Germination and seedling growth under anaerobic conditions in Echinochloa crus-galli (barnyard grass)*

Abstract Although rice has long been recognized to be uniquely adapted for growth in low oxygen environments of flooded rice fields, rice weeds of the Echinochloa crus-galli complex appear to be at least as well specialized for germination and growth under such unusual biological conditions. Seeds of two varieties of E. crus-galli germinate and grow for prolonged periods in a totally oxygen-free environment. E. crus-galli germinates as well as rice (Oryza sativa) under a total nitrogen atmosphere and produces as large a seedling in spite of its much smaller seed size. Like rice, the seedlings of E. crus-galli are unpigmented, the primary leaves do not emerge from the coleoptile and no root growth occurs without oxygen. Of particular interest is the ultrastructure of mitochondria from anaerobically-grown seedlings. Mitochondrial profiles from the primary leaf of seedlings grown continuously in nitrogen are very similar to those grown aerobically. The size and shape of the mitochondria are similar and the cristae are numerous and normal in appearance. This is in sharp contrast to previous studies of other species which have reported that mitochondria were vesiculate and tended to lose their normal fine-structure after similar periods without oxygen. Finally, based on ultrastructure and ¹⁴C labeling studies, anaerobically-grown seedlings are highly active metabolically, which may explain, at least for E. crus-galli var. oryzicola, its ability to germinate and emerge from flooded rice fields.     

Proteomic identification of differentially expressed proteins in the anoxic rice coleoptile

Rice is the staple food for more than fifty percent of the world's population, and is therefore an important crop. However, its production is hindered by several biotic and abiotic stresses. Although rice is the only crop that can germinate even in the complete absence of oxygen (i.e. anoxia), flooding (low oxygen) is one of the major causes of reduced rice production. Rice germination under anoxia is characterized by the elongation of the coleoptile, but leaf growth is hampered. In this work, a comparative proteomic approach was used to detect and identify differentially expressed proteins in the anoxic rice coleoptile compared to the aerobic coleoptile. Thirty-one spots were successfully identified by MALDI-TOF MS analysis. The majority of the identified proteins were related to stress responses and redox metabolism. The expression levels of twenty-three proteins and their respective mRNAs were analyzed in a time course experiment.     

Mitotic activity in rice seedlings germinating under oxygen deficiency.

Rice seedlings germinating in oxygen-deficient environments do not manifest visual evidence of seminal or adventitious root growth or normal leaf growth, the coleoptile being the only seedling structure to emerge under these environmental conditions. Microscopic evidence, however, shows that mitotic activity occurs in the different tissues comprising the shoots of the seedlings germinating under oxygen deficiency. Thus evidence is presented here which shows that mitotic activity in rice seedlings germinating in oxygen-deficient environments is not confined solely to the emerging coleoptiles of the germinating seedlings.     

Committee for the study of the Scottish Flora

Summary

Three different oxygen-containing germination environments demonstrate the profound influence exerted by environmental oxygen (0₂) on growth and plastogenesis in coleoptiles of light-germinated rice seedlings. Coleoptile greening is extensive in low numbers of seedlings germinated in a sealed, initially air-saturated, static water environment and in large numbers of seedlings germinated under unagitated water underambient gaseous exchange conditions. In seedlings germinated in air (?21% 0₂), coleoptile greening is sparse and extension growth is much reduced compared with coleoptile extension growth of the submerged seedlings. Coleoptile greening and shoot and root growth are completely inhibited under hypoxia resulting from large numbers of germinating seedlings competing for the limited 0₂ supply in the sealed, initially air-saturated, static water environment. Coleoptile extension growth is highest under hypoxia and lowest under ?21% 0₂. The observations presented here demonstrate that 0₂ stress and non- stress conditions serve as environmental signals which influence growth behaviour and plastogenesis in coleoptiles of light-germinated rice seedlings.     

