Transcriptional Analysis of Masson Pine (Pinus massoniana) under High CO2 Stress
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material and Experimental Conditions
2.2. Total RNA Isolation, Complementary DNA Library Preparation and Sequencing
2.3. Transcriptome Assembly and Functional Annotation
2.4. Differential Expression Analysis
2.5. Quantitative Real-Time PCR Validation
3. Results
3.1. Transcriptome Sequencing and De Novo Assembly
3.2. Gene Expression and KEGG Enrichment Analysis under CO2 Stress
3.3. Energy Metabolism under Elevated CO2 Stress
3.4. Biosynthesis of Sucrose, Starch and Cell Wall Components under Elevated CO2 Stress
3.5. Hormone Regulation under Elevated CO2 Stress
3.6. Validation by Quantitative Real-Time PCR
4. Discussion
4.1. Effects of Elevated CO2 on Masson Pine Energy Metabolism
4.2. Effects of Elevated CO2 on Masson Pine Carbohydrate and Cell Wall Component Synthesis
4.3. Effects of Elevated CO2 on Masson Pine Cell Wall Component Synthesis
4.4. Effects of Elevated CO2 on Masson Pine Hormones and Stomatal Regulation
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
Primer | Sequence (5′→3′) | |
---|---|---|
RHM | Forward | TACGAATAGTCTCTGGCTTGTGAG |
Reverse | TCTGGTTGTGTCCTTGACCTAATA | |
CYP | Forward | TCTATGGTGATCACTGGAGAAAGA |
Reverse | GATGAGAGAATGGTTGAGAATGTG | |
CPS | Forward | TACTCGGTGTTATAAGTGCAGCTC |
Reverse | CATGTAGCCCTTGACACAAAATAG | |
OPR | Forward | TACGATACGGGAACAACTACTGAA |
Reverse | TCGAGCTCTAAAAACTGAGGAGAT | |
PAL | Forward | GAAGCCTGAGTTTACAGATCCATT |
Reverse | CGTAAACCACTTCAATCACTTCAC | |
CesA | Forward | GGAAGGCTGTACTTTATCCTTCAA |
Reverse | ATGCAAGACCAGATACAAGAGACA | |
SBP | Forward | TACCAGCCCAATAACAATAACCAC |
Reverse | CTCTCATCCACGAAGCTAATAACC | |
TPI | Forward | CCCTCTGCCACTTTCTTTATGTC |
Reverse | TCTAAGACTTCCTCACTTCTCCG | |
Enolase | Forward | AAGAGCTGCAAGGTAAAGTCTGTT |
Reverse | TCTGATTCACCTTCAGCAGTAAAG | |
qRT-PCR-RHM | Forward | CCACATCCTCACAGTAGAGATAGC |
Reverse | CCGGTGATTACTACCAGAGGTAAC | |
qRT-PCR-CYP | Forward | AGGCCCTTCCTCAGAGGTTATCT |
Reverse | CCGGAGTTGGTACTAGTCTTGGTA | |
qRT-PCR-CPS | Forward | GTCTAGAGCGGTTCACTCAGAT |
Reverse | CCTCTCTCCAACTATCACTGTGTC | |
qRT-PCR-OPR | Forward | GGTACCGTTCTTACTGGTTTGAGG |
Reverse | GATCCTGTAGTTGGCTACACAGAC | |
qRT-PCR-PAL | Forward | CCCTCAGGTGGAGATTATCAG |
Reverse | CCTCCATGTAGAGCTTTGTCTC | |
qRT-PCR-CesA | Forward | CCTGTACGGAGTAAGTTTGGTG |
Reverse | ACCAGTGGAGGTAGATATGCTG | |
qRT-PCR-SBP | Forward | CTACAGAGATAGGAGAGGGGAAAC |
Reverse | CTCCTGTGTATCGGAGTGTGTACT | |
qRT-PCR-TPI | Forward | CCTCCCACTTCTACTAGGGTTT |
Reverse | ACCAGCCAGGAGTAGTTAAGAGTG | |
qRT-PCR-Enolase | Forward | GGCCAGACAGATTATAGACAGC |
Reverse | CTCTCATCTCTAGGGCCTCATA |
Abbr. | Full Name | Abbr. | Full Name |
---|---|---|---|
PS | Photosystem | LEC | Light Harvesting Complex |
PC | Plastocyanin | RuBP | Ribulose-1,5-Bisphosphate |
3-PG | 3-Phosphoglycerate | 1,3-DPG | 1,3-Diphosphoglycerate |
PGAL | 3-Phosphoglyceraldehyde | DHAP | Dihydroxy-Acetone Phosphate |
FBP | Fructose-1,6-Diphosphate | F-6-P | Fructose-6-Phosphate |
