Looking for the reason why lipids accumulate after induction to embryogenesis.
Is it a response to stress?
Is it because the gametophytic program is not completely suppressed?
Suggestions?
CV
CV of the Experienced Researcher
PERSONAL INFORNATION
FIRST NAME: Patricia
FAMILY NAME: Corral Martínez
DATE OF BIRTH: May 24, 1982.
PLACE OF BIRTH: Castellón (Spain)
MARITAL STATUS: Single. No children.
EMAIL: patriciacorralmartinez@gmail.com
EDUCATION
– 2013, July 26th. PhD: Institute for Conservation and Improvement of Valencian Agrodiversity (COMAV), Lab. Cell Biology/ Polytechnic University of Valencia/ Valencia,Spain.
– 2007, April-2013, October. Ph.D. Project: Production of doubled haploids in species of agronomic interest: analysis of factors and cellular mechanisms involved in androgenesis induction in eggplant, rapeseed and tomato. PhD supervisor: J.M Segui-Simarro.
– 2011, November 3rd. Master: Master in Plant Breeding. Dissertation for the Master: Production of doubled haploids in eggplant through isolated microspore culture. Institute for Conservation and Improvement of Valencian Agrodiversity (COMAV).
– 2006, July 26 th. Degree: Degree in Biology. University of Valencia, General study/ Valencia, Spain.
PREVIOUS POSITIONS
– 2005, July- 2006, June. Training Student: Biochemistry and Molecular Biology Department/ Valencian Institute of Agrarian Research (IVIA)/ Valencia, Spain.
– 2006, July-2006, September. Training Student: Faculty of Biology/ Genetics Department, Lab. Of Genetics, Biochemistry and Biotechnology/ University of Valencia/ Valencia, Spain.
– 2006, October- 2007, March. Training Student: Institute for Conservation and Improvement of Valencian Agrodiversity (COMAV), Lab. Cell Biology/ Polytechnic University of Valencia/ Valencia, Spain.
– 2007, April- 2011, April. PhD Student: Institute for Conservation and Improvement of Valencian Agrodiversity (COMAV), Lab. Cell Biology/ Polytechnic University of Valencia/ Valencia, Spain.
– 2011, May- 2013, October. Researcher: Institute for Conservation and Improvement of Valencian Agrodiversity (COMAV), Lab. Cell Biology/ Polytechnic University of Valencia/ Valencia,Spain.
– 2013, November- 2015, April. Unemployed, I used this time to finish writing papers and improve my English.
– 2015, May- 2017, May. Postdoc position: Plant Research International (PRI), Lab. Molecular Biology/ Wageningen University/ Wageningen, The Netherlands.
– 2017, June- 2017, Agoust. Unemployed, I have used this time to write the articles with the results obtained during my previous postdoc.
CURRENT POSITION
– 2017, September- 2018, February. Laboratory technician: Institute for Conservation and Improvement of Valencian Agrodiversity (COMAV), Lab. Cell Biology/ Polytechnic University of Valencia/ Valencia, Spain.
PUBLICATIONS
1- A. Fernández-San Millán, S.M. Ortigosa, S. Hervás-Stubbs, P. Corral-Martínez, J.M. Seguí-Simarro, J. Gaétan, P. Coursaget and J. Veramendi (2008). Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic. Plant Biotechnology Journal. 6 (5): 427-441. Cites: 129.
2- M.R. Abdollahi, P. Corral-Martínez, A. Mousavi, A.H. Salmanian, A. Moieni and J.M. Seguí-Simarro (2009). An efficient method for transformation of pre-androgenic, isolated Brassica napus microspores involving microprojectile bombardment and Agrobacterium-mediated transformation. Acta Physiologiae Plantarum. 31 (6): 1313-1317. Cites: 14.
3- Corral-Martínez, P., Nuez, F. and J.M. Seguí-Simarro (2011). Genetic, quantitative and microscopic evidence for fusion of haploid nuclei and growth of somatic calli in cultured ms1035 tomato anthers. Euphytica, 178 (2): 215-228. doi: 10.1007/s10681-010-0303-z. Cites: 15.
4- J.M. Seguí-Simarro, P. Corral-Martínez, E. Corredor, I. Raska, P.S. Testillano and M.C. Risueño (2011). A change of developmental program induces the remodelling of the interchromatin domain during microspore embryogenesis in Brassica napus L. Journal of Plant Physiology, 168 (8): 746-757. doi: 10.1016/j.jplph.2010.10.014. Cites: 19.
