The BON/CPN gene family represses cell death and promotes cell growth in Arabidopsis

Authors


*(fax +607 255 5407; e-mail: jh299@cornell.edu).

Summary

Copines are calcium-dependent membrane-binding proteins that are highly conserved among protozoa, plants, nematodes and mammals. Although they are implicated in membrane trafficking and signal transduction, the functions of these proteins are not well understood. The Arabidopsis copine gene BON1/CPN1 was previously shown to negatively regulate a disease resistance (R) gene SNC1. Here we report that in Arabidopsis, as in other organisms, there is a family of copine genes, BON1, 2 and 3. Using double and triple mutant combinations we show that these three copine genes have overlapping functions essential for the viability of plants. The loss of function of BON1 combined with that of BON2 or BON3 leads to extensive cell death phenotypes resembling the hypersensitive response (HR) in defense responses. The resulting lethality can be suppressed by mutations in PAD4 or EDS1 which are required for R gene signaling and cell death control. Accession-dependent phenotypes of the mutant combinations suggest that the BON/CPN genes may together repress several R genes other than SNC1. Moreover, the mutant combinations exhibit developmental defects when R-gene-mediated defense responses are largely suppressed in pad4 and eds1 mutants. Thus, the copine family in Arabidopsis may have effects in promoting growth and development in addition to repressing cell death, and these two processes might be intricately intertwined.

Introduction

Copines are evolutionarily conserved proteins present in protozoa, plants, nematodes and mammals (Creutz et al., 1998). Their ubiquitous presence in diverse organisms and their high sequence conservation indicate that copines have important functions in cellular processes. A role of copines in membrane trafficking and signal transduction is suggested by their structure and biochemical activities. The copine proteins are characterized by two C2 domains at the amino-terminus and an A domain at the C-terminus. C2 domains are calcium-dependent phospholipid-binding domains that confer calcium or lipid regulation activities on the proteins in which they reside (Rizo and Sudhof, 1998). The A domain of copines shows homology to the von Willebrand A domain found in integrin, and this domain is involved in protein–protein interaction (Williams et al., 1999). Indeed, potential interacting proteins of copine A domains have been isolated and these proteins are mostly signaling molecules often with coiled-coil domains (Tomsig et al., 2003). Recently, human copine III has been suggested to possess an intrinsic protein kinase activity in the A domain (Caudell et al., 2000), and copines may therefore have signaling activity. The biological functions of copine proteins are not well understood. The Arabidopsis BON1/CPN1 gene is the first in the copine family whose function has been revealed genetically.

bon1 was isolated as a mutant with a temperature-dependent growth defect and an enhanced disease resistance phenotype (Hua et al., 2001; Jambunathan et al., 2001). Further study of a natural modifier of bon1-1 revealed that BON1 acts as a repressor of a disease resistance (R) gene. The R gene interacts with a pathogen avirulence (Avr) gene in an allele-specific manner to trigger a hypersensitive response (HR) upon pathogen invasion. HR is a form of programmed cell death characterized by rapid calcium and ion fluxes, an extracellular oxidative burst and transcriptional reprogramming, followed by cell death. This form of defense responses halts pathogen growth rapidly and efficiently (Scheel, 1998). BON1 negatively regulates a haplotype-specific R gene, SNC1, present in the Columbia (Col) accession but not in the Wassilewskija (Ws) accession (Yang and Hua, 2004). The single mutant bon1-2 in the Ws accession and the double mutant bon1-1 and loss-of-function snc1-11 in Col have a wild-type appearance and do not exhibit enhanced disease resistance. Furthermore, the growth and defense defects of bon1 mutants can be suppressed either by lowering the amount of salicylic acid (SA) or by mutations in PAD4 or EDS1, two genes required for R gene function (Glazebrook, 2001). Thus, BON1 negatively regulates defense responses by repressing an R gene, SNC1, although the mechanism is not known.

This regulation supports a recent notion that R proteins do not necessarily function as receptors for pathogen effector Avr proteins as in the simple biochemical model postulated for ’gene-for-gene’ interaction (Flor, 1971). Rather, R proteins may ’guard’ cellular machinery, that is, they monitor the status of plant host proteins (Belkhadir et al., 2004b; Martin et al., 2003; Schneider, 2002; Van der Biezen and Jones, 1998). Pathogen effector Avr proteins may target (modify) host proteins to perturb cell physiology and cellular homeostasis in plants in favor of their own survival and propagation. Plants counteract this effect by recognizing modification of these few host proteins to monitor pathogen invasion. One such host protein supporting this ‘guard hypothesis’ is RIN4, which is guarded by at least two R proteins, RPM1 and RPS2 (Axtell and Staskawicz, 2003; Belkhadir et al., 2004a; Mackey et al., 2002, 2003). The loss of RIN4 function activates RPS2 and RPM1, leading to cell death and lethality. Thus, host genes such as RIN4 and BON1 could act as negative regulators of R genes and are repressors of cell death associated with defense responses. It is yet to be determined whether BON1 regulates R in a similar manner, biochemically, to the way in which RIN4 regulates RPM1 and RPS2. It is also yet to be determined what cellular functions these host proteins have, although it is speculated that they have important roles in basic cellular functions for growth and development.

We have characterized each member of the BON/CPN family to reveal potential overlapping and distinct functions of the gene family. This analysis not only allows further dissecting of the mechanisms of defense and growth regulation in plants, but also has broad implications because copines appear to exist as a gene family in many different organisms. Here we report the molecular analysis of single and multiple mutants of the BON/CPN family in Arabidopsis. Members of this gene family have overlapping functions essential for the viability of plants. They act as negative regulators of the cell death pathway, possibly through R-gene-mediated resistance, and they may have additional roles in promoting growth and development.

Results

BON1 has two closely related paralogs, BON2 and BON3, in Arabidopsis

The Arabidopsis genome contains two other copine genes in addition to BON1, and we designate them BON2 (At5g07300) and BON3 (At1g08860). Complimentary DNAs (cDNAs) of BON2 and BON3 were isolated by cDNA library screening and reverse transcriptase–polymerase chain reaction (RT-PCR), respectively. The GenBank number for BON2 cDNA is AY741135 and that for BON3 cDNA is AY741136.

