Cytoplasmic Male Sterility in Nicotiana, Restoration of Fertility, and the Nucleolus. II. N. DEBNEYI Cytoplasm

Previously, it was shown that a fragment chromosome, apparently derived from the Nicotiana repanda chromosomal complement, restores to normal the morphology and fertility of the abortive and feminized anthers produced by plants that possess the N. tabacum genome in cytoplasm from N. repanda. Furthermore, that restorer chromosome organizes the nucleolus and inhibits the nucleolus-forming activity of the nucleolar organizers of N. tabacum chromosomes present in the same cells, particularly in pollen mother cells. To determine whether these relations are basic or only coincidental, restorer chromosomes for other cytoplasms are now being investigated. The present paper describes a study of a chromosome, presumably derived from N. debneyi, with partial restoring power. Acting in the cytoplasm of N. debneyi, it directs formation of morphologically normal anthers, without, however, restoring pollen fertility. We find that this chromosome also has a functioning nucleolar organizer, but only slightly inhibits the nucleolus-forming capacity of N. tabacum chromosomes. The suggestion of a relationship between the nucleolar apparatus and restoration of normal anthers is thus strengthened by the observation that restorers are found on nucleolus-forming chromosomes from two very distinct Nicotiana species, as well as in several comparable cases cited from the Triticinae. The manner in which the nucleolus, or its organizer, may direct defeminization and restoration of anther morphology is not known; suggestions were offered in the preceding paper in this series (Gerstel, Burns and Burk 1978).     

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.     

Restoration of stamen development and production of functional pollen in an alloplasmic CMS tobacco line by ectopic expression of the Arabidopsis thaliana SUPERMAN gene

The alloplasmic male-sterile tobacco line Nta(rep)S, combining the nucleus of Nicotiana tabacum with the cytoplasm of Nicotiana repanda, exhibits cadastral-type anomalies due to a fusion of several stamens with the pistil. These anomalies share similarities with Arabidopsis superman mutants. SUPERMAN (SUP) is a cadastral gene controlling the boundary between whorls 3 (androecium) and 4 (gynoecium). Thus we hypothesized that the expression of the tobacco SUP orthologue might be impaired in the alloplasmic Nta(rep)S line, and that the deficiency could be complemented by the Arabidopsis SUP gene. Here we show that the ectopic expression of SUP in the alloplasmic male-sterile tobacco line Nta(rep)S significantly increases the frequency of flowers possessing free stamens, inducing the recovery of a proper structure for whorls 3 and 4. Furthermore, flowers of transgenic plants show a significant improvement of the morphology of stamens, and more particularly of the anthers, which are able to produce few but functional pollen. The data show that ectopic expression of Arabidopsis SUP reactivates the regulatory cascade of anther development. The plausible causes of the developmental defects of anthers in the alloplasmic male-sterile tobacco line are discussed in relation to the model of regulation of the Arabidopsis SUP gene.     

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.     

Interspecies compatibility of the anther specific cell wall invertase promoters from Arabidopsis and tobacco for generating male sterile plants

Histochemical GUS-staining and fluorometric analyses revealed strong tissue specific activities of the cell wall invertase promoters Nin88 from Nicotiana tabacum and AtcwINV2 from Arabidopsis thaliana that are restricted tightly to anthers and pollen, respectively. Both in A. thaliana and N. tabacum repression of invertase activity by anther specific RNA-interference turned out to be an efficient method to circumvent carbohydrate supply of the symplastically isolated pollen with subsequent strong decrease of pollen germination ability and seed setting. In the case of tobacco, comparable results were also obtained by expressing a proteinaceous invertase inhibitor, whereas this approach was less efficient in Arabidopis. The present study revealed that anther specific interference with invertase-activity in order to generate male sterile plants can be applied to members of the two different plant families Solanaceae (N. tabacum) and Brassicaceae (A. thalaina) and the strategy seems to be a general tool for practical application in hybrid breeding or as biological safety precautions. To elucidate the compatibility of the isolated promoters beyond plant families, we transferred the regulatory sequences into the respectively heterologous systems, i.e. the Nin88 promoter into Arabidopsis and the AtcwINV2 promoter into tobacco. The specificities of both promoters are maintained in the heterologous backgrounds, but their activities are strongly reduced as GUS-stainings of flowers and pollen revealed and fluorometrical quantification confirmed.     

Cell Specific,Cross-Species Expression of Myrosinases in Brassica Napus,Arabidopsis Thaliana and Nicotiana Tabacum

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.