Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum

Citation: 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 Academic Editor: Baohong Zhang Received: 19 May 2023 Revised: 20 June 2023 Accepted: 21 June 2023 Published: 23 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum Jesús Pérez-López 1, Ana B. Feria 1, Jacinto Gandullo 1, Clara de la Osa 1, Irene Jiménez-Guerrero 2, Cristina Echevarría1, JoséA. Monreal 1and Sofía García-Mauriño 1,* 1Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes nº 6, 41012 Seville, Spain; jper[email protected] (J.P.-L.); [email protected] (A.B.F.); [email protected] (J.G.); [email protected] (C.d.l.O.); [email protected] (C.E.); [email protected] (J.A.M.) 2Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes nº 6, 41012 Seville, Spain; [email protected] *Correspondence: [email protected] Abstract: 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 (SbPPCK1-3) by RNAi interference, obtaining 12 independent transgenic lines (Ppck1-12 lines), showing different degrees of SbPPCK1-3 silencing. Among them, two T2 homozygous lines (Ppck-2 and Ppck-4) were selected for further evaluation. Expression of SbPPCK1 was reduced by 65% and 83% in Ppck-2 and Ppck-4 illuminated leaves, respectively. Expression of SbPPCK2 was higher in roots and decreased by 50% in Ppck-2 and Ppck-4 in this tissue. Expression of SbPPCK3 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 Ppck lines had altered metabolic profiles and a general decrease in amino acid content. In addition, Ppck lines showed delayed flowering, and 20% of Ppck-4 plants did not produce flowers at all. The total amount of seeds was lowered by 50% and 36% in Ppck-2 and Ppck-4 lines, respectively. The quality of seeds was lower in Ppck 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. Keywords: phosphoenolpyruvate carboxylase; phosphoenolpyruvate carboxylase kinase; protein phosphorylation; seed production; seed quality; Sorghum bicolor 1. Introduction Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is a key enzyme in the metabolism of carbon and nitrogen, with central roles in photosynthesis, respiration, amino acid synthesis, and development and germination of seeds. This enzyme catalyzes the addition of bicarbonate to PEP to form oxaloacetate, which is reduced to malate by the enzyme malate dehydrogenase (MDH). PEPC is mostly recognized by its role in C 4 and CAM photosynthesis [ 1 , 2 ], though it has also key functions in C 3 plants and C 3 tissues, such as seeds, fruits, roots, stomata, legume nodules, and others [ 3 ]. Sorghum bicolor PEPC gene family (PPC genes) include five plant-type PEPC genes (SbPPC1-5: Sb10g021330, Sb02g021090, Sb04g00872, Sb07g014960, and Sb03g035090) and one bacterial-type PEPC gene (SbPPC6, Sb03g0084810) [4]. PEPC is subjected to different post-translational modifications (PTMs), such as phosphorylation, monoubiquitination, NO-related PTMs (S-nitrosylation, Tyr-nitration), and carbonylation [ 5 – 9 ]. In addition, PEPC from Arabidopsis thaliana has been reported to be persulfidated in a proteomic assay [ 10 ]. These PTMs regulate PEPC activity and the Plants 2023,12, 2426. https://doi.org/10.3390/plants12132426 https://www.mdpi.com/journal/plants Plants 2023,12, 2426 2 of 20 turnover of the protein [ 9 ], although the biological function of some of them is not yet fully understood. Protein phosphorylation is the best-known PTM of PEPC. This enzyme is phosphorylated at a conserved N-terminal serine residue by PEPC kinase (PEPCk), which belongs to the Ca 2+ /Calmodulin-dependent CDPK-SnRK protein kinase superfamily, although its activity is not Ca 2+ -dependent [ 11 ]. The phosphorylation of the photosynthetic isozymes occurs during the light period in C 4 plants [ 12 ] and throughout the dark period in CAM plants [ 2 ], which corresponds with the pattern of PPCK gene expression. The phosphorylation of PEPC positively affects its functional and regulatory properties, decreasing its sensitivity to feedback inhibition by L-malate and increasing its affinity for the allosteric activator glucose-6-phosphate and Vmax [ 5 ]. The relevance of this PTM has been questioned because Flaveria bidentis RNAi lines, in which PEPC was not phosphorylated in the light, showed no differences in the CO 2 assimilation rates with respect to the wild type [ 13 ]. On the contrary, a PEPCk-deficient Kalanchoë fedtschenkoi RNAi line showed perturbations of CAM photosynthesis, carbohydrate metabolism, and circadian rhythms [ 14 ]. Sorghum bicolor SbPPCK gene family includes three genes (SbPPCK1-3; Sb04g036570, Sb04g026490, and Sb06g022690) [ 15 ]. ZmPPCK1 expression is triggered by light in mesophyll cells from maize, and its transcript is more abundant in mesophyll than bundle sheath cells [ 16 ]. For these reasons, it is considered the photosynthetic isoform. In sorghum, SbPPCK1 expression is higher in leaves than in roots, and is increased in the light; meanwhile, SbPPCK2 and SbPPCK3 are similarly expressed in leaves and roots [6]. Most stressful conditions affect the photosynthetic activity of plants, thus causing C shortage. In these situations, PEPC activity can contribute to improving C balance. There are many reports that show the relevant role of PEPC in water stress; salinity; N, P, and Fe deficiency; ammonium, Al, and Cd toxicity; and other abiotic stresses [ 17 ]. In many of these situations, increased PEPC activity was accompanied by a rise in PEPCk activity, as is summarized below. Salinity