**2. PA-MRP Transporters**

MRP proteins are transmembrane transporters involved in several functions, such as organic ions transport, xenobiotic detoxification, oxidative stress tolerance, and transpiration control [23]. The first evidence of the involvement of an MRP-type ABC transporter in PA transport was reported for the maize ZmMRP4 protein from the analysis of the insertional *lpa1* mutant [24]. *ZmMRP4* gene is orthologous to *AtMRP5*, which had already been characterized some years ago as an anion transporter involved in root growth, lateral root formation, regulation of stomatal movement, guard cell hormonal signaling, and water use efficiency [25–27], aspects not immediately attributable to PA transport. The biochemical demonstration that PA transport was dependent on the presence of a functional MRP transporter in an ATP-dependent manner was given for AtMRP5, showing a very high affinity for PA (maximum reaction velocity -Vmax- values of about 1.6–2.5 μmol min−<sup>1</sup> mg<sup>−</sup><sup>1</sup> and Michaelis—Mentent constant -*K*m- ranging between 263 and 310 nM) and a vacuolar subcellular localization [16].

As summarized in Table 1, other *PA-MRP* genes and the corresponding mutants/transgenics have been hereafter characterized in rice, *Glycine max* (L.) Merr. (soybean), *Phaseolus vulgaris* L. (common bean) and *Triticum aestivum* L. (soft wheat) [28–33]. Moreover, the putative *Pennisetum glaucum* (L.) R.Br. (pearl millet) *PA-MRP* gene has recently been described [34].

The main difference between cereals and legumes for which PA-MRPs have been characterized so far is the gene number: While only one gene is present in diploid maize, pearl millet, and rice genomes, and three copies in the hexaploid *Triticum aestivum*, two and three paralogues are present in common bean and soybean, respectively [17,32,33]. The presence of more than one member of the *PA-MRP* genes seems to be a common feature of legumes, for example also in *Medicago truncatula* it is possible to predict two *PA-MRP* genes [35], unlike the situation in other dicotyledons, such as Arabidopsis and *Solanum lycopersicum* L. (tomato) in which only one *PA-MRP* gene was described [16,36]. As discussed below, the gene copy number has a significant influence on the *lpa* mutant phenotypes.

The gene structure of PA-MRP transporters is very highly conserved: All analyzed genes in the present study have 11 exons and 10 introns with very similar lengths, only some differences can be found in the lengths of some introns between cereals and legumes. In Figure 1a, the rice *OsMRP5* and the soybean *GmABCC1* gene schematic representations are given as examples.

PA-MRP proteins are full-length ABC transporters (length from 1501 aa of TaABCC13–4B to 1539 aa of GmABCC1) with three membrane-spanning domains (TMD0, containing five transmembrane α-helices and TMD1, TMD2, each with six α-helices) and two cytosolic nucleotide-binding domains (NBD1 and NBD2, containing the Walker A and B motifs), arranged in the TMD0-TMD1-NBD1\_TMD2-NBD2 so-called forward orientation (Figure 1b), as previously described [17]. Although it is not known which specific amino acids are involved in PA transport, a conserved lysine residues stretch, located in the cytosolic loop linking NBD1 and TMD2 and a number of charged amino acid residues (mostly lysine and arginine) found in other conserved stretches in TMD1 and TMD2 have been suggested to be involved in PA transport [17].

PA-MRP protein sequences are phylogenetically very highly conserved among different species, mainly in the TMD and NBD domains, but also outside, particularly among cereals or legumes (Figure 1 and Figure S1). As shown in Figure 1c, the degree of aminoacid identity among different PA-MRP proteins belonging to different species is very high, also between cereals and legumes where it ranges from 67 to 71.5% (similarity between cereals and legumes ranges from 86 to 89%, data not shown).


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**Figure 1.** (**a**) Gene structure of *OsMRP5* and *GmABCC1* genes, as an example of a cereal and a legume *PA-MRP* gene, respectively. Light and dark blue rectangles represent UTRs and coding exons, respectively, the black bars correspond to introns. Gene Structure Display Server [46] was used; (**b**) Predicted domains of the PA-MRP protein. The transmembrane domains (TMD) and the nucleotide-binding domains (NBD) are represented in red and green, respectively. The structure of the PA-MRP proteins was previously described [17]; (**c**) Distances between PA-MRP proteins, expressed as a percentage of identity. Phylogenies were constructed with the Geneious Tree Builder tool, using the Jukes—Cantor distance model, neighbor-joining tree build method.

In Figure 2, pictographic representations of the different organs' expression patterns of the *OsMRP5* and the soybean *PA-MRP* genes, taken as examples for cereals and legumes, are reported from the rice and soybean eFP Browsers [47]. The rice gene is expressed at high levels in different organs including the caryopsis, as previously reported [41]. The maize ortholog shows a similar expression pattern [24]. The *TaABCC13* genes are expressed in different plant organs, preferentially during grain developmental stages, with the transcript accumulation derived from the B genome the highest one, mainly at 14 days after anthesis [33,48].

The expression pattern of the different legume genes varies. As shown in Figure 2, *GmMRP3* and *GmMRP19* genes are expressed in different organs and highly expressed in seed, particularly at the late stage of development, while the *GmMRP13* gene is mainly expressed in root and flower and at a very low level in seed. As discussed below, when both *GmMRP3* and *GmMRP19* are mutated an *lpa* seed phenotype occurs [30]. It suggests that *GmMRP3* and *GmMRP19* have an important role in PA accumulation and their function is redundant, while *GmMRP13* is not active in the seed.

A similar diversified expression pattern was observed in common bean, where the *PvMRP1* gene, coding for a protein more similar to GmMRP3 and GmMRP19 (Figure 1c and Figure S1), is highly expressed in cotyledons, where its transcript levels continue to increase during seed development, reaching the highest levels at 28 days after flowering (DAF) with a similar kinetics to that reported for the accumulation of PA in the same organ. The *PvMRP2* gene, ortholog of *GmMRP13*, is expressed similarly to *PvMRP1* in vegetative organs, but at no appreciable level in cotyledons. Interestingly, both genes are expressed in root nodules, organs specialized in symbiosis with nitrogen-fixing bacteria, in which the role of PA is still unknown [35]. Recently, a detailed analysis was reported of GUS activity in *Arabidopsis thaliana* and *Medicago truncatula* plants, harboring a promoter sequence of *PvMRP1* and *PvMRP2* genes, fused upstream of the *GUS* reporter gene. The strongest GUS activity, driven by both constructs, in organs other than the seeds was present in the vascular tissues [35]. Similar patterns of reporter gene activity were previously shown in transgenic plants harboring the *AtMRP5* promoter [25] and promoters of different Arabidopsis genes coding for enzymes involved in different steps in PA pathway [49–56]. These data sugges<sup>t</sup> that vascular tissues are an important site for synthesis and

transport of PA involved in the regulation of different cellular processes, the so-called "signaling PA" [57].

**Figure 2.** PA-MRP gene expression data in various rice and soybean organs and tissues were obtained from the rice and soybean eFP Browsers [47]. Rice MAS and soybean Severin data sources were used. For rice, the default signal threshold was used, while for the three soybean genes the signal threshold was arbitrarily put to the same value (8.00) in order to compare expression data between different genes.
