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. 2020 Aug 20;184(2):895–908. doi: 10.1104/pp.20.00778

Cytosolic Invertase-Mediated Root Growth Is Feedback Regulated by a Glucose-Dependent Signaling Loop1

Lai-Sheng Meng a,2,3, Zhi-Qin Wei a,2, Xiao-Ying Cao a, Chen Tong a, Meng-Jiao Lv a, Fei Yu a, Gary J Loake b,c,4
PMCID: PMC7536704  PMID: 32820066

An Arabidopsis cytosolic Invertase is dynamically regulated by a glucose signaling feedback loop to control root growth

Abstract

The disaccharide Suc cannot be utilized directly; rather, it is irreversibly hydrolyzed by invertase to the hexoses Glc and Fru to shape plant growth. In this context, Glc controls the stability of the transcription factor Ethylene-Insensitive3 (EIN3) via the function of Hexokinase1 (HXK1), a Glc sensor. Thus, invertase, especially the major neutral cytosolic invertase (CINV), constitutes a key point of control for plant growth. However, the cognate regulatory mechanisms that modulate CINV activity remain unclear. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), EIN3 binds directly to both the promoters of Production of Anthocyanin Pigment1 (PAP1) and Phosphatidylinositol Monophosphate 5-Kinase 9 (PIP5K9), repressing and enhancing, respectively, their expression. Subsequently, PAP1 binds directly to and promotes transcription of the Cytosolic Invertase1 (CINV1) promoter, while PIP5K9 interacts with and negatively regulates CINV1. The accumulated CINV1 subsequently hydrolyzes Suc, releasing the sequestered signaling cue, Glc, which has been shown to negatively regulate the stability of EIN3 via HXK1. We conclude that a CINV1-Glc-HXK1-EIN3-PAP1/PIP5K9-CINV1 loop contributes to the modulation of CINV1 activity regulating root growth by Glc signaling.

Suc is the major source of carbon for plant cells, and consequently, Suc catabolism in plants is one of the largest metabolic fluxes in primary carbon assimilation. In Arabidopsis (Arabidopsis thaliana) seedlings, the shoot apex and roots are the main sinks, with assimilates being supplied by the cotyledons (Roitsch, 1999). Here, carbon is transported from source to sink in the form of the disaccharide Suc. However, Suc cannot be directly utilized in plants. Therefore, after arrival in the sink, Suc can either be irreversibly hydrolyzed by invertase, yielding Glc and Fru, or reversibly hydrolyzed via Suc synthase (SUS), yielding Fru and UDP-Glc (Barratt et al., 2009). The emerging evidence suggests that SUS functions in storage sinks, while invertase operates in growing sinks, such as Arabidopsis roots (Sergeeva et al., 2006). Moreover, SUS is not essential for plant growth and development (Barratt et al., 2009). Therefore, invertase, but not SUS, has a major function in roots. In invertase, vacuolar invertase (INV) regulates normal root growth in Arabidopsis via its impact on vacuolar osmotic potential (Sergeeva et al., 2006), whereas Cytosolic invertase1 (CINV1) and CINV2, two of the nine isoforms in Arabidopsis, regulate sugar signaling and/or metabolism-mediated root growth under routine growth conditions (Lou et al., 2007; Barratt et al., 2009).

Loss-of-function mutations in CINV1 and CINV2 decrease root Glc levels, leading to reduced primary root growth (Barratt et al., 2009), while exogenous Glc application promotes both primary and lateral root growth and root hair formation (Mishra et al., 2009). In the Arabidopsis root, CINV1 and CINV2 irreversibly hydrolyze the disaccharide Suc into the hexoses Glc and Fru, with Glc functioning as a hormone-like signaling molecule (Lou et al., 2007) as well as an energy source. CINV1 and CINV2 comprise the major route via which carbon from Suc is supplied to nonphotosynthetic cells in Arabidopsis (Barratt et al., 2009). CINV1 and CINV2 activity therefore provides a key point of control for coordinating Suc catabolism with root growth and development. In Arabidopsis, 15 Phosphatidylinositol monophosphate 5-kinase (PIP5K) isoforms have been identified (Mueller-Roeber and Pical, 2002), and PIP5K9 has been shown to interact with and regulate CINV1 at the posttranslational level by an unknown mechanism to regulate root growth and development (Lou et al., 2007). Anthocyanin Pigment 1 (PAP1), a putative MYB domain containing a transcription factor, is considered a key integration point for a variety of internal and external stimuli that influence the biosynthesis of anthocyanins (Teng et al., 2005). Significantly, Suc, but not other sugars, can induce PAP1 expression (Teng et al., 2005).

Glc negatively controls the stability of the ETHYLENE-INSENSITIVE3 (EIN3) transcriptional regulator via HEXOKINASE1 (HXK1), a Glc sensor, although the mechanistic link between Glc sensing and EIN3 degradation has not to date been demonstrated (Yanagisawa et al., 2003). EIN3 is a component associated with both sugar and ethylene function. In ethylene signaling, the EIN3 transcription factor is a key component (Chao et al., 1997). Besides EIN3, there are five other EIN3-like (EIL) transcription factors in Arabidopsis (Chao et al., 1997). The closest paralogs, EIN3 and EIL1, both function in ethylene-regulated processes (Alonso et al., 1999; Binder et al., 2004). EIN3 and EIL1 have overlapping functions and are degraded by the proteasome via the F-box proteins EIN3-BINDING F BOX PROTEIN1 (EBF1) and EBF2 (Guo and Ecker, 2003; Binder et al., 2007; An et al., 2010; Meng et al., 2018b; Zhang et al., 2018; Wang and Guo, 2019). Glc signaling promotes EIN3 degradation (Yanagisawa et al., 2003). However, it is unknown whether EIN3 influences the level of Glc.

It has been suggested that the absence of CNV1 function leads to a reduction in root length, possibly resulting from invertase feedback regulation following a reduction in the level of sugars (Lou et al., 2007). However, the molecular mechanism(s) underpinning these observations remain unclear. Here we show evidence for a CINV1-Glc-HXK1-EIN3-PAP1/PIP5K9-CINV1 signaling loop that contributes to the modulation of CINV activity. This regulatory mechanism participates in the coordination of Suc catabolism with the demands of both root growth and development.

