Search

Climate change and the accelerated rate of population growth are imposing a progressive degradation of natural ecosystems worldwide. In this context, the use of pioneer trees represents a powerful approach to reverse the situation. Among others, N2-fixing actinorhizal trees constitute important elements of plant communities and have been successfully used in land reclamation at a global scale. In this study, we have analyzed the transcriptome of the photosynthetic organs of Casuarina glauca (branchlets) to unravel the molecular mechanisms underlying salt stress tolerance. For that, C. glauca plants supplied either with chemical nitrogen (KNO3+) or nodulated by Frankia (NOD+) were exposed to a gradient of salt concentrations (200, 400, and 600 mM NaCl) and RNA-Seq was performed. An average of ca. 25 million clean reads was obtained for each group of plants, corresponding to 86,202 unigenes. The patterns of differentially expressed genes (DEGs) clearly separate two groups: (i) control- and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCl-treated plants. Additionally, although the number of total transcripts was relatively high in both plant groups, the percentage of significant DEGs was very low, ranging from 6 (200 mM NaCl/NOD+) to 314 (600 mM NaCl/KNO3+), mostly involving down-regulation. The vast majority of up-regulated genes was related to regulatory processes, reinforcing the hypothesis that some ecotypes of C. glauca have a strong stress-responsive system with an extensive set of constitutive defense mechanisms, complemented by a tight mechanism of transcriptional and post-transcriptional regulation. The results suggest that the robustness of the stress response system in C. glauca is regulated by a limited number of genes that tightly regulate detoxification and protein/enzyme stability, highlighting the complexity of the molecular interactions leading to salinity tolerance in this species. [ABSTRACT FROM AUTHOR]

The actinorhizal tree Casuarina glauca tolerates extreme environmental conditions, such as high salinity. This species is also able to establish a root-nodule symbiosis with N 2 -fixing bacteria of the genus Frankia . Recent studies have shown that C. glauca tolerance to high salt concentrations is innate and linked to photosynthetic adjustments. In this study we have examined the impact of increasing NaCl concentrations (200, 400 and 600 mM) on membrane integrity as well as on the control of oxidative stress in branchlets of symbiotic (NOD+) and non-symbiotic (KNO 3 +) C. glauca . Membrane selectivity was maintained in both plant groups at 200 mM NaCl, accompanied by an increase in the activity of antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase). Regarding cellular membrane lipid composition, linolenic acid (C18:3) showed a significant decline at 200 mM NaCl in both NOD+ and KNO 3 + plants. In addition, total fatty acids (TFA) and C18:2 also decreased in NOD+ plants at this salt concentration, resulting in malondialdehyde (MDA) production. Such initial impact at 200 mM NaCl is probably due to the fact that NOD+ plants are subjected to a double stress, i.e., salinity and low nitrogen availability. At 400 mM NaCl a strong reduction of TFA and C18:3 levels was observed in both plant groups. This was accompanied by a decrease in the unsaturation degree of membrane lipids in NOD+. However, in both NOD+ and KNO 3 + lipid modifications were not reflected by membrane leakage at 200 or 400 mM, suggesting acclimation mechanisms at the membrane level. The fact that membrane selectivity was impaired only at 600 mM NaCl in both groups of plants points to a high tolerance of C. glauca to salt stress independently of the symbiotic relation with Frankia . [ABSTRACT FROM AUTHOR]

Quantitative PCR ( q PCR) was used to follow population dynamics of indigenous Frankia populations in bulk soil, in leaf-litter-amended soil and in the rhizosphere of Alnus glutinosa or Casuarina equisetifolia at 2 matric potentials representing dry and wet conditions in soil microcosms. Analyses revealed between 10- and 100-fold increases of Frankia populations within the incubation period of 12 weeks independent of treatment. Numbers were generally higher under dry conditions and in the rhizosphere, with that of C. equisetifolia supporting highest abundance. Frankiae detected at any time and treatment belonged to either subgroup I of the Alnus host infection group or the Elaeagnus host infection group, with those of the Elaeagnus host infection group largely representing the genus in all samples under wet conditions, and in bulk and leaf litter amended soil under dry conditions. Subgroup I of the Alnus host infection group was most prominent in the rhizosphere of both plant species where it represented up to 95% of the genus with higher percentages in that of C. equisetifolia . [ABSTRACT FROM AUTHOR]

