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[The cDNA cloning of conarachin gene and its expression in developing peanut seeds].

In order to clone the genes of peanut seed storage proteins, a cDNA library of mid-maturation stage cotyledons of peanut (Arachis hypogaea L. Shanyou 523) was constructed. After immunoscreening with polyclonal antibody against conarachin from peanut seeds, two cDNA sequences named Ahy alpha and Ahy beta of conarachin genes were obtained. BLAST these sequences in GenBank showed that they share high homology with the genes of legume 7S globulins. One of the sequences (Ahy beta) was found to be identical to part of the published complete cDNA sequence of the peanut allergen, Ara h1 p17. The other fragment (Ahy alpha) was almost identical to parts of the published complete cDNA sequence of the peanut allergen, Ara h1 p41b. Results of southern analysis show that they belong to a small gene family (the result is not shown). These sequences were expressed only in developing peanut seeds. To isolate the genes involved in seed storage protein and to understand the gene expression during peanut seed development, the sequences of 414 ESTs were determined and further analyzed by BLAST searches and categorized functionally. Five rESTs (representative ones) of arahin, two rESTs of conarachin and six rESTs of conglutin-like proteins were isolated. 17.0% of total transcripts isolated in this study are involved in peanut seed storage proteins.

Allergens↗

TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor.

Mutants of a new gene, TRANSPARENT TESTA GLABRA2 (TTG2), show disruptions to trichome development and to tannin and mucilage production in the seed coat. The gene was tagged by the endogenous transposon Tag1 and shown to encode a WRKY transcription factor. It is the first member of this large, plant-specific family known to control morphogenesis. The functions of all other WRKY genes revealed to date involve responses to pathogen attack, mechanical stress, and senescence. TTG2 is strongly expressed in trichomes throughout their development, in the endothelium of developing seeds (in which tannin is later generated) and subsequently in other layers of the seed coat, and in the atrichoblasts of developing roots. TTG2 acts downstream of the trichome initiation genes TTG1 and GLABROUS1, although trichome expression of TTG2 continues to occur if they are inactivated. Later, TTG2 shares functions with GLABRA2 in controlling trichome outgrowth. In the seed coat, TTG2 expression requires TTG1 function in the production of tannin. Finally, TTG2 also may be involved in specifying atrichoblasts in roots redundantly with other gene(s) but independently of TTG1 and GLABRA2.

Amino Acid Sequence↗

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea).

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-14C]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparison of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

Amino Acid Sequence↗

Evaluation of soyasaponin, isoflavone, protein, lipid, and free sugar accumulation in developing soybean seeds.

A combination of analytical techniques was used to examine and quantify seed compositional components such as protein, lipid, free sugars, isoflavones, and soyasaponins during soybean development and maturation in two Korean soybean cultivars. Protein accumulation was rapid during reproductive stages, while lipid content was only relatively moderately increased. The major carbohydrate saccarides sucrose and stachyose constantly increased during the reproductive stage. Previously published results suggest that the free sugar and lipid content reached their maximal concentrations at a relatively early stage of seed development and remain constant in comparison to other chemical components. The malonylglucosides were the predominant isoflavone form followed by the glucosides, acetyl glucosides, and aglycone forms. As soybean seed matures, total soyasaponin concentration was constantly decreased until the R8 stage. Soyasaponin beta(g) was the major soyasaponin in DDMP-conjugated group B soyasaponins, followed by the non-DDMP counterpart soyasaponin I and soyasaponin A1. The ratio of total isoflavone to total soyasaponin in the developing soybean increased from 0.06 to 1.31. Protein, lipid, and free sugar contents in the developing soybean seeds showed significant positive correlations with conjugated isoflavones and total isoflavone concentration, while the lipid contents showed a negative correlation with the isoflavone aglycone. Protein, lipid, and free sugar contents showed a negative correlation with total group A and B soyasaponins and total soyasaponins; however, only the soyasaponin A content was significantly negatively correlated with free sugar content. Total soyasaponin content was negatively correlated with isoflavone content (r = -0.828 at p < 0.01).

Carbohydrates↗

Phospholipids in the developing soybean seed.

