Germination stimulation in wild oats (Avena fatua L.) by synthetic strigol analogs and gibberellic acid

At concentrations of 0.01–1 mM, five synthetic multiring analogs of strigol were effective germination stimulants of intact and dehulled wild oat (Avena fatua L.) seeds. The effect was concentration-dependent and equaled or exceeded that produced by equimolar gibberellic acid (GA₃). The most effective strigol analog treatments induced 55–80% germination within 7 days in intact wild oat seeds and resulted in 63–86% germination and normal seedling growth over 14 days. Intact wild oat controls germinated 14% after 14 days. The stimulation of wild oat germination by these synthetic strigol analogs demonstrates that these compounds, initially developed as germination stimulants for the seeds of the parasitic weed, witchweed (Striga asiatica L. Kuntz.), have bioregulatory activity in dormant seeds of monocots, as well as dicots. None of the compounds tested significantly affected the germination of nondormant cultivated oat seeds (Avena sativa L.). The commonly used dispersal agent, Tween 20 (0.1%), was found to inhibit germination of cultivated oats, alone and in the presence of 2% acetone.Names of companies or commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned.     

Terpenoid precursors of strigol as seed germination stimulants of broomrape (Orobanche ramosa) and witchweed (Striga asiatica)

Strigol and some of its synthetic precursors and analogs are known to be germination stimulants for broomrape (Orobanche ramosa) and witchweed (Striga asiatica). Fifteen synthetic terpenoids, similar in structure to one of the four rings of the strigol molecule, were evaluated in two bioassays as seed germination stimulants with broomrape, and nine were found to be active. Five of the more active compounds contained ester groups. Whereas the study was intended primarily to evaluate forced germination of broomrape by aqueous solutions, the results are almost qualitatively identical for broomrape and witchweed. Monocyclic compounds with chemical structures similar to two of the rings of strigol have now been shown to possess significant bioactivity as germination stimulants.     

Comparison of the seed germination effects of synthetic analogs of strigol,gibberellic acid,cytokinins, and other plant growth regulators

Four synthetic multiring analogs of strigol, a naturally occurring sesquiterpene lactone that promotes germination of dormant seeds ofStriga (witchweed), were found to stimulate germination of dormantLactuca (lettuce) seeds. The effects on light-sensitive and light-insensitive lettuce seeds were concentration-dependent and exceeded those produced by equimolar (0.1 mM) solutions of gibberellic acid. Strigol and epistrigol promoted lettuce seed germination to a lesser degree than did the synthetic analogs. The strigol group compounds had minimal effect on the germination of monocot seeds. The results indicate that the synthetic strigol analogs have plant growth regulatory activity in dormant seeds of genera beyondStriga in which germination stimulation by strigol and the synthetic analogs was first demonstrated.Names of companies of commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned.     

Conditioning duration and agents involved in broomrape seeds responding to germination stimulants

Weedy broomrape species, such as sunflower broomrape (Orobanche cumana Wallr.) and Egyptian broomrape [Phelipanche aegyptiaca Pers. (syn. O. aegyptiaca)], require a period of pre-conditioning before they can respond to germination stimulants. Thus, the sensitivity of weedy broomrape seeds to germination stimulants could be an important factor for broomrape control. In this study, the influence of conditioning agents, conditioning period (0–21 days) and germination stimulants on the germination of sunflower broomrape and Egyptian broomrape seeds was analyzed. Without conditioning, the sunflower and Egyptian broomrape seeds exhibited negligible germination responses to the stimulants. The germination rate of the broomrape seeds increased rapidly with conditioning period and reached a maximum under a conditioning period of 4–10 days; further prolonged conditioning resulted in a decrease in the germination rate. Gibberellic acid (GA₃) could not only break the dormancy of the sunflower and Egyptian broomrape seeds but also maintained the high sensitivity of these seeds even after 21 days of conditioning. Furthermore, 100 µM of GA₃ induced the germination of the Egyptian broomrape seeds. The stimulants that induced Egyptian broomrape germination were ranked in decreasing order as GR24 (76.8?%), strigol (76.1?%), tobacco root exudates (49.5?%), dehydrocostus lactones (DCL, 39.2?%), and maize root exudates (18?%). In contrast, GA₃ did not directly induce sunflower broomrape seed germination, which responded to strigol (62.8?%)?>?maize root exudates (58.2?%)?>?GR24 (57.9?%)?>?tobacco root exudates (41.6?%)?>?DCL (41.3?%). These results indicate specialized recognition of germination stimulants by sunflower and Egyptian broomrape. This study may contribute to a better understanding of parasitic weed germination and may lead to improved control strategies.     

