Programmed cell death promotes male sterility in the functional dioecious Opuntia stenopetala (Cactaceae)

Background and Aims

The sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described.

Methods

The spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD.

Key results

PCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption.

Conclusions

Temporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species.     

The effect of ultraviolet-depleted light on the flavonol contents of the cactus species Opuntia wilcoxii and Opuntia violacea

An early investigation at the Biosphere-2 Laboratory, an artificial ecosystem in the Arizona desert, had shown that the flavonoid content of cacti grown in glass-filtered solar light was lower than of cacti grown in normal solar light. This was attributed to the absence of ultraviolet (UV) radiation, which is required for flavonoid biosynthesis. In this study, two species of Opuntia cacti were grown in solar and UV-depleted light, and their flavonol contents of different tissues were determined by HPLC. O. wilcoxii, previously raised in the absence of UV light, was exposed to normal solar light. The flavonol content of young O. wilcoxii pads was 28-fold higher when grown in solar light as compared to UV-depleted light. The flavonol contents of mature outer tissues were only slightly higher. O. violacea, previously raised in solar light, was also maintained in the same UV-depleted artificial ecosystem. The flavonol content after hydrolysis of outer tissues was similar, whether grown in solar light or UV-depleted light. We attribute these responses to different biosynthetic and metabolic rates of young vs. mature plant tissues; slow-growing mature tissues neither produce nor metabolize compounds as quickly as immature tissues. These findings indicate that artificial ecosystems can influence the production of natural products in cultivated plants.     

Role of auxin in regulating Arabidopsis flower development

To elucidate the role of auxin in flower morphogenesis, its distribution patterns were studied during flower development in Arabidopsis thaliana (L.) Heynh. Expression of DR5::GUS was regarded to reflect sites of free auxin, while immunolocalization with auxin polyclonal antibodies visualized conjugated auxin distribution. The youngest flower bud was loaded with conjugated auxin. During development, the apparent concentration of free auxin increased in gradual patterns starting at the floral-organ tip. Anthers are major sites of high concentrations of free auxin that retard the development of neighboring floral organs in both the acropetal and basipetal directions. The IAA-producing anthers synchronize flower development by retarding petal development and nectary gland activity almost up to anthesis. Tapetum cells of young anthers contain free IAA which accumulates in pollen grains, suggesting that auxin promotes pollen-tube growth towards the ovules. High amounts of free auxin in the stigma induce a wide xylem fan immediately beneath it. After fertilization, the developing embryos and seeds show elevated concentrations of auxin, which establish their axial polarity. This developmental pattern of auxin production during floral-bud development suggests that young organs which produce high concentrations of free IAA inhibit or retard organ-primordium initiation and development at the shoot tip. Electronic Supplementary Material Supplementary material is available for this article at This paper is dedicated to Orna Aloni for continuous support and management over many years.     

Activities of auxin polar transport in inflorescence axes of flower mutants ofArabidopsis thaliana: Relevance to flower formation and growth

Relationships between the activity of auxin polar transport and flower formation were studied using several flower mutants ofArabidopsis thaliana. The activity of auxin polar transport in the upper portion of inflorescence axis of wildtype plants ofArabidopsis thaliana was significantly lower than that of the basal part. The activities of auxin polar transport in the upper portion of inflorescence axes ofap1 andclv1 mutants were significantly higher than that of wild-type plant. However, those of other flower mutants tested,ap3-1, ag, pi, Fl-40, Fl-54, Fl-89 andpin-formed, were extremely low as compared with that of wild one. We got some evidence that the reduction of the activity of auxin polar transport is concerned with the growth and development of plants. We could mimic it by the removal of all flowers and pods including mature or immature seeds. Moreover, artificial pollination inap3-1 andpi mutants, in which no seeds are found naturally, resulted in the partial recovery of the activity of auxin polar transport in inflorescence axis. Considering these results in this study together with the fact that inhibitors of auxin polar transport generated almost same disruptions ofpin-formed orpinoid mutants which normally had no flowers in inflorescence axis (Okadaet al. 1991, Uedaet al. 1992, Bennettet al. 1995), the systern of auxin polar transport and its activity in inflorescence axis seems to be essential for the development of flower bud in early stage ofArabidopsis thaliana, and the activity of auxin polar transport is also regulated by the formation of flowers and seeds in inflorescence axis.     

Local auxin production: a small contribution to a big field

Auxin is a plant growth regulator involved in diverse fundamental developmental responses. Much is now known about auxin transport, via influx and efflux carriers, and about auxin perception and its role in gene regulation. Many developmental processes are dependent on peaks of auxin concentration and, to date, attention has been directed at the role of polar auxin transport in generating and maintaining auxin gradients. However, surprisingly little attention has focussed on the role and significance of auxin biosynthesis, which should be expected to contribute to active auxin pools. Recent reports on the function of the YUCCA flavin monooxygenases and a tryptophan aminotransferase in Arabidopsis have caused us to look again at the importance of local biosynthesis in developmental processes. Many alternative and redundant pathways of auxin synthesis exist in many plants and it is emerging that they may function in response to environmental cues.     

