Plant science and human nutrition: challenges in assessing health-promoting properties of phytochemicals

The rise in noncommunicable chronic diseases associated with changing diet and lifestyles throughout the world is a major challenge for society. It is possible that certain dietary components within plants have roles both in reducing the incidence and progression of these diseases. We critically review the types of evidence used to support the health promoting activities of certain phytochemicals and plant-based foods and summarize the major contributions but also the limitations of epidemiological and observational studies and research with the use of cell and animal models. We stress the need for human intervention studies to provide high-quality evidence for health benefits of dietary components derived from plants.     

Effects of supplemental light quality on growth and phytochemicals of baby leaf lettuce

Using UV-A, blue (B), green (G), red (R), and far-red (FR) light-emitting diodes (LEDs), we investigated the effects of different supplemental light qualities on phytochemicals and growth of ‘Red Cross’ baby leaf lettuce (Lactuca sativa L.) grown at a high planting density under white fluorescent lamps as the main light source inside a growth chamber. Photon flux added by supplemental LEDs for UV-A, B, G, R and FR were 18, 130, 130, 130 and 160 μmol m^?2 s^?1, respectively. Photosynthetic photon flux (PPF, 400–700 nm), photoperiod, and air temperature (day/night) was 300 μmol m^?2 s^?1, 16 h, and 25 °C/20 °C in all treatments including white light control. After 12 days of light quality treatment (22 days after germination), phytochemical concentration and growth of lettuce plants were significant affected by light treatments. Anthocyanins concentration increased by 11% and 31% with supplemental UV-A and B, respectively, carotenoids concentration increased by 12% with supplemental B, phenolics concentration increased by 6% with supplemental R while supplemental FR decreased anthocyanins, carotenoids and chlorophyll concentration by 40%, 11% and 14%, respectively, compared to those in the white light control. The fresh weight, dry weight, stem length, leaf length and leaf width significantly increased by 28%, 15%, 14%, 44% and 15%, respectively, with supplemental FR light compare to white light, presumably due to enhanced light interception by enlarged leaf area under supplemental FR light. Although the mechanisms of changes in phytochemicals under different supplemental light quality are not well known, the results demonstrated that supplemental light quality could be strategically used to enhance nutritional value and growth of baby leaf lettuce grown under white light.     

Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.     

Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition

The potential of three arbuscular mycorrhizal fungi (AMF) to enhance the production of antioxidants (rosmarinic and caffeic acids, RA and CA) was investigated in sweet basil (Ocimum basilicum). After adjusting phosphorus (P) nutrition so that P concentrations and yield were matched in AM and non-mycorrhizal (NM) plants we demonstrated that Glomus caledonium increased RA and CA production in the shoots. Glomus mosseae also increased shoot CA concentration in basil under similar conditions. Although higher P amendments to NM plants increased RA and CA concentrations, there was higher production of RA and CA in the shoots of AM plants, which was not solely due to better P nutrition. Therefore, AMF potentially represent an alternative way of promoting growth of this important medicinal herb, as natural ways of growing such crops are currently highly sought after in the herbal industry.     

Thermal and physical stresses induce a short-term immune priming effect in Galleria mellonella larvae

Exposure of larvae of Galleria mellonella larvae to mild physical (i.e. shaking) or thermal stress for 24 h increased their ability to survive infection with Aspergillus fumigatus conidia however larvae stressed in a similar manner but incubated for 72 h prior to infection showed no elevation in their resistance to infection with A. fumigatus. Stressed larvae demonstrated an elevated haemocyte density 24 h after initiation of the stress event but this declined at 48 and 72 h. Larval proteins such as apolipophorin, arylophorin and prophenoloxidase demonstrated elevated expression at 24 h but not at 72 h. Larvae maintained at 37 °C showed increased expression of a range of antimicrobial and immune-related proteins at 24 h but these decreased in expression thereafter. The results presented here indicate that G. mellonella larvae are capable of altering their immune response following exposure to mild thermal or physical stress to mount a response capable of counteracting microbial infection which reaches a peak 24 h after the initiation of the priming event and then declines by 72 h. A short-term immune priming effect may serve to prevent infection but maintaining an immune priming effect for longer periods may be metabolically costly and unnecessary while living within the colony of another insect.     

