Mutation of the ALBOSTRIANS Ohnologous Gene HvCMF3 Impairs Chloroplast Development and Thylakoid Architecture in Barley.

Mingjiu Li,Michael Melzer,Henning Tschiersch,Goetz Hensel,Nils Stein,Astrid Junker,Hannes Ruwe,Thomas Börner,Jochen Kumlehn,Daniel Arend

doi:10.3389/fpls.2021.732608

Abstract

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed them as members of a subfamily of genes coding for CCT motif (CONSTANS, CONSTANS-LIKE and TIMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a yellowish/light green – xantha – phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus. Hvcmf3Hvcmf7 double mutants exhibited a xantha-albino or albino phenotype depending on the strength of molecular lesion of the HvCMF7 allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the Hvcmf3 mutants. Based on our observations, the genes HvCMF3 and HvCMF7 have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes.

Highlights

Chloroplasts are the photosynthetic active type of plastids
Most of the genes needed for plastid functions and in particular for the development of chloroplasts and their photosynthetic apparatus are, encoded in the nuclear genome and are targeted to the plastid/chloroplast; including genes involved in chloroplast transcription, RNA processing, RNA stability, and translation (Börner et al, 2014; Pogson et al, 2015)
Of specific interest are nuclear encoded genes which are dually targeted to both plastids and nucleus since they might be involved in the regulation of chloroplast biogenesis and in the communication between nucleus and plastids (Krupinska et al, 2020)

Summary

Introduction

Functional chloroplasts contain thylakoid membranes, which are the site of light-dependent photosynthesis reactions as mediated by four protein complexes – photosystem I (PSI), photosystem II (PSII), cytochrome b6f and ATPase (Dekker and Boekema, 2005). While PSII is mainly found in the grana thylakoids, PSI and the ATPase complex are enriched in the stroma lamellae, and the cytochrome b6f complex is distributed evenly between the two structures (Dekker and Boekema, 2005). Chloroplasts originated from photosynthetic cyanobacteria (Gould et al, 2008) They contain their own genome with a core set of approximately 100 genes inherited from the cyanobacterial ancestor and possess their own machinery for gene expression, i.e., for transcription, transcript processing and translation (Börner et al, 2014; Pogson et al, 2015). Extensive studies have demonstrated that chloroplast development and function require the import of nucleus-encoded proteins; more than 95% of the chloroplast proteins are encoded by the nuclear genome and subsequently targeted to the chloroplasts, in most cases with help of an N-terminal chloroplast transit peptide, cTP (Leister, 2003; Lee and Hwang, 2018)

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