Introduction
Iron (Fe) is an essential micronutrient for all living organisms. Fe plays a key role in electron transfer in both photosynthetic and respiratory reactions. In plants, Fe deficiency impairs chlorophyll synthesis and causes leaf yellowing due to Fe chlorosis, leading to low yield and poor nutritional quality (Marschner et al., Reference Marschner, Römheld and Kissel1986). In humans, Fe deficiency causes anaemia, which is associated with several comorbidities, including cardiovascular disease and is an independent risk factor for death (Groenveld et al., Reference Groenveld, Januzzi, Damman, van Wijngaarden, Hillege, van Veldhuisen and van der Meer2008; Parikh et al., Reference Parikh, Natarajan, Lipsitz and Katz2011; Klip et al., Reference Klip, Comin-Colet, Voors, Ponikowski, Enjuanes, Banasiak, Lok, Rosentryt, Torrens, Polonski, van Veldhuisen, van der Meer and Jankowska2013). Humans take in Fe, which is principally taken up by plants from the soil. Indeed, a decrease in the Fe concentrations in food was correlated with increasing Fe deficiency anaemia (Sun and Weaver, Reference Sun and Weaver2021). Therefore, increasing Fe uptake efficiency could contribute both to increasing plant productivity and improving human health.
Fe is abundant in the soil, but most of it is not available to plants, being oxidized to Fe(III) hydroxide (Fe(OH)3), which is poorly soluble in water. This phenomenon is exacerbated in high-pH soils, including calcareous soils, which account for an estimated 30% of the world's cultivated land, in which plants often exhibit severe chlorosis due to Fe deficiency constituting a major agricultural problem for crop production (Marschner, Reference Marschner1995; Mori, Reference Mori1999). Inappropriate irrigation in drylands leads to the accumulation of salts, increasing soil pH (FAO, 2022). Irrigated salt-affected soils were estimated as covering 20 to 50% of all irrigated land (FAO, 2022). In these regions, Fe deficiency is a common agronomic problem. Thus, Fe-deficiency-tolerant plants might stay green on calcareous soils.
Organisms have evolved methods to take up insoluble Fe(III). Grasses, including important staple crops such as wheat, rice and barley, secrete natural Fe chelators called mugineic acid family phytosiderophores (MAs) from their roots (Takagi, Reference Takagi1976) through the Transporter Of MAs (TOM1) (Nozoye et al., Reference Nozoye, Nagasaka, Kobayashi, Takahashi, Sato, Uozumi, Nakanishi and Nishizawa2011). MAs chelate and solubilize Fe(OH)3 in the soil, forming Fe(III)–MAs complexes, which are taken into root cells via Fe(III)–MAs transporters called yellow stripe 1 (YS1)/YS1-like (YSL) transporters, localized to the root cell membrane (Curie et al., Reference Curie, Panaviene, Loulergue, Dellaporta, Briat and Walker2001; Inoue et al., Reference Inoue, Kobayashi, Nozoye, Takahashi, Kakei, Suzuki, Nakazono, Nakanishi, Mori and Nishizawa2009). MAs are suggested to play an important additional role in the long-distance transport of metals (Mori et al., Reference Mori, Nishizawa, Hayashi, Chino, Yoshimura and Ishihara1991; Kawai et al., Reference Kawai, Kamei, Matsuda, Ando, Kondo, Ishizawa and Alam2001; Kakei et al., Reference Kakei, Yamaguchi, Kobayashi, Takahashi, Nakanishi, Yamakawa and Nishizawa2009; Nishiyama et al., Reference Nishiyama, Kato, Nagata, Yanagisawa and Yoneyama2012; Nozoye et al., Reference Nozoye, Nagasaka, Kobayashi, Sato, Uozumi, Nakanishi and Nishizawa2015) and can chelate not only Fe but also various transition metals such as zinc (Zn), manganese (Mn) and copper (Cu) (Murakami et al., Reference Murakami, Ise, Hayakawa, Kamei and Takagi1989; Mori et al., Reference Mori, Nishizawa, Hayashi, Chino, Yoshimura and Ishihara1991), being involved in metal homoeostasis, which is important for plant development.
The production and secretion of MAs increase markedly in response to Fe deficiency. Although wheat, rice and maize secrete 2′-deoxymugineic acid (DMA), other species, including barley and rye, hydroxylate DMA to other MAs. Takagi et al. (Reference Takagi, Nomoto and Takemoto1984) suggested that the kinds and amounts of MAs are positively correlated with plant tolerance to Fe deficiency. Indeed, transgenic enhancement of the production and secretion of MAs and increase of the kinds of MAs conferred Fe-deficiency-tolerance in plants (Takahashi et al., Reference Takahashi, Nakanishi, Kawasaki, Nishizawa and Mori2001; Suzuki et al., Reference Suzuki, Morikawa, Nakanishi, Takahashi, Saigusa, Mori and Nishizawa2008; Ogo et al., Reference Ogo, Itai, Kobayashi, Aung, Nakanishi and Nishizawa2011), suggesting that the abilities of MAs production and secretion are key factors in plant tolerance of Fe deficiency. We expect that the ability to make new MAs would further promote tolerance.
