Day Post Anthesis Research Articles

The functions of laccase gene GhLAC15 in fiber colouration and development in brown-colored cotton.

The monotonicity of color type in naturally colored cottons (NCCs) has become the main limiting factor to their widespread use, simultaneously coexisting with poor fiber quality. The synchronous improvement of fiber quality and color become more urgent and crucial as the demand for sustainable development increases. The homologous gene of wild cotton Gossypium stocksii LAC15 in G. hirsutum, GhLAC15, was also dominantly expressed in the developing fibers of brown cotton XC20 from 5 DPA (day post anthesis) to 25 DPA, especially at the secondary cell wall thickening stage (20 DPA and 25 DPA). In XC20 plants with downregulated GhLAC15 (GhLAC15i), a remarkable reduction in proanthocyanidins (PAs) and lignin contents was observed. Some of the key genes in the phenylpropane and flavonoid biosynthesis pathway were down-regulated in GhLAC15i plants. Notably, the fiber length of GhLAC15i plants showed an obvious increase and the fiber color was lightened. Moreover, we found that the thickness of cotton fiber cell wall was decreased in GhLAC15i plants and the fiber surface became smoother compared to that of WT. Taken together, this study revealed that GhLAC15 played an important role in PAs and lignin biosynthesis in naturally colored cotton fibers. It might mediate fiber color and fiber quality by catalyzing PAs oxidation and lignin polymerization, ultimately regulating fiber colouration and development.

A global survey of the gene network and key genes for oil accumulation in cultivated tetraploid cottons.

SummaryTo enrich our knowledge about gene network of fatty acid biosynthesis in cottonseed, we conducted comparative transcriptome to reveal the differences in gene expression between Gossypium hirsutum and Gossypium barbadense during cottonseed development. The prolonged expression period and increased expression abundance of oil‐related genes are the main reasons for producing high seed oil content (SOC) in G. barbadense, which manifested as the bias of homeologous gene expression in Dt‐subgenome after 25 day postanthesis (DPA). The dynamic expression profile showed that SAD6 and FATA are more important for oil biosynthesis in G. barbadense than that in G. hirsutum. Three key transcription factors, WRI1, NF‐YB6 and DPBF2, showed their elite roles in regulating seed oil in cotton. We observed that sequence variations in the promoter region of BCCP2 genes might contribute to its divergence in expression level between the two species. Based on the quantitative trait loci (QTL) information of the seed oil content and utilizing additional G. barbadense introgression lines (ILs), we propose 21 candidate genes on the basis of their differential expression level, of which the GbSWEET and the GbACBP6 showed the potential functional to improve the oil content. Taken together, studying the different expression of oil‐related genes and their genetic regulation mechanisms between G. hirsutum and G. barbadense provide new insights to understanding the mechanism of fatty acid biosynthesis network and fatty acid genetic improving breeding in cotton.

Long non-coding RNAs and their potential functions in Ligon-lintless-1 mutant cotton during fiber development

BackgroundLong non-coding RNAs (LncRNAs) are part of genes, which are not translated into proteins and play a vital role in plant growth and development. Nevertheless, the presence of LncRNAs and how they functions in Ligon-lintless-1 mutant during the early cessation of cotton fiber development are still not well understood. In order to investigate the function of LncRNAs in cotton fiber development, it is necessary and important to identify LncRNAs and their potential roles in fiber cell development.ResultsIn this work, we identified 18,333 LncRNAs, with the proportion of long intergenic noncoding RNAs (LincRNAs) (91.5%) and anti-sense LncRNAs (8.5%), all transcribed from Ligon-lintless-1 (Li1) and wild-type (WT). Expression differences were detected between Ligon-lintless-1 and wild-type at 0 and 8 DPA (day post anthesis). Pathway analysis and Gene Ontology based on differentially expressed LncRNAs on target genes, indicated fatty acid biosynthesis and fatty acid elongation being integral to lack of fiber in mutant cotton. The result of RNA-seq and RT-qPCR clearly singles out two potential LncRNAs, LNC_001237 and LNC_017085, to be highly down-regulated in the mutant cotton. The two LncRNAs were found to be destabilized or repressed by ghr-miR2950. Both RNA-seq analysis and RT-qPCR results in Ligon-lintless-1 mutant and wild-type may provide strong evidence of LNC_001237, LNC_017085 and ghr-miR2950 being integral molecular elements participating in various pathways of cotton fiber development.ConclusionThe results of this study provide fundamental evidence for the better understanding of LncRNAs regulatory role in the molecular pathways governing cotton fiber development. Further research on designing and transforming LncRNAs will help not only in the understanding of their functions but will also in the improvement of fiber quality.