The xanthophyll cycle and energy dissipation in differently oriented faces of the cactus Opuntia macrorhiza

  Diurnal changes in titratable acidity, photosynthesis, energy dissipation activity, and the carotenoid composition of differently oriented cladodes of the cactus Opuntia macrorhiza were characterized during exposure to full sunlight in the field. Four cladode faces were chosen such that each was exposed to maximum photon flux densities (PFD) at different times of the day in addition to receiving different daily integrated PFDs. The sum of all carotenoids per chlorophyll was found to increase with increasing exposure to PFD, with the carotenoids of the xanthophyll cycle present in the most exposed face at more than twice the concentration found in the least exposed face. All faces exhibited large increases in xanthophyll cycle-dependent energy dissipation as the sun rose in the morning, even those receiving only minimal levels of diffuse radiation. The transient high levels of energy dissipation in those faces that did not receive direct sunlight in the morning may have been due to low temperature inhibition of photosynthesis (predawn low of 2°C). For the two faces receiving peak PFDs in the morning hours (north and east faces), the level of energy dissipation activity increased rapidly during exposure to direct sunlight in the early morning, gradually declining in the late morning under warm temperatures, and was negligible during the afternoon low light conditions. Changes in the xanthophyll cycle paralleled the changes in energy dissipation with the majority of the cycle present as violaxanthin (V) prior to sunrise, largely de-epoxidized to zeaxanthin (Z) and antheraxanthin (A) during exposure to direct sunlight, and reconverted to V during the afternoon. For the two faces receiving peak PFDs in the afternoon (south and west faces), energy dissipation activity increased dramatically during the early morning low light period, subsequently decreasing during midday as decarboxylation of malic acid proceeded maximally (providing a high concentration of CO₂ for photosynthesis), and then increased to the highest level in the late afternoon as the supply of malic acid was depleted and rates of photosynthetic electron transport declined. The xanthophyll cycle, largely present as Z and A prior to sunrise in the south and west faces, was de-epoxidized to the greatest extent in the late afternoon, followed by epoxidation back to the predawn level by sunset. In all cladode faces high levels of energy dissipation activity were accompanied by decreases in the intrinsic efficiency of photosystem II (PSII), indicative of a regulatory process that diverted the excess energy away from the reaction centers during periods of excess light. Furthermore, the overnight retention of Z and A by the south and west faces was accompanied by a sustained reduction in PSII efficiency (i.e., “photoinhibition”). We suggest that this “photoinhibition” represents the sustained engagement of nocturnally retained Z and A in the photoprotective down-regulation of PSII. Received: 8 May 1996 / Accepted: 9 September 1996     

Seed bank dynamics of the desert cactus Opuntia rastrera in two habitats from the Chihuahuan Desert

In desert environments the main input to the seed bank of many succulents is the seed rain through zoochory while high levels of granivory by rodents, birds and ants are the main cause of subsequent losses. In the patchy environment of arid lands the characteristics of both processes may vary between habitats causing differences in the recruitment of new genets. To test this hypothesis we used populations of the desert cactus Opuntia rastrera which has different recruitment rates in the two adjacent habitats where it grows. In Opuntia-dominated scrublands (nopaleras, density ca. 4,000 plants/ha) 1 seedling out of 7,000,000 seeds establish, whereas in grasslands (density ca. 100 plants/ha) this ratio is 1:20,000. From 1996 until 1998 the seed rain, seed removal by granivores and seed abundance in the soil were monitored in both habitats. Results showed striking differences in the dynamics of the seed bank of both habitats. Seed rain was 8.5 times bigger in nopaleras than in grasslands. In nopaleras most seeds were removed by rodents while the quantities of seeds removed by rodents, birds and ants in grasslands were similar. One year after dispersal (the time necessary to break seed dormancy) only 6% of original nopalera seeds and 12% of grassland seeds remained. After germination trials only 1% (ca. 15,000 seeds/ha) and 2% (ca. 2,500 seeds/ha) respectively were viable. These differences in the effective seed bank (6 times bigger in nopaleras) can not explain the differences in genet recruitment (which is several orders of magnitude bigger in grasslands). Apparently the between habitat difference in nurse plant availability and in rodent density (which inflict a strong hervibory upon seedlings) can explain the differences in genet recruitment. It is speculated that this between habitat difference in genet recruitment suggests that the species evolved in less extreme environments (e.g. grasslands) than desert scrublands which, in turn, are colonised due to the singular ability of O. rastrera for vegetative propagation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Seasonal patterns of acid fluctuations and resource storage in the arborescent cactus Opuntia excelsa in relation to light availability and size

