JXB Advance Access originally published online on February 21, 2005
Journal of Experimental Botany 2005 56(414):1079-1091; doi:10.1093/jxb/eri099
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RESEARCH PAPER |
Genes encoding ADP-ribosylation factors in Arabidopsis thaliana L. Heyn.; genome analysis and antisense suppression
Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, ACT 2601, Australia
* To whom correspondence should be addressed. Fax: +61 2 6125 4331. E-mail: leigh.gebbie{at}anu.edu.au
Received 24 August 2004; Accepted 8 December 2004
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Abstract |
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Vesicle trafficking delivers proteins to intracellular and extracellular compartments, cellulose synthase to the plasma membrane, and non-cellulosic polysaccharides to the cell wall. The Arabidopsis genome potentially encodes 19 proteins with sequence similarities to ARFs (ADP-ribosylation factors) and its relatives such as ARLs (ARF-like proteins). ARFs are essential for vesicle coating and uncoating in all eukaryotic cells, while ARLs play more diverse roles. Nine proteins, six of them highly similar, are possible ARFs, three are putative ARL orthologues and the remainder were designated ARF-related proteins. The functions of the six highly similar, putative ARFs in whole plant development were probed by suppressing their expression with antisense. Antisense plants were severely stunted because cell production rate and final cell size were both reduced. Changed time-to-flowering, apical dominance, and fertility may reflect alterations to hormonal and other signalling pathways with which ARFs may interact. No gross changes in targeting or compartmentalization were seen in antisense plants containing GFP targeted to the ER and Golgi and changes in cell wall composition were limited to increases in some non-cellulosic polysaccharides and a relatively small decrease in cellulose. The reasons why these effects are less drastic than the effects on endomembranes and wall composition that are seen in short-term experiments with brefeldin A and with dominant negative ARF mutants are discussed.
Key words: ADP-ribosylation factor, antisense, Arabidopsis thaliana, cell division, cell expansion, vesicle trafficking
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Introduction |
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ADP-ribosylation factors (ARFs), a subfamily of the Ras superfamily of GTP-binding proteins, were first identified as cofactors required for cholera toxin-mediated ADP-ribosylation of a trimeric G protein
-chain (Kahn and Gilman, 1984
). They are now best known as GTP-dependent switches for the assembly/disassembly of the coat proteins driving vesicle budding/fusion. Guanine exchange factors (GEFs) convert inactive cytosolic GDP-ARF to active, membrane-associated GTP-ARF while GTPase activating proteins (GAPs) reform GDP-ARF. Coat proteins recruited from the cytosol by GTP-ARF sculpt the membrane into a bud and help capture specific membrane-bound receptors and cargo molecules. GTP hydrolysis dissociates ARF and then coat proteins from the membrane so that vesicles can dock and fuse with target membranes. Assembly of COPI-coated vesicles has received most attention in plants with their role in Golgi-to-ER and intra-Golgi transport (Pimpl et al., 2000; Ritzenthaler et al., 2002), but studies in other organisms show that ARFs take part in other forms of vesicle trafficking (Gaynor et al., 1998; Rudge et al., 1998; Yahara et al., 2001), activate phospholipase D (Brown et al., 1993), and regulate the actin cytoskeleton (Randazzo et al., 2000; Yahara et al., 2001). This gives ARFs a dynamic role in maintaining the integrity of organelles such as the Golgi and the ER so that there are far-reaching effects on endomembrane organization and dynamics (Nebenfuhr et al., 2002; Ritzenthaler et al., 2002) when the fungal metabolite brefeldin A (BFA) inhibits ARF GEFs (Steinmann et al., 1999). Mammalian and some other genomes encode several classes of functionally specialized ARFs (Moss and Vaughan, 1995).
ARF-like proteins (ARLs) show 30–60% amino acid identity with ARFs (which show >60% similarity amongst themselves), but cannot act as cofactors to activate the cholera toxin -subunit, cannot complement the arf1-arf2- yeast double mutant (Stearns et al., 1990), and probably play diverse roles in secretory and other pathways (Huang et al., 1999; Ingley et al., 1999; Lin et al., 2000). There are also ARF domain proteins (ARDs), which contain an ARF domain fused to a GAP domain (Vitale et al., 1996), and an ARF-related protein (ARP) of the plasma membrane (Schurmann et al., 1995).
