Plants have the astonishing ability to release their floral organs, leaves, fruit, and seeds at programmed points in their life cycle or in response to signals from their environment. Within Arabidopsis flowers, the series of events leading to organ abscission is genetically tractable. Analysis using this model system has revealed the influence of organ boundary genes in establishing the placement of abscission zones (Wang et al., 2006; González-Carranza et al., 2007; Gómez-Mena and Sablowski, 2008; McKim et al., 2008; Gubert et al., 2014), the critical roles played by hormones such as jasmonic acid (Kim et al., 2013) and managers of membrane traffic (Liljegren et al., 2009; Liu et al., 2013), and a signaling module that regulates the cell separation phase of organ abscission (Fang and Fernandez, 2002; Cho et al., 2008; Stenvik et al., 2008; Shi et al., 2011; Gubert and Liljegren, 2014; Patharkar and Walker, 2015; Santiago et al., 2016; Taylor et al., 2016). Central components in this module include a secreted peptide, INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and redundant leucine-rich repeat receptor-like kinases, HAESA (HAE) and HAESA-like2 (HSL2), that activate a MAP kinase cascade leading to organ abscission.

We have used suppression analysis as a genetic tool to identify additional genes that control the abscission process in Arabidopsis flowers. Starting with the nevershed (nev) mutant which blocks organ shedding due to defects in membrane traffic (Liljegren et al., 2009), we screened for second-site mutations that would restore organ abscission in the presence of the original mutation. The nev-3 allele chosen for this screen (Figure 1A) changes an invariant arginine in the encoded protein known to be essential for ADP-ribosylation factor GTPase-activating activity (Luo et al., 2007). Multiple alleles of genes encoding three receptor-like kinases—EVERSHED (EVR), SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1), and CAST AWAY (CST)—were found to rescue abscission in nev flowers (Leslie et al., 2010; Lewis et al., 2010; Burr et al., 2011). Mutations in these receptor-like kinases are also able to reverse nev-mediated alterations in the structure of the Golgi apparatus and associated trans-Golgi network. Additional analyses suggest that activation of organ abscission is modulated by inhibitory interactions between CST and EVR with HAE/HSL2 (Burr et al., 2011; Gubert and Liljegren, 2014). Recent studies have demonstrated that SERK1 and two related receptor-like kinases act as co-receptors of HAE/HSL2 (Meng et al., 2016; Santiago et al., 2016). We have proposed that CST and EVR may prevent the signaling that leads to organ abscission by sequestering HAE/HSL2 at the cell surface and promoting their internalization prior to activation by IDA (Burr et al., 2011). As NEV is thought to function in the cycling of HAE/HSL2 to the plasma membrane, disruption of CST or EVR activity may restore organ abscission in nev flowers by shifting the balance of stabilized HAE/HSL2 receptors at the cell surface from an excessive pool of internalized, inactive receptors in endosomal compartments (Burr et al., 2011; Bryan et al., 2012; Liljegren, 2012).

Contrasting behaviors are shown by the pair of cst mutant alleles we identified with regard to their ability to rescue abscission in nev flowers (Burr et al., 2011). The cst-1 allele introduces a missense mutation (G157R) near the ATP-binding site within the CST kinase domain (Figure 1A), abolishing the kinase activity of the mutant protein. Organ shedding in nev-3. nev-2, and nev-6 flowers is recessively rescued by two copies of the cst-1 allele (Figure 1B; Burr et al., 2011). The cst-2 allele contains a T-DNA insertion immediately upstream of the kinase domain, and is predicted to encode a truncated protein (Figure 1A). One copy of cst-2 dominantly restores organ abscission in nev-3 and nev-6 flowers, but nev-2 flowers retain their organs even if both copies of cst-2 are present (Figure 1B; Burr et al., 2011).

As these results were partially consistent with the allele-specific mechanism of conformational suppression, in which a suppressor mutation restores a physical interaction between two proteins, we designed a study to determine whether the location of a nev mutation would be predictive of its ability to be rescued by the cst alleles. Specifically, we tested whether alleles that independently affect either the ARF GAP domain or the C-terminal region of NEV would mimic the distinct interactions of nev-3 and nev-2 with cst-2.

Here we report further evidence that the cst-1 and cst-2 alleles differ in their ability to restore organ shedding in nev flowers. While cst-1 recessively suppresses each of the five nev alleles tested, cst-2 suppresses nev-3 and nev-6 dominantly, nev-4 recessively, and fails to rescue nev-1 and nev-2 (Burr et al., 2011; this study).

These results highlight the complexity of interpreting the mechanisms of allelic suppression. Although the ultimate goal for many geneticists carrying out suppression analysis is to find instances of conformational suppression, whereby allele-specific rescue reflects a restored physical interaction between two mutant proteins, this scenario is actually rare in practice (Manson, 2000). Indeed, our selection of the nev-3 missense allele (Figure 1A) as the genetic background for this screen was driven by an interest in identifying a mutant version of an unknown protein that might interact with and restore the ARF GAP enzymatic activity of the nev mutant protein.