Comparison of the genomic and cDNA sequences of the BON genes revealed that BON2 and BON3 share similar exon/intron structures to that of BON1 (Hua et al., 2001; Figure 1a), that is, they have the same number of introns at the same locations. BON1, BON2 and BON3 proteins are predicted to have 578, 587 and 585 amino acids, respectively (Figure 1b). BON1 and BON2 are most closely related, sharing 82% identity and 91% similarity in amino acid sequences over the entire encoded proteins. BON3 is more distantly related to BON1 and BON2, with 62% identity and 75% similarity to BON1. All three proteins contain three canonical domains found in copines: two C2 domains (C2A and C2B) and an A domain (Figure 1b).

Figure 1.

Structure and sequence comparison of the BON/CPN family.
(a) Schematic diagram of genomic fragments of the BON2 and BON3 genes. Exons are represented by open rectangles, and introns or intergenic regions are represented by lines. Dotted lines represent annotated untranslated region (UTR) sequences of BON3. The arrowhead indicates T-DNA insertion sites in bon2 and bon3 mutants.
(b) Amino acid sequence alignment of the BON/CPN proteins. C2A, C2B, and A domains are indicated by dotted lines above the sequences. Potentially essential residues for protein activities are indicated as follows: *, aspartates for calcium binding; x, residues for magnesium binding; ∧, glycine in the ATP-binding loop; ∼, lysine for kinase activity.
(c) Phylogenic tree of BON1/CPN1 homologs (copine genes) in Arabidopsis, rice (Oryza sativa) and tomato (Lycopersicon esculentum). The GenBank ID numbers of genes are given, except for the BON genes from Arabidopsis. Rice has three copine members, with GenBank IDs 2341.t00024, 6083.t00006, and 4496.t00018. Currently there are two copine members in tomato from the expressed sequence tag (EST) database: SGN-U145780 and U160222, and another entry U164635 appears to be the same as U160222. At, Arabidopsis thaliana; Le, Lycopersicon esculentum; Os, Oryza sativa; Cr, Chlamydomonas reinhardtii.

The BON proteins have all the characteristic residues of calcium-binding C2 domains. The aspartates postulated to coordinate calcium binding are found in both C2 domains in all three proteins (Figure 1b). They correspond to D63, D69, D122 and D124 in the first C2 domain and D209, D215, D269 and D271 in the second C2 domain of BON1. Consistent with sequence conservation, the BON1 protein has been shown to possess calcium-dependent lipid-binding activity and to be associated with the plasma membrane (Hua et al., 2001). A domains of these proteins also appear to be canonical. Residues coordinating the binding of magnesium demonstrated in the integrin A domain are found in all BON proteins, and they correspond to D347, T349, S351, T445 and D477 in BON1 (Figure 1b). The A domain of human copine III appears to possess intrinsic protein kinase activity, and structures that resemble the conserved kinase motifs and residues essential for kinase activity (Caudell et al., 2000). Sequence alignment also revealed the presence of these essential residues in BON proteins (Figure 1b). Most prominent is the FTASNG motif (G353 in BON1), probably representing the glycine loop found in most of the kinase domains responsible for ATP binding (Hardie and Hanks, 1995). An invariant lysine residue essential for maximal enzyme activity (Hardie and Hanks, 1995) is found in all BON proteins (K391 in BON1), suggesting that BON proteins may also possess kinase activities.

A comparison of the copine genes in Arabidopsis with those identified in Paramecium, Chlamydomonas, worms, humans, mice, Arabidopsis, tomato (Lycopersicon esculentum) and rice (Oryza sativa) suggests that there was probably one ancient copine gene in the ancestor of higher plants. We analyzed in more detail copines from three plant species: three from Arabidopsis, three from Oryza sativa and two from Lycopersicon esculentum, where relatively comprehensive sequence data are available. A molecular phylogenic tree was constructed for all eight genes using one of the copines from C. reinhardtii as an out-group (Figure 1c). BON1 and BON2 from Arabidopsis are closely clustered in a node, which itself clusters with a two-gene node from tomato and subsequently a two-gene cluster from rice. BON3 is outside the node of these three-gene pairs and it clusters with the third gene from rice. This tree suggests that there were two copine genes in the ancestral plant species before the divergence of monocots and dicots. One of them evolved into BON3 in Arabidopsis. The other underwent a gene duplication to become BON1 and BON2 in Arabidopsis after the divergence of Arabidopsis and tomato.

Single loss-of-function mutants of BON2 and BON3 are wild type in phenotype

To investigate the biological functions of the BON1 gene family, we isolated and characterized loss-of-function mutants of the BON2 and BON3 genes. bon2-2 and bon3-3 were identified in the Columbia (Col) accession and locations of T-DNA insertion in these mutants are shown in Figure 1a. We found that both bon2-2 and bon3-3represent loss-of-function mutants. bon2-2 has a T-DNA inserted in intron 14, and produces a BON2 transcript that is much weaker and larger than that of the wild type (Figure 2a). This aberrant transcript does not contain 3′ sequences of BON2 after the T-DNA insertion site (determined by RNA blot analysis), and it likely consists of 5′ sequences of BON2 followed by some T-DNA sequences. bon3-3 has a T-DNA insertion at nucleotides corresponding to amino acid 381 within exon 11, likely resulting in truncated protein even when transcribed.

Figure 2.

Single mutants of BON2 or BON3 do not have abnormal phenotypes.
(a) Expression of BON1, BON2 and BON3 in Columbia (Col), bon1-1, bon2-2 and bon3-3. BON1 and BON2 expression was analyzed by RNA blots. rRNA transcripts were used as controls. As BON3 cannot be detected by RNA blot analysis, reverse transcriptase–polymerase chain reaction (RT-PCR) was used to amplify BON3 transcripts from the wild type and the mutant. Two different sets of biological samples were analyzed and the tublin 2 (TUB2) gene was used as a control.
(b) bon1-1, but not bon2-2 or bon3-3, has a growth phenotype.

