Correction: 12 July 2012 www.sciencemag.org/cgi/content/full/336/6089/1711/DC1 Supplementary Material for Uniform ripening Encodes a Golden 2-like Transcription Factor Regulating Tomato Fruit Chloroplast Development Ann L.T. Powell,* Cuong V. Nguyen, Theresa Hill, KaLai Lam Cheng, Rosa Figueroa- Balderas, Hakan Aktas, Hamid Ashrafi, Clara Pons, Rafael Fernández-Muñoz, Ariel Vicente, Javier Lopez-Baltazar, Cornelius S. Barry, Yongsheng Liu, Roger Chetelat, Antonio Granell, Allen Van Deynze, James J. Giovannoni,* Alan B. Bennett *To whom correspondence should be sent. E-mail: [email protected] (A.L.T.P.); [email protected](J.J.G.) Published 29 June 2012, Science 336, 1711 (2012) DOI: 10.1126/science.1222218 This PDF file includes: Materials and Methods Figs. S1 to S8 Tables S1 to S3, S7, and S8 References (32–58) Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/336/6089/1711/DC1) Tables S4 to S6 as a separate Excel file Correction: On page 2, the accession number of Ailsa craig was corrected to “LA2838A” on the third line of Materials and Methods.
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Uniform ripening Encodes a Golden 2-like Transcription Factor Regulating Tomato Fruit Chloroplast Development
Ann L.T. Powell,* Cuong V. Nguyen, Theresa Hill, KaLai Lam Cheng, Rosa Figueroa-Balderas, Hakan Aktas, Hamid Ashrafi, Clara Pons, Rafael Fernández-Muñoz, Ariel Vicente, Javier Lopez-Baltazar, Cornelius S. Barry, Yongsheng Liu, Roger Chetelat,
Antonio Granell, Allen Van Deynze, James J. Giovannoni,* Alan B. Bennett
Published 29 June 2012, Science 336, 1711 (2012) DOI: 10.1126/science.1222218
This PDF file includes:
Materials and Methods
Figs. S1 to S8
Tables S1 to S3, S7, and S8
References (32–58) Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/336/6089/1711/DC1)
Tables S4 to S6 as a separate Excel file Correction: On page 2, the accession number of Ailsa craig was corrected to “LA2838A” on the third line of Materials and Methods.
2
Materials and Methods MATERIALS AND METHODS Plant materials, fruit staging and harvesting. Tomato plants (Solanum sps.) were
grown in fields and greenhouses in Davis, CA and Ithaca, NY, USA and Malaga, Spain.
The S. lycopersicum varieties ‘Ailsa Craig’ (LA2838A), the monogenic u mutant of
‘Ailsa Craig’ called ‘Craigella’ (LA3247) (36), ‘Castlemart’, ‘Fireball’, ‘E6203’ and
‘M82’ (LA3475) were germinated from stock seed collections (Tomato Genetics
Resource Center, UC Davis), transplanted and grown in two gallon pots in greenhouses
or in furrow irrigated fields. Seed for S. lycopersicum var. cerasiforme (PI114490) was
provided by A. Van Deynze and SolCAP and grown in greenhouses in Davis, CA, USA.
Seed for lines, N93 (u/u) and 73X (U/U), T91 (U/U) were provided by Hanoi University
of Agriculture, Viet Nam and grown in Ithaca, NY, USA. S. pennellii (LA0716), IL10-1
(LA4087), IL10-1-1 (LA4088) and IL10-2 (LA4089) lines from the S. lycopersicum
(‘M82’) x S. pennellii IL population were grown in greenhouses in Davis and Ithaca. The
BC2S1 and RIL S. lycopersicum (‘Moneymaker’) x S. pimpinellifolium (‘TO-937’)
population and additional IL10-1, IL10-1-1, IL10-2, and LA0716 plants were grown in
plastic greenhouses in Malaga, Spain. The ‘Cuatomate’ landrace was provided by J.
