Global expression analyses of genes involved in meristem organization and leaf initiation.
This project is an NSF Plant Genome-funded collaboration between five different labs:
Dr. Pat Schnable (Iowa State)
Dr. Marja Timmermans (Cold Spring Harbor Lab)
Dr. Brent Buckner and Dr. Diane Jannick Buckner (Truman State)
Dr. Dan Nettleton (Iowa State)
Dr. Mike Scanlon (Cornell/UGA),
and is aimed at understanding the function of shoot apical meristems (SAMs). All above ground organs of higher plants are ultimately derived from the SAM. The SAM exhibits distinctive structural organization, marked by tissue zonation and cell layering. The structure of plant SAMs is correlated with their function, such that new leaves are initiated from the peripheral zone of the SAM and the central zone replenishes new meristematic cells that are lost during organogenesis.
Experiments are ongoing to identify and analyze genes required for meristem function and early stages of leaf development in maize. Laser dissection microscopy is a powerful technique that permits the isolation of RNA from specific cell types within fixed plant tissues. RNA collected from 1,000-10,000 cells is sufficient for use in microarray analyses of gene expression, which permit the simultaneous examination of expression profiles of over 45,000 genes. The laser microdissection/microarray technique is being used to capture cells from specific domains of the maize meristem and newly-formed leaf primordia for use in comparative analyses of global gene expression. The differential expression patterns of candidate genes are verified by real time RT-PCR and in situ hybridization) of transcript accumulation in maize tissues. These experiments microdissect gene expression patterns in meristems and leaf primordia, and promise to provide novel insight into mechanisms of plant development.
More experimental details, including protocols, microarray platforms, links to data and project personnel are all available at the project website http://maize-meristems.plantgenomics.iastate.edu
BLADE KILLER1 is required for meristem maintainance.
Shoot meristems are ultimately responsible for development of all the organs in plant shoots, including leaves, branches, flowers and seed. To perform their essential functions, shoot meristems must maintain a balance of determinate and indeterminate cell types, by which the loss of meristematic cells during organogenesis is countered by the regeneration of stem cells required to sustain the meristem.
bladekiller1 (blk1) is recessive mutation affecting shoot meristem maintenance in maize. The blk1 phenotype is a variable yet progressive reduction in shoot meristem size that ultimately leads to premature termination of meristem structure and function. Vegetative, inflorescence and floral meristems are similarly affected, resulting in arrested and incomplete development of the vegetative shoot, the ear and the tassel. Curiously, the progressive decrease in vegetative SAM size is accompanied by a similar reduction in the length of the distal leaf blade. Preliminary qRT-PCR analyses of lateral shoot meristems reveal that the bladekiller1 mutation causes reduced transcript accumulation of specific genes implicated in meristem maintenance and function.
Our long term goal is to determine the precise mechanisms of BLADEKILLER1 function during shoot meristem and leaf development in maize; four short term goals are in progress.
- Fine scale genetic mapping of blk1 is used to identify molecular markers required to genotype double mutant plants (aim #2), and for map-based cloning of blk1 (aim #4).
- The blk1 mutant phenotype in diverse inbred genetic backgrounds and in combination with known maize mutants with defects in meristem development or phase change are analyzed in order to investigate genetic pathways involved in BLK1 function.
- Laser microdissection - microarray analyses of blk1 mutant and non-mutant lateral meristems are used to identify genes that are differentially expressed in blk1 mutants. Quantitative RT-PCR is used to verify microarray data, and in situ hybridization analyses will probe the expression patterns of selected, differentially expressed genes.
- A map-based cloning strategy is in progress to identify the blk1 gene and characterize its expression pattern.