Dehydration stress extends mRNA 3' untranslated regions with noncoding RNA functions in Arabidopsis.

The 3' untranslated regions (3' UTRs) of mRNAs play important roles in the regulation of mRNA localization, translation, and stability. Alternative cleavage and polyadenylation (APA) generates mRNAs with different 3' UTRs, but the involvement of this process in stress response has not yet been clarified. Here, we report that a subset of stress-related genes exhibits 3' UTR extensions of their mRNAs during dehydration stress. These extended 3' UTRs have characteristics of long noncoding RNAs and likely do not interact with miRNAs. Functional studies using T-DNA insertion mutants reveal that they can act as antisense transcripts to repress expression levels of sense genes from the opposite strand or can activate the transcription or lead to read-through transcription of their downstream genes. Further analysis suggests that transcripts with 3' UTR extensions have weaker poly(A) signals than those without 3' UTR extensions. Finally, we show that their biogenesis is partially dependent on a trans-acting factor FPA. Taken together, we report that dehydration stress could induce transcript 3' UTR extensions and elucidate a novel function for these stress-induced 3' UTR extensions as long noncoding RNAs in the regulation of their neighboring genes.

A noncoding RNA transcribed from the AGAMOUS (AG) second intron binds to CURLY LEAF and represses AG expression in leaves.

Dispersed H3K27 trimethylation (H3K27me3) of the AGAMOUS (AG) genomic locus is mediated by CURLY LEAF (CLF), a component of the Polycomb Repressive Complex (PRC) 2. Previous reports have shown that the AG second intron, which confers AG tissue-specific expression, harbors sequences targeted by several positive and negative regulators. Using RACE reverse transcription polymerase chain reaction, we found that the AG intron 2 encodes several noncoding RNAs. RNAi experiment showed that incRNA4 is needed for CLF repressive activity. AG-incRNA4RNAi lines showed increased leaf AG mRNA levels associated with a decrease of H3K27me3 levels; these plants displayed AG overexpression phenotypes. Genetic and biochemical analyses demonstrated that the AG-incRNA4 can associate with CLF to repress AG expression in leaf tissues through H3K27me3-mediated repression and to autoregulate its own expression level. The mechanism of AG-incRNA4-mediated repression may be relevant to investigations on tissue-specific expression of Arabidopsis MADS-box genes.

The role of miR156/SPLs modules in Arabidopsis lateral root development.

miR156 is an evolutionarily highly conserved miRNA in plants that defines an age-dependent flowering pathway. The investigations thus far have largely, if not exclusively, confined to plant aerial organs. Root branching architecture is a major determinant of water and nutrients uptake for plants. We show here that MIR156 genes are differentially expressed in specific cells/tissues of lateral roots. Plants overexpressing miR156 produce more lateral roots whereas reducing miR156 levels leads to fewer lateral roots. We demonstrate that at least one representative from the three groups of miR156 targets SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes: SPL3, SPL9 and SPL10 are involved in the repression of lateral root growth, with SPL10 playing a dominant role. In addition, both MIR156 and SPLs are responsive to auxin signaling suggesting that miR156/SPL modules might be involved in the proper timing of the lateral root developmental progression. Collectively, these results unravel a role for miR156/SPLs modules in lateral root development in Arabidopsis.

Analysis of non-coding transcriptome in rice and maize uncovers roles of conserved lncRNAs associated with agriculture traits.

Long non-coding RNAs (lncRNAs) have recently been found to widely exist in eukaryotes and play important roles in key biological processes. To extend our knowledge of lncRNAs in crop plants we performed both non-directional and strand-specific RNA-sequencing experiments to profile non-coding transcriptomes of various rice and maize organs at different developmental stages. Analysis of more than 3 billion reads identified 22 334 long intergenic non-coding RNAs (lincRNAs) and 6673 pairs of sense and natural antisense transcript (NAT). Many lincRNA genes were associated with epigenetic marks. Expression of rice lincRNA genes was significantly correlated with that of nearby protein-coding genes. A set of NAT genes also showed expression correlation with their sense genes. More than 200 rice lincRNA genes had homologous non-coding sequences in the maize genome. Much more lincRNA and NAT genes were derived from conserved genomic regions between the two cereals presenting positional conservation. Protein-coding genes flanking or having a sense-antisense relationship to these conserved lncRNA genes were mainly involved in development and stress responses, suggesting that the associated lncRNAs might have similar functions. Integrating previous genome-wide association studies (GWAS), we found that hundreds of lincRNAs contain trait-associated SNPs (single nucleotide polymorphisms [SNPs]) suggesting their putative contributions to developmental and agriculture traits.

