In this study, we isolated a WD40-repeat gene from Artemisia annua glandular trichomes. This gene shows 69.97% sequence similarity to Arabidopsis TTG1 at aminoacid level. Sub-cellular localization study shows that AaWD40 protein diffuses in both cell nucleus and cytosol. The correct nuclear localization of AaWD40 was observed when co-expressed with AabHLH, a putative A. thaliana AtTTG1 homologue cloned from Artemisia annua glandular trichomes. When AaWD40 gene was ectopically over expressed in Arabidopsis transparent testa glabrous1 -1 ( ttg1 -1) mutants of A. thaliana, PAs production in seeds was restored, and the trichomeless phenotypes of mutant were rescued. Real-time PCR analysis results revealed that ETC1, CPC, TTG2 and BAN (the downstream targets of AtTTG1 depend on regulatory complex), which regulate the epidermal differentiation and anthocyanin biosynthesis were differentially expressed as a result of AaWD40 over expression. Furthermore, the CLV1, CLV2, CLV3 and WUS, which are required to maintain the stem-cell niche of Arabidopsis shoot apex, were also modulated by AaWD40 and Arabidopsis TTG1. The transcriptions of AP2/ERF, bHLH, MYB, WRKY and NACs family proteins, which are mostly involved in defense, stress response and development regulation, were remarkably modulated by AaWD40 over expression. We hypothesize that WD40 repeat proteins act as a crucial factor in regulating a wide variety of cellular functions in A. thaliana.
TWD repeat (WDR) proteins are characterized by 4 - 16 tandem WD (also called Trp-Asp or WD-40) motifs, which comprise 44 - 60 residue sequence typically delineated by the Gly-His (GH) dipeptide 11 - 24 residues from its N-terminus and ending in Trp-Asp (WD) dipeptide. In between GH and WD, a core sequence locates, which exhibits only limited sequence conservation [
In this work, we focus on functional study of an A. annua WD40 repeat gene, AaWD40, using cell biology, molecular biology, and functional genomics approach. We found that over-expression of AaWD40 can fully rescue the trichomeless and PA phenotype in Arabidopsis ttg1-1 mutant. Subcellular localization study suggested that the translocation of AaWD40 into nuclear requires the assistance of bHLH family protein. Ectopic over expression of AaWD40 in Arabidopsis altered the expression of CLV1, CLV2, CLV3 and WUS transcripts, which are required to maintain the stem-cell niche of Arabidopsis shoot apex. Finally, microarray analysis revealed the possible new function of AaWD40 and Arabidopsis TTG1 in regulating plant development and response to biotic and abiotic stress.
Gene tag (Contig24501) encoding a putative WD40 protein was identified from an in-house-generated A. annua glandular trichome EST library. Tissue specific expression analysis showed that this contig was preferentially expressed in filamentous trichomess and roots, also in glandular trichomes and leaves, although with lower abundance [
Unlike most nuclear-bound WD40 proteins, no typical nuclear localization signal (NLS) was detected in the N-terminal region of AaWD40 using PSORT. In root tip cells of transgenic Arabidopsis plants over expressing 35S::AaWD40::GFP, strong fluorescent signals were detected in both nuclear and cytoplasmic were detected in both nuclear and cytoplasmic membrane (
nal was detected in both cytoplasm and nuclear. This result is somewhat contradictory to previous report that AtTTG1 was mostly located in the nuclear region [
Because AaWD40 shares high sequence similarity with Arabidopsis TTG1, we first examined if they are functional homologs using complementation test. The mutation of TTG1 results in severe defects in anthocyanin pigmentation, seed coat pigmentation, seed coat mucilage, root hair positioning, and trichome differentiation. When AaWD40 (35S::AaWD40::GFP fusion) was transformed into Arabidopsis ttg1-1 mutant, PA production in seeds was fully restored, and the trichomeless phenotypes was rescued (
In Arabidopsis, TTG1, GL1 and GL3 forms a trimericregulatory complex to activate the expression ofGLABRA2 (GL2), a homeo-domain Zip (HD-Zip) transcription factor, and TRANSPARENT TESTAGLABRA2 (TTG2), a WRKY factor [
Arabidopsis seeds are purchased from the Arabidopsis Biotechnology Resource Centre (Ohio State University). All seeds used in this study were newly harvested (within 3 months) and stored in airtight tubes after drying at 4˚C.Seeds were sterilized and germinated on MS Basal Medium (SigmaM5519) with 1% sucrose and 0.8% Agar. The plants were placed at 22˚C under long day condition (16 h light/8 dark). AaWD40 was isolated from A. annua by PCR (forward primer, 5’-AAAGCTTAACCGAGAATGTCTCCCGACTTCTAT-3’; reverse primer, 5’-AGTCGACTCAAACTCTAAGGAGCT GCATTT G-3’). Seedlings were harvested for real-time PCR analysis using SYBR green probeat 7 day following germination unless otherwise stated. The primers used are: Imaging of YFP fusions was performed on a FluoViewTM FV1000 confocal laser scanning biological microscope laser scanning microscope with excitation (488 or 514 nm) and emissions (530 - 600 nm for GFP and 675 - 800 for RFP). Collected images were processed for maximum intensity projection. Arabidopsis cell suspension cultures (ecotype Landsbergerecta) PSB-L were grown in 250 ml flasks at 22˚C in a 16 h light/8 h dark cycle and shaked at 130 r.p.m for 3 days. Cells were sub-cultured in MS medium supplemented with 4.3 g/L Murashige and Skoog Basal Salt Mixture, 100 mg/liter myo-inositol, 0.4 mg/liter (L?) thiamine hydrochloride, 50 mg/liter kinetin, 800 mg/liter 1-naphthaleneacetic acid and 30 g/(adjust pH to 5.7 with KOH). Sub-culture was conducted by transferring 5 ml of old cells into 45 ml of fresh Arabidopsis MS medium every 5 days. The cultures obtained on the 3rd day after sub-culture was used for protoplast preparation.
Transient expression of fluorescent fusion proteins in protoplasts of suspension cell cultures were performed as described by Miao etc. [
Imaging of YFP fusions was performed on Olympus FluoViewTM FV1000 confocal laser scanning biological microscope. Laser scanning microscope with excitation at 488 or 561 nm and emissions at 500 - 540 nm for GFP and 580 - 630 for RFP. Collected images were processed for maximum intensity projection.
In this study, we conducted functional analysis of a WD40 repeat protein (AaWD40) in Arabidopsis. Interestingly, in root tip cells of transgenic Arabidopsis plants over expressing 35S::AaWD40::GFP, strong fluorescent signals were detected in both nuclear and cytoplasmic membrane. Coexpression of AtTTG1 and AaWD40 resulted in an obvious nuclear pattern. This suggests that the protein-protein interactions between WD40-repeat protein and bHLH protein may facilitate their translocation into nucleus. The fact that ectopically over expressed AaWD40 can rescue the trichomeless phenotypes of Arabidopsis mutant indicates that besides the critical roles in regulation of trichome formation and anthocyanin biosynthesis, the AaWD40 is likely involved in multiple cellular functions in Artemisia annua.
This work was supported by grants from Hong Kong UGC Research Grant Council the Hong Kong Special Administrative Region, China (Project no. CUHK 465110).
Wei Wang,Qing Zhang,Dianjing Guo, (2016) An Artemisia WD40-Repeat Gene Regulates Multiple Cellular Functions in Arabidopsis. Journal of Biosciences and Medicines,04,30-36. doi: 10.4236/jbm.2016.45003