Polyamines and Nitric Oxide Link in Regulation of Dormancy Removal and Germination of Apple (Malus domestica Borkh.) Embryos

Polyamines (PAs) belong to plant growth regulators and in complex with classical phytohormones take part in regulation of seed dormancy and germination. Although the impact of reactive oxygen (ROS) and nitrogen (RNS) species on seed germination is well described, the cross talk of PAs with ROS/RNS has never been analyzed. Due to the close connection of PAs and ethylene biosynthetic pathways to arginine (Arg)-dependent NO biosynthesis we investigated production of nitric oxide (NO), peroxynitrite (ONOO^?) and the level of O ₂ ^?? or H₂O₂ in apple embryos, germination of which was PA regulated. PAs: putrescine (Put) and spermidine (Spd) in contrast to spermine (Spm) stimulated germination of apple embryos. Among amino acids, stimulation of germination was observed in Arg and ornithine (Orn) only. Dormancy removal of embryos by PAs was associated with increased accumulation of H₂O₂ and O ₂ ^?? in embryonic axes. At the same stage of completion of sensu stricto germination the stimulatory effect of PAs (Put and Spd) and amino acids, mainly Arg and Orn, was accompanied by enhanced NO and ONOO^? production in embryonic axis. The beneficial effect of PAs (Put and Spd) and their precursors on germination of apple embryos was removed by NO scavenging, suggesting a crucial role of NO in termination of embryo germination and radicle growth. Moreover, activity of polyamine oxidase in embryo axes was greatly enhanced by embryo fumigation with NO. Our data demonstrate the interplay of RNS/ROS with PAs and point to NO action as an integrator of endogenous signals activating germination.     

Seed Physiology: Its History from antiquity to the beginning of the 20th century

Seed physiology, especially seed germination, has intrigued the human mind since antiquity, partly out of curiosity, partly because of practical reasons. Theophrastus could be called the father of seed physiology since he already described many of the facts and problems which up to this day are being investigated by seed physiologists. Most of the other ancient authors writing about natural history or agriculture (Columella, Varro, Pliny the elder, Virgil) added little to Theophrastus’ all encompassing knowledge of seed physiology. The 1700 years from the first century A.D. to the end of the 18th century were a “black out” (Johansen, 1951) of seed physiology. At the beginning of the 19th century people like Schleiden, Amici, Brown and Hofmeister first understood structure and function of ovule, embryo sac and pollen tube whereas fertilization and double fertilization were only detected at the end of the 19th century by Strasburger, Nawaschin and Guignard. The physiology and biochemistry of seed maturation were first investigated by Sachs and Pfeffer, two great masters of modern plant physiology. In modern times our detailed knowledge of the effect of external factors on seed germination is due to the many authors working at the end of the 19th and the beginning of the 20th centuries. Our knowledge of the impact of temperature on germination derives mainly from the investigations of Sachs, De Vries and Haberlandt, and of light from those of Caspary, Cieslar, Heinricher and Kinzel. The action spectrum of light was first investigated by Cieslar, and Flint and McAllister whereas the most important discovery of the effect of light on germination (and on many other physiological phenomena), the R-FR photoreversible system, was made by Borthwick et al. leading later to the discovery of the phytochrome. The first germination inhibiting and stimulating substances were found by Siegmund and Lehman. The long history of seed physiology should make today’s seed physiologists conscious of the fact that they stand on the “shoulders of giants.”     

Seed-Specific Expression of AINTEGUMENTA in Medicago truncatula Led to the Production of Larger Seeds and Improved Seed Germination

The increase of seed size is of great interest in Medicago spp., to improve germination, seedling vigour and, consequently, early forage yield as well as for optimizing seeding techniques and post-seeding management. This study evaluated the effects of the ectopic expression of the AINTEGUMENTA (ANT) cDNA from Arabidopsis thaliana, under the control of the seed-specific USP promoter from Vicia faba, on seed size, germination and seedling growth in barrel medic (Medicago truncatula Gaertn.). All the transgenic T₂ barrel medic lines expressing ANT produced seeds significantly larger than those of control plants. Microscopic analysis on transgenic T₃ mature seeds revealed that cotyledon storage parenchyma cells were significantly larger and contained larger storage vacuoles than those of the untransformed control. Moreover, the percentage of germination was significantly higher and germination was more rapid in transgenic than in control seeds. Our results indicate that the seed-specific expression of ANT in barrel medic led to larger seeds and improved seed germination, and revealed a regulatory role for ANT in controlling seed size development.     

