Suitable nitrogen fertilizer application drives the endosperm development and starch synthesis to improve the physicochemical properties of common buckwheat grain.

The effects of nitrogen (N) fertilizer on endosperm development, starch component, key enzyme activity and grain quality of common buckwheat were investigated in this study. The results showed that N fertilization significantly enhanced the number and area of endosperm cells, and significant increases were also observed in the contents of amylose, amylopectin and total starch. With increasing N level, the activities of key enzyme significantly increased showing the maximum under the N2 level (180 kg N ha-1), and then decreased under high N level. As N level increased, the ash, crude protein and amylose content varied from 1.36 to 2.25 %, from 7.99 to 15.84 % and from 22.69 to 27.64 %, respectively. The gelatinization enthalpy significantly increased with the range of 3.46-5.66 J/g, while no change was found in crystalline structure of common buckwheat flour. These results indicated that appropriate N application could effectively improve the endosperm development, starch synthesis and accumulation, and grain properties of common buckwheat, with the best effect under the level of 180 kg N ha-1.

Mass spectrometry imaging of oligosaccharides following in situ enzymatic treatment of maize kernels.

In recent years enzymatic treatment of maize has been utilized in the wet-milling process to increase the yield of extracted starch, proteins, and other constituents. One of the strategies to obtain this goal is to add enzymes that break down insoluble cell-wall polysaccharides which would otherwise entrap starch granules. Due to the high complexity of maize polysaccharides, this goal is not easily achieved and more knowledge about the substrate and enzyme performances is needed. To gather information of both enzyme performance and increase substrate understanding, a method was developed using mass spectrometry imaging (MSI) to analyze degradation products from polysaccharides following enzymatic treatment of the maize endosperm. Different enzymes were spotted onto cryosections of maize kernels which had been pre-treated with an amylase to remove starch. The cryosections were then incubated for 17 h. before mass spectrometry images were generated with a MALDI-MSI setup. The images showed varying degradation products for the different enzymes observed as pentose oligosaccharides differing with regards to sidechains and the number of linked pentoses. The method proved suitable for identifying the reaction products formed after reaction with different xylanases and arabinofuranosidases and for characterization of the complex arabinoxylan substrate in the maize kernel. HYPOTHESES: Mass spectrometry imaging can be a useful analytical tool for obtaining information of polysaccharide constituents and enzyme performance from maize samples.

Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

Endosperm and Seed Transcriptomes Reveal Possible Roles for Small RNA Pathways in Wild Tomato Hybrid Seed Failure.

Crosses between the wild tomato species Solanum peruvianum and Solanum chilense result in hybrid seed failure (HSF), characterized by endosperm misdevelopment and embryo arrest. We previously showed that genomic imprinting, the parent-of-origin-dependent expression of alleles, is perturbed in the hybrid endosperm, with many of the normally paternally expressed genes losing their imprinted status. Here, we report transcriptome-based analyses of gene and small RNA (sRNA) expression levels. We identified 2,295 genes and 387 sRNA clusters as differentially expressed when comparing reciprocal hybrid seed to seeds and endosperms from the two within-species crosses. Our analyses uncovered a pattern of overdominance in endosperm gene expression in both hybrid cross directions, in marked contrast to the patterns of sRNA expression in whole seeds. Intriguingly, patterns of increased gene expression resemble the previously reported increased maternal expression proportions in hybrid endosperms. We identified physical clusters of sRNAs; differentially expressed sRNAs exhibit reduced transcript abundance in hybrid seeds of both cross directions. Moreover, sRNAs map to genes coding for key proteins involved in epigenetic regulation of gene expression, suggesting a regulatory feedback mechanism. We describe examples of genes that appear to be targets of sRNA-mediated gene silencing; in these cases, reduced sRNA abundance is concomitant with increased gene expression in hybrid seeds. Our analyses also show that S. peruvianum dominance impacts gene and sRNA expression in hybrid seeds. Overall, our study indicates roles for sRNA-mediated epigenetic regulation in HSF between closely related wild tomato species.

