2012. Genetic analysis of cell expansion during maize leaf development

2012. Genetic analysis of cell expansion during maize leaf development

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The growth and development of plants requires a finely tuned and exquisitely controlled sequence of events, including cell division and expansion. Efforts in the Sylvester lab have focused on understanding the mechanisms of cell expansion during maize leaf development. In this dissertation, the role of several proteins in cell expansion is investigated. First, evidence is presented that Z/77RAB2A1, a small guanosine-triphosphatase (GTPase) of the Ras superfamily, is localized to the Golgi in growing leaf cells. The localization pattern of this protein primarily to the Golgi and only secondarily to the ER, suggests a diversified, or altered, function from that observed in other eukaryotes and dicots. The research here shows that ZmRab2Al is differentially expressed in cells primarily undergoing cytokinesis and cell expansion, but is not highly expressed in fully differentiated, non-expanding tissue. Disruption of ZmRab2Al by transposon insertion correlates with an overexpansion phenotype in leaf epidermal cells. This report represents the first association of a RAB, or any portion of the vesicle transport machinery, with anisotropic cell expansion. The results presented here suggest a diversified function for the RAB2-family protein trafficking pathway in monocot cells when compared to either dicots or to animals.

A major goal of the research was to determine the subcellular localization of ZmRAB2Al, a critical step for determining its function. To accomplish this goal, an efficient method for transient transformation of maize leaf tissue was developed. This method is amenable to expressing genes that are encoded either by their cDNAs or in their intact genomic context (i.e., with regulatory sequences and introns present). The technique can also be used for expressing constructs that have been cloned into GATEWAY plasmids and so could be used for semi-high throughput expression studies, particularly due to the potential for simultaneous transient expression of multiple genes. This technique will simplify subcellular localization studies, and will also likely be useful for a wider variety of techniques, such as functional studies with rationally-designed, dominant-negative alleles or mislocalization studies with plant chromobodies. This technique is expected to be a major breakthrough for maize functional genetics.

Finally, the wartyl-0 allele was cloned using positional cloning techniques and shown shown here to encode ZmCSLD1, a member of the CELLULOSE SYNTHASE-LIKE superfamily predicted to be involved in the biosynthesis of cell wall material. Two other amino acid substitutions in this protein that result in mutant phenotypes, in the warty 1-bumpy and wartyl-rough liueate alleles, were also identified. In the absence of a crystal structure for any plant cellulose synthase protein, this work contributes to the understanding of amino acids that are essential for normal function in this protein. These alleles should also prove useful as material for further genetic screens, such as enhancer and suppressor screens that will identify additional key factors involved in cell wall biosynthesis and deposition.
 

The research presented here demonstrates that ZmRAB2Al and ZwCLSDl contribute to normal cell expansion in maize. These findings, along with the establishment of a novel method for transient gene expression in maize leaf tissue, provide insight into mechanisms of cell expansion and offer new tools that increase the experimental and engineering potential of maize as a major crop plant .