Identiﬁcation and Expression Analysis of Hexokinases Family in Saccharum spontaneum L. under Drought and Cold Stresses

: In plants, the multi-gene family of dual-function hexokinases (HXKs) plays an important role in sugar metabolism and sensing, that affects growth and stress adaptation. Sugarcane is an important sucrose crop and biofuel crop. However, little is known about the HXK gene family in sugarcane. A comprehensive survey of sugarcane HXKs , including physicochemical properties, chromosomal distribution, conserved motifs, and gene structure was conducted, identifying 20 members of the SsHXK gene family that were located on seven of the 32 Saccharum spontaneum L. chromosomes. Phylogenetic analysis showed that the SsHXK family could be divided into three subfamilies (group I, II and III). Motifs and gene structure were related to the classiﬁcation of SsHXKs . Most SsHXKs contained 8–11 introns which was consistent with other monocots. Duplication event analysis indicated that HXKs in S. spontaneum L. primarily originated from segmental duplication. We also identiﬁed putative cis-elements in the SsHXK promoter regions which were involved in phytohormone, light and abiotic stress responses (drought, cold et al.). During normal growth and development, 17 SsHXKs were constitutively expressed in all ten tissues. Among them, SsHXK2 , SsHXK12 and SsHXK14 had similar expression patterns and were more highly expressed than other genes at all times. The RNA-seq analysis showed that 14/20 SsHXKs had the highest expression level after cold stress for 6 h, especially SsHXK15 , SsHXK16 and SsHXK18 . As for drought treatment, 7/20 SsHXKs had the highest expression level after drought stress for 10 days, 3/20 ( SsHKX1 , SsHKX10 and SsHKX11 ) had the highest expression level after 10 days of recovery. Overall, our results revealed the potential biological function of SsHXKs , which may provide information for in-depth functional veriﬁcation.


Introduction
Sugars are not only major components of cellular architecture, but also the main energy sources of life activities [1]. In higher plants, sugars regulate plant growth and development by being involved in many vital processes, including glycolysis, tricarboxylic acid cycle, photosynthesis, growth, and stress responses [2,3]. Sugars can affect gene expression in plants not only through general metabolic effects, but also as signal molecules by interacting with sensor proteins [2]. Hexokinases (HXKs, EC 2.7.1.1) are the most important glucose signaling proteins and in higher plants, and are the essential enzymes in carbohydrate metabolism, which can regulate glycolytic flux by controlling the entry point of glucose into glycolysis [4][5][6][7]. HXKs also play important regulatory roles as the major enzymes in sugar metabolism and sugar signaling in plants through catalyzing irreversible reactions, such as the first irreversible catalytic enzyme in the glycolytic pathway [5,6]. In addition, HXKs can provide substrates for various pathways, such as starch synthesis and fatty acid synthesis [4,5,8].
Generally, hexokinases with both signal transduction and hexose phosphorylation functions are called HXK, while hexokinases with only glycol signal transduction functions are called hexokinase-like (HKL) [9]. In Arabidopsis thaliana, among the identified six AtHXKs proteins, three of them (AtHXK1, AtHXK2 and AtHXK3) can phosphorylate hexoses, while the other three (AtHKL1, AtHKL2 and AtHKL3) lack the ability of hexose phosphorylation and only have signal transduction functions [10]. AtHXK1 is the most representative glucose sensor involved in plant development and stress response [7,11]. Lugassi et al. found that overexpression of AtHXK1 in citrus was able to cause guard cells generated stomatal closure, which was consistent with the findings that increased expression of HXK in guard cells could accelerate stomatal closure by Kelly et al. [12]. Dai et al., showed that overexpression of AtHXK1 in Solanum lycopersicum L. led to reduced photosynthesis, slow growth and the induction of senescence, which indicated that AtHXK1 also plays an important role in photosynthetic tissues [13]. In Malus × domestica, Hu et al. found that MdHXK1 could regulate anthocyanin biosynthesis by phosphorylating MdbHLH3 [14], and Sun et al. found that MdHXK1 can also promote Na + accumulation in the vacuole and improve salt tolerance at least partially in a MdNHX1-dependent manner [15]. Li et al. found that suppression of SlHXK1 led to accelerated leaf senescence and stunted plant growth and that downregulation of SlHXK1 caused more starch to accumulate in tomato leaves [16]. However, the functions of HKL proteins are poorly understood. Due to its lack of glucose-phosphorylation activity, AtHKL1 proteins usually function as a negative regulator of plant growth [2].
Environmental stresses, especially those affecting water availability and temperature, such as drought and cold, are the major limitations of plant growth and development [17]. To survive these stresses, plants have developed sophisticated mechanisms, both on an external level (perception) and on an internal level (reaction) [18]. Non-enzymatic antioxidants such as glutathione are the first-line compounds against biotic/abiotic stress, which could be classified as the external perception level [19,20]. HXKs are involved in stress adaptation and defense regulation in plants as the internal level. Wang et al. reported that some Jatropha curcas HXK genes (JcHXK1, JcHXK2, JcHXK3 and JcHXK4) were up-regulated in the leaves after cold stress and JcHXK3 remarkably demonstrated cold-induced expression in the roots [21]. Han et al. discovered that HXK protein could significantly reduce the resistance to Monilinia fructicola infection in peach fruit [22]. Lugassi et al. found that the AtHXK1 transgenic tobacco can suffer salt and drought stresses due to lower transpiration rates [23]. Küstner et al. compared the gin2-1 mutant(∆HXK1) with its wildtype, and revealed that the mutant plants responded more slowly to highlight stress [24].
Sugarcane (Saccharum spp. hybrid) is a C4 Poaceae grass, which is widely grown in tropical and subtropical areas, due to its high yield of sugar [25,26]. Sugarcane is also one of the most important industrial crops, which accounts for approximately 80% of sucrose and 40% of the ethanol global production around the world [26]. The HXK activity is positively correlated with the sugar content and the sugar content significantly influences the sugar crop's and fruit's quality [1]. HXKs are functionally important and have been widely reported in many plant species; however, the sugarcane HXK gene family members have not yet been identified and characterized, and the functions of HXK-related features in sugarcane are still poorly understood. In the present study, a comprehensive survey of HXK encoding genes in S. spontaneum L. based on genome-wide analysis was conducted and the expression analysis of HXK genes was also performed using RNA-seq data and SRA data. Overall, our results revealed the potential biological function of SsHXKs, which may provide information for in depth functional verification.

