JXB Advance Access originally published online on February 3, 2008
Journal of Experimental Botany 2008 59(3):585-593; doi:10.1093/jxb/erm354
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
procera is a putative DELLA mutant in tomato (Solanum lycopersicum): effects on the seed and vegetative plant
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
* Present address and to whom correspondence should be sent: Department of Cell and Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2. E-mail: gbassel{at}csb.utoronto.ca
Received 12 October 2007; Revised 27 November 2007 Accepted 28 November 2007
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionConclusionReferences |
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The procera (pro) mutant of tomato exhibits a well-characterized constitutive gibberellic acid (GA) response phenotype. The tomato DELLA gene LeGAI in the pro mutant background contains a point mutation that results in an amino acid change in the conserved VHVID putative DNA-binding domain in LeGAI to VHEID. This same point mutation is in four different genetic backgrounds exhibiting the pro phenotype, suggesting that this mutation co-segregates with the pro phenotype. Complementation of the mutant with a constitutively expressed wild-type LeGAI gene sequence was not conclusive due to the infertility of transgenic plants. The pro mutation alters tomato branching architecture through differential suppression of axillary bud development, indicating a role for DELLA proteins in the regulation of plant structure. Isolated gib-1 pro double mutant embryo axes, which are unable to synthesize GA, germinate faster than their wild-type counterparts, and exert greater embryo growth potential. The pro mutation is therefore regulating GA responses within the tomato embryo. Transient expression of a LeGAI–GFP (green fluorescent protein) fusion protein in onion epidermis results in its location to the nucleus, and this protein is rapidly degraded by the proteasome in the presence of GA.
Key words: Branching pattern, DELLA, embryo growth potential, tomato seed germination
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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The hormone gibberellic acid (GA) mediates diverse developmental processes in plants, including stem elongation, leaf expansion, pollen development, flower and seed development, and seed germination (Olszewski et al., 2002; Cheng et al., 2004). Genes involved in the synthesis of GA have been discovered through the identification of dwarf mutant plants that are restored to the wild-type phenotype following GA application (Koornneef and van der Veen, 1980). Another class of GA mutant plants produce this hormone, but are insensitive to it and inhibitors of its synthesis (Olszewski et al., 2002). These mutations represent aberrations in genes encoding the GA response pathway.
Recessive mutations in a negatively acting GA signalling component produce a constitutive GA response, yielding tall, elongated plants that are insensitive to dwarfing by the GA synthesis inhibitor paclobutrazol (PAC) (Peng et al., 1999; Olszeweski et al., 2002). Recessive mutations in positively acting signalling components yield dwarf plants with compact dark green leaves, and fail to elongate when GA is applied (McGinnis et al., 2003; Sasaki et al., 2003; Dill et al., 2004).
Plants with increased stature are associated with recessive mutations in negatively acting GA response regulators. Such constitutive GA response mutants have been isolated from Arabidopsis gai (ga-insensitive) (Peng et al., 1997) and rga (repressor of ga1-3) (Silverstone et al., 1998), from barley sln1 (slender1) (Lanahan and Ho, 1988), and from rice slr1 (slender1) (Ikeda et al., 2001). These genes all code for DELLA proteins, nuclear-localized repressors of GA action that are central in the regulation of this hormonal response (Dill et al., 2001; Itoh et al., 2002; Alvey and Harberd, 2005). Another inhibitory protein in the GA response pathway is SPINDLY (SPY) (Jacobsen and Olszewski, 1993). The SPY gene encodes a serine and threonine O-linked N-acetylglucosamine transferase that is proposed to enhance DELLA action through its enzymatic activity (Silverstone et al., 2007).
GA exerts a profound influence over the stimulation of seed germination (Bewley, 1997; Kucera et al., 2005; Finch-Savage and Leubner-Metzger, 2006), and components of its signal transduction pathway have been suggested to act as regulators of this process (Lee et al., 2002; Peng and Harberd, 2002; Bassel et al., 2004). For instance, the Arabidopsis DELLA protein RGL2 has been proposed to be a repressor of germination in this species, since the absence of this protein confers GA-independent germination (Lee et al., 2002; Bassel et al., 2004). A comparable study, however, has not been carried out in a model species for the physiological study of seed germination.