Nitric oxide accelerates seed germination in warm-season grasses

The nitric oxide (NO) donor sodium nitroprusside (SNP) significantly promoted germination of switchgrass (Panicum virgatum L. cv Kanlow) in the light and in the dark at 25°C, across a broad range of concentrations. SNP also promoted seed germination in two other warm-season grasses. A chemical scavenger of NO inhibited germination and blocked SNP stimulation of seed germination. The phenolic (+)-catechin acted synergistically with SNP and nitrite in promoting seed germination. Acidified nitrite, an alternate NO donor also significantly stimulated seed germination. Interestingly, sodium cyanide, potassium ferricyanide and potassium ferrocyanide at 200 μM strongly enhanced seed germination as well, whereas potassium chloride was without effect. Ferrocyanide and cyanide stimulation of seed germination was blocked by an NO scavenger. Incubation of seeds with a fluorescent NO-specific probe provided evidence for NO production in germinating switchgrass seeds. Abscisic acid (ABA) at 10 μM depressed germination, inhibited root elongation and essentially abolished coleoptile emergence. SNP partially overcame ABA effects on radicle emergence but did not overcome the effects of ABA on coleoptile elongation. Light microscopy indicated extension of the radicle and coleoptiles in seeds maintained on water or on SNP after 2 days. In contrast, there was minimal growth of the radicle and coleoptile in ABA-treated seeds even after 3–4 days. These data indicate that seed germination of warm-season grasses is significantly influenced by NO signaling pathways and document that NO could be an endogenous trigger for release from dormancy in these species.     

Amylolytic activity and carbohydrate levels in relation to coleoptile anoxic elongation in Oryza sativa genotypes

Among starchy seeds, rice has the unique capacity to germinate successfully under complete anaerobiosis. In this conditions, starch degradation is supported by a complete set of starch-degrading enzymes that are absent or inactive in cereals except rice. A characterization of carbohydrate metabolism and starch-degrading enzyme activity across twenty-nine genotypes of Oryza sativa L. is presented here. The zymogram of amylolytic activities present in rice embryos and endosperms under anaerobic conditions seven days after sowing (DAS) revealed marked differences among cultivars. Coleoptile elongation was positively correlated with total amylolytic activities and α-amylase activity in embryos, and negatively correlated with α-amylase activity in endosperm. Moreover, carbohydrate content in embryos was found to be positively correlated with total amylolytic activities under anaerobic conditions, while a negative relationship was recorded in the endosperm. Carbohydrate status in rice seedlings has a primary importance in sustaining coleoptile elongation towards the surface. The relationship between carbohydrate level in embryo and anoxic germination, as well as with total amylolytic activities present in rice embryo under anaerobic condition 7 DAS, is consistent with the role of sugar metabolism to support rice germination under oxygen-deprived environment.     

Effect of periodic distribution of low irradiance upon accumulation of dry matter in different parts of the rice embryo and changes in the dry weight of the endosperm during post germination stages

Summary The work presented deals with the fact that continuous or periodic distribution of low irradiance significantly reduces the dry weight of coleoptile and second leaf with cell elongation in rice seedlings during the initial period of growth.Such reduction in dry matter accumulation under exposure to light is also accompanied by the reduction in the loss of dry weight of the endosperm, which apparently indicates that during germination of the seed the translocation of the hydrolysed organic solutes from endosperm to the embryo is also affected by the low irradiance.Treatment of seedlings with different temperatures indicates that light either continuous or periodic plays a significant role upon the emergence of leaves of the embryo.     

Effects of o(2) concentration on rice seedlings

The ability of rice, wheat, and oat seedlings to germinate and grow as the O₂ concentration was lowered to zero was compared. The germination of rice was completely unaffected by O₂ supply, whereas that of oats and wheat was strongly retarded at levels below 5% O₂. In contrast to the coleoptiles of oats and wheat and to roots of all three species where growth was progressively diminished as the O₂ concentration was lowered, that of the rice coleoptile was progressively increased. However, the dry weight and content of protein, sugars, and cellulose were all depressed in the rice coleoptile in anoxia, and the levels of several respiratory enzymes, particularly those of mitochondria, were also much lower than those of the coleoptiles grown in air. In 1% O₂, the growth of the rice coleoptile was similar to that in air. The effect of ethanol concentration on germination and growth of rice was measured. Coleoptile growth was reduced when the ethanol concentration exceeded 40 millimolarity, and root growth was somewhat more sensitive. Coleoptiles of all three species grown in air were transferred to N₂, and ethanol accumulation was measured over 24 hours. The rate of ethanol accumulation in oats was close to that in rice, and in all three species the amounts of ethanol lost to the surrounding medium were those expected from simple diffusion from the tissue. The ability of the rice coleoptile to grow in anoxia is apparently not due to a particularly low rate of ethanol formation or to unusual ethanol tolerance. Any explanation of the success of rice in anoxia must encompass the much lower rate of ATP synthesis than that in air and account for the biochemical deficiencies of the coleoptile.     

