Ecological and genetic factors contributing to the low frequency of male sterility in Chamaecrista Fasciculata (Fabaceae)

Bumble bee pollinated Chamaecrista fasciculata provides pollen as the sole reward to its pollinators. Male sterility, expressed as an absence or nearly complete absence of pollen production, occurs in low frequency in populations of C. fasciculata. Here we describe experiments, using C. fasciculata, to examine frequently cited determinants of the spread and maintenance of male sterility: compensation and the genetic basis of male sterility. In addition, we examine the role the pollination system plays in determining the reproductive success of the male steriles. Seventeen populations in Maryland, Illinois, and Kansas were surveyed and found to range from 0 to 6% male sterility per population. An artificial population of male-sterile simulants and hermaphrodites was created to examine how the local frequency of nonrewarding male steriles might affect male-sterile female reproductive success. Male steriles performed equally poorly, with respect to seed production, whether surrounded by other male-sterile simulants or hermaphrodites. Compensation was examined by comparison of male steriles and hermaphrodites with respect to several reproductive and nonreproductive characters. Male steriles outperformed hermaphrodites in terms of nonreproductive biomass, but performed equally in terms of ovule number and produced many fewer flowers. The genetic basis of male sterility was examined by performing both intra- and interpopulational crosses of male steriles to hermaphrodites and indicate that male sterility is not purely cytoplasmic. The low frequency of male sterility in C. fasciculata populations may reflect reduced female reproductive success because of pollinator avoidance, lack of reproductive compensation, and a mode of inheritance that is not purely cytoplasmic.     

Reproductive allocation and resource compensation in male-sterile and hermaphroditic plants of Plantago lanceolata (Plantaginaceae)

Gynodioecy is a breeding system in which hermaphrodites coexist with male steriles. Theoretical models predict that without any compensation in female fitness male steriles will disappear from a population due to their reproductive disadvantage. In the present study I investigated whether male-sterile (MS), partially male-sterile (IN), and hermaphroditic (H) plants of Plantago lanceolata differed in reproductive growth and allocation. Offspring of three interpopulation crosses segregating all three sex morphs were grown under nitrogen-limited conditions in a growth chamber. Independent of the genetic background MS plants attained a higher vegetative and reproductive dry mass and a higher reproductive output than H plants, whereas IN plants had intermediate values. When corrected for the mass of the pollen, the dry mass differences between the sex morphs were much reduced but still present. However, when whole-plant allocation was expressed on the basis of nitrogen, the differences between the sex morphs disappeared. Thus the sex morphs took up similar amounts of nitrogen but distributed them differently. The MS and IN plants used the nitrogen saved by not producing pollen for additional vegetative as well as reproductive growth. The data presented in this study suggest that resource compensation is one of the main mechanisms responsible for the maintenance of MS and IN plants in gynodioecious P. lanceolata.     

New CMS types in Plantago lanceolata and their relatedness

 Mitochondrial variation in Plantago lanceolata was used to detect new CMS types. Directional reciprocal crosses were made between plants which differed in mtDNA restriction patterns. Differential segregation of male steriles in reciprocal crosses indicated that the parents differed in CMS type. MtDNA variation revealed nine RFLP patterns, which could be categorised according to the sex phenotype of the plants as MS1 (brown-anther type), MS2 (petaloid flower type) and MS3 (more yellow anthers than MS1). A single mtDNA pattern was found within MS1, six mtDNA patterns were found within the MS2 group, and two mtDNA patterns were found within hermaphrodites which segregated MS3 in the crosses. MS1 and MS2 are known to represent different CMS types, CMSI and CMSII. In reciprocal crosses between plants with different mtDNA patterns within the MS2 group, different ratios of male steriles segregated in the crosses, indicating that the parents differed in CMS type. Within the MS2 group two CMS types were found, designated CMSIIa and b. Finally, the sex phenotype H/MS3 turned out to be a different type. From previous studies it was known that the MS3 phenotype can also occur in CMSI and CMSII types, hence it is unclear whether MS3 is diagnostic for CMSIII. Since the data in this study cannot distinguish between the new type being a fully restored new CMS type or a ‘Normal’ cytoplasm, it was denoted as CMSIII. In total, four CMS types were found in the material studied. CtDNA variation was screened and three chloroplast haplotypes were identified. Two haplotypes were associated with CMSI plants, and one haplotype with the other CMS types. The ctDNA variation indicated that the CMSI type is widespread within the species, due to migration rather than to recurrent mutation. This may lead to the conclusion that only a limited number of CMS types are successful. Received: 9 August 1996 / Accepted: 6 September 1996     

Evidence for effects of restorer genes on male and female reproductive functions of hermaphrodites in the gynodioecious species Thymus vulgaris L.