Transcriptional silencing of Arabidopsis endogenes by single-stranded RNAs targeting the promoter region.

Transcriptional gene silencing (TGS) of transgenes by promoter-related RNAs has been known for more than a decade. However, the effectiveness and efficiency of silencing of endogenes by single-stranded and inverted repeat (IR) RNA/silencers remain unclear. Here, we demonstrated that a single-stranded antisense (AS) silencer targeting the promoter region can efficiently silence four Arabidopsis endogenes, with comparable efficiency to an IR silencer. In the case of Too Many Mouths (TMM), single-stranded silencers generated mainly 24 nt small RNAs (smRNAs), whereas IR silencers produced a higher proportion of 21-23 nt smRNAs. Heavy CG, CHG and CHH methylations were detected on the TMM promoter in silenced plant lines. We also demonstrated that the silencing and DNA methylation of the TMM promoter was dependent on the presence of the silencer. Chromatin immunoprecipitation (ChIP) assays showed that DNA methylation was accompanied by formation of repressive chromatin structures. Our results suggest that single-stranded silencer transcripts are converted to double-stranded RNA to enter the RdRM (RNA-directed DNA methylation) pathway for TGS of endogenes.

Improving the effectiveness of artificial microRNA (amiR)-mediated resistance against Turnip mosaic virus by combining two amiRs or by targeting highly conserved viral genomic regions.

A drawback of recent antiviral therapies based on the transgenic expression of artificial microRNAs (amiRs) is the ease with which viruses generate escape mutations. Here, we show two alternative strategies for improving the effectiveness of resistance in plants. First, we expressed two amiRs complementary to independent targets in the viral genome, and second, we designed amiRs complementary to highly conserved RNA motifs in the viral genome.

Enhancing protein stability with retained biological function in transgenic plants.

The final expression level of a transgene-derived protein in transgenic plants depends on transcriptional and post-transcriptional processes. Here, we focus on methods to improve protein stability without comprising biological function. We found that the four isoforms of the Arabidopsis RAD23 protein family are relatively stable. The UBA2 domain derived from RAD23a can be used as a portable stabilizing signal to prolong the half-life of two unstable transcription factors (TFs), HFR1 and PIF3. The increased stability of the TF-UBA2 fusion proteins results in an enhanced phenotype in transgenic plants compared to expression of the TF alone. Similar results were obtained for the RAD23a UBA1 domain. In addition to UBA1/2 of RAD23a, the UBA domain from the Arabidopsis DDI1 protein also increased the half-life of the unstable protein JAZ10.1, which is involved in jasmonate signaling. Taken together, our results suggest that UBA fusions can be used to increase the stability of unstable proteins for basic plant biology research as well as crop improvement.

Tempo and mode of plant RNA virus escape from RNA interference-mediated resistance.

A biotechnological application of artificial microRNAs (amiRs) is the generation of plants that are resistant to virus infection. This resistance has proven to be highly effective and sequence specific. However, before these transgenic plants can be deployed in the field, it is important to evaluate the likelihood of the emergence of resistance-breaking mutants. Two issues are of particular interest: (i) whether such mutants can arise in nontransgenic plants that may act as reservoirs and (ii) whether a suboptimal expression level of the transgene, resulting in subinhibitory concentrations of the amiR, would favor the emergence of escape mutants. To address the first issue, we experimentally evolved independent lineages of Turnip mosaic virus (TuMV) (family Potyviridae) in fully susceptible wild-type Arabidopsis thaliana plants and then simulated the spillover of the evolving virus to fully resistant A. thaliana transgenic plants. To address the second issue, the evolution phase took place with transgenic plants that expressed the amiR at subinhibitory concentrations. Our results show that TuMV populations replicating in susceptible hosts accumulated resistance-breaking alleles that resulted in the overcoming of the resistance of fully resistant plants. The rate at which resistance was broken was 7 times higher for TuMV populations that experienced subinhibitory concentrations of the antiviral amiR. A molecular characterization of escape alleles showed that they all contained at least one nucleotide substitution in the target sequence, generally a transition of the G-to-A and C-to-U types, with many instances of convergent molecular evolution. To better understand the viral population dynamics taking place within each host, as well as to evaluate relevant population genetic parameters, we performed in silico simulations of the experiments. Together, our results contribute to the rational management of amiR-based antiviral resistance in plants.

Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing.

Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.

Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance.

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69(159) and amiR-HC-Pro(159) are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP(159) targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69(159) and amiR-HC-Pro(159) from a dimeric pre-amiR-P69(159)/amiR-HC-Pro(159) transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 degrees C, a temperature that compromises small interfering RNA-mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.

Marker-free transformation: increasing transformation frequency by the use of regeneration-promoting genes.

The generation of transgenic plants free of antibiotic resistance markers is a major challenge to plant biologists and plant breeders. Currently, there are two main strategies to achieve this goal: one approach is to excise or segregate marker genes from the host genome after regeneration of transgenic plants, and the second is based on so-called 'marker-free' transformation. Marker-free transformation has been successfully demonstrated by the use of several plant and non-plant genes that are capable of promoting explant regeneration. This approach appears not only to be effective for the generation of marker-free transgenic plants, but also has great potential to improve the transformation frequency of recalcitrant species.

Overexpression of PGA37/MYB118 and MYB115 promotes vegetative-to-embryonic transition in Arabidopsis.

Formation of somatic embryos from non-germline cells is unique to higher plants and can be manipulated in a variety of species. Previous studies revealed that overexpression of several Arabidopsis genes, including WUSCHEL (WUS)/PLANT GROWTH ACTIVATOR6 (PGA6), BABY BOOM, LEAFY COTYLEDON1 (LEC1), and LEC2, is able to cause vegetative-to-embryonic transition or the formation of somatic embryos. Here, we report that a gain-of-function mutation in the Arabidopsis PGA37 gene, encoding the MYB118 transcription factor, induced vegetative-to-embryonic transition, the formation of somatic embryos from root explants, and an elevated LEC1 expression level. Double mutant analysis showed that WUS was not required for induction of somatic embryos by PGA37/MYB118. In addition, overexpression of MYB115, a homolog of PGA37/MYB118, caused a pga37-like phenotype. A myb118 myb115 double mutant did not show apparent developmental abnormalities. Collectively, these results suggest that PGA37/MYB118 and MYB115 promote vegetative-to-embryonic transition, through a signaling pathway independent of WUS.

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method.

Collective efforts of several laboratories in the past two decades have resulted in the development of various methods for Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana. Among these, the floral dip method is the most facile protocol and widely used for producing transgenic Arabidopsis plants. In this method, transformation of female gametes is accomplished by simply dipping developing Arabidopsis inflorescences for a few seconds into a 5% sucrose solution containing 0.01-0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying the genes to be transferred. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. A transformation frequency of at least 1% can be routinely obtained and a minimum of several hundred independent transgenic lines generated from just two pots of infiltrated plants (20-30 plants per pot) within 2-3 months. Here, we describe the protocol routinely used in our laboratory for the floral dip method for Arabidopsis transformation. Transgenic Arabidopsis plants can be obtained in approximately 3 months.

The ENHANCER OF SHOOT REGENERATION 2 gene in Arabidopsis regulates CUP-SHAPED COTYLEDON 1 at the transcriptional level and controls cotyledon development.

