Membrane-associated proteomics of chickpea identifies Sad1/UNC-84 protein (CaSUN1), a novel component of dehydration signaling

Transcription

1 Membrane-associated proteomics of chickpea identifies Sad/UNC-84 (CaSUN), a novel component of dehydration signaling Dinesh Kumar Jaiswal, Poonam Mishra, Pratigya Subba, Divya Rathi, Subhra Chakraborty* and Niranjan Chakraborty National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-0067, India. Correspondence: Dr. Niranjan Chakraborty National Institute of Plant Genome Research Aruna Asaf Ali Marg, New Delhi-0067, India. nchakraborty@nipgr.ac.in Tel: Fax: *Correspondence: Dr. Subhra Chakraborty National Institute of Plant Genome Research Aruna Asaf Ali Marg, New Delhi-0067, India. Subhrac@hotmail.com Tel: Fax:

2 Dehydration (h) U Dehydration treatment and tissue collection Extraction of membrane s 2-DE analysis Dehydration-res ponsive membrane proteome Pre-processing of data and statistical analyses In-gel trypsin digestion and tandem mass spectrometry Protein identification Functional characterization of a novel dehydration-responsive Supplementary Fig. S. Experimental work flow used for analyzing the membrane proteome from the aerial tissue of chickpea and downstream analyses. The detailed experimental procedure is illustrated in SI Materials methods section. U represents the unstressed condition.

3 a b Unstressed Dehydration treatment (h) Supplementary Fig. S2. Dehydration-responsive comparative proteome of chickpea membrane and the representative 2-D gels. Three-week-old chickpea seedlings were subjected to dehydration and tissue were harvested every 24 h until 20 h. The membrane s were isolated from the unstressed and stressed tissue from each time point and 2-DE was performed. Three replicate gels for each time point (a) were computationally combined using PDQuest (version 7.2.0) software to generate the standard gels (b).

4 Relative fold change Relative fold change Relative fold change Relative fold change a U Dehydration time (h) b U Dehydration time (h) c U Dehydration time (h) d U 24 h 48 h 72 h 96 h 20 h Dehydration time (h) Supplementary Fig. S3. Relative abundance of few of the redundant DRPs. The expression profiles of the following s are represented. (a) Putative 60S acidic ribosomal P0, (b) Oxygen-evolving enhancer, (c) chlorophyll a/b binding type III, and (d) fructose-bisphosphate aldolase. The y-axis represents the fold change in expression of the across all the time points. The background value averaged across the replicates for a time point was used to calculate the fold changes. U represents unstressed condition.

5 U 24 h 48 h 72 h 96 h 20 h a -5:Trypsin inhibitor, chain B (fragments) -38:Chromosome chr7 scaffold_6-207: ATP synthase subunit alpha -208:Putative 60S acidic ribosomal P0-24:Glutamate-ammonia ligase (EC ) delta precursor -222:ATP synthase subunit beta -294:Putative PSII-P -46:Unknown 8-85:Calcineurin-like phosphoesterase family -99:Chaperone DnaK -240:40S ribosomal S2-263:OSJNBb0085H. -283:40S ribosomal S2-46:Glyceraldehyde-3-phosphate dehydrogenase -485:Carbonic anhydrase -39:Putative 60S ribosomal L -43:Ribulose bisphosphate carboxylase large chain -64:Hypothetical P045D :Chlorophyll a/b binding -94:Chaperonin 60 alpha subunit -47:ATP synthase subunit beta, chloroplastic -50:Chaperonin 60 alpha subunit -6:ATP synthase beta subunit (Fragment) -96:Oxygen-evolving enhancer -254:ATP synthase gamma chain -295:ATP synthase subunit alpha -30:Fructose-bisphosphate aldolase (EC ) precursor -3:ATP synthase CF alpha subunit -386:ATP synthase subunit alpha -26:Glutamate-ammonia ligase (EC ) delta precursor -54:Activator of spomin -80:ATP synthase beta subunit (Fragment) -4:Phosphoglycerate kinase -89:ATP synthase subunit alpha -205:ATP synthase subunit beta -209:Putative 60S acidic ribosomal P0-27:PSI type III chlorophyll a/b-binding (imported) -246:Putative serine/threonine kinase -247:Membrane CH-like -452:Ferredoxin: nadp+ reductase -9:Putative uncharacterized -25:Oxygen-evolving enhancer -55:Dynein--alpha heavy chain -62:ATP synthase beta subunit (Fragment) -22:ATP synthase beta subunit (Fragment) -223:Putative glutamate-trna ligase -257:Plastidic aldolase -323:Putative uncharacterized -346:Putative uncharacterized -376:Ferredoxin-NADP reductase -460:Glyceraldehyde 3-phosphate dehydrogenase -487:Guanine nucleotide-binding subunit beta-like -267:Hypothetical F26P :Histone acetyltransferase GCN5-435:Carbonic anhydrase -436:Putative quinone oxidoreductase -443:Predicted -20:Fructose-bisphosphate aldolase -56:Hypothetical OSJNBa0075N :Putative uncharacterized -83:Oxygen-evolving enhancer 2-28:Glutamine synthetase -269:ATP synthase subunit alpha -30:Proteasome subunit alpha type -39:Putative ferredoxin-nadp(h) oxidoreductase -322:Phosphoglycerate kinase -335:ATP synthase subunit alpha -407:Mitochondrial processing peptidase beta subunit -428:Beta-,3-glucanase -462:Glyceraldehyde-3-phosphate dehydrogenase A -7:light-harvesting complex II Lhcb3-73:Actin (Fragment) -57:Putative GroEL chaperonin 60kDa -7:Unknown -9:Ferritin -206:Phosphoribulokinase -27:H+-transporting two-sector ATPase-alpha chain -272:ATP synthase CF alpha subunit -36:ATP synthase subunit alpha -382:ATP synthase subunit alpha -396:ATP synthase subunit alpha -445:Putative uncharacterized -483:Luminal binding, BiP -2:Chlorophyll a/b-binding AB96-22:Chlorophyll a/b-binding 3 precursor -24:Putative uncharacterized -69:Chlorophyll A-B binding -74:Light-harvesting chlorophyll-a/b binding Lhcb -75:Chlorophyll a/b binding -22:Ribulose bisphosphate carboxylase large chain -82:Ferritin -87:Chlorophyll a/b binding -226:ATPase beta subunit (Fragment) -290:Intronic ORF; similar to LAGLIDADG endonuclease -308:Fructose-bisphosphate aldolase Cluster Cluster 2 Cluster 3 Cluster 4 Cluster 5 Cluster 6 Cluster 7 Cluster 8 Cluster 9 Cluster 0 Cluster b Supplementary Fig. S4. Hierarchical expression clustering of the identified DRPs in the membrane fraction of chickpea. Based on the expression profile, the DRPs were grouped into clusters. The SOTA cluster tree (heat map) is shown at the top (a), and the expression graphs are shown below (b). Each DRP is represented by a single row of colored box and each time point by a single column. In the expression graph, the number of s in each cluster is given in the left upper corner and the cluster number is given in the right lower corner. U represents unstressed condition.

