Abiotic stress poses a serious threat to agricultural production and leads to global environmental degradation and loss of crop yields, reducing the yield of most major crops by more than 50%(Dar et al,2021). In the past few decades, people have been paying attention to ozone depletion due to anthropogenic pollutants such as halogenated hydrocarbons and other ozone-depleting chemicals reaching the stratosphere(Lu et al,2018). One of the main reasons for the increase in UV-B radiation is the thinning of the ozone layer (Middleton et al,1994). Some crops are more severely affected by UV-B radiation, such as rice and maize (Thomas et al,2020) . miRNAs are considered to be important regulators of gene expression and are involved in the regulation of plant growth and development in many aspects, especially in response to stress in plants (Ghorecha et al,2017) . Studies have shown that miR408 is up-regulated in wild sweet potato and alfalfa under drought stress(Sunkarr et al,2004). The expression level of miR408 in Arabidopsis thaliana was up-regulated in response to low temperature stress(PANDA et al,2015) and copper deficiency(Zhou et al,2010). Osa-miR408 is also induced by drought(Macovei et al,2012)and salt damage(Jiawei et al,2018). We evaluated the photosynthesis and ultrastructure of the UV-B-treated miR408-OE rice and its yield traits to determine the mechanism of UV-B radiation impact on the crop. Our study showed that UV-B stress has significant inhibitory effects on rice plant growth, chlorophyll content, photosynthetic parameters and chloroplast development.
Ultrastructure of chloroplasts are damaged under UV-B stress
Li found that the chlorophyll content of wheat decreased significantly under UV-B stress(Zhijie et al,2019). Zu showed that the chlorophyll content of 12 varieties in 20 soybean varieties decreased by UV-B stress(Ranjbarfordoei et al,2011). UV-B stress inhibited the chlorophyll content of M1 and M2, which was consistent with previous studies(Fig. 2A,B). The decrease in Chl content caused by UV-B radiation may be due to inhibition of the biosynthesis of Chl or affecting the enzyme involved in the Chl biosynthetic pathway(Y.S.Wagh ,2019). In this study, the chloroplast structure of rice mesophyll cells changed under UV-B stress. The morphology and structure of rice chloroplasts were normal under normal illumination. Under the treatment of UV-B stress, the chloroplast of the control showed slight swelling, and the chloroplast oswelling of miR408-OE is intensified and changes from fusiform to circular, the number of steroids increased significantly, and the matrix sheet appeared to be broken and dispersed, and elongated and thinned. When the cell chloroplast ultrastructure is destroyed by stress, it leads to a change in the structure of the thylakoid membrane, which ultimately inhibits photosynthesis(Li et al,2014).
Effects of UV-B stress on photosynthesis of rice
Crop yield ultimately depends on photosynthetic performance. Therefore, improving the photosynthetic performance of crops has always been a long-term goal of biotechnology and breeding. Studies have shown that photosynthetic rate and stomatal conductance are significantly decreased under UV-B stress(Vinicius et al,2015). For example, Chen Zhanghe et al. showed that after UV-B radiation, the net photosynthetic rate and stomatal conductance of duck feet and monkey earrings decreased(Wu et al,2001). The results of this study are consistent with this. And compared with the control, UV-B significantly inhibited the net photosynthetic rate and stomatal opening of the miR408-OE plant and the knockout target gene UCL8, resulting in UV-B limiting its photosynthetic rate, thereby reducing the photosynthetic capacity of the plant. Changes in carbon dioxide concentration can affect plant growth and development, while cell gap carbon dioxide concentration is directly related to plant photosynthesis(Lawson et al,2019). UV-B stress significantly inhibited the intercellular carbon dioxide concentration of miR408 overexpressing plants, hindered the substrate required for photosynthesis, leading to a decrease in photosynthetic products, while photosynthesis played a direct role in crop yield(Mathobo et al,2017). These results indicate that UV-B is negatively correlated with photosynthetic rate, and UV-B stress leads to a decrease in rice yield by reducing the photosynthetic properties of miR408 overexpressing plants.
Chlorophyll fluorescence kinetic parameters can reflect the absorption and dissipation of light energy by leaf photosystems during plant photosynthesis and coping with stress(Zivcak et al,2017). In this study, under UV-B stress, compared with BWT and BWT1, the VJ of miR408 overexpressing plants and knocking out UCL8 plants increased significantly, this indicates that UV-B stress increases the variable fluorescence intensity at the J point of BM and BM1, resulting in a large accumulation of QA, which in turn impedes the electron transfer of QA and QB at the PSII receptor end(Jiawei et al,2018). Abiotic stress can directly affect PSII function(Wang et al,2017). UV-B stress significantly inhibited Sm and N of BM, indicating that enhanced UV-B radiation reduced the number of QA reductions of BM, thereby destroying the balance of electron transport transport in PSII internal networks(Mishra et al,2014). The fluorescence parameter FV/FM represents the efficiency of photosystem II, and its reduction is considered to be a reliable indicator of adversity(Yu et al,2013). This study showed that under UV-B stress, the FV/FM of M1 decreased more than that of CK1, indicating that the tolerance of UV-B radiation was reduced. Fluorescence kinetic curve analysis showed that miR408 overexpressing plants were affected by UV-B stress in I-P phase, indicating that the transfer of plastoquinone in PSII was significantly affected, suggesting that PSII structure may be destroyed(Yu et al,2013). UV-B stress increased the DI0/RC, φD0, and DI0/CSO of BM1, thus increasing energy dissipation against plant UV-B damage. At the same time, it will also lead to the inactivation of the PSII reaction center, which is consistent with previous studies(Casati et al,2005) . In this experiment, real-time fluorescent quantitative PCR of photosynthetic related genes was carried out. It was found that the relative expression levels of PPDK, PSII-PsbR and chlorophyll a/b-binding were lower than BCK1 under enhanced UV-B radiation. showed that pyruvate phosphate double kinase (PPDK) down-regulated expression under UV-B stress and regulated plant photosynthetic efficiency in response to stress(Heng et al,2014) . Studies have shown that decreased expression of PsbR leads to decreased photosynthesis and decreased photosynthetic efficiency of PSII(Weihua et al,2014) . The decrease in light-harvesting chlorophyll a/b binding protein leads to a decrease in the absorption and transmission of light energy(Xiang et al,2005). Therefore, under UV-B stress, the down-regulation of photosynthetic-related genes in BM1 resulted in a decrease in PSII light-energy conversion efficiency and a decrease in photosynthetic rate. UV-B inhibited photosynthetic gene expression in BM1. Therefore, it is speculated that UV-B stress leads to deactivation of the PSII reaction center by lowering the electron transport rate of PSII, which leads to a decrease in photosynthesis of BM1 and a decrease in rice yield.
UV-B stress destroys the internal balance of rice antioxidant enzyme system
When photosynthetic performance is reduced, it leads to a decrease in dry matter accumulation. The results of this study showed that compared with natural light, the dry matter weight, tiller number and 100-kernel weight of miR408 overexpressing rice under UV-B stress were significantly decreased, which eventually led to the decrease of biomass. Many studies have shown that UV-B stress inhibits crop yields, such as corn grain yield , cotton yield , and rice yield(Yin et al,2012) .
In this experiment, the SOD, POD and CAT of BM were significantly lower than BWT under UV-B stress, while H2O2 and MDA were significantly higher than BCK1. A decrease in the content of antioxidant enzymes such as SOD, POD, CAT, etc., causes an increase in superoxide anion. A large number of superoxide anions combine with water to produce hydrogen peroxide, which causes an increase in membrane lipid peroxides, which leads to a decrease in the ability of plants to scavenge free radicals and damage plants. This is consistent with the findings of previous studies that UV-B radiation enhancement hinders plant growth and development, reduces plant photosynthesis, and affects plant antioxidant enzyme content(Duan et al,2010) . This indicates that miR408 is responsive to UV-B radiation and that miR408 overexpression is less resistant to UV-B stress.
In this study, we report a conserved miRNA, OsmiR408, which negatively regulates rice yield under UV-B stress. UV-B stress has significant effects on miR408 overexpressing plants in terms of morphological structure, photosynthetic characteristics and yield traits. In this study, it was found that UV-B radiation can increase the expression of miR408 overexpressing rice in the middle and late growth stages, which is consistent with the results of Jia et al(Jia et al,2009). Studies have shown that miR408 overexpression can positively regulate grain yield under normal illumination(Zhang et al,2017), which is consistent with the results of this study. However, this study found that under UV-B stress, miR408 overexpression is negatively regulated in rice yield. Therefore, it is speculated that enhanced UV-B radiation destroys the antioxidant system of miR408-transformed plants, causing significant reduction of antioxidant enzymes such as SOD and POD to catalyze the formation of excessive and toxic hydrogen peroxide, causing damage to plant leaves. Due to the damage of the leaves of BM1 plants, the light-harvesting ability is reduced, resulting in a decrease in electron transport rate and photosynthetic performance, resulting in a decrease in rice yield. The results of this study indicate that overexpression of miR408 reduces the tolerance of rice to UV-B stress, and miR408 may negatively regulate rice photosynthesis by responding to UV-B radiation. This conclusion is the first report on the regulation of photosynthesis and grain yield of miR408 in rice under UV-B stress. Although these findings may provide a reference for miR408 in other abiotic stresses and in crop improvement, in addition to miR408, the miRNAs involved in the response to UV-B stress regulation of rice yield remains to be further studied.