Molecular Control of Oil Metabolism in the Endosperm of Seeds.

In angiosperm seeds, the endosperm develops to varying degrees and accumulates different types of storage compounds remobilized by the seedling during early post-germinative growth. Whereas the molecular mechanisms controlling the metabolism of starch and seed-storage proteins in the endosperm of cereal grains are relatively well characterized, the regulation of oil metabolism in the endosperm of developing and germinating oilseeds has received particular attention only more recently, thanks to the emergence and continuous improvement of analytical techniques allowing the evaluation, within a spatial context, of gene activity on one side, and lipid metabolism on the other side. These studies represent a fundamental step toward the elucidation of the molecular mechanisms governing oil metabolism in this particular tissue. In particular, they highlight the importance of endosperm-specific transcriptional controls for determining original oil compositions usually observed in this tissue. In the light of this research, the biological functions of oils stored in the endosperm of seeds then appear to be more diverse than simply constituting a source of carbon made available for the germinating seedling.

Intact endosperm cells in buckwheat flour limit starch gelatinization and digestibility in vitro.

The objective of this study was to determine the biophysical properties of buckwheat (BW) endosperm and their influences on detachment of intact cells, starch gelatinization and digestibility. The intact cells were isolated from BW kernels by dry milling and sieving. The microscopy and texture analysis showed intact endosperm cells could be detached easily due to the fragile structure and low hardness of BW endosperm. More than 70% intact cells were found in commercial light flour. The starch granules entrapped in intact cells exhibited a delay gelatinization and restricted swelling behavior (2-3 â„ƒ higher onset gelatinization temperature than isolated starch). Starch in BW flour had a markedly lower extent of digestion compared to the broken cells and isolated starch. This study provided a new mechanistic understanding of low glycemic index of BW food, and could guide the processing of BW flour to retain slow digestion properties.

Arabinoxylan, ß-glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo-SEM.

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of ß-glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo-SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with ß-glucan depositions on the inside. During germination, arabinoxylan and ß-glucan are hydrolysed, but unlike ß-glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.

Transitions in wheat endosperm metabolism upon transcriptional induction of oil accumulation by oat endosperm WRINKLED1.

BACKGROUND: Cereal grains, including wheat (Triticum aestivum L.), are major sources of food and feed, with wheat being dominant in temperate zones. These end uses exploit the storage reserves in the starchy endosperm of the grain, with starch being the major storage component in most cereal species. However, oats (Avena sativa L.) differs in that the starchy endosperm stores significant amounts of oil. Understanding the control of carbon allocation between groups of storage compounds, such as starch and oil, is therefore important for understanding the composition and hence end use quality of cereals. WRINKLED1 is a transcription factor known to induce triacylglycerol (TAG; oil) accumulation in several plant storage tissues. RESULTS: An oat endosperm homolog of WRI1 (AsWRI1) expressed from the endosperm-specific HMW1Dx5 promoter resulted in drastic changes in carbon allocation in wheat grains, with reduced seed weight and a wrinkled seed phenotype. The starch content of mature grain endosperms of AsWRI1-wheat was reduced compared to controls (from 62 to 22% by dry weight (dw)), TAG was increased by up to nine-fold (from 0.7 to 6.4% oil by dw) and sucrose from 1.5 to 10% by dw. Expression of AsWRI1 in wheat grains also resulted in multiple layers of elongated peripheral aleurone cells. RNA-sequencing, lipid analyses, and pulse-chase experiments using 14C-sucrose indicated that futile cycling of fatty acids could be a limitation for oil accumulation. CONCLUSIONS: Our data show that expression of oat endosperm WRI1 in the wheat endosperm results in changes in metabolism which could underpin the application of biotechnology to manipulate grain composition. In particular, the striking effect on starch synthesis in the wheat endosperm indicates that an important indirect role of WRI1 is to divert carbon allocation away from starch biosynthesis in plant storage tissues that accumulate oil.

Light induces gene expression to enhance the synthesis of storage reserves in Brassica napus L. embryos.

