Many multicellular organisms have remarkable capability to regenerate new organs after wounding. As a first step of organ regeneration, adult somatic cells often dedifferentiate to reacquire cell proliferation potential, but mechanisms underlying this process remain unknown in plants. Here we show that an AP2/ERF transcription factor, WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), is involved in the control of cell dedifferentiation in Arabidopsis. WIND1 is rapidly induced at the wound site, and it promotes cell dedifferentiation and subsequent cell proliferation to form a mass of pluripotent cells termed callus. We further demonstrate that ectopic overexpression of WIND1 is sufficient to establish and maintain the dedifferentiated status of somatic cells without exogenous auxin and cytokinin, two plant hormones that are normally required for cell dedifferentiation. In vivo imaging of a synthetic cytokinin reporter reveals that wounding upregulates the B-type ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin response and that WIND1 acts via the ARR-dependent signaling pathway to promote cell dedifferentiation. This study provides novel molecular insights into how plants control cell dedifferentiation in response to wounding.

A long awn is one of the distinct morphological features of wild rice species. This organ is thought to aid in seed dispersal and prevent predation by animals. Most cultivated varieties of Oryza sativa and Oryza glaberrima, however, have lost the ability to form long awns. The causal genetic factors responsible for the loss of awn in these two rice species remain largely unknown. Here, we evaluated three sets of chromosome segment substitution lines (CSSLs) in a common O. sativa genetic background (cv. Koshihikari) that harbor genomic fragments from Oryza nivara, Oryza rufipogon, and Oryza glaberrima donors. Phenotypic analyses of these libraries revealed the existence of three genes, Regulator of Awn Elongation 1 (RAE1), RAE2, and RAE3, involved in the loss of long awns in cultivated rice. Donor segments at two of these genes, RAE1 and RAE2, induced long awn formation in the CSSLs whereas an O. sativa segment at RAE3 induced long awn formation in O. glaberrima. These results suggest that the two cultivated rice species, O. sativa and O. glaberrima, have taken independent paths to become awnless.

The apicomplexan parasite Toxoplasma gondii produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite Plasmodium spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in P. berghei ANKA and T. gondii cell lysates. However, addition of salicylic acid to P. falciparum and T. gondii culture had no effect. We transfected P. falciparum 3D7 with the nahG gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting Pseudomonas sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E2, which potentially modulates host immunity as an adaptive evolution of Plasmodium spp. To investigate the function of salicylic acid and prostaglandin E2 on host immunity, we established P. berghei ANKA mutants expressing nahG. C57BL/6 mice infected with nahG transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The nahG-transfectant also significantly increased the mortality rate of mice. Prostaglandin E2 reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the nahG transfectant. Thus, salicylic acid of Plasmodium spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E2.

Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.

Grain number is an important agronomic trait. We investigated the roles of chromatin interacting factor Oryza sativa VIN3-LIKE 2 (OsVIL2), which controls plant biomass and yield in rice. Mutations in OsVIL2 led to shorter plants and fewer grains whereas its overexpression (OX) enhanced biomass production and grain numbers when compared with the wild type. RNA-sequencing analyses revealed that 1958 genes were up-regulated and 2096 genes were down-regulated in the region of active division within the first internodes of OX plants. Chromatin immunoprecipitation analysis showed that, among the downregulated genes, OsVIL2 was directly associated with chromatins in the promoter region of CYTOKININ OXIDASE/DEHYDROGENASE2 (OsCKX2), a gene responsible for cytokinin degradation. Likewise, active cytokinin levels were increased in the OX plants. We conclude that OsVIL2 improves the production of biomass and grain by suppressing OsCKX2 chromatin.

Deepwater rice possesses internode elongation ability to avoid drowning under deepwater conditions. Previous studies identified three QTLs regulating internode elongation ability on chromosomes 1, 3 and 12 using different populations. However, these QTLs only induce internode elongation in response to deepwater conditions from the 7-leaf stage and not during the early leaf stage. In this study, we detected two novel QTLs, qTIL2 and qTIL4 regulating deepwater response at the early leaf stage using an F(2) population derived from the cross between NIL1-3-12 carrying the three QTLs regulating deepwater response in T65 (O. sativa ssp. japonica) genetic background and C9285 (O. sativa ssp. indica, deepwater rice). Plants of the BC(2)F(2) population derived from NIL1-3-12/C9285 and the RILs of T65/Bhadua (O. sativa ssp. indica, deepwater rice) possessing these QTLs as well as the three QTLs previously identified also showed internode elongation during the early leaf stage. These results indicate that qTIL2 and qTIL4 regulate early internode elongation and function in coordination with the three major QTLs under deepwater conditions. The results presented here would not only help define the mechanism of deepwater response in rice but also contribute in the breeding of deepwater tolerant rice that is adapted to various water depths.

