Metabolomics Analyses Reveal Metabolites Affected by Plant Growth-Promoting Endophytic Bacteria in Roots of the Halophyte Mesembryanthemum crystallinum

Mesembryanthemum crystallinum L. (common ice plant) is an edible halophyte. However, if ice plants are used to phytoremediate salinity soil, there are problems of slow initial growth, and a long period before active NaCl uptake occurs under higher salinity conditions. Application of endophytic bacteria may improve the problem, but there remain gaps in our understanding of how endophytic bacteria affect the growth and the biochemical and physiological characteristics of ice plants. The aims of this study were to identify growth-promoting endophytic bacteria from the roots of ice plants and to document the metabolomic response of ice plants after application of selected endophytic bacteria. Two plant growth-promoting endophytic bacteria were selected on the basis of their ability to promote ice plant growth. The two strains putatively identified as Microbacterium spp. and Streptomyces spp. significantly promoted ice plant growth, at 2-times and 2.5-times, respectively, compared with the control and also affected the metabolome of ice plants. The strain of Microbacterium spp. resulted in increased contents of metabolites related to the tricarboxylic acid cycle and photosynthesis. The effects of salt stress were alleviated in ice plants inoculated with the endobacterial strains, compared with uninoculated plants. A deeper understanding of the complex interplay among plant metabolites will be useful for developing microbe-assisted soil phytoremediation strategies, using Mesembryanthemum species.

Proteomics of Homeobox7 Enhanced Salt Tolerance in Mesembryanthemum crystallinum

Mesembryanthemum crystallinum (common ice plant) is a halophyte species that has adapted to extreme conditions. In this study, we cloned a McHB7 transcription factor gene from the ice plant. The expression of McHB7 was significantly induced by 500 mM NaCl and it reached the peak under salt treatment for 7 days. The McHB7 protein was targeted to the nucleus. McHB7-overexpressing in ice plant leaves through Agrobacterium-mediated transformation led to 25 times more McHB7 transcripts than the non-transformed wild type (WT). After 500 mM NaCl treatment for 7 days, the activities of superoxide dismutase (SOD) and peroxidase (POD) and water content of the transgenic plants were higher than the WT, while malondialdehyde (MDA) was decreased in the transgenic plants. A total of 1082 and 1072 proteins were profiled by proteomics under control and salt treatment, respectively, with 22 and 11 proteins uniquely identified under control and salt stress, respectively. Among the 11 proteins, 7 were increased and 4 were decreased after salt treatment. Most of the proteins whose expression increased in the McHB7 overexpression (OE) ice plants under high salinity were involved in transport regulation, catalytic activities, biosynthesis of secondary metabolites, and response to stimulus. The results demonstrate that the McHB7 transcription factor plays a positive role in improving plant salt tolerance.

Impacts of Reduced Nitrate Supply on Nitrogen Metabolism, Photosynthetic Light-Use Efficiency, and Nutritional Values of Edible Mesembryanthemum crystallinum

Mesembryanthemum crystallinum (common ice plant), as a nutritious ready-to-eat salad in Singapore, has become popular in recent years. However, basic data about the impacts of NO3– supply on its NO3– accumulation and nutritional quality are lacking. In this study, all plants were first grown indoor hydroponically in 10% artificial seawater (ASW) with modified full-strength Netherlands Standard Composition nutrient solution for 11 days, before transferring them to different reduced NO3– solutions. All plants grew well and healthy after 7 days of treatment. However, plants grown with 3/4 N and 1/2 N were bigger with higher shoot and root fresh weight (FW), greater leaf number, and total leaf area (TLA) than those grown with full nitrogen (N), 1/4 N, and 0 N. Mesembryanthemum crystallinum grown with full N, 3/4 N, and 1/4 N had similar specific leaf area (SLA), while 0 N plants had significantly lower SLA. All plants had similar leaf succulence (LS). However, leaf water content (LWC) was lower, while leaf dry matter accumulation (LDMC) was higher in 0 N plants after 7 days of treatment. Compared with plants grown with full N, shoot NO3– concentrations in 3/4 N, 1/2 N, and 1/4 N plants were constant or slightly increased during the treatments. For 0 N plants, shoot NO3– concentration decreased significantly during the treatment compared with other plants. Shoot NO3– accumulation was associated with nitrate reductase activity (NRA). For instance, after 7 days of treatment, shoot NO3– concentration and NRA on a FW basis in 0 N plants were, respectively, 45 and 31% of full N plants. After transferring full N to 0 N for 7 days, all M. crystallinum had higher chlorophyll (Chl) content coupled with higher electron transport rate (ETR) and higher effective quantum yield of PSII, while full N plants had higher non-photochemical quenching (NPQ). The 0N plants had much higher concentrations of proline, total soluble sugar (TSS), and total ascorbic acid (ASC) than other plants. In conclusion, totally withdrawing NO3– from the growth media prior to harvest could be one of the strategies to reduce shoot NO3– concentration. Reduced NO3– supply further enhanced nutritional values as concentrations of proline, TSS, and ASC were enhanced markedly in M. crystallinum plants after transferring them from full N to 0 N.

Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes’ protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.