Is ethyl methane sulfonate induced mutation influence the KasI gene sequence and its expression?

A novel Chili pepper G1M1 mutant was produced from chili pepper Genotype 1 induced with 0.01% ethyl methane sulfonate (EMS). The aim of this research is to evaluate the effect of EMS induced mutation to the sequence of capsaicin synthesis gene KasI and its expression. Genetic profile of KasI genes of mutant plants and controls were analyzed by sequencing method and the results aligned and compared with the sequence of Capsicum annuum KasI (KM037709.1) on the NCBI. Capsaicin content was measured from fruit at 30-35 DPA (day post anthesis) using spectrophotometry method (Ȧ280 nm). The results of this study indicated that the capsaicin content of G1M1 mutant plant did not differ significantly with G1 control, but of G1M1 tended to have higher capsaicin content than control. The KasI gene of G1M1 mutant shows the presence of substitution at some sites which causes the replacement of amino acid composition. This may cause differences in the capsaicin content between plants.

SSR-Linkage map of interspecific populations derived from Gossypium trilobum and Gossypium thurberi and determination of genes harbored within the segregating distortion regions.

Wild cotton species have significant agronomic traits that can be introgressed into elite cultivated varieties. The use of a genetic map is important in exploring, identification and mining genes which carry significant traits. In this study, 188 F2mapping individuals were developed from Gossypium thurberi (female) and Gossypium trilobum (male), and were genotyped by using simple sequence repeat (SSR) markers. A total of 12,560 simple sequence repeat (SSR) markers, developed by Southwest University, thus coded SWU were screened out of which only 994 were found to be polymorphic, and 849 markers were linked in all the 13 chromosomes. The map had a length of 1,012.458 cM with an average marker distance of 1.193 cM. Segregation distortion regions (SDRs) were observed on Chr01, Chr02, Chr06, Chr07 Chr09, Chr10 and Chr11 with a large proportion of the SDR regions segregating towards the heterozygous allele. There was good syntenic block formation that revealed good collinearity between the genetic and physical map of G. raimondii, compared to the Dt_sub genome of the G. hirsutum and G. barbadense. A total of 2,496 genes were mined within the SSR related regions. The proteins encoding the mined genes within the SDR had varied physiochemical properties; their molecular weights ranged from 6.586 to 252.737 kDa, charge range of -39.5 to 52, grand hydropathy value (GRAVY) of -1.177 to 0.936 and isoelectric (pI) value of 4.087 to 12.206. The low GRAVY values detected showed that the proteins encoding these genes were hydrophilic in nature, a property common among the stress responsive genes. The RNA sequence analysis revealed more of the genes were highly upregulated in various stages of fiber development for instance; Gorai.002G241300 was highly up regulated at 5, 10, 20 and 25 day post anthesis (DPA). Validation through RT-qPCR further revealed that these genes mined within the SDR regions might be playing a significant role under fiber development stages, therefore we infer that Gorai.007G347600 (TFCA), Gorai.012G141600 (FOLB1), Gorai.006G024500 (NMD3), Gorai.002G229900 (LST8) and Gorai.002G235200 (NSA2) are significantly important in fiber development and in turn the quality, and further researches needed to be done to elucidate their exact roles in the fiber development process. The construction of the genetic map between the two wild species paves away for the mapping of quantitative trait loci (QTLs) since the average distance between the markers is small, and mining of genes on the SSR regions will provide an insight in identifying key genes that can be introgressed into the cultivated cotton cultivars.

