Mannans: Structural carbohydrates produced during seed maturation in Euterpe edulis Martius, an Atlantic Forest species vulnerable to extinction.

Palm seedlings are visually selected from mature fruits in a slow process that leads to nonuniform germination and high embryo mortality. In this study, we determined the levels of monosaccharides, their crystallinity, and their role in the formation of Euterpe edulis endosperm during seed maturation. Seeds harvested from 108 to 262 days after anthesis (DAA) were analyzed morphologically, physiologically, and chemically to measure soluble and insoluble lignins, ashes, structural carbohydrates, degree of crystallinity, and endo-ß-mannanase. The seeds achieved maximum germination and vigor at 164 DAA. During the early stages, only compounds with a low structural order were formed. The contents of soluble and insoluble lignins, ashes, glucans, and galactans decreased during maturation. Those of mannans, the main structural carbohydrate in the endosperm, increased along with the degree of crystallinity, as suggested by a mannan-I-type X-ray diffraction pattern. Similarly, endo-ß-mannanase activity peaked at 262 DAA. The superior physiological outcome of seeds and seedlings at 164 DAA implies a 98-day shorter harvesting time. The state of mannans during seed maturation could be used as a marker to improve seedling production by E. edulis.

Phytochemical composition of aerial parts and roots of Pfaffia glomerata (Spreng.) Pedersen and anticholinesterase, antioxidant, and antiglycation activities.

The Pfaffia glomerata, a plant popularly called Brazilian ginseng, is widely used in Brazil for the treatment of various pathologies, including those associated with the Central Nervous System. 20-hydroxyecdysone (20E), a phytosteroid present in this plant, can promote adaptogenic effects in the organism, providing greater body resistance to stressors. This study aimed to evaluate the phytochemical composition and the anticholinesterase, antioxidant, and antiglycation effects of extracts and fractions of aerial parts and roots of P. glomerata, also analyzing their possible cytotoxic effects. The fractions were obtained by partitioning methanol extracts from the aerial part and roots of P. glomerata with hexane, dichloromethane, ethyl acetate, n-butanol, and water. The samples were initially tested in anticholinesterase, antioxidant, and antiglycation assays, and the most promising samples were submitted for cytotoxicity and chromatographic analyses. Mass spectrometry and chromatography methods revealed that 20E was the main compound in the dichloromethane fractions, there being 35% more 20E in the aerial part (APD) than in the roots (RD). Added to the higher concentration of 20E, the APD fraction also presented more promising results than the RD fraction in anticholinesterase and antioxidant analyses, indicating that their effects may be related to the concentration of 20E. These same fractions showed no hemolytic effects but were cytotoxic in high concentrations. These new findings contribute to scientific information about P. glomerata and open more perspectives for the understanding of its therapeutic properties, allowing the association of biological activity with the presence of 20E.

Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis in Bixa orellana.

Age affects the production of secondary metabolites, but how developmental cues regulate secondary metabolism remains poorly understood. The achiote tree (Bixa orellana L.) is a source of bixin, an apocarotenoid used in diverse industries worldwide. Understanding how age-dependent mechanisms control bixin biosynthesis is of great interest for plant biology and for economic reasons. Here we overexpressed miRNA156 (miR156) in B. orellana to comprehensively study the effects of the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module on age-dependent bixin biosynthesis in leaves. Overexpression of miR156 in annatto plants (miR156ox) reduced BoSPL transcript levels, impacted leaf ontogeny, lessened bixin production, and increased abscisic acid levels. Modulation of expression of BoCCD4-4 and BoCCD1, key genes in carotenoid biosynthesis, was associated with diverting the carbon flux from bixin to abscisic acid in miR156ox leaves. Proteomic analyses revealed an overall low accumulation of most secondary metabolite-related enzymes in miR156ox leaves, suggesting that miR156-targeted BoSPLs may be required to activate several secondary metabolic pathways. Our findings suggest that the conserved BomiR156-BoSPL module is deployed to regulate leaf dynamics of bixin biosynthesis, and may create novel opportunities to fine-tune bixin output in B. orellana breeding programs.

Water stress modulates terpene biosynthesis and morphophysiology at different ploidal levels in Lippia alba (Mill.) N. E. Brown (Verbenaceae).

Monoterpenes are the main component in essential oils of Lippia alba. In this species, the chemical composition of essential oils varies with genome size: citral (geraniol and neral) is dominant in diploids and tetraploids, and linalool in triploids. Because environmental stress impacts various metabolic pathways, we hypothesized that stress responses in L. alba could alter the relationship between genome size and essential oil composition. Water stress affects the flowering, production, and reproduction of plants. Here, we evaluated the effect of water stress on morphophysiology, essential oil production, and the expression of genes related to monoterpene synthesis in diploid, triploid, and tetraploid accessions of L. alba cultivated in vitro for 40 days. First, using transcriptome data, we performed de novo gene assembly and identified orthologous genes using phylogenetic and clustering-based approaches. The expression of candidate genes related to terpene biosynthesis was estimated by real-time quantitative PCR. Next, we assessed the expression of these genes under water stress conditions, whereby 1% PEG-4000 was added to MS medium. Water stress modulated L. alba morphophysiology at all ploidal levels. Gene expression and essential oil production were affected in triploid accessions. Polyploid accessions showed greater growth and metabolic tolerance under stress compared to diploids. These results confirm the complex regulation of metabolic pathways such as the production of essential oils in polyploid genomes. In addition, they highlight aspects of genotype and environment interactions, which may be important for the conservation of tropical biodiversity.

Functional Fruit Trees from the Atlantic and Amazon Forests: Selection of Potential Chestnut Trees Rich in Antioxidants, Nutrients, and Fatty Acids.

The Amazon rainforest and the biodiversity hotspot of the Atlantic Forest are home to fruit trees that produce functional foods, which are still underutilized. The present study aimed to select potential functional nut donor trees from two Brazilian chestnuts, by evaluating the nutritional and antioxidant composition of the nuts and the fatty acid profile of the oil. The nutritional characteristics, antioxidants, oil fatty acid profile, and X-ray densitometry of the nuts were evaluated, as well as the characterization of leaf and soil nutrients for each parent tree. The nut oil was evaluated through Brix (%), mass (g), yield (%), and the fatty acid profile. For L. pisonis, the most nutritious nuts were produced by L. pisonis tree 4 (N > P > K > Mg > Ca > Zn > Fe) and L. pisonis tree 6 (P > Ca > Mg > Mn > Zn > Cu > Fe), and for the species L. lanceolata, L. lanceolata tree 6 (N > P > Ca > Mg > Zn > Fe > Cu) and L. lanceolata tree 2 (P > K > Mg > Zn > Cu). In L. pisonis, the highest production of anthocyanins, DPPH, total phenolics, and flavonoids was obtained from the nuts of L. pisonis tree 4 as well as for L. lanceolata, from L. lanceolata tree 1, except for flavonoids. The Brix of the oil from the nuts of both species showed no difference between the trees and the fatty acid profile with a similar amount between saturated (48-65%) and unsaturated (34-57%) fatty acids. Both species have nuts rich in nutrients and antioxidant compounds and can be considered unconventional functional foods. The data collected in the present study confirm that the nuts of these species can replace other foods as a source of selenium.

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.

Dynamics of annatto pigment synthesis and accumulation in seeds of Bixa orellana L. revealed by integrated chemical, anatomical, and RNA-Seq analyses.

Bixin is a commercially valuable apocarotenoid pigment found in the seed aril of Bixa orellana. The dynamics and regulation of its biosynthesis and accumulation during seed development remain largely unknown. Here, we combined chemical, anatomical, and transcriptomic data to provide stage-specific resolution of the cellular and molecular events occurring during B. orellana seed development. Seeds at five developmental stages (S1-S5) were used for analysis of bixin content and seed anatomy, and three of them (S1, S3, and S4) were selected for Illumina HiSeq sequencing. Bixin accumulated in large quantities in seeds compared with other tissues analyzed, particularly during the S2 stage, peaking at the S4 stage, and then decreasing slightly in the S5 stage. Anatomical analysis revealed that bixin accumulated in the large central vacuole of specialized cells, which were scattered throughout the developing mesotesta at the S2 stage, but enlarged progressively at later stages, until they occupied most of the parenchyma in the aril. A total of 13 million reads were generated and assembled into 73,381 protein-encoding contigs, from which 312 were identified as containing 1-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP), carotenoid, and bixin pathways genes. Differential transcriptome expression analysis of these genes revealed that 50 of them were sequentially and differentially expressed through the seed developmental stages analyzed, including seven carotenoid cleavage dioxygenases, eight aldehyde dehydrogenases, and 22 methyltransferases. Taken together, these results show that bixin synthesis and accumulation in seeds of B. orellana are a developmentally regulated process involving the coordinated expression of DOXP/MEP, carotenoid, and bixin biosynthesis genes.

