*3.3. Response to P Fertilization*

Seed PA content is a ffected by the amount of supplied P in various crops [18–20]. In this Issue, two articles investigate the e ffects of P fertilization in genotypes di ffering in their PA content, particularly in a soybean *lpa* mutant compared with two normal-phytate cultivars [10] and in two rice cultivars described for their contrasting grain PA content [9].

In the first article, Taliman et al. [10] compare di fferent parameters, such as dry weight, photosynthetic rate, dinitrogen fixation, mineral accumulation, and grain yield between a soybean *lpa* line and two varieties normally cultivated in Japan (wild type, wt) in response to high and low P fertilization. The authors generally observed increased plant performance and yield at higher P concentration in all three genotypes but very little di fference between *lpa* and wt genotypes. Seed yield was higher in the *lpa* line than in the normal-phytate cultivars at both fertilization doses. The results show that the already positive properties of *lpa* seeds in terms of increased mineral cations bioavailability can be also accompanied by good agronomic performance [10].

In the second article, Fukushima et al. [9] analyzed the response to P fertilization of two previously selected rice genotypes di ffering for their PA content with the World Rice Core Collection 5 (WRC 5) genotype showing the lowest and WRC 6 showing the highest PA content among di fferent accessions [21]. The authors reported that di fferences in PA content between the two contrasting cultivars were observed only under standard P fertilization conditions, while, if two di fferent doses of P fertilizer were applied at di fferent developmental stages (at seedling or heading stage) an increase in PA content was observed in both genotypes, highly reducing the di fferences between the two genotypes. Interestingly, the expression level of the *myo-inositol 3-phosphate synthase 1* (*INO1*) gene was suggested to be the genetic basis explaining the natural variation in PA accumulation in rice. Although the DNA sequences of the coding region and a putative promoter region of 1000 bp of the *INO1* gene were identical between WRC 5 and WRC 6, the gene is more expressed in the WRC 6 accession than in the WRC 5 one. Moreover, the *INO1* gene transcript accumulation increased in response to P fertilizer only in the WRC 6 accession. The authors hypothesized the existence of di fferent regulatory mechanisms of PA content besides the DNA mutation in the *INO1* gene [9].

### *3.4. lpa Mutants: Isolation and Characterization of New Mutants and Description of a Novel Screening Method in Maize*

The articles by Jiang et al. and Borlini et al. focus on the isolation and characterization of novel *lpa* mutants in rice and maize, respectively; in the second article, a particularly easy new screening method for maize *lpa* mutants is also described [7,8].

Most of the *lpa* mutants have been isolated by screening mutagenized populations and few examples using transgenic approaches have been also reported [12]. Some maize *lpa* mutant lines were obtained through a genome-editing based method, when this technology was not so popular as today [22] and very recently barley mutant lines have been isolated through the same technology [23]. In the article presented by Jiang et al. [8], the CRISPR/CAS9 method was used to generate four rice mutants in the *OsITPK6* gene, coding for inositol 1,3,4-triphosphate 5/6 kinase. Very recently barley *lpa* allelic variants have been isolated through the same technology. Jiang et al. described that the decrease in PA content and the severity of the negative pleiotropic e ffects depended on the induced mutations, with the three frameshift mutations resulting in a major reduction in PA content and in a stronger impact on plant germination, growth, reproduction, and abiotic stress tolerance compared to the e ffects due to the 6-bp in-frame mutation. There is a discrepancy between results obtained from the present study and from a previous one [24], where another mutant a ffected in the same gene showed a higher decrease in PA content than reported for the mutant in the article by Jiang et al., but normal

plant growth. Further studies on other *ositpk6* mutants could clarify this discrepancy and the role of this gene in plant growth and reproduction in addition to its role in PA biosynthesis [8].

Di fferent papers have reported the isolation of *lpa* mutants in di fferent species by the screening of F2 mutagenized populations through the disruption of the seeds analyzed by Chen's assay [12]. In the paper by Borlini et al. [7], it was proposed to directly identify the putative mutant seeds by a cheap and fast screening method based on the lower density of *lpa1* seeds with respect to the wild type, as reported in previous papers, where among the pleiotropic e ffects associated with the *lpa* mutation it was also shown that there was a reduction of seed density in maize and rice. This assay was able to identify the *lpa* mutant seeds because the *lpa1* seeds can float in a concentrated sugar solution (density 1.218–1.222 g/cm3) due to their lower density, unlike the wild sibs that sink [7]. Hence, this method could be used in massive screening of mutagenized populations with the aim to isolate allelic variants at the *lpa* locus.

### *3.5. Inositol Pyrophosphate: Suggested Strategies for the Development of Novel lpa Mutants*

In the cell, a small pool of PA can be further phosphorylated to form inositol pyrophosphates (PP-InsP), containing one or two diphosphate groups (InsP7 and InsP8), through the activity of inositol triphosphate kinase (ITPK) enzymes that phosphorylate PA to InsP7 and the diphosphoinositol-pentakisphosphate kinases (PPIP5Ks) that phosphorylate InsP7 to InsP8. PP-InsP have important roles in energy metabolism, hormone signaling (mainly jasmonate), and Pi sensing. It has been shown that di fferent Arabidopsis *lpa* mutations, a ffecting PA biosynthetic genes, also cause a reduction in the content of InsP8 and in some cases of InsP7. Starting from this point, Freed et al. recommend the breeders aiming at developing *lpa* mutants to take into account this aspect to avoid negative pleiotropic e ffects that may reduce pathogen defense, mediated by jasmonate, and a ffect phosphate homeostasis [11]. To overcome this possibility, one strategy is to develop transgenic *lpa* lines using tissue-specific promoters active only in the seed. However, also in the seed, InsP7 and InsP8 may have important roles in phosphate homeostasis not ye<sup>t</sup> investigated. On the other hand, the Arabidopsis *mrp5* mutant, a ffecting the PA-MRP vacuolar transporter, shows increased content of both InsP7 and InsP8, representing an interesting target for the development of new *lpa* mutants not compromised in Pi homeostasis and in jasmonate signaling. In this way, the review intends to bridge the gap between the basic science aspects of PP-InsP synthesis and function and the breeding/engineering strategies aimed at developing *lpa* crops [11].
