The main P resource for plants in soils is inorganic phosphate (Pi), which mostly can be retained or complexed by cations (e.g., Ca²⁺ and Mg²⁺) [29]. The other P pool in soil comprises organic P compounds derived from the degradation of plant litter, microbial detritus and organic matter [30]. Pi is involved in cellular bioenergetics and metabolic regulation, and it is also important as a structural component of essential biomolecules such as DNA, RNA, phospholipids, ATP and sugar-phosphates [2,31]. A decrease in cytosolic Pi may restrict oxidative phosphorylation, leading to an increased proton gradient and membrane potential. In turn, this prompts an over-reduction of the components of the electron transport chain, inhibiting oxygen consumption through the COX pathway, which is coupled with ATP synthesis. This creates a decrease in the re-oxidation of NADH produced in the TCA cycle [4,16]. Furthermore, the accumulation of NADH in the mitochondrial matrix also inhibits the TCA cycle dehydrogenases, decreasing the activity of the TCA cycle and limiting the production of important metabolic intermediates [32].

 

A plant trait that enhances the capacity to acquire P in the poorest P soils is the production of cluster roots in members of the Proteaceae family, most of which do not form mycorrhizal associations [33,34,35,36]. Cluster roots are very effective at acquiring P that is largely absorbed into soil particles, because of their pronounced capacity to exude carboxylates [9]. Cluster roots of Lupinus albus release much more citric and malic acid than lupin roots of plants grown under P sufficiency. Florez-Sarasa et al. [37] observed that growth under P limitation increased the activity of AOX in cluster roots of L. albus together with the synthesis of citrate and malate. This is in the line with previous studies describing an incremented AOX abundance in cluster roots of Hakea prostata [38] and in suspension cells of tobacco after exogenous supply of citrate [39]. It is thought that the production of vast amounts of citrate in cluster rootlets is inexorably associated with the production of NADH [37,38,39,40]. This led Florez-Sarasa et al. [37] to state that AOX allows the continuity of TCA cycle activity by re-oxidizing the high levels of NADH produced during citrate synthesis when COX activity is restricted due to the P-deficiency-induced adenylate restriction.

 

