Integrating computational and functional approaches to study maize cell expansion

Chase W. Nelson

Cell expansion is responsible for the majority of maize leaf growth, yet the mechanism of wall expansion is not yet fully understood. Wall expansion requires coordination between cellulose synthesis at the plasma membrane and secretion of wall matrix components into the enlarging wall space. Two proteins involved in these processes include RAB GTPases, which traffic vesicles for secretion in all eukaryotes, and xyloglucan endotransglucosylase/hydrolase (XTH), which contributes to wall loosening in dicots primarily. In this study, genetic, molecular and computational approaches were integrated to study the role of these two proteins in maize cell expansion. First, maize RAB2A1 was studied using computational methods, because the protein is currently believed to be encoded by the Warty1 gene. Mutations in warty1 cause abnormally expanded cells. Two potential lesions are hypothesized to be responsible for altered cell expansion seen in warty1 mutants. A single base pair change in the amino acid sequence is found in warty1-0 alleles and may cause the mutant phenotype. Alternatively, mutations in the gene promoter region may be causative. Computational methods were used in this study to test these two hypotheses. Second, ZmXTH was identified in the maize genome, suggesting its potential function in monocot cell expansion. To test for its potential role, ZmXTH is being tagged with a fluorescent protein (mRFP1) in order to localize the protein in expanding cells.

Homology modeling was used to test the impact of the single base pair change of N to T in warty1-0 mutants. Using the known structure of the homologous Homo sapiens Rab2A protein, the ZmRab2A1 protein was modeled using SwissModel, and the wild-type and warty1 structures compared. Additionally, the unsupervised motif-discovery tool WordSeeker was used to analyze the DNA (including UTRs) of the similarly-regulated genes ZmRab1A and ZmRab2A1, revealing any fully matching, significant motifs shared between them. Finally, a phylogenetic tree of all Rab2 proteins present in GenBank was constructed using RAxML. No significant differences were observed between the wild-type and warty1 mutant of ZmRab2A1 protein structures. Additionally, significant motifs were discovered in both UTRs of the gene. Finally, N’s presence at site 49 of the protein shows no clear pattern of common ancestry, suggesting that it is a polymorphism. This evidence suggests that the mutation is located in a nonprotein-coding region of the gene. Progress has been made toward a gene tree/species tree reconciliation, bootstrap calculations, and testing for positive (Darwinian) selection, and the completion of these calculations will be the next step in this process.

Whereas Rab proteins shuttle matrix components through vesicles, the XTH gene studied is proposed to exhibit xyloglucan endotransglycosylase activity, cutting and religating xyloglucan molecules in the cell wall matrix to allow controlled expansion. Using the triple-template PCR technique, the XTH gene was successfully tagged with RFP. After recombining the tagged gene into a pDONR vector, an attempt was made to transform the vector into E. coli; so far this has been unsuccessful. Future work will transform the vector into E. coli, recombine it into a binary vector, and transform the gene into Maize using Agrobacterium tumefaciens. This will allow localization of the XTH protein within the expanding tissue of the Maize leaf in order to better understand the expansion process. Computational and functional approaches will complement one another in the process of elucidating the details of plant cell wall expansion and guide future studies in this area.