In Thymus vulgaris L., sex determination involves both the nuclear and the cytoplasmic genomes: the cytoplasm is responsible for male-sterility (the female phenotype) while specific nuclear genes may restore male fertility (the hermaphrodite phenotype). Previous observations have shown high variation among hermaphrodites for pollen and seed production. In order to investigate the origin of this variation, 12 female plants, four from each of three populations, were hand-pollinated with pollen from hermaphrodites from three different paternal populations. The sex-ratio (i.e. the frequency of hermaphrodites) produced and the reproductive functions of these offspring were measured. A strong positive correlation was observed between the sex-ratio within a family and both female and male reproductive functions of its hermaphrodites. No such correlation was found for females. This result suggests that restorer genes may be directly or indirectly involved both in sex determination and in the efficiency of resource allocation to reproductive functions. As a consequence, female advantage, i.e. the relative fecundity of females to hermaphrodites, is larger in families with low sex-ratio, and this might affect the evolution of this gynodioecious breeding system.     

Possible role of adenylates in the engagement of the cyanideresistant pathway in nutrient-starved Catharanthus roseus Cells

In Cathuranthus roseus (L.) G. Don cells the cyanide-resistant pathway is engaged after phosphate or nitrogen starvation. Re-addition of these nutrients disengaged it again. Re-addition of phosphate leads to a transient disengagement which becomes only permanent after a second addition of phosphate. Disengagement after re-addition of nitrogen is slow: it takes 9 days before the activity has disappeared. In this system the mechanism of engagement of the cyanide-resistant pathway was studied. Addition of phosphate to phosphate-starved cells induced cell division within 24 h. The disengagement of the cyanide-resistant pathway was probably only an indirect effect of phosphate because the cellular P, content, which increased rapidly after addition, was low again before the cyanide-resistant pathway was disengaged. A better correlation was observed between high ADP and adenylate content of the cells and disengagement of the cyanide-resistant pathway. In addition it appeared that the engagement of the cyanide-resistant pathway was not the result of a limited carrier capacity of the cytochrome pathway. It is tentatively concluded that the engagement of the cyanide-resistant pathway in phosphate-starved cells was the result of a limited adenylate content. After nitrogen addition to N-starved cells, it took 5 days until the first growth occurred. Before the cyanide-resistant pathway was disengaged, its activity increased with the increased respiration rate which preceded growth. Within 72 h a higher ADP content was observed, which was still high after 10 days. The stimulation of the cytochrome pathway by uncoupler was small and more or less the same with and without added nitrogen, as long as the cyanide-resistant pathway was engaged. After disengagement the stimulation by uncoupler was significantly larger. It is suggested that the engagement during N-starvation was the result of a limited carrier capacity of the cytochrome pathway. Stimulation of the metabolism by re-addition of phosphate, nitrogen or sucrose resulted in a rapid increase in the levels of uracil nucleotides and uridine diphosphoglucose (UDPG) which are involved in sucrose metabolism.     

THE EFFECTS OF POPULATION SIZE AND PLANT DENSITY ON OUTCROSSING RATES IN LOCALLY ENDANGERED SALVIA PRATENSIS

Multilocus outcrossing rates were estimated in natural and experimental populations of Salvia pratensis, an entomophilous, gynodioecious, protandrous perennial. Male steriles were used to check the estimation procedure of outcrossing rates in hermaphrodites. Estimates of outcrossing rates in hermaphroditic plants ranged from 38.2% to 81.8% in natural populations and from 71.5% to 95.5% in experimental populations. No correlations were found between outcrossing rates and population size. However, outcrossing in hermaphrodites was promoted by high plant densities and low frequencies of male steriles. It is argued that effective management to preserve genetic variation in populations of S. pratensis should provide for the maintenance of high plant densities.     

