Functional Study Of LbMYB5 From Limonium Bicolor On Salt Gland Development And Salt Tolerance

There are approximately 9.6 billion hectares of salinized land in the world and 10%is distributed in China.Soil salinization is one of the major environmental factors limiting crop growth,development and global agricultural production.Halophytes can normally grow,develop and complete their life cycles in saline condition with greater than or equal to 200 m M Na Cl.While non-halophytes such as crops are sensitive to salt stress,and it seriously affects their growth and development,and even leads to death.More and more evidences show that the important ways to develop and to utilize salinized land,to expand the potential of agricultural production and to meet the demand of food are to unravel the molecular mechanism of salt resistance in halophytes,to screen and to identify the key genes of salt resistance,and then to improve the salt resistance ability of crops using biotechnology.Recretohalophyte have developed unique epidermal structures including salt gland and salt bladders to secret excess salt of the body.Salt gland can secrete excessive salt ions out of the plant cell to maintain ion homeostasis and avoid salt damage.However,the molecular mechanism of salt gland development is unknown.Limonium bicolor is an exo-recretohalophyte with multicellular salt glands in the leaf epidermis.In our laboratory,the RNA-seq of the five developmental stages of the first true leaves of L.bicolor（no trichome on the leaves）was performed and a lot of trichome development-related genes was screened and identified,among them,LBMYB5 has the higher relative expression in Stage A（Epidermal cell undifferentiated stage）and Stage B（stage of salt gland differentiation stage）.Moreover,LBMYB5 expression markedly increased after Na Cl and ABA treatments.Therefore,we speculate that LbMYB5 may play an important role in salt gland development and salt tolerance.In this thesis,we cloned the gene of LbMYB5 and studied the functions in salt gland development and salt tolerance.On the one hand,we have obtained the overexpression-and knockout-LbMYB5 or VIGS induced-LbMYB5 silenced lines of L.bicolor through genetic transformation to study the role in salt gland development and salt tolerance.On the other hand,we have obtained Atmyb5,LbMYB5 overexpression and complement lines of Arabidopsis thaliana through heterologous expression to examine the hypothesis of"salt glands and trichome have the common origin".The results indicate that LbMYB5 in deed participates in salt gland development and affects the salt tolerance of L.bicolor,and also affects trichome development of Arabidopsis.The main results are as follows:（1）LbMYB5 cloning and bioinformatics analysisLbMYB5 is 816 bp,encodes272 amino acids and the relative molecular mass is 31.13 k Da.LbMYB5 is hydrophilic protein without signal peptides and transmembrane domain,with two SANT domains,which is the typical R2R3 MYB transcription factor.Subcellular localization indicated that LbMYB5 is in the nucleus.The phylogenetic tree analysis showed that LBMYB5had high homology with AtMYB5 of Arabidopsis thaliana.LbMYB5 has transcriptional activity and the C-terminal determined the transcriptional activation activity.In situ hybridization showed that LbMYB5 mainly expressed in the cells of epidermal structures such as salt glands of L.bicolor.GUS staining showed that LbMYB5 expressed in the epidermal cells of Arabidopsis such as hypocotyl,cotyledon veins,roots and trichome of the true leaves.These results suggest that LbMYB5 is possibly involved in the structure development of plant epidermis such as salt gland.By analyzing the promoter sequence of LbMYB5,we found that LbMYB5 promoter has the cis-acting elements involved in phytohormone and abiotic stress responses in addition to the basic cis-acting regulatory elements.The relative expression levels of LbMYB5 showed a trend of first increases and then decreases under different hormones（ABA,GA₃,IAA,Me JA）and Na Cl treatment,and the relative expression level of Na Cl treatment was the fastest,which increased by 3 times in 3 hours.LbMYB5 highly expressed in early stage of salt gland development and induced by Na Cl and ABA,indicating that it plays an important role in salt gland development and salt tolerance.