Our ongoing research activity focuses on iron uptake and homeostasis by the maize plants and the relationship of the related mechanisms with the corresponding mechanisms of sulfate uptake and assimilation in these plants which follow the chelation strategy for iron uptake.

· Beyond Strategy II: Exploring alternative iron uptake mechanisms in maize

Ventouris YE¹, Charatsidou IE¹, Ionas K¹, Stylianidis GP¹, Pantazopoulou CK¹, Bouranis DL^1,2, Chorianopoulou SN^1,2

¹ Plant Physiology and Morphology Laboratory, Crop Science Department, Agricultural University of Athens, 11855 Athens, Greece

² PlanTerra – Institute for Plant Nutrition and Soil Quality, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

Despite its essential role in plant biology, iron (Fe) has limited bioavailability due to the chemical forms in which it’s usually found in the soil. To overcome this limitation, plants have evolved distinct mechanisms for Fe solubilization and uptake, known as Strategy I and Strategy II. Although maize is generally considered to rely exclusively on Strategy II, the presence of Strategy I-related genes in its genome suggests that alternative Fe acquisition pathways may also be present. To investigate this possibility, maize seedlings were grown hydroponically under Fe-sufficient (300 μM Fe) or Fe-deficient (10 μM Fe) conditions. Expression analysis of the two maize iron-regulated transporter genes, ZmIRT1 and ZmIRT2, revealed that ZmIRT1 was expressed under both Fe regimes, whereas ZmIRT2 expression occurred exclusively under low Fe supply. Both genes displayed distinct expression patterns between functionally different root regions, namely the root apex (A) and the lateral root zone (LR). In situ staining with the ferrous iron (Fe2+)-specific stain Turnbull blue confirmed the formation of Fe2+ on the surface of crown roots, while Fe3+ deposits in the cell walls of rhizodermal cells could function as a reservoir of readily available Fe. Even though the exact mechanism of Fe reduction remains unclear, this process could be carried out in a non-enzymatic, reactive oxygen species-dependent manner. This hypothesis is further supported by the lack of ZmFRO2 expression, the sole maize ferric-chelate reductase homolog gene. Overall, these findings provide strong evidence for a functional ferrous iron uptake mechanism in maize that operates alongside Strategy II-mediated ferric iron acquisition.

· Hormonal control of iron deficiency responses: Gibberellins modulate root architecture and Fe uptake in maize

Ventouris YE¹, Charatsidou IE¹, Ionas K¹, Stylianidis GP¹, Pantazopoulou CK¹, Bouranis DL^1,2, Chorianopoulou SN^1,2

¹ Plant Physiology and Morphology Laboratory, Crop Science Department, Agricultural University of Athens, 11855 Athens, Greece

² PlanTerra – Institute for Plant Nutrition and Soil Quality, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

Iron (Fe) deficiency strongly affects maize root system architecture, leading both to a marked reduction in root length and to the emergence of lateral roots in closer proximity to the root tip. These phenotypes have been associated with the quantitative trait loci (QTLs) “jpsb79-umc60” and “chrom7-glb1”, respectively. Bioinformatic analysis of these QTLs revealed a close genetic link between root morphological responses to Fe deficiency and gibberellins. However, the role of gibberellins in Fe homeostasis remains poorly understood. To investigate this relationship, maize seedlings were grown hydroponically under Fe-sufficient or Fe-deficient conditions and were additionally treated either with GA₃ or with mepiquat chloride, a gibberellin biosynthesis inhibitor. As expected, Fe deficiency induced both root phenotypes described above, while the respective shoots showed significantly reduced biomass accumulation and chlorosis, particularly in younger leaves. Exogenous GA₃ application under Fe-sufficient conditions resulted in a Fe deficiency-reminiscent phenotype in both the shoot and the roots. Conversely, inhibition of gibberellin biosynthesis in Fe-deficient plants rescued the -Fe phenotype in roots, but not in the shoot. Gibberellins were also found to be involved in the regulation of Strategy II-mediated Fe uptake. Specifically, the application of GA3 resulted in transcriptional repression of the phytosiderophore biosynthesis genes ZmNAS1 and ZmDMAS1, while GAs seem to positively regulate ZmIRT2 expression under Fe-deficient, but not Fe-sufficient, conditions. At the physiological level, Fe deficiency responses showed a clear spatial organization along the root. Responses observed in the lateral root zone (LR) were primarily associated with the nutritional status of the whole plant, whereas those occuring at the root apex (A) mainly reflected local Fe demands.