The distribution of phospholipids in developing soybean seeds [Glycine max (L.) Merr., var. "Chippewa 64," "Harosoy 63," "Wayne," and "Clark 63"] was followed. From 30 to 60 days after flowering expressed as mole per cent of phospholipid phosphorus phosphatidic acid decreased from 14.8 to 9.1; phosphatidylinositol increased from 0 to 9.1; phosphatidylcholine increased from 8.2 to 9.8; phosphatidylethanolamine increased from 5.3 to 8.6; phosphatidylglycerol increased from 3.2 to 4.8; diphosphatidylglycerol increased from 2.7 to 4.1; and N-acylphosphatidylethanolamine decreased from 65.8 to 54.6. However, from 60 days after flowering to maturity, phosphatidic acid decreased to 0; phosphatidylinositol increased roughly 2-fold; phosphatidylcholine increased roughly 4.7-fold; phosphatidylethanolamine increased 3-fold; N-acylphosphatidylethanolamine decreased 11-fold; whereas phosphatidylglycerol and diphosphatidylglycerol remained essentially constant. Percentages of individual phospholipid species were not statistically different between any two varieties at a given time period.Immature soybean cotyledons incubated with (14)C-acetate or -pyruvate demonstrated rapid incorporation into the phospholipid fraction. N-acylphosphatidylethanolamine was found to account for nearly 70% of the total radioactivity incorporated by the total polar lipid fraction and greater than 30% of the total radioactivity added.

Journal Article↗

Pyruvate orthophosphate dikinase gene expression in developing wheat seeds.

The amount of pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) protein in wheat (Triticum aestivum L. var Cheyenne) grains was determined at different stages of development by the protein blot method. The variation in PPDK protein with time in developing wheat grains was similar to that of the enzyme's activity reported by Meyer et al. (1982 Plant Physiol 69: 7-10). The variation in levels of PPDK mRNA with seed development was determined by analysis of polypeptides immunoprecipitated by anti-PPDK serum from in vitro translation products of extracted seed RNA. This mRNA variation was similar to that of the in vivo enzyme levels and the correlation is consistent with the regulation of PPDK gene expression by the level of its mRNA.The highest level of PPDK in developing wheat seeds occurs later than the highest levels of both ribulose bisphosphate carboxylase (EC 4.1.1.39) and of chlorophyll, which are located in the green pericarp tissue. PPDK was located in both endosperm and pericarp tissue of the seeds. The tissue location and developmental profile of seed PPDK are consistent with a metabolic role of providing phosphoenolpyruvate as a substrate for recapturing respiratory CO(2) in the seed, and possibly for amino acid interconversions during development.

Journal Article↗

Changes in oligosaccharide content and antioxidant enzyme activities in developing bean seeds as related to acquisition of drying tolerance and seed quality.

Seeds of bean (Phaseolus vulgaris cv. Vernel) were collected throughout their development on the plant and dried at 15 degrees C and 75% relative humidity to a final moisture content of about 16% (fresh weight basis) to determine whether the onset of tolerance to this drying condition was related to changes in soluble sugars or the activities of the main antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). Measurements of soluble sugars and enzyme activities were made after drying the seeds, and drying tolerance was evaluated by the ability of dried seeds to germinate and to produce normal seedlings. Seeds became tolerant to drying at 45 d after anthesis, a time marking physiological maturity. At physiological maturity, the moisture content of seeds was about 50-55% (fresh weight basis) and seed dry matter reached about 190 mg per seed. Seed vigour, evaluated by controlled deterioration and conductivity measurements, continued to increase after seed mass maturity, but decreased when seeds remained thereafter for more than 7 d on the plant. Acquisition of drying tolerance was coincident with an accumulation of raffinose and stachyose. Dried-tolerant seeds were also characterized by a high amount of sucrose, the most abundant sugar, and by a low content of monosaccharides. The (raffinose+stachyose)/sucrose ratio increased during seed filling, reaching a value close to 1 when all the seeds became tolerant to drying, and maintaining this proportion during the final stages of maturation. Acquisition of drying tolerance was also related to a reorientation of the enzymatic antioxidant defence system. Drying-tolerant dried seeds displayed high CAT and GR activities and low SOD and APX activities, while the opposite condition was observed in immature dried seeds. The shift in antioxidant enzymes corresponded to the beginning of the maturation-drying phase. These results suggest that oligosaccharide metabolism and enzymatic antioxidant defences may be involved in acquisition of drying tolerance during bean seed development, but are not related to seed vigour.

