MA Yu-xin, ZHOU Zhi-jun, CAO Hong-zhe, ZHOU Fan, Sl He-long, ZANG Jin-ping, XlNG Ji-hong,3#, ZHANG Kang,3#, DONG Jin-gao#

1 State Key Laboratory of North China Crop Improvement and Regulation, Hebei Agricultural University, Baoding 071000, P.R.China 2 Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Hebei Agricultural University, Baoding 071000, P.R.China

3 Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes, Hebei Agricultural University,Baoding 071000, P.R.China

4 Experimental Training Center of Hebei Agricultural University, Hebei Agricultural University, Baoding 071000, P.R.China

Abstract Sugar is an indispensable source of energy for plant growth and development, and it requires the participation of sugar transporter proteins (STPs) for crossing the hydrophobic barrier in plants.Here, we systematically identified the genes encoding sugar transporters in the genome of maize (Zea mays L.), analyzed their expression patterns under different conditions, and determined their functions in disease resistance.The results showed that the mazie sugar transporter family contained 24 members, all of which were predicted to be distributed on the cell membrane and had a highly conserved transmembrane transport domain.The tissue-specific expression of the maize sugar transporter genes was analyzed, and the expression level of these genes was found to be significantly different in different tissues.The analysis of biotic and abiotic stress data showed that the expression levels of the sugar transporter genes changed significantly under different stress factors.The expression levels of ZmSTP2 and ZmSTP20 continued to increase following Fusarium graminearum infection.By performing disease resistance analysis of zmstp2 and zmstp20 mutants, we found that after inoculation with Cochliobolus carbonum, Setosphaeria turcica, Cochliobolus heterostrophus, and F.graminearum, the lesion area of the mutants was significantly higher than that of the wild-type B73 plant.In this study, the genes encoding sugar transporters in maize were systematically identified and analyzed at the whole genome level.The expression patterns of the sugar transporter-encoding genes in different tissues of maize and under biotic and abiotic stresses were revealed, which laid an important theoretical foundation for further elucidation of their functions.

Keywords: maize, sugar transporter, gene expression, disease resistance

1.lntroduction

Maize is one of the three major grain crops in China, and it is also an important feed and industrial raw material.Disease has always been an important factor affecting maize yield and quality.There are more than 80 kinds of maize diseases have been reported in the world, and more than 40 kinds of maize diseases have been reported in China.Among them, there are more than 20 kinds of diseases, including northern leaf blight of corn, southern leaf blight of corn, corn curvularia leaf spot, gray leaf spot in maize, corn sheath blight, corn smut, corn stalk rot,maize ear rot, and so on (Farhan and Yan 2012).

The fungal pathogenCochliobolus carbonum(anamorph,Bipolaris zeicola) causes northern leaf spot,leading to a ubiquitous and devastating foliar disease of corn in Yunnan Province, China (Zhanget al.2014).In the early stage of corn northern leaf blight, small spots of water stains and bluish gray appeared on the leaves,and then gradually expanded along the veins to both ends, forming a dark brown edge, central tawny or taupe spindle-shaped spots, late lesions often longitudinal split(Xiaet al.2020).The hospital asexual type causing northern leaf blight isExserohilum turciumand the sexual type isSetosphaeria turcica(Zenget al.2020).The disease time of southern leaf blight disease of corn is earlier than that of northern leaf blight disease.The spots on the leaves are smaller and more.The asexual pathogen isBipolaris maydis, and the sexual pathogen isCochliobolus heterostrophus(Zuchmanet al.2021).Stalk rot is a general term for root and stem rot caused by a variety of pathogens alone or in combination, and the main pathogen isF.graminearum(Yu and Kim 2020).

