Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage.

Flavonols are the major class of flavonoids of green Chinese cabbage (Brassica rapa subsp. pekinensis). The B. rapa genome harbors seven flavonol synthase genes (BrFLSs), but they have not been functionally characterized. Here, transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage. Among them, only BrFLS1 showed major FLS activity and additional flavanone 3ß-hydroxylase (F3H) activity, while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities. We generated BrFLS1-knockout (BrFLS1-KO) Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T1 generation, which were further advanced to the T2 generation showing normal phenotype. UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 plants, while dihydroflavonol glycosides accumulated concomitantly to levels corresponding to the reduced levels of flavonols. Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were upregulated in the BrFLS1-KO plants. In accordance, total phenolic contents were slightly enhanced in the BrFLS1-KO plants, which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage. Phenotypic surveys revealed that the BrFLS1-KO Chinese cabbages showed normal head formation and reproductive phenotypes, but subtle morphological changes in their heads were observed. In addition, their seedlings were susceptible to osmotic stress compared to the controls, suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa seedling. In this study, we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.

Cryo-EM structures of the plant anion channel SLAC1 from Arabidopsis thaliana suggest a combined activation model.

The anion channel SLAC1 functions as a crucial effector in the ABA signaling, leading to stomata closure. SLAC1 is activated by phosphorylation in its intracellular domains. Both a binding-activation model and an inhibition-release model for activation have been proposed based on only the closed structures of SLAC1, rendering the structure-based activation mechanism controversial. Here we report cryo-EM structures of Arabidopsis SLAC1 WT and its phosphomimetic mutants in open and closed states. Comparison of the open structure with the closed ones reveals the structural basis for opening of the conductance pore. Multiple phosphorylation of an intracellular domain (ICD) causes dissociation of ICD from the transmembrane domain. A conserved, positively-charged sequence motif in the intracellular loop 2 (ICL2) seems to be capable of sensing of the negatively charged phosphorylated ICD. Interactions between ICL2 and ICD drive drastic conformational changes, thereby widening the pore. From our results we propose that SLAC1 operates by a mechanism combining the binding-activation and inhibition-release models.

PROTEIN PHOSPHATASE 2C08, a Negative Regulator of Abscisic Acid Signaling, Promotes Internode Elongation in Rice.

Clade A protein phosphatase 2Cs (PP2CAs) negatively regulate abscisic acid (ABA) signaling. Here, we investigated the functions of OsPP2CAs and their crosstalk with ABA and gibberellic acid (GA) signaling pathways in rice (Oryza sativa). Among the nine OsPP2CAs, OsPP2C08 had the highest amino acid sequence similarity with OsPP2C51, which positively regulates GA signaling in rice seed germination. However, OsPP2C08 was expressed in different tissues (internodes, sheaths, and flowers) compared to OsPP2C51, which was specifically expressed in seeds, and showed much stronger induction under abiotic stress than OsPP2C51. Transgenic rice lines overexpressing OsPP2C08 (OsPP2C08-OX) had a typical ABA-insensitive phenotype in a post-germination assay, indicating that OsPP2C08, as with other OsPP2CAs, negatively regulates ABA signaling. Furthermore, OsPP2C08-OX lines had longer stems than wild-type (WT) plants due to longer internodes, especially between the second and third nodes. Internode cells were also longer in OsPP2C08-OX lines than in the WT. As GA positively regulates plant growth, these results suggest that OsPP2C08 might positively regulate GA biosynthesis. Indeed, the expression levels of GA biosynthetic genes including gibberellin 20-oxidase (OsGA20ox4) and Ent-kaurenoic acid oxidase (OsKAO) were increased in OsPP2C08-OX lines, and we observed that GIBBERELLIN 2-OXIDASE 4 (OsGA2ox4), encoding an oxidase that catalyzes the 2-beta-hydroxylation of several biologically active GAs, was repressed in the OsPP2C08-OX lines based on a transcriptome deep sequencing and RT-qPCR analysis. Furthermore, we compared the accumulation of SLENDER RICE 1 (SLR1), a DELLA protein involved in GA signaling, in OsPP2C08-OX and WT plants, and observed lower levels of SLR1 in the OsPP2C08-OX lines than in the WT. Taken together, our results reveal that OsPP2C08 negatively regulates ABA signaling and positively regulates GA signaling in rice. Our study provides valuable insight into the molecular mechanisms underlying the crosstalk between GA and ABA signaling in rice.

Food web dynamics in the mangrove ecosystem of the Pearl River Estuary surrounded by megacities.

Global recognition that mangroves support coastal ecosystem services has increased; however, studies on trophic dynamics in mangrove ecosystems remain limited. We seasonally analysed the δ13C and δ15N of 34 consumers and 5 diets to elucidate the food web dynamics in the Pearl River Estuary (PRE). Fish had a large niche space during the monsoon summer, reflecting increased trophic roles. In contrast, the small niche space of benthos over seasons reflected consistent trophic positions. Consumers mainly utilized plant-derived organic matters in the dry season and particulate organic matters in the wet season. The present study with literature reviews revealed characteristics of the PRE food web with the depleted δ13C and enriched δ15N, indicating a high contribution of mangrove-derived organic carbon and sewage input, particularly in the wet season. Overall, this study confirmed the seasonal and spatial trophic dynamics in mangrove forests surrounding megacities for future sustainable mangrove ecosystem management.

A rice seed-specific glycine-rich protein OsDOR1 interacts with GID1 to repress GA signaling and regulates seed dormancy.

Seed dormancy is an important agronomic trait under the control of complex genetic and environmental interactions, which have not been yet comprehensively understood. From the field screening of rice mutant library generated by a Ds transposable element, we identified a pre-harvest sprouting (PHS) mutant dor1. This mutant has a single insertion of Ds element at the second exon of OsDOR1 (LOC_Os03g20770), which encodes a novel seed-specific glycine-rich protein. This gene successfully complemented the PHS phenotype of dor1 mutant and its ectopic expression enhanced seed dormancy. Here, we demonstrated that OsDOR1 protein binds to the GA receptor protein, OsGID1 in rice protoplasts, and interrupts with the formation OsGID1-OsSLR1 complex in yeast cells. Co-expression of OsDOR1 with OsGID1 in rice protoplasts attenuated the GA-dependent degradation of OsSLR1, the key repressor of GA signaling. We showed the endogenous OsSLR1 protein level in the dor1 mutant seeds is significantly lower than that of wild type. The dor1 mutant featured a hypersensitive GA-response of α-amylase gene expression during seed germination. Based on these findings, we suggest that OsDOR1 is a novel negative player of GA signaling operated in the maintenance of seed dormancy. Our findings provide a novel source of PHS resistance.

10 years long-term assessment on characterizing spatiotemporal trend and source apportionment of metal(loid)s in terrestrial soils along the west coast of South Korea.

Long-term trends in the spatial distributions and sources of metal(loid)s in soils adjacent to the west coastal areas of South Korea have been systematically investigated for 10 years (2010-2019). Monitoring in 17 sites clearly showed site- and region-specific distributions, being associated with land use type (significant differences, as road > agriculture > wild) (P < 0.05), rather than temporal variation. The great concentrations of all metal(loid)s were found near Lake Shihwa (LS) and Geum River (GG), near the road, indicating that transportation activity was the main source of metal(loid)s contamination in soil. Especially, Cd (0.5 mg kg-1), Hg (0.04 mg kg-1), Pb (65 mg kg-1), and Zn (184 mg kg-1), related to the transportation activity near the road, showed twice greater than other land use types, on average. The concentration of metal(loid)s in each site and with the same land use type did not greatly vary over the years, with no significant annual difference (P > 0.05). The degree of metal(loid)s contamination compared to the background levels was identified in the order of Pb > Zn > Cr > Cu > As>Cd > Ni > Hg, with the contaminated hotspots mostly in LS or GG. The potential ecological risk was evidenced for Cd and Hg, but such a trend was temporally irregular over the years, indicating site-specificity. The sources of metal(loid)s were carefully determined as natural (20%), fuel combustion & agricultural pollution (43%), and vehicular emissions (37%) using the Positive Matrix Factorization model. The relative contribution of each source to contamination over the last decade was found to be similar, supporting that site-dependent lesser variation in metal(loid)s contamination in the coastal areas of South Korea. Overall, the distribution of metal(loid)s in the soil near the west coastal areas over the last decade largely depended on land use activities, and contamination degree was associated with non-point sources, such as transportation and fuel combustion.

The first national scale evaluation of total nitrogen stocks and burial rates of intertidal sediments along the entire coast of South Korea.

The regulating ecosystem services, such as water purification, that tidal flats provide by nitrogen (N) burial are being increasingly recognized; yet, quantitative estimates remain limited. Here, we first present nationwide evaluation of total N stocks and burial rates in the Korean tidal flats, based on a 3 year long monitoring assessment combined with remote sensing approach. A total of 20 intertidal flats representing 7 provinces of South Korea were extensively surveyed in 2018-20. The sediment textural type (sand, mixed, and mud) classified from remotely sensed imagery was significantly correlated to that identified from field data (p < 0.01), warranting a nationwide estimate of total N stocks. The estimated total N stocks and burial rates in the tidal flats of Korea were 1.5 Tg N and 8,192 Mg N yr-1, respectively. Total N stocks significantly varied by region, province, morphology, salinity, and land use type adjacent to the corresponding tidal flats. In general, the N stocks of tidal flats were influenced by the degree of terrestrial N inputs to the ocean. The N stocks were significantly correlated with several physical parameters, such as precipitation (p < 0.05) and tide (p < 0.01). Among the sediment properties, the mud content was found to be the key factor determining the total N stocks across the 20 intertidal flats (p < 0.01). Finally, by applying the environmental value for N removal (USD km-2 yr-1) to the tidal flat area (km2), the economic value of the total N removal was estimated as ~233 Million USD yr-1 in Korea and ~15 Billion USD yr-1 globally. Overall, the present work confirms the valuable ecosystem service of tidal flat's cost-efficient N removal capacity, highlighting marine ecosystem service.

Reconstitution of the Core ABA Signaling in Protoplasts: Transcriptional Activators.

Transient expression of genes in protoplasts has been used widely for purposes ranging from subcellular localization to promoter activity analyses. Here, we describe methods for reconstituting the abscisic acid (ABA) signaling pathway using a transient expression system in rice protoplasts. ABA signaling is monitored via reporter systems consisting of synthetic promoters and luciferase. Thus, the effects of each signaling component as well as complexes involved in ABA signaling can be characterized in rice protoplasts, overcoming many of the limitations that hamper efforts to identify biological functions of effector genes in whole plants. This protoplast-based transient assay system for ABA signaling thus provides valuable tools and knowledge for understanding complicated ABA signaling networks.

Functional Characterization of BrF3'H, Which Determines the Typical Flavonoid Profile of Purple Chinese Cabbage.

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3'-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3'-hydroxylase BrF3'H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3'H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3'H protein catalyzes the 3'-hydroxylation of a wide range of 4'-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3'H among those tested. Transient expression of BrF3'H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3'H in planta. As the first functional characterization of BrF3'H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.

Modulation of Rice Leaf Angle and Grain Size by Expressing OsBCL1 and OsBCL2 under the Control of OsBUL1 Promoter.

Leaf angle and grain size are important agronomic traits affecting rice productivity directly and/or indirectly through modulating crop architecture. OsBC1, as a typical bHLH transcription factor, is one of the components comprising a complex formed with LO9-177 and OsBUL1 contributing to modulation of rice leaf inclination and grain size. In the current study, two homologues of OsBC1, OsBCL1 and OsBCL2 were functionally characterized by expressing them under the control of OsBUL1 promoter, which is preferentially expressed in the lamina joint and the spikelet of rice. Increased leaf angle and grain length with elongated cells in the lamina joint and the grain hull were observed in transgenic rice containing much greater gibberellin A3 (GA3) levels than WT, demonstrating that both OsBCL1 and OsBCL2 are positive regulators of cell elongation at least partially through increased GA biosynthesis. Moreover, the cell elongation was likely due to cell expansion rather than cell division based on the related gene expression and, the cell elongation-promoting activities of OsBCL1 and OsBCL2 were functional in a dicot species, Arabidopsis.

Spatiotemporal variation of extracellular polymeric substances (EPS) associated with the microphytobenthos of tidal flats in the Yellow Sea.

The physical functions of extracellular polymeric substances (EPS), viz., by-product of microphytobenthos (MPB), in tidal flat system are well documented, but some ecological aspects remain unknown. We investigated MPB biomass (Chl-a), EPS, diatom assemblage, and erodibility in two contrasting tidal flat environments (megatidal vs. macrotidal flat) in the Yellow Sea. Thick biofilms were observed when MPB bloomed, with high Chl-a and increased EPS concentrations. Among diatom genera, Navicula was the most dominant taxa found over the year (mean 41%) in both areas. Compared with non-bloom periods, the erodibility decreased by 54-73% as biofilm thickened during the blooms. It was attributed to the elevated abundance of large-sized (>40 µm) Navicula, which was expected to secrete large amounts of EPS. Overall, we successfully demonstrated spatiotemporal differences of sediment stabilization that significantly related to ecological variations of MPB, and identified the key diatom genus as a "sediment stabilizer" in the typical tidal flats of the Yellow Sea.

The OsERF115/AP2EREBP110 Transcription Factor Is Involved in the Multiple Stress Tolerance to Heat and Drought in Rice Plants.

The AP2/EREBP family transcription factors play important roles in a wide range of stress tolerance and hormone signaling. In this study, a heat-inducible rice ERF gene was isolated and functionally characterized. The OsERF115/AP2EREBP110 was categorized to Group-IIIc of the rice AP2/EREBP family and strongly induced by heat and drought treatment. The OsERF115/AP2EREBP110 protein targeted to nuclei and suppressed the ABA-induced transcriptional activation of Rab16A promoter in rice protoplasts. Overexpression of OsERF115/AP2EREBP110 enhanced thermotolerance of seeds and vegetative growth stage plants. The OsERF115/AP2EREBP110 overexpressing (OE) plants exhibited higher proline level and increased expression of a proline biosynthesis P5CS1 gene. Phenotyping of water use dynamics of the individual plant indicates that the OsERF115/AP2EREBP110-OE plant exhibited better water saving traits under heat and drought combined stress. Our combined results suggest the potential use of OsERF115/AP2EREBP110 as a candidate gene for genetic engineering approaches to develop heat and drought stress-tolerant crops.

The first national scale evaluation of organic carbon stocks and sequestration rates of coastal sediments along the West Sea, South Sea, and East Sea of South Korea.

Blue carbon science requires the estimates of organic carbon stock and sequestration rate; however, holistic data analysis remains limited in South Korea. The present study reports current organic carbon stocks and sequestration rates in the coastal areas of West Sea, South Sea, and East Sea of South Korea, encompassing entire intertidal areas using long-term field survey combined with remote sensing technology. Twenty-one intertidal flats were targeted across seven provinces (Gyeonggi, Chungnam, Jeonbuk, Jeonnam, Gyeongnam, Gyeongbuk, Gangwon). Out of the evaluated environmental parameters, mud content represented a significant factor controlling sedimentary organic carbon stocks across target areas, and was significantly positively correlated to the total organic carbon (p < 0.05). Organic carbon stocks measured in salt marshes (i.e., upper intertidal zone) reflected the high carbon fixation capacity of halophytes through primary production. Sediment textural type was classified using analysis of remotely sensed imagery, and was closely correlated to field-based classification data (p < 0.05). Using field and remote sensing results, we estimated total organic carbon stocks (13,142,149 Mg C) and sequestration rates (71,383 Mg C yr-1) in the tidal flats of South Korea. This study presents the first report on blue carbon potential in the Korean tidal flats, providing baseline information on the carbon dynamics of intertidal sediments in this region and, potentially, elsewhere.

Large-scale sediment toxicity assessment over the 15,000 km of coastline in the Yellow and Bohai seas, East Asia.

