Phenylalanine increases chrysanthemum flower immunity against Botrytis cinerea attack.

Flowers are the most vulnerable plant organ to infection by the necrotrophic fungus Botrytis cinerea. Here we show that pre-treatment of chrysanthemum (Chrysanthemum morifolium) flowers with phenylalanine (Phe) significantly reduces their susceptibility to B. cinerea. To comprehend how Phe treatment induces resistance, we monitored the dynamics of metabolites (by GC/LC-MS) and transcriptomes (by RNAseq) in flowers after Phe treatment and B. cinerea infection. Phe treatment resulted in accumulation of 3-phenyllactate and benzaldehyde, and in particular induced the expression of genes related to Ca2+ signaling and receptor kinases, implicating an induction of the defense response. Interestingly, the main effects of Phe treatment were observed in flowers exposed to B. cinerea infection, stabilizing the global fluctuations in the levels of metabolites and transcripts while reducing susceptibility to the fungus. We suggest that Phe-induced resistance is associated to cell priming, enabling rapid and targeted reprogramming of cellular defense responses to resist disease development. After Phe pre-treatment, the levels of the anti-fungal volatiles phenylacetaldehyde and eugenol were maintained and the level of coniferin, a plausible monolignol precursor in cell wall lignification, was strongly increased. In addition, Phe pre-treatment reduced ROS generation, prevented ethylene emission, and caused changes in the expression of a minor number of genes related to cell wall biogenesis, encoding the RLK THESEUS1, or involved in Ca2+ and hormonal signaling processes. Our findings point to Phe pre-treatment as a potential orchestrator of a broad-spectrum defense response which may not only provide an ecologically friendly pest control strategy but also offers a promising way of priming plants to induce defense responses against B. cinerea.

Abscisic acid mediates the reduction of petunia flower size at elevated temperatures due to reduced cell division.

MAIN CONCLUSION: Elevated temperatures suppress cell division in developing petunia buds leading to smaller flowers, mediated by ABA. Flower size is one of the most important showy traits in determining pollinator attraction, and a central factor determining the quality of floricultural products. Whereas the adverse effects of elevated temperatures on showy traits have been described in detail, its underlining mechanisms is poorly understood. Here, we investigated the physiological mechanism responsible for the reduction of flower size in petunia under elevated temperatures. We found that the early stages of flower-bud development were most sensitive to elevated temperatures, resulting in a drastic reduction of flower diameter that was almost independent of flower load. We demonstrated that the temperature-mediated flower size reduction occurred due to a shorter growth period, and a lower rate of corolla cell division. Consistently, local application of cytokinin, a phytohormone that promotes cell division, resulted in recovery of flower dimensions when grown under elevated temperatures. Hormone analysis of temperature-inhibited flower buds revealed no significant changes in levels of cytokinin, and a specific increase of abscisic acid (ABA) levels, known to inhibit cell division. Moreover, local application of ABA on flower buds caused a reduction of flower dimensions as a result of lower levels of cell division, suggesting that ABA mediates the reduction of flower size at elevated temperatures. Taken together, our results shed light on the mechanism by which elevated temperatures decrease petunia flower size, and show that temperature-mediated reduction of flower size can be alleviated by increasing the cytokinin/ABA ratio.

MIR172d Is Required for Floral Organ Identity and Number in Tomato.

