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PubMed1994

Chalcone synthase mRNA and activity are reduced in yellow soybean seed coats with dominant I alleles.

The seed of all wild Glycine accessions have black or brown pigments because of the homozygous recessive i allele in combination with alleles at the R and T loci. In contrast, nearly all commercial soybean (Glycine max) varieties are yellow due to the presence of a dominant allele of the I locus (either I or i) that inhibits pigmentation in the seed coats. Spontaneous mutations to the recessive i allele occur in these varieties and result in pigmented seed coats. We have isolated a clone for a soybean dihydroflavonol reductase (DFR) gene using polymerase chain reaction. We examined expression of DFR and two other genes of the flavonoid pathway during soybean seed coat development in a series of near-isogenic isolines that vary in pigmentation as specified by combinations of alleles of the I, R, and T loci. The expression of phenylalanine ammonia-lyase and DFR mRNAs was similar in all of the gene combinations at each stage of seed coat development. In contrast, chalcone synthase (CHS) mRNA was barely detectable at all stages of development in seed coats that carry the dominant I allele that results in yellow seed coats. CHS activity in yellow seed coats (I) was also 7- to 10-fold less than in the pigmented seed coats that have the homozygous recessive i allele. It appears that the dominant I allele results in reduction of CHS mRNA, leading to reduction of CHS activity as the basis for inhibition of anthocyanin and proanthocyanin synthesis in soybean seed coats. A further connection between CHS and the I locus is indicated by the occurrence of multiple restriction site polymorphisms in genomic DNA blots of the CHS gene family in near-isogenic lines containing alleles of the I locus.

Acyltransferases↗
PubMed1992

Phosphoenolpyruvate carboxylase activity and concentration in the endosperm of developing and germinating castor oil seeds.

Monospecific polyclonal antibodies against maize leaf phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were utilized to examine the subunit composition and developmental profile of endosperm PEPC in developing and germinating castor oil seeds (Ricinus communis L. cv Baker 296). PEPC from developing endosperm consists of a single type of 100-kilodalton subunit, whereas the enzyme from 2- to 5-day germinated endosperm appears to contain equal proportions of immunologically related 103- and 108-kilodalton subunits. The maximal activity of PEPC in developing endosperms (2.67 micromoles oxaloacetate produced per minute per gram fresh weight) is approximately 20-fold and threefold greater than that of fully mature (dry seed) and germinating endosperms, respectively. The most significant increase in the activity and concentration of endosperm PEPC occurs during the middle cotyledon to full cotyledon stage of seed development; this period coincides with the most active phase of storage oil accumulation by ripening castor oil seeds. The data are compatible with the recent proposal (RG Smith, DA Gauthier, DT Dennis, DH Turpin [1992] Plant Physiol 1233-1238) that PEPC plays a fundamental role in vivo in the cytosolic production of an important substrate (malate) for fatty acid biosynthesis by developing castor oil seed leucoplasts. Immediately following seed imbibition, PEPC activity and concentration increase in parallel, with the greatest levels attained by the third day of germination. It is suggested that during this early phase of seed germination PEPC has a critical function to build up cellular dicarboxylic acid pools required to initiate significant activities of both the tricarboxylic acid and glyoxylate cycles.

Journal Article↗
PubMed1994

Transcripts encoding an oleosin and a dormancy-related protein are present in both the aleurone layer and the embryo of developing barley (Hordeum vulgare L.) seeds.

