Dynamic formation and transcriptional regulation mediated by phytohormones during chalkiness formation in rice.

BACKGROUND: Rice (Oryza sativa L.) Chalkiness, the opaque part in the kernel endosperm formed by loosely piled starch and protein bodies. Chalkiness is a complex quantitative trait regulated by multiple genes and various environmental factors. Phytohormones play important roles in the regulation of chalkiness formation but the underlying molecular mechanism is still unclear at present. RESULTS: In this research, Xiangzaoxian24 (X24, pure line of indica rice with high-chalkiness) and its origin parents Xiangzaoxian11 (X11, female parent, pure line of indica rice with high-chalkiness) and Xiangzaoxian7 (X7, male parent, pure line of indica rice with low-chalkiness) were used as materials. The phenotype, physiological and biochemical traits combined with transcriptome analysis were conducted to illustrate the dynamic process and transcriptional regulation of rice chalkiness formation. Impressively, phytohormonal contents and multiple phytohormonal signals were significantly different in chalky caryopsis, suggesting the involvement of phytohormones, particularly ABA and auxin, in the regulation of rice chalkiness formation, through the interaction of multiple transcription factors and their downstream regulators. CONCLUSION: These results indicated that chalkiness formation is a dynamic process associated with multiple genes, forming a complex regulatory network in which phytohormones play important roles. These results provided informative clues for illustrating the regulatory mechanisms of chalkiness formation in rice.

Comparative proteomics reveals new insights into the endosperm responses to drought, salinity and submergence in germinating wheat seeds.

KEY MESSAGE: Beyond the role of a nutrient reservoir during germination, the endosperm of wheat seeds also responds to different abiotic stresses via modification of the protein profiles. The endosperm is the main component of wheat seeds. During seed germination, it provides nutrients to support the embryo development, and its constituents vary under environmental stresses such as drought, salinity and submergence that are associated with disordered water supply. However, the molecular mechanism of these stress responses remains unclear. In this study, a comparative label-free proteomic analysis was performed on endosperm from the germinating wheat seeds subjected to PEG, NaCl and submergence treatments. In total, 2273 high confidence proteins were detected, and 234, 207 and 209 of them were identified as differentially expressed proteins (DEPs) under the three stresses, respectively. Functional classification revealed that the DEPs were mainly involved in protein, amino acid and organic acid metabolic process in all stress treatments. While some other metabolic processes were highlighted in one or two of the stresses specifically, such as oxidative phosphorylation in PEG and submergence, and ß-alanine metabolism in PEG and NaCl treatments. The identification of a series of stress-related proteins and their biased expression in different stresses indicates the active stress-responding role of endosperm beyond a simple nutrient reservoir during germination, while the overall stress responses of the endosperm were found to be moderate and lag behind the embryo. Besides, some fundamental processes and DEPs shared by the three stresses could be selected priorly for future molecular breeding researches. Our results provide new insights into the mechanism of endosperm responses to abiotic stresses during seed germination.

Rice starch accumulation at different endosperm regions and physical properties under nitrogen treatment at panicle initiation stage.

The quality of rice grain is characterized by the component, structure and physicochemical properties of starch accumulated in endosperm cell. Nitrogen uptake strongly affects rice growth and starch development. In this study, Nangeng 9108 was used to investigated the accumulation of starch in different positions of the endosperm and physical properties of starch under nitrogen treatment of panicle initiation (PI) stage. Compared with the control group (CG), nitrogen treatment group (NTG) featured a higher number of grains per panicle and 1000-grain weight. Nitrogen treatment significantly increased starch accumulation among different regions during endosperm development, which was expressed as central endosperm cells > sub-aleurone cells of abdominal endosperm > sub-aleurone cells of dorsal endosperm. The amyloplast increased by constricting and budding-type division, generated a bead-like structure and derived some vesicles. The particle size of the starch granules obtained from the NTG was smaller and the apparent amylose content was lower than those of the CG, resulting in higher relative crystallinity. Nitrogen treatment promoted double helical components and provided a higher degree of order at short-rang scale for the starch granules. This study indicated that nitrogen significantly affected the accumulation and physicochemical properties of starch in the endosperm.