Terpenoid precursors of strigol as seed germination stimulants of broomrape (Orobanche ramosa) and witchweed (Striga asiatica)

Strigol and some of its synthetic precursors and analogs are known to be germination stimulants for broomrape (Orobanche ramosa) and witchweed (Striga asiatica). Fifteen synthetic terpenoids, similar in structure to one of the four rings of the strigol molecule, were evaluated in two bioassays as seed germination stimulants with broomrape, and nine were found to be active. Five of the more active compounds contained ester groups. Whereas the study was intended primarily to evaluate forced germination of broomrape by aqueous solutions, the results are almost qualitatively identical for broomrape and witchweed. Monocyclic compounds with chemical structures similar to two of the rings of strigol have now been shown to possess significant bioactivity as germination stimulants.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Regulation of Avena Fatua Seed Germination by Smoke Solutions, Gibberellin A3 and Ethylene

Dormant, intact Avena fatua L. (wild oat) seeds germinate poorly at 20 °C. Removing the hulls slightly increased germination. Treatment with smoke solutions increased the germination of both intact seeds and caryopses. Exogenous GA₃, alone or in the presence of smoke solution, increased the germination of caryopses, while ACC shows a tendency to increase germination of caryopses only when applied in combination with smoke solution. Results suggest that GA₃ and ethylene, but not smoke solutions, are involved in the regulation of α-amylase activity during germination. However, the participation of smoke solutions in the control of ACC oxidase activity cannot be excluded.     

Role of Endogenous Growth Regulators in Seed Dormancy of Avena fatua: I. Short Chain Fatty Acids

The hypothesis that endogenous short chain fatty acids (C 6-C 10) are important in maintaining seeds of wild oat (Avena fatua L.) in the dormant state by acting as natural germination inhibitors (Berrie, Buller, Don, Parker, 1979 Plant Physiol 63: 758-764) was investigated. When germination of nondormant seeds was inhibited by treatment with short chain fatty acids, the seeds did not revert to a similar biochemical and physiological state as exhibited by dormant seeds. First, nonanoic acid-induced inhibition of seed germination was not reversed by hormone treatments which normally break dormancy in wild oat seeds. Second, nondormant seeds treated with short chain fatty acids maintained similar relative proportions of the pentose phosphate pathway and the Embden-Meyerhoff-Parnas pathway for respiratory glucose metabolism as that found in the nondormant controls. Seeds imbibed in the presence of nonanoic acid lost more amino acids and proteins into the imbibition solution than did the untreated controls, suggesting membrane damage had occurred. Inasmuch as increasing concentrations of nonanoic acid also progressively reduced the growth of the coleoptile and roots of intact seedlings until all growth ceased and no germination occurred, the inhibition of seed germination could be due to a nonspecific inhibition of growth of the embryo, perhaps because of disruption of membrane structure and function. Finally, no correlation between endogenous levels of short chain fatty acids in seeds or isolated embryonic axes and seed dormancy could be demonstrated.     

