Xiofi Xu ,Shngyi Du ,Fuho Jio ,Mnghn Xi ,Aiguo Wng ,Hihng Xu ,Qiqing Jio ,Xin Zhng ,Ho Jing ,Jingtng Chn ,Ming Wng ,*

a College of Agronomy,Qingdao Agricultural University,Qingdao 266109,Shandong,China

b Administrative Committee of Yellow River Delta Agri-High-Tech Industry Demonstration Zone,Dongying 257347,Shandong,China

c Shandong Institute of Pomology,Tai’an 271000,Shandong,China

d Jinan Fruit Research Institute,All China Federation of Supply and Marketing Co-operatives,Jinan 250000,Shandong,China

e Productivity Promotion Center of Zoucheng,Jining 273500,Shandong,China

ABSTRACT Seed germination is the process by which an organism grows from a seed.It requires suitable conditions and environmental factors.Maize is one of the most important crops worldwide.Germination influences both final maize yield and quality.Seed germination is regulated by a complex gene network.It is also influenced by endogenous (phytohormones and nutrients) and exogenous (temperature and water)inputs,involving molecular networks only partly identified to date.This review describes current understanding of the influence of temperature,water,phytohormones,and nutrients in regulating maize seed germination,and indicates knowledge gaps that should be addressed.

Keywords:Germination of maize seed Temperature Water Phytohormones Nutrients

1.Introduction

Maize(Zea maysL.)is a staple cereal crop cultivated worldwide under a wide spectrum of soil and climatic conditions.Maize is a C4species in the Poaceae family and is moderately sensitive to stresses,such as salt,drought,and temperature [1–4].Maize was first domesticated by indigenous peoples in southern Mexico/Central America more than 5000 years ago [5].The seed contains approximately 72% starch,10% protein,and 4% fat,supplying an energy density of 1.53 MJ 100 g-1.The United States,China,and Brazil,the top three maize-producing countries,produce approximately 563 of the 717 million metric tons per year.The total production of maize surpasses that of wheat or rice [6,7].Maize is not only consumed directly by humans but also is widely used for the production of industrial products,biofuels,animal feed,and other products such as corn syrup and corn starch [7–9].

Seed germination may be defined as the fundamental process by which plant species grow from a single seed into a plant.It influences both crop yield and quality[10]and determines the efficient use of nutrients and water resources[11].The most common example of germination is the sprouting of a seedling from a seed of an angiosperm or gymnosperm [12,13].During the beginning stage of germination,called imbibition,the seeds take up water rapidly,resulting in swelling and softening of the seed coat at an optimum temperature.Rupturing of the seed coats allows the radicle and the plumule to emerge.The seed then activates its internal physiology and starts to respire[14].This is a lag phase of seed germination.Water,temperature,and oxygen are necessary factors for seed germination.Other factors including phytohormones,sugar,nutrients,and even magnetism influence germination[15–19].

In this context,and with a view to understanding the mechanisms involved in the germination of maize seed,it is desirable to identify and summarize the regulatory mechanisms behind the process The present review emphasizes the emerging understanding of the mechanisms by which temperature,water,phytohormones,and nutrients regulate maize germination.,and discusses the relevance of exogenous and endogenous factors to the process.

