Final Report Summary - CLONAL (Uncovering the postembryonic function of embryonic lethal genes in Arabidopsis thaliana)
Overview of the project
Genes with essential functions have been identified in plants through the isolation of gametophytic-lethal and embryonic-lethal mutations. If lethality is a sign of the important metabolic, cellular or developmental function of a given gene, then we might expect some of these genes to also play crucial roles later in plant development. Because homozygous plants are not viable or cannot be obtained, the function of lethal genes cannot normally be studied at post-embryonic stages or in the sporophyte. The "CLONAL" project aims at developing tools and resources for the systematic characterization of the post-embryonic functions of embryonic-lethal and gametophytic-lethal genes using clonal analysis.
Clonal analysis relies on the ability to induce sectors of clonally-related mutant cells in an otherwise normal individual, opening a window into the study of the effects of lethal mutations in the adult organism. Hundreds of embryonic-lethal and gametophytic-lethal mutants have been isolated in Arabidopsis thaliana and other plant species, but no systematic effort has been undertaken yet to characterize the functions of the corresponding genes in adult plants.
Comprehensive summary of scientific results
We have selected 35 embryo-lethal mutants of the model plant Arabidopsis thaliana for clonal analysis experiments, with a focus on their effects on leaf development. These mutants were selected based on the following criteria: (a) the expression of the corresponding genes in leaf tissues, which suggests that they normally function in adult stages; (b) the availability of T-DNA insertion lines that inactivate the correspondig genes and lead to embryonic and/or gametophytic lethality, and (c) the presence of evolutionary conserved domains in their protein products. As regards the latter criterion, we chose to focus on genes encoding proteins with putative regulatory functions, including some that encode transcription factors or participate in the chromatin-mediated regulation of gene expression. Other group of genes was selected based on their predicted roles in proteasome-mediated protein degradation.
We have followed two different approaches for the induction of mutant sectors in adult plants. The first approach involved the X-ray irradiation of so-called ‘cell autonomy’ (CAUT) lines, and the second involved the site-specific excision of transgenes mediated by Cre recombinase.
For the first approach (X-irradiation of CAUT lines), we generated strains for 27 different embryo-lethal mutations. These lines are homozygous for the pale-green chlorata-42 (ch-42) mutation and heterozygous for selected embryo-defective (emb) mutations. Individual lines were then crossed to appropriate CAUT lines carrying a transgene with a wild-type copy of CHLORATA-42 located between the centromere and the corresponding EMB gene . To induce hemizygous sectors for each emb mutation, we irradiated (with X-rays) seed samples from lines carrying 13 different emb mutations under two different irradiation conditions (1,000 rad on water imbibed seeds and 12,000 rad on dry seeds). This screen allowed us to identify numerous plants containing pale-green and albino sectors. These sectors also exhibited a variety of developmental phenotypes, which in some cases were suggestive of the cell lethality of the corresponding mutations. In three different families, the screen also allowed us to identify plants that were homozygous for an emb mutation but were able to grow despite their embryo-lethality (so-called "escapers"). These escapers were found at a very low frequency in the progeny of heterozygous plants, and exhibited reproducible phenotypes that point to the involvement of the corresponding gene in the regulation of gene expression during development. The logic of this strategy was thoroughly described in an article published in Trends in Plant Science (see reference #1 of the publications list).
For the second approach (the induction of mutant sectors through the site-specific excision of transgenes mediated by Cre recombinase), we first adapted the pCB1 vector for use with the Gateway technology [vectors pCB1-GW(+) and pCB1-GW(-)]. These new destination vectors are designed to use the Gateway cloning technology to place the wild-type copy of an EMB gene between two loxP sites, making it possible to induce GFP-marked mutant sectors when the site-specific Cre recombinase is expressed under the control of a heat shock promoter (HS:Cre).
We generated entry clones for 20 different lethal genes (carrying a wild-type copy of a given EMB gene). The inserts of these entry clones were mobilized into the pCB1-GW(+) and pCB1-GW(-) vectors to make constructs for clonal analysis. Constructs for 19 of these genes were successfully transferred to appropriate Agrobacterium strains, and have been used to generate doubly transgenic lines containing: (1) a pCB1-Gateway vector carrying a wild-type copy of the corresponding EMB gene, and (2) the construct HS:Cre, where Cre recombinase expression is controlled by a heat shock promoter. We crossed these doubly transgenic plants to heterozygous EMB/emb plants. Following this approach, we have generated 31 different lines that carry all the necessary elements for sector induction.
Part of the above work, including the screening of the lines, will be included in the PhD dissertation of a graduate student (co-supervised by the scientist in charge and the recruited researcher), who has recently been awarded a fellowship to continue working in this project during the next three years.
Reintegration goals of the IRG project
In line with the reintegration objectives of the project, the researcher has established his own laboratory, and has obtained independent grant funding to investigate on his own research project. During the reintegration period, the researcher was positively evaluated by ANECA (the government agency in charge of evaluations) and received the accreditation for university teaching and research positions. The researcher also filed a successful application to the Ramon-y-Cajal tenure-track program, which he declined when he was appointed for a faculty position at UMH. During the reintegration period, the researcher has supervised the work of four Master's students, and he is the co-advisor of two PhD students. The first of these two PhD theses will be defended in 2013 or early 2014.
In 2009 and 2011, the researcher played an important role in the organization of the Plant Growth Biology and Modelling Workshops (http://pgbm2011.umh.es) which took place in Elche (Spain). The latter workshop was recently featured in a review paper published in The Plant Cell (Eckardt and Bennett, 2012, In Silico Plant Biology Comes of Age. Plant Cell 24:3857-3858). In 2012, the researcher was invited to participate as a lecturer in a Master's programme at the Universite Joseph Fourier in Grenoble (France), where he also gave a research seminar. In 2012, the researcher participated in an international consortium that applied for an Initial Training Network (ITN) under EC FP7. The proposal reached the waiting list and will be re-submitted in the next call. In 2012, the researcher was appointed as an Academic Editor for the scientific journal PLOS ONE.
Genes with essential functions have been identified in plants through the isolation of gametophytic-lethal and embryonic-lethal mutations. If lethality is a sign of the important metabolic, cellular or developmental function of a given gene, then we might expect some of these genes to also play crucial roles later in plant development. Because homozygous plants are not viable or cannot be obtained, the function of lethal genes cannot normally be studied at post-embryonic stages or in the sporophyte. The "CLONAL" project aims at developing tools and resources for the systematic characterization of the post-embryonic functions of embryonic-lethal and gametophytic-lethal genes using clonal analysis.
Clonal analysis relies on the ability to induce sectors of clonally-related mutant cells in an otherwise normal individual, opening a window into the study of the effects of lethal mutations in the adult organism. Hundreds of embryonic-lethal and gametophytic-lethal mutants have been isolated in Arabidopsis thaliana and other plant species, but no systematic effort has been undertaken yet to characterize the functions of the corresponding genes in adult plants.
Comprehensive summary of scientific results
We have selected 35 embryo-lethal mutants of the model plant Arabidopsis thaliana for clonal analysis experiments, with a focus on their effects on leaf development. These mutants were selected based on the following criteria: (a) the expression of the corresponding genes in leaf tissues, which suggests that they normally function in adult stages; (b) the availability of T-DNA insertion lines that inactivate the correspondig genes and lead to embryonic and/or gametophytic lethality, and (c) the presence of evolutionary conserved domains in their protein products. As regards the latter criterion, we chose to focus on genes encoding proteins with putative regulatory functions, including some that encode transcription factors or participate in the chromatin-mediated regulation of gene expression. Other group of genes was selected based on their predicted roles in proteasome-mediated protein degradation.
We have followed two different approaches for the induction of mutant sectors in adult plants. The first approach involved the X-ray irradiation of so-called ‘cell autonomy’ (CAUT) lines, and the second involved the site-specific excision of transgenes mediated by Cre recombinase.
For the first approach (X-irradiation of CAUT lines), we generated strains for 27 different embryo-lethal mutations. These lines are homozygous for the pale-green chlorata-42 (ch-42) mutation and heterozygous for selected embryo-defective (emb) mutations. Individual lines were then crossed to appropriate CAUT lines carrying a transgene with a wild-type copy of CHLORATA-42 located between the centromere and the corresponding EMB gene . To induce hemizygous sectors for each emb mutation, we irradiated (with X-rays) seed samples from lines carrying 13 different emb mutations under two different irradiation conditions (1,000 rad on water imbibed seeds and 12,000 rad on dry seeds). This screen allowed us to identify numerous plants containing pale-green and albino sectors. These sectors also exhibited a variety of developmental phenotypes, which in some cases were suggestive of the cell lethality of the corresponding mutations. In three different families, the screen also allowed us to identify plants that were homozygous for an emb mutation but were able to grow despite their embryo-lethality (so-called "escapers"). These escapers were found at a very low frequency in the progeny of heterozygous plants, and exhibited reproducible phenotypes that point to the involvement of the corresponding gene in the regulation of gene expression during development. The logic of this strategy was thoroughly described in an article published in Trends in Plant Science (see reference #1 of the publications list).
For the second approach (the induction of mutant sectors through the site-specific excision of transgenes mediated by Cre recombinase), we first adapted the pCB1 vector for use with the Gateway technology [vectors pCB1-GW(+) and pCB1-GW(-)]. These new destination vectors are designed to use the Gateway cloning technology to place the wild-type copy of an EMB gene between two loxP sites, making it possible to induce GFP-marked mutant sectors when the site-specific Cre recombinase is expressed under the control of a heat shock promoter (HS:Cre).
We generated entry clones for 20 different lethal genes (carrying a wild-type copy of a given EMB gene). The inserts of these entry clones were mobilized into the pCB1-GW(+) and pCB1-GW(-) vectors to make constructs for clonal analysis. Constructs for 19 of these genes were successfully transferred to appropriate Agrobacterium strains, and have been used to generate doubly transgenic lines containing: (1) a pCB1-Gateway vector carrying a wild-type copy of the corresponding EMB gene, and (2) the construct HS:Cre, where Cre recombinase expression is controlled by a heat shock promoter. We crossed these doubly transgenic plants to heterozygous EMB/emb plants. Following this approach, we have generated 31 different lines that carry all the necessary elements for sector induction.
Part of the above work, including the screening of the lines, will be included in the PhD dissertation of a graduate student (co-supervised by the scientist in charge and the recruited researcher), who has recently been awarded a fellowship to continue working in this project during the next three years.
Reintegration goals of the IRG project
In line with the reintegration objectives of the project, the researcher has established his own laboratory, and has obtained independent grant funding to investigate on his own research project. During the reintegration period, the researcher was positively evaluated by ANECA (the government agency in charge of evaluations) and received the accreditation for university teaching and research positions. The researcher also filed a successful application to the Ramon-y-Cajal tenure-track program, which he declined when he was appointed for a faculty position at UMH. During the reintegration period, the researcher has supervised the work of four Master's students, and he is the co-advisor of two PhD students. The first of these two PhD theses will be defended in 2013 or early 2014.
In 2009 and 2011, the researcher played an important role in the organization of the Plant Growth Biology and Modelling Workshops (http://pgbm2011.umh.es) which took place in Elche (Spain). The latter workshop was recently featured in a review paper published in The Plant Cell (Eckardt and Bennett, 2012, In Silico Plant Biology Comes of Age. Plant Cell 24:3857-3858). In 2012, the researcher was invited to participate as a lecturer in a Master's programme at the Universite Joseph Fourier in Grenoble (France), where he also gave a research seminar. In 2012, the researcher participated in an international consortium that applied for an Initial Training Network (ITN) under EC FP7. The proposal reached the waiting list and will be re-submitted in the next call. In 2012, the researcher was appointed as an Academic Editor for the scientific journal PLOS ONE.