Genome-Wide Survey and Expression Analyses of Hexokinase Family in Poplar (Populus trichocarpa)

Hexokinase (HXK) family proteins exert critical roles in catalyzing hexose phosphorylation, sugar sensing, and modulation of plant growth and stress adaptation. Nevertheless, a large amount remains unknown about the molecular profile of HXK enzymes in Populus trichocarpa, a woody model tree species. A genome-wide survey of HXK-encoding genes, including phylogenies, genomic structures, exon/intron organization, chromosomal distribution, and conserved features, was conducted, identifying six putative HXK isogenes (PtHXK1-6) in the Populus genome. The evolutionary tree demonstrated that 135 homologous HXKs between 17 plant species were categorized into four major subfamilies (type A, B, C, and D), clustering one plastidic (PtHXK3) and five mitochondrial PtHXKs grouped into type A and B, respectively. The in silico deduction prompted the presence of the conserved sugar-binding core (motif 4), phosphorylation sites (motif 2 and 3), and adenosine-binding domains (motif 7). The transcriptomic sequencing (RNA-seq) and the quantitative real-time PCR (qRT-PCR) assays revealed that three isogenes (PtHXK2, 3, and 6) were abundantly expressed in leaves, stems, and roots, while others appeared to be dominantly expressed in the reproductive tissues. Under the stress exposure, PtHXK2 and 6 displayed a significant induction upon the pathogenic fungi (Fusarium solani) infection and marked promotions by glucose feeding in roots. In contrast, the PtHXK3 and 6 are ABA-responsive genes, following a dose-dependent manner. The comprehensive analyses of the genomic patterns and expression profiling provide theoretical clues and lay a foundation for unraveling the physiological and signaling roles underlying the fine-tuned PtHXKs responding to diverse stressors.


Introduction
Sucrose is the primary form of photosynthetic carbohydrates produced in source leaves. Depending on symplastic and apoplastic pathways, sucrose is translocated and unloaded to sink tissues (e.g., roots and stems), where it is further metabolized into glucose and fructose (hexoses), maintaining cellular metabolism in different compartmentations [1]. In plants, hexoses are the central carbon energy source and signaling molecules influencing the whole life cycle [2]. During the sucrose metabolism, the catalysis of hexose phosphorylation is intermediated by hexokinase (HXK, EC 2.7.1.1) or fructokinase (FRK, EC 2.7.1.4), involving multiple biological processes [3]. HXKs may evolve from the actin fold protein family, sharing a conserved ATP binding site, surrounded by more variable sequences that determine the substrate affinities and other biochemical properties [4]. The phosphorylated hexoses play fundamental functions in various metabolic processes, including intracellular Sugar signaling in regulating plant growth is coordinated with phytohormone signaling pathways [29,31,32]. Recent work in Arabidopsis demonstrated that AtHXK1 was involved in the regulation of the salicylic acid (SA)-dependent programmed cell death (PCD), which is mediated by the alternation of the myo-inositol biosynthesis [33]. In grape (Vitis vinifera), the exogenous abscisic acid (ABA) cannot block the glucose-induced repression of sucrose metabolic genes in CsHXK1or CsHXK2-silencing mutants that were insensitive to glucose treatment [34]. Ectopic expression of AtHXK1 in citrus (Citrus sinensis × Poncirus trifoliata) guard cells reduced the stomatal conductance and transpiration, leading to improved water-use efficiency (WUE) [35]. AtHXK1 could reduce hydraulic conductance in response to increased glucose levels via controlling aquaporin gene expression, preserving water levels in leaves [36]. Similarly, the constant induction of OsHXK1 in rice led to rapid leaf senescence and a reduced chlorophyll level, suggesting that OsHXK1 may modulate glucose homeostasis and reactive oxygen species (ROS) accumulation [37]. This finding supported the fact that HXKs might maintain a steady state of ADP recycling, which affect H 2 O 2 formation in the mitochondrion [38]. Suppression of OsHXK10 expression in rice led to the aberrant anther and impaired pollen development [13]. Surprisingly, silencing tomato SlHXK1 resulted in stunted plant growth and stimulated leaf senescence, associated with an altered starch turnover [39].
HXKs are also involved in stress adaptation and defense regulation in response to various environmental cues and pathogen infection [40,41]. Constantly expressing AtHXK1 in tobacco (Nicotiana tabacum), tomato, and potato (S. tuberosum) guard cells increased WUE, conferring tolerance to various abiotic stress (e.g., drought, salt, and heat) [42][43][44]. In a recent report, the different subcellular allocation of serine may be the reason for the retarded growth of the gin2-1 under high irradiant conditions [45]. It was shown that AtHXK1 acts as a positive regulator of plant immunity in leaves challenged by Pseudomonas syringae pv. tomato DC3000 through the glucose effect mediated by AtHXK1-related pathways [46]. Overexpression of native OsHXK1 in rice led to improved ROS accumulation and leaf resistance to virus infection [47]. Moreover, a recent report demonstrated that constant induction of MdHXK1 expression in apple (Malus domestica) enhanced resistance to ring rot fungi pathogen and ROS production related to glucose signaling [48]. In Populus, a GATA transcription factor (TF), PdGNC was characterized to modulate stomatal aperture and influence WUE and drought tolerance, resulting from activation of hexokinase that promoted NO and H 2 O 2 accumulation in guard cells [49].
The essential catalytic and signaling roles of HKXs in plant growth and development have been well attempted in Arabidopsis and other herbaceous crops. Nevertheless, the molecular conception in genomic patterns and expression profiles, particularly in response to various stress factors, remains largely unknown in forest trees. In this work, we performed a genome-wide survey and expression analyses of the HXKs family in Populus trichocarpa, a model woody plant species. The primary objective was to gain novel insights into the molecular aspects of the HXKs family in Populus. Our data provide a theoretical clue for further unveiling the physiological significance of PtHXKs in sucrose metabolism and signaling transduction during plant growth and stress acclimation.

