Molecular Aspects of Seed Development Controlled by Gibberellins and Abscisic Acids

Plants have evolved seeds to permit the survival and dispersion of their lineages by providing nutrition for embryo growth and resistance to unfavorable environmental conditions. Seed formation is a complicated process that can be roughly divided into embryogenesis and the maturation phase, characterized by accumulation of storage compound, acquisition of desiccation tolerance, arrest of growth, and acquisition of dormancy. Concerted regulation of several signaling pathways, including hormonal and metabolic signals and gene networks, is required to accomplish seed formation. Recent studies have identified the major network of genes and hormonal signals in seed development, mainly in maturation. Gibberellin (GA) and abscisic acids (ABA) are recognized as the main hormones that antagonistically regulate seed development and germination. Especially, knowledge of the molecular mechanism of ABA regulation of seed maturation, including regulation of dormancy, accumulation of storage compounds, and desiccation tolerance, has been accumulated. However, the function of ABA and GA during embryogenesis still remains elusive. In this review, we summarize the current understanding of the sophisticated molecular networks of genes and signaling of GA and ABA in the regulation of seed development from embryogenesis to maturation.     

Phenotypic variation in fruit and seed characteristics of buffalo gourd

Eighty-five accessions of Buffalo gourd,Cucurbita foetidissima HBK., were assayed for variation in seven fruit and seed characteristics: fruit diameter (−x = 65 mm); seed weight per 100 seeds (−x = 3.8 g); seed weight per fruit (−x = 8.4 g); seed number per fruit (−x = 225); percentage embryo in seed (−x = 67.3%); percent crude fat of whole seed (−x = 32.9%); and percent crude protein of defatted embryo (−x = 69.5%). The variation in amino acid distribution (12) accessions) and in oil composition (15 accessions) was obtained. Relationships among traits were explored. Agronomic improvement through breeding was also discussed. Arizona Agricultural Experiment Station No. 2805.     

Proteome Analysis of Dormancy-Released Seeds of Fraxinus mandshurica Rupr. in Response to Re-Dehydration under Different Conditions

Desiccation tolerance is the ability of orthodox seeds to achieve equilibrium with atmospheric relative humidity and to survive in this state. Understanding how orthodox seeds respond to dehydration is important for improving quality and long-term storage of seeds under low temperature and drought stress conditions. Long-term storage of seeds is an artificial situation, because in most natural situations a seed that has been shed may not remain in a desiccated state for very long, and if dormant it may undergo repeated cycles of hydration. Different types of seeds are differentially sensitive to desiccation and this directly affects long-term storage. For these reasons, many researchers are investigating loss of desiccation tolerance during orthodox seed development to understand how it is acquired. In this study, the orthodox seed proteome response of Fraxinus mandshurica Rupr. to dehydration (to a relative water content of 10%, which mimics seed dehydration) was investigated under four different conditions viz. 20 °C; 20 °C with silica gel; 1 °C; and 1 °C after pretreatment with Ca²⁺. Proteins from seeds dehydrated under different conditions were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2919 protein spots were detected, and high-resolution 2D-DIGE indicated there were 27 differentially expressed. Seven of these were identified using MALDI TOF/TOF mass spectrometry. Inferences from bioinformatics annotations of these proteins established the possible involvement of detoxifying enzymes, transport proteins, and nucleotide metabolism enzymes in response to dehydration. Of the seven differentially abundant proteins, the amounts of six were down-regulated and one was up-regulated. Also, a putative acyl-coenzyme A oxidase of the glyoxylate cycle increased in abundance. In particular, the presence of kinesin-1, a protein important for regulation and cargo interaction, was up-regulated in seeds exposed to low temperature dehydration. Kinesin-1 is present in all major lineages, but it is rarely detected in seed desiccation tolerance of woody species. These observations provide new insight into the proteome of seeds in deep dormancy under different desiccation conditions.     

Proteomic Analysis of Embryogenesis and the Acquisition of Seed Dormancy in Norway Maple (Acer platanoides L.)

