Plants have evolved different strategies to cope with high concentrations of Cu. The goal of this work was to identify novel genetic components involved in Cu tolerance. Up to now, forward genetics have only allowed the identification of one Cu2+-sensitive mutant, cup1-1, on high-cadmium medium, of which the responsible gene was not identified (van Vliet et al., 1995). Here we present the characterization of cmr1, a Cu2+-sensitive mutant, and the cloning of the corresponding gene. CMR1 is not only involved in Cu tolerance but also more generally in growth under normal, and especially under abiotic, stress conditions.
Identification of the cmr1 mutation and cloning of the CMR1 gene
The frequency of identified Cu2+-sensitive mutants appeared relatively low (4.7 × 10−5) as compared with those reported in the literature (Howden & Cobbett, 1992; Wu et al., 1996; Zhu et al., 1998). Several explanations can be found: mutants displaying weaker phenotype may have gone undetected; we can assume that the mechanisms of Cu tolerance are of such importance for plant survival that mutants affected in Cu detoxification mechanisms are lethal (Howden & Cobbett, 1992); and functional redundancy between the components of Cu detoxification mechanisms could have hampered the identification of sensitive mutants, a hypothesis also suggested by Howden & Cobbett (1992) to explain their low frequency observed in the screen for Cd-sensitive mutants. Nonetheless, our work describes the first successful identification of a mutant on high-Cu medium.
A map-based approach was undertaken to identify the position of the cmr1-1 mutation and the gene was eventually cloned using transcriptomics and next-generation sequencing on a mutant pool. Based on the read mappings, a large rearrangement in cmr1-1 involving a 65 kb inversion between At3g14190 and At3g14310 loci was identified. To our knowledge, such a genomic rearrangement after FN mutagenesis is quite exceptional in a plant genome. Deletions and inversions ranging from several bp to 30 kb have been described previously (Shirley et al., 1992; Li & Zhang, 2002; Belfield et al., 2012). However, another example, a large 460 kb deletion, was detected in a soybean supernodulation FN37 mutant (Men et al., 2002). As a consequence of the inversion, the At3g14190 CDS was placed under the activity of the At3g14310 promoter in cmr1-1. According to Genevestigator, the At3g14310 promoter activity is higher than the one of At3g14190, explaining why expression of the At3g14190 gene was higher in cmr1-1. Furthermore, the translation initiation codon of the At3g14310 gene was not in frame, implying that the At3g14310-encoded pectin methylesterase 3 was not functional in cmr1-1. However, the disruption of the At3g14310 gene had no impact on the cmr1-1 Cu2+-sensitive phenotype. This observation is consistent with the recent results of Weber et al. (2013) on pme3 loss-of-function mutants. An allelism test using cmr1-1 and cmr1-2, as well as complementation of the SALK_070337 line with the At3g14190 genomic fragment both validated the identity of At3g14190 as the CMR1 gene. Ogawa et al. (2011) have described an RSS1 protein that maintains meristematic activity in both shoots and roots under stress conditions in rice. RSS1 is homologous to the protein encoded by the At3g14190 gene. Like CMR1, RSS1 possesses a D-box in its N terminus and is localized both in the nucleus and in the cytosol.
