A.J. van Tunen

Regulation of plant gene expression by antisense RNA

Regulation of gene expression by antisense RNA was first discovered as a naturally‐occurring phenomenon in bacteria. Recently natural antisense RNAs have been found in a variety of eukaryotic organisms; their in vivo function is, however, obscure. Deliberate expression of antisense RNA in animal and plant systems has lead to successful down‐regulation of specific genes. We will review the current status of antisense gene action in plant systems. The recent discovery that ‘sense’ genes are able to mimic the action of antisense genes indicates that (anti)sense genes must operate by mechanisms other than RNA‐RNA interaction.

Chalcone Synthase Promoters in Petunia Are Active in Pigmented and Unpigmented Cell Types.

Chalcone synthase (CHS) catalyzes the first step in the biosynthesis of flavonoids that function in flower pigmentation, protection against stress, and induction of nodulation. The petunia genome contains eight complete chs genes, of which four are differentially expressed in floral tissues and UV-light-induced seedlings. The 5[prime]-flanking regions of these four chs genes were fused to the [beta]-glucuronidase (GUS) reporter gene and introduced into petunia plants by Agrobacterium-mediated transformation. We show that expression of each construct is identical to the expression of the authentic chs gene, implying that the differences in expression pattern between these chs genes are caused at least in part by their promoters. Histochemical analyses of GUS expression show that chs promoters are not only active in pigmented cell types (epidermal cells of the flower corolla and tube and [sub] epidermal cells of the flower stem) but also in a number of unpigmented cell types (mesophylic cells of the corolla, several cell types in the ovary and the seed coat). Comparison of chs-GUS expression and flavonoid accumulation patterns in anthers suggests that intercellular transport of flavonoids and enzymes occurs in this organ. Analysis of the flavonoids accumulated in tissues from mutant lines shows that only a subset of the genes that control flavonoid biosynthesis in the flower operates in the ovary and seed. This implies that (genetic) control of flavonoid biosynthesis is highly tissue specific.

Flavonoid gene expression follows the changes in tissue development of two Petunia hybrida homeotic flower mutants

TwoPetunia hybrida homeotic flower mutants,Blind (Bl) andGreen Petals (Gp), were characterized as a way to study floral morphogenesis. The influence of these homeotic mutations on the expression of the two genes encoding the flavonoid biosynthesis enzyme chalcone flavanone isomerase was investigated. TheBl mutation gives rise to highly modified flowers in which the limbs are transformed into antheroid limbs which develop on top of a normally formed tube. In wild type plants,chiA transcripts are detected in the flower limb. In antheroid limbs, however, expression of the anther-specificchiB gene was observed. This is in line with the homeotic nature of theBl mutation. TheGp mutation gives rise to mutated flowers in which the petals are transformed into sepaloid petals. An absence of CHI activity andchi transcripts in sepaloid petals was observed, which is in line with the homeotic nature of theGp gene.

Flavonoids and genetic modification of male fertility.

For their reproductive success higher plants depend on the functionality and efficacy of a specialized structure, the flower. This contains specialized organs (anther and carpel) in which the male and female reproductive cells are produced and non-reproductive organs (sepal and petal) which attract pollinators and protect the developing seed. In the anther the microspores develop which after maturation become pollen grains. A pollen grain can be regarded as a free living haploid organism. It contains one vegetative and one generative cell in bicellular pollen or one vegetative cell and two sperm cells in tricellular pollen. Its main task is to deliver the two sperm nuclei through the female reproductive tissues to the egg cell. The pollen can be transported to the female organs by a variety of mediators such as wind or insect pollinators. After arrival on a compatible and receptive stigma the pollen grains germinate by the extrusion of a tube through a germ pore in the pollen wall. The pollen tube grows through the transmitting tissue of the style to reach the ovary. After the ovary has been entered, the tube grows through the micropyle of the ovule towards the embryo sac. The tube enters one of the synergids of the embryo sac and ruptures. The sperm cells are then released into the synergid. In a process known as double fertilization one sperm cell fuses with the egg cell to form the diploid zygote while the other sperm cell fuses with the central cell to form the triploid endosperm. For most angiosperms, pollen germination, tube growth and fertilization are rapid events which are completed within 1 to 48 hours. At the morphological level a wealth of knowledge about the formation of the reproductive organs and cells, pollen germination, and the fertilization process has been gained during the last 50–100 years. This is in sharp contrast with the availability of data describing molecular processes regulating the different steps of plant reproduction. Only limited data exist describing the role of gene products or signal molecules which interact in and control the development of male and female reproductive cells and the fertilization process.

The Potential Use of the Glucuronidase Reporter Gene and Antisense Technologies to Unravel Biological Functions of Flavonoids

Flavonoids are higher plant secondary metabolites which have a key function in floral pigmentation (Harborne and Mabry 1982), defence against phytopathogens (Lamb et al 1989) and induction of nodulation (Firmin et al 1986; Zaat et al 1987). Furthermore, they have been implicated in UV-protection (Schmelzer et al 1988) and regulation of auxin transport (Jacobs and Rubery 1988). The multitude of functions suggest complex regulation and one might wonder if more functions are to be discovered. We propose that use of the s-glucuronidase (GUS) reporter gene in combination with antisense technologies may lead to a better understanding of the ‘signalling’ function(s) of flavonoid compounds.