Role of potassium transport in rice in securing high crop yields – RiceKTrans

The project is based on a reverse genetics approach: rice plants lacking functional versions of the targeted K+ channels and transporters, mutated by insertion of an endogenous transposon or generated using Crispr-Cas9 technology, will be obtained. Mutant lines for homologous genes in the model plant Arabidopsis (already available) will also be used. Phenotypic analyses of the mutant plants (loss of function) will concern growth, and physiological parameters such as water consumption, K+ content of tissues and ascending and descending saps, stomatal conductance, photosynthetic capacity, transpiration (analyzed under controlled growing conditions). In addition, yield field trials will be conducted on the non-transgenic mutant lines. We will express fluorescently tagged versions of the targeted K+ channels and transporters to study whether these transport proteins exhibit polarized subcellular localization in the plant. We will use Xenopus oocytes to express the rice efflux channels and K+ transporters and characterize their biophysical properties (ion selectivity, regulation by membrane electrical potential and environmental factors). The specific roles of these transporters will at last be further analyzed, at the cellular level, using ion-selective electrodes.

HAK/KUP transporters in rice. One of the main objectives of the project was to create and study a rice mutant that had lost the OsHAK9, 10, 24, and 25 genes, homologs of the Arabidopsis KUP2, 6, and 8 genes, described as K+ secretion systems in stomata (Osakabe et al. 2013, Plant Cell 25: 609-624). We obtained, using the crispr-Cas9 technology, the desired quadruple mutant (quadruple knockout “hak-q-ko”), which shows shifts in the reading frame of the nucleotide sequences of the four genes between positions 290 and 330. Stomatal conductance was indeed altered in the mutant (under K+ deficiency conditions). Furthermore (Figure 1), the loss of the four HAK genes led to an increase in root system biomass and a change in its architecture (less deep). An increase in tissue K+ content was observed in this quadruple knockout under conventional fertilization conditions, which could be a favorable trait for grain production.

Role of the OsK5.2 Shaker channel in K+ nutrition under high salinity conditions and plant salt tolerance. K+ transporters of the HAK family have a positive effect on salt tolerance, probably because they improve K+ uptake by the plant (Liu et al. 2022, Crop Journal 10:13-25). The role of K+ efflux channels in salt tolerance had not yet been studied. We have demonstrated the major role of the OsK5.2 K+ channel in salt tolerance in rice, through a dual mechanism: limiting Na+ flux toward the shoot (linked to transpiration control) and stimulating K+ secretion in ascending sap (Zhou et al. 2022 Plant Cell Environ 45:1734-1748) (Figure 2).

Evolution of K+ extrusion mechanisms in plants. Little attention had yet been paid to the evolution of K+ extrusion mechanisms in plants. Our analysis (Hmidi et al. 2025 New Phytologist 245:69-87) (Figure 3) revealed that these mechanisms must have developed very early in plant evolution, as charophyte algae possess several Shaker genes that encode K+ efflux channels, as well as large potassium channels (BK) with a similar function. During evolution, the molecular diversity of K+ efflux channels has decreased, but their role has diversified.

Software generated as part of the project, reusable and freely accessible to all. We have developed the SISE program, which enables the measurement and analysis of ion fluxes using ion-selective microelectrodes (Ahmad et al. 2025 Plant Methods 21:10). The source code is available (https://github.com/Rob-Roelfsema?tab=repositories) and the procedures for calculating ion fluxes can therefore be evaluated and modified.

Scientific event. We organized an international thematic school on mechanisms for studying ion and water transport in plants (Mistral, Montpellier 2022) to which attended the doctoral student recruited on RiceKTrans Florence Muraya and the post-doc Dorsaf Hmidi (Figure 4).

Rice is a major source of human nutrition, and identifying new breeding targets helps meet the demand of a growing global population. In the RiceKTrans project, we explored the potential of modifying the activity of rice K+ transport systems (Shaker channels and HAK transporters) on long-distance transport efficiency, stomatal movements, and salt tolerance.

The selected HAK genes (coding for four transporters from the same clade) proved to be a target of interest for breeders, as the simultaneous loss of activity of these systems made it possible to (i) obtain a more superficial root architecture, thereby capturing more nutrients, without suffering from drought periods when cultivated in paddies, (ii) increase plant biomass and K+ content, and thus probably grain yield.

Another potential new target of interest for breeders has been identified as the Shaker OsK5.2 gene, since it has been shown to be a strong positive determinant of salt tolerance in rice (Zhou et al. 2022 Plant Cell Environ 45:1734-1748).

The promising analyses of rice HAK genes need to be repeated and completed. New experiments are currently underway to analyze the root growth phenotype in greater detail. In addition to analyzing growth in controlled chambers, it will also be examined in greenhouses, and the impact of the loss of the four OsHAK genes on crop yield will be determined. At a later stage, this analysis will be continued under real conditions (in the field), if the amendment to European Union legislation on genetically modified crops (status of point mutations generated by CRISPR-Cas9) can be implemented. The hak-q-ko mutant exhibits a clear phenotype of growth and K+ accumulation, but it is not known whether the four affected genes are all equally important. As part of the following, the hak-q-ko mutant will be backcrossed with wild-type plants to obtain single mutants for each of the four genes. An analysis of the phenotype and genotype of the roots will reveal the relative impact of each of the four OsHAK genes on root growth. In addition, the expression profile of the OsHAK genes must be determined in order to predict their specific contribution to K+ transport in rice. Such an analysis is typically performed using constructs expressing β-glucuronidase (GUS) under the control of the OsHAK gene promoter region, but it can be supplemented by tissue expression data available for rice.

* Publication in peer-reviewer journal (impact factor: 7.2):
Zhou J, Nguyen TH, Hmidi D, Luu DT, Sentenac H, Véry A-A, 2022. The outward shaker channel OsK5.2 improves plant salt tolerance by contributing to control of both leaf transpiration and K+ secretion into xylem sap. Plant Cell Environ. 45(6): 1734-1748. doi: 10.1111/pce.14311 (the authors involved in the RiceKTrans project are in bold).

* Poster at an international meeting:
Zhou J, Hmidi D, Nguyen TH, Luu DT, Sentenac H, Véry AA, 2021. An outward Shaker channel improves rice salt tolerance by combining control of leaf transpiration and K+ secretion into xylem sap. 18th International Symposium on Rice Functional Genomics, Barcelona, Spain (3-5 Novembre).
The authors from the RiceKTans project, in bold, were present at the meeting; Dorsaf Hmidi, ccd recruited on the project presented the poster

Cereals in general, and rice in particular, are the major source of nutrition for a growing world population. Many of these crops are grown at intensively utilized fields, in which soil nutrients need to be constantly resupplied by fertilization. Because of the high costs and energy demand, there is a need to reduce the use of fertilizers and adapt to a more sustainable form of agriculture. Crop plants that use nutrients more efficiently as the currently available lines, can help to reach these future goals. Potassium (K+) is the most important cationic nutrient and its transport has been studied intensively for the model plant Arabidopsis., but little is known about the transport proteins that channel K+ fluxes in the cereals. Our previous study (Nguyen et al., 2017, Plant Physiology) has revealed important differences in tissue localization and activation mechanisms of K+ efflux channels, between rice plants and Arabidopsis. In the proposed project we will focus on Shaker type K+ efflux channels and HAK/KUP K+ efflux transporters, which enable transport of K+ from the root to the shoot of rice plants and within the stomatal complexes in the leaves. We will pinpoint the cell types in which the selected K+ transport proteins are expressed and generate rice plants that lack functional versions of these proteins. These mutant lines will be compared with wild type rice plants, for their ability to grow, consume water and produce crop yield, at green house and field conditions. Moreover, we will use Arabidopsis guard cells and Xenopus oocytes to express the rice K+ efflux channels and transporters and characterize their biophysical properties, like ion selectivity and voltage-dependent activation. The specific roles of the selected K+ channels and transporters in xylem function and stomatal movements will be at the centre of our attention. We will introduce fluorescence-tagged versions of the K+ channels and transporters to study if the transport proteins show a polarized subcellular localization. The specific roles of these transporters will be further uncovered with single cell techniques, in which ion selective electrodes are applied. Our studies will provide insights into the functions of specific K+ efflux channels and transporters at the cellular level, as well as their importance for growth of rice plants at field conditions. It is likely that this knowledge will be valuable for breeding rice plants with a lower demand for K+ fertilizers, while maintaining a good nutritional quality of the grains. Such traits will be of prime importance for sustainable agriculture and future food security.