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DOUBLE-STRANDED RNA BINDING (DRB) proteins DRB1, DRB2, and DRB4 are essential for microRNA (miRNA) production in Arabidopsis thaliana (Arabidopsis) with miR160, and its target genes, AUXIN RESPONSE FACTOR10 (ARF10), ARF16, and ARF17, forming an auxin responsive miRNA expression module crucial for root development. Methods: Wild-type Arabidopsis plants (Columbia-0 (Col-0)) and the drb1, drb2, and drb12 mutants were treated with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), and the miR160-mediated response of these four Arabidopsis lines was phenotypically and molecularly characterized. Results: In 2,4-D-treated Col-0, drb1 and drb2 plants, altered miR160 abundance and ARF10, ARF16, and ARF17 gene expression were associated with altered root system development. However, miR160-directed molecular responses to treatment with 2,4-D was largely defective in the drb12 double mutant. In addition, via profiling of molecular components of the miR160 expression module in the roots of the drb4, drb14, and drb24 mutants, we uncovered a previously unknown role for DRB4 in regulating miR160 production. Conclusions: The miR160 expression module forms a central component of the molecular and phenotypic response of Arabidopsis plants to exogenous auxin treatment. Furthermore, DRB1, DRB2, and DRB4 are all required in Arabidopsis roots to control miR160 production, and subsequently, to appropriately regulate ARF10, ARF16, and ARF17 target gene expression. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana (Arabidopsis) double-stranded RNA binding (DRB) proteins DRB1, DRB2 and DRB4 perform essential roles in microRNA (miRNA) production, with many of the produced miRNAs mediating aspects of the molecular response of Arabidopsis to abiotic stress. Exposure of the drb1, drb2 and drb4 mutants to mannitol stress showed drb2 to be the most sensitive to this form of osmotic stress. Profiling of the miRNA landscapes of mannitol-stressed drb1, drb2 and drb4 seedlings via small RNA sequencing, and comparison of these to the profile of mannitol-stressed wild-type Arabidopsis plants, revealed that the ability of the drb1 and drb2 mutants to mount an appropriate miRNA-mediated molecular response to mannitol stress was defective. RT-qPCR was next used to further characterize seven miRNA/target gene expression modules, with this analysis identifying DRB1 as the primary DRB protein required for miR160, miR164, miR167 and miR396 production. In addition, via its antagonism of DRB1 function, DRB2 was shown by RT-qPCR to play a secondary role in regulating the production of these four miRNAs. This analysis further showed that DRB1, DRB2 and DRB4 are all required to regulate the production of miR399 and miR408, and that DRB4 is the primary DRB protein required to produce the non-conserved miRNA, miR858. Finally, RT-qPCR was used to reveal that each of the seven characterized miRNA/target gene expression modules responded differently to mannitol-induced osmotic stress in each of the four assessed Arabidopsis lines. In summary, this research has identified mannitol-stress-responsive miRNA/target gene expression modules that can be molecularly manipulated in the future to generate novel Arabidopsis lines with increased tolerance to this form of osmotic stress. [ABSTRACT FROM AUTHOR]

Cannabis sativa (C. sativa L.) has garnered significant attention worldwide due to its widespread use as a pharmaceutical agent. With the increasing clinical application of C. sativa and cannabinoid therapeutics, there is strong interest in the development of superior plant varieties and optimisation of growth conditions to enhance secondary metabolite yield. Our RNA sequencing analysis revealed differential expression of hormone-related transcripts in developing C. sativa trichomes, suggesting the involvement of hormone signalling pathways in cannabinoid production. Leveraging the potency of exogenous hormones on plants, this study sought to determine if the application of cytokinin (CK), gibberellic acid (GA) and jasmonic acid (JA) modified trichome morphology and the cannabinoid profile over an 8-week period following the induction of flowering. Exogenous hormone application led to alterations in trichome morphology, with each treatment significantly reducing trichome head width by the final week of assessment. Interestingly, GA application also resulted in a significant reduction in the concentration of Δ-9-tetrahydrocannabinol (THC), Δ-9-tetrahydrocannabinolic acid (THCA), cannabidiol (CBD) and cannabidiolic acid (CBDA) by week 8 post floral induction, however, JA and CK treatment did not consistently modulate the accumulation of these cannabinoids. The minor cannabinoids, cannabidivaranic acid (CBDVA), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabichromene (CBC), cannabigerolic acid (CBGA) and cannabigerol (CBG), were also affected by hormone treatments, with varying degrees of accumulation observed. These findings underscore the intricate interplay between phytohormones and secondary metabolite biosynthesis in C. sativa. Our study highlights the potential of hormone modulation as a strategy to enhance cannabinoid yield and offers some insights into the regulatory mechanisms governing cannabinoid biosynthesis in C. sativa trichomes. [ABSTRACT FROM AUTHOR]

