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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.

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. Transcriptome analysis was performed under elevated CO2 condition in Arabidopsis thaliana. We identified small coding genes associated with signal molecules from elevated CO2 to circadian rhythm genes.

Increase in atmospheric carbon dioxide (CO

Plant leaves, harvesting light energy and fixing CO2, are a major source of foods on the earth. Leaves undergo developmental and physiological shifts during their lifespan, ending with senescence and death. We characterized the key regulatory features of the leaf transcriptome during aging by analyzing total- and small-RNA transcriptomes throughout the lifespan of Arabidopsis (Arabidopsis thaliana) leaves at multidimensions, including age, RNA-type, and organelle. Intriguingly, senescing leaves showed more coordinated temporal changes in transcriptomes than growing leaves, with sophisticated regulatory networks comprising transcription factors and diverse small regulatory RNAs. The chloroplast transcriptome, but not the mitochondrial transcriptome, showed major changes during leaf aging, with a strongly shared expression pattern of nuclear transcripts encoding chloroplast-targeted proteins. Thus, unlike animal aging, leaf senescence proceeds with tight temporal and distinct interorganellar coordination of various transcriptomes that would be critical for the highly regulated degeneration and nutrient recycling contributing to plant fitness and productivity.

Lettuce is a commercially important leafy vegetable that is cultivated worldwide, and it is also a target crop for plant factories. In this study, lettuce was selected as an alternative platform for recombinant miraculin production because of its fast growth, agronomic value, and wide availability. The taste-modifying protein miraculin is a glycoprotein extracted from the red berries of the West African native shrub Richadella dulcifica. Because of its limited natural availability, many attempts have been made to produce this protein in suitable alternative hosts. We produced transgenic lettuce with miraculin gene driven either by the ubiquitin promoter/terminator cassette from lettuce or a 35S promoter/nos terminator cassette. Miraculin gene expression and miraculin accumulation in both cassettes were compared by quantitative real-time PCR analysis, Western blotting, and enzyme-linked immunosorbent assay. The expression level of the miraculin gene and protein in transgenic lettuce was higher and more genetically stable in the ubiquitin promoter/terminator cassette than in the 35S promoter/nos terminator cassette. These results demonstrated that the ubiquitin promoter/terminator cassette is an efficient platform for the genetically stable expression of the miraculin protein in lettuce and hence this platform is of benefit for recombinant miraculin production on a commercial scale.

The E8 promoter, a tomato fruit-ripening-specific promoter, and the CaMV 35S promoter, a constitutive promoter, were used to express the miraculin gene encoding the taste-modifying protein in tomato. The accumulation of miraculin protein and mRNA was compared among transgenic tomatoes expressing the miraculin gene driven by these promoters. Recombinant miraculin protein predominantly accumulated in transgenic tomato lines using the E8 promoter (E8-MIR) only at the red fruit stage. The accumulations were almost uniform among all fruit tissues. When the 35S promoter (35S-MIR) was used, miraculin accumulation in the exocarp was much higher than in other tissues, indicating that the miraculin accumulation pattern can be regulated by using different types of promoters. We also discuss the potential of the E8-MIR lines for practical use.

Caffeine and catechins from the Yabukita, Yutakamidori, Saemidori, Okumidori, and Fushun varieties of tea leaves picked during different harvesting seasons from April to October were evaluated using HPLC. Total content of catechins increased greatly with the later harvesting time of tea leaves (i.e., picking the leaves in September versus in April) and decreased slightly after September. Yabukita tea leaves picked in August contained 43.1 mg% catechins including EGC, EC, ECG, and EGCG, with the ECGC levels constituting greater than 50% of those four compounds. Yutakamidori and Okumidori varieties picked in September contained the highest catechin values, at 43.6 mg% and 31.0 mg%, respectively. Fushun and Saemidori varieties contained lower catechin concentrations of 14.5 mg% (July) and 11.7 mg% (August) compared to other varieties. The EGCG levels gradually decreased in the late harvesting season, while levels of the other catechins, EC, EGC, and ECG, gradually increased. All varieties of green tea showed a gradual decrease in caffeine content toward the end of our harvesting efforts in October, with levels of 58～68 mg% in April and 28～57 mg% in October. Yabukita, Saemidori, and Okumidori varieties reached their highest caffeine levels in late spring/early summer, with Yabukita and Okumidori varieties reaching a high of 73.4% and 63.5% caffeine, respectively, in May, and Saemidori at 64.0% in June. In particular, Fushun still contained high caffeine of 66.8 mg% (September) during the late harvesting season.

Gene dosage and genetic background are factors that influence transgene expression in transgenic plants. In our previous studies, we produced transgenic tomato plants that accumulate miraculin, a taste-modifying protein, in a genetically stable manner. To elucidate the effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits, we generated hybrid tomato lines between the homozygous transgenic line 56B (background cultivar 'Moneymaker') and the pure cultivars 'Micro-Tom,' 'Moneymaker,' 'Ailsa Craig,' 'M82,' 'Rutgers' and 'Aichi-first' and analyzed them for miraculin mRNA expression and miraculin protein accumulation. The hybrid lines exhibited variation in their fruit structures. Among the hybrid lines heterozygous for the miraculin gene, miraculin accumulation in the fruits varied from 111.0 m gg �1 fresh weight (FW) to 159.4 m gg �1 FW. Furthermore, the homozygous line 56B showed higher miraculin accumulation and miraculin mRNA expression than the heterozygous line 56B�'Moneymaker.' These results demonstrate the profound effects of gene dosage and genetic background on miraculin accumulation in transgenic tomato fruits.

We previously developed a transgenic tomato that expresses the miraculin gene using a constitutive promoter. In this study, we profiled the developmental and spatial accumulation of the miraculin protein and mRNA in transgenic tomato fruits. Miraculin mRNA expression was almost constant up to orange stage, and then the expression increased at red stage. The miraculin protein accumulated gradually during fruit development and reached its highest level at the overripe stage. At the red stage of fruit, miraculin protein was accumulated at the highest level in the exocarp, and similar in other fruit tissues: mesocarp, dissepiment, upper placenta, lower placenta and jelly. Moreover, the pattern of miraculin accumulation in fruit tissues was the same regardless of genetic background and position at which the miraculin gene was inserted in the genome. We also discuss suitable tomato types expressing miraculin for their commercial use.

Plant leaves, harvesting light energy and fixing CO2, are a major source of foods on the earth. Leaves undergo developmental and physiological shifts during their lifespan, ending with senescence and death. We characterized the key regulatory features of the leaf transcriptome during aging by analyzing total- and small-RNA transcriptomes throughout the lifespan of Arabidopsis (Arabidopsis thaliana) leaves at multidimensions, including age, RNA-type, and organelle. Intriguingly, senescing leaves showed more coordinated temporal changes in transcriptomes than growing leaves, with sophisticated regulatory networks comprising transcription factors and diverse small regulatory RNAs. The chloroplast transcriptome, but not the mitochondrial transcriptome, showed major changes during leaf aging, with a strongly shared expression pattern of nuclear transcripts encoding chloroplast-targeted proteins. Thus, unlike animal aging, leaf senescence proceeds with tight temporal and distinct interorganellar coordination of various transcriptomes that would be critical for the highly regulated degeneration and nutrient recycling contributing to plant fitness and productivity.

Summary The EIN2-mediated senescence signalling pathway coordinates the expression of genes during leaf senescence via the gene regulatory network involving EIN3 and senescence-associated NAC TFs., Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs.