Light Controls Protein Localization through Phytochrome-Mediated Alternative Promoter Selection.

Alternative promoter usage is a proteome-expanding mechanism that allows multiple pre-mRNAs to be transcribed from a single gene. The impact of this mechanism on the proteome and whether it is positively exploited in normal organismal responses remain unclear. We found that the plant photoreceptor phytochrome induces genome-wide changes in alternative promoter selection in Arabidopsis thaliana. Through this mechanism, protein isoforms with different N termini are produced that display light-dependent differences in localization. For instance, shade-grown plants accumulate a cytoplasmic isoform of glycerate kinase (GLYK), an essential photorespiration enzyme that was previously thought to localize exclusively to the chloroplast. Cytoplasmic GLYK constitutes a photorespiratory bypass that alleviates fluctuating light-induced photoinhibition. Therefore, phytochrome controls alternative promoter selection to modulate protein localization in response to changing light conditions. This study suggests that alternative promoter usage represents another ubiquitous layer of gene expression regulation in eukaryotes that contributes to diversification of the proteome.

Alternative localization of HEME OXYGENASE 1 in plant cells regulates cytosolic heme catabolism.

Heme, an organometallic tetrapyrrole, is widely engaged in oxygen transport, electron delivery, enzymatic reactions, and signal transduction. In plants, it is also involved in photomorphogenesis and photosynthesis. HEME OXYGENASE 1 (HO1) initiates the first committed step in heme catabolism, and it has generally been thought that this reaction takes place in chloroplasts. Here, we show that HO1 in both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) has 2 transcription start sites (TSSs), producing long (HO1L) and short (HO1S) transcripts. Their products localize to the chloroplast and the cytosol, respectively. During early development or de-etiolation, the HO1L/HO1S ratio gradually increases. Light perception via phytochromes (Phys) and cryptochromes elevates the HO1L/HO1S ratio in the whole seedling through the functions of ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG and through the suppression of DE-ETIOLATED 1, CONSTITUTIVE PHOTOMORPHOGENESIS 1, and PHYTOCHROME INTERACTING FACTORs. HO1L introduction complements the HO1-deficient mutant; surprisingly, HO1S expression also restores the short hypocotyl phenotype and high pigment content and helps the mutant recover from the genomes uncoupled (gun) phenotype. This indicates the assembly of functional Phys within these lines. Furthermore, our findings support the hypothesis that a mobile heme signal is involved in retrograde signaling from the chloroplast. Altogether, our work clarifies the molecular mechanism of HO1 TSS regulation and highlights the presence of a cytosolic bypass for heme catabolism in plant cells.

Positive selective sweeps of epigenetic mutations regulating specialized metabolites in plants.

DNA methylation is an important factor regulating gene expression in organisms. However, whether DNA methylation plays a key role in adaptive evolution is unknown. Here, we show evidence of naturally selected DNA methylation in Arabidopsis thaliana In comparison with single nucleotide polymorphisms, three types of methylation-methylated CGs (mCGs), mCHGs, and mCHHs-contributed highly to variable gene expression levels among an A thaliana population. Such variably expressed genes largely affect a large variation of specialized metabolic quantities. Among the three types of methylations, only mCGs located in promoter regions of genes associated with specialized metabolites show a selective sweep signature in the A thaliana population. Thus, naturally selected mCGs appear to be key mutations that cause the expressional diversity associated with specialized metabolites during plant evolution.

A de novo gene originating from the mitochondria controls floral transition in Arabidopsis thaliana.

