Boosting pro-vitamin A content and bioaccessibility in leaves by combining engineered biosynthesis and storage pathways with high-light treatments.

Biofortification of green leafy vegetables with pro-vitamin A carotenoids, such as ß-carotene, has remained challenging to date. Here, we combined two strategies to achieve this goal. One of them involves producing ß-carotene in the cytosol of leaf cells to avoid the negative impacts on photosynthesis derived from changing the balance of carotenoids and chlorophylls in chloroplasts. The second approach involves the conversion of chloroplasts into non-photosynthetic, carotenoid-overaccumulating chromoplasts in leaves agroinfiltrated or infected with constructs encoding the bacterial phytoene synthase crtB, leaving other non-engineered leaves of the plant to sustain normal growth. A combination of these two strategies, referred to as strategy C (for cytosolic production) and strategy P (for plastid conversion mediated by crtB), resulted in a 5-fold increase in the amount of ß-carotene in Nicotiana benthamiana leaves. Following several attempts to further improve ß-carotene leaf contents by metabolic engineering, hormone treatments and genetic screenings, it was found that promoting the proliferation of plastoglobules with increased light-intensity treatments not only improved ß-carotene accumulation but it also resulted in a much higher bioaccessibility. The combination of strategies C and P together with a more intense light treatment increased the levels of accessible ß-carotene 30-fold compared to controls. We further demonstrated that stimulating plastoglobule proliferation with strategy P, but also with a higher-light treatment alone, also improved ß-carotene contents and bioaccessibility in edible lettuce (Lactuca sativa) leaves.

LsRTDv1, a reference transcript dataset for accurate transcript-specific expression analysis in lettuce.

Accurate quantification of gene and transcript-specific expression, with the underlying knowledge of precise transcript isoforms, is crucial to understanding many biological processes. Analysis of RNA sequencing data has benefited from the development of alignment-free algorithms which enhance the precision and speed of expression analysis. However, such algorithms require a reference transcriptome. Here we generate a reference transcript dataset (LsRTDv1) for lettuce (cv. Saladin), combining long- and short-read sequencing with publicly available transcriptome annotations, and filtering to keep only transcripts with high-confidence splice junctions and transcriptional start and end sites. LsRTDv1 identifies novel genes (mostly long non-coding RNAs) and increases the number of transcript isoforms per gene in the lettuce genome from 1.4 to 2.7. We show that LsRTDv1 significantly increases the mapping rate of RNA-seq data from a lettuce time-series experiment (mock- and Botrytis cinerea-inoculated) and enables detection of genes that are differentially alternatively spliced in response to infection as well as transcript-specific expression changes. LsRTDv1 is a valuable resource for investigation of transcriptional and alternative splicing regulation in lettuce.

The genome of Lactuca saligna, a wild relative of lettuce, provides insight into non-host resistance to the downy mildew Bremia lactucae.

Lactuca saligna L. is a wild relative of cultivated lettuce (Lactuca sativa L.), with which it is partially interfertile. Hybrid progeny suffer from hybrid incompatibility (HI), resulting in reduced fertility and distorted transmission ratios. Lactuca saligna displays broad-spectrum resistance against lettuce downy mildew caused by Bremia lactucae Regel and is considered a non-host species. This phenomenon of resistance in L. saligna is called non-host resistance (NHR). One possible mechanism behind this NHR is through the plant-pathogen interaction triggered by pathogen recognition receptors, including nucleotide-binding leucine-rich repeat (NLR) proteins and receptor-like kinases (RLKs). We report a chromosome-level genome assembly of L. saligna (accession CGN05327), leading to the identification of two large paracentric inversions (>50 Mb) between L. saligna and L. sativa. Genome-wide searches delineated the major resistance clusters as regions enriched in NLRs and RLKs. Three of the enriched regions co-locate with previously identified NHR intervals. RNA-seq analysis of Bremia-infected lettuce identified several differentially expressed RLKs in NHR regions. Three tandem wall-associated kinase-encoding genes (WAKs) in the NHR8 interval display particularly high expression changes at an early stage of infection. We propose RLKs as strong candidates for determinants of the NHR phenotype of L. saligna.

Integrated transcriptomic and metabolomic analysis provides insights into cold tolerance in lettuce (Lactuca sativa L.).