Lopez-Baltazar, Oaxaca, Mexico, and grown in greenhouses in Davis. Transgenic tomato
(S. lycopersicum cv. ‘T63’) lines expressing the transcription factors AtGLK1
(At2g20570) or AtGLK2 (At5g44190) regulated by the CaMV35S (p35S), the
Arabidopsis lipid transfer protein (pLTP) (37), the tomato rubisco small subunit 3b
(pRbcS) (38, 39) or the tomato phytoene desaturase (pPDS) (38, 40) promoters were
provided by Mendel Biotech. Inc., Hayward, CA, USA and Seminis Vegetable Seeds-
3
Monsanto, Woodland, CA, USA. The AtGLK expressing lines were obtained by crossing
two types of parental transgenic tomato lines. In the cross, one parent line was
transformed using Agrobacterium tumefaciens with a TDNA construct containing either
the AtGLK1 or the AtGLK2 coding sequence fused to the E. coli LexA operator binding
site (LexA:AtGLK1, LexA:AtGLK2) and the CaMV35S regulated sulfonamide selectable
marker (dihydropteroate synthase, SULII). The second parental lines used in the crosses
were transformed with the LexA-Gal4 activation domain coding sequence linked to the
CaMV35S, LTP, RbcS or PDS promoters (p35S:LexA-Gal4, pLTP:LexA-Gal4,
pRbcS:LexA-Gal4, pPDS:LexA-Gal4) and the CaMV35S regulated GFP and the
CaMV35S regulated kanamycin (NPTII) as a selectable marker (41). The LexA:GLK1 and
LexA:GLK2 parental lines were each crossed with each of the four promoter containing
Where Yijkl represents the lth observation on the ith type jth replicate kth sample genotype
μ is the common effect for the whole experiment. εijkl represents the random error present
in the lth observation on the ith type jth replicate kth sample genotype .The errors εijkl are
assumed to be normally and independently distributed with mean 0 and standard
deviation δ for all measurements.
To determine specific group differences in case of significant main effects (or
interaction), the ANOVA analysis was followed by Fisher’s LSD post hoc contrast to
generate p-values and fold changes for comparisons between type and sample type. Gene
lists of pair-wise contrasts were divided into up- and down-regulated genes (compared
with wildtype (WT) TControl). Genes whose expression changed as a consequence of
AtGLK expression were defined independently for each AtGLK expressing line using a
11
mean fold change ≥2 (relative to WT TControl samples) and a P value of ≤0.05. A total
of 3216 genes (≈ 10% of the EUTOM3 probes on the microarrays) were significantly up
or down regulated in AtGLK1 and/or AtGLK2 overexpressing tomato lines (Table S4).
Venn diagrams (Figure S6A) were used to identify sets of common and specifically
regulated genes. Genes differentially expressed (either up or down-regulated) in both
transgenic lines were defined as regulated by GLKs and genes up or down regulated only
in the material expressing either AtGLK1 or AtGLK2 were defined as specifically
regulated by GLK1 or GLK2, respectively. These classes of genes were used for
subsequent analyses. Two dimensional hierarchical agglomerative clustering using
Euclidean distance and average linkage were performed. The differentially expressed
genes identified genes were grouped into clusters to calculate Gene Ontology (GO)
enrichment scores for molecular function categories by applying Fisher Exact tests using
a local, customized version of the 'catscore.pl' Perl script (52) was used. Only GO terms
with a p<0.05, and three or more regulated genes for the GO-term were defined as over-
represented. Complete functional enrichment results are provided in Tables S5 and S6.
The EUTOM3 microarrays were designed and annotated by Stephane Romabauts (VIB
Department of Plant Systems Biology, Ghent University Technologiepark 927, 9052
Ghent, Belgium). The MIAME–compliant microarray data are available at
http://ted.bti.cornell.edu and at http://www.ebi.ac.uk/arrayexpress/ with the accession
number E-MEXP-3652.
