With the aim of clarifying the functional role of dihedral symmetry in GS functions, we first carried out a quest for GSs that share a high degree of sequence conservation, but demonstrate distinct quaternary structural assembly properties. We made use of the weak ring-ring interaction of GSII, a prominent structural difference between the type I and type II GSs (Unno et al., 2006; Torreira et al., 2014; Krajewski et al., 2008), built model structures for the candidate GSIIs based on the crystal structure of the maize GSII (pdb code:2D3B) and analyzed the amino acid variations in the context of model structures. Primary structure analysis reveals GSIIs from the plants of Camellia sinensis (CsGSIb) and Glycine max (GmGSβ2) share an overall very high sequence homology (~90 % identical and ~97 % conserved) and absolutely conserved substrate-binding and catalytic sites (Figure 1a), with, however, a significant portion of amino acid variations clustered at the interface between two pentamer rings (Figure 1a and b). We then recombinantly expressed both CsGSIb and GmGSβ2 in E. coli and purified these two GSII homologs. To assess the oligomerization status, we performed size-exclusion chromatography (SEC) coupled to both multi-angle light scattering (MALS) and quasi-elastic light scattering (QELS). MALS analysis shows the GSII from Glycine max being largely a homogeneous decamer in solution (Figure 1c). In contrast, under the same condition the majority fraction (~82%) of CsGSIb adopts a pentameric configuration, along with a minor fraction (~18%) being decameric (Figure 1d). We further show that CsGSIb exists in pentamer-decamer dynamic equilibrium in solution and a mixture of electrostatic and hydrophobic interactions is responsible for attaching of two pentameric rings; whereas substrates or ligands show no appreciable effect on the decamer-forming properties (Figure 1—figure supplement 1).

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We further employed negative-stain electron microscopy (EM) to directly visualize the distinct ring-ring packing propensities for CsGSIb and GmGSβ2. Two-dimensional (2D) class averages revealed that GmGSβ2 forms homogeneous, double stacked-ring shaped particles (Figure 1e), in line with the decameric organization pattern previously reported for other GSII species (Unno et al., 2006; Torreira et al., 2014; Krajewski et al., 2008). In contrast, CsGSIb adopted a mixture of two quaternary structural modes: detached pentamers (Figure 1f) and decamers composed of two stacked pentamer rings (Figure 1g), consistent with the above MALS analysis result (Figure 1d).

Analytical ultracentrifugation (AUC) was carried out to quantitively assess the thermodynamic parameters of CsGSIb pentamer-decamer transition, which demonstrated two species for CsGSIb in solution with molecular weights of 191 kDa and 395 kDa (Figure 2a), corresponding to the pentameric and decameric configurations, respectively. Sedimentation profiles at various protein concentrations were analyzed and a global analysis of the data yielded a pentamer-pentamer dissociation constant (Kd) of 0.27 ± 0.06 μM at room temperature. It is noteworthy that the dissociation constant within the sub-micromolar range allows CsGSIb to predominantly exist as isolated pentamers under the concentration assayed for enzymatic activities, laying a solid foundation for further probing the functional role of the decamer formation in modulating the enzyme activity of GS.

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We next sought to compare the glutamine synthesis activity of these two highly conserved GSIIs. As shown in Figure 2b and Figure 2—figure supplement 1a, when supplied with ammonium chloride, the stable decamer-adopting GmGSβ2 demonstrated significant GS activities. Further steady-state kinetic measurements yielded turnover numbers (k_cat) and Michaelis constants (K_m) of ~12.8 min^–1 and ~44 μM, respectively (Figure 2c). In sharp contrast, CsGSIb only demonstrated basal activity (Figure 2b and Figure 2—figure supplement 1a), consistent with previous observations that the isolated single-ringed GSII species is nonfunctional (Llorca et al., 2006; Mäck, 1998). These observations raised the question as to whether the drastic disparity in catalytic activities could be attributed to their distinct propensities for formation of a double-ringed architecture. If the above proposal holds true, a positive concentration-dependent cooperation of enzyme activity would be expected as increase in the concentration of CsGSIb favors decamer assembly (Figure 1—figure supplement 1a). Indeed, five times increase in the concentration of CsGSIb assayed (from 1 μM to 5 μM) resulted in ~30 folds increase in activity, that is ~6 folds activity increase per unit of enzyme, displaying a significant concentration-dependent simulation effect (Figure 2—figure supplement 1b).