Growth is characterized by the interplay between cell division and cell expansion, two processes that occur separated along the growth zone at the maize leaf. To gain further insight into the transition between cell division and cell expansion, conditions were investigated in which the position of this transition zone was positively or negatively affected. High levels of gibberellic acid (GA) in plants overexpressing the GA biosynthesis gene GA20-OXIDASE (GA20OX-1OE ) shifted the transition zone more distally, whereas mild drought, which is associated with lowered GA biosynthesis, resulted in a more basal positioning. However, the increased levels of GA in the GA20OX-1OE line were insufficient to convey tolerance to the mild drought treatment, indicating that another mechanism in addition to lowered GA levels is restricting growth during drought. Transcriptome analysis with high spatial resolution indicated that mild drought specifically induces a reprogramming of transcriptional regulation in the division zone. 'Leaf Growth Viewer' was developed as an online searchable tool containing the high-resolution data.

Plant organ growth is controlled by inter-cell-layer communication, which thus determines the overall size of the organism. The epidermal layer interfaces with the environment and participates in both driving and restricting growth via inter-cell-layer communication. However, it remains unknown whether the epidermis can send signals to internal tissue to limit cell proliferation in determinate growth. Very-long-chain fatty acids (VLCFAs) are synthesized in the epidermis and used in the formation of cuticular wax. Here we found that VLCFA synthesis in the epidermis is essential for proper development of Arabidopsis thaliana. Wild-type plants treated with a VLCFA synthesis inhibitor and pasticcino mutants with defects in VLCFA synthesis exhibited overproliferation of cells in the vasculature or in the rib zone of shoot apices. The decrease of VLCFA content increased the expression of IPT3, a key determinant of cytokinin biosynthesis in the vasculature, and, indeed, elevated cytokinin levels. These phenotypes were suppressed in ipt3;5;7 triple mutants, and also by vasculature-specific expression of cytokinin oxidase, which degrades active forms of cytokinin. Our results imply that VLCFA synthesis in the epidermis is required to suppress cytokinin biosynthesis in the vasculature, thus fine-tuning cell division activity in internal tissue, and therefore that shoot growth is controlled by the interaction between the surface (epidermis) and the axis (vasculature) of the plant body.

The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (2iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface-exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.

Wild and weedy relatives of domesticated crops harbor genetic variants that can advance agricultural biotechnology. Here we provide a genome resource for the wild plant green millet (Setaria viridis), a model species for studies of C4 grasses, and use the resource to probe domestication genes in the close crop relative foxtail millet (Setaria italica). We produced a platinum-quality genome assembly of S. viridis and de novo assemblies for 598 wild accessions and exploited these assemblies to identify loci underlying three traits: response to climate, a 'loss of shattering' trait that permits mechanical harvest and leaf angle, a predictor of yield in many grass crops. With CRISPR-Cas9 genome editing, we validated Less Shattering1 (SvLes1) as a gene whose product controls seed shattering. In S. italica, this gene was rendered nonfunctional by a retrotransposon insertion in the domesticated loss-of-shattering allele SiLes1-TE (transposable element). This resource will enhance the utility of S. viridis for dissection of complex traits and biotechnological improvement of panicoid crops.

Retained products of conception (RPOC) with hemorrhage need intervention when RPOC persist and remain symptomatic. The safety and efficacy of uterine artery embolization (UAE) for RPOC using gelatin sponge (GS) alone, and fertility after UAE for RPOC remain unknown. The purpose of this study is to retrospectively investigate the efficacy of UAE for RPOC with bleeding and future pregnancy outcomes.

