the control of seed dormancy and germination by

The Control of Seed Dormancy and Germinationby Temperature, Light and Nitrate

An Yan1& Zhong Chen1,2,3

1 Natural Sciences and Sciences Education, National Institute of Education, Nanyang TechnologicalUniversity, Singapore, Singapore

2M Grass International Institute of Smart Urban Greenology, 331 North Bridge Road, Singapore, Singapore3 Author for Correspondence; e-mail: [email protected]

# The New York Botanical Garden 2020

AbstractSeed dormancy and germination are two closely linked physiological traits that havegreat impacts on adaptation and survival of seed plants. Seed dormancy strengthen andgermination potential are comprehensively influenced by a variety of internal factorsand external environment cues. Environmental factors, such as water content, lightcondition, ambient temperature, and nitrogen availability, act as signal input to deter-mine whether seeds keep in a dormant state or start to germinate. Light, temperature,and nitrogen availability are the most critical environmental factors that have profoundimpacts on seed dormancy and germination. However, the mechanisms underlying theregulation of seed dormancy and germination by environmental signals are still poorlyunderstood. In this review, we summarize the current knowledge of signal transductionnetworks linking environmental stimulus to seed dormancy establishment, dormancybreak and germination, underscoring the dominating roles of temperature, light, andnitric oxide. We review temperature, light, and nitric oxide signaling pathway sepa-rately as well as the integration of these signaling pathways with phytohormoneabscisic acid (ABA) and gibberellins (GA) signaling pathway in the context of seeddormancy and germination.

Keywords Seeddormancy.Germination .Light .Temperature .Nitricoxide .DOG1.PIF1

Introduction

Flowering plants disperse their progeny by producing seeds. Upon seed production,orthodox seeds acquire desiccation tolerance and stay in a quiescent dehydrated statefor a certain period until ambient environment favor the growth of the new generation(Finkelstein, 2010). The arrested state of seeds refer to seed dormancy, which is definedas a quiescent state of a viable seed that is unable to germinate under favorableconditions (Bewley, 1997; Finch-Savage & Leubner-Metzger, 2006; Bentsink &Koornneef, 2008). Seed dormancy has an important adaptive role in preventing pre-harvest sprouting and optimizing seedling establishment. Seed dormancy is established

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during maturation (Chahtane et al., 2017), during which the dormancy level graduallyincreases and reaches a maximum in freshly matured seeds (Karssen et al., 1983).During subsequent dry storage, seed dormancy is gradually released until seeds candecide the timing of germination by sensing and integrating a series of environmentalsignals (Donohue et al., 2005). Temperature is the most crucial environmental signalexperienced by the mother plant that significantly influence the depth of seeddormancy.

When non-dormant seeds are imbibed under favorable environmental conditions,seed germination occurs. Seed germination is the most crucial developmental transitionin a plant’s life cycle, as it determines subsequent plant survival (Finch-Savage &Leubner-Metzger, 2006). Seed germination initiates upon imbibition, which can bedivided into three major phases: rapid water uptake (Phase I), plateau phase of wateruptake (Phase II), and resumption of water uptake with radicle protrusion (Phase III)(Bewley, 1997). Phase I and Phase II are classified as germination sensu stricto, whilePhase III falls into postgermination. To germinate at the appropriate time, seeds need tosense and integrate various environmental signals to precisely predict seasonal infor-mation (Bewley, 1997). Light, temperature, and nitrate are the most important envi-ronmental factors that greatly influence seed dormancy strength and germinationpotential. These environmental cues act as signal input perceived by plants andconverted into internal cues, further activate signal interplay among endogenousphytohormones, which in turn regulate physiological processes in seeds (Seo et al.,2009). ABA and GA play the most crucial roles as phytohormones in mediating light-and temperature-induced transition from seed dormancy establishment to seed germi-nation. ABA promotes dormancy establishment during seed maturation and inhibitsseed germination, while GA promotes seed germination.

In this review, we summarize the current understanding of mechanisms underlyingthe regulation of seed dormancy and germination by temperature, light and nitrate/nitricoxide, and review the roles of ABA and GA in temperature, light, and nitric oxidesignaling pathways with respect to seed dormancy establishment and germination.

Internal Factors Mediate Seed Dormancy and Germination

The Role of ABA

ABA is a phytohormone involved in a range of developmental and physiologicalprocesses in a plant’s life cycle (Finkelstein, 2013). ABA is the dominating hormoneinvolved in the induction of seed dormancy and control of germination (Gubler et al.,2005; Finkelstein et al., 2008; Yan & Chen, 2017). During seed development, ABA isgradually accumulated in the seed, leading to the establishment and maintenance ofseed dormancy, which is required for inhibition of precocious germination (Koornneefet al., 2002; Finkelstein, 2010). ABA levels in the seed correlate with the depth of seeddormancy. ABA-deficient mutants are often non-dormant or less dormant, even vivip-arous, such as mutants of ABA biosynthesis gene ABA DEFICIENT1 (ABA1), ABA2,ABA3, NINE-CIS-EPOXYCAROTENOID DEOXYGENASE 6 (NCED6) and NCED9(Léon-Kloosterziel et al., 1996; Raz et al., 2001; Lefebvre et al., 2006). Whereasoverexpression of ABA biosynthetic enzyme coding genes, or mutations at ABA

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catabolism genes such as CYP707As lead to enhanced seed dormancy (Kushiro et al.,2004; Okamoto et al., 2006; Lin et al., 2007).

ABA signaling pathway plays an important role in seed dormancy establishment andmaintenance. The ABA-insensitive mutants abi1-1 and abi2-1 show reduced levels ofseed dormancy (Koornneef et al., 1984; Finkelstein, 1994). Loss-of-function ofABSCISIC ACID INSENSITIVE 3 (ABI3), a positive regulator of ABA signalling,causes premature germination in Arabidopsis (Raz et al., 2001). SnRK2 triple mutantsrk2d srk2e srk2i, which is highly insensitive to ABA, exhibits a loss of dormancy(Nakashima et al., 2009). HONSU is one of the major negative regulator of ABAsignalling, honsu mutant shows deep seed dormancy, whereas overexpression ofHONSU results in shallow seed dormancy (Kim et al., 2013).

During dry storage of the seeds (termed after-ripening), seed dormancy graduallydecreases and seeds become sensitive to ambient environmental signals. The decline ofABA levels is a prerequisite for seed germination initiation (Weitbrecht et al., 2011).ABA levels in seed decrease rapidly within a few hours upon imbibition underfavorable conditions (Nambara & Marion-Poll, 2005; Piskurewicz et al., 2008; Linkieset al., 2009; Preston et al., 2009). Whereas unfavorable conditions activate de novoendogenous ABA accumulation and subsequently repress seed germination initiation(Kim et al., 2008; Toh et al., 2008; Piskurewicz et al., 2009; Chiu et al., 2012).Proteomic studies revealed that exogenous ABA application triggers proteolytic mech-anisms in imbibed seeds rather than impeding protein synthesis (Chibani et al., 2006).A major part of ABA-responsive proteins are down-accumulated, which are involvedmainly in protein destination, energy and protein metabolism (Chibani et al., 2006;Pawłowski, 2007). Recently, Footitt et al. (2019) reported that two key tonoplastaquaporins, TIP3;1 and TIP3;2, influenced seed dormancy and germination in responseto stress by antagonistically modulating the response to ABA, with TIP3;1 being apositive regulator while TIP3;2 a negative regulator. Consistent with a reduced re-sponse to ABA, TIP3;2 has a negative effect on primary dormancy induction, as theremoval or down-regulation of TIP3;2 enhanced seed dormancy. Whereas TIP3;1promotes secondary dormancy induction (Footitt et al., 2019). Collectively, ABA levelsand ABA signalling play pivotal roles in the regulation of seed dormancy andgermination.

The Role of GA

GA is another key phytohormone that positively mediates seed germination. Upon seedimbibition, the key GA biosynthetic genes GIBBERELLIN 3-OXIDASE 1 (GA3ox1)and GA3ox2 are strongly induced in the cortex and endodermis of the embryo axis ofgerminating seeds, leading to the de novo synthesis of GA in the embryo (Mitchumet al., 2006; Holdsworth et al., 2008). Bioactive GA in the seed induces synthesis ofhydrolytic enzymes that facilitate the breakage of endosperm and seed coat, mobilizesseed storage reserves in endosperm that support seedling growth, eventually promotesembryo growth and radicle protrusion (Groot & Karssen, 1987; Leubner-Metzger et al.,1996; Finkelstein et al., 2008). GA-deficient mutants fail to germinate without exog-enous GA application, such as GA biosynthesis mutant ga1-3 and ga2 (Mitchum et al.,2006; Shu et al., 2013). Whereas mutation in a GA catabolic genes GA2ox2 lead toelevated GA4 levels and enhanced seed germination potential (Yamauchi et al., 2007).

The Control of Seed Dormancy and Germination by Temperature, Light...

In addition, mutations in GA signalling components also have impacts on seedgermination. For example, loss-of-function mutant of DELLA gene RGA-LIKE 2(RGL2), a negative regulator of GA responses, exhibits strong resistance to GAbiosynthesis inhibitor paclobutrazol, it can also rescue the non-germinating phenotypeof ga1-3 (Lee et al., 2002). Moreover, simultaneous mutations of four DELLA genes(RGL1, RGL2, GAI, RGA) confer light and cold-independent seed germination even inGA-deficient mutant ga1-3 background (Cao et al., 2005), whereas triple mutants ofAtGID1 receptors are GA-insensitive and unable to germinate (Iuchi et al., 2007;Willige et al., 2007). Therefore, GA content in the seed and GA signalling play crucialroles in the control of seed germination.

Besides ABA and GA, other plant hormones have also been shown to participate inthe regulation of seed dormancy and germination, including auxin, brassinosteriods,ethylene, jasmonic acid, salicylic acid, cytokinins, and strigolactones (reviewed in (Huoet al., 2016; Shu et al., 2016b)).

Interplay Between ABA and GA

It has been demonstrated that the timing of seed germination is not determined by ABAor GA alone, but by the dynamic balance of endogenous ABA and GA levels in theseed (Finkelstein et al., 2008; Seo et al., 2009). This conclusion is supported by theobservation that when endogenous ABA levels are relatively low, less active GA isrequired for seed germination compared to conditions with high level of ABA content.High level of ABA biosynthesis associated with GA catabolism maintain the dormantstate of the seed, while active GA synthesis associated with ABA catabolism confergerminating potential.

In fact, ABA and GA act antagonistically to determine the timing of seed germina-tion. In ABA-deficient mutant aba2, the GA synthesis is improved and transcriptions ofGA3ox1 and GA3ox2 are enhanced in germinating seeds. Whereas in ABA over-accumulating mutant cyp707a2-1, PHYB-mediated induction of GA3ox1 and GA3ox2expression is inhibited, suggesting that GA biosynthesis is negatively regulated byABA (Seo et al., 2006, 2009).