2.Environmental factors

2.1.Temperature

Temperature is a necessary factor for seed germination.Moderate temperatures around 26–29°C are required for a maize seed to germinate [20].Maize cannot germinate when the temperature is too high (>45 °C) or too low (<6.2 °C) [21].Improving seed vigor in response to the change of temperature is a key breeding objective.Many studies have investigated the regulatory network by which temperature influences maize seed germination.As early as 1981,Riley et al.[20] showed that the cell energy status and the activity of some enzymes (such as lipase,alanine aminotransferase,aspartate aminotransferase,and especially ribonuclease)changed as the temperature increased from 23 °C to 41 °C.At the early stage of seed germination,the ATP content also increased significantly when the temperature increased from 28°C to 41°C.The rate of protein synthesis(a process with high energy demand)was severely reduced at high temperatures(>37°C)in contrast to 28°C.The change in temperature strongly influenced respiration and sugar metabolism during germination.Abnormal respiration interferes with the homeostasis of reactive oxygen species(ROS),which is critical for seed germination[22–25](Fig.1).ROS,which are generated by plant metabolism,are key regulators that mediate signaling pathways involved in developmental processes and plant responses to environmental fluctuations.These highly reactive metabolites can cause cellular damage when ROS homeostasis becomes unbalanced [26].The antioxidant system of the plant,which plays a key role in regulating ROS homeostasis,comprises a variety of antioxidant molecules and enzymes,including ascorbate,glutathione,superoxide dismutase (SOD),peroxidase (POD),malondialdehyde (MDA),and catalase (CAT).Temperature stress,such as heat,cold or freezing,can break ROS homeostasis,damaging cell membranes and proteins[27].Temperature stress in maize increased the transcription,translation,and activity of ROSscavenging enzymes and induced H2O2accumulation [28].The influence of temperature stress on antioxidant enzyme activity during maize germination is illustrated in Fig.1.Guan et al.[29]treated maize seed with chitosan under low-temperature stress(15°C).Chitosan is a linear polysaccharide composed of randomly distributed β-linked D-glucosamine and N-acetyl-D-glucosamine and has been indicated to accelerate growth and germination as well as improve the quality of flowers and fruit.The chitosan treatment had no significant effect on germination percentage under 15 °C,but increased germination index and reduced mean germination time.When seed was treated with various concentrations of chitosan,the content of MDA decreased and the activity of POD and CAT increased.The authors suggested that increased POD and CAT activity increases germination index and reduces mean germination time.Zhou et al.[30] also observed a change in antioxidant enzymes during maize germination.In their study,the hydrogen sulfide(H2S)signaling pathway improved maize seed germination and seedling growth by stimulating the activity of antioxidant enzymes (Fig.1),including guaiacol peroxidase(GPX),glutathione reductase (GR),SOD,ascorbate peroxidase(APX),and CAT.Pre-soaking with NaHS (stimulating the H2S signaling pathway)increased germination percentage compared with the control,showing that H2S signaling improved maize seed germination and seedling growth under high temperature by inducing antioxidant activity and osmolyte biosynthesis.Pre-soaking with NaHS also activated Δ1-pyrroline-5-carboxylate synthetase and ornithine aminotransferase(both rate-limiting enzymes in proline synthesis),betaine aldehyde dehydrogenase(a key enzyme in glycine betaine synthesis),and trehalose-6-phosphate phosphatase(a key enzyme in trehalose synthesis),indicating a complex regulatory network acting in response to temperature.In accord with these findings,phospholipid metabolism and ROS content,which have a tight relationship with antioxidant enzyme activity,also responded to temperature change during maize germination[31,32].ROS homeostasis is considered to be a key regulator of cell division and plant developmental programs,and can interact with abscisic acid (ABA) signaling,ABA degradation,and gibberellin biosynthesis to regulate seed germination [25,33,34].ROS homeostasis also exerts a direct effect on hormonal (ABA/GA) balance,which in turn affects germination [25].The antioxidant system involving SOD,POD,and CAT strongly influences seed germination of maize under temperature stress.These studies show a tight link between temperature stress,ROS homeostasis,and seed germination (Fig.1).

Fig.1.The reported regulatory network involving high temperature,water,and germination of maize seed.ROS homeostasis influences seed germination.High temperature exerts a negative effect on ROS homeostasis,causing abnormal respiration.Water deficit may also influence ROS homeostasis,probably via sugar metabolism.The activity of antioxidant enzymes involved in ROS homeostasis is regulated by H2S signaling during maize germination.Green line indicates positive effect;red line indicates negative effect;and blue line indicates protein interaction.

