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First published online August 19, 2005; 10.1104/pp.105.062695 Plant Physiology 139:115-126 (2005) © 2005 American Society of Plant Biologists Cosuppression of Eukaryotic Release Factor 1-1 in Arabidopsis Affects Cell Elongation and Radial Cell DivisionPlant Genetic Engineering Laboratory, Department of Botany, School of Integrative Biology, University of Queensland, Brisbane, Queensland 4072, Australia
The role of the eukaryotic release factor 1 (eRF1) in translation termination has previously been established in yeast; however, only limited characterization has been performed on any plant homologs. Here, we demonstrate that cosuppression of eRF1-1 in Arabidopsis (Arabidopsis thaliana) has a profound effect on plant morphology, resulting in what we term the broomhead phenotype. These plants primarily exhibit a reduction in internode elongation causing the formation of a broomhead-like cluster of malformed siliques at the top of the inflorescence stem. Histological analysis of broomhead stems revealed that cells are reduced in height and display ectopic lignification of the phloem cap cells, some phloem sieve cells, and regions of the fascicular cambium, as well as enhanced lignification of the interfascicular fibers. We also show that cell division in the fascicular cambial regions is altered, with the majority of vascular bundles containing cambial cells that are disorganized and possess enlarged nuclei. This is the first attempt at functional characterization of a release factor in vivo in plants and demonstrates the importance of eRF1-1 function in Arabidopsis.
Protein synthesis is an essential process for all living organisms. Translation of mRNA into protein basically consists of three stages: (1) initiation, involving the assembly of the ribosomal subunits at the 5' end of an mRNA, (2) elongation, the process of tRNA-mediated decoding of the mRNA to form a polypeptide chain, and (3) termination, during which a stop codon signals the end of translation, and the ribosomal subunits dissociate from the mRNA. Each stage requires specific accessory proteins or factors.
The signal that indicates the end of a polypeptide is the presence of an in-frame stop codon (UAA, UGA, or UAG) at the ribosomal A-site. In eukaryotes, termination of protein synthesis is carried out by two classes of release factors: eukaryotic release factor 1 (eRF1) and eRF3 (Frolova et al., 1994
Homologs of eRF1 have been identified in a wide range of eukaryotes, including human, frog (Xenopus laevis), nematode (Caenorhabditis elegans), ciliate (Tetrahymena theromophila), and fungus (Podospora anserina). Despite considerable differences between these organisms, a high degree of sequence similarity exists among homologs. Complementation studies have shown that this sequence similarity also translates into functional similarity. Xenopus eRF1 cDNA expressed in a yeast (Saccharomyces cerevisiae) mutant carrying a temperature-sensitive eRF1 allele, eRF1 (ts), rescued the conditional-lethal phenotype of the mutant at 36°C (Tassan et al., 1993
To date, three eRF1 genes (eRF1-1, eRF1-2, and eRF1-3) have been identified in Arabidopsis (Arabidopsis thaliana) by sequence homology (Brown et al., 1995
The Broomhead Phenotype Homozygous Arabidopsis transgenic lines were produced that overexpress an endogenous eRF1-1 cDNA (GenBank accession no. H37361). The eRF1-1 overexpression lines predominantly produced phenotypically normal plants, and we refer to these transgenics as nonphenotype plants. However, a limited percentage of plants (3%–60%) across multiple transgenic lines displayed an abnormal broomhead phenotype (Fig. 1A). The most distinctive feature of the phenotype is the reduction in internode spacing of the inflorescence stems, creating a broomhead-like structure. Compared to wild-type plants, broomhead plants are reduced in height (Table I), produce malformed siliques, and synthesize anthocyanin in nearly all mature plant tissues (Fig. 1, B and C). No significant difference in time to flowering was observed between broomhead and wild-type plants (Table I). However, broomhead plants flowered for longer, and there was an increase in the total number of inflorescence meristems formed and a reduction in number of seeds produced per pod (Table I). During juvenile rosette development, wild-type and broomhead plants are identical in appearance (Fig. 1, D and E). The onset of the broomhead phenotype primarily occurs during reproductive development, although the more severe transgenics exhibit signs of the phenotype in the adult vegetative stage through the production of anthocyanin in the veins of the rosette leaves (Fig. 1, F and G). In less severe transgenics, the onset of the phenotype is preceded by the production of anthocyanin in the inflorescence stem below the bud. As the phenotype progresses, the synthesis of anthocyanin subsequently occurs in the rosette and cauline leaves.