Casuarina glauca is a fast-growing multipurpose tree belonging to the Casuarinaceae family and native to Australia. It requires limited use of chemical fertilizers due to the symbiotic association with the nitrogen-fixing actinomycete Frankia and with mycorrhizal fungi, which help improve phosphorous and water uptake by the root system. C. glauca can grow in difficult sites, colonize eroded lands and improve their fertility, thereby enabling the subsequent growth of more demanding plant species. As a result, this tree is increasingly used for reforestation and reclamation of degraded lands in tropical and subtropical areas such as China and Egypt. Many tools have been developed in recent years to explore the molecular basis of the interaction between Frankia and C. glauca. These tools include in vitro culture of the host and genetic transformation with Agrobacterium, genome sequencing of Frankia and related studies, isolation of plant symbiotic genes combined with functional analyses (including knock-down expression based on RNA interference), and transcriptome analyses of roots inoculated with Frankia or Rhizophagus irregularis. These efforts have been fruitful since recent results established that many common molecular mechanisms regulate the nodulation process in actinorhizal plants and legumes, thus providing new insights into the evolution of nitrogen-fixing symbioses. [ABSTRACT FROM AUTHOR]

Indigenous species of actinorhizal plants of Casuarinaceae, Elaeagnaceae and Rhamnaceae are found in specific regions of Australia. Most of these plants belong to Casuarinaceae, the dominant actinorhizal family in Australia. Many of them have significant environmental and economical value. The other two families with their indigenous actinorhizal plants have only a minor presence in Australia. Most Australian actinorhizal plants have their native range only in Australia, whereas two of these plants are also found indigenously elsewhere. The nitrogen-fixing ability of these plants varies between species. This ability needs to be investigated in some of these plants. Casuarinas form a distinctive but declining part of the Australian landscape. Their potential has rarely been applied in forestry in Australia despite their well-known uses, which are being judiciously exploited elsewhere. To remedy this oversight, a programme has been proposed for increasing and improving casuarinas that would aid in greening more regions of Australia, increasing the soil fertility and the area of wild life habitat (including endangered species). Whether these improved clones would be productive with local strains of Frankia or they need an external inoculum of Frankia should be determined and the influence of mycorrhizal fungi on these clones also should be investigated. [ABSTRACT FROM AUTHOR]

The host range of seven Frankia strains isolated from the root nodules of five actinorhizal plants in four genera growing in Japan and their relatedness based on 16S rDNA were examined. The Ceq1 strain isolated from Casuarina equisetifolia formed many nodules with high nitrogen fixation activity on the source host, C. equisetifolia. The Ceq1 strain formed nodules on Alnus sieboldiana, Alnus hirsta and Myrica rubra, but their nodulation ratio and nitrogen fixation activity were very low. The Asi1 strain isolated from A. sieboldiana, the Ahi1 strain isolated from A. hirsta, and the Mru1, Mru2 and Mru8 strains isolated from M. rubra formed many nodules on both Alnus species and M. rubra. The Ema2 strain isolated from Elaeagnus macrophylla formed nodules on both Elaeagnus species and M. rubra. Based on the aspect of nodulation and nitrogen fixation activity, Ceq1 strain is an effective strain only for Casuarina, five strains of Asi1, Ahi1, Mru1, Mru2 and Mru8 are effective strains for both Alnus and Myrica, and Ema2 strain is an effective strain for both Elaeagnus and Myrica. The results of phylogenetic analyses based on 16S rDNA sequences showed that seven strains were separated into two major clusters. Cluster 1 was further grouped into two subclusters, 1a and 1b. The Asi1, Ahi1, Mru1, Mru2 and Mru8 strains belonged to 1a and the Ceq1 strain belonged to cluster 1b. The Ema2 strain was classified into cluster 2. This phylogenetic classification of Frankia based on 16S rDNA sequences appears to be consistent with the host range determined using an inoculation test. [ABSTRACT FROM AUTHOR]