Genome-Wide Identification and Evolutionary Relationship of HXKs
A systematic pBLAST search using homologs from Arabidopsis as queries was conducted in Phytozome v13.1. A total of six putative isogenes were retrieved, proposing to be PtHXK after the manual removal of redundant sequences. These gene isoforms were annotated from PtHXK1 to PtHXK6 based on the chromosome (Chr) ascending ID number. The gene name, DNA and transcript size, open reading frame (ORF) and protein length, molecular weight (MW), isoelectric point (pI), transit peptide (TP), and subcellular targets are in silico deduced (Table S1). The sequence length of six Populus HXK proteins varied from 494 to 508 amino acid (AA) residues, ranging the MW from 53.09 kDa (PtHXK3) to 54.97 kDa (PtHXK2). All PtHXKs displayed the theoretical acid pI from 5.63 to 6.54. The prediction of intracellular localization indicated that most PtHXKs were localized to the mitochondria, except for PtHXK3, targeting chloroplasts.
The homologous HXKs were identified in 16 other plants, including five monocots: Brachypodium distachyon, O. sativa, P. edulis, Sorghum bicolor, and Z. mays; eight eudicots: A. thaliana, Eucalyptus grandis, Glycine max, G. raimondii, M. esculenta, Medicago truncatula, N. tobacum, and S. lycopersicum, alongside one gymnosperm: Ginkgo biloba, one bryophyte: P. patens, and one lycophyte: Selaginella moellendorffii. One hundred and thirty-five homologous HXKs were characterized, varying the numbers from 4 to 14 in all selected plant species (Table 1 and Table S2). The phylogenetic tree revealed that HXKs were clustered into four major subfamilies: Type A, B, C, and D. Type A was typically composed of HXKs from monocots and eudicots with plastidic targets. Type B contained mitochondrial HXKs that were further clustered into three subgroups: Type B-I, type B-II, and type B-III ( Figure 1). Few of HXKs, particularly in eudicots, belong to type B-I. While other mitochondrial HXKs shared the common feature between monocots and eudicots, comprising the type B-II. In contrast, type B-III included mitochondrial HXKs, particularly in type B-III. The cytosolic HXKs in monocots were categorized into the type C subfamily. The remaining HXKs in bryophytes, lycophytes, and gymnosperms were grouped in type D, appearing to have variable subcellular patterns. In Populus, three members (PtHXK1, 4, and 6) were classified into the type B-I subfamily, two PtHXKs (PtHXK2 and 5) in type B-II, and one (PtHXK3) in type C. Analyses of the chromosomal location revealed that the six PtHXKs were mapped on five of the 19 Chrs in Populus. Two members (PtHXK1 and 2) of PtHXKs were located

