Oil and Protein Accumulation in Developing Seeds Is Influenced by the Expression of a Cytosolic Pyrophosphatase in Arabidopsis    

Abstract

This study describes a dominant low-seed-oil mutant (lo15571) of Arabidopsis (Arabidopsis thaliana) generated by enhancer tagging. Compositional analysis of developing siliques and mature seeds indicated reduced conversion of photoassimilates to oil. Immunoblot analysis revealed increased levels of At1g01050 protein in developing siliques of lo15571. At1g01050 encodes a soluble, cytosolic pyrophosphatase and is one of five closely related genes that share predicted cytosolic localization and at least 70% amino acid sequence identity. Expression of At1g01050 using a seed-preferred promoter recreated most features of the lo15571 seed phenotype, including low seed oil content and increased levels of transient starch and soluble sugars in developing siliques. Seed-preferred RNA interference-mediated silencing of At1g01050 and At3g53620, a second cytosolic pyrophosphatase gene that shows expression during seed filling, led to a heritable oil increase of 1% to 4%, mostly at the expense of seed storage protein. These results are consistent with a scenario in which the rate of mobilization of sucrose, for precursor supply of seed storage lipid biosynthesis by cytosolic glycolysis, is strongly influenced by the expression of endogenous pyrophosphatase enzymes. This emphasizes the central role of pyrophosphate-dependent reactions supporting cytosolic glycolysis during seed maturation when ATP supply is low, presumably due to hypoxic conditions. This route is the major route providing precursors for seed oil biosynthesis. ATP-dependent reactions at the entry point of glycolysis in the cytosol or plastid cannot fully compensate for the loss of oil content observed in transgenic events with increased expression of cytosolic pyrophosphatase enzyme in the cytosol. These findings shed new light on the dynamic properties of cytosolic pyrophosphate pools in developing seed and their influence on carbon partitioning during seed filling. Finally, our work uniquely demonstrates that genes encoding cytosolic pyrophosphatase enzymes provide novel targets to improve seed composition for plant biotechnology applications.

Predictive Modeling of Biomass Component Tradeoffs in Brassica napus Developing Oilseeds Based on in Silico Manipulation of Storage Metabolism

“…Manipulation of the cytosolic PPi balance could have an impact on carbon partitioning. Accordingly, overexpression of a cytosolic pyrophosphatase (At2g01050) in Arabidopsis has been found to reduce seed oil content, while silencing expression of the gene resulted in increased oil content at the expense of seed storage protein (Meyer et al, 2012). Our modeling approach might help to explain the effect of the manipulation of PPi on biomass composition.…”

Differential expression of soluble pyrophosphatase isoforms inArabidopsis upon external stimuli

“…The presence of a vacuolar membrane localized proton-pumping PPase (Geingenberger et al, 1998;Farre et al, 2001) and data implying the importance of a large cytosolic PP i pool for plant growth and development (Jelitto et al, 1992;Sonnewald, 1992) suggested lack of cytosolic sPPase activity. Recent data, however, imply possible roles of cytosolic sPPases in the regulation of plant metabolism (Meyer et al, 2012;Öztürk et al, 2014). In this study, we summarize real time PCR analyses of expressional changes of PPa isoforms in response to etiolation, different carbon sources, and environmental stresses including ABA, cold, salt, and phosphate deficiency.…”

“…genome has six sPPase isoforms, one of which was shown to be localized in the plastids and the others in the cytosol and/or nucleus (Schulze et al, 2004;George et al, 2010;Öztürk et al, 2014). Navarro-De la Sancha et al (2007) reported the possibility of ubiquitous expression of A. thaliana sPPase isoforms based on the GENEVESTIGATOR database and suggested that changes in cytosolic PP i concentration by differential expression of sPPase isoforms might be important in the regulation of plant metabolism (NavarroDe la Sancha et al, 2007;Meyer et al, 2012). Here, we aimed to analyze the differential expression of A. thaliana soluble pyrophosphatase isoforms in response to external stimuli to get further insight into their possible roles in the plant metabolism.…”

Roles of the vacuolar H⁺-PPase in seed storage oil mobilization and plant development

“…With the exception of the chloroplastic sPPase AtPPa6, the other five enzymes are predicted to be cytosolic. Interestingly, increased protein levels of AtPPa1 reduced the conversion of photo-assimilates to oil in developing fruits and mature seeds (Meyer et al 2012 (Maeshima 2001, Gaxiola et al 2007. Although the H + -PPase is known to be an important proton pump, the importance of its enzymatic hydrolysis of PPi has not been explored.…”

“…It is generally believed that sPPase activities are negligible in the plant cytosol (Weiner et al 1987). However, the Arabidopsis genome possesses six sPPase genes: AtPPa1 through AtPPa6, which correspond to At1g01050, At2g18230, At2g46860, At3g53620, At4g01480, and At5g09650, respectively (Meyer et al 2012, Ferjani et al 2014. These homologs are subdivided into prokaryotic (AtPPa1 to AtPPa5) and eukaryotic types (AtPPa6) (Rojas-Beltrán et al 1999, Gómez-García et al 2006.…”