Characteristic of the Ascorbate Oxidase Gene Family in Beta vulgaris and Analysis of the Role of AAO in Response to Salinity and Drought in Beet

Ascorbate oxidase, which is known to play a key role in regulating the redox state in the apoplast, cell wall metabolism, cell expansion and abiotic stress response in plants, oxidizes apo-plastic ascorbic acid (AA) to dehydroascorbic acid (DHA). However, there is little information about the AAO genes and their functions in beets under abiotic stress. The term salt or drought stress refers to the treatment of plants with slow and gradual salinity/drought. Contrastingly, salt shock consists of exposing plants to high salt levels instantaneously and drought shock occurs under fast drought progression. In the present work, we have subjected plants to salinity or drought treatments to elicit either stress or shock and carried out a genome-wide analysis of ascorbate oxidase (AAO) genes in sugar beet (B. vulgaris cv. Huzar) and its halophytic ancestor (B. maritima). Here, conserved domain analyses showed the existence of twelve BvAAO gene family members in the genome of sugar beet. The BvAAO_1–12 genes are located on chromosomes 4, 5, 6, 8 and 9. The phylogenetic tree exhibited the close relationships between BvAAO_1-12 and AAO genes of Spinacia oleracea and Chenopodium quinoa. In both beet genotypes, downregulation of AAO gene expression with the duration of salt stress or drought treatment was observed. This correlated with a decrease in AAO enzyme activity under defined experimental setup. Under salinity, the key downregulated gene was BvAAO_10 in Beta maritima and under drought the BvAAO_3 gene in both beets. This phenomenon may be involved in determining the high tolerance of beet to salinity and drought.


Introduction
Ascorbate oxidase (AAO) is an enzyme belonging to the blue copper oxidase family and regulates the redox state of the ascorbate pool in apoplasts [1,2]. AAOs are localized in the cell wall [3] and participate in the oxidation of apo-plastic ascorbate (AA) to monodehydroascorbic acid (MDHA), which is converted to dehydroascorbic acid (DHA), and then AA is generated by DHARs in the cytoplasm. The AA exchange between apoplast and cytoplasm is involved in the maintenance of redox homeostasis [1,4]. Although the AAO may be considered disadvantageous due to its role in the consumption of ascorbate levels [5], this enzyme has been found to play an important role in cell growth [5], cell wall metabolism [6] and stress responses [3,7,8]. Under abiotic stress conditions, AA is quickly oxidized to DHA and the redox state in the apoplast and cytoplasm is impaired, regulating in stress responses through ABA synthesis and H 2 O 2 accumulation [4,9]. Contrary to ascorbate peroxidases (APXs) that use AA to scavenge ROS and decrease oxidative stress, AAOs induce ROS production/synthesis in the apoplast. Thus, overexpression of the AAO [37][38][39][40]. Hence, understanding the abiotic stress response mechanisms at the molecular level for this crop is required. Recently, it has been demonstrated that epigenetic marks, including DNA methylation and histone modifications, play a crucial role in the regulation of gene expression in response to abiotic stress [41,42]. DNA cytosine methylation is a transcriptional switch, controlling the activity of effector genes [43]. It is correlated with the repression of gene expression. DNA methylation was altered in different regions of stress-responsive genes in Arabidopsis [44], soybean [45], rice [46] and tobacco [47], etc. In a recent study, salt stress brought about the alterations in DNA methylation levels of gene promoters and first exons of salt-inducible genes in sugar beet and sea beet [42]. DNA methylation levels were reduced under salt stress and shock in B. vulgaris, but increased in response to salt stress in B. maritima [42]. These findings suggest the involvement of DNA methylation in salt-tolerance and gene expression in beets under salt stress. However, there is little information about epigenetic regulation of abiotic stress response in sugar beet and Beta maritima [23,42,48]. Hence, in this paper, we have detected DNA methylation patterns along with AAO transcript levels under salt/drought stress and shock treatments.
The aim of the study was to characterize AAO genes in Beta vulgaris and to analyze the probable role of AAO in response to salinity and drought in Beta vulgaris cv. Huzar and Beta maritima. Firstly, AAO genes were identified in B. vulgaris and their chromosomal positions and conserved domain protein family were investigated. A phylogenetic tree and phylogram were constructed using AAO amino acid sequences from B. vulgaris, Spinacia oleracea and Chenopodium quinoa. In addition, the transcript levels of AAO genes BvAAO_3, BvAAO_4 and BvAAO_10 in leaves were determined under stress and shock. Moreover, DNA methylation patterns in CpG islands localized in promoter regions of BvAAO_3, BvAAO_4 and BvAAO_10 genes were compared in B. vulgaris and B. maritima after 3, 7 and 11 days of stress and shock. Furthermore, the concentration of total ascorbate (ASC) and the activity of the AAO enzyme were determined. This is the first study aimed at investigating the characteristics of AAO members in Beta vulgaris. The research results summarized in this study might contribute to better understanding the responses of AAO genes to salt and drought stress in sugar beet and its wild ancestor, Beta maritima.

Characteristics of Genes and Proteins of Beta Vulgaris Ascorbate Oxidases
Analysis of the RefBeet-1.2.2 beet reference genome deposited with GenBank allowed the identification of 12 ascorbate oxidase genes located on chromosomes 4, 5, 6, 8 and 9 ( Figure 1). The two genes encoding this enzyme have not been assigned a chromosomal location. The exact characteristics of individual genes and the proteins they encode are presented in Table 1. For the purposes of this study, the genes were marked with consecutive numbers that follow the abbreviation of the enzyme name.   Figure 2 shows the structure of individual beet AAO genes. They differ significantly in the number and length of exons and introns. The BvAAO_8 and BvAAO_9 genes are the longest. The shortest were BvAAO_2 and BvAAO_5. Figure 2 shows the structure of individual beet AAO genes. They differ significantly in the number and length of exons and introns. The BvAAO_8 and BvAAO_9 genes are the longest. The shortest were BvAAO_2 and BvAAO_5. The proteins encoded by the AAO genes are composed of 537-588 amino acids (Table  1). Their molecular weight falls within a range of 59.68-65.9 kDa and the isoelectric point is between 6.68-9.84. According to computer analysis, the signal peptide is detected in all proteins except BvAAO_9. According to the outcome of Plant-mPLoc analysis, all beet AAO proteins are located in the cell membrane. In turn, analyzes using the YLoc program indicated the cytoplasmic localization of the BvAAO_2, BvAAO_4, BvAAO_5, BvAAO_6, BvAAO_8, BvAAO_9 and BvAAO_10 proteins; peroxisome localization in the case of BvAAO_1, BvAAO_3. The BvAAO_7 and BvAAO_11 proteins were identified by the program in the extracellular space and the BvAAO_12 protein was characterized by the secretory pathway (Table 1).
Conserved protein domains of cupredoxins have been detected in beet AAO proteins. These domains contain copper centers and are involved in intermolecular electron transfer reactions. Depending on the copper-binding site, there are types I, II and III of such domains (CuRo_1_AAO_like_1; CuRo_2_AAO_like_1; CuRo_3_AAO_like_1). The domain of the cupredoxin superfamily (Cupredoxin) also has a type 1 copper center. Although the AAO proteins may have different functions, 3 cupredoxin domains were detected in each of them. The presence of the different types of domains has an equal configuration for the individual proteins ( Figure 2).