Chromosomal Location, Cis-Regulatory Elements, and Genomic Structure of HXKs
Analyses of the chromosomal location revealed that the six PtHXKs were mapped on five of the 19 Chrs in Populus. Two members (PtHXK1 and 2) of PtHXKs were located on Chr 1, whereas others showed individual distribution on Chr 5, 7, 9, and 18, respectively ( Figure 2a). The evolutionary relationship of PtHXK2 and 5 may propose the occurrence of segmental duplication events during the genome evolution. Comparative analyses of gene promotors between Arabidopsis and Populus showed that three potential cis-regulatory elements in PtHXKs represented the most widely spread elements, including the anaerobic induction (ARE), methyl jasmonate (MeJA) (CGTCA/TGACG), and low temperature (LTR). Within phytohormone-regulated elements, ABA-responsive elements (ABRE) and auxin response elements (TGA/AuxRR-core) were identified in three PtHXKs. The gibberellin (GA)-responsive elements (GARE/P-box/TATC-box) showed the presence in two PtHXKs. The SA-responsive elements (TCA), MYB TF binding site involved in drought response (MBS), and defense and stress response elements (TC-rich) were distributed in a few members of PtHXKs. Thereafter, analyses of genomic patterns revealed a more conserved organization of exon/intron in Populus, showing eight introns in all PtHXKs, whereas the number of introns varied from 6 to 8 in Arabidopsis (Figure 2c). PtHXK2 and 5 were clustered with the maximal length of the DNA sequence owing to the extended sizes of its introns. The high protein sequence identity to HXK homologs in Arabidopsis suggested that PtHXK2, 4, and 5 were postulated to be HKL proteins deficient in catalytic activity (Table S1 and Figure 2b). HXK homologs in Arabidopsis suggested that PtHXK2, 4, and 5 were postulated to be HKL proteins deficient in catalytic activity (Table S1 and Figure 2b).

Conserved Motifs and Domains in Protein Sequences of PtHXKs
Using the MEME web server, two essential HXK domains (HXK1 and HXK2) were typically present in HXKs between Arabidopsis and Populus ( Figure 3a). A total of ten individual motifs were programmed to vary the length of AA residues from 30 to 50 (Figure 3b). The commonly shared motifs showed a more conserved pattern in the HXK family, except for AtHLK2 and PtHXK4, lacking motif 4 due to the missing specific AA residues. A longer sequence distance between motif 6 and 7 was observed mainly for HXKs (e.g., AtHLK1, 2, PtHXK2, and 5). The multiple sequence alignments were conducted using the functional HXKs in various plants ( Figure 4). It was revealed the presence of several conserved fragments, including the N-terminal transmembrane anchor domain (5-24 AA), two phosphate domains (97-118 AA and 248-267 AA), sugar-binding core for substrate recognition (167-186 AA), adenosine phosphate-binding domain (425-461 AA), and C-terminal low complexity domain (477-491 AA). The divergent indel (6-10 AA) within the N-terminal adenosine-binding domain was verified as the critical sequences to distinguish the HXK and HLK subfamilies. These conserved features have been characterized widely in Arabidopsis, rice, tobacco, wheat, and Sorghum [1]. Two ac-

Conserved Motifs and Domains in Protein Sequences of PtHXKs
Using the MEME web server, two essential HXK domains (HXK1 and HXK2) were typically present in HXKs between Arabidopsis and Populus ( Figure 3a). A total of ten individual motifs were programmed to vary the length of AA residues from 30 to 50 ( Figure 3b). The commonly shared motifs showed a more conserved pattern in the HXK family, except for AtHLK2 and PtHXK4, lacking motif 4 due to the missing specific AA residues. A longer sequence distance between motif 6 and 7 was observed mainly for HXKs (e.g., AtHLK1, 2, PtHXK2, and 5). The multiple sequence alignments were conducted using the functional HXKs in various plants ( Figure 4). It was revealed the presence of several conserved fragments, including the N-terminal transmembrane anchor domain (5-24 AA), two phosphate domains (97-118 AA and 248-267 AA), sugar-binding core for substrate recognition (167-186 AA), adenosine phosphate-binding domain (425-461 AA), and Cterminal low complexity domain (477-491 AA). The divergent indel (6-10 AA) within the N-terminal adenosine-binding domain was verified as the critical sequences to distinguish the HXK and HLK subfamilies. These conserved features have been characterized widely in Arabidopsis, rice, tobacco, wheat, and Sorghum [1]. Two active amino acid residues (Asp104 and Ser177) labeled with stars were identified to possess HXK catalytic activity, demonstrated in rice and Arabidopsis [37,50]. tive amino acid residues (Asp104 and Ser177) labeled with stars were identified to possess HXK catalytic activity, demonstrated in rice and Arabidopsis [37,50].    tive amino acid residues (Asp104 and Ser177) labeled with stars were identified to possess HXK catalytic activity, demonstrated in rice and Arabidopsis [37,50].