6 Group CaSUN NCSD ScSLP NCSD ScMPS3 CCSD SpSAD CCSD SpSUNLIKE NCSD Atg22882 AtSUN NCSD CCSD Pps9_3V6 NCSD Pps3_89V6 CCSD O sgi CCSD O sgi NCSD Zmgi CCSD Zmgi NCSD Glyma5g35240 CCSD Gm09g00440 NCSD CeUNC84 CeR2E2 CCSD NCSD DmFBpp CCSD DmFBpp NCSD Hsgi CCSD Hsgi NCSD Supplementary Fig. S5. Analysis of plant and non-plant Sad/UNC-84 (SUN) domain family. In silico analysis of putative SUN domain s from different organisms showing Sad/UNC-84 (SUN) domain (InterProScan), transmembrane domain (TMPred), coiled-coil domain (Coils), galactose binding domain (InterProScan) and N-terminal signal peptide (SignalP). CCSD and NCSD denotes Canonical C-terminal SUN Domain and Non-Canonical SUN Domain, respectively.

7 ScMps3 ScSLP SpSad SpSUNLike Pps3_89V6 Pps9_3V6 Sm8985 Sm44228 AtSUN AtSUN3 CaSUN Glyma5g35240 Gm09g00440 Osgi Osgi Zmgi Zmgi DmFBpp DmFBpp CeUNC84 CeR2E2 Hsgi Hsgi Supplementary Fig. S6. Genomic organization of SUN gene family. A diagrammatic representation of the gene structures of SUN gene family of different taxa. The diagrams were drawn using SPIDEY program gov/spidey/ on the basis of alignments of cdna to genomic DNA sequences. Exons are represented by filled boxes, while introns are represented by diagonal lines.

8 a AtSUN3 Atg22882 LB TDNA b M RP Wild type Homozygous RP-LP RP-LB RP-LP RP-LB LP.6 kb.0 kb c Wt Mt AtSUN3 (.98 kb) Act Supplementary Fig. S7. Identification of homozygous AtSUN3 T-DNA mutants. a. Schematic representation of the AtSUN3 allele, with the T-DNA insertion shown as inverted triangle. Filled boxes indicate coding regions. b. Genotyping of wild type and AtSUN3 mutant. c. RT-PCR analysis for the presence of full-length AtSUN3 transcript. M: kb DNA ladder, RP and LP: gene specific right primer and left primer, LB: T-DNA left border primer, Wt: wild type, Mt: AtSUN3 mutant. Primers used in genotyping PCR and RT-PCR are listed in Supplementary Table S9.

9 Supplementary Table S. Reproducibility of two-dimensional gels. Time (h) Average No of spots a High quality spots b Reproducibility (%) Control Total a Average number of spots present in three replicate gels of each time points b Spots having a quality score more than 30 assigned by PDQuest (version 7.2.0)

10 Supplementary Table S2. List of dehydration-responsive membrane s identified by MS/MS analysis. Spot No a Identification gi No b Score NP c Covera % ge The MW/ pi Exp MW/ pi Time Kinetics d U GO annotation -246! Putative serine/threonine kinase / / 5.30 plastid -85! Calcineurin-like phosphoesterase family / / 4.72 peroxisome, hydrolase activity -487* Guanine nucleotide-binding subunit beta-like / /6.89 binding -247 Membrane CH-like / / 5.45 mitochondrion, membrane -299! Histone acetyltransferase GCN / / 5.63 flower development, transferase activity, biosynthetic process, carbohydrate metabolic process, cellular process, secondary metabolic process, response to extracellular stimulus, cell communication, response to stress, response to endogenous stimulus, binding, nucleoplasm, biological_process, anatomical structure morphogenesis, multicellular organismal development, DNA binding, response to abiotic stimulus, cellular component organization, cellular modification process, sequence-specific DNA binding transcription factor activity

11 -428* Beta-,3- glucanase / / 6.75 carbohydrate metabolic process, hydrolase activity, binding -483* Luminal binding, BiP / / 6.9 nucleotide binding -82* Ferritin / / 5.2 cellular homeostasis, catalytic activity, binding, transport, metabolic process -5! Trypsin inhibitor, chain B (fragments) / / 5.07 enzyme regulator activity, biological_process -9* Ferritin / / 5.23 cellular homeostasis, catalytic activity, binding, transport, metabolic process -27! H+-transporting two-sector ATPase-alpha chain / / 5.38 membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, transporter activity, mitochondrion, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process -94* Chaperonin 60 alpha subunit / / 4.7 metabolic process, cellular process, cytoplasm, binding, nucleotide binding

12 -99* Chaperone DnaK / / 4.69 metabolic process, cellular process, binding, nucleotide binding -57* Putative GroEL chaperonin 60kDa / / 5.00 metabolic process, cellular process, cytoplasm, binding, nucleotide binding -50! Chaperonin 60 alpha subunit / / 5.2 metabolic process, cellular process, cytoplasm, nucleotide bindin -73* Actin (Fragment) / /4.75 N/F -39* Putative 60S ribosomal L / / 4.29 ribosome, structural molecule activity, translation -240* 40S ribosomal S / /5.37 ribosome, structural molecule activity, translation -283* 40S ribosomal S / / ribosome, structural molecule activity, translation -223! Putative glutamate-trna ligase / / 5.23 ATP binding, cytoplasm, glutamate-trna ligase activity, glutamyl-trna aminoacylation

13 -208* Putative 60S acidic ribosomal P / / 5.0 ribosome, structural molecule activity, cellular process, translation -209* Putative 60S acidic ribosomal P / / 5.2 ribosome, structural molecule activity, cellular process, translation -407! Mitochondrial processing peptidase beta subunit / / 6.03 hydrolase activity, metabolic process, catabolic process, binding -30* Proteasome subunit alpha type / / 5.8 cytoplasm, intracellular, metabolic process, catabolic process, cellular process, nucleus, hydrolase activity -2! Chlorophyll a/bbinding AB / / 4.58 binding, generation of precursor metabolites and energy, photosynthesis, membrane, thylakoid, cellular modification process, plastid -87* Chlorophyll a/b binding / /5.0 membrane, generation of precursor metabolites and energy, photosynthesis, plastid -75* Chlorophyll a/b binding / / 4.78 membrane, generation of precursor metabolites and energy, photosynthesis, plastid -96* Oxygen-evolving enhancer / / 5.9 membrane, binding, generation of precursor metabolites and energy, photosynthesis, plastid, thylakoid

14 -294* Putative PSII-P / / 5.59 membrane, thylakoid, binding, photosynthesis -7* light-harvesting complex II Lhcb / / 4.77 membrane, generation of precursor metabolites and energy, photosynthesis, plastid -78* Chlorophyll a/b binding / / 4.77 membrane, generation of precursor metabolites and energy, photosynthesis, plastid -22! Chlorophyll a/bbinding 3 precursor / / 4.47 binding, generation of precursor metabolites and energy, photosynthesis, membrane, thylakoid, cellular modification process, plastid -69! Chlorophyll A-B binding / / 4.62 membrane, generation of precursor metabolites and energy, photosynthesis, plastid -27! PSI type III chlorophyll a/bbinding (imported) / / 4.98 plastid, generation of precursor metabolites and energy, photosynthesis, intracellular, binding, membrane, thylakoid -25! Oxygen-evolving enhancer / / 4.87 membrane, binding, generation of precursor metabolites and energy, photosynthesis, plastid, thylakoid -83! Oxygen-evolving enhancer / / 5.6 membrane, binding, photosynthesis, plastid, thylakoid