Environmental stresses inhibit splicing in the aquatic fungus Blastocladiella emersonii

Background  

Exposure of cells to environmental stress conditions can lead to the interruption of several intracellular processes, in particular those performed by macromolecular complexes such as the spliceosome.     

Environmental signals induce major changes in virulence of Shigella spp.

Growth conditions play a major role in expression of virulence by Shigella spp. both in vitro (adherence and internalization in eukaryotic host cells) and in vivo (keratoconjunctivitis). Optimized expression of virulence required anaerobic growth to log phase in particular media such as brain heart infusion broth. Kinetic studies of guinea pig eye infections showed that as few as 2 x 10(5) S. dysenteriae CG097 or S. flexneri M90T, grown under these optimized conditions, produced keratoconjunctivitis in 15 h. In vitro studies demonstrated that adherence to and invasion of Henle 407 cells, at 37 degrees C, by organisms grown under these optimized conditions, were significantly greater than when organisms were grown aerobically under the same conditions.     

Environmental light-darkness conditions induce changes in brain and peripheral pyroglutamyl-peptidase I activity

To evaluate the influence of light and darkness on brain pyroglutamyl-peptidase I (pGluPI) activity, four experimental groups of rats were compared at the same time-point (10.00 h). Two groups were designed with a standard 12-12 h light-dark cycle: In group A, the lights were on from 7.00 h to 19.00 h, and the experiment was done under light conditions; in group B, the lights were on from 19.00 h to 7.00 h, and the experiment was done under darkness conditions. Two additional groups were designed with nonstandard light-dark conditions: In group C, the animals were subjected to constant light, and the experiment was done under light conditions. In group D, animals were subjected to constant darkness, and the experiment was done under darkness conditions. Light (vs darkness) and standard (vs nonstandard) conditions produced significant changes on pGluPI activity in specific structures; the data suggested that endogenous substrates of pGluPI such as thyrotropin-releasing hormone and gonadotropin-releasing hormone, might be modified in parallel. There was left predominance in the retina under light conditions on a standard schedule (group A). The regional pattern of distribution of activity was similar in groups on a standard schedule (A vs B) and in groups tested under constant light-dark conditions (C vs D). However, this pattern differed between groups subjected to standard vs constant light-dark conditions (A and B vs C and D). These results support an influence of environmental light and darkness on pGluPI activity, which may reflect concomitant changes in its susceptible substrates and consequently in their functions.     

Ultraviolet and osmotic stresses induce and regulate the synthesis of mycosporines in the cyanobacterium Chlorogloeopsis PCC 6912

The cyanobacterium Chlorogloeopsis PCC 6912 was found to synthesize and accumulate two putative UV sunscreen compounds of the mycosporine (mycosporine-like amino acid; MAA) type: mycosporine-glycine and shinorine. These MAAs were not constitutively present in the cells; their synthesis could be induced specifically either by exposure to UVB radiation (280–320 nm) or by osmotic stress, but not by other stress factors such as heat or cold shock, nutrient limitation, or photooxidative stress. A significant synergistic enhancement of MAA synthesis was observed when both stress factors were applied in combination. Although osmotic stress could induce MAA synthesis, comparison of the intracellular contents of MAAs with those of sugar osmolytes (glucose and trehalose) indicated that MAAs play no significant role in attaining osmotic homeostasis. UVB strongly enhanced the accumulation of shinorine, whereas osmotic stress had a more pronounced effect on mycosporine-glycine. This differential effect on the steady-state contents of each MAA could be explained either by differential regulation of biosynthesis or by differential loss rates of MAAs (leakage) under each condition. A preferential leakage of mycosporine-glycine from the cells after a hypoosmotic shock was detected. The results are interpreted in terms of an adaptive necessity for a combined regulatory control responding to both UV and external osmotic conditions in organisms that accumulate water-soluble sunscreens intracellularly. Received: 26 March 1999 / Accepted: 13 July 1999     

Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block

We report here that importin alpha accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin alpha appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin alpha. In addition, nuclear retention and the importin beta/Ran-independent import of importin alpha also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin alpha from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin alpha is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin alpha can be involved in a common physiological response to various stress conditions.     

Metabolite fingerprinting in transgenic lettuce

Metabolite fingerprinting has been achieved using direct atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) and linked gas chromatography (GC-APCI/EI-MS) for transgenic lettuce (Lactuca sativa L. cv. Evola) plants expressing an IPT gene under the control of the senescence-specific SAG12 promoter from Arabidopsis thaliana (P(SAG12)-IPT). Mature heads of transgenic lettuce and their azygous controls were maintained under defined conditions to assess their shelf life. Transgenic lettuce plants exhibited delayed senescence and significant increases (up to a maximum of threefold) in the concentrations of three volatile organic compounds (VOCs), corresponding to molecular masses of 45, 47 and 63, when compared with heads from azygous plants. These VOCs were identified as acetaldehyde (45), ethanol (47) and dimethyl sulphide (63). The increase in dimethyl sulphide was paralleled by an accumulation of reactive oxygen species (ROS) in the heads of transgenic plants. These results demonstrate the applicability of metabolic fingerprinting techniques to elucidate the underlying pleiotropic responses of plants to transgene expression.     

Selenium accumulation in lettuce germplasm

Selenium (Se) is an essential micronutrient for animals and humans. Increasing Se content in food crops offers an effective approach to reduce the widespread selenium deficiency problem in many parts of the world. In this study, we evaluated 30 diverse accessions of lettuce (Lactuca sativa L.) for their capacity to accumulate Se and their responses to different forms of Se in terms of plant growth, nutritional characteristics, and gene expression. Lettuce accessions responded differently to selenate and selenite treatment, and selenate is superior to selenite in inducing total Se accumulation. At least over twofold change in total Se levels between cultivars with high and low Se content was found. Synergistic relationship between Se and sulfur accumulation was observed in nearly all accessions at the selenate dosage applied. The change in shoot biomass varied between lettuce accessions and the forms of Se used. The growth-stimulated effect by selenate and the growth-inhibited effect by selenite were found to be correlated with the alteration of antioxidant enzyme activities. The different ability of lettuce accessions to accumulate Se following selenate treatment appeared to be associated with an altered expression of genes involved in Se/S uptake and assimilation. Our results provide important information for the effects of different forms of Se on plant growth and metabolism. They will also be of help in selecting and developing better cultivars for Se biofortification in lettuce.     

Yeast cultivation in lettuce brine

The use of lettuce brine, a by-product of the vegetable fermentation industry, as a medium for yeast cultivation was investigated. Six strains of yeast, Saccharomyces sp., Pichia sp., Rhodotorula sp., Candida sp., Kluyveromyces sp. and Trichospora sp. grew well in diluted lettuce brine under aerobic conditions. The acid brine becomes neutral after yeast cultivation. The yeast strains reached the maximum growth after the first day of cultivation. Trichosporon sp. was found to grow best in the brine with the maximum specific growth rate at 0.09 h⁻¹ and growth yield of 67%. This revised version was published online in November 2006 with corrections to the Cover Date.     

