Source: WAKE FOREST UNIVERSITY submitted to
MECHANISMS OF ETHYLENE AND AUXIN CROSS TALK IN ROOT DEVELOPMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
0220756
Grant No.
2010-65116-20436
Project No.
NCR-2009-03459
Proposal No.
2009-03459
Multistate No.
(N/A)
Program Code
91414
Project Start Date
Jan 1, 2010
Project End Date
Dec 31, 2014
Grant Year
2014
Project Director
Muday, G. K.
Recipient Organization
WAKE FOREST UNIVERSITY
1834 WAKE FOREST ROAD
WINSTON SALEM,NC 27109-8758
Performing Department
(N/A)
Non Technical Summary
A highly branched root network is essential for efficient nutrient and moisture retention and, consequently, for maximal plant productivity. An understanding of the molecular mechanisms that control root architecture will guide strategies for crop improvement. This project examines the hormonal controls of root branching with a focus on the hormone auxin, whose importance in regulating lateral root initiation is well established, and ethylene, which is a hormone with a recently identified role in this processes. The proposed experiments combine genetic, molecular, and cell biological approaches to define the mechanisms for the crosstalk between auxin and ethylene that specify root architecture. These experiments will be performed in two plants, the crop species, Solanum lycopersicum (tomato), and the genetic model, Arabidopsis thaliana, which each have unique experimental advantages. Arabidopsis offers many well-characterized ethylene and auxin signaling and transport mutants, along with reporters and transgenic lines that can be used to examine the molecular mechanisms by which ethylene and auxin signaling interface. Tomato offers a more limited set of mutants and transgenics, but has a rapidly expanding community and set of molecular tools, and it represents a plant family with many agriculturally important members. We possess a collection of tomato mutants with altered root branching patterns and have developed tools, techniques, and resources to explore the mechanisms of hormonal control of root development in this crop species. Examining root development in two species will aid in understanding the universality of its regulation across the plant kingdom, providing vital insight into the crosstalk between hormones that control root architecture, which can be exploited to improve crop productivity.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20614601050100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1050 - Developmental biology;
Goals / Objectives
Lateral root formation is a highly plastic phenotype that is sensitive to nutrients, moisture, and other environmental parameters, with plant hormones acting as one important signaling mechanism to control this process. Auxin modulates root branching and this process is dramatically altered in plants with mutations in the genes encoding proteins that participate in auxin synthesis, transport, and signaling. With our prior USDA support, we identified a role for ethylene in adventitious and lateral root formation in both the model plant, Arabidopsis thaliana, and the crop plant, Solanum lysopersicum (tomato). In addition to the observations of root phenotype of ethylene signaling and synthesis mutants, we have shown that ethylene modulates root formation through alteration of auxin transport. We will extend these studies by using genetic approaches to explore the cross talk between auxin and ethylene that control lateral root formation in tomato. This work will focus on identification of the mechanisms by which ethylene alters auxin transport and this in turn alters root development. Identification of the mechanisms by which auxin transport proteins change in expression, localization, or activity will provide insight into the regulation of this process and how auxin distribution drives root formation. These mechanisms can be tested directly in Arabidopsis where molecular tools are in hand, while in tomato we will use several approaches to identify transport proteins and genetic controls of root development, to move toward experiments focused on parallel mechanisms in tomato. We have in hand tomato mutants isolated for altered root branching or synthesis of flavonoids, molecules that may regulate auxin transport and are working to characterize the mutant phenotypes. We also have identified candidates for genes encoding auxin transport proteins and will test their genetic function. Together, these experiments should allow us to test hypotheses for the regulation of auxin transport and root development and identify new genetic controls of this process.
Project Methods
These experiments will use a genetic approach to examine how cross-talk between auxin and ethylene define the architecture of roots and to identify genetic controls of this process. The experiments will utilize the genetic model plant, Arabidopsis thaliana, and the crop plant, Lycopersicon esculentum (tomato), which each offer unique experimental advantages. Arabidopsis offers many well-characterized ethylene-signaling mutants along with reporters and transgenic lines that can be used to examine the molecular mechanisms by which ethylene and auxin signaling interface. Although tomato offers fewer ethylene-insensitive mutants and transgenics it has a rapidly expanding set of molecular tools and it represents a plant family with many agriculturally important members. Additionally, the larger size of tomato roots will facilitate studies of local change in gene expression. We will explore auxin ethylene cross talk in greater detail, utilizing Arabidopsis mutants with defects in auxin transport proteins and with plants expressing transgenic auxin transport protein-fluorescent protein chimeras. These studies will inform our understanding of auxin transport proteins that function in this hormonal cross talk and provide insight into the orthologous tomato genes to dissect the controls of root branching in this crop species. We have in hand a collection of tomato mutants with altered root formation and have begun to characterize their responses to auxin and ethylene and will move toward isolation of the mutant genes. Finally, we propose to use a transgenic approach in tomato to test our hypotheses about genetic controls of root development. Together these experiments should provide insight into auxin and ethylene cross talk that regulates root architecture in this important crop species.

