Genetic Variation for Lettuce Seed Thermoinhibition Is ... · lettuce seed germination to inhibition by ABA is en-hanced at higher temperatures (Robertson and Berrie, 1977; Roth-Bejerano

Genetic Variation for Lettuce Seed Thermoinhibition IsAssociated with Temperature-Sensitive Expression ofAbscisic Acid, Gibberellin, and Ethylene Biosynthesis,Metabolism, and Response Genes1[C][W][OA]

Jason Argyris, Peetambar Dahal, Eiji Hayashi, David W. Still, and Kent J. Bradford*

Department of Plant Sciences, University of California, Davis, California 95616–8780 (J.A., P.D., K.J.B.);and Department of Plant Sciences, California State Polytechnic University, Pomona, California 91768(E.H., D.W.S.)

Lettuce (Lactuca sativa ‘Salinas’) seeds fail to germinate when imbibed at temperatures above 25�C to 30�C (termedthermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37�C inthe light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine themechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes wasdifferentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associatedwith these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germinationthermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability offluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved inABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in thetwo genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- andethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encodingan enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocatedwith Htg6.1. The temperature sensitivity of expression of LsNCED4may determine the upper temperature limit for lettuce seedgermination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.

The timing of seed germination is associated withthe presence or absence of seed dormancy, which is thetemporary failure of a viable seed to germinate after aspecified length of time in a particular environmentthat would allow germination if the seed were notdormant (Baskin and Baskin, 2004). Genetic and phys-iological approaches are revealing insights into thedeterminants of seed germination and dormancy(Bentsink et al., 2006; Finch-Savage and Leubner-Metzger, 2006; Finch-Savage et al., 2007). Mutationstudies, phenotypic screening, and surveys of natural

variation, largely in Arabidopsis (Arabidopsis thaliana),have identified dozens of genes that influence theimposition or release of dormancy or the ability ofseeds to transition from maturation to germination(Bentsink and Koornneef, 2002; Finkelstein et al., 2002;Bentsink et al., 2007). Many of these genes implicateplant hormones, including GA, abscisic acid (ABA),ethylene, cytokinins, brassinosteroids, and auxin (Alveyand Harberd, 2005; Kucera et al., 2005; Achard et al.,2006; Riefler et al., 2006; Seo et al., 2006; Feurtado andKermode, 2007), as well as sugars (Dekkers andSmeekens, 2007), light (Oh et al., 2007; Gubler et al.,2008; Sawada et al., 2008), temperature (Tamura et al.,2006; Toh et al., 2008), and other factors in complexregulatory webs that sense internal and external con-ditions and determine whether germination proceedsto completion. In many seeds, the endosperm andtesta play important roles in regulating dormancy(Lefebvre et al., 2006; Muller et al., 2006; Bethkeet al., 2007; Debeaujon et al., 2007; Nonogaki et al.,2007), and checkpoints for embryo growth after ger-mination sensu stricto have been described (Lopez-Molina et al., 2002; Kucera et al., 2005).

Seed dormancy is dependent on environmentalfactors. Dry storage (after-ripening) or moist chilling(stratification) are often required to alleviate dor-mancy, and the range of environmental conditions in

1 This work was supported by the U.S. National Science Founda-tion (grant no. 0421630) through the Compositae Genome Project(http://compgenomics.ucdavis.edu/) and by the California StateUniversity Agricultural Research Initiative (grant no. 07–4–160) to D.W.S. and K.J.B.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Kent J. Bradford ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.108.125807

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which germination will occur generally expands asdormancy is reduced (Allen et al., 2007). During theperiods when they aremost likely to germinate (i.e. areleast dormant), some seeds have additional require-ments for germination-stimulating signals, such aslight, GA, or nitrate, and increasing sensitivity to thesesignals is associated with dormancy loss (Fennimoreand Foley, 1998; Batlla and Benech-Arnold, 2005;Kucera et al., 2005; Hilhorst, 2007). Conversely, de-creased sensitivity to ABA, a strong germination inhib-itor, is associated with reduced dormancy (Koornneefet al., 1984; Walker-Simmons, 1987; Steinbach et al.,1997).Lettuce (Lactuca sativa) seeds exhibit thermoinhibi-

tion (fail to germinate) at temperatures well below thebiological upper limit for seedling growth (Cantliffeet al., 1981). Several factors influence the upper tem-perature limit for lettuce seed germination. Seedsmatured under warm temperatures have higher ger-mination temperature limits than do seeds maturedunder cooler temperatures (Kozarewa et al., 2006).Higher upper temperature limits are heritable, beingincreased by selection in high-temperature environ-ments (Guzman et al., 1992). The intact endospermenvelope enclosing the lettuce embryo is required forthe imposition of thermoinhibition (Borthwick andRobbins, 1928). Exposure to ethylene, GA, and redlight can increase the upper temperature limit oflettuce seed germination, and exposure to ABA candecrease it (Reynolds and Thompson, 1973; Saini et al.,1986, 1989; Dutta and Bradford, 1994; Kristie andFielding, 1994; Gonai et al., 2004). The sensitivity oflettuce seed germination to inhibition by ABA is en-hanced at higher temperatures (Robertson and Berrie,1977; Roth-Bejerano et al., 1999).Similar responses of germination to temperature

and light have also been documented in Lactucaserriola, the progenitor species of cultivated lettuce(Marks and Prince, 1982; Small and Gutterman, 1992).However, certain accessions of L. serriola have thermo-inhibitory temperature limits up to 10�C higher com-pared with both cultivated lettuce and other L. serriolaaccessions (Argyris et al., 2005; Argyris, 2008). Quan-titative trait loci (QTLs) associated with temperaturesensitivity of germination were identified in a core-mapping recombinant inbred line (RIL) populationfrom a cross between a thermosensitive cultivatedlettuce cultivar, ‘Salinas’, and thermotolerant L. serriolaaccession UC96US23 (Argyris et al., 2005). The paren-tal lines of the RIL population showed divergent ger-mination behavior at high temperature, with Salinasseeds exhibiting thermoinhibition above approxi-mately 30�C, while UC96US23 seeds germinated attemperatures up to 37�C in the light (Argyris et al.,2008). QTLs associated with seed dormancy have beenidentified in populations of Arabidopsis and otherspecies (Foley, 2001; Alonso-Blanco et al., 2003; Guet al., 2006; Bentsink et al., 2007), and the first genespecifically responsible for a seed dormancy QTL wasrecently identified (Bentsink et al., 2006).

Considerable physiological, gene expression, andmutant data support a role for the balance of GA andABA synthesis and catabolism in modulating thermo-inhibition (Gonai et al., 2004; Toh et al., 2008). Com-prehensive transcriptomic analyses of primary andsecondary dormancy of Arabidopsis and Nicotianaplumbaginifolia seeds indicate that genes associatedwith the synthesis and deactivation of GA and ABAare reciprocally regulated in association with the im-position and release of dormancy, with ABA accumu-lation favored during dormancy imposition and GAaccumulation favored during dormancy release (Boveet al., 2005; Cadman et al., 2006; Finch-Savage et al.,2007). Imbibition of Arabidopsis seeds at high tem-perature promotes the expression of genes involvedin ABA synthesis (e.g. AtNCED genes encoding 9-cis-epoxycarotenoid dioxygenases) and inhibits the ex-pression of genes involved in GA synthesis (e.g.AtGA3ox genes encoding GA 3b-hydroxylases; Tohet al., 2008). Seeds of ABA-deficient and ABA-insen-sitive mutants (aba1 and abi3) in Arabidopsis germi-nate at supraoptimal temperatures, suggesting a majorrole for ABA in the thermoinhibition mechanism(Tamura et al., 2006). Mutant analyses and gene ex-pression studies demonstrated that AtNCED6 andAtNCED9, which catalyze the first biochemical stepunique to ABA biosynthesis, are specifically requiredfor the induction of dormancy in Arabidopsis seeds(Tan et al., 2003; Lefebvre et al., 2006), with AtNCED9playing the major role in thermoinhibition (Toh et al.,2008). ABA catabolism is regulated primarily via ABA-8#-hydroxylases (e.g. CYP707A1 and CYP707A2),whose expression is limited to seeds and mutation orsuppression of which results in increased seed dor-mancy and higher ABA contents compared with thewild type (Kushiro et al., 2004; Okamoto et al., 2006;Gubler et al., 2008). ABA can suppress GA biosynthe-sis in Arabidopsis seeds, while red light reduces ABAcontent by down-regulating the expression ofAtNCED6and enhancing the expression of AtCYP707A2 (Seoet al., 2006; Oh et al., 2007). In lettuce seeds, ABAcontent decreases rapidly when seeds are imbibed atoptimal temperatures for germination, but it is main-tained at elevated levels when imbibition occurs athigh temperatures (Yoshioka et al., 1998; Toh et al.,2008). GA, in turn, promotes ABA catabolism andincreases the upper temperature limit for lettuce seedgermination (Gonai et al., 2004). Germination at hightemperature in lettuce can also be promoted by theapplication of fluridone, an inhibitor of ABA synthesis,suggesting that continued ABA synthesis is involvedin the maintenance of thermoinhibition (Yoshiokaet al., 1998; Gonai et al., 2004).

Ethylene is also involved in regulating thermoinhi-bition in lettuce seeds (Abeles, 1986; Saini et al., 1986,1989). Ethylene formation in plants occurs via theconversion of S-adenosylmethionine to 1-aminocyclo-propane-1-carboxylic acid (ACC) via ACC synthase(ACS) and of ACC to ethylene via ACC oxidase(ACO). The conversion of S-adenosylmethionine to

Genetic and Hormonal Regulation of Seed Thermoinhibition

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ACC by ACS is the rate-limiting step of ethyleneproduction, and two ACS genes (LsACS1 and LsACS2)have been reported in lettuce (Takahashi et al., 2003).Exogenously applied ethylene at least partially re-lieved thermoinhibition in lettuce, and seed ethyleneproduction was correlated positively with the abilityto germinate at high temperature (Saini et al., 1986,1989; Prusinski and Khan, 1990; Dutta and Bradford,1994; Kozarewa et al., 2006). Mutations in ethylenesignaling components altered the sensitivities of ger-minating seeds to exogenous ABA concentration andenhanced seed dormancy, providing strong evidencefor cross talk between ABA and ethylene signalingpathways (Beaudoin et al., 2000; Ghassemian et al.,2000; Matilla and Matilla-Vazquez, 2008).

The convergence of different signals regulating seeddormancy and germination occurs via the GRAS/DELLA proteins. These proteins, which in Arabidop-sis include GAI, REPRESSOR OF GA1-3 (RGA), RGA-LIKE1 (RGL1), RGL2, and RGL3, act as repressors ofGA-induced signaling and germination (Silverstoneet al., 2001; Tyler et al., 2004; Griffiths et al., 2006;Willige et al., 2007). Of these, RGL2 has the mostsignificant role in seed germination, and along withGAI and RGA, it may serve to integrate the influencesof light, GA, ABA, and ethylene in mediating theeffects of environmental factors on dormancy andgermination (Tyler et al., 2004; Achard et al., 2006,2007; Oh et al., 2006, 2007; Penfield et al., 2006;Ueguchi-Tanaka et al., 2007).