Transcriptomics of PtHXKs in Vegetative Tissues and upon Pathogenic Fungi Infection
The spatiotemporal expression patterns of PtHXKs were initially analyzed using transcriptomic sequencing (RNA-seq) retrieved from the Phytozome (v13.1). It was demon- strated that a varied transcript abundance was detected in seven selected vegetative tissues, including the root tips (RTP), roots (RT), stem internodes (STI), stem nodes (STN), leaf expanded fully (LFF), leaf immature (LFI), and leaf young (LFY). As depicted in the heat map, PtHXK2 and 6 were more abundantly expressed in all vegetative tissues, mainly showing the highest levels in the roots and stems (Figure 5a). While PtHXK3 displayed explicit transcript abundance in the stems, followed by the root tips. The PtHXK1 and 5 appeared to be not significantly expressed. However, along with PtHXK4, their transcripts were predominantly detected in the inflorescence ( Figure S1). The expression patterns of six PtHXKs in young leaves (YL), mature leaves (ML), stems (ST), and roots (RT) were further evaluated using the in vitro cultured plants by quantitative real-time PCR (qRT-PCR) that was compatible with the RNA-seq data except for PtHXK5, showing exceptional levels of low expression (Figure 5b). In addition, the responding PtHXKs were investigated in the roots under the time-coursed infection of the pathogenic fungi, Fusarium solani (Fs). Taken together, analyses of the tissue-specific expression confirmed that three PtHXKs (PtHXK2, 3, and 6) showed a higher expression level in the roots than other isogenes. These three genes also showed a strong induction upon the Fs infection, particularly after 24 h (h) post-inoculation (hpi) (Figure 5c). The PtHXK1 transcript appeared to be merely affected before 24 hpi. The time coursed expression evaluated via qRT-PCR assay confirmed that PtHXK3 and 6 were the Fs responsive genes.

Transcriptomics of PtHXKs in Vegetative Tissues and upon Pathogenic Fungi Infection
The spatiotemporal expression patterns of PtHXKs were initially analyzed using transcriptomic sequencing (RNA-seq) retrieved from the Phytozome (v13.1). It was demonstrated that a varied transcript abundance was detected in seven selected vegetative tissues, including the root tips (RTP), roots (RT), stem internodes (STI), stem nodes (STN), leaf expanded fully (LFF), leaf immature (LFI), and leaf young (LFY). As depicted in the heat map, PtHXK2 and 6 were more abundantly expressed in all vegetative tissues, mainly showing the highest levels in the roots and stems (Figure 5a). While PtHXK3 displayed explicit transcript abundance in the stems, followed by the root tips. The PtHXK1 and 5 appeared to be not significantly expressed. However, along with PtHXK4, their transcripts were predominantly detected in the inflorescence ( Figure S1). The expression patterns of six PtHXKs in young leaves (YL), mature leaves (ML), stems (ST), and roots (RT) were further evaluated using the in vitro cultured plants by quantitative real-time PCR (qRT-PCR) that was compatible with the RNA-seq data except for PtHXK5, showing exceptional levels of low expression (Figure 5b). In addition, the responding PtHXKs were investigated in the roots under the time-coursed infection of the pathogenic fungi, Fusarium solani (Fs). Taken together, analyses of the tissue-specific expression confirmed that three PtHXKs (PtHXK2, 3, and 6) showed a higher expression level in the roots than other isogenes. These three genes also showed a strong induction upon the Fs infection, particularly after 24 h (h) post-inoculation (hpi) (Figure 5c). The PtHXK1 transcript appeared to be merely affected before 24 hpi. The time coursed expression evaluated via qRT-PCR assay confirmed that PtHXK3 and 6 were the Fs responsive genes.  PtβActin, PtUBIC, and PtEF-α1 were used as the internal control. Data represent mean values standard error (±SE) of at least three independent biological replicates. Asterisks indicate significant differences relative to the control using the Student's t-test: *** p < 0.001, ** p < 0.01, and * p < 0.05.

Effects of PtHXKs Expression Responding to Sugars and ABA treatments
The deduced cis-regulatory elements on promoters reflect spatiotemporal expressions of specific genes that may be affected by various environmental cues and phytohormone exposures. The gene promoters' analyses revealed a few ABA-related response elements (Figure 2b). Under the input of different sugars (e.g., glucose, fructose, and sucrose) with gradient levels, the compared expression profile of PtHXKs was explored between 0 and 24 h by qRT-PCR assay ( Figure 6). The significant inductions of PtHXK2 and 6 were identified in the roots by adding glucose when the amount increased higher than 3%. In contrast to the control (0 h), the PtHXK3 appeared to be significantly promoted by 6% sucrose and fructose owing to the observed high expression levels after 24 h of inoculation. In addition, the marked upregulation of PtHXK6 expression was detected in response to sucrose feeding, following a dose-dependent manner. The HXK-mediated sugar signaling profoundly affects promoting or arresting plant morphogenesis in association with altered ABA levels [8]. In line with this, the effect on PtHXK transcripts was inspected in roots upon different dose feeding of ABA. Notably, the constant promotions of PtHXK3 and 6 were observed to be correlated with the increase in the ABA concentration. Nevertheless, detecting other PtHXKs expressions was unsuccessful due to the low transcript abundance in the selected tissues.
post-inoculation, hpi). (d) Validation of responsive PtHXKs compared to the control (0 h qRT-PCR. The RNA-seq data were given in the Log10 of the fragments per kilobase per m reads (FPKM) expression values. At least three independent biological replicates were con for qRT-PCR analyses. PtβActin, PtUBIC, and PtEF-α1 were used as the internal control. Da resent mean values standard error (±SE) of at least three independent biological replicates. isks indicate significant differences relative to the control using the Student's t-test: *** p < 0.0 < 0.01, and * p < 0.05.

