Final Report Summary - SHOOT APICAL MERISTEM (Stem cells specification and meristem maintenance at the shoot apical meristem of Arabidopsis thaliana)
A central question in biology is how cell fate is specified during development of a multicellular organism. This question can be divided into two. One, what are the signals and the microenvironment (niche) required for cell fate specification; and two, what is the mechanism through which cell fate specification is carried out?
Cell fate specification in the shoot apical meristem [SAM] is regulated by multiple transcription factors [TFs] acting in a complex gene regulatory network. Two genes are found at the center of the decision for stem cell specification and meristem maintenance, the WUCHEL (WUS) and SHOOTMERISTEMLESS (STM) encoding for transcription factors [TFs]. The WUS gene which required to specify and maintain stem cell population within the SAM, is expressed in a small set of cells at the center of the meristem, defining the domain referred to as the organizing center (OC) and the stem cell niche. STM is expressed throughout the SAM excluding the incipient leaf primordial and acts by antagonizing cell differentiation, allowing stem cell daughters to proliferate before being incorporated into organs. In the heart of this project we ask the questions of: How does WUS specify stem cell fate? What is the nature of the stem cell niche and what are the signals emanating from or perceived at the organizer center? How STM acts to antagonizing cell differentiation and to allow stem cell daughters to proliferate?
The main goal of this research is to study how WUS and STM function to confer meristematic fate thereby to gain insight into the genetic and molecular mechanisms underlying cell fate specification in the SAM.
The specific objectives of this proposed research are threefold: (I) first, to identify the direct target genes of WUS and STM TF in a genome wide scale. (II) The second aim is to study the role of WUS-binding and STM-binding in regulating the expression and function of their target genes. (III) To integrate the genes identified in aims I into regulatory model acting to specify cell fate in the SAM and to identify key genes within the model for further study.
A description of the work performed since the beginning of the project:
To identify the direct target genes of WUS and STM TF, we are combining chromatin immunoprecipitation (ChIP) with high-throughput Illumina sequencing technology. To this end, as was proposed, we tagged the WUS and STM with the BLRP tag which can be biotinylated in vivo following purification by strepavadin beads. We have generated transgenic plants carrying the WUS::WUS-BLRP and STM::STM-BLRP construct on wus-1 and stm-11 Arabidopsis mutants respectively, as well as on the clv3-2 mutant background which exhibits enlarged meristem. We grew five independent lines for each construct in each background for 3 generation to have homozygous plants and analyzed the expression of the transgene by RT-PCR with specific primes for the tagged genes. For each TF we selected two lines to proceed with in the ChIP analysis. In addition we preformed ChIP analysis on transgenic plants carrying the 35S::WUS-GR transgene using antibodies for GR. We preformed ChIP analysis both on 10-day-old seedlings and inflorescent with the result of enriched DNA for our control genes though with the pitfall of having low amount of DNA that can't be sequenced by Illumina sequencer. To solve this obstacle we calibrated an amplification method of Single-tube linear DNA amplification (LinDA) that was recently published in Nature Methods and currently we are preparing the samples for sequencing in the Technion Genome Center Israel.
To further deepen our understanding of cell specification in the meristem we added two TF to our analysis the KNAT6 and CORONA [CAN] and utilized other strategy to overcome the low amount of DNA by generating constructs of the CaMV 35 constitutive promoter. We selected lines that show phenotype [Fig 1] and high expression for further ChIP analysis.
To study the role of WUS in cell specification we used 35S::WUS-GR transgenic plants. We aim at testing WUS ability to change cell fate of different cells type. By using tissue culture approach we applied Dex to activate WUS on callus, leaf, seedlings, cotyledon and roots the results are summarized in Fig 2.
To gain additional insights on WUS and STM function in stem cell specification and meristem development we took a further genetic approach. Mapping the gene that enhances the jba1D meristem phenotype, we identified the ERECTA receptor like kinase as a regulator of WUS expression acting in a parallel pathway to the known CLAVATA and class III HDZIP regulators.
