Search

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.

The SCF complex is a widely studied multi-subunit ring E3 ubiquitin ligase that tags targeted proteins with ubiquitin for protein degradation by the ubiquitin 26S-proteasome system (UPS). The UPS is an important system that generally keeps cellular events tightly regulated by purging misfolded or damaged proteins and selectively degrading important regulatory proteins. The specificity of this post-translational regulation is controlled by F-box proteins (FBPs) via selective recognition of a protein-protein interaction motif at the C-terminal domain. Hence, FBPs are pivotal proteins in determining the plant response in multiple scenarios. It is not surprising that the FBP family is one of the largest protein families in the plant kingdom. In this review, the roles of FBPs, specifically in plants, are compiled to provide insights into their involvement in secondary metabolites, plant stresses, phytohormone signalling, plant developmental processes and miRNA biogenesis.

Alcohol dehydrogenases exist as a multigene family. Two isogenes, PmADHa and PmADHb were obtained from Persicaria minor via RACE-PCR were highly identical at the 5′-UTR and ORF region and highly divergent at the 3′-UTR. With the length of 1077 and 1163 bp respectively, PmADHa and PmADHb have 801 bp ORF length, encodes 266 amino acid residues. Four nucleotides are different between the two sequences in the ORF and caused only difference one of amino acid residue at the 107th position. PmADHa and PmADHb are categorized in short-chain dehydrogenase/reductase superfamily of Unassigned type, specifically in AT3G01980.1 family. Investigations of expression profile for both genes were conducted under the treatment of 100 μM exogenous ABA and drought stress using 20% PEG-8000 in leaves, stems and roots. Exogenous ABA treatment showed that the expression of PmADHa and PmADHb were highly up-regulated only in roots. Whereas, PmADHa and PmADHb showed up-regulation in response to drought stress in all organ tested. These indicate that PmADHa and PmADHb are involved in the ABA and drought signalling pathways. Peroxisome was predicted for the subcellular localization of PmADHa and PmADHb. The promoter analysis revealed that several stress-related elements were found in the promoter of PmADHa and PmADHb and these further strengthen its relation in the stress responses.

Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.