A combination of genetic, molecular, and physio-logical approaches, particularly in Arabidopsis, hasgreatly advanced our understanding of the mecha-nisms underlying the regulation of seed dormancyand germination (Holdsworth et al., 2008). Here, weutilized natural genetic variation, QTL analyses, genemapping, chemical genetics, hormonal profiling, andgene expression assays to analyze the mechanism(s)underlying the temperature sensitivity of germinationin lettuce genotypes. Our results are broadly consis-tent with prior work, indicating conservation of reg-ulatory mechanisms in seed germination, and providespecific physiological, genetic, andmolecular evidencethat the regulation of ABA synthesis may be a criticalcontrol point for lettuce seed thermoinhibition.

RESULTS

Germination of Cultivated Lettuce (Salinas) and

L. serriola UC96US23 Seeds in Response toLight and Temperature

Seeds of both Salinas and UC96US23 germinatedrapidly and uniformly at 20�C in the light, withgermination beginning at about 16 h and being com-plete within 30 h after sowing (Fig. 1A). In contrast,only UC96US23 seeds germinated at 35�C in the light,although germination was delayed and less uniform atthis temperature compared with 20�C, beginning after

30 h and being completed by 54 h (Fig. 1A). In the dark,Salinas seeds germinated rapidly and completely at20�C, with a time course similar to that in the light,compared with only 35% final germination forUC96US23 seeds (Fig. 1B). Seeds of both genotypeswere unable to germinate at 35�C in the dark (Fig. 1B).Salinas seeds are less dependent upon light than areUC96US23 seeds for germination at 20�C, and light isrequired for germination of UC96US23 seeds at 35�C.

Transfer experiments were conducted to determinethe times during which the imposition of or escapefrom thermoinhibition occurred. Seeds were imbibedat high (35�C) or low (20�C) temperature for varioustimes in the light or dark and then transferred to thealternative temperature in the same light or darkcondition (Supplemental Fig. S1). These experimentsestablished that the initial 6 to 12 h of imbibition is thecritical period for the induction of thermoinhibition inSalinas seeds, confirmed that the thermotolerance ofUC96US23 seeds requires light, and showed that ex-posures to 35�C for up to 24 h of imbibition did notinduce secondary dormancy.

Effects of Temperature and Light on Sensitivity ofGermination to ABA and GA

Seeds of Salinas and UC96US23 exhibited differen-tial sensitivity to exogenously applied ABA and GA

Figure 1. Germination time courses of Salinas (Sal; circles) andUC96US23 (UC; triangles) seeds at 20�C (open symbols) and 35�C(closed symbols) in the light (A) and in the dark (B). Error bars are notshown for clarity, but a pooled error for treatment comparisons isindicated in each panel. [See online article for color version of thisfigure.]

Argyris et al.

928 Plant Physiol. Vol. 148, 2008



with respect to thermoinhibition. Seeds of both geno-types germinated more than 95% at 29�C in the light inthe absence of ABA (Fig. 2A). UC96US23 seeds werealmost 3-fold less sensitive to ABA in comparison withSalinas seeds, with ABA concentrations reducing ger-mination to 50% (ABA50 values) of 7.7 and 2.8 mM,respectively (Fig. 2A). As GA synthesis in lettuce seedsis up-regulated by light via phytochrome (Toyomasuet al., 1998), germination tests for GA response wereconducted in the dark to reduce de novo GA synthesisand at a temperature just above the upper limit forSalinas seeds (32�C). Seeds of both lines were com-pletely thermoinhibited at this temperature in thedark, failing to germinate in water (Fig. 2B). Provisionof GA resulted in an approximately log-linear increasein germination of UC96US23 seeds, with restoration of100% germination in 100 mM GA; in contrast, Salinasseeds germinated only 31% in 100 mM GA (Fig. 2B).To assess the genetic basis of the differential sensi-

tivity of Salinas and UC96US23 seeds to ABA and GA,the same RIL population utilized for QTL mapping ofhigh-temperature germination capacity was screenedas described above. ABA50 values of RILs ranged from0 to 82 mM at 29�C in the light, and germination in100 mM GA ranged from 0% to 98% at 32�C in the

dark (data not shown; Argyris, 2008). Composite inter-val mapping analysis of ABA sensitivity (measuredas ABA50) and germination in response to 100 mM GA(probit-transformed percentage) revealed one highlysignificant QTL for each of these traits, termedABA506.1 and GA6.1, which accounted for 16% and22% of the phenotypic variation for each trait, respec-tively (Fig. 3). These QTLsmapped to the same 1-log ofthe odds (1-LOD) confidence interval as Htg6.1, amajor QTL associated with high-temperature germi-nation capacity (Argyris et al., 2005, 2008). ANOVA ofRIL genotypes at a marker locus closely associatedwith ABA506.1 showed that the UC96US23 allele con-ferred significantly reduced ABA sensitivity (P ,0.05). The mean ABA50 value for RILs homozygousfor UC96US23 alleles was 10.1 mM compared with 5.5mM for Salinas homozygotes at the same locus. Simi-larly, RILs homozygous for the UC96US23 allele atGA6.1 showed significantly higher mean germinationpercentages in 100 mM GA (65%) compared with thosehomozygous for the Salinas allele (12%). Thus, sensi-tivity of germination to ABA and GA at elevatedtemperatures is associated with the same genetic re-gion that confers high-temperature germination ca-pacity in UC96US23 seeds.

Effects of Fluridone on the Sensitivity of Germination toTemperature and GA

While neither Salinas nor UC96US23 seeds wereable to germinate when incubated in water at 32�C inthe dark, the addition of 30 mM fluridone to inhibitcarotenoid and ABA synthesis significantly increasedgermination to approximately 30% in both lines (Fig.4). Application of 100 mM fluridone alone was suffi-cient to restore over 90% germination of Salinas seedsbut not of UC96US23 seeds under these conditions(Fig. 4). However, adding even 1 mM GA4+7 to 100 mM

fluridone allowed 100% germination of UC96US23seeds (data not shown). Combining 10 or 100 mM GAwith 30 mM fluridone also allowed complete germina-tion of UC96US23 seeds, while germination of Salinasseeds did not exceed 60% (Fig. 4). These results sug-gest that the light requirement for GA biosynthesislimits germination of UC96US23 seeds in the dark,which can be replaced by exogenous GA. In Salinasseeds, on the other hand, the response of germinationto fluridone in the dark and the inability of GA tostimulate complete germination indicate that ABAmay be the primary factor preventing germination athigher temperatures.

Hormonal Contents of Seeds Imbibed under DifferentLight and Temperature Conditions

If ABA prevents germination of Salinas seeds at35�C in the light, the ABA content of Salinas seedswouldbe expected to be greater than that of UC96US23 seedswhen imbibed at high temperatures. The initial ABAcontent of dry Salinas seeds was significantly higher

Figure 2. Germination of Salinas (Sal; circles) and UC96US23 (UC;triangles) seeds in response to increasing ABA concentrations at 29�C inthe light (A) and increasing GA4+7 concentrations at 32�C in the dark(B). Hormone concentrations are plotted on a logarithmic scale. Errorbars indicate SE (n = 3). [See online article for color version of thisfigure.]





(284 ng g21) compared with that of UC96US23 seeds(108 ng g21) and remained so until 18 h after sowing at20�C (Fig. 5A). The ABA content in seeds of bothgenotypes declined rapidly from 2 to 18 h after sow-ing, decreasing from 296 to 10.5 ng g21 and from 95 to14.9 ng g21 in Salinas and UC96US23, respectively. Incomparison with 20�C, ABA amounts in Salinas seedsdeclined more slowly at 35�C and were significantlyhigher than in UC96US23 seeds between 2 and 48 h ofimbibition (Fig. 5A). Increased imbibition temperaturehad little effect on ABA contents of UC96US23 seeds(Fig. 5A). Between 24 and 48 h of imbibition at 35�C,ABA content of Salinas seeds was almost 5-fold greaterthan that of UC96US23 seeds (Fig. 5A), and the latterhad completed germination while the former werethermoinhibited (Fig. 1A).

Phaseic acid (PA) is the major metabolite of ABAdeactivation, formed by the action of ABA-8#-hydrox-ylases that are encoded in Arabidopsis and lettuce byCYP707A (ABA8ox) genes (Kushiro et al., 2004; Saitoet al., 2004; Yamaguchi et al., 2007; Sawada et al., 2008).Seed PA contents would be expected to increase asABA contents decreased following imbibition. PA con-tents were highest in Salinas seeds imbibed at 20�C,in which the decline in ABA content was quantita-tively greatest (Fig. 5B). PAwas also initially higher inSalinas seeds imbibed at 35�C but then declined tolower levels while ABA content remained elevated. PAcontents were relatively low at all times in UC96US23

seeds imbibed at either 20�C or 35�C (Fig. 5B). Quan-titatively, not all of the initial ABA was accounted foras PA, indicating that PA is further metabolized.

ABA and PA contents were also measured in seedsimbibed at 20�C and 35�C in the dark for 12 or 24 h.ABA content decreased in Salinas seeds imbibed at20�C during this time but remained relatively constantin UC96US23 seeds (Fig. 6A). ABA content was greaterin Salinas seeds imbibed at 35�C compared with 20�C,but it was similar at both temperatures in UC96US23seeds. Thus, the decline in ABA content in UC96US23seeds following imbibition is dependent upon light atboth low and high temperatures. PA contents werehigher in Salinas seeds than in UC96US23 seeds at bothtemperatures and were higher at 20�C than at 35�Cin both genotypes (Fig. 6B). These results are consis-tent with the germination responses in the dark atthese temperatures, with only Salinas seeds germinat-ing well at 20�C in the dark and neither genotypegerminating at 35�C in the dark (Fig. 1B).

Seed GA1, GA3, and GA4 contents were generallybelow the limits of confident detection with themethods employed (data not shown). GA7 was de-tected at low levels (,5 ng g21) in most samples anddid not differ significantly between the genotypes orimbibition temperatures (data not shown). Indole-3-acetic acid and indole-3-Asp contents increased withtime after imbibition at both 20�C and 35�C, but therewere no significant differences between the two geno-

Figure 3. Colocation of QTL for ABA sensitivity(ABA506.1; dotted line) and GA response (GA6.1;dashed line) with Htg6.1 (solid line), a QTL for high-temperature germination on linkage group 6.LsNCED4, an ABA biosynthetic gene, also maps tothe center of this QTL interval. Marker positions aregiven in centimorgan units. Horizontal bars representthe 1-LOD QTL confidence intervals. The dashedhorizontal line represents the average permutatedthreshold for all three traits for declaring a signi-ficant QTL.

