Effect of ectomycorrhizal fungi on survival and growth of micropropagated plants and seedlings of Castanea sativa mill.

 Four ectomycorrhizal fungi (Amanita muscaria, Laccaria laccata, Piloderma croceum and Pisolithus tinctorius) were used to produce mycorrhiza on seedlings and micropropagated plants of Castanea sativa in vitro. Pisolithus tinctorius was most effective in colonizing roots of both micropropagated plants and seedlings. A. muscaria and L. laccata only colonized a few feeder roots of some plants and Piloderma croceum did not form mycorrhizas. Mycorrhization of micropropagated plants increased survival and growth during weaning. Accepted: 27 February 1996     

Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth

Metabolic and physiological analyses of glutamate:glyoxylate aminotransferase 1 (GGT1) mutants were performed at the global leaf scale to elucidate the mechanisms involved in their photorespiratory growth phenotype. Air‐grown ggt1 mutants showed retarded growth and development, that was not observed at high CO₂ (3000 μL L^?1). When compared to wild‐type (WT) plants, air‐grown ggt1 plants exhibited glyoxylate accumulation, global changes in amino acid amounts including a decrease in serine content, lower organic acid levels, and modified ATP/ADP and NADP⁺/NADPH ratios. When compared to WT plants, their net CO₂ assimilation rates (A_n) were 50% lower and this mirrored decreases in ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) contents. High CO₂‐grown ggt1 plants transferred to air revealed a rapid decrease of A_n and photosynthetic electron transfer rate while maintaining a high energetic state. Short‐term (a night period and 4 h of light) transferred ggt1 leaves accumulated glyoxylate and exhibited low serine contents, while other amino acid levels were not modified. RuBisCO content, activity and activation state were not altered after a short‐term transfer while the ATP/ADP ratio was lowered in ggt1 rosettes. However, plant growth and RuBisCO levels were both reduced in ggt1 leaves after a long‐term (12 days) acclimation to air from high CO₂ when compared to WT plants. The data are discussed with respect to a reduced photorespiratory carbon recycling in the mutants. It is proposed that the low A_n limits nitrogen‐assimilation, this decreases leaf RuBisCO content until plants attain a new homeostatic state that maintains a constant C/N balance and leads to smaller, slower growing plants.     

Carob trees (Ceratonia siliqua L.) regenerated in vitro can acclimatize successfully to match the field performance of seed-derived plants

The use of in vitro regenerated plants in forestry and orchard depends ultimately on the development of efficient transplantation protocols, ensuring high survival rates and successful establishment under field conditions. We tested the performance of micropropagated carob trees (Ceratonia siliqua L.) throughout the acclimatization process in terms of survival, growth and physiological traits, including field comparisons with seed-derived and mother plants. The field trial was 100?% successful, i.e. we found no major differences between micropropagated, seed-derived and mother plants in terms of growth rate, height, number of leaves, photosynthetic efficiency, chlorophyll fluorescence, chlorophyll content and soluble protein content, although these parameters changed significantly during acclimatization. Stomatal conductance (g _s) was reduced by fourfold when plants were transferred from in vitro culture to the growth chamber, thus preventing uncontrolled wilting. The photosynthetic rate (P _N) was relatively low in vitro, in the growth chamber and the greenhouse, but increased to match seed-derived and mother plants in the field. The chlorophyll a/b ratio in leaves from in vitro and growth chamber plants was typical of shade plants (2.1) but became more characteristic of sun plants in the subsequent acclimatization stages (3.1–3.5). The maximum efficiency of photosystem II (F _v/F _m) remained mostly constant at?~0.80 throughout acclimatization, as is typical for healthy, non-stressed plants. We conclude that our micropropagation and acclimatization protocols provide a suitable alternative to traditional mass propagation techniques for the carob tree.     

Growth and photosynthetic activity of micropropagated strawberry plants inoculated with endomycorrhizal fungi (AMF) and growing under drought stress

The plants produced by in vitro methods are free of any microflora contrary to natural systems where plants are colonized by symbiotic fungi. The present paper reports the experiments carried out to evaluate the role of arbuscular endomycorrhizal fungi in development of micropropagated strawberries and their photosynthetic activity (measured by chlorophyll fluorescence) under drought conditions. Mycorrhization strongly affected growth and tolerance to water deficiency of the plants cultivated in greenhouse. Wilting of not-mycorrhized plants was accompanied by drastic increase of Fo and Tfm and decrease of Fm. At the same time, the value of these parameters for mycorrhized plants did not change. Drastic decrease in the value of parameters Fv/Fm, Fv/Fo and Fo/Fm for plants without AMF appeared at the end of dry period. Rise of Fs and decrease Rfd was noted only for not-mycorrhized plants. The plants colonized by fungi, fully recovered their photosynthetic activity when watering was restored.     

Effects of RuBisCO and CO2 concentration on cyanobacterial growth and carbon isotope fractionation

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO₂ concentrations), molecular, and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a specific range of genetic and environmental factors that may impact ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO across varying atmospheric CO₂ concentrations. We hypothesized that changes in RuBisCO expression would impact the net rates of intracellular CO₂ fixation versus CO₂ supply, and thus whole-cell carbon isotope discrimination. In particular, we investigated the impacts of RuBisCO overexpression under changing CO₂ concentrations on both carbon isotope biosignatures and cyanobacterial physiology, including cell growth and oxygen evolution rates. We found that an increased pool of active RuBisCO does not significantly affect the ¹³C/¹²C isotopic discrimination (ε_p) at all tested CO₂ concentrations, yielding ε_p of ≈ 23‰ for both wild-type and mutant strains at elevated CO₂. We therefore suggest that expected variation in cyanobacterial RuBisCO expression patterns should not confound carbon isotope biosignature interpretation. A deeper understanding of environmental, evolutionary, and intracellular factors that impact cyanobacterial physiology and isotope discrimination is crucial for reconciling microbially driven carbon biosignatures with those preserved in the geologic record.     

Comparative study of morphological parameters of Grand Nain banana (<Emphasis Type="Italic">Musa</Emphasis> AAA) after <Emphasis Type="Italic">in vitro</Emphasis> multiplication with growth retardants

Variation in morphological traits and increased disease susceptibility were observed in micropropagated plants of rhubarb (Rheum rhaponticum L.), PC49. Our investigations have demonstrated that micropropagated plants can vary substantially in morphological traits and the variation of quantitative traits was substantially greater than conventionally propagated plants. Micropropagated plants produced significantly more leaves than conventional plants under the same growth period, with a more bushy growth habit and/or recumbence. A higher incidence of disease (petiole spotting) was also observed in micropropagated plants. It is concluded that micropropagated PC49 had substantially higher incidence of somaclonal variation and regenerants were not suitable to establish an economic crop comparable to the conventional plants.     

How do vascular plants perform photosynthesis in extreme environments? An integrative ecophysiological and biochemical story

In this work, we review the physiological and molecular mechanisms that allow vascular plants to perform photosynthesis in extreme environments, such as deserts, polar and alpine ecosystems. Specifically, we discuss the morpho/anatomical, photochemical and metabolic adaptive processes that enable a positive carbon balance in photosynthetic tissues under extreme temperatures and/or severe water‐limiting conditions in C₃ species. Nevertheless, only a few studies have described the in situ functioning of photoprotection in plants from extreme environments, given the intrinsic difficulties of fieldwork in remote places. However, they cover a substantial geographical and functional range, which allowed us to describe some general trends. In general, photoprotection relies on the same mechanisms as those operating in the remaining plant species, ranging from enhanced morphological photoprotection to increased scavenging of oxidative products such as reactive oxygen species. Much less information is available about the main physiological and biochemical drivers of photosynthesis: stomatal conductance (g_s), mesophyll conductance (g_m) and carbon fixation, mostly driven by RuBisCO carboxylation. Extreme environments shape adaptations in structures, such as cell wall and membrane composition, the concentration and activation state of Calvin–Benson cycle enzymes, and RuBisCO evolution, optimizing kinetic traits to ensure functionality. Altogether, these species display a combination of rearrangements, from the whole‐plant level to the molecular scale, to sustain a positive carbon balance in some of the most hostile environments on Earth.     

Plant development and synthesis of essential oils in micropropagated and mycorrhiza inoculated plants of Origanum vulgare L. ssp. hirtum (Link) Ietswaart

Biomass production of micropropagated oregano was induced by inoculation with the fungus Glomus viscosum. The effects of arbuscular mycorrhizal (AM) symbiosis on morphological and metabolic variations of regenerated oregano plants were investigated at different growth stages. AM greatly increased parameters such as plant leaf area, fresh and dry weight, number of spicasters and verticillasters in infected plants. An increase of the gland density, especially on the upper leaf epidermis, was also observed following the physiological ageing of the tissues. The in vitro plants of O. vulgare ssp. hirtum described in this study provided a qualitatively and quantitatively good source of essential oils that have a chemical profile comparable to that of the control mother plants with carvacrol as the main compound.     

Overexpression of BUNDLE SHEATH DEFECTIVE 2 improves the efficiency of photosynthesis and growth in Arabidopsis

Bundle Sheath Defective 2, BSD2, is a stroma‐targeted protein initially identified as a factor required for the biogenesis of ribulose 1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) in maize. Plants and algae universally have a homologous gene for BSD2 and its deficiency causes a RuBisCO‐less phenotype. As RuBisCO can be the rate‐limiting step in CO₂ assimilation, the overexpression of BSD2 might improve photosynthesis and productivity through the accumulation of RuBisCO. To examine this hypothesis, we produced BSD2 overexpression lines in Arabidopsis. Compared with wild type, the BSD2 overexpression lines BSD2ox‐2 and BSD2ox‐3 expressed 4.8‐fold and 8.8‐fold higher BSD2 mRNA, respectively, whereas the empty‐vector (EV) harbouring plants had a comparable expression level. The overexpression lines showed a significantly higher CO₂ assimilation rate per available CO₂ and productivity than EV plants. The maximum carboxylation rate per total catalytic site was accelerated in the overexpression lines, while the number of total catalytic sites and RuBisCO content were unaffected. We then isolated recombinant BSD2 (rBSD2) from E. coli and found that rBSD2 reduces disulfide bonds using reductants present in vivo, for example glutathione, and that rBSD2 has the ability to reactivate RuBisCO that has been inactivated by oxidants. Furthermore, 15% of RuBisCO freshly isolated from leaves of EV was oxidatively inactivated, as compared with 0% in BSD2‐overexpression lines, suggesting that the overexpression of BSD2 maintains RuBisCO to be in the reduced active form in vivo. Our results demonstrated that the overexpression of BSD2 improves photosynthetic efficiency in Arabidopsis and we conclude that it is involved in mediating RuBisCO activation.     

The effect of growth at elevated CO2 concentrations on photosynthesis in wheat

Rising levels of atmospheric CO₂ will have profound, direct effects on plant carbon metabolism. In this study we used gas exchange measurements, models describing the instantaneous response of leaf net CO₂ assimilation rate (A) to intercellular CO₂ partial pressure (C_i), in vitro enzyme activity assay, and carbohydrate assay in order to investigate the photosynthetic responses of wheat (Triticum aestivum L., cv. Wembley) to growth under elevated partial pressures of atmospheric CO₂ (C_a). At flag leaf ligule emergence, the modelled, in vivo, maximum carboxylation velocity for RuBisCO was significantly lower in plants grown at elevated C_a than in plants grown at ambient C_a (70 Pa compared with 40 Pa). By 12 d after ligule emergence, no significant difference in this parameter was detectable. At ligule emergence, plants grown at elevated C_a exhibited reduced in vitro initial activities and activation states of RuBisCO. At their respective growth C_i values, the photosynthesis of 40-Pa-grown plants was sensitive to p(O₂) and to p(CO₂) whereas that of 70-Pa-grown plants was insensitive. Both sucrose and starch accumulated more rapidly in the leaves of plants grown at 70 Pa. At flag leaf ligule emergence, modelled non-photorespiratory respiration in the light (R_d) was significantly higher in 70-Pa-grown plants than in 40-Pa-grown plants. By 12 d after ligule emergence no significant differences in R_d were detectable.     

