A dual mechanism of cellulose deficiency in shv3svl1

ABSTRACT

SHAVEN3 (SHV3) and its homolog SHAVEN3-like 1 (SVL1) encode glycosylphosphatidylinositol (GPI)-anchored proteins (GAPs) that are involved in cellulose biosynthesis and hypocotyl elongation in Arabidopsis thaliana. In a recent report, we showed that the cellulose and hypocotyl elongation defects of the shv3svl1 double mutant are greatly enhanced by exogenous sucrose in the growth medium. Further investigation of this phenomenon showed that shv3svl1 exhibits a hyperpolarized plasma membrane (PM) proton gradient that is coupled with enhanced accumulation of sucrose via the PM sucrose/proton symporter SUC1. The resulting high intracellular sucrose concentration appears to favor starch synthesis at the expense of cellulose synthesis. Here, we describe our interpretation of these results in terms of 2 potential regulators of cellulose synthesis: intracellular sucrose concentration and a putative signaling pathway that involves SHV3-like proteins.

Two of the most abundant biopolymers on Earth are the plant-derived glucans cellulose and starch. Cellulose endows the cell wall with mechanical strength and is exceptionally resistant to enzymatic degradation, so its synthesis is thought to be an irreversible investment of carbon by the plant.¹ In contrast, starch is synthesized transiently and is readily degraded to provide energy when photosynthesis is limiting.² In heterotrophic, rapidly expanding cells, carbon partitioning must be tightly regulated in order to provide for cell wall synthesis while maintaining metabolic and osmotic homeostasis. Our recent work with the double mutant shv3svl1 has shown that intracellular sucrose can regulate carbon partitioning between cellulose and starch.³ We found that shv3svl1 exhibits a sucrose-conditional cellulose deficiency that is due to enhanced sucrose uptake in the mutant (Fig. 1A). The resulting high intracellular sucrose concentration appears to favor starch synthesis while slowing cellulose synthesis.

Figure 1.

SHV3-like proteins, sucrose, and cellulose synthesis. (A) The shv3svl1 double mutant exhibits a minor decrease in hypocotyl elongation (and cellulose amount) that is greatly enhanced by the presence of sucrose in growth media. (B) A hypothetical model for the role of SHV3-like proteins and sucrose in regulating cellulose synthesis. Putative regulatory relationships involving SHV3-like proteins (SHV) in response to an unknown factor “X” are shown in red. Blue indicates regulation by intracellular sucrose (Suc), inferred from our results, and gray arrows are previously discussed regulatory relationships involving Suc as discussed in the text. The cytosolic pool of interconvertible UDP-glucose and glucose-phosphate metabolites are highlighted in yellow. Additional abbreviations not given in the text are: Fru, fructose; Glc, glucose; PGM: phosphoglucomutase; UGPase, UDP-glucose pyrophosphorylase.

A genetic suppressor screen of shv3svl1 found that sucrose-conditional enhancement of cellulose deficiency was suppressed by mutations in SUC1, a gene encoding a sucrose uptake transporter (SUT). In plants, active sucrose transport across the plasma membrane (PM) is facilitated by SUTs that couple the transport of protons and sucrose from the apoplast.⁴ We found enhanced SUT activity in shv3svl1 that is associated with increased proton secretion. Thus, misregulation of proton/ATPase activity in shv3svl1 likely results in enhanced proton secretion and proton-coupled sucrose import. The resulting high intracellular sucrose concentration, in turn, induces starch accumulation while limiting cellulose synthase activity. We also found the same phenomenon in the unrelated receptor kinase mutant feronia, for which misregulated proton/ATPase activity⁵ and sucrose-sensitivity⁶ has been previously reported.

Cellulose is synthesized at the PM by large cellulose synthase complexes (CSCs) consisting of catalytic CESA subunits that convert UDP-glucose into β-1,4-glucan chains that crystallize into cellulose microfibrils in the cell wall.¹ Starch is synthesized from ADP-glucose in the plastid.² Both substrates are derived from a readily convertible cytosolic pool of UDP-glucose and sugar phosphates that can be replenished by catabolism of sucrose (Fig. 1B). Sucrose catabolism is primarily initiated by 2 sucrose-cleaving enzymes: Sucrose synthase (SuSy; Fig. 1B) catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose, while sucrose is hydrolyzed to glucose and fructose by invertase (Inv; Fig. 1B). The relative contribution of these activities to sucrose metabolism is largely unresolved and likely varies with species and ontogeny.⁷ For many years, localization of SuSy to the PM or cellulose synthase complex has been suggested as a means of substrate channeling for cellulose synthesis.⁸ However, the necessity for such channeling is questionable, as cytoplasmic UDP-glucose concentrations are likely well in excess of the K_M of cellulose synthase. Analysis of UDP sugars in various tissues of Arabidopsis indicate cytoplasmic UDP-glucose concentrations on the order of 5–10 mM,⁹ while the K_M of plant cellulose synthase is likely on the order of 0.5 mM, as has been observed for bacterial cellulose synthase.¹⁰