To further validate the above proposal, we performed mutagenesis to CsGSIb, aiming to convert its unstable pentamer-decamer equilibrium state to a stable decamer and then evaluate the impact on catalytic activity. According to the amino acid sequence of GmGSβ2, three CsGSIb mutants, that is EVK138DIQ, I143L and Y150F, respectively, were prepared as the wild-type (WT) protein. SEC-MALS measurements revealed both mutations of I143L and EVK138DIQ led to a drastic shift in the oligomerization equilibrium towards decamer assembly (Figure 2d and f), causing significant increase in the distribution of decameric state from ~18 % in the WT-CsGSIb (Figure 1d) to >95% and ~ 72%, respectively. These observations confirmed that both mutations introduced at the interface, albeit largely conservative, dramatically fortified the decamer edifice assembly. In contrast, substitution of the tyrosine at the residue 150 with a phenylalanine showed no appreciable change in its quaternary organization mode (Figure 2—figure supplement 2a), suggesting no critical role for the residue Y150 in maintaining two CsGSIb pentameric ring subcomplexes attached. Further AUC analysis revealed replacement of I143 or EVK at residues 138–140 with leucine or DIQ yielded ring-ring disassociation constants of ~0.01 or ~ 0.04 μM, respectively (Figure 2e and g), that is, ~ 27 or ~ 7 folds of increase in the ring-ring binding affinity compared with that of WT-CsGSIb. The Gibbs energy changes upon mutation (ΔΔG) for I143L and EVK138DIQ were calculated to be –8.1 kJ/mol and –4.7 kJ/mol, respectively.

We next set out to investigate the resulting impacts on their enzymatic activities. As shown in Figure 2h–j, both mutations of I143L and EVK138DIQ caused dramatic increase in catalytic activity of ~76 and ~ 64 folds, respectively. As all mutated amino acids are distal to either the catalytic site or substrate binding regions with distances > 20 Å and hence are unlikely to be directly involved in catalytic reaction, we infer that the stimulations of the enzymatic activity of CsGSIb upon residue perturbations are induced by remote contacts between two pentamer rings. As expected, the mutation of Y150F, which did not alter pentamer-decamer equilibrium of CsGSIb, showed no noticeable change in enzymatic activity (Figure 2—figure supplement 2b).

To elucidate the detailed mechanism of how the interactions between two GSII pentameric rings remotely trigger enzymatic activity, we next employed single-particle cryo-EM imaging technique and first determined the structures of GmGSβ2 decamer, as well as that of the CsGSIb that adopts decameric configuration (thereafter named as CsGSIb^Dec). 3D classifications of 104,717 and 43,876 particles for GmGSβ2 and CsGSIb^Dec, respectively, revealed that both molecules were arranged in D5 symmetry with two pentameric rings stacked in a head-to-head manner (Figure 3a–b), a strikingly conserved structural feature for type II GS species (Unno et al., 2006; Torreira et al., 2014; Krajewski et al., 2008). Refinement of the GmGSβ2 and CsGSIb^Dec structures yielded maps with an average resolution of 2.9 Å and 3.3 Å, respectively (Figure 3—figure supplements 1–3), with literally identical dimensions of 115 Å x 115 Å x 95 Å. As expected from the very high conservation in amino acid sequence (Figure 1a), decamer structures of GmGSβ2 and CsGSIb^Dec are strikingly similar to each other, as well as to that of the GSII of maize (pdb accession number 2D3A), as highlighted by the root-mean-square deviation (r.m.s.d.) of 0.66–0.80 Å for 328–352 aligned C_α atoms. The active sites of GmGSβ2, CsGSIb^Dec are structurally highly similar to each other, as well as to that of the maize GSII (Figure 3c), suggesting the catalysis mechanisms of these plant GSIIs are essentially identical. Indeed, structural alignments of GSs reveals a conservative catalytic site geometry across a wide range of species (Supplementary file 1).