Carbohydrates play an important role in plant growth and development as energy molecules, cell compound precursors, and signal transduction and environmental stress response signaling molecules (Zhanget al.2019).In plants, sugar transporters are categorized into monosaccharide transporters (MSTs) and sucrose transporters (SUTs) according to the types of sugars transported (Chenet al.2010).Sugar transporters can be subdivided into four different evolutionary branches (I to IV).Evolutionary branches I, II, and IV mainly transport hexose, while evolutionary branch III transports sucrose (Rozennet al.2015).The hexose transporter of evolutionary branch IV is located in vacuoles, and the sucrose transporter of evolutionary branch III has been identified as a candidate in the apoplast to promote sucrose diffusion into various plant tissues (Chardonet al.2013).The transport of glucose and fructose hydrolyzed from sucrose in the apoplast is regulated by the sugar transporter protein(STP) and hexose transporter (Choet al.2010).STP belongs to a branch of the MST superfamily and is considered to be an H+/sugar symporter.It usually contains 12 transmembrane domains and is localized on the cell membrane (Choet al.2010).STP uses an acid gradient to enable the passage of fructose, glucose,and other monosaccharides through the impermeable cell membrane (Yanet al.2013).STPs in plants have a higher affinity for sugars as compared to similar proteins in animals and bacteria, and their binding strength to sugars is 1 000-fold that of human-like proteins(Singhet al.2022).With the rapid development of the whole-genome sequencing technique, the genomewide identification of STP genes in various plants has been reported.The most thoroughly studied plant isArabidopsis thaliana(Buttneret al.2010).

To date, 14 STPs have been identified inA.thaliana.For example,AtSTP1is expressed in germinated seeds and young roots, and it has been shown to mediate extracellular hexose absorption (Otoriet al.2019).Overexpression ofAtSTP1can increase the expression levels ofYUC8andYUC9(genes involved in auxin metabolism), thereby expressing a large amount of auxin to inhibitA.thalianabud branching.It also directly stimulates the accumulation of the branchrelated protein BRC1, thereby inhibiting the branching ofA.thalianabuds.However, in the presence of hexose and sucrose,AtSTP1is involved in sugar transport,and the expression levels of YUC8, YUC9, and BRC1 are downregulated, resulting in branching (Cordobaet al.2015).AtSTP2is expressed in the early stage of pollen development and is most likely to mediate glucose production by callose degradation (Leachet al.2017).AtSTP4shows the highest expression in mature pollen grains (Leachet al.2017).AtSTP6is closely related to the absorption of fructose, which appears in the developmental stage of pollen and accumulates during pollen maturation (Scholzet al.2003).AtSTP11only appeared in fully mature pollen and pollen tubes(Scholzet al.2003).AtSTP13is a high-affinity hexose transporter gene expressed in vascular tissues, roots,guard cells, and cotyledons of newborn petals (Nour-Eldinet al.2006).

In addition to participating in plant growth and development, sugar transporter family members also actively respond to stress and are involved in plant disease resistance pathways.InA.thaliana, the AtSTP13 protein is located in the cytoplasm and transports glucose and fructose from the apoplast into the cytoplasm, and the extracellular sugars are utilized by pathogens.In the specific disease resistance pathway, following the infection of a cell by a pathogen, AtSTP13 binds to the leucine-rich repeat receptor kinase FLAGELLIN SENSING 2 (FLS2)and phosphorylates itself, thereby increasing glucose and fructose intake.Simultaneously, sugar in the apoplast for pathogen consumption is reduced, thus leading to disease resistance (Flütschet al.2020).AtSTP14is a galactose-specific transporter.AtSTP14plays a role in sugar supply during early embryonic development and late endosperm formation.Overexpression ofAtKIN10, a central regulator of plant stress and energy management,induces the expression ofAtSTP14to participate in stress resistance (Poschetet al.2010).

