Enhancing Plant Biomass

Michael M. Neff 
Enhancing Plant Biomass

ArabidopsisDr. Michael Neff’s research allows us to gain a better understanding of how multiple signaling pathways converge to regulate seedling responses to light as well as adult plant development. This research also addresses the biological mechanisms controlling multiple traits which have been proposed as targets for enhanced plant biomass production including; photoreceptor-mediated shade avoidance, leaf senescence, leaf size, flower/fruit size and stem diameter. By understanding these mechanisms, we are likely to generate leads for the transfer of fundamental knowledge learned using Arabidopsis towards applications that will develop increased biomass in crops.

Summary of research goals and accomplishments

One laboratory project focuses on the role that a pair of Arabidopsis cytochrome P450 enzymes, BAS1/CYP72B1/CYP734A1 and SOB7/CYP72C1, have on the inactivation of a family of growth-promoting hormones, the brassinosteroids. He and his colleagues have shown that hormone inactivation plays a role in both light- and brassinosteroid-mediated development in plants. They have shown that BAS1 and SOB7 interact synergistically and redundantly to negatively regulate seedling hypocotyl and cotyledon expansion in response to light, as well as the size of adult plants and fruits. Their future focus will be on understanding the similarities and differences between the biochemistries of BAS1 and SOB7, exploring which pathways regulate these P450s and characterizing interactions with other brassinosteroid-inactivation pathways.

A second laboratory project focuses on the role that a Dof-class transcription factor, SOB1/OBP3, has on modulating seedling and adult plant stature in various tissues and light conditions. Neff and his team have shown that SOB1 negatively regulates seedling hypocotyl and cotyledon expansion in response to light, acting through different photoreceptors in each tissue. SOB1 also represses the growth of adult plants. Their future focus will be to examine whether SOB1 is involved in hormone signaling, to address the possibility that related proteins are functional homologues of SOB1, and to identify which genes regulate and are regulated by this transcription factor.

Another focus is on characterizing a pair of AT-hook-domain containing DNA-binding proteins, SOB3 and ESC, and the role that they have in modulating seedling responsiveness to light. Neff and his team have shown that these two proteins act redundantly in light-mediated inhibition of hypocotyl elongation. They have also shown that the over-expression of SOB3 delays senescence and increases leaf biomass, stem thickness and flower size. Their future focus will be to uncover the mechanisms by which these and other AT-hook-domain containing proteins regulate plant development.

Yet another project focuses on characterizing a small family of plant-specific proteins related to SOB5 and the role that they have in modulating levels of the plant hormones, cytokinins. The SOB5-like (SOFL) family of proteins has not been previously characterized and has no homology to proteins or domains of known function. He and his colleagues have shown that the overexpression of SOB5, SOFL1 and SOFL2 confers an increase in the levels of specific cytokinins while delaying leaf senescence. Their future focus will be to uncover the biochemical action of this family of proteins and how they link cytokinin- and light-mediated plant development.

Background and future research

Each of the lines of research in Neff’s lab comes from a mutant screen designed to uncover novel, and potentially redundant components involved in signal transduction downstream of a major photoreceptor controlling plant development, phytochrome B (PHYB). This screen has allowed his team to identify components missed by other genetic approaches, and has led his team toward studying how interactions between light and hormone signaling affect seedling and adult plant development.

Their mutant screen involves transforming Arabidopsis with a transgene containing multimerized promoter-enhancer elements. These elements amplify the expression of nearby genes conferring a dominant, gain-of-function phenotype. By utilizing this approach, the team can specifically target groups of genes that may be functionally redundant and thus not readily identifiable in traditional loss-of-function mutant screens.