15 -376* Ferredoxin-NADP reductase / /6. 2 plastid -74! Light-harvesting chlorophyll-a/b binding Lhcb / / 4.93 binding, generation of precursor metabolites and energy, photosynthesis, membrane, thylakoid, cellular modification process, plastid -39! Putative ferredoxin- NADP(H) oxidoreductase / / 5.92 plastid -452 Ferredoxin: nadp+ reductase / / 6.49 nucleotide binding, binding, transport, plastid, catalytic activity, photosynthesis, membrane, thylakoid, generation of precursor metabolites and energy -226! ATPase beta subunit (Fragment) / / 5.2 membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, hydrolase activity, transporter activity, nucleotide binding, cellular process -47* -89* ATP synthase subunit beta, chloroplastic ATP synthase subunit alpha / / / / 5.6 membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, hydrolase activity, transporter activity, nucleotide binding, plastid, thylakoid, cellular process membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, transporter activity, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process

16 -22! -6! -80! -205* -62! -207* -222* -295* ATP synthase beta subunit (Fragment) ATP synthase beta subunit (Fragment) ATP synthase beta subunit (Fragment) ATP synthase subunit beta ATP synthase beta subunit (Fragment) ATP synthase subunit alpha ATP synthase subunit beta ATP synthase subunit alpha / / / / / / / / / / / / / / / / 5.76 membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, hydrolase activity, transporter activity, nucleotide binding, plastid, thylakoid, cellular process membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, hydrolase activity, transporter activity, nucleotide binding, plastid, thylakoid, cellular process membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, hydrolase activity, transporter activity, nucleotide binding, plastid, thylakoid, cellular process hydrolase activity, transporter activity, transport, cellular process, biosynthetic process, generation of precursor metabolites and energy, nucleobase-containing compound metabolic process, membrane, mitochondrion, catabolic process, nucleotide binding membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, hydrolase activity, transporter activity, plastid, nucleotide binding, cellular process membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, transporter activity, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, hydrolase activity, transporter activity, nucleotide binding, plastid, thylakoid, cellular process membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, transporter activity, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process

17 -36* -335* -382* -386* ATP synthase subunit alpha ATP synthase subunit alpha ATP synthase subunit alpha ATP synthase subunit alpha / / / / / / / / 6.8 membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, transporter activity, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, transporter activity, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, transporter activity, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, transporter activity, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process -254* ATP synthase gamma chain / / 5.48 membrane, intracellular, transporter activity, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobase-containing compound metabolic process, nucleotide binding, hydrolase activity -269* ATP synthase subunit alpha / / 5.34 membrane, intracellular, transporter activity, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobase-containing compound metabolic process, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process -3* ATP synthase CF alpha subunit / /5.89 ATP binding, ATP hydrolysis coupled proton transport, ATP synthesis coupled proton transport

18 -396* -272! ATP synthase subunit alpha ATP synthase CF alpha subunit / / / / 5.5 membrane, intracellular, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobasecontaining compound metabolic process, transporter activity, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process membrane, intracellular, transporter activity, transport, biosynthetic process, generation of precursor metabolites and energy, nucleobase-containing compound metabolic process, nucleotide binding, hydrolase activity, plastid, thylakoid, cellular process -28* Glutamine synthetase / / 5.32 catalytic activity, nucleotide binding, biosynthetic process, -308* Fructosebisphosphate aldolase / / 5.62 carbohydrate metabolic process, generation of precursor metabolites and energy, catabolic process, catalytic activity, plastid -30 Fructosebisphosphate aldolase (EC ) precursor / / 5.86 carbohydrate metabolic process, generation of precursor metabolites and energy, catabolic process, catalytic activity, plastid -46* Glyceraldehyde-3- phosphate dehydrogenase / /6.75 cytoplasm, nucleotide binding, carbohydrate metabolic process, generation of precursor metabolites and energy, catabolic process, catalytic activity, metabolic process -4* Phosphoglycerate kinase / / 4.90 phosphoglycerate kinase activity, glycolysis -322* Phosphoglycerate kinase / / 5.64 phosphoglycerate kinase activity, glycolysis

19 -257 Plastidic aldolase / / 5.6 carbohydrate metabolic process, generation of precursor metabolites and energy, catabolic process, catalytic activity -485* Carbonic anhydrase / / 6.95 binding, catalytic activity, metabolic process -20* Fructosebisphosphate aldolase / / 5.35 carbohydrate metabolic process, generation of precursor metabolites and energy, catabolic process, catalytic activity -460! Glyceraldehyde 3- phosphate dehydrogenase / / 6.66 catalytic activity, metabolic process -26 Glutamateammonia ligase (EC ) delta precursor / / 5.3 catalytic activity, metabolic process, nucleotide binding, plastid, biosynthetic process, 0-24 Glutamateammonia ligase (EC ) delta precursor / / 5.23 catalytic activity, metabolic process, nucleotide binding, plastid, biosynthetic process, 0-206! Phosphoribulokina se / / 5.8 carbohydrate metabolic process, kinase activity, nucleotide binding, metabolic process, cellular process -435* Carbonic anhydrase / / 6.47 binding, catalytic activity, metabolic process

20 -462* Glyceraldehyde-3- phosphate dehydrogenase A / / 6.8 biosynthetic process, carbohydrate metabolic process, photosynthesis, nucleotide binding, cellular process, plastid, metabolic process, catalytic activity -55! Dynein--alpha heavy chain / / 5. motor activity, cytoskeleton, cellular component organization, cellular process, intracellular, nucleobasecontaining compound metabolic process, catabolic process, nucleotide binding, cell, hydrolase activity -436* Putative quinone oxidoreductase / / 6.62 nucleotide binding, metabolic process, catalytic activity -54! Activator of spomin / / 4.69 sequence-specific DNA binding transcription factor activity, DNA binding, -38* Chromosome chr7 scaffold_ / / 4.97 ATP binding, binding, nucleoside-triphosphatase activity, metabolic process -43* Ribulose bisphosphate carboxylase large chain / / 4.29 catalytic activity, plastid, photosynthesis, binding, metabolic process -22* Ribulose bisphosphate carboxylase large chain / / 5.05 catalytic activity, plastid, photosynthesis, binding, metabolic process

21 -290! Intronic ORF; similar to LAGLIDADG endonuclease / / 6.00 DNA metabolic process, nuclease activity, DNA binding, mitochondrion -443* Predicted / / 6.64 transferase activity -46* Unknown / / 6.44 nucleotide binding, generation of precursor metabolites and energy, catabolic process, catalytic activity, molecular_function -263 OSJNBb0085H / / 5.6 RNA binding, RNA-directed DNA polymerase activity, RNA-dependent DNA replication, DNA integration, ribonuclease H activity -9* Putative uncharacterized / /4. 2 ribosome, structural molecule activity, translation -346* Putative uncharacterized / /5.75 transferase activity, metabolic process -24* Putative uncharacterized / / 4.47 ribosome, structural molecule activity, translation -323* Putative uncharacterized / / 5.66 nucleotide binding

22 -2* Putative uncharacterized / /4. 72 transport, membrane -445* Putative uncharacterized / / 6.5 transferase activity -7! Unknown / / 5. plastid -64! Hypothetical P045D / / 4.77 N/F -267! Hypothetical F26P / / 5.58 peptidyl-prolyl cis-trans isomerase activity, folding -56! Hypothetical OSJNBa0075N / / 4.49 N/F a Spot number as marked on 2-D gel images (Fig. a). The spot numbers were designated as -X, where Ca indicates the organism (Cicer arietinum), M denotes the membrane fraction, and X denotes the spot number as given on 2-D gel image. b Protein identification number as in GenBank. c NP represent the number of peptide.