No abnormal growth or development phenotypes were found in bon2 or bon3 loss-of-function mutants under standard growth conditions, in contrast to the bon1 mutant where a growth defect was observed (Figure 2b). Consistent with these findings, while the defense response marker gene PR1 was greatly up-regulated in bon1-1, no PR1 expression was observed in the bon2 or bon3 mutant under normal growth conditions (data not shown). In bon1-1, BON2 and BON3 were up-regulated, as indicated by Northern blot and RT-PCR analyses, respectively (Figure 2a). In contrast, BON1 expression was the same in bon2-2 and bon3-3 mutants as in the wild type (Figure 2a). In summary, the loss of either BON2 or BON3 function had no obvious effect on growth, development or defense responses.

BON3 overexpression rescues the bon1-1 mutant phenotype

The fact that the bon1 mutant has a dramatic mutant phenotype whereas bon2 and bon3 do not suggests that there may be differences in the biochemical activities, expression domains, or expression levels of these proteins. That BON1 and BON2, but not BON3, are detected by RNA blot analysis suggests that these three genes might be under differential transcriptional or post-transcriptional control. To determine whether up-regulating the expression level and expanding the expression domain can allow BON3 to substitute BON1 function, we expressed BON3 under the control of the promoter of CaMV 35S in bon1-1. This promoter confers both ubiquitous and higher expression in Arabidopsis (Odell et al., 1985). RNA blot analysis showed that BON3 is indeed overexpressed in these transgenic lines, while endogenous BON3 was not detected in either Col or bon1-1 (Figure 3a). Among 13 independent Pro35S:BON3 transgenic lines generated in bon1-1, five showed partial rescue and eight showed good rescue of the bon1-1 mutant phenotype in T2 transgenic plants (Figure 3b). This indicates that BON3 can substitute BON1 function when expressed at a higher level and/or in a more expanded domain.

Figure 3.

Overexpression of BON3 rescues the bon1-1 growth defect.
(a) BON3 RNA expression in Columbia (Col), bon1-1, and three independent Pro35S:BON3;bon1-1 lines (2, 8 and 9) analyzed by RNA blots. The BON3 transcript was not detected in Col or bon1-1 and was elevated in Pro35S:BON1;bon1-1 lines.
(b) Phenotypes of Pro35S:BON3 transgenic lines in bon1-1. The same three lines as in (a) are compared to Col and bon1-1 at 3-week-old stage.

The three BON genes are regulated differently

To investigate the expression pattern of the BON/CPN family members, we analyzed the transcriptional control of this gene family using the beta-glucoridinase (GUS) gene as a reporter. A previous study using the promoter-GUS fusion showed that BON1 is expressed mainly in young tissues including expanding leaves, elongating inflorescence stems, and root tips (Hua et al., 2001). GUS staining on ProBON2:GUS transgenic plants revealed that BON2 has a similar overall pattern to that of BON1, with expression mostly found in young tissues, including growing leaves, stems and roots (Figure 4a,b). In addition, BON2 has a unique pattern that is not observed in BON1: it is highly expressed in guard cells (Figure 4c). Most of the ProBON3:GUS transgenic lines (18 out of 25) had no GUS expression even with a long period of staining, indicating that BON3 is expressed at an extremely low level, which is consistent with RNA blot analysis.

Figure 4.

Expression patterns of the BON/CPN family.
(a, b) GUS expression pattern of ProBON1:GUS (a) and ProBON2:GUS (b) transgenic lines at the two-leaf stage (i), the four- to six-leaf stage (ii), the eight-leaf stage (iii), the early bolting stage (iv), and the mature stage (v).
(c) ProBON2:GUS expression in mature leaves. Strong expression was observed in guard cells.

The bon1bon2 and bon1bon3 double mutants are seedling-lethal at 22°C

To reveal possible overlapping functions among BON1, BON2 and BON3, we constructed double mutant combinations of these genes. Double mutants between the BON2 and BON3 genes in the Col background, bon2-2bon3-3, had no obviously abnormal phenotype (Figure 5a). Double mutants of bon1 and bon3 in Col, however, exhibited a stronger phenotype than bon1-1, and double mutants of bon1 and bon2 in Col exhibited an even more severe phenotype (Figure 5b). Both bon1-1bon2-2 and bon1-1bon3-3 produced normal cotyledons, as did bon1-1 shortly after germination. Approximately 6 days post-germination (dpg), bon1-1 produced the first two true leaves which had a wild-type appearance. At this stage, both double mutants started to show growth defects. bon1-1bon3-3 had slightly delayed or reduced leaf expansion compared with bon1-1, and bon1-1bon2-2 produced pin-like true leaves. At around 12 dpg, the wild type, bon1-1, and the two double mutants exhibited very distinct phenotypes. bon1-1 produced the normal number of visible leaves, but the leaves were reduced in size and curly in shape. bon1-1bon3-3 had more compact leaves than bon1-1. bon1-1bon2-2 again exhibited the most severe phenotype. The true leaves were barely visible, and the whole seedling started to become yellow. By 19 dpg, bon1-1 had small and curly leaves, but was otherwise normal looking. bon1-1bon3-3 became yellow, and its growth halted before it eventually died. bon1-1bon2-2 had died by this stage. These phenotypes indicate that BON2 and BON3 each have overlapping functions with BON1 and that these functions are essential for the viability of the plants.

Figure 5.

