Introduction
Potassium (K) is an essential nutrient (up to 5% of plant dry weight) involved in crucial physiological processes such as electrochemical homeostasis, stomatal aperture and enzyme catalysis (Wang & Wu, 2013; Anschütz et al. , 2014). K deficiency inhibits plant growth and primary production and therefore, intense efforts are being devoted to improve K acquisition by plants (Shin, 2014; Rawat et al. , 2016). K-deficient soils are relatively common in countries of the intertropical region, such as Brazil, Equatorial Africa, China and South-Eastern Asia (like Indonesia), and Australia (www.fao.org) and thus important crops like oil palm, sunflower, cotton, or rice are concerned. In addition, K-deficient areas have variable geological substratum and soil total base content (in particular exchangeable calcium), and as a result background nutrient (ionic) conditions that accompany K deficiency are highly variable. For example, in Sumatra (Indonesia) where oil palm is cultivated, both Ca-poor (histosol) and Ca-rich (cambisol) soils can be found, and such a difference in Ca has an important impact on K fertilization decisions.
Symptoms of K deficiency in plants have been studied for more than fifty years and include metabolic effects such as an inhibition of glycolysis due to a decrease in pyruvate kinase activity, as well as accumulation of typical metabolites such as putrescine (Jones, 1961; Jones, 1966; Okamoto, 1966; Freeman, 1967; Okamoto, 1967; Okamoto, 1968; Besford & Maw, 1976; Armengaud et al. , 2009; Hussain et al. , 2011; Sung et al. , 2015). Metabolomics analyses of K deficiency inArabidopsis showed a reconfiguration of amino acid and organic acid synthesis, consistent with a change in nitrogen assimilation (in favour of neutral amino acids), and an inhibition of the most K-sensitive enzyme, pyruvate kinase, thereby leading to altered glycolytic metabolism (Armengaud et al. , 2009). Recently, metabolic changes caused by low K availability have been described in details in sunflower, and it has been shown that in addition to the well-known build-up of putrescine, low K induced a strong increase in respiratory CO2 efflux and modified the flux through the C5-branched acid pathway; furthermore, the natural15N/14N isotope composition (δ15N) in leaf compounds showed that there was a change in nitrate circulation, with less nitrate influx to leaves under low K (Cui et al. , 2019a).
Importantly, as pointed out recently (Cui et al. , 2020), K deficiency is not associated with a general decrease, but actually leads to a significant increase in cation load, which comes from the considerable increase in Ca2+ (and occasionally in Mg2+) in tissues. In fact, there is an antagonism between K+ and Ca2+ (Dibb & Thompson, 1985; Jakobsen, 1993; Daliparthy et al. , 1994), and the K x Ca interference in fertilization has been documented for nearly 50 years in many crops (such as poplar, sugarcane, sunflower, rapeseed, tobacco, oil palm, soybean, castor bean or wheat). Also, if K-deficiency occurs under low Ca conditions, typical K symptoms such as putrescine accumulation are partly suppressed (Richards & Coleman, 1952; Coleman & Richards, 1956). Therefore, despite the role of Ca2+ in signaling to remobilize vacuolar K+ ions under low K conditions (Amtmann & Armengaud, 2007; Pandey et al. , 2007; Tang et al. , 2020), some of the commonly observed K deficiency symptoms probably reflect the response to a disequilibrium in external (soil) cation composition, in which Ca2+ is over-represented. To our knowledge, this question has never been tackled directly. In a recent meta-analysis of papers dealing with the effect of nutrient antagonism on yield, the antagonism between K and Ca appears to be understudied as compared to Kx Mg antagonism (Rietra et al. , 2017). Also, the detailed metabolic responses to K availability when Ca is varied have not been documented. In this context, putrescine is an interesting biomolecule, since its role under K deficiency has been suggested to be to both mitigate reactive oxygen species (ROS) production –and thus the down-regulation of mitochondrial damage– and participate in cellular Ca2+ homeostasis (Cui et al. , 2020).
To clarify these aspects, we cultivated sunflower plants under different K availability conditions (low, 0.2 mM, and high, 4 mM), under low or high Ca conditions, with or without addition of putrescine (experimental design detailed in Fig. S1). We carried out physiological, ionomics, metabolomics and proteomics analyses and also used15N-labelling to assess nitrate absorption. Our objectives were to assess whether (i) some of the typical metabolic symptoms of K deficiency could actually be suppressed by the use of low Ca conditions, and (ii) putrescine addition alleviates some metabolic effects of K deficiency or on the contrary, triggers a low-K response. Our results show a considerable effect of Ca on metabolism, low Ca conditions compensating for some of low-K symptoms. Putrescine addition is found to improve N nutrition but exaggerates glycolysis inhibition, suggesting that it plays a dual role under K deficiency.