JXB Advance Access originally published online on June 20, 2005
Journal of Experimental Botany 2005 56(418):2059-2069; doi:10.1093/jxb/eri204
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RESEARCH PAPER |
Identification and characterization of mutants capable of rapid seed germination at 10 °C from activation-tagged lines of Arabidopsis thaliana
Department of Horticulture, 434 Plant Science Building, University of Kentucky, Lexington, KY 40546-0312, USA
To whom correspondence should be addressed. Fax: +1 859 257 7874. E-mail: adownie{at}uky.edu
Received 24 November 2004; Accepted 19 April 2005
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Abstract |
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Five cold temperature germinating (ctg) mutants, completing germination at 10 °C faster than wild type, have been recovered from activation-tagged populations of Arabidopsis thaliana. Three (ctg10-D, 41-D, and 144-D) were tagged and segregated 3:1 for BASTA resistance in the F2 when crossed with wild type. None of the tagged ctg mutants was disturbed in sensitivity to abscisic acid or glucose but all were less sensitive to GA4+7 and osmoticum. The other two mutants (ctg156 and ctg225) were recessive, BASTA sensitive, and exhibited a transparent testa (tt) phenotype. They were more sensitive to abscisic acid, paclobutrazol, and GA4+7 than wild type but had similar sensitivity to osmoticum. Dimethylaminocinnamaldehyde staining of seeds from the two tt mutants, compared with stained seeds from the publicly available tt lines 1–10, suggested that ctg156 was a new allele of tt1, while ctg225 was similar to tt7-1. However, reciprocal crosses determined that ctg156 was not allelic to tt1 while ctg225 was a new allele of tt7. When the gene was sequenced from ctg225 it was missing 10 bp in the second exon, resulting in the incorporation of two spurious amino acids (G282E and D283A) followed by a stop. The screen successfully recovered mutants completing germination faster than wild type at 10 °C.
Key words: Activation tag, mean germination time, seed germination, transparent testa
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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There has been major progress in dissecting the antagonistic roles of abscisic acid (ABA) and gibberellic acid (GA) during germination (Koornneef et al., 1982
; Gomez-Cadenas et al., 2001). The interplay of these two hormones determines the alacrity with which seed germination progresses and even whether it is completed or not in dormant seeds (Koornneef et al., 2002). The influence which other plant hormones (ethylene and brassinolide) have on the ABA/GA antagonism during seed germination is being elucidated (Beaudoin et al., 2000; Ghassmian et al., 2000; Steber and McCourt, 2001; Mora-Garcia et al., 2004) as are the consequences of perturbations in the signal transduction pathways for ABA and GA during this event (Finkelstein et al., 2002). Additionally, the effect environmental factors have on controlling GA synthesis during germination (Ogawa et al., 2003) and dormancy alleviation are being described at the molecular level (Perez-Flores et al., 2003; Yamauchi et al., 2004) as are the roles of repressors of the GA response (Lee et al., 2002), and their selective degradation (McGinnis et al., 2003). However, our understanding of the mechanistic changes brought about by these hormones' actions and influencing radicle protrusion is rudimentary. Our ignorance stems, largely, from the fact that, although seed germination is under genetic control (Foolad and Jones, 1991, 1992), it is a complex trait under the influence of a wide variety of gene products (Foolad et al., 1998; Gallardo et al., 2001) increasing the possibility that it is served redundantly. This frustrates attempts to identify genes that are causally related to the stimulation of radicle protrusion through knockout, although a few notable exceptions exist (Dubreucq et al., 1996; Russell et al., 2000; Leubner-Metzger and Meins, 2001). Furthermore, those few successful screens identifying recessive mutants incapable of completing germination are lethal in the homozygous state unless it is known a priori what metabolic pathway has been affected and how to remedy the deficiency (Koornneef and van der Veen, 1980). Nevertheless, the socioeconomic value of seeds is of such overwhelming importance to modern society that a large effort has been expended to elucidate the underlying mechanisms controlling the completion of germination (Bewley, 1997). This effort has, with few exceptions (Dubreucq et al., 2000, Liu et al., 2005), used knockout mutation and, despite the caveats mentioned above, has begun to increase our understanding of the sophistication of seed germination (for a review, see Koornneef et al., 2002).