Chitinases (EC 3.2.1.14) catalyse the hydrolysis of chitin, a homopolymer of β-1,4-linked N-acetyl-d-glucosamine residues. Plant chitinases are involved in a wide variety of processes; in particular, their expression has been found to be enhanced in symbiotic and pathogenic plant–microbe interactions. During this work we have cloned and characterized a gene encoding a class III chitinase from actinorhizal nodules of Casuarina glauca ( cgchi3). CGCHI3 was found to be encoded by a single gene that was specifically activated in nodules as compared with uninoculated control roots and leaves. The expression of this gene was further enhanced in nodules after salicylic acid treatment and completely repressed after wounding. In situ hybridisation analysis revealed that cgchi3 is an early nodulin gene, being expressed in the meristem and in the uninfected cortical cells of young nodules. Based on the obtained results we suggest that this gene is involved in nodule development. This is the first report on a class III chitinase coding gene that is specifically activated during actinorhizal symbiosis. [ABSTRACT FROM AUTHOR]

Symbiotic association of the N[sub 2]-fixing actinomycete Frankia with the roots of more than 200 tree species from 24 genera of 8 families of angiosperms has been studied since 1829. The first successful isolation of the microsymbiont and reinfection in the host plant was achieved in 1978. Marked advances in research and understanding of Frankia biology, its actinorhizal hosts and their interactions have been made since then, although the studies on Frankia have been hampered by difficulties of isolation and their slow growth rate in vitro. Exponential growth with high biomass yields within three to four days has been obtained for a number of strains isolated from Casuarina spp. Use of BAP medium, supplemented with avian phospholipid mixtures and certain fatty acids at controled O[sub 2] access, optimizes growth. Monosporal cultures are scarce; recently a few became available for biochemical and genetic studies. Research using exponentially growing cultures has yielded information on a complex proteolytic system, including proteasomes, endo- and extracellular proteinases and aminopeptidases, and also on esterases, dehydrogenases, and extracellular DNAses. Molecular tools have revealed a marked genetic diversity of Frankia soil populations and have enabled the definition of four clades in the Frankia phylogenetic tree. Studies on Frankia-host plant interactions have detected molecular signal exchange preceeding the establishment of symbiosis. Similarly, there is progress in research on transgenic actinorhizal plants and on actinorhizal-specific genes and proteins (actinorhizins) involved in symbiotic interactions, infectivity, and host specificity. Actinorhizal plants are rapidly growing species, able to develop in N-poor soils, and for certain species, in harsh environmental stress conditions. They increase the fertility of agroforestry ecosystems, and have an economic potential for timber, fuelwood production, land reclamation, and amenity planting. The Casuarina spp. are of especial value in arid environments. [ABSTRACT FROM AUTHOR]

Actinorhizal plants have been regarded as promising species in the current climate change context due to their high tolerance to a multitude of abiotic stresses. While combined salt-heat stress effects have been studied in crop species, their impact on the model actinorhizal plant, Casuarina glauca, has not yet been fully addressed. The effect of single salt (400 mM NaCl) and heat (control at 26/22 °C, supra optimal temperatures at 35/22 °C and 45/22 °C day/night) conditions on C. glauca branchlets was characterised at the physiological level, and stress-induced metabolite changes were characterised by mass spectrometry-based metabolomics. C. glauca could withstand single salt and heat conditions. However, the harshest stress condition (400 mM NaCl, 45 °C) revealed photosynthetic impairments due to mesophyll and membrane permeability limitations as well as major stress-specific differential responses in C and N metabolism. The increased activity of enzymatic ROS scavengers was, however, revealed to be sufficient to control the plant oxidative status. Although C. glauca could tolerate single salt and heat stresses, their negative interaction enhanced the effects of salt stress. Results demonstrated that C. glauca responses to combined salt-heat stress could be explained as a sum of the responses from each single applied stress. [ABSTRACT FROM AUTHOR]