Many putative components of the plant secretory system are identifiable by comparative genomics, but few putative ARFs identified in this way or by homology cloning are characterized beyond demonstrated GTP-binding. Recently, however, Gebbie (2002) and Takeuchi et al. (2002) have shown that expression of At2g47170 complements yeast arf1-arf2- mutants pointing to strong functional similarities of the proteins. The underlying switch function in plant ARFs appears similar to that in other organisms: BFA inhibited GEF-dependent GDP/GTP exchange (Steinmann et al., 1999), blocked recruitment of ARF and coat protein to Golgi-enriched membranes (Pimpl et al., 2000) and dissociated ARF from Golgi bodies in vivo (Ritzenthaler et al., 2002). Consistent with essential functions for plant ARFs, mutations in a putative Arabidopsis GEF were embryo lethal (Shevell et al., 1994; Busch et al., 1996), although antisense suppression of potato ARF produced only limited morphological changes (Szopa and Sikorski, 1995) with reduced cAMP levels (Wilczynski et al., 1997). Dominant negative mutants of At1g23490 (AtARF1 of Lee et al., 2002) and At2g47170 (AtARF1 of Takeuchi et al., 2002) disrupted targeting of various GFP constructs and retained several other GFP-tagged Golgi or vacuolar markers in the ER of cultured cells. Mutations in the ARF GEF GNOM affect the endocytic pathway (Nebenfuhr et al., 2002; Geldner et al., 2003; Jürgens, 2004) suggesting that one or more ARFs are active here.
Plant vesicle trafficking will serve similar functions to those seen in animals and yeast as well as delivering non-cellulosic polysaccharides to the cell wall. The key developmental processes of cytokinesis and cell expansion require vesicle trafficking to deposit new wall material and increase plasma membrane area. At cytokinesis, numerous secretory vesicles fuse to form the cell plate whose composition gradually changes from callose-rich to cellulose-rich (Samuels et al., 1995). During cell expansion, trafficking delivers proteins to plasticize the wall and supplies new plasma membrane and cell wall material to keep pace with growth (Thiel and Battey, 1998; Cosgrove, 2000).
This study analyses sequences in the Arabidopsis genome that potentially encode proteins with the ARF family signature and clarifies relationships within the Arabidopsis ARF family and with mammalian and yeast ARFs, ARLs and ARF-related proteins. Six highly similar sequences encoding putative ARF1 family members were identified. To investigate their role in whole plant growth, and development, antisense plants were generated in which expression of all six genes was suppressed. The strong phenotype was analysed, paying particular attention to its dependence on cell production rates and final size reached by cell expansion. This genomic and whole plant approach extends previous studies that have not identified the full range of relevant sequences in the genome and have involved short-term analyses in cultured cells.
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Materials and methods |
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Identifying ARF genes and generating antisense plants
The EST 122C2DT7 was identified (before completion of the genome sequence) through BLAST searches using the Arabidopsis ARF1 gene (Regad et al., 1993
). Sequence analysis showed that it is a full-length cDNA derived from At2g47170 (ARF1b in the study of McElver et al., 2000, ARFA1c in the study of Vernoud et al., 2003 whose nomenclature is followed). The remaining 18 ARF-signature proteins were identified in the complete Arabidopsis genome sequence by performing similar BLAST searches using the AtARFA1b gene and through published studies (Lebas and Axelos, 1994; McElver et al., 2000). The 1 kb ARFA1c insert was excised from 122C2DT7 (SalI/BamHI) and ligated behind the CaMV 35S promoter in the vector pDH51 (Pietrzak et al., 1986). The entire expression cassette was excised (EcoRI/SacI) and subcloned into the binary vector pBin19 (Bevan, 1984). Constructs were transferred into Agrobacterium (strain AGL1) and Arabidopsis plants (Columbia wild type) transformed using vacuum infiltration (Bechtold et al., 1993).
Gene expression
The MPSS database was searched as described by Meyers et al. (2004). Total RNA for northern analysis was extracted from leaves, electrophoresed, and probed (Burn et al., 2002) with full-length copies of the ARFA1c cDNA. Blots were also probed with an antisense riboprobe of the ubiquitin gene as a control for RNA loading. Genomic DNA was extracted for Southern analysis using the CTAB protocol (Dean et al., 1992) with an additional precipitation with 5 M potassium acetate and two chloroform extractions. DNA (5–10 µg) was digested with three of the following restriction enzymes: EcoRI, BglII, HindIII, NsiI, and XbaI and blotted onto charged nylon filters (Hybond N+, Amersham, Buckinghamshire, UK). Probes were generated by radio-labelling restriction fragments containing the entire ARFA1c insert with 32P-dCTP. Southern blots were hybridized with the ARFA1c probe using Rapid Hyb buffer (Amersham) for 4 h or overnight at 65 °C.