Identification, Physicochemical Properties and Chromosomal Distribution of SsHXK Genes
In total, we identified 20 HXK genes from the S. spontaneum genome using HMMER software and blast program. The amino acid lengths ranged from 211 aa (SsHXK20) to 654 aa (SsHXK14), the molecular weight ranged from 23,062.57 Da (SsHXK20) to 70,491.44 Da (SsHXK14), and their isoelectric points ranged from 4.96 (SsHXK18) to 9.45 (SsHXK14) ( Table S1). In addition, S. spontaneum HXK genes prediction was localized in the chloroplast (ten SsHXKs), cytoplasmic (eight SsHXKs), mitochondrial (SsHXK8) and plasmalemma (SsHXK2) (Table S1). Based on their order on the chromosome, the SsHXK genes were named from SsHXK1 to SsHXK20. The chromosome distribution of SsHXKs was uneven, and only seven out of 32 chromosomes were distributed (Chr3A, Chr3B, Chr3C, Chr3D, Chr7A, Chr7B and Chr7D) ( Figure 1). Moreover, one tandem repeat gene pair (SsHXK15/SsHXK16) was identified. and the expression analysis of HXK genes was also performed using RNA-seq data and SRA data. Overall, our results revealed the potential biological function of SsHXKs, which may provide information for in depth functional verification.