Characterization of Strigolactones, Germination Stimulants for the Root Parasitic Plants Striga and Orobanche, Produced by Maize, Millet and Sorghum

The germination stimulants for root parasitic plants Striga and Orobanche produced by sorghum (Sorghum bicolor (L.) Moench), maize (Zea mays L.), and pearl millet (Pennisetum typhoideum Rich.) were examined. Characterization of strigolactones in the root exudates from the plants grown hydroponically was conducted by comparing retention times of germination stimulants on reverse phase high performance liquid chromatography (HPLC) with those of synthetic standards, and by using HPLC linked with tandem mass spectrometry (LC/MS/MS). All the plants tested, except for a sorghum cultivar Swarna, were found to exude two major stimulants, 5-deoxy-strigol, which is known as a branching factor for arbuscular mycorrhizal (AM) fungi, and an isomer of strigol, tentatively named sorghumol. Swarna was found to exude 5-deoxy-strigol and strigol. These results imply that 5-deoxy-strigol is one of major germination stimulants of gramineous plants and that major stimulants may differ even among cultivars within the same species.     

Water uptake and distribution in non-dormant and dormant wild oat (Avena fatua L.) caryopses

The influence of seed coat modification and light quality onwater uptake and distribution in caryopses of dormant and non-dormantlines of wild oat (Avena fatua L.) was determined using NMRmicroimaging. Non-dormant seeds absorbed water more rapidlythan dormant seeds during imbibition on distilled water. Thiseffect was detected first in the embryo-scutellar region (8h) and later in the proximal endosperm (12 h). Cutting the testaand pericarp close to the embryo or scarification with KOH promotedrapid embryo/scutellum hydration and germination. Cutting atthe middle part of the caryopsis did not enhance embryo hydrationnor did it greatly improve germination. The sensitivity of waterdistribution to the phytochrome germination effect was examined.Significant differences in imbibitional water uptake by embryos-scutellumtissue were detected by 18 h following red-light (germinationpromoter) compared with far-red (germination inhibitor) treatment.The results indicated that both the rate and the sequence ofembryo/scutellum hydration were important in initiating germinationin dormant seeds. A refinement of the model that describes waterimbibition in wild oat seeds during the early stages of germinationis discussed. Key words: Water uptake, water distribution, Avena fatua, seed coat modification, light quality, dormant and non-dormant seeds     

Role of mycorrhizal infection in the growth and reproduction of wild vs. cultivated plants

Summary We tested the hypothesis that mycorrhizal infection benefits wild plants to a lesser extent than cultivated plants. This hypothesis stems from two observations: (1) mycorrhizal infection improves plant growth primarily by increasing nutrient uptake, and (2) wild plants often possess special adaptations to soil infertility which are less pronounced in modern cultivated plants. In the first experiment, wild (Avena fatua L.) and cultivated (A. sativa L.) oats were grown hydroponically at four different phosphorus levels. Wild oat was less responsive (in shoot dry weight) to increasing phosphorus availability than cultivated oat. In addition, the root: shoot ratio was much more plastic in wild oat (varying from 0.90 in the low phosphorus solution to 0.25 in the high phosphorus solution) than in cultivated oat (varying from 0.44 to 0.17). In the second experiment, mycorrhizal and non-mycorrhizal wild and cultivated oats were grown in a phosphorus-deficient soil. Mycorrhizal infection generally improved the vegetative growth of both wild and cultivated oats. However, infection significantly increased plant lifespan, number of panicles per plant, shoot phosphorus concentration, shoot phosphorus content, duration of flowering, and the mean weight of individual seeds in cultivated oat, while it had a significantly reduced effect, no effect, or a negative effect on these characters for wild oat. Poor positive responsiveness of wild oat in these characters was thus associated with what might be considered to be inherent adaptations to nutrient deficiency: high root: shoot ratio and inherently low growth rate. Infection also increased seed phosphorus content and reproductive allocation.     

Selectivity of diclofop-methyl between wheat and wild oat: growth and herbicide metabolism

Abstract Growth of the second leaf of susceptible wild oat (Avena fatua L.) was inhibited within 2 days after treatment with the herbicide, diclofop-methyl, in the 1-1/2 leaf stage. Leaf growth of resistant wheat (Triticum aestivum L.) was unaffected by diclofop-methyl. In wild oat. chlorosis developed 1 day after leaf growth was inhibited. Foliar absorption of diclofop-methyl was similar between wild oat and wheat with 67 and 61% of the recovered radioactivity from [¹⁴C]diclofop-methyl being absorbed by wild oat and wheat, respectively, after 4 days. Wild oal was equally sensitive to the methyl ester and acid forms of the herbicide when the compounds were injected into the stem. Wheat was unaffected by both forms when treated similarly. Very little diclofop-methyl and diclofop (combined total of 10 to 12% in wild oat and 5 to 7% in wheat) remained in plant tissues 2 days after leaf treatment in both susceptible and resistant plants. Therefore, the active form of the herbicide must inhibit growth of susceptible plants very rapidly and at relatively low concentrations. Diclofop-methyl was rapidly hydrolyzed to diclofop by wild oat and wheat. Wild oat predominantly conjugated diclofop to an ester conjugate but wheat hydroxylated the 2,4-dichlorophenyl ring and formed a phenolic conjugate. The formation of the different conjugates between wild oat and wheat was the most significant difference in metabolism between the two species. Nearly 60 and 70% of the methanol-soluble radioactivity was present as water-soluble conjugates in wild oat and wheat, respectively, 4 days after treatment.     

Induction and release of secondary seed dormancy in genetically pure lines of Avena fatua

Induction and release of secondary dormancy in genetically pure dormant (AN-51, Mont 73) and non-dormant (CS-40, SH-430) lines of wild oat ( Avena fatua L.) were studied. These lines differed with regard to the optimal period of anaerobiosis necessary for induction of dormancy, and/or the degree (% of seeds acquiring dormancy) and duration of the dormancy induced. Secondary dormancy could be induced more effectively in the after-ripened seeds of dormant lines than in the non-dormant lines, where only a short-term dormancy could be induced (in 5–7 week-old-seeds). Higher anaerobiosis temperatures were more effective in inducing dormancy in all lines studied. Thus, as with primary dormancy, wild oat biotypes exhibit genetic variability in their secondary dormancy behaviour and factors like temperature can modify the expression of this trait.
The germination stimulants kinetin, isopentenyl adenine, sodium azide, potassium nitrate, ethanol and substituted phthalimides, which break primary dormancy in wild oats, stimulated germination of secondarily dormant seeds (line AN-51). Since these chemicals are structurally diverse, primary and secondary dormancies appear to be similar in part in their regulation.
Salicylhydroxamic acid, an inhibitor of cyanide-insensitive (alternative) respiration, did not inhibit: 1, spontaneous release of secondary dormancy in the line SH-430; and 2, stimulation of germination of secondarily dormant AN-51 seeds by various chemicals (except azide), suggesting that this respiratory pathway is not necessary for the release of induced dormancy.     

Effects of Temperature and dl-Strigol on Seed Conditioning and Germination of Witchweed (Striga asiatica)

Seed conditioning and germination in witchweed (Striga asiatica(L.) Kuntze) were temperature-dependent. With higher conditioningtemperatures, shorter conditioning time was required for germinationwith terminal dl-strigol (strigol) treatment at 30 °C. Maximumgermination (80–100%) was obtained by conditioning inwater at 20, 25, 30 and 35 °C for 14, 7, 5 and 3 d, respectively,and terminally treating with 10^–6 M strigol at 30 °C.Seeds conditioned in 10^–8 M strigol instead of water germinatedmuch less with the same terminal strigol treatment. Generally,conditioning was slower when seeds were conditioned in strigolrather than water. The reduction in germination rate by pretreatmentin strigol or pretreatment at low temperatures could be overcomeby increasing the terminal strigol concentration in the germinationtest. Conditioned seeds did not germinate at 10 and 15 °Cwith a terminal 10^–6 M strigol treatment but yielded closeto maximum germination at 25, 30 and 35 °C with the sameterminal strigol treatment. To obtain maximum germination, boththe minimum conditioning temperature and the minimum germinationtemperature for conditioned seeds were 20 °C. Factors suchas conditioning time, and strigol concentration and temperatureduring conditioning and/or germination determine whether seedsremain in the conditioning phase or shift to a germination phase. dl-Strigol, germination stimulation, parasitic plants, seed conditioning, seed germination, Striga asiatica, temperature, weed control     

