CONTROL OF CAROTENOID BIOSYNTHESIS BY A NOVEL REGULATORY GENE IDENTIFIED IN CAULIFLOWER

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0207945
• Grant No.: 2007-35318-17794
• Cumulative Award Amt.: $262,159.00
• Proposal No.: 2006-03288
• Project Start Date: Nov 15, 2006
• Project End Date: Nov 14, 2009
• Grant Year: 2007
• Program Code: [54.3]- (N/A)

Recipient Organization
Agricultural Research Service
600 E. Mermaid Lane Rm 2023
Glenside,PA 19038-8551

Performing Department
(N/A)

Non Technical Summary
Carotenoids play an indispensable role in human nutrition and health. They are important dietary precursors of vitamin A and provide protection against cancer, heart, and age related eye diseases. Diet-related diseases such as cancer and cardiovascular disease cost the economy approximately 200 billion dollars annually in this country. Production of plant foods altered with higher levels of carotenoids could result in significant economic benefits. Clearly, a comprehensive understanding of the mechanisms of carotenoid accumulation is essential for the development of carotenoid-enriched food crops. Using a unique Or mutant in cauliflower as a model system, we have identified a novel regulatory gene that controls carotenoid accumulation. This research will enable us to elucidate the functional role of Or in the control of carotenoid accumulation in the plant, and will provide a new strategy to enhance carotenoid levels in food crops, thus stimulating the development of new "healthy food" agriculture for better human nutrition and health.

Animal Health Component

10%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	1440	1000	50%
206	1440	1010	15%
206	1440	1040	35%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1440 - Cole crops;

Field Of Science
1040 - Molecular biology; 1010 - Nutrition and metabolism; 1000 - Biochemistry and biophysics;

Keywords

carotenoids

b-carotene

cauliflower

or gene

cysteine-rich zinc figure domain-containing protein

Goals / Objectives
Understanding of the regulation of carotenoid biosynthesis has become a key research focus in the area of plant biochemistry because carotenoids play indispensable roles in plants and have many potential benefits to human health. The high-b-carotene Or (Orange) gene mutation in cauliflower offers an ideal model to elucidate the control mechanisms. The Or gene has been cloned and encodes a protein containing a cysteine-rich zinc figure domain found in DnaJ-like proteins. Initial functional analysis of the Or gene suggests that Or represents a novel regulatory gene important for carotenoid accumulation. The research detailed in this proposal aims at elucidating the biochemical and molecular basis of Or in controlling carotenoid accumulation. The three specific objectives are: 1). To examine whether Or is a gain-of-function or a dominant negative mutation; 2) To identify proteins that interact with OR; and 3) To functionally study OR-protein association in regulating carotenoid accumulation.

Project Methods
Objective 1: Investigating the nature of Or mutation. The approaches will include to generate Arabidopsis and cauliflower Or RNAi transgenic lines and transformants overexpressing the wild type or gene, and to examine the phenotype and carotenoid levels of these transformants. Also, we will generate transformants overexpressing the Or variants and examine the role of the individual Or transcript in inducing carotenoid accumulation. Objective 2: Isolating OR-associated proteins. The experimental strategies will be to employ blue native gel electrophoresis (BN-PAGE) followed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF MS) to identify protein components associated with OR, or to use yeast two-hybrid and GST-tagged pull-down assays to isolate proteins that physically interact with OR. Objective 3: Functionally investigating OR-protein interaction. This will involve using molecular and biochemical approaches to examine the correlation of gene expression in RNAi or T-DNA insertion lines and analyze the functional relevance of OR-interacting protein in regulating carotenoid accumulation.