MicroRNA172 (miR172) functions as a central regulator of flowering time and flower development by post-transcriptional repression of APETALA2-LIKE transcription factors. In the model crop Solanum lycopersicum (tomato), the miR172 family is still poorly annotated and information about the functions of specific members is lacking. Here, de-novo prediction of tomato miR172 coding loci identified seven genes (SlMIR172a-g), that code for four unique species of miR172 (sly-miR172). During reproductive development, sly-miR172s are differentially expressed, with sly-miR172c and sly-miR172d being the most abundant members in developing flowers, and are predicted to guide the cleavage of eight APETALA2-LIKE transcription factors. By CRISPR-Cas9 co-targeting of SlMIR172c and SlMIR172d we have generated a battery of loss-of-function and hypomorphic mutants (slmir172c-dCR). The slmir172c-dCR plants developed normal shoot but their flowers displayed graded floral organ abnormalities. Whereas slmir172cCR loss-of-function caused only a slight greening of petals and stamens, hypomorphic and loss-of-function slmir172dCR alleles were associated with the conversion of petals and stamens to sepaloids, which were produced in excess. Interestingly, the degrees of floral organ identity alteration and proliferation were directly correlated with the reduction in sly-miR172d activity. These results suggest that sly-miR172d regulates in a dose-dependent manner floral organ identity and number, likely by negatively regulating its APETALA2-like targets.

GA as a regulatory link between the showy floral traits color and scent.

Emission of volatiles at advanced stages of flower development is a strategy used by plants to lure pollinators to the flower. We reveal that GA negatively regulates floral scent production in petunia. We used Agrobacterium-mediated transient expression of GA-20ox in petunia flowers and a virus-induced gene silencing approach to knock down DELLA expression, measured volatile emission, internal pool sizes and GA levels by GC-MS or LC-MS/MS, and analyzed transcript levels of scent-related phenylpropanoid-pathway genes. We show that GA has a negative effect on the concentrations of accumulated and emitted phenylpropanoid volatiles in petunia flowers; this effect is exerted through transcriptional/post-transcriptional downregulation of regulatory and biosynthetic scent-related genes. Both overexpression of GA20-ox, a GA-biosynthesis gene, and suppression of DELLA, a repressor of GA-signal transduction, corroborated GA's negative regulation of floral scent. We present a model in which GA-dependent timing of the sequential activation of different branches of the phenylpropanoid pathway during flower development may represent a link between the showy traits controlling pollinator attraction, namely color and scent.

Two showy traits, scent emission and pigmentation, are finely coregulated by the MYB transcription factor PH4 in petunia flowers.

The mechanism underlying the emission of phenylpropanoid volatiles is poorly understood. Here, we reveal the involvement of PH4, a petunia MYB-R2R3 transcription factor previously studied for its role in vacuolar acidification, in floral volatile emission. We used the virus-induced gene silencing (VIGS) approach to knock down PH4 expression in petunia, measured volatile emission and internal pool sizes by GC-MS, and analyzed transcript abundances of scent-related phenylpropanoid genes in flowers. Silencing of PH4 resulted in a marked decrease in floral phenylpropanoid volatile emission, with a concurrent increase in internal pool levels. Expression of scent-related phenylpropanoid genes was not affected. To identify putative scent-related targets of PH4, we silenced PH5, a tonoplast-localized H(+) -ATPase that maintains vacuolar pH homeostasis. Suppression of PH5 did not yield the reduced-emission phenotype, suggesting that PH4 does not operate in the context of floral scent through regulation of vacuolar pH. We conclude that PH4 is a key floral regulator that integrates volatile production and emission processes and interconnects two essential floral traits - color and scent.

The R2R3-MYB-like regulatory factor EOBI, acting downstream of EOBII, regulates scent production by activating ODO1 and structural scent-related genes in petunia.

