Gravitational stress on germinating Pinus pinea seeds

In the germination of lipid-rich seeds, the glyoxylate cycle plays a control role in that, bypassing the two decarboxylative steps of the Krebs cycle; it allows the net synthesis of carbohydrates from lipids. The activity of isocitrate lyase, the key enzyme of the glyoxylate cycle, is an indicator of the state of seed germination: stage of germination, growth of embryo, activation and progress of protein synthesis, depletion of lipidic supplies. In order to investigate the effects of gravity on seed germination, we carried out a study on the time pattern of germination of Pinus pinea seeds that were subjected to a hypergravitational stress (1000 g for 64 h at 4 degrees C), either in a dry or in a wet environment, before to be placed in germination plates. During the whole time of germination, we monitored the state of embryo growth and the most representative enzymes of the main metabolic pathways. In treated wet seeds, we observed an average germination of only 20% with a slowdown of the enzyme activities assayed and a noticeable degradation of lipidic reserves with respect to the controls. These differences in germination are not found for dry seeds.     

Changes in germination, growth and soluble sugar contents of Sorghum bicolor (L.) Moench seeds under various abiotic stresses

The effect of various abiotic stresses on germination rate, growth and soluble sugar content in Sorghum bicolor (L.) Moench cv. CSH 6 seed embryos and endosperm during early germination was investigated. Under stress conditions germination, water potential and tissue water content decreased markedly. Subsequently, this reduction resulted in marked decreases in fresh weight both in embryos and endosperm. Conversely, a substantial increase in dry weight was observed. Furthermore, a considerable increase in the sugar contents in both embryo and endosperm was detected. The fructose level was always higher than glucose and sucrose in response to various stresses. However, as compared to the control the level of glucose and sucrose was higher in embryos and endosperm after stress treatments. Based upon these results a possible physiological role of sugars in the germination of sorghum seeds is discussed.     

Micro X‐ray computed tomography suggests cooperative feeding among adult invasive bugs Leptoglossus occidentalis on mature seeds of stone pine Pinus pinea

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Occurrence of cyclic AMP and related enzymes during germination of Pinus pinea seeds

The occurrence of cAMP, adenylate cyclase and cAMP phosphodiesterase has been tested in Pinus pinea seed during germination. The study has been carried out on dormant and imbibed seeds, seedlings, endospermic residues, roots and cotyledons. cAMP has been detected by the protein binding method and its occurrence has been verified by HPLC detections. cAMP phosphodiesterase shows a very high activity at acidic pH, while being completely inactive at pH 7.4. At this pH value, well detectable levels of adenylate cyclase have been observed. Therefore, the classical pathway of synthesis and breakdown of cAMP, already accepted for animal and bacterial cells, seems to be operating in Pinus pinea plant too.     

Genome-wide survey of the RIP domain family in Oryza sativa and their expression profiles under various abiotic and biotic stresses

Ribosome-inactivating proteins (RIPs) are N-glycosidases that inhibit protein synthesis by depurinating rRNA. Despite their identification more than 25 years ago, little is known about their biological functions. Here, we report a genome-wide identification of the RIP family in rice based on the complete genome sequence analysis. Our data show that rice genome encodes at least 31 members of this family and they all belong to type 1 RIP genes. This family might have evolved in parallel to species evolution and genome-wide duplications represent the major mechanism for this family expansion. Subsequently, we analyzed their expression under biotic (bacteria and fungus infection), abiotic (cold, drought and salinity) and the phytohormone ABA treatment. These data showed that some members of this family were expressed in various tissues with differentiated expression abundances whereas several members showed no expression under normal growth conditions or various environmental stresses. On the other hand, the expression of many RIP members was regulated by various abiotic and biotic stresses. All these data suggested that specific members of the RIP family in rice might play important roles in biotic and abiotic stress-related biological processes and function as a regulator of various environmental cues and hormone signaling. They may be potentially useful in improving plant tolerance to various abiotic and biotic stresses by over-expressing or suppressing these genes.     