Double and triple mutant phenotypes of bon1, bon2 and bon3 and the wild-type Colombia (Col) phenotype.
(a) Single and double mutants of bon2 and bon3 had wild-type appearance.
(b) Phenotypes of Col, bon1-1, bon1-1bon2-2 and bon1-1bon3-3 grown at 22°C at 6, 12 and 19 days post-germination (dpg) and at 28°C at 19 dpg. The scale bar is 0.5 cm.
(c) Phenotypes of bon1-1, bon1-1bon2-2 and bon1-1bon3-3 at 18 dpg. Plants were grown at constant 22°C or at 28°C until 3 dpg or 6 dpg before shifting to 22°C.
(d, e) Phenotypes of bon1-1bon2-2bon3-3. The triple mutant can have normal seeds (not shown) that fail to develop after germination (d) or can have abnormal seeds that are incapable of germinating (e).

The overlapping functions between BON1 and BON2 and between BON1 and BON3 were essential for viability at 22°C but not 28°C. The bon1-1bon2-2 and bon1-1bon3-3 mutants germinated and grown at 28°C were largely indistinguishable from wild-type Col or bon1-1 (Figure 5b), although some of the bon1-1bon2-2 plants occasionally exhibited slightly reduced growth. Temperature shift experiments were carried out to determine whether the overlapping functions of the BON/CPN family are required throughout development. bon1-1bon3-3 and bon1-1bon2-2 mutant plants were germinated and grown at 28°C to several developmental stages including 3, 6 and 11 dpg as well as after bolting. All the shifted double mutants stopped wild-type growth, gradually turned yellow, and eventually died at 22°C, with bon1-1bon2-2 dying more rapidly than bon1-1bon3-3 (Figure 5c). This indicates that the overlapping functions between BON1 and BON2 and between BON1 and BON3 are not only essential for viability at early seedling stages, but are required throughout plant development at 22°C.

The bon1bon2bon3 triple mutant is seedling-lethal

To uncover the full extent of the overlapping functions of the BON/CPN family, we generated triple mutant combinations among BON1, BON2 and BON3. No bon1-1bon2-2bon3-3 triple mutant plants could be identified from the F2 progenies of a cross between bon1-1bon2-2 and bon2-2bon3-3. We confirmed that this was a result of a lethality of the triple mutants because a quarter (79 out of 321) of the progeny of bon1-1/+bon2-2bon3-3 plants (grown at 28°C) died immediately after germination at either 22 or 28°C (Figure 5d). None of the surviving seedlings was homozygous for bon1-1, indicating that the dead seedlings were bon1-1bon2-2bon3-3. The lethal phenotype of the triple mutants was sometimes expressed at an earlier stage. That is, seeds of triple mutant genotype sometimes had a shrunken phenotype (Figure 5e) and were not capable of germinating, suggesting even earlier defects during embryo or seed development. The variability of the onset of the triple mutant phenotype might derive from the slight variation of growth conditions of the bon1-1/+bon2-2bon3-3 plants at 28°C.

The bon1bon3 double mutant shows extensive cell death

To understand better the essential functions of the BON/CPN family, we characterized the bon1-1bon3-3 mutant in more detail because it has the mildest phenotype of all the lethal mutant combinations. At 14 dpg, the double mutant showed leaf chlorosis and its development was arrested (Figure 6a). Cell death occurred extensively in mature leaves of the double mutant. Using the vital stain trypan blue, we observed micro lesions in bon1-1, mostly on the margins of the leaves. More extensive cell death was also found in bon1-1bon3-3 on the lamina of leaves (Figure 6b). In addition, the double mutant had more extensive autofluorescence than bon1-1, possibly resulting from phenolic compounds in and around the dead cells (Figure 6b). Extensive cell death in bon1-1bon3-3 may have arisen from an overaccumulation of reactive oxygen species. A darker staining with diaminobenzidine (DAB) was observed in bon1-1bon3-3 than in the wild type or the bon1-1 single mutant at 6 dpg when the plants still had a wild-type appearance (Figure 6b).

Figure 6.

bon1-1bon3-3 undergoes cell death and growth arrest.
(a) The double mutant showed chlorosis 3 weeks after germination.
(b) The double mutant underwent more extensive cell death than bon1-1. Trypan blue staining, autofluorescence, and diaminobenzidine staining were stronger in the double mutant than in wild type or single mutants.
(c) Shoot apices of Columbia (Col), bon1-1 and bon1-1bon3-3 plants. Sections of seedlings at 5, 9, and 15 days post-germination (dpg) were stained with toluidine blue and examined under a microscope. bon1-1bon3-3 exhibited phenotypic variations among individuals; three representative seedlings are shown.
(d) bon1-1bon3-3 expressed defense-related genes. RNA transcript levels of PR1, SNC1, peroxidase C (PRXc) and glutathionine-S-transferase (GST) in Col, bon1-1, bon1-1bon3-3 and bon1-1bon3-3pad4-1 were analyzed by RNA blots. All four genes were further up-regulated in the bon1-1bon3-3 double mutant compared with the bon1-1 single mutant. The PAD4mutation suppressed the up-regulation of expression in bon1-1bon3-3. Equal amounts of RNA were loaded, this being confirmed by equal amounts of rRNA visualized by ethidium bromide staining (data not shown).

To characterize the defects of growth arrest in bon1-1bon3-3, we compared the morphology of shoot apices of the wild type, bon1-1 and bon1-1bon3-3 at 5, 9, and 15 dpg (Figure 6c). bon1-1bon3-3, but not bon1-1, exhibited a different structure to Col in both the shoot apical meristem and the leaf primordia at these developmental stages. At 5 dpg, some of the double mutant seedlings had a reduced number of leaf primordia compared with bon1-1 or the wild type. At 9 dpg, the phenotype of delayed leaf primordia formation was more pronounced and prevalent, being observed in the majority (17 of 20) of seedlings examined. At 15 dpg, Col and bon1-1 had formed inflorescence stems, while the double mutant arrested at the same stage as 9 dpg in all 20 seedlings analyzed. This analysis showed that the growth arrest of bon1-1bon3-3 starts at a very early developmental stage and is at least partly attributable to the halting of leaf primordium formation in the shoot apex.

