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Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation.

ABSTRACT: Rapid alkalinization factor (RALF) is a peptide signal that plays a basic role in cell biology and most likely regulates cell expansion. In this study, transgenic Arabidopsis thaliana lines with high and low levels of AtRALF1 transcripts were used to investigate this peptide's mechanism of action. Overexpression of the root-specific isoform AtRALF1 resulted in reduced cell size. Conversely, AtRALF1 silencing increased root length by increasing the size of root cells. AtRALF1-silenced plants also showed an increase in the number of lateral roots, whereas AtRALF1 overexpression produced the opposite effect. In addition, four AtRALF1-inducible genes were identified: two genes encoding proline-rich proteins (AtPRP1 and AtPRP3), one encoding a hydroxyproline-rich glycoprotein (AtHRPG2), and one encoding a xyloglucan endotransglucosylase (TCH4). These genes were expressed in roots and involved in cell-wall rearrangement, and their induction was concentration dependent. Furthermore, AtRALF1-overexpressing plants were less sensitive to exogenous brassinolide (BL); upon BL treatment, the plants showed no increase in root length and a compromised increase in hypocotyl elongation. In addition, the treatment had no effect on the number of emerged lateral roots. AtRALF1 also induces two brassinosteroid (BR)-downregulated genes involved in the BR biosynthetic pathway: the cytochrome P450 monooxygenases CONSTITUTIVE PHOTOMORPHISM AND DWARFISM (CPD) and DWARF4 (DWF4). Simultaneous treatment with both AtRALF1 and BL caused a reduction in AtRALF1-inducible gene expression levels, suggesting that these signals may compete for components shared by both pathways. Taken together, these results indicate an opposing effect of AtRALF1 and BL, and suggest that RALF's mechanism of action could be to interfere with the BR signalling pathway.

SUBMITTER: Bergonci T

PROVIDER: S-EPMC3991750 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation.

Bergonci Tábata T Ribeiro Bianca B Ceciliato Paulo H O PH Guerrero-Abad Juan Carlos JC Silva-Filho Marcio C MC Moura Daniel S DS

Journal of experimental botany 20140311 8

Rapid alkalinization factor (RALF) is a peptide signal that plays a basic role in cell biology and most likely regulates cell expansion. In this study, transgenic Arabidopsis thaliana lines with high and low levels of AtRALF1 transcripts were used to investigate this peptide's mechanism of action. Overexpression of the root-specific isoform AtRALF1 resulted in reduced cell size. Conversely, AtRALF1 silencing increased root length by increasing the size of root cells. AtRALF1-silenced plants also ...[more]

PMID: 24620000

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Project description:Background and aimsLATERAL ORGAN BOUNDARIES DOMAIN 29 (LBD29), an important molecule downstream of auxin response factors ARF7 and ARF19, has a critical role in lateral root formation in Arabidopsis thaliana. The cell cycle activation of pericycle cells and their speciﬁcation triggered by auxin are crucial for the initiation of lateral roots. In this study, we attempted to determine whether LBD29 is involved in auxin signalling and/or cell cycle regulation and to characterize the roles of LBD29 in these processes.MethodsThe impact of LBD29 on cell cycling progression in pericycle cells was investigated in lbd29 loss-of-function mutant or LBD29-over-expressing plants. The cell cycle was determined by measuring the expression of some cell cycle-related genes using in situ hybridization and quantitative real-time reverse transcription-PCR (qRT-PCR). Furthermore, the cell division in the root explants from either the lbd29 mutant, LBD29-over-expressing plants or the wild type grown in auxin-rich media was also analysed and compared by the distribution of DR5:β-glucuronidase (GUS) in the primordia or by the expression of PIN-FORMED (PIN) members and PLETHROA 1 (PLT1) which represented the auxin response by the pericycle cells.Key resultslbd29 mutation resulted in reduced numbers of lateral roots and primordia, whereas LBD29 over-expression resulted in more lateral root and primordia formation than in the wild type. More importantly, the level of LBD29 expression was found to be positively correlated with the level of expression of cell cycle-related genes and correlated with the numbers of subcellular organelles found in pericycle cells in the maturation zone. In addition, an in vitro experiment using root explants demonstrated that the presence of LBD29 was required for the maintenance of the cell division capacity of the pericycle. Furthermore, LBD29 appeared to modify PIN-dependent auxin signalling in the primordia since there was a correlated association between the expression of PINs, PLT1 and DR5:GUS and the expression of LBD29.ConclusionsThe ability of LBD29 to regulate lateral root initiation is associated with its maintenance of the cell division capacity of the pericycle in response to auxin and its involvement in the auxin signalling pathway.