Argyris et al.




types (Supplemental Fig. S2, A and B). cis-Zeatin ribosidecontent declined in both genotypes at both 20�C and35�C for the first 24 h of imbibition, then increasedonly in Salinas seeds imbibed at 35�C (SupplementalFig. S2C). A similar pattern was exhibited by dihy-drozeatin riboside content, although the increase inSalinas seeds at 35�C was quantitatively less (Supple-mental Fig. S2D). cis-Zeatin-O-glucoside content in-creased following imbibition in both genotypes and atboth temperatures and eventually reached higherlevels in UC96US23 seeds than in Salinas seeds im-bibed at 35�C (Supplemental Fig. S2E). These patternswere repeated in seeds imbibed in the dark for 12 and24 h (Supplemental Fig. S2, F–J); it is unknownwhether the delayed increase in cis-zeatin ribosidecontent would occur in the dark at longer imbibitiontimes. Levels of other hormones and their metabolitesthat were assayed were low and were not consistentlydetected, including ABA-Glc ester, neo-PA, 7#-hydroxy-ABA, indole-3-Glu, GA8, GA19, GA29, dihydrozeatin,isopentenyladenine, isopentenyladenosine, cis-zeatin,trans-zeatin, trans-zeatin riboside, and trans-zeatin-O-glucoside.

Expression of Genes Involved in ABA Synthesisand Metabolism

Differences in seed upper temperature limits, ABAand GA sensitivities, and ABA content between thelettuce genotypes may be due to differential expres-sion of genes in the hormone synthesis, metabolism,perception, and signaling pathways. Therefore, weassayed expression (relative mRNA levels) of genesrelated to ABA, GA, and ethylene biosynthesis andresponse in seeds of both genotypes across timecourses of germination at 20�C and 35�C in light anddarkness by quantitative reverse transcription-PCR(qRT-PCR) and bymultiplexedGeXP (Beckman-Coulter)analysis (genes and primers listed in SupplementalTable S1). Lettuce genes without reported full-length

clones will be referred to by the names of their closestArabidopsis homologs without the Ls prefix; nameswith the Ls prefix refer to genes that have been clonedand sequenced from lettuce.

Several genes in the ABA biosynthetic pathwaywere assayed, including LsZEP1 (for zeaxanthin epox-idase) and LsNCED (for 9-cis-epoxycarotenoid dioxy-genase) alleles. The former is highly homologous tothe Arabidopsis AtABA1 gene catalyzing the conver-sion of zeaxanthin to violaxanthin (Yamaguchi et al.,2007). Expression was analyzed for two of the fourNCED alleles in lettuce (LsNCED1 and LsNCED4),which have the highest homology to AtNCED2 andAtNCED6, respectively, among the ArabidopsisNCEDfamily (Sawada et al., 2008). Initially present at rela-tively low levels, the abundance of mRNAs for LsZEP1and LsNCED4 declined rapidly to very low or unde-tectable levels in seeds of both genotypes duringimbibition at 20�C in the light (Fig. 7, A and B). Incontrast, transcripts for these genes initially decreasedbut then increased in Salinas seeds after imbibition formore than 12 h at 35�C. In UC96US23 seeds, transcriptsfor these genes declined more slowly at 35�C than at20�C but were much lower than in Salinas seedsimbibed at 35�C. No expression was detected for theLsNCED1 allele (Supplemental Fig. S3W). A patternvery similar to that for LsZEP1 was observed for a

Figure 4. Germination of Salinas (Sal) and UC96US23 (UC) seeds inthe dark at 32�C in water, in fluridone, and in combinations of fluridoneplus GA4+7. Error bars indicate SE (n = 3). [See online article for colorversion of this figure.]

Figure 5. ABA (A) and PA (B) contents of Salinas (Sal; circles) andUC96US23 (UC; triangles) seeds during imbibition and germination at20�C (open symbols) and 35�C (closed symbols) in the light. ABAvaluesare plotted on a logarithmic scale due to their range and consistent withthe logarithmic germination response to ABA concentration. PA valuesare plotted on a linear scale. Error bars indicate SE (n = 3). DW, Dryweight. [See online article for color version of this figure.]





lettuce homolog of AtABA2 (Supplemental Fig. S3B), ashort-chain alcohol dehydrogenase/reductase (SDR1)converting xanthoxin to abscisic aldehyde subsequentto the step catalyzed by NCED (Yamaguchi et al.,2007). A homolog to ABA3, encoding a molybdenumcofactor to aldehyde oxidase that converts abscisicaldehyde to ABA (Yamaguchi et al., 2007), showed alow but more complex initial expression pattern, butits mRNA also increased in abundance at longerimbibition times at 35�C (Supplemental Fig. S3C).

In Arabidopsis seeds, AtCYP707A2 is responsiblefor the metabolism of ABA following imbibition(Kushiro et al., 2004). Transcript abundance of a lettucehomolog of this gene (termed LsABA8ox4; Sawadaet al., 2008) decreased in both genotypes followingimbibition at 20�C in the light but remained higherwhen imbibition occurred at 35�C (Fig. 7C; Supple-mental Fig. S3D).

When imbibed in the dark at 20�C or 35�C, expres-sion patterns of these genes differed much less be-tween genotypes (Fig. 8, A–C; Supplemental Fig. S4,A–D). Expression of both LsZEP1 and LsNCED4 washigher at 35�C than at 20�C. LsABA8ox4 mRNA de-creased with time in the dark at 20�C but showed anincreasing trend at 35�C in both genotypes.

Expression of Genes Involved in GA Synthesisand Metabolism

GA synthesis in lettuce seeds is induced by light viaphytochrome, which differentially affects the expres-sion of at least three genes involved in the synthesis ofactive GAs: LsGA20ox1 and LsGA20ox2, encoding GA20-oxidases involved in the penultimate steps, andLsGA3ox1 (previously termed Ls3h1), encoding a GA3b-hydroxylase that catalyzes the final step in GA1biosynthesis (Toyomasu et al., 1998; Yamauchi et al.,2004; Yamaguchi et al., 2007; Sawada et al., 2008). InArabidopsis, AtGA3ox1 and AtGA3ox2 act redundantlyand are essential for seed germination (Mitchum et al.,2006). LsGA20ox1 mRNA increased transiently inseeds of both genotypes at 8 to 12 h of imbibition at20�C in the light (Fig. 7F; Supplemental Fig. S5J). Whenseeds were imbibed at 35�C in the light, LsGA20ox1mRNA initially increased in UC96US23 seeds andthen declined, but remained relatively low in Salinasseeds (Fig. 7F; Supplemental Fig. S5J). Expression ofLsGA20ox2 and LsGA3ox1 increased at 20�C in bothgenotypes just prior to the initiation of germination(Fig. 7, G and H; Supplemental Fig. S5, H and J). Whenimbibed at 35�C in the light, mRNA of neither genewas detected in Salinas seeds, while in UC96US23seeds an increase was evident after a delay (Fig. 7, Gand H; Supplemental Fig. S5, H and J), consistent withthe delay in the initiation of germination (Fig. 1A).LsGA3ox2 exhibited a different expression pattern, withan initial increase after imbibition at 35�C followedby a decline and subsequent increase, particularly inUC96US23 seeds (Fig. 7I). No mRNAwas detected forLsGA3ox3 (Supplemental Fig. S5I). The genotypic dif-ferences in LsGA20ox1 expression observed in thelight (Fig. 7F; Supplemental Fig. S5J) were much lessevident in the dark (Fig. 8F; Supplemental Fig. S6J),suggesting that light is involved in regulating thisgene. Amounts of LsGA20ox2 and LsGA3ox1 transcriptswere higher in Salinas seeds than in UC96US23 seedsimbibed in the dark at 20�C (Fig. 8, G and H; Supple-mental Fig. S6, H and K).

Lettuce homologs of three genes early in the GAbiosynthetic pathway, ent-copalyl diphosphate syn-thase (LsCPS1; AtGA1), ent-kaurene synthase (LsKS1;AtGA2), and ent-kaurene oxidase (KO1; AtGA3), allexhibited similar expression patterns. Somewhat sur-prisingly, these genes were expressed more highly inseeds imbibed at 35�C than at 20�C in the light andmore highly in Salinas than in UC96US23 seeds (Sup-plemental Fig. S5, C, R, and S). This pattern is oppositeto that for the genes later in the GA biosynthetic

Figure 6. ABA (A) and PA (B) contents of Salinas (Sal) and UC96US23(UC) seeds during imbibition and germination at 20�C and 35�C in thedark for 12 or 24 h. C shows ABA and PA contents in seeds of the samegenotypes after 12 or 22 h of imbibition in the light at either 30�C or 33�C,respectively. Error bars indicate SE (n = 3). DW, Dry weight. [See onlinearticle for color version of this figure.]

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pathway described above. On the other hand, expres-sion of a lettuce homolog of a gene encoding ent-kaurenoic acid oxidase that converts ent-kaurenoicacid to GA12-aldehyde showed low and variable ex-pression at 20�C in both genotypes and was notdetected at 35�C (Supplemental Fig. S5Q). In seedsimbibed in the dark, abundance of LsCPS1, LsKS1, andKO1 mRNAs decreased with imbibition time at 20�C,but abundance increased at 35�C (Supplemental Fig.S6, C, R, and S). Genotypic differences were small,however, except in the case of LsCPS1, where expres-sion was greater in Salinas seeds at high temperaturein the dark (Supplemental Fig. S6C).GA levels are also regulated by expression of GA

2-oxidase (GA2ox) genes that metabolize active GAs

(Yamaguchi et al., 2007). Two alleles of GA2ox genesare known in lettuce, LsGA2ox1 and LsGA2ox2(Nakaminami et al., 2003). LsGA2ox1 mRNA abun-dance was high in dry seeds and declined rapidly inboth Salinas and UC96US23 seeds imbibed at 20�C inthe light (Fig. 7J). When imbibed at 35�C in the light,the initial decline was delayed in UC96US23 seedsand reversed in Salinas seeds, resulting in an increas-ing abundance in the latter (Fig. 7J). Only very low andtransient (Supplemental Figs. S5G and S6G) or unde-tectable (qRT-PCR; data not shown) levels of LsGA2ox2mRNA were present in these seeds under all condi-tions. In the dark, LsGA2ox1 mRNA abundance de-creased with time at 20�C and increased with time at35�C in seeds of both genotypes (Fig. 8J).

