Effect of miR408 on photosynthesis of rice under enhanced UV-B radiation

doi:10.21203/rs.3.rs-1655609/v1

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Effect of miR408 on photosynthesis of rice under enhanced UV-B radiation

https://doi.org/10.21203/rs.3.rs-1655609/v1

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UV-B stress has become one of the main abiotic stress factors threatening crop growth and development, and miR408 plays an important role in plant abiotic stress response. MiR408 is a type of endogenous non-coding small fragment single-stranded RNA, which is mainly found in eukaryotes and participates in the regulation of plant growth and development by responding to various stresses in a manner that negatively regulates the target gene. The effects of photosynthesis and expression levels of miR408-OE rice plants and knockout UCL8 plants under UV-B stress were investigated. The results showed that under UV-B enhanced radiation, the chlorophyll content, intercellular carbon dioxide concentration, net photosynthetic rate and stomatal conductance of the overexpressed miR408 plants were significantly reduced. Under UV-B enhanced radiation, chlorophyll fluorescence kinetic parameters such as Fv/Fm and qP overexpressing miR408 plants were reduced. UV-B significantly inhibited the activity of SOD, POD and CAT in overexpressing miR408 plants and significantly increased their H₂O₂ and MDA contents. Under UV-B stress treatment, the yield traits such as the number of grains, the number of tillers and the quality of dry matter of overexpressing miR408 plants were significantly reduced. Therefore, UV-B enhances the photosynthetic rate, photosynthetic performance and disruption of the antioxidant system balance of overexpressing miR408 plants, which leads to a decrease in yield. It is speculated that overexpression of miR408 reduced the tolerance of rice to UV-B radiation, that is miR408 may play a negative regulatory role in response to UV-B stress.

agronomic traits

miR408

photosynthesis

UV-B radiation

Rice(Oraza sativa L.)is one of the most important food crops in the world and one of the model plants for biological science research.In recent years, air pollution has been getting worse, ultraviolet rays are being enhanced due to the destruction of the ozone layer(Larin I.K,2021) .The ultraviolet rays in the solar spectrum can be classified into super strong effect waves (UV-C, 200–280 nm), strong effect waves (UV-B, 280–320 nm) and weak effect waves (UV-A, 320 ~ 400nm) according to biological effects. Under normal conditions, atmospheric stratospheric O₃ can absorb almost all UV-C and 90% UV-B(Vanhaelewyn et al,2020). However, as the concentration of O₃ decreases, it results in an increase in UV-B radiation, which poses a serious threat to animals, plants and even humans on Earth(Idris et al,2020). Studies have shown that UV-B radiation can destroy plant DNA(Tripathi et al,2011), protein(Zhong et al,2021) and membrane system(Han et al,2015), which has a negative impact on plant growth and development, physiological metabolism and other aspects(Bhandari et al,2011). In the physiological process of plants, the photosynthetic system is the most important target of UV-B radiation(Karsten et al,2003). UV-B plays an important role in the growth, development and maturation of plants(Arpita et al,2020). The impact of UV-B radiation enhancement on animals, plants and ecosystems as a whole is one of the most important topics in national research.

The UV-B of the plant passes through he light receptors and allows the introduction of optical signals into the nucleus(Yin et al,2016), the expression of the miRNA changes under the relevant action element and further regulates the expression of the target gene and the growth and development of the plant(Zhang et al,2017). MicroRNAs (miRNAs) are a class of endogenous small molecule non-coding RNAs that are widely found in animal and plant cells and cleave mRNA or inhibit the initiation of translation by inaccurate complementary pairing with target genes(Xing et al,2011), that is, negatively regulating the expression of target genes at the post-transcriptional level and translation level, and having the function of regulating gene expression ability(DING et al,2012). MiRNA plays an important role in regulating plant growth and development and adapting to various stresses(Biswas et al,2021). Furthermore, it is thought that miRNA itself can be regarded as a reservoir of resource genes to alter these difficult characteristics in agricultural production(Sanz et al,2020).

MiR408 is a highly conserved class of miRNA molecules consisting of 21 nucleotides with tissue specificity(Jiawei et al,2018). MiR408 not only regulates plant growth and development, but also is affected by environmental and abiotic stresses(Kantar et al,2010). In different plants, the expression patterns are completely different.For example, under drought stress, the expression level of miR408 is increased in alfalfa and barley, but decreased in peach trees(Eldem et al,2012). MiR408 acts as a powerful regulator of vegetative growth and enhances the growth of Arabidopsis seedlings and adult plants(Carrio et al,2019). Overexpression of miR408 plants can increase Arabidopsis anthocyanin content, rice yield, and salt and antioxidant tolerance(Zhang et al,2019;MAC et al,2015).

