JXB Advance Access originally published online on April 8, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 55, No. 399, pp. 961-973, May 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
A physical, enzymatic, and genetic characterization of perturbations in the seeds of the brownseed tomato mutants
Received 28 August 2003; Accepted 20 January 2004
1 Department of Horticulture, Plant Science Building, University of Kentucky, 1405 Veterans Drive, Lexington, KY 40546-0312, USA
2 Center of Membrane Sciences, Department of Chemistry, University of Kentucky, Lexington, KY 40546, USA
3 University of Kentucky, Advanced Genetics Technology Center, University of Kentucky, Lexington, KY 40546, USA
* To whom correspondence should be addressed. Fax: +1 859 257 7874. E-mail: adownie{at}uky.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The brownseed mutants (bs1, bs2, and bs4) of tomato all possess dark testae and deleteriously affect seed germination speed and/or final percentage. Poor germination performance of the bs1 but not the bs4 mutant, was due to greater impediment to radicle egress. Testa toughening (bs1) was prevented by drying in N2. However, poor germination speed was hardly affected by drying. GA4+7 did not ameliorate germination percentage or speed (bs1, bs2), whereas bs4 seeds commenced radicle protrusion sooner and had a greater germination percentage. bs1 mutant seeds have two times more catalase activity while those of bs4 contained six times more peroxidase and almost two times more catalase activity than WTs. bs4 release only half of the reactive oxygen species into the media than WT during imbibition. EPR detected the presence of free radicals in bs1 and its WT. bs mutants were epistatic to 12 anthocyaninless mutations, at least some of which produce seeds of lighter than usual testa colour. Macro-arrays of subtractive, suppressive PCR products identified differentially regulated transcripts between seeds of bs4 and WT. EST identity suggests bs4 does not exit the developmental programme upon attaining maturity.
Key words: Catalase, electron paramagnetic resonance (EPR), free radicals, germination, Lycopersicon esculentum, macro-array, peroxidase, seed, subtractive-suppressive PCR, testa.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The enclosure of the embryo and endosperm in integuments of strictly maternal origin (testa, pericarp) has led to interactions among tissues of different parental genetic contribution to establish seed longevity and control of radicle protrusion (Koornneef and Karssen, 1994; Léon-Kloosterziel et al., 1994; Debeaujon et al., 2000). Studies of arabidopsis and tomato mutants deficient in testa pigmentation (aberrations in the shikimic acid pathway) have underscored the importance of the testa (Atanassova et al., 1997a, b; 2001; Debeaujon and Koornneef, 2000; Downie et al., 2003b) in these processes. However, understanding of the testa attributes that confer high quality performance in such diverse functions remains poor (Koornneef et al., 2002). Nevertheless, transparent testa (tt; arabidopsis) and anthocyaninless (tomato) mutants have shown that control of radicle protrusion is exerted through the deposition of shikimate metabolites in the testa during development (Debeaujon et al., 2000; Atanassova et al., 1997a). Derivatives of the shikimate pathway have been found to crosslink extensin, cellulose, pectin, and other components of the cell wall. The assembly of the various members of the shikimate family lead to a diversity of polymers that have long been associated with increasing structural strength, providing defence against pathogens, and decreasing water permeability, attributes that are consistent with the final role of the testa (Mohamed-Yaseen et al., 1994).
The polymerization of shikimate metabolites is due to endogenous oxidative enzymatic action during testa maturation (Gillikin and Graham, 1991) coupled with an alteration in the physical environment surrounding the propagule. These changes profoundly alter testa permeability, oxygen availability to the embryo following imbibition, and/or testa physical strength (Egley et al., 1983; Gillikin and Graham, 1991; Qi et al., 1993). Studies using ethylene-insensitive mutants of Arabidopsis and tomato have implicated maternal tissue (fruit/testa) sensitivity to ethylene in the control of germination speed (Siriwitayawan et al., 2003).
Mutants with darker than usual testa appearance have been isolated in tomato on six different occasions (brownseed1, 2, 3, 4, lateral suppressor, blackseed; Soressi, 1967; Philouze, 1970; Yordanov and Stamova, 1971; Monti, 1972; Taylor, 1979; Downie et al., 2003b). In addition to bs1 and bs4 mutations’ negative impacts on seed germination percentage and rate, the BS1 (possibly BS4; Soressi, 1972) genes have been mapped to locations on chromosome one harbouring quantitative trait loci positively influencing salt-tolerant seed germination (Foolad et al., 1998).
With the exception of the lateral suppressor mutation, the darker-than-usual testa mutants are all inherited as monogenic, recessive, Mendelian traits that are not determined exclusively by the maternal genotype, indicative of a paternal contribution in the control of testa attributes (Downie et al., 2003b). Mutation of the genes encoding factors participating in the paternal control of testa attributes disrupts communication between the endosperm and the testa and results in altered testa composition and/or aberrations in the identity of the endosperm cell file immediately beneath the testa (Downie et al., 2003b). This report elucidates bs physiological and genetic perturbations, relating them to the delayed germination phenotype.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
Wild-type or mutant tomato (Lycopersicon esculentum Mill. cv. Moneymaker (MM and bs4) or Ailsa Craig (AC and bs1)) plants were grown, along with bs2, from seed in the University of Kentucky Horticulture Greenhouse complex (Buxton and Jia, 1999). Seeds were harvested, cleaned (0.1 M HCl for 1 h), washed in tap water, dried (5% moisture content on a fresh weight basis), and stored at –20 °C. The WT Pieralbo, the true background of the bs2 mutant, is unavailable. The bs2 mutant was included in this report to document its germination habit and physical attributes. Due to similarities in germination behaviour, bs2 seed attributes are reported in the figures and tables with those of bs1/wild-type Ailsa Craig (WT AC).
