*2.3. Gene Expression Studies*

Based on a microarray assay, the changes in the gene expression levels during the cold acclimation period under various light conditions have been analysed in our previous study [12]. In the present work—based on the same microarray database—gene expressions related to the above characterised changes in plant hormone metabolisms (Section 2.1.) and antioxidant capacity (Section 2.2.) have been further analysed in the leaves at the end of acclimation period.

A gene encoding a PAL enzyme was downregulated and genes encoding the enzyme related to the SA-MeSA (methyl-SA) conversation (salicylate carboxymethyltransferase), and enzymes related to the synthesis of ABA (zeaxanthin epoxidase (ZEP)) and JA (12-oxo-phytodienoic acid reductase7) and phenypropanoid metabolism (cinnamyl-alcohol dehydrogenase) were upregulated in GL compared to the control plants (Table S1). Chorismate mutase 1 (related to the SA biosynthesis as a part of the shikimic acid pathway) and violaxanthin de-epoxidase were downregulated, while enzymes of ABA (aldehyde oxidase, ZEP) and JA (12-oxo-phytodienoic acid reductase7, allene oxide synthase 1) biosynthesis were upregulated in LL compared to the control plants. In a comparison of GL and LL plants, salicylate carboxymethyltransferase, aldehyde oxidase, allene oxide synthase 1 were upregulated in GL plants, while chorismate mutase 1 and violaxanthin de-epoxidase were downregulated.

Gene expression levels related to the antioxidants were also examined at the end of the acclimation period in the leaves of the plants. Glutathione-S-transferase (GST) was downregulated and ascorbate peroxidases were upregulated in GL plants compared to the control (Table S2). Mainly the enzymes of the ascorbate–glutathione cycle (glutathione reductase, monodehydroascorbate reductase 5, dehydroascorbate reductase) were downregulated and GSTs were upregulated in LL compared to the control. Enzymes related to the flavonol/anthocyanin biosynthesis (isoflavone reductase, dihydroflavonol-4-reductase) were upregulated in GL and LL plants compared to the control (Table S3). In a comparison of GL–LL plants, GST and flavonol/anthocynin biosynthesis enzymes were upregulated and some enzymes of the ascorbate (ASC)–GSH cycle (glutathione reductase, monodehydroascorbate reductase 5) were downregulated in GL compared to the LL plants.

#### **3. Discussion**

Growing plants at suboptimal but non-lethal temperatures may help them to prepare for a subsequent, more severe cold stress. In the case of hardy winter cereals, it may also lead to development of frost tolerance, which enables plants to survive low temperatures well below 0 ◦C. Light during the hardening period is also essential for the development of adequate freezing tolerance. Its role and mode of action have been widely studied [6,20,21]. But the role of light during the cold acclimation period in the case of chilling sensitive plants is hardly documented. When light is accompanied by low growth temperature, usually, photoinhibitory damage can be detected, which may substantially contribute to the chilling injury in cold-sensitive plants. However, our previous study [12] indicated that, in spite of its photoinhibitory effects, light is also essential for the high level of cold acclimation in young maize plants. In order to better understand the molecular mechanisms of the light-related cold acclimation processes, in recent works, certain stress-related plant hormones, focusing mainly on SA and its related compounds, were also followed during the cold acclimation, chilling, and a subsequent recovery periods. SA is also a Janus-faced compound: its exogenous application provided protection against chilling injury in young maize plants [22], but its relatively high endogenous level inhibited the growth of *Arabidopsis* plants at low temperatures [23]. In the present work, the main changes could be observed in the free SA levels in the leaves, and it was lower in acclimated plants than in the controls. Although, the gene expression level of PAL enzyme was downregulated in GL plants under cold conditions, the PAL enzyme activity did not show remarkable differences, which could have been the reason for the lower SA contents in the leaves. However, at the end of the hardening period, the gene expression of the salicylate caboxymethyltransferase, an enzyme responsible for SA-MeSA conversation, increased in the leaves of GL plants both under cold and control conditions, suggesting the dominant effect of light, rather than the cold MeSA, is a volatile form of SA and is part of the SA signalling pathway [24] and the decreased level of free SA could at least be partly a result of this SA-MeSA conversation. Interestingly, parallel to this, the expression of chorismate mutase 1 was also downregulated in GL plants, suggesting that the branch point of the shikimate pathway to channel amino acid precursors to the biosynthesis of phenylalanine and tyrosine and away from that of tryptophan was lower at GL [25].

