| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online August 31, 2007; 10.1104/pp.107.104174 Plant Physiology 145:351-366 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Expression of Aberrant Forms of AUXIN RESPONSE FACTOR8 Stimulates Parthenocarpy in Arabidopsis and Tomato1,[W],[OA]Commonwealth Scientific and Industrial Research Organization, Plant Industry, Glen Osmond, South Australia 5064, Australia
Fruit initiation in Arabidopsis (Arabidopsis thaliana) is generally repressed until fertilization occurs. However, mutations in AUXIN RESPONSE FACTOR8 (ARF8) uncouple fruit initiation from fertilization, resulting in the formation of seedless, parthenocarpic fruit. Here we induced parthenocarpy in wild-type Arabidopsis by introducing either the mutant genomic (g) Atarf8-4 sequence or gAtARF8:  -glucuronidase translational fusion constructs by plant transformation. Silencing of endogenous AtARF8 transcription was not observed, indicating that the introduced, aberrant ARF8 transcripts were compromising the function of endogenous ARF8 and/or associated factors involved in suppressing fruit initiation. To analyze the role of ARF8 in tomato (Solanum lycopersicum) we initially emasculated 23 tomato cultivars to test for background parthenocarpy. Surprisingly, all had a predisposition to initiate fertilization-independent fruit growth. Expression of gAtarf8-4 in transgenic tomato (‘Monalbo’) resulted in a significant increase in the number and size of parthenocarpic fruit. Isolation of tomato ARF8 cDNA indicated significant sequence conservation with AtARF8. SlARF8 may therefore control tomato fruit initiation in a similar manner as AtARF8 does in Arabidopsis. Two SlARF8 cDNAs differing in size by 5 bp were found, both arising from the same gene. The smaller cDNA is a splice variant and is also present in Arabidopsis. We propose that low endogenous levels of the splice variant products might interfere with efficient formation/function of a complex repressing fruit initiation, thereby providing an explanation for the observed ovary expansion in tomato and also Arabidopsis after emasculation. Increasing the levels of aberrant Atarf8-4 transcripts may further destabilize formation/function of the complex in a dosage-dependent manner enhancing tomato parthenocarpic fruit initiation frequency and size and mimicking the parthenocarpic dehiscent silique phenotype found in homozygous Atarf8-4 mutants. Collectively these data suggest that similar mechanisms involving auxin signaling exist to inhibit parthenocarpic fruit set in tomato and Arabidopsis.
Signaling processes, initiated by pollination and fertilization, are normally required to initiate seed and fruit development (Raghavan, 2003  ). Subsequent fruit growth depends, in part, on the coordinated action of growth substances produced in the ovary and seed after pollination and/or fertilization (Gillaspy et al., 1993; Garcia-Martinez and Hedden, 1997). Various phytohormones, including auxin, cytokinin, and GAs, have been implicated in seed and fruit growth and development (Nitsch, 1952, 1970; Coombe, 1960; Garcia-Martinez and Hedden, 1997; Fos et al., 2000, 2001).
The effect of pollination and fertilization in stimulating fruit growth can be mimicked by hormone applications (Goodwin, 1978
Recent studies in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana) have revealed that the auxin signaling pathway is involved in controlling the early events of fruit initiation. Two components of the auxin signaling pathway, AUXIN RESPONSE FACTOR8 (ARF8) from Arabidopsis (Vivian-Smith et al., 2001
Parthenocarpy in mutant and wild-type Arabidopsis has been examined by emasculation, which involves removing all of the floral organs surrounding the carpel before the anthers open and shed pollen onto the stigma (Vivian-Smith and Koltunow, 1999
In this article, we directly examined whether the introduction of aberrant forms of ARF8 can induce parthenocarpic fruit initiation in Arabidopsis and tomato. These data together with the isolation and analysis of tomato ARF8 sequences suggest that ARF8 is also involved in the regulation of fruit initiation in tomato.
Induction of Parthenocarpy in Wild-Type Arabidopsis by gAtarf8-4
Preliminary results indicated that the Atarf8-4 gene can induce parthenocarpy in Arabidopsis (Vivian-Smith, 2001
The point mutation in Atarf8-4 creates a cleaved amplified polymorphic sequence (CAPS) marker (Vivian-Smith, 2001 ) and expression of the introduced mutant gene was confirmed by using the CAPS marker in reverse transcription (RT)-PCR assays and also by quantitative real-time PCR. A mixture of mutant Atarf8-4 and wild-type AtARF8 transcripts was observed using the CAPS marker in all transformants (data not shown) and levels of total AtARF8 transcript were also found to be higher in transgenic plants compared to wild-type plants (Table I), indicating that cosuppression or RNA silencing of the endogenous AtARF8 gene were not the cause of the induced parthenocarpic phenotype. These data indicate that expression of arf8-4 mutant genes seem to induce variable silique elongation and dehiscence zone formation responses. Although it is not possible to determine the exact ratios of expression levels between the mutant and wild-type mRNAs from these experiments, the observed variation in the extent of parthenocarpy is probably due to differences in expression levels of the introduced Atarf8-4 copies. Variable responses, as observed in the transgenic lines here, are expected if competitive interference occurs between the introduced aberrant Atarf8-4 products and the endogenous wild-type ARF8.
The genomic Atarf8-4 mutant sequence was translationally fused to GUS and introduced to Ler and arf8-4 Arabidopsis backgrounds by plant transformation to examine if a protein product is made. A total of eight independent, homozygous lines were generated in the Ler background and four independent, homozygous lines in the arf8-4 background and all the lines expressed GUS (Fig. 2 ). Since the GUS gene does not contain its own translation initiation codon, the detection of GUS expression indicates that translation of the fusion protein is initiated from an alternative start codon somewhere within the mutant Atarf8-4 gene. Consequently a protein fragment can be produced from the mutant allele despite the point mutation of the putative translation initiation codon of AtARF8 in Atarf8-4. The exact nature of this mutant protein is, however, unclear and remains to be determined in future experiments.
