Phloem-xylem water flow in developing cladodes of Opuntia ficus-indica during sink-to-source transition

Phloem versus xylem water and carbon flow between a developingdaughter cladode (flattened stem segment) and the underlyingbasal cladode of Opuntia ficus-indica was assessed using netCO₂ uptake, transpiration, phloem sap concentration, and waterpotential of both organs as well as phloem and apoplastic tracers.A 14-d-old daughter cladode was a sink organ with a negativedaily net CO₂ uptake; its water potential was higher than thatof the underlying basal cladode, implicating a non-xylem pathwayfor the water needed for growth. Indeed, the relatively dilutephloem sap (7.44% dry weight) of a basal cladode can supplyall the water (7.1 gd^–1) along with photosynthate neededfor the growth of a 14-d-old daughter cladode; about 3% of theimported water flowed back to the basal cladode via the xylem.In contrast, a 28-d-old daughter cladode was a source organwhose water potential was lower than that of its basal cladode,so the xylem can supply the water needed (25.7 g d^–1)for its growth; about 6% of the imported water flowed back tothe basal cladode along with photosynthate via the phloem. Thephloem tracer carboxyfluorescein occurred in the phloem of 14-d-olddaughter cladodes after its precursor was applied to basal cladodes.When applied to basal cladodes, the apoplastic tracers sulphorhodamineG (SR) and trisodium 8-hydroxy-1,3,6-pyrenetrisulphonate (PTS)failed to move into 14-d-old daughter cladodes within 5 h, butmoved into 28-d-old daughter cladodes within 2 h. SR and PTSmoved into basal cladodes within 2 h when applied to 14-d-olddaughter cladodes, but not within 5-6 h when applied to 28-d-olddaughter cladodes. The tracer experiments therefore confirmedthe patterns of water flow determined using water and carbonbudgets. Key words: Carboxyfluorescein, phloem-xylem water flow, source-sink water relations, suiphorhodamine G, trisodium 8-hydroxy-1,3,6-pyrenetnsulphonate     

Young daughter cladodes affect CO2 uptake by mother cladodes of Opuntia ficus-indica

BACKGROUND AND AIMS: Drought damages cultivated C3, C4 and CAM plants in the semi-arid lands of central Mexico. Drought damage to Opuntia is common when mother cladodes, planted during the dry spring season, develop young daughter cladodes that behave like C3 plants, with daytime stomatal opening and water loss. In contrast, wild Opuntia are less affected because daughter cladodes do not develop on them under extreme drought conditions. The main objective of this work is to evaluate the effects of the number of daughter cladodes on gas exchange parameters of mother cladodes of Opuntia ficus-indica exposed to varying soil water contents. METHODS: Rates of net CO2 uptake, stomatal conductance, intercellular CO2 concentration, chlorophyll content and relative water content were measured in mature mother cladodes with a variable number of daughter cladodes growing in spring under dry and wet conditions. KEY RESULTS: Daily carbon gain by mother cladodes was reduced as the number of daughter cladodes increased to eight, especially during drought. This was accompanied by decreased mother cladode relative water content, suggesting movement of water from mother to daughter cladodes. CO2 assimilation was most affected in phase IV of CAM (late afternoon net CO2 uptake) by the combined effects of daughter cladodes and drought. Rainfall raised the soil water content, decreasing the effects of daughter cladodes on net CO2 uptake by mother cladodes. CONCLUSIONS: Daughter cladodes significantly hasten the effects of drought on mother cladodes by competition for the water supply and thus decrease daily carbon gain by mother cladodes, mainly by inhibiting phase IV of CAM.     

Opuntia ficus-indica cladodes as feedstock for ethanol production by Kluyveromyces marxianus and Saccharomyces cerevisiae

The feasibility of ethanol production using an enzymatic hydrolysate of pretreated cladodes of Opuntia ficus-indica (prickly pear cactus) as carbohydrate feedstock was investigated, including a comprehensive chemical analysis of the cladode biomass and the effects of limited aeration on the fermentation profiles and sugar utilization. The low xylose and negligible mannose content of the cladode biomass used in this study suggested that the hemicellulose structure of the O. ficus-indica cladode was atypical of hardwood or softwood hemicelluloses. Separate hydrolysis and fermentation and simultaneous saccharification and fermentation procedures using Kluyveromyces marxianus and Saccharomyces cerevisiae at 40 and 35 °C, respectively, gave similar ethanol yields under non-aerated conditions. In oxygen-limited cultures K. marxianus exhibited almost double the ethanol productivity compared to non-aerated cultures, although after sugar depletion utilization of the produced ethanol was evident. Ethanol concentrations of up to 19.5 and 20.6 g l^?1 were obtained with K. marxianus and S. cerevisiae, respectively, representing 66 and 70 % of the theoretical yield on total sugars in the hydrolysate. Because of the low xylan content of the cladode biomass, a yeast capable of xylose fermentation might not be a prerequisite for ethanol production. K. marxianus, therefore, has potential as an alternative to S. cerevisiae for bioethanol production. However, the relatively low concentration of fermentable sugars in the O. ficus-indica cladode hydrolysate presents a technical constraint for commercial exploitation.     

Effects of drought on water relations and nonstructural carbohydrates in cladodes of Opuntia ficus-indica

Nocturnal acid accumulation, water content, osmotic pressure (π), and nonstructural carbohydrates were determined in the chlorenchyma and the water-storage parenchyma of Opuntia ficus-indica (L.) Miller for well-watered plants and those subjected to drought for 15 weeks. During the 15-week drought, total cladode water content decreased by 57%, the water-storage parenchyma losing a greater fraction of water than the chlorenchyma, which most likely helped maintain nocturnal acid accumulation in the latter tissue. Despite the preferential water loss from the water-storage parenchyma, it had a lower π than the chlorenchyma over the 15 weeks of drought, suggesting a substantial decrease in osmotically active solutes in the water-storage parenchyma. Also, the measured π increases of both tissues were much less than those predicted based on the loss of water during drought and the initial content of osmotically active solutes under well-watered conditions. A decrease in the amount of soluble sugars (glucose. fructose and sucrose) occurred in plants subjected to drought. accounting for 46% and 81% of the difference between the measured and the predicted increases in π of the chlorenchyma and the water-storage parenchyma. respectively. The decrease in soluble sugars was associated with an equivalenl increase in polysaccharides, presumably starch, in the water-storage parenchyma. but not in the chlorenchyma.     

Protective effect of Opuntia ficus-indica L. cladodes against UVA-induced oxidative stress in normal human keratinocytes

Opuntia ficus-indica L. is known for its beneficial effects on human health, but still little is known on cladodes as a potent source of antioxidants. Here, a direct, economic and safe method was set up to obtain water extracts from Opuntia ficus-indica cladodes rich in antioxidant compounds. When human keratinocytes were pre-treated with the extract before being exposed to UVA radiations, a clear protective effect against UVA-induced stress was evidenced, as indicated by the inhibition of stress-induced processes, such as free radicals production, lipid peroxidation and GSH depletion. Moreover, a clear protective effect against apoptosis in pre-treated irradiated cells was evidenced. We found that eucomic and piscidic acids were responsible for the anti-oxidative stress action of cladode extract. In conclusion, a bioactive, safe, low-cost and high value-added extract from Opuntia cladodes was obtained to be used for skin health/protection.     

Effects of shade, drought and daughter cladodes on the CO2 uptake by cladodes of Opuntia ficus-indica

The effects of shade on the physiology of opuntias have received little attention, notwithstanding that shade regularly occurs in both wild stands and cultivated populations. This research evaluates the effects of shade on the physiology of cladodes of Opuntia ficus‐indica, with and without daughter cladodes, as they are exposed to progressive drought. The stress caused by shade, drought and daughter cladodes reduced photosynthesis by mother cladodes and was associated with decreases in relative water content, parenchyma thickness and chlorophyll content. Shade exacerbated the physiological drought of mother cladodes imposed by daughter cladodes and by reduced soil water content.     

Carbon and water relations for developing fruits of Opuntia ficus-indica (L.) Miller, including effects of drought and gibberellic acid

Growth, gas exchange rates, and carbohydrate content were studied for developing fruits of the cultivated cactus Opuntia ficus-indica (L.) Miller, including effects of drought and exogenous gibberellic acid (GA3). Fruit development required 110 d from the time of bud differentiation to ripening at 80 d after anthesis, when the fruit mass averaged 67 g. Stomatal conductance and net CO2 uptake rates for fruits were higher during the night; they were maximal at 7 d before anthesis and decreased as development progressed. Fruits undergoing drought, imposed by detaching terminal stems bearing fruits, were 50% smaller than the control at 80 d after anthesis and did not ripen. Fruits injected with 2 ml of 500 ppm GA3 were 30% smaller than the control at 80 d after anthesis; they contained a large proportion of aborted seeds that produced a weak sink signal for dry mass accumulation. Gas exchange was higher at 21 d after anthesis for fruits treated with GA3. Total soluble sugars represented 40% of the fruit's dry mass until 45 d after anthesis, when the sugar content rapidly increased, reaching 90% at 73 d after anthesis. Such an increase was not observed for fruits treated with GA3, and the sugar content for fruits undergoing drought remained low throughout development. Starch content increased for developing fruits of O. ficus-indica until 14 d after anthesis and, except for the fruits undergoing drought, decreased thereafter. Fruit development for O. ficus-indica is apparently regulated by water availability as well as hormonal signals originating both within and outside the fruit.     

Metabolic profiling of the sink-to-source transition in developing leaves of quaking aspen

Profiles of small polar metabolites from aspen (Populus tremuloides Michx.) leaves spanning the sink-to-source transition zone were compared. Approximately 25% of 250 to 300 routinely resolved peaks were identified, with carbohydrates, organic acids, and amino acids being most abundant. Two-thirds of identified metabolites exhibited greater than 4-fold changes in abundance during leaf ontogeny. In the context of photosynthetic and respiratory measurements, profile data yielded information consistent with expected developmental trends in carbon-heterotrophic and carbon-autotrophic metabolism. Suc concentration increased throughout leaf expansion, while hexose sugar concentrations peaked at mid-expansion and decreased sharply thereafter. Amino acid contents generally decreased during leaf expansion, but an early increase in Phe and a later one in Gly and Ser reflected growing commitments to secondary metabolism and photorespiration, respectively. The assimilation of nitrate and utilization of stored Asn appeared to be marked by sequential changes in malate concentration and Asn transaminase activity. Principal component and hierarchical clustering analysis facilitated the grouping of cell wall maturation (pectins, hemicelluloses, and oxalate) and membrane biogenesis markers in relation to developmental changes in carbon and nitrogen assimilation. Metabolite profiling will facilitate investigation of nitrogen use and cellular development in Populus sp. varying widely in their growth and pattern of carbon allocation during sink-to-source development and in response to stress.     

Effect of polysaccharides from Opuntia ficus-indica (L.) cladodes on the healing of dermal wounds in the rat.

In traditional medicine extracts of polysaccharide-containing plants are widely employed for the treatment of skin and epithelium wounds and of mucous membrane irritation. The extracts of Opuntia ficus-indica cladodes are used in folk medicine for their antiulcer and wound-healing activities. The present study describes the wound-healing potential of two lyophilized polysaccharide extracts obtained from O. ficus-indica (L.) cladodes applied on large full-thickness wounds in the rat. When topically applied for 6 days, polysaccharides with a molecular weight (MW)>10(4)Da from O. ficus-indica cladodes induce a beneficial effect on cutaneous repair in this experimental model; in particular the topical application of O. ficus-indica extracts on skin lesions accelerates the reepithelization and remodelling phases, also by affecting cell-matrix interactions and by modulating laminin deposition. Furthermore, the wound-healing effect is more marked for polysaccharides with a MW ranging 10(4)-10(6)Da than for those with MW>10(6)Da, leading us to suppose that the fine structure of these polysaccharides and thus their particular hygroscopic, rheologic and viscoelastic properties may be essential for the wound-healing promoter activity observed.     

Changes in Osmotic Pressure and Mucilage during Low-Temperature Acclimation of Opuntia ficus-indica

Opuntia ficus-indica, a Crassulacean acid metabolism plant cultivated for its fruits and cladodes, was used to examine chemical and physiological events accompanying low-temperature acclimation. Changes in osmotic pressure, water content, low molecular weight solutes, and extracellular mucilage were monitored in the photosynthetic chlorenchyma and the water-storage parenchyma when plants maintained at day/night air temperatures of 30/20°C were shifted to 10/0°C. An increase in osmotic pressure of 0.13 megapascal occurred after 13 days at 10/0°C. Synthesis of glucose, fructose, and glycerol accounted for most of the observed increase in osmotic pressure during the low-temperature acclimation. Extracellular mucilage and the relative apoplastic water content increased by 24 and 10%, respectively, during exposure to low temperatures. These increases apparently favor the extracellular nucleation of ice closer to the equilibrium freezing temperature for plants at 10/0°C, which could make the cellular dehydration more gradual and less damaging. Nuclear magnetic resonance studies helped elucidate the cellular processes during ice formation, such as those revealed by changes in the relaxation times of two water fractions in the chlorenchyma. The latter results suggested a restricted mobility of intracellular water and an increased mobility of extracellular water for plants at 10/0°C compared with those at 30/20°C. Increased mobility of extracellular water could facilitate extracellular ice growth and thus delay the potentially lethal intracellular freezing during low-temperature acclimation.     

Aetiology of Opuntia ficus-indica malformations and stunting disease

The phytoplasma aetiology of epidemic stunting of cladodes and stunted growth observed in a cactus pear plantation in Carlentini (Sicily, Italy) was investigated with graft inoculation trials and PCR/restriction fragment length polymorphism analyses of 16S ribosomal RNA (rRNA) gene and S3 ribosomal protein gene. After sequencing of the 1525‐bp from 16S rRNA gene of both naturally infected and graft‐inoculated symptomatic cactus pear samples, phytoplasma TS belonging to ribosomal subgroup 16SrII‐C were identified as aetiological agents of this worldwide spread disease.     

Physiological responses of Opuntia ficus-indica to growth temperature

The influences of various day/night air temperatures on net CO₂ uptake and nocturnal acid accumulation were determined for Opuntia ficus-indica, complementing previous studies on the water relations and responses to photosynthetically active radiation (PAR) for this widely cultivated cactus. As for other Crassulacean acid metabolism (CAM) plants, net nocturnal CO₂ uptake had a relatively low optimal temperature, ranging from 11°C for plants grown at day/night air temperatures of 10°C/0°C to 23°C at 45°C/35°C. Stomatal opening, which occurred essentially only at night and was measured by changes in water vapor conductance, progressively decreased as the measurement temperature was raised. The CO₂ residual conductance, which describes chlorenchyma properties, had a temperature optimum a few degrees higher than the optimum for net CO₂ uptake at all growth temperatures. Nocturnal CO₂ uptake and acid accumulation summed over the whole night were maximal for growth temperatures near 25°C/15°C, CO₂ uptake decreasing more rapidly than acid accumulation as the growth temperature was raised. At day/night air temperatures that led to substantial nocturnal acid accumulation (25°C/15°C.). 90% saturation of acid accumulation required a higher total daily PAR than at non-optimal growth temperatures (10°C/0°C and 35°C/25°C). Also, the optimal temperature of net CO₂ uptake shifted downward when the plants were under drought conditions at all three growth temperatures tested, possibly reflecting an increased fractional importance of respiration at the higher temperatures during drought. Thus, water status, ambient PAR, and growth temperatures must all be considered when predicting the temperature response of gas exchange for O. ficus-indica and presumably for other CAM plants.     

Characterization of carbon metabolism in Opuntia ficus-indica Mill. exhibiting the idling mode of Crassulacean acid metabolism

Upon transfer from well-watered conditions to total drought, long-day-grown cladodes of Opuntia ficus-indica Mill. shift from full Crassulacean acid metabolism (CAM) to CAM-idling. Experiments using ¹⁴C-tracers were conducted in order to characterize the carbon-flow pattern in cladodes under both physiological situations. Tracer was applied by ¹⁴CO₂ fumigations and NaH¹⁴CO₃ injections during the day-night cycle. The results showed that behind the closed stomata, mesophyll cells of CAM-idling plants retained their full capacity to metabolize CO₂ in light and in darkness. Upon the induction of CAM-idling the level of the capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was maintained. By contrast, malate pools decreased, displaying finally only a small or no day-night oscillation. The capacity of NADP-malic enzyme (EC 1.1.1.40) decreased in parallel with the reduction in malate pools. Differences in the labelling patterns, as influenced by the mode of tracer application, are discussed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

Root growth, developmental changes in the apex, and hydraulic conductivity for Opuntia ficus-indica during drought