Mutant characterization
Seed weight, area, number per fruit, and fruit pH were determined as previously described (Downie et al., 2003b). Electron paramagnetic- and puncture force-analysis, the ontogeny of dark pigment accumulation within the bs mutant seeds, epistasis analysis with the anthocyaninless mutants, enzyme assays, and reactive oxygen species measurements were performed as previously described (Downie et al., 2003b).
Seed germination
Eight replicates of 25 seeds each were plated onto three layers of germination blotter paper in square germination trays for each genotype. Four replicate blotters per genotype were saturated with distilled, deionized water and four with 100 µM GA4+7. The seeds were scanned every 12 h for 14 d using the Paradigm Seedlot Vigor AssessmentTM system (Version 3.2; Paradigm Research Corp, South Haven, MN, USA) and mean percentage germination and mean germination time (MGT, Bewley and Black, 1994) for WT and mutant seed computed and compared using the Statistical Analysis Systems (1999; see below).
Seeds were harvested from fruit and fermented in the juice for 24 h to remove the sheath. The seeds were then apportioned into three equal fractions, and dried over activated alumina (Grabe, 1989) in an air-, nitrogen-, or oxygen-sparged desiccator. Following 3 d of dehydration, fresh seeds were harvested from fruit. Following 24 h fermentation they were cleaned and four replicates of 25 seeds each sown immediately on water-saturated blotters as described above. At this point four replicates of 25 seeds each that had been dried for 4 d in each of the atmospheres were also plated on water-saturated germination blotters (see above). The undried control in this experiment was possible because tomato seeds do not require dehydration to complete germination (Berry and Bewley, 1991). Seeds were scanned every 12 h using the Paradigm System and mean percentage germination and mean germination time calculated from the data.
Molecular genetic assessment of differentially expressed genes between seeds of wild-type Moneymaker and bs4
Subtracted library construction: Mature seeds of Moneymaker and bs4 mutant plants were harvested and ground directly in RNA isolation buffer (Cooley et al., 1999). Poly A+ RNA was procured using Dynabeads (Dynal Biotech Inc., Lake Success, NY, USA). The mRNA was reverse transcribed and cDNA used to perform two subtractive, suppressive PCR (Diatchenko et al., 1996) experiments between MoneyMaker and bs4 (forward and reverse selection) using a kit (PCR-select; BD Biosciences, Palo Alto, CA, USA). The resulting amplicons were size fractionated on a 1% (w/v) agarose gel and those fragments less than 500 bp, between 500 and 1000 bp, and greater than 1000 bp, were rescued from the gel. Size-fractionated amplicons were ligated into a homemade T/A cloning vector engineered to leave single, ‘T’ overhangs following XcmI cleavage (Q Xu and B Downie, unpublished data). Following transformation into DH5
, bacteria were titred on LB 100 µg ml–1 ampicillin. The libraries were diluted to 
4 cfu µl–1, and 750 µl spread on solid LB media containing 100 µg ml–1 ampicillin, 1.6% w/v agar, 37.5 µM IPTG, and 0.0075% (w/v) X-GAL. Those colonies containing inserts were picked using blue/white screening and arrayed in 384 well plates (Q-Pix II, Genetix Ltd., New Milton, Hants, UK) containing LB 100 µg ml–1 ampicillin and 15% glycerol. Libraries were duplicated, bacteria grown to high density overnight using a HiGro II (Genomic Instrumentation Service, Inc. San Carlos, CA, USA) at 520 rpm, 8.3 ml of humid, ultrapure air introduced every 30 s, and frozen at –80 °C.
Filter preparation, hybridization and analysis: Libraries were retrieved from –80 °C, warmed to room temperature over several hours and arrayed on the bed of the Q-Pix II (Genetix) fitted with a 384 pin arraying head. A 492 cm2 piece of HyBond N+ membrane (Amersham-Pharmacia, Piscataway, NJ, USA) was placed on an LB 100 µg ml–1 ampicillin-soaked 3MM filter paper (Whatman, Maidstone, Kent, UK) on the gridding block and bacteria transferred from the library and arrayed in a 4x4 G duplicate pattern as micro-dots on the membrane according to the manufacturer’s instructions (Genetix). The membrane was removed from the filter paper and placed on a Q-Tray (Genetix) containing 200 ml of LB 100 µg ml–1 ampicillin, 1.6% (w/v) agar. An LB 100 µg ml–1 ampicillin-soaked 3MM filter paper (Whatman) was placed in the lid to provide a moist atmosphere and the whole inverted and incubated for 20 h at 37 °C to permit colony growth. The membrane was removed from the LB, placed on 3MM filter paper soaked with denaturing solution (0.5 M NaOH, and 1.5 M NaCl) over a boiling water bath for 4 min. The filter was removed to a 3MM filter paper soaked in neutralization solution (1 M TRIS-HCl, pH 7.5, 1.5 M NaCl) for 4 min and then dried on 3MM filter paper for 1 min. Next the membrane was inverted (colony side down) in 100 ml, temperature pre-equilibrated proteinase K solution (0.1 mg ml–1 proteinase K [F-Hoffman-La Roche, Basel, Switzerland], 50 mM TRIS-HCl, pH 8.0, 1% w/v sarkosyl, 100 mM NaCl, 50 mM EDTA) at 37 °C for 1 h. The membrane was removed from the proteinase K solution, placed on a 3MM filter paper and a second filter paper placed on top of the membrane. A pipette was used to roll the filter paper onto the membrane and the whole was left to dry overnight. The following day, the top filter paper was peeled from the membrane and the dry membrane UV-crosslinked at 50 mJ (GS GENE LINKER, Bio-Rad Laboratories, Hercules, CA, USA).
