Chalkiness and premature controlled by energy homeostasis in OsNAC02 Ko-mutant during vegetative endosperm development.

BACKGROUND: Chalkiness is a common phenotype induced by various reasons, such as abiotic stress or the imbalance of starch synthesis and metabolism during the development period. However, the reason mainly for one gene losing its function such as NAC (TFs has a large family in rice) which may cause premature is rarely known to us. RESULTS: The Ko-Osnac02 mutant demonstrated an obviously early maturation stage compared to the wild type (WT) with 15 days earlier. The result showed that the mature endosperm of Ko-Osnac02 mutant exhibited chalkiness, characterized by white-core and white-belly in mature endosperm. As grain filling rate is a crucial factor in determining the yield and quality of rice (Oryza sativa, ssp. japonica), it's significant that mutant has a lower amylose content (AC) and higher soluble sugar content in the mature endosperm. Interestingly among the top DEGs in the RNA sequencing of N2 (3DAP) and WT seeds revealed that the OsBAM2 (LOC_Os10g32810) expressed significantly high in N2 mutant, which involved in Maltose up-regulated by the starch degradation. As Prediction of Protein interaction showed in the chalky endosperm formation in N2 seeds (3 DAP), seven genes were expressed at a lower-level which should be verified by a heatmap diagrams based on DEGs of N2 versus WT. The Tubulin genes controlling cell cycle are downregulated together with the MCM family genes MCM4 ( ↓), MCM7 ( ↑), which may cause white-core in the early endosperm development. In conclusion, the developing period drastically decreased in the Ko-Osnac02 mutants, which might cause the chalkiness in seeds during the early endosperm development. CONCLUSIONS: The gene OsNAC02 which controls a great genetic co-network for cell cycle regulation in early development, and KO-Osnac02 mutant shows prematurity and white-core in endosperm.

Multi-omic insights into the formation and evolution of a novel shell microstructure in oysters.

BACKGROUND: Molluscan shell, composed of a diverse range of architectures and microstructures, is a classic model system to study the relationships between molecular evolution and biomineralized structure formation. The shells of oysters differ from those of other molluscs by possessing a novel microstructure, chalky calcite, which facilitates adaptation to the sessile lifestyle. However, the genetic basis and evolutionary origin of this adaptive innovation remain largely unexplored. RESULTS: We report the first whole-genome assembly and shell proteomes of the Iwagaki oyster Crassostrea nippona. Multi-omic integrative analyses revealed that independently expanded and co-opted tyrosinase, peroxidase, TIMP genes may contribute to the chalky layer formation in oysters. Comparisons with other molluscan shell proteomes imply that von Willebrand factor type A and chitin-binding domains are basic members of molluscan biomineralization toolkit. Genome-wide identification and analyses of these two domains in 19 metazoans enabled us to propose that the well-known Pif may share a common origin in the last common ancestor of Bilateria. Furthermore, Pif and LamG3 genes acquire new genetic function for shell mineralization in bivalves and the chalky calcite formation in oysters likely through a combination of gene duplication and domain reorganization. CONCLUSIONS: The spatial expression of SMP genes in the mantle and molecular evolution of Pif are potentially involved in regulation of the chalky calcite deposition, thereby shaping the high plasticity of the oyster shell to adapt to a sessile lifestyle. This study further highlights neo-functionalization as a crucial mechanism for the diversification of shell mineralization and microstructures in molluscs, which may be applied more widely for studies on the evolution of metazoan biomineralization.

Proteomic Analyses Discern the Developmental Inclusion of Albumin in Pig Enamel: A New Model for Human Enamel Hypomineralization.