Phylogenetic and Evolutionary Analyses of AAO Gene Family
A phylogenetic tree was constructed based on the AAO Beta vulgaris protein sequences and according to the highest alignment index of the AAO protein sequences of Spinacia oleracea and Chenopodium quinoa. Based on the analysis of the reference genomes of these species, 13 spinach ascorbate oxidase sequences and 23 Chenopodium quinoa sequences were compared. Analyzing the evolutionary similarity of the beet AAO sequence, it can be noticed that the BvAAO_9 protein is evolutionarily most distant from the rest of the proteins. In addition, no close structural similarity to any of the AAO proteins of spinach and quinoa was demonstrated. Taking into account the next phylogenetic branch, we can distinguish here known groups, i.e., BvAAO_5 and the rest of the beet AAO proteins. Noticeable here also is the closer similarity of BvAAO_5 to the spinach protein sequence XP_021839772.1 and then to the two quinoa sequences XP_021752252.1 and XP_021714136.1. On the subsequent branches of the tree, individual beet AAO proteins are presented, which have their counterparts among the ascorbic oxidase proteins in the two plant species analyzed ( Figure 3). The proteins encoded by the AAO genes are composed of 537-588 amino acids (Table 1). Their molecular weight falls within a range of 59.68-65.9 kDa and the isoelectric point is between 6.68-9.84. According to computer analysis, the signal peptide is detected in all proteins except BvAAO_9. According to the outcome of Plant-mPLoc analysis, all beet AAO proteins are located in the cell membrane. In turn, analyzes using the YLoc program indicated the cytoplasmic localization of the BvAAO_2, BvAAO_4, BvAAO_5, BvAAO_6, BvAAO_8, BvAAO_9 and BvAAO_10 proteins; peroxisome localization in the case of BvAAO_1, BvAAO_3. The BvAAO_7 and BvAAO_11 proteins were identified by the program in the extracellular space and the BvAAO_12 protein was characterized by the secretory pathway (Table 1).
Conserved protein domains of cupredoxins have been detected in beet AAO proteins. These domains contain copper centers and are involved in intermolecular electron transfer reactions. Depending on the copper-binding site, there are types I, II and III of such domains (CuRo_1_AAO_like_1; CuRo_2_AAO_like_1; CuRo_3_AAO_like_1). The domain of the cupredoxin superfamily (Cupredoxin) also has a type 1 copper center. Although the AAO proteins may have different functions, 3 cupredoxin domains were detected in each of them. The presence of the different types of domains has an equal configuration for the individual proteins ( Figure 2).

Phylogenetic and Evolutionary Analyses of AAO Gene Family
A phylogenetic tree was constructed based on the AAO Beta vulgaris protein sequences and according to the highest alignment index of the AAO protein sequences of Spinacia oleracea and Chenopodium quinoa. Based on the analysis of the reference genomes of these species, 13 spinach ascorbate oxidase sequences and 23 Chenopodium quinoa sequences were compared. Analyzing the evolutionary similarity of the beet AAO sequence, it can be noticed that the BvAAO_9 protein is evolutionarily most distant from the rest of the proteins. In addition, no close structural similarity to any of the AAO proteins of spinach and quinoa was demonstrated. Taking into account the next phylogenetic branch, we can distinguish here known groups, i.e., BvAAO_5 and the rest of the beet AAO proteins. Noticeable here also is the closer similarity of BvAAO_5 to the spinach protein sequence XP_021839772.1 and then to the two quinoa sequences XP_021752252.1 and XP_021714136.1. On the subsequent branches of the tree, individual beet AAO proteins are presented, which have their counterparts among the ascorbic oxidase proteins in the two plant species analyzed ( Figure 3). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 23

Analysis of the AAO Beta Vulgaris Gene Expression Profile under Drought and Salinity Conditions
The expression level of AAO genes was determined in salt/drought shocked/stressed leaves of Beta vulgaris cv. Huzar and Beta maritima. Untreated controls were analyzed in line with the treated plants. Since only the BvAAO_3, BvAAO_4 and BvAAO_10 ascorbate

