**1. Introduction**

Due to its subtropical origin, chilling is one of the most important factors limiting the spread and production of maize plants. Long-term exposure to temperatures around 10–15 ◦C may already decrease the capacity for biomass production. Lower chilling temperatures (0–5 ◦C) may lead to severe irreversible damage and the death of the plants [1,2]. Especially at continental climates, chilling tolerance at early stages of growth is a critical part of resistance to low temperature stress in maize plants.

Exposure of plants to low, but non-lethal, acclimating temperatures may increase their tolerance to a subsequent severe chilling stress [3–5]. To some extent, this is also true for cold-sensitive species, including maize. Better understanding the mechanisms that play a role in cold acclimation processes may help us to develop crop plants with higher levels of cold tolerance. It has been known for a long time that, without enough light during the cold hardening period, winter cereals—even winter cereals with a potentially high level of frost hardiness—are incapable of achieving a high level of freezing tolerance [6,7]. Acclimation to low temperatures also responds to light and temperature signals [8,9]. Light has been shown to mediate the development of freezing tolerance via several biological processes. These include photosynthesis-related processes, the expression level of stress-related genes and the synthesis of various protective compounds [10]. In the case of chilling sensitive plants, light during the cold period is mainly known as an extra stress factor inducing photoinhibition of photosynthesis [11]. As a consequence, photoinhibition may also contribute to the development of the chilling injury. However, we have recently shown that, in spite of the photoinhibitory effects, light during the cold acclimation period could also enhance the effectiveness of acclimation processes in young maize plants [12]. Similarly, moderate photoinhibition could also protect Photosystem I from photodamage under low temperature conditions in tobacco plants [13]. Furthermore, photoinhition of Photosystem I in *Arabidopsis* also protected the chloroplasts from oxidative damage [14].

It seems that light is at least as important factor as the temperature during the cold acclimation period. During the exposure of maize plants to cold acclimating temperatures, light influenced various light-related cold acclimation processes not only directly, but also at the gene expression and metabolomics levels. A microarray study showed that complex regulation mechanisms and interactions between cold and light signaling processes exist during the acclimation period. Numerous significantly differentially expressed genes that are involved in most of the assimilation and metabolic pathways were detected [12]. However, the exact mechanisms regarding how light may regulate the cold acclimation processes are still poorly understood.

Plants often react to biotic or abiotic stresses with an increase in the secondary metabolite levels. In relation to this, the increased activity of phenylalanine ammonia lyase (PAL) and other related enzymes can be observed. Phenolic compounds are naturally occurring substances in plants, and many of them play important roles in defence mechanisms and the scavenging of oxidizing molecules [15]. Salicylic acid (SA) is also a phenolic compound, and it plays an important signalling role in plants in various abiotic and biotic stresses [16,17]. Among other effects, SA may also induce the production of plant defensive metabolites, including other phenolic compounds and antioxidant systems [18,19].

In the present work, based on our previous microarray experiment [12], we further analysed the differentially expressed genes, focusing on the phenylpropanoid pathways. Furthermore, the changes in certain phenolic compounds, and other plant growth regulators, such as plant hormones and thiol compounds were analysed, in order to reveal their possible role in light-regulating signalling during the cold acclimation processes in young maize plants.

#### **2. Results**

#### *2.1. Plant Hormones*

Growing young maize plants at cold acclimating temperatures (15 ◦C) did not cause significant changes in the free SA contents in the leaves compared to the control plants (Figure 1A). However, SA increased in a high manner during chilling at 5 ◦C in the non-acclimated plants, and it remained at this high level during the recovery period. Plants acclimated at low light (LL) had slightly higher free SA levels during recovery than the cold-acclimated one at growth light (GL). The bound SA level elevated only during recovery and only in non-acclimated plants (Figure 1B). While the bound SA was usually higher than the free SA in the leaves, this difference was less pronounced in the roots (Figure 1C,D). However, a substantial increase in the SA level could be detected in the free SA in the roots after a one-day recovery period. After a longer recovery, of 4 days or more, only low SA levels could be detected. The bound SA levels after the acclimation and chilling periods were significantly lower in the LL plants than in the control.

