Global DNA methylation changes in response to recurrent drought stress were investigated in two common Greek Medicago sativa L. varieties (Lamia and Chaironia-Institute of Ιndustrial and Forage Crops). The water deficit was implemented in two phases. At the end of the first phase, which lasted for 60 days, the plants were cut at the height of 5 cm and were watered regularly for two months before being subjected to the second drought stress, which lasted for two weeks. Finally, the following groups of plants were formed: CC (controls both in phase I and phase II), CD2 (Controls in phase I, experiencing drought in phase II), and D1D2 (were subjected to drought in both phase I and phase II). At the end of phase II, samples were taken for global DNA methylation analysis with the Methylation Sensitive Amplification Polymorphism (MSAP) method, and all plants were harvested in order to measure the fresh and dry weight of roots and shoots. The variety Lamia responded better, especially the D1D2 group, compared to Chaironia in terms of root and shoot dry weight. Additionally, the shoots of Lamia had a constant water status for CD2 and D1D2 group of plants. According to DNA methylation analysis by the MSAP method, Lamia had lower total DNA methylation percentage after the second drought episode (D1D2) as compared to the plants CD2 that had experienced only one drought episode. On the other hand, the total DNA methylation percentage of Chaironia was almost the same in plants grown under recurrent drought stress conditions compared to control plants. In conclusion, the decrease of DNA methylation of Lamia stressed plants probably indicates the existence of an epigenetic mechanism that may render drought tolerance.

Plants have developed sophisticated mechanisms for acquiring iron from the soil. In the graminaceous species, a chelation strategy is in charge, in order to take up ferric iron from the rhizosphere. The ferric iron chelation-strategy components may also be present in the aerial plant parts. The aim of this work was to search for possible roles of those components in maize leaves. To this end, the expression patterns of ferric iron chelation-strategy components were monitored in the leaves and roots of mycorrhizal and non-mycorrhizal sulfur-deprived maize plants, both before and after sulfate supply. The two levels of sulfur supply were chosen due to the strong impact of this nutrient on iron homeostasis, whilst mycorrhizal symbiosis was chosen as a treatment that forces the plant to optimize its photosynthetic efficiency, in order to feed the fungus. The results, in combination with the findings of our previous works, suggest a role for the aforementioned components in ferric chelation and/or unloading from the xylem vessels to the aerial plant parts. It is proposed that the gene expression of the DMA exporter ZmTOM1 can be used as an early indicator for the establishment of a mycorrhizal symbiotic relationship in maize.

Sulfur is an essential macronutrient for growth of higher plants. The entry of the sulfate anion into the plant, its importation into the plastids for assimilation, its long-distance transport through the vasculature, and its storage in the vacuoles require specific sulfate transporter proteins. In this study, mycorrhizal and non-mycorrhizal maize plants were grown for 60 days in an S-deprived substrate, whilst iron was provided to the plants in the sparingly soluble form of FePO₄. On day 60, sulfate was provided to the plants. The gene expression patterns of a number of sulfate transporters as well as sulfate assimilation enzymes were studied in leaves and roots of maize plants, both before as well as after sulfate supply. Prolonged sulfur deprivation resulted in a more or less uniform response of the genes’ expressions in the roots of non-mycorrhizal and mycorrhizal plants. This was not the case neither in the roots and leaves after the supply of sulfur, nor in the leaves of the plants during the S-deprived period of time. It is concluded that mycorrhizal symbiosis modified plant demands for reduced sulfur, regulating accordingly the uptake, distribution, and assimilation of the sulfate anion.

Iron (Fe) is an essential element for plant growth and productivity, and human and animal diets rely on Fe from plant sources. Despite the large number of studies on plants’ Fe deficiency responses, considerably less is known about the morphological and anatomical alterations that root systems of plants undergo, especially in the graminaceous plants following a chelation strategy to take up Fe³⁺ from the rhizosphere. A stress symptom observed in Fe-deprived maize plants is an ectopic lateral root branching occurring at the terminal 5 cm of the root. In order to understand this response, one-week-old maize seedlings were placed in containers with either full nutrient solution, or nutrient solution lacking an Fe source. Control and Fe-deprived plants were grown for another 14 days, and the trait of ectopic lateral root branching was observed both on roots that emerged before the onset of Fe deprivation, as well as on roots that emerged after the onset of the deprivation. Ongoing in silico analysis of a quantitative trait locus known to be related to this trait of maize grown under limited Fe, revealed several genes coding for known and unknown proteins, as well as long intergenic non-coding RNAs.

The aim of this work was to study maize root phenotype under sulfur deficiency stress towards revealing potential correlations between the altered phenotypic traits and the corresponding dry mass, sulfur, and iron allocation within plants at the whole-plant level. The dynamics of root morphological and anatomical traits were monitored. These traits were then correlated with plant foliage traits along with dry mass and sulfur and iron allocation dynamics in the shoot versus root. Plants grown under sulfate deprivation did not seem to invest in new root axes. Crown roots presented anatomical differences in all parameters studied; e.g., more and larger xylem vessels in order to maximize water and nutrient transport in the xylem sap. In the root system of S-deficient plants, a reduced concentration of sulfur was observed, whilst organic sulfur predominated over sulfates. A reduction in total iron concentration was monitored, and differences in its subcellular localization were observed. As expected, S-deprivation negatively affected the total sulfur concentration in the aerial plant part, as well as greatly impacted iron allocation in the foliage. Phenotypic adaptation to sulfur deprivation in maize presented alterations mainly in the root anatomy; towards competent handling of the initial sulfur and the induced iron deficiencies.

Iron (Fe) is an essential micronutrient for plant development and productivity. Nevertheless, the role of gibberellins (GAs) in the control of iron homeostasis is less studied compared to other growth regulators. We found that GAs modulate iron homeostasis in maize by inducing deficiency-like responses independent of rhizosphere iron availability. Plant phenotyping demonstrated that exogenous GA₃ application under iron-sufficient conditions phenocopied iron deprivation, while inhibiting GA biosynthesis with mepiquat chloride prevented the development of typical symptoms of Fe deficiency (–Fe). Gibberellins positively control strategy II Fe uptake genes, albeit indirectly, as opposed to the direct negative transcriptional regulation of phytosiderophore biosynthesis. Additionally, gibberellins disrupt iron partitioning by suppressing root-to-shoot Fe translocation, causing iron overaccumulation in roots of GA₃ treated plants. A functional ferrous iron uptake pathway was identified and was found to operate in conjunction with the strategy II uptake pathway via the differentially regulated Zea mays Iron-Regulated Transporter (IRT) paralogs ZmIRT1 and ZmIRT2. Root responses are spatially organized: gene expression in the lateral root sector reflects the shoot iron status, while transcriptional responses in the root apex correlate with local Fe demands. This study demonstrates that maize leverages a hybrid ferric/ferrous iron uptake strategy and establishes novel roles of GAs as pivotal regulators of iron homeostasis.