Flower scent is a highly dynamic trait, under developmental, spatial, and diurnal regulation. The mechanism governing scent production is only beginning to be unraveled. In petunia (Petunia hybrida), EMISSION OF BENZENOIDS II (EOBII) controls transcription of both the shikimate pathway-regulating MYB factor ODORANT1 (ODO1) and phenylpropanoid scent-related structural genes. A promoter-activation screen identified an R2R3-MYB-like regulatory factor of phenylpropanoid volatile biosynthesis acting downstream of EOBII, designated EOBI. EOBI silencing led to downregulation of ODO1 and numerous structural scent-related genes from both the shikimate and phenylpropanoid pathways. The ability of EOBI to directly activate ODO1, as revealed by electrophoretic mobility shift assay and yeast one-hybrid analysis, place EOBI upstream of ODO1 in regulating substrate availability for volatile biosynthesis. Interestingly, ODO1-silenced transgenic petunia flowers accumulated higher EOBI transcript levels than controls, suggesting a complex feedback loop between these regulatory factors. The accumulation pattern of EOBI transcript relative to EOBII and ODO1, and the effect of up/downregulation of EOBII on transcript levels of EOBI and ODO1, further support these factors' hierarchical relationships. The dependence of scent production on EOBI expression and its direct interaction with both regulatory and structural genes provide evidence for EOBI's wide-ranging involvement in the production of floral volatiles.

EOBII, a gene encoding a flower-specific regulator of phenylpropanoid volatiles' biosynthesis in petunia.

Floral scent, which is determined by a complex mixture of low molecular weight volatile molecules, plays a major role in the plant's life cycle. Phenylpropanoid volatiles are the main determinants of floral scent in petunia (Petunia hybrida). A screen using virus-induced gene silencing for regulators of scent production in petunia flowers yielded a novel R2R3-MYB-like regulatory factor of phenylpropanoid volatile biosynthesis, EMISSION OF BENZENOIDS II (EOBII). This factor was localized to the nucleus and its expression was found to be flower specific and temporally and spatially associated with scent production/emission. Suppression of EOBII expression led to significant reduction in the levels of volatiles accumulating in and emitted by flowers, such as benzaldehyde, phenylethyl alcohol, benzylbenzoate, and isoeugenol. Up/downregulation of EOBII affected transcript levels of several biosynthetic floral scent-related genes encoding enzymes from the phenylpropanoid pathway that are directly involved in the production of these volatiles and enzymes from the shikimate pathway that determine substrate availability. Due to its coordinated wide-ranging effect on the production of floral volatiles, and its lack of effect on anthocyanin production, a central regulatory role is proposed for EOBII in the biosynthesis of phenylpropanoid volatiles.

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa.

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The biosynthetic pathways for cannabinoid and volatile terpene metabolism are localized primarily in the Cannabis trichomes, and isolated trichomes are beneficial for transcriptome analysis. The existing protocols for isolating glandular trichomes for transcriptomic characterization are inconvenient and deliver compromised trichome heads and a relatively low amount of isolated trichomes. Furthermore, they rely on expensive apparatus and isolation media containing protein inhibitors to avoid RNA degradation. The present protocol suggests combining three individual modifications to obtain a large amount of isolated glandular capitate stalked and sessile trichomes from C. sativa mature female inflorescences and fan leaves, respectively. The first modification involves substituting liquid nitrogen for the conventional isolation medium to facilitate the passage of trichomes through the micro-sieves. The second modification involves using dry ice to detach the trichomes from the plant source. The third modification involves passing the plant material consecutively through five micro-sieves of diminishing pore sizes. Microscopic imaging demonstrated the effectiveness of the isolation technique for both trichome types. In addition, the quality of RNA extracted from the isolated trichomes was appropriate for downstream transcriptomic analysis.

Tobacco Rattle Virus as a Tool for Rapid Reverse-Genetics Screens and Analysis of Gene Function in Cannabis sativa L.

Medical cannabis (Cannabis sativa L.) is quickly becoming a central agricultural crop as its production has continued to increase globally. The recent release of the cannabis reference genomes provides key genetic information for the functional analysis of cannabis genes. Currently, however, the established tools for in vivo gene functional analysis in cannabis are very limited. In this study, we investigated the use of the tobacco rattle virus (TRV) as a possible tool for virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE). Using leaf photobleaching as a visual marker of PHYTOENE DESATURASE (PDS) silencing, we found that VIGS was largely restricted to the agro-infiltrated leaves. However, when agro-infiltration was performed under vacuum, VIGS increased dramatically, which resulted in intense PDS silencing and an increased photobleaching phenotype. The suitability of TRV as a vector for virus-aided gene expression (VAGE) was demonstrated by an analysis of DsRed fluorescence protein. Interestingly, a DsRed signal was also observed in glandular trichomes in TRV2-DsRed-infected plants, which suggests the possibility of trichome-related gene function analysis. These results indicate that TRV, despite its limited spread, is an attractive vector for rapid reverse-genetics screens and for the analysis of gene function in cannabis.