An alternative approach to knockout mutation analysis that has been successful in identifying genes involved in processes which are served by redundantly functioning gene products or for which knockouts are lethal at a particular stage of the life cycle is activation tagging (Weigel et al., 2000). Such ‘knock-on’ mutants can result in the hyper-expression of genes that are (over-expression) or are not (ectopic expression) usually expressed in the plant tissue which then provide a recognizable phenotype for the event or process under study. Additionally, the use of T-DNA to deliver the foreign promoters randomly throughout the genome includes the added bonus of generating tagged, insertional knockout mutations in some instances, thereby increasing the scope of the screen to knock-out as well as knock-on mutations.
Seeds from activation-tagged lines of Arabidopsis thaliana were used to identify mutants that were altered in their germination phenotype relative to wild type (WT). By using low temperatures (10 °C) to slow germination it was possible to accentuate differences between mutant and WT (Foolad and Lin, 2001) and select individuals that completed germination ahead of the earliest seeds to do so from a control, WT population. By re-screening populations of the putative mutant progeny and WT using machine vision time to radicle emergence was measured accurately, permitting calculation of an accurate mean germination time (MGT). Hence, it was possible to examine MGT as a measure of the whole population (Still and Bradford, 1997) to determine the validity of the mutant phenotype in the re-screen. This report provides a proof of concept for the use of activation tagging to recover Arabidopsis mutants completing germination at 10 °C prior to seeds of WT.
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Materials and methods |
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Mutant screen
Cold temperature germinating (ctg) mutant screens were conducted in a custom-built germinator (Hoffman Manufacturing Inc., Albany, OR, USA) at 10 ±1 °C on seeds from approximately 2000 M4 Arabidopsis thaliana activation-tagged lines (Weigel et al., 2000
) per experiment. Seeds were sown without sterilization on a disc of black filter paper on top of two discs of water-saturated germination blotter (both from Stults Scientific Engineering, Springfield, IL, USA) in Petri dishes (15 mm deep, 100 mm diameter; Fisher Scientific, Springfield, NJ, USA). Germination conditions always used constant light (135 µmol m–2 s–1). The base temperature for Arabidopsis seed germination is close to 0 °C and, using this base, the thermal time necessary for the completion of germination for the fastest WT Columbia seeds in a population was 50 degree days (DD; data not shown). Four days after the commencement of the test, seeds were examined under a dissecting scope for white radicles against the black filter paper. Any seed that had completed germination was considered a putative ctg mutant and transferred to soil in individual pots, and grown to maturity. WT Columbia was also planted at this time, selected from those seeds that had successfully completed germination from pools that had been placed in the germinator 1 d prior to the commencement of the mutant screen. Control seeds were selected in this manner to ensure the mutant phenotype was not due to directional selection.
Re-screening was performed using seeds from the putative ctg mutant and WT Columbia that had been grown at the same time, under the same conditions, and harvested on the same day. Five ctg mutants were confirmed in the re-test. Seeds from mutants ctg10, ctg41, and ctg144 were surface sterilized, along with the WT seeds against which they were to be tested. Seeds from mutants ctg156 and ctg225, exhibiting a transparent testa (tt) phenotype, were very susceptible to deterioration during surface sterilization (data not shown) and so were tested, along with WT seeds, without such treatment. Approximately 50 seeds of each mutant were sown on 1% (w/v) agarose solid media on one side of a line across a Petri dish (15 mm deep, 60 mm diameter; Sarstedt, Inc., Newton, NC, USA) and 50 seeds of the WT sown on the other side of the plate. The plate was sealed with 3M Micropore surgical tape (3M Consumer Health Care, St Paul, MN, USA) and four dishes (replications) per mutant line were arranged side-by-side on a flat bed scanner inside a germinator maintaining a constant 10±1 °C. A Visual Basic macro (SigmaScan Pro 5.0 for Windows; SPPC Science, Chicago, IL, USA), controlled the flat bed scanner permitting scanning to proceed automatically every hour. The resulting TIFF files (600 dpi), with time signature, were stored on the hard-drive. Seed germination time-courses were reconstituted from these data, graphed, and the MGT (in hours) calculated (Bewley and Black, 1994).
