The advent of plant cells in bioreactors.

Ever since agriculture started, plants have been bred to obtain better yields, better fruits, or sustainable products under uncertain biotic and abiotic conditions. However, a new way to obtain products from plant cells emerged with the development of recombinant DNA technologies. This led to the possibility of producing exogenous molecules in plants. Furthermore, plant chemodiversity has been the main source of pharmacological molecules, opening a field of plant biotechnology directed to produce high quality plant metabolites. The need for different products by the pharma, cosmetics agriculture and food industry has pushed again to develop new procedures. These include cell production in bioreactors. While plant tissue and cell culture are an established technology, beginning over a hundred years ago, plant cell cultures have shown little impact in biotechnology projects, compared to bacterial, yeasts or animal cells. In this review we address the different types of bioreactors that are currently used for plant cell production and their usage for quality biomolecule production. We make an overview of Nicotiana tabacum, Nicotiana benthamiana, Oryza sativa, Daucus carota, Vitis vinifera and Physcomitrium patens as well-established models for plant cell culture, and some species used to obtain important metabolites, with an insight into the type of bioreactor and production protocols.

How binomial (traditional rainfed olive grove-Crocus sativus) crops impact the soil bacterial community and enhance microbial capacities.

Intercropping can favour the yield of the main crop. However, because of the potential competition among woody crops, this system is rarely used by farmers. To increase knowledge about the intercropping system, we have explored three different combinations of alley cropping in rainfed olive groves compared to conventional management (CP): (i) Crocus sativus (D-S); (ii) Vicia sativa/Avena sativa in annual rotation (D-O); and (iii) Lavandula x intermedia (D-L). Different soil chemical properties were analyzed to evaluate the effects of alley cropping, while 16S rRNA amplification and enzymatic activities were determined to study the changes that occurred in soil microbial communities and activity. In addition, the influence of intercropping on the potential functionality of the soil microbial community was measured. Data revealed that the intercropping systems highly affected the microbial community and soil properties. The D-S cropping system increased soil total organic carbon and total nitrogen that were correlated with the bacterial community, indicating that both parameters were the main drivers shaping the structure of the bacterial community. The D-S soil cropping system had significantly higher relative abundances of the phyla Bacteroidetes, Proteobacteria, and Patescibacteria compared to the other systems and the genera Adhaeribacter, Arthrobacter, Rubellimicrobium, and Ramlibacter, related to C and N functions. D-S soil was also related to the highest relative abundances of Pseudoarthrobacter and Haliangium, associated with the plant growth-promoting effect, antifungal activity, and a potential P solubilizer. A potentially increase of C fixation and N fixation in soils was also observed in the D-S cropping system. These positive changes were related to the cessation of tillage and the development of a spontaneous cover crop, which increased soil protection. Thus, management practices that contribute to increasing soil cover should be encouraged to improve soil functionality.

Molecular mechanisms involved in fruit cracking: A review.

Several fleshy fruits are highly affected by cracking, a severe physiological disorder that compromises their quality and causes high economical losses to the producers. Cracking can occur due to physiological, genetic or environmental factors and may happen during fruit growth, development and ripening. Moreover, in fleshy fruits, exocarp plays an important role, acting as a mechanical protective barrier, defending against biotic or abiotic factors. Thus, when biochemical properties of the cuticle + epidermis + hypodermis are affected, cracks appear in the fruit skin. The identification of genes involved in development such as cell wall modifications, biosynthesis and transport of cuticular waxes, cuticular membrane deposition and associated transcription factors provides new insights to better understand how fruit cracking is affected by genetic factors. Amongst the major environmental stresses causing cracking are excessive water during fruit development, leading to imbalances in cations such as Ca. This review focus on expression of key genes in these pathways, in their influence in affected fruits and the potential for molecular breeding programs, aiming to develop cultivars more resistant to cracking under adverse environmental conditions.

Flower transcriptional response to long term hot and cold environments in Antirrhinum majus.

