Aethionema arabicum dimorphic seed trait resetting during transition to seedlings.

The transition from germinating seeds to emerging seedlings is one of the most vulnerable plant life cycle stages. Heteromorphic diaspores (seed and fruit dispersal units) are an adaptive bet-hedging strategy to cope with spatiotemporally variable environments. While the roles and mechanisms of seedling traits have been studied in monomorphic species, which produce one type of diaspore, very little is known about seedlings in heteromorphic species. Using the dimorphic diaspore model Aethionema arabicum (Brassicaceae), we identified contrasting mechanisms in the germination responses to different temperatures of the mucilaginous seeds (M+ seed morphs), the dispersed indehiscent fruits (IND fruit morphs), and the bare non-mucilaginous M- seeds obtained from IND fruits by pericarp (fruit coat) removal. What follows the completion of germination is the pre-emergence seedling growth phase, which we investigated by comparative growth assays of early seedlings derived from the M+ seeds, bare M- seeds, and IND fruits. The dimorphic seedlings derived from M+ and M- seeds did not differ in their responses to ambient temperature and water potential. The phenotype of seedlings derived from IND fruits differed in that they had bent hypocotyls and their shoot and root growth was slower, but the biomechanical hypocotyl properties of 15-day-old seedlings did not differ between seedlings derived from germinated M+ seeds, M- seeds, or IND fruits. Comparison of the transcriptomes of the natural dimorphic diaspores, M+ seeds and IND fruits, identified 2,682 differentially expressed genes (DEGs) during late germination. During the subsequent 3 days of seedling pre-emergence growth, the number of DEGs was reduced 10-fold to 277 root DEGs and 16-fold to 164 shoot DEGs. Among the DEGs in early seedlings were hormonal regulators, in particular for auxin, ethylene, and gibberellins. Furthermore, DEGs were identified for water and ion transporters, nitrate transporter and assimilation enzymes, and cell wall remodeling protein genes encoding enzymes targeting xyloglucan and pectin. We conclude that the transcriptomes of seedlings derived from the dimorphic diaspores, M+ seeds and IND fruits, undergo transcriptional resetting during the post-germination pre-emergence growth transition phase from germinated diaspores to growing seedlings.

The dimorphic diaspore model Aethionema arabicum (Brassicaceae): Distinct molecular and morphological control of responses to parental and germination temperatures.

Plants in habitats with unpredictable conditions often have diversified bet-hedging strategies that ensure fitness over a wider range of variable environmental factors. A striking example is the diaspore (seed and fruit) heteromorphism that evolved to maximize species survival in Aethionema arabicum (Brassicaceae) in which external and endogenous triggers allow the production of two distinct diaspores on the same plant. Using this dimorphic diaspore model, we identified contrasting molecular, biophysical, and ecophysiological mechanisms in the germination responses to different temperatures of the mucilaginous seeds (M+ seed morphs), the dispersed indehiscent fruits (IND fruit morphs), and the bare non-mucilaginous M- seeds obtained by pericarp (fruit coat) removal from IND fruits. Large-scale comparative transcriptome and hormone analyses of M+ seeds, IND fruits, and M- seeds provided comprehensive datasets for their distinct thermal responses. Morph-specific differences in co-expressed gene modules in seeds, as well as in seed and pericarp hormone contents, identified a role of the IND pericarp in imposing coat dormancy by generating hypoxia affecting ABA sensitivity. This involved expression of morph-specific transcription factors, hypoxia response and cell wall-remodeling genes, as well as altered abscisic acid (ABA) metabolism, transport, and signaling. Parental temperature affected ABA contents and ABA-related gene expression and altered IND pericarp biomechanical properties. Elucidating the molecular framework underlying the diaspore heteromorphism can provide insight into developmental responses to globally changing temperatures.

Salicylic acid and RNA interference mediate antiviral immunity of plant stem cells.

Stem cells are essential for the development and organ regeneration of multicellular organisms, so their infection by pathogenic viruses must be prevented. Accordingly, mammalian stem cells are highly resistant to viral infection due to dedicated antiviral pathways including RNA interference (RNAi). In plants, a small group of stem cells harbored within the shoot apical meristem generate all postembryonic above-ground tissues, including the germline cells. Many viruses do not proliferate in these cells, yet the molecular bases of this exclusion remain only partially understood. Here, we show that a plant-encoded RNA-dependent RNA polymerase, after activation by the plant hormone salicylic acid, amplifies antiviral RNAi in infected tissues. This provides stem cells with RNA-based virus sequence information, which prevents virus proliferation. Furthermore, we find RNAi to be necessary for stem cell exclusion of several unrelated RNA viruses, despite their ability to efficiently suppress RNAi in the rest of the plant. This work elucidates a molecular pathway of great biological and economic relevance and lays the foundations for our future understanding of the unique systems underlying stem cell immunity.