Sex allocation in a simultaneously hermaphroditic marine shrimp

Two fundamental questions dealing with simultaneous hermaphrodites are how resources are optimally allocated to the male and female function and what conditions determine shifts in optimal sex allocation with age or size. In this study, I explored multiple factors that theoretically affect fitness gain curves (that depict the relationship between sex-specific investment and fitness gains) to predict and test the overall and size-dependent sex allocation in a simultaneously hermaphroditic brooding shrimp with an early male phase. In Lysmata wurdemanni, sperm competition is absent as hermaphrodites reproducing in the female role invariably mated only once with a single other shrimp. Shrimps acting as females preferred small over large shrimps as male mating partners, male mating ability was greater for small compared to large hermaphrodites, and adolescent males were predominant in the population during the breeding season. In addition, brooding constraints were not severe and varied linearly with body size whereas the ability to acquire resources increased markedly with body size. Using sex allocation theory as a framework, the findings above permitted to infer the shape of the male and female fitness gain curves for the hermaphrodites. The absence of sperm competition and the almost unconstrained brooding capacity imply that both curves saturate, however the male curve levels off much more quickly than the female curve with increasing level of investment. In turn, the predominance of adolescent males in the population implies that the absolute gain of the female curve is greater than that of the male curve. Last, the size-dependent female preference and male mating ability of hermaphrodites determines that the absolute gain of the male curve is greater for small than for large hermaphrodites. Taking into consideration the inferred shape of the fitness gain curves, two predictions with respect to the optimal sex allocation were formulated. First, overall sex allocation should be female biased; it permits hermaphrodites to profit from the female function that provides a greater fitness return than the male function. Second, sex allocation should be size-dependent with smaller hermaphrodites allocating more than proportionally resources to male reproduction than larger ones. This size-dependent sex allocation permits hermaphrodites to profit from male mating opportunities that are the greatest at small body sizes. Size-dependent sex allocation is also expected because the male fitness gain curve decelerates more quickly than the female gain curve and experiments indicated that resources are greater for large than small hermaphrodites. These two predictions were tested when determining the sex allocation of hermaphrodites by dissecting their gonad and quantifying ovaries versus testes mass. Supporting the predictions above, hermaphrodites allocated, on average, 118 times more to the female than to the male gonad and the proportion of resources devoted to male function was higher in small than in large hermaphrodites. A trade-off between male and female allocation is assumed by theory but no negative correlation between male and female reproductive investment was observed. In L. wurdemanni, the relationship between sex-specific investment and fitness changes during ontogeny in a way that is consistent with an adjustment of sex allocation to improve size-specific reproductive success.     

The Dynamics of Gynodioecy in Plantago Lanceolata L. II. Mode of Action and Frequencies of Restorer Alleles

Male fertility in Plantago lanceolata is controlled by the interaction of cytoplasmic and nuclear genes. Different cytoplasmic male sterility (CMS) types can be either male sterile or hermaphrodite, depending on the presence of nuclear restorer alleles. In three CMS types of P. lanceolata (CMSI, CMSIIa, and CMSIIb) the number of loci involved in male fertility restoration was determined. In each CMS type, male fertility was restored by multiple genes with either dominant or recessive action and capable either of restoring male fertility independently or in interaction with each other (epistasis). Restorer allele frequencies for CMSI, CMSIIa and CMSIIb were determined by crossing hermaphrodites with ``standard' male steriles. Segregation of male steriles vs. non-male steriles was used to estimate overall restorer allele frequency. The frequency of restorer alleles was different for the CMS types: restorer alleles for CMSI were less frequent than for CMSIIa and CMSIIb. On the basis of the frequencies of male steriles and the CMS types an ``expected' restorer allele frequency could be calculated. The correlation between estimated and expected restorer allele frequency was significant.     

Effects of CMS types and restorer alleles on plant performance in Plantago lanceolata L.: an indication for cost of restoration

In gynodioecious species, male steriles co-occur with hermaphrodites. Usually, the male sterile trait is maternally inherited, hence it is called Cytoplasmic Male Sterility (CMS). Nuclear loci restore male fertility in combination with their ‘own’ specific cytoplasmic types. In theory, two fitness components are important for the maintenance of this breeding system: a fitness advantage of the male steriles, and costs of restoration. The costs of restoration are alleged negative pleiotropic effects of restorer alleles. In this study the effects of different CMS types on plant performance and the cost of restoration were assessed in two experiments with Plantago lanceolata L. Biomass production differed significantly between the CMS types studied. In order to assess the costs of restoration, hermaphrodites with or without restorer alleles for a CMS type other than its own were compared. The studied restorer alleles caused a reduction in weight per seed, but the number of seeds produced was unaffected. The estimated cost of restoration measured as reduction of seed biomass was 13% for restorer alleles for CMSI. However, in the second experiment no pleiotropic effects of restorer alleles were detected, either because the assumptions for the experimental set-up were not valid or the costs of restoration may not always be expressed.     

Mitochondrial activity during shoot formation and growth in tobacco callus

Mitochondria isolated from tobacco ( Nicotiana tabacum L. cv. Wisconsin 38) callus growing on either shoot-forming or non-shoot forming medium show an increase in state 3 and state 4 respiration and a drop in respiratory control and ADP/O ratios after subculture. the protein content of the mitochondria fraction and the activity of succinate dehydrogenase, malate dehydrogenase, cytochrome c oxidase and catalase also increase after subculture but there is no apparent difference between shoot-forming and non-shoot-forming tissue. For mitochondria assayed at their native osmolarities, a trend of higher respiration rates and respiratory control as well as lower levels of cyanide-resistant respiration was observed for shoot-forming tissue. Generally, differences were greatest after day 9 in culture, the time during which primordia formation occurred in the shoot-forming callus. These patterns are in concert with the view that the shoot-forming process has a high energy requirement which must be realized during the time of primordia formation.     

铵态氮和硝态氮营养与大豆幼苗的抗氰呼吸作用

在植物体内,NO云还原成NHI是一个耗能很多的生化过程,每还原一个NO。大约要消耗15个ATP分子（Salsac等1987）。植物直接吸收和利用NHi＋,可以减少能量消耗,所节约的能量可用于植物的生长。因此,从理论上讲,供NHI的植株要比供NOt的植株能够获得更高的生物产量。然而,对于大多数植物,供NOS的植株常具有更大的生长量和产量。对于这种现象,过去人们一直在矿物质和有机物积累的差异（Robin和Salsac1985）、根际的酸化程度（Ruftv等1983,Tolley－Henry和Rapen1986）、NHt有害浓度的积累（Haynes和Goh1978）、对光合作用的…     

Cyclosporin biosynthesis and dynamics of bioenergetic processes in cyclosporin-producing strain

An attempt was made to show a correlation between definite bioenergetic parameters of the cells of the cyclosporine-producing culture and biosynthesis of cyclosporine. It was found that the three strains producing cyclosporine used in the study had an alternative cyanide-resistant pathway along with the classical cytochrome chain. In the strain forming only traces of the cyclopeptide during fermentation of the cyanide-resistant respiration constituted 60 to 80%. In the isogenic highly productive strains the cyanide-resistant respiration appeared to be markedly decreased beginning from the 1st day of fermentation and during the maximum biosynthesis of cyclosporine (on day 4 or 5 of fermentation) it reached zero. The ATP content in the cells of the highly productive strain, despite its decrease by the antibiotic biosynthesis peak, remained at a much higher level than that in the strain producing only traces of cyclosporine. A procedure for isolating functionally active mitochondria from the protoplasts was developed and a bioenergetic characterization of the mitochondria isolated from the strains with different antibiotic productions is presented.     

