Variant mitochondrial protein and DNA patterns associated with cytoplasmic male-sterile lines of Nicotiana

Summary Variation in mitochondrial protein synthesis and genome organization was investigated. Three different alloplasmic cytoplasmic male-sterile Nicotiana tabacum cultivars, carrying N. repanda, N. suaveolens or N. debneyi cytoplasm, were analysed together with corresponding male-fertile parental and restored material. Although several differences were detected in the proteins synthesized by isolated mitochondria from the male-sterile and male-fertile plants, most of these were related to the origin of the mitochondria. However, a 23 kD protein was synthesized in the male-sterile cultivar carrying N. debneyi mitochondria, but not in other lines containing this cytoplasm. This protein was also present in the male-fertile parent containing N. tabacum mitochondria. Only the enhanced production of a 30 kD protein in the lines carrying mitochondria from N. repanda or N. debneyi was exclusively correlated with CMS. This protein was not present in any of the corresponding male-fertile parental and restored lines. Restriction enzyme analysis of mitochondrial DNA revealed a difference in abundance of a 5.6 kb XhoI fragment between lines containing N. debneyi mitochondria. No rearrangements of mitochondrial DNA was found between male-fertile and male-sterile lines carrying N. repanda or N. suaveolens cytoplasm. These results might indicate that CMS in alloplasmic Nicotiana cultivars is caused by alterations in the expression of mitochondrial genes, rather than by induced changes in the genome.     

A gene encoding a truncated large subunit of Rubisco is transcribed and salt-inducible in rice

Using the rice salt-tolerant mutant 20 as material, a cDNA library was constructed and two salt-inducible clones, SIR5.5 and SIR8.1, were isolated by differential screening. Homology analysis revealed that the two clones together constituted a chimeric rbcL which encoded a truncated large subunit of Rubisco with 337 amino-acids, plus 64 amino-acids of unknown origin. The expressions of both the normal and the chimeric locus appeared to be developmentally regulated and salt-inducible in shoots of the salt-tolerant mutant 20 and its original variety 77–170. In roots, their expressions were salt-inducible in the salt-tolerant mutant 20 whereas no, or only premature, forms were present in the salt-treated original variety 77–170. Higher concentrations of salt reduced the expressions of both normal rbcL and the chimeric locus. ABA showed no effect on their expression.     

Variation in mitochondrial translation products in fertile and cytoplasmic male-sterile sugar beets

Summary Intact and functional mitochondria were isolated from sugar beet plants (Beta vulgaris L.) containing normal fertile (F) or cytoplasmic male-sterile (S₁–S₄) cytoplasms. Incorporation of ³⁵S-methionine by mitochondria isolated from both roots and leaves showed approximately 20 major and ten minor translation products. Comparison of the polypeptide synthesis patterns produced by leaf mitochondria from fertile plants of three different species within the genus Beta revealed several taxonomically related differences. Contrary to this, the patterns of polypeptides synthesized by mitochondria from roots and leaves of sugar beet plants containing the F and S₁–S₄ cytoplasms were very similar; in the S₁ and S₂ cytoplasms no qualitative, and only a few quantitative, differences from the F cytoplasm were observed. Thus, in these cases, cytoplasmic male sterility in sugar beet is not correlated with the constitutive expression of variant polypeptides. In the S₃ cytoplasm, however, an additional 6 kDa polypeptide was synthesized and in the S₄ cytoplasm an additional 10 kDa polypeptide was observed when compared with the F cytoplasm. The expression of cytoplasmic male sterility in sugar beet may be associated with these variant polypeptides. The mitochondrial polypeptides synthesized were identical in plants with different nuclear backgrounds but with identical S₁ cytoplasms. Mitochondria from plants with variants of the S₄ cytoplasm in the same nuclear genotype also showed identical patterns of polypeptide synthesis, including the synthesis of the 10 kDa S₄-specific polypeptide. Pulse-chase experiments did not affect the synthesis of this polypeptide.     

A chimeric gene (orf256) is expressed as protein only in cytoplasmic male-sterile lines of wheat

Mitochondria derived from Triticum timopheevi have a chimeric gene, orf256, immediately upstream from coxI. Antibodies to a peptide corresponding to a part of the encoded amino acid sequence of orf256 detect a 7 kDa protein on western blots of mitochondrial proteins from cytoplasmic male-sterile (cms) wheat (T. aestivum nucleus, T. timopheevi mitochondria) but not in mitochondrial proteins from T. aestivum, T. timopheevi, or cms plants restored to fertility by introduction of nuclear genes for fertility restoration. The 7 kDa protein appears to serve as a marker for cms wheat. Its occurrence as an integral protein of the inner membrane may indicate a cms effect through an influence on mitochondrial membrane function.     

