| Soil contamination in lead-zinc tailings area is the main source of heavy metal pollution in Hunan province,and it is also a soil pollution problem that needs to be solved urgently.Recent studies have found that the woody oil plant Litsea cubeba has a well-developed root system,is tolerant to dry and infertile soils,and has strong tolerance to a variety of heavy metal pollution.In the remediation process of heavy metal contaminated soil,the individual growth and heavy metal enrichment and transport of male and female plants of L.cubeba show differences in response to heavy metal stress,but little is known about the mechanism of the differences in response to heavy metal stress in both sexes.In this study,we combined field observations and pot experiments under controlled conditions to analyze the differences of male and female plants in response to heavy metal stress in terms of morphology,heavy metal enrichment and transport,antioxidant defense physiology,photosynthetic physiology,and differential gene expression at the transcriptome level.The main results are as follows:1.L.cubeba male plants were better adapted than female plants on contaminated soil in Pb-Zn mining areas.1)Pb-Zn contamination inhibited the height growth of female plants of L.cubeba more than male plants(27.55%and 7.96%decrease in height of male and female plants compared with the control,respectively).Leaf area,leaf length and leaf width were significantly reduced in L.cubeba male leaves compared to the control,while there were no significant differences in female leaves;2)For enrichment and transport of heavy metals,the bioconcentration coefficients of Pb2+ and Zn2+ were significantly higher in the root system of female plants than in male plants(Pbfemaie:0.28,Pbmaie:0.13,Znfemaie:0.68,Znmale:0.37),while the transport coefficients of Pb2+and Zn2+were higher in male plants than in female plants;3)There were significant differences in the physiological responses between female and male plants,with females having higher photosynthetic pigment content,malondialdehyde content,and POD activity,while males had higher soluble protein content,SOD activity,CAD activity,and lignin content.2.In the pot stress test,there were sex differences in the effects of different concentrations of single and combined Pb,Zn stressors treatments on the growth,leaf morphological changes and Pb-Zn enrichment and translocation of L.cubeba female and male seedlings.1)In the range of stress concentrations in this test,single Pb stress had the greatest inhibitory effect on the growth indexes of both males and females,plant height and ground diameter were highly significantly negatively correlated with Pb stress concentration(R2were-0.427,-0.646,respectively),leaf area was positively correlated with Pb stress concentration(R2 was 0.299).The inhibition effect of Pb stress at 2 mmol·kg-1 was significantly greater for females than for males(40.63%and 16.18%decrease in height for males and females,respectively,compared with the control).The single Zn treatment had no inhibitory effect on the growth indexes of L.cubeba males and females.The effect of combined stressors on growth indexs was similar to that of single Pb stress.The leaf area of both sexes increased under Pb and Zn stress,and there were sex differences.2)There were sex differences in the enrichment and translocation of Pb2+and Zn2+ between males and females of L.cubeba,with the enrichment coefficient of Pb2+higher than that of females(maximum enrichment coefficient for male 4.66,for female 2.07)and the translocation coefficient lower than that of females;The enrichment coefficient of Zn2+in males was lower than that of females(maximum enrichment coefficient for female:3.39,for male:1.58),while the transport coefficients were lower than those of females.The results indicate that male seedlings of L.cubeba are more tolerant to Pb-Zn stress than female seedlings.3.The results of the pot stress experiments showed that there were significant sex differences in the physiological responses of male and female seedlings to Pb-Zn stress.1)Under the same ion concentration of single Pb and Pb-Zn combined stressors(3 mmol·kg-1),the accumulation rates of the four elements(K,P,Ca,and Mg)were higher in male seedlings than in female seedlings,and the most significant differences were found in the accumulation of K in the leaves of male and female plants(16.07%and 82.07%increase compared with the control,respectively).Under Zn stress,the accumulation rates of all four elements in leaves were higher in females than in males,and the sex differences in Ca and Mg accumulation in leaves were greater,with Ca and Mg contents increasing by 66.58%and 24.31%in females and decreasing by 2.55%and 13.55%in males,respectively,compared with the control.2)Under Pb stress,Fe contents in leaves were much higher in males than in females(Fe contents in females decreased by 28.20%and in males by 28.20%compared with the control),and the Mn content was higher in female plants than in male plants.The Mn content of male plants was higher than that of female plants under Zn stress.3)Under 3 mmol·kg-1 of single Pb,Zn and combined Pb-Zn stressors,the photosynthetic capacity and antioxidant activity of male seedlings were higher than those of females.The photosynthetic capacity and antioxidant activity were higher in male plants than in female plants,while the content of soluble protein and proline was higher in female seedlings than in male seedlings.Meanwhile,Pb stress at this concentration was more toxic to both male and female plants of L.cubeba than Zn stress.By principal component analysis,chlorophyll a,chlorophyll b,carotenoids,qP,ETR,Y(Ⅱ),SOD,and POD were found to be the main physiological factors for sex differences in response to Pb-Zn stress in male and female seedlings of L.cubeba.4.Transcriptome sequencing analysis of single and combined Pb-Zn stressors-treated male and female leaves at the same ion concentration(3 mmol·kg-1)showed that 1)A total of 11,421 genes were differentially expressed in male and female seedlings of L.cubeba under four stressors(control,single Pb,single Zn,and combined Pb-Zn),among which the control treatment(CKF vs.CKM)had the most differentially expressed genes,followed by Pb treatment(PF vs.PM),and the lowest number of differentially expressed genes was in the combined treatment(MF vs.MM),where the amount of differentially expressed genes between male and female seedlings decreased under heavy metal stress.2)The metabolic pathways involved in differentially expressed genes between male and female seedlings under different heavy metal stressors differed.The main metabolic pathways involved sugar metabolism,photosynthesis,phenylpropanoid biosynthesis,phytohormone signaling,heavy metal transport and signal perception,and glutathione metabolism.3)Comprehensive analysis of differentially expressed genes in the pathways related to heavy metal uptake and transport,photosynthesis and antioxidant enzymes in male and female seedlings of L.and preliminary screening of heavy metal transport proteins ABCB1,ABCC1,FER and The genes related to heavy metal transport proteins ABCB1,ABCC1,FER and signal perception ERF1,photosynthesis-related genes CYP707A,CAO,petE,petF,LHCB1,and the antioxidant enzyme POD gene were initially screened as candidates for improving tolerance to heavy metal stress.In summary,there were sex-specific responses between L.cubeba male and female seedlings at the individual,physiological and molecular levels under Pb-Zn stressors.The combined performance was that male plants of L.cubeba were more tolerant to heavy metals Pb and Zn than females.The main physiological factors and regulatory pathways underlying the differences between female and male seedlings were identified,and the sexually differentially expressed genes related to heavy metal tolerance were tentatively explored.To provide a reference for the construction,maintenance,and prediction of remediation potential of phytocommunities for remediation of heavy metal contaminated soils. |
