Ammonium protects rice against rice stripe virus by activating HDA703/OsBZR1-mediated BR signaling.

Ammonium (NH4+) is a major inorganic nitrogen source for plants and also as a signal regulates plant growth and defense. Brassinosteroids (BRs) are a class of steroid hormones that control plant developmental and physiological processes through its signaling pathway. Rice is a kind of NH4+-preferring plant which responds to virus infection involving in the regulation of BR biosynthesis and signaling. However, the BR-mediated regulatory mechanisms in rice-virus interactions are not fully understood. In addition, it remains unknown whether there is a direct link between NH4+ and BRs in regulating rice response to virus. HDA703, a histone deacetylase and OsBZR1, a transcription factor, are two positive regulator of BR signaling and interact with each other. In this study, we show that rice plants grown with NH4+ as the sole N source have enhanced resistance to rice stripe virus (RSV), one of the most devastating viruses of rice, than those grown with NO3- as the sole N source. We also show that in contrast to NO3-, NH4+ does not affect BR biosynthesis but promotes BR signaling by upregulating the expression of HDA703 and promoting the accumulation of OsBZR1 in rice shoots. We further show that BR biosynthesis and signaling is required for rice defense against RSV and BR-mediated resistance to RSV attributes to activating HDA703/OsBZR1 module, then decreasing the expression of Ghd7, a direct target of HDA703/OsBZR1. Consistently, increase of the expression of HDA703 or decrease of the expression of Ghd7 enhances rice resistance to RSV. Together, our study reveals that activation of HDA703/OsBZR1-Ghd7 signaling cascade is an undescribed mechanism conferring BR-mediated RSV resistance and NH4+ protects rice against RSV by activating HDA703/OsBZR1-Ghd7-mediated BR signaling in rice.

The histone deacetylase HDA703 interacts with OsBZR1 to regulate rice brassinosteroid signaling, growth and heading date through repression of Ghd7 expression.

The plant steroid hormones brassinosteroids (BRs) play crucial roles in plant growth and development. The BR signal transduction pathway from perception to the key transcription factors has been well understood in Arabidopsis thaliana and in rice (Oryza sativa); however, the mechanisms conferring BR-mediated growth and flowering remain largely unknown, especially in rice. In this study, we show that HDA703 is a histone H4K8 and H4K12 deacetylase in rice. Hda703 mutants display a typical BR loss-of-function phenotype and reduced sensitivity to brassinolide, the most active BR. Rice plants overexpressing HDA703 exhibit some BR gain-of-function phenotypes dependent on BR biosynthesis and signaling. We also show that HDA703 is a direct target of BRASSINAZOLE-RESISTANT1 (OsBZR1), a primary regulator of rice BR signaling, and HDA703 interacts with OsBZR1 in rice. We further show that GRAIN NUMBER, PLANT HEIGHT, and HEADING DATE 7 (Ghd7), a central regulator of growth, development, and the stress response, is a direct target of OsBZR1. HDA703 directly targets Ghd7 and represses its expression through histone H4 deacetylation. HDA703-overexpressing rice plants phenocopy Ghd7-silencing rice plants in both growth and heading date. Together, our study suggests that HDA703, a histone H4 deacetylase, interacts with OsBZR1 to regulate rice BR signaling, growth, and heading date through epigenetic regulation of Ghd7.

Promotion of BR Biosynthesis by miR444 Is Required for Ammonium-Triggered Inhibition of Root Growth.