For RT-PCR, RNA from aerial tissue of 3-week-old plants was treated with RQ1 RNase-free DNase (Promega, Madison, WI) following the manufacturer's instructions. Primers to amplify 18S rRNA were from Cho and Cosgrove (2000). PCR primers (Table 1) to amplify the six ARFA1 genes and three related genes (At2g15310, At3g03120, and At5g17060) were designed so that the PCR product spanned an intron and included the highly variable 3' region. Primer specificity was checked by BLAST searches and by sizing and sequencing the products. RT-PCR was carried out using the Invitrogen (Carlsbad, CA) Superscript One-step RT-PCR kit with platinum Taq using 1 µg of RNA and following the manufacturer's instructions. The PCR cycle was: 20 min at 45 °C; 2 min at 94 °C; (15 s at 94 °C; 30 s at 55 °C; 30 s at 72 °C) x X cycles (Table 1); 7 min at 72 °C. The amount of RNA from wild-type and antisense plants was adjusted to generate 18S rRNA bands of similar intensity after RT-PCR amplification. To ensure that amplification stayed within the log phase, at least five reactions for each gene using wild-type and antisense RNA were set up and removed from the thermocycler to determine the optimal number of cycles for each gene (Table 1). Product amounts were determined visually by running 40 µl on 4% agarose gels. RT-PCR was repeated three times on three different RNA samples from wild type and three independent antisense lines (1a5, 1e3, and 6h9).
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Other methods
Burn et al. (2002)
described the cryo-scanning electron microscopy (cryo-SEM) methods and those to measure epidermal cell lengths and cell flux (number of cells added per day to each longitudinal cell file). Cellulose was determined as glucose insoluble in 2 M trifluoroacetic acid which was measured by GC/MS of alditol acetates (Lane et al., 2001) and the neutral sugars in non-cellulosic polysaccharides were determined by similar analyses of the trifluoroacetic acid-soluble material. Seeds of Arabidopsis expressing GFP targeted to the ER by fusion with a dilysine signal (Benghezal et al., 2000) or to the trans-Golgi network by fusion with the transmembrane domain of rat sialyltransferase (Boevink et al., 1998) were kindly provided by Dr David Jones (Australian National University) and Professor Chris Hawes (Oxford Brookes University), respectively. Plants were transformed with the ARF antisense construct and fluorescent plants selected with kanamycin since the GFP-ER plants were basta-resistant and the GFP-Golgi plants were hygromycin-resistant. Young rosette leaves from bolting plants that showed extreme ARF1 antisense phenotypes were examined for GFP fluorescence by laser scanning confocal microscopy (Leica SP2, Wetzlar, Germany). |
Results |
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Proteins potentially encoded by the Arabidopsis genome that resemble ARFs
The Arabidopsis genome potentially encodes 19 proteins containing the ARF family signature (PROSITE PDOC00781). Figure 1A shows an unrooted, bootstrapped, tree containing them and selected human (H) and yeast (Sc) ARF-related proteins. ARFA1a through ARFA1f (nomenclature of Vernoud et al., 2003
) were considered as putative Arabidopsis ARFs of class 1 since they clustered near human class 1 ARFs (HARF1, HARF2, and HARF3) although it was recognized that more information is needed to extend such a classification to plants fully. Others have already studied aspects of some members of this group (Regad et al., 1993; Pimpl et al., 2000; Ritzenthaler et al., 2002; Takeuchi et al., 2002, for AtARFA1c; Lee et al., 2002, for ARFA1a). The six putative Arabidopsis ARFA1s share many features of known importance in other ARFs (Fig. 1B) including: four regions binding GTP (Kahn et al., 1995); the effector region binding GAPs (Amor et al., 1994; Greasley et al., 1995; Vitale et al., 1997); the switch 1 and switch 2 regions binding GEF Sec7 domains (Mossessova et al., 1998); a potential myristoylation site at Gly-2 (Kahn et al., 1992; Antonny et al., 1997); the N-terminal 17 amino acids which impart ARF properties to a Drosophila ARL (Kahn et al., 1992); residues 35–94 which activate phospholipase D and recruit adaptor protein AP-1 (Liang et al., 1997). ARFA1b lacks ESTs but ESTs for the others suggest wide expression.
