Endosperm-limited Brassicaceae seed germination: abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana

The endosperm is a barrier for radicle protrusion of many angiosperm seeds. Rupture of the testa (seed coat) and rupture of the endosperm are two sequential events during the germination of Lepidium sativum L. and Arabidopsis thaliana (L.) Heyhn. Abscisic acid (ABA) specifically inhibits the endosperm rupture of these two closely related Brassicaceae species. Lepidium seeds are large enough to allow the direct measurement of endosperm weakening by the puncture force method. We found that the endosperm weakens prior to endosperm rupture and that ABA delays the onset and decreases the rate of this weakening process in a dose-dependent manner. An early embryo signal is required and sufficient to induce endosperm weakening, which afterwards appears to be an organ-autonomous process. Gibberellins can replace this embryo signal; de novo gibberellin biosynthesis occurs in the endosperm and weakening is regulated by the gibberellin/ABA ratio. Our results suggest that the control of radicle protrusion during the germination of Brassicaceae seeds is mediated, at least in part, by endosperm weakening. We propose that Lepidium is an emerging Brassicaceae model system for endosperm weakening and that the complementary advantages of Lepidium and Arabidopsis can be used in parallel experiments to investigate the molecular mechanisms of endosperm weakening.     

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase

 Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination. Received: 28 September 1999 / Accepted: 27 December 1999     

Abscisic acid controls embryo growth potential and endosperm cap weakening during coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> cv. Rubi) seed germination

The mechanism and regulation of coffee seed germination were studied in Coffea arabica L. cv. Rubi. The coffee embryo grew inside the endosperm prior to radicle protrusion and abscisic acid (ABA) inhibited the increase in its pressure potential. There were two steps of endosperm cap weakening. An increase in cellulase activity coincided with the first step and an increase in endo--mannanase (EBM) activity with the second step. ABA inhibited the second step of endosperm cap weakening, presumably by inhibiting the activities of at least two EBM isoforms and/or, indirectly, by inhibiting the pressure force of the radicle. The increase in the activities of EBM and cellulase coincided with the decrease in the force required to puncture the endosperm and with the appearance of porosity in the cell walls as observed by low-temperature scanning electronic microscopy. Tissue printing showed that EBM activity was spatially regulated in the endosperm. Activity was initiated in the endosperm cap whereas later during germination it could also be detected in the remainder of the endosperm. Tissue printing revealed that ABA inhibited most of the EBM activity in the endosperm cap, but not in the remainder of the endosperm. ABA did not inhibit cellulase activity. There was a transient rise in ABA content in the embryo during imbibition, which was likely to be responsible for slow germination, suggesting that endogenous ABA also may control embryo growth potential and the second step of endosperm cap weakening during coffee seed germination.     

Class I chitinase and beta-1,3-glucanase are differentially regulated by wounding,methyl jasmonate,ethylene, and gibberellin in tomato seeds and leaves

Class I chitinase (Chi9) and beta-1,3-glucanase (GluB) genes are expressed in the micropylar endosperm cap of tomato (Lycopersicon esculentum) seeds just before radicle emergence through this tissue to complete germination. In gibberellin (GA)-deficient mutant (gib-1) seeds, expression of Chi9 and GluB mRNA and protein is dependent upon GA. However, as expression occurs relatively late in the germination process, we investigated whether the genes are induced indirectly in response to tissue wounding associated with endosperm cap weakening and radicle protrusion. Wounding and methyl jasmonate (MeJA) induced Chi9 expression, whereas ethylene, abscisic acid, sodium salicylate, fusicoccin, or beta-aminobutyric acid were without effect. Chi9 expression occurred only in the micropylar tissues when seeds were exposed to MeJA or were wounded at the chalazal end of the seed. Expression of Chi9, but not GluB, mRNA was reduced in germinating seeds of the jasmonate-deficient defenseless1 tomato mutant and could be restored by MeJA treatment. Chi9 expression during germination may be associated with "wounding" from cell wall hydrolysis and weakening in the endosperm cap leading to radicle protrusion, and jasmonate is involved in the signaling pathway for this response. Among these treatments and chemicals (other than GA), only MeJA and wounding induced a low level of GluB expression in gib-1 seeds. However, MeJA, wounding, and particularly ethylene induced both genes in leaves, whereas GA induced only Chi9 in leaves. Although normally expressed simultaneously during tomato seed germination, Chi9 and GluB genes are regulated distinctly and tissue specifically by hormones and wounding.     

Class I beta-1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomato seeds prior to radicle emergence.

beta-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. beta-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. beta-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of beta-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 microM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both beta-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.     