Phylogenetic Analysis and Synteny Analysis
To investigate the evolutionary relationships of the SsHXK proteins, an unrooted neighbor-joining phylogenetic tree were conducted based on complete protein sequences among S. spontaneum, Oryza sativa and A. thalian ( Figure 2). According to the phylogenetic tree of SsHXKs from three species, all SsHXK protein sequences were divided into four groups, named group I, group II, group III and other. Nine SsHXK proteins (SsHXK1, SsHXK2, SsHXK5, SsHXK10, SsHXK11, SsHXK12, SsHXK14, SsHXK17 and SsHXK19), four OsHXK proteins (OsHXK2, OsHXK5, OsHXK6 and OsHXK9) and two AtHXK proteins (AtHXK1 and AtHXK2) were classed into group I. Three SsHXK proteins (SsHXK4, SsHXK7, and SsHXK9) and three OsHXK proteins (OsHXK3, OsHXK4 and OsHXK10), two AtHKL proteins (AtHKL1 and AtHKL2) and one AtHXK protein (AtHXK3) were classed into group III. However, eight SsHXK proteins (SsHXK3, SsHXK6, SsHXK8, SsHXK13, SsHXK15, SsHXK16, SsHXK18 and SsHXK20) and three OsHXK proteins (OsHXK1, OsHXK7 and OsHXK8) were classed into group II, in which neither AtHXK protein no AtHKL protein was included. Even then, only one member, AtHKL3 independently forms a branch, which named other. classed into group III. However, eight SsHXK proteins (SsHXK3, SsHXK6, SsHXK8, SsHXK13, SsHXK15, SsHXK16, SsHXK18 and SsHXK20) and three OsHXK proteins (OsHXK1, OsHXK7 and OsHXK8) were classed into group II, in which neither AtHXK protein no AtHKL protein was included. Even then, only one member, AtHKL3 independently forms a branch, which named other. Segmental or tandem duplicates in many gene families are the main expanding pattern in plants. We predicted the SsHXK genes duplication events by the MCScanX ( Figure  3). Eleven HXK gene pairs were identified as fragment duplicates (SsHXK3/SsHXK6, SsHXK8/SsHXK13, SsHXK10/SsHXK14, SsHXK11/SsHXK19, SsHXK14/SsHXK17 and SsHXK18/SsHXK20), and one tandem duplication event (SsHXK15/SsHXK16). The results showed that the expansion of SsHXKs was mainly dependent on fragment repetition events. In addition, the Ka and Ks rates and the Ka/Ks ratio of these SsHXK gene pairs were calculated (Table S2). All Ka/Ks ratios were <1.0 and indicated that the purifying selection was the primary pressure during the SsHXKs evolution period. Segmental or tandem duplicates in many gene families are the main expanding pattern in plants. We predicted the SsHXK genes duplication events by the MCScanX (Figure 3). Eleven HXK gene pairs were identified as fragment duplicates (SsHXK3/SsHXK6, SsHXK3/ SsHXK8, SsHXK3/SsHXK13, SsHXK4/SsHXK9, SsHXK6/SsHXK8, SsHXK6/SsHXK13, SsHXK8/SsHXK13, SsHXK10/SsHXK14, SsHXK11/SsHXK19, SsHXK14/SsHXK17 and SsHXK18/SsHXK20), and one tandem duplication event (SsHXK15/SsHXK16). The results showed that the expansion of SsHXKs was mainly dependent on fragment repetition events. In addition, the Ka and Ks rates and the Ka/Ks ratio of these SsHXK gene pairs were calculated (Table S2). All Ka/Ks ratios were <1.0 and indicated that the purifying selection was the primary pressure during the SsHXKs evolution period.

Conserved Motifs and Gene Structures of the HXK Gene Family
To analyze the differences of protein sequences, we detected the conserved motifs using the MEME program ( Figure 5). Ten motifs were identified, and the motif length ranged from 21 to 50 aa (Table S3). All SsHXKs had motif 10 except SsHXK13. Motif 3 and motif 1 were found in all sequences except the SsHXK17. Most SsHXK members contain eight conserved motifs. In addition, we constructed an unrooted phylogenetic tree based on SsHXK protein sequences, suggesting that the motifs classification of HXK genes was consistent with the phylogenetic tree.
In addition, to better understand the gene structure of the SsHXK genes, exons-introns were analyzed by assessing the annotation information of the GFF files in the S. spontaneum genome through GSDS2.0. The results showed that all SsHXKs contained introns, four SsHXKs included both 5′-and 3′-UTRs (SsHXK1, SsHXK10, SsHXK18 and SsHXK20), three SsHXKs only contained 3′-UTR (SsHXK14, SsHXK16 and SsHXK17), three

Conserved Motifs and Gene Structures of the HXK Gene Family
To analyze the differences of protein sequences, we detected the conserved motifs using the MEME program ( Figure 5). Ten motifs were identified, and the motif length ranged from 21 to 50 aa (Table S3). All SsHXKs had motif 10 except SsHXK13. Motif 3 and Plants 2023, 12, 1215 6 of 15 motif 1 were found in all sequences except the SsHXK17. Most SsHXK members contain eight conserved motifs. In addition, we constructed an unrooted phylogenetic tree based on SsHXK protein sequences, suggesting that the motifs classification of HXK genes was consistent with the phylogenetic tree.