The Yellow and Bohai seas have long been contaminated by persistent toxic substances (PTSs) from numerous (un)known anthropogenic sources. In this study, we used Vibrio fischeri bioassay to evaluate ecotoxicological profiles associated with sedimentary PTSs contamination at a large marine ecosystem (LME) scale. A total of 125 surface sediments collected from the coastal areas of the Yellow and Bohai seas were analyzed both for aqueous and organic extracts. Not surprisingly, the results indicated site-dependent toxicities, but most sites were identified as non-toxic to V. fischeri. For aqueous extracts and organic extracts, 13% and 8% of samples, respectively exhibited marginal toxicity, while 0% and 2% of samples exhibited moderate toxicity. However, it should be noted that organic extracts (mean TU = 56) induced stronger toxicities than aqueous samples (mean TU = 0.4). This result generally back-supported the high toxicity potentials associated with sedimentary sink of organic pollutants. Several PTSs measured in the samples indicated a significant contribution to the observed V. fischeri toxicities. Of note, polycyclic aromatic hydrocarbons (PAHs; r = 0.28, p < 0.05), styrene oligomers (r = 0.41, p < 0.01), and alkylphenols (r = 0.38, p < 0.05) showed significant associations to the observed bacterial inhibition. Among PAHs, benzo[a]anthracene and phenanthrene exhibited a significant contribution to the observed V. fischeri toxicities. Meantime, salinity which reflects the distance from the point sources of land-driven pollutants along the rivers and estuaries in the Yellow and Bohai seas was a key environmental variable representing the sample toxicities. Overall, the present study provides baseline information for evaluating the potential sediment toxicity to implement responsible coastal management at an LME scale, and elsewhere.

Reconstitution of Cytokinin Signaling in Rice Protoplasts.

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

OsPP2C09 Is a Bifunctional Regulator in Both ABA-Dependent and Independent Abiotic Stress Signaling Pathways.

Clade A Type 2C protein phosphatases (PP2CAs) negatively regulate abscisic acid (ABA) signaling and have diverse functions in plant development and in response to various stresses. In this study, we showed that overexpression of the rice ABA receptor OsPYL/RCAR3 reduces the growth retardation observed in plants exposed to osmotic stress. By contrast, overexpression of the OsPYL/RCAR3-interacting protein OsPP2C09 rendered plant growth more sensitive to osmotic stress. We tested whether OsPP2CAs activate an ABA-independent signaling cascade by transfecting rice protoplasts with luciferase reporters containing the drought-responsive element (DRE) or ABA-responsive element (ABRE). We observed that OsPP2CAs activated gene expression via the cis-acting drought-responsive element. In agreement with this observation, transcriptome analysis of plants overexpressing OsPP2C09 indicated that OsPP2C09 induces the expression of genes whose promoters contain DREs. Further analysis showed that OsPP2C09 interacts with DRE-binding (DREB) transcription factors and activates reporters containing DRE. We conclude that, through activating DRE-containing promoters, OsPP2C09 positively regulates the drought response regulon and activates an ABA-independent signaling pathway.

The Rice Abscisic Acid-Responsive RING Finger E3 Ligase OsRF1 Targets OsPP2C09 for Degradation and Confers Drought and Salinity Tolerance in Rice.

Drought and salinity are major important factors that restrain growth and productivity of rice. In plants, many really interesting new gene (RING) finger proteins have been reported to enhance drought and salt tolerance. However, their mode of action and interacting substrates are largely unknown. Here, we identified a new small RING-H2 type E3 ligase OsRF1, which is involved in the ABA and stress responses of rice. OsRF1 transcripts were highly induced by ABA, salt, or drought treatment. Upregulation of OsRF1 in transgenic rice conferred drought and salt tolerance and increased endogenous ABA levels. Consistent with this, faster transcriptional activation of key ABA biosynthetic genes, ZEP, NCED3, and ABA4, was observed in OsRF1-OE plants compared with wild type in response to drought stress. Yeast two-hybrid assay, BiFC, and co-immunoprecipitation analysis identified clade A PP2C proteins as direct interacting partners with OsRF1. In vitro ubiquitination assay indicated that OsRF1 exhibited E3 ligase activity, and that it targeted OsPP2C09 protein for ubiquitination and degradation. Cell-free degradation assay further showed that the OsPP2C09 protein is more rapidly degraded by ABA in the OsRF1-OE rice than in the wild type. The combined results suggested that OsRF1 is a positive player of stress responses by modulating protein stability of clade A PP2C proteins, negative regulators of ABA signaling.

Stable isotope signatures reveal the significant contributions of microphytobenthos and saltmarsh-driven nutrition in the intertidal benthic food webs.

