- Open Access
Ubiquitin-Conjugating Enzyme OsUBC11 Affects the Development of Roots via Auxin Pathway
Rice volume 16, Article number: 9 (2023)
Rice has 48 ubiquitin-conjugating enzymes, and the functions of most of these enzymes have not been elucidated. In the present study, a T-DNA insertional mutant named R164, which exhibited a significant decrease in the length of primary and lateral roots, was used as the experimental material to explore the potential function of OsUBC11. Analysis using the SEFA-PCR method showed that the T-DNA insertion was present in the promoter region of OsUBC11 gene, which encodes ubiquitin-conjugating enzyme (E2), and activates its expression. Biochemical experiments showed that OsUBC11 is a lysine-48-linked ubiquitin chain-forming conjugase. OsUBC11 overexpression lines showed the same root phenotypes. These results demonstrated that OsUBC11 was involved in root development. Further analyses showed that the IAA content of R164 mutant and OE3 line were significantly lower compared with wild-type Zhonghua11. Application of exogenous NAA restored the length of lateral and primary roots in R164 and OsUBC11 overexpression lines. Expression of the auxin synthesis regulating gene OsYUCCA4/6/7/9, the auxin transport gene OsAUX1, auxin/indole-3-acetic acid (Aux/IAA) family gene OsIAA31, auxin response factor OsARF16 and root regulator key genes, including OsWOX11, OsCRL1, OsCRL5 was significantly down-regulated in OsUBC11 overexpressing plants. Collectively, these results indicate that OsUBC11 modulates auxin signaling, ultimately affecting root development at the rice seedling stage.
Root development is a vital process in plant growth. The functions of root include uptake of nutrients and water from soil, fixing in the soil to prevent logging, and serving as a major receptor interface between plants and various biotic and abiotic factors in the soil environment (Liu et al. 2005, 2009; Zhao et al. 2009; Kitomi et al. 2011). Therefore, exploring the molecules that regulate mechanisms associated with root development is essential in improving agricultural production and food security. The root system comprises primary root (PR), formed during embryo development, and crown roots (CR) which are formed after embryo development stage. Crown roots develop on non-root tissue such as the stems and tillering nodes (Bellini et al. 2014; Inukai et al. 2005), whereas lateral roots (LR) are formed on the surface of these roots. Development of these roots is modulated by various factors, such as phytohormones including auxin, ethylene, cytokinin, abscisic acid, and environmental stimuli (such as water and nutrients) (Gao et al. 2014; Liu et al. 2016a, b; Van et al. 2015; Huang et al. 2022). Although several phytohormones regulate root development, auxin is the main phytohormone that modulates development of roots (Overvoorde et al. 2010).
Auxin is involved in several plant growth and development processes, such as root and stem growth and development, vascular tissue formation, organ senescence and response to stress conditions (such as extreme temperatures, drought and flood, salt and alkali conditions). Root growth is modulated by the auxin pathway mainly through auxin biosynthesis, auxin transport and auxin signal transduction (Zhao et al. 2010). Overexpression of the auxin synthesis gene OsYUCCA1 increases density of lateral root and the length of primary root (Yuko et al. 2007). Mutants of the auxin influx transport pivotal factor OsAUX1/OsAUX3 and efflux carriers of the PIN family exhibit dysregulated root growth and density of lateral roots (Wang et al. 2009; Zhao et al. 2015a, b). T-DNA insertional mutant of the auxin respond factor OsARF16 have longer root compared with the wild type. In addition to auxin, other auxin-related genes affect root development. For example, OsWOX11 is a key gene involved in crown and root formation, which forms WOX11-ADA2-GCN5 complex and changes the chromatin status of target genes (Zhao et al. 2015a, b). Expression of downstream genes, implicated in auxin transport, cell composition and energy metabolism, is up-regulated in oswox11 mutant. The WOX11 gene regulates the development of roots through the interaction between cytokinin and auxin (Zhao et al. 2015a, b). Ostdd1 gene encodes anthranilate synthase β-subunit, and plants with a mutation in this gene showed severe root development defects, and overexpression of OsYUCCA1 reversed the defects (Sazuka et al. 2009). AtUBC13A/B encodes ubiquitin conjugation enzyme and the double-mutant affects root development by modulating auxin signaling (Wen et al. 2014).
The ubiquitination pathway is mainly involved in hormone signal transduction, flower development, photomorphogenesis and other biological processes (Moon et al. 2004; Sadanandom et al. 2012). The ubiquitin pathway mainly comprises ubiquitin-protein, E1 ubiquitin activator, E2 ubiquitin conjunction enzyme (abbreviated as E2 in this paper), E3 ubiquitin ligase and 26S protein degradation system. Approximately 6 E1s, 48 E2s, and 1300 E3s have been reported in rice (Bae and Kim 2014). The ubiquitin pathway involves tagging and degradation of substrate proteins through three enzymatic cascades, ultimately modulating transcriptional and post-translational activities in plants. The C-terminal glycine of the Ub molecule forms a bond with the ε-amino lysine residue of target proteins resulting in Ub enzymatic cascades (Wen et al. 2014). The lysine residue is linked through mono-ubiquitination or by forming a poly-ubiquitination chain. This poly-ubiquitination chain is formed through interaction with the lysine residues in Ub. Currently, K48-linked and K63-linked are the widely explored poly-ubiquitination types (Trempe et al. 2011). K48-linked poly-ubiquitination is a typical ubiquitination pathway involved in hormone signaling, pollen tube growth, pathogen defense, and the cell cycle (Smalle and Vierstra 2004). Studies have reported that K48-linked poly-ubiquitination is mainly associated with degradation-associated protein (Andrew et al. 2009). The SCFTIR1/AFB2 pathway is involved in degradation of Aux/IAA proteins, whereby auxin binds to TIR1/AFB2 to activate the SCFTIR1/AFB2 E3 complex resulting in ubiquitination of Aux/IAA proteins and their degradation through the 26S degradation pathway. Auxin response factor (ARF) is released, which enhances the auxin signaling (Maraschin et al. 2009). Previous findings indicate that K-63-linked poly-ubiquitination mainly plays several roles, such as restoration of DNA damage, kinase activation, protein synthesis, and regulation of root development (Jacobsen et al. 2009). Previous findings indicate that E2 contains a 150 aa conserved UBC domain, which is ubiquitous in eukaryotic cells. Studies report that 47 OsUBCc genes, with the exception of OsUBC38, have the conserved UBC domain (Liu et al. 2016a, b). UBC domain binds to the Ring domain of E3 to form a ubiquitin complex system, which mediates the downstream signaling. Studies have shown that E2 plays a vital role in plant stress resistance as well as plant growth and development (Wen et al. 2014). In Arabidopsis, AtUBC13 is implicated in response to iron deficiency stress (Li et al. 2010). The OsUBC6/8/9/22/25/43/45 gene family in rice modulates ABA stress (Zhiguo et al. 2015). OsUBC1/2 is implicated in drought resistance (Joungsu et al. 2019), whereas EL5 and OsUBC5b play roles in response to stress by inducing the ubiquitin/proteasome system (Hanae et al. 2007). These findings indicate a relationship between E3 and auxin levels and function. However, the correlation between OsUBCc and hormones has not been explored. Therefore, studies should be conducted to explore the mechanism of regulation of downstream auxin signaling by OsUBCc, and whether other pathways are implicated in regulating rice development.