Effects of PtHXKs Expression Responding to Sugars and ABA treatments
The deduced cis-regulatory elements on promoters reflect spatiotemporal e sions of specific genes that may be affected by various environmental cues and p hormone exposures. The gene promoters' analyses revealed a few ABA-related res elements (Figure 2b). Under the input of different sugars (e.g., glucose, fructose, a crose) with gradient levels, the compared expression profile of PtHXKs was exp between 0 and 24 h by qRT-PCR assay ( Figure 6). The significant inductions of Pt and 6 were identified in the roots by adding glucose when the amount increased h than 3%. In contrast to the control (0 h), the PtHXK3 appeared to be significantl moted by 6% sucrose and fructose owing to the observed high expression levels afte of inoculation. In addition, the marked upregulation of PtHXK6 expression was de in response to sucrose feeding, following a dose-dependent manner. The HXK-me sugar signaling profoundly affects promoting or arresting plant morphogenesis in ciation with altered ABA levels [8]. In line with this, the effect on PtHXK transcrip inspected in roots upon different dose feeding of ABA. Notably, the constant prom of PtHXK3 and 6 were observed to be correlated with the increase in the ABA conc tion. Nevertheless, detecting other PtHXKs expressions was unsuccessful due to th transcript abundance in the selected tissues. Figure 6. Expression effects of three PtHXKs in the roots feeding with various sugars and AB qRT-PCR analyses show the transcript responsiveness upon the altered concentration of (glucose, fructose, and sucrose) and ABA. The relative expression of the control was set as represent mean values ±SE of at least three independent biological replicates. PtβActin, PtUB PtEF-α1 were used as reference genes. Asterisks indicate significant differences in compariso the control using the Student's t-test: ** p < 0.001, * p < 0.01. Figure 6. Expression effects of three PtHXKs in the roots feeding with various sugars and ABA. The qRT-PCR analyses show the transcript responsiveness upon the altered concentration of sugars (glucose, fructose, and sucrose) and ABA. The relative expression of the control was set as 1. Data represent mean values ±SE of at least three independent biological replicates. PtβActin, PtUBIC, and PtEF-α1 were used as reference genes. Asterisks indicate significant differences in comparison with the control using the Student's t-test: ** p < 0.001, * p < 0.01.