Physiological, epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine.

Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the morphophysiological responses of Pfaffia glomerata, a medicinal plant, to salt stress and the demethylating agent 5-azacytidine (5-azaC). Moreover, we investigated how these changes affected the biosynthesis of 20-hydroxyecdysone (20-E), a pharmacologically important specialized metabolite. Plants were cultivated in vitro for 40 days in Murashige and Skoog medium supplemented with NaCl (50 mM), 5-azaC (25 µM), and NaCl + 5-azaC. Compared with the control (medium only), the treatments reduced growth, photosynthetic rates, and photosynthetic pigment content, with increase in sucrose, total amino acids, and proline contents, but a reduction in starch and protein. Comparative proteomic analysis revealed 282 common differentially accumulated proteins involved in 87 metabolic pathways, most of them related to amino acid and carbohydrate metabolism, and specialized metabolism. 5-azaC and NaCl + 5-azaC lowered global DNA methylation levels and 20-E content, suggesting that 20-E biosynthesis may be regulated by epigenetic mechanisms. Moreover, downregulation of a key protein in jasmonate biosynthesis indicates the fundamental role of this hormone in the 20-E biosynthesis. Taken together, our results highlight possible regulatory proteins and epigenetic changes related to salt stress tolerance and 20-E biosynthesis in P. glomerata, paving the way for future studies of the mechanisms involved in this regulation.

Transcriptome Analysis of Melocactus glaucescens (Cactaceae) Reveals Metabolic Changes During in vitro Shoot Organogenesis Induction.

Melocactus glaucescens is an endangered cactus highly valued for its ornamental properties. In vitro shoot production of this species provides a sustainable alternative to overharvesting from the wild; however, its propagation could be improved if the genetic regulation underlying its developmental processes were known. The present study generated de novo transcriptome data, describing in vitro shoot organogenesis induction in M. glaucescens. Total RNA was extracted from explants before (control) and after shoot organogenesis induction (treated). A total of 14,478 unigenes (average length, 520 bases) were obtained using Illumina HiSeq 3000 (Illumina Inc., San Diego, CA, USA) sequencing and transcriptome assembly. Filtering for differential expression yielded 2,058 unigenes. Pairwise comparison of treated vs. control genes revealed that 1,241 (60.3%) unigenes exhibited no significant change, 226 (11%) were downregulated, and 591 (28.7%) were upregulated. Based on database analysis, more transcription factor families and unigenes appeared to be upregulated in the treated samples than in controls. Expression of WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) and CALMODULIN (CaM) genes, both of which were upregulated in treated samples, was further validated by real-time quantitative PCR (RT-qPCR). Differences in gene expression patterns between control and treated samples indicate substantial changes in the primary and secondary metabolism of M. glaucescens after the induction of shoot organogenesis. These results help to clarify the molecular genetics and functional genomic aspects underlying propagation in the Cactaceae family.

Shaping the root system: the interplay between miRNA regulatory hubs and phytohormones.

The root system commonly lies underground, where it provides anchorage for the aerial organs, as well as nutrients and water. Both endogenous and environmental cues contribute to the establishment of the root system. Among the endogenous cues, microRNAs (miRNAs), transcription factors, and phytohormones modulate root architecture. miRNAs belong to a subset of endogenous hairpin-derived small RNAs that post-transcriptionally control target gene expression, mostly transcription factors, comprising the miRNA regulatory hubs. Phytohormones are signaling molecules involved in most developmental processes. Some miRNAs and targets participate in more than one hormonal pathway, thereby providing new bridges in plant hormonal crosstalk. Unraveling the intricate network of molecular mechanisms underlying the establishment of root systems is a central aspect in the development of novel strategies for plant breeding to increase yield and optimize agricultural land use. In this review, we summarize recent findings describing the molecular mechanisms associated with the interplay between miRNA regulatory hubs and phytohormones to ensure the establishment of a proper root system. We focus on post-embryonic growth and development of primary, lateral, and adventitious roots. In addition, we discuss novel insights for future research on the interaction between miRNAs and phytohormones in root architecture.

Irradiance-driven 20-hydroxyecdysone production and morphophysiological changes in Pfaffia glomerata plants grown in vitro.

