Search

Perturbations in ribosome biogenesis cause a type of cellular stress called nucleolar or ribosomal stress, which triggers adaptive responses in both animal and plant cells. The Arabidopsis ANAC082 transcription factor has been identified as a key mediator of the plant nucleolar stress response. The 5 '-untranslated region (5 '-UTR) of ANAC082 mRNA contains an upstream ORF (uORF) encoding an evolutionarily conserved amino acid sequence. Here, we report that this uORF mediates the upregulation of ANAC082 expression in response to nucleolar stress. When transgenic Arabidopsis plants containing a luciferase reporter gene under the control of the ANAC082 promoter and 5 '-UTR were treated with reagents that induced nucleolar stress, expression of the reporter gene was enhanced in a uORF sequence-dependent manner. Additionally, we examined the effect of an endoplasmic reticulum (ER) stress-inducing reagent on reporter gene expression because the closest homolog of ANAC082 in Arabidopsis, ANAC103, is involved in the ER stress response. However, the ANAC082 uORF did not respond to ER stress. Interestingly, although ANAC103 has a uORF with an amino acid sequence similar to that of the ANAC082 uORF, the C-terminal sequence critical for regulation is not well conserved among ANAC103 homologs in Brassicaceae. Transient expression assays revealed that unlike the ANAC082 uORF, the ANAC103 uORF does not exert a sequence-dependent repressive effect. Altogether, our findings suggest that the ANAC082 uORF is important for the nucleolar stress response but not for the ER stress response, and that for this reason, the uORF sequence-dependent regulation was lost in ANAC103 during evolution.

Perturbations in ribosome biogenesis cause a type of cellular stress called nucleolar or ribosomal stress, which triggers adaptive responses in both animal and plant cells. The Arabidopsis ANAC082 transcription factor has been identified as a key mediator of the plant nucleolar stress response. The 5′-untranslated region (5′-UTR) of ANAC082 mRNA contains an upstream ORF (uORF) encoding an evolutionarily conserved amino acid sequence. Here, we report that this uORF mediates the upregulation of ANAC082 translation in response to nucleolar stress. When transgenic Arabidopsis plants containing a luciferase reporter gene under the control of the ANAC082 promoter and 5′-UTR were treated with reagents that induced nucleolar stress, translation of the reporter gene was enhanced in a uORF sequence-dependent manner. Additionally, we examined the effect of an endoplasmic reticulum (ER) stress-inducing reagent on reporter gene expression because the closest homolog of ANAC082 in Arabidopsis, ANAC103, is involved in the ER stress response. However, the ANAC082 uORF did not respond to ER stress. Interestingly, although ANAC103 has a uORF with an amino acid sequence similar to that of the ANAC082 uORF, the C-terminal sequence critical for regulation is not well conserved among ANAC103 homologs in Brassicaceae. Transient expression assays revealed that unlike the ANAC082 uORF, the ANAC103 uORF does not exert a sequence-dependent regulatory effect. Altogether, our findings suggest that the ANAC082 uORF is important for the nucleolar stress response but not for the ER stress response, and that for this reason, the uORF sequence-dependent translational regulation was lost in ANAC103 during evolution.

We previously reported that ribosome stalling at AUG-stop sequences in the 5'-untranslated region plays a critical role in regulating the expression of Arabidopsis thaliana NIP5;1, which encodes a boron uptake transporter, in response to boron conditions in media. This ribosome stalling is triggered specifically by boric acid, but the mechanisms are unknown. Although upstream open reading frames (uORFs) are known in many cases to regulate translation through peptides encoded by the uORF, AUG-stop stalling does not involve any peptide synthesis. The unique feature of AUG-stops - that termination follows immediately after initiation - suggests a possible effect of boron on the translational process itself. However, the generality of AUG-stop-mediated translational regulation and the effect of boron on translation at the genome scale are not clear. Here, we conducted a ribosome profiling analysis to reveal the genome-wide regulation of translation in response to boron conditions in A. thaliana shoots. We identified hundreds of translationally regulated genes that function in various biological processes. Under high-boron conditions, transcripts with reduced translation efficiency were rich in uORFs, highlighting the importance of uORF-mediated translational regulation. We found 673 uORFs that had more frequent ribosome association. Moreover, transcripts that were translationally downregulated under high-boron conditions were rich in minimum uORFs (AUG-stops), suggesting that AUG-stops play a global role in the boron response. Metagene analysis revealed that boron increased the ribosome occupancy of stop codons, indicating that this element is involved in global translational termination processes.