Conversely, ABA biosynthesis is subjected to negative regulation by GA. Theoverall ABA levels in the seed of GA deficient mutant ga1 is elevated when comparedwith wild type. Meanwhile, expression levels of ABA biosynthetic genes ABA1,NCED6, and NCED9 are highly elevated in ga1 in comparison with wild type, whereasthe expression level of ABA catabolic gene CYP707A2 is lower than in wild type (Ohet al., 2007). Furthermore, direct application of exogenous GA could repress transcrip-tion of ABA biosynthetic genes and activate ABA catabolic gene (Oh et al., 2007; Songet al., 2019). In lettuce seeds, GA treatment could reduce ABA levels via down-regulation of LsNCED4 during induction of germination (Toyomasu et al., 1994;Sawada et al., 2008). In Arabidopsis dormant seeds, the expression of RGL2 is requiredfor constitutive production of ABA in the endosperm to maintain ABI-dependentrepression of embryo germination (Lee et al., 2010).

During seed germination initiation, ABA represses endosperm rupture, while GApromotes testa rupture and lowers ABA levels, thereby lifts the repression of ABA onendosperm rupture and ensures radicle protrusion. At low GA levels, stabilizedDELLA proteins (GAI, RGA and RGL2) overaccumulate in the seed, thus block testa

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rupture and promote ABA biosynthesis. ABA in turn blocks endosperm rupturethrough the actions of ABA signaling components ABI3 and ABI5 (Piskurewiczet al., 2008, 2009). A putative E3 ligase gene XERICO promotes accumulation ofABA to antagonize GA effects. DELLA proteins can induce transcription of XERICO,thus promote ABA accumulation (Zentella et al., 2007). ABI4, the key component ofABA signaling pathway, positively regulates primary seed dormancy by mediating thebiosynthesis of ABA and GA. The GA levels of abi4 seeds are higher than that of wildtype, suggesting that ABI4 represses GA biosynthesis (Shu et al., 2013). Further studyrevealed that ABI4 directly activates key GA catabolic gene GA2ox7, whereas GA canrepress expression of key ABA biosynthetic gene NCED6 in an ABI4-dependentmanner. In addition, GA can promote ABI4 degradation while ABA stabilizes it (Shuet al., 2016a). NUCLEAR FACTOR-Y C homologues NF-YC3, NF-YC4 and NF-YC9can interact with RGL2, further target ABI5 to form a NF-YC-RGL2-ABI5 module,thus integrating GA and ABA signalling crosstalk during seed germination (Liu et al.,2016; Bi et al., 2017).

Altogether, these observations demonstrate that ABA and GA mutually regulateeach other during seed germination. The complex interplay and dynamic balancebetween ABA and GA act as internal determinants for the transition from seeddormancy to germination, which are greatly influenced by external environment.Environmental signals, such as temperature, light and nitrate, have profound impactson seed dormant states and germinating potential.

Environmental Control of Dormancy Cycling

The depth of seed dormancy at shedding is not only determined genetically, but alsostrongly affected by the environmental conditions under which the mother plant isgrown during seed maturation (Finch-Savage & Footitt, 2017). These environmentalcues mainly include ambient temperature, light, water supply, and nitrate (Finch-Savage & Footitt, 2017). After shed from mother plant in the field, seeds enter intothe soil seed bank and may show a degree of primary dormancy. During their store inthe soil seed bank, seeds adjust their dormancy status by perceiving and integrating arange of environmental signals, cycle from deep dormancy to shallow dormancy untilpermissible environmental conditions arrive, and eventually germinate (Footitt et al.,2013; Finch-Savage & Footitt, 2017). They may also enter secondary dormancy whenthe favourable conditions for germination are absent or seeds encounter prolongedunfavourable conditions (Baskin & Baskin, 1998).

During dormancy cycling, soil environment, mainly soil temperature and moistureconditions, represent the seasonal change. Especially, the seasonal pattern of tempera-ture conveys information of a temporal window to seeds for the selection of right timeof the year to germinate (Footitt et al., 2013; Buijs et al., 2019). The depth of dormancyand the expression of dormancy-related genes are highly correlated with seasonalchange in soil temperature. When soil temperature declines in winter, the depth ofdormancy increases along with an increase in endogenous ABA, which is linked to theincreased expression of ABA synthesis genes. The ABA signalling and sensitivity alsoincrease during the time soil temperature declines (Finch-Savage & Footitt, 2017).When spring comes, soil temperature increases, dormancy level declines along with a


decrease in ABA levels and increase in expression of ABA catabolism genes and GAsynthesis genes (Finch-Savage & Footitt, 2017). At this time of year, the dormant stateswitch from deep to shallow dormancy, which can be rapidly removed by favourablelocal conditions such as light and nitrate. Hence, the roles of temperature, light, andnitrate on the control of seed dormancy and germination are discussed below.

The Effect of Temperature on Seed Dormancy and Germination

Temperature is considered as the most important environmental cue to influenceseed dormancy and germination timing (Chahtane et al., 2017). Seasonal vari-ation in the temperature during seed development strongly affects depth of seeddormancy at seed maturity (Footitt et al., 2011). In many species, low temper-ature experienced by the mother plant can increase final seed dormancy depthduring seed maturation, whereas seeds that develop at warmer temperature showreduced depth of seed dormancy at maturity (Fenner, 1991; Donohue et al.,2008; Contreras et al., 2009; Kendall et al., 2011; Graeber et al., 2012;Burghardt et al., 2016). Transcriptome during seed maturation in Arabidopsisthaliana is highly sensitive to temperature. Low temperature during seed mat-uration enhances seed dormancy through the promotion of DELAY OF GER-MINATION1 (DOG1) accumulation, as well as the influences on GA and ABAmetabolism and signalling in mature seeds (Kendall et al., 2011).

Temperature Regulates Seed Dormancy Through ABA and GA

The influences of temperature on seed dormancy are partially mediated by ABA/GAmetabolism and signalling. Low temperature during seed development can increaseABA content while decrease GA levels, Arabidopsis grown at 15°C has 2-fold higherABA content in seeds than those grown at 22°C, whereas GA4 contents are reducedaround 3-fold (Kendall et al., 2011; He et al., 2014).

In the field study, when soil temperature declines in winter, expression ofABA synthesis (NCED6) and GA catabolism (GA2ox2) genes is increased,resulting increased endogenous ABA levels and reduced GA levels, which areassociated with enhanced seed dormancy (Footitt et al., 2011; Finch-Savage &Footitt, 2017). SnrK 2.1 and SnrK 2.4 expression levels are also elevated,suggesting that ABA signalling and sensitivity are also enhanced by chill soiltemperature. Seed dormancy starts to decline in spring and summer as soiltemperature rises, associated with decreased ABA levels and signalling, as wellas increased GA metabolism. Consistently, expression of ABA signalling com-ponents ABI2 and ABI4 is reduced while expression of CYP707A2 and GA3ox1is elevated (Footitt et al., 2011; Finch-Savage & Footitt, 2017).

During Arabidopsis seed storage, the dormancy level of seeds is greatly affected byambient temperature. Dormancy release is associated with induction or repression ofkey genes related to ABA (NCED3, NCED6, NCED9, CYP707A2 and ABI5) and GA(GA20-oxidase 4 and GA3-oxidase 1) biosynthesis and signaling pathways (Basbouss-Serhal et al., 2016). Collectively, temperature regulates seed dormancy through medi-ating ABA and GA metabolism and signalling.

A. Yan, Z. Chen

Temperature Regulates Seed Dormancy Through DOG1

The Role of DOG1 During Seed Dormancy Establishment

DOG1 is a major quantitative trait locus (QTL) underlying natural variation in seedprimary dormancy of Arabidopsis ecotypes. It was initially identified in Arabidopsis byanalysis of a diallel cross between the highly dormant accession Cape Verde Islands(Cvi) and the weakly dormant accession Landsberg erecta (Ler) (Alonso-Blanco et al.,2003). DOG1 is exclusively expressed in seeds. Loss-of-function mutant of DOG1displays a completely non-dormant phenotype (Bentsink et al., 2006, 2010; Graeberet al., 2014), suggesting that DOG1 is exclusively involved in the control of seeddormancy.

Actually, the abundance of the DOG1 protein in freshly mature seeds highlycorrelates with the depth of primary dormancy levels (Nakabayashi et al., 2012).Deeper dormancy of freshly harvested Cvi seeds contain higher amount of DOG1,while less dormant Ler seeds have lower DOG1 abundance. DOG1 transcript andprotein levels show a strong correlation with temperature during seed maturation, withlower temperature leads to higher accumulation of DOG1, which is associated withenhanced dormancy of those seeds (Chiang et al., 2011; Kendall et al., 2011;Nakabayashi et al., 2012). Therefore, it is believed that DOG1 level is a good indicatorof the depth of seed dormancy imposed by maturation environment at the time of seedmaturation and also a timer for dormancy release (Chiang et al., 2011; Nakabayashiet al., 2012).

DOG1 transcript accumulates during the seed maturation stage with its highestexpression level during mid to late stages of seed maturation when dormancy isestablished (Bentsink et al., 2006). Thereafter, DOG1 transcript decreases duringafter-ripening and disappears quickly upon seed imbibition (Nakabayashi et al.,2012). DOG1 protein also accumulates during seed maturation, however, unlikeDOG1 mRNA, DOG1 protein does not decrease during after-ripening and imbibition.Instead, the protein levels remain stable throughout seed storage and even imbibition inboth dormant and after-ripened (non-dormant) seeds, which means relatively highlevels of DOG1 protein present in germinating seeds when seed dormancy is alreadyremoved (Nakabayashi et al., 2012). Therefore, despite the strong correlation betweenDOG1 protein levels and depth of dormancy in freshly mature seeds, the DOG1 proteinlevels in after-ripened seeds no longer correlate with dormancy levels, suggesting thatDOG1 protein loses its activity during storage (Nakabayashi et al., 2012). Further studyusing two-dimensional (2D) gel electrophoresis combined with immunoblotting re-vealed that the isoelectric focusing of DOG1 protein during after-ripening was changed,suggesting modifications in DOG1 structure and activity, which may render it non-functional (Nakabayashi et al., 2012). Further study indicated that post-translationalmodifications of DOG1 protein may prevent or reduce DOG1 self-binding, which isrequired for its full function (Nakabayashi et al., 2015).

Regulation of DOG1

The mechanism underlying regulation ofDOG1 during seed dormancy establishment iscomplex, involving alternative splicing and self-binding (Nakabayashi et al., 2015),


alternative polyadenylation (Cyrek et al., 2016), noncoding antisense transcript-mediated regulation (Fedak et al., 2016), transcription elongation factor TFIIS(Mortensen et al., 2011; Mortensen & Grasser, 2014) and epigenetic regulation(Zheng et al., 2012; Footitt et al., 2015).