The bon1bon3 mutant has up-regulated expression of defense-related genes

Because activation of defense responses can result in cell death, we investigated whether the lethal phenotype of bon1-1bon3-3 is caused by a stronger activation of defense responses than that in bon1-1. PR1, a defense response marker gene, was further up-regulated in the double mutant compared with bon1-1 (Figure 6d). Similarly, SNC1 had an even higher transcript level in bon1-1bon3-3 than in bon1-1 (Figure 6d), suggesting a greater accumulation of SA in the double mutant. Genes involved in oxygen metabolism were also elevated in the double mutant. Peroxidase C (PRXc) and glutathionine-S-transferase (GST) expression levels were elevated in bon1-1 and even more elevated in the double mutant (Figure 6d). Chlorosis observed in the double mutant was unlikely to have been a result of accelerated senescence because SAG12, a marker of senescence, was not expressed in the single or double mutant (data not shown). In summary, gene expression analysis suggested that the bon1bon3 double mutant in Col has a stronger activation of cell death pathways associated with defense responses than bon1-1.

The snc1 mutation partially suppresses the lethal phenotype of bon1bon3

Because the SNC1 gene is activated (through an increase in either RNA abundance or protein activity) in bon1-1, we asked whether the lethal phenotype of bon1bon3 results from a stronger activation of the R gene SNC1 by generating the bon1-1bon3-3snc1-11 triple mutant. In early development at 22°C (until approximately 14 dpg), the triple mutant grew normally with expanded true leaves (Figure 7a). However, bon1-1bon3-3snc1-11 exhibited an abnormal phenotype later in development (Figure 7a). Leaves emerging at approximately 21 dpg were yellowish and arrested. There also appeared to be growth arrest in the shoot apex and no bolting was observed. The triple mutant eventually died without any inflorescence stems. The lack of total rescue by snc1-11 indicates that the lethal phenotype of bon1-1bon3-3 at 22°C is not solely attributable to a stronger activation of the SNC1 gene than in bon1-1.

Figure 7.

Interaction of the bon double mutants with snc1, pad4, eds1 and ndr1.
(a) snc1-11 partially suppressed the bon1-1bon3-3 phenotype. bon1-1snc1-11 had a wild-type appearance throughout development at 22°C. bon1-1bon3-3snc1-11 had a wild-type appearance at early developmental stages [14 days post-germination (dpg) is shown], but showed chlorosis and growth arrest before bolting (29 dpg is shown). A close-up of the shoot apex of the arrested plant is shown in the insert.
(b) pad4-1 suppressed the bon1-1bon3-3 phenotype totally. bon1-1bon3-3pad4-1 plants grown at 22°C were wild-type in appearance throughout development (19 dpg is shown).
(c) eds1-1 suppressed the phenotypes of bon1-1bon3-3 and bon1-1bon2-2. Plants shown with eds1-1 were in a mixed background of Columbia (Col) and Wassilewskija (Ws) and contained homozygous SNC1 from Col.
(d) ndr1-1 partially suppressed the phenotypes of bon1-1 and bon1-1bon3-3. bon1-1ndr1-1 had a weaker phenotype than bon1-1. bon1-1bon3-3ndr1-1 rescued the lethal phenotype of bon1-1bon3-3 but was extremely dwarf when mature.

pad4 and eds1 mutations suppress the phenotypes of bon1bon3 and bon1bon2

The finding that the lethality of the bon1-1bon3-3 (Col) mutant is not completely suppressed by loss of SNC1 function while bon1-2bon3-1 (Ws) has a wild-type phenotype (see below) suggests that other Col-specific R genes are activated in bon1-1bon3-3. To test this hypothesis, we investigated whether this lethal phenotype can be reverted by the loss of PAD4, EDS1 or NDR1 whose functions are required for many of the R genes examined (Aarts et al., 1998; Glazebrook, 2001). We found that pad4 completely suppressed the lethal phenotype of bon1-1bon3-3. The bon1-1bon3-3pad4-1 triple mutant in the Col accession was wild type in appearance throughout development at 22°C (Figure 7b). Consistent with its morphological phenotype, up-regulation of PR1, SNC1, PRXc and GST in bon1-1bon3-3 was totally suppressed in bon1-1bon3-3pad4-1 (Figure 6d). Similarly to pad4, eds1 also suppressed the bon1-1bon3-3 phenotype. Because eds1-1 comes from the Ws accession, all mutant combinations containing eds1-1 have a mixed background of Col and Ws and only those with homozygous SNC1 from Col are described and characterized here. All bon1-1bon3-3eds1-1 plants had a wild-type phenotype (Figure 7c).

In contrast to pad4-1 and eds1-1, ndr1-1 did not totally suppress the bon1-1bon3-3 phenotype. ndr1-1 alleviated the bon1-1 single mutant phenotype especially at the early seedling stage before bolting, but it did not completely revert the bon1-1 single mutant phenotype, while pad4 and eds1 did (Figure 7d). ndr1-1 partially suppressed the phenotype of bon1-1bon3-3. bon1-1bon3-3ndr1-1 was not seedling-lethal but was extremely dwarf throughout development (Figure 7d).

To determine whether PAD4 and EDS1 also mediate the overlapping function between BON1 and BON2, we generated bon1-1bon2-2pad4-1 and bon1-1bon2-2eds1-1SNC1 triple mutants. The bon1-1bon2-2pad4-1 and bon1-1bon2-2eds1-1 plants behaved largely like the wild type (Figure 7c and data not shown). Occasionally, some of these mutants exhibited abnormal phenotypes late in development with shorter inflorescences, shorter internodes, and more lateral shoots than the wild type (data not shown).

