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First published online August 27, 2008; 10.1104/pp.108.125807 Plant Physiology 148:926-947 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Genetic Variation for Lettuce Seed Thermoinhibition Is Associated with Temperature-Sensitive Expression of Abscisic Acid, Gibberellin, and Ethylene Biosynthesis, Metabolism, and Response Genes1,[C],[W],[OA]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 (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37°C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.
The timing of seed germination is associated with the presence or absence of seed dormancy, which is the temporary failure of a viable seed to germinate after a specified length of time in a particular environment that would allow germination if the seed were not dormant (Baskin and Baskin, 2004  ). Genetic and physiological approaches are revealing insights into the determinants of seed germination and dormancy (Bentsink et al., 2006; Finch-Savage and Leubner-Metzger, 2006; Finch-Savage et al., 2007). Mutation studies, phenotypic screening, and surveys of natural variation, largely in Arabidopsis (Arabidopsis thaliana), have identified dozens of genes that influence the imposition or release of dormancy or the ability of seeds to transition from maturation to germination (Bentsink and Koornneef, 2002; Finkelstein et al., 2002; Bentsink et al., 2007). Many of these genes implicate plant hormones, including GA, abscisic acid (ABA), ethylene, cytokinins, brassinosteroids, and auxin (Alvey and Harberd, 2005; Kucera et al., 2005; Achard et al., 2006; Riefler et al., 2006; Seo et al., 2006; Feurtado and Kermode, 2007), as well as sugars (Dekkers and Smeekens, 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 complex regulatory webs that sense internal and external conditions and determine whether germination proceeds to completion. In many seeds, the endosperm and testa play important roles in regulating dormancy (Lefebvre et al., 2006; Muller et al., 2006; Bethke et al., 2007; Debeaujon et al., 2007; Nonogaki et al., 2007), and checkpoints for embryo growth after germination sensu stricto have been described (Lopez-Molina et al., 2002; Kucera et al., 2005).
Seed dormancy is dependent on environmental factors. Dry storage (after-ripening) or moist chilling (stratification) are often required to alleviate dormancy, and the range of environmental conditions in which germination will occur generally expands as dormancy is reduced (Allen et al., 2007
Lettuce (Lactuca sativa) seeds exhibit thermoinhibition (fail to germinate) at temperatures well below the biological upper limit for seedling growth (Cantliffe et al., 1981
Similar responses of germination to temperature and light have also been documented in Lactuca serriola, the progenitor species of cultivated lettuce (Marks and Prince, 1982
Considerable physiological, gene expression, and mutant data support a role for the balance of GA and ABA synthesis and catabolism in modulating thermoinhibition (Gonai et al., 2004
Ethylene is also involved in regulating thermoinhibition in lettuce seeds (Abeles, 1986
The convergence of different signals regulating seed dormancy and germination occurs via the GRAS/DELLA proteins. These proteins, which in Arabidopsis include GAI, REPRESSOR OF GA1-3 (RGA), RGA-LIKE1 (RGL1), RGL2, and RGL3, act as repressors of GA-induced signaling and germination (Silverstone et al., 2001
A combination of genetic, molecular, and physiological approaches, particularly in Arabidopsis, has greatly advanced our understanding of the mechanisms underlying the regulation of seed dormancy and germination (Holdsworth et al., 2008
Germination of Cultivated Lettuce (Salinas) and L. serriola UC96US23 Seeds in Response to Light and Temperature Seeds of both Salinas and UC96US23 germinated rapidly and uniformly at 20°C in the light, with germination beginning at about 16 h and being complete 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 at this 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 at 20°C, with a time course similar to that in the light, compared with only 35% final germination for UC96US23 seeds (Fig. 1B). Seeds of both genotypes were unable to germinate at 35°C in the dark (Fig. 1B). Salinas seeds are less dependent upon light than are UC96US23 seeds for germination at 20°C, and light is required for germination of UC96US23 seeds at 35°C.