Further resolution of the metabolic basis of the phenomenon that we described in shv3svl1 will require more detailed metabolomic characterization of the mutant, particularly with regard to UDP-glucose and sugar phosphates. On the other hand, sucrose may also act as a signaling molecule that initiates kinase-mediated regulation of enzymes involved in its own uptake and downstream metabolism. Waltraud Schulze and collaborators have studied the phosphoproteomic response of Arabidopsis to exogenous sucrose, and found rapid changes in phosphorylation of several kinases, transporters, and PM proton/ATPases when seedlings were treated with exogenous sucrose.¹¹ Directly relevant to our work with shv3svl1, SUC1 phosphorylation increased within 30 min of treatment, suggesting that sucrose uptake via SUC1 is self-regulating (Fig. 1B).¹¹ The function of a kinase that was identified to be rapidly phosphorylated in this work, SUCROSE-INDUCED RECEPTOR KINASE 1 (SIRK1), was further investigated by phosphoproteomic analysis of plants overexpressing SIRK1 that were treated with sucrose. In these experiments, CESA3 S171 was phosphorylated within 3 min of sucrose treatment.¹² Since the functional consequences of CESA3 S171 phosphorylation is unknown, it will be interesting to investigate its significance to the reduction of CSC activity that we observed in response to sucrose.

Even in the absence of exogenous sucrose, dark-grown seedlings of shv3svl1 exhibit a minor cellulose deficiency that indicates the general involvement of SHV3-like proteins in cellulose synthesis.³ However, assigning a biochemical function to SHV3-like proteins has been challenging. While they exhibit sequence similarity to glycerophosphodiester phosphodiesterase (GDPD) and phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes, they lack several conserved active site amino acids of these enzymes, and attempts to demonstrate either activity have been unsuccessful.^3,13 The localization of SHV3-like proteins may provide a general hint as to their function. SHV3-like proteins possess glycosylphosphatidylinositol (GPI) anchors that direct their localization to the outer leaflet of the PM.¹³ This subcellular localization suggests that they either play a relatively direct role in cellulose synthesis as it occurs, or alternatively, a more indirect role such as extracellular signaling that affects cellulose synthase activity.

We favor the latter hypothesis and note that it is consistent with several recent reports of GPI-anchored proteins (GAPs) functioning as coreceptors or components of PM signaling complexes that regulate diverse processes in plants. For example, LYM1 and LYM3 encode GAPs with a LysM domain that binds bacterial peptidoglycan. Together with the LysM domain-containing kinase CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1), these proteins contribute to chitin-triggered immune responses in Arabidopsis.¹⁴ SALT OVERLY SENSITIVE 5 (SOS5) encodes a GAP with similarity to the cell adhesion protein fasciclin that contributes to cellular expansion in roots.¹⁵ An epistatic relationship of sos5 and mutants of the receptor-like kinase-encoding genes FEI1 and FEI2 suggests they may function together in signaling at the PM to regulate cell expansion.¹⁶ Finally, LORELEI-LIKE GPI-ANCHORED PROTEIN1 (LLG1) was shown to form a complex with the receptor-like kinase FERONIA in order to facilitate perception of RALF peptide in seedlings.¹⁷ The authors of this report suggest that LLG1 may function as a coreceptor or chaperone of the FERONIA signaling complex.¹⁷

In this context, we have proposed that SHV3-like proteins may play a similar role in complex with a PM kinase that is presently unknown. Our analysis of the shv3svl1 mutant suggests that downstream output of this signaling module could include stimulus of cellulose synthase activity and inhibition of PM proton pumping activity (Fig. 1B). The coordination of these 2 processes would be logical, as acid-promoted cell wall loosening without sufficient cellulose synthesis could lead to catastrophic cell wall failure. To test this hypothesis, we are now working to identify physical and genetic interactions of SHV3-like proteins and their respective genes. By this means, we hope to identify ligands, PM kinases, and downstream components of this putative signaling pathway.

In summary, we believe we have identified a dual mechanism of cellulose deficiency in shv3svl1, wherein the mutant exhibits a primary, minor, cellulose deficiency that is greatly enhanced as a result of sucrose hyperaccumulation. While we think that high intracellular sucrose directing carbon toward starch at the expense of cellulose synthesis is a general phenomenon, this should also be tested in the absence of the additional cellulose-deficiencies that we observed in shv3svl1 and feronia. Thus, it will be interesting to see whether alternative methods of enhancing cytosolic sucrose accumulation, such as ectopic overexpression of SUTs, will similarly affect cellulose and starch synthesis. Many phosphorylation sites have been detected in CESA and accessory subunits of CSCs, indicating the likely mechanism for tight regulation of CSC function.¹⁸ A major challenge in the future will be the functional characterization of phosphorylation sites, kinases, and upstream signaling intermediates that integrate diverse inputs to regulate the conversion of sugars to cellulose.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

Funding for this work was from the Energy Biosciences Institute and the Philomathia Foundation.