A link between magnesium-chelatase H subunit and sucrose nonfermenting 1 (SNF1)-related protein kinase SnRK2.6/OST1 in Arabidopsis guard cell signalling in response to abscisic acid

Highlight A sucrose nonfermenting 1 (SNF1)-related protein kinase 2, SnRK2.6/ open stomata 1 (OST1), which plays critical role in abscisic acid (ABA) signalling in Arabidopsis guard cells, interacts directly with, and functions downstream of, the magnesium-chelatase H subunit in guard cell signalling in response to ABA.

7.4) was injected over flow cell 1 and flow cell 2 at different concentrations (500 nM, 50 nM, 125 nM, 62.5 nM, 31.25 nM) using glycine solution (pH 2.5) for regeneration. Data were collected and globally fitted to steady-state model available within Biacore Evaluation software v1.01.

Yeast one-hybrid assays
Yeast one-hybrid assays were performed as previously described (Shang et al., 2010). The yeast strain AH109 was chosen to be host strain. The primers used for constructing the related plasmids were listed in supplemental table 1.
Open reading frame of OST1, ABF4, ABI5 and cDNA sequence encoding the C-terminal half of ABAR (amino acid residues from 692 to 1381, ABAR c690 ) were constructed into pGADT7 prey vector, whereas the promoter fragment of RbohD, RbohF, GPX1, GPX2, GPX5, CAT1, CAT2 and CAT3 were cloned into the bait vector pHIS2. Protein linked pGADT7 prey vector and promoter linked pHIS2 bait vector were co-transformed into yeast cells. Yeast cells co-transformed with the empty pGADT7 vector and pHIS2 harbouring the corresponding promoter were used as negative controls. Yeast cells co-transformed with P53-pGADT7 and pHIS2 harbouring the promoter of P53 was used as positive control. Transformed yeast cells were first grown in SD-2 medium (lacking tryptophan and leucine) for 3 days to ensure that the yeast cells were successfully co-transformed and positive clones were cultured in SD-2 liquid medium overnight to OD600 of 0.1 and diluted in a 10× dilution series. 8μL yeast cells was dropped on SD-2 and SD-3 medium (lacking Trp, Leu, His) supplemented with different concentrations (20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 120 mM) of 3-aminotriazole (3-AT; Sigma). The plates were then incubated for 3 d at 30 °C prior to photograph.   prey vector harboring open reading frame (ORF) of OST1 or ABAR c690 coding for the C-terminal of ABAR, and pHIS2 bait vector harboring promoter sequence of RbohD, RbohF, GPX1, GPX2, GPX5 or CAT2 grew well in SD-2 medium and failed to grow in SD-3 medium with 40 mM 3-AT. These data suggest that neither OST1 nor ABAR c690 binds to the promoter of these genes. (B) Yeast cells co-transformed with pGADT7 prey vector harboring ORF of ABI5, ABF4, and pHIS2 bait vector harboring promoter sequence of RbohD, RbohF, GPX1, GPX2, GPX5, and CAT2 grew well in SD-2 medium, and failed to grow in SD-3 medium, suggesting that neither ABI5 nor ABF4 binds to the promoter of these genes.
Yeast cells co-transformed by the combinations of empty pGADT7 vector, OST1, ABAR c690 ABI5 or ABF4, with promoters of CAT1,CAT3, respectively, grew in SD-3 medium even in the presence of 120 mM 3-AT, due to the self-activation phenomenon in this yeast one hybrid system with these combinations (A, B). Therefore, it cannot be determined with the yeast one hybrid system whether OST1, ABAR c690 , ABI5 or ABF4 interact with the promoters of CAT1 or CAT3.
Transformation with empty vectors pGADT7 and pHIS2 harboring the corresponding promoters were used as negative controls. Given that P53 protein binds to its own promoter, co-transformation of pHIS2 harboring the promoter of P53 with pGADT7 harboring P53 (below the red lines, p53 control , +) were used as positive control.
Transformation of pHIS2 harboring the promoter of P53 with pGADT7 harboring P53 or empty pGADT7 were used as negative control (below the red lines, p53 control , -). All the experiments were repeated three times with the same results.