Auxin Acts Downstream of Ethylene and Nitric Oxide to Regulate Magnesium Deficiency-Induced Root Hair Development in Arabidopsis thaliana

Auxin Acts Downstream of Ethylene and Nitric Oxide to Regulate Magnesium Deficiency-Induced Root... Abstract This study examines the association of auxin with ethylene and nitric oxide (NO) in regulating the magnesium (Mg) deficiency-induced root hair development in Arabidopsis thaliana. With Mg deficiency, both ethylene and NO promoted the elevation of root auxin levels in roots by inducing the expression of AUXIN-RESISTANT1 (AUX1), PIN-FORMED 1 (PIN1) and PIN2 transporters. In turn, auxin stimulated ethylene and NO production by activating the activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO), ACC synthase (ACS), nitrate reductase (NR) and NO synthase-like (NOS-L). These processes constituted an NO/ethylene–auxin feedback loop. Interestingly, however, the roles of ethylene and NO in regulating Mg deficiency-induced root hair development required the action of auxin, but not vice versa. In summary, these results suggest that Mg deficiency induces a positive interaction between the accumulation of auxin and ethylene/NO in roots, with auxin acting downstream of ethylene and NO signals to regulate Mg deficiency-induced root hair morphogenesis. Introduction Magnesium (Mg) is critical for plant growth and development because of its essential roles in various physiological processes such as photosynthesis, carbon fixation, protein synthesis, Chl synthesis and enzyme activation (Lilley et al. 1974, Jezek et al. 2015). However, plants often suffer from Mg deficiency in acidic or sandy soils, because the soluble Mg easily leaches from these soils and its absorption by roots is intensively antagonized by the absorption of other cations such as ammonium, aluminum and potassium (Yang et al. 2007, Marschner and Rengel 2012). Mg deficiency reduces both the yield and nutritional quality of crops (Gerendás and Führs 2013); a greater understanding of the mechanisms by which plants mitigate the effects of Mg deficiency is, therefore, important to improve plant adaptation. In previous studies, we found that Mg deficiency significantly induced the initiation and elongation of root hairs (Niu et al. 2014, Liu et al. 2017). The stimulation of root hair growth increases the surface area of roots, allowing them to access greater volumes of soil; this facilitates the uptake of nutrients, especially in nutrient-limited soils (Genc et al. 2007, Nestler and Wissuwa 2016). This root hair stimulation probably allows the plant roots to acquire more Mg from Mg-limited soils. Elucidation of the mechanism regulating Mg deficiency-induced root hair development is, therefore, essential. In our most recent study, we showed that Mg deficiency stress elevated the levels of both ethylene and nitric oxide (NO) in the roots; interestingly, the ethylene activated the production of NO by enhancing the activities of nitrate reductase (NR) and nitric oxide synthase-like (NOS-L), while the NO in turn promoted the synthesis of ethylene by stimulating the activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) and ACC synthase (ACS) (Liu et al. 2017). This interaction is then involved in the regulation of development of Mg deficiency-induced root hairs (Liu et al. 2017). Several studies have shown strong genetic evidence that auxin, in addition to ethylene and NO, plays a crucial role in controlling root hair development under nutrient-adequate growth conditions (Lee and Cho 2013, Rigas et al. 2013). The auxin is mainly synthesized in young leaves and developing leaf primordia (Soeno et al. 2010), and is transported basipetally to other tissues including the roots (Habets and Offringa 2014). In Arabidopsis, auxin transport was mediated by AUXIN-RESISTANT1 (AUX1)/LAX (like AUX1) influx carriers and the PIN-FORMED (PIN) efflux carrier family, including PIN1, PIN2, PIN3, PIN4, PIN7 and P-glycoprotein (PGP) (Křeček et al. 2009, Péret et al. 2012). It had been suggested that PGP could interact with PIN to stabilize PINs at the membrane, and the PINs then provide the directionality to auxin efflux (Titapiwatanakun et al. 2009, Santos et al. 2010). It has been suggested that LAX1 and LAX2 are involved in the aerial development of Arabidopsis (Bainbridge et al. 2008, Péret et al. 2012). Although LAX3 is also involved in the regulation of root growth, the three genes perform distinct functions during Arabidopsis development. For example, it had been proposed that except for AUX1, no other member of the AUX/LAX family plays a role in the root gravitropic response (Péret et al. 2012). Interestingly, the endogenous level of auxin in plants has repeatedly been found to be elevated in response to nutrient deficiency stresses. For instance, iron deficiency quickly elevated auxin levels in the roots of Arabidopsis, red clover and tomato (Chen et al. 2010, Jin et al. 2007, Jin et al. 2011); phosphorus deficiency induced auxin accumulation in the roots of Arabidopsis and white lupin (Gilbert et al. 2000, Miura et al. 2011); nitrate deficiency increased auxin levels in the roots of Arabidopsis and soybean (Ma et al. 2014); and potassium deficiency increased root auxin accumulation in Arabidopsis (Ma et al. 2012). The similar responses observed in these different plant species under various nutrient deficiency stresses suggest that auxin generation in plant roots is probably also induced by Mg deficiency. If this is correct, auxin is likely to regulate Mg deficiency-induced root hair development, and it may be associated with ethylene and NO in this regulatory event. In this study, we used Arabidopsis plants as a model system to examine the above hypotheses. Our results revealed that while auxin, ethylene and NO accumulate similarly under Mg deficiency, auxin acts downstream of the other two chemical signals in the regulatory cascade leading to the induction of root hair development in response to Mg deficiency. Results Elevation of auxin level in roots is required for the regulation of Mg deficiency-induced root hair development As auxin was presumed to be involved in the regulation of Mg deficiency-induced root hair development, we first measured its level in roots of wild-type Col-0 plants in response to Mg deficiency stress. As shown in Fig. 1A, the root auxin level in the Mg-deficient treatment was approximately 1-fold higher than in the Mg-sufficient treatment. The activity of the synthetic auxin response reporter DR5::GFP (green fluorescent protein) had been shown to correlate with direct auxin measurements in plant tissues (Friml et al. 2002b). Here, we found that the intensity of GFP fluorescence in roots was clearly increased under Mg deficiency stress, providing evidence that Mg deficiency increases auxin response in roots (Fig. 1B). Interestingly, the auxin level in the excised roots was not affected by Mg deficiency (Fig. 1D). The result indicates that the auxin accumulation in roots induced by Mg deficiency probably resulted from an enhanced basipetal transport. Fig. 1 View largeDownload slide Effects of auxin on the Mg deficiency-induced growth of root hairs in Arabidopsis Col-0 plants. Auxin levels in the roots of the intact Col-0 plants (A), in the excised roots (D) of Col-0 plants and the root GFP fluorescence in DR5::GFP transgenic plants (B). Images of root hairs (C) and the corresponding length (E) and density (F) of root hairs on segments 0–1, 1–2 and 2–3 cm from the root tip upon auxin treatment. For (A) and (B), the plants were pre-cultured in nutrient solution for 4 weeks, and subsequently transferred to either complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 7 d. Thereafter, the analyses were performed. For (C), (E) and (F), the 4-week-old seedlings were grown in either +Mg or –Mg nutrient solutions for 5 d, and then transferred to +Mg or –Mg solution containing 0.1 μM NAA or 5 μM NPA for another 2 d. Values are means ± SD (n = 6). For (D), the 4-week-old plants were grown in either +Mg or –Mg nutrient solutions for 5 d, and then the roots were cut at the junction of the hypocotyl and the root (excised roots). The excised roots were incubated in +Mg or –Mg solutions for another 2 d. Different lower case letters above the bars indicate significant differences between treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Scale bar = 1 mm. Fig. 1 View largeDownload slide Effects of auxin on the Mg deficiency-induced growth of root hairs in Arabidopsis Col-0 plants. Auxin levels in the roots of the intact Col-0 plants (A), in the excised roots (D) of Col-0 plants and the root GFP fluorescence in DR5::GFP transgenic plants (B). Images of root hairs (C) and the corresponding length (E) and density (F) of root hairs on segments 0–1, 1–2 and 2–3 cm from the root tip upon auxin treatment. For (A) and (B), the plants were pre-cultured in nutrient solution for 4 weeks, and subsequently transferred to either complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 7 d. Thereafter, the analyses were performed. For (C), (E) and (F), the 4-week-old seedlings were grown in either +Mg or –Mg nutrient solutions for 5 d, and then transferred to +Mg or –Mg solution containing 0.1 μM NAA or 5 μM NPA for another 2 d. Values are means ± SD (n = 6). For (D), the 4-week-old plants were grown in either +Mg or –Mg nutrient solutions for 5 d, and then the roots were cut at the junction of the hypocotyl and the root (excised roots). The excised roots were incubated in +Mg or –Mg solutions for another 2 d. Different lower case letters above the bars indicate significant differences between treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Scale bar = 1 mm. We then investigated whether elevated auxin levels are required for the regulation of Mg deficiency-induced root hair morphogenesis by applying the auxin analog 1-naphthylacetic acid (NAA) and the auxin transport inhibitor naphthylphthalamic acid (NPA) (Mathesius et al. 1998). Both the length and density of root hairs of Mg-sufficient Col-0 plants were greatly increased by NAA treatment in all three measured root segments, resembling the phenotype of Mg-deficient plants. Conversely, the induction of root hair development in Col-0 plants by Mg deficiency was clearly restrained in the presence of NPA (Fig. 1C, E, F) or the auxin biosynthesis inhibitor l-kynurenine (Supplementary Fig. S1). These results indicated that auxin is required for the regulation of Mg deficiency-induced root hair development. Furthermore, we also examined the effects of auxin transport on root hair development under Mg deficiency by analyzing the auxin transport-defective mutants aux1-7 (Pickett et al. 1990), pin1-1 (Okada et al. 1991), pin2 (Luschnig et al. 1998), pin3-5 (Friml et al. 2002b), pin4-3 (Friml et al. 2002a) and pin7-2 (Friml et al. 2003). We found that the Mg deficiency-induced increase in root hair, including increases in both length and density, was strongly or completely inhibited in the auxin transport mutants aux1-7, pin1-1 and pin2 compared with the Col-0 plants (Fig. 2), in agreement with the above pharmacological study. However, Mg deficiency normally induced root hair development in three other auxin transport mutants pin3-5, pin4-3 and pin7-2; the phenotypes of these plants were similar to the phenotype observed in Col-0 plants (Supplementary Fig. S2). Fig. 2 View largeDownload slide Effects of Mg deficiency on the growth of root hairs in Arabidopsis Col-0, aux1-7, pin1-1 and pin2 plants. Images of root hairs (A) and the corresponding length (B) and density (C) on segments 0–1, 1–2 and 2–3 cm from the root tip. All seedlings were pre-cultured as described in Fig. 1 for 4 weeks and were subsequently transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 7 d. Values are means ± SD (n = 6). Different letters represent significant differences between treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Different lower case letters above the bars indicate significant differences between genotype and treatment interactions (two-way ANOVA, P < 0.05, Duncan’s test). Scale