Adaptation, Physiological↗

A simple and sensitive assay for distinguishing the expression of ricin and Ricinus communis agglutinin genes in developing castor seed (R. communis L.).

Castor oil is the only commercial source of ricinoleic acid and has numerous industrial applications. Among the factors limiting domestic production of castor oil is the presence of the toxin ricin and its less toxic homologue Ricinus communis agglutinin (RCA) in seeds. Although the sequences of ricin and RCA genes are known, their transcriptional expression patterns have not been distinguished due to their high degree of sequence similarity. As the information is critical for assessing success in developing a ricin-free castor crop using genetic silencing, we have designed a gene specific reverse transcription-polymerase chain reaction (RT-PCR) assay to examine the expression of the ricin and RCA genes in developing seeds. The results show that the ricin and RCA mRNA are highly abundant in seeds during the development of endosperm, and the expression pattern is similar to that observed in the Northern analysis. The RT-PCR results can be confirmed by a simple RT-PCR-based restriction fragment analysis.

Blotting, Northern↗

beta-Conglycinins in Developing Soybean Seeds.

The temporal sequence of development of the major proteins of seeds of soybean (Merr.) has been studied during development of cotyledons from flowering to maturity. A well-defined difference occurred in the times of appearance and the periods of maximum accumulation of alpha, alpha'-, and beta-subunits of betaconglycinin. Whereas alpha- and alpha'-subunits appeared 15 to 17 days after flowering, accumulation of beta-subunit did not commence until 22 days after flowering. Such alterations in subunit composition infer that changes also occurred in the amino acid composition of betaconglycinin during maturation, particularly in the content of methionine which is low in the beta-subunit.

Journal Article↗

The expression of a peroxiredoxin antioxidant gene, AtPer1, in Arabidopsis thaliana is seed-specific and related to dormancy.

We have isolated a gene, AtPer1, from the dicotyledon Arabidopsis thaliana, which shows similarity to the 1-cysteine (1-Cys) peroxiredoxin family of antioxidants. In higher plants, members of this group of antioxidants have previously only been isolated from monocotyledons. It has been suggested that seed peroxiredoxins protect tissues from reactive oxygen species during desiccation and early imbibition and/or are involved in the maintenance of/protection during dormancy. AtPer1 expression is restricted to seeds. Despite differences in seed development between monocots and dicots, AtPer1 shows an expression pattern during seed development and germination similar to the dormancy-related transcript Per1 in barley. In situ hybridization identifies AtPer1 as the first aleurone-expressed transcript characterized in developing Arabidopsis seeds. The transcript is also expressed in the embryo. AtPer1 expression in seeds is unaltered in an ABA-deficient mutant (aba-1) during seed development, while expression in seeds of an ABA-insensitive mutant (abi3-1) is reduced. The transcript is not induced in vegetative tissue in response to stress by ABA or drought. AtPer1 transcript levels are correlated to germination frequencies of wildtype seeds, but AtPer1 transcript abundance is not sufficient for expression of dormancy in non-dormant mutants. Hypotheses on peroxiredoxin function are discussed in view of the results presented here.

Amino Acid Sequence↗

Comparison of procedures to determine protein content of developing bean seeds (Phaseolus vulgaris).

Micro-Kjeldahl, Lowry and Bradford procedures were compared for determining the protein content of Phaseolus vulgaris seeds during their development. Micro-Kjeldahl and biuret techniques were also compared with mature seeds of a normal and a genetically-improved bean cultivar. The protein contents of casein and soy protein isolate were as well estimated by these four methods. For many samples of both bean and food protein products large disagreements were found between micro-Kjeldahl and the other three procedures.

Fabaceae↗

[Morphology and anatomy of the fruit and seed in development of Guarea macrophylla Vahl. (Meltaceae)].

Morphology and anatomy of the fruit and seed in development of Guarea macrophylla Vahl. were studied in flowers and fruits collected in secondary forests of Maringá, Paraná, Brazil. Pericarp originates from the wall of the ovary and is constituted of epidermic exocarp, parenchymatous mesocarp with brachysclereids and fibrous and lignified endocarp. The seed develops from the hemianatropous, bitegmic and crassinucellate ovule and it is classified as an exotegmic and exalbuminous one. Exotesta and mesotesta constitute the red sarcotesta, with oily content.

