GC-MS metabolomics of French lettuce (Lactuca Sativa L. var capitata) leaves exposed to bisphenol A via the hydroponic media.

INTRODUCTION: Bisphenol A (BPA), an organic compound used to produce polycarbonate plastics and epoxy resins, has become a ubiquitous contaminant due to its high-volume production and constant release to the environment. Plant metabolomics can trace the stress effects induced by environmental contaminants to the variation of specific metabolites, making it an alternative way to study pollutants toxicity to plants. Nevertheless, there is an important knowledge gap in metabolomics applications in this area. OBJECTIVE: Evaluate the influence of BPA in French lettuce (Lactuca Sativa L. var capitata) leaves metabolic profile by gas chromatography coupled to mass spectrometry (GC-MS) using a hydroponic system. METHODS: Lettuces were cultivated in the laboratory to minimize biological variation and were analyzed 55 days after sowing (considered the plant's adult stage). Hexanoic and methanolic extracts with and without derivatization were prepared for each sample and analyzed by GC-MS. RESULTS: The highest number of metabolites was obtained from the hexanoic extract, followed by the derivatized methanolic extract. Although no physical differences were observed between control and contaminated lettuce leaves, the multivariate analysis determined a statistically significant difference between their metabolic profiles. Pathway analysis of the most affected metabolites showed that galactose metabolism, starch and fructose metabolism and steroid biosynthesis were significantly affected by BPA exposure. CONCLUSIONS: The preparation of different extracts from the same sample permitted the determination of metabolites with different physicochemical properties. BPA alters the leaves energy and membrane metabolism, plant growth could be affected at higher concentrations and exposition times.

Effect of TiO2 Microparticles in Lettuce (Lactuca sativa L.) Seeds and Seedlings.

The particle size reduction technology is used in several segments, including sunscreens and new techniques and product improvement. One of the main particles used in the sunscreens formulation is titanium dioxide (TiO2). This formulation allows for better characteristics of these products. Perspectives like incorporation of the particles by other biological systems beyond humans and their effects should be observed. This work aimed to evaluate the titanium dioxide microparticles phytotoxicity on Lactuca sativa L. plants through tests of germination, growth, and weight analysis using microscopy techniques: optical microscopy (OM) and scanning electron microscopy (SEM). Some of the results showed cellular and morphological damage, mainly in the roots and 50 mg L-1 TiO2 concentration, confirmed by SEM. Additionally, anatomical damages like vascular bundle disruption and irregularity in the cortex cells were confirmed by SEM. Additionally, anatomical damages were observed on the three main organs (root, hypocotyl, and leaves) evidenced by the OM. Perspectives to confirm new hypotheses of the interaction of nanomaterials with biological systems are necessary.

Germination, cytotoxicity, and mutagenicity in Lactuca sativa L. and Passiflora alata Curtis in response to sewage sludge application.

The physical and chemical characteristics of the soil can influence plant growth. When sewage sludge (SS) is applied as a soil fertilizer, the accumulation of non-essential elements contained in it can be toxic for plants. The aim of this study was to understand the effect of SS dosage on the cell cycle of Lactuca sativa L. meristematic cells and on the initial growth of L. sativa and Passiflora alata Curtis. Nine concentrations of SS + distilled water (mg dm-3) corresponding to 0, 20, 40, 60, 80, 120, 160, 320, and 520 t ha-1 were tested in four replicates of 25 seeds. Chemical analysis showed an increase in pH of the sludge from 0 to 80 t ha-1 SS followed by its stabilization thereafter. The highest electrical conductivity was observed at 520 t ha-1 SS. SS negatively affected the germination and initial growth of seedlings from P. alata and L. sativa. Cytogenetic analysis on 6000 L. sativa meristematic cells for each treatment revealed that SS could adversely affect the genetic stability of this species. SS concentrations above 120 t ha-1 adversely affected the germination and early seedling growth of L. sativa and P. alata. At high concentrations (120 t ha-1), SS induced genetic lesions in L. sativa, along with chromosomal and nuclear alterations.