（2）LbMYB5 affects salt gland development and salt tolerance of L.bicolorIn order to examine whether LbMYB5 participates salt gland development and salt tolerance of L.bicolor,the overexpression and knockout vectors of LbMYB5 were constructed and transformed into agrobacterium to infect L.bicolor leaves to obtain the overexpression and knockout lines of LbMYB5 by hygromycin screening.However,no Chrisper-knockout LbMYB5L.bicolor lines were constructed.Therefore,VIGS-silenced LbMYB5 L.bicolor lines were constructed.The leaf epidermal structure was observed by DIC fluorescence microscopy.The salt glands of wild-type L.bicolor all possess 4 blue self-fluorescence points under 330-380nm UV light.The number of salt glands on the abaxial surface of the wild type leaf is twice of the adaxial surface.However,LbMYB5-overexpression lines showed a large number of abnormal salt glands（two,three or more than 4 blue self-fluorescence points of salt glands）on the leaves,and the number of salt gland complex cells increased or decreased.In particular,a small number of"sole-like salt glands"were found in LbMYB5-overexpression lines where two salt glands are linked to each other.In addition,the number of salt gland per leaf was significantly increased in the leave of LbMYB5-overexpression lines.No abnormal salt glands on the leaves of LbMYB5-Silenced lines were detected but number of salt glands per leaf markedly decreased.The results of q PCR showed that the expression levels of R3 MYB transcription factors LbCPC and LbTRY significantly decreased in LbMYB5-overexpression lines,while the expression levels of trichome development homologous genes LbGL2 and LbSAD2 markedly increased.RNA-seq Analysis of LbMYB5-Silenced lines showed that LbCPC significantly increased and LbGL2 and LbSAD2 markedly decreased.A total of 34 positive clones were obtained from yeast two-hybrid system of LbMYB5.In addition to 11 unknown proteins,most proteins were related to plant growth and development,among which actin and RPA32A were involved in the development of trichome.Many other genes were involved in responding abiotic stress such as cold,drought and salt.Meanwhile,LbMYB5 strongly interacts with Lbb HLH and slightly LbTTG1.These results suggest that LbMYB5 regulates the development of salt glands and salt tolerance of L.bicolor by reducing by promoting the expression of a repressor to reduce the expression levels of LbCPC and LbTRY and by promoting the expression of LbSDA2 and LbGL2.（3）LbMYB5 affects the trichome development of ArabidopsisTo examine whether LbMYB5 also regulates trichome development of Arabidopsis,LbMYB5-overexpression lines,atmyb5 and complement lines of Arabidopsis thaliana were constructed by heteroexpression.Unexpectedly,LbMYB5-overexpression significantly reduced leaf trichome number reflecting in no trichome in the middle part of rosette leaf and a little of trichome on the leaf marrgin compared with wild type.The trichome branches of LbMYB5-overexpression lines were also significantly less than those of wild type.LbMYB5partly restored the trichome phenotype of Atmyb5.However,the trichome phenotype of LbMYB5-overexpression lines on the cotyleoden and hypycotyl is not the same as the phenotype of AtMYB5-overexpression lines in Arabidopsis.These results indicate that LbMYB5 and AtMYB5 have a part role in trichome development.qPCR showed that the expression levels of trichome initiation repressor such as AtCPC,AtETC1 and trichome branching repressor such as AtMYB106 in LbMYB5-overexpression and complement lines of Arabidopsis thaliana lines were all significantly higher than those of wild type and atmyb5.These results suggest that LbMYB5 reduces trichome number by enhancing expression of AtCPC and AtETC1,and inhibits trichome branch by promoting expression of AtMYB106.In summary,the results indicate that LbMYB5 in deed participates in the development of L.bicolor salt gland and Arabidopsis trichome.This further clarified that our hypothesis of"salt glands and trichome have the common origin"However,the molecular mechanisms of LbMYB5regulating development of L.bicolor salt gland and Arabidopsis trichome are different,LbMYB5positively regulates salt gland development and negatively regulates the trichome development.The detail mechanism needs further study.