CEPCEB Members

Harley M.S. Smith Assistant Professor Assistant Plant Cell Biologist Botany and Plant Sciences 3121 Batchelor Hall University of California Riverside, California 92521 Phone: (951) 827-2643 Fax: (951) 827-4437   Areas of Expertise Cell and Developmental Biology Genetics
Background Regulation of Inflorescene Architecture in Arabidopsis Selected Publications (Bibliography page)

Background

My college education began at Cabrillo Community College in Aptos, CA (www.cabrillo.edu) after leaving the construction business to pursue my interests in biology. I transferred to the University of California, San Diego (www.ucsd.edu), where I received a B.S. in Biochemistry and Cellular Biology. I received a Ph.D. in Genetics at Michigan State University in the Department of Energy-Plant Research Laboratory (www.prl.msu.edu). I worked on my thesis project in Dr. Natasha Raikhel’s Laboratory (www.cepceb.ucr.edu/members/raikhel.htm) where I studied the function of a nuclear localization signal receptor, AT-IMPORTIN ALPHA.

After receiving my Ph.D., I pursued post-doctoral studies in plant development in Dr. Sarah Hake’s Laboratory (www.pgec.usda.gov/Hake/SHresearch1.html) at the University of California, Berkeley-USDA Plant Gene Expression Center (www.pgec.usda.gov). My background in cell biology allowed me to bring new approaches to basic problems in plant developmental biology that complemented the genetic approaches used in Dr. Hake’s laboratory. I was awarded a three year Post-doctoral Fellowship from the National Institute of Health (NIH) that involved developing a project to gain insight into the function of the KNOTTED1-like homeobox (KNOX) transcription factors in maize and Arabidopsis. During the first phase of my project, we showed that KNOX proteins form complexes with the BEL1-like ( BELL) family of homeodomain proteins. Studies from our laboratory at the CEPCEB indicate that inflorescence development requires the activities of specific KNOX-BELL heterodimers during reproductive development in Arabidopsis.

Currently, the research in my laboratory is focused on molecular mechanisms that control morphological changes in the shoot apical meristem (SAM) upon floral induction. Understanding restructuring of the SAM during the transition to flowering is essential for elucidating patterning events that control inflorescence architecture. To address these basic questions in plant development, we are incorporating a multidisciplinary approach using genetic, biochemical, genomic and cell biological methods to gain more insight into fundamental processes that regulate inflorescence architecture.

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Regulation of Inflorescence Architecture in Arabidopsis

Formation of the plant body is dependent upon the activity of self-organizing groups of cells called meristems located at the shoot and root apices. Shoot architecture results from activity of the SAM, which initiates primordia repetitively from its flanks. Maintenance of the SAM is essential for development of the shoot during vegetative and reproductive growth.

One of the major developmental phase changes in higher plants is the transition from vegetative to reproductive growth. The floral transition is controlled by environmental and/or intrinsic developmental cues that converge at the SAM. Upon floral induction, the vegetative meristem exhibits morphological changes, which initiates new patterns of growth essential for flowering and inflorescence development. The restructuring of the SAM is achieved by changes in the rate and patterning of cell division and has been referred to as floral evocation. In Arabidopsis, two paralogous BELL homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF) encode DNA-binding proteins that are essential for flowering and inflorescence development. Morphological and molecular evidence demonstrates that PNY and PNF regulators are necessary for restructuring the SAM during floral evocation. PNY and PNF also function in the proper allocation of cells into initiating organ primordia. Biochemical studies show that PNY and PNF associate with the KNOX proteins, SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). Interestingly, genetic studies indicate that PNY/PNF-STM and PNY/PNF-BP regulate early internode patterning events. In addition, PNY/PNF-STM are functionally redundant heterodimers that control floral specification and maintain boundaries between the inflorescence meristem and initiating floral primordia. Thus, inflorescence architecture is dependent upon the activity of PNY/PNF-STM and PNY/PNF-BP heterodimers.

The focus of my laboratory is to understand the biochemical and developmental functions of these KNOX-BELL heterodimers during inflorescence development. We are utilizing biochemical and proteomic approaches to purify PNY-transcriptional complexes from inflorescence meristems. We are also performing yeast two-hybrid studies to identify proteins that associate with PNY/PNF and STM during inflorescence development. To understand the role of PNY/PNF-STM in floral and internode specification, we are identifying target genes that are regulated by these homeodomain heterodimers. A better understanding of PNY and PNF function could provide the necessary framework to modify plant architecture as well as uncover the molecular mechanisms that regulate morphological diversity during reproductive development in higher plants.

Figure 1. Distinct patterns of growth are observed during (A) vegetative and (B) inflorescence development. During inflorescence development, the SAM initiates flowers and internodes. The inflorescence also initiates branches that mimic the same pattern as the central shoot.	Figure 2. This figure displays a cladogram of the 13 Arabidopsis BELL proteins. A maize BELL protein, KIP, is used as the outlying sequence. The Arabidopsis BELL cladogram shows that PNY and PNF are paralogous proteins that may share redundant functions during inflorescence development.
Figure 3. Inflorescence architecture requires the function of PNY and PNF in Arabidopsis. (A) During inflorescence development, mutations in PNY disrupt early internode patterning events in which shortened internodes are randomly dispersed along the reproductive shoot. (B) Although inflorescence development is normal in pnf plants, (C) pny pnf double mutants display a dramatic phenotype after floral induction in which flowers are not produced and internode development is severely impaired. Unlike flowering time mutants, which prolong the vegetative phase, pny pnf plants initiate axillary branches and produce cauline like-leaves after the floral transition.
Figure 4. PNY and PNF interacts with STM and BP. (A and B) Yeast two-hybrid studies in which cells were streaked on Trp-/Leu- media that selects for the GAL4-BD and AD plasmids (left plates). To detect interaction between the KNOX/BELL proteins used in this study, cells were streaked on His-/Ade- selection media (right plates). (A) PNY interacts with STM and BP. (B) PNF associates with STM and BP. (C) RT-PCR-expression profiles of PNY and PNF in wild-type inflorescence apices. (Lane 1) PNY and PNF were amplified from plasmids containing the appropriate cDNA. After isolation of mRNA from isolated inflorescence apices, cDNAs were synthesized. PCR was performed with 2 μl (Lane 2) and 5 μl (Lane 3) of inflorescence cDNA’s for 20 cycles. PCR products were separated by agarose electrophoresis and blotted to nylon and probed with the appropriate 32P-labeled probe. (D) Microarray experiments were used to examine the expression levels of genes in the vegetative meristem (VM) and inflorescence meristem (IM) (Schmid et al. 2005). The graph in displayed the absolute expression values for PNY and PNF in the VM and IM. The expression profiling was quantile-normalized using gcRMA. The absolute expression values for PNY and PNF were linearized gcRMA values (Schmid et al. 2005). Reference: Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Scholkopf B, Weigel D, Lohmann J (2005) A gene expression map of Arabidopsis development. Nature Genetics 37:501-506.
Figure 5. Inflorescence architecture requires the activities of PNY/PNF-BP and PNY/PNF-STM heterodimers. In this model, all four homeodomain complexes regulate early internode patterning events in the inflorescence meristem. Unlike PNY/PNF-BP heterodimers, PNY/PNF-STM complexes also regulate developmental programs required for floral specification and maintaining boundaries between initiating floral primordia and the inflorescence meristem. Thus, PNY/PNF-BP and PNY/PNF-STM heterodimers regulate early patterning events in the SAM that are essential for inflorescence architecture.

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Selected Publications (Bibliography page)

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