ABSTRACT
In plants, root hairs undergo a highly-polarized form of cell expansion called tip-growth, in which cell expansion is restricted to the root hair apex. In order to characterize cellular components playing a role in this specialized form of cellular expansion we screened for conditional temperature sensitive (ts) mutants by EMS mutagenesis. Here we describe one of these mutants, fer-ts (feronia-temperature sensitive). Mutant fer-ts seedlings grew normally at permissive temperatures (20°C), but failed to form root hairs at non-permissive temperatures (30°C). Map based-cloning and whole genome sequencing revealed that fer-ts resulted from a G41S substitution in the extracellular domain of FERONIA (FER). A functional fluorescent fusion of FER containing the fer-ts mutation maintained a plasma membrane localization at both permissive and non-permissive temperatures, but that the fer-ts allele was subject to enhanced protein turnover at elevated temperatures. Mutant fer-ts seedlings were resistant to added RALF1 peptide at non-permissive temperatures, supporting a role for FER in perception of this peptide hormone. Additionally, at non-permissive temperatures fer-ts seedlings displayed altered ROS accumulation upon auxin treatment and phenocopied constitutive fer mutant responses to a variety of plant hormone treatments. Molecular modeling and sequence comparison with other CrRLK1L receptor family members revealed that the mutated glycine in fer-ts is highly conserved, but significantly removed from recently characterized RALF23 and LORELI-LIKE-GLYCOPROTEIN (LLG2) binding domains, perhaps suggesting that fer-ts phenotypes may not be directly due to loss of binding to RALF1 peptides.
INTRODUCTION
In higher plants, root hairs are cellular protuberances resulting from the polarized outgrowth of specialized root epidermal cells, known as trichoblasts (Gilroy and Jones, 2000). Development of root hair can be divided into three phases: cell specification, initiation of bulge formation, and polarized tip growth (Cho and Cosgrove, 2002).
Polarized tip growth is precisely modulated due to the highly localized exocytosis of Golgi-derived vesicles and the deposition of cell wall material at a restricted area of the root hair tip region, and a tip-concentrated cytoplasmic calcium ion (Ca2+) gradient ensures correct targeting of this polarized membrane trafficking (Hepler et al., 2001; Smith et al., 2005; Cole and Fowler, 2006). This calcium ion gradient is established by localized generation of reactive oxygen species (ROS) by ROOT HAIR DEFECTIVE2 (RHD2) which encodes an NADPH oxidase in A. thaliana (Foreman et al., 2003). RHO OF PLANTS (ROP) small GTPases mediated signal transduction is involved in specifying the root hair initiation site, and future root hair elongation by stimulating RHD2 activity at the growing root hairs tip apex (Molendijk et al., 2001; Jones et al., 2002; Carol and Dolan, 2006). Recently, a number of receptor-like kinases (RLKs) have been identified that are involved in cellular growth regulatory mechanisms, especially in cell elongation associated with root hair tip growth in higher plants (Shiu and Bleecker, 2001; Lehti-Shiu et al., 2009; Lindner et al., 2012).
The plant RLK family has more than 600 members in Arabidopsis, divided into 44 subfamilies depending on their N-terminal domains (Shiu and Bleecker, 2001; Greeff et al., 2012). While RLKs have been implicated in many biologically important processes, a number of subfamilies within this superfamily have been implicated in monitoring of cell wall integrity and cell wall properties (Feng et al., 1995). In particular, CrRLK1L subfamily proteins, which includes FER (Huck et al., 2003), EREBUS (ERE) (Haruta et al., 2014), THESEUS1 (THE1) (Hematy et al., 2007), ANXUR1/2 (Miyazaki et al., 2009), have been implicated in cell wall sensing associated with a variety of cellular events such as female fertility, cell elongation, root-hair development, mechanosensing, and responses to hormones and pathogens (Boisson-Dernier et al., 2009; Cheung and Wu, 2011; Lindner et al., 2012).
The CrRLK1L subfamily is named after the first member functionally characterized in Catharanthus roseus cell cultures (Schulze-Muth et al., 1996), and Arabidopsis, contains 17 CrRLK1L subfamily members (Hematy and Hofte, 2008). The majority of CrRLK1L receptor-like kinase proteins are predicted serine/threonine kinases with a single transmembrane between an N-terminal extracellular domain and a C-terminal cytoplasmic kinase domain (Cheung and Wu, 2011). The CrRLK1L proteins have an extracellular domain with two domains showing limited homology to the carbohydrate-binding domain of animal malectin proteins (Schallus et al., 2008).
In the CrRLK1L subfamily, THE1 was discovered in a screen for suppressors that partially restored the dark-grown hypocotyl growth defect of procuste1-1(prc1-1), which is defective in the cellulose synthase catalytic subunit CESA6 (Hematy et al., 2007). THE1 is localized to the plasma membrane (PM) of elongating cells and in vascular tissues. THE1 loss- and gain-of-function plants do not appear to display significant growth defects in wild-type backgrounds, but these mutants altered the growth and ectopic lignification in a number of plants with defects in cell wall integrity (Hematy et al., 2007). Another member of CrRLK1L subfamily, HERCULES1 (HERK1), was identified as functionally redundant with THE1 in modulating cell elongation (Guo et al., 2009). While mutant herk1 plants displayed normal growth, the1 herk1 double mutants were severely stunted (Guo et al., 2009). In addition, HERK1 has an autophosphorylation activity and is highly phosphorylated in the kinase domain in planta (Guo et al., 2009). In addition, ANXUR1 and ANXUR2 (ANX1 and ANX2), are exclusively expressed in the male gametophyte (Boisson-Dernier et al., 2009). ANX1 and ANX2 are responsible for maintaining pollen tube wall integrity during migration through floral tissues, and their deactivation is thought to allow the pollen to burst during fertilization (Boisson-Dernier et al., 2009). These proteins are localized to growing pollen tube tips, and appeared to be associated with vesicles involved in polarized membrane trafficking during tip growth (Boisson-Dernier et al., 2009).