Resveratrol exerts beneficial effects on the growth and metabolism of Lactuca sativa L.

In order to assist sustainable agriculture, new strategies and methods are being used based on the utilization of new natural molecules. These natural compounds can be used as potential natural crop protectors and growth promoters, and the elucidation of their modes/mechanisms of action can represent a big step towards cleaner agriculture free of agrochemicals. In the present paper, the mechanisms underlying the effects of exogenous resveratrol (R), a natural phytoalexin found in plants, on Lactuca sativa metabolism were investigated through physiological and metabolomic approaches. The results highlighted that R stimulates the growth of lettuce. A reduction of the O2⋅- production in R-treated seedlings and an increase in the photosynthesis efficiency was observed, indicated by a higher Fv/Fm. The metabolomic analysis of lettuce seedlings treated with R identified 116 metabolites related to galactose, amino acids, sugar and nucleotide sugar, and ascorbate and aldarate metabolisms. Increased content of some polyamines and several metabolites was also observed, which may have contributed to scavenging free radicals and activating antioxidant enzymes, thus reducing oxidative damage and improving PSII protection in R-treated seedlings.

Synthesis of calcium borate nanoparticles and its use as a potential foliar fertilizer in lettuce (Lactuca sativa) and zucchini (Cucurbita pepo).

This study was carried out to evaluate the effects of foliar sprays containing boron (B) nano-fertilizer (NF) on the growth and physiology of lettuce (Lactuca sativa) and zucchini (Cucurbita pepo). Plants were grown under greenhouse conditions for 60 days on a modified Hoagland solution with the presence and absence of boron (+B or -B). A synthesized B-NF foliar spray and a commercial B foliar fertilizer (Bortrac™ 150, BT) was applied at a concentration of 30 mg B L-1 at 10-d intervals throughout the experiment. The B-NF treatment increased the growth of lettuce 2.7- and 1.9-fold for shoots and roots, respectively, with an average production of lettuce biomass by ~58%. Similarly, the NF increased the growth of zucchini by 18 and 66% compared with Control-B (the absence of B), and 13 and 36% compared with BT, both for shoots and roots, respectively. Nevertheless, NF + B mostly decreased lettuce growth with symptoms of B toxicity in leaves. In lettuce, addition of B did not affect concentrations of phenols; however, in zucchini, Control-B induced a higher production of phenolic compounds possibly related to B deficiency. The B addition in lettuce reduced the DPPH activity by 32 and 21% in NF and BT, respectively, compared to Control-B. These responses were similar in zucchini; however, the effect of B was product of its presence in mineral solution rather than due the foliar product applied. This suggests that a NF-based delivery system for B may be highly effective at boosting plant productivity on B-limited soils.

Do nitrogen sources and molybdenum affect the nutritional quality and nitrate concentrations of hydroponic baby leaf lettuce?