Progress 01/01/12 to 12/31/12

Outputs
Target Audience: This project focuses on basic research to understand the mechanisms that control root growth and development. Our studies of regulation of root development by hormones, flavonoid molecules, and wounding are communicated to other researchers through publication and presentation at scientific meetings. We have also used our tomato genetic resources to develop educational outreach activities that we have shared with middle and high school students. We worked with over 700 7th grade and high school biology students last year to teach plant genetics using mutant and heirloom tomatoes. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project supported the training of one graduate, two undergraduate, two high school students, and one post-doctoral research associates. Justin Watkins, a PhD student, is exploring the role of flavonoids as antioxidants that modulate guard cell signaling and thereby regulate stomatal aperture. The undergraduates and high school students helped in many aspects of the projects, from care and maintenance of tomato plants in the field and development of education outreach exercises that were used during visits to middle and high schools. These students have learned how to isolate DNA, phenotypically characterize root growth, and design experiments. Greg Maloney is a post-doc working on this project, who has examined adventitious root formation in Arabidopsis and tomato flavonoid biosynthetic mutants and introgression lines. He is examining the role of flavonoids in tomato root growth and development and using both mutants and introgression lines of tomato to ask if these molecules regulate root architecture in analogous ways to those reported in Arabidopsis. How have the results been disseminated to communities of interest? These results have been presented in publications and presentations at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals? As we enter our final year of funding we will be focusing on completing several manuscripts for which we have collected and analyzed data, but not yet submitted for review.

Impacts
What was accomplished under these goals? Duringthis yearwe completed our studies on the hormonal controls of adventitious root formation. Adventitious roots emerge from aerial plant tissues and the induction of adventitious root development is essential for clonal propagation of agriculturally important plant species. We have explored the role of auxin transport in this process in Arabidopsis and tomato seedlings in which adventitious root formation was induced by removing roots from low light grown hypocotyls. Root excision is accompanied by an increase in auxin transport and local changes in auxin responsive gene expression above the site of excision, consistent with local auxin accumulation preceding adventitious root formation. We examined excision-induced adventitious root formation in auxin influx and efflux mutants in Arabidopsis, and found the most profound reductions in abcb19 while an ABCB19 overexpression line formed more adventitious roots. Examination of both transcriptional and translational ABCB19-GFP fusions suggests both transcriptional and post-transcriptional regulation of ABCB19 in response to root excision that induces adventitious root formation. Together, these results suggest that polar auxin transport, mediated by ABCB19, is an essential regulator of excision-induced adventitious root formation in Arabidopsis. We have also explored the regulation of this process by ethylene. Ethylene negatively regulates ABCB19 expression in the hypocotyl and reduces adventitious root formation. Ethylene also increases flavonoid synthesis, which also negatively regulates auxin transport. The work on ABCB19’s role in this process was accepted for publication in Plant Physiology and a manuscript on the ethylene regulation is currently in preparation. The majority of our activity in year three focused on developing approaches to study flavonoid metabolism in tomato, as well as exploring the effects of mutations that alter flavonoid synthesis on growth and development. In contrast to Arabidopsis, which has a single gene encoding most enzymes in the flavonoid pathway, in tomato there are between 1 and 3 genes encoding each enzyme. We have examined the transcript abundance of each of these genes in young seedlings using quantitative RT-PCR separately in roots and shoots since these two tissues have differing flavonoid profiles and developmental processes. There are much higher levels of expression of these transcripts encoding flavonoid enzymesin hypocotyls than roots consistent with greater pathway activity and anthocyanin synthesis.We have also developed methods for quantification of flavonoid intermediates using mass spectroscopy. Although there are higher levels of all of the flavonoid intermediates in shoots, the difference is less profound than for transcripts. This observation is consistent with accumulation of flavonols in roots because of the absence of enzymes for later pathway steps, while in shoots, these molecules are converted into anthocyanins and other downstream metabolites. Although transcript abundance does not directly correlate with the amount of synthesis or activity of the enzymes they encode, they are a valuable proxy for predicting relative levels of enzymes. Together these results give baseline information to interpret mutant metabolic differences and regulation of flavonoid synthesis. We haveexamined the tomato mutant anthocyanin reduced (are), as this mutant should have accumulation of naringenin and depletion of flavonols and anthocyanins due to a point mutation in F3H and the absence