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Cell signaling mechanisms andmetabolic regulation

of germination and dormancy in barley seeds☆

Zhenguo Maa,b, Natalia V. Bykovac, Abir U. Igamberdieva,⁎aDepartment of Biology, Memorial University of Newfoundland, St. John's, NL A1B 3X9, CanadabPacific Forestry Centre, Natural Resources Canada, Victoria, BC V8Z 4N9, CanadacMorden Research and Development Centre, Agriculture and Agri-Food Canada, Morden, MB R6M 1Y5, Canada

A R T I C L E I N F O

Abbreviations: ABA, abscisic acid; ABI1,peroxidase; CAT, catalase; cPTIO, 2-(4-carbdehydroascorbate reductase; ELIPs, early lighGAMYB, GA-inducedMyb (myeloblastosis)-likeG protein-coupled receptors; GR, glutathionreductase; GSSG, glutathione disulfide; HvGAoxidase1; HvPTR, barley scutellar peptide translight, oxygen, or voltage-sensing domain 1; LOmalate synthase; NO, nitric oxide; PEP, phosABA-responsive protein kinase; PPDK, pyruvaPYR1-Like regulatory components; PYR1, PYRAspecies; ROS, reactive oxygen species; SCFSLY/

SLR1, SLENDER RICE1; SLY1, SLEEPY1; SNP,kinases; SOD, superoxide dismutase; SPS, suc☆ Peer review under responsibility of Crop S⁎ Corresponding author.E-mail addresses: a_igamberdiev@hotmail.

https://doi.org/10.1016/j.cj.2017.08.0072214-5141/© 2017 Crop Science Society of Chopen access article under the CC BY-NC-ND

A B S T R A C T

Article history:Received 26 May 2017Received in revised form23 August 2017Accepted 13 September 2017Available online 5 October 2017

During germination of barley (Hordeum vulgare L.) seeds, important morphological andphysiological changes take place, including development of organs and tissues andactivation of metabolic pathways. Germination and dormancy of seeds are regulated byabscisic acid, gibberellins, reactive oxygen species (ROS), reactive nitrogen species (RNS)and several other factors. Activities of ascorbate–glutathione cycle enzymes, responsible forscavenging ROS, strongly increase. Catalase and superoxide dismutase activities, alsoscavenging ROS, decrease at the onset of seed germination and then increase. With theincrease in aerobic metabolism after radicle protrusion, the activities of the fermentationenzymes lactate and alcohol dehydrogenase decline rapidly. The RNS-scavenging activity ofS-nitrosoglutathione reductase decreases in the course of seed germination, in concert withelevation of nitric oxide production and protein nitrosylation. This activity supports the roleof RNS in regulating seed germination. Transcription of various genes at different phases ofseed germination exhibits phase-specific changes. During imbibition, genes involved in cellwall metabolism are highly expressed; in the middle phase of seed germination beforeradicle protrusion, genes involved in amino acid synthesis, protein synthesis, and transportand nucleic acid synthesis are upregulated significantly, and after radicle protrusion, genesinvolved in photosynthetic metabolism are induced. In summary, signal transduction and

Keywords:Seed germinationReactive oxygen speciesReactive nitrogen speciesSignal transductionGene expression

ABA-Insensitive 1; ABI5, ABA-Insensitive 5; ADH, alcohol dehydrogenase; APX, ascorbateoxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DHA, dehydroascorbate; DHAR,t-inducible proteins; ETC, electron transport chain; FW, fresh weight; GA, gibberellic acid;protein; GID1, GIBBERELLIN INSENSITIVEDWARF1; GID2, GA-insensitive dwarf2; GPCRs/GCRs,e reductase; GSH, glutathione; GSNO, S-nitrosoglutathione; GSNOR, S-nitrosoglutathione2ox, barley Gibberellin 2-oxidase1; HvHY5, barley Elongated Hypocotyl5; HvKAO1, Kaurenoic acidporter; ICL, isocitrate lyase; LDH, lactate dehydrogenase;MDHA,monodehydroascorbate; LOV1,V2, light, oxygen, or voltage-sensing domain 2;MDHAR,monodehydroascorbate reductase;MS,phoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PHOT1, Phototropin 1; PKABA,te phosphate dikinase; PSSG, S-glutathionylated proteins; PYR1, Pyrabactin Resistance 1; PYLs,BACTIN RESISTANCE 1; RCAR, regulatory components of ABA receptor; RNS, reactive nitrogen

GID2, Skp1 (S-phase kinase-associated protein 1), Cullin, F-box; SDH, succinate dehydrogenase;sodium nitroprusside; SnRK2, subfamily 2 SNF1 (Sucrose-Nonfermenting Kinase 1)-relatedrose-phosphate synthase; SUT, sucrose transporter; TCA, tricarboxylic acidcience Society of China and Institute of Crop Science, CAAS.

com, igamberdiev@mun.ca (A.U. Igamberdiev)

ina and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. This is anlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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metabolic regulation of seed germination involve diverse reactions and complex regulationat different levels of metabolic organization.© 2017 Crop Science Society of China and Institute of Crop Science, CAAS. Production and

hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4602. Seed germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4603. Mobilization of reserves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

3.1. Metabolism of starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4623.2. Metabolism of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4623.3. Metabolism of storage oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

4. Tricarboxylic acid and glyoxylate cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4635. Change in morphology and water content during seed germination . . . . . . . . . . . . . . . . . . . . . . . . . . . 4646. Gene expression in barley seeds during germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4647. Effect of light on germination and dormancy of barley seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4668. Mechanism of GA and ABA in regulation of seed germination and dormancy . . . . . . . . . . . . . . . . . . . . . . 466

8.1. Effect of GA and ABA on seed germination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4668.2. Interaction between GA and its receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4678.3. Interaction between ABA and its receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

9. The production, scavenging and roles of ROS in regulation of seed dormancy and germination . . . . . . . . . . . . 46810. The role of nitric oxide in regulating seed dormancy and germination . . . . . . . . . . . . . . . . . . . . . . . . . . 47011. Perspectives: crosstalk between hormones, ROS, and RNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

1. Introduction

Germination and dormancy are vital elements in the plant lifecycle. Bewley [1] defined seed dormancy as the incapacity ofintact viable seeds to complete germination under favorableconditions. A more detailed definition of dormancy has beendeveloped by Baskin and Baskin [2], who claimed that dormantseeds lack a capacity to germinate in a specified period of timeunder any combination of normal physical environmentalfactors that favor germination. Seed germination is a complexprocess influenced by many physical factors such as water,temperature, and light and by a great number of chemicalfactors such as ABA, GA, ROS, and RNS that play vital roles inregulation of dormancy [3,4]. Seed germination is initiated bywater imbibition at appropriate temperature. During germina-tion, NO content and the level of protein nitrosylation increase[5], exerting amajor influence on seed germination, and variousS-nitrosylated proteins control many kinds of redox-basedregulation [6] such as reactions between protein thiols andthiol/disulfide exchange [7]. Although there are morphologicaldifferences between monocotyledonous and dicotyledonousseeds, the fundamental regulation of seed germination issimilar in both groups of plants. In this review, barley seeds,consisting of seed coat, endosperm and embryo and thustypicalmonocotyledonous seeds, are used as themain exampleto describe signal transduction and regulation mechanism inthe process of seed germination.