Excess albumin in enamel is a characteristic of the prevalent developmental dental defect known as chalky teeth or molar hypomineralization (MH). This study uses proteomic analyses of pig teeth to discern between developmental origin and post-eruptive contamination and to assess the similarity to hypomineralized human enamel. Here, the objective is to address the urgent need for an animal model to uncover the etiology of MH and to improve treatment. Porcine enamel is chalky and soft at eruption; yet, it hardens quickly to form a hard surface and then resembles human teeth with demarcated enamel opacities. Proteomic analyses of enamel from erupted teeth, serum, and saliva from pigs aged 4 (n = 3) and 8 weeks (n = 2) and human (n = 4) molars with demarcated enamel opacities show alpha-fetoprotein (AFP). AFP expression is limited to pre- and perinatal development and its presence in enamel indicates pre- or perinatal inclusion. In contrast, albumin is expressed after birth, indicating postnatal inclusion into enamel. Peptides were extracted from enamel and analyzed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) after tryptic digestion. The mean total protein number was 337 in the enamel of all teeth with 13 different unique tryptic peptides of porcine AFP in all enamel samples but none in saliva samples. Similarities in the composition, micro-hardness, and microstructure underscore the usefulness of the porcine model to uncover the MH etiology, cellular mechanisms of albumin inclusion, and treatment for demarcated opacities.

Artificial intelligence-based diagnostics of molar-incisor-hypomineralization (MIH) on intraoral photographs.

OBJECTIVE: The aim of this study was to develop and validate a deep learning-based convolutional neural network (CNN) for the automated detection and categorization of teeth affected by molar-incisor-hypomineralization (MIH) on intraoral photographs. MATERIALS AND METHODS: The data set consisted of 3241 intraoral images (767 teeth with no MIH/no intervention, 76 with no MIH/atypical restoration, 742 with no MIH/sealant, 815 with demarcated opacity/no intervention, 158 with demarcated opacity/atypical restoration, 181 with demarcated opacity/sealant, 290 with enamel breakdown/no intervention, 169 with enamel breakdown/atypical restoration, and 43 with enamel breakdown/sealant). These images were divided into a training (N = 2596) and a test sample (N = 649). All images were evaluated by an expert group, and each diagnosis served as a reference standard for cyclic training and evaluation of the CNN (ResNeXt-101-32 × 8d). Statistical analysis included the calculation of contingency tables, areas under the receiver operating characteristic curve (AUCs) and saliency maps. RESULTS: The developed CNN was able to categorize teeth with MIH correctly with an overall diagnostic accuracy of 95.2%. The overall SE and SP amounted to 78.6% and 97.3%, respectively, which indicate that the CNN performed better in healthy teeth compared to those with MIH. The AUC values ranging from 0.873 (enamel breakdown/sealant) to 0.994 (atypical restoration/no MIH). CONCLUSION: It was possible to categorize the majority of clinical photographs automatically by using a trained deep learning-based CNN with an acceptably high diagnostic accuracy. CLINICAL RELEVANCE: Artificial intelligence-based dental diagnostics may support dental diagnostics in the future regardless of the need to improve accuracy.

FLOURY ENDOSPERM11-2 encodes plastid HSP70-2 involved with the temperature-dependent chalkiness of rice (Oryza sativa L.) grains.

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature-sensitive floury endosperm11-2 (flo11-2) mutant was isolated from ion beam-irradiated rice of 1116 lines. The flo11-2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole-exome sequencing of the flo11-2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid-localized 70-kDa heat shock protein 2 (cpHSP70-2). The cpHSP70-2 of the flo11-2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11-2 mutants that express the wild-type cpHSP70-2 showed significantly lower chalkiness than the flo11-2 mutant. Moreover, the accumulation level of cpHSP70-2 was negatively correlated with the chalky ratio, indicating that cpHSP70-2 is a causal gene for the chalkiness of the flo11-2 mutant. The intrinsic ATPase activity of recombinant cpHSP70-2 was lower by 23% at Vmax for the flo11-2 mutant than for the wild type. The growth of DnaK-defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild-type DnaK. The results indicate that the lowered cpHSP70-2 function is involved with the chalkiness of the flo11-2 mutant.

Characterization of the chalky layer-derived EGF-like domain-containing protein (CgELC) in the pacific oyster, Crassostrea gigas.