Figure 7. Expression of genes asso-ciated with ABA, GA, and ethylenesynthesis, metabolism, and actionin Salinas (Sal; circles) andUC96US23 (UC; triangles) seedsimbibed at 20�C (open symbols)or 35�C (closed symbols) in thelight. Time courses were adjustedat each temperature to correspondto the timing of germination (Fig.1A). Results shown are as follows(see Supplemental Table S1): A, azeaxanthin epoxidase (LsZEP1 orLsABA1); B, a 9-cis-epoxycarot-enoid dioxygenase (LsNCED4); C,an ABA-8#-hydroxylase (LsABA8ox4);D, a bZIP transcription factor (ABI5);E, a subunit of the SNF-related kinasecomplex (SNF4); F, a GA 20-oxidase(LsGA20x1); G, a second GA 20-oxidase (LsGA20x2); H, a GA 3-b-hydroxylase (LsGA3ox1); I, a secondGA 3-b-hydroxylase (LsGA3ox2); J, aGA 2-oxidase (LsGA2ox1); K, an ACS(LsACS1); L, an ACO (ACO-A); M, asecond ACO (ACO-B); N, a proteinkinase in the ethylene signaling path-way (CTR1); and O, a member of theGRAS/DELLA family proteins associ-ated with the repression of GA re-sponses (GRAS-A). The inset in Bshows data on an expanded scalefor UC96US23 seeds only. Expressionis plotted relative to the geometricmean of three control genes assayedin the same samples (see “Materialsand Methods”); note the differentscales in each panel. Data are fromqRT-PCR assays except for C, I, J, L,andN,which are fromGeXPanalysis.Means are plotted 6SE (n = 3). [Seeonline article for color version of thisfigure.]





Expression of Genes Involved in Ethylene Synthesis

The mRNA abundance of a lettuce gene having highhomology to the ethylene biosynthetic enzyme ACS(LsACS1) increased between 8 and 24 h of imbibition inthe light at 20�C in both genotypes (Fig. 7K). Whileexpression was completely repressed in Salinas seedsat 35�C in the light, expression in UC96US23 seedswas only delayed (Fig. 7K), very similar to the expres-sion pattern for LsGA3ox1 (Fig. 7H). A lettuce genehighly homologous to Arabidopsis ACO (AtACO4 orAtEAT1; termed ACO-A here, as a full-length clone hasnot been reported in lettuce) was expressed in bothgenotypes in response to temperature in a patternsimilar to that of LsACS1 (Fig. 7L). A second ACO

homolog allele (ACO-B) was highly abundant inSalinas seeds at 20�C and somewhat less so inUC96US23; expression in both genotypes was delayedand reduced when imbibed at 35�C (Fig. 7M). LsACS1and ACO-A had similar expression patterns withrespect to temperature when seeds were imbibed inthe dark, although expression of LsACS1 was muchlower in both genotypes in the dark than in the light(Fig. 8, K and L). Expression of ACO-B in seedsimbibed in the dark was similar to its expression inthe light (Fig. 8M). Expression of all three genes wasalso consistently greater in Salinas seeds thanin UC96US23 seeds imbibed at 20�C in the dark (Fig.8, K–M).

Figure 8. Expression of genes asso-ciated with ABA, GA, and ethyl-ene synthesis and metabolism inSalinas (Sal) and UC96US23 (UC)seeds imbibed for 12 or 24 h at20�C or 35�C in the dark. Genesand assays are as described forFigure 7. Means are plotted 6SE

(n = 3). [See online article for colorversion of this figure.]

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Expression of Genes Involved in ABA, GA, and Ethylene

Signal Transduction and Action

High temperature could also influence the percep-tion of or sensitivity to ABA, GA, and ethylene, so theexpression of genes that are regulated by these hor-mones or are involved in their signal transductionpathways was also assayed. For example, ABI5 is abZIP transcription factor whose mutation results inABA insensitivity (Finkelstein and Lynch, 2000). Itsexpression is induced by ABA, and it can inhibit thelate stages of germination and early seedling growth(Lopez-Molina et al., 2001). mRNA abundance of alettuce homolog of Arabidopsis AtABI5 declined rap-idly after imbibition at 20�C in the light in both Salinasand UC96US23 seeds, but it remained high until 12 to18 h in both genotypes imbibed at 35�C before in-creasing in Salinas seeds and decreasing in UC96US23seeds (Fig. 7D). A similar pattern of expression wasalso observed for ABI3 and ABI4 homologs (Supple-mental Fig. S3, G and H), transcription factors alsoassociated with ABA signaling pathways (Feurtadoand Kermode, 2007). A very similar pattern of expres-sion was also evident for a lettuce homolog of SNF4(Fig. 7E), a GA-repressed and ABA-induced geneencoding a regulatory subunit of the SNF-relatedkinase complex involved in the regulation of reserveaccumulation and mobilization and seed dormancy(Bradford et al., 2003; Radchuk et al., 2006; Bolingueet al., 2007; Rosnoblet et al., 2007). Other genes thatalso exhibited a genotype- and temperature-specificexpression pattern similar to this included SDR1,AREB2, ERA1, LEA, PER1, PRL1, COP1, COP8,COP11, FUS5, and PHYA (Supplemental Figs. S3, B,K, O, U, Z, and CC, and S7, C, D, F, O, and T), as well asLsZEP1, LsABA8ox4, LsCPS1, KO1, and LsKS1, asnoted previously. This pattern correlated with seedABA content (Fig. 5A), suggesting that these genesmay be coordinately regulated by ABA.A somewhat different expression pattern was re-

corded for genes encoding homologs of GAI or GRASfamily proteins containing a DELLA domain that actas repressors of germination (Silverstone et al., 2001;Tyler et al., 2004). In Arabidopsis, AtRGL2 mRNAappears following imbibition and remains high indormant seeds while disappearing in germinatingseeds (Ariizumi and Steber, 2007). In lettuce, mRNAabundance of a GRAS family homolog (termedGRAS-A)decreased in seeds imbibed at 20�C, coincident withthe completion of germination, but increased andremained high in Salinas seeds imbibed at 35�C, whichexhibit thermodormancy (Fig. 7O). ABA has beenreported to stabilize DELLA proteins, and RGL2(along with DELLA proteins GAI and RGA) is re-quired for ABA inhibition of germination (Achardet al., 2006; Penfield et al., 2006). Somewhat surpris-ingly, GRAS-A mRNA also remained high inUC96US23 seeds imbibed at 35�C but eventually de-clined as germination occurred between 24 and 48 h(Fig. 7O). Two other GRAS family members, LsGAI2

and GRAS-B, as well as a homolog of the GA receptorGID1 (Ueguchi-Tanaka et al., 2007), maintained some-what higher mRNA levels in UC96US23 seeds than inSalinas seeds imbibed at high temperature (Supple-mental Fig. S5, M–O).

A lettuce homolog of a gene associated with ethyl-ene signal transduction (CTR1) exhibited generallydeclining mRNA abundance during imbibition, butmRNA levels remained higher in seeds of both gen-otypes imbibed at 35�C (Fig. 7N). Other genes associ-ated with ethylene signaling (e.g. EIN2 and LsETR1)showed similar patterns (Supplemental Fig. S5, D andF), although LsERS1 mRNA transiently increased inseeds of both genotypes imbibed at 20�C (Supplemen-tal Fig. S5E).

LsMAN1, which encodes an endo-b-mannanase in-volved in mobilization of the galactomannan cell wallstorage reserves of the lettuce endosperm after radicleemergence, is induced by GA and repressed by ABA(Halmer et al., 1975; Dulson et al., 1988; Wang et al.,2004a). LsMAN1 mRNA abundance was very highimmediately after radicle emergence in both geno-types at 20�C but remained much lower in seedsimbibed at 35�C, although a small increase occurredin UC96US23 seeds at 36 and 48 h (Supplemental Fig.S5T).

Expression of these genes was also assayed in seedsimbibed in the dark at 20�C and 35�C (Fig. 8, D, E, N,and O; Supplemental Fig. S6, D–F, M–O, and T).Overall, differences between genotypes were reducedin the dark relative to the light. For example, reduc-tions in mRNA abundance of ABI5 and SNF4 inUC96US23 seeds at 20�C in the dark (Fig. 8, D and E)were much less than those that occurred in the light(Fig. 7, D and E), suggesting that light was required forthese declines to occur, but the expression patterns inthe dark were still closely correlated with ABA content(Fig. 6A).

Expression of Genes in Relation to Light Perception,Signal Transduction, and Action

A panel of genes associated with light perceptionand action was assayed, and in general, they exhibitedrelatively small differences in expression patterns inrelation to genotype, temperature, and light (Supple-mental Figs. S7 and S8). A notable exception was aPIF3 homolog (Supplemental Figs. S7V and S8V) en-coding a protein involved in controlling the level ofPHYB protein (Leivar et al., 2008) and directly inter-acting with DELLA proteins (Feng et al., 2008), whichshowed an expression pattern very similar to that ofLsGA3ox1 or LsACS1 (Figs. 7, H and K, and 8, H and K)and correlated with germination responses (Fig. 1). Asnoted above, mRNA levels of COP1, COP8, COP11,FUS5, and PHYA were elevated in Salinas seeds afterlonger imbibition times at 35�C. For two genes, ELF3and SUB1 (Supplemental Figs. S7, K and Z, and S8, Kand Z), mRNAwas detected only from Salinas seeds; itis possible that polymorphisms between the genotypes





prevented the amplification of mRNAs fromUC96US23 seeds in these cases.

ANOVA and Cluster Analysis of Gene Expression

Patterns in Response to High Temperature and Light

Relative expression was compared by ANOVAwithrespect to light treatment (light or dark), genotype(Salinas or UC96US23), temperature (20�C or 35�C),and time of imbibition (12 and 24 h) as themain effects.A total of 796 light 3 genotype 3 temperature 3imbibition time 3 gene treatments had expressionabove the threshold level of detection and were eval-uated using linear contrasts. Of this number, 56 treat-ments (7.0%) exhibited differential expression (P ,0.0001), and among these, 36 genes were differentiallyregulated by light. Among the 56 treatments,UC96US23 seeds displayed a higher frequency ofdifferential transcription by light/dark treatmentsthan did Salinas seeds (41 and 15 significant treat-ments, respectively), and significant effects of lightregulation were detected more frequently at 24 h ofimbibition than at 12 h (41 and 15 significant treat-ments, respectively). A highly significant light3 geno-type interaction was detected for eight genes (P ,0.0001). Of these eight, LsZEP1, SDR1, ABI5, LEA, andKO1were up-regulated by dark in both genotypes, butmore strongly in UC96US23 than in Salinas (Figs. 7Aand 8A; Supplemental Figs. S3, B, I, and U, S4, B, I, andU, S5R, and S6R). This transcriptional pattern is con-sistent with the higher ABA content and apparentlylower GA content in UC96US23 seeds relative toSalinas seeds when imbibed in the dark (Figs. 5 and6). Only a single gene encoding the lipid mobilizationgene isocitrate lyase was significantly down-regulatedby dark in both genotypes, and more strongly inUC96US23 than in Salinas seeds, probably reflectingthe reduced germination in the dark (SupplementalFigs. S3S and S4S). ERA1 and PKL, negative regulatorsof ABA signaling that target ABI3 and ABI5 (Penfieldet al., 2005; Perruc et al., 2007), were down-regulated bydark in Salinas seeds but up-regulated in UC96US23(Supplemental Figs. S3O, S4O, S5U, and S6U). Elevengenes were identified as highly significant (P , 0.0001)in the three-way genotype3 light3 temperature inter-action: LsABA8ox4, ABI5, LsACS1, ACO-A, AREB2,LsGA3ox2, GRAS-A, HYL1, HY5, KO1, and PIP5K. Forthese genes, the genotypic differences in expression dueto light depended upon the imbibition temperature.This list reinforces the close interactions among ABA-,GA-, and light-related genes and points to relationshipsthat may warrant further investigation.

