Mecanismos implicados en la regulación de la actividad de la Fosfoenolpiruvato Carboxilasa (PEPC) y de la PEPC-quinasa en salinidad, carencia de hierro y estrés por amonio.

Resumen

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is a cytosolic enzyme well-known by its role in C4 and CAM photosynthesis. In addition, bicarbonate fixation by PEPC results in the anaplerotic production of organic acid skeletons in seeds, fruits, roots, legume nodules, other C3 tissues, and in leaves of C3 plants. C4-PEPC, the enzyme responsible for primary carboxylation in C4 photosynthesis, is phosphorylated by a light-dependent phosphoenolpyruvate carboxylase-kinase (PEPC-k). Increased phosphorylation of PEPC has been observed under nitrogen stress, phosphate starvation, oxidative stress and salinity. There is increasing evidence that supports a role for PEPC and PEPC-k in plant responses to stress. The main objectives of this research project were: To identify the mechanisms controlling the turnover of sorghum PEPC-k that contribute to enhance this enzyme activity in salinity. To investigate the regulation of PEPC-k activity by nitric oxide and to elucidate whether NO could mediate some of the effects of salinity on PEPC-k. To study the function of leaf PEPC in plant responses to iron deficiency, and its role in the synthesis of stress molecules in plant concurrently subjected to nutrient deficit and salt stress. To evaluate the participation of PEPC and PEPC-k in plant responses to ammonium stress. Factors involved in the rise of phosphoenolpyruvate carboxylase-kinase activity caused by salinity in sorghum leaves Salinity is the foremost environmental factor limiting plant growth and productivity. Many species displaying C4-type of photosynthesis are among the most salt-tolerant plants. Salinity increased PEPC-k activity in sorghum leaves, both in light and in the dark. This raise of PEPC-k activity was accompanied by an increase in the phosphorylation state of PEPC together with enlarged malate synthesis, and it could contribute to improve carbon deficiency under salinity. The physiological significance of the phenomenon is supported by the fact that both PEPC and PEPC-k are protected from degradation during aging and extended salinization. At least in part, the decreased rate of PEPC-k degradation is a consequence of the negative control of the ubiquitin-proteasome pathway by the phytohormone abscisic acid (ABA). This part of the research was aimed to identify the mechanisms involved in the rise of PEPC-k activity caused by salinity in sorghum, a C4 plant. The expression of three putative PPCK genes in sorghum leaves was analysed by quantitative RT-PCR (qPCR). Light increased SbPPCK1 level of transcripts, but had little effect on SbPPCK2 or SbPPCK3 expression. This result supported the role of SbPPCK1 in C4 photosynthesis. Salinity impacted differently on PEPC-k activity and on PPCK mRNA transcript levels. Salinity at 172 mM NaCl markedly increased PEPC-k activity and the phosphorylation state of PEPC, but had little effect on SbPPCK1, and SbPPCK2 mRNA transcript levels. At 258 mM NaCl, on the contrary, salt significantly increased SbPPCK1 and SbPPCK2 mRNA transcript level in the light. These results show that changes of PEPC-k activity caused by salt can not be attributed exclusively to changes of SbPPCK gene expression or to increased mRNA stability. At 172 mM NaCl changes are mainly due to a decreased rate of PEPC-k protein degradation. Meanwhile, increased SbPPCK gene expression and/or increased mRNA stability are contributing to enhance PEPC-k activity in 258 mM NaCl treated plants. Treatment with LiCl increased a calcium-dependent protein kinase activity (CDPK) while decreasing the rate of the degradation of PEPC-k protein in sorghum leaves. Recombinant PEPC-ks were produced as (10xHis)-proteins and were evaluated as substrates for phosphorylation by protein kinase A (PKA). The (10xHis)-PEPC-ks were efficiently phosphorylated by this mammalian kinase. Thus, it is possible that a phosphorylation event could be controlling (increasing) the stability of PEPC-k in salinity. These results propose a new mechanism of regulation of PEPC-k levels, and highlight the relevance of the preservation of key metabolic elements during the bulk degradation of proteins that is commonly associated to stress. Nitric oxide regulation of leaf phosphoenolpyruvate carboxylase-kinase activity: implication in sorghum responses to salinity Nitric oxide (NO) is a signaling molecule that mediates many plant responses to biotic and abiotic stresses, including salt stress. Interestingly, salinity increases NO production selectively in mesophyll cells of sorghum leaves, where photosynthetic C4 phosphoenolpyruvate carboxylase (C4-PEPC) is located. PEPC is regulated by a phosphoenolpyruvate carboxylase kinase (PEPC-k), which levels are greatly enhanced by salinity in sorghum. This part of the work investigated whether NO is involved in this effect. NO donors (SNP, SNAP), the inhibitor of NO synthesis NNA, and the NO scavenger cPTIO were used for long- and short-term treatments. Long-term treatments had multifaceted consequences on both PPCK gene expression and PEPC-k activity, and they also decreased photosynthetic gas exchange parameters and plant growth. Nonetheless, it could be observed that SNP increased PEPC-k activity, resembling salinity effect. Short-term treatments with NO donors, which did not change photosynthetic gas exchange parameters and PPCK gene expression, increased PEPC-k activity both in illuminated leaves and in leaves kept at dark. At least in part, these effects were independent on protein synthesis. PEPC-k activity was not decreased by short-term treatment with cycloheximide