Expression Pattern of SsHXK Genes during Saccharum Spontaneum Development
To understand spatiotemporal expression patterns of SsHXKs, we analyzed the transcriptomes of various tissues (Seedling-leaf, Seedling-stem, Pre-mature-leaf, Pre-maturestem-3, Pre-mature-stem-6, Pre-mature-stem-9, Mature-leaf, Mature-stem-3, Maturestem-6, Mature-stem-9) (Figure 7). In general, most of SsHXKs are expressed during growth and development but with a different expression. According to the expression profiles, there are 17 SsHXKs expressed in all ten tissues (FPKM > 0), except for three SsHXK genes (SsHXK8, SsHXK10 and SsHXK17). At the same time, there were seven SsHXK genes (SsHXK1, SsHXK2, SsHXK4, SsHXK5, SsHXK12, SsHXK14 and SsHXK20) whose FPKM values were greater than 1 in all tissues, indicated that these genes showed constitutive expression. Most of these SsHXK genes were found expressed in more than one detected organ. Three genes, SsHXK2, SsHXK12 and SsHXK14, had similar expression patterns and were highly expressed in all tissues at different development stages, which

Expression Pattern of SsHXK Genes during Saccharum Spontaneum Development
To understand spatiotemporal expression patterns of SsHXKs, we analyzed the transcriptomes of various tissues (Seedling-leaf, Seedling-stem, Pre-mature-leaf, Pre-maturestem-3, Pre-mature-stem-6, Pre-mature-stem-9, Mature-leaf, Mature-stem-3, Mature-stem-6, Mature-stem-9) (Figure 7). In general, most of SsHXKs are expressed during growth and development but with a different expression. According to the expression profiles, there are 17 SsHXKs expressed in all ten tissues (FPKM > 0), except for three SsHXK genes (SsHXK8, SsHXK10 and SsHXK17). At the same time, there were seven SsHXK genes (SsHXK1, SsHXK2, SsHXK4, SsHXK5, SsHXK12, SsHXK14 and SsHXK20) whose FPKM values were greater than 1 in all tissues, indicated that these genes showed constitutive expression. Most of these SsHXK genes were found expressed in more than one detected organ. Three genes, SsHXK2, SsHXK12 and SsHXK14, had similar expression patterns and were highly expressed in all tissues at different development stages, which indicated that these three genes maybe the key function genes in S. spontaneum. SsHXK2, SsHXK12 and SsHXK14 showed the highest expression levels in the pre-mature-stem-3 of S. spontaneum. Obviously, among SsHXK1, SsHXK2, SsHXK5, SsHXK12, SsHXK14 and SsHXK20 genes, the expression level in the stems were higher than in the leaves, which suggested that these six genes may play a more important role in the growth and development of stem. What's more, these six genes showed a decline from the top (pre-mature-stem-3) to bottom (pre-mature-stem-9) of the stem during sugar accumulation stage, which suggested that these six genes may be involved in the process of sugar metabolism in sugarcane stems. indicated that these three genes maybe the key function genes in S. spontaneum. SsHXK2, SsHXK12 and SsHXK14 showed the highest expression levels in the pre-mature-stem-3 of S. spontaneum. Obviously, among SsHXK1, SsHXK2, SsHXK5, SsHXK12, SsHXK14 and SsHXK20 genes, the expression level in the stems were higher than in the leaves, which suggested that these six genes may play a more important role in the growth and development of stem. What's more, these six genes showed a decline from the top (pre-maturestem-3) to bottom (pre-mature-stem-9) of the stem during sugar accumulation stage, which suggested that these six genes may be involved in the process of sugar metabolism in sugarcane stems.