The proteome of zygotic embryos of Acer platanoides L. was analyzed via high-resolution 2D-SDS-PAGE and MS/MS in order to: (1) identify significant physiological processes associated with embryo development; and (2) identify changes in the proteome of the embryo associated with the acquisition of seed dormancy. Seventeen spots were identified as associated with morphogenesis at 10 to 13 weeks after flowering (WAF). Thirty-three spots were associated with maturation of the embryo at 14 to 22 WAF. The greatest changes in protein abundance occurred at 22 WAF, when seeds become fully mature. Overall, the stage of morphogenesis was characterized by changes in the abundance of proteins (tubulins and actin) associated with the growth and development of the embryo. Enzymes related to energy supply were especially elevated, most likely due to the energy demand associated with rapid growth and cell division. The stage of maturation is crucial to the establishment of seed dormancy and is associated with a higher abundance of proteins involved in genetic information processing, energy and carbon metabolism and cellular and antioxidant processes. Results indicated that a glycine-rich RNA-binding protein and proteasome proteins may be directly involved in dormancy acquisition control, and future studies are warranted to verify this association.     

Changes in phospholipase D experession in soybeans during seed development and germination

Activation of phospholipase D (PLD) has been linked to accumulation of nonhydratable phosphatides and lipid degradation leading to soybean seed deterioration during preharvest and postharvest events. This study examined the changes in PLD activity, protein, and mRNA in soybeans during seed development and germination. RNA blotting analysis indicated that expression of the gene that encodes PLD was highest during the early and middle stages of seed development. However, the amount of PLD activity accumulated per cotyledon reached the highest level in mature seeds. During germination and early seedling growth, PLD mRNA was not detected one day after imbibition, while a significant increase in PLD expression occurred in the cotyledons of three- and seven-day seedlings. Similarly, PLD activity and protein concentration showed little change during the first day of imbibition and increased afterward in three- and seven-day seedlings. These results suggested that expression of PLD is developmentally regulated and that the changes in its amount of activity and protein are controlled primarily at the mRNA level. Immunoblotting analysis further revealed the presence of PLD variants that were associated with specific stages of seed development and seedling growth. The PLD variants present in the cotyledons of mature seeds appeared to be distinct from those observed in the early stage of seed development and in young seedlings.     

Sperm Energy Restriction and Recovery (SER) Alters Epigenetic Marks during the First Cell Cycle of Development in Mice

The sperm energy restriction and recovery (SER) treatment developed in our laboratory was shown to improve fertilization and blastocyst development following in vitro fertilization (IVF) in mice. Here, we investigated the effects of SER on early embryogenesis. Developmental events observed during the first cell cycle indicated that progression through the pronuclear stages of SER-generated embryos is advanced in comparison with control-generated embryos. These findings prompted further analysis of potential effects of SER on pronuclear chromatin dynamics, focusing on the key H3K4me3 and H3K27ac histone modifications. Nearly all the SER-generated embryos displayed H3K4me3 in the male pronuclei at 12 h post-insemination (HPI), while a subset of the control-generated embryos did not. Additionally, SER-generated embryos displayed a more homogenous intensity of H3K27ac at 8 and 12 HPI compared to control embryos. These changes in histone modifications during the first cell cycle were accompanied by differences in gene expression at the two-cell stage; both of these changes in early embryos could potentially play a role in the improved developmental outcomes of these embryos later in development. Our results indicate that sperm incubation conditions have an impact on early embryo development and can be useful for the improvement of assisted reproductive technology outcomes.     

