Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by
the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box
protein at the C-terminal domain functions to recognize the targeted protein to be ubiquitinated and degraded via UPS.
A stress-responsive F-box gene, PmF-box1 from Persicaria minor was categorized in the F-box containing kelch repeat
(FBK) family; a family that specific to plant kingdom. To identify the targeted protein of PmF-box1, yeast-two hybrid system
(Y2H) was used. In the Y2H screening process, mating efficiency is very important to fish out the interacting proteins.
Therefore, one modification was conducted to increase the mating efficiency. In this screening, PmF-box1 was used as a
bait to screen for the Y2H library which was constructed using RNA from plant samples treated with abscisic acid (ABA)
and polyethylene glycol (PEG)-8000 and control sample. Autoactivation and toxicity tests of bait were performed before
the Y2H screening. Tests on PmF-box1 showed that it is not toxic to the yeast and cannot autoactivate the yeast reporter
genes. Mating efficiency was improved from 2.07% to 9.15% after addition of PEG-4000 in the mating culture compared
to the original protocol, which it also increased the colony number in the screening step afterward. Additionally, bands
of gene with different sizes were observed on electrophoresis gel after colony PCR analysis from the improved technique.
Those genes may code for potential interacting proteins that needs further identification and confirmation.
F-box proteins containing variable C-terminal domains make an essential part of SKP1-Cullin-Ring box-F box (SCF)
complex. SCF complex catalyzes the final step to link the ubiquitin tag with the target protein, destined for degradation,
through F-box protein that confer overall substrate specificity to the complex. In this study, we analyzed the role of
At2g02870, a Kelch containing F-box protein from Arabidopsis thaliana, by using reverse genetics strategy. At2g02870
loss of function mutant lines (at2g02870) were analyzed and compared with wild type plants for the expression of genes
and products of hydroperoxide lyase (HPL) branch of oxylipin pathway. We found that the at2g02870 plants have enhanced
expression of HPL pathway genes and produce more green leaf volatiles (GLV) than the wild type plants. Our results
suggested that the gene is involved in the regulation of HPL pathway, possibly through the degradation of enzymes or/
and the regulatory factors of the pathway.