A tidal saltmarsh supporting marine ecosystem services becomes more recognized, but an understanding of its trophic dynamics is far limited. We investigated the benthic food-web dynamics in Ganghwa tidal flat, a representative tidal saltmarsh of South Korea. The stable carbon and nitrogen isotope ratio values were analyzed for the sixteen species of macrobenthos and their potential food sources such as microphytobenthos (MPB) and various organic matters. A year-round survey was conducted in the three sites at Ganghwa showing the different types of sediment bottom (sandy mud or mud) and/or vegetation (Phragmites or Suaeda). In general, the isotopic signature fairly well demonstrated that trophic structure is primarily influenced by sediment mud content and/or dominated vegetation. Among the four feeding types of consumers (filter feeder; deposit feeder; omnivore; carnivore), the deposit feeders such as mollusk, arthropod, and annelid showed increased dietary dependency on MPB at bare sites. Whilst they actively utilized 13C-depleted organic matters at vegetated sites. Meantime, significantly enriched stable nitrogen isotopic ratio of macrobenthos was evidenced in fall, which reflected the seasonal variation of food sources and physiological processes in survival strategy. Overall, the stable isotopic approach was useful to address the key factors (in)directly influencing the benthic food web structure and its functioning in a typical tidal saltmarsh of the Yellow Sea.

Comparison of MALDI-TOF-MS and RP-HPLC as Rapid Screening Methods for Wheat Lines With Altered Gliadin Compositions.

The wheat gliadins are a complex group of flour proteins that can trigger celiac disease and serious food allergies. As a result, mutation breeding and biotechnology approaches are being used to develop new wheat lines with reduced immunogenic potential. Key to these efforts is the development of rapid, high-throughput methods that can be used as a first step in selecting lines with altered gliadin contents. In this paper, we optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC) methods for the separation of gliadins from Triticum aestivum cv. Chinese Spring (CS). We evaluated the quality of the resulting profiles using the complete set of gliadin gene sequences recently obtained from this cultivar as well as a set of aneuploid lines in CS. The gliadins were resolved into 13 peaks by MALDI-TOF-MS. α- or γ-gliadins that contain abundant celiac disease epitopes and are likely targets for efforts to reduce the immunogenicity of flour were found in several peaks. However, other peaks contained multiple α- and γ-gliadins, including one peak with as many as 12 different gliadins. In comparison, separation of proteins by RP-HPLC yielded 28 gliadin peaks, including 13 peaks containing α-gliadins and eight peaks containing γ-gliadins. While the separation of α- and γ-gliadins gliadins achieved by RP-HPLC was better than that achieved by MALDI-TOF-MS, it was not possible to link peaks with individual protein sequences. Both MALDI-TOF-MS and RP-HPLC provided adequate separation of ω-gliadins. While MALDI-TOF-MS is faster and could prove useful in studies that target specific gliadins, RP-HPLC is an effective method that can be applied more broadly to detect changes in gliadin composition.

Ectopic Expression of OsPYL/RCAR7, an ABA Receptor Having Low Signaling Activity, Improves Drought Tolerance without Growth Defects in Rice.

Overexpression of abscisic acid (ABA) receptors has been reported to enhance drought tolerance, but also to cause stunted growth and decreased crop yield. Here, we constructed transgenic rice for all monomeric ABA receptors and observed that only transgenic rice over-expressing OsPYL/RCAR7 showed similar phenotype with wild type, without total yield loss when grown under normal growth condition in a paddy field. Even though transgenic rice over-expressing OsPYL/RCAR7 showed neither an ABA-sensitivity nor an osmotic stress tolerance in plate assay, it showed drought tolerance. We investigated the ABA-dependent interaction with OsPP2CAs and ABA signaling induction by OsPYL/RCAR7. In yeast two hybrid assay, OsPYL/RCAR7 required critically higher ABA concentrations to interact with OsPP2CAs than other ABA receptors, and co-immunoprecipitation assay showed strong interaction under ABA treatment. When ABA-responsive signaling activity was monitored using a transient expression system in rice protoplasts, OsPYL/RCAR7 had the lowest ABA-responsive signaling activity as compared with other ABA receptors. OsPYL/RCAR7 also showed weak suppression of phosphatase activity as compared with other ABA receptors in vitro. Transcriptome analysis of transgenic rice over-expressing OsPYL/RCAR7 suggested that only a few genes were induced similar to control under without exogenous ABA, but a large number of genes was induced under ABA treatment compared with control. We conclude that OsPYL/RCAR7 is a novel functional ABA receptor that has low ABA signaling activity and exhibits high ABA dependence. These results lay the foundation for a new strategy to improve drought stress tolerance without compromising crop growth.