of detectable F3H enzyme activity. Consistent with this prediction, the are mutant has more than 7-fold higher levels of naringenin than wild-type and no detectable levels of myricetin in roots. The reduced abundance of anthocyanins in the stem of are is apparent, as is the surprising observation that are formed less than 50% of the roots of wild-type , which contrasts with the increase in roots in the flavonol-deficient Arabidopsis mutant. The entirely anthocyaninless, or ae, mutant has a mutation linked to the ANS and have found that there are 6-fold higher levels of myricetin in ae over the parental line. This mutant also has a dramatic increase in lateral root formation. Since both are and ae have reduced anthocyanins, the difference in root formation seems more tightly correlated with flavonol levels. This is an intriguing contrast with Arabidopsis, where flavonols negatively affect root formation and clarifies the importance of studying flavonoid effects in a crop species, in addition to a model organism. A manuscript focused on the molecular and physiological characterization of the are mutant is currently in preparation. We have also been examining a tomato introgression line population. These introgression lines were made by crossing S. pennillii with cultivated tomato (S. lycopersicum). The resulting population was extensively backcrossed and regions of introgressed S. pennellii sequences were mapped to obtain a population with known regions of S. pennellii chromosomes introgressed into the S. lycopersicum background. We have examined the lateral root developmental pattern in this population of 75 lines in the presence and absence of sucrose, an important modulator of both lateral root formation and flavonoid synthesis, identifying 4-fold variation in lateral root formation among the population. We have asked if there is altered flavonoid metabolism in these lines, by performing a screen of these mutant lines examining flavonol accumulation using the dye, DPBA, which fluoresces when binding flavonols. DPBA has been shown to be specific for binding flavonols in vitro and in vivo, as the signal is absent in the Arabidopsis tt4 mutant and restored in vivo with quercetin treatment. We find more than 2-fold differences of DPBA fluorescence levels across the introgression lines. We have begun to quantify flavonoids by LCMS for these lines. We have observed that flavonoids accumulate in guard cells, but not in surrounding pavement cells using the dye DPBA in both Arabidopsis and tomato leaves. In Arabidopsis wild-type (Col) and tt4 guard cells examined by confocal imaging using spectral separation to resolve the DPBA and chlorophyll signals, this guard cell-specific flavonoid localization is evident in wild-type, but absent in tt4 imaged under identical conditions. The product of CHS promoter-driven GUS reporter also accumulates specifically in the guard cells, consistent with flavonoid synthesis localized to this tissue. One important second messenger in these cells is hydrogen peroxide, which is produced through ABA activation of an NADPH oxidase, which then signals for the closure of guard cells. We hypothesized that flavonoids might act as antioxidants in guard cells to keep hydrogen peroxide from reaching levels that cause oxidative damage and to modulate the rate of stomatal closure. We tested this hypothesis, with the dye DCF that fluoresces upon binding to a variety of reactive oxygen species (ROS) and examined fluorescence in wild-type and tt4. The levels of ROS in the cytoplasm are greatly elevated in tt4 and consistent with elevated ROS, the rate of stomatal closure is also elevated. The During our third year of funding we wrote a number of review articles and book chapters reviewing the interesting cross talk between auxin and ethylene in regulation of root growth and development. One of these was published in Trends in Plant Science and another appeared in the book entitled “Plant Roots: The Hidden Half”, which was a new addition of a classic book that has reviews on the very basic to very agriculturally applied aspects of root growth and development.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2012 Citation: Sukumar, P, Maloney, G, Muday, GK (In press) Localized induction of the ABCB19 auxin transporter enhances adventitious root formation in Arabidopsis. Plant Physiology
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Withers, JC, Shipp, MJ, Rupasinghe, SG, Sukumar, P, Clay, SL, Schuler, MA, Muday, GK, and Wyatt, SE (2012) Gravity Persistent Signal 1 (GPS1) reveals novel cytochrome P450s involved in gravitropism. American Journal of Botany: 100:183-193
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Muday, GK, Rahman, A, Binder, BM (2012) Auxin and ethylene: Collaborators or Competitors? Trends in Plant Science, 17: 181-195
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Chae, K, Isaacs, CG, Reeves, PH, Maloney, GS, Muday, GK, Nagpal,P, Reed, JW (2012) Arabidopsis SMALL AUXIN UP RNA63 promotes hypocotyl and stamen filament elongation. Plant Journal 71: 684-697.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2012 Citation: Muday, GK and Lewis, DR (In press) Ethylene regulates root growth and development. In Plant Roots: The Hidden Half. Eschel, A, Beeckman, T eds. CRC Press, Taylor and Francis Group, Boca Raton
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Maloney, G., Muday, G. K. , "Analysis of tomato flavonoid biosynthetic pathway and effects of flavonoids on tomato root and seed development," Plant Biology 2012, American Society of Plant Biologists, Austin, Texas, Invited podium presentation
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Lundy, S., Browne, C. L., Edwards, B., Muday, G. K., "Undergraduates learn genetics by teaching middle school and high school," Plant Biology 2012, American Society of Plant Biologists, Austin, Texas, Invited podium presentation (July 2012).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Muday, G. K. Lewis, D. R.), Lundy, S. Olex, A. Fetrow, J. S., "Transcriptional events that mediate the synergistic and antagonistic actions of auxin and ethylene on root growth and development," Ethylene 2012, Group of Researchers, Rotorua, New Zealand, Invited podium presentation (March 2012).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Maloney, G. Muday, G. K., "Understanding the role of flavonols in tomato root architecture using mutants and introgression lines," North Carolina Plant Molecular Biology Retreat, North Carolina Biotechnology Center, Wrightsville Beach, NC, Invited podium presentation (September 2012).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Muday, G. K. , Lewis, D. Lundy, S. Olex, A. Fetrow, J. S. "Transcriptional events that mediate the synergistic and antagonistic actions of auxin and ethylene on root growth and development.," Mini-Symposium on Molecular Plant Sciences, Virginia Tech, Blacksburg, Va, Invited podium presentation (March 2012).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Watkins, J and Muday, GK. Regulation of reactive oxygen species signaling in guard cells by flavonoid antioxidants. 29th New Phytologist Symposium Stomata 2012, Cambridge, UK
  • Type: Websites Status: Published Year Published: 2012 Citation: Internet - Text, Windows on Wake Forest, "Teaching with Tomatoes." (October 15, 2012).
  • Type: Other Status: Other Year Published: 2012 Citation: Muday, G. K., "Understanding the synergistic and antagonistic actions of auxin and ethylene on root growth and development," Invited Departmental Seminar, GregorMendel Research Institute, Vienna, Austria,(August 2012).
  • Type: Other Status: Other Year Published: 2012 Citation: Muday, G. K., "Understanding the synergistic and antagonistic actions of auxin and ethylene on root growth and development," Invited Departmental Seminar, University of Natural Resources and Life Sciences, Vienna, Austria, (July 2012).
  • Type: Other Status: Other Year Published: 2012 Citation: Muday, GK "Understanding the synergistic and antagonistic actions of auxin and ethylene on root growth and development," Invited Departmental Seminar, University of North Carolina at Charlotte, Department of Bioinformatics, April 2012


Progress 01/01/11 to 12/31/11

Outputs
Target Audience: This project explores the hormonal controls of tomato root growth and development and results are communicated to the scientific community by publication and presentation at scientific meetings. We also communicated the scientific process and how genes control plant growth and development of the plants we eat to middle and high school students, by taking more than 40 Wake Forest University students to 7th grade and high school biology classes to teach plant genetics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project supported the training of three graduate, two undergraduate, two high school students, and two post-doctoral research associates. A graduate student, who completed her PhD in 2010, Poornima Sukumar, worked on hormonal controls of adventitious root formation in tomato and Arabidopsis. Mary Beth Lovin also completed her PhD in 2010 on the environmental and hormonal controls on lateral root formation, with a focus on light quantity and temperature as signals modulating lateral root formation. A third PhD student, Justin Watkins, is exploring the role of flavonoids in parts of the plant beyond the roots. The undergraduates and high school students helped in many aspects of the projects, from care and maintenance of tomato plants in the field and development of education outreach exercises that were used during visits to elementary, middle and high schools as described above. These students have learned how to isolate DNA, phenotypically characterize root growth, and design experiments. Greg Maloney is a post-doc working on this project, who has examined adventitious root formation in Arabidopsis and tomato flavonoid biosynthetic mutants and introgression lines. He also is isolating the ABCB19 gene from tomato and examining its role in root growth and development. He is examining the role of flavonoids in tomato root growth and development and using both mutants and introgression lines of tomato to ask if these molecules regulate root architecture in analogous ways to those reported in Arabidopsis. A second post-doc, Hanya Chrispeels has focused on the characterization of the tomato erb1 mutant, with enhanced adventitious and lateral roots and on development of educational outreach exercises to teach plant genetics using heirloom tomatoes. How have the results been disseminated to communities of interest? Two of our primary aims of the project were completed in the first two years of the grant and publications in the journals Development and Plant Physiology communicated these findings to the scientific community. Lab members presented 5 talks and 7 posters at scientific meetings. Our educational reach activities in schools using mutant and heirloom tomatoes were also the subject of a newspaper article in the Winston Salem Journal, our local newspaper. What do you plan to do during the next reporting period to accomplish the goals? We have found that tomato mutants with altered flavonoid and anthocyanin levels have profound phenotypic differences in root growth and development. We will focus our activities in the next year on characterization of those mutants.

