Åke Strid

Gene regulation by low level UV-B radiation: identification by DNA array analysis

UV-B radiation alters transcript levels of various defence genes and photosynthetic genes in plants. Utilising a DNA array with 5000 ESTs and cDNAs from Arabidopsis thaliana, 70 genes were found to show a greater than two-fold induction or repression of transcript levels. Six genes (MEB5.2, PyroA, Ubq3, Lhcb6, F5D21.10 and the gene for an RNA polymerase II subunit) were tested for stress specific gene regulation on northern blots with RNA from plants exposed to low dose UV-B radiation, ozone or wounding. Transcript levels for PyroA, Uhq3 and the gene for a RNA polymerase II subunit were all specifically increased by UV-B. MEB5.2 mRNA levels also rose, whereas Lhcb6 and FSD21.10 transcript levels decreased under all stresses. The PyroA gene product in fungi is needed for biosynthesis of pyridoxine, and might have a role in protection against singlet oxygen. The Ubq3 gene encodes the ubiquitin protein that is attached to proteins destined for degradation. MEB5.2 and F5D21.10 represent novel gene products whose function have not yet been identified. Pairwise comparisons between the UV-B inducible promoters have identified a series of elements present in the MEB5.2 and PyroA promoters, absent from promoters of genes for early phenylpropanoid metabolism and that may be responsible for modulating their UV-B responses.

Multiple Roles for UV RESISTANCE LOCUS8 in Regulating Gene Expression and Metabolite Accumulation in Arabidopsis under Solar Ultraviolet Radiation

Summary: Under natural sunlight, this study demonstrates multiple and complex roles for the UV-B photoreceptor UV RESISTANCE LOCUS 8 in the acclimation of Arabidopsis plants to UV radiation. Photomorphogenic responses triggered by low fluence rates of ultraviolet B radiation (UV-B; 280–315 nm) are mediated by the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8). Beyond our understanding of the molecular mechanisms of UV-B perception by UVR8, there is still limited information on how the UVR8 pathway functions under natural sunlight. Here, wild-type Arabidopsis (Arabidopsis thaliana) and the uvr8-2 mutant were used in an experiment outdoors where UV-A (315–400 nm) and UV-B irradiances were attenuated using plastic films. Gene expression, PYRIDOXINE BIOSYNTHESIS1 (PDX1) accumulation, and leaf metabolite signatures were analyzed. The results show that UVR8 is required for transcript accumulation of genes involved in UV protection, oxidative stress, hormone signal transduction, and defense against herbivores under solar UV. Under natural UV-A irradiance, UVR8 is likely to interact with UV-A/blue light signaling pathways to moderate UV-B-driven transcript and PDX1 accumulation. UVR8 both positively and negatively affects UV-A-regulated gene expression and metabolite accumulation but is required for the UV-B induction of phenolics. Moreover, UVR8-dependent UV-B acclimation during the early stages of plant development may enhance normal growth under long-term exposure to solar UV.

Molecular markers for UV-B stress in plants: alteration of the expression of four classes of genes in Pisum sativum and the formation of high molecular mass RNA adducts.

Sixteen ultraviolet-B radiation-regulated pea genes were identified. Functionally, the corresponding proteins were divided into four groups. (i) Chloroplast-localized proteins. Genes for these proteins were down-regulated, underlining the deleterious effects of UV-B on this organelle. A novel down-regulated photosystem I light-harvesting chlorophyll a/b-binding protein gene (PsLhcA4), was cloned and sequenced. (ii) Protein turnover enzymes. Levels of mature mRNAs for the PU1 and PsUBC4 genes, encoding proteins of the ubiquitin protein degradation pathway, were up- and down-regulated, respectively, implying alteration of plant cell protein content by changes in both gene expression and protein degradation. (iii) Proteins involved in intracellular signalling. Expression of genes for small GTPases, rab and rho homologues, were altered. (iv) Phenylpropanoid or flavonoid biosynthesis. Expression of three genes encoding enzymes in these pathways were up-regulated and one of them, the novel PsC450R1, was cloned and sequenced. Moreover, unexpected high molecular mass psbA RNA adducts were found to appear after UV-B exposure. In addition, a large increase in corresponding high molecular mass adducts were also found for PsLhcA4, and PsUBC4 mRNA and 23S rRNA. These RNA species do not contain protein and probably appear due to cross-linking of two or more RNA molecules, or are the result of UV-B-induced failure of transcription termination.