23 d Time kinetics represents the average change in spot density at various time points (Unstressed to 20 h). The data were taken in terms of fold expression with respect to the control value and were log transformed to the base two in order to level the scale of expression. The spot volume of each spot during the course of dehydration is given in Table S3. Spots marked by * were identified by Q-TRAP, identified by QStar, and! identified by MALDI analysis.

24 Supplementary Table S3. Average normalized spot volume, standard deviation (SD) and coefficient of variance (CV) of identified spots and corresponding log transformed data are given below. Untransformed data Dehydration (h) Log transformed data Dehydration (h) Spot ID Identificatio n Putative uncharacteriz ed Chlorophyll a/b-binding AB96 Chlorophyll a/b-binding 3 precursor Putative uncharacteriz ed Putative 60S ribosomal L Ribulose bisphosphate carboxylase large chain Activator of spomin Hypothetical OSJNBa007 5N02.48 Hypothetical P045D Chlorophyll A-B binding lightharvesting complex II Lhcb3 Actin (Fragment) Lightharvesting chlorophylla/b binding Lhcb Chlorophyll a/b binding Chlorophyll a/b binding ATP synthase beta subunit (Fragment) Calcineurinlike phosphoester ase family Chaperonin 60 alpha subunit Chaperone DnaK Putative uncharacteriz ed Trypsin inhibitor, chain B (fragments) Ribulose bisphosphate carboxylase large chain Oxygenevolving enhancer PSI type III chlorophyll Av Norm Qty Unstressed Unstressed SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV % Av Norm Qty SD CV %

25 a/b-binding (imported) Chromosome chr7 scaffold_6 Phosphoglyc erate kinase ATP synthase subunit beta, chloroplastic Chaperonin 60 alpha subunit Dynein-- alpha heavy chain Putative GroEL chaperonin 60kDa ATP synthase beta subunit (Fragment) ATP synthase beta subunit (Fragment) Unknown Ferritin Oxygenevolving enhancer 2 Chlorophyll a/b binding ATP synthase subunit alpha Ferritin Oxygenevolving enhancer ATP synthase subunit beta Phosphoribul okinase ATP synthase subunit alpha Putative 60S acidic ribosomal P0 Putative 60S acidic ribosomal P0 Fructosebisphosphate aldolase Glutamateammonia ligase (EC ) delta precursor Glutamateammonia ligase (EC ) delta precursor Glutamine synthetase ATP synthase beta subunit (Fragment) ATP synthase subunit beta Putative glutamatetrna ligase ATPase beta subunit (Fragment)

26 S ribosomal S2 Putative serine/threon ine kinase Membrane CH- like ATP synthase gamma chain Plastidic aldolase OSJNBb008 5H. Hypothetical F26P2.80 ATP synthase subunit alpha H+transporting two-sector ATPasealpha chain ATP synthase CF alpha subunit 40S ribosomal S2 Intronic ORF; similar to LAGLIDAD G endonuclease Putative PSII-P ATP synthase subunit alpha Histone acetyltransfe rase GCN5 Proteasome subunit alpha type Fructosebisphosphate aldolase Fructosebisphosphate aldolase (EC ) precursor ATP synthase CF alpha subunit ATP synthase subunit alpha Putative ferredoxin- NADP(H) oxidoreducta se Phosphoglyc erate kinase Putative uncharacteriz ed ATP synthase subunit alpha Putative uncharacteriz ed Ferredoxin- NADP reductase ATP synthase subunit alpha ATP synthase subunit alpha ATP synthase subunit alpha Mitochondri

27 -407 al processing peptidase beta subunit Unknown Beta-, glucanase Carbonic anhydrase Putative quinone oxidoreducta se Predicted Putative uncharacteriz ed Ferredoxin: nadp+ reductase Glyceraldehy de 3- phosphate dehydrogena se Glyceraldehy de-3- phosphate dehydrogena se Glyceraldehy de-3- phosphate dehydrogena se A Luminal binding, BiP Carbonic anhydrase Guanine nucleotidebinding subunit betalike

28 Supplementary Table S4. One-way ANOVA of the data was done using MeV (Multi Experiment Viewer) software to determine the statistically significant (p< 0.05) differentially regulated spots. Spot ID Unstressed 24 h 48 h 72 h 96 h 20 h Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD F ratio E-06 Yes Putative uncharacterized SS Group SS Error df(groups) df (error) p value Are means signif. different? (P < 0.05) Identification E-07 Yes Chlorophyll a/bbinding AB E-06 Yes Chlorophyll a/bbinding 3 precursor E-6 Yes Putative uncharacterized E-6 Yes Putative 60S ribosomal L E-6 Yes Ribulose bisphosphate carboxylase large chain Yes Activator of spomin E-6 Yes Hypothetical OSJNBa0075N E-5 Yes Hypothetical P045D E-05 Yes Chlorophyll A-B binding E-06 Yes light-harvesting complex II Lhcb E-05 Yes Actin (Fragment) E-08 Yes Light-harvesting chlorophyll-a/b binding Lhcb E-06 Yes Chlorophyll a/b binding E-05 Yes Chlorophyll a/b binding E-6 Yes ATP synthase beta subunit (Fragment) E-4 Yes Calcineurin-like phosphoesterase family E-05 Yes Chaperonin 60 alpha subunit E- Yes Chaperone DnaK Yes Putative uncharacterized E-4 Yes Trypsin inhibitor, chain B (fragments) E-3 Yes Ribulose bisphosphate carboxylase large chain

29 E-07 Yes Oxygen-evolving enhancer E-05 Yes PSI type III chlorophyll a/bbinding (imported) E-5 Yes Chromosome chr7 scaffold_ E-6 Yes Phosphoglycerate kinase Yes ATP synthase subunit beta, chloroplastic E-4 Yes Chaperonin 60 alpha subunit Yes Dynein--alpha heavy chain E-4 Yes Putative GroEL chaperonin 60kDa Yes ATP synthase beta subunit (Fragment) E-06 Yes ATP synthase beta subunit (Fragment) E-2 Yes Unknown E-05 Yes Ferritin E-5 Yes Oxygen-evolving enhancer E-07 Yes Chlorophyll a/b binding E-5 Yes ATP synthase subunit alpha Yes Ferritin Yes Oxygen-evolving enhancer E-2 Yes ATP synthase subunit beta Yes Phosphoribulokinase Yes ATP synthase subunit alpha E- Yes Putative 60S acidic ribosomal P Yes Putative 60S acidic ribosomal P Yes Fructosebisphosphate aldolase E-5 Yes Glutamate-ammonia ligase (EC ) delta precursor Yes Glutamate-ammonia ligase (EC ) delta precursor E-3 Yes Glutamine synthetase E-07 Yes ATP synthase beta subunit (Fragment) E-06 Yes ATP synthase subunit beta Yes Putative glutamatetrna ligase E-07 Yes ATPase beta subunit (Fragment)

30 Yes 40S ribosomal S Yes Putative serine/threonine kinase E-07 Yes Membrane CH-like E-6 Yes ATP synthase gamma chain Yes Plastidic aldolase E-5 Yes OSJNBb0085H Yes Hypothetical F26P E-5 Yes ATP synthase subunit alpha E-05 Yes H+-transporting twosector ATPase-alpha chain E-05 Yes ATP synthase CF alpha subunit Yes 40S ribosomal S Yes Intronic ORF; similar to LAGLIDADG endonuclease Yes Putative PSII-P Yes ATP synthase subunit alpha E-05 Yes Histone acetyltransferase GCN E-05 Yes Proteasome subunit alpha type E-05 Yes Fructosebisphosphate aldolase Yes Fructosebisphosphate aldolase (EC ) precursor E-5 Yes ATP synthase CF alpha subunit E-5 Yes ATP synthase subunit alpha Yes Putative ferredoxin- NADP(H) oxidoreductase E-6 Yes Phosphoglycerate kinase E- Yes Putative uncharacterized E-3 Yes ATP synthase subunit alpha Yes Putative uncharacterized Yes Ferredoxin-NADP reductase E- Yes ATP synthase subunit alpha E-3 Yes ATP synthase subunit

