Silencing of Sb<i>PPCK1-3</i> Negatively Affects Development, Stress Responses and Productivity in Sorghum
Jesús Pérez-López | Ana B. Feria | Jacinto Gandullo | Clara de la Osa | Irene Jiménez-Guerrero | Cristina Echevarría | José A. Monreal | Sofía García-Mauriño
Phosphoenolpyruvate carboxylase (PEPC) plays central roles in photosynthesis, respiration, amino acid synthesis, and seed development. PEPC is regulated by different post-translational modifications. Between them, the phosphorylation by PEPC-kinase (PEPCk) is widely documented. In this work, we simultaneously silenced the three sorghum genes encoding PEPCk (Sb<i>PPCK1</i>-<i>3</i>) by RNAi interference, obtaining 12 independent transgenic lines (<i>Ppck1-12</i> lines), showing different degrees of Sb<i>PPCK1-3</i> silencing. Among them, two T2 homozygous lines (<i>Ppck-2</i> and <i>Ppck-4</i>) were selected for further evaluation. Expression of Sb<i>PPCK1</i> was reduced by 65% and 83% in <i>Ppck-2</i> and <i>Ppck-4</i> illuminated leaves, respectively. Expression of Sb<i>PPCK2</i> was higher in roots and decreased by 50% in <i>Ppck-2</i> and <i>Ppck-4</i> in this tissue. Expression of Sb<i>PPCK3</i> was low and highly variable. Despite the incomplete gene silencing, it decreased the degree of phosphorylation of PEPC in illuminated leaves, P-deficient plants, and NaCl-treated plants. Both leaves and seeds of <i>Ppck</i> lines had altered metabolic profiles and a general decrease in amino acid content. In addition, <i>Ppck</i> lines showed delayed flowering, and 20% of <i>Ppck-4</i> plants did not produce flowers at all. The total amount of seeds was lowered by 50% and 36% in <i>Ppck-2</i> and <i>Ppck-4</i> lines, respectively. The quality of seeds was lower in <i>Ppck</i> lines: lower amino acid content, including Lys, and higher phytate content. These data confirm the relevance of the phosphorylation of PEPC in sorghum development, stress responses, yield, and quality of seeds.
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