Non-photoperiodic transition of female cannabis seedlings from juvenile to adult reproductive stage.

KEY MESSAGE: Vegetative-to-reproductive phase transition in female cannabis seedlings occurs autonomously with the de novo development of single flowers. To ensure successful sexual reproduction, many plant species originating from seedlings undergo juvenile-to-adult transition. This phase transition precedes and enables the vegetative-to-reproductive shift in plants, upon perception of internal and/or external signals such as temperature, photoperiod, metabolite levels, and phytohormones. This study demonstrates that the juvenile seedlings of cannabis gradually shift to the adult vegetative stage, as confirmed by the formation of lobed leaves, and upregulation of the phase-transition genes. In the tested cultivar, the switch to the reproductive stage occurs with the development of a pair of single flowers in the 7th node. Histological analysis indicated that transition to the reproductive stage is accomplished by the de novo establishment of new flower meristems which are not present in a vegetative stage, or as dormant meristems at nodes 4 and 6. Moreover, there were dramatic changes in the transcriptomic profile of flowering-related genes among nodes 4, 6, and 7. Downregulation of flowering repressors and an intense increase in the transcription of phase transition-related genes occur in parallel with an increase in the transcription of flowering integrators and meristem identity genes. These results support and provide molecular evidence for previous findings that cannabis possesses an autonomous flowering mechanism and the transition to reproductive phase is controlled in this plant mainly by internal signals.

Architecture and Florogenesis in Female Cannabis sativa Plants.

The inflorescence is the main product of medical cannabis. Hundreds of specialized metabolites with potential bioactivity are produced and accumulated in the glandular trichomes that are highly abundant mainly on female inflorescences. Understanding the morphophysiological and genetic mechanisms governing flower and inflorescence development is therefore of high scientific and practical importance. However, in-depth investigations of cannabis florogenesis are limited. Cannabis producers and researchers consider long photoperiod to be "non-inductive" or "vegetative," but under these growth conditions, the development of solitary flowers and bracts in shoot internodes clearly indicates that the plant cannot be defined as vegetative or non-inductive in the classical sense. Most probably, induction of solitary flowers is age-dependent and controlled by internal signals, but not by photoperiod. Short photoperiod induces intense branching, which results in the development of a compound raceme. Each inflorescence consists of condensed branchlets with the same phytomer structure as that of the larger phytomers developed under long day. Each phytomer consists of reduced leaves, bracts, one or two solitary flowers, and an axillary shoot (or inflorescence). Therefore, the effect of short photoperiod on cannabis florogenesis is not flower induction, but rather a dramatic change in shoot apex architecture to form a compound racemose inflorescence structure. An understanding of the morphophysiological characteristics of cannabis inflorescence will lay the foundation for biotechnological and physiological applications to modify architecture and to maximize plant productivity and uniformity in medical Cannabis.

Virus-aided gene expression and silencing using TRV for functional analysis of floral scent-related genes.

Flower scent is a composite character determined by a complex mixture of low-molecular-weight volatile molecules. Despite the importance of floral fragrance, our knowledge on factors regulating these pathways remains sketchy. Virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE) are characterized by a simple inoculation procedure and rapid results as compared to transgenesis, allowing screening and characterization of scent-related genes. Here, we describe methods using TRV as a VIGS/VAGE vector for the characterization of scent-related genes, protein compartmentalization studies, and protein subcellular targeting.