1/25/2019 2. Where are chromoplasts come from? Genetic and Molecular Basis of Chromoplast Division Proplastid Tianhu Sun, Postdoctoral Associate 2 Cornell University Chloroplast Leucoplast PAG XXVII, San Diego Jan-13-2019 1 3 Chromoplast 1. Why we study chromoplasts? 3. How to control carotenoid content? • Plastids are main sites for carotenoid biosynthesis and storage in plants while chromoplast has highest carotenoid content. • Carotenoids are important for human nutrition and health. • Plant carotenoids provide dietary sources of provitamin A. Plastids (sink) Luke SW. livescience.com Sun et al. 2018 Mol. Plant Plastid type defines its capacity for carotenoid biosynthesis and accumulation proplastid etioplast amyloplast chloroplast chromoplast Can we increase carotenoid sequestration? Carotenoid content Sun et al. 2018 Mol Plant 11:58 1 1/25/2019 OR is a switch for chromoplast formation OR restricts chromoplast duplication An Or gene controls b-carotene accumulation in fruit and vegetable Only one or two large chromoplasts in each cell were observed in orange cauliflower curd. WT Or VC Or Tzuri, Zhou, Chayut et al. 2015 Plant J. Lu et al. 2006 Plant Cell Chayut et al., 2017 Plant Physiol. p p n Or encodes a DnaJ-like zinc finger protein with its function largely unknown. Paolillo et al. 2004 Li et al. 2001 Plant J. OR restricts chromoplast duplication Ectopic expression of OR in tomato induced chromoplast formation at early stage of fruit development. Melon Or also confers large chromoplast formation M82 ORWT ORHis Yazdani et al. 2018 Plant Biotech. J. VC Or Chayut et al., 2017 Plant Physiol. OR restricts chromoplast duplication Comparision of chromoplast numbers from orange cauliflower (BoOr), melon (ORHis) and tomato. A B C However… D E F Sun et al. unpublished data 2 1/25/2019 Mimiking OR induced chromoplast formation in Arabidopsis callus A Col-0 ORWT#6 ORWT#25 ORHis#8 ORHis#39 PSY-OE Mimic OR induced chromoplast formation in B Arabidopsis C D 25 * 5 117/28 FW) 1 20 - 4 15 * * 3 10 2 5 1 chromoplasts each cell each chromoplasts 13/36 23/50 Carotenoid content (μg·g content Carotenoid 0 0 Sun et al. unpublished data OR does not affect chloroplast division. A B 40 Col-0 ORWT ORHis 30 How does OR regulate chromoplast duplication? 20 ftsZ1 arc3 parc6 10 chloroplast number cell per number chloroplast 0 Sun et al. unpublished data Chloroplast division mechanism: Expression of ORHis causes chromoplast formation Col-0 ORWT ORHis Col-0 OR ORHis BoOR Col-0 OR ORHis AtPSY Yuan et al. 2015 Plant Physiol. Chen et al. 2018 Plant Physiol. 3 1/25/2019 Search for potential interactions between OR and plastid division machinary: -Leu/-Trp -Leu/-Trp/-His +10 mM 3-AT ORHis does not affect division gene expression in callus. AD-ORWT AD-ORHis AD-ORWT AD-ORHis BD-FtsZ1 Col-0 ORWT#6 ORWT#25 BD-FtsZ2 A 2.0 ORHis#8 B C ORHis#39 BD-ARC6ims 1.5 ARC3 FtsZ2 callus BD-ARC6stromal Ponceau S OR 1.0 BD-PARC6ims ARC3 Actin seedling 0.5 Ponceau S Relative gene expression gene Relative Ponceau S BD-PARC6stromal 0 BD-ARC3ΔMORN ARC3 PARC6 FtsZ2 BD-ARC3-MORN BD-MinE Sun et al. unpublished data ARC3 interact with ORHis through MORN domain, but not ORWT. Introducing