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At2g15310, At5g17060, and At3g03120 (Fig. 1A) could be additional ARFs with sufficiently divergent sequences to support the distinct functions associated with the three classes of mammalian ARFs (Moss and Vaughan, 1995
). Their N-terminal sequences, however, are not diagnostic for either ARFs or ARLs. At2g15310 has only one EST but several ESTs and full length cDNAs support the others.
The other ten putative proteins show lower levels of amino acid identity to ARFs and spread over areas of the tree containing ARLs, HARP (Schurmann et al., 1995) and HARD (Vitale et al., 1996) (Fig. 1A). Three are potential orthologues of individual human and yeast ARLs: At2g24765 (AtARF3 of Lebas and Axelos, 1994; Lee et al., 2002) and ScARL1 and HARL1; At3g22950 and HARL5; At2g18390 (TITAN5) and HARL2 (as concluded by McElver et al., 2000). At5g52210 (AtGB1 of Biermann et al., 1996) is a potential orthologue of HARP, a plasma membrane protein (Schurrman et al., 1995). All have EST support.
The other six proteins form two clusters not obviously associated with mammalian or yeast sequences. Cluster 1, the products of two adjacent genes (At1g02430 and At1g02440) is supported by only 1 EST (for At1g02430). Cluster 2, the products of two adjacent genes on chromosome 3 (At3g49860, 1 EST and At3g49870, 10 ESTs) and two non-adjacent genes on chromosome 5 (At5g67560, 12 ESTs and full length cDNA and At5g37680, 2 ESTs and full length cDNA). The GTP-binding domains of cluster 1 sequences more closely resemble the consensus sequence for other GTP-binding proteins such as Rabs than those for ARFs (Kahn et al., 1995). Cluster 2 proteins show some homology to HARD, but lack its GAP domain. At3g49860 and At3g49870 lack the 17 N-terminal amino acids (including the Gly-2 myristoylation site) that transfer an ARF function to a Drosophila ARL (Kahn et al., 1992). The switch regions of At5g37680, At5g67560, and At3g49870 differ from those of ARFs which may prevent them interacting with GEFs having the Sec7 domain present in all known ARF GEFs (Donaldson and Jackson, 2000).
In summary, the six Arabidopsis sequences (ARFA1a through ARFA1f) were considered as putative class 1 ARFs and three (At2g15310, At5g17060, and At3g03120) as potentially functionally differentiated ARFs that, when properly characterized, might be assigned to classes 2 onwards if appropriate. Three other proteins are probably ARL orthologues, one perhaps a HARD orthologue, but six other proteins cannot be plausibly linked to proteins known in other taxa.
To establish that members of the AtARFA1 class were authentic ARFs, the yeast arf1-arf2- mutant (RT166) of Kahn et al. (1991) was complemented with AtARFA1c (Gebbie, 2002). Takeuchi et al. (2002) published similar results. These findings show that a representative of the ARFA1 class can serve many of the important functions served by yeast ARFs. To explore their functions in intact Arabidopsis plants, expression was suppressed using an ARFA1c antisense construct.
Antisense suppression
The cDNA encoding AtARFA1c was cloned in antisense behind the 35S CaMV promoter and transformed into wild-type Columbia plants. About a third of T1 kanamycin-resistant transformants showed obviously reduced stature (Fig. 2A, B) and some plants with severe phenotypes died or were infertile. This, like other antisense phenotypes (Burn et al., 2002), was highly unstable. T1 plants with an antisense phenotype could produce all wild-type progeny or show non-Mendelian segregation for the presence or absence of a phenotype. Phenotype severity also varied considerably within each line and homozygous T3 lines still segregated wild-type individuals. Homozygotes were not obtained for some lines even with repeated screening, presumably because of lethality.
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Molecular basis of the antisense phenotype
The MPSS database (Meyers et al., 2004
) shows low ARFA1c expression and even lower ARFA1b expression in all tissues whereas ARFA1a, ARFA1d, ARFA1e, and ARFA1f are expressed widely with relatively small fold differences between different organs (Fig. 2C). Semi-quantitative RT-PCR (Fig. 2D) also showed expression of all except ARFA1b (not detected even after 40 PCR cycles). Expression of all five ARFA1 genes (>80% nucleotide identity) was strongly reduced in three antisense lines, but expression of the less closely related (<60% identity) putative ARFs (At2g15310, At3g03120, and At5g17060) was unaffected (Fig. 2D).