The DOG1 gene consists of three exon fragments, which can form five differenttranscript variants encoding three protein isoforms, suggesting that DOG1 gene issubjected to alternative splicing (Bentsink et al., 2006; Nakabayashi et al., 2015).Single DOG1 protein isoform is unable to accumulate efficiently in the seed and thusfails to complement non-dormant phenotype of dog1 mutant, suggesting that accumu-lation of DOG1 protein requires alternative splicing and additional isoforms arerequired to prevent protein degradation (Nakabayashi et al., 2015). Nakabayashiet al. (2015) further demonstrated that DOG1 protein can bind to itself and this self-binding is essential for full DOG1 activity to induce seed dormancy (Nakabayashiet al., 2015). In addition, Dolata et al. (2015) found that the Arabidopsis thaliana NTR1homologue (AtNTR1) is essential for DOG1 expression and splicing. AtNTR1 loss-of-function mutant atntr1-1 and atntr1-2 show low level of seed dormancy, which isassociated with reduced expression of DOG1 (Dolata et al., 2015). Further analysisindicated that AtNTR1 deficiency increases the rate of PolII elongation and leads to astrong bias towards downstream splice site of DOG1 gene, thereby essentially affectsthe splice isoforms of DOG1, especially the most abundant splice isoforms alpha andbeta (Bentsink et al., 2006; Dolata et al., 2015).

Interestingly, a transfer (T)-DNA insertion in the third exon of DOG1 leads toenhanced dormancy, suggesting that this region of DOG1 may negatively regulateseed dormancy (Cyrek et al., 2016; Fedak et al., 2016; Huo et al., 2016). Furtheranalysis demonstrated that this region of DOG1 gene contains a promoter that drive thetranscription of a noncoding antisense RNA, termed asDOG1 (Fedak et al., 2016).asDOG1 is both 5′ capped and polyadenylated. The expression pattern of asDOG1 isdifferent from all tested DOG1 mRNA isoforms during seed development. asDOG1can strongly suppress DOG1 expression in cis, thereby negatively regulate seeddormancy (Fedak et al., 2016). asDOG1 expression is strongly suppressed by ABA,resulting in the release of antisense-dependent silencing of DOG1 (Yatusevich et al.,2017), suggesting that ABA induce DOG1 expression through regulation of asDOG1.

Many alternatively spliced genes are subject to alternative polyadenylation (APA)(Di Giammartino et al., 2011), which can produce transcripts with different 3′ ends.DOG1 is also proved to be regulated by APA (Cyrek et al., 2016). Alternativepolyadenylation of DOG1 lead to the generation of two alternatively polyadenylatedisoforms of DOG1 transcript. The distally polyadenylated-mRNA is a three-exoniclong mRNA isoform, which is poorly expressed and translated in vivo. Whereas theproximally polyadenylated-mRNA short isoform, which contains only two-exon, istranslated in vivo and able to complement the non-dormant phenotype of the dog1mutant (Cyrek et al., 2016), suggesting that the short DOG1 protein isoform translatedfrom the proximally polyadenylatedDOG1mRNA is functional in the establishment ofseed dormancy (Cyrek et al., 2016). Further study revealed a reciprocal regulation ofthe DOG1-asDOG1 pair. More frequent polyadenylation at DOG1 proximal site leadsto the upregulation of asDOG1, while more frequent polyadenylation at DOG1 distalsite results in decreased asDOG1, indicating that the transcription of asDOG1 isregulated by differential APA site selection (Kowalczyk et al., 2017).

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DOG1 transcription is also enhanced by transcription elongation factor TFIIS, whichfacilitates RNA polymerase II-mediated transcription. tfIIs mutants grow and developnormally, but they have severe defect in seed dormancy, as fully developed tfIIs seedscan germinate efficiently without the requirement of after-ripening (Mortensen et al.,2011). Further study demonstrated that TFIIS mutation causes slowdown of PolIIelongation, thereby down-regulates DOG1 expression (Mortensen & Grasser, 2014).

Recently, Bryant et al. (2019) revealed that basic LEUCINE ZIPPER TRANSCRIP-TION FACTOR67 (bZIP67) is required for DOG1 expression and protein accumula-tion during primary dormancy establishment in Arabidopsis. When seeds mature incool conditions, bZIP67 protein abundance is increased and further activates DOG1expression by directly binding to the DOG1 promoter (Bryant et al., 2019). Li et al.(2019) reported that DOG1 is involved in ethylene-mediated reduction of seed dor-mancy during seed maturation. DOG1 acts downstream of ethylene receptor ETHYL-ENE RESPONSE1 (ETR1) and ethylene response factor ETHYLENE RESPONSEFACTOR12 (ERF12). ERF12 can bind directly to the DOG1 promoter and recruit co-repressor TOPLESS, thereby inhibiting DOG1 expression (Li et al., 2019).

Extensive studies have demonstrated that DOG1 is subjected to epigenetic regula-tion. Histone H3 lysine 9 methyltransferase KYP/SUVH4, which is required for histoneH3 lysine 9 dimethylation, negatively regulates DOG1 expression in Arabidopsis(Zheng et al., 2012). In kyp-2 mutant, DOG1 expression is increased, associated withenhanced seed dormancy, whereas KYP/SUVH4-overexpressing lines show decreasedseed dormancy, suggesting that KYP/SUVH4 can silence DOG1 expression via histonemethylation and thereby negatively regulate seed dormancy (Zheng et al., 2012).Arabidopsis histone demethylases, LYSINESPECIFIC DEMETHYLASE LIKE 1and 2 (LDL1 and LDL2) repress seed dormancy via negative regulation of DOG1.The ldl1 ldl2 double mutant exhibits increased seed dormancy, with enhanced expres-sion of DOG1 during seed dormancy establishment, while overexpression of LDL1 orLDL2 leads to reduced seed dormancy (Zhao, 2015). DOG1 expression is also regu-lated by Polycomb Repressive Complex (PRC). During relief of dormancy, DOG1expression decreases, partially resulting from changing histone methylation patterns onDOG1, with a decrease in H3K4me3 active marks onDOG1 chromatin and an increasein H3K27me3 repressive marks (Müller et al., 2012; Molitor et al., 2014; Footitt et al.,2015). Thus, Footitt et al. (2015) proposed that the changing ratios of H3K4me3 andH3K27me3 marks on DOG1 chromatin act as a thermal sensing mechanism inresponse to seasonally changing soil temperature (Footitt et al., 2015). Additionally,histone H2B ubiquitin transferase HUB is also required for DOG1 expression. Thehub1-2 (rdo4) mutant shows reduced seed dormancy, with a lower expression level ofDOG1 when compared to freshly harvested dormant wild-type seeds, suggesting thatchromatin remodeling by H2B monoubiquitination plays essential role in seed dor-mancy through epigenetic regulation of DOG1 (Liu et al., 2007).

Downstream Regulation of DOG1

Concerning the central role of DOG1 in the control of seed dormancy, understandingthe mechanism underlying DOG1 function is of great importance. Since ABA plays apivotal role in the regulation of seed dormancy during seed maturation, DOG1 mayaffect seed dormancy through its impacts on ABA levels (Nakabayashi et al., 2012).


The strong Cvi DOG1 allele is unable to confer seed dormancy in ABA deficientbackground (aba1-1) (Bentsink et al., 2006), indicating that DOG1 requires ABA toinduce dormancy. Indeed, ABA levels are reduced in dog1 mutants (Bentsink et al.,2006), suggesting that DOG1 may regulate seed dormancy primarily via changes inABA levels. However, Footitt et al. (2011) revealed that ABA is not quantitativelyrelated to the depth of dormancy (Footitt et al., 2011). Therefore, it is proposed thatDOG1 may be the dominant factor in the establishment of seed dormancy by influenc-ing ABA sensitivity (Finch-Savage & Footitt, 2017).

Nakabayashi et al. (2012) showed that both DOG1 and ABA are absolutely requiredto establish seed dormancy, as reduced dormancy is observed in a high ABA levelsbackground in the absence of DOG1 (dog1-2 cyp707a2-1) (Bentsink et al., 2006;Nakabayashi et al., 2012). Whereas genetic evidence demonstrated that DOG1 canfunction largely independent from ABA (Nakabayashi et al., 2012; Graeber et al.,2014). Interestingly, DOG1 can interfere with ABA signalling component ABI3 tomediate multiple aspects of seed maturation (Dekkers et al., 2016), whereas DOG1expression is upregulated in abi5mutants upon seed imbibition in the presence of ABA(Kinoshita et al., 2010; Chahtane et al., 2017), suggesting that a crosstalk existsbetween ABA signalling and DOG1 pathway (Chahtane et al., 2017).

Recent s tudies showed tha t four phosphatases , inc luding ABA-HYPERSENSITIVE GERMINATION 1 (AHG1), AHG3, REDUCED DORMAN-CY 5 (RDO5) and PROTODERMAL FACTOR 1 (PDF1), interact with DOG1 inseeds (Née et al., 2017). Two of them, AHG1 and AHG3, belonging to clade A oftype 2C protein phosphatases (PP2Cs), are involved in ABA singling pathway. Thetriple mutant dog1 ahg1 ahg3 show similar levels of seed dormancy and ABAsensitivity with the ahg1 ahg3 double mutant, suggesting that DOG1 acts upstreamof AHG1 and AHG3 (Née et al., 2017). Interaction of DOG1 with AHG1 and AHG3negatively regulates the PP2C activities of them in vitro (Née et al., 2017;Nishimura et al., 2018). It is proposed that ABA signalling and DOG1 pathwayconverge at clade A of type 2C protein phosphatases (AHG1 and AHG3 as majorcomponents) during the establishment of seed dormancy. Both ABA and DOG1 actupstream of AHG1 and AHG3 to suppress their actions, and further regulatedownstream components including SnRK2s and ABI5, thereby promote seed dor-mancy (Née et al., 2017; Nishimura et al., 2018). Therefore, AHG1/AHG3 andDOG1 constitute an important regulatory pathway in parallel with the PYL/RCARABA receptor-mediated regulatory system with the convergence at AHG1/AHG3(Née et al., 2017; Nishimura et al., 2018) (Fig. 1). In addition, Nishimura et al.(2018) found that DOG1 is an α-helical protein that is able to bind heme, which isessential for DOG1 function in vivo (Nishimura et al., 2018).

