Preliminary Study On The Function Of Coumarins And Related Genes In Iron Deficiency Tolerance In Citrus

Since 2019,citrus has become the fruit with the widest planting area and the largest yield in China.In Citrus producing areas such as Sichuan and Chongqing,many orchards are mainly purple soil,the soil pH value is high,and the phenomenon of iron deficiency（Fe）in citrus is common,which is difficult to be corrected by fertilization and other measures.As a trace element,Fe plays a vital role in the normal growth of citrus,which is mainly reflected in the participation of Fe as an enzyme cofactor in chlorophyll synthesis,redox reaction and electron chain transmission in vivo.Under Fe deficiency stress,physiological indexes such as root morphology,leaf number and dry fresh weight ratio of citrus will be affected,and fruit quality and yield will decline.Ziyang Xiangcheng（Citrus junos Sieb.ex Tanaka,CJ）and trifolicate orange（Poncirus trifoliata（L.）Raf.,PT）are two kinds of rootstocks commonly used in citrus production.Field investigation shows that the two rootstocks show obvious differences in Fe deficiency tolerance in alkaline soil:trifoliate orange is prone to Fe deficiency yellowing in alkaline soil,while Ziyang Xiangcheng is resistant to pH8 0 alkaline environment.The mechanism of trifoliate orange and Ziyang Xiangcheng in response to Fe deficiency and high pH is not clear.Through transcriptome and metabolome analysis in the early stage of the laboratory,it was found that there were differences in the number of metabolites between trifoliate orange and Ziyang Xiangcheng,and the number of phenols,lipids and alkanes was higher than that of other metabolites;In coumarin synthesis pathway,related genes were differentially expressed in trifoliate orange and Ziyang Xiangcheng,and the expression in Ziyang Xiangcheng was higher than that in trifoliate orange.Therefore,we speculate that there are great differences in coumarin synthesis and secretion between Ziyang Xiangcheng and trifoliate orange in response to Fe deficiency.The higher coumarin content and gene expression in Ziyang Xiangcheng may be one of the potential reasons for its Fe deficiency tolerance.In order to confirm our conjecture and clarify the function of coumarins in Citrus Fe tolerance,this study was carried out.The main results are as follows:（1）It is preliminarily proved that exogenous addition of p-coumaric acid improves the ability of citrus to withstand high pH.Exogenous addition 50μM p-coumaric acid reduced the degree of chlorosis of Pt under pH 8.0 stress,increased growth,root length and lateral root number less than 0.5cm.（2）Cloning and bioinformatics analysis of coumarin pathway related genes in citrus.The complete CDS sequence of coumarin pathway gene Cytochrome P450（CjCYP82C4）,Scopoletin8-Hydroxylase（CjS8H）and Feruloy-COA6’-Hydroxylase 1（CjF6’H1）were cloned from Ziyang Xiangcheng by PCR amplification.Sequence analysis showed that the molecular formula of CjCYP82C4 was C₂₆₇₉H₄₁₉₆N₇₂₄O₇₅₈S₂₁,the molecular weight was 5.93 k Da,the theoretical isoelectric point was 6.65,and there were two amino acid mutation sites compared with the amino acid sequence of CYP82C4gene of sweet orange.The phylogenetic tree showed that CjCYP82C4 had the highest homology with Cs3g241701.1,followed by AT4G319401.1（At CYP82C4）.The molecular formula of CjS8H protein is C₂₆₇₉H₄₁₉₆N₇₂₄O₇₅₈S₂₁,the molecular weight is4.04 k Da,and the theoretical isoelectric point is 5.96.The results of phylogenetic tree show that CjS8H has the highest homology with Cs4g12541 in sweet orange,followed by Cs4g12541.It has the highest homology with AT3G312900.1（At S8H）in Arabidopsis thaliana.The molecular formula of CjF6’H1 protein is C₁₈₁₉H₂₈₅₀N₄₇₄O₅₃₅S₁₁,the molecular weight is 4.03 k Da,and the theoretical isoelectric point is 6.1.The results of phylogenetic tree show that CjF6’H1 has the highest homology with Cs9G02931.1 in sweet orange,followed by Cs9g02891.1.It has the highest homology with AT3G1366.1（At F6’H1）in Arabidopsis thaliana.（3）The temporal and spatial expression patterns of coumarin related genes in trifoliate orange and Ziyang Xiangcheng were different.The expression analysis of different tissues showed that CYP82C4 was the highest in the stem of Ziyang Xiangcheng,followed by leaves,roots and flowers;However,in trifoliate orange,the highest expression was found in roots,followed by flowers,leaves and stems.The expression of S8H in Ziyang Xiangcheng leaves was the highest,followed by roots,stems and flowers;However,in trifoliate orange,the highest expression was found in roots,followed by flowers,leaves and stems.The expression of F6’H1was the highest in Ziyang Xiangcheng leaves,followed by stems,roots and flowers;The highest expression was also found in leaves,followed by roots,flowers and stems.The expression analysis under different stress treatments showed that both Ziyang Xiangcheng CjCYP82C4 and trifoliate