Physiological and metabolic alterations in basil (Ocimum basilicum L) varieties under distinct soil water levels / Alteraciones fisiológicas y metabólicas en variedades de albahaca (Ocimum basilicum L) bajo distintos niveles de agua del suelo

Basil (Ocimum basilicumL.) is a medicinal species used in several areas, such as food, medicines and cosmetics, and the understanding of its physiological behavior under environmental conditions is of paramount importance for the improvement of cultivation methods. The objective of this study was to evaluate the influence of different water availability under physiological, biochemical and metabolic characteristics, in three distinct genotypes: 'Alfavaca basilicão', 'Gennaro de menta' and 'Grecco à palla', during two different phenological stages (vegetative and reproductive). It was found that the water deficit promotes physiological changes to tolerate water stress, and the studied genotypes have different routes to achieve this physiological tolerance, which culminates in a distinct accumulation of metabolites in plants, and can be considered interesting if the final product is the production of essential oils.

La albahaca (Ocimum basilicum L.) es una planta medicinal utilizada en varias áreas: alimenticia, medicinal e industria cosmética; es de suma importancia el entendimiento de su comportamiento fisiológico bajo diferentes condiciones ambientales con el fin de mejorar los procesos del cultivo. El objetivo de este estudio fue evaluar la influencia de diferentes disponibilidades hídricas en las características fisiológicas, bioquímicas y metabólicas en tres genotipos de albahaca: "Alfavaca basilicão", "Gennaro de menta" y "Grecco à palla" durante dos etapas fenológicas (vegetativa y reproductiva). Fue encontrado que el déficit hídrico promueve cambios fisiológicos con el fin de tolerar el estrés hídrico. Los genotipos estudiados presentaron diferentes rutas para alcanzar esta tolerancia fisiológica, la cual culmina con distintas acumulaciones de metabolitos en las plantas, y puede ser considerado interesante si el producto final es la producción de aceites esenciales.

Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms.

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

Molecular cloning, functional characterization and expression of a drought inducible phenylalanine ammonia-lyase gene (ObPAL) from Ocimum basilicum L.

Phenylalanine ammonia-lyase (PAL) is a control point for branched phenylpropanoid and terpenoid pathways. It represents the first regulatory step to provide a metabolic flux to produce of the precursors needed for biosynthesizing main volatile phenylpropanoid compounds (methyleugenol and methylchavicol) in basil. It is crucial during the stage of the environmental and development stimulants. To obtain better knowledge of the biosynthesis of these phenylpropene compounds, characterization and cloning of Ocimum basilicum PAL (ObPAL) cDNA and its heterologous expression and enzyme activity were assessed. The almost full-length ObPAL was 2064 bp in size encoding a 687-amino-acid polypeptide with molecular weight of 74.642 kDa and theoretical pI of 8.62. Phylogenetic analysis revealed a significant evolutionary relatedness of ObPAL with the PAL sequence reported in different species of Lamiaceae. To further confirm its function, ObPAL was cloned into pET28a (+) vector and expressed in E. coli. The recombinant protein exhibited high PAL activity and could catalyze the L-Phe conversion to trans-cinnamic acid. Expression analysis of PAL gene showed that ObPAL manifested various transcription ratios exposed to drought stress. Overall, our results demonstrated the ObPAL regulation gene is possibly a mechanism dependent on cultivar and drought stress.

Changes in visual quality, physiological and biochemical parameters assessed during the postharvest storage at chilling or non-chilling temperatures of three sweet basil (Ocimum basilicum L.) cultivars.