Expression Pattern of SsHXK Genes under Cold and Drought Treatments
SsHXK genes contained numerous cis-elements responding to drought and cold stress in the promoter regions, so we analyzed the expression patterns of SsHXKs under drought and cold stress to further investigate the potential functions of SsHXK genes (Figure 8). Under cold stress, the SsHXKs had different expression profiles and were expressed at all time points. After cold treatment for six hours, the expression level of 14/20 SsHXKs was significantly higher than that of control group and other treatment groups, especially SsHXK18, SsHXK16 and SsHXK15. While SsHXK2, SsHXK3, SsHXK5 and SsHXK8 had the relatively stable expression levels after cold for six hours. The expression level of SsHXK6 and SsHXK12 genes had a continuous downward trend after cold treatment.
SsHXK18, SsHXK16 and SsHXK15. While SsHXK2, SsHXK3, SsHXK5 and SsHXK8 had the relatively stable expression levels after cold for six hours. The expression level of SsHXK6 and SsHXK12 genes had a continuous downward trend after cold treatment.
As for drought treatment, all genes were expressed except for SsHXK2, SsHXK5 and SsHXK12. Especially, these three genes were not expressed after ten days of drought treatment. After drought treatment for ten days, the expression level of seven SsHXKs (SsHXK3, SsHXK6, SsHXK13, SsHXK15, SsHXK16, SsHXK18 and SsHXK20) was higher than that other groups. After ten days of recovery, three genes (SsHKX1, SsHKX10 and SsHKX11) had a higher expression level than the control group and all of drought treatment groups. Moreover, seven SsHXKs (SsHXK4, SsHXK5, SsHXK7, SsHXK9, SsHXK12, SsHXK14 and SsHXK19) were found that the expression profiles had a continuous downward trend. These results showed that SsHXKs may play different roles in response to drought and cold stress.

Discussion
HXKs family proteins play a key regulatory role in sugar sensing, sucrose metabolism, catalyzing hexose phosphorylation, providing energy and modulation of plant growth and stress adaptation [27]. HXK belongs to a multigene family and HXK is usually encoded by a medium family of approximately 4-10 genes in higher plant, i.e., six members of the HXK family were identified in A. thaliana [2], while it is represented by 10 members in O. sativa [28], nine members in Z. mays [29], nine members in Nicotiana tabacum As for drought treatment, all genes were expressed except for SsHXK2, SsHXK5 and SsHXK12. Especially, these three genes were not expressed after ten days of drought treatment. After drought treatment for ten days, the expression level of seven SsHXKs (SsHXK3, SsHXK6, SsHXK13, SsHXK15, SsHXK16, SsHXK18 and SsHXK20) was higher than that other groups. After ten days of recovery, three genes (SsHKX1, SsHKX10 and SsHKX11) had a higher expression level than the control group and all of drought treatment groups. Moreover, seven SsHXKs (SsHXK4, SsHXK5, SsHXK7, SsHXK9, SsHXK12, SsHXK14 and SsHXK19) were found that the expression profiles had a continuous downward trend. These results showed that SsHXKs may play different roles in response to drought and cold stress.