EMS Mutagenesis of Maize Pollen.

Effective mutagenesis is critical for connecting traits of interest to specific plant genes. The development of site-directed mutagenesis and sequenced-indexed genetics resources in maize allows for targeted analysis of individual genes. These reverse genetics approaches have the potential for confirmation bias by only studying candidate genes for association with traits of interest. Genetic screens of induced, random mutations are important for identifying novel loci as well as interacting factors for known mutant loci. Chemical mutagenesis provides very high mutation rates and can be used for a variety of screen designs. This chapter provides an updated protocol for ethyl methanesulfonate (EMS) mutagenesis of maize pollen using paraffin or mineral oil. Mutagenesis occurs in mature pollen causing nonconcordant endosperm and embryo genotypes as well as sectored M1 plants. Considerations for these factors in genetic screens are discussed.

Transcriptomic and Metabolomics Analysis of Different Endosperm Region under Nitrogen Treatments.

Storage protein distribution in wheat-grain endosperm is heterogeneous, but the underlying molecular mechanism remains unclear. Two parts of the endosperm region, the innermost endosperm (IE) region and the remaining endosperm (RE) region, grown under low nitrogen (LN) and high nitrogen (HN) treatments were used to perform metabolomic and transcriptomic analysis. We identified 533 and 503 differentially expressed genes (DEGs) with at least a two-fold expression change (p < 0.05) between IE and RE, among which 81 and 78 transcripts under LN and HN, respectively, related to carbon and nitrogen metabolism, and encoded transcription factors or proteins involved in post-translational modification (PTM). The significantly differentially abundant metabolites between IE and RE were mainly amino acids, N-compounds, carbohydrates, and nucleic acids. More upregulated transcripts and metabolites were identified in RE than IE under HN conditions, indicating that HN activates metabolism in the endosperm periphery. In addition to carbon and nitrogen metabolism, transcription factors and protein PTMs, such as phosphorylation and acetylation, might determine the protein heterogeneous distribution between IE and RE and its response to nitrogen fertilizer supply.

Effects of inhibiting starch branching enzymes on molecular and crystalline structures of starches from endosperm different regions in rice.

Mature endosperm was separated regionally into different parts in three rice cultivars, Te-qing (TQ), Wu-xiang 9915 (WX9915) and Guang-ling-xiang-nuo (GLXN), and their transgenic lines with inhibition of starch branching enzyme I and IIb (SBEI/IIb-). Within the three wild-type cultivars, starches from endosperm different regions showed similar molecular and crystalline structures. However, in rices with inhibition of SBEs, amylopectin short branch-chain content and branching degree gradually decreased, but amylopectin B3+ chain content and average chain length increased gradually from the interior to exterior of endosperm. The amylose content gradually increased from the interior to exterior of endosperm in TQ- and WX9915-SBEI/II- lines. From the interior to exterior of endosperm, starch changed gradually from CC- to CB-type in TQ-SBEI/II- line and from CA- to CC-type in GLXN-SBEI/II- line, and remained CA-type in WX9915-SBEI/II- line. These results provided some information for quality breeding and utilizations of rice with inhibition of SBE.

Transcriptomic and metabolomic analysis of ZmYUC1 mutant reveals the role of auxin during early endosperm formation in maize.

Kernel size in cereal is an important agronomic trait controlled by the interaction of genetic and environmental factors. The endosperm occupies most of the kernel area; for this reason, the endosperm cells dimension, number and metabolic content strongly influence kernel properties. This paper presents the transcriptomic and metabolomic analysis of the maize defective endosperm 18 (de18) mutant, where auxin accumulation in the endosperm is impaired. This mutation, involving the ZmYuc1 gene, leads to a reduced kernel size compared to the wild-type line B37. Our results mainly indicate that IAA concentration controls sugar and protein metabolism during kernel differentiation and it is necessary for BETL formation. Furthermore, a fine tuning of different auxin conjugates is reported as the main mechanism to counteract the auxin deficit. Some candidates as master regulators of endosperm transcriptional regulation mediated by auxin are found between MYB and MADS-box gene families. A link between auxin and storage protein accumulation is highlighted, suggesting that IAA directly or indirectly, through CK or ABA, regulates the transcription of zein coding genes. This study represents a move forward with respect to the current knowledge about the role of auxin during maize endosperm differentiation thus revealing the genes that are modulated by auxin and that control agronomic traits as kernel size and metabolic composition.

