Decreased grain size1, a C3HC4-type RING protein, influences grain size in rice (Oryza sativa L.).

KEY MESSAGE: We reported that DGS1 plays a positive role in regulating grain size in rice and was regulated by OsBZR1. Grain size is an important agronomic trait that contributes to grain yield. However, the underlying molecular mechanisms that determine final grain size are still largely unknown. We isolated a rice mutant showing reduced grain size in a 60Co-irradiated variety Nanjing 35 population. We named the mutant decreased grain size1 (dgs1). Map-based cloning and subsequent transgenic CRISPR and complementation assays indicated that a mutation had occurred in LOC_Os03g49900 and that the DGS1 allele regulated grain size. DGS1 encodes a protein with a 7-transmembrane domain and C3HC4 type RING domain. It was widely expressed, especially in young tissues. DGS1 is a membrane-located protein. OsBZR1 (BRASSINAZOLE-RESISTANT1), a core transcription activator of BR signaling, also plays a positive role in grain size. We provided preliminary evidence that OsBZR1 can bind to the DGS1 promoter to activate expression of DGS1.

Btr1-A Induces Grain Shattering and Affects Spike Morphology and Yield-Related Traits in Wheat.

Spike brittleness represents an important domestication trait in crops. Although the brittle rachis of wild wheat was cloned, however, the molecular mechanism underlying spike brittleness is yet to be elucidated. Here, we identified a single dominant brittle rachis gene Br-Ab on chromosome arm 3AbS using an F2 population of diploid wheat and designated Btr1-Ab. Sequence analysis of the Btr1-A gene in 40 diploid wheat accessions, 80 tetraploid wheat accessions and 38 hexaploid wheat accessions showed that two independent mutations (Ala119Thr for diploid and Gly97* for polyploids) in the Btr1-A coding region resulting in the nonbrittle rachis allele. Overexpression of Btr1-Ab in nonbrittle hexaploid wheat led to brittle rachis in transgenic plants. RNA-Seq analysis revealed that Btr1-A represses the expression of cell wall biosynthesis genes during wheat rachis development. In addition, we found that Btr1-A can modify spike morphology and reduce threshability, grain size and thousand grain weight in transgenic wheat. These results demonstrated that Btr1-A reduces cell wall synthesis in rachis nodes, resulting in natural spikelet shattering, and that the transition from Btr1-A to btr1-A during wheat domestication had profound effects on evolution of spike morphology and yield-related traits.

Evidence for preservation of vacuolar compartments during foehn-induced chalky ring formation of Oryza sativa L.

MAIN CONCLUSION: Vacuolar compartments being sustained among the amyloplasts inadequately accumulated in rice endosperm cells are the main cause of chalky ring formation under dry wind conditions. Foehn-induced dry wind during the grain-filling stage induces shoot water deficit in rice (Oryza sativa L.) plants, which form a ring-shaped chalkiness in their endosperm that degrades milling quality and rice appearance. Air spaces formed in several inner cells cause significant transparency loss due to irregular light reflection. Although starch synthesis was suggested to be retarded by osmotic adjustment at foehn-induced moderately low water potential, the source of these air spaces remains unknown. We hypothesised that the preservation of vacuoles accompanied by a temporary reduction in starch biosynthesis in the inner cells leads to the chalky ring formation. Panicle water status measurement, light and transmission electron microscopic (TEM) observations, and an absolute qPCR analysis were conducted. Most starch synthesis-related genes exhibited temporarily reduced expression in the inner zone in accordance with the decrease in panicle water status. TEM observations provided evidence that vacuolar compartments remained among the loosely packed starch granules in the inner endosperm cells, where a chalky ring appeared after kernel dehydration. Taken together, we propose that vacuolar compartments sustained among the amyloplasts inadequately accumulated in rice endosperm cells and caused air space formation that leads to ring-shaped chalkiness under dry wind conditions.

A comprehensive overview of grain development in Brachypodium distachyon variety Bd21.