The popular leafy vegetable lettuce (Lactuca sativa L.) is susceptible to cold stress during the growing season, which slows growth rate, causes leaf yellowing and necrosis, and reduced yield and quality. In this study, transcriptomic and metabolomic analyses of two cold-resistant lettuce cultivars (GWAS-W42 and F11) and two cold-sensitive lettuce cultivars (S13K079 and S15K058) were performed to identify the mechanisms involved in the cold response of lettuce. Overall, transcriptome analysis identified 605 differentially expressed genes (DEGs), including significant enrichment of genes involved in the flavonoid and flavonol (CHS, CHI, F3H, FLS, CYP75B1, HCT, etc.) biosynthetic pathways related to oxidation-reduction and catalytic activity. Untargeted metabolomic analysis identified fifteen flavonoid metabolites and 28 other metabolites potentially involved in the response to cold stress; genistein, quercitrin, quercetin derivatives, kaempferol derivatives, luteolin derivatives, apigenin and their derivatives accumulate at higher levels in cold-resistant cultivars. Moreover, MYBs, bHLHs, WRKYs and Dofs also play positive role in the low temperature response, which affected the expression of structural genes contributing to the variation of metabolites between the resistant and sensitive. These results provide valuable evidence that the metabolites and genes involved in the flavonoid biosynthetic pathway play important roles in the response of lettuce to cold stress.

Lactuca super-pangenome reduces bias towards reference genes in lettuce research.

BACKGROUND: Breeding of lettuce (Lactuca sativa L.), the most important leafy vegetable worldwide, for enhanced disease resistance and resilience relies on multiple wild relatives to provide the necessary genetic diversity. In this study, we constructed a super-pangenome based on four Lactuca species (representing the primary, secondary and tertiary gene pools) and comprising 474 accessions. We include 68 newly sequenced accessions to improve cultivar coverage and add important foundational breeding lines. RESULTS: With the super-pangenome we find substantial presence/absence variation (PAV) and copy-number variation (CNV). Functional enrichment analyses of core and variable genes show that transcriptional regulators are conserved whereas disease resistance genes are variable. PAV-genome-wide association studies (GWAS) and CNV-GWAS are largely congruent with single-nucleotide polymorphism (SNP)-GWAS. Importantly, they also identify several major novel quantitative trait loci (QTL) for resistance against Bremia lactucae in variable regions not present in the reference lettuce genome. The usability of the super-pangenome is demonstrated by identifying the likely origin of non-reference resistance loci from the wild relatives Lactuca serriola, Lactuca saligna and Lactuca virosa. CONCLUSIONS: The super-pangenome offers a broader view on the gene repertoire of lettuce, revealing relevant loci that are not in the reference genome(s). The provided methodology and data provide a strong basis for research into PAVs, CNVs and other variation underlying important biological traits of lettuce and other crops.

Role of gamma-irradiated sodium alginate on growth, physiological and active components of iceberg lettuce (Lactuca sativa) plant.

BACKGROUND: One of the most widely recognized biostimulators of plant development; is oligoalginate, which regulates the biological processes of plants and was used in horticultural fields as a plant growth regulator. The plan of the current research was to study, however, the foliar application of un-irradiated and irradiated Na-alginate (UISA and ISA) to improve the growth, physiological activity, and other active components of the Egyptian iceberg lettuce plant. Degraded Na-alginate is equipped with exposure of sodium alginate in its solid state to gamma-rays at different dose levels (0.0, 25, 50, 75, and 100 kGy). The characterization of the oligo-alginates achieved by γ-radiation deprivation at different dose levels was performed by FTIR, XRD, TGA, SEM, and TEM. Different concentrations of irradiated sodium alginate at dose levels of 100 kGy (200, 400, 600, and 800 ppm, as well as deionized water used as a control) were sprayed with a hand sprayer every week after transplanting the iceberg lettuce seedlings in the field until the harvest stage. Morphological traits were evaluated, as well as pigments, ascorbic acid, phenols, flavonoids, soluble proteins, and antioxidant activity. RESULTS: Irradiated Na-alginate resulted in the depolymerization of Na-alginate into small molecular-weight oligosaccharides, and the best dose to use was 100 kGy. Certain chemical modifications in the general structure were observed by FTIR analysis. Two absorbed bands at 3329 cm-1 and 1599 cm-1, were recognized that are assigned to O-H and C-O stretching, respectively, and peaks achieved at 1411 cm-1 represent the COO-stretching group connected to the sodium ion. The peak obtained at 1028 cm-1 was owing to the stretching vibration of C-O. The results of TGA provided that the minimum weight reminder was in the ISA at 100 kGy (28.12%) compared to the UISA (43.39%). The images of TEM pointed out that the Na-alginate was globular in shape, with the particle distribution between 12.8 and 21.7 nm in ISA at 100 kGy. Irradiated sodium alginate caused a noteworthy enhancement in the vegetative growth traits (leaf area, stem length, head weight, and leaf number). By spraying 400 ppm, ISA showed a maximum increase in total pigments (2.209 mg/g FW), ascorbic acid (3.13 mg/g fresh weight), phenols (1.399 mg/g FW), flavonoids (0.775 mg/g FW), and antioxidant activities (82.14. %). Also, there were correlation coefficients (R values) between leaf area, stem length, head weight, and leaf number values with total pigment content, antioxidant activity, total soluble proteins, and ascorbic acid. CONCLUSIONS: The outcomes of the recent investigation demonstrated that the application of spraying irradiated Na-alginate (100 kGy) resulted in an improvement of the considered characters.