Morphological, physiological and oxidative stress markers during acclimatization and field transfer of micropropagated Tuberaria major plants

Tuberaria major (Willk.) P. Silva and Rozeira is a critically-endangered rock rose species endemic to Portugal. Because the species needs to be preserved, this study evaluated the morphological and physiological traits of micropropagated T. major plants during acclimatization and field transfer. There were no significant differences between wild and micropropagated plants in the field, although the latter underwent significant changes during acclimatization. Leaf pubescence and leaf mass per area increased during acclimatization whereas the chlorophyll content and chlorophyll/carotenoid ratio declined to eventually match those of wild plants. Stomatal conductance (g_s) and transpiration rates (E) also declined substantially during acclimatization, thus preventing uncontrolled wilting. Photosynthetic rate (P_N) was initially negative but increased during the later stages of acclimatization. Maximum quantum yield of PSII (F_v/F_m) remained constant at 0.78–0.85, showing that the plants were healthy and unstressed. PSII quantum efficiency (?_PSII) was initially low but increased during acclimatization along with photosynthetic performance as the energy partitioning in PSII was adjusted. This was balanced by the decline in non-regulated energy dissipation (?_NO) from an initially high value. Electrolyte leakage and malondialdehyde content remained constant at similar levels in both groups of plants, but H₂O₂ levels were higher in the field, perhaps indicating the early induction of antioxidant defense systems. The present study shows that T. major has enough phenotypic plasticity to adapt to changing environments and that the procedure described herein can be used for the restoration and preservation of this species.     

Stomatal physiology of a micropropagated CAM plant; Agave tequilana (Weber)

Experiments were designed to assess the capacity of an in vitro cultured CAM plant to control water loss and to examine the response of their stomata to various factors. Detached leaves of micropropagated Agave tequilana plants lost water at similar rates as did field-grown plantlets when dehydrated in air. This was consistent with the fact that stomata from micropropagated plants show similar morphology than field-grown plantlets. In addition, stomata from micropropagated plants responded to various factors in a manner similar to those from field-grown plantlets. It appears that in vitro culture does not affect the capacity of leaves to control water loss nor does it alters the nocturnal stomatal opening of this CAM plant.     

Physiological changes and growth of micropropagated chile ancho pepper plantlets during acclimatization and post-acclimatization

Little is known about physiological changes that occur with micropropagated chile ancho pepper (Capsicum annuum L. cv. San Luis) plantlets during acclimatization. Plantlets were transferred to ex vitro conditions to study selected physiological changes and growth performance during acclimatization and post-acclimatization. The physiology of the plantlets was characterized by measuring leaf gas exchange and water status. Plant growth was determined by assessing plant height, leaf number, total leaf area, relative growth rate (RGR), and leaf, root, and stem dry matter (DM). Chile pepper plantlets became acclimatized within 6 days after transplantation. During this period, physiological adjustments occurred, which were critical for plantlet survival. After initial ex vitro transplanting, plantlets experienced water deficit [leaf wilting and reduced relative water content (RWC)], which corresponded with reduced stomatal conductance (g _s) and transpiration (E), and an increase in stomatal resistance (r _s). Thus, leaf stomata that developed in vitro were functional ex vitro. Because of this stomatal control, plantlets minimized transplant shock, recovered and survived. Prior to transplanting, plantlets were photomixotrophic, as indicated by low photosynthetic rates (A). During acclimatization, RWC, g _s, E, and A were significantly lower two days after transplanting. However, within 6 days after transplanting, plantlets recovered and became photoautotrophic – attaining high A, g _s, and E. Water use efficiency was initially low during the first days after transplanting, but increased dramatically at the end of the acclimatization period in part due to increased A. The stabilization and improvement of plantlet water status and gas exchange during acclimatization and post-acclimatization closely correlated with increased plantlet growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Colonization with arbuscular mycorrhizal fungi improves salinity tolerance of tomato (Solanum lycopersicum L.) plants