Probe preparation, hybridization conditions, image analysis: The probe for colony macro-arrays and northern blot was prepared using PCR and adaptor primers in the presence of [32P]-dATP (New England Nuclear, Boston, MA, USA). The probe for macro-arrays was synthesized en masse from subtracted or un-subtracted cDNAs according to the kit manufacturer (BD Biosciences). Probes for northern blot used plasmid prepared from single colonies previously identified as being differentially expressed between the two populations by macro-array. Following PCR, the radiolabelled cDNAs for the macro-arrays were cleaved with RsaI to remove the adaptors (also present on the amplicons constituting the library). Thereafter, the probes for either the macro-arrays or the northern blot were run through a PCR purification column (Qiaquick, Qiagen, Valencia, CA), washed, and eluted in 50 µl. Aliquots were counted using scintillation, the probes boiled, quenched, and added to the hybridization solution.
Macro-array or northern blot membranes were blocked at 68 °C for 12 h in pre-hybridization solution (6x SSPE, 5x Denhardt’s solution (Denhardt, 1966), 0.5% SDS, and 100 µg ml–1 boiled, sheared salmon sperm DNA). Thereafter, the membranes were hybridized with (for macro-arrays) equal counts of boiled, quenched probe introduced into fresh pre-hybridization solution. Following hybridization, membranes were first washed twice, 15 min each time, at low stringency (2x SSC, 0.1% SDS at 68 °C) and exposed to a phosphor screen for 2 d. The image was captured using a PhosphorImager 445 SI (Molecular Dynamics, Sunnyvale, CA, USA) and the blots re-washed at high stringency (0.2x SSC, 0.1% SDS at 68 °C for 30 min) and re-exposed to the phosphor screen for 5 d.
Colonies harbouring ESTs significantly up- or down-regulated between the two genotypes were identified from images produced from the PhosphorImager and, for some, plasmid DNA was isolated for use in northern blot and sequencing.
Sequencing: Cycle sequencing reactions employed universal primers (Integrated DNA Technologies, Inc., Coralville, IA, USA) binding to the plasmid oriented toward the T/A cloning site and DTCS chemistry (Beckman Coulter, Inc., Fullerton, CA, USA) run on a GeneAmp 9700 PCR machine (Applied Biosystems, Foster City, CA, USA). Sequencing reactions were cleaned using magnetic bead technology (Agencourt Bioscience Corp., Beverly, MA, USA) and sequencing was performed at the Advanced Genetics Technologies Center [AGTC] (University of Kentucky, Lexington, KY, USA) using a Beckman Coulter CEQ 8000XL, eight capillary electrophoresis Genetic Analysis System. The proprietary UK-AGTC LIMS system was employed to perform automated sequence quality analysis, vector/adaptor masking, and database homology searches.
Statistical analysis
All comparisons of the effect of the mutations were performed within a cultivar (AC with bs1; MM with bs4). Seed weight, number of seed per fruit, fruit pH, enzyme activity, increases in moisture content during imbibition, and ROS release into the media were subjected to analysis of variance using the ANOVA procedure of Statistical Analysis Systems (1999). The analysis of the force necessary to puncture the micropylar tip, final percentage germination, and MGT was performed between genotypes (mutant versus WT) within a drying treatment. Because it was not possible to obtain the Pieralbo WT background of bs2, the mutant was left out of this analysis. The effect of drying treatment on puncture force, final percentage germination, and MGT were compared within each genotype (this included bs2). If the ANOVA indicated that there were significant differences among means, Tukey’s mean separation test was used to distinguish among them.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Testa toughness, germination percentage, and speed
The completion of germination of the afterripened, darker-than-usual testa mutants was significantly delayed relative to the afterripened seeds of the respective WTs except for bs1 and WT AC imbibed on water (Fig. 1A, B). The final germination percentage of bs1 and WT AC were not significantly different with or without GA4+7 (Fig. 1A, B). This was not the case for bs4 mutant seeds, although the percentage germination of these seeds after 10 d on GA4+7 was double that on water it was still significantly less than WT MM (Fig. 1A, B). The delay in completion of germination of the darker-than-usual testa mutants, when it occurred, was not due to a deficiency in imbibition rate (Fig. 2).
|
|
Previous work suggested a requirement for seed dehydration to enhance testa toughness in a blackseed mutant relative to the wild type (Downie et al., 2003b). To test this characteristic in the bs mutants, germination tests and puncture force analyses 24 HAI were conducted on freshly harvested mutant and respective WT seeds that had been: (1) harvested and dried in air; (2) harvested and placed directly on water without drying (fresh); (3) harvested and dried in N2; and (4) harvested and dried in O2.
Comparison of bs1 mutant seed with WT AC dried under a variety of conditions supported the contention that oxygen exacerbated testa toughness differences (Table 1). Dehydration in nitrogen eliminated differences in testa toughness between the two genotypes (Table 1). However, greater bs1 testa toughness did not necessarily result in a greater MGT or poorer final germination percentage (Table 1).
|
The presence of oxygen during dehydration did not result in greater testa toughness for bs4 (Table 1). However, bs4 seeds dried in oxygen completed germination significantly poorer than did WT MM seeds dried in oxygen. This was also the case for seeds germinated fresh after cleaning (Table 1), while seeds dried in air or nitrogen completed germination to the same extent as WT MM (Table 1). It was concluded that drying bs4 seeds in the presence of oxygen was no more detrimental than drying WT MM seeds in the same atmosphere.