Hierarchical clustering also was performed on thelog2 normalized gene expression values to revealpatterns of gene expression across all light and tem-perature treatments (Fig. 9). For each genotype, thetranscript level of each gene at 0 h (dry seed) was usedas the baseline level to which mRNA amounts duringimbibition were normalized. To prevent bias, genesthat were below the level of detection were removed,

as was the very highly expressed gene LsMAN1. Theclustering results indicate that the top five majorclades, representing the genes that are most stronglyup- or down-regulated across the 32 genotype, light,and temperature variables, contain only 10 differentgenes. The first two distinct clades each contained onlya single gene. LsNCED4 was down-regulated (relativeto dry seeds) across most light and temperature treat-ments but increased in Salinas seeds (compared withother treatments) at longer incubation times at 35�C.Conversely, relative to the dry seed transcript levels,LsACS1 was strongly up-regulated in both genotypesat 20�C but only in UC96US23 seeds at 35�C. A groupof three genes involved in ABA synthesis and signal-ing, AREB1, ABA3, and HK1, formed a clade subtend-ing LsACS1 that were strongly up-regulated in Salinasseeds in the dark. The next clade consisted of ACO-A,ACO-B, PIF3, and LsGA3ox1, which were strongly up-regulated following imbibition except in Salinas seedsat 35�C. Two ABA-responsive genes, ABH1 and PIP5K,clustered together and exhibited variable expression inboth genotypes at 20�C but higher expression inUC96US32 seeds at 35�C. The next major clade con-tained a group of 15 genes including ABA synthesis,catabolism, and signaling genes and genes early in theGA biosynthesis pathway. Another large clade of 28genes (SPT to SPA4) was largely composed of light-responsive genes. Between these two large groupswere the GA oxidase genes that are up-regulated whenconditions permit germination. Finally, a large clade of21 genes (LsGA20ox2 to GRAS-A-q) was not stronglyregulated or had complex expression patterns. Thesemajor clusters across all treatments were consistentwith the ANOVA results above using only the 12- and24-h time points. In addition, assays were conductedfor several genes on the same extracts using both qRT-PCR and GeXP. These clustered together in most cases(e.g. LsZEP1 and LsABA8ox4), confirming the consis-tency of the two methods.

Differential Expression Responses Occur over a Narrow

Temperature Range

To confirm that the differential patterns of geneexpression between 20�C and 35�C were specificallyassociated with the temperature sensitivity of germi-nation, gene expression in Salinas and UC96US23seeds imbibed in the light at 30�C, where both geno-types complete germination, was compared with ex-pression in seeds imbibed in the light at 33�C, whereonly UC96US23 seeds germinated. This 3�C increase inimbibition temperature was sufficient to induce con-trasting germination phenotypes in seeds of the twogenotypes and resulted in marked differences in geneexpression (Fig. 10). Genes associated with ABA syn-thesis and metabolism or induced by ABA (LsNCED4,LsABA8ox4, ABI5, and SNF4) were all up-regulated inSalinas seeds imbibed at the thermoinhibitory tem-perature (33�C), while no increase in expression oc-curred in UC96US23 seeds under this condition (Fig.

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10, A–D). Consistent with this, ABA contents in Salinasseeds were greater at 33�C than at 30�C, while ABAcontents of UC96US23 seeds were the same at bothtemperatures (Fig. 6C). A similar expression patternwas also evident for GRAS-A, which would be ex-pected to be repressed by GA (Fig. 10E). In contrast,genes associated with GA and ethylene synthesis(LsGA20ox1, LsGA3ox1, LsACS1, and ACO-B) wereexpressed more highly at 30�C than at 33�C andwere down-regulated in Salinas seeds imbibed at thehigher temperature (Fig. 10, F–I). Expression ofLsMAN1 was repressed only in Salinas seeds at 33�C,consistent with its promotion by GA and repression by

ABA (Fig. 10J). These contrasting gene expressionpatterns over only a 3�C temperature range, whichalso differentially affected germination of the twogenotypes, confirm that multiple genes are regulatedcoordinately and differentially over a narrow range ofimbibition temperatures in a manner consistent withtheir relationship to ABA or to GA and ethylene.

Candidate Gene Mapping: Colocalization of LsNCED4with Htg6.1

In related studies to be reported separately, a ge-nomic region containing the QTL Htg6.1 (Fig. 3) was

Figure 9. Heat map of gene expression patterns of ABA-, GA-, ethylene-, and light-related genes in Salinas (Sal) and UC96US23(UC) seeds imbibed at 20�C or 35�C in the light or dark. The data of Figures 7 and 8 and Supplemental Figures S3 to S8 wereanalyzed by hierarchical clustering of log2 ratios of expression relative to the dry seed for each genotype. Expression was assayedbyGeXP unless noted by –q after the gene name, indicating qRT-PCR. Genes are identified in Supplemental Table S1. HAI, Hoursafter imbibition.





introgressed into near-isogenic lines in the Salinasbackground. In BC3S2 progeny lines from multipleintrogressed families, those homozygous for theUC96US23 Htg6.1 allele germinated at temperatures2�C to 3�C higher than did seeds homozygous for theSalinas allele (i.e. derived from null segregants lacking

the introgression), confirming that this locus has a ma-jor effect on the upper temperature limit for germina-tion (data not shown; Argyris, 2008). To test whethergenes involved in the regulation of germination colo-calized with Htg6.1, 20 candidate genes were mappedusing DNA-based markers in the RIL population de-

Figure 10. Expression of genes associated with ABA,GA, and ethylene synthesis, metabolism, and re-sponse in Salinas (Sal) and UC96US23 (UC) seedsimbibed at 30�C or 33�C in the light for 12 or 22 h,respectively. These times were chosen to correspondto the times of initiation of radicle emergence (only ofUC seeds at 33�C). Genes are as described in thelegend of Figure 7, with the addition of LsMAN1 (J),an endo-b-mannanase expressed in the endospermfollowing radicle emergence. mRNA levels assayedby qRT-PCR are plotted relative to the geometricmean of three control genes assayed in the samesamples (see “Materials and Methods”); note thedifferent scales in each panel. Means are plotted6SE (n = 3). [See online article for color version of thisfigure.]

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veloped from Salinas and UC96US23. Several of thesemapped within confidence intervals of germination-related QTLs (Argyris et al., 2008), the most intriguingof which is LsNCED4, which mapped exactly with themaximum LOD peak marker identifying Htg6.1,ABA506.1, and GA6.1 (Fig. 3). When seeds from near-isogenic progeny lines homozygous for the Salinas orUC96US23 alleles at this locus were imbibed at hightemperature, expression of LsNCED4 was consistentlygreater in the lines with the Salinas allele (Argyris,2008). LsNCED4, therefore, is a strong candidate to beresponsible for the phenotypic effects of these QTLs.

DISCUSSION

Sensitivity of Lettuce Seed Germination to Light,Temperature, GA, and ABA

Light and temperature are critical regulators of seedgermination that largely determine the expression ofdormancy and the seasonality of germination andemergence in the field (Allen et al., 2007; Donohueet al., 2007). There is considerable evidence that lightand temperature interact through the phytochromes inregulating germination (Heschel et al., 2007) and that amajor effect of Pfr is to promote GA biosynthesis(Toyomasu et al., 1998). In lettuce seeds, the Pfr re-quirement for germination increases as the tempera-ture increases (Fielding et al., 1992; Kristie andFielding, 1994) and the seeds become more sensitiveto inhibition by ABA (Roth-Bejerano et al., 1999; Gonaiet al., 2004). In Salinas seeds, there is sufficient Pfr (orGA) to permit germination in the dark at lower tem-peratures but not at higher temperatures (Fig. 1B), andsupplemental GA alone does not restore completegermination (Fig. 2B). In UC96US23, on the otherhand, only about 30% of the seeds were capable ofgerminating in the dark at 20�C, although they couldall germinate at temperatures as high as 35�C in thelight (Fig. 1) and GAwas effective in replacing the lightrequirement (Fig. 2B). Thus, germination of UC96US23seeds at both low and high temperatures requires lightor GA, while germination of Salinas seeds at hightemperature is limited primarily by ABA, as the ABAbiosynthesis inhibitor fluridone overcomes thermo-inhibition (Fig. 4). This is also reflected in the greatersensitivity of Salinas seeds to ABA and of UC96US23seeds to GA (Fig. 2). The germination phenotypes ofSalinas and UC96US23 seeds in response to light,temperature, GA, and ABA indicate that there is agreater dependence upon light (or GA) in UC96US23seeds but that ABA blocks germination at high tem-perature only in Salinas seeds.

ABA and GA Contents and Expression of TheirBiosynthesis and Metabolism Genes

As predicted by this hypothesis, the ABA content ofSalinas seeds remained 5-fold higher than that ofUC96US23 seeds imbibed at 35�C in the light (Fig. 5)

or 3-fold higher when imbibed at 33�C in the light (Fig.6C). This compares well with the 2.75-fold differencein ABA50 values measured at 29�C (Fig. 2A). Differ-ences between the genotypes were reduced and totalABA contents were higher in seeds imbibed in thedark (Fig. 6A). However, the ABA content of Salinasseeds declined between 12 and 24 h in the dark at 20�Cbut remained higher in the dark at 35�C, similar toresults reported by Gonai et al. (2004) for ‘GrandRapids’ lettuce seeds. Light promoted the decline inABA contents of both Salinas and UC96US23 seeds(Figs. 5A and 6A), as has been reported in lettuce andArabidopsis seeds (Roth-Bejerano et al., 1999; Seoet al., 2006; Sawada et al., 2008). As light stimulatesGA biosynthesis (Toyomasu et al., 1998) and GApromotes ABAmetabolism (Gonai et al., 2004; Sawadaet al., 2008), it would be expected that light wouldcause a decline in ABA content. This is opposite to theeffect of light on ABA content and metabolism inbarley (Hordeum vulgare) embryos, in which light actsto inhibit rather than promote germination (Gubleret al., 2008).

