Successful Reproduction Requires the Function of Arabidopsis YELLOW STRIPE-LIKE1 and YELLOW STRIPE-LIKE3 Metal-Nicotianamine Transporters in Both Vegetative and Reproductive Structures

Several members of the Yellow Stripe-Like (YSL) family of proteins are transporters of metals that are bound to the metal chelator nicotianamine or the related set of mugineic acid family chelators known as phytosiderophores. Here, we examine the physiological functions of three closely related Arabidopsis (Arabidopsis thaliana) YSL family members, AtYSL1, AtYSL2, and AtYSL3, to elucidate their role(s) in the allocation of metals into various organs of Arabidopsis. We show that AtYSL3 and AtYSL1 are localized to the plasma membrane and function as iron transporters in yeast functional complementation assays. By using inflorescence grafting, we show that AtYSL1 and AtYSL3 have dual roles in reproduction: their activity in the leaves is required for normal fertility and normal seed development, while activity in the inflorescences themselves is required for proper loading of metals into the seeds. We further demonstrate that the AtYSL1 and AtYSL2 proteins, when expressed from the AtYSL3 promoter, can only partially rescue the phenotypes of a ysl1ysl3 double mutant, suggesting that although these three YSL transporters are closely related and have similar patterns of expression, they have distinct activities in planta. In particular, neither AtYSL1 nor AtYSL2 is able to functionally complement the reproductive defects exhibited by ysl1ysl3 double mutant plants.The transition metals iron (Fe), copper (Cu), and zinc (Zn) are among the most important and most problematic of all the micronutrients used by plants. The importance of these metals stems from their roles as essential cofactors for cellular redox reactions involved in photosynthesis, respiration, and many other reactions. The problematic nature of these metals stems from the same distinct chemical properties that make them so valuable to living systems. These metals, particularly Cu and Fe, are highly reactive and, if overaccumulated, can cause cellular redox damage. Fe presents an additional problem for plants, because it is also only sparingly soluble in aqueous solution and thus is typically not “bioavailable” in soil (Guerinot and Yi, 1994). As a response to these key properties, plants have evolved multifaceted systems to control metal uptake by the root, translocation through the plant body, storage within tissues, and remobilization during reproduction and times of nutrient stress.The nonproteinogenic amino acid nicotianamine (NA) is a strong complexor of various transition metals, particularly Fe(II) (Anderegg and Ripperger, 1989) and Fe(III) (von Wiren et al., 1999), as well as Cu(II), Ni(II), Co(II), Mn(II), and Zn(II) (Anderegg and Ripperger, 1989). NA is present in shoots and roots at concentrations ranging between 20 and 500 nm g⁻¹ fresh weight (Stephan et al., 1990) and is present in both xylem (approximately 20 μm Pich and Scholz, 1996]) and phloem (approximately 130 μm Schmidke and Stephan, 1995]), suggesting that it is a major complexor of metals throughout the plant. Much of what we know about NA function in plants comes from studies of a mutant of tomato (Solanum lycopersicum) called chloronerva, in which the single gene encoding NA synthase is disrupted (Herbik et al., 1999; Higuchi et al., 1999; Ling et al., 1999). The chloronerva phenotype is complex. Plants exhibit interveinal chlorosis in young leaves and constitutively activate their root Fe uptake systems, indicating that they have inadequate Fe. However, mature leaves of chloronerva mutants contain excess Fe, implying that the Fe that is present is not being properly localized in the absence of NA. These chloronerva plants also have severe defects in translocation of Cu in the xylem, indicating a clear role for NA in Cu transport. The plants are sterile, indicating that NA is important during plant reproduction. Complementing these classical studies on chloronerva, Takahashi et al. (2003) have developed tobacco (Nicotiana tabacum) plants that heterologously express a barley (Hordeum vulgare) gene encoding the enzyme NA aminotransferase, which converts NA into a nonfunctional intermediate. Recently, the phenotype of quadruple NA synthase mutants was described in Arabidopsis (Arabidopsis thaliana; Klatte et al., 2009). In both studies, the plants exhibited many of the defects caused by the chloronerva mutation, including chlorosis and an array of reproductive abnormalities.Several members of the well-conserved Yellow Stripe-Like (YSL) family of proteins function as metal-NA transporters (DiDonato et al., 2004; Koike et al., 2004; Roberts et al., 2004; Schaaf et al., 2004; Murata et al., 2006; Gendre et al., 2007). The founding member of the YSL family, maize (Zea mays) Yellow Stripe1 (ZmYS1), is the primary means by which roots of grasses take up Fe from the soil. The grasses, a group that includes most of the world’s staple grains (e.g. rice Oryza sativa], wheat Triticum aestivum], and maize), use