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. 2025 Sep 19;24(2):678–697. doi: 10.1111/pbi.70374

Strigolactones Regulate Sugar Allocation to Control Rice Tillering and Root Development via the OsSPL14‐OsSHR1‐OsSWEET16 Pathway

Miao Feng 1, Wenfan Luo 1, Sheng Luo 1, Rong Miao 2, Manpo Gu 1, Shuai Li 1, Xinxin Xing 1, Jinhui Zhang 1, Jinsheng Qian 1, Xin Liu 1, Chunlei Zhou 1, Qi Sun 1, Tong Luo 1, Nuo Chen 1, Yulong Ren 1, Zhijun Cheng 1,3, Cailin Lei 1, Zhichao Zhao 1, Shanshan Zhu 1, Xin Wang 1, Xiuping Guo 1, Qibing Lin 1,✉, Jianmin Wan 1,2,4,✉
PMCID: PMC12906830  PMID: 40974002

ABSTRACT

Strigolactones (SLs) are root‐to‐shoot phytohormones that regulate tillering (branching) and root development. Sugar, as an essential energy substance and signalling molecule, plays a fundamental role in the growth and development of plants. However, the molecular mechanisms by which SL directly regulates sugar allocation to control tillering and root development are still not fully understood. Here, we found that OsSHR1 operates directly downstream of OsSPL14 within the SL signalling pathway, facilitating root elongation while inhibiting tillering and crown root development. The expression of OsSHR1 is stimulated by OsSPL3/12/14 in vivo, and D53 can interact with these SPL proteins to suppress their transcriptional activities. Interestingly, we further demonstrate that OsSHR1 can directly bind to the promoters of OsSWEET2a/4/16, which encode sugar transporters that can control the allocation of sugar in plant growth and development. This binding facilitates the expression of sugar transporters, which in turn regulate sugar allocation and enable the plant's response to SLs. The results indicate that via the OsSPL14‐OsSHR1‐OsSWEET16 pathway, SLs orchestrate the distribution of sugars to ensure their effectiveness in stimulating root elongation while simultaneously suppressing tillering and crown root formation in rice.

Keywords: root development, strigolactones, sugar allocation, tillering

1. Introduction

Strigolactones (SLs), a class of carotenoid‐derived phytohormones, act as branching‐repressing hormones with highly conserved functions in both monocots and dicots (Gomez‐Roldan et al. 2008; Umehara et al. 2008). In addition to repressing shoot branching, SLs also regulate root development, leaf senescence and flower development (Snowden et al. 2005; Ueda and Kusaba 2015). For example, SLs promote the elongation of primary roots and adventitious (crown) roots, and suppress crown root and lateral root formation in Arabidopsis and rice (Arite et al. 2011; Ruyter‐Spira et al. 2011; Rasmussen et al. 2012; Sun et al. 2014, 2016; Kumar et al. 2015; Yuan et al. 2023).

The biosynthesis and signalling pathway of SLs has been generally elucidated. In rice, DWARF 3 (D3), DWARF14 (D14, HTD2 and D88) and DWARF53 (D53) are involved in the SL signalling (Ishikawa et al. 2005; Arite et al. 2009; Jiang et al. 2013; Zhou et al. 2013). D14 encodes a member of the α/β‐hydrolase fold family proteins, which binds and hydrolyses SLs (Arite et al. 2009; Gao et al. 2009; Yao et al. 2016; Hu et al. 2024). This triggers a conformational change of D14 to induce its binding with the F‐box protein D3 to form a Skp1–Cullin–F‐box (SCF) complex SCFD3‐D14 (de Saint Germain et al. 2016; Yao et al. 2016; Shabek et al. 2018). This complex then interacts with the SL signalling repressor D53, leading to its ubiquitination and subsequent degradation (Jiang et al. 2013; Zhou et al. 2013; Shabek et al. 2018). D53 degradation relieves inhibited activities of IDEAL PLANT ARCHITECTURE1 (IPA1), thereby triggering downstream SL responses that promote rice root elongation and suppress tillering (Song et al. 2017; Sun et al. 2021).

IPA1 belongs to SQUAMOSA promoter‐binding‐like transcription factors (SPLs), a class of plant‐specific transcription factors with a highly conserved DNA‐binding domain (SBP‐DBD), which enables them to primarily bind DNA sequences with a GTAC core sequence (Yamasaki et al. 2004; Birkenbihl et al. 2005). Rice contains 19 SPL encoding genes, which can be categorised into six subgroups (Xie et al. 2006). Among them, eight SPL genes (OsSPL3, OsSPL6, OsSPL8, OsSPL12, OsSPL13, OsSPL14, OsSPL16 and OsSPL17) play diverse roles in plant architecture, inflorescence architecture, leaf ligule development, panicle apical abortion, grain size, shape and quality, as well as root development (Lee et al. 2007; Jiao et al. 2010; Miura et al. 2010; Wang et al. 2012, 2018; Si et al. 2016; Shao, Zhou, et al. 2019; Sun et al. 2021). Among these SPL genes, OsSPL14 (IPA1) can repress tillering but promote inflorescence branching in rice; and its gain‐of‐function mutant ipa1‐2D with higher IPA1 mRNA levels can promote the big panicle formation, thus increasing the rice yield (Zhang et al. 2017). OsSPL14/17 act downstream of SL signalling to modulate rice root elongation in response to nitrate supply (Sun et al. 2021). In addition, the OsmiR156‐OsSPL3/OsSPL12 module directly activates OsMADS50 in the node to regulate crown root development in rice. The gain‐of‐function mutant lower crown root number 1 (lcrn1), which results from a point mutation in OsSPL3 that disrupts the OsmiR156‐mediated cleavage of OsSPL3 transcripts, exhibited reduced crown roots and tillers (Shao, Zhou, et al. 2019).

The Sugars Will Eventually be Exported Transporters (SWEET) gene family encodes a class of sugar transporters belonging to the MtN3/saliva family with multiple transmembrane domains, which can transport sugars, sugar alcohols and hormones (Chen et al. 2010; Yuan and Wang 2013; Singh et al. 2023). As the conserved transporters of hexoses and sucrose, SWEETs play a variety of functions in plant development and production by controlling the allocation of sugars, including leaf senescence (Quirino et al. 1999), sugar loading in phloem (Chen et al. 2012), nectar production (Lin et al. 2014), pollen viability (Chu et al. 2006; Yang et al. 2006), grain filling (Sosso et al. 2015; Ma et al. 2017; Yang et al. 2018), host‐pathogen interaction and various stress responses (Yang et al. 2006, 2023; Chen et al. 2010; Gao et al. 2018, 2021; Jeena et al. 2019; Breia et al. 2021; Kim et al. 2021; Mathan et al. 2021). Arabidopsis and rice genomes contain 17 and 22 SWEET genes, respectively and they can be grouped into four clades: clade I (OsSWEET1a/1b/2a/2b/3a/3b), clade II (OsSWEET4/5/6a/6b/7a/7b/7c/7d/7e), clade III (OsSWEET11a/11b/12/13/14/15) and clade IV (OsSWEET16) (Chen et al. 2010; Wu et al. 2022). In addition, OsSWEET3a, OsSWEET5 and OsSWEET13/15 are involved in the transport of GA20 to young leaves (Morii et al. 2020), the regulation of auxin signalling and auxin translocation (Zhou et al. 2014) and the response of ABA to abiotic stress (Mathan et al. 2021) in rice, respectively. A recent study has shown that ZmCCD8 promotes the accumulation of sugars in maize kernels by upregulating the activities of ZmSWEET10 and ZmSWEET13c, suggesting that SLs also regulate the expression of SWEET genes (Zhong et al. 2024). However, the specific mechanisms by which SWEET proteins participate in the SL signalling pathway and their precise roles in regulating plant architecture are largely unknown.

The SHORT ROOT (SHR) gene, which encodes a GRAS protein, plays a crucial role in root radial patterning and stem cell niche specification in Arabidopsis thaliana , in conjunction with the GRAS protein SCARECROW (SCR) (Benfey et al. 1993; Di Laurenzio et al. 1996; Helariutta et al. 2000; Nakajima et al. 2001). SHR protein is found to act as a signal from the stele to specify endodermal cell fate and activate SCR‐mediated asymmetric cell division, thus stimulating root ground tissue (endodermis and cortex) formation to promote root meristem development in Arabidopsis (Nakajima et al. 2001). SHR‐like genes also facilitate the development of root meristem in a variety of roots across rice, leguminous plants and maize (Henry et al. 2017; Dong et al. 2020; Lin et al. 2020; Ortiz‐Ramírez et al. 2021). Besides, SHR‐like genes regulate cell proliferation and differentiation in other organs such as the endodermis in hypocotyl and inflorescence stem (Fukaki et al. 1998) and leaf vascular, as well as bundle sheath development in Arabidopsis (Dhondt et al. 2010; Cui et al. 2014), stomatal patterns and minor vein differentiation in rice (Schuler et al. 2018; Wu et al. 2019; Liu et al. 2023), and the formation of Kranz anatomy in maize (Wang et al. 2013). As the key regulator of plant growth and development, SHR‐like genes or the proteins they encode, are tightly regulated by multiple hormones. In Arabidopsis, abscisic acid (ABA) and gibberellin (GA) coordinate the formation of the middle cortex in the root meristem by regulating the expression of the SHR and SCR genes at the transcriptional level (Choi and Lim 2016; Gong et al. 2016; Lee et al. 2016). In rice, ABA and GA antagonistically regulate the APC/CTE (an E3 ubiquitin ligase)‐mediated degradation of OsSHR1 to control the OsSHR1 protein level, thereby modulating root elongation (Lin et al. 2020). However, the relationship between SLs and SHR‐like genes remains unclear.

