Implication of NHX3 in flowering time and flower development

Wang et al. (2007) reported that AtNHX3 is mainly expressed in petals. In addition to this, the unusual behavior of this protein, which was unable to suppress the sensitivity of AXT3K cells to HygB and could not be expressed in onion cells as an GFP-translational fusion to determine subcellular localization (B. Cubero and JM Pardo, unpublished) indicated that this protein could have significant functional differences relative to other vacuolar AtNHXs. NHX proteins with specific expression in flowers or fruits have important roles in K⁺ accumulation in their locations (Yamaguchi et al. 2001; Hanana et al. 2007; Yoshida et al. 2009). Specifically, in the case of Ipomea nil and Ipomea tricolor, the NHX1 protein is expressed in the vacuolar

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membrane of petal limbs regulating vacuolar pH and petal color, an essential trait for the reproductive cycle of these plants. More interestingly, the activity of these proteins produced vacuolar alkalinization in petals, with a large pH shift of up to +1.1 units (Morita and Hoshino 2018). This is a large variation in comparison to the ones observed linked to AtNHX activity in Arabidopsis seedlings, that has been reported to account for pH shifts between 0.5 – 0.8 units by comparing the double mutant nhx1 nhx2 with wild-type plants (Bassil et al. 2011c;

Andres et al. 2014; Reguera et al. 2015). Taking all this into consideration, we hypothesized that AtNHX3 could have a mechanism of action similar to Ipomeoa’s NHX proteins with special function in flowering time or flower development. However, we demonstrated that neither nhx3 and nhx4 single mutants, nor the nhx3 nhx4 double mutant had any effect in flowering time r flower opening. As previously described for Ipomea nil, the lack of expression of the vacuolar NHX did not inhibit cell expansion and flower opening (Yamaguchi et al. 2001;

Pittman 2012), meaning even if AtNHX3 contributed to this process, other mechanisms are also involved. For instance, the reduced vacuolar NHX antiport activity in the nhx1 nhx2 mutant was detrimental for the opening and closing of the stomata. However, reduced and delayed stomatal movements remained in the mutant that were proposed to be mediated by the influx of osmotically active solutes such as sugars and organic acids (Andrés et al., 2014).

The double mutant nhx1 nhx2 has been described to display significant phenotypes in their reproductive organs (Bassil et al. 2011c), but rather than related to flower opening or petal size they were more related to reproductive traits, such as low number of siliques with few or no seeds, shorter and narrower flowers, stamens with shorter filaments, o non-dehiscent anthers.

To determine if AtNHX3 or AtNHX4 contributed to turgor generation for cell expansion in petals, the area of the petals was measured in nhx3 and nhx4 mutants. Although the result showed that the petal size of the single mutants nhx3 and nhx4 did not significantly differ from the wild type Col-0, the double mutant showed significantly largerpetal area then the wild-type and single mutant lines. Unfortunately, it was no possible to study weather this difference was due to a variation in the cells size or cell number in petals. However, there is no previous evidence that vacuolar NHX proteins could affect the number of cells in petals, but the nhx1 nhx2 mutant showed reduced cell sizes in leaves (Barragán et al., 2012).

Therefore, we suggest that the difference observed is due to increased cell/vacuolar size in the double nhx3 nhx4 mutant. Whether the lack of expression of AtNHX3 and AtNHX4 in flowers might induce the expression of other solute transporters that help in petal extension and flower opening should be studied. In guard cells the main solutes involved in the

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osmoregulation process are sucrose, K⁺, and accompanying anions (malate and chloride), depending on the environmental conditions and time of the day (Andres et al. 2014), and it can be assumed that these would be implicated in petals expansion as well. The Arabidopsis CHX20 is a putative K+/H+ exchanger that appears to play a role in guard cell osmoregulation through K+ fluxes and possibly pH modulation (Padmanaban et al. 2007). Little is known about the function of most CHX proteins, but it should be no ruled out the possibility of their implication in petal extension because they are expressed in flower organs and in pollen where they contribute to extension of the pollen tube (Sze et al. 2004)

Considering that the main function of Ipomea’s NHX1 in flower development is the change in vacuolar pH, pHvac was measured in Arabidopsis petals using the BCECF method. The results showed that nhx3 single mutant and double mutant nhx3 nhx4 had a more acidic pH in comparison with the wild-type line. Moreover, nhx4 single and the nhx1 nhx2 double mutant had intermediate pH values. This in accordance to the expected results considering the expression of AtNHX3 in flowers (Wang et al. 2007). The abscense of AtNHX3 would diminish K+ influx into the vacuole in petals, being these cells unable to alkalinize the lumen.

