Mol Biol Rep DOI 10.1007/s11033-014-3495-9

Isolation, structural analysis, and expression characteristics of the maize (Zea mays L.) hexokinase gene family Zhongbao Zhang • Jiewei Zhang • Yajuan Chen Ruifen Li • Hongzhi Wang • Liping Ding • Jianhua Wei

•

Received: 13 March 2013 / Accepted: 17 June 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Hexokinases (HXKs, EC 2.7.1.1) play important roles in metabolism, glucose (Glc) signaling, and phosphorylation of Glc and fructose and are ubiquitous in all organisms. Despite their physiological importance, the maize HXK (ZmHXK) genes have not been analyzed systematically. We isolated and characterized nine members of the ZmHXK gene family which were distributed on 3 of the 10 maize chromosomes. A multiple sequence alignment and motif analysis revealed that the maize ZmHXK proteins share three conserved domains. Phylogenetic analysis revealed that the ZmHXK family can be divided into four subfamilies. We identified putative cis-elements in the ZmHXK promoter sequences potentially involved in phytohormone and abiotic stress responses, sugar repression, light and circadian rhythm regulation, Ca2? responses, seed development and germination, and CO2-responsive transcriptional activation. To study the functions of maize HXK isoforms, we characterized the expression of the ZmHXK5 and ZmHXK6 genes, which are evolutionarily related to the OsHXK5 and OsHXK6 genes from rice. Analysis of tissue-

specific expression patterns using quantitative real timePCR showed that ZmHXK5 was highly expressed in tassels, while ZmHXK6 was expressed in both tassels and leaves. ZmHXK5 and ZmHXK6 expression levels were upregulated by phytohormones and by abiotic stress. Keywords Maize  Abiotic stress  Hexokinase  Expression behavior  Cis-elements Abbreviations 6-BA 6-Benzylaminopurine 2,4-D 2,4-Dichlorophenyloxyacetic acid ABA Abscisic acid Glc Glucose GA3 Gibberellin A3 HXK Hexokinase ORF Open reading frame PEG Polyethylene glycol

Introduction Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3495-9) contains supplementary material, which is available to authorized users. Z. Zhang  J. Zhang  Y. Chen  R. Li  H. Wang  L. Ding  J. Wei (&) Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China e-mail: [email protected] Z. Zhang e-mail: [email protected] Z. Zhang  J. Zhang  Y. Chen  R. Li  H. Wang  L. Ding  J. Wei Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Beijing 100097, China

Hexose sugars function in higher plants not only as sources of energy and carbon structural components for growth and development, but also as signaling molecules that control genes involved in many physiological processes, including germination, flowering, senescence, photosynthesis, and biotic and abiotic defense responses [1–11]. Accurate sugar signaling and sensing systems are essential for coordinating and regulating many vital metabolic pathways needed to maintain normal plant development and growth. Hexokinases (HXKs, EC 2.7.1.1) are ubiquitous proteins in all organisms that play important roles in metabolism, glucose (Glc) signaling, and phosphorylation of Glc and fructose [12, 13]. Biochemical and molecular investigations

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have distinguished HXKs in various plant species [14–16]. Among the plant HXK gene families that have been characterized thoroughly, the Arabidopsis HXK gene family has six members [17–19], rice has at least 10 expressed HXK genes [20], and tomato has at least four HXK genes [21]. Recent investigations have shown that HXKs associate with the membranes of various subcellular organelles including chloroplasts, mitochondria, Golgi complexes, plasma membranes, and endoplasmic reticula and with the cytosolic membrane [20–27]. Plant HXK proteins belong to one of two groups: type A HXKs, which have an N-terminal plastid signal peptide [21], and type B HXKs, which have an N-terminal hydrophobic membrane anchor [12, 17, 24, 28, 29]. The two types of plant HXKs can be distinguished by their N-terminal sequences [24]. The specific molecular mechanisms involved in sugar signaling remain mostly unknown, but some studies have shown a close interaction between sugar signaling and classical phytohormone signaling pathways [9], including the abscisic acid (ABA), cytokinin, gibberellin (GA), ethylene, and auxin pathways [7, 10, 30]. Glc regulates ABA signaling and biosynthesis [5], and HXK-dependent signaling interacts with auxin and cytokinin signaling [31]. Glc and ethylene often act antagonistically, and the two signaling pathways converge at the level of ETHYLENEINSENSITIVE3 protein stability [32]. These previous studies suggest that the HXK gene family may be vital not only for plant development but also for response and adaptation to stress. To illuminate the functions of the members of this gene family, we examined changes in the expression patterns of two HXK genes in various maize tissues in response to phytohormones and abiotic stress. The results presented in this study provide a reference for functional studies of HXK family genes in maize.