Pfaffia glomerata possesses potential pharmacological and medicinal properties, mainly owing to the secondary metabolite 20-hydroxyecdysone (20E). Increasing production of biomass and 20E is important for industrial purposes. This study aimed to evaluate the influence of irradiance on plant morphology and production of 20E in P. glomerata grown in vitro. Nodal segments of accessions 22 and 43 (Ac22 and Ac43) were inoculated in culture medium containing MS salts and vitamins. Cultures were maintained at 25 ± 2 °C under a 16-h photoperiod and subjected to irradiance treatments of 65, 130, and 200 µmol m-2 s-1 by fluorescent lamps. After 30 days, growth parameters, pigment content, stomatal density, in vitro photosynthesis, metabolites content, and morphoanatomy were assessed. Notably, Ac22 plants exhibited 10-fold higher 20E production when cultivated at 200 µmol m-2 s-1 than at 65 µmol m-2 s-1, evidencing the importance of light quantity for the accumulation of this metabolite. 20E production was twice as high in Ac22 as in Ac43 plants although both accessions responded positively to higher irradiance. Growth under 200 µmol m-2 s-1 stimulated photosynthesis and consequent biomass accumulation, but lowered carotenoids and anthocyanins. Furthermore, increasing irradiance enhanced the number of palisade and spongy parenchyma cells, enhancing the overall growth of P. glomerata. Graphical abstract.

A 20-hydroxyecdysone-enriched fraction from Pfaffia glomerata (Spreng.) pedersen roots alleviates stress, anxiety, and depression in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Pfaffia glomerata roots are widely used in Brazil to treat various pathological conditions, particularly psychological disorders. 20-hydroxyecdysone, a phytosteroid present in the plant, can promote greater body resistance against exogenous and endogenous stressors. The objective of this study was to evaluate the possible neuroprotective effect of a 20-hydroxyecdysone-enriched fraction (20E-EF), obtained from P. glomerata roots, in an acute murine stress model. MATERIAL AND METHODS: The 20E-EF was obtained by partitioning the methanol extract from P. glomerata roots with dichloromethane. Mice were treated by gavage with three doses of 20E-EF (3, 10, and 30 mg/kg) and parameters of stress, anxiety, and depression were evaluated. Biomarkers of oxidative stress (enzymes, antioxidant profile, and oxidized molecules) were evaluated in the cortex, striatum (basal ganglia), and hippocampus of animals treated with 30 mg/kg of 20E-EF. RESULTS: Mass spectrometry revealed that 20E was the main compound in the dichloromethane fraction. At a dose of 30 mg/kg, 20E-EF reduced stress, anxiety, and depression, while stimulating antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase), promoting antioxidant activity (antioxidant capacity, sulfhydryl groups, and reduced glutathione), and reducing oxidative markers (lipid peroxidation). In addition, 20E increased the concentration of NO in the striatum, possibly improving memory function and antioxidant activity. CONCLUSION: A 30 mg/kg dose of 20E-EF was able to reduce stress, anxiety, and depression, in addition to maintaining antioxidant defenses of the cortex and striatum. These findings open new perspectives for understanding the therapeutic properties of P. glomerata and the underlying mechanism(s).

Sapucaia nut: Morphophysiology, minerals content, methodological validation in image analysis, phenotypic and molecular diversity in Lecythis pisonis Cambess.

Sapucaia or Lecythis pisonis Cambess. is an ornamental tree that produces edible, tasty, and nutritious nuts, and can be used for timber production. Sapucaia has potential as a functional food, owing to optimal nutritional and particularly Se levels. The present study sought to characterize the seeds of 21 sapucaia matrices from forest remnants of a neotropical hotspot (Atlantic Forest) and farms in two Brazilian states. Biometrics, germination, vigor, lignin content in the teguments, minerals content, and phenotypic and molecular diversity were analyzed. The seeds of matrices 16 and 21 were the most vigorous. Nuts from matrices 21 and 17 contained the highest amounts of Se. Matrices 5, 8, and 21 were the most phenotypically distant; whereas matrix 21 was the most genetically distant. Importantly, we validated a new non-destructive and efficient X-ray-based methodology for internal and densitometric analysis of sapucaia seeds, and demonstrated a high genetic divergence among matrices.

Anatomy, Flow Cytometry, and X-Ray Tomography Reveal Tissue Organization and Ploidy Distribution in Long-Term In Vitro Cultures of Melocactus Species.

