Nucleotide diversity of 11S seed storage protein gene and its implications for ecological adaptation of Oryza nivara

The mechanism by which a new species arises and adapts to its environment is a fundamental question in evolutionary biology.Seed characteristics such as seed size and nutrient composition are important fitness-related traits and have been shown to vary greatly among populations and species.However,the significance of variation in seed traits in plant adaptation and speciation remains unclear.We carried out a population genetic study on nucleotide variation of one 11S seed storage protein gene(Pss) of Oryza rufipogon Griff,and O.nivara Sharma Shastry,two closely related wild rice species.By comparatively examining the genetic variation pattern of the regulatory and coding regions of Pss and fragments of six reference loci across different chromosomes,we found significantly lower polymorphisms at coding regions of the gene(PssI) in O.nivara relative to O.rufipogon.Neutrality tests indicated that the reduction of polymorphisms at PssI in O.nivara was caused by positive selection rather than population demography,implying a role of selection on the 11S seed protein gene.Further phylogenetic and principal component analyses also support the hypotheses that the origin of O.nivara was associated with the adaptive divergence on the coding region of Pss.It is most likely that higher reproductive effort would be favored when O.nivara arose from O.rufipogon populations and adapted to the environment change.     

苦荞全基因组11S种子储藏蛋白基因的鉴定及表达分析

以苦荞（Fagopyrum tataricum（L.）Gaertn）全基因组数据为平台,采用生物信息学方法,挖掘出9个11S种子储藏蛋白基因,并对其定位、蛋白结构、系统发育及表达模式进行了分析。结果表明,苦荞9个11S种子储藏蛋白基因编码的蛋白长度为189~914 aa,等电点位于5.18~9.82之间,分子量为21.27~103.33 kD;定位分析结果显示,这些成员位于苦荞基因组的6条连锁群上（Megascaffold2/5以及scaffold77/344/395/861）;序列比对分析发现,除了1个11S种子储藏蛋白sample1_00009513-RA具有1个cupin保守结构域外,其余8个都含有2个cupin结构域,并且在cupin保守结构域中,苦荞和拟南芥（Arabidopsis thaliana（L.）Heynh）共有14个保守的氨基酸残基;蛋白结构预测表明,苦荞11S种子储藏蛋白的结构具有2种类型;苦荞与其它6个物种[拟南芥、花生（Arachis hypogaea Linn.）、大豆（Glycine max（Linn.）Merr.）、杏仁（Armeniaca vulgaris Lam.）、胡桃（Juglans regia L.）和芝麻（Sesamum indicum Linn.）]11S种子储藏蛋白以及苦荞过敏蛋白（TBb和TBt）系统发育分析结果表明,这些蛋白可以分为3类,共具有4对旁系同源蛋白和3对直系同源蛋白;与已报道的苦荞过敏性储藏蛋白以及其它5个物种（花生、大豆、杏仁、胡桃和芝麻）的11S过敏蛋白比较发现,5个11S种子储藏蛋白（sample1_00013128-RA、sample1_00013130-RA、sample1_00021677-RA、sample1_00021668-RA和sample1_00021674-RA）与苦荞2个过敏蛋白的同源性较高,同时它们与胡桃11S过敏蛋白的同源性最高,但尚需进一步实验来确定这5个成员是否为食物过敏原;RNA-Seq转录组数据显示,4个基因（sample1_00018411-RA、sample1_00026786-RA、sample1_00021674-RA、sample1_00022718-RA）在2种荞麦属植物的灌浆期种子中表达水平较高,且在‘大苦1号’中的表达水平要高于‘大甜1号’。     

Effect of denaturants on the 11S protein of guar seed (Cyamopsis tetragonoloba)

The effect of sodium dodecyl sulphate, urea or guanidinium hydrochloride on the sedimentation velocity, viscosity, ultra violet spectra and fluorescence spectra of the 11S protein of guar seed has been determined. Sodium dodecyl sulphate dissociates the protein directly to the 2S protein, whereas urea or guanidinium hydrochloride produces an intermediate 7S protein. These reagents denature the protein also. Both the dissociative and the denaturation effect follow the order, sodium dodecyl sulphate > guanidinium hydrochloride > urea when the concentration are expressed as mols per litre. The denatured states in the three cases probably differ.     

