The sucrose non-fermenting 1-related protein kinase SAPK2 enhances rice yield under drought conditions

Background : Drought stress is an important factor limiting crop productivity worldwide. Rice is critical for food security because it is consumed by more than half of the global population. Thus, enhancing the drought tolerance of rice is crucial for ensuring the production of this important crop can satisfy the demands of future generations. Results : Compared with wild-type plants, the sapk2 rice mutant lines were shorter and produced fewer grains per panicle and smaller grains. Subsequent analyses suggested that SAPK2 considerably influences the nitrate, phosphorus, and potassium contents of rice grains. The examination of rice seedling growth and development under nutrient-deprived conditions (−K, −N, and −P) proved that SAPK2 can significantly affect rice seedling growth and root development in hydroponic cultures lacking N and K + . Moreover, the NO 3− influx rate and nitrate concentration of the analyzed plant materials indicated that SAPK2 promotes nitrate uptake and assimilation and influences the number of tillers and the number of grains per panicle by regulating nitrate-related transporters. Conclusion : These results suggest that SAPK2 is a key target gene for rice breeders aiming to increase yield.

A recent study confirmed that the overexpression of SAPK9 may significantly enhance drought tolerance, while also increasing the grain yield under drought conditions (Dey et al. 2016). Previously, SAPK2, a subclass II member, was confirmed as a positive regulator of drought resistance in rice (Lou et al. 2017). These reports indicate that SAPK2 may be useful for improving crop yields under drought conditions. However, the effect of SAPK2 on the productivity of drought-stressed plants remains unclear.
In this study, we characterized a rice sapk2 mutant, which produced fewer grains per panicle and smaller grains than the wild-type (WT) control plants. To confirm that the 5 SAPK2 function influences rice yield, we examined knock-out mutant lines (sapk2; sapk2-1 and sapk2-7), which we previously developed with the CRISPR/Cas9 system, and SAPK2overexpressing lines (OE; OES2-1 and OES2-2). Our analyses revealed that SAPK2 positively regulates rice grain number and size.

SAPK2 considerably affects plant height and grain number
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 reproductive stage drought stress (RS) 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 the WT controls under drought conditions (Fig. 1f). A subsequent investigation of the regulatory effect of SAPK2 on the number of effective tillers in rice revealed that the sapk2 mutant lines had considerably fewer effective tillers than the WT plants, but there were no significant differences in the two OE lines (Fig. 1e). This result further confirmed that the sapk2 mutant produces fewer grains than WT plants under drought conditions. Overall, our observations indicated that a lack of SAPK2 expression significantly decreases rice plant height and the number of grains per plant under drought conditions. Additionally, overexpressing SAPK2 does not appear to enhance rice plant growth or grain production.
M u t a t i o n s t o S A P K 2 d e c r e a s e g r a i n y i e l d The number of grains per panicle is one of the three key factors determining rice grain 6 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, and the 1,000-grain weight in response to RS. The panicles and grains of the sapk2 mutant lines were smaller than those of the WT controls (Fig. 2a).
Additionally, the average number of grains per panicle of the sapk2 mutant lines was 75% and 60.8% of that of the WT (Fig. 1e) and OE (Fig. 2b) plants. The seed setting rate of the OE lines did not differ from that of the WT plants, whereas the seed setting rate of the sapk2 mutant lines decreased significantly (76.9% of that of the WT plants) (Fig. 2c).
Compared with the WT grains, the grains of the 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 the WT grains, whereas the grain width of the OE lines was not significantly different (Fig. 2e).
Moreover, the 1,000-grain weight was much lower for the sapk2 mutant lines than for the WT and OE plants (Fig. 2f) (31.6% lower than that of WT). These results implied that SAPK2 influences panicle and grain sizes in rice. M u t a t i o n s t o S A P K 2 d e c r e a s e n i t r a t e , p h o s p h o r u s , a n d p o t a s s i u m c o n t e n t s i n r i c e g r a i n s u n d e r r e p r o d u c t i v e -s t a g e d r o u g h t 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, we measured the nitrate, phosphorus, and potassium contents of seeds. Under RS conditions, the seeds of the sapk2 mutant lines had lower nitrate, phosphorus, and potassium concentrations than the WT controls, with the biggest difference observed for the nitrate concentration ( Fig. 3a-c). However, the nitrate, phosphorus, and potassium concentrations 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 involving N, P, and K + . inhibited, resulting in roots that were shorter than those of WT seedlings (Fig. 4b). In contrast, the root phenotypes of the OE and WT plants were similar ( Fig. 4b; OE phenotype is not presented). The root and shoot dry weights of the sapk2 mutant lines were 8 significantly lower than the corresponding WT weights, but there were no significant differences in the OE lines (Fig. 4c, d). Similarly, compared with the WT plants, the sapk2 mutant lines had fewer roots, whereas there were no significant differences in the number of roots 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 the WT plants ( Fig. 5a-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 (40 days after germination; Additional file 1, Fig. S1a-f).
These findings suggested that SAPK2 can significantly influence rice seedling growth and root development in hydroponic cultures under N-and K + -deprived conditions.