Typically, only a small percentage of plants in a specific line show the broomhead phenotype, and not all of these plants display the phenotype to the same degree. Differences in plant height, number of inflorescences produced, total seed production, and the height of the primary bolt can be observed within a single homozygous line (Fig. 2, A–D). The broomhead structures also vary in the degree of clustering, and sometimes broomhead plants revert to producing wild-type inflorescences (Fig. 2, E–H). Plants displaying the most severe phenotype terminate growth of the primary inflorescence at the rosette level. A decrease in phenotype severity is correlated with an increase in the height of the primary inflorescence and the corresponding production of broomhead structures further away from the rosette.
The Broomhead Phenotype Is Caused by eRF1-1 Cosuppression Expression levels of the endogenous eRF1-1 gene as well as the transgene were determined by northern analysis performed on a variety of tissues from both broomhead and wild-type plants. In most tissues examined, eRF1-1 transcript levels were significantly higher in the transgenics than in the wild type (Fig. 3A). However, in the stem and bud tissue where the broomhead phenotype is most visible, significant degradation of the eRF1-1 transcript was observed. To exclude the possibility of poor RNA quality, the northern was reprobed with a second unrelated gene, AtDRG2, encoding a cytoplasmic G-protein (At4g39520). Although some degradation was observed in all broomhead samples, most of the AtDRG2 transcripts remained intact (Fig. 3A). Probing with a ribosomal gene also failed to reveal any significant degradation products. This suggests that the degradation of eRF1-1 in the stem and bud tissues of broomhead plants is transcript specific. The possibility that the phenotype was due to an insertional effect was ruled out by southern analysis on four independent broomhead lines that clearly shows different chromosomal integration sites for each line (Fig. 4). The degradation observed in broomhead plants was specific to plants showing the phenotype as eRF1-1 transcript levels were up-regulated in nonphenotype transgenic plants from six different lines with no significant degradation products observed (Fig. 3B).
To investigate the link between transgene degradation and the broomhead phenotype, an eRF1-1:  -glucuronidase (GUS) in-frame gene fusion was introduced into Arabidopsis plants under the control of the 35S promoter of cauliflower mosaic virus (CaMV 35S). A number of lines carrying this hybrid construct showed the characteristic broomhead phenotype, and homozygous lines were selected and analyzed in further detail. All severe broomhead phenotype transgenics failed to show GUS activity anywhere in the plant (Fig. 5A). These plants did not bolt above the rosette level, and no viable seed was produced. However, broomhead plants from less severe lines showed GUS activity in the developing seeds (Fig. 5B). This is in agreement with other cosuppression reports where gene silencing is reset during seed development (Hart et al., 1992 ; Deborne et al., 1994; Kunz et al., 1996; 2001). Staining of the nonphenotype plants was less consistent; approximately half of the plants exhibited extensive GUS staining (Fig. 5C), while the other half remained largely unstained (Fig. 5D). GUS activity in the mostly unstained plants was only apparent in the older rosette leaves either at the tips or in the area surrounding the veins (Fig. 5E) and in developing seeds (Fig. 5F). This GUS staining pattern is suggestive of a downward systemic silencing signal, as has been reported previously during cosuppression of GFP in tobacco (Nicotiana tabacum; Voinnet et al., 1998).
