Source: UNIVERSITY OF CALIFORNIA, RIVERSIDE submitted to NRP
DISSECTING ES1 ENDOSOMES IN PLANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0212396
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
Botany and Plant Sciences
Non Technical Summary
The Raikhel laboratory is involved in understanding the mechanisms of protein trafficking via the endomembrane system plant cell. The endomembrane system consists of many compartments that are involved in various pathways that constitute a complicated system. Plants cannot survive without a properly functioning endomembrane system. Transport through the endomembrane system requires multiple cellular elements (vesicles called endosomes) and players (proteins such as transporter ions and metabolites). Chemical genomics is an excellent tool that allows researchers to uncover previously unknown connections within and between cellular pathways. This approach uses small synthetic or natural compounds to perturb a particular biological function in a tunable and reversible manner. By combining chemical genomics, cell biology and genetics approaches, the Raikhel lab has characterized the small molecule Endosidin1 as a valuable probe for identifying so-called early endosomes. Identifying cognate target(s) is crucial for discovering the mechanistic detail of new gene function(s) and networks. It is important to understand the transport of proteins and metabolites through the endomembrane system because proteins are key components of nutritional value whereas metabolites like flavonoids, in addition to traits like color and flavor, are strong antioxidants that protect cells and their DNA from damage. Consequently this may increase the quality of plant-related food and reduce age-related diseases such as dementia and cancer.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2062420103050%
2062420104050%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
2420 - Noncrop plant research;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology;
Goals / Objectives
Objective 1.Genetic identification of ES1 resistance genes. With the goal of isolating the target(s) of ES1 associated with endocytosis arrest and ES1 body formation, a genetic screen for resistant mutants will be performed. Full characterization of the Arabidopsis mutants will provide insights into the effects of ES1 and its biological implications. We will utilize forward EMS resistance screens, followed by map-based cloning. We may also use Illumina sequencing to identify mutations directly within rough-mapped intervals. Objective 2.Characterization and Biological Role of ES1 target(s). Mutations leading to resistance to ES1 body formation are likely within the target involved in ES1-trafficking effects. Complementation experiments at the seedling and cellular levels will confirm this. Fluorescent protein fusions and antibodies will be prepared and the dynamics of the target and ES1 compartment will be analyzed in vivo. To discover crosstalk with other network compartments, fluorescent proteins fused to the target(s) will be co-localized with a panel of different endomembrane compartment markers including different endosome compartments using confocal microscopy and immunocytochemistry. Antibodies will also permit the identification of proteins interacting with the target. Objective 3. Dissection of the SYP61/ES1 endosome cargoes. The ES1/SYP61 compartment will be purified using our successful approach that couples immunoaffinity purification with quantitative proteomics. This will provide compartment-specific information, permitting an examination of the SYP61 compartment proteome at an equilibrium state (untreated) as well as following arrest (ES1 treated) to identify transient cargoes. This will permit the dissection vesicular trafficking through the SYP61 vesicles and identify PM proteins rapidly transiting through this endosome compartment. Some proteins may be part of interacting pathways such as brassinosteroid (BR) signaling, auxin transport and cell wall synthesis providing the beginning of a broader systems view of trafficking. As a result of this research, we will meet unique challenges and opportunities in plant chemical genomics such as visualizing chemically-induced intracellular phenotypes and developing high-throughput screening and informatics approaches to visualize and archive complex phenotypic datasets derived from screens, issues that otherwise would not have been addressed. As an indicator of the impact of our work to the plant community, we have been sought by collaborators in the UCR and other UC campuses and from all over the worlds in diverse areas such as small RNAs, plant pathology, stress tolerance, cellular polarity, long-distance signaling, and hormones such as auxin and BR: all that are extremely important for agriculture at large and California agriculture in particular. In addition, graduate and undergraduate students as well as postdoctoral fellows in my laboratory will participate in the experiments proposed below, and consequently, this grant will contribute to the formation of future leaders in Plant Sciences for California.
Project Methods
Objective 1. Genetic identification of ES1 resistance genes. We will screen Arabidopsis EMS populations available in our laboratory using the bri phenotype as a primary screen. Mutations resulting in resistance to ES1 body formation in the secondary screen will be mapped using the seedling resistance phenotype followed by deep sequencing using the Illumina HiSeq2000 at UCR. Objective 2. Characterization and Biological Role of ES1 target(s). Confirmed resistance genes will be examined in planta as fluorescent protein fusions to determine if they co-localize with SYP61 or other ES1-sensitive PM markers such as BRI1, PIN2, AUX1 and CESA6. This will be augmented by EM studies to examine the morphology of ES1 bodies and to determine their contents. For subcellular localization of putative target(s), we will utilize the method for "Fluorescent Tagging of Full-Length Proteins (FTFLP)" that was developed in several laboratories including ours as part of the NSF 2010 Arabidopsis Genomics project. We will use Citrine (YFP), a spectral variant of the GFP tag that will be inserted internally in the coding region with its 5' and 3' regulatory sequences to indicate native subcellular localization and tissue specificity. Proteins will also be tagged with Cyan Fluorescent Protein (CFP). This will be useful for co-localization with known endomembrane system markers available in the PI's lab. For subcellular localization, confocal microscopy will be utilized, and when necessary, EM immunocytochemistry will be performed. For EM localization we will either use monoclonal antibodies against GFP or fuse the proteins to the small tags T7 or HA and utilize existing monoclonal antibodies against these tags. Objective 3. Dissection of the SYP61/ES1 endosome cargoes. The SYP61 and SYP61/ES1 compartment will be purified using our successful approach (Drakakaki...Raikhel, 2011) that couples immunoaffinity purification with quantitative proteomics. This will provide compartment-specific information, permitting an examination of the proteome at an equilibrium state (untreated) as well as following arrest (ES1 treated) to identify transient cargoes. Thus, the goal of this proposal is focused on a functional and mechanistic understanding of the ES1/SYP61 compartment and pathways that depend on this endosome compartment. As a result of the proposed experiments, targets of ES1 will be identified and SYP61 endosomal cargoes will be analyzed. We predict that novel low-abundance regulatory proteins that reside transiently in these endosomes will be uncovered. This highly focused proposal is a part of a broader goal to unravel the complex network of interacting trafficking pathways and sorting mechanisms using the newest tools available. As we dissect individual compartments and discover novel factors and their inter-relationships, our vision is to gain a more expansive systems-wide knowledge of the dynamics of endosomes and their roles in fundamental development and responses to environment and pathogens.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Students and postdocs all over the world. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?An undergraduate student Ines Rabano Jimenez worked in our laboratory for three months as part of the Student Researcher, Postgraduate Diploma Program, University of California Extension. Ines characterized the compounds that were previous identified from our chemical library screening using different cellular markers. Ines tested the effects of different clusters of compounds in different steps of vesicle trafficking using confocal live cell imaging and drug treatment. During her staying at UCR Ines found out that our clustering analysis tools can be used to identify compounds that affect related vesicle trafficking process. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are working on designing new ES2 analogs that have higher binding affinity and better activity. We will further explore the application of ES2 on human disease control such as cancer and diabetes. We will test direct interaction between EXO70A1 N-terminus and proteins identified from co-IP Mass Spec using an alternative approach such as yeast two-hybrid and pull-down assay. At the same time, we are characterizing Arabidopsis mutants that are resistant to ES2. Further studies on the mutants will allow us to identify novel cellular components that regulate exocytosis process.

