Final Report Summary - ARABIGANS (Uncovering conserved proliferation pathways between plants and animals)
Plant leaves are the direct or indirect source of most of the food we eat, and of the oxygen that we breathe. The interest of the study, and eventual manipulation, of leaf development lays on the fact that leaves are the fundamental photosynthetic organ, of vital importance for life on our planet. Whereas plant products depend on the photosynthetic activity of leaves, animal products have their ultimate origin in animals that have been fed with plant products. The study of plant biology leads to results that make possible to improve the quality or the productivity of cultivated plant crops. In addition, plants are known to produce value-added chemicals with expanding applications in biotechnology and biomedicine, such as taxol for the treatment of cancer.
Development is among the most interesting aspects in the biology of any organism and its study makes its manipulation possible for the benefit of humankind. For this reason, an understanding of the genetic mechanisms that control growth and morphogenesis in plants, particularly in the cultivated ones, is very important. The most efficient approach to dissect leaf morphogenesis is the identification, characterization and manipulation of genes involved in the control of leaf development in a model plant such as Arabidopsis thaliana. Leaves develop from the shoot apical meristem, which contains a continuous source of stem cells that differentiate when meristematic gene expression is repressed by leaf identity genes. A phenotype of increased cell proliferation in leaves can be easily detected since leaves are larger. On the contrary, a decrease in cell proliferation should yield smaller leaves. Therefore, leaf development represents a good system for studying cell proliferation.
In our search for genes involved in leaf morphogenesis, we have identified 608 and 3 genes, whose knock out insertional T-DNA alleles and overexpression, respectively, cause aberrant leaves. In addition, a visible morphological phenotype in leaves was generated by the silencing of the target genes of 21 artificial microRNA (amiRNA)-expressing transgenes transferred into Arabidopsis. These amiRNA transgenes target the silencing of groups of 2-6 paralogous transcription factors.
We described the mutant phenotypes of our 608 mutants using a custom leaf phenotype ontology (Wilson-Sánchez et al., 2014). We genotyped 553 mutants finding that the indexed mutation is present in the annotated locus for 78% of the lines and that in half of these the annotated T-DNA is responsible for the phenotype. To quickly map non-annotated T-DNA insertions, we developed a cost-effective and easy method based on whole-genome sequencing, and proved its reliability. Mutant phenotypes, the genotyping results and visual phenotype information can be queried in the implemented public web application named PhenoLeaf (http://genetics.umh.es/phenoleaf). We have demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal-distal cell cycle-driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks, and examination of other traits in this fundamental photosynthetic organ.
Regarding the TRANSPLANTA overexpression lines (Coego et al., 2014), we have screened 1256 lines, from which 19 have a smaller lamina than the wild type. We selected 3 that showed a leaf morphological phenotype to characterize in detail. Two of them overexpress a transcription factor of the AP2-EREBP family, and display defects in the first pair of leaves, such as pointed epinastic leaves or elongated narrow leaves. The other line overexpressed a transcription factor of the NAC family, and shows a pale pigmentation in the primary and some secondary veins. All three genes are plant specific. The morphometric analysis of these three lines suggests a role for these genes in cell proliferation and leaf venation patterning establishment.
We have focused in the characterization of the mutant alleles of one of the previously mentioned 608 genes, of special interest since it causes deviation from the bilateral symmetry normally seen in the leaves of the wild type, and of its two putative paralogous genes, all three of unknown function. This insertion is located within a gene with a conserved DUF domain. We have named this gene desigual (deal). Bilateral asymmetry appears only in deal adult leaves and arises from a combination of outgrowths and growth defects along the proximal-distal axis, pointing to a role of the mutated gene in the coordination of lamina expansion. Bilateral asymmetry is already visible in mutant leaf primordia, confirming that cell division regulation is altered at very early stages of deal leaf development. We tracked cell division using the CYCB1;1pro:GUS marker, showing that proliferation is unbalanced between both lamina halves. Besides, we found by qRT-PCR that CYCB1;1 relative expression in the leaves as a whole is 1.6 fold higher in the mutant. TCP genes are a family of transcription factors that promote the transition from proliferation to differentiation states in the leaf. We measured TCP4 in deal leaves and found that its mRNA abundance is half the level of the wild type.
DEAL expression was detected in the shoot apical meristem, leaf primordia and during early leaf development using a DEALpro:GUS reporter. We looked at the cellular level and found that cells that were still cycling showed the highest blue staining, which faded basipetally, when cells start to differentiate and expand. These data together are consistent with a role for DEAL in cell proliferation-driven leaf morphogenesis.
We identified DEAL as a gene of unknown function exclusive of multicellular plants. We were able to rescue the mutant phenotype with a 35S:DEAL-CFP construct, confirming the identity of the gene responsible for the phenotype. The protein has four predicted transmembrane spanning domains and a secretory pathway signal peptide. We localized the fusion protein to a discrete organelle in the cell, frequently associated with the nucleus, which resembles an endomembrane system.
Screening of the AGRON-OMICS lines and the detailed characterization of DESIGUAL will be included in the PhD dissertation of the graduate student David Wilson-Sánchez (co-supervised by the scientist in charge and the recruited researcher) who was awarded a fellowship to work on this project until April 2015.
In line with the reintegration objectives of the project, Dr. Sara Jover Gil has been appointed to a teaching position (Profesora Asociada) in the Genetics area of the Applied Biology Department at the host institution, UMH.
Development is among the most interesting aspects in the biology of any organism and its study makes its manipulation possible for the benefit of humankind. For this reason, an understanding of the genetic mechanisms that control growth and morphogenesis in plants, particularly in the cultivated ones, is very important. The most efficient approach to dissect leaf morphogenesis is the identification, characterization and manipulation of genes involved in the control of leaf development in a model plant such as Arabidopsis thaliana. Leaves develop from the shoot apical meristem, which contains a continuous source of stem cells that differentiate when meristematic gene expression is repressed by leaf identity genes. A phenotype of increased cell proliferation in leaves can be easily detected since leaves are larger. On the contrary, a decrease in cell proliferation should yield smaller leaves. Therefore, leaf development represents a good system for studying cell proliferation.
In our search for genes involved in leaf morphogenesis, we have identified 608 and 3 genes, whose knock out insertional T-DNA alleles and overexpression, respectively, cause aberrant leaves. In addition, a visible morphological phenotype in leaves was generated by the silencing of the target genes of 21 artificial microRNA (amiRNA)-expressing transgenes transferred into Arabidopsis. These amiRNA transgenes target the silencing of groups of 2-6 paralogous transcription factors.
We described the mutant phenotypes of our 608 mutants using a custom leaf phenotype ontology (Wilson-Sánchez et al., 2014). We genotyped 553 mutants finding that the indexed mutation is present in the annotated locus for 78% of the lines and that in half of these the annotated T-DNA is responsible for the phenotype. To quickly map non-annotated T-DNA insertions, we developed a cost-effective and easy method based on whole-genome sequencing, and proved its reliability. Mutant phenotypes, the genotyping results and visual phenotype information can be queried in the implemented public web application named PhenoLeaf (http://genetics.umh.es/phenoleaf). We have demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal-distal cell cycle-driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks, and examination of other traits in this fundamental photosynthetic organ.
Regarding the TRANSPLANTA overexpression lines (Coego et al., 2014), we have screened 1256 lines, from which 19 have a smaller lamina than the wild type. We selected 3 that showed a leaf morphological phenotype to characterize in detail. Two of them overexpress a transcription factor of the AP2-EREBP family, and display defects in the first pair of leaves, such as pointed epinastic leaves or elongated narrow leaves. The other line overexpressed a transcription factor of the NAC family, and shows a pale pigmentation in the primary and some secondary veins. All three genes are plant specific. The morphometric analysis of these three lines suggests a role for these genes in cell proliferation and leaf venation patterning establishment.
We have focused in the characterization of the mutant alleles of one of the previously mentioned 608 genes, of special interest since it causes deviation from the bilateral symmetry normally seen in the leaves of the wild type, and of its two putative paralogous genes, all three of unknown function. This insertion is located within a gene with a conserved DUF domain. We have named this gene desigual (deal). Bilateral asymmetry appears only in deal adult leaves and arises from a combination of outgrowths and growth defects along the proximal-distal axis, pointing to a role of the mutated gene in the coordination of lamina expansion. Bilateral asymmetry is already visible in mutant leaf primordia, confirming that cell division regulation is altered at very early stages of deal leaf development. We tracked cell division using the CYCB1;1pro:GUS marker, showing that proliferation is unbalanced between both lamina halves. Besides, we found by qRT-PCR that CYCB1;1 relative expression in the leaves as a whole is 1.6 fold higher in the mutant. TCP genes are a family of transcription factors that promote the transition from proliferation to differentiation states in the leaf. We measured TCP4 in deal leaves and found that its mRNA abundance is half the level of the wild type.
DEAL expression was detected in the shoot apical meristem, leaf primordia and during early leaf development using a DEALpro:GUS reporter. We looked at the cellular level and found that cells that were still cycling showed the highest blue staining, which faded basipetally, when cells start to differentiate and expand. These data together are consistent with a role for DEAL in cell proliferation-driven leaf morphogenesis.
We identified DEAL as a gene of unknown function exclusive of multicellular plants. We were able to rescue the mutant phenotype with a 35S:DEAL-CFP construct, confirming the identity of the gene responsible for the phenotype. The protein has four predicted transmembrane spanning domains and a secretory pathway signal peptide. We localized the fusion protein to a discrete organelle in the cell, frequently associated with the nucleus, which resembles an endomembrane system.
Screening of the AGRON-OMICS lines and the detailed characterization of DESIGUAL will be included in the PhD dissertation of the graduate student David Wilson-Sánchez (co-supervised by the scientist in charge and the recruited researcher) who was awarded a fellowship to work on this project until April 2015.
In line with the reintegration objectives of the project, Dr. Sara Jover Gil has been appointed to a teaching position (Profesora Asociada) in the Genetics area of the Applied Biology Department at the host institution, UMH.