The purpose of this study was to investigate the mechanisms underlying alleviation of salt stress by mycorrhization. Solanum lycopersicum L. cultivars Behta and Piazar with different salinity tolerance were cultivated in soil without salt (EC?=?0.63 dSm^?1), with low (EC?=?5 dSm^?1), or high (EC?=?10 dSm^?1) salinity. Plants inoculated with the arbuscular mycorrhizal fungi Glomus intraradices (+AMF) were compared to non-inoculated plants (?AMF). Under salinity, AMF-mediated growth stimulation was higher in more salt tolerant Piazar than in sensitive Behta. Mycorrhization alleviated salt-induced reduction of P, Ca, and K uptake. Ca/Na and K/Na ratios were also better in +AMF. However, growth improvement by AMF was independent from plant P nutrition under high salinity. Mycorrhization improved the net assimilation rates through both elevating stomatal conductance and protecting photochemical processes of PSII against salinity. Higher activity of ROS scavenging enzymes was concomitant with lowering of H₂O_2, less lipid peroxidation, and higher proline in +AMF. Cultivar differences in growth responses to salinity and mycorrhization could be well explained by differences in ion balance, photochemistry, and gas exchange of leaves. Function of antioxidant defenses seemed responsible for different AMF-responsiveness of cultivars under salinity. In conclusion, AMF may protect plants against salinity by alleviating the salt-induced oxidative stress.     

CARBON FIXATION IN CULTURED MARINE BENTHIC DIATOMS1

The enzyme activity of ribulose 1, 5-bisphosphate carboxylase-oxygenase (RuBisCO) and phosphoenolpyruvate carboxylase (PEPC) was measured in four species of marine benthic diatoms isolated from subtidal sediments of Graveline Bayou, Mississippi. Enzyme activities were measured in cultures of Amphora micrometra Giffen, A. tenerrima Aleem and Hustedt, Nitzschia fontifuga Cholnoky, and Nitzschia vermicularis Grunow that were grown at light levels supporting μ_max and at light-limiting irradiances. All four species exhibited similar RuBisCO: PEP ratios (range = 1–1.8) at μ_max the lowest ratio (0.4) was observed in A. micrometra. Reduced light levels increased PEPC relative to that measured at μ_max in two species. Two-dimensional paper chromatography was used to determine the first products of carbon fixation in A. micrometra After a 15 s incorporation period, the first product of photosynthetic carbon fixation was 3-phosphoglycerate even though this alga had a PEPC activity that was three times higher than that of RuBisCO. After 30 s, over 50% of the recovered radioactivity was still in this compound. Stable carbon isotope analyses of a mixture of the four pennate diatoms also suggest the predominant carbon fixation pathway in these benthic diatoms was similar to C3 plants.     

Effect of rhizosphere bacteria <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> on the resistance of micropropagated plants to phytopathogens

Effects of colonization of micropropagated potato (Solanum tuberosum L.) and strawberry (Fragaria L.) plants by the rhizosphere bacterium Pseudomonas aureofaciens strain BS1393 (VKM B-2188 D) on plant growth and resistance to bacterial and fungal phytopathogens were studied. Pseudomonad colonization improved the physiological characteristics of plants and enhanced their adaptation to in vivo conditions. The presence of P. aureofaciens cells in various plant tissues (leaves, stems, and roots) in vitro was demonstrated on the background of plant cocolonization by two associative strains—P. aureofaciens strain BS1393 (VKM-B-2188 D) and Methylovorus mays (VKM-B-2221). The colonized plants displayed an increased resistance to the phytopathogens Erwinia carotovora, Sclerotinia sclerotiorum, and Phytophthora infestans. These results demonstrate that pseudomonades are promising for practical application in the microbial protection of plants against phytopathogens.     

