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

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.

Knock-down of phosphoenolpyruvate carboxylase 3 negatively impacts growth, productivity, and responses to salt stress in sorghum (Sorghum bicolor L.).

Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C4 plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, we analyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lines with the SbPPC3 gene silenced (Ppc3 lines). Ppc3 plants showed reduced stomatal conductance and plant size, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stress indicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity, even in control conditions. Salinity further affected stomatal conductance and yield and had a deeper impact on central metabolism in silenced plants compared to wild type, more notably in roots, with Ppc3 plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumulation of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is predominantly a root protein, its absence causes deep changes in plant physiology and metabolism in roots and leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses in sorghum plants. The consequences of SbPPC3 silencing suggest that this protein, and maybe orthologs in other plants, could be an important target to improve plant growth, productivity, and resistance to salt stress and other stresses where non-photosynthetic PEPCs may be implicated.

A conserved C-terminal peptide of sorghum phosphoenolpyruvate carboxylase promotes its proteolysis, which is prevented by Glc-6P or the phosphorylation state of the enzyme.

MAIN CONCLUSION: A synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect. Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context.

Phenotyping Tomato Root Developmental Plasticity in Response to Salinity in Soil Rhizotrons.

Plants have developed multiple strategies to respond to salt stress. In order to identify new traits related to salt tolerance, with potential breeding application, the research focus has recently been shifted to include root system architecture (RSA) and root plasticity. Using a simple but effective root phenotyping system containing soil (rhizotrons), RSA of several tomato cultivars and their response to salinity was investigated. We observed a high level of root plasticity of tomato seedlings under salt stress. The general root architecture was substantially modified in response to salt, especially with respect to position of the lateral roots in the soil. At the soil surface, where salt accumulates, lateral root emergence was most strongly inhibited. Within the set of tomato cultivars, H1015 was the most tolerant to salinity in both developmental stages studied. A significant correlation between several root traits and aboveground growth parameters was observed, highlighting a possible role for regulation of both ion content and root architecture in salt stress resilience.

Genetic and Pharmacological Inhibition of Autophagy increases the Monoubiquitination of Non-Photosynthetic Phosphoenolpyruvate Carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) is an enzyme with key roles in carbon and nitrogen metabolisms. The mechanisms that control enzyme stability and turnover are not well known. This paper investigates the degradation of PEPC via selective autophagy, including the role of the monoubiquitination of the enzyme in this process. In Arabidopsis, the genetic inhibition of autophagy increases the amount of monoubiquitinated PEPC in the atg2, atg5, and atg18a lines. The same is observed in nbr1, which is deficient in a protein that recruits monoubiquitinated substrates for selective autophagy. In cultured tobacco cells, the chemical inhibition of the degradation of autophagic substrates increases the quantity of PEPC proteins. When the formation of the autophagosome is blocked with 3-methyladenine (3-MA), monoubiquitinated PEPC accumulates as a result. Finally, pull-down experiments with a truncated version of NBR1 demonstrate the recovery of intact and/or fragmented PEPC in Arabidopsis leaves and roots, as well as cultured tobacco cells. Taken together, the results show that a fraction of PEPC is cleaved via selective autophagy and that the monoubiquitination of the enzyme has a role in its recruitment towards this pathway. Although autophagy seems to be a minor pathway, the results presented here increase the knowledge about the role of monoubiquitination and the regulation of PEPC degradation.

Changes to the functional traits of phosphoenolpyruvate carboxylase following hybridization in C-4 halophytes.

Hybridization is a relevant evolutionary mechanism linked to the invasiveness of plant species, but little is known about its effect on enzymatic activities in response to stress. We analyzed the effects of salinity on key mechanistic traits of phosphoenolpyruvate carboxylase (PEPC) enzyme for two hybrid taxa derived from native Spartina maritima (Curtis) Fernald and invasive Spartina densiflora Brongn. in comparison with their parental species. Parental species showed contrasted strategies at the PEPC level to cope with salinity. Spartina maritima showed its physiological optimum at 10 to 40 ppt salinity, with high PEPC activity (per unit leaf soluble protein), in contrast to the lower salinity optimum of 0.5 and 10 ppt for S. densiflora, where highest levels of PEPC apparent specific activity coincided with high light-induced activation of PEPC. Both hybrids showed constant PEPC apparent specific activity from fresh water to hypersalinity and exhibited higher net photosynthesis rates in fresh water than their parents. Spartina maritima × densiflora presented three transgressive PEPC-related traits, being the only taxon able to increase its PEPC activation in darkness at high salinity. Spartina densiflora × maritima showed most PEPC-related traits intermediate between its parents. Inheritance types operating differently in reciprocal hybrids determine key functional traits conditioning their ecological performance.

Anionic Phospholipids Induce Conformational Changes in Phosphoenolpyruvate Carboxylase to Increase Sensitivity to Cathepsin Proteases.

Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that serves a variety of functions in plants, acting as the primary form of CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). In a previous work we have shown that C4-PEPC bind anionic phospholipids, resulting in PEPC inactivation. Also, we showed that PEPC can associate with membranes and to be partially proteolyzed. However, the mechanism controlling this remains unknown. Using semi purified-PEPC from sorghum leaf and a panel of PEPC-specific antibodies, we analyzed the conformational changes in PEPC induced by anionic phospholipids to cause the inactivation of the enzyme. Conformational changes observed involved the exposure of the C-terminus of PEPC from the native, active enzyme conformation. Investigation of the protease activity associated with PEPC demonstrated that cysteine proteases co-purify with the enzyme, with protease-specific substrates revealing cathepsin B and L as the major protease species present. The anionic phospholipid-induced C-terminal exposed conformation of PEPC appeared highly sensitive to the identified cathepsin protease activity and showed initial proteolysis of the enzyme beginning at the N-terminus. Taken together, these data provide the first evidence that anionic phospholipids promote not only the inactivation of the PEPC enzyme, but also its proteolysis.

HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses.

Protein breakdown and mobilization from old or stressed tissues to growing and sink organs are some of the metabolic features associated with abiotic/biotic stresses, essential for nutrient recycling. The massive degradation of proteins implies numerous proteolytic events in which cysteine-proteases are the most abundant key players. Analysing the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and grain yield and quality. In this study, dark and nitrogen starvation treatments were selected to induce stress in barley. Results show that C1A proteases participate in the proteolytic processes triggered in leaves by both abiotic treatments, which strongly induce the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Differences in biochemical parameters and C1A gene expression were found when comparing transgenic barley plants overexpressing or silencing the HvPap-1 gene and wild-type dark-treated leaves. These findings associated with morphological changes evidence a lifespan-delayed phenotype of HvPap-1 silenced lines. All these data elucidate on the role of this protease family in response to abiotic stresses and the potential of their biotechnological manipulation to control the timing of plant growth.

New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination.

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.

Halotropism is a response of plant roots to avoid a saline environment.

Tropisms represent fascinating examples of how plants respond to environmental signals by adapting their growth and development. Here, a novel tropism is reported, halotropism, allowing plant seedlings to reduce their exposure to salinity by circumventing a saline environment. In response to a salt gradient, Arabidopsis, tomato, and sorghum roots were found to actively prioritize growth away from salinity above following the gravity axis. Directionality of this response is established by an active redistribution of the plant hormone auxin in the root tip, which is mediated by the PIN-FORMED 2 (PIN2) auxin efflux carrier. We show that salt-induced phospholipase D activity stimulates clathrin-mediated endocytosis of PIN2 at the side of the root facing the higher salt concentration. The intracellular relocalization of PIN2 allows for auxin redistribution and for the directional bending of the root away from the higher salt concentration. Our results thus identify a cellular pathway essential for the integration of environmental cues with auxin-regulated root growth that likely plays a key role in plant adaptative responses to salt stress.

Phylogenetically distant barley legumains have a role in both seed and vegetative tissues.

Legumains or vacuolar processing enzymes are cysteine peptidases (C13 family, clan CD) with increasingly recognized physiological significance in plants. They have previously been classified as seed and vegetative legumains. In this work, the entire barley legumain family is described. The eight members of this family belong to the two phylogenetic clades in which the angiosperm legumains are distributed. An in-depth molecular and functional characterization of a barley legumain from each group, HvLeg-2 and HvLeg-4, was performed. Both legumains contained a signal peptide and were located in the endoplasmic reticulum, were expressed in seeds and vegetative tissues, and when expressed as recombinant proteins showed legumain and caspase proteolytic activities. However, the role of each protein seemed to be different in their target tissues. HvLeg-2 responded in leaves to biotic and abiotic stimuli, such as salicylic acid, jasmonic acid, nitric oxide, abscisic acid, and aphid infestation, and was induced by gibberellic acid in seeds, where the protein is able to degrade storage globulins. HvLeg-4 responded in leaves to wounding, nitric oxide, and abscisic acid treatments, and had an unknown role in the germinating seed. From these results, a multifunctional role was assumed for these two phylogenetically distant legumains, achieving different physiological functions in both seed and vegetative tissues.

Factors involved in the rise of phosphoenolpyruvate carboxylase-kinase activity caused by salinity in sorghum leaves.

Salinity increases phosphoenolpyruvate carboxylase kinase (PEPCase-k) activity in sorghum leaves. This work has been focused on the mechanisms responsible for this phenomenon. The light-triggered expression of SbPPCK1 gene, accountable for the photosynthetic C4-PEPCase-k, is controlled by a complex signal transduction chain involving phospholipases C and D (PLC and PLD). These two phospholipase-derived signalling pathways were functional in salinized plants. Pharmacological agents that act on PLC (U-73122, neomycin) or PLD (n-butanol) derived signals, blocked the expression of SbPPCK1, but had little effect on PEPCase-k activity. This discrepancy was further noticed when SbPPCK1-3 gene expression and PEPCase-k activity were studied in parallel. At 172 mM, the main effect of NaCl was to decrease the rate of PEPCase-k protein turnover. Meanwhile, 258 mM NaCl significantly increased both SbPPCK1 and SbPPCK2 gene expression and/or mRNA stability. The combination of these factors contributed to maintain a high PEPCase-k activity in salinity. LiCl increased calcium-dependent protein kinase (CDPK) activity in illuminated sorghum leaves while it decreased the rate of PEPCase-k degradation. The latter effect was restrained by W7, an inhibitor of CDPK activity. Recombinant PEPCase-k protein was phosphorylated in vitro by PKA. A conserved phosphorylation motif, which can be recognized by PKA and by plant CDPKs, is present in the three PEPCase-ks proteins. Thus, it is possible that a phosphorylation event could be controlling (increasing) the stability of PEPCase-k in salinity. These results propose a new mechanism of regulation of PEPCase-k levels, and highlight the relevance of the preservation of key metabolic elements during the bulk degradation of proteins, which is commonly associated to stress.

A cathepsin F-like peptidase involved in barley grain protein mobilization, HvPap-1, is modulated by its own propeptide and by cystatins.

Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described.

Synergic effect of salinity and CO2 enrichment on growth and photosynthetic responses of the invasive cordgrass Spartina densiflora.

Spartina densiflora is a C(4) halophytic species that has proved to have a high invasive potential which derives from its clonal growth and its physiological plasticity to environmental factors, such as salinity. A greenhouse experiment was designed to investigate the synergic effect of 380 and 700 ppm CO(2) at 0, 171, and 510 mM NaCl on the growth and the photosynthetic apparatus of S. densiflora by measuring chlorophyll fluorescence parameters, gas exchange and photosynthetic pigment concentrations. PEPC activity and total ash, sodium, potassium, calcium, magnesium, and zinc concentrations were determined, as well as the C/N ratio. Elevated CO(2) stimulated growth of S. densiflora at 0 and 171 mM NaCl external salinity after 90 d of treatment. This growth enhancement was associated with a greater leaf area and improved leaf water relations rather than with variations in net photosynthetic rate (A). Despite the fact that stomatal conductance decreased in response to 700 ppm CO(2) after 30 d of treatment, A was not affected. This response of A to elevated CO(2) concentration might be explained by an enhanced PEPC carboxylation capacity. On the whole, plant nutrient concentrations declined under elevated CO(2), which can be ascribed to the dilution effect caused by an increase in biomass and the higher water content found at 700 ppm CO(2). Finally, CO(2) and salinity had a marked overall effect on the photochemical (PSII) apparatus and the synthesis of photosynthetic pigments.