Auxin and cytokinin control formation of the quiescent centre in the adventitious root apex of Arabidopsis.
ABSTRACT: BACKGROUND AND AIMS: Adventitious roots (ARs) are part of the root system in numerous plants, and are required for successful micropropagation. In the Arabidopsis thaliana primary root (PR) and lateral roots (LRs), the quiescent centre (QC) in the stem cell niche of the meristem controls apical growth with the involvement of auxin and cytokinin. In arabidopsis, ARs emerge in planta from the hypocotyl pericycle, and from different tissues in in vitro cultured explants, e.g. from the stem endodermis in thin cell layer (TCL) explants. The aim of this study was to investigate the establishment and maintenance of the QC in arabidopsis ARs, in planta and in TCL explants, because information about this process is still lacking, and it has potential use for biotechnological applications. METHODS: Expression of PR/LR QC markers and auxin influx (LAX3)/efflux (PIN1) genes was investigated in the presence/absence of exogenous auxin and cytokinin. Auxin was monitored by the DR5::GUS system and cytokinin by immunolocalization. The expression of the auxin-biosynthetic YUCCA6 gene was also investigated by in situ hybridization in planta and in AR-forming TCLs from the indole acetic acid (IAA)-overproducing superroot2-1 mutant and its wild type. KEY RESULTS: The accumulation of auxin and the expression of the QC marker WOX5 characterized the early derivatives of the AR founder cells, in planta and in in vitro cultured TCLs. By determination of PIN1 auxin efflux carrier and LAX3 auxin influx carrier activities, an auxin maximum was determined to occur at the AR tip, to which WOX5 expression was restricted, establishing the positioning of the QC. Cytokinin caused a restriction of LAX3 and PIN1 expression domains, and concomitantly the auxin biosynthesis YUCCA6 gene was expressed in the apex. CONCLUSIONS: In ARs formed in planta and TCLs, the QC is established in a similar way, and auxin transport and biosynthesis are involved through cytokinin tuning.
Project description:The elongator complex subunit 2 (ELP2) protein, one subunit of an evolutionarily conserved histone acetyltransferase complex, has been shown to participate in leaf patterning, plant immune and abiotic stress responses in Arabidopsis thaliana. Here, its role in root development was explored. Compared to the wild type, the elp2 mutant exhibited an accelerated differentiation of its root stem cells and cell division was more active in its quiescent centre (QC). The key transcription factors responsible for maintaining root stem cell and QC identity, such as AP2 transcription factors PLT1 (PLETHORA1) and PLT2 (PLETHORA2), GRAS transcription factors such as SCR (SCARECROW) and SHR (SHORT ROOT) and WUSCHEL-RELATED HOMEOBOX5 transcription factor WOX5, were all strongly down-regulated in the mutant. On the other hand, expression of the G2/M transition activator CYCB1 was substantially induced in elp2. The auxin efflux transporters PIN1 and PIN2 showed decreased protein levels and PIN1 also displayed mild polarity alterations in elp2, which resulted in a reduced auxin content in the root tip. Either the acetylation or methylation level of each of these genes differed between the mutant and the wild type, suggesting that the ELP2 regulation of root development involves the epigenetic modification of a range of transcription factors and other developmental regulators.
