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| 1 | Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots显示文摘Single-cell-type proteomics provides the capability to revealing the genomic and proteomics information at cell-level resolution.However,the methodology for this type of research has not been well-developed.This paper reports developing a workflow of laser capture microdissection(LCM)followed by gel-liquid chromatography-tandem mass spectrometry(GeLC-MS/MS)-based proteomics analysis for the identification of proteomes contained in individual cell layers of tomato roots.Thin-sections(~10-μm thick,10 sections per root tip)were prepared for root tips of tomato germinating seedlings.Epidermal and cortical cells(5000–7000 cells per tissue type)were isolated under a LCM microscope.Proteins were isolated and then separated by SDS–polyacrylamide gel electrophoresis followed by in-gel-tryptic digestion.The MS and MS/MS spectra generated using nanoLC-MS/MS analysis of the tryptic peptides were searched against ITAG2.4 tomato protein database to identify proteins contained in each single-cell-type sample.Based on the biological functions,proteins with proven functions in root hair development were identified in epidermal cells but not in the cortical cells.Several of these proteins were found in Al-treated roots only.The results demonstrated that the cell-type-specific proteome is relevant for tissue-specific functions in tomato roots.Increasing the coverage of proteomes and reducing the inevitable cross-contamination from adjacent cell layers,in both vertical and cross directions when cells are isolated from slides prepared using intact root tips,are the major challenges using the technology in proteomics analysis of plant roots. | Yingde Zhu Hui Li Sarabjit Bhatti Suping Zhou Yong Yang Tara Fish Theodore W Thannhauser | 2016 | Horticulture Research2016，3，1： | 2 |
| 2 | Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds显示文摘Staple grains with low levels of provitamin A carotenoids contribute to the global prevalence of vitamin A deficiency and therefore are the main targets for provitamin A biofortification.However,carotenoid stability during both seed maturation and postharvest storage is a serious concern for the full benefits of carotenoid biofortified grains.In this study,we utilized Arabidopsis as a model to establish car-otenoid biofortification strategies in seeds.We discovered that manipulation of carotenoid biosynthetic activity by seed-specific expression of Phytoene synthase(PSY)increases both provitamin A and total carotenoid levels but the increased carotenoids are prone to degradation during seed maturation and storage,consistent with previous studies of provitamin A biofortified grains.In contrast,stacking with Orange(OR^(His)),a gene that initiates chromopl ast biogenesis,dramatically enhances provitamin A and total carotenoid content and stability.Up to 65-and 10-fold increases of β-carotene and total car-otenoids,res pectively,with provitamin A carotenoids composing over 63%were observed in the seeds containing OR^(His) and PSY.Co-expression of Homogen tisate geranylgeranyl transferase(HGGT)with OR^(His) and PSY further increases carotenoid accumulation and stability during seed maturation and storage.Moreover,knocking-out of B-carotene hydroxylase 2(BCH2)by CRISPR/Cas9 not only potentially facilitates β-carotene accumulation but also minimizes the negative effect of carotenoid over production on seed germi nation.Our findings provide new insights into various processes on carotenoid accu-mulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis,turnover,and stable storage for carotenoid biofortification in crop seeds. | Tianhu Sun Qinlong Zhu Ziqing Wei Lauren AOwens Tara Fish Hyojin Kim Theodore W.Thannhauser Edgar B.Cahoon Li Li | 2021 | aBIOTECH2021，2，3： | 2 |
| 3 | Homopteran Vector Biomarkers for Efficient Circulative Plant Virus Transmission are Conserved in Multiple Aphid Species and the Whitefly Bemisia tabaci显示文摘Plant viruses in the families Luteoviridae and Geminiviridae are phloem restricted and are transmitted in a persistent,circulative manner by homopteran insects.Using fluorescence 2-D difference gel electrophoresis to compare the proteomes of F2 genotypes of Schizaphis graminum segregating for virus transmission ability,we recently discovered a panel of protein biomarkers that predict vector competency.Here we used aphid and whitefly nucleotide and expressed sequence tag database mining to test whether these biomarkers are conserved in other homopteran insects.S.graminum gene homologs that shared a high degree of predicted amino acid identity were discovered in two other aphid species and in the whitefly Bemisia tabaci.Selected reaction monitoring mass spectrometry was used to validate the expression of these biomarkers proteins in multiple aphid vector species.The conservation of these proteins in multiple insect taxa that transmit plant viruses along the circulative transmission pathway creates the opportunity to use these biomarkers to rapidly identify insect populations that are the most efficient vectors and allow them to be targeted for control prior to the spread of virus within a crop. | Michelle Cilia Michael Bereman Tara Fish Michael J MacCoss stewart Gray | 2012 | Journal of Integrative Agriculture2012，11，2： | 1 |
| 4 | Co-chaperoning of chlorophyll and carotenoid biosynthesis by ORANGE family proteins in plants显示文摘Chlorophylls and carotenoids are essential photosynthetic pigments.Plants spatiotemporally coordinate the needs of chlorophylls and carotenoids for optimal photosynthesis and fitness in response to diverse environmental and developmental cues.However,how the biosynthesis pathways of these two pigments are coordinated,particularly at posttranslational level to allow rapid control,remains largely unknown.Here,we report that the highly conserved ORANGE(OR)family proteins coordinate both pathways via posttranslationally mediating the first committed enzyme in each pathway.We demonstrate that OR family proteins physically interact with magnesium chelatase subunit I(CHLI)in chlorophyll biosynthesis pathway in addition to phytoene synthase(PSY)in carotenoid biosynthesis pathway and concurrently stabilize CHLI and PSY enzymes.We show that loss of OR genes hinders both chlorophyll and carotenoid biosynthesis,limits light-harvesting complex assembly,and impairs thylakoid grana stacking in chloroplasts.Overexpression of OR safeguards photosynthetic pigment biosynthesis and enhances thermotolerance in both Arabidopsis and tomato plants.Our findings establish a novel mechanism by which plants coordinate chlorophyll and carotenoid biosynthesis and provide a potential genetic target to generate climate-resilient crops. | Tianhu Sun Peng Wang Sombir Rao Xuesong Zhou Emalee Wrightstone Shan Lu Hui Yuan Yong Yang Tara Fish Theodore Thannhauser Jiping Liu Michael Mazourek Bernhard Grimm Li Li | 2023 | Molecular Plant2023，16，6： | 0 |
| 5 | Identification of heat-induced proteomes in meiotic pollen mother cells of tomato'Maxifort'using single-cell-type tandem mass tag(TMT)proteomics显示文摘Heat stress(HS)poses a significant challenge to tomato production due to disruption of the reproductive organs,especially the male gametophytes.This study reports HS-induced proteome changes in meiotic pollen mother cells during early stages of anther development.Tomato(Solanum lycopersicum L.×S.habrochaites)'Maxifort'were grown in a heated polytunnel in Nashville,Tennessee,USA.Plants at flowering stage were subjected to heat treatment at 40±2℃ for 4 hr(11:00−15:00 HR);and the non-heat-treated control was at 30±2℃(day/night)at the same period of time for 10 d.The size of the flower buds containing meiotic pollen mother cells was determined based on the histology of DAPI stained cross section of anthers.Flower buds were embedded in optimal cutting temperature solutions(OCT)and then cut into sections of 20μm thickness.Sections containing meiotic pollen mother cells were collected using laser capture microdissection(LCM).A protein extraction procedure was optimized for the LCM collected pollen samples which yielded 25−30μg protein from 150,000−200,000 pollen cells.The heatinduced proteomes of meiotic pollen mother cells were quantified using tandem mass tag(TMT)quantitative proteomics analysis.Among the 6,343 quantified proteins,254 differentially expressed proteins(DEPs)showed significant differences in abundance level from heat treated to non-heat-treated control conditions.The heat-up-regulated-DEPs(96 proteins)include heat shock proteins,calreticulin and exocytosis(synaptobrevin)which are involved in protein folding/refolding/targeting/removal and secretion of aggregated and damaged proteins/peptides.The heat-down-regulated-DEPs(158 proteins)were involved in pathways of ubiquitin-mediated protein degradation,antioxidant mechanism,and metabolic processes of carbohydrates and lipids.Proteins affecting apoptotic programmed cell death and pollen mother cell meiotic activity were significantly changed under HS.The identified proteins and the affected biological processes could represent the major heat tolerance mechanisms during early developmental stages of male gametophyte when exposed to daily periods of above 40°C HS condition. | Hui Li Yaoguo Qin Xingbo Wu Joshua O'Hair Jesse Potts Suping Zhou Yong Yang Tara Fish Theodore W.Thannhauser | 2022 | Vegetable Research2022，2，1： | 0 |
| 6 | Al-induced proteomics changes in tomato plants over-expressing a glyoxalase I gene显示文摘Glyoxalase I(Gly I)is the first enzyme in the glutathionine-dependent glyoxalase pathway for detoxification of methylglyoxal(MG)under stress conditions.Transgenic tomato‘Money Maker’plants overexpressing tomato SlGlyI gene(tomato unigene accession SGN-U582631/Solyc09g082120.3.1)were generated and homozygous lines were obtained after four generations of self-pollination.In this study,SlGlyI-overepxressing line(GlyI),wild type(WT,negative control)and plants transformed with empty vector(ECtr,positive control),were subjected to Al-treatment by growing in Magnavaca’s nutrient solution(pH 4.5)supplemented with 20μM Al3+ion activity.After 30 days of treatments,the fresh and dry weight of shoots and roots of plants from Al-treated conditions decreased significantly compared to the non-treated conditions for all the three lines.When compared across the three lines,root fresh and dry weight of GlyI was significant higher than WT and ECtr,whereas there was no difference in shoot tissues.The basal 5mm root-tips of GlyI plants expressed a significantly higher level of glyoxalase activity under both non-Al-treated and Al-treated conditions compared to the two control lines.Under Al-treated condition,there was a significant increase in MG content in ECtr and WT lines,but not in GlyI line.Quantitative proteomics analysis using tandem mass tags mass spectrometry identified 4080 quantifiable proteins and 201 Al-induced differentially expressed proteins(DEPs)in roottip tissues from GlyI,and 4273 proteins and 230 DEPs from ECtr.The Al-down-regulated DEPs were classified into molecular pathways of gene transcription,RNA splicing and protein biosynthesis in both GlyI and ECtr lines.The Alinduced DEPs in GlyI associated with tolerance to Al3+and MG toxicity are involved in callose degradation,cell wall components(xylan acetylation and pectin degradation),oxidative stress(antioxidants)and turnover of Al-damaged epidermal cells,repair of damaged DNA,epigenetics,gene transcription,and protein translation.A protein–protein association network was constructed to aid the selection of proteins in the same pathway but differentially regulated in GlyI or ECtr lines.Proteomics data are available via ProteomeXchange with identifiers PXD009456 under project title‘25Dec2017_Suping_XSexp2_ITAG3.2’for SlGlyI-overexpressing tomato plants and PXD009848 under project title‘25Dec2017_Suping_XSexp3_ITAG3.2’for positive control ECtr line transformed with empty vector. | Xudong Sun Hui Li Santosh Thapa Sasikiran Reddy Sangireddy Xiaobo Pei Wei Liu Yuping Jiang Shaolan Yang Dafeng Hui Sarabjit Bhatti Suping Zhou Yong Yang Tara Fish Theodore W.Thannhauser | 2020 | Horticulture Research2020，7，1： | 0 |
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