Removal of redox-sensitive Rubisco Activase does not alter Rubisco regulation in soybean.

Rubisco activase (Rca) facilitates the catalytic repair of Rubisco, the CO2-fixing enzyme of photosynthesis, following periods of darkness, low to high light transitions or stress. Removal of the redox-regulated isoform of Rubisco activase, Rca-α, enhances photosynthetic induction in Arabidopsis and has been suggested as a strategy for the improvement of crops, which may experience frequent light transitions in the field; however, this has never been tested in a crop species. Therefore, we used RNAi to reduce the Rca-α content of soybean (Glycine max cv. Williams 82) below detectable levels and then characterized the growth, photosynthesis, and Rubisco activity of the resulting transgenics, in both growth chamber and field conditions. Under a 16 h sine wave photoperiod, the reduction of Rca-α contents had no impact on morphological characteristics, leaf expansion rate, or total biomass. Photosynthetic induction rates were unaltered in both chamber-grown and field-grown plants. Plants with reduced Rca-α content maintained the ability to regulate Rubisco activity in low light just as in control plants. This result suggests that in soybean, Rca-α is not as centrally involved in the regulation of Rca oligomer activity as it is in Arabidopsis. The isoform stoichiometry supports this conclusion, as Rca-α comprises only ~ 10% of the Rubisco activase content of soybean, compared to ~ 50% in Arabidopsis. This is likely to hold true in other species that contain a low ratio of Rca-α to Rca-ß isoforms.

Phosphorylation-dependent subfunctionalization of the calcium-dependent protein kinase CPK28.

Calcium (Ca2+)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca2+ sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca2+ activation at physiological concentrations of Ca2+, possibly through stabilization of the Ca2+-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca2+] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca2+-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.

Arabidopsis plants expressing only the redox-regulated Rca-α isoform have constrained photosynthesis and plant growth.

Rubisco activase (Rca) facilitates the release of sugar-phosphate inhibitors from the active sites of Rubisco and thereby plays a central role in initiating and sustaining Rubisco activation. In Arabidopsis, alternative splicing of a single Rca gene results in two Rca isoforms, Rca-α and Rca-ß. Redox modulation of Rca-α regulates the function of Rca-α and Rca-ß acting together to control Rubisco activation. Although Arabidopsis Rca-α alone less effectively activates Rubisco in vitro, it is not known how CO2 assimilation and plant growth are impacted. Here, we show that two independent transgenic Arabidopsis lines expressing Rca-α in the absence of Rca-ß ('Rca-α only' lines) grew more slowly in various light conditions, especially under low light or fluctuating light intensity, and in a short day photoperiod compared to wildtype. Photosynthetic induction was slower in the Rca-α only lines, and they maintained a lower rate of CO2 assimilation during both photoperiod types. Our findings suggest Rca oligomers composed of Rca-α only are less effective in initiating and sustaining the activation of Rubisco than when Rca-ß is also present. Currently there are no examples of any plant species that naturally express Rca-α only but numerous examples of species expressing Rca-ß only. That Rca-α exists in most plant species, including many C3 and C4 food and bioenergy crops, implies its presence is adaptive under some circumstances.

The brassinosteroid receptor kinase, BRI1, plays a role in seed germination and the release of dormancy by cold stratification.

Seed dormancy is a critical mechanism that delays germination until environmental conditions are favorable for growth. Plant hormones gibberellin (GA) and abscisic acid (ABA) have long been recognized as key players in regulating dormancy and germination. Recent data have increased interest in brassinosteroid (BR) hormones that promote germination by activating GA downstream genes and inactivating ABA signaling. Exposure of imbibed seeds to low temperature (cold stratification) is widely used to release seed dormancy and to improve germination frequency. However, the mechanism by which cold stratification overcomes the inhibitory role of ABA is not completely understood. In the present study, we show delayed germination of seeds of the BR insensitive mutant, bri1-5, that was largely reversed by treatment with fluridone, an inhibitor of ABA biosynthesis. In addition, the bri1-5 seeds were markedly less sensitive to the cold stratification release of dormancy. These results suggest that BR locates upstream of ABA signaling and downstream of cold stratification signaling in dormancy and germination pathways. Consistent with this notion, BR biosynthetic genes, DWF4 and DET2, were upregulated by cold stratification. The transcripts of the GA biosynthesis gene, GA3ox1, and cold responsive genes, CBF1 and CBF2, increased in response to cold stratification in wild type seeds but not in bri1-5 seeds. Conversely, transgenic seeds overexpressing BRI1 germinated more rapidly than wild type in the absence of cold stratification. Thus, we propose that BR signaling plays a previously unrecognized role in the cold stratification pathway for seed dormancy and germination.

In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site.

Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine-78 (Thr78) in low light and in the dark, suggesting a potential regulatory role in photosynthesis, but this has not been directly tested. To do so, we transformed an rca-knockdown mutant largely lacking redox regulation with wild-type Rca-ß or Rca-ß with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site-directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-ß wild-type control, as evidenced by immunoblotting with custom antibodies and quantitative mass spectrometry. Moreover, plants expressing the T78S mutation had reduced photosynthesis and quantum efficiency of photosystem II (ϕPSII) and reduced growth relative to control plants expressing wild-type Rca-ß under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-ß with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ϕPSII relative to Rca-ß controls. While expression of the wild-type Rca-ß or the T78A mutant fully rescued the slow-growth phenotype of the rca-knockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-ß control plants at low light (30 µmol photons m-2 s-1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

Increased temperatures may safeguard the nutritional quality of crops under future elevated CO2 concentrations.

Iron (Fe) and zinc (Zn) deficiencies are a global human health problem that may worsen by the growth of crops at elevated atmospheric CO2 concentration (eCO2 ). However, climate change will also involve higher temperature, but it is unclear how the combined effect of eCO2 and higher temperature will affect the nutritional quality of food crops. To begin to address this question, we grew soybean (Glycine max) in a Temperature by Free-Air CO2 Enrichment (T-FACE) experiment in 2014 and 2015 under ambient (400 µmol mol-1 ) and elevated (600 µmol mol-1 ) CO2 concentrations, and under ambient and elevated temperatures (+2.7°C day and +3.4°C at night). In our study, eCO2 significantly decreased Fe concentration in soybean seeds in both seasons (-8.7 and -7.7%) and Zn concentration in one season (-8.9%), while higher temperature (at ambient CO2 concentration) had the opposite effect. The combination of eCO2 with elevated temperature generally restored seed Fe and Zn concentrations to levels obtained under ambient CO2 and temperature conditions, suggesting that the potential threat to human nutrition by increasing CO2 concentration may not be realized. In general, seed Fe concentration was negatively correlated with yield, suggesting inherent limitations to increasing seed Fe. In addition, we confirm our previous report that the concentration of seed storage products and several minerals varies with node position at which the seeds developed. Overall, these results demonstrate the complexity of predicting climate change effects on food and nutritional security when various environmental parameters change in an interactive manner.

Four tyrosine residues of the rice immune receptor XA21 are not required for interaction with the co-receptor OsSERK2 or resistance to Xanthomonas oryzae pv. oryzae.

Tyrosine phosphorylation has emerged as an important regulator of plasma membrane-localized immune receptors activity. Here, we investigate the role of tyrosine phosphorylation in the regulation of rice XANTHOMONAS RESISTANCE 21 (XA21)-mediated immunity. We demonstrate that the juxtamembrane and kinase domain of Escherichia coli-expressed XA21 (XA21JK) autophosphorylates on tyrosine residues. Directed mutagenesis of four out of the nine tyrosine residues in XA21JK reduced autophosphorylation. These sites include Tyr698 in the juxtamembrane domain, and Tyr786, Tyr907, and Tyr909 in the kinase domain. Rice plants expressing XA21-GFP fusion proteins or proteins with these tyrosine residues individually mutated to phenylalanine (XA21YF-GFP), which prevents phosphorylation at these sites, maintain resistance to Xanthomonas oryzae pv. oryzae. In contrast, plants expressing phosphomimetic XA21 variants with tyrosine mutated to aspartate (XA21YD-GFP) were susceptible. In vitro purified XA21JKY698F, XA21JKY907F, and XA21JKY909F variants are catalytically active, whereas activity was not detected in XA21JKY768F and the four XA21JKYD variants. We previously demonstrated that interaction of XA21 with the co-receptor OsSERK2 is critical for biological function. Four of the XA21JKYF variants maintain interaction with OsSERK2 as well as the XA21 binding (XB) proteins XB3 and XB15 in yeast, suggesting that these four tyrosine residues are not required for their interaction. Taken together, these results suggest that XA21 is capable of tyrosine autophosphorylation, but the identified tyrosine residues are not required for activation of XA21-mediated immunity or interaction with predicted XA21 signaling proteins.

Revisiting paradigms of Ca2+ signaling protein kinase regulation in plants.

Calcium (Ca2+) serves as a universal second messenger in eukaryotic signal transduction. Understanding the Ca2+ activation kinetics of Ca2+ sensors is critical to understanding the cellular signaling mechanisms involved. In this review, we discuss the regulatory properties of two sensor classes: the Ca2+-dependent protein kinases (CPKs/CDPKs) and the calcineurin B-like (CBL) proteins that control the activity of CBL-interacting protein kinases (CIPKs) and identify emerging topics and some foundational points that are not well established experimentally. Most plant CPKs are activated by physiologically relevant Ca2+ concentrations except for those with degenerate EF hands, and new results suggest that the Ca2+-dependence of kinase activation may be modulated by both protein-protein interactions and CPK autophosphorylation. Early results indicated that activation of plant CPKs by Ca2+ occurred by relief of autoinhibition. However, recent studies of protist CDPKs suggest that intramolecular interactions between CDPK domains contribute allosteric control to CDPK activation. Further studies are required to elucidate the mechanisms regulating plant CPKs. With CBL-CIPKs, the two major activation mechanisms are thought to be (i) binding of Ca2+-bound CBL to the CIPK and (ii) phosphorylation of residues in the CIPK activation loop. However, the relative importance of these two mechanisms in regulating CIPK activity is unclear. Furthermore, information detailing activation by physiologically relevant [Ca2+] is lacking, such that the paradigm of CBLs as Ca2+ sensors still requires critical, experimental validation. Developing models of CPK and CIPK regulation is essential to understand how these kinases mediate Ca2+ signaling and to the design of experiments to test function in vivo.

Allosteric Control of a Plant Receptor Kinase through S-Glutathionylation.

Growing evidence supports the importance of protein S-glutathionylation as a regulatory post-translational modification with functional consequences for proteins. Discoveries of redox-state-dependent protein kinase S-glutathionylation have fueled discussion of redox-sensitive signaling. Following previously published experimental evidence for S-glutathionylation induced deactivation of the Arabidopsis thaliana kinase BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR-LIKE KINASE 1 (BAK1), we investigated the consequences of S-glutathionylation on the equilibrium conformational ensemble of BAK1 using all-atom molecular dynamics simulations. We found that glutathionylation of C408 allosterically destabilizes the active-like state of BAK1 and stabilizes an inactive conformation known to recur in protein kinases. Glutathionylation of C408 also has structural consequences throughout the BAK1 kinase domain, whereas glutathionylation of C353 in the N-lobe and C374 near the ATP-binding site have few notable effects on BAK1 compared with the unmodified protein. Our results suggest an allosteric mechanism for inhibition of BAK1 by C408 S-glutathionylation, and more generally, support the notion of protein kinase S-glutathionylation as a means of redox signaling in plant cells.

Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity.

The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1) functions as a co-receptor with several receptor kinases including the brassinosteroid (BR) receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), respectively, which are involved in immunity. BAK1 is a dual specificity protein kinase that can autophosphorylate on serine, threonine and tyrosine residues. It was previously reported that phosphorylation of Tyr-610 in the carboxy-terminal domain of BAK1 is required for its function in BR signaling and immunity. However, the functional role of Tyr-610 in vivo has recently come under scrutiny. Therefore, we have generated new BAK1 (Y610F) transgenic plants for functional studies. We first produced transgenic Arabidopsis lines expressing BAK1 (Y610F)-Flag in the homozygous bak1-4 bkk1-1 double null background. In a complementary approach, we expressed untagged BAK1 and BAK1 (Y610F) in the bak1-4 null mutant. Neither BAK1 (Y610F) transgenic line had any obvious growth phenotype when compared to wild-type BAK1 expressed in the same background. In addition, the BAK1 (Y610F)-Flag plants responded similarly to plants expressing BAK1-Flag in terms of brassinolide (BL) inhibition of root elongation, and there were only minor changes in gene expression between the two transgenic lines as monitored by microarray analysis and quantitative real-time PCR. In terms of plant immunity, there were no significant differences between plants expressing BAK1 (Y610F)-Flag and BAK1-Flag in the growth of the non-pathogenic hrpA- mutant of Pseudomonas syringae pv. tomato DC3000. Furthermore, untagged BAK1 (Y610F) transgenic plants were as responsive as plants expressing BAK1 (in the bak1-4 background) and wild-type Col-0 plants toward treatment with the EF-Tu- and flagellin-derived peptide epitopes elf18- and flg22, respectively, as measured by reactive oxygen species production, mitogen-activated protein kinase activation, and seedling growth inhibition. These new results do not support any involvement of Tyr-610 phosphorylation in either BR or immune signaling.

