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Nitrogen (N) is a major limiting factor affecting crop yield in unfertilized soil. Thus, cultivars with a high N use efficiency (NUE) and good grain protein content (GPC) are needed to fulfill the growing food demand and to reduce environmental burden. This is especially true for rice (Oryza sativa L.) that is cultivated with a high input of N fertilizer and is a primary staple food crop for more than half of the global population. Here, we report that rice asparagine synthetase 1 (OsASN1) is required for grain yield and grain protein contents under both N-sufficient (conventional paddy fields) and N-limiting conditions from analyses of knockout mutant plants. In addition, we show that overexpression (OX) of OsASN1 results in better nitrogen uptake and assimilation, and increased tolerance to N limitation at the seedling stage. Under field conditions, the OsASN1 OX rice plants produced grains with increased N and protein contents without yield reduction compared to wild-type (WT) rice. Under N-limited conditions, the OX plants displayed increased grain yield and protein content with enhanced photosynthetic activity compared to WT rice. Thus, OsASN1 can be an effective target gene for the development of rice cultivars with higher grain protein content, NUE, and grain yield under N-limiting conditions., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Leaf senescence is regulated by diverse developmental and environmental factors to maximize plant fitness. The red to far-red light ratio (R:FR) detected by plant phytochromes is reduced under vegetation shade, thus initiating leaf senescence. However, the role of phytochromes in promoting leaf senescence under FR-enriched conditions is not fully understood. In this study, we investigated the role of phyA and phyB in regulating leaf senescence under FR in Arabidopsis thaliana (Arabidopsis). FR enrichment and intermittent FR pulses promoted the senescence of Arabidopsis leaves. Additionally, phyA and phyB mutants showed enhanced and repressed senescence phenotypes in FR, respectively, indicating that phyA and phyB antagonistically regulate FR-dependent leaf senescence. Transcriptomic analysis using phyA and phyB mutants in FR identified differentially expressed genes (DEGs) involved in leaf senescence-related processes, such as responses to light, phytohormones, temperature, photosynthesis and defense, showing opposite expression patterns in phyA and phyB mutants. These contrasting expression profiles of DEGs support the antagonism between phyA and phyB in FR-dependent leaf senescence. Among the genes showing antagonistic regulation, we confirmed that the expression of WRKY6, which encodes a senescence-associated transcription factor, was negatively and positively regulated by phyA and phyB, respectively. The wrky6 mutant showed a repressed senescence phenotype compared with the wild type in FR, indicating that WRKY6 plays a positive role in FR-dependent leaf senescence. Our results imply that antagonism between phyA and phyB is involved in fine-tuning leaf senescence under varying FR conditions in Arabidopsis., (� The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Leaf senescence, which constitutes the final stage of leaf development, involves programmed cell death and is intricately regulated by various internal and environmental signals that are incorporated with age-related information. ABA plays diverse and important physiological roles in plants, and is involved in various developmental events and stress responses. ABA has long been regarded as a positive regulator of leaf senescence. However, the cellular mediators of ABA-induced senescence have not been identified. We sought to understand the ABA-induced senescence signaling process in Arabidopsis by examining the function of an ABA- and age-induced gene, RPK1, which encodes a membrane-bound, leucine-rich repeat-containing receptor kinase (receptor protein kinase 1). Loss-of-function mutants in RPK1 were significantly delayed in age-dependent senescence. Furthermore, rpk1 mutants exhibited reduced sensitivity to ABA-induced senescence but little change to jasmonic acid- or ethylene-induced senescence. RPK1 thus mediates ABA-induced leaf senescence as well as age-induced leaf senescence. Conditional overexpression of RPK1 at the mature stage clearly accelerated senescence and cell death, whereas induction of RPK1 at an early developmental stage retarded growth without triggering senescence symptoms. Therefore, RPK1 plays different roles at different stages of development. Consistently, exogenously applied ABA affected leaf senescence in old leaves but not in young leaves. The results, together, showed that membrane-bound RPK1 functions in ABA-dependent leaf senescence. Furthermore, the effect of ABA and ABA-inducible RPK1 on leaf senescence is dependent on the age of the plant, which in part explains the mechanism of functional diversification of ABA action.

