Analysis of stylar self-incompatibility competence by use of heat induced inactivation

Summary Immersion of Lilium longiflorum pistils in 49 °C water for increasing durations of 1,2,3, or 4 minutes immediately prior to incompatible pollination resulted in a correspondingly progressive decrease in the stylar self-incompatibility competence, as determined from the lengths attained by pollen tubes during 48 hours growth in the styles at 24 °C. Neither pistils remaining on the plant nor those detached from the plant which were immersed after anthesis in 49 °C water for 5 minutes regained self-incompatibility competence during a 48 hour incubation at 24 °C prior to incompatible pollination. Heat treatment of detached pistils as early as 39 hours prior to bud anthesis also resulted in an inactivation of stylar self-incompatibility competence when incompatible pollination was made at 24 hours after anthesis. Experiments utilizing heat treatment of partial lengths of detached whole styles revealed that pollen tubes which have grown through as much at 45 millimeters of either a physiologically incompatible or compatible portion of the style are still capable of shifting to either a higher growth rate or lower growth rate upon entry into respectively either a physiologically compatible or incompatible portion of the style.     

Effect of pollen-style interaction on the pollen tube growth of Gossypium hirsutum

Summary All possible crosses among 5 strains of Gossypium hirsutum were made, and the pollen tubes were grown in vivo for 4 h before being fixed, stained and measured. Temperatures ranging from 18.5 to 40.0 °C were tested for pollen germination and pollen tube growth. The optimal temperature for pollen tube growth was 30.0 °C. Relative humidity levels of 0 to 100% were used as a pre-pollination treatment of the pollen. Significant differences among the mean pollen tube length of the strains occurred due to pollenXstyle interactions. The strains also differed in the number of styles which did not support pollen tube growth. These differences were also due to pollenXstyle interactions. Pollen and style strains could be ranked according to their relative contribution to pollen tube length.College of Agricultural Sciences Publication Number T-4-189     

Effects of high humidity and temperature stress on pollen membrane integrity and pollen vigour in Nicotiana tabacum

Summary The prolonged exposure of pollen Nicotiana tabacum to high humidity at both room temperature and 38° C did not affect membrane integrity as revealed by the fluorochromatic reaction (FCR) test, but did affect pollen vigour. At room temperature germination was not affected, although tube growth was reduced; at 38° C, there was both a reduction in tube growth and delayed germination. When the pollen was subjected to 1 h hydration followed by 1 h desiccation (up to a maximum of four cycles) at room temperature, a reduction in the FCR, germination and tube length after each desiccation treatment was observed. Subsequent hydration fully restored the FCR, but only partially restored germination and tube growth. At 38° C, however, FCR, germination, and tube growth were drastically reduced. The implications of these results on the relationship between FCR and germinability, the responses of pollen exposed to humidity and temperature stress in the field, and on pollen storage are discussed.     

Changes in pollen tube behaviour induced by carbon dioxide and their role in overcoming self-incompatibility in Brassica

Summary Comparative studies on self-pollination after the application of high concentrations of CO₂ gas, which is known to overcome the self-incompatibility reaction in Cruciferous species, have revealed significant changes in the pollen tubes of germinating grains prior to their penetration into the papilla cells. A remarkable increase in the width of the pollen tube was induced by treating the stigmatic papillae with high CO₂ concentrations. The width of the pollen tube appeared to be greatest with CO₂ concentrations ranging from 3% to 5%; these concentrations were also optimal for tube penetration. Callose accumulation was extensively induced in the stigmatic papilla with 10%–20% of CO₂, although a typical callosic reaction remained through the ranges appropriate for blocking self-incompatibility. Observations using the scanning electron microscope (SEM) after pollination revealed that compatible pollen tubes in cross-pollinations fused completely to the papular surface during tube penetration, while in self-pollination, pollen tubes remained on the papilla with some additional diffusate. In the case of CO₂ treatment for self-pollination, some pollen tubes behaved very similarly to the incompatible or compatible ones already described, while others were different from both of them: they showed a complete fusion, similar to compatible ones, with additional diffusate, similar to incompatible ones. These responses of the pollen and stigma to high CO₂ concentrations are discussed with respect to their effect upon the expression of self-incompatibility.     