Analysis of the AAO Beta vulgaris Gene Expression Profile under Drought and Salinity Conditions
The expression level of AAO genes was determined in salt/drought shocked/stressed leaves of Beta vulgaris cv. Huzar and Beta maritima. Untreated controls were analyzed in line with the treated plants. Since only the BvAAO_3, BvAAO_4 and BvAAO_10 ascor-bate oxidase genes were transcriptionally active in the analyzed tissue, the results of the expression analyzes are presented only for these genes.
When compared to the treatment starting day ("day 1") a downregulation of BvAAO_3 expression was observed, in the control, shock or salt stress treated Beta maritima leaves, the reduced expression level maintained over the treatment period. There were no significant differences in BvAAO_3 expression levels between the control samples and those from plants treated with stress and salt shock. On the 3rd day, in the leaves of Beta vulgaris cv. Huzar BvAAO_3 expression was observed to be downregulated in the control and salt shock treated samples relative to day 1. In turn, the expression of BvAAO_3 in the samples treated with salt stress was upregulated ( Figure 4). On the 5th day, the opposite effect was observed, i.e., an increase in BvAAO_3 expression in control plants and those representing salt shock and a significant decrease for the salt stress variant. This downregulation persisted until the 7th day of the treatment, after which an increase in the level of expression was observed until the end of the experiment. In the controls on the 7th day, there was a decrease in the expression level of BvAAO_3, which remained at a similar level on the 9th and 11th days. In the case of the samples representing salt shock, the upregulation of expression of this gene on the 5th day of treatment was maintained until the end of the experiment. oxidase genes were transcriptionally active in the analyzed tissue, the results of the expression analyzes are presented only for these genes. When compared to the treatment starting day ("day 1") a downregulation of BvAAO_3 expression was observed, in the control, shock or salt stress treated Beta maritima leaves, the reduced expression level maintained over the treatment period. There were no significant differences in BvAAO_3 expression levels between the control samples and those from plants treated with stress and salt shock. On the 3rd day, in the leaves of Beta vulgaris cv. Huzar BvAAO_3 expression was observed to be downregulated in the control and salt shock treated samples relative to day 1. In turn, the expression of BvAAO_3 in the samples treated with salt stress was upregulated ( Figure 4). On the 5th day, the opposite effect was observed, i.e., an increase in BvAAO_3 expression in control plants and those representing salt shock and a significant decrease for the salt stress variant. This downregulation persisted until the 7th day of the treatment, after which an increase in the level of expression was observed until the end of the experiment. In the controls on the 7th day, there was a decrease in the expression level of BvAAO_3, which remained at a similar level on the 9th and 11th days. In the case of the samples representing salt shock, the upregulation of expression of this gene on the 5th day of treatment was maintained until the end of the experiment.  Control−light gray lines, salt shock−gray lines, salt stress−dark gray lines. Different letters denote significant differences at p < 0.01 (ANOVA followed by Tukey's test). "Whiskers" indicate standard deviation.
When the plants were treated with drought, the expression profile of the BvAAO_3 gene was different. In control samples, the transcript level in the two beet species was similar and did not change much. On the other hand, in the samples representing plants treated with shock and drought stress, a significant downregulation of BvAAO_3 expression was observed, as compared to the controls, at each sampling point ( Figure 5).
When the plants were treated with drought, the expression profile of the BvAAO_3 gene was different. In control samples, the transcript level in the two beet species was similar and did not change much. On the other hand, in the samples representing plants treated with shock and drought stress, a significant downregulation of BvAAO_3 expression was observed, as compared to the controls, at each sampling point ( Figure 5).

Figure 5.
Expression profiles of AAO genes BvAAO_3, BvAAO_4 and BvAAO_10 determined in leaves of Beta vulgaris ssp. maritima and Beta vulgaris cv. Huzar subjected to drought shock or stress. Control−light gray lines, drought shock−gray lines, drought stress−dark gray lines. Different letters denote significant differences at p < 0.01 (ANOVA followed by Tukey's test). "Whiskers" indicate standard deviation.
Analyzing the effect of salinity, the expression of the BvAAO_4 gene in Beta maritima leaves was downregulated, relative to "day 1", at all time-points of the experiment for the samples representing both the salt shock and salt stress treatment. The lowest transcript level was observed on the 11th day of the treatment. In the samples coming from control plants, the effect was similar except on day 9 when a slight increase in expression was observed. In leaves of Beta vulgaris cv. Huzar BvAAO_4 expression level varied depending on the treatment day and variant of the experiment. Relative to "day 1", downregulation of this gene expression was observed on the 3rd, 7th and 11th day of the experiment, while on the 5th and 9th day a significant upregulation was observed in the control leaves. A similar profile of changes was observed under the salt shock treatment, except on the 7th day, in which the increase in the level of the BvAAO_4 transcript was maintained. In the case of samples representing salt stress on the 5th day, a decrease in the expression of this Control−light gray lines, drought shock−gray lines, drought stress−dark gray lines. Different letters denote significant differences at p < 0.01 (ANOVA followed by Tukey's test). "Whiskers" indicate standard deviation.
Analyzing the effect of salinity, the expression of the BvAAO_4 gene in Beta maritima leaves was downregulated, relative to "day 1", at all time-points of the experiment for the samples representing both the salt shock and salt stress treatment. The lowest transcript level was observed on the 11th day of the treatment. In the samples coming from control plants, the effect was similar except on day 9 when a slight increase in expression was observed. In leaves of Beta vulgaris cv. Huzar BvAAO_4 expression level varied depending on the treatment day and variant of the experiment. Relative to "day 1", downregulation of this gene expression was observed on the 3rd, 7th and 11th day of the experiment, while on the 5th and 9th day a significant upregulation was observed in the control leaves. A similar profile of changes was observed under the salt shock treatment, except on the 7th day, in which the increase in the level of the BvAAO_4 transcript was maintained. In the case of samples representing salt stress on the 5th day, a decrease in the expression of this gene was observed, while on the remaining days of treatment a significant increase occurred ( Figure 4).
Treating Beta maritima plants with drought, except for the stress variant on the 3rd day, brought about a decrease in the expression of the BvAAO_4 gene, observed in all experimental variants and on all treatment days. In leaves of Beta vulgaris cv. Huzar, the decrease in BvAAO_4 expression level was also observed in the stressed plants. The samples representing control plants showed unchanging transcript levels, except on the 11th day, when a slight increase was detected. Under drought shock, BvAAO_4 levels were similar to that observed in the stress variant on the 3rd, 5th and 7th days, but turned on the 11th day to the control level ( Figure 5).
Taking drought into account, the expression level of BvAAO_10 in Beta maritima did not change significantly, as compared to "day 1", in the samples representing control plants and those treated with salt shock up to the 7th day of the experiment. On the 9th and 11th days of treatment, a significant decrease in salt-shocked plants was observed. In contrast to the remaining variants, significant decreases in the level of the BvAAO_10 transcript were observed in plants treated with salt stress on each measurement day. In B. vulgaris cv. Huzar expression level of BvAAO_10 did not change compared to the control in the case of salt shock and on the 3rd, 9th and 11th days in the rest of the experimental variants. Significant downregulation of BvAAO_10 expression occurred on the 5th and 7th day of the experiment in control plants and plants treated with salt stress (Figure 4). In the drought testing experiment, the expression level of BvAAO_10 remained at a similar level compared to the control in all experimental variants in both beet genotypes ( Figure 5).  Analyzing the effect of salinity on the methylation level of the BvAAO_3 gene CpG island in Beta maritima on day 3 of treatment, it was revealed that in the control plants methylation level was above 63%, whereas on the 7th and 11th days the methylation level decreased to about 50%. In the case of plants treated with salt shock, the methylation level of the CpG BvAAO_3 island was about 49%, on the 7th treatment day it decreased to 28% and on the 11th day it increased to 56%. In plants treated with salt stress, on the 3 rd and 7th day of the experiment the methylation level was at the level of 76-78% and then on the 11th day of treatment it decreased to the level of the other variants. In Beta vulgaris cv. Huzar, on the 3rd day of treatment the level of CpG BvAAO_3 island methylation was Analyzing the effect of salinity on the methylation level of the BvAAO_3 gene CpG island in Beta maritima on day 3 of treatment, it was revealed that in the control plants methylation level was above 63%, whereas on the 7th and 11th days the methylation level decreased to about 50%. In the case of plants treated with salt shock, the methylation level of the CpG BvAAO_3 island was about 49%, on the 7th treatment day it decreased to 28% and on the 11th day it increased to 56%. In plants treated with salt stress, on the 3rd and 7th day of the experiment the methylation level was at the level of 76-78% and then on the 11th day of treatment it decreased to the level of the other variants. In Beta vulgaris cv. Huzar, on the 3rd day of treatment the level of CpG BvAAO_3 island methylation was 53% in the control plants, about 98% in shock-treated plants and 18% in salt-stressed. In the following days, the levels of methylation reached almost 100% in all variants of the experiment (Figure 7). In drought-treated Beta maritima plants, the level of methylation of the CpG BvAAO_3 island, measured on the 3rd day of the experiment was close to 100% in the control and drought-shock variants, while in the case of plants subjected to drought stress, it was 35%. On the 7th day of treatment, the methylation level was close to 100% in all experimental variants. It persisted on day 11 in plants treated with drought shock and stress. Contrastingly, in control plants, it decreased to the level of 14%. In Beta vulgaris cv. Huzar the methylation level of the CpG BvAAO_3 island was close to 100% on all measurement days and in all experimental variants (Figure 8). In drought-treated Beta maritima plants, the level of methylation of the CpG BvAAO_3 island, measured on the 3rd day of the experiment was close to 100% in the control and drought-shock variants, while in the case of plants subjected to drought stress, it was 35%. On the 7th day of treatment, the methylation level was close to 100% in all experimental variants. It persisted on day 11 in plants treated with drought shock and stress. Contrastingly, in control plants, it decreased to the level of 14%. In Beta vulgaris cv. Huzar the methylation level of the CpG BvAAO_3 island was close to 100% on all measurement days and in all experimental variants (Figure 8). The methylation levels of the CpG island of BvAAO_4 in Beta maritima on day 3 amounted to about 31% in the control plants and about 24-25% in the plants treated with salt shock or stress. On the 7th day of the experiment, the methylation level increased to about 50-55% in all experimental variants. This level was maintained on day 11 in the saltshock treated variant. In turn, in control plants, it decreased to the level of 13%, but in those treated with salt stress it increased to the level of 65%. In Beta vulgaris cv. Huzar observed a high percentage of methylation of the CpG BvAAO_3 island on the 7th and 11th day of treatment and on the 3rd day in the salt-shocked variant. In the remaining variants, on the 3rd day, the methylation amounted to 50% (Figure 7).