Using those three pathway we generated by crossing a set of five genetic backgrounds with increasing levels of WUS and STM expression and meristem size. We have performed comprehensive developmental and molecular analyses on the five genetic backgrounds as well as mRNA-seq analysis on mRNA extracted from their meristems at different developmental stages [Fig 3]. We are currently waiting for the data. In the scope of this grant we also generate a system to test what cells type can change their specification upon WUS expression as well as testing WUS movement from its expression cell to other cells [as it is known to act in a non- autonomous manner]. We transformed 12 plants of Jim Haselhoff's GAL4-GFP Enhancer-trap lines with UAS::WUS construct to derive the expression of WUS in specific tissues or cell type. We are currently growing the T1 plants with already amazing phenotypes that can teach us a lot on WUS function [Fig 4].
A description of the main results achieved so far:
The main goal of this research is to study how WUS and STM function to confer meristematic fate thereby to gain insight into the genetic and molecular mechanisms underlying cell fate specification in the shoot apical meristem. In order to identify the direct target genes of WUS and STM we have generated transgenic plant carrying a construct of WUS and STM tagged with the BLRP tag on their null mutants background as well as on clv3-2 mutant. We have checked homozygous T3 lines for expression and chose two individual lines for the ChIP analysis. To overcome the problem of low DNA amount we have calibrated amplification methods and currently we are preparing the samples for sequencing. Upon receiving the sequence data we hope to achieve our goal for identifying the direct target genes and to construct a genetic network for cell fate specification in the meristem. We performed an extensive analysis of WUS-GR and STM-GR activation in a tissue culture. We found that activation of WUS in callus with no additional hormones can lead to stem cell specification and establishing de novo meristems resulting in regeneration of shoot. However the induction of WUS in callus on a media with high auxin and low cytokinine [callus inducing media] block the ability of WUS to specify stem cell identity and generate new meristem [Fig 2]. The induction of WUS in the root led to the formation of embryo that later developed into a shoot.
With genetics approach we identified the ERECTA receptor like kinase as a regulator of WUS expression. This work is published in a Development ["The ERECTA receptor kinase regulates Arabidopsis shoot apical meristem size, phyllotaxy and floral meristem identity". Mandel T, Moreau F, Kutsher Y, Fletcher JC, Carles CC and Eshed Williams L. Development. 2014 Feb;141(4):830-41]. Using this finding we have generate generated by crossing a set of five genetic backgrounds with increasing levels of WUS and STM expression and meristem size. The meristems of five Arabidopsis mutant lines, from smallest to largest, showed a gradual shift from spiral to whorled phyllotaxis [Fig3], implying that meristem size regulates phyllotaxy. We are currently performing differential expression analysis on these meristems by mRNA-seq, in an attempt to elucidate how WUS and STM function to confer meristematic fate thereby to gain insight into the genetic and molecular mechanisms underlying cell fate specification in the shoot apical meristem. In addition we hope to reveal how the balance between the CZ and PZ cell populations affects phyllotaxy in these enlarged meristems. The results of ChIP analysis of the APETALA TF in tomato, under my supervision was published in Journal of Plant Cell [Burko Y, Shleizer-Burko S, Yanai O, Shwartz I, Zelnik ID, Jacob-Hirsch J, Kela I, Eshed-Williams L, Ori N. (2013). A role for APETALA1/fruitfull transcription factors in tomato leaf development. Plant Cell. 25(6):2070-83]
The expected final results and their potential impact and use:
We expect to get good results with the ChIP-seq analysis that will enable us to identify all the direct targets and to construct a model for the genetic network regulating cell specification at the shoot apical meristem. We expect to write a high quality paper that will summarize our conclusions. The results of the comprehensive developmental and molecular analyses on the five genetic backgrounds exhibiting gradual increase in WUS expression and meristem size is summarized into a paper that will be submitted upon receiving and integrating the mRNA-seq analysis. We plan to submit the paper to Development journal. For the WUS-GR and STM-GR project we are conducting one more follow-up experiment and starting to summarize all of our finding into a paper.