Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species.

Flavonoid metabolons (weakly-bound multi-enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein-protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4-reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3'-hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage-dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co-suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long-suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.

Evaluation of the effectiveness and salt stress of Pteris vittata in the remediation of arsenic contamination caused by tsunami sediments.

On March 11, 2011, one of the negative effects of the tsunami phenomenon that devastated the Pacific coast of the Tohoku district in Japan was the deposition of a wide range of arsenic (As) contamination to the soil. To remediate such a huge area of contamination, phytoremediation by Pteris vittata, an As-hyperaccumulator, was considered. To evaluate the efficacy of applying P. vittata to the area, the salt tolerance of P. vittata and the phytoextraction of As from soil samples were investigated. For the salt tolerance test, spore germination was considerably decreased at an NaCl level of more than 100 mM. At 200 mM, the gametophytes exhibited a morphological defect. Furthermore, the growth inhibition of P. vittata was observed with a salinity that corresponded to 66.2 mS/m of electric conductivity (EC) in the soil. A laboratory phytoremediation experiment was conducted using As-contaminated soils for 166 days. P. vittata grew and accumulated As at 264 mg/kg-DW into the shoots. Consequently, the soluble As in the soil was evidently decreased. These results showed that P. vittata was applicable to the phytoremediation of As-contaminated soil with low salinity as with the contamination caused by the 2011 tsunami.

Characterization of As efflux from the roots of As hyperaccumulator Pteris vittata L.

In some plant species, various arsenic (As) species have been reported to efflux from the roots. However, the details of As efflux by the As hyperaccumulator Pteris vittata remain unknown. In this study, root As efflux was investigated for different phosphorus (P) supply conditions during or after a 24-h arsenate uptake experiment under hydroponic growth conditions. During an 8-h arsenate uptake experiment, P-supplied (P+) P. vittata exhibited much greater arsenite efflux relative to arsenate uptake when compared with P-deprived (P-) P. vittata, indicating that arsenite efflux was not proportional to arsenate uptake. In the As efflux experiment following 24 h of arsenate uptake, arsenate efflux was also observed with arsenite efflux in the external solution. All the results showed relatively low rates of arsenate efflux, ranging from 5.4 to 16.1% of the previously absorbed As, indicating that a low rate of arsenate efflux to the external solution is also a characteristic of P. vittata, as was reported with arsenite efflux. In conclusion, after 24 h of arsenate uptake, both P+ and P- P. vittata loaded/effluxed similar amounts of arsenite to the fronds and the external solution, indicating a similar process of xylem loading and efflux for arsenite, with the order of the arsenite concentrations being solution ≪ roots ≪ fronds.

Effects of cultivation conditions on the uptake of arsenite and arsenic chemical species accumulated by Pteris vittata in hydroponics.

The physiological responses of the arsenic-hyperaccumulator, Pteris vittata, such as arsenic uptake and chemical transformation in the fern, have been investigated. However, a few questions remain regarding arsenic treatment in hydroponics. Incubation conditions such as aeration, arsenic concentration, and incubation period might affect those responses of P. vittata in hydroponics. Arsenite uptake was low under anaerobic conditions, as previously reported. However, in an arsenite uptake experiment, phosphorous (P) starvation-dependent uptake of arsenate was observed under aerobic conditions. Time course-dependent analysis of arsenite oxidation showed that arsenite was gradually oxidized to arsenate during incubation. Arsenite oxidation was not observed in any of the control conditions, such as exposure to a nutrient solution or to culture medium only, or with the use of dried root; arsenite oxidation was only observed when live root was used. This result suggests that sufficient aeration allows the rhizosphere system to oxidize arsenite and enables the fern to efficiently take up arsenite as arsenate. X-ray absorption near edge structure (XANES) analyses showed that long-duration exposure to arsenic using a hydroponic system led to the accumulation of arsenate as the dominant species in the root tips, but not in the whole roots, partly because up-regulation of arsenate uptake by P starvation of the fern was caused and retained by long-time incubation. Analysis of concentration-dependent arsenate uptake by P. vittata showed that the uptake switched from a high-affinity transport system to a low-affinity system at high arsenate concentrations, which partially explains the increased arsenate abundance in the whole root.

Production of tetraketide lactones by mutated Antirrhinum majus chalcone synthases (AmCHS1).

Chalcone synthase (CHS) is a key enzyme of flavonoid biosynthesis in higher plants, catalyzing the stepwise decarboxylative condensation of three acetate units from malonyl-CoA with p-coumaroyl-CoA to yield 2',4,4',6'-tetrahydroxychalcone (THC). Reaction (at pH 7.5) of a mutant (V196M/T197A) of Antirrhinum majus CHS (AmCHS1) with p-coumaroyl-CoA and malonyl-CoA yielded a significant amount of a non-chalcone product, along with a small amount of THC. The non-chalcone product was identified as p-coumaroyltriacetic acid lactone (CTAL), a tetraketide lactone produced due to derailment from the canonical THC-producing reaction pathway. In vitro, the wild-type AmCHS1 showed low CTAL-producing activity at pH 7.5, but an appreciable level at pH 10. Each of the amino acid substitutions, V196M, T197A and V196M/T197A, caused a shift toward neutrality of the optimum pH for CTAL-producing activity. The V196M substitution resulted in a loss of THC-producing activity, as well as a 12.6-fold enhancement of CTAL-producing activity (at pH 7.5); hence, AmCHS1 was converted to a p-coumaroyltriacetic acid synthase by this single amino acid substitution. The THC-producing activity of the V196M mutant appeared to be restored by additional T197A substitution, although a single T197A substitution caused no substantial enhancement of the CTAL-producing activity of the wild-type enzyme. The enhancement of the tetraketide producing activity upon V196M and V196M/T197A substitutions was most markedly observed when p-coumaroyl-CoA was used as the starter substrate, and only slightly with benzoyl-, caffeoyl- and hexanoyl-CoAs. These results show the importance of the two contiguous amino acids at positions 196 and 197 for product specificity of an AmCHS1-catalyzed reaction.

Localization of a flavonoid biosynthetic polyphenol oxidase in vacuoles.

Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+-translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.