Expression and characterization of the acidic subunit from 11S Amaranth seed protein

Amarantin acidic subunit has the potential to be employed as a functional and a nutraceutical protein. To evaluate both possibilities this protein was produced in recombinant Escherichia coli Origami (DE3) harboring the expression plasmid pET-AC6His. Three different expression factors were assayed: inductor concentration, temperature and time of the amarantin acidic subunit accumulation. The results indicated that a 0.3 mmol/L concentration of isopropyl-beta-D-thiogalactoside, at 37 degrees C and 6 h after induction were favorable for high expression of amarantin acidic subunit, mostly in the form of inclusion bodies. The protein was purified from soluble fraction by immobilized metal affinity chromatography, up to 30 mg amarantin acidic subunit/L Terrific broth culture were obtained. Sucrose density gradient ultracentrifugation analysis of the expressed soluble amarantin acidic subunit revealed that it was assembled in monomers. The expression of the amarantin acidic subunit, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.     

Antigenic relationship of legume seed proteins to the 7S seed storage protein of soybean

Extracts enriched for globulin proteins were prepared from the seeds of a large number of legume species and were tested for homology to antisera prepared against the glycosylated 7S seed storage protein of the soybean (Glycine max). Electrophoretic identification and subsequent analysis of proteins precipitated with 7S antisera was useful at relatively short taxonomic distances, particularly within the tribe Phaseoleae, to which G. max belongs. Glycine and most other members of the subtribe Glycininae are unusual within the Phaseoleae in having high molecular weight ($\text{> 70 000}$ dalton) subunit polypeptides. Seeds from other plants representing other subtribes of the Phaseoleae also contained proteins that cross-reacted with the G. max antisera; the molecular weights of these proteins varied from 30 000 to nearly 90 000 daltons. Homology was detected across a wider range of legume tribes within the subfamily Papilionoideae by enzyme-linked immunosorbent assay (ELISA). The results of these experiments suggest both that the 7S proteins of these tribes are evolutionarily related and that at least some features of these apparently rapidly-evolving proteins are under relatively strong selectional constraint.     

Vicilin-like seed storage proteins in the gymnosperm interior spruce (Picea glauca/engelmanii)

A seed storage protein cDNA was characterized from a library of interior spruce (Picea glauca/engelmanii complex) cotyledonary stage somatic embryos. The deduced amino acid sequence predicts a 448 amino acid (50 kDa) polypeptide with 28–38% identity with angiosperm vicilin-like 7S globulins. XXC/G codon usage is low (47%) relative to monocot angiosperms while pairwise comparisons show that spruce, monocot, and dicot vicilins are approximately equal in amino acid divergence. Although small by comparison, the spruce vicilin contains an N terminal hydrophilic region characteristic of angiosperm large vicilins. Genomic Southern blotting predicts that the cDNA is encoded by a gene family.     

Minireview: analysis of rape seed napin structure and potential roles of the storage protein

Structural and functional data on 2S albumins and particularly rape seed napins are reviewed and, based on the coordinates of the three-dimensional structure of napin-like albumin BnIb, are used to model different rape napins. Surprisingly, the modeled napins, despite great sequence homology, differ in tertiary arrangements of the polypeptide chains. It is proposed that these differences in 3D structures of the analyzed rape napins may reflect their functions, which may cover many other potential beneficial purposes besides simple storage.     

Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5 control region of the napA gene were fused to the reporter gene -glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between –1101 and –309 resulted in increased GUS activity. In contrast, deletion of sequences between –309 and –211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between –152 and +44. Further 5 deletion of the fragment to –126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between –148 to –120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to –152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.     

Development of protein bodies in cotyledons of Fagus sylvatica

Electron microscopic analysis of cotyledons of beech ( Fagus sylvatica ) at different stages of seed maturation indicates that protein bodies originate by gradual subdivision of the vacuoles in which reserve proteins are deposited. The majority of protein bodies show a proteinaceous matrix and a number of globoid inclusions of different sizes. In a small number of cells, druse-like inclusions were observed. Analysis by SDS-PAGE of proteins extracted from isolated protein bodies shows that the majority correspond to beech seed storage globulins.     

11 S seed storage proteins fromHelianthus species (Compositae): Biochemical,size and charge heterogeneity

The seeds of 19 sunflower species were compared on the basis of their protein contents and the relative proportions of their protein fractions. The globulin content varied from 50% to about 70% and the albumin content from 18% to 35% according to the species. The level of intermediateMr polypeptides showed a great variability (9.6 to 24.3%). Comparative studies onMr polymorphism were carried out by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of non reduced and/or reduced samples using both mono- and bidimensional procedures. Polypeptide constituents of helianthinin were compared including both number and molecular size (cultivatedH. annuus was used as a standard). Studies focused on differences observed between the major two (Mr 38 000), (Mr 32 000) and (Mr 25 500), (21 000) polypeptides families constituting the main A, B, and C subunits. and polypeptides analyses permit to discriminate easilyH. petiolaris from the other species. Charge polymorphism was studied using isoelectric focusing (IEF) and IEF-PAGE in mono and bidimensional procedures in the presence or absence of 2-mercaptoethanol (2-ME). Only a specific ₄ polypeptide enables an easy discrimination betweenH. petiolaris and all the other species. Detailed nomenclature of the , and , polypeptides constituting the different helianthinin globulin subunits is given via the results of pI andMr analyses. Monodimensional IEF patterns of the more basic albumins (pI > 8.0) appear to provide a more valuable approach to identifying specific protein markers.     

