Ken G. Ryan

An increase in the luteolin : Apigenin ratio in Marchantia polymorpha on UV-B enhancement

Abstract The effects of varying the UV-B component in ambient light on the liverwort, Marchantia polymorpha are reported. Plants grown under conditions of ambient light, ambient light lacking UV-B, and ambient light with a 25% enhancement of incident UV-B showed, with increasing levels of UV-B, a decrease in growth rate, a decrease in the production of gemmae cups and an increase in the proportion of dead thallus. Thallus surviving after three months of summer growth under these conditions showed no statistically significant increase in flavonoid levels with increasing UV-B levels. However, HPLC monitoring of individual flavonoids (all of which are apigenin and luteolin glucuronides) revealed a strong correlation between increasing UV-B levels and an increase in the ratio of luteolin to apigenin glycosides. It is considered unlikely that this change has significantly altered the UV-B screening effectiveness of the flavonoids. Rather, an improved level of antioxidant defence, or a more effective dissipation of absorbed UV energy, are proposed as possible UV-B protectant benefits to the plant.

UVB RADIATION INDUCED INCREASE IN QUERCETIN : KAEMPFEROL RATIO IN WILD-TYPE AND TRANSGENIC LINES OF PETUNIA

The use of genetically modified plants offers unique opportunities to study the role of specific flavonoids in plant UVB protection. Along with a parental wild‐type Mitchell Petunia, two transgenic lines with altered flavonoids were also examined; Lc with enhanced levels of antho‐cyanins due to the action of a maize flavonoid regulatory gene Leaf color, and AFLS that carries an antisense fla‐vonol synthase construct and is known to have reduced flavonol levels in flowers. All three lines were grown in near ambient sunlight, sunlight lacking UVB (280–320 nm) radiation and sunlight with 25% added UVB. Ultra‐violet‐B radiation induced significant reductions in the rates of leaf expansion and seedling growth in all three lines. The presence of anthocyanins did not appear to afford Lc plants any special protection from UVB. Ul‐traviolet‐B treatment induced increases in total flavonol content in young plants of all three lines, and this effect decreased with increasing leaf age. Notably, increasing UVB levels led to an increase in the ratio of quercetin: kaempferol with all three cultivars. The AFLS transgenic, contrary to expectations based on its genetic construction, had normal levels of flavonols in the leaves and the highest Q:K ratio of the three cultivars. This transgenic was the least susceptible to UVB, which may indicate an enhanced protective role for quercetin. Because both quercetin and kaempferol have similar UVB screening properties, quercetin may exert this role by other means.

Flavonoids and UV photoprotection in Arabidopsis mutants.

Abstract Wild-type Arabidopsis L. leaves exposed to low ultraviolet-B (U V B ) conditions contained predominantly kaempferol glycosides, with low levels of quercetin glycosides. The flavonoid level doubled on treatment with UVB and an increase in the ratio of quercetin: kaempferol was observed. These results suggest that flavonols protect Arabidopsis plants from UVB damage, and indicate that the flavonoid 3’-hydroxylase (F3’H) enzyme, which converts dihydrokaempferol to dihydroquercetin, may play a crucial role. The tt7 mutant lacks this gene and, after treatment with sub-ambient UVB, contained kaempferol glycosides exclusively, to a level of total flavonols similar to that in wild-type Arabidopsis. Total flavonols after enhanced UVB treatment were higher in tt7 than in similarly treated wild-type plants, and only kaempferol glycosides were detected. Despite this high level, tt7 plants were less tolerant of UVB radiation than wild-type plants. These observations suggests that kaempferol is a less effective photoprotectant than quercetin. The chalcone isomerase (CHI) mutant (tt5) surprisingly did not accumulate naringenin chalcone, and this suggests that the mutation may not be restricted to the CHI gene alone. The concentration of hydroxycinnamic acid derivatives did not change with UVB treatment in most varieties indicating that their role in UV photoprotection may be subordinate to that of the flavonoids.

Short-term effect of temperature on the photokinetics of microalgae from the surface layers of Antarctic pack ice

Microalgae growing within brine channels (85 psu salinity) of the surface ice layers of Antarctic pack ice showed considerable photosynthetic tolerance to the extreme environmental condition. Brine microalgae exposed to temperatures above −5°C and at irradiances up to 350 μmol photons·m−2·s−1 showed no photosynthetic damage or limitations. Photosynthesis was limited (but not photoinhibited) when brine microalgae were exposed to −10°C, provided the irradiance remained under 50 μmol photons·m−2·s−1. The highest level of photosynthetic activity (maximum relative electron transport rate [rETRmax]) in brine microalgae growing within the surface layer of sea ice was at approximately 18 μmol electrons·m−2·s−1, which occurred at −1.8°C. Effective quantum yield of PSII and rETRmax of the halotolerant brine microalgae exhibited a temperature‐dependent pattern, where both parameters were higher at −1.8°C and lower at −10°C. Relative ETRmax at temperatures above −5°C were stable across a wide range of irradiance.

Proteorhodopsin-Bearing Bacteria in Antarctic Sea Ice

ABSTRACT Proteorhodopsins (PRs) are widespread bacterial integral membrane proteins that function as light-driven proton pumps. Antarctic sea ice supports a complex community of autotrophic algae, heterotrophic bacteria, viruses, and protists that are an important food source for higher trophic levels in ice-covered regions of the Southern Ocean. Here, we present the first report of PR-bearing bacteria, both dormant and active, in Antarctic sea ice from a series of sites in the Ross Sea using gene-specific primers. Positive PR sequences were generated from genomic DNA at all depths in sea ice, and these sequences aligned with the classes Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria. The sequences showed some similarity to previously reported PR sequences, although most of the sequences were generally distinct. Positive PR sequences were also observed from cDNA reverse transcribed from RNA isolated from sea ice samples. This finding indicates that these sequences were generated from metabolically active cells and suggests that the PR gene is functional within sea ice. Both blue-absorbing and green-absorbing forms of PRs were detected, and only a limited number of blue-absorbing forms were found and were in the midsection of the sea ice profile in this study. Questions still remain regarding the proteins ecological functions, and ultimately, field experiments will be needed to establish the ecological and functional role of PRs in the sea ice ecosystem.

Polyphasic assessment of fresh‐water benthic mat‐forming cyanobacteria isolated from New Zealand

Mat-forming benthic cyanobacteria are widespread throughout New Zealand rivers, and their ingestion has been linked to animal poisonings. In this study, potentially toxic benthic cyanobacterial proliferations were collected from 21 rivers and lakes throughout New Zealand. Each environmental sample was screened for anatoxins using liquid chromatography-MS (LC-MS). Thirty-six cyanobacterial strains were isolated and cultured from these samples. A polyphasic approach was used to identify each isolate; this included genotypic analyses [16S rRNA gene sequences and intergenic spacer (ITS)] and morphological characterization. Each culture was analysed for anatoxins using LC-MS and screened for microcystin production potential using targeted PCR. The morphospecies Phormidium autumnale was found to be the dominant cyanobacterium in mat samples. Polyphasic analyses revealed multiple slight morphological variants within the P. autumnale clade and highlighted the difficulties in identifying Oscillatoriaceae. Only one morphospecies (comprising the two strains CYN52 and CYN53) of P. autumnale was found to produce anatoxins. These strains formed their own clade based on partial 16S rRNA gene sequences. These data indicate that benthic P. autumnale mats are composed of multiple morphospecies and toxin production is dependent on the presence of toxin-producing genotypes. Further cyanobacteria are also characterized, including Phormidium murrayi, which was identified for the first time outside of Antarctica.

Growth and Productivity of Antarctic Sea Ice Algae under PAR and UV Irradiances

Abstract A light perturbation experiment on sea ice at Cape Evans in 1996 found significant differences in biomass accumulation between ice algal assemblages exposed to additional UVB, UVA and PAR irradiances and those exposed to either control or elevated PAR and UVA without UVB. Sea ice with its snow cover removed (and thus exposed to additional UVB, UVA and PAR) had a 40% lower rate of biomass accumulation than sea ice with its snow cover removed but covered with UVB-absorbing mylar. Chlorophyll-specific production under PAR was up to 0.05 mg C (mg Chl a)−1 h−1 although it was approximately 0.02 mg C (mg Chl a)−1 h−1 at ambient under ice irradiances of 2–6 μmol photons m−2 s−1 PAR. UVB irradiation was found to have a greater impact on highly shade-adapted sea ice algal communities beneath thick ice with a thick snow cover than on thinner ice with less snow.

Cytoplasmic accumulation of flavonoids in flower petals and its relevance to yellow flower colouration

It is widely accepted that the mix of flavonoids in the cell vacuole is the source of flavonoid based petal colour, and that analysis of the petal extract reveals the nature and relative levels of vacuolar flavonoid pigments. However, it has recently been established with lisianthus flowers that some petal flavonoids can be excluded from the vacuolar mix through deposition in the cell wall or through complexation with proteins inside the vacuole, and that these flavonoids are not readily extractable. The present work demonstrates that flavonoids can also be compartmented within the cell cytoplasm. Using adaxial epidermal peels from the petals of lisianthus (Eustoma grandiflorum), Lathyrus chrysanthus and Dianthus caryophyllus, light and laser scanning confocal microscopy studies revealed a significant concentration of petal flavonoids in the cell cytoplasm of some tissues. With lisianthus, flavonoid analyses of isolated protoplasts and vacuoles were used to establish that ca 14% of petal flavonoids are located in the cytoplasm (cf. 30% in the cell wall and 56% in the vacuole). The cytoplasmic flavonoids are predominantly acylated glycosides (cf. non-acylated in the cell wall). Flavonoid aggregation on a cytoplasmic protein substrate provides a rational mechanism to account for how colourless flavonoid glycosides can produce yellow colouration in petals, and perhaps also in other plant parts. High vacuolar concentrations of such flavonoids are shown to be insufficient.

Cell wall sited flavonoids in lisianthus flower petals

Flavonoids are considered to be located predominantly in the vacuoles of epidermal cells and in the cuticular wax of terrestrial plants. However, recent reports have suggested that flavonoids may also reside elsewhere in the cells of green leaves. In the present study of lisianthus flower petals, it is demonstrated that ca. 30% of the whole petal flavonol glycosides are located in the cell wall. These flavonol glycosides are distinguished from the vacuolar glycosides in that they lack acylation. Evidence from light and confocal microscopy studies is corroborated by HPLC analyses of isolated protoplasts and cell wall digests, these having been produced by enzymic treatment of epidermal peels. This is the first report of the occurrence of flavonoids in petal cell walls, and it describes novel methodology for such studies.

Melting out of sea ice causes greater photosynthetic stress in algae than freezing in

Sea ice is the dominant feature of polar oceans and contains significant quantities of microalgae. When sea ice forms and melts, the microalgal cells within the ice matrix are exposed to altered salinity and irradiance conditions, and subsequently, their photosynthetic apparatuses become stressed. To simulate the effect of ice formation and melting, samples of sea‐ice algae from Cape Hallett (Antarctica) were exposed to altered salinity conditions and incubated under different levels of irradiance. The physiological condition of their photosynthetic apparatuses was monitored using fast and slow fluorescence‐induction kinetics. Sea‐ice algae exhibited the least photosynthetic stress when maintained in 35‰ and 51‰ salinity, whereas 16, 21, and 65‰ treatments resulted in significant photosynthetic stress. The greatest photosynthetic impact appeared on PSII, resulting in substantial closure of PSII reaction centers when exposed to extreme salinity treatments. Salinity stress to sea‐ice algae was light dependent, such that incubated samples only suffered photosynthetic damage when irradiance was applied. Analysis of fast‐induction curves showed reductions in J, I, and P transients (or steps) associated with combined salinity and irradiance stress. This stress manifests itself in the limited capacity for the reduction of the primary electron receptor, QA, and the plastoquinone pool, which ultimately inhibited effective quantum yield of PSII and electron transport rate. These results suggest that sea‐ice algae undergo greater photosynthetic stress during the process of melting into the hyposaline meltwater lens at the ice edge during summer than do microalgae cells during their incorporation into the ice matrix during the process of freezing.