Here, we have demonstrated that within the SL signalling pathway, a regulatory pathway OsSPL14‐OsSHR1‐OsSWEET16 alters sugar allocation in rice, effectively inhibiting tillering and crown root formation while promoting root elongation. Therefore, this study offers new insights into the specific regulatory mechanism of SL on rice plant architecture by regulating sugar allocation.

2. Results

2.1. OsSHR1 Regulates Root Development and Represses Tillering in Response to SLs

We previously showed that ABA and GA antagonistically regulate APC/CTE‐mediated degradation of OsSHR1 to regulate root length (Lin et al. 2020). During this study, we noticed that besides the varied root lengths (Figure 1a,c), the OsSHR1‐Ri seedlings also displayed more crown roots while the OsSHR1‐mD‐His (a stable OsSHR1 mutant with His marker) seedlings exhibited fewer crown roots than Nipponbare (Nip) seedlings (Figure 1a,b,d). Moreover, the OsSHR1‐Ri plants displayed more tillers while the OsSHR1‐mD‐His plants showed fewer tillers compared with Nip plants (Figure 1e,f). An expression analysis of OsSHR1 showed that OsSHR1 is widely expressed in different tissues with higher levels in the root at the early seedling stage and higher levels in the root and leaf at the heading stage (Figure S1). Taken together, these results suggest that OsSHR1 is a key plant architecture regulator with multiple functions in promoting root elongation, as well as inhibiting crown root formation and tillering.

FIGURE 1.

FIGURE 1

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OsSHR1 is involved in the SL‐mediated regulation of rice tiller and root development. (a) The seedling phenotype of 8‐day‐old Nipponbare (Nip), OsSHR1‐Ri and OsSHR1‐mD‐His. Bar = 1 cm. (b) The crown root phenotype of the boxed area in (a). Bar = 0.5 cm. (c, d) Statistical analyses of total root length (c) and crown root number (d) in (a). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (e) Plant morphology of Nip, OsSHR1‐Ri and OsSHR1‐mD‐His at the mature stage. Bar = 10 cm. (f) Statistical analyses of tiller number in (e). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (g) Root morphology of 8‐day‐old Nip, OsSHR1‐Ri, OsSHR1‐mD‐His, d27 and d53 mutants with or without GR245DS treatment. +, apply GR245DS with 1 μM (dissolved with DMSO); −, equal volume of DMSO. Bar = 1 cm. (h, i) Statistical analyses of total root length (h) and crown root number (i) in (g). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (j) Plant morphology of Nip, OsSHR1‐Ri, OsSHR1‐mD‐His, d27 and d53 seedlings with or without GR245DS treatment. +, apply GR245DS with 1 μM (dissolved with DMSO); −, equal volume of DMSO. Bar = 5 cm. (k) Statistical analyses of tiller number in (j). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA).

Like OsSHR1, SLs also repress shoot branching (Gomez‐Roldan et al. 2008; Umehara et al. 2008) and promote root elongation (Koltai 2011; Sun et al. 2014), suggesting a link between OsSHR1 and the SL signalling pathway. To test this, we performed a GR245DS treatment on seedlings of Nip, OsSHR1‐Ri and OsSHR1‐mD‐His lines, d27 and d53 mutants, respectively. After GR245DS treatment, the total root lengths of Nip, OsSHR1‐mD‐His and d27 seedlings were significantly increased while their crown root numbers were evidently reduced (Figure 1g–i). Notably, the stimulatory effect of GR245DS on the total root lengths and its inhibitory effect on crown root numbers of OsSHR1‐Ri and d53 seedlings were evidently attenuated (Figure 1g–i). In addition, the tiller number of OsSHR1‐Ri plants remained largely unchanged under GR245DS treatment, like that of d53 plants (Figure 1j,k). These findings indicate that OsSHR1 likely plays a key role within the SL signalling pathway regulating root development and tillering in rice.

2.2. D53 Inhibits OsSPL3/12/14‐Mediated Activation of OsSHR1 Expression

To explore how OsSHR1 is involved in the SL signalling pathway, we analysed the promoter of OsSHR1 and found that its promoter contains six GTAC motifs (A through F) recognised by SPLs (Figure S2a) (Birkenbihl et al. 2005), suggesting that OsSHR1 may be a direct downstream target of SPLs. Supporting this, a yeast one‐hybrid (Y1H) assay showed that OsSPL3/12/14 could bind to the last GTAC motif (F motif) in the promoter of OsSHR1 (Figure S2b), and this binding was confirmed by an electrophoretic mobility shift assay (EMSA) (Figure S2c–e). Chromatin immunoprecipitation‐quantitative PCR (ChIP‐qPCR) analyses showed the significant enrichment of the P2 segment containing the F motif within the OsSHR1 promoter sequence by OsSPL3/12/14 (Figure 2a–d). Furthermore, a transient dual‐LUC assay in rice protoplasts indicated that Pro35S:OsSPL3/12/14‐Flag significantly enhanced the expression of the luciferase (LUC) reporter gene driven by the promoter of OsSHR1 (Figure 2e,f). Together, these results showed that OsSPL3/12/14 proteins directly bind to the OsSHR1 promoter to activate its expression.

FIGURE 2.

FIGURE 2

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OsSPL3/12/14 and OsSHR1 bind to the promoters of OsSHR1 and OsSWEET2a/4/16, respectively. (a) Diagram of the OsSHR1 promoter region. The letters A to F indicate GTAC motifs. The red letter indicates the binding sites identified by EMSA. P1 and P2 represent the locations of ChIP‐enriched fragments. (b–d) ChIP‐qPCR analyses verify the binding of OsSPL3/12/14 to the promoter regions of OsSHR1. Cross‐linked chromatin samples were extracted from rice protoplasts co‐expressing Pro35S:OsSPL3/12/14‐Flag and ProOsSHR1:Luc‐Pro35S:Rluc, then were precipitated with anti‐Flag antibody. No Ab (No antibody) served as negative controls. Values are means ± SD (**p < 0.01, n = 3, two‐way ANOVA). (e) Effectors and reporter constructs used in the dual luciferase assay. (f) D53 represses the transcriptional activation activities of OsSPL3/12/14 on the OsSHR1 promoter in rice protoplasts. Relative LUC activity was calculated by LUC/REN. Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 3, two‐way ANOVA). (g–i) Diagrams of the OsSWEET2a/4/16 promoter region, respectively. The letters A to I indicate AATTT motifs. The red letters indicate the binding sites identified by EMSA. P1, P2 and P3 represent the locations of ChIP‐enriched fragments. (j) ChIP‐qPCR analyses verify the binding of OsSHR1 to the promoter regions of OsSWEET2a/4/16. Cross‐linked chromatin samples were extracted from rice protoplasts co‐expressing Pro35S:OsSHR1‐GFP and ProOsSWEET2a/4/16:Luc‐Pro35S:Rluc, then were precipitated with anti‐GFP antibody. No Ab (No antibody) served as negative controls. Values are means ± SD (**p < 0.01, n = 3, two‐way ANOVA). (k) Effector and reporter constructs used in the dual luciferase assay. (l) OsSHR1 activates the transcriptional activity of OsSWEET2a/4/16 in rice protoplasts. Relative LUC activity was calculated by LUC/REN. Values are means ± SD (**p < 0.01, n = 3, Student's t‐test).

Consistent with the previous report that D53 binds to OsSPL14, and together with TPL/TPR proteins represses the transcriptional activity of OsSPL14 (Song et al. 2017), we also found that besides OsSPL14, OsSPL3/12 also interact with D53 (Figure S3). Then, quantitative transactivation assays showed that the co‐transfection of ProOsSHR1:LUC and Pro35S:OsSPL3/12/14‐Flag more significantly up‐regulated LUC activity than the co‐transfection of ProOsSHR1:LUC, Pro35S:D53‐GFP and Pro35S:OsSPL3/12/14‐Flag (Figure 2e,f), indicating that D53 likely interacts with OsSPL3/12/14 to inhibit the transcription of OsSHR1 promoted by them.