KEY MESSAGE: In this manuscript, we disclosed the influence of light on the accumulation of storage reserves in B. napus embryos.1.Light induced the gene expression in the developing embryos of B. napus.2.Light promoted the starch synthesis in chloroplasts of B. napus embryos.3.Light enhanced the metabolic activity of storage reserve synthesis in B. napus embryos. Light influences the accumulation of storage reserves in embryos, but the molecular mechanism was not fully understood. Here, we monitored the effects of light on reserve biosynthesis in Brassica napus by comparing embryos from siliques grown in normal light conditions to those that were shaded or masked (i.e., darkened completely). Masked embryos developed more slowly, weighed less, and contained fewer proteins and lipids than control embryos. They also had fewer and smaller oil bodies than control embryos and lacked chloroplasts, where starch grains are usually synthesized. The levels of most amino acids, carbohydrates, and fatty acids were higher in masked embryos than in control or shaded embryos, whereas the levels of these metabolites in the masked endosperms were lower than those in control and shaded endosperm. Transcriptome analysis indicated that genes involved in photosynthesis (42 genes), amino acid biosynthesis (51 genes), lipid metabolism (61 genes), and sugar transport (13 genes) were significantly repressed in masked embryos. Our results suggest that light contributes to reserve accumulation in embryos by inducing the expression of metabolic genes, thereby enhancing the biosynthesis of storage reserves.

Characterization of a PLDζ2 Homology Gene from Developing Castor Bean Endosperm.

Castor oil contains approximately 90% ricinoleic acid (RA) which is stored mainly in the form of tri-ricinoleic acid containing triacylglycerols (TAG). Ricinoleate is synthesized from oleate (18:1n-9) esterified to the sn-2 position of phosphatidylcholine (PtdCho) catalyzed by oleoyl-12-hydroxylase. PtdCho-derived diacylglycerol (DAG) is an important substrate pool for TAG synthesis, and the interconversion between PtdCho and DAG has been shown to play a critical role in channeling hydroxy fatty acids (HFA) to TAG. Although phospholipase D (PLD) has been reported to catalyze the hydrolysis of PtdCho to produce phosphatidic acid which can then be converted to DAG, its potential functions in the channeling of RA from PtdCho to DAG and the assembly of RA on TAG is largely unknown. In the present study, 11 PLD genes were identified from the Castor Bean Genome Database. Gene expression analysis indicated that RcPLD9 is expressed at relatively high levels in developing seeds compared to other plant tissues. Sequence and phylogenetic analyses revealed that RcPLD9 is a homolog of Arabidopsis PLDζ2. Overexpression of RcPLD9 in the Arabidopsis CL7 line producing C18-HFA resulted in RA content reductions in the polar lipid fraction (mainly PtdCho) and mono-HFA-TAG, but increased RA content in di-HFA-TAG. Since part of RA in di-HFA-TAG is derived from HFA-DAG, the results indicated that RcPLD9 facilitates the channeling of RA from PtdCho to DAG for its assembly on TAG in developing seeds.

Genomic Characterization and Expressional Profiles of Autophagy-Related Genes (ATGs) in Oilseed Crop Castor Bean (Ricinus communis L.).

Cellular autophagy is a widely-occurring conserved process for turning over damaged organelles or recycling cytoplasmic contents in cells. Although autophagy-related genes (ATGs) have been broadly identified from many plants, little is known about the potential function of autophagy in mediating plant growth and development, particularly in recycling cytoplasmic contents during seed development and germination. Castor bean (Ricinus communis) is one of the most important inedible oilseed crops. Its mature seed has a persistent and large endosperm with a hard and lignified seed coat, and is considered a model system for studying seed biology. Here, a total of 34 RcATG genes were identified in the castor bean genome and their sequence structures were characterized. The expressional profiles of these RcATGs were examined using RNA-seq and real-time PCR in a variety of tissues. In particular, we found that most RcATGs were significantly up-regulated in the later stage of seed coat development, tightly associated with the lignification of cell wall tissues. During seed germination, the expression patterns of most RcATGs were associated with the decomposition of storage oils. Furthermore, we observed by electron microscopy that the lipid droplets were directly swallowed by the vacuoles, suggesting that autophagy directly participates in mediating the decomposition of lipid droplets via the microlipophagy pathway in germinating castor bean seeds. This study provides novel insights into understanding the potential function of autophagy in mediating seed development and germination.