This study identified four novel regulatory non-AUG-initiated upstream ORFs (uORFs) with evolutionarily conserved sequences in Arabidopsis and elucidated the mechanism by which a non-AUG-initiated uORF promotes main ORF translation. Upstream open reading frames (uORFs) are short ORFs found in the 5'-untranslated regions (5'-UTRs) of eukaryotic transcripts and can influence the translation of protein-coding main ORFs (mORFs). Recent genome-wide ribosome profiling studies have revealed that hundreds or thousands of uORFs initiate translation at non-AUG start codons. However, the physiological significance of these non-AUG uORFs has so far been demonstrated for only a few of them. In this study, to identify physiologically important regulatory non-AUG uORFs in Arabidopsis, we took an approach that combined bioinformatics and experimental analysis. Since physiologically important non-AUG uORFs are likely to be conserved across species, we first searched the Arabidopsis genome for non-AUG-initiated uORFs with evolutionarily conserved sequences. Then, we examined the effects of the conserved non-AUG uORFs on the expression of the downstream mORFs using transient expression assays. As a result, three inhibitory and one promotive non-AUG uORFs were identified. Among the inhibitory non-AUG uORFs, two exerted repressive effects on mORF expression in an amino acid sequence-dependent manner. These two non-AUG uORFs are likely to encode regulatory peptides that cause ribosome stalling, thereby enhancing their repressive effects. In contrast, one of the identified regulatory non-AUG uORFs promoted mORF expression by alleviating the inhibitory effect of a downstream AUG-initiated uORF. These findings provide insights into the mechanisms that enable non-AUG uORFs to play regulatory roles despite their low translation initiation efficiencies.

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which β-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the β-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.

Recent accumulation of genomic and transcriptomic information has facilitated genetic studies. Increasing evidence has demonstrated that translation is an important regulatory step, and the transcriptome does not necessarily reflect the profile of functional protein production. Deep sequencing of ribosome-protected mRNA fragments (ribosome profiling or Ribo-seq) has enabled genome-wide analysis of translation. Sorghum is a C4 cereal important not only as food but also as forage and a bioenergy resource. Its resistance to harsh environments has made it an agriculturally important research subject. Yet genome-wide translational profiles in sorghum are still missing. In this study, we took advantage of Ribo-seq and identified actively translated reading frames throughout the genome. We detected translation of 4,843 main open reading frames (ORFs) annotated in the sorghum reference genome version 3.1 and revealed a number of unannotated translational events. A comparison of the transcriptome and translatome between sorghums grown under normal and sulfur-deficient conditions revealed that gene expression is modulated independently at transcript and translation levels. Our study revealed the translational landscape of sorghum’s response to sulfur and provides datasets that could serve as a fundamental resource to extend genetic research on sorghum, including studies on translational regulation.

Let λ ∈ P+ be a level-zero dominant integral weight, and w the coset representative of minimal length for a coset in W/Wλ, where Wλ is the stabilizer of λ in a finite Weyl group W. In this paper, we give a module $$ {\mathbbm{K}}_w^{-}\left(\uplambda \right) $$ over the negative part of a quantum affine algebra whose graded character is identical to the specialization at t = ∞ of the nonsymmetric Macdonald polynomial Ewλ(q, t) multiplied by a certain explicit finite product of rational functions of q of the form (1 − q−r)−1 for a positive integer r. This module $$ {\mathbbm{K}}_w^{-}\left(\uplambda \right) $$ (called a level-zero van der Kallen module) is defined to be the quotient module of the level-zero Demazure module $$ {V}_w^{-}\left(\uplambda \right) $$ by the sum of the submodules $$ {V}_z^{-}\left(\uplambda \right) $$ for all those coset representatives z of minimal length for cosets in W/Wλ such that z > w in the Bruhat order < on W.

We prove a Chevalley formula for anti-dominant weights in the torus-equivariant K-group of semi-infinite flag manifolds, which is described explicitly in terms of semi-infinite Lakshmibai-Seshadri paths (or equivalently, quantum Lakshmibai-Seshadri paths); in contrast to the Chevalley formula for dominant weights in our previous paper [17] , the formula for anti-dominant weights has a significant finiteness property. Based on geometric results established in [17] , our proof is representation-theoretic, and the Chevalley formula for anti-dominant weights follows from a certain identity for the graded characters of Demazure submodules of a level-zero extremal weight module over a quantum affine algebra; in the proof of this identity, we make use of the (combinatorial) standard monomial theory for semi-infinite Lakshmibai-Seshadri paths, and also a string property of Demazure-like subsets of the set of semi-infinite Lakshmibai-Seshadri paths of a fixed shape, which gives an explicit realization of the crystal basis of a level-zero extremal weight module.