Project description:BACKGROUND:Adventitious root (AR) formation is a complex genetic trait, which is controlled by various endogenous and environmental cues. Auxin is known to play a central role in AR formation; however, the mechanisms underlying this role are not well understood. RESULTS:In this study, we showed that a previously identified auxin signaling module, AUXIN RESPONSE FACTOR(ARF)7/ARF19-LATERAL ORGAN BOUNDARIES DOMAIN(LBD)16/LBD18 via AUXIN1(AUX1)/LIKE-AUXIN3 (LAX3) auxin influx carriers, which plays important roles in lateral root formation, is involved in AR formation in Arabidopsis. In aux1, lax3, arf7, arf19, lbd16 and lbd18 single mutants, we observed reduced numbers of ARs than in the wild type. Double and triple mutants exhibited an additional decrease in AR numbers compared with the corresponding single or double mutants, respectively, and the aux1 lax3 lbd16 lbd18 quadruple mutant was devoid of ARs. Expression of LBD16 or LBD18 under their own promoters in lbd16 or lbd18 mutants rescued the reduced number of ARs to wild-type levels. LBD16 or LBD18 fused to a dominant SRDX repressor suppressed promoter activity of the cell cycle gene, Cyclin-Dependent Kinase(CDK)A1;1, to some extent. Expression of LBD16 or LBD18 was significantly reduced in arf7 and arf19 mutants during AR formation in a light-dependent manner, but not in arf6 and arf8. GUS expression analysis of promoter-GUS reporter transgenic lines revealed overlapping expression patterns for LBD16, LBD18, ARF7, ARF19 and LAX3 in AR primordia. CONCLUSION:These results suggest that the ARF7/ARF19-LBD16/LBD18 transcriptional module via the AUX1/LAX3 auxin influx carriers plays an important role in AR formation in Arabidopsis.
Project description:Root stem cell niche functioning requires the formation and maintenance of the specific "auxin-rich domain" governed by directional auxin transport and local auxin production. Auxin maximum co-localizes with the WOX5 expression domain in the quiescent center that separates mitotically active proximal and distal root meristems. Here we unravel the interconnected processes happening under WOX5 overexpression by combining in vivo experiments and mathematical modeling. We showed that WOX5-induced TAA1-mediated auxin biosynthesis is the cause, whereas auxin accumulation, PIN transporters relocation, and auxin redistribution between proximal and distal root meristems are its subsequent effects that influence the formation of the well-described phenotype with an enlarged root cap. These findings helped us to clarify the role of WOX5, which serves as a local QC-specific regulator that activates biosynthesis of non-cell-autonomous signal auxin to regulate the distal meristem functioning. The mathematical model with WOX5-mediated auxin biosynthesis and auxin-regulated cell growth, division, and detachment reproduces the columella cells dynamics in both wild type and under WOX5 dysregulation.
Project description:Somatic embryogenesis (SE) is induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but rarely in wild type Jemalong. The putative WUSCHEL (MtWUS), CLAVATA3 (MtCLV3) and the WUSCHEL-related homeobox gene WOX5 (MtWOX5) were investigated in M. truncatula (Mt) and identified by the similarity to Arabidopsis WUS, CLV3 and WOX5 in amino acid sequence, phylogeny and in planta and in vitro expression patterns. MtWUS was induced throughout embryogenic cultures by cytokinin after 24-48 h and maximum expression occurred after 1 week, which coincides with the induction of totipotent stem cells. During this period there was no MtCLV3 expression to suppress MtWUS. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localised to the embryo, and there was then the onset of MtCLV3 expression. This suggests that the expression of the putative MtCLV3 coincides with the WUS-CLAVATA feedback loop becoming operational. RNAi studies showed that MtWUS expression is essential for callus and somatic embryo production. Based on the presence of MtWUS promoter binding sites, MtWUS may be required for the induction of MtSERF1, postulated to have a key role in the signalling required for SE induced in 2HA. MtWOX5 expressed in auxin-induced root primordia and root meristems and appears to be involved in pluripotent stem cell induction. The evidence is discussed that the homeobox genes MtWUS and MtWOX5 are "hijacked" for stem cell induction, which is key to somatic embryo and de novo root induction. In relation to SE, a role for WUS in the signalling involved in induction is discussed.
Project description:Co-ordination of auxin and cytokinin activities determines root meristem size during post-embryonic development. Calcineurin B-like proteins (CBLs) and their interacting protein kinases (CIPKs) constitute signaling modules that relay calcium signals. Here we report that CIPK25 is involved in regulating the root meristem size. Arabidopsis plants lacking CIPK25 expression displayed a short root phenotype and a slower root growth rate with fewer meristem cells. This phenotype was rescued by restoration of CIPK25 expression. CIPK25 interacted with CBL4 and -5, and displayed strong gene expression in the flower and root, except in the cell proliferation domain in the root apical meristem. Its expression in the root was positively and negatively regulated by auxin and cytokinin, respectively. The cipk25 T-DNA insertion line was compromised in auxin transport and auxin-responsive promoter activity. The cipk25 mutant line showed altered expression of auxin efflux carriers (PIN1 and PIN2) and an Aux/IAA family gene SHY2. Decreased PIN1 and PIN2 expression in the cipk25 mutant line was completely restored when combined with a SHY2 loss-of-function mutation, resulting in recovery of root growth. SHY2 and PIN1 expression was partially regulated by cytokinin even in the absence of CIPK25, suggesting a CIPK25-independent cytokinin signaling pathway(s). Our results revealed that CIPK25 plays an important role in the co-ordination of auxin and cytokinin signaling in root meristem development.
Project description:Postembryonic de novo organogenesis represents an important competence evolved in plants that allows their physiological and developmental adaptation to changing environmental conditions. The phytohormones auxin and cytokinin (CK) are important regulators of the developmental fate of pluripotent plant cells. However, the molecular nature of their interaction(s) in control of plant organogenesis is largely unknown. Here, we show that CK modulates auxin-induced organogenesis (AIO) via regulation of the efflux-dependent intercellular auxin distribution. We used the hypocotyl explants-based in vitro system to study the mechanism underlying de novo organogenesis. We show that auxin, but not CK, is capable of triggering organogenesis in hypocotyl explants. The AIO is accompanied by endogenous CK production and tissue-specific activation of CK signaling. CK affects differential auxin distribution, and the CK-mediated modulation of organogenesis is simulated by inhibition of polar auxin transport. CK reduces auxin efflux from cultured tobacco cells and regulates expression of auxin efflux carriers from the PIN family in hypocotyl explants. Moreover, endogenous CK levels influence PIN transcription and are necessary to maintain intercellular auxin distribution in planta. Based on these findings, we propose a model in which auxin acts as a trigger of the organogenic processes, whose output is modulated by the endogenously produced CKs. We propose that an important mechanism of this CK action is its effect on auxin distribution via regulation of expression of auxin efflux carriers.
Project description:Understanding the mechanisms regulating root development under drought conditions is an important question for plant biology and world agriculture. We examine the effect of osmotic stress on abscisic acid (ABA), cytokinin and ethylene responses and how they mediate auxin transport, distribution and root growth through effects on PIN proteins. We integrate experimental data to construct hormonal crosstalk networks to formulate a systems view of root growth regulation by multiple hormones. Experimental analysis shows: that ABA-dependent and ABA-independent stress responses increase under osmotic stress, but cytokinin responses are only slightly reduced; inhibition of root growth under osmotic stress does not require ethylene signalling, but auxin can rescue root growth and meristem size; osmotic stress modulates auxin transporter levels and localization, reducing root auxin concentrations; PIN1 levels are reduced under stress in an ABA-dependent manner, overriding ethylene effects; and the interplay among ABA, ethylene, cytokinin and auxin is tissue-specific, as evidenced by differential responses of PIN1 and PIN2 to osmotic stress. Combining experimental analysis with network construction reveals that ABA regulates root growth under osmotic stress conditions via an interacting hormonal network with cytokinin, ethylene and auxin.