In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (^·OH)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance spectroscopy to show that ^·OH is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum; Brassicaceae) seeds. Endosperm weakening precedes radicle emergence, as demonstrated by direct biomechanical measurements. By ³H fingerprinting, we showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic ^·OH in radicles and endosperm caps: the production and action of ^·OH increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of ^·OH attack on polysaccharides were evident. In vivo ^·OH attack on cell wall polysaccharides were evident not only in germinating seeds but also in elongating maize (Zea mays; Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by ^·OH is a controlled action of this type of reactive oxygen species.     

Mechanism and control of Genipa americana seed germination

Genipa americana (Rubiaceae) is important for restoration of riparian forest in the Brazilian Cerrado. The objective was to characterize the mechanism and control of germination of G. americana to support uniform seedling production. Morphology and morphometrics of seeds, embryo and endosperm were assessed by light and scanning electron microscopy during germination. Imbibition and germination curves were generated and over the same time interval endosperm digestion and resistance were measured by puncture force analysis and activity assay of endo-β-mannanase (EBM) in water and in abscisic acid (ABA). The gene encoding for EBM was partially cloned and its expression monitored by quantitative real-time-polymerase chain reaction. Embryos displayed growth prior to radicle protrusion. A two-phase increase in EBM activity coincided with the two stages of weakening of the micropylar endosperm. The second stage also coincided with growth of the embryo prior to radicle protrusion. Enzyme activity was initiated in the micropylar endosperm but spread to the lateral endosperm. ABA completely inhibited germination by inhibiting embryo growth, the second stage of weakening and expression of the EBM gene, but EBM activity was not significantly inhibited. This suggests that a specific isoform of the enzyme is involved in endosperm weakening. EBM may cause a general 'softening' of micropylar endosperm cell walls, allowing the embryo to puncture the endosperm as the driving force of the decrease in puncture force.     

Expression of an expansin is associated with endosperm weakening during tomato seed germination

Expansins are extracellular proteins that facilitate cell wall extension, possibly by disrupting hydrogen bonding between hemicellulosic wall components and cellulose microfibrils. In addition, some expansins are expressed in non-growing tissues such as ripening fruits, where they may contribute to cell wall disassembly associated with tissue softening. We have identified at least three expansin genes that are expressed in tomato (Lycopersicon esculentum Mill.) seeds during germination. Among these, LeEXP4 mRNA is specifically localized to the micropylar endosperm cap region, suggesting that the protein might contribute to tissue weakening that is required for radicle emergence. In gibberellin (GA)-deficient (gib-1) mutant seeds, which germinate only in the presence of exogenous GA, GA induces the expression of LeEXP4 within 12 hours of imbibition. When gib-1 seeds were imbibed in GA solution combined with 100 microM abscisic acid, the expression of LeEXP4 was not reduced, although radicle emergence was inhibited. In wild-type seeds, LeEXP4 mRNA accumulation was blocked by far-red light and decreased by low water potential but was not affected by abscisic acid. The presence of LeEXP4 mRNA during seed germination parallels endosperm cap weakening determined by puncture force analysis. We hypothesize that LeEXP4 is involved in the regulation of seed germination by contributing to cell wall disassembly associated with endosperm cap weakening.     

The second step of the biphasic endosperm cap weakening that mediates tomato (Lycopersicon esculentum) seed germination is under control of ABA

The role of abscisic acid (ABA) in the weakening of the endosperm cap prior to radicle protrusion in tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds was studied. The endosperm cap weakened substantially in both water and ABA during the first 38 h of imbibition. After 38 h the force required for endosperm cap puncturing was arrested at 0.35 N in ABA, whereas in water a further decrease occurred until the radicle protruded. During the first 2 d of imbibition endo-beta-mannanase activity was correlated with the decrease in required puncture force and with the appearance of ice-crystal-induced porosity in the cell walls as observed by scanning electron microscopy. Prolonged incubation in ABA resulted in the loss of endo-beta-mannanase activity and the loss of ice-crystal-induced porosity, but not in a reversion of the required puncture force. ABA also had a distinct but minor effect on the growth potential of the embryo. However, endosperm cap resistance played the limiting role in the completion of germination. It was concluded that (a) endosperm cap weakening is a biphasic process and (b) inhibition of germination by ABA is through the second step in the endosperm cap weakening process.     