**Figure 1.** Changes in the salicylic acid (SA) contents during cold acclimation (15/13 ◦C), chilling (5 ◦C) and recovery in the leaves and roots of young maize plans: control plants (22/20 ◦C, 387 μmol m−<sup>2</sup> s<sup>−</sup>1). Light intensities during hardening: growth light (GL): 387μmol m−<sup>2</sup> s<sup>−</sup>1; low light (LL): 107μmol m−<sup>2</sup> s<sup>−</sup>1. \*, \*\*, \*\*\* significant differences compared to the control plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. **#**, **##**, **###** significant differences compared to the GL plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. (**A**: free SA in the leaves; **B**: bound SA in the leaves; **C**: free SA in the roots; **D**: bound SA in the roots).

The level of ortho-hydroxycinnamic acid (oHCA), a putative pre-cursor of SA, also increased during acclimation and chilling in the leaves and dropped back to the initial level during recovery (Figure 2A). A much higher increase was detected in non-acclimated plants at 5 ◦C and on the first day of recovery, but it also dropped later. No significant changes were detected in the roots, but the oHCA level was twice as high than in the leaves (Figure 2B).

The level of abscisic acid (ABA) did not change during the acclimation in the leaves of GL plants and it remained at the same level during the chilling and recovery period (Figure 3A). A slight increase could be detected in LL plants during the acclimation, but it was much more pronounced after 1d recovery. The ABA level of non-acclimated plants increased only during the recovery, especially on the first day, but it returned to the initial level in all the treatments on the seventh day. No changes were measured during acclimation in the roots (Figure 3B). Although the chilling increased the ABA level, no difference could be observed between the acclimated and non-acclimated plants. A slight increase in ABA level was determined in non-acclimated and GL plants during recovery, which dropped back at the end of recovery.

The jasmonic acid (JA) level did not change in the leaves during acclimation, but chilling significantly increased it in the cold-acclimated GL plants and especially in the non-acclimated ones (Figure 4A). On the first day of recovery, the JA level dropped back to the initial level only in the acclimated plants, but from the fourth day its level was sometimes even less than before the acclimation period. No effect of the light could be observed on the amount of JA during the acclimation, chilling and recovery periods. On the contrary, a slight elevation could be seen in the roots acclimated under GL conditions, but no changes in the LL plants (Figure 4B). The JA level in the roots increased during the chilling in non-acclimated plants, and more pronouncedly in GL, while no changes could be seen in LL plants. Interestingly, some rise could be observed in non-acclimated and LL plants on the first day of recovery, but a decrease in GL roots. On the seventh day of recovery, the JA level was similar to the initial one.

**Figure 2.** Changes in the ortho-hydroxycinnamic acid (oHCA) contents during cold acclimation (15/13 ◦C), chilling (5 ◦C) and recovery in the leaves and roots of young maize plants: control plants (22/20 ◦C, 387 μmol m−<sup>2</sup> s<sup>−</sup>1). Light intensities during hardening: GL: 387 μmol m−<sup>2</sup> s<sup>−</sup>1; LL: 107 μmol m−<sup>2</sup> s<sup>−</sup>1. \*, \*\*, \*\*\* significant differences compared to the control plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. **#**, **##** significant differences compared to the GL plants on the same day at the *p* < 0.05, and 0.01 levels, respectively. (**A**: free oHCA in the leaves; **B**: free oHCA in the roots).

