| Stigma exsertion is a major factor that can increase the opportunity for outcrossing pollination.The efficiency of hybrid seed production can be improved by increasing the percentage of exserted stigma.The stigma exsertion character is influenced by cytoplasmic type,but the mechanism of cytoplasmic genes and nuclear gene interaction on stigma exsertion is poorly understood.We found a stigma exsertion male sterile mutant in the field.Stigma exsertion traits showed cytoplasmic inheritance by classical genetic analysis.Three homonuclear heteroplasmic tobacco materials,MSK326 SE with stigma exsertion,MSK326 with normal stigma and K326 were obtained through continuous backcross.To explore the genetic development mechanism of tobacco cytoplasmic inheritance stigma exsertion,the morphological characteristics of the buds,the changes in physiological indicators,t he expression levels of related genes during flower buds development and the cytological characteristics at fixed length of flower buds of three tobacco varieties(lines)were examined.The main findings are as follows:The morphological characteristics of three tobacco varieties(lines)during flower buds development were detected.The result showed that the rapid development period of MSK326 SE style was earlier than that of flower buds.The stigma of the MSK326 SE began to expose in period 3(bud length 8.58±0.38mm).The length of flower buds of MSK326 SE was extremely less than that of K326 and MSK326,and the style length was significantly greater than that of K326 and MSK326.To facilitate the study of tobacco stigma exsertion,we analyzed the morphological development characteristics of buds of three tobacco varieties(lines)and divided the buds of the three materials into five development phases based on the main features of K326 buds developmental length.The CTK content,IAA content,antioxidant enzyme activity and soluble protein content of MSK326 SE in the early stages of buds development were significantly higher than those in K326 and MSK326.The content of CTK and IAA in MSK326 SE was highest in the early stage of buds development,indicating that stigma exsertion may be related to the increase of cells number.The highest content antioxidant enzyme activities,soluble protein content in the early stage of stigma development of MSK326 SE,indicating that stigma exsertion material physiological activity metabolism,soluble protein accumulation.By observing the cytological characteristics of the flower buds of three tobacco varieties(lines)at a fixed length,the results showed that the length of style cells and style cells of MSK326 SE were significantly smaller than those of K326 and MSK326.The total number of style cells in MSK326 SE was significantly higher than that of K326 and MSK326,indicating that the appearance of stigma head may be related to the increase of the number of style cells.Through the determination of cell division-related genes,hormone-related genes,active oxygen metabolism genes,stress protein genes,and polyamine synthesis genes in the five sampling stages of three tobacco varieties(lines),the experimental results showed that,The expression levels of the genes related to cell division(Nt CYCD3;1,Nt CDKB1-1,Nt ANTL)in the flower buds of MSK326 SE were significantly higher than those of K326 and MSK326 in the early stage of stigma development.The expression of hormone-related genes(Nt PR2,Nt CHN50,Nt EFE26,Nt NCED1)and stress protein genes(Nt ERD10 C,Nt ERD10D)in the flower buds of MSK326 SE was significantly higher than that of K326 and MSK326 in the early stage of stigma development.The expression of active oxygen metabolism genes(Nt GST1,Nt CAT1,Nt APX,and Nt SOD)in MSK326 SE buds was significantly higher than that of K326 and MSK326 in the early stage of stigma development.The expression of polyamine synthesis-related genes(Nt SAMDC,Nt ADC1)in the buds of MSK326 SE was significantly higher than that of K326 and MSK326 in the early stage of stigma development.The cell division-related genes(Nt CYCD3;1,Nt CDKB1-1,Nt ANTL)promote cell cycle progression and the hormone-related genes(Nt PR2,Nt CHN50,Nt EFE26,Nt NCED1)are salicylic acid responsive genes,ethylene biosynthetic genes,and abscisic acid synthesis,respectively.Genes,salicylic acid,ethylene,and abscisic acid can participate in signal transduction in plants and inhibit cell elongation.The high expression of genes associated with cell division(Nt CYCD3;1,Nt CDKB1-1,Nt ANTL)and hormone-related gene hormones(Nt PR2,Nt CHN50,Nt EFE26,and Nt NCED1)in the bud of MSK326 SE during the early stage of stigma development indicated that the cell extension of the stigma exposed material The length is inhibited and the cell division activity is vigorous.Polyamines are involved in the regulation of senescence and flower bud differentiation in plants.High expression of polyamine synthesizing genes(Nt SAMDC,Nt ADC1)in the buds of MSK326 SE during early stigma development suggests that polyamines may be involved in the regulation of stigma exsertion.The high expression of stress protein genes(Nt ERD10 C,Nt ERD10D)and active oxygen metabolism genes(Nt GST1,Nt CAT1,Nt APX,and Nt SOD)in MSK326 SE buds during the early stage of stigma development indicated that the stigma exsertion materials had a strong physiological metabolism activity.Stigma exsertion of MSK326 SE occurs during period 3.stigma exsertion were related to the strong physiological metabolism and the prosperous cell division activities.The appearance of the exposed traits of tobacco stigma may be related to the increase in the number of style cells. |