Discussion
HXKs family proteins play a key regulatory role in sugar sensing, sucrose metabolism, catalyzing hexose phosphorylation, providing energy and modulation of plant growth and stress adaptation [27]. HXK belongs to a multigene family and HXK is usually encoded by a medium family of approximately 4-10 genes in higher plant, i.e., six members of the HXK family were identified in A. thaliana [2], while it is represented by 10 members in O. sativa [28], nine members in Z. mays [29], nine members in Nicotiana tabacum [30], 17 members in Gossypium hirsutum L. [31] and 19 putative members in B. napus [32] (Table S5).
However, little information about the HXK gene family has been reported in sugarcane. The assembled genome of S. spontaneum AP85-441 served as convenient reference for analyzing the SsHXK gene family in sugarcane. In the present study, 20 SsHXK genes were identified from the whole genome of S. spontaneum AP85-441, which was more than that in O. sativa (10 HXKs) [28], Z. mays (9 HXKs) [29] and A. thaliana (6 HXKs) [2]. Is the number of HXKs gene family related to the genome size of the species? To answer this question, we compared the size of the selected plant species' genomes and the number of HXK family genes, i.e., O. sativa (466 Mb/10 HXKs), A. thaliana (125 Mb/6 HXKs), Z. mays (2.3 Gb/9 HXKs), and S. spontaneum (3.36 Gb/20 HXKs). The results showed that the number of members of the HXK gene family was not positively correlated with the size of the plant genome. The number of HXK genes varied widely among different plant species, suggesting that the HXKs were not conserved during the evolution progress. Moreover, the S. spontaneum AP85-441 was haploid, produced by the octoploid SES208, considering the expansion of gene families in polyploidy, the number of HXK genes in octoploid S. spontaneum most likely could be over 20.
Sugarcane is an allopolyploid and aneuploid perennial herbaceous crop with 100~120 chromosomes [26,33]. Zhang et al. have speculated that the sugarcane genome undergone whole-genome duplication, random duplication and segmental duplication, and during intergeneric hybridization or domestication [34].In the present study, 20 HXK members of sugarcane are located on seven of 32 chromosomes(Chr3A, Chr3B, Chr3C, Chr3D, Chr7A, Chr7B and Chr7D), while nine ZmHXKs of Z. mays [29], six AtHXKs of A. thaliana [2], and ten OsHXKs of O. sativa [28] are all located on three of their own chromosomes. ZmHXKs were located on chromosomes 3, 6 and 8 [29], AtHXKs were located on chromosomes 1, 5 and 7 [2], while OsHXKs were located on chromosomes 1, 5 and 7 [28]. The present study showed that the chromosome distribution of SsHXKs was uneven. For example, the chromosome Chr3D contained five SsHXK genes, while the chromosome Chr3A and Chr3B both have three SsHXK genes, respectively. Similar results were also obtained for the chromosome Chr7A, Chr7B and Chr7D. These findings indicated that whole-genome duplication and segmental duplication might have occurred on these seven chromosomes, which was consistent with the findings of Zhu et al. [3].
In addition, phylogenetic analysis of the HXKs among S. spontaneum, A. thaliana and O. sativa resulted in their clustering into four subgroups, named group I, group II, group III and other. AtHKL3 is a single branch, which is similar to the phenomenon of ZmHXK phylogenetic tree construction [29]. Moreover, according to the present evolutionary tree branch and HXK studies by Cho et al. and Zheng et al. [28,35], we speculated that SsHXK4, SsHXK7 and SsHXK9 are HKL proteins, and the rest are HXK proteins.
To determine the evolution of the SsHXK genes, gene structures and conserved motifs were conducted. The gene structure analysis suggested that most of SsHXK genes were found 8-11 exons, which is similar to those in Z. mays [29], G. hirsutum L. [31] and B. napus [32]. Conserved motif analysis showed that all SsHXK members had typically conserved region, which is consistent with that in other species, i.e., A. thaliana [2], Z. mays [29], G. hirsutum L. [31] and B. napus [32]. These findings indicated that the SsHXK genes in sugarcane were extremely conserved during the evolutionary process, intergeneric hybridization or domestication.
Cis-acting elements in promoters play a very important role in regulating phytohormone and stress response. We found that the SsHXK family members contain numerous elements related to phytohormone response, light response, and stress responses. All 20 SsHXKs contained ABA response elements except SsHXK1 and SsHXK12, which suggesting that most SsHXKs may regulate phytohormone sensitivity, which is consistent with the findings by Moore et al. that overexpression of AtHXK1 in A. thaliana can result in increased cytokinin sensitivity [7].
Segmental or tandem duplicates in many gene families are the main expanding pattern in plants [36]. We detected the SsHXK genes duplication events, and 11 HXK gene pairs were identified as fragment duplicates, while one tandem duplication event (SsHXK15/SsHXK16). These results clearly showed that the expansion of SsHXKs in sugarcane genome was mainly dependent on fragment repetition events, which is consistent with the results of a previous study [35]. Similar phenomenon of low tandem and high segmental duplication proportion for gene families were also found in BrVQ and ZmVQ gene family [37,38]. Ka/Ks ratio of SsHXks results indicated that the purifying selection were the primary pressure during the SsHXKs evolution period. Collinear maps of between S. spontaneum and O. sativa, S. spontaneum and Z. mays, S. spontaneum and S. bicolor, showed high homology of HXK family genes among monocots.
Tissue-specific expression patterns could provide a better understanding of gene function in plants [3,39]. In our study, the SsHXK genes showed diverse expression patterns in ten different tissues. Of the twenty SsHXK genes, seventeen genes were expressed in all the ten evaluated tissues, while three genes, SsHXK8, SsHXK10 and SsHXK17 were detected almost no expression in any evaluated tissues, which suggested that the expression of SsHXK genes is selective. The present study found that six genes (SsHXK1, SsHXK2, SsHXK5, SsHXK12, SsHXK14 and SsHXK20) were higher expressed in the stems than in the leaves, indicating that these genes may be more involved in stem metabolism and basic cellular activities, especially in the progress which may be related to sugar storage in stems of sugarcane [3]. A weak decrease of these six genes' expression was found from the top to bottom of the stem during the sugar accumulation stage, of which the same expression patterns of SsWRKYs were reported by Li et al. [40].
Expression profiles displayed by transcriptome data can provide a global perspective to examine gene family expression [26,40]. SRA data of transcriptomes of ten specific tissues of sugarcane ROC22 were employed to investigate the role of SsHXKs gene in response to low temperature and drought. The SsHXK expression levels from SRA showed that most SsHXKs were up-regulated or down-regulated, which is consistent with the prediction that the promoter region contained a large number of ABRE, ARE, G-Box, LTR and MBS response elements, which are important components in response to abiotic stresses such as drought, low temperature through ABA pathway or others [41,42]. The expression level of SsHXK15, SsHXK16 and SsHXK18 were higher than other groups after cold for 6 h. On the contrary, SsHXK4 and SsHXK9 had lower expression level than control. As for drought treatment, three SsHXKs (SsHKX1, SsHKX10 and SsHKX11) had the highest expression level after 10 days of recovery. Overall, our results primarily revealed the potential biological function of SsHXKs, which may provide information for in depth functional verification.