RESULTS

Glc Promotes Arabidopsis Root Growth by Enhancing Invertase Activity

We observed that increasing exogenous Suc promoted root elongation (Fig. 1, A, B, and E) by elevating the endogenous Glc level (Fig. 1G) resulting from enhanced neutral invertase activities (Fig. 1F). Further, with increasing age, endogenous Suc levels were enhanced (Fig. 1I), which in turn enhanced neutral invertase activities (Fig. 1J) and thus endogenous Glc levels (Fig. 1K). As a result, the root length increased (Fig. 1, C, D, and H). Thus, increased exogenous/endogenous Suc enhances neutral invertase activities, generating endogenous Glc, which in turn promotes root growth.

Figure 1.

Figure 1.

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Sugar promotes root growth and development. A and B, Seedlings of wild-type plants were grown on solid MS medium with 1%, 2%, or 3% Suc under standard light conditions for 8 (A) or 16 d (B). C and D, Seedlings of wild-type plants were grown on solid MS medium with 1% Suc under standard light conditions for 9 (C) or 18 d (D). Scale bars = 10 mm. E, Bar graph illustrating primary root length in A and B. Error bars represent the sd (n = 12F, Bar graph illustrating neutral invertase activity in the plants in A and B. Error bars represent sd (n = 3). G, Bar graph illustrating Glc levels in the plant lines in A and B. Error bars represent sd (n = 3). Asterisks indicate significant difference as determined by Student’s t test (*P < 0.05). H, Bar graph illustrating primary root length in the plant lines in C and D. Error bars represent sd (n = 16). I, Bar graph illustrating Suc levels in the plant lines in C and D. Error bars represent sd (n = 3). J, Bar graph illustrating neutral invertase activity in the plant lines in C and D. Error bars represent sd (n = 3). K, Bar graph illustrating Glc level in the plant lines given in C and D. Error bars represent sd (n = 3). Asterisks indicate significant difference as determined by Student’s t test (**P < 0.01 and ***P < 0.001).

Further, exogenous Glc application enhanced neutral invertase activities and in turn elongated root growth (Supplemental Fig. S1). To demonstrate that root growth regulated by endogenous Glc correlates with enhanced neutral invertase activities, we employed a cinv1 cinv2 double mutant, exhibiting loss of neutral invertase activities. When grown on solid Murashige and Skoog (MS) medium without sugar in high light, cinv1 cinv2 seedlings exhibited shortened root growth compared with the wild type, which results from a reduced endogenous Glc level due to decreased neutral invertase activity (Supplemental Fig. S2). Further, the short roots of cinv1 cinv2 seedlings can be restored to approximately one-half the length of wild-type roots following addition of exogenous Glc (Barratt et al., 2009).

Collectively, our data show that either exogenous or endogenous Suc promotes root growth by generating endogenous Glc derived from activated CINV function.

EIN3/EIL1 Functions Downstream of HXK1 to Negatively Regulate Root Growth

When seedlings were grown on MS medium with 1% Suc (Fig. 2, A and E) or 1% Glc (Supplemental Fig. S3, A, C, E, and G), but not 1% mannitol (Fig. 2, B and F; Supplemental Fig. S3, B, D, F, and H), both the EIN3 loss-of-function mutant lines ein3-1 and ein3 eil1 exhibited longer roots. In contrast, EIN3 overexpression resulted in shorter roots relative to the wild type. Consistent with these root phenotypes, neutral invertase activities and Glc levels of ein3-1 and ein3 eil1 seedlings increased, whereas those of EIN3 overexpression lines decreased (Fig. 2, G and H). Moreover, the elongated root length of ein3 eil1 seedlings was restored by exogenous Glc (Supplemental Fig. S4, A and B). Further, when grown on solid MS medium with 1% Suc (Fig. 2, C, D, and I–K) or 1% Glc (Supplemental Fig. S5), with increasing age, root length was enhanced in ein3-1 seedlings, due to the increased endogenous Glc level resulting from increased neutral invertase activities. Together, our data show that EIN3 negatively regulates Glc signaling/metabolism-mediated root growth, which correlates with neutral invertase activities.

Figure 2.

Figure 2.

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EIN3 negatively regulates Glc-mediated root growth and development. A, Seedlings of eil1-3, ein3-1, wild-type, ein3 eil1, 35S:EIN3, and ER:EIN3 plants were grown on solid MS medium with 1% Suc under high light conditions (230 μmol quanta PAR m−2 s−1) for 7 d. EIN3 overexpression lines include seedlings expressing either EIN3 under the 35S promoter (35S:EIN3) or EIN3-FLAG via an estradiol-inducible system. B, Seedlings of wild-type, ein3-1, eil1-3, 35S:EIN3, ER:EIN3, and ein3 eil1 plants were grown on solid MS medium with 1% mannitol under standard light conditions for 7 d. Scale bars = 1.0 cm. C and D, Seedlings of wild-type and ein3-1 plants were grown on solid MS medium with 1% Suc under standard light conditions for 9 (C) or 18 d (D). Scale bars = 10 mm. E, Bar graph illustrating root length of the plant lines in A. Error bars represent the sd (n = 14). F, Bar graph illustrating root length of the plant lines in B. Error bars represent the sd (n = 10). G, Bar graph illustrating neutral invertase activity in the plant lines in A. Error bars represent the sd (n = 3). H, Bar graph illustrating the Glc levels in the plant lines in A. Error bars represent the sd (n = 3). I, Bar graph illustrating root length of the plant lines in C and D. Error bars represent the sd (n = 16). J, Bar graph illustrating neutral invertase activity of the plant lines in C and D. Error bars represent the sd (n = 3). K, Bar graph illustrating the Glc levels in C and D. Error bars represent the sd (n = 3). Asterisks indicate significant difference as determined by Student’s t test (*P < 0.05 and **P < 0.01).

We then assayed whether EIN3 genetically acts downstream of HXK1 in the regulation of root growth. The primary roots of 7-d-old gin2-1 seedlings, defective in HXK1 activity, elongated more slowly than those of the corresponding wild-type seedlings (Fig. 3, A and C), which was caused by reduced endogenous Glc levels (Fig. 3F) due to decreased neutral invertase activity (Fig. 3E) in gin2-1 roots. Moreover, the shortened root length of gin2-1 seedlings was restored by exogenous Glc (Supplemental Fig. S4, C and D). Further, gin2-1/ein3 root length was enhanced, being similar to ein3-1 root length (Fig. 3, A and C). However, the root length of gin2-1, ein3-1, and ein3/gin2-1 seedlings was not significantly different following the exogenous addition of 1% mannitol (Supplemental Fig. S6, A and C).

Figure 3.

Figure 3.

