Acquisition Of SIZ1 And The Mutants Responding To UPR And Its Role In The Salt Tolerance Of Arabidopsis

Under the adverse environments,plants produce more secreted proteins than under normal condition.When the excessive proteins are synthesized but not folded properly in the endoplasmic reticulum(ER),unfolded or misfolded proteins will accumulate in the ER,leading to ER stress,which will activate unfolded protein response(UPR)signaling pathways to restore and maintain homeostasis within ER.Therefore,UPR is crucial for plant responses to abiotic and biotic stresses as well as plant growth and development.In this study,we screened a Arabidopsis mutant,atsiz1,which was sensitive to ER stress and showed the higher expression of UPR marker genes such as Bi P3 by q RT-PCR.To further investigate the roles of SIZ1 in response to ER stress,we crossed siz1 with the typical UPR-related mutants to obtain different mutants for revealing the genetic position of SIZ1 in UPR signaling pathways,including bzip28 bzip60,ire1a ire1b and bzip17.In addition,we found siz1 was also sensitive to salt stress.To investigate the roles of SIZ1 in response to salt stress,we measured some physiological indexes in siz1 compared to wild type,including the content of chlorophyll and the ion ratio of Na⁺/K⁺.Moreover,q RT-PCR was performed to test the expression of salt stress marker genes such as SOS1?HKT1?NHX1 and At P5CS1.The main results are as follows:(1)The siz1 mutant was used as the male parent to hybridize with bzip28bzip60,ire1aire1b,and bzip17 mutants,respectively.After screening by F1 and F2 generations,SIZ1(heterozygous)b ZIP28(homozygous)b ZIP60(homozygous),siz1 ire1a ire1b homozygous triple mutant,SIZ1(heterozygous)b ZIP17(heterozygous).The phenotype of the homozygous triple mutant of siz1 ire1a ire1b showed that the main root length of siz1 ire1a ire1b was significantly restored compared with ire1aire1b.(2)Short-term TM treatment and restoration of wild-type and siz1 mutant strains were performed.The results showed that the siz1 mutant is sensitive to short-term TM treatment.Statistical physiological indicators showed that the short-term TM treatment has a permanent effect on the siz1 mutant.Even when they were restored to culture without TM,the mutants did not grow as well as the wild type.In addition,various physiological indicators showed that fresh shoot weight and chlorophyll were significantly affected by short-term TM treatment.(3)After long-term TM treatment of wild-type and siz1 mutant strains,the siz1 mutant showed higher sensitivity than wild type,and various physiological indicators of the siz1 mutant such as main root length and fresh shoot weight were decreased more that those of wild type.(4)After treatment of 10-day-old wild-type and siz1 mutants with liquid TM,the expression of UPR marker gene BIP3 in siz1 mutant was significantly higher than that of wild-type and showed a trend of first increase and then decrease.(5)Effect of NaCl on the wild-type and siz1 mutant strains of Arabidopsis thaliana using 1/2MS medium(control)and 1/2MS medium containing100 mmol·L^-1 NaCl(NaCl stress)was performed to examine the responses of SIZ1 to salt stress.It is found that under NaCl treatment,siz1mutants were more sensitive than wild type,reflecting in shorter length of the main root and more decreases of the fresh shoot weight,the leaf area and the chlorophyll content.In addition,under salt stress,the Na⁺/K⁺of the siz1 mutant strain is significantly higher than that of wild-type Arabidopsis.These results suggest that the siz1 mutant is more sensitive than wild type to salt stress by regulating the expression levels of SOS1,HKT1,NHX1 and At P5CS1 genes.