The monosaccharide transporterOsGMST1in rice(Oryza sativa) is upregulated under salt stress, and the knockout of this gene causes hypersensitivity of rice to salt stress (Caoet al.2011).OsSWEET11,OsSWEET12, andOsSWEET14were upregulated duringFlavimonas oryzihabitansinfection (Yanget al.2006; Chuet al.2011; Qinet al.2020).The Pst-induced sugar transporterTaSTP3inTriticum aestivum, under the transcriptional regulation ofTaWRKY19/61/82, may promote wheat susceptibility through the synergistic effect of “sugar supply” and “sugar signaling” (Huaiet al.2022).Upregulation of the SWEET genes was also observed during pathogen attack in cassava (Manihot esculenta),apple (Malus pumilaMill.), citrus (Citrus paradisi), clover(Medicago truncatula), vine (Vitis vinifera), and sweet potato (Ipomoea batatas) (Chenet al.2010; Chonget al.2014; Cohnet al.2014; Huet al.2014; Liet al.2017;Luet al.2019).An increase in the number of sugar transporters results in sugar accumulation in the apoplast,which is used by pathogens as a source of carbon and energy (Lemoineet al.2013).

In recent years, studies have shown that SUTs play an important role in the growth of maize.ZmSut1mutant plants show growth retardation, leaf chlorosis, inability to transport radioactively labeled sucrose in source leaves,and reduced accumulation of sugar and starch in leaves(Poschetet al.2010).Studies have also shown that maize ZmSUT plays an important role in drought stress response (Tranet al.2017).

In the present study, we systematically identified and analyzed the genes encoding sugar transporters in maize at the whole genome level and revealed the expression patterns of these genes in different tissues of maize and under biotic and abiotic stresses.The results confirmed thatZmSTP2andZmSTP20play an important role in disease resistance, which lays an important theoretical foundation for further studies of the potential function of the maize STP family in molecular breeding.

2.Materials and methods

2.1.Plant materials and treatments

The plant materials used in this study included maize inbred line B73,zmstp2mutant seeds (EMS4-0C7022),andzmstp20mutant seeds (EMS4-14C93A) (Luet al.2018).Maize inbred lines and mutants were planted in a greenhouse in Baoding City (Hebei Agricultural University, Hebei Province, China).Methyl jasmonate(Sigma, Missouri, USA) and salicylic acid (Thermo Fisher Scientific, Massachusetts, USA) were sprayed on the entire seedlings at the V3 (3th leaf) growth stage (Liet al.2015).

The pathogen strains used in this study includedC.carbonum,S.turcica,C.heterostrophus, andF.graminearum.Cochlioboluscarbonum,S.turcica, andC.heterostrophuswere grown on potato dextrose agar(PDA) culture mediums for 6 days at 28°C.A punch with a diameter of 0.8 cm was used to punch holes.The fungal discs were picked up and inoculated on the leaves of the plant in the V5 growth stage.The inoculation site was covered with a plastic film to retain moisture and avoid other biological contaminants.Fusariumgraminearumwas grown on PDA plates for 6 days at 28°C.A punch with a diameter of 0.8 cm was used to punch the holes.The fungal discs were picked up and added to sterile distilled water containing 0.05% (v/v) Triton X-100 to make a conidial suspension.The concentration of conidia was quantified using a blood cell counter and diluted to 1×106spores mL–1for inoculation.A sterile micropipette tip (10 mm hole depth) was used to inoculate 20 μL of the conidial suspension on the 2nd or 3rd internode above the soil line of the corn stalk.The wound was covered with a sterile gauze to retain moisture and avoid contamination by other organisms.

The leaves infected by the lesion were cut and soaked in trypan blue dye solution for 3 h to make the lesion fully colored.After being placed in chloral hydrate for decolorization for more than 24 h, the leaves were finally taken out for photographing.

