Final Report Summary - JASMONATESTEMCELL (Studies on jasmonate modulation of distal stem cell activity in Arabidopsis root.)
Project summary
Typically, plant organogenesis and morphogenesis processes involve the timely accumulation of the pluripotent plant hormone auxin in subsets of susceptible cells that then changes their developmental status. Auxin accumulation is largely defined by a complex pattern of intracellular auxin transport during lateral root (LR) development. Our work identified a coherent feed-forward regulatory mechanism that capacitates the transcriptional auxin-sensitivity of the PIN-formed (PIN) auxin transporter PIN3. This feed-forward motif is defined by the direct binding to the PIN3 promoter of two transcription factors, Auxin Response transcription Factor7 (ARF7) and the ARF7-regulated FOUR LIPS/MYB124 (FLP). This mechanism endows the PIN3 circuitry with a temporal “memory” of auxin stimuli, potentially enhancing the robustness of auxin flux patterns. Moreover, we show that the joint recruitment of both transcription factors is necessary to generate the PIN3 expression levels in planta that are needed for LR development. The cooperative action between canonical auxin signalling and other transcription factors might constitute a general mechanism by which the transcriptional auxin-sensitivity of genes can be differentially modulated between tissues.
Project objectives, results, conclusions and impacts.
1. To determine at which stage of lateral root development the meristem becomes established, expression of quiescent center (QC) specific markers (WOX5:GFP, QC25, QC46 and QC184) and of some other meristem related marker genes (SHR:SHR-GFP and SCR:GFP-SCR) were checked during lateral root development. And also, we managed to set up an in vitro culture system that allows to determine at which point of development a lateral root primordium becomes independent from the parent root. All these experiments confirmed that most likely a new meristem becomes established round III.
2. We modified the lateral root induction system (LRIS) to make it more applicable for large scale sampling of well-defined lateral root developmental stages. We found out that the second section (1-2 mm from the primary root tip) was suitable for transcript profiling studies.
3. The experiment was setup for RNA Seq analysis. After NAA treatment, respectively for 12h, 18h and 24h, we could get enough synchronized primordia at stage I, stage II and stage III for RNA Seq analyses at each time point including three biological repetitions.
4. Based on the RNA Seq data we selected some candidate genes for further analysis. From this list of candidate genes, we will mainly focus on transcription factors FOUR LIPS/MYB124 (FLP) and some epigenetics components.
5. To analyse FLP's function during LR development, we ordered flp1 and flp1myb88 mutants and checked their LR phenotype. We found that flp1myb88 exhibited reduced numbers of LR initiation. Then, we prepared the transgenic line of proFLP:NLS-GFP, which show that FLP is specifically expressed in first stage of LR primordium. qRT-PCR results demonstrated that FLP is quickly induced by auxin, which is dependent on Auxin Response transcription Factor ARF7 and SOLITARY ROOT/IAA14 (SLR). All these results showed that FLP is an important components involved in auxin mediated early stage LR development.
6. We introgressed the synthetic auxin response output reporter proDR5::GUS into flp-1 myb88 and checked its response to auxin in LR primordia. We found that proDR5::GUS showed weak signal in flp-1 myb88, but respond to auxin normally compared with WT. Auxin induced LR related genes IAA19, ACR4 and GATA23 showed normal response to auxin by qRT-PCR assay. The LR defect in flp1 myb88 double mutants is due to abnormal auxin distribution.
7. To further dissect the contribution of defects in auxin transport to the LR phenotype in flp myb88, we focussed on the earliest stages of LR formation; LR founder cells (FC, undivided pericycle cells that exhibit proDR5::GFP expression) and LR initiation sites. The density of LR initiation sites was reduced in flp-1 myb88 compared to WT. In contrast, the FC density in flp-1 myb88 was dramatically increased compared to WT, resulting in a similar density of the total of FCs and LR initiation sites. Such a specific LR defect was recently also described for pin3-4, an auxin transport mutant that is also impaired in LR initiation. Importantly, the LR phenotype in the flp-7 myb88 pin3-4 triple mutants showed near identical reductions in LR initiation as the parental mutants.