Short term experiments have identified heat shock and cold response elements in many biological systems. However, the effect of long-term low or high temperatures is not well documented. To address this gap, we grew Antirrhinum majus plants from two-weeks old until maturity under control (normal) (22/16°C), cold (15/5°C), and hot (30/23°C) conditions for a period of two years. Flower size, petal anthocyanin content and pollen viability obtained higher values in cold conditions, decreasing in middle and high temperatures. Leaf chlorophyll content was higher in cold conditions and stable in control and hot temperatures, while pedicel length increased under hot conditions. The control conditions were optimal for scent emission and seed production. Scent complexity was low in cold temperatures. The transcriptomic analysis of mature flowers, followed by gene enrichment analysis and CNET plot visualization, showed two groups of genes. One group comprised genes controlling the affected traits, and a second group appeared as long-term adaptation to non-optimal temperatures. These included hypoxia, unsaturated fatty acid metabolism, ribosomal proteins, carboxylic acid, sugar and organic ion transport, or protein folding. We found a differential expression of floral organ identity functions, supporting the flower size data. Pollinator-related traits such as scent and color followed opposite trends, indicating an equilibrium for rendering the organs for pollination attractive under changing climate conditions. Prolonged heat or cold cause structural adaptations in protein synthesis and folding, membrane composition, and transport. Thus, adaptations to cope with non-optimal temperatures occur in basic cellular processes.

A novel ground truth multispectral image dataset with weight, anthocyanins, and Brix index measures of grape berries tested for its utility in machine learning pipelines.

BACKGROUND: The combination of computer vision devices such as multispectral cameras coupled with artificial intelligence has provided a major leap forward in image-based analysis of biological processes. Supervised artificial intelligence algorithms require large ground truth image datasets for model training, which allows to validate or refute research hypotheses and to carry out comparisons between models. However, public datasets of images are scarce and ground truth images are surprisingly few considering the numbers required for training algorithms. RESULTS: We created a dataset of 1,283 multidimensional arrays, using berries from five different grape varieties. Each array has 37 images of wavelengths between 488.38 and 952.76 nm obtained from single berries. Coupled to each multispectral image, we added a dataset with measurements including, weight, anthocyanin content, and Brix index for each independent grape. Thus, the images have paired measures, creating a ground truth dataset. We tested the dataset with 2 neural network algorithms: multilayer perceptron (MLP) and 3-dimensional convolutional neural network (3D-CNN). A perfect (100% accuracy) classification model was fit with either the MLP or 3D-CNN algorithms. CONCLUSIONS: This is the first public dataset of grape ground truth multispectral images. Associated with each multispectral image, there are measures of the weight, anthocyanins, and Brix index. The dataset should be useful to develop deep learning algorithms for classification, dimensionality reduction, regression, and prediction analysis.

Humans Share More Preferences for Floral Phenotypes With Pollinators Than With Pests.

Studies on the selection of floral traits usually consider pollinators and sometimes herbivores. However, humans also exert selection on floral traits of ornamental plants. We compared the preferences of bumblebees (Bombus terrestris), thrips (Frankliniella occidentalis), and humans for flowers of snapdragon. From a cross of two species, Antirrhinum majus and Antirrhinum linkianum, we selected four Recombinant Inbred Lines (RILs). We characterised scent emission from whole flowers and stamens, pollen content and viability, trichome density, floral shape, size and colour of floral parts. We tested the preferences of bumblebees, thrips, and humans for whole flowers, floral scent bouquets, stamen scent, and individual scent compounds. Humans and bumblebees showed preferences for parental species, whereas thrips preferred RILs. Colour and floral scent, in combination with other floral traits, seem relevant phenotypes for all organisms. Remarkably, visual traits override scent cues for bumblebees, although, scent is an important trait when bumblebees cannot see the flowers, and methyl benzoate was identified as a key attractant for them. The evolutionary trajectory of flowers is the result of multiple floral traits interacting with different organisms with different habits and modes of interaction.

gcProfileMakeR: An R Package for Automatic Classification of Constitutive and Non-Constitutive Metabolites.

