*2.1. Mineral Transport and Seed Accumulation*

Seed iron is mainly stored in vacuoles. Hence, improving iron uptake into the vacuole is a valuable alternative strategy to increase total iron content. For this reason, the role of vacuolar iron transporters needs to be addressed and exploited. The article by Sharma et al. describes the isolation and preliminary characterization of the family of the *VTL* genes in hexaploid wheat. The authors report data on phylogenetic analysis and on a quantitative expression analysis of *VTL* genes in response to iron surplus and deficiency, under zinc, manganese, and copper deficiency, and under heavy metals treatments [2]. Particularly, 23 wheat *VTL* gene sequences were identified that can be phylogenetically distinguished from the *Vacuolar Iron Transporters* (*VIT*) ones and are grouped as 4 *VTL* genes due to the occurrence of homeologs. The expression data in response to treatments with different concentrations of minerals sugges<sup>t</sup> that these genes have an important role in mineral homeostasis [2].

The knowledge of mechanisms involved in mineral accumulation, in terms of tissue-specificity, speciations, and ligand compounds, is very important to set up a precise biofortification program. In the wheat ear, awns (bristle-like structures extending from lemmas) have transpiration and photosynthetic activity. Hence, their presence could contribute to the translocation of elements taken up by roots on the one hand and/or to the phloem-driven (re)allocation of assimilates on the other hand, thereby affecting mineral element density in the grain. The study by Pongrac et al. presents a comparison of mineral element composition between awned and awnletted (those that have short or no awns) wheat cultivars. Moreover, tissue-specific iron speciation and iron ligands in the cultivars contrasting for seed iron content were also investigated using micro X-ray absorption near edge structure (micro-XANES) [3]. The authors found that among the 20 different cultivars, the awnletted ones showed lower whole-grain concentrations of calcium and manganese, but higher iron concentration, compared to the awned cultivars. Interestingly, no differences were observed either in iron speciation (the percentages of ferric and ferrous iron are similar in the four most contrasting analyzed awned and awnletted cultivars) or in terms of ligands, as on average 53% of the iron is in a phytate form. On the contrary, there was a distinct tissue-specificity in iron speciation and ligands, with the pericarp containing the largest proportion of ferric species with only non-phytate ligands, as also in the nucellar projection. In other tissues, such as the aleurone, scutellum, and embryo, iron was predominantly bound to phytate. The authors conclude that, as iron bioavailability is dependent on iron ligands, its bioavailability in wheat is tissue

specific. Further investigation on the genetic and/or metabolic reasons behind the observed di fferences is needed [3].

### *2.2. Germplasm Screening: Genetic Variation and Identification of Genomic Regions and Molecular Markers (Quantitative Trait Loci (QTLs), Single Nucleotide Polymorphisms (SNPs))*

The increase of seed micronutrient concentration can be achieved through agronomic interventions, genetic engineering, and plant breeding. The first two approaches are briefly summarized in the review by Jha and Warkentin [1]. However, a long section of the review is dedicated to conventional plant breeding approaches that have been used to biofortify pulses, particularly common bean, lentil, chickpea, mungbean, and pea, for some minerals (iron, zinc, and selenium), carotenoids, and folates. The advantages of using conventional plant breeding, compared to the genetic engineering strategy, are the relatively low costs and the high acceptability by consumers. The first step in this kind of approach is the screening for genetic variability for micronutrients' seed concentration. The authors report that a significant e ffect of the genotype in the determination of micronutrient seed concentration has been shown for the di fferent micronutrients in the various pulses, although the environment (weather and soil factors, such as aeration, water availability, pH, and texture) may have significant effects, for example, in the case of zinc and selenium seed concentrations. Moreover, for the pulses studied, they report the identification of genomic regions (Quantitative Trait Loci, QTLs) and molecular markers, mainly Single Nucleotide Polymorphisms (SNPs), which can be used in marker-assisted selection procedures, aimed at improving micronutrient seed concentration. In some cases, candidate genes involved in the accumulation of the di fferent micronutrients are described. The authors mention the di fferent crops, not only pulses, with increased micronutrient concentration that have been released in recent years in developing countries, mainly thanks to the activity of HarvestPlus, an initiative of the Consultative Group on International Agricultural Research (CGIAR), started in 2003 to enrich various major crops with iron, zinc, and vitamin A. Evidence that the introduction of some of these crops in the diet has helped in overcoming nutrient deficiency is well documented [1].

### **3. Decreasing Antinutritional Compounds Concentration: Four Decades of Research and Novel Perspectives for** *lpa* **Mutants**

### *3.1. Some Possibilities to Redeem the so far Neglected lpa Crops?*

PA is the most abundant form of phosphorus (P) occurring in seeds. However, it is a "non-available" form of P for monogastric animals devoid of phytase (poultry, swine, fish). Moreover, it is a strong cation chelator, reducing the bioavailability of cations important for nutrition. PA is also a very important signaling molecule involved in di fferent regulatory processes during plant development and responses to di fferent stimuli [12]. Di fferent contributions to the present Special Issue treat aspects related to PA and *lpa* mutants [4–10].

