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Figure 1.
cDNA-AFLP profiles using one EcoR I selective primer and eight Mes I selective primers. One EcoR I selective primer: EcoR I-AC; Eight Mes I selective primers: Mes I-AA, AG, AC, AT, CC, CG, CT, and CA; Lane 1: Normal juice sacs of C. grandis; Lane 2: Granulated juice sacs of C. grandis; Arrows indicate differentially expressed transcript-derived fragments.
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Figure 2.
Functional classification of differentially expressed transcript-derived fragments (TDFs) in normal and granulated juice sacs of C. grandis. Functional classification was performed based on information reported for each sequence in the NCBI database (
https://blast.ncbi.nlm.nih.gov/Blast.cgi ). -
Figure 3.
Relative expression levels of transcript-derived fragments (TDFs) in C. grandis normal and granulated juice sacs. (a) Relative expression levels of genes encoding β-amylase 4 (TDF #64-2); cytokinin-O-glucosyltransferase 1 (TDF #94-1); gibberellin 20 oxidase (TDF #13-2); galactose-1-phosphate guanylyltransferases (TDF #200-1); α-galactosidase precursor (TDF #160-1); cytochrome b5 (TDF #123-2); ADP-ribosylation factor 3 (TDF #246-4); 2-oxoglutarate-dependent dioxygenase (TDF #13-1); 1,4-alpha-glucan-maltohydrolase (TDF #20-3); ethylene insensitive 3-like protein (TDF #147-4). (b) Relative expression levels of genes encoding translation initiation factor 4A2 (TDF #14-3); cytochrome P450 (TDF #48-3); auxin down-regulated-like protein (TDF #5-3); cellulose synthase (TDF #249-3); transport protein SEC31 (TDF #34-2); Ca2+-transporting ATPase (TDF #252-3); heat shock protein (TDF #252-1); senescence-associated protein (TDF #5-1); dicer-like protein 4 (TDF #244-6); cell wall-associated hydrolase (TDF #216-3). Results are shown as the mean ± SD of three independent experiments. Different letters above bars indicate significant differences at P < 0.05.
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Figure 4.
Granulation led to alterations in hormone content in C. grandis juice sacs. The content of five hormones were measured by ultra-performance liquid chromatography mass-spectrometry (UPLC-MS). Normal juice sacs were considered as negative control. GA3: gibberellin 3; GA20: gibberellin 20, CK: cytokinins; ZT: zeatin; and IAA: indole-3-acetic acid. Error bars represent standard deviations calculated from three biological replicates. Different letters above bars indicate significant differences at P < 0.05.
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Figure 5.
Proposed regulatory network for the granulation phenomenon in C. grandis juice sacs. Red arrows indicate upregulated genes. HSP: heat-shock protein.
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Found only in normal Found only in granulated Found in both juice sacs Total Total TDFs detected 536 874 3,014 4,424 Total differentially expressed TDFs detected 68 89 26 183 TDFs produced useable sequence data 41 61 14 116 TDFs encoding known or putative proteins 38 57 11 106 TDFs encoding predicted, uncharacterized 8 9 2 19 TDFs without matches in the database 11 26 4 41 Table 1.
Summary of transcript-derived fragments (TDFs) in normal and granulated juice sacs of C. grandis.
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TDF number Size
