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Recent research on long noncoding RNAs (lncRNAs) has expanded our understanding of gene transcription regulation and the generation of cellular complexity. Depending on their genomic origins, lncRNAs can be transcribed from intergenic or intragenic regions or from introns of protein-coding genes. We have recently reported more than 6000 intergenic lncRNAs in Arabidopsis. Here, we systematically identified long noncoding natural antisense transcripts (lncNATs), defined as lncRNAs transcribed from the opposite DNA strand of coding or noncoding genes. We found a total of 37,238 sense–antisense transcript pairs and 70% of annotated mRNAs to be associated with antisense transcripts in Arabidopsis. These lncNATs could be reproducibly detected by different technical platforms, including strand-specific tiling arrays, Agilent custom expression arrays, strand-specific RNA-seq, and qRT-PCR experiments. Moreover, we investigated the expression profiles of sense–antisense pairs in response to light and observed spatial and developmental-specific light effects on 626 concordant and 766 discordant NAT pairs. Genes for a large number of the light-responsive NAT pairs are associated with histone modification peaks, and histone acetylation is dynamically correlated with light-responsive expression changes of NATs.

BackgroundStevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking.ResultsHere, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42–54% and 67–88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants.ConclusionThis study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1600-2) contains supplementary material, which is available to authorized users.

Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the mechanism is not understood. Here, we show that phyB is stable in darkness, but in R, a fraction of phyB translocates into nuclei and becomes degraded by 26S proteasomes. Nuclear phyB degradation is mediated by COP1 E3 ligase, which preferentially interacts with the PhyB N-terminal region (PhyB-N). PhyB-N polyubiquitination by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in vitro can be enhanced by different PHYTOCHROME INTERACTING FACTOR (PIF) proteins that promote COP1/PhyB interaction. Consistent with these results, nuclear phyB accumulates to higher levels in pif single and double mutants and in cop1-4. Our results identify COP1 as an E3 ligase for phyB and other stable phytochromes and uncover the mechanism by which PIFs negatively regulate phyB levels.

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight Ϫ1 . This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.is a nonreducing diglucoside that is found in various organisms, including bacteria, algae, fungi, yeast (Saccharomyces cerevisiae), insects, and some plants (Elbein, 1974). Trehalose serves not only as a carbohydrate reserve, but also as a protective agent against a variety of physical and chemical stresses in various organisms (van Laere, 1989;Wiemken, 1990;Eleutherio et al., 1993;Strøm and Kassen, 1993). Trehalose is known to have high water retention activity, which maintains the fluidity of membranes under dry conditions (Leslie et al., 1995). Thus, this sugar allows desert plants to tolerate naturally occurring stresses during cycles of dehydration and rehydration (Drennan et al., 1993;Mü ller et al., 1995).A role for trehalose in stress tolerance has been demonstrated for cryptobiotic plant species, such as the desiccation-tolerant Selaginella lepidophylla. In this case, trehalose accumulation represented 12% of the plant dry weight during dehydration, which probably protected the proteins and membrane structures. Upon rehydration, S. lepidophylla regained complete viability and the trehalose levels declined (Goddijn and van Dun, 1999). Plants accumulate a number of osmoprotective agents, such as Pro, in response to NaCl stress. During osmotic stress in rice (Oryza sativa), trehalose or similar carbohydrates appear to be more important than Pro. It has been shown that treatmen...

MicroRNAs (miRNAs) are 21 nt RNAs that regulate many biological processes in plants by mediating translational inhibition or cleavage of target transcripts. Arabidopsis mutants defective in miRNA biogenesis have overlapping and highly pleiotropic phenotypes including serrated leaves and ABA hypersensitivity. Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II (Pol II). Since Pol II transcripts are capped, we hypothesized that CBP (cap-binding protein) 20 and 80 may bind to capped primary miRNA (pri-miRNA) transcripts and play a role in their processing. Here, we show that cbp20 and cbp80 mutants have reduced miRNA levels and increased pri-miRNA levels. Co-immunoprecipitation experiments revealed that pri-miRNAs 159, 166, 168 and 172 could be associated with CBP20 and CBP80. We found that CBP20 and CBP80 are stabilized by ABA by a post-translational mechanism, and these proteins are needed for ABA induction of miR159 during seed germination. The lack of miR159 accumulation in ABA-treated seeds of cbp20/80 mutants leads to increased MYB33 and MYB101 transcript levels, and presumably higher levels of these positive regulators result in ABA hypersensitivity. Genetic and molecular analyses show that CBP20 and 80 have overlapping function in the same developmental pathway as SE and HYL1. Our results identify new components in miRNA biogenesis.

