Supplemental Data. Majeran et al. Plant Cell. (2010) /tpc

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1 A B Leaf 3 Leaf 3 Tip of leaf 3 labeled with 14 CO C-labeled sugar in major veins is being transported basipetally A small amount of 14 C has moved from the phloem to the xylem and is being carried to the tip of the leaf in major veins. 1 Ligule leaf 2 Ligule leaf 3 (hidden behind coleoptile) Ligule leaf 2 Sink zone. 14 C is beginning to unload from veins into the bundle sheath and mesophyll. The remainder is exported to other sinks. Sink zone. 14 C from leaf 2, an older exporting leaf, has been imported by leaf 3 and is unloading. Ligule leaf 3 Suppl. Figure 1 (A) Representative seedling, with numbered leaf, (B) 14C labeling of maize leaves to show sink source-transition in the 3rd leaf selected for proteome and image analysis. The leaf on the left hand side was labeled from its tip, whereas the leaf of the right hand side was labeled through the older leaf 2. 1

2 A -1 cm; base 6-7 cm SE B BSC VP VP SE VP 7-8 cm 2-3 cm BSC VP Peroxisomes or plastids 4-5 cm xylem 8-9 cm BSC MC See B 8-9 cm E (tip-1) 5-6 cm BSC F Grana area/ MC c chloroplast ( ) C D # thylakoid / BSC C chloroplast (o) See C 5-6 cm Suppl. Fig Distance from base (cm) 2

3 Supplemental Figure 2. Structural analysis of the maize leaf along the developmental gradient (A) TEM images with transverse section of 8 leaf zones along the developmental leaf axis. In the section -1 cm, the oval indicates where plasmodesmata are visible (visible as black spots), whereas arrows indicate starch particles. A close-up of the boxed area in section 4-5 cm is shown in panel B. (B) TEM of a vascular bundle with annotation at 4-5 cm from the leaf base. This is a close-up of a section of the image shown in panel A and Figure 1F. VP = vascular parenchyma cell, CC companion cell; SE sieve element. The organelles with the crystalline structure are either peroxisomes with catalase crystals or non-photosynthetic plastids with crystalline structure of unknown content. (C) Close-up of plasmodesmata at 4-5 cm section (D) Close-up of vesicles (marked by the oval) along the chloroplast inner envelope. (E) Close-up of plastoglobules in BSC chloroplasts (indicated by an arrow) (F) Quantification of the average number of thylakoid membranes per BSC chloroplast (open symbols) and average cross-section of grana per MC chloroplast (closed symbols) along the leaf developmental axis. The errors bars (s.e.) are indicated. 3

4 A 1. PPDK 2. SUSY 3. Meth. Synth. 4. TKL 5. ME 6. RBCL 7. RBCS BS-S strand cm Mass in kda B N NAdjSPC nicotianamine synthase lectin SUT1 Xylem serine protease Distance from base (cm) Supplemental Figure 3. BS strand analysis and subclusters (A) 1D gel analysis of soluble (B) BS strand proteomes. Equal amounts of proteins from each selected section separated by 1D Tricine gels and stained to visualize the protein patterns. A number of abundant proteins could be recognized (after MS analysis) and are indicated. (B) Expression of vascular marker proteins in the BS strands along the leaf gradient. SUT1 was also observed in the total leaf sections (a solid line connecting the open circles). 4

5 A clustered Tim17/Tim22 I-1 B.4 anion-atpase II NS SAF * OEP24-II II-3b.4.2 SAMT1 I-2b.4.3 PAA1 II Distance from base (cm) Distance from base (cm) Supplemental Figure 4. Plastid envelope transporters involved in transport of copper (PAA1), S-adenosylmethionine (SAMT1) or unknown substrates (Tim17/22, anion-atpase, OEP24-II) passing the minimum threshold for clustering. (A) Relative molar abundance in the leaf calculated from the normalized spectral abundance factor, NSAF. Proteins in grey are in cluster I and protein in black in cluster II. (B) Expression of proteins along the developmental gradient in leaves (closed squares) and BS strands (open squares). The cluster number is indicated. 5

6 12 A B ACC1 porin2 DTC2.12 I-1 I-1 I All clustered NSAF * PHT3 I DTC1 NRT.4 I-1 I PIP1 I-1 K-channel.4 I porin1 I-1.6 porin3 I Distance from base (cm) Distance from base (cm) Supplemental Figure 5. Mitochondrial transporters passing the minimum threshold for clustering. (A) Relative molar abundance in the leaf calculated from the normalized spectral abundance factor, NSAF. (B) Expression of proteins along the developmental gradient in leaves (closed squares) and BS strands (open squares). The cluster number is indicated. 6