Cacti have a highly specialized stem that enables survival during extended dry periods. Despite the ornamental value of cacti and the fact that stems represent the main source of explants in tissue culture, there are no studies on their morpho-anatomical and cytological characteristics in Melocactus. The present study seeks to address the occurrence of cells with mixed ploidy level in cacti tissues. Specifically, we aim to understand how Melocactus stem tissue is organized, how mixoploidy is distributed when present, and whether detected patterns of ploidy change after long periods of in vitro culture. To analyze tissue organization, Melocactus glaucescens and Melocactus paucispinus plants that had been germinated and cultivated in vitro were analyzed for stem structure using toluidine blue, Xylidine Ponceau, Periodic Acid Schiff, ruthenium red, and acid floroglucin. To investigate patterns of ploidy, apical, medial, and basal zones of the stem, as well as, periphery, cortex, and stele (vascular tissue and pith) regions of the stem and root apexes from four- and ten-year old cultured in vitro were analyzed by flow cytometry. X-ray micro-computed tomography (XRµCT) was performed with fragments of stems from both species. The scarcity of support elements (i.e., sclereids and fibers) indicates that epidermis, hypodermis, and wide-band tracheids present in cortical vascular bundles and stele, as well as water stored in aquifer parenchyma cells along the cortex, provide mechanical support to the stem. Parenchyma cells increase in volume with a four-fold increase in ploidy. M. glaucescens and M. paucispinus exhibit the same pattern of cell ploidy irrespective of topophysical region or age, but there is a marked difference in ploidy between the stem periphery (epidermis and hypodermis), cortex, stele, and roots. Mixoploidy in Melocactus is not related to the age of the culture, but is a developmental trait, whereby endocycles promote cell differentiation to accumulate valuable water.

Pfaffia glomerata hydroalcoholic extract stimulates penile tissue in adult Swiss mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Roots of Pfaffia glomerata are used in traditional medicine as aphrodisiacs and sexual stimulants. AIM OF THE STUDY: The aim of this study was to evaluate the action of the hydroalcoholic extract from the roots of Pfaffia glomerata on the Leydig cells, cavernous bodies and other penile constituents, as well as on serum testosterone and 17ß-estradiol levels of adult mice. MATERIALS AND METHODS: Mature male Swiss mice were divided into 6 groups: control (water), sildenafil citrate, 3 groups receiving daily doses of P. glomerata extract (100, 200 and 400 mg/kg) and one group receiving intermittent doses of P. glomerata (200 mg/kg/3-3d). RESULTS: The proportions of blood vessels, lymphatic space and estradiol levels were increased. On the other hand, reduction of testosterone levels due to Leydig cells death was observed. As for penile parameters, volumetric proportions of cavernous bodies, collagen and nitric oxide were increased, while smooth muscle content was decreased. CONCLUSIONS: Despite that the long term intake of P. glomerata extract was related to a stimulant action, reduction on Leydig cell viability induced decreased testosterone production.

Leaf development stages and ontogenetic changes in passionfruit (Passiflora edulis Sims.) are detected by narrowband spectral signal.

During shoot development, leaves undergo various ontogenetic changes, including variation in size, shape, and geometry. Passiflora edulis (passionfruit) is a heteroblastic species, which means that it experiences conspicuous changes throughout development, enabling a morphological distinction between the juvenile and adult vegetative phases. Quantification of heteroblasty requires a practical, inexpensive, reliable, and non-destructive method, such as remote sensing. Moreover, relationships among ontogenetic changes and spectral signal at leaf level can be scaled up to support precision agriculture in passion fruit crops. In the present study, we used laboratory spectroscopic measurements (400-2500 nm) and narrowband vegetation indexes (or hyperspectral vegetation indexes - HVIs) to evaluate ontogenetic changes related to development and aging in P. edulis leaves. We also assessed leaf pigment concentration to further support the application of biochemical-related narrowband indexes. We report that 30-d-old leaves can be discriminated into developmental stages through their spectral signals. MSI (Moisture Stress Index) and NDVI750 (Normalized Difference Vegetation Index ρ750) contribute most to the variation of age (15 to 30-d-old leaves) and developmental stage (phytomer positions along the plant axis) in passionfruit leaves. PRI (Photochemical Reflectance Index) played an important role in detecting age and development alterations, including heteroblasty. A biochemical and spectral comparison of pigments revealed that spectroscopy offered potential for diagnosing phenology in P. edulis, as some narrowband indexes correlated strongly with chlorophylls and carotenoids content. Narrowband vegetation indexes are found to be a suitable tool for monitoring passionfruit crops.