Other than MAs, plants also respond to Fe deficiency by enhancing root secretion of phenolic compounds (Römheld and Marschner, Reference Römheld and Marschner1983; Susín et al., Reference Susín, Abián, Sánchez-Baeza, Peleato, Abadía, Gelpí and Abadía1993; Rodríguez-Celma et al., Reference Rodríguez-Celma, Vázquez-Reina, Orduna, Abadía, Abadía, Álvarez-Fernández and López-Millán2011; Donnini et al., Reference Donnini, De Nisi, Gabotti, Tato and Zocchi2012). Phenolics are considered putative external Fe reductants or chelators (Brown and Ambler, Reference Brown and Ambler1973). In grasses, it is suggested that phenolics such as protocatechuic acid solubilize and chelate Fe(III) and then reduce it and translocate the precipitated insoluble Fe(III) in the plant body (Bashir et al., Reference Bashir, Ishimaru, Shimo, Kakei, Senoura, Takahashi, Sato, Sato, Uozumi and Nakanishi2011; Ishimaru et al., Reference Ishimaru, Kakei, Shimo, Bashir, Sato, Uozumi, Nakanishi and Nishizawa2011). In dicots, phenolics such as coumarins, which are produced via the phenylpropanoid pathway, are secreted from the roots and are involved in Fe uptake from the rhizosphere (Rodríguez-Celma et al., Reference Rodríguez-Celma, Lin, Fu, Abadía, López-Millán and Schmidt2013; Schmid et al., Reference Schmid, Giehl, Döll, Mock, Strehmel, Scheel, Kong, Hider and von Wirén2013; Fourcroy et al., Reference Fourcroy, Sisó-Terraza, Sudre, Savirón, Reyt, Gaymard, Abadía, Abadia, Álvarez-Fernández and Briat2014; Schmidt et al., Reference Schmidt, Günther, Weber, Spörlein, Loscher, Böttcher, Schobert and Clemens2014), in addition to its translocation in the plant body (Tsai et al., Reference Tsai, Rodríguez-Celma, Lan, Wu, Vélez-Bermúdez and Schmidt2018). An extensive study of plant diversity could find candidate genes that can improve Fe use efficiency.
Bread wheat (Triticum aestivum L.) is an important crop for global food security. It is grown on a greater area than any other crop and provides one-fifth of all calories consumed by humans (Simons et al., Reference Simons, Saha, Guillard, Clément, Armengaud, Cañas, Maranas, Lea and Hirel2014). Bread wheat originated through few events of natural hybridization between durum wheat (T. turgidum L. subsp. durum) and Aegilops tauschii Coss. (syn. Ae. squarrosa auct. non L.). Because these events involved few progenitors, the genetic diversity of durum wheat and Ae. tauschii is not fully represented in current bread wheat germplasm (Gill and Raupp, Reference Gill and Raupp1987; Dreisigacker et al., Reference Dreisigacker, Kishii, Lage and Warburton2008; Li et al., Reference Li, Wan and Yang2014). By introducing the genes of wild species, it might be possible to generate bread wheat which can take up more Fe from soils and which could grow under Fe-deficient conditions such as calcareous soils and drylands. Such genes could also be used to generate biofortified bread wheat with a high Fe content.
Ae. tauschii is a wild diploid self-pollinating species (2n = 2x = 14, DD) and was the donor of the D genome to the hexaploid bread wheat (2n = 6x = 42, AABBDD) (Kihara, Reference Kihara1944; McFadden and Sears, Reference McFadden and Sears1946). Molecular phylogenetic analysis divided Ae. tauschii into three groups in two main lineages (TauL1 and TauL2) and one minor lineage (TauL3), suggesting diversity among Ae. tauschii accessions (Matsuoka et al., Reference Matsuoka, Nasuda, Ashida, Nitta, Tsujimoto, Takumi and Kawahara2013; Mahjoob et al., Reference Mahjoob, Chen, Gorafi, Yamasaki, Kamal, Abdelrahman, Iwata, Matsuoka, Tahir and Tsujimoto2021). Since wild Ae. tauschii is widespread, it is speculated that it holds many useful genes that can improve the yield and quality of bread wheat (Ogbonnaya et al., Reference Ogbonnaya, Abdalla, Mujeeb-Kazi, Kazi, Xu, Gosman, Lagudah, Bonnett, Sorrells and Tsujimoto2013). Indeed, physiological traits such as heading date and mineral contents ranged widely in a population of synthetic bread wheat lines harbouring genomic fragments of Ae. tauschii (Gorafi et al., Reference Gorafi, Ishii, Kim, Elbashir and Tsujimoto2018a, Reference Gorafi, Kim, Elbashir and Tsujimoto2018b). Thus, physiological and genetic analyses of Ae. tauschii accessions might find new contributors to the efficient uptake and translocation of Fe.
Here, we report the comprehensive physiological analysis of Fe homoeostasis in Ae. tauschii, revealing genetic diversity that can be used to generate Fe-deficiency-tolerant or Fe-biofortified bread wheat.