Phytochromes mediate germination inhibition under red, far-red, and white light in Aethionema arabicum.

The view on the role of light during seed germination stems mainly from studies with Arabidopsis (Arabidopsis thaliana), where light is required to initiate this process. In contrast, white light is a strong inhibitor of germination in other plants, exemplified by accessions of Aethionema arabicum, another member of Brassicaceae. Their seeds respond to light with gene expression changes of key regulators converse to that of Arabidopsis, resulting in opposite hormone regulation and prevention of germination. However, the photoreceptors involved in this process in A. arabicum remain unknown. Here, we screened a mutant collection of A. arabicum and identified koy-1, a mutant that lost light inhibition of germination due to a deletion in the promoter of HEME OXYGENASE 1, the gene for a key enzyme in the biosynthesis of the phytochrome chromophore. koy-1 seeds were unresponsive to red- and far-red light and hyposensitive under white light. Comparison of hormone and gene expression between wild type and koy-1 revealed that very low light fluence stimulates germination, while high irradiance of red and far-red light is inhibitory, indicating a dual role of phytochromes in light-regulated seed germination. The mutation also affects the ratio between the 2 fruit morphs of A. arabicum, suggesting that light reception via phytochromes can fine-tune several parameters of propagation in adaptation to conditions in the habitat.

Aethionema arabicum genome annotation using PacBio full-length transcripts provides a valuable resource for seed dormancy and Brassicaceae evolution research.

Aethionema arabicum is an important model plant for Brassicaceae trait evolution, particularly of seed (development, regulation, germination, dormancy) and fruit (development, dehiscence mechanisms) characters. Its genome assembly was recently improved but the gene annotation was not updated. Here, we improved the Ae. arabicum gene annotation using 294 RNA-seq libraries and 136 307 full-length PacBio Iso-seq transcripts, increasing BUSCO completeness by 11.6% and featuring 5606 additional genes. Analysis of orthologs showed a lower number of genes in Ae. arabicum than in other Brassicaceae, which could be partially explained by loss of homeologs derived from the At-α polyploidization event and by a lower occurrence of tandem duplications after divergence of Aethionema from the other Brassicaceae. Benchmarking of MADS-box genes identified orthologs of FUL and AGL79 not found in previous versions. Analysis of full-length transcripts related to ABA-mediated seed dormancy discovered a conserved isoform of PIF6-ß and antisense transcripts in ABI3, ABI4 and DOG1, among other cases found of different alternative splicing between Turkey and Cyprus ecotypes. The presented data allow alternative splicing mining and proposition of numerous hypotheses to research evolution and functional genomics. Annotation data and sequences are available at the Ae. arabicum DB (https://plantcode.online.uni-marburg.de/aetar_db).

A blind and independent benchmark study for detecting differeally methylated regions in plants.

MOTIVATION: Bisulfite sequencing (BS-seq) is a state-of-the-art technique for investigating methylation of the DNA to gain insights into the epigenetic regulation. Several algorithms have been published for identification of differentially methylated regions (DMRs). However, the performances of the individual methods remain unclear and it is difficult to optimally select an algorithm in application settings. RESULTS: We analyzed BS-seq data from four plants covering three taxonomic groups. We first characterized the data using multiple summary statistics describing methylation levels, coverage, noise, as well as frequencies, magnitudes and lengths of methylated regions. Then, simulated datasets with most similar characteristics to real experimental data were created. Seven different algorithms (metilene, methylKit, MOABS, DMRcate, Defiant, BSmooth, MethylSig) for DMR identification were applied and their performances were assessed. A blind and independent study design was chosen to reduce bias and to derive practical method selection guidelines. Overall, metilene had superior performance in most settings. Data attributes, such as coverage and spread of the DMR lengths, were found to be useful for selecting the best method for DMR detection. A decision tree to select the optimal approach based on these data attributes is provided. The presented procedure might serve as a general strategy for deriving algorithm selection rules tailored to demands in specific application settings. AVAILABILITY AND IMPLEMENTATION: Scripts that were used for the analyses and that can be used for prediction of the optimal algorithm are provided at https://github.com/kreutz-lab/DMR-DecisionTree. Simulated and experimental data are available at https://doi.org/10.6084/m9.figshare.11619045. CONTACT: ckreutz@imbi.uni-freiburg.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Aethionema arabicum: a novel model plant to study the light control of seed germination.

