Petros P. Sigalas

SUMMARY: Metal tolerance protein 1 (MTP1) is essential for metal homeostasis and detoxification, but its roles in crop fertility and reproductive organ development are poorly understood. Here, we demonstrate that wheat TaMTP1 safeguards reproductive success and grain Zn allocation by mediating vacuolar Zn sequestration in stems. Heterologous expression in yeast and subcellular localization confirmed that TaMTP1 functions as a tonoplast Zn transporter. In wheat, the TaMTP1 gene was highly expressed in developing spikes and stems across all growth stages. Triple knockout mutants (aabbdd) from a TILLING population exhibited sterility, stunted growth, and abnormal pollen and stigma development, particularly under high Zn supply. ICP‐OES analysis revealed Zn hyperaccumulation in reproductive tissues due to impaired vacuolar sequestration in stems, leading to excessive Zn translocation to spikes. Fluorescence imaging showed Zn overaccumulation in pollen, and depletion in the phloem of stem vascular bundles. Fertility and normal growth were restored under low Zn condition, confirming that reproductive failure was driven by Zn toxicity rather than deficiency. Single functional TaMTP1 alleles were sufficient to maintain fertility, whereas partial mutants (Aa/AAbbdd and aaBbdd) accumulated elevated Zn in grain under excess Zn supply compared with null. Together, our findings uncover a stem‐based vacuolar Zn buffering mechanism that prevents reproductive Zn toxicity, establishing TaMTP1 as a key determinant of fertility and grain Zn partitioning in wheat, with potential for biofortification and Zn resilience breeding.

Wheat is an important source of minerals for human nutrition and increasing grain mineral content can contribute to reducing mineral deficiencies. Here, we identify QTLs for mineral micronutrients in grain of wheat by determining the contents of six minerals in a total of eleven sample sets of three biparental populations from crosses between A.E. Watkins landraces and cv. Paragon. Twenty-three of the QTLs are mapped in two or more sample sets, with LOD scores above five in at least one set with the increasing alleles for sixteen of the QTLs being present in the landraces and seven in Paragon. Of these QTLs, the number for each mineral varies between three and five and they are located on 14 of the 21 chromosomes, with clusters on chromosomes 5A (four), 6A (three), and 7A (three). The gene content within 5 megabases of DNA on either side of the marker for the QTL with the highest LOD score is determined and the gene responsible for the strongest QTL (chromosome 5A for Ca) identified as an ATPase transporter gene (TraesCS5A02G543300) using mutagenesis. The identification of these QTLs, together with associated SNP markers and candidate genes, will facilitate the improvement of grain nutritional quality.

SUMMARY: Strigolactones (SLs) are key regulators of shoot growth and responses to environmental stimuli. Numerous studies have indicated that nitrogen (N) limitation induces SL biosynthesis, suggesting that SLs may play a pivotal role in coordinating systemic responses to N availability, but this idea has not been clearly demonstrated. Here, we generated triple knockout mutants in the SL synthesis gene TaDWARF17 (TaD17) in bread wheat and investigated their phenotypic and transcriptional responses under N limitation, aiming to elucidate the role of SLs in the adaptation to N limitation. Tad17 mutants display typical SL mutant phenotypes, and fail to adapt their shoot growth appropriately to N. Despite exhibiting an increased tillering phenotype, Tad17 mutants continued to respond to N limitation by reducing tiller number, suggesting that SLs are not the sole regulators of tillering in response to N availability. RNA‐seq analysis of basal nodes revealed that the loss of D17 significantly altered the transcriptional response of N‐responsive genes, including changes in the expression profiles of key N response master regulators. Crucially, our findings suggest that SLs are required for the transcriptional downregulation of cytokinin (CK) synthesis and signalling in response to N limitation. Collectively, our results suggest that SLs are essential for the appropriate morphological and transcriptional adaptation to N limitation in wheat, and that the repressive effect of SLs on shoot growth is partly mediated by their repression of CK synthesis.

