Enhanced expression of OsNAC5 leads to up-regulation of OsNAC6 and changes rice (Oryza sativa L.) ionome.

NAC transcription factors are plant-specific proteins involved in many processes during the plant life cycle and responses to biotic and abiotic stresses. Previous studies have shown that stress-induced OsNAC5 from rice (Oryza sativa L.) is up-regulated by senescence and might be involved in control of iron (Fe) and zinc (Zn) concentrations in rice seeds. Aiming a better understanding of the role of OsNAC5 in rice plants, we investigated a mutant line carrying a T-DNA insertion in the promoter of OsNAC5, which resulted in enhanced expression of the transcription factor. Plants with OsNAC5 enhanced expression were shorter at the seedling stage and had reduced yield at maturity. In addition, we evaluated the expression level of OsNAC6, which is co-expressed with OsNAC5, and found that enhanced expression of OsNAC5 leads to increased expression of OsNAC6, suggesting that OsNAC5 might regulate OsNAC6 expression. Ionomic analysis of leaves and seeds from the OsNAC5 enhanced expression line revealed lower Fe and Zn concentrations in leaves and higher Fe concentrations in seeds than in WT plants, further suggesting that OsNAC5 may be involved in regulating the ionome in rice plants. Our work shows that fine-tuning of transcription factors is key when aiming at crop improvement.

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic factors is very relevant to help in understanding the processes of local adaptation. 1-Pyrroline-5-carboxylate synthetase (P5CS) is one interesting gene to study in this species since it encodes a key enzyme of proline biosynthesis, which is an osmoprotectant during abiotic stress. Applying in silico analysis, we identified one P5CS gene sequence of E. uniflora (EuniP5CS). Phylogenetic analysis, as well as, gene and protein structure investigation, revealed that EuniP5CS is a member of P5CS gene family. Plants of E. uniflora from two distinct environments (restinga and riparian forest) presented differences in the proline accumulation and P5CS expression levels under growth-controlled conditions. Both proline accumulation and gene expression level of EuniP5CS were higher in the genotypes from riparian forest than those from restinga. When these plants were submitted to drought stress, EuniP5CS gene was up-regulated in the plants from restinga, but not in those from riparian forest. These results demonstrated that EuniP5CS is involved in proline biosynthesis in this species and suggest that P5CS gene may be an interesting candidate gene in future studies to understand the processes of local adaptation in E. uniflora.

The Combined Strategy for iron uptake is not exclusive to domesticated rice (Oryza sativa).

Iron (Fe) is an essential micronutrient that is frequently inaccessible to plants. Rice (Oryza sativa L.) plants employ the Combined Strategy for Fe uptake, which is composed by all features of Strategy II, common to all Poaceae species, and some features of Strategy I, common to non-Poaceae species. To understand the evolution of Fe uptake mechanisms, we analyzed the root transcriptomic response to Fe deficiency in O. sativa and its wild progenitor O. rufipogon. We identified 622 and 2,017 differentially expressed genes in O. sativa and O. rufipogon, respectively. Among the genes up-regulated in both species, we found Fe transporters associated with Strategy I, such as IRT1, IRT2 and NRAMP1; and genes associated with Strategy II, such as YSL15 and IRO2. In order to evaluate the conservation of these Strategies among other Poaceae, we identified the orthologs of these genes in nine species from the Oryza genus, maize and sorghum, and evaluated their expression profile in response to low Fe condition. Our results indicate that the Combined Strategy is not specific to O. sativa as previously proposed, but also present in species of the Oryza genus closely related to domesticated rice, and originated around the same time the AA genome lineage within Oryza diversified. Therefore, adaptation to Fe2+ acquisition via IRT1 in flooded soils precedes O. sativa domestication.

Genotype Variation in Rice (Oryza sativa L.) Tolerance to Fe Toxicity Might Be Linked to Root Cell Wall Lignification.

Iron (Fe) is an essential element to plants, but can be harmful if accumulated to toxic concentrations. Fe toxicity can be a major nutritional disorder in rice (Oryza sativa) when cultivated under waterlogged conditions, as a result of excessive Fe solubilization of in the soil. However, little is known about the basis of Fe toxicity and tolerance at both physiological and molecular level. To identify mechanisms and potential candidate genes for Fe tolerance in rice, we comparatively analyzed the effects of excess Fe on two cultivars with distinct tolerance to Fe toxicity, EPAGRI 108 (tolerant) and BR-IRGA 409 (susceptible). After excess Fe treatment, BR-IRGA 409 plants showed reduced biomass and photosynthetic parameters, compared to EPAGRI 108. EPAGRI 108 plants accumulated lower amounts of Fe in both shoots and roots compared to BR-IRGA 409. We conducted transcriptomic analyses of roots from susceptible and tolerant plants under control and excess Fe conditions. We found 423 up-regulated and 92 down-regulated genes in the susceptible cultivar, and 42 up-regulated and 305 down-regulated genes in the tolerant one. We observed striking differences in root gene expression profiles following exposure to excess Fe: the two cultivars showed no genes regulated in the same way (up or down in both), and 264 genes were oppositely regulated in both cultivars. Plants from the susceptible cultivar showed down-regulation of known Fe uptake-related genes, indicating that plants are actively decreasing Fe acquisition. On the other hand, plants from the tolerant cultivar showed up-regulation of genes involved in root cell wall biosynthesis and lignification. We confirmed that the tolerant cultivar has increased lignification in the outer layers of the cortex and in the vascular bundle compared to the susceptible cultivar, suggesting that the capacity to avoid excessive Fe uptake could rely in root cell wall remodeling. Moreover, we showed that increased lignin concentrations in roots might be linked to Fe tolerance in other rice cultivars, suggesting that a similar mechanism might operate in multiple genotypes. Our results indicate that changes in root cell wall and Fe permeability might be related to Fe toxicity tolerance in rice natural variation.

AtGRP3 Is Implicated in Root Size and Aluminum Response Pathways in Arabidopsis.

AtGRP3 is a glycine-rich protein (GRP) from Arabidopsis thaliana shown to interact with the receptor-like kinase AtWAK1 in yeast, in vitro and in planta. In this work, phenotypic analyses using transgenic plants were performed in order to better characterize this GRP. Plants of two independent knockout alleles of AtGRP3 develop longer roots suggesting its involvement in root size determination. Confocal microscopy analysis showed an abnormal cell division and elongation in grp3-1 knockout mutants. Moreover, we also show that grp3-1 exhibits an enhanced Aluminum (Al) tolerance, a feature also described in AtWAK1 overexpressing plants. Together, these results implicate AtGRP3 function root size determination during development and in Al stress.

Increased senescence-associated gene expression and lipid peroxidation induced by iron deficiency in rice roots.

Iron deficiency is among the most common nutritional disorders in plants. Low iron supply causes decreased root growth and even plant death. However, there are no reports about the specific pathways that lead Fe-deficient roots to senescence and death. To investigate the molecular mechanisms that regulate rice roots response to Fe-deficiency, rice seedlings were grown for 3, 6 and 9 days in the presence or absence of Fe. Sequences of 28 induced genes in rice roots under Fe-deficiency were identified by representational difference analysis (RDA). About 40% of these sequences have been previously reported as senescence-related. Differential expression of selected genes was confirmed by semi-quantitative RT-PCR analysis. Classical senescence-related sequences, such as MYB and WRKY transcription factors, cysteine protease, ubiquitin-conjugating enzyme, lipid transfer protein, fatty acid hydroxylase, beta-glucosidase and cytochrome P450 oxydoreductase were identified. Fe-deficiency also resulted in decreased dry weight, increased lipid peroxidation (detected by TBA and histochemical methods) as well as evident membrane damage in Fe-deficient roots. Taken together, the results indicate that Fe-deficiency in roots is linked to typical senescence pathways, associated with lipid peroxidation.