Analysis of the Methylation Level of the CpG Islands of the Promoter Regions of the AAO Beta vulgaris Genes under Drought and Salinity Conditions
In an experiment aimed at analyzing the effect of drought on Beta maritima, the methylation level of the CpG island BvAAO_4 in the control plants was close to 100% on the 3rd and 7th days of treatment, but it decreased to about 12% on the 11th day. In plants treated with salt shock, the methylation level on the 3rd day of the experiment was about 75% and increased to nearly 100% on the 7th and 11th days. Similarly, in the droughtstressed variant it was 21% on the 3rd day of the experiment and increased to nearly 100% in the following days of treatment. In Beta vulgaris cv. Huzar, the percentage of methylation of CpG BvAAO_4 was very high in all experiment variants and all measurement times (Figure 8). The methylation levels of the CpG island of BvAAO_4 in Beta maritima on day 3 amounted to about 31% in the control plants and about 24-25% in the plants treated with salt shock or stress. On the 7th day of the experiment, the methylation level increased to about 50-55% in all experimental variants. This level was maintained on day 11 in the salt-shock treated variant. In turn, in control plants, it decreased to the level of 13%, but in those treated with salt stress it increased to the level of 65%. In Beta vulgaris cv. Huzar observed a high percentage of methylation of the CpG BvAAO_3 island on the 7th and 11th day of treatment and on the 3rd day in the salt-shocked variant. In the remaining variants, on the 3rd day, the methylation amounted to 50% (Figure 7).
In an experiment aimed at analyzing the effect of drought on Beta maritima, the methylation level of the CpG island BvAAO_4 in the control plants was close to 100% on the 3rd and 7th days of treatment, but it decreased to about 12% on the 11th day. In plants treated with salt shock, the methylation level on the 3rd day of the experiment was about 75% and increased to nearly 100% on the 7th and 11th days. Similarly, in the droughtstressed variant it was 21% on the 3rd day of the experiment and increased to nearly 100% in the following days of treatment. In Beta vulgaris cv. Huzar, the percentage of methylation of CpG BvAAO_4 was very high in all experiment variants and all measurement times ( Figure 8).
The level of CpG island methylation BvAAO_10 in Beta maritima, on day 3 of treatment, was about 50-56% in the control and salt shock-treated plants and about 15% in the salt stress-treated plants. In the case of the first two variants, methylation levels decreased slightly on day 7 of the experiment and increased significantly on day 11. In plants treated with salt stress, the level of CpG island methylation in the BvAAO_10 gene increased with the duration of the experiment, to 62% and 84%, respectively. In the control plants, Beta vulgaris cv. Huzar on the 3rd day showed an 81% methylation level of the CpG island, which increased to almost 100% on day 7 and then, on the 11th day, decreased to the level detected on the 3rd day. In plants treated with salt shock, the methylation level remained close to 100% during the course of the experiment. In the salt stress treatment variant, on the 3rd day of the experiment, the methylation level was 68% and on the 7th and 11th days it increased to almost 100% (Figure 7).
Analyzing the effect of drought revealed high methylation of CpG island in BvAAO_10 gene to levels reaching near 100% in Beta maritima on the 3rd and 7th days of treatment in the control variant and under drought-shock. Then, a decrease was observed on the 11th day of the experiment. In plants treated with drought stress, the methylation level increased with treatment time to 70%, 87% and 98%, respectively. In Beta vulgaris cv. Huzar high levels of near 100% CpG island methylation BvAAO_10 were observed in all variants and time points of the experiment, except for the control plants on the 3rd day (45%, Figure 8).

