Effects of different treatments of NaCl on L. ruthenicum
We observed the growth of L. ruthenicum seedlings under different concentrations of NaCl (0-400 mM). It was found that the growth of leaves and roots of L. ruthenicum under 50 and 100 mM NaCl treatments were significantly better than control and other treatments, and the effect of 50 mM NaCl was the most pronounced one among these treatments (Supplementary Fig. S1).
To further evaluate the effect of NaCl on growth of L. ruthenicum, the fresh weight, dry weight, tissue water content and relative growth rate was determined under NaCl treatments (0-400 mM) (Fig. 1). Results showed that the fresh weight, dry weight and relative growth rate of L. ruthenicum were significantly increased under 50 and 100 NaCl treatments, and 50 mM had the most obvious effect, these indexes above significantly increased by 126.4%, 60.4% and 47.2%, respectively. It is further suggesting that addition of appropriate NaCl could obviously promote plant growth.
The accumulation of Na+ and K+ in plants was also analyzed under 0-400 mM NaCl treatments (Fig. 2). Compared with the control, the concentrations of Na+ in roots, stems and leaves treatment were significantly increased (62.5%, 209.6%, 173.7% and 156.1%, 332.1%, 283.7% in roots, stems and leaves under 50 and 100 mM NaCl treatments, respectively) under 50-400 mM NaCl treatment. K+ in roots and leaves increased significantly (increased by 20.6%, 6.7% and 26.1%, 4.2% under 50 and 100 mM NaCl treatments, respectively), and remained stable in stems; however, with the increase of NaCl concentration, K+ concentration in various tissues decreased significantly.
To explore the pathway of coordinated regulation of Na+, K+ channels or transporters in L. ruthenicum, the expression patterns of LrAKT1, LrSKOR, LrSOS1, LrHKT, LrAPV1 and LrNHX in L. ruthenicum were analyzed. After NaCl treatment for 6 and 24 h, LrSOS1 and LrAKT1 expression increased gradually, LrSKOR decreased first and then increased, while LrHKT was down-regulated in roots; moreover, the expression of LrAPV1 and LrNHX were significantly up-regulated in leaves (Fig. 3).
Effects of drought treatment on growth and development of L. ruthenicum
As Supplementary Fig.S2 shows that compared with the control, the seedlings of L. ruthenicum grew normal under -0.25 MPa, and -0.5, -1.0, -1.5 MPa delayed plant growth. Furthermore, under -1.0 MPa treatment exhibiting poor plant performance and under -1.5 MPa stress treatment, the plant appeared obvious wilting phenomenon, thus -0.5 MPa was chosen as the followed drought treatment.
Moderate concentrations of NaCl alleviates the deleterious impact of water deficit
In order to explore whether 50 mM NaCl could alleviate the drought stress of L. ruthenicum seedlings, salt (S: 50 mM NaCl), drought (D: -0.5 MPa) and drought plus salt (D+S: 50 mM NaCl + - 0.5 MPa) treatments were carried out in this study (Fig. 4). Compared with the control, as same as the result of NaCl treatment above, plants still grew very well exposed 50 mM NaCl, but drought treatment exhibited a certain inhibitory effect on seedling growth. However, what really caught our attention was that drought plus salt treatment was better than drought treatment, therefore, 50 mM NaCl could alleviate the drought stress of plants.
The fresh and dry weight of L. ruthenicum plants increased significantly under 50 mM NaCl compared with control, and the fresh weight decreased significantly under -0.5 MPa and 50 mM NaCl plus -0.5MPa treatment, but the dry weight did not change significantly; However, the fresh weight under 50 mM NaCl plus -0.5MPa treatment was significantly higher than that of -0.5 MPa treatment alone (Fig. 5a, b).
Compared with control, the water content of tissues under salt treatment increased significantly, while under drought treatment which decreased significantly, and the water content of the plants under drought plus salt treatment was significantly higher than that under drought treatment alone (Fig. 5c). 50 mM NaCl significantly increased the relative growth rate of plants, drought and drought plus salt treatment obviously delayed the relative growth of plants compared to control; but compared with drought treatment, 50 mM NaCl plus drought treatment evidently increased the relative growth rate of plants by 1.8% (Fig. 5d), therefore NaCl could alleviate drought stress.
Compared with control, salt and drought plus salt treatments significantly increased the Na+ concentration in roots, stems and leaves of L. ruthenicum, but had no significant change under drought treatment; salt and drought plus salt treatment also increased K+ content in leaves and stems. Compared with drought, drought plus salt treatment increased Na+ (roots, stems and leaves: 54.8%, 385.7% and 155.4%, respectively) and K+ concentration (roots, stems and leaves: 95.3%, 3.7% and 59.4%, respectively) (Fig. 6a, b). Further analysis showed that the distribution ratio of Na+ and K+ in the root decreased and increased in the shoot when drought addited salt (Fig. 6c, d).
The proline content in roots and stems increased significantly under salt treatment, which were 5.1 and 5.7 times of the control, respectively; it was also significantly increased in stems under drought treatment, but there was no obviously change under drought plus salt treatment (Fig. 7a).
Compared with control, drought or drought plus salt treatment significantly increased the soluble sugar content in leaves, but evidently decreased the soluble sugar content in roots and stems; the soluble sugar content in roots also decreased significantly under salt treatment (Fig. 7b) .
Compared with control, the osmotic potential of plants decreased significantly under salt, drought or drought plus salt treatments, nevertheless, which under drought plus salt treatment was significantly lower than that of drought treatment (Fig. 8).
Salt treatment significantly increased the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr), and drought significantly decreased the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr), but significantly increased the water use efficiency (WUE). Compared with drought stress, Pn, Gs and Tr increased evidently under drought plus salt treatment, but WUE decreased significantly (Fig. 9).