Role of CMR1 in the regulation of growth in control and stress conditions
The fact that the root growth was slightly but significantly affected in control conditions supports a role for CMR1 in normal plant growth and development. Detailed observation of the root meristem showed an impairment in the establishment of the cell division plane, but only with a weak impact on root length and growth. There was no significant growth reduction of the cmr1 shoot in control conditions, suggesting that shoot apical meristem was not affected by the loss of function of CMR1. The analysis of shoot phenotype requires further studies. By contrast, the phenotype was more pronounced, but to a similar level, in both cmr1 mutant alleles under Cu excess, indicating that the presence of CMR1 is crucial for development during stress conditions. Similarly to cup1-1, stress sensitivity of cmr1 was not restricted to Cu. A strong growth reduction of PRs and LRs was observed upon all tested abiotic stresses (CdSO4, CoCl2, KCl, K2SO4, LiCl, MnCl2, NaCl, Na2SO4, ZnCl2 and sorbitol). Furthermore, upon exposure to CdSO4, CuSO4, NaCl and ZnCl2, the root phenotype of cmr1 mutants also included a swelling of the RT, an outgrowth of RHs close to the root apex and a lengthening of RHs. Several observations, such as the higher ethylene production, might account for the root phenotype. It was demonstrated that the ethylene-induced root growth inhibition was associated with a reduction in meristem size caused by a premature differentiation of cells (Thomann et al., 2009). Here we showed that upon Cu2+ excess, cmr1 overproduced ethylene in vitro. However, the inhibitors of ethylene biosynthesis were not able to restore the cmr1 phenotype significantly, suggesting that ethylene did not directly induce growth inhibition. GUS staining of pACS4::GUS lines indicated an induction of ACS4 expression in the cmr1 root apex 48 h after transfer onto Cu-enriched medium (Arteca & Arteca, 2007). This result showed a localized induction of ethylene production in the cmr1 root apex, which could be related to the RT swelling and the formation of RHs close to the root apex. In addition, ethylene may also be responsible for the RH elongation. Indeed, eto mutants which overproduce ethylene were shown to produce longer RHs than the WT (Pitts et al., 1998). Synergistic effects of auxin and ethylene on root growth have been extensively studied using Arabidopsis mutants defective in ethylene and auxin signalling (Růžička et al., 2007; Stepanova et al., 2007; Swarup et al., 2007). Ethylene was previously shown to promote auxin biosynthesis in roots by the activation of several auxin biosynthesis genes (Stepanova & Alonso, 2005). Accordingly, an increase in DR5::GUS staining in RTs of Cu2+-treated cmr1 may reflect the ethylene-induced IAA accumulation. In addition, mutants with altered responses to auxin also show defects in RH length, suggesting that, apart from ethylene, auxin also plays a role in controlling RH growth (Pitts et al., 1998; Rahman et al., 2002).
Since maintenance of cellular potassium concentrations is critical for salt tolerance (Zhu et al., 1998), the Na+-sensitive phenotype of the cmr1 mutant could be associated with impaired K+ homeostasis. Roots deprived of K+ were shown to induce the expression of genes involved in ethylene biosynthesis and signalling (Shin & Schachtman, 2004). More recently, Jung et al. (2009) demonstrated a role of ethylene signalling in low K+-induced plant responses in Arabidopsis. It was shown that the low K+-induced ethylene synthesis in turn stimulated the production of ROS in the RH-forming zone, resulting in RH elongation and induction of the high-affinity K+ uptake transporter HAK5, which contributes to the plant survival (Jung et al., 2009). Ethylene overproduction in cmr1 upon Cu stress is probably not directly induced by altered K concentration, as K concentration was also lower in control conditions without concomitantly increased ethylene concentration. Besides, the addition of K+ upon Cu excess did not restore WT pACS4::GUS expression in the RT (data not shown), strongly suggesting that elevated ethylene production in cmr1-1 was not a direct consequence of lower K+ content. The fact that the addition of ethylene inhibitors or Ca(NO3)2 only partially restored the root growth in cmr1 in stress conditions suggests that another affected process impairs root growth.
There are several features of the cmr1 phenotype pointing to a deregulation in the cell cycle: inhibition of root growth, cell division defects and the outgrowth of RHs close to RT, which is a sign of a root meristem shrinkage (Culligan et al., 2004; De Schutter et al., 2007). We have observed similarities between the phenotype of cmr1 and wee1. WEE1 is a cell cycle regulatory kinase that is activated upon cessation of DNA replication or DNA damage (De Schutter et al., 2007). WEE1 knockout plants displayed root growth arrest in the presence of DNA-damaging chemicals, similar to that of Cu- or salt-grown cmr1 (De Schutter et al., 2007; Cools et al., 2011). The phenotype in wee1 was attributable to a failure to block its cell cycle in response to DNA stress. The cells progressed into mitosis prematurely, resulting in a loss of genome integrity. The overexpression of WEE1 resulted in a strong reduction of the meristematic zone, cell cycle arrest, outgrowth of RHs close to RT and premature cell differentiation. It is thus not excluded that, analogously to WEE1, CMR1 is involved in mitosis regulation. An increased number of cells expressing the pCYCB1;1::GUS construct, especially upon Cu2+ excess, was observed in the PR meristem of cmr1. Considering growth inhibition, this result may reflect a G2/M delay or arrest, resulting in a higher number of cells expressing the mitotic marker CYCB1;1 (Zhu et al., 2006; De Schutter et al., 2007; Dohmann et al., 2008). Similar results were observed in csn affected in the COP9 signalosome, which plays a role in the G2 phase progression (Dohmann et al., 2008) or in plants overexpressing the WEE1 kinase (De Schutter et al., 2007). However, Wu et al. (2010) have recently suggested that an accumulation of CYCB1;1 could also be associated with a defect in microtubule organization rather than an arrest in the cell cycle. They noticed that many mutants which accumulated CYCB1;1 were affected in organ polarity and presented a root swelling. PI staining revealed severe alterations in cell division plane establishment in root meristems, which could indeed suggest a microtubule defect during formation of the mitotic spindle. Moreover, Wu et al. (2010) showed an accumulation of CYCB1;1 in a mutant affected in microtubule organization without any arrest in the cell cycle.