Brazil↗

The role of the acyl-CoA pool in the synthesis of polyunsaturated 18-carbon fatty acids and triacylglycerol production in the microsomes of developing safflower seeds.

Microsomes isolated from the developing cotyledons of the seeds of the safflower varieties, very-high-linoleate, Gila and high-oleate, were capable of exchanging the acyl groups in acyl-CoA with the fatty acids in position 2 of phosphatidylcholine. The specificity of the 'acyl-exchange' towards the acyl moiety in acyl-CoA was selective in the order: oleate greater than linoleate greater than linolenate. Stearoyl-CoA was completely selected against when presented in a mixed substrate with unsaturated 18-carbon acyl-CoAs. Microsomes, of the very-high-linoleate safflower variety, rapidly desaturated in situ-labelled [14C]oleoylphosphatidylcholine in the presence of NADH. Little oleate desaturation, however, was observed in the microsomes of the high-oleate variety. Microsomes of the Gila and high-oleate varieties of safflower rapidly synthesised phosphatidic acid by the acylation of glycerol 3-phosphate with acyl-CoA. The phosphatidic acid was metabolised to diacylglycerol, which was further acylated to triacylglycerol. A strong selectivity for linoleoyl-CoA was found for the acylation of glycerol 3-phosphate in both the Gila and high-oleate microsomes. On the basis of these results, we propose that the pattern of 18-carbon unsaturated fatty acids in the triacylglycerols of all 'oil'-producing seeds is a direct reflection of the fatty acids in the acyl-CoA pool. This, in turn, is governed by: A, the rate and specificity of the acyl exchange between acyl-CoA and phosphatidylcholine; B, the rate of oleate (and linoleate) desaturation in phosphatidylcholine; and C, the rate and specificity of the glycerophosphate acyltransferase.

Acyl Coenzyme A↗

Lysine metabolism is concurrently regulated by synthesis and catabolism in both reproductive and vegetative tissues.

The functional role of Lys catabolism in balancing Lys levels in plants has only been directly demonstrated in developing seeds. Seed-specific expression of a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in an Arabidopsis knockout mutant of the AtLKR/SDH gene that regulates Lys catabolism synergistically boosted Lys accumulation in mature seeds, but it also severely reduced the growth of seedlings derived from them. Here we further tested whether the inhibition of seedling growth was due to a negative physiological effect of excess Lys on seed maturation or to defective postgermination catabolism of Lys, which accumulated in the mature seeds. To address these questions, we coexpressed a bacterial DHPS gene with an RNAi construct of AtLKR/SDH, both under control of the same seed-specific promoter, to restrict Lys synthesis and catabolism to the developing seeds. Coexpression of these genes boosted seed Lys content and caused a significant, metabolically unanticipated increase in Met content, similarly to our previous report using plants expressing the bacterial DHPS on an AtLKR/SDH knockout background. However, postgermination seedling growth was significantly improved when the reduction of Lys catabolism was restricted to seed development, suggesting that defective postgermination Lys catabolism was responsible for inhibition of seedling growth in the AtLKR/SDH knockout plants expressing the bacterial DHPS gene in a seed-specific manner. Constitutive expression of the bacterial DHPS in the AtLKR/SDH knockout mutant boosted Lys levels in vegetative tissues in a similar manner to that observed in seeds, further demonstrating that Lys catabolism plays an important regulatory role in balancing Lys levels.

Arabidopsis↗

Both antisense and sense expression of biotin carboxyl carrier protein isoform 2 inactivates the plastid acetyl-coenzyme A carboxylase in Arabidopsis thaliana.