In cereal seeds, the aleurone layer and the embryo share several characteristics, including synthesis and accumulation of lipid bodies, desiccation tolerance and dormancy. A number of Balem transcripts present in both the barley aleurone layer and the embryo have been cloned by differential screening of a cDNA library from aleurone layers of immature barley grains. The Balem clones constitute two subgroups, one for which the transcripts are detectable in aleurone layers and embryos of developing seeds only (B23D and B15C), and another for which transcripts are present also in germinating embryos and in maternal tissues (B12D, B14E and B31E). Sequence analysis identified B23D and B15C as the barley homologues of the 18 kDa oleosin of maize embryos (72% amino acid identity) and the dormancy-associated transcript pBS128 from Bromus secalinus (95% identity), respectively. In situ hybridization experiments demonstrate that in the embryo, the B23D transcript is mainly present in the scutellum, whereas the B15C transcript is predominantly present in shoot and root apices. Using anther-derived embryos and embryogenic cell suspensions, it is demonstrated that the B23D and B15C transcripts can be used as molecular markers for somatic embryogenesis. The functions of the transcripts in the second Balem subgroup remain unknown. Further studies on the Balem transcripts may shed light on the molecular basis for the extensive similarities between the embryo and the aleurone layer of the endosperm in the grass family.

Amino Acid Sequence↗
PubMed2001

An unusual seed-specific 3-ketoacyl-ACP synthase associated with the biosynthesis of petroselinic acid in coriander.

Petroselinic acid (18:1 delta6) is the major component of the seed oil of Umbelliferae species such as coriander (Coriandrum sativum) as well as Araliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the delta4 desaturation of palmitoyl-acyl carrier protein (16:0-ACP) and subsequent elongation of delta4-hexadecenoyl (16:1 delta4)-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1 delta4-ACP and 16:0-ACP as substrates. Extracts from developing coriander seeds elongated 16:1 delta4-ACP in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroselinic acid, displayed a strong preference for the elongation of 16:0-ACP rather than 16:1 A4-ACP. In addition, the elongation of 16:1 A4-ACP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerulenin at concentrations as low as 10 microM. This finding suggested that the elongation of 16:1 A4-ACP and 16:0-ACP in coriander seed is catalyzed by a 3-ketoacyl-ACP synthase (KAS) 1-type enzyme(s), rather than a KAS II-type activity that is typically associated with 16:0-ACP elongation. Consistent with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterologous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, although the polypeptide corresponding to the coriander cDNA is more closely related to KAS I. its active site motif deviates from those found in both KAS I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an abundant 45 kDa polypeptide in coriander endosperm that is not detected in coriander leaves. These antibodies also recognize a major band of similar size in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1991

Level of Abscisic Acid in Integuments, Nucellus, Endosperm, and Embryo of Peach Seeds (Prunus persica L. cv Springcrest) during Development.

Free abscisic acid (ABA) in integuments, nucellus, endosperm, and embryo was determined throughout seed development of peach (Prunus persica L. cv Springcrest). Quantification of ABA was performed using combined high performance liquid chromatography-radioimmunoassay based on a monoclonal antibody raised against free (S)-ABA. In the integuments and endosperm, ABA concentration remained constant during the first 100 days after anthesis and rose in the following days when fresh weight was rapidly decreasing. In the nucellus, the ABA concentration variation pattern paralleled that of tissue growth. ABA concentration in the embryo increased constantly with the growth of the tissues to reach a maximum at the last growth stage. The role of ABA in peach seeds is discussed in relation to the development of the different seed tissues.

Journal Article↗
PubMed2005

Spatial and temporal development of chondrocyte-seeded agarose constructs in free-swelling and dynamically loaded cultures.

Dynamic deformational loading has been shown to significantly increase the development of material properties of chondrocyte-seeded agarose hydrogels, however little is known about the spatial development of the material properties within these constructs. In this study, a technique that combines video microscopy and optimized digital image correlation, was applied to assess the spatial development of material properties in tissue-engineered cartilage constructs cultured in free-swelling and dynamically-loaded conditions (3h/day, 5 days/week, and maintained in free-swelling conditions when not being loaded) over a 6-week period. Although homogeneous at day 0, both free-swelling and dynamically loaded samples progressively developed stiffer outer edges and a softer central region. The distribution of GAGs and collagens were shown to mimic this profile. These results indicate that although dynamic loading augments the development of bulk properties in these samples, possibly by overcoming some of the diffusion limitation and nutrient transport issues, the overall profile of construct properties in the axial direction remains qualitatively the same as in free-swelling culture conditions. Poisson's ratio of these constructs increased over time in culture with increased fixed charged density contributed by the GAGs, but this increase was significantly less in dynamically loaded samples by day 42. Polarized light microscopy of Picrosirius Red labeled samples, at an angle perpendicular to the direction of loading, suggests that these differences in Poisson's ratio may be due to improved organization of collagen network in the dynamically loaded samples.