Downregulation of γECS gene affects antioxidant activity and free radical scavenging system during pod development and maturation in Lablab perpureus L

Fruit development and maturation has been recognized as an oxidative phenomenon, which results in the generation of reactive oxygen species (ROSs). Plants cells contain several antioxidants which help to retained these ROS at low level by indulging them to enter in signalling pathways. The present work describes, the changes occurring in the components of enzymatic antioxidant defence systems during pod development and maturation in two hyacinth bean varieties viz. VRSEM-3 and VRSEM-8, which differ in their pod qualities. The experiment was conducted in randomized block design with three replications and pod samples were collected at the following day post anthesis (DPA) stages: immature (15 DPA), mature (25 DPA) and over mature (40 DPA). Activities of catalase (CAT) and ascorbate peroxidase (APX) reduced up to 1.5/2.0 folds, respectively, during the subsequent stages of pod maturation, indicating a higher accumulation of reactive oxygen species (ROSs) at the latter stages. Glutathione reductase (GR) and superoxide dismutase (SOD) activities decreased progressively during pod development in VRSEM-3 which continued towards the end of maturation as pods turn to the over-mature stage. The role of glutathione-mediated free-radical scavenging was also investigated at transcriptional level, as a plausible system for coping with oxidative stress. The transcript level of γECS (glutamylcysteine synthetase) gene remained relatively constant at the immature and mature stages, whereas it sharply decreased about 15–30 folds at the over-mature stage in both the varieties, which correlated well with the subsequent decrease in the levels of enzymatic antioxidants. Hence, the present study confirmed that the glutathione mediated free radical scavenging system was down-regulated at over-mature stage, conceivably in response to the growing oxidative stress resulting from the inflation of reactive oxygen species, increased solid content and concomitant with a decline in enzymatic and non-enzymatic antioxidants towards the end of maturation.

Distribution of XTH, expansin, and secondary-wall-related CesA in floral and fruit abscission zones during fruit development in tomato (Solanum lycopersicum).

After fruit development is triggered by pollination, the abscission zone (AZ) in the fruit pedicel strengthens its adhesion to keep the fruit attached. We previously reported that xyloglucan and arabinan accumulation in the AZ accompanies the shedding of unpollinated flowers. After the fruit has developed and is fully ripened, shedding occurs easily in the AZ due to lignin accumulation. Regulation of cell wall metabolism may play an important role in these processes, but it is not well understood. In the present report, we used immunohistochemistry to visualize changes in the distributions of xyloglucan and arabinan metabolism-related enzymes in the AZs of pollinated and unpollinated flowers, and in ripened fruits. During floral abscission, we observed a gradual increase in polyclonal antibody labeling of expansin in the AZ. The intensities of LM6 and LM15 labeling of arabinan and xyloglucan, respectively, also increased. However, during floral abscission, we observed a large 1 day post anthesis (DPA) peak in the polyclonal antibody labeling of XTH in the AZ, which then decreased. These results suggest that expansin and XTH play important, but different roles in the floral abscission process. During fruit abscission, unlike during floral abscission, no AZ-specific expansin and XTH were observed. Although lignification was seen in the AZ of over-ripe fruit pedicels, secondary cell wall-specific cellulose synthase signals were not observed. This suggests that cellulose metabolism-related enzymes do not play important roles in the AZ prior to fruit abscission.

Aberrant phenotype and transcriptome expression during fiber cell wall thickening caused by the mutation of the Im gene in immature fiber (im) mutant in Gossypium hirsutum L.