2.3. OsSHR1 Positively Regulates OsSWEET2a/4/16 Expression

Some recent studies have shown that sugars can affect the plant's response to SLs, including leaf senescence and tiller development (Tian et al. 2018; Bertheloot et al. 2019; Patil et al. 2021; Takahashi et al. 2021). As key transporters of sugars, SWEETs influence a wide range of plant development processes by regulating sugar partitioning (Chen et al. 2010, 2012; Yuan et al. 2014; Breia et al. 2021; Hu et al. 2021; Singh et al. 2023). Moreover, SLs can regulate the accumulation of sugars in grains (Zhong et al. 2024). These results suggest a connection between SLs and the distribution of sugars during plant development. In addition, the promoters of rice SWEET genes contain the AATTT motif that can be specifically recognised by GRAS proteins (Hirsch et al. 2009; Shi et al. 2019). Therefore, to test whether SWEETs are involved in the OsSHR1‐mediated regulation of tillering and root development, we performed a Y1H assay using the promoters of 22 OsSWEET genes. The results indicated that OsSHR1 directly binds to the C, G, I and C sites with the AATTT motif in the promoter regions of OsSWEET2a/4/16, respectively (Figure S4). This finding was confirmed by an EMSA (Figure S5). ChIP‐qPCR analyses with anti‐GFP antibodies also verified the recruitment of OsSHR1 by the promoter regions of OsSWEET2a/4/16 in vivo (Figure 2g–j). We further found that OsSHR1 significantly enhanced the expression of the luciferase (LUC) reporter gene driven by the promoters of OsSWEET2a/4/16 in rice protoplasts (Figure 2k,l). These results suggest that OsSHR1 can directly bind to the promoters of OsSWEET2a/4/16 to activate their expressions.

2.4. OsSHR1 Acts Downstream of OsSPL14 to Regulate Tillering and Root Development in Response to SLs

To test the genetic relationship between OsSHR1 and OsSPL3/12/14, we further generated osspl14 single mutants, OsSHR1‐Ri/osspl14 and OsSHR1‐mD‐His/osspl14 lines using the CRISPR/Cas9 genome‐editing method (Figures 3a and S6a,b). Upon observation, we found that the OsSHR1‐Ri, osspl14 and OsSHR1‐Ri/osspl14 lines all displayed a high tillering phenotype, compared with Nip (Figure S6a,c), which makes it difficult to define the genetic relationship between OsSHR1 and OsSPL14. However, the OsSHR1‐mD‐His/osspl14 lines showed a median tiller number between the OsSHR1‐mD‐His line and osspl14 single mutants, like that of Nip (Figure 3a,b), suggesting that OsSHR1‐mD‐His, a gain‐of‐function mutant of OsSHR1, could largely restore the high tillering phenotype of the osspl14 single mutant to a normal level. To confirm this, we further constructed the Actin1:OsSPL14‐overexpression vector and introduced it into Nip and OsSHR1‐Ri plants to obtain OsSPL14‐OE and OsSHR1‐Ri/OsSPL14‐OE lines (Figure S6d,e). The OsSPL14‐OE plants showed reduced tillers while the OsSHR1‐Ri/OsSPL14‐OE plants showed a high tillering phenotype like OsSHR1‐Ri plants, compared to Nip (Figure S6d–f). Similar genetic relationships were also observed between OsSHR1 and OsSPL3/12 in regulating tillering (Figures S7 and S8). These results indicate that OsSHR1 acts downstream of OsSPL3/12/14 to repress rice tillering.

FIGURE 3.

FIGURE 3

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OsSHR1 acts downstream of OsSPL14 in the SL signalling pathway to regulate rice tillering and root growth. (a) Plant morphology of Nip, OsSHR1‐mD‐His, osspl14 and OsSHR1‐mD‐His/osspl14 plants at the mature stage. Bar = 10 cm. (b) Statistical analyses of tiller number in (a). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (c) Root morphology of 7‐day‐old Nip, OsSHR1‐mD‐His, osspl14 and OsSHR1‐mD‐His/osspl14 plants. Bar = 1 cm. (d, e) Statistical analyses of total root length (d) and crown root number (e) in (c). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (f) Root morphology of 7‐day‐old Nip, OsSHR1‐Ri, OsSPL14‐OE and OsSHR1‐Ri/OsSPL14‐OE plants. Bar = 1 cm. (g, h) Statistical analyses of total root length (g) and crown root number (h) in (f). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (i) Root morphology of 8‐day‐old Nip, osspl14 and OsSPL14‐OE seedlings with or without GR245DS treatment. +, apply GR245DS with 1 μM (dissolved with DMSO); −, equal volume of DMSO. Bar = 1 cm. (j, k) Statistical analyses of total root length (j) and crown root number (k) in (i). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (l) Immunoblot analyses show the levels of OsSHR1 protein in the root of Nip, osspl14 and OsSPL14‐OE plants. ‘α‐HSP82’ antibody was used as the loading control. The numbers above the bands indicate the relative ratio of OsSHR1/HSP82. (m) Relative expression of OsSHR1 in Nip, osspl14 and OsSPL14‐OE roots. Values are means ± SD (**p < 0.01, n = 3, two‐way ANOVA).

Previous studies indicated that both SPL and SHR‐like genes act as key regulators of root development (Shao, Zhou, et al. 2019; Lin et al. 2020; Sun et al. 2021). To test the genetic relationships between OsSHR1 and OsSPL3/12/14 in root development regulation, we did additional analyses. The OsSHR1‐Ri, osspl14 and OsSHR1‐Ri/osspl14 lines all displayed shorter total root length and more crown roots, compared to Nip (Figure S9). However, the OsSHR1‐mD‐His/osspl14 lines displayed longer total root length and fewer crown roots, a root phenotype similar to that of the OsSHR1‐mD‐His line, compared to Nip (Figure 3c–e). The OsSPL14‐OE plants showed significantly longer total root length and fewer crown roots, while the OsSHR1‐Ri/OsSPL14‐OE plants showed shorter total root length and more crown roots like OsSHR1‐Ri plants, compared to Nip (Figure 3f–h). Similar genetic relationships were also observed between OsSHR1 and OsSPL3/12 in regulating root development (Figures S10 and S11). Taken together, these results indicate that OsSHR1 likely acts downstream of OsSPL3/12/14 to regulate root development.

To further test whether OsSPL14 regulates crown root formation in the SL signalling pathway, we analysed the total root length and crown root number under GR245DS treatments in Nip, osspl14 and OsSPL14‐OE plants (Figure 3i–k). Compared with the control group, the total root lengths of Nip after GR245DS treatment were significantly increased, and crown root formation was reduced, while the treatment effect on osspl14 mutants was significantly weakened, and the treatment effect on OsSPL14‐OE lines was not clearly different from that of Nip (Figure 3i–k). After GR245DS treatment, the crown root numbers of OsSPL14‐OE plants were not significantly reduced compared to Nip, possibly because the crown root numbers of untreated OsSPL14‐OE plants were already at a very low level. Notably, the GR245DS treatment effects on OsSHR1‐Ri (Figure 1g–i) and osspl14 plants (Figure 3i–k) were significantly weakened, suggesting that the regulation of SLs on rice root development is dependent on the OsSPL14‐OsSHR1 pathway.

Furthermore, immunoblot analyses showed that OsSPL14‐OE lines accumulated more while osspl14 mutants accumulated fewer OsSHR1 proteins compared to Nip in roots (Figure 3l). RT‐qPCR analyses revealed that the expression level of OsSHR1 was significantly increased in the OsSPL3/12/14‐OE lines and decreased in the osspl3/12/14 single mutants (Figures 3m and S12).

In summary, these findings support the conclusion that OsSHR1 acts downstream of OsSPL3/12/14 within the SL signalling pathway to promote root elongation, while inhibiting the formation of crown roots and tillers.

2.5. OsSHR1 Acts Upstream of OsSWEETs to Regulate Tillering in Response to SLs

To determine the genetic relationship of OsSHR1 and OsSWEETs, a series of ossweet16 mutants, OsSHR1‐Ri/ossweet16 and OsSHR1‐mD‐His/ossweet16 lines were generated using the CRISPR/Cas9 genome‐editing method (Figure 4a–c). Then we observed that both ossweet16 mutants and OsSHR1‐Ri/ossweet16 lines displayed increased tillers, compared to Nip (Figure 4a,c,d). In contrast to the fewer tillers of the OsSHR1‐mD‐His line, OsSHR1‐mD‐His/ossweet16 lines displayed more tillers, like ossweet16 single mutants (Figure 4b,c,e). To confirm this finding, we generated the Actin1:OsSWEET16‐OE lines (Figure 4f,g), and found that they showed reduced tiller number and plant height, a phenotype similar to that of OsSPL14‐OE lines (Figures 4f,h and S6d,f). Together, these results indicate that OsSWEET16 likely acts downstream of OsSHR1 to repress tillering in rice.