Progress 11/15/06 to 11/14/09

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The funding provided opportunities to train five undergraudate students and two postdoctoral research associates 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? Nothing Reported

Impacts
What was accomplished under these goals? Identification and investigation of the cauliflower Or-interacting proteins in controlling carotenoid biosynthesis and plant growth We have isolated a number of cauliflower OR-interacting proteins during the funding period. We show that one protein affects the plant's responses to sugars and hormones, resulting in controlling plant growth and development. Despite that we observed no obvious effect on carotenoid biosynthesis by this protein, our study provides a new functional role of this isolated protein and helps explain the polytrophic effect of Or in affecting plant growth and development. This work was published in New Phytologist (2011) and Journal of Experimental Botany (2010). In addition, we identified phytoene synthase, the key enzyme in the carotenoid biosynthesis pathway, as another OR-interacting protein. Our later work on the OR-phytoene synthase interaction uncovered a novel regulatory mechanism of carotenoid biosynthesis and led to a later publication in the Proceedings of the National Academy of Sciences of the United States of America (2015). Or serves as an effective genetic tool to enhance carotenoid content in crop We generated the Or transgenic potato lines and performed an in-depth characterization of these Or transgenic lines. We found that the Or transgene facilitated continuous carotenoid biosynthesis during long-term cold storage and the total carotenoid levels enhanced greatly in the Or transgenic lines over controls. The Or transgene conferred the formation of chromoplasts containing carotenoid sequestering structures in a heterologous system. These results provided further evidence demonstrating that the Or gene indeed controls chromoplast differentiation and that regulation of chromoplast formation can have a profound effect on carotenoid accumulation in plants. This work was published in Journal of Experimental Botany (2008) and formed the base for three invited reviews in Transgenic Research (2007), Biotechnology Annual Review (2008), Methods in Molecular Biology (2010). Carotenoid content in potato tubers is under multiple regulatory control Potato is the world's fourth largest crop and generally contains low levels of carotenoids. We employed biochemical, molecular, and genomics approaches to decipher the key factors controlling carotenoid content in potato tubers. We found that carotenoid levels in potato tubers were determined by a combination of increased metabolic flux into the carotenoid biosynthetic pathway as well as the transcriptional control of a number of carotenoid metabolic genes. The synergistic effect of elevated expression of upstream genes and suppressed expression of downstream genes could be responsible for specific carotenoid accumulation in yellow-flesh varieties. This work was published in Plant, Cell & Environment (2011). Carotenoid biosynthetic enzymes are in large protein complexes Carotenoid biosynthetic enzymes are believed to form multienzyme complexes in the plastid membranes. We demonstrated that phytoene desaturase in plants existed in two large protein complexes of approximately 660 kDa and 350 kDa in the stroma and membrane of plastids, respectively. The membrane associated enzyme complex may be critical to carry out carotenoid biosynthesis in plants. This work was published in Physiologia Plantarum (2008).

Publications

• Type: Journal Articles Status: Published Year Published: 2007 Citation: Li L, Van Eck J (2007) Perspectives: metabolic engineering of carotenoid accumulation by creating a metabolic sink. Transgenic Research 16:581-585.
• Type: Journal Articles Status: Published Year Published: 2008 Citation: Lopez, AB, Van Eck J, Conlin BJ, Paolillo DJ, ONeill J, Li L (2008) Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers. Journal of Experimental Botany 59:213-223.
• Type: Journal Articles Status: Published Year Published: 2008 Citation: Lopez AB, Yang Y, Thannhauser TW, Li L (2008) Phytoene desaturase is present in a large protein complex in plastid membrane. Physiologia Plantarum 133:190-198.
• Type: Journal Articles Status: Published Year Published: 2008 Citation: Lu S, Li L (2008) Carotenoid metabolism: the biosynthesis, regulation, and beyond. Journal of Integrative Plant Biology 50:778-785.
• Type: Book Chapters Status: Published Year Published: 2008 Citation: Zhou X, Van Eck J, Li L (2008) Use of the cauliflower Or gene to improve crop nutritional quality. Biotechnology Annual Review, Volume 14. Chapter 6, p171-190.
• Type: Book Chapters Status: Published Year Published: 2010 Citation: Van Eck J, Zhou X, Lu S, Li L (2010) Modulation of carotenoid accumulation in transgenic potato by inducing chromoplast formation with enhanced sink strength. Methods in Molecular Biology 643:77-93.
• Type: Journal Articles Status: Published Year Published: 2010 Citation: Zhou X, Cooke P, Li L (2010) Eukaryotic release factor 1-2 affects Arabidopsis responses to glucose and phytohormones during germination and early seedling development. Journal of Experimental Botany 61:357-367.
• Type: Journal Articles Status: Published Year Published: 2011 Citation: Zhou X, Sun TH, Wang N, Ling HQ, Lu S, Li L (2011) The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1. New Phytologist 190: 89-100
• Type: Journal Articles Status: Published Year Published: 2011 Citation: Zhou X, McQuinn R, Fei Z, Wolters AM, Van Eck J, Brown C, Giovannoni JJ, Li L (2011) Regulatory control of high levels of carotenoid accumulation in potato tubers. Plant, Cell & Environment 34:1020-1030

Progress 11/15/07 to 11/14/08

Outputs
OUTPUTS: Previous year we have isolated a protein that interacts with OR by yeast two-hybrid approach. To study its functional role in association with OR protein in regulation carotenoid accumulation and plant growth and development, we have generated overexpression and knockout lines in Arabidopsis as well as the dsRNAi transgenic lines in cauliflower. Examination of plants with altered expression of the OR-interacting protein appears to suggest that OR also involves in regulating plant growth and development by affecting GA metabolism and signaling. Further, to investigate whether OR is associated with carotenoid biosynthetic enzymes in controlling carotenoid accumulation, we examined the multienzyme complexes in the plastid membranes by employing Blue Native gel electrophoresis in conjunction with a second-dimension SDS-PAGE and Western blot analysis. We found that OR was not present at the carotenogenic enzyme complexes. Chromoplast is a structure that functions to sequester and store carotenoids. To identify proteins associated with carotenoid accumulation, we did comparative proteomic analysis of the proteins from carotenoid accumulating and non accumulating plastids. We are in the process to study the differential expressed proteins in order to unravel the molecular mechanism of OR-induced carotenoid accumulation. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Plant carotenoids are the primary dietary source of provitamin A for humans. The Or gene mutation in cauliflower exerts a unique mechanism in regulating carotenoid accumulation by inducing the formation of a metabolic sink. The research is expected to contribute our better understanding of a regulatory mechanism underlying carotenoid accumulation, and aid the development of new strategies to enhance carotenoid contents in staple crops to the levels required for optimal human nutrition and health.

Publications

• Zhou X, Van Eck J, Li L (2008) Use of the cauliflower Or gene to improve crop nutritional quality. Biotechnology Annual Review, Volume 14. Chapter 6, p171-190.
• Zhou X, Li L (2008) Enhancing plant carotenoids via manipulation of sink strength. Information Systems for Biotechnology, February 2008: 5-7 (Invited review)
• Lopez AB, Yang Y, Thannhauser TW, Li L (2008) Phytoene desaturase is present in a large protein complex in plastid membrane. Physiologia Plantarum 133:190-198.
• Salas-Fernandez MG, Hamblin M, Li L, Rooney WL, Tuinstra MR, Kresovich S (2008) Quantitative trait loci analysis of endosperm color and carotenoid content in sorghum grain. Crop Science 48:1732-1743.
• Lu S, Li L (2008) Carotenoid metabolism: the biosynthesis, regulation, and beyond. Journal of Integrative Plant Biology 50:778-785.