Materials and methods
Plant materials and analysis of plant physiological traits
We used 42 accessions of Ae. tauschii that have been used to generate synthetic wheat lines (Gorafi et al., Reference Gorafi, Ishii, Kim, Elbashir and Tsujimoto2018a), the tetraploid T. turgidum subsp. durum ‘Langdon’ and the hexaploid T. aestivum ‘Norin 61’. All plants were grown hydroponically in a growth chamber under a 14-h light / 10-h dark regimen at 20°C. The seeds were germinated on paper wetted with tap water for 7–12 days; then, they were moved to a 20-l plastic container containing the nutrient solution of the following composition: 0.7 mM K2SO4, 0.1 mM KCl, 0.1 mM KH2PO4, 2.0 mM Ca(NO3)2, 0.5 mM MgSO4, 10 μM H3BO3, 0.5 μM MnSO4, 0.2 μM CuSO4, 0.5 μM ZnSO4, 0.05 μM Na2MoO4 and 0.1 mM Fe-EDTA described by Nozoye et al. (Reference Nozoye, Nagasaka, Kobayashi, Takahashi, Sato, Uozumi, Nakanishi and Nishizawa2011). Fe deficiency was initiated 4–5 weeks after germination by transfer of the plants to an Fe-free culture medium. After 7 days, the root exudates were collected as described in the next section and plants were harvested to analyse chlorophyll (SPAD), phenylamides, phytoalexins and metals. SPAD values of the three most expanded leaves were measured with a SPAD-502 chlorophyll meter (Konica-Minolta, http://www.konicaminolta.com) and averaged. Dried shoots were ground and used for metal determination by inductively coupled plasma atomic emission spectrometry as described (Nozoye et al., Reference Nozoye, Aung, Masuda, Nakanishi and Nishizawa2017). All experiments were performed in triplicate.
Collection of root exudates
MAs secretion was measured as described (Nozoye et al., Reference Nozoye, Nagasaka, Bashir, Takahashi, Kobayashi, Nakanishi and Nishizawa2014). In brief, roots were rinsed with deionized water and then held in deionized water for 5 h from the start of illumination. The water was renewed once and an antimicrobial agent (Micropur; Katadyn Products Inc.) was added to prevent microbial degradation of the MAs. These root exudates were filtered through Amberlite IR(H+) 120 (Organo) cation exchange resin and the cationic fraction eluted with 2 M NH4OH was analysed by high-performance liquid chromatography (HPLC) as described (Nozoye et al., Reference Nozoye, Nagasaka, Kobayashi, Takahashi, Sato, Uozumi, Nakanishi and Nishizawa2011).
Analyses of phenylamides in root extracts
Phenylamides were prepared as described (Morimoto et al., Reference Morimoto, Ueno, Teraishi, Okumoto, Mori and Ishihara2018; Ube et al., Reference Ube, Harada, Katsuyama, Osaki-Oka, Tonooka, Ueno, Taketa and Ishihara2019a, Reference Ube, Yabuta, Tohnooka, Ueno, Taketa and Ishihara2019b). Plant materials were extracted in 10 vol. of 80% methanol for 24 h, and the extracts were subjected to liquid chromatography – tandem mass spectrometry (LC-MS/MS) analysis with multiple reaction monitoring on a triple-quadrupole LC-MS/MS system (Agilent 6420, Agilent) connected to an ultra-HPLC system (1290 Infinity II, Agilent), with the following LC conditions: column, Acquity UPLC BEH C18, 2.1 mm × 50 mm (1.7 μm; Waters); flow rate, 0.2 ml/min; column temperature, 40°C; solvents, 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B); gradient, 5–70% A/(A + B) within 10 min. The multiple reaction monitoring conditions were optimized using authentic compounds (online Supplementary Table S1).
We analysed 25 phenylamides – all 25 combinations of 5 acids (cinnamic acid, Cin; caffeic acid, Caf; p-coumaric acid, Cou; ferulic acid, Fer; and benzoic acid, Ben) and 5 amines (putrescine, Put; agmatine, Agm; tryptamine, Try; serotonin, Ser; and tyramine, Tyr) – and 2 phenylamide phytoalexins, triticamide (Tri) A (cinnamoyl-9-hydroxy-8-oxotryptamine) and TriB (cinnamoyl-8-oxotryptamine). We refer to each phenylamide by combining the abbreviations of their acid and amine parts, e.g. CouPut = p-coumaroylputrescine.
Genome-wide association study (GWAS), gene identification and statistical analysis
We performed GWAS as described (Elhadi et al., Reference Elhadi, Kamal, Gorafi, Yamasaki, Takata, Tahir, Itam, Tanaka and Tsujimoto2021) for DMA secretion from roots, leaf SPAD values, metal (Fe, Zn, Mn and Cu) concentrations in shoots and the six phenylamides and two phytoalexins abundant in the roots of Ae. tauschii. Best linear unbiased prediction (BLUP) values for each trait and a Mixed Linear Model (MLM) in TASSEL v. 5 software were used. To identify candidate genes, we used significant marker sequences for searches in Gramene (https://www.gramene.org/) with the BLAST option for comparison with the Ae. tauschii sequence (Aet_v4.0). To find the annotations of each marker, we performed BLAST analysis using the marker sequence as a query against the Ae. tauschii genome data set (Aet_v4.0) in Gramene and National Center for Biotechnology information (NCBI; National Center for Biotechnology Information (nih.gov)). When the sequence of a marker did not overlap a gene, the closest gene was retrieved. Multivariate analysis and principal component analysis (PCA) were performed in JMP Pro v. 16 software (SAS Institute Inc., https://www.jmp.com/en_us/home.html).
