Metabolic responses to P-limitation
Metabolic profiling previously revealed that P-limitation affects major metabolic processes, including primary metabolism, which results in significant changes in carbohydrates, storage compounds, phosphorylated intermediates, organic acids, amino acids, and lipids (Muller et al., 2007; Morcuende et al., 2007; Hernandez et al., 2007, 2009; Pant et al., 2015a,b). Shoots and roots of P-sufficient and P-stressed plants display distinct global metabolic phenotypes (Hernandez et al. 2007; Pant et al., 2015a), a trait also found here in switchgrass.
P-limitation impacts photosynthesis and carbon dioxide (CO2) fixation through repression of many genes involved in light reactions, chlorophyll synthesis, the Calvin cycle and photorespiration in Arabidopsis (Morcuende et al., 2007) and other plant species (Hammond et al., 2011; Carstensen et al., 2018). P-limitation also affects CO2 assimilation at the protein activity level by reducing the amount and carboxylation activity of RubisCO and other Calvin cycle enzymes, and the ATP-dependent regeneration of ribulose-1,5-bisphosphate (RuBP) (Rao & Terry, 1995). In switchgrass, the repression of genes encoding members of the RubisCO small subunit and RubisCO activase (Table S3 ), also suggests a decline of RubisCO activity. The reduction of photosynthetic energy production (ATP and NADPH) and CO2 fixation during P-stress decreases the levels of ATP-dependent glycolytic sugar phosphates (Plaxton & Tran, 2011; Carstensen et al., 2018). To maintain glycolytic carbon flux during P-limitation, plants can activate alternative metabolic pathway enzymes with lower P requirement to conserve ATP and recycle Pi (Plaxton & Tran, 2011). Such enzymes include cytosolic pyrophosphate (PPi)-dependent phosphofructokinase, non-phosphorylating NADP-dependent glyceraldehyde-3-P dehydrogenase, pyruvate Pi dikinase, PEP carboxylase (PEPC) or malate dehydrogenase. Increases in transcript levels of genes encoding some of these activities were found in switchgrass under P-stress (Table S3 ).
During P-stress, plant growth appears to be more inhibited than photosynthesis resulting in strongly reduced demand for carbon and accumulation of storage carbon components including di- and tri-saccharides (sucrose, maltose, and raffinose), starch (Plaxton & Tran, 2011) and sometimes triacylglycerides (TAGs) i.e. storage lipids (Pant et al., 2015b). With the exception of wheat and lupin (Muller et al., 2015; Nguyen et al., 2019), most plant species accumulate large amounts of sugars during P-stress (Morcuende et al., 2007; Hernandez et al., 2007; Pant et al., 2015b; Kc et al., 2018). P-stressed Arabidopsis plants accumulate starch, TAGs, large amounts of raffinose, maltose and also sucrose (Pant et al., 2015a,b). P-limited switchgrass, in contrast, accumulated sucrose as storage and/or transport form instead of maltose and raffinose (Figure 4; Table S1 ). Consistent with the accumulation of sucrose, but not maltose, several genes involved in starch breakdown to maltose and in the degradation of sucrose were downregulated in P-stressed switchgrass, suggesting that switchgrass also accumulates starch. TAG accumulation was not found in P-stressed switchgrass (Figure 3 ).
Enhanced expression and activity of PEPC during P deprivation has been associated with synthesis and exudation of organic acid/carboxylates (Shane et al., 2004; Gregory et al., 2009; Plaxton & Tran, 2011). P-stressed switchgrass displayed higher expression of severalPEPC genes and accumulated a range of organic acids (e.g. citramalic and malic acid), especially in roots. Although root exudates were not identified and quantified in this study, transcriptome analysis identified a suite of DEGs encoding enzymes and transporters conceivably involved in organic acid export from roots (Table S3 ).
Accumulation of free amino acids during P limitation has been reported for several plant species (Hernandez et al., 2007; Morcuende et al., 2007; Muller et al., 2015; Pant et al., 2015a, Kc et al. 2018; Nguyen et al., 2019) and appears to be dependent on species, age, organ and stress severity. For example, shoots and roots of Arabidopsis (Pant et al., 2015a) were found to accumulate many amino acids, including minor aromatic amino acids (e.g. Trp, Tyr), N-rich amino acids (e.g. Gln, Asn, Orn, Arg, Lys) and other major amino acids (Gly, Ala, Ser). Bean roots also accumulated a range of amino acids (especially Asn, Ser, Phe, Thr), while tea preferentially accumulated Trp, Phe and Tyr (Kc et al., 2018) and soybean roots accumulated Leu, Arg, His and Ile (Mo et al., 2019). In switchgrass, N-rich Asn increased consistently and strongly in shoots and roots and at different stress levels, while other amino acids (e.g. Glu, Ser, Thr, Phe, beta-Ala) increased in moderately stressed plants, but decreased in severely stressed plants, possibly indicating a general inhibition of primary metabolism and confirming that stress severity profoundly affects amino acid profiles.
Lipid remodeling is another metabolic response to P-deprivation in plants (Benning et al., 1998; Pant et al., 2015a). During severe P limitation, Pi is released from membrane phospholipids for critical cell activities, and degraded phospholipids are replaced by non-phosphorus containing glyco- and sulfolipids. Consistent with current models of membrane remodeling under P-limitation (Russo et al., 2007; Gaude et al., 2008), the relative abundance of phospholipids declined, while those of glycolipids and sulfolipids increased in perennial switchgrass (Figure 3 ). Underlying these metabolic changes, we found increases in the relative abundance of transcripts encoding phospholipase, glycerophosphodiester phosphodiesterase, ethanolaminephosphotransferase and galactosyltransferase processes, among others (Table S3 ). Interestingly, genes involved in the biosynthesis and modification of fatty acids were repressed while TAG lipases were induced under P-limitation (Table S3 ), suggesting that lipid recycling rather than de novo DAG and TAG biosynthesis may be the primary source of substrates for MGDG, DGDG and SQDG biosynthesis. The large induction of lipases could explain the limited accumulation (~1.5-fold during severe P stress in roots) and even decrease (~30% during severe P stress in shoots) of TAG in switchgrass (Figure 3 ) in comparison with Arabidopsis, which accumulates up to ~20-fold in shoots and ~13-fold in P-stressed roots (Pant et al., 2015b), and 40-fold in N-stressed cell cultures (Meï et al., 2017). Similar to Arabidopsis, diatoms accumulate large amounts of TAG under P and N limitations (Abida et al., 2015).