Temperature effects on self incompatibility in Lilium longiflorum

Summary Detached pistils of the clonal variety, Lilium longiflorum Aral No. 5, were submerged before pollination in 50°C water for 0, 1, 2, 3, 4, 5, 6 or 7 min and then immediately compatibly and incompatibly pollinated. Incompatibility, as indicated by pollen tube length after 48 h at 23.5°C, was eliminated by a 1–2 min submersion while compatibility was removed by a 4–5 min one. The window of incubation temperatures at which incompatible and compatible pollen tubes are clearly differentiated occurred between 15 and 30°C.     

STUDIES ON THE SELF-INCOMPATIBILITY RESPONSE OF LILIUM LONGIFLORUM

Removal of substances loosely associated with the wall of Lilium longiflorum pollen did not affect in vitro germination and tube growth, tube growth within compatible styles, or production of viable seeds. Nor was growth of pollen tubes within incompatible styles enhanced by prior removal of the loosely bound materials. Hence, these materials appear not to be recognition factors in the gametophytic self-incompatibility system of Lilium and to have no role in pollen germination. Heat treatment of selected portions of lily pistils prior to pollination indicated that the incompatibility factor(s) was located in the lower half of the style, and that the stigma plays no role in incompatibility.     

Induced ADH gene expression and enzyme activity in pollinated pistils of Solanum tuberosum

 The regulation of alcohol dehydrogenase (ADH) in relation to in vivo pollen tube growth of Solanum tuberosum was investigated. Adh gene expression as well as ADH enzyme activity were induced in pollinated pistils. The induced ADH isozyme in pollinated pistils is not present in pollen or anthers. The same ADH isozyme is induced in leaves submerged in water. The significance of the induction of ADH activity for pollen tube growth is discussed. Received: 13 November 1996 / Revision accepted: 8 January 1997     

High temperature limits in vivo pollen tube growth rates by altering diurnal carbohydrate balance in field-grown Gossypium hirsutum pistils

It has recently been reported that high temperature slows in vivo pollen tube growth rates in Gossypium hirsutum pistils under field conditions. Although numerous physical and biochemical pollen-pistil interactions are necessary for in vivo pollen tube growth to occur, studies investigating the influence of heat-induced changes in pistil biochemistry on in vivo pollen tube growth rates are lacking. We hypothesized that high temperature would alter diurnal pistil biochemistry and that pollen tube growth rates would be dependent upon the soluble carbohydrate content of the pistil during pollen tube growth. G. hirsutum seeds were sown on different dates to obtain flowers exposed to contrasting ambient temperatures but at the same developmental stage. Diurnal pistil measurements included carbohydrate balance, glutathione reductase (GR; EC 1.8.1.7), soluble protein, superoxide dismutase (SOD; EC 1.15.1.1), NADPH oxidase (NOX; EC 1.6.3.1), adenosine triphosphate (ATP), and water-soluble calcium. Soluble carbohydrate levels in cotton pistils were as much as 67.5% lower under high temperature conditions (34.6 °C maximum air temperature; August 4, 2009) than under cooler conditions (29.9 °C maximum air temperature; August 14, 2009). Regression analysis revealed that pollen tube growth rates were highly correlated with the soluble carbohydrate content of the pistil during pollen tube growth (r² = 0.932). Higher ambient temperature conditions on August 4 increased GR activity in the pistil only during periods not associated with in vivo pollen tube growth; pistil protein content declined earlier in the day under high temperatures; SOD and NOX were unaffected by either sample date or time of day; pistil ATP and water soluble calcium were unaffected by the warmer temperatures. We conclude that moderate heat stress significantly alters diurnal carbohydrate balance in the pistil and suggest that pollen tube growth rate through the style may be limited by soluble carbohydrate supply in the pistil.     

In vitro differentiation of embryogenic pollen,control by cold treatment and embryo formation inab initio pollen cultures ofNicotiana tabacum var. badischer burley

Summary InNicotiana cold treatment causes differentiation of embryogenic pollen. This differentiation initiates on the plant and is completed in culture. Differentiation on the plant results in pollen dimorphism and differentiation in culture leads to pollen embryogenesis. An increased number of pollen capable of embryogenesis is possible on plants induced to flower in short days and low temperature (8 hours light, 18 °C). These embryogenic pollen on selective isolation, from buds at a petal length of 3.4±0.1 cm, fail to form embryos but do so in the cultures which receive cold treatment at 10 °C for 10 days. To some extent the differentiation of embryogenic pollen can be completed on plants induced to flower at 15 °C and embryogenic pollen from such plants form embryos at a low frequency which can be substantially increased on giving the cultures a cold treatment. The frequency of embryogenesis is higher in cultures of 15 °C plants than those of 18 °C plants. Low temperature requirements at two stages—to the plant and to the culture—are essential and complimentary for embryogenesis inab initio pollen cultures.Cold treatment causes repression of gametophytic differentiation and this results in the differentiation of embryogenic potential. The embryogenic pollen, unlike gametophytic pollen, are not fully differentiated structures. They are unable to divide and form embryos in presence of metabolic inhibitors such as actinomycin-D and cycloheximide.     