5- J. M. Seguí-Simarro, P. Corral-Martínez, Verónica Parra-Vega and Beatriz González García (2011). Androgenesis in recalcitrant solanaceous crops. Plant Cell Reports, Special issue: Plant Biotechnology in Support of the Millenium Goals. 30 (5): 765-778. doi: 10.1007/s00299-010-0984-8. Cites: 55.
6- R. Sanz-Barrio, A. Fernández-San Millán, P. Corral-Martínez, José M. Seguí-Simarro and Inmaculada Farran (2011). Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts. Plant Biotechnology Journal, 9 (6): 639-650. doi: 10.1111/j.1467-7652.2011.00608.x. Cites: 18.
7- R. Sanz-Barrio, A. Fernández-San Millán, Jon Carballeda, P. Corral-Martínez, J. M. Seguí-Simarro and I. Farran (2012). Chaperone-like properties of two tobacco plastid thioredoxins. Journal of Experimental Botany, 63 (1): 365-379. doi: 10.1093/jxb/err282. Cites: 36.
8- M.R. Abdollahi, P. Ghazanfari, P. Corral-Martínez, A. Moieni and J.M. Seguí-Simarro (2012). Enhancing secondary embryogenesis in Brassica napus by selecting hypocotyl-derived embryos and using plant-derived smoke extract in culture medium. Plant Cell Tissue and Organ Culture (PCTOC), 110 (2): 307-315. doi: 10.1007/s11240-012-0152-7. Cites: 9.
9- P. Corral-Martínez and J. M. Seguí-Simarro (2012). Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). Euphytica, 187 (1): 47-61. doi: 10.1007/s10681-012-0715-z. Cites: 17.
10- R. Sanz-Barrio, P. Corral-Martínez, María Ancin, Jose M. Seguí-Simarro and Inmaculada Farran (2013). Overexpression of plastidial thioredoxin f leads to enhanced starch accumulation in tobacco leaves. Plant Biotechnology Journal, 11 (5): 618–627. doi: 10.1111/pbi.12052. Cites: 37.
11- P. Corral-Martínez, V. Parra-Vega and J.M. Seguí-Simarro. Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning. Journal of Experimental Botany. In press. doi: 10.1093/jxb/ert151. Cites: 15.
12- P. Corral-Martínez and J.M. Seguí-Simarro (2014). Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins. Euphytica, 195: 369-382. doi:10.1007/s10681-013-1001-4. Cites: 7.
13- V. Parra-Vega, P. Corral-Martínez, Alba Rivas-Sendra and Jose M. Seguí-Simarro. Formation and excretion of autophagic plastids (plastolysomes) in Brassica napus (2015). Frontiers in Plant Science, 6 – 94:1 – 13. doi: 10.3389/fpls.2015.00094. Cites: 5.
14- V. Parra-Vega, P. Corral-Martínez, Alba Rivas-Sendra and Jose M. Seguí-Simarro (2015). Induction of embryogenesis in Brassica napus microspores produces a callosic subintinal layer and abnormal cell walls with altered levels of callose and cellulose. Frontiers in Plant Science, 6 (1018): 1 – 17. doi: 10.3389/fpls.2015.01018. Cites: 4.
15- A. Rivas-Sendra, P. Corral-Martínez, C. Camacho-Fernandez and J. M. Seguí Simarro (2015). Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell, Tissue and Organ Culture (PCTOC),10: 2161 – 2174. doi: 10.1007/s11240-015-0791-6. Cites: 3.
16- P. Corral-Martinez, E. Garcia-Fortea, S. Bernard, A. Driouich and J. M. Seguí- Simarro (2016). Ultrastructural immunolocalization of arabinogalactan protein, pectin and hemicellulose epitopes through anther development in Brassica napus. Plant & Cell Physiology. 57 (10): 2161 – 2174. doi: 10.1093/pcp/pcw133. Cites: 2.
17- A. Rivas-Sendra, P. Corral-Martínez and J. M. Seguí-Simarro. Callose deposition in the cell Wall and subintinal layer during induction of microspore embryogenesis are independent processes, differently modulated by Calcium and variable among genotypes. Journal of Experimental Botany, Submitted.
18- P. Corral-Martinez; A. Driouich; J. M. Seguí Simarro. Ultrastructural immunolocalization of arabinogalactan protein, pectin and hemicellulose epitopes during microspore embryogenesis in Brassica napus. Manuscript in preparation.
19- A. Rivas-Sendra; C. Camacho-Fernandez; P. Corral-Martínez; E. Garcia-Fortea; J.M. Seguí-Simarro. Evaluation of the in vitro performance of microspores from DH36 a highly embryogenic eggplant doubled haploid line.
