Phaseolus vulgaris STP13.1 is an H+-coupled monosaccharide transporter, present in source leaves and seed coats, with higher substrate affinity at depolarized potentials.

Sugar transport proteins (STPs) are high-affinity H+-coupled hexose symporters. Recently, the contribution of STP13 to bacterial and fungal pathogen resistance across multiple plant species has garnered significant interest. Quantitative PCR analysis of source leaves, developing embryos, and seed coats of Phaseolus vulgaris L. (common bean) revealed that PvSTP13.1 was expressed in source leaves and seed coats throughout seed development. In contrast, PvSTP13.1 transcripts were detected at exceedingly low levels in developing embryos. To characterize the transport mechanism, PvSTP13.1 was expressed in Xenopus laevis oocytes, and inward-directed currents were analyzed using two-electrode voltage clamping. PvSTP13.1 was shown to function as an H+-coupled monosaccharide symporter exhibiting a unique high affinity for hexoses and aldopentoses at depolarized membrane potentials. Specifically, of the 31 assessed substrates, which included aldohexoses, deoxyhexoses, fructose, 3-O-methyl-D-glucose, aldopentoses, polyols, glycosides, disaccharides, trisaccharides, and glucuronic acid, PvSTP13.1 displayed the highest affinity (K 0.5) for glucose (43 µM), mannose (92 µM), galactose (145 µM), fructose (224 µM), xylose (1.0 mM), and fucose (3.7 mM) at pH 5.6 at a depolarized membrane potential of -40 mV. The results presented here suggest PvSTP13.1 contributes to retrieval of hexoses from the apoplasmic space in source leaves and coats of developing seeds.

Sugar loading of crop seeds - a partnership of phloem, plasmodesmal and membrane transport.

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.

Miniature Inverted-Repeat Transposable Elements: Small DNA Transposons That Have Contributed to Plant MICRORNA Gene Evolution.

Angiosperms form the largest phylum within the Plantae kingdom and show remarkable genetic variation due to the considerable difference in the nuclear genome size of each species. Transposable elements (TEs), mobile DNA sequences that can amplify and change their chromosome position, account for much of the difference in nuclear genome size between individual angiosperm species. Considering the dramatic consequences of TE movement, including the complete loss of gene function, it is unsurprising that the angiosperms have developed elegant molecular strategies to control TE amplification and movement. Specifically, the RNA-directed DNA methylation (RdDM) pathway, directed by the repeat-associated small-interfering RNA (rasiRNA) class of small regulatory RNA, forms the primary line of defense to control TE activity in the angiosperms. However, the miniature inverted-repeat transposable element (MITE) species of TE has at times avoided the repressive effects imposed by the rasiRNA-directed RdDM pathway. MITE proliferation in angiosperm nuclear genomes is due to their preference to transpose within gene-rich regions, a pattern of transposition that has enabled MITEs to gain further transcriptional activity. The sequence-based properties of a MITE results in the synthesis of a noncoding RNA (ncRNA), which, after transcription, folds to form a structure that closely resembles those of the precursor transcripts of the microRNA (miRNA) class of small regulatory RNA. This shared folding structure results in a MITE-derived miRNA being processed from the MITE-transcribed ncRNA, and post-maturation, the MITE-derived miRNA can be used by the core protein machinery of the miRNA pathway to regulate the expression of protein-coding genes that harbor homologous MITE insertions. Here, we outline the considerable contribution that the MITE species of TE have made to expanding the miRNA repertoire of the angiosperms.

Genetic Variants Associated with Long-Terminal Repeats Can Diagnostically Classify Cannabis Varieties.

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.

Molecular Manipulation of the miR396 and miR399 Expression Modules Alters the Response of Arabidopsis thaliana to Phosphate Stress.

In plant cells, the molecular and metabolic processes of nucleic acid synthesis, phospholipid production, coenzyme activation and the generation of the vast amount of chemical energy required to drive these processes relies on an adequate supply of the essential macronutrient, phosphorous (P). The requirement of an appropriate level of P in plant cells is evidenced by the intricately linked molecular mechanisms of P sensing, signaling and transport. One such mechanism is the posttranscriptional regulation of the P response pathway by the highly conserved plant microRNA (miRNA), miR399. In addition to miR399, numerous other plant miRNAs are also required to respond to environmental stress, including miR396. Here, we exposed Arabidopsis thaliana (Arabidopsis) transformant lines which harbor molecular modifications to the miR396 and miR399 expression modules to phosphate (PO4) starvation. We show that molecular alteration of either miR396 or miR399 abundance afforded the Arabidopsis transformant lines different degrees of tolerance to PO4 starvation. Furthermore, RT-qPCR assessment of PO4-starved miR396 and miR399 transformants revealed that the tolerance displayed by these plant lines to this form of abiotic stress most likely stemmed from the altered expression of the target genes of these two miRNAs. Therefore, this study forms an early step towards the future development of molecularly modified plant lines which possess a degree of tolerance to growth in a PO4 deficient environment.