Impacts
What was accomplished under these goals? 1,How does ethylene alter long distance auxin movement and local auxin gradients that control lateral and adventitious root formation in Arabidopsis? We utilized genetic and molecular approaches to identify the mechanisms by which the plant hormone ethylene reduces lateral root formation and regulates the synthesis of flavonoids, endogenous regulators of plant growth and development. Although ethylene inhibits lateral root formation, treatment with the ethylene precursor ACC enhances the polar transport of auxin. We have identified critical roles for two auxin transport proteins, PIN3 and PIN7, in the effects of ACC on both auxin transport and root formation. ACC increases the abundance of the transcripts encoding PIN3 and PIN7 in an ETR1 and EIN2 (ethylene signaling) dependent and TIR1 (auxin receptor) independent fashion. PIN3- and PIN7-GFP fluorescence increased along the length of ACC-treated roots, consistent with their role in ACC-elevated transport. ACC treatment also abolished a localized depletion of fluorescence of these two reporters normally found below the site of primordia formation. These results suggest that increased PIN3 and PIN7-mediated acropetal auxin transport after ACC treatment prevents localized accumulation of auxin needed to drive lateral root formation. This work is summarized in a manuscript that appeared in the journal Development (Lewis et al, 2011a). We also examined the hormonal cross talk regulating the synthesis of flavonols and examined their impact on root growth and development. We analyzed the effects of auxin and an ethylene precursor on roots of wild-type and hormone-insensitive Arabidopsis thaliana mutants at the transcript, protein, and metabolite levels at high spatial and temporal resolution. IAA and ACC differentially increased flavonol pathway transcripts and flavonol accumulation, altering the relative abundance of quercetin and kaempferol. The IAA, but not ACC response, is lost in the tir1 auxin receptor mutant; while ACC responses, but not IAA responses, are lost in ein2 and etr1 ethylene signaling mutants. Kinetic analyses identified auxin and ethylene mediated increases in transcripts encoding transcriptional regulators, which preceded increases in transcription of flavonoid biosynthetic enzymes. Physiological analyses revealed that tt4, which makes no flavonols, and tt7, which makes kaempferol, but not quercetin, have identical auxin transport and root gravitropism phenotypes. These results demonstrated that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth.Collectively, these experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. The study also provides new evidence that quercetin is the flavonol which modulates basipetal auxin transport. This work was recently published at Plant Physiology (Lewis et al., 2011b). Adventitious roots emerge from aerial plant tissues and the induction of adventitious root development is essential for clonal propagation of agriculturally important plant species. We have explored the role of auxin transport in this process in Arabidopsis and tomato seedlings in which adventitious root formation was induced by removing roots from low light grown hypocotyls. Root excision is accompanied by an increase in auxin transport and local changes in auxin responsive gene expression above the site of excision, consistent with local auxin accumulation preceding adventitious root formation. We examined excision-induced adventitious root formation in auxin influx and efflux mutants in Arabidopsis, and found the most profound reductions in abcb19 while an ABCB19 overexpression line formed more adventitious roots. Examination of both transcriptional and translational ABCB19-GFP fusions suggests both transcriptional and post-transcriptional regulation of ABCB19 in response to root excision that induces adventitious root formation. Together, these results suggest that polar auxin transport, mediated by ABCB19, is an essential regulator of excision-induced adventitious root formation in Arabidopsis. This work is the subject of the completed PhD thesis of Poornima Sukumar in 2010 and was submitted for publication during this year. 2. What are the mechanisms in tomato for ethylene and auxin cross talk in regulation of root development? We have explored this question using tomato mutants with altered ethylene signaling and synthesis. We have found that ethylene insensitive mutants form greater numbers of lateral roots, including the well characterized Never ripe mutant, which has a defect in an ETR receptor ortholog and the Greenripe mutant, which has a defect in the RTE gene encoding an ethylene signaling protein. We have also shown that ethylene positively regulates long distance transport in tomato in parallel to the effect in Arabidopsis. We have also explored the effect of ethylene on adventitious root formation and found that the response is opposite between Arabidopsis and tomato, perhaps occurring by different regulatory pathways. These results have been published (Negi et al. 2010), and we are currently exploring hormonal