The shells of the Pacific oyster Crassostrea gigas contain calcite crystals with three types of microstructures: prismatic, chalky, and foliated layers. Many shell matrix proteins were annotated from the shells of C. gigas; however, it is unclear which SMPs play important roles in their shell mineralization. The matrix proteins have never been reported from the chalky layer. In this study, we identified a chalky layer-derived EGF-like domain-containing protein (CgELC) from the chalky layer of C. gigas shells. The gene sequence of the CgELC was encoded under CGI_ 10,017,544 of the C. gigas genome database. Only peptide fragments in the N-terminal region of CGI_ 10,017,544 were detected by LC-MS/MS analyses, suggesting that CGI_ 10,017,544 was digested at the predicted protease digestion dibasic site by post-translational modification to become a mature CgELC protein. We produced three types of CgELC recombinant proteins, namely, the full length CgELC, as well as the N-terminal and C-terminal parts of CgELC (CgELC-N or -C, respectively), for in vitro crystallization experiments. In the presence of these recombinant proteins, the aggregation of polycrystalline calcite was observed. Some fibrous organic components seemed to be incorporated into the calcite crystals in the presence of the r-CgELC protein. We also noted different features in the crystallization between CgELC-N and CgELC-C; some crystals were inhibited crystal plane formation and contained many columnar prisms inside the crystals (CgELC-N) and formed numerous holes on their surfaces (CgELC-C). These results suggest that CgELC is involved in crystal aggregation and incorporated into calcite crystals.

GW2 Functions as an E3 Ubiquitin Ligase for Rice Expansin-Like 1.

Seed size is one of the most important traits determining the yield of cereal crops. Many studies have been performed to uncover the mechanism of seed development. However, much remains to be understood, especially at the molecular level, although several genes involved in seed size have been identified. Here, we show that rice Grain Width 2 (GW2), a RING-type E3 ubiquitin ligase, can control seed development by catalyzing the ubiquitination of expansin-like 1 (EXPLA1), a cell wall-loosening protein that increases cell growth. Microscopic examination revealed that a GW2 mutant had a chalky endosperm due to the presence of loosely packed, spherical starch granules, although the grain shape was normal. Yeast two-hybrid and in vitro pull-down assays showed a strong interaction between GW2 and EXPLA1. In vitro ubiquitination analysis demonstrated that EXPLA1 was ubiquitinated by GW2 at lysine 279 (K279). GW2 and EXPLA1 colocalized to the nucleus when expressed simultaneously. These results suggest that GW2 negatively regulates seed size by targeting EXPLA1 for degradation through its E3 ubiquitin ligase activity.

Rice grain yield and quality responses to free-air CO2 enrichment combined with soil and water warming.

Rising air temperatures are projected to reduce rice yield and quality, whereas increasing atmospheric CO2 concentrations ([CO2 ]) can increase grain yield. For irrigated rice, ponded water is an important temperature environment, but few open-field evaluations are available on the combined effects of temperature and [CO2 ], which limits our ability to predict future rice production. We conducted free-air CO2 enrichment and soil and water warming experiments, for three growing seasons to determine the yield and quality response to elevated [CO2 ] (+200 µmol mol(-1) , E-[CO2 ]) and soil and water temperatures (+2 °C, E-T). E-[CO2 ] significantly increased biomass and grain yield by approximately 14% averaged over 3 years, mainly because of increased panicle and spikelet density. E-T significantly increased biomass but had no significant effect on the grain yield. E-T decreased days from transplanting to heading by approximately 1%, but days to the maximum tiller number (MTN) stage were reduced by approximately 8%, which limited the panicle density and therefore sink capacity. On the other hand, E-[CO2 ] increased days to the MTN stage by approximately 4%, leading to a greater number of tillers. Grain appearance quality was decreased by both treatments, but E-[CO2 ] showed a much larger effect than did E-T. The significant decrease in undamaged grains (UDG) by E-[CO2 ] was mainly the result of an increased percentage of white-base grains (WBSG), which were negatively correlated with grain protein content. A significant decrease in grain protein content by E-[CO2 ] accounted in part for the increased WBSG. The dependence of WBSG on grain protein content, however, was different among years; the slope and intercept of the relationship were positively correlated with a heat dose above 26 °C. Year-to-year variation in the response of grain appearance quality demonstrated that E-[CO2 ] and rising air temperatures synergistically reduce grain appearance quality of rice.

