Volume 3, Issue 1, Pages 66-77 (January 2010) Developmental and Feedforward Control of the Expression of Folate Biosynthesis Genes in Tomato Fruit  Waller Jeffrey C. , Akhtar Tariq A. , Lara-Núñez Aurora , Gregory Jesse F. , McQuinn Ryan P. , Giovannoni James J. , Hanson Andrew D.   Molecular Plant  Volume 3, Issue 1, Pages 66-77 (January 2010) DOI: 10.1093/mp/ssp057 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 The Structure and Biosynthetic Pathway of Tetrahydrofolate. (A) Chemical structure of tetrahydrofolate. One-carbon units at various oxidation levels (methyl, methylene, formyl) can be enzymatically attached to the N5 and/or N10 positions. A γ-linked polyglutamyl tail of up to about six residues is coupled to the γ carboxyl of the glutamate moiety. (B) The folate biosynthesis pathway and its compartmentation in plants. Dotted lines indicate hypothetical feedback controls at the enzyme level; those in red are more probably physiologically significant than those in gray. Metabolite abbreviations: ADC, aminodeoxychorismate; p-aminobenzoate, pABA; DHN-PPP, dihydroneopterin triphosphate; DHN-P, dihydroneopterin phosphate; DHN, dihydroneopterin; HMDHP, hydroxymethyldihydropterin; HMDHP-PP, hydroxymethyldihydropterin pyrophosphate; DHP, dihydropteroate; DHF, dihydrofolate; THF, tetrahydrofolate; THF-Glun, tetrahydrofolate polyglutamate. Enzyme abbreviations: ADCS, ADC synthase; ADCL, ADC lyase; GCHI, GTP cyclohydrolase I; DPP, dihydroneopterin triphosphate pyrophosphatase; DHNA, dihydroneopterin aldolase; HPPK, hydroxymethylpterin pyrophosphokinase; DHPS, dihydropteroate synthase; DHFS, dihydrofolate synthase; DHFR, dihydrofolate reductase; FPGS, folylpolyglutamyl synthase. The conversion of dihydroneopterin phosphate to dihydroneopterin is mediated by non-specific phosphatase activity. HPPK and DHPS activities reside on a bifunctional protein. DHFR activity also resides on a bifunctional protein, the other activity being thymidylate synthase. Besides being present in mitochondria as shown, FPGS occurs in extramitochondrial compartments. Molecular Plant 2010 3, 66-77DOI: (10.1093/mp/ssp057) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 Changes in Transcript Abundance of Folate Synthesis Genes during Fruit Development. Measurements were made at mature green, breaker, and red stages for cultivars Micro-Tom and Ailsa Craig, and also at the red-ripe stage for Micro-Tom. Data are levels of mRNA determined by qPCR using the 2–ΔCT quantification method and an actin mRNA as internal reference. Values are means and SE from three independent samples. Molecular Plant 2010 3, 66-77DOI: (10.1093/mp/ssp057) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Analysis of Folates in Wild-Type and Engineered Micro-Tom Fruit at the Red Stage. Fruit from three wild-type (WT) plants and from three plants of each of two engineered high-folate lines (TG1 and TG2) were analyzed for total folate content (A), for one-carbon substituents (B), and for the length of the polyglutamate tail of 5-methyltetrahydrofolate, the most abundant folate in fruit (C). Folate content data are means and SE; other data are expressed as percentages. THF, tetrahydrofolate + 5,10-methylenetetrahydrofolate; CH3-THF, 5-methyltetrahydrofolate; 5,10 = CH-THF, 5,10-methenyltetrahhydrofolate + 10-formyltetrahydrofolate; 5-CHO-THF, 5-formyltetrahydrofolate. Molecular Plant 2010 3, 66-77DOI: (10.1093/mp/ssp057) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Comparison of Transcript Abundance of Folate Biosynthesis Genes in Wild-Type and Engineered High-Folate Micro-Tom Fruit at the Red Stage. Measurements were made on fruit from three plants of each genotype. Data are levels of mRNA determined by qPCR using the 2–ΔCT quantification method and an actin mRNA as internal reference. Values are means and SE. Blue bars indicate significant (P < 0.05) increases in transcript abundance. Molecular Plant 2010 3, 66-77DOI: (10.1093/mp/ssp057) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 Expression of Folate Biosynthesis Genes in Single-Transgenic Pterin- or pABA-Overproducing Fruit. Wild-type (WT) Micro-Tom fruit and fruit overexpressing either ADC synthase alone (T11, T14) or GTP cyclohydrolase I alone (M67, M75) were analyzed for pterin content (A), pABA content (B), and the transcript levels of DHNA, ADCL1, and FPGSm (C–E). mRNA levels were quantified by qPCR using the 2–ΔCT method and an actin mRNA as internal reference. Values are means and SE for three independent samples. Different letters above bars indicate a significant difference at P < 0.05 according to Duncan's multiple range test. Molecular Plant 2010 3, 66-77DOI: (10.1093/mp/ssp057) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 Verification by qPCR of Expression Changes in Three Non-Folate Pathway Genes in Engineered High-Folate Micro-Tom Fruit at the Red Stage. Measurements were made on fruit from three plants each of wild-type (WT), and the folate-accumulating double transgenics TG1 and TG2. Data are levels of mRNA determined by qPCR using the 2–ΔCT quantification method and an actin mRNA as internal reference. Values are means and SE. Different letters above bars indicate a significant difference at P < 0.05 according to Duncan's multiple range test. GO, glycolate oxidase; CHS, chalcone synthase; DAHPS, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase. Molecular Plant 2010 3, 66-77DOI: (10.1093/mp/ssp057) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 7 Scheme Showing Possible Developmental and Feedforward Regulation of Folate Synthesis Gene Expression in Tomato Fruit. The developmentally regulated genes specifying the two enzymes at the head of the folate pathway (GCHI, ADCS) are in red. The three genes subject to possible feedforward regulation by pathway metabolites are in blue; feedforward loops are blue arrows, and the most likely effector metabolites are boxed in blue (in dotted lines where there is more than one). Molecular Plant 2010 3, 66-77DOI: (10.1093/mp/ssp057) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions