Alleviation of norflurazon-induced photobleaching by overexpression of Fe-chelatase in transgenic rice.

We examined the effect of Bradyrhizobium japonicum FeCh (BjFeCh) expression on the regulation of porphyrin biosynthesis and resistance to norflurazon (NF)-induced photobleaching in transgenic rice. In response to NF, transgenic lines F4 and F7 showed lesser declines in chlorophyll, carotenoid, F v/F m, ϕPSII, and light-harvesting chlorophyll (Lhc) a/b-binding proteins as compared to wild-type (WT) plants, resulting in the alleviation of NF-induced photobleaching. During photobleaching, levels of heme, protoporphyrin IX (Proto IX), Mg-Proto IX (monomethylester), and protochlorophyllide decreased in WT and transgenic plants, with lesser decreases in transgenic plants. Most porphyrin biosynthetic genes were greatly downregulated in WT and transgenic plants following NF treatment, with higher transcript levels in transgenic plants. The expression of BjFeCh in transgenic rice may play a protective role in mitigating NF-induced photobleaching by maintaining levels of heme, chlorophyll intermediates, and Lhc proteins. This finding will contribute to understanding the resistance mechanism of NF-resistant crops and establishing a new strategy for weed control.

Immune Modulatory Activities of Arginyl-Fructose (AF) and AF-Enriched Natural Products in In-Vitro and In-Vivo Animal Models.

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF (p < 0.01) or GF (p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM (p < 0.01), IgG (p < 0.05), IL-2 (p < 0.01), IL-4 (p < 0.01), IL-6 (p < 0.01), and IFN-γ (p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

Natural variations at the Stay-Green gene promoter control lifespan and yield in rice cultivars.

Increased grain yield will be critical to meet the growing demand for food, and could be achieved by delaying crop senescence. Here, via quantitative trait locus (QTL) mapping, we uncover the genetic basis underlying distinct life cycles and senescence patterns of two rice subspecies, indica and japonica. Promoter variations in the Stay-Green (OsSGR) gene encoding the chlorophyll-degrading Mg++-dechelatase were found to trigger higher and earlier induction of OsSGR in indica, which accelerated senescence of indica rice cultivars. The indica-type promoter is present in a progenitor subspecies O. nivara and thus was acquired early during the evolution of rapid cycling trait in rice subspecies. Japonica OsSGR alleles introgressed into indica-type cultivars in Korean rice fields lead to delayed senescence, with increased grain yield and enhanced photosynthetic competence. Taken together, these data establish that naturally occurring OsSGR promoter and related lifespan variations can be exploited in breeding programs to augment rice yield.

Concurrent activation of OsAMT1;2 and OsGOGAT1 in rice leads to enhanced nitrogen use efficiency under nitrogen limitation.

Nitrogen (N) is a major factor for plant development and productivity. However, the application of nitrogenous fertilizers generates environmental and economic problems. To cope with the increasing global food demand, the development of rice varieties with high nitrogen use efficiency (NUE) is indispensable for reducing environmental issues and achieving sustainable agriculture. Here, we report that the concomitant activation of the rice (Oryza sativa) Ammonium transporter 1;2 (OsAMT1;2) and Glutamate synthetase 1 (OsGOGAT1) genes leads to increased tolerance to nitrogen limitation and to better ammonium uptake and N remobilization at the whole plant level. We show that the double activation of OsAMT1;2 and OsGOGAT1 increases plant performance in agriculture, providing better N grain filling without yield penalty under paddy field conditions, as well as better grain yield and N content when plants are grown under N llimitations in field conditions. Combining OsAMT1;2 and OsGOGAT1 activation provides a good breeding strategy for improving plant growth, nitrogen use efficiency and grain productivity, especially under nitrogen limitation, through the enhancement of both nitrogen uptake and assimilation.

OsASN1 Overexpression in Rice Increases Grain Protein Content and Yield under Nitrogen-Limiting Conditions.

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.

Perturbations in carotenoid and porphyrin status result in differential photooxidative stress signaling and antioxidant responses.

We examined differential photooxidative stress signaling and antioxidant responses in rice plants treated with norflurazon (NF) and oxyfluorfen (OF), which are inhibitors of carotenoid and porphyrin biosynthesis, respectively. Plants treated with OF markedly increased levels of cellular leakage and malondialdehyde, compared with NF-treated plants, showing that OF plants suffered greater oxidative damage with respect to membrane integrity. The enhanced production of H2O2 in response to OF, but not NF, indicates the important role of H2O2 in activation of photooxidative stress signaling in OF plants. In response to NF and OF, the increased levels of free salicylic acid as well as maintenance of the redox ratio of ascorbate and glutathione pools to a certain level are considered to be crucial factors in the protection against photooxidation. Plants treated with OF greatly up-regulated catalase (CAT) activity and Cat transcript levels, compared with NF-treated plants. Interestingly, NF plants showed no noticeable increase in oxidative metabolism, although they did show considerable increases in ascorbate peroxidase (APX) and peroxidase activities and transcript levels of APX, as in OF plants. Our results suggest that perturbations in carotenoid and porphyrin status by NF and OF can be sensed by differential photooxidative stress signaling, such as that involving H2O2, redox state of ascorbate and glutathione, and salicylic acid, which may be responsible for at least part of the induction of ROS-scavenging enzymes.

Perturbations in the Photosynthetic Pigment Status Result in Photooxidation-Induced Crosstalk between Carotenoid and Porphyrin Biosynthetic Pathways.