FIGURE 4.

FIGURE 4

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OsSHR1 acts upstream of OsSWEET16 in the SL signalling pathway to regulate rice tillering. (a, b) Plant morphology of Nip, OsSHR1‐Ri, ossweet16, OsSHR1‐Ri/ossweet16, OsSHR1‐mD‐His and OsSHR1‐mD‐His/ossweet16 plants at the mature stage. Bar = 10 cm. (c) Generation and sequence analyses of ossweet16, OsSHR1‐Ri/ossweet16 and OsSHR1‐mD‐His/ossweet16 lines produced using a CRISPR/Cas9 genome‐editing approach. The guide RNA targeting sites and PAMs are indicated. A black dash indicates the deletion of one base pair. Red letters represent inserted bases. (d, e) Statistical analyses of tiller number in (a) and (b), respectively. Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (f) Plant morphology of Nip and OsSWEET16‐OE plants at the mature stage. Bar = 10 cm. (g) Relative expression of OsSWEET16 in Nip and OsSWEET16‐OE plants. Values are means ± SD (**p < 0.01, n = 3, two‐way ANOVA). (h) Statistical analyses of tiller number in (f). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (i) Plant morphology of Nip, ossweet16 and OsSWEET16‐OE seedlings with or without GR245DS treatment. Seedlings were treated with 1.0 μM GR245DS (+) or mock (−). Bar = 5 cm. (j) Statistical analyses of tiller number in (i). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA).

To test whether OsSWEET16 functions in the SL signalling pathway, we examined the sensitivity of the ossweet16 mutants and OsSWEET16‐OE lines to SL. The results showed that the high tillering phenotype of ossweet16 mutants could not be rescued by exogenously applied GR245DS, in contrast to the evidently reduced tiller number of Nip by GR245DS (Figure 4i,j), indicating that ossweet16 mutants are insensitive to SL. Taken together, these results support the notion that OsSWEET16 can repress tillering in response to SL in rice.

However, we found that although the OsSWEET2a overexpression lines had reduced tiller numbers compared to Nip, the ossweet2a mutants displayed a similar tiller number to Nip (Figure S13). In addition, the OsSWEET4 overexpression lines also showed reduced tiller numbers compared to Nip; only the ossweet4‐2 mutant of the two mutants exhibited slightly increased tillering compared to Nip (Figure S14). These findings suggest that OsSWEET2a/4 exert limited effects on rice tillering regulation.

2.6. OsSWEET16 Functions Downstream of OsSPL14 ‐ OsSHR1 Pathway in the SL Signalling Pathway to Regulate Root Development

Besides more tillers, the ossweet16 mutants also exhibited shorter total root lengths and an increased number of crown roots compared to Nip (Figure 5a–c), suggesting that OsSWEET16 also regulates root development. The OsSHR1‐mD‐His/ossweet16 lines had significantly shorter total root lengths and more crown roots, a root phenotype like that of ossweet16 mutants (Figure 5d–f). The Actin1:OsSWEET16‐OE lines exhibited fewer crown roots, and two of three lines exhibited slightly longer total root length compared to Nip (Figure 5g–i). Further, the root phenotypes of ossweet2a/4 mutants showed similar results to ossweet16 mutants, which displayed shorter total root lengths and more crown roots, and the Actin1:OsSWEET2a‐OE lines showed longer root lengths and fewer crown roots compared with Nip (Figures S15 and S16). These results indicate that all OsSWEET2a/4/16 participate in regulating rice root elongation and crown root formation.

FIGURE 5.

FIGURE 5

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OsSHR1 acts upstream of OsSWEET16 in the SL signalling pathway to regulate root growth. (a) Root morphology of 7‐day‐old Nip, OsSHR1‐Ri, ossweet16 and OsSHR1‐Ri/ossweet16 plants. Bar = 1 cm. (b, c) Statistical analyses of total root length (b) and crown root number (c) in (a). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (d) Root morphology of 7‐day‐old Nip, OsSHR1‐mD‐His, ossweet16 and OsSHR1‐mD‐His/ossweet16 plants. Bar = 1 cm. (e, f) Statistical analyses of total root length (e) and crown root number (f) in (d). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (g) Root morphology of 7‐day‐old Nip and OsSWEET16‐OE plants. Bar = 1 cm. (h, i) Statistical analyses of total root length (h) and crown root number (i) in (g). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (j) Root morphology of 8‐day‐old Nip, ossweet16 and OsSWEET16‐OE seedlings with (+) or without (−) 1.0 μM GR245DS treatment. Bar = 1 cm. (k, l) Statistical analyses of total root length (k) and crown root number (l) in (j). Values are means ± SD. Different letters indicate significant differences (p < 0.05, n = 15, two‐way ANOVA). (m) Relative expression of OsSWEET16 in Nip, OsSHR1‐Ri and OsSHR1‐mD‐His roots. Values are means ± SD (**p < 0.01, n = 3, two‐way ANOVA). (n, o) Immunoblot analyses show the levels of OsSWEET16 protein in the root of Nip, OsSHR1 and OsSPL14 mutants. ‘α‐HSP82’ antibody was used as the loading control. The numbers above the bands indicate the relative ratio of OsSWEET16/HSP82.

To verify whether OsSWEET16 is also involved in the SL signalling pathway to regulate root development, we further treated ossweet16 mutants and OsSWEET16‐OE lines with GR245DS. The GR245DS treatment effects on root elongation and crown root formation in ossweet16 mutants were significantly diminished, and the treatment's impact on the root elongation of OsSWEET16‐OE lines did not markedly differ from the impact on Nip (Figure 5j–l), suggesting that ossweet16 mutants are insensitive to SLs.

Further, RT‐qPCR analyses showed that the expression levels of OsSWEET16 were increased in the OsSHR1‐mD‐His and OsSPL3/12/14‐OE plants but decreased in the OsSHR1‐Ri and osspl3/12/14 plants (Figures 5m and S17). In line with this, the OsSHR1‐mD‐His and OsSPL14‐OE plants accumulated more, while the OsSHR1‐Ri and osspl14 plants accumulated fewer OsSWEET16 proteins in their seedling roots compared to Nip (Figure 5n,o). Furthermore, the expression levels of OsSWEET2a/4 were also decreased in the OsSHR1‐Ri and osspl14 plants but increased in the OsSHR1‐mD‐His and OsSPL14‐OE plants (Figure S18).

In all, these results confirm that OsSWEET2a/4/16 acts downstream of the OsSPL14‐OsSHR1 pathway in the SL signalling pathway to regulate root development.

2.7. SL Regulates Sugar Allocation to Control Root Growth and Tillering via the OsSPL14‐OsSHR1‐OsSWEET16 Pathway

As shown above, OsSWEET16 acts downstream of the OsSPL14–OsSHR1 pathway of SL signalling (Figures 4 and 5), so we speculated that SLs likely regulate the expression of OsSWEET2a/4/16 to alter sugar distribution to affect tillering and root development in rice. As expected, the d53 mutant accumulated fewer OsSHR1 and OsSWEET16 proteins than its wild type Norin8 (Figure 6a); the transcription levels of OsSPL3/12/14, OsSHR1 and OsSWEET2a/4/16, and the protein levels of OsSHR1 and OsSWEET16 were induced by the GR245DS treatment in Nip roots (Figure 6b,c).

FIGURE 6.

FIGURE 6

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SLs modulate sugar allocation in rice by activating the expression of OsSWEETs. (a) Immunoblot analysis shows the levels of OsSHR1 and OsSWEET16 proteins in the roots of 7‐day‐old Norin8 and d53 plants. ‘α‐HSP82’ antibody was used as the loading control. The numbers above the bands indicate the relative ratio of OsSHR1/HSP82 or OsSWEET16/HSP82. (b) Relative expressions of OsSPL3/12/14, OsSHR1 and OsSWEET2a/4/16 in the roots of 7‐day‐old Nip plants with GR245DS treatment for 6 h. The gene expression levels before treatment with GR245DS were set as ‘1’. Values are means ± SD (**p < 0.01, *p < 0.05, n = 3, two‐way ANOVA). (c) Immunoblot analysis shows the levels of OsSHR1 and OsSWEET16 proteins in the roots of 7‐day‐old Nip plants with or without GR245DS treatment for 8 h. ‘α‐HSP82’ antibody was used as the loading control. The numbers above the bands indicate the relative ratio of OsSHR1/HSP82 or OsSWEET16/HSP82. (d) The transport activity analysis of OsSWEET2a/4/16 in yeast cells. Growth of the yeast mutant strain EBY.VW4000 expressing different genes in SD (‐Ura) media supplemented with different carbon sources (2% maltose, 2% glucose or 2% fructose). Yeast mutant strains transformed with the pDR196 empty vector were used as negative control. (e) Growth of the yeast mutant strain SUSY7/ura3 expressing different genes in SD (‐Ura) media supplemented with 2% glucose or 2% sucrose. Yeast mutant strains transformed with the pDR196 empty vector were used as negative control. (f, g) The concentrations of soluble sugars (fructose, glucose and sucrose) in the tiller buds and roots of 6‐week‐old Nip, OsSHR1‐Ri and ossweet2a/4/16 mutants with or without GR245DS treatment. The samples were collected at the end of the dark. Different letters indicate significant differences (p < 0.05, n = 3, one‐way ANOVA).