31 386 alpha E-2 Yes ATP synthase subunit alpha Yes Mitochondrial processing peptidase beta subunit E-6 Yes Unknown Yes Beta-,3-glucanase Yes Carbonic anhydrase Yes Putative quinone oxidoreductase E-08 Yes Predicted Yes Putative uncharacterized E-05 Yes Ferredoxin: nadp reductase E-08 Yes Glyceraldehyde 3- phosphate dehydrogenase Yes Glyceraldehyde-3- phosphate dehydrogenase E-2 Yes Glyceraldehyde-3- phosphate dehydrogenase A Yes Luminal binding, BiP Yes Carbonic anhydrase E-6 Yes Guanine nucleotidebinding subunit beta-like

32 Supplementary Table S5. List of identified s with their empai (exponentially Modified Protein Abundance Index) values. Spot No Identification No of identifications (peptide with ion score p < 0.05) empai Summation of empai mol fraction % mol fraction Putative uncharacterized (Fragment) Tax_Id=3827-2* Putative uncharacterized [Cicer arietinum] Chlorophyll A/B binding Tax_Id=4476 [Gardenia jasminoides] * Ribulose bisphosphate carboxylase large chain Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=28975 [Hypseocharis sp. Beck s.n] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id= [Anarthria prolifera] Ribulose bisphosphate carboxylase large chain (Fragment) 0.54 Tax_Id=60682 [Asterella lindenbergiana] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=2484 [Salacia undulata] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id= [Aspalathus shawii subsp. shawii] * Ribulose bisphosphate carboxylase large chain Ribulose bisphosphate carboxylase large chain Tax_Id=3827 [Cicer arietinum] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=60682 [Asterella lindenbergiana] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=26692 [Osmoxylon novoguineense] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=22308 [Trichomanes ankersii] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=52857 [Stephanostema stenocarpum] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=20586 [Dacrydium araucarioides] * Chromosome chr7 scaffold_6 Chromosome chr7 scaffold_6, whole genome shotgun sequence Tax_Id=29760 [Vitis vinifera] Putative uncharacterized Tax_Id=39946 [Oryza 0.2 sativa subsp. indica] ATP synthase subunit beta Tax_Id=37502 [Polytomella sp. Pringsheim 98.80] Phosphoglycerate kinase Tax_Id=3988 [Ricinus * Phosphoglycerate kinase communis

33 Phosphoglycerate kinase Tax_Id=3695 [Populus trichocarpa x Populus deltoides] Phosphoglycerate kinase (Fragment) Tax_Id=29760 [Vitis vinifera] Phosphoglycerate kinase Tax_Id=3880 [Medicago truncatula] ATP synthase subunit beta (Fragment) Tax_Id=85203 [Eucryphia milliganii] * ATP synthase subunit beta, chloroplastic sp Q9MUT5 ATP synthase subunit beta, chloroplastic Tax_Id=4882 [Mesostigma viride] ATP synthase subunit beta (Fragment) Tax_Id= [Arctagrostis latifolia] ATP synthase subunit beta (Fragment) Tax_Id=59303 [Triodanis coloradoensis] * Putative GroEL chaperonin 60kDa RuBisCO large subunit-binding subunit beta, chloroplastic Tax_Id=3888 [Pisum sativum] Beta chaperonin 60 Tax_Id=409 [Solanum commersonii] Putative GloEL :chaperonin, 60kDa (Fragment) Tax_Id=3755 [Prunus dulcis] * Ferritin Ferritin Tax_Id=3694 [Populus trichocarpa] * Chlorophyll a/b binding Chlorophyll a/b binding Tax_Id=3827 [Cicer arietinum] Putative uncharacterized Tax_Id=3847 [Glycine max] Putative uncharacterized Tax_Id=29760 [Vitis 5.0 vinifera] Light-harvesting complex II Lhcb Tax_Id=3694 [Populus trichocarpa] Chlorophyll a-b binding, chloroplast, putative, expressed Tax_Id=39947 [Oryza sativa subsp. japonica] * ATP synthase subunit alpha ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit alpha Tax_Id=3900 [Trifolium subterraneum] ATP synthase subunit alpha, chloroplastic Tax_Id=3885 [Phaseolus vulgaris] ATP synthase subunit alpha, chloroplastic Tax_Id=34305 [Lotus japonicus

34 AtpA Tax_Id=80498 [Jatropha curcas] ATP synthase subunit alpha, chloroplastic Tax_Id=3847 [Glycine max] ATP synthase subunit alpha, chloroplastic Tax_Id=408 [Solanum lycopersicum] ATP synthase subunit alpha (Fragment) Tax_Id=4733 [Typha latifolia] ATP synthase subunit alpha, chloroplastic Tax_Id=37657 [Silene latifolia] ATP synthase subunit alpha, chloroplastic Tax_Id=3942 [Oenothera biennis] ATP synthase subunit alpha, chloroplastic Tax_Id=27 [Citrus sinensis] ATP synthase subunit alpha (Fragment) Tax_Id=32095 [Lonchitis hirsuta] ATP synthase subunit alpha, chloroplastic Tax_Id=48387 [Anthoceros formosae] ATP synthase subunit alpha, chloroplastic Tax_Id=397 [Marchantia polymorpha] ATP synthase subunit alpha (Fragment) Tax_Id=33 [Ginkgo biloba] ATP synthase subunit alpha (Fragment) Tax_Id=28472 [Epipactis helleborine] ATP synthase subunit alpha (Fragment) Tax_Id= [Afrothismia hydra] * Ferritin Ferritin Tax_Id=3847 [Glycine max] Whole genome shotgun sequence of line PN40024, scaffold_49.assembly2x (Fragment) Tax_Id= [Vitis vinifera] Putative uncharacterized Tax_Id=39946 [Oryza -96* Oxygen-evolving enhancer sativa subsp. indica] Putative uncharacterized Tax_Id=3847 [Glycine max] Chloroplast manganese stabilizing -II (Fragment) Tax_Id=43 [Solanum tuberosum] Putative uncharacterized Tax_Id=3847 [Glycine max] Oxygen-evolving enhancer, chloroplastic Tax_Id=3888 [Pisum sativum]

