Linum lewisii Adventitious and Hairy-Roots Cultures as Lignan Plant Factories.

Plants synthesize specific secondary metabolites for survival, reproduction, environmental resilience, and defense. Among them, lignans are a class of polyphenols with several bioactive properties: chemopreventive, anti-inflammatory, antiviral, and antioxidant. These compounds are often extracted from field-grown plants with very low yields. To overcome these constraints, in vitro tissue cultures provide a tool to optimize large-scale production. Moreover, the use of elicitation to increase secondary metabolite production is gaining importance. The aim of this work was to develop adventitious (ARL) and hairy roots (HRL) from Linum lewisi, a species able to synthesize arylnaphthalene lignans such as justicidin B. The ARL and HRL were obtained for the first time and characterized for their phenol content, antioxidant activity, and the production of justicidin B after treatments with several elicitors and precursor feeding. Through NMR spectroscopy, other four lignans were highlighted and identified in the roots extracts. A pilot-scale bioreactor was adopted to assess the suitability of the developed root cultures for future large-scale production. The ARL and HRL cultures showed a justicidin B production higher than other Linum species cultures described up to now (75.8 mg/L and 82.2 g/L), and the production more than doubled after elicitation with MeJA.

Optimized Ultrasound-Assisted Extraction of Lignans from Linum Species with Green Solvents.

Lignans are plant phenols derived from phenylpropanoids. They play a significant role in plant defense and have features that make them appealing for pharmaceutical applications. Lignans can be obtained by plant in vitro cultures; their production by adventitious and hairy roots of Linum species seems to be a promising alternative to chemical synthesis. In the context of large-scale production, it is necessary to optimize their extraction from plants tissue by choosing the more suitable solvent and extraction procedure, paying attention to the use of green media and methods. With the aim to select the best conditions for the extraction of two interesting lignans (justicidin B and 6-methoxypodophyllotoxin) from Linum tissues, different green solvents and the method of ultrasound-assisted extraction were tested. The results showed that ethyl methyl ketone and dimethyl carbonate were the best media to extract justicidin B and 6-methoxypodophyllotoxin, respectively, in terms of purity and recovery. Moreover, we showed that ultrasound-assisted extraction presents different advantages compared to conventional methods. Finally, the optimal experimental conditions to extract justicidin B from L. austriacum hairy roots using methyl ethyl ketone were also determined by the response surface method. The models obtained are reliable and accurate to estimate the purity and recovery of justicidin B.

Enhanced Aryltetralin Lignans Production in Linum Adventi-Tious Root Cultures.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense molecules. They are also valuable for human health, in particular, for their potent antiviral and antineoplastic properties. In this study, the adventitious root cultures of three Linum species (L. flavum, L. mucronatum and L. dolomiticum) were developed to produce aryltetralin lignans. The effect of two elicitors, methyl jasmonate and coronatine, on aryltetralin lignans production was also evaluated. The adventitious root cultures from L. dolomiticum were obtained and analyzed for the first time and resulted as the best producer for all the aryltetralins highlighted in this system: Podophyllotoxin, 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside, the last showing a productivity of 92.6 mg/g DW. The two elicitors differently affected the production of the 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside.

New Insight into Justicidin B Pathway and Production in Linum austriacum.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense compounds but they are also valuable for human health, in particular, for novel therapeutics. In this work, Linum austriacum in vitro cultures, cells (Cc), adventitious roots (ARc) and hairy roots (HRc) were developed for the production of justicidin B through elicitation with methyl jasmonate (MeJA) and coronatine (COR). The performances of the cultures were evaluated for their stability, total phenols content and antioxidant ability. NMR was used to identify justicidin B and isojusticidin B and HPLC to quantify the production, highlighting ARc and HRc as the highest productive tissues. MeJA and COR treatments induced the synthesis of justicidin B more than three times and the synthesis of other compounds. RNA-sequencing and a de novo assembly of L. austriacum ARc transcriptome was generated to identify the genes activated by MeJA. Furthermore, for the first time, the intracellular localization of justicidin B in ARc was investigated through microscopic analysis. Then, HRc was chosen for small-scale production in a bioreactor. Altogether, our results improve knowledge on justicidin B pathway and cellular localization in L. austriacum for future scale-up processes.

Uniparental and transgressive expression of α-zeins in maize endosperm of o2 hybrid lines.

The α-zein gene family encodes the most abundant storage proteins of maize (Zea mays) endosperm. Members of this family are expressed in a parent-of-origin manner. To characterize this phenomenon further, we investigated the expression of a subset of α-zein polypeptides in reciprocal crosses between o2 lines that were characterized by a simplified α-zein pattern. Maize lines that suppressed the expression of α-zeins when used as female parents were identified. The suppression was cross-specific, occurring only when specific genetic backgrounds were combined. Four α-zein sequences that were sensitive to uniparental expression were isolated. Molecular characterization of these α-zeins confirmed that their expression or suppression depended on the genetic proprieties of the endosperm tissue instead of their parental origin. DNA methylation analysis of both maternally and paternally expressed α-zeins revealed no clear correlation between this epigenetic marker and parent-of-origin allelic expression, suggesting that an additional factor(s) is involved in this process. Genetic analyses revealed that the ability of certain lines to suppress α-zein expression was unstable after one round of heterozygosity with non-suppressing lines. Interestingly, α-zeins also showed a transgressive expression pattern because unexpressed isoforms were reactivated in both F2 and backcross plants. Collectively, our results suggest that parent-of-origin expression of specific α-zein alleles depends on a complex interaction between genotypes in a manner that is reminiscent of paramutation-like phenomena.