BackgroundThe immature fiber (im) mutant of Gossypium hirsutum L. is a special cotton fiber mutant with non-fluffy fibers. It has low dry weight and fineness of fibers due to developmental defects in fiber secondary cell wall (SCW).ResultsWe compared the cellulose content in fibers, thickness of fiber cell wall and fiber transcriptional profiling during SCW development in im mutant and its near-isogenic wild-type line (NIL) TM-1. The im mutant had lower cellulose content and thinner cell walls than TM-1 at same fiber developmental stage. During 25 ~ 35 day post-anthesis (DPA), sucrose content, an important carbon source for cellulose synthesis, was also significantly lower in im mutant than in TM-1. Comparative analysis of fiber transcriptional profiling from 13 ~ 25 DPA indicated that the largest transcriptional variations between the two lines occurred at the onset of SCW development. TM-1 began SCW biosynthesis approximately at 16 DPA, whereas the same fiber developmental program in im mutant was delayed until 19 DPA, suggesting an asynchronous fiber developmental program between TM-1 and im mutant. Functional classification and enrichment analysis of differentially expressed genes (DEGs) between the two NILs indicated that genes associated with biological processes related to cellulose synthesis, secondary cell wall biogenesis, cell wall thickening and sucrose metabolism, respectively, were significantly up-regulated in TM-1. Twelve genes related to carbohydrate metabolism were validated by quantitative reverse transcription PCR (qRT-PCR) and confirmed a temporal difference at the earlier transition and SCW biosynthesis stages of fiber development between TM-1 and im mutant.ConclusionsWe propose that Im is an important regulatory gene influencing temporal differences in expression of genes related to fiber SCW biosynthesis. This study lays a foundation for cloning the Im gene, elucidating molecular mechanism of fiber SCW development and further genetic manipulation for the improvement of fiber fineness and maturity.

The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower

Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development.

Changes in the distribution of cell wall polysaccharides in early fruit pericarp and ovule, from fruit set to early fruit development, in tomato (Solanum lycopersicum)

During fruit development in tomato (Solanum lycopersicum), cell proliferation and rapid cell expansion occur after pollination. Cell wall synthesis, alteration, and degradation play important roles during early fruit formation, but cell wall composition and the extent of cell wall synthesis/degradation are poorly understood. In this study, we used immunolocalization with a range of specific monoclonal antibodies to examine the changes in cell wall composition during early fruit development in tomato. In exploring early fruit development, the -1day post-anthesis (DPA) ovary and fruits at 1, 3, and 5 DPA were sampled. Paraffin sections were prepared for staining and immunolabeling. The 5 DPA fruit showed rapid growth in size and an increase in both methyl-esterified pectin and de-methyl-esterified pectin content in the pericarp, suggesting rapid synthesis and de-methyl esterification of pectin during this growth period. Labeling of pectic arabinan with LM6 antibody and galactan with LM5 antibody revealed abundant amounts of both, with unique distribution patterns in the ovule and premature pericarp. These results suggest the presence of rapid pectin metabolism during the early stages of fruit development and indicate a unique distribution of pectic galactan and arabinan within the ovule, where they may be involved in embryogenesis.

Expression of MicroRNAs in Cotton

MicroRNAs (miRNAs) are a class of newly identified non-protein-coding small RNAs. miRNAs post-transcriptionally regulate the expression of more than 30% genes, which control many biological and metabolic progresses, including plant growth, development, and response to environmental stresses. However, no studies have been performed on miRNA expression in cotton, one of the most important fiber and economic crops. In this study, we employed quantitative real-time PCR (qRT-PCR) to detect and compare miRNA expressions in eight different cotton organs at different developmental stages. Our results showed that miRNAs were differentially expressed in different cotton organs, with certain classes expressed preferentially in an organ-specific manner. The miR-156 was highly expressed in cotyledon, whereas the miR-172 was highly expressed in young leaves at fruit branch, young flower buds, 0 day post-anthesis (DPA) ovules, and 0 DPA petals. We also found that miR-172 was not highly expressed in all parts of flowers. In contrast, miR-172 was highly expressed in petal but not in stamen and carpel. Interestingly, miR-162 was highly expressed in immature fiber, 2 DPA ovules, and mixtures of 0 DPA stamen and carpel, suggesting miR-162 may play a role in cotton fiber differentiation and development. Our previous study showed that miR-396 may target a fiber-related gene the callose synthase catalytic subunit. In this study, the miR-396 expression was observed in all eight cotton organs. This study identified the expression of miRNAs that may regulate the development of cotton and cotton fiber.