Transfer experiments were conducted to determine the times during which the imposition of or escape from thermoinhibition occurred. Seeds were imbibed at high (35°C) or low (20°C) temperature for various times in the light or dark and then transferred to the alternative temperature in the same light or dark condition (Supplemental Fig. S1). These experiments established that the initial 6 to 12 h of imbibition is the critical period for the induction of thermoinhibition in Salinas seeds, confirmed that the thermotolerance of UC96US23 seeds requires light, and showed that exposures to 35°C for up to 24 h of imbibition did not induce secondary dormancy.
Seeds of Salinas and UC96US23 exhibited differential sensitivity to exogenously applied ABA and GA with respect to thermoinhibition. Seeds of both genotypes germinated more than 95% at 29°C in the light in the absence of ABA (Fig. 2A
). UC96US23 seeds were almost 3-fold less sensitive to ABA in comparison with Salinas seeds, with ABA concentrations reducing germination to 50% (ABA50 values) of 7.7 and 2.8 µM, respectively (Fig. 2A). As GA synthesis in lettuce seeds is up-regulated by light via phytochrome (Toyomasu et al., 1998
To assess the genetic basis of the differential sensitivity of Salinas and UC96US23 seeds to ABA and GA, the same RIL population utilized for QTL mapping of high-temperature germination capacity was screened as described above. ABA50 values of RILs ranged from 0 to 82 µM at 29°C in the light, and germination in 100 µM GA ranged from 0% to 98% at 32°C in the dark (data not shown; Argyris, 2008 ). Composite interval mapping analysis of ABA sensitivity (measured as ABA50) and germination in response to 100 µM GA (probit-transformed percentage) revealed one highly significant QTL for each of these traits, termed ABA506.1 and GA6.1, which accounted for 16% and 22% of the phenotypic variation for each trait, respectively (Fig. 3
). These QTLs mapped to the same 1-log of the odds (1-LOD) confidence interval as Htg6.1, a major QTL associated with high-temperature germination capacity (Argyris et al., 2005, 2008). ANOVA of RIL genotypes at a marker locus closely associated with ABA506.1 showed that the UC96US23 allele conferred significantly reduced ABA sensitivity (P < 0.05). The mean ABA50 value for RILs homozygous for UC96US23 alleles was 10.1 µM compared with 5.5 µM for Salinas homozygotes at the same locus. Similarly, RILs homozygous for the UC96US23 allele at GA6.1 showed significantly higher mean germination percentages in 100 µM GA (65%) compared with those homozygous for the Salinas allele (12%). Thus, sensitivity of germination to ABA and GA at elevated temperatures is associated with the same genetic region that confers high-temperature germination capacity in UC96US23 seeds.
Effects of Fluridone on the Sensitivity of Germination to Temperature and GA While neither Salinas nor UC96US23 seeds were able to germinate when incubated in water at 32°C in the dark, the addition of 30 µM fluridone to inhibit carotenoid and ABA synthesis significantly increased germination to approximately 30% in both lines (Fig. 4 ). Application of 100 µM fluridone alone was sufficient to restore over 90% germination of Salinas seeds but not of UC96US23 seeds under these conditions (Fig. 4). However, adding even 1 µM GA4+7 to 100 µM fluridone allowed 100% germination of UC96US23 seeds (data not shown). Combining 10 or 100 µM GA with 30 µM fluridone also allowed complete germination of UC96US23 seeds, while germination of Salinas seeds did not exceed 60% (Fig. 4). These results suggest that the light requirement for GA biosynthesis limits germination of UC96US23 seeds in the dark, which can be replaced by exogenous GA. In Salinas seeds, on the other hand, the response of germination to fluridone in the dark and the inability of GA to stimulate complete germination indicate that ABA may be the primary factor preventing germination at higher temperatures.
Hormonal Contents of Seeds Imbibed under Different Light and Temperature Conditions If ABA prevents germination of Salinas seeds at 35°C in the light, the ABA content of Salinas seeds would be expected to be greater than that of UC96US23 seeds when imbibed at high temperatures. The initial ABA content of dry Salinas seeds was significantly higher (284 ng g–1) compared with that of UC96US23 seeds (108 ng g–1) and remained so until 18 h after sowing at 20°C (Fig. 5A ). The ABA content in seeds of both genotypes declined rapidly from 2 to 18 h after sowing, decreasing from 296 to 10.5 ng g–1 and from 95 to 14.9 ng g–1 in Salinas and UC96US23, respectively. In comparison with 20°C, ABA amounts in Salinas seeds declined more slowly at 35°C and were significantly higher than in UC96US23 seeds between 2 and 48 h of imbibition (Fig. 5A). Increased imbibition temperature had 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 greater than that of UC96US23 seeds (Fig. 5A), and the latter had completed germination while the former were thermoinhibited (Fig. 1A).
