Characterizing the determinants of leaf patterning in maize

Abstract

Patterned growth is essential for proper plant development. However, the identity of the molecular signals that contribute to patterning remains incomplete. The maize leaf presents an excellent opportunity to study patterning due to its simplicity. The maize leaf is organized into four distinct tissues that are polarized in a proximal-distal (P-D) pattern: (1) sheath is the most proximal and wraps around the culm of the plant, (2) auricle and (3) ligule creating a hinge like structure that allows the leaf to bend away from the plant, and (4) blade is the most distal and acts as the main photosynthetic mechanisms. I use the semi-dominant mutant Hairy sheath frayed1 (Hsf1) to identify the molecular signals that control leaf patterning. The Hsf1 mutant develops abnormal ectopic outgrowths in the blade margin, called “prongs”, consisting of proximal tissue in the most distal compartment of the leaf. Map based cloning revealed that the Hsf1 phenotype is a result of a gain-of-function missense mutation in the CHASE domains of the Zea mays Histidine Kinase1 (ZmHK1), a cytokinin (CK) receptor. In the mutant, ZmHK1 has higher CK affinity as compared to the wild-type. The Hsf1 phenotype can be phenocopied by exogenous CK treatments on seeds. The picture emerging is that CK signaling influences P-D leaf patterning, an unknown function of CK. I hypothesize that CK hypersignaling causes cells to become dedifferentiated as meristematic tissue at the margin of the blade at some point in leaf development, resulting in the random distribution of “newly formed leaves” along the margin as the proximal tissue is not expressed consistently around the edge of the leaf. Although the determinants of CK perception and signaling have been well defined in plants, the identity and function of downstream affects are not well understood. Previous work to identify the downstream determinants of CK signaling that drive prong formation, used laser-capture microdissection (LCM) conducted by Jim Cahill at Cornell University coupled with whole transcriptome sequencing (RNA-seq), focused on emerging prongs (P), no-prong (N, WT margin in Hsf1), and wild-type (W) margin. The Muszynski’s lab at Iowa State University identified approximately 900 differentially expressed (DE) genes that were predominantly transcription factors (TFs) associated with developmental processes. This thesis project is focused on determining the function of seventeen DE TFs, by genetics and genomics approaches, along with characterization of a new possible genetic enhancer of Hsf1, enh. Histological methods were used on margin tissue to determine the prong developmental hallmarks based on normal leaf patterning. This led to the identification of three developmental stages for prongs, (1) emerging, (2) transitioning, and (3) mature. Quantitative PCR was used to calculate relative expression of seventeen genes of interest over each prong stage along with wild-type and no-prong margin. The relative expression and known gene function were compared to the previous RNA-seq data and revealed that not all genes were expressed as expected. Double mutant analysis of Hsf1/+ and delayed flowering1 (dlf1) provided further evidence for the relative expression pattern produced in objective 1 for dlf1. This data suggests a new role for dlf1 in leaf patterning. In the process creating Hsf1/+ and tru1 double mutants uncovered a possible genetic enhancer in the A619 inbred background. Further analysis will need to be done to determine the underlying gene to the mutation.