Effects of strigolactone signaling on Arabidopsis growth under nitrogen deficient stress condition

ABSTRACT

Strigolactones (SLs) are a group of terpenoid lactones found in plants that regulate diverse developmental phenomena. SLs are thought to be involved in the maintenance of phosphate homeostasis. In addition, SL signaling is required for the regulation of shoot branching by nitrogen supply in Arabidopsis. In this study, we evaluated the effects of SLs on nitrogen deficient-inducing phenomena (leaf senescence and reduction of plant weight) in Arabidopsis. SL-biosynthesis (max1-1) and SL-insensitive (atd14-1) mutants showed altered responses to nitrogen deficient in comparison with wild-type (WT) plants. Nitrogen deficient conditions led to alterations in the expression levels of SL biosynthesis genes (MAX3 and MAX4). These results indicate that SLs could be key mediators of plant growth response to nitrogen supply.

Strigolactones (SLs) are a new group of plant hormones that regulate various developmental processes.^1-3 The genetic analysis of a series of branching mutants in Arabidopsis, rice, petunia, and pea led to proposal of the SL biosynthetic and signaling pathways. The biosynthesis of SLs is mediated by a carotenoid isomerase (D27), 2 carotenoid cleavage dioxygenases (CCD7/MAX3 and CCD8/MAX4), and cytochrome P450 (CYP711A subfamily).^4,5 D27 catalyzes the isomerization at C-9 position of all-trans-β-carotene. CCD7 and CCD8 convert 9-cis-β-carotene to carlactone (CL) via sequential reaction.^6,7 Recently, it was reported that Os01g0700900 and Os01g0701400, which are members of the rice CYP711A subfamily, encode a CL oxidase and an ent-2′-epi-5-deoxystrigol-4-hydroxylase, respectively, and convert CL to orobanchol,⁸ whereas in Arabidopsis, CYP711A1 (AtMAX1) does not convert CL to SL but converts it to an SL-like chemical, carlactonoic acid.⁹ F-box protein/MAX2 and α/β hydrolase protein/D14 are involved in SL signal transduction,^10-12 and mutants in these genes render plants SL-insensitive.

SLs also serve as rhizosphere communication signals between plants and arbuscular mycorrhizal fungi.¹³ This symbiotic relationship provides the fungus with carbohydrates produced by the plants and provides the plants with phosphate taken up by the fungi. Recent studies have suggested that SLs likely serve as signals that mediate responses to phosphate deficient in various plant species.^14-17 In addition, the fact that SLs modulate the establishment of the symbiosis between nitrogen-fixing bacteria and legumes^18-22 and their biosynthesis is regulated by environmental nitrogen concentration in some plant species,²³ suggests the possible involvement of SLs in the nitrogen response. A recent study found that SL signaling is essential in root development in rice and shoot branching in Arabidopsis under low nitrogen condition.^24,25 However, the effects of SL signaling on the other nitrogen deficient responses including reduction of plant growth and chlorophyll synthesis have not been investigated.

In this study, we evaluated the effect of SL signaling on nitrogen deficient responses. We found that SL-biosynthesis and SL-insensitive mutants showed altered responses to nitrogen deficient. Furthermore, variations in nitrogen concentrations altered the expression levels of SL biosynthesis genes. These results suggest a potential overlap between SL signaling and nitrogen deficient signaling pathways in Arabidopsis.

Nitrogen deficient affects anthocyanin accumulation, plant growth, and leaf senescence.²⁶ Previously, it was shown that SL treatment in WT seedlings induced anthocyanin accumulation, reduction of plant growth and promotion of leaf senescence.^16,17, In addition, we have previously shown that SL signaling regulates nitrogen-deficient-induced anthocyanin accumulation.¹⁷ To clarify the role of SL signaling in the other nitrogen deficient responses, we estimated the effects of nitrogen deficient on plant weight. We measured the plant weight of WT (Col-0), max1-1 (SL biosynthesis mutant),¹¹ and atd14-1 (SL insensitive mutant)¹² under nitrogen-limited conditions. Control medium used for the assay contained 5 mM KNO₃, 1 mM KH₂PO₄, 1 mM MgSO₄, 1.5 mM Ca(NO₃)₂, 1 mM NH₄Cl, 50 µM Fe-EDTA, 46 µM HBO₃, 10 µM MnSO₄, 0.77 µM ZnSO₄, 0.32 µM CuSO₄, 0.58 µM Na₂MoO₄, 0.25 µM NH₄VO₃, 3 mM MES, 0.75% sucrose, and 0.7% agar (pH 5.7).²⁷ Nitrogen deficient medium (pH 5.7) was prepared by partially replacing KNO₃, Ca(NO₃)₂, and NH₄Cl in the control medium with KCl, CaCl₂, and NaCl, respectively. Plants grown on control medium for 3 days were transferred onto fresh media supplemented with or without nitrogen. After 3 weeks, plant weight was measured by using an AT261 Delta Range Analytical Balance (Mettler Toledo, Switzerland). The ratio of the weight of plants grown on nutrient-limited conditions to that grown on control medium was calculated.