A detailed and comprehensive understanding of seed reserve accumulation is of great importance for agriculture and crop improvement strategies. This work is part of a research programme aimed at using Brachypodium distachyon as a model plant for cereal grain development and filling. The focus was on the Bd21-3 accession, gathering morphological, cytological, and biochemical data, including protein, lipid, sugars, starch, and cell-wall analyses during grain development. This study highlighted the existence of three main developmental phases in Brachypodium caryopsis and provided an extensive description of Brachypodium grain development. In the first phase, namely morphogenesis, the embryo developed rapidly reaching its final morphology about 18 d after fertilization (DAF). Over the same period the endosperm enlarged, finally to occupy 80% of the grain volume. During the maturation phase, carbohydrates were continuously stored, mainly in the endosperm, switching from sucrose to starch accumulation. Large quantities of ß-glucans accumulated in the endosperm with local variations in the deposition pattern. Interestingly, new ß-glucans were found in Brachypodium compared with other cereals. Proteins (i.e. globulins and prolamins) were found in large quantities from 15 DAF onwards. These proteins were stored in two different sub-cellular structures which are also found in rice, but are unusual for the Pooideae. During the late stage of development, the grain desiccated while the dry matter remained fairly constant. Brachypodium exhibits some significant differences with domesticated cereals. Beta-glucan accumulates during grain development and this cell wall polysaccharide is the main storage carbohydrate at the expense of starch.

A plasma membrane transporter coordinates phosphate reallocation and grain filling in cereals.

Phosphate (Pi) is essential to plant growth and crop yield. However, it remains unknown how Pi homeostasis is maintained during cereal grain filling. Here, we identified a rice grain-filling-controlling PHO1-type Pi transporter, OsPHO1;2, through map-based cloning. Pi efflux activity and its localization to the plasma membrane of seed tissues implicated a specific role for OsPHO1;2 in Pi reallocation during grain filling. Indeed, Pi over-accumulated in developing seeds of the Ospho1;2 mutant, which inhibited the activity of ADP-glucose pyrophosphorylase (AGPase), important for starch synthesis, and the grain-filling defect was alleviated by overexpression of AGPase in Ospho1;2-mutant plants. A conserved function was recognized for the maize transporter ZmPHO1;2. Importantly, ectopic overexpression of OsPHO1;2 enhanced grain yield, especially under low-Pi conditions. Collectively, we discovered a mechanism underlying Pi transport, grain filling and P-use efficiency, providing an efficient strategy for improving grain yield with minimal P-fertilizer input in cereals.

Roles of FERONIA-like receptor genes in regulating grain size and quality in rice.

Grain yield and quality are critical factors that determine the value of grain crops. In this study, we analyzed the functions of 12 FERONIA-like receptor (FLR) family members in rice and investigated their effects on grain size and quality. We found that FLR1, FLR2 and FLR8 negatively regulated grain size, and FLR15 positively regulated grain size. flr1 mutants had a higher cell number and an accelerated rate of grain filling compared to wild-type plants, which led to grains with greater widths. A mechanism underlying the regulation of grain size by FLR1 is that FLR1 is associated with OsRac1 Rho-like GTPase, a positive regulator of grain size. Regarding grain quality, the flr1 mutant had a higher percentage of chalkiness compared with wild-type plants, and seeds carrying mutations in flr3 and flr14 had endosperms with white floury cores. To elucidate the possible mechanism underlying this phenomenon, we found that FLR1 was constitutively expressed during endosperm development. RNA-seq analysis identified 2,367 genes that were differentially expressed in the flr1 mutant, including genes involved in starch and sucrose metabolism and carbon fixation. In this study, we identified the roles played by several FLR genes in regulating grain size and quality in rice and provided insights into the molecular mechanism governing the FLR1-mediated regulation of grain size.

Ultrastructure of mature protein body in the starchy endosperm of dry cereal grain.

The development of the protein body in the late stage of seed maturation is poorly understood, because electron-microscopy of mature cereal endosperm is technically difficult. In this study, we attempted to modify the existing method of embedding rice grain in resin. The modified method revealed the ultrastructures of the mature protein body in dry cereal grains.

