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The paper deals with the seed morphology and anatomy of 32 wild Indian Musaceae by using light microscopy (LM) and scanning electron microscopy (SEM). This is the first comprehensive investigation into the seed micro-morphological and anatomical aspects of two genera of the family. Seed characters including shape, size, surface sculpturing (LM and SEM), seed coat thickness, layer characters and chalazal chamber column were considered. Seed characters play a significant role in the delimitation of taxa as they are more stable than vegetative (anatomical) and pollen features. Four types of seeds (oblate, ovoid, sub-globose and ellipsoid) were observed in the family, oblate seeds being the most common. LM revealed that most of the seeds have verrucate surface sculpturing, but some are smooth-verrucate or smooth. Seed size also plays an important role in the identification of the taxa to some extent. Number of layers and seed coat thickness are directly proportional to seed size. However, the thickness of endotesta, tegmen and inner endosperm layers (aleurone layers) differ in small and large seeds. The present study revealed eight types of outer sculpturing and three inner sculpturing patterns with diverse nature of anticlinal and periclinal cell walls. Based on the seed characters, a phenetic analysis was also carried out. [ABSTRACT FROM AUTHOR]

Several recent palaeobotanical studies claim to have found and described pre-Cretaceous angiosperm macrofossils. With rare exceptions, these papers fail to define a flower, do not acknowledge that fossils require character-based rather than group-based classification, do not explicitly state which morphological features would unambiguously identify a fossil as angiospermous, ignore the modern conceptual framework of phylogeny reconstruction, and infer features in the fossils in question that are interpreted differently by (or even invisible to) other researchers. This unfortunate situation is compounded by the relevant fossils being highly disarticulated two-dimensional compression-impressions lacking anatomical preservation. Given current evidence, all supposed pre-Cretaceous angiosperms are assignable to other major clades among the gymnosperms sensu lato. By any workable morphological definition, flowers are not confined to, and therefore cannot delimit, the angiosperm clade. More precisely defined character states that are potentially diagnostic of angiosperms must by definition originate on the phylogenetic branch that immediately precedes the angiosperm crown group. Although the most reliable candidates for diagnostic characters (triploid endosperm reflecting double fertilization, closed carpel, bitegmic ovule, and phloem companion cells) are rarely preserved and/or difficult to detect unambiguously, similar characters have occasionally been preserved in high-quality permineralized non-angiosperm fossils. The angiosperm radiation documented by Early Cretaceous fossils involves only lineages closely similar to extant taxonomic families, lacks obvious morphological gaps, and (as agreed by both the fossil record and molecular phylogenies) was relatively rapid—all features that suggest a primary radiation. It is unlikely that ancestors of the crown group common ancestor would have fulfilled a character-based definition of (and thereby required expansion of the concept of) an angiosperm; they would instead form a new element of the non-angiosperm members of the 'anthophyte' grade, competing with Caytonia to be viewed as morphologically determined sister group for angiosperms. Conclusions drawn from molecular phylogenetics should not be allowed to routinely constrain palaeobotanical inferences; reciprocal illumination between different categories of data offers greater explanatory power than immediately resorting to Grand Syntheses. The Jurassic angiosperm—essentially a product of molecular phylogenetics—may have become the holy grail of palaeobotany but it appears equally mythical. [ABSTRACT FROM AUTHOR]

Abstract. Oil bodies serve as a vital energy source of embryos during germination and contribute to sustaining the initial growth of seedlings until photosynthesis initiation. Despite high stability in chemical properties, how oil bodies break down and go into the degradation process during germination is still unknown. This study provides a morphological understanding of the mobilization of stored compounds in the seed germination of Cannabis. The achenes of fibrous hemp cultivar (Cannabis sativa cv. 'Chungsam') were examined in this study using light microscopy, scanning electron microscopy and transmission electron microscopy. Oil bodies in Cannabis seeds appeared spherical and sporadically distributed in the cotyledonary cells. Protein bodies contained electron-dense globoid and heterogeneous protein matrices. During seed germination, rough endoplasmic reticulum (rER) and high electron-dense substances were present adjacent to the oil bodies. The border of the oil bodies became a dense cluster region and appeared as a sinuous outline. Later, irregular hyaline areas were distributed throughout oil bodies, showing the destabilized emulsification of oil bodies. Finally, the oil bodies lost their morphology and fused with each other. The storage proteins were concentrated in the centre of the protein body as a dense homogenous circular mass surrounded by a light heterogeneous area. Some storage proteins are considered emulsifying agents on the surface region of oil bodies, enabling them to remain stable and distinct within and outside cotyledon cells. At the early germination stage, rER appeared and dense substances aggregated adjacent to the oil bodies. Certain proteins were synthesized within the rER and then translocated into the oil bodies by crossing the half membrane of oil bodies. Our data suggest that rER-associated proteins function as enzymes to lyse the emulsifying proteins, thereby weakening the emulsifying agent on the surface of the oil bodies. This process plays a key role in the degeneration of oil bodies and induces coalescence during seed germination. [ABSTRACT FROM AUTHOR]

Seed development in Arabidopsis is characterized by stereotypical division patterns, suggesting that coordinated control of cell cycle may be required for correct patterning and growth of the embryo and endosperm. D-type cyclins (CYCD) are key cell cycle regulators with roles in developmental processes, but knowledge regarding their involvement in seed development remains limited. Here, a family-wide gene expression, and loss- and gain-of-function approach was adopted to reveal additional functions for CYCDs in the development of seed tissues. CYCD genes have both discrete and overlapping tissue-specific expression patterns in the seed as revealed by GUS reporter gene expression. Analysis of different mutant combinations revealed that correct CYCD levels are required in seed development. The CYCD3 subgroup is specifically required as its loss caused delayed development, whereas overexpression in the embryo and endosperm of CYCD3;1 or a previously uncharacterized gene, CYCD7;1, variously leads to induced proliferation, abnormal phenotypes, and elevated seed abortion. CYCD3;1 overexpression provoked a delay in embryonic developmental progression and abnormalities including additional divisions of the hypophysis and suspensor, regions where CYCD3 genes are normally expressed, but did not affect endosperm development. Overexpression of CYCD7;1, not normally expressed in seed development, promoted overgrowth of both embryo and endosperm through increased division and cell enlargement. In contrast to post-germination growth, where pattern and organ size is not generally related to division, results suggest that a close control of cell division through regulation of CYCD activity is important during seed development in conferring both developmental rate and correct patterning. [ABSTRACT FROM PUBLISHER]

The wild grass Brachypodium distachyon has been proposed as an alternative model species for temperate cereals. The present paper reports on the characterization of B. distachyon grain, placing emphasis on endosperm cell walls. Brachypodium distachyon is notable for its high cell wall polysaccharide content that accounts for ∼52% (w/w) of the endosperm in comparison with 2–7% (w/w) in other cereals. Starch, the typical storage polysaccharide, is low [<10% (w/w)] in the endosperm where the main polysaccharide is (1–3) (1–4)-β-glucan [40% (w/w) of the endosperm], which in all likelihood plays a role as a storage compound. In addition to (1–3) (1–4)-β-glucan, endosperm cells contain cellulose and xylan in significant amounts. Interestingly, the ratio of ferulic acid to arabinoxylan is higher in B. distachyon grain than in other investigated cereals. Feruloylated arabinoxylan is mainly found in the middle lamella and cell junction zones of the storage endosperm, suggesting a potential role in cell–cell adhesion. The present results indicate that B. distachyon grains contain all the cell wall polysaccharides encountered in other cereal grains. Thus, due to its fully sequenced genome, its short life cycle, and the genetic tools available for mutagenesis/transformation, B. distachyon is a good model to investigate cell wall polysaccharide synthesis and function in cereal grains. [ABSTRACT FROM PUBLISHER]