GERMINATION AND SEEDLING GROWTH IN ECHINOCHLOA CRUS-GALLI VAR. ORYZICOLA UNDER ANOXIC CONDITIONS: STRUCTURAL ASPECTS

The morphological and cellular basis of anoxic germination in Echinochloa crus-galli var. oryzicola is reported. The embryo of E. crus-galli var. oryzicola is typically panicoid in its overall morphology, and is relatively large with a prominent coleoptile and mesocotyl. The response to anoxia is essentially the same in light and dark. Shoot growth occurs in both mesocotyl and coleoptile by cell elongation with no cell division. There is no emergence of the radicle without oxygen. Under anoxia the growth response is not the same as etiolation; there is no plumule elongation within the coleoptile, no protochlorophyll(ide) is found, and limited mesocotyl elongation occurs without oxygen. Air-dark treatment after anoxic germination results in a typical etiolated morphological response, including a resumption of mesocotyl growth, elongation of the plumule within the coleoptile, and initiation of pigment synthesis. These results indicate the effects of anoxia are not permanent but rather limiting and reversible.     

The rice coleoptile: an example of anaerobic nitrate assimilation

Nitrate present in rice caryopses can be reduced to ammonium and the ammonium subsequently assimilated by the coleoptile during anaerobic germination. All the enzymes of nitrate reduction and ammonia assimilation are present in the coleoptile. The supply of ¹⁵NO₃ confirms that the nitrate nitrogen is anaerobically incorporated into amino acids. Under anoxia, nitrate and nitrite reductase activities are increased in the coleoptile by exogenous nitrate. The importance of nitrate utilization during the anaerobic germination of rice caryopses is discussed.     

Incorporation of nitrate nitrogen in rice seedlings transferred to anaerobic conditions

Summary Incorporation of¹⁵NO₃- into amino acids was studied in 3-day-old aerobic rice seedlings (with coleoptile and root) subjected for 24h to anaerobic conditions. The incorporation of¹⁵N into glutamate, glutamine and alanine accounted for 89% and 84% of total incorporation in coleoptile and root, respectively. These findings indicate that, after the primary incorporation of¹⁵N into glutamate and glutamine, the main fate of nitrate nitrogen in rice seedlings subjected to anoxia is alanine.     

Seed vigour and water relations in wheat

The laboratory germination (criterion radicle emergence) of seven seed lots of winter wheat cv. Slejpner was similar. However, they differed in vigour as demonstrated by differences in germination after controlled deterioration carried out at a range of seed moisture contents, at two temperatures and for different times. A vigour assessment for each lot was quantified by calculating a value for the seed lot constant K_i, of the viability equation. Germination in lower water potentials reduced the uptake of water, radicle and coleoptile emergence and radicle and coleoptile extension. There was no difference in the water uptake of seed lots differing in vigour. However, seed lots of lower vigour showed less radicle emergence, less coleoptile emergence and shorter radicles than higher vigour seed lots in low water potentials. Similarly, controlled deterioration resulted in reduced radicle and coleoptile emergence and growth compared to unaged seed, and also to a greater sensitivity to low water potentials. The implications for field establishment are discussed.     

Preferential Induction of Alcohol Dehydrogenase in Coleoptiles of Rice Seedlings Germinated in Submergence Condition

Difference in the growth response to submergence between coleoptiles and roots of rice (Oryza sativa L.) was investigated in 9-d-old rice seedlings. The coleoptile length in the submergence condition was much greater than that in aerobic condition, whereas the root length in the submergence condition was less than that in the aerobic condition. Alcohol dehydrogenase (ADH) activity in the coleoptiles in the submergence condition was much greater than that in the aerobic condition, but ADH activity in the roots in the submergence condition increased slightly. These results suggest that the preferential ADH induction in rice seedlings may contribute to the difference in the growth response between the coleoptiles and roots under low oxygen conditions.     