The timing of seed germination is crucial for seed plants and is coordinated by internal and external cues, reflecting adaptations to different habitats. Physiological and molecular studies with lettuce and Arabidopsis thaliana have documented a strict requirement for light to initiate germination and identified many receptors, signaling cascades, and hormonal control elements. In contrast, seed germination in several other plants is inhibited by light, but the molecular basis of this alternative response is unknown. We describe Aethionema arabicum (Brassicaceae) as a suitable model plant to investigate the mechanism of germination inhibition by light, as this species has accessions with natural variation between light-sensitive and light-neutral responses. Inhibition of germination occurs in red, blue, or far-red light and increases with light intensity and duration. Gibberellins and abscisic acid are involved in the control of germination, as in Arabidopsis, but transcriptome comparisons of light- and dark-exposed A. arabicum seeds revealed that, upon light exposure, the expression of genes for key regulators undergo converse changes, resulting in antipodal hormone regulation. These findings illustrate that similar modular components of a pathway in light-inhibited, light-neutral, and light-requiring germination among the Brassicaceae have been assembled in the course of evolution to produce divergent pathways, likely as adaptive traits.

Expression of the eRF1 translation termination factor is controlled by an autoregulatory circuit involving readthrough and nonsense-mediated decay in plants.

When a ribosome reaches a stop codon, the eukaryotic Release Factor 1 (eRF1) binds to the A site of the ribosome and terminates translation. In yeasts and plants, both over- and underexpression of eRF1 lead to altered phenotype indicating that eRF1 expression should be strictly controlled. However, regulation of eRF1 level is still poorly understood. Here we show that expression of plant eRF1 is controlled by a complex negative autoregulatory circuit, which is based on the unique features of the 3΄untranslated region (3΄UTR) of the eRF1-1 transcript. The stop codon of the eRF1-1 mRNA is in a translational readthrough promoting context, while its 3΄UTR induces nonsense-mediated decay (NMD), a translation termination coupled mRNA degradation mechanism. We demonstrate that readthrough partially protects the eRF1-1 mRNA from its 3΄UTR induced NMD, and that elevated eRF1 levels inhibit readthrough and stimulate NMD. Thus, high eRF1 level leads to reduced eRF1-1 expression, as weakened readthrough fails to protect the eRF1-1 mRNA from the more intense NMD. This eRF1 autoregulatory circuit might serve to finely balance general translation termination efficiency.

Developmental Control and Plasticity of Fruit and Seed Dimorphism in Aethionema arabicum.

Understanding how plants cope with changing habitats is a timely and important topic in plant research. Phenotypic plasticity describes the capability of a genotype to produce different phenotypes when exposed to different environmental conditions. In contrast, the constant production of a set of distinct phenotypes by one genotype mediates bet hedging, a strategy that reduces the temporal variance in fitness at the expense of a lowered arithmetic mean fitness. Both phenomena are thought to represent important adaptation strategies to unstable environments. However, little is known about the underlying mechanisms of these phenomena, partly due to the lack of suitable model systems. We used phylogenetic and comparative analyses of fruit and seed anatomy, biomechanics, physiology, and environmental responses to study fruit and seed heteromorphism, a typical morphological basis of a bet-hedging strategy of plants, in the annual Brassicaceae species Aethionema arabicum Our results indicate that heteromorphism evolved twice within the Aethionemeae, including once for the monophyletic annual Aethionema clade. The dimorphism of Ae. arabicum is associated with several anatomic, biomechanical, gene expression, and physiological differences between the fruit and seed morphs. However, fruit ratios and numbers change in response to different environmental conditions. Therefore, the life-history strategy of Ae. arabicum appears to be a blend of bet hedging and plasticity. Together with the available genomic resources, our results pave the way to use this species in future studies intended to unravel the molecular control of heteromorphism and plasticity.

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes.

Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48A(NPL4) complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction.

Comparative functional analysis of full-length and N-terminal fragments of phytochrome C, D and E in red light-induced signaling.