Cadmium and copper change root growth and morphology of Pinus pinea and Pinus pinaster seedlings

Heavy metal loads in forest soils have been increasing over time due to atmospheric inputs. Accumulation in the upper soil layers could affect establishment of seedlings and forest regeneration. Mediterranean species show a high initial root development, allowing seedlings to reach the moisture of deeper soil layers. In the present work seedlings of stone pine ( Pinus pinea L.) and maritime pine ( Pinus pinaster Ait.), were grown in culture solution supplied with 0.0, 0.1, 1 or 5 μ M CdSO₄ or with 1 μ M CdSO₄ and 1 μ M CuSO₄ combined. In both species tap-root elongation was drastically reduced in the 5 μ M Cd²⁺ and in the (Cd²⁺+ Cu²⁺) treatments. A supply of 0.1 or 1 μ M Cd²⁺, however, enhanced root elongation in Pinus pinea without significantly influencing root elongation in Pinus pinaster . In both species the root density (weight per unit length) and the width of the cortex increased in response to Cd²⁺ exposure. In Pinus pinaster the mitotic index decreased at the higher Cd²⁺ concentrations and when Cd²⁺ and Cu²⁺ were combined. The data suggest that cell elongation is more sensitive to Cd²⁺ than cell division. The number and length of the lateral roots were also affected by Cd²⁺ treatment to a higher degree in Pinus pinaster than in Pinus pinea, reflecting the different Cd- tolerance of the two species.     

Dynamics of indole-3-acetic acid and indole-3-ethanol during development and germination of Pinus sylvestris seeds

Indole-3-acetic acid (IAA) and indole-3-ethanol (IEt) were identified in immature seeds of Pinus sylvestris L. by combined gas chromatography-mass spectrometry. Indole-3-methanol was tentatively identified using multiple ion monitoring. Anatomical investigations of seeds, as well as measurements of free and alkali-hydrolysable IAA and IEt, were made during seed development and germination. Levels of free IAA and IEt decreased during seed development. In the later stages of seed maturation most IAA and IEt were present in alkali-hydrolysable forms. Bound IAA and bound IEt rapidly decreased during germination, while levels of free IAA and IEt increased dramatically for a short period.     

Beneficial effects of enhanced UV-B radiation under field conditions: improvement of needle water relations and survival capacity of Pinus pinea L. seedlings during the dry Mediterranean summer

The possible mechanism(s) by which supplemental UV-B radiation alleviates the adverse effects of summer drought in Mediterranean pines (Petropoulou et al. 1995) were investigated with seedlings of Pinus pinea. Plants received ambient or ambient plus supplemental UV-B radiation (biologically equivalent to a 15% ozone depletion over Patras, 38.3° N, 29.1° E) and natural precipitation or additional irrigation. Treatments started on 1 February, 1994 and lasted up to the end of the dry period (29 September). In well-watered plants, UV-B radiation had no influence on photosystem II photochemical efficiency and biomass accumulation. Water stressed plants suffered from needle loss and reduced photosystem II photochemical efficiency during the summer. These symptoms, however, were less pronounced in plants receiving supplemental UV-B radiation, resulting in higher total biomass at plant harvest. Laboratory tests showed that enhanced UV-B radiation did not improve the tolerance of photosystem II against drought, high light, high temperature and oxidative stress. Enhanced UV-B radiation, however, improved the water economy of water stressed plants, as judged by measurements of needle relative water content. In addition, it caused an almost two-fold increase of cuticle thickness. No such UV-B radiation effects were observed in well-watered pines. The results indicate that the combination of water stress and UV-B radiation may trigger specific responses, enabling the plants to avoid excessive water loss and, thereby, maintain a more efficient photosynthetic apparatus during the summer. The extent of this apparently positive UV-B radiation effect would depend on the amount of summer precipitation. Abbreviations: DW – dry weight, F_v/F_m – ratio of variable to maximum fluorescence, A ₃₀₀ – absorbance at 300 nm, PAR – photosynthetically active radiation, PS II – photosystem II, RWC – relative water content, TCA – trichloroacetic acid, UV-B_BE – biologically effective ultraviolet-B radiation     

Chlorophyll accumulation in cotyledons, hypocotyls and primary needles of Pinus pinea seedlings in light and dark

Seeds of Pinus pinea L. were germinated and grown in darkness or under natural photoperiodic (day/night) conditions for 8 weeks. Samples were taken for analysis at frequent intervals and chlorophyll (Chl) and protochlorophyllide (Pchlide) contents of cotyledons, hypocotyls and primary needles determined spectrophotometrically alter separating the esterified from unesterified pigments by partitioning between ammoniacal acetone and petroleum ether. Chlorophyll accumulated in cotyledons, hypocotyls and primary needles of dark as Well as light grown seedlings. The maximum Chl concentrations achieved in darkness in these organs were approximately 60, 20 and 10% respectively, of concentrations achieved in light. The tinsel of rapid Chl accumulation by cotyledons in darkness coincided with the emergence of the radicle from the seed. Light had no effect on the timing of this process or on the duration of the lag period. However, it extended the exponential growth phase of cotyledons by at least a week and. once the seedlings were about 2 weeks old, enabled Chl to accumulate at a faster rate. Time courses of Chi and Pchlide accumulation by cotyledons in darkness were similar. Pchlide did not accumulate prior to the onset of Chi accumulation and Chl slopped accumulating once the Pchlide pool reached maximum size. The implications of these findings are discussed and a hypothesis to account for the inability of primary needles of smaller seeded pines to accumulate Chl in darkness is proposed.     