Distinct gene networks modulate floral induction of autonomous maize and photoperiod-dependent teosinte.

Temperate maize was domesticated from its tropical ancestor, teosinte. Whereas temperate maize is an autonomous day-neutral plant, teosinte is an obligate short-day plant that requires uninterrupted long nights to induce flowering. Leaf-derived florigenic signals trigger reproductive growth in both teosinte and temperate maize. To study the genetic mechanisms underlying floral inductive pathways in maize and teosinte, mRNA and small RNA genome-wide expression analyses were conducted on leaf tissue from plants that were induced or not induced to flower. Transcriptome profiles reveal common differentially expressed genes during floral induction, but a comparison of candidate flowering time genes indicates that photoperiod and autonomous pathways act independently. Expression differences in teosinte are consistent with the current paradigm for photoperiod-induced flowering, where changes in circadian clock output trigger florigen production. Conversely, differentially expressed genes in temperate maize link carbon partitioning and flowering, but also show altered expression of circadian clock genes that are distinct from those altered upon photoperiodic induction in teosinte. Altered miRNA399 levels in both teosinte and maize suggest a novel common connection between flowering and phosphorus perception. These findings provide insights into the molecular mechanisms underlying a strengthened autonomous pathway that enabled maize growth throughout temperate regions.

Florigen-Encoding Genes of Day-Neutral and Photoperiod-Sensitive Maize Are Regulated by Different Chromatin Modifications at the Floral Transition.

The activity of the maize (Zea mays) florigen gene ZEA CENTRORADIALIS8 (ZCN8) is associated with the floral transition in both day-neutral temperate maize and short-day (SD)-requiring tropical maize. We analyzed transcription and chromatin modifications at the ZCN8 locus and its nearly identical paralog ZCN7 during the floral transition. This analysis was performed with day-neutral maize (Zea mays ssp. mays), where flowering is promoted almost exclusively via the autonomous pathway through the activity of the regulatory gene indeterminate1 (id1), and tropical teosinte (Zea mays ssp. parviglumis) under floral inductive and noninductive photoperiods. Comparison of ZCN7/ZCN8 histone modification profiles in immature leaves of nonflowering id1 mutants and teosinte grown under floral inhibitory photoperiods reveals that both id1 floral inductive activity and SD-mediated induction result in histone modification patterns that are compatible with the formation of transcriptionally competent chromatin environments. Specific histone modifications are maintained during leaf development and may represent a chromatin signature that favors the production of processed ZCN7/ZCN8 messenger RNA in florigen-producing mature leaf. However, whereas id1 function promotes histone H3 hyperacetylation, SD induction is associated with increased histone H3 dimethylation and trimethylation at lysine-4. In addition, id1 and SD differently affect the production of ZCN7/ZCN8 antisense transcript. These observations suggest that distinct mechanisms distinguish florigen regulation in response to autonomous and photoperiod pathways. Finally, the identical expression and histone modification profiles of ZCN7 and ZCN8 in response to floral induction suggest that ZCN7 may represent a second maize florigen.

The WD40-repeat proteins NFC101 and NFC102 regulate different aspects of maize development through chromatin modification.

The maize (Zea mays) nucleosome remodeling factor complex component101 (nfc101) and nfc102 are putative paralogs encoding WD-repeat proteins with homology to plant and mammalian components of various chromatin modifying complexes. In this study, we generated transgenic lines with simultaneous nfc101 and nfc102 downregulation and analyzed phenotypic alterations, along with effects on RNA levels, the binding of NFC101/NFC102, and Rpd3-type histone deacetylases (HDACs), and histone modifications at selected targets. Direct NFC101/NFC102 binding and negative correlation with mRNA levels were observed for indeterminate1 (id1) and the florigen Zea mays CENTRORADIALIS8 (ZCN8), key activators of the floral transition. In addition, the abolition of NFC101/NFC102 association with repetitive sequences of different transposable elements (TEs) resulted in tissue-specific upregulation of nonpolyadenylated RNAs produced by these regions. All direct nfc101/nfc102 targets showed histone modification patterns linked to active chromatin in nfc101/nfc102 downregulation lines. However, different mechanisms may be involved because NFC101/NFC102 proteins mediate HDAC recruitment at id1 and TE repeats but not at ZCN8. These results, along with the pleiotropic effects observed in nfc101/nfc102 downregulation lines, suggest that NFC101 and NFC102 are components of distinct chromatin modifying complexes, which operate in different pathways and influence diverse aspects of maize development.