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The overall buried inter-ring surfaces for both GmGSβ2 and CsGSIb^Dec amount to ~2000 Ų, that is approximately only 400 Ų per individual monomer–monomer interaction. This highlights the weakness of the inter-ring contacts, characteristic of type II GS. Indeed, in both structures the inter-ring contacts are established by only a limited number of hydrophobic and polar interactions provided by the residues 136–141 and 146–152 segments of each of the intervening subunits (Figure 3—figure supplement 4), which behave as two gear teeth (thereafter named as tooth-1 and tooth-2, respectively) interlocking the two pentameric rings (Figure 3d–f). This observation is in line with the above result that mutations to tooth-1 resulted in drastic change in oligomeric states behavior (Figure 2d–g), and the mixed nature of the inter-ring interactions is consistent with the MALS analysis result of CsGSIb under various buffer conditions (Figure 1—figure supplement 1). Intriguingly, the local structure of the teeth regions that mediate inter-ring interactions remains largely the same in GmGSβ2 and CsGSIb^Dec (Figure 3d–e), suggesting the dramatically different propensities of GmGSβ2 and CsGSIb for decamer formation are due to the nature of the amino acids involved in inter-ring contacts, rather than the structure. Although the residue of I143 is not directly involved in inter-ring contact, we argue that its replacement with leucine may stabilize the conformation of tooth-1 via its interaction with the residue of L134, thus playing an important role in stabilizing decamer architecture (Figure 2d and e).

In order to elucidate the mechanism of how the pentameric CsGSIb (thereafter named as CsGSIb^Pen) demonstrates distinct enzymatic properties than CsGSIb^Dec (Figure 2h), we next determined the structure of CsGSIb^Pen. Lowering the sample concentration, which favored pentamer dissociation (Figure 1—figure supplement 1a), allowed us to obtain sufficient number of CsGSIb^Pen particles (Figure 3—figure supplements 2 and 3), which, in turn, enabled us to solve the cryo-EM structure of the inactive single-ringed GSII for the first time. Interestingly, we observed additional class averages in which the two masses of density attributed to the pentameric rings are no longer parallel (Figure 3—figure supplement 5). These nonparallel ring particles may reflect intermediate assembly stages in the formation/disruption of the enzyme decamer, again confirming the flexibility of the inter-ring interactions of the type II GS.

Unexpected, CsGSIb^Pen exhibited high conformational heterogeneity and 3D particles classification generated three similar structures with the r.m.s.d. ranging from 0.6 to 0.8 Å, differing in a few peripheral regions (Figure 4—figure supplement 1). The most striking difference between CsGSIb^Pen and CsGSIb^Dec is that several regions are missing in the electron density map of all three classifications of CsGSIb^Pen particles, with only 229–255 out of 356 residues electron densities in presence. As a result, CsGSIb^Pen demonstrated a decagram-shaped density map with a few regions missing at the rim (Figure 4a and Figure 4—figure supplement 1a), in sharp contrast to a pentagon-shaped map yielded by the CsGSIb^Dec particles (Figure 4b). For the 229–255 residues that show clear density in CsGSIb^Pen particles, the conformation of each subunit in three CsGSIb^Pen EM structures, as well as the arrangement pattern, closely resembles that of the CsGSIb^Dec, as evidenced by r.m.s.d. in the range of 0.7–1.0 Å (Figure 4—figure supplement 1b-d). The density-missing regions include the segments around residues of 110–117, 140–166, and 260–334, among which, the fragment around residues 260–334 is a major component making up an integral catalytic site (Figure 4c), while the segment of residues 140–166 comprising of the two gear teeth is responsible for ring-ring interaction (Figures 3d–f ,–4d). As electron density missing often reflects the conformational heterogeneity arising from internal motions Armache and Cheng, 2019, these observation strongly suggest that the conformation of CsGSIb^Pen active site is highly dynamic, contrasting sharply to the conformationally largely homogeneous CsGSIb^Dec. In support this, thermal shift assays show the melting temperature (T_m) of wild-type CsGSIb is significantly lower than that of its mutants of I143L or EVK138DIQ, indicating of structural instability for the pentameric GSII (Figure 4—figure supplement 2). We therefore conclude that the dramatically difference in the dynamic property of catalytic sites accounts for the distinct activities of pentameric and decameric CsGSIb.

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Taken together, our cryo-EM structures allow us to propose a disorder-order transition mechanism of how the GSII activity is regulated by changes in oligomeric state: (1) The active sites within isolated CsGSIb^Pen rings are highly disordered and the unstable catalytic environments render it catalytically inactive; (2) Upon stacking of two pentameric rings and formation of a decamer, the signals of interactions mediated by the gear teeth of each intervening subunit are propagated to the active sites and induce folding, which, in turn, unlock the catalytic potential of GSII (Figure 4e).