were downloaded from the Maize Sequence or NCBI databases. Structural analysis of maize HXK genes Chromosomal locations, open reading frame (ORF) lengths, and full-length cDNA sequences for all of the ZmHXK genes were obtained from the B73 MaizeSequence database. The exonic and intronic structures of the ZmHXK genes were confirmed using the Genes Structure Display Server (GSDS; http://gsds.cbi.pku.edu.cn/) [34]. Motif display and phylogenetic analysis of HXK proteins The Multiple Expectation Maximization for Motif Elicitation (MEME) multipurpose program [35] was used to identify sequence motifs in the HXK proteins from maize (ZmHXK), rice (OsHXK), Arabidopsis (AtHXK), and sorghum (SbHXK). The matrix used for phylogenetic analysis included 6, 10, 7, and 9 HXK genes from Arabidopsis, rice, sorghum, and maize, respectively. The amino acid (aa) sequences of all the proteins were aligned using ClustalX 2.0 software [36], and an unrooted phylogenetic tree was constructed by the neighbor joining method using MEGA4 software [37]. Analysis of ZmHXK promoter regions In order to identify cis-elements in the promoter sequences of ZmHXK genes, B73 genomic DNA sequences extending 2 kb upstream of the translation initiation codons (ATG) were downloaded from NCBI. Promoter cis-elements were predicted using Promoter 2.0 (http://www.cbs.dtu.dk/) and Plant cis-acting Regulatory DNA Elements (PLACE) database [38].

Materials and methods Plant stress treatments Isolation of HXK gene sequences from maize The sequences of 6 Arabidopsis thaliana [18] and 10 rice (Oryza sativa) [20] HXK proteins were downloaded from the TAIR and TIGR databases (http://rice.plantbiology. msu.edu). To obtain all HXK gene sequences from maize (Zea mays), BLASTP searches were performed against the Maize-Sequence database (http://www.maizesequence.org) using the Arabidopsis and rice HXK protein sequences as queries. The protein sequences of putative maize HXK family members with expectation values less than 10-10 were downloaded and then tested by the Pfam database (http://www.sanger.ac.uk) [33]. Full-length cDNA sequences corresponding to maize HXK (ZmHXK) genes

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B73 maize seeds were surface-sterilized, germinated, and grown hydroponically until the three-leaf stage in pots filled with vermiculite with four seeds per pot. The pots were grown in a greenhouse under a daily cycle of 16 h light at 28 °C and 8 h dark at 22 °C and watered once every 3 days. Plants were treated with the hormones ABA, auxin, cytokinin, and GA by spraying the leaves with (?)-cis, trans-ABA, 2,4-dichlorophenyloxyacetic acid (2,4-D), 6-benzylaminopurine (6-BA), or gibberellin A3 (GA3), respectively, at a 100-lM concentration and then wrapped with preservative film followed by sampling at 0, 1, 3, 6, 12, and 24 h. Abiotic stress treatments included exposure to cold temperatures, sodium chloride (NaCl),

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and polyethylene glycol (PEG). Cold stress treatment was performed by placing seedlings in a growth chamber at 4 °C and collecting samples at 0, 1, 3, 6, 12, and 24 h. For the NaCl and PEG treatments, seedlings were carefully removed from the vermiculite, and the roots were washed with running water and dried on filter paper. Then the roots of the seedlings were submerged in 200 mM-NaCl or 20 %-PEG (molecular weight 6,000) solutions, airdried using a pump, and sampled at 0, 1, 3, 6, 12, and 24 h [39]. Samples were collected from the leaves, stems, and roots of trefoil seedlings and from tassels, silks, and young ears at the silking stage to determine organ-specific HXK expression. All fresh samples were preserved at -80 °C after freezing in liquid nitrogen for subsequent RNA extraction.

using the Pfam database indicated that all nine maize HXK genes belonged to the HXK gene family. The PSORT software tool (http://psort.nibb.ac.jp) was used to analyze the ZmHXK protein sequences for the presence of transmembrane domains. With the exception of ZmHXK7 and ZmHXK8, each of the ZmHXK proteins had a single 16-aa transmembrane domain (Table 1). The nine ZmHXK genes were distributed on three maize chromosomes: four genes on chromosome 3, three on chromosome 6, and two on chromosome 8 (Fig. 1). The numbers and positions of exons and introns within each ZmHXK gene were detected by the GSDS (Fig. 2) [34]. Most of the ZmHXK genes contained nine exons, except for ZmHXK7, which had seven (Fig. 2). Most Arabidopsis and rice HXK genes also contain nine exons [18, 20].

