Ohelo Research Articles

Isolation of oligomeric proanthocyanidins from flavonoid-producing cell cultures

The extraction, fractionation, and chromatographic separation of a series of proanthocyanidin monomers and oligomers were facilitated using a flavonoid-rich cell culture of Vaccinium pahalae Skottsberg as the donor tissue. The cell cultures, after exposure to light, readily accumulated anthocyanin pigments and other flavonoids in relatively large amounts, with minimal concurrent production of pectins, enzymes, and complex sugars produced in field-grown Vaccinium berries. The absence of these interfering compounds greatly simplified the isolation and purification of proanthocyanidins and other phenolic compounds from cell cultures, primarily using vacuum chromatography. Subsequently, the structures and molecular weights of several individual compounds and the general composition of unresolved fractions were established with 1H- and 13C-NMR and MS. The initial extract of V. pahalae cell cultures was readily fractionated on silica gel to yield a series of fractions containing proanthocyanidin B-2, a series of increasingly polar proanthocyanidin oligomers ranging from dimers to heptamers largely based on (−)-epicatechin structures (some with A-type linkages), a mixture of E- and Z-p-coumaric acid, the corresponding 4-O-glucoside, and other compounds containing E- and Z-p-coumaric acid moieties. Cell culture extracts demonstrated broad antioxidant capacity and significant ability to inhibit tumor promotion in vitro, as indicated in an ornithine decarboxylase assay.

Application of imaging tools to plant cell culture: Relationship between plant cell aggregation and flavonoid production

Image analysis tools were developed to measure biomass concentration, aggregate size and distribution, and pigmentation from anthocyanin-producing cell suspension cultures of ohelo (Vaccinium pahalae). The ex situ imaging system could image cell aggregates from 30 µm to 2 mm in diameter. The image analysis algorithm was based on extracted geometric features and morphological methods for biomass volume estimates, and hue, saturation, and intensity color characteristics for pigmentation estimates. Detailed information available from sampled cell culture images was validated by comparison to standard destructive manual measurements. Image analysis measurements revealed that pigment accumulation was negatively correlated with aggregate size. Although a substantial proportion of small aggregates remained colorless, the highly-pigmented small aggregates, 18 to 238 µm in breadth, contributed over 70% of the culture anthocyanin production (mg L−1), despite their minor contribution to the overall biomass. The relative frequency of pigmented aggregates was higher in large-size aggregate classes; however, the pigmented sectors were mostly confined to only the periphery of the aggregates. As a result, large aggregate classes had only a minor contribution to overall culture anthocyanin yield.

Effects of exogenous methyl jasmonate in elicited anthocyanin-producing cell cultures of ohelo (Vaccinium phalae)

Elicitation of anthocyanin-producing cells of ohelo (Vaccinium pahalae) by both biotic (purified β-glucan and chitosan) and abiotic [sodium ferric ethylenediamine di-(o-hydroxyphenylacetate) FeEDDHA, and CuSO4] elicitors resulted in significant enhancement of anthocyanin accumulation. Anthocyanin production increased up to 1.8 and 1.5-fold over the control in the presence of abiotic elicitors (90 µM FeEDDHA and 20 µM CuSO4, respectively), and increased 1.9 and 1.6-fold in the presence of biotic elicitors (10 mg L−1 β-glucan and 100 mg L−1 chitosan). Maximum anthocyanin production with the two most effective elicitors was achieved when cultures were treated on Day 3 (β-glucan) or Day 0 (FeEDDHA) after the initiation of fresh cell cultures. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). The addition of 0.5 µM MJ alone provoked a 2–3-fold increase in anthocyanin production over that of the control; however, no additive effect on anthocyanin production was observed in any treatments which combined MJ and β-glucan or FeEDDHA. Conditioning of the cells with a preculture in either MJ, β-glucan, or FeEDDHA similarly did not enhance anthocyanin production. Inoculation of cultures elicited by MJ or β-glucan with ibuprofen, a reported inhibitor of jasmonate biosynthesis, dramatically stimulated, rather than inhibited, anthocyanin production, resulting in levels of accumulation beyond any of the tested elicitor combinations. Hypotheses for the observed influence of ibuprofen in this system are discussed.

Pigment recovery from encapsulated-dehydrated Vaccinium pahalae (ohelo) cryopreserved cells

Pigment producing in vitro cells of Vaccinium pahalae (ohelo) were tested for their ability to survive cryopreservation and retain pigment-production capacity after encapsulation-dehydration. Preculture of cells for 6 to 8 days in a medium containing 1.0 M sucrose was essential before dehydration. Reduction of bead water content before quenching in liquid nitrogen was highly correlated (r = 0.94) with increased survival rate in cells after cryopreservation. Dehydration of beads for 4 h was satisfactory for survival of cells. After quenching in liquid nitrogen, colored cells became pale, but pigment content was recovered once cells resumed growth. After three subcultures, cells regained their maximum capacity for pigment accumulation. The percentage of colored-to-total cell volume was not influenced by cryopreservation. Encapsulation-dehydration and cryopreservation did not diminish the capacity of cells to produce anthocyanins and other flavonoids.