controls of expression of genes encoding auxin transport proteins that mediate both lateral and adventitious root formation in tomato to compare and contrast responses between species. We have focused on identification of potential auxin transport proteins from the newly complete tomato genome, isolation of these genes, and qRT-PCR analyses of their expression. We have identified an ABCB19 ortholog and are examining the expression of this gene in response to treatments that alter its expression in Arabidopsis to ask if similar hormonal controls are present in tomato. We are also collaborating with the lab of Carmen Catala to characterize the auxin transport levels in transgenic tomatoes engineered to have altered levels of expression of genes encoding auxin transport proteins. 3. Do tomato lateral and adventitious root mutants have altered ethylene and/or auxin signaling, synthesis, or transport? We are also focusing on examination of a set of mutants with altered root development. The enhanced root branching1 (erb1) mutant was isolated in a screen by Sangeeta Negi, a former lab member, and has enhanced root formation and branching. This mutant has strong alterations in auxin dependent growth and transcriptional responses, with weaker alterations in ethylene response, which appear to be the indirect result of hormone cross talk. We have found that this mutant has altered expression of genes linked to auxin regulated transcription, including ARF and IAA genes. We are currently writing a manuscript describing the erb1 phenotype and its hormone signaling defects. We are also examining tomato mutants with defects in anthocyanin and flavonoid accumulation to determine if there is regulation of root architecture and auxin transport by flavonols analogous to Arabidopsis. In particular, the anthyocyanin reduced (are) mutant has a defect in flavonoid biosynthesis, which we have confirmed both biochemically and by mass spectroscopy of metabolite profiles. This mutant has an array of phenotypes tied to auxin dependent growth processes, including lateral root, trichome, and embryo development. We have also completed a screen of a population of introgression lines in which regions of the Solanum penellii chromosomes have been introgressed into S. lycopersicum. We have found interesting lines with strongly enhanced and reduced lateral or adventitious roots. We are focusing on several chromosome regions to identify candidate genes that may contribute to these altered phenotypes. This work wasthe subject of an invited minisymposia presentations at the Plant Biology meeting in 2011.

Publications

  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Lewis, DR, Negi, S, Sukumar, P, Muday, GK (2011) Ethylene inhibits lateral root development and enhances IAA transport by altered expression and localization of auxin transport proteins. Development 138: 3485-3495
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Lewis, DR, Ramirez, MV, Valbuena, P, Miller, ND, Keith, R, Helm, R, Winkel, BSJ, Muday, GK (2011) Auxin and ethylene induce distinct flavonol accumulation patterns through independent transcriptional networks: Plant Physiol. 156: 144-164
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Negi, S, Sukumar, P, Liu, X, Cohen, JD, and Muday, GK (2010) Genetic dissection of the role of ethylene in regulating auxin dependent lateral and adventitious root formation in tomato. Plant Journal 61: 3-15
  • Type: Journal Articles Status: Under Review Year Published: 2011 Citation: Muday, GK, Rahman, A, Binder, BM (In review) Auxin and ethylene: Collaborators or Competitors? Trends in Plant Science.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2011 Citation: Maloney, GS, Muday, GK The role of flavonoids in regulating tomato root architecture. ASPB Plant Biology Meeting, Invited Minisymposium presentation, July 2011, Minneapolis, MN.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Chrispeels, H, Muday, GK. Using heirloom tomatoes for educational outreach: teaching plant biology with a familiar food. ASPB Plant Biology meeting, Montreal, July 2010
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Chrispeels, H, Negi, S, and Muday, GK. The tomato mutant erb1 (enhanced root branching1) has altered auxin and ethylene responses. ASPB Plant Biology meeting, Montreal, July 2010
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Cooper, K, Lewis, DR, Muday, GK. Auxin-induced flavonoid gene expression and root architecture. ASPB Plant Biology meeting, Montreal, July 2010
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Lewis, DR, Miller, N, Winkel, BSJ, Muday, GK. Auxin and ethylene induce distinct flavonol accumulation patterns through independent transcriptional networks. ASPB Plant Biology meeting, Montreal, July 2010
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Maloney, G, Lewis, DR., Muday, GK. The role of ABCB19 from Arabidopsis and tomato in lateral and adventitious root formation. Gordon Conference on Plant Molecular Biology, New Hampshire, July, 2010


Progress 01/01/10 to 12/31/10

Outputs