Results
Analysis of MAs in root exudates of Ae. tauschii
The main peaks detected in all root exudate samples were consistent with DMA (Fig. 1a). Small peaks were detected before the peak of DMA in accessions KU-2109, IG126387, KU-2075, KU-2092, KU-2093, KU-2096, KU-2097 and KU-2156, but these peaks differed from mugineic acid (MA) and 3′-hydroxymugineic acid (HMA). In KU-2093, KU-2097, KU-2156, there was a peak between DMA and asparagine (ASP). The variation in the peaks seemed not to be linked to lineage (TauL1, 2, or 3). DMA concentration was estimated at 0.004 to 56.9 μg/g FW per 5 h (Fig. 1b).
Figure 1. HPLC analysis of Ae. tauschii root exudates. (a) Chromatograms of root exudates of 42 Ae. tauschii lines and wheat ‘Norin 61’ and ‘Langdon’. Standard solutions of 2′-deoxymugineic acid (DMA), mugineic acid (MA), 3′-hydroxymugineic acid (HMA), avenic acid and aspartate (ASP) were used as controls. The x-axis represents the retention time (min). Retention times: DMA standard, 12.48 min; MA, 10.08 min; HMA, 7.95 min; avenic acid, 11.63 min; ASP, 19.13 min. (b) DMA secretion from roots calculated from the peak area of DMA of each accession. Lineages by molecular phylogenetic analysis are shown in colour: blue, TauL1; red, TauL2; teal, TauL3.
Analysis of phenolics in roots of Ae. tauschii
In the roots of Ae. tauschii and wheat plants, we detected 15 phenylamides – CafPut, CouPut, FerPut, CinAgm, CouAgm, FerAgm, BenAgm, CafAgm, BenTry, CinTry, CouTry, FerTry, FerSer, CouTyr and FerTyr – and both phytoalexins (TriA and TriB) (Fig. 2). Among them, CouPut, FerPut, CouAgm and FerAgm were abundant, though FerPut and FerAgm were variable, and CafPut, CinAgm, TriA and TriB were low and highly variable. Differences among TauL1, TauL2 and TauL3 were not clear.
Figure 2. Analysis of phenylamides in roots of Ae. tauschii accessions and wheat ‘Norin 61’ and ‘Langdon’. LC-MS/MS detected 15 phenylamides – caffeoylputrescine (CafPut), p-coumaroylputrescine (CouPut), feruloylputrescine (FerPut), cinnamoylagmatine (CinAgm), p-coumaroylagmatine (CouAgm), feruloylagmatine (FerAgm), benzoylagmatine (BenAgm), caffeoylagmatine (CafAgm), benzoyltryptamine (BenTry), cinnamoyltryptamine (CinTry), p-coumaroyltryptamine (CouTry), feruloyltryptamine (FerTry), feruloylserotonin (FerSer), p-coumaroyltyramine (CouTyr), feruloyltyramine (FerTyr) – and the phenylamide phytoalexins triticamide A (TriA) and triticamide B (TriB). Lineages by molecular phylogenetic analysis are shown in colour: blue, TauL1; red, TauL2; teal, TauL3.
Analysis of SPAD values and metal concentrations in shoots of Ae. tauschii
The SPAD values and metal concentrations varied among the lines of Ae. tauschii, although there were no clear differences among TauL1, TauL2 and TauL3 (Fig. 3).
Figure 3. Analysis of SPAD values and concentrations of Fe, Zn, Cu and Mn in shoots of Ae. tauschii accessions and wheat ‘Norin 61’ and ‘Langdon’. Lineages by molecular phylogenetic analysis are shown in colour: blue, TauL1; red, TauL2; teal, TauL3.
Multivariate analysis and PCA
SPAD values were correlated positively with Fe concentrations in the shoots and negatively with DMA secreted from the roots (Fig. 4a). They were correlated slightly positively with Cu, Zn, Mn, CouPut, CinAgm, CouAgm and FerAgm and slightly negatively with CafPut, FerPut, TriA and TriB.
Figure 4. (a) Multivariate analysis and (b) principal component analysis of physiological traits involved in Fe homoeostasis. SPAD, leaf chlorophyll content; DMA, amount of 2′-deoxymugineic acid secreted from roots; Fe, iron in shoots; Cu, copper in shoots; Zn, zinc in shoots; Mn, manganese in shoots; CafPut, caffeoylputrescine in roots; CouPut, p-coumaroylputrescine in roots; FerPut, feruloylputrescine in roots; CinAgm, cinnamoylagmatine in roots; CouAgm, p-coumaroylagmatine in roots; FerAgm, feruloylagmatine in roots; TriA, triticamide A in roots; TriB, triticamide B in roots. Lineages by molecular phylogenetic analysis are shown in colour: red, TauL1; green, TauL2; blue, TauL3.
Fe concentrations in the shoots were correlated positively with Cu, Mn, Zn, CouPut, FerPut and CouAgm, in addition to SPAD value. Fe was correlated more strongly with Cu than with Mn and Zn. Cu, Mn and Zn were correlated strongly positively with each other and moderately positively with CouPut.
There were several positive correlations among phenylamides and phytoalexins. CafPut was correlated strongly with CinAgm and weakly with CouPut, CouAgm, TriA and TriB. CouPut was correlated strongly with CouAgm and CinAgm. FerPut was correlated strongly with CouAgm and FerAgm. CinAgm was correlated strongly with CafPut, CouPut, CouAgm, TriA and TriB. TriA and TriB were correlated with each other and with DMA secreted from the roots.