Segregation analysis, seed staining, and allelism tests
Each ctg mutant was crossed with WT Columbia, F1 plants were selfed, and F2 seeds surface sterilized (except for transparent testa mutants) and sown on BASTA-containing media [4.3 g MS basal salts (Sigma), 2.6 mM MES/KOH pH 5.6, 0.7% (w/v) agar, 10 µg ml–1 gluphosinate ammonium (Crescent Chemical Co., Inc., Hauppague, NY, USA)]. The ratio of resistant to sensitive seedlings was determined as a measure of the number of complete T-DNA insertions present in each of the mutant plants. Resistant seedlings were recovered from the media, planted in soil, single plants grown to maturity and approximately 50 seeds from each plant tested again for complete BASTA resistance. Seedlings were selected from plates in which all seedlings were BASTA resistant (homozygous for the transgene), planted in soil, and grown to maturity. Seeds from these homozygotes were tested for rapid completion of germination in comparison to WT Columbia.
To determined the penetrability of the testa, approximately 50 seeds of each genotype were stained at 25 °C with 1% (w/v) 2,3,5-triphenyltetrazolium chloride (Sigma) in water for 24 h in the dark. The tt mutants (ctg156 and ctg225) were also stained with 1% (w/v) vanillin (Sigma) in 6 N HCl for 3 h to test for the presence of proanthocyanidins, or with 0.03% (w/v) ruthenium red (Sigma) in water for 30 min at 25 °C to test for aberrant mucilage production (Debeaujon et al., 2000).
All publicly available tt lines (1–10) were acquired from the Arabidopsis Biological Resource Center. Seeds from each line were sown along with ctg156 and ctg225 and seedlings grown at the same time under identical conditions and harvested on the same day. Thereafter, approximately 50 seeds from each line were stained for 3 d in the dark, along with WT Columbia, with 1% p-dimethylaminocinnamaldehyde (DMACA) as described in Abrahams et al. (2002). Seeds were then washed in 70% ethanol and photographed using a Stemi SV11 dissecting scope (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA) attached to an AxioCam MRc5 digital camera (Carl Zeiss MicroImaging, Inc.) and AxioVision 40 V 4.1.1.0
[EC]
software (Carl Zeiss MicroImaging, Inc.). Based on the pattern of staining of the ctg mutants, they were crossed with WT Columbia and with the respective tt mutant, whose staining pattern they most closely resembled.
Seed and seedling attributes
Seed and seedling size were measured from high resolution (600 dpi) images of mature seeds or seedlings 8 d after imbibition (DAI). The images were acquired on a flat bed scanner with transparency adaptor engaged to eliminate shadows. Images and a paper square of known dimensions (standard) were scanned and saved as greyscale TIFF files. Images were opened in SigmaScan Pro 5.0 for Windows (SPPC Science, Chicago, IL, USA) and the threshold function used to select the pixels comprising each seed's (seedling's) image and that of the paper standard. These were then enumerated using the measure function of the same software, sorted in descending order and exported to Excel (Microsoft Corporation, Redmond, WA, USA). Excel values were converted to square millimetres relative to the pixel count of the standard and imported into SAS (Statistical Analysis Systems V8; SAS Institute, 1988) where they were analysed for significant differences (see below). The 1000 seed weight for each genotype was calculated from four replications of 50 seeds each. The average length of the seedling shoot and root 15 DAI were obtained from 20 seedlings measured using a batch counting chamber (Hausser Scientific, Blue Bell, PA, USA) under a dissecting microscope.
Hormonal sensitivity, glucose sensitivity, and imbibition speed
In all instances, three replications of 50 mutant and WT Columbia seeds each were used in the germination tests described below. Seeds were sown on 1% (w/v) agarose containing a range of concentrations of cis-trans abscisic acid (Sigma) (0, 0.5, 1.0, 2.0, 5.0, and 10 µM). In another test, seeds were sown on germination blotter (Stults Scientific Engineering) soaked in the GA biosynthetic inhibitor paclobutrazol (Yamaguchi and Kamiya, 2002) at 0, 5, 10, 20, 50, and 100 µM concentrations. Seeds were also sown on germination blotter soaked in ABA but only those on 0 and 0.5 µM ABA completed germination to any extent after 1 month. In a separate experiment, WT and mutant seeds were sown on germination blotter soaked in 100 µM paclobutrazol and varying concentrations of GA4+7 (0, 0.01, 0.1, 1.0, 10, and 100 µM; Debeaujon and Koornneef, 2000). Percentage germination was assessed at 7 and 14 DAI at 25 °C.
To ascertain whether the ctg phenotype was conferred upon the mutants due to perturbations in carbohydrate catabolism, seeds were sown on different concentrations of glucose [0, 2, 4, and 7% (w/v)] in 1% (w/v) agarose and monitored for seeds that could complete germination and subsequently become chlorophyllous (Zhou et al., 1998; Arenas-Huertero et al., 2000). To determine imbibition speed, three replications of 50 seeds each were weighed before being sown on water-saturated filter paper and allowed to imbibe at 10 °C. Twelve hours after imbibition, seeds were removed from the filter paper, blotted dry, and re-weighed. The relative increase in weight was calculated and converted to a percentage increase over initial weight. This value was considered as a measure of the amount of water taken up by the seed in the initial 12 h of imbibition.
DNA isolation and analysis
Genomic DNA was isolated from young rosette leaves of mutant and WT Columbia using a kit (DNAEasy; Qiagen Corp., Valencia, CA, USA). Gene-specific primers amplifying 1000 bp of the promoter region of TT1 (At1g34790, encoding a WIP ZINC FINGER; Sagasser et al., 2002) or TT7 (At5g07990, encoding a P450 with a FLAVONOID 3'-HYDROXYLASE activity; Schoenbohm et al., 2000), as well as the respective genes, were used with genomic DNA from ctg156 or ctg225, respectively. Primers amplifying 1750 bp of the CaMV35S quadruple promoter array at the right border or 585 bp of a ß-TUBULIN gene (Table 1) were used with genomic DNA from each mutant. Amplification used a proof-reading enzyme that still permitted T/A cloning (Easy-A; Stratagene La Jolla, CA, USA). Amplicons from the TT1 and TT7 promoter and gene were cloned into a T/A cloning vector, transformed into Escherichia coli, purified, and both strands were sequenced. The presence or absence of an amplicon encompassing the CaMV35S array from mutant genomic DNA was used to ascertain whether the right border of the activation tag T-DNA had transferred to the mutants.
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Sequence analysis
Sequencing was performed at the Advanced Genetics Technologies Center (University of Kentucky, Lexington, KY, USA) using a Beckman Coulter CEQ 8000, eight capillary electrophoresis genetic analysis system. Cycle-sequencing reactions employed a combination of universal and gene-specific primers (Integrated DNA Technologies, Inc., Coralville, IA, USA). Sequences were viewed and assembled using Sequencer V4.0 (Gene Codes Corp., Madison, WI, USA).
Statistical analysis
All comparisons were performed between a particular mutant and WT Columbia, because the purpose of this work was verification of mutant phenotypes relative to WT. Seed and seedling parameters, relative increases in moisture content during imbibition, differences in percentage germination at representative time points after imbibition, and MGT (both assessed at 10 and 25 °C) were subjected to analysis of variance using the ANOVA procedure of SAS (SAS Institute, 1988). If the ANOVA indicated that there were significant differences among means, Dunnett's mean separation test was used to distinguish between WT Columbia and each mutant at 
=0.05. Dunnett's test controls the experiment-wise error for comparisons of all mutants against the control.
To ascertain whether there were multiple, independent, complete T-DNA cassette insertions present in the mutants, an analysis of segregation ratios of BASTA resistance among F2 progeny from a cross between the mutants and WT Columbia was conducted. A chi-square analysis of goodness-of-fit to a 3:1 segregation ratio for a single resistance gene was calculated using Excel and a single degree of freedom.