Starch synthesis and programmed cell death during endosperm development in triticale (x Triticosecale Wittmack).

Triticale (x Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source. Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase, soluble starch synthases, granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development. There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale. Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development. Dead cells within the endosperm were detected at 6 d post anthesis (DPA), and evidence of DNA fragmentation was first observed at 21 DPA. The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development. Cell death occurred stochastically throughout the whole endosperm, meanwhile, the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling. These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.

Mashes to Mashes, Crust to Crust. Presenting a novel microstructural marker for malting in the archaeological record.

The detection of direct archaeological remains of alcoholic beverages and their production is still a challenge to archaeological science, as most of the markers known up to now are either not durable or diagnostic enough to be used as secure proof. The current study addresses this question by experimental work reproducing the malting processes and subsequent charring of the resulting products under laboratory conditions in order to simulate their preservation (by charring) in archaeological contexts and to explore the preservation of microstructural alterations of the cereal grains. The experimentally germinated and charred grains showed clearly degraded (thinned) aleurone cell walls. The histological alterations of the cereal grains were observed and quantified using reflected light and scanning electron microscopy and supported using morphometric and statistical analyses. In order to verify the experimental observations of histological alterations, amorphous charred objects (ACO) containing cereal remains originating from five archaeological sites dating to the 4th millennium BCE were considered: two sites were archaeologically recognisable brewing installations from Predynastic Egypt, while the three broadly contemporary central European lakeshore settlements lack specific contexts for their cereal-based food remains. The aleurone cell wall thinning known from food technological research and observed in our own experimental material was indeed also recorded in the archaeological finds. The Egyptian materials derive from beer production with certainty, supported by ample contextual and artefactual data. The Neolithic lakeshore settlement finds currently represent the oldest traces of malting in central Europe, while a bowl-shaped bread-like object from Hornstaad-Hörnle possibly even points towards early beer production in central Europe. One major further implication of our study is that the cell wall breakdown in the grain's aleurone layer can be used as a general marker for malting processes with relevance to a wide range of charred archaeological finds of cereal products.

Analysis of development, accumulation and structural characteristics of starch granule in wheat grain under nitrogen application.

Nitrogen is one of the most important nutrients for wheat growth and has a critical influence on yield and quality. This study aims to examine how medium nitrogen level (240 kg/hm2) affects the starch granule development, starch accumulation, and structural characteristics of wheat starch. The results showed that nitrogen treatment could reduce the biosynthesis of starch and amylose, delay the degradation of starch in pericarp, and promote the proportion of B-type small starch granule in endosperm compared with those in the N0. In addition, the composition and distribution of starch granules were changed, the crystal structure in the inner lamellae and ordered structure in the external region of starch granules were affected, and the swelling power and solubility of starch during wheat development were increased. The effect of nitrogen treatment on the mRNA expression of enzymes related to starch biosynthesis or degradation varied in different developmental stages. During middle and later grain filling stages, AGPase, GBSSI, and GBSSII were lower, and SSS, SBE, and DBE were higher in N240 than in N0. This study indicated that nitrogen application at booting stage significantly affected the structural characteristics of starch, and ultimately determines its quality.

GWC1 is essential for high grain quality in rice.

Appearance quality is an important determinant of rice quality. Many genes that affect grain appearance quality have been identified, but the regulatory mechanisms that contribute to this trait remain unclear. Here, two grains with chalkiness (gwc1) mutants, gwc1-1 and gwc1-2, were identified from an EMS-mutagenized population of indica rice cultivar Shuhui498 (R498). The gwc1 mutants had poor grain appearance quality consistent with the measured values for the percentage of grains with chalkiness, square of chalky endosperm, the total starch, amylose and sucrose contents. Milling quality and grain size were also affected in the gwc1 mutants. The gwc1-1 and gwc1-2 were found to be loss-of-function allelic mutants. GWC1 was mapped to the long arm of rice chromosome 8 using the MutMap strategy and incorrectly annotated in the reference genome for Nipponbare (MSU). The GWC1 gene corresponds to the WTG1/OsOTUB1 gene, which encodes an otubain-like protease with deubiquitinating activity that is homologous to human OTUB1. GWC1 transcripts accumulated to high levels in early endosperm after fertilization and developing inflorescences, and GWC1-green fluorescent protein (GFP) signal was detected in the nucleus and cytoplasm. GWC1 is likely to regulate grain appearance quality through genes involved in sucrose metabolism and starch biosynthesis. Overall, the present findings reveal that GWC1 is important for grain quality and yield due to its effects on grain chalkiness and size.