Time of arrival during plant disease progression and humidity additively influence Salmonella enterica colonization of lettuce.

The interplay between plant hosts, phytopathogenic bacteria, and enteric human pathogens in the phyllosphere has consequences for human health. Salmonella enterica has been known to take advantage of phytobacterial infection to increase its success on plants, but there is little knowledge of additional factors that may influence the relationship between enteric pathogens and plant disease. In this study, we investigated the role of humidity and the extent of plant disease progression on S. enterica colonization of plants. We found that high humidity was necessary for the replication of S. enterica on diseased lettuce, but not required for S. enterica ingress into the UV-protected apoplast. Additionally, the Xanthomonas hortorum pv. vitians (hereafter, X. vitians)-infected lettuce host was found to be a relatively hostile environment for S. enterica when it arrived prior to the development of watersoaking or following necrosis onset, supporting the existence of an ideal window during X. vitians infection progress that maximizes S. enterica survival. In vitro growth studies in sucrose media suggest that X. vitians may allow S. enterica to benefit from cross-feeding during plant infection. Overall, this study emphasizes the role of phytobacterial disease as a driver of S. enterica success in the phyllosphere, demonstrates how the time of arrival during disease progress can influence S. enterica's fate in the apoplast, and highlights the potential for humidity to transform an infected apoplast into a growth-promoting environment for bacterial colonizers. IMPORTANCE: Bacterial leaf spot of lettuce caused by Xanthomonas hortorum pv. vitians is a common threat to leafy green production. The global impact caused by phytopathogens, including X. vitians, is likely to increase with climate change. We found that even under a scenario where increased humidity did not enhance plant disease, high humidity had a substantial effect on facilitating Salmonella enterica growth on Xanthomonas-infected plants. High humidity climates may directly contribute to the survival of human enteric pathogens in crop fields or indirectly affect bacterial survival via changes to the phyllosphere brought on by phytopathogen disease.

A WOX homolog disrupted by a transposon led to the loss of spines and contributed to the domestication of lettuce.

The loss of spines is one of the most important domestication traits for lettuce (Lactuca sativa). However, the genetics and regulation of spine development in lettuce remain unclear. We examined the genetics of spines in lettuce using a segregating population derived from a cross between cultivated and wild lettuce (Lactuca serriola). A gene encoding WUSCHEL-related homeobox transcription factor, named as WOX-SPINE1 (WS1), was identified as the candidate gene controlling the spine development in lettuce, and its function on spines was verified. A CACTA transposon was found to be inserted into the first exon of the ws1 allele, knocking out its function and leading to the lack of spines in cultivated lettuce. All lettuce cultivars investigated have the nonfunctional ws1 gene, and a selection sweep was found at the WS1 locus, suggesting its important role in lettuce domestication. The expression levels of WS1 were associated with the density of spines among different accessions of wild lettuce. At least two independent loss-of-function mutations in the ws1 gene caused the loss of spines in wild lettuce. These findings provide new insights into the development of spines and facilitate the exploitation of wild genetic resources in future lettuce breeding programs.

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.

Genome assembly and transcriptomic analysis to elucidate the ability of Nasonovia ribisnigri to break host plant resistance.

