Mutations to SAPK2 Decrease Plant Height and Grain Yield
Under drought conditions, the two sapk2 lines were shorter than the WT control plants (Fig. 1a–c). To further investigate the SAPK2 roles influencing rice yield, we analyzed OE lines (OES2–1 and OES2–2) and sapk2 knock-out mutant lines (sapk2–1 and sapk2–7). An analysis of plants exposed to RDS indicated that the two sapk2 lines had substantially more tillers than the WT plants (Fig. 1a, d), whereas there were no significant differences in the two OE lines (Fig. 1b–d). However, although they had more tillers, the sapk2 mutant plants produced fewer grains per plant than WT under RDS (Fig. 1f). A subsequent investigation of the regulatory effect of SAPK2 on the number of effective tillers revealed that the sapk2 mutant lines had considerably fewer effective tillers than WT, but there were no significant differences in the OE lines (Fig. 1e). This result further confirmed that the sapk2 mutant produces fewer grains than WT under RDS.
The number of grains per panicle is one of the three key factors determining rice grain yield (Xing and Zhang 2010). Thus, we investigated the SAPK2 roles related to panicle and grain development by analyzing the number of grains per panicle, the seed setting rate, the grain length and width, the 1000-grain weight and the grain yield per plant in response to RDS. Under RDS, the panicles and grains of the sapk2 mutant lines were smaller than those of WT (Fig. 2a). Additionally, the grain number per panicle of the sapk2 mutant lines was 75% and 60.8% of that of WT and OE lines (Fig. 2b). The setting rate of OE lines did not differ from that of WT, whereas the setting rate of the sapk2 mutant lines decreased significantly (76.9% of that of WT) (Fig. 2c). Compared with WT, the grains of OE lines were significantly longer, whereas there was no significant difference in the grain length of the sapk2 mutant lines (Fig. 2d). In contrast, the grains of the sapk2 mutant lines were significantly thinner than WT, whereas the grain width of OE lines was not significantly different (Fig. 2e). Moreover, the 1000-grain weight and grain yield per plant of the sapk2 mutant lines were much lower than WT and OE plants (Fig. 2f, g). These results implied that SAPK2 influences panicle and grain sizes in rice under RDS.
Overall, our observations indicated that knocking out of SAPK2 significantly decreases rice plant height and grain yield per plant under drought condition. Additionally, overexpressing SAPK2 does not appear to enhance rice plant growth or grain production.
Mutations to SAPK2 Decrease Nitrate, Phosphorus, and Potassium Contents in Rice Grains under RDS
Umezawa et al. (2004) reported that the expression of SnRK2.8, which is a homolog of rice SAPK2, is down-regulated by potassium deprivation. This down-regulation is associated with a substantial decrease in the growth of A. thaliana under nutrient-deprived conditions. As mentioned earlier, SAPK2 influences rice panicle and grain sizes. To clarify the mechanisms by which SAPK2 influences panicle and grain sizes in rice under RDS, we measured total nitrogen, nitrate, phosphorus, and potassium contents of seeds. Under RDS conditions, the seeds of the sapk2 mutant lines had lower total nitrogen, nitrate, phosphorus, and potassium content than WT, with the biggest difference observed for the total nitrogen (Fig. 3a–c, Additional file 2: Figure S1). However, the total nitrogen, nitrate, phosphorus, and potassium content were relatively consistent between the OE and WT plants (Fig. 3a–c).
Next, we investigated the SAPK2 expression profiles under control conditions (i.e., sufficient nutrients) and nutrient-deficient conditions [i.e., lacking K (−K), N (−N), and P (−P)]. The qRT-PCR analyses revealed that the SAPK2 transcript levels in the roots decreased in the absence of N, P, and K (Fig. 3d–f).
These findings confirmed that in rice, the seed nitrate, phosphorus, and potassium contents are largely affected by SAPK2. Therefore, we hypothesized that SAPK2 influences panicle and grain sizes by modulating metabolic processes of N, P, and K.
SAPK2 Affects Seedling Growth and Root Development in Response to N and K Deprivation
To further validate our hypothesis, we investigated the effects of knocking out and overexpressing SAPK2 on rice seedling growth and development in hydroponic cultures under different nutrient-deprived (−K, −N, and − P) conditions.
The sapk2 mutant seedlings under N-deprived conditions produced weaker culms than WT, whereas the OE lines were phenotypically similar to WT (Fig. 4a, f; In Fig. 4f, the OE phenotype is not presented). A previous study proved that the root morphology influences plant interactions with soil nitrates, making it important for N absorption (Hachiya and Sakakibara 2016). Accordingly, we examined the root development of the SAPK2 transgenic lines. In the sapk2 mutant lines, root growth was inhibited, resulting in roots than WT seedlings (Fig. 4b, f). In contrast, the root phenotypes of the OE and WT plants were similar (Fig. 4b, f; In Fig. 4f, OE phenotype is not presented). The root and shoot dry weights of the sapk2 mutant lines were significantly lower than WT, but there were no significant differences in the OE lines (Fig. 4c, d). Similarly, compared with WT, the sapk2 mutant lines had fewer roots, whereas there were no significant differences in the OE lines (Fig. 4e).
The effects of the K-deprived conditions were similar to those of the N-deprived conditions. For example, the sapk2 mutant seedlings produced weaker culms and had lower root and shoot dry weights than WT (Additional file 3: Figure S2a–f). In contrast, the exposure to P-deprived conditions did not result in any significant differences in the seedling growth and root development of the WT, OE and sapk2 seedlings (Additional file 4: Figure S3a–f).
These findings suggested that SAPK2 can significantly influence rice seedling growth and root development in hydroponic cultures under N- and K-deprived conditions.
SAPK2 Influences the NO3− Influx Rate and Nitrate Concentration under Drought RDS
To explore the potential mechanism underlying the effects of SAPK2 on rice seedling growth and root development under N-deprived condition, we investigated the NO3− influx rate and nitrate concentration of the WT, OE, and sapk2 plants under control and drought conditions. Under control conditions, there were no significant differences among the WT, OE, and sapk2 plants (Fig. 5a, c). However, in response to drought stress, the NO3− influx rate and nitrate concentration significantly decreased in the WT, OE, and sapk2 plants (Fig. 5a–d). Additionally, the rate of NO3− influx into the roots was lower for the sapk2 mutant lines than for WT (Fig. 5b), suggesting that knocking out of SAPK2 weakens the nitrate uptake by the roots. Regarding the sapk2 mutant lines, we also detected a lower rate of NO3− influx into the leaf sheath and leaf blade, implying that SAPK2 promotes the translocation of NO3− from the roots to the leaf sheath (Fig. 5b). Moreover, the root, leaf sheath, and leaf blade nitrate concentrations were consistent with the NO3− influx rates in the different lines (Fig. 5d). These results demonstrated that SAPK2 enhances nitrate influx and increases the nitrate concentration by promoting the translocation of nitrate from the roots to the leaf sheath.
Nitrogen use efficiency is an important trait for the development of sustainable agricultural production (Xu et al. 2012). Plants have diverse transporters facilitating N uptake and internal distribution (Rentsch et al. 2007). In higher plants, members of the NPF family (previously called the PTR/NRT1 family) can take up and translocate nitrate or small peptides. Of the rice NPF family members, only a few have been studied. For example, OsNPF7.2, which encodes a positive regulator of nitrate influx and concentration, helps control the allocation of nitrate between the roots and shoots (Wang et al. 2018). In rice, the peptide transporter OsNPF7.3 (OsPTR6) mediates the transport of organic N from the leaves to the grains and increases the grain yield (Fang et al. 2017). Additionally, OsNPF6.5 (OsNRT1.1B) is predominantly expressed in the root hairs, epidermis, and stellar cells adjacent to the xylem in roots. The osnrt1.1b mutant is reportedly defective in both nitrate uptake and root-to-shoot nitrate transport, suggesting that OsNRT1.1B is involved in nitrate uptake and transport (Hu et al. 2015). Down-regulating OsNRT2.3a expression impairs the loading of nitrate into the xylem and inhibits plant growth under low-nitrate conditions, implying OsNRT2.3a contributes to the long-distance transport of nitrate from the roots to the shoots (Tang et al. 2012). The silencing of OsNPF2.4 diminishes the low-affinity nitrate acquisition by roots, disrupts the K-coupled root-to-shoot nitrate transport, and inhibits the redistribution of nitrate from old leaves to N-starved roots or young leaves (Xia et al. 2015).
To further validate our hypothesis that SAPK2 influences panicle and grain sizes by modulating N metabolic processes, we determined the expression levels of genes crucial for the absorption, transport, and assimilation of nitrate among the WT, OE, and sapk2 plants cultured under control and drought stress condition (PEG). The OsNPF7.2, OsNPF7.3, OsNPF5.6, OsNPF2.2, OsNRT2.3a, and OsNPF2.4 expression levels were significantly lower in the sapk2 mutant lines than in WT under drought stress condition (Fig. 6a–f). However, the opposite expression patterns were detected for the OE lines (Fig. 6a–f). These results implied that SAPK2 promotes nitrate uptake and assimilation by regulating nitrate-related transporters.