A Maternally Deposited Endosperm Cuticle Contributes to the Physiological Defects of transparent testa Seeds.

Mature dry seeds are highly resilient plant structures where the encapsulated embryo is kept protected and dormant to facilitate its ultimate dispersion. Seed viability is heavily dependent on the seed coat's capacity to shield living tissues from mechanical and oxidative stress. In Arabidopsis (Arabidopsis thaliana), the seed coat, also called the testa, arises after the differentiation of maternal ovular integuments during seed development. We recently described a thick cuticle tightly embedded in the mature seed's endosperm cell wall. We show here that it is produced by the maternal inner integument 1 layer and, remarkably, transferred to the developing endosperm. Arabidopsis transparent testa (tt) mutations cause maternally derived seed coat pigmentation defects. TT gene products encode proteins involved in flavonoid metabolism and regulators of seed coat development. tt mutants have abnormally high seed coat permeability, resulting in lower seed viability and dormancy. However, the biochemical basis of this high permeability is not fully understood. We show that the cuticles of developing tt mutant integuments have profound structural defects, which are associated with enhanced cuticle permeability. Genetic analysis indicates that a functional proanthocyanidin synthesis pathway is required to limit cuticle permeability, and our results suggest that proanthocyanidins could be intrinsic components of the cuticle. Together, these results show that the formation of a maternal cuticle is an intrinsic part of the normal integumental differentiation program leading to testa formation and is essential for the seed's physiological properties.

Biochemical and molecular characterisation of exogenous cytokinin application on grain filling in rice.

BACKGROUND: Poor filling of grains in the basal spikelets of large size panicles bearing numerous spikelets has been a major limitation in attempts to increase the rice production to feed the world's increasing population. Considering that biotechnological intervention could play important role in overcoming this limitation, the role of cytokinin in grain filling was investigated based on the information on cell proliferating potential of the hormone and reports of its high accumulation in immature seeds. RESULTS: A comparative study considering two rice varieties differing in panicle compactness, lax-panicle Upahar and compact-panicle OR-1918, revealed significant difference in grain filling, cytokinin oxidase (CKX) activity and expression, and expression of cell cycle regulators and cytokinin signaling components between the basal and apical spikelets of OR-1918, but not of Upahar. Exogenous application of cytokinin (6-Benzylaminopurine, BAP) to OR-1918 improved grain filling significantly, and this was accompanied by a significant decrease in expression and activity of CKX, particularly in the basal spikelets where the activity of CKX was significantly higher than that in the apical spikelets. Cytokinin application also resulted in significant increase in expression of cell cycle regulators like cyclin dependent kinases and cyclins in the basal spikelets that might be facilitating cell division in the endosperm cells by promoting G1/S phase and G2/M phase transition leading to improvement in grain filling. Expression studies of type-A response regulator (RR) component of cytokinin signaling indicated possible role of OsRR3, OsRR4 and OsRR6 as repressors of CKX expression, much needed for an increased accumulation of CK in cells. Furthermore, the observed effect of BAP might not be solely because of it, but also because of induced synthesis of trans-zeatin (tZ) and N6-(Δ2-isopentenyl)adenine (iP), as reflected from accumulation of tZR (tZ riboside) and iPR (iP riboside), and significantly enhanced expression of an isopentenyl transferase (IPT) isoform. CONCLUSION: The results suggested that seed-specific overexpression of OsRR4 and OsRR6, and more importantly of IPT9 could be an effective biotechnological intervention towards improving the CK level of the developing caryopses leading to enhanced grain filling in rice cultivars bearing large panicles with numerous spikelets, and thereby increasing their yield potential.