a chelation strategy for primary Fe uptake. In response to Fe starvation, grasses secrete phytosiderophores (PS): derivatives of the mugineic acid family that are structurally similar to NA and that form stable Fe(III) chelates in soil (Tagaki et al., 1984). This accomplishes solubilization of the otherwise nearly insoluble soil Fe. The YS1 protein, located at the root surface, then moves the Fe(III)-PS complexes from the rhizosphere into root cells (Romheld and Marchner, 1986; Curie et al., 2001; Roberts et al., 2004)Arabidopsis has eight YSL genes. Three of these (AtYSL1, At4g24120; AtYSL2, At5g24380; and AtYSL3, At5g53550) are expressed strongly in the xylem parenchyma of leaves and are down-regulated during Fe deficiency (DiDonato et al., 2004; Waters et al., 2006). We have previously shown that double mutant plants with lesions in both AtYSL1 and AtYSL3 display strong interveinal chlorosis. We have hypothesized that the function of these YSL transporters in vegetative tissues is to take up Fe that arrives in leaves via the xylem (Waters et al., 2006). All of the defects displayed by ysl1ysl3 double mutants can be alleviated if excess Fe is applied to the soil, demonstrating that these growth defects are caused primarily by a lack of Fe. Intriguingly, although Fe deficiency appears to be the basis of the double mutant phenotype, the concentrations of several metals are specifically altered in the double mutants (Waters et al., 2006). AtYSL1 single mutant plants have subtle phenotypes, the most striking of which is a decrease in both NA and Fe in seeds (Le Jean et al., 2005). Interestingly, leaves of these mutants contain excess NA, while Fe levels are normal. These observations are consistent with the more obvious and extensive phenotypes exhibited by the ysl1ysl3 double mutant and highlight the idea that AtYSL proteins affect the homeostasis of both Fe and NA.In addition to the vegetative defects mentioned above, the ysl1ysl3 double mutant has multiple defects in reproduction. Double mutant flowers produce few functional pollen grains and thus exhibit greatly reduced fertility. Many of the seeds that these plants do manage to produce are small and contain embryos arrested at various immature stages, which often fail to germinate. These fertility defects can be reversed by application of Fe-ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) solution to the soil, again demonstrating that these growth defects are caused by a lack of Fe (Waters et al., 2006). Expression of YSL1 and YSL3 is very limited in flowers and developing siliques; furthermore, the patterns of expression of YSL1 and YSL3 are distinct and largely nonoverlapping in these structures. However, expression of AtYSL1 and AtYSL3 increases markedly during leaf senescence, a period in which many minerals are remobilized from leaves, presumably for delivery into developing seeds (Himelblau and Amasino, 2001). This model is in good agreement with the accepted model for nutrient loading into seeds proposed originally by Hocking and Pate (1977, 1978), which suggests that metals mobilized from vegetative structures account for 20% to 30% of the content in seeds. Direct measurements of metals in senescing and younger leaves demonstrated that double mutants failed to mobilize Zn and Cu from leaves. Seeds produced by the double mutant plants contained reduced levels of Zn and Cu, the same metals that failed to be mobilized out of the leaves (Waters et al., 2006). This led us to propose a model in which the activity of AtYSL1 and AtYSL3 in leaves was required for correct localization of metals into the seeds. However, seeds also had low Fe levels, even though Fe appeared to be mobilized normally from leaves of the double mutants.Here, we further investigate the role(s) of AtYSL1 and AtYSL3 in the allocation of metals into various organs of Arabidopsis. AtYSL1 and AtYSL3 are localized to the plasma membrane, and each is capable of suppressing the growth defect of yeast lacking normal Fe uptake, indicating that the most likely biochemical function for these proteins is in uptake of Fe(II)-NA complexes. We have used inflorescence grafting to determine the relative roles of AtYSL1 and AtYSL3 in leaves and inflorescences during seed development. These proteins are found to have dual roles: activity in the leaves is required for normal inflorescence development, while activity in the inflorescences themselves is required for proper loading of metals into the seeds. We have further examined the effect of overexpressing AtYSL3, which resulted in a small increase in Cu in shoots, and have demonstrated that the AtYSL1 protein, when expressed from the AtYSL3 promoter, can only partially rescue the phenotypes of the ysl1ysl3 double mutant, indicating that these proteins have distinct biochemical activities. A third AtYSL from the same subgroup of the YSL family, AtYSL2, also only partially complements the phenotypes of ysl1ysl3 double mutants, suggesting that although these three YSL transporters are closely related, they have distinct activities in planta.