In tomato, a monogenic recessive constitutive GA response mutant named procera (pro) has been identified (Stubbe, 1957; Jones, 1987; Jupe et al., 1988; Van Tuinen et al., 1999). procera is Latin for noble, perhaps in reference to the increased height of the pro mutant relative to the wild type (Stubbe, 1957; Jones, 1987). The pro mutant mimics GA-treated tomato with both elongated and increased number of internodes, thinner leaves, and reduced lobing of the main leaflets (Jones, 1987). The pro mutation also phenocopies GA-treated plants at the cellular level in terms of decreased peroxidase activity (Jupe and Scott, 1992) and response to fusicoccin (Woodhead et al., 1997). As is the case in other constitutive GA response mutants, pro contains reduced concentrations of GAs despite its constant response to this hormone (Jupe et al., 1988; Van Tuinen et al., 1999).
The elongated internodes of the pro mutant can further lengthen following GA application, indicating that the constitutive GA response conferred by the pro mutation is not saturated (Jupe et al., 1988; Van Tuinen et al., 1999). Null DELLA gene mutants sln1 in barley (Lanaham and Ho, 1988) and slr1 in rice (Ikeda et al., 2001) exhibit a saturated GA response phenotype, and do not respond when GA is applied. Barley and rice each have only one DELLA gene, suggesting that GA responses leading to their growth are all regulated by their single DELLA protein. The Arabidopsis genome encodes five DELLA genes, and stem elongation is controlled by the concerted action of two of these, RGA and GAI. Consequently, plants carrying a mutation in either gai or rga are capable of additional stem elongation in the presence of GA (Dill and Sun, 2001). Similar to pro, the spy phenotype in Arabidopsis does not exhibit a saturated GA response (Jacobsen and Olszewski, 1993). The SPY gene from tomato (LeSPY) was isolated from the wild type and the pro mutant to investigate whether mutations in this gene are responsible for the non-saturated constitutive GA response phenotype in pro (Greb et al., 2002). It was concluded that no obvious mutation in the LeSPY gene sequence leads to the production of a non-functional protein. LeSPY was mapped to chromosome 9 (Greb et al., 2002), whereas the pro mutation maps to chromosome 11 (Van Tuinen et al., 1998), further supporting the hypothesis that pro does not represent a mutation in LeSPY.
In this study, the hypothesis that pro carries a mutation in the DELLA gene of tomato (LeGAI) was tested. The effect of this mutation on branching architecture was examined, as well as its effect on seed germination; a model species for the study of this process. In addition, the post-translational stability of LeGAI fused to green fluorescent protein (GFP) was followed in a transient transformation system. This was carried out to determine whether the proteasome degrades this protein in the presence of GA.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Plant material and germination conditions
Tomato (Solanum lycopersicum L. cv. ‘Ailsa Craig’) seeds were obtained from the C. M. Rick Tomato Genetics Resource Center (Davis, CA) (accession LA2838A), as well as the four mutant lines exhibiting the pro phenotype. The pro accession numbers and genetic backgrounds are: LA3283 cv. Ailsa Craig (Van Tuninen et al., 1999), LA0565 cv. Condine Red (Stubbe, 1957), and LA0803 and LA0730 both of unknown backgrounds. The LA3283 accession was used in these analyses in conjunction with the cv. Ailsa Craig. gib-1 mutant tomato seeds cv. Moneymaker and abscisic acid (ABA)-deficient sitiens seeds cv. Rheinlands Ruhm were obtained from Dr Henk Hilhorst (Wageningen University, The Netherlands; Koornneef et al., 1990). Double mutant gib-1 pro seeds were obtained from Dr Maarten Koornneef (Wageningen University, The Netherlands; Van Tuinen et al., 1999).
