Photosynthesis: Assimilate partitioning Getting the building blocks for growth and cell wall biosynthesis from the leaf tissue to actively growing organs. Bob Turgeon Vacuole Mesophyll pH = 5.5 pH = 7.3 ∆E = -180 mV H+ Plasmodesmata Sucrose Sucrose H+ amino acids PM-ATPase ATP Sucrose amino acids chloroplast proton-coupled symporters Apoplastic phloem loading Phloem CO2 Water mass flow pH jump forms trans-membrane pH gradient potassium & valinomycin form defined membrane potential 14C-substrate pH = 8.0 Κ + pH = 6.0 14C-substrate pHi = pHo Negative membrane potential 1200 Sucrose Transport pmol/ mg p CCCP 800 400 0 0.0 2.0 4.0 Time (min) 6.0 Plasma Membrane Cytoplasm Extracellular DEPC Binds to a substrate protectable site (∆ Ψ sensitive?) CHSuc CHSuc H + Sucrose Proton-sucrose symporter C Sucrose + H C (∆ Ψ sensitive?) SH PCMBS Properties of the Proton-Sucrose Symporter Apparent Kmm Sucrose Protons Inhibitors DEPC PCMBS pH Optimum Stoichiometry 1.0 mM µM 0.7 I 50 50= 750 µM I 5050= 30 µM < 6.0 1:1 Location Plasma Membrane Specificity Low Affinity For: glucose, fructose raffinose, maltose mannose, lactose and melibose Amino Acid Import Developing leaves, fruit, and seed Amino acids are the currency of nitrogen allocation in multicellular plants. Amino Acid Import Mature leaves Am ino Acid Export Primary Assimilation Amino Acid Cycling (xylem to phloem) Root Amino Acid Import Amino Acid Export Primary Assimilation Amino Acid Cycling (phloem to xylem) Developing roots and meristem Amino Acid Import Phloem Xylem Strategies for identifying symporter proteins and genes …. • Biochemical Differential labeling Photoaffinity labeling Solubilization & reconstitution • Molecular PCR Mutants Functional complementation Molecular Cloning via Functional Complementation Positive Transformant Yeast Mutant Transform mutants with a plant cDNA library constructed in a yeast expression vector Biochemically limitedand transport incompetent H Expression Vector + ATP With successful transcription, translation, and insertion, we select for plant transporterdependent growth. Screen for restored growth on a limiting medium Limiting Substrate 0.8 A B 2.0 Alanine Transport (nmol/ mg cells) Histidine Transport (nmol/ mg cells) 0.6 0.4 0.2 1.0 0.0 0.0 0.0 4.0 8.0 Time (min) 12.0 16.0 0.0 2.0 4.0 Time (min) 6.0 3 400 300 200 100 Hydrophobic 3 2 1 0 -1 -2 2 1 0 -1 -2 -3 * * Hydrophilic * 200 Amino Acid Position, N to C 100 -3 400 300 Terminal C 1 2 3 4 5 6 7 8 9 10 11 N Structure no. 1 CYTOPLASM 1 2 3 4 5 6 7 8 9 10 C N CYTOPLASM Structure no. 2 Proton-Sucrose Symporter 1 2 3 4 5 N 6 7 8 9 10 11 12 C CYTOPLASM Cytoplasmic side Random Mutagenesis silent mutations, nonsense mutations, substitutions, premature stops Transform into yeast Screen for altered transport phenotypes 0.6 Maltose transport (nmol / mg FW . min) 0.5 0.2 mM 0.4 0.5 mM 0.3 1 mM 0.2 2 mM 0.1 0 Wt Mal9 (G334S) Construct H65 1 2 G334 3 4 N155 N 5 6 7 8 9 L461 10 11 12 Q249 W250 Cytoplasmic C Mesophyll Vacuole NO3 H Plasmodesmata + Sucrose amino acids Sucrose ATP Sucrose + H Amino acids amino acids chloroplast 1. Sucrose symporters serve both source and sink tissues Sink Tissue Vacuole amino acids hexose Phloem CO2 water mass flow symplastic Sucrose sucrose Amino acids amino acids Vacuole water 2. BUT, there is at least one other way to generate the hydrostatic pressure difference Phloem Loading in a “Symplastic System” Also depends on accumulating high concentrations of solutes, but in this case, the “sugar” is synthesized in place, not transported into the CC/SE complex. Sucrose diffuses into the companion cell X H2O Synthesize high concentrations of raffinose &/or stachyose in the companion cell (intermediary cell). Raffinose is too large to diffuse back into bundle sheath cell via plasmodesmata. This generates a high osmotic concentration that results in water influx and high hyrodstatic pressure that drives phloem transport to sinks. Direct evidence of pressure flow: the aphid. 