Phaseic acid (PA) is the major metabolite of ABA deactivation, formed by the action of ABA-8'-hydroxylases that are encoded in Arabidopsis and lettuce by CYP707A (ABA8ox) genes (Kushiro et al., 2004 ; Saito et al., 2004; Yamaguchi et al., 2007; Sawada et al., 2008). Seed PA contents would be expected to increase as ABA contents decreased following imbibition. PA contents were highest in Salinas seeds imbibed at 20°C, in which the decline in ABA content was quantitatively greatest (Fig. 5B). PA was also initially higher in Salinas seeds imbibed at 35°C but then declined to lower levels while ABA content remained elevated. PA contents were relatively low at all times in UC96US23 seeds imbibed at either 20°C or 35°C (Fig. 5B). Quantitatively, not all of the initial ABA was accounted for as PA, indicating that PA is further metabolized. ABA and PA contents were also measured in seeds imbibed at 20°C and 35°C in the dark for 12 or 24 h. ABA content decreased in Salinas seeds imbibed at 20°C during this time but remained relatively constant in UC96US23 seeds (Fig. 6A ). ABA content was greater in Salinas seeds imbibed at 35°C compared with 20°C, but it was similar at both temperatures in UC96US23 seeds. Thus, the decline in ABA content in UC96US23 seeds following imbibition is dependent upon light at both low and high temperatures. PA contents were higher in Salinas seeds than in UC96US23 seeds at both temperatures and were higher at 20°C than at 35°C in both genotypes (Fig. 6B). These results are consistent with the germination responses in the dark at these temperatures, with only Salinas seeds germinating well at 20°C in the dark and neither genotype germinating at 35°C in the dark (Fig. 1B).
Seed GA1, GA3, and GA4 contents were generally below the limits of confident detection with the methods employed (data not shown). GA7 was detected at low levels (<5 ng g–1) in most samples and did not differ significantly between the genotypes or imbibition temperatures (data not shown). Indole-3-acetic acid and indole-3-Asp contents increased with time after imbibition at both 20°C and 35°C, but there were no significant differences between the two genotypes (Supplemental Fig. S2, A and B). cis-Zeatin riboside content declined in both genotypes at both 20°C and 35°C for the first 24 h of imbibition, then increased only in Salinas seeds imbibed at 35°C (Supplemental Fig. S2C). A similar pattern was exhibited by dihydrozeatin riboside content, although the increase in Salinas seeds at 35°C was quantitatively less (Supplemental Fig. S2D). cis-Zeatin-O-glucoside content increased following imbibition in both genotypes and at both temperatures and eventually reached higher levels in UC96US23 seeds than in Salinas seeds imbibed at 35°C (Supplemental Fig. S2E). These patterns were repeated in seeds imbibed in the dark for 12 and 24 h (Supplemental Fig. S2, F–J); it is unknown whether the delayed increase in cis-zeatin riboside content would occur in the dark at longer imbibition times. Levels of other hormones and their metabolites that were assayed were low and were not consistently detected, 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.
Differences in seed upper temperature limits, ABA and GA sensitivities, and ABA content between the lettuce genotypes may be due to differential expression of genes in the hormone synthesis, metabolism, perception, and signaling pathways. Therefore, we assayed expression (relative mRNA levels) of genes related to ABA, GA, and ethylene biosynthesis and response in seeds of both genotypes across time courses of germination at 20°C and 35°C in light and darkness by quantitative reverse transcription-PCR (qRT-PCR) and by multiplexed GeXP (Beckman-Coulter) analysis (genes and primers listed in Supplemental Table S1). Lettuce genes without reported full-length clones will be referred to by the names of their closest Arabidopsis homologs without the Ls prefix; names with the Ls prefix refer to genes that have been cloned and sequenced from lettuce.