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A Glc signal loop genetically regulates root growth. A and B, The indicated seedlings were grown on solid MS medium with 1% Suc for 7 d. Scale bars = 1.0 cm. C, Bar graph illustrating the differences in root length between the indicated plant lines in A. Error bars represent the sd (n = 15). D. Bar graph illustrating root length of the plant lines in B. Error bars represent the sd (n = 13). E and F, Bar graph illustrating invertase activity (E) and Glc level (F) in 7-d-old wild-type (Ler) and gin2-1 seedlings. Error bars represent the sd (n = 3). Asterisks indicate significant difference as determined by Student’s t test (*P < 0.05 and **P < 0.01).

Taken together, our data show that EIN3 and EIL1 functions downstream of HXK1 to negatively regulate Glc signaling/metabolism-mediated root growth.

EIN3 Directly Binds to the PAP1 Promoter Repressing PAP1 Expression to Negatively Regulate Signaling/Metabolism-Mediated Root Growth

Our results show that transcript levels corresponding to the transcriptional regulators PAP1 and PAP2 were dramatically upregulated in ein3 eil1 plants relative to the wild type (Fig. 4A). This is in agreement with previous findings (Jeong et al., 2010), implying that the transcription factor EIN3 represses the expression of these genes.

Figure 4.

Figure 4.

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EIN3 directly binds the PAP1 promoter, inhibiting PAP1 expression. A, Bar graph based on RT-qPCR analysis exhibiting differential expression of PAP1 and PAP2 between wild-type and ein3 eil1 roots. Independent seedlings of 8-d-old lines were analyzed. Quantifications were normalized to the expression of UBQ5. Error bars represent the sd (n = 3). Asterisks indicate significant difference as determined by Student’s t test (**P < 0.01). B, Schematic of PAP1 promoter loci and amplicons for chromatin immunoprecipitation (ChIP) analysis. P1, −600 to −700 bp; P2, −1,600 to −1,700 bp; P3, −1,900 to −2,100 bp; P4, −2,400 to −2,500 bp; P5, −2,700 to −2,900 bp. C, ChIP analysis of P1 in the PAP1 promoter. Enrichment of specific PAP1 chromatin regions with anti-GFP antibody in the roots of 35S:EIN3-GFP/ein3-1 transgenic plants as detected by RT-qPCR analysis. Quantifications were normalized to the expression of UBQ5. Quantification of the coding sequence in the anti-GFP antibody is set as 1.0. At least 20 independent seedlings of 8-d-old 35S:EIN3-GFP/ein3-1 lines grown on MS medium with 1% or 3% Glc were analyzed. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student’s t test (**P < 0.01). D, Western blotting showing reduced stability of EIN3-GFP in at least 10 independent 8-d-old 35S:EIN3-GFP/ein3-1 seedlings grown on MS medium with 3% Glc as compared to 1% Glc. E, EMSA was performed to detect binding of EIN3 to the PAP1 promoter using a biotin-labeled P1 probe. Unlabeled probes were used as competitors. F, A mutant version of the PAP1 promoter (caaa/cttt) was labeled with biotin and used for EMSA with recombinant EIN3. G, LUC activity from PAP1-LUC and PAP1-P1Δ-LUC reporter genes in protoplasts isolated from ein3 eil1 double mutant (iE/dm) seedlings treated or not with 25 μm estradiol for 1.5 d. Luminescence units are represented by relative LUC activity. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student's t test (**P < 0.01). The sequence of the PAP1 promoter was fused to the LUC reporter gene (PAP1-LUC), and this construct was transfected subsequently into protoplasts derived from a ER:EIN3-FLAG transgenic line in an ein3 eil1 double mutant (iE/dm) background. The P1 element was removed from the PAP1-LUC construct (PAP1P1Δ-LUC) and the resulting construct was transfected into the same iE/dm protoplasts as described above.

There are a few putative EIN3 binding sites (EBSs) in the PAP1 promoter (Fig. 4B; Li et al., 2013). To explore whether EIN3 interacts with any of these EBSs, we performed chromatin immunoprecipitation (ChIP) experiments. Indeed, EIN3 bound strongly to one PAP1 promoter sequence (P1) but not to three other PAP1 promoter sequences (P2–P4; Fig. 4C) or the PAP2 promoter (Supplemental Fig. S7). Further, 3% Glc treatment reduced EIN3 binding to the PAP1 promoter (Fig. 4C). As EIN3 degradation is enhanced by Glc treatment (Yanagisawa et al., 2003), this implies that EIN3 binding to the P1 sequence is decreased following EIN3 degradation (Fig. 4D). Moreover, an electrophoretic mobility shift assay (EMSA) showed that EIN3 bound exclusively to the labeled P1 EBS element in vitro (Fig. 4E). Excess unlabeled competitor DNA effectively abolished this binding ability in a dose-dependent manner (Fig. 4E). However, EIN3 did not bind the mutated P1 (mP1) sequence, confirming the specificity of this interaction (Fig. 4F). In a transactivation assay, PAP1-LUCIFERASE (LUC) expression, but not LUC expression driven by the PAP1 promoter deleted for the P1 sequence (PAP1P1Δ-LUC), was reduced with estradiol treatment (Fig. 4G), indicating that the P1 element plays a key role in transcriptional repression of the PAP1 promoter via EIN3.

We then explored whether EIN3 functions upstream of PAP1 in the regulation of root growth. pap1-d (a dominant activation tagged mutant; Borevitz et al., 2000) seedlings exhibited elongated roots (Fig. 3, B and D), whereas a loss of PAP1 function (pap1-KO) resulted in decreased root length (Supplemental Fig. S8, A and C). Further, the shorter roots of pap1-KO seedlings were restored to wild-type levels under exogenous Glc supply (Supplemental Fig. S8, E and F). Also, roots of the 35S:EIN3 pap1-d line were comparable in length to those of pap1-d seedlings and thus significantly longer than those of 35S:EIN3 seedlings (Fig. 3, B and D). However, the root lengths of pap1-d, 35S:EIN3, and 35S:EIN3 pap1-d seedlings were not different following application of exogenous 1% mannitol (Supplemental Fig. S6, B and D). Therefore, these findings indicate that EIN3 is upstream of PAP1 in the regulation of Glc-mediated root growth.

Collectively, our results indicate that EIN3 directly binds to the PAP1 promoter, suppressing PAP1 expression to negatively regulate Glc signaling/metabolism-mediated root growth.