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Results |
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Using the ctg screen, 52 000 independent lines were examined and five mutants recovered in the re-screen that completed germination faster than the WT at 10 °C. Seedlings of ctg10, ctg41, and ctg144 were BASTA resistant whereas ctg156 and ctg225 were not. Plants from the three resistant lines were crossed with WT and the F2 progeny exhibited BASTA resistance segregating in a three resistant to one sensitive ratio consistent with the acquisition of a single (or several tightly linked) dominant BASTA-resistance gene residing at the transferred DNA left border from the pSKI015 binary vector (Weigel et al., 2000
). Henceforth, these mutants were designated with the suffix ‘D’ to indicate that they are dominant for the resistance gene (Table 2). Progeny from these plants were selected for complete BASTA resistance (homozygous for the transgene) and re-tested for speed of germination at 10 °C. The ctg10-D, ctg41-D, and ctg144-D mutants all had statistically significantly greater percentage germination than WT at 10 °C at 144 h after imbibition (HAI) (Fig. 1). ctg144-D had statistically greater percentage germination than WT from 132 to 180 HAI (Fig. 1). As a result, the three dominant mutants had statistically significantly shorter MGTs at 10 °C than WT (Fig. 1, upper panel). The recessive mutants ctg156 and ctg225 also commenced radicle protrusion ahead of WT (at 120 HAI ctg156=1.5±0.5, ctg225=1.0±0.6, and WT=0% germination; at 132 HAI ctg156=28.0±3.5, ctg225=4.0±1.4, and WT=0.5±0.5% germination), although only ctg156 was statistically significantly different and ctg225 was soon surpassed by WT (Fig. 1, lower panel). The ctg156 germination percentage was superior to that of WT from 132 to 144 HAI and the ctg156 MGT was statistically significantly shorter (Fig. 1). However, the poor germination performance of the population of ctg225 mutant seeds resulted in a statistically significantly longer MGT than WT despite the fact that some ctg225 seeds commenced radicle protrusion earlier (Fig. 1 and text above).
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At 25 °C, only the percentage germination of ctg10-D and ctg144-D from 30–36 HAI were statistically superior to WT (Fig. 2, upper panel). The percentage germination of ctg41-D was indistinguishable from that of WT while both ctg156 and ctg225 were statistically inferior to WT (Fig. 2). At 25 °C, there was insufficient resolution of the germination rates to permit statistically significant differences in MGT to register for the dominant mutants, while the recessive mutants (ctg156 and ctg225) had statistically significantly longer MGTs than WT (Fig. 2, lower panel).
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Once faster completion of germination at 10 °C (in some instances at 25 °C) had been established, additional attributes of the mutants were catalogued. ctg10-D produced heavier, larger seeds than WT (Table 3). Both ctg156 and ctg225 had significantly lighter seeds than WT although the seed size was not significantly altered (Table 3). Eight days after imbibition at 25 °C, seedling size was significantly greater for ctg10-D and ctg41-D and significantly smaller for ctg156 and ctg225, relative to WT (Table 3). In a separate experiment, the shoot and root lengths of seedlings 15 DAI at 10 °C were longer than those of WT for all the dominant mutants (Table 3). The increases were, however, proportional so that there were no significant differences in the root:shoot ratio between any dominant mutant and WT (Table 3). The recessive mutants were comparable in size to WT, except for shorter shoot length for ctg225, which led to a lower root:shoot ratio for this mutant relative to WT (Table 3).
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Each mutant line was tested for sensitivity to ABA. None of the dominant mutants differed in ABA response relative to WT, while the ctg156 and ctg225 mutants were hypersensitive to supplied ABA over a range of concentrations (Fig. 3). Another possible explanation for the faster completion of seed germination exhibited by the mutants is that they were hypersensitive to GA. However, the dominant mutants exhibited less rather than more sensitivity to GA4+7 when simultaneously imbibed on 100 µM of the GA biosynthetic inhibitor, paclobutrazol (Fig. 4). However, this difference was only at a single GA4+7 concentration and was not observed when seeds were sown on varying concentrations of paclobutrazol (Fig. 5A). The tt mutants were hypersensitive to exogenous GA when simultaneously imbibed on 100 µM paclobutrazol (Fig. 4), but were also hypersensitive to paclobutrazol over a range of concentrations (Fig. 5A). None of the mutants was resistant to glucose supplied in the media at up to 7% (w/v) (data not shown).