In Arabidopsis thaliana (Arabidopsis), microRNA160 (miR160) regulates the expression of AUXIN RESPONSE FACTOR10 (ARF10), ARF16 and ARF17 throughout development, including the development of the root system. We have previously shown that in addition to DOUBLE-STRANDED RNA BINDING1 (DRB1), DRB2 is also involved in controlling the rate of production of specific miRNA cohorts in the tissues where DRB2 is expressed in wild-type Arabidopsis plants. In this study, a miR160 overexpression transgene (MIR160B) and miR160-resistant transgene versions of ARF10 and ARF16 (mARF10 and mARF16) were introduced into wild-type Arabidopsis plants and the drb1 and drb2 single mutants to determine the degree of requirement of DRB2 to regulate the miR160 expression module as part of root development. Via this molecular modification approach, we show that in addition to DRB1, DRB2 is required to regulate the level of miR160 production from its precursor transcripts in Arabidopsis roots. Furthermore, we go on to correlate the altered abundance of miR160 or its ARF10, ARF16 and ARF17 target genes in the generated series of transformant lines with the enhanced development of the root system displayed by these plant lines. More specifically, promotion of primary root elongation likely stemmed from enhancement of miR160-directed ARF17 expression repression, while the promotion of lateral and adventitious root formation was the result of an elevated degree of miR160-directed regulation of ARF17 expression, and to a lesser degree, ARF10 and ARF16 expression. Taken together, the results presented in this study identify the requirement of the functional interplay between DRB1 and DRB2 to tightly control the rate of miR160 production, to in turn ensure the appropriate degree of miR160-directed ARF10, ARF16 and ARF17 gene expression regulation as part of normal root system development in Arabidopsis. [ABSTRACT FROM AUTHOR]

Summary: Sugar loading of developing seeds comprises a cohort of transport events that contribute to reproductive success and seed yield. Understanding these events is most advanced for grain crops (Brassicaceae, Fabaceae and Gramineae) and Arabidopsis. For these species, 75–80% of their final seed biomass is derived from phloem‐imported sucrose. Sugar loading consecutively traverses three genomically distinct, and symplasmically isolated, seed domains: maternal pericarp/seed coat, filial endosperm and filial embryo. Sink status of each domain co‐ordinately transitions from growth to storage. The latter is dominated by embryos (Brassicaceae and Fabaceae) or endosperms (Gramineae). Intradomain sugar transport occurs symplasmically through plasmodesmata. Interdomain sugar transport relies on plasma‐membrane transporters operating in efflux (maternal and endosperm) or influx (endosperm and embryo) modes. Discussed is substantial progress made in identifying, and functionally evaluating, sugar symporters (STPs, SUTs or SUCs) and uniporters (SWEETs). These findings have underpinned a mechanistic understanding of seed loading. Less well researched are possible physical limitations imposed by hydraulic conductivities of differentiating protophloem and of subsequent plasmodesmal transport. The latter is coupled with sugar homeostasis within each domain mediated by sugar transporters. A similar conclusion is ascribed to fragmentary understanding of regulatory mechanisms integrating transport events with seed growth and storage. [ABSTRACT FROM AUTHOR]

Cannabis sativa (Cannabis) has recently been legalized in multiple countries globally for either its recreational or medicinal use. This, in turn, has led to a marked increase in the number of Cannabis varieties available for use in either market. However, little information currently exists on the genetic distinction between adopted varieties. Such fundamental knowledge is of considerable value and underpins the accelerated development of both a nascent pharmaceutical industry and the commercial recreational market. Therefore, in this study, we sought to assess genetic diversity across 10 Cannabis varieties by undertaking a reduced representation shotgun sequencing approach on 83 individual plants to identify variations which could be used to resolve the genetic structure of the assessed population. Such an approach also allowed for the identification of the genetic features putatively associated with the production of secondary metabolites in Cannabis. Initial analysis identified 3608 variants across the assessed population with phylogenetic analysis of this data subsequently enabling the confident grouping of each variety into distinct subpopulations. Within our dataset, the most diagnostically informative single nucleotide polymorphisms (SNPs) were determined to be associated with the long-terminal repeat (LTRs) class of retroelements, with 172 such SNPs used to fully resolve the genetic structure of the assessed population. These 172 SNPs could be used to design a targeted resequencing panel, which we propose could be used to rapidly screen different Cannabis plants to determine genetic relationships, as well as to provide a more robust, scientific classification of Cannabis varieties as the field moves into the pharmaceutical sphere. [ABSTRACT FROM AUTHOR]