PAP1 Directly Binds to the CINV1 Promoter to Activate CINV1 Expression to Positively Regulate Glc Signaling/Metabolism-Mediated Root Growth

PAP1 has previously been implicated in sugar signaling (Teng et al., 2005; Solfanelli et al., 2006). In this context, the expression of INV (Lou et al., 2007; Barratt et al., 2009), which encodes a key enzyme integral to sugar homeostasis, was enhanced by a potato (Solanum tuberosum) PAP1 homolog (Payyavula et al., 2013). Indeed, CINV1 expression was markedly enhanced in the roots of PAP1-overexpressing lines, suggesting that CINV1 is a downstream target of PAP1 (Fig. 5A).

Figure 5.

Figure 5.

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The CINV1 promoter is a target for PAP1 binding. A, Bar graph showing differential expression of CINV1 and CINV2 among wild-type, 35S:PAP1, and pap1-d seedlings grown under standard light conditions. Wild-type data were set as 1.0. Quantification was normalized to the expression of UBQ5. At least 20 independent seedlings of 7-d-old lines were analyzed. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student’s t test (**P < 0.01). B, Schematic of the promoter loci of CINV1 and their amplicons for ChIP analysis. C1, −2,707 to −2,478 bp; C2, −1,901 to −1,580 bp; C3, −400 to −167 bp. C, ChIP analysis of the promoter of CINV1. Enrichment of particular chromatin regions of the CINV1 promoter with anti-HA antibody in 35S:PAP1-HA transgenic seedlings was detected by RT-qPCR analysis. At least 20 independent seedlings of 8-d-old 35S:PAP1-HA transgenic lines were analyzed. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student’s t test (**P < 0.01). D and E, EMSA was performed to detect the binding of PAP1 protein to the CINV1 promoter using a biotin-labeled C1 probe. Unlabeled CINV1 promoter or unlabeled probes were used as competitors to determine the specificity of DNA-binding activity for PAP1 (D). A mutant version of the CINV1 promoter (GGTT/GGAA) was labeled with biotin and used for EMSA with recombinant PAP1 (E). Free probe and PAP1 protein complexes are indicated by an asterisk and arrows, respectively. F and G, Bar graph showing expression levels of PAP1 (F) and CINV1 (G). Ten-day-old 35S:EIN3-GFP wild-type seedlings were grown on solid MS medium with 1% Suc, then treated with 3% Glc for 0, 30, 60, and 90 mins. The expression levels of PAP1 (F) and CINV1 (G) at 0 min are set as 1.0. Quantification was normalized to the expression of UBQ5. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student’s t test (**P < 0.01). G, Western blotting showing reduced stability of EIN3-GFP in at least 10 independent 10-d-old 35S:EIN3-GFP ein3-1 seedlings grown on MS medium with 3% Glc versus 1% Glc. I, Bar graph illustrating a transactivation assay showing how PAP1 promotes LUC activity from a CINV1pro-LUC reporter gene. Activity of firefly LUC was normalized to Renilla luciferase activity as an internal control. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student’s t test (*P < 0.05).

We found PAP1 binding sites (PBSs) AACCTAAC and TATCCAACC in the promoter of CINV1 (Fig. 5B; Tian et al., 2015). ChIP analysis indicated that PAP1 only bound to one PBS (C1 sequence) of CINV1 (Fig. 5C) in vivo. EMSA experiments further indicated that PAP1 bound to the labeled C1 sequence in vitro (Fig. 5D). Excessive unlabeled competitor DNA effectively abolished this binding in a dose-dependent manner (Fig. 5D). Further, PAP1 did not bind to the corresponding mutated PBS (mC1; Fig. 5E). Treatment with 3.0% exogenous Glc led to a significant enhancement in expression of both PAP1 and CINV1 (Fig. 5, F and G), suggesting that Glc treatment decreases EIN3 stability (Fig. 5H), which in turn promotes PAP1 expression, enhancing CINV1 expression. Finally, transient expression data in Nicotiana benthamiana showed that coexpression of PAP1 elevated the expression of the ProCINV1-LUC reporter gene (Fig. 5I).

We then examined whether PAP1 is upstream of CINV1 in the regulation of root growth. cinv1 roots were shorter and pap1-d roots were longer relative to wild-type roots (Fig. 3, B and D). Root length in pap1-d cinv1 seedlings was similar to that in cinv1 seedlings (Fig. 3, B and D). However, the root length of cinv1, pap1-d, and pap1-d/cinv1 seedlings were indistinguishable following exogenous 1% mannitol application (Supplemental Fig. S6, B and D). Thus, PAP1 acts upstream of CINV1 in the regulation of Glc signaling/metabolism-mediated root growth.

Collectively, our results establish that PAP1 directly binds to the CINV1 promoter to activate CINV1 expression to positively regulate Glc signaling/metabolism-mediated root growth.

EIN3 Directly Binds to the PIP5K9 Promoter Activating PIP5K9 Expression to Negatively Regulate Glc Signaling/Metabolism-Mediated Root Growth

PIP5K9 directly and negatively interacts with CINV1, repressing its activity and thereby decreasing root growth (Lou et al., 2007). Therefore, we investigated whether EIN3 might also bind directly to the promoter of PIP5K9 to enable regulation at the transcriptional level.

PIP5K9 expression was reduced in both ein3-1 and ein3 eil1 seedlings (Fig. 6A). ChIP analysis further indicated that EIN3 binds in vivo to the P2 sequence of the PIP5K9 promoter, which contains an EBS (TTCAAA; Fig. 6B), but not to other sections of the PIP5K9 promoter (P1 and P3) that contain EBS sequences (Fig. 6C). Further, 3% Glc treatment reduced specific EIN3 binding to the P2 sequence of the PIP5K9 promoter (Fig. 6C), suggesting that EIN3 binding to this sequence is decreased due to EIN3 degradation (Fig. 6D). Furthermore, EIN3 bound exclusively to the labeled PIP5K9 promoter P2 sequence, as determined by EMSA (Fig. 6E). An excess of unlabeled P2 sequence utilized as competitor DNA effectively abolished this binding in a dose-dependent manner (Fig. 6E). EIN3 did not bind a mutated P2 (mP2) DNA sequence (Fig. 6F). Finally, the expression of the PIP5K9-LUC reporter gene, but not of a reporter gene possessing a deletion of P2 (PIP5K9 P2Δ-LUC), was increased under estradiol treatment (Fig. 6G). Together, our findings indicate that EIN3 binds the P2 sequence within the PIP5K9 promoter to activate its expression.

Figure 6.

Figure 6.