The HXKs Family and Conserved Profiles in Plant Species
Recent advances in high-throughput technologies and multi-omics-based strategy have boosted the framework in functional genomics and metabolic research in plants [51]. As a model perennial woody plant species, the entire genome of Populus has been released for over 15 years, whereas a thorough survey of the PtHXKs family remains unknown. The putative HXK-encoding genes were identified within 17 selected plant species, including 6-14 members (Table 1). Along with other plant species, the phylogenies of 135 HXK homologs revealed a classification of four subfamilies (type A, B, C, and D), showing one plastidic PtHXK3 in type A and five members of mitochondrial PtHXKs in type B [1,8]. However, there were no cytosolic HXKs identified in the Populus genome. Based on phylogenetic relationships and the protein sequence identity to Arabidopsis, three members (PtHXK2, 4, and 5) most likely belong to HKL-like proteins, while other gene isoforms are HXK candidates in P. trichocarpa. The genome duplication facilitates woody perennials to variable environments adaption during million years of evolution [52]. One gene pair (PtHXK2 and 6) was speculated to undergo a segmental duplication combined with the sequence analyses, suggesting notable physiological roles associated with evolved mechanisms to adapt to stress stimuli (Figure 2a). The cis-regulatory elements predicted in promoter regions indicated the dynamic regulation and evolution of specific gene expression upon various environmental cues [53]. Therefore, the deduction of principal diverse cis-regulatory elements related to phytohormone response (ABA, GA, MeJA, and Auxin) and stress factors (e.g., MBS, TC-rich, and LRT), along with TF binding sites, provided a hint that the molecular regulation of PtHXKs depends significantly on the crosstalk between phytohormone and glucose signaling pathways [31]. In addition, the analyses of the genomic structure of PtHXKs compared with homologs in Arabidopsis suggested a similar exon and intron organization (e.g., eight introns included) within the same subgroup (Figure 2c).
Furthermore, approximately ten conserved motifs (30-50 AA) of HXKs have predicted distribution between Arabidopsis and Populus, which was in line with the previous reports [1,15,16,34]. The multiple alignments of PtHXK protein sequences with other functional homologs led to the identification of six hallmarks, including 19-50 AA transit peptide sequences (membrane anchor domain) at the N-terminus for the plastidic or mitochondrial targets (Table S1). The prediction of theoretical core sites for glucose and phosphate binding demonstrated that all homologous HXKs possessed the conserved AA residues (e.g., Asp-101, Gly-104, and Ser-177) except for AtHKL1 and PtHXK4, suggesting that PtHXK4 may be a catalytically inactive glucose sensor (Figure 4). These results were supported by several recent works performed on Cassava, bamboo, and cotton [16][17][18]. These conserved features of HXKs may be more variable in gymnosperms, bryophytes, and lycophytes, suggesting that the plant-derived HXKs probably evolved from prokaryotic ancestors, common primordial actin fold protein [54,55]. Nevertheless, despite the advanced molecular information concerning functional HXKs in various plants, hitherto, no strategy was potentially used to predict the sensor role of a specific HXK, and not all HXKs played identical functions in Arabidopsis [8].
Investigating gene expression patterns is one of the strategies to imply whether candidates may be involved in specific metabolic processes or signaling roles. The significant challenge for the tree functional study of a particular gene family is to overpass the relationships between the transcript abundance and corresponding variation of the enzyme kinetics [56]. In our work, the depicted gene expression profiling revealed that a few genes (e.g., PtHXK2 and 6) displayed specific expression patterns in vascular tissues, including the roots and stems, reflecting the potential role during plant growth and development. However, the expression of gene pairs (PtHXK2 and 5) with segmental duplication was observed to show a divergent pattern ( Figure 5). The gene alternative splicing (AS) was prevalent in generating variation in protein structure, functional diversity, and stress adaptation among different plant tissues, cell types, and treatments [57]. Therefore, it was hypothesized that the expression variation of gene duplication might be due to the overlapped levels of transcript variants derived from AS. It is worthwhile to note that when focusing on the effects of PtHXKs in the roots responding to the F. solani infection, the PtHXK3 and 6 showed significant increases in expression, particularly at Fs 48 hpi. In contrast to the control, the PtHXK2 and 6 identified showed a marked promotion in roots upon the gradient glucose (3% and 6%) feeding at 24 h, suggesting the role of the sugar sensor. Only the PtHXK2 was expressed significantly by adding the highest amount (6%) of fructose.
Moreover, the PtHXK3 and 6 were highly responsive to the sucrose and ABA treatments, suggesting the crosstalk regulation between the sugar and phytohormone under the biotic stress conditions. In contrast, a much lower number of HXK transcripts were significantly influenced in mature leaves than in roots under the above conditions, indicating that this chemical concentration was a significant dosage for roots, but not for aerial leaf tissues within 24 h (data not shown). Overall, transcriptional and post-transcriptional interferences of HXK isogenes in specific tissue types indicate the subsequent exploration of physiological and signaling functions under different stress regimes. Analysis of cis-elements and gene expression revealed that PtHXKs contained various defense/stress-related response sequences that might be modulated by phytohormone (e.g., ABA), indicating the crosstalk between HXKs and sugar/phytohormone signals during the root development and stress tolerance.