To account for the greater ABA content in Salinasseeds imbibed at high temperature, the expression ofgenes involved in ABA biosynthesis or metabolismshould differ between the genotypes. This was ob-served for LsZEP1, LsNCED4, and SDR1 gene expres-sion, where mRNA abundance of these genes inSalinas seeds fell rapidly to low or undetectable levelswithin 8 to 12 h after imbibition at 20�C, but after aninitial decrease relative to the dry seeds, it remainedrelatively constant or increased at 35�C (Fig. 7, A andB; Supplemental Fig. S3B). mRNA abundance of thesegenes also decreased rapidly in UC96US23 seeds im-bibed at 20�C, but it decreased more slowly beforeeventually falling to low levels at 35�C, consistent withthe 24-h delay in initiation of germination under theseconditions. The higher and continued expression ofthese ABA biosynthetic genes in Salinas seeds imbibedat 35�C was consistent with their elevated ABA con-tent relative to UC96US23 seeds (Fig. 5A). In Arabi-dopsis, AtNCED6 and AtNCED9, two of severalNCEDgene family members controlling the first biochemicalsteps unique to ABA biosynthesis, are required for theinduction of dormancy during seed development (Tanet al., 2003; Lefebvre et al., 2006). mRNA levels ofAtNCED2, AtNCED5, and AtNCED9 among theNCED family members are elevated when Arabidop-sis seeds are imbibed at high temperatures, withAtNCED9 apparently playing the primary role inthermoinhibition (Toh et al., 2008). LsNCED4 showshighest sequence homology to AtNCED6 (Sawadaet al., 2008), which in Arabidopsis is expressed specif-ically in the endosperm of developing seeds whileAtNCED9 is expressed in both the embryo and theendosperm (Lefebvre et al., 2006). The expression ofLsNCED4 in Salinas lettuce seeds imbibed at 35�C waslocalized exclusively in the embryo, based upon qRT-PCR results from seeds separated into embryo andendosperm + testa + pericarp samples (data not





shown). A central role for LsNCED4 in regulatingthermoinhibition in lettuce is supported also by itscolocation with Htg6.1, a QTL accounting for over 60%of the genotypic variance for high-temperature germi-nation in these genotypes (Fig. 3; Argyris, 2008), andits unique expression pattern in relation to genotypeand temperature (Figs. 9 and 10).

ABA content can also be controlled by metabolismof ABA, primarily to PA via the action of ABA8#-hydroxylase enzymes encoded by AtCYP707A1 andAtCYP707A2 in Arabidopsis (Kushiro et al., 2004; Saitoet al., 2004; Okamoto et al., 2006). In some cases, ABAaccumulation is enhanced by concerted up-regulationof NCED genes and down-regulation of CYP707Agenes (Seo et al., 2006; Oh et al., 2007; Sawada et al.,2008; Toh et al., 2008). In contrast, we found thatexpression of a lettuce gene (LsABA8ox4) havinghigh homology to AtCYP707A2 (Sawada et al., 2008)was greater in genotypes and conditions in whichLsNCED4 expression was enhanced and ABA contentwas higher (Fig. 7C). However, the relative abundanceof LsABA8ox4mRNAwas lower than that of LsNCED4at longer imbibition times when differences in ABAcontent weremost apparent (Fig. 7, B and C). Synthesisof ABA evidently exceeded metabolism, as Salinasseeds maintained higher ABA levels at high temper-ature (Fig. 5A). PA content did not show a consistentrelationship with LsABA8ox4 expression (Fig. 5B). PAcontent was greatest in Salinas seeds imbibed at 20�C,which also exhibited the greatest absolute decrease inABA content during imbibition. However, PA levelswere also higher in Salinas seeds imbibed at 35�C thanin UC96US23 seeds imbibed at either 20�C or 35�C.Thus, the elevated expression of ABA biosyntheticenzymes and the accumulation of ABA are apparentlyaccompanied by higher expression of LsABA8ox4 andongoing metabolism of both ABA and PA. A similarsituation was reported for a GA-metabolizing gene(AtGA2ox6), whose expression was up-regulated byGA in a feed-forward manner (Wang et al., 2004b). Asnoted previously, ABA contents were higher in thedark than at corresponding times in the light (Figs. 5Aand 6A), and in Arabidopsis seeds, expression ofAtCYP707A2 was increased by red light (Oh et al.,2007). However, this was evident in our data only forlettuce seeds imbibed at 20�C (compare Figs. 7C and8C). Different alleles of ABA synthesis andmetabolismgenes may be capable of independently integratingand responding to multiple inputs, including light,temperature, GA, and ABA.

In Grand Rapids lettuce seeds, light acted via phy-tochrome to promote the expression of a GA 3b-hydroxylase (LsGA3ox1) catalyzing the final step inthe synthesis of active GA1, but it either had no effecton (LsGA20ox1) or inhibited (LsGA20ox2) GA 20-oxidases catalyzing earlier steps in the biosyntheticpathway (Toyomasu et al., 1998). We also found thatLsGA20ox1 and LsGA20ox2 exhibited different expres-sion patterns in response to temperature in the light(Fig. 7, F and G). LsGA20ox1 mRNA increased tran-

siently at 8 h of imbibition and then declined in seedsof both genotypes imbibed at 20�C, while LsGA20ox2expression increased somewhat later. At 35�C, expres-sion of both GA20ox genes was greater in UC96US23seeds than in Salinas seeds. Thus, while LsGA20ox1and LsGA20ox2 had differing and relatively complexexpression patterns, their expression tended to begreater in genotypes and under conditions wheregermination will occur. GA20ox family members alsoexhibited differing expression patterns in Arabidopsisseeds, but expression of all alleles was repressed athigh temperature, as was expression of AtGA3ox1 andAtGA3ox2 genes (Toh et al., 2008). In both Salinas andUC96US23 seeds imbibed in the light at either low orhigh temperature, expression of LsGA3ox1 was closelyassociated with germination (Fig. 7H), while expres-sion of LsGA3ox2 was not as clearly correlated to thecompletion of germination (Fig. 7I).

We were unable to confirm whether GA1 contentsincreased with expression of LsGA3ox1, as expected, asthe levels were generally below the limits of detectionof the method used. Neither Chiwocha et al. (2003) norGonai et al. (2004) reported consistent effects of imbi-bition temperature on lettuce seed GA1 content, butboth of those experiments were conducted in the dark,where expression of LsGA3ox1 and LsGA3ox2 wasmuch lower than in the light, particularly at hightemperatures (Fig. 8, H and I).

Interestingly, genes encoding enzymes active in theearly steps of the GA biosynthetic pathway, includingLsCPS1, KO1, and LsKS1, were up-regulated in Salinasseeds imbibed at high temperature (Supplemental Fig.S5, C, R, and S). In fact, their expression patterns mostclosely resembled those of ABA-inducible genes suchas ABI5 or SNF4 (Figs. 7, D and E, and 9). This recip-rocal regulation by temperature of genes early and latein the GA biosynthetic pathway (Fig. 11) may reflectfeedback relationships regulating GA content. Underhigh-temperature conditions, when active GA synthe-sis is repressed due to down-regulation of LsGA20oxand LsGA3ox alleles, low GA levels may derepress theexpression of genes earlier in the pathway. Alterna-tively, these genes may be responding to the increasedABA levels resulting from up-regulation of the ABAbiosynthetic pathway.

GA metabolism via GA 2-oxidase genes may also beinvolved in regulating GA content and action duringgermination (Wang et al., 2004b; Yamaguchi et al.,2007). Two GA2ox genes have been identified in let-tuce, one of which (LsGA2ox2) was expressed primar-ily in seeds and showed some inhibition by red light(Nakaminami et al., 2003). However, we were unableto detect the expression of LsGA2ox2 consistently viaqRT-PCR, and only very low levels were detectedtransiently by GeXP in our experiments (SupplementalFig. S5G). LsGA2ox1 mRNA, however, was abundantin dry seeds and declined during imbibition, except inSalinas seeds imbibed at 35�C (Fig. 7J). In fact, itsexpression pattern closely matched that of genes earlyin the GA biosynthetic pathway described above,

Argyris et al.




particularly KO1, and of ABA-inducible genes (Sup-plemental Fig. S5R; Fig. 9). ABA induced the expres-sion of AtGA2ox6 in Arabidopsis seedlings (Zentellaet al., 2007), consistent with higher expression ofLsGA2ox1 in Salinas lettuce seeds at high temperaturewhen ABA content is high. ABA-deficient aba2mutantArabidopsis seeds also had higher GA4 levels thanwild-type seeds, suggesting that endogenous ABA cansuppress GA biosynthesis or enhance its metabolism(Seo et al., 2006). On the other hand, mRNA levels ofAtGA2ox2 in Arabidopsis seeds were relatively unaf-fected by imbibition at 22�C or 34�C, despite higherABA content in the latter (Toh et al., 2008). SpecificGA2ox gene family members may be differentiallyregulated by temperature and ABA.These contrasting patterns of expression of genes

involved in ABA and GA biosynthesis and metabo-lism are particularly evident when seeds imbibed at30�C and 33�C in the light are compared (Fig. 10). Thehigh expression of LsNCED4 only in Salinas seedsimbibed at 33�C is particularly striking (Fig. 10A), and

LsABA8ox4 shows a similar pattern (Fig. 10B).LsGA20ox1 and LsGA3ox1, on the other hand, showexactly the opposite pattern, with Salinas seeds im-bibed at 33�C having the lowest expression (Fig. 10, Fand G). These relationships likely reflect the intercon-nected effects of ABA and GA on the synthesis andmetabolism of the other hormone. For example, ABAenhances the catabolism of GA and can inhibit theexpression of GA biosynthesis genes (Gonai et al.,2004; Seo et al., 2006; Zentella et al., 2007), while GAhas reciprocal effects to inhibit ABA biosynthesis andpromote its catabolism (Oh et al., 2007). Thus, inlettuce seeds imbibed in the light at low temperature,GA biosynthesis is favored, ABA biosynthetic genesare repressed, and ABA content declines rapidly fol-lowing imbibition. When imbibed at high temperature,expression of LsNCED4 and other ABA biosyntheticgenes is stimulated, resulting in an increase in ABA,which may then repress the expression of GA biosyn-thetic genes even in the light. In UC96US23 seeds,which do not exhibit the promotion of LsNCED4 ex-pression by high temperature (Fig. 10A), expressionof GA-related genes is reduced (Fig. 10, F and G), butapparently it is sufficient to allow the completion ofgermination after a delay.

Expression of ABA- and GA-Responsive Genes inRelation to Thermoinhibition

While the hypothesis proposed above is attractiveand consistent with the available data, we do not havedata on protein levels or enzyme activities associatedwith these genes, and as noted earlier, the sensitivity oftissues to phytohormones is equally as important asthe hormone levels. Estimating hormone sensitivityfrom dose-response curves can be confounded bydifferences in uptake or deactivation of the appliedcompounds. An additional way to gain insight intoendogenous hormonal action is to monitor the expres-sion of genes known to be responsive to the hormonesas in vivo reporters of the net hormonal sensitivity andcontent balance. Therefore, we assayed the expressionof a selection of ABA- and GA-responsive genes underthe same conditions described above.