Isolation and molecular characterization of a novel homogalacturonan galacturonosyl- transferase gene (GbGAUT1) from Gossypium barbadense

A novel gene, designated as GbGAUT1 (Gossypium barbadense homogalacturonan galacturonosyltransferase gene 1), was successfully cloned from G. barbadense (GenBank accession No. EF552432) by bioinformatics methods, RT-PCR and RACE. The open reading frame (ORF) was 1797 bp long and putatively encoded a polypeptide of 598 amino acids, with a predicted molecular mass of 69 kDa. GbGAUT1 could be considered as a homogalacturonan (HG) galacturonosyltransferase protein since the conserved GAUT1-related supperfamily motif had been found and it was clustered into GAUT-A group in the phylogenetic tree. Semi quantitative RT-PCR showed that GbGAUT1 preferentially expressed in the fiber secondary cell wall thickening, especially in the fibers of 35 day post anthesis (DPA). These results suggested that the novel cotton GbGAUT1 might play an important role in fiber development.

Cloning and expression of two sterol C-24 methyltransferase genes from upland cotton ( Gossypium hirsuturm L.)

Brassinosteroids (BRs) are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. Two kinds of intermediates, sitosterol and campesterol, play a crucial role in cell elongation, cellulose biosynthesis, and accumulation. To illuminate the effects of sitosterol and campesterol on the development of cotton ( Gossypium hirsuturm L.) fibers through screening cotton fiber EST database and contigging the candidate ESTs, two key genes GhSMT2-1 and GhSMT2-2 controlling the sitosterol biosynthesis were cloned from developing fibers of upland cotton cv. Xuzhou 142. The full length of GhSMT2-1 was 1,151 bp, including an 8 bp 5′-untranslated region (UTR), a 1,086 bp open reading frame (ORF), and a 57 bp 3′-UTR. GhSMT2-1 gene encoded a polypeptide of 361 amino acid residues with a predicted molecular mass of 40 kDa. The full length of GhSMT2-2 was 1,166 bp, including an 18 bp 5′-UTR, a 1,086 bp ORF, and a 62 bp 3′-UTR. GhSMT2-2 gene encoded a polypeptide of 361 amino acid residues with a predicted molecular mass of 40 kDa. The two deduced amino acid sequences had high homology with the SMT2 from Arabidopsis thaliana and Nicotiana tabacum. Furthermore, the typical conserved structures characterized by the sterol C-24 methyltransferase, such as region I (LDVGCGVGGPMRAI), region II (IEATCHAP), and region III (YEWGWGQSFHF), were present in both deduced proteins. Southern blotting analysis indicated that GhSMT2-1 or GhSMT2-2 was a single copy in upland cotton genome. Quantitative real-time RT-PCR analysis revealed that the highest expression levels of both genes were detected in 10 DPA (day post anthesis) fibers, while the lowest levels were observed in cotyledon and leaves. The expression level of GhSMT2-1 was 10 times higher than that of GhSMT2-2 in all the organs and tissues detected. These results indicate that the homologue of sterol C-24 methyltransferase gene was cloned from upland cotton and both GhSMT2 genes play a crucial role in fiber elongation. The role of GhSMT2-1 may be more important than that of GhSMT2-2.

Cloning and Expression Analysis of a Brassinosteroid Biosynthetic Enzyme Gene, GhDWF1, from Cotton ( Gossypium hirsuturm L.)