A root development deficiency mutant was established and R164 was obtained from T-DNA insertional mutant bank to explore the association between E2 and auxin. Molecular and genetic analyses revealed that the mutant phenotype was associated with upregulation of a putative gene that encodes a ubiquitin conjunction enzyme, OsUBC11. In this study, a detailed analysis of OsUBC11 was conducted and its role in modulating rice root development was explored.
Phenotypes of R164 Mutant
A root development deficiency mutant denoted as R164 was obtained from the T-DNA insertional mutant bank. The results showed that the primary and lateral roots had a lower development rate than the wild-type Zhonghua11 and the shoot length was affected at the seedling stage (Fig. 1a–f). Characterization of R164 mutant showed that the length of primary roots reduced by about 20% compared with WT plant (Fig. 1e) and the length of lateral root found was only 10% compared with WT by microscopic observation (Fig. 1c, d, f). Further analysis showed that the primary root of a 3-day-old WT presented a lateral root primordium, which was absent in the R164 mutant (Fig. 1g, h). In addition, R164 mutant exhibited several different phenotypic variations throughout the reproductive life, such as differences in plant height during the heading date stage compared with the WT (Additional file 1: Fig. S1c). The uppermost internode of the R164 mutant was shorter than that of the wild-type plant (Additional file 1: Fig. S1a, b). R164 mutant also showed shriveled pollen and increased number of aborted pollen grains (Additional file 1: Fig. S1d–f), as well as reduced seed setting rate and panicle length (Additional file 1: Fig. S1g–i).
The Relative Expression of OsUBC11 was Activated in R164 Mutant
Self-formed adaptor PCR (SEFA-PCR) method was performed to confirm the T-DNA insertional site (Gao et al. 2014). The right border of T-DNA exhibited four 35S enhanced elements, which can activate the genes in the insertional site. Analysis showed that this T-DNA insertional site was present in Chr1 36023995 site (National Center for Biotechnology Information). Two genes were observed on the left and right border of this locus. The two significantly may affected genes were LOC_01g62230 and LOC_Os01g62244 on the right border and LOC_Os01g62260 and LOC_Os01g62290 on the left border (Fig. 2a). Expression of LOC_Os01g62244 was significantly up-regulated, whereas semi-quantitative PCR analysis showed that the expression of the other three genes was not significantly different compared with WT (OsUBI5 treated with reference gene) (Fig. 2b). LOC_Os01g62244 gene showed 200% higher expression level compared with WT (OsUBI5 treated with reference gene) (Fig. 2c). Analysis of rice database (https://rapdb.dna.affrc.go.jp/) showed that LOC_Os01g62244 encodes ubiquitin conjugation enzyme (E2) named OsUBC11, which has a conserved UBCc domain (Fig. 2d). A total of 48 genes encode E2 in rice and are classified into 15 groups. The OsUBC11 gene is classified in the V group (Bae and Kim 2014). Arabidopsis has three homologous genes (AtUBC7, AtUBC13 and AtUBC14) (Fig. 2e). These genes may participate in response to stress conditions by modulating the plant endoplasmic reticulum (ER)- associated degradation (ERAD) pathway (Feng et al. 2020).
The Phenotypes of OsUBC11 Overexpression Lines Were Similar to R164
The coding region of OsUBC11 was cloned into the binary vector pCAMBIA2300-actin1-ocs to generate an OsUBC11 overexpression vector in which OsUBC11 was driven by the actin1 promoter, then transformed into Zhonghua11, to verify that overexpression of OsUBC11 was responsible for the R164 phenotypes. Five independent T0 transgenic plants were identified in this study, with higher expression levels of lines 3 and 5 relative to the WT (Additional file 1: Fig. S2e). These two lines were selected for subsequent experiments (denoted as OE3 and OE5 in the subsequent sections). The OsUBC11 overexpression plants and R164 mutant showed a slower rate of root development (Fig. 3a, c, g, h) and significantly shorter primary and lateral root length compared with the WT. The lateral root primordium was absent in OE3 at 3 DAG and in the R164 mutant (Fig. 3d, e). Auxin is associated with root development; thus, auxin concentration in R164, OE3 and WT plants was determined. The results showed that the auxin concentrations of R164 and OE3 were significantly lower than WT (Fig. 3i). Treatment with exogenous auxin increased the length of primary roots in the overexpression lines or the R164 mutant (Fig. 3b, h). Lateral root elongation in OsUBC11 overexpression lines and R164 mutant was also recovered after treatment with 10 nmol NAA (Fig. 3c, g). These results indicate that the phenotype of root was associated with a decrease in auxin concentration. The rate of inhibition of the primary root of WT after treatment with NAA was significantly higher than that of OsUBC11 overexpression lines (Fig. 3f). This finding implies that OsUBC11 overexpression decreased the sensitivity of roots to auxin. OsUBC11 overexpression plants exhibited similar aerial phenotypes as the R164 mutant, such as plant height, the length of the uppermost internode, shriveled pollen and the number of aborted pollen grains (Additional file 1: Fig. S2a–d, f, g). The results further indicate that OsUBC11 modulated root, plant height and pollen development. The osubc11 mutant was constructed by CRISPR technology. The T1 transgenic plants were harvested and sequencing was conducted. Three mutants, named osubc11-1, osubc11-4 and osubc11-9, were identified (Additional file 1: Fig. S3a). The results did not show significant phenotype differences between osubc11 mutants at the seedling stage and the wild-type (Additional file 1: Fig. S3b, c). We speculated that OsUBC11 could have homologous gene, these genes had redundancy. The next step we would screened out the homologous gene and constructed the double mutant to observed the phenotype.