Okadaic acid causes breakdown of self-incompatibility in Brassica oleracea: evidence for the involvement of protein phosphatases in the incompatible response

Summary Detached pistils from inbred lines of Brassica oleracea L. var alboglabra were fed with okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases, via the transpiration stream. Following self-pollination, pollen tubes were observed to have grown into or through the styles of pistils treated with OA, but not those of untreated controls. Treatment with 1 M OA was sufficient to completely overcome self-incompatibility (SI) in an inbred line homozygous for the S63 allele, though an OA concentration of 5 M was required to cause breakdown of SI in an inbred line homozygous for the S29 allele. At the higher concentration used, pollen tube growth was arrested before the pollen tubes reached the ovary, but this effect was also noted in cross-pollinated styles treated in the same manner. These data provide evidence for the involvement of type 1 and/or type 2A protein phosphatases in the Brassica SI signal transduction mechanism. Present address after November 1993: Department of Biology, Colorado State University, Fort Collins, Colorado, USA     

Temperature dependence of horse heart cytochromec oxidation by membrane preparations ofAnacystis nidulans: Characterization of two distinct arrhenius discontinuities

The oxidation of reduced horse heart cytochromec by membranes isolated from the cyanobacteriumAnacystis nidulans after growth at different temperatures was studied between 4°C and 41°C in the light and the dark using both spectrophotometric and polarographic techniques. Arrhenius plots of the temperature dependence of cytochromec photooxidation showed a single discontinuity at 25°C, 15°C, and 12°C in membranes derived from cells grown at 40°C, 30°C, and 25°C, respectively. By contrast. Arrhenius plots of the temperature dependence of dark respiratory cytochromec oxidation always displayed two distinct breaks at 25 and 18°C, 15 and 8.5°C, and 12 and 5.5°C in membranes isolated from cells grown at 40°C, 30°C, and 25°C, respectively. The results are discussed in terms of the thermotropic lipid-phase transitions known to take place in the membranes ofA. nidulans. Special reference will be made to possibly distinct localizations of the membrane-bound cytochromec oxidase complexes in respiration and photosynthesis.     

Breakdown of self-incompatibility during pistil development in Lycopersicon peruvianum by modified bud pollination

Summary Stylar self-incompatibility barriers in L. peruvianum can be avoided if pollen germination and growth through immature pistils is promoted under specific environmental conditions approximately 2–3 days before the initiation of anthesis. Since immature stigmas lack sufficient exudate for pollen germination, the sandwiching of a thin layer of pollen germination medium between the stigma and a mineral oil layer containing pollen allows precocious pollen germination and some compatible pollen tube growth through the style. This procedure is rapid, inexpensive, applicable in the field, and makes efficient use of pollen. Consistent though low seed yields have been obtained. A high proportion of aborted seed, seedling lethals, and aberrant seedling phenotypes in selfed progeny indicate the presence of strong post-zygotic barriers to such selfing. No evidence for a reduction in the strength of the SI response with increasing pistil age was observed.     