Leaves of three different sweet basil (Ocimum basilicum L.) cultivars (Italico a foglia larga, Cammeo, and Italiano classico) packed in macro-perforated polyethylene bags were stored at chilling (4°C) or non-chilling temperature (12°C) for 9days. During storage, visual quality, physiological (respiration rate, ethylene production, ammonium content) and chemical (antioxidant activity, total polyphenols and polyphenol profile) parameters were measured. Detached leaves stored at chilling temperature showed visual symptoms related to chilling injury, while ethylene production and ammonium content resulted associated to cultivar sensibility to damage at low temperature. Storage at 4°C caused a depletion in polyphenols content and antioxidant capability, which was preserved at 12°C. Regarding the polyphenols profile, stressful storage conditions did not enhance the phenolic metabolism. However, leaves stored at 12°C did not loss a significant amount of metabolites respect to fresh leaves, suggesting the possibility to extend the storability after the expiration date, for a possible recovery of bioactive compounds.

Dissecting molecular and physiological response mechanisms to high solar radiation in cyanic and acyanic leaves: a case study on red and green basil.

Photosynthetic performance and the expression of genes involved in light signaling and the biosynthesis of isoprenoids and phenylpropanoids were analysed in green ('Tigullio', TIG) and red ('Red Rubin', RR) basil. The aim was to detect the physiological and molecular response mechanisms to high sunlight. The attenuation of blue-green light by epidermal anthocyanins was shown to evoke shade-avoidance responses with consequential effects on leaf morpho-anatomical traits and gas exchange performance. Red basil had a lower mesophyll conductance, partially compensated by the less effective control of stomatal movements, in comparison with TIG. Photosynthesis decreased more in TIG than in RR in high sunlight, because of larger stomatal limitations and the transient impairment of PSII photochemistry. The methylerythritol 4-phosphate pathway promoted above all the synthesis and de-epoxidation of violaxanthin-cycle pigments in TIG and of neoxanthin and lutein in RR. This enabled the green leaves to process the excess radiant energy effectively, and the red leaves to optimize light harvesting and photoprotection. The greater stomatal closure observed in TIG than in RR was due to enhanced abscisic acid (ABA) glucose ester deglucosylation and reduced ABA oxidation, rather than to superior de novo ABA synthesis. This study shows a strong competition between anthocyanin and flavonol biosynthesis, which occurs at the level of genes regulating the oxidation of the C2-C3 bond in the dihydro-flavonoid skeleton.

A cold-tolerant evergreen interspecific hybrid of Ocimum kilimandscharicum and Ocimum basilicum: analyzing trichomes and molecular variations.

Ocimum (Lamiaceae) is an important source of essential oils and aroma chemicals especially eugenol, methyl eugenol, linalool, methyl chavicol etc. An elite evergreen hybrid has been developed from Ocimum kilimandscharicum and Ocimum basilicum, which demonstrated adaptive behavior towards cold stress. A comparative molecular analysis has been done through RAPD, AFLP, and ISSR among O. basilicum and O. kilimandscharicum and their evergreen cold-tolerant hybrid. The RAPD and AFLP analyses demonstrated similar results, i.e., the hybrid of O. basilicum and O. kilimandscharicum shares the same cluster with O. kilimandscharicum, while O. basilicum behaves as an outgroup, whereas in ISSR analysis, the hybrid genotype grouped in the same cluster with O. basilicum. Ocimum genotypes were analyzed and compared for their trichome density. There were distinct differences on morphology, distribution, and structure between the two kinds of trichomes, i.e., glandular and non-glandular. Glandular trichomes contain essential oils, polyphenols, flavonoids, and acid polysaccharides. Hair-like trichomes, i.e., non-glandular trichomes, help in keeping the frost away from the living surface cells. O. basilicum showed less number of non-glandular trichomes on leaves compared to O. kilimandscharicum and the evergreen cold-tolerant hybrid. Trichomes were analyzed in O. kilimandscharicum, O. basilicum, and their hybrid. An increased proline content at the biochemical level represents a higher potential to survive in a stress condition like cold stress. In our analysis, the proline content is quite higher in tolerant variety O. kilimandscharicum, low in susceptible variety O. basilicum, and intermediate in the hybrid. Gene expression analysis was done in O. basilicum, O. kilimandscharicum and their hybrid for TTG1, GTL1, and STICHEL gene locus which regulates trichome development and its formation and transcription factors WRKY and MPS involved in the regulation of plant responses to freezing and cold. The analysis showed that O. kilimandscharicum and the hybrid were very close to each other but O. basilicum was more distinct in all respects. The overexpression of the WRKY coding gene showed high expression in the hybrid as compared to O. kilimandscharicum and O. basilicum and the transcription factor microspore-specific (MPS) promoter has also shown overexpression in the hybrid for its response against cold stress. The developed evergreen interspecific hybrid may thus provide a base to various industries which are dependent upon the bioactive constituents of Ocimum species.

