A proteomic analysis of peanut seed at different stages of underground development to understand the changes of seed proteins.

In order to obtain more valuable insights into the protein dynamics and accumulation of allergens in seeds during underground development, we performed a proteomic study on developing peanut seeds at seven different stages. A total of 264 proteins with altered abundance and contained at least one unique peptide was detected by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). All identified proteins were classified into five functional categories as level 1 and 20 secondary functional categories as level 2. Among them, 88 identified proteins (IPs) were related to carbohydrate/ amino acid/ lipid transport and metabolism, indicating that carbohydrate/amino acid/ lipid metabolism played a key role in the underground development of peanut seeds. Hierarchical cluster analysis showed that all IPs could be classified into eight cluster groups according to the abundance profiles, suggesting that the modulatory patterns of these identified proteins were complicated during seed development. The largest group contained 41 IPs, the expression of which decreased at R 2 and reached a maximum at R3 but gradually decreased from R4. A total of 14 IPs were identified as allergen-like proteins by BLAST with A genome (Arachis duranensis) or B genome (Arachis ipaensis) translated allergen sequences. Abundance profile analysis of 14 identified allergens showed that the expression of all allergen proteins was low or undetectable by 2-DE at the early stages (R1 to R4), and began to accumulate from the R5 stage and gradually increased. Network analysis showed that most of the significant proteins were involved in active metabolic pathways in early development. Real time RT-PCR analysis revealed that transcriptional regulation was approximately consistent with expression at the protein level for 8 selected identified proteins. In addition, some amino acid sequences that may be associated with new allergens were also discussed.

The dynamic process of dietary soybean ß-conglycinin in digestion, absorption, and metabolism among different intestinal segments in grass carp (Ctenopharyngodon idella).

The present study aimed to investigate the dynamic process of soybean ß-conglycinin in digestion, absorption, and metabolism in the intestine of grass carp (Ctenopharyngodon idella). Fish fed with 80 g ß-conglycinin/kg diet for 7 weeks, the intestinal digestive enzyme was extracted to hydrolyze ß-conglycinin in vitro, the free amino acid and its metabolism product contents in intestinal segments were analyzed. The present study first found that ß-conglycinin cannot be thoroughly digested by fish intestine digestive enzyme and produces new products (about 60- and 55-kDa polypeptides). The indigestible ß-conglycinin further caused the free amino acid imbalance, especially caused free essential amino acid deficiency in the proximal intestine but excess in the distal intestine. Moreover, these results might be partly associated with the effect of ß-conglycinin in amino acid transporters and tight junction-regulated paracellular pathway. Finally, dietary ß-conglycinin increased the content of amino acid catabolism by-product ammonia while decreased the amino acid anabolism product carnosine content in the proximal intestine and distal intestine. Thus, the current study first and systemically explored the dynamic process of ß-conglycinin in digestion, absorption, and metabolism, which further supported our previous study that dietary ß-conglycinin suppressed fish growth and caused intestine injure.

A new type of specialized morphophysiological dormancy and seed storage behaviour in Hydatellaceae, an early-divergent angiosperm family.

BACKGROUND AND AIMS: Recent phylogenetic analysis has placed the aquatic family Hydatellaceae as an early-divergent angiosperm. Understanding seed dormancy, germination and desiccation tolerance of Hydatellaceae will facilitate ex situ conservation and advance hypotheses regarding angiosperm evolution. METHODS: Seed germination experiments were completed on three species of south-west Australian Hydatellaceae, Trithuria austinensis, T. bibracteata and T. submersa, to test the effects of temperature, light, germination stimulant and storage. Seeds were sectioned to examine embryo growth during germination in T. austinensis and T. submersa. KEY RESULTS: Some embryo growth and cell division in T. austinensis and T. submersa occurred prior to the emergence of an undifferentiated embryo from the seed coat ('germination'). Embryo differentiation occurred later, following further growth and a 3- to 4-fold increase in the number of cells. The time taken to achieve 50 % of maximum germination for seeds on water agar was 50, 35 and 37 d for T. austinensis, T bibracteata and T. submersa, respectively. CONCLUSIONS: Seeds of Hydatellaceae have a new kind of specialized morphophysiological dormancy in which neither root nor shoot differentiates until after the embryo emerges from the seed coat. Seed biology is discussed in relation to early angiosperm evolution, together with ex situ conservation of this phylogenetically significant group.

Protein storage vacuoles of Brassica napus zygotic embryos accumulate a BURP domain protein and perturbation of its production distorts the PSV.