What Is Chalky? Investigating Consumer Language and Perception of Fine Particles in Beverages Containing Pea and Potato Starch.

Despite its importance as an undesirable food texture, the phenomenon of chalkiness remains understudied. Chalky sensations presumably arise from fine particulates found in foods, but semantic overlap with other common descriptors of small particles, like gritty or sandy, is unclear. Here, we compare the usage of Chalky with related descriptors, and determine the effect of particle size, concentration, and xanthan content on Chalky ratings in a model beverage. A 23 factorial design with starch particle size (D90 = 33.8 and 64.6 µm), starch concentrations (10 and 20% w/v), and xanthan content (0.075 and 0.15% w/v) was used. Participants' salivary flow rate was also assessed. A multi-sip taste test was performed where naïve consumers (n = 82; 39% men, 60% women; age range = 18-79 years) rated the intensity of Chalky, Powdery, Gritty, Sandy, Mouthdrying, and Residual mouthcoating at 0, 30, and 60 s after each of three consecutive sips. All attribute ratings were highly correlated, with Chalky, Powdery, and Residual Mouthcoating being more closely correlated with each other than Gritty or Sandy. Although Chalky was still reported 60 s after consumption, no evidence of build-up was found with repeated sips. A larger size and higher concentration increased Chalky ratings, with the low-salivary-flow group reporting greater ratings for Chalky relative to the high-flow group. Our results suggest consumer percepts of small particles are overlapping but not entirely redundant. This suggests researchers and product developers should carefully distinguish between these descriptors when trying to understand consumer perception of food products containing fine particles.

A dataset for analyzing the climate change response of grain quality of 48 Japanese rice cultivars with contrasting levels of heat tolerance.

Climate change has a significant impact on rice grain appearance quality; in particular, high temperatures during the grain filling period increase the rate of chalky immature grains, reducing the marketability of rice. Heat-tolerant cultivars have been bred and released to reduce the rate of chalky grain and improve rice quality under high temperatures, but the ability of these cultivars to actually reduce chalky grain content has never been demonstrated due to the lack of integrated datasets. Here, we present a dataset collected through a systematic literature search from publicly available data sources, for the quantitative analysis of the impact of meteorological factors on grain appearance quality of various rice cultivars with contrasted heat tolerance levels. The dataset contains 1302 field observations of chalky grain rates (%) - a critical trait affecting grain appearance sensitive to temperature shocks - for 48 cultivars covering five different heat-tolerant ranks (HTRs) collected at 44 sites across Japan. The dataset also includes the values of key meteorological variables during the grain filling period, such as the cumulative mean air temperature above the threshold temperature (TaHD), mean solar radiation, and mean relative humidity over 20 days after heading, obtained from a gridded daily meteorological dataset with a 1-km resolution developed by the National Agriculture and Food Research Organization. The dataset covers major commercial rice cultivars cultivated in Japan in different environmental conditions. It is a useful resource for analyzing the climate change impact on crop quality and assess the effectiveness of genetic improvements in heat tolerance. Its value has been illustrated in the research article entitled "Effectiveness of heat tolerance rice cultivars in preserving grain appearance quality under high temperatures - A meta-analysis", where the dataset was used to develop a statistical model quantifying the effects of high temperature on grain quality as a function of cultivar heat tolerance.

Morphological, Molecular Structural and Physicochemical Characterization of Starch Granules Formed in Endosperm of Rice with Ectopic Overexpression of α-Amylase.

