Rolf Knoth

Struktur und Funktion der genetischen Information in den Plastiden: X. Das Fehlen von Fraktion-I-Protein in den weißen Plastiden einiger Sorten von Pelargonium zonale Ait.

Summary Soluble proteins were extracted from pure green leaves, and from pure white leaves containing white plastids of the varieties “Mrs. Parker”, “Flower of Spring”, “Gnom”, and “Madame Salieron (BBC)” of Pelargonium zonale. The green plastids contain normal amounts of ribosomes; in the white leaves plastid ribosomes are absent. The proteins were electrophoretically separated on polyacrylamide gels. In extracts obtained from white leaves fraction I protein is absent (or only present in very small quantities). Not only the large subunit of fraction I protein is lacking; also the small subunit which is generally assumed to be synthesized on cytoplysmic ribosomes could not be detected.

[Structure and function of the genetic information in plastids : V. The absence of ribosomal rna from the plastids of the plastom-mutant 'mrs. parker' of Pelargonium zonale].

Summary1.The variety ‘Mrs. Parker’ of Pelargonium zonale (fig. 1) is a periclinal chimera of the constitution white-over-green (LI and L II: white, L III: green). Reciprocal crosses with the green variety ‘Trautlieb’ demonstrate a biparental, extranuclear inheritance of the character green.- white. The F1 consists of green, green-white variegated and white seedlings (table 1).2.In green-white variegated F1-plants “mixed cells” (fig. 2) have been found containing two genetically different types of plastids: green plastids (from ‘Trautlieb’) and white plastids (from ‘Mrs. Parker’). The white cells of ‘Mrs. Parker’ represent a white plastid mutant (= plastom mutant); its genetic designation is “extranuclear: alba-1”, symbol en: alba-1.3.Leaf material for biochemical studies was obtained from pure white and entirely green shoots of variegated F1 hybrids (fig. 3). The ultrastructure of the white plastids was studied by electron microscopy.4.From normal green cells of Pelargonium zonale four bands of high molecular weight ribosomal RNA can be isolated: 25 S and 18 S RNA of the cytoplasmic ribosomes and 23 S and 16 S RNA of plastid ribosomes (fig. 5a).5.The mutation of the plastid DNA in the plastids of ‘Mrs. Parker’ causes an altered RNA pattern: The 23 S and 16 S RNA of the plastid ribosomes cannot be detected in polyacrylamid gels (whereas 25 S and 18 S RNA are present) (fig. 5b).6.In cells of white leaves numerous plastids are present. They are smaller than normal chloroplasts and have a double-layered envelope. However, the formation of normal internal membrane structures is blocked.7.In mutated plastids of ‘Mrs. Parker’ ribosomes cannot be detected in electron micrographs (fig. 6).8.From these findings we conclude that protein synthesis cannot be performed in mutated plastids. The multiplication of the plastids — and presumably also the replication of plastid DNA — is not impaired by the deficiency in plastid protein synthesis.9.These results indicate that protein synthesis within the plastids is necessary for full development and differentiation of the chloroplasts, although an essential part of the plastidal proteins are synthesized on cytoplasmic ribosomes.Zusammenfassung1.Die Sorte ‘Mrs. Parker’ von Pelargonium zonale (Abb. 1) ist eine Periklinalchimäre der Konstitution Weiß-über-Grün (LI und LII weiß, LIII grün). Reziproke Kreuzungen mit der grünen Sorte ‘Trautlieb’ zeigen einen biparentalen, extranukleären Erbgang des Unterschiedes grün — weiß. Die F1 besteht aus grünen, grün-weiß gescheckten und weißen Keimlingen (Tabelle 1).2.In grün-weiß gescheckten F1-Pflanzen konnten echte Mischzellen nachgewiesen werden, die nebeneinander normal grüne Plastiden (von ‘Trautlieb’) und mutierte weiße Plastiden (von ‘Mrs. Parker’) enthalten (Abb. 2). Die weißen Zellen von ‘Mrs. Parker’ repräsentieren somit eine weiße Plastommutante; sie erhält die Bezeichnung „extranukleär: alba-1”; Symbol en: alba-1.3.Entmischte weiße und grüne Triebe von F1-Bastarden (Abb. 3) dienten als Objekte für biochemische Untersuchungen. Von weißen Plastiden wurden elektronenmikroskopische Aufnahmen angefertigt.4.Aus normal grünen Zellen von Pelargonium zonale sind vier Banden hochmolekularer rRNS zu gewinnen: 25S und 18S RNS der Cytoplasma-Ribosomen und 23S und 16S RNS der Plastiden-Ribosomen (Abb. 5a).5.Als Folge einer Mutation der Plastiden-DNS treten in den weißen Plastiden von ‘Mrs. Parker’ Veränderungen im RNS-Muster auf (Abb. 5b): Die 23S und 16S rRNS lassen sich in Polyacrylamid-Gelen nicht nachweisen (die 25S und 18S RNS der Cytoplasma-Ribosomen sind unverändert vorhanden).6.In den Zellen der weißen Blätter sind zahlreiche Plastiden vorhanden. Sie sind kleiner als normale Chloroplasten und haben eine doppelschichtige Hüllmembran; hingegen ist eine normale Ausbildung innerer Membranstrukturen blockiert.7.In den mutierten Plastiden von ‘Mrs. Parker’ konnten Ribosomen elektronenmikroskopisch nicht nachgewiesen werden (Abb. 6).8.Aus diesen Befunden kann geschlossen werden, daß in den mutierten Plastiden keine Proteinsynthese stattfinden kann. Die Vermehrung der Plastiden — und wahrscheinlich auch die Replikation der Plastiden-DNS — wird durch diesen Defekt nicht beeinträchtigt.9.Die Ergebnisse weisen darauf hin, daß die Proteinsynthese innerhalb der Plastiden für eine vollständige Entwicklung und Differenzierung der Chloroplasten notwendig ist, auch wenn ein wesentlicher Teil der Plastiden-Proteine an den Ribosomen des Cytoplasmas synthetisiert wird.

