Veronica Volpe

Two putative-aquaporin genes are differentially expressed during arbuscular mycorrhizal symbiosis in Lotus japonicus

BackgroundArbuscular mycorrhizas (AM) are widespread symbioses that provide great advantages to the plant, improving its nutritional status and allowing the fungus to complete its life cycle. Nevertheless, molecular mechanisms that lead to the development of AM symbiosis are not yet fully deciphered. Here, we have focused on two putative aquaporin genes, LjNIP1 and LjXIP1, which resulted to be upregulated in a transcriptomic analysis performed on mycorrhizal roots of Lotus japonicus.ResultsA phylogenetic analysis has shown that the two putative aquaporins belong to different functional families: NIPs and XIPs. Transcriptomic experiments have shown the independence of their expression from their nutritional status but also a close correlation with mycorrhizal and rhizobial interaction. Further transcript quantification has revealed a good correlation between the expression of one of them, LjNIP1, and LjPT4, the phosphate transporter which is considered a marker gene for mycorrhizal functionality. By using laser microdissection, we have demonstrated that one of the two genes, LjNIP1, is expressed exclusively in arbuscule-containing cells. LjNIP1, in agreement with its putative role as an aquaporin, is capable of transferring water when expressed in yeast protoplasts. Confocal analysis have demonstrated that eGFP-LjNIP1, under its endogenous promoter, accumulates in the inner membrane system of arbusculated cells.ConclusionsOverall, the results have shown different functionality and expression specificity of two mycorrhiza-inducible aquaporins in L. japonicus. One of them, LjNIP1 can be considered a novel molecular marker of mycorrhizal status at different developmental stages of the arbuscule. At the same time, LjXIP1 results to be the first XIP family aquaporin to be transcriptionally regulated during symbiosis.

An AM‐induced, MYB‐family gene of Lotus japonicus (LjMAMI) affects root growth in an AM‐independent manner

The interaction between legumes and arbuscular mycorrhizal (AM) fungi is vital to the development of sustainable plant production systems. Here, we focus on a putative MYB-like (LjMAMI) transcription factor (TF) previously reported to be highly upregulated in Lotus japonicus mycorrhizal roots. Phylogenetic analyses revealed that the protein is related to a group of TFs involved in phosphate (Pi) starvation responses, the expression of which is independent of the Pi level, such as PHR1. GUS transformed plants and quantitative reverse transcription PCR revealed strong gene induction in arbusculated cells, as well as the presence of LjMAMI transcripts in lateral root primordia and root meristems, even in the absence of the fungus, and independently of Pi concentration. In agreement with its putative identification as a TF, an eGFP-LjMAMI chimera was localized to the nuclei of plant protoplasts, whereas in transgenic Lotus roots expressing the eGFP-LjMAMI fusion protein under the control of the native promoter, the protein was located in the nuclei of the arbusculated cells. Further expression analyses revealed a correlation between LjMAMI and LjPT4, a marker gene for mycorrhizal function. To elucidate the role of the LjMAMI gene in the mycorrhizal process, RNAi and overexpressing root lines were generated. All the lines retained their symbiotic capacity; however, RNAi root lines and composite plants showed an important reduction in root elongation and branching in the absence of the symbiont. The results support the involvement of the AM-responsive LjMAMI in non-symbiotic functions: i.e. root growth.

The phosphate transporters LjPT4 and MtPT4 mediate early root responses to phosphate status in non mycorrhizal roots

Arbuscular mycorrhizal (AM) symbiosis improves host plant phosphorous (P) status and elicits the expression of AM-inducible phosphate transporters (PTs) in arbuscule-containing cells, where they control arbuscule morphogenesis and P release. We confirmed such functions for LjPT4 in mycorrhizal Lotus japonicus. Promoter-GUS experiments showed LjPT4 transcription not only in arbusculated cells but also in root tips, in the absence of the fungus: here LjPT4 transcription profile depended on the phosphate level. In addition, quantitative RT-PCR confirmed the expression of Lotus and Medicago truncatula PT4 in the tips of non-mycorrhizal roots. Starting from these observations, we hypothesized that AM-inducible PTs may have a regulatory role in plant development, irrespective of the fungal presence. Firstly, we focused on root development responses to different phosphate treatments in both plants demonstrating that phosphate starvation induced a higher number of lateral roots. By contrast, Lotus PT4i plants and Medicago mtpt4 mutants did not show any differential response to phosphate levels, suggesting that PT4 genes affect early root branching. Phosphate starvation-induced genes and a key auxin receptor, MtTIR1, showed an impaired expression in mtpt4 plants. We suggest PT4 genes as novel components of the P-sensing machinery at the root tip level, independently of AM fungi.

LjLHT1.2—a mycorrhiza-inducible plant amino acid transporter from Lotus japonicus

In mycorrhizal associations, the fungal partner assists its plant host by providing nitrogen (N) in addition to phosphate. Arbuscular mycorrhizal (AM) fungi have access to inorganic and organic forms of N and translocate them, via arginine, from the extra- to the intraradical mycelium, where N is transferred to the plant as inorganic N compounds such as ammonium. However, several putative amino acid transporters (AATs) with an altered expression in Lotus japonicus mycorrhizal roots were recorded in a previous microarray-based investigation, which led to the question of whether a transfer of organic N, mainly in the form of amino acids, could occur in AM roots. Here, we have characterized an AAT gene (LjLHT1.2) that encodes for lysine–histidine–transporter (LHT)-type amino acid transporter. We show that it is induced in mycorrhizas, but not in nodulated roots. By using in situ hybridization and laser microdissection technology, the corresponding transcripts have been demonstrated to be located above all in arbusculated cells but also in the non-colonized cells of the root cortex. The gene expression resulted to be differentially regulated by the availability of the N sources. Furthermore, functional experiments, via heterologous expression in yeast, have demonstrated that the protein was a high-affinity amino acid transporter. Taken together, the results show that LjLHT1.2 may allow the uptake of energy-rich N compounds, such as amino acids, towards the cortical cells. We suggest that LjLHT1.2 could be involved in complex mechanisms that guarantee the re-uptake and recycle of amino acids and which are particularly efficient in mycorrhizal roots.