Phytochromes (phy) C, D and E are involved in the regulation of red/far-red light-induced photomorphogenesis of Arabidopsis thaliana, but only limited data are available on the mode of action and biological function of these lesser studied phytochrome species. We fused N-terminal fragments or full-length PHYC, D and E to YELLOW FLUORESCENT PROTEIN (YFP), and analyzed the function, stability and intracellular distribution of these fusion proteins in planta. The activity of the constitutively nuclear-localized homodimers of N-terminal fragments was comparable with that of full-length PHYC, D, E-YFP, and resulted in the regulation of various red light-induced photomorphogenic responses in the studied genetic backgrounds. PHYE-YFP was active in the absence of phyB and phyD, and PHYE-YFP controlled responses, as well as accumulation, of the fusion protein in the nuclei, was saturated at low fluence rates of red light and did not require functional FAR-RED ELONGATED HYPOCOTYL1 (FHY-1) and FHY-1-like proteins. Our data suggest that PHYC-YFP, PHYD-YFP and PHYE-YFP fusion proteins, as well as their truncated N-terminal derivatives, are biologically active in the modulation of red light-regulated photomorphogenesis. We propose that PHYE-YFP can function as a homodimer and that low-fluence red light-induced translocation of phyE and phyA into the nuclei is mediated by different molecular mechanisms.

Plant nonsense-mediated mRNA decay is controlled by different autoregulatory circuits and can be induced by an EJC-like complex.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3'untranslated region (UTR). In yeasts, unusually long 3'UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3'UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3'UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD.

Phosphorylation of phytochrome B inhibits light-induced signaling via accelerated dark reversion in Arabidopsis.

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λmax = 730 nm) and inactive Pr (λmax = 660 nm) forms in a red/far-red-dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein-protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB-yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB(Ser86Asp) Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light-induced nuclear import and interaction of phyB(Ser86Asp)-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB-green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.

The late steps of plant nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and degrades mRNAs containing premature termination codons (PTCs). If translation terminates at a PTC, the UPF1 NMD factor binds the terminating ribosome and recruits UPF2 and UPF3 to form a functional NMD complex, which triggers the rapid decay of the PTC-containing transcript. Although NMD deficiency is seedling lethal in plants, the mechanism of plant NMD remains poorly understood. To understand how the formation of the NMD complex leads to transcript decay we functionally mapped the UPF1 and SMG7 plant NMD factors, the putative key players of NMD target degradation. Our data indicate that the cysteine-histidine-rich (CH) and helicase domains of UPF1 are only essential for the early steps of NMD, whereas the heavily phosphorylated N- and C-terminal regions play a redundant but essential role in the target transcript degradation steps of NMD. We also show that both the N- and the C-terminal regions of SMG7 are essential for NMD. The N terminus contains a phosphoserine-binding domain that is required for the early steps of NMD, whereas the C terminus is required to trigger the degradation of NMD target transcripts. Moreover, SMG7 is a P-body component that can also remobilize UPF1 from the cytoplasm into processing bodies (P bodies). We propose that the N- and C-terminal phosphorylated regions of UPF1 recruit SMG7 to the functional NMD complex, and then SMG7 transports the PTC-containing transcripts into P bodies for degradation.

Functional analysis of the grapevine paralogs of the SMG7 NMD factor using a heterolog VIGS-based gene depletion-complementation system.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and eliminates transcripts having a premature translation termination codon (PTC). NMD is also involved in the control of several wild-type mRNAs. The NMD core machinery consists of three highly conserved NMD factors (UPF1, UPF2 and UPF3) and at least one less conserved 14-3-3-like domain containing protein (SMG7). A PTC is identified by UPF factors, and then SMG7 triggers rapid transcript decay. UPF factors are generally encoded by a single gene, whereas SMG7 has duplicated several times during evolution. Recently it was reported that the plant SMG7 is autoregulated through NMD and that SMG7 has two relatively divergent paralogs in dicots, SMG7 and SMG7L. In mammals all three SMG7 related genes (SMG5, SMG6 and SMG7) are essential in NMD, so we hypothesized that in plants the SMG7 and SMG7L duplicates may also play distinct roles in NMD. To test this possibility, we have analyzed the evolution and the function of plant SMG7 homologs. We show that SMG7L is not required for plant NMD. Interestingly, we found that the grapevine and poplar genomes contain two quite divergent SMG7 paralogs which may have derived from an ancient duplication event. Using heterolog depletion/complementation assays we demonstrate that both grapevine SMG7 copies retained the complete NMD activity and both of them are under NMD control, whilst SMG7L has lost NMD activity and NMD control.