Several genes in the ABA biosynthetic pathway were assayed, including LsZEP1 (for zeaxanthin epoxidase) and LsNCED (for 9-cis-epoxycarotenoid dioxygenase) alleles. The former is highly homologous to the Arabidopsis AtABA1 gene catalyzing the conversion of zeaxanthin to violaxanthin (Yamaguchi et al., 2007
In Arabidopsis seeds, AtCYP707A2 is responsible for the metabolism of ABA following imbibition (Kushiro et al., 2004 ). Transcript abundance of a lettuce homolog of this gene (termed LsABA8ox4; Sawada et al., 2008) decreased in both genotypes following imbibition at 20°C in the light but remained higher when imbibition occurred at 35°C (Fig. 7C; Supplemental Fig. S3D). When imbibed in the dark at 20°C or 35°C, expression patterns of these genes differed much less between genotypes (Fig. 8, A–C ; Supplemental Fig. S4, A–D). Expression of both LsZEP1 and LsNCED4 was higher at 35°C than at 20°C. LsABA8ox4 mRNA decreased with time in the dark at 20°C but showed an increasing trend at 35°C in both genotypes.
Expression of Genes Involved in GA Synthesis and Metabolism
GA synthesis in lettuce seeds is induced by light via phytochrome, which differentially affects the expression of at least three genes involved in the synthesis of active GAs: LsGA20ox1 and LsGA20ox2, encoding GA 20-oxidases involved in the penultimate steps, and LsGA3ox1 (previously termed Ls3h1), encoding a GA 3β-hydroxylase that catalyzes the final step in GA1 biosynthesis (Toyomasu et al., 1998 Lettuce homologs of three genes early in the GA biosynthetic pathway, ent-copalyl diphosphate synthase (LsCPS1; AtGA1), ent-kaurene synthase (LsKS1; AtGA2), and ent-kaurene oxidase (KO1; AtGA3), all exhibited similar expression patterns. Somewhat surprisingly, these genes were expressed more highly in seeds imbibed at 35°C than at 20°C in the light and more highly in Salinas than in UC96US23 seeds (Supplemental Fig. S5, C, R, and S). This pattern is opposite to that for the genes later in the GA biosynthetic pathway described above. On the other hand, expression of a lettuce homolog of a gene encoding ent-kaurenoic acid oxidase that converts ent-kaurenoic acid to GA12-aldehyde showed low and variable expression at 20°C in both genotypes and was not detected at 35°C (Supplemental Fig. S5Q). In seeds imbibed in the dark, abundance of LsCPS1, LsKS1, and KO1 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 expression was greater in Salinas seeds at high temperature in 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
The mRNA abundance of a lettuce gene having high homology to the ethylene biosynthetic enzyme ACS (LsACS1) increased between 8 and 24 h of imbibition in the light at 20°C in both genotypes (Fig. 7K). While expression was completely repressed in Salinas seeds at 35°C in the light, expression in UC96US23 seeds was only delayed (Fig. 7K), very similar to the expression pattern for LsGA3ox1 (Fig. 7H). A lettuce gene highly homologous to Arabidopsis ACO (AtACO4 or AtEAT1; termed ACO-A here, as a full-length clone has not been reported in lettuce) was expressed in both genotypes in response to temperature in a pattern similar to that of LsACS1 (Fig. 7L). A second ACO homolog allele (ACO-B) was highly abundant in Salinas seeds at 20°C and somewhat less so in UC96US23; expression in both genotypes was delayed and reduced when imbibed at 35°C (Fig. 7M). LsACS1 and ACO-A had similar expression patterns with respect to temperature when seeds were imbibed in the dark, although expression of LsACS1 was much lower in both genotypes in the dark than in the light (Fig. 8, K and L). Expression of ACO-B in seeds imbibed in the dark was similar to its expression in the light (Fig. 8M). Expression of all three genes was also consistently greater in Salinas seeds than in UC96US23 seeds imbibed at 20°C in the dark (Fig. 8, K–M).