Identification and characterization of transcription factor ZmEREB94 involved in starch synthesis in maize.

Maize is an important food crop and industrial material owing to its high starch content. However, the mechanism of starch synthesis is not fully elucidated, especially with regard to the expression and regulation of starch synthetic genes. The APETALA2/Ethylene Responsive Factor (AP2/ERF) family plays a crucial role in various biological processes via regulating gene expression. In this study, the ZmEREB94 gene was identified through co-expression analysis. Bioinformatics analysis confirmed that ZmEREB94 belongs to the AP2/ERF family. Expression pattern analysis showed that this protein is strongly expressed in the maize endosperm. A ZmEREB94-GFP fusion protein was localized in the nuclei of onion epidermal cells, and ZmEREB94 showed strong transcriptional activation activity, which indicated that this protein is a transcription factor. In addition, yeast-one hybrid assays and transient expression in maize endosperm showed that ZmEREB94 could directly bind to the ZmSSI promoter and indirectly regulate ZmSh2 and ZmGBSSI expression. Our results revealed that ZmEREB94 might act as a key regulator of starch synthesis in maize.

Regulation of maize kernel weight and carbohydrate metabolism by abscisic acid applied at the early and middle post-pollination stages in vitro.

Abscisic acid (ABA) accumulates in plants under drought stress, but views on the role of ABA in kernel formation and abortion are not unified. The response of the developing maize kernel to exogenous ABA was investigated by excising kernels from cob sections at four days after pollination and culturing in vitro with different concentrations of ABA (0, 5, 10, 100µM). When ABA was applied at the early post-pollination stage (EPPS), significant weight loss was observed at high ABA concentration (100µM), which could be attributed to jointly affected sink capacity and activity. Endosperm cells and starch granules were decreased significantly with high concentration, and ABA inhibited the activities of soluble acid invertase and acid cell wall invertase, together with earlier attainment of peak values. When ABA was applied at the middle post-pollination stage (MPPS), kernel weight was observably reduced with high concentration and mildly increased with low concentration, which was regulated due to sink activity. The inhibitory effect of high concentration and the mild stimulatory effect of low concentration on sucrose synthase and starch synthase activities were noted, but a peak level of ADP-glucose pyrophosphorylase (AGPase) was stimulated in all ABA treatments. Interestingly, AGPase peak values were advanced by low concentration and postponed by high concentration. In addition, compared with the control, the weight of low ABA concentration treatments were not statistically significant at the two stages, whereas weight loss from high concentration applied at EPPS was considerably obvious compared with that of the MPPS, but neither led to kernel abortion. The temporal- and dose-dependent impacts of ABA reveal a complex process of maize kernel growth and development.

Reactive Oxygen Species Generated by NADPH Oxidases Promote Radicle Protrusion and Root Elongation during Rice Seed Germination.

Seed germination is a complicated biological process that requires regulation through various enzymatic and non-enzymatic mechanisms. Although it has been recognized that reactive oxygen species (ROS) regulate radicle emergence and root elongation in a non-enzymatic manner during dicot seed germination, the role of ROS in monocot seed germination remains unknown. NADPH oxidases (NOXs) are the major ROS producers in plants; however, whether and how NOXs regulate rice seed germination through ROS generation remains unclear. Here, we report that diphenyleneiodinium (DPI), a specific NOX inhibitor, potently inhibited embryo and seedling growth-especially that of the radicle and of root elongation-in a dose-dependent manner. Notably, the DPI-mediated inhibition of radicle and root growth could be eliminated by transferring seedlings from DPI to water. Furthermore, ROS production/accumulation during rice seed germination was quantified via histochemistry. Superoxide radicals (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) accumulated steadily in the coleorhiza, radicle and seedling root of germinating rice seeds. Expression profiles of the nine typical NOX genes were also investigated. According to quantitative PCR, OsNOX5, 7 and 9 were expressed relatively higher. When seeds were incubated in water, OsNOX5 expression progressively increased in the embryo from 12 to 48 h, whereas OsNOX7 and 9 expressions increased from 12 to 24 h and decreased thereafter. As expected, DPI inhibits the expression at predetermined time points for each of these genes. Taken together, these results suggest that ROS produced by NOXs are involved in radicle and root elongation during rice seed germination, and OsNOX5, 7 and 9 could play crucial roles in rice seed germination. These findings will facilitate further studies of the roles of ROS generated by NOXs during seed germination and seedling establishment and also provide valuable information for the regulation of NOX family gene expression in germinating seeds of monocot cereals.

