Effect of nutrient conditions on selected proteins
A focus on enzymes involved in pyruvate metabolism is provided in Fig. 6. As mentioned above and shown in Table 1, pyruvate kinase was down-regulated under low K and this effect could be alleviated by low Ca conditions in both roots and leaves. Putrescine addition tended to increase the content in pyruvate kinase under low K but decreased it at high K. For other enzymes (NADP-dependent malic enzyme, aconitase, phosphoenol pyruvate carboxylase), both low Ca and putrescine tended to attenuate the increase in protein content observed under low K in leaves. In roots, low Ca has little effect on these enzymes while putrescine increased their content at high K.
Transporters and channels that were both detected and associated with significant changes are shown in Figs. S3 and S4. As expected, a high affinity K transporter was induced at low K in roots, and in both leaves and roots, the subunit β2 of the voltage dependent aldo/keto reductase-potassium channel was downregulated at low K + high Ca and upregulated at low K + low Ca. In leaves, low K caused a decline in two ABC cassette containing proteins (TAP1 and cABCI6) and an ammonium/urea transporter, suggesting changes in nitrogen/protein homeostasis. In roots, nitrate transporters were upregulated at low K + high Ca, high K + low Ca and were clearly downregulated by putrescine.
The two-way ANOVAs carried out to look at significant proteins were used to visualize proteins associated with specific interactions effects (Fig. S5). In the group of roots proteins upregulated under high K + low Ca, proteins involved in nitrogen assimilation appeared clearly (group 1, Fig. S5a). In leaves, some enzymes of catabolism (isocitrate dehydrogenase, aconitase) were specifically enhanced at low K + high Ca (group 1, Fig 5b). Interestingly, many proteins of the photosynthetic machinery (including magnesium chelatase, Rubisco and proteins of the chloroplastic electron transfer chain) were downregulated in this specific condition, suggesting that the effect of low K was mostly driven by high Ca (group 2, Fig. 5b). K x putrescine interaction analysis clearly showed that, as found above, the downregulation of pyruvate kinase was strong under low K + high Ca, and that putrescine addition minimized differences between low K and high K for key catabolic enzymes (Figs. 5c-d).