Stomatal density and metabolic determinants mediate salt stress adaptation and water use efficiency in basil (Ocimum basilicum L.).

Increasing salinity tolerance and water-use efficiency in crop plants are two major challenges that agriculture must face in the next decades. Many physiological mechanisms and molecular components mediating crop response to environmental stresses have been identified. However, the functional inter-links between stress adaptation responses have not been completely understood. Using two basil cultivars (Napoletano and Genovese) with contrasting ability to respond to salt stress, here we demonstrate that reduced stomatal density, high ascorbate level and polyphenol oxidase (PPO) activity coordinately contribute to improve basil adaptation and water use efficiency (WUE) in saline environment. The constitutively reduced stomatal density was associated with a "delayed" accumulation of stress molecules (and growth inhibiting signals) such as abscisic acid (ABA) and proline, in the more tolerant Genovese. Leaf volatile profiling also revealed cultivar-specific patterns, which may suggest a role for the volatile phenylpropanoid eugenol and monoterpenes in conferring stress tolerance via antioxidant and signalling functions.

Superação de dormência em sementes de manjericão (Ocimum basilicum L. ) / Overcoming dormancy in seeds of basil (Ocimum basilicum L. )

O objetivo do trabalho foi avaliar a eficiência de tratamentos pré-germinativos na superação da dormência de sementes de manjericão, produzidas no Horto de Plantas Medicinais da Unimontes, em fevereiro de 2011. Foram realizadas as seguintes determinações para avaliação da qualidade fisiológica das sementes: teor de água, germinação, primeira contagem de germinação, emergência de plântulas e índice de velocidade de emergência. O delineamento experimental utilizado foi inteiramente casualizado com quatro repetições de 50 sementes por tratamento, sendo T1- testemunha; T2 - pré esfriamento das sementes em câmara tipo BOD sob temperatura de 10ºC por 4 dias; T3 - embebição das sementes em água destilada por 24 horas; T4 - embebição das sementes em solução contendo KNO3 a 0,2 % por 5 minutos e T5 - sementes submetidas em água destilada a temperatura de 70ºC por 5 minutos. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste Scott - Knott a 5% de probabilidade. O tratamento pré esfriamento em câmara tipo BOD a 10ºC por 4 dias reduz a dormência e promove incrementos na qualidade fisiológica das sementes do manjericão.

Aiming in order to assess the effectiveness of treatments to overcome dormancy in seeds of basil, an experiment was conducted at the Laboratory of Seed Analysis of Unimontes. Following determinations were performed to evaluate the physiological quality of seeds, water content, germination, first count germination, seedling emergence and emergence speed index. The experimental design was completely randomized design with four replications of 50 seeds per treatment, which consisted of: T1 - control, T2 - pre-cooling of the seed chamber BOD at a temperature of 10ºC for 4 days, T3 - soaking the seeds in water distilled for 24 hours, T4 - soaking the seeds in a solution containing 0,2% for 5 minutes and T5 - submitted seeds in distilled water at 70ºC for 5 minutes. Data were subjected to analysis of variance and the averages compared by Scott-Knott 5% probability. The pre-cooling treatment in BOD chamber at 10ºC for 4 days reduced dormancy and promotes increases in the physiological quality of seeds of basil.