Regulation of Small RNAs and Corresponding Targets in Nod Factor-Induced Phaseolus vulgaris Root Hair Cells.

A genome-wide analysis identified the set of small RNAs (sRNAs) from the agronomical important legume Phaseolus vulgaris (common bean), including novel P. vulgaris-specific microRNAs (miRNAs) potentially important for the regulation of the rhizobia-symbiotic process. Generally, novel miRNAs are difficult to identify and study because they are very lowly expressed in a tissue- or cell-specific manner. In this work, we aimed to analyze sRNAs from common bean root hairs (RH), a single-cell model, induced with pure Rhizobium etli nodulation factors (NF), a unique type of signal molecule. The sequence analysis of samples from NF-induced and control libraries led to the identity of 132 mature miRNAs, including 63 novel miRNAs and 1984 phasiRNAs. From these, six miRNAs were significantly differentially expressed during NF induction, including one novel miRNA: miR-RH82. A parallel degradome analysis of the same samples revealed 29 targets potentially cleaved by novel miRNAs specifically in NF-induced RH samples; however, these novel miRNAs were not differentially accumulated in this tissue. This study reveals Phaseolus vulgaris-specific novel miRNA candidates and their corresponding targets that meet all criteria to be involved in the regulation of the early nodulation events, thus setting the basis for exploring miRNA-mediated improvement of the common bean-rhizobia symbiosis.

Role of gibberellins during arbuscular mycorrhizal formation in tomato: new insights revealed by endogenous quantification and genetic analysis of their metabolism in mycorrhizal roots.

Gibberellins (GAs) are key regulators of plant growth and development and recent studies suggest also a role during arbuscular mycorrhizal (AM) formation. Here, complementary approaches have been used to obtain a clearer picture that correlates AM fungal development inside roots with GA metabolism. An extensive analysis of genes associated with GA metabolism as well as a quantification of GA content in roots was made. Application of GA3 and its biosynthesis inhibitor prohexadione calcium (PrCa) combined with a GA-constitutive response mutant (procera) were used to determine whether fungal colonization is altered by the level of these hormones or by changes in the GA-signaling pathway. The increased levels of specific GAs from the 13-hydroxylation pathway in mycorrhizal roots correlate closely with the increased expression of genes coding enzymes from the GA biosynthetic trail. The imbalance of GAs in tomato roots caused by exogenous applications of GA3 or PrCa affects arbuscules in both negative and positive ways, respectively. In addition, procera plants were adversely affected by the mycorrhization process. Our findings demonstrate that an imbalance in favor of an increased amount of GAs negatively affects the frequency of mycorrhization and particularly the arbuscular abundance in tomato mycorrhizal roots and the results point out that AM formation is associated with a change in the 13-hydroxylation pathway of GAs.

Ethylene-dependent/ethylene-independent ABA regulation of tomato plants colonized by arbuscular mycorrhiza fungi.

We investigated the relationship between ABA and ethylene regulating the formation of the arbuscular mycorrhiza (AM) symbiosis in tomato (Solanum lycopersicum) plants and tried to define the specific roles played by each of these phytohormones in the mycorrhization process. We analysed the impact of ABA biosynthesis inhibition on mycorrhization by Glomus intraradices in transgenic tomato plants with an altered ethylene pathway. We also studied the effects on mycorrhization in sitiens plants treated with the aminoethoxyvinyl glycine hydrochloride (AVG) ethylene biosynthesis inhibitor and supplemented with ABA. In addition, the expression of plant and fungal genes involved in the mycorrhization process was studied. ABA biosynthesis inhibition qualitatively altered the parameters of mycorrhization in accordance with the plant's ethylene perception and ethylene biosynthesis abilities. Inhibition of ABA biosynthesis in wild-type plants negatively affected all the mycorrhization parameters studied, while tomato mutants impaired in ethylene synthesis only showed a reduced arbuscular abundance in mycorrhizal roots. Inhibition of ethylene synthesis in ABA-deficient sitiens plants increased the intensity of mycorrhiza development, while ABA application rescued arbuscule abundance in the root's mycorrhizal zones. The results of our study show an antagonistic interaction between ABA and ethylene, and different roles of each of the two hormones during AM formation. This suggests that a dual ethylene-dependent/ethylene-independent mechanism is involved in ABA regulation of AM formation.

Functional Analysis of Root microRNAs by a Constitutive Overexpression Approach in a Composite Plant System.

Agrobacterium-mediated transformation is a fast and efficient method for genome modification in plants. In this protocol, we apply this technique for the analysis of root microRNA functionality. The induction of hairy roots constitutively overexpressing a given microRNA precursor allows us, in a simple way, to modify the accumulation of specific mature microRNA and analyze the consequence of this alteration on a phenotype of interest. This method generates ready-to-phenotype "composite plants" with untransformed aerial part and microRNA-overexpressing root system, in about 20 days.

Identification and expression analysis of the arbuscular mycorrhiza-inducible Rieske non-heme oxygenase Ptc52 gene from tomato.

Arbuscular mycorrhizal (AM) formation enhances plant growth and fitness through improved uptake of water and mineral nutrients in exchange for carbon compounds to the AM fungus. The fungal structure for the reciprocal exchange of nutrients in the symbiosis is the arbuscule, and defence genes expressed in cells containing arbuscules could play a role in the control of hyphal spread and arbuscule formation in the root. We characterized and analyzed the Ptc52 gene from tomato (SlPtc52), a member of the gene family of non-heme oxygenases, whose function has been related to the lethal leaf spot 1 (Lls1) lesion mimic phenotype in plants which is sometimes associated with enhanced disease resistance. Sequence analysis of the SlPTC52 protein revealed conserved typical motifs from non-heme oxygenases, including a Rieske [2Fe-2S] motif, a mononuclear non-heme iron-binding motif and a C-terminal CxxC motif. The level of transcript accumulation was low in stem, flower and green fruits, and high in leaves. Although SlPtc52 expression was perceptible at low levels in roots, its expression increased concomitantly with AM fungus root colonization. Tomato non-mycorrhizal hairy roots expressing the GUS protein under the control of promoter SlPtc52 exhibited GUS activity in the endodermis, the apical meristem of the root tip and in the lateral root primordium. AM fungal colonization also resulted in intensive GUS activity that clearly corresponds to cortical cells containing arbuscules. SlPtc52 gene silencing led to a delay in root colonization and a decrease in arbuscular abundance, suggesting that SlPTC52 plays a regulatory role during AM symbiosis.