Metabolomes comprise constitutive and non-constitutive metabolites produced due to physiological, genetic or environmental effects. However, finding constitutive metabolites and non-constitutive metabolites in large datasets is technically challenging. We developed gcProfileMakeR, an R package using standard Excel output files from an Agilent Chemstation GC-MS for automatic data analysis using CAS numbers. gcProfileMakeR has two filters for data preprocessing removing contaminants and low-quality peaks. The first function NormalizeWithinFiles, samples assigning retention times to CAS. The second function NormalizeBetweenFiles, reaches a consensus between files where compounds in close retention times are grouped together. The third function getGroups, establishes what is considered as Constitutive Profile, Non-constitutive by Frequency i.e., not present in all samples and Non-constitutive by Quality. Results can be plotted with the plotGroup function. We used it to analyse floral scent emissions in four snapdragon genotypes. These included a wild type, Deficiens nicotianoides and compacta affecting floral identity and RNAi:AmLHY targeting a circadian clock gene. We identified differences in scent constitutive and non-constitutive profiles as well as in timing of emission. gcProfileMakeR is a very useful tool to define constitutive and non-constitutive scent profiles. It also allows to analyse genotypes and circadian datasets to identify differing metabolites.

Gigantea: Uncovering New Functions in Flower Development.

GIGANTEA (GI) is a gene involved in multiple biological functions, which have been analysed and are partially conserved in a series of mono- and dicotyledonous plant species. The identified biological functions include control over the circadian rhythm, light signalling, cold tolerance, hormone signalling and photoperiodic flowering. The latter function is a central role of GI, as it involves a multitude of pathways, both dependent and independent of the gene CONSTANS(CO), as well as on the basis of interaction with miRNA. The complexity of the gene function of GI increases due to the existence of paralogs showing changes in genome structure as well as incidences of sub- and neofunctionalization. We present an updated report of the biological function of GI, integrating late insights into its role in floral initiation, flower development and volatile flower production.

ATR-MIR spectroscopy as a tool to assist 'Tempranillo' clonal selection process: Geographical origin and year of harvest discrimination and oenological parameters prediction.

The present study evaluated the ability of Attenuated Total Reflectance - Mid-Infrared (ATR-MIR) spectroscopy combined with Partial Least Squares Discriminant Analysis (PLS-DA) to discriminate the origin and harvest year of 'Tempranillo' grape clones and with Partial Least Squares (PLS) regressions to predict its contents in soluble solids (SS), pH and titratable acidity (TA). Normalized spectra of grape homogenates and normalized plus 1st Derivative spectra of grape skins allowed an overall percentage of correct classifications of 99.6% and 96.7% in validation, according to origin, and 98.3% and 90.0% in validation, according to harvest year, respectively. The normalized spectra of grape homogenates allowed a calibration and validation determination coefficients (R2) of 0.92 and 0.90 for SS, 0.90 and 0.84 for pH, 0.88 and 0.84 for TA, respectively. The ATR-MIR combined with multivariate analysis showed to be an appropriate tool to assist the clonal selection process of 'Tempranillo'.

The clock gene Gigantea 1 from Petunia hybrida coordinates vegetative growth and inflorescence architecture.

The gene GIGANTEA (GI) appeared early in land plants. It is a single copy gene in most plants and is found in two to three copies in Solanaceae. We analyzed the silencing of one GI copy, Petunia hybrida GI1 (PhGI1), by hairpin RNAs in Petunia in order to gain knowledge about its range of functions. Decreased transcript levels of PhGI1 were accompanied also by a reduction of PhGI2. They were further associated with increased time period between two consecutive peaks for PhGI1 and CHANEL (PhCHL), the orthologue of the blue light receptor gene ZEITLUPE (ZTL), confirming its role in maintaining circadian rhythmicity. Silenced plants were bigger with modified internode length and increased leaf size while flowering time was not altered. We uncovered a new function for PhGI1 as silenced plants showed reduction of flower bud number and the appearance of two flower buds in the bifurcation point, were normally one flower bud and the inflorescence meristem separate. Furthermore, one of the flower buds consistently showed premature flower abortion. Flowers that developed fully were significantly smaller as a result of decreased cell size. Even so the circadian pattern of volatile emission was unchanged in the silenced lines, flowers emitted 20% less volatiles on fresh weight basis over 24 hours and showed changes in the scent profile. Our results indicate a novel role of PhGI1 in the development of reproductive organs in Petunia. PhGI1 therefore represses growth in vegetative plant parts, maintains the typical cymose inflorescence structure, and inhibits premature flower abortion.

The Effect of Post-harvest Conditions in Narcissus sp. Cut Flowers Scent Profile.

Narcissus flowers are used as cut flowers and to obtain high quality essential oils for the perfume industry. As a winter crop in the Mediterranean area, it flowers at temperatures ranging between 10 and 15°C during the day and 3-10°C during the night. Here we tested the impact of different light and temperature conditions on scent quality during post-harvest. These two types of thermoperiod and photoperiod. We also used constant darkness and constant temperatures. We found that under conditions of 12:12 Light Dark and 15-5°C, Narcissus emitted monoterpenes and phenylpropanoids. Increasing the temperature to 20°-10°C in a 12:12 LD cycle caused the loss of cinnamyl acetate and emission of indole. Under constant dark, there was a loss of scent complexity. Constant temperatures of 20°C caused a decrease of scent complexity that was more dramatic at 5°C, when the total number of compounds emitted decreased from thirteen to six. Distance analysis confirmed that 20°C constant temperature causes the most divergent scent profile. We found a set of four volatiles, benzyl acetate, eucalyptol, linalool, and ocimene that display a robust production under differing environmental conditions, while others were consistently dependent on light or thermoperiod. Scent emission changed significantly during the day and between different light and temperature treatments. Under a light:dark cycle and 15-5°C the maximum was detected during the light phase but this peak shifted toward night under 20-10°C. Moreover, under constant darkness the peak occurred at midnight and under constant temperature, at the end of night. Using Machine Learning we found that indole was the volatile with a highest ranking of discrimination followed by D-limonene. Our results indicate that light and temperature regimes play a critical role in scent quality. The richest scent profile is obtained by keeping flowers at 15°-5°C thermoperiod and a 12:12 Light Dark photoperiod.

Transcriptional Structure of Petunia Clock in Leaves and Petals.

The plant circadian clock coordinates environmental signals with internal processes including secondary metabolism, growth, flowering, and volatile emission. Plant tissues are specialized in different functions, and petals conceal the sexual organs while attracting pollinators. Here we analyzed the transcriptional structure of the petunia (Petunia x hybrida) circadian clock in leaves and petals. We recorded the expression of 13 clock genes in petunia under light:dark (LD) and constant darkness (DD). Under light:dark conditions, clock genes reached maximum expression during the light phase in leaves and the dark period in petals. Under free running conditions of constant darkness, maximum expression was delayed, especially in petals. Interestingly, the rhythmic expression pattern of PhLHY persisted in leaves and petals in LD and DD. Gene expression variability differed among leaves and petals, time of day and photoperiod. The transcriptional noise was higher especially in leaves under constant darkness. We found that PhPRR7, PhPRR5, and PhGI paralogs showed changes in gene structure including exon number and deletions of CCT domain of the PRR family. Our results revealed that petunia petals presented a specialized clock.

Evaluating stress responses in cowpea under drought stress.

Drought impact on plants is an increasing concern under the climate change scenario. Cowpea (Vigna unguiculata L. Walp.) is considered as one of the most tolerant legume crops to drought, being the search for the best well-adapted genotypes crucial to face the future challenges. Different approaches have been used for differentiating plant responses to drought stress. Plants of four cowpea genotypes were submitted to three watering regimens (a severe and moderate drought stress, and well-watered control) during 15 days, and several physiological, biochemical and molecular parameters were evaluated. Stressed plants revealed commonly-described drought stress characteristics, but not all assayed parameters were useful for discriminating plants with different drought severities or genotypes. The analyses which have contributed most to genotype discrimination were those related with stomatal function, and biochemical markers such as proline and anthocyanin contents. Antioxidant enzymes activities and related genes expression did not differed among genotypes or upon drought stress treatments, suggesting that scavenging enzymes are not involved in the differential ability of cowpea plants to survive under drought stress. This information will be useful to evaluate and use genetic resources, as well as design strategies for breeding cowpea resistance to drought stress.