In recent years, it has become readily accepted among interdisciplinary agriculturalists that the current global crop yield to land capability ratio is significantly insufficient to achieve food security for the predicted population of 9.5 billion individuals by the year 2050. This issue is further compounded by the: (1) food versus biofuel debate; (2) decreasing availability of arable land; (3) required reductions to the extensive and ongoing environmental damage caused by either poor agricultural practices or agriculture expansion, and; (4) increasingly unfavorable (duration and severity) crop cultivation conditions that accompany man-made climate change, driven by ever-expanding urbanization and its associated industrial practices. Mounting studies are repeatedly highlighting the critical importance of linking genotypes to agronomically beneficial phenotypes and/or using a molecular approach to help address this global crisis, as "simply" clearing the remaining natural ecosystems of the globe for the cultivation of additional, non-modified crops is not efficient, nor is this practice sustainable. The majority of global food crop production is sourced from a small number of members of the Poaceae family of grasses, namely; maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.). It is, therefore, of significant concern that all three of these Poaceae grass species are susceptible to a range of abiotic stresses, including drought and salt stress. Highly conserved among monocotyledonous and dicotyledonous plant species, microRNAs (miRNAs) are now well-established master regulators of gene expression, influencing all aspects of plant development, mediating defense responses against pathogens and adaptation to environmental stress. Here we investigate the variation in the abundance profiles of six known abiotic stress-responsive miRNAs, following exposure to salt and drought stress across these three key Poaceae grass crop species as well as to compare these profiles to those obtained from the well-established genetic model plant species, Arabidopsis thaliana (L.) Heynh. Additionally, we outline the variables that are the most likely primary contributors to instances of differential miRNA abundance across the assessed species following drought or salt stress exposure, specifically; (1) identifying variations in the experimental conditions and/or methodology used to assess miRNA abundance, and; (2) the distribution of regulatory transcription factor binding sites within the putative promoter region of a MICRORNA (MIR) gene that encodes the highly conserved, stress-responsive miRNA. We also discuss the emerging role that non-conserved, species-specific miRNAs play in mediating a plant's response to drought or salt stress. [ABSTRACT FROM AUTHOR]

We previously demonstrated that microRNA396 (miR396) abundance is altered in 15-day-old Arabidopsis thaliana (Arabidopsis) whole seedlings following their exposure to a 7-day salt stress treatment regime. We, therefore, used a molecular modification approach to generate two new Arabidopsis transformant populations with reduced (MIM396 plants) and elevated (MIR396 plants) miR396 abundance. The exposure of 8-day-old wild-type Arabidopsis whole seedlings and a representative plant line of the MIM396 and MIR396 transformant populations to a 7-day salt stress treatment regime revealed unique phenotypic and physiological responses to the imposed stress by unmodified wild-type Arabidopsis plants and the MIM396 and MIR396 transformat lines. A quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) approach was, therefore, applied to demonstrate that the plant line specific responses to salt stress likely stemmed from the unique molecular profile of each of the GROWTH REGULATING FACTOR (GRF) transcription factor gene family members which form posttranscriptional targets of miR396-directed expression regulation. RT-qPCR additionally revealed that, in 15-day-old Arabidopsis whole seedlings, the three previously identified putative target genes of miR396 belonging to the NEUTRAL/ALKALINE NONLYSOSOMAL CERAMIDASE-LIKE (NCER) gene family, including NCER1, NCER2, and NCER3, do not form targets of miR396-directed expression regulation at the posttranscriptional level. Taken together, the phenotypic and molecular analyses presented here demonstrate that alteration of the miR396/GRF expression module is central to the molecular response of Arabidopsis to salt stress. [ABSTRACT FROM AUTHOR]

