albostrians is a classical variegation mutant of barley

The albostrians mutant originated from the two-rowed spring barley variety ‘Haisa’ by means of X-ray irradiation (Hagemann and Scholz, 1962). Albino, green-white striped and pure green seedlings can be observed when growing progeny of a fully green but homozygous albostrians mutant (as/as) (Figure 1-2). This pattern of segregation is following a ratio of 1:8:1 (green / striped / albino). Based on the genetic segregation analysis in F2 generation obtained by crossing the mutant to a wild-type genotype and self pollinating the obtained F1, however, it could be shown that the phenotype was caused by a single recessive, nucleus-encoded gene (Hagemann and Scholz, 1962). In contrast, the inheritance of the chlorophyll deficiency is following a purely maternal pattern, as was revealed by reciprocal crosses between Haisa and the mutant line M4205 (Figure 1-3) (Hagemann and Scholz, 1962). The albostrians mutant was therefore initially considered as a nuclear gene induced plastome mutation, i.e. a mutation of chloroplast DNA induced by action of a nuclear mutator gene (Hagemann and Scholz, 1962).

Figure 1-2: Examples of variegation of coloration in homozygous albostrians mutant plants.

The leaves exhibited here were collected from different seedlings of the mutant line M4205.

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Figure 1-3: Pattern of inheritance of the albostrians mutant phenotype based on chloroplast constitution. The inheritance of leaf color variegation is depending on the chloroplast population in the maternal plant as can be shown in offspring of reciprocal crosses. If WT (As/As) or a green spike of M4205 (as/as) is used as the female parent, all F1 plants are green (A, B, C, E). A striped spike as female parent given green, striped and albino F1 plants (D), and a white spike as female parent produces only albino F1 plants because the plastid aberration is irreversible and cannot be rescued (F). The figure is drawn on the basis of the crossing experiments done by Hagemann and Scholz (1962).

1.3.2 Status of research towards characterizing the albostrians mutant

The most prominent characteristic of the albostrians mutant is the absence of 70S ribosomes in plastids of white sectors. This was supported by five lines of evidence:

1) neither 16S nor 23S rRNA could be found in preparations of total RNA from white leaves; 2) no ribosomes were observed by transmission electron microscopy in the white plastids; 3) the CF1-ATPase consisting of nuclear and plastid encoded subunits was lacking in the white albostrians leaves; 4) ribosomal protein L2 was never

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detected by means of western blot in extracts from white albostrians seedlings; 5) white leaves lack RUBISCO the large subunit of which is synthesized on chloroplast ribosomes (Börner et al., 1976; Reichenbächer et al., 1978; Börner et al., 1979; Hess et al., 1993; Hess et al., 1994a). Since the functional ribosome is an essential apparatus involved in the process of translation, theoretically, translation of all chloroplast genes coding for proteins is entirely abolished in the white sectors of the mutant. Because of this aspect, the ribosome-free plastids of the albostrians mutant were considered an excellent system for studying regulatory interactions between the three DNA-containing compartments, for instance, the impact of protein synthesis deficiency in plastids on gene expression in nucleus and mitochondria as well as on plastids themselves (Hess et al., 1994a).

The first significant achievement reached by studying the albostrians mutant was the first evidence for the existence of plastid to nucleus signals (Bradbeer and Börner, 1978; Bradbeer et al., 1979), now known as retrograde signaling (Pesaresi et al., 2007). Initially, instead of using the term ‘retrograde signals’, a ‘chloroplast control principle’ (Hagemann and Börner, 1978) was postulated. Plastids were thought to influence nucleo-cytoplasmic gene expression. This could be the result of affecting the accumulation of nuclear gene-encoded chloroplast polypeptides, such as LIGHT-HARVESTING CHLOROPHYLL A/B-BINDING PROTEIN (LHCP), the small subunit of RUBISCO (Hagemann and Börner, 1978), the GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE (NADP+) and PHOSPHORIBULOKINASE (Bradbeer and Börner, 1978). All these proteins are known to be of nucleocytoplasmic origin and were drastically reduced to the limit of detection in their activities and/or quantities in white leaves of the albostrians mutant. Plastids were also shown to affect the accumulation of mRNAs transcribed from nuclear genes, or the accumulation of the non-chloroplast enzyme NITRATE REDUCTASE (Börner, 1986; Börner et al., 1986).

The proposed ‘chloroplast control principle’ was in agreement with the ‘multi-subunit completion principle’ (Ellis, 1977) supporting the idea that proteins within the chloroplast are synthesized on both plastid and cytoplasmic ribosomes. However, the

‘chloroplast control principle’ was not compatible with the ‘cytoplasmic control principle’ postulated by Ellis who insisted on the requirement that cytoplasmic products control organellar protein synthesis, but not the other way around (Ellis, 1977). On the contrary, the ‘chloroplast control principle’ proposed that the

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nucleocytoplasmic compartment and the plastid behave as cooperation partners, i.e.

plastid gene expression is under the control of nuclear genes and vice versa (Hagemann and Börner, 1978). Interestingly, the ‘signaling factor’ was initially postulated to be RNA transcribed from the plastid genome (Bradbeer and Börner, 1978); a new hypothesis was subsequently assumed by accounting the intermediates of chlorophyll biogenesis serving as the connection between nuclear gene expression and chloroplast development (Hess et al., 1992; Hess et al., 1994b).

Furthermore, it was considered the initial steps of Mg-porphyrin biosynthesis to contribute to plastid-derived signaling towards the nucleus (Yaronskaya et al., 2003).

The latter hypothetical scenario was supported by the finding that Mg-PROTOPORPHYRIN IX acts as a negative regulator of photosynthesis gene expression in the nucleus and the chloroplast (Strand et al., 2003; Ankele et al., 2007). Besides the plastid to nucleus retrograde signaling, it is noteworthy to mention that studies on albostrians mutant of barley provided also for the first time evidence for an influence of the plastids/chloroplasts on the expression of mitochondrial genes and mitochondrial gene copy numbers (Hedtke et al., 1999).

The second milestone reached by investigating the albostrians mutant was finding the first evidence for the existence of two different chloroplast RNA polymerase systems, i.e. plastid-encoded plastid RNA polymerase (PEP) and nucleus-encoded plastid RNA polymerase (NEP) (Hess et al., 1993). As early as in 1970, the idea was proposed for maize where chloroplasts contained two different types of DNA-dependent RNA polymerase (Bogorad and Woodcock, 1970). Studies with respect to the albostrians mutant clearly revealed that the plastid genes, RNA polymerase B, C1 and C2 (rpoB/C1/C2) as well as 40S ribosome protein S15 (rps15), were transcribed despite the lack of PEP subunits in the ribosome-deficient plastids. The rps15 gene and rpo genes showed high gene expression in ribosome-deficient plastids, in contrast to photosynthetic genes. On the contrary, the functional chloroplast contained abundant transcripts of the photosynthetic genes but not of the rpo genes.

It was speculated that PEP has the preference for expression of the photosynthetic and bioenergetic genes, while, the NEP has preference for transcription of the housekeeping genes (Hess et al., 1993). This assumption was consistent with observations in tobacco and Arabidopsis showing that genes of the two photosystems completely relied on PEP transcription, while, transcription of