Benzyl adenine restores anthocyanin pigmentation in suspension cultures of wild Vaccinium pahalae

The overriding influence of cytokinin source on flavonoid production in vitro was explored using a suspension culture system for Vaccinium pahalae. The substitution of kinetin by 20 μM benzyl adenine (BA) in the suspension culture media resulted in a three-fold increase in total anthocyanin yield, and a more rapid production during the cell culture cycle. Anthocyanin production reached a maximum after a 16–20 day interval in cultures containing an optimal kinetin concentration, but pigment accumulation peaked at only 12–16 days when BA was used as the sole cytokinin source. Unlike some other production systems which increase secondary metabolite production at the expense of cell growth, BA-supplementation promoted both increased growth and increased anthocyanin productivity. In BA-supplemented medium, cultures were not susceptible to typical osmotically-induced cell growth suppression. When, after multiple subcultures in kinetin-containing media, anthocyanin production capability was lost or diminished, productivity could be restored within 3 days after transfer of cells to a BA-supplemented medium.

Continuous cell culture and product recovery from wild Vaccinium pahalae germplasm

Summary The productivity of Vaccinium pahalae (ohelo) cell cultures as a resource for natural food colorants and bioactive phytochemicals was examined. Ohelo callus and suspension cultures, established from leaf expiants, produced vivid anthocyanin pigments, identified as cyanidin-3-galactoside, cyanidin-3-arabino-side, and peonidin-3-galactoside. Ohelo plant foliage produced a comparable anthocyanin composition. Both pigment yield and cell biomass continued to increase over a 30 d cell-culture cycle. Pigment yield was enhanced by increasing sucrose concentration in the liquid suspension medium (up to 70 g · L −1 ) and by manipulating the initial inoculum density. A hexane extract from the pigmented ohelo cells demonstrated a strong quinone reductase-inducing activity in vitro , indicating that compounds effective in inhibiting the initiation stages of chemically-induced carcinogenesis were also accumulated in the cell cultures.

Headspace ethylene accumulation effects on secondary metabolite production in Vaccinium pahalae cell culture

The influence of headspace ethylene on anthocyanin, anthocyanidin, and carotenoid accumulation was studied in suspension cultures of Vaccinium pahalae. Exogenous application of ethrel (an ethylene-releasing compound) significantly reduced growth and secondary metabolite production, whereas incorporation of 5.0 or 10.0 mg l-1 CoCl2 or NiCl2 effectively reduced ethylene accumulation and improved product accumulation, but AgNO3was toxic to cells. This study showed an overall negative impact of increased ethylene levels in the vessel headspace on phytochemical production in ohelo cell cultures.

Freezing tolerance and avoidance in high‐elevation Hawaiian plants

ABSTRACTFreezing resistance mechanisms were studied in five endemic Hawaiian species growing at high elevations on Haleakala volcano, Hawaii, where nocturnal subzero (°C) air temperatures frequently occur. Extracellular freezing occurred at around ‐5°C in leaves of Argyroxiphium sandwicense and Sophora chrysophylla, but these leaves can tolerate extracellular ice accumulation to ‐15°C and ‐12°C, respectively. Mucilage, which apparently acted as an ice nucleator, comprised 9 to 11% of the dry weight of leaf tissue in these two species. Leaves of Vaccinium reticulatum and Styphelia tameiameiae were also found to tolerate substantial extracellular freezing. Dubautia menziesii, on the other hand, exhibited the characteristics of permanent supercooling; a very rapid decline in liquid water content associated with simultaneous intracellular and extracellular freezing. However, in those species that tolerate extracellular freezing, the decline in liquid water content during freezing is relatively slow. Osmotic potential was lower at pre‐dawn than at midday in four of the species studied. Nocturnal production of osmotically active solutes may have helped to prevent intracellular freeze dehydration as well as to provide non‐colligative protection of cell membranes. Styphelia tameiameiae supercooled to ‐9·3°C and tolerated tissue freezing to below ‐15°C, a unique combination of physiological characteristics related to freezing. Tolerance of extracellular ice formation after considerable supercooling may have resulted from low tissue water content and a high degree of intracellular water binding in this species, as determined by nuclear magnetic resonance studies. The climate at high elevations in Hawaii is relatively unpredictable in terms of the duration of subzero temperatures and the lowest subzero temperature reached during the night. It appears that plants growing in this tropical alpine habitat have been under selective pressures for the evolution of freezing tolerance mechanisms.

Plant-soil interactions in primary succession at Hawaii Volcanoes National Park.

Plant nutrient status and physiological processes were examined in relation to soil nutrient characteristics under individuals of five species colonizing a young cinder deposit in Hawaii Volcanoes National Park. Two exotic species, Buddeleja asiatica and Myrica faya, had high photosynthetic rates and high nitrogen concentrations and relatively easily decomposed leaves; soils under them had high concentrations of nitrogen, cations, and organic matter and high rates of net nitrogen mineralization. At the other extreme, the natives Metrosideros polymorpha and Vaccinium reticulatum had low plant concentrations and photosynthetic rates, and low concentrations and turnover rates of N in the soil. Thus, a strong correlation exists between soil processes and plant processes, suggesting a positive feedback cycle.

ULTRASTRUCTURE OF THE CUTICLE OF OHELO (VACCINIUM RETICULATUM)

When viewed by electron microscopy of thin sections, the inner two‐thirds of the petiolar cuticle of Vaccinium reticulatum appears dense and is penetrated by a channel‐like reticulum which emanates from the primary cell wall. The outer one‐third of the cuticle stains lightly and is homogeneous. This arrangement is compared with the ultrastructure of cuticle previously reported for other plant species.