Manuscript in preparation.
20- P. Corral-Martinez, A. Horstman, N. Ruijter and K. Boutiler. Time lapse imaging and trasncriptome analysis to define the fate of the embryogenic structures in B.napus microspore culture. Manuscript in preparation.
21- P. Corral-Martinez, A. Horstman and K. Boutiler. Histone deacetylase inhibitors promote embryogenic divisions in recalcitrants crops: tomato and arabidopsis. Manuscript in preparation.
22- P. Corral-Martinez, C. Camacho-Fernandez, and K. Boutiler. High level of stress induce alteration in the cell walls, endoplasmic reticulum stress and starch and lipid accumulation in rapeseed during microspore embryogenesis. Manuscript in preparation.
BOOK CHAPTERS
1-.P. Corral-Martínez, F. Nuez and J.M. Seguí-Simarro. (2008). Recent advances in eggplant microspore cultures for production of androgenic doubled haploids. In: Prohens, J and Badenes, M.L.(eds). Modern variety breeding for present and future needs. UPV Press, Valencia, Spain. Pp. 104-108. ISBN: 978-84-8363-302-1.
2- P. Corral-Martínez, V. Parra Vega, B. González-García, N. Palacios-Calvo and J.M. Seguí-Simarro (2010). Androgénesis en solanáceas recalcitrantes: tomate, pimiento y berenjena. In: E. Benavente Barzana y J.M. Carrillo Becerril (eds.), Actas de Horticultura, 55. Centro de Publicaciones de la Secretaría General Técnica del Ministerio de Medio ambiente y Medio Rural y Marino, Madrid, Spain. pp 179-180. ISBN: 978-84-491-1004-7.
3- V. Parra-Vega, N. Palacios Calvo, P. Corral-Martínez and J.M. Seguí-Simarro (2010). Establishment of isolated microspore cultures in pepper of the California and Lamuyo types. In: Prohens, J and Rodriguez-Burruezo, A. (eds.). Advances in genetics and breeding of Capsicum and eggplant. UPV Press, Valencia, Spain. pp. 411-415. ISBN: 978-84-693-4139-1.
4- P. Corral-Martínez and J.M. Seguí-Simarro (2010). Improvement in doubled haploid production through in vitro culture of isolated eggplant microspores. In: Prohens, J and Rodriguez-Burruezo, A. (eds.). Advances in genetics and breeding of Capsicum and eggplant. UPV Press, Valencia, Spain. pp. 369-374. ISBN: 978-84-693-4139-1.
5- A. Rivas-Sendra, P. Corral-Martínez, P. Salas, J. M. Seguí-Simarro (2013). Influence of the stage for anther excision in embryogenesis induction from eggplant anther cultures and isolated microspore cultures. In: S. Lanteri y G.L. Rotino (eds.). Breakthroughs in the genetics and breeding of Capsicum and eggplant. Comitato per l´organizzazione degli eventi (COE) DIFASA, Università deg li Studi di Torino. pp. 545-548. ISBN: 978-88-97239-16-1.
CONTRIBUTIONS TO CONGRESSES
– 31 contributions to congresses, being 13 national (Spanish) and 18 international. Posters: 23 and oral presentations: 8.
CONFERENCES/SEMINARS/WORKSHOPS GIVEN BY INVITATION
1- International Conferences of Winter. Institute for Plant Molecular and Cellular Biology (IBMCP). Valencia, Spain. February, 2013. Why changes the microspore fate?.
2- Seminário Internacional sobre o Desenvolvimiento do Programa da Biotecnologia Vegetal na Universidade Agostinho Neto.
Luanda, Angola, September, 2013. Basis of in vitro culture. Applications of the in vitro culture in Plant Breeding.
3- TKI Chemical Genomics Workshop. Laboratory demonstration. Plant Research International. Wageningen University. July, 2015.
PROJECTS PARTICIPATION
1-Ultrastructural localization of movement proteins from two viruses affecting agronomically interesting crops. Universidad Politécnica de Valencia. 01/01/2010 – 31/12/2010. P.I: Dr. Vicente Pallás Benet.
2- Efficient production of doubled haploid lines in eggplant and pepper through in vitro culture of isolated microspores. Cellular and molecular analysis of androgenic development. Spanish Ministry of Science and Innovation, Ref: AGL2010-17895. 01/01/2011 – 31/12/2013.PI: Dr. J.M. Seguí-Simarro.
3- Implementation and set up of a laboratory for in vitro culture at the Ibn Khaldoun University of Tiaret (Algeria). Spanish Agency for International Cooperation and development (AECID). 01/01/2012 – 30/06/2013.