· Effect of foliar iron application on root system architecture of maize plants

Charatsidou IE¹, Stylianidis GP¹, Ventouris YE¹, Nikolopoulos D¹, Pantazopoulou CK¹, Chorianopoulou SN^1,2

¹ Plant Physiology and Morphology Laboratory, Crop Science Department, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

² PlanTerra – Institute for Plant Nutrition and Soil Quality, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

Iron is an essential micronutrient for plant growth and development. Plants acquire iron from the rhizosphere through various mechanisms, resulting in species-specific variation in their ability to adapt to conditions of limited iron supply. However, the responses of the root system of maize (Zea mays) to foliar iron application have not been studied and for this reason, the present experiment was conducted. Maize plants were grown under iron-sufficient and iron-deficient conditions in hydroponics, while simultaneously receiving foliar iron fertilization. Three treatments were applied: 300-0, 10-0 and 10-300. In the first two treatments, plants were grown under iron sufficiency (300 μM Fe) and iron deficiency (10 μM Fe), respectively, while foliar fertilization was performed with 0 μM Fe. In the 3rd treatment, plants were grown under iron deficiency (10 μM Fe) and received foliar fertilization with 300 μM Fe. Seven days after the initiation of the treatments, phenotyping and anatomical analysis of the root system were performed, along with measurements of fresh and dry biomass. Lateral roots were divided in two categories: fully developed (LR1) and partially developed (LR2). In treatments 10-300 and 10-0, a greater number of LR2 lateral roots developed on the primary and secondary embryonic roots, whereas more LR1 lateral roots developed on the first-row crown roots. In first-row crown roots, the lateral roots’ (LR1 and LR2) length was decreased gradually from treatment 300-0, to 10-300 and lastly, to 10-0. Stele surface, metaxylem vessels number and total surface showed at the apex part of the root a decrease from treatment 300-0, to 10-300 and finally, to 10-0. The consistent gradation among the three treatments in many phenotypic characteristics leads to the conclusion that the iron foliar application influences root system architecture, highlighting the need for further research to elucidate the underlying mechanism.

· Beyond Chlorosis: An Integrated Investigation of Photosynthetic Function and Stomatal Anatomy in Iron-Deficient Maize

Stylianidis GP¹, Charatsidou IE¹, Michailidis I¹, Ventouris YE¹, Nikolopoulos D¹, Liakopoulos G¹, Bresta P², Pantazopoulou CK¹, Bouranis DL^1,3, Chorianopoulou SN^1,3

¹Plant Physiology and Morphology Laboratory, Crop Science Department, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece

²Laboratory of Electron Microscopy, Crop Science Department, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece

³PlanTerra - Institute for Plant Nutrition and Soil Quality, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece

Iron (Fe) is an essential micronutrient for plant photosynthetic function, and its deficiency causes severe physiological and developmental changes. However, the relationship between physiological responses and anatomical alterations in leaves remains poorly understood. The aim of the present study was to investigate the physiological and anatomical responses of young maize (Zea mays L.) plants grown hydroponically under conditions of Fe sufficiency (300 μM Fe) and deficiency (10 μM Fe). The assessment was performed on leaves at different developmental stages, which had expanded either before or during the onset of Fe deficiency. Plant physiological status was evaluated through the SPAD index, as a non-destructive estimation of leaf chlorophyll content, and chlorophyll a fluorescence induction analysis (JIP-test). In parallel, stomatal density, stomatal index (SI), stomatal morphometric traits, and relative stomatal area were determined. Iron deficiency primarily affected leaves that developed after the onset of stress, which exhibited pronounced chlorosis and significantly lower SPAD values. Spatial analysis of SPAD measurements revealed that the effects of Fe deficiency were more severe in both younger leaves and younger tissues within the same leaf, particularly at the leaf base. This pattern is consistent with the basipetal developmental gradient characteristic of maize leaf growth. These changes were accompanied by increased stress-related parameters (VK/VJ, ABS/RC, and TR0/RC) and decreased quantum yields associated with photochemical activity and electron transport (φP0, φE0, φR0, ψE0 and δR0), together with an increased energy dissipation yield (φD0). These responses indicate a progressive damage of photosystem II functionality and electron transport towards photosystem I. The same leaves exhibited increased stomatal density without a corresponding change in stomatal index, suggesting that this response was mainly associated with reduced leaf expansion during development rather than enhanced differentiation of new stomata. The findings demonstrate that Fe deficiency extends beyond the induction of chlorosis, disrupting the normal developmental patterning of maize leaves and coordinately affecting photosynthetic performance and epidermal anatomical organization. The combined evaluation of physiological and anatomical responses provides a framework for the development of non-destructive indicators for the early diagnosis of Fe deficiency in maize