2. Seed germination

Seed germination is a complex process, starting from wateruptake of dry seeds and continuing to elongation of theembryonic axis [8]. Weitbrecht et al. [9] separate the process ofseed germination into three phases. Phase I, the early phase,includes imbibition of dry seeds and the early plateau phase ofwater uptake. Phase II, the middle phase, includes the plateauphase ofwater uptake andvisible radicle protrusion through seedcovering layers. Phase III, the later phase, is seedling develop-ment, also called the post-germination phase (Fig. 1-A). Inmonocotyledonous seed germination, the coleorhiza is the firstpart to growout of the seed coat, whereas in dicotyledonous seedgermination, roots (radicles) grow out of the seed coat first.During these processes, many physical and chemical reactionsoccur, including the rupture of endosperm and testa; leakage ofcellular solutes; repair of organelles, membranes and DNA; andsynthesis of DNA, RNA, and proteins. With imbibition, germina-tion signal GAs stored in the embryo,mainlyGA3, are transportedto the aleurone layer of the endosperm, which is rich in protein(Fig. 2). Hydrolytic enzymes including acid cysteine endopepti-dases, serine carboxypeptidases, and neutral aminopeptidases[10,11], become activated in the endosperm, which containsaleurone cells that degrade storageproteins into amino acids (Fig.2). GA3 activates the expression of DNA encoding α-amylase inthe aleurone cells, given that the GA-induced transcription factorGAMYB (GA-induced Myb (myeloblastosis)-like protein) can bind

A

B

Fig. 1 – Key processes during germination of typical endospermic monocot seeds (A) and corresponding morphology ofgerminating barley (cv. Harrington) grains at different periods of the germination process (B). Photo by Zhenguo Ma.

Fig. 2 – Metabolic processes occurring in embryo and endosperm during barley seed germination.

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promoters of the genes encoding α-amylase to increase theexpression of this enzyme [12].

3. Mobilization of reserves

3.1. Metabolism of starch

Starch is the most abundant reserve carbohydrate in cerealseeds. The two components of starch, amylose and amylo-pectin, are first hydrolyzed by α-amylase, breaking the α-(1-4)glycosidic bond between the glucose residues from thenonreducing end of the large oligomers to produce glucoseand maltose from amylose and dextrins from amylopectin.Maltose is converted to glucose by maltase. Debranchingenzymes such as dextrinase hydrolyze the α-(1-6) branchpoints to generate short chains, which are further hydrolyzedby amylases. α-Amylase, debranching enzyme, and maltaseare produced in the embryo (in the scutellum which is aspecialized cotyledon of cereal plants) and aleurone layer andare secreted into the aleurone or endosperm in the process ofseed germination [13]. In some types of dicotyledonous seeds,starch is degraded by phosphorolysis, catalyzed by phosphor-ylase, instead of amylolysis. The enzymes breaking downstarch can be released directly in the storage organs [13].α-Amylase transported by the endoplasmic reticulum into theGolgi complex is packaged into vesicles and then exportedinto the endosperm through the cell membrane by exocytosis.With the degradation of starch, sucrose is the major sugar inthe endosperm in the early period of germination, whereasmaltose and glucose become predominant in the late periodof germination [14]. GA3 also increases the synthesis ofα-amylase in the embryo, where α-amylase has the sameisoforms as in the endosperm [15]. α-Amylase in the embryocan be secreted or transported directly into the endosperm todigest starch. Both GA production and amylase secretion areinhibited by sugar influx from the endosperm when germi-nation develops [16]. The synthesis of α-amylase in thescutellum is activated by both GAs and sugar demand orstarvation, given that carbohydrates including starch in theembryo are quickly exhausted with imbibition [17].

In the endosperm, α-amylase, β-amylase, debranchingenzyme, and α-glucosidase combine to digest starch [18].α-Amylase plays the main role in digesting storage starch,followedbyα-glucosidase.β-Amylase anddebranching enzymeare not important at the beginning of germination, butcontribute in a later stage of germination [19]. The main endproduct from degradation of starch is maltose [20]. α-Glucosi-dase in the endosperm can convert maltose into glucose [21].These sugars from degradation of starch are absorbed into thescutellum, where they are converted into sucrose, loaded intophloem, and transported to the embryonic axis [22]. Theenzymes responsible for conversion of these hexoses to sucrosein the scutellum of barley seeds have been identified [23]. Forexample, sucrose-phosphate synthase (SPS) has been identifiedin the scutellum of rice for synthesis of sucrose [24]. In addition,there is probably a sucrose transporter (SUT) in the scutellum ofbarley seeds, given that the SUT protein, responsible for loadingsucrose from the scutellum to the embryonic axis through thephloem, is found in the scutellum of wheat seeds [14]. The

endosperm plays two roles during the germination of cerealseeds, one being the supply of carbohydratesmainly in the formof sucrose for transport to the embryo to fuel the growth of theembryonic axis, and the other the secretion of hydrolyticenzymes to break down cell walls of the endosperm and seedcoat, a process by which the mechanical barriers to radicleemergence are removed [25].

Barley seeds are similar to other cereal seeds in which theenergy reserves are stored mainly in the endosperm, which isdegraded for embryo growth during germination [26]. Oncethe process of seed germination begins, germination is fueledby soluble carbohydrates. Hydrolysis and mobilization of themain reserves in storage organs of seeds generally occurconcurrently [13]. One of the factors influencing digestion ofstarch is the pH of the endosperm. In barley seeds, this pH isabout 4.5 because of organic acid secretion from surroundingtissue in the late phase of seed maturation [27]. This pH isfavorable to the solubilization of starch [28] and the stability ofα-amylase [29]. In addition, transportation of peptides fromthe endosperm to the scutellum is pH-dependent, with theoptimal value 3.8–5.0 [30]. In the tricarboxylic acid (TCA) andglyoxylate cycles, several organic acids including isocitric,citric, malic, and succinic acids are produced. During seedgermination and seedling establishment, these intermediatesand their transport to the endosperm are crucial to the growthof embryos and seedlings. With the release of organic acidsand phosphoric acids from the aleurone layer to the endo-sperm, the pH value in the endosperm of cereal seeds reaches4.5–5.0 [31]. Thus, organic acids play an important role inregulating pH value in the endosperm. Acidification of theendosperm favors several physiological processes includingstarch mobilization, peptide transportation, phytate solubili-zation, hydrolytic activity of secreted enzymes, cell wallexpansion, and nutrient transportation [28,30,32]. The cellsof the embryo in seeds are compartmented in such a way thata low pH in the endosperm will not influence the activities ofenzymes in other areas, even though the endosperm occupiesthe greatest volume of cereal seeds.