is the major environmental factor restraining plant growth and productivity, and C 4 plants are recognized as salt-tolerant plants. In sorghum, both PEPC and PEPCk proteins were conserved during prolonged salinity [ 18 ]. The main effect of salt stress in sorghum was a marked increase in leaf PEPCk activity [ 19 ], mainly as a consequence of decreased PEPCk protein degradation due to negative control of the ubiquitin–proteasome pathway by the phytohormone abscisic acid (ABA) [ 20 ]. The increase in the phosphorylation state of PEPC caused augmented malate synthesis, and it could contribute to alleviating carbon deficiency under salinity. In sorghum roots, salinity increased PEPC and PEPCk activity, and SbPPC3 and SbPPCK2 gene expression [ 21 ]. Likewise, salinity increased AtPPC3 expression, PEPC activity and its phosphorylation state, and L-malate content in Arabidopsis roots [22]. Despite being a common intermediate in plant metabolism, ammonium can produce toxicity symptoms in plants, causing leaf chlorosis, net photosynthesis decrease, and a reduction in growth [ 23 ]. PEPC activity, by supplying carbon skeletons for ammonium assimilation, plays a pivotal role in tolerance to ammonium stress. Ammonium increased sorghum PEPCk activity and the phosphorylation state of PEPC in leaves and roots [ 6 ]. In leaves, increased PEPCk activity was associated with PPCK1, the photosynthetic isozyme. In roots, increased PEPCk activity was dependent on the PPCK2 isozyme. Orthophosphate (Pi) is an essential macronutrient required for many fundamental processes in plants, including photosynthesis and respiration, as well as nucleic acid, protein, and membrane phospholipid synthesis. Nevertheless, it is often a limiting nutrient in soils, and P deficiency is one of the greatest limitations in agricultural production [ 24 ]. To overcome low Pi availability, plants have evolved an array of responses. Among them, excreting organic acids, mainly malate and citrate, to acidify the rhizosphere and chelate metal ions has been related to increased PEPC [ 3 , 25 ] and PEPCk [ 26 ] activities. In sorghum, we have found that P deficiency increased the phosphorylation of PEPC in leaves, and both PEPC activity and PEPCk activity in roots. In Arabidopsis, phosphate starvation increased Plants 2023,12, 2426 3 of 20 the expression of all AtPPC and AtPPCK genes in shoots, but only and specifically AtPPC3 and AtPPCK2 in roots [22]. Root synthesis and the excretion of organic acids also have relevant roles in Al and Cd tolerance. As in P deficiency, the role of PEPC contributing to organic acid synthesis is well documented. For example, a transgenic rice overexpressing C 4 -PEPC showed increased tolerance towards Al toxicity [ 27 ]. In sorghum, Al increased the synthesis of malic acid, more in tolerant than in Al-sensitive cultivars [ 28 ]. With respect to SbPPCK gene expression, Al treatment increased SbPPCK2 and SbPPCK3 expressions in roots, and SbPPCK1 in leaves [ 29 ]. Similarly, Cd stress enhanced the activity of PEPC in several plant species (wheat, bean, maize). Organic acid anions are important chelators of toxic metals, including cadmium. In sorghum, Cd increased the expression of SbPPCK2 in roots and leaves [ 29 ]. Likewise, in Arabidopsis, Cd increased PEPC activity and its phosphorylation, and AtPPC and AtPPCK gene expression [30]. There is experimental evidence that sorghum PEPC is phosphorylated both in developing and germinating seeds [ 8 , 31 ]. Seed formation needs very active primary C/N metabolisms to build up structures, including embryo formation and endosperm cellularization, together with storage of stock resources for the forthcoming germination. Due to the key role of PEPC in C and N metabolisms, an impact of the lack of PEPC or PEPCk on the ability of seeds to germinate is expected. Arabidopsis SALK lines deficient in AtPPCK1 (ppck1 − )or AtPPCK2 (ppck2 − )showed reduced weight and delayed flowering [ 22 ]. Seeds from these plants had a marked deficiency in global seed nitrogen, and changes in lipid quantity and identity [ 32 ]. This shows that PEPC and its phosphorylation state impacts not only seed production but also its composition, modifying its value for human and animal food. This study aimed to assess the impact of decreased PEPC kinase activity, and subsequent reduced phosphorylation of PEPC, in sorghum. This has been carried over with sorghum lines in which the expression of all three SbPPCK genes was silenced by RNAi interference (Ppck lines). The impacts of silencing on growth, stress responses, and yield were investigated. The impact on vegetative growth was evaluated by measuring growth and biomass production, and responses to light, salinity, and phosphate deficiency. The impact on reproductive growth and yield was evaluated by the time of flowering and by the number and weight of seeds. In addition, several parameters related to the metabolomics and nutritional quality of both forage and seeds have been quantified: N and C content, metabolic profiles of amino acids and fatty acids, and, in seeds, tannins and phytates. Despite the uncompleted silencing of SbPPCK genes, silenced Ppck lines showed alterations in metabolomics, growth, stress responses, flowering time, and amount and quality of seeds. 