Boron (B) is an essential element in plants but is toxic when it accumulates to high levels. In root cells of Arabidopsis (Arabidopsis thaliana), the borate exporter BOR1 is polarly localized in the plasma membrane toward the stele side for directional transport of B. Upon high-B supply, BOR1 is rapidly internalized and degraded in the vacuole. The polar localization and B-induced vacuolar sorting of BOR1 are mediated by endocytosis from the plasma membrane. To dissect the endocytic pathways mediating the polar localization and vacuolar sorting, we investigated the contribution of the clathrin adaptor protein, ADAPTOR PROTEIN2 (AP2) complex, to BOR1 trafficking. In the mutants lacking µ- or σ-subunits of the AP2 complex, the polar localization and constitutive endocytosis of BOR1 under low-B conditions were dramatically disturbed. A coimmunoprecipitation assay showed association of the AP2 complex with BOR1, while it was independent of YxxΦ sorting motifs, which are in a cytosolic loop of BOR1. A yeast two-hybrid assay supported the interaction of the AP2 complex µ-subunit with the C-terminal tail but not with the YxxΦ motifs in the cytosolic loop of BOR1. Intriguingly, lack of the AP2 subunit did not affect the B-induced rapid internalization/vacuolar sorting of BOR1. Consistent with defects in the polar localization, the AP2 complex mutants showed hypersensitivity to B deficiency. Our results indicate that AP2-dependent endocytosis maintains the polar localization of BOR1 to support plant growth under low-B conditions, whereas the B-induced vacuolar sorting of BOR1 is mediated through an AP2-independent endocytic pathway.

We prove an explicit inverse Chevalley formula in the equivariantK-theory of semi-infinite flag manifolds of simply laced type. By an ‘inverse Chevalley formula’ we mean a formula for the product of an equivariant scalar with a Schubert class, expressed as a$\mathbb {Z}\left [q^{\pm 1}\right ]$-linear combination of Schubert classes twisted by equivariant line bundles. Our formula applies to arbitrary Schubert classes in semi-infinite flag manifolds of simply laced type and equivariant scalars$e^{\lambda }$, where$\lambda $is an arbitrary minuscule weight. By a result of Stembridge, our formula completely determines the inverse Chevalley formula for arbitrary weights in simply laced type except for type$E_8$. The combinatorics of our formula is governed by the quantum Bruhat graph, and the proof is based on a limit from the double affine Hecke algebra. Thus our formula also provides an explicit determination of all nonsymmetricq-Toda operators for minuscule weights in ADE type.

In this paper, we give an explicit formula of Chevalley type, in terms of the Bruhat graph, for the quantum multiplication with the class of the line bundle associated to the anti-dominant minuscule fundamental weight $- \varpi_{k}$ in the torus-equivariant quantum $K$-group of the partial flag manifold $G/P_{J}$ (where $J = I \setminus \{k\}$) corresponding to the maximal (standard) parabolic subgroup $P_{J}$ of minuscule type in type $A$, $D$, $E$, or $B$. This result is obtained by proving a similar formula in a torus-equivariant $K$-group of the semi-infinite partial flag manifold $\mathbf{Q}_{J}$ of minuscule type, and then by making use of the isomorphism between the torus-equivariant quantum $K$-group of $G/P_{J}$ and the torus-equivariant $K$-group of $\mathbf{Q}_{J}$, recently established by Kato., 30 pages

Background Upstream open reading frames (uORFs) in the 5′-untranslated regions (5′-UTRs) of certain eukaryotic mRNAs encode evolutionarily conserved functional peptides, such as cis-acting regulatory peptides that control translation of downstream main ORFs (mORFs). For genome-wide searches for uORFs with conserved peptide sequences (CPuORFs), comparative genomic studies have been conducted, in which uORF sequences were compared between selected species. To increase chances of identifying CPuORFs, we previously developed an approach in which uORF sequences were compared using BLAST between Arabidopsis and any other plant species with available transcript sequence databases. If this approach is applied to multiple plant species belonging to phylogenetically distant clades, it is expected to further comprehensively identify CPuORFs conserved in various plant lineages, including those conserved among relatively small taxonomic groups. Results To efficiently compare uORF sequences among many species and efficiently identify CPuORFs conserved in various taxonomic lineages, we developed a novel pipeline, ESUCA. We applied ESUCA to the genomes of five angiosperm species, which belong to phylogenetically distant clades, and selected CPuORFs conserved among at least three different orders. Through these analyses, we identified 89 novel CPuORF families. As expected, ESUCA analysis of each of the five angiosperm genomes identified many CPuORFs that were not identified from ESUCA analyses of the other four species. However, unexpectedly, these CPuORFs include those conserved across wide taxonomic ranges, indicating that the approach used here is useful not only for comprehensive identification of narrowly conserved CPuORFs but also for that of widely conserved CPuORFs. Examination of the effects of 11 selected CPuORFs on mORF translation revealed that CPuORFs conserved only in relatively narrow taxonomic ranges can have sequence-dependent regulatory effects, suggesting that most of the identified CPuORFs are conserved because of functional constraints of their encoded peptides. Conclusions This study demonstrates that ESUCA is capable of efficiently identifying CPuORFs likely to be conserved because of the functional importance of their encoded peptides. Furthermore, our data show that the approach in which uORF sequences from multiple species are compared with those of many other species, using ESUCA, is highly effective in comprehensively identifying CPuORFs conserved in various taxonomic ranges.