orange Pt CYP82C4 were significantly up-regulated under iron deficiency and pH 8.0 stress,but the expression level of CjCYP82C4was significantly higher than that of Pt CYP82C4 at most treatment time points;Under Cd stress,the expression of CjCYP82C4 was significantly up-regulated,while the expression of Pt CYP82C4 was significantly higher than that of the control only after 1 day of treatment;Under high salt and drought stress,the up-regulated expression multiples of CjCYP82C4 and Pt CYP82C4 were less than 2 times at most treatment time points.The expression of CjS8H in Ziyang Xiangcheng and Pt S8H in trifoliate orange under iron deficiency and pH 8.0 stress showed an overall upward trend,and the expression of CjS8H was significantly higher than that of Pt S8H under iron deficiency;Under Cd stress,the expression of CjS8H was down-regulated and Pt S8H was up-regulated;Under high salt and drought stress,the expression of CjS8H and Pt S8H had no obvious regularity.The expression of CjF6’H1 in Ziyang Xiangcheng was significantly up-regulated under iron deficiency stress,but Pt F6’H1 in trifoliate orange did not change significantly;Under pH 8.0,heavy cadmium,high salt and drought stress,the expression levels of CjF6’H1 and Pt F6’H1 were only differentially expressed at individual sampling time points,and there was no obvious change rule in general.（4）The gene functions of CjCYP82C4,CjS8H and CjF6’H1 were preliminarily analyzed.Subcellular localization analysis showed that CjCYP82C4 was mainly located on the nucleus and cell membrane,while CjS8H and CjF6’H1 were located on the cell membrane.Through heterologous overexpression of CjCYP82C4 in Arabidopsis,the CjCYP82C4 overexpression lines OE-CjCYP82C4-8,OE-CjCYP82C4-11,OE-CjCYP82C4-12 and OE-CjCYP82C4-13 were obtained.The expression of CjCYP82C4in the four overexpression lines was up-regulated 276-75893 times.Under iron deficiency stress,the growth of CjCYP82C4 overexpressing Arabidopsis plants was better than that of wild-type and cyp82c4 mutant plants.85%of the new leaves did not appear yellowing,while the new leaves of wild-type and cyp82c4 mutant Arabidopsis plants appeared yellowing;Except OE-CjCYP82C4-8-6-13,the chlorophyll content of other overexpression lines was significantly higher than that of wild-type Arabidopsis and cyp82c4 Arabidopsis mutants;In addition,the growth of OE-CjCYP82C4-8-6-13 was significantly higher than that of the mutant.Under pH 8.0 stress,the growth of CjCYP82C4 overexpression Arabidopsis plants was also better than that of wild-type and cyp82c4 mutant plants.When treated with pH 8.0 for 20 days,the number of lateral roots of CjCYP82C4 overexpression plants was significantly more than that of wild-type and cyp82c4 mutant plants,and 57%of the overexpression lines had significantly higher fresh weight per plant than that of wild-type Arabidopsis and cyp82c4 mutant plants.The above results showed that overexpression of CjCYP82C4 in Arabidopsis improved the iron deficiency tolerance of transgenic plants.Through heterologous overexpression of CjS8H in Arabidopsis,the CjS8H overexpression Arabidopsis strains OE-CjS8H-2,OE-CjS8H-3,OE-CjS8H-4,OE-CjS8H-5,OE-CjS8H-6,OE-CjS8H-7,OE-CjS8H-8 and OE-CjS8H-13 were obtained.The expression of CjS8H in each strain was 110-171833 times that of the control.Under the treatment of iron deficiency,the growth of CjS8H overexpression Arabidopsis plants was better than that of wild-type plants,and 71.4%of the overexpression plants did not appear yellowing;Except that OE-CjS8H-2-6 and OE-CjS8H-13-1 strains had low chlorophyll content,the chlorophyll content of other cjs8h overexpression lines was significantly higher than that of wild-type Arabidopsis.In addition,except OE-CjS8H-2-6,the fresh weight per plant of other overexpression Arabidopsis strains was significantly higher than that of wild-type Arabidopsis.Under pH 8.0 stress,the growth of CjS8H overexpressing Arabidopsis plants was also better than that of wild-type plants.On the20th day under pH 8.0 stress,the number of lateral roots of CjS8H overexpressing Arabidopsis plants was significantly higher than that of wild-type plants.The chlorophyll content of OE-CjS8H-2-4,OE-CjS8H-2-5,OE-CjS8H-2-6,OE-CjS8H-3-2 and OE-CjS8H-3-6 strains were significantly higher than that of wild-type Arabidopsis.The fresh weight per plant of OE-CjS8H-2-5,OE-CjS8H-3-1 and OE-CjS8H-13-1 was significantly higher than that of wild-type plants.The above results showed that overexpression of CjS8H in Arabidopsis improved the iron deficiency tolerance of transgenic plants.The CjF6’H1 overexpression lines OE-CjF6’H1-1,OE-CjF6’H1-2,OE-CjF6’H1-3,OE-CjF6’H1-4,OE-CjF6’H1-6,OE-CjF6’H1-9 and OE-CjF6’H1-15 were obtained by heterologous overexpression of CjF6’H1 in Arabidopsis.The expression of CjF6’H1 in each strain was up-regulated 356 times at least and 7682 times at most.It provides experimental materials for the follow-up study.