Previous studies have found that osa-miR408 (rice) has high homology with ptc-miR408 (Populus candicans Ait.)(Grobhans et al,2008), and ptc-miR408 increases in expression under UV-B radiation(Jia et al,2009).Howover the research mechanism of rice miR408 has not been reported yet. In view of this, this study analyzes the effects of miR408 on photosynthesis in rice under UV-B enhanced radiation, and provides reference for further study of its function in the future.This provides a reliable scientific basis for crop variety improvement and agricultural production.

Plant material and stress treatments

Using "Nipponbare"(Oryza sativa subsp. Japonica cv.) as a receptor, overexpressing miR408 plants as test materials.And Using "Zhonghua11" as a receptor, knock out the target gene UCL8 plant as a test material.The emergence of flag leaves in rice began UV-B treatment, and the UV-B radiation intensity was increased by 40% compared with normal illumination.UV-B radiation treatment time is 8h/d (9:00–17:00). Until the end of the mature harvest period Under normal light, the Nipponbare is CK1, the overexpressing miR408 plant is recorded as M1, Zhonghua 11 is recorded as CK2, Knock out miR408 target gene plant(UCL8), recorded as M2. UV-B radiation treatment, the Nipponbare is BCK1, the overexpressing miR408 plant is recorded as BM1,Zhonghua 11 is recorded as BCK2, knockout target gene plant (ucl8), recorded as BM2.

Determination of chlorophyll content

The SPAD value of the flag leaf of the test material was measured using a Spad-502 instrument.More than 6 leaves were determined for each replication, leaves of the same breed were the same for each treatment.

Observation of chloroplast structure

First, the middle of the fully expanded rice leaves was cut into 0.5 mm×0.5 mm segments, fixed with 4% glutaraldehyde fixative at 4℃for 2 h, then washed with phosphate buffer for 3 times, and then added with 1% citric acid. The fixative was fixed at 4℃ for 2 h, washed 3 times with phosphate buffer, dehydrated with acetone, and embedded in epoxy resin Epon 812. Finally, it was sliced with an LKB-V ultrathin slicer and observed and photographed under a Hitachi TEM 600A-2 electron microscope.

Determination of Chlorophyll metabolism intermediate synthesis product

Relevant indicators were determined using an ELISA kit for Shanghai ruishuo Biotechnology Co., Ltd. For instance,ALA,PBG,Urogen Ⅲ,ProtoⅨ,Mg-ProtoⅨ,Pchlide etc.

Determination of photosynthetic performance

Referring to the method of Li et al(Li et al,2011), the LI-6400 portable photosynthetic tester manufactured by LI-COR Company of the United States is used.Using the red and blue light source of the instrument, the atmospheric CO₂ concentration is 400 µmol·L^− 1, the photosynthetic photon quanta flux density(PPFD) is 800 µmol· m^− 2· s^− 1, and the flow rate is set to 500 µmol· s^− 1. At 8:30 − 11:30 am on a sunny day, the inverted leaves of the plants were taken and measured.At 8:30 − 11:30 am on sunny days,the penultimate leaf leaves of the plants were taken and measured net photosynthesis rate (Pn), stomatal conductance (Gs) and intercellular CO₂ concentration (Ci) were measured. Each independent experiment was repeated 6 times.

Chlorophyll fluorescence parameter determination

According to the method of Strasser et al(Strasser et al,2004), the chlorophyll fluorescence of rice leaves was determined by HandyPEA continuous excitation fluorometer (Hansatech, UK), and the fluorescence data within 1 s was recorded.Repeat 6 times for each treatment.After 20 min dark adaptation, the leaves were challenged with 800 ms saturated light (1500 umol·m^− 2·s^− 1) at 650 nm provided by HandyPEA.Light-induced chlorophyll fluorescence was digitized by HandyPEA equipment, and JIP-test analysis was performed with PEP Plus and Biolyzer HP3 to calculate the structural and performance parameters of the photoreactive tissue.