DOG1 has also been proposed to enhance seed dormancy by a temperature-dependent alteration of the GA metabolism. Both DOG1 and GA2ox are induced inresponse to low temperature. GA levels are elevated in dog1 mutant (Bentsink et al.,2006; Nakabayashi et al., 2012). Further study indicated that DOG1 promotes GAcatabolism during maturation, mutants with altered GA synthesis exhibit reduced seeddormancy in response to low temperature during seed maturation (Kendall et al., 2011).In addition, DOG1 can inhibit the expression of GA-regulated cell-wall remodelingprotein coding genes, which are required for endosperm and seed coat weakeningduring seed germination in Arabidopsis and other Brassicaceae family members

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(Graeber et al., 2013, 2014). Thus, it is proposed that DOG1-mediated coat-dormancymechanism controls seed germination through regulation of GA metabolism in atemperature-dependent manner (Graeber et al., 2014).

Another mechanism underlying DOG1-mediated seed dormancy regulation hasbeen revealed recently. Huo et al. (2016) found that DOG1 regulates seed dor-mancy through affecting expression levels of miRNAs (miR156 and miR172)(Huo et al., 2016). In Arabidopsis, higher miR156 levels lead to enhanced seeddormancy. The expression levels of a few genes associated with miRNA process-ing are lower in dry seeds of loss-of-function dog1-3 mutant or DOG1-RNAi lines,whereas the transcriptions of these genes increase in DOG1 gain-of-function dog1-5 mutant, which displays enhanced seed dormancy (Huo et al., 2016). In contrast,suppression of LsDOG1 expression lead to reduced miR156 and increasedmiR172 levels associated with decreased seed dormancy level in lettuce. Theseobservations suggest that DOG1 controls seed dormancy through regulation ofmiRNA processing (Huo et al., 2016).

Most knowledge regarding the functional characterization ofDOG1 in the control ofseed dormancy originates from studies on model plant Arabidopsis. It is thus veryinteresting that whether DOG1 genes from other species act in a similar manner as inArabidopsis. Reciprocal gene-swapping experiments between Brassicaceae speciesdemonstrated that the mechanism of DOG1-mediated dormancy is conserved amongBrassicaceae species (Graeber et al., 2014). Ashikawa et al. (2010) showed that ectopicoverexpression of either of wheat and barley homologues of the AtDOG1 (TaDOG1L1and HvDOG1L1) in transgenic Arabidopsis markedly increase seed dormancy, despitethe low sequence similarities to AtDOG1 and different tissue-specific expressionpattern (Ashikawa et al., 2010). Further study introduced these two DOG1 homologues(TaDOG1L4 and HvDOG1L1) into the wheat cultivar Fielder and found that seeddormancy level was enhanced, whereas knockdown of endogenous TaDOG1L4 inFielder remarkably reduced seed dormancy level (Ashikawa et al., 2014). Altogether,these experiments demonstrate that the function of DOG1 in seed dormancy regulationis conserved in many different plant species.

Fig. 1 Simplified model showing regulatory network of DOG1-mediated seed dormancy under low temper-ature. During seed maturation, cold temperature can promote accumulation of DOG1 and enhance ABAsignaling. Both DOG1 and ABA are required to establish seed dormancy. They can act coordinately orindependently from each other to regulate seed dormancy. Arrows indicate positive regulation and T-barsindicate negative regulation. Factors that positively regulate DOG1 are shown in red, factors that negativelyregulate DOG1 are shown in blue


Effect of Temperature on Germination Potential During Seed Imbibition

After harvest, seed dormancy status reduces during after-ripening, the states of seedsshift from dormant to quiescent. At this stage, seeds are able to initiate germinationwhen imbibed under favourable conditions. The ambient temperature surround seedsduring imbibition has significant impacts on seed germination potential.

Stratification

In Arabidopsis, low temperature during seed maturation enhances depth of seeddormancy, while low temperature during seed imbibition stimulates germination(Footitt et al., 2011; Kendall et al., 2011; Graeber et al., 2012; He et al., 2014).Therefore, incubation of imbibed seeds at low temperature (usually at 4°C) for a fewdays, termed cold stratification, is widely used to break seed dormancy and promotegermination. It has been shown that cold stratification breaks seed dormancy andimproves the frequency of germination by modulating ABA/GA balance.

Yamauchi et al. (2004) demonstrated that a subset of GA biosynthesis genes areupregulated by cold stratification (Yamauchi et al., 2004; Kim et al., 2019), especiallyGA3ox1, a rate-limiting gene for GA biosynthesis, which triggers expansion of cortexcells in the radicle/hypocotyl region and subsequently increases growth potential of theembryo during germination (Ogawa et al., 2003). Loss-of-function mutant of GA3ox1does not respond to cold treatment. Meanwhile, expression level of GA catabolismgene GA2ox2 decreases at low temperature. These expression patterns of GA metab-olism genes are consistent with the elevated levels of bioactive GAs during seedimbibition at low temperature (Yamauchi et al., 2004). in situ RNA hybridizationanalysis suggested that GA3ox1 is expressed in the entire embryonic axes and stronglyaccumulates in the micropylar endosperm after cold stratification (Yamauchi et al.,2004), suggesting that the cellular distribution of GAs may be changed after coldstratification.

Besides GA metabolism genes, a set of GA-inducible genes are also upregulated inimbibed seeds during moist cold stratification, such as EXPA1 and EXPA2, which show30-fold increase in expression level at 4°C compared that at 22°C (Yamauchi et al.,2004; Weitbrecht et al., 2011). Many α-expansin genes are GA-inducible and involvedin seed germination (Yan et al., 2014). In addition, α-expansins also localize in themicropylar endosperm, further supporting that cold stratification promotes seed germi-nation through induction of GA biosynthesis and signalling (Weitbrecht et al., 2011).

Cold stratification releases seed dormancy also via the impact on ABA metabolismand signalling. ABA content is declined upon cold imbibition, gene expression levelsof several ABA signalling components including PYL6, ABI4, ABI5, PP2Cs andSnRK2s are also changed (Weitbrecht et al., 2011). In the natural environment, soiltemperature is the major environmental factor which determines the timing of transitionfrom dormancy to germination in the soil seed bank. The oscillations in temperatureassociated with soil depth and changing seasons act as an important instructive signalfor seeds. Topham et al. (2017) identifed the presence of a decision-making centerwithin embryonic radicle of dormant seeds. In the decision-making center, GA andABA metabolic interactions and response occur within spatially separated compart-ments, providing seeds with an efficient way to process variable temperature inputs to

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promote the breaking of dormancy (Topham et al., 2017). Altogether, these studiesdemonstrate that cold stratification promotes seed germination by exerting impact onthe ABA/GA metabolism and signalling.

At physiological and metabolic level, seed imbibition activates a series of processes,including mRNA metabolism and protein synthesis, storage reserve mobilization anddetoxification, amino acid metabolism, energy production, proteolysis, enzymaticweakening of testa and endosperm, and cell and embryo expansion (Nonogaki et al.,2010; Weitbrecht et al., 2011). A proteomic study by Arc et al. (2012) investigated seedproteome dynamics during Arabidopsis seed dormancy release caused by cold strati-fication (Arc et al., 2012). It is observed that cold stratification promotes accumulationof enzymes involved in reserve mobilization. Energy consumption processes associatedwith seed germination and preparation for subsequent seedling establishment are alsoenhanced. 2D electrophoresis analysis combined with differential proteomic toolsidentified differentially abundant protein spots, which are related to the translationmachinery, energetic pathways, amino acid biosynthesis and recyclingafter coldstratification.

Thermoinhibition

When seeds are imbibed at temperature above optimum, the germination potential isoften inhibited. The suppression of germination at supraoptimal temperature is termedthermoinhibition (Reynolds & Thompson, 1971; Abeles, 1986; Gallardo et al., 1991).Thermoinhibition is of ecological importance in the determination of the appropriateseasonal timing for seed germination of winter annual plants (Baskin & Baskin, 1998),since delaying germination at high temperature has a protective role to the newgeneration.

In lettuce, seed germination is inhibited at 28°C, which can be prevented by ABAbiosynthesis inhibitor fluridone (Gonai et al., 2004). An earlier study showed thatapplication of ABA can lower the threshold temperature for lettuce seed germination(Reynolds & Thompson, 1971), suggesting that ABA is required for thermoinhibition.The sensitivity of lettuce seeds to ABA is enhanced by high temperature, as applicationof fluridone is no longer effective to prevent thermoinhibition at 33°C (Gonai et al.,2004). However, further application of GA along with fluridone can preventthermoinhibition and promote germination at 33°C, suggesting that GA is also involvedin thermoinhibition. Actually, exogenous GA exerts its impact via lowering endoge-nous ABA content in the seeds, suggesting that ABA plays a crucial role in theregulation of thermoinhibition of lettuce seed germination, whereas GA regulates seedgermination in response to high temperature through affecting ABA metabolism(Gonai et al., 2004). The important roles of ABA and GA in the regulation ofthermoinhibition have also been revealed in extensive studies and in other species(Tamura et al., 2006; Argyris et al., 2008; Toh et al., 2008; Chiu et al., 2012).

At high temperature, ABA content in imbibed seeds is elevated, resulting from up-regulation of ABA biosynthetic genes (ABA1, NCED2, NCED5, and NCED9) anddown-regulation of ABA catabolic genes (CYP707A1, CYP707A2, and CYP707A3)(Toh et al., 2008; Liu et al., 2019). Meanwhile, GA content in imbibed seeds isdecreased by high temperature and this inhibition effect is correlated with down-regulation of GA biosynthetic genes, such as GA20ox1, GA20ox2, GA20ox3, GA3ox1,


and GA3ox2 (Toh et al., 2008). In line with the roles of ABA in thermoinhibition,ABA-deficient mutants, such as aba1, aba2, and nced2 nced5 nced9, as well as ABA-insensitive mutants, such as abi1 and abi3, are all thermoinhibition-tolerant and exhibithigher germination frequency (Tamura et al., 2006; Toh et al., 2008). GA signalling isalso involved in thermoinhibition. As loss-of-function mutants of SPINDLY (SPY) andRGL2 are thermoinhibition-tolerant, suggesting that repression of GA signalling isrequired for thermoinhibition.

