A genetic approach to understanding the responses of Arabidopsis thaliana to ultraviolet-B light

A decrease in stratospheric ozone over the past few decades has resulted in an increase in the quantity of UV-B (280-320 nm) reaching the surface of the Earth. Paradoxically, plants can both suffer from UV-B induced damage and benefit from utilizing UV-B as a trigger for developmental responses and gene expression. There is almost certainly more than one pathway by which plants transmit a signal after perceiving UV-B, in order to effect appropriate responses. Although it is not known how serious the implications of increased ambient UV-B will be for the yield of important crops in the future, our lack of understanding of the mechanisms by which UV-B elicits different responses from plants depending on fluence rate, wavelength and developmental stage ought to be redressed. In order to try to identify some of the proteinaceous components involved in UV-B perception and signal transduction in Arabidopsis we adopted a genetic approach. Various responses to UV-B were carefully investigated with a view to identifying those most suitable to form the bases for genetic screens. It was demonstrated that UV-B wavelengths are capable of stimulating positive phototropic hypocotyl curvature even in the absence of the phototropins (phot1 and phot2), although a screen for mutants lacking this response to UV-B proved unsuccessful. By contrast, a screen for mutants altered in UV-B induced CHALCONE SYNTHASE (CHS) gene expression facilitated by a CHS promoter - Luciferase (Luc) reporter gene, produced four under-expressing and two over-expressing mutants. The four under-expressing mutants were each shown to have single base pair changes in the UVRESISTANCE LOCUS 8 (UVR8) gene. An Arabidopsis uvrS mutant had previously been isolated by Kliebenstein et al. in a screen for plants exhibiting hypersensitivity to supplementary UV-B induced damage. Reduced levels of CHS transcript accumulation in response to UV-B had also been previously observed in the mutant. However, characterization of uvr8 mutants was substantially extended in the work described here. The involvement of UVRS in a UV-B specific branch of photoreception / signal transduction is strongly supported by the uvr8 mutants' retention of CHS expression in response to UV-A and cold treatment in mature plants and to far-red and sucrose treatment in seedlings. UVR8 was shown to be necessary for UV-B (but not UV-A) induced transcript accumulation of the LONG HYPOCOTYL 5 (HY5) transcription factor, and the expression of genes important in UV-B photoprotection (eg. EARLY LIGHT INDUCIBLE PROTEINS, PHOTOREACTIVATING ENZYME 1 and flavonoid biosynthesis genes) is apparently stimulated via a signalling pathway which requires both UVR8 and HY5. Accordingly, in the absence of either UVR8 or HY5 plants show an increased susceptibility to supplementary UV-B induced growth inhibition and leaf damage. Microarray analyses of UV-B induced gene expression patterns in both the wr8 and hy5 mutants, compared to wild- type controls, have identified a subset of genes regulated by a UV-B signalling pathway in which UVR8 and HY5 each plays a critical role. The loss of this signalling pathway probably places the plant at a significant selective disadvantage in the wild. In addition to this work, the 30e5 mutant which had previously been isolated, using a CHS promoter beta-GLUCURONIDASE (GUS) reporter gene, as deficient in its response to UV-B was characterized. Although 30e5 was subsequently found to accumulate CHS transcripts normally in response to UV-B and UV-A, it proved to be more sensitive to supplementary UV-B induced growth inhibition and leaf damage than was uvr8. Crosses with the corresponding mutants demonstrated that the 30e5 mutant gene is an allele of the DIFFERENTIAL DEVELOPMENT OF VASCULAR-ASSOCIATED CELLS (DOV1) gene. Map-based cloning delimited the 30E5 gene to a 40 kb region of Arabidopsis chromosome IV which contained 12-14 genes. One of these candidate genes, ATase DEFICIENT 2 (ATD2), encodes 5-PHOSPHORIBOSYL-1-PYROPHOSPHATE AMIDOTRANSFERASE 2 (ATase2), an isoenzyme which catalyzes the first step of de novo purine biosynthesis in Arabidopsis. The atd2 mutant is strikingly similar in phenotype to 30e5 and crosses are being initiated to determine whether 30e5 is an allele of the ATD2 gene