Lectin AtGAL1 interacts with high‐mannose glycoform of the purple acid phosphatase AtPAP26 secreted by phosphate‐starved Arabidopsis

Abstract

Among 29 predicted Arabidopsis purple acid phosphatases (PAPs), AtPAP26 functions as the principle extracellular and intracellular PAP isozyme that is upregulated to recycle and scavenge Pi during Pi‐deprivation or leaf senescence. Our companion paper documented the copurification of a secreted, high‐mannose AtPAP26‐S2 glycoform with AtGAL1 (At1g78850), a Pi starvation‐inducible (PSI), and Galanthus nivalis agglutinin‐related (mannose‐binding) and apple domain lectin. This study tests the hypothesis that AtGAL1 binds AtPAP26‐S2 to modify its enzymatic properties. Far‐western immunodot blotting established that AtGAL1 readily associates with AtPAP26‐S2 but not the low mannose AtPAP26‐S1 glycoform nor other secreted PSI PAPs (i.e., AtPAP12 or AtPAP25). Analytical gel filtration indicated that 55‐kDa AtGAL1 and AtPAP26‐S2 polypeptides associate to form a 112‐kDa heterodimer. Microscopic imaging of transiently expressed, fluorescent protein‐tagged AtGAL1, and associated bimolecular fluorescence complementation assays demonstrated that (a) like AtPAP26, AtGAL1 also localizes to lytic vacuoles of Pi‐deprived Arabidopsis and (b) both proteins interact in vivo. AtGAL1 preincubation significantly enhanced the acid phosphatase activity and thermal stability of AtPAP26‐S2 but not AtPAP26‐S1. We hypothesize that AtGAL1 plays an important role during Pi deprivation through its interaction with mannose‐rich glycans of AtPAP26‐S2 and consequent positive impact on AtPAP26‐S2 activity and stability.

Purple acid phosphatase 10c encodes a major acid phosphatase that regulates plant growth under phosphate-deficient conditions in rice

“…Since their discovery in mammals, many PAP isoforms have also been purified from plant tissues ( del Pozo et al , 1999 ; Miller et al , 2001 ; Bozzo et al , 2004 ), and the physiological functions of a number of them have been characterized, most of which are related to P nutrition ( Tran et al , 2010b ; Wang et al , 2011 ; Robinson et al , 2012 ; Lu et al , 2016 ; Li et al , 2017 ; Mehra et al , 2017 ; Wu et al , 2018 ). In Arabidopsis, AtPAP12 and AtPAP26 have been shown to be the predominant PAP isozymes that are secreted into the medium under Pi starvation ( Tran et al , 2010b ; Ghahremani et al , 2019 ). Mutations of AtPAP12 and AtPAP26 result in a significant reduction in the activity of secreted APases and hence in the ability to utilize extracellular Po ( Robinson et al , 2012 ).…”

Phosphate and phosphite have a differential impact on the proteome and phosphoproteome of Arabidopsis suspension cell cultures

“…A combination of systemic 'omic' (Li et al, 2008;Tran and Plaxton, 2008;Plaxton and Tran, 2011;Plaxton and Shane, 2015;Pan et al, 2019) and targeted biochemical (Gregory et al, 2009;Liang et al, 2012;Shane et al, 2013;Ghahremani et al, 2019) studies have indicated that multiple PTMs play important roles in the PSR. These PTMs include phosphorylation, ubiquitination and glycosylation signatures that affect gene expression profiles and the activities of intra-and extracellular metabolic enzymes (Shane et al, 2013;Plaxton and Shane, 2015;Ghahremani et al, 2019;Pan et al, 2019). Targeted studies involving protein phosphorylation indicate that calcium-dependent protein kinases (CDPKs) are probably protein-level PTM regulators of the PSR (Saito and Uozumi, 2020), which aligns with observed fluctuations in calcium levels under changing Pi conditions (Matthus et al, 2019).…”

A glycoform of the secreted purple acid phosphatase AtPAP26 co‐purifies with a mannose‐binding lectin (AtGAL1) upregulated by phosphate‐starved Arabidopsis

“…An obvious hypothesis is that AtGAL1 binds AtPAP26‐S2's high‐mannose glycans and that this association modulates the catalytic activity and physico‐chemical properties of AtPAP26‐S2. Indeed, our companion paper (Ghahremani et al, ) documents that (a) AtGAL1 specifically interacts with AtPAP26‐S2 and (b) AtGAL1 preincubation significantly enhances the APase activity and thermal stability of purified AtPAP26‐S2, but not AtPAP26‐S1. AtGAL1 's induction by numerous stresses combined with the broad distribution of AtGAL1‐like lectins in phylogenetically diverse species (Table , Figure S4) implies an important and conserved function for AtGAL1 orthologs within the plant kingdom.…”

“…An obvious hypothesis is that AtGAL1 binds AtPAP26-S2's high-mannose glycans and that this association modulates the catalytic activity and physico-chemical properties of AtPAP26-S2. Indeed, our companion paper (Ghahremani et al, 2018) documents that (a) AtGAL1 specifically interacts with AtPAP26-S2 and (b) AtGAL1 preincubation significantly enhances the APase activity and thermal stability of purified AtPAP26-S2, but not AtPAP26-S1.…”

“…AtGAL1 belongs to Arabidopsis ' Galanthus nivalis agglutinin (GNA)‐related and apple domain lectin family, whose GNA domain binds high‐mannose glycans (Van Damme et al, ). Our companion paper reports that AtGAL1 specifically associates with AtPAP26‐S2 and that this interaction significantly enhances the APase activity and thermal stability of AtPAP26‐S2 (Ghahremani et al, ). To our knowledge, the current study provides the first definitive evidence for involvement of secreted glycoforms, lectins, or a phosphotyrosylated protein in plant Pi‐starvation responses, while representing the first purification and characterization of a GNA‐apple domain lectin from any species.…”