De novo genes created in the plant mitochondrial genome have frequently been transferred into the nuclear genome via intergenomic gene transfer events. Therefore, plant mitochondria might be a source of de novo genes in the nuclear genome. However, the functions of de novo genes originating from mitochondria and the evolutionary fate remain unclear. Here, we revealed that an Arabidopsis thaliana specific small coding gene derived from the mitochondrial genome regulates floral transition. We previously identified 49 candidate de novo genes that induce abnormal morphological changes on overexpression. We focused on a candidate gene derived from the mitochondrial genome (sORF2146) that encodes 66 amino acids. Comparative genomic analyses indicated that the mitochondrial sORF2146 emerged in the Brassica lineage as a de novo gene. The nuclear sORF2146 emerged following an intergenomic gene transfer event in the A. thaliana after the divergence between Arabidopsis and Capsella. Although the nuclear and mitochondrial sORF2146 sequences are the same in A. thaliana, only the nuclear sORF2146 is transcribed. The nuclear sORF2146 product is localized in mitochondria, which may be associated with the pseudogenization of the mitochondrial sORF2146. To functionally characterize the nuclear sORF2146, we performed a transcriptomic analysis of transgenic plants overexpressing the nuclear sORF2146. Flowering transition-related genes were highly regulated in the transgenic plants. Subsequent phenotypic analyses demonstrated that the overexpression and knockdown of sORF2146 in transgenic plants resulted in delayed and early flowering, respectively. These findings suggest that a lineage-specific de novo gene derived from mitochondria has an important regulatory effect on floral transition.

Enhanced growth rate under elevated CO2 conditions was observed for transgenic lines of genes identified by intraspecific variation analyses in Arabidopsis thaliana.

KEY MESSAGE: Using the whole genome and growth data of Arabidopsis thaliana ecotypes, we identified two genes associated with enhancement of the growth rate in response to elevated CO2 conditions. Improving plant growth under elevated CO2 conditions may contribute to enhanced agricultural yield under future global climate change. In this study, we examined the genes implicated in the enhancement of growth rates under elevated CO2 conditions by analyzing the growth rates of Arabidopsis thaliana ecotypes originating from various latitudes and altitudes throughout the world. We also performed a genome-wide association study and a transcriptome study to identify single nucleic polymorphisms that were correlated with the relative growth rate (RGR) under elevated CO2 conditions or with CO2 response of RGR. We then selected 43 candidate genes and generated their overexpression and/or RNA interference (RNAi) transgenic mutants for screening. After screening, we have found that RNAi lines of AT3G4000 and AT5G50900 showed significantly higher growth rates under the elevated CO2 condition. As per our findings, we conclude that natural variation includes genetic variation associated with the enhancement of plant productivity under elevated CO2 conditions.

Degree of Functional Divergence in Duplicates Is Associated with Distinct Roles in Plant Evolution.

Gene duplication is a major mechanism to create new genes. After gene duplication, some duplicated genes undergo functionalization, whereas others largely maintain redundant functions. Duplicated genes comprise various degrees of functional diversification in plants. However, the evolutionary fate of high and low diversified duplicates is unclear at genomic scale. To infer high and low diversified duplicates in Arabidopsis thaliana genome, we generated a prediction method for predicting whether a pair of duplicate genes was subjected to high or low diversification based on the phenotypes of knock-out mutants. Among 4,017 pairs of recently duplicated A. thaliana genes, 1,052 and 600 are high and low diversified duplicate pairs, respectively. The predictions were validated based on the phenotypes of generated knock-down transgenic plants. We determined that the high diversified duplicates resulting from tandem duplications tend to have lineage-specific functions, whereas the low diversified duplicates produced by whole-genome duplications are related to essential signaling pathways. To assess the evolutionary impact of high and low diversified duplicates in closely related species, we compared the retention rates and selection pressures on the orthologs of A. thaliana duplicates in two closely related species. Interestingly, high diversified duplicates resulting from tandem duplications tend to be retained in multiple lineages under positive selection. Low diversified duplicates by whole-genome duplications tend to be retained in multiple lineages under purifying selection. Taken together, the functional diversities determined by different duplication mechanisms had distinct effects on plant evolution.

AtPep3 is a hormone-like peptide that plays a role in the salinity stress tolerance of plants.

Peptides encoded by small coding genes play an important role in plant development, acting in a similar manner as phytohormones. Few hormone-like peptides, however, have been shown to play a role in abiotic stress tolerance. In the current study, 17 Arabidopsis genes coding for small peptides were found to be up-regulated in response to salinity stress. To identify peptides leading salinity stress tolerance, we generated transgenic Arabidopsis plants overexpressing these small coding genes and assessed survivability and root growth under salinity stress conditions. Results indicated that 4 of the 17 overexpressed genes increased salinity stress tolerance. Further studies focused on AtPROPEP3, which was the most highly up-regulated gene under salinity stress. Treatment of plants with synthetic peptides encoded by AtPROPEP3 revealed that a C-terminal peptide fragment (AtPep3) inhibited the salt-induced bleaching of chlorophyll in seedlings. Conversely, knockdown AtPROPEP3 transgenic plants exhibited a hypersensitive phenotype under salinity stress, which was complemented by the AtPep3 peptide. This functional AtPep3 peptide region overlaps with an AtPep3 elicitor peptide that is related to the immune response of plants. Functional analyses with a receptor mutant of AtPep3 revealed that AtPep3 was recognized by the PEPR1 receptor and that it functions to increase salinity stress tolerance in plants. Collectively, these data indicate that AtPep3 plays a significant role in both salinity stress tolerance and immune response in Arabidopsis.

