Progress 01/15/24 to 01/14/25
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate and two postdoctoral research associates were involved in this project. The project provided training in plant genetics, molecular biology and biochemistry analyses. It also offered the opportunity for developing skills with effective scientific communications by presenting findings in writing and verbal presentations. How have the results been disseminated to communities of interest?Oral presentation of "Genetic factors and mechanisms underlying phytonutrient carotenoid accumulation in crops". The International Plant & Animal Genome PAG 32 Conference, San Diego, CA January 10-15, 2025 What do you plan to do during the next reporting period to accomplish the goals?We will proceed with our ongoing experiments to achieve the set objectives.
Impacts
What was accomplished under these goals?
During the past year, we further examined various cauliflower genotypes with green and white curd colors for pigment content and for sequence difference of the candidate gene for Gr. The study confirmed that the two distinct alleles encode proteins differing by a single amino acid substitution in the chloroplast transit peptide (cTP) region and the addition of one amino acid in the mature protein between white and green cauliflower genotypes. To investigate the function of Gr, we generated transgenic lines overexpressing either the white or green allele of the cauliflower Gr gene in the backgrounds of Arabidopsis ap1/cal mutant which forms green, curd-like meristem structures and of Arabidopsis Gr homolog mutant that gives severely retarded growth and pale-yellow phenotype. We observed greener phenotype in ap1/cal mutant expressing the green allele of the cauliflower Gr gene comparing to expressing the white allele, which also functionally confirmed the identity of Gr. Furthermore, we observed that overexpression of the green allele successfully rescued the phenotype of Arabidopsis Gr homolog mutant, whereas the white allele did not. These findings support the role of Gr in chlorophyll accumulation. The Gr protein was reported to be involved in plastid pyrimidine metabolism to regulate chloroplast development. We have previously demonstrated that Gr influences protein levels rather than transcription. To further investigate the reason behind the higher protein accumulation in the green allele compared to the white allele, we are conducting an in vitro phosphorylation assay. Specifically, we aim to determine whether the green allele exhibits greater phosphorylation activity than the white allele. This experiment is currently underway. In addition, the Gr protein was reported to impact RNA metabolism which in turn influences chloroplast transcription. To investigate this, we performed RNA-Seq and Ribo-Seq (ribosome profiling) analyses on white and green cauliflower curds, as well as on Arabidopsis transgenic lines expressing the white and green Gr alleles. These experiments aim to uncover differences in translation rate between these samples. These experiments are currently in progress, and we expect to identify notable differences.
Publications
- Type:
Other Journal Articles
Status:
Accepted
Year Published:
2025
Citation:
Sun T, Hazra A, Lui A, Zeng S, Wang X, Rao S, Owens L, Fei Z, Zhao Y, Mazourek M, Giovannoni JJ, Li L (2025) GLKs directly regulate carotenoid biosynthesis via interacting with GBFs in in plants. New Phytologist accepted
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Progress 01/15/23 to 01/14/24
Outputs
Target Audience:The output of this research would benefit agricultural, horticultural and biological scientists following revealing novel genes and molecular mechanisms, which regulate chloroplast biogenesis in agronomically important crops. Breeders may also utilize such information to generate novel germplasms with more appealing visual as well as more nutrients associated with chloroplast development, which would consequently benefit the general public as consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate and one postdoctoral research associate were involved in this project. The project provided training in plant genetics, molecular biology and biochemistry analyses. It also offered the opportunity for developing skills with effective scientific communications by presenting findings in writing and verbal presentations. How have the results been disseminated to communities of interest?Oral presentation entitled"Orchestration of photosynthetic pigment synthesis in plants" at The 19th International Symposium on Carotenoids. July 9-14, 2023 Toyama, Japan What do you plan to do during the next reporting period to accomplish the goals?We will proceed with our ongoing experiments to achieve the set objectives. We will try to demonstrate the distinguished role and mechanism of Gr protein from green allele in mediating chloroplast development.
Impacts
What was accomplished under these goals?