Phytoferritin-accumulating Plastids in the Male Generative Cell of Pelargonium X hortorum Bailey

Summary Generative cells of Pelargonium X hortorum have been investigated electron microscopically after fixation with glutaraldehyde/OsO 4 . These cells bear numerous proplastids which contain in all developmental stages unusually large amounts of phytoferritin, whose possible function as an iron supply for the young embryo is briefly discussed.

Struktur und Funktion der genetischen Information in den Plastiden: XI. DNA in normalen und mutierten Plastiden der Sorte „Mrs. Parker‟ von Pelargonium zonale

Summary As a consequence of a plastid mutation the white plastids en: alba-1 of the variety “Mrs. Parker” of Pelargonium zonale do not possess ribosomes, and therefore they are not able to perform plastidal protein synthesis. By means of biochemical and electron microscopic methods the question was studied, whether the mutant plastids contain DNA. The DNA of isolated wild type chloroplasts of Pelargonium zonale has a buoyant density in CsCl of 1,697 ± 0.001 g/cm3 (fig. 1), and a melting temperature of 85°C (fig. 2). The chloroplast DNA of Pelargonium cannot be separated by buoyant density centrifugation in CsCl and melting temperature from the nuclear DNA. The base composition of both chloroplast and nuclear DNA was calculated to be 37–38% GC. However, the chloroplast DNA renatures almost completely, whereas nuclear DNA renatures only to a low extend. In the DNA of pure white mutant leaves of en: alba-1 there is no indication of the presence of any other component differing in the density (fig. 1c). The electron microscopic study of plastid DNA by means of three independent techniques gave the following results for both normal chloroplasts and mutated en: alba-1 plastids: In situ studies show an intraplastidal localization of DNA (fig. 4 b) in nucleoplasmatic areas, occupying 39.3 ± 8.2 % of total plastid capacity (fig. 4a) in the case of mutant plastids. The identity of the fibrillar structures with DNA was proved in positive DNase-tests. Spreaded DNA from osmotically disrupted plastids is associated with membranes in a consistent way. It was possible to find good visible, loop-like moleeules from wild type (fig. 8) and from the mutant plastids (fig. 6 a). Apart from this there are large “displays” of DNA with numerous membrane attachment points (fig. 7 a). Thereby the DNA has contact with the membrane either directly in polyfibrillar form or by building solid stick-like structures (fig. 7 b). A single DNA molecule from en: alba-1 plastids is presented with partially distinct diameters of its strand. This phenomenon was interpreted as a possible result of replication (fig. 6 b). Evidence for the ability for replication of en: alba-1 DNA was obtained by electron microscopic autoradiography (fig. 8). As in wild type chloroplasts, about four labelled centres are scattered over the plastid sections. DNase-treated samples do not show any specific labelling. It is discussed, whether the DNA is present in the plastid as a single molecule or as a multitude of independent molecules.