Chlorophyll. Chlorophyll was measured in the youngest fully expanded apical leaves
in a truss and in immature green (15 dpa) fruit. Tissues from the outer fruit pericarp and
epidermis (~50 mg each) and leaf (~7 mg) from well irrigated plants were extracted into
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1 ml N,N dimethylformamide (DMF) overnight at 4 oC. The amounts of chlorophyll a
and chlorophyll b, were determined spectrophotometrically using published equations
(53). Total chlorophyll was calculated as chlorophyll a + chlorophyll b. Results were
expressed as µg chlorophyll per mg of tissue and the results agreed with chlorophyll
determinations made with material extracted in 80% acetone. A minimum of five
biologically replicated samples was used for each genotype and tissue.
Starch measurements. For starch quantitation, two grams of outer fruit pericarp
were ground in 10 mL ethanol. The samples were centrifuged and the pellet was re-
extracted two more times with 10 mL ethanol. After centrifugation, the pellet was dried at
50 oC and resuspended in 5 mL of 50 mM NaAc buffer (pH 5.0). 100 µL containing 10
units of amylase and 3 units of amylo-glucosidase were added and samples were
incubated at 30 oC with stirring overnight. The samples were centrifuged and adjusted to
6 mL with water. The content of reducing sugars was determined using a modification of
the Somogyi-Nelson method and measured with a spectrophotometer at 520 nm (54).
Transmission electron microscopy. Pericarp fragments were excised from fruit at
the mature green stage and from fully expanded leaves. Fragments were fixed in
Karnovsky’s fixative using vacuum-microwave combination as described by Russin and
Trivett (55) and washed in 0.1 M sodium phosphate buffer, pH 7.2, microwaved under
vacuum at 450 W for 40 seconds, post-fixed for 2 hours in 1 % (w/v) osmium tetroxide
buffered in 0.1 M sodium phosphate buffer and microwaved a second time at 450 W for
40 seconds. After incubation in 0.1% (w/v) tannic acid in water for 30 minutes on ice and
in 2 % (w/v) aqueous uranyl acetate for 1 hour, samples were dehydrated in acetone and
embedded in Epon/Araldite resin. Ultrathin sections were examined with a Philips
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CM120 Biotwin Lens transmission electron microscope (FEI Company). Images in
Figure 4A were taken at 11,000 magnification and 0.5 µm scale bars are shown.
Measurement of soluble solids. Soluble solid contents (oBRIX) were of total fruit
juice from freshly harvested red ripe fruit were measured with a digital refractometer
(PR100, Atago Co., Ltd.).
Sugar analysis. For simple sugar analysis, 5 to 7 g of total mature green and red fruit
tissue was extracted with 20 mL 95% (v/v) ethanol. The samples were centrifuged and
the pellets re-extracted with 10 mL 95% (v/v) ethanol. The supernatants were pooled and
adjusted with 95% (v/v) ethanol to a final volume of 45 mL. From these pooled
supernatants, 200 µL samples were dried and resuspended in 1 mL water. Forty
microliters of the resuspended sample were diluted to 10 mL with water and 200 µL were
injected in the HPLC for sugar analysis. Sugar profiles were analyzed using a DX-500
HPLC system (Dionex Corp.) equipped with an analytical Carbopac PA1 column and an
ED-40 electrochemical detector for pulsed amperimetric detection (PAD). A linear
sodium carbonate gradient at a flow rate of 0.6 mL min-1 was used. Glucose and fructose
were identified and quantified by using authentic standards. Results were expressed in
grams of sugar per 100 g of fresh fruit.
Carotenoid analysis. Carotenoid compounds were extracted and measured by
HPLC from three independent biological samples of red ripe (42 dpa) fruit epidermis and
pericarp as described previously (56). For spectrophotometric measurements, 0.25 g of
pericarp and epidermis from red ripe fruit (42 dpa) were ground in liquid N2, and
extracted with 8 ml hexane:ethanol:acetone (2:1:1) with shaking at room temperature
overnight. 1 ml water was added to each sample which was mixed by vortexing and the
14
solvent layers separated. The absorbance of the organic phase at 503 nm was used to
calculate the amount of lycopene (57, 58).