Lettuce is one of the most popular vegetables, and the market niche of the baby leaf variety is expanding. The manner in which nitrogen (N) is supplied and the available concentration of the micronutrient molybdenum (Mo) affect N metabolism, with reflects on the nutritional quality of that vegetable. Here, two Mo concentrations (0.06 and 0.12 mg/L) and four proportions (%) of nitrate (NO3 - )/ammonium (NH4 + )(100/0, 75/25, 50/50, and 25/75) were supplied to hydroponic baby leaf lettuce floating type to evaluate their effects on plant growth and leaf mineral and NO3 - compositions. Shoot dry mass did not differ among the different treatments, although fresh mass was lower in treatments with larger proportions of NH4 + . Higher leaf concentrations of NO3 - were observed in plants treated with 100% N-NO3 - , but they were still below tolerable limits for human health. The enzyme nitrate reductase was not found to be sensitive to the nitrogen sources or to Mo concentrations. N proportions and Mo concentrations differently affected macro- (C, N, P, K, Ca, Mg, and S) and micronutrients (Cu, Fe, Mn, Mo, and Zn) leaf concentrations. Although treatment with 100% N-NO3 - favored higher mineral concentrations in lettuce leaves, the addition of 25% N-NH4 + allowed fresh mass production with the lowest NO3 - concentrations. As such, and considering the healthy reduction of NO3 - consumption by humans and the maintenance of plant productivity, the 75/25 NO3 - /NH4 + proportion is recommended for the hydroponic cultivation of baby leaf var. Mimosa lettuce. PRACTICAL APPLICATION: We demonstrate a direct link between the constitution of nutrient solution with nitrate accumulation by hydroponic lettuce and indicate the best source of N as well as the concentration of Mn to healthy reduction of NO3 - consumption by humans and the maintenance of plant productivity.

Antarctic root endophytes improve physiological performance and yield in crops under salt stress by enhanced energy production and Na+ sequestration.

Climatic change is pointed as one of the major challenges for global food security. Based on current models of climate change, reduction in precipitations and in turn, increase in the soil salinity will be a sharp constraint for crops productivity worldwide. In this context, root fungi appear as a new strategy to improve plant ecophysiological performance and crop yield under abiotic stress. In this study, we evaluated the impact of the two fungal endophytes Penicillium brevicompactum and P. chrysogenum isolated from Antarctic plants on nutrients and Na+ contents, net photosynthesis, water use efficiency, yield and survival in tomato and lettuce, facing salinity stress conditions. Inoculation of plant roots with fungal endophytes resulted in greater fresh and dry biomass production, and an enhanced survival rate under salt conditions. Inoculation of plants with the fungal endophytes was related with a higher up/down-regulation of ion homeostasis by enhanced expression of the NHX1 gene. The two endophytes diminished the effects of salt stress in tomato and lettuce, provoked a higher efficiency in photosynthetic energy production and an improved sequestration of Na+ in vacuoles is suggested by the upregulating of the expression of vacuolar NHX1 Na+/H+ antiporters. Promoting plant-beneficial interactions with root symbionts appears to be an environmentally friendly strategy to mitigate the impact of climate change variables on crop production.

Thermal disruption of the food matrix of biofortified lettuce varieties modifies absorption of carotenoids by Caco-2 cells.

Amongst green leafy vegetables, new varieties of lettuce enriched in lutein and ß-carotene are being developed to provide increased supply of dietary carotenoids. We investigated the effect of lettuce genotypes (varieties) and thermal treatments on lutein and ß-carotene bioaccessibility to the micellar fraction (and also carotenoid bioavailability) using a human Caco-2 cell model system. Carotenoid absorption by mammalian cells is not correlated with initial carotenoid concentration in fresh lettuce leaves. While thermal treatment of lettuce leaves increases carotenoid availability, resulting in higher lutein and ß-carotene absorption, disruption of the food matrix by prior cooking results in reduced carotenoid levels and transfer to the micellar fraction. Unless the food matrix is disrupted through breeding or post-harvest treatments, absorption of carotenoids from biofortified lettuce remains similar to lettuce cultivars with low carotenoid levels. Genetic improvement programs for biofortified lettuce varieties need to focus on increasing the carotenoid bioavailability from the food matrix.

Bioavailability Assessment of Copper, Iron, Manganese, Molybdenum, Selenium, and Zinc from Selenium-Enriched Lettuce.

Cu, Fe, Mn, Mo, Selenium (Se), and Zn bioavailability from selenate- and selenite-enriched lettuce plants was studied by in vitro gastrointestinal digestion followed by an assay with Caco-2 cells. The plants were cultivated in the absence and presence of two concentrations (25 and 40 µmol/L of Se). After 28 days of cultivation, the plants were harvested, dried, and evaluated regarding the total concentration, bioaccessibility, and bioavailability of the analytes. The results showed that biofortification with selenate leads to higher Se absorption by the plant than biofortification with selenite. For the other nutrients, Mo showed high accumulation in the plants of selenate assays, and the presence of any Se species led to a reduction of the plant uptake of Cu and Fe. The accumulation of Zn and Mn was not strongly influenced by the presence of any Se species. The bioaccessibility values were approximately 71%, 10%, 52%, 84%, 71%, and 86% for Cu, Fe, Mn, Mo, Se, and Zn, respectively, and the contribution of the biofortified lettuce to the ingestion of these minerals is very small (except for Se and Mo). Due to the low concentrations of elements from digested plants, it was not possible to estimate the bioavailability for some elements, and for Mo and Zn, the values are below 6.9% and 3.4% of the total concentration, respectively. For Se, the bioavailability was greater for selenite-enriched than selenate-enriched plants (22% and 6.0%, respectively), because selenite is biotransformed by the plant to organic forms that are better assimilated by the cells.

Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2.

The bone morphogenetic protein BMP2 plays a crucial role in the formation and regeneration of bone and cartilage, which is critical for maintaining skeletal integrity and bone fracture repair. Because of its important role in osteogenic properties it has been commercially produced for clinical use. Here we report attempts to express human BMP2 using two plant systems (lettuce chloroplast and soybean seeds). The rhBMP2 gene (coding for the 13 kDa active polypeptide) was introduced in two regions of the lettuce chloroplast genome. Two homoplasmic events were achieved and RT-PCR demonstrated that the BMP2 gene was transcribed. However, it was not possible to detect accumulation of rhBMP2 in leaves. Two soybean events were achieved to express a full-length hBMP2 gene (coding for the 45 kDa pro-BMP2) fused with the α-coixin signal peptide, under control of the ß-conglycinin promoter. Pro-BMP2 was expressed in the transgenic seeds at levels of up to 9.28% of the total soluble seed protein as determined by ELISA. It was demonstrated that this recombinant form was biologically active upon administration to C2C12 cell cultures, because it was able to induce an osteogenic cascade, as observed by the enhanced expression of SP7 (osterix) and ALPI (alkaline phosphatase) genes. Collectively, these results corroborated our previous observation that soybean seeds provide an effective strategy for achieving stable accumulation of functional therapeutic proteins, such as BMP2.

Mercury and other trace metals in lettuce (Lactuca sativa) grown with two low-salinity shrimp effluents: Accumulation and human health risk assessment.

Shrimp farming effluents from two sources of low-salinity water, well water (WW) and diluted seawater (DSW) (salinity, 1.7 g L-1; electrical conductivity, 2.7 dS m-1), were used to grow lettuce (L. sativa) in order to assimilate the nutrients present in shrimp effluents and produce edible biomass. The two treatments, WW and DSW, were tested in triplicate. Additionally, one hydroponic system in triplicate was constructed to grow lettuce using a nutritive solution as the control treatment (HS). The production variables of lettuce in the two crop varieties (Parris Island (VPI) and Tropicana M1 (VTM1)) showed a general trend of DSW > HS > WW with regards to the size, weight and total foliage, except for the number of leaves, which was higher with HS treatment than with WW and DSW treatments. The accumulation of Cu, Hg, Mn and Zn in edible lettuce tissue and the health risk by the intake of lettuce were evaluated. Heavy metal concentrations in edible lettuce tissue for the three treatments showed the same trend of Mn > Zn > Cu > Hg, with concentration ranges of 47.1 to 188.7, 35.7 to 66.2, 4.1 to 6.4, and 0.01 to 0.02 mg kg-1 (dry weight), respectively. Such concentrations did not exceed the safe limits (CAC, 1984). The health risk index and target hazard quotient were <1, which indicates that the population exposed to these metals due to intake from lettuce consumption is unlikely to have adverse health effects when shrimp farming effluents are used to grow lettuce plants.