High temperature could also influence the perception of or sensitivity to ABA, GA, and ethylene, so the expression of genes that are regulated by these hormones or are involved in their signal transduction pathways was also assayed. For example, ABI5 is a bZIP transcription factor whose mutation results in ABA insensitivity (Finkelstein and Lynch, 2000
A somewhat different expression pattern was recorded for genes encoding homologs of GAI or GRAS family proteins containing a DELLA domain that act as repressors of germination (Silverstone et al., 2001 A lettuce homolog of a gene associated with ethylene signal transduction (CTR1) exhibited generally declining mRNA abundance during imbibition, but mRNA levels remained higher in seeds of both genotypes imbibed at 35°C (Fig. 7N). Other genes associated with ethylene signaling (e.g. EIN2 and LsETR1) showed similar patterns (Supplemental Fig. S5, D and F), although LsERS1 mRNA transiently increased in seeds of both genotypes imbibed at 20°C (Supplemental Fig. S5E).
LsMAN1, which encodes an endo-β-mannanase involved in mobilization of the galactomannan cell wall storage reserves of the lettuce endosperm after radicle emergence, is induced by GA and repressed by ABA (Halmer et al., 1975 Expression of these genes was also assayed in seeds imbibed 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 reduced in the dark relative to the light. For example, reductions in mRNA abundance of ABI5 and SNF4 in UC96US23 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 for these declines to occur, but the expression patterns in the dark were still closely correlated with ABA content (Fig. 6A).
A panel of genes associated with light perception and action was assayed, and in general, they exhibited relatively small differences in expression patterns in relation to genotype, temperature, and light (Supplemental Figs. S7 and S8). A notable exception was a PIF3 homolog (Supplemental Figs. S7V and S8V) encoding a protein involved in controlling the level of PHYB protein (Leivar et al., 2008
Relative expression was compared by ANOVA with respect 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 the main effects. A total of 796 light x genotype x temperature x imbibition time x gene treatments had expression above the threshold level of detection and were evaluated using linear contrasts. Of this number, 56 treatments (7.0%) exhibited differential expression (P < 0.0001), and among these, 36 genes were differentially regulated by light. Among the 56 treatments, UC96US23 seeds displayed a higher frequency of differential transcription by light/dark treatments than did Salinas seeds (41 and 15 significant treatments, respectively), and significant effects of light regulation were detected more frequently at 24 h of imbibition than at 12 h (41 and 15 significant treatments, respectively). A highly significant light x genotype interaction was detected for eight genes (P < 0.0001). Of these eight, LsZEP1, SDR1, ABI5, LEA, and KO1 were up-regulated by dark in both genotypes, but more strongly in UC96US23 than in Salinas (Figs. 7A and 8A; Supplemental Figs. S3, B, I, and U, S4, B, I, and U, S5R, and S6R). This transcriptional pattern is consistent with the higher ABA content and apparently lower GA content in UC96US23 seeds relative to Salinas seeds when imbibed in the dark (Figs. 5 and 6). Only a single gene encoding the lipid mobilization gene isocitrate lyase was significantly down-regulated by dark in both genotypes, and more strongly in UC96US23 than in Salinas seeds, probably reflecting the reduced germination in the dark (Supplemental Figs. S3S and S4S). ERA1 and PKL, negative regulators of ABA signaling that target ABI3 and ABI5 (Penfield et al., 2005 Hierarchical clustering also was performed on the log2 normalized gene expression values to reveal patterns of gene expression across all light and temperature treatments (Fig. 9 ). For each genotype, the transcript level of each gene at 0 h (dry seed) was used as the baseline level to which mRNA amounts during imbibition were normalized. To prevent bias, genes that were below the level of detection were removed, as was the very highly expressed gene LsMAN1. The clustering results indicate that the top five major clades, representing the genes that are most strongly up- or down-regulated across the 32 genotype, light, and temperature variables, contain only 10 different genes. The first two distinct clades each contained only a single gene. LsNCED4 was down-regulated (relative to dry seeds) across most light and temperature treatments but increased in Salinas seeds (compared with other treatments) at longer incubation times at 35°C. Conversely, relative to the dry seed transcript levels, LsACS1 was strongly up-regulated in both genotypes at 20°C but only in UC96US23 seeds at 35°C. A group of three genes involved in ABA synthesis and signaling, AREB1, ABA3, and HK1, formed a clade subtending LsACS1 that were strongly up-regulated in Salinas seeds 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 seeds at 35°C. Two ABA-responsive genes, ABH1 and PIP5K, clustered together and exhibited variable expression in both genotypes at 20°C but higher expression in UC96US32 seeds at 35°C. The next major clade contained a group of 15 genes including ABA synthesis, catabolism, and signaling genes and genes early in the GA biosynthesis pathway. Another large clade of 28 genes (SPT to SPA4) was largely composed of light-responsive genes. Between these two large groups were the GA oxidase genes that are up-regulated when conditions permit germination. Finally, a large clade of 21 genes (LsGA20ox2 to GRAS-A-q) was not strongly regulated or had complex expression patterns. These major clusters across all treatments were consistent with the ANOVA results above using only the 12- and 24-h time points. In addition, assays were conducted for 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 consistency of the two methods.