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It was possible that the mutant seeds were capable of imbibing water faster than WT at 10 °C leading to faster rehydration and commencement of germination. The mutants ctg41-D, ctg144-D, and ctg225 were all capable of significantly faster water uptake than control WT seeds (Fig. 5B) but, unlike in the tt mutants, this was not due to enhanced testa permeability to tetrazolium salts (Fig. 6A).
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The dominant mutant capacity to imbibe water faster than WT translated into an enhanced capacity to complete germination on media of greater osmotic potential than WT (Fig. 6B). The recessive tt mutants were not significantly different from WT in this respect (Fig. 6B).
Analysing the tt mutant seeds for tannins using DMACA, results in distinguishable, mutant-specific staining patterns (Abrahams et al., 2002). The two recessive mutants (ctg156 and ctg225) and the publicly available transparent testa lines tt1 through tt10 were stained, along with WT Columbia. Seeds were cleared and compared with unstained seeds using a dissecting scope (Fig. 7). Similar staining was noted for seeds of the ctg156 mutant and tt1 (Fig. 7E, H) and for seeds of the ctg225 mutant and tt7 (Fig. 7F, I). Plants for ctg156, ctg225, tt1, tt7, and WT Columbia were grown and reciprocal crosses made. F1 seeds were sown and F2 seeds examined for complementation. Crosses between ctg156 and tt1 complemented (Fig. 7K) while crosses using ctg225 failed to complement the tt7 mutant (Fig. 7L).
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Examination of the ctg156 and the ctg225 mutant BASTA resistance (Table 2), testa appearance of F2 seeds following a cross with WT (Fig. 7G, J), and lack of an amplicon using pSKI015 right border specific primers (Table 1 and Fig. 8A), suggested that the activation tag T-DNA had simply disrupted the genes responsible for, or controlling, anthocyanin production and condensed tannin accumulation without tagging the mutagenic site with any portion of the T-DNA. Following up on the complementation analysis between ctg225 and tt7, when the TT7 gene was amplified from ctg225 and sequenced, it was missing 10 bp in the second exon (Fig. 8B). This deletion resulted in the loss of four amino acids, a frame shift introducing two spurious amino acids, before culminating in a premature stop (Fig. 8B). The TT1 gene was amplified, cloned, and sequenced from ctg156 mutant prior to the results from the complementation test becoming available. Although the TT1 gene was not the cause of the transparent testa phenotype in ctg156, the intron was missing 2 bp relative to the sequence present in Genbank. Both the 10 bp deletion resulting in tt7-3 and the 2 bp deletion in TT1 were catalogued as polymorphisms in the dbSNP (NCBI_ss# ctg225 28515374 and NCBI_ss# ctg156 28515375, respectively). Seed stocks of both mutants (CS6509: ctg225/tt7-3; CS6510: ctg156) have been released to the Arabidopsis Biological Resource Center, the Ohio State University, Columbus, OH, USA.
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Discussion |
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Prior to screening the activation-tagged lines, two mutants potentially capable of germination faster than WT at cold temperatures were tested. First, reports suggested that some point mutations in acetolactate synthase (als) also serendipitously resulted in faster than usual completion of germination under sub-optimal temperatures (Gasquez et al., 1981
). This is thought to be due to abnormally early commencement of the cell cycle during germination through branched amino acid accumulation and/or precursor depletion (Rost, 1984; Rost and Reynolds, 1985; Robbins and Rost, 1987). The cold temperature germinating phenotype of the als mutant is possibly related to this stimulation (Dyer et al., 1993). Under the present conditions, als mutant seeds (obtained from the Arabidopsis Biological Resource Center; Haughn and Somerville, 1986) did not exhibit an enhancement of germination speed at 10 °C. Secondly, the ABA/GA antagonism discussed in the Introduction suggested that ABA-deficient or -insensitive seeds might be recovered in the screen. Seeds of the ABA-deficient mutant, aba1, were tested but did not complete germination faster than WT at 10 °C. Hence, mutants unresponsive to, or deficient in, ABA would not be selected in the ctg screen.