Effect of small coding genes on the circadian rhythms under elevated CO2 conditions in plants.

Increase in atmospheric carbon dioxide (CO2) has a significant effect on plant growth and development. To explore the elevated-CO2 response, we generated transcriptional profiles over a time course (2 h-14 days) of exposure to elevated CO2 in Arabidopsis thaliana. Genes related to photosynthesis were down-regulated and circadian rhythm-related genes were abnormally regulated in the early to middle phase of elevated CO2 exposure. To understand the novel mechanism of elevated CO2 signaling, we focused on 42 unknown small coding genes that showed differential expression patterns under elevated CO2 conditions. Four transgenic plants overexpressing the small coding gene exhibited a growth-defective phenotype under elevated CO2 but not under current CO2. Transcriptome analysis showed that circadian rhythm-related genes were commonly regulated in four transgenic plants. These circadian rhythm-related genes were transcribed in the dark when CO2 concentrations in the leaf was high. Taken together, our identified four small coding genes are likely to participate in elevated CO2 signaling to the circadian rhythm.

Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening.

Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions.

Tyr82 Amino Acid Mutation in PB1 Polymerase Induces an Influenza Virus Mutator Phenotype.

In various positive-sense single-stranded RNA viruses, a low-fidelity viral RNA-dependent RNA polymerase (RdRp) confers attenuated phenotypes by increasing the mutation frequency. We report a negative-sense single-stranded RNA virus RdRp mutant strain with a mutator phenotype. Based on structural data of RdRp, rational targeting of key residues, and screening of fidelity variants, we isolated a novel low-fidelity mutator strain of influenza virus that harbors a Tyr82-to-Cys (Y82C) single-amino-acid substitution in the PB1 polymerase subunit. The purified PB1-Y82C polymerase indeed showed an increased frequency of misincorporation compared with the wild-type PB1 in an in vitro biochemical assay. To further investigate the effects of position 82 on PB1 polymerase fidelity, we substituted various amino acids at this position. As a result, we isolated various novel mutators other than PB1-Y82C with higher mutation frequencies. The structural model of influenza virus polymerase complex suggested that the Tyr82 residue, which is located at the nucleoside triphosphate entrance tunnel, may influence a fidelity checkpoint. Interestingly, although the PB1-Y82C variant replicated with wild-type PB1-like kinetics in tissue culture, the 50% lethal dose of the PB1-Y82C mutant was 10 times lower than that of wild-type PB1 in embryonated chicken eggs. In conclusion, our data indicate that the Tyr82 residue of PB1 has a crucial role in regulating polymerase fidelity of influenza virus and is closely related to attenuated pathogenic phenotypes in vivoIMPORTANCE Influenza A virus rapidly acquires antigenic changes and antiviral drug resistance, which limit the effectiveness of vaccines and drug treatments, primarily owing to its high rate of evolution. Virus populations formed by quasispecies can contain resistance mutations even before a selective pressure is applied. To study the effects of the viral mutation spectrum and quasispecies, high- and low-fidelity variants have been isolated for several RNA viruses. Here, we report the discovery of a low-fidelity RdRp variant of influenza A virus that contains a substitution at Tyr82 in PB1. Viruses containing the PB1-Y82C substitution showed growth kinetics and viral RNA synthesis levels similar to those of the wild-type virus in cell culture; however, they had significantly attenuated phenotypes in a chicken egg infection experiment. These data demonstrated that decreased RdRp fidelity attenuates influenza A virus in vivo, which is a desirable feature for the development of safer live attenuated vaccine candidates.

