Leaf Variegation and Impaired Chloroplast Development Caused by a Truncated CCT Domain gene inalbostriansBarley

Abstract

Chloroplasts fuel plant development and growth by converting solar into chemical energy. They mature from proplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genes impair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Such mutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplast biogenesis. Characterization of the green-white variegated albostrians mutant of barley has greatly broadened the field of chloroplast biology including the discovery of retrograde signaling. Here, we report the identification of the ALBOSTRIANS gene HvAST by positional cloning as well as its functional validation based on independently induced mutants by TILLING and RNA-guided Cas9 endonuclease mediated gene editing. The phenotypes of the independent HvAST mutants imply residual activity of HvAST in the original albostrians allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation. HvAST is a homolog of the Arabidopsis thaliana CCT Motif transcription factor gene AtCIA2, which was reported to be involved in the expression of nuclear genes essential for chloroplast biogenesis. Interestingly, in barley we localized HvAST to the chloroplast indicating novel without any clear evidence of nuclear localization.One-sentence summaryLeaf variegation in the barley mutant albostrians is caused by mutation of a single CCT-domain containing gene with residual activity, which is directed to the chloroplast.

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

“…Similar to its ohnolog HvCMF7, which is allocated to barley chloroplasts (Li et al, 2019), in silico analysis by PredSL (Petsalaki et al, 2006) predicted the presence of a 95 AA chloroplast transit peptide at the HvCMF3 N-terminus (Supplemental Table 7). To test its function, we performed transient subcellular localization in barley epidermis of green fluorescent protein (GFP) fusion constructs with either the complete wild-type HvCMF3 allele (HvCMF3:GFP) or the putative cTP of HvCMF3 only (cTP_95AA_HvCMF3:GFP) (Figure 8A and Supplemental Table 7).…”

“…One of the most prominent characteristics of the albostrians mutant is the lack of ribosomes in plastids of albino leaves and albino sections of striped leaves (Hess et al, 1993; Li et al, 2019). We checked therefore whether mutation of HvCMF3 has also an effect on plastid ribosomes.…”

“…The lack of plastid ribosomes and the albino phenotype of the albostrians mutant indicate that the presence of the wild-type allele of the ohnologous gene HvCMF3 cannot rescue the effects of the mutation in HvCMF7 suggesting that the two ohnologs do not act at redundancy. Strikingly, the ALBOSTRIANS protein HvCMF7 was localized to chloroplasts and the phenotype of the albostrians mutant implies that HvCMF7 plays a role in the biogenesis and/or stability of chloroplast ribosomes, i.e., has a function and location entirely different from all previously investigated CCT domain proteins (Li et al, 2019). In contrast, the Arabidopsis homolog of HvCMF7 and HvCMF3 , AtCIA2, codes for a nuclear transcription factor regulating genes for the transport of nuclear encoded proteins into chloroplasts and for the biogenesis of chloroplast ribosomes (Sun et al, 2009).…”

“…Among the genes with a proposed ohnologous relationship, the genes HvCMF7 ( ALBOSRIANS ) and its paralog HvCMF3 ( ALBOSTRIANS-LIKE ) are representing ohnologs within the CCT domain gene family of barley (Cockram et al, 2012; Li et al, 2019). A mutation in HvCMF7 confers the variegated “ albostrians” phenotype (Li et al, 2019).…”

“…Among the genes with a proposed ohnologous relationship, the genes HvCMF7 ( ALBOSRIANS ) and its paralog HvCMF3 ( ALBOSTRIANS-LIKE ) are representing ohnologs within the CCT domain gene family of barley (Cockram et al, 2012; Li et al, 2019). A mutation in HvCMF7 confers the variegated “ albostrians” phenotype (Li et al, 2019). Besides incomplete penetrance of its variegation phenotype (Hagemann and Scholz, 1962) one of the most prominent characteristics of the albostrians mutant are the ribosome-free plastids leading to albino leaves and albino sectors of striped leaves (Hess et al, 1993).…”