To our knowledge the present paper shows for the first time the kinetic parameters of all the three starch branching enzyme (BE) isozymes, BEI, BEIIa and BEIIb, from rice with both amylopectin and synthetic amylose as glucan substrate. The activities of these BE isozymes with a linear glucan amylose decreased with a decrease in the molar size of amylose, and no activities of BEIIa and BEIIb were found when the degree of polymerization (DP) of amylose was lower than at least 80, whereas BEI had an activity with amylose of a DP higher than approximately 50. Detailed analyses of debranched products from BE reactions revealed the distinct chain length preferences of the individual BE isozymes. BEIIb almost exclusively transferred chains of DP7 and DP6 while BEIIa formed a wide range of short chains of DP6 to around DP15 from outer chains of amylopectin and amylose. On the other hand, BEI formed a variety of short chains and intermediate chains of a DP ≤40 by attacking not only outer chains but also inner chains of branched glucan while BEIIa or BEIIb could only scarcely or could not attack inner chains, respectively. The comprehensive in vitro studies revealed different enzymatic characteristics of the three BE isozymes and give a new insight into the distinct roles of individual BE isozymes in amylopectin biosynthesis in the endosperm. Based on these results, the functional distinction and interaction of BE isozymes during amylopectin biosynthesis in cereal endosperm is discussed. [ABSTRACT FROM PUBLISHER]

Background and Aims: Decaisnea insignis, known as ‘dead man's fingers’ (Lardizabalaceae), is widely distributed in China and the Himalayan foothill countries. This economically important plant, which is the only species in the genus, has not been the subject of any embryological studies aside from one brief, older paper that lacks micrographs. Data on Decaisnea are also important because its systematic position has been unstable since the genus was established in 1855. Therefore, the objectives of this study were: (a) to use modern microscopy to document early reproductive anatomical development in Decaisnea; and (b) to compare qualitatively these early embryological characters with allied taxa in a systematic context. [ABSTRACT FROM PUBLISHER]

Many endosperm traits are related to grain quality in cereal crops. Endosperm traits are mainly controlled by the endosperm genome but may be affected by the maternal genome. Studies have shown that maternal genotypic variation could greatly influence the estimation of the direct effects of quantitative trait loci (QTLs) underlying endosperm traits. In this paper, we propose methods of interval mapping of endosperm QTLs using seeds of F2 or BC1 (an equal mixture of F1 × P1 and F1 × P2 with F1 as the female parent) derived from a cross between 2 pure lines (P1 × P2). The most significant advantage of our experimental designs is that the maternal effects do not contribute to the genetic variation of endosperm traits and therefore the direct effects of endosperm QTLs can be estimated without the influence of maternal effects. In addition, the experimental designs can greatly reduce environmental variation because a few F1 plants grown in a small block of field will produce sufficient F2 or BC1 seeds for endosperm QTL analysis. Simulation studies show that the methods can efficiently detect endosperm QTLs and unbiasedly estimate their positions and effects. The BC1 design is better than the F2 design. [ABSTRACT FROM AUTHOR]

The article discusses several papers published within the issue including one about the statue "Napoleon as Mars the Peacemaker" and another regarding Napoleonic propaganda.

If we wish to understand the uneven mobilization for the Anti-Nepoleonic wars of 1813–15 in different German territories, it is not enough to analyse the political, social and cultural disparities and the diverse experiences of French warfare and occupation. We also have to look at the political discourse that responded to, reflected and formed these experiences with its images. Protestant northern and eastern Germany, and particularly Prussia, suffered more than other German regions under French rule following the dramatic Prussian–Saxon defeat of 1806–07. The hatred of France and all things French was more developed in political discourse there than elsewhere in Germany. The patriotic–national mobilization for the ‘War of Liberation’ in 1813 extended beyond the small elite of the educated strata. This paper will therefore analyse, not only the images of Napoleon and the French in Prussia and northern Germany, but also their social, political and cultural context. [ABSTRACT FROM PUBLISHER]

Starch is made up of amylose (linear α-1,4-polyglucans) and amylopectin (α-1,6-branched polyglucans). Amylopectin has a distinct fine structure called multiple cluster structure and is synthesized by multiple subunits or isoforms of four classes of enzymes: ADPglucose pyrophosphorylase, soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE). In the present paper, based on analyses of mutants and transgenic lines of rice in which each enzyme activity is affected, the contribution of the individual isoform to the fine structure of amylopectin in rice endosperm is evaluated, and a new model referred to as the “two-step branching and improper branch clearing model” is proposed to explain how amylopectin is synthesized. The model emphasizes that two sets of reactions, α-1,6-branch formation and the subsequent α-1,4-chain elongation, are catalyzed by distinct BE and SS isoforms, respectively, are fundamental to the construction of the cluster structure. The model also assesses the role of DBE, namely isoamylase or in addition pullulanase, to remove unnecessary α-1,6-glucosidic linkages that are occasionally formed at improper positions apart from two densely branched regions of the cluster. [ABSTRACT FROM PUBLISHER]

This study investigates carbon partitioning in the developing endosperm of a European variety of spring wheat subjected to moderately elevated daytime temperatures (27°C/16°C d/night) from anthesis to grain maturity. Elevated daytime temperatures caused significant reductions in both fresh and dry weights and reduced the starch content of harvested grains compared to plants grown under a 20°C/16°C d/night regimen. Accelerated grain development caused by elevated temperatures was accounted for by representing plant development as thermal time (°C DPA). We examined the effects of high-temperature stress (HTS) on the uptake and partitioning of [U-14C]-sucrose supplied to isolated endosperms. HTS caused reduced sucrose uptake into developing endosperms from the second major grain-filling stage (approximately 260°C DPA) up to maturity. Enzymes involved in sucrose metabolism were unaffected by HTS, whereas key enzyme activities involved in endosperm starch deposition such as ADP-glucose pyrophosphorylase and soluble isoforms of starch synthase were sensitive to HTS throughout grain development. HTS caused a decrease in other major carbon sinks such as evolved CO2, ethanol-soluble material, cell walls and protein. Despite reductions in the labeling of carbon pools caused by HTS, the relative proportions of sucrose taken up by endosperm cells allocated to each cellular pool remain unchanged, except for evolved CO2, which increased under HTS and may reflect enhanced respiratory activity. The results of this study show that moderate temperature increases can cause significant yield reductions in some temperate wheat cultivars chiefly through three effects: reduced sucrose uptake by the endosperm, reduced starch synthesis and increased partitioning of carbon into evolved CO2. [ABSTRACT FROM AUTHOR]

