Allelic variations of Vrn-1 and Ppd-1 genes in Japanese wheat varieties reveal the genotype-environment interaction for heading time.

The timing of heading is largely affected by environmental conditions. In wheat, Vrn-1 and Ppd-1 have been identified as the major genes involved in vernalization requirement and photoperiod sensitivity, respectively. To compare the effects of Vrn-1 and Ppd-1 alleles on heading time under different environments, we genotyped Vrn-1 and Ppd-1 homoeologues and measured the heading time at Morioka, Tsukuba and Chikugo in Japan for two growing seasons. A total of 128 Japanese and six foreign varieties, classified into four populations based on the 519 genome-wide SNPs, were used for analysis. Varieties with the spring alleles (Vrn-D1a or Vrn-D1b) at the Vrn-D1 locus and insensitive allele (Hapl-I) at the Ppd-D1 locus were found in earlier heading varieties. The effects of Vrn-D1 and Ppd-D1 on heading time were stronger than those of the other Vrn-1 and Ppd-1 homoeologues. Analysis of variance revealed that heading time was significantly affected by the genotype-environment interactions. Some Vrn-1 and Ppd-1 alleles conferred earlier or later heading in specific environments, indicating that the effect of both alleles on the timing of heading depends on the environment. Information on Vrn-1 and Ppd-1 alleles, together with heading time in various environments, provide useful information for wheat breeding.

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis.

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants.

Suppression of phospholipase D genes improves chalky grain production by high temperature during the grain-filling stage in rice.

High temperature (HT) during the grain developing stage causes deleterious effects on rice quality resulting in mature grains with a chalky appearance. Phospholipase D (PLD) plays an important role in plants, including responses to environmental stresses. OsPLDα1, α3 and ß2-knockdown (KD) plants showed decreased production of chalky grains at HT. HT ripening increased H2O2 accumulated in the developing grains. However, the increase was canceled by the knockdown of OsPLDß2. Expression levels of OsCATA which is one of three rice catalase genes, in developing grains of OsPLDß2-KD plants at 10 DAF were increased compared with that in vector-controls in HT growth conditions. Overexpression of OsCATA markedly suppressed the production of chalky grains in HT growth conditions. These results suggested that OsPLDß2 functions as a negative regulator of the induction of OsCATA and is involved in the production of chalky grains in HT growth conditions.

MutMapPlus identified novel mutant alleles of a rice starch branching enzyme IIb gene for fine-tuning of cooked rice texture.

Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine-tune cooked rice textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a japonica cultivar Nipponbare and found two independent mutant lines, altered gelatinization (age)1 and age2, with moderate changes in starch gelatinization property. A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of starch branching enzyme IIb (BEIIb) gene. In age1, amino acid substitution of Met-723 to Lys completely abolished BEIIb enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of BEIIb gene reduced BEIIb protein level and activity in age2. Production of a series of the mutant lines by combining age alleles and indica-type starch synthase IIa allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α-amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.

An Activity-Staining Method on Filtration Paper Enables High-Throughput Screening of Temperature-Sensitive and Inactive Mutations of Rice α-Amylase for Improvement of Rice Grain Quality.

α-Amylase is a starch-hydrolyzing enzyme (EC 3.2.1.1) indispensable for germination of cereal seeds, but it is also expressed during the ripening stage. Previous studies demonstrated that the enzyme is activated in developing rice seeds under extremely hot weather and triggers a loss of grain quality by hindering the accumulation of storage starch in the endosperm. Since inactive or, preferably, heat-labile α-amylases are preferable for breeding premium rice, we developed a method for rapid screening of inactive and temperature-sensitive mutants of the enzyme by combining the random mutagenesis by error-prone PCR and an on-filter activity test of the recombinant enzyme expressed by Escherichia coli. This technique was applied to a major α-amylase in the developing seed, Amy3D, and the activity of the isolated mutant enzymes was verified with both the bacteria-expressed recombinant proteins and the extract from the endosperm overexpressing each of them. Then, we identified several substitutions leading to loss of the activity of amino acid residues (Leu28, Asp112, Cys149, Trp201, Asp204, Gly295, Leu300 and Cys342), as well as a variety of heat-sensitive substitutions of Asp83, Asp187 and Glu252. Furthermore, variations of the heat-labile enzymes were created by combining these heat-sensitive mutations. The effects of the respective mutations and their relationship to the structure of the enzyme molecule are discussed.

A bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate jasmonate signaling in arabidopsis.

Jasmonates (JAs) are plant hormones that regulate the balance between plant growth and responses to biotic and abiotic stresses. Although recent studies have uncovered the mechanisms for JA-induced responses in Arabidopsis thaliana, the mechanisms by which plants attenuate the JA-induced responses remain elusive. Here, we report that a basic helix-loop-helix-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1 (JAM1), acts as a transcriptional repressor and negatively regulates JA signaling. Gain-of-function transgenic plants expressing the chimeric repressor for JAM1 exhibited substantial reduction of JA responses, including JA-induced inhibition of root growth, accumulation of anthocyanin, and male fertility. These plants were also compromised in resistance to attack by the insect herbivore Spodoptera exigua. Conversely, jam1 loss-of-function mutants showed enhanced JA responsiveness, including increased resistance to insect attack. JAM1 and MYC2 competitively bind to the target sequence of MYC2, which likely provides the mechanism for negative regulation of JA signaling and suppression of MYC2 functions by JAM1. These results indicate that JAM1 negatively regulates JA signaling, thereby playing a pivotal role in fine-tuning of JA-mediated stress responses and plant growth.

Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation.

A molecular mechanism to ensure signaling specificity is a scaffold. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in gibberellin feedback regulation. The 14-3-3 proteins negatively regulate RSG by sequestering it in the cytoplasm in response to gibberellins. The N. tabacum Ca2+-dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of RSG. CDPKs are unique sensor responders of Ca2+ that are only found in plants and some protozoans. Here, we report a scaffolding function of CDPK. 14-3-3 proteins bound to NtCDPK1 by a new mode. Autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14-3-3 but not for its maintenance. NtCDPK1 formed a heterotrimer with RSG and 14-3-3. Furthermore, we found that NtCDPK1 transfers 14-3-3 to RSG after phosphorylation of RSG and that RSG dissociates from NtCDPK1 as a complex with 14-3-3. These results suggest that NtCDPK1 is an interesting scaffolding kinase that increases the specificity and efficiency of signaling by coupling catalysis with scaffolding on the same protein.

Alteration of substrate specificity: the variable N-terminal domain of tobacco Ca(2+)-dependent protein kinase is important for substrate recognition.

Protein kinases are major signaling molecules that are involved in a variety of cellular processes. However, the molecular mechanisms whereby protein kinases discriminate specific substrates are still largely unknown. Ca(2+)-dependent protein kinases (CDPKs) play central roles in Ca(2+) signaling in plants. Previously, we found that a tobacco (Nicotiana tabacum) CDPK1 negatively regulated the transcription factor REPRESSION OF SHOOT GROWTH (RSG), which is involved in gibberellin feedback regulation. Here, we found that the variable N-terminal domain of CDPK1 is necessary for the recognition of RSG. A mutation (R10A) in the variable N-terminal domain of CDPK1 reduced both RSG binding and RSG phosphorylation while leaving kinase activity intact. Furthermore, the R10A mutation suppressed the in vivo function of CDPK1. The substitution of the variable N-terminal domain of an Arabidopsis thaliana CDPK, At CPK9, with that of Nt CDPK1 conferred RSG kinase activities. This chimeric CDPK behaved according to the identity of the variable N-terminal domain in transgenic plants. Our results open the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways.

Agent-based model using GPS analysis for infection spread and inhibition mechanism of SARS-CoV-2 in Tokyo.