RAF2 is a RuBisCO assembly factor in Arabidopsis thaliana

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the reaction between gaseous carbon dioxide (CO₂) and ribulose‐1,5‐bisphosphate. Although it is one of the most studied enzymes, the assembly mechanisms of the large hexadecameric RuBisCO is still emerging. In bacteria and in the C4 plant Zea mays, a protein with distant homology to p terin‐4α‐c arbinolamine d ehydratase (PCD) has recently been shown to be involved in RuBisCO assembly. However, studies of the homologous PCD‐like protein (RAF2, RuBisCO assembly factor 2) in the C3 plant Arabidopsis thaliana (A. thaliana) have so far focused on its role in hormone and stress signaling. We investigated whether A. thalianaRAF2 is also involved in RuBisCO assembly. We localized RAF2 to the soluble chloroplast stroma and demonstrated that raf2 A. thaliana mutant plants display a severe pale green phenotype with reduced levels of stromal RuBisCO. We concluded that the RAF2 protein is probably involved in RuBisCO assembly in the C3 plant A. thaliana.     

Large-scale field acclimatization of Olea maderensis micropropagated plants: morphological and physiological survey

Micropropagation allows large-scale plant multiplication and germplasm preservation, representing an added value in forest breeding strategies to combat desertification and/or protect endangered species. We developed a large-scale micropropagation protocol of Olea maderensis (a native endangered wild olive of Madeira Archipelago) using OMG medium (rich in Fe, Mg and Mn) supplemented with zeatin for elongation and with NAA for rooting. We now describe the performance of micropropagated plants during five-period field acclimatization: (1) in vitro, (2) growth-cabinet, (3) greenhouse, (4) open-greenhouse, and (5) field mountain in Porto Santo Island. One hundred OG4 plants were acclimatized, showing >95% surviving rates. During acclimatization, several physiological parameters were evaluated; water content remained higher in in vitro/greenhouse conditions, decreased in field leaves. Soluble protein contents decreased during the first acclimatization periods increasing thereafter. Membrane permeability slightly increased during the field acclimatization. Chlorophylls content increased in in vitro leaves, while during acclimatization, mostly chl b decreased, increasing chl a/chl b ratio. F ₀ decreased in first acclimatization periods, increasing thereafter, while the other parameters (F _v; F _m; F _v/F _m) decreased. Nutrient contents decreased in plants transferred to poor field soil conditions, reaching values similar to mother plant leaves. Overall, with the exception of PSII fluorescence, field acclimatized plants had similar values to mother plants, showing a good adjustment to stressful field conditions. This protocol is being used in large-scale micropropagation within a reforestation program, and is an example of R&D technologies with immediate application on protection of endangered ecosystems.     

Elevated pCO2 Affects C and N Metabolism in Wild Type and Transgenic Tobacco Exhibiting Altered C/N Balance in Metabolite Analysis

Abstract: Diurnal changes in starch, sugar and amino acid concentrations in source leaves, sink leaves and roots of tobacco plants were determined. In addition to wild type tobacco, transformed plants deficient in root nitrate reductase and exhibiting decreased rates of growth were employed. Further, the growth rates of tobacco plants were modulated by exposure to elevated pCO₂. From the diurnal alterations in metabolite concentrations, the daily turnover of starch and amino N was estimated in order to: (i) elucidate whether turnover rates can be related to growth rates, and (ii) identify individual amino compounds with the potential to indicate nitrogen fluxes and the C/N status of plants. Elevated pCO₂ increased growth rates and daily turnover of starch in both wild type and transformed plants, indicating enhanced rates of photosynthesis. In wild type plants, elevated pCO₂ increased the turnover of amino N, notably glutamine and alanine, in mature source leaves, indicating enhanced nitrate reduction. By contrast, amino N turnover in source leaves of transformed plants was not affected by elevated pCO₂, although nitrate reduction was presumably enhanced. Apparently, export of amino N was increased from the source leaves of transformed plants. This assumption was supported by a significantly increased turnover of amino N in young sink leaves compared to mature source leaves, indicating a preference for acropetal amino N allocation and import into the young leaves of the transformed plants. Further, elevated pCO₂ increased the allocation of leaf‐derived amino N to the roots of transformed plants. This led to increased levels of amino compounds during the entire day, notably glutamate, but did not affect root growth of the transformed plants. The suitability of individual amino compounds as markers for major N fluxes, such as nitrate reduction, photorespiration, and amino N export and import is discussed.