Comparing air-dried seeds with fresh seeds, there was no consistent trend in germination performance. Placing WT seeds directly on water did not affect or significantly decreased mean germination time, was deleterious or inconsequential to the final percentage germination, and did not affect the puncture force attained by WT AC and WT MM, respectively (Table 1). Drying under any atmosphere exacerbated the deleterious effect of the dark testa colouration on the final percentage germination for bs2, while air-dried bs4 seeds completed germination to a greater percentage than O2-dried or fresh seeds (Table 1). Fresh seeds from bs1 plants attained a faster MGT than did air- or N2-dried seeds while most fresh bs4 seeds failed to complete germination (Table 1).
Treatments tending to produce low puncture forces also tended to have faster MGTs for all genotypes except bs1 (Table 1). The converse relationship was also true for all genotypes except bs1 where tougher seeds had slower MGTs (Table 1). In addition, except for bs2, treatments with slower MGTs and tougher seeds also completed germination poorer (Table 1).
General characterization
A description of the ontogeny of pigment accumulation and localization in the bs mutants can be found in Downie et al. (2003b).
The seeds of the bs mutants were all heavier and larger than seeds from their respective WTs (Table 2). The number of seeds per fruit was unaffected by the mutations (Table 2). The pH of the fruit was negatively affected, being greater than that of WT fruit for the bs4 mutant (Table 2). No discernible difference in fruit pH was determined for bs1 relative to WT AC.
|
Ailsa Craig seeds, both WT and bs1, contained unpaired electrons, as determined using EPR (Table 3). The peak was at the same frequency (3475 G) as that for blackseed (bks) mutant tomato seeds (Downie et al., 2003b), black sunflower (Helianthus annuus L.), and niger (Guizotia abyssinica (L.S.) Cass.). The latter two species and the recently characterized mutant tomato are known to produce seeds with testae comprised of melanic compounds. However, unlike the bks seeds (Downie et al., 2003b), hyperproduction of a melanic compound does not appear to be the chemical alteration of the bs mutants resulting in aberrations in seed germination because both wild-type Ailsa Craig and bs1 seeds had similar EPR spectra. Neither wild-type Moneymaker nor bs4 exhibited appreciable EPR signals, which is attributed to a lack of unpaired electrons, a hallmark of melanic compounds (Table 3).
|
Reactive oxygen species (ROS) scavenging enzymes were up-regulated in bs testa mutants
Shikimate compounds accumulate in testae and may be activated by peroxidase-mediated reduction of peroxide into oxidative polymerization producing compounds that increase testa strength (Gillikin and Graham, 1991; D’Ischia et al., 1991; Todd and Vodkin, 1993; Gijzen, 1997). Previous work has demonstrated an up-regulation of peroxidase in bks mutant seeds relative to WT (Downie et al., 2003b). The hypothesis was therefore tested that oxygen or a ROS was interacting with some testa component in bs1, bs2, and bs4, to crosslink and darken (in some instances, toughen) the testa. Toughening could be enzymatically mediated by either peroxidase (PRX) or polyphenol oxidase (PPO). There was no difference in catalase-insensitive PPO activity among the genotypes (data not shown). However, the bs4 mutants exhibited considerable PRX activity relative to WT (Fig. 3A, B). In-gel activity staining revealed that the bs1 mutant seed also had detectable PRX activity while WT AC did not, but activity assays were unable to document a difference (Fig. 3A, B). Catalase (CAT) activity was statistically significantly greater (
=0.05) in bs1 and bs4 mutant seeds relative to WT AC and WT MM, respectively (Fig. 3C). In-gel staining for CAT activity following native PAGE did not reveal obvious differences in CAT (Fig. 3D). There were no significant differences in superoxide dismutase (SOD) activity among the genotypes (Fig. 3E). Using SDS-PAGE, without boiling the sample in SDS-loading buffer, it was determined that similar SOD isoforms were present in the seeds of all genotypes (Fig. 3F).
|
Statistically significantly greater amounts (
=0.05) of reactive oxygen species (ROS) were released from intact, dry WT MM seeds than from those of bs4 (Fig. 4). This difference was substantially mitigated 24 HAI, but WT MM seeds still released statistically significant greater amounts (=0.05) of ROS into the media than bs4 early during the test (Fig. 4). No such statistical difference was apparent, regardless of imbibition status of the seed, for bs1 and its wild type.
|
bs testa mutants were epistatic to anthocyaninless mutants
Testa attributes of the tomato anthocyaninless mutants anthocyaninless of Hoffman (ah), without anthocyanin (aw), and baby lea syndrome (bls) include: (1) a lighter than WT testa colour; (2) a pattern of inheritance dependent on the genotype of the testa (Atanassova et al., 1997a); and (3) more rapid completion of germination than WT (Atanassova et al., 1997a, b). These mutants along with anthocyaninless (a), anthocyanin absent (aa), entirely anthocyaninless (ae), anthocyanin free (af), anthocyanin gainer (ag), anthocyanin gainer-2 (ag-2), anthocyanin loser (al), and anthocyanin reduced (are) were all tested for epistasis with the bs mutants. Double mutant plants (seedlings with green hypocotyls from bs seeds) were produced (except for bs2xare, despite several attempts) and F3 seed collected and compared with seeds from WT and bs plants. In every case examined, the bs mutants were epistatic to the anthocyaninless mutants (Fig. 5).
|
Many ESTs were differentially expressed between WT MM and bs4 seeds
Seeds of the bs4 mutants were considerably delayed in radicle protrusion and did not attain the percentage germination of WT MM seeds (Fig. 1; Table 1). Furthermore, bs4 mutant seeds were perturbed in aspects of ROS scavenging activities (Fig. 3) and mis-identified the outermost cell wall of the endosperm as testa (Downie et al., 2003b). These aberrations suggested that there might be considerable differences in the transcriptional activity between bs4 and WT MM seeds.