Having mechanistically established the functional coupling between GSII activity and its quaternary assembly status, we next asked whether there exist cellular factors that may regulate the CsGSIb activity, potentially via favoring its decamer assembly. 14-3-3 proteins are an important family of scaffold proteins that bind and regulate many key proteins involved in diverse intracellular processes in all eukaryotic organisms (Tzivion et al., 2001; Chevalier et al., 2009; Obsilova and Obsil, 2020). In particular, self-dimerization of 14-3-3 proteins, which induces dimerization of their clients, plays a key role in its functional scaffolding and subsequent activity regulation (Tzivion et al., 2001; Chevalier et al., 2009; Kondo et al., 2019). It has been reported that 14-3-3 proteins act as an activator of GSs in various plants (Finnemann and Schjoerring, 2000; Pozuelo et al., 2001; Lima et al., 2006; Riedel et al., 2001), although the detailed activation mechanism remains unclear. Based on these findings, here we tentatively provide the missing link in mechanistically assigning the role 14-3-3 proteins play in regulating GS activity: One protomer of the 14-3-3 protein recognizes one phosphorylated GSII pentamer, and its self-dimerization brings two pentamer rings in close proximity and therefore promotes decamer assembly, which, in turn, switches on the GS activity via rigidification of the catalytic sites (Figure 5a). One prerequisite for this proposal is that, for the GS species whose activities being 14-3-3 protein-dependent, they must have an intrinsically weak decamer-forming propensity that is to be overcome by 14-3-3. In support of this, the GS from Medicago truncatula, whose activity is simulated upon binding to 14-3-3 protein (Lima et al., 2006), has been shown to exhibit a dynamic pentamer-decamer transition (Torreira et al., 2014), similar to the CsGSIb presented here (Figure 1d). Moreover, it has been shown that only the higher order complex of tobacco GS-2 that is bound to 14-3-3 is catalytically active (Riedel et al., 2001).

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To further support the above proposal, we then explored whether the activity of the weak-decamer forming CsGSIb could also be regulated by 14-3-3 scaffold protein. Homology search against tea plant genome revealed several candidate tea plant Cs14-3-3 proteins (Figure 5—figure supplement 1). Analysis of their coding genes' expression patterns in tea plant tissues and in nitrogen assimilation or metabolism-related processes allowed us to identify Cs14-3-3-1a and Cs14-3-3-1b genes that displayed expression patterns highly similar to CsGSI genes (Figure 5a and Figure 5—figure supplement 2). Moreover, the expression levels of Cs14-3-3-1a and Cs14-3-3-1b genes were regulated upon changes in the availability of ammonia (Figure 5b and c), the substrate of GS, suggesting both Cs14-3-3-1a and Cs14-3-3-1b are physiologically related to GS. We then examined the in vivo interactions between Cs14-3-3-1a and CsGSIb using the bimolecular fluorescence complementation (BiFC) technique, which is based on complementation between two non-fluorescent fragments of a fluorescent protein when they are brought together by interactions between proteins fused to each fragment Kerppola, 2006. Cs14-3-3-1a or CsGSIb was fused in frame with N-terminal half of a yellow florescence protein (NYFP) or C-terminal half of a yellow florescence protein (CYFP), respectively, and expressed in tobacco leaf epidermal cells alone or in various combinations, such as CsGSIb ^CYFP alone or together with Cs14-3-3-1a^NYFP. As expected, Cs14-3-3-1a and 1b could self-dimerize or form heterodimers in plant cells (Figure 5e), consistent with 14-3-3 scaffold proteins adopting a dimeric structure (Tzivion et al., 2001; Chevalier et al., 2009; Kondo et al., 2019). Importantly, formation of the fluorescent complex clearly demonstrated the interaction of Cs14-3-3-1a with CsGSIb (Figure 5f). In order to further establish the functional relevance, we performed the RNA interference (RNAi) technique to knock down the transcript level of Cs14-3-3-1a gene in hairy roots of chimerical transgenic tea seedlings (Figure 5h), and evaluated the impact on GS activity by measuring the contents of GS catalysis product, glutamine. We show that, along with the reduction in Cs14-3-3-1a transcript level, the glutamine contents (Figure 5i), as well as the crude enzyme activity (Figure 5j), were drastically reduced, indicating the 14-3-3 protein in Camellia sinensis functions as an activator molecule of CsGSIb. Further work is needed to elucidate the detailed mechanism of how Cs14-3-3-1a recognizes phosphorylated CsGSIb.