Chalky endosperm negatively affects the appearance, milling, and eating qualities of rice (Oryza sativa L.) grains. Here, we report the role of two receptor-like kinases, FERONIA-LIKE RECEPTOR 3 (FLR3) and FERONIA-LIKE RECEPTOR 14 (FLR14), in grain chalkiness and quality. Knockouts of FLR3 and/or FLR14 increased the number of white-core grains caused by aberrant accumulation of storage substances, resulting in poor grain quality. Conversely, the overexpression of FLR3 or FLR14 reduced grain chalkiness and improved grain quality. Transcriptome and metabolome analyses showed that genes and metabolites involved in the oxidative stress response were significantly up-regulated in flr3 and flr14 grains. The content of reactive oxygen species was significantly increased in flr3 and flr14 mutant endosperm but decreased in overexpression lines. This strong oxidative stress response induced the expression of programmed cell death (PCD)-related genes and caspase activity in endosperm, which further accelerated PCD, resulting in grain chalkiness. We also demonstrated that FLR3 and FLR14 reduced grain chalkiness by alleviating heat-induced oxidative stress in rice endosperm. Therefore, we report two positive regulators of grain quality that maintain redox homeostasis in the endosperm, with potential applications in breeding rice for optimal grain quality. [ABSTRACT FROM AUTHOR]

Pseudogamous apomixis in Paspalum simplex generates seeds with embryos genetically identical to the mother plant and endosperms deviating from the canonical 2(maternal):1(paternal) parental genome contribution into a maternal excess 4m:1p genome ratio. In P. simplex , the gene homologous to that coding for subunit 3 of the ORIGIN OF RECOGNITION COMPLEX (PsORC3) exists in three isogenic forms: PsORC3a is apomixis specific and constitutively expressed in developing endosperm whereas PsORCb and PsORCc are up-regulated in sexual endosperms and silenced in apomictic ones. This raises the question of how the different arrangement and expression profiles of these three ORC3 isogenes are linked to seed development in interploidy crosses generating maternal excess endosperms. We demonstrate that down-regulation of PsORC3b in sexual tetraploid plants is sufficient to restore seed fertility in interploidy 4 n ×2 n crosses and, in turn, its expression level at the transition from proliferating to endoreduplication endosperm developmental stages dictates the fate of these seeds. Furthermore, we show that only when being maternally inherited can PsORC3c up-regulate PsORC3b. Our findings lay the basis for an innovative route—based on ORC3 manipulation—to introgress the apomictic trait into sexual crops and overcome the fertilization barriers in interploidy crosses. [ABSTRACT FROM AUTHOR]

Rabl organization is a type of interphase chromosome arrangement with centromeres and telomeres clustering at opposite nuclear poles. Here, we analyzed nuclear morphology and chromosome organization in cycling and endoreduplicated nuclei isolated from embryo and endosperm tissues of developing barley seeds. We show that endoreduplicated nuclei have an irregular shape, less sister chromatid cohesion at 5S rDNA loci, and a reduced amount of centromeric histone CENH3. While the chromosomes of the embryo and endosperm nuclei are initially organized in Rabl configuration, the centromeres and telomeres are intermingled within the nuclear space in the endoreduplicated nuclei with an increasing endoreduplication level. Such a loss of chromosome organization suggests that Rabl configuration is introduced and further reinforced by mitotic divisions in barley cell nuclei in a tissue- and seed age-dependent manner. [ABSTRACT FROM AUTHOR]

In this study, the efficacy of different nonstarch polysaccharide (NSP) enzyme sources on wheat ingredients and wheat basal diets in vitro were evaluated by simulating the avian digestive tract. In Exp. 1, pH level was increased from 2.0 to 8.0 by simulating the avian digestive tract. The relative enzyme activities of xylanase A, B, and C and β-glucanase X at pH 3.0–3.5 were higher (P < 0.05) than those at pH 2.0 or 7.0–8.0. The optimal pH levels of 3.5 and 7.0 were screened by simulating the proventriculus and small intestine, respectively to evaluate the efficacy of NSP enzyme on wheat sources. In Exp. 2, wheat 1 contained the highest content of NSP fractions and the lowest digestibility in vitro dry matter (IVDMD) and energy (IVED) in wheat samples. Therefore, wheat 1 was selected for hydrolysis research under different NSP enzyme sources and levels (1,500, 4,500, 13,500, 40,500, 121,500 U xylanase/kg and 250, 500, 1,000, 2,000, 4,000 U β-glucanase/kg) in vitro. The hydrolysis of wheat on the basis of the released reducing sugar content was determined by xylanase sources A > B > C (P < 0.05) and β-glucanase sources of X > Y (P < 0.05). On the basis of the hydrolysis, the optimum dose of xylanase A and β-glucanase X were 40,500 U/kg and 2,000 U/kg, respectively. Subsequently, the completely randomized designs involving 2 NSP enzymes treatments × 2 endogenous digestive enzymes treatments (Exp. 3), as well as 2 wheat basal diets × 2 NSP enzymes treatments (Exp. 4) were used to evaluate the efficacy of NSP enzymes on dietary nutrient digestibility. The addition of NSP enzymes (40,500 U xylanase A/kg and 2,000 U β-glucanase X/kg) increased the IVDMD and IVED of wheat 1 without endogenous enzymes (P < 0.05), while the IVDMD and IVED of wheat 1 with endogenous enzyme were only slightly increased (P > 0.05). The addition of NSP enzymes could increase the IVDMD and IVED of corn–wheat–soybean meal diet (P < 0.05), but had no effect on those of wheat–cottonseed meal rapeseed meal diet (P > 0.05). In conclusion, xylanase and β-glucanase additions could effectively eliminate the adverse effects on wheat and wheat basal diets at the optimal pH levels of 3.5 and 7.0 by simulating the proventriculus and small intestine parts, respectively. The efficacy of NSP enzymes was influenced by the enzyme sources, dietary type, and the interaction of endogenous enzymes. [ABSTRACT FROM AUTHOR]

Apomixis, defined as the transfer of maternal germplasm to offspring without fertilization, enables the fixation of F1-useful traits, providing advantages in crop breeding. However, most apomictic plants require pollination to produce the endosperm. The endosperm is essential for embryogenesis, and its development is suppressed until fertilization. We show that the expression of a chimeric repressor of the Elongation of Siliques without Pollination 3 (ESP3) gene (Pro35S:ESP3-SRDX) induces ovule enlargement without fertilization in Arabidopsis thaliana. The ESP3 gene encodes a protein similar to the flowering Wageningen homeodomain transcription factor containing a StAR-related lipid transfer domain. However, ESP3 lacks the homeobox-encoding region. Genes related to the cell cycle and sugar metabolism were upregulated in unfertilized Pro35S:ESP3-SRDX ovules similar to those in fertilized seeds, while those related to autophagy were downregulated similar to those in fertilized seeds. Unfertilized Pro35S:ESP3-SRDX ovules partially nourished embryos when only the egg was fertilized, accumulating hexoses without central cell proliferation. ESP3 may regulate nutrient flow during seed development, and ESP3-SRDX could be a useful tool for complete apomixis that does not require pseudo-fertilization. [ABSTRACT FROM AUTHOR]