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EIN3 directly binds the PIP5K9 promoter, activating PIP5K9 expression. A, Bar graph exhibiting PIP5K9 expression in 7-d-old wild-type, ein3-1, and ein3 eil1 roots of plants grown under standard light conditions. Quantifications were normalized to the expression of UBQ5. At least 20 independent seedlings were analyzed. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student’s t test (**P < 0.01). B, Schematic of PAP1 promoter loci and amplicons for ChIP analysis. P1, −83 to −445 bp; P2, −910 to −1,207 bp; P3, −1,343 to −1,693 bp. C, ChIP analysis of P1 in the PIP5K9 promoter. Enrichment of specific PIP5K9 chromatin regions with anti-GFP antibody in the roots of 35S:EIN3-GFP transgenic plants as detected by RT-qPCR analysis. Quantifications were normalized to the expression of UBQ5. At least 20 independent seedlings of 7-d-old lines grown on MS medium with 1% or 3% Suc were analyzed. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student’s t test (**P < 0.01). D, Western blotting showing reduced stability of EIN3-GFP in at least 10 independent 35S:EIN3-GFP/ein3-1 seedlings of 7-d-old lines grown on MS medium with 3% Glc as compared to 1% Glc. E, EMSA was performed to detect binding of EIN3 to the PIP5K9 promoter using a biotin-labeled P2 probe. Excess unlabeled P2 fragments and unlabeled probes were used as competitors. F, A mutant version of the PIP5K9 promoter (caaa/cttt) was labeled with biotin and used for EMSA with recombinant EIN3. G, LUC activity from PIP5K9-LUC and PIP5K9-P2Δ-LUC reporter gene expression in protoplasts isolated from iE/dm seedlings treated or not with 25 μm estradiol for 2 d. Luminescence units are represented by relative LUC activity. Two biological replicates were performed. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student's t test (*P < 0.05). The sequence of the PIP5K9 promoter was fused to the LUC reporter gene (PIP5K9-LUC). Subsequently, this construct was transfected into protoplasts derived from an ein3 eil1 double mutant (iE/dm) containing an ER:EIN3-FLAG transgene. The P2 sequence was removed from the PIP5K9-LUC construct (PIP5K9 P2Δ-LUC).

We then examined whether EIN3 is epistatic to PIP5K9 in the regulation of root growth. Root length was elongated in pip5k9 seedlings but shortened in the 35S:EIN3 line relative to that of wild-type seedlings (Supplemental Fig. S9, A and C). Further, elongated roots of pip5k9 seedlings were restored to the wild-type root length under exogenous Glc supply (Supplemental Fig. S9, E and F). Moreover, roots of the 35S:EIN3 pip5k9 line were comparable in length to those of pip5k9 seedlings and thus significantly longer than those of 35S:EIN3 seedlings (Supplemental Fig. S9, A and C), suggesting that EIN3 acts upstream of PIP5K9. However, there was no significant difference in the root length of pip5k9, 35S:EIN3, and 35S:EIN3 pip5k9 seedlings following treatment with 1% mannitol (Supplemental Fig. S9, B and D). These results indicate that EIN3 acts upstream of PIP5K9 in Glc signaling/metabolism-mediated regulation of root growth.

EIN3 Acts Upstream of CINV1 But Does Not Directly Bind the CINV1 Promoter

EIN3 directly binds to the PAP1 promoter and PAP1 directly binds to the CINV1 promoter. Further, EIN3 acts upstream of PAP1 and PAP1 acts upstream of CINV1 to regulate root growth. Therefore, we examined whether EIN3 directly binds to the CINV1 promoter and also whether EIN3 acts upstream of CINV1 in the regulation of root growth.

There are 11 TTCAAA EIN3 binding sequences in the CINV1 promoter. However, CINV1 expression was only slightly enhanced in ein3 eil1 plants (Supplemental Fig. S10, A and B). Further, ChIP analysis indicated that EIN3 cannot directly bind to the CINV1 promoter (Supplemental Fig. S10C).

Analysis of root phenotypes revealed that ein3 eil1 roots were elongated and cinv1 roots shortened relative to wild-type roots (Supplemental Fig. S10, D and F). Further, ein3 eil1 cinv1 seedlings all exhibited short roots, similar to those of cinv1 seedlings (Supplemental Fig. S10, D and F). However, the root length of ein3 eil1, cinv1, and ein3 eil1 cinv1 seedlings was not significantly different in the presence of exogenous 1% mannitol (Supplemental Fig. S10, E and G), indicating that EIN3 acts upstream of CINV1 in Glc signaling/metabolism-mediated regulation of root growth.

Since EIN3 acts upstream of PAP1 (Fig. 3, B and D) and PAP1 acts upstream of CINV1 (Fig. 3B and D), EIN3 directly binds to the PAP1 promoter (Fig. 4), and PAP1 directly binds to the CINV1 promoter (Fig. 5), collectively these molecular components form a Glc-HXK1-EIN3-PAP1-CINV1-Glc loop. Moreover, since EIN3 acts upstream of PIP5K9 (Supplemental Fig. S9) and PIP5K9 acts upstream of CINV1 (Lou et al., 2007), EIN3 directly binds to the PIP5K9 promoter (Fig. 6) and PIP5K9 directly interacts with CINV1 at a posttranslational level (Lou et al., 2007). The data support the presence of a Glc-HXK1-EIN3-PIP5K9-CINV1-Glc loop.

HXK1 Signaling Activity within the Glc-HXK1-EIN3-PAP1/PIP5K9-CINV1-Glc Loop

HXK1 has both signaling and enzymatic functions (Moore et al., 2003; Cho et al., 2006). We thus examined whether Glc-induced regulation of the Glc-HXK1-EIN3-PAP1/PIP5K9-CINV1-Glc loop is dependent on the signaling activity of HXK1. Ser-177 was identified as key for the catalytic activity of HXK1 but not for its sugar signaling activity (Moore et al., 2003). Thus, the gin2-1 mutant expressing HXK1S177A (S177A/gin2-1, a mutation of Ser-177) has been utilized to discern whether HXK1 plays a metabolic or a signaling role (Moore et al., 2003).

