Chemically Induced Conditional Rescue of the Reduced Epidermal Fluorescence8 Mutant of Arabidopsis Reveals Rapid Restoration of Growth and Selective Turnover of Secondary Metabolite Pools

Abstract

The phenylpropanoid pathway is responsible for the biosynthesis of diverse and important secondary metabolites including lignin and flavonoids. The reduced epidermal fluorescence8 (ref8) mutant of Arabidopsis (Arabidopsis thaliana), which is defective in a lignin biosynthetic enzyme p-coumaroyl shikimate 39-hydroxylase (C39H), exhibits severe dwarfism and sterility. To better understand the impact of perturbation of phenylpropanoid metabolism on plant growth, we generated a chemically inducible C39H expression construct and transformed it into the ref8 mutant. Application of dexamethasone to these plants greatly alleviates the dwarfism and sterility and substantially reverses the biochemical phenotypes of ref8 plants, including the reduction of lignin content and hyperaccumulation of flavonoids and p-coumarate esters. Induction of C39H expression at different developmental stages has distinct impacts on plant growth. Although early induction effectively restored the elongation of primary inflorescence stem, application to 7-week-old plants enabled them to produce new rosette inflorescence stems. Examination of hypocotyls of these plants revealed normal vasculature in the newly formed secondary xylem, presumably restoring water transport in the mutant. The ref8 mutant accumulates higher levels of salicylic acid than the wild type, but depletion of this compound in ref8 did not relieve the mutant's growth defects, suggesting that the hyperaccumulation of salicylic acid is unlikely to be responsible for dwarfism in this mutant.Phenylpropanoids including flavonoids, hydroxycinnamate esters, and lignin have been shown to play important roles in many aspects of plant growth and development. Flavonoids are important for flower pigmentation and pollen viability in some species (Coe et al., 1981;Mo et al., 1992;Taylor and Jorgensen, 1992;Mol et al., 1998), and sinapate esters, a class of hydroxycinnamate esters found in Arabidopsis (Arabidopsis thaliana) and related members of the Brassicaceae, are important UV protectants (Landry et al., 1995). Lignin is a major component of the plant cell wall, where it confers mechanical strength to plants, and is important for the vascular system to conduct long-distance water transport. Reducing lignin content or manipulating its composition is of great interest in an applied context because of the polymer's negative impact on the utilization of cellulosic biomass for feed, paper manufacture, and biofuel production (Li et al., 2008).The lignin biosynthetic pathway has been largely elucidated during the last two decades (for review, see Bonawitz and Chapple, 2010;Vanholme et al., 2013). In Arabidopsis and other species, down-regulation or mutation of genes and enzymes early in the pathway leads to drastic lignin reduction and a concomitant inhibition of plant growth. For example, knocking out four Phe ammonia-lyase genes (PAL) in Arabidopsis decreases lignin content by 75% and results in stunted and sterile plants (Rohde et al., 2004;Huang et al., 2010). Arabidopsis reduced epidermal...

Transcriptional networks governing plant metabolism

“…Mutating genes within the lignin/phenylpropanoid network frequently lead to plants with diminished growth, which had been presumed to be a side effect of the altered lignin [98]. This hypothesis has begun to be overturned by the application of a suppressor screen wherein Arabidopsis ref8 mutants deficient in p-coumaroylshikimate 3 -hydroxylase (C3 H) was screened for second site mutations that rescued growth of this genotype.…”

“…Key evidence that metabolites and/or their direct consequences have the capacity to generate a feedback loop to regulate transcription and properly coordinate a regulatory network has come from the study of metabolic mutants affecting plant phenylpropanoid metabolism [70,98,99]. Mutating genes within the lignin/phenylpropanoid network frequently lead to plants with diminished growth, which had been presumed to be a side effect of the altered lignin [98].…”

Arabidopsis thaliana β-glucosidase BGLU15 attacks flavonol 3-O-β-glucoside-7-O-α-rhamnosides

“…Alternatively, BGLU15 mediated turnover of flavonol bisglycosides may have important consequences for plant growth and development. Hyper-accumulation of kaempferol bisglycosides, including K3G7R, in an Arabidopsis mutant deficient in the lignin biosynthesis enzyme p-coumaroyl shikimate 3 0 -hydroxylase is associated with dwarfism; transformation of the mutant with a chemically inducible form of this enzyme confers a rapid loss of flavonol bisglycosides, followed by renewed stem inflorescence development due to increased lignin deposition (Kim et al, 2014). (1) and/or Q3G7R (2) levels.…”

“…Notably, peaks identified as flavonol 3-O-b-rutinoside-7-O-arhamnosides are consistent with nomenclature devised by Olsen et al (2009), although the possibility remains for their identification as flavonol 3-O-b-neohesperidoside-7-O-a-rhamnosides (Saito et al, 2013;Yonekura-Sakakibara et al, 2008) since UHPLC-DAD-MS n technology yields identical ion fragments for either conjugate. Kim et al (2014) established that minor degradation of kaempferol bisglycosides occurs during the development of the rosette habit stage in Arabidopsis when cultivated under non-stressed conditions. No such phenomena were observed here, as flavonol bisglycoside levels remained low and stable under constant NSHT (control; Fig.…”

Advances in Genetic Manipulation of Lignocellulose to Reduce Biomass Recalcitrance and Enhance Biofuel Production in Bioenergy Crops

“…For example in transgenic maize have been reported that overexpression of GH11 (xynB) gene lead to enhances biomass enzymatic digestibility [93], while in poplar have showed that GH10 (xyn10A) gene by RANi silencing increasing biomass yield [94].…”