7 .8.7 ) NAdjSPC (X II-3c.2.1 II-1b Distance from base (cm) Supplemental Figure 6. Accumulation patterns of unusual LHC proteins, LIL3 (GRMZM2G477236_P1; filled square), ELIP1/2 (GRMZM2G355752_P1; open square), SEP4 (GRMZM2G162451_P1; filled circle) and OHP2- like (GRMZM2G143469_P1; open circle), along the leaf developmental gradient. Each of these proteins passed the threshold for cluster analysis. LIL3 and ELIP followed the same developmental expression pattern, peaking in the sink region before the 4 cm point, and decreasing gradually towards the tip and with consistent higher levels in the BS strands (cluster II-1b). In contrast, SEP4 and OHPs-like proteins had very similar expression patterns and were part of cluster II-3c, similar to PSII proteins and other MC chloroplast markers. 7

8 Supplemental Figure 7. The expression patterns of enzymes involved in N- assimilation. This diagram illustrates the incorporation of inorganic nitrogen into amino acids. The abbreviation and EC number are given for each catalytic step as well as transporters. The abbreviations are also included in Supplemental Data set 1C. The expression profiles along the leaf gradient are shown in line plots. The color of the enzyme name indicates the localization. Green, plastids. Blue, mitochondria. Red, cytosol. Black, unknown. the color of the lines in the plots indicates the total adjspc. Black, >2adjSPC. Gray, 5-2adjSPC. light gray, <5adjSPC. NR, nitrate reductase. NiR, nitrite reductase. GS, glutamine synthase. GOGAT, glutamate synthase. AspT, aspartate aminotransferase. NADP-MDH, NADP-malate dehydrogenase. NAD-MDH, NAD-malate dehydrogenase. GDH, glycine dehydrogenase. NAGS, N- acetylglutamate synthase. NAGK, N- acetylglutamate kinase. NAGPR, N-acetyl- gamma-glutamyl-phosphate reductase. NAOAT, N2-acetylornithine aminotransferase. NAOGAcT, N2-acetylornithine:glutamate acetyltransferase. CPS, carbamoyl phosphate synthetase. OTC, ornithine transcarbamylase. ASS, argininosuccinate synthase. AL, argininosuccinate lyase. NIRT, nitrate transporter. DiT1, 2-oxoglutarate/malate translocator 1. DiT2, 2-oxoglutarate/malate translocator 2. DTC, dicarboxylate-tricarboxylate carrier. Cit cytosol ASS N metabolism OTC ASS AL NT OTC CPS NAOGAcT NAOAT CP Orn NAO NAGSA NAGPR* NAGK NAGS DiT1 T DiT1 BS OMT1 T OMT1 BS DTC 1 T DTC 1 BS DTC 2 T DTC 2 BS NAG 5P NAG Glu AS plastid NAGPR NAGK Gln 2 OG DiT1 2 OG AL Arg Fd GOGAT NADH GOGAT NH 4 + GS GS2 DTC GDH OG GOGAT Malate Malate NiR GDH NO 3 _ NO 2 _ NIRT NO 2 _ Glu Glu NR NiR purine Asp AspT DiT2 Glu DTC Glu mitochondria NADP MDH NAD MDH AspAT mit AspAT cyt AtAAT T AtAAT BS ASP5 T ASP5 BS DiT2 OAA GS AA Gln NO 3 _ NH 4 + 8

9 Supplemental Figure 8. The expression patterns of enzymes involved in S-assimilation and the SAM cycle.this diagram illustrates the flow of sulfur from sulfate to cysteine and methionine. Biosynthesis of serine and glutathione, the SAM cycle and SMM cycle are also included. The color scheme is the same as in Supplemental Fig.7. ATPS, ATP sulfurylase. APR, APS reductase. SiR, sulfite reductase. OASTL, cysteine synthase/o-acetylserine (thiol)lyase. CGS, cystathionine gamma-synthase. CBL, cystathionine beta-lyase. MetS, methionine synthase. -ECS/GSH1, gamma-glutamylcysteine synthetase. GSHS, glutathione synthetase. GR, glutathione reductase. GPX, glutathione peroxidase. 3-PGDH, 3-PGA dehydrogenase. PSAT, 3-phosphoserine aminotransferase. PSP, 3- phosphoserine phosphatase. SAT, serine acetyltransferase. MAT, methionine adenosyltransferase. AdoHcyase, adenosylhomocysteinase. MMT, methionine S-methyltransferase. OASTL II 1 T OASTL II 1 BS OASTL II 3 T OASTL II 3 BS OASTL T OASTL BS SiR SO 3 2 S 2 SiR APR-3 APR OAS OASTL SO 4 2 SULTR SO 4 2 APS ATPS ATPS2 T ATPS2 BS ATPS3 BS SAT plastid PSP Ser PSP SO 4 2 SAM cycle methylated methyl acceptor AdoHcy H 2 O AdoHcyase AdoHcyase HOG11 adenosine HCys MS methyltetrahyd ropteroyltri L glutamate Cystathionine MTO1 T MTO1 BS CBL T CBL BS MS2 T MS2 BS CIMS T CIMS BS HCys CBL MS CGS Cys EC Glu ECS(GSH1) GSHS GSH1 Gly GR PSAT PGDH PSer 3 PHPyr 3 PGA GR NADP + NADPH + H + PSAT 3 PGDH T 3 PGDH BS EDA9 1 T EDA9 1 BS EDA9 2 T EDA9 2 BS calvin cycle glycolysis methyl acceptor AdoMet Pi + PPi MAT SAM1 1 T SAM1 1 BS SAM1 1 T SAM1 2 BS SAM2 T SAM2 BS H 2 O + ATP Met tetrahydropter oyltri L glutamate Met GSH H 2 O 2 GPX GPX2 2H 2 O GSSG MMT AdoMet MMT EC GSH cytosol HCys SMM AdoHcy SMM cycle 9

10 Nucleotide-sugar interconversion Plasma- membrane Cell Wa all UDP-L-Ara UXE UDP-D-Xyl UXS UDP-D-GlcAD GAE UDP-D-GalA UDP-D-Apiose INO Myo-inositol D-GlcA AXS GAK UDP-D-Xyl UXS UDP-D-GlcA UGD D-GlcA-1-P UDP-L-Rha 2NADH RHM/ UER UGD 2NAD+ NADP+ NADPH UGE UDP-D-Glc UGP PGM D-Glc-1-P Cytosol UDP-D-Gal 1,4- -D-glucan PPi UTP SUS CEL1 CESA lose Cellul Cellulose (Primary Wall) Cellulose (Secondary Wall) Endomembrane system Hemi cellulose Pectin Glycoprotein Glycosylation site Ascorbate NADP+ NADPH GDP-L-Fuc GER GMD GDP-L-Gal GDH GDP-D-Man GME PMM PGI D-Frc-6-P Sucrose PMI Photosynthesis s Glycolysis Plasma- membrane Ce ell Wall Supplemental Figure 9. The expression patterns of quantified enzymes in cellulose biosynthesis and nucleotide-sugar conversions. PGI, Phosphoglucose isomerase. PGM, Phosphoglucomutase. UGP, UDP-D-glucoseD pyrophosphorylase. RHM, UDP-L-rhamnose synthase. UER, dtdp-4-dehydrorhamnose reductase. UGE, UDP-glucose 4-epimerase. CEL1, endo-1,4-beta-glucanase. CESA, cellulose synthase. SUS, sucrose synthase. UGD, UDP-glucose 6-dehydrogenase. PMI, phosphomannose isomerase. PMM, Phosphomannomutase. GMD, GDP-mannose 4,6 dehydratase 2. GER, GDP-L-fucose synthase 1. GME, GDP-L-mannose epimerase. GDH, L-galactose dehydrogenase. UXS, UDP-glucuronic acid decarboxylase 1. AXS, UDP-D-APIOSE/UDP-D-XYLOSE SYNTHASE 2. GAK, D-glucuronokinase. INO, inositol oxygenase. UXE, UDP-Dxylose- 4 epimerase. GAE, UDP-D-glucoronic acid 4-epimerase. 1

11 Salicylic acid Flavonoid Suberin Cinnamic acid PAL Phe Chorismate Shikimate pathway C4H p-coumaric acid p-coumaroyl shikimic acid C3H HCT 4CL p-coumaraldehyde CCR p-coumaroyl -CoA CAD HCT CCoAOMT CCR CAD Caffeoyl shikimic acid Caffeoyl-CoA Feruloyl-CoA Coniferaldehyde Monolignol biosynthesis F5H 5-hydroxyconiferaldehyde COMT Sinapaldehyde CAD p-coumaryl alcohol Coniferyl alcohol Sinapyl alcohol Cytosol Syringyl lig gnin Guaiacyl lig gnin p-hydroxyp phenyl lignin Cell Wall Supplemental Figure 1. The expression patters of enzymes involved in lignin biosynthesis. PAL, phenylalanine ammonia-lyse. C4H, cinnamate 4-hydroxylase. 4CL, 4-coumarate-CoA ligase 1. HCT, p-hydroxycinnamoyl-coa. C3H, p-coumarate 3-hydroxylase. CCoAOMT, caffeoyl-coa O-methyltransferase. CCR, cinnamoyl-coa reductase. F5H, Ferulate-5-hydroxylase COMT, caffeic acid O-methyltransferase. CAD, cinnamyl alcohol dehydrogenase. 11

12 Supplemental Results N, S and amino acid metabolism shows distinct developmental and differentiation patterns Assimilation of nitrogen starts with uptake of nitrate (NO - 3 ) through the vascular system, followed by a 2-step reduction into ammonium (NH + 4 ) by cytosolic nitrate reductase (NR) leading to nitrite, and further reduction in the chloroplast by nitrite reductase (NiR) to ammonium (NH + 4 ). NH + 4 is also produced in the mitochondria by glycine dehydrogenase (GDH) during conversion of Glu to 2-oxoglutarate (Supplemental Figure 7) and during photorespiration through conversion of Gly to Ser by Gly decarboxylase (GDC). The NH + 4 is primarily used in the synthesis of Gln in the glutamine synthase 2/glutamate synthase (GS2/GOGAT) cycle and in the synthesis of Arg (Supplemental Figure 7) in plastids. A second GS (GS1) is non-plastidic and belonged to cluster II-1. Glu is used to generate Asp, catalyzed by the aspartate aminotransferase (AAT), represented by enzymes in different subcellular compartments, with the most abundant AAT in the plastid. NiR and Fd-GOGAT belonged to cluster II-3c, whereas GS2 and AAT belonged to cluster II-3d, indicative of increase in expression from base to tip, following the same expression pattern as the build-up of the photosynthetic light reaction. NiR and Fd- GOGAT were preferentially expressed in MSC indicative of the tight link to linear electron transport, whereas AAT clustered with MSC-enriched Calvin cycle enzymes and C4 malate shuttle enzymes PPDK and PEPC (II-3d). The expression levels of the Dit1 and Dit2 transporters were relatively low, but were most abundant in the tip. GDH was only observed in the BS strands and showed the same induction kinetics as the photorespiratory markers, GOX and 2PGP discussed above (Figure 1). In contrast, the two mitochondrial 2-oxoglutarate/malate transporters (DTC1,2) showed expression highest at the base (in cluster I-1) (Supplemental Figure 5B). However, the additional BS strand enrichment of DTC2 in the leaf tip, suggests a key function of DTC2 in the photorespiratory pathway late in leaf development (Supplemental -1 Figures 5,7). NRT1, a putative transporter of NO 3 (GRMZM2G86496_P1) in cluster I-1 showing pronounced peak expression ~2.5 cm from the base (Supplemental Figure 5). Upon uptake of sulfur as sulfate (SO 2-4 ) through the vascular system, SO 2-4 is imported into the chloroplast and first activated by ATP sulfurylase (ATPS) to adenosine 5 -phoshosulfate (APS) and then reduced in 2 steps by APS reductase (APR) and sulfite reductase (SiR) leading to sulfide (S 2- ) (Supplemental Figure 8). The envelope sulfate transporter (SULTR) (GRMZM2G395114_P1) with 1 predicted transmembrane domains was exclusively identified in the BS strands near the leaf tip. ATPS was part of cluster II-3e, showing near exclusive 12