Germination studies ofMolucella laevis

Seed of Molucella laevis (L.) was gathered in the years 1963-66 and germinated soon after harvest and at various intervals subsequently. All seeds showed dormancy on gathering. There was a noticeable fluctuation in the percentage of seeds germinating during storage. Dormancy persisted throughout the years of the experiment. It appears that a number of factors are operative in the inhibition of germination of M. laevis seeds. One factor is an inhibitor which may be adsorbed by active charcoal or heavy loam, involving some process which requires fluctuating temperature. Rupture of the seed-coat also improved the germination of dormant seed, and a subsequent water rinse for 24 hrs. further increased the percent of germinating seed. Immersion of the seed for 48 hrs. in gibberellic acid (G.A.), 400 ppm, greatly improved germination, but it did not completely overcome dormancy. The maximum effect (93% germinating seed) was obtained when seed pre-treated with G.A. was germinated on top of active charcoal. The optimal germination temperature was found to be a daily alternating one of 16 hrs. at 15°C and 8 hrs. at 30°C with light supplied at the latter temperature. Treated seed was sown in the screen house and found to develop normal plants and seed. The dormancy-breaking effect of G.A. lasts for at least 180 days. The fluctuations in germination of seed pre-treated with gibberellin were similar to those of the untreated ones. The effect of dormancy-breaking factors differed with year of gathering and date of application. Thus, M. laevis seeds display much heterogeneity in their germination potential.     

Legumin and albumin of pea cotyledons during seed germination

During 6 days of pea seed germination the depletion of legumin with mol. m. 390 000 from protein bodies was observed. SDS-PAGE indicated that the legumin subunits with mol. m. 41 700 and 21 000 were prevailing. Only the former of these, probably corresponding to α-subunit, was degraded rapidly during 6 days of germination. Water-soluble proteins (albumins) prepared from pea cotyledons were separated by preparative IEF into proteins with pI 7.1, 6.5, 6.0, 5.4, 5.0, and 4.6. During 6 days the components of albumin with pI 7.1, and 6.5 were dramatically depleted. Major fractions with pI 6.5, 6.0, and 5.4 were subjected to SDS-PAGE and their subunit composition was determined. Moreover, albumin of pea cotyledons was resolved into 13 components by SDS-PAGE. Mobilization of albumin began from the degradation of components with higher mol. m. during germination.     

Arabidopsis thaliana for testing the phytotoxicity of volatile organic compounds

Geosmin and C-8 hydrocarbons are among the major volatile organic compounds (VOC) responsible for the distinctive, musty odor of filamentous fungi. In this study, we developed a plant bioassay for testing the possible toxicity of these compounds, as well as four air freshener products sometimes used to mask their odor. Seeds and vegetative plants of Arabidopsis thaliana were exposed to 1 ppm of 14 different volatile treatments (both single compounds and mixtures) for 72 h and monitored for germination rate, seedling formation, vegetative plant vigor and chlorophyll concentration. All VOCs tested had some inhibitory effect on seed germination or seedling formation; 1-octen-3-one was the most active, giving almost complete inhibition of germination. Geosmin did not prevent germination (radicle protrusion) but seedling formation was arrested 90 %. Of solvents and fragrance products tested, only the scented oil product was as active as the C-8 biogenic compounds in inhibiting seed germination and seedling formation. Two-week-old plants exposed to 1 ppm of individual fungal VOCs for 72 h all exhibited some degree of stress symptoms including smaller leaf size and weight, discoloration, leaf curling, small necrotic lesions, and reduced chlorophyll concentration. Two-week-old vegetative plants exposed to solvents and air freshener products were generally smaller in size. The single most phytotoxic compound tested was 1-octen-3-one which almost completely inhibited seed germination and was lethal to vegetative phase plants. Formaldehyde at 1 ppm killed 2-week-old seedlings but had little effect on seed germination. In conclusion, the A. thaliana bioassay provides an inexpensive approach for testing the toxicity of gas phase molecules.     