Project description:The phytohormones auxin and cytokinin control development and maintenance of plant meristems and stem cell systems. Fluorescent protein reporter lines that monitor phytohormone controlled gene expression programmes have been widely used to study development and differentiation in the model species Arabidopsis, but equivalent tools are still missing for the majority of crop species. Barley (Hordeum vulgare) is the fourth most abundant cereal crop plant, but knowledge on these important phytohormones in regard to the barley root and shoot stem cell niches is still negligible. We have now analysed the role of auxin and cytokinin in barley root meristem development, and present fluorescent protein reporter lines that allow to dissect auxin and cytokinin signalling outputs in vivo. We found that application of either auxin or cytokinin to barley seedlings negatively impacts root meristem growth. We further established a barley cytokinin reporter, TCSnew, which revealed significant cytokinin signalling in the stele cells proximal to the QC, and in the differentiated root cap cells. Application of exogenous cytokinin activated signalling in the root stem cell niche. Commonly employed auxin reporters DR5 or DR5v2 failed to respond to auxin in barley. However, analysis of putative auxin signalling targets barley PLETHORA1 (HvPLT1) is expressed in a similar pattern as its orthologue AtPLT1 from Arabidopsis, i.e. in the QC and the surrounding cells. Furthermore, the PINFORMED1 (HvPIN1) auxin efflux carrier was found to be expressed in root and shoot meristems, where it polarly localized to the plasma membrane. HvPIN1 expression is negatively regulated by cytokinin and its intracellular localisation is sensitive to brefeldinA (BFA). With this study, we provide the first fluorescent reporter lines as a tool to study auxin and cytokinin signalling and response pathways in barley.
Project description:In Arabidopsis, stem cells maintain the provision of new cells for root growth. They surround a group of slowly dividing cells named the quiescent center (QC), and, together, they form the stem cell niche (SCN). The QC acts as the signaling center of the SCN, repressing differentiation of the surrounding stem cells and providing a pool of cells able to replace damaged stem cells. Maintenance of the stem cells depends on the transcription factor WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the QC. However, the molecular mechanisms by which WOX5 promotes stem cell fate and whether WOX5 regulates proliferation of the QC are unknown. Here, we reveal a new role for WOX5 in restraining cell division in the cells of the QC, thereby establishing quiescence. In contrast, WOX5 and CYCD3;3/CYCD1;1 both promote cell proliferation in the nascent columella. The additional QC divisions occurring in wox5 mutants are suppressed in mutant combinations with the D type cyclins CYCD3;3 and CYCD1;1. Moreover, ectopic expression of CYCD3;3 in the QC is sufficient to induce cell division in the QC. WOX5 thus suppresses QC divisions that are otherwise promoted by CYCD3;3 and CYCD1;1, in part by interacting with the CYCD3;3 promoter to repress CYCD3;3 expression in the QC. Therefore, we propose a specific role for WOX5 in initiating and maintaining quiescence of the QC by excluding CYCD activity from the QC.
Project description:In Arabidopsis thaliana, besides several key transcription factors and chromatin modifiers, phytohormones auxin and cytokinin play pivotal role in shoot and root meristem maintenance, and lateral root (LR) development. Sirtinol, a chemical inhibitor of Sir2 proteins, is known to promote some auxin induced phenotypes in Arabidopsis. However, its effect on plant stem cell maintenance or organ formation remained unaddressed. Here we show that sirtinol affects meristem maintenance by altering the expression of key stem cell regulators, cell division and differentiation by modulating both auxin and cytokinin signaling in Arabidopsis thaliana. The expression of shoot stem cell niche related genes WUSCHEL (WUS) and CLAVATA3 (CLV3) was upregulated, whereas SHOOT MERISTEMLESS (STM) was downregulated in sirtinol treated seedlings. The expression level and domain of key root stem cell regulators PLETHORA (PLTs) and WUS-Related Homeobox 5 (WOX5) were altered in sirtinol treated roots. Sirtinol affects LR development by disturbing proper auxin transport and maxima formation, similar to 2,4-dichlorophenoxyacetic acid (2,4-D). Sirtinol also affects LR formation by altering cytokinin biosynthesis and signaling genes in roots. Therefore, sirtinol affects shoot and root growth, meristem maintenance and LR development by altering the expression of cytokinin-auxin signaling components, and regulators of stem cells, meristems, and LRs.