Fifteen physiological traits related to osmoregulation and reactive oxygen species metabolism in two life form aquatic plants under a natural water salinity gradient on the Tibetan Plateau and Northwest China

Aquatic plants, as the primary producers, determine the community structure and ecological function of freshwater ecosystems. However, salinization threatens inland freshwater wetlands and thus the survival of aquatic plants. Exploring the plant physiological responses to increasing water salinity could enhance our understandings of plant adaptive strategies under future climate change regimes in wetlands. We measured 15 physiological traits of 49 aquatic plant species along a large environmental gradient in alpine and arid regions of western China, to explore the physiological adaptions and compare the similarities and differences in adaptive strategies between the two life forms to natural water salinity. We found that both water salinity and low temperature were key factors affecting aquatic plants in alpine and arid regions. Aquatic plants adapt to saline habitats by accumulating proline and sulfur (S) concentrations, and to cold habitats by increasing ascorbate peroxidase activity. Plant trait network analysis showed that the hub trait in emergent plants was S, but in submerged plants was proline, suggesting that emergent plants balanced osmoregulation and reactive oxygen metabolism via S-containing compounds, while submerged plants prioritizing the regulation of osmotic balance via proline.

Methods
We first bottled 500 ml water from an intermediate depth below the water surface for laboratory analysis. Three sediment columns were dug randomly from the upper 0-20 cm under the water-sediment interface and then mixed as the sediment sample for each site. All sediment samples were air-dried, ground, and sieved through a 0.15 mm sieve for further analysis. In total, we investigated 178 sites, including 90 sites on the Tibetan Plateau and 88 sites in the Northwest Region. We measured the salinity of undisturbed water in situ using a hand-held multi-parameter meter (PROPLUS, YSI, USA). The total Cl-, SO42-, Na+, K+, Mg2+, and Ca2+ contents in water were determined by an ion chromatograph (ICS-1000, DIONEX, USA). The total Ca, K, Mg, and Na concentrations in sediments were measured by inductively coupled plasma mass spectrometer (ICPMS-2030, Shimadzu, Japan) using the samples from the field, and the total sediment S concentration was measured by the elemental analyzer (UNICUBE, Elementar, Germany).

Two life forms of aquatic plants were sampled. Different sampling methods were adopted according to different plant life forms. For emergent plants, 30-50 fully expanded and intact leaves were randomly selected for each species. For submerged plants, we collected 30-50 segments of the plants (20 cm from the tip) and then removed all leaves from these segments. All plant samples of each species were divided into two parts: one for elemental measurements after oven dried at 70°C for 72 h and the other part for measuring the plant physiological traits using fresh samples that have been frozen by liquid nitrogen in the field. In total, we collected 527 plant samples (251 on the Tibetan Plateau and 276 in the Northwest Region) belonging to 49 species across the whole study area. The frozen fresh leaves were used to determine the contents of proline, soluble protein, soluble sugar, and glutathione (GSH), and the activity of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in plants. Detailed methods for measuring the traits were described in Supplementary materials. Oven-dried plant samples were used to measure the element concentrations in plants. The samples were ground to powder and divided into two parts: one for measuring total plant S concentrations by Vario MACRO cubic elemental analyzer (UNICUBE Elementar, Germany), and the other for total Calcium (Ca), Potassium (K), Magnesium (Mg) and Sodium (Na) concentrations by inductively coupled plasma mass spectrometer (ICPMS-2030, Shimadzu Japan).