Analyzing the SARS-CoV-2 pandemic outbreak based on actual data while reflecting the characteristics of the real city provides beneficial information for taking reasonable infection control measures in the future. We demonstrate agent-based modeling for Tokyo based on GPS information and official national statistics and perform a spatiotemporal analysis of the infection situation in Tokyo. As a result of the simulation during the first wave of SARS-CoV-2 in Tokyo using real GPS data, the infection occurred in the service industry, such as restaurants, in the city center, and then the infected people brought back the virus to the residential area; the infection spread in each area in Tokyo. This phenomenon clarifies that the spread of infection can be curbed by suppressing going out or strengthening infection prevention measures in service facilities. It was shown that pandemic measures in Tokyo could be achieved not only by strong control, such as the lockdown of cities, but also by thorough infection prevention measures in service facilities, which explains the curb phenomena in real Tokyo.

The transcription factor RSG regulates negative feedback of NtGA20ox1 encoding GA 20-oxidase.

Gibberellins (GAs) are phytohormones that regulate growth and development throughout the life cycle of plants. RSG (REPRESSION OF SHOOT GROWTH) is a tobacco (Nicotiana tabacum) transcriptional activator with a basic leucine zipper domain that regulates the endogenous amount of GAs by control of GA biosynthetic enzymes. Negative feedback contributes to homeostasis of the GA levels. Previous studies suggested that RSG is directly or indirectly involved in the GA negative feedback of NtGA20ox1 encoding GA 20-oxidase. Using transgenic tobacco plants, we have identified a cis-acting region that is responsible for the feedback regulation of NtGA20ox1. This region contains an RSG-binding sequence. A mutation in the RSG-binding sequence abolished negative feedback of NtGA20ox1 in transgenic plants. Chromatin immunoprecipitation (ChIP) assays showed that RSG binds to the NtGA20ox1 promoter in vivo in response to a decrease in GA levels, and that this binding is abolished within 3 h after GA treatment. Furthermore, decreases in GA levels promote modifications of active histone marks in the promoter of NtGA20ox1. Our results suggest that RSG plays a role in the homeostasis of GAs through direct binding to the NtGA20ox1 promoter.

High Temperature-Induced Expression of Rice α-Amylases in Developing Endosperm Produces Chalky Grains.

Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by α-amylases. Moreover, the chalky grains contained increased amounts of soluble sugars including maltooligosaccharides at the expense of starch. The integrated analyses proposed that expression of Amy1A, Amy3C, and Amy3D at the specific regions of the developing endosperm could generate the chalkiness. This finding provides the fundamental knowledge to narrow down the targets for the development of high temperature-tolerant premium rice.

Phosphorylation-independent binding of 14-3-3 to NtCDPK1 by a new mode.

14-3-3 pproteins play essential roles in diverse cellular processes through the direct binding to target proteins. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in gibberellin (GA) feedback regulation. The 14-3-3 proteins bind to RSG depending on the RSG phosphorylation of Ser-114 and negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The Ca(2+)-dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of RSG. 14-3-3 weakly binds to NtCDPK1 by a new mode. The autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14-3-3 but not for its maintenance. In this study, we showed that 14-3-3 binding to NtCDPK1 does not require the autophosphorylation when RSG was bound to NtCDPK1. These data suggest that 14-3-3 binds to an unphosphorylated motif in NtCDPK1 exposed by a conformational change in NtCDPK1 but not to a phosphate group generated by autophosphorylation of NtCDPK1.

Two bHLH-type transcription factors, JA-ASSOCIATED MYC2-LIKE2 and JAM3, are transcriptional repressors and affect male fertility.

The jasmonate (JA) plant hormones regulate responses to biotic and abiotic stress and aspects of plant development, including male fertility in Arabidopsis thaliana. The bHLH-type transcription factor JA-ASSOCIATED MYC2-LIKE1 (JAM1) negatively regulates JA signaling and gain-of-function JAM1 transgenic plants have impaired JA-mediated male fertility. Here we report that JAM2 and JAM3, 2 bHLHs closely related to JAM1, also act as transcriptional repressors. Moreover, overexpression of JAM2 and JAM3 also results in reduced male fertility. These results suggest that JAM1, JAM2, and JAM3 act redundantly as negative regulators of JA-mediated male fertility.