Similar to ANX1 and ANX2, and FER, which is allelic to SIRÈNE (SRN), was initially identified in the regulation of female control of fertility (Huck et al., 2003). Interestingly, FER is highly expressed in the synergid cells of the female gametophyte and in a variety of vegetative tissues, but not in the male gametophyte (Escobar-Restrepo et al., 2007; Guo et al., 2009). In the female gametophyte, FER is involved in sensing pollen tube arrival and promoting its rupture, (Huck et al., 2003; Rotman et al., 2003), in the initiation of programmed cell death of one of two synergid cells during this double fertilization event (Ngo et al., 2014), and in the inhibition of polyspermy through regulation of demethylesterified pectin accumulation in the filiform apparatus of the ovule (Duan et al., 2020). In addition to important roles during fertilization, FER has also been shown to regulate aspects of root hair elongation (Duan et al., 2010; Huang et al., 2013), calcium signaling during mechanical stimulation of roots (Shih et al., 2014), and cell wall responses to both abiotic and biotic stress (Huck et al., 2003; Rotman et al., 2003; Lindner et al., 2012; Duan et al., 2014; Ngo et al., 2014; Shih et al., 2014; Li et al., 2016). FER was identified as a ROP guanidine exchange factor 1 (ROPGEF1) interacting partner by yeast two-hybrid screening for root hair tip-growth in Arabidopsis (Duan et al., 2010). More recently, FER, and other members of the CrRLK1L receptor families have been proposed to bind to secreted RALF (rapid alkalinization factor) peptide ligands (Haruta et al., 2014), with RALF1, binding the FER extracellular domain to suppress cell elongation of the primary root (Haruta et al., 2014), and RALF23 binding FER during plant immune responses (Xiao et al., 2019).
Although several mutants of FER have been previously described, we have identified a new temperature-sensitive mutation (fer-ts) in a highly conserved glycine residue (G41S) present in the extracellular domain of the FER receptor kinase, as well as other members of the CrRLK1L receptor-like kinase family and mammalian malectin sequences. The fer-ts mutant exhibited rapid and dramatically decreased root hair tip-growth upon transferal from permissive temperature to non-permissive growth temperature. Additionally, fer-ts mutants were partially insensitive to RALF1 peptide induced root elongation inhibition at non-permissive temperatures, showed altered root growth characteristics compared to wild-type plants when exposed to auxin, and displayed reduces ROS accumulation. Cessation of root hair tip growth occurred within five minutes of transfer to non-permissive temperatures, and observation that a fluorescently-tagged version of the temperature-sensitive FER(G41S)-EYFP mutant was still correctly targeted to the plasma membrane at these early time points, suggests that the primary defect of this mutant is due to failure to properly transmit extracellular signals at non-permissive temperatures.
RESULTS
Isolation of a temperature-sensitive mutant that inhibits root hair tip growth
The regulatory GTPase, RabA4b, participates in membrane trafficking associated with the polarized secretion of cell wall components in plant cells. In addition, loss of tip localization of EYFP-RabA4b is highly correlated with inhibition of root hair tip growth (Preuss et al., 2004). In order to understand molecular mechanisms that control root hair tip growth, EMS-mutagenized seeds of a stable transgenic Arabidopsis line expressing EYFP-RabA4b were screened for seedlings with wild-type root hairs at permissive temperatures (20°C), but which displayed impaired root hair growth when grown at non-permissive temperatures (30°C). The progeny of approximately 6,000 EMS-mutagenized seeds were screened. From the screening, four temperature-sensitive (ts-) root hair growth defect mutants were isolated, which we initially termed Loss-of-Tip-Localization mutants (ltl1 to ltl4). Among these ltl ts mutants, ltl2, (subsequently referred to as fer-ts) root hair growth characteristics were examined under permissive and non-permissive temperature conditions. In permissive growth conditions (20°C), fer-ts root hairs displayed normal growth, however, both root hair growth and apical accumulation of EYFP-RabA4b of fer-ts root hairs were dramatically inhibited at 30°C (Figure 1a and 1d).
To quantify root hair elongation in fer-ts under permissive and non-permissive temperature conditions, both root hair lengths and root hair density were measured. No significant differences were found between wild-type and fer-ts either in mature root hair length, or in the number of root hairs per unit root length when plants were grown at 20°C. However, both length of root hairs and root hair density were greatly reduced in fer-ts in plants grown at 30°C (Figure 1c and 1d). Primary root length and root growth rates of fer-ts seedlings were only slightly reduced compared with those of wild-type at 30°C (See Supplemental Figure S1). These results indicated that, at least in early stages of seedling growth and development fer-ts temperature sensitive defects are largely specific to root hair elongation in non-permissive temperature conditions.