Table 1

Selected JIP parameters from the fast OJIP fluorescence induction used in the present study
Terms and formulas	Illustrations
F₀	First reliable fluorescence value after the onset of actinic illumination; used as the initial value of the fluorescence
V_I = (F_I – F₀)/(F_M – F₀)	Relative variable fluorescence at phase I of the fluorescence induction curve
Sm = Area/F_V	Normalized total complementary area above the OJIP transient
N	Number of Q_A redox turnovers until Fm is reached
M₀	Net rate of PSII closure
F_M	Maximum value under saturating illumination
F_V/F_M	Maximum quantum yield of PSII photochemistry
VJ = (FJ – F0)/(FM – F0)	Relative variable fluorescence at phase J of the fluorescence induction curve
PI_ABS = (RC/ABS) × (φP₀/(1 − φP₀)) × (ψ₀/(1 − ψ₀))	Performance index on absorption basis
φP₀ = TR₀/ABS = F_V/F_M = [1 – (F₀/F_M)]	Maximum quantum yield of primary photochemistry
φE₀ = ET₀/ABS = [1 – (F₀/F_M)] × (1 – V_J)	Quantum yield of electron transport
ψ₀ = 1 – V_J	Efficiency with which a trapped exaction can move an electron into the electron transport chain further than Q_A
φD₀ = DI₀/ABS = 1 – φP₀ = (F₀/F_M)	Thermal dissipation quantum yield
φR₀ = φP₀. ψE₀. δR₀	Quantum yield of reduction of end electron acceptors of PSI
ABS/RC = (M₀/V_J)/[1 – (F₀/F_M)]	Light absorption flux (for PSII antenna chlorophylls) per reaction center (RC)
DI₀/RC = ABS/RC – TR₀/RC	Dissipation energy flux per PSII RC (at t = 0)
ET₀/RC = (M₀/V_J) × (1 – V_J)	Maximum electron transport flux (further than Q_A⁻) per PSII RC (at t = 0)
TR₀/RC = (M₀/V_J)	Trapped (maximum) energy flux (leading to Q_A reduction) per RC (at t = 0)
RE₀/RC = M₀(1/V_J) (1 – V_I)	Electron flux reducing end electron acceptors at the PSI acceptor side per RC
ABS/CSO ≈ FO	Absorption flux of photons per cross section, approximated by Fo
TRO/CSO = φPo•(ABS/CSO)	Light energy captured per unit area (at t = 0)
ETO/CSO = φEo•(ABS/CSO)	Quantum yield per unit area of electron transfer (at t = 0)
DIO/CSO= (ABS/CSO) - (TRO/CSO)	Heat dissipation per unit area (at t = 0)
RC/CSO = φPo•(VJ/MO)•(ABS/CSO	The number of reaction centers per unit area (at t = 0)

Determination of SOD,POD,CAT content

Determination of SOD activity by measuring the absorbance of each tube at 560 nm by recording nitroblue tetrazolium (NBT) according to the method of Stewert and Bewley(Stewert et al,1980). According to the method of Fu et al.(Fu et al,2014), the activity of POD was determined by monitoring the change of absorbance of guaiacol at 470 nm by using guaiacol oxidation method.CAT activity was determined by monitoring the degradation of H₂O₂ absorbance at 240 nm according to the method of Luna (Luna et al,2005) .

Determination of H₂O₂ content

H₂O₂ content was determined using the H₂O₂ kit (Suzhou Comin Biorechnology) according to the instructions.H₂O₂ and titanium sulfate to form a yellow complex titanium peroxide, and characteristic absorption at 415nm. Each material was repeated 3 times.

Determination of MDA content

The MDA content was determined using an MDA kit (Suzhou Comin Biorechnology) according to the instructions of the specification. MDA condenses with thiobarbituric acid (TBA) to form a red product,the maximum absorption peak at 532nm, the content of lipid peroxide in the sample can be estimated after colorimetry;Simultaneous determination of absorbance at 600 nm,calculate the content of MDA by using the difference between the absorbance at 532 nm and 600 nm. Each material was repeated 3 times.