35 ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit beta Tax_Id=2807 [Dimocarpus * ATP synthase subunit beta longan] ATP synthase subunit alpha Tax_Id=3827 [Cicer -207* ATP synthase subunit alpha arietinum] ATP synthase subunit alpha, chloroplastic Tax_Id=48387 [Anthoceros formosae] ATP synthase subunit alpha, chloroplastic Tax_Id= [Marchantia polymorpha] ATP synthase subunit alpha (Fragment) Tax_Id=4733 [Typha latifolia] ATP synthase subunit alpha (Fragment) Tax_Id=32095 [Lonchitis hirsuta] Putative 60S acidic ribosomal Putative 60S acidic ribosomal P0 Tax_Id= * P0 [Trifolium pratense] Putative 60S acidic ribosomal Putative 60S acidic ribosomal P0 Tax_Id= * P0 [Trifolium pratense] Fructose-bisphosphate aldolase Tax_Id=57577 [Trifolium -20* Fructose-bisphosphate aldolase pratense] Fructose bisphosphate aldolase Tax_Id=3879 [Medicago sativa] * Glutamine synthetase Glutamine synthetase Tax_Id=6934 [Beta vulgaris] * ATP synthase subunit beta ATP synthase subunit beta Tax_Id=3827 [Cicer arietinum] Putative uncharacterized Tax_Id=3880 [Medicago -24* Putative uncharacterized truncatula] Putative uncharacterized Tax_Id=3880 [Medicago truncatula] * 40S ribosomal S2 40S ribosomal S2 Tax_Id=3847 [Glycine max] * ATP synthase gamma chain ATP synthase gamma chain Tax_Id=3880 [Medicago truncatula] ATP synthase gamma chain Tax_Id=3847 [Glycine max] ATP synthase gamma chain Tax_Id=3988 [Ricinus.33 communis] Putative uncharacterized Tax_Id=4577 [Zea mays] Putative uncharacterized Tax_Id=3880 [Medicago truncatula]

36 -269* ATP synthase subunit alpha ATP synthase subunit alpha, chloroplastic Tax_Id=34305 [Lotus japonicus] ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit beta Tax_Id=3827 [Cicer arietinum] ATP synthase CF alpha subunit Tax_Id=5779 [Vigna radiata] ATP synthase subunit beta (Fragment) Tax_Id=20340 [Bischofia javanica] * 40S ribosomal S2 40S ribosomal S2 Tax_Id=3847 [Glycine max] Putative PSII-P (Fragment) Tax_Id= * Putative PSII-P [Trifolium pratense] ATP synthase subunit alpha Tax_Id=3827 [Cicer * ATP synthase subunit alpha arietinum] Proteasome subunit alpha type Tax_Id=3694 [Populus -30* Proteasome subunit alpha type trichocarpa] Proteasome subunit alpha type Tax_Id=3988 [Ricinus communis] Proteasome subunit alpha type (Fragment) Tax_Id=3750 [Malus domestica] Proteasome subunit alpha type-6 Tax_Id=3847 [Glycine max] Fructose-bisphosphate aldolase, chloroplastic * Fructose-bisphosphate aldolase Tax_Id=3888 [Pisum sativum] * ATP synthase CF alpha subunit ATP synthase CF alpha subunit Tax_Id=5779 [Vigna radiata] ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit alpha Tax_Id= [Geranium palmatum] ATP synthase subunit alpha, chloroplastic Tax_Id=55564 [Chara vulgaris] * ATP synthase subunit alpha ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit alpha, chloroplastic Tax_Id= [Citrus sinensis] ATP synthase subunit alpha, chloroplastic Tax_Id=48387 [Anthoceros formosae]

37 -322* -323* -335* -346* -376* -382* Phosphoglycerate kinase precursor like Putative uncharacterized ATP synthase subunit alpha Putative uncharacterized Ferredoxin-NADP reductase ATP synthase subunit alpha ATP synthase subunit alpha Tax_Id=3369 [Cryptomeria japonica] Phosphoglycerate kinase Tax_Id=43 [Solanum tuberosum] Phosphoglycerate kinase Tax_Id=3988 [Ricinus communis] Putative uncharacterized Tax_Id=4577 [Zea mays] Phosphoglycerate kinase Tax_Id=3880 [Medicago truncatula] Phosphoglycerate kinase Tax_Id=29760 [Vitis vinifera] Putative uncharacterized Tax_Id=3332 [Picea sitchensis Putative uncharacterized Tax_Id=3694 [Populus 0.36 trichocarpa] Ribulose bisphosphate carboxylase activase B Tax_Id=4565 [Triticum aestivum] ATP synthase subunit alpha Tax_Id=3827 [Cicer 0.07 arietinum] Putative uncharacterized Tax_Id=3694 [Populus trichocarpa] Transketolase (Fragment) Tax_Id=3827 [Cicer arietinum] Putative uncharacterized Sb06g Tax_Id=4558 [Sorghum bicolor] Ferredoxin--NADP reductase Tax_Id=39946 [Oryza sativa subsp. indica] ATP synthase subunit alpha, chloroplastic Tax_Id=3443 [Coffea arabica] ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit alpha (Fragment) Tax_Id=4733 [Typha latifolia] Fructose-bisphosphate aldolase, cytoplasmic isozyme Tax_Id=3888 [Pisum sativum] ATP synthase subunit alpha Tax_Id=3369 [Cryptomeria japonica] Fructose-bisphosphate aldolase Tax_Id=3847 [Glycine max] ATP synthase subunit alpha (Fragment) Tax_Id=

38 -386* -39* -394* -396* ATP synthase subunit alpha Putative 60S ribosomal L ATP synthase subunit alpha ATP synthase subunit alpha [Lonchitis hirsuta] ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit alpha (Fragment) Tax_Id=4733 [Typha latifolia] ATP synthase subunit alpha Tax_Id=3369 [Cryptomeria japonica] ATP synthase subunit alpha, chloroplastic Tax_Id= [Anthoceros formosae] ATP synthase subunit alpha, chloroplastic Tax_Id=397 [Marchantia polymorpha] ATP synthase subunit alpha (Fragment) Tax_Id=32095 [Lonchitis hirsuta] ATP synthase subunit alpha, chloroplastic Tax_Id=27 [Citrus sinensis] Putative uncharacterized Tax_Id=3880 [Medicago 0.08 truncatula] Ribulose-,5-bisphosphate carboxylase/oxygenase activase (Fragment) Tax_Id=74825 [Pachysandra terminalis] ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit alpha (Fragment) Tax_Id=4733 [Typha latifolia] Ribulose-,5-bisphosphate carboxylase activase 0.93 (Fragment) Tax_Id=4565 [Triticum aestivum] Putative uncharacterized Tax_Id=3694 [Populus trichocarpa] Putative uncharacterized Tax_Id=3332 [Picea sitchensis] ATP synthase subunit alpha (Fragment) Tax_Id=32095 [Lonchitis hirsuta] ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit alpha (Fragment) Tax_Id=4733 [Typha latifolia] ATP synthase subunit alpha, chloroplastic Tax_Id=48387 [Anthoceros formosae] ATP synthase subunit alpha, chloroplastic Tax_Id=

39 -46* Succinate dehydrogenase flavo subunit,mitochondrial [Marchantia polymorpha ATP synthase subunit alpha Tax_Id=3369 [Cryptomeria japonica] ATP synthase subunit alpha (Fragment) Tax_Id=49509 [Pilularia globulifera] ATP synthase subunit alpha, chloroplastic Tax_Id=344 [Chlorokybus atmophyticus] Unknown 8 (Fragment) Tax_Id=3357 [Pseudotsuga menziesii] Putative uncharacterized Tax_Id=4577 [Zea mays] Predicted Tax_Id= [Micromonas pusilla CCMP545] * Beta-,3-glucanase Beta-,3-glucanase Tax_Id=895 [Musa paradisiaca] * Ribulose bisphosphate carboxylase large chain Ribulose bisphosphate carboxylase large chain Tax_Id=3827 [Cicer arietinum] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id= [Triodanis perfoliata] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=32576 [Delonix brachycarpa] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=9389 [Orthilia secunda] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=28475 [Lasia spinosa] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=7468 [Levenhookia leptantha] Ribulose bisphosphate carboxylase large chain (Fragment) 3.4 Tax_Id=37395 [Reboulia hemisphaerica] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id= [Plagiochila arbuscula] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=62704 [Caesalpinia calycina] Ribulose bisphosphate carboxylase large chain Tax_Id=8404 [Comarostaphylis arbutoides] Ribulose-,5-bisphosphate carboxylase/oxygenase large subunit (Fragment) Tax_Id= [Desmidium aptogonum] Ribulose bisphosphate carboxylase large chain (Fragment)