In order to characterize effects of the fer-ts mutation on root hair growth dynamics, elongating root hairs were visualized by time-lapse microscopy for two hours. Seven day-old fer-ts seedlings were placed in a temperature-controlled plant growth chamber at 20°C for 50 min and then the temperature of the chamber was rapidly transitioned to 30°C (Figure 2). While root hair growth was unaffected by temperature transition in wild-type plants, transition from permissive to non-permissive temperatures resulted in rapid cessation of tip-growth in the fer-ts mutant (Figure 2 and Movie S1). The rapid kinetics of inhibition of fer-ts root hair growth within 5-10 minutes would be consistent with rapid inactivation of fer-ts protein function at the plasma membrane.
The polarized cell expansion that occurs in root hairs is driven by specific targeting of newly-synthesized cell wall cargo to the growing apex of the root hair cell (Nielsen, 2008; Cheung and Wu, 2011). Delivery of this cell wall cargo, which occurs by polarized membrane trafficking, is associated with the tip-localized accumulation of membrane compartments labeled by the small regulatory GTPase, RabA4b (Preuss et al., 2004). EYFP-RabA4b was detected in the apical region of growing fer-ts root hairs at 20°C, but tip localization of EYFP-RabA4b was rapidly lost upon transition to 30°C (Figure 3). Because the apical accumulation of EYFP-RabA4b compartments has been tightly linked to polarized expansion in root hair cells (Preuss et al., 2004; Preuss et al., 2006; Thole et al., 2008), we examined how these compartments were affected by the transition from permissive to non-permissive growth temperatures (Figure 3a). EYFP-RabA4b accumulation was examined in fer-ts plants in a temperature-controlled chamber at 20°C for 14 min, and then the chamber was rapidly transitioned to 30°C (Figure 3b). Images of growing root hairs were collected at one minute intervals by time-lapse confocal microscopy, and tip-localized EYFP signal was quantified. While tip-localized EYFP-RabA4b signal was unaffected by transition from 20°C to 30°C in wild-type root hairs (See supplemental Figure S2), tip-localized EYFP-RabA4b was significantly reduced within one minute of the transition from 20°C to 30°C (Figure 3b and 3c). Significantly, this reduction coincided with both elevated chamber temperature and cessation of tip-growth (Figure 3c).
Map-based cloning and full-genome sequencing of the fer-ts locus
To identify the mutant locus responsible for the rapid, temperature-sensitive loss of root hair elongation and tip-localized EYFP-RabA4b, map-based cloning and full-genome sequencing was performed. F2 mapping populations were obtained by reciprocal crosses of back-crossed mutants (Col-0) with Ler wild-type plants (Bell and Ecker, 1994). Segregating F2 populations were used for the subsequent map-based cloning. The temperature-sensitive mutant lesion was initially located on chromosome 3 between the SSLP markers NIT1.2 and NGA6 (Figure 4a). Low-resolution mapping narrowed the location of the mutant locus to an approximately 2 Mb region of chromosome 3. We then performed whole-genome sequencing, and determined that the fer-ts mutant locus within this 2 Mb region of chromosome 3 was due to a G121A nucleotide replacement resulting in a G41S substitution mutation within the extracellular domain of the previously characterized FER receptor-like kinase (Figure 4b and 4c). In order to eliminate the possibility that the G➔A substitution that gave rise to the fer-ts G41S mutation influenced accumulation of FER mRNA at the transcriptional level, we performed RT-PCR analysis. FERONIA transcript levels were unchanged from those in wild-type plants (Figure 4d).
fer-ts phenotypes were confirmed by reciprocal crossing with FERONIA mutants and complementation assays
To confirm that the temperature sensitive root hair defects and loss of tip-localized EYFP-RabA4b were causally linked to the G41S mutation in the FER locus, fer-ts mutant plants were reciprocally crossed with two previously characterized FER mutants, fer-4 and fer-5 (Duan et al., 2010). In previous reports, fer-4 was shown to fully abolish FER protein accumulation, while fer-5 was shown to accumulate a truncated FER protein missing a functional cytosolic protein kinase domain, although both fer-4 and fer-5 mutants displayed constitutive root hair growth defects (Duan et al., 2010). The F1 generation of fer-ts crossed to wild-type plants displayed normal root hair growth in both permissive and non-permissive temperature conditions. However, F1 progeny of either fer-ts (paternal line) crossed with fer-4 and fer-5 mutants (maternal lines; Figure 5a), or fer-ts (maternal line) crossed with fer-4 and fer-5 mutants (paternal line; Supplemental Figure S3a) displayed ts-phenotypes at non-permissive temperatures, respectively. The F1 generations of reciprocally crossed plants were confirmed by genomic DNA PCR analysis with primers that discriminated between the fer-ts (or wild-type) FER loci and fer-4 and fer-5 T-DNA insertion mutant loci (Figure 5b-c, and Supplemental Figure S3b).
To verify that the FER G41S mutation specifically conferred the temperature-sensitive root hair phenotype, a fluorescently tagged FER-EYFP containing the G41S mutation, FER(G41S), driven by endogenous FER promoter sequences, was transformed into fer-4 and fer-5 mutant plants. Transgenic fer-4 and fer-5 plants, expressing mutant FER(G41S)-EYFP proteins rescued root hair growth defects in these two fer mutant backgrounds in a temperature-sensitive manner (Figure 5d). Further, a wild-type fluorescently-tagged FER-EYFP, driven by endogenous FER promoter sequences, was able to fully rescue fer-ts root hair defects. FER(WT)-EYFP protein was successfully detected in plasma membranes and pFER-FER(WT)-EYFP in fer-ts transgenic lines displayed the normal root hair growth both 20°C and 30°C (See Supplemental Figure S4). Taken together, these data strongly support that fer-ts phenotype is the result of the G41S mutation of the FERONIA protein.
