We have assessed ultraviolet-B (UV-B)-induced injury in wildtype Arabidopsis thaliana and two mutants with altered aromatic secondary product biosynthesis. Arabidopsis mutants defective in the ability to synthesize UV-6-absorbing compounds (flavonoids in transparent testa 5 [ttfl and sinapate esters in ferulic acid hydroxylase 7 [fahl]) are more sensitive to UV-B than is the wild-type Landsberg erecta. Despite its ability to accumulate UV-absorptive flavonoid compounds, the ferulic acid hydroxylase mutant fahl exhibits more physiological injury (growth inhibition and foliar lesions) than either wild type or tt5. l h e extreme UV-B sensitivity of fahl demonstrates the importance of hydroxycinnamate esters as UV-B protectants. Consistent with the whole-plant response, the highest levels of lipid and protein oxidation products were seen in fahl. Ascorbate peroxidase enzyme activity was also increased in the leaves of UV-B-treated plants in a,dose-and genotype-dependent manner. These results demonstrate that, in A. thaliana, hydroxycinnamates are more effective UV-B protedants than flavonoids. The data also indicate that A. tbaliana responds to UV-B as an oxidative stress, and sunscreen compounds reduce the oxidative damage caused by UV-B.

To further our understanding of how plants defend against the harmful effects of ultraviolet (UV) light, we characterized an Arabidopsis mutant hypersensitive to UV-B. This mutant, UV resistance locus 8-1 (uvr8-1), contains a single recessive mutation at the bottom of chromosome 5. Fine-scale mapping localized uvr8-1 to a 21-kb locus containing five predicted open reading frames. Sequencing of this entire region revealed that the uvr8-1 allele contains a 15-nucleotide deletion in a gene similar to the human guanine nucleotide exchange factor regulator of chromatin condensation 1. This mutation reduces the UV-B-mediated induction of flavonoids and blocks chalcone synthase mRNA and protein induction. In contrast, uvr8-1 has enhanced induction of PR1 and PR5 proteins in response to UV-B, an indication of increased UV-B injury. These results suggest that UVR8 acts in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis.Plants must resist the deleterious effects of UV light because they are dependent on sunlight for photosynthesis and cannot avoid UV light exposure. Although UV is defined as the region of the spectrum from 200 to 400 nm, only the levels of UV-B (280-320 nm) reaching the earth's surface are increased by the thinning of the stratospheric ozone layer (Caldwell et al., 1989; Frederick et al., 1989;Stolarski et al., 1992; Kerr and McElroy, 1993). Thus, studies have focused on UV-B tolerance mechanisms because plants are directly affected by changes in terrestrial UV-B fluence.UV-B is known to cause DNA damage predominantly through cyclobutyl pyrimidine dimer formation and, to a lesser extent, pyrimidine-pyrimidinone (6, 4) photoproducts, both of which form by covalent bonding of adjacent pyrimidines (for review, see Britt, 1995;Landry et al., 1997;Nakajima et al., 1998). Failure to repair these DNA lesions interferes with DNA synthesis and transcription, and can result in heritable mutations (for review, see Britt, 1995). Studies of Arabidopsis have identified a variety of UV-Bhypersensitive mutants deficient in DNA repair (uvr1 [Britt et al., 1993], uvr2 [Jiang et al., 1997;Landry et al., 1997], uvr3 [Jiang et al., 1997;Nakajima et al., 1998], and uvh1 [Harlow et al., 1994]). For example, photolyases, enzymes that use blue light energy to repair pyrimidine dimers (Todo et al., 1993;Sancar, 1994), are critical for plant survival under UV-B in the laboratory (Ahmad et al., 1997;Landry et al., 1997;Nakajima et al., 1998). Other light-independent DNA repair mechanisms in plants are currently under study.In addition to directly causing DNA damage, UV-B generates oxidative stress through the formation of reactive oxygen species (ROS;Strid, 1992;Krizek et al., 1993 ; Doke et al., 1994; Foyer et al., 1994b), which in turn causes enhanced lipid and protein oxidation (Kramer et al., 1991;Landry et al., 1995). Plants counteract this increased ROS by increasing antioxidant enzymes (Foyer et al., 1994a; Kangasjarvi et al., 1994). For example, exposure to UV-B induces guaiacolperoxidases...

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing β-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19–35, and two clones from separate donors responded to insulin B-chain amino acids 9–23 (B:9–23), which are known to be a critical self-antigen–driving disease progress in animal models of autoimmune diabetes. These B:9–23–specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9–23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment.

Photolyases are DNA repair enzymes that use energy from blue light to repair pyrimidine dimers. We report the isolation of an Arabidopsis thaliana mutant (uvr2-1) that is defective in photorepair of cyclobutylpyrimidine dimers (CPDs). Whereas uvr2-1 is indistinguishable from wild type in the absence of UV light, low UV-B levels inhibit growth and cause leaf necrosis. uvr2-1 is more sensitive to UV-B than wild type when placed under white light after UV-B treatment. In contrast, recovery in darkness or in light lacking photoreactivating blue light results in equal injury in uvr2-1 and wild type. The uvr2-1 mutant is unable to remove CPDs in vivo, and plant extracts lack detectable photolyase activity. This recessive mutation segregates as a single gene located near the top of chromosome 1, and is a structural gene mutation in the type II CPD photolyase PHR1. This mutant provides evidence that CPD photolyase is required for plant survival in the presence of UV-B light.

The biosynthesis of capsaicinoids in the placenta of chilli fruit is modelled to require components of the fatty acid synthase (FAS) complex. Three candidate genes for subunits in this complex, Kas, Acl, and Fat, isolated based on differential expression, were characterized. Transcription of these three genes was placental-specific and RNA abundance was positively correlated with degree of pungency. Kas and Acl were mapped to linkage group 1 and Fat to linkage group 6. None of the genes is linked to the pungency locus, C, on linkage group 2. KAS accumulation was positively correlated with pungency. Western blots of placental extracts and histological sections both demonstrated that the accumulation of this enzyme was correlated with fruit pungency and KAS was immunolocalized to the expected cell layer, the placental epidermis. Enzyme activity of the recombinant form of the placental-specific KAS was confirmed using crude cell extracts. These FAS components are fruit-specific members of their respective gene families. These genes are predicted to be associated with Capsicum fruit traits, for example, capsaicinoid biosynthesis or fatty acid biosynthesis necessary for placental development.

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The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated. Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHR1 (for photoreactivating enzyme). It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors. Instead, the PHR1 gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems. PHR1 is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants. The PHR1 protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity. In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence. We conclude that PHR1 represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis.

Cytotoxic CD8 T lymphocytes play a central role in the tissue destruction of many autoimmune disorders. In type 1 diabetes (T1D), insulin and its precursor preproinsulin are major self-antigens targeted by T cells. We comprehensively examined preproinsulin specificity of CD8 T cells obtained from pancreatic islets of organ donors with and without T1D and identified epitopes throughout the entire preproinsulin protein and defective ribosomal products derived from preproinsulin messenger RNA. The frequency of preproinsulin-reactive T cells was significantly higher in T1D donors than nondiabetic donors and also differed by individual T1D donor, ranging from 3 to over 40%, with higher frequencies in T1D organ donors with HLA-A*02:01. Only T cells reactive to preproinsulin-related peptides isolated from T1D donors demonstrated potent autoreactivity. Reactivity to similar regions of preproinsulin was also observed in peripheral blood of a separate cohort of new-onset T1D patients. These findings have important implications for designing antigen-specific immunotherapies and identifying individuals that may benefit from such interventions.