The Snapdragon LATE ELONGATED HYPOCOTYL Plays A Dual Role in Activating Floral Growth and Scent Emission.

The plant circadian clock controls a large number of internal processes, including growth and metabolism. Scent emission displays a circadian pattern in many species such as the snapdragon. Here we show that knocking down LATE ELONGATED HYPOCOTYL in Antirrhinum majus affects growth and scent emission. In order to gain an understanding of the growth kinetics, we took a phenomic approach using in-house artificial vision systems, obtaining time-lapse videos. Wild type flowers showed a higher growth speed than knockdown plants. The maximal growth rate was decreased by 22% in plants with lower LHY expression. Floral volatiles were differentially affected as RNAi plants showed advanced emission of compounds synthesized from cinnamic acid and delayed emission of metabolites of benzoic acid. The monoterpenes myrcene and ocimene were delayed, whereas the sesquiterpene farnesene was advanced. Overall, transgenic lines showed an altered volatile emission pattern and displayed a modified scent profile. Our results show that AmLHY plays an important role in the quantitative and qualitative control of floral growth and scent emission.

The Petunia CHANEL Gene is a ZEITLUPE Ortholog Coordinating Growth and Scent Profiles.

The floral perianth, comprising sepals and petals, conceals the sexual organs and attracts pollinators. The coordination of growth and scent emission is not fully understood. We have analyzed the effect of knocking down CHANEL (PhCHL), the ZEITLUPE ortholog in petunia (PhCHL) by hairpin RNAs. Plants with low PhCHL mRNA had overall decreased size. Growth evaluation using time lapse image analysis showed that early leaf movement was not affected by RNAi:PhCHL, but flower angle movement was modified, moving earlier during the day in knockdown plants than in wild types. Despite differences in stem length, growth rate was not significantly affected by loss of PhCHL. In contrast, petal growth displayed lower growth rate in RNAi:PhCHL. Decreased levels of PhCHL caused strongly modified scent profiles, including changes in composition and timing of emission resulting in volatile profiles highly divergent from the wild type. Our results show a role of PhCHL in controlling growth and development of vegetative and reproductive organs in petunia. The different effects of PhCHL on organ development indicate an organ-specific interpretation of the down regulation of PhCHL. Through the control of both timing and quantitative volatile emissions, PhCHL appears to be a major coordinator of scent profiles.

A comparison of semi-quantitative methods suitable for establishing volatile profiles.

BACKGROUND: Full scent profiles emitted by living tissues can be screened by using total ion chromatograms generated in full scan mode and gas chromatography-mass spectrometry technique using Headspace Sorptive Extraction. This allows the identification of specific compounds and their absolute quantification or relative abundance. Quantifications ideally should be based on calibration curves using standards for each compound. However, the unpredictable composition of Volatile Organic Compounds (VOCs) and lack of standards make this approach difficult. Researchers studying scent profiles therefore concentrate on identifying specific scent footprints i.e. relative abundance rather than absolute quantities. We compared several semi-quantitative methods: external calibration curves generated in the sampling system and by liquid addition of standards to stir bars, total integrated peak area per fresh weight (FW), normalized peak area per FW, semi-quantification based on internal standard abundance, semi-quantification based on the nearest n-alkane and percentage of emission. Furthermore, we explored the usage of nearest components and single calibrators for semi-quantifications. RESULTS: Any of the semi-quantification methods based on a standard produced similar or even identical results compared to quantification by a true-standard for a compound, except for the method based on standard addition. Each method beholds advantages and disadvantages regarding level of accuracy, experimental variability, acceptance and retrieved quantities. CONCLUSIONS: Our data shows that, except for the method of standard addition to the biological sample, the rest of the semi-quantification methods studied give highly similar statistical results. Any of the methodologies presented here can therefore be considered as valid for scent profiling. Regarding relative proportions of VOCs, the generation of calibration curves for each compound analysed is not necessary.