OsERF2 controls rice root growth and hormone responses through tuning expression of key genes involved in hormone signaling and sucrose metabolism
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Root determines plant distribution, development progresses, stress response, as well as crop qualities and yields, which is under the tight control of genetic programs and environmental stimuli. Ethylene responsive factor proteins (ERFs) play important roles in plant growth and development. Here, the regulatory function of OsERF2 involved in root growth was investigated using the gain-function mutant of OsERF2 (nsf2857) and the artificial microRNA-mediated silenced lines of OsERF2 (Ami-OsERF2). nsf2857 showed short primary roots compared with the wild type (WT), while the primary roots of Ami-OsERF2 lines were longer than those of WT. Consistent with this phenotype, several auxin/cytokinin responsive genes involved in root growth were downregulated in nsf2857, but upregulated in Ami-OsERF2. Then, we found that nsf2857 seedlings exhibited decreased ABA accumulation and sensitivity to ABA and reduced ethylene-mediated root inhibition, while those were the opposite in Ami-ERF2 plants. Moreover, several key genes involved in ABA synthesis were downregulated in nsf2857, but unregulated in Ami-ERF2 lines. In addition, OsERF2 affected the accumulation of sucrose and UDPG by mediating expression of key genes involved in sucrose metabolism. These results indicate that OsERF2 is required for the control of root architecture and ABA- and ethylene-response by tuning expression of series genes involved in sugar metabolism and hormone signaling pathways.
KeywordsOsERF2 Rice Root growth ABA Ethylene Sugar
Root systems are central to the acquisition of water and nutrients, plant anchorage, seedling vigor, and responses to various stresses, which have pivotal effects on crop distribution, maximal productivity and yield stability, particularly in arid regions. Root architecture is under the tight control of genetic programs and environmental stimuli. It is well known that all phytohormones synergistically or antagonistically control root growth and development (Cuesta et al. 2013; Jung and McCouch 2013). In Arabidopsis, numerous crucial genes and intricate signaling pathways involved in root development have been well clarified, but many plant species also show their unique molecular and cellular regulatory mechanisms governing root development (Coudert et al. 2010).
Rice (Oryza sativa) is one of the most common crops worldwide. A series of mutants, genes, and genetic mechanisms governing root development have been characterized in rice, some of which are controlled by auxin and cytokinin (Mai et al. 2014). For examples, OsAUX1 controls auxin-mediated lateral root initiation through mediating polar auxin transport in rice (Yu et al. 2015). Mutation of OsXXT1 causes abnormal root hair development with the reduction of XyG content and the tensile strength of the cell wall (Wang et al. 2014). Increasing auxin-related mutants including crl1 (crownless root1), arl1 (adventitious rootless1), crl4/osgnom1, and oscand1, exhibit abnormal root formation and growth due to the disorders of auxin biosynthesis, polar transport or signaling transduction in rice (Inukai et al. 2005; Liu et al. 2005, 2009; Wang et al. 2011). Moreover,auxin–cytokinin crosstalk plays curial roles in the control of root growth and development in rice. For examples, auxin-induced CRL5 promotes crown root initiation through repressing cytokinin signaling (Kitomi et al. 2011). OsCKX4 and EL5 are essential for crown and lateral root development by mediating crosstalk between auxin and cytokinin pathways (Gao et al. 2014; Koiwai et al. 2007; Mochizuki et al. 2014). In addition, OsMPK3/6 and OsMKK4/5 are involved in the regulation of root architecture through tuning the interplay of auxin and cytokinin (Singh et al. 2015).
Sugar signals are central in determining plant growth and development by interacting with other signalling pathways (Lastdrager et al. 2014). In Arabidopsis, sucrose affects root architecture through regulating endogenous flavonols accumulation, which can suppress the effect of ABA on root growth (Nguyen et al. 2013). In rice, ABA mediates grain-filling rate by controlling the activities of key enzymes involved in sucrose-to-starch conversion in spikelet (Wang et al. 2015; Zhu et al. 2011). Moreover, several genes involved in sucrose synthesis and metabolism or transport including OsSPS1, OsSUT1, OsSUT2, OsCYT-INV1, and OscFBP1 have been proved to be required for the control of rice growth and development (Eom et al. 2011; Hirose et al. 2010, 2014; Jia et al. 2008; Lee et al. 2008). For examples, mutation of OsSUT2 affected sucrose and other sugars transport and caused growth retardation including tiller number, plant height, and root dry weight (Eom et al. 2011). Disruption of OsCYT-INV1 impaired normal sucrose metabolism and root growth in rice (Jia et al. 2008).
Recent researches have proved that ethylene and its interaction with ABA play important roles in the control of rice growth and development. For examples, MHZ4/ABA4- and MHZ5-mediated ABA signaling is required for ethylene-induced inhibition of root growth (Ma et al. 2014; Yin et al. 2015). Moreover, mhz6/Oseil1 and mhz7/ein2 exhibit longer root, while overexpression of MHZ6 and MHZ7 represses rice root elongation (Ma et al. 2013; Yang et al. 2015). Importantly, the inducible expression of OsERF2 (Os06g08340) by ethylene in roots was impaired in these mutants (Ma et al. 2013, 2014; Yang et al. 2015; Yin et al. 2015). In this paper, we found that OsERF2 was required for the regulation of primary root growth in rice. The gain-of-function mutation of OsERF2 (nsf2857) shows short root, while artificial microRNA-OsERF2 (Ami-OsERF2) lines exhibit longer root, this phenotype is consentient with that of OsEIL1. At the transcript level, OsERF2 negatively regulates expression of candidate genes including ARF1, MKK4/5, MPK3/6 and PIN1b/2/9, which are involved in root growth and development through mediating auxin and cytokinin signaling. Furthermore, OsERF2 affects expression of key genes involved in ABA synthesis, which results in the enhanced accumulation of ABA in Ami-OsERF2 lines associated with root growth hypersensitivity to ABA, while those are opposite in nsf2857. Moreover, OsERF2 influence accumulation of sucrose and UDPG through mediating expression of genes related to sucrose metabolism, including INV (Os01g0332100) and OsCYT-INV1, OsSPS2, OsSUS3 and OsSUS6. Particularly, OsCYT-INV1 is closely related to the accumulation of sucrose and root growth in rice (Jia et al. 2008). Thus, our results indicate that OsERF2 is required for the control of root architecture and ABA response by regulating expression of pivotal genes involved in root development, ABA synthesis and sucrose metabolism.
OsERF2 negative regulates primary root growth
OsERF2 regulates expression of key genes related to root architecture
OsERF2 affects ABA accumulation and root growth response to ABA and ethylene
OsERF2 transcriptionally regulates accumulation of sucrose and UDPG
Sucrose play central roles in the regulation of plant root development by generating a range of signal molecules and interacting with various hormones (Ljung et al. 2015). Here, the contents of sucrose and UDPG (a key precursor for sucrose metabolism) were compared between nsf2857, Ami-OsERF2 and WT plants. Compared with corresponding WT, the sucrose of nsf2857 increased to 112 %, while that of Ami-OsERF2 lines decreased to about 84.7 % (Fig. 5a). In contrast to sucrose, the UDPG of nsf2857 (22.3 μg/mg) was 88 % of ZH11 (25.1 μg/mg), while that of Ami-OsERF2 (about 26.2 μg/mg) was higher than that of NiP (24.2 μg/mg) (Fig. 5b), suggesting that OsERF2 is involved in regulation of sucrose metabolism. Then, we checked transcripts of several key genes involved in sucrose metabolism including INV (Os01g0332100) and OsCYT-INV1 (encoding neutral invertase), OsSPS2 (encoding sucrose phosphate synthase), OsSUS3 and OsSUS6 (encoding sucrose synthase) between nsf2857, Ami-OsERF2 and WT plants. As shown in Fig. 5c, d, the expression of candidate genes was upregulated in Ami-OsERF2 plants, but downregulated in nsf2857. Particularly, OsCYT-INV1 has been reported to be closely related to the accumulation of sucrose and root growth in rice (Jia et al. 2008). Thus, these results suggest that OsERF2 transcriptionally regulates the accumulation of sucrose and UDPG, which partially contribute to its regulatory roles in rice root growth.
The root system is essential for plants to absorb nutrients and water from soils, which determines plant development progress, response to drought and salinity, as well as crop qualities and yield. Rice is very susceptible to water deficit for its shallow root architecture, but the control of root system can increase its yield under drought conditions (Uga et al. 2013). Recently, many genes and mutants involved in root growth and development have been characterized in rice. Some genes are involved in auxin and cytokinin biosynthesis, transport and homeostasis, and their signal transduction (Azizi et al. 2015; Balzan et al. 2014; Coudert et al. 2010; Mai et al. 2014). For example, the mutation of CRL5, encoding an ERF transcription factor, impairs crown root initiation through repression of cytokinin signaling (Kitomi et al. 2011).