MADS78 and MADS79 Are Essential Regulators of Early Seed Development in Rice.

MADS box transcription factors (TFs) are subdivided into type I and II based on phylogenetic analysis. The type II TFs regulate floral organ identity and flowering time, but type I TFs are relatively less characterized. Here, we report the functional characterization of two type I MADS box TFs in rice (Oryza sativa), MADS78 and MADS79 Transcript abundance of both these genes in developing seed peaked at 48 h after fertilization and was suppressed by 96 h after fertilization, corresponding to syncytial and cellularized stages of endosperm development, respectively. Seeds overexpressing MADS78 and MADS 79 exhibited delayed endosperm cellularization, while CRISPR-Cas9-mediated single knockout mutants showed precocious endosperm cellularization. MADS78 and MADS 79 were indispensable for seed development, as a double knockout mutant failed to make viable seeds. Both MADS78 and 79 interacted with MADS89, another type I MADS box, which enhances nuclear localization. The expression analysis of Fie1, a rice FERTILIZATION-INDEPENDENT SEED-POLYCOMB REPRESSOR COMPLEX2 component, in MADS78 and 79 mutants and vice versa established an antithetical relation, suggesting that Fie1 could be involved in negative regulation of MADS78 and MADS 79 Misregulation of MADS78 and MADS 79 perturbed auxin homeostasis and carbon metabolism, as evident by misregulation of genes involved in auxin transport and signaling as well as starch biosynthesis genes causing structural abnormalities in starch granules at maturity. Collectively, we show that MADS78 and MADS 79 are essential regulators of early seed developmental transition and impact both seed size and quality in rice.

Characterization of Oil Palm Acyl-CoA-Binding Proteins and Correlation of Their Gene Expression with Oil Synthesis.

Acyl-CoA-binding proteins (ACBPs) are involved in binding and trafficking acyl-CoA esters in eukaryotic cells. ACBPs contain a well-conserved acyl-CoA-binding domain. Their various functions have been characterized in the model plant Arabidopsis and, to a lesser extent, in rice. In this study, genome-wide detection and expression analysis of ACBPs were performed on Elaeis guineensis (oil palm), the most important oil crop in the world. Seven E. guineensis ACBPs were identified and classified into four groups according to their deduced amino acid domain organization. Phylogenetic analysis showed conservation of this family with other higher plants. All seven EgACBPs were expressed in most tissues while their differential expression suggests various functions in specific tissues. For example, EgACBP3 had high expression in inflorescences and stalks while EgACBP1 showed strong expression in leaves. Because of the importance of E. guineensis as an oil crop, expression of EgACBPs was specifically examined during fruit development. EgACBP3 showed high expression throughout mesocarp development, while EgACBP1 had enhanced expression during rapid oil synthesis. In endosperm, both EgACBP1 and EgACBP3 exhibited increased expression during seed development. These results provide important information for further investigations on the biological functions of EgACBPs in various tissues and, in particular, their roles in oil synthesis.

Active DNA Demethylation in Plants.

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.

A Combined Metabolomics and Fluxomics Analysis Identifies Steps Limiting Oil Synthesis in Maize Embryos.

