Abstract
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.
Highlights
In plants from temperate climates such as Arabidopsis thaliana low, non-freezing temperatures lead to increased freezing tolerance in a process termed cold acclimation
The content of different flavonols is correlated with leaf freezing tolerance in various Arabidopsis accessions and their crosses[19,20] and in addition the expression of some flavonoid biosynthesis genes and freezing tolerance are correlated in diverse Arabidopsis accessions[20,23]
We used a collection of 20 mutants in two different Arabidopsis thaliana accessions (Col-0 and Landsberg erecta (Ler)) that cover all major regulatory and biosynthetic steps in flavonoid biosynthesis (Fig. 1) to investigate the role of flavonols and anthocyanins in plant freezing tolerance and cold acclimation
Summary
In plants from temperate climates such as Arabidopsis thaliana low, non-freezing temperatures lead to increased freezing tolerance in a process termed cold acclimation. 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. Plant species from temperate regions, among them Arabidopsis thaliana, increase in freezing tolerance during exposure to low, but non-freezing temperatures in a process termed cold acclimation[25,26] This involves a massive reprogramming of the transcriptome, proteome and metabolome (see refs 27 and 28 for reviews) including flavonols and anthocyanins[20]. A direct causal relationship between the accumulation of flavonoids and freezing tolerance or the ability to cold acclimate has not been shown
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