Statistical analysis. Experiments were performed according to a factorial design.
Data were analyzed by ANOVA, if the number of experimental replicates was equal, or
by General Linear Model (GLM) if the number of replicates was unequal followed by
post hoc testing using Tukey’s Honestly Significant Difference (HSD) or Bonferonni
Multiple Comparison Test (MCT) with JMP 9.0 (SAS, Cary, NC). Genetic linkage
analysis was performed by using JoinMap 4.0 software (Van Ooijen, J. W., Kyazma
B.V., Wageningen, the Netherlands, 2006).
15
Fig. S1.
Figure S1. Total soluble solids as measured by oBRIX of juice from red ripe (42 days post anthesis, dpa) fruit that had dark green shoulders (‘Ailsa Craig’ U/U) or were uniformly light green (‘Craigella’ u/u) prior to ripening. n=100 fruit of each genotype. Significant statistical differences determined by means of ANOVA and Tukey’s HSD at p<0.05 are indicated by different letters.
16
Fig. S2 A
17
B
18
C
19
D BLOSUM50 BLOSUM62 PAM250
Name Length Name Length Identity Similarity Identity Similarity Identity Similarity
SlGLK2 310.0 SlGLK1 464.0 43.4 53.2 43.4 51.3 41.9 55.2 Figure S2. Amino acid alignments showing the sequences of the predicted GLK1 and GLK2 proteins from Arabidopsis (AtGLK1, AtGLK2), domesticated tomato (S. lycopersicum, (A) cv. Ailsa Craig (B) cv.’Craigella’) (SlGLK1, SlGLK2), diploid potato (S. phureja) (SpGLK1, SpGLK2) and pepper (Capsicum annuum) (CaGLK1, CaGLK2) cDNA sequences. To identify potato GLK genes, homology searches of the potato transcriptome assembly and genomes using Arabidopsis GLK1 and GLK2 were used to identify two GLK-like genes in potato (SpGLK1 and SpGLK2). The exon structures are based on comparisons with Arabidopsis, tomato and potato genomic sequences. Sequence alignment was generated by using Geneious Pro v 5.3. (C) A phylogenetic tree
20
of GLKs generated using a Bayesian inference of phylogeny. In addition to the Arabidopsis, tomato, potato and pepper GLKs, BLAST searches against protein and EST databases at NCBI and EMBL were used to identify multiple GLK sequences from Monocot, Rosid Dicot and Asterid Dicot groups. Predicted amino acid sequences were aligned with T-Coffee using the PSI-Coffee method followed by an additional alignment evaluation using Core (http://tcoffee.crg.cat/). Sequences were trimmed to remove regions that showed inconsistent alignment (0-5 reliability score out of 10). Trimmed sequences were used to construct the tree using MrBayes 3.2.1 (http://mrbayes.sourceforge.net/index.php) with mixed amino acid and co-varion models run for 300,000 iterations at 2 runs by 1 chain per run. The branch lengths indicate the evolutionary distances, and numbers indicate percent probabilities for each node. Abbreviations and accession numbers used are found in Table S8. (D) Pairwise sequence identity and similarity between tomato, potato, pepper and Arabidopsis GLKs was calculated using MatGAT 2.02 (http://bitincka.com/ledion/matgat/) run with BLOSUM50, BLOSUM62 and PAM250 alignment matrices.
Figure S3. Maturing fruit from Tcontrol (A) and transgenic plants expressing p35S::AtGLK1 (B,) or p35S::AtGLK2 (C). From left to right fruit are green (4, 6, 12, 18, 25, 32 dpa) turning (35 dpa) and fully red ripe (42 dpa).