A single band of approximately 0.8 kb was detected by northern analysis with the ARFA1c EST probe. Because Southern analysis with the same probe detected at least some bands of the predicted size for each of the six ARFA1 genes (Gebbie, 2002), it is thought that the band in northern analysis also reflects expression of all ARFA1 genes. The severity of the antisense phenotype broadly correlated with the degree to which ARFA1 expression seen on northern analysis was reduced in that particular line. A T3 antisense line showing a strong phenotype (6h5) and one which had reverted to wild type (6h3) showed very low and near wild-type levels of ARFA1 expression, respectively (Fig. 2E) and seven different T2 antisense lines expressed ARFA1 at between 12% (line 1d4) and 45% (line 5d14) of wild type, with 5d14 showing the weakest phenotype (Fig. 2F). No lines expressed antisense mRNA at high levels (not shown), consistent with antisense RNA degrading together with endogenous ARFA1 mRNA.
Taken together, it is believed that the antisense phenotype reflects reduced expression of ARFA1a, ARFA1c, ARFA1d, ARFA1e, and ARFA1f without reduced expression of the three, potentially functionally differentiated, ARFs (At2g15310, At3g03120, and At5g17060).
Morphology of antisense plants
Antisense plants grown on soil showed cotyledons and rosette leaves whose blades and petioles were smaller than wild type (Fig. 3A). Leaf blade epidermal cells were smaller but remained lobed (Fig. 3B, C). Stomates (Fig. 3B, C) and trichomes (not shown) were apparently unaffected except that spacing was reduced, presumably by the intervening epidermal cells being smaller. Palisade and spongy mesophyll differentiation persisted although air spaces and leaf thickness were reduced (Fig. 3D, E). Rosette leaf production rates and the transition to flowering (at day 21 in the wild type) were slowed in antisense plants, which began bolting at various times up to day 43. Rosette leaf number increased until bolting (Fig. 4A, B). Stem elongation varied widely in different antisense lines (Fig. 4C); high growth rates were maintained for only short periods whereas slow-growing plants (as low as 3 mm d–1 versus 23 mm d–1 for the wild type) might still be growing at day 90, some 40 d after the wild type finished. Even at day 90, however, they might only be about 20 mm tall versus 350 mm for the wild type. Apical dominance was often lost; some antisense plants initiated three or four bolts almost simultaneously with the primary bolt and, by senescence, could have up to 10 inflorescences compared with three or four for the wild type (Fig. 4D).
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Antisense flowers resembled those of antisense CesA plants (Burn et al., 2002
) in being smaller than the wild type (Fig. 5A–D) with highly reduced sepals and petals leaving a protruding stigma that appeared normal (Fig. 5B) except in the most severe phenotypes (Fig. 5C, D). Flower and so silique spacings were irregular (Fig. 5E, F), fewer flowers initiated and few produced fertile siliques (Fig. 5G, H) because shortened stamen filaments left the anthers below the receptive stigma and/or anthers failed to dehisce (Fig. 5B–D) or dehisced only after the stigma became unreceptive. Stamen filament cells were shorter than those in the wild type (Fig. 5I, J) as were epidermal cells in petals and sepals (not shown). Pollen was abundant and appeared normal.
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The antisense construct produced only mild changes in seedlings even when later development was severely affected. Seedling root growth on agar plates was not consistently and obviously reduced so it could not be predicted which plants would subsequently develop an antisense phenotype in aerial organs. Older, soil-grown plants showing an aerial phenotype had smaller root systems (not shown), but it was unclear to what extent this was a secondary consequence of reduced shoot growth.
Cellular and subcellular aspects of the phenotype
Finally we measured whether the antisense construct affected cell division, cell expansion, and cell wall deposition—processes underlying growth and strongly dependent on vesicle trafficking—and whether endomembrane arrangement and protein targeting were disrupted as reported with BFA and dominant negative mutants.
Stem elongation rate strongly correlated with both cell flux (a measure of cell production rate; r=0.94 for linear regression) and cell length (r=0.93) showing that antisense reduced both cell division and expansion (Fig. 4E, F). Antisense stems were also thinner than the wild type, with fewer epidermal cell files rather than cells with smaller cross-sectional areas (not shown).
Deposition of non-cellulosic wall polysaccharides and delivery of cellulose synthase to the plasma membrane require vesicle trafficking (Samuels et al., 1995; Thiel and Battey, 1998; Cosgrove, 2000) and so might be reduced in antisense plants. However, it was found (Fig. 6) that, although cellulose in rosette leaves (nmol glucose mg–1 tissue dry weight) declined slightly with increasingly severe antisense phenotypes, non-cellulosic sugars increased (rhamnose, fucose, arabinose, xylose, galactose) or remained unchanged (mannose).