Expression patterns of a rice (Oryza sativa) cytochrome c gene OsCc1 and its promoter activity were characterized in transgenic rice plants. OsCc1 transcripts accumulate in most cell types, but to varying levels. Large amounts of OsCc1 transcripts are found in the roots, calli, and suspension cells, but relatively lower in mature leaves, demonstrating its higher levels of expression in non-photosynthetic tissues. Unlike the human cytochrome c gene, which is responsive to cAMP, OsCc1 expression is not enhanced in various rice tissues after dibutyryl cAMP treatments. OsCc1 promoter was linked to the sgfp gene and its activities in different tissues and cell types of transgenic rice plants were analyzed in comparison with the Act1 and RbcS promoters. OsCc1 promoter directs expression in virtually all organs of transgenic plants including roots, leaves, calli, embryos, and suspension cells, showing a particularly high activity in calli and roots. Activity of the OsCc1 promoter was 3-fold higher than Act1 in calli and roots and comparable with RbcS in leaves, representing a useful alternative to the maize (Zea mays) Ubi1 and the rice Act1 promoters for transgene expression in monocots.Several factors increase the expression of chimeric genes in transgenic plants. The choice of promoters affects transgene transcription, resulting in changes not only in concentration, but also in the stage, tissue, and cell specificity of its expression. Over the years, several well-characterized promoters have been made available for transgene expression in plants. However, most of these promoters are from dicot plants. Presently, the cauliflower mosaic virus (CaMV) 35S promoter and its derivatives are among the most commonly used. The CaMV 35S promoter is active in monocots, but its relative strength is substantially lower in monocot than in dicot cells. In addition, it is inactive in some cell types; for example, pollen (Bruce et al., 1989;Christensen et al., 1992; McElroy and Brettell, 1994). Other dicot promoters have also been used for monocot transformation, but activity tends to be lower than for monocot promoters (Wilmink et al., 1995). For example, Kyozuka et al. (1993) reported that expression of the gusA gene driven by a tomato (Lycopersicon esculentum) RbcS promoter is induced by light. However, the expression of the rice (Oryza sativa) RbcS::gusA gene is significantly higher than expression of the tomato RbcS::gusA gene. Conversely, the use of monocot promoters has resulted in a high degree of gene expression in monocots, including rice (McElroy et al., 1991;Cornejo et al., 1993; Kyozuka et al., 1993). Several promoters have been investigated as useful alternatives to drive a high level of expression in monocot transgenesis; for example, the rice Act1 promoter (McElroy et al., 1991), the rice RbcS promoter (Kyozuka et al., 1993; Jang et al., 1999), and the maize (Zea mays) Ubi1 promoter (Uchimiya et al., 1993). In many cases, introns strongly enhance transgene expression in transgenic plants (Wilmink et al., 1995). In ...

Among the five phytochromes in Arabidopsis thaliana, phytochrome A (phyA) plays a major role in seedling deetiolation. Mutant analyses have identified more than 10 positive components acting downstream of phyA to inhibit hypocotyl elongation. However, their sites of action and their hierarchical relationships are poorly understood. Here, we investigated the genetic and molecular relationship between two homologous proteins, FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and FHY1-LIKE (FHL), and two transcription factors, LONG AFTER FAR-RED LIGHT1 (LAF1) and LONG HYPOCOTYL IN FAR-RED1 (HFR1). Analyses of double and triple mutants showed that LAF1, a myb factor, and HFR1, a basic helix-loop-helix factor, independently transmit phyA signals downstream of FHY1 and FHL. Coimmunoprecipitation experiments showed that phyA, FHY1, FHL, LAF1, and HFR1 are components of protein complexes in vivo. In vitro pull-down assays demonstrated direct interactions between partner proteins with the N-terminal region of FHY1, as well as that of FHL, interacting with the LAF1 N-terminal portion and the HFR1 C-terminal region. These results suggest that, in addition to assisting phyA nuclear accumulation, FHY1 and FHL are required to assemble photoreceptor/transcription factor complexes for phyA signaling.