Interestingly, the spatial and temporal expression patterns of the translational gAtarf8-4:GUS construct in both the Ler and arf8-4 backgrounds during flower and seed initiation were identical to those previously published for gAtARF8:GUS in the Ler and arf8-4 backgrounds (compare Fig. 2 with Goetz et al., 2006 ). As for gAtARF8:GUS, expression of gAtarf8-4:GUS showed reduced expression levels in arf8-4 plants in some tissues (e.g. petals and anther filaments) and a temporal change in expression was observed, especially in the carpel walls, the septum, the funiculi, and the ovules, where GUS activity was detected earlier and persisted in the mutant background. A detailed description of the expression patterns is provided in Goetz et al. (2006). Thus, as for the gAtARF8:GUS translational construct, expression of the gAtarf8-4:GUS translational fusion is spatially and temporally altered in the arf8-4 background compared to the expression of the same constructs in the Ler background (Fig. 2), indicating that the regulation of their expression is impaired in arf8-4. Therefore the arf8-4 mutant genetic background influences the spatial and temporal expression pattern of the introduced genes.
We previously observed that a dehiscence zone formed in some of the emasculated gAtARF8:GUS plants examined at that time (Goetz et al., 2006
These data show that expression of the translational gAtARF8:GUS and gAtarf8-4:GUS fusion constructs induce dehiscence zone formation but variable silique elongation responses. These data together with those obtained from plants transformed with the Atarf8-4 mutant gene indicate that the introduced aberrant constructs are able to destabilize the function of endogenous ARF8 and/or interacting factors that restrict fertilization-dependent fruit initiation in some way, leading to the induction of parthenocarpic phenotypes.
Arabidopsis produces a dry, dehiscent fruit. To examine the role of ARF8 during the initiation of fleshy fruit growth we selected tomato for further experiments. We emasculated a total of 23 different cultivars to examine their potential for parthenocarpic fruit initiation (see "Materials and Methods"). All of the cultivars tested exhibited some ovary growth after emasculation and most were able to initiate seedless fruit formation to varying degrees where the parthenocarpic fruits colored up and ripened. Data concerning eight indicative cultivars is provided in Table II .
The extent of fruit set observed varied between the different tomato cultivars in terms of the percentage of emasculated buds that initiated fruit development, final fruit size, and in particular the time lag between emasculation and fruit initiation. Naturally smaller fruited cultivars were most efficient in setting parthenocarpic fruit (Fig. 4A ; Table II). In general, the size of ripe parthenocarpic fruit was smaller than fertilization-induced fruit size (Table II), although we did not carry out an exhaustive analysis in this regard. In cultivars with poor fruit initiation, many emasculated flowers abscised at the abscission zone in the pedicel. If the emasculated flowers were retained some ovary expansion and fruit development was generally observed with very slow growth or early arrest in the varieties with poor fruit initiation (Fig. 4B). In some cultivars the parthenocarpic fruit had a different shape (Fig. 4C) and pulp formed better in the seedless fruit of some cultivars compared to others. Emasculated flowers of ‘Monalbo’ took between 63 and 143 d to form red, ripe parthenocarpic fruit with an average development time of 90 d (±18.4 d). When multiple flowers on a single truss were emasculated on the same day, different rates of fruit initiation and growth were detected on nine individual trusses observed. Figure 4D shows an example of such a truss from ‘Sweet Cherry Gold’ where a ripe fruit is senescing and different sized fruits have developed to varying degrees. Collectively, these data demonstrate that in most tomato cultivars examined after emasculation some limited ovary growth up to full fruit development occurs.
‘Monalbo’ was chosen for subsequent experiments. It has been selfed for many generations and is frequently used experimentally. It has moderate parthenocarpic ability and there was a substantial time difference between the initiation of fertilization-induced fruits at 8 d after pollination compared to parthenocarpic fruit development at around 35 d postemasculation. Fertilization-induced fruits in ‘Monalbo’ were larger than the smaller seedless fruit obtained after emasculation (Fig. 5, A and B ; Table II).
gAtarf8-4 Sequences Enhance Parthenocarpy in the Tomato ‘Monalbo’
We transgenically introduced the gAtarf8-4 mutant allele as a genomic construct into the tomato ‘Monalbo’ to examine if this would influence parthenocarpic fruit initiation in tomato. We chose the Atarf8-4 allele because it stimulated fertilization-independent fruit initiation in Arabidopsis in the experiments described above via a non-gene-silencing mechanism. Phylogenetic studies suggested a close relationship between ARFs from different plant species, implying that they derived from a common ancestor (Wang et al., 2007
Emasculation of untransformed ‘Monalbo’ plants showed that the majority of parthenocarpic fruit developing on the control plants were small (Fig. 5A), while wild-type fruits that developed after fertilization were much larger (Fig. 5B). All three of the primary transgenic plants showed enhanced parthenocarpy with respect to an increase in the number of emasculated flowers that initiated fruit development, and in terms of final fruit size compared with the emasculated, untransformed control plants (Table III; Fig. 5C). The extent of this enhanced parthenocarpic capability correlated with an increase in the steady-state pool of introduced mutant Atarf8-4 transcripts (Table III). Enhanced parthenocarpic fruit set and growth was maintained in segregating plants containing one to five copies of the Atarf8-4 gene obtained from self-pollinated primary transgenics. Retention of emasculated flowers and subsequent parthenocarpic fruit initiation increased up to 79% in the transgenic lines compared to 50% in wild-type plants (Table IV ). Larger fruits were generally observed in plants containing more copies of the introduced Atarf8-4 gene. Many of the parthenocarpic fruit from transgenic lines were similar in size and weight to seeded fruit from untransformed control plants and a greater percentage of fruit grew much larger than the average small sized seedless fruit formed after the emasculation of untransformed ‘Monalbo’ flowers (Table IV; Fig. 5, A–C). The addition of the gAtarf8-4 mutant allele has resulted in an enhanced pathenocarpic phenotype in terms of increased parthenocarpic fruit set and also size, thus we conclude that fruit initiation in tomato is controlled in a similar manner as found previously in Arabidopsis and that a S. lycopersicum ARF8 (SlARF8) ortholog has a role in controlling fruit initiation in tomato.
Larger Parthenocarpic Fruit Contain Seed-Like Structures (Pseudoembryos) Tomato fruit from the control and transgenic plants were also examined for seed, pulp, and flesh content. Emasculated flowers of wild-type ‘Monalbo’ predominantly formed small fruits with small locules and little pulp (Fig. 5D). After fertilization ‘Monalbo’ formed large fruits with large locules that were filled with pulp, in which the seeds were embedded (Fig. 5E). Fruits that developed after emasculation in the transgenic plants had pulp-filled locules, a thinner mesocarp, and enlarged central columella (Fig. 5F).
When assessed for seed content, the small parthenocarpic fruits from both emasculated control and transgenic flowers predominantly contained enlarged ovules (Fig. 5G), compared to the fully developed, large, hairy seeds found in fertilization-induced fruit (Fig. 5H). In the larger parthenocarpic fruit from emasculated control ‘Monalbo’ and transgenic plants (generally 20 g and above) many of the enlarged ovules had further differentiated into well-developed seed-like structures often called pseudoembryos (Fig. 5J). The size and level of differentiation of these seed-like structures varied, but was generally more advanced the larger the parthenocarpic fruit were in which they formed. Fully differentiated pseudoembryos were about one-eighth of fertilization-induced seed size (compare Fig. 5, H and J). Seed coat differentiation had occurred producing soft, seed-like structures with thickened lignified radial walls and protruding hairs. Sections of the fertilization-induced tomato seeds contained embryo and endosperm (Fig. 5I), whereas the well-developed pseudoembryos from the large parthenocarpic transgenic fruit lacked both embryo and endosperm and the collapsed embryo sac was surrounded by deeply staining multilayered endothelium (Fig. 5K). A correlation between the frequency and extent of pseudoembryo development with fruit growth and final fruit size has been observed previously (Kataoka et al., 2003
The tomato ortholog of ARF8 was isolated from ‘Monalbo’ to investigate potential links to parthenocarpy given that mutations in Arabidopsis ARF8 induce parthenocarpic fruit initiation and that expression of gAtarf8-4 was able to enhance parthenocarpy in transgenic tomatoes. Screening of tomato EST databases from flowers and fruits identified three overlapping unigene sequences with high homology to AtARF8. Based on these sequences, PCR primers were designed and RT-PCR and 5' and 3' RACE were used to amplify and clone the full-length mRNA clones of SlARF8. Amplifications and cloning using these PCR primers led to the isolation of two nearly identical cDNA sequences. The longer ARF8 clone of 2,872 bp (SlARF8 [full]) had 74% sequence identity with AtARF8 at the DNA level (Supplemental Fig. S1A). The similarity between the predicted protein sequence from tomato and AtARF8 was 76%, while the predicted protein identity was 66% (Table V ; Supplemental Fig. S1B), reflecting the observed conservation of ARFs across various plant species (Table V). The next closest related AtARF protein sequence was AtARF6 with 65% similarity and 55% identity, suggesting that the isolated sequence was a strong candidate for SlARF8.
The second SlARF8 cDNA sequence (SlARF8 [–5 bp]) was identical to SlARF8 (full) except for a 5 bp deletion within the DNA-binding domain of the predicted protein (compare SlARF8 [–5 bp] and SlARF8 [full]; Fig. 6A ). This 5 bp deletion is predicted to introduce an early stop codon immediately after the deletion within the DNA-binding domain (boxed in Fig. 6A) potentially resulting in a truncated ARF8 protein containing 185 amino acids. We considered that the shorter cDNA might arise from alterations in splicing at an exon/exon boundary of the same gene from initial comparison of both SlARF8 cDNA sequences with AtARF8 mRNA and the AtARF8 genomic sequence. Alternatively, given the tomato genome is not yet sequenced, it could have originated from mutations or sequence deletions during recent gene duplication events. DNA gel-blot analysis using a 359 bp fragment comprising part of the Q-rich middle region and the beginning of the C-terminal protein interaction domain of SlARF8 (Fig. 6B) as a probe to ‘Monalbo’ genomic DNA showed the presence of multiple bands (data not shown). Given the high sequence similarity between ARFs, especially in the DNA-binding domain and the C-terminal protein interaction domain, a cross-reaction with other ARFs in the ‘Monalbo’ genome is to be expected.
To directly examine the origin of the shorter transcript, we used PCR to isolate an 1,856 bp genomic fragment of the SlARF8 gene spanning exons IV to VIII and including the region around the 5 bp deletion (Fig. 6B). A total of five cloned PCR products were examined and found to be identical in sequence. This tends to argue against multiple genes giving rise to the two SlARF8 mRNAs as intron sequences are rarely conserved to this extent even in recently duplicated genes and duplicate genes have been found to exhibit dramatically accelerated rates of protein evolution (Castillo-Davis et al., 2004 ).
Comparison of the two SlARF8 mRNA sequences with the SlARF8 genomic sequence, the intron/exon structure of AtARF8, and consensus splice donor and acceptor sites confirmed that the shorter mRNA is likely to be a product of alternative splicing. Intron/exon structure comparison of the AtARF8 gene, the SlARF8 gene fragment, and the ARF8 gene from Brassica rapa (Br) showed that the positions and sizes of introns IV to VIII identified in the SlARF8 genomic fragment are very similar to those in the AtARF8 and BrARF8 genes, except that intron VI is significantly larger in SlARF8 at 1,083 bp compared with 281 bp in AtARF8 and 132 bp in BrARF8 (Supplemental Fig. S1C). A splice donor site (GC) marking the end of exon VI exists at 479 bp in the isolated SlARF8 genomic sequence (arrow in Fig. 6C). This indicates that there is an exon/exon boundary after 553 bp of the mRNA, immediately 5' to the 5 bp deletion found in SlARF8 (–5 bp). The consensus sequence for splice acceptor sites is (T/C) AG (Simpson and Filipowicz, 1996 To test this hypothesis we developed a dCAPS marker to examine the presence or absence of the two SlARF8 mRNA variants in ‘Monalbo’ and some of the other tomato cultivars known to form parthenocarpic fruit after emasculation. Using the dCAPS marker with control SlARF8 plasmid DNA resulted in a single band as expected. When mRNA from ‘Monalbo’ flowers collected around anthesis was used, the dCAPS marker showed two bands, indicating both SlARF8 (full) and SlARF8 (–5 bp) mRNA variants were present (Fig. 6D). Twelve additional tomato cultivars were tested with the dCAPS marker. Both mRNA variants were found in flowers of all cultivars collected at anthesis as indicated by the presence of two bands (Fig. 6D). We also tested the expression of SlARF8 in flowers of the primary transgenic lines transformed with the gAtarf8-4 construct. There was no detectable decrease in the levels of endogenous SlARF8 (full) and SlARF8 (–5 bp) variant transcripts, indicating that cosuppression or silencing of the endogenous SlARF8 mRNAs did not occur in these plants (Fig. 6E). In addition, various ‘Monalbo’ tissues were sampled and tested and all samples contained the two different SlARF8 mRNA variants (Fig. 5E). The factors that give rise to the alternatively spliced mRNAs in tomato are thus not floral specific. These observations in tomato prompted us to examine whether an alternative splice acceptor sequence is also present in the ARF8 gene sequence of Arabidopsis (Ler, Col, and Ws ecotypes). We found that the alternative splice acceptor site (CAG) is present at the same position in Arabidopsis (see position 553–555 of AtARF8 sequence in Fig. 6A) as previously identified in tomato. To determine if it is recognized by the splice machinery of Arabidopsis to produce an alternatively spliced AtARF8 mRNA variant, we performed the dCAPS marker test for the 5 bp deletion of the alternative splice variant with Arabidopsis sequence-specific primers using cDNAs from ecotypes Ler, Col, and Ws. The results show two bands were present for all three ecotypes tested (Fig. 6E), indicating the presence of the splice variant. As there is a single ARF8 gene in the Arabidopsis genome, both transcripts clearly arise from the same gene.
These data indicate that in both Arabidopsis and tomato low levels of endogenous, aberrant ARF8 transcripts exist in the form of the shorter splice variant mRNA that is predicted to give rise to a truncated protein. In most tomato cultivars we examined, we observed some limited ovary growth up to full fruit development following emasculation. Similarly, some carpel elongation occurs in emasculated Arabidopsis ecotypes (Vivian-Smith and Koltunow, 1999
Transgenic Introduction of Aberrant ARF8 Transcripts Induces Parthenocarpy in Arabidopsis
Recent work in Arabidopsis has shown that ARF8 might be part of a regulatory complex that negatively regulates fruit initiation. Lesions in AtARF8 lead to parthenocarpic fruit formation (Goetz et al., 2006
The extent of parthenocarpy induced in Arabidopsis varied depending on the construct used and individual lines analyzed, which is most likely due to differences in expression levels and effectiveness of the products of the various constructs. Variable responses like this are expected if competitive interference occurs between the introduced aberrant ARF8 protein and the regulatory complex. The stronger effects with the gAtARF8:GUS construct over the gAtarf8-4 and gAtarf8-4:GUS constructs seem to support this. Due to the previously identified point mutation in the translation initiation codon (Goetz et al., 2006 There were also differences between the translational GUS constructs and the gAtarf8-4 allele itself in terms of efficiency of parthenocarpic induction in Arabidopsis. Clear parthenocarpic phenotypes were more readily observed in lines containing the translational GUS constructs. Although the elongation response was variable with all constructs, dehiscence zone formation was only variable in lines transformed with the gAtarf8-4 construct, while it was present in all lines transformed with the translational GUS constructs. We suggest that one reason for this is that elongation and dehiscence zone formation are two separate pathways that might be controlled through the same complex, but activated in different ways.
Since the GUS protein is attached to the C-terminal protein-protein interaction domain (Fig. 6B) of Atarf8-4 and AtARF8, the greater effectiveness of the translational GUS constructs in inducing the dehiscence zone could be due to the presence of the GUS protein in that position. ARFs can homodimerize and also heterodimerize with other proteins including Aux/IAA proteins via the C-terminal protein-protein interaction domain (Liscum and Reed, 2002
Evidence for dosage-related ARF8 function in flower development is provided in a study analyzing combinations of Atarf6 and Atarf8 mutants. Nagpal et al. (2005)
Collectively the data presented show that in addition to the genetic induction of parthenocarpy, as demonstrated in our previous work (Goetz et al., 2006
Parthenocarpy is a desirable trait in horticultural crops, as it enables fruit set and growth to be independent of pollination, fertilization, and seed development. Therefore it may circumvent the environmental constraints on fruit production and ensure yield stability. Marketable parthenocarpy in tomato would provide the possibility to produce seedless fruits with high consumer appeal and could also be a valuable trait for industrial tomatoes because parthenocarpic fruit can have a higher percentage of soluble solids, improving yield and flavor of paste and reducing processing costs.
Fruit set in tomato is temperature dependent and commercial fruit quantities are obtained when night temperatures are maintained between 15°C to 21°C. Problems in pollen formation limit fruit set if night temperature is lower than 13°C or when the day temperature is higher than 38°C (Baksh et al., 1978
Emasculation has rarely been used to score parthenocarpic fruit growth in tomato, which is understandable in the plant breeding context where the intention is to provide the grower with varieties that are facultative for parthenocarpy in that they set fruit under normal growth conditions and also temperatures that limit pollen formation. In our study plants were emasculated and subsequently monitored for long periods of time. Our analysis showed that many commercially available tomato cultivars and also experimentally used lines that have been selfed for many generations show some degree of natural parthenocarpy following emasculation ranging from ovary expansion to varying sized seedless fruits. Some of these small and misshapen fruits are likely to be similar to those referred to as pseudofruits, puffs, or nuts in the literature (Foster and Tatman, 1937
Here we have established that ARF8 sequences are highly conserved between Arabidopsis and tomato at the mRNA and predicted protein level. When we introduced the mutant genomic Atarf8-4 sequence to ‘Monalbo’ a significant increase in parthenocarpic fruit initiation, fruit size, and weight was observed following emasculation above the background levels in control plants, supporting a role for ARF8 in regulating fruit initiation in tomato. Moreover, silencing of SlAux/IAA9 has been demonstrated to induce parthenocarpic fruit initiation in tomato (Wang et al., 2005
When we isolated SlARF8 cDNA sequences from tomato, we found a second variant of the mRNA that is most likely formed as a result of alternative splicing. Analysis of ARF8 expression in wild-type Arabidopsis ecotypes Ler, Col, and Ws confirmed the presence of the same alternative splice form and both plants show ovary expansion and growth following floral emasculation. This is in contrast to other plants where unfertilized flowers abscise. The use of other splice sites resulting in the alternative splicing of plant genes has been reported previously and heat stress or temperature-dependent splicing has been described as the cause in several of these cases (Burr et al., 1996
Our data suggests that ARF8 has a role in modulating fruit initiation in both Arabidopsis and tomato. Given the large amount of biochemical data available about the auxin signal transduction pathway (Ulmasov et al., 1999a
Obviously additional signals and activators are required following removal of the block in fruit initiation to promote fruit development and growth. In fertilization-induced fruits, seeds have long been known to produce growth-promoting hormones. Work in garden peas (Pisum sativum) suggests that seeds produce a modified auxin that stimulates GA production in the surrounding carpel tissues (Ozga et al., 2002 ; Ozga and Reinecke, 2003). Parthenocarpic fruits are likely to have other sources of growth hormones, but a link between auxin and GA in Arabidopsis is supported by the dependence of Atarf8 parthenocarpy on GA activity (Vivian-Smith et al., 2001) and increased GA levels are associated with growth of parthenocarpic tomato fruit (Fos et al., 2000, 2001). In tomato, the pat-2 and pat-3/pat-4 mutations, for example, have been found to independently accumulate high concentrations of GAs in unpollinated tomato ovaries via different pathways. It was therefore suggested that natural parthenocarpy induced by pat-2 and pat-3/pat-4 is due to increased synthesis of active GAs (e.g. GA1) in unpollinated ovaries as a result (Fos et al., 2000, 2001).
The pseudoembryos observed in parthenocarpic tomato may contribute to early growth of fruits as a substitute for developing seeds, either by producing phytohormones or by creating a sink for hormones to be transported into the growing fruits (Nitsch, 1952
Groot et al. (1987)
In our study, the development of ovules into pseudoembryos in parthenocarpic tomato fruit contrasted with the observed senescence of Arabidopsis ovules around 5 d after emasculation in both wild-type and arf8-4 plants. Arabidopsis ovules similarly senesced when parthenocarpy was induced by application of various growth hormones (Vivian-Smith, 2001
Arabidopsis Growth and Scoring Parthenocarpy
Arabidopsis (Arabidopsis thaliana) seeds (Ler, Col, and Ws) were surface sterilized and grown as described previously (Vivian-Smith and Koltunow, 1999
The cloning of the arf8-4 mutant gene in the pGEM-T easy vector (Promega) was described earlier (Goetz et al., 2006
The cloning of the translational ARF8:GUS and arf8-4:GUS constructs and the plant transformations have been detailed earlier (Goetz et al., 2006 The arf8-4 mutation creates a CAPS marker. Amplification with the primers ARF8-143F (5'-AGG AGA TGG AGA AAG ACG AG-3') and ARF8 + 48R (5'-CTC TCC TTC ATG ACC CTG TTG-3') and subsequent digest with Hsp92 II (Promega, NSW) resulted in bands of 142 bp + 41 bp + 8 bp from Ler wild-type plants, whereas 183 bp + 8 bp bands were present in arf8-4 plants.
Total RNA was extracted from Arabidopsis and tomato (Solanum lycopersicum) plant tissues using the RNeasy Plant Mini kit (Qiagen) according to manufacturer's instructions. One microgram of total RNA treated with an on-column RNase-free DNase protocol (Qiagen) was used as template for cDNA synthesis with the ThermoScript RT-PCR system (Invitrogen).
Quantitative real-time PCR was performed as described before (Goetz et al., 2006
The tomato cultivars used were either obtained as young plants from nurseries and repotted or purchased as seed and grown as described below. ‘Roma’, ‘LA1563’, ‘LA1714’, ‘LA3130’, and the male sterile lines ‘LA1222’ and ‘UC82b’ were obtained from the C.M. Rick Tomato Genetics Resource Centre (University of California). ‘Monalbo’ and ‘ChicoIII’ were kindly donated by Andrea Mazzucato (University of Viterbo). ‘Moneymaker’, ‘Beefsteak’, ‘BigBoy’, ‘Roma’, and ‘Margold’ were obtained from Eden Seeds. ‘Mighty Red’, ‘Roma’, ‘Cocktail Supreme’, ‘Health Kick’, ‘Red Mamma’, ‘Sweet Cherry Gold’, ‘Grosse Lisse’, ‘Top Dog’, and ‘Patio Prize’ were obtained from local nurseries and ‘Cherry Ripe’, ‘Greek’, and ‘LM’ were obtained from a local private garden. Flowers from each cultivar were emasculated, tagged, and checked after 3 to 4 weeks for signs of fruit growth. Most cultivars showed signs of fruit development after the indicated time. Seeds of tomato (‘Monalbo’ [‘LA2818’ in Charles Rick collection, UC Davis]) were sown in a peat-based seed and modular compost and were germinated in a glasshouse compartment. Temperatures in the glasshouse varied between 10°C to 19°C minimum temperature at night and 24°C to 34°C maximum temperature during the day. After 3 weeks, seedlings were pricked out into 1-L pots containing peat-based potting compost. At first flowering, plants were transplanted into 10-L pots containing peat-based potting compost and plants were supported by canes.
The arf8-4 allele from Arabidopsis was used for transformation of ‘Monalbo’. Tomato transformations were done essentially as described by Fillatti et al. (1987)
EST databases from Solanum esculentum were screened for sequences with high homology to AtARF8. The three UniGene sequences SGN-U228441, SGN-U238776, and SGN-U227556 were identified and analyzed further. Alignments of the three UniGenes showed that SGN-U227556 and SGN-U238776 overlap and that after a 70 bp gap SGN-U238776 and SGN-U228441 also overlap with high identity. Based on the consensus sequences, the primers SlARF8_SF1 to SlARF8_SF12 (Supplemental Table S1) were designed to amplify and clone this sequence and perform 5'- and 3'-RACE experiments to amplify and clone the full-length mRNA sequence of SlARF8. 5'- and 3'-untranslated regions were isolated with the 5'-RACE system for amplification of cDNA ends, version 2.0 (Invitrogen), and the 3'-RACE system for amplification of cDNA ends (Invitrogen), respectively. The gene-specific primers used were SlARF8_SF11 and SlARF8_SF12 (Supplemental Table S1) for 5'-RACE and SlARF8_SF6 and SlARF8_SF7 (Supplemental Table S1) for 3'-RACE.
The two SlARF8 variants can be distinguished with a dCAPS marker (Neff et al., 2002
Seeds, ovule traces, and pseudoembryos were collected from tomato fruits and fixed in 3.1% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4. Fixed tissues were dehydrated in an acetone series and infiltrated with acetone:Spurrs resin (1/1) overnight at room temperature in a vacuum chamber. They were then transferred to 100% Spurrs resin overnight in a vacuum chamber, embedded in fresh Spurrs resin, and polymerized at 65°C overnight. Specimen were cut into 2 µm thin sections and then stained with 0.1% toluidine blue in 0.02% sodium carbonate to determine cell identity.
Photographs of whole and cut fruits were taken with a Nikon Coolpix 995 digital camera. Whole-mount seeds and seed sections were viewed with Stemi2000C or Axioskop microscopes (Carl Zeiss). Digital images were captured using a Spot II camera (Diagnostic Instruments Inc.). Image processing and reproduction were performed with Auto Montage Essentials (Syncroscopy) and Photoshop 7.0 (Adobe Systems). Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AT5G37020 (AtARF8) and EF667342 (SlARF8).
The following materials are available in the online version of this article.
We thank Horticulture Australia Limited for funding the research and our colleagues involved in the two other projects in the Key Genes for Horticultural Markets initiative for their comments and feedback. Particular thanks to Dr. Steve Swain (CSIRO Plant Industry, Merbein) who coordinated the initiative and provided critical comments on the manuscript. Part of the Arabidopsis work was supported by an Australian Postgraduate Award to A.V.-S. as a student of the University of Adelaide. We thank the C.M. Rick Tomato Genetics Resource Centre (University of California, Davis) and Professor Andrea Mazzucato (University of Viterbo) for kind donations of tomato seeds and Dr. Rivka Barg (The Volcani Center, Bet Dagan) for very helpful discussions concerning parthenocarpy and fruit growth in tomato. Received June 19, 2007; accepted August 22, 2007; published August 31, 2007.
1 This work was supported by funding from a Horticulture Australia Limited Grant as part of the Key Genes for Horticultural Markets Project. 
2 Present address: Institute Biology Leiden, Section 2A, Molecular and Developmental Genetics, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Anna M. Koltunow (anna.koltunow{at}csiro.au).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.104174 * Corresponding author; e-mail anna.koltunow{at}csiro.au.
Adams SR, Cockshull KE, Cave CRJ (2001) Effect of temperature on the growth and development of tomato fruits. Ann Bot (Lond) 88: 869–877 Asahira T, Takeda Y, Nishio T, Hirabayashi M, Tsukamoto Y (1967) Studies on fruit development in tomato. I. Ovule development and content of diffusible auxin- and gibberellin-induced parthenocarpic tomato fruits in relation to their development. Mem Res Inst Food Sci Kyoto Univ 28: 47–74 Baksh S, Jamal A, Iqbal M (1978) A new report of environmental parthenocarpy in Solanaceae. Isr J Bot 27: 62–65 Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12: 8711–8721 Burr FA, Burr B, Scheffler BE, Blewitt M, Wienand U, Matz EC (1996) The maize repressor-like gene intensifier1 shares homology with the r1/b1 multigene family of transcription factors and exhibits missplicing. Plant Cell 8: 1249–1259[Abstract] Carmi N, Salts Y, Dedicova B, Shabtai S, Barg R (2003) Induction of parthenocarpy in tomato via specific expression of the rolB gene in the ovary. Planta 217: 726–735[CrossRef][Web of Science][Medline] Castillo-Davis CI, Hartl DL, Achaz G (2004) cis-regulatory and protein evolution in orthologous and duplicate genes. Genome Res 14: 1530–1536 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][Web of Science][Medline] Colot HV, Loros JJ, Dunlap JC (2005) Temperature-modulated alternative splicing and promoter use in the circadian clock gene frequency. Mol Biol Cell 16: 5563–5571 Coombe B (1960) Relationship of growth and development to changes in sugars, auxins, and gibberellings in fruit of seeded and seedless varieties of vitis vinifera. Plant Physiol 35: 241–250 Ficcadenti N, Sestili S, Pandolfini T, Cirillo C, Rotino GL, Spena A (1999) Genetic engineering of parthenocarpic fruit development in tomato. Mol Breed 5: 463–470[CrossRef] Fillatti J, Kiser J, Rose R, Comai L (1987) Efficient transfer of glyphosate tolerance gene into tomato using a binary Agrobacterium-tumefaciens vector. Biotechnology 5: 726–730[CrossRef] Fos M, Nuez F, Garcia-Martinez JL (2000) The gene pat-2, which induces natural parthenocarpy, alters the gibberellin content in unpollinated tomato ovaries. Plant Physiol 122: 471–480 Fos M, Proaño K, Nuez F, García-Martínez JL (2001) Role of gibberellins in parthenocarpic fruit development induced by the genetic system pat-3/pat-4 in tomato. Physiol Plant 111: 545–550[CrossRef][Medline] Foster AC, Tatman EC (1937) Environmental conditions influencing the development of tomato pockets or puffs. Plant Physiol 12: 875–880 Garcia-Martinez JL, Hedden P (1997) Gibberellins and fruit development. In FA Tomás-Barberán, RJ Robins, eds, Phytochemistry of Fruit and Vegetables, Vol 1. Clarendon Press, Oxford, pp 263–286 George WL, Scott JW, Splittstoesser WE (1984) Parthenocarpy in tomato. Hortic Rev (Am Soc Hortic Sci) 6: 65–84 Gillaspy G, Ben-David H, Gruissem W (1993) Fruits: a developmental perspective. Plant Cell 5: 1439–1451 Goetz M, Vivian-Smith A, Johnson SD, Koltunow AM (2006) AUXIN RESPONSE FACTOR8 is a negative regulator of fruit initiation in Arabidopsis. Plant Cell 18: 1873–1886 Goodwin PB (1978) Phytohormones and fruit growth. In DS Letham, PB Goodwin, TJV Higgins, eds, Phytohormones and Related Compound. A Comprehensive Treatise, Vol II. Elsevier, Amsterdam, pp 175–214 Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M (2001) Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Nature 414: 271–276[CrossRef][Medline] Groot SPC, Bruinsma J, Karssen CM (1987) The role of endogenous gibberellin in seed and fruit development of tomato: studies with a gibberellin-deficient mutant. Physiol Plant 71: 184–190[CrossRef] Hardtke CS, Ckurshumova W, Vidaurre DP, Singh SA, Stamatiou G, Tiwari SB, Hagen G, Guilfoyle TJ, Berleth T (2004) Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4. Development 131: 1089–1100 Ho LC, Hewitt JD (1986) Fruit Development. In JG Atherton, J Rudich, eds, The Tomato Crop. A Scientific Basis for Improvement. Chapman and Hall, London, pp 201–239 Kataoka K, Uemachi A, Yazawa S (2003) Fruit growth and pseudoembryo development affected by uniconazole, an inhibitor of gibberellin biosynthesis, in pat-2 and auxin-induced parthenocarpic tomato fruit. Sci Hortic (Amsterdam) 98: 9–16[CrossRef] Kepinski S, Leyser O (2004) Auxin-induced SCFTIR1-Aux/IAA interaction involves stable modification of the SCFTIR1 complex. Proc Natl Acad Sci USA 101: 12381–12386 Kepinski S, Leyser O (2005) The Arabidopsis F-box protein TIR1 is an auxin receptor. Nature 435: 446–451[CrossRef][Medline] King GN (1947) Artificial parthenocarpy in Lycopersicon esculentum: tissue development. Plant Physiol 22: 572–581 Lazar G, Goodman HM (2000) The Arabidopsis splicing factor SR1 is regulated by alternative splicing. Plant Mol Biol 42: 571–581[CrossRef][Web of Science][Medline] Lin S, Splittstoesser WE, George WL (1983a) Factors controlling the expression of parthenocarpy in ‘Severianin’ tomato. Sci Hortic (Amsterdam) 19: 45–53[CrossRef] Lin S, Splittstoesser WE, George WL (1983b) A comparison of normal seeds and pseudoembryos produced in parthenocarpic fruit of ‘Severianin’ tomato. HortScience 18: 75–76[Web of Science] Liscum E, Reed JW (2002) Genetics of Aux/IAA and ARF action in plant growth and development. Plant Mol Biol 49: 387–400[CrossRef][Web of Science][Medline] Lukyanenko AN (1991) Parthenocarpy in Tomato. In G Kalloo, ed, Genetic Improvement of Tomato, Monographs on Theoretical and Applied Genetics, Vol 14. Springer-Verlag, Berlin, pp 167–178 Mapelli S, Frova C, Torti G, Soressi GP (1978) Relationship between set, development and activities of growth regulators in tomato fruits. Plant Cell Physiol 19: 1281–1288 McKibbin RS, Wilkinson MD, Bailey PC, Flintham JE, Andrew LM, Lazzeri PA, Gale MD, Lenton JR, Holdsworth MJ (2002) Transcripts of Vp-1 homeologues are misspliced in modern wheat and ancestral species. Proc Natl Acad Sci USA 99: 10203–10208 Mezzetti B, Landi L, Pandolfini T, Spena A (2004) The defH9-iaaM auxin-synthesizing gene increases plant fecundity and fruit production in strawberry and raspberry. BMC Biotechnol 4: 4[CrossRef][Medline] Nagpal P, Ellis CM, Weber H, Ploense SE, Barkawi LS, Guilfoyle TJ, Hagen G, Alonso JM, Cohen JD, Farmer EE, et al (2005) Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation. Development 132: 4107–4118 Neff MM, Turk E, Kalishman M (2002) Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 18: 613–615[CrossRef][Web of Science][Medline] Nitsch JP (1952) Plant hormones in the development of fruits. Q Rev Biol 27: 33–57[CrossRef][Medline] Nitsch JP (1970) Hormonal factors in growth and development. In AC Hulme, ed, The Biochemistry of Fruits and their Products, Vol 1. Academic Press, New York, pp 427–472 O'Neill SD (1997) Pollination regulation of flower development. Annu Rev Plant Physiol Plant Mol Biol 48: 547–574[CrossRef][Web of Science][Medline] Ozga JA, Reinecke DM (2003) Hormonal interactions in fruit development. J Plant Growth Regul 22: 73–81 Ozga JA, van Huizen R, Reinecke DM (2002) Hormone and seed-specific regulation of pea fruit growth. Plant Physiol 128: 1379–1389 Pandolfini T, Rotino G, Camerini S, Defez R, Spena A (2002) Optimisation of transgene action at the post-transcriptional level: high quality parthenocarpic fruits in industrial tomatoes. BMC Biotechnol 2: 1[Medline] Raghavan V (2003) Some reflections on double fertilization, from its discovery to the present. New Phytol 159: 565–583[CrossRef][Web of Science] Reddy ASN (2007) Alternative splicing of pre-messenger RNAs in plants in the genomic era. Annu Rev Plant Biol 58: 267–294[CrossRef][Medline] Rogg LE, Bartel B (2001) Auxin signaling: derepression through regulated proteolysis. Dev Cell 1: 595–604[CrossRef][Web of Science][Medline] Rotino GL, Perri E, Zottini M, Sommer H, Spena A (1997) Genetic engineering of parthenocarpic plants. Nat Biotechnol 15: 1398–1401[CrossRef][Web of Science][Medline] Rylski I (1979) Effect of temperatures and growth regulators on fruit malformation in tomato. Sci Hortic (Amsterdam) 10: 27–35[CrossRef] Sablowski RWM, Meyerowitz EM (1998) Temperature-sensitive splicing in the floral homeotic mutant apetala3-1. Plant Cell 10: 1453–1464 Simpson GG, Filipowicz W (1996) Splicing of precursors to mRNA in higher plants: mechanisms, regulation and sub-nuclear organisation of the spliceosomal machinery. Plant Mol Biol 32: 1–41[CrossRef][Web of Science][Medline] Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis. Plant Cell 2: 755–767 Swain SM, Koltunow AM (2006) Auxin and Fruit Initiation. In L Taiz, E Zeiger, eds, Plant Physiology, Ed 4. Sinauer Associates, Inc., Sunderland, MA Talon M, Hedden P, Primo-Millo E (1990) Gibberellins in Citrus sinensis: a comparison between seeded and seedless varieties. J Plant Growth Regul 9: 201–206 Talon M, Zacarias L, Primomillo E (1992) Gibberellins and parthenocarpic ability in developing ovaries of seedless mandarins. Plant Physiol 99: 1575–1581 Tatematsu K, Kumagai S, Muto H, Sato A, Watahiki MK, Harper RM, Liscum E, Yamamoto KT (2004) MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana. Plant Cell 16: 379–393 Tian C, Muto H, Higuchi K, Matamura T, Tatematsu K, Koshiba T, Yamamoto KT (2004) Disruption and overexpression of Auxin Response Factor 8 gene of Arabidopsis affect hypocotyl elongation and root growth habit, indicating its possible involvement in auxin homeostasis in light condition. Plant J 40: 333–343[CrossRef][Web of Science][Medline] Tiwari SB, Hagen G, Guilfoyle T (2003) The roles of Auxin Response Factor domains in auxin-responsive transcription. Plant Cell 15: 533–543 Tiwari SB, Wang X-J, Hagen G, Guilfoyle TJ (2001) Aux/IAA proteins are active repressors, and their stability and activity are modulated by auxin. Plant Cell 13: 2809–2822 Ulmasov T, Hagen G, Guilfoyle TJ (1999a) Activation and repression of transcription by Auxin-Response Factors. Proc Natl Acad Sci USA 96: 5844–5849 Ulmasov T, Hagen G, Guilfoyle TJ (1999b) Dimerization and DNA binding of Auxin Response Factors. Plant J 19: 309–319[CrossRef][Web of Science][Medline] Vardy E, Lapushner D, Genizi A, Hewitt JD (1989a) Genetics of parthenocarpy in tomato under a low temperature regime. I. Line RP 75/59. Euphytica 41: 1–8[CrossRef][Web of Science] Vardy E, Lapushner D, Genizi A, Hewitt JD (1989b) Genetics of parthenocarpy in tomato under a low temperature regime. II. Cultivar ‘Severianin’. Euphytica 41: 9–15[CrossRef][Web of Science] Varga A, Bruinsma J (1976) Roles of seeds and auxins in tomato fruit growth. Z Pflanzenphysiol 80: 95–104 Vivian-Smith A (2001) The Molecular Basis for the Initiation of Fruit Development & Parthenocarpy. University of Adelaide, Adelaide, Australia Vivian-Smith A, Koltunow AM (1999) Genetic analysis of growth-regulator-induced parthenocarpy in Arabidopsis. Plant Physiol 121: 437–452 Vivian-Smith A, Luo M, Chaudhury A, Koltunow A (2001) Fruit development is actively restricted in the absence of fertilization in Arabidopsis. Development 128: 2321–2331[Web of Science][Medline] Wang D, Pei K, Fu Y, Sun Z, Li S, Liu H, Tang K, Han B, Tao Y (2007) Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa). Gene 394: 13–24[CrossRef][Web of Science][Medline] Wang H, Jones B, Li Z, Frasse P, Delalande C, Regad F, Chaabouni S, Latche A, Pech J-C, Bouzayen M (2005) The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. Plant Cell 17: 2676–2692 Zensner N, Ellsmore A, Leasure C, Callis J (2001) Auxin modulates the degradation rate of Aux/IAA proteins. Proc Natl Acad Sci USA 98: 11795–11800 This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
