Periodic Reporting for period 1 - ITRABAE (Investigating the transcriptional regulation of auxin biosynthesis in Arabidopsis embryo)
Okres sprawozdawczy: 2021-01-01 do 2022-12-31
Seeds have considerable biological and commercial importance. They are a major source of food and nutrition for most of the world’s population. In plants, seeds protect and nourish the developing embryo, thus ensuring reproductive success. Therefore, improvement of seed quality and quantity is a key issue. Moreover, this challenge is compounded by climate changes, with an increase in average temperatures over the past decades already negatively impacting seed production.
Seed development proceeds through two stages, (i) morphogenesis and (ii) maturation. During embryogenesis, a single-cell zygote divides into a multicellular embryo. The mature embryo encloses all the features of a new plantlet that will develop after germination. Among various hormones, phytohormone auxin plays an essential role in embryo development. Auxin defines embryo patterning through its concentration gradients, maintained by auxin biosynthesis combined with transport. However, how local embryonic auxin biosynthesis is transcriptionally regulated is not known. In this project, we aimed to elucidate the transcriptional regulation of auxin biosynthetic genes during embryo development with an overall goal of improving seed development using auxin as a tool.
The Objectives of this project were 1) to elucidate the transcriptional regulation of auxin biosynthetic genes (TAA1 and YUCs) by the identified AGL transcription factors (TFs) during embryo development and 2) to characterise the impact of high temperatures on this regulation.
In conclusion, we identified the AGL5 transcription factor as a novel regulator of auxin biosynthesis. It interacted the promoters of auxin biosynthetic genes (TAA1, YUC8, and YUC9) and activated their transcription. Over-expression of AGL5 could up-regulate the expression of auxin biosynthetic genes. It acted redundantly with SHP1 and STK to control ovule and seed development. Overall, this work will pave the way for understanding a key mechanism for auxin production to mediate proper ovule and seed development, a finding widely relevant to flowering plants.
Seed development proceeds through two stages, (i) morphogenesis and (ii) maturation. During embryogenesis, a single-cell zygote divides into a multicellular embryo. The mature embryo encloses all the features of a new plantlet that will develop after germination. Among various hormones, phytohormone auxin plays an essential role in embryo development. Auxin defines embryo patterning through its concentration gradients, maintained by auxin biosynthesis combined with transport. However, how local embryonic auxin biosynthesis is transcriptionally regulated is not known. In this project, we aimed to elucidate the transcriptional regulation of auxin biosynthetic genes during embryo development with an overall goal of improving seed development using auxin as a tool.
The Objectives of this project were 1) to elucidate the transcriptional regulation of auxin biosynthetic genes (TAA1 and YUCs) by the identified AGL transcription factors (TFs) during embryo development and 2) to characterise the impact of high temperatures on this regulation.
In conclusion, we identified the AGL5 transcription factor as a novel regulator of auxin biosynthesis. It interacted the promoters of auxin biosynthetic genes (TAA1, YUC8, and YUC9) and activated their transcription. Over-expression of AGL5 could up-regulate the expression of auxin biosynthetic genes. It acted redundantly with SHP1 and STK to control ovule and seed development. Overall, this work will pave the way for understanding a key mechanism for auxin production to mediate proper ovule and seed development, a finding widely relevant to flowering plants.
The project was organised into four work packages, and each work package (WP) contained 3-4 tasks. We have completed most of the tasks and achieved the milestones for the project.
In WP1, we employed a dual luciferase assay in N. benthamiana leaves that revealed AGL5/SHATTERPROOF2 (SHP2) as an activator of YUC8, YUC9 and TAA1 promoters. Also, in the β-estradiol inducible lines of AGL5, the transcripts of TAA1 and YUC9 were enhanced after AGL5 induction. AGL5 shares sequence similarities with AGL1/SHP1 and AGL11/SEEDSTICK (STK), and these genes interact with each other genetically. Therefore, we selected SHP1, AGL5/SHP2 and STK as potential candidates for further studies.