Evaluation of chemical extractants to assess metals phytoavailability in Brazilian municipal solid waste composts.

Municipal solid wastes (MSW) can be composted to become an organic fertilizer. However, besides plant nutrients, it can also contain high concentration of some toxic metals than can pollute agricultural soils, contaminate food, animals and human being. A greenhouse experiment was carried out for two purposes: i) to evaluate the concentrations of cadmium, copper, chromium, nickel, lead and zinc in four Brazilian MSW composts, and, ii) to know which is the best solution for extracting those metals in phytoavailable form from the composts. In order to evaluate the phytoavailability of metals, they were extracted with six chemical extractants: i) water, ii) 0.05 mol L-1 Ca(NO3)2, iii) 0.1 mol L-1 HCl, iv) 0.005 mol L-1 DTPA at pH 7.3, v) 0.05 mol L-1 CaCl2 and vi) Mehlich 3 solution. In addition, lettuces were cultivated as a test plant in pots containing 1.8 kg of MSW compost as substrate. Fifty-six days later, lettuce plants were harvested. New lettuces were then planted for a second cycle, and then harvested after fifty-six days. Semi-total concentration of metals in composts and total in plants was also determined through an extraction with nitric-perchloric acid. Semi-total concentration of Cd and Pb exceeded the intervention limits from Brazil in the four studied composts, and lettuce plants were polluted by those two elements. Therefore, compost made of MSW must be characterized before being used for agricultural soils. Copper and nickel in phytoavailable were effectively extracted with the strongest chelating agents used, HCl and Mehlich 3, probably because most metal is bound to organic matter in the compost. Cadmium, chromium, lead and zinc were no efficiently extracted with any of the tested extractants.

Toxic effects of environmental pollutants: Comparative investigation using Allium cepa L. and Lactuca sativa L.

Studies that help understand the mechanisms of action of environmental pollutants are extremely important in environmental toxicology. In this context, assays using plants as models stand out for their simplicity and low performance cost. Among the plants used for this purpose, Allium cepa L. is the model most commonly applied for cytogenotoxic tests, while Lactuca sativa L., already widely used in phytotoxic investigations, has been gaining prominence in cytotoxic analyses. The present study aimed to compare the responses of A. cepa and L. sativa via macroscopic (root growth) and microscopic analyses (cell cycle and DNA fragmentation via TdT-mediated deoxy-uracil nick and labeling (TUNEL) and comet assays) after exposure of their roots to environmental pollutants with known cytogenotoxic mechanisms. Both species presented sensitive and efficient response to the applied tests after exposure to the DNA-alkylating agent Methyl Methanesulfonate (MMS), the heavy metal Cadmium, the aluminum industry waste Spent Potliner (SPL) and the herbicide Atrazine. However, they differed regarding the responses to the evaluated endpoints. Overall, A. cepa was more efficient in detecting clastogenic changes, arising from DNA breakage, while L. sativa rather detected aneugenic alterations, related to chromosome segregation in mitosis. In the tests applied to verify DNA fragmentation (comet and TUNEL assays), A. cepa presented higher sensitivity. In conclusion, both models are efficient to evaluate toxicological risks of environmental pollutants.

Evaluation of the toxic potential of coffee wastewater on seeds, roots and meristematic cells of Lactuca sativa L.