FERONIA is localized to the plasma membrane and G41S substitution does not alter its subcellular localization at non-permissive temperatures
Previously, GFP-fused FERONIA was shown to localize to plasma membranes in various plant tissues (Duan et al., 2010). In order to confirm the plasma membrane localization of our fluorescently-tagged FER fusions, and examine whether the presence of the G41S mutation affected FER(G41S) subcellular localization, we examined the subcellular distributions of the FER(WT)-EYFP and FER(G41S)-EYFP fusion proteins in stably transformed plants (Figure 6). At permissive temperatures, FER(WT)-EYFP was observed primarily in plasma membranes in various tissues such as leaf, root and root hairs (Figure 6a). Interestingly, FER(WT)-EYFP protein was observed both in plasma membranes and an apical vesicle population in growing root hairs (Movie S2). Magnified images of FER(WT)-EYFP indicated that this fusion protein is almost exclusively plasma membrane localized, and does not display any significant accumulation in intracellular compartments (Figure 6b).
To determine whether the introduction of the G41S substitution in the fer-ts mutant might affect its protein stability, we blocked new protein synthesis by treating five-day-old Arabidopsis seedlings with cycloheximide, and then compared protein turnover rates of the FER(WT)-EYFP and FER(G41S)-EYFP proteins when grown at 30°C. Overall accumulation of the FER(G41S)-EYFP was reduced significantly during the time course, but no significant reduction in protein accumulation was not observed for either EYFP(WT)-EYFP or an actin loading control (Figure 6c). These results suggest that fer-ts mutant phenotypes may be associated with enhanced turnover due to protein misfolding.
Since the FER(G41S)-EYFP appeared to be less stable than FER(WT)-EYFP we wanted to check whether might affect the accumulation or subcellular distribution of this protein in plants subjected to non-permissive temperatures. We therefore compared the subcellular distributions of FER(WT)-EYFP and FER(G41S)-EYFP at both 20°C and 30°C (Figure 6d and 6e). While no changes in accumulation or distribution of FER(WT)-EYFP were observed in roots and root hairs between 20°C and 30°C conditions (Figure 6c), at 30°C some FER(G41S)-EYFP fluorescence could be observed in internal subcellular membranes, although significant levels of the FER(G41S)-EYFP remained at the plasma membranes in these cells even after incubation at 30°C for 6 hours (Figure 6e). Furthermore, FER(WT)-EYFP and FER(G41S)-EYFP subcellular distributions were visualized in cells in the root elongation zone every 30 s by confocal microscopy at 20°C for 10 minutes and then subsequently at 30°C for an additional 50 minutes. In both FER(WT)-EYFP and FER(G41S)-EYFP seedlings, significant fluorescent signal remained associated with the plasma membranes in the cells in these tissues at both permissive and non-permissive temperatures (Movies S3 and S4). Interestingly, FER(G41S)-EYFP signal detected in internal subcellular membranes was significantly higher when plants were continuously incubated at 30°C for 24 hours (Supplemental Figure S5). These data, when taken together with the rapid onset (<5 minutes) of mutant root hair growth defects, are consistent with a model in which the mutant FER(G41S) temperature-sensitive phenotypes are caused by inactivation of the receptor-like activities associated with this protein, and are not simply due to destabilization of the protein, or its removal from the plasma membrane.
The fer-ts displays impaired sensitivity to RALF1 peptides in non-permissive temperature conditions
Signaling in the CrRLK1L family of receptor kinases have been linked to a family of small extracellular peptide hormones called rapid alkalinization factors (RALFs; (Haruta et al., 2014; Stegmann et al., 2017). RALF1, which was previously demonstrated to suppress cell elongation of the primary root in Arabidopsis and other plants (Pearce et al., 2001), has now been shown to directly bind with the FER extracellular domain (Haruta et al., 2014; Stegmann et al., 2017). Because the G41S mutation appears to affect protein stability at non-permissive temperatures, perhaps by destabilizing the structure of the extracellular domain of this protein (Figure 6c), we were curious whether RALF1 suppression of primary root elongation would be affected in fer-ts mutants. As shown in Figure 7, both wild-type seedlings and fer-ts mutants were highly sensitive to active RALF1 peptide under permissive temperature conditions. However, as previously described, the sensitivity of root growth to RALF1 in fer-5 mutant was reduced in comparison to wild-type plants at 20°C (Figure 7a and 7c). Importantly, sensitivity of fer-ts seedlings to RALF1 peptide treatment was dramatically reduced at 30°C, even though wild-type plants and fer-5 mutants still responded to RALF1 peptide treatment with similar levels of root elongation inhibition (Figure 7b and 7d). These results support the previous determination that RALF1 peptide signaling occurs through the FER receptor-like kinase, and would be consistent with a model in which the G41S mutation results in temperature-sensitive inactivation of the extracellular ligand-binding domain of the FERONIA protein.