13 Supplemental Results expression in the BS strand and clustering with the majority of the Calvin cycle enzymes and photosynthetic cyclic electron flow. Consistently, APR and SiR in the BS strand followed the same expression pattern as ATPS. Surprisingly, SiR showed also a high expression in the very base of the leaf, suggesting an additional role of SiR in the base of the leaf; possibly for sulfite reduction using OPPP as electron source. Sulfide is converted into cysteine, which is used for protein synthesis, to generate Met or the abundant redox regulator glutathione (see previous section Redox regulation and ROS defense ). Synthesis of Cys, Met and glutathione were distributed across multiple subcellular localizations. We identified three homologues of plastid localized cysteine synthase (OASTL); the two most abundant clustered to II-1a,b. Methionine is used in the SAM cycle to provide methyl groups (C1 metabolism) for various metabolic pathways, with a particular high demand for synthesis of nucleic acids and abundant structural components of the cell wall (eg lignin, choline, pectin) and chlorophyll. We quantified all enzymes of the SAM cycle (Supplemental Figure 8), as well as the inner envelope membrane transporter SAMT1 (Supplemental Figure 4B) which transports SAM from the cytosol into the plastid. Impaired function of SAMT1 led to decreased accumulation of prenyllipids and mainly affected the chlorophyll pathway in tobacco {Bouvier, 26 #12783}. SAM cycle proteins were members of cluster I-1 or I-2a,b, gradually decreasing along the developmental axis. Methionine synthase 1 was by far the most abundant protein. The high expression in the sink zone is consistent with high rates of synthesis of structural components such as cell walls and chlorophyll. Gly and Ser are synthesized by the plastid-localized phosphorylated pathway or by the mitochondrial glycolate pathway as part of photorespiration {Ho, 21 #12966}Bourgulgnon et al., 1999). The phosphorylated pathway utilizes 3-PGA (from glycolysis or Calvin cycle) to make Ser, which is then converted to Gly by the enzyme serine hydroxymethyltransferase (SHMT). We identified all three enzymes in the phosphorylated pathway for Ser biosynthesis, 3- PGA dehydrogenase (3-PGDH), 3-phosphoserine aminotransferase (PSAT) and 3-phosphoserine phosphatase (PSP) (Supplemental Figure 8). The two most abundant isoforms of 3-PGDH and PSAT fell in cluster I-1, I-2a and I-2b, respectively. Both 3-PGDH and PSAT show decreased protein level towards the leaf tip, implying they may be more crucial for Ser biosynthesis in the sink tissue. Interestingly, the third enzyme PSP was only found in BS strands, with increased accumulation towards the tip, opposite to the trends of 3-PGDH and PSAT. The reason is unclear 13

14 Supplemental Results but it is consistent with transcript profiling for these three genes in Arabidopsis {Ho, 1999 #2497}. In photosynthetic tissues, Ser and Gly are predominantly metabolized in the peroxisomes and mitochondria as part of photorespiration. Three enzymes, GGAT, GDC and SHM1,4 (three isoforms) are responsible for Ser and Gly metabolism. GGAT level increased from sink to source tissue, as expected for enzymes involved in photorespiration. The level of the most abundant GDC isozyme increased from base to tip in BS strands, but the levels in total leaf and of other isozymes peaked at the transition zone. Similarly, SHMT level is highest in transition zone except for one of the isoforms (GRMZM2G135283_P2), which increased from base to tip in the BS strand. Since SHMT participate in both Ser biosynthesis pathways, it is not surprising that the expression profile of SHMT does not follow that of the other photorespiratory enzymes. We identified 28 enzymes in the plastid-localized biosynthesis of aromatic amino acids (Phe, Tyr, Trp) which included six out of seven enzymatic steps (1 proteins) in chorismate synthesis. Biosynthesis starts with the condensation of erythrose 4-phosphate (from OPPP/Calvin) and PEP, followed by the conversion of shikamate into chorismate, the branch point of Trp synthesis from Tyr and Phe synthesis. DHAP synthase, the first step was by far the most abundant step in chorismate synthesis and belonged to cluster I-1 (high expression in the base). Subsequent steps in chorismate synthesis were enriched in BS strands and increased from base to tip, probably because shikimate and chorismate are respectively important precursors for phenylpropapoid biosynthesis (for lignin) and vitamin K1 (phylloquinone) in PSI. Synthesis of the aromatic biosynthetic enzymes after chorismate decreased from base to tip. We identified four enzymes of the linear His pathway, albeit at relatively low levels, with expression levels decreasing from base to tip. Ala is synthesized from pyruvate and Glu outside the plastid by Ala aminotransferase and several isoforms belonged to clusters I-2b and II-1a, peaking quite broadly around the sink-source transition zone, with modest enrichment in the BS strands. We identified all enzymes involved in branched amino (Val, Leu and Ile) biosynthesis except one (dihydroxyacid dehydratase). Most of them belonged to cluster I, some to cluster II-1 and II-2, with highest expression level at the base or towards the transition zone. This is consistent with previous findings that this pathway is most active in young developing tissues (Keeler et al., 1993; Hofgen et al., 1995; Singh and Shaner, 1995). 14