2. Results 2.1. Effects of SbPPCK1-3 Silencing on SbPPCK Gene Expression and PEPCk Activity The effect of silencing on SbPPCK gene expression was measured in leaves of T2 homozygous lines Ppck-2 and Ppck-4 (Figure 1). PPCK1 is the C 4 photosynthetic isozyme, expressed in leaves but not in roots. SbPPCK1 gene expression was reduced by about 65% and 83% in Ppck-2 and Ppck-4 illuminated leaves, respectively. In addition, the expression of this gene significantly decreased by 25% in leaves of Ppck-2 and Ppck-4 kept in the dark. The impact of SbPPCK1-3 silencing was probably higher in illuminated leaves, due to the remarkable enhancement of SbPPCK1 expression by light (15-fold with respect to dark). SbPPCK2 is expressed in both leaves and roots. Because SbPPCK1 does not express in roots, and the expression of SbPPCK3 is very low in this tissue, PPCK2 is the main isozyme accounting for root PEPCk activity. Root SbPPCK2 gene expression was decreased by about 50% in Ppck-2 and Ppck-4. Finally, the SbPPCK3 level of expression was low and highly variable in both leaves and roots. Plants 2023,12, 2426 4 of 20 Plants 2023, 12, x FOR PEER REVIEW 4 of 21 Figure 1. Expression of SbPPCK1 (a), SbPPCK2 (b), and SbPPCK3 (c) in leaves and roots of sorghum lines. Leaves were illuminated (2 h, 700 μmol m−2 s−1 PAR) or kept in the dark prior to RNA extraction and cDNA synthesis. Data are % of Wt in the dark. Data are means ± SE (n = 4). a p < 0.05 versus Wt. * p < 0.05 versus dark. The in vitro PEPCk activity in leaf extracts was quantified by the Pro-Q/SYPRO method (Table 1). This method allows the fluorescent detection of phosphoproteins directly in SDS-PAGE gels. Fluorescence signal intensity correlates with the number of phosphorylated residues, and the results are normalized to total protein with SYPRO Ruby gel stain. Protein extracts containing PEPCk were incubated with purified dephosphorylated sorghum C4 PEPC and ATP. The amount of phosphorylated exogenous PEPC at the end of the incubation period, referred to as the amount of protein in the same band, is considered the in vitro PEPCk activity. Similar activities were measured in Wt and Ppck lines. This indicates that the partial silencing of SbPPCK1-3 may not have a great impact on PEPCk activity. We cannot rule out that the incomplete silencing of SbPPCK genes could allow the synthesis of similar quantities of PEPCk protein in Wt and Ppck lines. However, the phenotype found in Ppck lines indicates that the partial silencing of SbPPCK genes in these lines might be enough to affect the PEPCk activity in planta. Table 1. In vitro PEPCk activity of sorghum lines. 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 PEPCk activity of illuminated leaves (2 h, 700 μmol m−2 s−1 PAR), or leaves kept in the dark, was measured in protein extracts (25 μg) with 0.1 U of purified C4 PEPC. Data are % of Wt in the dark. Data are means ± SE (n = 3). 2.2. Impact of SbPPCK1-3 Silencing on Phosphorylation of PEPC and Vegetative Development Plant growth and biomass production and the phosphorylation state of PEPC were assessed in different conditions that have been associated with increased PEPCk activity, Figure 1. Expression of SbPPCK1 ( a ), SbPPCK2 ( b ), and SbPPCK3 ( c ) in leaves and roots of sorghum lines. Leaves were illuminated (2 h, 700 µ mol m −2 s −1 PAR) or kept in the dark prior to RNA extraction and cDNA synthesis. Data are % of Wt in the dark. Data are means ± SE (n= 4). a p< 0.05 versus Wt. * p< 0.05 versus dark. The in vitro PEPCk activity in leaf extracts was quantified by the Pro-Q/SYPRO method (Table 1). This method allows the fluorescent detection of phosphoproteins directly in SDS-PAGE gels. Fluorescence signal intensity correlates with the number of phosphorylated residues, and the results are normalized to total protein with SYPRO Ruby gel stain. Protein extracts containing PEPCk were incubated with purified dephosphorylated sorghum C 4 PEPC and ATP. The amount of phosphorylated exogenous PEPC at the end of the incubation period, referred to as the amount of protein in the same band, is considered the in vitro PEPCk activity. Similar activities were measured in Wt and Ppck lines. This indicates that the partial silencing of SbPPCK1-3 may not have a great impact on PEPCk activity. We cannot rule out that the incomplete silencing of SbPPCK genes could allow the synthesis of similar quantities of PEPCk protein in Wt and Ppck lines. However, the phenotype found in Ppck lines indicates that the partial silencing of SbPPCK genes in these lines might be enough to affect the PEPCk activity in planta. Table 1. In vitro PEPCk activity of sorghum lines. 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 PEPCk activity of illuminated leaves (2 h, 700 µ mol m −2 s −1 PAR), or leaves kept in the dark, was measured in protein extracts (25 µ g) with 0.1 U of purified C 4 PEPC. Data are % of Wt in the dark. Data are means ± SE (n= 3). 2.2. Impact of SbPPCK1-3 Silencing on Phosphorylation of PEPC and Vegetative Development Plant growth and biomass production and the phosphorylation state of PEPC were assessed in different conditions that have been associated with increased PEPCk activity, Plants 2023,12, 2426 5 of 20 such as salinity and phosphate deficiency. In control conditions, Ppck-2 length, shoot weight, and root weight were lower than in Wt (about 60, 30, and 40% of Wt, respectively) (Table 2). Salinity decreased these parameters to the same extent in the three lines: by 60% plant length and by 90% and 88% shoot and root weight. Due to the reduction in the growth of Ppck-2 in the control condition, plants of this line were the smallest under salinity (Table 2). Table 2. Effect of salinity on growth and biomass production by Ppck lines. 