Developmental changes in the root apex and accompanying changes in lateral root growth and root hydraulic conductivity were examined for Opuntia ficus-indica (L.) Miller during rapid drying, as occurs for roots near the soil surface, and more gradual drying, as occurs in deeper soil layers. During 7 d of rapid drying (in containers with a 3-cm depth of vermiculite), the rate of root growth decreased sharply and most root apices died; such a determinate pattern of root growth was not due to meristem exhaustion but rather to meristem mortality after 3 d of drying. The length of the meristem, the duration of the cell division cycle, and the length of the elongation zone were unchanged during rapid drying. During 14 d of gradual drying (in containers with a 6-cm depth of vermiculite), root mortality was relatively low; the length of the elongation zone decreased by 70%, the number of meristematic cells decreased 30%, and the duration of the cell cycle increased by 36%. Root hydraulic conductivity ( L _P) decreased to one half during both drying treatments; L _P was restored by 2 d of rewetting owing to the emergence of lateral roots following rapid drying and to renewed apical elongation following gradual drying. Thus, in response to drought, the apical meristems of roots of O. ficus-indica near the surface die, whereas deeper in the substrate cell division and elongation in root apices continue. Water uptake in response to rainfall in the field can be enhanced by lateral root proliferation near the soil surface and additionally by resumption of apical growth for deeper roots.     

The farnesoid X receptor modulates hepatic carbohydrate metabolism during the fasting-refeeding transition

The liver plays a central role in the control of blood glucose homeostasis by maintaining a balance between glucose production and utilization. The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor. Hepatic FXR expression is regulated by glucose and insulin. Here we identify a role for FXR in the control of hepatic carbohydrate metabolism. When submitted to a controlled fasting-refeeding schedule, FXR(-/-) mice displayed an accelerated response to high carbohydrate refeeding with an accelerated induction of glycolytic and lipogenic genes and a more pronounced repression of gluconeogenic genes. Plasma insulin and glucose levels were lower in FXR(-/-) mice upon refeeding the high-carbohydrate diet. These alterations were paralleled by decreased hepatic glycogen content. Hepatic insulin sensitivity was unchanged in FXR(-/-) mice. Treatment of isolated primary hepatocytes with a synthetic FXR agonist attenuated glucose-induced mRNA expression as well as promoter activity of L-type pyruvate kinase, acetyl-CoA carboxylase 1, and Spot14. Moreover, activated FXR interfered negatively with the carbohydrate response elements regions. These results identify a novel role for FXR as a modulator of hepatic carbohydrate metabolism.     

Carbohydrate Partitioning and Compartmental Analysis for a Highly Productive CAM Plant, Opuntia ficus-indica

Carbon partitioning patterns of Opuntia ficus-indica, a widelycultivated crassulacean acid metabolism species, were analysedto estimate carbon fluxes. After labelling a cladode with ¹⁴CO₂,activities of ¹⁴C in various organs were measured for 6 weeks;the observed ¹⁴C time-courses for ¹⁴C in the labelled cladodeand for transfer into other organs were simulated with a compartmentmodel. Within the first week, half of the newly synthesizedcarbohydrate in the labelled cladode was either converted intostructural material in that cladode, lost by respiration ofthat cladode, or moved to other organs. In the non-labelledcladode and the roots, such newly synthesized carbohydrate initiallyincreased, reached maxima, and then declined. The basal cladodeand the daughter cladode used 65 and 96%, respectively, of theirown assimilate. Roots imported 12 and 2 % of carbohydrate fromthe basal cladode and the daughter cladode, respectively. Whenthe whole plant was shaded, the daughter cladode incorporatednearly threefold more carbohydrate from the basal cladode intostructural material compared with the control. When plants weredroughted, roots incorporated 23 % more and the daughter cladodeincorporated 68 % less carbohydrate from the basal cladode intotheir structural material than for the control. The basal cladodesof the 18-month-old plants exported 60% more carbon than thoseof the 6-month-old plants. Carbon flux rates derived from compartmentalanalysis can be used as parameter values in plant productionmodels. Carbon partitioning, compartment model, drought, plant age, shading     

Genetic transformation of prickly-pear cactus (Opuntia ficus-indica) by Agrobacterium tumefaciens

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l⁻¹ kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus.