Nitrogen (N) fertilization is one of the most crucial factors that contribute to increasing food production requiring the generation of rice cultivars with improved N use efficiency (NUE) to maintain yield during low N fertilizer application. To assay NUE extent, we developed a screening system to evaluate shoot growth of each rice cultivar under gradient changes in N concentrations. This system comprises a gradient hydroponic culture and growth visualization systems. The former allows gradient changes in ammonium concentrations, while the latter records the increment in shoot length of individual rice seedlings at given time periods using a fixed-point camera. We chose 69 cultivars including two controls (Oryza sativa L. cv. Nipponbare [WRC01] and Kasalath [WRC02]) from the World Rice Core Collection to investigate shoot growth responses under ammonium-sufficient, ammonium-limited, and low ammonium concentration gradients without transplanting stress. We observed three growth patterns in response to different ammonium concentrations. Subsequently, we selected three representative cultivars (Kasalath, WRC03, and WRC05) for the characteristic responses under the different ammonium environments. Distinct expression patterns of glutamine synthetase 1;2 (OsGS1;2) but OsGS1;1 were observed in response to varying ammonium concentration regimes, indicating that the expression patterns of OsGS1;2 may be a growth marker in terms of shoot growth when transitioning from ammonium-limited to low ammonium concentrations. This system with the level of OsGS1;2 allows us to screen for candidate cultivars that return high NUE in low N environments.

In plants, mitogen activated protein kinases (MPKs) are involved in various signaling pathways that lead to biotic and abiotic responses as well as that regulate developmental processes. Among them, MPK6 and its closely related homologue, MPK3, act redundantly and are known to be involved in asymmetric cell divisions of meristemoid mother cells in stomata development and of zygotes in Arabidopsis. Loss-of-function mutants of GLE4/OsMPK6, which is an orthologue of MPK6 in rice, showed a defect in polarity establishment in early stage of embryogenesis. However, because of the embryo lethality of the mutations, the function of GLE4/OsMPK6 in post-embryonic development is not clarified. Here, we report the analysis of post embryonic function of GLE4/OsMPK6 in vegetative stage of rice using regenerated gle4/osmpk6 homozygous plants from tissue culture. The regenerated plants are dwarf and produce multiple shoots with small leaves. These shoots never develop into reproductive stage, instead, proliferate vegetative shoots repeatedly. Leaves of gle4/osmpk6 have small leaf blade at the tip and blade-sheath boundary become obscure. Stomata arrangement is also disturbed in gle4/osmpk6 leaf blade. The shape of shoot apical meristem of gle4/osmpk6 become disorganized. Thus, GLE4/OsMPK6 functions in shoot organization and stomata patterning in the post embryonic development in rice.

Carotenoids are isoprenoid pigments produced typically in plants, algae, and part of bacteria and fungi. Violaxanthin, neoxanthin, and lutein are xanthophylls biosynthesized specifically in land plants and part of algae. Nowadays, it is feasible to produce violaxanthin and lutein in Escherichia coli by pathway engineering, whereas there is no report to synthesize neoxanthin in E. coli. So far, several genes have been reported to code for neoxanthin synthases, e.g., NSY (NXS), ABA4 and VDL, which were assigned to catalyze a reaction for forming neoxanthin from violaxanthin. However, neither gene of these was common in plants or algae that biosynthesize neoxanthin, nor was confirmed by the E. coli complementation system. This study showed that the algal VDL gene (PtVDL1) was functional in recombinant E. coli cells accumulating violaxanthin to produce neoxanthin, whereas the E. coli cells failed to generate neoxanthin, when the NSY or ABA4 gene was introduced there instead of VDL. This result notes that VDL is one of veritable neoxanthin synthase genes.

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.

Pre-harvest sprouting of cereals greatly reduces yield and quality of the grains. Abscisic acid (ABA) is an essential phytohormone for the induction and maintenance of seed dormancy. In this study, the ABA responsive promoter-driven ABA biosynthesis gene system was introduced to common wheat (Triticum aestivum L.) to enhance ABA production in the embryos and pre-harvest sprouting tolerance of the grains. This system consists of a wheat ABA responsive element containing Early-Methionine-labelled (EM) promoter and a sorghum 9-cis-epoxycarotenoid dioxygenase (SbNCED) gene which encodes an ABA biosynthesis rate-limiting enzyme. Twenty-three independent single-insertion lines were obtained, from which five homozygous lines showing various SbNCED expression levels were selected. Correlations were observed between SbNCED expression, ABA accumulation in the embryos and enhanced dormancy levels of the grains. The engineered wheat grains exhibited a few day-delay in germination, which should be effective in reducing pre-harvest sprouting damage. However, the increase in ABA levels in the recombinant grains was moderate, which explains why germination was not completely suppressed. Further analysis indicated a concomitant increase in the expression of the ABA catabolic enzyme gene TaABA8′OH1 and in the levels of isoleucine-conjugated jasmonic acid, implying the presence of possible negative feedback regulation in the innate system, which should be overcome for future technology development. These findings advance an understanding of the regulatory mechanisms of hormone metabolism in seeds and facilitate the development of pre-harvest sprouting tolerance in cereal grains.

Seed vigor is an important trait for tobacco production. However, the evaluation of seed vigor using molecular biomarkers is scarcely reported in tobacco. In this study, the development of molecular marker isopropylmalate synthase NtIPMS was conducted to detect seed ageing degree and seed priming effect in tobacco. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of NtIPMS was significantly induced at the initial imbibition stage during seed germination. The NtIPMS expression was positively correlated with the degree of seed ageing in non-pelleted and pelleted seeds. The mRNA level of NtIPMS was gradually increased with the increasing degree of seed ageing. The early best effect of gibberellin priming was observed in 30-h primed seeds, and the highest expression of NtIPMS was observed in 12-h primed seeds. The best stop time-point of seed priming is likely at the time 18 h after the relatively higher NtIPMS expression occurred during seed priming process. The NtIPMS mRNA detection has the potential usage as a potential molecular marker for the evaluation of seed vigor in tobacco.

Shortening the juvenility is a burning issue in breeding fruit trees such as Satsuma mandarin (Citrus unshiu Marc.). Decreasing the breeding period requires a comprehensive understanding of the flowering process in woody plants. Throughout the Arabidopsis flowering system, FLOWERING LOCUS T (FT) interacts with other transcription factors (TFs) and functions as a transmissible floral inducer. In a previous study, a VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1)-like TF from the Satsuma mandarin, CuVOZ1, showed protein–protein interaction with two citrus FTs in a yeast two-hybrid (Y2H) system and precocious flowering in Arabidopsis. In this study, another VOZ, CuVOZ2, was isolated from the Satsuma mandarin ‘Aoshima’ and protein–protein interaction was confirmed between CuVOZ2 and CuFTs. No apical meristem (NAM) and zinc coordination motifs were identified within the N-terminal of CuVOZ2. Docking simulation predicted that interactions between CuVOZ2 and CuFTs might occur in domain B of CuVOZ2, which contains a zinc finger motif. According to docking predictions, the distances between the amino acid residues involved ranged from 1.09 to 4.37 Å, indicating weak Van der Waals forces in the interaction. Cys216, Cys221, Cys235, and His239 in CuVOZ2 were suggested to bond with a Zn²⁺ in the Zn coordination motif. Ectopic expression of 35SΩ:CuVOZ2 in Arabidopsis affected the flowering time, length of inflorescence and internode, and number of siliques, suggesting that CuVOZ2 might regulate both vegetative and reproductive development, act as a trigger for early flowering, and be involved in the elongation of inflorescence possibly in a slightly different way than CuVOZ1.

Lactoferrin is a non-hematic iron-binding 80-kDa protein that exhibits antimicrobial activity. Higher plants function as “green bioreactors” for large-scale recombinant protein production. In this study, we transiently expressed recombinant human lactoferrin (rhLF) in Nicotiana benthamiana at a yield of approximately 40 µg g⁻¹ fresh mass (gFM) using the Tsukuba system. Additionally, the expression level of rhLF increased when it was fused with KDEL, an endoplasmic reticulum retention motif. Purified plant-derived rhLF possesses antibacterial activity that inhibits the growth of Escherichia coli. These results indicated that rhLF containing antimicrobial activity can be produced in N. benthamiana using the Tsukuba system.

A group of anti-nutritional specialized metabolites called steroidal glycoalkaloids (SGAs) are produced in Solanum species such as tomato, potato, and eggplant. The transcription factor JASMONATE-RESPONSIVE ETHYLENE RESPONSE FACTOR 4 (JRE4) regulates many SGA biosynthesis genes in tomato and potato. Here we report that the expression of a cluster of genes encoding nitrate transporter 1/peptide transporter family (NPF) members is downregulated in the jre4-1 loss-of-function tomato mutant, which has a low-SGA phenotype compared to the wild type. NPFs are a large family of plant membrane transporters that transport a wide range of substrates, including specialized metabolites. We found that the JRE4-regulated NPF genes are induced by the defense-related phytohormone jasmonate. Conversely, jasmonate-mediated induction of gene expression was attenuated by ethylene treatment of the leaves. The co-regulation of the NPF genes with SGA biosynthesis genes by JRE4 suggests that NPF transporters are involved in the SGA pathway.