ABA is a stress hormone, playing a role in the responses to various environmental stimuli [26]. In our experiment, the level of ABA did not correlate with the level of tolerance. Its level mainly increased in the leaves during the post-chilling recovery period. It seems that it is mainly related to the level of the stress, and less related to the level of the acclimation. At the end of acclimation, the synthesis gene of ABA (ZEP) was upregulated both in GL and LL plant compared to the non-acclimated plants, but there was no difference between the GL and LL plants. In addition, the expression of violaxanthin de-epoxidase, which is responsible for the catalysation of the reaction in the opposite direction, was downregulated in LL compared to the control under cold conditions, while it was upregulated in the GL compared to the LL plants. It has been found that the gene expression of ZEP showed mainly circadian rhythms in the leaves rather than changes in the daily periods of stress, and no changes were detected in the amount of the enzyme [27]. On the other hand, light was required for the gene expression of ZEP, because cold treatment only induced the genes of the ABA biosynthesis in light, as was demonstrated in *Arabidopsis* plants [28].

A similar trend could be observed in the JA levels, which mainly increased in the leaves of non-acclimated plants. In addition, at the end of acclimation, the gene of JA biosynthesis (12-oxo-phytodienoic acid reductase7) was upregulated both in GL and LL plants compared to the non-acclimated plants, but there was no difference between GL and LL plants, while another gene encoding allene oxide synthase 1 was shown to exhibit higher upregulation in GL plants. Chilling temperatures may increase the generation of the reactive oxygen species (ROS), especially in light. Plants generally respond to such conditions by activating antioxidant systems. oHCA, a precursor of SA, belongs to the hydroxycinnamic acid family, and it also has antioxidant properties. Although the oHCA level was lower in the acclimated plants, and no pronounced differences were observed between the GL and LL plants, the gene of cinnamyl-alcohol dehydrogenase, related to the cinnamic acid metabolism, was upregulated in GL plants. This latter result is in accordance with earlier findings, where two genes playing roles in the synthesis of hydroxycinnamic acids were also upregulated at a low temperature in light in *Arabidopsis* plants [28]. Parallel to the induction of the gene, a cold-induced increase in the oHCA level was also detected in young maize plants. However, this increase also occurred in the dark. This response was entirely different from that which was earlier found during the cold hardening period in winter wheat plants, where a substantial increase in the oHCA level only occurred in light, but not in the dark [29]. However, the pattern of leaf oHCA content under the recovery period showed similar differences and changes, as was found for the SA. It seems that the exact role of oHCA as a precursor and/or antioxidant compound during the cold acclimation period still requires intensive further research.

Hydroxycinnamic acid biosynthesis, like SA metabolism, is part of the phenylpropanoid pathway which involves the synthesis of various antioxidants, such as flavonols, anthocyanins, etc., and one of the key enzymes of this pathway is PAL. It was found earlier that light had a role not only in the development of chilling injury, but also in the appearance of the post-chilling symptoms in young maize plants [30]. Furthermore, light was also required for the synthesis of anthocyanins when plants were transferred from chilling conditions to optimum growth temperatures [31]. In the present work, although the recovery was in light, and only the acclimation was carried out at different illuminations, the differences in anthocyanin level could be seen both in the leaves and roots. Plants acclimated at low light had much lower anthocyanin content compared to the GL plants. Interestingly, differences in the anthocyanin levels did not follow the changes in the activity of the PAL enzyme. Furthermore, the changes in the various flavonols, such as K, Q, M or R, either in the leaves or in the roots, were more temperature- than light-dependent. This was in accordance with the gene expression changes, as the transcript level of the genes related to flavonoid metabolism was also upregulated in GL and LL plants under cold conditions.

GSH also acts as an antioxidant by quenching ROS. The ASC–GSH cycle plays an important role in this respect. In *Arabidopsis*, cold treatment in light induced the expression of genes of two GSH-dependent peroxidases, while the genes of the ASC–GSH cycle were not induced [28]. In our case similar changes were found in maize plants, the genes of the ASC–GSH cycle enzymes were downregulated, while genes encoding GST were upregulated in GL plants compared to the LL conditions during cold acclimation. However, the accumulation of GSH was also light dependent. The synthesis of GSH is a two-step reaction: the first step is the formation of γEC from Cys catalysed by γEC synthetase then the GSH synthetase catalyses the addition of a glycine. The rate-limiting step is the γEC synthesis [32]. In a recent study, the highest GSH level was found at optimum light conditions in wheat plants. Either the higher or lower illumination caused lower GSH accumulation [33]. In the present work, the highest GSH level was found in the leaves of GL plants on the first day of recovery, but the pre-cursors of GSH (Cys, γEC) started to accumulate during chilling in GL plants. However, this accumulation did not cause an increase in the GSH level, because the enhancement of the degradation processes led to high levels of CysGly. In contrast to the GL plants, under low light conditions, higher GSH accumulation occurred during the acclimation period.