(bp)Homologous protein Organism of origin E-value Degree of similarity (%) Genbank ID Fold change Hormone and biological regulation 48-2 292 CAAX amino terminal protease family Cucumis melo 7E-18 90 ADN33781.1 + 5-3 259 Auxin down-regulated-like protein, partial Picea sitchensis 5E-05 55 ADM77850.1 + 6-2 158 Protein embryo defective 2752 Arabidopsis thaliana 2E-12 61 NP_567830.1 0 118-3 290 Expressed protein Oryza sativa 5E-27 63 ABF95726.1 0 94-1 358 Cytokinin-O-glucosyltransferase 1 Aegilops tauschii 9E-25 48 EMT28784.1 + 103-1 211 Nitrate reductase Cucumis sativus 1E-30 89 ADK77877 + 219-5 244 ARF-GAP domain 2 Arabidopsis lyrata subsp. lyrata 5E-09 43 NP_176283.1 0 13-2 269 Gibberellin 20 oxidase Medicago truncatula 4E-42 81 AES62614.2 + 147-4 271 EIN3-like (Ethylene insensitive 3) protein C. melo 8E-41 100 BAB64345 + 195-6 197 MATE efflux family protein Theobroma cacao 2E-19 79 EOX90702.1 0 115-1 355 Transmembrane emp24 domain-containing protein p24delta9-like Crocus sativus 3E-60 98 XP_004143772.1 + 195-1 455 Multicatalytic endopeptidase complex A. thaliana 8E-69 84 CAA74030.1 + Carbohydrate and energy metabolism 134-1 192 ATP-binding protein-like A. thaliana 2E-10 75 BAB09414.1 + 249-1 311 UDP-glucosyltransferase family 1 protein Camellia sinensis 2E-25 93 ACS87991.1 + 6-1 214 Fructose-bisphosphate aldolase Lemna minor 1E-33 94 ACD10928.1 0 5-4 148 ATP synthase subunit beta Medicago truncatula 2E-13 95 XP_003627732.1 + 118-2 304 Glyceraldehyde-3-phosphate dehydrogenase, partial Vernicia fordii 4E-11 94 AFJ04516.1 0 118-1 441 Glycosyltransferase, CAZy family GT8 Populus trichocarpa 5E-75 90 XP_002312381.1 + 64-2 220 β-amylase 4 Citrus trifoliata 2E-22 69 AFQ33616 + 249-3 217 Cellulose synthase Populus tremula × Populus tremuloides 5E-12 91 AAT09895.1 + 141-3 499 Mitochondrial benzaldehyde dehydrogenase Antirrhinum majus 8E-89 80 ACM89738.1 + 160-1 174 α-galactosidase precursor Coffea arabica 2E-04 62 CAJ40777.1 + 13-1 269 2-oxoglutarate-dependent dioxygenase Populus trichocarpa 8E-48 89 XP_002330269.1 7.07 ± 0.52 200-1 389 Galactose-1-phosphate guanylyltransferases T. cacao 1E-65 83 EOY12255.1 + 20-3 220 1,4-alpha-glucan-maltohydrolase Solanum lycopersicum 1E-14 60 NP_001234052.1 + 246-3 192 ABC transporter family protein P. trichocarpa 1E-12 80 XP_002310031.2 0 221-3 123 Diacylglycerol kinase-like protein A. thaliana 6E-11 69 BAB09587.1 + 246-4 172 ADP-ribosylation factor 3 A. lyrata subsp. lyrata 4E-98 96 XP_002869315.1 0 65-1 363 Methylenetetrahydrofolate reductase family protein isoform 3 T. cacao 7E-49 72 EOY04345.1 0 Protein and nucleic acid metabolism 228-1 243 Ribonucleoside-diphosphate reductase
subunit M1A. thaliana 2E-33 89 AEC07222.1 0 251-1 188 BET1P/SFT1P-like protein 14A A. thaliana 6E-07 78 NP_191376.1 0 253-2 306 Class II aaRS and biotin synthetases
superfamily proteinA. thaliana 5E-41 83 NP_186925.4 + 130-3 193 Ribosomal protein L5 Citrullus lanatus 1E-30 96 YP_003587255.1 0 119-3 254 Nuclear transport factor 2 family protein T. cacao 1E-22 62 EOY06196.1 + 27-3 224 Glutathione S-transferase family protein T. cacao 1E-07 62 EOY27562.1 + 195-5 268 BRCA1-associated protein M. truncatula 8E-39 73 XP_003609376.2 0 54-2 169 Pre-mRNA splicing factor PRP38 family protein P.trichocarpa 2E-22 92 ERP53525.1 0 151-4 347 Ribonuclease II family protein A. thaliana 2E-51 80 NP_565418.1 + 127-1 180 Mitochondrial substrate carrier family protein isoform 2 T. cacao 2E-28 61 EOY07093.1 + 15-2 349 Nuclear transcription factor Y subunit B18 M. truncatula 1E-09 68 AFK49668.1 + 15-1 392 RRNA intron-encoded homing endonuclease M. truncatula 6E-13 88 XP_003614385.1 + 20-1 280 Solute carrier family 25 member M. truncatula 6E-23 63 XP_003615848.2 0 221-1 256 TPA: heterogeneous nuclear ribonucleoprotein A3-like protein 2 isoform 1 Zea mays 1E-29 82 DAA58966.1 0.56 ± 0.06 217-5 194 Adenine nucleotide alpha hydrolases-like superfamily protein T. cacao 2E-06 96 EOY06709.1 1.86 ± 0.34 215-1 363 Transcription regulator A. thaliana 1E-06 88 NP_171710.4 + 195-4 333 PLP-dependent transferases superfamily protein A. thaliana 2E-37 60 NP_191772.1 + 21-2 210 60S ribosomal protein L24-1 T. cacao 1E-37 97 EOY23121.1 + 155-3 187 Valyl-tRNA synthetase/valine-tRNA ligase T. cacao 3E-19 75 EOY31957.1 2.23 ± 0.11 252-2 299 ACT domain-containing protein ACR8 Ricinus communis 2E-30 86 XP_002509632.1 0.79 ± 0.03 151-5 347 Exosome complex exonuclease RRP44
homolog AR. communis 5E-53 82 XP_002521738.1 + 217-2 369 Transcription initiation factor TFIID subunit A A. thaliana 1E-22 77 NP_564023.1 + 218-1 239 Chaperonin 60 alpha subunit Arachis diogoi 1E-20 86 ACA23472.1 0 31-1 293 Aspartyl protease family protein A. thaliana 3E-23 57 XP_002891474.1 + 236-3 251 Protein kinase domain-containing protein A. thaliana 3E-30 80 AEE27605.1 0 247-1 324 Importin beta-2 subunit family protein A. thaliana 2E-16 76 XP_002867489.1 + 253-1 263 Serine/threonine protein kinase TNNI3K M. truncatula 2E-09 76 XP_003601186.1 0 5-2 151 Amino acid adenylation protein Calothrix sp. PCC 6303 9.9 40 YP_007137552.1 + 119-1 290 Ricin B-like lectin EULS3 A. lyrata subsp. lyrata 4E-36 73 XP_002862306.2 + 119-2 190 Spl1-Related 2 protein A. thaliana 2E-18 76 CAB56773.1 + 128-1 286 Chloroplast elongation factor TuB (EF-TuB) Nicotiana sylvestris 7E-12 90 BAA01975.1 + 94-2 242 Clone 6F8 eukaryotic initiation factor 4A-14 gene Nicotiana benthamiana 2E-47 89 JN688263.1 + 34-2 343 Transport protein SEC31 T. cacao 2E-06 81 EOY23302.1 + 91-1 199 Phosphatase 2C family protein isoform 2 T. cacao 2E-83 87 EOY06499.1 + 160-6 271 Kinase superfamily protein isoform 1 T. cacao 3E-34 78 EOY06443.1 0 209-3 337 Ser/Thr phosphatase-containing Kelch repeat domain protein, partial N. benthamiana 1E-50 90 AFN44702.1 0 220-3 208 Pentatricopeptide repeat (PPR) superfamily protein isoform 2 T. cacao 5E -08 51 EOY04957.1 2.25 ± 0.22 197-1 398 Ubiquitin-specific protease family C19-related protein A. thaliana 9E-68 79 NP_564009.1 + 87-2 220 Tetratricopeptide repeat (TPR)-like superfamily protein isoform 1 T. cacao 3E-27 86 EOY33236.1 2.55 ± 0.06 209-5 319 Serine/threonine-protein phosphatase, partial Genlisea aurea 3E-52 88 EPS64063.1 0 194-1 474 Proteasome subunit beta type-4 Solanum nigrum 6E-73 91 ADW66147.1 0 150-9 199 Serine/threonine-protein kinase AtPK2/AtPK19 R. communis 2.8 51 XP_002528702.1 + 123-4 246 IFA binding protein Lilium longiflorum 2E-37 74 ABM68547.1 0 123-3 302 Dual specificity kinase 1 Arabidopsis thaliana 3E-27 93 AEE27635 0 26-2 324 Dephospho-CoA kinase A. thaliana 1E-43 69 NP_180318.1 0 14-3 222 Translation initiation factor 4A2 Z. mays 7E-37 97 AAD20980.1 0.65 ± 0.01 244-1 302 Translation elongation factor, partial Ammopiptanthus mongolicus 1E-05 88 AFC01200.1 + 246-2 165 Eukaryotic translation initiation factor 5
isoform 2T. cacao 3E-05 52 EOX90767.1 0 Lipid metabolism 123-1 278 Patellin-5 A. lyrata subsp. lyrata 3E-13 67 XP_002872438.1 0 141-4 209 Patellin-5 Triticum urartu 8E-27 71 EMS62735
XP_0036230.27 ± 0.02 195-3 347 Non-specific lipid-transfer protein M. truncatula 1E-05 92 596.3 + 197-3 327 Pleckstrin (PH) and lipid-binding START domains-containing protein isoform 2 T. cacao 1E-47 78 EOY34386.1 + 125-2 278 Glyoxylate/hydroxypyruvate reductase