The Rice Small Auxin-Up RNA Gene OsSAUR33 Regulates Seed Vigor via Sugar Pathway during Early Seed Germination

Seed vigor affects seed germination and seedling emergence, and therefore is an important agronomic trait in rice. Small auxin-up RNAs (SAURs) function in a range of developmental processes, but their role in seed vigor remains unclear. Here, we observed that disruption of OsSAUR33 resulted in reduced germination rates and low seed uniformity in early germination. Expression of OsSAUR33 was higher in mature grains and early germinating seeds. RNA-seq analysis revealed that OsSAUR33 modulated seed vigor by affecting the mobilization of stored reserves during germination. Disruption of OsSAUR33 increased the soluble sugar content in dry mature grains and seeds during early germination. OsSAUR33 interacted with the sucrose non-fermenting-1-related protein kinase OsSnRK1A, a regulator of the sugar signaling pathway, which influences the expression of sugar signaling-related genes during germination. Disruption of OsSAUR33 increased sugar-sensitive phenotypes in early germination, suggesting OsSAUR33 likely affects seed vigor through the sugar pathway. One elite haplotype of OsSAUR33 associated with higher seed vigor was identified mainly in indica accessions. This study provides insight into the effects of OsSAUR33 on seed vigor in rice.     

Localisation of Storage Reserves in Developing Seeds of Pongamia pinnata (L.) Pierre, a Potential Agroforestry Tree

Pongamia pinnata is an important oil yielding perennial tree species. The aim of the present study was to document the histological and ultrastructural change that is occurring during pongamia seed development. The seeds were sampled at five stages of development at intervals of 3 weeks starting from 30 weeks after flowering up to 42 weeks. The seed development was followed microscopically using toluidine blue staining. The seed coat was made up of an external layer of palisade cells, an internal layer of hourglass cells followed by a parenchymatous cell layer and aleurone cell layer. The seed reserve compounds such as polysaccharides, proteins and starch showed distinct histochemical characterisation. Lignin was mainly found in the seed coat cell layers, while polysaccharides, proteins and starch granules in the cotyledon cells. The ultrastructural studies showed marked cellular changes during the seed development. The cell size varied from 9.4 to 78 μm during the seed development. The number of oil bodies per cell ranged from 200 to 300 at 42 weeks after flowering. Protein storage vacuoles were observed during the later stages of seed development. The plastids contained electron-dense starch granules. The seeds harvested after 42 weeks after flowering had maximum physiological maturity with high oil content and other seed reserve materials. This basic knowledge on pongamia seed development could invariably be used for further understanding of biochemical changes that might be involved in the biosynthetic pathway of oil.     

Cleavage of Early Mouse Embryo with Damaged DNA

The preimplantation period of embryogenesis is crucial during mammalian ontogenesis. During this period, the mitotic cycles are initiated, the embryonic genome is activated, and the primary differentiation of embryonic cells occurs. All cellular abnormalities occurring in this period are the primary cause of fetal developmental disorders. DNA damage is a serious cause of developmental failure. In the context of DNA damage response on the cellular level, we analyzed the course of embryogenesis and phenotypic changes during the cleavage of a preimplantation embryo. Our results document that DNA damage induced before the resumption of DNA synthesis in a zygote can significantly affect the preimplantation development of the embryo. This developmental ability is related to the level of the DNA damage. We showed that one-cell embryos can correct the first cleavage cycle despite low DNA damage and incomplete replication. It seems that the phenomenon creates a predisposition to a segregation disorder of condensed chromatin that results in the formation of micronuclei in the developmental stages following the first cleavage. We conclude that zygote tolerates a certain degree of DNA damage and considers its priority to complete the first cleavage stage and continue embryogenesis as far as possible.     

How green is green? Sampling and perception in assessing green seeds and chlorophyll in canola

Green seeds are used as a grading factor in estimating chlorophyll in canola and rapeseed in the Canadian and U.S.A. grading systems. This work examines the effect that sampling and perception have on the estimation of green seeds as well as the effect that sampling has on the determination of chlorophyll. Individual seed analysis indicated that in order to be considered as green, seeds needed to contain between 200 and 400 mg/kg chlorophyll. Variation due to binomial sampling played a predominant role in the error in determining the green seed levels in canola. Sampling of large numbers of seeds, as in the loading of export shipments, reduced the error. Binomial sampling also contributed to the error in chlorophyll determination even with sample sizes as large as 500. Differences in perception of green also were noted between individuals with coefficients of variation as high as 50% at the 1% green seed level. The combination of perception error and sampling error may result in samples of 1,000 seeds drawn from a mass with 2% green seeds having green seed counts ranging from 0.96 to 3.04%, 19 times out of 20.     