Phylogenetic Analysis and Gene Duplication Analysis of SsHXK Proteins
The neighbor-joining (N-J) phylogenetic tree among A. thaliana, O. sativa and S. spontaneum was constructed using MEGA X software with 1000 bootstrap replications [43]. AtHXK and OsHXK proteins were downloaded from phytozomes (http://www.phytozome. org, accessed on 20 May 2022). The chromosomal distribution of the identified SsHXK genes was obtained and visualized using TBtools software (v.1.0971) [44]. The gene duplication events were conducted by using MCScanX [45]. Non-synonymous (ka) and synonymous (ks) substitutions of each duplicated SsHXK genes were calculated using KaKs Calculator 2.0 [46].

Expression Analysis of SsHXKs Based on RNA-Seq
Firstly, sugarcane different tissue expression data were download from Saccharum Genome database (http://sugarcane.zhangjisenlab.cn/sgd/html/index.html, accessed on 10 August 2022). In addition, the public data (PRJNA590595 and PRJNA636260) were download from the SRA database about drought and cold stress in ROC22. There are ten transcriptomes of tissues from Seedling-leaf, Seedling-stem, Pre-mature-leaf, Pre-maturestem-3, Pre-mature-stem-6, Pre-mature-stem-9, Mature-leaf, Mature-stem-3, Mature-stem-6, Mature-stem-9 (Seedling, Pre-mature and Mature represented 35-day-old, 9-month-old and 12-month-old S. spontaneum, respectively. Stem-3, stem-6, stem-9 means the thirdly, sixth and ninth nodal stem of sugarcane from the top to bottom, respectively). Then, Fastq and hisat2 tools were applied to get clean reads and map sequence data on the reference genome (S. spontaneum AP85-441), respectively [50,51]. The featurCounts in Subread package and trimmed mean of M-values (TMMs) were conducted for count read and normalization. Finally, all the expression values (FPKMs or TMMs) were used to make heat maps and cluster analysis by TBtools.

Conclusions
In the present study, 20 SsHXK genes were identified from the S. spontaneum genome and were classified into four groups. Most SsHXKs contained 8-11 introns which was consistent with other monocots. Duplication event analysis indicated that HXKs in S. spontaneum L. primarily originated from segmental duplication. Promoter analysis and expression analysis suggested that SsHXK genes may play an important role in the process of development and response to abiotic stress. These results provide valuable resources to better understand the biological role of the sugarcane HXK genes.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12061215/s1, Table S1: The detailed information of HXK genes included in this study; Table S2: Syntenic relationships of HXK genes in Saccharum spontaneum; Table S3: The detail information of HXK conserved motif; Table S4: Promoter cis-element analysis of the HXK genes from Saccharum spontaneum; Table S5: The HXK gene family from different species; Figure S1: the domain of the SsHXKs family; Figure S2: Amino acid sequence alignment of the glucose-binding domain in Arabidopsis thaliana (At), AtHXK1, AtHXK2, and SsHXKs.