Project description:Lateral root (LR) formation is a vital organogenetic process that determines the root architecture in plants. The number of root branches governs the degree of anchorage, efficiency of nutrients acquisition, and water uptake. The molecular pathways involved in LR formation have been extensively studied in Arabidopsis thaliana (At). A plant hormone, Auxin, is a key regulator of root development and promotes LR formation in plants. A plethora of Arabidopsis genes have been identified to regulate LR initiation, patterning, and emergence processes. Recently, the involvement of flowering time control pathways and circadian clock pathways in LR development has come to light, but the connecting link between these processes is still missing. We have established that GIGANTEA (GI), a key component of photoperiodic flowering, can regulate the formation of LRs in Arabidopsis. GI is known to be involved in red light signaling and circadian clock signaling pathways. Here, we report that over-expression of GI enhances LR formation in red light in At. Real-time PCR analysis shows that GI positively regulates the transcription of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) which is an upstream component of auxin signaling. Furthermore, gi-100 mutant downregulates the LR initiation signaling gene, AUXIN RESPONSE FACTOR 7 (ARF7), and its downstream target gene, LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16). Hence, GI acts as a positive regulator of IAA14-ARF7-LBD16 modules during LR initiation. We have also checked the effect of GI on the expression of NAC1 and AIR3 genes which are controlled by TIR1 during LR formation. Our results show that GI induces the NAC1 transcription and its downstream gene, AIR3 expression, which leads to the enhancement of LR initiation. Taken together, our results suggest that GI controls the expression of TIR1 to govern auxin signaling during LR formation in presence of red light and GI can act as a link between circadian clock signaling, flowering time control pathways, light signaling, and lateral root development pathways.

Project description:Brassinosteroids (BRs) are signaling molecules that play essential roles in the spatial regulation of plant growth and development. In contrast to other plant hormones BRs act locally, close to the sites of their synthesis, and thus homeostatic mechanisms must operate at the cellular level to equilibrate BR concentrations. Whilst it is recognized that levels of bioactive BRs are likely adjusted by controlling the relative rates of biosynthesis and by catabolism, few factors, which participate in these regulatory events, have as yet been identified. Previously we have shown that the UDP-glycosyltransferase UGT73C5 of Arabidopsis thaliana catalyzes 23-O-glucosylation of BRs and that glucosylation renders BRs inactive. This study identifies the closest homologue of UGT73C5, UGT73C6, as an enzyme that is also able to glucosylate BRs in planta.In a candidate gene approach, in which homologues of UGT73C5 were screened for their potential to induce BR deficiency when over-expressed in plants, UGT73C6 was identified as an enzyme that can glucosylate the BRs CS and BL at their 23-O-positions in planta. GUS reporter analysis indicates that UGT73C6 shows over-lapping, but also distinct expression patterns with UGT73C5 and YFP reporter data suggests that at the cellular level, both UGTs localize to the cytoplasm and to the nucleus. A liquid chromatography high-resolution mass spectrometry method for BR metabolite analysis was developed and applied to determine the kinetics of formation and the catabolic fate of BR-23-O-glucosides in wild type and UGT73C5 and UGT73C6 over-expression lines. This approach identified novel BR catabolites, which are considered to be BR-malonylglucosides, and provided first evidence indicating that glucosylation protects BRs from cellular removal. The physiological significance of BR glucosylation, and the possible role of UGT73C6 as a regulatory factor in this process are discussed in light of the results presented.The present study generates essential knowledge and molecular and biochemical tools, that will allow for the verification of a potential physiological role of UGT73C6 in BR glucosylation and will facilitate the investigation of the functional significance of BR glucoside formation in plants.

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Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation.

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Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation.

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