ATK researchers studied the effect of plant free radicals on maize

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The effect of plant free radicals on cold-tolerant and cold-sensitive maize cultivars was investigated as part of the research led by senior scientist Imre Majláth of the ELKH Centre for Agricultural Research, Agricultural Institute (ATK MGI). During germination at low temperatures, methylglyoxal pretreatment improved germination rates, growth parameters, shoot lengths and shoot fresh weights, especially in the cold-sensitive genotype. The researchers found that the defense response to cold was not directly caused by the effect of the methylglyoxal itself, but instead was induced by the secondary stress response in the plant cells. The more sensitive maize cultivar gave an antioxidant protective response, while the more tolerant one responded to the treatment more hormonally. The new findings, published in the prestigious journal Physiologia Plantarum, contribute to a deeper understanding of the biology of plant free radicals.

Compared to reactive oxygen species (ROS), reactive carbonyl and aldehyde species (RAS) are less known as free radicals. The effects of these former three groups are similar in many respects, but at the same time they are somewhat ambivalent with regard to plant metabolic processes. These free radicals oxidize, (i.e. damage) the structure of macromolecules ‒ such as DNA, RNA, and enzymes for example ‒ and impair their functioning, but in smaller concentrations play a primary and important role as signal transmitters within cells, they are important mediating members of cellular signaling networks. Their signal transduction role is extremely important to the development of the abiotic stress response.

The current research focused on a form of RAS called the methylglyoxal (MG) molecule. The researchers were interested in how external, dosed MG treatment affects maize plants under low temperature stress. MG is also naturally present in plants. It is mainly produced from the intermediate products of glycolysis and photosynthesis in non-enzymatic pathways, during deprotonation and β-elimination chemical reactions of triose phosphate groups. Maize is fundamentally a cold-sensitive species, so the subjects of the experiments were two free-use maize cultivars: the cold-tolerant A654 and the cold-sensitive Cm174.

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Effect of soaking seeds in methylglyoxal (control, 2.5 mM, 5 mM, and 10 mM) in the A654 cultivar on day 10 after germination

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Effect of soaking seeds in methylglyoxal (control, 2.5 mM, 5 mM, and 10 mM) in the Cm174 cultivar on day 10 after germination

The researchers created an MG-induced artificial stress response in maize plants using a variety of treatments. Pre-soaking the seeds in MG solution proved to be the most successful method of treatment. The method of administration and the effect of the applied MG concentrations were also decisive factors. While higher concentrations either damaged or outright destroyed the embryos, medium and lower concentrations created a successful foundation in the metabolism of the tested plants. During germination at a low temperature (13 °C), MG pretreatment improved the germination rates, growth parameters, shoot lengths and shoot fresh weights, especially in the cold-sensitive genotype. The light utilization of their photochemical systems also proved superior, for which their higher photosynthetic pigment content was also probably a contributing factor. With regard to the activity of photosynthesis, the most marked improvement was exhibited by the values of net photosynthetic rate. In this case, too, the Cm174 plants, which started with a significant disadvantage compared to the A654 line, responded better.

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The net photosynthetic rate values (Pn) of the control (distilled water containing 0 mM methylglyoxal) and the maize seedlings grown at 13 °C (cultivars A654 and Cm174) treated with a 10 mM methylglyoxal solution show carbon dioxide uptake as measured over one square meter of leaf surface for one second. The measurements were made at atmospheric (390 ppm) and saturation (1000 ppm) carbon dioxide levels. The saturation Pn values were higher in all cases, indicating the simple regulation affecting the movement of the stomatal closure of the cold.

The question was raised as to whether the plant takes up the MG. So, was it the MG molecule itself that had an effect on the plants, or did the cold tolerance develop as a result of the signaling initiated by the molecule? Using high-performance liquid chromatography, the researchers clarified that on the seventh day after germination, the endogenous MG was already below the detection limit, meaning that the plants – even if they absorbed all the MG at the time of soaking the seeds – probably did not develop the cold stress due to the endogenous MG protection against the cold. Another piece of evidence for the abundance of otherwise cytotoxic MG tissue was the low level of malondialdehyde. The amount of this is directly proportional to the degree of damage to the cell membranes. On this basis, the researchers concluded that the defense response to cold was not a de facto effect of the MG, but rather of the MG inducing a secondary stress response in the plant cells.

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First explored during the weighted correlation network analysis (WGCNA) of the transcriptomes were the individual modules (clusters). In terms of resolution, various modules have surfaced with different color codes. Several subsequent steps were required in order to analyze the relationships between co-expression modules and to compare different network topologies.

An m-RNA transcriptome-sequencing and WGCNA co-expression network analysis, a comprehensive modern genomic analysis capable of determining the presence or absence of many thousands of gene products, confirmed the expression of important genes specialized in protection against cold and helped to reveal some functionally meaningful gene modules co-expressed at low germination temperature as an effect of MG treatment. Some of these were the genes of enzymes involved in the metabolism of hormones and flavonoid compounds (abscisic acid 8-hydroxylase and phenylalanine ammonia lyase), while others were the genes of proteins that accumulate during general stress protection (glutathione S-transferases, "late embryogenesis abundant" protective proteins, and UDP-glycosyltransferases).

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Detail of the functional mapping of significantly differentially expressed transcriptomes (genes). The analysis was performed using KEGG metabolic pathway maps. The intensity of the colors is proportional to the magnitude of the fold change values expressing the degree of expression (red: stimulation, green: inhibition).

The biochemical and molecular effects of antioxidant flavonoid compounds and major plant hormones (gibberellic acid, abscisic acid (ABA), and indole-3-acetic acid) are important factors during germination. Determination of their precise quantitative levels was carried out in the Metabolomics Laboratory of ATK's Agricultural Institute, which possesses the only equipment in Hungary capable of measuring them with ultra-high-efficiency liquid chromatography and mass spectrometry technology. To summarize the changes in the measured flavonoids, it was again only the individual samples of the Cm174 cultivar that showed a stronger antioxidant response to the cold, while the hormonal response was more pronounced in the case of the A654 cultivars. In their case, the amount of indole-3-acetic acid, an auxin important for growth, was higher. Although the level of ABA, which promotes aging and inhibits germination, was more significant in the A654 plants than in the Cm174 cultivars, the proportion of its degradation products showed that the ABA degradation was also more intense in the former case. This means that the degradation of ABA, which inhibits germination, was more intense in the case of the cold-tolerant cultivars. The more cold-tolerant maize cultivars responded more hormonally, while the more cold-sensitive ones exhibited an antioxidant defense in response to MG treatment. In the future, the research group will also try to gain a better understanding of the effects of other reactive forms of aldehyde.