Plants use nitrate, ammonium, and organic nitrogen in the soil as nitrogen sources. Since the elevated CO2 environment predicted for the near future will reduce nitrate utilization by C3 species, ammonium is attracting great interest. However, abundant ammonium nutrition impairs growth, i.e., ammonium toxicity, the primary cause of which remains to be determined. Here, we show that ammonium assimilation by GLUTAMINE SYNTHETASE 2 (GLN2) localized in the plastid rather than ammonium accumulation is a primary cause for toxicity, which challenges the textbook knowledge. With exposure to toxic levels of ammonium, the shoot GLN2 reaction produced an abundance of protons within cells, thereby elevating shoot acidity and stimulating expression of acidic stress-responsive genes. Application of an alkaline ammonia solution to the ammonium medium efficiently alleviated the ammonium toxicity with a concomitant reduction in shoot acidity. Consequently, we conclude that a primary cause of ammonium toxicity is acidic stress.

In grafted plants, inorganic ions and plant hormones in the xylem exudate transported from the rootstock to the scion directly or indirectly affect the scion, thereby improving the traits. Therefore, the concentration of these components in the xylem exudate of grafted plants may be an indicator for rootstock selection. On the other hand, few reports have presented a comprehensive analysis of substances transferred from the rootstock to the scion in plants grafted onto different rootstocks, primarily commercial cultivars. In this study, we measured inorganic ions and plant hormones in the xylem exudate from the rootstock to the scion in various grafted plants of tomato and eggplant. The results revealed that the concentrations of inorganic ions and plant hormones in the xylem exudate significantly differed depending on the type of rootstock. In addition, we confirmed the concentration of the inorganic ions and plant hormones in the xylem exudate of plants grafted onto the same tomato rootstock cultivars as rootstock with tomato or eggplant as the scions. As a result, the concentrations of inorganic ions and plant hormones in the xylem exudate were significantly different in the grafted plants with eggplant compared with tomato as the scion. These results suggest that signals from the scion (shoot) control the inorganic ions and plant hormones transported from the rootstock (root).

Plasma membrane (PM) proton-translocating adenosine triphosphatase (H+-ATPase) is a pivotal enzyme for plant growth and development that acts as a primary transporter and is activated by phosphorylation of the penultimate residue, threonine, at the C-terminus. Small Auxin-Up RNA family proteins maintain the phosphorylation level via inhibiting dephosphorylation of the residue by protein phosphatase 2C-D clade. Photosynthetically active radiation activates PM H+-ATPase via phosphorylation in mesophyll cells of Arabidopsis thaliana, and phosphorylation of PM H+-ATPase depends on photosynthesis and photosynthesis-related sugar supplementation, such as sucrose, fructose and glucose. However, the molecular mechanism and physiological role of photosynthesis-dependent PM H+-ATPase activation are still unknown. Analysis using sugar analogs, such as palatinose, turanose and 2-deoxy glucose, revealed that sucrose metabolites and products of glycolysis such as pyruvate induce phosphorylation of PM H+-ATPase. Transcriptome analysis showed that the novel isoform of the Small Auxin-Up RNA genes, SAUR30, is upregulated in a light- and sucrose-dependent manner. Time-course analyses of sucrose supplementation showed that the phosphorylation level of PM H+-ATPase increased within 10 min, but the expression level of SAUR30 increased later than 10 min. The results suggest that two temporal regulations may participate in the regulation of PM H+-ATPase. Interestingly, a 15NO3- uptake assay in leaves showed that light increases 15NO3- uptake and that increment of 15NO3- uptake depends on PM H+-ATPase activity. The results opened the possibility of the physiological role of photosynthesis-dependent PM H+-ATPase activation in the uptake of NO3-. We speculate that PM H+-ATPase may connect photosynthesis and nitrogen metabolism in leaves.

Two species of rice have been independently domesticated from different ancestral wild species in Asia and Africa. Comparison of mutations that underlie phenotypic and physiological alterations associated with domestication traits in these species gives insights into the domestication history of rice in both regions. Asian cultivated rice, Oryza sativa, and African cultivated rice, Oryza glaberrima, have been modified and improved for common traits beneficial for humans, including erect plant architecture, nonshattering seeds, nonpigmented pericarp, and lack of awns. Independent mutations in orthologous genes associated with these traits have been documented in the two cultivated species. Contrary to this prevailing model, selection for awnlessness targeted different genes in O. sativa and O. glaberrima. We identify Regulator of Awn Elongation 3 (RAE3) a gene that encodes an E3 ubiquitin ligase and is responsible for the awnless phenotype only in O. glaberrima. A 48-bp deletion may disrupt the substrate recognition domain in RAE3 and diminish awn elongation. Sequencing analysis demonstrated low nucleotide diversity in a ~600-kb region around the derived rae3 allele on chromosome 6 in O. glaberrima compared with its wild progenitor. Identification of RAE3 sheds light on the molecular mechanism underlying awn development and provides an example of how selection on different genes can confer the same domestication phenotype in Asian and African rice.

Bispecific antibodies (bsAbs) have emerged as promising therapeutics. A bispecific diabody (bsDb) is a small bsAb consisting of two distinct chimeric single-chain components, with two possible arrangements of the domains. We previously reported the effect of domain order on the function of a humanized bsDb targeting the epidermal growth factor receptor (EGFR) on cancer cells, and CD3 on T cells. Notably, the co-localization of a T-cell receptor (TCR) with CD3 is bulky, potentially affecting the cross-linking ability of bsDbs, due to steric hindrance. Here, we constructed and evaluated humanized bsDbs, with different domain orders, targeting EGFR and CD16 on natural killer (NK) cells (hEx16-Dbs). We predicted minimal effects due to steric hindrance, as CD16 lacks accessory molecules. Interestingly, one domain arrangement displayed superior cytotoxicity in growth inhibition assays, despite similar cross-linking abilities for both domain orders tested. In hEx16-Dbs specifically, domain order might affect the agonistic activity of the anti-CD16 portion, which was supported by a cytokine production test, and likely contributed to the superiority of one of the hEx16-Dbs. Our results indicate that both the target antigen and mode of action of an antibody must be considered in the construction of highly functional bsAbs.

Conferring drought resistant traits to crops is one of the major aims of current breeding programs in response to global climate changes. We previously showed that exogenous application of acetic acid to roots of various plants could induce increased survivability under subsequent drought stress conditions, but details of the metabolism of exogenously applied acetic acid, and the nature of signals induced by its application, have not been unveiled. In this study, we show that rice rapidly induces jasmonate signaling upon application of acetic acid, resulting in physiological changes similar to those seen under drought. The major metabolite of the exogenously applied acetic acid in xylem sap was determined as glutamine-a common and abundant component of xylem sap-indicating that acetic acid is not the direct agent inducing the observed physiological responses in shoots. Expression of drought-responsive genes in shoot under subsequent drought conditions was attenuated by acetic acid treatment. These data suggest that acetic acid activates root-to-shoot jasmonate signals that partially overlap with those induced by drought, thereby conferring an acclimated state on shoots prior to subsequent drought.

Plants have a high ability to cope with changing environments and grow continuously throughout life. However, the mechanisms by which plants strike a balance between stress response and organ growth remain elusive. Here, we found that DNA double-strand breaks enhance the accumulation of cytokinin hormones through the DNA damage signaling pathway in the Arabidopsis root tip. Our data showed that activation of cytokinin signaling suppresses the expression of some of the PIN-FORMED genes that encode efflux carriers of another hormone, auxin, thereby decreasing the auxin signals in the root tip and causing cell cycle arrest at G2 phase and stem cell death. Elevated cytokinin signaling also promotes an early transition from cell division to endoreplication in the basal part of the root apex. We propose that plant hormones spatially coordinate differential DNA damage responses, thereby maintaining genome integrity and minimizing cell death to ensure continuous root growth.

Drought severely damages crop production, even under conditions so mild that the leaves show no signs of wilting. However, it is unclear how field-grown plants respond to mild drought. Here, we show through six years of field trials that ridges are a useful experimental tool to mimic mild drought stress in the field. Mild drought reduces inorganic phosphate levels in the leaves to activate the phosphate starvation response (PSR) in soybean plants in the field. Using Arabidopsis thaliana and its mutant plants grown in pots under controlled environments, we demonstrate that PSR occurs before abscisic acid response under progressive mild drought and that PSR plays a crucial role in plant growth under mild drought. Our observations in the field and laboratory using model crop and experimental plants provide insight into the molecular response to mild drought in field-grown plants and the relationship between nutrition and drought stress response.