Mutation of theALBOSTRIANSOhnologous GeneHvCMF3Impairs Chloroplast Development and Thylakoid Architecture in Barley due to Reduced Plastid Translation

Abstract

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained only if at least one gene of the pair undergoes neo- or subfunctionalization. Sequence-based phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed that they belong to a newly identified subfamily of genes encoding CCT domain proteins with putative N-terminal chloroplast transit peptides. 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 xantha phenotype and successively develop pale green leaves. Compared to the wild type, plastids of the mutant seedlings show decreased PSII efficiency and lower 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 region, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs localized to plastids. Hvcmf3Hvcmf7 double mutants indicated epistatic activity of HvCMF7 over HvCMF3. The chloroplast ribosome deficiency is discussed as the primary defect of the Hvcmf3 mutants. Our data suggests that HvCMF3 and HvCMF7 have similar but not identical functions.One-sentence summaryPhylogenetic and mutant analyses of the barley protein HvCMF3 (ALBOSTRIANS-LIKE) identified, in higher plants, a subfamily of CCT domain proteins with essential function in chloroplast development.

The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs involved in Chloroplast Development

“…The more serious effect of the two mutations in cia2cil compared to cia2 on 16S and 23SrRNA processing, chlorophyll content, thylakoid/grana structure and, in particular, photosynthetic parameters may be responsible for the slower growth of cia2cil vs. cia2, cil and wild type. Mutant analyses demonstrated that inefficient chloroplast translation is often associated with altered organization of the thylakoid membrane system (Fristedt et al, 2014;Liu et al, 2015;Zhang et al, 2017;Li et al, 2019a). The delayed greening phenotype of cia2cil resembles the rbf1-1 mutant with defects in chloroplast translation due to inefficient processing of 16S rRNA (Fristedt et al, 2014).…”

“…After antibiotic screening with hygromycin, 24 plants were selected from each of the Col-0, cia2 mutant and HvCMF3 families, and 96 plants were selected from each of the HvCMF7 and ▲cTP_CIA2 families. The selected plants were transferred into the imaging system and phenotyping was performed following the protocols described previously (Li et al, 2019a) with the following modifications. For experiment I, measurement of PSII operating efficiency (ΦPSII) and electron transport rate (ETR) were performed for light adapted plants.…”

“…Recently, we identified a small class of nuclear-encoded proteins in seed plants (Li et al, 2019b;Li et al, 2019a) representing a subfamily of CMF (CCT MOTIF FAMILY) proteins (Cockram et al, 2012). The CCT domain [from the three Arabidopsis (Arabidopsis thaliana) proteins CONSTANS, CONSTANS-LIKE and TIMING OF CAB1] is found near the C-terminus of numerous proteins.…”

“…The CCT domain is described to support transport into the nucleus and protein-protein interactions (Kurup et al, 2000;Strayer et al, 2000;Robson et al, 2001). The members of the newly identified subfamily have only the single CCT domain in common with other CMF proteins, but share several conserved regions including a putative N-terminal cTP (Li et al, 2019a). Based on the three more intensively studied genes/proteins of this subfamily, we call this group of CCT domain containing proteins the AAC protein family [for: ALBOSTRIANS/HvAST/HvCMF7 (Li et al, 2019b), ALBOSTRIANS-LIKE/HvASL/HvCMF3 (Li et al, 2019a), and CHLOROPLAST IMPORT APPARATUS 2/CIA2 (Sun et al, 2001)] (Li et al, 2019a).…”

ATP-Dependent Clp Protease Subunit C1, HvClpC1, Is a Strong Candidate Gene for Barley Variegation Mutant luteostrians as Revealed by Genetic Mapping and Genomic Re-sequencing

“…Primary leaves were collected from 7-day-old seedlings of wild type and variegated mutant (luteostrians-P1). Preparation of 1-2 mm 2 leaf cuttings for ultrastructure analysis including aldehyde/osmium tetroxide fixation, dehydration, resin infiltration and ultramicrotomy were performed as previously described (Li et al, 2019a).…”