油菜素内酯浸种对盐胁迫番茄种子萌发的影响及其生理机制

为揭示油菜素甾醇类化合物提高作物耐盐的效应和机理,研究了10^-11、10^-10、10^-9、10^-8、10^-7、10^-6、10^-5 mol/L 2,4-表油菜素内酯（EBL）浸种处理对0、50、100、150、175 mmol/L NaCl胁迫7 d的番茄种子萌发、生长、溶质积累、抗氧化代谢的影响。结果显示：NaCl浓度越高的盐胁迫下,10^-9 mol/L EBL浸种可体现出越显著的促进番茄种子萌发的效应;在所有处理下,EBL浸种浓度过高,即10^-6、10^-5 mol/L EBL,均表现出对种子萌发的抑制效应。盐胁迫下种子萌发后,一定浓度的EBL浸种可表现出明显的增加种子胚根和下胚轴长,提高萌发种子鲜重和种子活力指数,其中10^-9 mol/L EBL浸种处理促进效果最适;EBL浸种浓度过高,则表现出抑制效应。150 mmol/L NaCl胁迫或非盐胁迫下,10^-9 mol/L EBL浸种均可降低萌发种子体内的O₂^·-、H₂O₂、丙二醛（MDA）和脯氨酸（Pro）含量;盐胁迫下,10^-9 mol/L EBL浸种可显著提高萌发种子可溶性糖（SS）和可溶性蛋白（SP）的含量。150 mmol/L NaCl胁迫或非盐胁迫下,10^-9 mol/L EBL处理可不同程度促进番茄种苗超氧化物歧化酶（SOD）和过氧化物酶（POD）活性的上升。综上所述,盐胁迫下,一定浓度范围内的EBL浸种可明显促进番茄种子萌发或成苗,其中以10^-9 mol/L EBL浸种的效果最好,主要是因为EBL施用可积极促进番茄种子萌发中物质转化,SS和SP等溶质积累增多,增强其渗透调节能力;同时SOD和POD酶活增强,缓解盐胁迫导致番茄种子萌发中的次生氧化胁迫。     

The effect of phenols on respiratory enzymes in seed germination

Low molecular weight phenolic compounds were identified in two soilswith different vegetative cover, Fagus sylvatica, L. andPinus laricio, Poiret, spp. calabrica, and were tested atdifferent concentrations on seed germination of Pinuslaricio, and on respiratory and oxidative pentose phosphate pathwayenzymes involved in the first steps of seed germination. The data obtained showthat there are marked differences in the phenolic acid composition of the twoinvestigated soils. All the phenolic compounds bioassayed inhibited seedgermination and those extracted from Pinus laricio soilwere particularly inhibitory. We also found that the non-germination of seedsisstrongly correlated to the inhibition of the activities of enzymes ofglycolysisand the oxidative pentose phosphate pathway.     

Morphometric and chemical fruit variability of selected stone pine trees (Pinus pinea L.) grown in non-native environments

Few studies have been conducted on Stone pine (Pinus pinea) morphology, cone to kernel yield (KY) and composition. To study the species variability, a morphometric and chemical characterization of pine nuts was performed across the species distribution in Chile. Seventy-six highly productive trees were selected in three macrozones. Ten cones per tree were harvested, and cone weight, in-shell pine nut number cone^?1 and size and weight of in-shell pine nuts and kernels were measured; KY was determined. Chemical and nutritional characterization of pine nuts was performed in 38 trees. Differences among macrozones were tested with mixed linear models. Partial correlation was used to test for correlation between cone and pine nut morphometric data, chemical features, and climate. Canonical correlation coefficients between groups were tested. Selected trees differed in cone and in-shell pine nut size, kernel morphometry, and KY, with lowest trait values being generally found in the coast. KY was higher than in Europe. Chemical composition was similar to values reported in producer countries, homogeneous among macrozones, with the exception of oleic acid and potassium. Detected differences would respond to the environment and to the interaction genotype?×?environment given the restricted origins of the genetic material, confirming the species adaptability.     