To verify the function of OsSWEET2a/4/16 as sugar transporters, we explored their subcellular localization in rice protoplasts and sugar transport activity in yeast cells. Subcellular localization analyses showed that both OsSWEET2a and OsSWEET4 are located at the plasma membrane and tonoplast, while OsSWEET16 is only expressed at the tonoplast (Figure S19). The heterologous expression of OsSWEET2a enabled the growth of EBY.VW4000, a hexose transport‐deficient yeast strain, on the medium supplemented with fructose, while its growth on the medium supplemented with glucose was relatively slow; and the expression of OsSWEET4/16 enabled the growth of EBY.VW4000 on the medium supplemented with glucose and fructose (Figure 6d). In addition, compared with the corresponding cells transformed with the control empty vector, the sucrose uptake‐deficient strain SUSY7/ura3 cells transformed with pDR196 containing OsSWEET4/16 showed faster growth on the medium with sucrose as the sole carbon source, whereas the SUSY7/ura3 yeast cells transformed with OsSWEET2a grew more slowly (Figure 6e). These results indicate that OsSWEET2a/4/16 all have the ability to transport sugars in vivo: OsSWEET2a has a relatively strong ability to transport fructose, while OsSWEET4/16 can transport glucose, fructose and sucrose.

Sucrose is enzymatically hydrolyzed into glucose and fructose for respiration to maintain plant growth at night (Schleucher et al. 1998). To verify whether OsSWEET2a/4/16 has rhythmic expression patterns, we performed the RT‐qPCR and found that OsSWEET2a/4/16 exhibits circadian changes at the mRNA level in tiller buds and the roots of 6‐week‐old Nip seedlings. Under light, the gene expression levels of OsSWEET2a/4/16 gradually decreased, while in the dark, their expression levels showed a gradually increasing trend. Additionally, in the Nip seedlings treated with GR245DS, the expression levels of OsSWEET2a/4/16 in tiller buds and roots all showed an upward trend (Figure S20).

Next, to verify whether OsSWEET2a/4/16 affects sugar distribution in rice root and tiller buds. Given the higher expression levels of OsSWEET2a/4/16 at the end of dark, we measured the soluble sugar levels in Nip, OsSHR1‐Ri and ossweet2a/4/16 mutants with or without GR245DS treatment at the end of dark. Results showed that compared with Nip, the OsSHR1‐Ri accumulated more fructose, glucose and sucrose in tiller buds and more fructose and sucrose in roots; and ossweet2a/4/16 mutants accumulated more fructose, glucose and sucrose in both tiller buds and roots (Figure 6f,g). Further, fructose, glucose and sucrose treatment all promoted the growth of rice tiller buds to varying degrees (Figure S21), and treatments with 0.5% to 2% fructose, glucose and sucrose all significantly inhibited the elongation of rice roots, while treatments with fructose and sucrose promoted the formation of crown roots (Figure S22). These findings indicate that the accumulation of sugars in the tiller buds and roots of OsSHR1‐Ri and ossweet2a/4/16 mutants likely results in their shorter roots and more crown roots and tillers. Notably, only the accumulation of fructose and glucose in tiller buds, as well as the accumulation of fructose and sucrose in roots were repressed by GR245DS treatment in Nip when compared with the control group, and OsSHR1‐Ri and ossweet2a/4/16 mutants did not exhibit significant changes in the sugar contents in tiller buds and roots after GR245DS treatment (Figure 6f,g). Based on the colour changes: the stained colours of amylose and amylopectin shift from a vivid reddish‐brown to a rich blue‐black as starch concentration increases (Figure S23), we found that GR245DS treatment reduced starch accumulation in Nip root tips and the regions at the stem base where crown roots and tiller buds develop, and the OsSHR1‐Ri and ossweet16 plants exhibited significant starch accumulation in these regions, which were unaffected by GR245DS (Figure 7a,b). These results suggest that the regulation of different sugar contents in rice plants by the SL signalling depends on the functions of OsSWEET proteins to varying degrees.

FIGURE 7.

FIGURE 7

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OsSPL14‐OsSHR1‐OsSWEET16 regulatory pathway regulates root development and tillering by modulating sugar allocation in response to SLs. (a) Iodine staining of temporary starch accumulation in the primary root tip of the Nip, OsSHR1‐Ri and ossweet16 7‐day‐old plants with or without GR245DS treatment. Bar = 100 μm. (b) Iodine staining of temporary starch accumulation in the stem base of the Nip, OsSHR1‐Ri and ossweet16 4‐week‐old plants with or without GR245DS treatment. The red arrow indicates the tiller bud, the white arrow indicates the emerging crown root. Bar = 500 μm. (c, d) RNA in situ hybridization of OsSHR1 and OsSWEET16 in crown root primordia and tiller buds. The sense probes were used as negative controls. Bar = 100 μm. (e) A proposed working model for the regulation of rice tillering and root development by the OsSPL14‐OsSHR1‐OsSWEET16 pathway in SL signalling. In the absence of SLs, D53 binds to OsSPL14 and together with TPL/TPR proteins represses the transcriptional activation of OsSPL14 on downstream genes such as OsTB1 and OsSHR1 and its feedback on D53 transcription. Repressed OsSHR1 expression thus inhibits the transcription of OsSWEET16, leading to the fructose and glucose accumulation in tiller buds, fructose and sucrose accumulation in roots, finally resulting in increased tillers and crown roots and reduced root length. In response to SLs, D53 is degraded by the proteasome system, thus releasing OsSPL14 to induce transcription of D53 and OsTB1, thereby inhibiting rice tiller development. Simultaneously, OsSPL14 also activates the expression of OsSHR1; the expressed OsSHR1 further promotes the transcription of OsSWEET16, thus reducing the glucose and fructose accumulation in tiller buds, fructose and sucrose accumulation in roots, finally resulting in reduced tillers and crown roots but an increased root length.

In addition, RNA in situ hybridization revealed that both OsSHR1 and OsSWEET16 were evidently expressed throughout the root meristem zone of crown root primordium, as well as in the tiller buds (Figure 7c,d), which is consistent with their roles in regulating root growth and tillering.

In summary, these results support the notion that the OsSPL14‐OsSHR1‐OsSWEET16 pathway in the SL signalling pathway can alter sugar distribution in vivo to regulate tillering and root development in rice.

3. Discussion

Many studies have shown that SLs promote the elongation of primary and crown roots while suppressing the formation of crown and lateral roots in rice and Arabidopsis (Arite et al. 2011; Kapulnik et al. 2011; Ruyter‐Spira et al. 2011; Rasmussen et al. 2012; Kumar et al. 2015; Yuan et al. 2023). Notably, as a key hormone in response to low soil nutrients, SLs are induced by low soil nutrient conditions such as low phosphorus and low nitrogen conditions, to promote root elongation and inhibit lateral roots to absorb more soil nutrients in deeper soil (Sun et al. 2014; Yuan et al. 2023). Further investigation revealed that SLs suppress the density of lateral roots by downregulating the expression of CROWN ROOTLESS 1 (CRL1), a key gene that facilitates the development of crown roots and lateral roots (Inukai et al. 2005; Yuan et al. 2023). In addition, as the key SL signalling genes, some SPL genes including OsSPL14 can significantly promote root elongation and repress tillering (Jiao et al. 2010; Kerr and Beveridge 2017; Song et al. 2017; Sun et al. 2021). Although IPA1 (OsSPL14) can directly promote the expression of OsTB1 to repress tillering (Lu et al. 2013), whether there is a direct downstream regulatory pathway regulated by these SPL genes to promote root elongation remains unclear.