The capacity to synthesize acidifying and/or chelating compounds is not restricted to species with morphological structures such as cluster roots and dauciform roots, although it is less abundant [9]. In roots without these adaptations, and in the absence of mycorrhiza, the levels of enzymes involved in organic acid biosynthesis, such as PEP carboxylase, often increase in response to P starvation in pea, tomato and Brassica nigra [41]. This increase in enzyme levels was related to a higher amount of organic acids being produced for root exudation. This capacity is not only present in roots; leaves of plants grown under P limitation may accumulate carboxylates such as citrate, malate and fumarate [41,42]. Carboxylates in leaves can be transported via the phloem and directed to roots for exudation [41,42]. Pioneering studies reported an adaptive response of respiratory metabolism and the mitochondrial electron transport chain to P limitation in NM roots [43,44,45,46], including increased AOX capacity [44,46,47,48,49]. This in the line with previous studies reporting imbalances of C/N ratio and ROS levels in AOX-deficient cells under P deficiency [46,48], although the situation at tissue level has been recognized to be more complex [18]. Recent studies have observed increases of AOX activity in roots of non-cluster roots for species grown under P limitation, such as Nicotiana tabacum and Solanum lycopersicum in the absence of mycorrhiza [13,50]. In these species, increments of AOX activity were observed, coinciding with a higher synthesis of carboxylates citrate and malate. In leaves, there were reports of pioneer studies reported increases of AOX activity in Phaseolus vulgaris and Gliricidia sepium plants grown under P limitation, but a decrease of foliar AOX activity was observed in Nicotiana tabacum, although this disparity was not related to any respiratory metabolite [51]. A recent study in Solanum lycopersicum plants grown at P-sufficient and limiting conditions, and exposed to sudden short-term (24 h) P-sufficient pulse, observed foliar respiratory bypasses via AOX and an increased accumulation of citrate, together with an enhanced expression of high-affinity P transporters LePT1 and LePT2 in conditions of limited P concentration [50]. These observations suggest that P concentration in plant organs regulates AOX activity in coordination with biochemical and molecular adjustments, functioning as a mechanism directed to maximize P acquisition [50]. Despite these findings, there is still a lack of understanding about the entire metabolic puzzle leading to the synthesis of citrate and increases in AOX activity. Studies combining metabolite profiling and measurements of electron partitioning between COX and AOX in P deficient plants could certainly shed light on the metabolic role of AOX in plant species adapted to P deficiency, which increase carbon use efficiency by decreasing Pi consumption in leaves as represented in Figure 1, below. The rate of photosynthesis and the export of its products from the chloroplast are determined by the availability of Pi in both chloroplast and cytosol [9,52]. Low chloroplast Pi availability induces a decrease in the rate of photosynthesis by decreasing both ATP synthesis and Calvin-Benson cycle activity, which results in a reduced availability of intermediates, e.g., ribulose 1,5-biphosphate (RuBP), and decreased carboxylation activity of Rubisco [53]. Low cytosolic Pi availability decreases the export rate of the products of the Calvin-Benson cycle, leading to increasing amounts of triose-phosphate and starch in the chloroplast [52,53]. Consequently, sucrose formation and glycolysis can be reduced, which may limit carbon supply into mitochondria, thus decreasing both TCA cycle activity and respiration [5,30], and therefore, plant growth and yield. In order to save Pi, leaves reduce Pi consumption in phosphorylation of sugar metabolites by converting phosphorylated metabolites (glucose-6-P, fructose-6-P, inositol-1-P and glycerol-3-P) to non-P-containing di- and tri-saccharides, as observed in Hordeum vulgare and Eucalyptus globulus P-deficient plants [54,55]. In these studies, such changes coincided with reduced levels of organic acid intermediates of the TCA cycle, suggesting a short entry of carbon into mitochondria. Bearing in mind that the conversion of di- and tri-saccharides to organic acids requires Pi, it is unlikely that they can be further respired [54]. Under this circumstance, the use of alternative carbon resources would allow the continuity of TCA cycle reactions to produce organic acids, e.g., citrate for secretion and to sustain the mitochondrial electron transport chain. In this sense, changes in levels of amino acids glutamine, arginine and asparagine was observed in P-deficient plants [54,55]. A similar response was recently observed in Hordeum vulgare [42]. These amino acids were suggested to provide carbon skeletons to mitochondria when plants reduce the consumption of Pi [42]. It is known that plants can metabolize proteins and lipids as alternative respiratory substrates when carbohydrates are scarce in plant cells [56,57,58]. Carbon consumption of these alternative respiratory substrates could be associated with the generation of NADH in the TCA cycle, whose re-oxidation would be favored by AOX activity when COX is restricted under P deficiency (Figure 1).

Nitrogen is a major component of the photosynthetic apparatus and is required by plants in greater quantities than any other mineral element. Almost all the N available for plants is present in the reduced form of nitrate (NO₃⁻), ammonium (NH₄⁺), organic compounds and molecular nitrogen (N₂) in the air [91,92]. The major source of N in soils resides in the atmosphere, through both biological N₂ fixation and the deposition of NO₃⁻ and NH₄⁺ in precipitation. In soils, NH₄⁺ and NO₃⁻ move towards roots through transpiration-driven mass flow because they are water soluble [93]. Both NH₄⁺ and NO₃⁻ enter the plant cells via specific transporters [94,95]. In order to be incorporated, NO₃⁻ is reduced to NH₄⁺ by nitrate reductase (NR) and nitrite reductase [96]. Then, NH₄⁺ is further converted into glutamine and glutamate, in a reaction catalyzed by glutamine synthetase/glutamine 2-oxoglutarate amino-transferase (GS/GOGAT) cycle [95]. At the cellular level, nitrogen assimilation is finely regulated according to its supply and demand. Nitrogen controls the regulation of nitrate transporters, activities of nitrate and nitrite reductase, the functioning of primary metabolic pathways associated with the production of reducing equivalents and the production of organic acids required for N assimilation into amino acids [2].