bar = 1 mm. Fig. 2 View largeDownload slide Effects of Mg deficiency on the growth of root hairs in Arabidopsis Col-0, aux1-7, pin1-1 and pin2 plants. Images of root hairs (A) and the corresponding length (B) and density (C) on segments 0–1, 1–2 and 2–3 cm from the root tip. All seedlings were pre-cultured as described in Fig. 1 for 4 weeks and were subsequently transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 7 d. Values are means ± SD (n = 6). Different letters represent significant differences between treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Different lower case letters above the bars indicate significant differences between genotype and treatment interactions (two-way ANOVA, P < 0.05, Duncan’s test). Scale bar = 1 mm. The Mg deficiency-induced elevation of auxin is controlled by both ethylene and NO As previously hypothesized, auxin may function together with ethylene and NO in the regulation of Mg deficiency-induced root hair growth. Therefore, it is necessary to clarify whether the Mg deficiency-induced elevation of auxin in roots is associated with ethylene and NO. Application of either the ethylene precursor ACC (Songstad et al. 1988) or the exogenous NO donor sodium nitroprusside (SNP) (Garthwaite et al. 1988) to Mg-sufficient Col-0 plants mimicked the effects of Mg deficiency on auxin accumulation in roots, whereas treatment with either the ethylene inhibitor silver thiosulfate (STS) (Veen and Van de Geijn 1978) or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; Akaike et al. 1993) completely abolished the elevation of auxin in roots in response to Mg deficiency (Fig. 3A). Additionally, the DR5::GFP expression increased upon treatment with ACC or SNP under Mg sufficiency, whereas it decreased upon treatment with STS or c-PTIO under Mg deficiency (Fig. 3B). Furthermore, we also found that the application of l-kynurenine slightly reversed the effects of either the ACC or SNP under normal Mg levels in Col-0 plants, but not in the pin1-1 mutant (Supplementary Fig. S3). These results indicate that both ethylene and NO are required to regulate the elevation of auxin in roots of Mg-deficient plants. In the excised roots, however, the auxin level was barely affected by ACC and SNP under Mg sufficiency, and was not significantly affected by STS and c-PTIO under Mg deficiency (Fig. 3C). The result indicated that the accumulation of auxin in roots induced by ethylene and NO was probably caused by auxin transport instead of local biosynthesis under Mg deficiency. Fig. 3 View largeDownload slide Effects of ethylene and nitric oxide on auxin accumulation in the roots of Arabidopsis. The auxin levels in roots of Col-0 plants (A), the excised roots of Col-0 plants (C) and the root fluorescence images of DR5::GFP plants (B). For (A) and (B), the seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to +Mg or –Mg solutions containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. For (C), the plants were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either +Mg or –Mg nutrient solution for 5 d. The roots were cut at the junction of the hypocotyl, and these excised roots were incubated in +Mg or –Mg solutions containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 3 View largeDownload slide Effects of ethylene and nitric oxide on auxin accumulation in the roots of Arabidopsis. The auxin levels in roots of Col-0 plants (A), the excised roots of Col-0 plants (C) and the root fluorescence images of DR5::GFP plants (B). For (A) and (B), the seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to +Mg or –Mg solutions containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. For (C), the plants were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either +Mg or –Mg nutrient solution for 5 d. The roots were cut at the junction of the hypocotyl, and these excised roots were incubated in +Mg or –Mg solutions containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments (one-way ANOVA, P < 0.05, Duncan’s test). We then examined the expression of auxin transporter genes by quantitative real-time PCR analysis (Fig. 4). The root expression of AUX1, PIN1 and PIN2 in Mg-sufficient Col-0 plants was increased by either SNP or ACC to levels similar to, or higher than, in Mg-deficient roots, whereas the induction of their expression by Mg deficiency was reversed by both STS and c-PTIO. However, Mg deficiency had little effect on the expression of PIN3, PIN4 and PIN7; the expression of these three genes in the roots of both Mg-sufficient and Mg-deficient plants was barely affected by SNP, ACC or STS, although the root expression of PIN3 and PIN7 in Mg-deficient plants was inhibited by c-PTIO. Therefore, it appears that AUX1, PIN1 and PIN2, but not PIN3, PIN4 and PIN7, are associated with ethylene and NO in response to Mg deficiency. This speculation was confirmed by analyzing the GFP-, yellow fluorescent protein (YFP)- or β-glucuronidase (GUS)-tagged auxin transporters in transgenic plants. As shown in Fig. 5 and Supplementary Fig. S4, YFP fluorescence, GUS staining and GFP fluorescence in the roots of AUX1::AUX1-YFP, PIN1::PIN1-GUS and PIN2::PIN2-GFP (Friml et al. 2003) transgenic plants were increased by ACC and SNP under Mg sufficiency, whereas the opposite was true for the treatments with either STS or c-PTIO under Mg deficiency. As expected, root GUS staining in PIN3::PIN3-GUS, PIN4::PIN4-GUS and PIN7::PIN7-GUS (Friml et al. 2003) transgenic lines was not affected by either ACC, SNP, STS or c-PTIO (Supplementary Fig. S5). Fig. 4 View largeDownload slide Effects of ethylene and nitric oxide on the expression of AUX1, PIN1, PIN2, PIN3, PIN4 and PIN7 in the roots of Arabidopsis Col-0. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 4 View largeDownload slide Effects of ethylene and nitric oxide on the expression of AUX1, PIN1, PIN2, PIN3, PIN4 and PIN7 in the roots of Arabidopsis Col-0. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 5 View largeDownload slide Effects of ethylene and nitric oxide on auxin transport components. The YFP fluorescence images of AUX1::AUX1-YFP roots (A), GFP fluorescence images of PIN2::PIN2-GFP roots (B) and GUS staining of PIN1::PIN1-GUS roots (C). All Arabidopsis seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Fig. 5 View largeDownload slide Effects of ethylene and nitric oxide on auxin transport components. The YFP fluorescence images of AUX1::AUX1-YFP roots (A), GFP fluorescence images of PIN2::PIN2-GFP roots (B) and GUS staining of PIN1::PIN1-GUS roots (C). All Arabidopsis seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Auxin facilitates ethylene and NO production in roots under Mg-deficient conditions As both ethylene and NO facilitated auxin accumulation in roots, we sought to determine whether there is a positive feedback loop from auxin to ethylene and NO production under Mg deficiency. We first investigated the effect of auxin on ethylene response and production. In the roots of the EBS::GUS transgenic line, which contains the GUS reporter gene driven by a synthetic EIN3-responsive promoter (Stepanova and Ecker 2000), GUS staining was enhanced by NAA under Mg sufficiency, but was decreased by NPA under Mg deficiency (Supplementary Fig. S6). Consistently, the ethylene production of Mg-sufficient roots increased significantly by NAA treatment, while the Mg deficiency-induced elevation in ethylene production was clearly reversed by NPA treatment (Fig. 6A). These results indicate that the elevated auxin level in Mg-deficient roots facilitates ethylene production. We also analyzed the activities of ACS and ACO, both of which are critical for ethylene biosynthesis in plants (Kende 1993). The activities of ACO and ACS in roots were increased by NAA by about 80% and 250%, respectively, under Mg sufficiency, while the induction of these enzymes under Mg deficiency was substantially inhibited by NPA (Fig. 6C, D). These results provide further evidence that auxin promotes ethylene production under Mg deficiency. Fig. 6 View largeDownload slide Effects of auxin on ethylene and nitric oxygen (NO) production in the roots of Arabidopsis Col-0 plants. Ethylene production (A) and the images of NO-dependent fluorescence (B) of roots. The activities of 1-aminocyclopropane-1-carboxylate synthase (ACS) (C) and oxidase (ACO) (D), nitrate reductase (NR) (E) and nitric oxide synthase-like enzyme (NOS-L) (F) in the roots of Col-0 plants. The Col-0 plants were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 0.1 μM NAA or 5 μM NPA for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 6 View largeDownload slide Effects of auxin on ethylene and nitric oxygen (NO) production in the roots of Arabidopsis Col-0 plants. Ethylene production (A) and the images of NO-dependent fluorescence (B) of roots. The activities of 1-aminocyclopropane-1-carboxylate synthase (ACS) (C) and oxidase (ACO) (D), nitrate reductase (NR) (E) and nitric oxide synthase-like enzyme (NOS-L) (F) in the roots of Col-0 plants. The Col-0 plants were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 0.1 μM NAA or 5 μM NPA for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). We then examined the effect of auxin on NO generation under Mg deficiency. The level of NO was analyzed using the fluorescent NO indicator dye DAF-FM DA (4,5-diaminofluorescein diacetate). As shown in Fig. 6, the Mg deficiency induced stronger NO-dependent fluorescence in the roots compared with Mg sufficiency; this induction was inhibited in the presence of NPA. Conversely, application of NAA increased NO-dependent fluorescence in Mg-sufficient roots to a level comparable with that in Mg-deficient roots. Consistent with these results, the Mg deficiency-stimulated activities of NR and NOS-L, two enzymes that have been recognized as the major enzymes involved in NO generation in plants (Desikan et al. 2002, Bethke et al. 2004), could be inhibited by NPA (Fig. 6E, F). Furthermore, NAA treatment strongly stimulated the activities of NR and NOS-L under Mg sufficiency. Therefore, we proposed that an elevation in root auxin level also favors NO generation under Mg deficiency. The regulatory roles of ethylene and NO in Mg deficiency-induced root hair development depend on the action of auxin Further to our results that show a positive feedback loop between the accumulation of auxin and ethylene/NO, we examined whether there is a linkage between the roles of these signals in regulating the induction of root hair development by Mg deficiency. Recently, we showed that both the initiation and elongation of root hairs in ethylene-insensitive ein2-5 (Alonso et al. 1999) and ein3-1 mutants (Guo and Ecker 2003), and the NO synthesis-defective nia1,2 (Desikan et al. 2002) and noa1 mutants (Guo 2006) were abolished under Mg deficiency, compared with those in Col-0 plants (Liu et al. 2017). This was also true in the present study (Fig. 7). Nevertheless, the application of NAA to Mg-deficient medium stimulated both the length and the density of root hairs in the ein2-5, etr1-3 (Hall et al. 1999), nia1,2 and noa1 mutants to levels similar to those of Col-0 plants in all three measured root segments (Fig. 7). Consistent with this result, we also found that the inhibition of Mg deficiency-induced root hair development in Col-0 plants by either STS or c-PTIO was completely reversed by NAA (Supplementary Fig. S7). Furthermore, NAA also significantly stimulated the length and density of root hairs in either ein2-5 mutants fed with c-PTIO or nia1,2 and noa1 mutants fed with STS (Supplementary Fig. S8). Conversely, both the length and density of root hairs in all three measured root segments in aux1-7, pin1-1 and pin2 mutants were not affected or only slightly increased by either ACC or SNP under Mg deficiency; consequently, they were still much more reduced than those of Col-0 plants (Fig. 8). We also analyzed the root hair growth in the tir1-1 mutant, which shows an impaired auxin receptor (Dharmasiri et al. 2005). The root hair growth in the tir1-1 mutant was comparably resistant to auxin, ethylene and NO under a normal Mg supply (Supplementary Fig. S9). The above results suggested that the regulatory roles of ethylene and NO in Mg deficiency-induced root hair morphogenesis required the action of auxin, but not vice versa. Therefore, auxin is likely to act downstream of ethylene and NO in regulating Mg deficiency-induced root hair development. Fig. 7 View largeDownload slide Effects of auxin on root hair length (A) and density (B) on segments of 0–1, 1–2 and 2–3 cm from root tips in Arabidopsis Col-0 plants, ethylene-insensitive ein2-5 and etr1-3, and nitric oxide synthesis-defective nia1,2 and noa1 mutants. The seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solutions with or without 0.1 μM NAA for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Asterisks indicate significant differences between genotype and treatment interactions (two-way ANOVA, P < 0.05, Duncan’s test). Fig. 7 View largeDownload slide Effects of auxin on root hair length (A) and density (B) on segments of 0–1, 1–2 and 2–3 cm from root tips in Arabidopsis Col-0 plants, ethylene-insensitive ein2-5 and etr1-3, and nitric oxide synthesis-defective nia1,2 and noa1 mutants. The seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solutions with or without 0.1 μM NAA for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Asterisks indicate significant differences between genotype and treatment interactions (two-way ANOVA, P < 0.05, Duncan’s test). Fig. 8 View largeDownload slide Effects of ethylene and nitric oxide on root hair length and density on segments of 0–1, 1–2 and 2–3 cm from root tips in Arabidopsis Col-0 plants and auxin transport-defective aux1-7, pin1-1 and pin2 mutants. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP or 3 μM ACC for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 8 View largeDownload slide Effects of ethylene and nitric oxide on root hair length and density on segments of 0–1, 1–2 and 2–3 cm from root tips in Arabidopsis Col-0 plants and auxin transport-defective aux1-7, pin1-1 and pin2 mutants. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP or 3 μM ACC for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). It is clear that the CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC (ETC1) and RHO-RELATED PROTEIN FROM PLANTS 2 (ROP2) genes are required for root hair initiation, while the WEREWOLF (WER), TRANSPARENT TESTA GLABRA1 (TTG1), GLABRA2 (GL2) and GLABRA3 (GL3) genes inhibit the formation of root hairs (Schiefelbein 2000, Jones et al. 2002, Wachsman et al. 2015). Previously, we found that Mg deficiency induced expression of CPC, TRY and ROP2, and inhibited the expression of TTG, GL2 and GL3, but did not affect the expression of ETC1 or WER (Liu et al. 2017). Consequently, we also investigated whether auxin was linked with ethylene and NO to regulate the expression of CPC, TRY, ROP2, TTG1, GL2 and GL3 in Mg-deficient roots. Here, we observed that the expression of CPC, TRY and ROP2 was decreased, whereas that of TTG1, GL2 and GL3 was increased, in response to the treatment with NPA, STS or c-PTIO. Interestingly, the above effects of STS and c-PTIO could be substantially attenuated by NAA, whereas the effects of NPA were not affected or only partially attenuated by either ACC or SNP (Fig. 9). These results provide further evidence that auxin is required for ethylene- and NO-mediated regulation of Mg deficiency-induced root hair morphogenesis. Fig. 9 View largeDownload slide Interactions between the roles of auxin and ethylene/nitric oxide in regulating the expression of CPC, TRY, ROP2, TTG1, GL2 and GL3 in the roots of Arabidopsis Col-0 plants. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 0.1 μM NAA, 5 μM NPA, 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Relative expression levels were normalized to levels of UBQ10. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 9 View largeDownload slide Interactions between the roles of auxin and ethylene/nitric oxide in regulating the expression of CPC, TRY, ROP2, TTG1, GL2 and GL3 in the roots of Arabidopsis Col-0 plants. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 0.1 μM NAA, 5 μM NPA, 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Relative expression levels were normalized to levels of UBQ10. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Discussion The identification of key regulators of Mg deficiency-induced root hair formation is crucial for the improvement of crop growth in acidic or sandy soils, where Mg deficiency frequently limits crop growth. Previously, we demonstrated that ethylene and NO interactively regulated Mg deficiency-induced root hair growth in Arabidopsis (Liu et al. 2017). In the present study, we reveal that auxin acts downstream of ethylene and NO in the regulatory cascade leading to the induction of root hairs under Mg deficiency. Mg deficiency-induced root hair development is regulated by auxin The process of a chemical compound regulating a physiological process usually depends on an alteration of its endogenous level in plant tissue. Here, we found that the induction of root hair development by Mg deficiency was accompanied by an increase in auxin in the roots and that this increase was required for regulating the induction of root hair development under Mg deficiency (Fig. 1). As already mentioned previously, auxin is mainly synthesized in shoots and is transported basipetally to the roots (Habets and Offringa 2014). In addition, several studies demonstrated that auxin is locally synthesized in the root (Swarup et al. 2007, Stepanova et al. 2008, Tao et al. 2008, Yamada et al. 2009). Nevertheless, our results indicated that the local auxin synthesis may not contribute to an elevation of auxin level in roots owing to Mg deficiency (Fig. 1D). Thus, we emphasized the action of auxin transport. As stated previously, the process of auxin transport in plants is complex and highly regulated, involving many identified transporter proteins (Křeček et al. 2009, Péret et al. 2012). Here, we found that Mg deficiency induced the expression of AUX1, PIN1 and PIN2 genes and their encoded proteins in roots, but had little effect on the expression of PIN3, PIN4 and PIN7 genes and their encoded proteins (Figs. 4, 5; Supplementary Fig. S5). This selective induction of auxin transporters by Mg deficiency is highly correlated with evidence that Mg deficiency-induced root hair growth is restrained in mutants lacking AUX1, PIN1 or PIN2, but not in mutants lacking PIN3, PIN4 or PIN7 (Fig. 2; Supplementary Fig. S2). Therefore, we propose that Mg deficiency-induced root hair morphogenesis is probably controlled by a relatively specific auxin transport pathway. Auxin, ethylene and NO mutually facilitate each other’s accumulation under Mg deficiency Previously, we have shown that ethylene and NO were also involved in the regulation of Mg deficiency-induced root hair development (Liu et al. 2017). It was not known, however, whether ethylene or NO was associated with auxin accumulation in roots in response to Mg deficiency. Pharmacological evidence from Mg-deficient plants treated with STS or c-PTIO, and Mg-sufficient plant treated with ACC or SNP showed that the elevation of ethylene and NO levels is associated with an increase in the root auxin level under Mg deficiency (Fig. 3). This may be caused by enhanced auxin transport in plants. Ethylene has been shown to regulate auxin transporters positively at both the transcriptional and post-transcriptional levels (Muday et al. 2012, Li et al. 2015). However, little information is available regarding the effects of NO on auxin transport in plants. Of six known auxin transporters, i.e. AUX1, PIN1, PIN2, PIN3, PIN4 and PIN7 (Grieneisen et al. 2007), we found that only the first three transporters induced by Mg deficiency required the actions of both ethylene and NO (Figs. 4, 5; Supplementary Fig. S5). This result was highly correlated with the finding that AUX1, PIN1 and PIN2, but not PIN3, PIN4 and PIN7, are involved in auxin-mediated Mg deficiency-induced root hair development (Fig. 2; Supplementary Fig. S2). Further, neither ethylene nor NO induced auxin accumulation in the aux1-7 and pin2 mutants (Supplementary Fig. S10). Therefore, we suggest that the regulatory effects of ethylene and NO on the elevation of auxin accumulation in roots in response to Mg deficiency could be associated with enhanced expression of the AUX1, PIN1 and PIN2 transporters. Interestingly, the Mg deficiency-induced increases in both ethylene and NO levels required the action of auxin in roots (Fig. 6), indicating that there may be a positive feedback loop from auxin to ethylene and NO production under Mg deficiency. Several previous studies had shown that auxin stimulated the enzymes involved in ethylene or NO synthesis. For instance, auxin induced de novo synthesis of the ACS enzyme, thus stimulating its activity (Muday et al. 2012); auxin also elevated the activity of NR that produces NO via nitrite reduction (Kolbert et al. 2008). In the present study, we found that the activities of ACS, ACO, NR and NOS-L in roots, four enzymes that are involved in the synthesis of ethylene or NO, were induced by Mg deficiency and that these inductions were all highly associated with the action of elevated auxin (Fig. 6). This result supports a role for auxin in the production of ethylene and NO under Mg deficiency. Previously, we have shown that ethylene and NO stimulated each other’s production under Mg deficiency (Liu et al. 2017). This mechanism, together with the above findings, suggests that auxin, ethylene and NO mutually favor each other’s accumulation in roots in response to Mg deficiency, thus forming an NO–ethylene–auxin positive feedback loop. Auxin acts downstream of ethylene and NO in regulating Mg deficiency-induced root hair development As explained in this and in a previous study (Liu et al. 2017), the three signals, i.e. auxin, ethylene and NO, regulate Mg deficiency-induced root hair development, with the latter two signals acting in parallel. It is therefore of interest to clarify how auxin co-ordinates with ethylene and NO in the above regulation. Our genetic and pharmacological evidence showed that both ethylene and NO required the action of auxin to regulate Mg deficiency-induced root hair morphogenesis, but that ethylene and NO were not required for the action of auxin in this process (Figs. 7, 8); this suggests that auxin acts downstream of ethylene and NO in this process. Consistent with this conclusion, we observed that auxin also acts downstream of ethylene and NO in regulating Mg deficiency-induced expression changes in CPC, TRY, ROP2, TTG1, GL2 and GL3 (Fig. 9), the genes involved in controlling root hair formation. The co-ordinate action of auxin and ethylene/NO has also been found to regulate other physiological processes in plants. In many cases, auxin also acts downstream of ethylene in the regulatory cascades controlling these physiological events, such as in the inhibition of root cell elongation (Rahman et al. 2001, Růzicka et al. 2007, He et al. 2011), apical hook formation (Mazzella et al. 2014) and lateral root development in Arabidopsis (Negi et al. 2010, Lewis et al. 2011). Notably, however, NO has previously been reported to function downstream of auxin in various physiological processes in plants, such as the induction of root ferric-chelate reductase under Fe deficiency (Chen et al. 2010, Jin et al. 2011), the development of lateral roots (Jin et al. 2011, Correa-Aragunde et al. 2015) and the activation of cell division and embryogenic cell formation (Ötvös et al. 2005). Therefore, it seems that the signaling cascade of NO upstream of auxin in regulating Mg deficiency-induced root hair development might be relatively specific to this process. In summary, based on this study and our previous work (Liu et al. 2017), we propose the following model for the mechanism regulating Mg deficiency-induced root hair development (Fig. 10). In this model, Mg deficiency elevates the levels of auxin, ethylene and NO in roots, with each positively influencing the accumulation of the other two; auxin acts downstream of ethylene and NO in the regulatory cascade controlling Mg deficiency-induced root hair morphogenesis (Fig. 10). Fig. 10 View largeDownload slide Model of auxin, ethylene and NO in regulating Mg deficiency-induced root hair development. The Mg deficiency elevates the levels of auxin, ethylene and NO in roots; each facilitates the accumulation of the other two by stimulating the activities of synthesis-related enzymes (ACO and ACS for ethylene; NR and NOS-L for NO) or the expression of transporters (AUX1, PIN1 and PIN2 for auxin), thus forming an ethylene–NO–auxin positive feedback loop. Auxin acts downstream of ethylene and NO in the regulatory cascade leading to the induction of root hairs under Mg deficiency. Fig. 10 View largeDownload slide Model of auxin, ethylene and NO in regulating Mg deficiency-induced root hair development. The Mg deficiency elevates the levels of auxin, ethylene and NO in roots; each facilitates the accumulation of the other two by stimulating the activities of synthesis-related enzymes (ACO and ACS for ethylene; NR and NOS-L for NO) or the expression of transporters (AUX1, PIN1 and PIN2 for auxin), thus forming an ethylene–NO–auxin positive feedback loop. Auxin acts downstream of ethylene and NO in the regulatory cascade leading to the induction of root hairs under Mg deficiency. Materials and Methods Plant materials and growth conditions All Arabidopsis mutants used in this study were of the Col-0 background. The pin3-5, pin4-3, pin7-2, pin2 and etr1-3 mutants and the transgenic lines EBS::GUS, PIN3::PIN3-GUS, PIN4::PIN4-GUS and PIN7::PIN7-GUS were obtained from the Arabidopsis Biological Resource Center. The nia1,2 and noa1 seeds were purchased from the Nottingham Arabidopsis Stock Centre. The ein2-5 mutant was provided by H.W. Guo (Tsinghua University, China). The transgenic lines AUX1::AUX1-YFP, PIN1::PIN1-GUS and PIN2::PIN2-GFP were provided by Y.F. Niu (Zhejiang University, China). All seeds were surface-sterilized and germinated in half-strength nutrient solution. After 7 d, uniform seedlings were transferred to full-strength nutrient solution of the following composition (μM): 1,500 KNO3, 500 MgSO4, 1,000 CaCl2, 500 NaH2PO4, 250 (NH4)2SO4, 10 H3BO3, 0.5 MnSO4, 0.5 ZnSO4, 0.1 CuSO4, 0.1 (NH4)6Mo7O24 and 25 Fe-EDTA. The nutrient solution was renewed every 3 d and the pH was adjusted to 6.0 using 1 M NaOH. All the plants were grown in a controlled environment with 70% humidity and a daily cycle of 25°C/14 h day and 22°C/10 h night. After the seedlings were grown in nutrient solution for another 14 d, they were transferred into 0.4 liter pots containing full-strength nutrient solution for 7 d. Thereafter, the plants were transferred to nutrient solutions that either contained (+Mg) or did not contain MgSO4 (–Mg) for 5 d. These plants were used in the experiments and treated as follows for another 2 d in the earlier mentioned +Mg or –Mg solutions. For the chemical reagent treatment, 0.1 μM NAA (Catalog number 86-87-3, Sangon Biotech), 5 μM NPA (Catalog number 132-66-1, Sigma-Aldrich), 10 μM SNP (Catalog number 13755-38-9, Sangon Biotech), 100 μM c-PTIO (Catalog number 148819-94-7, Santa Cruz), 3 μM ACC (Catalog number 22059-21-8, Sigma-Aldrich) and 1 μM l-kynurenine (Catalog number 2922-83-0, Santa Cruz) or 5 μM STS (Catalog number 7772-98-7, Sigma-Aldrich) was added to the nutrient solution. The NAA stock solution was prepared by dissolving NAA in ethanol to a final concentration of 1 mM, and the NPA stock solution was prepared by dissolving NPA in dimethyl sulfoxide (DMSO; Catalog number 67-68-5, Sangon Biotech). The ACC stock solution was prepared by dissolving ACC in ultrapure water to a concentration of 1 mM, and the STS stock solution was prepared by dissolving STS in ultrapure water to a concentration of 5 mM. The SNP stock solution was prepared by dissolving SNP in ultrapure water to a final concentration of 10 mM SNP, and the c-PTIO stock solution was prepared by dissolving c-PTIO in ultrapure water to a final concentration of 10 mM. The l-kynurenine stock solution was prepared by dissolving l-kynurenine in DMSO to a final concentration of 50 mM. Microscopy and fluorescence measurement Seedlings were harvested and the individual roots were divided into three segments, 0–1, 1–2 and 2–3 cm from the root tips. The length and density of root hairs in each segment were measured with microscopy and recorded with a charge-coupled device camera (Eclipse E600; Nikon). In situ measurement of GFP was conducted with an epifluorescence microscope (Nikon Eclipse E600, Nikon, excitation at 488 nm and emission at 495–575 nm). GUS staining GUS staining was conducted according to Mao et al. (2014). Briefly, the root was rinsed three times with GUS staining buffer {50 mM potassium phosphate buffer (pH 7.0), 0.1 mM K3[Fe(CN6)], 0.5 mM K4[Fe(CN6)], 0.1 mM EDTA and 0.01% Triton X-100}, and then incubated overnight with GUS buffer supplemented with 10 mg ml–1 5-bromo-4-chloro-3-indolyl β-d-glucuronic acid at 37°C in the dark. GUS expression was observed under a microscope (Nikon Eclipse E600; Nikon) with an attached camera. Measurement of IAA concentration in roots The measurement of the IAA level in the roots was performed according to the method described by Jin et al. (2011) and Swarup et al. (2007). Briefly, 0.2 g of roots was finely ground in liquid nitrogen and homogenized in 1.5 ml of pre-chilled 80% methanol containing 1 mM 2,6-di-tert-butyl-p-methylphenol in weak light conditions, followed by centrifugation at 5,000 r.p.m. for 10 min at 4°C. The supernatant was purified with C18 columns. Next, 500 μl of the extract was dried with N2 gas, dissolved in 200 μl methanol and dried in a vacuum freeze-dryer (Christ ALPHA 1-4). The extract was then dissolved in 300 μl of phosphate buffer solution (pH 7.4) for enzyme-linked immunosorbent assay (ELISA). The IAA concentration was measured at 490 nm using a microplate reader. Determination of ethylene production in roots The ethylene production in roots was measured according to the method of Tian et al. (2009). Briefly, 0.2 g of roots was cut and immediately placed in 6 ml vials with 1 ml of agar medium (0.7% agar) for 1 h to minimize wounding effects, and then the vials were sealed for 8 h. Thereafter, 1 ml of the headspace gas was collected, and subsequently injected into a gas chromatograph with an alumina column (GDX502) and a flame ionization detector (GC-7AG; Shimadzu). Determination of ACO and ACS activities ACO and ACS activities were determined according to the methods of Tian et al. (2009). For ACO determination, 0.2 g of roots was ground to a fine powder in liquid nitrogen and added to 1.5 ml of extract mixture [100 M Tris–HCl buffer (pH 7.2), 30 mM sodium ascorbate, 10% (v/v) glycerol and 5% polyvinylpolypyrrolidone], followed by centrifugation at 15,000×g for 20 min at 4°C. A total of 800 μl of supernatant was added to gas-tight vials containing 2 ml of extraction buffer (without polyvinylpolypyrrolidone, 50 μM FeSO4 and 2 mM ACC), and incubated for 1 h at 30°C. The ethylene was measured as described earlier. To examine ACO activity, 0.2 g of roots was ground in liquid nitrogen and added to 1 ml of extract mixture [200 mM phosphate buffer (pH 8.0), 2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM dithiothreitol (DTT), 10 μM pyridoxal phosphate and 1 mM EDTA], followed by centrifugation at 15,000×g for 20 min at 4°C. A total of 0.6 ml of supernatant was added to a 6 ml vial containing 0.2 ml of 5 mM S-(5′-adenosyl)-l-methionine (Catalog number 29908-03-0, Sangon Biotech) and left to react for 1 h at 22°C. The ACC formed was converted to ethylene by adding 0.2 ml of 1:1 (v/v) NaOH:bleach and 100 mM HgCl2. The vials were kept on ice for 20 min, and the ethylene content was determined as described above. Histochemical analysis of NO The NO level was determined using the fluorescence indicator DAF-FM DA (Catalog number S0019, Beyotime) according to the method described by Jin et al. (2009b) with some modifications. Briefly, the roots were loaded with 10 μM DAF-FM DA in 20 μM HEPES-NaOH buffer (pH 7.4), washed three times with fresh buffer and observed under a microscope (Nikon Eclipse E600, Nikon), with excitation and emission wavelengths of 488 and 515 nm, respectively. The fluorescence intensity of root tips in the images was quantified using Image J. Measurement of NR and NOS-L activities The activity of NR was determined according to the method of Jin et al. (2011). Briefly, 0.2 g of roots was ground in liquid nitrogen and added to 1.5 ml of extract mixture [100 mM HEPES-KOH (pH 7.5), 10% (v/v) glycerol, 1 mM EDTA, 0.1% Triton X-100, 5 mM DTT, 0.5 mM PMSF, 20 μM FAD, 25 μM leupeptin, 5 μM Na2MoO4 and 1% polyvinylpyrrolidone), followed by centrifugation at 13,000×g for 20 min at 4°C. A 0.2 ml aliquot of supernatant was added to 0.4 ml of assay mixture with 100 μM HEPES-KOH (pH 7.5), 0.25 mM NADH and 5 mM KNO3, and left to react for 1 h at 30°C. The reaction was stopped by the addition of 100 μl of 0.1 M zinc acetate. NR activity was determined colorimetrically at 540 nm by the addition of 1 ml of 1% sulfanilamide and 1 ml of 0.02% N-(1-naphthyl)-ethylenediamine. The activity of NOS-L was measured as described by Tian et al. (2007) with some modifications. Briefly, 0.2 ml of supernatant was prepared as described above and added to 0.1 ml of assay mixture with NADH, l-arginine, DAF-FM DA and NOS-L assay buffer. The reaction was conducted in the dark at 37°C for 1 h. NO content was detected under a microscope as described earlier. Quantitative PCR analyses Total RNA in roots was extracted using RNAiso Plus (Catalog number 9109, TAKARA). All RNA samples were checked for DNA contamination before cDNA synthesis. Then, quantitative real-time PCR analyses were performed with pairs of gene primers (Supplementary Table S1) and a PrimeScript™ RT reagent kit (Catalog number RR037A, TAKARA). The housekeeping gene UBQ10 was used as a reference. Relative expression was calculated using an efficiency-corrected ΔΔCt formula as described previously (Jin et al. 2009a, Fang et al. 2016). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Key R&D Program of China [2016YFD0200103]; the National Key Project on Science and Technology of China [2015BAC02B03]; the Agricultural Technology Extension Funds of Zhejiang University [2016NTZX002]; the Natural Science Foundation of China [31622051]; and the Fundamental Research Funds for the Central Universities [2017XZZX002–06]. Acknowledgments We sincerely thank Ms. Y.F. Niu (Zhejiang University, China) for the gifts of aux1-7, PIN1::PIN1-GUS, AUX::AUX-YFP, PIN2::PIN2-GFP seeds, Professor H.W. Guo (Tsinghua University, China) for the seeds of ein2-5 and ein3-1 mutants, Professor H.W. Xue (Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China), and X. Huang (Hainan University, China) for the tir1-1 mutant seeds. Disclosures The authors have no conflicts of interest to declare. References Akaike T., Yoshida M., Miyamoto Y., Sato K., Kohno M., Sasamoto K. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations ACC 1-aminocyclopropane-1-carboxylate ACO ACC oxidase ACS ACC synthase AUX1 AUXIN-RESISTANT1 c-PTIO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide CPC CAPRICE DAF-FM DA 4,5-diaminofluorescein diacetate EBS ETHYLENE BIS STEARAMIDE ETC1 ENHANCER OF TRY AND CPC GFP green fluorescent protein GL2 GLABRA2 GL3 GLABRA3 GUS β-glucuronidase Mg magnesium NAA 1-naphthylacetic acid NO nitric oxide NOS-L NO synthase-like NPA naphthylphthalamic acid NR nitrate reductase PIN1, 2, 3, 4, 7 PIN-FORMED1, 2, 3, 4, 7 ROP2 RHO-RELATED PROTEIN FROM PLANTS 2 SNP sodium nitroprusside STS silver thiosulfate TTG1 TRANSPARENT TESTA GLABRA1 TRY TRIPTYCHON UBQ10 UBIQUITIN10 WER WEREWOLF YFP yellow fluorescent protein © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Auxin Acts Downstream of Ethylene and Nitric Oxide to Regulate Magnesium Deficiency-Induced Root Hair Development in Arabidopsis thaliana

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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy078
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Abstract

Abstract This study examines the association of auxin with ethylene and nitric oxide (NO) in regulating the magnesium (Mg) deficiency-induced root hair development in Arabidopsis thaliana. With Mg deficiency, both ethylene and NO promoted the elevation of root auxin levels in roots by inducing the expression of AUXIN-RESISTANT1 (AUX1), PIN-FORMED 1 (PIN1) and PIN2 transporters. In turn, auxin stimulated ethylene and NO production by activating the activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO), ACC synthase (ACS), nitrate reductase (NR) and NO synthase-like (NOS-L). These processes constituted an NO/ethylene–auxin feedback loop. Interestingly, however, the roles of ethylene and NO in regulating Mg deficiency-induced root hair development required the action of auxin, but not vice versa. In summary, these results suggest that Mg deficiency induces a positive interaction between the accumulation of auxin and ethylene/NO in roots, with auxin acting downstream of ethylene and NO signals to regulate Mg deficiency-induced root hair morphogenesis. Introduction Magnesium (Mg) is critical for plant growth and development because of its essential roles in various physiological processes such as photosynthesis, carbon fixation, protein synthesis, Chl synthesis and enzyme activation (Lilley et al. 1974, Jezek et al. 2015). However, plants often suffer from Mg deficiency in acidic or sandy soils, because the soluble Mg easily leaches from these soils and its absorption by roots is intensively antagonized by the absorption of other cations such as ammonium, aluminum and potassium (Yang et al. 2007, Marschner and Rengel 2012). Mg deficiency reduces both the yield and nutritional quality of crops (Gerendás and Führs 2013); a greater understanding of the mechanisms by which plants mitigate the effects of Mg deficiency is, therefore, important to improve plant adaptation. In previous studies, we found that Mg deficiency significantly induced the initiation and elongation of root hairs (Niu et al. 2014, Liu et al. 2017). The stimulation of root hair growth increases the surface area of roots, allowing them to access greater volumes of soil; this facilitates the uptake of nutrients, especially in nutrient-limited soils (Genc et al. 2007, Nestler and Wissuwa 2016). This root hair stimulation probably allows the plant roots to acquire more Mg from Mg-limited soils. Elucidation of the mechanism regulating Mg deficiency-induced root hair development is, therefore, essential. In our most recent study, we showed that Mg deficiency stress elevated the levels of both ethylene and nitric oxide (NO) in the roots; interestingly, the ethylene activated the production of NO by enhancing the activities of nitrate reductase (NR) and nitric oxide synthase-like (NOS-L), while the NO in turn promoted the synthesis of ethylene by stimulating the activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) and ACC synthase (ACS) (Liu et al. 2017). This interaction is then involved in the regulation of development of Mg deficiency-induced root hairs (Liu et al. 2017). Several studies have shown strong genetic evidence that auxin, in addition to ethylene and NO, plays a crucial role in controlling root hair development under nutrient-adequate growth conditions (Lee and Cho 2013, Rigas et al. 2013). The auxin is mainly synthesized in young leaves and developing leaf primordia (Soeno et al. 2010), and is transported basipetally to other tissues including the roots (Habets and Offringa 2014). In Arabidopsis, auxin transport was mediated by AUXIN-RESISTANT1 (AUX1)/LAX (like AUX1) influx carriers and the PIN-FORMED (PIN) efflux carrier family, including PIN1, PIN2, PIN3, PIN4, PIN7 and P-glycoprotein (PGP) (Křeček et al. 2009, Péret et al. 2012). It had been suggested that PGP could interact with PIN to stabilize PINs at the membrane, and the PINs then provide the directionality to auxin efflux (Titapiwatanakun et al. 2009, Santos et al. 2010). It has been suggested that LAX1 and LAX2 are involved in the aerial development of Arabidopsis (Bainbridge et al. 2008, Péret et al. 2012). Although LAX3 is also involved in the regulation of root growth, the three genes perform distinct functions during Arabidopsis development. For example, it had been proposed that except for AUX1, no other member of the AUX/LAX family plays a role in the root gravitropic response (Péret et al. 2012). Interestingly, the endogenous level of auxin in plants has repeatedly been found to be elevated in response to nutrient deficiency stresses. For instance, iron deficiency quickly elevated auxin levels in the roots of Arabidopsis, red clover and tomato (Chen et al. 2010, Jin et al. 2007, Jin et al. 2011); phosphorus deficiency induced auxin accumulation in the roots of Arabidopsis and white lupin (Gilbert et al. 2000, Miura et al. 2011); nitrate deficiency increased auxin levels in the roots of Arabidopsis and soybean (Ma et al. 2014); and potassium deficiency increased root auxin accumulation in Arabidopsis (Ma et al. 2012). The similar responses observed in these different plant species under various nutrient deficiency stresses suggest that auxin generation in plant roots is probably also induced by Mg deficiency. If this is correct, auxin is likely to regulate Mg deficiency-induced root hair development, and it may be associated with ethylene and NO in this regulatory event. In this study, we used Arabidopsis plants as a model system to examine the above hypotheses. Our results revealed that while auxin, ethylene and NO accumulate similarly under Mg deficiency, auxin acts downstream of the other two chemical signals in the regulatory cascade leading to the induction of root hair development in response to Mg deficiency. Results Elevation of auxin level in roots is required for the regulation of Mg deficiency-induced root hair development As auxin was presumed to be involved in the regulation of Mg deficiency-induced root hair development, we first measured its level in roots of wild-type Col-0 plants in response to Mg deficiency stress. As shown in Fig. 1A, the root auxin level in the Mg-deficient treatment was approximately 1-fold higher than in the Mg-sufficient treatment. The activity of the synthetic auxin response reporter DR5::GFP (green fluorescent protein) had been shown to correlate with direct auxin measurements in plant tissues (Friml et al. 2002b). Here, we found that the intensity of GFP fluorescence in roots was clearly increased under Mg deficiency stress, providing evidence that Mg deficiency increases auxin response in roots (Fig. 1B). Interestingly, the auxin level in the excised roots was not affected by Mg deficiency (Fig. 1D). The result indicates that the auxin accumulation in roots induced by Mg deficiency probably resulted from an enhanced basipetal transport. Fig. 1 View largeDownload slide Effects of auxin on the Mg deficiency-induced growth of root hairs in Arabidopsis Col-0 plants. Auxin levels in the roots of the intact Col-0 plants (A), in the excised roots (D) of Col-0 plants and the root GFP fluorescence in DR5::GFP transgenic plants (B). Images of root hairs (C) and the corresponding length (E) and density (F) of root hairs on segments 0–1, 1–2 and 2–3 cm from the root tip upon auxin treatment. For (A) and (B), the plants were pre-cultured in nutrient solution for 4 weeks, and subsequently transferred to either complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 7 d. Thereafter, the analyses were performed. For (C), (E) and (F), the 4-week-old seedlings were grown in either +Mg or –Mg nutrient solutions for 5 d, and then transferred to +Mg or –Mg solution containing 0.1 μM NAA or 5 μM NPA for another 2 d. Values are means ± SD (n = 6). For (D), the 4-week-old plants were grown in either +Mg or –Mg nutrient solutions for 5 d, and then the roots were cut at the junction of the hypocotyl and the root (excised roots). The excised roots were incubated in +Mg or –Mg solutions for another 2 d. Different lower case letters above the bars indicate significant differences between treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Scale bar = 1 mm. Fig. 1 View largeDownload slide Effects of auxin on the Mg deficiency-induced growth of root hairs in Arabidopsis Col-0 plants. Auxin levels in the roots of the intact Col-0 plants (A), in the excised roots (D) of Col-0 plants and the root GFP fluorescence in DR5::GFP transgenic plants (B). Images of root hairs (C) and the corresponding length (E) and density (F) of root hairs on segments 0–1, 1–2 and 2–3 cm from the root tip upon auxin treatment. For (A) and (B), the plants were pre-cultured in nutrient solution for 4 weeks, and subsequently transferred to either complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 7 d. Thereafter, the analyses were performed. For (C), (E) and (F), the 4-week-old seedlings were grown in either +Mg or –Mg nutrient solutions for 5 d, and then transferred to +Mg or –Mg solution containing 0.1 μM NAA or 5 μM NPA for another 2 d. Values are means ± SD (n = 6). For (D), the 4-week-old plants were grown in either +Mg or –Mg nutrient solutions for 5 d, and then the roots were cut at the junction of the hypocotyl and the root (excised roots). The excised roots were incubated in +Mg or –Mg solutions for another 2 d. Different lower case letters above the bars indicate significant differences between treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Scale bar = 1 mm. We then investigated whether elevated auxin levels are required for the regulation of Mg deficiency-induced root hair morphogenesis by applying the auxin analog 1-naphthylacetic acid (NAA) and the auxin transport inhibitor naphthylphthalamic acid (NPA) (Mathesius et al. 1998). Both the length and density of root hairs of Mg-sufficient Col-0 plants were greatly increased by NAA treatment in all three measured root segments, resembling the phenotype of Mg-deficient plants. Conversely, the induction of root hair development in Col-0 plants by Mg deficiency was clearly restrained in the presence of NPA (Fig. 1C, E, F) or the auxin biosynthesis inhibitor l-kynurenine (Supplementary Fig. S1). These results indicated that auxin is required for the regulation of Mg deficiency-induced root hair development. Furthermore, we also examined the effects of auxin transport on root hair development under Mg deficiency by analyzing the auxin transport-defective mutants aux1-7 (Pickett et al. 1990), pin1-1 (Okada et al. 1991), pin2 (Luschnig et al. 1998), pin3-5 (Friml et al. 2002b), pin4-3 (Friml et al. 2002a) and pin7-2 (Friml et al. 2003). We found that the Mg deficiency-induced increase in root hair, including increases in both length and density, was strongly or completely inhibited in the auxin transport mutants aux1-7, pin1-1 and pin2 compared with the Col-0 plants (Fig. 2), in agreement with the above pharmacological study. However, Mg deficiency normally induced root hair development in three other auxin transport mutants pin3-5, pin4-3 and pin7-2; the phenotypes of these plants were similar to the phenotype observed in Col-0 plants (Supplementary Fig. S2). Fig. 2 View largeDownload slide Effects of Mg deficiency on the growth of root hairs in Arabidopsis Col-0, aux1-7, pin1-1 and pin2 plants. Images of root hairs (A) and the corresponding length (B) and density (C) on segments 0–1, 1–2 and 2–3 cm from the root tip. All seedlings were pre-cultured as described in Fig. 1 for 4 weeks and were subsequently transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 7 d. Values are means ± SD (n = 6). Different letters represent significant differences between treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Different lower case letters above the bars indicate significant differences between genotype and treatment interactions (two-way ANOVA, P < 0.05, Duncan’s test). Scale bar = 1 mm. Fig. 2 View largeDownload slide Effects of Mg deficiency on the growth of root hairs in Arabidopsis Col-0, aux1-7, pin1-1 and pin2 plants. Images of root hairs (A) and the corresponding length (B) and density (C) on segments 0–1, 1–2 and 2–3 cm from the root tip. All seedlings were pre-cultured as described in Fig. 1 for 4 weeks and were subsequently transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 7 d. Values are means ± SD (n = 6). Different letters represent significant differences between treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Different lower case letters above the bars indicate significant differences between genotype and treatment interactions (two-way ANOVA, P < 0.05, Duncan’s test). Scale bar = 1 mm. The Mg deficiency-induced elevation of auxin is controlled by both ethylene and NO As previously hypothesized, auxin may function together with ethylene and NO in the regulation of Mg deficiency-induced root hair growth. Therefore, it is necessary to clarify whether the Mg deficiency-induced elevation of auxin in roots is associated with ethylene and NO. Application of either the ethylene precursor ACC (Songstad et al. 1988) or the exogenous NO donor sodium nitroprusside (SNP) (Garthwaite et al. 1988) to Mg-sufficient Col-0 plants mimicked the effects of Mg deficiency on auxin accumulation in roots, whereas treatment with either the ethylene inhibitor silver thiosulfate (STS) (Veen and Van de Geijn 1978) or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; Akaike et al. 1993) completely abolished the elevation of auxin in roots in response to Mg deficiency (Fig. 3A). Additionally, the DR5::GFP expression increased upon treatment with ACC or SNP under Mg sufficiency, whereas it decreased upon treatment with STS or c-PTIO under Mg deficiency (Fig. 3B). Furthermore, we also found that the application of l-kynurenine slightly reversed the effects of either the ACC or SNP under normal Mg levels in Col-0 plants, but not in the pin1-1 mutant (Supplementary Fig. S3). These results indicate that both ethylene and NO are required to regulate the elevation of auxin in roots of Mg-deficient plants. In the excised roots, however, the auxin level was barely affected by ACC and SNP under Mg sufficiency, and was not significantly affected by STS and c-PTIO under Mg deficiency (Fig. 3C). The result indicated that the accumulation of auxin in roots induced by ethylene and NO was probably caused by auxin transport instead of local biosynthesis under Mg deficiency. Fig. 3 View largeDownload slide Effects of ethylene and nitric oxide on auxin accumulation in the roots of Arabidopsis. The auxin levels in roots of Col-0 plants (A), the excised roots of Col-0 plants (C) and the root fluorescence images of DR5::GFP plants (B). For (A) and (B), the seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to +Mg or –Mg solutions containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. For (C), the plants were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either +Mg or –Mg nutrient solution for 5 d. The roots were cut at the junction of the hypocotyl, and these excised roots were incubated in +Mg or –Mg solutions containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 3 View largeDownload slide Effects of ethylene and nitric oxide on auxin accumulation in the roots of Arabidopsis. The auxin levels in roots of Col-0 plants (A), the excised roots of Col-0 plants (C) and the root fluorescence images of DR5::GFP plants (B). For (A) and (B), the seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to +Mg or –Mg solutions containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. For (C), the plants were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either +Mg or –Mg nutrient solution for 5 d. The roots were cut at the junction of the hypocotyl, and these excised roots were incubated in +Mg or –Mg solutions containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments (one-way ANOVA, P < 0.05, Duncan’s test). We then examined the expression of auxin transporter genes by quantitative real-time PCR analysis (Fig. 4). The root expression of AUX1, PIN1 and PIN2 in Mg-sufficient Col-0 plants was increased by either SNP or ACC to levels similar to, or higher than, in Mg-deficient roots, whereas the induction of their expression by Mg deficiency was reversed by both STS and c-PTIO. However, Mg deficiency had little effect on the expression of PIN3, PIN4 and PIN7; the expression of these three genes in the roots of both Mg-sufficient and Mg-deficient plants was barely affected by SNP, ACC or STS, although the root expression of PIN3 and PIN7 in Mg-deficient plants was inhibited by c-PTIO. Therefore, it appears that AUX1, PIN1 and PIN2, but not PIN3, PIN4 and PIN7, are associated with ethylene and NO in response to Mg deficiency. This speculation was confirmed by analyzing the GFP-, yellow fluorescent protein (YFP)- or β-glucuronidase (GUS)-tagged auxin transporters in transgenic plants. As shown in Fig. 5 and Supplementary Fig. S4, YFP fluorescence, GUS staining and GFP fluorescence in the roots of AUX1::AUX1-YFP, PIN1::PIN1-GUS and PIN2::PIN2-GFP (Friml et al. 2003) transgenic plants were increased by ACC and SNP under Mg sufficiency, whereas the opposite was true for the treatments with either STS or c-PTIO under Mg deficiency. As expected, root GUS staining in PIN3::PIN3-GUS, PIN4::PIN4-GUS and PIN7::PIN7-GUS (Friml et al. 2003) transgenic lines was not affected by either ACC, SNP, STS or c-PTIO (Supplementary Fig. S5). Fig. 4 View largeDownload slide Effects of ethylene and nitric oxide on the expression of AUX1, PIN1, PIN2, PIN3, PIN4 and PIN7 in the roots of Arabidopsis Col-0. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 4 View largeDownload slide Effects of ethylene and nitric oxide on the expression of AUX1, PIN1, PIN2, PIN3, PIN4 and PIN7 in the roots of Arabidopsis Col-0. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 5 View largeDownload slide Effects of ethylene and nitric oxide on auxin transport components. The YFP fluorescence images of AUX1::AUX1-YFP roots (A), GFP fluorescence images of PIN2::PIN2-GFP roots (B) and GUS staining of PIN1::PIN1-GUS roots (C). All Arabidopsis seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Fig. 5 View largeDownload slide Effects of ethylene and nitric oxide on auxin transport components. The YFP fluorescence images of AUX1::AUX1-YFP roots (A), GFP fluorescence images of PIN2::PIN2-GFP roots (B) and GUS staining of PIN1::PIN1-GUS roots (C). All Arabidopsis seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Auxin facilitates ethylene and NO production in roots under Mg-deficient conditions As both ethylene and NO facilitated auxin accumulation in roots, we sought to determine whether there is a positive feedback loop from auxin to ethylene and NO production under Mg deficiency. We first investigated the effect of auxin on ethylene response and production. In the roots of the EBS::GUS transgenic line, which contains the GUS reporter gene driven by a synthetic EIN3-responsive promoter (Stepanova and Ecker 2000), GUS staining was enhanced by NAA under Mg sufficiency, but was decreased by NPA under Mg deficiency (Supplementary Fig. S6). Consistently, the ethylene production of Mg-sufficient roots increased significantly by NAA treatment, while the Mg deficiency-induced elevation in ethylene production was clearly reversed by NPA treatment (Fig. 6A). These results indicate that the elevated auxin level in Mg-deficient roots facilitates ethylene production. We also analyzed the activities of ACS and ACO, both of which are critical for ethylene biosynthesis in plants (Kende 1993). The activities of ACO and ACS in roots were increased by NAA by about 80% and 250%, respectively, under Mg sufficiency, while the induction of these enzymes under Mg deficiency was substantially inhibited by NPA (Fig. 6C, D). These results provide further evidence that auxin promotes ethylene production under Mg deficiency. Fig. 6 View largeDownload slide Effects of auxin on ethylene and nitric oxygen (NO) production in the roots of Arabidopsis Col-0 plants. Ethylene production (A) and the images of NO-dependent fluorescence (B) of roots. The activities of 1-aminocyclopropane-1-carboxylate synthase (ACS) (C) and oxidase (ACO) (D), nitrate reductase (NR) (E) and nitric oxide synthase-like enzyme (NOS-L) (F) in the roots of Col-0 plants. The Col-0 plants were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 0.1 μM NAA or 5 μM NPA for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 6 View largeDownload slide Effects of auxin on ethylene and nitric oxygen (NO) production in the roots of Arabidopsis Col-0 plants. Ethylene production (A) and the images of NO-dependent fluorescence (B) of roots. The activities of 1-aminocyclopropane-1-carboxylate synthase (ACS) (C) and oxidase (ACO) (D), nitrate reductase (NR) (E) and nitric oxide synthase-like enzyme (NOS-L) (F) in the roots of Col-0 plants. The Col-0 plants were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 0.1 μM NAA or 5 μM NPA for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). We then examined the effect of auxin on NO generation under Mg deficiency. The level of NO was analyzed using the fluorescent NO indicator dye DAF-FM DA (4,5-diaminofluorescein diacetate). As shown in Fig. 6, the Mg deficiency induced stronger NO-dependent fluorescence in the roots compared with Mg sufficiency; this induction was inhibited in the presence of NPA. Conversely, application of NAA increased NO-dependent fluorescence in Mg-sufficient roots to a level comparable with that in Mg-deficient roots. Consistent with these results, the Mg deficiency-stimulated activities of NR and NOS-L, two enzymes that have been recognized as the major enzymes involved in NO generation in plants (Desikan et al. 2002, Bethke et al. 2004), could be inhibited by NPA (Fig. 6E, F). Furthermore, NAA treatment strongly stimulated the activities of NR and NOS-L under Mg sufficiency. Therefore, we proposed that an elevation in root auxin level also favors NO generation under Mg deficiency. The regulatory roles of ethylene and NO in Mg deficiency-induced root hair development depend on the action of auxin Further to our results that show a positive feedback loop between the accumulation of auxin and ethylene/NO, we examined whether there is a linkage between the roles of these signals in regulating the induction of root hair development by Mg deficiency. Recently, we showed that both the initiation and elongation of root hairs in ethylene-insensitive ein2-5 (Alonso et al. 1999) and ein3-1 mutants (Guo and Ecker 2003), and the NO synthesis-defective nia1,2 (Desikan et al. 2002) and noa1 mutants (Guo 2006) were abolished under Mg deficiency, compared with those in Col-0 plants (Liu et al. 2017). This was also true in the present study (Fig. 7). Nevertheless, the application of NAA to Mg-deficient medium stimulated both the length and the density of root hairs in the ein2-5, etr1-3 (Hall et al. 1999), nia1,2 and noa1 mutants to levels similar to those of Col-0 plants in all three measured root segments (Fig. 7). Consistent with this result, we also found that the inhibition of Mg deficiency-induced root hair development in Col-0 plants by either STS or c-PTIO was completely reversed by NAA (Supplementary Fig. S7). Furthermore, NAA also significantly stimulated the length and density of root hairs in either ein2-5 mutants fed with c-PTIO or nia1,2 and noa1 mutants fed with STS (Supplementary Fig. S8). Conversely, both the length and density of root hairs in all three measured root segments in aux1-7, pin1-1 and pin2 mutants were not affected or only slightly increased by either ACC or SNP under Mg deficiency; consequently, they were still much more reduced than those of Col-0 plants (Fig. 8). We also analyzed the root hair growth in the tir1-1 mutant, which shows an impaired auxin receptor (Dharmasiri et al. 2005). The root hair growth in the tir1-1 mutant was comparably resistant to auxin, ethylene and NO under a normal Mg supply (Supplementary Fig. S9). The above results suggested that the regulatory roles of ethylene and NO in Mg deficiency-induced root hair morphogenesis required the action of auxin, but not vice versa. Therefore, auxin is likely to act downstream of ethylene and NO in regulating Mg deficiency-induced root hair development. Fig. 7 View largeDownload slide Effects of auxin on root hair length (A) and density (B) on segments of 0–1, 1–2 and 2–3 cm from root tips in Arabidopsis Col-0 plants, ethylene-insensitive ein2-5 and etr1-3, and nitric oxide synthesis-defective nia1,2 and noa1 mutants. The seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solutions with or without 0.1 μM NAA for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Asterisks indicate significant differences between genotype and treatment interactions (two-way ANOVA, P < 0.05, Duncan’s test). Fig. 7 View largeDownload slide Effects of auxin on root hair length (A) and density (B) on segments of 0–1, 1–2 and 2–3 cm from root tips in Arabidopsis Col-0 plants, ethylene-insensitive ein2-5 and etr1-3, and nitric oxide synthesis-defective nia1,2 and noa1 mutants. The seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solutions with or without 0.1 μM NAA for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Asterisks indicate significant differences between genotype and treatment interactions (two-way ANOVA, P < 0.05, Duncan’s test). Fig. 8 View largeDownload slide Effects of ethylene and nitric oxide on root hair length and density on segments of 0–1, 1–2 and 2–3 cm from root tips in Arabidopsis Col-0 plants and auxin transport-defective aux1-7, pin1-1 and pin2 mutants. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP or 3 μM ACC for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 8 View largeDownload slide Effects of ethylene and nitric oxide on root hair length and density on segments of 0–1, 1–2 and 2–3 cm from root tips in Arabidopsis Col-0 plants and auxin transport-defective aux1-7, pin1-1 and pin2 mutants. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 10 μM SNP or 3 μM ACC for another 2 d. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). It is clear that the CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC (ETC1) and RHO-RELATED PROTEIN FROM PLANTS 2 (ROP2) genes are required for root hair initiation, while the WEREWOLF (WER), TRANSPARENT TESTA GLABRA1 (TTG1), GLABRA2 (GL2) and GLABRA3 (GL3) genes inhibit the formation of root hairs (Schiefelbein 2000, Jones et al. 2002, Wachsman et al. 2015). Previously, we found that Mg deficiency induced expression of CPC, TRY and ROP2, and inhibited the expression of TTG, GL2 and GL3, but did not affect the expression of ETC1 or WER (Liu et al. 2017). Consequently, we also investigated whether auxin was linked with ethylene and NO to regulate the expression of CPC, TRY, ROP2, TTG1, GL2 and GL3 in Mg-deficient roots. Here, we observed that the expression of CPC, TRY and ROP2 was decreased, whereas that of TTG1, GL2 and GL3 was increased, in response to the treatment with NPA, STS or c-PTIO. Interestingly, the above effects of STS and c-PTIO could be substantially attenuated by NAA, whereas the effects of NPA were not affected or only partially attenuated by either ACC or SNP (Fig. 9). These results provide further evidence that auxin is required for ethylene- and NO-mediated regulation of Mg deficiency-induced root hair morphogenesis. Fig. 9 View largeDownload slide Interactions between the roles of auxin and ethylene/nitric oxide in regulating the expression of CPC, TRY, ROP2, TTG1, GL2 and GL3 in the roots of Arabidopsis Col-0 plants. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 0.1 μM NAA, 5 μM NPA, 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Relative expression levels were normalized to levels of UBQ10. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Fig. 9 View largeDownload slide Interactions between the roles of auxin and ethylene/nitric oxide in regulating the expression of CPC, TRY, ROP2, TTG1, GL2 and GL3 in the roots of Arabidopsis Col-0 plants. Seedlings were pre-cultured as described in Fig. 1 for 4 weeks, and were then transferred to either a complete (+Mg) or MgSO4-removed (–Mg) nutrient solution for 5 d, and subsequently to a +Mg or –Mg solution containing 0.1 μM NAA, 5 μM NPA, 10 μM SNP, 100 μM c-PTIO, 3 μM ACC or 5 μM STS for another 2 d. Relative expression levels were normalized to levels of UBQ10. Values are means ± SD (n = 6). Different lower case letters above the bars indicate significant differences among treatments in a genotype (one-way ANOVA, P < 0.05, Duncan’s test). Discussion The identification of key regulators of Mg deficiency-induced root hair formation is crucial for the improvement of crop growth in acidic or sandy soils, where Mg deficiency frequently limits crop growth. Previously, we demonstrated that ethylene and NO interactively regulated Mg deficiency-induced root hair growth in Arabidopsis (Liu et al. 2017). In the present study, we reveal that auxin acts downstream of ethylene and NO in the regulatory cascade leading to the induction of root hairs under Mg deficiency. Mg deficiency-induced root hair development is regulated by auxin The process of a chemical compound regulating a physiological process usually depends on an alteration of its endogenous level in plant tissue. Here, we found that the induction of root hair development by Mg deficiency was accompanied by an increase in auxin in the roots and that this increase was required for regulating the induction of root hair development under Mg deficiency (Fig. 1). As already mentioned previously, auxin is mainly synthesized in shoots and is transported basipetally to the roots (Habets and Offringa 2014). In addition, several studies demonstrated that auxin is locally synthesized in the root (Swarup et al. 2007, Stepanova et al. 2008, Tao et al. 2008, Yamada et al. 2009). Nevertheless, our results indicated that the local auxin synthesis may not contribute to an elevation of auxin level in roots owing to Mg deficiency (Fig. 1D). Thus, we emphasized the action of auxin transport. As stated previously, the process of auxin transport in plants is complex and highly regulated, involving many identified transporter proteins (Křeček et al. 2009, Péret et al. 2012). Here, we found that Mg deficiency induced the expression of AUX1, PIN1 and PIN2 genes and their encoded proteins in roots, but had little effect on the expression of PIN3, PIN4 and PIN7 genes and their encoded proteins (Figs. 4, 5; Supplementary Fig. S5). This selective induction of auxin transporters by Mg deficiency is highly correlated with evidence that Mg deficiency-induced root hair growth is restrained in mutants lacking AUX1, PIN1 or PIN2, but not in mutants lacking PIN3, PIN4 or PIN7 (Fig. 2; Supplementary Fig. S2). Therefore, we propose that Mg deficiency-induced root hair morphogenesis is probably controlled by a relatively specific auxin transport pathway. Auxin, ethylene and NO mutually facilitate each other’s accumulation under Mg deficiency Previously, we have shown that ethylene and NO were also involved in the regulation of Mg deficiency-induced root hair development (Liu et al. 2017). It was not known, however, whether ethylene or NO was associated with auxin accumulation in roots in response to Mg deficiency. Pharmacological evidence from Mg-deficient plants treated with STS or c-PTIO, and Mg-sufficient plant treated with ACC or SNP showed that the elevation of ethylene and NO levels is associated with an increase in the root auxin level under Mg deficiency (Fig. 3). This may be caused by enhanced auxin transport in plants. Ethylene has been shown to regulate auxin transporters positively at both the transcriptional and post-transcriptional levels (Muday et al. 2012, Li et al. 2015). However, little information is available regarding the effects of NO on auxin transport in plants. Of six known auxin transporters, i.e. AUX1, PIN1, PIN2, PIN3, PIN4 and PIN7 (Grieneisen et al. 2007), we found that only the first three transporters induced by Mg deficiency required the actions of both ethylene and NO (Figs. 4, 5; Supplementary Fig. S5). This result was highly correlated with the finding that AUX1, PIN1 and PIN2, but not PIN3, PIN4 and PIN7, are involved in auxin-mediated Mg deficiency-induced root hair development (Fig. 2; Supplementary Fig. S2). Further, neither ethylene nor NO induced auxin accumulation in the aux1-7 and pin2 mutants (Supplementary Fig. S10). Therefore, we suggest that the regulatory effects of ethylene and NO on the elevation of auxin accumulation in roots in response to Mg deficiency could be associated with enhanced expression of the AUX1, PIN1 and PIN2 transporters. Interestingly, the Mg deficiency-induced increases in both ethylene and NO levels required the action of auxin in roots (Fig. 6), indicating that there may be a positive feedback loop from auxin to ethylene and NO production under Mg deficiency. Several previous studies had shown that auxin stimulated the enzymes involved in ethylene or NO synthesis. For instance, auxin induced de novo synthesis of the ACS enzyme, thus stimulating its activity (Muday et al. 2012); auxin also elevated the activity of NR that produces NO via nitrite reduction (Kolbert et al. 2008). In the present study, we found that the activities of ACS, ACO, NR and NOS-L in roots, four enzymes that are involved in the synthesis of ethylene or NO, were induced by Mg deficiency and that these inductions were all highly associated with the action of elevated auxin (Fig. 6). This result supports a role for auxin in the production of ethylene and NO under Mg deficiency. Previously, we have shown that ethylene and NO stimulated each other’s production under Mg deficiency (Liu et al. 2017). This mechanism, together with the above findings, suggests that auxin, ethylene and NO mutually favor each other’s accumulation in roots in response to Mg deficiency, thus forming an NO–ethylene–auxin positive feedback loop. Auxin acts downstream of ethylene and NO in regulating Mg deficiency-induced root hair development As explained in this and in a previous study (Liu et al. 2017), the three signals, i.e. auxin, ethylene and NO, regulate Mg deficiency-induced root hair development, with the latter two signals acting in parallel. It is therefore of interest to clarify how auxin co-ordinates with ethylene and NO in the above regulation. Our genetic and pharmacological evidence showed that both ethylene and NO required the action of auxin to regulate Mg deficiency-induced root hair morphogenesis, but that ethylene and NO were not required for the action of auxin in this process (Figs. 7, 8); this suggests that auxin acts downstream of ethylene and NO in this process. Consistent with this conclusion, we observed that auxin also acts downstream of ethylene and NO in regulating Mg deficiency-induced expression changes in CPC, TRY, ROP2, TTG1, GL2 and GL3 (Fig. 9), the genes involved in controlling root hair formation. The co-ordinate action of auxin and ethylene/NO has also been found to regulate other physiological processes in plants. In many cases, auxin also acts downstream of ethylene in the regulatory cascades controlling these physiological events, such as in the inhibition of root cell elongation (Rahman et al. 2001, Růzicka et al. 2007, He et al. 2011), apical hook formation (Mazzella et al. 2014) and lateral root development in Arabidopsis (Negi et al. 2010, Lewis et al. 2011). Notably, however, NO has previously been reported to function downstream of auxin in various physiological processes in plants, such as the induction of root ferric-chelate reductase under Fe deficiency (Chen et al. 2010, Jin et al. 2011), the development of lateral roots (Jin et al. 2011, Correa-Aragunde et al. 2015) and the activation of cell division and embryogenic cell formation (Ötvös et al. 2005). Therefore, it seems that the signaling cascade of NO upstream of auxin in regulating Mg deficiency-induced root hair development might be relatively specific to this process. In summary, based on this study and our previous work (Liu et al. 2017), we propose the following model for the mechanism regulating Mg deficiency-induced root hair development (Fig. 10). In this model, Mg deficiency elevates the levels of auxin, ethylene and NO in roots, with each positively influencing the accumulation of the other two; auxin acts downstream of ethylene and NO in the regulatory cascade controlling Mg deficiency-induced root hair morphogenesis (Fig. 10). Fig. 10 View largeDownload slide Model of auxin, ethylene and NO in regulating Mg deficiency-induced root hair development. The Mg deficiency elevates the levels of auxin, ethylene and NO in roots; each facilitates the accumulation of the other two by stimulating the activities of synthesis-related enzymes (ACO and ACS for ethylene; NR and NOS-L for NO) or the expression of transporters (AUX1, PIN1 and PIN2 for auxin), thus forming an ethylene–NO–auxin positive feedback loop. Auxin acts downstream of ethylene and NO in the regulatory cascade leading to the induction of root hairs under Mg deficiency. Fig. 10 View largeDownload slide Model of auxin, ethylene and NO in regulating Mg deficiency-induced root hair development. The Mg deficiency elevates the levels of auxin, ethylene and NO in roots; each facilitates the accumulation of the other two by stimulating the activities of synthesis-related enzymes (ACO and ACS for ethylene; NR and NOS-L for NO) or the expression of transporters (AUX1, PIN1 and PIN2 for auxin), thus forming an ethylene–NO–auxin positive feedback loop. Auxin acts downstream of ethylene and NO in the regulatory cascade leading to the induction of root hairs under Mg deficiency. Materials and Methods Plant materials and growth conditions All Arabidopsis mutants used in this study were of the Col-0 background. The pin3-5, pin4-3, pin7-2, pin2 and etr1-3 mutants and the transgenic lines EBS::GUS, PIN3::PIN3-GUS, PIN4::PIN4-GUS and PIN7::PIN7-GUS were obtained from the Arabidopsis Biological Resource Center. The nia1,2 and noa1 seeds were purchased from the Nottingham Arabidopsis Stock Centre. The ein2-5 mutant was provided by H.W. Guo (Tsinghua University, China). The transgenic lines AUX1::AUX1-YFP, PIN1::PIN1-GUS and PIN2::PIN2-GFP were provided by Y.F. Niu (Zhejiang University, China). All seeds were surface-sterilized and germinated in half-strength nutrient solution. After 7 d, uniform seedlings were transferred to full-strength nutrient solution of the following composition (μM): 1,500 KNO3, 500 MgSO4, 1,000 CaCl2, 500 NaH2PO4, 250 (NH4)2SO4, 10 H3BO3, 0.5 MnSO4, 0.5 ZnSO4, 0.1 CuSO4, 0.1 (NH4)6Mo7O24 and 25 Fe-EDTA. The nutrient solution was renewed every 3 d and the pH was adjusted to 6.0 using 1 M NaOH. All the plants were grown in a controlled environment with 70% humidity and a daily cycle of 25°C/14 h day and 22°C/10 h night. After the seedlings were grown in nutrient solution for another 14 d, they were transferred into 0.4 liter pots containing full-strength nutrient solution for 7 d. Thereafter, the plants were transferred to nutrient solutions that either contained (+Mg) or did not contain MgSO4 (–Mg) for 5 d. These plants were used in the experiments and treated as follows for another 2 d in the earlier mentioned +Mg or –Mg solutions. For the chemical reagent treatment, 0.1 μM NAA (Catalog number 86-87-3, Sangon Biotech), 5 μM NPA (Catalog number 132-66-1, Sigma-Aldrich), 10 μM SNP (Catalog number 13755-38-9, Sangon Biotech), 100 μM c-PTIO (Catalog number 148819-94-7, Santa Cruz), 3 μM ACC (Catalog number 22059-21-8, Sigma-Aldrich) and 1 μM l-kynurenine (Catalog number 2922-83-0, Santa Cruz) or 5 μM STS (Catalog number 7772-98-7, Sigma-Aldrich) was added to the nutrient solution. The NAA stock solution was prepared by dissolving NAA in ethanol to a final concentration of 1 mM, and the NPA stock solution was prepared by dissolving NPA in dimethyl sulfoxide (DMSO; Catalog number 67-68-5, Sangon Biotech). The ACC stock solution was prepared by dissolving ACC in ultrapure water to a concentration of 1 mM, and the STS stock solution was prepared by dissolving STS in ultrapure water to a concentration of 5 mM. The SNP stock solution was prepared by dissolving SNP in ultrapure water to a final concentration of 10 mM SNP, and the c-PTIO stock solution was prepared by dissolving c-PTIO in ultrapure water to a final concentration of 10 mM. The l-kynurenine stock solution was prepared by dissolving l-kynurenine in DMSO to a final concentration of 50 mM. Microscopy and fluorescence measurement Seedlings were harvested and the individual roots were divided into three segments, 0–1, 1–2 and 2–3 cm from the root tips. The length and density of root hairs in each segment were measured with microscopy and recorded with a charge-coupled device camera (Eclipse E600; Nikon). In situ measurement of GFP was conducted with an epifluorescence microscope (Nikon Eclipse E600, Nikon, excitation at 488 nm and emission at 495–575 nm). GUS staining GUS staining was conducted according to Mao et al. (2014). Briefly, the root was rinsed three times with GUS staining buffer {50 mM potassium phosphate buffer (pH 7.0), 0.1 mM K3[Fe(CN6)], 0.5 mM K4[Fe(CN6)], 0.1 mM EDTA and 0.01% Triton X-100}, and then incubated overnight with GUS buffer supplemented with 10 mg ml–1 5-bromo-4-chloro-3-indolyl β-d-glucuronic acid at 37°C in the dark. GUS expression was observed under a microscope (Nikon Eclipse E600; Nikon) with an attached camera. Measurement of IAA concentration in roots The measurement of the IAA level in the roots was performed according to the method described by Jin et al. (2011) and Swarup et al. (2007). Briefly, 0.2 g of roots was finely ground in liquid nitrogen and homogenized in 1.5 ml of pre-chilled 80% methanol containing 1 mM 2,6-di-tert-butyl-p-methylphenol in weak light conditions, followed by centrifugation at 5,000 r.p.m. for 10 min at 4°C. The supernatant was purified with C18 columns. Next, 500 μl of the extract was dried with N2 gas, dissolved in 200 μl methanol and dried in a vacuum freeze-dryer (Christ ALPHA 1-4). The extract was then dissolved in 300 μl of phosphate buffer solution (pH 7.4) for enzyme-linked immunosorbent assay (ELISA). The IAA concentration was measured at 490 nm using a microplate reader. Determination of ethylene production in roots The ethylene production in roots was measured according to the method of Tian et al. (2009). Briefly, 0.2 g of roots was cut and immediately placed in 6 ml vials with 1 ml of agar medium (0.7% agar) for 1 h to minimize wounding effects, and then the vials were sealed for 8 h. Thereafter, 1 ml of the headspace gas was collected, and subsequently injected into a gas chromatograph with an alumina column (GDX502) and a flame ionization detector (GC-7AG; Shimadzu). Determination of ACO and ACS activities ACO and ACS activities were determined according to the methods of Tian et al. (2009). For ACO determination, 0.2 g of roots was ground to a fine powder in liquid nitrogen and added to 1.5 ml of extract mixture [100 M Tris–HCl buffer (pH 7.2), 30 mM sodium ascorbate, 10% (v/v) glycerol and 5% polyvinylpolypyrrolidone], followed by centrifugation at 15,000×g for 20 min at 4°C. A total of 800 μl of supernatant was added to gas-tight vials containing 2 ml of extraction buffer (without polyvinylpolypyrrolidone, 50 μM FeSO4 and 2 mM ACC), and incubated for 1 h at 30°C. The ethylene was measured as described earlier. To examine ACO activity, 0.2 g of roots was ground in liquid nitrogen and added to 1 ml of extract mixture [200 mM phosphate buffer (pH 8.0), 2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM dithiothreitol (DTT), 10 μM pyridoxal phosphate and 1 mM EDTA], followed by centrifugation at 15,000×g for 20 min at 4°C. A total of 0.6 ml of supernatant was added to a 6 ml vial containing 0.2 ml of 5 mM S-(5′-adenosyl)-l-methionine (Catalog number 29908-03-0, Sangon Biotech) and left to react for 1 h at 22°C. The ACC formed was converted to ethylene by adding 0.2 ml of 1:1 (v/v) NaOH:bleach and 100 mM HgCl2. The vials were kept on ice for 20 min, and the ethylene content was determined as described above. Histochemical analysis of NO The NO level was determined using the fluorescence indicator DAF-FM DA (Catalog number S0019, Beyotime) according to the method described by Jin et al. (2009b) with some modifications. Briefly, the roots were loaded with 10 μM DAF-FM DA in 20 μM HEPES-NaOH buffer (pH 7.4), washed three times with fresh buffer and observed under a microscope (Nikon Eclipse E600, Nikon), with excitation and emission wavelengths of 488 and 515 nm, respectively. The fluorescence intensity of root tips in the images was quantified using Image J. Measurement of NR and NOS-L activities The activity of NR was determined according to the method of Jin et al. (2011). Briefly, 0.2 g of roots was ground in liquid nitrogen and added to 1.5 ml of extract mixture [100 mM HEPES-KOH (pH 7.5), 10% (v/v) glycerol, 1 mM EDTA, 0.1% Triton X-100, 5 mM DTT, 0.5 mM PMSF, 20 μM FAD, 25 μM leupeptin, 5 μM Na2MoO4 and 1% polyvinylpyrrolidone), followed by centrifugation at 13,000×g for 20 min at 4°C. A 0.2 ml aliquot of supernatant was added to 0.4 ml of assay mixture with 100 μM HEPES-KOH (pH 7.5), 0.25 mM NADH and 5 mM KNO3, and left to react for 1 h at 30°C. The reaction was stopped by the addition of 100 μl of 0.1 M zinc acetate. NR activity was determined colorimetrically at 540 nm by the addition of 1 ml of 1% sulfanilamide and 1 ml of 0.02% N-(1-naphthyl)-ethylenediamine. The activity of NOS-L was measured as described by Tian et al. (2007) with some modifications. Briefly, 0.2 ml of supernatant was prepared as described above and added to 0.1 ml of assay mixture with NADH, l-arginine, DAF-FM DA and NOS-L assay buffer. The reaction was conducted in the dark at 37°C for 1 h. NO content was detected under a microscope as described earlier. Quantitative PCR analyses Total RNA in roots was extracted using RNAiso Plus (Catalog number 9109, TAKARA). All RNA samples were checked for DNA contamination before cDNA synthesis. Then, quantitative real-time PCR analyses were performed with pairs of gene primers (Supplementary Table S1) and a PrimeScript™ RT reagent kit (Catalog number RR037A, TAKARA). The housekeeping gene UBQ10 was used as a reference. Relative expression was calculated using an efficiency-corrected ΔΔCt formula as described previously (Jin et al. 2009a, Fang et al. 2016). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the National Key R&D Program of China [2016YFD0200103]; the National Key Project on Science and Technology of China [2015BAC02B03]; the Agricultural Technology Extension Funds of Zhejiang University [2016NTZX002]; the Natural Science Foundation of China [31622051]; and the Fundamental Research Funds for the Central Universities [2017XZZX002–06]. Acknowledgments We sincerely thank Ms. Y.F. Niu (Zhejiang University, China) for the gifts of aux1-7, PIN1::PIN1-GUS, AUX::AUX-YFP, PIN2::PIN2-GFP seeds, Professor H.W. Guo (Tsinghua University, China) for the seeds of ein2-5 and ein3-1 mutants, Professor H.W. Xue (Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China), and X. Huang (Hainan University, China) for the tir1-1 mutant seeds. Disclosures The authors have no conflicts of interest to declare. References Akaike T., Yoshida M., Miyamoto Y., Sato K., Kohno M., Sasamoto K. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations ACC 1-aminocyclopropane-1-carboxylate ACO ACC oxidase ACS ACC synthase AUX1 AUXIN-RESISTANT1 c-PTIO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide CPC CAPRICE DAF-FM DA 4,5-diaminofluorescein diacetate EBS ETHYLENE BIS STEARAMIDE ETC1 ENHANCER OF TRY AND CPC GFP green fluorescent protein GL2 GLABRA2 GL3 GLABRA3 GUS β-glucuronidase Mg magnesium NAA 1-naphthylacetic acid NO nitric oxide NOS-L NO synthase-like NPA naphthylphthalamic acid NR nitrate reductase PIN1, 2, 3, 4, 7 PIN-FORMED1, 2, 3, 4, 7 ROP2 RHO-RELATED PROTEIN FROM PLANTS 2 SNP sodium nitroprusside STS silver thiosulfate TTG1 TRANSPARENT TESTA GLABRA1 TRY TRIPTYCHON UBQ10 UBIQUITIN10 WER WEREWOLF YFP yellow fluorescent protein © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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