Impacts
What was accomplished under these goals? We crystallized EXO70A1 protein and obtained the structure at 3.1Å resolution. We used molecular docking to predict ES2 binding site on EXO70A1. We confirmed the binding site by testing the interaction between ES2 and EXO70A1 mutant protein with altered amino acids at the binding pocket. We also confirmed direct interaction between mammalian EXO70 protein and ES2. At the cellular level, ES2 inhibits exocytosis in mammalian cells and arrests EXO70 in Rab8-positive compartments. We identified proteins that interact with N-terminus of EXO70A1 using co-IP Mass Spec analysis. This will help us identify the functions of different regions in EXO70A1 in regulating exocytosis.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang C, Hicks GR, Raikhel NV. Plant vacuole morphology and vacuolar trafficking. Front Plant Sci. 2014 Sep 24;5:476. doi: 10.3389/fpls.2014.00476.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Li R, Sun R, Hicks, G, Rakhel NV (2015) Arabidopsis Ribosomal Proteins Control Vacuole Trafficking and Developmental Programs through the Regulation of Lipid Metabolism. Proc Natl Acad Sci USA 112 (1): E89-E98, doi:10.1073/pnas.1422656112.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang, C.; Hicks, G.R.; Raikhel, N.V. Molecular Composition of Plant Vacuoles: Important but Less Understood Regulations and Roles of Tonoplast Lipids. Plants 2015, 4, 320-333.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang C, et al., (2015) Endosidin2 Targets Conserved EXO70 to Inhibit Exocytosis. Proc Natl Acad Sci USA, 2015. pii: 201521248


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Students and postdocs all over the world. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A graduate student,Ying Zhu, worked in our laboratory for three months in 2014 as part of the collaboration with Dr. Hongwei Guo at Peking University of China and as part of a grant from the National Science Foundation of China to discover novel compounds that involve in ethylene signaling in plants. During her staying at UC Riverside, Ying Zhu learned to screen UCR small molecule collections, including those discovered by our group. She found several promising molecules that reverse the growth defects in EIN3 overexpression lines. Further identification of the endogenous target proteins of these molecules will give novel genes involved in ethylene signaling. Another graduate student, Nolan Ung, developed software to qualify endomembrane phenotypes in plants. Nolan Ung will be defending his thesis in 2015. How have the results been disseminated to communities of interest? Dr. Natasha Raikhel gave research seminars in different meetings and research institutes: Tri-Plant Seminar Series, Department of Biology, Center for Genomics & Systems Biology, New York University January 6th, 2014; HUCK Distinguished Lecturer (honorary lectureship given by leaders in the field of Life Science), Dept. Biochemistry & Molecular Biology, Pennsylvania State University, University Park, PA. January 9th, 2014. Crop Metabolomics International Symposium, Fuzhou Agricultural and Forestry University, Fuzhou, China, April 14th, 2014; Colloquium lecture, IST Austria (Institute of Science and Technology Austria), May 5, 2014; 2014 Joseph E. Varner Lecturer, Washington University, St. Louis, MO, May 9th, 2014; Phenomics Symposium, Max-Planck-Institut für Molekulare Pflanzenphysiologie. Golm Germany, June 26-27th, 2014; Keynote talk at Institute of Plant Physiology & Ecology at Shanghai Institute for Biological Sciences (SIPPE), Shanghai, China, July 1st, 2014; Seminar at Fuzhou Agricultural and Forestry University, Fuzhou, China, July 8th, 2014; Invited lecture at the Sichuan University, Chengdu, China, July 16th, 2014; Lecture at the Institute of Biosynthesis and Biotechnology, Milan, Italy, September 17th, 2014; DOE meeting in Annapolis, Maryland, September 23th, 2014; International Symposium on Horticultural Plant Biology and Metabolomics, Fujian Agriculture and Forestry University (FAFU), Fuzhou, China, November 8th, 2014. Graduate student Nolan Ung presented his current research in International Conference on Arabidopsis Research, Vancouver, Canada, July 28th-August 1st. Postdoctoral fellow, Dr. Ruixi Li presented a research poster in American Society of Plant Biology Meeting, Portland, Oregon, July 12-16th. Postdoctoral fellow, Dr. Chunhua Zhang gave a seminar inEuropean Network for Plant Endomembrane Research (ENPER), Lecce, Italy, Sep 8-11th, 2014. What do you plan to do during the next reporting period to accomplish the goals? We will use EXO70A1 and ES2 co-crystallization and small molecule docking approaches to identify the binding sites of ES2. We will study the mechanisms of ES2 inhibits exocytosis by studying the roles of the binding sites on EXO70A1 protein function. We will study the conservation of essential amino acids for ES2 binding among EXO70 family in Arabidopsis, which has 23 members. This will help us understand the specialized functions of EXO70 proteins in plant. Our previous genetic screening for mutant plants that are resistant to ES2 identified genes that are involved in plant exocytosis. We can further understand the mechanisms of exocytosis regulation by characterizing functions of these genes.