Tomato seeds, intact except for having their micropylar endosperm removed, were imbibed in 90 mm diameter plastic Petri dishes (Fisher Scientific, Ottawa, ON, Canada) on two layers of Whatman No. 1 filter paper (Fisher) with 
5 ml of double-deionized water (ddH2O) at 25 °C. Polyethylene glycol 8000 (PEG) at an osmotic strength of –0.5 MPa at 25 °C was prepared as a 22% (w/v) solution (Chen et al., 2001) and used at this concentration and temperature. Petri dishes were sealed with Parafilm (Fisher) and placed in the dark at 25 °C.
Tomato axes were excised with a scalpel following 4 h imbibition on water, as described above. Germination of an axis was scored as complete when the radicle began to show cell expansion; that is, the observation of radicle curvature.
RNA extraction, reverse transcription, and PCR
RNA from the second fully expanded true leaf of tomato seedlings was isolated using the RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada). Reverse transcriptase reactions were performed using 5 µg of total RNA along with 1 µl of oligo(dT)18 primer (0.5 µg µl–1), with the volume adjusted to 11 µl with RNase-free water. This mixture was heated at 70 °C for 5 min and then chilled on ice for 5 min. A 4 µl aliquot of reverse transcriptase 5x reaction buffer [250 mM TRIS-HCl pH 8.3, 250 mM KCl, 20 mM MgCl2, 50 mM dithiothreitol (DTT)] (Fermentas, Burlington, ON, Canada), 1 µl of ribonuclease inhibitor (20 U µl–1) (Fermentas), 2 µl of 10 mM dNTPs, and 2 µl of M-MuLV reverse transcriptase (20 U µl–1) (Fermentas) were mixed together by pipetting and incubated at 42 °C for 1 h. A 5 µl aliquot of the reverse transcriptase reaction mixture was used in a PCR consisting of the following reagents: 1 µl each of 50 pmol µl–1 forward and reverse primer, 1 µl of 10 mM dNTP mix, 5 µl of 10x reaction buffer (Fermentas), 3 µl of 25 mM MgCl2, and 1.5 U of Taq polymerase (Fermentas). Primers used for semi-quantitative PCR to examine LeGAI expression were LeGAI-fwd 5'-CCCGAGTTTACAACTCGACTTCTCC-3' and LeGAI-rev 5'-CCAGCACTTGTCATTCTTACCCAATC-3'. Primers to amplify ubiquitin were Ubi-fwd 5'-TCCAAAAAGAGTCTACCCTTCATC-3' and Ubi-rev 5'-CTTTTGGATGTTGTAATCAGCAAG-3'. The same primers were used for semi-quantitative RT-PCR to amplify the LeGAI gene sequence from the various pro lines for DNA sequencing.
Recombinant DNA construction and DNA sequencing
To complement the pro mutant, the wild-type LeGAI cDNA sequence (GenBank accession no. AY269087
[GenBank]
) was placed under the control of the 35S cauliflower mosaic virus promoter in the binary vector pROK2 using the XbaI and KpnI sites in the multiple cloning site (MCS) following PCR amplification using the primers LeGAI-pROK2-f 5'-GGCGGCTCTAGAATGAAGAGAGATCGAGATCGAGATC-3' and LeGAI-pROK2-r 5'-GCGGGCTCTAGATTACAACTCGACTTCTCCG-3'.
For the transient transformation of onion epidermis, the LeGAI cDNA was cloned into pUC18-GFP using the BamHI restriction site. This vector contains a duplicated 35S promoter before the MCS, followed by the gene coding for the GFP. Restriction sites were introduced into LeGAI following PCR amplification using the primers LeGAI-GFP-f 5'-GCGCGCGGATCCATGAAGAGAGATCGAGATCGAGA-3' and LeGAI-GFP-r 5'-GCGGGCGGATCCCAACTCGACTTCTCCGGCGCCGG-3'.
DNA sequencing was performed using the BigDye Terminator Cycle Sequencing protocol with an ABI 377 DNA Sequencer (Applied Biosystems, Foster City, CA, USA) at the Guelph Molecular Supercenter (University of Guelph).
Stable transformation of tomato
Tomato was stably transformed using standard Agrobacterium-mediated procedures (McCormick et al., 1986). Transformed cells were selected based on the kanamycin resistance conferred by the pROK2 vector. The transformation of regenerated plants was confirmed by PCR amplification of the kanamycin selectable marker gene from genomic DNA using the primers KAN-fwd 5'-AAGAACTCGTCAAGAAGGCGATAG-3' and KAN-rev 5'-GATGGAAGCCGGTCTTGTCGATC-3'.
