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The covalent coupling of porphine molecules on Au(111) is studied by scanning probe microscopy experiments and density functional theory. At sufficient temperatures, dehydrogenative C–C coupling of the unfunctionalized molecules occurs directly on the surface. Characteristic dimer structures between individual porphine molecules are observed and assigned to various binding motifs that are distinguished by specific intermolecular connections. Different preparations show that the relative abundance of these motifs depends on the temperature of the gold sample during deposition and is explained by calculated free energies and kinetic aspects that are relevant during the linking process. Observations on the gold terraces are completed by studying polymerization at step edges, giving insight into their role during the covalent linking process.

The cytoplasmic male sterility (CMS) trait of at least one line of Vicia fabaplants is always associated with the presence of high molecular weight double-stranded RNA in the leaf tissue extracts. Subcellular fractions of leaf tissue from CMS and fertile maintainer plants were initially analyzed in an attempt to locate, identify, and characterize the genetic material involved with the sterility trait. This CMS-associated high molecular weight RNA was found only in the cytosol of the “447” male sterile line of V.fabaplants and could not be isolated from the recurrent parent (maintainer), from lines that had been fertility-restored, or from lines that had reverted from the sterile condition. We have been able to move the CMS-associated RNA from donor to fertile host plants through a dodder bridge. These hots not only contain the RNA but now exhibit a male sterile phenotype, as detected by visual examination of the flower, the pollen, morphological characteristics, and pollen staining ability.

Molecular hybridization with 125I-labeled citrus exocortis viroid RNA has been used to survey nucleic acid preparations from Gynura aurantiacafor viroid complementary molecules. A differential hybridization effect was detected between nucleic acid extracts from healthy and infected tissue in which significant RNase-resistant 125I-labeled citrus exocortis viroid resulted in hybridization studies with the infected tissue extracts. Subsequent characterization indicated that RNA from infected tissue was involved in the formation of a duplex molecule with citrus exocortis viroid RNA and had properties of an RNA·RNA hybrid. Subcellular fractionation of infected tissue indicates that the complementary RNA is present in nuclear and soluble RNA fractions. This RNA may represent an intermediate molecule in the replication of the viroid or a pathogenic expression and may have a regulatory role in the host cell.

The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.

Viroids, pathogenic, low molecular weight (105) single-stranded circular RNA molecules, represent a unique class of minimal infectious agents. Physical characterisation has culminated in the primary sequence of the 359 nucleotides comprising the potato spindle tuber viroid (PSTV)1. However, other than the dramatic expression of disease symptoms in plants2, little is understood of the synthesis and biological activity of the viroid RNA. Molecular hybridisation techniques resulted in the detection of both viroid complementary RNA3and DNA4sequences. The putative template role of the DNA sequences was reinforced by the reported inhibition of viroid synthesis by actinomycin D5–7. However, viroid-specified proteins do not seem to function in replication or pathogenesis, for in vitro translation has not been accomplished8,9. Furthermore, no AUG initiation triplets have been found in the primary sequence of PSTV or in the hypothetical complementary sequence. Viroid synthesis, therefore, may be characterised as a markedly host-dependent process and consequently potentially very sensitive to inhibition of DNA-directed functions by agents such as actinomycin D. The accumulation of viroid progeny in nuclei5,10and nuclear-rich subcellular fractions11has provided clues to the mechanism of viroid synthesis. What was reported as a viroid complementary DNA sequence from DNA-rich extracts of Citrus exocortis viroid (CEV)-infected tissue6has subsequently been positively identified as complementary RNA3that may be associated with DNA-rich preparations. With further purification and treatment, no significant levels of viroid-complementary DNA can be detected in diseased Gynura aurantiaca or tomato. Therefore, we feel an RNA intermediate must assume a predominant role in any scheme for CEV replication and/or pathogenesis. We now report that synthesis of CEV and PSTV are not inhibited by actinomycin D concentrations which reduce host 5S RNA synthesis by 80%. Thus, the primary role of actinomycin D in viroid synthesis may reflect either inhibition of a host-specified function or a general toxic effect on cell metabolism, and not evidence for the replication through a DNA intermediate.