraces and modern wheat varieties, providing a valuable insight for better understanding of the molecular mechanism underlying the genotypic differences in root morphology of wheat in the future.     

Cyclin‐dependent kinase activity maintains the shoot apical meristem cells in an undifferentiated state

As the shoot apex produces most of the cells that comprise the aerial part of the plant, perfect orchestration between cell division rates and fate specification is essential for normal organ formation and plant development. However, the inter‐dependence of cell‐cycle machinery and meristem‐organizing genes is still poorly understood. To investigate this mechanism, we specifically inhibited the cell‐cycle machinery in the shoot apex by expression of a dominant negative allele of the A‐type cyclin‐dependent kinase (CDK) CDKA;1 in meristematic cells. A decrease in the cell division rate within the SHOOT MERISTEMLESS domain of the shoot apex dramatically affected plant growth and development. Within the meristem, a subset of cells was driven into the differentiation pathway, as indicated by premature cell expansion and onset of endo‐reduplication. Although the meristem structure and expression patterns of the meristem identity genes were maintained in most plants, the reduced CDK activity caused splitting of the meristem in some plants. This phenotype correlated with the level of expression of the dominant negative CDKA;1 allele. Therefore, we propose a threshold model in which the effect of the cell‐cycle machinery on meristem organization is determined by the level of CDK activity.     

SHY2 as a node in the regulation of root meristem development by auxin,brassinosteroids, and cytokinin

In multicellular organisms, the balance between cell division and differentiation determines organ size, and represents a central unknown in developmental biology. In Arabidopsis roots, this balance is mediated between cytokinin and auxin through a regulatory circuit converging on the IAA3/SHORT HYPOCOTYL 2 (SHY2) gene. Here, we show that crosstalk between brassinosteroids (BRs) and auxin occurs in the vascular transition zone to promote root meristem development. We found that BR increases root meristem size by up‐regulating expression of the PINFORMED 7 (PIN7) gene and down‐regulating expression of the SHY2 gene. In addition, BES1 could directly bind to the promoter regions of both PIN7 and SHY2, indicating that PIN7 and SHY2 mediate the BR‐induced growth of the root meristem by serving as direct targets of BES1. Moreover, the PIN7 overexpression and loss‐of‐function SHY2 mutant were sensitive to the effects of BR and could partially suppress the short‐root phenotypes associated with deficient BR signaling. Interestingly, BRs could inhibit the accumulation of SHY2 protein in response to cytokinin. Taken together, these findings suggest that a complex equilibrium model exists in which regulatory interactions among BRs, auxin, and cytokinin regulate optimal root growth.     

DAR2 acts as an important node connecting cytokinin,auxin, SHY2 and PLT1/2 in root meristem size control

Cytokinin and auxin antagonistically affect cell proliferation and differentiation and thus regulate root meristem size by influencing the abundance of SHORT HYPOCOTYL2 (SHY2/IAA3). SHY2 affects auxin distribution in the root meristem by repressing the auxin-inducible expression of PIN-FORMED (PIN) auxin transport genes. The PLETHORA (PLT1/2) genes influence root meristem growth by promoting stem cells and transit-amplifying cells. However, the factors connecting cytokinin, auxin, SHY2 and PLT1/2 are largely unknown. In a recent study, we have shown that the DA1-related protein 2 (DAR2) acts downstream of cytokinin and SHY2 but upstream of PLT1/2 to affect root meristem size. Here, we discuss the possible molecular mechanisms by which Arabidopsis DAR2 controls root meristem size.     

Auxin biosynthetic gene TAR2 is involved in low nitrogen‐mediated reprogramming of root architecture in Arabidopsis

In plants, the plasticity of root architecture in response to nitrogen availability largely determines nitrogen acquisition efficiency. One poorly understood root growth response to low nitrogen availability is an observed increase in the number and length of lateral roots (LRs). Here, we show that low nitrogen‐induced Arabidopsis LR growth depends on the function of the auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 was expressed in the pericycle and the vasculature of the mature root zone near the root tip, and was induced under low nitrogen conditions. In wild type plants, low nitrogen stimulated auxin accumulation in the non‐emerged LR primordia with more than three cell layers and LR emergence. Conversely, these low nitrogen‐mediated auxin accumulation and root growth responses were impaired in the tar2‐c null mutant. Overexpression of TAR2 increased LR numbers under both high and low nitrogen conditions. Our results suggested that TAR2 is required for reprogramming root architecture in response to low nitrogen conditions. This finding suggests a new strategy for improving nitrogen use efficiency through the engineering of TAR2 expression in roots.     