**Figure 3.** Changes in the abscisic acid (ABA) contents during cold acclimation (15/13 ◦C), chilling (5 ◦C) and recovery in the leaves and roots of young maize plans: control plants (22/20 ◦C, 387 μmol m−<sup>2</sup> s<sup>−</sup>1). Light intensities during hardening: GL: 387 μmol m−<sup>2</sup> s<sup>−</sup>1; LL: 107 μmol m−<sup>2</sup> s<sup>−</sup>1. \*, \*\*, \*\*\* significant differences compared to the control plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. **##**, **###** significant differences compared to the GL plants on the same day at the *p* < 0.05 and 0.001 levels, respectively. (**A**: free ABA in the leaves; **B**: free ABA in the roots).

**Figure 4.** Changes in the jasmonic acid (JA) contents during cold acclimation (15/13 ◦C), chilling (5 ◦C) and recovery in the leaves and roots of young maize plans: control plants (22/20 ◦C, 387 μmol m−<sup>2</sup> s<sup>−</sup>1). Light intensities during hardening: GL: 387 μmol m−<sup>2</sup> s<sup>−</sup>1; LL: 107 μmol m−<sup>2</sup> s<sup>−</sup>1. \*, \*\*, \*\*\* significant differences compared to the control plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. **#**, **###** significant differences compared to the GL plants on the same day at the *p* < 0.05 and 0.001 levels, respectively. (**A**: free JA in the leaves; **B**: free JA in the roots).

### *2.2. Oxidative Stress and Antioxidants*

For detection of the oxidative stress, the malondialdehyde (MDA) level was measured in the leaves and roots of plants during acclimation, chilling and recovery. No changes were detected in the leaves during the acclimation and chilling periods, and only a slight increase was detected in the leaves of hardened plants on the fourth day of recovery (Figure 5A). However, the MDA level increased significantly in the roots of GL plants (Figure 5B).

**Figure 5.** Changes in the malondialdehyde (MDA) contents during cold acclimation (15/13 ◦C), chilling (5 ◦C) and recovery in the leaves and roots of young maize plans: control plants (22/20 ◦C, 387 μmol m−<sup>2</sup> s<sup>−</sup>1). Light intensities during hardening: GL: 387 μmol m−<sup>2</sup> s<sup>−</sup>1; LL: 107 μmol m−<sup>2</sup> s<sup>−</sup>1. \*, \*\*, \*\*\* significant differences compared to the control plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. **#**, **##**, **###** significant differences compared to the GL plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. (**A**: leaves; **B**: roots).

Thiols, especially glutathione (γ-L-glutamyl-L-cysteinyl–glycine (GSH)), have an important role to play in the defence processes against oxidative stress. Thus, thiol compounds, namely GSH and its precursors, cysteine (Cys) and γ-L-glutamyl-L-cysteine (γEC) were also analysed. Cys levels increased after chilling in the leaves of GL plants and was still higher on the first day of recovery (Figure 6A). Similar tendencies could be seen in the amount of γEC (Figure 6B). Compared to these, the GSH level increased in LL plants after acclimation and decreased during the chilling (Figure 6C), and a dramatic rise was observed on the first day of recovery, both in GL and LL plants. Cysteinyl–glycine (CysGly), a degradation product of GSH, had the highest amount after the chilling in GL plants, but dropped back to the initial level during the recovery (Figure 6D). Cys increased after acclimation in the roots of LL plants, but after chilling it was at the initial level (Figure 6E). An increased level of it was detected in non-acclimated plants after chilling and it was still high on the first day of recovery. The GL plants had elevated levels of root Cys on the first and fourth days of recovery, while root γEC increased only in LL plants after chilling and it was also higher on the fourth and seventh days of recovery (Figure 6F). Elevated levels could be also seen in the non-acclimated and GL plants during recovery, but this level was still lower than in LL plants. A big enlargement in the GSH amount was measured in LL plants after acclimation, but after the chilling, and during recovery, the initial level was detected in the roots of the plants (Figure 6G). The amount of CysGly increased during acclimation and remained at the same level in LL plants after chilling and during the recovery (Figure 6H). It dropped to the initial level in GL plants after chilling, but it started to increase during the recovery, and the highest level was detected after seven days.