Aphid genomic resources enable the study of complex life history traits and provide information on vector biology, host adaption and speciation. The currant-lettuce aphid (Nasonovia ribisnigri (Hemiptera: Aphididae) (Mosley)) is a cosmopolitan pest of outdoor lettuce (Lactuca sativa (Asterales: Asteraceae) (Linnaeus)). Until recently, the use of resistant cultivars was an effective method for managing N. ribisnigri. A resistant cultivar containing a single gene (Nr-locus), introduced in the 1980s, conferred complete resistance to feeding. Overreliance of this Nr-locus in lettuce resulted in N. ribisnigri's ability to break resistance mechanism, with first reports during 2003. Our work attempts to understand which candidate gene(s) are associated with this resistance-breaking mechanism. We present two de novo draft assembles for N. ribisnigri genomes, corresponding to both avirulent (Nr-locus susceptible) and virulent (Nr-locus resistant) biotypes. Changes in gene expression of the two N. ribisnigri biotypes were investigated using transcriptomic analyses of RNA-sequencing (RNA-seq) data to understand the potential mechanisms of resistance to the Nr-locus in lettuce. The draft genome assemblies were 94.2% and 91.4% complete for the avirulent and virulent biotypes, respectively. Out of the 18,872 differentially expressed genes, a single gene/locus was identified in N. ribisnigri that was shared between two resistant-breaking biotypes. This locus was further explored and validated in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) experiments and has predicted localisations in both the cytoplasm and nucleus. This is the first study to provide evidence that a single gene/locus is likely responsible for the ability of N. ribisnigri to overcome the Nr-locus resistance in the lettuce host.

Public perception of folate-biofortified genetically modified lettuce varieties in Brazil.

Lettuce is one of the most widely consumed vegetables in the world, commonly eaten fresh in salads, sandwiches, wraps, and as a garnish in various dishes. Consequently, it is a very promising vehicle to deliver vitamins, such as folate (vitamin B9), to a specific population using biofortified varieties generated by conventional or molecular breeding. A new genetically modified lettuce was generated with increased folate content. However, some issues related to public perception regarding this technology should still be evaluated. The aim of this study was to analyze whether consumers are willing to accept a folate-biofortified GM lettuce that could become available to the Brazilian market. A questionnaire involving several issues regarding lettuce consumption was answered by 2,391 people from almost all Brazilian states. When informed that the folic acid biofortified lettuce is a transgenic plant, 46.1% of respondents stated that they would eat it and 30.5% stated that it would be a possibility. This study demonstrated that if there is any explanation regarding the advantage in relation to the use of biotechnology, like enrichment with folic acid, the number of people who accept it increases.

Effects of dietary nitrate supplementation on oral health and associated markers of systemic health: a systematic review.

Poor oral health can impact an individual's ability to eat and has been associated with an increased risk of non-communicable diseases. While the benefits of nitrate consumption on oral health were first proposed more than 20 years ago, no systematic review has been published examining effects of dietary nitrate on oral health. This systematic review investigated the effects of dietary nitrate on markers of oral health in vivo in randomized controlled trials (RCTs). Five databases (PubMed, The Cochrane Library, CINAHL, MEDLINE, and SPORTDiscus) were searched from inception until March 2023. Nine articles reporting data on 284 participants were included. Dietary nitrate was provided via beetroot juice in most studies. The duration of the interventions ranged from one day to six weeks. Dietary nitrate supplementation increased the relative abundance of several individual bacterial genera including Neisseria and Rothia. Dietary nitrate supplementation increased salivary pH and decreased salivary acidification following consumption of a sugar-sweetened beverage. Furthermore, dietary nitrate supplementation resulted in a decrease in the gingival inflammation index. The results of this systematic review suggest that dietary nitrate could represent a potential nutritional strategy to positively modify oral health by impacting the oral microbiome, altering salivary pH, and minimizing gingival inflammation.

Arbuscular Mycorrhizal Fungus Alleviates Charged Nanoplastic Stress in Host Plants via Enhanced Defense-Related Gene Expressions and Hyphal Capture.

