Source: UNIV OF WISCONSIN submitted to
GENETIC ANALYSIS OF SPIRAL GROWTH AND ITS IMPLICATIONS FOR ROOT SHAPE
 
PROJECT DIRECTOR: Masson, P.
 
PERFORMING ORGANIZATION
GENETICS
UNIV OF WISCONSIN
MADISON,WI 53706
 
NON TECHNICAL SUMMARY: Plant roots have a tendency to spiral under defined conditions, and this root growth behavior has been shown to plague high-density vegetable-root productions such as carrots, where altered root shape is a major problem that reduces marketability and is selected against in plant breeding programs. While genetic variation is often observed in those crops, breeding to improve this trait is complicated because its genetic basis remains poorly understood. It would be difficult to analyze this process directly in the root crops because the genetic tools that are needed for such studies have not been developed in these plants. Therefore, there is considerable interest in using a model plant system such as the mouse-ear cress (Arabidopsis thaliana) to investigate some of the molecular mechanisms that govern root shape. When known, this information can be translated into temperate root crops such as carrot and beet, and tropical crops such as cassava, arracacha and sweet potato, by isolating the corresponding genes in these species and testing their potential role in modulating root shape in the corresponding crop. We recently showed that roots of the mouse-ear cress do spiral when forced to grow on a tilted hard-agar surface. Considering the large array of tools available for molecular genetic studies in this plant, we adopted this system to uncover some of the molecular mechanisms that govern root spiraling. This project is aimed at untilizing the natural genetic variation that exists between Arabidopsis strains to identify and characterize genes that contribute to the control of spiral root growth. These data will then be used to identify the corresponding carrot genes and test their involvement in conditioning important agricultural traits such as carrot splitting and twisting. Initial studies of the molecular mechanisms that modulate spiral root growth in the mouse-ear cress have led to a better understanding of the processes that control the synthesis and composition of plant cell walls, which constitute an important fraction of the material that can be fermented into biofuels. We have all reasons to believe that further investigations will continue shedding light on this important aspect of plant growth and development. Our lack of knowledge in this area of plant biochemistry and cell biology is recognized as a critical factor that limits our ability to generate crops that are optimized for biofuel production, a recognized top priority for the upcoming decades. It is anticipated that our work will, in the long run, also have important applications in this new area of agriculture.
 
OBJECTIVES: Most plant organs display oscillatory spiraling movements that appear to facilitate exploration of their environment. However, these behaviors often alter marketability of root-crop products, and a lack of molecular genetic tools in these crops prevents efficient selection against these unwanted traits. Initial studies with Arabidopsis thaliana have identified ecotype-specific variation in helical root growth, and a Quantitative Trait Loci (QTL) analysis was initiated to elucidate the underlying molecular mechanisms. The specific objectives of this project are: 1) Identify QTLs associated with variation in helical root growth within the Cvi x Ler2 Recombinant Inbred Line (RIL) population; 2) Clone and characterize two of these QTLs; 3) Clone carrot orthologs to arabidopsis genes that modulate helical growth. Identified loci will subsequently be used to investigate the genetic variability that underlies root shape in vegetable root crops, with the ultimate objective of improving this trait. The tools derived from this research will also be used to design instructional materials that address K-12 curricular needs, providing an educational network that values basic research while fostering direct application in agriculture.
 
APPROACH: To identify QTLs associated with spiral root growth on agar surfaces, we germinated Arabidopsis seeds from the Ler2/Cvi recombinant inbred lines (RIL) population on vertical, hard-agar media, and grew them for 3 days. Plates were then tilted 300 backward and incubated for 2 more days. Pictures were taken and used to quantify root length and straightness, along with horizontal and vertical growth indices, two parameters that estimate root skewing from the vertical. The data derived from this experiment and from biological repeats were entered into QTL Cartographer to identify and map significant QTLs. The same images will be quantified for traits related to root waving and analyzed for QTLs. Two of the identified QTLs will then be chosen for further molecular analysis. These QTLs will be verified using available Near Isogenic Lines (NILs). These NILs will be crossed to plants of the opposite parental ecotype. The corresponding F1 progeny will be selfed to generate 1,500 segregating F2 plants, which will be selfed to generate F3 families. These families will be analyzed for root waving and skewing. Parallel molecular analysis of linked polymorphic markers will allow identification of possible recombination breakpoints between polymorphic chromosomes, which will be used to fine-map the QTL. When a small chromosomal segment has been shown to carry the QTL of interest, we will use a combination of gene sequencing, expression profiling and transformation-rescue experiments to identify and characterize the QTL. If the cloned QTL is a protein-coding gene, translational fusions with YFP will be generated to localize the corresponding protein in vivo, using confocal microscopy. However, some QTLs may not encode proteins. Instead, they could encode structural or regulatory RNAs, such as miRNAs. miRNA loci can be identified by their defined, predictable structures. Many miRNAs have already been identified and recorded in a publicly accessible database that can be used to identify potential target(s). In order to translate results from our analysis of spiral growth into potential applications in agriculture, we will initiate the cloning and characterization of carrot orthologs to previously isolated Arabidopsis genes, using a combination of Bioinformatics, low stringency hybridization and RT-PCR approaches. Cloned carrot cDNAs and genomic DNAs will be handed out to professor Philipp Simon, whose lab will locate the corresponding loci on the carrot map, define possible linkage with genes that affect root shape and twisting in this system, and generate constructs for expression in transgenic carrot. In the long term, such material could be used to speed up the selection of carrot varieties that are less prone to bifurcation and twisting. The results from these studies will be written up for submission to high-impact scientific journals, and evaluated by the traditional peer-review process. Furthermore, the data will be presented at National and International Conferences, and they will be used to develop instructional materials for K-12 education.
 