High-affinity nitrate transporters (NRT) are key components for nitrogen (N) acquisition and distribution within plants. However, insights on these transporters in wheat are scarce. This study presents a comprehensive analysis of the NRT2 and NRT3 gene families, where the aim is to shed light on their functionality and to evaluate their responses to N availability. A total of 53 NRT2s and 11 NRT3s were identified in the bread wheat genome, and these were grouped into different clades and homoeologous subgroups. The transcriptional dynamics of the identified NRT2 and NRT3 genes, in response to N starvation and nitrate resupply, were examined by RT-qPCR in the roots and shoots of hydroponically grown wheat plants through a time course experiment. Additionally, the spatial expression patterns of these genes were explored within the plant. The NRT2s of clade 1, TaNRT2.1-2.6, showed a root-specific expression and significant upregulation in response to N starvation, thus emphasizing a role in N acquisition. However, most of the clade 2 NRT2s displayed reduced expression under N-starved conditions. Nitrate resupply after N starvation revealed rapid responsiveness in TaNRT2.1-2.6, while clade 2 genes exhibited gradual induction, primarily in the roots. TaNRT2.18 was highly expressed in above-ground tissues and exhibited distinct nitrate-related response patterns for roots and shoots. The TaNRT3 gene expression closely paralleled the profiles of TaNRT2.1-2.6 in response to nitrate induction. These findings enhance the understanding of NRT2 and NRT3 involvement in nitrogen uptake and utilization, and they could have practical implications for improving nitrogen use efficiency. The study also recommends a standardized nomenclature for wheat NRT2 genes, thereby addressing prior naming inconsistencies.

Strigolactones (SLs) are a class of phytohormones regulating branching/tillering and their biosynthesis has been associated with nutritional signals and plant adaptation to nutrient-limiting conditions. The enzymes in the SL biosynthetic pathway downstream of carlactone are of interest as they are responsible for structural diversity in SLs, particularly cytochrome P450 CYP711A subfamily members, such as MORE AXILLARY GROWTH1 (MAX1) in Arabidopsis. We identified 13 MAX1 homologues in wheat, clustering in four clades and five homoeologous subgroups. The utilization of RNA-sequencing data revealed a distinct expression pattern of MAX1 homologues in above- and below-ground tissues, providing insights into the distinct roles of MAX1 homologues in wheat. In addition, a transcriptional analysis showed that SL biosynthetic genes were systematically regulated by nitrogen supply. Nitrogen limitation led to larger transcriptional changes in the basal nodes than phosphorus limitation, which was consistent with the observed tillering suppression, as wheat showed higher sensitivity to nitrogen. The opposite was observed in roots, with phosphorus limitation leading to stronger induction of most SL biosynthetic genes compared to nitrogen limitation. The observed tissue-specific regulation of SL biosynthetic genes in response to nutritional signals is likely to reflect the dual role of SLs as rhizosphere signals and branching inhibitors.

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.

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.

The granules of conventional fertilizers have been enriched recently with 2% elemental sulfur (S⁰) via a binding material of organic nature and such fertilizers are suitable for large scale agriculture. In a previous work, we demonstrated that a durum wheat crop that received the enriched fertilization scheme (FBS⁰-crop) accumulated a higher amount of Fe compared to the durum wheat crop fertilized by the corresponding conventional fertilization scheme (F-crop). In this study, we investigated the effect of S⁰ on the contingent mobilization of iron from the iron pools of the calcareous field that affiliated the durum wheat crop and the corresponding effect on the crop’s iron nutrition and sulfur nutrition. A sequential extraction of Fe from root zone soil (rhizosoil) was applied and the fluctuations of these fractions during crop development were monitored. The fertilization with FBS⁰ at sowing affected the iron fractions of the rhizosoil towards iron mobilization, thus providing more iron to the crop, which apart from the iron nutrition fortified the crop’s sulfur nutrition, too. No iron was found as iron attached to carbonates of the rhizosoil. Fluctuations of the iron pool, bound or adsorbed to the organic matter, were exactly the opposite to those of the iron pool associated with the clay particles in both treatments, suggesting iron exchange between the two pools. Replenishment of the F-crop’s Fe content and a deficit in the FBS⁰-crop’s Fe content in the rhizosoil were found at the end of the cultivation period. Furthermore, the initiation of the fast stem elongation stage (day 125) constituted a turning point. Before day 125, the use of FBS⁰ increased the iron concentration in the main stems and this was an early fortification effect, followed by an increase in the organic S concentration. Following day 125, the FBS⁰-crop consisted of plants with higher main stems and less tillers. A late fortification effect was observed in the iron concentration of the main stems and their heads after the stage of complete flowering. Prior to harvesting in the FBS⁰-crop, all plant parts were heavier, with more iron and organic sulfur accumulated in these plant parts, and the obtained commercial yield of the FBS⁰-crop was higher by 27.3%.