The phenotype observed in the double mutant nhx1 nhx2 could be explained by the reported expression of AtNHX1 and AtNHX2 in the stomata of petals, which might me altering somehow the size of petals (Shi and Zhu 2002; Barragán et al. 2012). Apse and Blumwald (2003) also identified AtNHX1 mRNA in petal epidermis, but no reference to the cell type in which it was expressed or their size was given. A similar situation was described for Ipomoea nil and Ipomoea tricolor. In the nhx1 mutant of I.nil, the petals reddish-purple buds become purple open flowers instead of blue flowers, and the cells’ vacuolar pH have a partial increase, indicating that there must be genes other than InNHX1 that mediate vacuolar alkalization in the flowers (Yamaguchi et al. 2001). Ohnishi et al. (2005) showed that in I. nil the protein InNHX2, which is expressed mainly in leaves, stems and roots, has a low and time limited expression in petals before flower opening. This protein accumulates Na⁺ and K⁺ in the vacuole, being the responsible for the partial alcalinization observed in the nhx1 mutant.

Based in this, it is probable that other vacuolar AtNHX o AtCHX have a role in the pHvac of petals, which could explain the intermediate pHvac of XXX. However, AtNHX3 seems to be InNHX1 orthologue which has evolved to specifically modify petals pH, with a pollination and reproduction role (Morita and Hoshino 2018). In summary, our results indicate that AtNHX3 is the most critical protein responsible for the control of the vacuolar pH in the petals of Arabidopsis.

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CONCLUSIONS

1- The topological and ternary models of AtNHX1 indicate that this plant protein conserves the structural features characteristic of microbial and mammalian members of the CPA superfamily, consisting of cytosolic N- and C-terminal ends, twelve transmembrane segments arranged in an antiparallel manner and the distinctive Nha-fold at the active center.

2- Several conserved amino acids that are essential for the activity of AtNHX1 have been identified at the active site and the Nha-fold. These residues are the T156 and D157 of the TD-motif in TM4, D185 of the ND-motif in TM5,, and arginines R353 and R390 in in TM10 and TM11, respectively. Residue N184 at the ND-motif that is highly conserved in electroneutral antiporters of the CPA1 family is not essential for the activity of AtNHX1, at least in the heterologous system used to validate the functionality of mutated AtNHX1 proteins.

3- In agreement with the proposed function in planta, AtNHX1 can regulate vacuolar pH in yeasts cells, and the expression of mutant mutant in the conserved amino acids of the Nha-fold generate pH differences in the yeast vacuole.

4- Calmodulin-binding domains that mediate interaction with CML18 have been identified at the C-terminal cytosolic extensions of AtNHX1 and AtNHX2, to two major K+/H+

exchangers in Arabidopsis vacuoles. This interaction takes places in the cytosol and not in the vacuolar lumen as previously reported.

5- The calmodulin-binding domain of AtNHX1 is essential for protein activity. The highly conserved residue D506, but not H499, at the core of the calmodulin binding domain is essential for the interaction of AtNHX1 and CML18 and for protein activity.

6- A model is proposed in which the calmodulin binding domain of AtNHX1 integrates a cis-regulation by cytosolic pH and the trans-regulation by CML18 and calcium. According to this model, CML18 binding stimulates K+/H+ antiport at the tonoplast as long as the cytosolic pH is not exceedingly acidic, in which case protonation of H499 promotes the formation of a salt bridge with D506, thereby hindering the binding of CML18 to restrain further ion exchange and the release of protons into the cytosol.

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7- Genetically encoded ratiometric pH-sensors based on pHGFP fused to markers of different cellular membranes is a suitable to generate cytosolic pH-maps of Arabidopsis thaliana seedling that excellent resolution in time and space.

8- The SOS3/CBL4 is generally regarded as a critical sensor protein of salinity stress, and the pH-maps of sos3 mutant demonstrate that dynamic variations of cytosolic pH induced by salinity in wild-type seedlings are largely absent in the mutant.

9- AtNHX3 and AtNHX4 are vacuolar proteins with specific roles in Arabidopsis development. AtNHX3 has an important role in the regulation of vacuolar pH of petals cells.