I enjoy research immensely along with the collaborative exchange and development of ideas with my colleagues. I am fascinated by how life works and the more complex interactions that occur in biology. I think that is why I have been so drawn in my early student years to whole plant physiology and later to systems biology.
My research has spanned 37 years and focused primarily on salinity stress during the first two decades of my career. I wanted to make more salt tolerant crop plants, crops that can even tolerate seawater irrigation. In the last decade, I have changed my focus to abiotic stress tolerance (drought, salinity and cold) of grapes using a systems biology approach.
A plant is a complex organism made up of many organelles, cells, tissues and organs, all of which work in harmony with each other. There are more than 250,000 plant species displaying a wide diversity of traits. Complex traits are influenced by many small quantitative trait loci (QTLs) indicating complex interactions with a lot of factors. Plant phenotypes are dependent upon genotype x environment interactions. With so many genes, proteins, metabolites and environmental variables the possible interactions are nearly infinite. Systems biology approaches are necessary to study such complexity.
Early Salinity Studies
The inhibition of plant growth by salinity involves two components. Initially, the plant experiences a water stress, but with time, salts accumulate in the plant creating an additional ionic stress. In my laboratory, we have focused on how salinity inhibits plant growth by these two components.
I have investigated the ionic stress component since the start of my Master’s thesis. I focused on the effects of Ca on the salt tolerance of plants including research on ion toxicity, growth, ion transport and cytosolic Ca. With the new focus on grapevines (described below), my attention is now directed at understanding and manipulating Cl- transport processes.
Originally in my thesis work, I focused on ion transport mechanisms and ion toxicity. However, I soon discovered that the water-stress component was more significant and poorly understood. This started a series of studies to understand the mechanisms that regulate plant growth and what growth component was restricted by salinity.
The water stress component was investigated by studying the immediate effects of salinity on the growth parameters regulating leaf elongation. Scientists in my lab and I found that salinity increases the apparent yield threshold of the cell walls in the growing region of the leaf, which results in lower growth rates. We have found interesting correlations of the plant response to salinity with the plant hormone, abscisic acid (ABA), particularly on growth, the apparent yield threshold and cytosolic calcium. Analysis with ABA mutants, however, has shown that ABA is not regulating growth significantly in salt-stressed plants but may act in protection against stress, particularly photoinhibition. Salt stress appears to be affecting plant growth by altering metabolic and signaling factors in response to the lower water potential and/or by altering water flow through aquaporins, plasmodesmata and long-distance hydraulic conductance mechanisms.
Recent Grape Research and Systems Biology
I have begun a research program since 1995 that has focused on how abiotic stress affects wine grapes. Grapevine is certainly the most complex and interesting plant I have ever worked on. Initially, the research program started off small with a simple varietal trial investigation on the feasibility of growing quality wine grapes in Northern Nevada. One off-shoot of that program was the development of regulated-deficit irrigation to improve grape quality without significant loss of production. Regulated deficit irrigation has reduced water usage by 80% compared with previous years and significantly improved the quality of grape musts and wines, producing more intense flavors, colors and fruity aromas. Our research has shown that grapevines use 12 times less water than local production methods for alfalfa, the major crop plant of the region. Another off-shoot has been the development of two additional experimental vineyards with private landowners in the state. One clear threat to a successful Nevada wine grape industry became obvious: cold temperatures. The region is susceptible to the occasional spring frost or a severe artic cold front in the middle of winter.
In addition, I established an experimental winery at the University in January of 2004 (see http://www.ag.unr.edu/cramer/nevadawinegrapes.htm). We have fermented the juice of 13 varieties of well-watered and drought-stressed grapes from our experimental vineyard on Valley Road for approximately 20 years in order to determine the effects of water deficit on grape quality and wines. We held weekly wine tastings at the winery or at other venues in the area to test the public reaction to our wines. The public reaction was highly positive and many persons are now planting grapes for wine production in the area. In addition, I hold classes and workshops to teach interested people in the local practices necessary for successful viticulture and enology in our area. My web page hosts grape and wine information for the interested grower and consumer.
My research goals quickly expanded to the development of more stress tolerant plants producing better quality wines. This development was facilitated by very useful collaborations with John Cushman, and David Schooley in the Biochemistry and Molecular Biology Department at the University of Nevada, Reno. Since that time, I have expanded collaborations with many scientists from all over the world. I have been very successful in obtaining substantial funds (approximately 9 million dollars during my career) from the American Vineyard Foundation (supported by most of the growers and wineries of California), the USDA, NSF and a large private winery (J. Lohr).