Cannabis sativa: Interdisciplinary Strategies and Avenues for Medical and Commercial Progression Outside of CBD and THC.

Cannabis sativa (Cannabis) is one of the world's most well-known, yet maligned plant species. However, significant recent research is starting to unveil the potential of Cannabis to produce secondary compounds that may offer a suite of medical benefits, elevating this unique plant species from its illicit narcotic status into a genuine biopharmaceutical. This review summarises the lengthy history of Cannabis and details the molecular pathways that underpin the production of key secondary metabolites that may confer medical efficacy. We also provide an up-to-date summary of the molecular targets and potential of the relatively unknown minor compounds offered by the Cannabis plant. Furthermore, we detail the recent advances in plant science, as well as synthetic biology, and the pharmacology surrounding Cannabis. Given the relative infancy of Cannabis research, we go on to highlight the parallels to previous research conducted in another medically relevant and versatile plant, Papaver somniferum (opium poppy), as an indicator of the possible future direction of Cannabis plant biology. Overall, this review highlights the future directions of cannabis research outside of the medical biology aspects of its well-characterised constituents and explores additional avenues for the potential improvement of the medical potential of the Cannabis plant.

MicroRNA-Mediated Responses to Cadmium Stress in Arabidopsis thaliana.

In recent decades, the presence of cadmium (Cd) in the environment has increased significantly due to anthropogenic activities. Cd is taken up from the soil by plant roots for its subsequent translocation to shoots. However, Cd is a non-essential heavy metal and is therefore toxic to plants when it over-accumulates. MicroRNA (miRNA)-directed gene expression regulation is central to the response of a plant to Cd stress. Here, we document the miRNA-directed response of wild-type Arabidopsis thaliana (Arabidopsis) plants and the drb1, drb2 and drb4 mutant lines to Cd stress. Phenotypic and physiological analyses revealed the drb1 mutant to display the highest degree of tolerance to the imposed stress while the drb2 mutant was the most sensitive. RT-qPCR-based molecular profiling of miRNA abundance and miRNA target gene expression revealed DRB1 to be the primary double-stranded RNA binding (DRB) protein required for the production of six of the seven Cd-responsive miRNAs analyzed. However, DRB2, and not DRB1, was determined to be required for miR396 production. RT-qPCR further inferred that transcript cleavage was the RNA silencing mechanism directed by each assessed miRNA to control miRNA target gene expression. Taken together, the results presented here reveal the complexity of the miRNA-directed molecular response of Arabidopsis to Cd stress.

Molecular Manipulation of MicroRNA397 Abundance Influences the Development and Salt Stress Response of Arabidopsis thaliana.

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.

Molecular Manipulation of the miR399/PHO2 Expression Module Alters the Salt Stress Response of Arabidopsis thaliana.

In Arabidopsis thaliana (Arabidopsis), the microRNA399 (miR399)/PHOSPHATE2 (PHO2) expression module is central to the response of Arabidopsis to phosphate (PO4) stress. In addition, miR399 has been demonstrated to also alter in abundance in response to salt stress. We therefore used a molecular modification approach to alter miR399 abundance to investigate the requirement of altered miR399 abundance in Arabidopsis in response to salt stress. The generated transformant lines, MIM399 and MIR399 plants, with reduced and elevated miR399 abundance respectively, displayed differences in their phenotypic and physiological response to those of wild-type Arabidopsis (Col-0) plants following exposure to a 7-day period of salt stress. However, at the molecular level, elevated miR399 abundance, and therefore, altered PHO2 target gene expression in salt-stressed Col-0, MIM399 and MIR399 plants, resulted in significant changes to the expression level of the two PO4 transporter genes, PHOSPHATE TRANSPORTER1;4 (PHT1;4) and PHT1;9. Elevated PHT1;4 and PHT1;9 PO4 transporter levels in salt stressed Arabidopsis would enhance PO4 translocation from the root to the shoot tissue which would supply additional levels of this precious cellular resource that could be utilized by the aerial tissues of salt stressed Arabidopsis to either maintain essential biological processes or to mount an adaptive response to salt stress.

DRB1, DRB2 and DRB4 Are Required for Appropriate Regulation of the microRNA399/PHOSPHATE2 Expression Module in Arabidopsis thaliana.

Adequate phosphorous (P) is essential to plant cells to ensure normal plant growth and development. Therefore, plants employ elegant mechanisms to regulate P abundance across their developmentally distinct tissues. One such mechanism is PHOSPHATE2 (PHO2)-directed ubiquitin-mediated degradation of a cohort of phosphate (PO4) transporters. PHO2 is itself under tight regulation by the PO4 responsive microRNA (miRNA), miR399. The DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2 and DRB4, have each been assigned a specific functional role in the Arabidopsis thaliana (Arabidopsis) miRNA pathway. Here, we assessed the requirement of DRB1, DRB2 and DRB4 to regulate the miR399/PHO2 expression module under PO4 starvations conditions. Via the phenotypic and molecular assessment of the knockout mutant plant lines, drb1, drb2 and drb4, we show here that; (1) DRB1 and DRB2 are required to maintain P homeostasis in Arabidopsis shoot and root tissues; (2) DRB1 is the primary DRB required for miR399 production; (3) DRB2 and DRB4 play secondary roles in regulating miR399 production, and; (4) miR399 appears to direct expression regulation of the PHO2 transcript via both an mRNA cleavage and translational repression mode of RNA silencing. Together, the hierarchical contribution of DRB1, DRB2 and DRB4 demonstrated here to be required for the appropriate regulation of the miR399/PHO2 expression module identifies the extreme importance of P homeostasis maintenance in Arabidopsis to ensure that numerous vital cellular processes are maintained across Arabidopsis tissues under a changing cellular environment.