25 µm Sievetube member Sap droplet Aphid feeding Stylet Stylet in sieve-tube member (LM) Sap droplet Severed stylet exuding sap What happens when sucrose gets to the importdependent organs? It is unloaded from the companion cell/ sieve element complex via apoplastic and symplastic pathways. The movement of sucrose from sources to sinks is called assimilate partitioning. Controlling this process could have a profound impact on crop yield. Mesophyll Vacuole NO3 water H Plasmodesmata amino acids ATP Sucrose Sucrose + H TP Sucrose Amino acids amino acids chloroplast Sink Tissue Vacuole Phloem CO2 + mass flow amino acids H+ hexose symplastic Sucrose sucrose H+ ? Vacuole Apoplastic model of phloem loading ? Amino acids amino acids water SUCROSE SYMPORTER PHOTOSYNTHETIC ACTIVITY SINK ACTIVITY H2 O CO2 time transpirational feeding (pmol / min/ mg protein) Transport Activity 2500 2000 1500 Suc Gluc Ala 1000 500 0 0 100 200 mM Sucrose Fed 24 Hours • Are changes in sucrose transport activity due to osmotic effects? No. KCl, sorbitol, mannitol have no effect. • Is the observed regulation sucrose-specific? Yes. Hexoses and hexose analogs are not effective. • What happens to sucrose transport activity? Km remains unchanged. Vmax decreases. • How? BvSUT1 mRNA and protein decrease with 2 hr half-lives Transcription is controlled by a sucrose sensing protein-phospho relay and overall transport capacity is directly proportional to transcription Jen Chiou, Matt Vaughn, Wendy Ransom-Hodgkins, Greg Harrington Sucrose Sensor Sucrose Kinase Protein Turnover mRNA Turnover B A Phloem Loading Sucrose Symporter Transcription Phosphatase All within the companion cell High rate of symporter transcription Sucrose Less symporter protein Sucrose H+ Down-regulation of symporter transcription Abundant symporter protein High rate of sucrose export PHLOEM PHLOEM H+ High Sink Demand Sucrose-signaling response pathway Sucrose accumulation in the phloem Low rate of sucrose export Low Sink Demand How can we identify players in the sucrose signaling pathway? 1. Mutagenize SUT1promoter::reporter plants and screen for mutants that lack the sucrose signaling pathway 2. Use expression profiling of companion cells to identify specific kinases and phosphatases, then look at phenotypes of knockouts 3. Look for a sucrose response that is more tractable for genetic analysis Water Four day old seedlings induced with 90 mM sugar treatments and photographed three days later. 8 7 6 5 4 3 2 Sorbitol sorbitol Maltose Mannose Gluc + Fruc Fructose maltose mannose Glucose H20 PAP1 (Production of Anthocyanin Pigment 1) Transcription factor ‐ known “master” regulator of anthocyanin biosynthesis and part of a multi‐peptide complex Sucrose gluc+fruc fructose glucose sucrose 1 0 water A530-A657/gFW Anthocyanin Content PAP1 ACTIN Pro 8 H2O Pro 62 SUC H2O Col-0 Pro 63 SUC H2O SUC 35S-GUS 1 mm H2O SUC H2O SUC Pro 8 H2O Pro 62 SUC Wg 34 H2O H2O Pro 63 SUC H2O Wg 35 SUC Col-0 H2O Wg 43 SUC H2O 35S-GUS 1 mm H2O SUC H2O SUC SUC SUC ΔIntron1 2A H2O SUC ΔIntron2 3A H2O SUC ΔIntron1&2 4A H2O SUC ΔIntron1 3B H2O SUC ΔIntron2 5B H2O SUC ΔIntron1&2 6A H2O SUC SURE-1 5’-upstream sequence ATG I I 200 bps SURE-2 II = exon III PAP1 gene = intron SURE-1 (TTTTCTATT) mutated to TTTGAGATT I II III GUS PAP1wg_mut1-GUS