Identification and characterization of microRNAs in maize endosperm response to exogenous sucrose using small RNA sequencing.

Sucrose acts as a signaling molecule for genes critical to starch biosynthesis in maize endosperm. Previously, we showed that sucrose could regulate starch biosynthesis in maize via transcription factors. To better understand the complex regulation of starch biosynthesis, the 10days after pollination endosperm from Zea mays L. B73 inbred line was collected and treated with sucrose for small RNA sequencing. The sequencing results revealed that 24 known miRNAs and 190 novel miRNAs were significantly differentially expressed in response to sucrose. In addition, most of target mRNAs were characterized as transcription factors, mainly including, MYB, ARF, NAC, AP2/ERF, WRKY, and GRAS, which play important roles in starch biosynthesis and seed development in maize endosperm. The expression profiles of miR398a/b and miR159b/j/k followed opposite expression trends to their target genes when analyzed by qPCR. In conclusion, these results show that sucrose regulates the expression of starch synthetic genes through miRNAs.

Grain Filling Characteristics and Their Relations with Endogenous Hormones in Large- and Small-Grain Mutants of Rice.

This study determined if the variation in grain filling parameters between two different spikelet types of rice (Oryza sativa L.) is regulated by the hormonal levels in the grains. Two rice mutants, namely, a large-grain mutant (AZU-M) and a small-grain mutant (ZF802-M), and their respective wild types (AZU-WT and ZF802-WT) were grown in the field. The endosperm cell division rate, filling rate, and hormonal levels: zeatin + zeatin riboside (Z+ZR), indo-3-acetic acid (IAA), polyamines (PAs), and abscisic acid (ABA) were determined. The results showed that there was no significant difference between the filling and endosperm cell division rates. These rates were synchronous between the superior and inferior spikelets for both mutants. However, the abovementioned parameters were significantly different between the two spikelet types for the two wild types. The superior spikelets filled faster and their filling rate was higher compared to the inferior ones. Changes in the concentrations of plant hormones were consistent with the observed endosperm cell division rate and the filling rate for both types of spikelets of mutant and wild type plants. Regression analysis showed a significant positive correlation between cell division and filling rates with the concentrations of the investigated hormones. Exogenous chemical application verified the role of ABA, IAA, and PAs in grain filling. The results indicate that poor filling of inferior spikelets in rice occurs primarily due to the reduced hormone concentrations therein, leading to lower division rate of endosperm cells, fewer endosperm cells, slower filling rate, and smaller grain weight.

Cell wall degradation is required for normal starch mobilisation in barley endosperm.

Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and ß-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth.

Abscisic acid and ethephon regulation of cellulase in the endosperm cap and radicle during lettuce seed germination.

The purpose of this study was to investigate the role of cellulase in endosperm cap weakening and radicle elongation during lettuce (Lactuca sativa L.) seed germination. The application of abscisic acid (ABA) or ethephon inhibits or promotes germination, respectively, by affecting endosperm cap weakening and radicle elongation. Cellulase activities, and related protein and transcript abundances of two lettuce cellulase genes, LsCEL1 and LsCEL2, increase in the endosperm cap and radicle prior to radicle protrusion following imbibition in water. ABA or ethephon reduce or elevate, respectively, cellulase activity, and related protein and transcript abundances in the endosperm cap. Taken together, these observations suggest that cellulase plays a role in endosperm cap weakening and radicle elongation during lettuce seed germination, and that the regulation of cellulase in the endosperm cap by ABA and ethephon play a role in endosperm cap weakening. However, the influence of ABA and ethephon on radicle elongation may not be through their effects on cellulase.