My recent work has substantiated published research that exposure of Vitis vinifera vines to water-deficits can enhance the aroma, flavor, and color characteristics of grape juice and wine. I have developed an integrated systems biology approach to study the effects of abiotic stress on transcript, protein and metabolite abundance in vegetative and fruit tissues of Vitis vinifera. This work has been supported by two consecutive but separate systems biology grants by the NSF Plant Genome Program starting in 2002. Using this approach, I have characterized qualitatively and quantitatively, the effects of abiotic stresses on alterations in the chemical composition of aroma, flavor, and color components of grape juice that form the foundation for improved wine quality and health benefits. The first NSF Plant Genome project focused on abiotic stress responses of grapevines (2002-2006). The second project investigated the influence of light and temperature on bud endodormancy (2006-2011). The PI of this project, Anne Fennell, has developed a unique population of grapes that segregate for regulation of bud dormancy for light and temperature. In addition, the second project involved a collaboration with Julie Dickerson at Iowa State University to develop customized integrated bioinformatics systems to track and analyze genomic data.
Cold tolerance is an issue for grapes in Nevada, especially for frost damage during the spring. To better understand cold tolerance mechanisms, grapes were transformed with a Vitis CBF transcription factor with assistance from the Ralph M. Parsons Foundation Plant Transformation Facility at UC Davis. CBF is known to regulate the cold-responsive regulon of many plants. Regenerated plants were shown to be more cold tolerant.
Currently, I am focusing my research in five major areas. One major project is on the effects of water deficit and ABA on water-use-efficiency and ABA signaling in grapes. We screened more than 30 genotypes (wild and cultivated) for differences in induction of NCED (nine-cis epoxycarotenoid dioxygenase), the rate-limiting step in ABA biosynthesis, and ABA hydroxlase, an enzyme that catabolizes ABA. The hypothesis is that ABA concentrations are correlated with drought tolerance and WUE. ABA sensitivity is another interest and this has been explored at the transcriptomic level. A more recent focus has been on root sensitivity to ABA and its effect on growth and water flow through the root.
A second focus is on the effects of ABA on berry development. Previous work indicated that ABA affects berry development at the veraison stage. Data indicate that ABA signaling is quite different in berry skins as compared to shoot tips. The nature of these differences is under examination at both the transcriptomic and proteomic levels.
In my third major project, we recently analyzed whole-genome microarrays of Cabernet Sauvignon berries that span a narrow developmental window when fruit flavors appear in mature fruit, as determined by sensory analysis by Hildegarde Heymann at UC Davis. More recently we have surveyed 7 grape cultivars at this stage using RNA-seq. We have identified a common set of biochemical pathways that are involved. Most interesting we have found a role for the circadian clock. In addition, we have identified a transcription factor that sharply rises and decreases in transcript abundance just at the developmental stage that these fruit flavors appear. This occurs specifically in the berry skin, the source of most fruit flavors. One of my students is actively determining the function of this transcription factor. It may play a role in biotic defense responses in the plant.
In the fourth project, we have sequenced and assembled the Cabernet Sauvignon genome us long-read PacBio technology. This is a collaborative project with Dario Cantu at UC Davis and Massimo Delledonne at the University of Verona. The quality of this genome sequencing is exceptional and will likely become a reference genome for this species. One paper has been published in Nature Methods, another paper is expected to be published in PNAS or Nature this year.
In the fifth project, we are investigating the drought and salt-tolerance of 4 grapevine genotypes, one a native species of Southern Nevada. The focus is on the roots to identify markers for breeding improved grapevine rootstock. This is in collaboration with Dylan Kosma (Department of Biochemistry and Molecular Biology), Beth Leger (Department of Natural Resources and Environmental Science), and Felipe Masias Barrios (Agriculture, Nutrition and Veterinary Sciences) at UNR. In addition, ML Robinson and Angela O’Callaghan (University of Nevada Cooperative Extension), Andy Walker (UC Davis), and Andy McElrone (USDA-ARS) are involved as Co-PIs.
There are several other on-going projects in the lab involving ion transport, wine quality, bud endodormancy, and water deficit and light effects on wine and vine growth, but the major focus is on the five projects described above.
of genes is a major challenge of the post-genomic era. To
traits, one requires a large number of genotypes of a
population in order to
apply association mapping, network analysis and systems
biology approaches to
help elucidate plant physiology and phenotype. The theory is
in place, the
tools are well developed and the data sets are emerging. It
won’t be long
before these efforts will result in significant progress in
plant breeding and
crop production. This is the major goal of my future research.