GSH is not only an antioxidant, it can also interact with plant hormones. GSH regulates SA accumulation via the expression of genes of isochorismate synthetase 1 in *Arabidopsis* plants and it can lead to an increase in the intracellular hydrogen peroxide level for the activation of SA signalling [34]. In our case, GSH accumulation was not accompanied by SA accumulation either in the free or bound forms. In another study, the GSH content was higher after SA treatment in maize plants, while it was lower after the application of ABA [35]. In the present work, it was also found that a higher GSH content was coupled with a lower ABA level. However, under the present conditions, the antagonistic relationship between SA and ABA, or between SA and JA, could not be detected.

### **4. Materials and Methods**

## *4.1. Plant Material and Growth Conditions*

Maize plants were grown for 11 days at 22/20 ◦C at higher photosynthetic photon flux density (PPFD), (growth light, GL = 387 μmol m−<sup>2</sup> s−1) with 16/8 h light/dark periods. Some of the plants were then cold acclimated at 15/13 ◦C for 3 days, either at GL or at moderate low light intensity (LL) 107 μmol m−<sup>2</sup> s<sup>−</sup>1. Afterwards, all plants were transferred to 5 ◦C at a continuous growth light (GL) for 3 days, followed by a seven-day recovery period at 22 ◦C under GL conditions. Samples were collected for biochemical analysis from the control plants, and from treated plants after the acclimation (GL and LL plants), chilling and during recovery periods (non-acclimated, GL and LL plants).

All the chemicals were purchased from Sigma-Aldrich or Merck (Merck Group, Darmstadt, Germany).

#### *4.2. Estimation of Lipid Peroxidation*

The lipid peroxidation analysis was based on the measurement of the MDA level according to Gondor et al. [36] using 0.5 g plant material. Leaves and roots were extracted with 1.5 mL 0.1% (*w*/*v*) tricholoracetic acid and, after incubation with 0.5% (*w*/*v*) thiobarbituric acid at 90 ◦C for 30 min, the MDA equivalent compounds were measured spectrophotometrically at 532 nm, with the subtraction of non-specific absorption at 600 nm. MDA was then quantified using an extinction coefficient of 155 mM−1·cm−<sup>1</sup> and expressed as nM·g−<sup>1</sup> fresh weight. Five replicates were measured from each treatment and at least three leaves were used for one replicate.

#### *4.3. Extraction and Analytical Procedure of Salicylic Acid and Flavonols*

Flavonoids, JA, ABA, SA and its precursors were measured according to Meuwly and Métraux [37] and Pál et al. [38] using 1 g plant material. The leaves and the roots were ground in liquid nitrogen in a mortar and pestle, in the presence of 0.5 g quartz sand. The tissue powder was transferred to a centrifugation tube and mixed with 2 mL of 70% methanol. The extract was centrifuged at 10,000× *g* for 20 min. The pellet was resuspended in 2 mL of 90% methanol, vortexed and centrifuged as above. The methanol content was evaporated from the mixed supernatants at room temperature under a vacuum. One millilitre of 5% (*w*/*v*) trichloroacetic acid was added to the residual aqueous phase, and the mixture was centrifuged at 15,000× *g* for 10 min. The supernatant was gently partitioned against 3.6 mL of a 1:1 (*v*/*v*) mixture of ethyl acetate/cyclohexane. The upper organic layers contained the free phenolic portion. The aqueous phases containing the methanol-soluble bound phenolics were acid-hydrolysed. 1.3 mL of 8 N HCl was added to the aqueous phase and incubated for 60 min at 80 ◦C, before partitioning as above. The organic phases were evaporated to dryness under a vacuum and stored at −20 ◦C. Just prior to the HPLC analysis, the evaporated samples were resuspended in 0.5 mL 15% acetonitrile and filtered through a 0.45 m pore size polytetrafluoroethylene (PTFE) membrane filter (Millipore, Merck Group, Darmstadt, Germany). The detailed description of the HPLC analysis was described by Janda et al. [39].