Besides physiological experiments,genetic studies have been conducted to identify key genes in germination response to temperature.Huang et al.[35] performed a genome-wide association analysis under low-temperature at the seed germination stage using 125 maize inbreds and identified 43 single-nucleotide polymorphisms associated with chilling tolerance.They identified 40 candidate genes involved in this process,encoding ascorbate peroxidase(flavin adenine dinucleotide is a cofactor of it),WRKY transcription factor,chromatin regulatory factor,and other products[35].Ascorbate peroxidase is the main enzyme responsible for hydrogen peroxide removal in the chloroplasts and cytosol of higher plants and plays an important role in regulating ROS homeostasis[36].WRKY transcription factor is involved in the regulation of various plant physiological processes and acts as a key component in ABA signaling [37].WRKY transcription factors coordinate with ABA to regulate seed germination [38–40].Chromatin regulatory factor plays an important role in cell division,and cell division is the basis of seed germination [41].Hu et al.[42] used a recombinant inbred line (RIL) population for a QTL (quantitative trait locus) analysis of low-temperature germination ability and identified three QTL for optimum-temperature germination rate and six QTL for low-temperature germination rate [42].These QTL were involved in meristem development,physiological process of radicle protrusion,key enzymes in respiratory metabolism such as malate dehydrogenase,energy production,and auxinmediated organogenesis.Using 2,271,584 SNPs (single-nucleotide polymorphisms),a genome-wide association study identified 17 genetic loci involved in cold tolerance during maize germination[43].Li et al.[44]performed a QTL analysis,identifying 43 QTL that explained a phenotypic variance of 0.62%–39.44%.Seventeen QTL explained more than 10% of phenotypic variance.These QTL were involved in cold resistance,including freezing tolerance,ascorbate biosynthesis,ABA signaling,energy metabolism,and seedling photosynthesis.These genetic studies support the notion that energy metabolism,ROS homeostasis,and ABA signaling act as regulators of maize seed germination.The expression of MAPK signalingassociated genes,GA biosynthesis genes,and genes encoding SOD and CAT also changed dramatically under temperature stress.These studies provide valuable resources for future studies aimed at increasing our understanding of the genetic basis of maize germination [45–47].However,how maize germination responds to changes in temperature is poorly understood.The key genes and the associated regulatory network await discovery.

2.2.Water

Water is necessary for seed germination.It provides hydration for the vital activities of protoplasm,provides dissolved oxygen for the growing embryo,softens the seed coat,and increases seed permeability[48,49].It also helps in the rupturing of seed and converts the insoluble food into soluble form for its translocation to the embryo[48,49].Many studies have investigated the molecular regulation of maize seed germination under water-deficit conditions.Liu et al.[50]reported that maize seed germination depends on osmotic adjustment under water-deficit conditions.In plants,basic leucine zipper (bZIP) transcription factors regulate diverse functions,including plant development and stress response [51].Ying et al.[52] clonedZmbZIP72,a bZIP transcription-factor gene from maize.Its expression was induced by ABA,high salinity,and water-deficit treatment.The germination ofZmbZIP72-overexpressingArabidopsisseeds was sensitive to osmotic stress,because expression of many ABA-inducible genes was stimulated byZmbZIP72during seed germination.This finding showed that moderate expression ofZmbZIP72could influence seed germination under osmotic stress.Jiang et al.[53] showed that calciummediated signal transductions coordinate with ABA signaling to regulate seed germination in response to water-deficit stress.They isolated a subgroup I calcium-dependent protein kinase (CDPK)gene,ZmCPK4,whose transcription level is induced by water deficit(Fig.1).Overexpression ofZmCPK4inArabidopsisincreased drought stress tolerance and ABA sensitivity.Their result suggested thatZmCPK4might be involved in ABA-mediated regulatory of stomatal closure in response to drought stress.A WRKY transcription factor,ZmWRKY58,also participated in calcium signalingassociated germination.In rice,overexpression ofZmWRKY58resulted in delayed germination [54].ZmWRKY58-overexpressing transgenic plants showed higher survival rates under water deficit stress.Yeast two-hybrid assay showed that ZmWRKY58 interacted with ZmCaM2 (a calmodulin in maize),suggesting that ZmWRKY58 may function as a calmodulin-binding protein and may act as a positive regulator involved in drought and salt stress response(Fig.1).Physiological experiments revealed[55]that glycine betaine could play an important role in increasing drought tolerance during maize germination.Recently,Yao et al.[56]reported that exogenous application of ABA improved maize seed germination rate under water-deficit conditions,with increased endogenous ABA content,osmotic substances,antioxidant enzyme activities,andAsr1(abscisic acid stress ripening-1)expression level,but MDA content dropped significantly.These studies provided a potential link between water deficit and ABA,and suggested that ABA could play an important role in maize seed germination under water deficit.However,key pathways and genes in the molecular regulatory network of maize adaptation to water deficit await discovery.