The Regulatory Role of HXKs in Defense and Stress Acclimation
According to the previous research, Arabidopsis AtHXK1 was identified as a glucose sensor to interrelate nutrient, light, and phytohormone signaling networks for regulating growth and development by responding to various environmental cues [50]. Increasing evidence implicated that HXKs are primary enzymes and exert numerous regulatory actions other than merely catalyzing the phosphorylation of hexoses, the central energy nutrients, and signaling molecules in the effect of sugars on morphogenesis and stress adaptation [7,58]. The glucose acts as a signal molecule through interactions with IAA, GA, and ABA, while the HXK-catalyzed G-6P was regarded as the core intermediate in glucose signal transduction [59]. HXKs with catalytic activity significantly impact multi-cellular processes and sugar signaling, accounting for the metabolite biosynthesis on the glycolytic pathways, providing energy for cell growth [8]. During the plant-pathogen interaction, the host defense and immune responses are mounted with a profound modulation of the primary plant metabolism, including biosynthesis of carbohydrates, amino acids, and the derived secondary metabolites [60].
Feeding experiments elucidated the link between carbohydrate metabolism and defense mechanisms and revealed the induction of pathogenesis-related (PR) genes by sugars, suggesting that carbohydrate metabolism positively regulates the expression of defenserelated genes [61]. As an evolutionarily conserved glucose sensor, the AtHXK1 played dual functions in sugar metabolism, sensing, and defense response [62]. In transgenic tobacco, overexpressing invertase led to sugar accumulation, and the induction of PR-1 and PR-5 by glucose in correlation with AtHXK1 mediated the signaling and catalytic activities, suggesting the positive regulation of defense-related genes through carbohydrate metabolism [24]. Constant induction of AtHXK1 and 2 transcripts in Arabidopsis resulted in elevated resistance to the infections of necrotrophic fungi (Alternaria brassicicola) and bacterial P. syringae, indicating the defense and immunity roles of HXK against various pathogens [40,46]. The underlying mechanisms of HXK involved in regulating immunity and defense appeared to be related to altered ROS accumulation, further supported by several recent works performed on rice and apples [47,48].
Nevertheless, the AtHXK1 might play a negative role as the suppression/deletion of AtHXK1 led to the increased H 2 O 2 production associated with defense-related genes and accelerated SA-dependent PCD, prompting that AtHXK1 might exert dual regulatory roles mediating up-and downregulation of sugar responsive genes [33]. The HXKderived metabolic processes regulate plant defense and immunity to the pathogen, reaching far beyond phytohormone cues and crosstalk between signaling pathways and sucrose metabolism. Recently, two grape HXK isoforms and sucrose metabolic enzyme genes increased concomitantly with elevated levels of endogenous glucose and ABA during grape berry development, postulating the regulatory role of glucose and ABA on HXK-dependent sugar metabolism [34]. Emerging reports revealed that ABA signaling pathways regulate water status and mediate drought tolerance by controlling stomatal aperture, water conductance, and gene expression, which were conserved in tree species [63,64]. In line with the previous work, the transcriptional activation of PdHXK1 via the GATA transcription factor (PdGNC) resulted in the ABA-induced NO and H 2 O 2 accumulation, mediating the stomatal closure and drought tolerance in woody Populus [35,49,65]. In summary, the HXK-derived crosstalk of phytohormone signaling and sugar metabolism deploys defense-related genes and yields profound compounds associated with metabolic conversion, ROS control, and stressor scavenging while remaining a blank in Populus.

Plant Materials, Growth Conditions, and Treatments
The P. trichocarpa (genotype Nisqually-1) grows under long-day conditions (25 • C, 16/8 h day/night photoperiod, 50 µE), culturing in vitro on a traditional woody plant medium (WPM) with 30 g L −1 sucrose, 0.1 mg L −1 IBA, and solidified with 8 g L −1 plant agar. The Fs culture, the fungal spore calculation (1.0 × 10 6 spores/mL), and the root infection were conducted according to the report [66]. For feeding experiments, the 4-week in vitro cultured plants were transferred to conical flasks containing 40 mL gradient concentrations (0%, 1%, 3%, and 6%) of solution and incubated in an artificial chamber for 24 h. For RNA-seq data, different vegetative tissues and roots with time-course (0, 24, 48, and 72 hpi) of Fs infection were sampled based on the previous research [67]. The roots of eight randomly selected plants with Fs infection and 24-h-feeding by sugars (glucose, fructose, and sucrose) and ABA (0, 50, 100, and 200 µM) were pooled for one biological replication to evaluate gene expressions using the 0 h treatment as the control.

Sequence Mining, Identification, and Genomic Analyses of HXKs
The Arabidopsis HXK homologs were collected in TAIR. Available online: https: //www.arabidopsis.org (accessed on 24 October 2000) as queries to search for candidate isogenes in P. trichocarpa genome assembly (v3.1) from the JGI gene catalog. Available online: https://phytozome-next.jgi.doe.gov/info/Ptrichocarpa_v3_1 (accessed on 30 November 2018) with the E-value cutoff set as 1e-5. Incomplete protein sequences with short lengths (<300 AA) were eliminated. The upstream 1.5 kb sequences of gene promotors were predicted by the program PlantCARE. Available online: http://bioinformatics.psb.ugent. be/webtools/plantcare/html (accessed on 11 September 2000) to obtain overviews of cis-regulatory elements associated with the responsiveness of biotic and abiotic stresses, according to the previous reports [60]. The gene structure was deducted by comparing CDS and the corresponding DNA sequence in GSDS. Available online: http://gsds.gao-lab.org (accessed on 1 January 2015). The chromosomal distribution of PtHXKs was obtained from the PopGenIE. Available online: http://popgenie.org/chromosome-diagram (accessed on 1 January 2021), and physical locations were drawn by the program MapInspect. Available online: http://www.softsea.com/review/MapInspect.html (accessed on 9 November 2010) [68].