Analysis of ZmHXK5 and ZmHXK6 expression

Motif analysis and protein architecture

Total RNA was extracted using TRIzol reagent (Invitrogen, Karlsruhe, Germany) and treated with DNase I (RNase free; TaKaRa, Dalian) according to the manufacturers’ protocols. Total RNA integrity and amounts were tested by NanoDrop2000 (Thermo). Real-time PCR (RT-PCR) was performed in an optical 96-well plate using an ABI StepOnePlus RT-PCR System. Gene-specific primers used for ZmHXK5 and ZmHXK6 were ZmHXK5-FP, 50 -CAGAGGGTAGGCA GAGAGAACT-30 ; ZmHXK5-RP, 50 -CAGCCAATGTGCC AACTGTATC-30 ; ZmHXK6-FP, 50 -CCACCTACCAGATA GCAAGCAG-30 ; and ZmHXK6-RP, 50 -AGCCACAACATC TTCTCCAACC-30 . ZmGAPDH transcript levels served as a control, and the primers used for ZmGAPDH were FP, 50 -CC CTTCATCACCACGGACTAC-30 , and RP, 50 -AACCTTC TTGGCACCACCCT-30 [40]. Each reaction contained 10 ll SYBR Premix Ex Taq (TaKaRa, Dalian), 1.0 ll cDNA, 0.4 ll ROX Reference Dye II, and 10 lM gene-specific primers in a final volume of 20 ll. The thermal cycle and melting curve determination acquired according to the manufacturers’ protocols. The RT-PCR data were analyzed using the 2-DDCT method [41].

We used the MEME web server to analyze the HXK protein sequences from Arabidopsis, rice, sorghum, and maize to identify motifs conserved among the HXK proteins. We identified three conserved motifs, each containing 49 aas. Motifs 1, 2, and 3 contained 21, 21, and 20 conserved aa residues, respectively. Each of these motifs was present in all HXK proteins of maize, rice, Arabidopsis, and sorghum (Fig. 3). Motif 1 is a nucleotide-binding domain belonging to the sugar kinase/HSP70/ actin superfamily. Motifs 2 and 3 belong to the HXK2 and HXK1 superfamilies, respectively. The crystal structures of several HXK proteins from yeast [42] and Escherichia coli [43] have been determined. HXKs generally contain conserved aa residues involved in the binding of hexose and ATP [42, 44]. Alignment of the ZmHXK aa sequences indicated that all of them contain the hexose- and ATP-binding sites (Supplementary Fig. 1).

Results The maize HXK gene family After a careful survey of the maize genome, we identified nine ZmHXK gene family members (Table 1). None of the ZmHXK genes were homologous to the rice OsHXK1 or OsHXK2 genes, but two of the maize genes, ZmHXK3a and ZmHXK3b, were homologous to OsHXK3. The ZmHXK proteins were 459–509 aas in length. BLAST analysis by

Phylogenetic analysis To investigate the patterns of evolution and the phylogenic relationships among the HXK genes in maize (9 genes), rice (10 genes), Arabidopsis (6 genes), and sorghum (7 genes), the aa sequences encoded by the HXK genes were aligned using ClustalX 2.0 software, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA4 software (Fig. 4). The proteins grouped into four clusters (I–IV). Cluster I contained six proteins with one from maize, three from rice, two from Arabidopsis, and one from sorghum. Cluster II included seven proteins with two from maize, two from rice, and two from sorghum. Cluster III contained 12 proteins with 3 from maize, 3 from rice, 4 from Arabidopsis, and 2 from sorghum. Cluster IV

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Mol Biol Rep Table 1 List of HXK genes in maize

