Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum
Abstract
:1. Introduction
2. Results
2.1. Effects of SbPPCK1-3 Silencing on SbPPCK Gene Expression and PEPCk Activity
2.2. Impact of SbPPCK1-3 Silencing on Phosphorylation of PEPC and Vegetative Development
2.3. Metabolic Changes in Ppck Lines
2.4. Impact of SbPPCK1-3 Silencing on Reproductive Development and Seed Production
2.5. Impact of SbPPCK1-3 Silencing on the Composition of Seeds
3. Discussion
4. Materials and Methods
4.1. Plant Material and Growth Conditions
4.2. Generation of Sorghum Ppck Lines
4.3. Selection of Transformed Ppck Plants
4.4. Site of Insertion of the T-DNA into Ppck Lines Genome
4.5. Determination of Enzyme Activity and Protein Quantification
4.6. RNA Extraction and cDNA Synthesis
4.7. Quantitative Real-Time PCR (qPCR)
4.8. Quantification of Amino Acids
4.9. Determination of C/N Ratio
4.10. Quantification of Starch
4.11. Quantification of Fatty Acids
4.12. Quantification of Tannins
4.13. Quantification of Phytates
4.14. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Line | Pro-Q/SYPRO (%) |
---|---|
Wt dark | 100 |
Wt light | 138 ± 22 |
Ppck-2 dark | 134 ± 29 |
Ppck-2 light | 141 ± 36 |
Ppck-4 dark | 119 ± 25 |
Ppck-4 light | 125 ± 24 |
Line | Treatment | Plant Length (cm) | Shoot Weight (g) | Root Weight (g) |
---|---|---|---|---|
Wt | Control | 54.3 ± 0.5 | 5.4 ± 0.2 | 4.1 ± 0.3 |
NaCl | 20.9 ± 0.3 * | 0.5 ± 0.1 * | 0.5 ± 0.1 | |
Ppck-2 | Control | 34.2 ± 3.6 a | 1.6 ± 0.1 a | 1.7 ± 0.2 |
NaCl | 12.4 ± 0.1 *a | 0.2 ± 0.1 *a | 0.2 ± 0.1 | |
Ppck-4 | Control | 50.8 ± 1.1 a | 5.4 ± 0.7 | 3.8 ± 0.7 |
NaCl | 19.0 ± 2.4 * | 0.4 ± 0.1 * | 0.3 ± 0.1 |
Line | Treatment | Plant Length (cm) | Shoot Weight (g) | Root Weight (g) |
---|---|---|---|---|
Wt | Control | 61.5 ± 0.7 | 22.9 ± 1.8 | 11.5 ± 1.1 |
-P | 29.7 ± 3.1 * | 3.0 ± 0.2 * | 4.0 ± 0.1 * | |
PCa | 51.6 ± 2.0 * | 12.1 ± 3.1 * | 9.4 ± 2.2 | |
Ppck-2 | Control | 56.6 ± 1.2 a | 22.6 ± 3.9 | 11.2 ± 1.4 |
-P | 26.5 ± 0.2 * | 2.8 ± 0.1 * | 3.8 ± 0.1 * | |
PCa | 48.8 ± 1.5 *a | 10.8 ± 2.8 | 8.1 ± 1.4 |
Amino Acid (mg g−1 fw) | Wt | Ppck-2 | Ppck-4 |
---|---|---|---|
Asp | 1.82 ± 0.02 | 1.78 ± 0.04 | 1.57 ± 0.10 |
Glu | 2.11 ± 0.02 | 2.00 ± 0.04 | 1.70 ± 0.08 a |
Ala | 1.36 ± 0.05 | 1.40 ± 0.02 | 1.22 ± 0.08 |
Arg | 1.01 ± 0.02 | 0.93 ± 0.02 a | 0.86 ± 0.05 a |
Cys | 0.07 ± 0.01 | 0.06 ± 0.01 | 0.07 ± 0.01 |
Phe | 1.28 ± 0.02 | 1.23 ± 0.02 | 1.14 ± 0.05 a |
Gly | 1.04 ± 0.07 | 1.01 ± 0.06 | 0.97 ± 0.07 |
His | 0.60 ± 0.01 | 0.57 ± 0.01 | 0.52 ± 0.03 a |
Ile | 0.78 ± 0.01 | 0.75 ± 0.02 | 0.67 ± 0.03 a |
Leu | 1.79 ± 0.03 | 1.70 ± 0.05 | 1.47 ± 0.10 a |
Lys | 1.46 ± 0.02 | 1.36 ± 0.03 a | 1.25 ± 0.08 a |
Met | 0.32 ± 0.01 | 0.34 ± 0.01 | 0.22 ± 0.03 a |
Pro | 1.30 ± 0.01 | 1.29 ± 0.06 | 1.17 ± 0.02 a |
Ser | 0.79 ± 0.01 | 0.78 ± 0.01 | 0.73 ± 0.04 |
Tyr | 0.52 ± 0.01 | 0.49 ± 0.02 | 0.45 ± 0.04 |
Hyp | 0.09 ± 0.01 | 0.09 ± 0.01 | 0.08 ± 0.01 |
Thr | 0.91 ± 0.01 | 0.89 ± 0.01 | 0.81 ± 0.05 |
Val | 0.89 ± 0.04 | 0.75 ± 0.01 a | 0.73 ± 0.01 a |
Line | Amino Acids (mg g−1 fw) | Total C (%) | Total N (%) | C/N Ratio |
---|---|---|---|---|
Wt | 18.10 ± 0.18 | 39.73 ± 2.58 | 3.92 ± 0.19 | 10.12 ± 0.26 |
Ppck-2 | 17.39 ± 0.40 | 42.93 ± 0.40 | 4.17 ± 0.07 | 10.30 ± 0.11 |
Ppck-4 | 15.62 ± 0.81 a | 42.55 ± 0.14 | 3.76 ± 0.08 | 11.32 ± 0.26 a |
Fatty Acid (%) | Wt | Ppck-2 | Ppck-4 |
---|---|---|---|
Palmitic acid | 26.88 ± 0.68 | 25.55 ± 0.38 | 28.01 ± 0.63 |
Palmitoleic acid | 1.26 ± 0.04 | 1.55 ± 0.06 a | 1.59 ± 0.06 a |
Suberic acid | 0.18 ± 0.01 | 0.29 ± 0.01 a | 0.34 ± 0.03 a |
Azelaic acid | 0.16 ± 0.01 | 0.30 ± 0.02 a | 0.29 ± 0.02 a |
Stearic acid | 12.54 ± 1.48 | 12.59 ± 0.25 | 14.17 ± 0.71 |
Oleic acid | 7.73 ± 2.98 | 2.96 ± 0.39 | 2.29 ± 0.63 |
Linoleic acid | 6.67 ± 0.59 | 8.17 ± 0.48 | 5.86 ± 0.22 |
γ-Linolenic acid | 0.37 ± 0.05 | 0.26 ± 0.04 | 0.34 ± 0.04 |
Arachidic acid | 0.53 ± 0.04 | 0.53 ± 0.07 | 0.60 ± 0.08 |
α-Linolenic acid | 28.64 ± 3.61 | 34.85 ± 0.58 | 32.44 ± 1.06 |
Eicosenoic acid | 0.74 ± 0.26 | 0.33 ± 0.04 | 0.17 ± 0.02 a |
Arachidonic acid | 0.11 ± 0.02 | 0.25 ± 0.04 a | 0.33 ± 0.04 a |
Line | Panicle Length (cm) | Panicle Weight (g) | Seed Weight (g) | Seed Number |
---|---|---|---|---|
Wt | 9.3 ± 0.8 | 6.9 ± 0.8 | 5.7 ± 0.6 | 204 ± 20 |
Ppck-2 | 7.3 ± 0.1 a | 4.0 ± 0.5 a | 3.4 ± 0.4 a | 101 ± 20 a |
Ppck-4 | 8.2 ± 0.6 | 5.2 ± 1.1 | 4.3 ± 0.6 | 131 ± 22 |
Amino Acid (mg g−1 fw) | Wt | Ppck-2 | Ppck-4 |
---|---|---|---|
Asp | 4.86 ± 0.40 | 3.45 ± 0.18 a | 2.44 ± 0.39 a |
Glu | 16.01 ± 1.55 | 9.91 ± 0.62 a | 6.64 ± 1.12 a |
Ala | 6.62 ± 0.46 | 4.23 ± 0.26 a | 2.89 ± 0.57 a |
Arg | 2.56 ± 0.30 | 1.94 ± 0.02 | 0.29 ± 0.04 a |
Cys | 0.54 ± 0.03 | 0.41 ± 0.01 a | 0.07 ± 0.01 a |
Phe | 3.50 ± 2.61 | 2.55 ± 0.16 a | 1.85 ± 0.27 a |
Gly | 2.30 ± 0.21 | 1.64 ± 0.10 a | 1.20 ± 0.22 a |
His | 2.09 ± 0.17 | 1.49 ± 0.08 a | 1.01 ± 0.15 a |
Ile | 2.38 ± 0.21 | 1.59 ± 0.06 a | 1.15 ± 0.19 a |
Leu | 10.40 ± 0.95 | 6.76 ± 0.40 a | 4.25 ± 0.69 a |
Lys | 1.95 ± 0.18 | 1.47 ± 0.08 a | 1.08 ± 0.17 a |
Met | 0.43 ± 0.09 | 0.25 ± 0.02 | 0.28 ± 0.02 |
Pro | 7.85 ± 0.60 | 5.02 ± 0.25 a | 3.81 ± 0.72 a |
Ser | 2.99 ± 0.22 | 2.01 ± 0.13 a | 1.31 ± 0.20 a |
Tyr | 0.82 ± 0.01 | 0.71 ± 0.03 | 0.48 ± 0.08 a |
Hyp | 0.12 ± 0.01 | 0.11 ± 0.01 a | 0.09 ± 0.01 a |
Thr | 2.23 ± 0.18 | 1.57 ± 0.07 a | 1.10 ± 0.18 a |
Val | 3.42 ± 0.35 | 2.12 ± 0.18 a | 1.43 ± 0.25 a |
Line | Amino Acids (mg g−1 fw) | Starch (µmol mg−1) | Tannins (mg g−1) | Phytates (g%) |
---|---|---|---|---|
WT | 71.05 ± 6.13 | 1.43 ± 0.02 | 1.39 ± 0.36 | 4.37 ± 0.08 |
Ppck-2 | 47.23 ± 2.66 | 1.41 ± 0.03 | 2.36 ± 0.25 a | 4.78 ± 0.07 a |
Ppck-4 | 32.52 ± 5.46 a | 1.23 ± 0.04 a | 1.50 ± 0.36 | 5.47 ± 0.08 a |
Line | Total C (%) | Total N (%) | C/N Ratio |
---|---|---|---|
Wt | 41.90 | 1.51 | 27.75 |
Ppck-2 | 40.64 | 1.32 | 30.81 |
Ppck-4 | 40.07 | 1.13 | 35.59 |
Fatty Acid (%) | Wt | Ppck-2 | Ppck-4 |
---|---|---|---|
Palmitic acid | 19.20 | 24.81 | 19.91 |
Palmitoleic acid | 0.49 | 1.24 | 0.16 |
Suberic acid | 0.05 | 0.05 | 0.05 |
Azelaic acid | 0.05 | 0.05 | 0.04 |
Stearic acid | 5.69 | 11.09 | 7.40 |
Oleic acid | 26.62 | 22.21 | 25.45 |
Linoleic acid | 34.00 | 33.64 | 39.40 |
γ-Linolenic acid | 0.16 | 0.20 | 0.31 |
Arachidic acid | 0.16 | 0.00 | 0.00 |
α-Linolenic acid | 1.52 | 1.43 | 2.40 |
Eicosenoic acid | 1.31 | 0.18 | 0.07 |
Arachidonic acid | 0.10 | 0.06 | 0.36 |
Line | Plant Length (cm) | Panicle Length (cm) | Panicle Weight (g) | Seed Weight (g) | Seed Number |
---|---|---|---|---|---|
Wt | 142.1 ± 3.2 | 13.5 ± 0.6 | 12.0 ± 2.6 | 9.9 ± 2.2 | 365.4 ± 64.7 |
Ppck-1 | 138.9 ± 3.2 | 14.4 ± 1.0 | 10.8 ± 0.8 | 9.9 ± 1.2 | 300.0 ± 48.0 |
Ppck-2 | 128.7 ± 4.2 a | 12.1 ± 0.7 | 9.2 ± 1.7 | 8.3 ± 1.4 | 271.2 ± 52.7 |
Ppck-3 | 119.7 ± 4.5 a | 13.8 ± 0.8 | 17.0 ± 2.2 a | 12.8 ± 2.3 | 478.9 ± 67.0 |
Ppck-4 | 150.8 ± 3.8 | 11.7 ± 0.3 a | 9.0 ± 0.8 | 8.2 ± 0.7 | 268.3 ± 23.7 |
Ppck-5 | 147.0 ± 7.6 | 13.2 ± 1.3 | 11.2 ± 2.2 | 13.0 ± 5.2 | 453.3 ± 133.1 |
Ppck-6 | 144.7 ± 4.5 | 11.4 ± 0.5 a | 9.4 ± 0.7 | 7.5 ± 0.8 | 245.8 ± 23.7 |
Ppck-7 | 137.8 ± 8.6 | 15.1 ± 0.7 | 18.2 ± 3.0 | 15.4 ± 2.2 a | 587.3 ± 89.6 a |
Ppck-8 | 138.0 ± 5.1 | 12.5 ± 1.4 | 11.4 ± 2.3 | 9.4 ± 1.8 | 391.8 ± 98.4 |
Ppck-9 | 92.5 ± 7.4 a | 10.9 ± 0.7 a | 3.5 ± 0.9 a | 1.6 ± 0.7 a | 42.7 ± 18.0 a |
Ppck-10 | 135.4 ± 9.8 | 12.1 ± 1.1 | 8.0 ± 2.3 | 7.1 ± 2.0 | 309.8 ± 89.3 |
Ppck-11 | 126.6 ± 6.0 a | 12.5 ± 0.9 | 10.0 ± 1.4 | 8.2 ± 1.0 | 300.2 ± 47.5 |
Ppck-12 | 139.6 ± 5.6 | 14.6 ± 0.8 | 13.5 ± 1.4 | 11.0 ± 1.2 | 365.4 ± 64.7 |
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Pérez-López, J.; Feria, A.B.; Gandullo, J.; de la Osa, C.; Jiménez-Guerrero, I.; Echevarría, C.; Monreal, J.A.; García-Mauriño, S. Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum. Plants 2023, 12, 2426. https://doi.org/10.3390/plants12132426
Pérez-López J, Feria AB, Gandullo J, de la Osa C, Jiménez-Guerrero I, Echevarría C, Monreal JA, García-Mauriño S. Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum. Plants. 2023; 12(13):2426. https://doi.org/10.3390/plants12132426
Chicago/Turabian StylePérez-López, Jesús, Ana B. Feria, Jacinto Gandullo, Clara de la Osa, Irene Jiménez-Guerrero, Cristina Echevarría, José A. Monreal, and Sofía García-Mauriño. 2023. "Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum" Plants 12, no. 13: 2426. https://doi.org/10.3390/plants12132426