A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa

Main conclusion

Co-expression of a lesquerella fatty acid elongase and the castor fatty acid hydroxylase in camelina results in higher hydroxy fatty acid containing seeds with normal oil content and viability. Producing hydroxy fatty acids (HFA) in oilseed crops has been a long-standing goal to replace castor oil as a renewable source for numerous industrial applications. A fatty acid hydroxylase, RcFAH, from Ricinus communis, was introduced into Camelina sativa, but yielded only 15 % of HFA in its seed oil, much lower than the 90 % found in castor bean. Furthermore, the transgenic seeds contained decreased oil content and the germination ability was severely affected. Interestingly, HFA accumulation was significantly increased in camelina seed when co-expressing RcFAH with a fatty acid condensing enzyme, LfKCS3, from Physaria fendleri, a native HFA accumulator relative to camelina. The oil content and seed germination of the transgenic seeds also appeared normal compared to non-transgenics. LfKCS3 has been previously characterized to specifically elongate the hydroxylated ricinoleic acid to lesquerolic acid, the 20-carbon HFA found in lesquerella oil. The elongation reaction may facilitate the HFA flux from phosphatidylcholine (PC), the site of HFA formation, into the acyl-CoA pool for more efficient utilization in triacylglycerol (TAG) biosynthesis. This was demonstrated by increased HFA accumulation in TAG concurrent with reduced HFA content in PC during camelina seed development, and increased C20-HFA in HFA-TAG molecules. These effects of LfKCS3 thus may effectively relieve the bottleneck for HFA utilization in TAG biosynthesis and the feedback inhibition to fatty acid synthesis, result in higher HFA accumulation and restore oil content and seed viability.     

The action of exogenous gibberellic acid on polysome formation and translation of mRNA in germinating castor-bean seeds

The amount of protein synthesis in germinating castor-bean seeds has been estimated by the quantitative and qualitative exmainatin of polysomes from the seeds in the presence and absence of gibberellic acid (GA₃). Careful optimisation of polysome extraction procedures was required to minimise the ribonuclease activity in the extracts. Ribonuclease activity in seed extracts increased fourfold over the first 5 d of germination. Gibberellic acid stimulated polysome formation about twofold during the first 4 d of germination. It also stimulated the amount of mRNA associated with polysomes by about twofold during the first 3 d of germination. Between days 1 and 5 of germination, polysome formation was primarily limited by mRNA availability. During the period 0–24 h, polysome formation was independent of mRNA levles. The increase in enzyme activities stimulated by GA₃ was probably the result of an increase in the amount of cellular mRNA. No evidence was obtained for an action of GA₃ on translation other than on the increased production of RNA. Examination of the recruitment of isocitrate-lyase mRNA into polysomes showed that GA₃ did not specifically stimulate production of this enzyme.     

Proteolytic activities in the developing seeds of ×Haynaldoticum sardoum

Aminopeptidase, carboxypeptidase and proteinase activities were measured in endosperms from unripe and ripe seeds of ×Haynaldoticum sardoum. Aminopeptidase and proteinase activities were high during the early maturation stages and then decreased. In contrast, carboxypeptidase activity increased with maturation. Localization studies demonstrated that aminopeptidase and carboxypeptidase activities were present in the three tissues examined (pericarp, green layer plus aleurone, and starchy endosperm). Proteinase activity against gliadin was located in the pericarp and in the green layer plus aleurone, but was absent in the starchy endosperm. The presence of proteolytic activities in the outer kernel layers might be correlated to the hydrolysis of transitory protein reserves during the senescence of the seed coat?. Aminopeptidase and carboxypeptidase activities located in the starchy endosperm could participate in the breakdown of protein reserves during the early phases of seed germination.     