When plasmid cDNA libraries of size-fractionated, subtractive, suppression PCR amplicons (Fig. 6A) were arrayed and identical filters challenged with cDNA probes of radiolabelled subtracted and unsubtracted libraries, many putatively differentially expressed cDNAs between the two genotypes were identified (Fig. 6B, C). Northern blot analysis of poly A+ RNA from developing seeds from maturing fruit staged as ‘pink’ (Fig. 6D) or total RNA from mature, quiescent or 24 HAI bs4 and WT MM seeds (Fig. 7), independently verified that some of the genes tested were differentially expressed between the two genotypes at some stage. Those ESTs up-regulated in bs4 relative to WT MM and identifiable with orthologues in public repositories, were associated with seed development (Fig. 7).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Pleiotropic effects of the bs mutants
The fruit from the bs4 mutants had greater than usual pH, a phenotype reported for fruit from bs1 mutants (Martiniello et al., 1985), but not observed in this study. Many phenolic acids (shikimate metabolites) accumulate in tomato fruit tissue as maturation progresses (Walker, 1962; Buta and Spaulding, 1997). Disruption of the normal accumulation of one or more of these compounds in the fruit could alter fruit pH. The resultant accumulation of this precursor or alternative shikimate compound in the testa could participate in darkening the testa more extensively than usual.
Seed germination
The poor germination rate and percentage (bs4) of bs seeds relative to smaller, lighter AC and MM WT seeds is consistent with previously published conclusions (Whittington and Fierlinger, 1972) that, for tomato, small seed size leads to faster completion of germination. However, Atanassova et al. (1997a) and Downie et al. (2003b) could not find a consistent relationship between seed weight and rate of germination for anthocyaninless or blackseed tomato mutants, respectively. Afterripened bs1 mutant seeds attained a final percentage germination comparable with WT AC on both water and 100 µM GA4+7 (Fig. 1A, B). These results argue against the poor germination of this mutant being due to the effects of the testa on light quality. The dark pigment in the mutant testa was also unlikely to be a germination inhibitor (Walker, 1962) because, when the testa and endosperm cap opposing the radicle were removed from bs mutant seeds, the radicle protruded readily and most established as seedlings (data not shown). Finally, although dark testa coloration has frequently been associated with inhibition of water uptake in seeds during imbibition (Egley et al., 1983) this is not the cause of delayed germination in the bs mutants (Fig. 2). Based on puncture force analysis, one of the reasons the bs1 mutant seed is delayed in the completion of germination is due to a greater testa mechanical restraint (Table 1). This is not, however, the case for the bs4 mutant seeds which had a mechanical restraint equal to or less than that of all but fresh WT MM seeds (Table 1).
Many seeds of both WT MM and bs4, when harvested fresh, were dormant, resulting in low percentage germination even for the WT (Table 1). Dormancy is a trait of greenhouse-grown MM seeds that has been observed previously (Downie et al., 2003a). Afterripening WT MM seeds at 25 °C for 4 months prior to imbibition on water alleviated dormancy and allowed 100% completion of germination. The final germination percentage of bs4 seeds on water was also marginally improved by afterripening and a combination of afterripening and imbibition on GA4+7 greatly improved bs4 germination percentage (Fig. 1B).
Reactive oxygen species scavenging enzymes were up-regulated in the bs mutants
It is difficult to determine whether the dark testa colour of the mutants is associated with greater ROS scavenging enzyme activity or if the two are divorced consequences of a pleiotropic mutation. Enzymes associated with ROS scavenging have been implicated previously with developmental processes in the testa and/or seed germination processes (Gross, 1977; Cassab and Varner, 1987; McClung, 1997; Downie et al., 2003b). One report has suggested that inhibition of CAT is required for dormancy alleviation by redirecting H2O2 to participate in the oxidation of NADPH thus permitting the pentose phosphate pathway to proceed (Hendricks and Taylorson, 1975). In maize, constitutive CAT activity during seed development can be stimulated, and during germination, inhibited, by the flavonoid-derived phytohormone, salicylic acid (Guan and Scandalios, 1995). However, there is no evidence of CAT affecting the testa directly while NADPH availability rather than NADP amounts appear limiting for successful dormancy alleviation (Lozano et al., 1996).
It is possible that ROS generation is greater in the bs1 and/or bs4 mutants, but that, in the first instance, the up-regulation of CAT may decrease the steady-state amounts of ROS to WT levels while in the second, up-regulation of both PRX and CAT decreases ROS amounts below that of WT MM. Conversely, ROS generation may be normal in mutant seed and the up-regulation of CAT or PRX activity may be a peripheral consequence of the lesion, divorced from ROS generation.
The epistatic relationship between bks and anthocyaninless mutants
Precedence exists for the colour of seed coats (testa, pericarp) to be imparted by flavonoid metab olites (Mol et al., 1998). Reports in the literature of tomato mutants affected in anthocyanin production producing seeds with lighter than usual testa colour (ah, aw, bls; Atanassova et al., 1997a) led to the speculation that the flavonoid biosynthetic pathway was functioning to produce condensed tannins in the tomato testa in much the same way it does in Arabidopsis.
The dark testa colour of the double mutants was in no case even mitigated, let alone eliminated, when combined with the anthocyaninless mutants. Hence, the anthocyaninless mutants are not epistatic to the bs mutants. However, for reasons developed previously (Downie et al., 2003b) it is not possible to determine if the bs mutants are truly epistatic to the anthocyaninless mutants or simply masking the lighter anthocyaninless testa colour.