Phytochrome regulates various physiological and developmental processes throughout the life cycle of plants. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates the far-red light high irradiance response (FR-HIR), which is elicited by continuous far-red light. In FR-HIR, nuclear accumulation of phyA, which precedes physiological responses, is proposed to be required for the response. In contrast to FR, red light induces rapid degradation of phyA to suppress undesirable long-term photomorphogenic responses of phyA. In the present study, we compared biological activities between phyA derivatives to which either a nuclear localization (NLS) or export (NES) signal sequence was attached. Those derivatives were expressed under the control of the PHYA promoter in the Arabidopsis phyA mutant. Detailed microscopic observation revealed that the phyA-green fluorescent protein (GFP) without a signal sequence is localized exclusively in the cytoplasm in darkness. Rapid nuclear entry was observed after exposure to both red and far-red light. Interestingly, both phyA-GFP-NLS and phyA-GFP-NES were rapidly degraded under continuous red light. Furthermore, a proteasome inhibitor delayed degradation equally under these two conditions. Therefore, similar mechanisms for phyA degradation may exist in the cytoplasm and nucleus. As expected from previous reports, phyA-GFP-NLS, but not phyA-GFP-NES, mediated different aspects of FR-HIR, such as inhibition of hypocotyl elongation and rapid induction of gene expression, confirming that phyA nuclear localization is required for FR-HIR. In addition, a detailed time course analysis of phyA-GFP and phyA-GFP-NLS responses revealed that they were almost indistinguishable, raising the question of the physiological relevance of phyA cytoplasmic retention in darkness.

A gene-trapping vector carrying a GUS/Luciferase dual reporter gene was developed to establish an efficient and convenient screening system for T-DNA-based gene trapping in plants. A key feature of this gene trap scheme is to place two different types of reporters, luciferase (Luc) and beta-glucuronidase (GUS), as a fusion protein within a trapped gene to probe the activity of the gene. Luc is then utilized as a non-invasive, vital and highly sensitive screening reporter to identify trapped lines, including direct screening of the trapped lines from the primary T-DNA mutant pools. GUS is utilized as a histochemical assay reporter to analyze detailed cellular expression patterns. Transgenic expression studies in Arabidopsis showed that this fusion reporter protein retains functional enzyme activity for both GUS and Luc. Using this system in Arabidopsis, we were able to identify 3,737 trapped lines from 26,900 individual T-DNA insertion lines. Sequence determination of the T-DNA insertion loci in the genome of 78 trapped lines identified GUS/Luc fusions with 27 annotated Arabidopsis genes which included a subset of transcription factors, protein kinases, regulatory proteins and metabolic enzymes. Of these, particular expression patterns of four tagged genes were further confirmed by analyzing putative promoter regions of the corresponding wild-type genes. Furthermore, the protein stability of the GUS/Luc fusion reporter was controlled by application of luciferase substrate (luciferin), overcoming the excessive stability problem of GUS that causes misrepresentation of the transcriptional activity of a promoter. These results demonstrate the utility of the GUS/Luc dual reporter system as a gene trap reporter for studying plant genome function and also as a convenient dual reporter system for study of gene expression.

The BLADE-ON-PETIOLE1 (BOP1) gene of Arabidopsis thaliana is required for proper leaf morphogenesis. BOP1 regulates leaf differentiation in a proximal-distal manner, and represses the expression of three class I knotted-like homeobox (knox) genes during leaf formation. Utilizing a map-based approach, we identified the molecular nature of the BOP1 gene, which encodes a BTB/POZ domain protein with ankyrin repeats. BOP1 is a member of a small gene family in Arabidopsis that includes the disease resistance regulatory protein NPR1. Insertions in and around BOP1 cause distinct lesions in leaf morphogenesis, revealing complex regulation of the locus. BOP1 transcripts are initially detectable in embryos, where they specifically localize to the base of the developing cotyledons near the SAM. During vegetative development, BOP1 is expressed in young leaf primordia and at the base of the rosette leaves on the adaxial side. During reproductive development, BOP1 transcripts are detected in young floral buds, and at the base of the sepals and petals. Our results indicate that BOP1 encodes a putative regulatory protein that modulates meristematic activity at discrete locations in developing lateral organs. This is the first report on a plant protein that plays a key role in morphogenesis with the distinctive combinatorial architecture of the BTB/POZ and ankyrin repeat domains.

Reactive oxygen species play a critical role in mediating the oxidative damage that causes senescence in a variety of aerobic organisms, from yeast to mammals. Genetic studies of these organisms have revealed that extended longevity is frequently associated with an increased resistance to stress. However, the relationship between life span and oxidative stress tolerance in plants is poorly understood. We have investigated the responses to oxidative stress in the delayed leaf senescence mutants of Arabidopsis thaliana, ore1, ore3, and ore9. The detached leaves of these mutants exhibit increased tolerance to various types of oxidative stress. The ore1, ore3, and ore9 mutants were also more tolerant to oxidative stress at the level of the whole plant, as determined by measuring physiological and molecular changes associated with oxidative stress. However, the activities of antioxidant enzymes were similar or lower in the mutants, as compared to wild type. These results suggest that the increased resistance to oxidative stress in the ore1, ore3, and ore9 mutants is not due to enhanced activities of these antioxidant enzymes. Taken together, our findings provide genetic evidence that oxidative stress tolerance is linked to control of leaf longevity in plants.

We report the characterization of a semi-dominant mutation fin5-1 (far-red insensitive 5-1) of Arabidopsis, which was isolated from genetic screening of phytochrome A (phyA) signaling components. Plants with the fin5-1 mutation exhibited a long hypocotyl phenotype when grown under far-red (FR) light, but not under red light. Physiological analyses implied that FIN5 might be differentially involved in diverse responses that are regulated by phyA under continuous FR light. Anthocyanin accumulation, gravitropic response of hypocotyl growth, and FR light-preconditioned blocking of greening were also impaired in the fin5-1 mutant, whereas photoperiodic floral induction was not, if at all, significantly affected. Moreover, light-regulated expression of the CHS, PORA and PsbS genes was attenuated in fin5-1 mutant plants, while the light-induced expression of CAB was normal. The mutation exhibited semi-dominance regarding control of hypocotyl growth in FR light. We suggest that FIN5 defines a novel branch in the network of phyA signaling in Arabidopsis.

A dominant mutant line characterized by abnormal leaf venation pattern was isolated from a transgenic Arabidopsis plant pool that was generated with Agrobacterium culture harboring an Arabidopsis antisense cDNA library. In the mutant line, the phenotype was due to antisense suppression of a gene we named VEP1 (Vein Patterning). The predicted amino acid sequence of the gene contained a motif related to the mammalian death domain that is found in the apoptotic machinery. Reduced expression of the VEP1 gene resulted in the reduced complexity of the venation pattern of the cotyledons and foliar leaves, which was mainly due to the reduced number of the minor veins and their incomplete connection. The analysis of mutant embryos indicated that the phenotype was originated, at least in part, from a defect in the procambium patterning. In the mutant, the stem and root were thinner than those in wild type. This phenotype was associated with reduced vascular development. The promoter activity of the VEP1 gene was detected preferentially in the vascular regions. We propose that the death domain-containing protein VEP1 functions as a positive element required for vascular strand development in Arabidopsis thaliana.

We have identified a homolog of the mammalian ionotropic glutamate receptor genes in Arabidopsis thaliana (AtGluR2). This gene was found to alter Ca2+ utilization when overexpressed in A. thaliana. These transgenic plants displayed symptoms of Ca2+ deficiency, including browning and death of the shoot apex, necrosis of leaf tips, and deformation of leaves. Supplementation with Ca2+ alleviated these phenotypes. Overall levels of Ca2+ in tissues of control plants were not significantly different from those of transgenic plants, suggesting that overexpression of the AtGluR2 gene did not affect Ca2+ uptake. However, the relative growth yield as a function of Ca2+ levels revealed that the critical deficiency content of Ca2+ in transgenic plants was three times higher than that of control plants. The transgenic plants also exhibited hypersensitivity to Na+ and K+ ionic stresses. The ion hypersensitivity was ameliorated by supplementation with Ca2+. The results showed that overexpression of the AtGluR2 gene caused reduced efficiency of Ca2+ utilization in the transgenic plants. The promoter of the AtGluR2 gene was active in vascular tissues, particularly in cells adjacent to the conducting vessels. This suggests that AtGluR2 encodes a functional channel that unloads Ca2+ from the xylem vessels. The results together suggest that appropriate expression of the AtGluR2 protein may play critical roles in Ca2+ nutrition by controlling the ion allocation among different Ca2+ sinks both during normal development and during adaptation to ionic stresses.

We have isolated a male-sterile mutant from a pool of T-DNA insertional lines of Arabidopsis thaliana generated by an in planta transformation procedure [Chang et al. (1994) Plant J. 5: 551]. Pollen in this mutant is not effectively released from anther locules after cleavage of the stomium. Most mutant pollen grains are round, in contrast to the tricolpate wild-type pollen, and some pollen grains show an abnormal surface structure. Manually released mutant pollen grains are not fertile and show defects in pollen tube germination in vitro. Genetic analysis disclosed that this lesion is due to a single recessive nuclear mutation located on chromosome 3 closely linked to the gll locus. The mutation locus is tightly linked to the inserted T-DNA.

To characterize the molecular structure of the S alleles in Lycopersicon peruvianum, we isolated two genomic fragments that corresponded to cDNAs for S11 and S12 RNases from a genomic library of a plant with the S11S12 genotype. The coding regions and the flanking regions in both fragments were sequenced. A single short intron was found in each gene for RNase, and it was located in the previously identified HV2 region [Chung et al. (1994) Plant Mol. Biol. 26: 757]. The site of initiation of transcription of the gene for S11 RNase was determined by a primer extension analysis and a putative TATA box was identified. Three short sequences that were highly homologous in the two S alleles were found within 360 bp upstream of the ATG initiation codon. It is suggested that these sequences might be involved in the regulation of transcription required for the appropriate-level or tissue-specific expression of the S genes in L. peruvianum.