| Limonium bicolor belongs to the Limonium of the Plumbaginaceae,and is a dicotyledonous salt-secreting halophyte with a multicellular salt gland structure.The salt glands will directly secrete excess salt absorbed into the body of the body,allowing for normal growth and development in saline alkali soil and completing life cycles.All nonhalophytes and crops do not have salt glands.Therefore,studying the molecular mechanism of salt gland development has significant theoretical significance and application value for revealing the development of epidermal cells of Limonium bicolor and the transformation of nonhalophytic plants,in order to increase the salt tolerance of crops.By studying the developmental stages and distribution patterns of trichomes in nonhalophytic plants such as Arabidopsis and tomato,as well as the salt glands of Limonium bicolor,it is speculated that the salt gland and trichome may have similar developmental molecular mechanisms.Numerous studies have shown that the C2H2 zinc finger protein transcription factors play a crucial role in determining the fate of plant trichome and root hair development.We identified a C2H2 type zinc finger protein gene with high expression specificity in the early stage of salt gland development,and found that this gene is a homologous gene to At ZFP5,a key gene controlling the development of Arabidopsis trichome,and named it LbZFP5.It is speculated that this gene may play a crucial role in the development of salt glands in Limonium bicolor.We have studied the role of this gene in the development of salt glands and salt resistance of plants in Limonium bicolor and Arabidopsis.The main results are as follows:1)The expression pattern of LbZFP5 was analyzed through bioinformatics and molecular biology.Subcellular localization showed that LbZFP5 was localized in the nucleus,and in situ hybridization revealed that LbZFP5 was specifically overexpressed in the salt gland.The transcriptional activation activity validation experiment found that LbZFP5 had no transcriptional activation activity.2)LbZFP5 overexpression vector and VIGS gene silencing vector were constructed and transformed into Limonium bicolor.It was found that the LbZFP5 overexpression lines showed a significant increase in salt gland density and total volume of salt gland secretion,while the salt secretion rate of a single salt gland did not change significantly;after the LbZFP5 was silenced via VIGS,the density of salt glands and the total volume of salt gland secretion significantly decreased,but the salt secretion rate of a single salt gland did not change significantly;the detection of salt tolerance physiological indicators indicated that the LbZFP5 overexpression lines significantly improved the salt resistance of Limonium bicolor,while the silent lines significantly decreased its salt tolerance.3)Yeast two-hybrid experiments found that LbZFP5 interacted with the eukaryotic translation initiation factor Lbe IF3 h and calcineurin B-like protein Lb CBL2 of Limonium bicolor.The RNA-seq,yeast one-hybrid,and EMSA experiments showed that LbZFP5 directly and negatively regulates the expression of downstream genes Lb CPC-Like and Lb ETC1,controlling the development of salt glands.Further VIGS experiments found that Lb CPC-Like and Lb ETC1 are negative regulatory factors for salt gland development,salt secretion,and salt tolerance in Limonium bicolor.4)The LbZFP5 overexpression Arabidopsis lines has the phenotype of significantly increased leaf trichome,decreased root hair,and improved salt tolerance.Further,yeast one-hybrid experiments demonstrated that LbZFP5 may positively regulate the development of trichome by inhibiting the expression of TRY;Yeast two-hybrid,yeast one-hybrid,and EMSA experiments showed that LbZFP5 may inhibit CPC expression and negatively regulate root hair development through its interaction with Ate IF3h;RNA-Seq analysis showed that LbZFP5 positively regulates Arabidopsis salt tolerance through regulating the expression of target genes of SOS and ABA signaling pathway.These results showed that Limonium bicolor gene LbZFP5 plays an important role in the development of plant epidermal structures such salt gland,trichome and root hair and plant salt tolerance. |
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