The Expression of Auxin-Related Genes Were Altered in OsUBC11 Overexpressing Lines
The expression profiles of genes implicated in auxin biosynthesis and degradation were explored to verify the association between OsUBC11 and auxin levels. The expressions of auxin synthesis genes, OsYUCCA4, OsYUCCA6, OsYUCCA7 and OsYUCCA9 were down-regulated in OsUBC11 overexpressing lines (Fig. 4a-d), but up-regulated in osubc11 mutant lines (osubc11-1 was used as the experimental material). The expression levels of OsYUCCA1, OsYUCCA2 and auxin degradation genes OsGH3.2 and OsGH3.11 were not significantly different compared with wild type (Additional file 1: Fig. S4a–d). The results indicated that overexpression of OsUBC11 inhibited auxin synthesis by partly reducing transcription levels of auxin synthesis genes, which was consistent with the decreased auxin concentrations observed in R164 and OE3 lines. Analysis was also conducted to determine the expression levels of the auxin transport genes, OsPIN1, OsAUX1 and OsAUX3 (Fig. 4e and Additional file 1: Fig. S4e–f). The findings showed that the expression of the auxin influx carrier encoded gene, OsAUX1 was down-regulated in OsUBC11 overexpressing lines compared with the WT. Expression of OsAUX1 in osubc11 mutant was up-regulated, indicating that OsUBC11 repressed auxin transport. The levels of transcriptional repressors OsIAA1 (Song et al. 2009), OsIAA11 (Zhu et al. 2012), OsIAA23 (Ni et al. 2011), OsIAA31 (Zhang et al. 2019), and auxin response factor OsARF12 (Qi et al. 2012) and OsARF16 (Shen et al. 2013) were also evaluated (Additional file 1: Fig. S4g–l and Fig. 4f–g), to determine the effect of overexpression of OsUBC11 gene on the development of crown roots and lateral roots. The results indicated that expression of OsIAA31 and OsARF16 was down-regulated in OsUBC11 overexpression lines relative to the WT (Fig. 4f, g). These findings indicate that OsUBC11 modulated auxin biosynthesis, auxin distribution and auxin signaling.
Further, the expression levels of crown root development marker genes in rice roots, including, OsCRL1 (Inukai et al. 2005; Liu et al. 2005), OsCRL5 (Kitomi et al. 2011), and OsWOX11 (Zhao et al. 2015a, b) (Fig. 4h–j) were determined. Knockout or knockdown of these genes resulted in a shorter root phenotype consistent with the phenotypes of R164 and OsUBC11 overexpression lines.
OsUBC11 is a Lysine-48-Linked Ubiquitin Chain-Forming Conjugase
OsUBC11-pET32b recombinant vector was constructed to explore the ubiquitin activity of OsUBC11. The conserved cysteine residue at position 92 of OsUBC11 protein sequence was mutated to alanine (UBC11-Mu) by point mutation technique. The recombinant proteins OsUBC11 and OsUBC11-Mu were expressed in large quantities through prokaryotic expression, tested and purified (Fig. 5a, b). Analysis of the ubiquitin cascade showed that OsUBC11 lost ubiquitination activity after mutation of cysteine at position 92 to alanine, indicating that OsUBC11 binds to the ubiquitination chain through the conserved cysteine residue (Fig. 5c, d). Subsequently, K48 and K63 residues of the ubiquitin chain were mutated to explore the mechanism of formation of polyubiquitin chain of OsUBC11. The results showed that the ubiquitin polymerization chain could not be formed after mutation of K48 residue (Fig. 5e–f), implying that OsUBC11 formed a polyubiquitination system through K48. Previous studies report that K48 ubiquitination is associated with protein degradation (Smalle and Vierstra 2004). These findings imply that OsUBC11 marks related substrate proteins through ubiquitination and degrades them through the ubiquitination pathway to regulate gene expression.
Expression Pattern of OsUBC11 and its Subcellular Localization
Total RNA was extracted from roots, stems, leaves of 1-week-old seedlings, sheath, glumes and rice anthers at the reproductive stage to explore the expression pattern of OsUBC11. The results showed that OsUBC11 was mainly expressed in root but was also expressed at high levels in anthers (Fig. 6a). A 2 kb fragment upstream of the translation start site of OsUBC11 was fused to the GUS reporter gene and transgenic plants were generated to evaluate the expression pattern of OsUBC11 gene. Histochemical staining was conducted on samples obtained from T1 transgenic plants. Histochemical staining results of samples from 1-week-old transgenic lines and the heading stage tissues showed deep staining of root and internode, indicating consistent findings with the qRT-PCR results (Fig. 6c–i). These results may provide some understanding of the root development phenotype of R164 mutant. In addition, OsUBC11 expression response to auxin was determined through treatment of plants with 1 µmol NAA treatment. qRT-PCR analysis revealed that the expression level of OsUBC11 was significantly down-regulated within 0.75 h compared with the expression at 0 h (Fig. 6b). GUS staining of root samples showed auxin treatment reduced the degree of root staining (Fig. 6c, d), further indicating that the expression of OsUBC11 was suppressed by auxin treatment.
GFP-OsUBC11 recombinant plasmid was constructed and transformed to the protoplast of rice to evaluate OsUBC11 subcellular localization. A vector expressing GFP alone was used as control. Microscopic analysis showed that OsUBC11 gene was only expressed in cell nucleus (Additional file 1: Fig. S5), indicating that OsUBC11 functions in the nucleus.
OsUBC11 Regulates Root Development and Plant Growth
Plant roots obtain water and nutrients from the soil. These resources are transported through the vascular system to other plant tissues and are essential for plant growth (Petricka et al. 2012; Smith et al. 2012). Several genes have been recently reported to regulate the growth of roots, most of which exhibit functional redundancy. The functions of these genes can be explored using root development mutants developed through the T-DNA insertion method (Gao et al. 2014). In the present study, a root development mutant, R164, was evaluated by SEFA-PCR method. The mutated gene encodes an ubiquitin conjunction enzyme (https://rapdb.dna.affrc.go.jp/) implicated in the ubiquitin cascade. Overexpression of OsUBC11 inhibited development of primary roots and lateral roots. This finding implied that OsUBC11 overexpression was the main cause of slow root development. Previous studies report that slow rate of root development is significantly correlated with auxin levels (Overvoorde et al. 2010). YUC (YUCCA encodes a flavin monooxygenase) and TAA/TAR (Tryptophan Aminotransferase of Arabidopsis) are gene families involved in the indole pyruvate pathway (IPA) associated with auxin synthesis (Zhao et al. 2012). Mutation of indole acetonitrile pathway associated genes, including AtTGG4 and AtTGG5, causes defects in establishing an auxin gradient in the root tips resulting in suppressed root growth (Fu et al. 2016). The AtUBC13A/B mutation in the E2 gene family of Arabidopsis thaliana affects the auxin signaling factor, and down-regulates the expression of YUC family genes and TAA family genes, ultimately resulting in short roots (Wen et al. 2014). In this study, OsUBC11 overexpression lines exhibited down-regulated expression of auxin synthesis regulator gene OsYUCCA4/6/7/9, although there is a degree of functional redundancy in these genes (Yuko et al. 2007). Triple- or quadruple- mutants exhibit root dysplasia (Stepanova et al. 2008; Cheng et al. 2006). The levels of auxin were significantly lower in the mutant compared with the WT, indicating that the function of OsUBC11 is similar to that of the Atubc13a/b gene. This implies that OsUBC11 down-regulates expression of YUC4, YUC6, YUC7 and YUC9 genes, which affects the synthesis of the auxin hormone, reduces the auxin content, and inhibits PR growth and LR development.