Possible crosstalk between the carotenoid and porphyrin biosynthetic pathways under photooxidative conditions was investigated by using their biosynthetic inhibitors, norflurazon (NF) and oxyfluorfen (OF). High levels of protoporphyrin IX (Proto IX) accumulated in rice plants treated with OF, whereas Proto IX decreased in plants treated with NF. Both NF and OF treatments resulted in greater decreases in MgProto IX, MgProto IX methyl ester, and protochlorophyllide. Activities and transcript levels of most porphyrin biosynthetic enzymes, particularly in the Mg-porphyrin branch, were greatly down-regulated in NF and OF plants. In contrast, the transcript levels of GSA, PPO1, and CHLD as well as FC2 and HO2 were up-regulated in NF-treated plants, while only moderate increases in FC2 and HO2 were observed in the early stage of OF treatment. Phytoene, antheraxanthin, and zeaxanthin showed high accumulation in NF-treated plants, whereas other carotenoid intermediates greatly decreased. Transcript levels of carotenoid biosynthetic genes, PSY1 and PDS, decreased in response to NF and OF, whereas plants in the later stage of NF treatment exhibited up-regulation of BCH and VDE as well as recovery of PDS. However, perturbed porphyrin biosynthesis by OF did not noticeably influence levels of carotenoid metabolites, regardless of the strong down-regulation of carotenoid biosynthetic genes. Both NF and OF plants appeared to provide enhanced protection against photooxidative damage, not only by scavenging of Mg-porphyrins, but also by up-regulating FC2, HO2, and Fe-chelatase, particularly with increased levels of zeaxanthin via up-regulation of BCH and VDE in NF plants. On the other hand, the up-regulation of GSA, PPO1, and CHLD under inhibition of carotenogenic flux may be derived from the necessity to recover impaired chloroplast biogenesis during photooxidative stress. Our study demonstrates that perturbations in carotenoid and porphyrin biosynthesis coordinate the expression of their biosynthetic genes to sustain plastid function at optimal levels by regulating their metabolic flux in plants under adverse stress conditions.

Perturbations of carotenoid and tetrapyrrole biosynthetic pathways result in differential alterations in chloroplast function and plastid signaling.

In this study, we used the biosynthetic inhibitors of carotenoid and tetrapyrrole biosynthetic pathways, norflurazon (NF) and oxyfluorfen (OF), as tools to gain insight into mechanisms of photooxidation in rice plants. NF resulted in bleaching symptom on leaves of the treated plants, whereas OF treatment developed a fast symptom of an apparent necrotic phenotype. Both plants exhibited decreases in photosynthetic efficiency, as indicated by Fv/Fm. NF caused severe disruption in thylakoid membranes, whereas OF-treated plants exhibited disruption of chloroplast envelope and plasma membrane. Levels of Lhca and Lhcb proteins in photosystem I (PSI) and PSII were reduced by photooxidative stress in NF- and OF-treated plants, with a greater decrease in NF plants. The down-regulation of nuclear-encoded photosynthesis genes Lhcb and rbcS was also found in both NF- and OF-treated plants, whereas plastid-encoded photosynthetic genes including RbcL, PsaC, and PsbD accumulated normally in NF plants but decreased drastically in OF plants. This proposes that the plastids in NF plants retain their potential to develop thylakoid membranes and that photobleaching is mainly controlled by nuclear genes. Distinct photooxidation patterns between NF- and OF-treated plants developed differential signaling, which might enable the plant to coordinate the expression of photosynthetic genes from the nuclear and plastidic genomes.

Porphyrin biosynthesis control under water stress: sustained porphyrin status correlates with drought tolerance in transgenic rice.

A controlled flow of porphyrin metabolites is critical for organisms, but little is known about the control of porphyrin biosynthesis under environmental stress. We monitored transgenic rice (Oryza sativa) plants expressing Myxococcus xanthus protoporphyrinogen oxidase (PPO) for their response to drought stress. Transgenic plants showed significantly improved drought tolerance, as indicated by a higher shoot water potential, less oxidative damage, and a more favorable redox balance compared with wild-type plants. Both transgenic and wild-type plants responded to the onset of drought stress, even prior to changes in shoot water potential and oxidative metabolism, by drastically scavenging porphyrin intermediates in leaves, which was crucial for alleviating reactive oxygen species-induced stress. Protoporphyrin IX, protochlorophyllide, magnesium-protoporphyrin IX, and its methyl ester were absent or hardly detected with the intensification of water stress (-3.1 MPa) in the wild type, whereas transgenic plants retained these intermediates to some extent. Additionally, the expression and activity of most enzymes involved in porphyrin biosynthesis, particularly in the chlorophyll branch, were primarily down-regulated under dehydrating conditions, with stronger repression in the wild type than in transgenic plants. There was up-regulation of Glutamate 1-Semialdehyde Aminotransferase, PPO1, and Fe Chelatase2 transcripts in drought-stressed transgenic plants, enabling the transgenic plants to make larger pools of 5-aminolevulinic acid and protoporphyrin IX available for subsequent steps in the heme branch. Overexpression of PPO ultimately protected the transgenic plants from drought-induced cytotoxicity, demonstrating clearly that manipulation of porphyrin biosynthesis can produce drought-tolerant plants. Our results support a possible role for tetrapyrroles in signaling their metabolic state and in plant protection under drought stress conditions.