Determination of yield component

The plant height per plant, the actual yield per plant, and the 100-grain weight were determined.Each treatment was repeated ≥ 3 times.

qRT-PCR analysis

Total RNA from rice was reverse transcribed using the Vazyme Reverse Transcription Kit. Real-time PCR was carried out using SYBR Premix Ex Taq (Vazyme) to detect PCR products.ACTIN2 was chosen as the reference genes. Real-time PCR was performed according to the manufacturer’s in-structions (Vazyme). The data represent means ± SD of three independent experiments. The primers were as follows:

Table 2

RT-PCR primers
PRIMER	SEQUENCE
ACTIN2	5’-TCTTACGGAGGCTCCACTTAAC-3’ 5’-TCCACTAGCATAGAGGGAAAGC-3’
miR408	5’-GGAGACAGGGATGAGGCAGAG-3’ 5’-GGAGCCAGGGAAGAGGCAGT-3’
OsUCL8(UF)	5’-TCGTCGTGAGCTGCATCTTC-3’ 5’-ACCTGAACACGAGGATGTCG-3’
PPDK	5’-TGCGGCTGCTGGAATTCTTA-3’ 5’-TGACCTCTCCAGTCGAACCA-3’
PSII-PsbR	5’-GATTGGCCATGCTGGCTTTT-3 5’-CTCGGGATGATGATGTCGCC-3’
chlorophylla/b-binding	5’-TGGTGGTCTGTGGTTTGACC-3’ 5’-TGGAACCAAGCTCCCATGAC-3’
PSI-PsaO	5’-GTCATGGCCTCTTGTGCTCT-3’ 5’-ACCGTCTTTCAGGTTCGGTC-3’
HXK	5’-ATCGCCAAGCTACACCCATC-3’ 5’-TCGTCGAACTCCGTCAATGG-3’

Statistical Analysis

The data were analyzed for LSD and DUNCAN significance using SPSS 19.0.In all of our data analyses, P < 0.05 was considered to have significant differences.

Effect of UV-B stress on the growth of miR408 overexpressing rice plants

To investigate the function of OsmiR408, we used to high expression level of OsmiR408 (miR408-OE) and knockout of its target gene OsUCL8 (ucl8) plants to evaluate the effect of OsmiR408 on rice growth and development under UV-B stress (Fig. 1). The growth of rice plants under UV-B stress was slightly slower than under normal light, among them, miR408-OE and ucl8 grew worse under UV-B stress. It indicates that miR408 might negatively regulate plant growth under UV-B stress.

The expression level of miR408 decreased firstly, then increased and then decreased under UV-B stress(Fig. 2). The expression level of miR408 was highest at the 40th day (booting stage) after UV-B stress treatment, the most significant. The relative expression level of miR408 increased by 49.3% under UV-B stress at booting stage compared with normal light. The expression of miR408 in the heading and maturity stages increased by 2.48% and 0.59% under UV-B treatment. Therefore, the expression of miR408 in the middle and late growth stages of rice was up-regulated under UV-B stress, indicating that miR408 can respond to UV-B stress.

To further determine whether miR408 negatively regulates rice yield under UV-B stress, we measured yield-related traits in the miR408-OE and ucl8 plants. UV-B radiation significantly inhibited the number of tillers of plants compared to normal light plants. Compared with the CK1, the number of tillers per plant in BCK1 was reduced by 29.8%, while that of BmiR408-OE was significantly reduced by 45% compared with miR408-OE (P = 0.000). Intriguingly, BmiR408-OE was lower than BCK1 19.1% (P = 0.000), the difference was extremely significant (Fig. 1A). The yield per plant of UV-B treated plants was significantly lower than that of the normal light group. Compared with CK1 and miR408-OE, the yield per plant of BCK1 and BmiR408-OE decreased by 38.3% and 68.7%, respectively. The miR408-OE was significantly higher than CK1 10.5% (p = 0.000). BmiR408-OE was significantly lower than BCK1 43.9% (p = 0.000). The results showed that the yield per plant of miR408 overexpressing plants under UV-B stress decreased more than that of CK1(Fig. 1B). Compared with CK1 and miR408-OE, the 100-grain weight of BCK1 and BmiR408-OE decreased by 5% and 23.2%, respectively. Compared with CK1, miR408-OE increased significantly by 3.1% (p = 0.000). BmiR408-OE decreased significantly by 16.6% compared with BCK1 (p = 0.000). It shows that miR408-OE can increase the 100-grain weight of rice under normal illumination, and its decline was more obvious under UV-B radiation(Fig. 1C). Compared with CK1 and miR408-OE, BCK1 and BmiR408-OE decreased by 11.6% and 12.5%, respectively. UV-B inhibits miR408-OE rice plant height more obvious(Fig. 1D). These results demonstrated the negative regulation of OsmiR408 on the growth and development of rice plants under UV-B stress. Compared with CK2 and M2, the yield per plant of BCK2 and BM2 decreased by 54.7% and 67.8%, respectively. Compared with CK2, M2 increased by 18.9%. As shown Fig. 1E, compared with M2, BM2 decreased by 15.4%, indicating UV-B. The yield per plant was significantly inhibited, and BM2 was the most obvious. Under normal illumination, the knockout target gene increased the yield per plant compared with the control, which was consistent with the miR408 overexpressing plant. As shown in Figure.1F, compared with CK2, the 100-grain weight of M2 increased by 8.13% (P = 0.000), BCK2 decreased by 7.21%, and compared with M2, BM2 decreased by 10.13%. Therefore, under normal illumination, knocking out target gene plants can increase rice yield, but under UV-B stress, the knockout target gene UCL8 plants grow even worse.

Defective photosynthetic pigmentation and chloroplast development in Overexpression of miR408

The Fig. 4 shows, compared with CK1 and M1, the chlorophyll content of BCK1 and BM1 decreased by 0.6% and 12.8%(p = 0.000), respectively. This shows that UV-B reduces the chlorophyll content after radiation enhancement. The chlorophyll content of M1 is higher than CK1 5.9% and the BM1 is lower than BCK1 7%. The chlorophyll content of BM1 is significantly lower than that of BCK1. UV-B significantly reduced the chlorophyll content of overexpressing miR408 plants. Compared to CK2, the chlorophyll content of BCK2 decreased by 4.6%. Compared to M2, BM2 decreased by 9.8%. The decrease of BM2 was greater than that of BCK2, and UV-B stress significantly reduced the chlorophyll content of BM2. Decreased Chl a (p < 0.05), Chl b (p < 0.05) were observed with rice plants of UV-B stress. Among them, the decrease of BM1 is larger. Compared with BCK1, the Chl a of BM1 decreased by 16.2% (P < 0.01), and the decrease of Chl b by 15.5% (P < 0.01).

The chlorophyll synthesis pathway is a complex process. Analysis of anabolic intermediates from chlorophyll. The contents of ALA, PBG and Urogen III in BM1 were significantly lower than those in BCK1, while the contents of ProtoIX, Mg-ProtoIX and Pchlide were significantly higher than those in BCK1. It is speculated that the transformation reaction from UrogenIII to ProtoIX in chlorophyll anabolism of BM1 is suppressed, resulting in abnormal accumulation of ProtoIX.

In order to investigate a role of miR408-OE in chloroplast development, we compared the ultrastructure of plastids in the M1 and CK1 plants under normal light and UV-B stress by using TEM(Fig. 6 ). Under normal light, the chloroplasts of CK1 and M1 were close to the cell wall, long oval shape, with clear inner lamellae, exhibite an orderly arrangement of granal and stromal thylakoids and small amount of starch small particles. Under UV-B stress, the chloroplasts of CK1 plants changed from spindle to round and slightly swollen, the matrix lamellae expanded slightly, but arranged neatly, stacked orderly, the number of osmiophils increased significantly, and the bending of individual chloroplasts tended to detach from the inside of plasma membrane. After UV-B treatment, the chloroplast of M1 expanded into an abnormal shape and the structure was loosely distorted. The cytoplasmic membrane of the M1 was collapsed and invaded, the separation of the plasmolysis was serious, and the matrix sheet is elongated and thinned to exhibit a filamentous sheet structure and the starch granules become large and have a large amount of steroidal distribution. UV-B stress significantly increased the damage of the chloroplast structure of miR408-OE, causing severe damage to the internal structure of its chloroplast.

UV-B stress significantly inhibited the photosynthetic rate of flag leaf of miR408 overexpressing rice

As can be seen from the Fig. 1, compared with CK1 and M1, the Ci of BCK1 and BM1 decreased by 11.2% and 5.2%, respectively, and the BM1 decreased by less than BCK1. Compared with CK1, the intercellular carbon dioxide concentration of M1 decreased by 3%, while the BM1 of UV-B treatment increased by 3.4% (P = 0.02) compared with BCK1, showing a significant difference. Therefore, UV-B stress causes a large accumulation of intercellular carbon dioxide concentration in BM1 leaves, which leads to the obstruction of CO₂ transport to mesophyll cells required for photosynthesis, which may lead to a decrease in photosynthesis carbon assimilation efficiency.

In Fig. 3.4.B, UV-B stress significantly reduced stomatal conductance in rice leaves. Compared with CK1 and M1, the Gs of BCK1 and BM1 decreased by 5.9% and 26.5%. There was no significant difference in stomatal conductance between M1 under normal illumination and CK1 (P = 0.85), but under UV-B enhanced radiation, BM1 decreased by 22.22% (P = 0.000) compared with BCK1, showing a very significant difference. It is indicated that after UV-B enhanced radiation, the Gs of BM1 is drastically reduced, which reduces the degree of stomatal opening of the leaf, limits the transmission of moisture and atmospheric CO₂ to the intercellular space, and makes UV-B significantly reduce the moisture exchange rate and CO₂ exchange capacity of BM1, there by reducing the photosynthetic rate of BM1.

As shown the figure C, UV-B radiation reduces the net photosynthetic rate of rice leaves. Compared with CK1 and M1, the Pn of BCK1 and BM1 decreased by 21.1% and 30%, respectively, and BM1 decreased more than BCK1. Compared with CK1, M1 decreased by 9% (P = 0.048). Compared with BCK1, BM1 decreased by 19.6% (P = 0.0005), which was extremely significant. It is indicated that UV-B radiation more significantly inhibits the net photosynthetic rate of BM1 plants.

The figure D shows, compared with CK1, the transpiration rate of BCK1 decreased by 2.4% (P = 0.03). Compared with M1, BM1 decreased by 6% (P = 0.000), which was extremely significant. BM1 increased by 2% compared to BCK1. It shows that miR408 is most sensitive to UV-B response and resists UV-B stress by regulating its ability to lose water.

As can be seen from the Fig. 3.4.E, compared with CK1 and M1 under normal illumination, the water use efficiency of BCK1 and BM1 decreased by 9.3% and 21%, respectively (P = 0.000). M1 is reduced by 13.5% compared to CK1. Under UV-B enhanced radiation, BM1 is reduced by 24% compared to BCK1, and the decrease of BM1 was greater than that of BCK1. From the above results, UV-B radiation can reduce the water use efficiency of rice, especially for miR408 overexpressing plants.

Chlorophyll a fluorescence induction kinetics

The OJIP fluorescence kinetic curves at various stages of rice growth under UV-B stress showed characteristic differences(Fig. 1). The chlorophyll fluorescence kinetics curves of CK1 and M1 showed significant differences with the increase of UV-B radiation days. On the 20th day of UV-B stress treatment, compared with BCK1, F₀ (50 µs), F_J (2 ms), F_I (30 ms) and F_M of BM1 increased by 7.76%, 10.1%, 10.0%, and 9.2%, respectively. The UV-B treatment showed significant changes in the fluorescence kinetics curve after 20 days, and the BM1 group was most affected. Analysis of the fluorescence transients revealed that the effects of UV-B radiation on BM1 mainly appeared in the I-P phase. Under UV-B conditions, the content of chlorophyll a and b in miR408-OE leaves also decreased significantly, resulting in reduced ability to absorb and transmit light energy, which may have a certain effect on the fluorescence peak. On the 60th and 80th day of UV-B treatment, there was a significant transient rise in the miR408-OE leaves. It is possible that UV-B radiation inhibits the oxygen-evolving complex (OEC), causing the PSII electron transfer to be blocked and the photosynthetic function to decrease.

Effects of UV-B stress on PSII performance index, electron acceptor and its expression level in photosynthetic related genes in miR408-OE leaves

To further clarify the effect of UV-B on photosynthesis in rice, photosynthetic properties of PSII were determined by Chl-a fluorimetry. In Fig. 4, the initial fluorescence Fo was the fluorescence level when the PSII reaction center was completely open, that is, when the QA is completely oxidized. Fo of BCK1 has no significant change compared with CK1(P = 0.96 > 0.05). However, compared to M1, BM1's Fo is significantly increased(P = 0.000 < 0.05). Therefore, under UV-B stress, Fo significantly increases, which makes the PSII antenna dissipate energy enhancement. FV/FM is a common indicator used when plants encounter stress, and it indicates the original photochemical efficiency of PSII. As can be seen from the Fig. 3.5.B, BCK1 has no significant change compared with CK1(P = 0.80 > 0.05). However, compared to M1, Fv/Fm of BM1 is significantly decreased(P = 0.000 < 0.05). It is indicated that UV-B radiation inhibits the primary photochemical efficiency of PSII of M1 plants.It is indicated that M1 is most sensitive to UV-B, and UV-B radiation inhibits Fv/Fm of M1 plants. Photosynthetic performance parameters PI_abs can better reflect PSII performance. PI_abs showed a similar tendency. BM1 decreased by 28.7% compared with M1(p = 0.0002 < 0.05). The results showed that UV-B significantly inhibited the photosynthetic capacity of miR408-OE plants.