Phylogenetic Tree Construction and Analyses of Conserved Motifs
The alignments of multiple protein sequences were conducted by Clustal Omega. Available online: https://www.ebi.ac.uk/Tools/msa/clustalo (accessed on 23 May 2018). The reciprocal pBLAST was conducted to establish the genetic relationship between gene pairs. The phylogenetic tree was constructed by MEGA X. Available online: https://www. megasoftware.net/v10.2.2 (accessed on 1 October 2020), using the Maximum Likelihood method with 1000 bootstrap replicates [69]. The evolutionary distances were computed using the Poisson correction method and the number of AA substitutions per site. The conserved motifs were analyzed by MEME. Available online: http://meme-suite.org/ index.html (accessed on 1 August 2022), setting the maximum numbers and widths of motifs to 10 and 50, respectively [70]. Transmembrane region and low complexity were predicted in the SMATE database. Available online: https://smart.embl-heidelberg.de (accessed on 26 October 2020) based on AtHXK1. The motif was annotated by CDD in NCBI. Available online: https://www.ncbi.nlm.nih.gov/cdd (accessed on 5 November 2021) and ScanProsite. Available online: http://prosite.expasy.org/scanprosite (accessed on 25 May 2022) [71].

Transcriptome and Expression Validation by qRT-PCR
Transcriptome and data processing were performed on the Phytozome (v13.1) and based on a previous report [67]. Gene transcript levels in various tissues were valued by fragments per kilobase of exon model per million mapped reads (FPKM). For Fs infection, the significance of differentially expressed genes (DEGs) (FPKM > 5) was judged by the p < 0.05. Both Fisher's exact test (p < 0.05) and multi-test adjustment (false discovery rate (FDR) < 0.05) were applied in DEGs identification based on the report [67]. For the qRT-PCR assay, the RNA extraction and cDNA synthesis were performed, according to the previous report [72]. The primer amplification efficiency was evaluated with dilutions of cDNA, producing an R 2 ≥ 0.99. The cDNA samples were loaded to a TB green Premix ExTap™ Tli RNaseH Plus (Takara, China). The mixture was subjected to StepOnePlus™ Real-Time PCR System (AB, USA). The relative gene expression was normalized by the geometric mean of three housekeeping genes (Ptβ-Actin, PtUBIC, and PtEF-1α). The primers used for targeting specific genes are listed in Table S3. Heatmaps were constructed by the program CIMminer. Available online: http://discover.nci.nih.gov/cimminer/home.do (accessed on 19 July 2018).

Conclusions
HXKs are multifaceted enzymes, playing essential roles in various metabolic processes and signaling that significantly impact the whole plant cycle, including regulating vegetative growth and reproduction, male fertility, and senescent signals. Despite the substantial evidence of the regulatory role of HXK in plant defense and stress adaptation, still more studies are needed to integrate molecular information on the biochemical properties, cellular localization, and sensor capacities with other sugar-sensing pathways to improve performance in woody plants. Among the six putative PtHXKs identified in Populus, three isogenes (PtHXK2, 3, and 6) showed predominant expressions in the vascular tissues (e.g., roots and stems). PtHXK3 and 6 were significantly induced upon the sugar and ABA treatment, suggesting potential in vivo activities for catalyzing the hexose phosphorylation and signaling effects on Populus growth and development. Moreover, the genomic characterization of HXK families is primary for the fine-tuning of HXK-dependent pathways by engineering the activities or expressions of critical HXKs, which might be sufficient to achieve pathogen resistance and abiotic stress tolerance without compromising plant biomass. Therefore, the inspected PtHXKs with dominant responsive features to selected stimuli will be the modifying target for functional analyses under stress exposure in Populus.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants11152025/s1. Figure S1: The heatmap shows the transcript in reproductive tissues of P. trichocarpa. Table S1: List of HXK candidate genes identified in P. trichocarpa; Table S2: Phylogenetic relationship related species and gene IDs; Table S3: List of primers used for qRT-PCR analyses.

Data Availability Statement:
The raw sequence reads of RNA-seq were deposited in the NCBI database with the accession BioProject of PRJNA680933 and the accession BioSample, SAMN16927537, including twelve accession numbers of SRR13347970-981 for triplicate data of each F. solani treatment (Fs0, Fs24, Fs48, and Fs72).