The emergence of embryos from hard seeds is related to the structure of the cell walls of the micropylar endosperm, and not to endo-beta-mannanase activity

BACKGROUND AND AIMS: Seeds of carob, Chinese senna, date and fenugreek are hard due to thickened endosperm cell walls containing mannan polymers. How the radicle is able penetrate these thickened walls to complete seed germination is not clearly understood. The objective of this study was to determine if radicle emergence is related to the production of endo-beta-mannanase to weaken the mannan-rich cell walls of the surrounding endosperm region, and/or if the endosperm structure itself is such that it is weaker in the region through which the radicle must penetrate. METHODS: Activity of endo-beta-mannanase in the endosperm and embryo was measured using a gel assay during and following germination, and the structure of the endosperm in juxtaposition to the radicle, and surrounding the cotyledons was determined using fixation, sectioning and light microscopy. KEY RESULTS: The activity of endo-beta-mannanase, the major enzyme responsible for galactomannan cell wall weakening increased in activity only after emergence of the radicle from the seed. Thickened cell walls were present in the lateral endosperm in the hard-seeded species studied, but there was little to no thickening in the micropylar endosperm except in date seeds. In this species, a ring of thin cells was visible in the micropylar endosperm and surrounding an operculum which was pushed open by the expanding radicle to complete germination. CONCLUSIONS: The micropylar endosperm presents a lower physical constraint to the completion of germination than the lateral endosperm, and hence its structure is predisposed to permit radicle protrusion.     

Mechanisms of hormonal regulation of endosperm cap-specific gene expression in tomato seeds

The micropylar region of endosperm in a seed, which is adjacent to the radicle tip, is called the 'endosperm cap', and is specifically activated before radicle emergence. This activation of the endosperm cap is a widespread phenomenon among species and is a prerequisite for the completion of germination. To understand the mechanisms of endosperm cap-specific gene expression in tomato seeds, GeneChip analysis was performed. The major groups of endosperm cap-enriched genes were pathogenesis-, cell wall-, and hormone-associated genes. The promoter regions of endosperm cap-enriched genes contained DNA motifs recognized by ethylene response factors (ERFs). The tomato ERF1 (TERF1) and its experimentally verified targets were enriched in the endosperm cap, suggesting an involvement of the ethylene response cascade in this process. The known endosperm cap enzyme endo-β-mannanase is induced by gibberellin (GA), which is thought to be the major hormone inducing endosperm cap-specific genes. The mechanism of endo-β-mannanase induction by GA was also investigated using isolated, embryoless seeds. Results suggested that GA might act indirectly on the endosperm cap. We propose that endosperm cap activation is caused by the ethylene response of this tissue, as a consequence of mechanosensing of the increase in embryonic growth potential by GA action.     

Effects of ethylene and some other environmental factors on different stages of germination in red root pigweed (Amaranthus retroflexus L.) seeds*

Abstract. Ethylene was found to promote two distinct processes during germination of redroot pigweed (Amarantus retroflexus L.) seeds: embryo expansion that splits the seed coat (incomplete germination), and radicle penetration through the more elastic endosperm (complete germination). The two events can be separated in time by subjecting seeds to low water potential or low CO₂ levels, which arrest germination of some seeds at the incomplete stage. Ethylene applications to incompletely germinated seeds promote complete germination, with a response threshold near 0.02 cm³ m^?3 and saturation near 0.5 cm³ m^?3. Higher ethylene concentrations (0.5 to 50 cm³ m^?3) given during the first day of seed imbibition also increase the percentage of seeds which initiate embryo expansion and split the seed coat. Light and elevated CO₂ also promote radicle penetration of the endosperm in seeds incubated under water stress. The results support the view that the germination pause at the incomplete stage is an adaptation to environmental stresses that can be overcome with exogenous ethylene or certain other stimuli.     

Myrigalone A inhibits Lepidium sativum seed germination by interference with gibberellin metabolism and apoplastic superoxide production required for embryo extension growth and endosperm rupture

Myrica gale L. (sweet gale) fruit leachate contains myrigalone A (MyA), a rare C-methylated dihydrochalcone and putative allelochemical, which is known to be a phytotoxin impeding seedling growth. We found that MyA inhibited Lepidium sativum L. seed germination in a dose-dependent manner. MyA did not affect testa rupture, but inhibited endosperm rupture and the transition to subsequent seedling growth. MyA inhibited micropylar endosperm cap (CAP) weakening and the increase in the growth potential of the radical/hypocotyl region (RAD) of the embryo, both being key processes required for endosperm rupture. We compared the contents of abscisic acid (ABA) and gibberellins in the tissues and found that the major bioactive forms of gibberellin in L. sativum seed tissues were GA(4) and GA(6), while GA(8) and GA(13) were abundant inactive metabolites. MyA did not appreciably affect the ABA contents, but severely interfered with gibberellin metabolism and signaling by inhibiting important steps catalyzed by GA3 oxidase, as well as by interfering with the GID1-type gibberellin signaling pathway. The hormonally and developmentally regulated formation of apoplastic superoxide radicals is important for embryo growth. Specific zones within the RAD were associated with accumulation of apoplastic superoxide radicals and endoreduplication indicative of embryo cell extension. MyA negatively affected both of these processes and acted as a scavenger of apoplastic reactive oxygen species. We propose that MyA is an allelochemical with a novel mode of action on seed germination.