Enhancing fatty acid synthesis (FAS) in maize (Zea mays) has tremendous potential nutritional and economic benefits due to the rapidly growing demand for vegetable oil. In maize kernels, the endosperm and the embryo are the main sites for synthesis and accumulation of starch and oil, respectively. So far, breeding efforts to achieve elevated oil content in maize have resulted in smaller endosperms and therefore lower yield. Directly changing their carbon metabolism may be the key to increasing oil content in maize kernels without affecting yield. To test this hypothesis, the intracellular metabolite levels were compared in maize embryos from two different maize lines, ALEXHO S K SYNTHETIC (Alex) and LH59, which accumulate 48% and 34% of oil, respectively. Comparative metabolomics highlighted the metabolites and pathways that were active in the embryos and important for oil production. The contribution of each pathway to FAS in terms of carbon, reductant, and energy provision was assessed by measuring the carbon flow through the metabolic network (13C-metabolic flux analysis) in developing Alex embryos to build a map of carbon flow through the central metabolism. This approach combined mathematical modeling with biochemical quantification to identify metabolic bottlenecks in FAS in maize embryos. This study describes a combination of innovative tools that will pave the way for controlling seed composition in important food crops.

WRINKLED1 homologs highly and functionally express in oil-rich endosperms of oat and castor.

Oat (Avena sativa) and castor (Ricinus communis) accumulate a large amount of lipids in their endosperms, however the molecular mechanism remains unknown. In this study, differences in oil regulators between oat and wheat (Triticum aestivum) as well as common features between oat and castor were tested by analyzing their transcriptomes with further q-PCR analysis. Results indicated that WRINKLED1 (WRI1) homologs and their target genes highly expressed in the endosperms of oat and castor, but not in the starchy endosperms of wheat. Expression pattern of WRI1s was in agreement with that of oil accumulation. Three AsWRI1s (AsWRI1a, AsWRI1b and AsWRI1c) and one RcWRI1 were identified in the endosperms of oat and castor, respectively. AsWRI1c lacks VYL motif, which is different from the other three WRI1s. Expressions of these four WRI1s all complemented the phenotypes of Arabidopsis wri1-1 mutant. Overexpression of these WRI1s in Arabidopsis and tobacco BY2 cells increased oil contents of seeds and total fatty acids of the cells, respectively. Moreover, this overexpression also resulted in up-regulations of WRI1 target genes, such as PKp-ß1. Taken together, our results suggest that high and functional expression of WRI1 play a key role in the oil-rich endosperms and the VYL motif is dispensable for WRI1 function.

A rapid and universal method for isolating starch granules in plant tissues.

Starch is the major form of carbohydrate storage in plants and exists as discrete starch granules (SGs). Isolation of high-quality SGs in different plant tissues is a prerequisite for studying the roles of SGs during plant growth, development, and responses to abiotic stress. However, it is difficult to isolate transitory SGs from leaves and storage SGs from pollen grains due to their small sizes and low quantities. Herein, we develop a novel method for isolating SGs by using the aqueous two-phase system (ATS) of ethanol/NaH2 PO4 . The ATS method efficiently separated SGs from contaminants based on their differences in density, solubility, and polarity. Using this method, we first isolated and purified three kinds of SGs from maize seeds, pollen, and leaves. The biochemical, microscopic, and proteomic analyses demonstrated the high purity of the isolated SGs. Proteomic analysis revealed distinct differences in SG-bound proteins between seed SGs and pollen SGs. As a simple, rapid, and low-cost method, the ATS-based method exhibits highly universal and reproducible results for starch-containing tissues in various plant species.

The overexpression of rice ACYL-CoA-BINDING PROTEIN2 increases grain size and bran oil content in transgenic rice.