Diverse cell stresses induce unique patterns of tRNA up- and down-regulation: tRNA-seq for quantifying changes in tRNA copy number

Emerging evidence points to roles for tRNA modifications and tRNA abundance in cellular stress responses. While isolated instances of stress-induced tRNA degradation have been reported, we sought to assess the effects of stress on tRNA levels at a systems level. To this end, we developed a next-generation sequencing method that exploits the paucity of ribonucleoside modifications at the 3′-end of tRNAs to quantify changes in all cellular tRNA molecules. Application of this tRNA-seq method to Saccharomyces cerevisiae identified all 76 expressed unique tRNA species out of 295 coded in the yeast genome, including all isoacceptor variants, with highly precise relative (fold-change) quantification of tRNAs. In studies of stress-induced changes in tRNA levels, we found that oxidation (H₂O₂) and alkylation (methylmethane sulfonate, MMS) stresses induced nearly identical patterns of up- and down-regulation for 58 tRNAs. However, 18 tRNAs showed opposing changes for the stresses, which parallels our observation of signature reprogramming of tRNA modifications caused by H₂O₂ and MMS. Further, stress-induced degradation was limited to only a small proportion of a few tRNA species. With tRNA-seq applicable to any organism, these results suggest that translational control of stress response involves a contribution from tRNA abundance.     

Photomanipulation of phytochrome in lettuce seeds

Seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) were imbibed and given either short irradiation with red or far red light prior to drying or dried under continuous red or far red light. Seeds treated with either short or continuous red germinate in darkness, whereas seeds treated with either short or continuous far red require a short exposure to red light, after a period of imbibition, to stimulate germination. Irradiation of dry red seeds with far red light immediately before sowing results in a marked inhibition of germination. This result was predicted since far red-absorbing form phytochrome can be photoconverted to the intermediate P650 (absorbance maximum 650 nm) in freeze-dried tissue. A similar far red treatment to continuous red seeds is less effective and it is concluded that in these seeds a proportion of total phytochrome is blocked as intermediates between red-absorbing and far red-absorbing form phytochrome, which only form the far red-absorbing form of phytochrome on imbibition. The inhibition of dry short red seeds by far red light can be reversed by an irradiation with short red light given immediately before sowing, confirming that P650 can be photoconverted back to the far red-absorbing form of phytochrome. The results are discussed in relation to seed maturation (dehydration) on the parent plant.     

The metabolism of fluoroacetate in lettuce

1. Whole lettuce plants were incubated with (1) [1-(14)C]acetate, (2) fluoroacetate followed by [1-(14)C]acetate, (3) fluoro[1-(14)C]acetate, (4) fluoro[2-(14)C]acetate or (5) S-carboxy[(14)C]methylglutathione. 2. Fluoroacetate did not affect the expiration of (14)CO(2) from [1-(14)C]acetate and only a small amount of (14)CO(2) was produced from either fluoro[1-(14)C]-acetate or fluoro[2-(14)C]acetate in 43h. 3. Fluoroacetate at 50mg/kg wet wt. doubled the plant citrate concentration after 43h incubation, and depending on the age and size of the plant 50-100% of the compound was metabolized. 4. With both fluoro[1-(14)C]acetate and fluoro[2-(14)C]acetate all the radioactivity except that in the CO(2) was found in the water-soluble acid fraction. About 2% was in fluorocitrate and the remainder, apart from unchanged fluoroacetate, was in a number of compounds devoid of fluorine but containing nitrogen and sulphur. These were peptide-like and could be separated by chromatography on an amino acid analyser. 5. Identical compounds were obtained from the spontaneous reaction between iodo[2-(14)C]acetate and glutathione, the major product being S-carboxymethylglutathione. 6. S-Carboxymethylcysteine was also isolated and its mass spectrum compared with a commercial sample. 7. Reaction rates of all the monohaloacetates with glutathione were studied at pH7 at 25 degrees C. No reaction was observed with fluoroacetate. 8. The metabolism of fluoroacetate by lettuce is discussed in relation to that of aliphatic and aromatic halogen compounds, including fluoroacetate, by mammalian liver and to the metabolism of fluoroacetate by different plants reported by other workers.