2.2.Screening and identification of maize histone coding genes

The gene and protein sequences of the maize STP family were downloaded from MaizeGDB (https://www.maizegdb.org).During screening of the STP family, the HMM configuration file of Sugar_tr domain (PF00083)was obtained from the Pfam Database (http://pfam.xfam.org/).All STPs were searched by HMMER 3.2.1 (Finnet al.2015) (using default parameters) and BLASTP(Jacobet al.2008) (E-value

2.3.Analysis of physicochemical properties of maize histone

MaizeGDB was used to obtain the location information,sequence length, and amino acids of the encoded proteins of the maize STP gene family.The bioinformatics software ExPASy-Prot Param tool in Expasy (http://web.expasy.org/protparam/) was used to analyze the molecular weight and isoelectric point of the proteins encoded by the maize STP family.

2.4.Phylogenetic tree construction and domain analysis of the STP family

MEGA 7 (Kumaret al.2016) was used to construct the phylogenetic tree based on the protein sequences ofOryza sativa,B.distachyon,S.bicolor, andZ.maysSTP families, and phylogenetic tree analysis was then performed.According to the neighbor-joining method(bootstrap value was set to 1 000), the phylogenetic tree was obtained using the Jones-Taylor-Thornton model.The iTOL (https://itol.embl.de/) online tool was then used to manipulate the phylogenetic tree.The phylogenetic tree of the maize STP family was obtained using the same method.The protein sequence of the maize STP family was obtained, and the domain location information of the family was determined by the online website SMART(http://smart.embl-heidelberg.de/) and Pfam (http://pfam.xfam.org).The gene domain was drawn by IBS (http://ibs.biocuckoo.org).

2.5.Chromosome positioning analysis

According to the position of the maize STP family members in the genome, we used Rideogram, an R language package, to map the maize STP family genes on chromosomes (Haoet al.2020).

2.6.Analysis of gene expression in different tissues and under biotic and abiotic stresses

By using the data downloaded from the SRA Database in NCBI (https://www.ncbi.nlm.nih.gov) (Appendix A).Hisat2 (Perteaet al.2016) with default parameters was used to align the transcriptome dataset to the downloaded maize reference genome.StringTie Software (Perteaet al.2016) was used to calculate gene expression values through the standardized parameters of gene length and read number, and FPKM (read fragments per thousand base transcripts per million mappings) represented the gene expression levels.Heml (https://hemi.biocuckoo.org) was used to draw the expression heat map of maize sugar transporter members in different tissues and under biotic and abiotic stresses and to determine the expression pattern of the maize sugar transporter encoding genes.

2.7.Real-time quantitative PCR analysis

All samples were stored in liquid nitrogen before RNA extraction.Total RNA was extracted from the sample using TRIzol (Invitrogen, Massachusetts, USA) and purified using a RNeasy column (Qiagen, Dusseldorf,Germany).Actin was used as a reference for qRT-PCR,and TransStart Tip Green qPCR SuperMix was used according to the manufacturer’s instructions.cDNA from the sample was collected at different time points as a template.Moreover, each gene in the wild-type B73 plant and its relative expression levels at different time points were compared by Ct analysis (2-ΔΔCt), and the quantitative data were expressed as mean±SEM.The primers used in this study are listed in Appendix B.

3.Results

3.1.ldentification of STP family members in maize

HMMER 3.2.1 and BLASTP (E-value

3.2.Construction of maize STP phylogenetic tree and domain analysis

Multiple sequence alignment and phylogenetic tree analysis of the protein sequences of the STP family inO.sativa,B.distachyon,S.bicolor, andZ.mayswere performed.According to the classification of rice OsSTP,the STP family can be divided into four subfamilies:CLASS I, CLASS II, CLASS III, and CLASS IV (Fig.1).Each subfamily contains proteins fromO.sativa,B.distachyon,S.bicolor, andZ.mays, indicating that these grassy STP genes share a common ancestor.