Target of rapamycin (TOR) regulates essential processes associated with plant growth, development, and cell death by modulating metabolic activities and translation in response to environmental signals. The ATP-competitive TOR inhibitor AZD8055 suppressed the hypersensitive response (HR) cell death in Nicotiana benthamiana infected with the incompatible Ralstonia solanacearum. The induced expression of the HR marker gene hin1 was also inhibited by the AZD8055 treatment. To further clarify the mechanisms underlying TOR-regulated HR cell death, we focused on TOR-related ErbB3-binding protein 1 (EBP1) in N. benthamiana (NbEBP1). We found four EBP1 orthologs in the N. benthamiana genome. The expression levels of all four EBP1 orthologs in N. benthamiana were up-regulated by the R. solanacearum infection. The silencing of the four NbEBP1 orthologs suppressed the induction of HR cell death, hin1 expression, and the production of reactive oxygen species. These results suggest that the TOR signaling pathway helps regulate HR cell death along with reactive oxygen species-related signaling in N. benthamiana.

A binary vector carrying two WUSCHEL-related homeobox (WOX) genes, WOX2 and WOX8, under the control of a chemical-inducible expression system, worked in the transformation in N. paniculata, a recalcitrant species of Nicotiana. The resulting transformants exhibited improved culture performance in regeneration from leaf segments and suspended cells. Multicellular masses generated from freely suspended cells showed a specific cell division pattern similar to that of somatic embryo, likely owing to the function of the two WOX genes.

Phospholipid signaling plays an important role in plant immune responses. Here, we isolated two phospholipase C4 (PLC4) orthologs in the Nicotiana benthamiana genome, designated as N. benthamiana PLC4-1 and PLC4-2 (NbPLC4-1 and NbPLC4-2). We created NbPLC4-1- and NbPLC4-2- silenced plants. Induction of the hypersensitive response (HR), including HR cell death and bacterial population reduction, was accelerated in both NbPLC4-1- and NbPLC4-2-silenced plants challenged with N. benthamiana-incompatible Ralstonia solanacearum 8107. The NbPLC4-1- and NbPLC4-2-silenced plants also showed enhanced expression of Nbhin1, a HR marker gene. Expressions of genes for salicylic acid (SA) and jasmonic acid (JA) signaling were drastically increased in NbPLC4-1- and NbPLC4-2-silenced plants by R. solanacearum inoculation. In addition, NbPLC4-1 and NbPLC4-2 silencing triggered reactive oxygen species (ROS) hyper-production. These results suggest that NbPLC4s are closely associated with JA, SA, and ROS signaling and act as negative regulators of the HR in N. benthamiana.

Nitrogen (N) availability is one of the most important factors regulating plant metabolism and growth as it affects global gene expression profiles. Dynamic changes in chromatin structure, including histone modifications and nucleosome assembly/disassembly, have been extensively shown to regulate gene expression under various environmental stresses in plants. However, the involvement of chromatin related changes in plant nutrient responses has been demonstrated only in a few studies to date. In this study, we investigated the function of histone chaperone NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) proteins under N deficient conditions in Arabidopsis. In the nap1;1 nap1;2 nap1;3 triple mutant (m123-1), the expression of N-responsive marker genes and growth of lateral roots were decreased under N deficient conditions. In addition, the m123-1 plants showed a delay in N deficiency-induced leaf senescence. Taken together, these results suggest that NAP1s affect plant growth under N deficient conditions in Arabidopsis.

Root hairs play vital roles in plant growth since they enable the efficient absorption of water and nutrients from the soil. Recent advances in Arabidopsis research have provided a deeper understanding of the molecular genetic mechanisms underlying root hair differentiation. CAPRICE (CPC) and its four homologs, which belong to the CPC gene family and encode R3 MYB transcription factors, play central roles in root hair differentiation. In this study, to better understand the functional specificity and contribution of these five CPC family genes, we conducted phenotypic and expression analyses of the CPC family proteins in a cpc mutant background. As a result, ENHANCER OF TRY AND CPC1 (ETC1) and ETC3 were found to complement the hairless root phenotype of the cpc mutant, as did CPC, whereas TRIPTYCHON (TRY) and ETC2 did not rescue the cpc phenotype. Protein expression analysis revealed that GFP fluorescence was nearly undetectable in pCPC::TRY:GFP/cpc and pCPC::ETC2:GFP/cpc plants, supporting the incapability of root hair formation in these plants. Interestingly, the fluorescence intensity of the CPC:GFP fusion protein was weaker than that of ETC1:GFP and ETC3:GFP fusion proteins. These results were inconsistent with the result of the phenotypic analysis, in which the three genes promoted root hair formation to almost the same degree in the cpc mutant background. We further discuss the discrepancy between the root hair phenotypes and the expression levels of CPC family proteins.