Target Audience: This project is focused on understanding the hormonal controls of tomato root growth and development and the primary audience for publication of this work is the scientific community. We are also working with middle and high school students using the genetic resources generated by this project to teach plant genetics to these students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project lead to the training of two graduate, two undergraduate, two high school students, and two post-doctoral research associates. A graduate student, who completed her PhD this year, Poornima Sukumar, worked on hormonal controls of adventitious root formation in tomato and Arabidopsis. She is a coauthor on two papers that are currently in review and one in preparation. Mary Beth Lovin also completed her PhD in 2010 on the environmental and hormonal controls on lateral root formation, with a focus on light quantity and temperature as signals modulating lateral root formation. This work has yielded two manuscripts that are in preparation. The undergraduates and high school students helped in many aspects of the projects, from care and maintenance of tomato plants in the field and development of education outreach exercises that were used during visits to elementary, middle and high schools as described above. These students have learned how to isolate DNA, phenotypically characterize root growth, and design experiments. Greg Maloney is a post-doc working on this project, who has examined adventitious root formation in Arabidopsis and tomato and is a co-author on one paper on this topic that is in review. He also is isolating the ABCB19 gene from tomato and examination of its role in root growth and development. He is examining the role of flavonoids in tomato root growth and development and using both mutants and introgression lines of tomato to ask if these molecules regulate root architecture in analogous ways to those reported in Arabidopsis. A second post-doc, Dr. Hanya Chrispeels has focused on the characterization of the tomato erb mutant, with enhanced adventitious and lateral roots and on development of educational outreach exercises to teach plant genetics using heirloom tomatoes. We are currently writing a paper describing the erb mutant and the altered auxin and ethylene signaling in this mutant. How have the results been disseminated to communities of interest? This work was reported at three scientific meetings during this first year of the project, with the first journal publication submitted and in review. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. How does ethylene alter long distance auxin movement and local auxin gradients that control lateral and adventitious root formation in Arabidopsis? We utilized genetic and molecular approaches to identify the mechanisms by which the gaseous plant hormone ethylene reduces lateral root formation and regulates the synthesis flavonoids, which are endogenous auxin transport regulators. Although ethylene inhibits lateral root formation, treatment with the ethylene precursor ACC enhances the polar transport of auxin. We have identified critical roles for two auxin transport proteins, PIN3 and PIN7, in the effects of ACC on both auxin transport and root formation. ACC increases the abundance of the transcripts encoding PIN3 and PIN7 in an ETR1 and EIN2 (ethylene signaling) dependent and TIR1 (auxin receptor) independent fashion. PIN3- and PIN7-GFP fluorescence increased along the length of ACC-treated roots, consistent with their role in ACC-elevated transport. ACC treatment also abolished a localized depletion of fluorescence of these two reporters normally found below the site of primordia formation. These results suggest that increased PIN3 and PIN7-mediated acropetal auxin transport after ACC treatment prevents localized accumulation of auxin needed to drive lateral root formation. This work is summarized in a manuscript that is in review at Development. We also examined the hormonal cross talk regulating the synthesis of flavonols and examined their impact on root growth and development. We analyzed the effects of auxin and an ethylene precursor on roots of wild-type and hormone-insensitive Arabidopsis thaliana mutants at the transcript, protein, and metabolite levels at high spatial and temporal resolution. IAA and ACC differentially increased flavonol pathway transcripts and flavonol accumulation, altering the relative abundance of quercetin and kaempferol. The IAA, but not ACC response, is lost in the tir1 auxin receptor mutant; while ACC responses, but not IAA responses, are lost in ein2 and etr1 ethylene signaling mutants. Kinetic analyses identified auxin and ethylene mediated increases in transcripts encoding transcriptional regulators, which preceded increases in transcription of flavonoid biosynthetic enzymes. Physiological analyses revealed that tt4, which makes no flavonols, and tt7, which makes kaempferol, but not quercetin,have identical basipetal transport-dependent phenotypes. These results demonstrated that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth. Studies are underway to determine the relative roles of