22
Fig. S4 A
B
Figure S4. Chlorophyll contents of green fruit as a function of exposure to light during maturation. A. Phenotypes of ‘Ailsa Craig’ U/U and ‘Craigella’ u/u mature green (32 dpa) fruit after maturation in the absence (Dark) and presence (Light) of light. B. Chlorophyll contents of pericarp from the pedicel (shoulders) or style (stylar) region of mature green fruit. Significant statistical differences determined by means of GLM and Tukey’s HSD at p<0.05 are indicated by different letters.
23
Fig. S5
Figure S5. Expression and phenotypes of fruit expressing p35S::SlGLK2. A. SlGLK2 expression as determined by hybridization of SlGLK2 specific probes to gel blots of RNA from ‘Ailsa Craig’ U/U or ‘M82’ u/u transformed with p35S::SlGLK2. B. Fruit phenotypes from representative plants of lines overexpressing (OE) or with co-suppressed expression (CS) of SlGLK2. Five transformed U/U lines and more than 5 transformed u/u lines showed the overexpression fruit phenotype. Four other transformed U/U lines showed the co-suppression phenotype.
24
Fig. S6
Figure S6. Summary of transcript abundance analysis by hybridization to EUTOM3 microarrays. A. Comparison of 3216 genes differentially expressed in AtGLK expressing lines relative to Tcontrol lines. B. Cellular components Gene Ontology (GO) terms of significantly (p<0.05, fold change >2) down-regulated genes in IM green fruit identified in EUTOM3 microarray hybridizations. The total number of genes with known GO terms is shown below bars. C. Hierarchical average linkage clustering of 3216 genes differentially expressed in AtGLK expressing lines relative to Tcontrol. Red and blue correspond to up- and down-regulation, respectively. D. 672 genes differentially expressed in both AtGLK1 and AtGLK2 expressing lines. E. Genes differentially expressed only in the AtGLK1 expressing lines. F. Genes differentially expressed only in the AtGLK2 expressing lines.
25
Fig. S7
Figure S7. Exposure of fruit to light determines soluble solids in ripe fruit. A. Ripe fruit (42 dpa) fruit phenotype of u/u ‘T63’ fruit that developed in normal light (Light) and in light blocking bags (Dark). B. Total soluble solids of juice from red ripe (42 dpa) fruit. Significant statistical differences determined by means of GLM and Tukey’s HSD at p<0.05 are indicated by different letters.
26
Fig. S8
A
B Phytoene Phytofluene Lutein γ-Carotene β-Carotene cis-Lycopene trans-Lycopene Total Carotenoids
Average ± SE Average ± SE Average ± SE Average ± SE Average ± SE Average ± SE Average ± SE Average ± SE
Tcontrol 4.23 ± 0.10 a 2.83 ± 0.44 a 0.95 ± 0.20 a 2.44 ± 0.09 a 4.35 ± 0.33 a 2.25± 0.21 a 121.58 ± 0.39 a 138.18 ± 0.81 a
p35S::AtGLK1 6.45 ± 0.87 a 4.41 ± 0.40 b 1.56 ± 0.14 a 3.42 ± 0.10 a 6.47 ± 0.51 a 3.58 ± 0.42 a 252.29 ± 17.28 b 278.51 ± 17.68 b
p35S::AtGLK2 4.61 ± 0.35 a 2.68 ± 0.20 a 1.80 ± 0.19 a 3.18 ± 0.14 a 6.72 ± 0.35 a 4.04 ± 0.73 a 166.28 ± 14.51 a 189.43 ± 16.18 a Figure S8. Carotenoid compounds in ripe fruit. A. Lycopene content of red fruit pericarp and epidermis measured as the absorbance at 503 nm of ethanol/hexane/acetone extracts of pulverized pericarp tissue. B. Carotenoid compound contents of red fruit measured by HPLC. Statistical significance determined by means of GLM and Bonferonni MCT at p<0.05 are indicated by different letters.