The objective of this study was to characterize the endosperm starch in rice that ectopically overexpressed the α-amylase. Transgenic rice plants, transformed with cauliflower mosaic virus 35S promoter driven AmyI-1 (35S::AmyI-1) and AmyII-4 (35S::AmyII-4), and 10 kDa prolamin promoter driven AmyI-1 (P10::AmyI-1), were cultivated under standard conditions (23 °C, 12 h in the dark/ 26 °C, 12 h in the light), and brown grains were subsequently harvested. Each grain displayed characteristic chalkiness, while electron microanalyzer (EPMA)-SEM images disclosed numerous small pits on the surface of the starch granules, attributable to α-amylase activity. Fluorescence labeling and capillary electrophoresis analysis of starch chain length distribution revealed no significant alterations in the starches of 35S::AmyI-1 and 35S::AmyII-4 transgenic rice compared to the wild-type. Conversely, the extremely short α-glucan chains (DP 2-8) exhibited a dramatic increase in the P10::AmyI-1 starch. Rapid visco-analyzer analysis also identified variations in the chain length distribution of P10::AmyI-1 starch, manifesting as changes in viscosity. Moreover, 1H-NMR analysis uncovered dynamic modifications in the molecular structure of starch in rice grain transformed with P10::AmyI-1, which was found to possess unprecedented structural characteristics.

Effects of Hard Water Boiling on Chalky Rice in Terms of Texture Improvement and Ca Fortification.

In the present paper, we investigated the characteristics of chalky rice grains generated by ripening under high temperature and compared them with whole grains. We evaluated 14 unpolished Japonica rice grains harvested in Japan in 2021, and these samples (original grains) were divided into two groups (a whole grain group and a chalky grain one). We found that not only activities of endogenous amylase and proteinase, but also cell wall-degrading enzymes, such as xylanase and cellulase, changed markedly between chalky grains and whole grains. Using rice grains blended with 30% of chalky grains as the material, we compared the sugar and mineral contents and textural properties of the rice grains soaked and boiled in either ordinary water or hard water, such as Evian or Contrex. It was shown that xylanase, in addition to amylase and proteinase, may play an important role in changing the texture of the boiled chalky rice grains. For the sake of preventing the above-mentioned deterioration in the texture of boiled grains of chalky rice, we tried to use hard water, such as Evian or Contrex, to soak and cook the chalky rice grains. It was shown that the hard water was useful for the prevention of texture deterioration of the boiled rice grains due to inhibition of the activities of endogenous hydrolytic enzymes, such as α-amylase, ß-amylase, proteinase, and xylanase. Furthermore, we found that the hard water was useful in increasing the calcium absorption through the meal by 2.6 to 16.5 times.

Identification of a new allele of soluble starch synthase IIIa involved in the elongation of amylopectin long chains in a chalky rice mutant.

A chalky endosperm mutant (GM03) induced from an indica rice GLA4 was used to investigate the functional gene in starch biosynthesis. Bulked segregant analysis and sanger sequencing determined that a novel mutation in soluble starch synthase IIIa (SSIIIa) is responsible for the chalky phenotype in GM03. Complementary test by transforming the active SSIIIa gene driven by its native promoter to GM03 recovered the phenotype to its wildtype. The expression of SSIIIa was significantly decreased, while SSIIIa protein was not detected in GM03. The mutation of SSIIIa led to increased expression of most of starch synthesis related genes and elevated the levels of most of proteins in GM03. The CRISPR/Cas9 technology was used for targeted disruption of SSIIIa, and the mutant lines exhibited chalky endosperm which phenocopied the GM03. Additionally, the starch fine structure in the knockout mutant lines ss3a-1 and ss3a-2 was similar with the GM03, which showed increased amylose content, higher proportions of B1 and B2 chains, much lower proportions of B3 chains and decreased degree of crystallinity, leading to altered thermal properties with lower gelatinization temperature and enthalpy. Collectively, these results suggested that SSIIIa plays an important role in starch synthesis by elongating amylopectin long chains in rice.

Genetic architecture of head rice and rice chalky grain percentages using genome-wide association studies.