Role of Endogenous Plant Growth Regulators in Seed Dormancy of Avena fatua: II. Gibberellins

Gibberellin A₁ (GA₁) was identified by combined gas chromatographymass spectrometry as the major biologically active gibberellin (GA) in seeds of wild oat (Avena fatua L.) regardless of the depth of dormany or stage of imbibition. Both unimbibed dormant and nondromant seeds contained similar amounts of GA₁ as estimated by the d5-maize bioassay. During imbibition, the level of GA₁ declined in both dormant and non-dormant seeds, although the decline was more rapid in dormant seeds. Only in imbibing nondormant seeds did the GA biosynthesis inhibitor, 2-chloroethyltrimethyl ammonium chloride (CCC), cause a reduction in the level of GA₁ from that observed in control seeds. These results are interpreted as an indication that while afterripening does not cause a direct change in the levels of GAs during dry storage, it does induce a greater capacity for GA biosynthesis during imbibition.

Nondormant seeds imbibed in the presence of 50 millimolar CCC germinated equally as well as untreated seeds. When wild oat plants were fed CCC throughout the entire life cycle, viable seeds were produced that lacked detectable GA-like substances. These seeds afterripened at a slightly slower rate than the controls. Moreover, completely afterripened (nondormant) seeds from plants fed CCC continuously contained no detectable GA-like substances, and when these seeds germinated, dwarf seedlings were produced, indicating GA biosynthesis was inhibited during and after germination. In total, these results suggest that the increased capacity for GA biosynthesis observed in imbibing nondormant seeds is not a necessary prerequisite for germination. It is therefore possible that GA biosynthesis in imbibing nondormant seeds is one of many coordinated biochemical events that occur during germination rather than an initiator of the processes leading to germination.

     

Ethylene biosynthesis and strigol-induced germination of Striga asiatica

Germination of witchweed [ Striga asiatica (L.) Kuntze], an important parasite on cereal crops, is stimulated by several natural and synthetic compounds. In the present study the role of ethylene in germination of Striga asiatica in response to strigol was examined. Unconditioned seeds and those conditioned for 3 days produced negligible amounts of ethylene in response to strigol. However, extending the conditioning period to 5 and 8 days increased ethylene evolution by more than 10-fold. Ethylene production preceded radicle protrusion and was detectable within 3h after treatment. No germination was observed in the first 6 h of exposure to strigol. Germination and ethylene production increased with strigol concentration. Strigol-induced germination was considerably reduced by the ethylene action inhibitors. 2. 5-norbornadiene, silver thiosulphate and CO₂. The ethylene precursor 1-aminocyclopropane-1-carboxyac acid (ACC) at 5 to 200 μ M elicited neither germination nor ethylene production. However, a combination of strigol and ACC resulted in a high germination rate and copious ethylene production. Both germination and ethylene production were reduced by CoCl₂ and cyclobeximide, inhibitors of the ethylene-forming enzyme and of protein synthesis, respectively. The results are consistent with a model in which conditioning and strigol are required to remove a restriction on the ethylene biosynthetic pathway and in which the ethylene-forming enzyme is rate limiting.     