ROS accumulation of fer-ts was greatly reduced at 30 °C and fer-ts phenotype was not rescued by various hormone treatments
In previous reports, ROS accumulation is highly reduced in fer-4 and fer-5 mutants especially in root hair tips and primary roots (Duan et al., 2010). In order to investigate the ROS accumulation, WT and fer mutants were treated with H2DCF-DA to monitor ROS levels. In WT plants, ROS accumulation was observed in primary roots and root hairs, and these levels increased slightly upon NAA treatment at both 20°C and 30°C (Figure 8). While fer-ts plants showed similar ROS accumulation patterns as those observed in wild-type plants at 20°C, at 30°C ROS accumulation was dramatically reduced both in the absence and presence of NAA (Figure 8a). However, while ROS accumulation in the fer-ts mutant was strictly temperature dependent (compare Figures 8b and 8c), these reduced ROS levels were similar to those observed at both temperatures for the constitutive fer-4 and fer-5 mutants (Figure 8a; compare Figures 8b and 8c)
In order to investigate how broadly the temperature-sensitive fer-ts mutant phenocopied fer-4 and fer-5 mutants, these three mutants were treated with several different concentrations of hormones, and then root hair lengths and densities were measured either at 20°C or 30°C. Root hair lengths and densities displayed the similar patterns for both wild-type and fer-ts at 20°C, while both fer-4 and fer-5 root hairs were consistently shorter and less dense (Figure 9a and 9b; upper panels). However at 30°C, fer-ts root hair lengths and densities largely resembled the fer-4 and fer-5 phenotypes (Figure 9a and 9b; lower panels). Primary root length, total lateral root number, fresh weight, and total leaf numbers of fer-ts mutants also displayed a similar temperaturedependent trend; resembling wild-type plants with various hormone treatments at 20°C, but resembling fer-4 and fer-5 mutants at 30°C (See supplemental Figure S6).
The fer-ts G41S mutation reveals a functionally important role for this highly conserved glycine residue in CrRLK1L subfamily proteins
The profound effects of the G41S substitution of the fer-ts mutant on FER protein stability, RALF1 perception, ROS accumulation, and responses to a variety of hormones, suggested this mutation rapidly inactivates FER signaling at non-permissive temperatures. In addition, a similar G37D mutation is responsible for inactivation of THE1, another member of the CrRLK1L family (Hematy et al., 2007), and multiple sequence alignment analysis with other Arabidopsis CrRLK1L family members and animal malectin sequences showed that the G41 residue of FERONIA is absolutely conserved in these malectins and malectin-like 1 (ML1) domains (Figure 10a, and Supplemental Figure S7). Interestingly, based on structural studies of animal malectin proteins, five key residues (Y67, Y89, Y116, F117, D186; Figure 10b, red residues) were found to form contacts with a bound disaccharide ligand, nigerose, in the active site as determined by structural analysis of the X. laevis malectin protein (Schallus et al., 2008; Muller et al., 2010). In this malectin structure these surface exposed residues extend from the malectin fold forming the nigerose binding pocket, with the conserved glycine (G40) the bottom of this structural region (Figure 10b). While several of the tyrosine and phenylalanine residues shown to be important for interaction with carbohydrates in animal malectin proteins are maintained in plant malectin-like domains (e.g. FERONIA Y88, Y114, F115, D197) (Figure 10a, and Supplemental Figure S7), these are not surface exposed in the ML1 domain of the recently described crystal structure of FER (Figure 10c, in green) with its co-receptor LLG2 (Figure 10b, in blue) and a RALF23 ligand (Figure 10c, in magenta) (Xiao et al., 2019). It is however notable that in this structure the invariant glycine (G41; Figure 10c, red residue) of the FER ML1 domain is structurally remote from the RALF23 and LLG2 binding surfaces in the ML2 domain.
DISCUSSION
In eukaryotes, receptor like kinases (RLKs) have been implicated to play an important role in many crucial eukaryotic cellular processes, such as cell cycle progression, cell signaling, embryogenesis, abiotic and biotic stress responses (Shiu and Bleecker, 2001; Morillo and Tax, 2006; Lehti-Shiu et al., 2009). In this study, we isolated and identified a temperature-sensitive root hair elongation mutant, which we have determined is a new mutant FER allele that we have called fer-ts. The fer-ts mutant displays normal overall growth characteristics at permissive temperature (20°C), but root hair initiation and elongation are specifically and rapidly inhibited within approximately five minutes upon transfer of these plants to non-permissive temperature (30°C). We have shown that the fer-ts mutant is the result of a substitution mutation in which a highly conserved glycine residue in the FER extracellular domain is changed to serine (G41S). FERONIA is a member of the CrRLK1L subfamily of receptor-like kinases (RLKs) in Arabidopsis and the mutated glycine residue (G41S) is highly conserved in multiple members of the CrRLK1L family of receptor proteins as well as in animal malectin proteins.
While both FER(WT)-EYFP and FER(G41S)-EYFP fusion proteins displayed a plasma membrane localization at both permissive and non-permissive temperatures, FER(G41S)-EYFP displayed significantly increased protein turnover 30°C (Figure 6), that might be consistent with protein misfolding. Increased accumulation of the FER(G41S)-EYFP in internal membranes upon extended incubation at non-permissive temperatures would be consistent with at least some of the FER(G41S)-EYFP protein being retained in the ER due to misfolding (Supplemental Figure S5). However, this increased protein turnover did not appear to result in loss of accumulation of FER(G41S)-EYFP in plasma membranes. This is likely due to continued protein synthesis and at least some secretion of these proteins at the non-permissive temperature. These results, along with the rapid cessation of root hair elongation (<5 min) in non-permissive temperatures would be consistent with rapid inactivation of FER signaling activity due to protein inactivation rather than simply depletion of FER activity from the plasma membrane due to increased turnover.