Role of auxin and gibberellin in regenerative differentiation of tracheids in Pinus pinea seedlings

A new experimental system was developed for studying the hormonal mechanisms which control tracheid differentiation. In this system the tracheids redifferentiated from parenchyma cells in the hypocotyl of young Pinus pinea L. seedlings. The experimentally induced tracheids have unique shapes and patterns, and are therefore easily distinguished from the primary and secondary tracheids formed before the experiments. Auxin (0·1–1% NAA) alone sufficed to cause the redifferentiation of short tracheids, usually in discontinuous patterns across the hypocotyl. Gibberellin by itself did not induce redifferentiated tracheids. Combinations of auxin with gibberellin (0·1% NAA+0·1–1% GA₃) promoted the differentiation of long tracheids (up to threefold greater than those induced by auxin) in continuous patterns along the stem axis. Gibberellin in the presence of auxin promoted tracheid elongation by stimulating intrusive growth of both the upper and lower ends of the differentiating tracheids. The role of auxin and gibberellin in controlling the evolution of tracheary elements, from tracheids to vessels and fibres, is discussed.     

Canola seed germination and seedling emergence from pre-hydrated and re-dried seeds subjected to salt and water stresses at low temperatures

Low soil temperatures and low water potentials reduce and delay the seed germination of canola (Brassica rapa L., B. napus L.) in western Canada. Germination is also very sensitive to the salinity effects of nitrogen fertiliser placed with the seed, especially when the seed bed is relatively dry. The effects of pre-hydration and re-drying treatment on canola (Brassica rapa L. cv. Tobin) seed germination and seedling emergence at 10°C subjected to either a water or salt stress were determined. Low water potentials, induced by polyethylene glycol (PEG 8000), low soil moisture, or high concentrations of salts, reduced both germination and seedling emergence, and increased the time to 50% germination and emergence of seeds at 10°C. At equal osmotic potentials, Na₂SO₄ was less inhibitory on low temperature germination than either NaCl or PEG, suggesting that the sulphate ion partially alleviated the inhibitory effects of low water potential. Solutions of NaCI produced more abnormal seedlings compared to Na₂SO₄, suggesting that NaCl was more toxic than Na₂SO₄ during seedling development. Pre-hydration and re-drying partially overcame the inhibitory effects of both low water potential and salts on seed germination and seedling emergence at 10°C. The seed treatment increased the germination rate in Petri dishes and seedling emergence from a sandy loam soil. Water potentials or soil water contents required to inhibit 50% germination or emergence at 10°C were lower for treated seeds compared to control seeds. Salt concentrations inhibiting 50% emergence were higher for treated seeds than control seeds. Neither treated nor control seeds produced seedlings which emerged if the soil water content was lower than 9% or when the soil was continuously irrigated with salt solutions of 100 mmol kg^-1 of NaCl or 50 mmol kg^-1 of Na₂SO₄. These results suggest that the pre-hydration and re-drying treatment did not lower the base water potentials at which seedling emergence could occur. Abnormal seedlings were observed in both treated and control seeds, particularly if the soil was watered with NaCl solutions; however, the seed treatment reduced the number of abnormal seedlings.     

Changes in mRNA populations during loblolly pine (Pinus taeda) seed stratification, germination and post-germinative growth

Changes in the patterns of gene expression were examined during loblolly pine ( Pinus taeda L.) seed stratification, germination, and post-germinative growth. In both the megagametophyte and the embryo, DNA contents remained relatively constant at all stages examined. RNA contents, however, increased in both tissues following seed germination, particularly in the embryo where a 7-fold increase in the RNA content was observed 5 days after germination. Poly(A)⁺ RNA, extracted from megagametophytes and embryos, was translated in vitro in a rabbit reticulocyte lysate cell-free system. Analysis of [³⁵S]-methionine-labelled translation products by two-dimensional electrophoresis/fluorography indicated that there were changes in the populations of mRNAs during all developmental stages examined. In both the megagametophyte and the embryo several distinct mRNA populations, including one constitutively present at all stages examined, were identified. One mRNA population, present in the mature seed, decreased during seed stratification. Another population, not present in the mature seed, rose during the period of stratification that coincided with an increase in seed germinability. A third population, which appeared during seed germination, increased steadily during post-germinative growth. Besides these similarities, specific differences between megagametophyte and embryo were noted. For example, one mRNA population, which was present in the megagametophyte of the mature seed and remained constant during the stratification period, disappeared immediately following seed germination. In the embryo, one set of messages was germination specific. In total, these results show that mRNA populations change in a temporal fashion that is consistent with the patterns of de novo protein synthesis known to occur in loblolly pine during the same developmental periods.     

Role of ethylene and auxin in regenerative differentiation and orientation of tracheids in Pinus pinea seedlings

A low auxin concentration (0.1% naphthalene acetic acid) induced tracheids with longitudinal polarity parallel to the hypocotyl axis in young Pinus pinea seedlings. Application of 0.1% ethrel laterally and 0.1% naphthalene acetic acid apically disturbed axial tracheid polarity and promoted the differentiation of tracheids with a lateral orientation. Ethrel by itself, with no auxin background, did not affect tracheid differentiation. Apical application of 1% gibberellic acid with the low auxin, reversed the polarity disorder induced by ethrel. Disturbance of axial tracheid polarity was observed under a high auxin concentration (0.5% naphthalene acetic acid) which was similar to the combined effect of ethrel and auxin. The high auxin concentration increased tracheid number significantly. This effect was curtailed following treatment with inhibitors of ethylene formation (Co²⁺; 1-aminoethoxy- vinylglycine) and action (Ag²⁺). The role of ethylene in controlling the differentiation of radial tracheids, which characterize the vascular rays of pines, is discussed.     

光温条件对不同种源红松种子萌发的影响

种子萌发受种源和环境条件(如光照和温度条件等)的影响。种子萌发决定红松(Pinus koraiensis)的天然更新能力, 因此也是恢复地带性顶极群落阔叶红松林的关键。该研究以我国3个主要种源地(辽宁清原、吉林长白山和黑龙江伊春)当年的红松种子为对象, 监测自然光、温(不同季节、林窗和林下)和控制光、温(3个光照强度: 200、20和0 μmol·m^-2·s^-1, 记为L200、L20和L0; 2个温度: 25和15 ℃)条件下红松种子的萌发特征。结果表明, 自然越冬后, 春季(5月)所有种源种子均未萌发, 夏季(7月)和秋季(9月)各种源种子萌发率(GP)均较低(1.8%-33.7%), 但夏季林窗内各种源种子GP均显著高于林下。对林下各季节未萌发的种子, 带回室内给予适宜的光温条件(L200、25/15 ℃)后发现: 春季和夏季种子均能大量萌发(32%-77%), 而秋季种子GP极低(<2%)。25 ℃下, 3个种源地种子GP和萌发指数(GV)均呈现在L200条件下显著高于L20和L0处理。15 ℃条件下, 清原种子GP和GV对光照的响应与25 ℃条件下相一致, 长白山和伊春种子GP和GV分别在L0和L20处理下最高。所有光照强度下, 3个种源地种子GP和GV均为25 ℃处理显著高于15 ℃处理。可见, 适宜的温度是红松种子萌发的必要先决条件, 长白山和伊春红松种子可能需要更高的积温解除休眠; 适宜温度下(25 ℃), 红松种子表现出萌发需光性; 红松种子萌发对光、温的响应存在种源特异性, 长白山和伊春红松种子在相对低温下萌发需光性弱或消失。林下光、温条件不适宜红松种子萌发是造成红松林天然更新慢或更新差的重要原因。     

Light and nitrate interaction in phytochrome-controlled germination ofPaulownia tomentosa seeds

Pulsed light and nitrate exhibit an interactive effect on the germination ofPaulownia tomentosa Steud. seeds that require long periods of light irradiation. Two pulses of red light (R), separated by an adequately long dark interval, substitute for continuous prolonged irradiation. A far-red (FR) pulse given at the beginning of the dark interval inhibits germination, while it has no effect if given at the end. The requirement for certain ratios of the far-red-absorbing form of phytochrome/total phytochrome (P_fr/P_tot) differs when a FR+R-pulse is given as the first or second of two pulses (FR+R or R) separated by a dark interval. An equal decrease of the P_fr/P_tot ratio leads to a more pronounced decrease in germination when the pulse of the same FR+R ratio is given as the second pulse at the end of the dark interval. The length of dark interval between light pulses needed for maximal germination, differed in (i) seeds with a natural requirement for long periods of light irradiation from that in (ii) seeds with their long light requirement imposed by two weeks of imbibition in darkness or by (iii) imbibition in 40% heavy water. However, a single R pulse was sufficient to induce a high percentage of germination if the seeds were supplied with KNO₃ (10 mM) from the onset of imbibition up to the onset of light. This effect decreased with a delayed time of application, and was prevented if FR preceded the KNO₃ application. We dedicate this paper to Professor Hans Mohr on the occasion of his 60th birthday