REGULATORY NETWORKS IN THE SEED: INTEGRATION OF DEVELOPMENT, METABOLISM AND ENVIRONMENTAL CONDITIONS
- González Ceballos, Mar - Technician
- Guerrero Galán, Carmen - Postdoctoral Fellow
- Iglesias Fernández, Raquel - Assistant Lecturer
- Medina Alcázar, Joaquín - Researcher INIA
- Nayak, Shweta - Student
- Pastor Mora, Elena - Technician
- Sousaraei, Naeimeh - Visiting Scientist
- Yang, Lu - PhD Student
Our main research interest is transcriptional control of gene expression, primarily in the field of seed development. In this framework our group identified and characterized several transcription factors participating in the regulation of gene expression programmes associated to the seed maturation and germination phases. In addition, we are interested in the study of transcription factors affecting metabolic adjustment to carbon/nitrogen partitioning and stress responses.
We are investigating regulatory networks, based mainly on transcription factors (TFs) of the bZIP, DOF, MYB and B3 classes. Our group is currently performing transcriptomic analyses of Arabidopsis transgenic plants affected in the expression of transcriptions factors controlling key aspects of seed development and the validation of potentially regulated genes in transient expression assays. Other techniques relevant to our research include the study of protein-DNA and protein-protein interactions, in situ RNA hybridisation and comparative bioinformatic analyses of TF families, mostly in Arabidopsis and cereal species.
Function and Biotechnological potential of seed Transcription Factors
Transcription factors (TFs) are regulatory proteins that have a key role in evolution and have also a great biotechnological potential. They are crucial in regulating gene expression in a combinatorial way and they have been important in domestication and plant breeding; some examples are Teosinte Branched 1 that affects plant architecture (branching), the Green Revolution Genes (GRAS) responsible of the semi-dwarf cultivars of presently grown cereals, the DOF1 gene that improve nitrogen assimilation and growth under low nitrogen conditions, etc. The identification of key regulatory transcription factors (TFs) involved in the maturation and germination of seeds from crops (barley) and from model plants (Arabidopsis thaliana and Brachypodium distachyon) has been an aim of our group in the past years and a number of TF genes belonging to several TF families, such as bZIP, DOF and MYB have been demonstrated to be important in both processes. However, it is still unclear to what extent these TF genes are regulated in monocot- and dicotyledonous seeds. The AFL sub-family of B3 TFs (VP1/ABI3, FUS3, LEC2) has been recently studied and its origin, through the course of evolution, traced back to non-vascular plants such as Physcomitrella patens and its function explored taking into account expression patterns and interactions with other TFs. The first member of this family to be described was the maize Viviparous-1 (Vp-1) as a central player of Pre-Harvest Sprouting and as a regulator of the C1 gene encoding a MYB-like TF regulator of the anthocyanin biosynthesis pathway. Its ortholog from barley (HvVP1) has been demonstrated to play essential roles in the regulation of genes upon maturation and germination through interaction with GAMYB, BPBF and BLZ2. HvFUS3 from barley unveils a common transcriptional regulation of seed specific genes between cereals and Arabidopsis (AtFUS3).
During germination of Arabidopsis thaliana seeds, the AtMAN7 gene that encodes an endo-β-mananase (MAN; EC. 126.96.36.199) is critical for hydrolyzing the seed covering layers that are rich in mannans. The bioinformatic analysis of the promoter sequences of orthologous genes of AtMAN7 in several species of the Brassicaceae family (phylogenomics) discovered highly conserved motives. These conserved motives were used as baits in a yeast 1-hybrid screening of an arrayed yeast library of circa 1,200 TFs from A. thaliana (Castrillo et al., 2011). AtbZIP44 was identified as a putative regulator of the AtMAN7 expression and later validated by several molecular and physiological techniques. Co-expression in the micropylar endosperm of the putative regulator gene (AtbZIP44) and the regulated one (AtMAN7) was corroborated by microscopic techniques. A post-germination gene AtCathB3, encoding a Cathepsin B-like protease has been shown to be transcriptionally represed by the basic leucine zipper protein GBF1.
Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysacharides in the root embryo and in the coleorhiza in the early stages of germination (12 h of imbibition; hoi); mannans decrease drastically thereafter (27 hoi). Concurrently, the activity of endo-beta-mannanases increases as germination progresses. From the MAN gene family (six members) in Brachypodium, the transcripts of three of them (BdMAN2, 4 and 6) accumulate in the germinating embryos and their expression is detected in the coleorhiza and in the root, by mRNA-in situ hybridization techniques before root protrusion. These data indicate that these three genes are important for germination sensu stricto and that the coleorhiza in monocots and the micropylar endosperm in dicots may have similar functions. The BdGAMYB protein from Brachypodium distachyon interacting with BdDOF24 regulates transcription of the BdCathB, gene enconding a Cathepsin-like proteinase, during reserve mobilization in post-germination.
Besides our own research, we have had external collaborations with Spanish scientific groups from IBMCP (UPV-CSIC; Valencia), CEBAS (CSIC; Murcia), Department of Plant Physiology (USC; Santiago de Compostela), Department of Biochemistry and Molecular Biology (UC, Córdoba). Most important , through our participation in the TRILATERAL project (Transcriptional networks and their evolution in the Brassicaceae; MICINN-EUI2008-03716), Germany-France-Spain Action, we have collaborated with important European groups, such as those of George Coupland (Max Planck Institute, MPI-Köln, Germany), Detlef Weigel (MPI-Tubingen, Germany), Vincent Colot (CNRS-INSERM, France), Haidi Quednesville (INRA-Versalles, France), Pilar Carbonero (CBGP-UPM, Spain) and Javier Paz-Ares (Centro Nacional de Biotecnología, CNB-CSIC, Spain).
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