Contrasting patterns of inheritance between anthocyaninless and the bs mutants
The manifestation in the seed of all reported tt, ats, mum (Arabidopsis), and anthocyaninless (tomato) mutants affecting the biochemical and physical properties of the testa depends solely on the genotype of the maternal parent (i.e. the genetic composition of the maternally derived testa; Atanassova et al., 1997a). However, the bs1,2,4 and bks mutants are all inherited as recessive, monogenic, Mendelian traits (i.e. their phenotype is not determined strictly by the genotype of the testa). There appears to be antagonism between the maternal testa and the endosperm/embryo with their paternal genetic contribution that influences testa colour and, in some instances, toughness (Ellner, 1986). This necessitates communication between the testa and the underlying endosperm/embryo with the latter mitigating the full potential of the testa to darken and, in some instances, toughen (Downie et al., 2003b).
Differences among bs/bks mutants
Although manifestations of the bs/bks mutations are similar (slow completion of germination, dark testa colour, inheritance independent of the testa genotype), differences are also evident. All the bs seeds were larger and heavier than their respective WT while bks mutant seeds are smaller and lighter (Downie et al., 2003b). The bs4, but not the other mutant seeds, accumulate pigment in the periclinal, secondarily thickened cell walls of the first endosperm cell file (Downie et al., 2003b), and increased germination percentage in response to 100 µM GA4+7. The force required to penetrate the micropylar endosperm and testa was significantly greater for air-dried bs1 and bks seeds 24 HAI on water relative to their respective WTs (this report and Downie et al., 2003b). However, such was not the case for air-dried bs4 seeds (Table 1). There was no consistent effect of the atmosphere under which the bs seeds were dried on germination rate, final germination percentage, or testa toughness. This contrasts with bks mutant seeds for which the germination percentage and speed increased, and the puncture force decreased, when drying in air was avoided (Downie et al., 2003b). The different lesions varied in the degree of severity of their effect on final germination percentage and speed despite a similar testa appearance. Different combinations of ROS scavenging enzymes were significantly up- or down-regulated in the different mutants relative to their respective WTs (Fig. 3) and bks mutant seeds accumulate free radicals while WT MT did not. In conclusion, the bs1, bs4, and bks mutants do not represent aberrations that are manifest through the same biochemical/physiological processes.
Perturbations in the transcriptome of bs4 seeds: failing to exit late development?
The wide range of ESTs recovered from the subtractive, suppressive PCR libraries and their differential expression between the two genotypes, confirmed hypotheses that the bs4 mutation represents a pleiotropic diversion from the normal developmental program suggested by: (1) the mutation’s effects on seed attributes (size, weight, colour); (2) differences in ROS scavenging enzyme activity; and (3) fruit attributes.
Only one of the differentially expressed transcripts identified as up-regulated in bs4 mutant seed was retrieved from a differential cDNA display (DCD) analysis of gibberellic acid deficient (gib-1) seeds imbibed on water or 100 µM GA4+7 for 40 h (Cooley et al., 1999; KJ Bradford, personal communication). The LeSNF4, the regulatory subunit of the sucrose non-fermenting protein kinase subfamily, was up-regulated in gib-1 seeds on water relative to these seeds on GA4+7 (Bradford et al., 2003) and is up-regulated in bs4 mutants seeds relative to WT MM (data not shown). Other than LeSNF4, the paucity of identical cDNAs between the two analyses might be because the DCD analysis examined seeds during germination while the current study examined seeds that had completed development. Although, in tomato, the two processes of development and germination can be closely associated in time, with no requirement for maturation desiccation and the incidence of precocious germination in the fruit fairly common, the corresponding change in gene expression pattern is thought to be profound (Kermode, 1990; Ogas et al., 1999). In addition, while gib-1 seeds are prevented from timely completion of germination, relative to WT or gib-1 with exogenous GA4+7, by mechanical restraint (Groot and Karssen 1987), bs4 seeds are probably not. The gibberrellic acid biosynthetic pathway is intact in bs4 mutants producing plants of normal stature, and the testa was not significantly tougher than that of WT MM seeds.
To date, no clones encoding a PRX or CAT have been retrieved from the bs4-enriched library. However, sequence data for clones from the bs4 library subtracted by MM (i.e. present in greater abundance in bs4) have identified ESTs (including LeSNF4, Bradford et al., 2003) whose products are associated with late seed development rather than germination (Table 1). The greater expression in quiescent and 24 HAI bs4 relative to WT MM seeds of genes encoding an oleosin (Aalen et al., 1994), storage protein (Bewley and Black, 1994), and a dehydrin (Han et al., 1997) suggest that the bs4 mutant continues in the developmental programme following maturation desiccation. A substantial CAT activity, associated with seed development (Suzuki et al., 1995) and normally transiently repressed during germination (McClung, 1997) supports this contention as does the response of the bs4 seeds to GA4+7, a hormone known to stimulate the germinative program (Cooley et al., 1999; Bradford et al., 2003). A failure to exit the late developmental programme may also explain why the bs4 seeds are larger and heavier (a longer duration/more intense period of seed filling). The darker testa colour in bs4 mutant seeds may be due to the prolonged production of testa precursors. Once the testa dies, these precursors may be trapped in the endosperm and polymerize in the outermost periclinal wall (Downie et al., 2003b). These suppositions are all harmonious with the recessive, Mendelian inheritance of the bs4 lesion. Whether the other bs mutants or the bks mutant also fail to exit the developmental programme is under investigation.