The bon1bon2bon3 mutant has growth defects in the presence of pad4, eds1 or ndr1

To determine whether the loss-of-function phenotype of the entire BON/CPN family is mediated by PAD4 and EDS1, we further constructed quadruple mutants of bon1-1bon2-2bon3-3pad4-1 and bon1-1bon2-2bon3-3eds1-1. Viable normal seeds were obtained for the quadruple mutants, indicating that pad4 and eds1 rescue the embryo- or seedling-lethal phenotype of bon1-1bon2-2bon3-3. At 22°C, the quadruple mutants had wild-type appearance shortly after germination. However, seedlings of bon1-1bon2-2bon3-3pad4-1 and bon1-1bon2-2bon3-3eds1-1 started to exhibit abnormal phenotypes from as early as the four-leaf stage to as late as before bolting (Figure 8a). Early leaves had elongated petioles and were darker green with purple hues. Leaves that emerged later in development were very small with a darker green and purple color. The primary shoot apex appeared to be arrested in growth and many small curly leaves were subsequently produced (Figure 8a). Some of the quadruple mutants recovered later from the arrest: they put out green leaves and bolted with multiple lateral shoots (Figure 8b). The percentage of seedlings recovering varied from batch to batch, probably as a result of variations in growth conditions. The recovered quadruple mutants had shorter inflorescence stems and shorter internodes between flowers than the wild type (Figure 8b). The siliques of the quadruple mutants were more or less normal and viable seeds were produced.

Figure 8.

Interaction of the bon triple mutants with pad4, eds1, and ndr1 mutants.
(a) pad4, eds1 and ndr1 could not totally rescue the defects in the bon1bon2bon3 mutant. The quadruple and quintuple mutants of bon1-1bon2-2bon3-3pad4-1, bon1-1bon2-2bon3-3eds1-1, and bon1-1bon2-2bon3-3pad4-1ndr1-1 were delayed in development with small purple leaves [plants at 22 days post-germination (dpg) are shown]. They had arrested growth when the wild- type was bolting (plants at 40 dpg are shown). Occasionally, some plants (indicated by the arrow) recovered from the arrested state and produced greener leaves before putting out lateral shoots. Plants with eds1-1 were of mixed Columbia (Col) and Wassilewskija (Ws) background and contained SNC1from Col.
(b) bon1-1bon2-2bon3-3pad4-1 had growth defects late in development. The quadruple mutant had a dwarf stature, multiple shoots, and clustered flowers when mature (plants at 60 dpg are shown).
(c) PRXc, GST and PR1transcript levels in bon1-1bon2-2bon3-3pad4-1.
(d) bon1-1bon2-2bon3-3pad4-1 had a wild-type appearance at 28°C. The wild-type Col and the quadruple mutant grown at 28°C are shown.

Because NDR1 and EDS1/PAD4 are in general required for different types of R genes, we further generated the quintuple mutant bon1-1bon2-2bon3-3pad4-1ndr1-1. This quintuple mutant behaved similarly to bon1-1bon2-2bon3-3pad4-1 and bon1-1bon2-2bon3-3eds1-1 (Figure 8a). Thus, ndr1 cannot further rescue the growth defect of these two quadruple mutants.

To assess the extent of defense response activation in bon1-1bon2-2bon3-3pad4-1, we analyzed expression of several marker genes in the quadruple mutant (Figure 8c). PR1 expression was greatly reduced compared with that in bon1-1, suggesting that the SA-mediated pathway is largely suppressed in the quadruple mutant. GST was expressed in the quadruple mutant at a level slightly higher than in the wild type but not as high as in the bon1-1 mutant. PRXc was more strongly expressed in the quadruple mutant than in the wild type, at a level comparable to that in bon1-1, suggesting an up-regulation of the oxidative stresses in the quadruple mutant.

The growth defects of bon1bon2bon3pad4 or bon1bon2bon3eds1 were temperature dependent. Mutant plants grown at 28°C were largely wild type in appearance from germination to maturation (Figure 8d).

Phenotype of the bon combination mutants is accession dependent

T-DNA insertion mutant lines of BON2 and BON3 (bon2-1 and bon3-1) were isolated in the Ws accession; they are both loss-of-function mutants (Figure 1a and data not shown). bon2 and bon3 single mutants and bon2bon3 double mutants in Ws did not exhibit an obvious abnormal phenotype (Figure 9a). In contrast to bon1-1bon2-2 and bon1-1bon3-3 in Col, bon1-2bon2-1 and bon1-2bon3-1 in the Ws accession did not have obvious growth or developmental defects at 22°C (Figure 9a), suggesting that natural variations between Col and Ws accessions modulate the overlapping functions between BON1 and BON2 and between BON1 and BON3. The triple homozygous bon1-2bon2-1bon3-1 mutant arrested at the germinating stage and died 4 days after germination before the production of any visible true leaves at either 22°C or 28°C (Figure 9b).

Figure 9.

Phenotype of the bon mutants in the Wassilewskija (Ws) accession.
(a) bon double mutants in Ws had wild-type appearance. Wild-type Ws and three bon double mutant combinations grown at 22°C are shown.
(b) bon1-2bon2-1bon3-1 in Ws was seedling-lethal. Seedlings a few days after germination are shown. The bon1-2bon2-1bon3-1 seedling wilted immediately after germination while bon1-2bon2-1bon3-1 continued to grow after germination.

Discussion

Overlapping and distinct functions of the BON/copine gene family

Copines are evolutionarily conserved proteins implicated in signaling and membrane trafficking. They exist as gene families in a given organism, which potentially makes it difficult to uncover overlapping functions between family members. Arabidopsis has three copine genes (BON1, BON2 and BON3), and our molecular and genetic analyses of the BON/CPN family indicate that they have both distinct and overlapping functions. BON1 appears to play a dominant role among the copine genes in Arabidopsis. An obvious mutant phenotype was observed in bon1 but not in bon2 or bon3 single mutants. The difference likely results from differential gene expression and protein activity. BON3 is expressed at an extremely low level, and the rescue of the bon1-1 mutant phenotype by overexpression of BON3 suggests a conservation of biochemical activities of the BON/CPN proteins. The overlapping functions revealed by the double and triple mutant analyses further indicate that the BON/CPN proteins possess largely similar biochemical activities.

