Sphingadienine-1-phosphate levels are regulated by a novel glycoside hydrolase family 1 glucocerebrosidase widely distributed in seed plants

Abstract

Long-chain base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as abscisic acid (ABA)-mediated signaling molecules that regulate stomatal closure in plants. Recently, a glycoside hydrolase family 1 (GH1) β-glucosidase, Os3BGlu6, was found to improve drought tolerance by stomatal closure in rice, but the biochemical functions of Os3BGlu6 have remained unclear. Here we identified Os3BGlu6 as a novel GH1 glucocerebrosidase (GCase) that catalyzes the hydrolysis of glucosylceramide to ceramide. Phylogenetic and enzymatic analyses showed that GH1 GCases are widely distributed in seed plants and that pollen or anthers of all seed plants tested had high GCase activity, but activity was very low in ferns and mosses. Os3BGlu6 had high activity for glucosylceramides containing (4E,8Z)-sphingadienine, and GCase activity in leaves, stems, roots, pistils, and anthers of Os3BGlu6-deficient rice mutants was completely absent relative to that of wild-type rice. The levels of ceramides containing sphingadienine were correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. The levels of LCBPs synthesized from ceramides, especially the levels of sphingadienine-1-phosphate, were also correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. These results indicate that Os3BGlu6 regulates the level of ceramides containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice. Long-chain base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as abscisic acid (ABA)-mediated signaling molecules that regulate stomatal closure in plants. Recently, a glycoside hydrolase family 1 (GH1) β-glucosidase, Os3BGlu6, was found to improve drought tolerance by stomatal closure in rice, but the biochemical functions of Os3BGlu6 have remained unclear. Here we identified Os3BGlu6 as a novel GH1 glucocerebrosidase (GCase) that catalyzes the hydrolysis of glucosylceramide to ceramide. Phylogenetic and enzymatic analyses showed that GH1 GCases are widely distributed in seed plants and that pollen or anthers of all seed plants tested had high GCase activity, but activity was very low in ferns and mosses. Os3BGlu6 had high activity for glucosylceramides containing (4E,8Z)-sphingadienine, and GCase activity in leaves, stems, roots, pistils, and anthers of Os3BGlu6-deficient rice mutants was completely absent relative to that of wild-type rice. The levels of ceramides containing sphingadienine were correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. The levels of LCBPs synthesized from ceramides, especially the levels of sphingadienine-1-phosphate, were also correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. These results indicate that Os3BGlu6 regulates the level of ceramides containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice. Glucocerebrosidase (GCase; EC 3.2.1.45) catalyzes the hydrolysis of glucosylceramide to glucose and ceramide (Fig. 1A) and is critical in regulating animal physiology. Four human GCases are known: lysosomal GCase (GBA1, glycoside hydrolase family [GH] 30), cytosolic GCase (GBA2, GH116), cytosolic GCase (GBA3, GH1), and intestinal lactase/phlorizin hydrolase (GH1). Gaucher's disease, the most prevalent lysosomal storage disorder, is caused by mutations in the gene encoding a lysosomal enzyme GBA1 (GH30), leading to glucosylceramide accumulation in the liver, spleen, and bone marrow and to severe complications such as thrombocytopenia and skeletal deterioration (1Grabowski G.A. Hopkin R.J. Enzyme therapy for lysosomal storage disease: Principles, practice, and prospects.Annu. Rev. Genomics Hum. Genet. 2003; 4: 403-436Crossref PubMed Scopus (89) Google Scholar, 2Jmoudiak disease: and PubMed Scopus Google a human GBA1 as enzyme therapy to of glucocerebrosidase in a with PubMed Scopus Google Scholar, therapy for enzyme glucocerebrosidase for PubMed Scopus Google GCases have but in GCases were found and functions unclear. the was identified from as a of human cytosolic The is a that PubMed Google activity in and ceramide levels in mutants were significantly with the wild type. a GCase such as to function in plants. Os3BGlu6 was from rice as a GH1 β-glucosidase, and the was by but and enzymatic of Os3BGlu6, a rice and and PubMed Scopus Google Recently, Os3BGlu6 significantly to and drought tolerance and in PubMed Scopus Google found that a of Os3BGlu6 gene in rice plants to in abscisic acid levels and to stomatal in the leaves, to a of Os3BGlu6 gene to the Os3BGlu6-deficient to stomatal closure in the leaves, in to that Os3BGlu6 drought tolerance by stomatal closure in rice but the biochemical functions of Os3BGlu6 have remained unclear. the we identified Os3BGlu6 as a novel GH1 GCase that regulates the level of have that base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as signaling molecules that regulate stomatal closure in plants in 2003; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, and the of phytosphingosine-1-phosphate stomatal PubMed Scopus Google Scholar, closure by phytosphingosine-1-phosphate and sphingosine-1-phosphate and of in PubMed Scopus Google we that levels Os3BGlu6 and the LCBPs as signaling molecules for stomatal closure in rice, LCBPs are synthesized from ceramides by we the biochemical of Os3BGlu6 and the regulation of levels by Os3BGlu6 Os3BGlu6-deficient rice plants with such as and a acid of and accumulation in PubMed Scopus Google Scholar, of PubMed Scopus Google These are by molecules from the of of in PubMed Scopus Google novel from rice with the and for the activity of a from Scopus Google Scholar, and novel from rice, Scopus Google The most of in rice were from and were identified as and (Fig. glucosylceramides for activity in rice Google Scholar, and of and accumulation in rice PubMed Scopus Google of rice with the glucosylceramide the of and to subsequent by and of and accumulation in rice PubMed Scopus Google Scholar, activity of a in of PubMed Scopus Google Scholar, found in in rice PubMed Scopus