A standardized bioassay for evaluation of potential germination stimulants for seeds of parasitic weeds

A standard bioassay for the evaluation of the biological activity of potential germination stimulants for seeds of parasitic weeds has been designed by carefully reconsidering all steps of a literature procedure. Addition of a surfactant to the solution for sterilization of the seeds gave a considerable reduction of contamination during the conditioning and stimulation process. Furthermore, it was shown that the water/seed ratio during conditioning affects the germination results. For the seed conditioning and stimulation process, the sandwich technique was introduced, whereby the seeds were placed between two layers of glass fiber filter paper discs. The standardized bioassay has been used for the evaluation of the stimulatory activity of numerous (new) synthetic analogues of strigol (the natural germination stimulant). It is essential to include a reference compound—GR24, an analogue of strigol, is recommended—in every test series, because it was observed that, although a standard bioassay was used, germination percentages obtained with GR24 solutions vary from test to test. For seeds of Striga hermonthica, seasonal effects were found in the germination percentages obtained with GR24 as stimulant. Test results with strigol analogues having modifications in ring D reveal that structural variations in this part of the molecule have dramatic effects on the biological activity of strigol analogues.     

Possible role of volatile Fatty acids and abscisic Acid in the dormancy of oats

Species of Avena differ markedly in their levels of pre- and post-harvest dormancy. These species offer the opportunity of determining if dormancy is related to the endogenous level of growth inhibitor. Germinability in two species of differing levels of dormancy, common oat Avena sativa L., and wild oat Avena fatua L. was assessed as were the contents of abscisic acid and volatile fatty acids of chain length C₆-C₁₀. In A. sativa which did not possess postharvest dormancy there was no correlation between germination and inhibitor levels but in A. fatua the relationship between the content of fatty acid and dormancy was good. The loss of these fatty acids in dry storage by evaporation could explain after ripening.     

Influence of Temperature and Seed Ripening on the in-vivo Incorporation of CO(2) into the Lipids of Oat Grains (Avena sativa L.)

To elucidate the influence of growth temperature and of stage of maturity on lipid synthesis in seeds, oat plants (Avena sativa nuda L., variety NOS) were fed with ¹⁴CO₂ at different stages after flowering, and the ¹⁴C-incorporation into the grain lipids was determined at 2, 24, and 48 hours after the end of ¹⁴CO₂-application. By changing growth temperature from 12 C to 28 C after the application of ¹⁴CO₂ to intact plants, a higher ¹⁴C-labeling of saturated fatty acids was found at the higher temperature. At 28 C, palmitic and stearic acids contained 23% and 9% respectively of total fatty acid-¹⁴C shortly after the ¹⁴CO₂-application, whereas at 12 C the corresponding values were 19% and 4%, respectively. Within 2 days ¹⁴C-activity of saturated fatty acids decreased at both temperatures, but to a lesser degree at 28 C. The higher ¹⁴C-labeling of saturated fatty acids and its lower decrease within 2 days at 28 C clearly show a direct influence of temperature on fatty acid biosynthesis in oat grains.     

ATP Synthesis during Water Imbibition in Caryopses of Genetically Dormant and Non-dormant Lines of Wild Oat (Avena fatua L.)

Levels of ATP in dry caryopses of wild oats (Avena fatua L.)were much lower than in imbibed seeds of the seven geneticallypure lines surveyed. The ATP content of the lines with highgenetic dormancy was consistently lower than the ATP contentof genetically non-dormant lines, but no significant correlationwith depth of dormancy was found apart from this. Massive increasesin ATP content occurred within 30 min of water uptake by caryopsesof both dormant and non-dormant lines. The synthetic pathwaystudied utilized inorganic phosphate with great avidity to formATP. The ability to form ATP upon imbibition was present inboth embryo and de-embryonated caryopsis. The ATP levels attainedin imbibing caryopses appeared sufficient to support considerablesynthetic activity, and this reduced the possibility that adeficiency in ATP was responsible for the maintenance of dormancyin such imbibed seeds. The low levels of inorganic phosphatein the embryos of genetically dormant lines of wild oat couldrepresent a limiting factor, if the active formation of ATPupon water imbibition resulted in a scarcity of phosphate forother reactions essential to germination. Key words: Avena fatua, ATP synthesis, Inorganic phosphorus, Seed dormancy, Germination, Water uptake