CRIS NUMBER: 0214668 SUBFILE: CRIS
PROJECT NUMBER: WIS01338 SPONSOR AGENCY: NIFA
PROJECT TYPE: HATCH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2008 TERMINATION DATE: Sep 30, 2011

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
206242010802.270%
206145210802.210%
201145210802.220%

CLASSIFICATION HEADINGS
KA201 - Plant Genome, Genetics, and Genetic Mechanisms
KA206 - Basic Plant Biology
S2420 - Noncrop plant research
S1452 - Carrot
F1080 - Genetics
G2.2 - Increase Efficiency of Production and Marketing Systems


RESEARCH EFFORT CATEGORIES
BASIC 90%
APPLIED 10%
DEVELOPMENTAL (N/A)%

KEYWORDS: spiral root growth~arabidopsis thaliana~root waving and skewing~natural genetic variation~quantitative trait loci~carrot root bifurcation and spiraling~carrot orthologs~transgenic carrot

PROGRESS: Jan 1, 2010 TO Dec 31, 2010
OUTPUTS: The roots of Arabidopsis seedlings exposed to tilted hard-agar surfaces develop a wavy and skewing pattern of growth that results from responses to gravity, touch and other surface-derived stimuli. These complex growth behaviors are also accompanied by rotations of the root tips about their axes, developing spirals that are reminiscent of growth behaviors often displayed by crop vegetables such as carrot and are responsible for economical loss. To better understand this process, we developed a Quantitative Trait Loci (QTL) analysis of root waving and skewing on hard surfaces using a mapping population of Recombinant Inbred Lines (RILs) derived from a cross between Cvi and Ler Arabidopsis plants. This analysis allowed us to identify several QTLs, spread along the five Arabidopsis chromosomes, for biometrical parameters associated with root spiraling. Last year, we reported having used two Near Isogenic Lines (NILs) carrying a piece of Cvi genomic segment introgressed into a Ler2 genomic background to fine-map a strong QTL for root skewing located on chromosome 2. After crossing each NIL with Ler, new recombination breakpoints were obtained in the segregating F2 population, and the corresponding recombinants were phenotypically characterized to determine if the Cvi allele at this QTL was present in the plants. Using this fine-mapping strategy, we were able to map the QTL to a 1-Mb region of chromosome 2. We then used a microarray analysis of gene expression to identify genes that are differentially expressed in the root tips of seedlings growing on tilted hard-agar surfaces between Cvi and Ler. 699 genes were found to display distinct hybridization signals under these conditions, reflecting either a differential expression between ecotypes or differential hybridization intensity due to ecotype polymorphisms (the microarray probes were generated using sequence information from the Col ecotype). Of these, nine were found to fall within the fine-mapped interval and constitute strong candidates for the QTL under investigation. Further studies are underway to determine which of these candidates is truly responsible for the QTL. PARTICIPANTS: Laura Vaughn, Graduate student (PhD) in the Genetics program. TARGET AUDIENCES: This project is yielding important information that increases our understanding of the molecular mechanisms that govern root growth behavior in response to mechanical stimulation. This is likely to be of interest to researchers in the fields of plant growth, development and response to the environment. In the long term, this project may also generate the knowledge and tools needed to engineer root crops that are better adapted to growth in rocky environments without the unwanted consequence of developing organ spiraling, a process that decreases appeal from the consumer and affects accessibility to food-processing equipment. PROJECT MODIFICATIONS: NA

IMPACT: 2010-01-01 TO 2010-12-31 Spiral growth of plant organs leads to morphologies that may be advantageous or disadvantageous to the crop depending on its utilization. In Horticulture, this trait may be advantageous if esthetically pleasing, or allowing the plant to explore new environments (climbing shrubs; plant cover; etc). On the other hand, this trait may also be disadvantageous if it leads to heterogeneously shaped products that are poorly suited for mechanical conditioning. This is especially true for root crops, where altered root shape due to spiraling or splitting is responsible for major production losses every year. A better understanding of the molecular mechanisms underlying this growth behavior should, in the long term, allow the development of new strategies to improve this trait for better crop utilization in Agriculture and Horticulture.

PUBLICATION INFORMATION: 2010-01-01 TO 2010-12-31
Vaughn, L.M., Baldwin, K.L., Jia, G., Verdonk, J.C., Strohm, A.K., and Masson, P.H. (2010). Chapter 4: The Cytoskeleton and Root Growth Behavior. In The Plant Cytoskeleton. Advances in Plant Biology 2. (B. Liu, Ed.). Springer Verlag, pp 307-326.

PROJECT CONTACT INFORMATION
NAME: Masson, P.
PHONE: 608-265-2312
FAX: 608-262-2976