3.2. Metabolism of proteins

The major storage proteins present in the endosperm arehydrolyzed by proteinases during germination. Proteins orpolypeptides are hydrolyzed into shorter oligopeptides byendopeptidases and then further broken down into aminoacids by peptidase, or proteins or peptides are hydrolyzeddirectly by carboxypeptidases or aminopeptidases from thecarboxyl or amino ends, respectively, into shorter peptides oramino acids. The proteolytic enzymes are synthesized in theendoplasmic reticulum and then transported into proteinbodies to be released to contact storage proteins [13]. Theprotein content in barley seeds is not very high, about 15% ofseed dry weight [33]. The content of water-soluble proteins islow, only about 1.5% in dry barley seeds, and decreasesgradually during seed germination (Fig. 3). In dormant seeds,the content of water-soluble proteins is stabilized at approxi-mately 1.2 mg g−1 FW, while in germinated seeds it furtherdecreases (Fig. 3). In the embryo, the water-soluble proteincontent is much higher, about 6% [5]. There are at least foursystems for uptake of amino acids, including two nonspecific

Fig. 3 – Changes in content of water-soluble proteins duringgermination of barley seeds. Based on quantitative dataobtained by Ma [39]. Blue line, non-dormant cultivar(Harrington); Red line, moderately dormant cultivar (Sundre);Dotted line, seeds of Sundre remaining dormant.

Fig. 4 – Changes in content of ATP + ADP and ATP/ADP ratioduring germination of barley seeds. Based on quantitativedata obtained by Ma [39]. Blue line, non-dormant cultivar(Harrington); Red line, moderately dormant cultivar (Sundre);Dotted line, seeds of Sundre remaining dormant.

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systems, one system specific for proline, and another onespecific for basic amino acids [34]. Phosphorylation of proteinsplays an important role in the regulation of the barley scutellarpeptide transporter (HvPTR) [35]. The nutrients from metabo-lism of storage proteins are absorbed into the scutellum, wheremost peptides are further hydrolyzed into amino acids [36].

3.3. Metabolism of storage oil

Themain storage oil in seeds is triacylglycerol stored in the oilbody, where it is hydrolyzed into fatty acids and glycerol bylipases. The fatty acids are transported into glyoxysomes to bedegraded by β-oxidation, which is coupled with the glyoxylatecycle to yield succinate. The synthesized succinate istransported into the mitochondria to enter the TCA cycle toform malate or oxaloacetate, which are transported into thecytosol to produce sucrose [13].

In summary, water absorption by quiescent dry seedsactivates many kinds of hydrolysis and synthesis. The storedstarch, proteins, oil, and other complex nutrients in endo-sperm are hydrolyzed to sugars, amino acids, and organicacids, which are absorbed by the scutellum and transported tosupport the growth of the embryonic axis.

4. Tricarboxylic acid and glyoxylate cycles

Once seeds are imbibed, metabolic activities resume. A keymetabolic pathway supplying intermediates and energy tosupport seed germination and seedling growth is the TCAcycle, also called the citric acid cycle [37], which providesmany organic intermediates and much energy via oxidationof acetyl residues into carbon dioxide. Among the organicacids, the content of citric acid is high but the accumulation ofsuccinic acid and isocitric acid is very low in the embryo ofbarley seeds [38]. This cycle is coupled with oxidativephosphorylation to produce ATP. In the process of barley

seed germination, the activity of the TCA cycle increasesgradually [38], in a pattern different from that of ATPaccumulation. The content of ATP + ADP in whole barleyseeds reaches its peak value within the first 3 h, but later itscontent decreases rapidly to almost the same level as in dryseeds (Fig. 4). Thus, active synthesis of adenylates is observedwithin 3 h after imbibition. Also during this time, the ATP/ADP ratio increases from slightly greater than 3 to a valuearound 4 and then becomes stabilized at the stage whenoxygen is depleted (the early phase of stage 2). A furtherincrease of ATP/ADP ratio is observed after radicle protrusion(Fig. 4) with increased oxygen availability, but does notproceed further in dormant seeds. However, in the period ofoxygen limitation, the ATP/ADP ratio does not drop, suggest-ing that there are mechanisms that contribute to ATPsynthesis under highly hypoxic conditions. One such mech-anism (the cycle of phytoglobin and nitric oxide) will bediscussed later. Compared with the content of ATP and ADP inproceed embryo [5], the content in whole barley seeds [39] ismuch lower, indicating that ATP and ADP production occursmainly in the embryo.

In addition, with the development of seed germination,aerobic metabolism becomes more and more intense, asshown by increasing activities of succinate dehydrogenase(SDH, EC 1.3.5.1) and fumarase (EC: 4.2.1.2) in the process ofseed germination [38,39]. SDH, catalyzing conversion of

Fig. 5 – Changes in activities of alcohol dehydrogenase (ADH),lactate dehydrogenase (LDH), and pyruvate phosphatedikinase (PPDK) during germination of barley seeds. Based onquantitative data obtained by Ma [39]. Blue line, non-dormantcultivar (Harrington); Red line, moderately dormant cultivar(Sundre); Dotted line, seeds of Sundre remaining dormant.

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succinate to fumarate in the TCA cycle, is crucial, as the onlyenzyme joining the TCA cycle with the electron transportchain (ETC) [40]. In contrast, anaerobic activities, such asfermentation, are active mostly before radicle protrusion (first10–20 h after imbibition), as illustrated by the activities ofalcohol dehydrogenase (ADH, EC 1.1.1.1) and lactate dehydro-genase (LDH, EC 1.1.1.27) (Fig. 5). The ADH pathway accountsfor the main fermentation flux, given that ADH is muchhigher than LDH activity, and the activity of pyruvatephosphate dikinase (PPDK; EC 2.7.9.1) participating inpyrophosphate-dependent reactions linked to anaerobic gly-colysis [41] is also maximal during the first hour afterimbibition but remains low, indicating that it contributesinsignificantly to glycolytic flux (Fig. 5). The content of PPDK inC3 plants is usually low [42]. In seeds remaining dormant, theactivities of ADH, LDH and PPDK are higher than in

germinating seeds, indicating some level of fermentationduring the dormancy period.

A variant of the TCA cycle in seeds is the glyoxylate cycleoccurring in glyoxysomes, which is an anabolic pathwayconverting acetyl-CoA to succinate [43]. It has two specificenzymes, malate synthase (MS, EC 2.3.3.9) and isocitrate lyase(ICL, EC 4.1.3.1). Three other enzymes, malate dehydrogenase,citrate synthase and aconitase, are shared with the TCA cycle(Fig. 6). The product of the glyoxylate cycle, succinate, is used tosynthesize malate in the TCA cycle [44,45]. By this pathway,plants canmetabolize simple carbon sources such as fatty acidsand acetate to synthesize complex carbohydrates via succinateand produce NADH, which can contribute to ATP synthesis [46]even though this cycle is active only at certain developmentalstages such as seed germination [47,48]. Using the glyoxylatecycle, seeds are able to use lipids for formation of organic acidsand carbohydrates to support the development of shoots [46].Thus, the glyoxylate cycle is involved in gluconeogenesis usingstorage lipids [49]. Thus, the glyoxylate cycle bridges lipid andcarbohydratemetabolism in germinating seeds, and its presenceis indicated by the activities of ICL and MS [50]. The reactioncatalyzed by phosphoenolpyruvate carboxykinase (PEPCK) con-nects theTCAand glyoxylate cycles to gluconeogenesis and thusplays a key role in plant metabolism, as it links the metabolismof organic acids and sugars.