Analyzing seed weight,fatty acid composition,oil, and protein contents in <Emphasis Type="Italic">Vernonia galamensis</Emphasis> germplasm by near-infrared reflectance spectroscopy

Vernonia galamensis is a potential new industrial oilseed crop from the Asteraceae family. The interest in this species is due to the presence of a high vernolic acid content of its seed oil, which is useful in the oleochemical industry for paints and coatings. The development of a rapid, precise, robust, nondestructive, and economical method to evaluate quality components is of major interest to growers, processors, and breeders. NIR reflectance spectroscopy (NIRS) is routinely used for the prediction of quality traits in many crops. This study was conducted to establish a rapid analytical method for determining the quality of intact seeds of V. galamensis. A total of 114 Vernonia accessions were scanned to determine seed weight, FA composition, oil, and protein contents using NIRS. Conventional chemical analysis for FA composition, total oil, and protein contents were performed by GC, Soxhlet extraction, and the Dumas combustion method, respectively. Calibration equations were developed and tested through cross-validation. The coefficient of determination in cross-validation for FA ranged from 0.47 (linoleic acid) to 0.55 (vernolic acid), and for oil, protein, and seed weight from 0.71 (oil) to 0.86 (seed protein). It was concluded that NIRS calibration equations developed for seed weight and seed quality traits can be satisfactorily used as early screening methods in V. galamensis breeding programs.     

Relationships among the A Genomes of Triticum L. Species as Evidenced by SSR Markers, in Iran

The relationships among 55 wheat accessions (47 accessions collected from Iran and eight accessions provided by the Institute of Plant Biology of the University of Zurich, Switzerland) belonging to eight species carrying A genome (Triticum monococcum L., T. boeoticum Boiss., T. urartu Tumanian ex Gandilyan, T. durum Desf., T. turgidum L., T. dicoccum Schrank ex Schübler, T. dicoccoides (Körn. ex Asch. & Graebner) Schweinf. and T. aestivum L.) were evaluated using 31 A genome specific microsatellite markers. A high level of polymorphism was observed among the accessions studied (PIC = 0.77). The highest gene diversity was revealed among T. durum genotypes, while the lowest genetic variation was found in T. dicoccoides accessions. The analysis of molecular variance (AMOVA) showed a significant genetic variance (75.56%) among these accessions, representing a high intra-specific genetic diversity within Triticum taxa in Iran. However, such a variance was not observed among their ploidy levels. Based on the genetic similarity analysis, the accessions collected from Iran were divided into two main groups: diploids and polyploids. The genetic similarity among the diploid and polyploid species was 0.85 and 0.89 respectively. There were no significant differences in A genome diversity from different geographic regions. Based on the genetic diversity analyses, we consider there is value in a greater sampling of each species in Iran to discover useful genes for breeding purposes.     

The Histone Chaperone HIRA Is a Positive Regulator of Seed Germination

Histone chaperones regulate the flow and dynamics of histone variants and ensure their assembly into nucleosomal structures, thereby contributing to the repertoire of histone variants in specialized cells or tissues. To date, not much is known on the distribution of histone variants and their modifications in the dry seed embryo. Here, we bring evidence that genes encoding the replacement histone variant H3.3 are expressed in Arabidopsis dry seeds and that embryo chromatin is characterized by a low H3.1/H3.3 ratio. Loss of HISTONE REGULATOR A (HIRA), a histone chaperone responsible for H3.3 deposition, reduces cellular H3 levels and increases chromatin accessibility in dry seeds. These molecular differences are accompanied by increased seed dormancy in hira-1 mutant seeds. The loss of HIRA negatively affects seed germination even in the absence of HISTONE MONOUBIQUITINATION 1 or TRANSCRIPTION ELONGATION FACTOR II S, known to be required for seed dormancy. Finally, hira-1 mutant seeds show lower germination efficiency when aged under controlled deterioration conditions or when facing unfavorable environmental conditions such as high salinity. Altogether, our results reveal a dependency of dry seed chromatin organization on the replication-independent histone deposition pathway and show that HIRA contributes to modulating seed dormancy and vigor.     