in NaCl-treated plants; on the contrary, it was decreased by cPTIO. In summary, NO donors mimicked salt effect on PEPC-k activity, and scavenging of NO abolished it. Collectively, these results indicate that NO is involved in the complex control of PEPC-k activity, and it may mediate some of the plant responses to salinity. Role of leaf phosphoenolpyruvate carboxylase in barley tolerance to iron deficiency and on subsequent proline synthesis triggered by salinity The main objective of this research was to evaluate the role of leaf PEPC in plant responses to Fe deficiency, and on the subsequent synthesis of the stress metabolite proline caused by salinity. The working hypothesis was that the supply of reduced carbon by PEPC could alleviate the shortage due to reduced photosynthesis caused by Fe deficiency, and that it could allow a more efficient synthesis of stress molecules, such as proline, in response to salinity. Fe deficiency reduced growth and photosynthetic pigments, leading to development of chlorosis, in sugar beet, sorghum and barley. Symptoms appeared at 10 mM NaFe-EDTA in sorghum, and at 0.5 mM NaFe-EDTA in sugar beet and barley. The increase of PEPC activity in roots of Strategy I plants (non-graminaceous monocots and dicots) in response to Fe deficiency is a well known phenomenon, but little is known about leaf PEPC in this nutritional stress. Leaf PEPC activity increased in leaves of sugar beet, sorghum and barley in response to Fe deficiency. This increase was positively correlated with the shoot growth of six barley varieties, with comparable tolerance to Fe deficiency, supplied with 0.5 mM NaFe-EDTA. In addition, when barley plants were subjected to salinity, a positive and statistically significant correlation between shoot PEPC and shoot proline was found at 0.5 mM NaFe-EDTA. The results confirmed the working hypothesis that the supply of reduced carbon by leaf PEPC could alleviate the shortage due to reduced photosynthesis caused by Fe deficiency, and that it could allow a more efficient synthesis of proline in response to salinity. As suggested for root PEPC, this parameter could help to screen for more tolerant genotypes. Effect of ammonium stress on sorghum PEPC: monoubiquitination as a novel mechanism of regulation. Nitrogen is quantitatively the most required element for plants, which reduce nitrate to ammonium in order to incorporate this nutrient in its reduced form to essential organic molecules, such as amino acids, proteins, nucleic acids and others. Thus, nitrogen nutrition is a major limiting factor for growth. On the other hand, NH4+ can result toxic for plants when it becomes the main source of N instead of nitrate. Carbon depletion, acidification, deficiencies of mineral cations, and useless NH4+ cycling, are some of the effects of excess of NH4+. The tolerance to NH4+ depends in large part on the N assimilation capacity. The enzyme phosphoenolpyruvate carboxylase (PEPC) is good known for its important role in this process. Bicarbonate fixation by PEPC results in the anaplerotic production of organic acid skeletons for amino acid synthesis. Previous studies have revealed that PEPC activity and gene expression are affected by ammonium supply in many species. This part of the work studies the effect of ammonium nutrition on PEPC activity, its phosphorylation, and PEPC gene expression in roots and leaves of Sorghum bicolor. Plants supplied with 5mM NH4+ as sole nitrogen source showed alteration of the morphology of roots and shoots. Manifest symptoms of ammonium toxicity included slowed growth rate, reduced biomass production, and decreased root to shoot ratio, as well as acidification of the culture medium. C3-PEPC activity from sorghum roots was increased in ammonium treated plants, and it was observed a general rise of the expression of the genes encoding the different isoforms of root PEPC. Quantitative RT-PCR (qPCR) showed that Sb02g021090, Sb07g014960, Sb03g035090, Sb04g008720 and Sb03g008410 level of transcripts were higher in plants fed with ammonium. In addition, preliminary results indicated that ammonium promoted the phosphorylation of the C4-PEPC in leaves, which decreases the sensibility of the enzyme to its allosteric inhibitor L-malate. Postraductional modifications of root PEPC were investigated by SDS/PAGE and immunoblot analysis. Polyclonal antibodies against C4-PEPC immunodecorated two polypeptides (103-kD and 108-kD) in extracts from control and ammonium plants. The signal corresponding to the 108-kD polypeptide was more intense in ammonium plants, and it was recognized by anti-ubiquitin antibodies. The identity of PEPC was further confirmed by immunoprecipitation. These results suggested that ammonium stress triggered the monoubiquitination of root PEPC, a process that has been shown to regulate PEPC activity in castor oil seeds. Further studies will clarify this mechanism of regulation of PEPC, and its relevance for such an important crop as sorghum in ammonium rich soils. Phosphatidic Acic (PA) has been shown to bind to C4-PEPC. In addition, C4-PEPC activity, unlike its C3 counterpart, was inhibited by addition of PA. It was found that ammonium treatment increased the affinity of PA towards root C3-PEPC in sorghum. Further experiments will clarify whether this fact is related to the monoubiquitination of the PEPC in ammonium treated plants. Finally, preliminary experiments were carried out with Arabidopsis thaliana to visualize changes in the architecture of the roots caused by ammonium. This will open a research line studying Arabidopsis lines lacking specific PEPC isoenzymes, and, in this way, it would contribute to clarify the role of these specific PEPC isoenzymes in plant responses to ammonium stress.