Profiling the Abiotic Stress Responsive microRNA Landscape of Arabidopsis thaliana.

It is well established among interdisciplinary researchers that there is an urgent need to address the negative impacts that accompany climate change. One such negative impact is the increased prevalence of unfavorable environmental conditions that significantly contribute to reduced agricultural yield. Plant microRNAs (miRNAs) are key gene expression regulators that control development, defense against invading pathogens and adaptation to abiotic stress. Arabidopsis thaliana (Arabidopsis) can be readily molecularly manipulated, therefore offering an excellent experimental system to alter the profile of abiotic stress responsive miRNA/target gene expression modules to determine whether such modification enables Arabidopsis to express an altered abiotic stress response phenotype. Towards this goal, high throughput sequencing was used to profile the miRNA landscape of Arabidopsis whole seedlings exposed to heat, drought and salt stress, and identified 121, 123 and 118 miRNAs with a greater than 2-fold altered abundance, respectively. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was next employed to experimentally validate miRNA abundance fold changes, and to document reciprocal expression trends for the target genes of miRNAs determined abiotic stress responsive. RT-qPCR also demonstrated that each miRNA/target gene expression module determined to be abiotic stress responsive in Arabidopsis whole seedlings was reflective of altered miRNA/target gene abundance in Arabidopsis root and shoot tissues post salt stress exposure. Taken together, the data presented here offers an excellent starting platform to identify the miRNA/target gene expression modules for future molecular manipulation to generate plant lines that display an altered response phenotype to abiotic stress.

The Plant microRNA Pathway: The Production and Action Stages.

Plant microRNAs are an endogenous class of small regulatory RNA central to the posttranscriptional regulation of gene expression in plant development and environmental stress adaptation or in response to pathogen challenge. The plant microRNA pathway is readily separated into two distinct stages: (1) the production stage, which is localized to the plant cell nucleus and where the microRNA small RNA is processed from a double-stranded RNA precursor transcript, and (2) the action stage, which is localized to the plant cell cytoplasm and where the mature microRNA small RNA is loaded into an effector complex and is used by the complex as a sequence specificity guide to direct expression repression of target genes harboring highly complementary microRNA target sequences. Historical research indicated that the plant microRNA pathway was a highly structured, almost linear pathway requiring a small set of core machinery proteins. However, contemporary research has demonstrated that the plant microRNA pathway is highly dynamic, and to allow for this flexibility, a large and highly functionally diverse set of machinery proteins is now known to be required. For example, recent research has shown that plant microRNAs can regulate target gene expression via a translational repression mechanism of RNA silencing in addition to the standard messenger RNA cleavage-based mechanism of RNA silencing: a mode of RNA silencing originally assigned to all plant microRNAs. Using Arabidopsis thaliana as our model system, here we report on both the core and auxiliary sets of machinery proteins now known to be required for both microRNA production and microRNA action in plants.

Roles of Aquaporins in Setaria viridis Stem Development and Sugar Storage.

Setaria viridis is a C4 grass used as a model for bioenergy feedstocks. The elongating internodes in developing S. viridis stems grow from an intercalary meristem at the base, and progress acropetally toward fully expanded cells that store sugar. During stem development and maturation, water flow is a driver of cell expansion and sugar delivery. As aquaporin proteins are implicated in regulating water flow, we analyzed elongating and mature internode transcriptomes to identify putative aquaporin encoding genes that had particularly high transcript levels during the distinct stages of internode cell expansion and maturation. We observed that SvPIP2;1 was highly expressed in internode regions undergoing cell expansion, and SvNIP2;2 was highly expressed in mature sugar accumulating regions. Gene co-expression analysis revealed SvNIP2;2 expression was highly correlated with the expression of five putative sugar transporters expressed in the S. viridis internode. To explore the function of the proteins encoded by SvPIP2;1 and SvNIP2;2, we expressed them in Xenopus laevis oocytes and tested their permeability to water. SvPIP2;1 and SvNIP2;2 functioned as water channels in X. laevis oocytes and their permeability was gated by pH. Our results indicate that SvPIP2;1 may function as a water channel in developing stems undergoing cell expansion and SvNIP2;2 is a candidate for retrieving water and possibly a yet to be determined solute from mature internodes. Future research will investigate whether changing the function of these proteins influences stem growth and sugar yield in S. viridis.