FER has been implicated in a variety of plant processes, including roles in root hair tip growth as well as crucial plant processes, such as pollen tube reception, hypocotyl elongation, regulation of ABA signaling and controlling seed size (Escobar-Restrepo et al., 2007; Deslauriers and Larsen, 2010; Duan et al., 2010; Yu et al., 2012; Yu et al., 2014). In many of these processes, FER signaling appears to regulate ROS production. In constitutive fer mutants, ROS levels are reduced, and FER overexpression results in increased ROS levels. The observation that the fer-ts mutant also displays reduced ROS levels only at non-permissive temperatures suggests that this mutation affects FER signaling in a similar fashion as other fer mutants, perhaps providing a powerful tool for elucidation of downstream signaling events associated with FER function, and indicating that at least one important downstream effect of FER signal transduction is regulation of ROS production. This was elegantly explained by the discovery that FER recruits ROPGEFs, which in turn activate ROP GTPases, leading to the stimulation of RHD2 NADPH oxidase dependent ROS production (Duan et al., 2010). Therefore, FER mediated regulation of ROS production is likely important and tightly controlled for many cellular functions.
Based on sequence comparison, the extracellular domains of members of the CrRLK1L subfamily of plant RLK proteins might be predicted to share some structural similarity to the mammalian malectin protein (Schallus et al., 2008). Malectin was first identified and characterized in X. laevis as carbohydrate binding protein of the endoplasmic reticulum where it plays an important role in the early steps of protein N-glycosylation for biogenesis of glycoproteins (Schallus et al., 2008). Based on NMR structure analysis, there are five key residues in the malectin domain (Y67, Y89, Y116, F117, D186) that are located in pocket-shaped structure and these aromatic residues and the aspartate mediate interactions with the glucose residues of maltose and nigerose disaccharide ligands (Schallus et al., 2008). In plants, malectin-like domains are mainly found in CrRLK1L subfamily with a low overall sequence identity with animal malectins (Shiu and Bleecker, 2003). In FER, two malectin-like domains, ML1 and ML2, are found as a tandem-repeat in the extracellular domain. Interestingly, several key residues found in the ligand-binding pocket of the animal malectin structure are maintained in the malectin-like domains of FER and other plant CrRLK1L family members (Schallus et al., 2008). However, the discovery that members of a family of small secreted peptides, RALFs, rather than cell wall polysaccharides or oligosaccharides, serve as important ligands for FER and other CrRLK1L family receptors (Haruta et al., 2014; Ge et al., 2017; Stegmann et al., 2017; Gonneau et al., 2018) might indicate that these extracellular domains may interact with ligands in a manner distinct from their animal counterparts. Indeed the recent structural characterization of ANX1/2 extracellular domains (Du et al., 2018) and the FER extracellular domain in complex with RALF23 and the FER co-receptor, LLG2 (Xiao et al., 2019) has shown that the RALF23 binding domain and interaction with LLG2 occurs primarily with the ML2 domain, and that conserved tyrosine and phenylalanine residues in CrRLK1L malectin folds in these structures appear to be buried within the ML1 fold, and therefore likely unavailable to interact with cell wall carbohydrates in a manner similar to animal malectins.
On the other hand, analysis of the animal and plant malectin domains, reveals an additional invariant glycine residue, that is present in all animal and plant malectin sequences, and which is also found in close proximity to pocket-shape ligand-binding cleft determined in the structure of the animal ML1 domain. This invariant glycine is replaced with a serine (G41S) in the fer-ts mutation described in this paper. The highly conserved nature of this glycine residue, and the rapid elimination of FER signaling at non-permissive temperatures, suggests a critical role for the FER ML1 domain in ligand binding or transduction of a ligand-binding signal in members of the CrRLK1L family of receptor-like kinases. Indeed, mutation of an analogous glycine residue to aspartic acid (G37D) in the extracellular domain of THESEUS in the the1-1 mutant also results in a loss of function mutation in this RLK (Hematy et al., 2007). The the1-1 mutation also results in its insensitivity to its specific RALF ligand, RALF34 (Gonneau et al., 2018). Similarly, the response of fer-ts mutant to treatment with RALF1 peptide was dramatically reduced under non-permissive temperature conditions (Figure 7). Precisely how the G41S fer-ts mutation, which is structurally distant from the RALF23 peptide binding surface in the FER ML2 domain, would directly block RALF peptide perception and signaling is unclear. One potential explanation may involve the recent discovery of links between FER signaling and pectin dynamics during salt stress (Chen et al., 2016; Feng et al., 2018) and fertilization events (Duan et al., 2020). During salt stress, FER appears to sense cell wall softening and both FER ML1 and FER ML2 domains were shown to directly interact with pectin in vitro (Feng et al., 2018). More recently, FER function was shown to be required in order to maintain de-esterified pectin levels in the filiform apparatus during pollination and fertilization events (Duan et al., 2020), Whether the G41S mutation in fer-ts, or other analogous mutations of this invariant glycine residue in other CrRLK1L receptors affect the ability of these receptors to interact with or regulate pectin dynamics in plant cell walls is an intriguing possibility that warrants future investigation.
