Abstract
Low temperature (LT) negatively affects plant growth and development via the alteration of the metabolism of reactive oxygen and nitrogen species (ROS and RNS). Among RNS, tyrosine nitration, the addition of an NO2 group to a tyrosine residue, can modulate reduced nicotinamide-dinucleotide phosphate (NADPH)-generating systems and, therefore, can alter the levels of NADPH, a key cofactor in cellular redox homeostasis. NADPH also acts as an indispensable electron donor within a wide range of enzymatic reactions, biosynthetic pathways, and detoxification processes, which could affect plant viability. To extend our knowledge about the regulation of this key cofactor by this nitric oxide (NO)-related post-translational modification, we analyzed the effect of tyrosine nitration on another NADPH-generating enzyme, the NADP-malic enzyme (NADP-ME), under LT stress. In Arabidopsis thaliana seedlings exposed to short-term LT (4 °C for 48 h), a 50% growth reduction accompanied by an increase in the content of superoxide, nitric oxide, and peroxynitrite, in addition to diminished cytosolic NADP-ME activity, were found. In vitro assays confirmed that peroxynitrite inhibits cytosolic NADP-ME2 activity due to tyrosine nitration. The mass spectrometric analysis of nitrated NADP-ME2 enabled us to determine that Tyr-73 was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. The in silico analysis of the Arabidopsis NADP-ME2 protein sequence suggests that Tyr73 nitration could disrupt the interactions between the specific amino acids responsible for protein structure stability. In conclusion, the present data show that short-term LT stress affects the metabolism of ROS and RNS, which appears to negatively modulate the activity of cytosolic NADP-ME through the tyrosine nitration process.
Highlights
Low temperature (LT) is an environmental issue that affects plant physiology and biochemistry at different levels, including antioxidant enzymes, photosynthesis, gene expression, nutrients, and water uptake [1,2,3,4,5,6,7,8,9]
As NADP-malic enzyme (NADP-ME) was the only NADP-dehydrogenase to be modulated under low temperature (LT) conditions, we focused on this enzyme to study the potential regulatory effect of tyrosine nitration by an in vitro approach
The fresh weight of these seedlings reduced by 50% as a result of LT (Figure 1B), which indicates that the LT conditions caused significant drastically reduced by 50% as a result of LT (Figure 1B), which indicates that the LT conditions stress to Arabidopsis thaliana seedlings
Summary
Low temperature (LT) is an environmental issue that affects plant physiology and biochemistry at different levels, including antioxidant enzymes, photosynthesis, gene expression, nutrients, and water uptake [1,2,3,4,5,6,7,8,9]. The impact of LT on plant crops, such as pepper, rice, tomato, maize, and olive, and tropical and subtropical fruits worldwide, is very significant as it influences both their production and quality [1,10,11] Model plants, such as Arabidopsis thaliana, act as a very useful tool to decipher the molecular mechanism of response to LT stress [12,13,14,15,16]. Several NADPH-generating systems come into play, such as ferredoxin-NADP reductase as a component of photosystem I, and a group of NADP-dehydrogenases that have been found in different subcellular locations This group of enzymes includes NADP-isocitrate dehydrogenase (NADP-ICDH), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and the NADP-malic enzyme (NADP-ME), called NADP-malate dehydrogenase
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