Boron (B) is an essential element for plants; however, as high B concentrations are toxic, B transport must be tightly regulated. BOR1 is a borate exporter in Arabidopsis (Arabidopsis thaliana) that facilitates B translocation into shoots under B deficiency conditions. When the B supply is sufficient, BOR1 expression is down-regulated by selective degradation of BOR1 protein, while additional BOR1 regulatory mechanisms are proposed to exist. In this study, we identified a novel B-dependent BOR1 translational suppression mechanism. In vivo and in vitro reporter assays demonstrated that BOR1 translation was reduced in a B-dependent manner and that the 5'-untranslated region was both necessary and sufficient for this process. Mutational analysis revealed that multiple upstream open reading frames in the 5'-untranslated region were required for BOR1 translational suppression, and this process depended on the efficiency of translational reinitiation at the BOR1 open reading frame after translation of the upstream open reading frames. To understand the physiological significance of BOR1 regulation, we characterized transgenic plants defective in either one or both of the BOR1 regulation mechanisms. BOR1 translational suppression was induced at higher B concentrations than those triggering BOR1 degradation. Plants lacking both regulation mechanisms exhibited more severe shoot growth reduction under high-B conditions than did plants lacking BOR1 degradation alone, thus demonstrating the importance of BOR1 translational suppression. This study demonstrates that two mechanisms of posttranscriptional BOR1 regulation, each induced under different B concentrations, contribute to the avoidance of B toxicity in plants.

We give a representation-theoretic (or rather, crystal-theoretic) proof of Cherednik-Orr's recursion formula of Demazure type for the specialization at t = ∞ of the nonsymmetric Macdonald polynomials Ewλ(q, t), w ∈ W, where λ is a dominant integral weight and W is a finite Weyl group.

Specific sequences of certain nascent peptides cause programmed ribosomal arrest during mRNA translation to control gene expression. In eukaryotes, most known regulatory arrest peptides are encoded by upstream open reading frames (uORFs) present in the 5′-untranslated region of mRNAs. However, to date, a limited number of eukaryotic uORFs encoding arrest peptides have been reported. Here, we searched for arrest peptide-encoding uORFs among Arabidopsis thaliana uORFs with evolutionarily conserved peptide sequences. Analysis of in vitro translation products of 22 conserved uORFs identified three novel uORFs causing ribosomal arrest in a peptide sequence-dependent manner. Stop codon-scanning mutagenesis, in which the effect of changing the uORF stop codon position on the ribosomal arrest was examined, and toeprint analysis revealed that two of the three uORFs cause ribosomal arrest during translation elongation, whereas the other one causes ribosomal arrest during translation termination. Transient expression assays showed that the newly identified arrest-causing uORFs exerted a strong sequence-dependent repressive effect on the expression of the downstream reporter gene in A. thaliana protoplasts. These results suggest that the peptide sequences of the three uORFs identified in this study cause ribosomal arrest in the uORFs, thereby repressing the expression of proteins encoded by the main ORFs.

In 1980, Lusztig introduced the periodic Kazhdan-Lusztig polynomials, which are conjectured to have important information about the characters of irreducible modules of a reductive group over a field of positive characteristic, and also about those of an affine Kac-Moody algebra at the critical level. The periodic Kazhdan-Lusztig polynomials can be computed by using another family of polynomials, called the periodic $R$-polynomials. In this paper, we prove a (closed) combinatorial formula expressing periodic $R$-polynomials in terms of the "doubled" Bruhat graph associated to a finite Weyl group and a finite root system., Comment: 33 pages

Background Upstream open reading frames (uORFs) are present in the 5′-untranslated regions of many eukaryotic mRNAs, and some peptides encoded in these regions play important regulatory roles in controlling main ORF (mORF) translation. To comprehensively identify uORFs encoding functional peptides, genome-wide searches for uORFs with conserved peptide sequences (CPuORFs) have been conducted in various organisms using comparative genomic approaches. However, in animals, CPuORFs have been identified only by comparing uORF sequences between a limited number of closely related species, and how many previously identified CPuORFs encode regulatory peptides is unclear. Results Here, we conducted exhaustive genome-wide searches for animal CPuORFs conserved in various taxonomic ranges, using the ESUCA pipeline, which we recently developed for efficient comprehensive identification of CPuORFs. ESUCA can efficiently compare uORF sequences between an unlimited number of species using BLAST and automatically determine the taxonomic ranges of uORF sequence conservation. By applying ESUCA to human, chicken, zebrafish, and fruit fly genomes, 1,517 (1,425 novel and 92 known) CPuORFs were identified. We examined the effects of 17 human CPuORFs on mORF translation using transient expression assays. Through these analyses, we identified seven novel regulatory CPuORFs that repressed mORF translation in a sequence-dependent manner, including the one conserved only among Eutheria. Conclusions We discovered a much higher number of animal CPuORFs than previously identified. Since most human CPuORFs identified in this study are conserved across a wide range of Eutheria or a wider taxonomic range, many CPuORFs encoding regulatory peptides are expected to be found in the identified CPuORFs.