Figure 2 shows the variation of the PSII acceptor side. Compared with the normal light group, the V_J of the UV-B radiation group increased significantly, and both S_M and N decreased. The VJ of the BM is always higher than the BCK1 during the entire UV-B stress process and the VJ of BM1 plants increased significantly within 40 d of UV-B radiation, indicating an excessive accumulation of QA. The number of QA redox reductions is indicated by N before reaching maximum fluorescence. Compared with CK1, N of BCK1 was 10.9% lower, while BM1 was 31.3% lower than M1 (P = 0.000), and BM1 has a greater decline than BCK1. At the 20th to 40th day of UV-B treatment, the Sm of BM1 plants decreased by 29.3% (P = 0.000), which was extremely significant. These results confirmed that UV-B has a greater inhibitory effect on the receptor side and electron transport of QA and QB in miR408-OE plants.

As can be seen from the Fig. 3.5.D, compared with CK1 and M1, Qp of BCK1 and BM1 decreased by 12.2% and 27.4%, respectively. Compared with BCK1, qp of BM1 was reduced by 24.7%. It indicated that under UV-B radiation, miR408 responded to UV-B stress, which affected the PSII electron transport activity of plants and reduced photosynthetic efficiency. The Fig. 3.5.E shows, compared with CK1, qn of M1 increased by 5.7%. Compared with BCK1, qn of BM1 was increased by 7%. It indicated that the overexpression of miR408 in rice can make the ability to dissipate excess light energy into heat energy stronger, which is not conducive to plant growth.

We further studied the expression of photosynthetic-related genes in BCK1 and BM1 under UV-B treatment by qRT-PCR. The expression level of photosynthetically related genes was found to be significantly different between BCK1 and BM1 plants. BM1 pyruvate phosphate double kinase (PPDK), photosystem II subunit protein R (PSII-PsbR), and chlorophyll a/b-binding protein were significantly lower than BCK1. However, its photosystem I (PSI-PsaO) and hexokinase (HXK) did not change significantly. These results indicate that UV–B significantly inhibits the PSII photosystem of miR408-OE, which is consistent with effect of UV-B stress on photosynthesis of miR408 overexpressing plants.

Effects of UV-B stress on PSII efficiency and excitation energy dissipation of miR408 overexpressing plants

It can be seen from Fig. 3.4 that as the number of days of UVB radiation increases, the energy flow of BM1 becomes more and more significant. The light energy absorbed by the unit reaction center of the BM1 (ABS/RC) is first increased and then decreased during the andmaturation period. There is no significant change in the energy (ET0/RC) captured by the unit reaction center for electron transfer. On the 60th day of UV-B treatment, the heat dissipation per unit area of BM1 (DI0/CSo) increased by 9.2% compared with BCK1, and the energy dissipated by unit reaction center of BM1 (DI0/RC) increased by 12%. However, at 80 d, BM1's (DI0/CSo) and (DI0/RC) were significantly reduced. It indicated that UV-B radiation significantly increased the heat dissipation energy of miR408 overexpressing plants, causing excessive heat energy in the PSII reaction center of BM1, resulting in blocked electron transfer and decreased photosynthesis.

UV-B treatment increased the MDA and H₂O₂ content of miR408 overexpression

MDA is a product of membrane lipid peroxidation induced by oxidative stress in plant tissues under stress, and it is an indicator that reflects the degree of cell membrane lipid peroxidation and the strength of plants in response to adverse conditions.It can be seen from the figure that compared with CK1 and M1 under normal illumination, after UV-B radiation, BCK1 increased by 23.8% and BM1 increased by 22.4%.Under UV-B enhanced radiation, the MDA content of BM1 plants increased by 11.5% compared to BCK1. It indicated that under the stress of UV-B radiation, the MDA content of membrane lipid peroxidation products in plants increased, causing injury. In particular, M1 plants are more sensitive to UV-B response, accumulating a large amount of membrane lipid peroxidation product MDA, so that UV-B inhibits miR408 plants growth.