Although plant growth was also inhibited in a nitrogen concentration-dependent manner in max1-1 and atd14-1 mutants, the ratio of plant weight under nitrogen-limited conditions tended to increase in max1-1 and atd14-1 compared to WT (Fig. 1A). Especially, when the plants were grown on 1/5 N media, the ratio of plant weight was significantly increased in both max1-1 and atd14-1. Thus, nitrogen deficiency had a lower impact on the growth of max1-1 and atd14-1 mutants than WT seedlings. Based on this result, we used the 1/5 N medium as a nitrogen deficient medium for subsequent study. Under nitrogen-sufficient conditions (1 N), SL treatment significantly reduced the weight of WT plant, as previously demonstrated.¹⁷ On the other hand, max1-1 mutant also showed reduced weight by SL treatment, but the difference was not significant, whereas atd14-1 did not (Fig. 1B), suggesting that SLs regulate plant weight. Under nitrogen-deficient conditions, SL treatment significantly reduced plant weight in max1-1, but not in atd14-1. These results suggest that SL signaling is required for the growth defect under nitrogen-deficient conditions.

Figure 1.

Comparison of weights of WT, max1-1, and atd14-1 plants. (A) Ratio of plant weight of WT, max1-1, and atd14-1 seedlings grown on nitrogen-limited medium. The weight of WT, max1-1 and atd14-1 grown under 1 N condition are 41.9 ± 8.2, 43.1 ± 1.7 and 45.1 ± 2.0 mg/seedling, respectively. (B) Effect of GR24 (synthetic SL analog) on plant weight under 1 N and 1/5 N conditions. 5 µM GR24 was added to 1 N or 1/5 N media. The weight of WT, max1-1 and atd14-1 grown under 1 N condition are 40.9 ± 1.8, 51.1 ± 6.5 and 60.3 ± 4.6 mg/seedling, respectively. Data represent means ± SD of 4 biological replicates (each replicate contained 16 seedlings). * and ** indicate significant differences from WT seedlings (A), and 0 µM GR24 treatment (B) (Student's t-test, *:P < 0.05, **: P < 0.01). Black, white, and gray bars represent WT, max1-1, and atd14-1 plants, respectively.

Nitrogen deficient also accelerates the reduction of chlorophyll content, which is induced by leaf senescence. In addition, SLs regulate dark-induced leaf senescence.²⁸ Based on this information, we measured the chlorophyll content in the leaves of WT and max1-1 and atd14-1 mutants under nitrogen-sufficient (1 N) and nitrogen-deficient (1/5 N) conditions to examine the effect of SL signaling on nitrogen–deficient–induced leaf senescence (Fig. 2). Plants grown on control medium for 7 days were transferred onto fresh media supplemented with or without nitrogen. After 1 week, chlorophyll was extracted from each sample in dimethylformamide. Absorbance values at 663.8 and 646.8 nm were measured using a DU530 spectrometer (Beckman Coulter). Chlorophyll content was estimated as described previously.²⁹

Figure 2.

Comparison of chlorophyll content in WT, max1-1, and atd14-1 plants. 5 µM GR24 was added to 1 N or 1/5 N media. Data represent means ± SD of 4 biological replicates each replicate contained 16 seedlings). * and ** indicate significant differences from WT seedlings (Student's t-test, *: P < 0.05, **: P < 0.01). Black, white, and gray bars represent WT, max1-1, and atd14-1 plants, respectively.

When the seedlings were grown on 1 N medium, no differences were observed in the chlorophyll content in max1-1 and atd14-1 mutants as well as in WT, regardless of SL treatment. In contrast, under 1/5 N condition, the chlorophyll content in max1-1 and atd14-1 mutants was about 2-fold higher than that in WT (Fig. 2). In addition, SL treatment of max1-1 mutant induced the reduction of chlorophyll content to a level similar to that in WT under 1/5 N condition. Moreover, the chlorophyll content in atd14-1 mutant was not affected by SL treatment under this condition. These results suggest that SLs modulate chlorophyll content under nitrogen-deficient as well as under dark conditions.

SL biosynthesis is up-regulated by nitrogen deficiency in some plants.^30,31 However, the effect of nitrogen deficient on SL biosynthesis in Arabidopsis has not been studied thus far. To examine this, we performed the expression analysis of SL biosynthesis genes in WT roots, using real-time RT-PCR. Plants grown on control medium for 7 days were transferred onto fresh media supplemented with or without nitrogen. After 2 weeks, total RNA was extracted from roots and cDNA synthesis was performed. qRT-PCR was performed on a Takara Thermal Cycler Dice Real Time System using a SYBR premix Ex Taq (Takara). Specific primers used for qRT-PCR are designed as follows: MAX1 (forward: 5′-GGCCCTATTTTCAGATTTCAG-3′, reverse: 5′-TGGTGAAGAAGAGGCCTTTC-3′), AtD27 (forward: 5′-TCGCGTAACTTTGACACCAAGC-3′, reverse: 5′-CGAGACTTTTGAAGGTGGAAATGC-3′), MAX3 (forward: 5′-GTGTATTTAAGATGCCACCGA-3′, reverse: 5′-CTTGAATTCCGAATCATACTCAC-3′), MAX4 (forward: 5′-GTTTTACCCGATGCTAGGATC-3′, reverse: 5′-TGATGCTGCACATATCCATCG-3′), and UBC (encoding ubiquitin-conjugating enzyme, At5g25760) (forward: 5′-TAGCATTGATGGCTCATCCT-3′, reverse: 5′-GGCGAGGCGTGTATACATTT-3′). Expression of UBC was used as an internal standard.³²

MAX3 and MAX4 transcripts were significantly upregulated under 1/5 N condition as compared with 1 N condition (Fig. 3). However, the transcript levels of the other SL biosynthesis genes (MAX1 and D27) were not up-regulated under 1/5 N conditions. These data indicate that nitrogen-deficient treatment elevates the endogenous levels of SL via the up-regulation of the levels of the specific SL biosynthesis genes, MAX3 and MAX4. As shown in Fig. 1B and Fig. 2, SL treatment of WT seedlings did not result in reductions of plant weight and chlorophyll content under 1/5 N condition. Nitrogen-deficient condition enhanced the expression levels of SL biosynthesis genes, suggesting that endogenous SL levels were increased in WT seedlings (Fig. 3).^30,31 These results suggest that nitrogen deficient up-regulates the endogenous levels of CL and its metabolites (carlactonoic acid, its methyl ester and SLs), and elevated levels of these chemicals partially induce the nitrogen deficient responses.

Figure 3.

Expression analysis of SL biosynthesis and signaling genes. Roots of 3-week-old Arabidopsis seedlings were used for expression analysis of SL biosynthesis and signaling genes. The expression level of each transcript was normalized with UBC and is displayed relative to the expression level in plant grown on the 1N condition (= 1). Data represent means ± SD of 3 biological replicates. ** indicates significant differences from the expression of plants grown on 1 N condition (Student's t-test, **: P < 0.01).

To clarify the role of SL signaling in nitrogen-deficient response, we examined morphological changes in both SL biosynthesis and signaling mutants under nitrogen-sufficient and nitrogen-deficient conditions. Under nitrogen-deficient conditions, the deficiencies in SL biosynthesis and signaling repressed the reductions of plant weight and chlorophyll content, which are responses indicative of nitrogen deficiency. In addition, we have previously shown that SLs modulate anthocyanin accumulation under nitrogen-limited conditions.¹⁷ Furthermore, in the present study, we found that nitrogen deficiency enhanced the expression levels of some SL biosynthesis genes. Our results indicate that SL signaling is involved in multiple responses to nitrogen as well as phosphate deficiency.

Disclosure of potential conflict of interest

No potential conflicts of interest were disclosed.

Acknowledgment

This work was supported by the grants from the Kato memorial Bioscience Foundation (S. I.); and the Agricultural Chemical Research Foundation (S.I.)