Shoot has important roles in strigolactone production of rice roots under sulfur deficiency

ABSTRACT

Strigolactones (SLs) are a class of plant hormones that control plant architecture. SL levels in roots are determined by the nutrient conditions in the rhizosphere, especially the levels of nitrogen (N) and phosphorus (P). Our previous research showed that SL production is induced in response to deficiency of sulfur (S) as well as of N and P, and inhibits shoot branching, accelerates leaf senescence, and regulates lamina joint angle in rice. Here we show biomass, total S contents, and SL levels in rice under S-sufficient and S-deficient conditions using a split-root system. When one part of the root system was cultured in S-sufficient medium and the other in S-deficient medium (+S/−S), shoot fresh weight was unaffected relative to the +S/+S condition. The shoot weight significantly decreased in −S/−S condition. In contrast, there was no significant difference in root fresh weight between +S and −S conditions. In +S/−S condition, SL levels were systemically reduced in both parts, the shoot S content increased, but the root S content in S-deficient medium was unaffected relative to the −S/−S condition. These results suggest that shoots, not roots, recognize S deficiency, which induces SL production in roots.

Sulfur (S) is a major macronutrient required for plant development and growth, being an essential component of S-containing amino acids such as cysteine and methionine, co-enzyme A, the antioxidant tripeptide glutathione, the ethylene biosynthesis precursor S-adenosyl methionine, sulfolipids, and many secondary metabolites.¹ Plant roots take up sulfate ion as oxidized sulfur from the rhizosphere through several sulfate transporters and then assimilate it into S-containing compounds.² S deficiency reduces chlorophyll content in young leaves, tiller number in grasses, and plant height.³ It also strongly suppresses growth and reduces grain yield of rice.⁴

Strigolactones (SLs) were first discovered as communication signals used in parasitism and symbiosis in the rhizosphere.^5,6 Later, they were identified as a class of plant hormones that inhibit shoot branching; stimulate stem thickening, leaf senescence, root hair elongation, and primary root growth; and suppress lateral root growth and adventitious root formation.⁷ SL levels in roots dramatically change in response to nutrient deficiencies. SLs are highly produced in roots under phosphorus (P) deficiency in alfalfa (Medicago sativa), tomato (Solanum lycopersicum), red clover (Trifolium pratense), Chinese milk vetch (Astragalus sinicus), wheat (Triticum aestivum), sorghum (Sorghum bicolor), marigold (Tagetes patula), lettuce (Lactuca sativa), maize (Zea mays), and rice (Oryza sativa).⁸ Under P deficiency in rice, endogenous SL levels are highly elevated, inhibit tiller bud outgrowth, and accelerate leaf senescence.^9,10 Thus, SLs are thought to be key regulators for efficient P allocation and adaptation to P deficiency. SL levels also increase under nitrogen (N) deficiency in Chinese milk vetch, wheat, sorghum, marigold, lettuce, maize, and rice.⁸ In white lupine (Lupinus albus), SL levels in roots do not increase under N and P deficiencies.⁸ In addition, SL levels increased under S deficiency, as well as under N and P deficiency, in rice (Oryza sativa ssp. japonica).¹¹ Under S deficiency, SL production in rice was caused mainly by upregulation of an SL biosynthesis gene, DWARF27 (D27), but expression of other SL biosynthesis genes was unchanged.¹¹ Numbers of tiller buds and chlorophyll content were higher in a d27 rice mutant than in the wild type under S deficiency, but lower than in two other dwarf mutants, d10 and d14.¹¹ These results indicate that D27 plays an important role in adaptation to S deficiency in rice. Notably, some symptoms of S deficiency are similar to the physiological effects of SLs. In rice, SL levels increase to regulate shoot branching, leaf senescence, and lamina joint angle under S deficiency.^11,12 In this study, we investigated the relationships among biomass, S contents, and SL levels in S-sufficient and S-deficient conditions.