For the genetic background in their screen, Neff and his team use a mis-sense mutation in PHYB, phyB-4. phyB-4 mutant seedlings that don’t respond normally to light. They display longer/taller hypocotyls relative to the wild type. Adult phyB-4 mutants are leggy or flimsy and reminiscent of plants avoiding deep shade. They screen specifically for suppressor mutations (i.e. bas1-D and sob1-D through sob7-D) that restored these phyB-4 seedlings’ ability to respond to light while also repressing the adult mutant phenotypes related to stature and shade avoidance. By using this genetic background with reduced PHYB signaling the team has identified components that regulate plant development via both light- and hormone-mediated signaling pathways. This mutant screen in Arabidopsis has also generated leads that may be useful for increasing biomass in crop plants.

Undergraduate contribution to research

Neff with studentsThis mutant screen has been performed primarily by undergraduates working directly with Dr. Neff, or one of his postdocs in the lab. Six of the eight suppressor mutants were cloned and initially characterized by undergraduates from Washington University in St. Louis during either the school year or as summer research fellows. One of these, Elizabeth Wrage, was an American Society of Plant Biology Summer Undergraduate Research Fellow (SURF). Six others (four of whom were women) were Howard Hughes SURF recipients. In addition, many of the 32 undergraduates working in Neff’s lab over the past seven years were involved in other aspects of characterizing these genes. A major portion of the funding for this research has come from Research Experience for Undergraduate (REU) supplements that were awarded to Neff’s previous NSF grant. To date he has raised $30,000 in REU supplements.

With Neff’s renewed grants from the NSF and DOE, he will continue this tradition of undergraduate education. He intends to recruit at least one post doc who is interested in undergraduate education. This post doc would be able to work directly with undergraduates and possibly gain formal teaching experience while establishing a research program that she or he can take when starting their own lab. This formula has worked well for a former postdoctoral fellow in his lab, Dr. Leeann Thornton, who is now an Assistant Professor at the College of New Jersey, an academic institution with an emphasis on undergraduate education.

The role of brassinosteroid inactivation in plant development

One of the most important contributions that Neff and his colleagues have made to plant biology comes from studying a group of hormones, the brassinosteroids. Brassinosteroids are growth promoting steroid hormones critical for seedling and adult plant development. Most brassinosteroid investigations have concentrated on either the biosynthesis of these hormones or on their receptor-mediated signal transduction pathways. Neff’s team has demonstrated that the inactivation of these steroid hormones plays an important role in the regulation of plant development, including responses to light.

Their studies on brassinosteroid inactivation focus on two genes, BAS1/CYP72B1/CYP734A1 and SOB7/CYP72C1, encoding proteins belonging to a large family, the cytochrome P450s. His team has shown that BAS1 is a brassinosteroid-inactivating carbon-26 hydroxylase (Neff et al. 1999; Turk et al. 2003). They have also shown that SOB7 is a brassinosteroid-inactivating enzyme, though it is not a C-26 hydroxylase (Turk et al. 2005). The removal of BAS1 and SOB7 elevates levels of active brassinosteroids and confers an increase in adult rosette size and fruit length (Turk et al. 2005), suggesting that these genes may be good targets for modification to increase biomass in crops.

Their future focus will be on understanding the similarities and differences between the biochemistries of BAS1 and SOB7. Homology-modeling studies support our hypothesis that these two enzymes can interact with multiple brassinosteroids via different docking mechanisms. Heterologous expression of these proteins in yeast or baculovirus systems will allow Neff and his colleagues to examine the kinetics of enzyme/substrate interactions and possibly identify the products of SOB7 activity. Expression of translational fusions between these proteins and reporter enzymes or epitope tags will allow them to explore the degree of overlapping activity between these two brassinosteroid-inactivating enzymes during plant development. The testing of genetic interactions between BAS1/SOB7 and other recently identified brassinosteroid-inactivating pathways will allow them to gain a better understanding of how this method of modulating levels of active hormones contributes to plant development. This research has been funded by a grant from the NSF that has been recently renewed for an additional three years of support.