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ARFA1c antisense suppression in plants expressing GFP in the ER (dilysine ER-retention sequence; Benghezal et al., 2000
) or Golgi (rat sialytransferase; Boevink et al., 1998) did not change ER or Golgi organization or targeting of the tested proteins. Upper epidermal and palisade mesophyll cells from rosette leaves of varying ages showed ER and Golgi resembling those seen in wild-type leaves, even when the antisense phenotype was severe (Fig. 7A, B).
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Discussion |
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We identified six, closely related Arabidopsis proteins as putative class 1 ARFs (ARFA1s in the terminology of Vernoud et al., 2003
), three additional putative ARFs, three potential orthologues of ARLs and of ARD, and six proteins not obviously related to proteins outside plants. One of the six ARFA1s complements a yeast ARF mutant and, in antisense, reduces Arabidopsis growth (affecting both cell division and cell expansion) and produces morphological abnormalities. The severe, at times lethal, effects on Arabidopsis contrast with the mild effects of a similar construct on potato morphology (Szopa and Sikorski, 1995; Wilczynski et al., 1997). Changes in wall composition and endomembrane arrangements are much milder in antisense Arabidopsis plants than those seen with short BFA treatments or dominant negative mutants.
ARF-related sequences in the Arabidopsis genome
Size, amino acid sequence, phylogenetic analysis, and gene structure place mammalian (Moss and Vaughan, 1995) and Drosophila (Lee et al., 1994a) ARFs in three classes. Six closely related Arabidopsis ARFA1 proteins lie close to the three class 1 mammalian ARFs (HARF1, HARF2, and HARF3) in the unrooted tree whereas At5g17060, At3g03120, and At2g15310 lie further from them but still within a region suggesting that they may be genuine ARFs. The six ARFA1s are so similar as to preclude the functional differentiation seen in the more divergent mammalian and Drosophila ARFs, but the other three putative ARFs of Arabidopsis certainly could be, since they diverge more from the ARFA1s than the human class 2 (HARF4 and HARF5) and class 3 (HARF6) ARFs diverge from human class 1 ARFs. Further work is needed to see if the Arabidopsis classes have a functional basis. MPSS data suggest that the five strongly expressed ARFA1 genes have wide and often overlapping expression patterns that, together with their likely lack of functional specialisation, may lead to redundancy.
Sequence analyses suggest there are potential Arabidopsis orthologues of HARL1 (At2g24765), HARL5 (At3g22950), HARL2 (At2g18390; TITAN5 of McElver et al., 2000), and HARP (At5g52210). Only HARL2 and TITAN5 have functional information to support the assignment (Tzafrir et al., 2002). The putative HARL1 orthologue At2g24765 was first described by Lebas and Axelos (1994) who noted its similarities to ARLs, but designated it ARF3. Takeuchi et al. (2002) found that it failed to complement the yeast arf mutant, consistent with it not being a true ARF. Some findings of Lee et al. (2002) are also consistent with this although they were not interpreted as such. They compared the effects of dominant negative mutations in this gene (At2g24765, their ARF3) with the effects of the corresponding dominant negative mutation of ARFA1a (At1g23490, their ARF1). They found that mutating At2g24765 did not change plasma membrane and Golgi marker protein trafficking and did not disassemble Golgi or drastically remodel the ER in the way that corresponding mutants of At1g23490 (ARFA1a) did. It is suggested that these differences probably reflect differences between ARLs (e.g. At2g24765) and ARFs (e.g. ARFA1a) rather than differentiation amongst ARFs as their discussion and nomenclature (ARF3 and ARF1) implies.
The other Arabidopsis proteins with ARF signatures have no obvious relationships with known ARLs or similar proteins. Homology to the ARF family suggests likely roles in membrane trafficking, signalling or cytoskeleton rearrangement, but these will require direct functional analysis. No ESTs are known for some sequences although full-length cDNAs are being found for some genes previously lacking EST support (e.g. At2g15310 and At1g02430) and strong sequence conservation suggests continuing selective pressure consistent with restricted or low expression undetected by ESTs rather than their being pseudogenes.