Northern analysis confirmed the presence of the eRF1-1:GUS transcript in the blue nonphenotype plants (Fig. 5G, top panel, lane 3). In contrast, only a faint signal was observed for the transgene in broomhead transgenic bud tissue (Fig. 5G, top panel, lane 5), and no transcript was detected in GUS negative nonphenotype bud tissue (Fig. 5G, top panel, lane 4). When the endogenous levels of eRF1-1 were detected using the 3' untranslated region of the gene (absent in the eRF1-1:GUS transgene) as a probe, only the broomhead tissue displayed down-regulation of the transcript (Fig. 5G, middle panel). This suggests that posttranscriptional silencing of both the transgene and endogenous gene occurs in broomhead plants, even though down-regulation of eRF1-1 is not complete. The presence of a single band followed by a smear in the blue nonphenotype and broomhead RNA samples (Fig. 5G, top panel, lanes 3 and 5) may be indicative of a cosuppression event, although the blue nonphenotype samples still exhibit high levels of the transgene and wild-type levels of the endogenous eRF1-1 gene. It is possible that cosuppression has been triggered in these plants but has not yet had the chance to progress very far. The absence of the eRF1-1:GUS transgene in GUS negative nonphenotype plants is also consistent with a silencing effect, although it appears that only the transgene is silenced in these plants, as the endogenous eRF1-1 transcript levels were unaffected.
eRF1-1 is part of a gene family that includes two other members, eRF1-2 (At1g12920) and eRF1-3 (At3g26618). At the nucleotide level, these family members show 76% (eRF1-2) and 77% (eRF1-3) identity to eRF1-1 and 87% identity to each other. To determine if either the eRF1-2 or eRF1-3 endogenous gene is affected in broomhead phenotype plants, quantitative real-time PCR was performed on bud tissue with specific primers for each family member and compared to wild-type controls. In wild-type and nonphenotype transgenics, eRF1-2 and eRF1-3 transcript levels were almost identical (Fig. 6A, white bars). However, in broomhead transgenics, eRF1-2 and eRF1-3 transcript levels were down-regulated to approximately half of the wild-type levels (Fig. 6A, black bars). When total eRF1-1 (endogenous gene and transgene) transcript levels were quantified, an average of 7-fold and 3.8-fold overexpression was detected in the nonphenotype and broomhead transgenics, respectively (Fig. 6B). This shows down-regulation of eRF1-1 in broomhead plants relative to nonphenotype plants, although not below wild-type levels. Down-regulation of eRF1-2 and eRF1-3 indicates that the silencing effect is not restricted to eRF1-1 but also affects the family members, most likely due to their high sequence homology.
Cellular Architecture of the Broomhead Phenotype To study the broomhead phenotype at the anatomical level, histological analyses were performed on longitudinal and transverse sections of the broomhead inflorescence stem and the corresponding region in wild-type plants. Microscopic examination revealed that a reduction in cell elongation rather than cell division was responsible for the decrease in plant height. In general, the average pith cell length was reduced approximately 10-fold in broomhead inflorescence stems (17.33 µm) relative to the wild type (174.5 µm; Fig. 7, A and B), and the cells were more cytoplasmically dense. No morphological aberrations were observed in the apical meristem, suggesting that cell division in this region is unaffected (Fig. 7, D and E). However, the majority of cambial cells were disorganized and hypertrophic and contained enlarged nuclei (Fig. 7C). The nuclei were visualized with safranin-O, which stains chromosomes and nucleoli in addition to lignified tissue. Further analysis of transverse stem sections (Fig. 7, F–J) revealed that most fascicular cambial regions exhibit this disorganized cell growth, although a few fascicular cambial regions appeared more active and contained an increased number of cell layers (Fig. 7I). Several layers of interfascicular fiber precursor cells were also identified in broomhead stems that were absent in the wild-type stems (Fig. 7, F and G). In addition, ectopic lignification of the phloem cap cells and some phloem sieve cells as well as enhanced lignification of the interfascicular fibers were observed in broomhead stems (Fig. 7, G, I, and J). Some areas of the fascicular cambium that had become disorganized also showed evidence of lignification (Fig. 7, G and J). To confirm that the safranin-O stain was in fact detecting the presence of ectopic lignin, wild-type and broomhead stem sections were treated with the lignin-specific stain phloroglucinol-HCl (Fig. 7, K and L). As expected, broomhead stem sections stained pink/red around the walls of the phloem cap cells and phloem sieve cells and also in some regions of the disorganized fascicular cambium (Fig. 7L), indicating the presence of lignin. Compared to wild-type sections, enhanced lignification was also observed in the interfascicular fibers. These results confirm the presence of enhanced and ectopically lignified cells in broomhead plants, as observed with the safranin-O stain.