Recombinant protein yield in rice seed is enhanced by specific suppression of endogenous seed proteins at the same deposit site

Human IL‐10 (hIL‐10) is a therapeutic treatment candidate for inflammatory allergy and autoimmune diseases. Rice seed‐produced IL‐10 can be effectively delivered directly to gut‐associated lymphoreticular tissue (GALT) via bio‐encapsulation. Previously, the codon‐optimized hIL‐10 gene was expressed in transgenic rice with the signal peptide and endoplasmic reticulum (ER) retention signal (KDEL) at its 5′ and 3′ ends, respectively, under the control of the endosperm‐specific glutelin GluB‐1 promoter. The resulting purified hIL‐10 was biologically active. In this study, the yield of hIL‐10 in transgenic rice seed was improved. This protein accumulated at the intended deposition sites, which had been made vacant through the selective reduction, via RNA interference, of the endogenous seed storage proteins prolamins or glutelins. Upon suppression of prolamins that were sequestered into ER‐derived protein bodies (PB‐I), hIL‐10 accumulation increased approximately 3‐fold as compared to rice seed with no such suppression and reached 219 μg/grain. In contrast, reducing the majority of the glutelins stored in protein‐storage vacuoles (PB‐II) did not significantly affect the accumulation of hIL‐10. Considering that hIL‐10 is synthesized in the ER lumen and subsequently buds off in ER‐derived granules called IL‐10 granules in a manner similar to PB‐Is, these results indicate that increases in the available deposition space for the desired recombinant proteins may be crucial for improvements in yield. Furthermore, efficient dimeric intermolecular formation of hIL‐10 by inhibiting interaction with Cys‐rich prolamins also contributed to the enhanced formation of IL‐10 bodies. Higher yield of hIL‐10 produced in rice seeds is expected to have broad application in the future.     

Conservation and divergence on plant seed 11S globulins based on crystal structures

The crystal structures of two pro-11S globulins namely: rapeseed procruciferin and pea prolegumin are presented here. We have extensively compared them with the other known structures of plant seed 11S and 7S globulins. In general, the disordered regions in the crystal structures among the 11S globulins correspond to their five variable regions. Variable region III of procruciferin is relatively short and is in a loop conformation. This region is highly disordered in other pro-11S globulin crystals. Local helical and strand variations also occur across the group despite general structure conservation. We showed how these variations may alter specific physicochemical, functional and physiological properties. Aliphatic hydrophobic residues on the molecular surface correlate well with T_m values of the globulins. We also considered other structural features that were reported to influence thermal stability but no definite conclusion was drawn since each factor has additive or subtractive effect. Comparison between proA3B4 and mature A3B4 revealed an increase in r.m.s.d. values near variable regions II and IV. Both regions are on the IE face. Secondary structure based alignment of 11S and 7S globulins revealed 16 identical residues. Based on proA3B4 sequence, Pro60, Gly128, Phe163, Phe208, Leu213, Leu227, Ile237, Pro382, Val404, Pro425 and Val 466 are involved in trimer formation and stabilization. Gly28, Gly74, Asp135, Gly349 and Gly397 are involved in correct globular folding.     

The glycinin box: a soybean embryo factor binding motif within the quantitative regulatory region of the 11S seed storage globulin promoter

The soybean embryo factor binding sequence in the glycinin A₂B_1a gene promoter was delimited to an A/T-rich 9 bp sequence, 5-TAATAATTT-3, designated as the glycinin box, by DNA footprinting and gel mobility shift assay using synthetic oligonucleotides. It was shown that the interaction with the factor takes place at a defined DNA sequence rather than at random A/T-rich sequence blocks in the glycinin 5 flanking region. There are four glycinin boxes in the quantitative regulatory region between positions – 545 and – 378 of the glycinin A₂B_1a promoter. Multiple nonamer motifs similar to the glycinin box were also found in the equivalent regions of other glycinin and legumin promoters, suggesting that they must be conserved as a binding site for the embryo factor that activates the differential and stage-specific expression of seed 11S globulin genes in leguminous plants.