1. Muhammad Naeem-ul-Hassana, Ismanizan Ismaila, b, Zamri Zainala,b
To study the role of F-box proteins in general and SKIP11 specifically in the metabolism and development of Arabidopsis.
To analyze the effects of impaired SCF complex , i.e., due to depletion of SKIP11, the “molecular glue”, on the growth and development of Arabidopsis.
To identify the “missing link” that enhance or reduce the protein-protein interactions by using known substrates.
OBJECTIVES
Protein degradation is a common regulatory process in multi-cellular organisms to control various metabolic pathways. Specific target proteins, destined for degradation are first labelled by a tag for recognition. Ubiquitination of target proteins is one of the labelling process by which target proteins undergo repeated Ubiquitin conjugation to give polyubiquitin and subsequently degraded by 26 proteasome complex. SCF is the largest and the best characterized of all E3 complexes studied so far. SCF complex is composed of four proteins including SKP1, Cullin 1, Ring box protein and an F-box protein (1).
F-box proteins, with a conserved F-box domain and one or more highly variable secondary motifs, are an essential component of SCF complex (2). F-box proteins have been reported to participate in many regulatory pathways of different physiological phenomena in plants (3, 4). Role of most of these proteins in molecular mechanisms are still unexplored. SKIP11 is an Arabidopsis F-box protein, containing 467 amino acids and with two kelch-repeat motifs.
In the present study seeds of T-DNA inserted skip11 mutant lines were obtained from uNASC. Homozygous lines were screened and the growth pattern and metabolic profiling analysis was compared with WT plants.
INTRODUCTION
Role of SKIP11 in Seed germination and development of Arabidopsis thaliana
Muhammad Naeem-ul-Hassana, Ismanizan Ismaila, b, Zamri Zainala,b
aDepartment of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor
bInstitute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia
RESULTS
GC-MS Analysis of VOCs
Some esters of long chain carboxylic acids were identified by library search and matching. The retention time of these compounds on the non polar column was between 38 and 39 minutes in both WT and mutant plants, though the compounds were different.
This preliminary study on the role of SKIP11 in the growth and development of Arabidopsis presented some very valuable information. Especially, the seed germination stage and first 6 weeks of plant development are important to be considered. Germination of 60% of the seeds show that SKIP11 has some significant, though not vital role at this stage. While, during the development stage the role of this F-box protein cannot be ignored as only 40% of the seeds spread crossed the 6 weeks of age. However, the protein does not seem to be involved in the development of the plants after 6 weeks, because there was no difference in the development of mutant plants and the WT plants. In future a more detailed and comprehensive study is needed for the evaluation of SKIP11 protein.
CONCLUSION
1. Petroski, M.D. and Deshaies, R.J Function and regulation of cullin-RING ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 6, 9–20.
2. Sun, Y., Zhou, X., & Ma, H Genome-wide analysis of Kelch repeat-containing F-box family. ournal of integrative plant biology, 49: 940–952.
3. Kipreos E, T., & Pagano, M The F-box protein family. Genome Biology, 1: –
4. Lechner, E. e. a F-box proteins everywhere. Current Opinion in Plant Biology, 9:
REFERENCES
2 Weeks
WT SKIP11 mutants
WT SKIP11 mutant
6 weeks
Screening of plants for homozygous lines
Two plants were found with T-DNA insertions in both chromosomes by running the PCR products on 1% agarose gel electrophoresis of gDNAs of 6 weeks old plants. A single band of 700bps represents a homozygous mutant.
Growth pattern comparison
Forty seeds spread for each WT and SKIP11 mutant plants in different pots. Most of the mutant seeds could not grow, some seedlings died before the age of 2 weeks and a few could not survive till 6 weeks.
Figure: Comparison of plant growth pattern, WT vs SKIP11 mutants
Figure: Comparison of VOCs from WT plants vs SKIP11 mutant plants
Figure: Electrophoretograms of PCR products of gDNAs from mutant plants with WT
MATERIALS AND METHODS
Seed germination and plant growth
Seed stock number “SALK_ x” containing T-DNA insertion in the SKIP11 region of chromosome 2 was searched from the website of uNASC. 40 seeds were spread in two 4” X 4” pots with commercial soil and vermiculite and the plants grown.
Screening of Homozygous plants
Leaf material (about 100mg) was used to isolate the gDNA from each plant with Qiagen kit. Using primers specific for this mutant line, PCR of gDNA was run and checked by 1% agarose gel electrophoresis and visualized by Sony gel-doc system.
Growing and Analysis of Homozygous Plants
Seeds of homozygous plants were placed in pots, plants grown for 4 to 6 weeks along with wild type plants. 2gm of leaves were cut and analyzed by GC-MS in splitless mode on non-polar column.
ACKNOWLEDGEMENT
Figure: Polyubiquitation via SCF A representative F-box protein
This work was funded by Research University Grant “GUP ”