Phosphorylation of Phosphoenolpyruvate Carboxylase Is Not Essential for High Photosynthetic Rates in the C₄ Species Flaveria bidentis

Abstract

Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) plays a key role during C 4 photosynthesis. The enzyme is activated by metabolites such as glucose-6-phosphate and inhibited by malate. This metabolite sensitivity is modulated by the reversible phosphorylation of a conserved serine residue near the N terminus in response to light. The phosphorylation of PEPC is modulated by a protein kinase specific to PEPC (PEPC-PK). To explore the role PEPC-PK plays in the regulation of C 4 photosynthetic CO 2 fixation, we have transformed Flaveria bidentis (a C 4 dicot) with antisense or RNA interference constructs targeted at the mRNA of this PEPC-PK. We generated several independent transgenic lines where PEPC is not phosphorylated in the light, demonstrating that this PEPC-PK is essential for the phosphorylation of PEPC in vivo. Malate sensitivity of PEPC extracted from these transgenic lines in the light was similar to the malate sensitivity of PEPC extracted from darkened wild-type leaves but greater than the malate sensitivity observed in PEPC extracted from wild-type leaves in the light, confirming the link between PEPC phosphorylation and the degree of malate inhibition. There were, however, no differences in the CO 2 and light response of CO 2 assimilation rates between wild-type plants and transgenic plants with low PEPC phosphorylation, showing that phosphorylation of PEPC in the light is not essential for efficient C 4 photosynthesis for plants grown under standard glasshouse conditions. This raises the intriguing question of what role this complexly regulated reversible phosphorylation of PEPC plays in C 4 photosynthesis.

Evolution of the Phosphoenolpyruvate Carboxylase Protein Kinase Family in C3 and C4 Flaveria spp.

“…Thus, phosphorylation seems to broaden the conditions under which PEPC can be active and seems to decrease the K m , while the V max of PEPC is only modestly affected Tovar-Méndez et al, 2000;Takahashi-Terada et al, 2005). Interestingly, the knockdown of PEPC phosphorylation in Flaveria bidentis (C 4 ) by RNA interference inhibition of PPCK did not affect the CO 2 assimilation rate, although the response to malate was observed as in previous studies (Furumoto et al, 2007). The latter indicates that phosphorylation is probably required to adjust PEPC activity in response to various signals or fluctuating conditions or to potentially mediate the interaction with another protein, such as 14-3-3 proteins (O'Leary et al, 2011;Grieco et al, 2012).…”

“…The genus Flaveria is an exception, with only one characterized PPCK gene, encoding a 31.8-kD protein identified in F. trinervia and F. bidentis (Tsuchida et al, 2001;Furumoto et al, 2007). This raises the question of whether these species are a true exception in possessing only a single PPCK gene or if the nucleic acid sequences for further isoforms simply have been missed so far.…”

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Essential for Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Species Kalanchoë fedtschenkoi

“…All Flaveria species also possessed a PPCKB gene that displayed peak transcript abundance in the dark period and that was downregulated in the C 4 species relative to the C 3 species. Aldous et al (2014) suggested that the C 4 -recruited ppcA and PPCKA formed a coevolving substrate-kinase pair, and yet the findings of Furumoto et al (2007) argued that PPCKA in F. bidentis was dispensable for optimal CO 2 fixation via the C 4 pathway. Why would there be strong evolutionary selection acting on the C 4 ppcA/PPCKA substrate-kinase pair if the kinase and its phosphorylation of PPC were dispensable for optimal CO 2 fixation via the C 4 pathway?…”

“…Previous work had shown that the PPCK gene responsible for light period phosphorylation and in vivo activation of photosynthetic PPC in the C 4 species Flaveria bidentis was not essential for high C 4 photosynthetic rates in this species (Furumoto et al, 2007). This work on a C 4 species surprised the wider community because decades of prior work had argued that PPC phosphorylation by PPCK was vital for alleviating malate/aspartate inhibition of PPC in planta and thus for optimizing photosynthetic CO 2 fixation in C 4 and CAM species (Vidal and Chollet, 1997;Nimmo, 2003).…”

Antisense Reduction of NADP-Malic Enzyme in Flaveria bidentis Reduces Flow of CO2 through the C4 Cycle

“…The nature of the controlling mechanisms for balance and coordination between the C 3 and C 4 cycles is still unclear, however, and concrete evidence for the coordinated regulation of primary carboxylation in the mesophyll and decarboxylation of C 4 acids in the bundle sheath has not been forthcoming. A key approach to revealing these mechanisms has been the use of antisense RNA in the C 4 dicot F. bidentis to reduce levels of key photosynthetic enzymes, including Rubisco (Furbank et al, 1996), NADP-malate dehydrogenase and pyruvate phosphate dikinase , Rubisco activase , carbonic anhydrase (Cousins et al, 2006), and PEPC protein kinase (Furumoto et al, 2007). This has proven to be a valuable method to help gain insight into enzyme function and regulation during C 4 photosynthesis and to potentially alter the balance between the C 3 and C 4 cycles.…”