Gene names

Chromosomesa

ORF length

Protein length

Protein ID

TD

PLb

ZmHXK3a

3

1,494

497

GRMZM2G068913_P01

8–24

Chloroplast

ZmHXK3b

8

1,494

497

GRMZM2G467069_P01

8–24

Chloroplast

ZmHXK4

8

1,543

509

GRMZM2G058745_P01

5–21

Cytoplasmic Vacuolar

ZmHXK5

6

1,524

507

GRMZM2G432801_P01

5–21

TD transmembrane domain

ZmHXK6

3

1,521

506

GRMZM5G856653_P01

12–28

Vacuolar

a

Chromosome number in which the gene resides

ZmHXK7

6

1,380

459

GRMZM2G051806_P01

No

Cytoplasmic

ZmHXK8

3

1,512

503

GRMZM2G104081_P01

No

Cytoplasmic

b

ZmHXK9

3

1,515

504

GRMZM2G171373_P01

7–23

Chloroplast

ZmHXK10

6

1,512

503

GRMZM2G046686_P01

9–25

Chloroplast

Localization of ZmHXK protein supported by PSORT (http://psort.nibb.ac.jp)

Fig. 1 Chromosomal localization of maize HXK genes. Segmental duplicates, including ZmHXK3a/ ZmHXK3b, ZmHXK4/ZmHXK5 and ZmHXK7/ZmHXK8, are indicated by dotted arrows

had seven proteins with three from maize, two from rice, and two from sorghum. Cis-element analysis We analyzed the promoter regions contained in the B73 genomic DNA sequences extending 2 kb upstream of the translation start sites of the ZmHXK genes by searching the PLACE database. A total of 89 putative cis-elements greater than 6 bp in length were identified (Supplementary Table S1). The functions of the cis-elements included responses to

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phytohormones and abiotic stress, sugar repression, light and circadian rhythm regulation, Ca2? responses, seed development and germination, and CO2-responsive transcriptional activation. The cis-elements included ABRERATCAL, ANAERO1CONSENSUS, ARR1AT, BOXLCOREDCPAL, CAATBOX1, CARGCW8GAT, CGCGBOXAT, CIACADI ANLELHC, CTRMCAMV35S, DPBFCOREDCDC3, EBOXBNNAPA, EECCRCAH1, E2FCONSENSUS, GT1CON SENSUS, INRNTPSADB, MYBCORE, MYBPLANT, MY CCONSENSUSAT, POLASIG2, PRECONSCRHSP70A, RYREPEATGMGY2, RYREPEATLEGUMINBOX, SE

Mol Biol Rep Fig. 2 Structures of maize HXK gene family members. Exons and introns are indicated by the filled boxes and lines, respectively. Intron phases 0, 1, and 2 are indicated by the numbers 0, 1, and 2 above the introns, respectively

Fig. 3 Motif distribution among Arabidopsis, rice, sorghum, and maize HXK proteins. Motifs in the HXK proteins were identified using the MEME web server

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Fig. 4 Phylogenetic tree for Zea mays, Arabidopsis, Oryza sativa, and sorghum HXK genes. A joint unrooted tree was generated using the neighbor joining method with MEGA4 software

BFCONSSTPR10A, SEF4MOTIFGM7S, TATABOX2, and TATABOX5.

their expression patterns were analyzed in three-leaf stage shoots treated with exogenous ABA, cytokinin (6-BA), GA, auxin (2,4-D), PEG, sodium chloride (NaCl), and low temperature (4 °C) using qRT-PCR. Expression levels of both ZmHXK5 and ZmHXK6 were responsive to all seven treatments. Both ZmHXK5 and ZmHXK6 expression was upregulated in shoots in response to phytohormone treatment (Fig. 6). In response to ABA treatment, the maximum levels of upregulated ZmHXK5 and ZmHXK6 expression were reached after 1 h of treatment. In response to GA treatment, the maximum level of ZmHXK5 expression was observed after 24 h and that of ZmHXK6 after 6 h. ZmHXK5 and ZmHXK6 expression levels were highest after 1 h of treatment with 6-BA or 2,4-D. In response to abiotic stressors (cold temperature, NaCl, and PEG), both ZmHXK5 and ZmHXK6 exhibited upregulated expression in shoots (Fig. 6), as well as in response to cold temperature, with maximum expression reached 1 h after exposure at 4 °C. PEG treatment resulted in upregulated expression of ZmHXK5 and ZmHXK6 in as soon as 1 h, with the highest levels occurring after 6 and 3 h of treatment, respectively. Under NaCl stress, ZmHXK5 expression exhibited sustained upregulation that reached a maximum after 24 h, while maximum ZmHXK6 expression was seen after 6 h.