EXPERIMENTAL PROCEDURES
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia (Col-0), the fer-ts mutant was isolated from an EMS mutagenized population of wild-type (Col-0) stably transformed with a single copy of EYFP-RabAb4 driven by a 35S promoter (Preuss et al., 2004; Weigel and Glazebrook, 2006), and two FERONIA T-DNA insertional mutants (designated as fer-ts and two T-DNA insertion mutants, GABI-GK106A06 (designated as fer-4) and SALK_029056c (designated as fer-5) (Duan et al., 2010) were used in this study. Seeds were sterilized by soaking in 1% bleach solution for 10 min; after washing five times with sterilized water, they were sown onto agar plates for germination. Five-to seven-day-old A. thaliana seedlings used in the root-hair growth assays were grown vertically on plates containing 0.25x Murashige and Skoog (Sigma-Aldrich) medium at pH 5.7 supplemented with 0.6% (w/v) phytagel at 20°C under long day conditions (16 h light/8 h dark cycle). For harvesting seeds, seedling plants were transferred to soil and grown to maturity at 20°C under long day conditions.
Quantification of root hair elongation in fer-ts under permissive and non-permissive temperatures
To characterize root hair growth defective phenotypes in the fer-ts mutant, bright-field microscopy was carried out using a Nikon Eclipse E600 wide-field microscope with a x 10 Plan Apo (0.45 NA) lens as previously described in (Preuss et al., 2004). The fer-ts mutants were germinated and grown vertically on plates containing 0.25x MS medium at pH 5.7 supplemented with 0.6% (w/v) phytagel at 20°C for 7 days and then transferred to 30°C and grown for 6 hours before returning back to 20°C growth conditions for an additional 24 hours, when images of roots and root hairs were then collected. Time-lapse video microscopic analysis was carried out under permissive and non-permissive temperature conditions in wild-type and fer-ts mutants as described previously (Preuss et al., 2004). Images of growing root hairs were collected every 5 s from seedlings by time-lapse video microscopy. The temperatures of MS medium from permissive to non-permissive temperatures were controlled by an inline single-channel automatic temperature controller (Werner Instruments, Hamden, CT, model:TC-324B) controlled by a dry air thermostat inserted into the growth chamber and situated approximately 2 mm from the ROI. Temperatures were actively recorded using an Infrared Thermometer (Kintrex Inc., Vienna, VA, model:IRT0424). Raw image sequences were cropped with Adobe Photoshop and imported into Fiji-ImageJ (Schindelin et al., 2012) to generate time projections using the Stacks function. Quantification of root-hair lengths, growth rates, and densities were quantified by using calibrate and measure functions.
Map-based cloning and full genome sequencing of fer-ts
Self-fertilized, backcrossed fer-ts (ecotype; Columbia) mutants were crossed with Landsberg wild-type plants to generate a mapping population. F1 crossed plants were checked for heterozygosity with the SSLP marker “nga8” that is polymorphic between Col-0 and Ler (Bell and Ecker, 1994). Homozygous fer-ts plants were selected from the segregating F2 population by germination on MS media plates at 20°C, and subsequent analysis of root hair tip growth defective phenotypes in non-permissive growth temperatures (30°C). Homozygous plants displaying fer-ts phenotypes were grown to maturity at 20°C and seed were collected. Genomic DNA was isolated using Qiagen Plant DNA mini kits, and SSLP markers were used for rough mapping the fer-ts mutant lesion, which was initially located on chromosome 3 between the SSLP markers NIT1.2 and NGA6. Low-resolution mapping narrowed the location of the fer-ts mutant locus to an approximately 2 Mb region of chromosome 3, and full genome sequencing was performed to further determine the fer-ts mutation within this region. Libraries were generated for both the fer-ts and wild-type (ecotype Columbia) extracted DNA using Illumina TruSeq DNA kits and barcoded for multiplexing by the University of Michigan DNA Sequencing Core. Samples were sequenced on an Illumina MiSeq platform with paired-end 150 bp cycles. Sequence reads were checked for quality using FastQC then aligned to the TAIR9 genome using Bowtie2. Potential SNPs were identified using Freebayes. Additional analysis of sequence variants within the low-resolution mapped 2 Mb region of chromosome 3 to eliminate SNPs common to our resequenced Col-0 population and the fer-ts allele were sorted for context and predicted effect using a custom PERL script.
Fluorescence microscopic analysis
For pFER-FER(WT)-EYFP and pFER-FER(G41S)-EYFP transgenic plants, full-length of FERONIA including approximately 2 kb promoter was prepared by PCR reaction and sub cloned into the pCAMBIA-EYFP-C1 expression vector (Preuss et al., 2004). Wild-type and mutant FERONIA sequences were amplified from genomic DNA isolated from wild-type and fer-ts mutant plants using PCR. To produce pFER-FER(WT)-EYFP and pFER-FER(G41S)-EYFP transgenic plants, these constructs were introduced into A. tumefaciens strain GV3101, and Arabidopsis plants were transformed with A. tumefaciens using the ‘floral-dip’ method (Clough and Bent, 1998). The transgenic plants were selected by germination on 25 mg/L of hygromycin-containing medium (Duchefa, Haarlem, The Netherlands) under long day conditions (16 h light/8 h dark cycle) at 20°C. Confocal images were generated using a laser confocal microscope (Zeiss Observer.A1) connected to a CSU10 confocal scanner unit (Yokogawa, Japan) and a 10x Plan-Neofluar (0.3 NA lens), 40x Plan-Apochromat (1.3 NA lens) or 100x Plan-Apochromat (1.46 NA lens) oil objective with 491 nm laser excitation and a 535 nm emission filter for EGFP and EYFP fluorescence. Images were collected with a Hamamatsu C9100-50 camera operated using the Volocity software version 5 (the electron-multiplying (EM)-CCD detector gain settings were 123, 116 and 190 for images collected with x10, x40 and x100 objectives, respectively). Time-lapse fluorescent images were taken every 5 s using temperature controlled chambers (DeBolt et al., 2007).
