Validation of the Interaction Between Stress-Responsive LncRNAs and RNA-Binding Proteins In Vitro.

Plant long non-coding RNAs (lncRNAs) have been implicated in many biological processes, including responses to abiotic stresses, yet their detailed functions and especially their modes of action are still underexplored. lncRNAs often interact with proteins to participate in multiple levels of gene regulation. Therefore, identifying the RNA-binding proteins and validating their interaction with lncRNAs will be instrumental in revealing the functions of lncRNAs. Here, we describe two major methods to determine the interaction between lncRNA and proteins in vitro, RNA pull-down, and RNA EMSA.

Nodulation Signaling Pathway 1 and 2 Modulate Vanadium Accumulation and Tolerance of Legumes.

Vanadium (V) pollution potentially threatens human health. Here, it is found that nsp1 and nsp2, Rhizobium symbiosis defective mutants of Medicago truncatula, are sensitive to V. Concentrations of phosphorus (P), iron (Fe), and sulfur (S) with V are negatively correlated in the shoots of wild-type R108, but not in mutant nsp1 and nsp2 shoots. Mutations in the P transporter PHT1, PHO1, and VPT families, Fe transporter IRT1, and S transporter SULTR1/3/4 family confer varying degrees of V tolerance on plants. Among these gene families, MtPT1, MtZIP6, MtZIP9, and MtSULTR1; 1 in R108 roots are significantly inhibited by V stress, while MtPHO1; 2, MtVPT2, and MtVPT3 are significantly induced. Overexpression of Arabidopsis thaliana VPT1 or M. truncatula MtVPT3 increases plant V tolerance. However, the response of these genes to V is weakened in nsp1 or nsp2 and influenced by soil microorganisms. Mutations in NSPs reduce rhizobacterial diversity under V stress and simplify the V-responsive operational taxonomic unit modules in co-occurrence networks. Furthermore, R108 recruits more beneficial rhizobacteria related to V, P, Fe, and S than does nsp1 or nsp2. Thus, NSPs can modulate the accumulation and tolerance of legumes to V through P, Fe, and S transporters, ion homeostasis, and rhizobacterial community responses.

Rhizobium symbiosis modulates the accumulation of arsenic in Medicago truncatula via nitrogen and NRT3.1-like genes regulated by ABA and linalool.

Arsenic (As) contamination is a worldwide problem and threatens human health. Here, we found that Rhizobium symbiosis can improve the tolerance to arsenate [As(V)], and a wild type R. meliloti Rm5038 symbiosis can significantly decrease the accumulation of As in Medicago truncatula shoots. The As content in plants could be decreased by nitrogen and the mutation of nitrate transporter NRT3.1. The expression of M. truncatula NRT3.1-like gene NRT3.1L1 could reverse the As(V)-tolerance phenotype of the Arabidopsis nrt3.1 mutant. Rm5038 symbiosis significantly increased the level of nitrogen in the shoot and reduced the expression of NRT3.1Ls in plants afflicted by As(V). The genetic analyses of aba2-1, pyr1/pyl1/2/4/5/8, and abi1-2/abi2-2/hab1-1/pp2ca-1 mutants revealed that abscisic acid (ABA) signaling regulates the tolerance of plants to As(V). ABA and linalool could promote the expression of NRT3.1Ls, however, their root biosynthesis was inhibited by ammonium, the first form of nitrogen fixed by Rhizobium symbiosis. Moreover, ABA and linalool may also control As and nitrate accumulation in Rhizobium symbionts via signaling pathways other than ammonia and NRT3.1Ls. Thus, Rhizobium symbiosis modulates the accumulation of As in plants via nitrogen and NRT3.1Ls regulated by ABA and linalool, which provides novel approaches to reduce As accumulation in legume crops.

Putrescine metabolism modulates the biphasic effects of brassinosteroids on canola and Arabidopsis salt tolerance.

Brassinosteroids (BRs) are known to improve salt tolerance of plants, but not in all situations. Here, we show that a certain concentration of 24-epibrassinolide (EBL), an active BR, can promote the tolerance of canola under high-salt stress, but the same concentration is disadvantageous under low-salt stress. We define this phenomenon as hormonal stress-level-dependent biphasic (SLDB) effects. The SLDB effects of EBL on salt tolerance in canola are closely related to H2 O2 accumulation, which is regulated by polyamine metabolism, especially putrescine (Put) oxidation. The inhibition of EBL on canola under low-salt stress can be ameliorated by repressing Put biosynthesis or diamine oxidase activity to reduce H2 O2 production. Genetic and phenotypic results of bri1-9, bak1, bes1-D, and bzr1-1D mutants and overexpression lines of BRI1 and BAK1 in Arabidopsis indicate that a proper enhancement of BR signaling benefits plants in countering salt stress, whereas excessive enhancement is just as harmful as a deficiency. These results highlight the involvement of crosstalk between BR signaling and Put metabolism in H2 O2 accumulation, which underlies the dual role of BR in plant salt tolerance.