Impacts
What was accomplished under these goals? Our further characterization of ES1 was limited by the availability of ES1 small molecule. We thus focused on another small molecule that inhibits plant exocytosis named Endosidin2 (ES2). Using biochemical approach combined with genetic analysis, we found that ES2 targets EXO70 proteins in plants. EXO70 is one subunit of the exocyst complex. The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its disfunction is correlated with several significant diseases such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that ES2 transiently arrests the EXO70 subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N-terminus and C-terminus of the protein. This study provides not only new insights into the novel features of the exocyst complex but also offers a potentially new target for drugs aimed at addressing human disease.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, R, Hicks, G.R. and Raikhel, N.V. 2015. Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism. Proc Natl Acad Sci U S A. 112(1):E89-98.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang , C. , Hicks, G.R. and Raikhel, N.V. 2014. Plant vacuole morphology and vacuolar trafficking. Frontiers in Plant Sciences. 5:476.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Doyle, S.M., Haeger, A, Vain, T, Rigal, A, Viotti, C, ?angowska, M, Ma, Q, Friml, J, Raikhel, N.V., Hicks, G.R., Robert, S. 2015. An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 17;112(7):E806-15.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Hicks, G.R, Raikhel, N.V. 2014. Plant Chemical Biology: Are We Meeting the Promise? Frontiers Perspective. 5:455.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhang, C., Raikhel, N.V., Hicks, G.R. 2013. CLASPing microtubules and auxin transport. Dev Cell. 24(6):569-71.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Ung, N., Brown, M.Q., Hicks, G.R., and Raikhel, N.V. 2013. An approach to quantify endomembrane dynamics in pollen utilizing bioactive chemicals. Mol. Plant 6(4):1202-13.
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Li, R., Raikhel, N.V. and Hicks G.R. 2013. Chemical effectors of plant endocytosis and endomembrane trafficking. In Endocytosis in plants, Ed. Jozef Samaj, Springer, pp 37-61.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Ponts N., Fu, L., Harris, E.Y., Zhang, J., Chung, D.W., Cervantes, M.C., Prudhomme, J., Atanasova-Penichon, V., Zehraoui, E., Bunnik, E.M., Rodrigues, E.M., Lonardi, S., Hicks, G.R., Wang, Y., and Le Roch, K.G. 2013. Genome-wide Mapping of DNA Methylation in the Human Malaria Parasite Plasmodium falciparum. Cell Host Microbe. 14(6):696-706.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2013 Citation: Zhang C, Hicks GR, Raikhel NV. 2013.Chemical biology in plants: Finding new connections between pathways using the small molecule Sortin1


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Students and postdocs around the world. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Dr. Cecilia Rodriguez visited our laboratory for five months in 2013 as part of collaboration with Dr. Lorena Norambuena at the University of Chile and as a component of a grant from the Chilean funding agency Fondef to discover novel compounds that can modify growth of agricultural species. During her time at UCR Dr. Rodriguez screened UCR small molecule collections, including those discovered by our group. She found several promising molecules that altered growth morphology and also provided tools to gain insight in to basic growth processes that can be applied to crops. Collaborator Dr. Hicks from our lab recently visited Chile to interact with the group and to report to the Chilean funding organization. In another component of the grant we are collaborating within the grant to understand transcriptional responses during the ripening of strawberries. We plan to host a PhD student for about 4 months in 2014 to learn next-gen sequencing and bioinformatics. How have the results been disseminated to communities of interest? Graduate student, Nolan Ung presented his current research to an audience of 6th to 8th grade students at the 2012 GATE-together in the Chino Hills school district. He showed movies of fluorescent labeled organelles and had the students track them and draw lines on the board. He projected the videos and explained why scientists need computers and tracking algorithms and how computers could help educate others about cell biology. He also touched on why it is important to research fundamental biology in plants and went on to demonstrate how plant biology is enriching young students’ everyday lives. Drs. Raikhel and Hicks gave several seminars to wide audiences: 2013 Illumina Core Directors Meeting, Los Angeles, CA (Sept 18); 2013 European Network for Plant Endomembrane Research (ENPER), Ghent Belgium (Aug 27-31); 2013 Botaniker Tatung 2013, Tuebingen, Germany (Sept 30-Oct 4); 2013 Plant Biology Meeting, Pucan, Chile (Dec 2-5); 2013 Plant Molecular Biology Laboratory, Biology Dept., University of Chile, Santiago, Chile (Dec 7); 2013; Anton Lang Memorial Lecture, Plant Biology Seminar Series, Department of Plant Biology and the MSU-DOE Plant Research Laboratory, East Lansing, (March 11). Postdoctoral Fellow, Dr. Chunhua Zhang gave a plenary lecture at the International Conference on Plant Growth Substances, Shanghai, China, June 17-22, 2013. What do you plan to do during the next reporting period to accomplish the goals? This proposal will permit the continued training of graduate students and postdocs and produce a new generation of diverse scientists who can form complementary teams required for 21st century research and education. The inclusion of undergraduate students to our research program will continue to contribute to the undergraduate experience of UCR and local community college students.