Coffee wastewater (CWW) is an effluent produced through wet processing of coffee containing high concentration of organic matter, nutrients, salts and also agrochemicals. It is released directly into the argillaceous soil or into decantation tanks for later disposal into soils, by fertigation, subsurface infiltration or superficial draining. However, this practice is not followed by the monitoring the toxicity potential of this effluent. In this sense, the present work aimed to evaluate the phytotoxic, cytogenotoxic and mutagenic potential of CWW on seed germination, root elongation and cell cycle alterations in the plant model Lactuca sativa L. The effluent (CWW) collected was diluted in distilled water into six concentrations solutions (1.25%, 1.66%, 2.5%, 5.0%, 10%, 20%). A solution of raw CWW (100%) was also applied. Distilled water was used as negative control), and the DNA alkylating agent, metilmetano sulfonate (4×10(-4)M) as positive control. Physico-chemical parameters of the CWW was accessed and it was found that the effluent contained total phenols and inorganic matter in amounts within the limits established by the National Environment Council (CONAMA). Nevertheless, the biologicals assays performed demonstrated the phytotoxicity and cytogenotoxicty of CWW. Seed germination was totally inhibited after exposure of raw CWW. In addition, a decrease in seed germination speed as well as in root growth dose-dependently manner was noticed. Moreover, nuclear and chromosomal alterations were observed in the cell cycle, mostly arising from aneugenic action.

Effects of co-cropping Bidens pilosa (L.) and Tagetes minuta (L.) on bioaccumulation of Pb in Lactuca sativa (L.) growing in polluted agricultural soils.

Polluted agricultural soils are a serious problem for food safety, with phytoremediation being the most favorable alternative from the environmental perspective. However, this methodology is generally time-consuming and requires the cessation of agriculture. Therefore, the purpose of this study was to evaluate two potential phytoextractor plants (the native species Bidens pilosa and Tagetes minuta) co-cropped with lettuce growing on agricultural lead-polluted soils. The concentrations of Pb, as well as of other metals, were investigated in the phytoextractors, crop species, and in soils, with the potential risk to the health of consumers being estimated. The soil parameters pH, EC, organic matter percentage and bioavailable lead showed a direct relationship with the accumulation of Pb in roots. In addition, the concentration of Pb in roots of native species was closely related to Fe (B. pilosa, r = 0.81; T. minuta r = 0.75), Cu (T. minuta, r = 0.93), Mn (B. pilosa, r = 0.89) and Zn (B. pilosa, r = 0.91; T. minuta, r = 0.91). Our results indicate that the interaction between rhizospheres increased the phytoextraction of lead, which was accompanied by an increase in the biomass of the phytoextractor species. However, the consumption of lettuce still revealed a toxicological risk from Pb in all treatments.

Transcriptome analysis and anthocyanin-related genes in red leaf lettuce.

This study aimed to analyze the transcriptome profile of red lettuce and identify the genes involved in anthocyanin accumulation. Red leaf lettuce is a popular vegetable and popular due to its high anthocyanin content. However, there is limited information available about the genes involved in anthocyanin biosynthesis in this species. In this study, transcriptomes of 15-day-old seedlings and 40-day-old red lettuce leaves were analyzed using an Illuminia HiseqTM 2500 platform. A total of 10.6 GB clean data were obtained and de novo assembled into 83,333 unigenes with an N50 of 1067. After annotation against public databases, 51,850 unigene sequences were identified, among which 46,087 were annotated in the NCBI non-redundant protein database, and 41,752 were annotated in the Swiss-Prot database. A total of 9125 unigenes were mapped into 163 pathways using the Kyoto Encyclopedia of Genes and Genomes database. Thirty-four structural genes were found to cover the main steps of the anthocyanin pathway, including chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase. Seven MYB, three bHLH, and two WD40 genes, considered anthocyanin regulatory genes, were also identified. In addition, 3607 simple sequence repeat (SSR) markers were identified from 2916 unigenes. This research uncovered the transcriptomic characteristics of red leaf lettuce seedlings and mature plants. The identified candidate genes related to anthocyanin biosynthesis and the detected SSRs provide useful tools for future molecular breeding studies.

Identifying N fertilizer regime and vegetable production system in tropical Brazil using (15) N natural abundance.