The light-harvesting chlorpohyll-protein complex of photosystem II. Its location in the photosynthetic membrane.

We have investigated the structure of the photosynthetic membrane in a mutant of barley known to lack a chlorophyll-binding protein. This protein is thought to channel excitation energy to photosystem II, and is known as the "light-harvesting chlorophyll-protein complex." Extensive stacking of thylakoids into grana occurs in both mutant and wild-type chloroplasts. Examination of membrane internal structure by freeze-fracturing indicates that only slight differences exist between the fracture faces of mutant and wild-type membranes. These differences are slight reductions in the size of particles visible on the EFs fracture face, and in the number of particles seen on the PFs fracture face. No differences can be detected between mutant and wild-type on the etched out surface of the membrane. In contrast, tetrameric particles visible on the etched inner surface of wild-type thylakoids are extremely difficult to recognize on similar surfaces of the mutant. These particles can be recognized on inner surfaces of the mutant membranes when they are organized into regular lattices, but these lattices show a much closer particle-to-particle spacing than similar lattices in wild-type membranes. Although several interpretations of these data are possible, these observations are consistent with the proposal that the light-harvesting chlorophyll-protein complex of photosystem II is bound to the tetramer (which is visible on the EFs face as a single particle) near the inner surface of the membrane. The large tetramer, which other studies have shown to span the thylakoid membrane, may represent an assembly of protein, lipid, and pigment comprising all the elements of the photosystem II reaction. A scheme is presented which illustrates one possibility for the light reaction across the photosynthetic membrane.

[Pharmacognostical study on Fructus Hordei germinatus, Fructus Oryzae germinatus and Fructus Setariae germinatus].

OBJECTIVE: To establish the standard for quality control of Fructus Hordei germinatus, Fructus Oryzae germinatus and Fructus Setariae germinatus. METHODS: The digital microscope and infrared spectroscopy were used in the pharmacognostical study. RESULTS: Distinguished differences were found on morphological and microscopical features of these three crude drugs. Whereas, their infrared spectrums were basically all the same. CONCLUSION: The study provides a convenient, effect method for the identification of these three medicinal materials.

Shedding Light on Penetration of Cereal Host Stomata by Wheat Stem Rust Using Improved Methodology.

Asexual urediniospore infection of primary cereal hosts by Puccinia graminis f. sp. tritici (Pgt), the wheat stem rust pathogen, was considered biphasic. The first phase, spore germination and appressoria formation, requires a dark period and moisture. The second phase, host entry by the penetration peg originating from the appressoria formed over the guard cells, was thought to require light to induce natural stomata opening. Previous studies concluded that inhibition of colonization by the dark was due to lack of penetration through closed stomata. A sensitive WGA-Alexa Fluor 488 fungal staining, surface creation and biovolume analysis method was developed enabling visualization and quantification of fungal growth in planta at early infection stages surpassing visualization barriers using previous methods. The improved method was used to investigate infection processes of Pgt during stomata penetration and colonization in barley and wheat showing that penetration is light independent. Based on the visual growth and fungal biovolume analysis it was concluded that the differences in pathogen growth dynamics in both resistant and susceptible genotypes was due to light induced pathogen growth after penetration into the substomatal space. Thus, light induced plant or pathogen cues triggers pathogen growth in-planta post penetration.

Multiparametric high-resolution imaging of barley embryos by multiphoton microscopy and magnetic resonance micro-imaging.

