Characterization of mannitol metabolism genes inSaccharinaexplains its key role in mannitol biosynthesis and evolutionary significance in Laminariales

Abstract

As a unique photosynthetic product in brown algae, mannitol exhibits high synthesis and accumulation in Saccharina japonica. Mannitol acts as a carbon-storage compound and is an osmoprotectant, imparting increased tolerance to osmotic stress. However, the underlying biochemical and molecular mechanisms in macroalgae have not been studied. Analysis of genomic and transcriptomic data has shown that mannitol metabolism in S. japonica is a circular pathway composed of four steps. In this study, one S. japonica mannitol-1-phosphate dehydrogenase (M1PDH2) and two mannitol-1-phosphatase (M1Pase) proteins were recombinantly expressed to analysis enzyme biochemical properties. RNA sequencing and droplet digital polymerase chain reaction were used to analyze the gene expression patterns of mannitol metabolism in different generations, tissues, sexes, and abiotic stresses. Our findings revealed insights into the mannitol synthesis pathways in brown algae. All genes were constitutively expressed in all samples, allowing maintenance of basic mannitol anabolism and dynamic maintenance of the “saccharide pool” in vivo as the main storage and antistress mechanism. Enzyme assays confirmed that the recombinant proteins produced mannitol, with the specific activity of SjaM1Pase1 being 1.8–4831 times that of other algal enzymes. Combined with the transcriptional analysis, SjaM1Pase1 was shown to be the dominant gene of mannitol metabolism. Mannitol metabolism genes in multicellular filamentous (gametophyte) and large parenchyma thallus (sporophyte) generations had different expression levels and responded differently under environmental stresses (hyposaline and hyperthermia) in gametophytes and sporophytes. The considerable variation in enzyme characteristics and expression of mannitol synthesis genes suggest their important ecophysiological significance in the evolution of complex systems (filamentous and thallus) and environmental adaptation of Laminariales.

Characterization of redox sensitive algal mannitol-1-phosphatases of the haloacid dehalogenase superfamily of proteins

“…As indicated in Table 2, similar biochemical properties were observed when comparing kinetic constants of SjaM1Pase1 and SjaM1Pase2 on mannitol-1P. In S. japonica, it was suggested, based on gene expression analysis, that SjaM1Pase1 was the main M1Pase responsible for production of mannitol, whereas SjaM1Pase2 may support mannitol synthesis under changes in environmental conditions (Chi et al, 2018b). Interestingly, in Ectocarpus sp., the gene coding for EsM1Pase2 was not found to be differentially expressed under short-term abiotic stress conditions, while EsM1Pase1 was downregulated under oxidative and hyposaline conditions (Dittami et al, 2009).…”

“…In contrast, in the brown alga S. japonica, significant phosphatase activity for both SjaM1Pases was also detected in presence of others substrates, such as glucose-1P, glucose-6P, and fructose-6P. Furthermore, the highest activity of SjaM1Pase2 was measured with glucose-1P (Table 1) (Chi et al, 2018b), as also observed for the M1Pase of the red alga Dixoniella grisea (Eggert et al, 2006). However, this latter result should be taken with caution because activities were measured on algal crude extracts, and not with (partially) purified proteins.…”

“…that is active as a tetramer (Groisillier et al, 2014). The two HAD type M1Pases from Saccharina japonica (SjaM1Pases) have also been purified to homogeneity (Chi et al, 2018b), but no information related to quaternary structure were given.…”

“…Presence of the reductant in the assay mixture permitted to maintain enzymatic activity of the EsM1Pase2 tetramer for at least five days after purification (data not shown). Interestingly, none of the purified native and recombinant M1Pases biochemically characterized so far have been shown to be redox sensitive (Ikawa et al, 1972;Liberator et al, 1998;Iwamoto et al, 2001;Sand et al, 2015;Chi et al, 2018b).…”

“…In this alga, one M1PDH (Rousvoal et al, 2011;Bonin et al, 2015) and one M1Pase (EsM1Pase2, Groisillier et al, 2014) have been previously biochemically characterized by heterologous expression in Escherichia coli. Very recently, Chi et al (2018b) have identified and characterized homolog genes in S. japonica. Other M1Pases have been characterized from diverse organisms.…”