Pollen irradiation and possible gene transfer in Nicotiana species

Summary Progeny from crosses of Nicotiana langsdorffii with gamma irradiated pollen of Nicotiana alata Crimson Bedder showed skewed segregation in the F₂ favoring the maternal parent. This is probably not gene transfer in a strict sense, rather just an extreme case of reduced transmission of irradiated chromosomes, leading to massive overrepresentation of maternal genes. Gene transfer or mutational loss may explain some anomalous F₁ plants. Segregation in the F₂ progeny showed the presence of several genes from the irradiated pollen. Crosses of Nicotiana sylvestris, N. plumbaginifolia N. paniculata, and Petunia parodii with irradiated pollen from N. alata and Petunia hybrida showed no evidence of gene transfer, nor did experiments with irradiated mentor pollen. This indicates that gene transfer with irradiated pollen between non-crossing species or between species giving sterile hybrids is probably a rare phenomenon.     

Functional cybrid plants possessing a Nicotiana genome and an Atropa plastome

Summary Mesophyll protoplasts of plastome chlorophyll-deficient, streptomycin-resistant Nicotiana tabacum were fused with those of wild type Atropa belladonna using the polyethylene-glycol/high pH/high Ca⁺⁺/dimethylsulfoxide method. Protoplasts were cultured in nutrient media suitable for regeneration of tobacco but not Atropa cells. In two experiments, a total of 41 cell lines have been selected as green colonies. Cytogenetic (chromosomal number and morphology) and biochemical (isozyme analyses of esterase, amylase and peroxidase) studies were used to evaluate the nuclear genetic constitution of regenerated plants. To study plastid genetic constitution, restriction endonuclease analysis of chloroplast DNA was performed. Three groups of regenerants have been identified: (a) nuclear hybrids (4 cell lines); (b) Atropa plants, most probably arising from rare surviving parental protoplasts (4 lines) and (c) Nicotiana/Atropa cybrids possessing a tobacco genome and an Atropa plastome (33 lines). Most of cybrids obtained were diploid, morphologically normal plants phenotypically similar to tobacco. Some plants flowered and yielded viable seeds. Part of cybrid regenerants were variegated, variegation being transmitted to sexual progeny. Electron microscopic analysis of the mesophyll cells of variegated leaves revealed the presence of heteroplastidic cells. Analysis of thylakoid membrane polypeptides shows that in the cybrids the content of at least one of the major polypeptides, presumably a chlorophyll a/b binding protein is drastically reduced.     

Investigation of Nicotiana tabacum (+) N. suaveolens cybrids with carpelloid stamens

To investigate cytoplasmic effects on homeotic floral morphology, Nicotiana tabacum and N. suaveolens protoplasts were fused and cybrids obtained to contrast with the sexual alloplasmic line Nta(sua)S. Nta(sua)S contains the nucleus of N. tabacum and cytoplasm of N. suaveolens while cybrids derive from fused cells where the cytoplasms can interact. The three male-sterile somatic cybrid plants analyzed contained mitochondria with N. tabacum and N. suaveolens mtDNA sequences, but not all the N. tabacum or all the N. suaveolens mtDNA sequences were present. The flowers were N. tabacum-like but with a split corolla (not observed in Nta(sua)S) and the whorl of stamens replaced by a whorl of carpel-like structures. Based on scanning electron microscopy the carpelloid stamens had a characteristic N. tabacum stigma, a style of variable length and a pseudo-ovary with ovule-like structures. The Southern blot data were consistent with mtDNA recombination. These genomic changes were maternally inherited. Chloroplasts were either of the N. tabacum or N. suaveolens type. AFLP analysis showed transfer of variable amounts of N. suaveolens nuclear DNA. However, it is the presence of the N. suaveolens sequences and/or absence of N. tabacum sequences in the mitochondria that correlates with the homeotic floral morphology. These cybrids will facilitate the analysis of the role of mitochondrial DNA sequences in floral organ identity; which has received limited attention in genetic flowering models based primarily on Arabidopsis research.