The mechanism of substrate recognition of Ca2+-dependent protein kinases.

Ca2+-dependent protein kinases (CDPKs) are encoded by a multigene family and are thought to play central roles in Ca2+ signaling in plants. Although the primary structures of CDPK isoforms are highly conserved, several studies suggested a distinct physiological function for each CDPK isoform in plants. Hence, there should be mechanisms by which individual CDPK specifically recognizes its substrate. Recently, the variable N-terminal domain of NtCDPK1 was shown to play an essential role in the specific recognition of the substrate. Because the variable N-terminal domain of other CDPKs may also be involved in the substrate recognition, the search for interacting proteins of the variable N-terminal domain would provide important clues to identify the physiological substrates of each CDPK. Additionally, manipulation of the variable N-terminal domain may enable us to engineer the substrate specificity of CDPK, leading a rational rewiring of cellular signaling pathways.

bZIP transcription factor RSG controls the feedback regulation of NtGA20ox1 via intracellular localization and epigenetic mechanism.

Gibberellins (GAs) are phytohormones that regulate growth and development throughout the life cycle of plants. Negative feedback contributes to homeostasis of GA levels. DELLA proteins are involved in this process. Since DELLA proteins do not have apparent DNA binding motifs, other DNA binding proteins might act as a mediator downstream of DELLA proteins in the GA feedback regulation. In this review, we highlight the mechanisms of GA feedback regulation, specifically the differential regulation of GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox) by transcription factors. RSG (REPRESSION OF SHOOT GROWTH) is a tobacco (Nicotiana tabacum) transcriptional activator with a basic leucine zipper domain that controls the levels of endogenous GAs through the regulation of GA biosynthesis genes. Recently we reported that RSG not only regulates the expression of ent-kaurene oxidase gene but is also involved in the negative feedback of NtGA20ox1 by GAs. RSG plays a role in the homeostasis of GAs through direct binding to the NtGA20ox1 promoter triggered by a decrease in GA levels in the cell. Furthermore, decreases in GA levels promote modifications of active histone marks on the NtGA20ox1 promoter. We have developed a hypothetical model to explain how RSG regulates dual target genes via epigenetic regulation.

CRES-T, an effective gene silencing system utilizing chimeric repressors.

Chimeric REpressor gene Silencing Technology (CRES-T) is a useful tool for functional analysis of plant transcription factors. In this system, a chimeric repressor that is produced by fusion of a transcription factor to the plant-specific EAR-motif repression domain (SRDX) suppresses target genes of a transcription factor dominantly over the activity of endogenous and functionally redundant transcription factors. As a result, the transgenic plants that express a chimeric repressor exhibit phenotypes similar to loss-of-function of the alleles of the gene encoding the transcription factor. This system is simple and effective and can be used as a powerful tool not only for functional analysis of redundant transcription factors but also for the manipulation of plant traits by active suppression of the gene expression. Strategies for construction of the chimeric repressors and their expression in transgenic plants are described. Transient effector-reporter assays for functional analysis of transcription factors and detection of protein-protein interactions using the trans-repressive activity of SRDX repression domain are also described.

CDPK1, a calcium-dependent protein kinase, regulates transcriptional activator RSG in response to gibberellins.

The homeostasis of gibberellins (GAs) is maintained by negative-feedback regulation in plant cells. REPRESSION OF SHOOT GROWTH (RSG) is a transcriptional activator with a basic Leu zipper domain suggested to contribute GA feedback regulation by the transcriptional regulation of genes encoding GA biosynthetic enzymes. The 14-3-3 signaling proteins negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The phosphorylation on Ser-114 of RSG is essential for 14-3-3 binding of RSG; however, the kinase that catalyzes the reaction is unknown. Recently a Ca(2+)-dependent protein kinase (CDPK) was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of the Ser-114 of RSG. Our results suggest that CDPK decodes the Ca(2+) signal produced by GAs and regulates the intracellular localization of RSG in plant cells.

Identification of negative cis-acting elements in response to copper in the chloroplastic iron superoxide dismutase gene of the moss Barbula unguiculata.

Superoxide dismutases (SODs) are ubiquitous metalloenzymes that catalyze the dismutation of superoxide radicals. Chloroplasts have two isozymes, copper/zinc SOD (Cu/ZnSOD) and iron SOD (FeSOD), encoded by nuclear genes. Because bryophytes are considered as the earliest land plants, they are one of the most interesting plant models for adaptation against oxidative stress. In a previous study, we found that the FeSOD gene was expressed under Cu-deficient conditions and repressed under high-Cu-supply conditions; on the other hand, the Cu/ZnSOD gene was induced by Cu in a moss, Barbula unguiculata. The expression of Cu/ZnSOD and FeSOD is coordinately regulated at the transcriptional level depending on metal bioavailability. Here, using transgenic moss plants, we determined that the GTACT motif is a negative cis-acting element of the moss FeSOD gene in response to Cu. Furthermore, we found that a plant-specific transcription factor, PpSBP2 (for SQUAMOSA promoter-binding protein), and its related proteins bound to the GTACT motif repressed the expression of the FeSOD gene. The moss FeSOD gene was negatively regulated by Cu in transgenic Nicotiana tabacum plants, and the Arabidopsis thaliana FeSOD gene promoter containing the GTACT motif was repressed by Cu. Our results suggested that molecular mechanisms of GTACT motif-dependent transcriptional suppression by Cu are conserved in land plants.

A tobacco calcium-dependent protein kinase, CDPK1, regulates the transcription factor REPRESSION OF SHOOT GROWTH in response to gibberellins.

The homeostasis of gibberellins (GAs) is maintained by negative feedback in plants. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcriptional activator that has been suggested to play a role in GA feedback by the regulation of GA biosynthetic enzymes. The 14-3-3 signaling proteins negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The phosphorylation on Ser-114 of RSG is essential for 14-3-3 binding of RSG. Here, we identified tobacco Ca(2+)-dependent protein kinase (CDPK1) as an RSG kinase that promotes 14-3-3 binding to RSG by phosphorylation of Ser-114 of RSG. CDPK1 interacts with RSG in a Ca(2+)-dependent manner in vivo and in vitro and specifically phosphorylates Ser-114 of RSG. Inhibition of CDPK repressed the GA-induced phosphorylation of Ser-114 of RSG and the GA-induced nuclear export of RSG. Overexpression of CDPK1 inhibited the feedback regulation of a GA 20-oxidase gene and resulted in sensitization to the GA biosynthetic inhibitor. Our results suggest that CDPK1 decodes the Ca(2+) signal produced by GAs and regulates the intracellular localization of RSG.

Salt stress-induced dissociation from cells of a germin-like protein with Mn-SOD activity and an increase in its mRNA in a moss, Barbula unguiculata.

To estimate the physiological roles of a germin-like protein (BuGLP) with Mn-SOD activity isolated newly from a moss, Barbula unguiculata, BuGLP mRNA levels during cell growth and the effects of methyl viologen and salt stress were studied. BuGLP mRNA levels were at their peak during the exponential phase of growth and decreased thereafter, but SOD activity was held at the same level as that during the exponential phase. When methyl viologen was present as a generator of superoxide the amount of BuGLP transcripts decreased, but that of SOD activity of BuGLP bound to the cell wall was not affected. The addition of NaCl to the cells during the logarithmic phase increased both the BuGLP mRNA levels and total SOD activity of BuGLP, but decreased the SOD activity bound to the cell wall due to release of most of the SOD activity into the medium. On the other hand, the addition of NaCl to the cells during the stationary phase hardly affected BuGLP mRNA levels or SOD activity levels bound to the cell wall. These results suggest that the induction of BuGLP gene by salt stress is caused by dissociation of BuGLP protein from the cell wall into the medium in the cells during the logarithmic phase.