Salt acclimation in sorghum plants by exogenous proline: physiological and biochemical changes and regulation of proline metabolism.

KEY MESSAGE: Mitigation of deleterious effects of salinity promoted by exogenous proline can be partially explained by changes in proline enzymatic metabolism and expression of specific proline-related genes. Proline accumulation is a usual response to salinity. We studied the ability of exogenous proline to mitigate the salt harmful effects in sorghum (Sorghum bicolor) leaves. Ten-day-old plants were cultivated in Hoagland's nutrient solution in either the absence or presence of salinity (NaCl at 75 mM) and sprayed with distilled water or 30 mM proline solution. Salinity deleterious effects were alleviated by exogenous proline 14 days after treatment, with a return in growth and recovery of leaf area and photosynthetic parameters. Part of the salinity response reflected an improvement in ionic homeostasis, provided by reduction in Na+ and Cl- ions and increases in K+ and Ca2+ ions as well as increases of compatible solutes. In addition, the application of proline decreased membrane damage and did not increase relative water content. Proline-treated salt-stressed plants displayed increase in proline content, a response counterbalanced by punctual modulation in proline synthesis (down-regulation of Δ1-pyrroline-5-carboxylate synthetase activity) and degradation (up-regulation of proline dehydrogenase activity) enzymes. These responses were correlated with expression of specific proline-related genes (p5cs1 and prodh). Our findings clearly show that proline treatment results in favorable changes, reducing salt-induced damage and improving salt acclimation in sorghum plants.

Polymorphisms in plastoquinol oxidase (PTOX) from Arabidopsis accessions indicate SNP-induced structural variants associated with altitude and rainfall.

Plant plastoquinol oxidase (PTOX) is a chloroplast oxidoreductase involved in carotenoid biosynthesis, chlororespiration, and response to environmental stresses. The present study aimed to gain insight of the potential role of nucleotide/amino acid changes linked to environmental adaptation in PTOX gene/protein from Arabidopsis thaliana accessions. SNPs in the single-copy PTOX gene were identified in 1190 accessions of Arabidopsis using the Columbia-0 PTOX as a reference. The identified SNPs were correlated with geographical distribution of the accessions according to altitude, climate, and rainfall. Among the 32 identified SNPs in the coding region of the PTOX gene, 16 of these were characterized as non-synonymous SNPs (in which an AA is altered). A higher incidence of AA changes occurred in the mature protein at positions 78 (31%), 81 (31.4%), and 323 (49.9%). Three-dimensional structure prediction indicated that the AA change at position 323 (D323N) leads to a PTOX structure with the most favorable interaction with the substrate plastoquinol, when compared with the reference PTOX structure (Columbia-0). Molecular docking analysis suggested that the most favorable D323N PTOX-plastoquinol interaction is due to a better enzyme-substrate binding affinity. The molecular dynamics revealed that plastoquinol should be more stable in complex with D323N PTOX, likely due a restraint mechanism in this structure that stabilize plastoquinol inside of the reaction center. The integrated analysis made from accession geographical distribution and PTOX SNPs indicated that AA changes in PTOX are related to altitude and rainfall, potentially due to an adaptive positive environmental selection.

In silico identification of alternative oxidase 2 (AOX2) in monocots: A new evolutionary scenario.

We identified AOX2 genes in monocot species from Lemnoideae (Spirodela polyrhiza, Lemna gibba and Landoltia punctata), Pothoideae (Anthurium andraeanum and Anthurium amnicola) and Monsteroideae (Epipremnum aureum) subfamilies within the Araceae, an early-diverging monocot family. These findings highlight the presence of AOX2 in the most ancient monocot ancestor and also that at least partial loss of this gene occurred during speciation events within several monocot orders. The presence of AOX2 in monocot species challenges (1) new understanding of the evolutionary history of the AOX gene family in angiosperms and (2) drives experimental and bioinformatics efforts to explore functional relevance of the two AOX gene family members for plant growth and development. Knowledge gain in this field will impact running strategies on AOX-derived functional marker candidate development for plant breeding.

Phylogenetic analysis and differential expression of EF1α genes in soybean during development, stress and phytohormone treatments.

The EF1α is a multifunctional protein with additional unrelated activities to its primary function in translation. This protein is encoded by a multigene family and few studies are still available in plants. Expression of six EF1α genes in Glycine max was performed using RT-qPCR and RNA-seq data to advance in the function of each gene during plant development, stress conditions and phytohormone treatments. A phylogenetic classification in Phaseoleae tribe was used to identify the G. max EF1α genes (EF1α 1a1, 1a2, 1b, 2a, 2b and 3). Three EF1α types (1-3) were found in Phaseoleae revealing duplications in G. max types 1 and 2. EF1α genes were expressed in all studied tissues, however, specific amount of each transcript was detected. In plant development, all EF1α transcripts were generally more expressed in younger tissues, however, in unifoliolate leaves and cotyledons a higher expression occurred in older tissues. Five EF1α genes (except 2a) were up-regulated under stress in a response tissue/stress/cultivar-dependent. EF1α 3 was the most stress-induced gene linked to cultivar stress tolerance mainly in aerial tissues. Auxin, salicylate and ethylene induced differentially the EF1α expression. Overall, this study provides a consistent EF1α classification in Phaseoleae tribe to better understand their functional evolution. The RT-qPCR and RNA-seq EF1α expression profiles were consistent, both exhibiting expression diversification of each gene (spatio-temporal, stress and phytohormone stimuli). Our results point out the EF1α genes, especially EF1α 3, as candidate for developing a useful tool for future G. max breeding.

Identification of duplicated and stress-inducible Aox2b gene co-expressed with Aox1 in species of the Medicago genus reveals a regulation linked to gene rearrangement in leguminous genomes.

In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is reported as a stress-responsive gene, but in the leguminous Vigna unguiculata Aox2b is also induced by stress. In this work we investigated the Aox genes from two leguminous species of the Medicago genus (Medicago sativa and Medicago truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M. sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed germination. Similar analyses in leaves and roots under different treatments (SA, PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of expression under control conditions and were induced by stress in leaves and roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress conditions in leaves, and showed basal expression levels in roots that were induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and during seed germination than in roots and appeared to be not responsive to stress. The Aox expression profiles obtained from a M. truncatula microarray dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2 in leaves and roots. These results reinforce the stress-inducible co-expression of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates that this regulation is linked to Aox1/Aox2b proximity in the genome as a result of the gene rearrangement that occurred in some leguminous plants during evolution. The differential expression of Aox2b1/2b2 suggests that a second gene has been originated by recent gene duplication with neofunctionalization.