Hydrogen metabolism was studied in three Casuarina species, C. equisetifolia Forst., C. glauca Sieb. ex. Spreng. and C. obesa Miq., either inoculated with the pure Frankia culture HFP CcI3 or inoculated with a crushed nodule inoculum made from C. glauca nodules. Nitrogenase (EC 1.7.99.2) activity and hydrogen evolution was measured on intact plants, while hydrogen uptake was measured on excised nodules arid in nodule homogenates. Nitrogenase activity was highest in C. glauca inoculated with C. glauca nodules, while no hydrogen evolution was detected. Hydrogen evolution was highest in the symbiosis between C. equisetifolia and HFP CcI3, but the nitrogenase activity showed intermediate values compared to the other symbioses. Measured at a concentration of 93 μM H2, H2 uptake was highest in C. glauca inoculated with the C. glauca inoculum. H2 uptake activity in homogenates was 83% of the intact nodule rate. With phenazine methosulfate as the electron acceptor. H2 uptake by nodule homogenates showed typical Michaelis-Menten kinetics with a Km of 21.3 μM for H2. The data presented here indicate a host plant effect on the endobiont which alters the hydrogen metabolism. [ABSTRACT FROM AUTHOR]

Frankia strains have been isolated from actinorhizal nodules of Alnus (2 strains), Casuarina (5 strains), Coriaria (1 strain), Datisca (3 strains), Elaeagnus (1 strain) and Hippophaë (1 strain). The isolates were characterized for their growth on various carbon and nitrogen sources, nitrogen-fixing ability in culture and nodulation of seedlings of the original host plant. [ABSTRACT FROM AUTHOR]

Carbon isotope composition (δ13C) was measured in a glasshouse experiment with N2-fixing and NO3–- or NH4+-fed Casuarina equisetifolia Forst. & Forst plants, both under well-watered and drought conditions. The abundance of 13C was higher (more positive δ13C) for NH4+- than for NO3– -grown plants and was lowest for N2-fixing plants. NH4+-fed plants had more leaf area and dry weight and higher water use efficiency (on a biomass basis) than N2- and NO3–-grown plants and had lower water consumption than plants supplied with NO3–, either with high or low water supply. Specific leaf areas and leaf area ratios were higher with NH4+ than with NO3– or N2 as the N source. The difference observed in δ13C between plants grown with different N sources was higher than that predicted by theory and was not in the right direction (NH4+-grown plants with a more negative δ13C) to be explained by differences in plant composition and engagement of the various carboxylation reactions. The more positive δ13C in NH4+- than in NO3–-grown plants is probably due to a decreased ratio of stomatal to carboxylation conductances, which accounts for the lower water cost of C assimilation in NH4+-grown plants. [ABSTRACT FROM AUTHOR]

Casuarina glauca displays high levels of salt tolerance, but very little is known about how this tree adapts to saline conditions. To understand the molecular basis of C. glauca response to salt stress, we have analyzed the proteome from branchlets of plants nodulated by nitrogen-fixing Frankia Thr bacteria (NOD+) and non-nodulated plants supplied with KNO3 (KNO3+), exposed to 0, 200, 400, and 600 mM NaCl. Proteins were identified by Short Gel, Long Gradient Liquid Chromatography coupled to Tandem Mass Spectrometry and quantified by Sequential Window Acquisition of All Theoretical Mass Spectra -Mass Spectrometry. 600 proteins were identified and 357 quantified. Differentially Expressed Proteins (DEPs) were multifunctional and mainly involved in Carbohydrate Metabolism, Cellular Processes, and Environmental Information Processing. The number of DEPs increased gradually with stress severity: (i) from 7 (200 mM NaCl) to 40 (600 mM NaCl) in KNO3+; and (ii) from 6 (200 mM NaCl) to 23 (600 mM NaCl) in NOD+. Protein–protein interaction analysis identified different interacting proteins involved in general metabolic pathways as well as in the biosynthesis of secondary metabolites with different response networks related to salt stress. Salt tolerance in C. glauca is related to a moderate impact on the photosynthetic machinery (one of the first and most important stress targets) as well as to an enhancement of the antioxidant status that maintains cellular homeostasis. [ABSTRACT FROM AUTHOR]