Modulation of enzyme activities and expression of genes related to primary and secondary metabolism in response to UV-B stress in cucumber (Cucumis sativus L.)

Abstract Exposure to UV-B at ambient or enhanced levels is known to trigger a variety of responses in all living organisms, including higher plants. Here we show that in Cucumis sativus L. UV-B radiation affects enzyme activity of key oxydative pentose phosphate pathway (OPPP) enzymes glucose-6-phosphate dehydrogenase (G6P-DH) and 6-phosphogluconate dehydrogenase (6-PGlu-DH), of key phenolic compounds enzyme phenylalanine ammonia lyase (PAL) as well as erythrose-4-phosphate, tryptophan and tyrosine levels. Furthermore, we found an increased activity of antioxidant enzymes such as peroxidase (POX) and catalase (CAT) in treated plants, with respect to the controls. In order to confirm the biochemical results, we isolated total RNA from both controls and UV-B treated plants to be used for gene expression analysis. We demonstrated that UV-B increases the gene expression level of peroxidase (POX), catalase (CAT) and phenylalanine ammonia lyase (PAL). Finally, our results are useful for understanding protective strategies against UV-B radiation and for elucidating what components are involved in stress-induced signals within the plant.

The Lotus japonicus MAMI gene links root development, arbuscular mycorrhizal symbiosis and phosphate availability

The arbuscular mycorrhizal-induced LjMAMI gene is phylogenetically related to GARP transcription factors involved in Pi-starvation responses such as AtPHR1. The gene is strongly upregulated in arbusculated cells from mycorrhizal plants and in root meristems, irrespectively of the fungal presence. A further expression analysis revealed a similar expression pattern for LjPT4, considered a marker gene for mycorrhizal functionality. Here we show that the LjPT4 promoter contains two conserved cis-acting elements typically found in Pi-starvation induced Pi transporters. One of these is strongly related to the binding site of AtPHR1, suggesting a direct regulation of LjPT4 by LjMAMI. The expression of both genes in non-mycorrhizal tissues leads to the hypothesis that these symbiosis-inducible genes are also involved in Pi starvation responses in root meristems in an AM-independent manner.

Ectopic activation of cortical cell division during the accommodation of arbuscular mycorrhizal fungi

Arbuscular mycorrhizas (AMs) between plants and soil fungi are widespread symbioses with a major role in soil nutrient uptake. In this study we investigated the induction of root cortical cell division during AM colonization by combining morphometric and gene expression analyses with promoter activation and protein localization studies of the cell-plate-associated exocytic marker TPLATE. Our results show that TPLATE promoter is activated in colonized cells of the root cortex where we also observed the appearance of cells that are half the size of the surrounding cells. Furthermore, TPLATE-green fluorescent protein recruitment to developing cell plates highlighted ectopic cell division events in the inner root cortex during early AM colonization. Lastly, transcripts of TPLATE, KNOLLE and Cyclinlike 1 (CYC1) are all upregulated in the same context, alongside endocytic markers Adaptor-Related Protein complex 2 alpha 1 subunit (AP2A1) and Clathrin Heavy Chain 2 (CHC2), known to be active during cell plate formation. This pattern of gene expression was recorded in wild-type Medicago truncatula roots, but not in a common symbiotic signalling pathway mutant where fungal colonization is blocked at the epidermal level. Altogether, these results suggest the activation of cell-division-related mechanisms by AM hosts during the accommodation of the symbiotic fungus.

The Association With Two Different Arbuscular Mycorrhizal Fungi Differently Affects Water Stress Tolerance in Tomato

Arbuscular mycorrhizal (AM) fungi are very widespread, forming symbiotic associations with ∼80% of land plant species, including almost all crop plants. These fungi are considered of great interest for their use as biofertilizer in low-input and organic agriculture. In addition to an improvement in plant nutrition, AM fungi have been reported to enhance plant tolerance to important abiotic and biotic environmental conditions, especially to a reduced availability of resources. These features, to be exploited and applied in the field, require a thorough identification of mechanisms involved in nutrient transfer, metabolic pathways induced by single and multiple stresses, physiological and eco-physiological mechanisms resulting in improved tolerance. However, cooperation between host plants and AM fungi is often related to the specificity of symbiotic partners, the environmental conditions and the availability of resources. In this study, the impact of two AM fungal species (Funneliformis mosseae and Rhizophagus intraradices) on the water stress tolerance of a commercial tomato cultivar (San Marzano nano) has been evaluated in pots. Biometric and eco-physiological parameters have been recorded and gene expression analyses in tomato roots have been focused on plant and fungal genes involved in inorganic phosphate (Pi) uptake and transport. R. intraradices, which resulted to be more efficient than F. mosseae to improve physiological performances, was selected to assess the role of AM symbiosis on tomato plants subjected to combined stresses (moderate water stress and aphid infestation) in controlled conditions. A positive effect on the tomato indirect defense toward aphids in terms of enhanced attraction of their natural enemies was observed, in agreement with the characterization of volatile organic compound (VOC) released. In conclusion, our results offer new insights for understanding the molecular and physiological mechanisms involved in the tolerance toward water deficit as mediated by a specific AM fungus. Moreover, they open new perspectives for the exploitation of AM symbiosis to enhance crop tolerance to abiotic and biotic stresses in a scenario of global change.