Contamination of small-sized plastics is recognized as a factor of global change. Nanoplastics (NPs) can readily enter organisms and pose significant ecological risks. Arbuscular mycorrhizal (AM) fungi are the most ubiquitous and impactful plant symbiotic fungi, regulating essential ecological functions. Here, we first found that an AM fungus, Rhizophagus irregularis, increased lettuce shoot biomass by 25-100% when exposed to positively and negatively charged NPs vs control, although it did not increase that grown without NPs. The stress alleviation was attributed to the upregulation of gene expressions involving phytohormone signaling, cell wall metabolism, and oxidant scavenging. Using a root organ-fungus axenic growth system treated with fluorescence-labeled NPs, we subsequently revealed that the hyphae captured NPs and further delivered them to roots. NPs were observed at the hyphal cell walls, membranes, and spore walls. NPs mediated by the hyphae were localized at the root epidermis, cortex, and stele. Hyphal exudates aggregated positively charged NPs, thereby reducing their uptake due to NP aggregate formation (up to 5000 nm). This work demonstrates the critical roles of AM fungus in regulating NP behaviors and provides a potential strategy for NP risk mitigation in terrestrial ecosystems. Consequent NP-induced ecological impacts due to the affected AM fungi require further attention.

Toxic and non-toxic cyanobacterial biomass as a resource for sustainable agriculture: A lettuce cultivation experiment.

Cyanobacteria represent a promising resource for sustainable agriculture, as they have demonstrated the ability to restore soil fertility even after death and decay. However, several cyanobacteria can also release secondary metabolites, such as cyanotoxins, which may compromise the quality of agricultural products and pose a potential risk to human health. Depending on the concentration of exposure, few studies reported deleterious effects on plant species when irrigated with cylindrospermopsin (CYN) contaminated water, impairing plant growth and leading to food product contamination, while other studies show promoting effects on plant yield. To evaluate the potential of cyanobacterial biomass (cyanotoxin-containing or not) as a sustainable resource for soil amendment, biostimulants or fertilizers for lettuce cultivation, a study was carried out that consisted of the culture of lettuce plants under controlled conditions, in soil: (1) with no extra nutrient addition (control) and supplemented with 0.6 g of freeze-dried Raphidiopsis raciborskii biomass of (2) a non-CYN-producing strain, (3) a CYN-producing strain, and (4) the same CYN-producing strain pasteurized. Results showed no significant differences in photosystem II efficiency with the amendment addition. On the contrary, shoot fresh weight significantly increased in lettuce plants grown with the cyanobacterial biomass addition, especially in condition (3). In addition, there were significant differences in mineral concentrations in lettuce leaves after the cyanobacterial biomass addition, such as K, Na, Ca, P, Mg, Mn, Zn, Cu, Mo, and Co. CYN accumulation was detected under conditions (3) and (4), with concentrations observed in descending order from roots > soil > shoot. Nevertheless, the CYN concentration in edible tissues did not exceed the WHO-proposed tolerable daily intake of 0.03 µg/kg/day. These findings suggest that incorporating cyanobacterial biomass as a soil amendment, biostimulant or fertilizer for lettuce cultivation, even with trace amounts of CYN (1-40 µg/g), may enhance plant yield without leading to cyanotoxin accumulation in edible tissues above the WHO-recommended tolerable daily intake.

Research on precise phenotype identification and growth prediction of lettuce based on deep learning.

In recent years, precision agriculture, driven by scientific monitoring, precise management, and efficient use of agricultural resources, has become the direction for future agricultural development. The precise identification and assessment of phenotypes, which serve as external representations of a crop's growth, development, and genetic characteristics, are crucial for the realization of precision agriculture. Applications surrounding phenotypic indices also provide significant technical support for optimizing crop cultivation management and advancing smart agriculture, contributing to the efficient and high-quality development of precision agriculture.This paper focuses on lettuce and employs common nutritional stress conditions during growth as experimental settings. By collecting RGB images throughout the lettuce's complete growth cycle, we developed a deep learning-based computational model to tackle key issues in the lettuce's growth and precisely identify and assess phenotypic indices. We discovered that some phenotypic indices, including custom ones defined in this study, are representative of the lettuce's growth status. By dynamically monitoring the changes in phenotypic traits during growth, we quantitatively analyzed the accumulation and evolution of phenotypic indices across different growth stages. On this basis, a predictive model for lettuce growth and development was trained.The model incorporates MSE, SSIM, and perceptual loss, significantly enhancing the predictive accuracy of the lettuce growth images and phenotypic indices. The model trained with the reconstructed loss function outperforms the original model, with the SSIM and PSNR improving by 1.33% and 10.32%, respectively. The model also demonstrates high accuracy in predicting lettuce phenotypic indices, with an average error less than 0.55% for geometric indices and less than 1.7% for color and texture indices. Ultimately, it achieves intelligent monitoring and management throughout the lettuce's life cycle, providing technical support for high-quality and efficient lettuce production.