Progress 11/15/06 to 11/14/07

Outputs
OUTPUTS: A comprehensive understanding of the regulatory mechanisms of carotenogenesis is essential for both plant biology and the development of carotenoid-enriched food crops. Significant progress has been made on continual characterization of the isolated cauliflower Or gene and elucidation of its function in controlling carotenoid accumulation. To obtain some clues on the nature of Or mutation and study the effects of Or on plant growth and development, we generated cauliflower and Arabidopsis transformants which overexpressed the Or gene and Or variants of alternatively spliced transcripts as well as the wild type or transcript. Additionally, we also generated double-stranded Or RNAi transgenic lines in cauliflower and Arabidopsis. Except the mutant Or gene overexpressing transgenic lines, the other transformants exhibited no observed mutant phenotype or increased level of carotenoid accumulation in cauliflower, suggesting that Or may be a gain-of-function mutation that positively controls carotenoid accumulation. Interestingly, detailed examination of the Or transformants in Arabidopsis revealed that the wild type or gene controls other aspects of plant growth and development such as root growth and flower development. The underlying mechanisms for its effect on plant growth and development are under investigation. To further elucidate the molecular and biochemical basis of Or in conferring carotenoid accumulation, a yeast two-hybrid approach was employed to isolate the proteins that interact with OR. A truncated form of the Or gene containing the DnaJ cysteine-rich zinc figure domain was cloned and expressed as a fusion protein to the GAL4 DNA binding domain and used to screen an Arabidopsis cDNA library expressed as fusions to the GAL4 activation domain. A protein that interacts with OR was identified and such interaction was confirmed by pulling down assay. This protein belongs to a three member protein family. We are in the process to generate triple knockout lines in Arabidopsis and the dsRNAi transgenic lines in cauliflower to investigate the association of OR and the OR-interacting protein in regulating carotenoid accumulation and plant growth and development. In addition, to examine whether the cauliflower Or gene can enhance carotenoid accumulation in a major staple crop, we have successfully transformed the Or gene into potato and demonstrated that the Or gene can be used to increase carotenoid accumulation in a major staple crop. Examination of the Or potato transgenic lines confirmed that Or acts as a bona fide molecular switch to confer chromoplast differentiation for carotenoid accumulation in plants. PARTICIPANTS: PDs: Dr. Li Li oversees the overall experiment design and progress, and Dr. Ted Thannhauser guides all aspects of protein identification experiments. Postdoctoral associates: Dr. Xiangjun Zhou is responsible for studying the nature of Or mutation and identifying proteins interacting with OR. Dr. Alex Lopez examined the effects of the Or transgene on platid development and carotenoid accumulation in potato. Collaborators: Dr. Joyce Van Eck's lab at Boyce Thompson Institute for Plant Research at Cornell performed the transformation of the Or gene into potato. Training or professional development: The project provided training opportunities for addtional two undergraduates and one high school student.

Impacts
The Or gene mutation in cauliflower exerts a unique mechanism in regulating carotenoid accumulation by inducing the formation of a metabolic sink. The research is expected to contribute our understanding of a regulatory mechanism underlying carotenoid accumulation, and perhaps a general mechanism to regulate metabolite accumulation in plants. Furthermore, from a practical point of view, a better understanding of the effects of Or on plant growth and development can aid the development of new strategies for using the novel molecular tool to enrich carotenoid content in food crops for improving human nutrition and health.

Publications

• 4. Lopez, AB, Van Eck J, Conlin BJ, Paolillo DJ, ONeill J, Li L (2007) Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers. Journal of Experimental Botany doi:10.1093/jeb/erm299.
• 1. Lu S, Van Eck J, Zhou X, Lopex AB, O'Halloran DM, Cosman KM, Conlin B, Paolillo DJ, Garvin DF, Vrebalov J, Kochian L, V, Kupper H, Earle ED, Cao J, and Li L (2006) The cauliflower Or gene encodes a Dnaj cysteine-rich domain-containing protein that mediates high-levels of beta-carotene accumulation. Plant Cell 18: 3594-3605.
• 2. Yang Y, Thannhauser TW, Li L, Zhang S (2007) Comparison of protein identifications by MALDI-TOF/TOF and LC-ESI-MS/MS for evaluation of 2-D gel iImage analysis: impact of multiple proteins in single spots on comparative analysis. Electrophoresis 28:2080-2094.
• 3. Li L, Van Eck J (2007) Perspectives: metabolic engineering of carotenoid accumulation by creating a metabolic sink. Transgenic Research 16:581-585.