Transcriptomic analysis of manganese toxicity and manganese leaf-tissue tolerance in cowpea (Vigna unguiculata (L.) Walp.)
Zusammenfassung der Projektergebnisse
Part A: The aim of this initial transcriptomic study was the analysis of short-term excess Mn induced transcriptomic changes in leaves ofthe Mn-sensitive cowpea cv. TVu 91. Additionally, Si was applied to study early changes of the leaf-gene expression possibly leading to Sienhanced Mn tolerance in the Mn-sensitive cowpea genotype. Mineral and physiological analysis revealed typical physiological markers being to insensitive to indicate changes in the apoplast of cowpea plants after one-day excess Mn treatment. Therefore, apoplastic mechanisms promoting Mn toxicity were not detectable after one day of high Mn supply particulariy in the Mn-sensitive cowpea genotype. The SSH based comparison of mRNA populations extracted from Mn-sensitive cowpea plants created subtractive cDNA libraries which revealed a short-term Mn coupled Si-induced strong downregulation of photosynthesis/respiration related transcripts but an upregulation of metabolism and transport/cell motility related sequences. A constitutive influence of Si promoted a downregulation of photosynthesis/respiration related transcripts as well as a downregulation of sequences belonging to signal transduction, transport/cell motility and transcription/translation. A total of 23 putative candidate genes, connected to transport, stress response, signal transduction, metabolism, photosynthesis and cell wall organization, was selected from the subtractive cDNA libraries. Because of the abundance of cDNA clones showing highest homology to RubisCO Acfivase mRNA from V. radiata (AFI26870.2), this gene was chosen as a candidate for a further expression analysis. Relative changes in the RubisCO Activase transcript level were detected in two qRT-PCR assays. Compared to an untreated control variant, short-term Mn supply caused a 11.5 fold downregulation ofthe transcript amount, whereas a consfitutive Si influence promoted a 7.5 fold downregulation and the combined treatment resulted in an only 1.2 fold downregulafion. In turn, Si was able to cause a 2298.8 fold increase of RubisCO Aclivase transcripts in Mntreated cowpea plants and in comparison to Si-treated plants a.combined treatmenl caused a 6.6 fold increase of transcripts. Part B: The aim of this continuative transcriptomic work was the analysis of early excess Mn induced transcriptomic changes in Mn tolerant cowpea cv. TVu 1987 and Mn sensitive cowpea cv. TVu 1987. The SSH based cultivar-specific comparison of mRNA populations generated subtractive cDNA libraries containing sequences induced by one day excess Mn supply for both cowpea genotypes. In general, the results for both genotypes were quite similar further emphasizing the early stage of Mn toxicity development. A total of six candidate genes were selected from subtractive cDNA libraries and were subjected to qRT-PCR. This method allowed analyzing the Mn and Si treatment dependent changes in expression level of selected candidate genes, leading to possible identification of interaction effecis of symplasfic and apoplastic processes involved in Mn tolerance, Simediated Mn toxicity alleviation and/or Mn stress. The expression analysis of candidate genes did not reveal insights inlo Mn-induced effects contributing to possible Mn-tolerance mechanisms. However, different expression levels of candidate genes between the Mn-tolerant cowpea cv. TVu 1987 and the Mn-sensitive cowpea cv. TVu 91 were detected. In TVu 1987, the amount of glutathione peroxidase transcripts was 4 fold' higher, the amount of aquaporin transcripts was 34 fold higher and the amount of RuBisCO activase transcripts was 9 fold higher than in TVu 91. Additionally, the gene expression of cationic peroxidase, an enzyme known to play a significant role in Mn toxicity development, "was 474 fold lower in the Mn-tolerant cultivar. These findinigs lead to the conclusion that mechanisms probably playing a role in genolypical Mn-tolerance or Mn sensitivity originate from a constitutively higher or lower expression of corresponding genes in TVu 91 and/or TVu 1987 and these genes might not be solely modulated by Mn and Si supply.