Auxin plays a central role in regulating root growth through modulation of auxin biosynthesis, transport, and signaling (Wang et al. 2009). Auxin transport and auxin signal transduction affect the distribution mode of auxins such as asymmetric distribution of auxins and intercellular transport (Zhao et al. 2015a, b; Qi et al. 2012). These effects ultimately determine the rate of cell division, cell differentiation, and cell expansion in plants (Friml 2021). The activities modulate auxin signaling intensities, thus affecting root development by altering auxin concentration in roots. Auxin influx carriers of the AUXIN1/LIKE AUX1 (AUX1/LAX) family and efflux carriers of the PIN-FORMED (PIN) family mediate polar auxin transport in plants and regulate root growth (Liu et al. 2016a, b). The AUX1/LAX family influx carriers in Arabidopsis comprises 4 genes, which encode four highly conserved transmembrane proteins denoted as AtAUX1, AtLAX1, AtLAX2 and AtLAX3 (Marchant et al. 2002; Swarup et al. 2004; Yang et al. 2006; Péret et al. 2012). Previous studies report that AtAUX1 and AtLAX3 genes modulate lateral root development (Marchant et al. 2002; Swarup et al. 2004). The homolog of AtAUX1 gene, OsAUX1 has a similar function, and its mutants are associated with reduced auxin transport. The homolog and mutants dysregulate the expression of cell cycle genes implicated in mobilizing auxin to the target root tissue to enhance elongation of roots (Huang et al. 2022), ultimately affecting development of lateral roots (Zhao et al. 2015a, b). OsAUX3 functions as a negative regulator resulting in a decrease in rice Al-tolerance in roots (Wang et al. 2018). Inhibition of OsPIN1 expression increases the tiller number and decreases crown root number. Overexpression of OsPIN1 causes significant changes in the root-shoot ratio, implying that OsPIN1 plays an important role in plant type establishment and development of adventitious roots in rice (Xu et al. 2005). OsPIN2 overexpression changes the number of adventitious roots and lateral root density in rice (Chen et al. 2012). In this current study, expression of OsAUX1 was repressed in OE3 and OE5 lines, whereas the expression levels of OsPIN1 and OsAUX3 were not significantly different compared with the WT, indicating that OsUBC11 may be the upstream gene of OsAUX1.
The key components of the auxin signaling pathway include an E3 ligase complex, SCFTIR1, downstream proteins comprising two gene families, auxin response factor (ARF) and auxin/indole acetic acid (AUX/IAAs) (Guilfoyle and Hagen 2007). Auxin signal through the TIR receptor promotes degradation of AUX/indole acetic acid (IAA) proteins, which induces ARF7/19 to regulate the expression of a series of downstream genes implicated in LR development (Pérez-Torres et al. 2008). For instance, SOR1-OsIAA26 complex acts downstream of OsTIR1/AFB2-OsIAA9 cascade to regulate the root growth of rice seedlings (Hui et al. 2018). OsIAA1 overexpression enhances root development (Song et al. 2009). Osiaa11 mutant inhibits development of lateral roots (Zhu et al. 2012). OsARF12 and OsARF25 and auxin response factors and their knockout decrease primary root length in rice (Qi et al. 2012). The phenotype of OsARF16 is similar to OsARF12 phenotype and OsPIN1b, OsPIN4 and OsPIN9 are the downstream genes, which regulate auxin distribution, ultimately affecting root growth (Shen et al. 2013). The findings of the present study showed that overexpression of OsUBC11 gene down-regulated expression of the auxin signal repressor gene OsIAA31 (Zhang et al. 2019) and the auxin response factor OsARF16 (Shen et al. 2013). OsAUX1 and OsARF16 genes may play a synergistic role in regulating the development of root hair (Jia et al. 2022). OsIAA31 is associated with disease resistance and is mainly expressed in roots with a similar expression pattern as OsUBC11 gene (Ayako et al. 2006; Zhang et al. 2019). A recent study showed that E2 modulates stress resistance and acts in response to other hormones, such as the hormone encoded by AtUBC27, thus positively regulating ABA signaling and response to drought (Pan et al. 2020). Most E2 are affected by at least two hormones (Zhiguo et al. 2015). The homologous of OsUBC11 in Arabidopsis are AtUBC7, AtUBC13 and AtUBC14, representing single, double, or triple mutants, which do not exhibit the phenotype presented by OsUBC11 overexpressed lines. Notably, plants with the three mutants or triple mutant of AtUBC7, AtUBC13 and AtUBC14 respond to stress conditions and ABA in mutation dependent manner. These genes may be part of the plant endoplasmic reticulum (ER)- associated degradation (ERAD) pathway. ERAD removes misfolded proteins to maintain protein homeostasis (Feng et al. 2020). These findings imply that OsUBC11 may respond to other hormones and is implicated in ERAD pathway through the K48-linked pathway, thus, it degrades associated proteins, which ultimately affects plant development. Further studies should be conducted to explore whether other hormones affect the expression profile of OsUBC11 and the response of OsUBC11 expression to abiotic stress.
In addition to root development, R164 mutant and OsUBC11 overexpression lines were associated with aerial growth phenotypes, such as shortened top internode, shrinkage of anthers, and increased number of abortive pollen (Additional file 1: Figs. S1 and S2). GUS staining results were consistent with real-time PCR results (Fig. 6). Expression pattern analysis showed that OsUBC11 gene was mainly expressed in roots (Fig. 6a). These results indicate that OsUBC11 plays a critical role in rice development, especially in regulation of root development.
OsUBC11 Down-Regulates Expression of Genes Involved in Root Development
Our previous findings showed that OsUBC11 overexpression affects root development, thus further analysis was conducted to explore whether OsUBC11 could modulate expression of genes implicated in root growth. The results showed that expression of these genes was down-regulated in the plants overexpressing OsUBC11 gene. OsWOX11 gene is important for root development (Zhao et al. 2009). Loss-of-function mutation or down-regulation of OsWOX11 gene results in reduced number and decreased growth rate of crown roots. In this study, the expression level of OsWOX11 was lower in OsUBC11 overexpression lines relative to the WT (Fig. 4j). WOX11-ADA2-GCN5 is a ternary complex in rice crown root formation. WOX11 plays a key role in crown root formation by modulating the cytokinin pathway (Zhao et al. 2015a, b). WOX11 interacts with ADA2 to recruit acetylase GCN5 by binding to specific downstream target genes. GCN5 acetylates histones at target gene loci and ultimately promotes expression of downstream genes implicated in auxin transport, cell composition and energy metabolism by changing the chromatin status of target genes (Zhou et al. 2017). Notably, down-regulation of OsWOX11 expression was associated with low expression levels of OsIAA31 and OsIAA11 genes. OsIAA11 gene regulates root development (Zhu et al. 2012; Jing et al. 2015). Overexpression of OsWOX11 up-regulates expression of OsIAA31 and OsIAA11 genes, implying that OsUBC11 inhibits the expression of OsWOX11, which further inhibits the expression of OsIAA31 and OsIAA11 genes. Previous findings demonstrated that the root development phenotypes of plants overexpressing OsYUC are dependent on the presence of WOX11, indicating that WOX11 is a potential downstream gene for OsYUC genes (Zhang et al. 2018).