We analyzed the expression of EIN3, PAP1, CINV1, and PIP5K9 genes through quantitative PCR (qPCR). Roots grown on exogenous Glc (Fig. 7, G–J) showed increased EIN3 and PIP5K9 expression but decreased PAP1 and CINV1 expression in the gin2-1 mutant relative to wild-type Landsberg erecta (Ler) plants. Therefore, HXK1 contributes to the decreased level of EIN3 and PIP5K9 transcripts and the increased level of PAP1 and CINV1 transcripts in roots. Further analysis of S177A/gin2-1 and HXK1/gin2-1 lines revealed an expression pattern for these genes similar to that observed in wild-type Ler plants grown in media with or without Glc (Fig. 7, G–J), indicating that the enzymatic function of HXK1 is not responsible for the change in expression of EIN3, PIP5K9, PAP1, and CINV1. Consistent with these data, the gin2-1 root phenotype was completely restored through both the wild-type HXK1 and S177A transgenes (Fig. 7, A–F).

Figure 7.

Figure 7.

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The signaling role of HXK1 is integral to the root Glc-mediated feedback loop. A, Seedlings of Ler, gin2-1, HXK1/gin2-1, and S177A/gin2-1 plants grown on solid MS medium without sugar under low light (60 μmol quanta PAR m−2 s−1) for 7 d. B, Seedlings of Ler, gin2-1, HXK1/gin2-1, and S177A/gin2-1 plants grown on solid MS medium with 1% Glc under standard light conditions (150 μmol quanta PAR m−2 s−1) for 7 d. C. Seedlings of Ler, gin2-1, HXK1/gin2-1, and S177A/gin2-1 plants grown on solid MS medium with 1% Suc under standard light conditions for 7 d. Scale bars = 10 mm. D to F, Analysis of primary root length in the plant lines in A to C, respectively. Error bars represent the SD (n = 12). G to J, RT-qPCR analysis of EIN3 (G), PIP5K9 (H), PAP1 (I), and CINV1 (J) expression in 7-d-old Ler, gin2-1, HXK1/gin2-1, and S177A/gin2-1 roots grown on solid MS medium without sugar or with 3% Glc. Ler roots were set as 1.0 in the RT-qPCR analysis. Gene expression was normalized to the expression of UBQ5 for quantification. Error bars represent the SD (n = 3). ein3 eil1 double mutant determined by Student’s t test (**P < 0.01).

Taken together, the Glc-HXK1-EIN3-PAP1/PIP5K9-CINV1-Glc loop promotes root growth by HXK1-mediated Glc signaling.

The Glc-HXK1-EIN3-PAP1/PIP5K9-CINV1-Glc Loop Is under Dynamic Regulation

Our model predicts that a dynamic change in EIN3 activities in response to Glc signaling will result in transcriptional changes of PAP1, CINV1, and PIP5K9 expression through the Glc feedback loop, which in turn will dynamically impact Glc levels.

EIN3 function is reduced in the presence of Glc or Suc but not mannitol (Fig. 8, A and C). We also employed a transgenic line containing the GUS reporter gene driven by five tandem repeats of the EBS followed by a minimal 35S promoter (Li et al., 2013). GUS staining intensity in the elongated and mature root regions of a 5×EBS:GUS transgenic line treated with 3% Glc or Suc was significantly reduced. In contrast, there was no impact on GUS activity following application of mannitol (Fig. 8, B and D), implying that the downregulation of GUS activity might be caused by reduced EIN3 abundance. As a result, this reduced EIN3 function resulted in elevated transcripts of PAP1 and CINV1, together with reduced PIP5K9 expression (Fig. 8, E and F). Consistent with these data, the endogenous Glc level is dynamically elevated (Fig. 8G). Collectively, these data show that the identified Glc feedback loop is under dynamic regulation.

Figure 8.

Figure 8.

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The Glc feedback loop is dynamically induced by Glc. A, Roots of 35S:EIN3-GFP were grown on solid MS medium with 1% Suc for 10 d and then treated with 3% Glc or 3% mannitol for 4 or 8 h. Subsequently, GFP analysis was performed. Scale bar = 10 μm. B, Roots of the 5×EBS (EIN3 binding site)-GUS line were grown on solid MS medium with 1% Suc for 10 d and then treated with 3% Glc or 3% mannitol for 4 or 8 h. Subsequently, GUS staining was performed and mature regions of roots were compared. Scale bar = 100 μm. C. Images showing the differential stability of EIN3 treated with 3.0% Glc or 3.0% mannitol for 4 or 8 h. D. Expression of the 5×EBS-GUS reporter gene in roots (B) was assayed quantitatively at the indicated time points by determining GUS activity. This activity was measured in picomoles of 4-methyl umbelliferone per milligram protein per minute. Error bars represent the SD (n = 3). E and F, Bar graphs based on RT-qPCR analysis exhibiting differential expression of PAP1, CINV1, and PIP5K9 in the roots in A. Expression of the 35S:EIN3-GFP transgene in roots at 0.0 h was set at 1.0. Quantification was normalized to the expression of UBQ5. Error bars represent the SD (n = 3). G, Bar graph illustrating Glc levels of the roots in A. Error bars represent the SD (n = 3). Asterisks indicate significant difference as determined by Student’s t test (*P < 0.05 and **P < 0.01).

DISCUSSION

Feedback suppression of photosynthesis due to reduced sink demand is a well-established phenomenon (Roitsch, 1999; Lou et al., 2007). Therefore, CINV1/2 can be considered as one of a series of enzymes linked to photosynthesis that are feedback inhibited in response to changes of endogenous Suc and the hexoses Glc and Fru. Thus, changes in the endogenous levels of these sugars may result in feedback suppression of INV activity in the absence of CINV1 function (Roitsch, 1999; Lou et al., 2007). However, the cognate regulatory mechanism(s) that modulate CINV activity remain to be determined.

As seedlings develop, endogenous Suc gradually accumulates under typical photosynthetic conditions (Fig. 1I; Yu et al., 2013). Subsequently, CINV1 can irreversibly convert Suc to Glc (Barratt et al., 2009), which can be subsequently perceived through HXK1 inhibiting the abundance of EIN3, presumably via the activity of the 26S proteasome (Yanagisawa et al., 2003; Cho et al., 2006; Binder et al., 2007). Thus, direct binding of EIN3 to the PAP1 promoter at a specific EBS, repressing the transcription of PAP1, is reduced, promoting PAP1 expression (Fig. 4). Direct PAP1 binding to the CINV1 promoter is thus enhanced, increasing CINV1 expression and INV activity (Fig. 5). Simultaneously, there is also a reduction of direct EIN3 binding to the PIP5K9 promoter (Fig. 6), decreasing PIP5K9 expression. Consequently, the ability of PIP5K9 to directly interact with CINV1, thus decreasing INV activity (Lou et al., 2007), is reduced. Therefore, CINV1 activity is enhanced by both an increase in CINV1 transcription and a decrease in PIP5K9 inhibition of this enzyme. Our data hence suggest the existence of a Glc-HXK1-EIN3-PAP1/PIP5K9-CINV1-Glc loop that generates Glc and thus promotes root growth (Fig. 9).