yeast three-hybrid system to test the effect of ORHis-ARC3 interaction on ARC3 function. -Leu/-Trp/-His -Leu/-Trp AD BD 10 mM 3-AT 4 * WT N-OR ARC3-MORN 3 * N-ORR90H ARC3-MORN N-ORR90F ARC3-MORN 2 R90K N-OR ARC3-MORN 1 R88H unit) (miller activity gal N-OR ARC3-MORN - N-ORR102H ARC3-MORN 0 Beta N-ORR90H E.V Z as a ‘Bridge’ protein: facilitate or inhibit protein interaction? Sun et al. unpublished data ORHis interferes interaction between PARC6 and ARC3. Confirm the interaction by pulldown and BiFC assay -Leu/-Trp/-His +10 mM 3AT -Leu/-Trp AD BD Bridge -Met +Met FtsZ1 ARC3 N-ORWT FtsZ1 ARC3 N-ORHis YFP Bright field Merge FtsZ2 ARC3 N-ORWT ARC3-cY FtsZ2 ARC3 N-ORHis His Ponceau S +OR -nY WT PARC6stromal ARC3 N-OR ARC3 N-ORHis input (MORN-His) PARC6stromal pulldown ARC3-cY (anti-His) +ORWT-nY AD BD Bridge PARC6stromal ARC3598-741 w/o N-ORWT PARC6stromal ARC3598-741 w/o N-ORHis Sun et al. unpublished data PARC6stromal ARC3598-741 with N-ORWT * PARC6stromal ARC3598-741 with N-ORHis __ ARC3598-741 __ 0 1 2 3 4 5 Beta-gal activity (miller unit) Sun et al. unpublished data 4 1/25/2019 Over-expression of FtsZ1, FtsZ2, PDV1 and PDV2 in ORHis background: Only PDV2 could increase carotenoid level in callus. Col-0 ORHis ORHis ORHis FtsZ2#11 FtsZ1#1 Can we manipulate chromoplast duplication? ORHis ORHis FtsZ2#3 FtsZ1#4 25 * FW) * 1 - 20 15 Col-0 ORHis 10 ORHis ORHis 5 * PDV2#18 PDV1#1 0 Carotenoid content (μg·g content Carotenoid ORHis ORHis PDV2#5PDV1#4 Sun et al. unpublished data The increase of carotenoid content is caused by increased number Over-expression and knock-out of ARC3 in ORHis background: of chromoplasts other than increased biosynthesis ability. Col-0 ORHis ORHis PDV1#1 ORHis PDV1#4 A B Col-0 ORHis ORHis arc3#1 ORHis arc3 #2 ORHis arc3 #3 ORHis arc3 #4 C arc3 ORHis ARC3 ORHis ORHis ORHis PDV1 #1 ORHis PDV1 #4 #1 #2 #3 #4 #1 #2 #6 4 anti-OR 3 ORHis ARC3 #1 ORHis ARC3 #2 ORHis ARC3 #6 2 anti-myc each cell each 1 Ponceau S Chromoplasts 0 ORHis ORHis His OR PDV1 PDV1 #1 #4 Sun et al. unpublished data Sun et al. unpublished data Proposed model for chromoplast division Alteration of ARC3 could affect carotenoid accumulation. OEM OEM PDV1 PARC6 ORHis IEM IEM ARC3 24 ORHis * Col-0 FW) 20 * 1 - * 16 12 * * * * 8 4 * Normal chromoplast OR induced chromoplast Carotenoid content (μg·g content Carotenoid 0 #1 #2 #3 #4 #1 #2 #6 ORHis arc3 ORHis ARC3 Sun et al. unpublished data 5 1/25/2019 Perspectives How does chromoplast duplicate? Budding Binary fission tomato suffulta mutant with abnormal chromoplast of nearly mature orange fruit plastid division machinary l, globules; st, starch; m, mitochondria Green, tpGFP; Red, chlorophyll Forth and Pyke. 2006. JXB William W. Thomson. 1966. Botanical Gazette Perspectives • Non-green plastid duplications are less understood • The major duplication mechanism of chromoplast: binary fission? • Are there more essential factors for chromoplast division? • Provide an alternative way to increase carotenoid sink Acknowledgement Dr. Li Li group Kobi Tadmor— Israel Hui Yuan Hongbo Cao Kathy Osteryoung—MSU Huaqiang Tan Cheng Chen Andre Reis Michael Miller Franklin Santiago Michael Mazourek—Cornell Meghna Prasd 6.