27
Table S1. Table S1. Eight predicted genes in the 60,507 bp region of S. lycopersicum chromosome 10 (ITAG2.4 Release: genomic annotations, http://solgenomics.net) between SL2.40ch10:2275056 and SL2.40ch10:2335463. SlGLK2 (yellow highlight) is within this region, specifically between Sl2.40chr10:2291209 and Sl240chr10:2295578. Start Stop Gene ID Identifier 2276303 2278610 Solyc10g008140 Unidentified, length=2308
2281545 2281874 Solyc10g008150 Glutaredoxin (AHRD V1 ***- B9MYC1_POPTR); B contains Interpro domain(s) IPR011905 Glutaredoxin-like%2C plant II
Table S2. Table S2. Relative expression of SlGLK1 and SlGLK2 as determined by qRT-PCR of cDNA prepared from young fully expanded leaves and from the pedicellar shoulder (Pedicel) and stylar (Style) portions of ‘Ailsa Craig’ U/U or ‘Craigella’ u/u green fruit (25 dpa) that developed in normal light conditions. qRT-PCR reactions were done on 4 - 5 biological replicated samples. Values were normalized to the expression of actin and expression of SlGLK1 and SlGLK2 is relative to the expression of SlGLK1 in the pedicellar portion of u/u. Standard errors are indicated.
Table S3 Table S3. Relative expression of SlGLK1 and SlGLK2 as determined by qRT-PCR of cDNA prepared from the pedicellar shoulder (Pedicel) or the blossom stylar (style) portions of green ‘Ailsa Craig’ U/U or ‘Craigella’ u/u fruit (25 dpa) that developed in normal light conditions (Light) or in light blocking bags (Dark). qRT-PCR reactions were done on 4-5 biologically replicated samples. Values were normalized to the expression of actin and expression of SlGLK1 and SlGLK2 is relative to the expression of SlGLK1 in the pedicellar portion of u/u fruit that developed in the light. Standard errors are indicated.
Table S4 – attached separately Table S4 (ST.4). Genes (3215 genes) identified as differentially expressed in p35S::AtGLK expressing lines versus wild-type (WT, TControl). Functional annotations are based on ITAG2.3 and ANOVA 3 way-LSD statistics. The sample types are WT (Tcontrol), GLK1 (expressing p35S::AtGLK1) and GLK2 (expressing p35S::AtGLK2). The annotations in this file are: Gene identity (ID) corresponding to the mRNA ID (Tomato whole genome sequence (WGS) cDNA ITAG 2.3) and Cluster based on Venn diagrams. Gene Functional Annotations:GO terms, Tomato WGS cDNA ITAG 2.3 hit name (exons); ITAG description; nearest 3-prime marker; nearest 5-prime marker; pseudo-molecule and position; tomato cDNA TAIR10 best hit; Arabidopsis gene symbol; Arabidopsis gene description; Arabidopsis component GO term and general cellular component. Matches with previous work with GLKs, photomorphogenesis regulators: Genes reported in Waters et al. (32), Savitch et al. (33), Rohrmann et al. (34) and Enfissi et al. (35), Kolotilin et al. (2007). Statistics: probe id, p-value(type), p-value(replicate), p-value(sample type(type)), p-value(WT * WT vs. transgenic * GLK1), Fold-Change(WT * WT vs. transgenic * GLK1), Fold-Change (WT * WT vs. transgenic * GLK1) (Description), p-value (WT * WT vs. transgenic * GLK2), Fold-Change (WT * WT vs. transgenic * GLK2), Fold-Change (WT * WT vs. transgenic * GLK2) (Description), F (type), SS (type), F (sample type(type)), SS (sample type(type)), F (replicate), SS (replicate), SS (Error), F (Error). Red cells indicate p<0.05.
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Table S5- attached separately
Table S5 (ST.5). Results of GO term enrichment analysis of AtGLK expressing lines vs WT (Tcontrol). For enrichment all Venn diagram sectors are considered.
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Table S6- attached separately
Table S6 (ST-6). GO term description for functional categories with p-value <0.1
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