Here, we report that within the SL signalling pathway, SPLs and D53 directly regulate the expression of OsSHR1 to promote root elongation and inhibit crown root formation and tillering (Figures 1 and 2a–f; Figures S2 and S3). Our previous research revealed that OsSHR1 promotes root development in rice by sustaining the activity and size of the root meristem (Lin et al. 2020). This study further showed that OsSHR1 was also induced by SL (Figure 6a–c), acting as a positive regulator of SL signalling to promote root elongation and inhibit tillering and crown root formation in rice (Figure 1). Notably, the repressive effect of OsSHR1 on tillering is different from the promoting effects of other GRAS genes such as MONOCULM1 (MOC1), DWARF AND LOW‐TILLERING (DLT) and SLENDER RICE1 (SLR1) (Li et al. 2003; Tong et al. 2009, 2012; Liao et al. 2019; Shao, Lu, et al. 2019), indicating that the SHR clade of the GRAS gene family plays a distinct role in regulating rice tillering, differing from other clades, offering novel insights into the functional diversification of the GRAS gene family.

At present, it is known that OsSWEET2a (clade I) and OsSWEET4 (clade II) respectively regulate sheath blight (ShB) resistance and grain filling (Sosso et al. 2015; Gao et al. 2021; Yang et al. 2023), and there is no report on the function of OsSWEET16 yet. Here, we found that three SWEET genes, OsSWEET2a/4/16, are all involved in regulating root elongation, crown root formation and tillering to varying degrees (Figures 4 and 5; Figures S13–S16). Further, we found that SLs modulate the sugar distribution to shape plant architecture by regulating the expression of SWEET genes in rice (Figures 4, 5, 6; Figures S13–S16). Notably, OsSWEET16 belongs to clade IV, along with AtSWEET16 and AtSWEET17 from Arabidopsis thaliana (Chen et al. 2010), and all of them are located at the tonoplast, serving as fructose, glucose and sucrose transporters (Figures 6d,e and S19c) (Chardon et al. 2013; Klemens et al. 2013; Guo et al. 2014; Valifard et al. 2021). AtSWEET17 can promote primary root elongation and reduce the fructose‐mediated inhibition on the primary root elongation in Arabidopsis thaliana (Valifard et al. 2021). Besides, AtSWEET16 regulates germination, growth and stress tolerance in Arabidopsis, and transports out glucose, fructose and sucrose from the leaves at the end of the night (Klemens et al. 2013). Consistent with these findings, the ossweet16 mutants also display shorter roots and more tillers, alongside striking accumulation of fructose and glucose in the tiller buds and roots, while sucrose accumulation was less pronounced (Figures 4, 5 and 6f,g). Therefore, the members of the SWEET family in clade IV may conservatively play an important role in the transport of fructose, glucose and sucrose during plant development. Besides, there may be functional redundancy and/or diversification among OsSWEET2a, OsSWEET4 and OsSWEET16 proteins. Consistent with this hypothesis, we observed that OsSWEET2a/4/16 exhibited different expression patterns, among which OsSWEET2a/16 showed high expression in rice seedlings and seedling roots, while OsSWEET4 displayed high expression in young panicles at the heading stage (Figure S24). Additionally, OsSWEET2a/4/16 showed distinct subcellular localization patterns (Figure S19). Among them, OsSWEET4 and OsSWEET16 seem to play a more crucial role in the sugar transport of tiller buds, and the mutants show a more significant accumulation of fructose and glucose (Figure 6f,g), which may be the reason why the tiller phenotypes of the ossweet4/16 mutants are more pronounced than those of ossweet2a (Figure 4a–e; Figures S13 and S14).

Recent studies have revealed that sugars play key roles in regulating plant growth and development by affecting the SL signalling pathway. Sugars appear to be consistent with the SL pathway in promoting grain development. In ZmCCD8 overexpression lines, enhanced ZmCCD8 transcription promotes SL biosynthesis, upregulates the expression of ZmSWEET10, ZmSWEET13c and ZmLHT14, leading to enhanced accumulation of sugar and amino acids in maize kernels (Zhong et al. 2024). However, in leaves and tillers, sugars negatively regulate plant responses to SL. For instance, glucose reduces SL‐induced leaf senescence in rice and bamboo (Tian et al. 2018; Takahashi et al. 2021), and sucrose alleviates SL‐mediated inhibition of axillary bud outgrowth in rose and pea (Bertheloot et al. 2019). In rice seedlings, sucrose inhibits the degradation of the repressor protein D53 of SL signalling to alleviate the SL‐induced inhibition of tiller bud outgrowth (Patil et al. 2021). Similarly, we found that the mutation of OsSWEET16 also causes sugar accumulation in tiller buds and roots (Figure 6f,g), and renders insensitivity to SLs, thus promoting the formation of tillers and crown roots (Figures 4i,j and 5j–l). Conversely, our study also showed that SL can orchestrate sugar allocation to effectively regulate root growth and tillering, thus discovering a complex crosstalk between sugar and SL.

In all, we discovered an important regulatory pathway comprising OsSPLs, OsSHR1 and OsSWEETs that works directly downstream of OsSPL14 within the SL signalling pathway to regulate root development and tillering (Figure 7e). In the absence of SLs, D53 binds to OsSPL14 and together with TPL/TPR proteins represses the transcriptional activation of OsSPL14 on downstream genes such as OsTB1 and OsSHR1 and its feedback on D53 transcription. Repressed OsSHR1 expression thus inhibits the transcription of OsSWEET16, leading to the fructose and glucose accumulation in tiller buds, fructose and sucrose accumulation in roots, finally resulting in increased tillers and crown roots and reduced root length. In response to SLs, D53 is degraded by the proteasome system, thus releasing OsSPL14 to induce transcription of D53 and OsTB1, thereby inhibiting rice tiller development. Simultaneously, OsSPL14 also activates the expression of OsSHR1; the expressed OsSHR1 further promotes the transcription of OsSWEET16, thus reducing the glucose and fructose accumulation in tiller buds, fructose and sucrose accumulation in roots, finally resulting in reduced tillers and crown roots but an increased root length. Thus, our study enhances the comprehension of the regulatory mechanisms by which SLs oppositely affect rice root elongation and the formation of crown roots and tillers, offering a novel regulatory pathway to balance the rice root system and tiller number for improving rice yield.

4. Experimental Procedures

4.1. Plant Materials and Growth Conditions

The RNA interference (RNAi) transgenic lines of OsSHR1 and transgenic plants expressing the undegradable OsSHR1‐mD‐His protein (driven by its endogenous promoter) in the Nip background used in this study were previously described (Lin et al. 2020).

Rice plants were cultivated in paddy fields during the normal growing seasons in Beijing (40°13′ N, 116°13′ E). Hydroponically cultured rice seedlings were grown in the climate chamber (HP1500GS; Ruihua) at 70% humidity, under short‐day conditions with daily cycles of 10 h of light at 30°C and 14 h of dark at 25°C. Light was provided by white‐light emitting diode tubes (400–700 nm, 250 μmol m−2 s−1).

4.2. Plasmid Construction and Transformation

For knocking out OsSPL3/12/14 and OsSWEET2a/4/16, 19‐bp gene‐specific sequences targeting the exons were inserted into the sgRNA/Cas9 vector (Biogle, BGK01‐G418) to generate the OsSPL3/12/14‐Cas9 and OsSWEET2a/4/16‐Cas9 constructs. Then the constructs were introduced into the calli of Nip, OsSHR1‐Ri and OsSHR1‐mD‐His via Agrobacterium‐mediated transformation. T0 transgenic lines were analysed by sequencing. T2 homozygous transgenic lines of each knockout line were selected and used for phenotypic observation.

For the OsSPL3/12/14 and OsSWEET2a/4/16 overexpression constructs, the full‐length coding sequences were amplified and ligated to the pCAMBIA2300 vector (at the SmaI site) to generate the overexpression constructs. Then the constructs were introduced into Nip or OsSHR1‐Ri calli via Agrobacterium‐mediated transformation. All transgenic lines were analysed with stable T2 to T3 progeny. All primers used in this assay are listed in Table S1.

4.3. RNA Extraction and RT‐qPCR Analysis

Total RNA of different tissues or genotypes was extracted using the ZR Plant RNA MiniPrep Kit (Zymo Research, R2024) following the manufacturer's recommendations. Then mRNAs were reverse transcribed using a Reverse Transcription Kit (Qiagen, 205 311). RT‐qPCR analyses were performed on the CFX96 Real‐Time System (BIO‐RAD) with the SYBR Premix Ex Taq Kit (TaKaRa, RR820). The rice Ubiquitin (UBQ) gene was used as an internal control. Primer pairs used for RT‐qPCR analysis are listed in Table S2.

4.4. Y1H Assay

Full‐length coding regions of OsSPLs and OsSHR1 were cloned into the pB42AD vector; promoter regions of OsSHR1 and OsSWEETs were cloned into the pLacZi reporter vector. Various combinations of plasmids were then co‐transformed into the yeast ( S. cerevisiae ) strain EGY48, with combinations with empty pB42AD used as negative controls. Transformants were grown on SD/−Trp/−Ura agar medium at 30°C until colonies appeared. Colonies were then plated onto X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactopyranoside) agar medium for blue colour development. Primers used for this assay are listed in Table S1.