There is a correlation between leaf N content and rates of respiration [97,98,99,100]. Short supply of N leads to decreasing rates of both photosynthesis and respiration by reducing protein turnover and increasing breakdown of nucleic acids and enzymes [9,101]. Under these circumstances, a minimum supply of TCA metabolites (e.g., 2-oxoglutarate, isocitrate, and citrate) may maintain optimum N assimilation and amino acid biosynthesis [102,103]. Indeed, TCA cycle enzymes fumarase, NAD-dependent isocitrate dehydrogenase, and NAD-dependent malic enzyme are up-regulated under low N [98,104], and protein degradation acts as an alternative respiratory substrate [56]. In this situation, AOX activity could play a role in maintaining the functioning of primary metabolism by allowing adjustments in energy efficiency of respiration. However, there are no studies that test the regulation of AOX activity in vivo under N limitation. Such a hypothetical role should be evaluated in plant species with constitutively high levels of both AOX protein and activity. Plants possess an AOX overcapacity [8,26,105,106,107], which is variable depending on the plant species and environmental conditions [26,107]. It is thought that this overcapacity eliminates the need for de novo AOX protein synthesis, granting alternative respiration the ability to respond to sudden changes in levels of reducing equivalents [8,26,107]. In this sense, a short supply of N in plant species with high levels of AOX protein, such as legumes, could induce a decrease of both AOX protein and capacity to some extent without compromising the accuracy of AOX activity measurements, allowing us to evaluate the in vivo role of AOX under stress. Preliminary results from our laboratory in Lotus japonicus have shown that total respiration decreases (via COX and AOX) in leaves and roots when plants grow under short supply of KNO₃, in comparison with plants grown at sufficient KNO₃ supply. Interestingly, a short supply of KNO₃ induces an increase of the energy efficiency of respiration (via decreased contribution of AOX activity to total respiration) only in leaves (unpublished), which suggests the existence of a differential regulation between organs directed to maximize ATP synthesis in leaves, most likely for maintenance purposes.

On the other hand, the source of N could be another regulatory factor of AOX activity in leaves. Classic studies have observed that the expression and activity of several glycolytic and TCA cycle enzymes were differentially affected following NH₄⁺ or NO₃⁻ uptake [102,103,108], which could be accompanied with respiratory adjustments. It is known that nitrate uptake and its conversion to ammonium require large amounts of ATP and reducing equivalents [109,110,111]. However, respiration increases when ammonium is present as the main N source [112,113,114]. This has been explained as a consequence for the lack of the important reductant sink exerted by nitrate reductase, thus leading to an increase of reducing equivalents in cytosol, that are dissipated by mitochondrial electron transport chain. Under this scenario, AOX could play a significant role during this dissipation of reductants, considering its roles in maintaining the cell redox balance [8,115,116]. In this way, the accumulation of NH₄⁺ and its associated toxicity is prevented by the action of the GS/GOGAT cycle activity [117] in parallel to the mitochondrial dissipation of reductants. In fact, previous studies have shown an increase in AOX capacity and enhanced of several AOX isoforms in plants grown under NH₄⁺ supply [114,115,118,119,120]. Moreover, negative correlations between AOX capacity and nitrate concentrations were observed [114], although the electron flow through AOX under aerobic conditions can be important for the reduction of NO generation associated to nitrate reduction [121]. Interestingly, the growth of AOX-overexpressing plants is less restricted as compared to wild type (WT) Arabidopsis plants grown under NH₄⁺ nutrition, although the metabolic causes of this phenotype remain uncertain [118]. The hypothetical role of AOX in conferring metabolic flexibility during NH₄⁺ nutrition still needs to be tested by in vivo activity measurements (Figure 3).