E-4-P | Erythrose 4-Phosphate | SBP | Sedoheptulose-1,7-Diphosphate |
S-7-P | Sedoheptulose-7-Phosphate | R-5-P | Ribose-5-Phosphate |
Ru-5-P | Ribulose-1,5-Phosphate | Xu-5-P | Xylulose-5-Phosphate |
6-PG | 6-Phosphogluconate | 6-PG-L | Gluconolactone-6-Phosphate |
G-6-P | Glucose-6-Phosphate | 2-PG | 2-Phosphoglycerate |
PEP | Phosphoenolpyruvic acid | EP | Enolpyruvic Acid |
Pyr | Pyruvic Acid | AcCoA | Acetyl CoA |
ICL | Isocitric Acid | KGA | α-Ketoglutaric Acid |
SUA | Succinate Acid | H2MA | Malic Acid |
OAA | Oxaloacetic Acid | RCA | Rubisco Activase |
PGK | Phosphoglycerate Kinase | GAPD | Glyceraldehyde-3-Phosphate Dehydrogenase |
TPI | Triose-Phosphate Isomerase | TRA | Transaldolase |
FBPase | Fructose-1,6-Bisphosphatase | TKL | Transketolase |
RPI | Ribulose Phosphate Isomerase | SBPase | Sedoheptulose-1,7-Bisphosphatase |
RPK | Phosphoribulokinase | RPE | Ribulose Phosphate Epimerase |
6-PGDH | 6-Phosphogluconate Dehydrogenase | G-6-PD | Glucose-6-Phosphate-Dehydrogenase |
G-6-Pase | Glucose-6-Phosphatase | HK | Hexokinase |
PK | Pyruvate Kinase | PGAM | Phosphoglycerate Mutase |
CS | Citrate Synthase | ICD | Isocitrate Dehydrogenase |
OGDC | α-Ketoglutarate Dehydrogenase | SDH | Succinate Dehydrogenase |
MDH | Malate Dehydrogenase | TP | Triose Phosphate |
ADP-Glc | Adenosine Diphosphate Glucose | UDP-Glc | Uridine Diphosphate Glucose |
M-6-P | Mannose-6-Phosphate | M-1-P | Mannose-1-Phosphate |
GDP-Man | Guanosine Diphosphate Mannose | GDP-Fuc | Guanosine Diphosphate Fucose |
UDP-Rha | Uridine Diphosphate Rhamnose | UDP-Gal | Uridine Diphosphate Galacturonate |
UDP-GlcA | Uridine Diphosphate Glucuronate | UDP-Xyl | Uridine Diphosphate Xylose |
UDP-Api | Uridine Diphosphate Apiose | UDP-Arap | Uridine Diphosphate Arabinose, Pyranose form |
UDP-Araf | Uridine Diphosphate, Furanose form | FDA | Fructose-Bisphosphate Aldolase |
GPI | Phosphoglucose Isomerase | PGM | Phosphoglucomutase |
SP | Starch Phosphorylase | AGP | Adenosine Diphosphoglucose Pyrophosphorylase |
SSS | Soluble Starch Synthase | GBSS | Granule-bound Starch Synthase |
SBE | Starch Branching Enzyme | UGP | UDP-Glucose Pyrophosphorylase |
CesA | Cellulose Synthase Catalytic Subunit | Csl | Cellulose Synthase |
MPI | Phosphomannose Isomerase | PMM | Phosphomannomutase |
MGP | Mannose-1-Phosphate Guanylyltransferase | GMD | GDP-Mannose 4,6-Dehydratase |
INV | Invertase | cwINV | Cell Wall Invertase |
SPS | Sucrose-Phosphate Synthase | SUS | Sucrose Synthase |
RHM | Rhamnose Biosynthetic Enzyme | UGE | UDP-Glucose 4-Epimerase |
UGD | UDP-Glc Dehydrogenase | UXS/AXS | UDP-Glucuronate Decarboxylases |
UAE | UDP-Arabinose 4-Epimerase | UAM | UDP-Arabinose Mutase |
ABA | Abscisic Acid | GA | Gibberellin |
SA | Salicylic Acid | JA | Jasmonate |
BR | Brassinolide | MVA | Mevalonic Acid |
IPP | Isopentenyl Pyrophosphate | Zx | Zeaxanthin |
ATV | All-trans Violaxanthin | 9-cis-Nx | 9-cis-Neoxanthin |
GPP | Geranyl- Pyrophosphate | FPP | Farnesyl Pyrophosphate |
GGPP | Geranylgeranyl Pyrophosphate | CPP | Cuban Pyrophosphate |
KA | Kaurenoic Acid | Sha | Shikimic Acid |