Dynamics of auxin-dependent Ca2+ and pH signaling in root growth revealed by integrating high-resolution imaging with automated computer vision-based analysis

Plants adapt to a changing environment by entraining their growth and development to prevailing conditions. Such 'plastic' development requires a highly dynamic integration of growth phenomena with signal perception and transduction systems, such as occurs during tropic growth. The plant hormone auxin has been shown to play a key role in regulating these directional growth responses of plant organs to environmental cues. However, we are still lacking a cellular and molecular understanding of how auxin-dependent signaling cascades link stimulus perception to the rapid modulation of growth patterns. Here, we report that in root gravitropism of Arabidopsis thaliana, auxin regulates root curvature and associated apoplastic, growth-related pH changes through a Ca2+-dependent signaling pathway. Using an approach that integrates confocal microscopy and automated computer vision-based image analysis, we demonstrate highly dynamic root surface pH patterns during vertical growth and after gravistimulation. These pH dynamics are shown to be dependent on auxin, and specifically on auxin transport mediated by the auxin influx carrier AUX1 in cells of the lateral root cap and root epidermis. Our results further indicate that these pH responses require auxin-dependent changes in cytosolic Ca2+ levels that operate independently of the TIR1 auxin perception system. These results demonstrate a methodology that can be used to visualize vectorial auxin responses in a manner that can be integrated with the rapid plant growth responses to environmental stimuli.     

Specialized functions of the PP2A subfamily II catalytic subunits PP2A‐C3 and PP2A‐C4 in the distribution of auxin fluxes and development in Arabidopsis

Protein phosphorylation is a key molecular switch used to transmit information in biological signalling networks. The output of these signalling circuits is governed by the counteracting activities of protein kinases and phosphatases that determine the direction of the switch. Whereas many kinases have been functionally characterized, it has been difficult to ascribe precise cellular roles to plant phosphatases, which are encoded by enlarged gene families that may provide a high degree of genetic redundancy. In this work we have analysed the role in planta of catalytic subunits of protein phosphatase 2A (PP2A), a family encoded by five genes in Arabidopsis. Our results indicate that the two members of subfamily II, PP2A‐C3 and PP2A‐C4, have redundant functions in controlling embryo patterning and root development, processes that depend on auxin fluxes. Moreover, polarity of the auxin efflux carrier PIN1 and auxin distribution, determined with the DR5_pro:GFP proxy, are affected by mutations in PP2A‐C3 and PP2A‐C4. Previous characterization of mutants in putative PP2A regulatory subunits had established a link between this class of phosphatases and PIN dephosphorylation and subcellular distribution. Building on those findings, the results presented here suggest that PP2A‐C3 and PP2A‐C4 catalyse this reaction and contribute critically to the establishment of auxin gradients for proper plant development.     

OsMOGS is required for N‐glycan formation and auxin‐mediated root development in rice (Oryza sativa L.)

N‐glycosylation is a major modification of glycoproteins in eukaryotic cells. In Arabidopsis, great progress has been made in functional analysis of N‐glycan production, however there are few studies in monocotyledons. Here, we characterized a rice (Oryza sativa L.) osmogs mutant with shortened roots and isolated a gene that coded a putative mannosyl‐oligosaccharide glucosidase (OsMOGS), an ortholog of α‐glucosidase I in Arabidopsis, which trims the terminal glucosyl residue of the oligosaccharide chain of nascent peptides in the endoplasmic reticulum (ER). OsMOGS is strongly expressed in rapidly cell‐dividing tissues and OsMOGS protein is localized in the ER. Mutation of OsMOGS entirely blocked N‐glycan maturation and inhibited high‐mannose N‐glycan formation. The osmogs mutant exhibited severe defects in root cell division and elongation, resulting in a short‐root phenotype. In addition, osmogs plants had impaired root hair formation and elongation, and reduced root epidemic cell wall thickness due to decreased cellulose synthesis. Further analysis showed that auxin content and polar transport in osmogs roots were reduced due to incomplete N‐glycosylation of the B subfamily of ATP‐binding cassette transporter proteins (ABCBs). Our results demonstrate that involvement of OsMOGS in N‐glycan formation is required for auxin‐mediated root development in rice.     