Particle bombardment of onion epidermis
Particle bombardment of onion epidermal cells was conducted as described by McCartney et al. (2004). Peeled onion epidermis was placed abaxial side down on agar Petri plates containing MS medium. A 10 µg aliquot of pUC18::LeGAI-GFP plasmid DNA was precipitated onto M-17 tungsten particles using isopropyl alcohol, and transformed into the onion peels through a particle delivery system –1000/He (Bio-Rad) as described by Banjoko and Trelease (1995). Transformed peels were kept in the dark for 18 h at 25 °C. The peel was placed on a microscope slide without a coverslip, and GFP fluorescence was viewed using an epifluorescence microscope. Hormone and inhibitor applications were performed by pipetting 25 µl of liquid onto onion peels on top of a transformed cell. GA and ABA (10 µM) were dissolved in water. The proteasome inhibitor MG132 was dissolved in dimethylsulphoxide (DMSO) at a concentration of 100 µM.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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The procera mutant exhibits altered branching architechture
The elongated phenotype of the pro mutant (Fig. 1A) has been reported previously (Jones, 1987; Jupe et al., 1988; Van Tuinen et al., 1999). A striking phenotype of this mutant noted here is its altered branching architecture. In wild-type tomato plants, the oldest axillary buds (those furthest from the apex) commence growth before the newer buds closer to the apex. In the pro mutant, the opposite is the case. The growth of axillary buds furthest from the apex is repressed, while those closest to the apex grow normally. This phenomenon is shown in Fig. 1A, with the black arrows pointing to the third node in each plant. The axillary bud in the wild-type third node had commenced growth (Fig. 1B); the equivalent node in pro has developed, but has failed to grow to the extent of that of the wild type (Fig. 1C). Interestingly, the pro axillary meristems closest to the apical meristem at the 11th and 12th nodes elongated, such that branching occurred at the top of the plant (arrowhead, Fig. 1A). Increased axillary bud growth in these younger nodes resulted in an obvious change in the branching architecture of the tomato plant.
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The altered branching pattern is probably due to the constitutive GA response within the axillary bud meristem. Doust et al. (2004) have identified quantitative trait loci in foxtail millet for branching architecture, including genes encoding GA biosynthetic enzymes. In the LATERAL SUPPRESSOR (ls) mutant of tomato, in which axillary bud growth is repressed, the GA content of these axillary buds is higher than in those of the wild type (Tucker, 1976). The author concluded that the LS protein acts negatively on the GA response pathway, consistent with observations in this constitutive GA response mutant.
procera contains a mutation in the tomato DELLA gene LeGAI
Greb et al. (2002) concluded that a mutation in LeSPY is not responsible for the pro phenotype; genes encoding DELLA proteins are the alternative logical candidates (Van Tuinen et al., 1999). A DELLA gene named LeGAI has been isolated from tomato (Bassel et al., 2004), and its sequence compared here with that from pro in the genetic background from the cv. Ailsa Craig. A point mutation was identified within the region of LeGAI coding for the VHIID domain, a conserved putative DNA-binding domain present in proteins that belong to the GRAS protein family, which includes DELLA proteins (Pysh et al., 1999). This amino acid domain in DELLA proteins is a conserved VHVID, and the predicted wild-type tomato LeGAI sequence is VHVID based on the sequencing of this gene from cv. ‘Trust’, cv. ‘Glamour’, the dwarf variety cv. ‘Micro Tom’ (Martí et al., 2006), and the GA response mutant 7B-1 (Fellner et al., 2001; data not shown). The point mutation in pro results in the third residue of this domain being changed from a valine to a glutamate such that the VHVID domain becomes VHEID (Fig. 2). The same point mutation was identified in three other tomato accessions with differing genetic backgrounds (see Materials and methods) exhibiting the pro phenotype (data not shown), suggesting a single origin for the introgression of this single allele into various cultivars (Stubbe, 1957; Van Tuinen et al., 1999).
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The third residue in the VHVID domain in other DELLA proteins including GAI, RGA, and RGL2 in Arabidopsis, SLN1 in barley, and SLR1 in rice is the conserved hydrophobic valine (Fig. 2), and the change to glutamate, an acidic and positively charged residue, suggests the production of a non-functional LeGAI protein. In support of this, a study performing a deletion analysis of the rice DELLA protein SLR1 indicated that the VHVID domain is required for the repression of GA responses (Itoh et al., 2002).
Complementation of the procera mutation
To determine whether this point mutation in the LeGAI gene from pro is responsible for the constitutive GA response phenotype, the pro mutant was transformed with the wild-type version of LeGAI under the control of the constitutive 35S cauliflower mosaic virus promoter (Odell et al., 1985). Transformed lines of pro expressing 35S::LeGAI were regenerated, and overexpression of this gene was confirmed by semi-quantitative RT-PCR using leaf tissue RNA as the template.
Complementation analysis of transformed pro was confounded by the fact that all T1 lines failed to produce viable seeds, and hence no T2 lines could be established. In certain lines, transgenic floral development was impaired, leading to premature abortion prior to anthesis (compare Fig. 3A and B), although the pistil elongated in certain flowers (Fig. 3C). Even though there was fruit set in other lines, the seeds aborted at an early stage of development, resulting in seedless fruit (Fig. 3D, E). These results are consistent with a study by Martí et al. (2007) that examined DELLA signalling in tomato by silencing the endogenous LeGAI gene in the wild type using RNA interference, and introducing the gain-of-function gene Atgaidel from Arabidopsis. Herein, elongation of the style and anthers was concluded to be due to the modulation of DELLA, leading to self-sterility (Martí et al., 2007).
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The disruption of hormone action by the constitutive expression of the GA-inhibitory protein LeGAI may explain the infertility of the pro plants overexpressing LeGAI. Transgenic expression of the Arabidopsis GAI gene controlled by the 35S promoter in tobacco demonstrated that DELLA genes can act in a dose-dependent manner (Hynes et al., 2003). Transgenic tobacco lines with low GAI expression had no discernible effect on GA responsiveness, while lines that exhibited high GAI expression had an impaired GA response (Hynes et al., 2003). In a similar fashion, overexpression of LeGAI may have resulted in reduced GA responses, leading to defective floral and seed development. DELLA proteins can mediate floral development in Arabidopsis (Cheng et al., 2004), and applied GA is required to prevent seed abortion in the gib-1 GA-deficient tomato mutant (Groot and Karssen, 1987).
The phenotypic variability observed in flowers of the T1 complemented pro lines was also present with respect to the altered branching phenotype of the pro mutant. It was difficult to conclude, however, whether this phenotype reverted back to that of the wild type, as the culture-derived T1 plants showed aberrant growth. An examination of the T2 generation would probably clarify these observations.
LeGAI expression and the procera mutation
The effect of the pro mutation on the expression of LeGAI was investigated. To separate the constitutive GA response phenotype due to the pro mutation from GA responses due to GA synthesis, the gib-1 pro double mutant (Van Tuinen et al., 1999) was included in this analysis. Using semi-quantitative RT-PCR, LeGAI expression was examined in the second fully expanded leaf from wild-type, gib-1, pro, and gib-1 pro plants (Fig. 4).
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In leaves of the wild type, LeGAI transcript abundance was higher than in those of the gib-1 mutant and gib-1 pro double mutant leaves. Expression in pro mutant leaves was higher than in the wild type and gib-1 mutant. This suggests that GA is responsible for an increase in expression of LeGAI, since both of the hormone-deficient mutants gib-1 and gib-1 pro show relatively low LeGAI transcript abundance. Therefore, the pro mutation promotes LeGAI transcript abundance, but only in the presence of GA synthesis.
These observations are in part consistent with gel blots using embryo RNA from the gib-1 mutant. LeGAI transcript abundance is low in water-imbibed mutant embryos, and increases strongly in the presence of GA (Bassel et al., 2004).