BackgroundUpstream open reading frames (uORFs) in the 5′-untranslated regions (5′-UTRs) of certain eukaryotic mRNAs encode evolutionarily conserved functional peptides, such as cis-acting regulatory peptides that control translation of downstream main ORFs (mORFs). For genome-wide searches for uORFs with conserved peptide sequences (CPuORFs), comparative genomic studies have been conducted, in which uORF sequences were compared between selected species. To increase chances of identifying CPuORFs, we previously developed an approach in which uORF sequences were compared using BLAST betweenArabidopsisand any other plant species with available transcript sequence databases. If this approach is applied to multiple plant species belonging to phylogenetically distant clades, it is expected to further comprehensively identify CPuORFs conserved in various plant lineages, including those conserved among relatively small taxonomic groups.ResultsTo efficiently compare uORF sequences among many species and efficiently identify CPuORFs conserved in various taxonomic lineages, we developed a novel pipeline, ESUCA. We applied ESUCA to the genomes of five angiosperm species, which belong to phylogenetically distant clades, and selected CPuORFs conserved among at least three different orders. Through these analyses, we identified 88 novel CPuORF families. As expected, ESUCA analysis of each of the five angiosperm genomes identified many CPuORFs that were not identified from ESUCA analyses of the other four species. However, unexpectedly, these CPuORFs include those conserved in wide taxonomic ranges, indicating that the approach used here is useful not only for comprehensive identification of narrowly conserved CPuORFs but also for that of widely conserved CPuORFs. Examination of the effects of 11 selected CPuORFs on mORF translation revealed that CPuORFs conserved only in relatively narrow taxonomic ranges can have sequence-dependent regulatory effects, suggesting that most of the identified CPuORFs are conserved because of functional constraints of their encoded peptides.ConclusionsThis study demonstrates that ESUCA is capable of efficiently identifying CPuORFs likely to be conserved because of the functional importance of their encoded peptides. Furthermore, our data show that the approach in which uORF sequences from multiple species are compared with those of many other species, using ESUCA, is highly effective in comprehensively identifying CPuORFs conserved in various taxonomic ranges.

We prove that the action of an $\omega$-root operator on the set of all paths fixed by a diagram automorphism $\omega$ of a Kac–Moody algebra $\mathfrak{g}$ can be identified with the action of a root operator for the orbit Lie algebra $\breve{\mathfrak{g}}$. Moreover, we prove that there exists a canonical bijection between the elements of the crystal base $\mathcal{B}(\infty)$ for $\mathfrak{g}$ fixed by $\omega$ and the elements of the crystal base $\breve{\mathcal{B}}(\infty)$ for $\breve{\mathfrak{g}}$. Using this result, we give twining character formulas for the "negative part" of the quantized universal enveloping algebra $U_q(\mathfrak{g})$ and for certain modules of Demazure type.

Let $\lambda$ be a (level-zero) dominant integral weight for an untwisted affine Lie algebra, and let $\mathrm{QLS}(\lambda)$ denote the quantum Lakshmibai-Seshadri (QLS) paths of shape $\lambda$. For an element $w$ of a finite Weyl group $W$, the specializations at $t = 0$ and $t = \infty$ of the nonsymmetric Macdonald polynomial $E_{w \lambda}(q, t)$ are explicitly described in terms of QLS paths of shape $\lambda$ and the degree function defined on them. Also, for (level-zero) dominant integral weights $\lambda$, $\mu$, we have an isomorphism $\Theta : \mathrm{QLS}(\lambda + \mu) \rightarrow \mathrm{QLS}(\lambda) \otimes \mathrm{QLS}(\mu)$ of crystals. In this paper, we study the behavior of the degree function under the isomorphism $\Theta$ of crystals through the relationship between semi-infinite Lakshmibai-Seshadri (LS) paths and QLS paths. As an application, we give a crystal-theoretic proof of a recursion formula for the graded characters of generalized Weyl modules., Comment: arXiv admin note: text overlap with arXiv:1802.06339

In this paper, we give a representation-theoretic interpretation of the specialization E w ∘ λ ( q , ∞ ) E_{w_\circ \lambda } (q,\infty ) of the nonsymmetric Macdonald polynomial E w ∘ λ ( q , t ) E_{w_\circ \lambda }(q,t) at t = ∞ t=\infty in terms of the Demazure submodule V w ∘ − ( λ ) V_{w_\circ }^- (\lambda ) of the level-zero extremal weight module V ( λ ) V(\lambda ) over a quantum affine algebra of an arbitrary untwisted type. Here, λ \lambda is a dominant integral weight, and w ∘ w_\circ denotes the longest element in the finite Weyl group W W . Also, for each x ∈ W x \in W , we obtain a combinatorial formula for the specialization E x λ ( q , ∞ ) E_{x \lambda } (q, \infty ) at t = ∞ t=\infty of the nonsymmetric Macdonald polynomial E x λ ( q , t ) E_{x \lambda } (q,t) and also a combinatorical formula for the graded character gch ⁡ V x − ( λ ) \operatorname {gch} V_{x}^- (\lambda ) of the Demazure submodule V x − ( λ ) V_{x}^- (\lambda ) of V ( λ ) V(\lambda ) . Both of these formulas are described in terms of quantum Lakshmibai-Seshadri paths of shape λ \lambda .