Dephosphorylation of clustered phosphoserine residues in human Grb14 by protein phosphatase 1 and its effect on insulin receptor complex formation.

The physical interaction of the human growth factor receptor-bound protein 14 (hGrb14) and the insulin receptor (IR) represses insulin signaling. With respect to the recruiting mechanism of hGrb14 to IR respond to insulin stimulus, our previous reports have suggested that phosphorylation of Ser358 , Ser362 , and Ser366 in hGrb14 by glycogen synthase kinase-3 repressed hGrb14-IR complex formation. In this study, we investigated phosphatase-mediated dephosphorylation of the hGrb14 phosphoserine residues. An in vitro phosphatase assay with hGrb14-derived synthetic phosphopeptides suggested that protein phosphatase 1 (PP1) is involved in the dephosphorylation of Ser358 and Ser362 . Furthermore, coimmunoprecipitation experiments suggested that insulin-induced hGrb14-IR complex formation was repressed by the substitution of Ser358 or Ser362 with glutamic acid. These findings suggested that phosphate groups on Ser358 and Ser362 in hGrb14 are dephosphorylated by PP1, and the dephosphorylation facilitates hGrb14-IR complex formation.

A Highly Specific Genome-Wide Association Study Integrated with Transcriptome Data Reveals the Contribution of Copy Number Variations to Specialized Metabolites in Arabidopsis thaliana Accessions.

Lineage-specific gene duplications contribute to a large variation in specialized metabolites among different plant species. There is also considerable variability in the specialized metabolites within a single plant species. However, it is unclear whether copy number variations (CNVs) derived from gene duplication events contribute to the diversity of specialized metabolites within species. We identified metabolome quantitative trait genes (mQTGs) associated with quantitative metabolite variations and examined the relationship between mQTGs and CNVs. We obtained 1,335 specialized metabolite signals from 53 worldwide A. thaliana accessions using liquid chromatography-quadrupole time-of-flight mass spectrometry. In this study, genes associated with specialized metabolites were inferred by either a generally authorized genome-wide association study (GWAS) approach or a novel analysis of the association between gene expression and metabolite accumulation. Genes qualified by both analyses are defined to be mQTGs. The integrated method enabled us to detect mQTGs with a low false positive rate (=5.71 × 10-4). We also identified 5,654 genes associated with 1,335 specialized metabolites. Of these genes, 4.4% were affected by CNVs, which was more than expected (χ2 test: P < 0.01). This result suggests that CNVs contribute to variations in specialized metabolites within a species. To assess the contribution of CNVs to adaptive evolution in A. thaliana, we examined the selective sweeps around the mQTGs. We observed that the mQTGs with CNVs tended to undergo selective sweeps. These observations imply that variations in specialized metabolites caused by CNVs contribute to the adaptive evolution of A. thaliana.

Distinct gene expression signatures induced by viral transactivators of different HTLV-1 subgroups that confer a different risk of HAM/TSP.

BACKGROUND: Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, there is an association between HTLV-1 tax subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. To investigate the role of HTLV-1 subgroups in viral pathogenesis, we studied the functional difference in the subgroup-specific viral transcriptional regulators Tax and HBZ using microarray analysis, reporter gene assays, and evaluation of viral-host protein-protein interaction. RESULTS: (1) Transcriptional changes in Jurkat Tet-On human T-cells that express each subgroup of Tax or HBZ protein under the control of an inducible promoter revealed different target gene profiles; (2) the number of differentially regulated genes induced by HBZ was 2-3 times higher than that induced by Tax; (3) Tax and HBZ induced the expression of different classes of non-coding RNAs (ncRNAs); (4) the chemokine CXCL10, which has been proposed as a prognostic biomarker for HAM/TSP, was more efficiently induced by subgroup-A Tax (Tax-A) than subgroup-B Tax (Tax-B), in vitro as well as in unmanipulated (ex vivo) PBMCs obtained from HAM/TSP patients; (5) reporter gene assays indicated that although transient Tax expression in an HTLV-1-negative human T-cell line activated the CXCL10 gene promoter through the NF-κB pathway, there was no difference in the ability of each subgroup of Tax to activate the CXCL10 promoter; however, (6) chromatin immunoprecipitation assays showed that the ternary complex containing Tax-A is more efficiently recruited onto the promoter region of CXCL10, which contains two NF-κB binding sites, than that containing Tax-B. CONCLUSIONS: Our results indicate that different HTLV-1 subgroups are characterized by different patterns of host gene expression. Differential expression of pathogenesis-related genes by subgroup-specific Tax or HBZ may be associated with the onset of HAM/TSP.