Apomixis, or asexual seed formation, is prevalent in Citrinae via a mechanism termed nucellar or adventitious embryony. Here, multiple embryos of a maternal genotype form directly from nucellar cells in the ovule and can outcompete the developing zygotic embryo as they utilize the sexually derived endosperm for growth. Whilst nucellar embryony enables the propagation of clonal plants of maternal genetic constitution, it is also a barrier to effective breeding through hybridization. To address the genetics and evolution of apomixis in Citrinae , a chromosome-level genome of the Hongkong kumquat (Fortunella hindsii) was assembled following a genome-wide variation map including structural variants (SVs) based on 234 Citrinae accessions. This map revealed that hybrid citrus cultivars shelter genome-wide deleterious mutations and SVs into heterozygous states free from recessive selection, which may explain the capability of nucellar embryony in most cultivars during Citrinae diversification. Analyses revealed that parallel evolution may explain the repeated origin of apomixis in different genera of Citrinae. Within Fortunella , we found that apomixis of some varieties originated via introgression. In apomictic Fortunella , the locus associated with apomixis contains the FhRWP gene, encoding an RWP-RK domain-containing protein previously shown to be required for nucellar embryogenesis in Citrus. We found the heterozygous SV in the FhRWP and CitRWP promoters from apomictic Citrus and Fortunella , due to either two or three miniature inverted transposon element (MITE) insertions. A transcription factor, FhARID , encoding an AT-rich interaction domain-containing protein binds to the MITEs in the promoter of apomictic varieties, which facilitates induction of nucellar embryogenesis. This study provides evolutionary genomic and molecular insights into apomixis in Citrinae and has potential ramifications for citrus breeding. [ABSTRACT FROM AUTHOR]

Intrinsic improvement of iron (Fe) concentration in rice grains, called rice Fe biofortification, is a promising countermeasure against widespread human Fe deficiency. In this study, two novel rice Fe biofortification approaches are reported. The first approach (Y approach) involved the expression of maize YELLOW STRIPE 1 controlled by the HEAVY METAL ATPASE 2 promoter. The Y approach increased the polished grain Fe concentrations up to 4.8-fold compared with the non-transgenic (NT) line. The second approach (T approach) involved the expression of rice TRANSPORTER OF MUGINEIC ACID 1 controlled by the FERRIC REDUCTASE DEFECTIVE LIKE 1 promoter. The T approach increased the polished grain Fe concentrations by up to 3.2-fold. No synergistic increases in the polished grain Fe concentrations were observed when Y and T approaches were combined (YT approach). However, the polished grain Fe concentrations further increased by 5.1- to 9.3-fold compared with the NT line, when YT approach was combined with the endosperm-specific expression of FERRITIN (YTF approach), or when YTF approach was combined with the constitutive expression of NICOTIANAMINE SYNTHASE (YTFN approach). Total grain weight per plant in most Y, T, YT, and YTFN lines was comparable to that in the NT line, while it was significantly decreased in most YTF lines. The novel approaches reported in this study expand the portfolio of genetic engineering strategies that can be used for Fe biofortification in rice. [ABSTRACT FROM AUTHOR]

In cereal crops, starch synthesis and storage depend mainly on a specialized class of plastids, termed amyloplasts. Despite the importance of starch, the molecular machinery regulating starch synthesis and amyloplast development remains largely unknown. Here, we report the characterization of the rice (Oryza sativa) floury endosperm7 (flo7) mutant, which develops a floury-white endosperm only in the periphery and not in the inner portion. Consistent with the phenotypic alternation in flo7 endosperm, the flo7 mutant had reduced amylose content and seriously disrupted amylopectin structure only in the peripheral endosperm. Notably, flo7 peripheral endosperm cells showed obvious defects in compound starch grain development. Map-based cloning of FLO7 revealed that it encodes a protein of unknown function. FLO7 harbors an N-terminal transit peptide capable of targeting functional FLO7 fused to green fluorescent protein to amyloplast stroma in developing endosperm cells, and a domain of unknown function 1338 (DUF1338) that is highly conserved in green plants. Furthermore, our combined β-glucuronidase activity and RNA in situ hybridization assays showed that the FLO7 gene was expressed ubiquitously but exhibited a specific expression in the endosperm periphery. Moreover, a set of in vivo experiments demonstrated that the missing 32 aa in the flo7 mutant protein are essential for the stable accumulation of FLO7 in the endosperm. Together, our findings identify FLO7 as a unique plant regulator required for starch synthesis and amyloplast development within the peripheral endosperm and provide new insights into the spatial regulation of endosperm development in rice. [ABSTRACT FROM AUTHOR]

Wheat is one of the Neolithic founder crops domesticated ~10 500 years ago. Following the domestication episode, its evolution under domestication has resulted in various genetic modifications. Grain weight, embryo weight, and the interaction between those factors were examined among domesticated durum wheat and its direct progenitor, wild emmer wheat. Experimental data show that grain weight has increased over the course of wheat evolution without any parallel change in embryo weight, resulting in a significantly reduced (30%) embryo weight/grain weight ratio in domesticated wheat. The genetic factors associated with these modifications were further investigated using a population of recombinant inbred substitution lines that segregated for chromosome 2A. A cluster of loci affecting grain weight and shape was identified on the long arm of chromosome 2AL. Interestingly, a novel locus controlling embryo weight was mapped on chromosome 2AS, on which the wild emmer allele promotes heavier embryos and greater seedling vigour. To the best of our knowledge, this is the first report of a QTL for embryo weight in wheat. The results suggest a differential selection of grain and embryo weight during the evolution of domesticated wheat. It is argued that conscious selection by early farmers favouring larger grains and smaller embryos appears to have resulted in a significant change in endosperm weight/embryo weight ratio in the domesticated wheat. Exposing the genetic factors associated with endosperm and embryo size improves our understanding of the evolutionary dynamics of wheat under domestication and is likely to be useful for future wheat-breeding efforts. [ABSTRACT FROM AUTHOR]

Starch biosynthesis during rice endosperm development is important for grain quality, as it influences grain size and physico-chemical properties, which together determine rice eating quality. Cereal starch biosynthetic pathways have been comprehensively investigated; however, their regulation, especially by transcriptional repressors remains largely unknown. Here, we identified a DUF1645 domain-containing protein, STRESS_tolerance and GRAIN_LENGTH (OsSGL), that participates in regulating rice starch biosynthesis. Overexpression of OsSGL reduced total starch and amylose content in the endosperm compared with the wild type. Chromatin immunoprecipitation sequencing and RNA-seq analyses indicated that OsSGL targets the transcriptional activity of several starch and sucrose metabolism genes. In addition, ChIP-qPCR, yeast one-hybrid, EMSA and dual-luciferase assays demonstrated that OsSGL directly inhibits the expression of SUCROSE SYNTHASE 1 (OsSUS1) in the endosperm. Furthermore, OsSUS1 interacts with OsSGL to release its transcriptional repression ability. Unexpectedly, our results also show that knock down and mutation of OsSGL disrupts the starch biosynthetic pathway, causing lower starch and amylose content. Therefore, our findings demonstrate that accurate control of OsSGL homeostasis is essential for starch synthesis and grain quality. In addition, we revealed the molecular mechanism of OsSGL in regulating starch biosynthesis-related genes, which are required for grain quality. [ABSTRACT FROM AUTHOR]

