The Original Form of C4-Photosynthetic Phospho enol pyruvate Carboxylase Is Retained in Pooids but Lost in Rice

Abstract

Poaceae is the most prominent monocot family that contains the primary cereal crops wheat, rice, and maize. These cereal species exhibit physiological diversity, such as different photosynthetic systems and environmental stress tolerance. Phosphoenolpyruvate carboxylase (PEPC) in Poaceae is encoded by a small multigene family and plays a central role in C₄-photosynthesis and dicarboxylic acid metabolism. Here, to better understand the molecular basis of the cereal species diversity, we analyzed the PEPC gene family in wheat together with other grass species. We could designate seven plant-type and one bacterial-type grass PEPC groups, ppc1a, ppc1b, ppc2a, ppc2b, ppc3, ppc4, ppcC₄, and ppc-b, respectively, among which ppc1b is an uncharacterized type of PEPC. Evolutionary inference revealed that these PEPCs were derived from five types of ancient PEPCs (ppc1, ppc2, ppc3, ppc4, and ppc-b) in three chromosomal blocks of the ancestral Poaceae genome. C₄-photosynthetic PEPC (ppcC₄ ) had evolved from ppc1b, which seemed to be arisen by a chromosomal duplication event. We observed that ppc1b was lost in many Oryza species but preserved in Pooideae after natural selection. In silico analysis of cereal RNA-Seq data highlighted the preferential expression of ppc1b in upper ground organs, selective up-regulation of ppc1b under osmotic stress conditions, and nitrogen response of ppc1b. Characterization of wheat ppc1b showed high levels of gene expression in young leaves, transcriptional responses under nitrogen and abiotic stress, and the presence of a Dof1 binding site, similar to ppcC₄ in maize. Our results indicate the evolving status of Poaceae PEPCs and suggest the functional association of ppc1-derivatives with adaptation to environmental changes.

Keywords: Pooideae; abiotic stress; gene function and evolution; grass genome evolution; nitrate response; phosphoenolpyruvate carboxylase; positive selection; ppc1b.

Figure 1

Schematic representation of cross-species genome-wide analysis of grass Phosphoenolpyruvate carboxylases (PEPCs).

Figure 2

An maximum likelihood (ML) phylogenetic tree dissects grass PEPC proteins. Out-grouped PEPCs were shown as follows; Chlamydomonas reinhardtii (P81831, A0A2K3CVJ4, A0A2K3D8D0, Q6R2V6, and A0A2K3DX44), Ostreococcus tauri (A0A1Y5IAS9, A0A090M703), Anabaena sp. (K7VXZ2), Synechocystis sp. (P74299), Marchantia polymorpha (A0A2R6X663), Escherichia coli (P00864, E2QIY7), and Ferroglobus placidus (D3S0D1). The log likelihood for this tree was “−37030.92.”

Figure 3

Inferred molecular inheritance of PEPC isogenes in the ancestral grass genome model. The molecular clock information was referred from a study by The International Brachypodium Initiative (2010). The present status of the rice genome, the O. officinalis genome, the wheat genome, the sorghum genome, and the maize genome were also shown. The asterisk represents “pseudogene.”

Figure 4

A partial NJ tree represents the orthologous relationship between ppc1b and ppcC₄.

Figure 5

An evolutionary model for ppc1b and ppcC₄ in Eleusine coracana.

Figure 6

A NJ phylogenetic tree with JTT model for monocot PEPCs. Key PEPC protein identifiers are follows: Joasc.17G077400, Joasc.09G001100, Joasc.06G112900, and Joasc.07G069900 (Joinvillea ascendens), Phala.12G050000, Phala.04G0141900, and Phala.02G088600 (Pharus latifolius), Spipo19G0008900 and Spipo14G0030900 (Spirodela polyrhiza), Acora.02G073100 and Acora.02G021300 (Acorus americanus), Aco018093, Aco010025, and Aco016429 (Ananas comosus), Dioal.05G101700, Dioal.04G055800, and Dioal.17G104400 (Dioscorea alata), evm.model.AsparagusV1 05.1847 and evm.model.AsparagusV1 01.440 (Asparagus officinalis), GSMUA Achr9P06420, GSMUA Achr6P26850, GSMUA Achr4P08030, GSMUA Achr6P14490, GSMUA Achr10P25410, and GSMUA Achr1P20720 (Musa acuminata), Zosma04g04800, Zosma06g00180, Zosma05g25910, and Zosma05g32230 (Zostera marina). Black triangles represent sub-lineages of PEPC types. The bar shows the branch length of 0.2.

Figure 7

Detected positive selection in the ppc1b and ppcC₄ lineages. (A) A partial phylogenetic tree showing ω values of branches. The ω values of the ppc1b lineage were underlined. Bold ω values represent statistical significance. (B) The positive selection site in the ppc1b branch. The amino acid positions are maize C₄ PEPC coordinate’s. α_c represents the physicochemical amino acid property related to the substitution.

Figure 8

Abiotic stress responses of Tappc1a, Tappc1b, and Tappc4 isogenes. Asterisks represent statistical significance at 5% level in Student’s t-test. (A) Drought stress responses in flag leaves, (B) salt stress (100 mM NaCl) responses in roots and leaves.

Figure 9

Enzyme activity and gene expressions of PEPCs in response to nitrate. Error bars indicate SEs. Double and single asterisks indicate statistical significance at 5 and 10% level, respectively. (A) PEPC activity per g fresh weight, (B) PEPC activity per mg protein, (C–G) Relative expression levels of wheat PEPC isogene groups, and (H–P) Relative expression levels of Tappc1b, Tappc2, and Tappc4 isogenes in the A, B, and D genomes.

Figure 10

A predicted cis-motif associated with the nitrate responses of Tappc1b isogenes. The black bar above the motif logo represents the position of Dof1 binding core motif.