Regulation of cell death by the BON/CPN family

Analysis of mutant combinations of bon1, bon2 and bon3 revealed an important function of the BON/CPN family in suppressing cell death. An increasing degree of severity of phenotype at 22°C was observed in the following mutant series: bon1-1, bon1-1bon3-3, bon1-1bon2-2 and bon1-1bon2-2bon3-3 from a mild phenotype of reduced cell size and cell number to a severe phenotype of extensive cell death.

We propose that the BON/CPN family regulates cell death associated with defense responses by repressing a number of R genes, and the loss of an increasing number of copine genes leads to the activation of an increasing number of R genes. It has been suggested that there is a threshold responding to R-gene-derived signals, above which a cell undergoes cell death (Shirasu and Schulze-Lefert, 2000). Low levels of activation of R genes induce micro lesions and reduced cell expansion and cell division. Stronger activation leads to more extensive cell death and consequent lethality in seedlings. Even stronger activation results in cell death at very early developmental stages including embryogenesis.

Our previous study showed that BON1 is a negative regulator of a Col-specific R gene SNC1 and that the snc1 loss-of-function mutation can suppress the bon1 phenotype. The lethal phenotype in the bon1bon3 double mutant is not caused by further de-repression of the SNC1 function, because the snc1 mutation cannot totally rescue the bon1bon3 phenotype. Because the bon1bon3 phenotype is accession dependent, we hypothesize that another Col-specific component(s) is required, and this could well be another R gene(s). That bon1bon2bon3 has a stronger phenotype than bon1bon3 or bon1bon2 in Col suggests that either an additional R gene(s) is activated in the triple mutant compared with the double mutant or that the R gene(s) regulated together by BON1 and BON3 is further activated in the triple mutant. In summary, an R gene(s) other than SNC1 might be co-regulated by BON1, BON2 and BON3.

The role of the BON/CPN family in negative regulation of several R genes is supported by our finding that pad4 and eds1 can totally suppress the lethal phenotype of bon1-1bon3-3 and largely suppress the lethal phenotype of the bon1-1bon2-3bon3-3 triple mutant. PAD4 and EDS1 are required for disease resistance mediated by many TIR-NB-LRR types of R genes, while NDR1 is mostly required for the CC-NB-LRR type of R genes (Glazebrook, 2001). Because this requirement might not be exclusive, it is still to be determined whether the R genes regulated by BON genes are of the TIR type rather than the CC type.

The biochemical mechanism by which BON genes regulate R genes is yet to be determined. One possibility is that the BON genes regulate the transcript level of R genes either at the transcription or at the post-transcription level. BON mutations might induce stresses or perturb membrane homeostasis and therefore turn on R gene transcription. The finding that the SNC1 transcript level is elevated in bon1-1 and even more elevated in bon1-1bon3-3 is consistent with this hypothesis. However, our data suggest that this regulation at the RNA transcript level is likely to occur through the accumulation of SA in the bon1 mutant and is therefore probably indirect (Yang and Hua, 2004). The other possibility is that BON proteins regulate the activities of R proteins. BON proteins could directly or indirectly bind to R proteins such as SNC1 and therefore keep them in an inactive state. However, it is yet to be determined whether the activities of R proteins are altered in bon mutants and whether there is a physical association of BON proteins with R proteins.

Regulation of other processes by the BON/CPN family

The finding that the bon1bon2bon3pad4ndr1 mutant exhibits severe growth defects raises the possibility that the BON/CPN family in Arabidopsis has functions other than regulating R-gene-mediated defense responses. The following two scenarios are equally possible regarding the function of the BON/CPN genes.

  • (i) They are solely involved in regulating cell death and defense responses. The BON/CPN proteins may regulate the protein activity or the transcript level of R genes or they may regulate an R-independent cell death pathway mediated by EDS1 and PAD4 (Rusterucci et al., 2001). The growth defect in the quintuple mutant is attributable to an activation of a defense/cell death pathway not totally suppressed by pad4 and ndr1.
  • (ii) They have a primary role in regulating an important process in growth and development. The phenotype of this quintuple mutant reflects this primary defect, with the loss of the whole BON/CPN family. Defense response or cell death is triggered by a perturbation of this process.

The potential role of the BON/CPN family in growth and development may be closely related to the ancient and conserved function of copines across kingdoms. The exact biochemical process that copines regulate is yet to be identified. In Chlamydomonas elegans, a copine gene, GEM-4, was discovered to antagonize cation channel GON-2 activity in the promotion of gonadal cell division (Church and Lambie, 2003). In mammalian cells, copines have been found to interact with a diverse groups of signaling molecules in yeast two-hybrid assays (Tomsig et al., 2003). The dominant negative form of copine was found to reduce the effect of calcium on the activation of the transcription factor nuclear factor (NF)-κB in human cell lines (Tomsig et al., 2004). It is thus possible that copines confer calcium or membrane localization modulation on various signaling pathways. Alterations of the calcium state and the membrane system could modify the localization and activity of copines, which may in turn regulate the activities of copines and/or their interaction with membrane-associated proteins such as GON-2 and R proteins. In both scenarios, the processes that BON/CPNs regulate are temperature dependent, as the defects of bon1bon2bon3pad4 seen at 22°C were suppressed at 28°C.

In the second scenario, there is an intriguing connection between defense responses and growth/development. In the bon mutant combinations, a partial loss of the ancestral function in an important pathway leads to perturbation of homeostasis, and a total loss of this function leads to lethality. The perturbed state in bon mutants is likely recognized by R proteins which in turn trigger cell death and defense responses. Consistent with the emerging ‘guard hypothesis’, the basic function of copines could be targeted by pathogen effector proteins, as pathogen invasion has been shown to alter calcium concentration and modify membrane systems and membrane trafficking. Plants could counteract this invasion by evolving R genes to detect such modification through monitoring BON/CPN genes. Thus, regulators of R genes could be primarily involved in important cellular functions rather than solely modulating defense responses as suggested by the duel role of AtTIP49a in development and defense (Holt et al., 2002). Further investigation of copine function will enhance our understanding of the regulation of plant defense responses as well as the evolution of plant disease resistance.