Animals↗
PubMed1996

Evolution of soluble carbohydrates during the development of pea, faba bean and lupin seeds.

Seeds of pea (Pisum sativum L. cv. Ergo), faba bean (Vicia faba ssp. minor Harz., cv. Tibo) and yellow pea lupin (Lupinus luteus L. cv. Juno) were sampled at different days after flowering (DAF) and their content of soluble carbohydrates was determined. Analysis of samples showed that myo-inositol, fructose, glucose, galactose and sucrose were found in high abundance early in development and their content decreased gradually during maturation. alpha-Galactosides, which includes the content of raffinose, stachyose and verbascose, started to appear later in seed development, at 37 DAF in peas, 40 DAF in faba beans and 45 DAF in lupins. Their accumulation increased considerably during seed growth, and the maximum content was obtained in mature seeds; 3.8% in peas, 4.5% in faba beans and 10.4% in lupins. Results obtained for these sugars during seed development were fitted to modelling curves in order to predict sugar content at different development stages.

Carbohydrates↗
PubMed2004

Arabidopsis trehalose-6-phosphate synthase 1 is essential for normal vegetative growth and transition to flowering.

In resurrection plants and yeast, trehalose has a function in stress protection, but the absence of measurable amounts of trehalose in other plants precludes such a function. The identification of a trehalose biosynthetic pathway in angiosperms raises questions on the function of trehalose metabolism in nonresurrection plants. We previously identified a mutant in the Arabidopsis trehalose biosynthesis gene AtTPS1. Plants homozygous for the tps1 mutation do not develop mature seeds (Eastmond et al., 2002). AtTPS1 expression analysis and the spatial and temporal activity of its promoter suggest that this gene is active outside the seed-filling stage of development as well. A generally low expression is observed in all organs analyzed, peaking in metabolic sinks such as flower buds, ripening siliques, and young rosette leaves. The arrested tps1/tps1 embryonic state could be rescued using a dexamethasone-inducible AtTPS1 expression system enabling generation of homozygous mutant plants. When depleted in AtTPS1 expression, such mutant plants show reduced root growth, which is correlated with a reduced root meristematic region. Moreover, tps1/tps1 plants are retarded in growth and remain generative during their lifetime. Absence of Trehalose-6-Phosphate Synthase 1 in Arabidopsis plants precludes transition to flowering.

Arabidopsis↗
PubMed2002

Early expression of grain hardness in the developing wheat endosperm.

Seeds from near-isogenic hard and soft wheat lines were harvested at regular intervals from 5 days post-anthesis to maturity and examined for hardness using the single kernel characterisation system (SKCS). SKCS analysis revealed that hard and soft lines could be distinguished from 15 days post-anthesis (dpa). This trend continued until maturity where the difference between the hard and soft lines was most marked. SKCS could not be applied to the small 5- and 10-dpa wheat kernels. Fresh developing endosperm material was examined using light microscopy and no visible differences between the cultivars were detected. When air-dried material was examined using scanning electron microscopy (SEM) differences between soft and hard lines were visible from as early as 5 dpa. Accumulation of puroindoline a and puroindoline b was investigated in developing seeds using both Western blotting and ELISA. Low levels of puroindoline a could be detected in the soft cultivar from 10 dpa, reaching a maximum at 32 dpa. In the hard cultivar, puroindoline a levels were negligible throughout grain development. Puroindoline b accumulates in both the soft and hard cultivars from 15 dpa, but overall contents were higher in the soft cultivar. These findings indicate that endosperm hardness is expressed very early in developing grain when few starch granules and storage proteins were deposited in the endosperm cells. Further, the near-isogenic soft and hard Heron lines could be differentiated by SEM at a stage in development when the accumulation of puroindolines could not be detected by the methods used in this study.

