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Final Report Summary - RAM (Role of ABA in root meristem maintenance)

Introduction and Overview:
The ability to maintain groups of stem cells after embryogenesis represents an important characteristic of higher plants and is fundamental to plant development, in particular post-embryonic plant development. A key challenge in plant sciences is to understand how stem cells are maintained in plants. Previous studies have indicated that plant hormones, such as auxin and cytokinine, play very important roles in this important process. This project focuses on the role of another plant hormone, abscisic acid (ABA), in stem cell maintenance in root meristems. The root system of high plants plays vital biological functions such as the acquisition of water and mineral nutrients. Its development is controlled by root meristems located at the tip of every individual root. Vital to the function of a root meristem is a group of stem cells, known as the stem cell niche (Fig. 1). This group of cells must be maintained in order the root to maintain its capacity to grow and develop. This project seeks further insights about the molecular mechanisms underlying stem cell maintenance in Arabidopsis root meristems and is based on two previous observations of the host lab that externally applied ABA could rescue the root meristem failure phenotype of a root meristem defective mutant, alf3-1, and that this ABA rescue occurred in known ABA-insensitive mutants. These observations led to the hypothesis that ABA might play a role in stem cell maintenance in Arabidopsis root meristems via novel ABA signalling mechanism. The aim of the project is to test the above-mentioned hypothesis by characterising the molecular mechanism of this novel ABA function and thereby to. The project utilises alf3-1 and a mutant that is in the alf3-1 background but is insensitive to the ABA’s rescue, designated as root meristem ABA insensitive 8 (mabi8) and contains the following three approaches: 1). characterization of the root meristem failing process in the alf3-1 and the mabi8 mutants; 2). identification of MABI8 gene; 3) expressional and functional characterization of MABI8. According to the original proposal, the project includes an incoming phase of 24 moths and a returning phase of 12 months.

Summary of results and conclusions:
Root meristem failing process in alf3-1/mabi8: According to the original plan, we used a range of cell specific markers to examine cellular indicators of the root meristem falling process in alf3-1 and mabi8. We have established that the root meristem failing process occurs between 3-5 days after germination (DAG) in alf3-1 and mabi8 and that the earliest detectable cellular indicator of this process is CSC stem cell differentiation, as indicated by the accumulation of starch granules. We also found that the expression domains of two molecular markers, SHRp::SHR-GFP and PIN1p::PIN1-GFP, change significantly during the root meristem failing process.
We also noticed that cell division activities, as monitored by the expression of pCYCB1::CYCB1CDB-GUS, in alf3-1/mabi8 root meristems increases at earlier stage of the root meristem failing process (3-4 DAG), but diminish gradually then after and disappeared almost completely after 8 DAG. alf3-1 mutation has been suggested to cause defect in either auxin synthesis or transportation based on the fact that exogenous auxin application can rescue the mutant phenotype in roots, our analyses using two auxin responsive markers, pDR5:GFP and pIAA2::GUS, reveal elevated auxin response in alf3-1 and mabi8 root meristems, indicating that the root meristem failure of alf3-1 is unlikely due to auxin deficiency or auxin transport defect.
Molecular identification of MABI8: We have crossed mabi8 with Landsberg WT and carried out a mapping analysis using a population of about 5000. This resulted in the mutation being mapped to a 340Kb region (between AGI 19475826 & 19826237) on Chromosome V. Attempt to map the mutation to a more defined region has been unsuccessful due to a lack of recombination within this region even with an larger mapping population (>7000). To overcome this problem, we used complementation with selected JAtY clones (which contains large inserts in a transformation ready vector) from the Jon Innes Centre and have found two (with no overlapping) clones within the mapped region that can complement the root meristem failing phenotype of mabi8 (Fig. 3). We have carried out some sequencing analyses of the regions covered by both complementing clones and have identified some candidate genes with confirmed mutations in the mabi8 genome for further analyses, which could be carried out in the returning phase of the fellow (Dr Chhun) according to the original plan. However, the fellow has now accepted a position in Tamasek Life Sciences Laboratory in the National University of Singapore following the incoming phase and therefore will no longer work on the project. As a consequence, we are currently seeking funding to support the continuation of the project in the host laboratory.

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