| Plant xylem is able to transport water and minerals,but also provide mechanical support for plant upright growth.In addition,secondary xylem in woody plants is the main component of wood,which is widely used in construction,papermaking,and biofuel production.The secondary xylem formation consists of sequential development events,such as vascular cambium proliferation,xylem cell expantion,secondary cell wall deposition,and programmed cell death(PCD),which involving the expression variation of a large number of genes.Therefore,understanding the regulatory mechanism of transcription factors in the xylem development is crucial to manipulate xylem formation and improve the plant biological and economic value.In this study,we analyzed the function and regulatory mechanism of Arabidopsis transcription factors MYB2,MYB48,and b HLH68 in xylem development.The main results are as follows:(1)MYB2 was involved in the xylem PCD and secondary wall deposition by activating the expression of protease genes γVPE and CEP1 as well as secondary wall component biosynthesis-related genes.The expression pattern of MYB2 was analyzed in Arabidopsis stem using RT-q PCR,the result showed that MYB2 expression was positively correlated with the stem development.The paraffin sections and transmission electro microscopy were used to observe the stem phenotype of myb2 mutants and MYB2 complementation at different development stages.The results showed that the insertion mutation of MYB2 led to reduced vessel number,delayed fiber PCD,and decreased secondary wall thickness.On the one hand,the promoter sequence analysis,EMSA,dual luciferase assay,and RT-q PCR proved that MYB2 bind to the promoter of xylem PCD-related protease genes γVPE and CEP1 and activate their expression,resulting in a significant decrease of both γVPE and CEP1 genes expression in myb2 mutants.These results indicated MYB2 activated proreases γVPE and CEP1 genes expression and formed transcription factor-proteases cascade pathway to participate in xylem fiber PCD.On the other hand,the loss of MYB2 expression resulted in the decrease of secondary wall thickness,the promoter prediction and RT-q PCR were used to analyze the secondary wall biosynthesis-related genes,which data showed that the promoter of the secondary wall biosynthesis-related genes contained MYB2 binding elements,and the expression of these genes was significantly down-regulated in the myb2 mutant.These results suggested that MYB2 was involved in the secondary wall deposition by regulating the expression of secondary wall component biosynthesis-related genes.(2)MYB48 and b HLH68 were target genes of MYB2 and interacted with each other.The promoter analisis showed MYB2 binding site were observed in MYB48 and b HLH68 promoter,which indicted that MYB48 and b HLH68 might be regulated by MYB2.The regulatory relationships among these three transcription factors were analyzed by EMSA and dual luciferase assay,which results showed that MYB2 was function as the upstream of MYB48 and b HLH68 and activated their expression in stem.The relationship between MYB48 and b HLH68 was analyzed using Yeast-2-Hybrid and Bi FC,which results showed that MYB48 could interact with b HLH68 in vitro.(3)MYB48 was involved in xylem development by regulating the expression of secondary wall biosynthesis-related genes.Detection of GFP fluorescence signal in stems of Pro MYB48: GFP transgenic plants and RT-PCR analysis showed that MYB48 was expressed in xylem and related to xylem development.The results of phenotypic observation about the myb48 mutant using paraffin sections and transmission electron microscopy showed that the secondary wall thickness was no significant difference in myb48 mutants compared with wild type.Nevertheless,RT-q PCR results showed that the loss of MYB48 expression led to a significant increase of secondary wall biosynthesis-related genes expression,and complementation MYB48 restored their expression to normal level.These results demonstrated that MYB48 could negatively regulate the expression of secondary wall biosynthesis-related genes.(4)b HLH68 was involved in xylem development by regulating the expression of secondary wall biosynthesis-related genes.The expression pattern of b HLH68 at different developmental stages in stems was analyzed by GFP fluorescence signal detection and RT-q PCR experiments,which results showed that b HLH68 was expressed in xylem,and the expression was increased with the development of stems.The plant morphology and related gene expression of the T-DNA insertion mutation in b HLH68 gene were analyzed,which results showed that the expression loss of b HLH68 caused the decreased expression of secondary wall biosynthesis-related genes and the weak reduction of secondary wall thickness.These results showed that b HLH68 play a positive regulatory role in the xylem secondary wall synthesis pathway.(5)MYB2 activated the gene expression of proteases βVPE and CEP1 to regulate tapetal PCD and affect pollen development.The myb2 mutants resulted in delayed tapetal PCD and aborted pollen.To investigate the molecular mechanism of MYB2 involoving in tapetal PCD,The expression pattern of MYB2 in anther was analyzed by GUS staining and RT-q PCR experiments,which results showed that MYB2 was expressed in tapetal cell.Dual luciferase assay and RT-q PCR experiments showed that MYB2 was able to activate the genes expression of tapetal PCD-related proteases βVPE and CEP1,resulting in a significant decrease of proteases βVPE and CEP1 genes expression in myb2 mutants.The complementation of βVPE and CEP1 expression in myb2 mutants could restore defected pollen phenotype of myb2 mutants to varing degress.These results revealed that MYB2 regulated tapetal PCD and pollen development by activating the genes expression of proteases βVPE and CEP1.In summary,MYB2 not only activated the expression of PCD and secondary wall biosynthesis related genes,but also activated the genes expression of secondary wall-related transcription factors MYB48 and b HLH68 to involoving in Arabidopsis xylem PCD and secondary wall deposition.These results revealed the regulatory mechanism of three Arabidopsis thaliana transcription factors in xylem development,which not only improves the transcriptional regulatory network of xylem formation,but also provides new insights for the regulatory mechanism of transcription factors in the formation of xylem PCD and secondary walls deposition. |