In WP2, we aimed to identify the interacting proteins of selected AGL TF to explore the regulatory modules underpinning auxin biosynthesis. Screening of the string database revealed that ten proteins were predicted to interact with AGL5. Among them, BEL1 and VERDANDI (VDD) are known to play essential roles in ovule identity. Another protein, SEPALLATA3 (SEP3), is required for inflorescence and floral organ development. In addition, we observed that the promoter of AGL5 can strongly drive the GUS expression in developing ovules and embryos, indicating its involvement during ovule and embryo development. However, in the translational lines (proAGL5::AGL5-GFP), AGL5-GFP was uniformly detected in the ovules and valve margin in stage 12 pistils. In the seed, GFP expression was found to be restricted in the outermost layer of the seed coat. Unfortunately, no GFP fluorescence could be detected in the embryos.
In WP3, we performed the microscopic phenotyping of the mutants (shp1, shp2, shp1shp2, shp1shp2stk). Embryos of single mutants (shp1, shp2, and stk) and double mutants (shp1shp2) revealed no deviant phenotype relative to Col-0 wild-type plants. In contrast, normal ovule and seed development was disrupted in the triple mutant (shp1shp2stk). It indicates that SHP2 acts redundantly with SHP1 and STK. We also checked the expression of TAA1 and YUCs in shp1shp2 and shp1shp2stk mutant pistils (stage 12) through RT-qPCR. The expression analysis revealed that the expression of TAA1 was lower in shp1shp2 and shp1shp2stk ovules than in the wild type. To test whether the reduced transcript levels of auxin biosynthetic genes lead to reduced auxin activity in shp1shp2 ovules and seeds, a reporter DR5::Venus was crossed into the shp1shp2 mutant. However, we didn’t observe any changes in the intensity of expression of DR5::Venus in shp1shp2 mutant ovules compared with the wild type.
In WP4, we investigated the effect of high temperatures (28°C and 34°C) on the activity of AGL5. RT-qPCR analysis using stage 12 pistils revealed that AGL5 does not respond to high temperatures. Furthermore, we performed a ChIP experiment to test the binding of STK on TAA1 and YUC promoters. Two regions from YUC8 and YUC9 promoters harbouring CArG-box were screened through qPCR after immunoprecipitation for enrichment analysis. The ChIP assay revealed significant enrichment of binding to the regions spanning CArG-box in the YUC8 and YUC9 promoter in pSTK::STK-GFP compared with the wild-type control.
Exploitation and dissemination of the results
During the project, exploitation and dissemination of the results were done by presenting and discussing the work in weekly lab meetings, conferences, and retreats. Some of the performed activities for effectively disseminating the project results are listed below.
Flash talk on “Plant embryo development and impact of climate change” at Falling Walls Lab Czech Republic. September 2021.
Poster presentation on “Role of MADS-box TFs in regulating auxin biosynthesis during embryo development” at Auxin 2022, Cavtat, Croatia, October 2-7.
Poster presentation on “Role of MADS-box TFs in regulating auxin biosynthesis during embryo development” at 32nd International Conference on Arabidopsis research ICAR:2022 -Belfast, June 20-24.
Poster presentation on “Role of MADS-box TFs in regulating auxin biosynthesis during embryo development” at 3rd Annual CEITEC postdoc retreat 2022. Křtiny Castle, September 22-23.
Visit to Prof Lucia Colombo’s lab, Department of Biosciences, University of Milan, Italy, to perform experiments and discuss work progress (20th Nov – 2nd Dec 2022)
In WP1, we employed a dual luciferase assay in N. benthamiana leaves that revealed AGL5/SHATTERPROOF2 (SHP2) as an activator of YUC8, YUC9 and TAA1 promoters. Also, in the β-estradiol inducible lines of AGL5, the transcripts of TAA1 and YUC9 were enhanced after AGL5 induction. AGL5 shares sequence similarities with AGL1/SHP1 and AGL11/SEEDSTICK (STK), and these genes interact with each other genetically. Therefore, we selected SHP1, AGL5/SHP2 and STK as potential candidates for further studies.