Brassinosteroids (BRs) are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. To determine the effects of BRs on the development of cotton fibers, through screening cotton fiber EST database and contigging the candidate ESTs, a key gene ( GhDWF1) involved in the upstream biosynthetic pathway of BRs was cloned from developing fibers of upland cotton ( Gossypium hirsutum L.) cv. Xuzhou 142. The full length of the cloned cDNA is 1849 bp, including a 37 bp 5′-untranslated region, an ORF of 1692 bp, and a 120 bp 3′-untranslated region. The cDNA encodes a polypeptide of 563 amino acid residues with a predicted molecular mass of 65 kD. The deduced amino acid sequence has high homology with the BR biosynthetic enzyme, DWARF1/DIMINUTO, from rice, maize, pea, tomato, and Arabidopsis. Furthermore, the typical conserved structures, such as the transmembrane domain, the FAD-dependent oxidase domain, and the FAD-binding site, are present in the GhDWF1 protein. The Southern blot indicated that the GhDWF1 gene is a single copy in upland cotton genome. RT-PCR analysis revealed that the highest level of GhDWF1 expression was detected in 0 DPA (day post anthesis) ovule (with fibers) while the lowest level was observed in cotyledon. The GhDWF1 gene presents high expression levels in root, young stem, and fiber, especially, at the fiber developmental stage of secondary cell wall accumulation. Moreover, the expression level was higher in ovules (with fibers) of wildtype (Xuzhou 142) than in ovules of fuzzless-lintless mutant at the same developmental stages (0 and 4 DPA). The results suggest that the GhDWF1 gene plays a crucial role in fiber development.

Cloning and characterization of cottonGhBGgene encoding β-glucosidase

Beta-1,4-glucosidase (BG, EC3.2.1.21), one of three cellulases, is a widespread family of enzymes involved in the metabolism of cell wall polysaccharides in both prokaryocytes and eukaryotes. Here, we report the isolation of a full-length cDNA encoding beta-1,4-glucosidase protein (designated as GhBG) and its putative function in the process of fiber development and in yeast. Through random sequencing of the cotton fiber cDNA library from 7235 germplasm line, with elite fiber quality in Gossypium hirsutum L. and utilizing the 5' rapid amplification of cDNA ends (RACE) technique, a 2133 bp cDNA clone encoding a cotton fiber specifically expressed protein (accession number: DQ103699) was isolated. GhBG was composed of a 1884 bp open reading frame (ORF) encoding 627 amino acid residues. This putative protein had an isoelectric point of 8.17, a calculated molecular weight of 68.78 KD and a signal peptide with 23 amino acid residues at the N-terminal. RT-PCR analysis indicated GhBG was specifically expressed in fiber cells and was highly abundant in 5-17 day post anthesis (DPA). It was not, however, expressed in root, hypocotyls or leaves. Southern blotting analysis showed there were two copies of GhBG in the upland cotton genome; most likely contained in sub-genome A and sub-genome D. GhBG was then integrated into a yeast expression vector, pREP-5N and electro-transformed into fission yeast Schizosaccharomyces pombe Q-01. The results demonstrated that GhBG led to a significant increase in cell length and width and a remarkable decrease of the length/width ratio. Compared to vector control transformants, cells were significantly larger and rounder and their growth velocity was also reduced.