Efficient anchoring of alien chromosome segments introgressed into bread wheat by new Leymus racemosus genome-based markers.

BACKGROUND: The tertiary gene pool of bread wheat, to which Leymus racemosus belongs, has remained underutilized due to the current limited genomic resources of the species that constitute it. Continuous enrichment of public databases with useful information regarding these species is, therefore, needed to provide insights on their genome structures and aid successful utilization of their genes to develop improved wheat cultivars for effective management of environmental stresses. RESULTS: We generated de novo DNA and mRNA sequence information of L. racemosus and developed 110 polymorphic PCR-based markers from the data, and to complement the PCR markers, DArT-seq genotyping was applied to develop additional 9990 SNP markers. Approximately 52% of all the markers enabled us to clearly genotype 22 wheat-L. racemosus chromosome introgression lines, and L. racemosus chromosome-specific markers were highly efficient in detailed characterization of the translocation and recombination lines analyzed. A further analysis revealed remarkable transferability of the PCR markers to three other important Triticeae perennial species: L. mollis, Psathyrostachys huashanica and Elymus ciliaris, indicating their suitability for characterizing wheat-alien chromosome introgressions carrying chromosomes of these genomes. CONCLUSION: The efficiency of the markers in characterizing wheat-L. racemosus chromosome introgression lines proves their reliability, and their high transferability further broadens their scope of application. This is the first report on sequencing and development of markers from L. racemosus genome and the application of DArT-seq to develop markers from a perennial wild relative of wheat, marking a paradigm shift from the seeming concentration of the technology on cultivated species. Integration of these markers with appropriate cytogenetic methods would accelerate development and characterization of wheat-alien chromosome introgression lines.

A Genome Scan for Genes Underlying Microgeographic-Scale Local Adaptation in a Wild Arabidopsis Species.

Adaptive divergence at the microgeographic scale has been generally disregarded because high gene flow is expected to disrupt local adaptation. Yet, growing number of studies reporting adaptive divergence at a small spatial scale highlight the importance of this process in evolutionary biology. To investigate the genetic basis of microgeographic local adaptation, we conducted a genome-wide scan among sets of continuously distributed populations of Arabidopsis halleri subsp. gemmifera that show altitudinal phenotypic divergence despite gene flow. Genomic comparisons were independently conducted in two distinct mountains where similar highland ecotypes are observed, presumably as a result of convergent evolution. Here, we established a de novo reference genome and employed an individual-based resequencing for a total of 56 individuals. Among 527,225 reliable SNP loci, we focused on those showing a unidirectional allele frequency shift across altitudes. Statistical tests on the screened genes showed that our microgeographic population genomic approach successfully retrieve genes with functional annotations that are in line with the known phenotypic and environmental differences between altitudes. Furthermore, comparison between the two distinct mountains enabled us to screen out those genes that are neutral or adaptive only in either mountain, and identify the genes involved in the convergent evolution. Our study demonstrates that the genomic comparison among a set of genetically connected populations, instead of the commonly-performed comparison between two isolated populations, can also offer an effective screening for the genetic basis of local adaptation.

Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis.

Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer's disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase.

Phytochrome controls alternative splicing to mediate light responses in Arabidopsis.

Plants monitor the ambient light conditions using several informational photoreceptors, including red/far-red light absorbing phytochrome. Phytochrome is widely believed to regulate the transcription of light-responsive genes by modulating the activity of several transcription factors. Here we provide evidence that phytochrome significantly changes alternative splicing (AS) profiles at the genomic level in Arabidopsis, to approximately the same degree as it affects steady-state transcript levels. mRNA sequencing analysis revealed that 1,505 and 1,678 genes underwent changes in their AS and steady-state transcript level profiles, respectively, within 1 h of red light exposure in a phytochrome-dependent manner. Furthermore, we show that splicing factor genes were the main early targets of AS control by phytochrome, whereas transcription factor genes were the primary direct targets of phytochrome-mediated transcriptional regulation. We experimentally validated phytochrome-induced changes in the AS of genes that are involved in RNA splicing, phytochrome signaling, the circadian clock, and photosynthesis. Moreover, we show that phytochrome-induced AS changes of SPA1-RELATED 3, the negative regulator of light signaling, physiologically contributed to promoting photomorphogenesis. Finally, photophysiological experiments demonstrated that phytochrome transduces the signal from its photosensory domain to induce light-dependent AS alterations in the nucleus. Taking these data together, we show that phytochrome directly induces AS cascades in parallel with transcriptional cascades to mediate light responses in Arabidopsis.