Impacts
What was accomplished under these goals? This project is the unprecedented opportunity to precisely identify components and cargoes involved in endosomal trafficking and understand mechanisms controlling the sorting of PM proteins for recycling or vacuole degradation. This resulted from the innovative use of bioactive chemicals that arrest transport on a biological timescale (minutes), permitting the identification of transient PM cargoes. The quantification of endomembrane dynamics permitted an in vivo understanding of endosomal sorting that controls the distribution of cellular receptors, transporters and other proteins critical for plant development or responses to pathogens and the environment. The broader impact of unraveling these fundamental cell biological processes was the enhanced development, yield, and biotic and abiotic stress tolerance in agriculture. In addition, the discovery of bioactive compounds that affect cellular processes in mammalian cells can lead to novel drugs.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhang, C., Raikhel, N.V., Hicks, G.R. (2013) CLASPing microtubules and auxin transport. Dev Cell. 24(6):569-71.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Ung, N., Brown, M.Q., Hicks, G.R., and Raikhel, N.V. (2013) An approach to quantify endomembrane dynamics in pollen utilizing bioactive chemicals. Mol. Plant 6(4):1202-13.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Li, R., Raikhel, N.V. and Hicks G.R. (2013) Chemical effectors of plant endocytosis and endomembrane trafficking. In Endocytosis in plants, Ed. Jozef Samaj, Springer, pp 37-61.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Ponts N., Fu, L., Harris, E.Y., Zhang, J., Chung, D.W., Cervantes, M.C., Prudhomme, J., Atanasova-Penichon, V., Zehraoui, E., Bunnik, E.M., Rodrigues, E.M., Lonardi, S., Hicks, G.R., Wang, Y., and Le Roch, K.G. (2013) Genome-wide Mapping of DNA Methylation in the Human Malaria Parasite Plasmodium falciparum. Cell Host Microbe. 14(6):696-706.
  • Type: Book Chapters Status: Accepted Year Published: 2013 Citation: Zhang C, Hicks GR, Raikhel NV. (2013) Chemical biology in plants: Finding new connections between pathways using the small molecule Sortin1.


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

Outputs
OUTPUTS: Our collection of compounds, which we have shared openly with many labs in the US and abroad, provides a deep toolbox for additional useful chemicals, if necessary. In spite of several very productive years (the list of publications since 2005 acknowledging NSF funding is listed above) and a very positively reviewed grant application last year, our proposal was not renewed and the work was hindered by lack of funds. In response to past advice, the current proposal is strongly focused on detailed characterization of the target and cell biology of ES2. The proposal focuses on ultimately understanding the endosome sorting that directs proteins to the PM for recycling or toward the vacuole. ES2 will permit us to begin to dissect a specific recycling endosome compartment at the intersection of two major circuits of the endosomal-trafficking network: recycling and vacuole targeting. We hope we will be given an opportunity to continue our work in endomembrane trafficking and the cell biological aspects of plant endosomes. PARTICIPANTS: Dr. Raikhel talked about her experience in science and excitement with scientific discoveries. She also introduced students to opportunities in the field of Biology. At the end of the quarter, five female students declared biology as their major and asked to volunteer in Dr. Raikhel's lab, and two additional students expressed an interest in Biology as their major. Drs. R. Li and Glenn Hicks performed a lot of experimental work and presented their research at meetings. Dr. Hicks also held regular workshops on next generation sequencing where he regularly introduced students and postdocs to state-of-the art sequencing facilities at the Institute for Integrative Genome Biology where he is also the Academic Administrator. He is currently mentoring a Chilean graduate student as part of a collaboration with Dr. Norambuena at the University of Santiago. We maintain an up-to-date website (www.cepceb.ucr.edu), where most of our findings and publications are registered. In addition, we implemented ChemMine(63, 64) (http://bioweb.ucr.edu/ChemMine/search.php), a very useful in-house chemical compound and screening database. ChemMine supports our current screening efforts by allowing users to query and annotate more than a million compounds. This database is available to the scientific community at large and contains information regarding all our hits. Several scientists have used this data in their own research and will acknowledge our contributions in resulting publications. During the last year, postdocs from the University of Indiana and the University of Heidelberg in Germany came to our laboratory to perform chemical genomics screens. We taught them how to conduct screens, use confocal microscopes and analyze data. We also shared our numerous GFP/CFP marker lines with them. During their stay in Riverside (from two to five months), these young scientists presented their data and participated actively in lab seminars. These projects provide an excellent international and interdisciplinary experience in science to graduate students, postdocs and professional researchers alike. In general, young scientists associated with our lab are well-versed and experienced in cell biology, chemistry, genetics, molecular biology and the computational sciences. These young scientists are adept practitioners in the new field of chemical genomics and are very competitive in the job market. The majority of postdocs that were working on chemical genomics project now have faculty positions at universities worldwide. TARGET AUDIENCES: Students and postdoc all over the world. PROJECT MODIFICATIONS: There are no major changes.

Impacts
This project is the unprecedented opportunity to precisely identify components and cargoes involved in endosomal trafficking and understand mechanisms controlling the sorting of PM proteins for recycling or vacuole degradation. This will result from the innovative use of bioactive chemicals that arrest transport on a biological timescale (minutes), permitting the identification of transient PM cargoes. The quantification of endomembrane dynamics will permit an in vivo understanding of endosomal sorting that controls the distribution of cellular receptors, transporters and other proteins critical for plant development or responses to pathogens and the environment. The broader impact of unraveling these fundamental cell biological processes is the potential for enhanced development, yield, and biotic and abiotic stress tolerance in agriculture. An addition, the discovery of bioactive compounds that affect cellular processes in mammalian cells can lead to novel drugs. This proposal will permit the continued training of graduate students and postdocs and produce a new generation of diverse scientists who can form complementary teams required for 21st century research and education. The inclusion of undergraduate students to our research program will continue to contribute to the undergraduate experience of UCR and local community college students.