PCA separated the data into two main groups, one with SPAD values and Fe, Zn, Mn and Cu in the shoots and the other with phenylamides and phytoalexins (Fig. 4b). CouPut and DMA did not belong to either group.
GWAS analysis
Manhattan plots showed that some markers were correlated with each physiological trait (Fig. 5; Table 1, online Supplementary Table S2). Among the markers most closely associated with each trait, those associated with Zn, DMA, CafPut, CinAgm, TriA and TriB overlapped a gene coding region (Table 1). For other traits, overlapping genes were not found, so the closest genes are listed. Among the gene extracted, some traits shared the same genes (online Supplementary Table S2). Among those associated with Cu, Mn and Zn, five were the same as each other. In addition, among 10 genes associated with Cu, 2 were also associated with Zn and 1 was also associated with Cu. Such duplications were also found between FerAgm and FerPut, CinAgm and TriB and TriA and TriB.
Figure 5. Manhattan plots representing seven chromosomes carrying significant markers detected by Mixed Linear Model using BLUP value in accessions. x-axis, genomic coordinates; y-axis, negative logarithm of the association P value for each single nucleotide polymorphism (SNP). Each dot signifies a SNP.
Table 1. Most significant marker loci-associated traits based on BLUP values in the analysed accessions
‘Trait’: plant phenotype analysed; ‘Marker’: name of the gene marker; ‘Chr’: number of the Ae. tauschii chromosome where the marker is located; ‘−log10(P)’: the negative logarithm of the association P value for each marker values based on BLUPs; ‘Overlapping or closest gene’: gene located at or closest to the position in Gramene (most similar to the marker in Aet_v4.0: https://www.gramene.org/); ‘Description’: annotation of the listed gene.
Discussion
New MAs in Ae. tauschii might be useful to enhance Fe homoeostasis in bread wheat
Both the amounts of MAs secreted from roots and the number of MAs types are positively correlated with tolerance to Fe deficiency (Takagi et al., Reference Takagi, Nomoto and Takemoto1984). MAs are produced from S-adenosylmethionine to DMA, the first MAs, via four sequential steps catalysed by S-adenosylmethionine synthetase, nicotianamine synthase, nicotianamine aminotransferase and DMA synthase. (Mori and Nishizawa, Reference Mori and Nishizawa1987; Shojima et al., Reference Shojima, Nishizawa, Fushiya, Nozoe, Irifune and Mori1990; Higuchi et al., Reference Higuchi, Suzuki, Nakanishi, Yamaguchi, Nishizawa and Mori1999; Takahashi et al., Reference Takahashi, Yamaguchi, Nakanishi, Shioiri, Nishizawa and Mori1999; Bashir et al., Reference Bashir, Inoue, Nagasaka, Takahashi, Nakanishi, Mori and Nishizawa2006). As in wheat, rice produces and secretes only DMA. Introducing the barley Fe-deficiency-specific clone no. 3 gene (IDS3) (Nakanishi et al., Reference Nakanishi, Yamaguchi, Sasakuma, Nishizawa and Mori2000) for the hydroxylase which converts DMA to MA conferred tolerance to Fe deficiency in calcareous soil (Suzuki et al., Reference Suzuki, Morikawa, Nakanishi, Takahashi, Saigusa, Mori and Nishizawa2008). Wheat with a rye genome added to it became able to produce HMA and MA in addition to DMA (Mori et al., Reference Mori, Nishizawa and Fujigaki1990). Although the D genome is already present in cultivated wheat, the intraspecific diversity of the D genome is in wild Ae. tauschii accessions may carry novel and useful genes for involvement in new MAs biosynthesis. We expect that Fe-deficiency tolerance or Fe biofortification of bread wheat could be generated by introducing Ae. tauschii genes into bread wheat to produce additional MAs or increase MA contents.
The root exudates of 42 accessions of Ae. tauschii contained mainly DMA (Fig. 1). These results are consistent with previous reports that some accessions of Ae. tauschii produce and secrete only DMA (Singh et al., Reference Singh, Sasakuma, Bughio, Takahashi, Nakanishi, Yoshimura, Nishizawa and Mori2000; Tolay et al., Reference Tolay, Erenoglu, Römheld, Braun and Cakmak2001). In the exudates of accessions KU-2109, IG126387, KU-2075, KU-2092, KU-2093, KU-2096, KU-2097 and KU-2156, there were small unknown peaks that differed from known MAs (Fig. 1). These compounds seem to be new MAs, but their amounts were lower than those of DMA, suggesting that their effect on Fe nutrition in the accessions might be small. Barley produces and secretes larger amounts and more kinds of MAs than wheat (Takagi et al., Reference Takagi, Nomoto and Takemoto1984). Since it is speculated that barley was domesticated earlier than wheat and has a different history of breeding, it is hypothesized that few lines of Ae. tauschii became ancestors of bread wheat (Ogbonnaya et al., Reference Ogbonnaya, Abdalla, Mujeeb-Kazi, Kazi, Xu, Gosman, Lagudah, Bonnett, Sorrells and Tsujimoto2013). There are many wild lines of Ae. tauschii whose genome is not reflected in bread wheat. Further analysis with more accessions might find accessions that produce and secrete other kinds of MAs.