Cytokinins stimulate shoot regeneration in tissue culture but the genes required for this developmental process are not well understood. Here we show that the Arabidopsis gene, ENHANCER OF SHOOT REGENERATION 2 (ESR2), encoding an AP2-domain transcriptional factor, functions in the regeneration of shoots in tissue culture. ESR2 overexpression conferred cytokinin-independent shoot regeneration from cre1/ahk4 mutant explants, suggesting that CRE1 is not required for ESR2-mediated shoot regeneration. ESR2 directly targeted CYCLIN D1;1, ARABIDOPSIS PHOSPHOTRANSMITTER 6 (AHP6) and CUP-SHAPED COTYLEDON 1 (CUC1) in assays involving the translocation of ESR2-ER (estradiol receptor) fusions to the nucleus. Knock-down of ESR2 expression by RNA interference (RNAi) reduced CUC1 expression and resulted in altered cotyledon phenotypes at a low frequency, including partially fused cotyledons and triple cotyledons. Phenotypes of induced ESR2 expression in a cuc1-1 mutant background were suppressed. Our results suggest that ESR2 plays a role in shoot regeneration through transcriptional regulation of CUC1.

Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development.

mRNA turnover in eukaryotes involves the removal of m7GDP from the 5' end. This decapping reaction is mediated by a protein complex well characterized in yeast and human but not in plants. The function of the decapping complex in the development of multicellular organisms is also poorly understood. Here, we show that Arabidopsis thaliana DCP2 can generate from capped mRNAs, m7GDP, and 5'-phosphorylated mRNAs in vitro and that this decapping activity requires an active Nudix domain. DCP2 interacts in vitro and in vivo with DCP1 and VARICOSE (VCS), an Arabidopsis homolog of human Hedls/Ge-1. Moreover, the interacting proteins stimulate DCP2 activity, suggesting that the three proteins operate as a decapping complex. Consistent with their role in mRNA decay, DCP1, DCP2, and VCS colocalize in cytoplasmic foci, which are putative Arabidopsis processing bodies. Compared with the wild type, null mutants of DCP1, DCP2, and VCS accumulate capped mRNAs with a reduced degradation rate. These mutants also share a similar lethal phenotype at the seedling cotyledon stage, with disorganized veins, swollen root hairs, and altered epidermal cell morphology. We conclude that mRNA turnover mediated by the decapping complex is required for postembryonic development in Arabidopsis.

The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis.

Formation of somatic embryos in plants is known to require high concentrations of auxin or 2,4-dichlorophenoxyacetic acid (2,4-D), which presumably acts to trigger a signalling cascade. However, very little is known about the molecular mechanism that mediates the vegetative-to-embryogenic transition. We have employed a genetic approach to dissect the signal transduction pathway during somatic embryogenesis. In a functional screen using a chemical-inducible activation-tagging system, we identified two alleles of Arabidopsis gene PGA6 whose induced overexpression caused high-frequency somatic embryo formation in all tissues and organs tested, without any external plant hormones. Upon inducer withdrawal, all these somatic embryos were able to germinate directly, without any further treatment, and to develop into fertile adult plants. PGA6 was found to be identical to WUSCHEL (WUS), a homeodomain protein previously shown to be involved in specifying stem cell fate in shoot and floral meristems. Transgenic plants carrying an estradiol-inducible XVE-WUS transgene can phenocopy pga6-1 and pga6-2. Our results suggest that WUS/PGA6 also plays a key role during embryogenesis, presumably by promoting the vegetative-to-embryogenic transition and/or maintaining the identity of the embryonic stem cells.

The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis.

Cytokinin plays a critical role in plant growth and development by stimulating cell division and cell differentiation. Despite many years' research efforts, our current understanding of this hormone is still limited regarding both its biosynthesis and signaling. To genetically dissect the cytokinin pathway, we have used a functional screen to identify Arabidopsis gain-of-function mutations that enable shoot formation in the absence of exogenous cytokinins. By using a chemical-inducible activation tagging system, we have identified over 40 putative mutants, designated as pga (plant growth activators), which presumably were affected in key components of cytokinin biosynthesis and signaling pathway. Here, we report a detailed characterization of pga22, a representative mutant from this collection. A gain-of-function mutation in the PGA22 locus resulted in typical cytokinin responses. Molecular and genetic analyses indicated that PGA22 encodes an isopentenyl transferase (IPT) previously identified as AtIPT8. Plants of the pga22 mutant accumulated at remarkably higher levels of isopentenyladenosine-5'-monophosphate and isopentenyladenosine when analyzed by mass spectrometry, suggesting that AtIPT8/PGA22 is a functional IPT that may direct the biosynthesis of cytokinins in planta via an isopentenyladenosine-5'-monophosphate-dependent pathway.