Effect of the procera mutation on germination and embryo growth potential
gib-1 pro double mutant seeds are unable to produce GA, and only 20% of them germinate when imbibed in water (Van Tuinen et al., 1999). Thus, in the absence of this hormone, the pro mutation is not fully capable of mediating the events leading to the release from coat dormancy (Bewley, 1997). Consistent with this observation, endo-β-mannanase activity was not detected in protein extracts from germinating whole seeds of the gib-1 pro double mutant (data not shown). These data suggest that the pro mutation does not mediate the release from coat dormancy in tomato seeds.
The effect of the pro mutation on the speed of germination and embryo growth potential was investigated to determine whether the constitutive GA response conferred by the pro mutation acted within the tomato embryo.
To determine the speed of germination, embryo axes consisting of the radicle and hypocotyl were dissected from their endosperms after 4 h of imbibition, and left to germinate on water. Wild-type axes germinated at a greater speed than those from the gib-1 mutant, and at a similar speed to axes from ABA-deficient sitiens (sit) seeds (Fig. 5A). The pro axes germinated fastest of all, reaching 35% as early as 12 HAD (hours after dissection). Germination of gib-1 pro mutant axes was faster than those of the wild type, but slower than the pro axes, even at 12 HAD. Thus the increased speed of germination was a result of the pro mutation and not the combined effect of responding to GA and pro. Also, the GA-mediated stimulation of the germination response is not saturated in the pro mutant because the GA-deficient gib-1 pro axes germinated at a slower speed than the GA-synthesizing pro genotype.
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The effect of the pro mutation on embryo growth potential was investigated by imbibing tomato seeds for 4 h on water, dissecting away their micropylar endosperm to remove this physical barrier to germination, then placing the partially dissected seeds on –0.5 MPa PEG 8000 and measuring how long it took for their radicles to elongate. The growth potential of wild-type dissected seeds in the presence of the osmoticum was greater than that of the gib-1 mutant, but less than that of the ABA-deficient sit mutant seeds (Fig. 5B); pro mutant dissected seeds had the greatest growth potential, followed by the gib-1 pro double mutant seeds. Therefore, the increased growth potential of the radicle was due to the pro mutation, and was not a saturated GA response in this tissue.
These results demonstrate that the pro mutation acts in the tomato embryo by increasing both the speed of germination and embryo growth potential. In addition, these enhanced GA responses in the embryo due to the pro mutation are not saturated.
The pro mutation and constitutive GA response phenotype in tomato
Assuming that pro is indeed a tomato LeGAI mutant, and that this is the only DELLA gene in tomato (Bassel et al., 2004), the question remains as to why the pro mutant is capable of responding to GA. There are three possible explanations. The first is that an unidentified DELLA gene in tomato acts to regulate the GA responses that are not under the control of LeGAI in the pro phenotype. The second is that the pro mutation represents a weak or ‘leaky’ mutation such that the LeGAI protein is still able to retain some of its GA-repressing activity. This is supported by the observation that only a small percentage of the gib-1 pro double mutant seeds are capable of germinating on water (Van Tuinen et al., 1999), while gib-1 mutant seeds do not germinate in the absence of GA (Groot et al., 1988). If pro were a complete null mutation, all of the double mutant seeds would be expected to germinate in the absence of GA. The final possibility is that the pro mutation is a null allele for LeGAI and LeGAI is the sole DELLA protein in tomato, but not all GA responses are regulated by this protein. Cao et al. (2006) have presented evidence for DELLA-independent changes in gene expression that are mediated by GA in Arabidopsis seeds and flowers.
LeGAI–GFP localizes to the nucleus and is degraded by the proteasome in the presence of GA
The cellular localization of the LeGAI protein was examined by generating a protein fusion between LeGAI and the GFP (Cormack et al., 1996) under the control of a duplicated constitutive 35S cauliflower mosaic virus promoter (Odell et al., 1985; Kay et al., 1987). This LeGAI–GFP fusion construct was transiently transformed in onion epidermis peels using particle bombardment, and the GFP product viewed using epifluorescence microscopy (McCartney et al., 2004).