In suspension cultures of Catharanthus roseus the cyanide-resistant pathway is engaged in respiration by excess sugar in combination with phosphate or nitrogen starvation

Suspension cultured cells of Catharanthus roseus were used to study the effects of excess sugar, and phosphate or nitrogen starvation on growth and respiration. Excess sugar resulted in a higher dry weight but the cell number was not significantly higher than in the controls. After inoculation in phosphate-free medium almost no growth was observed, whereas in medium without nitrogen some growth still occurred, indicating that the medium contains more than enough nitrogen and only a limited amount of phosphate. The maintenance respiration in the nitrogen-free medium was lower than in the control, probably as a result of a lower protein production. In phosphate-free medium, however, the maintenance respiration was higher, because the cyanide-resistant pathway was engaged and the P/O ratio was decreased. In Catharanthus roseus cells the cyanide-resistant pathway is engaged upon phosphate or nitrogen starvation in combination with excess sugar, while excess sugar, as such, does not engage the cyanide-resistant pathway. In the presence of excess sugar the rate of glycolysis is adenylate-controlled. It is only in combination with mineral starvation that the rate of glycolysis does not match the capacity of the cytochrome pathway, resulting in an overflow via the alternative pathway. After combination of mineral starvation with a low sugar supply the rate of glycolysis is controlled by both the substrate supply and the adenylates, and the cyanide-resistant pathway is not engaged.     

The relationship between phosphate status and cyanide-resistant respiration in bean roots

Bean ( Phaseolus vulgaris L.) seedlings were cultured on complete or phosphate-deficient nutrient medium. After 14 days of culture on phosphate-deficient medium the visible symptoms of P_i deficiency were observed only in the shoot, the fresh and dry weights of the roots were slightly higher than in control plants. The decreased P_i content in the roots had little effect on total respiration rate but had an effect on the level of inhibition of respiration by cyanide. The high resistance of respiration to cyanide observed in P_i-deficient roots was the result of the suppression of cytochrome path activity and an increased participation of the alternative, cyanide-resistant pathway. The cytochrome pathway activity increased when inorganic phosphate was supplied to P_i-deficient roots for 1 or 3.5 h. It is speculated that the suppression of cytochrome pathway in P_i-deficient roots may result from restriction of the phosphorylating capacity or a partial inhibition of cytochrome oxidase activity.     

Self-Incompatibility and Male Fertilization Success in Phillyrea angustifolia (Oleaceae)

Androdioecy is a rare breeding system in which low male frequency is expected in populations because males require a strong increase in their fertility to be maintained by selection. Phillyrea angustifolia L. has previously been reported as possibly functionally androdioecious. However, 1&rcolon;1 sex ratios have been reported and suggest functional dioecy. In this article, we compared both pollen tube growth and siring success of male and hermaphrodite pollen in two single-donor pollination experiments. We verified at both pre- and postzygotic levels that hermaphrodites produce functional pollen. Self-incompatibility was also clearly established. However, pollen from hermaphrodites was less efficient than male pollen. The probability of a pollen tube growing through the style was higher for male than for hermaphrodite pollen donors, and males sired twice as many fruits as hermaphrodites. The twofold male advantage in relative fecundity was mainly because of lower pollen fertility of hermaphrodites and possible cross-incompatibility among hermaphrodites.     

干旱胁迫对小麦幼苗抗氰呼吸和活性氧代谢的影响

研究了干旱胁迫对抗旱性强弱不同的两种小麦幼苗的抗氰呼吸和活性氧代谢的影响。干旱胁迫导致了两种小麦抗氰呼吸活性及基因转录水平的下降,但抗旱品种在轻度干旱胁迫下表现出一定的适应能力,其抗氰呼吸活性及基因转录水平均高于不抗旱品种。干旱胁迫下,对干旱敏感的小麦幼苗叶片中活性氧含量高于抗旱小麦;3种抗氧化酶的活性低于抗旱小麦的3种抗氧化酶的活性。据此认为,严重的干旱胁迫引起活性氧含量的增加扰动了活性氧与抗氰呼吸之间的应答平衡,但抗氰呼吸可能通过清除活性氧等机制而起了抗旱的作用。     

Photosynthetic characteristics of leaves of male-sterile and hermaphrodite sex types of Plantago lanceolata grown under conditions of contrasting nitrogen and light availabilities