Evidence of High •OH Radical Quenching Efficiency by Vitamin B6

Molecules acting as antioxidants capable of scavenging reactive oxygen species (ROS) are of the utmost importance in the living cell. The antioxidative properties of pyridoxine (vitamin B6) have recently been discovered. Previous theoretical calculations have shown a high reactivity of pyridoxine toward hydroxyl radicals, where the latter preferably abstract H from either carbon of the two methanol substituents (C8 or C9). In this study, we have explored the reactivity of pyridoxine toward further hydroxyl radicals, considering as the first step the H abstraction from either C8 or C9, also including addition reactions and cyclization. Many of the reactions display similar DeltaG, and hence, the quenching of hydroxyl radicals by pyridoxine may undergo different pathways leading to a mix of products. In addition, we observe that pyridoxine, under high hydroxyl radical concentrations, may scavenge up to eight radicals, supporting its observed high antioxidant activity.

The mRNA-binding ribosomal protein S26 as a molecular marker in plants: molecular cloning, sequencing and differential gene expression during environmental stress.

One condition for using a gene as a transcriptional marker for environmental stress is its specific and differential expression. In order to be used as such a marker, the ribosomal protein S26 cDNA from pea (Pisum sativum L.) was cloned and fully sequenced. The gene (PsRPS26) was shown to be differentially regulated by ozone and UV-B radiation in opposite ways. Ozone gave rise to increased mRNA levels, whereas UV-B led to a decrease in S26 transcript abundance. Thus, the expression of PsRPS26 can be used as a molecular marker to differentiate between these two environmental stresses.

The role of the pyridoxine (vitamin B6) biosynthesis enzyme PDX1 in ultraviolet-B radiation responses in plants

Ultraviolet-B radiation regulates plant growth and morphology at low and ambient fluence rates but can severely impact on plants at higher doses. Some plant UV-B responses are related to the formation of reactive oxygen species (ROS) and pyridoxine (vitamin B(6)) has been reported to be a quencher of ROS. UV-B irradiation of Arabidopsis Col-0 plants resulted in increased levels of PDX1 protein, compared with UV-A-exposed plants. This was shown by immunoblot analysis using specific polyclonal antibodies raised against the recombinant PDX1.3 protein and confirmed by mass spectrometry analysis of immunoprecipitated PDX1. The protein was located mainly in the cytosol but also to a small extent in the membrane fraction of plant leaves. Immunohistochemical analysis performed in pea revealed that PDX1 is present in UV-B-exposed leaf mesophyll and palisade parenchyma but not in epidermal cells. Pyridoxine production increased in Col-0 plants exposed to 3 days of UV-B, whereas in an Arabidopsis pdx1.3 mutant UV-B did not induce pyridoxine biosynthesis. In gene expression studies performed after UV-B exposure, the pdx1.3 mutant showed elevated transcript levels for the LHCB1*3 gene (encoding a chlorophyll a/b-binding protein of the photosystem II light-harvesting antenna complex) and the pathogenesis-related protein 5 (PR-5) gene, compared with wild type.

Induction of early light-inducible protein gene expression in Pisum sativum after exposure to low levels of UV-B irradiation and other environmental stresses

Plants are constantly subjected to environmental changes and have developed various defence mechanisms to facilitate their continued existence. Pisum sativum plants were exposed to low levels of UV-B radiation and ELIP (early light-inducible proteins) mRNA, with a probable protective function, was rapidly and strongly induced during this type of stress. To our knowledge, this is the only photosynthetic gene that is up-regulated following exposure to UV-B, and this result has to be compared with studies predominantly reporting down-regulation by UV-B of genes encoding proteins localised in the plastid. The expression pattern of ELIP mRNA in pea was also investigated during salt, wounding and ozone stress. The transcript levels of ELIP were induced after the salt and wounding treatments but not during ozone fumigation.

Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs

An optimized gene expression construct was designed in order to increase the accumulation of the HIV-1 subtype C p24 protein in Arabidopsis thaliana and carrot (Daucus carota) plants. An ER retention signal was introduced into the genetic construct generating a p24 protein containing a SEKDEL amino acid sequence at its C-terminus. Mature A. thaliana plants and carrot cells were transformed using Agrobacterium tumefaciens carrying the improved pGreen0229/p24_SEKDEL vector. Several transgenic plant lines were obtained from both plant species by growth on selective medium and confirmed by PCR. Transformed lines were analyzed for p24 protein content by western blotting using anti-p24-specific antibodies and by Southern blotting to establish the number of copies of the insert in the plant nuclear genome. To estimate the accumulation levels of p24 protein in the plants, ELISA was run using soluble plant extracts. By comparing these results with our previous findings, the ER retention signal increased the level of p24 protein fivefold in the A. thaliana plants. In carrot taproot, the content of p24_SEKDEL protein was approximately half of that in Arabidopsis on a fresh weight basis and was stable in planta for several months. However, on a total soluble protein basis, carrots produced considerable higher levels of the p24_SEKDEL protein than Arabidopsis.

Feeding of mice with Arabidopsis thaliana expressing the HIV-1 subtype C p24 antigen gives rise to systemic immune responses

Development of transgenic edible plants, to be used as production, storage and delivery systems for recombinant vaccine antigens, is a promising strategy to obtain cost effective vaccines against infectious diseases, not least for use in developing countries. Therefore, we used Agrobacterium tumefaciens‐mediated gene transfer to introduce the p24 gag gene encoding the nucleocapsid protein from HIV‐1 subtype C into the Arabidopsis thaliana plant genome. Eighteen plant lines were confirmed positive for the p24 gene by PCR; four of these lines showed an apparent homozygous phenotype when grown on selective medium and these lines also showed transcription of the p24 gene into its corresponding mRNA. The mRNA in all four cases generated the p24 protein in plants, as verified by Western blot analysis. The plants were shown to contain between 0.2 μg and 0.5 μg p24 protein per g of fresh tissue. Analysis of the localisation of the p24 protein showed that stem tissue contained the largest amount of protein, more than twice as much as leaf tissue, whereas no p24 protein was detected in roots. By using Southern blotting, we found that 4, 2–3, 2 and 1 T‐DNA insertion events took place in the four lines 1, 2, 7, and 10, respectively. The genetic insertions of line 1 were stable from the T2 to the T5 generation and gave rise to the p24 protein in all cases, as verified by Western blotting. In mice fed with fresh transgenic A. thaliana (line 10), anti‐gag IgG was obtained in serum after a booster injection with recombinant p37Gag. No immune response was observed after equal booster injection of untreated mice or mice fed with A. thaliana WT plants.

Catalytic roles of active-site residues in 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase : an ONIOM/DFT study

The catalytic mechanism of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) has been systematically studied using DFT and ONIOM(DFT:MM) methods. MHPCO catalyzes the hydroxylation and subsequent ring-opening of the aromatic substrate MHPC to give the aliphatic product α-(N-acetylaminomethylene)succinic acid (AAMS). Our calculations show that the active-site residues Arg211 and Tyr223 have a minor effect on the reaction, while the peptide bond of Pro295-Ala296, the side chain of Tyr82 and several crystal water molecules affect the reaction energy profile considerably. Both DFT and ONIOM calculations show that the ring-opening pathway B, in which an epoxy transition state is formed, is more favored than the direct C2-C3 cleavage pathway A. Different QM/MM partitioning schemes have been used to study the enzymatic reaction, and the results show that both the reaction barriers for the hydroxylation and the ring-opening pathways are sensitive to the QM/MM partitioning.