The chloroplast-located HKT transporter plays an important role in fertilization and development in Physcomitrium patens

Abstract

Cell survival depends on the maintenance of cell homeostasis that involves all the biochemical, genomic and transport processes that take place in all the organelles within a eukaryote cell. In particular, ion homeostasis is required to regulate the membrane potential and solute transport across all membranes, any alteration in these parameters will reflect in the malfunctioning of any organelle, and consequently, in the development of the organism. In plant cells, sodium transporters play a central role in keeping the concentrations of this cation across all membranes under physiological conditions to prevent its toxic effects. HKT transporters are a family of membrane proteins exclusively present in plants, with some homologs present in prokaryotes. HKT transporters have been associated to salt tolerance in plants, retrieving any leak of the cation into the xylem, or removing it from aerial parts, including the flowers, to be transported to the roots along the phloem. This function has been assigned as most of the HKT transporters are located at the plasma membrane. Here, we report the localization of the HKT from Physcomitrium patens to the thylakoid membrane, reminiscent of the prokaryote origin of these family of transporters. Mutation of PpHKT leads to several alterations in the phenotype of the organism, including the lack of sporophyte formation, and changes in expression of many genes. These alterations suggest that the breakdown in chloroplast ion homeostasis triggers a signalling cascade to the nucleus to communicate its status, being important for the moss to complete its life cycle.

Keywords: HKT; Physcomitrium patens; chloroplast; sporophyte; thylakoid.

Figure 1

Deletion of PpHKT generates pleiotropic changes at the gametophyte and sporophyte stages. (a) Wild‐type (WT) gametophores grew on PpNH₄ medium formed normal adult gametophores after 30 days under a long day photoperiod (LD) condition (left panel); WT gametophores grew on Jiffy pellets after sporophyte induction under a short‐day photoperiod (SD) condition showed the development of normal sporophyte (right panel, arrow). (b) The PpΔhkt gametophores grew on PpNH4 medium formed larger gametophores after 30 days under a LD condition (left panel); PpΔhkt gametophores grew on Jiffy pellets after sporophyte induction under SD conditions did not produce sporophyte (right panel, arrow). (c) PpΔhkt gametophores (n = 34) were longer than WT (n = 38), independently of the growth conditions. t‐test, P‐value <0.001 (**) for both LD, and SD. (d) Fifty percent of WT gametophores (n = 412, data from four independent experiments) produced a sporophyte, while those from the PpΔhkt mutant (n = 474, data from five independent experiments) did not produce any sporophyte. t‐test P‐value <0.001. (e) Gametophores from the PpΔhkt line (n = 30) contain less chlorophyll than the wild type. Data from nine independent experiments, bars indicate standard deviation: t‐test P‐value <0.05. (f) Co‐cultivation of WT and PpΔhkt lines caused the formation of sporophytes in both lines (arrows). (g) Close to 20% of the gametophores from the PpΔhkt mutant produced sporophytes, while in the WT, 60% did. (h) Sporophytes from the PpΔhkt line were smaller and deformed, in comparison to WT.

Figure 2

PpHKT resides at the chloroplast. (a) In protonema cells, PpHKT‐3XmNeon (HKT) localizes in dots inside the chloroplast (Chloroplast), as shown in the merged images (Merge). (b) In phyllids, PpHKT‐3XmNeon is observed in cells at the centre (top) or edge (bottom) of the lamina. (c) In arquegonia, PpHKT‐3XmNeon localizes at plastids in cells of the neck (top); the square areas are shown as enlarge images (bottom). (d) In antheridia, PpHKT‐3XmNeon localizes in the sperm cells (HKT), as indicated by staining of the nuclei (DAPI) and confirmed in the merged images. Images in a, b and c correspond to Z‐stack images showing HKT in green and chloroplasts in magenta. Images in d are stacks of 10 confocal slices from the central part of the antheridium.

Figure 3

Chloroplast ultrastructure is modified in the PpΔhkt mutant. Chloroplast morphology from WT (a) and PpΔhkt (b) moss lines. Observe the unstructured thylakoid membrane and the presence of plastoglobuli in the PpΔhkt mutant.

Figure 4

PpHKT functions as a high affinity K⁺ transporter and as a low affinity Na⁺ transporter. (a) BYT12 yeast cells (Δtrk1 Δtrk2) did not grow at μM concentrations of K⁺ when transformed with the empty vector but grew when expressing PpHKT. The sensitivity of the BYT45 yeast strain to Na⁺ (b) or Li⁺ (c) was increased by the expression of PpHKT. (d) Location of the pore‐located G residues in PpHKT1 (top line) and their conservation in plant homologs (sequence logo). (e) Sodium sensitivity of the BYT45 yeast strain was decreased in the presence of increasing concentrations of K⁺.

Figure 5

Downregulated biological processes in PpΔhkt. (a) Affected pathways were mainly associated to photosynthesis, but also microtubule‐based process associated to the flagellum (yellow arrows) or translation (red arrows). (b) Downregulated genes related to photosynthesis. (c) Downregulated genes related to generation of precursor metabolites and energy. (d) Downregulated genes related to translation in chloroplast or mitochondria. (e) Downregulated genes related to cell projection organization.

Figure 6

Downregulated molecular functions in PpΔhkt. (a) Most of the molecular functions downregulated in PpΔhkt were mainly associated to chlorophyll binding or oxidoreductase activities. (b) Downregulated genes associated with oxidoreductase activity.

Figure 7

Downregulated cellular components in PpΔhkt. (a) Most of the cellular components downregulated in PpΔhkt were associated to the thylakoid membrane. (b) Downregulated genes associated to the thylakoid.

Figure 8

Upregulated biological processes in PpΔhkt. (a) Most of the upregulated biological processes in PpΔhkt were associated to (b) transmembrane transport, (c) oxoacid metabolic process, (d) carbohydrate metabolic process or (e) organic acid metabolic processes.