Publications

  • Perez-Henriquez P, Raikhel NV, Norambuena L (2012) Endocytic Trafficking towards the Vacuole Plays a Key Role in the Auxin Receptor SCFTIR Independent Mechanism of Lateral Root Formation in A. thaliana. Mol Plant (in press).
  • Ung N, Brown MQ, Hicks G, Raikhel N (2012) An approach to quantify endomembrane dynamics in pollen utilizing bioactive chemicals. Molecular Plant (in press).
  • Hicks GR, Raikhel NV (2012) Small Molecules Present Large Opportunities in Plant Biology. Annu. Rev. Plant Biol 63:261-282.
  • Li R, Hicks G, Raikhel N (2012) Chemical Effectors of Plant Endocytosis and Endomembrane Trafficking. In, Endocytosis in Plants. Verlag Berlin Heidelberg. J. Samaj, ed. pp 37-61.DOI:10.1007/978-3-642-32463-5_2.


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

Outputs
OUTPUTS: Occupying up to 90% of the volume of an individual cell, the vacuole is the most prominent compartment of the plant cell. It is responsible for storing sugars, pigments, ions, proteins, and volatile compounds, as well as for cellular homeostasis maintenance and regulation of the cytosolic pH. In plants, two types of vacuoles with different properties have been identified, the lytic vacuoles that carry out many of the same processes as yeast and mammalian lysosomes such as the breakdown and recycling of cellular components, and the protein storage vacuoles (PSVs) that carry out an additional number of plant-specific functions such as the accumulation and sequestration of toxic compounds and the storage of defense molecules such as alkaloids, phenolics, and protease inhibitors. Although components of the trafficking machinery to lytic vacuoles are well defined and appear to be conserved in yeast and multicellular eukaryotes, very little is known about the cellular machinery required for transport to PSVs and the mechanisms that regulate the transit of vacuolar soluble proteins containing CTPP and NTPP vacuolar sorting determinants (VSDs) to the PSV are only starting to be unveiled. To determine the nature of the molecular mechanisms that mediate plant-specific trafficking to the vacuoles, the so-called CTPP pathway we used a T-DNA-mutagenized population in the Vac2 background (Vac2 T-DNA) (Sanmartin et al., 2007; Sohn et al., 2007). The screen of the T-DNA-mutagenized Vac2 plants and subsequent TAIL-PCR of the mutant lines isolated allowed us to identify and characterize different elements involved in the delivery of vacuolar targeted cargoes through the CTPP-pathway. The new elements in the pathway include an endosomal FYVE containing Zinc finger (C3HC4-type RING finger) family protein, a plasma membrane H+ ATPase AHA1 interactor, and a cytosolic ribosomal protein (r-protein) L4/L1 (RPL4A). The rpl4a mutant identified in our Vac2 s screen was of particular interest since it belongs to a totally different type of protein than the plant trafficking determinants previously identified. Subsequent experiments confirmed that rpl4a was not a bypass suppressor of clv3-2 and that multiple alleles displayed similar vacuolar trafficking defects (Rosado et al., 2010). The identification of a ribosomal mutation causing specific missorting of CTPP-containing suggested that a novel regulatory mechanism involving RPL4 and specific propeptides was activated while the vacuolar CTPP proteins are newly synthesized within the ribosome. To further confirm that hypothesis a multidisciplinary approach including physiology, biochemistry, pharmacology, proteomics and genetics was taken. Thus, RPL4A provides new biological insights in the field of intracellular trafficking processes, specifically CTPP-mediated plant protein sorting to the vacuole and demonstrate that the T-DNA Vac2 system is extremely powerful for the genetic dissection of ctVSD trafficking pathways. PARTICIPANTS: Raikhel Lab: Raikhel NV - Professor/PI Hicks GR -Professional Researcher Rosado A -Postdoctoral Fellow Sohn EJ- Postdoctoral Fellow Norambuena L- Postdoctoral Fellow van de Ven, W -Staff Research Associate Brown MQ- Graduate Student Boirsdore MP- REU Undergraduate Student Swidergal A- REU undergraduate student UCR: Cutler SR -Professor Pan S- Academic Coordinator Proteomics Puckrin RS- Graduate Student Partner Organizations: Rojo E -Professor- National Center for Biotechnology, Madrid, Spain Aharoni A -Professor, Weizmann Institute, Israel Bressan RA- Professor. Purdue University Kang BH- Assistant Professor.University of Florida Rogachev I- Postdoctoral Fellow, Weizmann Institute, Israel Meir S- Postdoctoral Fellow, Weizmann Institute, Israel Pourcel L- Postdoctoral Fellow , Weizmann Institute, Israel Zouhar J- Postdoctoral Fellow - National Center for Biotechnology, Madrid, Spain Xiong Y- Postdoctoral Fellow. University of Florida TARGET AUDIENCES: Two undergraduate students working on this project were selected from the NSF Center for Plant Cell Biology Research Experiences for Undergraduates (REU) Program. The REU program specifically targets underrepresented populations from two- and four-year colleges with limited research infrastructure to the excitement and career options that studies of plant and plant pathogen biology offers. One student (Boirsdore MP) is now a graduate student pursuing her MS degree in science. The other former-REU student (Swidergal A) is currently pursuing a PhD is science. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
a. Sortin1 Hypersensitive Mutants Link Vacuolar Trafficking Defects and Flavonoid Metabolism in Arabidopsis Vegetative Tissues. The modified transport to the vacuole 6 / Chalcone Synthase mutant (mtv6/ tt5) is a flavonoid defective mutant isolated from an EMS mutagenized population in the Vac2 background (Vac2 T-DNA) in a screen funded by the current DOE project. Interestingly, different flavonoid defective mutants were also isolated from a chemical screen for mutants that cause specific mislocalization of the tonoplast marker δ-TIP:GFP in a screen funded by a NSF project. The multidisciplinary approaches taken in both screens uncovered the function of flavonoid metabolites as important elements for vacuolar biogenesis and transport of vacuolar cargoes in Arabidopsis. Our study also highlights the importance of metabolomic analyses for the dissection of vacuolar trafficking pathways. b.Auxin-Mediated Ribosomal Biogenesis Regulates Vacuolar Trafficking in Arabidopsis. In plants, the mechanisms that regulate the transit of vacuolar soluble proteins containing C-terminal and N-terminal vacuolar sorting determinants (VSDs) to the vacuole are largely unknown. To isolate new components of the plant-specific CtVSD sorting machinery in Arabidopsis, we used a T-DNA mutagenized population in the Vac2 background (Vac2 T-DNA). The Vac2 line contains a genetically engineered CLV3 fused to the barley lectin C-terminal vacuolar sorting signal (CLV3:CtVSDBL) in the clv3-2 mutant background. As a result, we isolated the ribosomal biogenesis mutant rpl4a characterized by its partial secretion of different vacuolar targeted proteins and a plethora of developmental phenotypes derived from its aberrant auxin responses. We propose that the influence of auxin on ribosomal biogenesis acts as a regulatory mechanism for auxin mediated developmental processes, and we demonstrate the involvement of this regulatory mechanism in the sorting of vacuolar targeted proteins in Arabidopsis. c. Application of the Gene Dosage Balance Hypothesis to Auxin-Related Ribosomal Mutants in Arabidopsis. Several proteomic studies in Arabidopsis have shown the presence of heterogeneous ribosomal populations in different tissues. However, the phenotypic consequences of the imbalance of those ribosomal populations, and the regulatory mechanisms activated to control specific ratios between them, have yet to be evaluated. In a previous report we show that the maintenance of proper auxin-regulated developmental responses requires the presence of RPL4A- and RPL4D-containing ribosomes. Based on the compensatory effects within the RPL4 family proteins, we proposed the Gene Dosage Balance Hypothesis (GDBH) as a regulatory mechanism for ribosomal complexes in Arabidopsis. By using the concepts of dosage compensation and hierarchy, GDBH is able to explain the severity and specificity of different ribosomal mutant phenotypes associated with the same ribosomal complex. d. As a result of this work, Federal Funding from DOE has been obtained (Award #DE-FG03-02ER15295).

