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.     

Synergistic action of auxin and cytokinin mediates aluminum‐induced root growth inhibition in Arabidopsis

Auxin acts synergistically with cytokinin to control the shoot stem‐cell niche, while both hormones act antagonistically to maintain the root meristem. In aluminum (Al) stress‐induced root growth inhibition, auxin plays an important role. However, the role of cytokinin in this process is not well understood. In this study, we show that cytokinin enhances root growth inhibition under stress by mediating Al‐induced auxin signaling. Al stress triggers a local cytokinin response in the root‐apex transition zone (TZ) that depends on IPTs, which encode adenosine phosphate isopentenyltransferases and regulate cytokinin biosynthesis. IPTs are up‐regulated specifically in the root‐apex TZ in response to Al stress and promote local cytokinin biosynthesis and inhibition of root growth. The process of root growth inhibition is also controlled by ethylene signaling which acts upstream of auxin. In summary, different from the situation in the root meristem, auxin acts with cytokinin in a synergistic way to mediate aluminum‐induced root growth inhibition in Arabidopsis.     

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 UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCF(AUF1/2) provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.     

Melatonin Antagonizes Cytokinin Responses to Stimulate Root Growth in Arabidopsis

Melatonin functions as the key growth regulator in various plant species. The mechanisms of the interactions between melatonin and cytokinins remain largely unknown. In this study, the kinetic effects of melatonin over a range of concentrations were investigated. The results showed that melatonin functioned as a positive regulator of root growth ranged from 0.1 to 100 nM. In contrast, exogenous cytokinin at 0.5–1 nM and overexpression of cytokinin biosynthesis gene ISOPENTENYLTRANSFERASE 8 (IPT8) inhibited primary root growth. Combined treatments with melatonin and cytokinin indicated that melatonin antagonized the inhibitory effect of cytokinin 6-benzylaminopurine (6-BA) on primary root elongation. Further analysis revealed that melatonin promotes primary root growth by modulating expression and distribution of auxin efflux transporters PIN2/3 and influx transporter AUX1. Moreover, the cytokinin signaling components AHK4, AHP2/3/5, and type-A ARR15 were down-regulated after melatonin treatment. The polar auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) impaired the promotive effect of melatonin on primary root growth, indicating that auxin transport is essential in melatonin-mediated root growth. Taken together, our data provided evidence to show that melatonin regulates primary root growth in coordination with cytokinin partially through auxin-dependent pathway.

     

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.     

The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development

Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.     

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.     

Arabidopsis cytokinin-resistant mutant, cnr1, displays altered auxin responses and sugar sensitivity

Based upon the phenotype of young, dark-grown seedlings, a cytokinin-resistant mutant, cnr1, has been isolated, which displays altered cytokinin- and auxin-induced responses. The mutant seedlings possess short hypocotyls and open apical hooks (in dark), and display agravitropism, hyponastic cotyledons, reduced shoot growth, compact rosettes and short roots with increased adventitious branching and reduced number of root hairs. A number of these features invariably depend upon auxin/cytokinin ratio but the cnr1 mutant retains normal sensitivity towards auxin as well as auxin polar transport inhibitor, TIBA, although upregulation of primary auxin-responsive Aux/IAA genes is reduced. The mutant shows resistance towards cytokinin in hypocotyl/root growth inhibition assays, displays reduced regeneration in tissue cultures (cytokinin response) and decreased sensitivity to cytokinin for anthocyanin accumulation. It is thus conceivable that due to reduced sensitivity to cytokinin, the cnr1 mutant also shows altered auxin response. Surprisingly, the mutant retains normal sensitivity to cytokinin for induction of primary response genes, the type-A Arabidopsis response regulators, although the basal level of their expression was considerably reduced as compared to the wild-type. The zeatin and zeatin riboside levels, as estimated by HPLC, and the cytokinin oxidase activity were comparable in the cnr1 mutant and the wild-type. The hypersensitivity to red light (in hypocotyl growth inhibition assay), partial photomorphogenesis in dark, and hypersensitivity to sugars, are some other features displayed by the cnr1 mutant. The lesion in the cnr1 mutant has been mapped to the top of chromosome 1 where no other previously known cytokinin-resistant mutant has been mapped, indicating that the cnr1 mutant defines a novel locus involved in hormone, light and sugar signalling.     

The Interaction between Rice ERF3 and WOX11 Promotes Crown Root Development by Regulating Gene Expression Involved in Cytokinin Signaling

Crown roots are the main components of the fibrous root system in rice (Oryza sativa). WOX11, a WUSCHEL-related homeobox gene specifically expressed in the emerging crown root meristem, is a key regulator in crown root development. However, the nature of WOX11 function in crown root development has remained elusive. Here, we identified a rice AP2/ERF protein, ERF3, which interacts with WOX11 and was expressed in crown root initials and during crown root growth. Functional analysis revealed that ERF3 was essential for crown root development and acts in auxin- and cytokinin-responsive gene expression. Downregulation of ERF3 in wox11 mutants produced a more severe root phenotype. Also, increased expression of ERF3 could partially complement wox11, indicating that the two genes functioned cooperatively to regulate crown root development. ERF3 and WOX11 shared a common target, the cytokinin-responsive gene RR2. The expression of ERF3 and WOX11 only partially overlapped, underlining a spatio-temporal control of RR2 expression and crown root development. Furthermore, ERF3-regulated RR2 expression was involved in crown root initiation, while the ERF3/WOX11 interaction likely repressed RR2 during crown root elongation. These results define a mechanism regulating gene expression involved in cytokinin signaling during different stages of crown root development in rice.     

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild‐type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss‐of‐function mutations, or in a dominant (gain‐of‐function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.