Molecular basis for the antisense phenotype
The ARFA1c antisense construct reduced expression of the five ARFA1s detected by semi-quantitative RT-PCR. Given >80% nucleotide identity, this conforms with experience in other gene families (Waterhouse et al., 1999; Stam et al., 2000; Lally et al., 2001). Consistent with reduced expression causing the phenotype, northern analysis, thought to detect all expressed ARFA1 genes, correlated reduced ARFA1 gene expression and the severity of the morphological phenotype. Given the lack of organ-specific expression of different ARFA1s and suppression of all five genes by antisense, a widespread phenotype is not surprising. Seedlings are a surprising exception showing no clear phenotype even though they will have active trafficking, express ARFA1 genes and 35S-driven constructs (Burn et al., 2002). One possible explanation is that lines showing strong antisense suppression were inadvertently discarded at the seedling stages when vigorous and healthy seedlings were selected as kanamycin-resistant. Whatever the explanation, lack of seedling phenotype with ARF antisense should not be taken to indicate lack of seedling function for ARF gene products, since antisense suppression of CesA1 also produced no phenotype (Burn et al., 2002), whereas a mutant CesA1 allele (rsw1) has a strong seedling phenotype (Baskin et al., 1992).
Reduced cell division and cell expansion
Cell division and cell expansion both depend on vesicle trafficking to deposit new wall material, to secrete proteins (e.g. expansins) that promote wall yielding and to deliver new plasma membrane proteins for cellulose synthesis and other needs (Samuels et al., 1995; Thiel and Battey, 1998; Cosgrove, 2000). Kinematic analysis quantified cell production rates and final cell size in intact plants and showed that reductions in both strongly correlate with reduced stem elongation rates in antisense plants. An effect on cell division is consistent with gnom (affecting an ARF GEF) blocking embryo cell divisions (Shevell et al., 1994; Steinmann et al., 1999). gnom's drastic, early phenotype may obscure cell expansion effects. Incomplete cell walls in antisense plants comparable with those seen in tobacco cells treated with BFA were not seen (Yasuhara et al., 1995), although they were not sought exhaustively.
Reducing cell division and expansion could directly reflect changes in the rate at which trafficking delivers materials to expand the plasma membrane and to construct and plasticize walls. However, many links exist between secretory pathways and hormonal or other signalling pathways. For example, antisensing potato ARF changed cAMP levels (Wilczynski et al., 1997); gnom (defective ARF GEF) mistargeted the PIN1 auxin transporter through effects on endocytosis (Steinmann et al., 1999; Grebe et al., 2002; Nebenfuhr et al., 2002; Geldner et al., 2003; Jürgens, 2004); antisense suppression of a rab11-related protein altered ethylene production, reduced apical dominance and changed floral structure (Lu et al., 2001); an ARF GAP bound phosphatidylinositol 3-monophosphate (Jensen et al., 2000); BFA affected pectin endocytosis and so perhaps pectin signalling (Baluska et al., 2002). BFA also affects the actin cytoskeleton in some cells (Randazzo et al., 2000). Such less direct effects of ARFA1c antisense may also impact on cell division and cell expansion less directly and could explain the changes in higher levels of organization such as the delayed flowering onset with greatly increased leaf number and reduced apical dominance.
Subcellular changes in antisense plants differ from changes by short-term secretory blocks
The arrangement of Golgi and ER membranes and protein targeting changed drastically in many yeast ARF mutants (Gaynor et al., 1998; Yahara et al., 2001) and in at least some plant cell types (Baluska et al., 2002) when BFA inhibited ARF GEF(s) (discussed in Ritzenthaler et al., 2002) or when transiently expressed, dominant negative mutations of ARFA1a and ARFA1c competed for interacting factors or regulators such as GEFs and effectors (Lee et al., 2002). However, no changes in membrane arrangement or protein targeting even in severely dwarfed ARF antisense plants were detected in this work. Such differences from BFA and dominant negative mutations may arise because antisense plants probably have less ARF, but what remains probably cycles normally through the GDP/GTP changes underlying function. This may support reduced but balanced fluxes of vesicles between the various compartments whereas dominant negative mutations will disturb that cycling by increasing competition for GEFs (Lee et al., 2002) and BFA will selectively release ARF from Golgi membranes (Ritzenthaler et al., 2002).