Rice (Oryza sativa), the staple food for almost half of the world's population, prefers ammonium (NH4 +) as the major nitrogen resource, and while NH4 + has profound effects on rice growth and yields, the underlying regulatory mechanisms remain largely unknown. Brassinosteroids (BRs) are a class of steroidal hormones playing key roles in plant growth and development. In this study, we show that NH4 + promotes BR biosynthesis through miR444 to regulate rice root growth. miR444 targeted five homologous MADS-box transcription repressors potentially forming homologous or heterogeneous complexes in rice. miR444 positively regulated BR biosynthesis through its MADS-box targets, which directly repress the transcription of BR-deficient dwarf 1 (OsBRD1), a key BR biosynthetic gene. NH4 + induced the miR444-OsBRD1 signaling cascade in roots, thereby increasing the amount of BRs, whose biosynthesis and signaling were required for NH4 + -dependent root elongation inhibition. Consistently, miR444-overexpressing rice roots were hypersensitive to NH4 + depending on BR biosynthesis, and overexpression of miR444's target, OsMADS57, resulted in rice hyposensitivity to NH4 + in root elongation, which was associated with a reduction of BR content. In summary, our findings reveal a cross talk mechanism between NH4 + and BR in which NH4 + activates miR444-OsBRD1, an undescribed BR biosynthesis-promoting signaling cascade, to increase BR content, inhibiting root elongation in rice.

A Signaling Cascade from miR444 to RDR1 in Rice Antiviral RNA Silencing Pathway.

Plant RNA-DEPENDENT RNA POLYMERASE1 (RDR1) is a key component of the antiviral RNA-silencing pathway, contributing to the biogenesis of virus-derived small interfering RNAs. This enzyme also is responsible for producing virus-activated endogenous small interfering RNAs to stimulate the broad-spectrum antiviral activity through silencing host genes. The expression of RDR1 orthologs in various plants is usually induced by virus infection. However, the molecular mechanisms of activation of RDR1 expression in response to virus infection remain unknown. Here, we show that a monocot-specific microRNA, miR444, is a key factor in relaying the antiviral signaling from virus infection to OsRDR1 expression. The expression of miR444 is enhanced by infection with Rice stripe virus (RSV), and overexpression of miR444 improves rice (Oryza sativa) resistance against RSV infection accompanied by the up-regulation of OsRDR1 expression. We further show that three miR444 targets, the MIKC(C)-type MADS box proteins OsMADS23, OsMADS27a, and OsMADS57, form homodimers and heterodimers between them to repress the expression of OsRDR1 by directly binding to the CArG motifs of its promoter. Consequently, an increased level of miR444 diminishes the repressive roles of OsMADS23, OsMADS27a, and OsMADS57 on OsRDR1 transcription, thus activating the OsRDR1-dependent antiviral RNA-silencing pathway. We also show that overexpression of miR444-resistant OsMADS57 reduced OsRDR1 expression and rice resistance against RSV infection, and knockout of OsRDR1 reduced rice resistance against RSV infection. In conclusion, our results reveal a molecular cascade in the rice antiviral pathway in which miR444 and its MADS box targets directly control OsRDR1 transcription.

Comparative profiling of miRNA expression in developing seeds of high linoleic and high oleic safflower (Carthamus tinctorius L.) plants.

Vegetable oils high in oleic acid are considered to be advantageous because of their better nutritional value and potential industrial applications. The oleic acid content in the classic safflower oil is normally 10-15% while a natural mutant (ol) accumulates elevated oleic acid up to 70% in seed oil. As a part of our investigation into the molecular features of the high oleic (HO) trait in safflower we have profiled the microRNA (miRNA) populations in developing safflower seeds expressing the ol allele in comparison to the wild type high linoleic (HL) safflower using deep sequencing technology. The small RNA populations of the mid-maturity developing embryos of homozygous ol HO and wild type HL safflower had a very similar size distribution pattern, however, only ~16.5% of the unique small RNAs were overlapping in these two genotypes. From these two small RNA populations we have found 55 known miRNAs and identified two candidate novel miRNA families to be likely unique to the developing safflower seeds. Target genes with conserved as well as novel functions were predicted for the conserved miRNAs. We have also identified 13 miRNAs differentially expressed between the HO and HL safflower genotypes. The results may lay a foundation for unraveling the miRNA-mediated molecular processes that regulate oleic acid accumulation in the HO safflower mutant and developmental processes in safflower embryos in general.

Comparative transcriptomic analysis of developing cotton cotyledons and embryo axis.