Mepiquat chloride seed treatment and germination temperature effects on cotton growth,nutrient partitioning,and water use efficiency

Cotton seed (Gossypium hirsutum L. cv. “Stoneville 825”), treated with 0, 0.2, 1.0, and 2.0 g active ingredient (a.i.) mepiquat chloride (MC) kg^?1, was evaluated for the effect of MC on early plant growth. Emergence rate and total emergence of MC-treated seed and control were similar regardless of germination temperature. However, the number of leaves and squares and the dry weight of leaves, stems, and roots for hydroponically grown cotton plants were significantly lower at lower germination temperatures (15°C for 3 day/30°C for 1 day and 15°C for 4 days) than at higher germination temperatures (30°C for 4 days and 30°C for 3 days/15°C for 1 day). All MC treatments significantly decreased the number of nodes, leaves, and squares, as well as dry weight of leaves, stems, and roots, as compared to control plants at 28 days after emergence. MC seed treatments also significantly reduced plant height and total leaf area compared to controls. Water-use efficiency (WUE) was significantly lower for the 1.0 g a.i. MC treatment than for control plants. In general, the highest rate of MC seed treatment resulted in greater concentrations of calcium, phosphorus, and nitrogen in plant leaves and stems and also in greater concentrations of magnesium, phosphorus, and nitrogen in roots than in controls.     

A model of seed dormancy

Seed dormancy is considered as an aspect of growth cessation. It is characterized by partial metabolic arrest with its inception and termination under endogenous hormonal control. Although several distinct types of seed dormancy have been recognized traditionally, an examination of the experimental literature suggests that a common regulatory mechanism exists. Dormancy onset, control, and termination are apparently regulated by a balance of growth inhibitors and promotors. At the onset of dormancy this balance is shifted in favor of the inhibitor component; at its termination it is shifted back in favor of the promotor component. The experimental evidence indicates that the levels of growth-promoting hormones decrease markedly during seed maturation, imposing dormancy. A trigger mechanism stimulates hormone activation and/or synthesis, thus terminating dormancy and culminating in resumption of embryo growth (germination). A model is proposed that embodies a hormonal regulation of four phases of dormancy: an inductive mechanism, a maintenance mechanism, a trigger mechanism, and a germination mechanism. This hormonal regulation, in conjunction with specific metabolic inhibitors, is considered to mediate the activation and/or synthesis of hydrolytic enzymes. From this model the dormant seed is viewed as a cybernetic system. Particular emphasis is given to the regulatory role of gibberellins in seed dormancy and germination.     

A genome-wide screen for non-template nucleotides and isomiR repertoires in miRNAs indicates dynamic and versatile microRNAome

miRNA variants (termed isomiRs) have been reported as potential functional molecules that may affect miRNA stability or target selection. The aims of the present study were to comprehensively survey and characterize non-template nucleotides (NTNs) and isomiR repertoires in miRNAs. Over 50 % of the NTNs were located in the 3′ ends (also termed 3′ additions), followed by the 5′ ends and adjacent positions to 5′ and 3′ ends. The similar distributions of NTNs and isomiR repertoires might be detected between homologous or clustered miRNAs. miRNA might be stably expressed based on the typical analysis, but its isomiRs might be strongly up- or down-regulated. IsomiRs with novel seed sequences were mainly derived from “seed shifting” events in 5′ isomiRs, NTNs in 5′ ends or in seed sequences. IsomiRs from a miRNA locus or homologous miRNA loci maybe have the same seed sequences, but they would have various enrichment levels and 3′ ends. Interestingly, isomiR species with novel seed sequences via NTNs in the seed region were always stably expressed. These novel seed sequences could lead to novel functional roles through driving the potential novel target mRNAs. Integrated predicted target mRNAs and further microarray validation showed that these isomiRs have versatile biological roles. Collectively, multiple isomiR products and miRNA maturation processes provide opportunities to perform versatile roles in the regulatory network, which further enriches and complicates the regulation of biological processes.     

Amylases during different stages of seed development in wheat

The β-Amylase activity was high in seeds of pre-Milk and milk stages but low in seeds of half seed and dough stages and very low at fully ripe stage of seed development. Zymogen (latent β-Amylase) was absent in half seed stage but appeared at pre-Milk stage. The content of zymogen increased progressively with advance in ripening time. No α-Amylase activity was detected at any stages of seed development in wheat.