Mutant selection in the self-incompatible plant radish (Raphanus sativus L. var. sativus) using two-step TILLING.

Radish (Raphanus sativus L. var. sativus), a widely cultivated root vegetable crop, possesses a large sink organ (the root), implying that photosynthetic activity in radish can be enhanced by altering both the source and sink capacity of the plant. However, since radish is a self-incompatible plant, improved mutation-breeding strategies are needed for this crop. TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful method used for reverse genetics. In this study, we developed a new TILLING strategy involving a two-step mutant selection process for mutagenized radish plants: the first selection is performed to identify a BC1M1 line, that is, progenies of M1 plants crossed with wild-type, and the second step is performed to identify BC1M1 individuals with mutations. We focused on Rubisco as a target, since Rubisco is the most abundant plant protein and a key photosynthetic enzyme. We found that the radish genome contains six RBCS genes and one pseudogene encoding small Rubisco subunits. We screened 955 EMS-induced BC1M1 lines using our newly developed TILLING strategy and obtained six mutant lines for the six RsRBCS genes, encoding proteins with four different types of amino acid substitutions. Finally, we selected a homozygous mutant and subjected it to physiological measurements.

Which plant trait explains the variations in relative growth rate and its response to elevated carbon dioxide concentration among Arabidopsis thaliana ecotypes derived from a variety of habitats?

Elevated atmospheric carbon dioxide (CO2) concentration ([CO2]) enhances plant growth, but this enhancement varies considerably. It is still uncertain which plant traits are quantitatively related to the variation in plant growth. To identify the traits responsible, we developed a growth analysis model that included primary parameters associated with morphology, nitrogen (N) use, and leaf and root activities. We analysed the vegetative growth of 44 ecotypes of Arabidopsis thaliana L. grown at ambient and elevated [CO2] (800 µmol mol(-1)). The 44 ecotypes were selected such that they were derived from various altitudes and latitudes. Relative growth rate (RGR; growth rate per unit plant mass) and its response to [CO2] varied by 1.5- and 1.7-fold among ecotypes, respectively. The variation in RGR at both [CO2]s was mainly explained by the variation in leaf N productivity (LNP; growth rate per leaf N),which was strongly related to photosynthetic N use efficiency (PNUE). The variation in the response of RGR to [CO2] was also explained by the variation in the response of LNP to [CO2]. Genomic analyses indicated that there was no phylogenetic constraint on inter-ecotype variation in the CO2 response of RGR or LNP. We conclude that the significant variation in plant growth and its response to [CO2] among ecotypes reflects the variation in N use for photosynthesis among ecotypes, and that the response of PNUE to CO2 is an important target for predicting and/or breeding plants that have high growth rates at elevated [CO2].

Small open reading frames associated with morphogenesis are hidden in plant genomes.

It is likely that many small ORFs (sORFs; 30-100 amino acids) are missed when genomes are annotated. To overcome this limitation, we identified ∼8,000 sORFs with high coding potential in intergenic regions of the Arabidopsis thaliana genome. However, the question remains as to whether these coding sORFs play functional roles. Using a designed array, we generated an expression atlas for 16 organs and 17 environmental conditions among 7,901 identified coding sORFs. A total of 2,099 coding sORFs were highly expressed under at least one experimental condition, and 571 were significantly conserved in other land plants. A total of 473 coding sORFs were overexpressed; ∼10% (49/473) induced visible phenotypic effects, a proportion that is approximately seven times higher than that of randomly chosen known genes. These results indicate that many coding sORFs hidden in plant genomes are associated with morphogenesis. We believe that the expression atlas will contribute to further study of the roles of sORFs in plants.