3.Phytohormones

Plant hormones are signal molecules produced within plants,and function in extremely low concentrations.Plant hormones contribute to almost all aspects of plant growth and development[57–59].Many plant hormones participate in the seed germination process [13].In maize,most previous studies have focused on the relationship between seed germination and ABA,auxin,and gibberellin.

3.1.Abscisic acid

ABA functions in many developmental processes in plants,including seed and bud dormancy,control of organ size,and stomatal closure[60,61].It also functions in plant response to environmental stresses [62].ABA is required for regulation of seed dormancy and germination in maize.A mutant blocked in ABA synthesis germinated earlier than the wild type[63].Interestingly,the addition of a low concentration of H2O2can promote maize seed germination by increasing NADPH oxidase and ROS levels in seed[31].Both NADPH oxidase and ROS mediate ABA signaling in plants[64–66].Wu et al.[67]compared proteins differentially expressed between a viviparous-5(deficient in ABA biosynthesis)mutant and wild type,using 2-D proteomics and mass spectrometry.They found that the expression of late embryogenesis-abundant proteins,small heat shock proteins,stress-related proteins,antioxidant enzymes,and storage-related proteins was regulated by ABA.Embryos from germinating seeds after 24 h of soaking,from five elite maize hybrids and their parents,were selected by Fu et al.[68] to identify differentially expressed proteins during seed germination.They identified 54 differentially expressed proteins included F-box proteins,ubiquitin-activating enzyme,Ca2+-ATPase,ROS-related proteins,14-3-3 proteins,and GTP-binding proteins,and classified them into several functional groups,including cell metabolism,cell detoxification,signal transduction,chaperones,development process,transporters,and stress response.They suggested that chaperones,ABA and gibberellin signal transduction,ROS-related proteins,and cell detoxification function in seed germination.He et al.[69]reported that the three PYL family members in maize(ZmPYL8,ZmPYL9,andZmPYL12),which encode ABA receptors and mediate ABA signaling,regulated seed germination under drought stress.They overexpressed these three ABA receptors inArabidopsis.The overexpressingArabidopsismutant showed high proline accumulation,increased expression of drought-related marker genes,and increased sensitivity of seed germination to ABA[69].In plants,proline is considered an important osmolyte that protects subcellular structures and macromolecules under osmotic stress,and its biosynthesis is mediated by ABA signaling [70].Proline also acts as a signaling molecule to modulate mitochondrial function,influence cell proliferation or cell death,and trigger specific gene expression,which can be essential for plant recovery from stress [71].During germination,ABA delays the maize seed germination process by affecting cell cycle advance through influencing many cell cycle-associated proteins,such as CDKA (cyclin-dependent kinase A) protein,proliferating cell nuclear antigen,and cyclin D2 [72,73].CycD3;1,a protein that participates in cell cycle and cell division,also influences the seed germination process in maize [74,75].Maize CycD3;1 can interact with CDKA protein or CDKB1;1 protein to form a complex that has kinase activity.ABA can inhibit the level of CDKA(Fig.2),while stimulating the level of CDKB1;1 to regulate the activity of the protein complex during early germination [76].The presence of ABA can also lead to reduce the abundance of the CycD3;1 protein[76].These results indicate that ABA regulates maize germination through a variety of regulatory pathways,including ABA signaling,gibberellin signaling,cell cycle,energy metabolism,and ROS homeostasis.