Summary We investigated relationships between light availability, diel acid fluctuation, and resource storage in the arborescent cactus Opuntia excelsa growing in western Mexico. We compared canopy and understory individuals from a deciduous forest as well as open-grown plants of the same approximate size as those in the understory. During the wet season light availability and daily fluctuations in titratable acidity (an index of carbon uptake) were lower in the understory than in unshaded habitats. In the dry season all plants had reduced levels of acid fluctuation, with the smallest individuals, regardless of habitat, showing the greatest reduction. These data suggest that light availability in the forest understory constrains carbon assimilation during the wet season, but that a factor associated with plant size, possibly water status, limits carbon gain during the dry season. Plants in all habitats remained physiologically active for at least five months into the dry season. We suggest that this was possible due to the maintenance of constant concentrations of water and nitrogen in the photosynthetically active chlorenchyma. Parenchyma in terminal cladodes showed a different seasonal pattern of resource storage; water content and nitrogen concentration were reduced from the wet to the dry season in the parenchyma. Using the parenchyma to supply photosynthetic tissues during times of reduced resource availability allows O. excelsa to assimilate carbon during times of the year when most other trees in the forest are leafless.     

胭脂虫寄生对仙人掌生长的影响

从印榕仙人掌(Opuntia ficus-indica Mill)植株高度、新增茎片数量和新增茎片面积3个方面研究不同密度胭脂虫Dactylopius coccus Costa寄生对仙人掌生长量的影响。结果表明:1)胭脂虫寄生对寄主仙人掌有明显的抑制作用,未被寄生的仙人掌株高、茎片数量和茎片面积的增加均明显大于被寄生的仙人掌;2)随着胭脂虫寄生密度的增加,仙人掌植株生长量的增加相应减少,生长高峰期的出现也会相应推迟;3)胭脂虫的寄生密度应控制在1000头/株以下,过大会导致仙人掌的死亡。     

The origins of an important cactus crop, Opuntia ficus-indica (Cactaceae): new molecular evidence

Opuntia ficus-indica is a long-domesticated cactus crop that is important in agricultural economies throughout arid and semiarid parts of the world. The biogeographic and evolutionary origins of this species have been obscured through ancient and widespread cultivation and naturalization. The origin of O. ficus-indica is investigated through the use of Bayesian phylogenetic analyses of nrITS DNA sequences. These analyses support the following hypotheses: that O. ficus-indica is a close relative of a group of arborescent, fleshy-fruited prickly pears from central and southern Mexico; that the center of domestication for this species is in central Mexico; and that the taxonomic concept of O. ficus-indica may include clones derived from multiple lineages and therefore be polyphyletic.     

Pollinator responses to variation in floral display and flower size in dioecious Sagittaria latifolia (Alismataceae)

In animal-pollinated plants with unisexual flowers, sexual dimorphism in floral traits may be the consequence of pollinator-mediated selection. Experimental investigations of the effects of variation in flower size and floral display on pollinator visitation can provide insights into the evolution of floral dimorphism in dioecious plants. Here, we investigated pollinator responses to experimental arrays of dioecious Sagittaria latifolia in which we manipulated floral display and flower size. We also examined whether there were changes in pollinator visitation with increasing dimorphism in flower size. In S. latifolia, males have larger flowers and smaller floral displays than females. Visitation by pollinators, mainly flies and bees, was more frequent for male than for female inflorescences and increased with increasing flower size, regardless of sex. The number of insect visits per flower decreased with increasing floral display in males but remained constant in females. Greater sexual dimorphism in flower size increased visits to male inflorescences but had no influence on the number of visits to female inflorescences. These results suggest that larger flower sizes would be advantageous to both females and males, and no evidence was found that females suffer from increased flower-size dimorphism. Small daily floral displays may benefit males by allowing extended flowering periods and greater opportunities for effective pollen dispersal.     

Isolation and characterization of polymorphic microsatellite markers in Galapagos prickly pear (Opuntia) cactus species

The Opuntia (prickly pear) genus contains over 200 species. Six of them are endemic to the Galapagos archipelago. Although these cacti are ‘keystone’ species of the Galapagos’ semi‐arid ecosystem, they have never been studied in detail. Because of their current threatened status and their important role in the ecosystem, we developed 16 microsatellite markers to study the population genetic structure of some of these species. These markers display a high level of polymorphism with numbers of alleles per locus ranging from six to 53. Results also revealed possible polyploidy in these cacti.     