A HPR2A. lyrata subsp. lyrata 1E-29 70 XP_002889322.1 + Stress response and defense 48-1 263 Trehalose 6-phosphate synthase Nicotiana tabacum 3E-18 91 BAI99252.1 + 220-2 208 Transcription factor bHLH130 M. truncatula 4E-22 75 XP_003590427.1 1.41 ± 0.06 123-2 189 Cytochrome b5 N. tabacum 9E-26 80 CAA50575 + 48-3 268 Cytochrome P450 Citrus sinensis 2E-28 95 AAL24049.1 + 64-1 297 Cytochrome P450 A. thaliana 1E-23 81 NP_176086.1 + 221-2 451 Cytochrome oxidase subunit 1 Curcuma longa 1E-10 56 ABY83898.1 + 218-2 341 DNA damage-binding protein, partial M. truncatula 3E-64 67 XP003638090.1 0 252-1 353 Heat shock protein M. truncatula 6E-07 45 XP_003621962.1 0.36 ± 0.04 115-2 290 Stress responsive protein Z. mays 5E-32 64 NP_001149550.1 + 5-1 230 Senescence-associated protein Picea abies 2E-45 96 ACA04850.1 + 154-5 184 Dehydration-induced 19-like protein Gossypium hirsutum 8E-05 56 ADP30960.1 + 27-1 271 B-box zinc finger protein Bambusa oldhamii 7E-09 57 ACF35275.1 0 244-6 248 Dicer-like protein 4 A. thaliana 9E-09 63 NP_197532.3 + Cell metabolism 252-3 355 Ca2+-transporting ATPase A. thaliana 2E-04 72 NP_195479 + 130-1 240 Plasma membrane isoform 4 T. cacao 3E-29 83 EOY10146.1 0 244-2 287 Cinnamyl-alcohol dehydrogenase A. thaliana 3E-05 91 AAA99511.1 + 40-1 163 Clathrin adaptor complexes medium subunit family protein A. lyrata subsp. lyrata 3E-22 93 XP_002886592.1 0 154-3 429 RAB GTPase homolog A5A A. thaliana 1E-17 90 NP_199563.1 0.67 ± 0.01 217-6 323 Receptor-like kinase binding protein P. trichocarpa 4E-28 58 XP_002325092.1 0.87 ± 0.05 147-3 168 Calreticulin-like protein Solanum melongena 6E-22 88 BAA85118.1 3.35 ± 0.12 16-3 238 Ycf2 (chloroplast) Gossypium raimondii 3E-37 96 YP_005087735.1 + 143-4 350 Tetraspanin8 T. cacao 9E-24 69 EOY31574.1 + 216-3 443 Cell wall-associated hydrolase Vibrio cholerae 5E-39 76 ACX81677.1 0 200-2 283 Nucleic acid binding protein Z. mays 5E-43 89 NP_001152488.1 + Unknown biological processes 217-3 323 Hypothetical protein AT5G07270 A. thaliana 4E-09 59 NP_196344 0 236-1 256 Choline/ethanolamine kinase, putative Ricinus communis 4E-35 83 XP_002525542.1 + 236-2 322 Uncharacterized protein LOC8268581 R. communis 3E-31 82 XP_002530954.1 0 249-2 196 Amino acid transporter, putative R. communis 3E-03 39 XP_002531860.1 + 8-2 168 Predicted: monoacylglycerol lipase abhd6-B-like Fragaria vesca subsp. vesca 1E-18 80 XP_004303453.1 0 119-4 310 Domain of uncharacterized protein function 724 6, putative isoform 1 T. cacao 2E-22 68 EOX95351.1 0 57-1 205 Predicted: Vitis vinifera peroxidase 3-like mRNA Vitis vinifera 2E-21 91 XM_002280238.4 + 100-1 318 Hypothetical protein CICLE_v10006049mg Citrus clementina 6E-14 100 ESR32793.1 0 17-3 206 Hypothetical protein MTR_2g077840 M. truncatula 2E-3 76 XP_003596462.1 0 197-2 335 Mitochondrial protein, putative M. truncatula 3E-04 88 XP_003588355.1 0 217-4 261 Putative ATP synthetase alpha chain Oryza sativa subsp. japonica 3E-13 63 AAO72570.1 0 154-1 202 Hypothetical protein CICLE_v10022616mg Citrus clementina 7E-22 95 ESR54213.1 0 89-3 215 Hypothetical protein CICLE_v10033239mg C. clementina 3E-19 100 ESR51519.1 + TDFs: Transcript-derived fragments. Results are shown as the mean ± SD of at least three independent experiments. Fold change: 0 indicates TDFs only detected in normal juice sacs; + indicates TDFs only detected in granulated juice sacs. Relative expression ratio was obtained by analyzing gel images using PDQuest version 8.0.1 (Bio-Rad, Hercules, CA, USA). Table 2.
Homology of differentially expressed cDNA-AFLP fragments with known gene sequences in the database using BLASTX algorithm along with their expression patterns in granulation juice sacs of C. grandis.
Figures
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Tables
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