1-MCP treatment enhanced expression of genes controlling endosperm cell division and starch biosynthesis for improvement of grain filling in a dense-panicle rice cultivar.

High ethylene production in dense-panicle rice cultivars impacts grain filling. 1-MCP (ethylene action inhibitor) treatment increased assimilates partitioning, cell number and size and expression of starch synthesizing enzyme genes of developing caryopses mostly in the basal spikelets of panicle at early post-anthesis stage. The gain in cell number was less compared to the increase of size. High ethylene production in spikelets matched with greater expression of ethylene receptor and signal transducer genes. Genes encoding cell cycle regulators CDK, CYC and CKI expressed poorly on 9 DAA. 1-MCP treatment enhanced their expression; the increase of expression was higher for CDKs and lower for CKIs in basal compared to apical spikelets. Greater expression of CDKB2:1 might have lifted cytokinesis of nascent peripheral cells of endosperm, while promotion of CDKAs, CYCD2:2 and inhibition of CYCB2:2 expression contributed to endoreduplication of central cells increasing cell size and DNA ploidy level. It is concluded that the process of endoreduplication, which begins at mid-grain filling stage, is crucially linked with the final caryopsis size of rice grain. The enhanced endosperm growth brought about by repressed ethylene action during the first few days after anthesis seems to be associated with the overall increased cell cycle activity and sink strength.

OsMPK6 plays a critical role in cell differentiation during early embryogenesis in Oryza sativa.

The formation of body axes is the basis of morphogenesis during plant embryogenesis. We identified embryo-lethal mutants of rice (Oryza sativa) in which T-DNAs were inserted in OsMPK6 Embryonic organs were absent because their development was arrested at the globular stage. Similar to observations made with gle4, shootless, and organless, the osmpk6 mutations affected the initial step of cell differentiation. Expression of an apical-basal axis marker gene, OSH1, was reduced in the mutant embryos while that of the radial axes marker genes OsSCR and OsPNH1 was not detected. The signal for ROC1, a protodermal cell marker, was weak at the globular stage and gradually disappeared. Transcript levels of auxin and gibberellin biosynthesis genes were diminished in osmpk6 embryos. In addition, phytoalexin biosynthesis genes were down-regulated in osmpk6 and a major diterpene phytoalexin, momilactone A, did not accumulate in the mutant embryos. These results indicate that OsMPK6 begins to play a critical role during early embryogenesis, especially when the L1 radial axis is being formed.

Iminosugar inhibitors of carbohydrate-active enzymes that underpin cereal grain germination and endosperm metabolism.

Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively limited knowledge about the nature and control of starch degradation in plants. Increased societal and commercial demand for enhanced yield and quality in starch crops requires a better understanding of starch metabolism as a whole. Here we review recent advances in understanding the roles of carbohydrate-active enzymes in starch degradation in cereal grains through complementary chemical and molecular genetics. These approaches have allowed us to start dissecting aspects of starch degradation and the interplay with cell-wall polysaccharide hydrolysis during germination. With a view to improving and diversifying the properties and uses of cereal grains, it is possible that starch degradation may be amenable to manipulation through genetic or chemical intervention at the level of cell wall metabolism, rather than simply in the starch degradation pathway per se.

Basic Techniques to Assess Seed Germination Responses to Abiotic Stress in Arabidopsis thaliana.

The model organism Arabidopsis thaliana has been extensively used to unmask the molecular genetic signaling pathways controlling seed germination in plants. In Arabidopsis, the normal seed to seedling developmental transition involves testa rupture soon followed by endosperm rupture, radicle elongation, root hair formation, cotyledon expansion, and greening. Here we detail a number of basic procedures to assess Arabidopsis seed germination in response to different light (red and far-red pulses), temperature (seed thermoinhibition), and water potential (osmotic stress) environmental conditions. We also discuss the role of the endosperm and how its germination-repressive activity can be monitored genetically by means of a seed coat bedding assay. Finally we detail how to evaluate germination responses to changes in gibberellin (GA) and abscisic acid (ABA) levels by manipulating pharmacologically the germination medium.