Struktur und Funktion der genetischen Information in Piastiden XV. Beziehungen zwischen Chlorophyllgehalt, Photosyntheseverhalten und Piastidenfeinstruktur in Kerngen-bedingten Aureamutanten von Antirrhinum majus (Mutante „Aurea“) und Pelargonium zonale (Sorte „Cloth of Gold“)

Summary The electron microscopic observations of yellow-green and green plastids of Antirrhinum and Pelargonium demonstrate direct connections between fine structure, chlorophyll content and photo-synthetic activity. It can be shown that in young yellow-green plastids a significant decreased level of chlorophyll b is correlated with a very low amount of stacked thylakoids. A normalization of the chlorophyll a/b-ratio in the course of the development and increased greening of the plastids is connected with a preferential building of partitions. This finding supports the conception of a direct participation of chlorophyll b in the formation and maintenance of grana stacks. Young Aurea plastids accumulate only a small amount of chlorophyll, but they are photosynthetically active. Plastom mutants of the same species have an impaired photosynthesis in spite of a higher chlorophyll content (Herrmann 1971; Herrmann and Hagemann 1971). Besides of magnograna only perforated primary thylakoids are present in the mutant plastids (Knoth 1975). The Aurea plastids, however, posses always continous stromal membranes. This structurally detectable difference between both kinds of plastids is based probably on lacking or presence of chlorophyll-proteincomplex I and possibly represents the cause for a blocked or intact photosynthesis.

Phänotypische Normalisierung der Mutante albina2 von Lycopersicon pimpinellifolium (Jusl.) Mill.

Summary 1. The homozygous recessive mutant albina 2 ( ala 2 ) of Lycopersicon pimpinellifolium has white cotyledons and is lethal in the seedlings stage. 2. Grafting of these white seedlings on normal green tomato stocks results in the compensation of the metabolic deficiency in the mutant scion: The grafted mutant becomes able to form a green shoot with typical chloroplast (Fig. 1 and 2). 3. Seeds from normalized green mutant shoots give typical white albina 2 seedlings. 4. Grafting of albina 2 on green stocks thus leads to the occurence of a practically complete phenotypic no rmalization. 5. Reciprocal grafting (green seedlings grafted on mutant stocks) also leads to phenotypic normalization of the side branches formed by the mutant stock. 6. Attempts to achieve normalization of mutant seedlings by various other treatments (organic and anorganic compounds, sterile culture) have been without success. 7. Branches of the normalized mutant scion have been cut off and caused to regenerate roots. These cuttings, which tend to die under usual conditions, are able to normal continuous growth under artificial light of 20 hours per day (Fig. 3). Decisive for the normalization is the length of daily exposure to light, and not the intensity or the quality of light or the temperature. All other treatments of the cuttings did not result in a continuous normalization (Table 1, Fig. 2). 8. The normalized mutant cuttings have an almost normal chlorophyll content (Table 2, 3); however, their rate of photosynthesis is clearly reduced in comparison with the control. 9. The relation between the reduced rate of photosynthesis in normalized leaves and the requirement of a very long daily light period is discussed.

Struktur und Funktion der genetischen Information in den Plastiden: XIII. Lamellarproteine bleicher Plastiden von Plastom- und Genmutanten von Hordeum und Lycopersicon

Summary The plastid lamellar proteins of two plastom mutants of Hordeum vulgare and two gene mutants of Lycopersicon esculentum were investigated by electrophoresis in Polyacrylamide gels. The plastidal protein synthesis in the plastom mutants of the mutant lines “Saskatoon” and “albostrians” og Hordeum is impaired The deficiency of plastidal protein synthesis causes distinct changes in the pattern of the lamellar proteins as compared with the green control. The chlorophyll-protein complex I is absent. But numerous lamellar proteins are found in the white mutant leaves. These proteins — among them presumably also the protein of the chlorophyll-protein complex II — are expected to be synthesized on cytoplasmic ribosomes. In the gene mutants albina-2 and sulfureapura of Lycopersicon the chlorophyll-protein complex I is also absent. These results suggest that the formation of the chlorophyll-protein complex I is not only controlled by the genetic information of the plastids (plastom), but also by the genetic information of the nucleus (genome). Two reasons for the absence of the chlorophyll-protein complex I in these different mutant are discussed: (1) The protein component of the chlorophyll-protein complex I consists of difieren proteins of lower molecular weight, coded partly by the genome, partly by the plastom and synthesized on cytoplasmic and on plastid ribosomes, respectively. (2) The protein of the chlorophyll-protein complex I will not be synthesized and/or will not be incorporated into the plastid lamellar structure before the differentiation of the chloroplast has reached a particular stage.