Analysis of Total Ascorbate Concentration in Beta vulgaris Leaves under Drought and Salinity Conditions
Changes in ascorbate content in leaves of Beta vulgaris ssp. maritima and Beta vulgaris cv. Huzar were tested over a 11-day-long in plants subjected to stress-generating factors or in those maintained under control conditions, which did not involve exposing plants to stress factors. The total ascorbate levels, i.e., the sum of its oxidized and reduced forms, were determined. Leaf total ascorbate concentrations were expressed as µg ASC per gram fresh weight (FW) of leaves and are presented in Figure 9.
A similar trend of changes in ascorbate content was observed in Beta maritima leaves treated with both salinity and drought, irrespective of the intensity of the treatment. From the 3rd to the 9th day of the experiment, a significant decrease in ASC concentration was observed, compared to the control. The exception was the salt -treated Beta maritima leaves on day 9, where the ASC level was similar to that in the control. On the 11th day, in the case of salt shock and salt stress, as well as drought shock, a significant increase in ASC concentration was observed ( Figure 9). In Beta vulgaris cv. Huzar a profile of changes was different. The leaves of plants treated with salt stress on day 5 displayed a significant increase in ASC which was maintained until the end of the experiment. In leaves treated with salt shock, similar increase was observed only from the 7th day of the experiment. Changes in ASC concentration under drought conditions were comparable to those observed in Beta maritima. With respect of the untreated to the control, a significant decrease in ASC concentration occurred by the 9th day of treatment, except for the leaves of plants treated with salt stress. On the 11th day of the experiment, a significant increase in ASC concentration was observed in this variant, as compared to the control. The ASC concentration for the drought shock treatment variant on day 11th was not determined due to the death of the plants (Figure 9).

Figure 9.
Total ascorbate concentration determined in leaves of Beta vulgaris ssp. maritima and B vulgaris cv. Huzar subjected to salt shock or stress and drought shock or stress. Control−light g bars, salt shock−gray bars, salt stress−black bars. Different letters denote significant differences < 0.01 (ANOVA followed by Tukey's test). "whiskers" indicate standard deviation.

Analysis of Ascorbate Oxidase Activity in Beta Vulgaris Leaves in Drought and Saline Conditions
The experiments also included the determination of AAO activity in two beet su species ( Figure 10). In Beta maritima, an increase in enzyme activity was observed in leav treated with both, salt shock and stress, on the 3rd day of the experiment, compared the control. On day 5, this increase was still visible in the samples representing salt str treatment, although the difference was statistically insignificant. Similarly, in the leav representing the salt shock-treated leaves, there was a decrease in activity relative to control on the 5th day of treatment, although these differences were not statistically s nificant. On the 7th and 11th days of the experiment, AAO activity did not differ betwe the variants. On day 9, a significant decrease in AAO activity was observed, relative control, in leaves of plants treated with salt stress. The leaves treated with salt shock a showed a decrease in AAO activity, although the difference was not statistically sign cant when compared to untreated control. Under the drought treatment, the AAO activ was similar in all variants on the 3rd and 11th days of the experiment. On the 5th da significant decrease in AAO activity relative to the control were observed in the samp representing drought shock-treated and on days 7th and 9th by drought shock a drought stress-treated plants. In Beta vulgaris cv. Huzar a significant decrease in AAO tivity was observed between the 5th and 11th day of the experiment, in leaves treated w both saline and drought. Furthermore, a decrease in activity occurred in the leaves trea with drought shock on the 3rd day of treatment (Figure 10). Figure 9. Total ascorbate concentration determined in leaves of Beta vulgaris ssp. maritima and Beta vulgaris cv. Huzar subjected to salt shock or stress and drought shock or stress. Control−light gray bars, salt shock−gray bars, salt stress−black bars. Different letters denote significant differences at p < 0.01 (ANOVA followed by Tukey's test). "whiskers" indicate standard deviation.

Analysis of Ascorbate Oxidase Activity in Beta vulgaris Leaves in Drought and Saline Conditions
The experiments also included the determination of AAO activity in two beet subspecies ( Figure 10). In Beta maritima, an increase in enzyme activity was observed in leaves treated with both, salt shock and stress, on the 3rd day of the experiment, compared to the control. On day 5, this increase was still visible in the samples representing salt stress treatment, although the difference was statistically insignificant. Similarly, in the leaves representing the salt shock-treated leaves, there was a decrease in activity relative to the control on the 5th day of treatment, although these differences were not statistically significant. On the 7th and 11th days of the experiment, AAO activity did not differ between the variants. On day 9, a significant decrease in AAO activity was observed, relative to control, in leaves of plants treated with salt stress. The leaves treated with salt shock also showed a decrease in AAO activity, although the difference was not statistically significant when compared to untreated control. Under the drought treatment, the AAO activity was similar in all variants on the 3rd and 11th days of the experiment. On the 5th day a significant decrease in AAO activity relative to the control were observed in the samples representing drought shock-treated and on days 7th and 9th by drought shock and drought stress-treated plants.
In Beta vulgaris cv. Huzar a significant decrease in AAO activity was observed between the 5th and 11th day of the experiment, in leaves treated with both saline and drought. Furthermore, a decrease in activity occurred in the leaves treated with drought shock on the 3rd day of treatment (Figure 10).

Discussion
AAO is a member of the polymorphic oxidase family that catalyzes redox reaction and plays an important role in many plant physiological processes. The enzyme coul also have a role during stress responses modifying the ascorbic acid levels and therefo changing the cellular redox balance, which might provide the plant cell with an excellen system to sense the environment and react appropriately by contributing to the stres related control. The expression of AAO is regulated by complex transcriptional and pos transcriptional controls, with transcript levels induced by growth promoters, e.g., auxi jasmonates, and reduced by growth suppressors, e.g., salicylic acid [49]. On the oth hand, experiments with many plants show that suppression of the AAO gene increase their stress tolerance, while overexpression has the opposite effect [50,51]. Overexpressio of the AAO gene in tobacco inhibited germination and seed yield upon salt stress [51]. I another report, upregulation of cucumber AAO gene expression reduced the apo-plast AA redox state and oxidative stress tolerance in tobacco plants, which is associated wit the lower transcription of genes involved in AA recycling under oxidative stress cond tions [52]. Transgenic tobacco overexpressing the AAO gene from cucumber displayed a increase in apo-plastic DHA accumulation, lower stomatal conductance and higher wat content as compared to WT [4]. In contrast, suppression of the tomato AAO gene resulte in AA accumulation in fruits and enhanced drought stress tolerance by higher photosyn thetic capacity [53]. Similarly, Yamamoto et al. (2005) reported that the transgenic tobacc plants that have suppressed AAO gene expression in antisense orientation showed in creased germination, photosynthetic activity and seed yield and therefore salt toleranc compared to WT and sense plants under high salt concentrations [51].
This study aimed to characterize the ascorbate oxidase genes in Beta vulgaris, as we as to evaluate selected elements of the salinity and drought response at the epigenetic an Figure 10. AAO activity determined in leaves of Beta vulgaris ssp. maritima and Beta vulgaris cv. Huzar subjected to salt shock or salt stress and drought shock or drought stress. Control−light gray bars, salt shock−gray bars, salt stress−black bars. Different letters denote significant differences at p < 0.01 (ANOVA followed by Tukey's test). "Whiskers" indicate standard deviation.