BACKGROUND: As a by product of higher value cotton fibre, cotton seed has been increasingly recognised to have excellent potential as a source of additional food, feed, biofuel stock and even a renewable platform for the production of many diverse biological molecules for agriculture and industrial enterprises. The large size difference between cotyledon and embryo axis that make up a cotton seed results in the under-representation of embryo axis gene transcript levels in whole seed embryo samples. Therefore, the determination of gene transcript levels in the cotyledons and embryo axes separately should lead to a better understanding of metabolism in these two developmentally diverse tissues. RESULTS: A comparative study of transcriptome changes between cotton developing cotyledon and embryo axis has been carried out. 17,384 unigenes (20.74% of all the unigenes) were differentially expressed in the two adjacent embryo tissues, and among them, 7,727 unigenes (44.45%) were down-regulated and 9,657 unigenes (55.55%) were up-regulated in cotyledon. CONCLUSIONS: Our study has provided a comprehensive dataset that documents the dynamics of the transcriptome at the mid-maturity of cotton seed development and in discrete seed tissues, including embryo axis and cotyledon tissues. The results showed that cotton seed is subject to many transcriptome variations in these two tissue types and the differential gene expression between cotton embryo axis and cotyledon uncovered in our study should provide an important starting point for understanding how gene activity is coordinated during seed development to make a seed. Further, the identification of genes involved in rapid metabolite accumulation stage of seed development will extend our understanding of the complex molecular and cellular events in these developmental processes and provide a foundation for future studies on the metabolism, embryo differentiation of cotton and other dicot oilseed crops.

Rolling circle amplification-mediated hairpin RNA (RMHR) library construction in plants.

Long hairpin RNA (lhRNA) construct-induced gene silencing facilitates the study of gene function in plants and animals, but constructing multiple lhRNA vectors using traditional approaches is both time-consuming and costly. Also, most of the existing approaches are based on sequence-specific cloning of individual sequences, and are therefore not suitable for preparing hpRNA libraries from a pool of mixed target sequences. Here we describe a rolling-circle amplification (RCA)-mediated hpRNA (RMHR) construction system suitable for generating libraries of lhRNA constructs from any gene of interest or pool of genes. Using RMHR we successfully generated a lhRNA library from a Arabidopsis cDNA population containing known and unknown genes, with an average size of 500-800 bp for the inverted-repeat inserts. To validate the RMHR system, lhRNA constructs targeting the beta-glucuronidase (GUS) gene were tested using Agrobacterium infiltration and shown to be effective at inducing GUS silencing in tobacco leaves. Our results indicate that the RMHR technique permits rapid, efficient and low-cost preparation of genome-wide lhRNA expression libraries.

Au nanoparticle micropatterns prepared from self-assembled films.

A kind of hybrid multilayer film based on mercaptobenzoic acid-capped Au nanoparticles (MBA-Au-NPs) and photoreactive nitrodiazoresin (NDR) has been fabricated via electrostatic self-assembly. Upon exposure to UV light, the initial ionic bonds between the layers of the film convert into covalent bonds and the film stability toward polar solvents, salt, or surfactant solutions increases significantly. The micropatterned NDR/MBA-Au-NP film with the covalently linked architecture was formed by selecting exposure of the film through a photomask and later developed in sodium dodecyl sulfate (SDS) aqueous solution. The metallic Au-NP micropatterns, furthermore, are produced by sintering the micropatterned NDR/MBA-Au-NP film at 550 degrees C, at which the organic components are removed completely. The well-defined micropatterns were characterized with atomic force microscopy (AFM), field emission scanning electron microscopy (FE-SEM), microscope with a charge-coupled device (CCD) camera, and X-ray photoelectron spectroscopy (XPS).

Micropatterns constructed from Au nanoparticles.

Covalently linked Au-NPs micropatterns have been successfully fabricated from the self-assembly film composed of 4-mercaptophenol-capped Au nanoparticles (Au-NPs) and -N2+ containing polymers of nitro-diazoresin (NDR) by selective exposure to UV light and development in sodium dodecyl sulfate (SDS) aqueous solution. The resultant well-defined micropatterns were characterized with AFM and XPS.