COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis

Abstract

Background: Trichomes and phenylpropanoid-derived phenolics are structural and chemical protection against many adverse conditions. Their production is regulated by a network that includes a TTG1/bHLH/MYB tri-protein complex in Arabidopsis. CSN5a, encoding COP9 signalosome subunit 5a, has also been implicated in trichome and anthocyanin production; however, the regulatory roles of CSN5a in the processes through interaction with the tri-protein complex has yet to be investigated.

Results: In this study, a new csn5a mutant, sk372, was recovered based on its altered morphological and chemical phenotypes compared to wild-type control. Mutant characterization was conducted with an emphasis on trichome and phenylpropanoid production and possible involvement of the tri-protein complex using metabolite and gene transcription profiling and scanning electron microscopy. Seed metabolite analysis revealed that defective CSN5a led to an enhanced production of many compounds in addition to anthocyanin, most notably phenylpropanoids and carotenoids as well as a glycoside of zeatin. Consistent changes in carotenoids and anthocyanin were also found in the sk372 leaves. In addition, 370 genes were differentially expressed in 10-day old seedlings of sk372 compared to its wild type control. Real-time transcript quantitative analysis showed that in sk372, GL2 and tri-protein complex gene TT2 was significantly suppressed (p < 0.05) while complex genes EGL3 and GL3 slightly decreased (p > 0.05). Complex genes MYB75, GL1 and flavonoid biosynthetic genes TT3 and TT18 in sk372 were all significantly enhanced. Overexpression of GL3 driven by cauliflower mosaic virus 35S promotor increased the number of single pointed trichomes only, no other phenotypic recovery in sk372.

Conclusions: Our results indicated clearly that COP9 signalosome subunit CSN5a affects trichome production and the metabolism of a wide range of phenylpropanoid and carotenoid compounds. Enhanced anthocyanin accumulation and reduced trichome production were related to the enhanced MYB75 and suppressed GL2 and some other differentially expressed genes associated with the TTG1/bHLH/MYB complexes.

Keywords: Anthocyanin; Arabidopsis; COP9 signalosome; CSN5a; MYB75; Trichomes.

Fig. 1

Morphological alterations of the sk372 mutant compared to wild-type plants. a 45-day old plants; b 17-day old plants; c 4-day old plants in MS media containing 4% sucrose; d 4-day old plants grown in the dark; e leaf trichomes of 7-day old sk372 plants; f leaf trichomes of 7-day old WT plants; g, h leaf trichomes of 7-day old sk372 plants under scanning electronic microscopy; i leaf trichomes of 7-day old WT plants under scanning electronic microscopy

Fig. 2

Leaf pigment abundances in 14-day old plants of sk372 and WT. Significant difference (*) (p < 0.05) in the pigment abundance (total abundance for carotenoids) was found between sk372 and WT. All measurements were made from triplicate experiments and the data was analysed by ANOVA for significance (LSD at P < 0.05) using SAS 9.0. viola, violaxanthin; zea, zeaxanthin; cantha, canthaxanthin; crypto, cryptoxanthin

Fig. 3

Transcript levels of genes related to trichome production, flavonoid metabolism, and tri-protein complexes in sk372 mutant relative to WT control. Values for WT were set to an arbitrary number of 1. a Trichome production. b Flavonoid metabolism. c MBW tri-protein complex genes in Arabidopsis seedlings which were germinated and grown for 8 days in the MS media containing 500 ng/mL of the histone deacetylation inhibitor TSA

Fig. 4

Molecular characterization of sk372. a Schematic representation of the CSN5a locus in sk372 showing T-DNA insertion and primer locations. UTRs and exons are indicated by the grey and black boxes, respectively and introns are represented by lines. The CSN5a JAMM/MPN domain is underlined; b CSN5a truncation analysis using cDNA as a template. Lanes 1 and 2, full length coding sequence (938 bp) of CSN5a in WT and sk372, respectively; Lanes 3 and 4, amplified fragment from 162 bp to 886 bp of in WT and sk372; Lanes 5 and 6, amplified fragment starting from bp 1034 to 3′ end in WT and sk372

Fig. 5

Genetic complementation of sk372 with CSN5a. a Transgene (CSN5a) expression in the complemented sk372; b Restored phenotypes of the complemented sk372 plants

Fig. 6

Alteration of metabolites and trichome development in sk372 and their restoration in the CSN5a complementation lines. a Seed metabolites largely comprising phenylpropanoids and flavonoids. Numbers in the brackets after the compounds in the heatmap are the probabilities of relative abundance difference analysis; b Seed carotenoid levels, ‘*’ indicates significantly different from WT, p < 0.05; c Close-up examination of seedling trichomes in sk372 complemented with CSN5a or GL3. WT, non transgenic plant of Col-0; sk372, T-DNA activation tagged mutant; 10b2I, -II, -III, three biological replicates of CSN5a complementation line 10b2 of T3 generation; 2a3, 6a3, 6b2, 6b4, and 8b3, and 8b4, CSN5a complementation lines of T3 generation. Columns with * and ** were significantly different from WT, p < 0.05 and p < 0.01