Differential Expression Responses Occur over a Narrow Temperature Range To confirm that the differential patterns of gene expression between 20°C and 35°C were specifically associated with the temperature sensitivity of germination, gene expression in Salinas and UC96US23 seeds imbibed in the light at 30°C, where both genotypes complete germination, was compared with expression in seeds imbibed in the light at 33°C, where only UC96US23 seeds germinated. This 3°C increase in imbibition temperature was sufficient to induce contrasting germination phenotypes in seeds of the two genotypes and resulted in marked differences in gene expression (Fig. 10 ). Genes associated with ABA synthesis and metabolism or induced by ABA (LsNCED4, LsABA8ox4, ABI5, and SNF4) were all up-regulated in Salinas seeds imbibed at the thermoinhibitory temperature (33°C), while no increase in expression occurred in UC96US23 seeds under this condition (Fig. 10, A–D). Consistent with this, ABA contents in Salinas seeds were greater at 33°C than at 30°C, while ABA contents of UC96US23 seeds were the same at both temperatures (Fig. 6C). A similar expression pattern was also evident for GRAS-A, which would be expected to be repressed by GA (Fig. 10E). In contrast, genes associated with GA and ethylene synthesis (LsGA20ox1, LsGA3ox1, LsACS1, and ACO-B) were expressed more highly at 30°C than at 33°C and were down-regulated in Salinas seeds imbibed at the higher temperature (Fig. 10, F–I). Expression of LsMAN1 was repressed only in Salinas seeds at 33°C, consistent with its promotion by GA and repression by ABA (Fig. 10J). These contrasting gene expression patterns over only a 3°C temperature range, which also differentially affected germination of the two genotypes, confirm that multiple genes are regulated coordinately and differentially over a narrow range of imbibition temperatures in a manner consistent with their relationship to ABA or to GA and ethylene.
Candidate Gene Mapping: Colocalization of LsNCED4 with Htg6.1
In related studies to be reported separately, a genomic region containing the QTL Htg6.1 (Fig. 3) was introgressed into near-isogenic lines in the Salinas background. In BC3S2 progeny lines from multiple introgressed families, those homozygous for the UC96US23 Htg6.1 allele germinated at temperatures 2°C to 3°C higher than did seeds homozygous for the Salinas allele (i.e. derived from null segregants lacking the introgression), confirming that this locus has a major effect on the upper temperature limit for germination (data not shown; Argyris, 2008
Sensitivity of Lettuce Seed Germination to Light, Temperature, GA, and ABA
Light and temperature are critical regulators of seed germination that largely determine the expression of dormancy and the seasonality of germination and emergence in the field (Allen et al., 2007
As predicted by this hypothesis, the ABA content of Salinas seeds remained 5-fold higher than that of UC96US23 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 difference in ABA50 values measured at 29°C (Fig. 2A). Differences between the genotypes were reduced and total ABA contents were higher in seeds imbibed in the dark (Fig. 6A). However, the ABA content of Salinas seeds declined between 12 and 24 h in the dark at 20°C but remained higher in the dark at 35°C, similar to results reported by Gonai et al. (2004)