In WP2, we aimed to identify the interacting proteins of selected AGL TF to explore the regulatory modules underpinning auxin biosynthesis. Screening of the string database revealed that ten proteins were predicted to interact with AGL5. Among them, BEL1 and VERDANDI (VDD) are known to play essential roles in ovule identity. Another protein, SEPALLATA3 (SEP3), is required for inflorescence and floral organ development. In addition, we observed that the promoter of AGL5 can strongly drive the GUS expression in developing ovules and embryos, indicating its involvement during ovule and embryo development. However, in the translational lines (proAGL5::AGL5-GFP), AGL5-GFP was uniformly detected in the ovules and valve margin in stage 12 pistils. In the seed, GFP expression was found to be restricted in the outermost layer of the seed coat. Unfortunately, no GFP fluorescence could be detected in the embryos.
In WP3, we performed the microscopic phenotyping of the mutants (shp1, shp2, shp1shp2, shp1shp2stk). Embryos of single mutants (shp1, shp2, and stk) and double mutants (shp1shp2) revealed no deviant phenotype relative to Col-0 wild-type plants. In contrast, normal ovule and seed development was disrupted in the triple mutant (shp1shp2stk). It indicates that SHP2 acts redundantly with SHP1 and STK. We also checked the expression of TAA1 and YUCs in shp1shp2 and shp1shp2stk mutant pistils (stage 12) through RT-qPCR. The expression analysis revealed that the expression of TAA1 was lower in shp1shp2 and shp1shp2stk ovules than in the wild type. To test whether the reduced transcript levels of auxin biosynthetic genes lead to reduced auxin activity in shp1shp2 ovules and seeds, a reporter DR5::Venus was crossed into the shp1shp2 mutant. However, we didn’t observe any changes in the intensity of expression of DR5::Venus in shp1shp2 mutant ovules compared with the wild type.
In WP4, we investigated the effect of high temperatures (28°C and 34°C) on the activity of AGL5. RT-qPCR analysis using stage 12 pistils revealed that AGL5 does not respond to high temperatures. Furthermore, we performed a ChIP experiment to test the binding of STK on TAA1 and YUC promoters. Two regions from YUC8 and YUC9 promoters harbouring CArG-box were screened through qPCR after immunoprecipitation for enrichment analysis. The ChIP assay revealed significant enrichment of binding to the regions spanning CArG-box in the YUC8 and YUC9 promoter in pSTK::STK-GFP compared with the wild-type control.
Exploitation and dissemination of the results
During the project, exploitation and dissemination of the results were done by presenting and discussing the work in weekly lab meetings, conferences, and retreats. Some of the performed activities for effectively disseminating the project results are listed below.
Flash talk on “Plant embryo development and impact of climate change” at Falling Walls Lab Czech Republic. September 2021.
Poster presentation on “Role of MADS-box TFs in regulating auxin biosynthesis during embryo development” at Auxin 2022, Cavtat, Croatia, October 2-7.
Poster presentation on “Role of MADS-box TFs in regulating auxin biosynthesis during embryo development” at 32nd International Conference on Arabidopsis research ICAR:2022 -Belfast, June 20-24.
Poster presentation on “Role of MADS-box TFs in regulating auxin biosynthesis during embryo development” at 3rd Annual CEITEC postdoc retreat 2022. Křtiny Castle, September 22-23.
Visit to Prof Lucia Colombo’s lab, Department of Biosciences, University of Milan, Italy, to perform experiments and discuss work progress (20th Nov – 2nd Dec 2022)
The work accomplished in this project has significantly improved fellows’ scientific skills and expertise in microscopy, molecular biology techniques, data analysis, etc. Apart from this, he has also developed time management and project management skills. The conferences attended by the fellow improved his public speaking and presentation skills. In addition, he managed to make some fruitful collaborations during the project.
The standardised protocols, constructs, and transgenic lines generated to fulfil this project will be helpful to fellow researchers and the wider plant science community working on ovule and seed development. As the work carried out is related to seed development, it will act as a model to address the issue of food security, hence directly impacting societal needs at the global level. Our data identified key genes involved in ovule and/or seed development. The identified genes can be utilised to improve crop production through translational research in crop plants.
The standardised protocols, constructs, and transgenic lines generated to fulfil this project will be helpful to fellow researchers and the wider plant science community working on ovule and seed development. As the work carried out is related to seed development, it will act as a model to address the issue of food security, hence directly impacting societal needs at the global level. Our data identified key genes involved in ovule and/or seed development. The identified genes can be utilised to improve crop production through translational research in crop plants.