During the past year, we were focused on characterizing the Gr protein and understanding its basis in conferring chloroplast development. We expanded Gr candidate gene expression analysis to a panel of white and green curd cauliflower varieties. In general, the Gr gene expresses higher in the green curd cultivars than the white ones, although some green cultivars show similar gene expression levels like white ones as we observed previously when comparing one green and one white cultivar. However, when we analyzed Gr protein level in curd tissues of these cultivars by western blot analysis, all green curd varieties showed high abundant Gr protein levels and all white curd varieties had very low Gr protein levels. These results indicate that the white cauliflower varieties lose the ability to accumulate the Gr protein in the curd tissue despite its normal transcription. To understand the basis underlying such Gr protein accumulation difference between white and green curd cauliflower varieties, we examined the transgenic Arabidopsis lines overexpressing either the cauliflower white or green allele of Gr gene. Although the three independent transgenic lines with white allele showed similar transgene expression levels as the three independent lines with green allele, they had much lower Gr protein abundance than those lines with green allele, consistent with what was observed between white and green curd cauliflower varieties. These results confirm the difference of the two alleles of Gr in affecting its protein accumulation. To examine whether the two Gr alleles encode proteins that alter their subcellular localization, we examined their subcellular localizations by expressing individual allele fused with YEP in tobacco leaves. Both were found to localize in chloroplasts. However, very strong signals were observed for the green allele protein and very faint signals were noted for white allele protein, further suggesting the Gr protein stability difference between the green allele vs the white allele protein. The two different alleles encode Gr protein with one amino acid change in the transit peptide (cTP) region and one amino acid addition in the mature protein of Gr green protein. To check whether the alteration of one amino acid in cTP affects Gr abundance in plants, we performed cTP import assay by fusing cTP from white and green protein to GFP and transient expression in tobacco leaves. While GFP fused to Green cTP produced predominantly mature protein, the GFP fused to white cTP generated mainly premature protein, indicating the single amino acid change greatly affects Gr protein targeting into chloroplast to influence its protein abundance in chloroplasts. At the same time, we also generated transgenic lines deleting the extra amino acid in Gr green protein. These lines showed greatly reduced Gr protein accumulation. These results indicate that both the amino acid change in cTP and the single amino acid addition contribute to Gr protein stability in the green curd cauliflower varieties, subsequently affecting chloroplast development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Sun T, Wang P, Rao S, Zhou X, Wrightstone E, Lu S, Yuan H, Yang Y, Fish T, Thannhauser T, Liu J, Mazourek M, Grimm B, Li L (2023) Co-chaperoning of chlorophyll and carotenoid biosynthesis by ORANGE family proteins in plants. Molecular Plant 16:10481065. http://dx.doi.org/10.1016/j.molp.2023.05.006
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:The output of this research would benefit agricultural, horticultural and biological scientists following revealing novel genes and molecular mechanisms, which regulate chloroplast biogenesis in agronomically important crops. Breeders may also utilize such information to generate novel germplasms with more appealing visual as well as more nutrients associated with chloroplast development, which would consequently benefit the general public as consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate and one postdoctoral research associate was involved in this project. The project provided the training in plant genetics, molecular biology and bioinformatics analyses. It also offered the opportunity for developing skills with effective scientific communications by presenting findings in writing and verbal presentations. How have the results been disseminated to communities of interest?Oral presentation: "Colored cauliflower, molecular basis, and genetic tools and strategies to breed nutrient dense crops". Genomic advances in fruit and vegetable breeding workshop. The International Plant & Animal Genome XXX Conference, San Diego, CA January 13-18, 2023 What do you plan to do during the next reporting period to accomplish the goals?We will proceed with our ongoing experiments to achieve the set objectives. We hope to functionally confirm the Gr gene leading to the green curd trait in cauliflower. We will further characterize this gene in green and white cauliflower and start to dissection of the mechanism of Gr in mediating chloroplast development.
Impacts
What was accomplished under these goals?