The renowned cactus moth, Cactoblastis cactorum: its natural history and threat to native Opuntia floras in Mexico and the United States of America

Abstract. The cactus moth, Cactoblastis cactorum (Berg) (Phycitidae) is native to South America. It was released as a biological control agent against alien Opuntia- cacti in Australia in the 1920s, then in southern Africa, and latterly on several islands, including those in the Caribbean. In 1989, the cactus moth was discovered in Florida, in the United States of America, where it is now threatening the survival of indigenous Opuntia species. In this paper we identify some of the attributes that have contributed to the success of C. cactorum as a weed biological control agent. Many of these same qualities account for the problems that C. cactorum has caused in Florida and predispose it as a major threat to the speciose, native Opuntia- floras of Central and North America. An estimated 79 platyopuntia (prickly pear) species are at risk: 51 species endemic to Mexico; nine species endemic to the United States; and 19 species common to both countries. Many cultivated and wild Opuntia species, that are used in various ways, are also vulnerable to attack by C. cactorum , including at least 25 species in Mexico and three species in the United States, particularly the widely exploited and culturally important cultivars of O. ficus-indica . Some control strategies are suggested that may minimize the risk and consequences of invasion by the cactus moth. The wider implications of this threat to the practice of weed biological control and to conservation are discussed.     

Analysis of the community structure of yeasts associated with the decaying stems of cactus. II.Opuntia species

A survey was made of yeast species associated with the decaying pads of 3 prickly pear cacti (Opuntia phaeacantha, O. ficus-indica, andO. lindheimeri) in Arizona and Texas. Yeast communities from 12 localities were compared among localities, amongOpuntia species, and with previous data on yeast communities associated with columnar cacti. The results indicate thatOpuntia necroses contain relatively more yeast species with broader physiological abilities in their communities than columnar necroses. It is argued that differences in chemistry of the opuntias and columnar forms in concert with the insect vectors specific for these cacti account for the differences in yeast community structure. It is further hypothesized that the differences in yeast community structure have been important in the evolution and maintenance of species diversity forDrosophila species which live in the decaying stems or cladodes of various cacti. Most of the yeast community evolution in the cacti is postulated to have proceeded by evolution in situ and not by additions and replacements from outside of the system.     

The allometry of flower size dimorphism in dioecious Dombeya species on La Réunion

In this paper we examine whether variation in male and female flower size follows an allometric relation for 10 dioecious species in the genus Dombeya endemic to La Réunion. Male flowers are significantly larger than female flowers and the degree of dimorphism varies significantly across species. There is a significant allometric relationship between male and female flower size: as mean flower size decreases the degree of flower size dimorphism increases. This is the first study to document an allometric relationship between male and female flower size in unisexual plants. We discuss this allometric relationship in relation to two characteristics frequently observed in dioecious flowers: flower size dimorphism is common in unisexual plants and dioecious species often have smaller flowers than hermaphrodites.     

Crossed graft of embryonic testis between quail and chick embryos: contribution to the study of the mechanism of female bird embryo masculinization]

Quail, or chick embryonic testes grafted respectively in the extraembryonic coelom of chick or quail embryos induce both a Mullerian duct regression and a masculinization of the female host gonads up to the differentiation of two testes, in some cases. Such a result confirms the fact evidenced previously in other bird species (chick and duck) that the testis-inducer is interspecific. Quail cells are not observed in histological sections of embryonic gonads of testis-grafted chicks. This allows to discard a possible influence of cells migrating from the graft to the host. The grafted testes act then through substance/s secreted in the blood stream. Present and previous experimental data strongly suggest that the same substance, i.e. the so-called anti-Mullerian hormone, is responsible for both MD regression and gonadal sex reversal.     

Drought-stress-induced up-regulation of CAM in seedlings of a tropical cactus, Opuntia elatior, operating predominantly in the C3 mode

Immediately after unfolding, cotyledons of the tropical platyopuntoid cactus, Opuntia elatior Mill., exhibited a C(3)-type diel CO(2) exchange pattern characterized by net CO(2) uptake in the light. Significant nocturnal increases in titratable acidity typical of crassulacean acid metabolism (CAM) were not detected at this early developmental stage. As cotyledons matured and the first cladode (flattened stem) developed, features of CAM were observed and the magnitude of CAM increased. Nonetheless, in well-watered seedlings up to 10 cm tall, C(3) photosynthetic CO(2) fixation in the light remained the major pathway of carbon fixation. Reduced soil water availability led to an up-regulation of net dark CO(2) fixation and greater nocturnal increases in tissue acidity, consistent with facultative CAM. These observations demonstrate that C(3) photosynthesis, drought-stress-related facultative CAM, and developmentally controlled constitutive CAM can all contribute to the early growth of O. elatior. The strong C(3) component and facultative CAM features expressed in young O. elatior contrast with mature plants in which obligate CAM is the major pathway of carbon acquisition.