A Possible Mechanism for Seed Coat Rupture by the Embryo in Germinating Wheat

The process of seed-coat splitting, terminating germination,in red wheats is described. We show that sequential expansionof the coleorhiza and coleoptile is required for germinativegrowth to be normal. Normal emergence requires the seed-coatsto be split by the expanding coleorhiza. We here describe agroup of cells associated with the coleorhizal tip forming apapilla-like projection which could aid this process. Embryo, germination, development, wheat, Triticum aestivum     

Primary and Lateral Root Development of Dark- and Light-Grown Cotton Seedlings under Salinity Stress*

Primary root growth dynamics and lateral root development of dark- and light grown cotton seedlings (Gossypium hirsutum L., cv. Acala SJ-2) were studied under control and salinity stress conditions. The seedlings were grown by two methods: A) in paper-lined, vermiculite-filled beakers with the plants growing between the paper and the glass wall (Gladish and Rost, 1993), and B) in hydroponics after germination and initial growth in germination paper rolls saturated with the treatment solutions (Kent and Läuchli, 1985). After germination, daily primary root elongation rate gradually incrased to a maximum, then gradually declined to close to zero for dark-grown seedlings, or to sustained rates of about 10 mm per day for light-grown control plants. Salinity stress delayed primary root growth and reduced peak elongation rates, without changing the general primary root growth pattern. These results suggest that salinity changed the time-scale, but did not modify the normal developmental sequence. Lateral root growth was more inhibited by salinity than primary root growth. In addition, elongation of lateral roots was more inhibited by salinity than their initiation and emergence. Light exposure of the shoot favored both sustained primary root growth from 7 days after planting, and lateral root emergence and growth. Salinity effects were more severe on seedlings germinated and grown in hydroponics (method B) than on vermiculite-grown plants (method A). These results emphasize the importance of growing conditions for the NaCl-induced effects on cotton root development. In addition, the differential effects of salinity on primary and lateral roots became evident, pointing to diverse control mechanisms for the development of these root types.     

The Effect of Oxygen and Turbulence on Elongation of Coleoptiles of Submergence-Tolerant and -Intolerant Rice Cultivars

Coleoptiles of rice (Oryza saliva L. ) elongated more rapidlyin stagnant solution than in water-saturated air: elongationrates in aerated solution were intermediate. Elongation wasstimulated between 170% to 390% in stagnant solution in 17 cultivarsscreened. On this basis, a relatively submergence-intolerantcultivar and two submergence-tolerant cultivars were studiedfurther. When seedlings were grown in a range of oxygen concentrationsimposed by bubbling solutions rapidly with gas, germinationand early coleoptile growth (up to 5.0 mm) were inhibited atlow oxygen concentrations, particularly in the submergence-intolerantcultivar. Weight per unit length declined with time in thiscultivar, even in mild oxygen deficits. However, later stagesof coleoptile elongation were unaffected by low oxygen supply:elongation proceeded at control rates even in anoxia. It isconcluded that processes unique to germination and early stagesof coleoptile elongation (possibly cell division) have greateroxygen requirements than cell extension. Rates of elongation in stagnant solution were much faster thanthose in any other treatment, including bubbled solutions atsimilar oxygen concentrations (0.125 mmol O₂ I_–1). Therefore,elongation appears to be stimulated by accumulation of endogenouscompounds which are dispersed in bubbled solutions and in air.The principal volatile compound was almost certainly ethylene,since Ag+ (an antagonist of ethylene) reduced the rate of elongationin stagnant solution by 53%. Differences in turgor pressure were only partly responsiblefor rapid coleoptile elongation observed in stagnant solution:there must have also been differences in cell wall propertiesbetween coleoptiles grown in stagnant and bubbled solution.The rapid elongation in stagnant solution hastens coleoptileemergence from the solution, thus establishing an adequate oxygensupply for the seedling. Leaf and root growth then proceeds.     

渗透胁迫下水稻种子萌发特性及抗旱性鉴定指标研究

用聚乙二醇（PEG）-6000作为渗透剂模拟干旱胁迫,探讨在4种不同浓度的渗透胁迫下水稻种子的萌发特性,并确立间接评价水稻苗期抗旱性的萌发期鉴定指标。结果表明,高浓度渗透液对种子萌发的抑制作用明显大于低浓度,萌发率的干旱胁迫反应指数随渗透液浓度的降低和萌发进程而增加。在不同浓度的渗透胁迫处理中,20％浓度下萌发6d后的萌发率表现较大的变异幅度和变异系数以及较均衡的品种频率分布,较好地反映了水稻种质对渗透胁迫的不同反应,认为20％／6d条件较适合作为萌发率鉴定的渗透液模拟干旱胁迫;经反复干旱后的幼苗存活率与萌发胁迫指数和20％胁迫浓度下萌发8d后芽鞘长的干旱胁迫反应指数（DRI）表现极显著或显著正相关,认为萌发胁迫指数和芽鞘长的DRI可作为评价水稻苗期抗旱性的间接鉴定指标。