8. The promoter sequence of PIN3 was analysed, and we found some potential binding sites for ARF and MYB transcription factors, which gave us some cues that PIN3 maybe a direct target of ARF7 and FLP. To verify whether PIN3 is a direct target of FLP and ARF7, we carried out yeast-one-hybrid and CHIP-PCR assay, which demonstrated that FLP and ARF7 could bind cis-elements in the PIN3 promoter in vitro and in vivo.
9. We evaluated the role of FLP- and/or ARF7-mediated regulation of PIN3 on LR development. For this purpose, we designed proPIN3 variants of 1.8 kb, containing specific mutations predicted to abrogate activation by FLP (mF) or by ARF7 (mA), then fused them to PIN3-YFP and transformed them into pin3-4. Five independent lines were selected per construct, to minimise user-biased pre-selection for transgenic lines, and analysed YFP positive individuals for auxin-responsive PIN3-YFP amplitude and LR density. Only PIN3 promoter without mutations drived PIN3-YFP could respond to auxin normally, and rescue LR defects in pin3-4. PIN3 promoter with mutations (mA, mF, or both of mA and mF) could not respond to auxin, and also, could not rescue LR defects in pin3-4. These results demonstrate that the regulation of auxin sensitive PIN3 expression as defined by FLP as well as ARF7 is a crucial parameter for LR development.
All the data demonstrated that the feed-forward-motif regulation of auxin signalling provides a temporary memory of cellular auxin levels to specific specialized cells, potentially enhancing the robustness of auxin flux patterns and focussing auxin maxima during post-embryonic patterning processes, such as LR initiation and development. In addition, our simulations indicate that delayed PIN3 down-regulation might temporarily capacitate the system for mitigating the effects of subsequent auxin stimuli on ARF7 activity and downstream auxin signalling. Together our data represent a first example of a mechanism by which plant endogenous cues such as auxin can become “memorized” to temporarily sustain transcriptional auxin signalling in selected cells for specific transcripts.
With the support of Marie Curie Fellowship, the project was successfully finished, and published in the well-known journal Nature Communications (DOI: 10.1038/ncomms9821) this year. And more importantly, several of my colleagues in China tried to apply this famous fellowship to support their research in Europe.
Typically, plant organogenesis and morphogenesis processes involve the timely accumulation of the pluripotent plant hormone auxin in subsets of susceptible cells that then changes their developmental status. Auxin accumulation is largely defined by a complex pattern of intracellular auxin transport during lateral root (LR) development. Our work identified a coherent feed-forward regulatory mechanism that capacitates the transcriptional auxin-sensitivity of the PIN-formed (PIN) auxin transporter PIN3. This feed-forward motif is defined by the direct binding to the PIN3 promoter of two transcription factors, Auxin Response transcription Factor7 (ARF7) and the ARF7-regulated FOUR LIPS/MYB124 (FLP). This mechanism endows the PIN3 circuitry with a temporal “memory” of auxin stimuli, potentially enhancing the robustness of auxin flux patterns. Moreover, we show that the joint recruitment of both transcription factors is necessary to generate the PIN3 expression levels in planta that are needed for LR development. The cooperative action between canonical auxin signalling and other transcription factors might constitute a general mechanism by which the transcriptional auxin-sensitivity of genes can be differentially modulated between tissues.
Project objectives, results, conclusions and impacts.
1. To determine at which stage of lateral root development the meristem becomes established, expression of quiescent center (QC) specific markers (WOX5:GFP, QC25, QC46 and QC184) and of some other meristem related marker genes (SHR:SHR-GFP and SCR:GFP-SCR) were checked during lateral root development. And also, we managed to set up an in vitro culture system that allows to determine at which point of development a lateral root primordium becomes independent from the parent root. All these experiments confirmed that most likely a new meristem becomes established round III.
2. We modified the lateral root induction system (LRIS) to make it more applicable for large scale sampling of well-defined lateral root developmental stages. We found out that the second section (1-2 mm from the primary root tip) was suitable for transcript profiling studies.
3. The experiment was setup for RNA Seq analysis. After NAA treatment, respectively for 12h, 18h and 24h, we could get enough synchronized primordia at stage I, stage II and stage III for RNA Seq analyses at each time point including three biological repetitions.