Although natural variations in Ae. tauschii are separated into three lineages (TauL1, 2, 3) by molecular phylogenetic analysis (Sohail et al., Reference Sohail, Shehzad, Kilian, Eltayb, Tanaka and Tsujimoto2012; Matsuoka et al., Reference Matsuoka, Nasuda, Ashida, Nitta, Tsujimoto, Takumi and Kawahara2013; Mahjoob et al., Reference Mahjoob, Chen, Gorafi, Yamasaki, Kamal, Abdelrahman, Iwata, Matsuoka, Tahir and Tsujimoto2021), the phenotypic traits were not associated with any specific group. Fe solubility differs with soil pH: it is high in acidic soils and low in alkaline soils. TauL3 is found only in Georgia, while TauL2 and TauL1 are found over larger areas (Mahjoob et al., Reference Mahjoob, Chen, Gorafi, Yamasaki, Kamal, Abdelrahman, Iwata, Matsuoka, Tahir and Tsujimoto2021). TauL2 is found mainly along the Caspian Sea in Azerbaijan and Iran. TauL1 is most widespread and is found in Georgia, Turkey, Armenia, Azerbaijan, Syria, Iraq, Iran, Uzbekistan, Tajikistan, Kyrgyzstan, Afghanistan, Pakistan, India and China. Soil pH is alkaline in all of these regions except Georgia (IGBP-DIS, 1998). We used only three accessions of TauL3. Further analysis with more accessions could reveal the connection between Fe homoeostasis in Ae. tauschii and soil pH.
Sensitivities to Fe deficiency might differ among Ae. tauschii accessions
SPAD values (chlorophyll content) and Fe, Zn, Mn and Cu concentrations in leaves differed among accessions (Fig. 3). Under Fe deficiency, interveinal chlorosis occurs and the SPAD value decreases. It was suggested that chlorosis occurs through the impairment of chlorophyll biosynthesis, since Fe is a cofactor of the several enzyme in this process. The SPAD values and Fe concentrations in leaves were correlated positively with each other but negatively with DMA secretion (Fig. 4a). Fe deficiency transcriptionally induces DMA production and secretion (Kobayashi et al., Reference Kobayashi, Nozoye and Nishizawa2019). These results suggest that sensitivity to Fe deficiency varies among accessions, some of which sense it more strongly than others and enhance MAs production and Fe translocation to the leaves.
MAs may be involved in Fe transport to chloroplasts and may influence the efficiency of photosynthesis (Yoneyama, Reference Yoneyama2021). Chlorophylls capture solar energy and transfer excited electrons to the photosystem I and II complexes, which contain Fe ions arranged on the chloroplast thylakoids (Raven et al., Reference Raven, Evans and Korb1999). Fe concentrations in leaves were positively correlated with Cu, Zn and Mn (Fig. 4a), which are also important in photosynthesis (Yruela, Reference Yruela2013). We speculate that the systems of Fe, Cu, Zn and Mn transport to chloroplasts might overlap.
Phenolics are involved in Fe homoeostasis in Ae. tauschii
In Fe-deficient roots, we detected 15 phenylamides and 2 phytoalexins at varying concentrations among accessions (Fig. 2). CouPut, FerPut and CouAgm in the roots were correlated positively with Fe in the leaves (Fig. 4a). Coumarins are involved in Fe uptake from the rhizosphere and Fe translocation in the plant body in Arabidopsis thaliana (Rodríguez-Celma et al., Reference Rodríguez-Celma, Lin, Fu, Abadía, López-Millán and Schmidt2013; Schmid et al., Reference Schmid, Giehl, Döll, Mock, Strehmel, Scheel, Kong, Hider and von Wirén2013; Fourcroy et al., Reference Fourcroy, Sisó-Terraza, Sudre, Savirón, Reyt, Gaymard, Abadía, Abadia, Álvarez-Fernández and Briat2014; Schmidt et al., Reference Schmidt, Günther, Weber, Spörlein, Loscher, Böttcher, Schobert and Clemens2014). They are biosynthesized via the phenylpropanoid pathway by ortho-hydroxylation of cinnamates including Cou and Fer (Shimizu, Reference Shimizu2014). In this pathway, A. thaliana ortho-hydroxylase (AtF6′H1) is induced at the transcriptional level by Fe deficiency, and its disruption impairs Fe translocation to leaves (Fourcroy et al., Reference Fourcroy, Sisó-Terraza, Sudre, Savirón, Reyt, Gaymard, Abadía, Abadia, Álvarez-Fernández and Briat2014). Ferulic acid can also chelate Fe and is effective at rescuing mouse brains from Fe overload (AAlikhani et al., Reference AAlikhani, Khalili and Jahanshahi2022). We speculate that coumarin and ferulic acid are also involved in Fe homoeostasis in Ae. tauschii. Fe concentrations in leaves were positively correlated with both phytoalexins (Fig. 4a). Phytoalexins provide a chemical defence against pathogen infection (Ahuja et al., Reference Ahuja, Kissen and Bones2012). Triticamides are novel phytoalexins identified in barley and wheat that accumulate in roots in response to microbial infection (Ube et al., Reference Ube, Harada, Katsuyama, Osaki-Oka, Tonooka, Ueno, Taketa and Ishihara2019a, Reference Ube, Yabuta, Tohnooka, Ueno, Taketa and Ishihara2019b). Phytoalexins are regulated by jasmonate (JA) and salicylic acid (SA) (Miyamoto et al., Reference Miyamoto, Shimizu and Okada2014). Triticamides are induced by JA and cytokinins but not by SA (Ube et al., Reference Ube, Harada, Katsuyama, Osaki-Oka, Tonooka, Ueno, Taketa and Ishihara2019a, Reference Ube, Yabuta, Tohnooka, Ueno, Taketa and Ishihara2019b). Endogenous JA rapidly increases in roots in response to Fe deficiency treatment in rice (Kobayashi et al., Reference Kobayashi, Itai, Senoura, Oikawa, Ishimaru, Ueda, Nakanishi and Nishizawa2016). Moreover, a basic leucine zipper transcription factor, OsbZIP83, which is involved in Fe homoeostasis, positively regulates genes related to phytoalexin biosynthesis and the SA pathway (Kobayashi et al., Reference Kobayashi, Shinkawa, Nagano and Nishizawa2022). We speculate that triticamides are involved in Fe homoeostasis in Ae. tauschii under the control of JA.
Candidate genes in Ae. tauschii accessions to improve Fe homoeostasis of bread wheat
The insertion of alien chromosome segments is an attractive method to improve the physiological and agronomic traits of wheat (Ogbonnaya et al., Reference Ogbonnaya, Abdalla, Mujeeb-Kazi, Kazi, Xu, Gosman, Lagudah, Bonnett, Sorrells and Tsujimoto2013). For example, the improvement of Zn efficiency, the ability to grow and yield better under Zn deficiency, in synthetic wheat by genes from Ae. tauschii (DD) suggests the presence of genes for high Zn efficiency in the D genome of Ae. tauschii (Cakmak et al., Reference Cakmak, Cakmak, Eker, Ozdemir, Watanabe and Braun1999). And the insertion of Aegilops chromosomes increased seed Fe and Zn concentrations by 50 to 248%, respectively, relative to the recipient wheat cultivar (Wang et al., Reference Wang, Yin, Tanaka, Tanaka and Tsujimoto2011). Here, GWAS analysis supported the association of several markers with DMA secretion from roots, leaf SPAD values, shoot metal concentrations and root phenylamides and phytoalexins (Table 1, online Supplementary Table S2).
For SPAD, AET1Gv20067800, annotated as a putative F-box/LRR-repeat protein 23, was closest to the marker. In humans, F-box/LRR-repeat protein 5 (FBXL5) acts as an Fe sensor by degrading iron regulatory protein 2 under Fe-replete conditions through the ubiquitin–proteasome system (Salahudeen et al., Reference Salahudeen, Thompson, Ruiz, Ma, Kinch, Li, Grishin and Bruick2009; Vashisht et al., Reference Vashisht, Zumbrennen, Huang, Powers, Durazo, Sun, Bhaskaran, Persson, Uhlen, Sangfelt, Spruck, Leibold and Wohlschlegel2009). Although plants do not contain this animal Fe-deficiency response system, the protein contains a putative Fe-binding haemerythrin domain as in FBXL5, which acts as an Fe sensor in Arabidopsis (Long et al., Reference Long, Tsukagoshi, Busch, Lahner, Salt and Benfey2010) and rice (Kobayashi et al., Reference Kobayashi, Nagasaka, Senoura, Itai, Nakanishi and Nishizawa2013). Since chlorophyll contents in the leaves of Fe-deficient Ae. tauschii accessions were diverse and were correlated with Fe deficiency, differences in AET1Gv20067800 might influence susceptibility to Fe deficiency through its role as an Fe sensor in Ae. tauschii.
For Fe, AET1Gv20678900, which has homology to Os05g0458300, a laccase-13 (OsLAC13) similar to l-ascorbate oxidase, was closest to the marker. Ascorbate can donate electrons and scavenge free radicals to reduce Fe3+ to Fe2+ (Smirnoff, Reference Smirnoff2018). Laccase could be involved in processes that affect Fe homoeostasis such as lignification, ferroxidase activity and oxidative stress, which are speculated to be regulated through microRNAs upregulated under Cu deficiency in Arabidopsis and rice (Liu et al., Reference Liu, Luo, Wang, Shen and Zheng2017; Carrió-Seguí et al., Reference Carrió-Seguí, Ruiz-Rivero, Villamayor-Belinchón, Puig, Perea-García and Peñarrubia2019). AET1Gv20678900 could be involved in Fe accumulation in shoots under Fe deficiency in Ae. tauschii.
For Zn, AET2Gv21156300, which has homologies to Os04g0640500, an ABC-1 domain-containing protein, and to AT1G71810, a protein kinase, lay in the marker region. The ABC-1 domain is found in yeast ABC1 proteins required for the function of the mitochondrial bc1 complex (Bousquet et al., Reference Bousquet, Dujardin and Slonimski1991), in which ubiquinone functions as an essential cofactor. Although the function of these proteins is not clear, the ABC-1 domain is found in a eukaryotic-type protein kinase, so a role in the regulation of phosphorylation has been speculated. The ABC-1-like protein AtOSA1 (A. thaliana oxidative stress–related ABC1-like protein) is involved in cadmium, oxidative and high-light stresses (Jasinski et al., Reference Jasinski, Sudre, Schansker, Schellenberg, Constant, Martinoia and Bovet2008). AET2Gv21156300 is associated with not only Zn but also Mn and Cu (online Supplementary Table S2), suggesting its involvement in the response to stress caused by metal imbalances.