S A P K 2 i n f l u e n c e s t h e N O 3 − i n f l u x r a t e a n d n i t r a t e c o n c e n t r a t i o n u n d e r d r o u g h t s t r e s s c o n d i t i o n s
To explore the potential mechanism underlying the effects of SAPK2 on rice seedling growth and root development under N-deprived conditions, we investigated the NO 3 − 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. 6a, c). However, in response to drought stress, the NO 3 − influx rate and nitrate concentration significantly decreased (relative to the values under control conditions) in the WT, OE, and sapk2 plants ( Fig. 6a-d). Additionally, the rate of NO 3 − influx into the roots was lower for the sapk2 mutant lines than for the WT plants ( Fig. 6b), suggesting that silencing SAPK2 expression weakens the nitrate uptake by the roots. Regarding the sapk2 mutant lines, we also detected a lower rate of NO 3 − influx into the leaf sheath and leaf blade, implying that SAPK2 promotes the translocation of NO 3 − from the roots to the leaf sheath (Fig. 6b). Moreover, the root, leaf sheath, and leaf blade nitrate concentrations were consistent with the NO 3 − influx rates in the different lines (Fig. 6d). 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.  . 7a-f).
However, the opposite expression patterns were detected for the OE lines ( Fig. 7a-f).
These results implied that SAPK2 promotes nitrate uptake and assimilation by regulating nitrate-related transporters. To identify additional rice grain yield-related genes affected by drought stress, we functionally characterized SAPK2 by examining sapk2 mutant lines exposed to drought We also investigated the SAPK2 roles associated with panicle and grain development in response to RS conditions. Specifically, the panicle size, number of grains per panicle, grain size, seed setting rate, and 1,000-grain weight were significantly lower for the sapk2 mutant lines than for the WT plants (Fig. 2a-f). These results indicated that SAPK2 increases rice yields under RS conditions by influencing panicle and grain sizes. concentrations. The data indicated the sapk2 mutant seeds had lower nitrate, phosphorus, and potassium concentrations than the WT seeds, with the difference especially pronounced for the nitrate concentration (Fig. 3a-c). Moreover, SAPK2 expression in the roots was down-regulated in response to N, P, and K + deprivation ( Fig. 3d-f). These findings indicate that SAPK2 influences panicle and grain sizes via its effects on metabolic processes involving N, P, and K + .
The roots, which are responsible for the uptake of water and nutrients from the soil, are Additionally, we previously determined that SAPK2 regulates the expression of genes related to Na + and K + homeostasis, including OsSOS1, OsNHX1, OsHKT1;1, and OsHKT1;5 (lou et al. 2018). In the current study, the sapk2 mutant seedlings deprived of K + produced weaker culms and had lower root and shoot dry weights than the WT controls ( Fig. 5a-f).
Therefore, the data presented herein further confirm that SAPK2 is important for increasing the rice grain yield through its effects on the metabolic processes related to N and K + . and also into the leaf sheath and leaf blade (Fig. 6b). Besides, the detected nitrate concentration was consistent with the rate of NO 3 − influx in different lines (Fig. 6d)  . Our study revealed that the sapk2 mutant lines produced significantly fewer tillers than the WT plants (Fig. 1d).
Moreover, the sapk2 mutant seeds had a lower nitrate concentration than the WT seeds under RS conditions (Fig. 3a).
On the basis of our results, we conclude that under drought conditions, SAPK2 promotes nitrate uptake and assimilation and influences the number of tillers and the number of grains per panicle by regulating nitrate-related transporters.

Conclusions
We analyzed transgenic rice lines that differed regarding their SAPK2 expression levels and determined that SAPK2 positively affects the number of tillers, the number of grains per panicle, the seed setting rate, and grain size under drought conditions by regulating the expression of nitrate transporter genes to enhance the N use efficiency, ultimately leading to an increase in the rice grain yield.

Generation of transgenic rice lines
We employed knock-out mutant lines (sapk2, sapk2-1 and sapk2-7) which we built previously by the CRISPR/Cas9 system and over-expression lines (OE, OES2-1 and OES2-2)  For the qRT-PCR analysis, we used the same method as described (Jiang et al., 2016).
Total RNA was isolated from whole seedlings using the TriZol reagent (Invitrogen). The cDNAs were obtained by using Superscript II in accordance with manufacturer's instructions (Invitrogen). The qRT-PCR analysis was performed using SYBR Premix Ex Taq kit (Takara).
At least three independent biological experiments were conducted (three independent samples were conducted for each experiment and three technological replications in every independent experiment). One representative result was displayed here. Gene-specific primers used in qRT-PCR analysis were listed in Additional file 2: Table S1.

Statistical analysis
The experiments were arranged in a completely randomized design with at least three replicates for each treatment. Excel 2010 was used for making charts. Two-tailed Student's t tests were performed using the SPSS 10 software (IBM, Inc.). "* and **" indicate significance at P < 0.05 and P < 0.01, respectively. The data represent mean ± as means ± SD (n = 20) from three replicates. A student's t-test was used to generate P values; "**" indicate significance at P < 0.01.
Additional file 2 Table S1. Primers used in this study.     from three replicates. A student's t-test was used to generate P values; "**" indicate significance at P < 0.01.

Supplementary Files
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