High head rice and low chalky grain percentages are key grain quality traits selected in developing rice cultivars. The objectives of this research were to characterize the phenotypic variation of head rice and chalky grain percentages in a diverse collection of rice accessions, identify single nucleotide polymorphism (SNP) markers associated with each of these traits using genome-wide association studies (GWAS), and identify putative candidate genes linked to the SNPs identified by GWAS. Diverse rice varieties, landraces, and breeding lines were grown at the Texas A&M AgriLife Research Center in Beaumont. Head rice percentages (HRP) and chalky grain percentages (CGP) of 195 and 199 non-waxy accessions were estimated in 2018 and 2019, respectively. Phenotypic data were analyzed along with 854,832 SNPs using three statistical models: mixed linear model (MLM), multi-locus mixed model (MLMM), and fixed and random model circulating probability unification (FarmCPU). Significant variations in HRP and CGP were observed between rice accessions. Two significant marker-trait associations (MTAs) were detected on chromosomes 1 and 2, respectively, based on best linear unbiased prediction (BLUP) values in 2018, while in 2019, one SNP was significantly associated with HRP in each of chromosomes 6, 8, 9, and 11, and two in chromosome 7. CGP was significantly associated with five SNPs located in chromosomes 2, 4, 6, and 8 in the 2018 study and ten SNPs in chromosomes 1, 2, 3, 4, 7, 8, 11, and 12 in the 2019 study. The SNPs are located within or linked to putative candidate genes involved in HRP and CGP. This study reports five and ten novel MTAs for HRP and CGP, respectively, while three and five MTAs co-located with previously reported quantitative trait loci for HRP and CGP, respectively. The validation of candidate genes for their roles in determining HRP and CGP is necessary to design functional molecular markers that can be used to effectively develop rice cultivars with desirable grain quality.

Palatability and Bio-Functionality of Chalky Grains Generated by High-Temperature Ripening and Development of Formulae for Estimating the Degree of Damage Using a Rapid Visco Analyzer of Japonica Unpolished Rice.

Global warming inhibits grain filling in rice and leads to chalky grains, which are damaged in physical and cooking qualities. In the present paper, we evaluated 54 Japonica unpolished rice grains harvested in Japan in 2020, and these samples (original grains) were divided to two groups (whole grains and chalky grains). Using rice grains of 100% whole grains or those blended with 30% of chalky grains, we measured contents of sugars and amino acids, and textural properties of boiled rice grains. It was shown that the α-amylase activity and proteinase activity of raw chalky rice were significantly higher than those of whole rice grains, which led to the significant increase of low-molecular-weight sugars and free amino acids after boiling. Furthermore, hardness and toughness of the boiled rice grains were decreased markedly by blending chalky grains. The ratio of α-amylase activity of chalky grains to that of whole grains was shown to be a useful indicator for damage degree by high-temperature ripening. It became possible to estimate the degree of high-temperature damage of rice grains based on only the pasting properties of unpolished rice.

Microstructure, Water Migration and Texture of Thai Chalky Rice Varieties.

Chalk is an inferior characteristic of rice because it leads to the poor milling quality. In addition, chalky rice is generally considered to have poor cooking and eating qualities. However, the data on the poor cooking and eating qualities of chalky rice is limited, especially in high amylose Thai chalky rice varieties. Therefore, this work aimed to compare the microstructure, water migration during cooking, texture and sensorial qualities of four Thai chalky rice varieties, Prachin Buri2 (PB2), Khao Bahn Nah432 (KBN432), Ayutthaya1 (AY1), and Plai Ngahm Prachin Buri (PNPB), with the translucent Thai rice variety, Phitsanuplok2 (PSL2). The loosely packed cell structure in all chalky rice varieties was observed under scanning electron microscope. KBN 432 had the highest degree of chalkiness. Water migrated in the KBN432 with the fastest speed. All cooked chalky rice had lower hardness than the translucent rice, except AY1. Softness scores of chalky rice samples were higher than translucent rice. However, there was no difference in overall acceptability between chalky rice and translucent rice. Therefore, chalky rice varieties studied in this work may be used for consumption with the same quality to the translucent rice variety.

A High-Yielding Rice Cultivar "Takanari" Shows No N Constraints on CO2 Fertilization.