H₂O₂ is one of the active oxygen species and is one of the harmful products in the process of plant metabolism, which can cause oxidative stress in plants.Compared with CK1, there was no significant change in BCK1 (p = 0.34 > 0.05).Compared with M1, BM1 increased by 50.1% (p = 0.001 < 0.05), showing a significant difference.UV-B radiation causes excessive accumulation of H₂O₂ in BM1 plants, causing toxicity to rice.

UV-B treatment reduces the antioxidant enzyme activity of rice

UV-B stress has a significant effect on the antioxidant enzyme activity of miR408-OE. Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) act as key protective enzymes for scavenging and reducing free radicals in plants, which can effectively scavenge free radicals. As can be seen from the figure,the SOD, POD and CAT of the plants after UV-B enhanced radiation decreased significantly. Under UV-B enhanced radiation, BM1's SOD decreased by 18.2%, POD decreased by 21.7%, and CAT decreased by 12.6% compared with BCK1. The decrease in the antioxidant enzyme index of BM1 was greater than that of BCK1, and there were significant differences. It is indicated that the anti-free radical ability of miR408-OE is more significant under UV-B radiation stress, that is, the tolerance of BM1 plants to UV-B stress stress was reduced, which affected the growth.

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 V_J 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 F_V/F_M 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 F_V/F_M 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 H₂O₂and 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.

Materials described in the manuscript, including all relevant raw data, will be freely available to any researcher wishing to use them for non-commercial purposes, without breaching participant confidentiality.

“The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.”

“The authors have no relevant financial or non-financial interests to disclose.”

“All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Meiping Zhang, Dan Li, Jiayu Zhang, Yongliang Zhang and Lirong Ji. The first draft of the manuscript was written by Lirong Ji and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”

“The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.”

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Effect of miR408 on photosynthesis of rice under enhanced UV-B radiation

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Plant material and stress treatments

Determination of chlorophyll content

Observation of chloroplast structure

Determination of Chlorophyll metabolism intermediate synthesis product

Determination of photosynthetic performance

Chlorophyll fluorescence parameter determination

Determination of SOD,POD,CAT content

Determination of H₂O₂ content

Determination of MDA content

Determination of yield component

qRT-PCR analysis

Statistical Analysis

Results

Effect of UV-B stress on the growth of miR408 overexpressing rice plants

Defective photosynthetic pigmentation and chloroplast development in Overexpression of miR408

UV-B stress significantly inhibited the photosynthetic rate of flag leaf of miR408 overexpressing rice

Chlorophyll a fluorescence induction kinetics

Effects of UV-B stress on PSII efficiency and excitation energy dissipation of miR408 overexpressing plants

UV-B treatment increased the MDA and H₂O₂ content of miR408 overexpression

UV-B treatment reduces the antioxidant enzyme activity of rice

Discuss

Ultrastructure of chloroplasts are damaged under UV-B stress

Effects of UV-B stress on photosynthesis of rice

UV-B stress destroys the internal balance of rice antioxidant enzyme system

Declarations

References

Status:

Version 1

Effect of miR408 on photosynthesis of rice under enhanced UV-B radiation

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Plant material and stress treatments

Determination of chlorophyll content

Observation of chloroplast structure

Determination of Chlorophyll metabolism intermediate synthesis product

Determination of photosynthetic performance

Chlorophyll fluorescence parameter determination

Determination of SOD,POD,CAT content

Determination of H2O2 content

Determination of MDA content

Determination of yield component

qRT-PCR analysis

Statistical Analysis

Results

Effect of UV-B stress on the growth of miR408 overexpressing rice plants

Defective photosynthetic pigmentation and chloroplast development in Overexpression of miR408

UV-B stress significantly inhibited the photosynthetic rate of flag leaf of miR408 overexpressing rice

Chlorophyll a fluorescence induction kinetics

Effects of UV-B stress on PSII efficiency and excitation energy dissipation of miR408 overexpressing plants

UV-B treatment increased the MDA and H2O2 content of miR408 overexpression

UV-B treatment reduces the antioxidant enzyme activity of rice

Discuss

Ultrastructure of chloroplasts are damaged under UV-B stress

Effects of UV-B stress on photosynthesis of rice

UV-B stress destroys the internal balance of rice antioxidant enzyme system

Declarations

References

Status:

Version 1

Determination of H₂O₂ content

UV-B treatment increased the MDA and H₂O₂ content of miR408 overexpression