The cold temperature germinating (ctg) screen was a positive screen for mutants capable of radicle protrusion faster than WT at 10 °C. The 10 °C screening temperature was a compromise that permitted discrimination between potentially subtle differences in the time-to-radicle protrusion (Foolad and Lin, 2001) while still allowing high throughput. The test conditions also alleviated dormancy (not a serious impediment in the Columbia ecotype) through moist chilling which can occur at 10 °C in some species requiring this treatment (Downie et al., 1998), including the A. thaliana ecotype Columbia (RK Kar and AB Downie, unpublished observation). Previous work screening populations of EMS mutagenized lines (unsuccessful) had determined that the base temperature (Gummerson, 1986) for Arabidopsis germination is close to 0 °C and, using this base, the thermal time necessary for the completion of germination for the fastest WT Columbia seeds in a population was 50 DD; data not shown). This value correlates well with data collected for this ecotype at 3 °C (17 d to commencement of protrusion, or 51 DD) but not with published accounts (Miquel and Browse, 1994; 15 d at 6 °C or 90 DD). Nor was 50 DD for WT Columbia seed germination consistent among the various generations of WT seeds assessed in the various germination tests used to evaluate the mutant phenotype (Figs 1 and 2). It is well known that the environment in which the maternal plant develops and sets seed plays an important role in the germination attributes of the progeny (Miquel and Browse, 1994; Debeaujon et al., 2000; Munir et al., 2001) and could be one reason for the observed discrepancy.
Two of the five ctg mutants that completed germination faster than WT at 10 °C in the primary screen exhibited a transparent testa (tt) phenotype (Fig. 7A–C). The anthocyaninless mutants of tomato, some of which are known to be homologues of Arabidopsis tt mutants (anthocyanin without and transparent testa3 both encode DIHYDROFLAVONOL 4-REDUCTASE; Shirley et al., 1992; Goldsbrough et al., 1994), produce seeds with a lighter than usual testa colour. This has been attributed to a paucity of condensed tannins in the testa of at least three of the anthocyaninless mutants (Atanassova et al., 2004). These anthocyaninless mutant seeds can also complete germination under stressful conditions (including cold temperatures) faster than WT (Atanassova et al., 1997a, b, 2001). The weaker testa presents less of a barrier to imbibition (Debeaujon and Koornneef, 2000; Fig. 5B) and to radicle protrusion and hence these mutants can complete germination faster than the WT under abiotic stress (Debeaujon and Koorneef, 2000). The greater permeability of the mutant testa to exogenous chemicals (Fig. 6A) resulted in hypersensitivity to ABA and paclobutrazol but also increased sensitivity to exogenous GA (Debeaujon and Koornneef, 2000; this report). The greater testa permeability also made the tt mutants particularly susceptible to damage during surface sterilization (data not shown). In addition, the smaller seed and seedling size (Table 3) and greater testa permeability (Fig. 6A) negatively affected seed vigour (Debeaujon et al., 2000) such that both tt mutants completed germination slower, to a lower percentage, and with less uniformity than WT Columbia at 25 °C (Fig. 2). Despite these negative attributes, under cold- or osmotic-stress (Figs 1 and 6B, respectively) the speed of germination of ctg156, compared with WT, improved considerably. The population of ctg225 mutant seeds under cold stress had individuals that commenced radicle protrusion ahead of WT (see Results) but the bulk of the population were slower to complete germination (Fig. 1). Nevertheless, comparing the time-course of germination of ctg225 mutant seeds to WT with (Figs 1 and 6B) or without (Fig. 2) stress, ctg225 seeds were most similar to WT under stress.