In Arabidopsis, ABI5 is a bZIP transcription factorthat can inhibit the late stages of germination and earlyseedling growth and whose expression is induced byABA (Lopez-Molina et al., 2001). In tomato (Solanumlycopersicum), LeSNF4, encoding an activating sub-unit of the SnRK1 protein kinase complex, was up-regulated by ABA and down-regulated by GA duringgermination (Bradford et al., 2003). Thus, high expres-sion of these genes should be indicative of a higheffective ABA/GA ratio in the seeds. Lettuce homo-logs of these and a number of other genes exhibitedvery similar expression patterns in response to geno-type and imbibition temperature (Figs. 7, D and E, and9). For all of these genes, mRNA abundance declinedrapidly following imbibition of either genotype at

Figure 11. Diagram illustrating the effects of high temperature on theexpression of genes in the ABA, GA, and ethylene synthesis, metabo-lism, and response pathways. Arrows indicate promotion and barsindicate inhibition of expression of the indicated genes or of germina-tion. Solid arrows in biochemical pathways indicate single steps, whiledashed arrows indicate that multiple steps are involved (not all areshown). The responses of Salinas (cultivated) lettuce seeds are shown;in general, UC96US23 seeds had a reduced response to high temper-ature compared with Salinas seeds. Not shown are reciprocal effects ofABA, GA, or ethylene on the expression of genes in their respectivesynthesis or response pathways. Genes are identified in SupplementalTable S1. GGDP, Geranyl geranyl diphosphate. [See online article forcolor version of this figure.]





20�C but remained high and increased in Salinas seedsand declined slowly in UC96US23 seeds imbibed at35�C. Thus, the expression patterns of these geneslikely report the net ABA effect, which is consistentwith the seed ABA content (Fig. 5A). However, ex-pression of ABA-responsive genes in UC96US23 seedsimbibed at 35�C was elevated longer than would beexpected based on ABA content alone (Fig. 5A),suggesting that coincident with the higher ABA con-tent, sensitivity to ABA may have been increased athigh temperature.

The GRAS/DELLA proteins act to repress germina-tion, and transcription of their genes is repressed byGA (Lee et al., 2002; Tyler et al., 2004; Ariizumi andSteber, 2007). Thus, the levels of their transcriptsshould report in vivo GA activity, with low levelsindicating high GA action. This is consistent with theexpression pattern of one lettuce GRAS family gene(GRAS-A), which declined at the time that visiblegermination began in seeds imbibed at 20�C andremained high in seeds imbibed at 35�C, consistentwith a repressor of germination (Fig. 7O). Somewhatsurprisingly, however, transcript levels for GRAS-Aremained high in UC96US23 seeds imbibed at 35�C,even after germination had occurred. This was alsoevident for LsGAI2 and GRAS-B, additional GRASfamily alleles in lettuce (Supplemental Fig. S5, M andO). ABA has been shown to stabilize other DELLAproteins (Achard et al., 2006; Penfield et al., 2006), butwhether transcription of these genes is up-regulatedby ABA or high expression is a response to low GAlevels is unknown. In Arabidopsis and tomato, regu-lation of RGL2 transcription and protein stability arecomplex, and germination can occur in the presenceof RGL2 transcript and protein (Bassel et al., 2004;Ariizumi and Steber, 2007). In addition, DELLA pro-teins may up-regulate ABA biosynthesis via expres-sion of XERICO, an H2-type RING E3 protein thatpromotes ABA accumulation (Ko et al., 2006; Zentellaet al., 2007). Thus, GRAS family proteins may beinvolved in the reciprocal regulation of both GA andABA biosynthesis and action. Also intriguing in thisconnection is the expression pattern of PIF3, encodinga phytochrome-interacting protein involved in tran-scriptional regulation by light and GA (Castillon et al.,2007). Although mutant experiments in Arabidopsisindicated that PIF3 is not involved in regulating ger-mination (Oh et al., 2004), PIF3 mRNA abundance inlettuce seeds correlated with germination and with theexpression of LsACO1 and LsGA3ox1, consistent withthe promotion of PIF3 transcription by ethylene and ofPIF3 action by the degradation of DELLA proteins inthe presence of GA (Castillon et al., 2007; Feng et al.,2008).

LsMAN1 encodes an endo-b-mannanase that mobi-lizes the galactomannan reserves in the cell wallsof the lateral endosperm of lettuce (Nonogaki andMorohashi, 1999; Wang et al., 2004a). It is expressedcoincident with or immediately after radicle emer-gence and is induced by GA and repressed by ABA

(Halmer et al., 1975; Dulson et al., 1988). This patternwas reflected in the expression pattern of LsMAN1 inresponse to temperature, with very high expressionin seeds imbibed at 20�C but much lower expression inseeds imbibed at 35�C (Supplemental Fig. S5T). How-ever, a low level of expression of LsMAN1 accompa-nied germination of UC96US23 seeds imbibed at 35�C.This differential response to temperature was partic-ularly clear in the comparison of seeds imbibed at 30�Cversus 33�C (Fig. 10J) and in seeds imbibed in the dark(Supplemental Fig. S6T). While the in vivo GA activityappears to be reduced in UC96US23 seeds imbibed inthe light at high temperature, it is nonetheless suffi-cient to result in germination in the light when com-bined with low ABA content.

Expression of Ethylene Biosynthetic and Signaling

Genes in Relation to Thermoinhibition

Ethylene is known to stimulate the germination ofseeds of a wide variety of species, particularly understressful conditions (Kepczynski and Kepczynska,1997; Matilla and Matilla-Vazquez, 2008). Ethylenecan increase the upper temperature limit of lettuceseeds, especially when combined with GA or a cyto-kinin (Abeles, 1986; Saini et al., 1986; Khan andPrusinski, 1989), and ethylene production is reducedin thermoinhibited seeds (Prusinski and Khan, 1990;Nascimento et al., 2000). While ACS is generally therate-limiting enzyme in the ethylene biosyntheticpathway, there also is evidence that ACO activity islower in seeds imbibed at high temperatures (Khanand Prusinski, 1989) and may be important in thetransition from dormancy to germination (Calvo et al.,2004). Here, we found distinct expression patterns forLsACS1 and two ACO loci depending upon lettucegenotype and imbibition temperature (Fig. 7, K–M).LsACS1 and ACO-A exhibited expression patternssimilar to that of LsGA3ox1 (Fig. 7E), increasing justbefore visible germination, consistent with the generalconclusion that ethylene acts in concert with GA andantagonistically to ABA in seeds (Kucera et al., 2005;Feurtado and Kermode, 2007; Matilla and Matilla-Vazquez, 2008). ACO-B mRNA, however, increasedbetween 2 and 8 h of imbibition at 20�C, then de-creased or remained constant; expression was delayedand reduced in both genotypes imbibed at 35�C (Fig.7M). Assuming that the ACO-B gene is translated andthe enzyme is active, ethylene production prior togermination is likely to be dependent upon the activityof ACS, which closely paralleled the time course ofgermination. Expression of genes associated with eth-ylene signal transduction and repression of ethyleneaction (CTR1, EIN2, and LsETR1) was less affected bytemperature, although their mRNA levels tended toremain higher at high temperature (Fig. 7N; Supple-mental Fig. S5, D and F). There is abundant geneticand transcriptional evidence indicating regulatoryinteractions among ethylene, ABA, and GA in seed

Argyris et al.




germination (Matilla and Matilla-Vazquez, 2008). Ourdata indicate that in lettuce seeds, some key GA andethylene biosynthetic genes are coordinately regulatedby temperature in a reciprocal pattern to that of ABAbiosynthetic genes (Figs. 10 and 11). The regulation ofthe synthesis and action of all three of these hormonesis so tightly integrated that environmental influencesmay cause coordinated adjustments throughout theregulatory network (Oh et al., 2007; Matilla andMatilla-Vazquez, 2008).

Temperature-Sensitive Expression of LsNCED4 May

Determine Lettuce Seed Thermoinhibition

Genetic analyses of a RIL population derived fromSalinas and UC96US23 revealed that QTLs for ABAsensitivity and GA response mapped to the samegenomic interval as Htg6.1, a highly significant QTLfor high-temperature germination (Argyris et al., 2005,2008). LsNCED4 is a strong candidate to be the generesponsible for the Htg6.1 phenotype based on itscolocation with these QTLs and its elevated expressionin response to high temperature only in the thermo-sensitive genotype (Figs. 3, 7B, and 10A). This isconsistent with results of a mutant screen for high-temperature germination capacity in Arabidopsis thatidentified alleles of abi3 and aba1, mutations in genesimparting ABA insensitivity and decreased ABA syn-thesis (Tamura et al., 2006), and with the role of NCEDgene family members in thermoinhibition of Arabi-dopsis seed germination (Toh et al., 2008). Thus, inthermosensitive genotypes of lettuce such as Salinas,imbibition at high temperature results in an increase inABA biosynthesis and accumulation and a decrease inGA and ethylene biosynthesis and accumulation, result-ing in inhibition of germination (Fig. 11). The LsNCED4allele from UC96US23, on the other hand, does notincrease in expression in response to high temperature,maintaining lower ABA levels and permitting germina-tion in the light (Figs. 1A, 5A, 7B, and 10A). In theabsence of elevated ABA levels, light promotes theexpression of GA (and possibly ethylene) biosyntheticgenes that remove the repression of germination by theGRAS/DELLA system. This hypothesis remains to beconfirmed through fine mapping of Htg6.1 in advancedNIL generations and functional analyses of LsNCED4alleles in Salinas and UC96US23 to determine the basisof their differential expression. If LsNCED4 is the pri-mary gene underlying Htg6.1, it will be of furtherinterest to understand how a change in expression ofthis gene is propagated through biochemical and regu-latory networks to result in the diverse transcriptionalchanges observed in response to high temperature.

MATERIALS AND METHODS

Seeds of cultivated lettuce (Lactuca sativa ‘Salinas’) and Lactuca serriola

(accession UC96US23) were grown in the field in the summers of 2002

and 2005 at Davis, California. Seeds of RILs were produced in 2002. Details

of seed production and storage were described previously (Argyris et al.,

2005).

Seed Germination Tests

Germination tests for seeds from Salinas and UC96US23 were conducted

with three replications of 25 seeds sown onto two layers of absorbent blotter

paper discs (VWR Scientific Products) in 4.7-cm petri dishes moistened with

4mLof deionizedwater. Germinationwas scoredwhen the radicle had emerged

from the enclosing tissues (endosperm membrane and fused testa/pericarp).

For hormonal and temperature sensitivity assays, Salinas and UC96US23 seeds

were germinated in GA4+7 (Abbott Labs), ABA (Gibco-Invitrogen), or GA4+7 +

fluridone (Elanco) at the indicated concentrations and temperatures.

For temperature transfer experiments to determine the times of induction

and escape from thermoinhibition, seeds were transferred to 35�C after 4, 6, 8,

10, 12, and 14 h of imbibition at 20�C and transferred to 20�C after 4, 8, 12, 18,

and 24 h of imbibition at 35�C. In each case, imbibition was in either light or

dark and seeds were transferred to the same light or dark conditions.

