Physiological And Molecular Regulation Mechanisms Of Plant Response To Mn Toxicity And Cu Deficiency

Mn and Cu are essential micronutrients that required for the proper growth and development of plants.Deficiency or over-accumulation of Mn and Cu may have adverse effects on plants.With the development of industry and agriculture,soil Mn pollution is becoming more and more serious.Soil Mn pollution not only cause damage to plants,but also the human body through the food chain,posing a great threat to human health.Wheat(Triticum aestivum L.)as the main food crop for more than 35%of the world's population,its production and safety are threatened by heavy metals pollution,including Mn.Therefore,improving the tolerance of heavy metal stress and reducing the accumulation of heavy metals in wheat plants is of great significance to Chinese food security.The application of exogenous substances(such as exogenous hormones,plant nutrients and antioxidants)to improve the tolerance of plants to heavy metal stress has been widely studied worldwide.In addition to being stressed by heavy metals,wheat is highly sensitive to Cu deficiency,and it has been known for decades that Cu deficiency can reduce wheat fertility.But so far,it is still unclear how Cu affects the fertility of wheat and the regulation mechanism of Cu homeostasis in wheat.Brachypodium distachyon L.is genetically close to wheat,because of its simple genome and easy transformation technique,it has gradually become an ideal model plant for wheat gene function study.In our study,phenotypic analysis,photosynthetic and antioxidant system analysis,gene expression and metal content determination were utilized to explore the role of exogenous salicylic acid and sulfur in polish wheat(Triticum polonicum L.)plants response to Mn stress;In addition,CRISPR/Cas9 gene editing and XRF techniques were used to analyze the function of the potential Cu regulatory genes BdYSL3 and SPL7 in Brachypodium and to elucidate the molecular mechanism of how Cu affecting the fertility of Brachypodium,providing reference for wheat Cu homeostasis study.The results are as follow:For the effect of salicylic acid(SA)on manganese toxicity in polish wheat seedlings grown hydroponically.Our findings showed Mn stress could decrease plant growth,cause serious chlorosis and injury the photosynthetic apparatus.An increase of Mn accumulation and the inhibition of the K and Ca absorption and the Mg,Fe and Zn translocation were observed under Mn stress.Also,there was a considerable increase in H₂O₂ and TBARS(thiobarbituric acid-reactive substances)content in both the roots and leaves under Mn condition.The combination of SA and Mn treatment decreased the transport of Mn,Fe and Zn from roots to shoots and increased the Ca absorption and Mg translocation.In antioxidant system,such as CAT,APX,GR,DHAR,GSH and As A,the combined treatment significantly increased the antioxidant content and antioxidative enzyme activities compared to the Mn stress alone.The level of ROS and lipid peroxidation significantly decreased under the combination of SA and Mn.These results suggested that SA-induced Mn tolerances in polish wheat are mainly by inhibiting Mn translocation,enhancing enzymatic activities and nonenzymatic antioxidants contents,and regulating nutrient absorption and distribution in plants.Sulfur(S)is an essential macronutrient that has been proved to play an important role in regulating plant responses to various biotic and abiotic stresses.For the effect of S status on polish wheat plant response to Mn toxicity.Results showed that Mn stress inhibited plant growth,disturbed photosynthesis and induced oxidative stress.In response to Mn stress,polish wheat plant activated several detoxification mechanisms to counteract Mn toxicity,including enhanced antioxidant defense system,increased Mn distribution in the cell wall and up-regulated genes involved in S assimilation.Moderate S application was found to alleviate Mn toxicity mainly by sequestering excess Mn into vacuoles,inhibiting Mn translocation from roots to shoots,stimulating activities of antioxidant enzymes and enhancing GSH production via up-regulating genes involved in S metabolism.However,application of high-level S to Mn-stressed plants did not significantly alleviated Mn toxicity likely due to osmotic stress.In conclusion,moderate S application is beneficial to polish wheat plant against Mn toxicity,S exerts its effects via stimulating the antioxidant defense system and regulating the translocation and subcellular distribution of Mn,in which processes GSH plays an indispensable role.Here,we used Brachypodium distachyon as a wheat proxy to explore the molecular mechanisms of copper transport processes and their relationship to Brachypodium fertility and grain set.We selected a gene encoding a yellow stripe-like transporter,BdYSL3 for its homolog in rice has been shown to function in copper delivery to the developing leaves and grains.BdYSL3 was highly expressed in young,mature and flag leaves,and was transcriptionally up-regulated in most of the examined tissues under copper deficiency.GUS staining mediated tissue localization showed that BdYSL3 was primarily detected in the phloem region of vascular bundles of whole plants and ovaries and stigmas of flowers.Loss of BdYSL3 function significantly increased the sensitivity of Brachypodium to copper deficiency.ICP-MS and synchrotron x-ray fluorescence(SXRF)microscopy data showed that the ysl3 knockout mutant fails to deliver copper from mature leaves to sink tissues including young leaves,flag leaves,ovaries and anthers.Because of altered copper transport,the ysl3 mutant manifested delayed flowering time and increased flower production.However,the mutant showed significant reduction in fertility and grain yield.Collectively,our data show that BdYSL3 is essential for copper transport processes that are fundamental for the plant fertility and grain production.For the functional study of BdSPL7,we found that BdSPL7 can fully rescue the sensitivity of Arabidopisis spl7 to Cu deficiency,and the expression level of COPT2 and FSD1(down stream targets of At SPL7)in spl7 mutant.BdSPL7 was found to be localized in nuclear of protoplast.The expression pattern of BdSPL7 was found to be expressed in all tissues of Brachypodium at different stages,but the expression was higher in leaves.The expression of BdSPL7 was not induced by Cu and Fe deficiency,but was induced by Cd treatment.The loss of function of SPL7 resulted in Brachypodium plants sensitive to Cu deficiency.Knocking out of SPL7 also affected the Cu translocation from old leaves to the young tissues and reproductive organs.Combining the expression level of BdYSL3 in the spl7mutant and the DNA binding site analysis of the BdYSL3 promoter region,we found that BdYSL3 is the downstream target of SPL7.Knocking out of SPL7 also decreased the plant fertility,but did not affect the flowering period and grain size.Our results indicated that SPL7plays important roles in regulating Cu homeostasis in Brachypodium.It regulates Cu transporting to the reproductive organs by transcriptionally regulating downstream genes including BdYSL3 to ensure the normal fertility of plants.