Figure 6A shows that the fluorescence due to the LeGAI–GFP fusion protein is localized within the nucleus of a transformed onion epidermal cell, consistent with previous reports of this subcellular localization of DELLA proteins in rice and Arabidopsis (Dill et al., 2001; Itoh et al., 2002). The addition of GA to the onion peel resulted in a rapid degradation (within 5 min) of the LeGAI–GFP protein, shown by the loss of fluorescence (Fig. 6C). To determine whether the degradation of LeGAI–GFP was proteasome mediated, the proteasome inhibitor MG132 (Rock et al., 1994) was added to the onion peel 5 min prior to the addition of GA. In its presence, the LeGAI–GFP protein was not degraded when GA was added, as indicated by the maintenance of GFP fluorescence in the nucleus (Fig. 6E). The MG132 was dissolved in DMSO, and when this solvent alone was added to the peel it had no effect on the stability of the LeGAI–GFP fusion protein (Fig. 6G). When DMSO was added prior to GA, the hormone was still capable of inducing the proteasome-mediated degradation of LeGAI–GFP (Fig. 6I), demonstrating that MG132 and not DMSO is responsible for the maintenance of LeGAI–GFP in the presence of GA. MG132 alone was also insufficient to destabilize LeGAI–GFP since this protein persisted in the nucleus following its application (Fig. 6K). Collectively, these data demonstrate that LeGAI–GFP is localized to the nucleus in transiently transformed onion epidermis cells, and the protein is specifically and rapidly degraded by the proteasome in the presence of GA. This is consistent with the results reported for barley SLN1 (Fu et al., 2002), SLR1 in rice (Ikeda et al., 2001; Sasaki et al., 2003), and RGA in Arabidopsis (Dill et al., 2001; McGinnis et al., 2003).
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The application of ABA had no effect on the presence of LeGAI–GFP fusion protein in the nucleus of onion epidermal cells (Fig. 6M). When both GA and ABA were added together to the epidermis, the LeGAI–GFP protein fusion was degraded (Fig. 6O), as when GA alone was added (Fig. 6C). Therefore, ABA does not affect LeGAI–GFP protein stability and GA is capable of degrading this fusion protein, even in the presence of ABA. This observation is consistent with protein gel blot data using barley aleurone layer protein extracts and an antibody against SLN1 (Gubler et al., 2002).
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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The procera mutation of tomato has a profound affect on both the vegetative plant and the seed. The architecture of the plant is altered so that the branching pattern is abnormal, with growth of the lower rather than the upper branches being suppressed, and hence is the opposite of the anticipated pattern normally imposed by apical dominance. The mutation also strongly enhances the speed at which the embryo completes germination and embryo growth potential, and even the gib-1 pro double mutant embryo exhibits faster germination and has a higher embryo growth potential than the wild type. The pro mutation therefore regulates GA responses within the embryo.
In wild-type plants, for GA to be effective, the synthesis of DELLA protein must cease and/or it must be degraded to allow for completion of the hormone signal transduction pathway. That the DELLA protein of tomato is degraded by the proteasome is shown in the transient expression experiments; this is not affected by ABA.
Several lines of evidence support the hypothesis that pro is a tomato DELLA (LeGAI) mutant. These include: (i) the constitutive GA response phenotype of the pro mutant plant is partially resistant to GA synthesis inhibitors (Van Tuinen et al., 1999); and, as shown here, (ii) a point mutation in pro changes a conserved hydrophobic residue (valine) in the VHVID domain of LeGAI to a positively charged acidic residue (glutamate); and (iii) the identification of the identical point mutation in four different genetic backgrounds exhibiting the pro phenotype suggests that this potentially deleterious mutation co-segregates with the pro phenotype.
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Acknowledgements |
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We thank Dr Henk Hilhorst (Wageningen University, The Netherlands) for providing the gib-1 and sit mutant tomato seeds, and Dr Maarten Koornneef (Wageningen University, The Netherlands) for the gib-1 pro double-mutant seed. The four pro lines were obtained from the Charles M. Rick Tomato Genetics Resource Center (Davis, CA). We also thank Alanna Aspinall for taking the photos used for Fig. 3.
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References |
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