Diurnal O2 and carbohydrate levels in wheat kernels during embryony

In vitro zygotic and somatic embryogenesis procedures for wheat have been improved by simulating in ovulo nutritional, hormonal and dissolved oxygen (dO2) conditions. However, diurnal fluctuations in these conditions during early embryony are not well characterized. In this study, dO2 and water-soluble carbohydrate levels in wheat kernels were determined after 8h of light and 8h of dark at approximately 6, 12 and 18 day post anthesis (DPA). Clark style O2 microelectrodes, having a tip diameter of approximately 115μm, were inserted into intact kernels immediately distil to the developing embryo, and dO2 levels were recorded at 50μm intervals into the center of kernels. High-performance anion exchange chromatography with pulsed amperometric detection was used to quantify carbohydrate levels in endosperm sap. dO2 levels in the chlorophyllous layer of the pericarp reached 190mmolm−3 during the day, which probably represents, because of photosynthesis, a supersaturated O2 condition relative to the external environment (21% O2). At the embryo surface, dO2 levels at 6DPA ranged from 135 to 170mmolm−3. At 12 and 18DPA, dO2 levels at the embryo axis ranged from 100 to 150mmolm−3. At all three stages, dO2 levels in the center of the endosperm were below 13mmolm−3. Extreme fluctuations in carbohydrate levels were observed diurnally during rapid seed fill (12DPA). Levels of sucrose and short-chain fructans were much higher during the day than during the night. In contrast, fructose, glucose, and myo-inositol levels were much higher during the night than during the day. By 18DPA (hard dough stage), carbohydrate levels tended to be similar during the day and night. These dynamic fluctuations may assist in regulating embryony in ovulo, and their simulation might improve the development of somatic and zygotic embryos in vitro.

Characterization of the natural chemical and osmotic environment of early wheat embryogenesis

Soluble carbohydrates, amino acids, and major inorganic ions were quantified and compared with the total osmolarity of the ovule. Most of the analyzed components increased rapidly in concentration, reaching a peak at 1 day post anthesis (DPA) and decreasing quickly 1 day later. Within this time period, the osmolarity decreased from 1.49 to 1.18 MPa. The osmolarity then increased to 1.39 MPa at 4 DPA, and yet the overall concentration of analyzed molecules decreased. At 1 DPA, inorganic ions were observed at levels that could produce a high osmolarity (1.11 MPa), followed by the carbohydrates (0.37 MPa) and amino acids (0.07 MPa). Maltose was detected in ovules only from 0 to 2 DPA. Inositol was abundant in the ovules on the day of anthesis, but decreased to non‐detectable levels after 4 DPA. This suggested that ovules allow the rapid entry of solutes into the syncytial endosperm after fertilization, but are osmotically buffered through the increase and decrease of partly identified substances. Specific gravity tests on the liquid squeezed from young endosperm sukpported this new hypothesis. Osmolarity in ovules seems only indirectly related to morphogenetic regulation mechanisms, and it may play a lesser role than the fluctuations of certain specific substances. The observed chemical changes offer insights into the rapidly varying nutritional needs of proembryos. Thus, in this research yielded a useful complex amino acid formula, derived from knowledge of the natural environment of the proembryo, and also an improved proembryo and ovule culture medium.

Characterization of the natural chemical and osmotic environment of early wheat embryogenesis

Soluble carbohydrates, amino acids, and major inorganic ions were quantified and compared with the total osmolarity of the ovule. Most of the analyzed components increased rapidly in concentration, reaching a peak at 1 day post anthesis (DPA) and decreasing quickly 1 day later. Within this time period, the osmolarity decreased from 1.49 to 1.18 MPa. The osmolarity then increased to 1.39 MPa at 4 DPA, and yet the overall concentration of analyzed molecules decreased. At 1 DPA, inorganic ions were observed at levels that could produce a high osmolarity (1.11 MPa), followed by the carbohydrates (0.37 MPa) and amino acids (0.07 MPa). Maltose was detected in ovules only from 0 to 2 DPA. Inositol was abundant in the ovules on the day of anthesis, but decreased to non-detectable levels after 4 DPA. This suggested that ovules allow the rapid entry of solutes into the syncytial endosperm after fertilization, but are osmotically buffered through the increase and decrease of partly identified substances. Specific gravity tests on the liquid squeezed from young endosperm sukpported this new hypothesis. Osmolarity in ovules seems only indirectly related to morphogenetic regulation mechanisms, and it may play a lesser role than the fluctuations of certain specific substances. The observed chemical changes offer insights into the rapidly varying nutritional needs of proembryos. Thus, in this research yielded a useful complex amino acid formula, derived from knowledge of the natural environment of the proembryo, and also an improved proembryo and ovule culture medium.