Boron (B) is an essential element for plants; however, as high B concentrations are toxic, B transport must be tightly regulated. BOR1 is a borate exporter in Arabidopsis (

Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments.

Upstream open reading frames (uORFs) are often found in the 5′-leader regions of eukaryotic mRNAs and can negatively modulate the translational efficiency of the downstream main ORF. Although the effects of most uORFs are thought to be independent of their encoded peptide sequences, certain uORFs control translation of the main ORF in a peptide sequence-dependent manner. For genome-wide identification of such peptide sequence-dependent regulatory uORFs, exhaustive searches for uORFs with conserved amino acid sequences have been conducted using bioinformatic analyses. However, whether the conserved uORFs identified by these bioinformatic approaches encode regulatory peptides has not been experimentally determined. Here we analyzed 16 recently identified Arabidopsis thaliana conserved uORFs for the effects of their amino acid sequences on the expression of the main ORF using a transient expression assay. We identified five novel uORFs that repress main ORF expression in a peptide sequence-dependent manner. Mutational analysis revealed that, in four of them, the C-terminal region of the uORF-encoded peptide is critical for the repression of main ORF expression. Intriguingly, we also identified one exceptional sequence-dependent regulatory uORF, in which the stop codon position is not conserved and the C-terminal region is not important for the repression of main ORF expression.

Arabidopsis bZIP11 is a transcription factor that modulates amino acid metabolism under high-sucrose conditions. Expression of bZIP11 is downregulated in a sucrose-dependent manner during translation. Previous in vivo studies have identified the second upstream open reading frame (uORF2) as an essential regulatory element for the sucrose-dependent translational repression of bZIP11. However, it remains unclear how uORF2 represses bZIP11 expression under high-sucrose conditions. Through biochemical experiments using cell-free translation systems, we report on sucrose-mediated ribosome stalling at the stop codon of uORF2. The C-terminal 10 amino acids (29-SFSVxFLxxLYYV-41) of uORF2 are important for ribosome stalling. Our results demonstrate that uORF2 encodes a regulatory nascent peptide that functions to sense intracellular sucrose abundance. This is the first biochemical identification of the intracellular sucrose sensor.

Boron (B) cross-links the pectin polysaccharide rhamnogalacturonan II (RG-II) and thus is important for cell wall structure in plants. B deficiency is an agricultural problem that causes significant losses of crop productivity worldwide. To address this, B deficiency-tolerant plants have been generated using B transporters. With the goal of further improving plant tolerance to low-B conditions, we generated transgenic Arabidopsis thaliana (L.) Heynh. with enhanced expression of BOR2, a B transporter that promotes cross-linking of RG-II and root elongation under low B supply. We generated a DNA construct containing the cauliflower mosaic virus 35S RNA promoter, a native promoter of BOR2, a BOR2 gene and green fluorescent protein (GFP) (Pro35S-BOR2: BOR2-GFP), and obtained three independent transgenic lines with relatively high levels of BOR2-GFP expression. In the transgenic lines, BOR2-GFP was expressed mainly in the lateral root caps in the meristem zone and in the epidermal cells in the elongation zone, similar to its expression when driven only by its native promoter. In the Pro35S-BOR2: BOR2-GFP lines, BOR2-GFP was also expressed in various cells in the maturation zone of roots and epidermal cells of shoots, where the expression was hardly detectable in ProBOR2: BOR2-GFP lines. The transgenic lines were cultured under various B concentrations on solid media and it was found that root growth of three lines and shoot growth of two lines were enhanced compared to wild-type plants under low-B conditions. This finding established that enhanced expression of BOR2 leads to improved root growth under low-B conditions. We also examined growth of the transgenic lines under hydroponic conditions. One of the three lines showed better growth and fertility under a low-B condition, while the wild type did not set seeds under the same condition, suggesting the potential utility of BOR2 expression in agricultural applications.

Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.