Effects of vermicompost on soil microbiological properties in lettuce rhizosphere: An environmentally friendly approach for sustainable green future.

The aim of this study is to investigate the effects of vermicompost on the biological and microbial properties of lettuce rhizosphere in an agricultural field in Samsun, Turkey. The experiment was conducted in a completely randomised design (CRD) and included four vermicompost dosages (0%, 1%, 2%, and 4%) and two application methods (with and without plants). Batavia lettuce was selected as the test plant due to its sensitivity to environmental conditions and nutrient deficiencies. The study evaluated the changes in organic matter (OM), pH, electrical conductivity (EC), carbon dioxide (CO2), dehydrogenase activity (DHA), microbial biomass carbon (MBC), and catalase activity (CA) in the rhizosphere of lettuce plants treated with different vermicompost levels (0%, 1%, 2%, and 4%). The findings showed that vermicompost application significantly increased chlorophyll content in lettuce plants, with the highest content observed in plants treated with V1 compared to the control. Different vermicompost concentrations also influenced chlorophyll b and total chlorophyll levels, with positive effects observed at lower concentrations than the control. Plant height and fresh weight were highest in plants treated with V2, indicating the positive impact of vermicompost on plant growth. Additionally, vermicompost application increased plant dry weight and improved soil properties such as pH, organic matter content, and microbial activity. The findings showed that vermicompost increased the rhizosphere's microbial biomass and metabolic activity, which can be beneficial for plant growth and disease suppression. The study highlights the importance of understanding the effects of organic amendments on soil properties and the microbial community in the rhizosphere, which can contribute to sustainable agricultural practices. Overall, the results suggest that vermicompost can be used as an effective organic amendment for enhancing plant growth and improving soil properties in agricultural fields. Moreover, based on the data, it can be suggested that a dose between 1% and 2% vermicompost is beneficial for the overall growth of plants.

Silencing of RDR1 and RDR6 genes by a single RNAi enhances lettuce's capacity to express recombinant proteins in transient assays.

KEY MESSAGE: Enhanced recombinant protein expression was achieved in Salinas lettuce and commercial lettuce by designing a unique RNAi that knockdown the gene-silencing mechanism in transient assays. Improved yields of recombinant proteins (RP) are necessary for protein-production efficiency and ease of purification. Achieving high yield in non-tobacco plants will enable diverse plants to be used as hosts in transient protein-expression systems. With improved protein yield, lettuce (Lactuca sativa) could take the lead as a plant host for RP production. Therefore, this study aimed to improve RP production in lettuce var. Salinas by designing a single RNA interference (RNAi) construct targeting LsRDR1 and LsRDR6 using the Tsukuba system vector. Two RNAi constructs, RNAi-1 and RNAi-2, targeting common regions of LsRDR1 and LsRDR6 with 75% and 76% similarity, respectively, were employed to evaluate simultaneous gene silencing. Quantitative transcription analysis demonstrated that both RNAi constructs effectively knocked down LsRDR6 and LsRDR1, but not LsRDR2, at both 3 and 5 days post-infiltration (dpi), with RNAi-1 exhibited slightly higher efficiency. Based on the protein yield, co-expression of RNAi-1 with enhanced green fluorescent protein (EGFP) increased EGFP expression by approximately 4.9-fold and 3.7-fold at 3 dpi and 5 dpi, respectively, compared to control. A similar but slightly lower increase (2.4-fold and 2.33-fold) was observed in commercial lettuce at 3 and 5 dpi, respectively. To confirm these results, co-infiltration with Bet v 1, a major allergen from birch pollen, resulted in a 2.5-fold increase in expression in Salinas lettuce at 5 dpi. This study marks a significant advancement in enhancing transient protein production in lettuce, elevating its potential as a host for recombinant protein production.

LsMybW-encoding R2R3-MYB transcription factor is responsible for a shift from black to white in lettuce seed.