Interestingly, the At3g14190 gene was differentially expressed across several microarrays related to the cell cycle (Beemster et al., 2005; Menges et al., 2005; Dewitte et al., 2007; Cools et al., 2011; Heyndrickx & Vandepoele, 2012). The analysis of cis-acting regulatory elements, by means of PLACE (www.dna.affrc.go.jp/PLACE/), in the upstream region of CMR1 revealed several motifs found in the promoter of genes involved in the cell cycle or expressed in meristematic regions (Table S8; Planchais et al., 2002; Ramirez-Parra et al., 2003; Trémousaygue et al., 2003). According to Genevestigator, CMR1 was shown to be highly expressed in shoot and root meristems, which are the main sites of dividing cells. Moreover, the presence of a D-box supports the involvement of the protein in cell cycle-dependent protein turnover. We also observed that the cell cycle category was overrepresented in genes differentially regulated in cmr1 relative to WT (Table S9). Finally, the transient expression of CMR1 fused to GFP in tobacco protoplasts confirmed the predicted nuclear localisation.
The resemblance between the cmr1 phenotype and that of rss1 is also significant. The latter was shown to be NaCl-, LiCl- and sorbitol-sensitive. Root growth and meristem size were strongly reduced under salinity conditions, so that the differentiation zone appeared close to RT. However, in contrast to cmr1, rss1 has no particular phenotype in normal growth conditions. RSS1 is predominantly expressed in root and shoot meristems, most abundantly during the G1 and S phases. RSS1 was proposed to antagonize the G1/S checkpoint in response to stress and induce a slower cell cycle progression to equilibrate cell division with cell differentiation. It is therefore not excluded that CMR1 plays a similar role to RSS1 in maintaining meristematic activity under saline conditions, but CMR1 also plays a role, though to a lesser extent, in root growth under control conditions.
In contrast to rss1, a defect in K homeostasis in cmr1 was shown. By using K+ channel blockers on synchronized BY-2 cells, Sano et al. (2007) demonstrated that a cellular K+ threshold was required for cells to re-enter the cell cycle from the G1 to the S phase. This K+ threshold is thought to be necessary for the proper turgor regulation of cycling cells. Interestingly, they showed that the expression of some K+ transporters was modulated during the cell cycle progression. For example, the inward-rectifying channel gene NKT1 was shown to be predominantly expressed in the G1 phase and responsible for K+ uptake during the G1-to-S phase transition. An Arabidopsis NKT1 ortholog, AKT1, is involved in K+ uptake by roots (Hirsch et al., 1998). It is therefore possible that the putative cell cycle defect in cmr1 has an impact on the K+ uptake detected in normal and stress conditions. In agreement with this, our microarrays revealed several differences in the expression of K+ transporters between the WT and cmr1-1. For instance, KCO1 and AKT5, outward- and inward-rectifying K channels, respectively, were more strongly expressed in Cu2+-treated roots of cmr1-1 than in the WT (Table S10), which was confirmed by RT-PCR (data not shown). In addition, the Na+ transporter AtHKT1, which controls Na+ homeostasis and in turn affects K+ acquisition (Rus et al., 2004), was repressed in Cu2+-treated roots of cmr1-1.
In summary, the present work describes a novel genetic factor involved in plant growth and stress response. Impairment of cmr1 activity alters root growth, meristem activity, K content, and ethylene and IAA accumulation. Research into the role of CMR1 in maintaining meristematic activity is ongoing and will contribute to understanding the plasticity of plants in response to changing environments.