Desiccation↗
PubMed1998

A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed.

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Delta9) plus cis-vaccenic acid (18:1Delta11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Delta9-18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Delta9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Delta9-18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Delta9-18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Delta9-18:0-ACP desaturase.

Amino Acid Sequence↗
PubMed1989

Endothelial cell seeding on PTFE vascular prostheses using a standardized seeding technique.

A standardized method was developed for seeding endothelial cells (EC) in tubular vascular grafts. A rotational cell seeding device for tubular prostheses is presented and parameters influencing the kinetics of cell adhesion (rotation speed, graft diameter, cell suspension level, inoculated cell number) are reported. Seeding EC in 14 mm ID PTFE vascular grafts with rotation rate of 10 rph gave an adhesion rate of 80% in a homogeneous monolayer.

Blood Vessel Prosthesis↗
PubMed2002

Temperature and oxygen regulation of oleate desaturation in developing sunflower (Helianthus annuus) seeds.

The effect of low (10 degrees C) and high (30 degrees C) temperature on in vivo oleate desaturation has been studied in developing sunflower (Helianthus annuus L.) seeds under conditions of different oxygen availability (capitulum, detached achenes or peeled seeds). In seeds remaining in the capitulum, only a part of the oleate newly synthesized at high temperature was desaturated to linoleate, whereas more oleate than that synthesized de novo was desaturated at low temperature. Achenes were only able to significantly desaturate oleate at low temperatures. In contrast, oleate desaturation was detected in peeled seeds incubated at low and high temperatures, showing the highest rate at 20 degrees C. Hull removing dramatically increased the activity of the microsomal oleate desaturase (FAD2, EC 1.3.1.35) at all studied temperatures, although a long-term inactivation of the enzyme was observed at high temperatures. Low oxygen concentration (1-2%) obtained by respiration of peeled seeds incubated in sealed vials, brought about the inactivation of the enzyme. All these data suggest that temperature regulates oleate desaturation controlling the amount of oleate and the FAD2 activity. In addition, this enzyme seems to be also regulated by the availability of oxygen, which is affected inside the achene by its diffusion through the hull, and the competition with respiration, both factors being temperature-dependent.

Journal Article↗
PubMed2003

Elevated levels of N-lauroylethanolamine, an endogenous constituent of desiccated seeds, disrupt normal root development in Arabidopsis thaliana seedlings.

N-Acylethanolamines (NAEs) are prevalent in desiccated seeds of various plant species, and their levels decline substantially during seed imbibition and germination. Here, seeds of Arabidopsis thaliana (L.) Heynh. were germinated in, and seedlings maintained on, micromolar concentrations of N-lauroylethanolamine (NAE 12:0). NAE 12:0 inhibited root elongation, increased radial swelling of root tips, and reduced root hair numbers in a highly selective and concentration-dependent manner. These effects were reversible when seedlings were transferred to NAE-free medium. Older seedlings (14 days old) acclimated to exogenous NAE by increased formation of lateral roots, and generally, these lateral roots did not exhibit the severe symptoms observed in primary roots. Cells of NAE-treated primary roots were swollen and irregular in shape, and in many cases showed evidence, at the light- and electron-microscope levels, of improper cell wall formation. Microtubule arrangement was disrupted in severely distorted cells close to the root tip, and endoplasmic reticulum (ER)-localized green fluorescent protein (mGFP5-ER) was more abundant, aggregated and distributed differently in NAE-treated root cells, suggesting disruption of proper cell division, endomembrane organization and vesicle trafficking. These results suggest that NAE 12:0 likely influences normal cell expansion in roots by interfering with intracellular membrane trafficking to and/or from the cell surface. The rapid metabolism of NAEs during seed imbibition/germination may be a mechanism to remove this endogenous class of lipid mediators to allow for synchronized membrane reorganization associated with cell expansion.