Publications

  • Rosado A, Hicks GR, Norambuena L, Rogachev I, Meir S, Pourcel L, Zouhar J, Brown MQ, Boirsdore MP, Puckrin RS, Cutler SR, Rojo E, Aharoni A, Raikhel NV. (2011) Sortin1 Hypersensitive Mutants Link Vacuolar Trafficking Defects and Flavonoid Metabolism in Arabidopsis Vegetative Tissues. Chem & Biol. 18:187-197.
  • Rosado A, Sohn EJ, Drakakaki G, Pan S, Swidergal A, Xiong Y, Kang BH, Bressan RA, and Raikhel NV. (2010) Auxin-Mediated Ribosomal Biogenesis Regulates Vacuolar Trafficking in Arabidopsis Plant Cell, 22:143-158.
  • Rosado A, Raikhel NV (2010a) Understanding Plant Vacuolar Trafficking from a Systems Biology Perspective. Plant Physiology 154: 545-550.
  • Rosado A, Raikhel NV (2010b) Application of the Gene Dosage Balance Hypothesis to Auxin-Related Ribosomal Mutants in Arabidopsis. Plant Sig & Behavior 5(4): 450-452.


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

Outputs
OUTPUTS: Our lab is interested in studying the molecular mechanisms that mediate plant-specific trafficking to the vacuoles, the so-called CTPP pathway. To isolate new components of the plant-specific CTPP sorting machinery in Arabidopsis thaliana we used a T-DNA-mutagenized population in the Vac2 background (Vac2 T-DNA) (Sanmartin et al., 2007; Sohn et al., 2007). The screen of the T-DNA-mutagenized Vac2 plants and subsequent TAIL-PCR of the mutant lines isolated allowed us to identify and characterize different elements involved in the delivery of vacuolar targeted cargoes through the CTPP-pathway. The new elements in the pathway include an endosomal FYVE containing Zinc finger (C3HC4-type RING finger) family protein, a plasma membrane H+ ATPase AHA1 interactor, and a cytosolic ribosomal protein (r-protein) L4/L1 (RPL4A). The rpl4a mutant identified in our Vac2 s screen was of particular interest since it belongs to a totally different type of protein than the plant trafficking determinants previously identified. Subsequent experiments confirmed that rpl4a was not a bypass suppressor of clv3-2 and that multiple alleles displayed similar vacuolar trafficking defects (Rosado et al., 2010). The identification of a ribosomal mutation causing specific missorting of CTPP-containing suggested that a novel regulatory mechanism involving RPL4 and specific propeptides was activated while the vacuolar CTPP proteins are newly synthesized within the ribosome. To further confirm that hypothesis a multidisciplinary approach including physiology, biochemistry, pharmacology, proteomics and genetics was taken. As a result, our pharmacological and genetic approaches identified specific regions of the ribosomal complex involved in protein sorting processes; our physiological approach determined that the primary effects of the ribosomal mutation were changes in the auxin responses, and our proteomic and biochemistry approaches identified the first antibiotic-resistant mutant family in Arabidopsis, providing evidences of the presence of heterogeneous populations of ribosomes in metabolically active tissues (Rosado and Raikhel, 2010). Thus, RPL4A provides new biological insights in the field of intracellular trafficking processes, specifically CTPP-mediated plant protein sorting to the vacuole and demonstrate that the T-DNA Vac2 system is extremely powerful for the genetic dissection of ctVSD trafficking pathways. PARTICIPANTS: Publication 1. Rosado A- Postdoctoral Fellow Sohn EJ- Postdoctoral Fellow Pan S- Academic Coordinator Proteomics Swidergal A- REU student Xiong Y- Postdoctoral Fellow. University of Florida Kang BH- Assistant Professor.University of Florida Bressan RA- Professor. Purdue University Raikhel NVR -Professor Publication 2 Rosado A - Postdoctoral Fellow Raikhel NVR -Professor TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
a.A thorough analysis of the RPL4A-mediated trafficking pathway. We are currently focused on the identification of novel regulatory mechanisms required for RPL4A function. Our preliminary results suggest that the phytohormone auxin is involved in the regulation of vacuolar trafficking by inhibiting the transfer of vacuolar cargoes from the prevacuolar compartment to the vacuole (Rosado et al., 2010). In this context, several elements of the ribosomal machinery has been proposed to be involved in the regulation of this process through the translational regulation of Auxin Responsive transcription factors, a process dependent of Upstream Open Reading Frames (uORFs). To demonstrate the role of ribosomal proteins in the translational regulation mediated by uORFs the ribosomal proteins rpl5b and rpl24a were crossed with the Vac2 parental line and their vacuolar trafficking defects are being analyzed. We are also using these mutant backgrounds to analyze the presence or absence of ribosomal stalling, an indicator of translational regulation defects. We anticipate at least one manuscript describing various aspects of ribosomal regulation of vacuolar trafficking mediated by uORFs early in 2011. b. Further characterization of PSV trafficking mutants from the collection of T-DNA-tagged Vac2 plants. The combination of TAIL-PCR techniques and deep sequencing approaches for recalcitrant T-DNA mutants has allowed us to identify multiple elements belonging to the PSV trafficking pathway. In this moment we are performing the primary analysis of the most interesting mutants isolated from our T-DNA mutant collection, the endosomal FYVE containing Zinc finger (C3HC4-type RING finger) family protein, and the plasma membrane H+ ATPase AHA1 interactor. Once we demonstrate that those mutants are not bypassing the cvl3-2 mutation, we will perform complementation analysis transforming the Vac2 mutant lines with the genomic fragments encoding those proteins under endogenous and 35S promoters. Next, we will characterize the specificity of the vacuolar defects by analyzing the transport of C-terminal and N-terminal propeptide containing cargoes and finally, we will confirm the subcellular localization of the proteins by using colocalization studies of GFP-tagged proteins with known markers belonging to the endomembrane system. We plan to submit a manuscript describing the identification and associated phenotypes of the Arabidopsis T-DNA mutants by the second quarter of 2011.

