Developmental biochemistry of cottonseed embryogenesis and germination XVIII cDNA and amino acid sequences of members of the storage protein families

Summary We have sequenced cDNA clones representing each of the three distinct groups of storage proteins of the cotton seed. Characteristics of their mRNAs and derived proteins are given. Dot matrix analysis of the nucleotide and amino acid sequences shows that 2 of these groups of proteins have a great deal of vestigial homology at low stringency and should be considered subfamilies of a single storage protein gene family. The remaining group is quite distinct and should be considered a separate multigene family. It also can be divived into 2 subfamilies based on the presence or absence of glycosyl residues and other sequence differences.These proteins are processed to smaller species during embryogenesis, and all of the mature storage proteins of cotton can be traced back to these 2 gene families.In view of these relationships we propose that these 2 families be called the and globulins of cotton storage proteins, each comprised of an A and B subfamily.     

Developmental biochemistry of cotton seed embryogenesis and germination: x. Nitrogen flow from arginine to asparagine in germination

The enzymic basis for the flow of nitrogen from arginine to asparagine during the first 3 days of germination has been measured in extracts from cotton (Gossypium hirsutum) cotyledons. Evidence that asparagine synthetase regulates asparagine accumulation in germination (for transport to the axis) is presented. Further, evidence that the bulk of the nitrogen passed from one generation to the next in dicots is through an asparagine cycle involving the following sequence asparagine → arginine → storage protein → arginine → asparagine is discussed.     

Developmental biochemistry of cotton seed embryogenesis and germination. VII. Characterization of the cotton genome.

The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, base composition, and genetic complexity. The haploid genome size is found to bo 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due to endoreduplication of the entire genome.Non-repetitive deoxynucleotide sequences account for approximately 60.5% of the cotton genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$pure⁵ = 437); highly repetitive sequences (> 10,000 repetition frequency) constitute about 7.7% of the genome. ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 4.6 × 10}{}^{\mathrm{?4}}$) and intermediately repetitive sequences constitute the remaining 27% of the genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 1.46}$). Hybridization of ¹²⁵I-labeled cytoplasmic ribosomal RNA to whole-cell DNA on filters and in solution indicate approximately 300 to 350 copies of the rRNA cistrons per haploid genome.The interspersion of repetitive sequences that reassociate between C₀t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined by determining the reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the genome consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 20% of the genome may be involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence divergence is evident among the members of this component.Highly repetitive sequence elements that are reassociated by a C₀t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of this highly repetitive component reassociates by C₀t = 10^?4, zero-time binding DNA, and accounts for less than 3% of the genome. At least a third of these sequences appear by electron microscopy to be intramolecular duplexes (palindromes) of 150 to 200 base-pairs and to occur in clusters.     

Developmental pathways of somatic embryogenesis

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue. Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Although great progress has been made in improving the protocols used, it has been revealed that some treatments, coinciding with increased yield of somatic embryos, can cause adverse effects on the embryo quality, thereby impairing germination and ex vitro growth of somatic embryo plants. Accordingly, ex vitro growth of somatic embryo plants is under a cumulative influence of the treatments provided during the in vitro phase. In order to efficiently regulate the formation of plants via somatic embryogenesis it is important to understand how somatic embryos develop and how the development is influenced by different physical and chemical treatments. Such knowledge can be gained through the construction of fate maps representing an adequate number of morphological and molecular markers, specifying critical developmental stages. Based on this fate map, it is possible to make a model of the process. The mechanisms that control cell differentiation during somatic embryogenesis are far from clear. However, secreted, soluble signal molecules play an important role. It has long been observed that conditioned medium from embryogenic cultures can promote embryogenesis. Active components in the conditioned medium include endochitinases, arabinogalactan proteins and lipochitooligosaccharides.     

Inhibition of cottonseed germination with abscisic Acid and its reversal

Germination of cottonseed (Gossypium hirsutum L.) was inhibited by abscisic acid. Inhibition was greater when seeds were soaked in abscisic acid for 5 hours and dried prior to germination than when abscisic acid was applied in the germination medium. (2-Chloroethyl)phosphonic acid, gibberellic acid, and kinetin partially overcame the inhibitory action of abscisic acid. Combinations of (2-chloroethyl)phosphonic acid with gibberellic acid or kinetin were more effective than the individual substances. Germination also was partially restored by removal of seed coats. Fusicoccin completely restored germination of abscisic acidtreated seeds.     

The developmental biochemistry of cotton seed embryogenesis and germination. VI. Levels of cytosol and chloroplast aminoacyl-tRNA synthetases during cotyledon development.