BNM2is a prototypical member of the enigmatic BURP domain protein family whose members contain the signature FX6-7GX10-28PX25-31CX11-12X2SX45-56CHX10 CHX25-29CHX2TX15-16PX5CH in the C-terminus. This protein family occurs only in plants, and the cognate genes vary very widely in their expression contexts in vegetative and reproductive tissues. None of theBURP family members has been assigned any biochemical function. BNM2 was originally discovered as a gene expressed in microspore derived embryos (MDE) of Brassica napus but we found that MDE do not contain the corresponding protein. We show that BNM2 protein production is confined to the seeds and localized to the protein storage vacuoles (PSV) even though the transcript is found in vegetative parts and floral buds as well. In developing seeds, transcript accumulation precedes protein appearance by more than 18 days. RNA accumulation peaks at approximately 20 days post anthesis (DPA) whereas protein accumulation reaches its maximum at approximately 40 DPA. Transgenic expression of BNM2 does not abrogate this regulation to yield ectopic protein production or to alter the temporal aspect ofBNM2 accumulation. Overexpression ofBNM2 led to spatial distortion of storage protein accumulation within PSV and to some morphological alterations of PSVs. However, the overall storage protein content was not altered.

The BURP domain protein AtUSPL1 of Arabidopsis thaliana is destined to the protein storage vacuoles and overexpression of the cognate gene distorts seed development.

BURP domain proteins comprise a broadly distributed, plant-specific family of functionally poorly understood proteins. VfUSP (Vicia faba Unknown Seed Protein) is the founding member of this family. The BURP proteins are characterized by a highly conserved C-terminal protein domain with a characteristic cysteine-histidine pattern. The Arabidopsis genome contains five BURP-domain encoding genes. Three of them are similar to the non-catalytic beta-subunit of the polygalacturonase of tomato and form a distinct subgroup. The remaining two genes are AtRD22 and AtUSPL1. The deduced product of AtUSPL1 is similar in size and sequence to VfUSP and that of the Brassica napus BNM2 gene which is expressed during microspore-derived embryogenesis. The protein products of BURP genes have not been found, especially that of VfUSP despite a great deal of interest arising from copious transcription of the gene in seeds. Here, we demonstrate that VfUSP and AtUSPL1 occur in cellular compartments essential for seed protein synthesis and storage, like the Golgi cisternae, dense vesicles, prevaculoar vesicles and the protein storage vacuoles in the parenchyma cells of cotyledons. Ectopic expression of AtUSPL1 leads to a shrunken seed phenotype; these seeds show structural alterations in their protein storage vacuoles and lipid vesicles. Furthermore, there is a reduction in the storage protein content and a perturbation in the seed fatty acid composition. However, loss of AtUSP1 gene function due to T-DNA insertions does not lead to a phenotypic change under laboratory conditions even though the seeds have less storage proteins. Thus, USP is pertinent to seed development but its role is likely shared by other proteins that function well enough under the laboratory growth conditions.

Vacuolar sorting behaviors of 11S globulins in plant cells.

Plant seed cells amass storage proteins that are synthesized on the endoplasmic reticulumn (ER) and then transported to protein storage vacuoles (PSVs). Many dicotyledonous seeds contain 11S globulin (11S) as a major storage protein. We investigated the accumulation behaviors of pea and pumpkin 11S during seed maturation and compared them with soybean 11S biogenesis (Mori et al., 2004). The accumulation of pea 11S in seeds was very similar to that of soybean 11S at all the development stages we examined, whereas pumpkin 11S condensed in the ER. The determinant of accumulation behavior might be the surface hydrophobicity of 11S. Further, we examined the accumulation of 11Ss in tobacco BY-2 cells to analyze behavior in the same environment. 11Ss expressed in BY2 cells were all observed in precursor form (pro11S). Pro11S with high surface hydrophobicity might be transported to vacuoles in a multivesicular body-mediated pathway when the expression level remains low.

Allergens to wheat and related cereals.

Wheat is one of the major crops grown, processed and consumed by humankind and is associated with both intolerances (notably coeliac disease) and allergies. Two types of allergy are particularly well characterized. The first is bakers' asthma, which results from the inhalation of flour and dust during grain processing. Although a number of wheat proteins have been shown to bind IgE from patients with bakers' asthma, there is no doubt a well-characterized group of inhibitors of alpha-amylase (also called chloroform methanol soluble, or CM, proteins) are the major components responsible for this syndrome. The second well-characterized form of allergy to wheat proteins is wheat-dependent exercise-induced anaphylaxis (WDEIA), with the omega(5)-gliadins (part of the gluten protein fraction) being the major group of proteins which are responsible. Other forms of food allergy have also been reported, with the proteins responsible including gluten proteins, CM proteins and non-specific lipid transfer proteins. Processing of wheat and of related cereals (barley and rye, which may contain related allergens) may lead to decreased allergenicity while genetic engineering technology offers opportunities to eliminate allergens by suppressing gene expression.