For the Objective 1: From a list of 25 potential Gr candidates, we have narrowed down to a promising gene that likely confers the green curd trait in cauliflower. An SNP and a deletion mutation of the same gene were identified between white and green curd cauliflower. We are currently generating transgenic cauliflowers that overexpress the green allele of the candidate gene for phenotypic complementation. So far, we have established an in-house protocol to regenerate cauliflower seedlings from calli after Agrobacterium-mediated transformation. We expect to obtain positive transformants soon. In addition, we obtained a corresponding Arabidopsis knockout mutant of our candidate gene. The mutant is deficient in chloroplast biogenesis, appeared pale yellow, and showed dwarfism. We had generated transgenic Arabidopsis plants that overexpress either the white or green allele of our candidate gene in the mutant background. Transgenic lines overexpressing the green allele fully complemen the phenotypic deficiencies (displayed by the Arabidopsis mutant), while those overexpressing the white allele could only partially complement the phenotypes. These transgenic studies show the difference of the two alleles of our candidate gene in conferring chloroplast development. For the Objective 2: We first examined the candidate gene expression in the white and green curd tissue by quantitative real-time polymerase chain reactions. The gene expression analysis revealed that the candidate gene's expression level in white and green curds is similar. We then performed western blot experiments. The analysis showed that their protein levels are significantly different. We are currently examining how the differences in amino acid sequence (from the SNP and/or deletion mutations) would affect its protein stability in the curd tissue. Molecular characterization of Arabidopsis transgenic lines overexpressing the white and green allele of the candidate gene is now in progress to account for their phenotypic differences.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
1. Sun T, Zhou X, Rao S, Liu J, Li L (2022) Protein-protein interaction techniques to investigate post-translational regulation of carotenogenesis. Methods in Enzymology Volume 671: Carotenoids: Carotenoid and apocarotenoid biosynthesis, metabolic engineering and synthetic biology. pp 301-325
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The output of this research would benefit agricultural, horticultural and biological scientists following revealing novel genes and molecular mechanisms, which regulate chloroplast biogenesis in agronomically important crops. Breeders may also utilize such information to generate novel germplasms with more appealing visual as well as more nutrients associated with chloroplast development, which would consequently benefit the general public as consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate and one postdoctoral research associate was involved in this project. The project provided the training in plant genetics, molecular biology and bioinformatics analyses. It also offered the opportunity for developing skills with effective scientific communications by presenting findings in writing and verbal presentations. 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 will proceed with our ongoing experiments to achieve the set objectives. We will continue functional confirmation of the Gr gene leading to the green curd trait in both Arabidopsis mutant and in white curd cauliflower. We will start to characterize this gene in green and white cauliflower and to dissection of the mechanism of Gr in mediating chloroplast development.
Impacts
What was accomplished under these goals?
The COVID pandemic greatly delayed the start of the project due to the late arrival of newly hired researchers and limited number of peoplewho could work in the lab. To accomplish the above objectives, in this rating period we thoroughly examined the 35 candidate genes located within the mapped major QTL locus (named Gr5.1; on Chromosome 5) responsible to the green curd trait. By further fine mapping the Gr5.1 locus and remapping the genome region to the recently published white cauliflower genome, we narrowed down the candidates to 25 genes (excluding pseudogenes and long non-coding RNAs). We then analyzed these candidates via combination of bioinformatics, computational analyses, and literature review. We reasoned that gene products localized to either chloroplast or nucleus would most likely affect chloroplast biogenesis. Their corresponding peptide sequences were hence subjected to computational analyses and identified 14 candidates with subcellular localization signals. We then compared their gene expression level between white and green cauliflower curds and examined the INDELs and SNPs of genes expressed in curd tissues. We identified one gene to be of particular interest with the presence of INDELs within its coding sequence by comparing all white cauliflower sequences publically available with the green gene. Concurrently, we have confirmed its predicted subcellular localization and noticed a difference in the suborganelle localization within the chloroplast between the gene product from white and green cauliflowers. We are now generating the CRISPR/Cas9-mediated knockout construct and will first transform into Arabidopsis mutants in the ap1/cal background which forms green, curd-like meristem structure. We expect to observe a loss of green curd color in these mutants, which would allow us to tentatively identify the candidate Gr gene. We have also established a regeneration platform for Agrobacterium-mediated cauliflower transformation. We are in progress to transform the green allele into white curd cauliflower to confirm the identity of the Gr gene.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
Sun T, Rao S, Zhou X, Li L (2022) Plant carotenoids: recent advances and future perspectives. Molecular Horticulture (in press)
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