Publications

  • Rosado, A., Sohn, E.J., Drakakaki, G., Pan, S., Swidergal, A., Xiong, Y., Kang, B.H., Bressan, R.A. and Raikhel, N.V. (2010) Auxin-mediated ribosomal biogenesis regulates vacuolar trafficking in Arabidopsis. Plant Cell, 22, 143- 158.
  • Rosado, A. and Raikhel, N.V. (2010) Application of the gene dosage balance hypothesis to auxin-related ribosomal mutants in Arabidopsis. Plant Signal Behav, 5, 450-452.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The mechanisms of sorting and trafficking of newly synthesized proteins with C-terminal vacuolar sorting determinants (ctVSDs) in plant tissues remain unclear. Our laboratory has begun to address this important question by developing tools for the experimental analysis of ctVSD protein transport pathways. We created an Arabidopsis screening line, Vac2, in a clv3-2 mutant background. CLV3 is an extracellular ligand that is a negative regulator of shoot stem cell proliferation, and clv3 mutants display increased growth at apical and floral meristems. When a ctVSD is fused to CLV3 (Vac2 construct) and expressed in a clv3-2 mutant background, the ligand is targeted to the vacuole. Vac2 plants that have mutations in the ctVSD trafficking machinery secrete CLV3 to the extracellular space. These plants are complemented for the clv3-2 mutation and have reduced meristem phenotypes. Using this approach, we have identified the Qb-SNARE protein VTI12 as a component of ctVSD, and not N-terminal VSD (ntVSD), trafficking (Sanmartin et al., 2007). A screen of EMS-mutagenized Vac2 plants and subsequent map-based cloning of one mutant line, mtv5, identified the shoot meristem identity gene TFL1 as a component of ctVSD trafficking, that is, vacuole-localized proteins are secreted in this mutant, and trafficking of ntVSD proteins is not affected. Subsequent experiments demonstrated that mtv5 was not a bypass suppressor of clv3-2, that complementation with a native TFL1 construct restored the parental phenotype, and that the TFL1 protein was localized to the endomembrane system (Sohn et al., 2007). In addition to the EMS-mutagenized Vac2 seed collection, we have prepared a T-DNA collection of Vac2 plants. All these results demonstrate that the Vac2 system is extremely powerful for the genetic dissection of ctVSD trafficking pathways. [a] A thorough analysis of the TFL1-mediated trafficking pathway. In Sohn et al. (2007), we demonstrated that a mutant in the Arabidopsis TERMINAL FLOWER 1 (TFL1) gene, a shoot meristem identity gene, was specifically defective in trafficking of proteins in the C-terminal vacuolar sorting determinant (ctVSD) pathway. We showed that the TFL1 protein is localized to endomembrane compartments and that it co-localizes with adaptin, a putative subunit of the AP-3 adapter complex. [b] Isolation of PSV trafficking mutants from a collection of T-DNA-tagged Vac2 plants. In collaboration with Dr. Bressan of Purdue University, we have created a population of T-DNA tagged plants in the Vac2 background. 164 T2 pools of transformed seed that contain progeny of 50 independent insertion mutants was analyzed. In the primary screen, we selected 450 lines that exhibited complementation of the clv3 phenotype for further characterization. Fifty lines passed the secondary screen. After the secondary screen, twenty lines were selected for TAIL-PCR experiments and ten lines were obtained. The identities of the genes in which we detected T-DNA insertions fall into three categories: (I) putative endomembrane system related genes, (2) putative direct suppressors of the CLV3 pathway, and (3) genes that have no obvious characterization. PARTICIPANTS: Publication 1. Raikhel NVR -Professor (PI); Sohn EJ- Postdoctoral Fellow; Roberts S- Postdoctoral Fellow; Surpin M- Assistant Plant Cell Biologist. Publication # 1 is a collaboration with the Lab of my former postdoc Dr. Enrique Rojo at CNB-CSIC, Cantoblanco, E-28049 Madrid, Spain. Publication 2 Raikhel NVR -Professor (PI); Sohn EJ - Postdoctoral Fellow; Rojas-Pierce M- Postdoctoral Fellow; Carter C- Postdoctoral Fellow; Surpin M Assistant Plant Cell Biologists. Publication # 2 is a collaboration with the Lab of my former postdoc Dr. Enrique Rojo at CNB-CSIC, Cantoblanco, E-28049 Madrid, Spain and Instituto de Biologia Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas, Universidad Politecnica de Valencia, Valencia 46022, Spain TARGET AUDIENCES: 1. Although components of the trafficking machinery to lytic vacuoles are well defined and appear to be conserved in yeast and multicellular eukaryotes, very little is known about the cellular machinery required for transport to protein storage vacuoles (PSVs). The PSV is a plant-specific organelle that accumulates reserve proteins, which are amongst the main agricultural products obtained from crops. Interfering with transport to PSVs has been shown to result in secretion of cargo. Therefore, secretion of a suitable marker could be used as an assay to identify mutants in this pathway. In Sanmartin et al (2007) we determined that trafficking of Vac2 required the SNARE VTI12 but not its close homologue, the conditionally redundant VTI11 protein. Furthermore, the vti12 mutant is specifically altered in transport of storage proteins, whereas vti11 is affected in transport of a lytic vacuole marker. These results demonstrate the