Embryo and endosperm development are two well co-ordinated developmental processes in seed formation; however, signals involved in embryo and endosperm interactions remain poorly understood. It has been shown before that CLAVATA3/ESR-RELATED 19 (CLE19) peptide is able to trigger root meristem consumption in a CLV2-dependent manner. In this study, the role of CLE19 in Arabidopsis seed development was explored using antagonistic peptide technology. CLE19 is expressed in the epidermal layers of the cotyledon primordia, hypocotyl, and root cap in the embryo. Transgenic plants carrying an antagonistic CLE19G6T construct expressed under the control of CLE19 regulatory elements exhibited a dominant seed abortion phenotype, with defective cotyledon establishment in embryos and delayed nuclear proliferation and cellularization in endosperms. Ectopic expression of CLE19G6T in Arabidopsis under the control of an endosperm-specific ALE1 promoter led to a similar defect in cotyledon establishment in embryos but without an evident effect on endosperm development. We therefore propose that CLE19 may act as a mobile peptide co-ordinating embryo and endosperm development. [ABSTRACT FROM AUTHOR]

Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein-protein interactions in maize and wheat amyloplasts. This study investigated whether protein-protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700 kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200-400 kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs-BEs, and, among BE isozymes, BEIIa-Pho1, and pullulanase-type DBE-BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch. [ABSTRACT FROM AUTHOR]

Towards the aim of examining the potential function of KORRIGAN (KOR), a highly conserved membrane-bound endoglucanase, in reproductive development, here transgenic evidence is provided that a cotton (Gossypium hirsutum) endoglucanase, GhKOR1, plays significant roles in endosperm and embryo development. RNA interference (RNAi)- and co-suppression-mediated down-regulation of GhKOR1 resulted in smaller filial tissue and reduced seed weight, which were characterized by disrupted endosperm cellularization and delayed embryo development, leading to a delayed germination and a weak growth of seedlings early in development. The transgenic seeds exhibited fewer and smaller endosperm cells with irregular and brittle cell walls, and their embryos developed only to the globular stage at 10 days post-anthesis (DPA) when the wild-type endosperm has become highly cellularized and the embryo has progressed to the heart stage. The transgenic seed also displayed a significant reduction of callose in the seed coat transfer cells and reduced cellulose content both in the seed coat and in mature fibres. These findings demonstrate that GhKOR1 is required for the developmental of both seed filial and maternal tissues and the establishment of seedling vigour. [ABSTRACT FROM AUTHOR]

A thorough study of starch biosynthesis and deposition in a non-domesticated wild grass was performed using Brachypodium distachyon as a model.Brachypodium distachyon is a non-domesticated cereal. Nonetheless, Brachypodium was recently introduced as a model plant for temperate cereals. This study compares grain starch metabolism in Brachypodium and barley (Hordeum vulgare). In Brachypodium, we identified and annotated 28 genes involved in starch metabolism and identified important motifs including transit peptides and putative carbohydrate-binding modules (CBMs) of the families CBM20, CBM45, CBM48, and CBM53. Starch content was markedly lower in Brachypodium grains (12%) compared to barley grains (47%). Brachypodium starch granules were doughnut shaped and bimodally distributed into distinct small B-type (2.5–10 µm) and very small C-type (0.5–2.5 µm) granules. Large A-type granules, typical of cereals, were absent. Starch-bound phosphate, important for starch degradation, was 2-fold lower in Brachypodium compared with barley indicating different requirements for starch mobilization. The amylopectin branch profiles were similar and the amylose content was only slightly higher compared with barley cv. Golden Promise. The crystallinity of Brachypodium starch granules was low (10%) compared to barley (20%) as determined by wide-angle X-ray scattering (WAXS) and molecular disorder was confirmed by differential scanning calorimetry (DSC). The expression profiles in grain for most genes were distinctly different for Brachypodium compared to barley, typically showing earlier decline during the course of development, which can explain the low starch content and differences in starch molecular structure and granule characteristics. High transitory starch levels were observed in leaves of Brachypodium (2.8% after 14h of light) compared to barley (1.9% after 14h of light). The data suggest important pre-domesticated features of cereals. [ABSTRACT FROM PUBLISHER]

Arabinoxylans (AX) and (1→3),(1→4)-β-glucans (BG) are the major components of wheat grain cell walls. Although incompletely described at the molecular level, it is known that the chemical and distributional heterogeneity of these compounds impacts the quality and use of wheat. In this work, an emerging technique based on MALDI mass spectrometry imaging (MSI) was employed to map variations in the quantity, localization, and structure of these polysaccharides in the endosperm during wheat maturation. MALDI MSI couples detailed structural information with the spatial localization observed at the micrometer scale. The enzymic hydrolysis of AX and BG was performed directly on the grain sections, resulting in the efficient formation of smaller oligosaccharides that are easily measurable through MS, with no relocation across the grain. The relative quantification of the generated oligosaccharides was achieved. The method was validated by confirming data previously obtained using other analytical techniques. Furthermore, in situ analysis of grain cell walls through MSI revealed previously undetectable intense acetylation of AX in young compared to mature grains, together with findings concerning the feruloylation of AX and different structural features of BG. These results provide new insights into the physiological roles of these polysaccharides in cell walls and the specificity of the hydrolytic enzymes involved. [ABSTRACT FROM PUBLISHER]

Rice seed storage proteins glutelin and α-globulin are synthesized in the endoplasmic reticulum (ER) and deposited in protein storage vacuoles (PSVs). Sar1, a small GTPase, acts as a molecular switch to regulate the assembly of coat protein complex II, which exports secretory protein from the ER to the Golgi apparatus. To reveal the route by which glutelin and α-globulin exit the ER, four putative Sar1 genes (OsSar1a/b/c/d) were cloned from rice, and transgenic rice were generated with Sar1 overexpressed or suppressed by RNA interference (RNAi) specifically in the endosperm under the control of the rice glutelin promoter. Overexpression or suppression of any OsSar1 did not alter the phenotype. However, simultaneous knockdown of OsSar1a/b/c resulted in floury and shrunken seeds, with an increased level of glutelin precursor and decreased level of the mature α- and β-subunit. OsSar1abc RNAi endosperm generated numerous, spherical, novel protein bodies with highly electron-dense matrixes containing both glutelin and α-globulin. Notably, the novel protein bodies were surrounded by ribosomes, showing that they were derived from the ER. Some of the ER-derived dense protein bodies were attached to a blebbing structure containing prolamin. These results indicated that OsSar1a/b/c play a crucial role in storage proteins exiting from the ER, with functional redundancy in rice endosperm, and glutelin and α-globulin transported together from the ER to the Golgi apparatus by a pathway mediated by coat protein complex II. [ABSTRACT FROM AUTHOR]

Pollen tube growth and endosperm development are important for fertilization and seed formation. The genetic mechanism of the processes remains poorly understood. This study reports the functional characterization of AtTFIIB1 in pollen tube growth and endosperm development. AtTFIIB1 shares 86% and 44% similarity with AtTFIIB2 and AtTFIIB3/AtpBRP2, respectively. It is expressed in many tissues including vegetative nuclei and generative cells of pollen grains and pollen tubes, endosperm, and embryos. It is thus different from AtTFIIB2, whose expression is not found in the endosperm and vegetative nucleus of mature pollen, and AtTFIIB3/AtpBRP2, which is expressed mostly in male gametophytes and weakly in seeds. Mutations in AtTFIIB1 caused a drastic retardation of pollen tube growth and endosperm development, as well as impaired pollen tube guidance and reception, leading to disruption of fertilization and seed development. Expression of AtTFIIB2 driven by the AtTFIIB1 promoter could restore the defective pollen tube growth, guidance, and reception completely, but only partially recovered the seed development in attfiib1, whilst expression of AtTFIIB3/AtpBRP2 driven by the AtTFIIB1 promoter could rescue only the defective attfiib1 seeds. All these results suggest that AtTFIIB1 plays important roles in pollen tube growth, guidance, and reception as well as endosperm development and is partially functionally different from AtTFIIB2 and AtTFIIB3/AtpBRP2. [ABSTRACT FROM AUTHOR]