Di fferent *lpa* mutants have been isolated and characterized so far in di fferent species, starting from the first ones, the maize *lpa1-1* and *lpa2-1*, isolated in the early 1990s in the USDA-ARS laboratory of Raboy, but none of them has been commercialized, as the scientist underlines, with a certain regret, in his very interesting commentary [4]. The commentary is strongly felt, as the author is "a person intimately involved in the entire process across a 40-year period," as written in his/her report by one of the anonymous reviewers of the manuscript (the review reports are publicly available at the webpage of the commentary). As still observed by one of the two reviewers, the view presented in this commentary is multidisciplinary, as scientific, economic, and social aspects of the subject are discussed, "encompassing the issues faced by all breeders (and probably agronomists as well) attempting to develop new materials with significant social benefit but di fficult to capture short term economic benefit when favored alternatives exist with the reverse tendencies". The author underlines that these mutants have some potential advantages, mainly (i) improving phosphorus managemen<sup>t</sup> in non-ruminant production, contributing to enhance sustainability and reduce animal waste P, and (ii) increasing mineral bioavailability as a strategy to combat mineral deficiencies, as shown by di fferent studies. Nevertheless, these mutants have received very little interest. The author thinks that the reasons for this are primarily due to the reduced yield (5–10% decrease) and field performance that characterize some of these mutants, together with the criticism that reducing PA is not wholly advantageous as it may also have positive nutritional benefits (antioxidant and anticancer properties, shown through *in vitro* studies [13], although it has been shown that no phytate is present in human biofluids [14]) that might be lost in the *lpa* mutants. Moreover, another simple explanation may be the tendency to use a conservative approach to crop improvement strategies for crops that provide staple foods to at-risk populations in developing countries. However, no support or time to improve the agronomic performance of these mutants has been provided. Some alternatives that have been preferred in recent years exist. Concerning methods to increase P for feed, it can be directly added or phytase can be used to increase the component of available P. The author says that these methods have been preferred to the use of *lpa* crops, without calculating the possible long-term money-saving deriving from using the *lpa* crops. Moreover, the positive results from animal nutrition studies when animals are fed *lpa* crops (for example: leaner pigs, with enhanced muscle density and less backfat when fed *lpa* maize; eggs with reduced cholesterol from hens fed *lpa* maize) ironically pushed farmers to apply phytase superdoses, also in this case not considering the long-term money-saving if, instead, *lpa* crops might have been used [4]. These savings would have overcome the reduced yield problem with the further advantages of having more nutritious crops. Raboy is also quite critical of the HarvestPlus program and the international agricultural centers participating in the Gates Foundation that concentrated all their efforts only to promote biofortification through breeding crops for elevated micronutrient density and, therefore, pushed the development and promotion of *lpa* crops to the sidelines, although a combination of both approaches would likely give the most promising results.

After the publication of this Special Issue, an article has been published by different authors, including scientists from HarvestPlus and CIAT (a CGIAR institute), comparing the retention of iron and zinc when preparing common household recipes with conventional, biofortified, or *lpa* common beans [15]. The retention of iron was very high and similar using the different bean genotypes, while *lpa* beans exhibited lower retention for zinc. Further studies are needed to understand this difference. However, the authors encourage the development of beans with an increased mineral content combined with a low PA trait, and also with low concentrations of specific polyphenolic compounds, as the research target for the next generation of biofortified beans [15]. This publication can lay the foundation for a brighter future for *lpa* crops.

### *3.2. lpa Mutants in Di*ff*erent Classes of Transporters: Not Always so Obvious*

PA reduction can be achieved with mutations in different types of transporters that control PA transport to the vacuole (MRP-type ABC transporters), or by modifying inorganic P (Pi) availability for PA synthesis through mutations in transporters involved in Pi loading and organ/intracellular distribution (SULFATE TRANSPORTER 3;3 -SULTR3;3- and SULTR3;4, members of the group 3 of putative sulfate transporters) or by Pi acquisition and mobilization during seed development (PHOSPHATE TRANSPORTER 1;4, PHT1;4). The review by Cominelli et al. is focused on the description of genes, proteins, and mutants of these different transporters in cereals and legumes [5]. Particular attention is dedicated to those mutants devoid of negative pleiotropic effects, such as mutants affected by the common bean *MRP1* gene and by the rice and barley *SULTR3;3* and rice *SULTR3;4* genes, suggesting strategies to develop useful *lpa* mutants in other species as well [5].

Sacchi and Nocito in their opinion paper propose a deeper discussion on group 3 putative sulfate transporters and sugges<sup>t</sup> some hypotheses to unveil the links between sulfate and P accumulation in seeds [6]. The fact that genes predicted to encode for sulfate transporters, when mutated, cause a *lpa* phenotype is anything but obvious. Differently from other sulfate transporters, for which capability to transport sulfate has mainly been proven through complementation of yeas<sup>t</sup> mutants, the function of SULTR3s has only been hypothesized based on their sequence homologies. The rice and Arabidopsis SULTR3;4 proteins are able to transport phosphate and not sulfate, as recently shown [16,17], explaining the *lpa* phenotype of the rice mutant. However, the OsSULTR3;3 protein does not show either phosphate or sulfate transport activity. Some hypotheses on the link between sulfate and phosphate homeostasis and the development of the *lpa* phenotype in *sultr3;3* mutants are proposed in the opinion paper [6].