Setaria viridis has recently emerged as an ideal model species to genetically characterize the C4 monocotyledonous grasses via a molecular modification approach. Soil salinization has become a compelling agricultural problem globally with salinity adversely impacting the yield potential of many of the major cereals. Small regulatory molecules of RNA, termed microRNAs (miRNAs), were originally demonstrated crucial for developmental gene expression regulation in plants, however, miRNAs have since been shown to additionally command a central regulatory role in abiotic stress adaptation. Therefore, a small RNA sequencing approach was employed to profile the salt stress responsive miRNA landscapes of the shoot and root tissues of two Setaria viridis accessions (A10 and ME034V) amenable to molecular modification. Small RNA sequencing-identified abundance alterations for miRNAs, miR169, miR395, miR396, miR397, miR398 and miR408, were experimentally validated via RT-qPCR. RT-qPCR was further applied to profile the molecular response of the miR160 and miR167 regulatory modules to salt stress. This analysis revealed accession- and tissue-specific responses for the miR160 and miR167 regulatory modules in A10 and ME034V shoot and root tissues exposed to salt stress. The findings reported here form the first crucial step in the identification of the miRNA regulatory modules to target for molecular manipulation to determine if such modification provides S. viridis with an improved tolerance to salt stress. [ABSTRACT FROM AUTHOR]

The previous assignment of functional roles for AGO7, and the DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2, and DRB4, in either microRNA (miRNA) or trans-acting small-interfering RNA (tasiRNA) production allowed for use of the loss-of-function mutant lines, drb1, drb2, drb4, and ago7, to further functionally characterize the TAS3 pathway in Arabidopsis thaliana (Arabidopsis). Towards achieving this goal, we also describe the developmental and molecular phenotypes expressed by three newly generated Arabidopsis lines, the drb1ago7, drb2ago7, and drb4ago7 double mutants. We show that the previously reported developmental abnormalities displayed by the drb1, drb2, drb4, and ago7 single mutants, are further exacerbated in the drb1ago7, drb2ago7, and drb4ago7 double mutants, with rosette area, silique length, and seed set all impaired to a greater degree in the double mutants. Molecular assessment of the TAS3 pathway in the floral tissues of the seven analyzed mutants revealed that DRB1 is the sole DRB required for miR390 sRNA production. However, DRB2 and DRB4 appear to play secondary roles at this stage of the TAS3 pathway to ensure that miR390 sRNA levels are tightly maintained. We further show that the expression of the TAS3-derived tasiARF target genes, AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4, was altered in drb1ago7, drb2ago7, and drb4ago7 flowers. Altered ARF2, ARF3, and ARF4 expression was in turn demonstrated to lead to changes in the level of expression of KAN1, KAN3, and KAN4, three KANADI transcription factor genes known to be transcriptionally regulated by ARF2, ARF3, and ARF4. Taken together, the demonstrated relationship between altered ARF and KAN gene expression in drb1ago7, drb2ago7 and drb4ago7 flowers, could, in part, explain the more severe developmental defects displayed by the double mutants, compared to milder impact that loss of only a single piece of TAS3 pathway protein machinery was demonstrated to have on drb1, drb2, drb4 and ago7 reproductive development. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana (Arabidopsis) has been used extensively as a heterologous system for molecular manipulation to genetically characterize both dicotyledonous and monocotyledonous plant species. Here, we report on Arabidopsis transformant lines molecularly manipulated to over-accumulate the small regulatory RNA microRNA397 (miR397) from the emerging C4 monocotyledonous grass model species Setaria viridis (S. viridis). The generated transformant lines, termed SvMIR397 plants, displayed a range of developmental phenotypes that ranged from a mild, wild-type-like phenotype, to a severe, full dwarfism phenotype. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)-based profiling of the SvMIR397 transformant population revealed a strong correlation between the degree of miR397 over-accumulation, repressed LACCASE (LAC) target gene expression, reduced lignin content, and the severity of the developmental phenotype displayed by SvMIR397 transformants. Further, exposure of SvMIR397 transformants to a 7-day regime of salt stress revealed the SvMIR397 transformant lines to be more sensitive to the imposed stress than were wild-type Arabidopsis plants. Taken together, the findings reported here via the use of Arabidopsis as a heterologous system show that the S. viridis miR397 small regulatory RNA is able to repress the expression of three Arabidopsis LAC genes which led to reduced lignin content and increased salt stress sensitivity. [ABSTRACT FROM AUTHOR]