SEUSS and SEUSS‐LIKE 2 coordinate auxin distribution and KNOXI activity during embryogenesis

In Arabidopsis, SEUSS (SEU) and SEUSS‐LIKE 2 (SLK2) are components of the LEUNIG (LUG) repressor complex that coordinates various aspects of post‐embryonic development. The complex also plays a critical role during embryogenesis, as seu slk2 double mutants have small, narrow cotyledons and lack a shoot apical meristem (SAM). Here we show that seu slk2 double mutant embryos exhibit delayed cotyledon outgrowth and that this is associated with altered PIN‐FORMED1 (PIN1) expression and localisation during the early stages of embryogenesis. These observations suggest that SEU and SLK2 promote the transition to bilateral symmetry by modulating auxin distribution in the embryonic shoot. This study also shows that loss of SAM formation in seu slk2 mutants is associated with reduced expression of the class I KNOX (KNOXI) genes SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS and KNAT2. Furthermore, elevating STM expression in seu slk2 mutant embryos was sufficient to restore SAM formation but not post‐embryonic activity, while both SAM formation and activity were rescued when SLK2 expression was restored in either the cotyledons or boundary regions. These results demonstrate that SEU and SLK2 function redundantly to promote embryonic shoot development and likely act through a non‐cell autonomous pathway to promote KNOXI activity.     

Effect of abscisic acid on endogenous IAA, auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata cuttings

Endogenous levels of free and conjugated IAA, auxin protectors (Prs) and peroxidase (PER) activity and their relation to adventitious root initiation (ARI) were investigated at the potential sites of adventitious rooting in relation to exogenous application of 250 μM ABA during the first 120 h after treatment. Cuttings from 7-day-old mung bean [Vigna radiata (L.) Wilcz.] seedlings were treated with 125, 250, and 500 μM ABA for 24 h. ABA significantly stimulated ARI but extremely inhibited epicotyl growth as compared to control. Free and conjugated IAA were measured by reversed-phase high performance liquid chromatography while Prs and PER activities were measured spectrophotometrically. The present results also indicate that endogenous free IAA levels peaked later in ABA-treated cuttings than that in control, suggesting that ABA extended the length of the induction phase of rooting process in treated cuttings and that might explain the significant delay of the appearance of roots at the treated cuttings. Higher level of IAA conjugates was found in ABA-treated cuttings than that in untreated ones. Pr level also peaked later in ABA-treated cuttings than that in control, indicating that ABA extended the period of Pr activity. An initial temporary decrease of PER activity was found in associating with high levels of free IAA and Prs during most of the primary events, while the opposite occurred during the secondary events of adventitious rooting process in both treated and untreated cuttings. Thus, ABA may stimulate ARI in mung bean Vigna radiata cuttings by regulating the concentration and /or activities of endogenous IAA, Prs, and PER activity in favor of inducing a large number of adventitious roots at their potential sites of adventitious rooting.     

AUXIN RESPONSE FACTOR 3 integrates the functions of AGAMOUS and APETALA2 in floral meristem determinacy

In Arabidopsis, AUXIN RESPONSE FACTOR 3 (ARF3) belongs to the auxin response factor (ARF) family that regulates the expression of auxin‐responsive genes. ARF3 is known to function in leaf polarity specification and gynoecium patterning. In this study, we discovered a previously unknown role for ARF3 in floral meristem (FM) determinacy through the isolation and characterization of a mutant of ARF3 that enhanced the FM determinacy defects of agamous (ag)‐10, a weak ag allele. Central players in FM determinacy include WUSCHEL (WUS), a gene critical for FM maintenance, and AG and APETALA2 (AP2), which regulate FM determinacy by repression and promotion of WUS expression, respectively. We showed that ARF3 confers FM determinacy through repression of WUS expression, and associates with the WUS locus in part in an AG‐dependent manner. We demonstrated that ARF3 is a direct target of AP2 and partially mediates AP2's function in FM determinacy. ARF3 exhibits dynamic and complex expression patterns in floral organ primordia; altering the patterns spatially compromised FM determinacy. This study uncovered a role for ARF3 in FM determinacy and revealed relationships among genes in the genetic network governing FM determinacy.