Figure 9.

Figure 9.

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Model illustrating how CINV activity is feedback regulated by a Glc signal loop to control root growth. (1) HXK1-mediated Glc signaling induces EIN3 degradation (Yanagisawa et al., 2003). (2) EIN3 directly binds to the PAP1 promoter, suppressing PAP1 expression. (3) PAP1 directly binds the CINV1 promoter, enhancing CINV1 expression. (4) EIN3 directly binds the PIP5K9 promoter and enhances PIP5K9 expression. (5) PIP5K9 directly inhibits CINV activity (Lou et al., 2007). (6) CINV activity enhances primary root elongation. Arrows and bars represent positive and negative regulation, respectively.

However, once the photosynthetic rate declines under disadvantageous conditions, the resulting Suc deficiency decreases CINV1 expression and by extension INV activity (Lou et al., 2007). This decline in INV activity results in the accumulation of Glc (Fig. 9) due to the decreased demand for hexoses under disadvantageous conditions. This position is supported by the finding that Glc accumulation is enhanced in cinv1 seedlings (Lou et al., 2007; Barnes and Anderson., 2018). The decreased demand for hexoses feedback-inhibits photosynthesis (such as INV activity) and thus leads to Suc deficiency caused by this Glc feedback loop (Roitsch, 1999; Lou et al., 2007). As a result, under disadvantageous photosynthesis conditions, Suc deficiency delays root growth by this Glc feedback loop (Fig. 9). Collectively, our findings establish a molecular link between the major photosynthate, Suc, the predominant control point of carbon metabolism to sugar, and root growth. Further, we identify some of the cognate molecular components of this Glc feedback loop.

Interestingly, while the Suc level is decreased, the Glc level is increased in the cinv1 mutant due to feedback inhibition (Lou et al., 2007). Moreover, the Glc level in the cinv1 cinv2 mutant was enhanced under standard light conditions (Barnes and Anderson, 2018) but declined under high light (Supplemental Fig. S2C; Barratt et al., 2009). Consequently, the short roots of cinv1 cinv2 seedlings can be restored to approximately one-half the length of wild-type roots by addition of exogenous Glc (Barratt et al., 2009). Thus, it remains uncertain whether total sugar levels are enhanced or depleted in seedlings of this double mutant. Similarly, as in the gin2-1 line, loss of HXK1 function in this line leads to different Glc concentrations under either high or low light conditions (Moore et al., 2003). This indicates that HXK1 regulates root growth through its signaling function rather than through metabolic activity (Moore et al., 2003). Therefore, it is unlikely that the root growth defect exhibited in cinv1 cinv2 seedlings grown under standard light conditions is caused by Glc deficiency or the absence of Glc metabolism. In cinv1 cinv2 seedlings, the levels of hexoses were substantially enhanced under standard light conditions (Lou et al., 2007), implying possible coordination between signaling and endogenous Glc signals. In this context, we have uncovered a Glc-HXK1-EIN3-PAP1/PIP5K9-CINV1-Glc loop that is dependent on the signaling activity of HXK1 (Fig. 7).

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) ein3-1, eil1-3, ein3 eil1 (An et al., 2010), pap1-KO (myb75-1; pst16228; Bhargava et al., 2010), pap1-d (Jeong et al., 2010), cinv1 (Lou et al., 2007), and gin2-1 (CS6383; Moore et al., 2003) mutants, ER (estradiol-inducible):EIN3–FLAG and 35S:EIN3-GFP transgenic plants with wild-type background (An et al., 2010), and 5×EBS-GUS/wild-type plants (Li et al., 2013) have been described previously. The term wild-type refers to ecotype Columbia (Col-0) of Arabidopsis unless Ler is specified. In all experiments, three biological replicates were performed with similar results, and error bars represent the sd.

The 35S:EIN3/pap1-d mutant was obtained from F2 plants grown on solid MS medium with 6.0 μm 1-aminocyclopropane-1-carboxylic acid (ACC; An et al., 2010) that had shortened hypocotyls and plants grown on solid MS medium with 3% (w/v) Suc (Teng et al., 2005) that had stems and leaves of dark purple (accumulated anthocyanin pigments). The gin2/ein3 mutant was obtained from F2 seedlings of gin2-1/ein3-1 grown on solid MS medium with 6.0 μm ACC that had dark green cotyledons and small leaves (Moore et al., 2003) and elongated hypocotyls (An et al., 2010). The genotype was confirmed by PCR and specific primers for EIN3 as described previously (An et al., 2010). The pap1-d cinv1 mutant was obtained from F2 plants grown on solid MS medium with 3% (w/v) Suc that have stems and leaves of dark purple (accumulated anthocyanin pigments; Teng et al., 2005), and the absence of CINV1 transcript was confirmed by RT-PCR, as described (Barratt et al., 2009). The cinv1 cinv2 mutant was obtained by crossing the cinv1 mutant with the cinv2 mutant, and the absence of CINV1 and CINV2 transcripts was confirmed by RT-PCR, as described (Barratt et al., 2009). The pip5k9-KO (SALK_147578.31.10.X)/35S:EIN3 line was obtained from F2 plants that produced shortened hypocotyls on solid MS medium with 6.0 μm ACC (An et al., 2010), and the absence of PIP5K9 transcripts was verified by RT-PCR, as described (Lou et al., 2007). The ein3 eil1 cinv1 mutant was obtained from F2 seedlings of ein3 eil1 cinv1 plants grown on solid MS medium with 6.0 μm ACC that had elongated hypocotyls (An et al., 2010), and the absence of CINV1 transcripts was confirmed by RT-PCR, as described (Barratt et al., 2009). EIN3pER–FLAG/wild-type was crossed with the ein3 eil1 mutant and confirmed by RT-PCR to produce the EIN3pER–FLAG ein3 eil1 line. PAP1-LUC, PAP1-P1Δ-LUC, PIP5K9-LUC, and PIP5K9-P2Δ-LUC transgenes were introduced into the ER:EIN3–FLAG ein3 eil1 line by the Agrobacterium-mediated floral dip method (Meng, 2015).