Google that the in plants is a of and that ceramides in in 2003; PubMed Scopus Google Scholar, by PubMed Scopus Google Scholar, of for PubMed Scopus Google we that glucosylceramide to ceramide by rice GCase and that ceramide a glucosylceramide (Fig. 1A) was with enzyme from rice leaves, a 1 in a (Fig. and 1 was and to a The and the of 1 in were to of a ceramide that the enzyme from rice GCase with and was to the enzyme to as a by with to of (Fig. The of the rice GCase was from the with and the of were by of showed that the of from the was to that of the of Os3BGlu6 that was and found to GH1 from rice and enzymatic of Os3BGlu6, a rice and and PubMed Scopus Google and and the rice GCase is Os3BGlu6, the Os3BGlu6 was and the was in was to ceramide by the (Fig. and that Os3BGlu6 functions as a novel GCase to from identified as GCase from and we the containing the Os3BGlu6 in to as a by with to of (Fig. The and for the glucosylceramide of the enzyme were and The of the Os3BGlu6 was very to that of Os3BGlu6 from rice plants the of the Os3BGlu6 was than that of Os3BGlu6 from rice plants the Os3BGlu6 in is the to Os3BGlu6 in the for the of Os3BGlu6 for hydrolysis of from rice plants and of were in of for The were from The results are of for are from is a novel cytosolic PubMed Scopus Google in a Os3BGlu6 from rice plants and of were in of for The were from The results are of for are from is a novel cytosolic PubMed Scopus Google biochemical of Os3BGlu6 from rice plants. 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GCase activity in leaves, stems, roots, pistils, and anthers of the rice Os3BGlu6-deficient mutants was completely absent relative to that of the wild (Fig. that all GCase activity in rice is to GCase activity in anthers was found to than in (Fig. we the regulation of ceramide levels by Os3BGlu6 in rice. to high of ceramides by Os3BGlu6, high activity in we the of ceramides in rice as of glucosylceramide and ceramide with and of the PubMed Scopus Google in the of ceramides in anthers than in the ceramides containing sphingadienine such as or were most in the with the but ceramides containing such as were most in the with the (Fig. The of ceramides containing sphingadienine in each rice organ was correlated with the GCase activity in each rice organ (Fig. and that the level of ceramides containing sphingadienine by the level of ceramides containing sphingadienine is by Os3BGlu6, we the of ceramides in anthers of Os3BGlu6-deficient rice mutants by as of glucosylceramide and ceramide with and of the PubMed Scopus Google The of ceramides containing sphingadienine in anthers of Os3BGlu6-deficient rice mutants and were lower than in the wild but the of ceramides containing in the anthers were lower than in the wild (Fig. that the level of ceramides containing sphingadienine is by Os3BGlu6 in rice plants. we the of Os3BGlu6 in rice. to the of Os3BGlu6 in we the glucosylceramide and of and accumulation in rice PubMed Scopus Google is to ceramide by Os3BGlu6 and the ceramide the of for the activity of a from Scopus Google Scholar, and novel from rice, Scopus Google of the a ceramide from the glucosylceramide in the of the wild but was in the of Os3BGlu6-deficient rice mutants and (Fig. the of in the of Os3BGlu6-deficient rice mutants with the were the as of the wild with the (Fig. These results indicate that the ceramide from the glucosylceramide by Os3BGlu6 is in the such as results and the enzymatic results that Os3BGlu6 a of the a glucosylceramide we that Os3BGlu6 a function than the in Os3BGlu6 significantly to and drought tolerance and in PubMed Scopus Google that Os3BGlu6 in rice drought tolerance via stomatal have that LCBPs such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as signaling molecules that regulate stomatal closure in plants in 2003; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, and the of phytosphingosine-1-phosphate stomatal PubMed Scopus Google Scholar, closure by phytosphingosine-1-phosphate and sphingosine-1-phosphate and of in PubMed Scopus Google we that levels Os3BGlu6 and the LCBPs as signaling molecules for stomatal closure in rice, LCBPs are synthesized from ceramides, and the level of ceramides is by Os3BGlu6 as in the level of LCBPs in rice is correlated with Os3BGlu6 activity, we the of are as in each organ as of in of PubMed Scopus Google Scholar, of from by to PubMed Scopus Google Scholar, of and phosphates in by PubMed Scopus Google The of LCBPs in anthers to than in the (Fig. The levels of especially were correlated with GCase activity in each organ and that the level by to the level is by Os3BGlu6, we the in the and the anthers of Os3BGlu6-deficient rice mutants and and found that the of LCBPs in each organ of the mutants were significantly lower than in the wild (Fig. and the of sphingadienine-1-phosphate in mutants were significantly lower than in the wild but the of phytosphingosine-1-phosphate sphingosine-1-phosphate the mutants and the wild (Fig. and and the of Os3BGlu6 significantly to and drought tolerance and in PubMed Scopus Google indicate that Os3BGlu6 regulates the level of ceramide containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice (Fig. containing sphingadienine are to by are to sphingadienine-1-phosphate by (Fig. to and of are by Os3BGlu6, we the activity of in Os3BGlu6-deficient rice The activity of and significantly the Os3BGlu6-deficient rice mutants and and the wild (Fig. and that are by we the of sphingadienine in Os3BGlu6-deficient rice mutants by The of sphingadienine in the of Os3BGlu6-deficient rice mutants and were significantly lower than in the wild (Fig. that the for of in is and the of phytosphingosine-1-phosphate stomatal PubMed Scopus Google than the of sphingadienine in Os3BGlu6-deficient rice mutants The enzymatic activity to with the the of the enzyme is than the results indicate that the in the level of sphingadienine-1-phosphate in each Os3BGlu6-deficient rice is to the in the level of sphingadienine, a of we GH1 GCase is widely distributed in the plants. 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