To analyze which plant organs recognize S deficiency in terms of its effect on SL production, we grew rice ‘Shiokari’ seedlings under the S-sufficient (+S) (310 µM) or S-deficient (−S) (0 µM) condition using a split-root assay. After preculture under S-sufficient conditions for 7 days, the root system was divided into two parts, each part was placed into a separate container of hydroponic culture medium,¹³ and the seedlings were further cultured for 7 days under 16 h fluorescent white light (130–180 µmol m⁻² s⁻¹) at 25°C and 8 h dark at 23°C (Figure 1a,b).¹² When one part of the root system was cultured in S-sufficient medium and the other in S-deficient medium (+S/−S), shoot fresh weight was unaffected relative to the +S/+S condition, and decreased by about 60% in −S/−S condition (Figure 1c,Figure 1d). In contrast, there was no significant difference in root fresh weight between +S and −S conditions (Figure 1c,e). A canonical SL, 4-deoxyorobanchol (4DO) levels were analyzed using LC-MS/MS according to methods described previously.¹² 4DO levels in root exudates and roots significantly increased when both root parts were in S-deficient medium (−S/−S) relative to S-sufficient medium (+S/+S), but were much lower when one or both parts were in S-sufficient medium (+S/−S, +S/+S) (Figure 2a,b). To measure total S contents, we wet-decomposed samples in nitric acid/hydrogen peroxide (HNO₃:H₂O₂ = 6:1) in a Titan MPS (PerkinElmer, Waltham, MA, USA) microwave sample preparation system, then measured S contents by inductively coupled plasma–mass spectroscopy (ICP-MS) analysis (X series 2, Thermo Scientific, Pittsburgh, PA, USA). S contents in shoots were consistent with the S conditions of the media (Figure 2c). S contents in roots were relatively high in S-sufficient medium and low in S-deficient medium (Figure 2d). In the +S/−S treatment, the S content in −S roots was unaffected relative to the −S/−S condition, yet the SL level did not increase (Figure 2b,Figure 2d), indicating that SL production in roots increases in response to S content in shoots. Nutrient levels in shoots are important for SL production: under N and P deficiency, SL production in roots also increased in response to N and P contents in shoots.¹⁴ Therefore, shoots have an important role in SL production of roots in response to nutrient deficiency.

Figure 1.

Analysis of biomass in rice seedlings by split-root assay. (a) Schematic diagram showing experimental conditions. After preculture, the root systems of wild-type seedlings were separated into two parts and each part was put into either S-sufficient (+S) or S-deficient (−S) hydroponic culture medium. (b) A rice seedling in the split-root assay system. Arrowheads: white, rice seedling; orange, bamboo stake; red, split roots. Bar: 1 cm. (c) A photograph of rice seedlings after cultivated using split-root assay. Bar: 5 cm. (d and e) Fresh weight of shoots (d) and roots (e). Data are means ± S.E. (n = 3). Different letters indicate significant differences (Tukey’s HSD, P < .05)

Figure 2.

Analysis of SL and S contents in rice seedlings by split-root assay. (a and b) SL (4DO) levels in root exudates (a) and split roots (b). Data are means ± S.E. (n = 3). (c and d) Total S contents in shoots (c) and split roots (d). FW, fresh weight. DW, dry weight. Data are means ± S.E. (n = 3). Different letters indicate significant differences (Tukey’s HSD, P < .05)

Finally, what is the long-distance signal from the shoots? An unknown second messenger probably moves from shoots to roots, where it induces SL production in roots. P supply in a split-root system of pea systematically downregulated SL production.¹⁵ Yoneyama et al. (2015) showed that auxin derived from the shoot apex is not involved in the regulation of SL production in roots.¹⁴ A shoot-derived polypeptide, C-TERMINALLY ENCODED PEPTIDE DOWNSTREAM (CEPD), moves to roots and activates expression of nitrate transporter in response to N deficiency.¹⁶ However, it is unknown whether CEPD interacts with SL signaling. Sugars produced in rice leaves are transported to roots, where they might inhibit SL production through circadian clock regulation.¹⁷ In fact, sugar signals can promote tiller bud outgrowth. Further analysis is required to identify the shoot-derived signal that stimulates SL production in roots.

Funding Statement

This work was in part supported by a grant from Toyo University (Inoue Enryo Memorial Foundation for Promoting Sciences to M.S.), by a grant from the Japan Society for the Promotion of Science (KAKENHI, 20K05776 to M.U.).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.