specialization of pathways to plant vacuoles and validate the Vac2 secretion assay as a robust method to isolate genes that mediate trafficking to the PSV. 2. In Sohn et al (2007) using the Vac2 assay line we developed a novel genetic screening approach where we can identify genes whose products comprise the trafficking machinery to the plant PSV. One of the mutants that disrupted trafficking to the PSV was identified as the TERMINAL FLOWER 1 (TFL1) gene, a shoot meristem identity gene. Further experiments demonstrated that it is a component of the PSV trafficking machinery. These results indicate a dramatic and unexpected developmental role for the protein storage vacuole in vegetative tissues. The demonstration that TFL1 is indeed involved in protein trafficking leads to two major conclusions. First, that the localization of TFL1 to the endomembrane system adds a new layer of complexity to the study of plant development, which has traditionally been more focused on the regulation of gene expression. Second, our results establish a function for the PSV in development. The model we put forward in our conclusion is that protein factors that are important for development are stored in the PSV until the proper combination of developmental and environmental cues trigger secretion. The conclusions of this manuscript represent significant advances for the fields of developmental and cellular biology and will present fresh perspectives for problems of development and trafficking. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
[a] A thorough analysis of the TFL1-mediated trafficking pathway. We are currently focused on examining the spatial relationship between the TFL1 and VTI12 proteins. Both proteins are involved in ctVSD trafficking and have been co-localized with adaptin (Sohn et al., 2007). We co-transformed protoplasts with GFP:VTI12 and HA:TFL1 constructs. Fluorescent confocal microscopy examination of transformed protoplasts showed that the two proteins were co-localized to punctate structures. We are currently attempting to confirm this co-localization with additional constructs with different reporters, and also examining the potential co-localization of TFL1 and SYP41, which is an interacting SNARE partner of VTI12. We crossed vti12 with tfl1-1 and selected double homozygous mutants; we are actively investigating the phenotype of the vti12 tfl1-1 double mutant. We created lines in which to perform co-immunoprecipitation experiments to determine the identities of proteins that interact with TFL1.We have multiple lines that complement the mutation with respect to the flowering time and shoot growth phenotypes. We anticipate one manuscript describing various aspects of TFL1 biology in early 2010. [b1] Isolation of PSV trafficking mutants from a collection of T-DNA-tagged Vac2 plants. We screened a T-DNA-mutagenized Vac2 population and determined that trafficking of the CLV3:T7:VSS-BL reporter protein to the vacuole requires the proper function of the large subunit ribosomal protein family RPL4, which includes two nearly identical members, RPL4a and RPL4d. Characterization of the RPL4 family has shown that it is essential for proper protein trafficking and auxin-dependent developmental responses in metabolically active cells. We demonstrated that the impaired translational deficiency in rpl4 single mutants is due to defective ribosomal peptidyl transferase center architecture, and we identified a regulatory mechanism at the translational level where the lack of one member of the RPL4 family evokes the activation of the second member as a compensatory mechanism. The findings of this project will be submitted in the next few months. [b2] SEC1B. We identified a complemented Vac2 line, 14-8, which had a partially restricted floral meristem and silique morphology that was essentially wild type. Sequencing of the TAIL-PCR product, generated using genomic DNA from this line, showed the presence of a T-DNA insert in the eighteenth exon of the SEC1B gene. However, subsequent generations of plants from this line no longer showed a complemented phenotype, a phenomenon we observed in over half of the complemented Vac2 Supertag lines. We are currently investigating whether this loss of complementation is due to trans-silencing of the Vac2 construct. We are in the process of carrying out RT-PCR studies of the expression of the Vac2 construct in the original line and progeny lines in order to test this hypothesis. We are also in the process of validating T-DNA mutants for SEC1B from the Salk collection. We plan to submit a manuscript describing the identification and associated phenotypes of the Arabidopsis sec1b and sec1a mutants during the second quarter of 2010.

Publications

  • Sanmartin, M., Ordonez, A., Sohn, E.J., Robert, S., Sanchez-Serrano, J.J., Surpin, M., Raikhel, N.V., and Rojo, E. 2007. Divergent functions of VTI12 and VTI11 in trafficking to storage and lytic vacuoles in Arabidopsis. Proceedings of the National Academy of Science USA.104: 3645-3650.
  • Sohn, E.J., Rojas-Pierce, M., Carter, C., Serrano-Mislata, A., Madueno, F., Rojo, E., Surpin, M.A., and Raikhel, N.V. 2007. The shoot meristem identity gene TFL1 is involved in flower development and trafficking to the protein storage vacuole. Proceedings of the National Academy of Science USA. 104: 18801-18806.