The expression level of OsCRL1 and OsCRL5 in this study was lower in OsUBC11 overexpression lines compared with the WT. OsCRL1 is downstream gene of AUX/IAA and ARF-mediated pathway and is regulated by OsARF16 (Inukai et al. 2005; Liu et al. 2005). OsCRL5 is a member of auxin signaling cascade and promotes root development, thus the mutant did not exhibit initiation of crown root primordia. OsARF1 binds to the promoter of OsCRL5 and further modulates the cytokinin signaling pathway through type-A response (ARRS) regulators (Kitomi et al. 2011). These results indicate that overexpression of OsUBC11 inhibits these genes and further inhibits the growth of roots at the seedling stage.
OsUBC11 overexpression reduces the concentration of auxin and inhibits root development by modulating expression of genes involved in auxin synthesis and transport. The present findings show that administration of exogenous auxin can down-regulate expression of OsUBC11 gene (Fig. 7).
Materials and Methods
Plant material and Growth Conditions
The R164 T-DNA insertional mutants and the wild type Zhonghua11 were stored in our laboratory. Seeds of R164 and WT were surfaced-sterilized and sowed in 1/2 strength MS medium, the 7-day-old seedlings were washed off the remnant medium, observing and statistics the length of shoot and root. For phenotype complement experiment, various seeds of OsUBC11 were immersed in water at 37 °C for 24 h, then transformed hydroponic box, was floating on Kimura nutrient solution in a growth chamber with a 14 h light:10 h dark photoperiod at 28 °C for 7 days. Auxin treatment was NAA supplemented in distilled water, distilled water was treated as control. The observation of root and flower structure are in stereomicroscope (Olympus SZX16).
To observe the phenotype of reproductive stage, the overexpression line of OsUBC11, the CRISPER mutant of OsUBC11 and wild type Zhonghua11 were grown in artificial shading outdoors in Harbin, 10 h light/14 h darkness.
Identification of R164 T-DNA Insertion Mutant
The insert site was confirmed by Self-Formed Adaptor PCR method (Wang et al. 2007). T-DNA right border three primers were listed in Additional file 2: Table S1, Genomic DNA was extracted from leaves of R164 mutant using the CTAB method. By agarose gel electrophoresis found about 5 kb band, sequenced blast by NCBI (www.ncbi.nlm.nih.gov). The expression levels of OsUBC11 in R164 were determined by semiquantitative PCR and Real time PCR. Semiquantitative PCR prime and real-time PCR prime were amplified as a control treatment, UBI5 (Os01g0328400) was chosen as internal control. All primes in this chapter were listed in Additional file 2: Table S1.
Vector Constructions and Phenotype Observation
Vector construction was by using ClonExpress® technology (Vazyme Biotech Co., Ltd), or Gateway® technology (Thermo Fisher Scientific-CN).
To constructed the OsUBC11 overexpression lines, a 510-bp fragment of OsUBC11 CDS region was amplified from the cDNA. The restriction sites (Xma I and Pst I) were incorporated into the primers to facilitate cloned into the pCAMBIA2300-actin1 to form overexpression recombinant vector.
To constructed the osubc11 crisper mutant, we designed the crisper target spot primer, primer listed in Additional file 2: Table S1, the target sequences was ligated in sgRNA, and subsequently inserted into CRISPR/Cas9 vector pYLCRISPR/Cas9Pubi‐H (Ma et al. 2015).
To constructed the OsUBC11-GUS recombinant vector, a 2000-bp fragment of upstream OsUBC11 encoding region was amplified from genomic DNA of Zhonghua11, recombined with intermediate vector pQB-V3, then with the technology of LR reaction, take fragment recombined into pHGWFS7.0 plasmid.
Above recombined plasmid were transferred to Agrobacterium tumefaciens strain EHA105 and then transformed into wild-type Zhonghua11 as described previously (Karimi 2015). All of the primers used to generate the above-mentioned constructs are listed in Additional file 2: Table S1.
To constructed the OsUBC11-GFP recombined vector, a 510-bp fragment of OsUBC11 CDS region was amplified, the method of the constructed was the same as OsUBC11-GUS. Then transform into protoplast of rice for further observation (The vector is pH7WGF2.0).
The extracted of Protein and Ubiquitin Cascade Experiment
To confirm the ubiquitin activity of OsUBC11, the OsUBC11-His and site mutant protein-His all constructed by pET-32b vector, the proteins were purified by His-Bind resign (EMD Milillipore Corp, USA), the reagent and ubiquitin cascade experiment and K48/63 polyubiquitin confirm experiment is supported by YOUBI biology. Separated by 10% SDS‐PAGE, transferred to Immobilon®-P transfer membrane (Merck Millipore, IPVH00010), and detected by immunoblot analysis using anti-His (Abmart) and ubiquitin antibody Mab (Beytone), respectively.
Measurement of Auxin and Sensitivity Analysis to Auxin
IAA concentration from roots of 7-day-old seedlings in WT and R164 plants was measured by High-performance liquid chromatography method, each sample was set three repetition.
The equation of sensitivity analysis to auxin is (the length of primary root in 0 nM NAA-the length of primary root in 10 nM NAA)/the length of primary root in 10 nM NAA. Experiments were repeated at least three times.
GUS staining of OsUBC11-GUS lines were harvested at different growth stages was performed using phosphate buffer with 2 mM X-Gluc, vacuum pump to drain, then immersed in these buffers for 48 h. After staining, the samples were observed under an Olympus SZX16 stereo microscope and photographed with a digital camera (Cannon 720S).
Subcellular Localization of OsUBC11
To examine the subcellular localization of OsUBC11, the coding sequence of OsUBC11 was fused in frame to of pH7WGF2.0. The OsUBC11-GFP fusion construct and empty GFP vector control were transfected into protoplast of rice were observed with a confocal laser scanning microscope (Leica TCSSP5).
Assay of Real-Time PCR
Entire root of WT and other OsUBC11 transgenic lines at 7-day-old were used for isolation total RNA (Takara 9109), reverse transcriptase was chosen ReverTra Ace qPCR RT Kit (TOYOBO, FSQ-301), Real-Time PCR system was conducted on LightCycler® 96 System (Roche) using TransStart® Top Green qPCR SuperMix(TransGene,AQ131). The amplification of UBI5 (Os01g0328400) was chosen as an internal control, and the relative expression of genes was calculated via the 2−ΔΔCT method. Other alternative genes primer are listed in Additional file 2: Table S1. Experiments were repeated at least three times.