Nonlinear optical microscopy and magnetic resonance imaging (MRI) address different properties of the sample and operate on different geometrical scales. MRI maps density and mobility of molecules tracking specific molecular signatures. Multiphoton imaging profits from the nonlinear absorption of light in the focus of a femtosecond laser source stimulating the autofluorescence of biomolecules. As this effect relies on a high light intensity, the accessible field of view is limited, but the resolution is very high (a few hundred nanometers). Here, we aim to link the different accessible scales and properties addressed in the different techniques to obtain a synoptic view. As model specimen we studied embryos of barley. Multiphoton stimulated autofluorescence images and images of second harmonic generation are achieved even down to low magnification (10x), low numerical aperture (N.A. 0.25) conditions. The overview images allowed morphological assignments and fluorescence lifetime imaging provides further information to identify accumulation of endogenous fluorophores. The second, complementary contribution from high-resolution MR images provides a 3D model and shows the embedding of the embryo in the grain. Images of the proton density were acquired using a standard 3D spin-echo imaging pulse sequence. Details directly comparable to the low magnification optical data are visible. Eventually, passing from the MR images of the whole grain via low magnification to high resolution autofluorescence data bridges the scale barrier, and might provide the possibility to trace transport and accumulation of, e.g., nutrients from large structure of the plant to the (sub-) cellular level.

Biophysical features of cereal endosperm that decrease starch digestibility.

The influence of the physical structure of cereal endosperm on the natural structural integrity (intact cells) and starch bioaccessibility of the resultant flours was studied using maize as example. Endosperm hardness, defined by its intracellular (protein matrix) and extracellular (cell walls) constituents, affected the granular and molecular damage of the starch of the resultant flours leading to higher digestibility of raw hard than soft endosperm flours, but comparatively lower digestibility after cooking. After milling, hard endosperm possessed more damaged starch (radial splitting of amylopectin clusters) in the periphery of the resultant particles that increased in vitro starch digestibility of raw flours. Conversely, the hard endosperm plant tissue matrix significantly limited water availability and heat transfer on starch gelatinisation, thereby decreasing the digestion rate after hydrothermal processing (in particle size flours >80µm). This study provides a unique mechanistic understanding to obtain cereal flours with slow digestion property for commercial utilisation.

Enzymes enhance degradation of the fiber-starch-protein matrix of distillers dried grains with solubles as revealed by a porcine in vitro fermentation model and microscopy.

Effects of treating corn and wheat distillers dried grains with solubles (DDGS) with a multicarbohydrase alone or in combination with a protease on porcine in vitro fermentation characteristics and the matrix structure of the DGGS before and after the fermentation were studied. Three DDGS samples (wheat DDGS sample 1 [wDDGS1], wheat DDGS sample 2 [wDDGS2], and corn DDGS [cDDGS]) were predigested with pepsin and pancreatin. Residues were then subjected to in vitro fermentation using buffered mineral solution inoculated with fresh pig feces without or with a multicarbohydrase alone or in combination with protease in a 3 × 3 factorial arrangement. Accumulated gas production was measured for up to 72 h. Concentration of VFA was measured in fermented solutions. The matrix of native DDGS and their residues after fermentation was analyzed using confocal laser scanning microscopy and scanning electron microscopy to determine internal and external structures, respectively. On a DM basis, wDDGS1, wDDGS2, and cDDGS contained 35.5, 43.4, and 29.0% CP; 2.23, 0.51, and 6.40% starch; 0.82, 0.80, and 0.89% available Lys; and 24.8, 22.5, and 23.0% total nonstarch polysaccharides, respectively. The in vitro digestibility of DM for wDDGS1, wDDGS2, and cDDGS was 67.7, 72.1, and 59.6%, respectively. The cDDGS had greater ( < 0.05) total gas and VFA production than both wheat DDGS. The wDDGS2 had lower ( < 0.05) total gas production than wDDGS1. Multicarbohydrase increased ( < 0.05) total gas production for cDDGS and total VFA production for wDGGS1 but did not increase gas or VFA production for wDDGS2. Addition of protease with multicarbohydrase to DDGS reduced ( < 0.05) total gas and VFA productions and increased ( < 0.05) branched-chain VFA regardless of DDGS type. Confocal laser scanning microscopy and scanning electron microscopy revealed that DDGS were mainly aggregates of resistant and nonfermentable starchy and nonstarchy complexes formed during DDGS production. After in vitro fermentation with porcine fecal inoculum, particles of enzyme-treated DDGS were generally smaller than those of the untreated DDGS. In conclusion, cDDGS had a more porous matrix that was more fermentable than the wheat DDGS. The wDDGS2 was less fermentable than wDDGS1. Multicarbohydrase increased fermentability of cDDGS and wDDGS1 but not wDDGS2, indicating that its efficacy in DDGS is dependent on matrix porosity and DDGS source. Protease hindered efficacy of multicarbohydrase.