Plants have duplicate versions of the oxidative pentose phosphate pathway (oxPPP) enzymes with a subset localized to the chloroplast. The chloroplast oxPPP provides NADPH and pentose sugars for multiple metabolic pathways. This study identified two loss-of-function alleles of the Zea mays (maize) chloroplast-localized oxPPP enzyme 6-phosphogluconate dehydrogenase (6PGDH). These mutations caused a rough endosperm seed phenotype with reduced embryo oil and endosperm starch. Genetic translocation experiments showed that pgd3 has separate, essential roles in both endosperm and embryo development. Endosperm metabolite profiling experiments indicated that pgd3 shifts redox-related metabolites and increases reducing sugars similar to starch-biosynthetis mutants. Heavy isotope-labelling experiments indicates that carbon flux into starch is altered in pgd3 mutants. Labelling experiments with a loss of cytosolic 6PGDH did not affect flux into starch. These results support the known role for plastid-localized oxPPP in oil synthesis and argue that amyloplast-localized oxPPP reactions are integral to endosperm starch accumulation in maize kernels. [ABSTRACT FROM AUTHOR]

The relationship between the solubility, crystallinity, and length of the unit chains of plant storage α-glucan was investigated by manipulating the chain length of α-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIaI, coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total α-glucan in the isa1 mutant, whereas it was only 35–70% in the transgenic SSIIaI/isa1 lines. Thus, the α-glucans from the SSIIaI/isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the α-glucans of the insoluble fraction, while no crystallinity was confirmed for α-glucans in isa1. Concerning the degree of polymerization (DP) ≤30, the chain lengths of these α-glucans differed significantly in the order of SSIIaI/isa1 insoluble > SSIIaI/isa1 soluble > α-glucans in isa1. The amount of long chains with DP ≥33 was higher in the insoluble fraction α-glucans than in the other two α-glucans. No difference was observed in the chain length distributions of the β-amylase limit dextrins among these α-glucans. These results suggest that in the SSIIaI/isa1 transgenic lines, the unit chains of α-glucans were elongated by SSIIaI, whereas the expression of SSIIaI did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIaI. [ABSTRACT FROM PUBLISHER]

Starch synthase I (SSI) contributes the majority of the starch synthase activity in developing maize endosperm. In this work, the effects of various plant hormones and sugars on the expression of the starch synthase I gene (ZmSSI) in developing maize endosperms were examined. The accumulation of ZmSSI mRNA was induced using abscisic acid (ABA) but not with glucose, sucrose, or gibberellin treatment. To investigate the molecular mechanism underlying this effect, the ZmSSI promoter region (–1537 to +51) was isolated and analysed. A transient expression assay in maize endosperm tissue showed that the full-length ZmSSI promoter is activated by ABA. The results of deletion and mutation assays demonstrated that a CACCG motif in the ZmSSI promoter is responsible for the ABA inducibility. The results of binding shift assays indicated that this CACCG motif interacts with the maize ABI4 protein in vitro. The overexpression of ABI4 in endosperm tissue enhanced the activity of a promoter containing the CACCG motif in the absence of ABA treatment. Expression pattern analysis indicated that the transcription pattern of ABI4 in the developing maize endosperm was induced by ABA treatment but was only slightly affected by glucose or sucrose treatment. Taken together, these data indicate that ABI4 binds to the CACCG motif in the ZmSSI promoter and mediates its ABA inducibility. [ABSTRACT FROM PUBLISHER]

The biotechnological approach to improve performance or yield of crops or for engineering metabolic pathways requires the expression of a number of transgenes, each with a specific promoter to avoid induction of silencing mechanisms. In maize (Zea mays), used as a model for cereals, an efficient Agrobacterium tumefaciens-mediated transformation system has been established that is applied for translational research. In the current transformation vectors, the promoters of the 35S gene of the cauliflower mosaic virus and of the ubiquitin gene of maize are often used to drive the bialaphos-selectable marker and the transgene, respectively. To expand the number of promoters, genes with either constitutive or seed-specific expression were selected in Brachypodium distachyon, a model grass distantly related to maize. After the corresponding Brachypodium promoters had been fused to the β-glucuronidase reporter gene, their activity was followed throughout maize development and quantified in a fluorimetric assay with the 4-methylumbelliferyl β-D-glucuronide substrate. The promoters pBdEF1α and pBdUBI10 were constitutively and highly active in maize, whereas pBdGLU1 was clearly endosperm-specific, hence, expanding the toolbox for transgene analysis in maize. The data indicate that Brachypodium is an excellent resource for promoters for transgenic research in heterologous cereal species. [ABSTRACT FROM AUTHOR]

House dust mites (HDM) are the most common source of indoor allergens and are associated with allergic diseases worldwide. To benefit allergic patients, safer and non-invasive mucosal routes of oral administration are considered to be the best alternative to conventional allergen-specific immunotherapy. In this study, transgenic rice was developed expressing derivatives of the major HDM allergen Der f 2 with reduced Der f 2-specific IgE reactivity by disrupting intramolecular disulphide bonds in Der f 2. These derivatives were produced specifically as secretory proteins in the endosperm tissue of seeds under the control of the endosperm-specific glutelin GluB-1 promoter. Notably, modified Der f 2 derivatives aggregated in the endoplasmic reticulum (ER) lumen and were deposited in a unique protein body (PB)-like structure tentatively called the Der f 2 body. Der f 2 bodies were characterized by their intracellular localization and physico-chemical properties, and were distinct from ER-derived PBs (PB-Is) and protein storage vacuoles (PB-IIs). Unlike ER-derived organelles such as PB-Is, Der f 2 bodies were rapidly digested in simulated gastric fluid in a manner similar to that of PB-IIs. Oral administration in mice of transgenic rice seeds containing Der f 2 derivatives encapsulated in Der f 2 bodies suppressed Der f 2-specific IgE and IgG production compared with that in mice fed non-transgenic rice seeds, and the effect was dependent on the type of Der f 2 derivative expressed. These results suggest that engineered hypoallergenic Der f 2 derivatives expressed in the rice seed endosperm could serve as a basis for the development of viable strategies for the oral delivery of vaccines against HDM allergy. [ABSTRACT FROM PUBLISHER]

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80–90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published Km values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective Km values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass–action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways. [ABSTRACT FROM PUBLISHER]