Data were analysed by GraphPad Prism 8, every single piece of data are shown as a black spot in the figure.
The sequence data of this article can be found in the Rice Genome Annotation Project Database and Resource (http://rice.plantbiology.msu.edu) under the following accession numbers:OsUBC11(LOC_Os01g62244), UBI5(Os01g0328400), OsGH3.2(LOC_Os01g55940), OsGH3.11(LOC_Os07g47490), OsYUCCA1(LOC_Os01g45760), OsYUCCA2(LOC_Os05g45240), OsYUCCA4(LOC_Os01g12490), OsYUCCA6(LOC_Os07g25540), OsYUCCA7(LOC_Os04g03980), OsYUCCA9(LOC_Os01g16714), OsAUX1(LOC_Os01g63770), OsAUX3(LOC_Os05g37470), OsPIN1(LOC_Os04g02830), OsIAA1(LOC_Os01g08320), OsIAA11(LOC_Os03g43400), OsIAA19(LOC_Os05g48590), OsIAA23(LOC_Os06g39590), OsIAA31(LOC_Os12g40900), OsARF1(LOC_Os11g32110), OsARF12(LOC_Os04g57610), OsARF16(LOC_Os06g09660), OsCRL1(LOC_Os03g05510), OsCRL5(LOC_Os07g03250) and OsWOX11(LOC_Os07g48560).
Availability of Data and Materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
OsUBC11 Overexpression line 3
OsUBC11 Overexpression line 5
Ubiquitin conjunction enzyme
E3 ubiquitin ligase
Quantitative real-time PCR
Green fluorescent protein
Days after germination
Self-formed adaptor PCR
Andrew DJ, Zhang NY, Xu P, Han K, Seth N, Peng J, Liu CW (2009) The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26S proteaso-me. J Biolo Chem 284(51):35485–35494. https://doi.org/10.1074/jbc.M109.052928
Ayako N, Iichiro U, Kenji G, Yasuko H, Hidemi K, Takashi S, Makoto M (2006) Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein. Plant J 46:297–306. https://doi.org/10.1111/j.1365-313X
Bae H, Kim WT (2014) Classification and interaction modes of 40 rice E2 ubiquitin-conjugating enzymes with 17 rice ARM-U-box E3 ubiquitin ligases. Biochem Bio-Phys Res Commun 444(4):575–580. https://doi.org/10.1016/j.bbrc.2014.01.098
Bellini C, Pacurar DI, Perrone I (2014) Adventitious roots and lateral roots: similarities and differences. Annu Rev Plant Biol 65:639–666. https://doi.org/10.1146/annurev-arplant-050213-035645
Chen Y, Fan X, Song W, Zhang Y, Xu G (2012) Over-expression of OsPIN2 leads to increased tiller numbers angle and shorter plant height through suppression of OsLAZY1. Plant Biotechnol J 10(2):139–149. https://doi.org/10.1111/j.1467-7652.2011.00637.x
Cheng Y, Dai X, Zhao Y (2006) Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes Dev 20(13):1790–1799. https://doi.org/10.1101/gad.1415106
Feng H, Wang S, Dong D, Zhou RY, Hong W (2020) Arabidopsis ubiquitin- conjugating enzymes UBC7, UBC13, and UBC14 are required in plant responses to multiple stress conditions. Plants 9:723. https://doi.org/10.3390/plants9060723
Friml J (2021) Fourteen stations of auxin. Cold Spr Har Pers Biol 16:a039859. https://doi.org/10.1101/cshperspect.a039859
Fu LL, Wang M, Han BY, Tan DG, Sun XP, Zhang JM (2016) Arabidopsis myrosinase genes AtTGG4 and AtTGG5 are root-tip specific and contribute to auxin biosynthesis and root-growth regulation. Int J Mol Sci 17(6):892. https://doi.org/10.3390/ijms17060892
Gao SP, Fang J, Xu F, Wang W, Sun XL, Chu JF, Cai BD, Feng YQ, Chu CX (2014) CYTOKININ OXIDASE/DEHYDROGENASE4 Integrates cytokinin and auxin signaling to control rice crown root formation. Plant Physiol 165:1035–1046. https://doi.org/10.1104/pp.114.238584
Guilfoyle TJ, Hagen G (2007) Auxin response factors. Curr Opin Plant Biol 10:453–460. https://doi.org/10.1016/j.pbi.2007.08.014
Hanae K, Akemi T, Shizue K, Etsuko K, Hiroaki I, Eiichi M, Yoko N (2007) RING-H2 type ubiquitin ligase EL5 is involved in root development through the maintenance of cell viability in rice. Plant J 51:92–104. https://doi.org/10.1111/j.1365-313X
Huang GQ, Azad K, Michal K, Zhang J, Poonam M, Song XY, Craig JS, Zhu WW, Qin H, Sjon H, Hannah MS, Rahul B, Ian CD, Robert ES, Huang RF, Sacha JM, Liang WQ, Malcolm JB, Zhang DB, Bipin KP (2022) Ethylene inhibits rice root elongation in compacted soil via ABA- and auxin-mediated mechanisms. Proc Natl Acad Sci USA 119(30):e2201072119. https://doi.org/10.1073/pnas.2201072119
Hui C, Biao M, Yang Z, Si-Jie H, San-Yuan T, Xiang L, Qi X, Shou-Yi C, Jin-Song Z (2018) E3 ubiquitin ligase SOR1 regulates ethylene response in rice root by modulating stability of Aux/IAA protein. Proc Natl Acad Sci U S A 115(17):4513–4518. https://doi.org/10.1073/pnas.1719387115
Inukai Y, Sakamoto T, Ueguchi-Tanaka M, Shibata Y, Gomi K, Umemura I, Hasegawa Y, Ashikari M, Kitano H, Matsuoka M (2005) Crown rootless1, which is essential for crown root formation in rice, is a target of an AUXIN RESPONSE FACTOR in auxin signaling. Plant Cell 17(1387–1396):154. https://doi.org/10.1105/tpc.105.030981
Jacobsen AD, Zhang NY, Xu P, Han KJ, Noone S, Peng J (2009) The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26S proteasome. J Biol Chem 284:35485–35494. https://doi.org/10.1074/jbc.M109.052928
Jia ZT, Giehl FH, Nicolaus VW (2022) Nutrient-hormone relations: driving root plasticity in plants. Mol Plant 15(1):86–103. https://doi.org/10.1016/j.molp.2021.12.004
Jing HW, Yang XL, Zhang J, Liu XH, Zheng HK, Dong GJ, Nian JQ, Feng J, Xia B, Qian Q, Li JY, Zuo JR (2015) Peptidyl-prolyl isomerization targets rice Aux/IAAs for proteasomal degradation during auxin signaling. Nat Commun 6:7395. https://doi.org/10.1038/ncomms8395
Joungsu J, Dong HC, Youn HL, Hak SS, Sang IS (2019) The rice SUMO conjugating enzymes OsSCE1 and OsSCE3 have opposing effects on drought stress. J Plant Physiol 240:152993. https://doi.org/10.1016/j.jplph.2019.152993
Karimi M, De MeyerHilson B (2015) Modular cloning and expression of tagged fluorescent protein in plant cells. Trends Plant Sci 10(3):103–105. https://doi.org/10.1007/s11103-005-0340-5