Chiu et al. (2012) found FUS3, a master regulator of seed maturation, is involved inthermoinhibition in Arabidopsis (Chiu et al., 2012). FUS3 overexpression lines showhypersensitivity in response to high temperature and do not germinate. Application offluridone can partly recover hypersensitivity of FUS3 overexpression lines to hightemperature, suggesting that FUS acts downstream of ABA to inhibit seed germinationat supraoptimal temperature (Chiu et al., 2012). Lim et al. (2013) found that SOMNUS(SOM), a regulator of light-mediated seed germination, plays an important role inthermoinhibition. At high temperature, som mutant seeds germinate more frequentlythan wild type, while SOM-overexpressing lines germinate at lower frequencies,suggesting that SOM negatively regulates seed germination at high temperature. SOMexpression is induced by high temperature in ABA- and GA-dependent manner. Furtheranalysis demonstrated that ABI3, ABI5, and DELLAs all bind to the SOM promoterand interact with each other to form a complex, thereby activate SOM expression athigh temperature (Lim et al., 2013). Yang et al. (2019) identified the epigenetic factorPowerdress (PWR) as the ABI3 interaction protein.in this pathway. PWR enhancesseeds germination tolerance to high temperature by accelerating histone H3deacetylation level and H2A.Z deposition at SOM locus, thereby suppressing ABI3-dependent SOM transcription. Overexpressing PWR enhanced seeds germination ther-motolerance, while pwr mutant showed reduced thermotolerance (Yang et al., 2019).Hence, these findings provide a mechanism by which high temperature inhibits seedgermination via ABA- and GA-mediated activation of SOM. Recently, Chen et al.(2019) found that high temperature accelerates the efflux of E3 ligase CONSTITU-TIVE PHOTOMORPHOGENESIS 1 (COP1) from the nucleus to the cytosol, leadingto increased nuclear accumulation of ELONG HYPCOTYL 5 (HY5), which canactivate the expression of ABI5 and thereby suppress seed germination. Furthermore,they found a small gas messenger hydrogen sulfide (H2S) reverses the effect ofthermoinhibition by restraining COP1 in the nuclear to increase degradation of HY5,thereby reduces ABI5 expression and enhances seed thermotolerance (Chen et al.,2019). Altogether, ABA and GA metabolism and signalling play a central role toinhibit seed germination at unfavorable supraoptimal temperature. In addition, otherplant hormones, such as ethylene and strigolactone, are also involved inthermoinhibition, partially in ABA- and GA-dependent manner (Matilla, 2000;Kozarewa et al., 2006; Toh et al., 2012).

At protein level, high temperature during seed imbibition can decrease the abun-dance of proteins which are involved in methionine metabolism, amino acid biosyn-thesis, protein folding, energy metabolism, and reserve degradation (Liu et al., 2015). Aproteomic study on lettuce seed thermoinhibition (Wang et al., 2015a, b, c) identified20 proteins showing higher abundance in germinated seeds after imbibition at optimaltemperature than in ungerminated seeds imbibed at thermoinhibitory temperature.These proteins are involved in various biological processes, including amino acid

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and lipid metabolism, cellular structure establishment, and detoxification. Abundancesof three proteins (AACT1, HMGS2 and MDPC2) that involved in the mevalonatepathway for isoprenoid biosynthesis are markedly higher at optimal temperature than atthermoinhibitory temperature, suggesting that mevalonate pathway is involved in theregulation of lettuce seed thermoinhibition.

The Effect of Light on Seed Dormancy and Germination

Light is not only absorbed by plants as a source of energy to produce organiccompounds, but also perceived as a source of information for plants to adjust theirgrowth and development in response to the changing environment. When buried in thesoil, seeds can judge whether they are close enough to the surface of the ground bysensing the quality and intensity of light signal. If seeds are buried too deep, germina-tion will be inhibited, otherwise the emerging seedlings will not be able to reach thesurface to initiate photosynthesis before the energetic compounds stored in the seedsare exhausted (Ballaré et al., 1992; Batlla & Benech-Arnold, 2014). Therefore, light isan important environmental factor for seeds to determine whether surrounding envi-ronment is suitable for germination and subsequent seedlings emergence.

Light Regulates Seed Germination Through Phytochrome

Plants use photoreceptors to perceive light. There are four different types of photore-ceptors in Arabidopsis, including phytochromes, cryptochromes, phototropins andzeitlupes (Bae & Choi, 2008). Among these photoreceptors, phytochromes are theprimary photoreceptors that are responsible for seed germination in response to red andfar-red light. Phytochromes are photoreversible biliproteins, which are composed of anapoprotein and a chromophore. They have two photoreversible forms: the far-red light-absorbing Pfr form and red light-absorbing Pr form . Phytochromes are synthesized inthe Pr form in darkness. Upon exposure to red light, the Pr form of phytochrome switchisomeric conformation into the Pfr form, which is the bioactive form (Quail, 2002).This reaction is photoreversible and the final proportion of active Pfr is determined bythe ratio of the red light/far-red light.

The Arabidopsis genome encodes five phytochromes (PHYA-PHYE) (Clack et al.,1994; Mathews & Sharrock, 1997). They have both specific and partially overlappingfunctions in seed germination in response to different light conditions (Casal &Sánchez, 1998). PHYA and PHYB are the best characterized phytochromes inArabidopsis. PHYA mediates the very low fluence response (VLFR) and the highirradiance response in far red light (FR-HIR). It is irreversibly activated by both red andfar-red light. PHYA is the main photoreceptor promoting seed germination in responseto wide spectra of light and continuous FR light after long period of imbibition in thedark. Conversely, PHYB mediates red/far-red-reversible low fluence response (LFR).PHYB plays a prominent role to promote red light-induced seed germination(Shinomura et al., 1996). Therefore, FR light has opposite effects on PHYA and PHYBactivity, activating PHYAwhile deactivating PHYB. As a consequence, a pulse of FRlight at early stage of seed imbibition deactivates PHYB and thus leads to repression ofPHYB-dependent seed germination. However, after long period of imbibition, PHYA


accumulates at high levels, a second pulse of FR light converts PHYA into its active Pfrform and thus promote PHYA-dependent germination (Reed et al., 1994; Shinomuraet al., 1994). Lee et al. (2012) demonstrated that a pulse of FR light early uponimbibition inactivates PHYB in the endosperm and leads to accumulation of highABA levels in the endosperm. Although the FR pulse activates PHYA-mediatedgermination in the embryo at the same time, ABA synthesized in endosperm isdispersed towards the embryo and overrides the positive effect of PHYA signallingthrough ABI5, thereby prevents seed germination. After 2 days of imbibition, theinhibitory effect of ABA originating from the endosperm decreases over time, a FRpulse at this moment activates PHYA signalling and eventually promotes germination(Lee et al., 2012).

In addition to PHYA and PHYB, the rest phytochromes, PHYC, PHYD, and PHYE,also play roles in seed germination (Hennig et al., 2002; Dechaine et al., 2009; Aranaet al., 2014). PHYE has been shown to be necessary for PHYA-mediated seedgermination under continuous FR light (Hennig et al., 2002), both PHYE and PHYDcan stimulate seed germination at very low R/FR ratios, probably via promoting PHYAsignalling (Arana et al., 2014). In addition, PHYD is required for completely germina-tion of seeds that exposed to high temperature during imbibition (Heschel et al., 2008;Martel et al., 2018). PHYC has been shown to negatively regulate seed germination inresponse to light (Arana et al., 2014). These observations collectively indicate thatphytochromes have diverse and overlapping functions to cope with different lightconditions imposed on seed during seed germination.

The Effect of Light on ABA and GA

It has been demonstrated that light controls seed dormancy and germination throughintegrating ABA and GA metabolism and signalling. Phytochrome-mediated seedgermination is associated with decreased ABA content and reduced ABA signallingstrength, as well as increased GA content and enhanced GA responsiveness.

Red light decreases endogenous ABA content in lettuce and Arabidopsis seedsduring imbibition (Toyomasu et al., 1994; Seo et al., 2006; Sawada et al., 2008). Thedecreased ABA content is associated with down-regulation of ABA biosynthesis genes(ABA1, NCED6, and NCED9), and with up-regulation of ABA catabolic gene(CYP707A2) in Arabidopsis (Seo et al., 2006; Oh et al., 2007; Kim et al., 2008). Inlettuce seeds, the expression of LsNCED2 and LsNCED4 is repressed by red light,whereas expression of LsABA8ox4 is upregulated by red light (Sawada et al., 2008).Recently, Gu et al. (2019) identified a histone H3 lysine 9 methyltransferase SUVH5 asa positive regulator of light-mediated transcriptional regulatory network in seed germi-nation in Arabidopsis. SUVH5 regulates 24.6% of the light-responsive transcriptome.SUVH5 promotes PHYB-dependent seed germination by repressing the transcriptionof ABA biosynthesis and signal transduction-related genes, as well as DOG genes viadimethylation of H3K9 (Gu et al., 2019).

In addition to ABA metabolism, crosstalk between ABA signalling and lightsignalling has also been implied. FAR-RED ELONGATED HYPOCOTYL3 (FHY3)and FAR-RED IMPAIRED RESPONSE1 (FAR1), two key components in the PHYApathway in Arabidopsis, directly bind to the promoter of ABI5 and activate its expres-sion (Tang et al., 2013), suggesting that FHY3 and FAR1 positively regulate ABA

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signaling. fhy3 mutant is less sensitive to ABA-mediated inhibition of seed germina-tion, which can be restored by overexpression of ABI5. Additionally, FHY3 and FAR1transcriptions are upregulated by ABA. These observations suggest that light and ABAsignalling converge at FHY3 and FAR1 during seed germination (Tang et al., 2013).

Red light promotes PHYB-dependent seed germination partially through increasingendogenous GA levels and GA responsiveness. Consistent with the increased GAlevels, the expression of GA anabolic genes (GA3ox1 and GA3ox2) is enhanced byred light, whereas GA catabolic gene GA2ox2 is repressed (Yamauchi et al., 2004; Ohet al., 2006; Seo et al., 2006; Yamauchi et al., 2007). The induction of GA biosyntheticgenes can be canceled by FR light, suggesting a photo-reversible regulation of GAbiosynthesis by phytochrome. Jiang et al. (2016) identified two transcription factorsRVE1 and RVE2 that promote seed dormancy and repress R/FR light-mediatedgermination in Arabidopsis (Jiang et al., 2016). RVE1 can directly bind to the promoterof GA3ox2 to inhibit its transcription, subsequently suppresses bioactive GA biosyn-thesis. RVE1 and RVE2 expression is down-regulated by PHYB, suggesting that RVE1and RVE2 act downstream of PHYB (Jiang et al., 2016). In addition, GA signallingcomponents DELLA proteins are also involved in phytochrome-mediated seed germi-nation. The crosstalk between light signalling and GA signalling is discussed in thefollowing section.

PIF1 is Involved in Phytochrome-Mediated Seed Germination

Phytochromes Regulate Seed Germination in Response to Light Through Phytochrome

INTERACTING FACTOR1 (PIF1, also known as PIL5) (Oh et al., 2007). PIF1 isa basic helix-loop-helix (bHLH) transcription factor belonging to the bHLHsubfamily 15 of Arabidopsis (Toledo-Ortiz et al., 2003; Leivar & Quail, 2011).It contains an active phytochrome binding (APB) domain, which is necessary forits direct interaction with phytochromes (Khanna et al., 2004). In the dark, asphytochromes are inactive, the stabilized PIF1 inhibits germination of imbibedseeds. Whereas in the light, Pfr forms of PHYA and PHYB are translocated intothe nucleus and interact with PIF1, thereby degrade PIF1 protein via the ubiquitin-proteasome system and promote seed germination (Oh et al., 2006). Loss-of-function mutant of PIF1 can germinate in the dark, whereas PIF1 overexpressionlines require high fluence of light for seed germination, suggesting that phyto-chromes promote seed germination by deactivating PIF1 (Oh et al., 2004, 2006;Shen et al., 2005).