As Oryza sativa (rice) seeds represent food for over three billion people worldwide, the identification of genes that enhance grain size and composition is much desired. Past reports have indicated that Arabidopsis thaliana acyl-CoA-binding proteins (ACBPs) are important in seed development but did not affect seed size. Herein, rice OsACBP2 was demonstrated not only to play a role in seed development and germination, but also to influence grain size. OsACBP2 mRNA accumulated in embryos and endosperm of germinating seeds in qRT-PCR analysis, while ß-glucuronidase (GUS) assays on OsACBP2pro::GUS rice transformants showed GUS expression in embryos, as well as the scutellum and aleurone layer of germinating seeds. Deletion analysis of the OsACBP2 5'-flanking region revealed five copies of the seed cis-element, Skn-I-like motif (-1486/-1482, -956/-952, -939/-935, -826/-822, and -766/-762), and the removal of any adversely affected expression in seeds, thereby providing a molecular basis for OsACBP2 expression in seeds. When OsACBP2 function was investigated using osacbp2 mutants and transgenic rice overexpressing OsACBP2 (OsACBP2-OE), osacbp2 was retarded in germination, while OsACBP2-OEs performed better than the wild-type and vector-transformed controls, in germination, seedling growth, grain size and grain weight. Transmission electron microscopy of OsACBP2-OE mature seeds revealed an accumulation of oil bodies in the scutellum cells, while confocal laser scanning microscopy indicated oil accumulation in OsACBP2-OE aleurone tissues. Correspondingly, OsACBP2-OE seeds showed gain in triacylglycerols and long-chain fatty acids over the vector-transformed control. As dietary rice bran contains beneficial bioactive components, OsACBP2 appears to be a promising candidate for enriching seed nutritional value.

Spatial and temporal distribution of cell wall polysaccharides during grain development of Brachypodium distachyon.

Brachypodium distachyon (Brachypodium) is now well considered as being a suitable plant model for studying temperate cereal crops. Its cell walls are phylogenetically intermediate between rice and poaceae, with a greater proximity to these latter. By microscopic and biochemical approaches, this work gives an overview of the temporal and spatial distribution of cell wall polysaccharides in the grain of Brachypodium from the end of the cellularization step to the maturation of grain. Variation in arabinoxylan chemical structure and distribution were demonstrated according to development and different grain tissues. In particular, the kinetic of arabinoxylan feruloylation was shown occuring later in the aleurone layers compared to storage endosperm. Mixed linked ß-glucan was detected in whole the tissues of Brachypodium grain even at late stage of development. Cellulose was found in both the storage endosperm and the outer layers. Homogalacturonan and rhamnogalacturonan I epitopes were differentially distributed within the grain tissues. LM5 galactan epitope was restricted to the aleurone layers contrary to LM6 arabinan epitope which was detected in the whole endosperm. A massive deposition of highly methylated homogalacturonans in vesicular bodies was observed underneath the cell wall of the testa t2 layer at early stage of development. At maturity, low-methylated homogalacturonans totally fulfilled the lumen of the t2 cell layer, suggesting pectin remodeling during grain development. Xyloglucans were only detected in the cuticle above the testa early in the development of the grain while feruloylated arabinoxylans were preferentially deposited into the cell wall of t1 layer. Indeed, the circumscribed distribution of some of the cell wall polysaccharides raises questions about their role in grain development and physiology.

Specific region affects the difference in accumulation levels between apple food allergen Mal d 1 and birch pollen allergen Bet v 1 which are expressed in vegetative tissues of transgenic rice.

KEY MESSAGE: Specific domain of the Mal d 1 was identified to be mainly involved in higher accumulation level in vegetative tissues of transgenic rice than the Bet v 1. Apple food allergen Mal d 1 and birch pollen allergen Bet v 1 belong to the same pathogen related protein 10 (PR10) family. When green fluorescent protein (GFP) fused to either of these allergens was expressed as a secretory protein in transgenic rice by ligating an N terminal signal peptide and a C terminal KDEL ER retention signal under the control of the maize ubiquitin constitutive promoter, the GFP:Mald1 highly accumulated in various tissues, whereas accumulation level of the GFP:Betv1 was remarkably reduced in vegetative tissues except for seed. Analysis by RT-PCR exhibited that there was little difference in their transcript levels, indicating the involvement of post-transcriptional regulation. To investigate the cause of such difference in accumulation levels, deletion analysis of the Mal d 1 and domain swapping between them were carried out in transgenic rice. The results showed that the region between positions 41-90 in the Mal d 1 is predominantly implicated in higher level accumulation in vegetative tissues as well as seed as compared with the Bet v 1. The GFP:Mald1 was localized in oligomeric form within ER lumen or ER-derived particles in vegetative tissues, whereas in seed mainly deposited into novel huge ER-derived protein bodies with the size of 5-10 µm in aleurone cells.