Enhancing crop yield response to elevated CO2 concentrations (E-[CO2]) is an important adaptation measure to climate change. A high-yielding indica rice cultivar "Takanari" has recently been identified as a potential candidate for high productivity in E-[CO2] resulting from its large sink and source capacities. To fully utilize these traits, nitrogen should play a major role, but it is unknown how N levels influence the yield response of Takanari to E-[CO2]. We therefore compared grain yield and quality of Takanari with those of Koshihikari, a standard japonica cultivar, in response to Free-Air CO2 enrichment (FACE, +200 µmol mol-1) under three N levels (0, 8, and 12 g m-2) over three seasons. The biomass of both cultivars increased under E-[CO2] at all N levels; however, the harvest index decreased under E-[CO2] in the N-limited treatment for Koshihikari but not for Takanari. The decreased harvest index of Koshihikari resulted from limited enhancement of spikelet number under N-limitation. In contrast, spikelet number increased in E-[CO2] in Takanari even without N application, resulting in significant yield enhancement, averaging 18% over 3 years, whereas Koshihikari exhibited virtually no increase in yield in E-[CO2] under the N-limited condition. Grain appearance quality of Koshihikari was severely reduced by E-[CO2], most notably in N-limited and hot conditions, by a substantial increase in chalky grain, but chalky grain % did not increase in E-[CO2] even without N fertilizer. These results indicated that Takanari could retain its high yield advantage over Koshihikari with limited increase in chalkiness even under limited N conditions and that it could be a useful genetic resource for improving N use efficiency under E-[CO2].

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.

First description of 'Chalky back' phenomenon in banana prawns (Fenneropenaeus merguiensis) and its possible association with Vibrio and Photobacterium species.

Here we report a newly identified 'Chalky back' phenomenon in banana prawns (Fenneropenaeus merguiensis) farmed in North Queensland, Australia. This was characterized by localized white discoloured segmentation of the cervical groove, moreover, after cooking the prawns exploded, making them unfit for commercial sale. Histological examination revealed breakdown of gut and abdominal muscle tissue in some moribund specimens. We selectively isolated Vibrio spp., which are known prawn pathogens, from healthy and Chalky back specimens. Isolated bacteria were identified, typed and tested for the presence of eight virulence genes (VGs), biofilm formation, adherence and cytotoxicity to fish cells. In all, 32 isolates were recovered and identified as Vibrio harveyi, V. owensii, V. sinaloensis-like, V. campbellii, V. shilonii, Vibrio sp. and Photobacterium damselae using 16S rRNA gene sequencing. All V. harveyi carried VGs coding for haemolysin, toxR and flagella; formed biofilm; and adhered to both cell lines. This was similar to the V. sinaloensis-like strains that were only isolated from Chalky back specimens. Our data suggest that Vibrio spp. may play a role in the pathogenesis of Chalky back. This study is the first report of Chalky back phenomenon in farmed banana prawns that needs to be closely monitored by the industry.

QTL analysis for chalkiness of rice and fine mapping of a candidate gene for qACE9.

BACKGROUND: An ideal appearance is of commercial value for rice varieties. Chalkiness is one of the most important appearance quality indicators. Therefore, clarification of the heredity of chalkiness and its molecular mechanisms will contribute to reduction of rice chalkiness. Although a number of QTLs related to chalkiness were mapped, few of them have been cloned so far. RESULTS: In this study, using recombinant inbred lines (RILs) of PA64s and 9311, we identified 19 QTLs associated with chalkiness on chromosomes 1, 4, 6, 7, 9 and 12, which accounted for 5.1 to 30.6 % of phenotypic variations. A novel major QTL qACE9 for the area of chalky endosperm (ACE) was detected in Hainan and Hangzhou, both mapped in the overlapping region on chromosome 9. It was further fine mapped to an interval of 22 kb between two insertion-deletion (InDel) markers IND9-4 and IND9-5 using a BC4F2 population. Gene prediction analysis identified five putative genes, among which only one gene (OsAPS1), whose product involved in starch synthesis, was detected two nucleotide substitutions causing amino acid change between the parents. Significant difference was found in apparent amylose content (AAC) between NILqACE9 and 9311. And starch granules were round and loosely packed in NILqACE9 compared with 9311 by scanning electron microscopy (SEM) analysis. CONCLUSIONS: OsAPS1 was selected as a novel candidate gene for fine-mapped qACE9. The candidate gene not only plays a critical role during starch synthesis in endosperm, but also determines the area of chalky endosperm in rice. Further cloning of the QTL will facilitate the improvement of quality in hybrid rice.