The role of DNA-binding proteins in plant development

Neff in the labA second project in Neff’s lab focuses on the role that a Dof-class transcription factor, SOB1/OBP3, has on modulating seedling and adult plant stature in various tissues and light conditions. They have shown that SOB1 negatively regulates seedling hypocotyl and cotyledon expansion in response to light, acting through different photoreceptors in each tissue (Ward et al., 2005). SOB1 also represses the growth of adult plants, making it a good target, like BAS1 and SOB7, for increasing yield in various crops.

Their future focus will be to identify and characterize SOB1-interacting proteins using yeast-two-hybrid and/or Co-IP techniques coupled with heterologous expression and biochemical kinetic analysis. Neff and his team are currently utilizing DNA-chip analysis to identify which genes are regulated by this transcription factor. They will also examine the possibility that related proteins are functional homologues of SOB1. This research was funded by a grant from the DOE that has recently been renewed for an additional three years of support.

A third project in Neff’s lab focuses on characterizing a pair of AT-hook-domain containing DNA-binding proteins, SOB3 and ESC, and the role that they have in modulating seedling responsiveness to light. He and his colleagues have shown that these two proteins act redundantly in light-mediated inhibition of hypocotyl elongation. They have also shown that the over-expression of SOB3 and ESC delays senescence and increases leaf biomass, stem thickness and flower size, all important targets for elevating yield in crops (Street et al., in revision). Their future focus will be to uncover the biochemical mechanisms by which these and other AT-hook-domain containing proteins regulate plant development. Funding from the DOE supports this research.

The role of a novel class of proteins in plant development

 A fourth project in Neff’s lab focuses on characterizing a small family of plant-specific proteins related to SOB5 and the role that they have in modulating levels of the plant hormones, cytokinins. The SOB5-like (SOFL) family of proteins has not been previously characterized and has no homology to proteins or domains of known function. Neff and his colleagues have shown that the overexpression of SOB5 (Zhang et al., 2006), SOFL1 and SOFL2 confers an increase in the levels of specific cytokinins while delaying leaf senescence, an important target trait for increasing biomass in certain crops.

Their future focus will be to uncover the biochemical action of this family of proteins. We will identify and characterize SOB5/SOFL1/SOFL2-interacting proteins using yeast-two-hybrid and/or Co-IP techniques coupled with heterologous expression and biochemical kinetic analysis. Another approach will be to express, with an inducible promoter, these genes in planta, followed by the identification of potential targets of activity using 2-D protein gel electrophoresis. This research has been funded by the Monsanto Corporation and is the subject of a pending proposal to the USDA.

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Contact Information
Michael M. Neff, Ph.D.
Associate Professor, Crop Biotechnology
Crop and Soil Sciences

Washington State University
PO Box 646420
Johnson 371
Pullman, WA 99164-6420

Telephone: 509-335-7705
Fax: 509-335-8674
E-mail: mmneff@wsu.edu

Michael M. Neff, Ph.D.
Dr. Neff grew up on the East Coast in Baltimore, Maryland where he attended the  Friends (Quaker) school for his K-12 education. He moved to Colorado Springs in 1981 and was a biology major focusing on ecology at the Colorado College. At the same time, his parents moved to Seattle, Washington. Visits to the Northwest convinced Dr. Neff to move further west where he became more interested in bench-related research. It was while working in a virology lab at the University of Washington that he met his wife, Sandy O’Keefe. A fascination with plants led Dr. Neff to pursue a B.S. degree in Botany at the University of Washington. After working as a lab technician in a yeast genetics lab with Dr. Trisha Davis, Dr. Neff rejoined the University of Washington Department of Botany to pursue his Ph.D., studying plant physiology with Dr. Elizabeth Van Volkenburgh. After graduating in 1995, Dr. Neff was a NIH postdoctoral fellow studying plant molecular genetics with Dr. Joanne Chory at the Salk Institute for Biological Studies in La Jolla California. He was a faculty member in the Department of Biology at Washington University in St. Louis, Missouri from 1999 to 2007 before accepting the new Crop Biotechnology faculty position in the Department of Crop and Soil Sciences at Washington State University.