PIF1 Regulates Seed Germination by Affecting ABA and GA Pathway

It has been proved that PIF1 exerts repressive effect on seed germination by reducingGA content while enhancing ABA accumulation in imbibed seeds (Oh et al., 2006,2007; Kim et al., 2008). Specifically, PIF1 reduces GA content by repressing theexpression of GA biosynthetic genes (GA3ox1 and GA3ox2) and promoting theexpression of GA catabolic gene GA2ox2 (Oh et al., 2006). Meanwhile, PIF1 increasesABA accumulation through activating ABA biosynthetic genes (ABA1, NCED6, andNCED9) and repressing ABA catabolic gene CYP707A2 (Oh et al., 2007).


PIF1 also exerts its repression on seed germination through affecting GA and ABAresponsiveness. PIF1 directly binds to the promoters of GAI and RGA and upregulatestheir transcript levels, thereby reduces GA responsiveness (Oh et al., 2007). Mean-while, PIF1 can enhance ABA responsiveness by activating the expression of ABI3 andABI5, the positive components of ABA signaling pathway (Oh et al., 2009; Kim et al.,2016). Moreover, lower GA levels in dark imbibed seeds can increase GAI and RGAprotein stability, further reducing GA responsiveness. Whereas higher ABA levelsenhance ABA responsiveness by stimulating ABI3 and ABI5 transcription. Therefore,PIF1 coordinately regulates ABA and GA metabolism and signalling, leading to theABA/GA balance towards the inhibitory effect on seed germination in the dark (Fig. 2).In addition, PIF1 represses a few genes required for cell wall loosening, such asEXPANSIN (EXP) genes and XYLOGLUCANENDO-TRANSGLYCOSYLASE/HYDRO-LASE (XTH) genes (Oh et al., 2009), which are essential for seed germination.

Downstream Targets of PIF1 During Light-Mediated Seed Germination

As there is no evidence to support the direct regulation of ABA and GA metabolism byPIF1, it is reasonable that PIF1 acts indirectly upstream of ABA and GA through theactions of its direct downstream target genes. SOMNUS (SOM) has been identified as a

Fig. 2 A model for phytochrome-mediated control of seed germination. Left, in the dark. The phytochromesare in the inactive Pf form. PIF1 accumulates to high levels and activates various downstream factors,including SOM, DAG1, GAI, RGA, ABI3, and ABI5. Their actions result in increased ABA biosynthesisand enhanced ABA signalling, as well as decreased GA biosynthesis and reduced GA responsiveness, leadingto the repression of seed germination. Right, in the light. Upon light exposure, the Pr form of phytochromeswitch isomeric conformation into the biologically active Pfr form. Pfr phytochromes bind to PIF1 and triggerrapid degradation of PIF1, leading to reduced ABA content and elevated GA levels in the seeds. Ultimately,seed germination initiates. Arrows indicate positive regulation and T-bars indicate negative regulation. Redelements and lines indicate active factors and regulations

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direct target gene of PIF1 (Kim et al., 2008). SOM encodes a CCCH-type zinc fingerprotein, negatively regulates light-dependent seed germination downstream of PIF1 byactivating the expression of ABA biosynthetic genes (ABA1, NCED6, and NCED9) andGA catabolic gene GA2ox2, as well as decreasing the expression of GA biosyntheticgenes (GA3ox1 and GA3ox2) and ABA catabolic gene CYP707A2 (Kim et al., 2008).som mutant seeds contain higher levels of GA4 and lower levels of ABA. Similar topif1, som mutant seeds can germinate in the dark (Kim et al., 2008). Therefore, PIF1inhibits seed germination by directly activating the transcription of SOM (Fig. 2).

SOM is also activated by ABI3 through directly binding of ABI3 to two RY motifswhich present in the SOM promoter. ABI3 interacts with PIF1 and directly bind to theSOM promoter, collaboratively activate SOM expression (Park et al., 2011). Therefore,SOM also acts downstream of ABI3.

Cho et al. (2012) showed that two histone arginine demethylases, JMJ20 and JMJ22,act downstream of SOM to positively regulate seed germination in response to light(Cho et al., 2012). In the dark, JMJ20 and JMJ22 are directly repressed by SOM.Whereas in the light, when PHYB is activated, light-activated PHYB-PIF1-SOMpathway leads to the downregulation of SOM, JMJ20/JMJ22 are thereby derepressedand target to GA3ox1 and GA3ox2 chromatin, further remove histone arginine meth-ylations at the promoters of GA3ox1 and GA3ox2. As a consequence, GA3ox1 andGA3ox2 expression increase, resulting in accumulation of GA in seeds, and germina-tion occurs (Cho et al., 2012).

DOF AFFECTING GERMINATION 1 (DAG1) is another downstream target ofPIF1, acting as a negative regulator in the PHYB-mediated seed germination inArabidopsis (Gabriele et al., 2010). dag1 knockout mutant require lower R light fluencerates and GA to germinate when compared to wildtype (Gualberti et al., 2002; Papiet al., 2002). DAG1 expression is reduced in seeds after red light irritation, and thisreduction is PIF1-dependent (Gabriele et al., 2010). DAG1 acts downstream of PIF torepresse seed germination by directly repressing GA biosynthetic gene GA3ox1(Gabriele et al., 2010). Further analysis indicated that GAI cooperates with DAG1 inrepressing GA3ox1. Additionally, GAI directly interacts with DAG1 and mutuallyregulates each other (Boccaccini et al., 2014).

In addition to SOM and DAG1, another Dof protein DAG2 has been shown to actdownstream of PIF1. Santopolo et al. (2015) demonstrated that DAG2 acts as a positiveregulator of the light-mediated seed germination (Santopolo et al., 2015). DAG2transcription is positively regulated by light, while negatively regulated by PIF1 inthe dark. In addition, DAG1 represses DAG2 expression by directly binding DAG2promoter (Papi et al., 2002). Though DAG2 shares a high degree of amino acidicidentity with DAG1, they act in opposite ways to regulate PHYB-mediated seedgermination. dag2 mutant seeds require more light and GA for germination, whereasdag1 germination is less dependent on light and GA (Gualberti et al., 2002; Papi et al.,2002). Altogether, these results suggest that DAG2 acts downstream of PIF1 andDAG1 in light-mediated seed germination (Fig. 2).

Mechanism Underlying PIF1 Action

Despite the identification of several PIF1 target genes, the signalling network underly-ing the PHYB-PIF1-mediated seed germination is still not fully understood. Recently, a


Groucho family transcriptional corepressor, LEUNIG_HOMOLOG (LUH), has beenproved to act as a corepressor of PIF1 in imbibed seeds (Lee et al., 2015). Tran-scriptome analysis indicated that luh mutant seeds share similar transcriptional profilewith pif1. Additionally, more than 80% of pif1 seeds and luh seeds are able togerminate in the dark. In vitro Binding Assay demonstrated that LUH interacts withPIF1 at the protein level and co-regulate a subset of its targets by binding to thepromoters of PIF1 targets (Lee et al., 2015). Therefore, LUH acts as a transcriptionalcorepressor of PIF1 to repress seed germination in the dark.

Previous study revealed that PIF1 inhibits light-dependent germination by bindingG-box elements presenting in promoters of its target genes. Kim et al. (2016) reportedthat PIF1 not only binds G-box, but also binds other hexameric sequence elements,such as G-box coupling elements (GCEs) (Kim et al., 2016). ABI5 can interact withPIF1 and facilitate its binding to target genes that possess multiple G-boxes or the GCE.Additionally, the targeting of PIF1 to specific binding sites is determined by itsinteraction with PIF1-interacting transcription factors (PTFs) and their binding to GCEs(Kim et al., 2016).

Gu et al. (2017) found that histone deacetylase15 (HDA15) is involved in PHYB-PIF1-mediated seed germination in Arabidopsis (Gu et al., 2017). Overexpression ofHDA15 leads to strong germination inhibition, whereas loss-of-function of HDA15show increased seed germination, suggesting that HDA15 is a negative regulator ofPHYB-dependent seed germination. HDA15 and PIF1 co-regulate the expression oflight-responsive genes in germinating seeds. Further analysis confirmed that PIF1interacts with HDA15 and recruits HDA15 to the promoters of target genes, decreasethe histone H3 acetylation levels, thereby represses their expression. Therefore, PIF1can also function as a transcriptional repressor via HDA15 in phytochrome-mediatedseed germination (Gu et al., 2017).

Shi et al. (2013) identified LONG HYPOCOTYL IN FAR-RED1 (HFR1) as apositive regulator of PHYB-dependent seed germination (Shi et al., 2013). HFR1functions upstream of PIF1 and forms a heterodimer with PIF1 to impede PIF1transcriptional activity, thereby prevents PIF1 from binding to its target genes. HFR1and PIF1 oppositely mediate transcriptome in response to light in imbibed seeds. In thedark, PIF1 accumulates to a high level and overrides the inhibitory effect of HFR1,leading to dominant repression of seed germination. Upon low light exposure, PHYB isactivated and induces degradation of PIF1, HTR1 start to sequesters PIF1, leading tothe reverse of seed germination inhibition by PIF1. Therefore, it is proposed that HFR1-PIF1 regulatory module enacts a fail-safe mechanism to fine-tune seed germination inresponse to low illumination (Shi et al., 2013).

A recent study suggested that MOTHER-OF-FT-AND-TFL1 (MFT), a member ofthe phosphatidylethanolamine-binding protein (PEBP) family, is also involved in PIF1-dependent repression of seed germination (Vaistij et al., 2018). MFT has been previ-ously reported to regulate seed germination via ABA and GA signaling pathways inArabidopsis (Xi et al., 2010) and act downstream of SPATULA (SPT) (Vaistij et al.,2013). In wheat, MFT is involved in low temperature-induced seed dormancy duringseed development (Nakamura et al., 2011). Vaistij et al. (2018) showed that MFTrepresses seed germination through the regulation of ABA and GA signaling pathwaysunder FR light. MFT expression is regulated in a light quality-dependent manner. Itsexpression is induced by FR light via the PIF1/SOM/ABI5/DELLA pathway, while

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repressed by R light through SPT pathway. In addition, SPT expression is alsorepressed by FR light in a PIF1-dependent manner. Therefore, MFT is an importantcomponent of PIF1-mediated repression of seed germination (Vaistij et al., 2018).