4.5. Y2H Assay

Full‐length coding regions of D53 and OsSPL3/12/14 were cloned into pGBKT7 and pGADT7, respectively, to form BD‐D53 and AD‐OsSPL3/12/14. Various combinations of plasmids were then co‐transformed into the yeast strain AH109; the combination of BD‐D53 and AD‐OsDLT2 was used as a negative control. Transformants were grown on SD/−Leu/−Trp agar medium at 30°C until colonies appeared. Colonies were then plated onto SD/−Ade/−His/−Leu/−Trp dropout screen medium to test protein interactions. Primers used for this assay are listed in Table S1.

4.6. Expression and Purification of Fusion Proteins

The full‐length CDS of OsSPL3/12/14 and OsSHR1 was cloned into the pGEX4T‐1 vector, generating a fusion with the GST protein. The constructs of OsSPL3/12/14‐GST, OsSHR1‐GST and empty GST vectors were transformed into Escherichia coli BL21 (DE3) to induce protein expression. GST and GST labelled proteins were eluted with 50 mM glutathione. Primers used for this assay are listed in Table S1.

4.7. Electrophoretic Mobility Shift Assay

The EMSA assay was performed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, 20 148), according to the manufacturer's instructions. Oligonucleotide probes were synthesised and labelled with biotin by Thermo Fisher Scientific. The probe sequences are listed in Table S3.

4.8. LUC Activity Determination

Promoter regions of OsSHR1 and OsSWEET2a/4/16 were cloned into the vector pGreenII0800‐LUC to generate the ProOsSHR1:LUC and ProOsSWEET2a/4/16:LUC reporters. Full‐length coding regions of D53 and OsSHR1 were cloned into the pAN580‐GFP vector to generate the Pro35S:D53‐GFP and Pro35S:OsSHR1‐GFP effectors. Full‐length coding regions of OsSPL3/12/14 were cloned into the vector pCAMBIA1300‐Flag to generate the Pro35S:OsSPL3/12/14‐Flag effectors. The combined reporter and effector plasmids were then co‐transformed into rice protoplasts; combinations with empty vectors were used as negative controls. The LUC gene from Renilla reniformis (Ren) under the control of the CaMV35S promoter was used as an internal control. The LUC activities were determined by the Dual‐luciferase Assay Kit (Promega, E1910) following the manufacturer's recommendations, and the relative LUC activity was calculated as the ratio of LUC/REN. Primers used for this assay are listed in Table S1.

4.9. ChIP‐qPCR Assay

Rice protoplasts co‐transfected with Pro35S:OsSPL3/12/14‐Flag and ProOsSHR1:LUC vectors were used to test the enrichment of OsSPL3/12/14 on the promoter regions of OsSHR1. The Flag antibodies were used for detection (Figure 2a–d). Rice protoplasts co‐transfected with Pro35S:OsSHR1‐GFP and ProOsSWEET2a/4/16:LUC vectors were used to test the enrichment of OsSHR1 on the promoter regions of OsSWEET2a/4/16. The GFP antibodies were used for detection (Figure 2g–j). No addition of antibodies (No Ab) served as negative controls. Detailed procedures for ChIP assays were performed according to the previously reported method (Duan et al. 2019). Primers used for this assay are listed in Tables S1 and S2.

4.10. Luciferase Complementation Imaging Assay

Full‐length coding regions of D53 and OsSPL3/12/14 were cloned into the pCAMBIA1300‐nLUC and pCAMBIA1300‐cLUC vectors, respectively. These vectors were introduced into A. tumefaciens strain EHA105, and various combinations of EHA105 strains were co‐infiltrated into N. benthamiana leaves. The combination of pCAMBIA1300‐D53‐nLUC and pCAMBIA1300‐OsDLT2‐cLUC was used as a negative control. After 36 to 48 h, leaves were treated with 1 mM luciferin (E1601, Promega) for 5 min, and luciferase activities were measured using an imaging apparatus (NightShade LB 985, Berthold). Primers used for this assay are listed in Table S1.

4.11. Bimolecular Fluorescence Complementation Assays

Full‐length coding regions of OsSPL3/12/14 were ligated into the C‐terminal fragment of yellow fluorescent protein (YFP) in p2YC vector to generate the YC‐OsSPL3/12/14 constructs, and the full‐length coding region of D53 was ligated into the N‐terminal fragment of YFP in p2YN vector to generate the YN‐D53 construct. These constructs were introduced into A. tumefaciens strain EHA105, and various combinations of EHA105 strains were co‐infiltrated into N. benthamiana leaves. The combination of YN‐D53 and YC‐OsDLT2 was used as a negative control. After 36 to 48 h, leaves were harvested for fluorescence signal capture using a laser scanning confocal microscope (ZEISS LSM 980). Primers used for this assay are listed in Table S1.

4.12. Antibody Preparation and Immunoblot Analyses

The antibody against OsSHR1 was previously described (Lin et al. 2020). For the detection of OsSWEET16, a SUMO‐fused protein‐specific polypeptide (amino acids 216–328 of OsSWEET16 protein) was expressed in E.coli Rosetta cells, and then affinity purified. Subsequently, the recombination protein was injected into rabbits to produce polyclonal antibodies against OsSWEET16 at ABclonal Technology. Fractionation of proteins and the immunoblotting assay were performed as previously described (Duan et al. 2023), and immunoblotted with anti‐OsSHR1 antibody (1:2000), anti‐OsSWEET16 antibody (1:2000) and anti‐HSP82 antibody (AbM51099‐31‐PU, Beijing Protein Innovation, 1:5000).

4.13. Co‐Immunoprecipitation Assay

Full‐length coding regions of D53 and OsSPL3/12/14 were ligated into the pCAMBIA1305‐GFP and pCAMBIA1300‐Flag vectors to produce the Pro35S:D53‐GFP and Pro35S:OsSPL3/12/14‐Flag constructs, respectively. The constructs were introduced into A. tumefaciens strain EHA105, and then co‐infiltrated into N. benthamiana leaves. After 36 h treatment, total protein was extracted from infiltrated N. benthamiana leaves. Immunoblots were analysed with anti‐Flag antibody (M185‐7, MBL, 1:5000) and anti‐GFP antibody (11 814 460 001, Roche, 1:5000). Primers used for this assay are listed in Table S1.

4.14. GR24 and Sugar Treatment

Germinated seeds were sown on floating nets and grown in hydroponic culture with GR245DS (1 μM) or sugar (0.5%, 1% and 2% w/v). Then the root lengths and crown root numbers were measured after treatment. Samples were harvested at indicated time points. RNA isolation, cDNA synthesis, RT‐qPCR, protein extraction and immunoblotting were performed as described above.

4.15. Subcellular Localization Analysis

Subcellular localization of OsSWEET2a/4/16 was detected using the cDNA (without stop codons) sequence of OsSWEET2a/4/16 fused in‐frame into the pAN580‐GFP vector driven by the 35S promoter. Rice protoplasts were isolated from Nip stem base. A total of 10 μg plasmids (pOsSWEET2a/4/16‐GFP and OsSCAMP1‐mCherry or OsVIT1‐mCherry) were transformed into rice protoplasts by the polyethylene‐glycol‐mediated method. After incubation in the dark for 12 ~ 15 h at 28°C, a confocal laser scanning microscope (STELLARIS 5, Leica; Germany) was used to observe the fluorescence signals in transformed protoplasts. The OsSCAMP1‐mCherry and OsVIT1‐mCherry were selected as the plasma membrane and tonoplast localization markers, respectively.

4.16. Complementation Assays for OsSWEET2a/4/16 in Yeast

For complementation assays in yeast cells, the ORFs of the three OsSWEETs with XhoI were cloned into the yeast expression vector pDR196. Subsequently, the resulting constructs were transformed into the hexose transport‐deficient yeast strain EBY.VW4000 and the sucrose uptake‐deficient yeast strain SUSY7/ura3. Transformants of the hexose transport‐deficient strain were grown on liquid SD/−uracil media supplemented with 2% maltose (glucose was used for the sucrose uptake‐deficient strain). Serial dilutions of yeast cell suspensions of EBY.VW4000 were added dropwise onto solid SD/uracil media consisting of either 2% maltose or 2% glucose/fructose. Similarly, serial dilutions of yeast cell suspensions of SUSY7/ura3 were added dropwise onto solid SD/−uracil media consisting of 2% glucose or 2% sucrose. Growth was documented via imaging after 3 to 4 days of growth at 30°C.