5. Change in morphology and water content duringseed germination

During seed germination, barley seeds show distinctive char-acteristics of physiology and morphology. With imbibition, drybarley seeds absorb water quickly, resulting in an increase inseed volume, and the rough surface of barley seeds becomessmooth [51]. Generally, in 14 to 18 h of germination, coleorhizaeemerge from seeds at room temperature (25 to 30 °C) and rootsappear within 24 h. When a part of the embryo, generally thecoleorhizae in monocotyledonous plants or the radicle indicotyledonous plants, protrudes from the seed coat, theprocess of germination is completed [1]. The roots extendfurther and the coleoptile grows rapidly within 48 h (Fig. 1-B).

Initially, thewater absorption rate is 7.1% per hour, but speeddrops markedly to 1.2% per hour until the coleorhizae emerge.After the low point, the rate of water uptake rises gradually to5.2% after barley seeds germinate. α-Amylase activity showslittle change until coleorhizae emerge, but then rises dramati-cally from 24 to 48 h [51]. Mature dry barley seeds exhibit highresistances to desiccation and abiotic and biotic stress, but theresistance declines gradually with the progress of germination.Before the coleorhizae emerge, the desiccation resistance ofbarley seeds shows no noticeable decrease, whereas after theemergence of coleorhizae the survival rate of germinated barleyseeds decreases significantly under stress [51].

6. Gene expression in barley seeds duringgermination

During seed germination, the observed increase of organelleactivity can be caused by the activation of pre-existing

Fig. 6 – Scheme of coordination of metabolic processes via the glyoxylate cycle in the scutellum and endosperm of barley seedsduring germination. OAA, oxaloacetate; PEP, phosphoenolpyruvate.

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organelles or formation of new ones. In starch-storing seeds,the initial activity of mitochondria relies mainly on theactivation of metabolism in pre-existing organelles [52]. Thisassertion is confirmed on the transcriptional level duringgermination of barley seeds by the observation that genesencodingmRNA synthesis and degradationmachinery proteinsare not upregulated at the early stage of germination, indicatingthat the germinating barley seeds activate pre-existing RNAsynthesis and degradation machinery upon imbibition [51].However, in lipid-storing seeds such as pumpkin, cucumber,and castor bean, de novo synthesis of mitochondrial proteinsusing newly synthesized RNA is predominantly responsible forincrease of the organelle activities [52].

In a study of An and Lin [51], the transcription level of genesduring germination of barley seeds displayed significantdifferences. In the early phase of seed germination, thetranscription of genes involved in regulatory components andcell wall metabolismwas upregulated, and such genes as thoseencoding zinc finger proteins and glutaredoxin reached theirhighest abundance within the first 3 h. The genes encodingWRKY family transcription factors and serine/threonine phos-phatase 2C (PP2C) reached their highest level of expression after9 h of imbibition. The transient high expression of these genesimplies that they are important in initiating or regulatinggermination of barley seeds. For example, the gene encodingserine/threonine PP2C, a negative regulator of ABA sensitivityresponse [53], is upregulated by about 3.5 times within 9 h, anevent favorable to seed germination owing to inhibition of ABA

signaling in the early period of seed germination [51]. Withinthe first 9 h, except for the upregulation of genes involved insignal transduction and regulatory components, the genesinvolved in synthesis, degradation and modification of cellwalls are also upregulated to activate various reactions totrigger seed germination. For instance, most of the genesinvolved in cell wall metabolism such as cell wall precursorsynthesis and cell wall degradation and modification areupregulated [51]. In some seeds, embryo is encompassed byendosperm tissue. Endosperm weakening, which is the maincharacteristic of endosperm rupture caused by radicle expan-sion, is caused by cell wall-remodeling enzymes includingendo-β-mannanase, β-1,3-glucanases, expansins, xyloglucanendotransglycosylase, polygalacturonase, and others, whichare encoded by the upregulated genes at the early germinationphase. The GATA zinc finger transcription factor is an essentialpositive regulator of endosperm rupture and is activatedspecifically in the embryonic axis in Arabidopsis seeds [54]. Inbarley seeds, GATA zinc finger transcription factors areupregulated by more than 5 times in the early phase of seedgermination, displaying that they are vital to root protrusionthrough seed coat [51]. In contrast, several genes encoding lightsignaling and heat-shock transcription factors are downregu-lated, meaning that heat and light-stress responses areinhibited in the early phase of barley seed germination. Thegenes encoding proteins involved in mRNA synthesis anddegradation are not upregulated, suggesting that the RNAsynthesis and degradation machinery becomes active but that

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no new proteins responsible for RNA metabolism are synthe-sized in the early phase of barley seed germination [51].

In themiddle phase of germination, expression ofmore thana thousand genes at the transcription level is adjusted indifferent patterns. Germinating barley seeds undergo markedmorphological and physiological changes, including emergenceof coleorhizae and decrease of desiccation resistance. The genesinvolved in amino acid synthesis, protein synthesis and trans-portation, and synthesis and structure of DNA are upregulated,whereas no upregulation is observed for genes responsible foramino acid degradation. One of the most significantupregulations in the middle phase is that of the transcriptionalactivity of chromatin, consisting of DNA wrapping aroundhistone proteins [55]. Genes encoding histones are upregulatedstrikingly, by 50 times or evenmore, and genes encodingproteinsassociated with chromatin structure are also upregulated in themiddle phase of germination. The dramatic upregulation ofhistone genes in this phase demonstrates that they play pivotalroles in chromatin remodeling and transcription. They may beessential for barley seeds for switching from themiddle phase tothe late phase of germination (post-germination phase). Acety-lation of histones in the nucleosome is vital for regulating thetranscriptional activity of DNA and can increase gene expression[56]; for example, the gene encoding histone acetyl transferaseincreases more than 5 times. In the middle phase of barley seedgermination, some genes are downregulated, including genesinvolved in stress, ABA metabolism and synthesis of lateembryogenesis abundant (LEA) proteins associated with loss ofstress tolerance such as cold and desiccation tolerance [51].

In the late (post-germination) phase several other genes areupregulated. Expression of genes involved in photosynthesisdoes not change much in the early and middle phases, but theyare upregulated significantly in the late phase of germination,meaning that germinating seeds become prepared for thetransition from heterotrophic growth to photo-autotrophicgrowth even though germination occurs in the dark. Further-more, in this phase, expression of the genes involved inmobilization of storage in endosperm is upregulated, whereasno changes or even a decrease in transcripts of the genesinvolved in protein degradation are observed. For instance,transcription of the gene encoding serine protease is downregu-lated in this phase, while expression of the gene encodingglutamine synthase is upregulated. Downregulation of thesegenes is attributed to the decreased demands of their function inseed germination [51].

During the whole process of seed germination, mRNAcomplexity increases gradually. No gene transcription is upreg-ulated inmore than twophases, but the expression of geneswithunnecessary functions is downregulated in more than twogermination phases. In addition, regulation of synthesis anddegradation of GA and ABA in different phases of seedgermination control the processes of germination anddormancy,suggesting that plant seeds have evolved a highly efficientregulatory mechanism for transcription of DNA that can preventfunctionally antagonistic expression of genes in the samegermination phase [51]. More than half of expressed genes arelocated in coleorhizae in the early phase of barley seedgermination, while in the middle phase of seed germination,although the number of expressed genes in roots is greater thanthat in the early phase of germination, it is still less than that in

coleorhizae, indicating that coleorhizae play a vital role inregulation of barley seed germination [57].

