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This article belongs to the Special Issue Research on Tropical Food Crop Genomics., Pineapple (Ananas comosus (L.) Merr.) is the second most important tropical fruit crop globally, and ‘MD2’ is the most important cultivated variety. A high-quality genome is important for molecular-based breeding, but available pineapple genomes still have some quality limitations. Here, PacBio and Hi-C data were used to develop a new high-quality MD2 assembly and gene prediction. Compared to the previous MD2 assembly, major improvements included a 26.6-fold increase in contig N50 length, phased chromosomes, and >6000 new genes. The new MD2 assembly also included 161.6 Mb additional sequences and >3000 extra genes compared to the F153 genome. Over 48% of the predicted genes harbored potential deleterious mutations, indicating that the high level of heterozygosity in this species contributes to maintaining functional alleles. The genome was used to characterize the FAR1-RELATED SEQUENCE (FRS) genes that were expanded in pineapple and rice. Transposed and dispersed duplications contributed to expanding the numbers of these genes in the pineapple lineage. Several AcFRS genes were differentially expressed among tissue-types and stages of flower development, suggesting that their expansion contributed to evolving specialized functions in reproductive tissues. The new MD2 assembly will serve as a new reference for genetic and genomic studies in pineapple., This research was supported by the Dole Nutrition Institute at Dole Food Company Inc., and M.I. and J.C. were also supported by the United States Department of Agriculture National Institute of Food and Agriculture, Hatch project 1008691.

Purple or black carrots (Daucus carota ssp. sativus var. atrorubens Alef) are characterized bytheir dark purple- to black-colored roots, owing their appearance to high anthocyanin concentrations. In recent years, there has been increasing interest in the use of black carrot anthocyanins as natural food dyes. Black carrot roots contain large quantities of mono-acylated anthocyanins, which impart a measure of heat-, light- and pH-stability, enhancing the color-stability of food products over their shelf-life. The genetic pathway controlling anthocyanin biosynthesis appears well conserved among land plants; however, different variants of anthocyanin-related genes between cultivars results in tissue-specific accumulations of purple pigments. Thus, broad genetic variations of anthocyanin profile, and tissue-specific distributions in carrot tissues and organs, can be observed, and the ratio of acylated to non-acylated anthocyanins varies significantly in the purple carrot germplasm. Additionally, anthocyanins synthesis can also be influenced by a wide range of external factors, such as abiotic stressors and/or chemical elicitors, directly affecting the anthocyanin yield and stability potential in food and beverage applications. In this study, we critically review and discuss the current knowledge on anthocyanin diversity, genetics and the molecular mechanisms controlling anthocyanin accumulation in carrots. We also provide a view of the current knowledge gaps and advancement needs as regards developing and applying innovative molecular tools to improve the yield, product performance and stability of carrot anthocyanin for use as a natural food colorant. Fil: Iorizzo, Massimo. North Carolina State University. Department Of Food, Bioprocessing And Nutrition Sciences. Plants For Human Health Institute.; Estados Unidos Fil: Curaba, Julien. North Carolina State University. Department Of Food, Bioprocessing And Nutrition Sciences. Plants For Human Health Institute.; Estados Unidos Fil: Pottorff, Marti. North Carolina State University. Department Of Food, Bioprocessing And Nutrition Sciences. Plants For Human Health Institute.; Estados Unidos Fil: Ferruzzi, Mario G.. North Carolina State University. Department Of Food, Bioprocessing And Nutrition Sciences. Plants For Human Health Institute.; Estados Unidos Fil: Simon, Pihilipp W.. United States Department of Agriculture. Agricultural Research Service; Argentina. University of Wisconsin; Estados Unidos Fil: Cavagnaro, Pablo Federico. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Departamento de Producción Agropecuaria. Cátedra de Horticultura y Floricultura; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria La Consulta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina

Anthocyanins are natural health promoting pigments that can be produced in large quantities in some purple carrot cultivars. Decoration patterns of anthocyanins, such as acylation, can greatly influence their stability and biological properties and use in the food industry as nutraceuticals and natural colorants. Despite recent advances made toward understanding the genetic control of anthocyanin accumulation in purple carrot, the genetic mechanism controlling acylation of anthocyanin in carrot root have not been studied yet. In the present study, we performed fine mapping combined with gene expression analyses (RNA-Seq and RT-qPCR) to identify the genetic factor conditioning the accumulation of non-acylated (Cy3XGG) versus acylated (Cy3XFGG and Cy3XSGG) cyanidin derivatives, in three carrot populations. Segregation and mapping analysis pointed to a single gene with dominant effect controlling anthocyanin acylation in the root, located in a 576kb region containing 29 predicted genes. Orthologous and phylogenetic analyses enabled the identification of a cluster of three SCPL-acyltransferases coding genes within this region. Comparative transcriptome analysis indicated that only one of these three genes, DcSCPL1, was always expressed in association with anthocyanin pigmentation in the root and was co-expressed with DcMYB7, a gene known to activate anthocyanin biosynthetic genes in carrot. DcSCPL1 sequence analysis, in root tissue containing a low level of acylated anthocyanins, demonstrated the presence of an insertion causing an abnormal splicing of the 3rd exon during mRNA editing, likely resulting in the production of a non-functional acyltransferase and explaining the reduced acylation phenotype. This study provides strong linkage-mapping and functional evidences for the candidacy of DcSCPL1 as a primary regulator of anthocyanin acylation in carrot storage root.

RNA-dependent RNA polymerases (RDRPs) are encoded by RNA viruses as well as eukaryotic organisms such as plants. The function of these cellular RDRPs has been associated with the synthesis of short interfering RNAs (siRNAs), which are essential regulators of genomic integrity and plant viral defense. The multiple gene copies, and functional diversities, of the plant RDRPs raise the question of whether their intrinsic properties differ. This chapter describes protocols to extract and test in vitro, the activity of plant RDRPs.

As rice is both an important crop and a well-developed model for monocotyledonous plants, the study of miRNAs and their functions in this species is an active area of research. In this chapter, we summarize the progress that has been made in identifying miRNAs and their targets in rice. The identified miRNAs and their target mRNAs suggest that miRNAs function in a wide range of biological processes. The expression patterns of the miRNAs support their roles not only in development but also in response to environmental stresses. Demonstrations of biological functions for rice miRNAs are beginning to emerge and are detailed in this chapter.

RNA-dependent RNA polymerases (RDRPs) are encoded by RNA viruses as well as eukaryotic organisms such as plants. The function of these cellular RDRPs has been associated with the synthesis of short interfering RNAs (siRNAs), which are essential regulators of genomic integrity and plant viral defense. The multiple gene copies, and functional diversities, of the plant RDRPs raise the question of whether their intrinsic properties differ. This chapter describes protocols to extract and test in vitro, the activity of plant RDRPs.

In Arabidopsis, genetic evidence demonstrates that RNA-dependent RNA polymerase 6 (RDR6) plays a fundamental role in at least four RNA silencing pathways whose functions range from defense against transgenes or viruses to endogene regulation in development and in stress responses. Despite its critical role in RNA silencing, the biochemical activities of RDR6 have yet to be characterized. In this study, we transiently expressed Arabidopsis RDR6 in Nicotiana benthamiana and investigated the biochemical activities of immunopurified RDR6 in vitro. We showed that RDR6 possesses terminal nucleotidyltransferase activity as well as primer-independent RNA polymerase activity on single-stranded RNAs. We found that RDR6 cannot distinguish RNAs with or without a cap or poly(A) tail. We also demonstrated that RDR6 has strong polymerase activity on single-stranded DNA. All these activities require the conserved catalytic Asp(867) residue. Our findings have important implications on the processes involving RDR6 in vivo and provide new biochemical insights into the mechanisms of RNA silencing in Arabidopsis.

Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both β-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.

Trichomes of Arabidopsis are single-celled epidermal hair that are a useful model for studying plant cell fate determination. Trichome initiation requires the activity of the GLABROUS1 (GL1) gene whose expression in epidermal and trichome cells is dependent on the presence of a 3'-cis-regulatory element. Using a one-hybrid screen, we have isolated a cDNA, which encodes for a protein, GL1 enhancer binding protein (GeBP), that binds this regulatory element in yeast and in vitro. GeBP and its three homologues in Arabidopsis share two regions: a central region with no known motifs and a C-terminal region with a putative leucine-zipper motif. We show that both regions are necessary for trans-activation in yeast. A translational fusion with the Yellow Fluorescent Protein (YFP) indicates that GeBP is a nuclear protein whose localization is restricted to, on average, 3-5 subnuclear foci that might correspond to nucleoli. Transcriptional fusion with the GUS reporter indicates that GeBP is mainly expressed in vegetative meristematic tissues and in very young leaf primordia. We looked at GeBP expression in plants mutated in or misexpressing KNAT1, a KNOX gene, expressed in the shoot apical meristem and downregulated in leaf founder cells, and found that GeBP transcript level is regulated by KNAT1 suggesting that KNAT1 is a transcriptional activator of GeBP. This regulation suggests that GeBP is acting as a repressor of leaf cell fate.

Background The transitions from juvenile to adult and adult to reproductive phases of growth are important stages in the life cycle of plants. The regulators of these transitions include miRNAs, in particular miR156 and miR172 which are part of a regulatory module conserved across the angiosperms. In Arabidopsis miR171 represses differentiation of axillary meristems by repressing expression of SCARECROW-LIKE(SCL) transcription factors, however the role of miR171 has not been examined in other plants. Results To investigate the roles of mir171 and its target genes in a monocot, the Hvu pri-miR171a was over-expressed in barley (Hordeum vulgare L. cv. Golden promise) leading to reduced expression of at least one HvSCL gene. The resulting transgenic plants displayed a pleiotropic phenotype which included branching defects, an increased number of short vegetative phytomers and late flowering. These phenotypes appear to be the consequence of changes in the organisation of the shoot meristem. In addition, the data show that miR171 over-expression alters the vegetative to reproductive phase transition by activating the miR156 pathway and repressing the expression of the TRD (THIRD OUTER GLUME) and HvPLA1 (Plastochron1) genes. Conclusions Our data suggest that some of the roles of miR171 and its target genes that have been determined in Arabidopsis are conserved in barley and that they have additional functions in barley including activation of the miR156 pathway.

Background During the early stages of seed development many genes are under dynamic regulation to ensure the proper differentiation and establishment of the tissue that will constitute the mature grain. To investigate how miRNA regulation contributes to this process in barley, a combination of small RNA and mRNA degradome analyses were used to identify miRNAs and their targets. Results Our analysis identified 84 known miRNAs and 7 new miRNAs together with 96 putative miRNA target genes regulated through a slicing mechanism in grain tissues during the first 15 days post anthesis. We also identified many potential miRNAs including several belonging to known miRNA families. Our data gave us evidence for an increase in miRNA-mediated regulation during the transition between pre-storage and storage phases. Potential miRNA targets were found in various signalling pathways including components of four phytohormone pathways (ABA, GA, auxin, ethylene) and the defence response to powdery mildew infection. Among the putative miRNA targets we identified were two essential genes controlling the GA response, a GA3oxidase1 and a homolog of the receptor GID1, and a homolog of the ACC oxidase which catalyses the last step of ethylene biosynthesis. We found that two MLA genes are potentially miRNA regulated, establishing a direct link between miRNAs and the R gene response. Conclusion Our dataset provides a useful source of information on miRNA regulation during the early development of cereal grains and our analysis suggests that miRNAs contribute to the control of development of the cereal grain, notably through the regulation of phytohormone response pathways.