eRF1-1 Expression Is Localized to the Vascular Tissues and Actively Growing Regions To examine the endogenous expression pattern of eRF1-1, approximately 2 kb of the eRF1-1 promoter was fused to the GUS reporter gene, and a number of Arabidopsis transgenic lines carrying the construct were analyzed. Under normal growth conditions, GUS expression was detected at all developmental stages, including germination and seedling development, and in mature plants (Fig. 8, A–F). The most obvious expression was seen in the vascular tissues at all growth stages, particularly in the phloem and also in the cambium, xylem parenchyma, and interfascicular parenchyma cells (Fig. 8, G and H). However, in the elongating stem region just below the buds, a more uniform pattern of expression was observed across all cells types (Fig. 8I). Other areas of GUS positive staining included pollen grains, trichomes, and guard cells (Fig. 8, J–L). Younger and more actively growing tissues, i.e. germinating seeds, the shoot apical meristem, and root meristem, exhibited the strongest GUS staining (Fig. 8, A, B, and F). These results are consistent with the expression pattern observed in the northern analysis of wild-type plants (Fig. 8M), where high levels of the eRF1-1 transcript were seen in closed and open buds and in the roots. The basal level of eRF1-1 expression in the rosette and cauline leaves also correlates with the low level of GUS activity detected in the leaf veins. Promoter-GUS fusions of the two family members (eRF1-2 and eRF1-3) were also constructed, and Arabidopsis transgenic lines were produced and characterized. The patterns of GUS expression found in these lines were identical to the ones observed for eRF1-1 (data not shown), indicating that there is no complementarity in expression patterns between family members and suggesting a possible redundancy in function.
The Broomhead Phenotype Is a Result of Gene Silencing
Our evidence indicates that the broomhead phenotype in Arabidopsis lines engineered to overexpress eRF1-1 is a result of gene silencing. First, northern analysis on broomhead plants revealed that eRF1-1 transcripts were partially degraded in tissues showing the phenotype. In contrast, those plants not exhibiting a phenotype displayed overexpression of the gene with no significant degradation products. A similar degradation smear below the full-length transcript on a northern has been reported for transgenic N. benthamiana plants in which the LUCIFERASE gene had been silenced (Kjemtrup et al., 1998
Second, all broomhead plants containing the 35S-eRF1-1:GUS gene fusion failed to show GUS activity in any plant tissues, except for the developing seeds (Fig. 5A). This indicates that at least the transgene is being silenced in broomhead phenotype plants, and the GUS activity in the developing seeds represents the post-meiotic resetting of the silencing event and has been described in a number of reports (Hart et al., 1992
Third, quantitative real-time results revealed that the transcript levels for family members eRF1-2 and eRF1-3 were also reduced relative to nonphenotype and wild-type plants. It has been suggested that at least 60% to 70% identity in the coding sequence is the threshold necessary to produce posttranscriptional gene silencing of homologous genes (Kunz et al., 2001
The failure to detect overall lower levels of the eRF1-1 transcript in 35S-eRF1-1 broomhead transgenics does not necessarily imply that all the eRF1-1 transcripts are functional and able to be translated. In fact, one of the models proposed to explain how posttranscriptional gene silencing is induced is via the production of aberrant RNA from the homologous transgene. The aberrant RNA is suggested to act as a template for the degradation of itself and other homologous RNAs. Because we were unable to distinguish between the endogenous eRF1-1 transcript and the transgene, due to their similarity in length and sequence, we could not confirm whether or not the endogenous gene was down-regulated. However, based on the findings that (1) a significant portion of the eRF1-1 transcript is already partially degraded in broomhead plants, (2) both family members are down-regulated, and (3) 35S-eRF1-1:GUS broomhead transgenics show down-regulation of the endogenous eRF1-1 transcript, it seems likely that the broomhead phenotype is a result of cosuppression. In addition to the molecular evidence presented so far, the phenotypic characteristics of the broomhead phenotype, such as the variability within and between lines, the low percentage of progeny showing a phenotype, and the differences in the onset of silencing, are also consistent with other reports of transgenic plants showing cosuppresion effects (Elkind et al., 1990
By down-regulating an Arabidopsis eukaryotic release factor, we have produced an unexpected yet interesting phenotype that is predominantly defective in inflorescence stem cell elongation. The majority of broomhead transgenics resemble wild-type plants in the vegetative stage of development, but during reproductive growth, the inflorescence stems cease to elongate properly, producing a cluster of mostly malformed siliques. Histological analysis on the broomhead stem revealed that the clustering of siliques is a result of a reduction in cell elongation. In addition, ectopic lignification of the phloem cap cells, some phloem sieve cells, and regions of the disorganized fascicular cambium was observed as well as enhanced lignification of the interfascicular fibers. This is in contrast to what occurs in wild-type plants, where lignin deposition is limited to xylem and fully elongated interfascicular parenchyma cells (Dharmawardhana et al., 1992
Several Arabidopsis mutants that are defective in cell expansion and display ectopic lignification have been reported (Caño-Delgado et al., 2000 In addition to the cell elongation defect, cell division is also affected in broomhead plants. In some fascicular cambial regions, cell division is more prolific, although in the majority of vascular bundles, the cambium exhibits disorganized cell growth. This disorganization of the cambium is also associated with a corresponding enlargement of the nuclei, suggesting that the cell cycle has been disrupted such that DNA replication has become uncoupled from cell division. However, it is also possible that the nuclei appear enlarged because they are decondensed and actively transcribing. The fact that some fascicular cambial regions appear to be more active, while others exhibit disorganized cell growth and contain enlarged nuclei, even within the same stem section, may be directly related to the level of eRF1-1 down-regulation.
The eRF1-1 promoter-GUS experiments revealed that the expression pattern of eRF1-1 is primarily observed in the vascular tissues and the actively growing and elongating zones of Arabidopsis plants. The presence of GUS activity in the meristems and elongating zones appears consistent for a protein involved in translation, as it would be expected that protein synthesis is up-regulated to accommodate for new cell growth in these tissues. However, the observation that GUS activity was mostly restricted to the vascular tissues in the inflorescence stems and leaves was unexpected, although this may be due to a lack of sensitivity in the GUS staining method. Previously it has been shown that a number of promoter-GUS transgenics exhibit preferential staining in the vascular tissues (Gallagher, 1992
It has been previously established that a reduction in eRF1 protein levels decreases the efficiency of translation termination and therefore causes an increase in the translational readthrough of proteins (Stansfield et al., 1996
Further support for an alternative function comes from the various yeast eRF1 mutants that are recessive and have allele-specific pleiotropic effects such as temperature sensitivity (Inge-Vechtomov and Andrianova, 1975
So far, the most compelling evidence for an additional role for eRF1 has come from a study by Valouev et al. (2002) In conclusion, we have established that cosuppression of eRF1-1 in Arabidopsis has a dramatic effect on plant morphology, ultimately causing a reduction in internode elongation. The broomhead phenotype is the first report of any functional characterization of a release factor in vivo in Arabidopsis. However, the specific effects down-regulating eRF1 has on certain cell types, e.g. the cambial cells and the ectopically lignified cells, allow for speculation as to whether Arabidopsis eRF1-1 also functions in additional processes to that of translation termination. Examining the subcellular localization of eRF1-1, measuring the ploidy levels in broomhead plants, and screening for interacting proteins in Arabidopsis will provide further insight into the functions of eRF1-1 and may establish whether or not translation termination is its only role.
Plant Material and Growth Conditions For all phenotypic analyses, Arabidopsis (Arabidopsis thaliana) seeds were sown on soil and stratified for 2 to 5 d at 4°C. Trays of seed were then transferred to a temperature-controlled growth room and grown at 21°C under 50 µE/m2/s of long-day light (16/8 light/dark).
The eRF1-1 overexpression construct was produced by inserting the full-length eRF1-1 cDNA (GenBank accession no. H37361) into the EcoRI and BamHI sites of the binary vector pSOV2 (Mylne and Botella, 1998
eRF1-1, eRF1-2, and eRF1-3 promoter-GUS fusions were generated by cloning approximately 2 kb of sequence upstream of each of the translational start sites into the promoterless vector pAOV-intronGUS
Wild-type Arabidopsis plants (ecotype Columbia) were transformed using the floral dip method (Clough and Bent, 1998
Tissue samples were collected and immediately frozen in liquid nitrogen. RNA was extracted as described by Etheridge et al. (1999)
Genomic DNA was isolated from 2-week-old broomhead seedlings as previously described (Etheridge et al., 1999
Three biological replicates were performed on three independent broomhead lines. Total RNA was extracted from bud tissue (Promega SV Total RNA Extraction kit), and 1 µg was used to synthesize cDNA using reverse transcriptase and random hexamers in a reaction volume of 20 µL. Prior to quantitative PCR, cDNA samples were diluted 5-fold in water and then amplified using the ABI PRISM 7700 sequence detector and SYBR Green Master mix (Applied Biosystems). Gene-specific primers were designed to regions of low homology between the gene family members using Primer Express 1.5 software (Applied Biosystems). Primers used and locus identifiers are as follows: eRF1-1 forward, 5'-ACTGCCTTTGATTCCGAGGA-3'; eRF1-1 reverse, 5'-GCGATGGTGAGGATTTGATTG-3'; eRF1-2 forward, 5'-TTGATTTCTCCTTCTCCATCTTCG-3'; eRF1-2 reverse, 5'-CAGATCTCGATGTTCGTATCCG-3'; eRF1-3 forward, 5'-TTAAAGAACAACACGACCGGAGA-3'; eRF1-3 reverse, 5'-GCATCATGGAAATTGCTTTGGT-3'. Locus IDs are as follows: eRF1-1 (At5g47880); eRF1-2 (At1g12920); eRF1-3 (At3g26618). Expression detected from three
Stem sections from areas of reduced internode elongation in broomhead plants and the corresponding regions in wild-type plants were selected and fixed in ethanol:acetic acid (3:1) overnight. Samples were then dehydrated through a tertiary-butyl alcohol series, and flakes of Paraplast plus (Sigma-Aldrich) were added to the final step. Once the flakes dissolved at room temperature, liquefied Paraplast plus was added, and samples were transferred to an oven at 58°C. The medium was replaced after approximately 8 h, and samples were put back in the oven overnight. Stem samples were positioned in Paraplast plus, and microtome sections of 10 µm were cut, mounted on slides, and stained with safranin-O and fast green. Stained sections were photographed under bright-field illumination. To specifically detect the presence of lignin, deparaffinized and rehydrated tissue sections were stained for 5 min with phloroglucinol (2.5% [w/v] phloroglucinol in 6 N HCl) and rinsed in water. The slides were then mounted in 50% glycerol, 6 N HCl and visualized under a light microscope.
Whole plant specimens and small tissue sections were incubated at room temperature in chloroform for 15 min, then rinsed approximately two to three times in rinse solution (staining solution lacking 5-bromo-4-chloro-3-indolyl-
We thank Dr. Richard Moyle and Professor Robert Birch for critically commenting on the manuscript and past and present members of the Plant Genetic Engineering Laboratory for helpful discussions. Received March 13, 2005; returned for revision May 30, 2005; accepted May 30, 2005.
1 Present address: John Innes Centre, Colney, Norwich NR4 7UH, UK.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.062695. * Corresponding author; e-mail j.botella{at}uq.edu.au; fax 61–7–3365–1699.
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ABSTRACT
RESULTS
et al., 1996
35S. This vector is based on pAOV-intronGUS but lacks the CaMV 35S promoter. The primers used to clone all three promoter regions are as follows: eRF1-1, 5'-CGCTCGAGGGCTGATATGCGACAACCATAAAG-3' and 5'-CGAAGCTTCTTAACAATAGAACAAAAACC-3'; eRF1-2, 5'-CGAAGCTTCTTAGTTTATTGACCAGCCAGTTG-3' and 5'-CGAAGCTTTTTTAGCTTCTCTCGAGGTCG-3'; eRF1-3, 5'-CGCTCGAGCTGGCTCACCGAAGTTCGCGTAAG-3' and 5'-CGATCGATGTTTTTCCTCCTTCAAGC-3'.