BACKGROUND: This study was conducted in areas of vegetable production in tropical Brazil, with the objectives of (i) measuring the variation in δ(15) N in soils, organic N fertilizer sources and lettuce (Lactuca sativa L.) from different farming systems, (ii) measuring whether plant δ(15) N can differentiate organic versus conventional lettuce and (iii) identifying the factors affecting lettuce δ(15) N. RESULTS: Samples of soil, lettuce and organic inputs were taken from two organic, one conventional and one hydroponic farm. The two organic farms had different N-sources with δ(15) N values ranging from 0.0 to +14.9‰ (e.g. leguminous green manure and animal manure compost, respectively), and differed significantly (P < 0.05) in lettuce δ(15) N (+9.2 ± 1.1‰ and +14.3 ± 1.0‰). Conventional lettuce δ(15) N (+8.5 ± 2.7‰) differed from hydroponic lettuce δ(15) N (+4.5 ± 0.2‰) due to manure inputs. The N from leguminous green manure made a small contribution to the N nutrition of lettuce in the multi-N-source organic farm. CONCLUSION: To differentiate organic versus conventional farms using δ(15) N the several subsets of mode of fertilization should be considered. Comparisons of δ(15) N of soil, organic inputs and lettuce allowed a qualitative analysis of the relative importance of different N inputs.

Silencing the lettuce homologs of small rubber particle protein does not influence natural rubber biosynthesis in lettuce (Lactuca sativa).

Natural rubber, cis-1,4-polyisoprene, is an important raw material in chemical industries, but its biosynthetic mechanism remains elusive. Natural rubber is known to be synthesized in rubber particles suspended in laticifer cells in the Brazilian rubber tree (Hevea brasiliensis). In the rubber tree, rubber elongation factor (REF) and its homolog, small rubber particle protein (SRPP), were found to be the most abundant proteins in rubber particles, and they have been implicated in natural rubber biosynthesis. As lettuce (Lactuca sativa) can synthesize natural rubber, we utilized this annual, transformable plant to examine in planta roles of the lettuce REF/SRPP homologs by RNA interference. Among eight lettuce REF/SRPP homologs identified, transcripts of two genes (LsSRPP4 and LsSRPP8) accounted for more than 90% of total transcripts of REF/SRPP homologs in lettuce latex. LsSRPP4 displays a typical primary protein sequence as other REF/SRPP, while LsSRPP8 is twice as long as LsSRPP4. These two major LsSRPP transcripts were individually and simultaneously silenced by RNA interference, and relative abundance, polymer molecular weight, and polydispersity of natural rubber were analyzed from the LsSRPP4- and LsSRPP8-silenced transgenic lettuce. Despite previous data suggesting the implications of REF/SRPP in natural rubber biosynthesis, qualitative and quantitative alterations of natural rubber could not be observed in transgenic lettuce lines. It is concluded that lettuce REF/SRPP homologs are not critically important proteins in natural rubber biosynthesis in lettuce.

Capture and accumulation of perchlorate in lettuce. Effect of genotype, temperature, perchlorate concentration, and competition with anions.

Various studies have evaluated the accumulation of ClO4(-) in lettuce (Lactuca sativa), but very few have dealt with the variables that can interfere with its capture. The present study evaluates the transfer of ClO4(-) in two L. sativa varieties: butter head (L. sativa var. capitata) and cos lettuce (L. sativa var. crispa) under hydroponic conditions. The ClO4(-) concentrations used correspond to levels (1 and 2mgL(-1)), measured in irrigation water in the Iquique region in the north of Chile. Results indicate that the capture of ClO4(-) is dependent on its concentration, lettuce genotype, and temperature. The butter head variety accumulates the highest perchlorate concentrations. Anion competition involving NO3(-) (16 and 48mM), Cl(-) (23 and 56mM), and SO4(2-) (10 and 20mM) was evaluated, being NO3(-) (48mM), the most significant competition reducing the concentration of ClO4(-) in tissues of L. sativa varieties.

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.