Light-induced degradation of PIF1 is very important for plants to determine appro-priate timing for seed germination during transition from dark to light. Zhu et al. (2015)showed that CUL4COP1–SPA E3 ubiquitin ligase is essential for light-induced degrada-tion of PIF1 in Arabidopsis (Zhu et al., 2015). SPA1 acts as a serine/threonine kinase todirectly phosphorylate PIF1. In the light, phyB interacts with SPA1 through its C-terminus, thereby enhances the recruitment of PIF1 for phosphorylation (Paik et al.,2019). COP1, SPA1 and CUL4 preferentially interact with the phosphorylated forms ofPIF1 and form a complex, PIF1 is thereby subjected to ubiquitylation and subsequentdegradation via the 26S proteasome pathway (Zhu et al., 2015). In the cop1-4 and spaQmutants, the light-induced ubiquitylation and degradation of PIF1 is decreased, sug-gesting that COP1 and SPA promote seed germination by mediating degradation ofPIF1 in response to R and FR light (Zhu et al., 2015). Majee et al. (2018) indentified aF-BOX protein, COLD TEMPERATURE-GERMINATING (CTG)-10, as a positiveregulator of light-dependent seed germination. CTG10 overexpression lines showhigher germination efficiency while ctg10 mutant seeds less germinate. In the light,CTG10 can recognize and interact with PIF1, thereby negatively affect PIF1 stabilityand accelerate the degradation of PIF1. Genetic evidence suggests that PIF1 is epistaticto CTG10. Whereas PIF1 can down-regulate CTG10 expression, indicating a feedbackloop of CTG10/PIF1 regulation. These evidences provide another mechanism by whichlight induces the removal of PIF1 to promote seed germination through CTG10 action(Majee et al., 2018).

A New Role of Phytochrome as Thermosensor

Although light-mediated and temperature-mediated seed germination has been exten-sively studied, how seeds integrate light and temperature cues to determine theappropriate timing of germination has still remained unclear. In 2016, two publicationscomplementarily demonstrated that light sensor PHYB also acts as a thermosensor (ortemperature sensor) in the elongation growth of Arabidopsis organs (Jung et al., 2016;Legris et al., 2016). Legris et al. (2016) showed that warm temperature reduced theabundance of bioogically active Pfr-Pfr dimer pool of PHYB. The reversion of activePfr to inactive Pr, termed dark or thermal reversion, was particularly sensitive to hightemperature under low light conditions. Further more, the size of PHYB containingnuclear bodies which accumulate mainly Pfr-Pfr homodimers was significantly reducedby increasing temperatures higher than 20°C (Legris et al., 2016). Jung et al. (2016)found that high temperature accelerated thermal reversion of PHYB during night time,and the rate of PHYB inactivation was proportional to temperature. Bioactive PHYBdirectly associated with promoters of temperature-responsive genes which are alsotargeted by PIFs. During warm nights, faster thermal reversion of Pfr back to inactivePr at higher temperature resulted in the loss of PHYB occupancy at these target genepromoters, therefore, the repression of PIF-dependent elongation growth is relieved,and elongation growth was promoted to protect plants from adverse temperatures (Junget al., 2016). Taken together, both studies support that PHYB is physiologicallyresponsive to light and temperature in the process of elongation growth in Arabidopsis.


Hence, whether PHYB acts as a thermosensor to integrate both light and temperaturesignals in the processes of seed dormancy and germination remain to be an openquestion. hy2-1 mutant, which is deficient in the phytochrome chromophore, requirescold stratification for germination, while the background genotype Ler lacks dormancyin most treatments and does not respond to post-stratification temperature, suggestingthat phytochrome-mediated pathways are involved in germination responses to tem-perature (Donohue et al., 2007). Arana et al. (2017) found that incubation of dormantArabidopsis seeds at 15°C/23°C alternating temperature cycles enhance germination inresponse to light. The promotion of germination by alternating temperatures requiredPHYB, as phyB mutant seeds did not respond to such alternating temperatures. Duringalternating temperature cycles, the expression of genes involved in GA/ABA pathwayand cell expansion were adjusted to facilitate seed germination. This study furtherdemonstrated that promotion of germination by daily alternating temperatures requiresthe expression of specific clock components, such as TOC1 and PRR7, revealing amechanism that synergic action of PHYB and circadian clock integrate light andtemperature inputs to fine adjust dormancy relief (Arana et al., 2017). Interestingly,phyB mutants are still able to respond to thermal-dependent growth, suggesting thatother phytochromes might replace its function under different temperature conditions(Arana et al., 2017). Thus, it is possible that specific phytochromes contribute to seeddormancy and germination differently, which rely on specific thermosensory pathwaysat different temperatures (Donohue et al., 2008). Indeed, Heschel et al. (2007) foundthat different phytochrome mutants respond differently to a arrange of temperaturesduring seed germinaition. PHYB is important for germination across a range oftemperatures (7°C to 28°C), PHYA plays an important role at warmer temperatures,while PHYE is more important at colder temperatures (Heschel et al., 2007). Inaddition, Martel et al. (2018) found that PHYD is required for secondary dormancyacquisition and germination in seeds exposed to high temperature (Martel et al., 2018).Hence, phytochromes may act as thermosensor to integrate seasonal cues includinglight and temperature signal inputs to regulate seasonal timing of germination. Futurestudies are required to examine the role of PHYB in mediating temperature-dependentseed germination and identify other phytochromes which are involved in temperaturesensory in the processes of seed dormancy and germination.

The Effect of Nitrate and Nitric Oxide on Seed Dormancyand Germination

Nitrate Promotes the Release of Seed Dormancy

Seeds need to integrate the environmental signals in the soil that represent theirnutritional status in order to detect vegetation gaps and germinate under the appropriatenutrients conditions, which ensure the successful seedling establishment after germi-nation (Bewley et al., 2012; Osuna et al., 2015). Nitrogen is not only a key structuralcomponent of macromolecules, but also an essential macronutrient in plants (Canaleset al., 2014). Nitrate is a major nitrogen source for plants, but it also acts as a signalmolecule to promote seed dormancy release and stimulate seed germination in manyplant species (Bewley & Black, 1994; Alboresi et al., 2005). When applied

A. Yan, Z. Chen

exogenously in the germination medium, nitrate stimulates the release of dormancy andpromote germination in Arabidopsis. A maternal effect of nitrate on seed dormancywas also evidenced that feeding of mother plants with high nitrate lead to higher nitrateaccumulation and lower depth of dormancy of the seed progeny, indicating a negativecorrelation between nitrate content and the depth of seed dormancy (Alboresi et al.,2005; He et al., 2014). The effect of nitrate on seed dormancy is independent of nitratereduction and assimilation, as nitrate reductase (NR) deficient seeds which are impairedin nitrate assimilation still show reduced dormancy, while glutamine, another nitrogensource, dose not relieve dormancy, suggesting that nitrate acts as a signal molecule tocontrol seed dormancy (Alboresi et al., 2005).

Nitrate controls seed dormancy probably by affecting ABA metabolism. Exogenousnitrate application during Arabidopsis seed imbibition lead to lower levels of ABA inimbibed seeds (Ali-Rachedi et al., 2004; Matakiadis et al., 2009). When nitrate issupplied to the mother plants during seed development, it can also reduce ABA contentin dry mature seeds (Matakiadis et al., 2009). Transcriptome analyses in nitrate-treatedseeds revealed that CYP707A2 is responsive to nitrate and plays a key role in thecontrol of ABA content during nitrate-mediated seed dormancy and germination(Matakiadis et al., 2009). The CYP707A2 mRNA levels are positively correlated withnitrate dosage supplied to the mother plants, whereas the cyp707a2-1 mutant is lesssensitive to exogenous nitrate and fails to lower seed ABA content in response to nitrate(Matakiadis et al., 2009). Yan et al. (2016) reported that Arabidopsis NIN-like protein 8(NLP8) is essential for nitrate-mediated seed dormancy release. NLP8 is activated bynitrate signaling and regulates nitrate-promoted germination by reducing ABA levels ina nitrate-dependent manner. Further analysis demonstrated that NLP8 directly binds tothe promoter of CYP707A2 and thereby activates its expression (Yan et al., 2016).Hence, the stimulating effect of nitrate on dormancy release is mainly mediated throughthe actions on ABA metabolism via CYP707A2 expression.

Nitrate is assimilated via its reduction by NR and other enzymes, resulting in theproduction of nitrite, nitric oxide (NO), amino acids and other nitrogenous compounds(Crawford, 1995; Arc et al., 2012). These nitrogen-containing compounds, especiallyNO, have also been shown to promote seed germination (Bethke et al., 2006a, b).Interestingly, although both of nitrate and nitrite can reduce Arabidopsis seed dorman-cy, their effects on seed dormancy removal are prevented by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (Bethke et al.,2006a). Therefore, it is argued that nitrate may not regulate seed dormancy on its ownbut rather through NO signaling.

The Effect of Nitric Oxide on Seed Dormancy and Germination

Nitric oxide (NO) is a small gaseous molecule emitted by plant cells that diffusesreadily through biomembrane. It functions as an important developmental signal thatregulates a wide range of physiological processes from development to environmentaladaptation in plants. The effect of NO on seed dormancy alleviation has been demon-strated in a variety of plant species, including Arabidopsis (Bethke et al., 2004, 2006a,b), barley (Bethke et al., 2004), lettuce (Beligni & Lamattina, 2000), Pauloniatomentosa (Giba et al., 1998), apple (Gniazdowska et al., 2007, 2010), and evenwarm-season (C4) grasses (Sarath et al., 2006). Incubation of dormant seeds with


NO donor compounds such as sodium nitroprusside (SNP) or S-nitroso, N-acetylpenicillamine (SNAP) can release dormancy and promote seed germination in aconcentration-dependent manner (Giba et al., 1998; Beligni & Lamattina, 2000;Bethke et al., 2004). Conversely, application of NO scavengers such as cPTIO duringseed imbibition strengthens seed dormancy and block the effects of NO donors on seedgermination (Beligni & Lamattina, 2000; Bethke et al., 2004, 2006a, b). Theseobservations suggests that NO is an endogenous regulator of seed dormancy.

NO Regulates Seed Germination Through Crosstalk with ABA Signaling Pathway

NO Promotes ABA Catabolism

Similar to nitrate, it has been shown that the effect of NO on the removal of seeddormancy is associated with the stimulation of ABA catabolism. In Arabidopsis, NO isproduced at the endosperm layer in the early hours during seed imbibition, subsequent-ly leading to a rapid increase of ABA catabolism, which is essential for dormancybreaking (Liu et al., 2009). The NO-induced rapid decrease of ABA contents ismediated by an increase of CYP707A2 transcription and CYP707A2 protein accumu-lation. Seed treated with SNP shows enhanced CYP707A2 transcription and proteinaccumulation, whereas c-PTIO treatment represses CYP707A2 expression and reversesthe NO donor effect on seed dormancy (Liu et al., 2009). Hence, NO promotes seeddormancy alleviation through enhancing CYP707A2 expression and subsequentlyreducing ABA contents in seeds.