Cell wall composition presents one opportunity to see differences in a structure assembled by vesicle trafficking. It was found that several monosaccharides from non-cellulosic polysaccharides increased, but cellulose decreased in severe antisense plants. Increases in non-cellulosic polysaccharides delivered in Golgi-derived vesicles were unexpected, but might reflect reduced endocytosis of pectins (Baluska et al., 2002), mislocalization of pectin (as seen in gnom) or other polysaccharides (Shevell et al., 2000), or reduced secretion of enzymes degrading matrix polysaccharides. Cellulose deficiency produced by mutations in the KORRIGAN endo-1,4-ß-glucanase (Lane et al., 2001) reduces cell division and cell expansion, just as ARFA1c antisense does. The ARF antisense phenotype is not, however, a typical cellulose deficiency phenotype (Lane et al., 2001; Burn et al., 2002) in that, for example, leaf epidermal cells remain lobed and changes in flowering onset and leaf number occur in ARFA1c antisense plants, but not in CesA or KOR cellulose-deficient mutants. Compared with the ARFA1c antisense plants, short-term BFA treatments more strongly reduced incorporation of cellulose into both primary and secondary walls and reduced (rather than increased) incorporation of monosaccharides into matrix polysaccharides (Driouich et al., 1993; Lanubile et al., 1997; Piro et al., 1999; Rojas et al., 1999; Thompson and Fry, 2001). Differences between antisense ARFA1c and BFA responses could reflect BFA's strong effects on endomembrane architecture and protein targeting. Plants also may accommodate to long-term antisense inhibition as they apparently do to BFA or low-temperature treatments (Boevink et al., 1999). An inducible ARFA1 antisense construct could test whether plants adapt to constitutive antisense expression.
Experimental strategies for studying ARFs in vivo
Arabidopsis ARFA1s have now been studied by complementing yeast mutants, in cultured cells of Arabidopsis by dominant negative mutations and in whole Arabidopsis plants by antisense. All approaches have value, as will conventional single gene mutants whose properties have not yet been reported.
ARFA1c (Gebbie, 2002; Takeuchi et al., 2002) joins genes encoding yeast ARF2, members of all three classes of mammalian ARFs and genes from species such as Giardia and Drosophila in complementing the yeast arf1-arf2- mutant (Kahn et al., 1991; Moss and Vaughan, 1998) whereas yeast ARF3 (Lee et al., 1994b) and ARL1 (Lee et al., 1997) do not. This suggests ARFA1s serve enough of the functions performed by yeast ARF1 and ARF2 (which act at several points in the secretory pathway and not just ER–Golgi steps; Gaynor et al., 1998; Rudge et al., 1998; Yahara et al., 2001) to make arf1-arf2- viable. This requires meeting known constraints acting on yeast ARF such as the myristoylation requirement (Kahn et al., 1995; Click et al., 2002) and the conservation of the region interacting with yeast ARF GEF (Click et al., 2002), a conclusion reinforced by complementation of a yeast ARF GEF mutant by an Arabidopsis gene (Steinmann et al., 1999). Complementation does not test whether ARFA1c activates phospholipase D or interacts with G proteins, properties characteristic of mammalian but not yeast ARFs (Dietzel and Kurjan, 1987; Nakafuku et al., 1988; Rudge et al., 1998). It is probably significant, however, that the exact sequence needed for mammalian ARF1 to bind phospholipase D (Liang et al., 1997) is conserved in ARFA1s but not in yeast ARFs (Fig. 1B).
Dominant negative ARF mutants expressed in cultured plant cells (Lee et al., 2002; Takeuchi et al., 2002) confirm the involvement of ARFA1s in trafficking between ER and Golgi, but have not yet settled whether they also function at other steps in the secretory pathway, such as vacuolar transport (Pimpl et al., 2003). Antisense plants allow analysis of whole plant phenotypes, some of whose features may reflect reduced vesicle trafficking whereas others may reflect links between trafficking and signalling or cytoskeleton events. Insertional or classical mutants will best separate the functions of the individual, possibly redundant Arabidopsis ARFA1s since antisense suppresses all five expressed ARFA1s and dominant negative constructs (Lee et al., 2002) will also not discriminate among them if all compete for the same regulators and effectors. Potential paralogues of ARF GEFs and GAPs could allow specific interactions with individual ARFA1s, but the putative paralogues may interact with some of the 13 other ARF-signature proteins rather than specific ARFA1s.
In conclusion, ARFA1 antisense reduces cell division, cell expansion, and cellulose production, processes that directly depend on vesicle trafficking for processes such as cell wall construction. Other changes (delayed flowering, reduced apical dominance) are more plausibly related to changed hormonal/signalling pathway activities. These striking changes in growth rate and morphology in antisense plants occur without the major disruptions to endomembrane architecture, protein targeting, or wall composition caused by short-term exposure to BFA and, in the case of endomembranes and targeting, by transient expression of dominant negative ARFA1 constructs.
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We thank Roger Heady for cryo-SEM, Rosemary Birch for statistical analysis, Ann Cork for carbohydrate analyses, and Daryl Webb for confocal microscopy. This work was supported by an Australian Postgraduate Award for Industry from the Australian Research Council and by North Forest Products.
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