Experimental procedures

Plant growth conditions

Arabidopsis plants were grown at 22 or 28°C under continuous fluorescent light (100 μmol m−2 sec−1) and 50–70% relative humidity. Arabidopsis seeds were either directly sown on soil or grown on Petri dishes containing half-strength Murashige–Skoog (MS) medium (Murashige and Skoog, 1962; Sigma, St. Louis, MO, USA) with 2% sucrose and 0.7% agar.

RNA blot analysis

Total RNA was extracted from 3-week-old plants (unless specified otherwise) with TRI Reagents (Molecular Research Inc., Cincinnati, OH, USA) according to the manufacturer's protocol. Ten to 30 μg of RNA was resolved on a 1% agarose gel containing 1.8% formaldehyde. Ethidium bromide was used to visualize the rRNA bands to ensure equal loading. RNA gel blots were hybridized with gene-specific, 32P-labeled, single-stranded DNA probes.

RT-PCR

Total RNA was isolated from 3-week-old plants with TRI Reagents, followed by treatment with RNase-free DNase I (Promega, Madison, WI, USA) at 37°C for 1 h to degrade genomic DNA. Treated RNA samples (1 μg each) were used as templates for the first-strand cDNA synthesis (Clontech, Palo Alto, CA, USA). A portion of the resulting cDNAs was then subjected to PCR amplification using gene-specific primers.

Plasmid construction and plant transformation

A 6-kb XhoI genomic fragment containing the whole BON3 coding region and the 3′-untranslated region (UTR) was cloned from BAC clone F7G19 into the binary vector pKYLX (http://www.uky.edu/aghunt00/kylx.html) to generate the Pro35S:BON3 construct.

A 2.5-kb BamHI–SacI genomic fragment from BAC clone T2I1 (comprising 2.2 kb of the sequence upstream of the translation start site) and a 1.5-kb XbaI genomic fragment (comprising 1.5 kb of the sequence upstream of the translation start site) from BAC clone F7G19 were transcriptionally fused with the GUS reporter gene respectively in the binary vector PZP212 (Hua et al., 2001) to create ProBON2:GUS and ProBON3:GUS fusion constructs. Agrobacterium tumefaciens strains GV3101 (Koncz and Schell, 1986) carrying different constructs were used to transform the wild type or the bon1-1 mutant via floral dip transformation (Clough and Bent, 1998).

Isolation and generation of single, double, triple and quadruple mutants

bon2-1 and bon3-1 were isolated from T-DNA line collections from the Arabidopsis Biological Resource Center and Wisconsin Knockout Facility by PCR-based screening. bon2-2 and snc1-11 were screened from the SALK T-DNA line collections (http://signal.salk.edu/cgi-bin/tdnaexpress). bon3-3 was obtained from SAIL collections (http://www.tmri.org/en/partnership/sail_collection.aspx) generated by Syngenta Biotechnology (Research Triangle Park, NC, USA).

To create the bon1-1bon3-3 mutant, bon1-1 was crossed with bon3-3. In the F2 population, approximately one out of 16 plants was tiny and died before it matured. The progeny of bon1-1bon3-3/+ were grown at 28°C and bon1-1bon3-3 was selected by PCR-based genotyping.

bon1-1bon3-3snc1-11 and bon1-1bon3-3pad4-1 triple mutants were generated by crossing bon1-1bon3-3 with bon1-1snc1-11 and bon1-1pad4-1 respectively and genotyping their F2 progeny at the BON3, SNC1 and PAD4 loci.

bon1-1/+bon2-2bon3-3 was selected from the F2 progeny of bon1-1bon3-3 crossed with bon2-2bon3-3.

The bon1-1bon2-2bon3-3pad4-1 quadruple mutant was generated by crossing bon1-1/+bon2-2bon3-3 with bon1-1pad4-1 and genotyping its F2 progeny at the BON1, BON2, BON3 and PAD4 loci.

bon1-1bon2-2bon3-3eds1-1 was generated by crossing bon1-1bon2-2bon3-3pad4-1 with bon1-1eds1-1SNC1 and genotyping its F2 progeny at the BON2, BON3, PAD4 and EDS1 loci.

bon1-1ndr1-1 was generated by crossing bon1-1 with ndr1-1 and genotyping its F2 progeny.

bon1-1bon2-2ndr1-1, bon1-1bon3-3ndr1-1 and bon1-1bon2-2bon3-3pad4-1ndr1-1 were screened from the F2 progeny of a cross between bon1-1bon2-2bon3-3pad4-1 and bon1-1ndr1-1.

Mutant phenotypes were confirmed in a population of approximately of 100 plants segregating for one locus. Sequences of primers for genotyping are available upon request.

Histochemistry and microscopy

Histochemical detection of GUS activity was performed as previously described (Hua et al., 2001). Briefly, tissues for GUS staining were incubated in staining solution [50 mm sodium phosphate, pH 7.0, 10 mm ethylenediaminetetraacetic acid (EDTA), 2 mm 5-bromo-4-chloro-3-indoyl glucuronide, 1 mm potassium ferricyanide, and 1 mm potassium ferrocyanide] at 37°C overnight. After incubation, stained tissues were cleared of chlorophyll in an ethanol series.

Tissues for histological analysis were fixed with 5% formaldehyde [volume/volume (v/v)] and 5% acetic acid (v/v) in 70% ethanol solution. After dehydration in ethanol and infiltration with butanol, the tissues were embedded in paraffin. Microtome sections (5 μm thick) were stained with 0.1% toluidine blue.

Examination of autofluorescence of leaf tissue was performed as described by Adam and Somerville (1996). Trypan blue staining was performed as described by Bowling et al. (1997).

Acknowledgements

We thank the Arabidopsis Bioresource Research Center, Wiscosin Knockout Facility, Syngenta, B. Staskawicz, and J. Dewdney for mutant seeds. We also thank anonymous reviewers for critical reading of the manuscript. This research was supported by a grant from the National Science Foundation to JH.

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