Blue and red light affects morphogenesis and 20-hydroxyecdisone content of in vitro Pfaffia glomerata accessions.

The combination of different colors from light-emitting diodes (LEDs) may influence growth and production of secondary metabolites in plants. In the present study, the effect of light quality on morphophysiology and content of 20-hydroxyecdysone (20E), a phytoecdysteroid, was evaluated in accessions of an endangered medicinal species, Pfaffia glomerata, grown in vitro. Two accessions (Ac22 and Ac43) were cultured in vitro under three different ratios of red (R) and blue (B) LEDs: (i) 1R:1B, (ii) 1R:3B, and (iii) 3R:1B. An equal ratio of red and blue light (1R:1B) increased biomass accumulation, anthocyanin content, and 20E production (by 30-40%). Moreover, 1R:1B treatment increased the size of vascular bundles and vessel elements, as well as strengthened xylem lignification and thickening of the cell wall of shoots. The 1R:3B treatment induced the highest photosynthetic and electron transport rates and enhanced the activity of oxidative stress-related enzymes. Total Chl content, Chl/Car ratio, and NPQ varied more by accession type than by light source. Spectral quality affected primary metabolism differently in each accession. Specifically, in Ac22 plants, fructose content was higher under 1R:1B and 1R:3B treatments, whereas starch accumulation was higher under 1R:3B, and sucrose under 3R:1B. In Ac43 plants, sugars were not influenced by light spectral quality, but starch content was higher under 3R:1B conditions. In conclusion, red and blue LEDs enhance biomass and 20E production in P. glomerata grown in vitro.

miR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses.

Root growth is modulated by different factors, including phytohormones, transcription factors, and microRNAs (miRNAs). MicroRNA156 and its targets, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, define an age-dependent pathway that controls several developmental processes, including lateral root emergence. However, it remains unclear whether miR156-regulated SPLs control root meristem activity and root-derived de novo shoot regeneration. Here, we show that MIR156 and SPL genes have opposing expression patterns during the progression of primary root (PR) growth in Arabidopsis, suggesting that age cues may modulate root development. Plants with high miR156 levels display reduced meristem size, resulting in shorter primary root (PRs). Conversely, plants with reduced miR156 levels show higher meristem activity. Importantly, loss of function of SPL10 decreases meristem activity, while SPL10 de-repression increases it. Meristem activity is regulated by SPL10 probably through the reduction of cytokinin responses, via the modulation of type-B ARABIDOPSIS RESPONSE REGULATOR1(ARR1) expression. We also show that SPL10 de-repression in the PRs abolishes de novo shoot regenerative capacity by attenuating cytokinin responses. Our results reveal a cooperative regulation of root meristem activity and root-derived de novo shoot regeneration by integrating age cues with cytokinin responses via miR156-targeted SPL10.

Irradiance and light quality affect two annatto (Bixa orellana L.) cultivars with contrasting bixin production.

Light is a key factor influencing growth and development in plants. Specific irradiance and light quality can improve development and production of secondary compounds such as carotenoids during plant tissue culture. Bixin and norbixin, two apocarotenoids obtained from the seeds of Bixa orellana L. (annatto), are used as natural dyes in various industries. While annatto tissue culture has been successful, the effect of light in this species remains poorly understood. Here, we analyze for the first time the effect of irradiance regime (50, 150, 50 + 150, 200, 50 + 200 µmol m-2 s-1) and light spectral quality (fluorescent, white, blue/red LED) on in vitro development of apexes and bixin content in two contrasting bixin-producing varieties of B. orellana, namely 'Piave Vermelha' and 'UESB74'. The number of leaves per plant, stomatal density, leaf area, leaf expansion, chlorophylls and carotenoids content, malondialdehyde and bixin content were analyzed in the leaves of both cultivars. 'Piave Vermelha' produced 1.6-fold more bixin than 'UESB74'. Stomata cells of both cultivars had a paracytic arrangement with peltate trichomes along the adaxial and abaxial leaf surfaces. 'Piave Vermelha' preferred blue/red LED light; whereas fluorescent light was optimal for 'UESB74'. Under fluorescent light, an irradiance of 50 µmol m-2 s-1 is indicated for both cultivars. LED light increased bixin content only in 'Piave Vermelha', suggesting that the dye biosynthetic pathway is genotype-dependent. The present findings suggest the possibility of using light to modulate the bixin biosynthetic pathway.