7. Effect of light on germination and dormancy ofbarley seeds

White light—mainly the blue part of its spectrum—canpromote dormancy in freshly harvested barley seeds butdark and after-ripening can reduce it [58]. The mechanism ofafter-ripening in reducing seed dormancy is largely unknown.After-ripening does not increase content of GAs duringstorage but does enhance the ability of seeds to generateGAs during imbibition [59]. Light has no effect on germinationof after-ripened barley seeds and these seeds germinatesimilarly in dark and light [57]. The content of ABA in embryosof dormant barley seeds during 24 h imbibition in dark ismuch lower than in seeds imbibed under white or blue light.However, red light and far red light have no obvious effect onthe content of ABA in the embryo, and the expression ofHvNCED1 (Nine-cis-epoxycarotenoid dioxygenase 1), responsiblefor ABA synthesis, in dormant and non-dormant barley seedsimbibed under blue or white light is much higher than that inseeds imbibed under red and far red light [58].

Barley ELIPs (Early Light-Inducible Proteins) such as ELIP Hv58and ELIP Hv90, encoding early light-inducible proteins, play aregulatory role in photomorphogenesis of thylakoids [60], andhave a photoprotective function against light stress [61]; theyare severely inhibited by after-ripening in barley seeds. Howev-er, their expression is induced by blue light in dormant ratherthan after-ripened seeds in the early andmiddle phases of seedgermination [57,58]. Barley ElongatedHypocotyl5 (HvHY5), playinga role in inhibition of hypocotyl and cell elongation under bluelight [62], is upregulated markedly in coleorhizae but not inroots of dormant barley seeds. Its expression is suppressed inboth roots and coleorhizae of non-dormant barley seeds in theearly and middle phases of seed germination under blue light[57]. In addition, PHOT1 (Phototropin 1) encoding phototropin 1, ablue light receptor with motifs designated LOV1 (light, oxygen,or voltage-sensing domain 1) and LOV2 mediating phototro-pism, chloroplast movements, and stomatal opening inArabidopsis, is suppressed in coleorhizae and roots of non-dor-mant barley seeds during the period of germination from dryseeds to emergenceof coleorhizaeunder blue light.However, itstranscription is not inhibited in dormant barley seeds under thesame conditions [57].

8. Mechanism of GA and ABA in regulation of seedgermination and dormancy

8.1. Effect of GA and ABA on seed germination

Seed dormancy and germination are regulated by concentra-tions of ABA and GA and their ratios which are changing inthe process of seed germination. In our experiments [39], GAstimulated germination of dormant barley seeds, whereasABA inhibited growth of radicles. The level of GA is low in drybarley seeds and reaches its highest level after imbibition fortwo days, but the level of ABA is highest in dry seeds and

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decreases to its lowest level after one day of imbibition [63].Expression of HvNCED1, encoding 9-cis-epoxycarotenoiddioxygenase 1, a rate-limiting regulatory step responsible forsynthesis of ABA, is higher in dormant barley seeds than innon-dormant ones, a finding consistent with the ABA content inthe embryos of dormant and non-dormant barley seeds [64].Differential expression of genes involved in ABA metabolism inthe process of seed germination occurs much earlier than that ofGA-associated genes [57]. GA induces expression of genesencoding α-amylase, which is suppressed by ABA, but ABAinduces expression of protein kinase, PKABA1 [65]. GA signalingfactors such as GAMyb (GA-induced Myb (myeloblastosis)-likeprotein) transactivating promoter of α-amylase and other genesencoding hydrolytic enzymes in aleurone cells in barley seeds[12,66] can enhance expression of genes encoding proteins thatmobilize storage reserves in endosperm, but these factors do notdetermine seed germinationdirectly [67]. InArabidopsis seeds, theGA/ABA ratio is crucial for regulation of seed dormancy andgermination. When the ratio is higher, dormancy is maintained.In contrast, when the ratio is lower, dormancy is released andgermination is favored [68]. Testa rupture and endospermrupture are sequential steps during germination of many kindsof seeds. Endosperm rupture was promoted by GA and inhibitedby ABA, but ABA had no effect on testa rupture [69]. ABA inhibitsendosperm rupture and delays endospermweakening [70]. Thus,from this observation it appears that ABA has more obvioussuppressing effects on seeds with embryo surrounded byendosperm (such as Arabidopsis seeds) than on seeds withembryo not surrounded by endosperm (such as barley seeds).

Coleorhiza, a tissue protecting the embryo's radicle duringdevelopment and germination ofmonocotyledonous seeds, is abarrier to germination of seeds that is functionally similar to theblocking of Brassicaceae seed germination by the aleuronelayer(s) [3]. Its content ofABA is vital in regulating the dormancyand germination of barley seeds [57]. Expression of HvABA8′OH-1, encoding ABA 8′ hydroxylase, responsible for catabolismof ABA, is upregulated and is much higher in embryos ofnon-dormant barley seeds than in embryos of dormant barleyseeds during germination, especially in its early phase, and itsexpression is located mainly in coleorhizae of barley seeds.

GA putatively enhances seed germination, but expressionof the genes involved in its metabolism is not markedlyupregulated in coleorhizae of either dormant or non-dormantbarley seeds in the early phase of germination, showing thatGA does not play a key role in regulation of dormancy releaseof coleorhizae or coleorhiza growth in early phase ofgermination [57]. For example, barley Kaurenoic acid oxidase 1(HvKAO1), responsible for generation of GA, is upregulatedonly in coleorhizae of dormant barley seeds after imbibitionfor 18 h, and shows no upregulation in non-dormant barleyseeds within 18 h. In contrast, barley Gibberellin 2-oxidase1(HvGA2ox1), responsible for GA inactivation, is upregulated incoleorhizae of non-dormant barley seeds after 18 h ofimbibition [57], indicating no new production of GA3 in theearly phase of seed germination.

8.2. Interaction between GA and its receptors

Only few reports of interaction between GA and its receptorsin barley seeds have appeared. Thus, research results for rice

and Arabidopsis seeds are used to describe this interaction,and should be generally applicable to barley, whose genes arelargely homologous to the corresponding genes of rice and, tolesser extent, of Arabidopsis. The GA receptor in rice cells isGIBBERELLIN INSENSITIVE DWARF1 (GID1), which is a nuclearsoluble protein encoded by GID1 [71]. In Arabidopsis, threehomologous genes, GID1a, GID1b, and GID1c, have been clonedand the protein encoded by each of them can bind GA in vitro.Expression of these genes individually in a rice gid1mutant canrescue the GA-insensitive dwarf phenotype [72]. A singlemutation has a slight inhibition effect on seed germinationand seedling development, double mutants show more inhibi-tion of seed germination and seedling development, and triplemutants show complete inhibition of seed germination ofArabidopsis and significant inhibition of seedling growth [73–75]. This result shows that the functions of these three genesoverlap and that they work coordinately in GA response as GAreceptors. GA binds GID1, and GA-GID1 interacts with theDELLA domain in SLENDER RICE1 (SLR1) in rice [76]. TheGID1-GA-DELLA complex is then recognized by the SCFSLY/GID2

(Skp1 (S-phase kinase-associated protein 1), Cullin, F-boxcontaining) complex catalyzing the ubiquitination of proteinsfor subsequent protein degradation in a proteasomal pathway[77]. The affinity of GAs for binding GID1 in plants is different.Even though GA3 is the most active in leaf sheath elongation, itbinds to GID only at the intermediate level in a yeast two-hybridtest. GA4 in rice cells has the highest affinity for GID1. In theabsence of GA-inactivating enzymes, GA4 is the most bioactiveamong GA1, GA3, and GA4 [76].

8.3. Interaction between ABA and its receptors

Most of the findings presented here concerning the interac-tion between ABA and its receptors are from Arabidopsis;however, the homologs of genes encoding the correspondingreceptors can be found in cereal genomes. ABA signaltransduction is regulated by some of PP2C genes encodingprotein phosphatase 2C, a negative regulator of ABA signaling,highly expressed in coleorhizae, especially in seeds ofdormant cultivars, but less in radicles of barley seeds inearly and middle phases of germination [57]. The PP2C genesare inducible by ABA treatment even though they confer ABAinsensitivity to seeds during germination [78]. PP2Cs play acentral role in regulating signal transduction of ABA. If ABA isabsent in tissue, PP2Cs interact with SnRK2 (subfamily 2 SNF1(Sucrose-Non-Fermenting Kinase1)-related kinases), positiveregulators of ABA [79], to inhibit activity of SnRK2 bydephosphorylation, which stops further signal transductionof ABA. If ABA is present in tissue, ABA binds with itsreceptors PYR1 (Pyrabactin Resistance 1)/PYLs (PYR1-Likeregulatory components)/RCAR (regulatory components ofABA receptor). The ABA-receptor complex combines withPP2C and suppresses its phosphatase activity, leading to theactive function of SnRK2. Furthermore, SnRK2 phosphorylatestranscriptors of ABA-response genes, resulting in furthersignal transduction [80].

One ABA receptor, PYR protein, encoded by PYRABACTINRESISTANCE 1 (PYR1), has been identified and 13 similarproteins encoded by similar genes called PYR1-like (PYL) fromPYL1 to PYL13 have been found in Arabidopsis [81]. The 14

Fig. 7 – Changes in levels of superoxide (O2U−) and hydrogen

peroxide (H2O2) during germination of barley seeds. Based onquantitative data obtained by Ma [39]. Blue line, non-dormantcultivar (Harrington); Red line, moderately dormant cultivar(Sundre); Dotted line, seeds of Sundre remaining dormant.

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proteins encoded by these genes are located in the nucleusand belong to a family of steroidogenic acute regulatoryprotein (StAR)-related lipid transfer (START) proteins [81,82].They have also been identified as regulatory components ofABA receptor (RCAR), from RCAR1 to RCAR14 [82]. ABA bindsto its receptors, PYR/PYL proteins, within START-domainligand-binding pockets. When the complex anchors to PP2C'snearby active centers, it prevents access by substrates ofPP2Cs. Thus, ABA-PYR/PYL becomes a competitive inhibitor ofPP2Cs [77]. Interaction between PP2C and SnRK2s is constitu-tive and ABA-independent. PP2Cs inactivate SnRK2 activitythrough dephosphorylation when ABA is absent. When ABA ispresent, ABA-PYR/PYL-PP2C binds ABA-activated SnRK2 dif-ferently and activates it [83]. SnRK2 phosphorylates R-x-x-S/Tsites of ABA-responsive element (ABRE)-binding proteins,resulting in upregulation of gene expression [84].

Trans-membrane G protein-coupled receptors (GPCRs/GCRs) are also possible receptors of ABA. GCR1 is one memberof GPCRs with 7-transmembrane (7TM) domains and itsmutants show hypersensitivity to ABA. Thus, it may act as anegative regulator of ABA signaling [85,86]. However, GCR1does not appear to bind ABA. GCR2 is a protein with the 7TMdomains and has high binding affinity for ABA Seeds of thegcr2 mutant of Arabidopsis lose their dormancy and plantswith GCR2 overexpression show hypersensitivity to ABA. Inaddition, GCR2 interacts with G protein α unit (GPA1), butinteraction of ABA with GCR2 causes dissociation of theGCR2-GPA1 complex. All of these results indicate that GCR2and GPA1 work together to transduce ABA signal [87].However GCR2 has been found not to be a 7TM protein andthe gcr2 mutant has similar ABA sensitivity to the wild type[88].

ABA-INSENSITIVE1 (ABI1) and ABI2 are two members ofPP2Cs, negative regulators of ABA responses [89,90] that caninteract with several members of PYR/PYL/RCAR family inArabidopsis. The interaction is induced by ABA, supplementingABA signal transduction [91]. ABI3, ABI4, and ABI5 aredifferent transcription factors with diverse functions in plantsand are different from ABI1 and ABI2 functionally [92–94].ABI3 promotes both seed dormancy and maturation [93],although it does not regulate seed germination [95]. ABI4, animportant transcription factor involved in glucose signalingduring seedling development, binds the promoter of ABI5 anditself to activate their expression [94].

9. The production, scavenging and roles of ROS inregulation of seed dormancy and germination

ROS such as hydrogen peroxide (H2O2), hydroxyl radical (OHU),and superoxide anion (O2U

−) are continuously produced duringseed germination and also in dry seeds. They are generated inall stages of development and germination of seeds. ROSaccumulation during imbibition is important for breakingseed dormancy [96]. In addition, seed imbibition increasesH2O2 content and production of ROS occurs in imbibed bothdormant and non-dormant seeds [5,38,97]. The content ofH2O2 in barley seeds increases at the beginning and content ofO2U

− decreases sharply at the beginning, and later the contentof both H2O2 and O2U

− increases gradually [39]. Also important

is that the content of H2O2 in dormant Sundre seeds is higherthan that in non-dormant Harrington seeds and there is atransient ROS burst in dormant Sundre seeds at the beginningof germination (Fig. 7). Several pathways contribute to ROSproduction during seed germination, including metabolism inmitochondria [98], chloroplast [99], peroxisomes [100] andnitrogen-fixing nodules [101]. First, mitochondrial respirationis probably a major source of ROS produced from incompleteoxygen reduction mainly at respiratory complex I (NADHdehydrogenase) and complex III (ubiquinol-cytochrome c reduc-tase) in the electron transport chain [100]. Second, the peroxi-some is another important organelle producing ROS, generatingboth H2O2 and O2U

−. Electrons from NADH oxidation by trans--membrane monodehydroascorbate reductase (MDHAR, EC1.6.5.4) on the matrix side of peroxisomal membrane aretransferred to their acceptor monodehydroascorbate (MDHA)on the cytoplasmic side of themembrane [102], where O2 can actas an alternative electron acceptor to form O2U

− [103]. Threemembrane polypeptides with molecular masses of 18, 29 and32 kDa have been identified as being involved in generation ofO2U

− [103,104]. Reactions catalyzed by flavin oxidases anddismutation of O2U

− also can produce H2O2 in peroxisomes[105]. Glyoxysomes, which are specialized peroxisomes in seeds,are another source of O2U

− and H2O2 produced from β-oxidationof fatty acids and other biochemical reactions [106].