To further characterize the role of biotin carboxyl carrier protein isoform 2 (BCCP2) in acetyl-coenzyme A carboxylase (ACCase) function and fatty acid biosynthesis, plants with reduced or increased expression of this protein were characterized. Analysis of 38 independent Arabidopsis lines expressing antisense BCCP2 transcript behind a constitutive promoter showed no significant phenotype, though antisense transcript was highly expressed. In developing seed, BCCP2 protein was reduced by an average of 38% resulting in a 9% average decrease in fatty acid content in mature seed. Over-expression of BCCP2 behind a seed-specific napin promoter increased the amount of holo-BCCP2 in developing seed by an average of twofold, as determined with anti-biotin antibodies. Surprisingly, the average fatty acid content of T2 seed from over-expression lines was 23% lower than wild-type seed. These plants also exhibited reduced seed setting in 18 of 20 T1 lines which was coincident with increased individual seed mass. Quantification of total BCCP2 in developing siliques using anti-BCCP2 antibodies indicated that as much as 60% of total plastidial BCCP2 was in the non-biotinylated form (apo-BCCP2). Although apo-BCCP2 was highly over-expressed in developing seed, accumulation of other ACCase subunits was unaffected. The specific activity of ACCase was up to 65% lower in developing seed of over-expression lines versus wild type. This was attributed to the assembly of biologically inactive (non-biotinylated) ACCase complexes. Consistent with ACCase exerting control over de novo fatty acid synthesis, reduced activity in developing seed resulted in lower oil content, altered fatty acid composition and reduced seed setting.

Acetyl-CoA Carboxylase↗

Developmental and growth temperature regulation of two different microsomal omega-6 desaturase genes in soybeans.

The polyunsaturated fatty acid content is one of the major factors influencing the quality of vegetable oils. Edible oils rich in monounsaturated fatty acid provide improved oil stability, flavor, and nutrition for human and animal consumption. In plants, the microsomal omega-6 desaturase-catalyzed pathway is the primary route of production of polyunsaturated lipids. We report the isolation of two different cDNA sequences, FAD2-1 and FAD2-2, encoding microsomal omega-6 desaturase in soybeans and the characterization of their developmental and temperature regulation. The FAD2-1 gene is strongly expressed in developing seeds, whereas the FAD2-2 gene is constitutively expressed in both vegetative tissues and developing seeds. Thus, the FAD2-2 gene-encoded omega-6 desaturase appears to be responsible for production of polyunsaturated fatty acids within membrane lipids in both vegetative tissues and developing seeds. The seed-specifically expressed FAD2-1 gene is likely to play a major role in controlling conversion of oleic acid to linoleic acid within storage lipids during seed development. In both soybean seed and leaf tissues, linoleic acid and linolenic acid levels gradually increase as temperature decreases. However, the levels of transcripts for FAD2-1, FAD2-2, and the plastidial omega-6 desaturase gene (FAD 6) do not increase at low temperature. These results suggest that the elevated polyunsaturated fatty acid levels in developing soybean seeds grown at low temperature are not due to the enhanced expression of omega-6 desaturase genes.

Amino Acid Sequence↗

Regulated expression of three alcohol dehydrogenase genes in barley aleurone layers.

Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity.Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O(2) deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O(2) levels were reduced below 5%, and were most intense at 0% O(2).In vivo(35)S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O(2), synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O(2), the synthesis of all three ADH peptides increased markedly.Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O(2) deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O(2), and about 100-fold after 1 day at 0% O(2).We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O(2), that regulation of mRNA level is likely to be a major controlling factor, and that whereas the ADH system of barley has strong similarities to that of maize, it also has some distinctive features.

Journal Article↗

Changes in molecular species of triacylglycerols in developing cotton seeds under salt stress.

Cotton plants (Gossypium hirsutum L. cv. S4) were grown and irrigated with increasing salt concentrations: 0, 50, 100 and 150 mM NaCl. Lipids from developing seeds, at stages of 3, 4, 5 and 6 weeks after anthesis, were analysed. High salt dose (150 mM NaCl) affected triacylglycerol levels drastically at the end of ripening. HPLC analysis of triacylglycerols in control seeds showed ten molecular species. Palmitodilinolein (PLL) is the major fraction, representing about 25% of total triacylglycerols. The levels of trilinolein (LLL), palmitolinoleo-olein (PLO) and oleodilinolein (OLL) molecules varied between 13 and 16% of the total. The other molecular species: triolein (OOO), dioleopalmitin (POO) and dipalmito-olein (POP) are minor and do not exceed 5%. Moreover, the composition of triacylglycerol molecular species was almost constant during developing stages of control seeds. However, amounts of triacylglycerol species LLL, OLL and palmitodilinolein (PLL), as expressed on a dry-matter basis (mg/g), decreased severely under the highest NaCl concentration, while contents of triacylglycerol species OOO, POO, POP were unchanged. These findings confirm our previous results, which indicated that the amount of linoleic acid in cotton seeds was reduced by salt stress and that the amount of oleic acid remained unchanged.

Chromatography, Thin Layer↗