The phylogenetic tree of the maize STP family was obtained by the same method.The gene domain of the maize STP family was drawn (Fig.2).As shown in the figure, the maize STP family can be divided into four subfamilies.All four subfamilies contain transmembrane domains.The number of subfamily II and IV domains shows the least changes, and these domains are strongly conserved.Although the number of subfamily I and III domains shows high changes, except forZmSTP9with two structural domains, the number of other domains is restricted to 10.The results reveal that the maize STP family is a structurally conserved gene family.Previous studies have shown that the STP is a complete membrane protein with multiple transmembrane domains and is regarded as an H+/sugar cotransporter located on the plasma membrane(Nakadet al.2022).We analyzed the subcellular localization of maize STP family proteins.The results showed that corn STPs had multiple transmembrane domains and were localized on the cell membrane; this finding was consistent with the results of conserved domain analysis.

3.3.Positioning analysis of maize STP family members

To study the distribution of maize STP family members in chromosomes, we created the chromosome distribution map by mapping ZmSTPs in the genome.The results showed that ZmSTPs were evenly distributed in multiple chromosomes of maize (Fig.3); this distribution pattern may be closely related to their important function of transporting sugar.ZmSTPs did not appear in chromosomes 6 and 8, while they were distributed in the other eight chromosomes.We also predicted the subcellular localization of maize STP family members and found that all the members were located on the cell membrane.Thus, the maize STP gene family is widely distributed in maize chromosomes, and the protein is concentrated on the cell membrane.

3.4.Expression analysis of maize STP family members in different tissues

Fig.2 Phylogenetic tree and domain analysis of the maize STP family members.The phylogenetic tree of the maize STP family is shown in the left figure, and the different color patterns represent different subfamilies.Hexagon represents the transmembrane domain of the maize STP protein.

The expression levels of maize STP family members in different tissues were determined using the data obtained from NCBI.The results showed that the four subfamily members were involved in both vegetative and reproductive growth processes.Among the 24 genes,15 genes were found in all stages of maize, especially in leaves, such asZmSTP2,ZmSTP10,ZmSTP20, andZmSTP22(Fig.4).We speculate that these genes may be closely related to photosynthesis.Additionally, some ZmSTPs genes were highly expressed in panicles and anthers, such asZmSTP8,ZmSTP11,ZmSTP12, andZmSTP14, among whichZmSTP12showed the highest expression level (Fig.4).We speculate that these genes may play an important role in reproductive growth.

3.5.Abiotic and biotic stress analysis of maize STP family members

To study the expression pattern of ZmSTPs in response to abiotic stress, we analyzed the transcriptome data obtained under abiotic stress conditions such as high temperature, cold, high salt, ultraviolet irradiation, and drought.According to our results, the expression values forZmSTP12,ZmSTP13,ZmSTP17, andZmSTP24in the abiotic stress data were all zero and were not presented on the graph.Moreover, the expression levels of the maize STP family genes were significantly different under abiotic stress (Fig.5).Under high temperature stress,the expression of most genes was downregulated, while the expression ofZmSTP2,ZmSTP8, andZmSTP14was upregulated.Under cold stress, the expression of most genes remained unchanged; a few genes showed decreased expression, while the expression ofZmSTP6increased.In high salt stress, the expression of most genes decreased, the expression of a few remained unchanged, while the expression ofZmSTP4increased.Under UV stress, most genes showed no change in expression; however, a few genes showed decreased expression, while the expression ofZmSTP2andZmSTP20increased.In drought stress, the expression of most genes decreased, and the expression ofZmSTP3,ZmSTP4,ZmSTP6,ZmSTP9, andZmSTP19increased(Fig.5).

Fig.4 Tissue expression heat map of the maize STP family genes.The logarithm of the gene expression value is based on 2.Color marks indicate changes in gene expression; blue represents high expression and white represents low expression.

Fig.5 Abiotic stress heat map of the maize STP family genes.The logarithm of the gene expression value is based on 2.Color marks indicate changes in gene expression; blue represents high expression and white represents low expression.