Seeds were treated with jarovization at 4°C overnight and then sown on solid MS medium (Duchefe Biochemie) supplemented with different sugar levels at pH 5.8 and 0.8% (w/v) agar. Seedlings grown on agar were maintained in a growth room under a 16 h/8 h light/dark cycle with white fluorescent light (60 [low light], 160 [standard light conditions], and 230 [high light] μmol quanta PAR m−2 s−1) at 21 ± 2°C. Unless otherwise stated, seedlings grown on agar were under standard light conditions. Plants grown in soil were maintained in a controlled environment growth chamber under a 16 h/8 h light/dark cycle with cool white fluorescent light at 21 ± 2°C (Meng 2015).

Quantification of Sugar Metabolites

Seedlings were ground in liquid nitrogen. Subsequently, the powder was dissolved with 1 mL of 80% (v/v) ethanol for 1 h and samples were centrifuged at 12,000g for 10 to 15 min. The ethanol buffer and supernatant were transferred to a fresh tube and evaporated under vacuum to dry for 40 to 60 min, and the residues were redissolved in 600 μL of distilled, deionized water and maintained at 70°C for 10 to 15 min.

The aqueous fraction was extracted three times before HPLC analysis using chloroform:isoamyl alcohol (24:1 [v/v]). This assay has been described in detail previously (Meng et al., 2016).

Assay of Neutral Invertase Activities via ELISA

Neutral Invertase in Arabidopsis plants was extracted, purified, and assayed as previously reported (Meng et al., 2016).

Western Blotting

Western blotting of Glc treatments was performed as described previously (Meng et al., 2018b).

Assay of Root Length and Root Cell Length

The length of the primary roots was directly determined using a ruler. The elongation region of primary roots was photographed using a three-dimensional video microscope (HIROX) and root cell length was calculated using Image J software (http://rsbweb.nih.gov/ij/).

GUS Assay and Histochemical Assay of GUS Activity

The GUS assay (Meng et al., 2015a, 2015b) and the histochemical assay for GUS activity (Li et al., 2013) have been described previously.

GFP Imaging

GFP visualization was undertaken as described in Meng et al. (2016).

Plasmid Constructs

The relevant primers for obtaining pCB308R:EIN3-GUS, pCB308R:CINV1-GUS, and pCB2004:PAP1.

RT-qPCR

Total RNA was extracted from tissues indicated in the figures by the TRIZOL reagent (Invitrogen), as described by Meng (2015) and Meng and Yao (2015). SYBR green was used to monitor the kinetics of PCR products in real-time RT-qPCR, as described previously (Meng, 2015; Meng et al., 2018a, 2018b).

ChIP-PCR

ER (estradiol-inducible):EIN3-FLAG, 35S:EIN3-GFP, and 35S:PAP1-HA constructs were used for ChIP analysis. ChIP was performed using the indicated seedlings, as described previously (Meng et al., 2015a, b). GFP and hemagglutinin (HA) tag-specific monoclonal antibody and Anti-FLAG M2 antibody were used for ChIP analysis.

The ChIP DNA products interacting with EIN3 were analyzed through qPCR using a few pairs of primers that were synthesized to amplify ∼100- to 300-bp DNA fragments in the promoter of a few genes. The primer sequences of PAP1, PAP2, CINV1, and PIP5K9 can be found in the Supplemental Methods.

Transactivation Assay

The sequence of the PAP1 and PIP5K9 promoters can be found in the Supplemental Methods. To generate PAP1-LUC and PIP5K9-LUC, the resulting promoter sequence products were inserted into the cloning site of the pGreen0800-LUC vector. The PAP1 P1Δ-LUC construct containing a deletion in the P1 sequence of the PAP1 promoter and the PIP5K9 P2Δ-LUC construct containing a deletion in the P2 sequence of the PIP5K9 promoter were produced utilizing overlap elongation PCR and inserted into the pGreen0800-LUC vector. Protoplasts of Arabidopsis were obtained and transfected with target DNA constructs, and activities of the LUC reporter were determined (Li et al., 2013).

To generate CINV1-LUC, the CINV1 promoter was amplified via PCR (see Supplemental Methods for the relevant primers). The CINV1 promoter was inserted into the cloning site of the pGreen0800-LUC vector. The Agrobacterium tumefaciens strain with the effector or reporter constructs was coincubated for 3 h and then infiltrated into 20-d-old Nicotiana benthamiana leaf blades. Leaf blades of these seedlings were incubated under standard light (160 μmol quanta PAR m−2 s−1) for 2 to 4 d postinfiltration. Light released from firefly LUC activity was photographed after spraying with 1 mm luciferin. Dual luciferase activity resulting from either firefly luciferase or Renilla luciferase was measured as previously described (Li et al., 2010).

Protein Expression and Purification

The plasmids pGEX-5X-1-EIN3 and pGEX-5X-1-PAP1 were used for EIN3 and PAP1 expression. The relevant primer sequences can be found in the Supplemental Methods. The products were cloned into pGEX-5X-1 to generate the final plasmid. Recombinant glutathione S-transferase binding protein (GST)-tagged EIN3 and PAP1 were extracted from transformed Escherichia coli (Rosetta2) after 10 h of incubation at 16°C following induction with 10 μm isopropylß-d-1-thiogalactopyranoside. These recombinant proteins were purified using GST-agarose affinity and ultrafiltrated into Tris-buffered saline plus 10% (v/v) glycerol.

EMSA

EMSA was performed as described previously in Meng (2015).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: AT3G20770 (EIN3), AT2G27050 (EIL1), AT1G56650 (PAP1), AT1G35580 (CINV1), AT4G09510 (CINV2), AT3G09920 (PIP5K9), and AT4G29130 (HXK1).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

The pap1-D (CS3884), cinv2 (CS821919), and pip5k9-KO (SALK_147578.31.10.X) lines were obtained from the Arabidopsis Biological Resource Center (Ohio State University) and the pap1-KO (myb75-1; pst16228) line was obtained from RIKEN Bioresource. The estradiol-inducible ER:EIN3-FLAG, eil1-3, ein3-1, ein3 eil1, and 35S:EIN3-GFP seeds were kindly provided by Hong-Wei Guo (South University of Science and Technology of China). The cinv1 mutant was kindly provided by Hong-Wei Xue (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). The pGreen0800-LUC vector and 5×EBS:GUS seeds were kindly provided by Ziqiang Zhu (Nanjing Normal University). gin2-1 (CS6383), gin2-1/HXK1, and gin2-1/S177A seeds were kindly provided by R. Scott Poethig (University of Pennsylvania).

Footnotes

1

This work was supported by the Natural Science Foundation of China (grant no. 31401443) and the National Natural Science Foundation of JiangSu Province (grant no. BK20170236).

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