Zhang et al.[77] identified another ABA-regulated seed germination process.They showed that the expression of both histone deacetylases (HDACs) and histone acetyltransferases (HATs)increased during maize seed germination with an increased acetylation level of histone H3.ABA repressed the expression of HATs and HDACs,therefby delaying histone acetylation.The promoter region of an embryogenesis-related gene,viviparous 1 (VP1,a key transcription factor involved in regulating seed development in maize) [78],was deacetylated (Fig.2).ABA inhibited the deacetylation of the VP1 promoter.These results showed that ABA could regulate acetylation status during maize seed germination.

3.2.Gibberellin and auxin

It has long been known that plant hormones are involved in the seed germination process.Besides ABA,several other hormones can break seed dormancy and promote maize germination,including auxin,gibberellins,and cytokinin [79].For example,CKX (cytokinin oxidase/dehydrogenase) activity changed dramatically during the germination of maize seed,suggesting a potential role of cytokinin in regulating maize germination [80].Several reports have described the role of auxin and gibberellin in regulating seed germination.Song et al.[81] performed expression profiling analysis in maize seed.The transcript levels of 15 gibberellin metabolism-related genes changed during germination.Among them,the transcription level of the genes that encode kaurene oxidase and kaurenoic acid oxidase increased after seed imbibition.After 72 h,the transcript levels of GA20 oxidase,GA3 oxidase,and GA2 oxidase were higher than their levels at the beginning of seed germination.This analysis revealed the complexity of the mechanism underlying gibberellin-regulated seed germination.In maize,gibberellin also interacts with ABA to participate in the seed germination process.White et al.[63] showed that a gibberellin deficiency during seed development suppressed vivipary in ABAdeficient maize kernels.A temporal analysis of gibberellin accumulation revealed that the accumulation of bioactive GA1and GA3negatively influenced ABA content (Fig.2).This study indicated that the balance of ABA with gibberellin is a determinant in seed germination.White and Rivin [82] showed that gibberellin can antagonize ABA signaling to control maize embryo development and that inhibition of gibberellin synthesis mimicked the effects of exogenous ABA during germination.

Besides gibberellin,auxin also regulates seed germination in maize.Cyclins are a family of proteins that control the progression of a cell through the cell cycle by activating cyclin-dependent kinase (CDK) enzymes or a group of enzymes required for the cell cycle.In maize,every cyclin showed a different expression pattern during seed germination [83].During germination of maize seed,cytokinins,and auxins,which stimulate the germination process,regulate the expression pattern of all cyclins,with cytokinins promoting an increase in expression during the early hours of germination,whereas auxins promote the transcription level ofCycD2;1,CycD4;1,andCycD5;2[83] (Fig.2).Lara-Nú?ez et al.[75] reported that auxin cannot stimulate cyclin accumulation of CycD5 and CycD4;1 (the two D-type cyclins) during germination,but modify the kinase activity associated with D-type cyclins at the early stage of germination.In 2017,further research[74,84]revealed a regulatory mechanism linking auxin,ABA,and D-type cyclin.CycD3;1 is a D-type cyclin protein that associates with CDKA or CDKB1;1 to regulate seed germination.Garza-Aguilar et al.[76]showed that auxin can cooperate with ABA to influence the activity of CycD3;1.The transcription levels of CDKA increased with auxin,but decreased with ABA.However,during the early germination stage,both auxin and ABA increased the level of CDKB1;1 to regulate maize germination [77].

Fig.2.The relationship between plant hormones(ABA,GA,and auxin)and germination of maize seed.CycD,D-type cyclin;VP1,viviparous1;CDKA,cyclin-dependent kinase A;HAT,histone acetyltransferases;HDAC,histone deacetylases.Broken line indicates indirect regulation.Green line indicates positive effect;red line indicates negative effect.