ZmHXK5 and ZmHXK6 expression patterns

35 30 25 20 15 10 5 0

ZmHXK5

Roots

Stems Leaves

Silks

Discussion The maize HXK family HXK is a component of various eukaryotic Glc response pathways [45]. In this study, we identified nine genes belonging to the maize HXK gene family. Among the better characterized plant HXK gene families, Arabidopsis has 6 HXK genes [17, 18], rice at least 10 expressed HXK genes [20], and tomato at least 4 HXK genes [21]. The nine ZmHXK genes are distributed among chromosomes 3, 6, and 8 (Fig. 1). The 10 OsHXK genes are located on rice chromosomes 1, 5, and 7 [20]. The six AtHXK genes are located on Arabidopsis chromosomes 1, 5, and 7 [18]. Most

Relative expression

Relative expression

To investigate the functions of maize HXKs, we characterized the expression patterns of the ZmHXK5 and ZmHXK6 genes, which are evolutionarily related to the OsHXK5, OsHXK6, and AtHXK1 genes. Expression profiling was performed using quantitative RT-PCR (qRTPCR) with primers specific to ZmHXK5 and ZmHXK6 to determine their expression patterns in roots, leaves, stems, silks, ears, and tassels. ZmHXK5 exhibited high expression in tassels, while ZmHXK6 was expressed in both leaves and tassels (Fig. 5), suggesting that ZmHXK5 expression may be pollen-specific. Many plant gene families are involved in both development and stress responses. To determine whether the ZmHXK5 and ZmHXK6 genes were responsive to stress,

Ears

Tassels

ZmHXK6

20 15 10 5 0

Roots

Stems

Leaves

Silks

Ears

Tassels

Fig. 5 Relative levels of ZmHXK5 and ZmHXK6 transcripts in various tissues based on quantitative real-time PCR analysis

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ZmHXK5 3 2 1 0

0

1

3

6

12

24

Relative expression

Relative expression

Mol Biol Rep ZmHXK6 3 2 1 0

0

3

6

12

24

100µM GA treatment time(h) ZmHXK5

3 2 1 0

0

1

3

6

12

24

100µM 6-BA treatment time (h) ZmHXK5

3 2 1 0

0

1

3

6

12

24

100µM 2,4-D treatment time (h) ZmHXK5

6 4 2 0

0

1

3

6

12

24

4°C treatment time (h) ZmHXK5

3 2 1 0

0

1

3

6

12

24

20% PEG treatment time (h)

4 3 2 1 0

ZmHXK5

0

1

3

6

12

24

200mM NaCl treatment time (h)

Relative expression

1

Relative expression

0

Relative expression

1

Relative expression

2

0

3

6

12

24

ZmHXK6

3 2 1 0

0

1

3

6

12

24

100µM GA treatment time(h) ZmHXK6

3 2 1 0

0

1

3

6

12

24

100µM 6-BA treatment time (h) ZmHXK6

3 2 1 0

0

1

3

6

12

24

100µM 2,4-D treatment time (h) ZmHXK6

6 4 2 0

0

1

3

6

12

24

12

24

4°C treatment time (h)

Relative expression

ZmHXK5

3

1

100µM ABA treatment time(h)

4 3 2 1 0

Relative expression

Relative expression

Relative expression

Relative expression

Relative expression

Relative expression

Relative expression

100µM ABA treatment time (h)

4 3 2 1 0

ZmHXK6

0

1

3

6

20% PEG treatment time (h) ZmHXK6

0

1

3

6

12

24

200mM NaCl treatment time (h)

Fig. 6 Relative ZmHXK5 and ZmHXK6 expression levels in response to hormone treatment (ABA, 2,4-D, 6-BA, or GA) and abiotic stress (cold temperature, NaCl, or PEG) based on real-time quantitative

PCR analysis. Samples were collected for analysis after 0, 1, 3, 6, 12, and 24 h of treatment. The transcript level at the 0 h time point (untreated) was set equal to 1 and used to normalize expression values

maize (Fig. 2), Arabidopsis, and rice HXK genes have nine exons [17, 18], the same is true of most SbHXK genes, except for SbHXK7 and SbHXK9, which have eight and six exons, respectively (Supplementary Fig. 2).