4. Based on the RNA Seq data we selected some candidate genes for further analysis. From this list of candidate genes, we will mainly focus on transcription factors FOUR LIPS/MYB124 (FLP) and some epigenetics components.
5. To analyse FLP's function during LR development, we ordered flp1 and flp1myb88 mutants and checked their LR phenotype. We found that flp1myb88 exhibited reduced numbers of LR initiation. Then, we prepared the transgenic line of proFLP:NLS-GFP, which show that FLP is specifically expressed in first stage of LR primordium. qRT-PCR results demonstrated that FLP is quickly induced by auxin, which is dependent on Auxin Response transcription Factor ARF7 and SOLITARY ROOT/IAA14 (SLR). All these results showed that FLP is an important components involved in auxin mediated early stage LR development.
6. We introgressed the synthetic auxin response output reporter proDR5::GUS into flp-1 myb88 and checked its response to auxin in LR primordia. We found that proDR5::GUS showed weak signal in flp-1 myb88, but respond to auxin normally compared with WT. Auxin induced LR related genes IAA19, ACR4 and GATA23 showed normal response to auxin by qRT-PCR assay. The LR defect in flp1 myb88 double mutants is due to abnormal auxin distribution.
7. To further dissect the contribution of defects in auxin transport to the LR phenotype in flp myb88, we focussed on the earliest stages of LR formation; LR founder cells (FC, undivided pericycle cells that exhibit proDR5::GFP expression) and LR initiation sites. The density of LR initiation sites was reduced in flp-1 myb88 compared to WT. In contrast, the FC density in flp-1 myb88 was dramatically increased compared to WT, resulting in a similar density of the total of FCs and LR initiation sites. Such a specific LR defect was recently also described for pin3-4, an auxin transport mutant that is also impaired in LR initiation. Importantly, the LR phenotype in the flp-7 myb88 pin3-4 triple mutants showed near identical reductions in LR initiation as the parental mutants.
8. The promoter sequence of PIN3 was analysed, and we found some potential binding sites for ARF and MYB transcription factors, which gave us some cues that PIN3 maybe a direct target of ARF7 and FLP. To verify whether PIN3 is a direct target of FLP and ARF7, we carried out yeast-one-hybrid and CHIP-PCR assay, which demonstrated that FLP and ARF7 could bind cis-elements in the PIN3 promoter in vitro and in vivo.
9. We evaluated the role of FLP- and/or ARF7-mediated regulation of PIN3 on LR development. For this purpose, we designed proPIN3 variants of 1.8 kb, containing specific mutations predicted to abrogate activation by FLP (mF) or by ARF7 (mA), then fused them to PIN3-YFP and transformed them into pin3-4. Five independent lines were selected per construct, to minimise user-biased pre-selection for transgenic lines, and analysed YFP positive individuals for auxin-responsive PIN3-YFP amplitude and LR density. Only PIN3 promoter without mutations drived PIN3-YFP could respond to auxin normally, and rescue LR defects in pin3-4. PIN3 promoter with mutations (mA, mF, or both of mA and mF) could not respond to auxin, and also, could not rescue LR defects in pin3-4. These results demonstrate that the regulation of auxin sensitive PIN3 expression as defined by FLP as well as ARF7 is a crucial parameter for LR development.
All the data demonstrated that the feed-forward-motif regulation of auxin signalling provides a temporary memory of cellular auxin levels to specific specialized cells, potentially enhancing the robustness of auxin flux patterns and focussing auxin maxima during post-embryonic patterning processes, such as LR initiation and development. In addition, our simulations indicate that delayed PIN3 down-regulation might temporarily capacitate the system for mitigating the effects of subsequent auxin stimuli on ARF7 activity and downstream auxin signalling. Together our data represent a first example of a mechanism by which plant endogenous cues such as auxin can become “memorized” to temporarily sustain transcriptional auxin signalling in selected cells for specific transcripts.
With the support of Marie Curie Fellowship, the project was successfully finished, and published in the well-known journal Nature Communications (DOI: 10.1038/ncomms9821) this year. And more importantly, several of my colleagues in China tried to apply this famous fellowship to support their research in Europe.