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 a1.6 ±0.1 a1.7 ±0.2 NaCl 12.4 ±0.1 *a0.2 ±0.1 *a0.2 ±0.1 Ppck-4 Control 50.8 ±1.1 a5.4 ±0.7 3.8 ±0.7 NaCl 19.0 ±2.4 * 0.4 ±0.1 * 0.3 ±0.1 Plants were grown with increasing amounts of NaCl: 0 mM (7 days), 86 mM (3 days), 172 mM (7 days), and 257 mM (5 days), and measurements were made at the end of treatments. Each sample was the mean of the measurements of the 3 plants of the same hydroponic. Data are means ± SE of 3 hydroponics (9 plants). a p< 0.05 versus Wt. * p< 0.05 versus Control. The phosphorylation of PEPC in sorghum leaves was estimated with the malate test (IC 50 , malate concentration that causes 50% of inhibition of PEPC activity at pH 7.3). The IC 50 of the inhibition of PEPC activity by malate is higher when PEPC is phosphorylated and lower for dephosphorylated PEPC. It reflects the in vivo PEPCk activity. Light increased the phosphorylation of PEPC in Wt, both in control and NaCl-treated plants (Figure 2). This effect was lost in Ppck-2, and greatly reduced in Ppck-4. Plants 2023, 12, x FOR PEER REVIEW 5 of 21 such as salinity and phosphate deficiency. In control conditions, Ppck-2 length, shoot weight, and root weight were lower than in Wt (about 60, 30, and 40% of Wt, respectively) (Table 2). Salinity decreased these parameters to the same extent in the three lines: by 60% plant length and by 90% and 88% shoot and root weight. Due to the reduction in the growth of Ppck-2 in the control condition, plants of this line were the smallest under salinity (Table 2). Table 2. Effect of salinity on growth and biomass production by Ppck lines. 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 Plants were grown with increasing amounts of NaCl: 0 mM (7 days), 86 mM (3 days), 172 mM (7 days), and 257 mM (5 days), and measurements were made at the end of treatments. Each sample was the mean of the measurements of the 3 plants of the same hydroponic. Data are means ± SE of 3 hydroponics (9 plants). a p < 0.05 versus Wt. * p < 0.05 versus Control. The phosphorylation of PEPC in sorghum leaves was estimated with the malate test (IC 50 , malate concentration that causes 50% of inhibition of PEPC activity at pH 7.3). The IC 50 of the inhibition of PEPC activity by malate is higher when PEPC is phosphorylated and lower for dephosphorylated PEPC. It reflects the in vivo PEPCk activity. Light increased the phosphorylation of PEPC in Wt, both in control and NaCl-treated plants (Figure 2). This effect was lost in Ppck-2, and greatly reduced in Ppck-4. Figure 2. Effect of light and NaCl on the phosphorylation of leaf PEPC of sorghum lines. Plants were grown with increasing amounts of NaCl: 0 mM (7 days), 86 mM (3 days), 172 mM (7 days), and 257 mM (5 days), and measurements were made at the end of treatments. Leaves were illuminated (700 μmol m −2 s −1 PAR), or kept in the dark for 2 h before the preparation of protein extracts. Data are means ± SE (n = 3). a p < 0.05 versus Wt. * p < 0.05 versus dark. Phosphorous deficiency is known to increase root PEPC activity and PEPC phosphorylation [3,25,26]. The effect of P deficiency on plant growth and PEPC phosphorylation was evaluated in the absence of P (-P) and with treatment with insoluble P (calcium phosphate, PCa) due to the known role of PEPC in the synthesis and secretion of root exudates that increase P availability. In the absence of P, growth and biomass decreased, and the treatment with PCa partially recovered control values (Table 3). No remarkable differences were observed between lines, although lower values were measured in Ppck- 2. Figure 2. Effect of light and NaCl on the phosphorylation of leaf PEPC of sorghum lines. Plants were grown with increasing amounts of NaCl: 0 mM (7 days), 86 mM (3 days), 172 mM (7 days), and 257 mM (5 days), and measurements were made at the end of treatments. Leaves were illuminated (700 µ mol m −2 s −1 PAR), or kept in the dark for 2 h before the preparation of protein extracts. Data are means ±SE (n= 3). ap< 0.05 versus Wt. * p< 0.05 versus dark. Phosphorous deficiency is known to increase root PEPC activity and PEPC phosphorylation [ 3 , 25 , 26 ]. The effect of P deficiency on plant growth and PEPC phosphorylation was evaluated in the absence of P (-P) and with treatment with insoluble P (calcium phosphate, PCa) due to the known role of PEPC in the synthesis and secretion of root exudates that increase P availability. In the absence of P, growth and biomass decreased, and the treatment with PCa partially recovered control values (Table 3). No remarkable differences were observed between lines, although lower values were measured in Ppck-2. Plants 2023,12, 2426 6 of 20 Table 3. Effect of phosphate deficiency on growth and biomass production by Ppck lines. 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 a22.6 ±3.9 11.2 ±1.4 -P 26.5 ±0.2 * 2.8 ±0.1 * 3.8 ±0.1 * PCa 48.8 ±1.5 *a10.8 ±2.8 8.1 ±1.4 Plants were grown for 3 weeks with Hewitt medium (control), without phosphate (-P), or with 0.67 mM Ca 3 (PO 4 ) 2 (PCa). Each sample was the mean of the measurements of the 3 plants of the same hydroponic. Data are means ±SE of 4 hydroponics (12 plants). ap< 0.05 versus Wt. * p< 0.05 versus Control. P deficiency increased PEPC activity in Wt and Ppck-2 roots (Figure 3a), and this increase was higher in Ppck-2. This result shows that the treatment was effectively triggering a response in the plant, and the higher increase in PEPC activity in Ppck-2 leaves may reflect compensation for the lack of phosphorylation of the protein. In leaves, P deficiency increased the phosphorylation of PEPC (IC50 malate) in Wt, but not in Ppck-2 (Figure 3b). Plants 2023, 12, x FOR PEER REVIEW 6 of 21 Table 3. Effect of phosphate deficiency on growth and biomass production by Ppck lines. 