KEY MESSAGE: We identified LsMybW as the allele responsible for the shift in color from black to white seeds in wild ancestors of lettuce to modern cultivars. Successfully selected white seeds are a key agronomic trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black-in its wild ancestor-to white seeds remains uncertain. We aimed to identify the gene/s responsible for white seed trait in lettuce. White seeds accumulated less proanthocyanidins than black seeds, similar to the phenotype observed in Arabidopsis TT2 mutants. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F2 population derived from a cross between "ShinanoPower" (white) and "Escort" (black). The white seed trait was controlled by a single recessive locus (48.055-50.197 Mbp) in linkage group 7. Using five PCR-based markers and numerous cultivars, eight candidate genes were mapped in the locus. Only the LG7_v8_49.251Mbp_HinfI marker, employing a single-nucleotide mutation in the stop codon of Lsat_1_v5_gn_7_35020.1, was completely linked to seed color phenotype. In addition, the coding region sequences for other candidate genes were identical in the resequence analysis of "ShinanoPower" and "Escort." Therefore, we proposed Lsat_1_v5_gn_7_35020.1 as the candidate gene and designated it as LsMybW (Lactuca sativa Myb White seeds), an ortholog encoding the R2R3-MYB transcription factor in Arabidopsis. When we validated the role of LsMybW through genome editing, LsMybW knockout mutants harboring an early termination codon showed a change in seed color from black to white. Therefore, LsMybW was the allele responsible for the shift in seed color. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds have implications for future breeding technology and physiological analysis.

Lettuce seed pelleting with Pseudomonas sp. 31-12: plant growth promotion under laboratory and greenhouse conditions.

Plant growth promotion by Pseudomonas sp. 31-12 incorporated into a lettuce seed pelleting matrix was studied. We examined (1) the effect of five rhizosphere derived bacterial strains on green oak lettuce (Lactuca sativa L.) seed germination, root and shoot growth, as a strain selection step for seed coating and seed pelletizing studies, (2) population stability of Pseudomonas sp. 31-12 incorporated into a pelleting matrix on lettuce seed stored three months at 4 °C, and (3) lettuce growth promotion in the laboratory and greenhouse by Pseudomonas sp. 31-12 coated and pelletized seed. A spontaneous streptomycin mutant of Pseudomonas sp. 31-12 (str) was used to determine population size on seed and roots of 15- and 30-day-old lettuce. The population of Pseudomonas sp. 31-12str on coated and pelleted seed decreased from 104 cfu/seed to 103 cfu/seed after 3 months storage at 4 °C. However, the population exceeded 104 cfu/g root dry mass and 105/g root dry mass after 15 days and 30 days in the greenhouse. Leaf fresh mass was significantly increased (P ≤ 0.05) with Pseudomonas sp. 31-12 seed treatment as compared to noninoculated seed. In conclusion, pelletized lettuce seed with Pseudomonas sp. 31-12 promoted growth and yield in the greenhouse.

Effects of a S-metolachlor based herbicide on two plant models: Zea mays L. and Lactuca sativa L.

Corn is the second most cultivated crop in Brazil, the number-one country in pesticide consumption. Chemical control of weeds is performed using herbicides such as S-metolachlor with pre- and post-emergence action and thus the toxicity of herbicides constitutes a matter of great concern. The present investigation aimed to examine the effects of an S-metolachlor-based herbicide on Lactuca sativa L. (lettuce) and Zea mays L. (maize) utilizing various bioassays. The test solutions were prepared from commercial products containing the active ingredient. Seeds from the plant models were exposed in petri dishes and maintained under biochemical oxygen demand (BOD) at 24°C. Distilled water was negative and aluminium positive control. Macroscopic analyses (germination and growth) were conducted for both plant species, and microscopic analysis (cell cycle and chromosomal alterations) were performed for L. sativa root tip cells. Detrimental interference of S-metolachlor-based herbicide was noted with lettuce for all parameters tested reducing plant germination by over 50% and the germination speed by over 45% and showing a significant decrease in mitotic index, from 16.25% to 9,28% even on the lowest concentration tested. In maize, there was no significant interference in plant germination; however, speed of germination was significantly hampered, reaching a 51.22% reduction for the highest concentration tested. Data demonstrated that the herbicide was toxic as evidenced by its phyto- and cytotoxicity in L. sativa L. and Z. mays L.