40 Tax_Id=43553 [Pinckneya pubens] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=2484 [Salacia undulata] Ribulose-,5-bisphosphate carboxylase/oxygenase large subunit (Fragment) Tax_Id= [Campanulastrum americanum] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=25047 [Pentaphylax euryoides] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=3296 [Ammannia latifolia] * Carbonic anhydrase Carbonic anhydrase Tax_Id=396 [Phaseolus aureus] Carbonic anhydrase Tax_Id=3635 [Gossypium hirsutum] Putative quinone Putative quinone oxidoreductase Tax_Id=3827 [Cicer * oxidoreductase arietinum] * Predicted Predicted Tax_Id=3694 [Populus trichocarpa] Putative uncharacterized Tax_Id=3847 [Glycine -445* Putative uncharacterized max] Putative uncharacterized (Fragment) Tax_Id=3880 [Medicago truncatula] * Fructose-bisphosphate aldolase ATP synthase subunit alpha Tax_Id=3827 [Cicer arietinum] ATP synthase subunit alpha (Fragment) Tax_Id=4733 [Typha latifolia] Fructose-bisphosphate aldolase, cytoplasmic isozyme 0.8 Tax_Id=3827 [Cicer arietinum] ATP synthase subunit alpha (Fragment) Tax_Id=32095 [Lonchitis hirsuta] Fructose-bisphosphate aldolase Tax_Id=337 [Metasequoia glyptostroboides] * Glyceraldehyde-3-phosphate dehydrogenase Putative uncharacterized Tax_Id=3880 [Medicago truncatula] Glyceraldehyde 3-phosphate dehydrogenase (Fragment) Tax_Id= [Scopellaria marginata] utative uncharacterized Tax_Id=4577 [Zea mays] Glyceraldehyde 3-phosphate dehydrogenase (Fragment) Tax_Id=38699 [Mukia maderaspatana] Glyceraldehyde-3-phosphate Glyceraldehyde-3-phosphate dehydrogenase A, * dehydrogenase A chloroplastic Tax_Id=3888 [Pisum sativum]

41 -483* Mitochondrial outer membrane porin of 34 kda Whole genome shotgun sequence of line PN40024, scaffold_25.assembly2x (Fragment) Tax_Id=29760 [Vitis vinifera] Luminal-binding Tax_Id=3562 [Spinacia oleracea] Outer plastidial membrane porin Tax_Id= [Pisum sativum] Porin Tax_Id=3544 [Mesembryanthemum crystallinum] Luminal binding, BiP Tax_Id=390 [Scherffelia dubia] Carbonic anhydrase Tax_Id=3635 [Gossypium hirsutum] Carbonic anhydrase Tax_Id=74825 [Pachysandra terminalis] Putative uncharacterized Tax_Id=3880 [Medicago -485* Carbonic anhydrase Carbonic anhydrase Tax_Id=3885 [Phaseolus vulgaris] * -7* Guanine nucleotide-binding subunit beta-like light-harvesting complex II Lhcb3 truncatula] Putative uncharacterized Tax_Id=3847 [Glycine max] Guanine nucleotide-binding subunit beta-like Tax_Id=3879 [Medicago sativa] Putative uncharacterized Tax_Id=3332 [Picea sitchensis] Whole genome shotgun sequence of line PN40024, scaffold_4.assembly2x (Fragment) Tax_Id=29760 [Vitis vinifera] Putative uncharacterized Tax_Id=3695 [Populus trichocarpa x Populus deltoides] Putative uncharacterized Tax_Id=3847 [Glycine 0.4 max] tr Q0PWS5 Chloroplast pigment-binding CP26 Tax_Id=4097 [Nicotiana tabacum] * Actin (Fragment) Actin (Fragment) Tax_Id=69334 [Chara contraria] Actin (Fragment) Tax_Id=38836 [Pterosperma cristatum] Chlorophyll a/b binding Tax_Id=3827 [Cicer -75* Chlorophyll a/b binding arietinum] Chlorophyll a-b binding AB80, chloroplastic 3.78 Tax_Id=3888 [Pisum sativum] Light-harvesting chlorophyll a/b-binding

42 -78* -9* -94* Chlorophyll a/b binding Ribulose bisphosphate carboxylase large chain Chaperonin 60 alpha subunit Tax_Id=3760 [Prunus persica] Putative uncharacterized Tax_Id=29760 [Vitis vinifera] Chlorophyll a-b binding, chloroplast, putative, expressed Tax_Id=39947 [Oryza sativa subsp. japonica] Chlorophyll a/b binding Tax_Id=3827 [Cicer arietinum] Putative uncharacterized Tax_Id=29760 [Vitis 2.92 vinifera] Putative chloroplast chlorophyll a/b-binding Tax_Id=39927 [Carya cathayensis] Ribulose bisphosphate carboxylase large chain (Fragment) Tax_Id=22300 [Orobanche coerulescens] Putative uncharacterized Tax_Id=3880 [Medicago truncatula] Putative uncharacterized Tax_Id=3880 [Medicago truncatula] tr Q2PEP Putative rubisco subunit binding- alpha subunit Tax_Id=57577 [Trifolium pratense] RuBisCO large subunit-binding subunit alpha, chloroplastic Tax_Id=3888 [Pisum sativum] Chaperonin 60 alpha subunit Tax_Id=28957 [Canavalia lineata] Predicted Tax_Id=3694 [Populus trichocarpa] Predicted Tax_Id=3694 [Populus trichocarpa] Putative rubisco subunit binding- alpha subunit Tax_Id=3702 [Arabidopsis thaliana] RuBisCO large subunit-binding subunit alpha, chloroplastic Tax_Id=3708 [Brassica napus] Putative uncharacterized Tax_Id=39946 [Oryza sativa subsp. indica] RuBisCO large subunit-binding subunit alpha, chloroplastic Tax_Id=4565 [Triticum aestivum] Putative uncharacterized Tax_Id=39946 [Oryza sativa subsp. indica] Chaperonin-60 alpha subunit Tax_Id=82927 [Avicennia marina] Predicted Tax_Id= [Micromonas pusilla

43 CCMP545] Plastid 60 kda chaperonin alpha subunit (Fragment) Tax_Id=3 [Prototheca wickerhamii] * Chaperone DnaK Chaperone DnaK Tax_Id=3880 [Medicago truncatula] Chloroplast heat shock 70-2 Tax_Id=35883 [Ipomoea nil] Chloroplast heat shock 70- Tax_Id= [Ipomoea nil] Os03g (Fragment) Tax_Id=39947 [Oryza sativa subsp. japonica]