4.17. Glucose, Fructose and Sucrose Measurements

At the end of the dark, samples were collected from the rice seedlings of Nip, OsSHR1‐Ri and ossweet2a/4/16 mutants with or without GR245DS treatment. All samples were frozen and ground into powder, and then 0.1 g of the fresh sample was weighed and placed into a 2 mL tube. Subsequently, 1 mL of 50% ethanol was added to each tube, and the mixture was sonicated for 30 min. The samples were centrifuged at 11 500 g for 5 min, and the supernatant was aspirated. The soluble sugar content was determined by using high performance liquid chromatography (UHPLC3600, Wayeal, China). The standard curve was prepared with glucose, fructose and sucrose standards.

4.18. Starch Iodine Staining

The stem bases were soaked in 100% (v/v) ethanol to remove chlorophyll, then rinsed in water to remove excess ethanol (root tips do not need this treatment). This was followed by staining in Lugol solution (Sigma) for 10 min as previously reported (Seung et al. 2015). The samples were then destained in water for several hours for optimal visualisation.

4.19. RNA In Situ Hybridization

RNA in situ hybridization was performed as previously described (Lin et al. 2012). Shoot bases of rice seedlings at the third and fourth leaf stages were fixed in cold, freshly prepared FAA overnight at 4°C, then dehydrated, clarified and embedded in paraffin (Paraplast Plus; Sigma‐Aldrich) in sequence. Thin sections of paraffin‐embedded shoot bases of 8 to 12 μm thickness were generated for the hybridization. Specific encoding fragments of OsSHR1 and OsSWEET2a/4/16 were selected for synthesis of digoxigenin (DIG)‐labelled RNA sense and antisense probes in vitro by DIG RNA Labeling Kit (Roche). The hybridization reaction was conducted for 16 h at 50°C. Anti‐DIG alkaline phosphatase‐conjugated antibody and the NBT/BCIP staining method were used to visualise the tissue‐specific localization of gene expression in shoot base sections.

4.20. Statistical Analysis

The data were collected by Microsoft Excel. All collected data were analysed using GraphPad Prism 8. Two‐tailed Student's t‐test, one‐way ANOVA or two‐way ANOVA tests were performed to test the statistical significance. p value < 0.05 was considered statistically significant. Graphs were plotted by GraphPad Prism 8 and edited by Adobe Illustrator.

4.21. Accession Numbers

Genome sequence data from this study can be found in the EMBL/GenBank data libraries under the following accession numbers: OsSHR1, Os07g0586900; D53, Os11g0104300; OsSPL3, Os02g0139400; OsSPL12, Os06g0703500; OsSPL14, Os08g0509600; OsSWEET2a, Os01g0541800; OsSWEET4, Os02g0301100; OsSWEET16, Os03g0341300.

Author Contributions

Jianmin Wan and Qibing Lin supervised the project. Miao Feng and Qibing Lin designed the research and wrote the paper. Miao Feng, Wenfan Luo, Sheng Luo, Rong Miao performed the experiments. Manpo Gu, Shuai Li, Xinxin Xing, Jinhui Zhang, Jinsheng Qian, Xin Liu, Chunlei Zhou, Qi Sun, Tong Luo, Nuo Chen, Yulong Ren, Zhijun Cheng, Cailin Lei, Zhichao Zhao, Shanshan Zhu, Xin Wang, Xiuping Guo provided technical assistance. All authors read and approved the final article.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: The expression pattern of OsSHR1 in seedlings and heading stage plants.

Figure S2: OsSPL3/12/14 directly binds to the promoter of OsSHR1.

Figure S3: OsSPL3/12/14 interacts with D53 in vivo and in vitro.

Figure S4: OsSHR1 directly binds to the promoters of OsSWEET2a/4/16 in yeast.

Figure S5: OsSHR1 directly binds to the promoters of OsSWEET2a/4/16 in EMSA.

Figure S6: OsSHR1 acts downstream of OsSPL14 to regulate tillering.

Figure S7: OsSHR1 acts downstream of OsSPL3 to regulate tillering.

Figure S8: OsSHR1 acts downstream of OsSPL12 to regulate tillering.

Figure S9: Root morphology of OsSHR1‐‐Ri, osspl14 and OsSHR1‐Ri/osspl14 plants.

Figure S10: OsSHR1 acts downstream of OsSPL3 to regulate root development.

Figure S11: OsSHR1 acts downstream of OsSPL12 to regulate root development.

Figure S12: Reduced expression levels of OsSHR1 in OsSPL3/12 mutants.

Figure S13: The relationship between OsSHR1 and OsSWEET2a in regulating tillering.

Figure S14: The relation between OsSWEET4 and OsSHR1in regulating tillering.

Figure S15: OsSHR1 acts upstream of OsSWEET2a to regulate root development.

Figure S16: The relation between OsSWEET4 and OsSHR1 in regulating root development.

Figure S17: The expression of OsSWEET16 in OsSPL3/12/14 mutants.

Figure S18: The expression of OsSWEET2a/4 in OsSHR1 and OsSPL14 mutants.

Figure S19: Subcellular localization of OsSWEET2a/4/16‐GFP fusion proteins in rice protoplasts.

Figure S20: Diurnal rhythmic expression of OsSWEET16/2a/4 in tiller buds and roots under GR245DS treatment.

Figure S21: Sugar treatments promote the growth of tiller buds.

Figure S22: Sugar treatments regulate the root development of rice.

Figure S23: Starch reacts with iodine to yield distinct colours that vary with its concentration.

Figure S24: Expression pattern of OsSWEET2a/4/16 in rice.

Table S1: Primers used in this study for vector constructions.

Table S2: Primers used for RT‐qPCR and ChIP‐qPCR analyses.

Table S3: Oligonucleotides used for EMSA assays.

PBI-24-678-s001.docx (39.8MB, docx)

Acknowledgements

This research was supported by grants from the National Key Research and Development Program of China (2022YFD1200104) and the Innovation Program of Chinese Academy of Agricultural Sciences (CAAS‐CSCB‐202401), the Central Public‐interest Scientific Institution Basal Research Fund (No. Y2024YJ13) and the National Natural Science Foundation of China (31671769).

Funding: This work was supported by National Key Research and Development Program of China, 2022YFD1200104; Chinese Academy of Agricultural Sciences, CAAS‐CSCB‐202401; National Natural Science Foundation of China, 31671769.

Contributor Information

Qibing Lin, Email: linqibing@caas.cn.

Jianmin Wan, Email: wanjianmin@caas.cn.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: The expression pattern of OsSHR1 in seedlings and heading stage plants.

Figure S2: OsSPL3/12/14 directly binds to the promoter of OsSHR1.

Figure S3: OsSPL3/12/14 interacts with D53 in vivo and in vitro.

Figure S4: OsSHR1 directly binds to the promoters of OsSWEET2a/4/16 in yeast.

Figure S5: OsSHR1 directly binds to the promoters of OsSWEET2a/4/16 in EMSA.

Figure S6: OsSHR1 acts downstream of OsSPL14 to regulate tillering.

Figure S7: OsSHR1 acts downstream of OsSPL3 to regulate tillering.

Figure S8: OsSHR1 acts downstream of OsSPL12 to regulate tillering.

Figure S9: Root morphology of OsSHR1‐‐Ri, osspl14 and OsSHR1‐Ri/osspl14 plants.

Figure S10: OsSHR1 acts downstream of OsSPL3 to regulate root development.

Figure S11: OsSHR1 acts downstream of OsSPL12 to regulate root development.

Figure S12: Reduced expression levels of OsSHR1 in OsSPL3/12 mutants.

Figure S13: The relationship between OsSHR1 and OsSWEET2a in regulating tillering.

Figure S14: The relation between OsSWEET4 and OsSHR1in regulating tillering.

Figure S15: OsSHR1 acts upstream of OsSWEET2a to regulate root development.

Figure S16: The relation between OsSWEET4 and OsSHR1 in regulating root development.

Figure S17: The expression of OsSWEET16 in OsSPL3/12/14 mutants.

Figure S18: The expression of OsSWEET2a/4 in OsSHR1 and OsSPL14 mutants.

Figure S19: Subcellular localization of OsSWEET2a/4/16‐GFP fusion proteins in rice protoplasts.

Figure S20: Diurnal rhythmic expression of OsSWEET16/2a/4 in tiller buds and roots under GR245DS treatment.

Figure S21: Sugar treatments promote the growth of tiller buds.

Figure S22: Sugar treatments regulate the root development of rice.

Figure S23: Starch reacts with iodine to yield distinct colours that vary with its concentration.

Figure S24: Expression pattern of OsSWEET2a/4/16 in rice.

Table S1: Primers used in this study for vector constructions.

Table S2: Primers used for RT‐qPCR and ChIP‐qPCR analyses.

Table S3: Oligonucleotides used for EMSA assays.

PBI-24-678-s001.docx (39.8MB, docx)

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Articles from Plant Biotechnology Journal are provided here courtesy of Society for Experimental Biology (SEB) and the Association of Applied Biologists (AAB) and John Wiley and Sons, Ltd

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