Molecular markers have been widely used in plant breeding to improve the accuracy and efficiency of trait selection. In particular, molecular markers are powerful in facilitating the introgression of resistance genes by circumventing costly and time-consuming infection assays. To achieve their practical use, it is important to ensure the tight linkage between the markers and the traits. Here we report a new cleaved amplified polymorphic sequence (CAPS) marker, Mi1713, for the root-knot nematode resistance gene, Mi-1.2, in cultivated tomato. The Mi1713 marker is designed in the conserved region of Mi-1.2 and its homologs in tomato and other nightshade species. Combined with a single-step procedure for preparing PCR templates, the Mi1713 marker enables rapid and reliable screening for the presence of Mi-1.2. The approach described in this study is applicable in designing CAPS markers for various genes or alleles of interest in tomato and other crops.

A highly contiguous mitochondrial and plastid genome sequences of a japonica rice cultivar, Taichung 65, were determined by a hybrid approach with long- and short-read sequences. The assembled mitochondrial genome was 465,453 bases in length with an overall GC content of 43.8%. It was predicted to harbor 62 protein-encoding genes, 16 kinds (33 copies) of transfer RNA, and three kinds (six copies) of ribosomal RNA genes. The mitochondrial genome structure in Taichung 65 is largely the same as that of Nipponbare, but the first ∼9.5 kb sequence in Nipponbare (DQ167400) is replaced with a ∼27 kb sequence duplicated from other parts of the mitochondrial genome. Phylogenetic and sequence polymorphism analysis indicated that Taichung 65 is classified as typical japonica. The assembled plastid genome sequence was 134,551 bases in length and completely identical to the previously reported Nipponbare sequence. These near-complete organelle genome sequences will serve as fundamental resources for investigating alloplasmic cytoplasmic male sterile lines and other organelle-controlled phenomena in rice.

Pea (Pisum sativum) is an agriculturally important leguminous crop cultivated worldwide. It is also the plant from which phytoalexin was isolated for the first time. Several studies have investigated gene functions using pea hairy root culture systems. However, the procedures for producing hairy roots are relatively complicated and only a few pea cultivars and Rhizobium strains have been used. In this study, we established a simple method for generating transgenic hairy roots using a pea cultivar and a Rhizobium strain available in Japan. The transformation efficiency for the transgenic hairy roots (approximately 14%) was calculated on the basis of GFP fluorescence because the binary vector used in this study carried a GFP cassette as a marker. Furthermore, we confirmed that the production of the phytoalexin (+)-pisatin was induced by a copper dichloride treatment, indicating that this system can be used to characterize the biosynthesis of (+)-pisatin, which is a compound with a unique pterocarpan structure. Interestingly, some of the hairy roots turned into crown galls during the culture period. In summary, our simple method enables the production of transgenic pea hairy roots using biological materials accessible in Japan. The generated hairy roots can be used to elucidate the molecular mechanisms underlying (+)-pisatin biosynthesis as well as hairy root/crown gall formation.

Piriformospora indica, which is an endophytic fungus that grows on various media in the absence of a host, emits plant growth promoting volatile organic compounds (VOCs). Kaefer medium (KF) has been shown to be the most suitable medium for P. indica growth; however, different media may differentially affect fungal metabolism which may in turn influence the VOC profiles of P. indica. To date, how the VOCs emitted from P. indica cultured on different media affect plant growth has not been well characterized. Here, we show that poor nutrient medium (PNM) promoted the growth of P. indica more effectively than potato dextrose agar (PDA) or KF medium. By contrast, plant total biomass and root fresh weight were increased 1.8-fold and 2.1-fold, when co-cultivated with P. indica cultured on PDA medium in comparison with KF or PNM medium, respectively. Furthermore, sucrose in the plant culture medium downregulated the fold-induction ratio of the plant growth promoted by P. indica VOCs.