Plant-to-Plant Variability in Root Metabolite Profiles of 19 Arabidopsis thaliana Accessions Is Substance-Class-Dependent

Natural variation of secondary metabolism between different accessions of Arabidopsis thaliana (A. thaliana) has been studied extensively. In this study, we extended the natural variation approach by including biological variability (plant-to-plant variability) and analysed root metabolic patterns as well as their variability between plants and naturally occurring accessions. To screen 19 accessions of A. thaliana, comprehensive non-targeted metabolite profiling of single plant root extracts was performed using ultra performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS). Linear mixed models were applied to dissect the total observed variance. All metabolic profiles pointed towards a larger plant-to-plant variability than natural variation between accessions and variance of experimental batches. Ratios of plant-to-plant to total variability were high and distinct for certain secondary metabolites. None of the investigated accessions displayed a specifically high or low biological variability for these substance classes. This study provides recommendations for future natural variation analyses of glucosinolates, flavonoids, and phenylpropanoids and also reference data for additional substance classes.     

Extraction and methanolysis of oil from whole or crushed rapeseed for fatty acid analysis

Three methods are described for extraction of oil from rapeseed for routine determinations of fatty acid composition. In the “whole-seed method,” ca. 50% of the total seed oil is extracted, without prior crushing of the seeds, by soaking the dried seeds in petroleum ether and benzene at room temperature for 2 days. For certain types of rapeseed with a “less permeable seed coat,” a presoaking in water is required to rupture the seed coat. The extracted oil has practically the same fatty acid composition as the total seed oil, and can therefore be used as a representative sample for determination of the fatty acid composition of the total seed oil. In the “crushing method,” the seeds are lightly crushed before the oil is extracted. In the “half-seed method,” the outer cotyledon of a single seed is dissected from the embryo; the oil is extracted from this cotyledon for fatty acid analysis, while the remaining part of the embryo can be germinated and planted to produce the progeny of the seed. In all three methods the extracted oil is converted to fatty acid methyl esters by a rapid reaction with sodium in methanol at room temperature. Presented in part at the Joint Conference of the Chemical Institute of Canada and the American Chemical Society, Toronto, May 1970. Contribution No. 329, Department of Plant Science, University of Manitoba.     

Anatomy and Histochemistry of Seed Coat Development of Wild (Pisum sativum subsp. elatius (M. Bieb.) Asch. et Graebn. and Domesticated Pea (Pisum sativum subsp. sativum L.)

In angiosperms, the mature seed consists of embryo, endosperm, and a maternal plant-derived seed coat (SC). The SC plays a role in seed filling, protects the embryo, mediates dormancy and germination, and facilitates the dispersal of seeds. SC properties have been modified during the domestication process, resulting in the removal of dormancy, mediated by SC impermeability. This study compares the SC anatomy and histochemistry of two wild (JI64 and JI1794) and two domesticated (cv. Cameor and JI92) pea genotypes. Histochemical staining of five developmental stages: 13, 21, 27, 30 days after anthesis (DAA), and mature dry seeds revealed clear differences between both pea types. SC thickness is established early in the development (13 DAA) and is primarily governed by macrosclereid cells. Polyanionic staining by Ruthenium Red indicated non homogeneity of the SC, with a strong signal in the hilum, the micropyle, and the upper parts of the macrosclereids. High peroxidase activity was detected in both wild and cultivated genotypes and increased over the development peaking prior to desiccation. The detailed knowledge of SC anatomy is important for any molecular or biochemical studies, including gene expression and proteomic analysis, especially when comparing different genotypes and treatments. Analysis is useful for other crop-to-wild-progenitor comparisons of economically important legume crops.     

Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop,Plukenetia volubilis,by Real-Time Quantitative PCR

Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔC_t), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.