**Figure 6.** Changes in the thiol contents during cold acclimation (15/13 ◦C), chilling (5 ◦C) and recovery in the leaves and roots of young maize plants. Control plants (22/20 ◦C, 387 μmol m−<sup>2</sup> s<sup>−</sup>1). Light intensities during hardening: GL: 387 μmol m−<sup>2</sup> s<sup>−</sup>1; LL: 107 μmol m−<sup>2</sup> s<sup>−</sup>1. \*, \*\*, \*\*\* significant differences compared to the control plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. **#**, **##**, **###** significant differences compared to the GL plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. (**A**: cysteine in the leaves; **B**: γ-glutamyl-cysteine in the leaves; **C**: gluthathione in the leaves; **D**: cysteinyl–glycine in the leaves; **E**: cysteine in the roots; **F**: γ-glutamyl-cysteine in the roots; **G**: gluthathione in the roots; **H**: cysteinyl–glycine in the roots).

PAL is a key enzyme of the phenylpropanoid metabolism. It has a role either in the SA or flavonol/anthocyanin biosynthesis. The PAL activity increased in the leaves of LL plants after acclimation, but showed a much higher amount on the first day of recovery (Figure 7A). Its activity was higher in the roots than in the leaves and an increase could be seen after acclimation, chilling and 1 d recovery, mainly in LL plants (Figure 7B).

**Figure 7.** Changes in the phenylalanine ammonia lyase (PAL) contents during cold acclimation (15/13 ◦C), chilling (5 ◦C) and recovery in the leaves and roots of young maize plans: control plants (22/20 ◦C, 387 μmol m−<sup>2</sup> s<sup>−</sup>1). Light intensities during hardening: GL: 387 μmol m−<sup>2</sup> s<sup>−</sup>1; LL: 107 μmol m−<sup>2</sup> s<sup>−</sup>1. \*, \*\*, \*\*\* significant differences compared to the control plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. **#**, **##** significant differences compared to the GL plants on the same day at the *p* < 0.05 and 0.01 levels, respectively. (**A**: leaves; **B**: roots).

Flavonols (kaempferol (K), quercetin (Q), myricetin (M) and rutin (R)) and anthocyanins were also analysed. K, Q and M were in a much lower amount (0.1–2 μg g−<sup>1</sup> FW) both in the leaves and roots than rutin (100–130 μg g−<sup>1</sup> FW). Only the R level increased in the leaves, especially in LL plants, but after 4 d recovery it was lower than the initial level (Table 1). There were no substantial changes in the roots, only the M increased at 3 d and at 15 ◦C in GL and LL plants (Table 1).


*Int. J. Mol. Sci.* **2020**, *21*, 1942

**Table 1.** Changes in the flavonol contents during cold acclimation

22/20 ◦C, 387 μmol m−2 s−1. NA:

 (15/13 ◦C), chilling (5 ◦C) and recovery in the leaves and roots of young maize plants. Control:

 GL: 387 μmol m−2 s−1; LL: 107 μmol m−2 s−1. \*, \*\*, \*\*\* significant

non-acclimated

 plants. Light intensities during hardening:

Anthocyanins could only be detected during the recovery period. It accumulated mainly in the roots of GL plants but changes in the leaves were also observed (Figure 8). The highest amount was detected on the fourth day of recovery, but it was still high on the seventh day in the acclimated plants.

**Figure 8.** Changes in the anthocyanin contents during cold acclimation (15/13 ◦C), chilling (5 ◦C) and recovery in the leaves and roots of young maize plants. Light intensities during hardening: GL: 387 μmol m−<sup>2</sup> s<sup>−</sup>1; LL: 107 μmol m−<sup>2</sup> s<sup>−</sup>1. \*, \*\*, \*\*\* significant differences compared to the control plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively. **#**, **##**, **###** significant differences compared to the GL plants on the same day at the *p* < 0.05, 0.01 and 0.001 levels, respectively.