Molecular dynamics simulations reveal the conformational dynamics of Arabidopsis thaliana BRI1 and BAK1 receptor-like kinases.

The structural motifs responsible for activation and regulation of eukaryotic protein kinases in animals have been studied extensively in recent years, and a coherent picture of their activation mechanisms has begun to emerge. In contrast, non-animal eukaryotic protein kinases are not as well understood from a structural perspective, representing a large knowledge gap. To this end, we investigated the conformational dynamics of two key Arabidopsis thaliana receptor-like kinases, brassinosteroid-insensitive 1 (BRI1) and BRI1-associated kinase 1 (BAK1), through extensive molecular dynamics simulations of their fully phosphorylated kinase domains. Molecular dynamics simulations calculate the motion of each atom in a protein based on classical approximations of interatomic forces, giving researchers insight into protein function at unparalleled spatial and temporal resolutions. We found that in an otherwise "active" BAK1 the αC helix is highly disordered, a hallmark of deactivation, whereas the BRI1 αC helix is moderately disordered and displays swinging behavior similar to numerous animal kinases. An analysis of all known sequences in the A. thaliana kinome found that αC helix disorder may be a common feature of plant kinases.

Autophosphorylation-based Calcium (Ca2+) Sensitivity Priming and Ca2+/Calmodulin Inhibition of Arabidopsis thaliana Ca2+-dependent Protein Kinase 28 (CPK28).

Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming.

Canopy position has a profound effect on soybean seed composition.

Although soybean seeds appear homogeneous, their composition (protein, oil and mineral concentrations) can vary significantly with the canopy position where they were produced. In studies with 10 cultivars grown over a 3-yr period, we found that seeds produced at the top of the canopy have higher concentrations of protein but less oil and lower concentrations of minerals such as Mg, Fe, and Cu compared to seeds produced at the bottom of the canopy. Among cultivars, mean protein concentration (average of different positions) correlated positively with mean concentrations of S, Zn and Fe, but not other minerals. Therefore, on a whole plant basis, the uptake and allocation of S, Zn and Fe to seeds correlated with the production and allocation of reduced N to seed protein; however, the reduced N and correlated minerals (S, Zn and Fe) showed different patterns of allocation among node positions. For example, while mean concentrations of protein and Fe correlated positively, the two parameters correlated negatively in terms of variation with canopy position. Altering the microenvironment within the soybean canopy by removing neighboring plants at flowering increased protein concentration in particular at lower node positions and thus altered the node-position gradient in protein (and oil) without altering the distribution of Mg, Fe and Cu, suggesting different underlying control mechanisms. Metabolomic analysis of developing seeds at different positions in the canopy suggests that availability of free asparagine may be a positive determinant of storage protein accumulation in seeds and may explain the increased protein accumulation in seeds produced at the top of the canopy. Our results establish node-position variation in seed constituents and provide a new experimental system to identify genes controlling key aspects of seed composition. In addition, our results provide an unexpected and simple approach to link agronomic practices to improve human nutrition and health in developing countries because food products produced from seeds at the bottom of the canopy contained higher Fe concentrations than products from the top of the canopy. Therefore, using seeds produced in the lower canopy for production of iron-rich soy foods for human consumption could be important when plants are the major source of protein and human diets can be chronically deficient in Fe and other minerals.

The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State.

Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAß isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

Functional analysis of the BRI1 receptor kinase by Thr-for-Ser substitution in a regulatory autophosphorylation site.

BRI1 becomes highly phosphorylated in vivo upon perception of the ligand, brassinolide, as a result of autophosphorylation and transphosphorylation by its co-receptor kinase, BAK1. Important autophosphorylation sites include those involved in activation of kinase activity and those that are inhibitory, such as Ser-891. The inhibitory sites are autophosphorylated after kinase activation has been achieved and are postulated to contribute to deactivation of the kinase. The function of phosphosites is usually tested by substituting a non-phosphorylatable residue or an acidic residue that can act as a phosphomimetic. What has typically not been examined is substitution of a Thr for a Ser phosphosite (or vice versa) but given that Thr and Ser are not equivalent amino acids this type of substitution may represent a new approach to engineer regulatory phosphorylation. In the present study with BRI1, we substituted Thr at the Ser-891 phosphosite to generate the S891T directed mutant. The recombinant Flag-BRI1 (S891T) cytoplasmic domain protein (the S891T protein) was catalytically active and phosphorylation occurred at the engineered Thr-891 site. However, the S891T recombinant protein autophosphorylated more slowly than the wild-type protein during expression in E. coli. As a result, activation of peptide kinase activity (measured in vitro) was delayed as was transphosphorylation of bacterial proteins in situ. Stable transgenic expression of BRI1 (S891T)-Flag in Arabidopsis bri1-5 plants did not fully rescue the brassinosteroid (BR) phenotype indicating that BR signaling was constrained. Our working model is that restricted signaling in the S891T plants occurs as a result of the reduced rate of activation of the mutant BRI1 kinase by autophosphorylation. These results provide the platform for future studies to critically test this new model in vivo and establish Ser-Thr substitutions at phosphosites as an interesting approach to consider with other protein kinases.