Starch synthase (SS) I and IIIa are the first and second largest components of total soluble SS activity, respectively, in developing japonica rice (Oryza sativa L.) endosperm. To elucidate the distinct and overlapping functions of these enzymes, double mutants were created by crossing the ss1 null mutant with the ss3a null mutant. In the F2 generation, two opaque seed types were found to have either the ss1ss1/SS3ass3a or the SS1ss1/ss3ass3a genotype. Phenotypic analyses revealed lower SS activity in the endosperm of these lines than in those of the parent mutant lines since these seeds had different copies of SSI and SSIIIa genes in a heterozygous state. The endosperm of the two types of opaque seeds contained the unique starch with modified fine structure, round-shaped starch granules, high amylose content, and specific physicochemical properties. The seed weight was ∼90% of that of the wild type. The amount of granule-bound starch synthase I (GBSSI) and the activity of ADP-glucose pyrophosphorylase (AGPase) were higher than in the wild type and parent mutant lines. The double-recessive homozygous mutant prepared from both ss1 and ss3a null mutants was considered sterile, while the mutant produced by the leaky ss1 mutant×ss3a null mutant cross was fertile. This present study strongly suggests that at least SSI or SSIIIa is required for starch biosynthesis in rice endosperm. [ABSTRACT FROM PUBLISHER]

Germination of endospermic seeds is partly regulated by the micropylar endosperm, which acts as constraint to radicle protrusion. Gibberellin (GA) signalling pathways control coat-dormancy release, endosperm weakening, and organ expansion during seed germination. Three GIBBERELLIN INSENSITIVE DWARF1 (GID1) GA receptors are known in Arabidopsis thaliana: GID1a, GID1b, and GID1c. Molecular phylogenetic analysis of angiosperm GID1s reveals that they cluster into two eudicot (GID1ac, GID1b) groups and one monocot group. Eudicots have at least one gene from each of the two groups, indicating that the different GID1 receptors fulfil distinct roles during plant development. A comparative Brassicaceae approach was used, in which gid1 mutant and whole-seed transcript analyses in Arabidopsis were combined with seed-tissue-specific analyses of its close relative Lepidium sativum (garden cress), for which three GID1 orthologues were cloned. GA signalling via the GID1ac receptors is required for Arabidopsis seed germination, GID1b cannot compensate for the impaired germination of the gid1agid1c mutant. Transcript expression patterns differed temporarily, spatially, and hormonally, with GID1b being distinct from GID1ac in both species. Endosperm weakening is mediated, at least in part, through GA-induced genes encoding cell-wall-modifying proteins. A suppression subtraction hybridization (SSH) cDNA library enriched for sequences that are highly expressed during early germination in the micropylar endosperm contained expansins and xyloglucan endo-transglycosylases/hydrolases (XTHs). Their transcript expression patterns in both species strongly suggest that they are regulated by distinct GID1-mediated GA signalling pathways. The GID1ac and GID1b pathways seem to fulfil distinct regulatory roles during Brassicaceae seed germination and seem to control their downstream targets distinctly. [ABSTRACT FROM PUBLISHER]

Starch within the endosperm of most species of the Triticeae has a unique bimodal granule morphology comprising large lenticular A-type granules and smaller near-spherical B-type granules. However, a few wild wheat species (Aegilops) are known to lack B-granules. Ae. peregrina and a synthetic tetraploid Aegilops with the same genome composition (SU) were found to differ in B-granule number. The synthetic tetraploid had normal A- and B-type starch granules whilst Ae. peregrina had only A-granules because the B-granules failed to initiate. A population segregating for B-granule number was generated by crossing these two accessions and was used to study the genetic basis of B-granule initiation. A combination of Bulked Segregant Analysis and QTL mapping identified a major QTL located on the short arm of chromosome 4S that accounted for 44.4% of the phenotypic variation. The lack of B-granules in polyploid Aegilops with diverse genomes suggests that the B-granule locus has been lost several times independently during the evolution of the Triticeae. It is proposed that the B-granule locus is susceptible to silencing during polyploidization and a model is presented to explain the observed data based on the assumption that the initiation of B-granules is controlled by a single major locus per haploid genome. [ABSTRACT FROM PUBLISHER]

After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions. [ABSTRACT FROM PUBLISHER]

Grain development and its evolution in grasses remains poorly understood, despite cereals being our most important source of food. The grain, for which many grass species have been domesticated, is a single-seeded fruit with prominent and persistent endosperm. Brachypodium distachyon, a small wild grass, is being posited as a new model system for the temperate small grain cereals, but little is known about its endosperm development and how this compares with that of the domesticated cereals. A cellular and molecular map of domains within the developing Brachypodium endosperm is constructed. This provides the first detailed description of grain development in Brachypodium for the reference strain, Bd21, that will be useful for future genetic and comparative studies. Development of Brachypodium grains is compared with that of wheat. Notably, the aleurone is not regionally differentiated as in wheat, suggesting that the modified aleurone region may be a feature of only a subset of cereals. Also, the central endosperm and the nucellar epidermis contain unusually prominent cell walls that may act as a storage material. The composition of these cell walls is more closely related to those of barley and oats than to those of wheat. Therefore, although endosperm development is broadly similar to that of temperate small grain cereals, there are significant differences that may reflect its phylogenetic position between the Triticeae and rice. [ABSTRACT FROM PUBLISHER]

Haem oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury in animals and plants. In this report, it is confirmed that programmed cell death (PCD) in wheat aleurone layers is stimulated by GA and prevented by ABA. Meanwhile, HO activity and HO-1 protein expression exhibited lower levels in GA-treated layers, whereas the hydrogen peroxide (H2O2) content was apparently increased. The pharmacology approach illustrated that scavenging or accumulating H2O2 either delayed or accelerated GA-induced PCD. Furthermore, pretreatment with the HO-1 specific inhibitor, zinc protoporphyrin IX (ZnPPIX), before exposure to GA, not only decreased HO activity but also accelerated GA-induced PCD significantly. The application of the HO-1 inducer, haematin, and the enzymatic reaction product of HO, carbon monoxide (CO) aqueous solution, both of which brought about a noticeable induction of HO expression, substantially prevented GA-induced PCD. These effects were reversed when ZnPPIX was added, suggesting that HO in vivo played a role in delaying PCD. Meanwhile, catalase (CAT) and ascorbate peroxidase (APX) activities or transcripts were enhanced by haematin, CO, or bilirubin (BR), the catalytic by-product of HO. This enhancement resulted in a decrease in H2O2 production and a delay in PCD. In addition, the antioxidants butylated hydroxytoluene (BHT), dithiothreitol (DTT), and ascorbic acid (AsA) were able not only to delay PCD but also to mimic the effects of haematin and CO on HO up-regulation. Overall, the above results suggested that up-regulation of HO expression delays PCD through the down-regulation of H2O2 production. [ABSTRACT FROM PUBLISHER]

In plants, the amino acids tyrosine and phenylalanine are synthesized from arogenate by arogenate dehydrogenase and arogenate dehydratase, respectively, with the relative flux to each being tightly controlled. Here the characterization of a maize opaque endosperm mutant (mto140), which also shows retarded vegetative growth, is described The opaque phenotype co-segregates with a Mutator transposon insertion in an arogenate dehydrogenase gene (zmAroDH-1) and this led to the characterization of the four-member family of maize arogenate dehydrogenase genes (zmAroDH-1–zmAroDH-4) which share highly similar sequences. A Mutator insertion at an equivalent position in AroDH-3, the most closely related family member to AroDH-1, is also associated with opaque endosperm and stunted vegetative growth phenotypes. Overlapping but differential expression patterns as well as subtle mutant effects on the accumulation of tyrosine and phenylalanine in endosperm, embryo, and leaf tissues suggest that the functional redundancy of this gene family provides metabolic plasticity for the synthesis of these important amino acids. mto140/arodh-1 seeds shows a general reduction in zein storage protein accumulation and an elevated lysine phenotype typical of other opaque endosperm mutants, but it is distinct because it does not result from quantitative or qualitative defects in the accumulation of specific zeins but rather from a disruption in amino acid biosynthesis. [ABSTRACT FROM PUBLISHER]