RT-PCR analysis
For detection of FERONIA expression in wild-type, fer-4, fer-5 and fer-ts plants, plants frozen immediately in liquid nitrogen. Two microgram aliquots of total RNA extracted from the wild-type or mutant seedlings were used for reverse transcription primed by oligo(dT). Superscript III (Invitrogen, USA) was used for the reverse transcription reaction according to the manufacturer’s instructions. One microliter aliquot of the reaction mixture was used for subsequent PCR analysis. Actin was used as a quantifying control.
Detection of ROS in root
ROS detection by using H2DCF-DA in root hair and primary root was performed following the protocol described previously (Duan et al., 2014). Briefly, Arabidopsis seedlings were germinated grown vertically on 1/4x MS media plates for seven days at permissive (20°C) or non-permissive (30°C) temperatures. Plates were bathed with five ml of 50 uM in H2DCF-DA (Sigma-Aldrich) suspended in 1/4xMS liquid media for 5 min, followed by two gentle washes with 10 ml of 1/4xMS. Fluorescence images were collected with a Zeiss Axio Imager Z1 fluorescence microscope with 2.5x objective and green (GFP) filter set.
Effect of RALF1 peptide for root growth inhibition in fer-ts mutant
Synthetic Arabidopsis RALF1 polypeptide was synthesized by using 9-fluorenylmethyl chloroformate solid-phase chemistry with a peptide synthesizer from Thermo Scientific company and confirmed by MALD-TOF analysis (Applied Biosystems Voyager System 2098, USA). After synthesis, 5 mg of reduced synthetic polypeptide was oxidized by dissolving in 25 ml of degassed 0.1 M ammonium bicarbonate and incubating for 2 days in an opened flask under 4°C, then lyophilized. Lyophilized RALF1 powder was re-suspended in 10 ml of PBS buffer followed by two buffer exchange steps using Amicon Ultra centrifugal filter (Ultracel-3K, 3000g for 45 min each) to remove any residual ammonium bicarbonate. Seedling germination was performed in 1/2x MS liquid medium at 20°C for 3 days in long-day conditions (16-h days with 150 μE·m-2·s-1 (E, Einstein; 1 E = 1 mol of photons) light intensity. After 3 days, germinated Arabidopsis seeds were transferred to 6 well Falcon tissue culture plate with 3 ml of 1/2xMS liquid media containing 1 μM RALF1 or an equal volume of PBS and agitated on a shaker at 100 rpm (Model VS2010, Vision Scientific CO.,LTD) for an additional 3 days at 20°C or 30°C. All solutions were filter-sterilized (0.2 μm pores, Minisart 16534), and the seedlings were photographed 3 days after being transferred to the media. Quantification of primary root length was measured using ImageJ software.
Accession numbers
Sequences of the genes in this paper may be found in the GeneBank/EMBL database library under the following accession numbers: At3g51550 (FER), At4g39990 (RabA4b), At3g18780 (Actin2), At3g04690 (ANX1), At5g28680 (ANX2), At3g46290 (HERK1), At1g30570 (HERK2), At5g54380 (THE1), NP_001085212.1 (X. laevis Malectin), NP_055545.1 (H. sapiens Malectin).
SUPPORTING INFORMATION
Additional supporting Information may be found in the online version of this article.
Figure S1. Primary root growth of fer-ts mutants under permissive and non-permissive temperature conditions.
Figure S2. Subcellular dynamics of EYFP-RabA4b labeled compartments in growing root hairs in wild-type plants in permissive and non-permissive temperature conditions.
Figure S3. Confirmation of fer-ts phenotype by crossing with fer-4 and fer-5 mutant.
Figure S4. Complementation of fer-ts mutant by pFER::FER(WT)-EYFP.
Figure S5. Subcellular localization of pFER::FER(G41S)-EYFP grown in extended on-permissive temperature conditions.
Figure S6. Sequence alignment of Arabidopsis CrRLK1L subfamily receptor kinases.
Figure S7. Hormonal effects on primary root length, total lateral root number, fresh weight and total leaf number in permissive and non-permissive temperature conditions.
Movie S1. Time-lapse imaging of growing root hairs of wild-type and ltl2 mutant in permissive and non-permissive temperatures
Movie S2. Dynamics of EYFP fused FERONIA protein localization in growing root hairs.
Movies S3 and S4. FER(WT)-EYFP and FER(G41S)-EYFP protein localization in growing primary roots in permissive and non-permissive temperature conditions.
Table S1. List of primers used in this study.
ACKNOWLEDGEMENTS
We thank to Dr. Hen-Ming Wu and Dr. Alice Cheung (University of Massachusetts) for providing fer-4 and fer-5 mutants, and Jiyuan Yang for assistance in assembling and editing the manuscript. This research was supported by the U.S. Department of Energy Office of Science, Office of Basic Energy Sciences, Physical Biosciences program (DE-FG02-07ER15887; S.P., F.G., J.C., and E.N,), the National Science Foundation under Grant No. 1817697 (A.A., H.M, and E.N.), and the BK21plus program of the Ministry of Education, Science and Technology in Korea (D.K., and J-D.B.). This work used the Extreme Science and Engineering Discovery Environment (XSEDE; (John W Towns, 2014), which is supported by National Science Foundation grant number ACI-1548562.