Kitomi Y, Ito H, Hobo T, Aya K, Kitano H, Inukai Y (2011) The auxin responsive AP2/ERF transcription factor CROWN ROOTLESS5 is involved in crown root initiation in rice through the induction of OsRR1, a type-A response regulator of cytokinin signaling. The Plant J 67:472–484. https://doi.org/10.1111/j.1365-313X.2011.04610.x
Li WF, Wolfgang S (2010) A lysine-63-linked ubiquitin chain-forming conjugase, UBC13, promotes the developmental responses to iron deficiency in Arabidopsis roots. Plant J 62:330–343. https://doi.org/10.1111/j.1365-313X.2010.04150.x
Liu S, Wang J, Wang L, Wang X, Xue Y, Wu P, Shou H (2009) Adventitious root formation in rice requires OsGNOM1 and is mediated by the OsPINs family. Cell 19:1110–1119. https://doi.org/10.1038/cr.2009.70
Liu H, Wang S, Yu X, Yu J, He X, Zhang S, Shou H, Wu P (2005) ARL1 a LOB-domain protein required for adventitious root formation in rice. Plant J 43(1):47–56. https://doi.org/10.1111/j.1365-313X.2005.02434.x
Liu X, Zhang H, Kan HF, Zhou LS, Huang H, Song LL, Zhai HC, Zhang J, Lu GD (2016a) Bioinformatics and expression analysis of rice Ubiquitin binding enzyme gene family. Chin Rice Sci 30(03):223–231
Liu YY, Wang RL, Zhang P, Chen Q, Luo Q, Zhu YY, Jin Xu (2016b) The nitrification inhibitor methyl 3-(4-Hydroxyphenyl) propionate modulates root development by interfering with auxin signaling via the NO/ROS pathway. Plant Physiol 171(3):1686–1703. https://doi.org/10.1104/pp.16.00670
Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B et al (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284. https://doi.org/10.1016/j.molp.2015.04.007
Maraschin FDS, Memelink J, Offringa R (2009) Auxin-induced, SCF(TIR1): mediated poly-ubiquitination marks AUX/IAA proteins for degradation. The Plant J 59(1):100–109. https://doi.org/10.1111/j.1365-313X.2009.03854.x
Marchant A, Bhalerao R, Casimiro I, Eklöf J, Casero PJ, Bennett M, Sandberg G (2002) AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling. Plant Cell 14(3):589–597. https://doi.org/10.1105/tpc.010354
Moon J, Parry G, Estelle M (2004) The ubiquitin-proteasome pathway and plant development. Plant Cell 16(12):3181–3195. https://doi.org/10.1105/tpc.104.161220
Ni J, Wang GH, Zhu ZX, Zhang HH, Wu YR, Wu P (2011) OsIAA23-mediated auxin signaling defines postembryonic maintenance of QC in rice. Plant J 68(3):433–442. https://doi.org/10.1111/j.1365-313X.2011.04698.x
Overvoorde P, Fukaki H, Beeckman T (2010) Auxin control of root development. Cold Spring Harb Perspect Biol 2:15–20. https://doi.org/10.1101/cshperspect.a001537
Pan WB, Lin BY, Yang XY, Liu LJ, Xia R, Li JG, Wu YR, Xie Q (2020) The UBC27–AIRP3 ubiquitination complex modulates ABA signaling by promoting the degradation of ABI1 in arabidopsis. Pros Nati Acad Sci USA 117(44):27694–27702. https://doi.org/10.1073/pnas.2007366117
Péret B, Swarup K, Ferguson A, Seth M, Yang YD, Stijn D, Nicholas J, Ilda C, Paula P, Adnan S, Swarup S (2012) AUX1/LAX genes encode a family of auxin influx transporters that perform distinct functions during Arabidopsis development. Plant Cell 24:2874–2885. https://doi.org/10.1105/tpc.112.097766
Pérez TCA, Lo’pez-Bucio J, Cruz-Ramı’rez A, Ibarra-laclette E, Dharmasiri S, Estelle M, Herrera-Estrella L (2008) Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor. Plant Cell 20:3258–3272. https://doi.org/10.1105/tpc.108.058719
Petricka JJ, Winter CM, Benfey PN (2012) Control of arabidopsis root development. Annu Rev Plant Biol 63:563–590. https://doi.org/10.1146/annurev-arplant-042811-105501
Qi YH, Wang SK, Shen CJ, Zhang SN, Chen Y, Xu YX, Liu Y, Wu YR, Jiang DA (2012) OsARF12, a transcription activator on auxin response gene, regulates root elongation and affects iron accumulation in rice (Oryza sativa). New Phytol 193:109–120. https://doi.org/10.1111/j.1469-8137.2011.03910.x
Sadanandom A, Bailey M, Ewan R, Lee J, Nelis S (2012) The ubiquitin-proteasome system: central modifier of plant signaling. New Phytol 196(1):13–28. https://doi.org/10.1111/j.1469-8137.2012.04266.x
Sazuka T, Noriko K, Takeshi N, Kozue O, Yutaka S, Kohei I, Yasuo N, Tomokazu K, Yoshiaki N, Motoyuki A, Hidemi K, Makoto M (2009) A rice tryptophan deficient dwarf mutant, tdd1, contains a reduced level of indole acetic acid and develops abnormal flowers and organless embryos. Plant J 60(2):227–241. https://doi.org/10.1111/j.1365-313X.2009.03952.x
Shen CJ, Wang SK, Zhang SN, Xu YX, Qian Q, Qi YH, Jiang DA (2013) OsARF16, a transcription factor, is required for auxin and phosphate starvation response in rice (Oryza sativa L.) plant. Cell Environ 36:607–620. https://doi.org/10.1111/pce.12001
Smalle J, Vierstra RD (2004) The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol 55:555–590. https://doi.org/10.1146/annurev.arplant.55.031903.141801
Smith S, De SI (2012) Root system architecture: insights from arabidopsis and cereal crops. Philos Trans R Soc Lond B Biol Sci 367:1441–1452. https://doi.org/10.1098/rstb.2011.0234
Song YL, You J, Xiong LZ (2009) Characterization of OsIAA1 gene, a member of rice Aux/IAA family involved in auxin and brassinosteroid hormone responses and plant morphogenesis. Plant Mol Biol 70:297–309. https://doi.org/10.1007/s11103-009-9474-1
Stepanova AN, Robertson-Hoyt J, Yun J, Benavente LM, Xie DY, Doležal K, Schlereth A, Jürgens G, Alonso JM (2008) TAA1-Mediated auxin biosynthesis is essential for hormone crosstalk and plant development. Cell 133(1):177–191. https://doi.org/10.1016/j.cell.2008.01.047