In summary,ABA may affect maize seed germination through various biological processes including proline biosynthesis,cell cycle,energy metabolism,and ROS homeostasis.ABA may be one of the key point for gibberellin and auxin to regulate maize seed germination.Questions that await investigation are whether gibberellin and auxin can affect seed germination independent of ABA and whether other phytohormone-mediated regulatory networks are active in maize during seed germination.

4.Nutrients

Many chemical elements and compounds are necessary for plant growth and plant metabolism.Nitrogen (N) is one of the key factors in the control of seed germination.In 2002,a physiological study[88]highlighted the central role of glutamine(Gln)synthetase (GS) and Gln synthesis,which is important for nitrogen metabolism during the germination of maize seed.The activity of glutamine synthetase and the amino acid content increased during maize germination.Maize seeds showing a faster germination rate showed higher GS activity.Two genes,gln3(glutamine synthetase 3) andgln4,played a major role in the regulation of maize germination.These results suggested that increased glutamine(Gln)synthetase (GS) and Gln synthesis stimulate and stabilize nitrogen metabolism in the germination process.The nitrogen metabolism is one of the most important metabolic events during germination,and is important for amino acid synthesis.

Besides N,sugar also shows both trophic and signaling activity in plant growth and development.Carbohydrate metabolismrelated enzymes,such as pyruvate dehydrogenase,2-oxoglutarate dehydrogenase,NAD-malic enzyme,citrate synthase,α-amylase,β-amylase,and starch branching enzyme,participate in the regulation of maize seed germination [85–87].In addition to N and sugar,phosphorus is necessary for seed germination[89].After germination,98% of seed phytate was hydrolyzed,revealing a high demand for phosphorus during the germination of maize seed [90].Although N,sugar,and phosphorus are important nutrient factors during maize germination,there have been few studies of the molecular network regulating their participation in maize germination.Together with N and phosphorus,potassium,sodium,calcium,and other elements can also act as nutrient factors in seed germination.Their roles await further study.

5.Conclusions

Germination of maize seed plays an important role in maize growth and development and strongly influences maize yield.However,the detailed mechanisms whereby various factors regulate maize seed germination remain unknown,and many mechanistic scenarios are plausible.For example,we do not understand how temperature and water,the main two environmental factors,regulate maize seed germination,and which molecular actors are involved.The detailed mechanism and the specific regulatory network in maize are still missing,despite studies inArabidopsisand rice.Similar questions concern the molecular mechanism behind the effect of plant hormones on the germination of maize seed.The regulation of gene expression includes many aspects,such as epigenetic regulation,transcriptional regulation,posttranscriptional regulation,and translational and posttranslational regulation.The relevance of these mechanisms in the regulation of maize seed germination deserves investigation in different biological contexts.In a recent study[91],the ERF,bZIP,MYB,WRKY,and bHLH transcription factor families were involved in driving seed germination in maize.But the target genes of these transcription factors during maize germination are still unknown.Many biological processes including amino acid metabolism,sugar metabolism,and ribosome biosynthesis,are involved in maize germination[91,92].A microRNA is a small non-coding RNA molecule,that functions in RNA silencing and post-transcriptional regulation of gene expression.Some microRNAs also participate in the maize germination process[93,94].But the target mRNAs of these micro-RNAs are still unknown.Our knowledge about the molecular network and signaling transduction pathway governing the germination of maize seed is still limited,and the only available studies have shown that maize germination could be regulated by different biological processes.All these findings invite further investigation of the regulatory network behind the germination of maize seed and identification of a specific regulatory mechanism in maize germination.

CRediT authorship contribution statement

All authors listed have made direct contributions to the work and approved it for publication.Ming Wang,Xiaofei Xue,Shangyi Du,Fuchao Jiao,Menghan Xi,and Aiguo Wang:wrote parts of the manuscript.Ming Wang,Haicheng Xu,Qiqing Jiao,Xin Zhang,Hao Jiang,and Jingtang Chen:contributed to language revision and figure design.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgment

This work was supported by Talent Introduction Special Funds of Qingdao Agricultural University (663/1120070).