The separation of isotransferring aminoacyl-tRNA synthetase activities (amino acid: tRNA ligases, EC 6.1.1.x) for several amino acids extracted from tissues of embryonic and germinating cotton seeds was carried out by DEAE-cellulose column chromatography. Evidence was obrained that the separated activities represent discrete enzymes, and could be defined as cytosol or chloroplast enzymes by several criteria. The levels of the cytosol enzymes per cell were found to be constant in germinated and ungerminated cotyledons. Chloroplast enzymes were found to be present in immature embryonic cotyledons and in roots at constant levels relative to the cytosol enzymes, but found to increase markedly in germinating cotyledons. This increase takes place to the same extent in etiolated cotyledons as in greened cotyledons indicating that the chloroplast synthetase increase is analogous to the simultaneous increase in chloroplast tRNA and rRNA which also is not light dependent. The separated cytosol and chloroplast enzymes show varying degrees of specificity for isoaccepting tRNA species from homologous and heterologous sources.     

Optimization of somatic embryogenesis and embryo germination in soybean

Summary Somatic embryos of soybean [Glycine max (L.) Merr.] are induced on immature cotyledons explanted onto a medium containing moderately high levels of auxin. Germinability of embryos is related to morphologic normality, and both are reduced by excessive exposure to auxin during the induction process. Shoot meristem development was improved by reducing exposure of cotyledonary explants from 30 to 10 to 14 d on 10 mg/liter α-naphthaleneacetic acid (NAA). A 3-d exposure was sufficient to induce embryos, and embryo frequency was not significantly increased by exposures to NAA for more than 1 wk. Embryo frequency was enhanced, however, by transfer after 9 d to fresh medium containing 10 mg/liter NAA. Germination of morphologically normal embryos was achieved without growth regulators, after maturation for 1 mo. on hormone-free medium and desiccation for 1 wk in a sealed, dry container. This research was funded by Lubrizol Genetics, Inc., Madison, WI.     

The controls of late dicot embryogenesis and early germination

During seed formation, the embryo appears to be germinable as soon as cell division is completed; however, it continues development on the plant. This review describes the stages of development after cell division and provides a summary of important observations and recent use of molecular markers as they apply to the regulation of dicot seed formation. Genetic evidence suggests that abscisic acid may help initiate late embryogenesis, although no evidence firmly establishes that abscisic acid controls any other aspect of late dicot development. Previous studies utilizing cultured embryos have implicated abscisic acid and water potential as endogenous promoters of late embryogenesis and inhibitors of germination. However, these embryo culture experiments have been misinterpreted. The experiments show that both immature and mature embryos respond to environmental water stress by expressing a developmental program that is normally induced in late embryogenesis by abscission of the vascular connection. This postabscission program probably prepares the embryo for its forthcoming desiccation during normal development and is predicted to be important in protecting the embryo from water stress during germination.     

Lipid biochemistry of echinochloa crus-galli during anaerobic germination

Seven day old seedlings of Echinochloa crus-galli var. oryzicola (Vasing) had a higher total lipid content when germinated under N₂ than in air, although ungerminated seeds contained more lipid than either seedling. The triacylglycerol pool was not depleted under anaerobiosis as it was in air and only air-grown seedlings showed a net increase in free fatty acids and polar lipids. Concentrations of most of the individual acids of the total fatty acid profile declined during germination in air and in the free acid and polar lipid fractions of these seedlings the relative proportion of polyunsaturated fatty acids increased. Compared to air-grown seedlings, ungerminated seeds and N₂-grown seedlings had a similar qualitative and quantitative lipid composition. Our results show that mobilization of storage lipids was apparently severely inhibited under anoxia. The importance of lipid metabolism to the germination and growth of Echinochloa during anoxia is discussed in terms of maintaining membrane integrity and serving (indirectly) to reoxidize pyridine nucleotides.     

Developmental expression of CagMdkb during gibel carp embryogenesis

Midkine (Mdk) genes have been revealed to have different expression patterns in vertebrates and therefore, additional studies on Mdk expression patterns are required in more species. In this study, CagMdkb has been cloned and characterized from a SMART cDNA library of 10-somite stage embryos of Carassius auratus gibelio. Its full length cDNA is 1091 bp and encodes a sequence of 147 amino acids, which shows 97.3% identity to zebrafish Mdkb on the amino acid level. RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during subsequent embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band and the zygotic CagMdkb protein is expressed from gastrula stage. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein was found to be localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal was detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein was also extended to numerous small neurons in the forebrain, midbrain and hindbrain, as well as to nerve fibers in the spinal cord. Co-localization with 3A10 antibody revealed CagMdkb immunoreactivity on developing Mauthner neurons, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppressed head formation and CagMdkb function was found to depend on secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish head formation.