Analysis of companion cell and phloem metabolism using a transcriptome-guided model of Arabidopsis metabolism

Abstract

Companion cells and sieve elements play an essential role in vascular plants, and yet the details of the metabolism that underpins their function remain largely unknown. Here, we construct a tissue-scale flux balance analysis (FBA) model to describe the metabolism of phloem loading in a mature Arabidopsis (Arabidopsis thaliana) leaf. We explore the potential metabolic interactions between mesophyll cells, companion cells, and sieve elements based on the current understanding of the physiology of phloem tissue and through the use of cell type-specific transcriptome data as a weighting in our model. We find that companion cell chloroplasts likely play a very different role to mesophyll chloroplasts. Our model suggests that, rather than carbon capture, the most crucial function of companion cell chloroplasts is to provide photosynthetically generated ATP to the cytosol. Additionally, our model predicts that the metabolites imported into the companion cell are not necessarily the same metabolites that are exported in phloem sap; phloem loading is more efficient if certain amino acids are synthesized in the phloem tissue. Surprisingly, in our model predictions, the proton-pumping pyrophosphatase (H+-PPiase) is a more efficient contributor to the energization of the companion cell plasma membrane than the H+-ATPase.

Figure 1.

Schematic of the diel–multicell–FBA model of leaf tissue showing the inputs, outputs, and exchanges of metabolites, gases, and minerals between the 4 cell types considered. Each cell contains a core stoichiometric model of central metabolism, and fluxes are scaled to account for the different proportions of each cell type in the leaf. Each cell model is further replicated to account for the light phase (above) and the dark phase (below) with certain metabolites and minerals allowed to be passed between the phases as indicated. Since sieve elements do not have vacuoles, only starch is allowed to accumulate in them. The cells are all connected via the apoplast (this is also split into light phase apoplast and dark phase apoplast). However, only the companion cell, sieve element, and petiole sieve element cells are symplastically connected; sugars and amino acids must transit through the apoplast in order to move between the mesophyll and companion cells. CC, companion cell; MC, mesophyll cell; SE, sieve element (in the leaf); petiole SE, sieve element in the petiole, fru, fructose, glu, glucose.

Figure 2.

A flux map of the principal reactions involved in companion cells during the light phase. Arrow thickness indicates reaction flux value. The key on the right explains the colours used to indicate the production/consumption of key metabolites. The reactions shown are 1, GAP-3–PGA shuttle; 2, (NADP) GAP dehydrogenase; 3, 3-PGA kinase; 4, malate dehydrogenase; 5, GAP–Pi shuttle; 6, DHAP–Pi shuttle; 7, FBP aldolase; 8, FBP phosphotransferase; 9, Glu6P isomerase; 10, Glu6P dehydrogenase; 11, 6-phosphogluconolactonase; 12, gluconate-6-phosphate dehydrogenase; 13, Ru5P epimerase; 14, X5P–phosphate shuttle; 15, ribose-5-phosphate isomerase; 16, 3-PGA dehydrogenase; 17, triosephosphate isomerase; 18, transketolase; 19, H⁺-pyrophosphatase; 20, malate–oxaloacetic acid shuttle; 21, ATP–AMP shuttle; 22, plastidial ATP synthase; 23, fructose kinase; 25, mitochondrial ATP synthase; 26, adenylate kinase; 30, transaldolase; 31, pyruvate orthophosphate dikinase; 32, transketolase; 33, pyruvate channel; 34, phosphoenol pyruvate channel; and 35, plastidial ATP synthase. The metabolites shown are GAP, glyceraldehyde-3-phosphate; DPGA, 3-phospho-d-glyceroyl phosphate; 3-PGA, 3-phosphoglycerate; DHAP, Dihydroxy acetone phosphate; MAL, malate; OAA, oxaloacetic acid; Glu1P, glucose-1-phosphate; Glu6P, glucose-6-phosphate; d-6PGL, 6-phosphoglucono-∂-lactone; 6-PGA, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; X5P, xylulose-5-phosphate; and Ri5P, ribose-5-phosphate.

Figure 3.

Predicted fluxes of metabolite transport via the apoplast between mesophyll and companion cells in the transcriptome-weighted model solution. Abbreviations are as in Fig. 2.

Figure 4.

Amino acid synthesis by compartment in each cell in the light phase. Arrow thickness indicates magnitude of synthesis flux. Note that flux is scaled by cell ratios to better compare the activity between individual cells. MC, mesophyll cell; CC, companion cell; SE, sieve element; pSE, petiolar sieve element.

Figure 5.