In addition, Bethke et al. (2006a) showed that SNP enhanced the positive effect ofABA biosynthesis inhibitor norfluorazon on germination of Arabidopsis C24 seeds.Moreover, SNP vapour reduces the sensitivity of Arabidopsis seeds to exogenous ABA(Bethke et al., 2006a). Conversely, NO scavenger cPTIO prevent stimulating effect ofanother ABA biosynthesis inhibitor fluridone on seed germination in tomato seeds(Piterková et al., 2012). Therefore, NO mediates seed dormancy by affecting both ABAaccumulation and sensitivity.

NO impairs ABA Signaling Through Protein Post-Translational Modification

NO signaling may mainly rely on protein post-translational modification (PTM), suchas cysteine S-nitrosylation and tyrosine nitration (Delledonne, 2005; Moreau et al.,2010). A parallel increase in NO production and S-nitrosylation was observed duringseed germination in wheat and barley (Sen, 2010; Ma et al., 2016). During NO actionover seed dormancy, it has been reported that key components of ABA signalingpathway are targeted for NO-dependent PTM, reflecting a crosstalk between ABAsignaling and NO pathway in this process (Albertos et al., 2015; Castillo et al., 2015;Wang et al., 2015a, b).

S-nitrosylation of ABA signaling components SnRK2.6 was firstly reported in guardcells, resulting to the inactivation of its function in the ABA signaling pathway andconsequent impairment of ABA-induced stomatal closure (Wang et al., 2015a). Furtherstudy extend to regulation of seed germination, Wang et al. (2015b) found thatSnRK2.2 and SnRK2.6 were inactivated by S-nitrosoglutathione (GSNO) treatmentthrough S-nitrosylation (Wang et al., 2015b). SnRK2.3 is closely related to SnRK2.2

A. Yan, Z. Chen

and plays redundant roles in ABA inhibition of seed germination. Application of NOdonor SNP during seed germination phenocopies the snrk2.2 snrk2.3 double mutant,which shows insensitivity to exogenous ABA (Wang et al., 2015b). Therefore, NOattenuates ABA signaling through S-nitrosylation of SnRK2s, a process S-nitrosylatingcysteine residue adjacent to the kinase catalytic site of SnRK2s, further blocking theirkinase activities during seed germination (Fig. 3).

ABI5, a key regulator of the ABA transduction pathway, is also regulated by NO-dependent S-nitrosylation during seed germination. In Arabidopsis, application of theNO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) quickly reduces ABI5 expres-sion in the seeds (Albertos et al., 2015). Conversely, NO-deficient atnoa1-2 nia1 nia2triple mutant, which is defective in NIA/NR- and AtNOA1-dependent NO biosynthesispathways, accumulates higher ABI5 protein levels and shows hypersensitivity to ABAwith strengthened seed dormancy (Albertos et al., 2015). Further studies demonstratedthat NO directly affects ABI5 stability by S-nitrosylation. NO-mediated S-nitrosylationof ABI5 at cysteine-153 facilitates its interaction with CULLIN4-based and KEEP ONGOING E3 Ligases, targeting it to the proteasome and subsequently leading to itsdegradation (Albertos et al., 2015). In contrast, mutation at Cys 153 of ABI5 impedesits protein degradation, therefore deregulates the inhibition of seed germination by NOdepletion (Albertos et al., 2015).

Besides directly regulated by NO-dependent S-nitrosylation, ABI5 is also indirectlyregulated by NO. Gibbs et al. (2014) identified group VII ET response transcriptionfactors (ERFVII) as new NO sensors in NO-mediated seed germination. NO promotesthe degradation of the ERFVII via the N-end rule pathway of proteolysis (Gibbs et al.,2014). Mutations at genes involved in N-end rule pathway or overexpression ofERFVII lead to hypersensitivity to ABA during seed germination, which result from

Fig. 3 Simplified model representing the regulatory network of NO-mediated seed germination. NO exerts itscontrol on seed germination mainly through impairing ABA signaling pathway, specifically, by inactivatingcomponents of ABA signaling pathway. NO can also reduce ABA levels either by upregulation ofCYCP707A2 or by downregulation of ABA3. Arrows indicate positive regulation and T-bars indicate negativeregulation


non-degradation and overaccumulation of ERFVII proteins, suggesting that ERFVIItranscription factors play essential roles during ABA-mediated repression of seedgermination (Holman et al., 2009; Gibbs et al., 2014). As these ERFVII are positiveregulators of ABI5, they can induce ABI5 expression and trigger ABA inhibitoryeffects on seed germination. Hence, NO-mediated degradation of group VII ERFsblocks induction of ABI5 transcription, subsequently inhibits ABA responses andpromotes seed germination. Collectively, these findings suggest a molecular mecha-nism for the antagonistic action of ABA and NO in the control of seed germinationthrough affecting ABI5 transcription and protein stability (Fig. 3).

Nitration of tyrosine residues, another NO-mediated PTM, is also involved in thecross talk between ABA and NO signaling. Tyrosine nitration is a covalent posttrans-lational modification which derived from the reactions between proteins andperoxynitrite (ONOO-) or other nitrating agents, leading to inactivation of targetedproteins. Tyrosine nitration modulates signaling functions by interfering with thetyrosine phosphorylation and dephosphorylation signaling pathways (Monteiro et al.,2008). Under conditions in which NO and reactive oxygen species are simultaneouslyproduced, ABA receptors PYR/PYL/RCAR are nitrated at tyrosine residues. Thesereceptors with nitrated tyrosines are subsequently polyubiquitylated and subjected toproteasome-mediated degradation (Castillo et al., 2015). Thus, NO negatively regulatesABA signaling by interfering with ABA receptors and reduction of receptor activitiesthrough tyrosine nitration. In addition, a proteomic analysis of nitrated proteins inArabidopsis identified molybdenum cofactor (MoCo) sulfurase ABA3 to be a target oftyrosine nitration (Lozano-Juste et al., 2011). ABA3 catalyzes the conversion of Mocofrom de-sulfo form to sulfo form , which is essential for the last step of ABA synthesis(Mendel, 2007). Therefore, nitration of ABA3 may inactivate it and further block ABAbiosynthesis, negatively regulate the inhibitory effect of ABA on seed germination(Fig. 3).

Conclusions and Future Perspectives

Seed dormancy is an adaptive trait with far-reaching significance for the evolution ofentire species. It exerts as a safety machinery to prevent germination in an unfavorableseason when environmental conditions are detrimental for subsequent seedling estab-lishment and survival. In agricultural industry, seed dormancy is undesired for cropsthat require fast and synchronous germination after sowing in the soil, because longperiod of dormancy affects uniform germination and delay agricultural production,which is disadvantageous to crop yield. Whereas as for crops for which rapid germi-nation is not required, lack of dormancy may cause pre-harvest sprouting and lead tothe reduction of seed quality. Hence, the management of seed dormancy is of funda-mental importance for the seed agricultural performance and industry. It is imperative toincrease our knowledge of the intrinsic mechanisms underlying seed dormancy.

Seed dormancy and germination are regulated by both internal and environmentalfactors. Among various environmental factors, temperature, light, and nitrogen avail-ability play the mostly dominant role in determination of the depth of dormancy andtiming of germination (Fig. 4). Temperature has profound impacts on seed dormancyestablishment and release. DOG1 has been identified to play a pivotal role downstream

A. Yan, Z. Chen

of temperature pathway in the regulation of seed dormancy establishment, its proteinabundance in seeds is proposed as an indicator of dormancy strength and a timer fordormancy release. Light is perceived mainly by phytochromes. Light exerts its controlon seed germination mainly through PHYs-PIF1 module, which acts in a derepressionmanner to mediate germination potential in response to various light environments.More than nutrient source, nitrate and other nitrogenous compounds such as NO act assignal molecules to mediate seed dormancy and germination. After perceived by seeds,these environmental factors are converted into internal signals, leading to the dynamicchange of endogenous factors (mainly ABA and GA), eventually resulting in thealterations of physiological processes in seeds.

Although some key factors that involved in environmental control of seed dormancyand germination have been identified, there are still many open questions remain to beaddressed. For example, what components act upstream to regulate DOG1 and ABA/GA balance in temperature-mediated pathway? Most importantly, what is thethermosensor that acts in the first step of signal transduction pathway to bridge soiltemperature with the timing of germination? Although PHYB has been demonstrated toact as thermosensor in elongation growth in Arabidopsis, whether it has similarfunction in the process of seed dormancy and germination remain unknown. Inaddition, are other phytochromes involved in the temperature sensing? Do they playdistinct or redundant roles during dormancy cycling in response to a range of temper-atures? In light-mediated pathway, what is the exact mechanism of PIF1 repression byphytochromes? Besides PHYB-triggered degradation of PIF1 via ubiquitin-protea-some, are there any other mechanisms such as competition for binding sites ofdownstream genes exist? At last, how different environmental inputs are integrated atphysiological and molecular level to synergically fine tune dormancy relief andgermination? Hence, identification of shared pathway components, such as receptorsof environmental inputs, downstream network, will greatly improve our understandingof mechanisms underlying environmental control of dormancy and germination.

Fig. 4 Schematic diagram illustrating the environmental control of seed dormancy and germination duringseed maturation and imbibition. Arrows indicate positive regulation and T-bars indicate negative regulation


In the field, the environmental conditions are diverse and more complicated (Fig. 4).Beside temperature, light, and nitrogen availability, other environmental cues such aswater content also play important roles in the control of seed dormancy and germina-tion. Actually, crosstalks between signaling pathways of different environmental factorsexist. Therefore, in addition to single environmental factor study, field study is alsorequired for better understanding of how seed dormancy is regulated in the variablefield environment.

Different plant species have different depth of seed dormancy, which is influencedand released by different environmental conditions. The intrinsic mechanisms under-lying seed dormancy in different species might be distinct from each other, our currentknowledge regarding the regulation of seed dormancy and germination originatesmainly from studies based on model plant Arabidopsis. Therefore, how the knowledgeobtained from studies on Arabidopsis contributes to seed industry and agricultureperformance is of great concern. In the future, comparative genomics studies betweencrops and Arabidopsis will be very informative for the comprehensive understanding ofseed dormancy, which will greatly benefit practical applications in crops.

Author contributions Z.C. conceived and initiated the work. A.Y. and Z.C. wrote and revised themanuscript.

Funding Authors thank the research grant (NTU-MSE-Facile Non-T) to Z.C. and NIE AcRF funding (RI 8/16 CZ) to support A.Y.

Compliance with ethical standards

Conflict of interest The authors declare no conflict of interest.

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