For investigating the effect of biological stress, we infected maize stem withF.graminearumand observed the changes in the expression level ofZmSTPgenes.Six maize STP family members (ZmSTP8,ZmSTP11,ZmSTP14,ZmSTP15,ZmSTP17, andZmSTP24) showed no change under biotic stress; hence, they are not shown in the Fig.5.The expression levels of the maize STP family genes showed different trends under biotic stress.ZmSTP2,ZmSTP7,ZmSTP16, andZmSTP20showed increased expression levels gradually at 48 h.Therefore,these genes, especiallyZmSTP2andZmSTP20,were predicted to be involved in resistance toF.graminearuminfection (Fig.6).

3.6.Expression analysis of maize STP family members under different hormonal treatments

Fig.6 Expression heat map of maize STP family genes following Fusarium graminearum infection.The logarithm of the gene expression value is based on 2.Color marks indicate changes in gene expression; blue shows high expression and white shows low expression.

Real-time qPCR was used to detect the expression of the maize STP family genes during treatment with jasmonic acid and SA.Following jasmonic acid treatment, the changes in the expression of the maize STP family genes were mainly divided into three categories.In the first category, the expression level ofZmSTP2andZmSTP20increased rapidly after 3 h of jasmonic acid treatment,then decreased slowly at 9 h, and continued to decrease or increase slowly at 24 h.In the second category, the expression level ofZmSTP13increased slowly within 3 h of the treatment and then continued to increase,reaching the highest level at 24 h.In the third category,the expression level ofZmSTP21rapidly declined at 3 h of jasmonic acid treatment and then increased.On the basis of these results, it is speculated that the maize STP family genes are involved and play different roles in the jasmonic acid signaling pathway (Fig.7).

Following SA treatment, the changes in the expression level of the maize STP family members were mainly divided into three categories.In the first category, the expression ofZmSTP2increased rapidly after 3 h of SA treatment and then decreased slowly at 9 h.In the second category,the expression level ofZmSTP4continued to increase after SA treatment, reaching the highest value at 24 h.In the third category,ZmSTP5showed a rapid decrease in expression at 3 and 9 h after SA treatment, reached the lowest expression level, and then showed an increase in expression.From these findings, it is speculated that the maize STP family genes are involved and play different roles in the SA signaling pathway (Fig.8).

Fig.7 Expression levels of the ZmSTP family genes following jasmonic acid treatment.Expression pattern of each ZmSTP gene after methyl jasmonate (MeJA) treatment from 0 to 24 h.qRT-PCR was performed using gene-specific primers.Data are expressed as mean±SE of three independent experiments.＊ and ＊＊, significant at P<0.05 and P<0.01, respectively by the student’s t-test.

Fig.8 Expression levels of the ZmSTP family genes following salicylic acid (SA) treatment.Expression pattern of each ZmSTP gene after SA treatment from 0 to 24 h.qRT-PCR was performed using gene-specific primers.Data are expressed as mean±SE of three independent experiments.＊ and ＊＊, significant at P<0.05 and P<0.01, respectively by the student’s t-test.

3.7.Functional study of ZmSTP2 and ZmSTP20 against F.graminearum

The homozygous plants ofzmstp2andzmtp20mutants were selected as experimental materials, and the mutation sites were detected by PCR to determine the homozygous plants (Appendix C).To determine whetherzmstp2andzmtp20are involved in the disease resistance of maize, we inoculated maizezmstp2andzmtp20mutant plants and the wild-type B73 plant withF.graminearumand compared the lesion area ofzmstp2andzmtp20relative to B73 (Fig.9).The results showed that 7 days after inoculation, the symptoms ofzmstp2andzmtp20mutants were more obvious than that of the wild-type B73 plant, indicating thatZmSTP2andZmSTP20may be involved in the resistance of maize toF.graminearum.

