Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus- and chloroplast-encoded proteins of higher plant chloroplasts

Emine Dinç, Szilvia Z. Tóth, G. Schansker, F. Ayaydin, László Kovács, D. Dudits, G. Garab, S. Bottka

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Selective inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) is widely applied in gene function analyses; however, experiments with ODNs in plants are scarce. In this work, we extend the use of ODNs in different plant species, optimizing the uptake, stability, and efficiency of ODNs with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. We targeted the nucleus-encoded phytoene desaturase (pds) gene, encoding a key enzyme in carotenoid biosynthesis, the chlorophyll a/b-binding (cab) protein genes, and the chloroplast-encoded psbA gene, encoding the D1 protein. For pds and psbA, the in vivo stability of ODNs was increased by phosphorothioate modifications. After infiltration of ODNs into juvenile tobacco (Nicotiana benthamiana) leaves, we detected a 25% to 35% reduction in mRNA level and an approximately 5% decrease in both carotenoid content and the variable fluorescence of photosystem II. In detached etiolated wheat (Triticum aestivum) leaves, after 8 h of greening, the mRNA level, carotenoid content, and variable fluorescence were inhibited up to 75%, 25%, and 20%, respectively. Regarding cab, ODN treatments of etiolated wheat leaves resulted in an up to 59% decrease in the amount of chlorophyll b, a 41% decrease of the maximum chlorophyll fluorescence intensity, the cab mRNA level was reduced to 66%, and the protein level was suppressed up to 85% compared with the control. The psbA mRNA and protein levels in Arabidopsis (Arabidopsis thaliana) leaves were inhibited by up to 85% and 72%, respectively. To exploit the potential of ODNs for photosynthetic genes, we propose molecular design combined with fast, noninvasive techniques to test their functional effects.

Original languageEnglish
Pages (from-to)1628-1641
Number of pages14
JournalPlant Physiology
Volume157
Issue number4
DOIs
Publication statusPublished - Dec 2011

Fingerprint

Chloroplast Proteins
Oligodeoxyribonucleotides
Chloroplasts
chloroplasts
chlorophyll
Carotenoids
proteins
carotenoids
Triticum
Messenger RNA
fluorescence
Fluorescence
genes
leaves
Arabidopsis
Genes
Tobacco
Chloroplast Genes
Chlorophyll Binding Proteins
gene expression

ASJC Scopus subject areas

  • Plant Science
  • Genetics
  • Physiology

Cite this

Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus- and chloroplast-encoded proteins of higher plant chloroplasts. / Dinç, Emine; Tóth, Szilvia Z.; Schansker, G.; Ayaydin, F.; Kovács, László; Dudits, D.; Garab, G.; Bottka, S.

In: Plant Physiology, Vol. 157, No. 4, 12.2011, p. 1628-1641.

Research output: Contribution to journalArticle

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abstract = "Selective inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) is widely applied in gene function analyses; however, experiments with ODNs in plants are scarce. In this work, we extend the use of ODNs in different plant species, optimizing the uptake, stability, and efficiency of ODNs with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. We targeted the nucleus-encoded phytoene desaturase (pds) gene, encoding a key enzyme in carotenoid biosynthesis, the chlorophyll a/b-binding (cab) protein genes, and the chloroplast-encoded psbA gene, encoding the D1 protein. For pds and psbA, the in vivo stability of ODNs was increased by phosphorothioate modifications. After infiltration of ODNs into juvenile tobacco (Nicotiana benthamiana) leaves, we detected a 25{\%} to 35{\%} reduction in mRNA level and an approximately 5{\%} decrease in both carotenoid content and the variable fluorescence of photosystem II. In detached etiolated wheat (Triticum aestivum) leaves, after 8 h of greening, the mRNA level, carotenoid content, and variable fluorescence were inhibited up to 75{\%}, 25{\%}, and 20{\%}, respectively. Regarding cab, ODN treatments of etiolated wheat leaves resulted in an up to 59{\%} decrease in the amount of chlorophyll b, a 41{\%} decrease of the maximum chlorophyll fluorescence intensity, the cab mRNA level was reduced to 66{\%}, and the protein level was suppressed up to 85{\%} compared with the control. The psbA mRNA and protein levels in Arabidopsis (Arabidopsis thaliana) leaves were inhibited by up to 85{\%} and 72{\%}, respectively. To exploit the potential of ODNs for photosynthetic genes, we propose molecular design combined with fast, noninvasive techniques to test their functional effects.",
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