Germination was scored until 96 h of imbibition.

To assess ABA sensitivity, seeds were imbibed in water and in 1, 3, 10, and

30 mM ABA in the light at a temperature below the thermoinhibitory threshold

for most RILs (29�C). The percentage of germinated seeds was scored at 96 h

after the start of incubation. To determine the ABA50, probit-transformed

germination percentages were plotted against log ABA concentration and

regression analysis was performed. The intersection of regression lines with

probit = 0 (50% of the total seed population) determined ABA50 values.

For the germination tests of GA alone or GA + fluridone, seeds were sown

in a darkroom under a green light-emitting diode (557 nm) safelight

(LEDtronics), wrapped in aluminum foil, and transferred to an incubator.

Seeds were incubated in darkness at 32�C and scored under the safelight at 24,

48, 72, and 96 h. Percentage germination values were transformed to probits to

normalize variances in germination percentages for statistical analyses. Seeds

of the RIL population were screened for germination percentage in 100 mM

GA4+7 in the dark at 32�C. Probit-transformed percentage under these condi-

tions was mapped as GA sensitivity.

Mapping Population, QTL Analysis, and CandidateGene Mapping

Details on the production of the Salinas 3 UC96US23 F2 population, from

which RILs were descended, and the linkage map used for QTL analysis from

RILs grown in three different environments have been described (Johnson

et al., 2000; Argyris et al., 2005). Map construction, plant material preparation,

and DNA extraction were as described previously (Kesseli et al., 1994; Johnson

et al., 2000; Truco et al., 2007). QTL analysis of germination data utilizing

probit-transformed data was performed using Windows QTL Cartographer

version 2.0 (Basten et al., 2001). A subset of 486 markers approximately 2 to

3 cM apart were chosen as the framework map for QTL analysis (http://

cgpdb.ucdavis.edu/cgpdb2/lettuce_map/december_2006/).ANOVAwas con-

ducted to estimate additive effects of QTLs as described previously (Argyris

et al., 2005; Gandhi et al., 2005).

Germination and dormancy candidate genes that were identified and placed

on the lettuce consensus map are described by Argyris et al. (2008). For

LsNCED4, amplicons of approximately 1.4 kb from Salinas and UC96US23 were

obtained using nondegenerate primers designed to the ends of the full-length

coding region found in GenBank (AB120110). The PCR amplicons were purified

with ExoSAP-IT reagent (USB). Sequencing reactions were performed using the

Big Dye Terminator version 3.1 cycle sequencing kit (Applied Biosystems), and

sequencing analysis was conducted on an ABI Prism-3730 DNA analyzer

(Applied Biosystems). PCR products were sequenced to 83 redundancy to

ensure accurate single-nucleotide polymorphism (SNP) calls. Nondegenerate

primer sets were then designed around a putative SNP detected between the

Salinas and UC96US23 sequences at 843 bp, and PCRwas performed on the RIL

population described above. Subsequent 330-bp PCR products were first

screened on SSCP gels to ensure that SNPs were codominant and scorable

and then sequenced using the above methods to confirm the SSCP results.

Hormonal and Metabolomic Profiling

Seeds (100 mg dry weight) representing the same time points and tem-

peratures and from the same seed lots as those used for gene expression

analysis were collected, frozen, and lyophilized. Hormonal metabolomic

profilingwas conducted at the National Research Council Plant Biotechnology

Institute in Saskatoon, Saskatchewan, Canada, according to the methods

described by Chiwocha et al. (2005).





Gene Expression Assays

Salinas and UC96US23 lettuce seeds (three biological replicates of 0.5 g

each) were imbibed at the desired temperatures on two germination blotters in

9-cm petri dishes with 14 mL of water. Total RNA was isolated from dry or

imbibed lettuce seeds using a phenol-chloroform method (Cooley et al., 1999)

with the following modifications. Following overnight LiCl precipitation of

RNA, the carbohydrate contaminants were minimized by adding 0.5 mL of 10

mM Tris, pH 8.0, and 50 mL of 2 M KOAc to the pellet for 15 min on ice. The

resulting supernatant collected after centrifugation (11,000g, 10 min at 4�C)was precipitated overnight at 220�C after adding 1.3 mL of 100% ethanol.

RNA was dissolved in 50 to 100 mL of diethyl pyrocarbonate water after

complete drying. DNA in RNA samples was digested with DNase I following

the manufacturer’s protocol (Invitrogen). RNA quality was assessed by gel

analysis and 260:280-nm and 260:230-nm spectrophotometric ratios.

Candidate and reference (constitutive) gene sequences corresponding to

the top BLAST hit were identified within the Compositae Genome Project EST

database (http://cgpdb.ucdavis.edu/) through sequence homology to known

germination/dormancy-related candidates in Arabidopsis (Arabidopsis thali-

ana) and from existing lettuce sequence data in GenBank. Primer sequences

were designed using Primer Express (Applied Biosystems) to amplify 50- to

150-bp PCR amplicons for qRT-PCR analyses. PCR products for genes of

interest were sequenced to confirm their identity. Genes and primers used for

qRT-PCR analyses are shown in Supplemental Table S1.

Total RNA (4.8 mg) was reverse transcribed using random hexamers

(SuperScript First Strand Synthesis System; Invitrogen). cDNA from house-

keeping genes and genes of interest was PCR amplified in an Applied

Biosystems 7300 Real-Time PCR System using Sybergreen detection. The

change in fluorescence for each sample was analyzed by DART PCR 1.0

(www.gene-quantification.de/peirson-dart-version-1). Analysis of expression

data for reference genes by geNorm software (http://medgen.ugent.be/

~jvdesomp/genorm) identified homologs of a set of three housekeeping

genes (ubiquitin ligase, 18s1, PP2A; Supplemental Table S1) that were rela-

tively uniformly expressed across time, temperature, and light treatments

(Vandesompele et al., 2002). The geometric mean of the expression levels of

these genes was used to normalize the expression value for each gene of

interest. Gene expression experiments were conducted independently on

Salinas and UC96US23 seeds produced in 2002 and 2005 for most of the genes

shown in Figure 7 with essentially identical results; data for seeds produced in

2005 are shown.

Expression patterns were also confirmed using northern blotting for

LsNCED4, LsABA8ox4, LsGA20ox1, LsGA3ox1, and LsMAN1. Probes were

derived utilizing the same sequence sources as above. PCR primers were

designed with the Primer3 program (http://frodo.wi.mit.edu/) to generate

approximately 500-bp amplicons that were ligated into the pCRII vector and

cloned into competent Escherichia coli cells using the TOPO TA cloning kit

(Invitrogen). Cloned fragments were sequenced to determine the orientation

of insertion and to confirm their identity, and digoxigenin-labeled RNA

probes (Roche) were synthesized by either Sp6 or T7 RNA polymerase

(Ambion) from the antisense DNA strand. Total RNA (5 mg) corresponding to

a single replication of each treatment (32 samples) was loaded onto agarose

gels and subsequently transferred to nylon membranes (Amersham Pharma-

cia Biotech). Northern hybridizations were conducted as described previously

(Cooley et al., 1999), and expression patterns were consistent with qRT-PCR

results (data not shown).

The GenomeLab GeXP Genetic Analysis System (Beckman-Coulter) was

also used to assay mRNA levels in the same samples that were used for qRT-

PCR (2005 seed production lots). The GeXP system is a capillary gene

expression analysis system in which a total of 94 genes of interest were

assayed in three separate multiplexed reactions (Hayashi et al., 2007). Three

multiplex panels were designed and designated as ABA, GA/ethylene, and

light panels, consisting of 31, 32, and 31 genes of interest, respectively. Each

gene and primer sequence is listed in Supplemental Table S1. In addition to

the genes of interest, each panel contained an internal control gene (Kanr) and

three normalization genes (ACT2/7, APT1, and TUB2). For each gene target,

Arabidopsis sequences were used to identify lettuce homologs from the

Compositae Genome Project Database (http://cgpdb.ucdavis.edu/cgpdb2/).

Primers were designed using the GenomeLab eXpress Profiler software,

which produced fragment sizes ranging from 122 to 424 nucleotides with a

seven-nucleotide minimum separation size between each fragment. All PCR

products were sequenced to confirm gene identity. cDNA for GeXP was

synthesized from 100 ng of total RNA using the GenomeLab GeXP Start Kit.

PCR andmultiplex detection were performed according to the manufacturer’s

instructions (Hayashi et al., 2007).

For analysis of GeXP data, all genes of interest were normalized against the

geometric mean of the three normalization genes (Vandesompele et al., 2002).

Hierarchical gene clustering was performed on both GeXP and qRT-PCR data

with Genesis software release 1.7.2 using average linkage clustering and

Euclidean distances (Sturn et al., 2002). The clustering data are expressed as

log2 ratios using a given gene’s dry seed (0 imbibition time) normalized value

as the baseline level. For some genes, no transcripts were detected at 0 h after

imbibition but were present at a later sampling time. In those instances,

relative expression values of 0.0065 (GeXP) or 0.0002 (qRT-PCR; the estimated

minimum detection levels) were used for the dry seed data in order to

calculate expression ratios.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Germination responses to temperature transfers

between 20�C and 35�C.

Supplemental Figure S2. Seed contents of auxin, cytokinin, and their

derivatives.

Supplemental Figure S3. Expression of genes associated with ABA

biosynthesis, regulation, or response in seeds imbibed at 20�C or 35�Cin the light.

Supplemental Figure S4. Expression of genes associated with ABA

biosynthesis, regulation, or response in seeds imbibed at 20�C or 35�Cin the dark.

Supplemental Figure S5. Expression of genes associated with GA and

ethylene biosynthesis, regulation, or response in seeds imbibed at 20�Cor 35�C in the light.

Supplemental Figure S6. Expression of genes associated with GA and

ethylene biosynthesis, regulation, or response in seeds imbibed at 20�Cor 35�C in the dark.

Supplemental Figure S7. Expression of genes associated with responses to

light in seeds imbibed at 20�C or 35�C in the light.

Supplemental Figure S8. Expression of genes associated with responses to

light in seeds imbibed at 20�C or 35�C in the dark.

Supplemental Table S1. Gene descriptions, accessions numbers, and

primers used in expression assays.

ACKNOWLEDGMENTS

We appreciate the assistance of Dr. Suzanne Abrams at the Plant Biotech-

nology Institute, National Research Council, Saskatoon, Saskatchewan, Can-

ada in performing the hormone profiling assays. We also thank our

collaborators Dr. Richard Michelmore, Dr. Maria Truco, and Mr. Oswaldo

Ochoa for providing the RIL population and for assistance with the genetic

mapping and QTL analyses.

Received July 3, 2008; accepted August 25, 2008; published August 27, 2008.

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Genetic Variation for Lettuce Seed Thermoinhibition Is ... · lettuce seed germination to inhibition by ABA is en-hanced at higher temperatures (Robertson and Berrie, 1977; Roth-Bejerano