Discussion
AAO is a member of the polymorphic oxidase family that catalyzes redox reactions and plays an important role in many plant physiological processes. The enzyme could also have a role during stress responses modifying the ascorbic acid levels and therefore changing the cellular redox balance, which might provide the plant cell with an excellent system to sense the environment and react appropriately by contributing to the stressrelated control. The expression of AAO is regulated by complex transcriptional and posttranscriptional controls, with transcript levels induced by growth promoters, e.g., auxin, jasmonates, and reduced by growth suppressors, e.g., salicylic acid [49]. On the other hand, experiments with many plants show that suppression of the AAO gene increases their stress tolerance, while overexpression has the opposite effect [50,51]. Overexpression of the AAO gene in tobacco inhibited germination and seed yield upon salt stress [51]. In another report, upregulation of cucumber AAO gene expression reduced the apo-plastic AA redox state and oxidative stress tolerance in tobacco plants, which is associated with the lower transcription of genes involved in AA recycling under oxidative stress conditions [52]. Transgenic tobacco overexpressing the AAO gene from cucumber displayed an increase in apo-plastic DHA accumulation, lower stomatal conductance and higher water content as compared to WT [4]. In contrast, suppression of the tomato AAO gene resulted in AA accumulation in fruits and enhanced drought stress tolerance by higher photosynthetic capacity [53]. Similarly, Yamamoto et al. (2005) reported that the transgenic tobacco plants that have suppressed AAO gene expression in antisense orientation showed increased germination, photosynthetic activity and seed yield and therefore salt tolerance compared to WT and sense plants under high salt concentrations [51].
This study aimed to characterize the ascorbate oxidase genes in Beta vulgaris, as well as to evaluate selected elements of the salinity and drought response at the epigenetic and transcriptional levels associated with AAO in the leaves of Beta vulgaris and its halophytic ancestor Beta maritima. Literature data show only two studies presenting the characteristics of ascorbate oxidase genes in other plant species [11,49]. Thus, there is no study of this enzyme in Beta vulgaris. The conducted analyses allowed distinguishing of 12 genes of beet ascorbate oxidase. Genomic characterization of other plant species revealed five AAO genes in Oryza sativa, three AAO genes in Arabidopsis thaliana, seven AAO genes in Glycine max, four AAO genes in Zea mays, six AAO genes in Sorghum bicolor and eight AAO genes in Gossypium hirsutum [11,49]. In each of the 12 beet AAO protein sequences, three from the cupredoxin domain were localized. This is consistent with the presented data of other studies characterizing the genes of ascorbate oxidases in other plant species [11,49]. Interestingly, individual proteins contain different configuration combinations of domain types, which can presumably be associated with different functions of individual AAO proteins and/or their different localization. Different computer programs point to different locations of the same beet AAO. The literature data is also inconsistent in this matter. Caputo et al. indicate that immunogold transmission electron microscopy analysis showed labeling of AAO in the cytosol, walls and chloroplasts of Chaetomorpha linum cells, while in angiosperms this enzyme is almost exclusively limited to the extracellular matrix [54]. According to other literature data, AAO activity is mainly related to the cell wall fraction. It has been suggested that it may play an important role in regulating the oxygen balance and controlling the growth of plant cells, perhaps by controlling transport processes through the plasma membrane [55]. Phylogenetic analysis shows that all beet AAO genes, except BvAAO_9, have structural equivalents in the genes of Spinacia oleracea and Chenopodium quinoa. Interestingly, this is the only beet protein sequence in which the program failed to detect a signal peptide.
The conducted experiments showed that the BvAAO_3, BvAAO_4 and BvAAO_10 genes were transcriptionally active in the leaves of two beet species. Sanmartin et al. also showed that of at least four AAO genes in Cucumis melo, only CmAO1 and CmAO4 were transcriptionally active. Moreover, researchers proved that their expression was regulated under the influence of wounding and heat stress [9]. The expression profiles of the BvAAO_3, BvAAO_4 and BvAAO_10 genes are different under salinity and drought treatments. The effects of shock and stress in both treatments also have a different effect on the expression level of AAO genes. Moreover, the transcriptional response of these three genes differs in the leaves of Beta vulgaris and its halophytic ancestor Beta maritima.
When analyzing the effect of salinity on the level of gene expression in Beta maritima, a decrease in the expression of all three genes was observed and, in the case of salt stress, a significant decrease in BvAAO_10 expression was observed compared to the control and salt shock-treated samples up to the 7th day of the experiment. A different situation was observed in Beta vulgaris cv. Huzar. Here, increases and decreases in expression were observed alternately and their directions were mostly opposite for the salt stress treated and non-salt stress treatments. Taking into account the fact that the comparison concerned the halophyte and the beet cultivar, it can be assumed that the important element of higher salinity tolerance is the suppression of the expression of ascorbate oxidase genes during the stress. It is also noticeable that the expression profile of the tested genes in the salt shock-treated samples is similar to that in the control, suggesting that the salt shock treatment of plants does not initiate changes in the regulation of transcription of AAO genes in most cases. Analyzing the impact of drought on the expression level of BvAAO_3, BvAAO_4 and BvAAO_10 genes, we observe its decrease in the treated samples or no effect. The BvAAO_3 gene in both beet species is characterized by a decrease in expression compared to the control, similar to the BvAAO_4 gene in Beta vulgaris cv. Huzar. We can see that, in contrast to salinity, a cultivated beet genotype downregulates the expression of two AAO genes during drought, while Beta maritima only one. The sole literature data on the regulation of the expression level under salt and drought stress were presented by Batth et al. [49]. The researchers also showed different expression profiles depending on the AAO gene and the type of stress. Similar observations were made in the case of Oryza sativa. Here, the downregulation of the OsAAO2 gene under salt stress and the downregulation of the OsAAO2, OsAAO3 and OsAAO5 genes under drought conditions were observed. Conversely, in the case of most AAO genes in Arabidopsis thaliana and Zea Mays, salt stress and drought caused their over-expression [49]. These observations may also be due to differences in salt and drought stress tolerance by these three plant species, which result in different regulations of AAO gene expression.
Analyzing the effect of salinity on the level of methylation of the CpG islands of the promoter regions of the BvAAO_3, BvAAO_4 and BvAAO_10 genes, we also observe differences depending on the intensity and type of stress, as well as the beet genotype. In the case of salinity, the methylation level of the CpG islands of the studied genes in Beta vulgaris cv. Huzar was much higher than in the halophytic ancestor. This observation does not translate into a decrease in the expression level of AAO genes in this genotype. The explanation for this may be the involvement of other epigenetic factors, which may directly or indirectly affect gene expression and/or modify the effects of DNA methylation [42,56,57]. Taking into account the salt stress, in all cases there was an increase in the level of CpG island methylation with the duration of the experiment. When analyzing the treatment of plants with drought, a higher degree of methylation of the CpG islands of the AAO genes is also observed in the beet cultivar. In Beta martima, on the other hand, these values are significantly higher than the levels of methylation in the salinity treatment. As there is no literature data on the regulation of AAO gene methylation levels in beet and other plant species, we cannot compare the obtained results. However, there is no doubt that, during salinity or drought, selected epigenetic elements are regulated to increase the level of DNA methylation and thus silence the expression of the ascorbate oxidase genes of two beet species.
Ascorbate is a very important component of plant cells due to its numerous functions. One of them is ROS detoxification, which is especially important under oxidative stressgenerating conditions. ASC scavenges singlet oxygen, superoxide anion and hydroxyl radical. Moreover, it is electron donor in many necessary redox reactions [10]. Studies on transgenic plants with reduced ascorbate production indicate a significant increase in the amount of hydrogen peroxide, which exerts a destructive effect on many areas of cellular functioning [58]. In turn, it has been confirmed that exogenous application of ASC mitigates the effects of salinity and stimulates plant regeneration after stress, [59,60]. In our study, we observed an initial decrease in ASC concentration in leaves of Beta maritima treated with salinity and in leaves of both drought-treated beet genotypes, compared to the control. After this time, in these experimental systems, a significant increase in ASC concentration occurred, compared to the control. In turn, in leaves of Beta vulgaris cv. Huzar treated with salinity, an increase in ASC was observed, lasting from the 5th day of the experiment. The initial decrease in ASC could indicate that the pool of this type of antioxidant was partially used up, as a result of the ROS detoxification. Similar results were observed, by Chen et al., 2021 [61]. The increase in total ASC concentration over time may indicate that mechanisms leading to an increase in its amount under stress conditions are activated. One such mechanism may be the reduction in the expression level of genes encoding AAO, which results in a decrease in the amount and activity of this enzyme. The increase in ASC concentration under abiotic stress conditions was also confirmed in other studies [62,63]. Analyses aimed at the determination of AAO activity in our experimental system showed a decrease in the activity of this enzyme over time of treatments with stress-inducing factors. This correlated with our results, demonstrating the reduction of AAO gene expression under stress conditions. AAO regulates the redox state of the apoplast [10]. It has been proven that reducing the activity of this enzyme is promotes increase in plant resistance to oxidative stress since, under limited AAO activity, larger amounts of reduced ascorbate is maintained and this may be used for ROS scavenging [4,51]. Consequently, increasing the ascorbic acid concentration allows for a reduction in the amount of H 2 O 2 and, consequently, reduces sensitivity to stress [10,64]. It should be kept in mind that the response to salinity differs between the two beet genotypes analyzed. Beta maritima is a halophytic plant and thus is more resistant to salinity than Beta vulgaris cv. Huzar. Therefore, it is possible to observe the activation of other mechanisms of the response to salinity, which involve different rate of ascorbate production and/or consumption.

Identification of Ascorbate Oxidase Sequences in Beta vulgaris
The source of the gene sequences analyzed in the study of Beta vulgaris subsp. vulgaris was the reference genome of this plant with the number RefBeet-1.2.2 deposited at GenBank (https://www.ncbi.nlm.nih.gov/gene/?term=ascorbate%20oxidase%20beta% 20vulgaris; accessed on 5 January 2021).

Sequence Alignment and Phylogenetic Analysis
Alignment and phylogenetic trees were constructed in CLC Sequence Viewer 8.0 (Qiagen, CLC bio A/S, Aarhus, Denmark). Phylogenetic and molecular evolutionary analyses were conducted using the neighbor-joining method according to the amino acids sequence with 1000 bootstrap replicates.

Plant Material
Beta vulgaris ssp. maritima (B. maritima) and sugar beet, Beta vulgaris ssp. vulgaris cv. Huzar were used as plant material. B. maritima seeds were obtained from National Germplasm Resources Laboratory (Beltsville, MD, USA) and the sugar beet seeds from Greater Poland Sugar Beet Breeding-WHBC (Poznań, Poland). The seeds (5 per pot) were sown into pots filled with sand and vermiculite (1/1 v/v) and plants were watered regularly with half-strength Hoagland solution. Plants were cultured for four weeks in a growth chamber with a photoperiod of 16 h of light and 8 h of darkness with standard irradiation of 30 ± 5 µmol m −2 s −1 , provided by T8 15 W 6500 K "Daylight" tubes (POLAMP, Ełk, Poland). The temperature regime was 25 • C during the day and 18 • C at the night. Subsequently, treatments with salinity or drought were performed.

Exposing Plants to Salinity
Salt treatment was started when the first pair of mature leaves were fully developed. Salt shock-Plants were watered, over an 11-day-long treatment period, in two-day-long intervals, with half-strength Hoagland medium supplemented with 150 mM NaCl. Untreated controls were watered with a NaCl-unsupplemented medium. The leaf samples representing intact leaf blades were collected for analysis on first, third, fifth, seventh, ninth and eleventh day of the experiment (Skorupa et al., 2019). Salt stress-Over the first 11 days of treatment, plants were watered in two-day-long intervals, with half-strength Hoagland medium supplemented with increasing concentrations of NaCl, until the final concentration of 150 mM NaCl was reached. (first day of treatment-25 mM NaCl, third day-50 mM NaCl, fifth day-75 mM NaCl, seventh day-100 mM NaCl, ninth day-125 mM NaCl, eleventh day-150 mM NaCl). Untreated controls were watered with a NaCl-unsupplemented medium. Plants were watered with 200 mL of solution per 2 l of sand/vermiculite mixture. Leaves representing the second pair of true leaves were collected for analysis.