• Secondary metabolites were identified in C. glauca plants exposed to salt stress. • Increasing salt concentrations led to an accumulation of secondary metabolites. • At 600 mM NaCl, C. glauca activates a flavonoid-based secondary antioxidant system. • Flavonols play a key role in mitigating oxidative stress in C. glauca plants. The model actinorhizal plant Casuarina glauca is characterized by its capacity to establish nitrogen-fixing root-nodule symbiosis with Frankia bacteria and by its resilience to thrive under extreme environments, such as high salinity. Previous studies have shown that salt tolerance in C. glauca is linked to photosynthetic, primary metabolic adjustments and to an effective antioxidant machinery. This study aimed at analyzing the secondary metabolite composition of tissues from nodulated (NOD+) and non-nodulated (KNO 3 +) C. glauca plants (nodules, roots, and branchlets) with liquid-chromatography-high resolution mass spectrometry (LC-HRMS) to evaluate the effect of three NaCl concentrations (200, 400 and 600 mM) on the secondary metabolite composition of these two plant groups. In the branchlets, significant accumulation of almost all annotated secondary metabolites, including flavonoids and their glycosides, was mainly induced by the highest salt concentration. Furthermore, the different secondary metabolite levels between roots and nodules were associated to the previously reported impairment of symbiotic activity from 200 mM NaCl onwards. Ionome analysis in C. glauca branchlets revealed salt stress-induced changes in ion homeostasis, which in turn, affected the K+/Na+ ratio. Nevertheless, despite these changes C. glauca plants can still maintain its primary metabolic activity. Moreover, our results suggest that C. glauca plants activate a flavonoid-based secondary antioxidant system to mitigate the impacts of oxidative stress, thereby enhancing their salt stress tolerance. [ABSTRACT FROM AUTHOR]

The activity of the promoter of a metallothionein gene expressed in actinorhizal nodules of Casuarina glauca Sieber ex Spreng., CgMT1, has previously been analysed in Casaurinaceae and in tobacco (Nicotiana tabacum L.), Arabidopsis and rice. In all these plants, the promoter showed high activity in the root cortex and epidermis, making it a useful tool for the expression of transgenes. Therefore, its activity was now analysed in transgenic root systems of Datisca glomerata (C. Presl) Baill, an actinorhizal plant from a different phylogenetic group than C. glauca, using the same CgMT1::GUS fusion as in previous studies. However, in contrast with all other plant species examined previously, the CgMT1::GUS construct showed no activity at all in D. glomerata hairy roots: the expression pattern in nodules resembled that found in C. glauca nodules. This is probably due to the changed hormone balance in hairy roots since experiments on the CgMT1::GUS construct in transgenic Arabidopsis showed that CgMT1 promoter activity was repressed by auxin or cytokinin, respectively. Yet, in hairy roots of the model legume Lotus japonicus L. induced by the same Agrobacterium rhizogenes strain, the CgMT1 promoter was active in roots and not in nodules. These results indicate that although the expression of pRi T-DNA genes leads to changes in root hormone balance, these changes do not abolish the differences in phytohormone levels or sensitivity between plant species. Therefore, gene expression data obtained using transgenic hairy root systems have to be viewed with care, not only due to the disturbed hormone balance, but also because the effects of the pRI-T-DNA genes can differ between species [ABSTRACT FROM AUTHOR]

In recent years, our understanding of the plant side of actinorhizal symbioses has evolved rapidly. No homologues of the common nod genes from rhizobia were found in the three Frankia genomes published so far, which suggested that Nod factor-like molecules would not be used in the infection of actinorhizal plants by Frankia. However, work on chimeric transgenic plants indicated that Frankia Nod factor equivalents signal via the same transduction pathway as rhizobial Nod factors. The role of auxin in actinorhizal nodule formation differs from that in legume nodulation. Great progress has been made in the analysis of pathogenesis-related and stress-related gene expression in nodules. Research on nodule physiology has shown the structural and metabolic diversity of actinorhizal nodules from different phylogenetic branches. The onset of large-scale nodule transcriptome analysis in different actinorhizal systems will provide access to more information on the symbiosis and its evolution. [ABSTRACT FROM AUTHOR]