#### *4.4. Measurement of Thiols*

A total of 200 mg plant material was extracted with 1 mL of 0.1 M HCl and centrifuged for 20 min with 10,000 rpm. Total thiol content was determined after the supernatant was reduced (120 μL) with dithiothreitol, and derivatization was carried out with monobromobimane [40]. The samples were analysed after the separation of Cys, γEC, CysGly (a degradation product of GSH) and GSH by reverse-phase HPLC (W996, Waters, Milford, MA, USA) using a scanning fluorescence detector (W2474, Waters, Milford, MA, USA). The detailed parameters of the analysis were described by Janda et al. [39].

#### *4.5. Determination of Phenylalanine Ammonia Lyase (PAL) Activity*

PAL activity was measured according to Gao et al. [41] using 1 g leaves and roots homogenised with 4 mL 50 mM TRIS-HCl buffer (pH 8.8 containing 5 mM β–mercaptoethanol and 4% (*v*/*v*) polyvinylpyrrolidone). After centrifugation (10 min 10,000× *g*), 250 μL supernatant was used for enzyme activity determination in a 3 mL total volume of reaction mixture containing 50 mM phosphate buffer (pH 8.8) and 50 mM phenylalanine. The reaction was stopped with 10% trichloroacetic acid after a one-hour incubation at 37 ◦C and the produced *trans*-cinnamic acid was detected at 290 nm spectrophotometrically and expressed as enzyme units per g fresh weight (U g−<sup>1</sup> FW).

#### *4.6. Determination of Anthocyanin Content*

The method described by Kho et al. [42] was used for the anthocyanin content determination using 0.5 g plant tissue extracting with 2 × 300 μL of methanol containing 1% HCl and was kept at 4 ◦C for overnight. After removing the chlorophylls with 500 μL chloroform and centrifugation at 10,000× *g* for 30 min, the total anthocyanin content was determined from the supernatant at 530 nm spectrophotometrically.

#### *4.7. Statistical Analysis*

The experiments were repeated three times and representative data are shown. The results were the means of five measurements. The data were statistically evaluated using the standard deviation, ANOVA and *t*-test methods.

#### **5. Conclusions**

Different light conditions during the cold acclimation period differentially affected certain stress-related mechanisms in young maize plants (Figure 9). Interestingly, changes were also light dependent in the root, not only in the leaves. PAL has a key role in the synthesis of various secondary metabolic routes, including compounds with antioxidant activities, and was mainly activated under limited light conditions. However, light-induced anthocyanin accumulation occurred not only in the leaves, but in the roots, too. Chilling induced the accumulation of SA and one of its putative precursors, oHCA. However, this accumulation was moderate in the cold-acclimated plants. Low temperature reduced the accumulation of JA in the leaves, but induced it in the roots. The highest GSH level was found on the first day of the recovery period in the leaves of GL plants, but its pre-cursors started to accumulate even during the chilling. The level of ABA is mainly related to the level of stress, and less related to the level of the acclimation. According to these results, it was demonstrated that LL condition partly modifies the acclimation processes after only 1 day at the gene expression level, which, in turn, may result in hormonal and metabolic shifts, sometimes in different ways, in the leaves and roots of the plants.

**Figure 9.** Schematic representation of the effect of the light on phenolic metabolism in young maize plants during cold acclimation. JA: jasmonic acid; LL: low light intensity (107 μmol m−<sup>2</sup> s<sup>−</sup>1) during cold acclimation; ortho-hydroxycinnamic acid (oHCA); phenylalanine ammonia lyase (PAL).

**Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/6/1942/s1. Table S1: BLASTX search hits of cold- and light-induced significantly differentially expressed transcripts related to hormone biosynthesis. Table S2: BLASTX search hits of cold- and light-induced significantly differentially expressed transcripts related to thiol metabolism. Table S3: BLASTX search hits of cold- and light-induced significantly differentially expressed transcripts related to flavonoid metabolism.

**Author Contributions:** Conceptualization, G.S. and T.J.; methodology, M.P.; formal analysis, I.M.; investigation, G.S. and M.P.; resources, G.S.; writing—original draft preparation, G.S. and T.J.; writing—review and editing, M.P. and I.M.; visualization, G.S. and I.M; supervision, G.S.; project administration, G.S.; funding acquisition, G.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Research, Development and Innovation Office, Hungary, grant number K124430 and 2018-2.1.14-TÉT-CN2018-00010.

**Acknowledgments:** Thanks are due to Timea Oláh for her technical assistance.

**Conflicts of Interest:** There is no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**