Arabidopsis↗
PubMed1986

Seed germination and seedling development in the mango (Mangifera indica L.).

Mango (Mangifera indica L., cv Ruby) seeds taken from ripe fruit showed no dormancy. They germinated at temperatures between 5 and 40 degrees C, but germination was most rapid near the upper end of this range (25-40 degrees C). The fresh seeds had a high moisture content (85%, dry weight basis) and quickly died on dehydration. The optimal temperature for growth of the seedlings was close to 30 degrees C. High temperatures (40 degrees C) and temperatures below 15 degrees C were lethal. Growth of the stem occurred in successive flushes separated by rest periods. When the leaves of the preceding flush finished growing, the axis lengthened beneath the apical bud.

Journal Article↗
PubMed2025

Large-scale screening of genes responsible for silique length and seed size in Brassica Napus via pooled CRISPR library.

BACKGROUND: Enhancing rapeseed (Brassica napus, B. napus) yield is critical for ensuring global vegetable oil security. However, yield is heavily influenced by silique development and seed size, the enhancement of which is limited by scarce genetic resources. The CRISPR/Cas9 system has emerged as a powerful tool for constructing genome-wide mutant libraries, even in polyploid crops with complex genomes. RESULTS: The transcriptome-wide association study (TWAS) data, tissue-specific expression profiles data and reported genes were integrated to identify candidate genes regulating silique development and seed size. We constructed a sgRNA library targeting these genes and generated a CRISPR/Cas9 editing mutant library through genetic transformation. Specifically, 6124 sgRNAs were designed for 1739 candidate genes with ≦ 4 orthologues. 681 T0 plants were obtained through genetic transformation, which harbor 453 sgRNAs. Of 408 T0 plants analyzed, 151 (37.00%) exhibited successful gene editing events, targeting 84 candidate genes. Ten homozygous mutant plants were isolated and preliminary phenotypic analysis was performed in mutants targeting the BnaHRDs. The results suggest that mutations in BnaHRD.A03 and BnaHRD.C03 may modulate plant height (PH), main inflorescence length (MIL), silique length (SL), effective silique number per plant (ENS), seed number per silique (SNPS), and thousand-seed weight (TSW). CONCLUSIONS: This study harnessed the CRISPR/Cas9 technology to establish a preliminary library of gene-edited mutants in B. napus, thereby laying a robust foundation for the future screening of candidate genes pertaining to silique development and seed size. Furthermore, this study provides a methodological framework for rapid functional gene discovery in B. napus through CRISPR-based approaches.

Brassica napus↗
PubMed2004

Della proteins and gibberellin-regulated seed germination and floral development in Arabidopsis.

RGA (repressor of ga1-3) and GAI (gibberellin insensitive) are negative regulators of plant hormone gibberellin (GA) signaling in Arabidopsis. The GA-deficient mutant ga1-3 is a nongerminating, extreme dwarf that flowers late and produces male-sterile flowers. The rga and gai null alleles interact synergistically to rescue vegetative growth and floral initiation in ga1-3, indicating that RGA and GAI are major repressors for these processes. However, rga and gai in combination cannot rescue seed germination or floral development in ga1-3. RGA and GAI belong to the DELLA subfamily within the GRAS family of plant regulatory proteins. Three additional DELLA proteins RGL1, RGL2, and RGL3 are present in Arabidopsis. Previous studies provided evidence that RGL2 and possibly RGL1 control seed germination. To investigate further the function of the RGL genes, we examined the expression profiles of all 5 DELLA protein genes by real-time PCR. RGA and, to a lesser extent, GAI mRNAs were expressed ubiquitously in all tissues, whereas RGL1, 2, and 3 transcripts were present at high levels only in germinating seeds and/or flowers and siliques. Using the newly isolated rgl1, rgl2, and rgl3 T-DNA insertion mutants, we demonstrated that RGL2 is the major repressor in seed germination. We further provided evidence that RGA, RGL1, and RGL2 are all involved in modulating floral development. Interestingly, RGL2 expression is regulated not only at the transcript level. We showed that RGL2 protein in imbibed seeds is rapidly degraded by GA treatment and that the F-box protein SLY1 is required for RGL2 degradation to occur.