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When plants produce ROS, they have antioxidant systemsthat scavenge ROS, one of which is the ascorbate–glutathionecycle. In this cycle, ascorbate peroxidase (APX, EC1.11.1.11)reduces H2O2 to H2O by consuming two molecules of ascorbate,generating two molecules of MDHA. MDHA can be reduced toascorbate directly by MDHAR with consumption of NAD(P)H[107]. Another way of regenerating ascorbate is reduction ofdehydroascorbate (DHA), catalyzed by dehydroascorbate reduc-tase (DHAR, EC1.8.5.1) with consumption of two molecules ofglutathione (GSH). Glutathione disulfide (GSSG) is produced inthis reaction, where DHA is supplied from non-enzymaticdisproportionation of MDHA [107]. GSSG is reduced to GSH byNADPH, catalyzed by glutathione reductase (GR, EC1.8.1.7). Insummary, H2O2 is reduced to H2O by the electrons derived fromNADPH and there is no net consumption of ascorbate and GSH,but they participate in transient redox reactions during theprocess [108]. A high content of antioxidants supports ahomoeostatic redox environment in cells of barley seeds [109].Besides this cycle, superoxide dismutase (SOD, EC1.15.1.1), whichtransforms superoxide (O2U

−) to O2, and H2O2 [110], and catalase(CAT, EC1.11.1.6), which transforms H2O2 into H2O and O2 [111],are the important enzymes for detoxifying ROS. In plants, thereare three classes of SOD: Cu/Zn-SOD in chloroplasts and cytosol,Mn-SOD inmitochondria and peroxisomes [112,113], and Fe-SOD

Fig. 8 – Changes in activities of catalase, total SOD, Cu/Zn-SOD,quantitative data obtained by Ma [39]. Blue line, non-dormant cu(Sundre); Dotted line, seeds of Sundre remaining dormant.

in chloroplast [114], though probably not all of these are presentin seeds. Activities of all the enzymes involved in scavengingROS, such as APX, DHAR, and GR, increase in germinating barleyseeds, but activity of DHAR decreases after imbibition for 24 h. Inseeds remaining dormant, their activities do not increasesignificantly (Fig. 8). Activity of CAT increases in the first fewhours of non-dormant Harrington seed germination but laterdecreases. In germinated seeds of a more dormant cultivar itsactivity gradually decreases, whereas it remains at a high level inseeds remaining dormant (Fig. 9). The activities of MDHAR, totalSOD, Mn-SOD, and Cu/Zn-SOD decline within the first few hoursof seed germination but later increase. In seeds remainingdormant, these activities do not change significantly (Figs. 8and 9). In antioxidant metabolism, ascorbate and GSH play animportant role in scavenging ROS, and the change of theircontent has an obvious major effect on redox status in cells.During barley seed germination, the content of ascorbate andGSH decreases, but in seeds remaining dormant their contentdoes not change significantly (Fig. 10).

The content of DHA is higher than that of ascorbate, butthe content of GSH is higher than that of GSSG in the twokinds of barley seeds (Fig. 10). During seed germination, thechange in their content directly influences the ratios ofascorbate/DHA and GSH/GSSG. The ratio of ascorbate/DHA

and Mn-SOD during germination of barley seeds. Based onltivar (Harrington); Red line, moderately dormant cultivar

Fig. 9 – Changes in activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbatereductase (DHAR), and glutathione reductase (GR) during germination of barley seeds. Based on the quantitative data obtainedby Ma [39]. Blue line, non-dormant cultivar (Harrington); Red line, moderately dormant cultivar (Sundre); Dotted line, seeds ofSundre remaining dormant.

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decreases slightly and then increases to peak value within thefirst 9 h but declines later (Fig. 10). The ratio of GSH/GSSGdeclines quickly within the first 9 h. As a result, the reductionpotential of the GSSG/2GSH pair increases at the beginning ofseed germination but shows no obvious change later exceptfor a decrease in the reduction potential within the first 3 h inHarrington seeds.

ROS play a dual role in the process of seed germination.Their beneficial role is participation in cellular signal trans-duction, in endosperm weakening, disease resistance, redoxregulation, and programmed cell death in the aleurone layerin barley seeds [106]. Their detrimental role is in excessiveROS accumulation, causing oxidative damage of cell compo-nents. Bailly et al. [97] introduced a term for the content ofROS during germination of seeds: the “oxidative window”, todescribe the threshold of their concentration during seedgermination. If the concentration of ROS is within theoxidative window, namely between the upper and lowerlimits of ROS concentration, dormancy of seeds will be brokenand germination will start. Within this oxidative window, ROSmay interact with associated hormones to trigger geneexpression or redox reactions. If the concentration of ROS islower than the oxidative window, seeds will remain dormant

and germination will not occur. In contrast, if the content ofROS is higher than the oxidative window, they will causeoxidative damage, inhibition of germination, or abnormalseedlings. Thus, antioxidant defenses are required forprotecting seeds from oxidative damage, and in addition, thethiol redox level of proteins during seed germination in-creases rapidly [115]. Also, signal transduction through redoxreactions plays an important role in seed dormancy release,and thiol–disulfide proteins play an key role in regulatingredox-dependent metabolism and development of seed cellsduring germination [116].

10. The role of nitric oxide in regulating seeddormancy and germination

RNS include nitric oxide (NO) and a group of relatedmoleculessuch as peroxynitrite (ONOO−), dinitrogen trioxide (N2O3),S-nitrosoglutathione (GSNO), and nitrogen dioxide (NO2U). NOitself is present in different forms, such as nitrosyl cation(NO+), NO radical (NOU) and nitroxyl (NO−). NO is synthesizedby nitrate reductase (NR) [117], NO2

−/NO-reductase [118], NOreductase of mitochondrial ETC [119], and non-enzymatic

Fig. 10 – Changes in the ratios of reduced and oxidized ascorbate (ASC/DHA), reduced and oxidized glutathione (GSH/GSSG),total ascorbate (ASC + DHA), and total glutathione (GSH + GSSG) during germination of barley seeds. Based on quantitativedata obtained by Ma [39]. Blue line, non-dormant cultivar (Harrington); Red line, moderately dormant cultivar (Sundre); Dottedline, seeds of Sundre remaining dormant.

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conversion of nitrite [120,121]. NR can reduce both nitrate andnitrite to produce NO with addition of NAD(P)H in vivo and invitro [117,122], the first confirmed mechanism generating NO[123]. However, NO production in plants is still controversial.

In plants, NO plays a vital role in regulating seed dormancyand germination. Results from many experiments demon-strate that NO is involved in germination [124] and dormancyrelease [125] of seeds. The content of NO during seedgermination increases [5,6,39,126]. The content of NO ingerminating seeds of barley sharply increases to a peakvalue within 9 h after imbibition, then decreases to its lowestat 15 h, i.e., at the time of radicle protrusion. This findingindicates that the formation of NO is an important processduring germination, and that NO is actively used even in theanaerobic stage of phase II (Fig. 1-A) in an active metabolicprocess that may be associated with expression of thehypoxically induced plant hemoglobin (phytoglobin) [96,127].In seeds remaining dormant, NO content remains lower thanin germinated seeds (Fig. 11).

Nitrite, azide, and hydroxylamine promote germination ofseeds via NO production from these chemicals [124], and theirstimulation effect is similar to that exerted by gaseous nitrogen

oxides [128]. Dormancy of barley seeds is released by sodiumnitroprusside (SNP), an NO donor, but NO-dependent stimula-tion of germination is blocked by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an NO scaven-ger, during imbibition of seeds, enhancing dormancy of barleyseeds [129]. SNP increases the quantity and types of nitrosylatedproteins and NO concentration in the embryo, but cPTIOreduces the level of protein nitrosylation and NO concentrationduring imbibition of 24 h. S-Nitrosylated proteins can preserveNO and protect it from being scavenged, an effect that plays animportant role in regulating seed dormancy and germination byreleasing NO to the signal transduction chain. It is possible thatnitrosylation of proteins plays a key role in regulating seedgermination and dormancy directly instead of that NOpredom-inantly regulates the process [6]. However, SNP cannot over-come the inhibition by ABA on germination of seeds [129].

In embryo of barley seeds, the level of protein nitrosylationincreases [5], which is also shown in whole barley seeds duringthe first 9 h after imbibition [39], then it decreases and becomesstabilized at time point of 20 h (Fig. 11). In the seeds remainingdormant it remains low. The level of nitrosylation correspondsto theprofile of nitric oxide (Fig. 11). S-Nitrosoglutathione can be

Fig. 11 – Changes in levels of nitric oxide (NO) and totalS-nitrosylation (R-SNO) during germination of barley seeds.Based on quantitative data obtained by Ma [39]. Blue line,non-dormant cultivar (Harrington); Red line, moderatelydormant cultivar (Sundre); Dotted line, seeds of Sundreremaining dormant.

Fig. 12 – Schemeof thephytoglobin–nitric oxide (Pgb/NO) cycle, a seqby its regeneration via metphytoglobin reductase (MetPgb-R) proteinMetPgb-R, NR, and nitrite:NO reductase (Ni:NOR) reactions. The latte

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formed in several ways [7,130–133] such as reaction betweenprotein thiols and intermediate S-nitrosothiols, direct interac-tion between protein sulfhydryls, thiol/disulfide exchangereactions between protein thiols and GSSG orS-glutathionylated proteins (PSSG) and direct interaction be-tween a free protein cysteinyl residue and GSH. Nitrosothiolsare vulnerable and easy to react with intracellular reducingagents such as ascorbic acid or GSH with reduced metal ions,especially Cu+. The instability of S-nitrosothiols leads to theirhalf-lives of seconds to a fewminutes in tissue. S-Nitrosylationregulates protein functions from development to defenseresponses by S-nitrosylation of a single critical Cys residue.Hypoxic conditions lead to the increase of protein nitrosylationin root tissue. For example, transcription of genes encodingclass 1 hemoglobin (GLB1) and NR is expressed several timesmore than that under aerobic conditions in Arabidopsis roots[134]. Nitrate in medium is taken up by tomato roots, nitrite isaccumulated in tissue by oxygen deprivation, activity of NR ismuch higher in anaerobic than in aerobic conditions [135], andNR can reduce nitrate to nitrite using NAD(P)H [136].

External cPTIO has been applied to scavenge NO in manyexperiments [129,137], where it acts by oxidizing NO to formUNO2 radical: NO + cPTIO → UNO2 + cPTI. UNO2 can react withNO to form N2O3: NO2 + NO → N2O3 [138]. In plants, NO can bescavenged by GSNOR and hemoglobin, in plants now calledphytoglobin (Pgb) [139]. Pgb interacts with NO, by which NO istransformed into nitrate and NO is removed, in a reactionfacilitated by MDHAR and ascorbate [127,140,141].Igamberdiev et al. [140] described a Pgb/NO cycle (Fig. 12),where oxyphytoglobin [Pgb(Fe2+)O2] oxygenates NO intonitrate, being reduced itself to metphytoglobin [Pgb(Fe3+)](MetPgb). MetPgb reductase catalyzes the transformation ofMetPgb to [Pgb(Fe2+)] by consuming NAD(P)H. Pgbs in barleyseeds are hypoxia-inducible, and their expression plays a keyrole in maintaining the energy status of cells under hypoxic

uenceof reactionsofNOscavengingbyoxyphytoglobin followed. The cycle is linked to the oxidation of NADH and NADPH inr can be putatively linked to ATP synthesis. Modified from [96].

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conditions [142]. Specifically, if the internal environment inbarley seeds is hypoxic during germination, the producedPgbs will scavenge excessive NO, and ascorbate and activeMDHAR in barley seeds will facilitate the reaction.

Besides reduction of NO by Pgbs, denitrosylation of GSNO isanother way to metabolize NO, which is catalyzed by GSNOreductase (GSNOR). Ma [39] has shown that GSNOR activity inbarley seeds declined quickly from 0 to 24 h and thenstabilized. It is higher in seeds remaining dormant than ingerminated seeds. The decrease of GSNOR activity mayindicate that seed germination needs NO, in particular in theform of nitrosothiols. GSNO is a storage and transport form forNO in plants and seeds [143], having a crucial impact on NOcontent in cells and on seed germination [143–145]. WhenGSNO is decomposed to release NO, GSNO can be convertedinto GSSG or glutathione sulfinic acid, depending on theconcentrations of GSH and oxygen in the environment [146]. IfGSNO is reduced by GSNOR, one source of NO disappears, oneway in which the content of NO in tissue is regulated.

11. Perspectives: crosstalk between hormones,ROS, and RNS

The process of seed germination includes crosstalk betweenNO, ABA, GA, and H2O2. H2O2 treatment increases germinationof dormant Arabidopsis seeds and upregulates ABA catabolismgenes and GA biosynthesis genes. The whole process ismediated by NO signal transduction, given that an NOscavenger, cPTIO, eliminates the stimulation effect of H2O2.H2O2 production is induced by GA in the aleurone layer butsuppressed by ABA, and H2O2 represses transcription ofABA-responsive protein kinase (PKABA). H2O2 induced pro-duction of GAMyb mRNA by inhibition of PKABA expression,given that H2O2 suppressed production of PKABA mRNA [65].H2O2 represses autophosphorylation of PKABA. PKABA isinvolved in signal transduction that suppresses expressionof α-amylase [65,147]. GA and ABA are regulated coordinatelyby GA but antagonistically by ABA, demonstrating that theydisplay antagonistic roles in regulating seed germination onthe transcription level [51]. Thus, they jointly regulatedormancy and germination of seeds [148]. Interaction be-tween ABA and GA has been described in detail, but thecrosstalk between the other elements involved in germinationhas not yet been thoroughly explained. In addition, althoughit is known that levels of NO and nitrosylated proteinsincrease during imbibition for 24 h, the contribution ofdifferent sources of NO release should be further investigated.In conclusion, although much progress has been achieved inunderstanding the processes of germination and dormancyrelease, there are still unresolved questions, including specificaspects of the crosstalk between the action of hormones, ROS,and RNS at the different stages of germination.

Acknowledgments

Thisworkwas supportedby the grant of theNatural Sciences andEngineering Research Council of Canada to A.U.I. (355753/2013).

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