To account for the greater ABA content in Salinas seeds imbibed at high temperature, the expression of genes involved in ABA biosynthesis or metabolism should differ between the genotypes. This was observed for LsZEP1, LsNCED4, and SDR1 gene expression, where mRNA abundance of these genes in Salinas seeds fell rapidly to low or undetectable levels within 8 to 12 h after imbibition at 20°C, but after an initial decrease relative to the dry seeds, it remained relatively constant or increased at 35°C (Fig. 7, A and B; Supplemental Fig. S3B). mRNA abundance of these genes also decreased rapidly in UC96US23 seeds imbibed at 20°C, but it decreased more slowly before eventually falling to low levels at 35°C, consistent with the 24-h delay in initiation of germination under these conditions. The higher and continued expression of these ABA biosynthetic genes in Salinas seeds imbibed at 35°C was consistent with their elevated ABA content relative to UC96US23 seeds (Fig. 5A). In Arabidopsis, AtNCED6 and AtNCED9, two of several NCED gene family members controlling the first biochemical steps unique to ABA biosynthesis, are required for the induction of dormancy during seed development (Tan et al., 2003
ABA content can also be controlled by metabolism of ABA, primarily to PA via the action of ABA 8'-hydroxylase enzymes encoded by AtCYP707A1 and AtCYP707A2 in Arabidopsis (Kushiro et al., 2004
In Grand Rapids lettuce seeds, light acted via phytochrome to promote the expression of a GA 3β-hydroxylase (LsGA3ox1) catalyzing the final step in the synthesis of active GA1, but it either had no effect on (LsGA20ox1) or inhibited (LsGA20ox2) GA 20-oxidases catalyzing earlier steps in the biosynthetic pathway (Toyomasu et al., 1998
We were unable to confirm whether GA1 contents increased with expression of LsGA3ox1, as expected, as the levels were generally below the limits of detection of the method used. Neither Chiwocha et al. (2003) Interestingly, genes encoding enzymes active in the early steps of the GA biosynthetic pathway, including LsCPS1, KO1, and LsKS1, were up-regulated in Salinas seeds imbibed at high temperature (Supplemental Fig. S5, C, R, and S). In fact, their expression patterns most closely resembled those of ABA-inducible genes such as ABI5 or SNF4 (Figs. 7, D and E, and 9). This reciprocal regulation by temperature of genes early and late in the GA biosynthetic pathway (Fig. 11 ) may reflect feedback relationships regulating GA content. Under high-temperature conditions, when active GA synthesis is repressed due to down-regulation of LsGA20ox and LsGA3ox alleles, low GA levels may derepress the expression of genes earlier in the pathway. Alternatively, these genes may be responding to the increased ABA levels resulting from up-regulation of the ABA biosynthetic pathway.
GA metabolism via GA 2-oxidase genes may also be involved in regulating GA content and action during germination (Wang et al., 2004b ; Yamaguchi et al., 2007). Two GA2ox genes have been identified in lettuce, one of which (LsGA2ox2) was expressed primarily in seeds and showed some inhibition by red light (Nakaminami et al., 2003). However, we were unable to detect the expression of LsGA2ox2 consistently via qRT-PCR, and only very low levels were detected transiently by GeXP in our experiments (Supplemental Fig. S5G). LsGA2ox1 mRNA, however, was abundant in dry seeds and declined during imbibition, except in Salinas seeds imbibed at 35°C (Fig. 7J). In fact, its expression pattern closely matched that of genes early in the GA biosynthetic pathway described above, particularly KO1, and of ABA-inducible genes (Supplemental Fig. S5R; Fig. 9). ABA induced the expression of AtGA2ox6 in Arabidopsis seedlings (Zentella et al., 2007), consistent with higher expression of LsGA2ox1 in Salinas lettuce seeds at high temperature when ABA content is high. ABA-deficient aba2 mutant Arabidopsis seeds also had higher GA4 levels than wild-type seeds, suggesting that endogenous ABA can suppress GA biosynthesis or enhance its metabolism (Seo et al., 2006). On the other hand, mRNA levels of AtGA2ox2 in Arabidopsis seeds were relatively unaffected by imbibition at 22°C or 34°C, despite higher ABA content in the latter (Toh et al., 2008). Specific GA2ox gene family members may be differentially regulated by temperature and ABA.