ctg225 was allelic to tt7, a mutation in FLAVONOID 3'-HYDROXLASE (Schoenbohm et al., 2000). Such an untagged mutant (tt7-3) has been reported previously from the Weigel activation-tagged lines (Abrahams et al., 2002), but no information concerning the nature of the mutation was provided. It is suspected that in the present research the same mutant has been recovered and it has been determined that it is due to a 10 bp deletion in the second exon resulting in a premature stop (Fig. 8B) and it has been catalogued in the dbSNP accordingly. The hypersensitivity to exogenous ABA and GA, relative to WT, was similar between tt7-1 and tt7-3 (Figs 3B and 4B). Although there was an apparent resemblance between ctg156 and tt1 based on testa staining with DMACA, complementation tests of F2 progeny from reciprocal crosses proved that the ctg156 mutant was not allelic with any of the publicly available tt mutants.
In accordance with pre-screens of ABA-deficient mutant seeds, none of the dominant mutants isolated in the screen was insensitive to ABA (Fig. 3). Neither were the dominant ctg mutants overly sensitive to GA, rather exhibiting somewhat less sensitivity on 100 µM paclobutrazol plus increasing concentrations of GA4+7 relative to WT (Fig. 4).
It is exciting that neither the ABA nor the GA sensitivity of the mutant seeds was less or greater, respectively, relative to WT. Presumably, whatever metabolic pathways have been altered as a result of the insertions are divorced from those associated with the classical GA/ABA conflict known to influence radicle protrusion (Gomez-Cadenas et al., 2001).
In accordance with results from selections of tomato lines with faster completion of germination at cold temperature (Foolad and Lin, 1999), two of the three ctg-D mutants that completed germination faster than WT at 10 °C were also able to do so at 25 °C. Furthermore, all dominant mutants completed germination to a greater percentage than WT on media of negative osmotic potential (–0.25 MPa; Fig. 6B). Considerable evidence exists indicating that physiological processes resulting in faster completion of germination under one set of stress conditions results in faster completion of germination when seeds are unstressed (Scott and Jones, 1982) or are stressed by seemingly divorced agents (Foolad et al., 2003). The presence of a single, complete T-DNA insert in each of the dominant mutants, based on the segregation ratio of BASTA resistance (Table 2), the presence of the CaMV35S promoter array (Fig. 8A), and the recovery of faster germination speed at 10 °C in F2 BASTA-resistant plants following a cross to WT Columbia strongly suggests that the mutations are due to hyper-expression of a gene(s) that is(are) capable of hastening seed germination to its culmination.
Based on additional phenotypic attributes shared by the three dominant mutants (greater shoot and root lengths and faster imbibition for two of the three) it is possible that the cells of the three mutants are predisposed to elongate. This would be a general physiological mechanism, controlled or served by the same set of gene products, used to explain why selection for rapid germination speed under one stress leads to rapid germination speed under other, unrelated stresses (Foolad et al., 2003). Efforts are underway to walk to the regions tagged by the T-DNA insertions.
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This research was supported by a pilot project and a full research grant to ABD from the Kentucky Tobacco Research and Development Center. The mutant screen is part of a project conducted by Mr Louai Salaita performed in partial fulfillment of the requirements for the Undergraduate Agricultural Biotechnology Program, University of Kentucky, Lexington, KY, 40546-0312, USA. The activation-tagged lines, transparent testa mutants, tt1 to tt10, and the Columbia WT were all acquired from The Arabidopsis Biological Resource Center, The Ohio State University, Columbus, OH, USA. Dr Robert Geneve and Mr Manjul Dutt developed the software to run the flatbed scanner. Dr Sharyn Perry kindly permitted the use of her dissecting scope, digital camera, computer, and paclobutrazol. Thanks to Dr Hiro Nonogaki, Oregon State University, for making available some unpublished research findings. An anonymous reviewer provided many suggestions for the improvement of this manuscript. This article (04-11-152) is published with the approval of the Director of the Kentucky Agricultural Experiment Station and has benefited from comments by members of the University of Kentucky, Seed Biology Program.
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* Present address: College of Medicine, Department of Physiology, University of Arizona, Ina Gittings Room 101, PO Box 210093, Tucson, AZ 85721, USA.
Present address: Department of Botany, Visva-Bharati University, Santiniketan 731 235 West Bengal, India.
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