PI: Dr. J.M. Seguí-Simarro.
4- Efficient production of doubled haploid lines in eggplant and pepper through in vitro culture of isolated microspores. Cellular and molecular analysis of androgenic development. Consellería d’Educació, Formació i Ocupació. Valencian Government. 01/01/2012 – 31/12/2012. PI: Dr. J.M. Seguí-Simarro.
5- High throughput live imaging to identify embryogenic cells in culture. Enabling Technologies Hotels. The Netherlands Government. 01/04/201501/04/2016. PI: Dr. Kim Boutilier.
PARTICIPATION IN INDUSTRIAL INNOVATION
– Establishment of a method for isolated microspore culture in eggplant.
Company: Vilmorin Clause & Cie. France. 2011 – 2014. PI: J.M. Seguí-Simarro.
FELLOWSHIPS AND AWARDS
– 2005, September- 2006, June. Collaboration Scholarship, Spanish Ministry of Education and Science/ Spain.
– 2007, April- 2011, April. PhD Grant, FPI Generality of the Valencian Community/ Valencia, Spain.
– 2015, May- 2017, May. MARIE SkŁodowska-CURIE ACTIONS. Individual Fellowships (IF) Call: H2020-MSCA-IF-2014.
SUPERVISING AND MENTORING ACTIVITIES: degree/master thesis
1- Title: New ways to obtain doubled haploid individuals in pepper (Capsicum annuum): In vitro culture of isolated microspores. Student: Beatriz González García. Bachelor’s Degree in Biotechnology, School of Agricultural Engineering and Environment. Polytechnic University of Valencia. Valencia, Spain. 2010. Qualification: 10.
2- Title: Regeneration of doubled haploid plants from calli derived from eggplant isolated microspore cultures. Student: Desireé González Blasco. Degree in Agricultural Engineering, School of Agricultural Engineering and Environment. Polytechnic University of Valencia. Valencia, Spain. 2012. Qualification: 10.
1- Title: Reprogramming towards embryogenesis in sweet cultivars of pepper (Capsicum annuum) through in vitro culture of isolated microspores. Student: Verónica Parra Vega. Master in Plant Breeding (COMAV). Polytechnic University of Valencia. Valencia,Spain. 2010. Qualification: 10.
2- Title: Inmunolocalization of human serum albumin in chloroplasts of genetically modified tobacco (Nicotiana tabacum) plants. Student: Everlyn Dayana Pierce. Master in Plant Breeding (COMAV). Polytechnic University of Valencia. Valencia,Spain. 2011. Qualification: 10.
3- Title: Optimization of isolated microspore cultures in eggplant (Solanum melongena) for production of doubled haploids. Student: Alba Rivas i Sendra. Master in Plant Breeding (COMAV). Polytechnic University of Valencia. Valencia,Spain. Date: 2012. Qualification: 10.
4- Title: Aspects of microspore embryogenesis in Brassica napus. Student: Martha van Os. Plant Research International. Wageningen University. 2016.
TEACHING ACTIVITIES
1- Teaching (practical classes) at the “Molecular Genetics” course of the Degree in Biotechnology of Polytechnic University of Valencia, Spain. Year 2009-2010.
2- Teaching (practical classes) at the “Plant Breeding” course of the Degree in Agronomical Engineering of Polytechnic University of Valencia, Spain. Year 2010.
3- Teaching (practical classes) at the “Plant Reproductive Biology and Biotechnology” of the “Masters on Plant Breeding”of Polytechnic University of Valencia, Spain. Years 2009-2010, 2010-2011, 2011-2012.
SHORT STAYS IN FOREIGN RESEARCH INSTITUTIONS
Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale. Head: Dr. Azeddine Driouich. Université de Rouen. Mont Saint Aignan. France. 1st June. – 31th August 2010/ 7th July. – 22th july 2011/ 12th Sept. – 18th Sept 2012.
Dra. Patricia Corral Martínez
“A Molecular Framework for Plant Cell Totipotency”
1. Define and collect the different embryogenic cell types present in microspore culture using GFP-based reporters, time-lapse imaging, and cell sorting.
2. Define the transcriptional landscape of embryogenic cells using high throughput mRNA sequencing.
3. Determine the function of candidate microspore embryogenesis genes.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far (For the final period please include an overview of the results and their exploitation and dissemination)
Objective 1: Define and collect the different embryogenic cell types present in microspore culture using GFP-based reporters, time-lapse imaging, and cell sorting. The three GFP reporters mark an overlapping set of embryogenic cells, each with different fates i) differentiated embryos (DR5, LEC1 and GRP) and ii) unorganized callus (LEC1 and GRP) (Figure 1).