Alkyl and Aryl Transferases↗
PubMed2004

Asymmetric localization of seed storage protein RNAs to distinct subdomains of the endoplasmic reticulum in developing maize endosperm cells.

Plant storage proteins are synthesized and stored in different compartments of the plant endomembrane system. Developing maize seeds synthesize and accumulate prolamin (zein) and 11S globulin (legumin-1) type proteins, which are sequestered in the endoplasmic reticulum (ER) lumen and storage vacuoles, respectively. Immunofluorescence studies showed that the lumenal chaperone BiP was not randomly distributed within the ER in developing maize endosperm but concentrated within the zein-containing protein bodies. Analysis of the spatial distribution of RNAs in maize endosperm sections by in situ RT-PCR showed that, contrary to the conclusions made in an earlier study [Kim et al. (2002) Plant Cell 14: 655-672], the zein and legumin-1 RNAs are not symmetrically distributed on the ER but, instead, targeted to specific ER subdomains. RNAs coding for 22 kDa alpha-zein, 15 kDa beta-zein, 27 kDa gamma-zein and 10 kDa delta-zein were localized to ER-bounded zein protein bodies, whereas 51 kDa legumin-1 RNAs were distributed on adjacent cisternal ER proximal to the zein protein bodies. These results indicate that the maize storage protein RNAs are targeted to specific ER subdomains in developing maize endosperm and that RNA localization may be a prevalent mechanism to sort proteins within plant cells.

Endoplasmic Reticulum↗
PubMed2006

Development of weaning food from sorghum supplemented with legumes and oil seeds.

BACKGROUND: The development of low-cost, high-protein food supplements for weaning infants from local and readily available raw materials is a constant challenge for developing countries. OBJECTIVE: To formulate, develop, and assess the nutritive value, acceptability, and keeping quality of sorghum-based staple dried flakes as instant weaning foods. METHODS: Weaning blends composed of 42% sorghum supplemented with 20% legumes, 10% oil seeds, and 28% additives (sugar, oil, skim milk powder, and vanillin) were prepared according to FAO/WHO/UNU recommendations and processed in a twin-roller drum dryer. The effects of fermentation and of the addition of malt on the nutritive value and functional properties of the blends were investigated. RESULTS: The blends were found to contain 16.6% to 19.3% protein, 68.7% to 72.7% carbohydrate, 0.9% to 1.3% fiber, and 405.8 to 413.2 kcal of energy per 100 g. The iron content of the blends ranged from 5.3 to 9.1 mg/100 g, and the calcium content ranged from 150 to 220 mg/100 g. All blends reconstituted well and formed a soft paste when stirred with hot or cold water. The water-holding capacity, wettability, and bulk density were within the ranges of corresponding values of commercial weaning foods. Sensory attributes, viscosity values, and in vitro digestibility varied among the blends, whereas lysing content improved considerably (p < or = .05) for all blends. All blends had similar keeping quality, with no signs of spoilage or development of off-flavors or colors after 10 months of storage. Most blends remained free of aflatoxins. CONCLUSIONS: Legumes and oil seeds can be effectively used in sorghum-based weaning foods as an acceptable protein and mineral supplement.

Dietary Proteins↗