Functional analysis of amino-terminal domains of the photoreceptor phytochrome B.

At the core of the circadian network in Arabidopsis (Arabidopsis thaliana), clock genes/proteins form multiple transcriptional/translational negative feedback loops and generate a basic approximately 24-h oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances the fitness of plants only if it corresponds to the natural day/night cycles. Light, absorbed by photoreceptors, is the most effective signal in synchronizing the oscillator to environmental cycles. Phytochrome B (PHYB) is the major red/far-red light-absorbing phytochrome receptor in light-grown plants. Besides modulating the pace and phase of the circadian clock, PHYB controls photomorphogenesis and delays flowering. It has been demonstrated that the nuclear-localized amino-terminal domain of PHYB is capable of controlling photomorphogenesis and, partly, flowering. Here, we show (1) that PHYB derivatives containing 651 or 450 amino acid residues of the amino-terminal domains are functional in mediating red light signaling to the clock, (2) that circadian entrainment is a nuclear function of PHYB, and (3) that a 410-amino acid amino-terminal fragment does not possess any functions of PHYB due to impaired chromophore binding. However, we provide evidence that the carboxyl-terminal domain is required to mediate entrainment in white light, suggesting a role for this domain in integrating red and blue light signaling to the clock. Moreover, careful analysis of the circadian phenotype of phyB-9 indicates that PHYB provides light signaling for different regulatory loops of the circadian oscillator in a different manner, which results in an apparent decoupling of the loops in the absence of PHYB under specific light conditions.

Plant upstream ORFs can trigger nonsense-mediated mRNA decay in a size-dependent manner.

Nonsense-mediated decay (NMD) is a quality control mechanism that identifies and degrades aberrant mRNAs containing premature termination codons (PTC). NMD also regulates the expression of many wild-type genes. In plants, NMD identifies a stop codon as a PTC and initiates the rapid degradation of the transcript if the 3'untranslated region (UTR) is unusually long or if it harbors an intron. Approximately 20% of plant transcripts have an upstream ORF (uORF) in the 5'UTR. In theory, if a uORF is translated, the 3'UTR downstream of the uORF will be long and harbor introns, thus these transcripts might be degraded by NMD. Therefore, if uORFs can trigger NMD, uORF containing transcripts would be a major group of NMD regulated wild-type plant mRNAs. The aim of this study was to clarify whether plant uORFs could activate NMD. Here we demonstrate that plant uORFs induce NMD in a size-dependent manner, a 50 amino acid (aa) long uORF triggered NMD efficiently, whereas similar but shorter (31 and 15 aa long) uORFs failed to activate NMD response. We have found that only ~2% of annotated Arabidopsis genes contain a first uORF that is longer than 35 aa, thus we propose that NMD regulates only a small fraction of uORF containing transcripts. However, as mRNAs having uORF that is longer than the critical size are strongly overrepresented within the up-regulated transcripts of NMD deficient plants, it is likely that this subset of natural NMD targets induces NMD because of containing a relatively long translatable uORF.

Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3'UTRs, whereas a distinct pathway degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2 and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. The molecular mechanism of long 3'UTR-based plant NMD resembled yeast NMD, whereas the intron-based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG-7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms.

Both introns and long 3'-UTRs operate as cis-acting elements to trigger nonsense-mediated decay in plants.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control mechanism that identifies and eliminates aberrant mRNAs containing a premature termination codon (PTC). Although, key trans-acting NMD factors, UPF1, UPF2 and UPF3 are conserved in yeast and mammals, the cis-acting NMD elements are different. In yeast, short specific sequences or long 3'-untranslated regions (3'-UTRs) render an mRNA subject to NMD, while in mammals' 3'-UTR located introns trigger NMD. Plants also possess an NMD system, although little is known about how it functions. We have elaborated an agroinfiltration-based transient NMD assay system and defined the cis-acting elements that mediate plant NMD. We show that unusually long 3'-UTRs or the presence of introns in the 3'-UTR can subject mRNAs to NMD. These data suggest that both long 3'-UTR-based and intron-based PTC definition operated in the common ancestors of extant eukaryotes (stem eukaryotes) and support the theory that intron-based NMD facilitated the spreading of introns in stem eukaryotes. We have also identified plant UPF1 and showed that tethering of UPF1 to either the 5'- or 3'-UTR of an mRNA results in reduced transcript accumulation. Thus, plant UPF1 might bind to mRNA in a late, irreversible phase of NMD.