Plantago lanceolata is a gynodioecious species: In natural populations male steriles (MS) coexist with hermaphrodites (H). Since male steriles have a reproductive disadvantage, without any compensation for their loss in male function by an increase in female function, they are expected to disappear from the population. In this study we investigated the possibility that differences in ecologically important photosynthetic characteristics, between MS and H lines of P. lanceolata. play a role in maintaining gynodioecy. One MS line and two H lines were grown under conditions of high N and light availability, as well as under either N limitation or light limitation, to investigate whether the sex types respond differently to environmental constraints. Photosynthetic light-response and CO₂-response curves were made, together with leaf organic N and chlorophyll determinations. There were only few small differences between the lines and since the MS line did not differ in any of the determined photosynthetic characteristics from either H line, it is unlikely that these differences are involved in maintaining male sterility in populations of P. lanceolata. The low-light-grown plants showed a high degree of acclimation as shown by a two-fold higher leaf area to leaf weight ratio (SLA), a two-fold higher investment of N in light harvesting, and higher net photosynthetic rates under low-light conditions, as compared to the high-light-grown plants. The low-N-grown plants used their organic N more efficiently in photosynthesis compared to plants grown at an optimal N supply. This was mainly due to the N-limited plants having leaves with a lower organic N content and thus lower photosynthetic capacities. To a lesser extent it was due to the higher value for the curvature factor of the light-response curves of the N-limited plants, to their decreased rates of photorespiration and possibly to their relatively higher allocation of organic N to photosynthetic functions.     

On the absence of correlation between cyanide-resistant respiration and cytochrome d content in bacteria

The regularity of appearance of cyanide-resistant respiration and cytochrome d in various bacteria as well as the relationship between the degree of resistance of respiration to cyanide and cytochrome d content was studied. Bacteria able to synthesize cyanide-resistant respiration were shown to appear during transition of culture to the stationary phase of growth caused by the exhaustion of carbon source. No regulatory of appearance of cytochrome d was observed. There is no correlation between the degree of resistance to cyanide and cytochrome d content. It was concluded that the cyanide-resistant respiration of bacteria and eukaryotic microorganisms may be associated with the functioning of a non-cytochrome nature oxidase.     

Possible role of a 36 kDa protein induced by respiratory inhibitors in cyanide-resistant respiration in Hansenula anomala

The antimycin A or cyanide-dependent appearance of a 36 kDa protein in the particulate fraction was observed in L-[35S]methionine pulse-labeling experiments on cells of Hansenula anomala, in which cyanide-resistant respiration was induced. The combined addition of cycloheximide or anaerobiosis, which block the induction of cyanide-resistant respiration, repressed the synthesis of this protein. These results suggest the involvement of the particulate 36 kDa protein in cyanide-resistant respiration.     

MICROSPOROGENESIS IN MALE-STERILE AND HERMAPHRODITIC PLANTS OF NINE GYNODIOECIOUS TAXA OF HAWAIIAN BIDENS (ASTERACEAE)

Microsporogenesis was investigated in hermaphroditic and male-sterile plants in nine gynodioecious taxa of Hawaiian Bidens. Normal microsporogenesis in hermaphrodites and the onset of abortion in male steriles were similar in all taxa and in a hybrid between two gynodioecious species. The early abnormal vacuolation of tapetal cells is the first visible evidence leading to premeiotic abortion of microsporogenesis in male steriles. The sporogenous cells disintegrate rapidly after the vacuolation of the tapetum, resulting in a shrunken, indehiscent anther which is composed of only the epidermal layer with some remnant cells of the endothecium and the connective at anthesis. In hermaphrodites, the tapetal cells remain dense and undergo karyokinesis to become binucleate during meiosis I. The tapetum becomes plasmodial after microspores are released from tetrads and gradually disappears during pollen formation. The genetic factor(s) which cause the abortion act with remarkable precision and consistency in all taxa investigated. This suggests that gynodioecy in all Hawaiian Bidens is homologous and the establishment of male sterility in Hawaiian Bidens occurred only once. The spread of the genetic male-sterile factor(s) may be the result of adaptive radiation of the original gynodioecious species or natural interspecific hybridization.