Fig.9 Comparison of lesions of maize mutants inoculated with pathogens.A, lesion comparison of wild-type B73 and zmstp2 and zmstp20 mutants inoculated with Fusarium graminearum suspension.Scale bar indicates 1 cm.B, lesion comparison of wild-type B73 and zmstp2 and zmstp20 mutants inoculated with Cochliobolus heterostrophus, Cochliobolus carbonum, and Setosphaeria turcica.Scale bar indicates 1 cm.Green leaves were before destaining, lesions were yellow, white leaves were after destaining with chloral hydrate, lesions were blue after trypan blue staining.C, lesion comparison of wild-type B73 and zmstp2 and zmstp20 mutant stems inoculated with F. graminearum suspension.D, lesion statistics of wild-type B73 and zmstp2 and zmstp20 mutant leaves inoculated with C. heterostrophus, C. carbonum, and S. turcica.Data are expressed as mean±SE of three independent experiments.

To confirm whetherZmSTP2andZmSTP20have broad-spectrum disease resistance, we conducted leaf inoculation experiments usingC.heterostrophus,C.carbonum, andS.turcica.The leaves were photographed at 4 days after the inoculation ofC.heterostrophusandS.turcicaand at 5 days after the inoculation ofC.carbonum.The results of the inoculation test showed significant differences in the disease spots.The area of disease spots in the leaves ofzmstp2andzmtp20mutants was significantly larger than that in the leaves of the B73 plant, thus indicating thatZmSTP2andZmSTP20play an important role in the resistance of maize toC.heterostrophus,C.carbonum, andS.turcica.

4.Discussion

The STP gene family plays an important role in plant monosaccharide distribution and participates in many metabolic processes during growth and development(Liuet al.2018).The STP genes have been shown to play an important role in plant growth and development inO.sativa,B.distachyon,S.bicolor, andZ.mays.However, the expression pattern of the STP genes in maize has not yet been studied.In the present study,the physicochemical properties, conserved domains,phylogenetic evolution, and expression patterns of the maize STP gene family members were analyzed by bioinformatics methods.

In this study, 24 ZmSTP genes were identified by screening the maize genome sequence, and these 24 ZmSTPs were evenly distributed on eight chromosomes.All ZmSTP conserved domains contain only transmembrane domains; only five ZmSTPs have 12 transmembrane domains.Most ZmSTPs have 11 transmembrane domains, of which four ZmSTPs have 10 transmembrane domains and two have nine transmembrane domains.Specifically,ZmSTP24has eight transmembrane domains andZmSTP23the least,with two transmembrane domains.Phylogenetic analysis revealed that all the proteins ofO.sativa,B.distachyon,S.bicolor, andZ.mayscould be divided into four groups; each group contained OsSTP, BdSTP, SbSTP,and ZmSTP, indicating that the STP proteins of the four species were closely related.In the evolutionary tree, the number distribution of ZmSTP and SbSTP in each branch was similar, thus indicating that maize STP has a closer relationship with sorghum STP.SbSTP showed different expression patterns in different sorghum tissues.For example,SbSTP7was highly expressed in leaves, whileSbSTP6,SbSTP10,SbSTP9, andSbSTP17were highly expressed in inflorescences (Bihmidineet al.2016).Similarly, ZmSTP also showed tissue-specific expression.ZmSTP1,ZmSTP2,ZmSTP14,ZmSTP17,ZmSTP18,ZmSTP22, andZmSTP24were highly expressed in leaf tip and seedling leaves;ZmSTP10andZmSTP12were highly expressed in pollen and anther;ZmSTP9was highly expressed in the spikelet; whileZmSTP20was highly expressed during the initial development of the spikelet and embryo.It is speculated that STP is involved in the entire process of maize growth and development,especially in leaves before emergence and after maturity.