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 Plants were grown for 3 weeks with Hewitt medium (control), without phosphate (-P), or with 0.67 mM Ca3(PO4)2 (PCa). Each sample was the mean of the measurements of the 3 plants of the same hydroponic. Data are means ± SE of 4 hydroponics (12 plants). a p < 0.05 versus Wt. * p < 0.05 versus Control. P deficiency increased PEPC activity in Wt and Ppck-2 roots (Figure 3a), and this increase was higher in Ppck-2. This result shows that the treatment was effectively triggering a response in the plant, and the higher increase in PEPC activity in Ppck-2 leaves may reflect compensation for the lack of phosphorylation of the protein. In leaves, P deficiency increased the phosphorylation of PEPC (IC50 malate) in Wt, but not in Ppck-2 (Figure 3b). Figure 3. Root PEPC activity (a) and leaf PEPC phosphorylation (b) of sorghum plants with different phosphorous nutrition. Plants were grown for 3 weeks with Hewitt medium (control), without phosphate (-P), or with 0.67 mM Ca3(PO4)2 (PCa). Data are means ± SE (n = 3). a p < 0.05 versus Wt. * p < 0.05 versus Control. Taken together, these results show that SbPPCK gene silencing, although without great effect on growth, significantly decreased the phosphorylation of PEPC associated with stress responses. 2.3. Metabolic Changes in Ppck Lines The effect of SbPPCK gene silencing on the amino acid profile was investigated in leaves of sorghum lines after acid hydrolysis (Table 4). A general decrease in amino acid quantities was observed in Ppck lines, more notably in Ppck-4 (Table 5). The amounts of Arg, Lys, and Val were significantly decreased by 8%, 7%, and 16% in Ppck-2 and by 15%, 14%, and 18% in Ppck-4, respectively. Other amino acids were also significantly lower in Ppck4 (Glu, Phe, His, Leu, Met, Ser). Total amino acid content was 4% and 14% lower in Ppck-2 and Ppck-4 lines, respectively (Table 5). In Ppck-4, the C/N ratio was significantly higher than Wt due to increased C and decreased N content (Table 5). Table 4. Amino acid content in leaves of sorghum lines. Amino Acid (mg g−1fw) Wt Ppck-2 Ppck-4 Asp 1.82 ± 0.02 1.78 ± 0.04 1.57 ± 0.10 Figure 3. Root PEPC activity ( a ) and leaf PEPC phosphorylation ( b ) of sorghum plants with different phosphorous nutrition. Plants were grown for 3 weeks with Hewitt medium (control), without phosphate (-P), or with 0.67 mM Ca 3 (PO 4 ) 2 (PCa). Data are means ± SE (n= 3). a p< 0.05 versus Wt. *p< 0.05 versus Control. Taken together, these results show that SbPPCK gene silencing, although without great effect on growth, significantly decreased the phosphorylation of PEPC associated with stress responses. 2.3. Metabolic Changes in Ppck Lines The effect of SbPPCK gene silencing on the amino acid profile was investigated in leaves of sorghum lines after acid hydrolysis (Table 4). A general decrease in amino acid quantities was observed in Ppck lines, more notably in Ppck-4 (Table 5). The amounts of Arg, Lys, and Val were significantly decreased by 8%, 7%, and 16% in Ppck-2 and by 15%, 14%, and 18% in Ppck-4, respectively. Other amino acids were also significantly lower in Ppck4 (Glu, Phe, His, Leu, Met, Ser). Total amino acid content was 4% and 14% lower in Ppck-2 and Ppck-4 lines, respectively (Table 5). In Ppck-4, the C/N ratio was significantly higher than Wt due to increased C and decreased N content (Table 5). Plants 2023,12, 2426 7 of 20 Table 4. Amino acid content in leaves of sorghum lines. Amino Acid (mg g−1fw) 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 a0.86 ±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 a1.25 ±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 a0.73 ±0.01 a Data are means ±SE of 3 hydroponics (9 plants). ap< 0.05 versus Wt. Table 5. Total amino acid, C, and N content in leaves of sorghum lines. Line Amino Acids (mg g−1fw) 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 a42.55 ±0.14 3.76 ±0.08 11.32 ±0.26 a Total amino acid is the sum of the values in Table 4. Data are means ±SE. ap< 0.05 versus Wt. Leaf fatty acids were about 50% saturated, 15% monounsaturated, and 35% polyunsaturated in Wt plants. The amount of several fatty acids was increased in Ppck lines (palmitoleic, suberic, azelaic, α -linolenic, and arachidonic acid), and the amount of other acids was decreased (oleic and eicosenoic acid) (Table 6). Globally, a tendency towards decreased amounts of monounsaturated fatty acids in Ppck lines was observed (Figure 4). Table 6. Fatty acid content in leaves of sorghum lines. 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 a1.59 ±0.06 a Suberic acid 0.18 ±0.01 0.29 ±0.01 a0.34 ±0.03 a Azelaic acid 0.16 ±0.01 0.30 ±0.02 a0.29 ±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 a0.33 ±0.04 a Data are means ±SE (n= 3). ap< 0.05 versus Wt. Plants 2023,12, 2426 8 of 20 Plants 2023, 12, x FOR PEER REVIEW 8 of 21 Figure 4. Total saturated, monounsaturated, and polyunsaturated fatty acids in sorghum leaves. Data are means ± SE (n = 3). 2.4. Impact of SbPPCK1-3 Silencing on Reproductive Development and Seed Production The time of flowering was delayed in Ppck lines (Figure 5). On day 49, 50% of Wt plants had flowers, but only 3% of Ppck-2 and 0% of Ppck-4. At day 56, 100% of Wt plants had flowers, but only 66% of Ppck-2 and 50% of Ppck-4. In addition, about 20% of Ppck-4 plants did not produce flowers at all. In summary, 100% of Wt plants had flowers on day 56, 100% of Ppck-2 plants on day 66, and 80% of Ppck-4 plants on day 73. Panicle length and weight and seed weight and number were decreased in Ppck lines (Table 7). Ppck-2 number of seeds was 50% of Wt. In Ppck-4 plants, it was 60% of Wt in plants with flowers, but it is worth noting that, as mentioned above, 20% of plants of this line did not form flowers. These results clearly show that PEPC phosphorylation has a great impact on sorghum reproductive development and productivity. Figure 5. Flowering rate of sorghum lines. Data are % of plants with flowers at the indicated time. Table 7. Seed production of sorghum lines. 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 Data are means ± SE (n = 4) per panicle. a p < 0.05 versus Wt. 2.5. Impact of SbPPCK1-3 Silencing on the Composition of Seeds Seed composition was analyzed first to clarify the impact of SbPPCK gene silencing on seed metabolites and second to evaluate changes related to the nutritional quality of Figure 4. Total saturated, monounsaturated, and polyunsaturated fatty acids in sorghum leaves. Data are means ±SE (n= 3). 2.4. Impact of SbPPCK1-3 Silencing on Reproductive Development and Seed Production The time of flowering was delayed in Ppck lines (Figure 5). On day 49, 50% of Wt plants had flowers, but only 3% of Ppck-2 and 0% of Ppck-4. At day 56, 100% of Wt plants had flowers, but only 66% of Ppck-2 and 50% of Ppck-4. In addition, about 20% of Ppck-4 plants did not produce flowers at all. In summary, 100% of Wt plants had flowers on day 56, 100% of Ppck-2 plants on day 66, and 80% of Ppck-4 plants on day 73. Panicle length and weight and seed weight and number were decreased in Ppck lines (Table 7). Ppck-2 number of seeds was 50% of Wt. In Ppck-4 plants, it was 60% of Wt in plants with flowers, but it is worth noting that, as mentioned above, 20% of plants of this line did not form flowers. These results clearly show that PEPC phosphorylation has a great impact on sorghum reproductive development and productivity. Plants 2023, 12, x FOR PEER REVIEW 8 of 21 Figure 4. Total saturated, monounsaturated, and polyunsaturated fatty acids in sorghum leaves. Data are means ± SE (n = 3). 2.4. Impact of SbPPCK1-3 Silencing on Reproductive Development and Seed Production The time of flowering was delayed in Ppck lines (Figure 5). On day 49, 50% of Wt plants had flowers, but only 3% of Ppck-2 and 0% of Ppck-4. At day 56, 100% of Wt plants had flowers, but only 66% of Ppck-2 and 50% of Ppck-4. In addition, about 20% of Ppck-4 plants did not produce flowers at all. In summary, 100% of Wt plants had flowers on day 56, 100% of Ppck-2 plants on day 66, and 80% of Ppck-4 plants on day 73. Panicle length and weight and seed weight and number were decreased in Ppck lines (Table 7). Ppck-2 number of seeds was 50% of Wt. In Ppck-4 plants, it was 60% of Wt in plants with flowers, but it is worth noting that, as mentioned above, 20% of plants of this line did not form flowers. These results clearly show that PEPC phosphorylation has a great impact on sorghum reproductive development and productivity. Figure 5. Flowering rate of sorghum lines. Data are % of plants with flowers at the indicated time. Table 7. Seed production of sorghum lines. 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 Data are means ± SE (n = 4) per panicle. a p < 0.05 versus Wt. 2.5. Impact of SbPPCK1-3 Silencing on the Composition of Seeds Seed composition was analyzed first to clarify the impact of SbPPCK gene silencing on seed metabolites and second to evaluate changes related to the nutritional quality of Figure 5. Flowering rate of sorghum lines. Data are % of plants with flowers at the indicated time. Table 7. Seed production of sorghum lines. 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 a4.0 ±0.5 a3.4 ±0.4 a101 ±20 a Ppck-4 8.2 ±0.6 5.2 ±1.1 4.3 ±0.6 131 ±22 Data are means ±SE (n= 4) per panicle. ap< 0.05 versus Wt. Plants 2023,12, 2426 9 of 20 2.5. Impact of SbPPCK1-3 Silencing on the Composition of Seeds Seed composition was analyzed first to clarify the impact of SbPPCK gene silencing on seed metabolites and second to evaluate changes related to the nutritional quality of seeds. The quantity of all the measured amino acids, except Met, was decreased in Ppck lines (Table 8). Accordingly, total amino acid content was lowered by 34% and 55% in these lines (Table 9). Meanwhile, starch was only decreased by 12% in Ppck-4 (Table 9). On the contrary, two parameters that affect the nutritional quality of seeds (tannins and phytates) were increased in Ppck lines (Table 9). Although both total C and total N were lower in Ppck lines, the ratio C/N was higher in these lines due to a lesser N content (Table 10). Table 8. Amino acid content in seeds of sorghum lines. Amino Acid (mg g−1fw) Wt Ppck-2 Ppck-4 Asp 4.86 ±0.40 3.45 ±0.18 a2.44 ±0.39 a Glu 16.01 ±1.55 9.91 ±0.62 a6.64 ±1.12 a Ala 6.62 ±0.46 4.23 ±0.26 a2.89 ±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 a0.07 ±0.01 a Phe 3.50 ±2.61 2.55 ±0.16 a1.85 ±0.27 a Gly 2.30 ±0.21 1.64 ±0.10 a1.20 ±0.22 a His 2.09 ±0.17 1.49 ±0.08 a1.01 ±0.15 a Ile 2.38 ±0.21 1.59 ±0.06 a1.15 ±0.19 a Leu 10.40 ±0.95 6.76 ±0.40 a4.25 ±0.69 a Lys 1.95 ±0.18 1.47 ±0.08 a1.08 ±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 a3.81 ±0.72 a Ser 2.99 ±0.22 2.01 ±0.13 a1.31 ±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 a0.09 ±0.01 a Thr 2.23 ±0.18 1.57 ±0.07 a1.10 ±0.18 a Val 3.42 ±0.35 2.12 ±0.18 a1.43 ±0.25 a Data are means ±SE (n= 3). Samples were composed of homogenates of 5 g of seeds. ap< 0.05 versus Wt. Table 9. Effect of SbPPCK gene silencing on seed composition. Line Amino Acids (mg g−1fw) 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 a4.78 ±0.07 a Ppck-4 32.52 ±5.46 a1.23 ±0.04 a1.50 ±0.36 5.47 ±0.08 a Total amino acid is the sum of the values in Table 8. Samples were composed of homogenates of 5 g of seeds. Data are means ±SE (n= 3). ap< 0.05 versus Wt. Table 10. Total C and N content in seeds of sorghum lines. 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 Samples were composed of homogenates of 5 g of seeds. Fatty acid composition was different in seeds in comparison to leaves, with similar amounts (30–35%) of saturated, monounsaturated, and polyunsaturated fatty acids in seeds of Wt. Palmitic, stearic, γ - and α -linolenic, and arachidonic acids were increased, and oleic, arachidic, and eicosenoic acids were lowered in Ppck lines (Table 11). The same than in leaves, it was observed a tendency to a diminution of monounsaturated fatty acids (Figure 6). Plants 2023,12, 2426 16 of 20 4.5. Determination of Enzyme Activity and Protein Quantification Protein extracts were obtained by grinding 0.2 g fresh weight of leaf or root tissue in 1 mL of extraction buffer containing 0.1 M Tris–HCl pH 7.5, 20 % (v/v) glycerol, 1 mM EDTA, 10 mM MgCl 2 , a protease inhibitor cocktail (Sigma), 10 mM potassium fluoride, and 14 mM β -mercaptoethanol. The homogenate was centrifuged at 15.000 × gfor 2 min and PEPC activity was quantified in the supernatant. Protein concentrations were determined using the method of Bradford [51] with BSA as a standard. PEPC activity was measured spectrophotometrically at optimal pH 8.0 using the NADMDH-coupled assay at 2.5 mM PEP [ 52 ]. A single enzyme unit (U) is defined as the amount of PEPC that catalyzes the carboxylation of 1 µ mol of phosphoenolpyruvate per minute at pH 8 and 30 ◦C. PEPCk activity was measured by staining phosphorylated proteins with Pro-Q Diamond. The in vitro PEPCk activity of sorghum leaves or roots was measured in aliquots of protein extracts (25 µ g) that were incubated in a reaction medium containing 100 mM Tris- HCl, pH 7.5, 20% (v/v) glycerol, 5 mM MgCl 2 , 0.25 mM P1P5-di(adenosine-5 0 )-pentaphosphate (adenylate kinase inhibitor), 1 mM EGTA, and 0.1 units of nonphosphorylated purified sorghum C 4 PEPC. The phosphorylation reaction was initiated by the addition of 1 mM ATP. The reaction was stopped by boiling the samples for 5 min at 90 ◦ C in the presence of a loading buffer. The denatured proteins were separated by SDS-PAGE. Fluorescent staining of SDS-PAGE gels was performed using Pro-Q Diamond phosphoprotein stain and SYPRO Ruby protein stain (Invitrogen-Molecular Probes, Carlsbad, CA, USA), following the manufacturer’s instructions. After electrophoresis, the gels were stained with Pro-Q Diamond phosphoprotein stain and analyzed with a phosphor imager (Fuji FLA-5100; Fuji, Tokyo), and then stained with SYPRO Ruby and re-imaged. The positive control for the Pro-Q Diamond stain was phosphorylated ovalbumin, which is included in molecular weight markers and is the single protein stained by Pro-Q in this lane. 4.6. RNA Extraction and cDNA Synthesis Total RNA was extracted from 100 mg of frozen powdered leaves or roots using the Plant RNA Isolation Mini Kit (Agilent Technologies). Extracted nucleic acids were DNase-treated to wipe out genomic DNA. RNA concentration was determined using Nanodrop 2000 (Thermo). Reverse transcription reactions were performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and HISCRIPT II 1st Strand cDNA Synthesis Kit (Vazyme), following the manufacturers’ instructions, with 1 µ g of purified total RNA. cDNA synthesized was used for qPCR experiments. 4.7. Quantitative Real-Time PCR (qPCR) Quantitative PCR reactions (qPCR) were performed in a final volume of 20 µ L consisting of 1 µ L of the cDNA, 15 µ M of the specific primers, and 10 µ L of SensiFAST SYBR No-ROX kit (Bioline). PCR was conducted on the Light Cycler 480 II Real-Time PCR System (Roche), and the threshold cycles (Ct) were determined using the Light Cycler 480 software for all treatments. To normalize the obtained values, actin was used as an internal control in each sample. Relative gene transcript abundance data were obtained through the Livak method [ 53 ] (2 −∆∆Ct ), assigning the relative value of 1 for transcript abundance in Wt leaves or roots in control conditions. 4.8. Quantification of Amino Acids Lyophilized samples (0.1 g) were ground in liquid nitrogen with 1 mL of 6N HCl for hydrolyzing proteins. Then, they were dried at 100 ◦ C and kept overnight in a rotary evaporator. Subsequently, they were resuspended in 1 mL of 70% methanol, incubated at 4 ◦ C overnight, and the supernatant was analyzed by gas chromatography–mass spectrometry (GC-MS) using a Thermo Scientific instrument at the Mass Spectrometry Services (CITIUS, University of Sevilla, Seville, Spain). Plants 2023,12, 2426 17 of 20 4.9. Determination of C/N Ratio Analyses of the total C and N were performed by a LECO Elemental CNS-Trumac Autoanalyzer, version 1.3x (2014), at the Mass Spectrometry Services (CITIUS, University of Sevilla). 4.10. Quantification of Starch The quantification of starch was carried out by digestion with amylolytic enzymes (amyloglucosidase and amylase) and subsequent quantification of reducing sugars [ 54 ]. Previously, free sugars were removed with successive 80% ethanol washings. After digestion, an aliquot was assayed with 3,5-dinitrosalicylic acid (DNS) and Na/K tartrate measuring the absorbance at 570 nm in a VERSA max microplate spectrophotometer (Molecular Devices, San Jose, CA, USA), using glucose for the standard curve. 4.11. Quantification of Fatty Acids Lyophilized samples (0.1 g) were ground in liquid nitrogen. Fatty acids (FAs) were quantified at the Agricultural Research Service (CITIUS, University of Sevilla). Fat extraction and direct methylation of FAs were performed in a single-step procedure based on the method by Sukhija and Palmquist [ 55 ]. Separation and quantification of FA methyl esters (FAMEs) were carried out using a gas chromatograph (Agilent 6890N Network GS System, Agilent, Santa Clara, CA, USA) equipped with a flame ionization detector (FID) and automatic sample injector HP 7683, and fitted with an HP-88 J&W fused silica capillary column (100 m, 0.25 mm intern diameter, 0.2 µ m film thickness; Agilent Technologies Spain, S.L., Madrid, Spain). Nonanoic acid methyl ester (C9:0 ME) was used as an internal standard (Sigma Chemical Co., Ltd., Poole, UK). Individual FAs were identified by comparing their retention times with those of the authenticated standard FA mix Supelco 37 (Sigma Chemical Co., Ltd., Poole, UK). 4.12. Quantification of Tannins The ferric reagent was used for the quantification of condensed tannins according to the method of Porter et al. [ 56 ]. Samples were tested with 2% ferric ammonium sulfate and butanol:HCl (95:5 v/v), and absorbance was measured at 550 nm in a VERSA max microplate spectrophotometer (Molecular Devices). 4.13. Quantification of Phytates Phytates were quantified with the Phytic Acid Assay Kit (K-PHYT_Megazyme). The samples were mixed with 0.66 M HCl and incubated at room temperature overnight in darkness to extract phytic acid and other inositol phosphates. Samples were centrifuged and supernatants were neutralized by adding 0.5 mL of 0.75 M NaOH. The extraction of phytic acid was performed in duplicate, and from each of the neutralized samples, two replicate analyses were carried out. Samples were dephosphorylated or not in the presence (for total Pi measurement) or absence (for free Pi measurement) of phytase and alkaline phosphatase, respectively. Pi was quantified as phosphorus from a calibration curve generated using standards of known phosphorous concentrations. For all the samples, free and total phosphorus contents were determined spectrophotometrically (VERSA max microplate spectrophotometer) at 655 nm, and their difference was calculated for the phytic acid content (g/g). 4.14. Statistical Analysis All data were analyzed by ANOVA. Then, differences between group means were compared by the Duncan or Games–Howell multiple range test. A pvalue of <0.05 was considered to be statistically significant. All analyses were conducted using SPSS Statistics 21 (IBM, Armonk, NY, USA). Different letters indicate statistically significant differences. Plants 2023,12, 2426 18 of 20 5. Conclusions This work shows that SbPPCK gene silencing negatively impacts sorghum growth, stress responses, reproductive development, yield, and quality of forage and seeds. As a whole, it highlights the key role of the phosphorylation of PEPC in all these physiological contexts. Author Contributions: J.P.-L. performed most of the experiments with the collaboration of C.d.l.O., J.G. and A.B.F. I.J.-G. obtained the plasmid for sorghum transformation. A.B.F., J.A.M. and S.G.-M. designed the research. A.B.F., C.E. and S.G.-M. obtained funding. A.B.F., C.E., J.A.M. and S.G.-M. performed critical revision of results. S.G.-M. wrote the original manuscript and revised it with the input of all the authors. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Ministerio de Economía, Industria y Competitividad (AGL2016- 75413-P), by the Junta de Andalucía (P12-FQM-489 and PAI group BIO298), and Fondo Europeo de Desarrollo Regional (FEDER) (Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía within Programa Operativo FEDER 2014-2020, US-1251626). Jesús Pérez-López received a grant from Universidad de Sevilla (VI PPIT-US-2018-IV.3). Data Availability Statement: All relevant data can be found within the manuscript. Acknowledgments: The authors thank Maria Eugenia Soria and Rocío Valderrama (Servicio de Espectrometría de Masas, Centro de Investigación, Tecnología e Innovación (CITIUS) of the University of Seville), and Inmaculada Pajuelo and Oliva Polvillo (Agronomic Research Services, CITIUS, University of Sevilla) for providing technical assistance with amino acid and fatty acid analysis. We thank Zhanyuan Zhang and Hyeyoung Lee from the Plant Transformation Core Facility, University of Missouri, for advice on the selection of transformed plants. Conflicts of Interest: The authors declare no conflict of interest. References 1. Chollet, R.; Vidal, J.; O’Leary, M.H. Phosphoenolpyruvate carboxylase: A ubiquitous, highly regulated enzyme in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996,47, 273–298. [CrossRef] [PubMed] 2. Nimmo, H.G. The regulation of PEPC in CAM plants. Trends Plant. Sci. 2000,5, 75–80. 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