Exposing Plants to Drought
Drought treatment was started when the first pair of mature leaves were fully developed. Drought shock-Over 11 days of treatments, plants were not watered. Untreated controls were watered with a 30 mL half-strength Hoagland medium. The leaf samples representing intact leaf blades were collected for analysis on the first, third, fifth, seventh, ninth and eleventh days of the experiment. Drought stress-Over 11 days of treatment, plants were watered in two-day-long intervals, with half-strength Hoagland medium with decreasing water volume. (1st day of treatment-30 mL/per pot, third day-25 mL/per pot, fifth day-20 mL/per pot, seventh day-15 mL/per pot, ninth day-10 mL/, eleventh day-5 mL/per pot). Untreated controls were watered with a 30 mL NaCl-unsupplemented medium. Plants were watered with 200 mL of solution per 2 l of sand/vermiculite mixture. Leaves representing the second pair of true leaves were collected for analysis.
62 experimental variants were analyzed in the experiments. Two variants were represented by Beta maritima and Beta vulgaris cv. Huzar leaves on the day of starting the experiment, before applying the stresses. Sixty experimental variants were represented by the leaves of two beet species grown under control conditions or treated with drought or salinity on the respective leaf harvest days. In the case of RT qPCR analyses and methylation analyses, the number of analysis samples was: 62 experimental variants × 3 biological replicates × 3 technical repetitions = 558. For the determination of total ascorbate and AAO activity, the number of analyzed samples was: 62 experimental variants × 6 biological replicates × 3 repetitions technical = 1116.

RT qPCR Analysis
Total RNA was isolated from the same leaf samples using the TriPure reagent (Roche Diagnostics GmbH, Mannheim, Germany). Total RNA preparations were subjected to DNase treatment, which was carried out using DNase I (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer's instructions. The first-strand cDNA was synthesized from 1 µg of total RNA using random hexamers and Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany), following the manufacturer's instructions. Gene-specific primers were designed using Primer3Plus software. To ensure the correctness of the quantification, the expression was normalized to the β-actin reference gene. To determine the PCR efficiencies, standard curves for both target and control genes were obtained using a series of cDNA dilutions as a template. RT qPCR was performed in three biological replicates, each of which consisted of three technical replicates, using FastStart SYBR Green Master Mix, following the standard PCR program suggested by the manufacturer (Roche Diagnostics GmbH, Mannheim, Germany). The relative level of gene expression was calculated according to the Pfaffl method. The control against which the calculations were performed was represented by the leaves of the two beet genotypes at the start of the experiment, before the start of the stress treatment. Blank controls without a cDNA template were included in every experimental setup. A list of PCR primers used for experimental is provided in Table S1. The ordinal number of the primer sequences in Table S1 for the BvAAO_3, BvAAO_4, BvAAO_10 0 genes, respectively, are 3, 4 and 10. The qPCR efficiency values for the genes analyzed are summarized in Table S2.

Analysis of the CpG Island Methylation Level of Selected Beet AAO Genes
The level of methylation of the CpG islands of the promoter regions of the BvAAO_3, BvAAO_4 and BvAAO_10 genes was analyzed. Measurements were taken on the 3rd, 7th and 11th day of treating plants with saline or drought. The location of the islands and the analysis of their methylation level was determined according to Skorupa et al. 2021 [24]. The primer sequences for analysis are listed in the Table S1. The qPCR efficiency values for the genes analyzed are summarized in Table S2.

Determination of the Concentration of Total Ascorbate
150 mg of leaf tissue was extracted in a mortar with 1 mL of 5% TCA. The samples were then centrifuged at 14,000× g and 4 • C for 10 min. 16.8 µL of 10 mM DTT and 80 mM K 2 HPO 4 were added to 135 µL of supernatant and incubated for 5 min at RT. In the next step, 40 µL of 85% H3PO4, 685 µL of 0.5% α, α -dipyridyl and 140 µL of 1% FeCl 3 were added successively, followed by incubation for 30 min at RT and centrifugation at 14,000× g for 5 min. The absorbance values were measured spectrophotometrically at a wavelength of 525 nm (Analytik Jena, Jena, Germany). The blank was a sample containing water with 5% TCA, instead of a plant extract. Ascorbate concentrations (µg/mL) were read from a standard curve prepared from a series of ascorbic acid concentrations. Leaf ascorbate concentrations are expressed as µg ascorbate/gFW.

Measurement of the Enzymatic Activity of Ascorbate Oxidase
200 mg of plant tissue was homogenized in 1 mL of extraction buffer (66 mM Na + /K + phosphate buffer pH 6.1, 0.5 mM EDTA) and centrifuged for 10 min at 10,000× g at 4 • C. The AAO activity was calculated on the basis of the change in absorbance at the wavelength of 265 nm for 60 s, in the reaction mixture with the following composition: 960 µL of the buffer (66 mM Na + /K + phosphate buffer pH 6.1), 20 µL of the sample and 20 µL of 2.5 mM acid ascorbic acid. The calculations were made using the millimolar absorption coefficient for ascorbic acid at 265 nm, which is 14 mM −1 cm −1 . The enzyme activity was expressed in AAO, nmol ASC mg of total protein −1 min −1 ].

Statistics
Statistical significance of differences between mean values were determined with twoway ANOVA followed by Tukey's HSD test in SigmaPlot 2008 (Systat Software, Palo Alto, Santa Clara, CA, USA). Differences at the level of p < 0.01 were considered as significant. Mean and standard deviation were calculated. Error bars shown in all figures represent the standard deviation calculated from all repetitions of each experiment.

Conclusions
The response of a sugar beet cultivar Beta vulgaris cv. Huzar and its halophytic ancestor Beta maritima to drought and salinity is manifested by downregulating the expression of ascorbate oxidase genes. This correlated with a decrease in AAO enzyme activity in a given experimental setup. Silencing of the AAO gene expression is most pronounced in the downregulation of AAO transcripts in Beta maritima under salinity, compared to a crop and conversely, under drought, in Beta vulgaris cv. Huzar. Considering the high level of drought and salinity stress tolerance in wild beet, Beta maritima and relatively moderate tolerance to these stresses in a beet crop variety, suppression of AAO gene expression in beet leaves under stress conditions may contribute to the mechanism of increasing salinity and drought tolerance. Moreover, it can be concluded that the downregulation of BvAAO_10 expression plays a key role in response to salinity treatment in Beta maritima, whereas downregulation of the BvAAO_3 gene is predominant under drought in both beet genotypes. This phenomenon may be involved in determining the high tolerance of beet to salinity and drought.