SURE-1 (TTTTCTATT) mutated to SURE-2 (AATACTAAT) I II III GUS PAP1wg_mut2-GUS GUS PAP1wg_mut3-GUS GUS PAP1wg_mut4-GUS 205 bp deletion (includes SURE-1) I II III 205 bp deletion (excludes SURE-1) I II III GLU SUC + SURE-1, - 5’ H20 GLU SUC Minus SURE-1 H20 GLU SUC ∆ SURE-1 H20 GLU SUC H20 ∆ SURE-1 PAP1 GUS ACTIN 4 day old plants, 4 hr induction Biotech Approach to Increase Yield: Use sugar beet as a model biofuel crop by by manipulating sugar allocation in the plant’s vascular system. Why sugar beet as a model for carbon partitioning • Root yields of over 40 tons per hectare at 15.5-18% sucrose content (6-7 tons of sugar per hectare) Record yields approach 25% sucrose, so there is somewhere to put “extra” sucrose • Approach: Constitutively express hyper-active symporter Hypothesis: This will draw down sugars in photosynthetic cells which will, 1) Increase photosynthesis 2) Delay senescence vacuole Leaf mesophyll Phloem Plasmodesmata H+ Sucrose CO2 ATP Sucrose Sucrose ? H+ chloroplast mass flow CmGAS1p AtSUC1H65K TT Sucrose ? 257 248 239 230 226 202 139 121 46 43 13 Independent transgenic lines 8 Control 2 Control 1 The modified SUT is expressed in the transgenic lines * Independent transgenic lines p-value ≤ 0.01 SUT-257 SUT-248 SUT-239 SUT-226 SUT-202 SUT-139 SUT-121 SUT-46 SUT-43 SUT-13 SUT-230 * * SUT-8 900 800 700 600 500 400 300 200 100 0 Control average tuber FW (g) Average tuber FW of control and transgenic lines 35 * * * * 30 25 20 15 10 5 Independent transgenic lines p-value ≤ 0.01 p-value ≤ 0.05 SUT-257 SUT-248 SUT-239 SUT-230 SUT-226 SUT-202 SUT-139 SUT-121 SUT-46 SUT-43 SUT-13 SUT-8 0 Control average above ground biomass (g) Average above ground biomass of control and transgenic lines average tuber FW/leaf DW 50 45 40 35 30 25 20 15 10 5 0 Independent transgenic lines SUT-257 SUT-248 SUT-239 SUT-230 SUT-226 SUT-202 SUT-139 SUT-121 SUT-46 SUT-43 SUT-13 SUT-8 Control Average tuber FW/leaf DW of control and transgenic lines Rice as a model crop for identifying biomass genes Rationale: The Green Revolution was about seed production, not biomass Rice is a great model for new energy crops because ………. Simple grass Gene synteny Ag-infrastructure well established Powerful genetic, molecular, and genomic tools OryzaSNP set 20 diverse rice lines • resequenced for SNP discovery PNAS 106:12273-12278 2009 Approach: • Define morphological and physiological differences • Forward genetic screens • QTL mapping • Association mapping • Transgenic manipulation of candidate genes knockout, over‐expression, truncated proteins OryzaSNP set • Sampling – Morphological • leaf length & width, plant height, tiller number, and total above ground biomass (3‐fold) and seed yield (inverse of biomass) – Physiological • Leaf area index, photosynthetic rates, cellulose and lignin content of leaves and stems OryzaSNP set • Significant genetic variation for both morphological and physiological data • Heritability • Photosynthesis – M2O2 and Cypress • Highest photosynthetic rate – Pokkali • Lowest photosynthetic rate, largest biomass – Why PS not correlated to total biomass Mutant populations: chemical and fast neutron Amino Acid Transporters & Nitrogen regulation of gene expression Lishan Chen Hui-Chu Chang Adriana Ortiz-Lopez Aaron Schmitz Ekrem Dundar Mengjuan Guo Xianan Liu Vince Stoerger Christian Hermans Silvana Porco Sucrose Transporters and gene regulation Tzyy-Jen Chiou Jade Lu Matt Vaughn Wendy Ransom-Hodgkins Greg Harrington Anshuman Kumar RICE Jan Leach John McKay Hei Leung - IRRI Bettina Broeckling Courtney Jahn USDA-ARS, DOE, & NSF
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