Temperature effects on solute accumulation by Candida utilis

Summary A study was made of the effect of temperature on accumulation of glucosamine and 2-aminoisobutyrate by Candida utilis NCYC 321 grown at 30° C or 10° C. Exponential-phase cells contained greater proportions of C_16:1 and C_18:3 acids, and smaller proportions of C_13:1 and C_18:2 acids, when grown in a defined medium at 10° C compared with 30° C. Cells grown at 30° C or 10° C were able to accumulate extracellular (10 mM) glucosamine and 2-aminoisobutyrate against concentration gradients. 2-Aminoisobutyrate was not metabolised by the cells; glucosamine was accumulated probably as a mixture of glucosamine 1- and 6-phosphates. Rates of accumulation of glucosamine and 2-aminoisobutyrate by cells grown at 30° C or 10° C decreased markedly when the test temperature was decreased from 30° C to 15° C. The rate of accumulation of glucosamine by cells grown at 10° C was considerably lower at each of the test temperatures compared with the corresponding rates for cells grown at 30° C; the rate of accumulation of 2-aminoisobutyrate was much less affected by the temperature at which the cells were grown and then only when measured at temperatures below about 20° C. Apparent K _m values for accumulation of glucosamine by cells grown at 30° C or 10° C decreased considerably when the test temperature was lowered from 20° C to 15° C. The extent of the decrease in K _m value was approximately the same for cells grown at 30° C or 10° C. Apparent K _m values for accumulation of 2-aminoisobutyrate were hardly affected by test temperature. Apparent V _max values for accumulation of glucosamine or 2-aminoisobutyrate were much lower when measured at 15° C than at 30° C. When measured at 30° C, apparent V _max values for accumulation of either solute were slightly lower with cells grown at 10° C compared with cells grown at 30° C; when measured at 15° C, the values were slightly greater with cells grown at 10° C. Net accumulation of glucosamine, at 30° C or 20° C, by cells grown at 30° C or 10° C ceased after 4–6 h. Cells grown at either temperature continued to accumulate 2-aminoisobutyrate at 30° C or 20° C for at least 12 h. The rate of efflux of glucosamine by cells grown at 30° C was slower when measured at 20° C compared with 30° C. With cells grown at 10° C, the rate of efflux at 30° C was slower than with cells grown at 30° C; when measured at 20° C, the rates were about equal. The temperature at which the cells were grown did not affect the ability of d-glucose, d-mannose or d-ribose to compete with d-glucosamine, or with the ability of l-alanine to compete with 2-aminoisobutyrate, when tested at 30° C or 20° C. Cells grown 30° C or 10° C had very similar ATP contents. The results are discussed in relation to the effect of temperature on the rate of solute accumulation by micro-organisms.Abbreviation AIB 2-Aminoisobutyrate     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Longevity and temperature response of pollen as affected by elevated growth temperature and carbon dioxide in peanut and grain sorghum

It is important to understand the effects of environmental conditions during plant growth on longevity and temperature response of pollen. Objectives of this study were to determine the influence of growth temperature and/or carbon dioxide (CO₂) concentration on pollen longevity and temperature response of peanut and grain sorghum pollen. Plants were grown at daytime maximum/nighttime minimum temperatures of 32/22, 36/26, 40/30 and 44/34 °C at ambient (350 μmol mol⁻¹) and at elevated (700 μmol mol⁻¹) CO₂ from emergence to maturity. At flowering, pollen longevity was estimated by measuring in vitro pollen germination at different time intervals after anther dehiscence. Temperature response of pollen was measured by germinating pollen on artificial growth medium at temperatures ranging from 12 to 48 °C in incubators at 4 °C intervals. Elevated growth temperature decreased pollen germination percentage in both crop species. Sorghum pollen had shorter longevity than peanut pollen. There was no influence of CO₂ on pollen longevity. Pollen longevity of sorghum at 36/26 °C was about 2 h shorter than at 32/22 °C. There was no effect of growth temperature or CO₂ on cardinal temperatures (T_min, T_opt, and T_max) of pollen in both crop species. The T_min, T_opt, and T_max identified at different growth temperatures and CO₂ levels were similar at 14.9, 30.1, and 45.6 °C, respectively for peanut pollen. The corresponding values for sorghum pollen were 17.2, 29.4, and 41.7 °C. In conclusion, pollen longevity and pollen germination percentage was decreased by growth at elevated temperature, and pollen developed at elevated temperature and/or elevated CO₂ did not have greater temperature tolerance.     

Pollen-tube behavior and embryo development in interspecific crosses among the genus<Emphasis Type="Italic">Fagopyrum</Emphasis>

Common buckwheat (Fagopyrum esculentum Moench) is an agriculturally and pharmaceutically valuable crop due to its wellbalanced essential amino acids and rutin content. However, global mass production of buckwheat is limited because its genetic self-incompatibility results in low seed sets and poor grain yield. Therefore, this study was conducted to classify the modes of pistil-pollen interaction between species belonging to the genusFagopyrum and to determine the optimal combination of outcrosses for the most successful pollinations. Based on the interaction between pistils and pollen, we classified the modes of pollen tube growth during interspecific crosses ofFagopyrum species into five categories: (i) Highly compatible: normal pollen tube elongation and style penetration within 6~24 hours of pollination, (ii) Slightly compatible: delayed (for 1~6 hours) pollen tube elongation and normal style penetration, (iii) Incompatible type I: pollen tube inhibition at the stigma, (iv) Incompatible type II: pollen tube inhibition at the style, and (v) Incompatible type III: pollen tube inhibition at the stylodium. Based on the observed pollen tube elongation and the following embryo development, highly compatible pollinations were found to be crosses betweenF. esculentum x F. cymosum and betweenF. esculentum (thrum)x F. homotropicum.     

Sperm cells of the pollen tubes of Brassica: Ultrastructure and isolation

Summary Sperm cells of pollen tubes grown both in vivo and in vitro form a male germ unit. Extensions from both sperm cells of each pollen tube are closely associated with the tube nucleus. A high yield (2.7 × 10⁴. 20 mg^–1 pollen grains germinated) of intact sperm cells was obtained following release by osmotic shock from pollen tubes grown in vitro. Structural integrity of isolated sperm was maintained by isolation at low temperature in an osmotically balanced medium. At 4° C many isolated sperm pairs were still enclosed within the pollentube inner plasma membrane. Sperm cells not enclosed within this membrane no longer remained connected as a pair. During isolation vesicles formed on the sperm cell surface from disruption of the fibrillar components bridging the periplasmic space. Both in the pollen tube and after isolation the sperm nucleus is in close association with at least one region of the sperm plasma membrane. Sperm isolated at room temperature showed the presence of nucleopores, and nuclei were euchromatic, instead of heterochromatic as in intact sperm in the pollen tube.     

Androgenesis in isolated pollen cultures ofNicotiana tabacum: Dependence upon pollen development

Summary The influence of the stage of pollen development and of the growth conditions of donor plants on the performance of cultures of isolated pollen fromNicotiana tabacum, var. Badischer Burley has been studied. The method described includes cold treatment (4–5 °C for 3 days) and a pre-culture of the anthers for 7 days at 24 °C before the pollen is isolated. With this system reproducible results were obtained with pollen at the early binucleate stage collected from plants 11–13 weeks old. Another prerequisite for reproducibility is that the donor plants must have been grown for eight weeks in soil with an additional supply of mineral salts. Furthermore, the production of haploids by these pollen cultures was strongly influenced by the photoperiodic and temperature regime experienced by the donor plants; it was best (0.07%) with pollen from short-day plants (8 hours light per day at 18 °C) and rather weak (0.015%) with pollen from long-day plants (16 hours light per day at 24 °C). In contrast to other reports, haploid production from anther cultures was not influenced by the photoperiod or temperature.Cytological studies undertaken at the end of the pre-culture period showed that there were no differences in the percentage of potential embryos for the stages of the late uninucleate, 1. pollen mitosis and early binucleate pollen of long-day plants (1.5%). This value was considerably higher with pollen from short-day plants (7–9%), indicating that short-day conditions at 18 °C of the donor plants are favourable for the induction of androgenesis. However, only the potential embryos formed by the pollen at the initial binucleate stage were able to continue androgenetic development after isolation.     

Pollen selection for low temperature adaptation in tomato

Summary Pollen selection experiments were conducted in tomato to determine the effects of low temperature conditions during pollination on the rate of root elongation of the progeny. Pollen was harvested from an F₁ interspecific hybrid between a high altitude Lycopersicon hirsutum accession and the cultivated tomato L. esculentum. The pollen was applied to stigmas of malesterile L. esculentum plants maintained in growth chambers set at either 12°C/7°C or 24°C/18°C. BC₁ seeds from the low and normal temperature crosses were germinated and root elongation rate was measured at either 9°C or 24°C. At 9°C, the rate of root elongation for progeny of the low temperature crosses was higher than for progeny of crosses at normal temperatures; at 24°C the rate of root elongation was similar for the two crossing treatments. To compare the temperature responses of the two backcross populations we also calculated the relative inhibitory effect of low temperature on the rate of root elongation: the ratio between the rate of root elongation at 9°C to that at 24°C. Root elongation of seedlings from the low temperature crosses was less inhibited by the cold than root elongation for progeny of the normal temperature crosses. These results suggest a relationship between pollen selection at low temperatures and the expression of a sporophytic trait under the same environmental stress.