Cha | Chorismic acid | ICA | Isochorismic Acid |
L-Aro | L-Arogenate | Ca | Cinnamic Acid |
Ba | Benzoic Acid | ALA | Linolenic Acid |
HPALA | 13-Hydrogen Peroxide Linolenic Acid | EALA | 12,13-Epoxylinolenic Acid |
OPDA | 12-Oxophytodienoic Acid | CR | Campesterol |
CHOLK | Cholesten-3-Ketone | CN | Campestanol |
OCN | 6-Oxocampestanol | CT | Cathasterone |
6DCT | 6-Deoxycathasterone | TE | Teasterone |
6DTE | 6-Deoxyteasterone | 3DT | 3-Dehydrocathasterone |
6D3DT | 6-Deoxy-3-Dehydrocathasterone | TY | Typhasterol |
6DTY | 6-Deoxytyphasterol | CS | Castasterone |
6DCS | 6-Deoxycastasterone | OTS1 | Open Stomata 1 |
SnRK2 | Sucrose Non-fermenting 1-related Kinase | MAPK | Mitogen Activated Protein Kinase |
ZEP | Zeaxanthin Epoxidase | NCED | 9-cis-Epoxycarotenoid Dioxygenase |
SDR | Short-chain Dehydrogenase/reductase | AAO | ABA Aldehyde Oxidase |
ICS | Isochorismate Synthase | PAL | Phenylalanine Ammonialyase |
GGPS | Geranylgeranylpyrophosphate | CPS | Copalyl Pyrophosphate Synthase |
KS | Kaurene Synthase | KO | Kaurene Oxidase |
GA | Gibberellin Oxidase | LOX | Lipoxygenase |
AOS | Allene Oxide Synthase | AOC | Allene Oxide Cyclase |
OPR | 12-Oxophytodienoate Reductase | DWF | Trans-Cinnamate 4-Hydroxylase |
ROT | C-23 Hydroxylase | CPD | Coumarate-3-Hydroxylase |
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CK | 6 h | 12 h | 24 h | |
---|---|---|---|---|
Raw reads | 49,314,299 | 47,459,322 | 45,980,036 | 60,876,932 |
Raw bases | 7,446,459,199 | 7,166,357,723 | 6,942,985,436 | 9,192,416,732 |
Clean reads | 48,795,571 | 46,976,134 | 45,496,760 | 60,205,674 |
Clean bases | 7,292,383,178 | 7,017,302,463 | 6,802,876,221 | 9,000,454,730 |
Error rate (%) | 0.02 | 0.02 | 0.03 | 0.03 |
Mapped reads | 16,879,027 | 16,459,249 | 15,859,340 | 21,108,414 |
Mapped ratio (%) | 0.69 | 0.70 | 0.70 | 0.70 |
GC content (%) | 47.47 | 46.74 | 46.24 | 46.32 |
Q20 (%) | 98.12 | 98.20 | 98.06 | 97.86 |
Q30 (%) | 94.04 | 94.22 | 93.86 | 93.40 |
Type | Transcript | Unigenes |
---|---|---|
<500 bp | 61,696 | 48,592 |
501~1000 bp | 37,586 | 22,267 |
1001~2000 bp | 25,994 | 12,887 |
>2000 bp | 15,587 | 8,678 |
Total | 140,863 | 92,424 |
Min length (bp) | 201 | 201 |
Max length (bp) | 15,491 | 15,491 |
Mean length (bp) | 891 | 935 |
N50 (bp) | 1463 | 1550 |
TransRate score | 0.20045 | 0.30498 |
BUSCO score | 77.7% | 74.2% |
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Wu, F.; Sun, X.; Zou, B.; Zhu, P.; Lin, N.; Lin, J.; Ji, K. Transcriptional Analysis of Masson Pine (Pinus massoniana) under High CO2 Stress. Genes 2019, 10, 804. https://doi.org/10.3390/genes10100804
Wu F, Sun X, Zou B, Zhu P, Lin N, Lin J, Ji K. Transcriptional Analysis of Masson Pine (Pinus massoniana) under High CO2 Stress. Genes. 2019; 10(10):804. https://doi.org/10.3390/genes10100804
Chicago/Turabian StyleWu, Fan, Xiaobo Sun, Bingzhang Zou, Peihuang Zhu, Nengqing Lin, Jingquan Lin, and Kongshu Ji. 2019. "Transcriptional Analysis of Masson Pine (Pinus massoniana) under High CO2 Stress" Genes 10, no. 10: 804. https://doi.org/10.3390/genes10100804