44 Supplementary Table S6. In silico analyses of SUN domain s from different organisms. Gene/ Accession No. Organism Sequence length (aa) Position of SUN domain No. of TMD CaSUN C. arietinum N-terminus outside 3 strong transmembrane helices SLP (YOR54W) S. cerevisae N-terminus inside 2 strong transmembrane helices MPS3 (YJL09W) S. cerevisae N-terminus inside strong transmembrane helices SAD ( SPBC2D2) S. pombe N-terminus outside 2 strong transmembrane helices Sad-UNC-like (SPBC3E7.09) AtSUN3 (ATG22882 ) AtSUN (AT5G04990) Hypothetical (Pps9_3V6 ) Hypothetical (Pps3_89V6 ) Hypothetical (gi ) Mmembrane CH-like (gi ) S. pombe N-terminus outside 2 strong transmembrane helices A. thaliana N-terminus inside 3 strong transmembrane helices A. thaliana N-terminus inside strong transmembrane helices P. patens N-terminus outside 3 strong transmembrane helices P. patens N-terminus outside 2 strong transmembrane helices O. sativa N-terminus outside 3 strong transmembrane helices O. sativa N-terminus inside 3 strong transmembrane helices gi Z. mays N-terminus inside strong transmembrane helices No. of CC domain and position (474 L-487 Q) (362 N-375 E) 3 (245 N-260 I) (366 I-380 I ) (386 N-40Q) 2 (82 R-95 N) (263 L- 282 K) 3 (336 I-349 Q ) (458 Y-473 K) (525 A-538 E) 2 (00 N-3 E) (574 R-598 V) 2 (8 K-97 K) (203 S-27 K) 2 (35 Q-48 K ) (73 L-726 N) Position of signal peptide Nonsecretory Nonsecretory Nonsecretory Nonsecretory (82 L-206 D) 2 (50 A-56 S) (550 S-565 R) 2 (72 L-96 K) (206 E-29 L) 0 Nonsecretory Nonsecretory Nonsecretory Nonsecretory

45 gi Z. mays N-terminus outside 4 strong transmembrane helices Glyma5g35240 G. max N-terminus outside strong transmembrane helices Gm09g00440 G. max N-terminus outside 4 strong transmembrane helices UNC-84 C. elegans N-terminus inside 9 strong transmembrane helices gi: C. elegans N-terminus inside 2 strong transmembrane helices gi D. melanogaster N-terminus outside 4 strong transmembrane helices DrossoFBpp D. melanogaster N-terminus outside strong transmembrane helices gi H. sapiens N-terminus inside 3 strong transmembrane helices gi H. sapiens N-terminus inside 4 strong transmembrane helices (5 E-524 E) 2 (6 K-77 K) (85 L-209 E) Nonsecretory Nonsecretory (546 K-559 Q) 3 (89 E-207 K) (755 I-770 L) (775 L-797 T) 3 (83 S-200 I) (246 L-260 S) (30 L-323 N ) 2 (279 R-293 E) (349 R-368 Q) 3 (235 D-248 K) (872 M-888 S) ( 892 L-90 F) (949 V-964 S) (43 I-426 Q) (46 E-474 N) 0 Nonsecretory Nonsecretory Nonsecretory 29-30

46 Supplementary Table S7. Genomic organization of SUN gene family. Gene ID/Accession number Organism No of introns Size and position of introns CaSUN C. arietinum (8-222) ( ) 3. 07( ) ( ) SLP S. cerevisae 0 - MPS3 S. cerevisae 0 - SAD S. pombe 0 - SUNLIKE S. pombe 0 - ATG22882 A. thaliana 2. 8(70-349) 2. 89( ) AtSUN A. thaliana. 658( ) Pps9_3V6 P. patens 0 - Pps3_89V6 P. patens 0 - Selaginella_44228 S. moellendorffii 3. 70(34-382) 2. 70( ) 3. 68(53-579) Selaginella_8985 S. moellendorffii. 24( ) Osgi O. sativa. 08( ) Osgi O. sativa ( ) 2. 86( ) ( ) Zmgi Z. maize ( ) ( ) Zmgi Z. maize 3. 0( ) 2. 76( ) 3. 95( ) Glyma5g35240 G. max 2. 49( ) ( ) Gm09g00440 G. max 3. 87(256-34) 2. ( ) 3. 4( ) DmFBpp D. melanogaster 4. 82(35-25) 2. 69( ) ( ) 4. 64( ) DmFBpp D. melanogaster 2. 58( ) 64( ) UNC-84 C. elegans (78-69) 2. 53( ) 3. 60(854-92) 4. 59( ) 5. 48( ) 6. 52( ) 7. 76( )

47 8. 53( ) 9. 6( ) CeR2E2 C. elegans 8. 4(30-42) 2. 53( ) 3. 46(368-42) 4. 53( ) 5. 53( ) 6. 58( ) 7. 47( ) 8. 53( ) Hsgi H. sapiens 6. 34(23-255) 2. 75( ) ( ) ( ) ( ) ( ) ( ) ( ) (36-578) ( ). 324( ) ( ) ( ) 4. 9( ) ( ) ( ) Hsgi H. sapiens ( ) ( ) ( ) ( ) ( ) ( ) ( ) 8. 36( ) (40564-

48 42082) 0. 24( ). 479( ) 2. 83( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2. 04( )

49 Supplementary Table S8. Detail of the mass spectra of membrane s identified with single peptide. Spot No. Identification Observed Mr(expt) Mr(calc) Delta Miss Score Expect Rank Peptide Instrument Taxonomy Database Peptide mass tolerance Ludwig -73 Actin (Fragment) K.SYELPDGQVITIGNER.F itrap Viridiplantae NR.2 Da 0.6 Da Fragment mass tolerance -82 Ferritin K.IAEYVTQLR.M itrap Viridiplantae Ludwig NR.2 Da 0.6 Da -24 Glutamate-ammonia ligase (EC ) delta precursor E-05 R.AAEIFSNPK.V Qstar Viridiplantae MSDB 00 ppm 0.4 Da Glutamate-ammonia ligase (EC ) delta -26 precursor R.AAEIFSNPK.V Qstar Viridiplantae MSDB 00 ppm 0.4 Da Ludwig -28 Glutamine synthetase K.AAEIFSNPK.V itrap Viridiplantae NR.2 Da 0.6 Da OSJNBb0085H K.GATVEAPR.E Qstar Viridiplantae MSDB 00 ppm 0.4 Da -428 Beta-,3-glucanase E- 05 R.NSNIQVLLDVPR.S itrap Viridiplantae -443 Predicted K.LAVAINDLSK.T itrap Viridiplantae Ludwig NR.2 Da 0.6 Da Ludwig NR.2 Da 0.6 Da

50 -73 # b b ++ b* b* ++ b 0 b 0++ Seq. y y ++ y* y* ++ y 0 y 0++ # S Y E L P D G Q V I T I G N E R

51 -82 # b b ++ b* b* ++ b 0 b 0++ Seq. y y ++ y* y* ++ y 0 y 0++ # I A E Y V T Q L R

52 -24 # b b ++ b* b* ++ b 0 b 0++ Seq. y y ++ y* y* ++ y 0 y 0++ # A A E I F S N P K

53 -26 # b b ++ b* b* ++ b 0 b 0++ Seq. y y ++ y* y* ++ y 0 y 0++ # A A E I F S N P K

54 -28 # b b ++ b* b* ++ b 0 b 0++ Seq. y y ++ y* y* ++ y 0 y 0++ # A A E I F S N P K

55 -263 # b b ++ b 0 b 0++ Seq. y y ++ y* y* ++ y 0 y 0++ # G A T V E A P R

56 -428 # b b ++ b* b* ++ b 0 b 0++ Seq. y y ++ y* y* ++ y 0 y 0++ # N S N I Q V L L D V P R