Struktur und Funktion der genetischen Information in den Plastiden@@@Structure and function of the genetic information in plastids: V. Das Fehlen von ribosomaler RNS in den Plastiden der Plastommutante ?Mrs. Parker? von Pelargonium zonale Ait.@@@V. The absence of ribosomal rna from the plastids of the plastom-mutant ?mrs. parker? of Pelargonium zonale

Summary1.The variety ‘Mrs. Parker’ of Pelargonium zonale (fig. 1) is a periclinal chimera of the constitution white-over-green (LI and L II: white, L III: green). Reciprocal crosses with the green variety ‘Trautlieb’ demonstrate a biparental, extranuclear inheritance of the character green.- white. The F1 consists of green, green-white variegated and white seedlings (table 1).2.In green-white variegated F1-plants “mixed cells” (fig. 2) have been found containing two genetically different types of plastids: green plastids (from ‘Trautlieb’) and white plastids (from ‘Mrs. Parker’). The white cells of ‘Mrs. Parker’ represent a white plastid mutant (= plastom mutant); its genetic designation is “extranuclear: alba-1”, symbol en: alba-1.3.Leaf material for biochemical studies was obtained from pure white and entirely green shoots of variegated F1 hybrids (fig. 3). The ultrastructure of the white plastids was studied by electron microscopy.4.From normal green cells of Pelargonium zonale four bands of high molecular weight ribosomal RNA can be isolated: 25 S and 18 S RNA of the cytoplasmic ribosomes and 23 S and 16 S RNA of plastid ribosomes (fig. 5a).5.The mutation of the plastid DNA in the plastids of ‘Mrs. Parker’ causes an altered RNA pattern: The 23 S and 16 S RNA of the plastid ribosomes cannot be detected in polyacrylamid gels (whereas 25 S and 18 S RNA are present) (fig. 5b).6.In cells of white leaves numerous plastids are present. They are smaller than normal chloroplasts and have a double-layered envelope. However, the formation of normal internal membrane structures is blocked.7.In mutated plastids of ‘Mrs. Parker’ ribosomes cannot be detected in electron micrographs (fig. 6).8.From these findings we conclude that protein synthesis cannot be performed in mutated plastids. The multiplication of the plastids — and presumably also the replication of plastid DNA — is not impaired by the deficiency in plastid protein synthesis.9.These results indicate that protein synthesis within the plastids is necessary for full development and differentiation of the chloroplasts, although an essential part of the plastidal proteins are synthesized on cytoplasmic ribosomes.Zusammenfassung1.Die Sorte ‘Mrs. Parker’ von Pelargonium zonale (Abb. 1) ist eine Periklinalchimäre der Konstitution Weiß-über-Grün (LI und LII weiß, LIII grün). Reziproke Kreuzungen mit der grünen Sorte ‘Trautlieb’ zeigen einen biparentalen, extranukleären Erbgang des Unterschiedes grün — weiß. Die F1 besteht aus grünen, grün-weiß gescheckten und weißen Keimlingen (Tabelle 1).2.In grün-weiß gescheckten F1-Pflanzen konnten echte Mischzellen nachgewiesen werden, die nebeneinander normal grüne Plastiden (von ‘Trautlieb’) und mutierte weiße Plastiden (von ‘Mrs. Parker’) enthalten (Abb. 2). Die weißen Zellen von ‘Mrs. Parker’ repräsentieren somit eine weiße Plastommutante; sie erhält die Bezeichnung „extranukleär: alba-1”; Symbol en: alba-1.3.Entmischte weiße und grüne Triebe von F1-Bastarden (Abb. 3) dienten als Objekte für biochemische Untersuchungen. Von weißen Plastiden wurden elektronenmikroskopische Aufnahmen angefertigt.4.Aus normal grünen Zellen von Pelargonium zonale sind vier Banden hochmolekularer rRNS zu gewinnen: 25S und 18S RNS der Cytoplasma-Ribosomen und 23S und 16S RNS der Plastiden-Ribosomen (Abb. 5a).5.Als Folge einer Mutation der Plastiden-DNS treten in den weißen Plastiden von ‘Mrs. Parker’ Veränderungen im RNS-Muster auf (Abb. 5b): Die 23S und 16S rRNS lassen sich in Polyacrylamid-Gelen nicht nachweisen (die 25S und 18S RNS der Cytoplasma-Ribosomen sind unverändert vorhanden).6.In den Zellen der weißen Blätter sind zahlreiche Plastiden vorhanden. Sie sind kleiner als normale Chloroplasten und haben eine doppelschichtige Hüllmembran; hingegen ist eine normale Ausbildung innerer Membranstrukturen blockiert.7.In den mutierten Plastiden von ‘Mrs. Parker’ konnten Ribosomen elektronenmikroskopisch nicht nachgewiesen werden (Abb. 6).8.Aus diesen Befunden kann geschlossen werden, daß in den mutierten Plastiden keine Proteinsynthese stattfinden kann. Die Vermehrung der Plastiden — und wahrscheinlich auch die Replikation der Plastiden-DNS — wird durch diesen Defekt nicht beeinträchtigt.9.Die Ergebnisse weisen darauf hin, daß die Proteinsynthese innerhalb der Plastiden für eine vollständige Entwicklung und Differenzierung der Chloroplasten notwendig ist, auch wenn ein wesentlicher Teil der Plastiden-Proteine an den Ribosomen des Cytoplasmas synthetisiert wird.