Expresión de los genes de la fosfoenolpiruvato Carboxilasa (PEPC) y PEPC-Quinasa (PPCK) de la semilla de sorgofunción y regulación por fosforilación y ubiquitinación de estas proteínas en el desarrollo y germinación de la semilla

Resumen

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important cytosolic regulatory enzyme that plays a pivotal photosynthetic role in primary CO2 fixation by C4 and Crassulacean acid metabolism leaves but also has a variety of additional important functions in plants, including seed development and germination (O�Leary et al., 2011b). PEPC was characterized in the life cycle of sorghum seeds, a mono-cotyledonous C4 plant, focusing on the post-translational control of the enzyme. The activity of the enzyme was investigated in developing and germinating grains. During seed development, total PEPC activity increased to reach a maximum level in the stage III. During imbibition, total PEPC activity in embryo was increasing and was found to be higher at 96 h while in aleuron/endosperm PEPC activity did not change significantly and it was lower than embryos. Immunolocalization studies in 24 h germinated seeds revealed the presence of PEPC in tissues with a high metabolic activity. The phosphorylation state in crude extract, as judged by L-malate 50% inhibition of initial activity values, Pro-Q Diamond phosphoprotein staining, immunodetection with pSer13-IGgs against the phosphorylated N terminus was found to be high between the stages 7 and 27 days postanthesis (DPA) and between 24-72 h of germinated seed at 25 ºC. In contrast, the enzyme appeared to be in a low phosphorylation state from 27 DPA up to 14 h and in the late stages of germination. At 48 h, in vivo radiolabeling, followed by PEPC immunoprecipitation, SDS-PAGE and autoradiography further demonstrated 32Pi incorporation into immunoprecipitated p110 and p107. In sorghum, PEPC is encoded by a small multigene family with six PEPC genes: Sb8720-CP21, Sb1090-CP28, Sb4960, Sb5090 (C3 type) and Sb1330 (C4 photosynthetic type) encode closely related plant type PEPC (PTPCs), whereas Sb1330 encodes the distantly related bacterial type PEPC4 (BTPC) (Paterson et al., 2009). In the present work, we describe the analysis by real time PCR of the expression of these PTPCs genes. CP21, CP28, 4960 transcripts were found in sorghum seed. MS studies of PEPC purified by immunoaffinity chromatography and immunoprecipitation of clarified extracts revealed that those different PTPC isoforms and Sb5090 as well, co-exists in germinating sorghum seeds, however CP21 was the most abundant. In addition, all of these PTPCs were in vivo phosphorylated at their conserved N-terminal phosphorylation site. PEPC is subjected to in vivo regulatory phosphorylation by a specific Ca2+-independent serine/threonine kinase known as PPCK (PEPC kinase) (Nimmo et al., 2003). The genome of sorghum contains three putative PPCK genes: SbPPCK1, SbPPCK2 and SbPPCK3 (Paterson et al., 2009) and we found expression for all of them in seeds. We also described new phosphorylation sites in PTPCs, that never had been seen before. In developed and germinated seeds, q-PCR analysis confirmed the low accumulation of BTPC transcripts. In addition, Class-2 PEPC p107/p118 hetero-octamer was not detected when the extracts were subjected to in-gel PEPC activity staining. Two immunoreactive PTPC polypeptides having molecular masses of 110 and 108- or 103-kD have frequently been observed on immunoblots of extracts from diverse C3 plant tissues including species such as barley, wheat and sorghum (Osuna et al., 1996, 1999; Gonzalez et al., 1998; Nhiri et al., 2000; Feria et al., 2008). In order to establish the biochemical basis for this observation, a 460 kDa PEPC heterotetramer composed of an equivalent ratio of p110 and p107 subunits was purified to near homogeneity from 48 h germinated seeds. Mass spectrometry established that p110 and p107 are both encoded by the same plant-type PEPC gene (8720-CP21), but that p107 was in vivo monoubiquitinated at Lys624 to form p110. This residue is absolutely conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Anti-ubiquitin IgG immunodetected p110 but not p107, whereas incubation with a deubiquitinating enzyme (USP-2 core) efficiently converted p110 into p107, while relieving the enzyme�s feedback inhibition by L-malate. The purified PEPC was not phosphorylated, as indicated by immunoblotting with phospho-site specific antibodies. However, both subunits were phosphorylated in vitro by PEPC kinase 2 (PPCK2) and PPCK3. By contrast, immunoaffinity chromatography of clarified extracts, coupled with immunoblotting and MS indicated that this fraction contained different PEPCs among which the CP21, CP28 and 4960 were phosphorylated in vivo at the consensus N-terminal Ser in both p110 and p107 subunits. Among them CP21 was shown not to be phosphorylated at p107 during germination in sorghum seeds. Ms also revealed the monoubiquitination site at Lys624 in CP21 PEPC and at Lys630 in 1090-CP28 PEPC isoenzyme. Partial PEPC monoubiquitination was also detected during sorghum seed development for the first time. Results of the current study suggest a novel pattern of post-translational modification of plant PEPC in seeds, as it appears that the same p110 subunit can be simultaneously phosphorylated and monoubiquitinated in vivo in a starch-storing cereal seed. It is apparent that monoubiquitination at Lys is opposed to phosphorylation at Ser in terms of regulating the catalytic activity of sorghum seed PEPC. Monoubiquitination inhibited sorghum PEPC by sensitizing the enzyme to the allosteric inhibitor L-malate, lowers IC50, while phosphorylation actived the enzyme increasing its IC50 value. PEPC monoubiquitination is hypothesized to fine-tune anaplerotic carbon flux according to the cell�s immediate physiological requirements for tricarboxylic acid cycle intermediates needed in support of biosynthesis and carbon-nitrogen interactions. In this thesis we also propose various models acting in sorghum seed that will be analyzed with futures studies. PEPC interactome of sorghum seeds was assessed using coimmunopurification followed by proteomic analysis. Peptide mass fingerprinting confirmed that 438 proteins coimmunopurificates with PEPC but the most interesting result was the interaction with sucrose synthase (SUS), also confirmed by immunoblotting with SUS-IgGs. An extensive copurification of PEPC with SUS also occurs in developing COS and in proteoid roots from harsh hakea (Gennidakis et al., 2007; Shane et al., 2013). Future research will need to know how these two key enzymes of carbohydrate metabolism might interact in vivo in the cytosol of the cells. Finally, new results in this thesis showed for the first time that monoubiquitination occurred earlier and the time taken was reduced when seeds were germinated at 35 °C instead of 25 ºC and it was an early step that always happened from stage II to IV of the germinating seeds.