Plants show several types of responses to stress, such as increase in hydrolase activity, increase in cell wall structural proteins, production of antibacterial substances,and increase in sugar transporter content (Bihmidineet al.2016).The selection of resistant varieties is an important means to deal with plant stresses and plant diseases.For example,Ht2,Ht3, andHtn1genes have certain resistance to northern leaf blight of corn (Rashidet al.2020).ZmAPX1positively regulates southern leaf blight resistance by reducing H2O2accumulation and activating JA-mediated defense signaling pathways(Zhanget al.2022).At present, there are also no immune maize varieties for northern leaf blight of corn and southern leaf blight of corn.The main prevention methods are to strengthen cultivation management and use chemical agents (Redinbaugh and Stewart 2018).However, maize plants are tall and difficult to operate in the field, so it is particularly important to cultivate excellent disease-resistant varieties.Corn stalk rot is a soil-borne disease, so the implementation of comprehensive control measures based on breeding and application of diseaseresistant varieties, supplemented by a series of health cultivation measures is the most effective measure.The common maize inbred line resistant toF.graminearumis X178.Two independent dominant genesRpiX178-1andRpiX178-2carried by X178 played a major role in its resistance (Duanet al.2019).Previous studies have reported thatAtSTP13contributes toA.thalianaresistance toBotrytis cinerea, wherein AtSTP13is phosphorylated and then recovers sugar produced by the cell wall invertase CWINV to limit bacterial proliferation in apoplasts (Lemonnieret al.2014).In the present study,the gene expression ofZmSTP2increased gradually afterF.graminearuminfection; the gene expression also fluctuated after jasmonic acid and SA treatment.Bioinformatics analysis revealed thatZmSTP2in maize is a homologous gene ofAtSTP13; therefore, ZmSTP2 may also be phosphorylated and then absorb sugar to resist pathogen infection.Pathogens often compete with the host for energy sources, resulting in changes in the distribution of photosynthetic products (Wanget al.2008).STP4expression is rapidly induced in both suspensionculturedA.thalianacells stimulated withPseudomonas syringaeor treated with chitin and inA.thalianaplants exposed to fungal attack (Truernitet al.1999).Protein sequence alignment shows thatZmSTP20in maize is the homologous gene ofAtSTP4.In the present study, the gene expression ofZmSTP20gradually increased afterF.graminearuminfection.Moreover, after treatment with jasmonic acid, the gene expression decreased first and then increased.LikeAtSTP4,ZmSTP20may recover sugar from apoplasts after plant injury or pathogen infection to reduce the loss caused by pathogens.In this study, the resistance ofzmstp2andzmstp20mutants toC.carbonum,S.turcica,C.heterostrophus, andF.graminearumwas significantly weakened.Continuous study ofZmSTP2andZmSTP20is of great significance for cultivating new resistant varieties.

5.Conclusion

In summary, the maize STP family contains 24 members.They are widely distributed in the cell membrane and possess a highly conserved transmembrane transporter domain, and their expression levels are significantly different in different tissues.They actively participate in the resistance of maize to biotic and abiotic stress.Two representative genes, namelyZmSTP2andZmSTP20, were selected for analyzing disease resistance.After inoculation withC.carbonum,S.turcica,C.heterostrophus, andF.graminearum, the lesion area ofzmstp2andzmstp20mutants was significantly smaller than that of the wild-type B73 plant.In this study,the genes encoding sugar transporters in maize were identified, and their expression patterns in different tissues and under biotic and abiotic stresses were revealed.In future studies, we will investigate the disease resistance pathways ofZmSTP2andZmSTP20and clarify the function of the maize STP family genes.

AcknowledgementsThis work was supported by the National Natural Science Foundation of China (31901864), the State Key Laboratory of North China Crop Improvement and Regulation (NCCIR2020ZZ-9), the Research Project of Science and Technology in Universities of Hebei Province, China (BJK2022006), the earmarked fund for China Agriculture Research System (CARS-02),the Key Research and Development Projects of Hebei(19226503D), and the Central Government Guides Local Science and Technology Development Projects, China(216Z6501G and 216Z6502G).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2022.12.014