Characterization of ZmHXK promoter cis-elements By controlling promoter efficiency, cis-elements play a vital role in the regulation of gene expression. Analysis of cis-

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elements could provide a foundation for further functional analysis of the ZmHXK gene family. In this study, we identified cis-elements involved in responses to phytohormones and abiotic stress, sugar repression, light and circadian rhythm regulation, Ca2? responses, seed development and germination, and CO2-responsive transcriptional activation. Among these cis-elements, eight types were present in all nine ZmHXK genes, including dehydration-stress responsive elements (CBFHV, MYBCORE and MYCCONSENSUSAT), a cold-responsive element (DRECRTCOREAT), ABA-responsive elements (MYB2CONSENSUSAT and DPBFCOREDCDC3), a salicylic acid-responsive element (GT1CONSENSUS), a pathogen- and NaCl-responsive element (GT1GMSCAM4), light-responsive elements (EBOXBNNAPA and INRNTPSADB), a Ca2?-responsive element (CGCGBOXAT) [46], and a fermentation pathwayresponsive element (ANAERO1CONSENSUS) [47]. Five types of elements related to sugar repression (PYRIMIDINEBOXOSRAMY1A, SREATMSD, TATCCAOSAMY, TATCCAYMOTIFOSRAMY3D, and WBOXH VISO1) [48, 49] were present in most of the ZmHXK promoters. The Ca2?-responsive ABRERATCAL and CO2responsive EECCRCAH1 cis-elements were present in eight of the nine ZmHXK promoters. The protein storage-related element, RYREPEATLEGUMINBOX [50], and the circadian-regulation element, CIACADIANLELHC [51], were also present in most of the ZmHXK promoters. ZmHXK5 and ZmHXK6 expression patterns To examine the functions of different maize HXK isoforms, we characterized the expression patterns of the ZmHXK5 and ZmHXK6 genes, which are evolutionarily related to the rice OsHXK5 and OsHXK6 genes. The members of HXK gene families in rice and Arabidopsis were shown previously to exhibit tissue-specific expression. In Arabidopsis, AtHKL1, AtHKL2, and AtHXK1 transcripts were abundant in young leaves, mature leaves, roots, flowers, siliques, and stems. AtHXK2 transcripts were relatively abundant in leaves, while those of AtHXK3 were abundant in roots and siliques. In contrast, AtHKL3 mRNA was detected only in flowers [18]. The OsHXK genes, except for OsHXK1 and OsHXK10, were expressed in leaves, roots, flowers, and immature seeds, while OsHXK10 was expressed only in flowers. OsHXK2, OsHXK4, OsHXK5, OsHXK6, and OsHXK8 transcripts were expressed preferentially in endosperm, while OsHXK3 and OsHXK7 transcripts were abundant in seed coats [20]. In maize, ZmHXK5 was expressed at high levels in tassels, while ZmHXK6 was expressed in leaves and tassels (Fig. 5), suggesting that ZmHXK5 expression may be pollen-specific. Sugar signaling pathways in plants interact with various plant hormone signaling networks, including the ABA,

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cytokinin, GA, ethylene, and auxin pathways [7, 10, 30]. Glc signaling during early vegetative development requires numerous ABA-associated genes and processes [9, 52]. Glc represses genes associated with ethylene biosynthesis [53, 54]. A recent study indicated that HXK1 was a positive effector of several Glc and ethylene interactions in plants [55]. In this study, we showed that the expression of both ZmHXK5 and ZmHXK6 was upregulated in shoots in response to treatment with exogenous ABA, 6-BA, GA, or 2,4-D (Fig. 6). In maize and tomato roots, HXK activity was elevated by hypoxia [56, 57]. In the flood-tolerant species, Echinochloa phyllopogon, anaerobic stress stimulated HXK activity, but heat shock and salt treatment restrained activity in shoots and roots. Cold stress up regulated HXK activity only in roots. In the flood intolerant species (Echinochloa crus-pavonis) HXK activity was stimulated to varying degrees in roots by anaerobic and heat shock conditions and by salt stress and chilling [58]. In this study, both ZmHXK5 and ZmHXK6 exhibited upregulated expression in shoots in response to cold, NaCl, and PEG treatments (Fig. 6). Acknowledgments We are grateful to editors and reviewers for their helpful comments. This work was supported partly by Beijing Nova Program (No. Z121105002512031), Youth Foundation of Beijing Academy of Agriculture and Forestry Sciences (No. QNJJ201303), Beijing Academy of Agriculture and Forestry Sciences (No. KJCX20140202), the Beijing Municipal Science and Technology Commission (No. Z121100001512012).

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