For Cu, AET1Gv20974700, which has homology to root phototropism protein 2 (RPT2), was closest to the marker. RPT2 is a signal transducer of the phototropic response and photo-induced movements and is involved in adaptation to high-intensity light (Haga et al., Reference Haga, Tsuchida-Mayama, Yamada and Sakai2015). AET1Gv20974700 was also associated with Mn. Since AtOSA1, which is associated with both Cu and Mn, is also involved in responses to high light along with RPT2, high light might be involved in the accumulation of Cu and Mn in Ae. tauschii shoots under Fe deficiency.
For Mn, AET7Gv20131100, which has homologies to BTB/POZ and MATH domain-containing protein 3 (BPM3)-like, was closest to the gene marker. BPM3 acts as a substrate-specific adapter of an E3 ubiquitin–protein ligase complex that mediates the ubiquitination and subsequent proteasomal degradation of target proteins (Weber et al., Reference Weber, Bernhardt, Dieterle, Hano, Mutlu, Estelle, Genschik and Hellmann2005) and assembles with members of the ethylene response factor/Apetala 2 transcription factor family, which is induced by abiotic stresses (Weber and Hellmann, Reference Weber and Hellmann2009). AET7Gv20131100 was also associated with Zn and Cu (online Supplementary Table S2), so it might be involved in the induction of gene expression to cope with an imbalance in metal homoeostasis caused by Fe deficiency.
Genes involved in responses to disease and pathogens were included in the list of gene markers that could be related to phenylamides and phytoalexins. For CinAgm, AET2Gv21172900, which has homology to diacylglycerol kinase (OsBIDK1), lay in the marker region. OsBIDK1 has a role in disease resistance responses, and its overexpression enhances disease resistance in transgenic tobacco (Zhang et al., Reference Zhang, Chen, Zhang and Song2008). For CouAgm, AET5Gv20365900, which has homology to Os12g0131100, which is similar to glucosamine-fructose-6-phosphate aminotransferase (GFAT), was closest to the marker. GFAT is the first rate-limiting enzyme of the hexosamine biosynthesis pathway and is suggested to be involved in chitin biosynthesis in insects (Kato et al., Reference Kato, Dasgupta, Smartt and Christensen2002), although its function in plants has not been identified.
For CouPut, AET3Gv20996200, which has homology to Os01g0931600 and Tubby (C-terminal domain-containing protein), was closest to the marker. Tubby proteins are found in multicellular plants and animals and are suggested to have a role in transcription (Carroll et al., Reference Carroll, Gomez and Shapiro2004). GWAS analysis listed Os01g0931600 among markers associated with chilling-tolerant germination in maize (Ma et al., Reference Ma, Yao, Zhang, Su, Wang, Song, Li and Zhao2022). For TriA, AET1Gv20604600, which has homology to a putative rhomboid homolog (OsRhmbd3), lay in the marker region. Rhomboid-like is a widespread superfamily of intramembrane serine proteases conserved in all kingdoms and is suggested to be involved in signal transduction of several biological functions (Freeman, Reference Freeman2014). For TriB, AET3Gv20081800, which has homology to Os01g0134900, or ‘(1,4)-beta-xylan endohydrolase, isoenzyme X-II’, lay in the gene marker region. ‘(1,4)-Beta-xylan endohydrolase, isoenzyme X-II’ is implied to be involved in cell wall degradation in germinated barley grain and has similarities to pathogenesis-related glucanases in plants that hydrolyse the polysaccharides that are abundant in fungal cell walls (Hrmova et al., Reference Hrmova, Banik, Harvey, Garrett, Varghese, Høj and Fincher1997). AET3Gv20081800 was also associated with CinAgm (online Supplementary Table S2). These genes might be involved in the biosynthesis of phenylamides and phytoalexins under Fe deficiency. Further analysis is needed to clarify the functions associated with the markers and their contribution to tolerance to Fe deficiency.
Conclusions
We analysed physiological traits under Fe deficiency in 42 accessions of Ae. tauschii. All accessions secreted mainly DMA from their roots under Fe deficiency. Eight of them secreted unknown products, which may be new MAs, from their roots under Fe deficiency, suggesting that there are new genes involved in MAs biosynthesis in Ae. accessions. We identified 15 phenylamides and 2 phytoalexins, amounts of which varied among the accessions. Several correlations among traits suggest that these traits are important to cope with Fe deficiency. The GWAS analysis showed that several markers correlated with the examined traits studied, suggesting that Ae. tauschii accessions contain novel useful genes to improve Fe nutrition. Further analysis with more accessions is needed to reveal the most effective markers that confer tolerance to Fe deficiency in Ae. tauschii, which will lead to the breeding of Fe-deficiency-tolerant and Fe-biofortified bread wheat.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262123000424
Acknowledgements
This work was funded by the Joint Research Program of the Arid Land Research Center, Tottori University (31C2011 and 03C2005 to T.N.), the Japan Society for the Promotion of Science (JSPS) for the KAKENHI Grant 15K18658, 15KK0286 and JP20K05777 (to T.N.) and Uragami-zaidan (27s22 to T.N.).
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