Being a key enzyme implicated in many aspects of the complex matrix of nitrogen metabolism, GS must be strictly regulated. Decades of efforts have been applied to understand how GS is controlled at gene, transcript and protein levels (Bernard and Habash, 2009). Studies have demonstrated positive cooperativity of GS with regard to different substrates and cofactors, such as L-glutamate (Montanini et al., 2003) and metal cations (Sakakibara et al., 1996); and GS functions are regulated by multiple post-translational mechanisms including nitration, oxidative turnover and phosphorylation (Seabra and Carvalho, 2015), and by the 14-3-3 protein (Finnemann and Schjoerring, 2000; Pozuelo et al., 2001; Lima et al., 2006; Riedel et al., 2001). Our results reconcile with many of the above observations, and allow us to gain a more complete picture of how GS activity is regulated in cells by an exquisite machinery (Figure 6). While the protein turnovers machineries to adjust cellular enzyme level can always provide means to modulate pentamer-decamer transitions and thus deactivation-activation conversion of GSII, we argue that phosphorylation-dephosphorylation processes, coupled with 14-3-3 binding and subsequent activation, may enable a more efficient regulatory way. When sufficient reaction products are available demanding low glutamine synthesis activity, GSII is kept in the dephosphorylated state and exists as isolated inactive single-ringed pentamers. In the physiological context of high demand of glutamine, phosphorylation of GSII by certain kinase prompts 14-3-3 protein binding, and the intrinsic dimerization property of 14-3-3 recruits two GSII pentamer rings in close proximity and in doing so, result in a rapid transition of quaternary assembly from the pentamer to decamer, and eventually enzymatic activation. In this manner, the poised GSII pentamer ring itself acts as a positive effector and the functional ring-ring association offers a great advantage of immediate response to precisely meet the ever-changing metabolic needs, whereas the reversible assembly-disassembly behavior enables a tunable mode for activity modulation. Indeed, the dynamic association-disassociation of GSII subcomplexes, a prerequisite for this modulatory machinery, have been widely observed in various species including humans (Denman and Wedler, 1984), plants other than Camellia sinensis reported here (Torreira et al., 2014; Llorca et al., 2006) and fungi (Mora, 1990). Therefore, the activation mechamism shown here may represent a general regulatory machinery harnessed by many eukaryotes to ensure optimal utilization of nitrogen sources, and the infrastructure of fragile ring-ring contacts evolutionarily chosen by many eukaryotes offers a convenient and robust avenue for activity regulation.

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As a crucial enzyme to all living organisms, which is involved in all aspects of nitrogen metabolism, GS has emerged as an attractive target for drug design (Mowbray et al., 2014) and herbicidal compounds development, as well as a suitable intervention point for the improvement of crop yields (Thomsen et al., 2014). However, because the overall geometry of the active site is the most conserved structural element amongst GS enzymes (Eisenberg et al., 2000; van Rooyen et al., 2011; Unno et al., 2006), the traditional strategy of selective inhibition, which relies heavily on the subtle difference in the active sites from different species, has only achieved limited success. Thus, the regulatory mechanism discovered here will help guide the search for specific inhibitors of potential therapeutic interest. For example, inhibition of the GS in Mycobacterium tuberculosis has long been recognized as a novel antibiotic strategy to treat tuberculosis (Harth and Horwitz, 2003; Gising et al., 2012; Kumari and Subbarao, 2020). Our result opens new possibilities to develop chemicals to target the drugable ring-ring interface region and specifically interrupt the interactions between two GS subcomplexes in pathogens or unwanted plants to develop new types of herbicide. Moreover, although overexpression of GS has been investigated extensively for decades with the goal of improving crop nitrogen use efficiency, the outcome has not been consistent (Thomsen et al., 2014). The modulatory ‘hot-spots’ identified here, which mediate inter-ring communication and in turn stimulate GS activity, will guide engineering catalytically more powerful GSs for crops in which pentamer units only weakly associate, and thus increase plant nitrogen use efficiency and crop production.

The target genes encoding CsGSIb from Camellia sinensis (Genbank accession No. MK716208) and GmGSβ2 from Glycine max (Genbank accession No. NM001255403) were cloned into the pET-16b vector (Novagen) containing a His₆-tagged-MBP tag followed by a tobacco etch virus (TEV) protease cleavage site at the N-terminus. All constructs were transformed into E. coli Rosetta (DE3) cells, which were cultured in Luria-Bertani (LB) medium at 37 °C supplemented with ampicillin (100 µg/ml) and chloramphenicol (35 µg/ml) to an OD600 ~0.8. Cells were induced by the addition of isopropyl-β-D-1- thiogalactopyranoside (IPTG) to the concentration of 0.3 mM, and incubated for additional 16 hr at 18 °C. Cells were harvested by centrifugation at 5000 g for 20 min and resuspended in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, pH 8 and 1 mM PMSF).

Cells were subjected to a high-pressure homogenizer, named JN-Mini Pro Low-temperature Ultra-high-pressure cell disrupter (JNBIO) and then centrifuged at 50,000 g for 30 min at 4°C. Proteins were initially purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare). The protein tags were cleaved with His-tagged TEV-protease overnight at 4 °C while dialyzing against TEV cleavage buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM β-mercaptoethanol, pH 8). Cleaved sample was collected and run over Ni-NTA column to remove His-tagged TEV and protein tags. Flow-through was collected, concentrated and passed over Hiload 16/600 Superdex 200 column (GE Healthcare) in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5 mM MgCl₂ and 1.5 mM β-mercaptoethanol.

MALS was measured using a DAWN HELEOS-II system (Wyatt Technology Corporation) downstream of a GE liquid chromatography system connected to a Superdex 200 10/300 GL (GE Healthcare) gel filtration column. The running buffer for the protein samples contained 50 mM KPi (pH 7.0), 100 mM NaCl, 1 mM β-mercaptoethanol and 0.05 % NaN₃. The flow rate was set to 0.5 mL min⁻¹ with an injection volume of 200 μL, and the light scattering signal was collected at room temperature (~23 °C). The data were analyzed with ASTRA version 6.0.5 (Wyatt Technology Corporation).

GS activity assay was performed described previously (Gawronski and Benson, 2004; Masalkar and Roberts, 2015) and reactions were performed for 30 min at 37 °C in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5 mM MgCl₂ and 1.5 mM β-mercaptoethanol. Enzymatic activity comparison was conducted with 1 μM (monomer) enzyme in the presence of 0.5 mM NH₄HCl, 2 mM L-glutamate, 0.5 mM ATP. Steady-state kinetic analysis was performed under the same conditions except with the variable concentration of ammonium chloride from 0.05 mM to 4 mM. Steady-state kinetic parameters were determined by double reciprocal Lineweaver-Burk plot for reactions that followed Michaelis-Menten kinetics. All experiments were repeated independently at least three times.

Reactants containing 2 uM CsGSIb (monomer) and 1000-fold diluted Sypro Orange in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl and 1.5 mM β-mercaptoethanol were performed using an iCycler thermocycler (Bio-Rad) as previous described (Krajewski et al., 2008). Briefly, CsGSIb in presence of various concentrations of the following ligands were tested, alone and in combination: 10 mM glutamate, 20 mM MgCl₂ and 1 mM ATP. The temperature of the reactions was increased from 20°C to 90 °C in increments of 0.2 °C/12 s, coincident with a fluorescent measurement at each step. The wavelengths for excitation and emission were set to 490 and 575 nm, respectively. Fluorescence changes were monitored simultaneously with a charge-coupled device (CCD) camera. To obtain the temperature midpoint for the protein unfolding transition, Tm, a Boltzmann model was used to fit the fluorescence imaging data obtained by the CCD detector using the curve-fitting software GraphPad Prism 7.0.

Sedimentation velocity experiments were carried out with a Proteomelab XL-A analytical ultracentrifuge (Beckman Coulter, USA) using a four-hole An-60 Ti analytical rotor. An aliquot of 410 μL of buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl and 1.5 mM β-mercaptoethanol) as the reference and 400 μL of protein solution (0.1/0.25/0.5 mg⋅mL⁻¹) were loaded into a double-sector cell. A centerpiece with a path length of 12 mm was used. The speed of rotor was 35,000 rpm. The operation temperature of rotor was 20°C. The time dependence of the absorbance at different radial positions was monitored at a wavelength of 280 nm by an UV–Vis absorbance detector, and the data were analyzed by the software SEDFIT (version 15.01b) using c(s) model to obtain the sedimentation coefficient distribution. Viscosity and density of the buffer solution were calculated by the Sednterp software.

Purified protein samples of CsGSIb (4 μL, 0.02 mg/mL) and GmGSβ2 (4 μL, 0.02 mg/mL) in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 1.5 mM β-mercaptoethanol were negatively stained with uranyl acetate 1 % (w/v) on carbon-film 400 mesh copper grids. Samples were imaged using a FEI T12 operated at 120 keV with a 3.236 Å pixel size, 68,000× nominal magnification, and defocus range about 1.5 μm. For cryo-EM, 3 μL of CsGSIb (0.1 mg/mL and 0.5 mg/mL) and GmGSβ2 (0.1 mg/mL) were added onto glow-discharged Quantifoil R1.2/1.3 100 holey-carbon Cu grids with a Vitrobot Mark IV (Thermo Fisher Scientific). The grids were blotted for 3.5 s at 8 °C with 100 % humidity, and then plunged frozen into liquid ethane cooled by liquid nitrogen. Cryo-grids were first screened on a FEI TF20 operated at 200 keV. Images of CsGSIb and GmGSβ2 were collected using Titan Krios G3i microscope (FEI) operated at 300 kV with a Gatan K2 Summit direct detection camera. Two datasets were acquired using the SerialEM in super-resolution mode with a nominal magnification of 29,000 x, yielding a pixel sizes of 0.505 Å with a total dose of 51 e/ Ų. The defocus ranges were set from −1.6 μm to −2.3 μm.

All processing steps were performed using cryoSPARC (Punjani et al., 2017). A total of 4051 raw movie stacks acquired for CsGSIb and 1777 raw movie stacks for GmGSβ2 were subjected to patch motion correction and patch CTF estimation. An initial set of about 500 particles were manually picked to generate 2D templates for auto-picking. The auto-picked particles were extracted by a box size of 512 pixel and then subjected to reference-free 2D classification. After particle screening using 2D and 3D classification, the final 355,289 particles for CsGSIb and 115,795 particles for GmGSβ2 were subjected to Ab-Initio Reconstitution and followed by 3D Refinement with C5 symmetry imposed. Four different conformational states were obtained for CsGSIb, resulting in a 3.3 Å density map for CsGSIb^Dec, 3.5 Å density map for CsGSIb^Pen State I, 3.6 Å density map for CsGSIb^Pen State II, and 3.4 Å density map for CsGSIb^Pen State III. Only one major conformation was obtained with 2.9 Å density map for GmGSβ2. The global resolution of the map was estimated based on the gold-standard Fourier shell correlation (FSC) using the 0.143 criterion.

Homology models of CsGSIb and GmGSβ2 were generated with the I-TASSER server (Yang and Zhang, 2015) and docked into the cryoEM maps using UCSF Chimera (Pettersen et al., 2004). The sequences were mutated with corresponding residues in CsGSIb and GmGSβ2, followed by rebuilding in Coot (Emsley et al., 2010). The missing residues of CsGSIb^Pen were not built due to the lack of corresponding densities. Real-space refinement of models with geometry and secondary structure restraints applied was performed using PHENIX (Afonine et al., 2018). The final model was subjected to refinement and validation in PHENIX. The statistics of cryo-EM data collection, refinement and model validation are summarized in Supplementary file 2.

Two-year-old hydroponic tea cuttage seedlings were grown in a greenhouse at 20–25°C until new tender roots emerged. These healthy tea seedlings were then transferred into hydroponic solutions with different nitrogen sources, namely, 0 mM NH₄⁺ (Shigeki Konishi solution), 5 mM NH₄⁺ (Shigeki Konishi solution with 5 mM ammonium nitrogen), 10 mM NH₄⁺ (Shigeki Konishi solution with 10 mM ammonium nitrogen), and a control (Shigeki Konishi solution alone). All these tea seedling root samples were cleaned and collected in liquid nitrogen after treatment for RNA analysis.

Tea plant tissues or root materials were ground in liquid nitrogen into fine powders for total RNA extraction with an RNA extraction kit (Tiangen Biotech Co., Ltd.) according to the manufacturer’s instructions. RNA quality and purity were assessed by a NanoDrop 2000 spectrophotometer (Thermo Scientific). The integrity of the RNA samples was rapidly checked by 1.0 % agarose gel electrophoresis. The total RNA was reverse-transcribed to single-stranded cDNAs using SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using cDNA synthesized by the Prime Script RT Reagent Kit (Takara). Each qRT-PCR was conducted in a 20 μL reaction mixture containing 2 μL of diluted template cDNA, 0.4 μL of each specific primer, 10 μL of SYBR Premix Ex-Taq (Takara), and 7.2 μL of H₂O. All qRT-PCR assays were performed on the Bio-Rad CFX96 fluorescence-based quantitative PCR platform. The program used was as follows: 95 °C for 5 min; 40 cycles of 95 °C for 5 s for denaturation and 60 °C for 30 s for annealing and extension; and 61 cycles of 65 °C for 10 s for melting curve analysis. All experiments were independently repeated three times, and relative expression levels were measured using the 2−ΔCt method.

Construction of the Cs14-3-3-1a-GFP, CsGSI1b-GFP, and Cs14-3-3-1b-GFP fusions were performed using gateway recombination systems. The corresponding ORFs for CsGSIb and Cs14-3-3-1a, 1b were subcloned into pK7WGF2 in frame with a GFP tagged at the N-terminus. Determination of the subcellular localization of these GFP fusions was performed using tobacco leaf infiltration as previously described (Zhao et al., 2011). Briefly, the pK7WGF2-Cs14-3-3-1a-GFP, pK7WGF2-Cs14-3-3-1b-GFP, and pK7WGF2-CsGSIb-GFP plasmids were transformed into A. tumefaciens strain EHA105, and selected positive colonies harboring these constructs were used for plant transformation by infiltration. Acetosyringone-activated Agrobacterium cells were infiltrated into the Nicotiana benthamiana leaves leaf abaxial epidermal surface, and the tobacco plants were grown at room temperature for 3 days before imaging. Imaging of these GFP fusion proteins was performed using a confocal microscope with a 100× water immersion objective and appropriate software. The excitation wavelength was 488 nm, and emissions were collected at 500 nm.

The ORFs of Cs14-3-3-1a, Cs14-3-3-1b, and CsGSIb were amplified and subcloned into pCAMBIA1300-eYFPN (YFP N-terminal portion) and pCAMBIA1300-eYFPC (YFP C-terminal portion) (CAMBIA) by the in-fusion technology. The resulting constructs were transformed into A. tumefaciens strains GV3101, which were infiltrated into Nicotiana benthamiana leaves individually or in different pair combinations. A Leica DMi8 M laser scanning confocal microscopy system was used for fluorescence observation, according to the method described previously (Zhao et al., 2011). If the fluorescence signal could be detected with any interaction pair, the pair of half YFP-fusion proteins should interact.

Approximately 400 bp of the gene-specific fragments from Cs14-3-3-1a were amplified and subcloned into the final RNA interference (RNAi) destination vector pB7GWIWG by BP and LR clonase-based recombination reactions (Invitrogen). The resulting binary vectors pB7GWIWG-Cs14-3-3-1a were transformed into A. rhizogenes strain ATCC 15834 by electroporation. The selected positive transformants harboring pB7GWIWG-Cs14-3-3-1a were used to transform 3-month-old tea seedlings, which were pretreated with acetosyringone. The positive transgenic hairy root lines were verified with qRT-PCR for examination of transgene expression. At least three independent hairy root lines were selected for further analysis.

The free amino acids in tea plant samples were analyzed by using an amino acid analyzer (L-8900, Hitachi) according to manufacture instruction. The free amino acids were extracted from 120 mg leaves with 1 mL of 4 % sulfosalicylic acid in water bath sonication for 30 min and then centrifuged at 13,680 x g for 30 min. The debris was re-extracted once again and the supernatants from two extractions were combined as previously described (Lu et al., 2019). The supernatants were filtered through a 0.22 µm Millipore filter before analysis. A mobile phase containing lithium citrate for amino acid derivatization and UV–Vis detection at 570 and 440 nm were used in the Hitachi High-Speed Amino Acid Analyzer system. The flow rates were set at 0.35 mL/min for the mobile phase and 0.3 mL/min for the derivatization reagent. The temperature for separation column was set to 38 °C, and for the post-column reaction equipment was maintained at 130 °C. The temperature of the autosampler was kept at 4 °C. The peak areas of amino acids were quantified in comparison with the amino acid standards.

To determine the total GS activity, 100 mg of frozen plant samples were grounded into fine powder in liquid nitrogen. Samples were homogenized in extraction buffer (50 mmol/L Tris-HCl, pH 8.0, 2 mmol/L MgSO₄, 4 mmol/L dithiothreitol, and 0.4 mmol/L sucrose). Plant extracts were centrifuged at 13,680 x g (4 °C) for 25 min and the supernatants of extracts were analyzed for the soluble protein content using the Bradford assay. GS activity was determined after incubating the enzyme extracts in a reaction buffer (100 mmol/L Tris-HCl, 80 mmol/L MgSO₄, 20 mmol/L sodium glutamate, 80 mM NH₄OH, 20 mmol/L cysteine, 2 mmol/L EGTA and 40 mmol/L ATP) at 37 °C for 30 min (Husted et al., 2002). A stop solution containing 0.2 mol/L Trichloride acetic acid, 0.37 mol/L FeCl₃ and 0.6 mol/L HCl was added; and the absorbance of enzyme reactions at 540 nm was recorded. A standard curve was made in an identical way for calculation of the specific enzyme activity.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

© 2021, Chen et al.

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