|
Acknowledgements |
|---|
The gibberellic acid (GA4+7) used in these experiments was the kind gift of Abbott Biochemicals, Chicago, IL. The lateral suppressor, brownseed, and anthocyaninless mutant lines were provided by Roger Chetelat from stock maintained at the Charles M Rick Tomato Genetics Resource Center, UC Davis, CA, USA. Janet A Pfeiffer expertly maintained the capillary mat bench, the health of the plants, and the greenhouse facility. Mr David McNertney kindly allowed the use of a Paradigm Seedlot Vigor AssessmentTM system (version 3.2; Paradigm Research Corp, South Haven, MN, USA) for performing and documenting the mutant evaluations. Daryl Slone, Kay Oakley, and Dave Lowrey maintained the plants at the University of Kentucky Horticulture Experimental Farm. Ms Love Gill (UK-Advanced Genetics Technologies Center, AGTC) performed the plasmid preparation, cycle sequencing reactions, clean-up, and analysis. Mr Venu-Gopal Puram (UK–Kentucky Biomedical Research Infrastructure Network, UK–KBRIN, Department of Biology) oversaw sequence quality, vector/adaptor masking, and batch BLAST analysis using the UK-AGTC data pipeline. This work was supported by the Department of Horticulture, University of Kentucky, Hatch funds, a Kentucky NSF EPSCoR grant, and CSREES/USDA Special Research Grant (2003-34457-13114).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Aalen RB, Opsahl-Ferstad HG, Linnestad C, Olsen OA. 1994. Transcripts encoding an oleosin and a dormancy-related protein are present in both the aleurone layer and the embryo of developing barley (Hordeum vulgare L.) seeds. The Plant Journal 5, 385–396.[CrossRef][ISI][Medline]
Atanassova B, Shtereva L, Molle E. 1997a. Effect of three anthocyaninless genes on germination in tomato (Lycopersicon esculentum Mill.). I. Seed germination under optimal conditions. Euphytica 95, 89–98.[CrossRef]
Atanassova B, Shtereva L, Molle E. 1997b. Effect of three anthocyaninless genes on germination in tomato (Lycopersicon esculentum Mill.). II. Seed germination under stress conditions. Euphytica 97, 31–38.[CrossRef]
Atanassova B, Daskalov S, Shtereva L, Balatcheva E. 2001. Anthocyanin mutations improving tomato and pepper tolerance to adverse climatic conditions. Euphytica 120, 357–365.[CrossRef]
Berry T, Bewley JD. 1991. Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement of desiccation. Planta 186, 27–34.
Bewley JD, Black M. 1994. Seeds: physiology of development and germination. New York: Plenum Press, 83–84.
Bradford KJ, Downie AB, Gee OH, Alvarado V, Yang H, Dahal P. 2003. Abscisic acid and gibberellin differentially regulate expression of genes of the SNF1-related kinase complex in tomato seeds. Plant Physiology 132, 1560–1576.
Buta JG, Spaulding DW. 1997. Endogenous levels of phenolics in tomato fruit during growth and maturation. Journal of Plant Growth Regulation 16, 43–46.[CrossRef]
Buxton JW, Jia W. 1999. A controlled water table irrigation system for hydroponic lettuce production. Proceedings of the International Symposium on Growing Media and Hydroponics. Acta Horticulturae 481, 281–298.
Cassab GI, Varner JE. 1987. Immunocytolocalization of extensin in developing soybean seed coats by immunogold-sliver staining and by tissue printing on nitrocellulose paper. Journal of Cell Biology 105, 2581–2588.
Cooley MB, Yang H, Dahal P, Mella RA, Downie AB, Haigh AM, Bradford KJ. 1999. Vacuolar H+-ATPase is expressed in response to gibberellin during tomato seed germination. Plant Physiology 121, 1339–1347.
Debeaujon I, Koornneef M. 2000. Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid. Plant Physiology 122, 415–424.
Debeaujon I, Léon-Kloosterziel KM, Koornneef M. 2000. Influence of the testa on seed dormancy, germination, and longevity in arabidopsis. Plant Physiology 122, 403–414.
Denhardt DT. 1966. A membrane-filter technique for the detection of complementary DNA. Biochemistry and Biophysics Research Communications 23, 641–646.
Diatchenko L, Lau Y-FC, Campbell AP, et al. 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences, USA 93, 6025–6030.
D’Ischia M, Napolitano A, Prota G. 1991. Peroxidase as an alternative to tyrosinase in the oxidative polymerization of 5,6-dihydroxyindoles to melanins. Biochimica et Biophysica Acta 1073, 423–430.[Medline]
Downie B, Gurusinghe S, Dahal P, Thacker RR, Snyder JC, Nonogaki H, Yim K, Fukanaga K, Alvarado V, Bradford KJ. 2003a. Expression of a GALACTINOL SYNTHASE gene in tomato seeds is upregulated prior to maturation desiccation and again following imbibition whenever radicle protrusion is prevented. Plant Physiology 131, 1347–1359.
Downie B, Zhang D, Dirk LMA, Thacker RR, Pfeiffer J, Drake J, Levy A, Butterfield DA, Buxton JW, Snyder JC. 2003b. Communication between the maternal testa and the embryo and/or endosperm affect testa attributes in tomato. Plant Physiology 133, 145–160.
Egley GH, Paul Jr RN, Vaughn KC, Duke SO. 1983. Role of peroxidase in the development of water-impermeable seed coats in Sida spinosa L. Planta 157, 224–232.[CrossRef]
Ellner S. 1986. Germination dimorphisms and parent-offspring conflict in seed germination. Journal of Theoretical Biology 123, 173–185.[CrossRef]
Foolad MR, Chen FQ, Lin GY. 1998. RFLP mapping of QTLs conferring salt tolerance during germination in an interspecific cross of tomato. Theoretical and Applied Genetics 97, 1133–1144.[CrossRef]
Gijzen M. 1997. A deletion mutation at the ep locus causes low seed coat peroxidase activity in soybean. The Plant Journal 12, 991–998.[CrossRef][ISI][Medline]
Gillikin JW, Graham JS. 1991. Purification and developmental analysis of the major anionic peroxidase from the seed coat of Glycine max. Plant Physiology 96, 214–220.
Grabe DF. 1989. Measurement of seed moisture. In: Seed moisture. Crop Science Society of America, Special Publication 14, 69–92.
Groot SPC, Karssen CM. 1987. Gibberellin regulates seed germination in tomato by endosperm weakening: a study with gibberellin-deficient mutants. Planta 171, 525–531.[CrossRef]
Gross GG. 1977. Biosynthesis of lignin and related monomers. Recent Advances in Phytochemistry 11, 141–184.
Guan LM, Scandalios JG. 1995. Developmentally related responses of maize catalase genes to salicylic acid. Proceedings of the National Academy of Sciences, USA 92, 5930–5934.
Han B, Hughes DW, Galau GA, Bewley JD, Kermode AR. 1997. Changes in late-embryogenesis-abundant (LEA) messenger RNAs and dehydrins during maturation and premature drying of Ricinus communis L. seeds. Planta 201, 27–35.[CrossRef][ISI][Medline]
Hendricks SB, Taylorson RB. 1975. Breaking of seed dormancy by catalase inhibition. Proceedings of the National Academy of Sciences, USA 72, 306–309.
Kermode AR. 1990. Regulatory mechanisms involved in the transition from seed development to germination. Critical Reveiws in Plant Science 9, 155–195.
Koornneef M, Karssen CM. 1994. Seed dormancy and germination. In: Meyerowitz EM, Somerville CR, eds. Arabidopsis. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 313–334.
Koornneef M, Bentsink L, Hilhorst H. 2002. Seed dormancy and germination. Current Opinions in Plant Biology 5, 33–36.[CrossRef][ISI][Medline]
Léon-Kloosterziel KM, Keijzer CJ, Koornneef M. 1994. A seed shape mutant in Arabidopsis thaliana that is affected in integument development. The Plant Cell 6, 385–392.[Abstract]
Lozano RM, Wong JH, Yee BC, Peters A, Kobrehel K, Buchanan BB. 1996. New evidence for a role for thior edoxin h in germination and seedling development. Planta 200, 100–106.
Martiniello P, Falavigna A, Soressi GP. 1985. Influence of the tomato Lycopersicon esculentum brown seed gene on plant and fruit characteristics. Genetica Agraria 39, 417–422.
McClung CR. 1997. Regulation of catalases in Arabidopsis. Free Radical Biology and Medicine 23, 489–496.[CrossRef][ISI][Medline]
Mohamed-Yaseen Y, Barringer SA, Splittstoesser WE, Costanza S. 1994. The role of seed coats in seed viability. The Botanical Review 60, 246–260.
Mol J, Grotewold E, Koes R. 1998. How genes paint flowers and seeds. Trends in Plant Science 3, 212–217.[CrossRef][ISI]
Monti LM. 1972. Three ‘brown seed’ mutations induced in tomato by neutron treatments. Report of the Tomato Genetics Cooperative 22, 18.
Ogas J, Kaufmann S, Henderrson J, Somerville C. 1999. PICKLE is a CHD3 chromatin-remodeling factor that regulates the transition from embryonic to vegetative development in Arabidopsis. Proceedings of the National Academy of Sciences, USA 96, 13839–13844.
Philouze J. 1970. A second gene for brown color of tomato seed. Report of the Tomato Genetics Cooperative 20, 44.
Qi MQ, Upadhyaya M, Furness NH, Ellis BE. 1993. Mechanism of seed dormancy in Cynoglossum officinale L. Journal of Plant Physiology 142, 325–330.
Statistical Analysis Systems. 1999. Version 8. Cary, NC, USA: SAS Institute Inc.
Siriwitayawan G, Geneve RL, Downie AB. 2003. Seed germination of ethylene perception mutants of tomato and arabidopsis. Seed Science Research 13, 303–314.[CrossRef]
Soressi GP. 1967. Brown seed (bs), a tomato seed character which behaves as an endosperm trait. Report of the Tomato Genetics Cooperative 17, 50.
Soressi GP. 1972. Allelism test for different brown seed (bs) genes. Report of the Tomato Genetics Cooperative 22, 25–26.
Suzuki M, Miyamoto R, Hattori T, Nakamura K, Asahi T. 1995. Differential regulation of the expression in transgenic tobacco of the gene for beta-glucuronidase under the control of the 5'-upstream regions of two catalase genes from castor bean. Plant and Cell Physiology 36, 273–279.
Taylor IB. 1979. The effect of the lateral suppressor gene on seed germination in tomato. Euphytica 28, 93–97.[CrossRef]
Todd JJ, Vodkin LO. 1993. Pigmented soybean (Glycine max) seed coats accumulate proanthocyanidins during development. Plant Physiology 102, 663–670.[Abstract]
Yordanov M, Stamova L. 1971. A new brown seed mutant. Report of the Tomato Genetics Cooperative 21, 41.
Walker JRL. 1962. Phenolic acids in ‘cloud’ and normal tomato fruit wall tissue. Journal of the Science of Food and Agriculture 13, 363–367.[CrossRef]
Whittington WJ, Fierlinger P. 1972. The genetic control of time to germination in tomato. Annals of Botany 36, 873–880.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Shi, Q. Xu, S. V. Seemanapalli, K. McShan, and F. T. Liang The dbpBA Locus of Borrelia burgdorferi Is Not Essential for Infection of Mice Infect. Immun., November 1, 2006; 74(11): 6509 - 6512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Xu, S. V. Seemanapalli, L. Lomax, K. McShan, X. Li, E. Fikrig, and F. T. Liang Association of Linear Plasmid 28-1 with an Arthritic Phenotype of Borrelia burgdorferi Infect. Immun., November 1, 2005; 73(11): 7208 - 7215. [Abstract] [Full Text] [PDF]
|
|