Time lapse imaging:Time-lapse imaging of the embryo-expressed GFP reporter lines was used to determine the fate of the different embryogenic cell types in culture. We designed an efficient immobilization system to fix the position ofembryogenic structures and to follow them in time without interfering with their development. We optimized the imaging parameters by testing different microscopes/imaging platforms and different software packages for data handling and image processing. Time-lapse imaging of GFP-positive embryogenic structures at day 5 indicated that only the compact structures develop into differentiated embryos (Figure 2). Embryogenic callus-like structures never form differentiated embryos; the majority of these both stop growing and lose their embryo identity, visualized by a loss GFP embryo marker expression.
Cell sorting: We tested two systems for their utility in sorting GFP-positive and GFP-negative cells from microspore culture: 1) the BioSorter (Union Metrica); and 2) Fluorescence Activated Cell Sorting or FACS. In addition, we tested filtering as a means to enrich for differentiated embryos in older cultures. The BioSorter was not useful for sorting GFP-positive and negative populations in microspore embryo cultures. Using FACS, we could separate 100% GFP-positive and 100% GFP-negative cell populations in the GRP:GFP and LEC1:GFP lines until day 6 of culture (Figure 3). However, DR5: GFP expression was too weak to allow cell sorting. Filtering was the most efficient method for isolating older embryogenic structures, around 94% of the filtrate corresponded to GFP-positive embryogenic structures (compact and callus-like), while the remaining 6% was pollen(Figure 4).
Objective 2: Transcriptome analysis
We used freshly isolated microspores, which can be easily isolated in large numbers, to optimise different steps in the mRNA-seq protocol, including mRNA isolation, mRNA amplification and mRNA-seq library construction before proceeding with the FACS samples.
After many trials we decided to use the Picopure kit (Thermo Fisher)) for RNA isolation in combination with the MessageAmp kit (Thermo fisher) for mRNA amplification. 40,000 cells/sample was set as the lower limit for each sample. We collected GFP-positive and GFP-negative cells by FACS or filtering during 6 months. Samples were collected from microspore culture with and without TSA, at day 0, 2, 4 and six days after stress treatment, using three replicates from two different Brassica napus lines. Most samples are ready for sequencing.
Objective 3: Determine the function of candidate genes
Due to the large amount of time needed to set up the FACS system and to collect the samples we did not have time to study the function of candidate genes.
New objective: Study the cellular characteristics of embryogenic cells.
We prepared samples for light and transmission electron microscopy (Figure 5). We observed differences between the different types of embryogenic structures. Embryogenic calli, which do not form embryos, are characterized by loss of cell adhesion (Figure 6), most likely resulting from a decrease in the amount of pectins, arabinogalactan proteins and callose in the cell wall. Moreover in embryogenic calli we observed a massive increase in endoplasmic reticulum, ER stress and autopgahy-related markers (Figure 7), including lipohagy (Figure 8), as well as cell death. These abnormalities may explain why these structures do not continue with the embryogenic pathway.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
There is very little known at a mechanistic level about how plant cells regenerate, due to the difficulty in linking the cellular/molecular observations to specific cell fates. I have developed a time lapse imaging system and used it to show identify the fate of the different embryogenic structures found in microspore culture. We now know that of all the initially embryogenic structures only the compact structures form differentiated embryos. However less than half of these compact structures form embryos, and although the remainder of the compact structures retain their embryo identity, they fail to differentiate. By contrast, most callus-like structures tend to lose their embryo identity as they age. It is not only important to understand the molecular-cellular processes in cells that successfully form haploid embryos in culture, but also to study the processes that take place in cells that are initially converted to embryos, but fail to complete this developmental pathway.. This knowledge can help us to understand what goes wrong and what we have to modify to help these structures to continue with the embryogenic pathway. Such knowledge can be directly translated to agronomically important crops that are recalcitrant for microspore embryogenesis.
The cell-sorting system that we have set up will allow us to specifically analyse the transcriptome of these different cell types to unambiguously assign specific signalling pathways to specific cell fates. These data are unique in the plant community and will allow us to answer a classical plant tissue culture question: how does a single differentiated cell regenerate in the absence of stem cells.
The output of this project can also be used in applied crop research to understand genotype-specific bottlenecks underlying recalcitrance for DH production and to develop biomarkers for marker assisted breeding of responsive germplasm.
Microspore embryogenesis in Brassica napus and species of agronomic interest 