GroEL-related molecular chaperones are present in the cytosol of oat cells.

In eukaryotic cells GroEL-related molecular chaperones (cpn 60) are considered to be restricted to plastids and mitochondria. Re-evaluation of the intracellular localization of chaperonins by electron microscopy, using two different anti-chaperonin antisera, revealed additionally their presence in the cytosol of oat primary leaf and coleoptile cells. The distribution of cpn 60 is not influenced by heat or light treatments.

An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique.

A new variation of the antimonate precipitation technique, employing tannic acid in the primary aldehyde-antimonate fixative, is described for use in the subcellular localization of calcium in various tissues. Chelation studies and electron microscopic, X-ray microanalytical studies of antimonate precipitates in etiolated oat tissues indicate that calcium is the major cation localized using the present experimental protocol. Preservation of ultrastructural morphology in these tissues is greatly improved over that observed in tissues fixed with conventional antimonate-aldehyde or antimonate-osmium fixatives. The regularity and reproducibility of tissue precipitate patterns suggests that 1) penetration of the tissue by the fixative, and subsequent precipitation of calcium, is rapid and uniform and 2) ion displacement during sample preparation is negligible. Calcium appears to be immobilized efficiently in situ, with greater than 90% 45Ca retention in radiolabeled tissues prepared for electron microscopy. Quantitative aspects of calcium precipitation by antimonate in 45Ca-labeled CaCl2 solutions were examined over a wide range of calcium concentrations. Precipitation was essentially linear over the expected range of biological concentrations of calcium. Furthermore, the 3:1 antimonate to calcium ratio estimated for test tube precipitates was also established for Sb/Ca in tissue precipitates analyzed using energy dispersive x-ray microanalytical (EDX) techniques. These observations suggest that the present technique is potentially useful in the semiquantitative estimation of tissue calcium levels.

Leaf sheath cuticular waxes on bloomless and sparse-bloom mutants of Sorghum bicolor.

Leaf sheath cuticular waxes on wild-type Sorghum bicolor were approximately 96% free fatty acids, with the C28 and C30 acids being 77 and 20% of these acids, respectively. Twelve mutants with markedly reduced wax load were characterized for chemical composition. In all of the 12 mutants, reduction in the amount of C28 and C30 acids accounted for essentially all of the reduction in total wax load relative to wildtype. The bm2 mutation caused a 99% reduction in total waxes. The bm4, bm5, bm6, bm7 and h10 mutations caused more than 91% reduction in total waxes, whereas the remaining six mutants, bm9, bm11, h7, h11, h12 and h13, caused between 35 and 78% reduction in total wax load. Relative to wild-type, bm4 caused a large increase in the absolute amount of C22, C24 and C26 acids, and reduction in the C28 and longer acids, suggesting that bm4 may suppress elongation of C26, acyl-CoA primarily. The h10 mutation increased the absolute amounts of the longest chain length acids, but reduced shorter acids, suggesting that h10 may suppress termination of acyl-CoA elongation. The bm6, bm9, bm11, h7, h11, h12 and h13 mutations increased the relative amounts, but not absolute amounts, of longer chain acids. Based on chemical composition alone, it is still uncertain which genes and their products were altered by these mutations. Nevertheless, these Sorghum cuticular wax mutants should provide a valuable resource for future studies to elucidate gene involvement in the biosynthesis of cuticular waxes, in particular, the very-long-chain fatty acids.