Endomembrane organization is important for various aspects of cell physiology, including membrane protein trafficking. To explore the molecular mechanisms regulating the trafficking of plasma membrane-localized proteins in plants, we screened for Arabidopsis mutants with defective localization of green fluorescent protein (GFP)–nodulin 26-like intrinsic protein (NIP)5;1. Fluorescence imaging-based screening led to the isolation of a mutant which accumulated abnormal intracellular aggregates labeled by GFP–NIP5;1. The aggregates appeared in epidermal cells in the root elongation zone and included the trans -Golgi network/early endosomes. Rough mapping and whole-genome sequencing identified the mutant as an allele of UDP-glucose 4-epimerase 4 (uge4) /root hair defective 1 (rhd1) / root epidermal bulgar 1 (reb 1), which was originally defined as a cell wall mutant. The responsible gene encodes UDP-glucose 4-epimerase 4 (UGE4), which functions in the biosynthesis of d -galactose, especially for the synthesis of the cell wall polysaccharide xyloglucan and arabinogalactan proteins (AGPs). The endomembrane aggregates in the mutants were absent in the presence of d -galactose, indicative of a requirement for a d -galactose-containing component in endomembrane organization. Genetic and pharmacological analyses suggested that the aggregates were not caused by the disruption of cell wall polysaccharides or the cytoskeleton. Overall, our results suggest that UGE4 activity in d -galactose synthesis is required for the structure of cell wall polysaccharides and endomembrane organization.

Methionine (Met) is a sulfur-containing amino acid that is essential in mammals and whose low abundance limits the nutritional value of grain legumes. Cystathionine γ-synthase (CGS) catalyzes the first committed step of Met biosynthesis, and the stability of its mRNA is autoregulated by the cytosolic concentration of S-adenosyl-l-methionine (SAM), a direct metabolite of Met. The mto1-1 mutant of Arabidopsis thaliana harbors a mutation in the AtCGS1 gene that renders the mRNA resistant to SAM-dependent degradation and therefore results in the accumulation of free Met to high levels in young leaves. To manipulate Met biosynthesis in soybean and azuki bean, we introduced the AtCGS1 mto1-1 gene into the two grain legumes under the control of a seed-specific glycinin gene promoter. Transgenic seeds of both species accumulated soluble Met to levels at least twice those apparent in control seeds. However, the increase in free Met did not result in an increase in total Met content of the transgenic seeds. In transgenic azuki bean seeds, the amount of cystathionine, the direct product of CGS, was markedly increased whereas the total content of Met was significantly decreased compared with control seeds. Similar changes were not detected in soybean. Our data suggest that the regulation of Met biosynthesis differs between soybean and azuki bean, and that the expression of AtCGS1 mto1-1 differentially affects the metabolic stability of sulfur amino acids and their metabolites in the two grain legumes.

Cystathionine γ-synthase (CGS) catalyzes the first committed step of methionine (Met) biosynthesis in plants. Expression of the Arabidopsis thaliana CGS1 gene is negatively feedback-regulated in response to the direct Met metabolite S-adenosyl-L-methionine (AdoMet). This regulation occurs at the step of mRNA stability during translation and is coupled with AdoMet-induced CGS1-specific translation arrest. In general, mRNA decay is initiated by a shortening of the poly(A) tail. However, this process has not been studied in detail in cases where regulatory events, such as programmed translation arrest, are involved. Here, we report that the poly(A) tail of the full-length CGS1 mRNA showed an apparent increase from 50-80 nucleotides (nt) to 140-150 nt after the induction of CGS1 mRNA degradation. This finding was unexpected because mRNAs that are destined for degradation harbored longer poly(A) tail than mRNAs that were not targeted for degradation. The results suggest that poly(A) shortening is inhibited or delayed during AdoMet-induced translation arrest of CGS1 mRNA. We propose an explanation for this phenomenon that remains consistent with the recent model of actively translating mRNA. We also found that CGS1 mRNA degradation intermediates, which are 5'-truncated forms of CGS1 mRNA, had a short poly(A) tail of 10-30 nt. This suggests that poly(A) shortening occurs rapidly on the degradation intermediates. The present study highlights CGS1 mRNA degradation as a useful system to understand the dynamic features of poly(A) shortening.

Boron uptake in Arabidopsis thaliana is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that is located preferentially on the soil side of the plasma membrane in root cells. However, the mechanism underlying this polar localization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cells is mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region of NIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasma membrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1. Consistent with this, the polar localization was compromised in µ subunit mutants of the clathrin adaptor AP2. The Thr-to-Ala substitutions did not affect the boron transport activity of GFP-NIP5;1 in Xenopus laevis oocytes but did inhibit the ability to complement boron translocation to shoots and rescue growth defects in nip5;1-1 mutant plants under boron-limited conditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, is dependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots.

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat-binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

Upstream open reading frames (uORFs) are often translated ahead of the main ORF of a gene and regulate gene expression, sometimes in a condition-dependent manner, but such a role for the minimum uORF (hereafter referred to as AUG-stop) in living organisms is currently unclear. Here, we show that AUG-stop plays an important role in the boron (B)-dependent regulation of NIP5;1, encoding a boric acid channel required for normal growth under low B conditions in Arabidopsis thaliana High B enhanced ribosome stalling at AUG-stop, which was accompanied by the suppression of translation and mRNA degradation. This mRNA degradation was promoted by an upstream conserved sequence present near the 5'-edge of the stalled ribosome. Once ribosomes translate a uORF, reinitiation of translation must take place in order for the downstream ORF to be translated. Our results suggest that reinitiation of translation at the downstream NIP5;1 ORF is enhanced under low B conditions. A genome-wide analysis identified two additional B-responsive genes, SKU5 and the transcription factor gene ABS/NGAL1, which were regulated by B-dependent ribosome stalling through AUG-stop. This regulation was reproduced in both plant and animal transient expression and cell-free translation systems. These findings suggest that B-dependent AUG-stop-mediated regulation is common in eukaryotes.

RNA viruses use various strategies to condense their genetic information into small genomes. Potyviruses not only use the polyprotein strategy, but also embed an open reading frame, pipo, in the P3 cistron in the –1 reading frame. PIPO is expressed as a fusion protein with the N-terminal half of P3 (P3N-PIPO) via transcriptional slippage of viral RNA-dependent RNA polymerase (RdRp). We herein show that clover yellow vein virus (ClYVV) produces a previously unidentified factor, P3N-ALT, in the +1 reading frame via transcriptional slippage at a conserved G1–2A6–7 motif, as is the case for P3N-PIPO. The translation of P3N-ALT terminates soon, and it is considered to be a C-terminal truncated form of P3. In planta experiments indicate that P3N-ALT functions in cell-to-cell movement along with P3N-PIPO. Hence, all three reading frames are used to produce functional proteins. Deep sequencing of ClYVV RNA from infected plants endorses the slippage by viral RdRp. Our findings unveil a virus strategy that optimizes the coding capacity.

A Newton-Okounkov convex body is a convex body constructed from a projective variety with a valuation on its homogeneous coordinate ring; this is deeply connected with representation theory. For instance, the Littelmann string polytopes and the Feigin-Fourier-Littelmann-Vinberg polytopes are examples of Newton-Okounkov convex bodies. In this paper, we prove that the Newton-Okounkov convex body of a Schubert variety with respect to a specific valuation is identical to the Nakashima-Zelevinsky polyhedral realization of a Demazure crystal. As an application of this result, we show that Kashiwara's involution (*-operation) corresponds to a change of valuations on the rational function field., Comment: 21 pages

The purpose of this paper is to prove that the Mirkovic–Vilonen (MV) polytope corresponding to the tensor product of two arbitrary MV polytopes is contained, as a set, in the Minkowski sum of these two MV polytopes. This result generalizes the one in our previous paper, which was obtained under the assumption that the first tensor factor is an extremal MV polytope.

Boron (B) is essential for plant growth but is toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, are required for efficient B uptake and subsequent translocation into the xylem, respectively. However, under high-B conditions, BOR1 activity is repressed through endocytic degradation, presumably to avoid B toxicity. In this study, we investigated the localization of GFP-tagged NIP5;1 and BOR1 expressed under the control of their native promoters. Under B limitation, GFP-NIP5;1 and BOR1-GFP localized preferentially in outer (distal) and inner (proximal) plasma membrane domains, respectively, of various root cells. The polar localization of the boric acid channel and boric acid/borate exporter indicates the radial transport route of B toward the stele. Furthermore, mutational analysis revealed a requirement of tyrosine residues, in a probable cytoplasmic loop region of BOR1, for polar localization in various cells of the meristem and elongation zone. The same tyrosine residues were also required for vacuolar targeting upon high B supply. The present study of BOR1 and NIP5;1 demonstrates the importance of selective endocytic trafficking in polar localization and degradation of plant nutrient transporters for radial transport and homeostasis of plant mineral nutrients.

特定の品種の中の遺伝変異やその発生過程については，殆ど知見がない．本研究では全国15府県から集められたコシヒカリ86集団を材料に，遺伝的な変異についてトランスポゾンの挿入位置を指標に調査した．3種のトランスポゾンの挿入位置を調べると，4集団の例外をのぞいて，コシヒカリ集団間での多型はきわめて少なく，品種の遺伝的同一性が保持されていることが示された．しかし，トランスポゾン mPingを指標にした場合，多くの多型が現れた．多数を占めたのは，コシヒカリの親である農林1号と農林22号にはない固有バンドからなる多型で， mPingがコシヒカリで転移していることを示すものである．その他の多型には，新潟以外の集団にのみ存在し，新潟由来の集団にはない2つのバンドが検出され，コシヒカリを新潟タイプと非新潟タイプ（福井タイプ）で明瞭に分けた．2つの多型バンドは両親に1つずつ存在するので，コシヒカリの育成過程で発生した遺伝的分離に基づくと考えられる．新潟では福井県農業試験場から分譲された育成過程のコシヒカリを独自で選抜したとされ，この期間に新潟タイプと福井タイプのコシヒカリの間に遺伝的な差異が発生したと思われる．