These contrasting patterns of expression of genes involved in ABA and GA biosynthesis and metabolism are particularly evident when seeds imbibed at 30°C and 33°C in the light are compared (Fig. 10). The high expression of LsNCED4 only in Salinas seeds imbibed at 33°C is particularly striking (Fig. 10A), and LsABA8ox4 shows a similar pattern (Fig. 10B). LsGA20ox1 and LsGA3ox1, on the other hand, show exactly the opposite pattern, with Salinas seeds imbibed at 33°C having the lowest expression (Fig. 10, F and G). These relationships likely reflect the interconnected effects of ABA and GA on the synthesis and metabolism of the other hormone. For example, ABA enhances the catabolism of GA and can inhibit the expression of GA biosynthesis genes (Gonai et al., 2004
While the hypothesis proposed above is attractive and consistent with the available data, we do not have data on protein levels or enzyme activities associated with these genes, and as noted earlier, the sensitivity of tissues to phytohormones is equally as important as the hormone levels. Estimating hormone sensitivity from dose-response curves can be confounded by differences in uptake or deactivation of the applied compounds. An additional way to gain insight into endogenous hormonal action is to monitor the expression of genes known to be responsive to the hormones as in vivo reporters of the net hormonal sensitivity and content balance. Therefore, we assayed the expression of a selection of ABA- and GA-responsive genes under the same conditions described above.
In Arabidopsis, ABI5 is a bZIP transcription factor that can inhibit the late stages of germination and early seedling growth and whose expression is induced by ABA (Lopez-Molina et al., 2001
The GRAS/DELLA proteins act to repress germination, and transcription of their genes is repressed by GA (Lee et al., 2002
LsMAN1 encodes an endo-β-mannanase that mobilizes the galactomannan reserves in the cell walls of the lateral endosperm of lettuce (Nonogaki and Morohashi, 1999
Ethylene is known to stimulate the germination of seeds of a wide variety of species, particularly under stressful conditions (Kepczynski and Kepczynska, 1997
Genetic analyses of a RIL population derived from Salinas and UC96US23 revealed that QTLs for ABA sensitivity and GA response mapped to the same genomic interval as Htg6.1, a highly significant QTL for high-temperature germination (Argyris et al., 2005
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 ).
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 4 mL of deionized water. Germination was scored when 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 µM 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 µM GA4+7 in the dark at 32°C. Probit-transformed percentage under these conditions was mapped as GA sensitivity.
Details on the production of the Salinas x 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
Germination and dormancy candidate genes that were identified and placed on the lettuce consensus map are described by Argyris et al. (2008)
Seeds (100 mg dry weight) representing the same time points and temperatures and from the same seed lots as those used for gene expression analysis were collected, frozen, and lyophilized. Hormonal metabolomic profiling was conducted at the National Research Council Plant Biotechnology Institute in Saskatoon, Saskatchewan, Canada, according to the methods described by Chiwocha et al. (2005)
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 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 thaliana) 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 µg) was reverse transcribed using random hexamers (SuperScript First Strand Synthesis System; Invitrogen). cDNA from housekeeping 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/
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 µg) corresponding to a single replication of each treatment (32 samples) was loaded onto agarose gels and subsequently transferred to nylon membranes (Amersham Pharmacia Biotech). Northern hybridizations were conducted as described previously (Cooley et al., 1999
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
For analysis of GeXP data, all genes of interest were normalized against the geometric mean of the three normalization genes (Vandesompele et al., 2002
The following materials are available in the online version of this article.
We appreciate the assistance of Dr. Suzanne Abrams at the Plant Biotechnology Institute, National Research Council, Saskatoon, Saskatchewan, Canada 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.
1 This work was supported by the U.S. National Science Foundation (grant no. 0421630) through the Compositae Genome Project (http://compgenomics.ucdavis.edu/) and by the California State University Agricultural Research Initiative (grant no. 07–4–160) to D.W.S. and K.J.B.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kent J. Bradford (kjbradford{at}ucdavis.edu).
[C] Some figures in this article are displayed in color online but in black 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 subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.125807 * Corresponding author; e-mail kjbradford{at}ucdavis.edu.
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