Instead, our results suggest that the cst-1 and cst-2 alleles restore organ abscission in nev flowers through distinct suppression mechanisms. We have found that the kinase-dead CST protein encoded by cst-1 recessively suppresses the abscission defects of all nev alleles tested, including nev-6, which is predicted to produce an abbreviated protein missing the ARF GAP domain (Figure 1A; Supplementary Figure S1B). Suppression of a deletion (or null) allele of the original gene by an extragenic suppressor is considered strong evidence of bypass suppression (Prelich, 1999). Bypass suppression occurs when a second site mutation creates an alternate opportunity to cover the function disabled by the first mutation (Manson, 2000; Michels, 2002). Another hallmark of bypass suppression is that it is not allele-specific (Manson, 2000), which fits with the observed behavior of the nev cst-1 double mutants (Figure 1B). Considering that interactions between CST and HAE were detected in subdomains of the plasma membrane via biomolecular fluorescence complementation assays (Burr et al., 2011), the cst-1 allele may consistently rescue organ shedding in nev flowers due to the failure of the kinase-deficient cst-1 protein to promote the internalization of the HAE/HSL2 receptors from the cell surface. The recessive nature of the cst-1 suppression suggests that the reduced amount of the functional CST kinase in nev flowers heterozygous for cst-1 is sufficient to remove enough of the HAE/HSL2 receptors from this plasma membrane pool to prevent activation of the MAP kinase module leading to organ abscission. Redelivery of HAE/HSL2 to the cell surface after internalization is predicted to be disrupted in each of the nev mutant alleles tested (Figure 1A).

We have found that cst-2, which may produce reduced levels of an abbreviated, membrane-associated protein without a kinase domain (Figure 1A; Supplementary Figure S2B), rescues organ abscission in nev-3. nev-4, and nev-6 flowers but not in nev-1 or nev-2 flowers (Figure 1B). This allele-specific outcome may result from a dominant-negative mutation enacting a gradient of suppression (Manson, 2000; Burr et al., 2011). Under this scenario, the predicted strength of the nev alleles tested would range from nev-6 and nev-3 (relatively weak; rescued by one copy of cst-2) to nev-4 (intermediate, rescued by two copies of cst-2) to nev-1 and nev-2 (strong, not rescued by cst-2). Relative differences in the activities of nev mutant proteins may impact the ratio of HAE/HSL2 receptors trapped in the endosomal compartments and thereby influence the ease of cst-2 mediated suppression (Figure 1B). While a truncated nev-6 mutant protein without an ARF GAP domain would not be expected to retain more function than the nev-2 mutant protein (Figure 1), intronic T-DNA insertions can be spliced out in a fraction of the transcripts produced, leading to synthesis of functional protein (Chehab et al., 2011; Rodriguez et al., 2014). Although we did not detect notable levels of correctly spliced NEV transcripts in nev-6 flowers (Supplementary Figure S1A), it is likely that even a small amount of functional protein is sufficient to promote abscission. Indeed, it has been previously observed that the petals of nev-7 flowers detach more readily than those of nev-3 flowers (Liu et al., 2013). Like nev-6, the nev-7 allele contains a T-DNA insertion in the first intron (Liljegren et al., 2009).

It is striking that the nev-1 and nev-2 alleles can be recessively rescued by cst-1 but not by cst-2. These results, in addition to the dominant suppression of nev-3 and nev-6 by cst-2, suggest that the truncated cst-2 protein may exhibit an altered set of interactions with receptor-like kinase complexes than the cst-1 protein. Future analysis of the expression, localization, and ability of the cst-2 and cst-1 mutant proteins to form heteromeric complexes with EVR and HAE may reveal additional clues to the unique mechanisms underlying their restoration of the signaling leading to organ abscission in nev flowers.

With the growing accessibility of approaches to identify the transcriptomes of abscission zone cells in model as well as crop plants using laser capture microdissection (Cai and Lashbrook, 2006, 2008; Agustí et al., 2009) and RNA sequencing (Niederhuth et al., 2013; Kim et al., 2016; Sundaresan et al., 2016), the agronomic value of using model plants to study abscission is under debate (Patterson et al., 2015). The recent discovery that drought-triggered leaf abscission is dependent on the activities of IDA, HAE/HSL2, and NEV (Patharkar and Walker, 2016) significantly enhances the usefulness of Arabidopsis as a model system. Furthermore, until analysis of gene function is feasible in crop plants, parallel approaches to investigate the functions of abscission zone-enriched genes in model plants with reverse genetic approaches will be crucial.

Forward genetic screens, when carefully designed, are also expected to provide novel insights regarding the regulation of organ abscission. In addition to our discovery of a set of receptor-like kinases that modulate organ abscission via proposed interactions with HAE and HSL2, the homeodomain transcription factor BREVIPEDICELLUS (BP) was found to act downstream of the IDA-HAE/HSL2 signaling module through suppression analysis of ida flowers (Shi et al., 2011). While bp mutants display enlarged abscission zones (Wang et al., 2006), it is noteworthy that the cst. evr, and serk1 mutants do not present phenotypes on their own, yet alleles of each are able to rescue organ shedding in the context of nev flowers (Leslie et al., 2010; Lewis et al., 2010; Burr et al., 2011). Suppression analysis of a weak hae hsl2 mutant has revealed that mutations in either of two mannosyltransferases that normally mediate degradation of the mutant hsl2 protein in the ER may restore abscission by allowing this partially functioning receptor to escape to the cell surface (Baer et al., 2016). Understanding the threshold levels at which organs are released in sensitized mutants like nev. ida, and hae hsl2 may inform the future design of nuanced solutions to control abscission in crop plants.

SL designed the experiments. All authors performed the experiments and contributed in preparing the figures.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.