quercetin and kaempferol in lateral root formation. Collectively, these experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. The study also provides new evidence that quercetin is the flavonol which modulates basipetal auxin transport. This work was submitted during year 1 of this project. Adventitious roots emerge from aerial plant tissues and the induction of adventitious root development is essential for clonal propagation of agriculturally important plant species. We have explored the role of auxin transport in this process in Arabidopsis and tomato seedlings in which adventitious root formation was induced by removing roots from low light grown hypocotyls. Root excision is accompanied by an increase in auxin transport and local changes in auxin responsive gene expression above the site of excision, consistent with local auxin accumulation preceding adventitious root formation. We examined excision-induced adventitious root formation in auxin influx and efflux mutants in Arabidopsis, and found the most profound reductions in abcb19 while an ABCB19 overexpression line formed more adventitious roots. Examination of both transcriptional and translational ABCB19-GFP fusions suggest both transcriptional and post-transcriptional regulation of ABCB19 in response to root excision that induces adventitious root formation.Our results implicate a change in the protein phosphorylation state of ABCB19 as a mechanism by which excision drives adventitious root development. Together, these results suggest that polar auxin transport, mediated by ABCB19, is an essential regulator of excision-induced adventitious root formation in Arabidopsis. This work is the subject of the completed PhD thesis of Poornima Sukumar in 2010 and a manuscript is in preparation. What are the mechanisms in tomato for ethylene and auxin cross talk in regulation of root development? 3) Do tomato lateral and adventitious root mutants have altered ethylene and/or auxin signaling, synthesis, or transport? We are examining the hormonal controls of lateral and adventitious roots in both wild-type and mutant tomatoes. We previously reported that ethylene has similar negative effects on lateral root formation and positive effects on auxin transport in roots of both tomato and Arabidopsis. In contrast, adventitious root formation shows opposite responses to ethylene treatment with stimulation of this process in tomato and inhibition in Arabidopsis. We have focused on identification of potential auxin transport proteins from the newly complete tomato genome, isolation of these genes, and development of qRT-PCR techniques to look at the controls of their expression. We have identified an ABCB19 ortholog and are examining the expression of this gene in response to treatments that alter its expression in Arabidopsis to ask if similar hormonal controls are present in tomato. We are also focusing on examination of a set of mutants with altered root development. The erb mutant was isolated in a screen by Sangeeta Negi, a former lab member, and has enhanced root formation and branching. This mutant has strong alterations in auxin signaling, with weaker alterations in ethylene signaling. We are using these mutants to explore the hormonal controls of root development and working to identify the primary genetic defect of erb. We are currently working on a manuscript describing the erb phenotype and the causal relationship with its hormone signaling defects. We are also examining tomato mutants with defects in anthocyanin and flavonoid accumulation to determine if there is regulation of root architecture and auxin transport by flavonols analogous to Arabidopsis. We have also completed a screen of a population of introgression lines in which regions of the S. penellii chromosomes have been introgressed into S. lycopersicum. We have found interesting lines with strongly enhanced and reduced lateral or adventitious roots. We are focusing on several chromosome regions to identify candidate genes that may contribute to these altered phenotypes.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2010 Citation: Lewis, DR, Ramirez, MV, Miller, ND, Keith, R, Helm, R, Winkel, BSJ, Muday, GK (In review) Auxin and ethylene induce distinct flavonol accumulation patterns through independent transcriptional networks: Plant Physiol
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Lewis, DR, Miller, N, Winkel, BSJ, Muday, GK. Auxin and ethylene induce distinct flavonol accumulation patterns through independent transcriptional networks. ASPB Plant Biology meeting, Montreal, Minisymposium presentation. 2010
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Muday, GK, Negi, S, Lewis, DR, Sukumar, P. Ethylene inhibits lateral root formation via altered auxin transport, International Plant Growth Substances Meeting, Barcelona, Spain, Root Development Symposium speaker and session organizer.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2010 Citation: Lewis, DR, Muday, GK. Hormonal Controls of Flavonoid Synthesis North Carolina Biotechnology Center, Plant Molecular Biology Retreat, Asheville, NC,Invited speaker.