An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana.

Leucine-rich repeat receptor-like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single-pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high-throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His-tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho-Ser autophosphorylation.

Glutaredoxin AtGRXC2 catalyses inhibitory glutathionylation of Arabidopsis BRI1-associated receptor-like kinase 1 (BAK1) in vitro.

Reversible protein phosphorylation, catalysed by protein kinases, is the most widely studied post-translational modification (PTM), whereas the analysis of other modifications such as S-thiolation is in its relative infancy. In a yeast-two-hybrid (Y2H) screen, we identified a number of novel putative brassinosteroid insensitive 1 (BR1)-associated receptor-like kinase 1 (BAK1) interacting proteins including several proteins related to redox regulation. Glutaredoxin (GRX) C2 (AtGRXC2) was among candidate proteins identified in the Y2H screen and its interaction with recombinant Flag-BAK1 cytoplasmic domain was confirmed using an in vitro pull-down approach. We show that BAK1 peptide kinase activity is sensitive to the oxidizing agents H2O2 and diamide in vitro, suggesting that cysteine oxidation might contribute to control of BAK1 activity. Furthermore, BAK1 was glutathionylated and this reaction could occur via a thiolate-dependent reaction with GSSG or a H2O2-dependent reaction with GSH and inhibited kinase activity. Surprisingly, both reactions were catalysed by AtGRXC2 at lower concentrations of GSSG or GSH than reacted non-enzymatically. Using MALDI-TOF MS, we identified Cys353, Cys374 and Cys408 as potential sites of glutathionylation on the BAK1 cytoplasmic domain and directed mutagenesis suggests that Cys353 and Cys408 are major sites of GRXC2-mediated glutathionylation. Collectively, these results highlight the potential for redox control of BAK1 and demonstrate the ability of AtGRXC2 to catalyse protein glutathionylation, a function not previously described for any plant GRX. The present work presents a foundation for future studies of glutathionylation of plant receptor-like protein kinases (RLKs) as well as for the analysis of activities of plant GRXs.

Oligomerization, membrane association, and in vivo phosphorylation of sugarcane UDP-glucose pyrophosphorylase.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.

A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

The Carboxy-terminus of BAK1 regulates kinase activity and is required for normal growth of Arabidopsis.

Binding of brassinolide to the brassinosteroid-insenstive 1(BRI1) receptor kinase promotes interaction with its co-receptor, BRI1-associated receptor kinase 1 (BAK1). Juxtaposition of the kinase domains that occurs then allows reciprocal transphosphorylation and activation of both kinases, but details of that process are not entirely clear. In the present study we show that the carboxy (C)-terminal polypeptide of BAK1 may play a role. First, we demonstrate that the C-terminal domain is a strong inhibitor of the transphosphorylation activity of the recombinant BAK1 cytoplasmic domain protein. However, recombinant BAK1 lacking the C-terminal domain is unable to transactivate the peptide kinase activity of BRI1 in vitro. Thus, the C-terminal domain may play both a positive and negative role. Interestingly, a synthetic peptide corresponding to the full C-terminal domain (residues 576-615 of BAK1) interacted with recombinant BRI1 in vitro, and that interaction was enhanced by phosphorylation at the Tyr-610 site. Expression of a BAK1 C-terminal domain truncation (designated BAK1-ΔCT-Flag) in transgenic Arabidopsis plants lacking endogenous bak1 and its functional paralog, bkk1, produced plants that were wild type in appearance but much smaller than plants expressing full-length BAK1-Flag. The reduction in growth may be attributed to a partial inhibition of BR signaling in vivo as reflected in root growth assays but other factors are likely involved as well. Our working model is that in vivo, the inhibitory action of the C-terminal domain of BAK1 is relieved by binding to BRI1. However, that interaction is not essential for BR signaling, but other aspects of cellular signaling are impacted when the C-terminal domain is truncated and result in inhibition of growth. These results increase the molecular understanding of the C-terminal domain of BAK1 as a regulator of kinase activity that may serve as a model for other receptor kinases.