Flux map showing fluxes of adenylate kinases (AK) and related reactions in the transcriptome-weighted model solution. The net effect of adenylate kinase fluxes is a consumption of ATP. The reactions depicted other than adenylate kinase are 1, pyruvate kinase; 2, ATP synthase; 3, ATP–ADP exchanger; 4, protein/RNA turnover; 6, succinyl-coA synthetase; 7, pyruvate orthophosphate dikinase; and 8, phosphoglycerate kinase and acetyl-glutamate kinase. Arrow thickness is scaled to flux value as indicated. Other than adenylate kinase, only fluxes >0.05 µmol m⁻² s⁻¹ are shown. The total ATP consumption by adenylate kinase in phloem cells is 0.2390 µmol m⁻² s⁻¹. CC, companion cell; SE, sieve element. The ranges on the adenylate kinase fluxes are CC_m, 0 to 0.1657; CC_c, 0 to 0.1657; CC_p, 0 to 0.1405; SE_m, 0 to 0.1349; SE_c, 0 to 0.1349; SE_p, 0 to 0.1403; pSE_m, 0 to 0.0299; pSE_c, 0 to 0.0299; and pSE_p, 0 to 0 µmol m⁻² s⁻¹ ATP consumed where the subscripts m, c, and p indicate the mitochondrion, cytosol, and plastid, respectively.

Figure 6.

Energy metabolism in companion cell chloroplasts. A) A flux map of the transcript-guided model solution in the light phase companion cell chloroplast. The internal, dashed arrows indicate electron transport rather than mass flow. B) A flux map of the transcript-guided model solution in the light phase companion cell chloroplast when PSII is disabled. Asterisks next to reaction numbers indicate a lower transcript abundance in companion cells when compared with mesophyll cells. Reactions with flux less than 0.005 µmol m⁻² s⁻¹ were excluded. The thickness of the lines of each solid arrow (excluding electron transport reactions) is scaled linearly to flux. The scale is shown on the left hand side of each diagram. The reactions shown are 1, GAP–3-PGA shuttle; 2, (NADP) GAP dehydrogenase; 3, 3-PGA kinase; 4, NADP⁺ malate dehydrogenase; 5, GAP–Pi shuttle; 6, DHAP–Pi shuttle; 7, FBP aldolase; 8, FBP phosphotransferase; 9, Glu6P isomerase; 10, Glu6P dehydrogenase; 11, 6-phosphogluconolactonase; 12, gluconate-6-phosphate dehydrogenase; 13, Ru5P epimerase; 14, X5P–phosphate shuttle; 15, Ri5P isomerase; 16, transketolase; 17, triosephosphate isomerase; 18, GDP kinase; 19, H⁺-pyrophosphatase; 20, malate–oxaloacetic acid shuttle; 21, ATP–AMP shuttle; 22, plastidial ATP synthase; 23, GDP-glucose pyrophosphorylase; 24, inorganic pyrophosphate; 26, adenylate kinase; 27, plastoquinol-plastocyanin reductase; 28, PSI; 29, ferredoxin–plastoquinone reductase; 30, transaldolase; 31, pyruvate orthophosphate dikinase; 32, transketolase; 33, pyruvate channel; 34, PEP channel; 35, glutamate synthase ferredoxin reaction; 36, aspartate aminotransferase; 37, PSII; 38, citrulline channel; 39, aspartate channel; 40, ornithine–citrulline shuttle; 41, ornithine synthesis pathway (from glutamate); 42, glucose channel; 43, malate–glutamate and malate–2-oxoglutarate shuttles; 44, serine biosynthesis pathway; and 45, serine channel. The metabolites shown are GAP, glyceraldehyde-3-phosphate; DPGA, 3-phospho-d-glyceroyl phosphate; 3-PGA, 3-phosphoglycerate; DHAP, dihydroxy acetone phosphate; MAL, malate; OAA, oxaloacetic acid; Glu1P, glucose-1-phosphate; GDP-Glu, GDP-glucose; Glu6P, glucose-6-phosphate; d-6PGL, 6-phosphoglucono-∂-lactone; 6-PGA, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; X5P, xylulose-5-phosphate; Ri5P, ribose-5-phosphate; Fd, ferredoxin; PC, plastocyanin; PQ, plastoquinol; Glu, glucose; GLT, glutamate; GLN, glutamine; PYR, pyruvate; PEP, phosphoenol pyruvate; CLN, citrulline; ORN, ornithine; and SER, serine.

Figure 7.

Synthesis and expenditure of energy and reducing power in companion cells during the light phase. A) A plot of the major reactions involved in ATP synthesis and consumption in companion cells during the light phase. B) A plot of the major reactions involved in PPI synthesis and consumption in companion cells during the light phase. C) A plot of the major reactions involved in NAD(P)H synthesis and consumption in companion cells during the light phase. (m), (p), and (c) indicate reaction occurs in the mitochondrion, chloroplast, and cytosol, respectively.