Swarup R, Joanna K, Alan M, Daniel Z, Abidur R, Rebecca M, Anthony Y, Sean M, Lorraine W, Paul M, Seiji T, Ian M, Richard N, Ian DK, Malcolm JB (2004) Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1. Plant Cell 16(11):3069–3083. https://doi.org/10.1105/tpc.104.024737
Trempe JF (2011) Reading the ubiquitin postal code. Curr Opin in Struc Biol 21:792–801. https://doi.org/10.1016/j.sbi.2011.09.009
Van PB, Smet D, Van DSD (2015) Ethylene and hormonal cross talk in vegetative growth and development. Plant Physiol 169(1):61–72. https://doi.org/10.1104/pp.15.00724
Wang SM, He J, Cui ZL, Li SP (2007) Self-formed adaptor PCR: a simple and efficient method for chromosome walking. Appl Environ Microbiol. https://doi.org/10.1128/AEM.02973-06
Wang YN, Li KX, Li X (2009) Auxin redistribution modulates plastic development of root system architecture under salt stress in Arabidopsis thaliana. J Plant Physiol 166(15):1637–1645. https://doi.org/10.1016/j.jplph.2009.04.009
Wang M, Qiao J, Yu CL, Chen H, Sun CD, Huang LZ, Li CY, Markus G, Qian Q, Jiang DA, Qi YH (2018) The auxin influx carrier, OsAUX3, regulates rice root development and responses to aluminum stress. Plant Cell Environ 42:1125–1138. https://doi.org/10.1111/pce.13478
Wen R, Wang S, Xiang D, Prakash V, Shi XZ, Zang YP, Datla R, Xiao W, Wang H (2014) UBC13, an E2 enzyme for Lys63-linked ubiquitination, functions in root development by affecting auxin signaling and Aux/IAA protein stability. Plant J 80:424–436. https://doi.org/10.1111/tpj.12644
Xu M, Zhu L, Shou H, Wu P (2005) A PIN1 Family Gene OsPIN1 involved in Auxin-dependent adventitious root emergence and tillering in rice. Plant Cell Physiol 46(10):1674–1681. https://doi.org/10.1093/pcp/pci183
Yang YD, Hammes UZ, Taylor CG, Schachtman DP, Nielsen E (2006) High affinity auxin transport by the AUX1 influx carrier protein. Curr Biol 16:1123–1127. https://doi.org/10.1016/j.cub.2006.04.029
Yuko Y, Noriko K, Yoichi M, Makoto M, Takashi S (2007) Auxin Biosynthesis by the YUCCA Genes in Rice. Plant Physiol 143:1362–1371. https://doi.org/10.1104/pp.106.091561
Zhiguo E, Zhang YP, Li TT, Wang L, Zhao HM (2015) Characterization of the ubiquitin- conjugating enzyme gene family in rice and evaluation of expression profiles under abiotic stresses and hormone treatments. PLoS ONE 10(4):e0122621. https://doi.org/10.1371/journal.pone.0122621
Zhang T, Li R, Xing J, Yan L, Wang R, Zhao Y (2018) The YUCCA- auxin- WOX11 module controls crown root development in rice. Front Plant Sci 9:523. https://doi.org/10.3389/fpls.2018.00523
Zhang HH, Tan XX, Li LL, He YQ, Hong GJ, Li JM, Lin L, Cheng Y, Fei Y, Chen JP, Sun ZT (2019) Suppression of auxin signalling promotes rice susceptibility to rice black streaked dwarf virus infection. Mol Plant Pathol 20(8):1093–1104. https://doi.org/10.1111/mpp.12814
Zhao YD (2010) Auxin biosynthesis and its role in plant development. Annu Rev Plant Biol 61:49–64. https://doi.org/10.1146/annurev-arplant-042809-112308
Zhao YD (2012) Auxin biosynthesis: a simple two-step pathway converts tryptophan to indole-3-acetic acid in plants. Mol Plant 5(2):334–338. https://doi.org/10.1093/mp/ssr104
Zhao Y, Hu YF, Dai MQ, Huang LM, Zhou DX (2009) The WUSCHEL-related homeobox gene WOX11 Is required to activate shoot-borne crown root develo- pment in rice. Plant Cell 21:736–748. https://doi.org/10.1105/tpc.108.061655
Zhao HM, Ma TF, Wang X, Deng YT, Ma HL, Zhang RS, Zhao J (2015a) OsAUX1 controls lateral root initiation in rice. Plant Cell Environ 38:2208–2222. https://doi.org/10.1111/pce.12467
Zhao Y, Cheng S, Song Y, Huang Y, Zhou S, Liu X, Zhou DX (2015b) The interaction between rice ERF3 and WOX11 promotes crown root development by regulating gene expression involved in cytokinin signaling. Plant Cell 27:2469–2483. https://doi.org/10.1105/tpc.15.00227
Zhou SL, Jiang W, Long F, Cheng SF, Yang WJ, Zhao Y, Zhou DX (2017) Rice homeodomain protein WOX11 recruits a histone acetyltransferase complex to establish programs of cell proliferation of crown root meristem. Plant Cell 29:1088–1104. https://doi.org/10.1105/tpc.16.00908
Zhu ZX, Liu Y, Liu SJ (2012) A gain-of-function mutation in OsIAA11 affects lateral root development in rice. Mol Plant 5(1):154–161. https://doi.org/10.1093/mp/ssr074
We are grateful to Professor Qingyun Bu, most of strains, empty vectors, instrument and some equipments. Grateful to all members in our laboratory, give me many suggestions.
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA28100301), the National Natural Science Foundation of China(U20A2025), Major research and development project of Inner Mongolia (2020ZD0023), Chinese Postdoctoral Science Foundation (2021M693157), Natural Science Foundation of Heilongjiang Province (JJ2021YX0477) and the Opening Foundation of Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Cold Region, Ministry of Education, Northeast Agricultural University (CXSTOP2021006).
The authors declare no conflict of interest.
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Additional file 1
. Fig S1: Phenotype observation of R164 and ZH11. Fig S2: Phenotype observation of OsUBC11 overexpression lines and ZH11. Fig S3: Phenotype observation of osubc11 mutant. Fig S4: Determination of auxin relative genes. Fig S5: Subcellular location of OsUBC11.
Additional file 2
. Table S1: Primers used in this study.
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Han, Y., Zhang, C., Sha, H. et al. Ubiquitin-Conjugating Enzyme OsUBC11 Affects the Development of Roots via Auxin Pathway. Rice 16, 9 (2023). https://doi.org/10.1186/s12284-023-00626-3
- Root development
- Ubiquitin-conjugating enzyme
- Auxin transport
- Ubiquitin degradation signal