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

PhMYB4 fine-tunes the floral volatile signature of Petunia×hybrida through PhC4H

In Petunia×hybrida cv ‘Mitchell Diploid’ (MD), floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis is controlled spatially, developmentally, and daily at molecular, metabolic, and biochemical levels. Multiple genes have been shown to encode proteins that either directly catalyse a biochemical reaction yielding FVBP compounds or are involved in metabolite flux prior to the formation of FVBP compounds. It was hypothesized that multiple transcription factors are involved in the precise regulation of all necessary genes, resulting in the specific volatile signature of MD flowers. After acquiring all available petunia transcript sequences with homology to Arabidopsis thaliana R2R3-MYB transcription factors, PhMYB4 (named for its close identity to AtMYB4) was identified, cloned, and characterized. PhMYB4 transcripts accumulate to relatively high levels in floral tissues at anthesis and throughout open flower stages, which coincides with the spatial and developmental distribution of FVBP production and emission. Upon RNAi suppression of PhMYB4 (ir-PhMYB4) both petunia CINNAMATE-4-HYDROXYLASE (PhC4H1 and PhC4H2) gene transcript levels were significantly increased. In addition, ir-PhMYB4 plants emit higher levels of FVBP compounds derived from p-coumaric acid (isoeugenol and eugenol) compared with MD. Together, these results indicate that PhMYB4 functions in the repression of C4H transcription, indirectly controlling the balance of FVBP production in petunia floral tissue (i.e. fine-tunes)

Microgametophyte development in Cannabis sativa L. and first androgenesis induction through microspore embryogenesis

[EN] Development of double haploids is an elusive current breeding objective in Cannabis sativa L. We have studied the whole process of anther and pollen grain formation during meiosis, microsporogenesis, and microgametogenesis and correlated the different microgametophyte developmental stages with bud length in plants from varieties USO31 and Finola. We also studied microspore and pollen amyloplast content and studied the effect of a cold pretreatment to excised buds prior to microspore in vitro culture. Up to 476,903 microspores and pollen grains per male flower, with in vivo microspore viability rates from 53.71 to 70.88% were found. A high uniformity in the developmental stage of microspores and pollen grains contained in anthers was observed, and this allowed the identification of bud length intervals containing mostly vacuolate microspores and young bi-cellular pollen grains. The starch presence in C. sativa microspores and pollen grains follows a similar pattern to that observed in species recalcitrant to androgenesis. Although at a low frequency, cold-shock pretreatment applied on buds can deviate the naturally occurring gametophytic pathway toward an embryogenic development. This represents the first report concerning androgenesis induction in C. sativa, which lays the foundations for double haploid research in this species.Galán-Ávila, A.; García-Fortea, E.; Prohens Tomás, J.; Herraiz García, FJ. (2021). Microgametophyte development in Cannabis sativa L. and first androgenesis induction through microspore embryogenesis. Frontiers in Plant Science. 12:1-22. https://doi.org/10.3389/fpls.2021.669424S1221

A Regulatory Network for Coordinated Flower Maturation

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs

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

Identification of transcription factors controlling floral morphology in wild Petunia

Adaptation to different pollinators is an important driver of speciation in the angiosperms. Genetic approaches such as QTL mapping have been successfully used to identify the underlying speciation genes. However, these methods are often limited by widespread suppression of recombination due to divergence between species. While the mutations that caused the interspecific differences in floral color and scent have been elucidated in a variety of plant genera, the genes that are responsible for morphological differences remain mostly unknown. Differences in floral organ length determine the pollination efficiency of hawkmoths and hummingbirds, and therefore the genes that control these differences are potential speciation genes. Identifying such genes is challenging, especially in non‐model species and when studying complex traits for which little prior genetic and biochemical knowledge is available. Here we combine transcriptomics with detailed growth analysis to identify candidate transcription factors underlying interspecific variation in the styles of Petunia flowers. Starting from a set of 2284 genes, stepwise filtering for expression in styles, differential expression between species, correlation with growth‐related traits, allele‐specific expression in interspecific hybrids, and/or high‐impact polymorphisms resulted in a set of 43 candidate speciation genes. Validation by virus‐induced gene silencing identified two MYB transcription factors, EOBI and EOBII, that were previously shown to regulate floral scent emission, a trait associated with pollination by hawkmoths