Since the cereal endosperm is a dead tissue in the mature grain, β-oxidation is not possible there. This raises the question about the use of the endosperm oil in cereal grains during germination. In this study, mobilization of lipids in different tissues of germinating oat grains was analysed using thin-layer and gas chromatography. The data imply that the oat endosperm oil [triacylglycerol (TAG)] is not a dead-end product as it was absorbed by the scutellum, either as free fatty acids (FFAs) released from TAG or as intact TAG immediately degraded to FFAs. These data were supported by light and transmission electron microscopy (LM and TEM) studies where close contact between endosperm lipid droplets and the scutellum was observed. The appearance of the fused oil in the oat endosperm changed into oil droplets during germination in areas close to the aleurone and the scutellar epithelium. However, according to the data obtained by TEM these oil droplets are unlikely to be oil bodies surrounded by oleosins. Accumulation of FFA pools in the embryo suggested further transport of FFAs from the scutellum. Noticeably high levels of TAG were also accumulated in the embryo but were not synthesized by re-esterification from imported FFAs. Comparison between two oat cultivars with different amounts of oil and starch in the endosperm suggests that an increased oil to starch ratio in oat grains does not significantly impact the germination process. [ABSTRACT FROM PUBLISHER]

The roles of starch branching enzyme (SBE, EC 2.4.1.18) IIa and SBE IIb in defining the structure of amylose and amylopectin in barley (Hordeum vulgare) endosperm were examined. Barley lines with low expression of SBE IIa or SBE IIb, and with the low expression of both isoforms were generated through RNA-mediated silencing technology. These lines enabled the study of the role of each of these isoforms in determining the amylose content, the distribution of chain lengths, and the frequency of branching in both amylose and amylopectin. In lines where both SBE IIa and SBE IIb expression were reduced by >80%, a high amylose phenotype (>70%) was observed, while a reduction in the expression of either of these isoforms alone had minor impact on amylose content. The structure and properties of the high amylose starch resulting from the concomitant reduction in the expression of both isoforms of SBE II in barley were found to approximate changes seen in amylose extender mutants of maize, which result from lesions eliminating expression of the SBE IIb gene. Amylopectin chain length distribution analysis indicated that both SBE IIa and SBE IIb isoforms play distinct roles in determining the fine structure of amylopectin. A significant reduction in the frequency of branches in amylopectin was noticed only when both SBE IIa and SBE IIb were reduced, whereas there was a significant increase in the branching frequency of amylose when SBE IIb alone was reduced. Functional interactions between SBE isoforms are suggested, and a possible inhibitory role of SBE IIb on other SBE isoforms is discussed. [ABSTRACT FROM PUBLISHER]

During rice (Oryza sativa L.) seed development, the primary endosperm nucleus undergoes a series of divisions without cytokinesis, producing a multinucleate cell, known as a syncytium. After several rounds of rapid nuclear proliferation, the syncytium ceases to undergo mitosis; thereafter, the syncytium is partitioned into individual cells by a specific type of cytokinesis called cellularization. The transition between syncytium and cellularization is important in determining the final seed size and is a model for studying the cell cycle and cytokinesis. The involvement of cyclin-dependent kinase (CDK) inhibitors (CKIs) in cell cycle control was investigated here during the transition between syncytium and cellularization. It was found that one of the rice CKIs, Orysa;KRP3, is strongly expressed in the caryopsis at 2 d after flowering (DAF), and its expression is significantly reduced at 3 DAF. The other CKI transcripts did not show such a shift at 2 DAF. In situ hybridization analysis revealed that Orysa;KRP3 is expressed in multinucleate syncytial endosperm at 2 DAF, but not in cellularized endosperm at 3 DAF. Two-hybrid assays showed that Orysa;KRP3 binds Orysa;CDKA;1, Orysa;CDKA;2, Orysa;CycA1;1, and Orysa;CycD2;2. By contrast, Orysa;CDKB2;1 and Orysa;CycB2;2 do not show binding to Orysa;KRP3. Orysa;KRP3 was able to rescue yeast premature cell division due to the dominant positive expression of mutant rice CDKA;1 indicating that Orysa;KRP3 inhibited rice CDK. These data suggest that Orysa;KRP3 is involved in cell cycle control of syncytial endosperm. [ABSTRACT FROM PUBLISHER]

The amylose extender (ae–) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein–protein interactions in amyloplasts of ae– mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein–protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae– amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae– endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae–), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis. [ABSTRACT FROM PUBLISHER]

The movement of water into harvest-ripe grains of dormant and non-dormant genotypes of wheat (Triticum aestivum L.) was investigated using Magnetic Resonance Micro-Imaging (MRMI). Images of virtual sections, both longitudinal and transverse, throughout the grain were collected at intervals after the start of imbibition and used to reconstruct a picture of water location within the different grain tissues and changes over time. The observations were supplemented by the weighing measurements of water content and imbibition of grains in water containing I2/KI which stains starch and lipid, thereby acting as a marker for water. In closely related genotypes, with either a dormant or a non-dormant phenotype, neither the rate of increase in water content nor the pattern of water distribution within the grain was significantly different until 18 h, when germination became apparent in the non-dormant genotype. Water entered the embryo and scutellum during the very early stages of imbibition through the micropyle and by 2 h water was clearly evident in the micropyle channel. After 12 h of imbibition, embryo structures such as the coleoptile and radicle were clearly distinguished. Although water accumulated between the inner (seed coat) and outer (pericarp) layers of the coat surrounding the grain, there was no evidence for movement of water directly across the coat and into the underlying starchy endosperm. [ABSTRACT FROM PUBLISHER]

Kernel weight is an important factor determining grain yield and nutritional quality in sorghum, yet the developmental processes underlying the genotypic differences in potential kernel weight remain unclear. The aim of this study was to determine the stage in development at which genetic effects on potential kernel weight were realized, and to investigate the developmental mechanisms by which potential kernel weight is controlled in sorghum. Kernel development was studied in two field experiments with five genotypes known to differ in kernel weight at maturity. Pre-fertilization floret and ovary development was examined and post-fertilization kernel-filling characteristics were analysed. Large kernels had a higher rate of kernel filling and contained more endosperm cells and starch granules than normal-sized kernels. Genotypic differences in kernel development appeared before stamen primordia initiation in the developing florets, with sessile spikelets of large-seeded genotypes having larger floret apical meristems than normal-seeded genotypes. At anthesis, the ovaries for large-sized kernels were larger in volume, with more cells per layer and more vascular bundles in the ovary wall. Across experiments and genotypes, there was a significant positive correlation between kernel dry weight at maturity and ovary volume at anthesis. Genotypic effects on meristem size, ovary volume, and kernel weight were all consistent with additive genetic control, suggesting that they were causally related. The pre-fertilization genetic control of kernel weight probably operated through the developing pericarp, which is derived from the ovary wall and potentially constrains kernel expansion. [ABSTRACT FROM PUBLISHER]