Natural Variation among Arabidopsis Accessions in the Regulation of Flavonoid Metabolism and Stress Gene Expression by Combined UV Radiation and Cold.

Plants are constantly exposed to stressful environmental conditions. Plant stress reactions were mainly investigated for single stress factors. However, under natural conditions plants may be simultaneously exposed to different stresses. Responses to combined stresses cannot be predicted from the reactions to the single stresses. Flavonoids accumulate in Arabidopsis thaliana during exposure to UV-A, UV-B or cold, but the interactions of these factors on flavonoid biosynthesis were unknown. We therefore investigated the interaction of UV radiation and cold in regulating the expression of well-characterized stress-regulated genes, and on transcripts and metabolites of the flavonoid biosynthetic pathway in 52 natural Arabidopsis accessions that differ widely in their freezing tolerance. The data revealed interactions of cold and UV on the regulation of stress-related and flavonoid biosynthesis genes, and on flavonoid composition. In many cases, plant reactions to a combination of cold and UV were unique under combined stress and not predictable from the responses to the single stresses. Strikingly, all correlations between expression levels of flavonoid biosynthesis genes and flavonol levels were abolished by UV-B exposure. Similarly, correlations between transcript levels of flavonoid biosynthesis genes or flavonoid contents, and freezing tolerance were lost in the presence of UV radiation, while correlations with the expression levels of cold-regulated genes largely persisted. This may indicate different molecular cold acclimation responses in the presence or absence of UV radiation.

Flavonoids are determinants of freezing tolerance and cold acclimation in Arabidopsis thaliana.

In plants from temperate climates such as Arabidopsis thaliana low, non-freezing temperatures lead to increased freezing tolerance in a process termed cold acclimation. This process is accompanied by massive changes in gene expression and in the content of primary metabolites and lipids. In addition, most flavonols and anthocyanins accumulate upon cold exposure, along with most transcripts encoding transcription factors and enzymes of the flavonoid biosynthetic pathway. However, no evidence for a functional role of flavonoids in plant freezing tolerance has been shown. Here, we present a comprehensive analysis using qRT-PCR for transcript, LC-MS for flavonoid and GC-MS for primary metabolite measurements, and an electrolyte leakage assay to determine freezing tolerance of 20 mutant lines in two Arabidopsis accessions that are affected in different steps of the flavonoid biosynthetic pathway. This analysis provides evidence for a functional role of flavonoids in plant cold acclimation. The accumulation of flavonoids in the activation tagging mutant line pap1-D improved, while reduced flavonoid content in different knock-out mutants impaired leaf freezing tolerance. Analysis of the different knock-out mutants suggests redundancy of flavonoid structures, as the lack of flavonols or anthocyanins could be compensated by other compound classes.

Natural variation in flavonol and anthocyanin metabolism during cold acclimation in Arabidopsis thaliana accessions.

In plants from temperate climates such as Arabidopsis thaliana, low, non-freezing temperatures lead to increased freezing tolerance in a process termed cold acclimation. During cold acclimation, massive changes in gene expression and in the content of primary metabolites and lipids have been observed. Here, we have analysed the influence of cold acclimation on flavonol and anthocyanin content and on the expression of genes related to flavonoid metabolism in 54 Arabidopsis accessions covering a wide range of freezing tolerance. Most flavonols and anthocyanins accumulated upon cold exposure, but the extent of accumulation varied strongly among the accessions. This was also true for most of the investigated transcripts. Correlation analyses revealed a high degree of coordination among metabolites and among transcripts, but only little correlation between metabolites and transcripts, indicating an important role of post-transcriptional regulation in flavonoid metabolism. Similarly, levels of many flavonoid biosynthesis genes were correlated with freezing tolerance after cold acclimation, but only the pool sizes of a few flavonols and anthocyanins. Collectively, our data provide evidence for an important role of flavonoid metabolism in Arabidopsis freezing tolerance and point to the importance of post-transcriptional mechanisms in the regulation of flavonoid metabolism in response to cold.

Clinal variation in the non-acclimated and cold-acclimated freezing tolerance of Arabidopsis thaliana accessions.

Arabidopsis thaliana is a geographically widely spread species consisting of local accessions differing both genetically and phenotypically. These differences may constitute environmental adaptations and a latitudinal cline in freezing tolerance has been shown previously. Many plants, including Arabidopsis, exhibit increased freezing tolerance after cold exposure (cold acclimation). Here we present evidence for geographical clines (both latitudinal and longitudinal) in acclimated (ACC) and non-acclimated (NA) freezing tolerance, estimated from electrolyte leakage measurements on 54 accessions. Leaf Pro contents were not correlated with freezing tolerance, while sugar contents (Glc, Fru, Suc, Raf) were in the ACC, but not the NA state. Expression levels of 14 cold-induced genes were investigated before and after 2 weeks of cold acclimation by quantitative RT-PCR. Expression of the CBF1, 2 and 3 genes was not correlated with freezing tolerance. The expression of some CBF-regulated (COR) genes, however, was correlated specifically with ACC freezing tolerance. A tight correlation between CBF and COR gene expression was only observed under non-acclimating conditions, where CBF and COR expression were also correlated with the expression of PRR5, a component of the circadian clock. Collectively, this study sheds new light on the molecular determinants of plant-freezing tolerance and cold acclimation and their geographical dependence.

Natural variation in the freezing tolerance of Arabidopsis thaliana: effects of RNAi-induced CBF depletion and QTL localisation vary among accessions.

Plants from temperate regions are able to withstand freezing temperatures and to increase their freezing tolerance during exposure to low, but non-freezing, temperatures through a process known as cold acclimation. Key regulatory proteins in this process are the cold-induced CBF1, 2 and 3 transcription factors which control many cold regulated genes. Although much work has focused on this signal transduction pathway, the details of its regulation and of its quantitative contribution to cold acclimation are still unclear. Here, we have used the large natural variation present in the 48 accessions of the Versailles core collection of Arabidopsis thaliana to further elucidate the function of the CBF transcription factors. CBF gene expression studies showed that the freezing sensitive accessions had mostly low expression levels 2h after transfer of plants to 5°C, while the most tolerant accessions showed a wide range of CBF expression levels. To investigate the quantitative contribution of CBF expression to plant freezing tolerance and low temperature growth performance, RNAi lines targeting all three CBF genes were produced in eight different accessions. We observed striking differences between different accessions in the effects that reduced CBF expression had on freezing tolerance, while effects on growth were generally too small to draw firm conclusions. Analysis of CBF expression indicated a tight co-regulation between CBF1 and CBF3, while the relationship between the expression levels of CBF2 and CBF1 or CBF3 strongly depended on the genetic background of the RNAi lines. In agreement with the observed differences between the different accessions, QTL analyses with two different RIL populations indicated that QTL localisation varies strongly between populations. Collectively, these results show that both the regulation of the CBF genes and their relative contribution to freezing tolerance strongly depend on the accession studied. In addition, natural variation is suggested to be an interesting source of novel regulatory pathways and genes that may be useful in the future for improving plant freezing tolerance.