ERF family factors play multiple roles in the control of plant metabolism, growth and development, and stress response (Licausi et al. 2013). In rice, many ERFs have been reported to be involved in the regulation of response to various stresses by mediating multiple signaling pathways and expression of stress-related genes (Kazan 2015). Importantly, increasing ERF members are involved in the control of development progress in rice. For examples, OsEATB influences plant height, panicle length and branching by regulating gibberellin biosynthesis (Qi et al. 2011). MFS1, SNB and OsIDS1 control inflorescence architecture and floral meristem establishment through tuning expression of spikelet meristem genes (Lee and An 2012; Ren et al. 2013). Moreover, auxin-induced CRL5 promotes crown root initiation via repressing cytokinin signaling (Kitomi et al. 2011). In this study, our findings reveal that OsERF2 negatively regulates expression of pivotal genes related to root growth. Among of these candidate genes, OsMPK3/6, OsMKK4/5 and OsPIN 1b/9 are reported to be involved in root growth by tuning auxin and cytokinin signaling (Singh et al. 2015).
ABA affects root growth and development in rice very different from how it does in Arabidopsis, and endogenous ABA positively regulates early root growth and is required for the maintenance of root elongation in rice (Chen et al. 2006; Ma et al. 2014). Here, our results suggest that several genes involved in ABA synthesis, including MZH4, OsPDS, OsZDS, OsCRTISO and β-OsLCY, are downregulated in nsf2857 seedlings, but upregulated in Ami-OsERF2 seedlings. These transcription changes are consistent with the increased ABA accumulation in Ami-OsERF2, and the decreased ABA level in nsf2857. Importantly, it has been proved that MHZ4 is responsible for ABA biosynthesis and involved in the control of ethylene response, whose mutation and overexpression causes reduced and enhanced ethylene-inhibited root growth, respectively (Ma et al. 2014). In this paper, we found that Ami-OsERF2 lines exhibited longer primary roots and enhanced sensitivity to ABA- and ethylene-treatment, while these phenotypes were the opposite in nsf2857. Moreover, our results showed that upregulation of OsERF2 caused the decreased expression of MHZ4 and ABA accumulation in nsf2857, which displayed similar phenotypes with mhz4 including the shorter roots and reduced ABA- and ethylene-responses. However, Ami-OsERF2 plants exhibited similar phenotypes with transgenic rice overexpressing MHZ4. Taken together, we speculate that OsERF2 is required for the interplay between ethylene and ABA in control of rice root growth, which might be partially related with its transcriptional control of MHZ4 expression and ABA accumulation.
As the main assimilated carbon of photosynthesis, sucrose play central roles in plant growth and development, as well as stress response by generating series of sugars as metabolites or as signaling molecules interacting the cross talks of metabolic, hormonal, and stress signals (Ljung et al. 2015; Ruan 2014). In Arabidopsis, sucrose inhibits hypocotyl elongation and promotes root growth by tuning auxin signal and cotyledon-derived long-distance signal (Kircher and Schopfer 2012; Stokes et al. 2013). In rice, mutation of OsSPS1, OsSUT1, OsSUT2, and OscFBP1 causes growth retardation and abnormal pollen development by affecting sucrose synthesis or transporter (Eom et al. 2011; Hirose et al. 2010, 2014; Lee et al. 2008). Similarly, UDP-glucose (UDPG) is a key metabolite for the synthesis of sucrose, polysaccharides, glycoproteins, glycolipids and myriads of glycosylated secondary metabolites (Decker et al. 2012). UGPase plays important roles in carbohydrate metabolism through catalyzing the reversible conversion of glucose-1-phosphate to UDPG, which is essential for plant growth, pollen development, and male sterility in rice and Arabidopsis (Chen et al. 2007; Park et al. 2010; Woo et al. 2008). In rice, expression levels of genes involved in sucrose metabolism are correlated with sucrose accumulation (Maruyama et al. 2014). In this paper, we found that upregulation of OsERF2 resulted in the decreased sucrose and the increased UDPG in nsf2857 plants, while it was the opposite in Ami-OsERF2 plants. Consistent with these physiological changes, expression of key genes closely related to sucrose metabolism including INV and OsCYT-INV1, OsSPS2, OsSUS3, and OsSUS6, were downregulated in nsf2857, but upregulated in Ami-OsERF2 lines. Particularly, mutation of OsCYT-INV1 has been proved to cause the enhanced sucrose level and the reduced root growth in rice (Jia et al. 2008). Consistent with this result, we found that OsCYT-INV1 was downregulated in nsf2857, which exhibited short primary root and increased accumulation of sucrose. Thus, these findings suggest that OsERF2 transcriptionally controls the accumulation of sucrose and UDPG, which might further trigger the complex alteration of carbohydrate metabolism and multiple signaling pathways, and partially account for the effect of OsERF2 on rice root growth.
Materials and methods
Rice materials and treatments
In this study, rice (O. sativa L. subsp. japonica cv Zhonghua 11 (ZH11) and O. sativa L. subsp. japonica cv nipponbare (Nip) were used as the wild-type (WT) for nsf2857 and amiERF2, respectively. For root growth and ABA response, more 40 geminated seeds from all samples were planted in 1/2 Murashige and Skoog (MS) medium solution supplemented with 0, 0.5 and 1 μM ABA. After growth for 7 days, root lengths were measured and photographed. For measurement of ABA, sucrose and UDPG, geminated seeds from all samples were planted in 1/2 Murashige and Skoog (MS) medium solution for 2 weeks. All data are presented as means of three times. For ethylene treatment, rice seedlings were grown on a stainless steel sieve placed in an air-tight plastic box with 10 ppm ethylene and in air as control. The seedlings were incubated at 28 °C in the dark for 3 days.
Artificial microRNA (amiRNA-OsERF2) construct was generated using specific primer sets (Table S1) designed through programs available on the (http://wmd3.weigelworld.org/), and cloned into the binary vectors pCAMBIA5300. amiRNA-OsERF2 plants were generated by Agrobacterium-mediated transformation and examined by qRT-PCR with specific primers (Table S1). The seeds of oseil1 mutant and OsEIL1-OX were obtained from Dr. Jin-song Zhang.
Measurements of ABA, sucrose and UDPG
ABA extraction was according to Zhu et al. (2010) measured using the ELISA kit (CSB-E09159Pl, China Agricultural University, China). 0.2 g leaves of 2-week-old seedlings were homogenized and extracted overnight at 4 °C in 80 % methanol by vortex spinning. After centrifuged and concentrated to approximately 50 μl, these extracts were suspended with 1 ml TBS buffer (25 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM MgCl2, and 3 mM NaN3) and used for ELISA according to the manufacturer’s instructions. ABA concentrations were calculated as ng/g fresh weight. Each measurement was replicated three times.
Sucrose and UDPG content was measured by HPLC according to Bahaji et al. (2015). 0.1 g 2-week-old seedlings were ground in liquid nitrogen. For measurement of sucrose, the powder was suspended in 1 ml of 90 % ethanol, left at 70 °C for 90 min and centrifuged at 13,000×g for 10 min. For measurement of UDPG, the powder was suspended in 1 ml of 1 M HClO4, left at 4 °C for 2 h and centrifuged at 13,000×g for 10 min. The supernatant was neutralized with K2CO3 and centrifuged at 13,000×g for 10 min. Sucrose was determined by HPLC with UPLC WATERS Acquity system by gradient separation with BEH Amide 2.1 × 100 mm 1.7 μm. UDPG was measured by HPLC on a system obtained from Waters Associates fitted with ZORBAX Carbohydrate 4.6 × 250 mm 5 μm. Standard sucrose and UDPG were purchased from Sigma.
Gene expression analysis
After geminated seeds were planted in 1/2 MS medium solution for 7 days, total RNA of roots or leaves was isolated from all samples using TRIzol reagent (Invitrogen, Beijing, China) and used to synthesize first-strand cDNA with All-in-One™ First-Strand cDNA Synthesis Kit (Transgen, China). RT-qPCR was performed using gene-specific primers (Table S1) with TransStart Green qPCR SuperMix (Transgen, China). OsActin1 was used as the internal control. All experiments were repeated three times, and the average was calculated.
We are grateful to Dr. Jin-Song Zhang from Institute of Genetics and Developmental Biology (Chinese Academy of Sciences) for his kind sharing oseil1 mutant and OsEIL1-OX seeds. This work was supported by the Major Special Foundation of Transgenic Plants in China (2013ZX08001003 and 2014ZX08009-15B), and by National Natural Science Foundation of China (Grant No. 31172025).
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