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Cambium genetics in Arabidopsis and Aspen: basic science meets wood production

Periodic Reporting for period 1 - WOODSofCHANGE (Cambium genetics in Arabidopsis and Aspen: basic science meets wood production)

Reporting period: 2015-09-01 to 2017-08-31

Meristematic cells give rise to various organs of the plant and keep the plant growing. Among meristems, the vascular cambium plays a fundamental role in growth, because it promotes the thickening of stems and roots conferring girth to the plants. The vascular cambium continuously produces phloem towards the outside of stems and roots and xylem (or wood) towards the inside of stems and roots. This radial growth confers the structural support for other organs, such as leaves, flowers and fruits and provides a network of transport of water and nutrients and photo-assimilates throughout the plant body. This growth in girth (also named secondary growth) is crucial for plant biology. In addition, in trees, the cambium-mediated growth brings about large amounts of wood. Therefore, understanding the regulation of cambium activity is not only crucial to understand plant biology but also to enhance our possibilities for biomass formation. Yet, little is known about the genetic control of the cambium activity. With the goal of understanding of the genetics of secondary growth in plants, in WOODSofCHANGE we used advanced genomic technology to discover new genes involved in the regulation of the process. In concrete, we used an approach based on 166 natural strains of the model plant Arabidopsis thaliana collected from different locations of the planet and, therefore genetically adapted to specific environmental conditions. Such genetic adaptation is not only reflected in the genomes of the strains (which are different to one another) but also in the anatomical and morphological characteristics. Based on the rationale that the genetic differences between the strains should associate with the morphological and anatomical differences that we can observe among them, we used publicly available software to associate the differences between the strains in terms of secondary growth with variations in their genomes, to identify genomic regions containing genes that control secondary growth. This is an approach that is called Genome Wide Association Study (GWAS) that has been proved as a great gene discovery tool.
We assessed secondary growth by quantifying the amount of xylem production at the hypocotyl, which is located between the main stem and the root and is the most analogous organ in Arabidopsis to the woody stem of trees (Figure). By associating differences in terms of xylem development with the differences within the genomes of the analysed strains, we found a region of the genome significantly associated with cambial activity. Using further experimentation, we tested all the genes that were contained in the identified genomic region and identified one as a new regulator of secondary growth. To learn more about the specific activity of the new regulator of secondary growth, we performed experimentation that allowed us to determine that such new gene negatively regulates the production of one specific cell-type within the xylem (namely, the fibres) by preventing its precocious formation, thereby ensuring the correct pace of xylem development. In this way, mutants for the gene initiate the formation of fibres earlier in the development than normal plants (the latter usually called wild-type plants). Furthermore, our results suggest that this gene acts through transcriptional repression of previously described xylem fiber differentiation identity genes. The results from WOODSofCHANGE have advanced our understanding about the complex regulation of plant development in general and the genetic control of secondary growth in particular.
An overview of the work performed, and main results achieved during the MSCA period, is hereby described:
1) Natural variation of Arabidopsis secondary growth was assessed by quantifying secondary xylem area, by taking measurements of secondary xylem area in transverse sections of 21-day-old hypocotyls from 164 natural accessions.
2) To identify genomic loci that control secondary xylem in Arabidopsis thaliana hypocotyl a Genome Wide Association Study (GWAS) to secondary xylem area was performed. We looked for known regulators of secondary growth amongst genes found in the significant peaks. We identified the already known regulators of secondary growth PIN3 and WOX14, which validated the results. We explored the most significant peak in the GWA analysis to determine the genetic variation causing the highly associated phenotypic variation.
3) We performed bioinformatics and gene expression analysis with the genes within the most significant peak. Together with phenotypic analysis to loss of function mutants for genes within the peak, we identified a causal gene for the phenotypic variation found in secondary xylem area. The loss of function mutant for the candidate gene showed impaired secondary growth at the hypocotyl. Whole hypocotyl area, secondary xylem area and cambium width were found significantly reduced in the mutant when compared to the wild-type.
4) Functional characterization of the causal gene expression by qRT-PCR showed strong repression in several tissues with secondary growth, which is coherent with the expected expression profile of a negative regulator of secondary growth. By analyzing the anatomy of secondary growth performing developmental-time-courses at the hypocotyl we observed that precocious fibers differentiation occurred when the causal gene was dysfunctional. These results suggest that the causal gene is involved in preventing the precocious xylem expansion at the hypocotyl of Arabidopsis.
5) To position the causal gene in the known regulatory pathways of secondary growth we analysed the gene expression of the causal gene in mutants for genes already known to have roles in secondary growth control and of already known secondary growth regulators in the mutant for our newly found secondary growth regulator. We found that the causal gene acts downstream of other known secondary growth regulators, and upstream of fiber differentiation genes, which agrees with the role hereby described in xylem differentiation.
Finding one of the negative regulators of secondary growth is an important contribution to the research field, given that these are rarely found in literature so far. The impact can be translated into society by developing breeding programs that consider the manifestation of the regulatory gene in different woody species, and enrol in fundamental discovery of the mode of action of the regulatory gene by exploring its interactions with other regulatory element involved in secondary growth deepening the knowledge in the field.
WOODSofCHANGE is contributing to European excellence by presenting a novel regulator of wood formation in this way providing a fundamental novelty in the field of plant developmental genetics. It is worth mentioning that wood formation is an understudied topic in biology for which little is known in terms of genetic regulation and, thus, our discovery is of great significance for the field. The beginning of a tree breeding program that would generate new tree varieties with enhanced wood production might be a follow up project in the future. These broader objectives are reliant on the discovery of candidate genes involved in wood formation, which was accomplished by the work developed during WOODSofCHANGE.
Figure. Comparison between the stem of a Populus tree and the hypocotyl in Arabidopsis