Calcium-dependent protein kinases CPK21 and CPK23 phosphorylate and activate the iron-regulated transporter IRT1 to regulate iron deficiency in Arabidopsis

Iron(Fe)is an essential micronutrient for all organisms.Fe availability in the soil is usually much lower than that required for plant growth,and Fe deficiencies seriously restrict crop growth and yield.Calcium(Ca2+)is a second messenger in all eukaryotes;however,it remains largely unknown how Ca2+regulates Fe deficiency.In this study,mutations in CPK21 and CPK23,which are two highly homologous calcium-dependent protein kinases,conferredimpaired growth and rootdevelopment under Fe-deficient conditions,whereas constitutively active CPK21 and CPK23 enhanced plant tolerance to Fe-deficient conditions.Furthermore,we found that CPK21 and CPK23 interacted with and phosphorylated the Fe transporter IRONREGULATED TRANSPORTER1(IRT1)at the Ser149 residue.Biochemical analyses and complementation of Fe transport in yeast and plants indicated that IRT1 Ser149 is critical for IRT1 transport activity.Taken together,these findings suggest that the CPK21/23-IRT1 signaling pathway is critical for Fe homeostasis in plants and provides targets for improving Fe-deficient environments and breeding crops resistant to Fe-deficient conditions.

A Calcium-Dependent Protein Kinase Interactswith and Activates A Calcium Channel toRequlate Pollen Tube Growth

ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neu-rons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code anddecode Ca2＋ signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32,controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca2＋accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activationof CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity.Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, simi-lar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading tomore severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in whichcalcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca2＋ oscilla-tions in the polar growth of pollen tubes.

Salt Stress Triggers Phosphorylation of the Arabidopsis Vacuolar K＋ Channel TPK1 by Calcium-Dependent Protein Kinases （CDPKs）

14-3-3 proteins play an important role in the regulation of many cellular processes. The Arabidopsis vacuolar two-pore K＋ channel 1 （TPK1） interacts with the 14-3-3 protein GRF6 （GF14-λ）. Upon phosphorylation of the putative binding motif in the N-terminus of TPK1, GRF6 binds to TPK1 and activates the potassium channel. In order to gain a deeper understanding of this 14-3-3-mediated signal transduction, we set out to identify the respective kinases, which regulate the phosphorylation status of the 14-3-3 binding motif in TPK1. Here, we report that the calcium-dependent protein kinases （CDPKs） can phosphorylate and thereby activate the 14-3-3 binding motif in TPK1. Focusing on the stress-activated kinase CPK3, we visualized direct and specific interaction of TPK1 with the kinase at the tonoplast in vivo. In line with its proposed role in K＋ homeostasis, TPK1 phosphorylation was found to be induced by salt stress in planta, and both cpk3 and tpkl mutants displayed salt-sensitive phenotypes. Molecular modeling of the TPK1-CPK3 interaction domain provided mechanistic insights into TPK1 stress-regulated phosphorylation responses and pinpointed two arginine residues in the N-terminal 14-3-3 binding motif in TPK1 critical for kinase interaction. Taken together, our studies provide evidence for an essential role of the vacuolar potassium channel TPK1 in salt-stress adaptation as a target of calcium-regulated stress signaling pathways involving Ca2＋, Ca2＋-dependent kinases, and 14-3-3 proteins.

The calcium-dependent kinase OsCPK24 functions in cold stress responses in rice

Calcium-dependent protein kinases（CPKs）are serine/threonine protein kinases that function in plant stress responses. Although CPKs are recognized as key messengers in signal transduction, the specific roles of CPKs and the molecular mechanisms underlying their activity remain largely unknown. Here, we characterized the function of Os CPK_（24）, a cytosol-localized calciumdependent protein kinase in rice. Os CPK_（24） was universally and highly expressed in rice plants and was induced by cold treatment. Whereas Os CPK_（24） knockdown plants exhibited increased sensitivity to cold compared to wild type（WT）, Os CPK_（24）-overexpressing plants exhibited increased cold tolerance. Plants overexpressing Os CPK_（24） exhibited increased accumulation of proline（an osmoprotectant） and glutathione（an antioxidant） and maintained a higher GSH/GSSG（reduced glutathione to oxidized glutathione） ratio during cold stress compared to WT. In addition to these effects in response to cold stress, we observed the kinase activity of Os CPK_（24） varied under different calcium concentrations. Further,Os CPK_（24） phosphorylated Os Grx_（10）, a glutathionedependent thioltransferase, at rates modulated by changes in calcium concentration. Together, our results support the hypothesis that Os CPK_（24） functions as a positive regulator of cold stress tolerance in rice, a process mediated by calcium signaling and involving phosphorylation and the inhibition of Os Grx_（10） to sustain higher glutathione levels.

Cellular localization and biochemical characterization of a novel calciumdependent protein kinase from tobacco

By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 （accession number AY971376）, which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526nmol/min/mg and 210μg/ml respectively with calf thymus histone （fraction Ⅲ, abbreviated to histone Ⅲs） as substrate. For substrate syntide-2, NtCPK5 showed a higher Vmax of 2008 nmol/min/mg and a lower Km of 30μM. The K0.5 of calcium activation was 0.04μM or 0.06μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.

A Highly Sensitive Detection Method, Phos-tag^TMAffinity SDS-PAGE, Used to Analyze a Possible Substrate of CDPK-Related Protein Kinase5 in <i>Arabidopsis</i>

Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 in vitro. Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.

Tonoplast-associated calcium signaling regulates manganese homeostasis in Arabidopsis

Manganese(Mn)is an essential micronutrient in plants.However,excessive Mn absorption in acidic and waterlogged soils can lead to Mn toxicity.Despite their essential roles in Mn homeostasis,transcriptional and post-transcriptional modifications of Mn transporters remain poorly understood.Here,we demonstrated that high-Mn stress induces an obvious Ca^(2+) signature in Arabidopsis.We identified four calcium-dependent protein kinases,CPK4/5/6/11,that interact with the tonoplast-localized Mn and iron(Fe)transporter MTP8 in vitro and in vivo.The cpk4/5/6/11 quadruple mutant displayed a dramatic high-Mn-sensitive phenotype similar to that of the mtp8 mutant.CPKs phosphorylated the N-terminal domain of MTP8 primarily at the Ser31 and Ser32 residues.Transport assays combined with multiple physiological experiments on phospho-dead variant MTP8^(S31/32A)and phospho-mimetic variant MTP8^(S31/32D)plants under different Mn and Fe conditions suggested that Ser31 and Ser32 are crucial for MTP8 function.In addition,genetic analysis showed that CPKs functioned upstream of MTP8.In summary,we identified a tonoplast-associated calcium signaling cascade that orchestrates Mn homeostasis and links Mn toxicity,Ca^(2+) signaling,and Mn transporters.These findings provide new insight into Mn homeostasis mechanisms and Ca^(2+) signaling pathways in plants,providing potential targets for engineering heavy metal toxicity-tolerant plants.

Ca^(2+)-dependent successive phosphorylation of vacuolar transporter MTP8 by CBL2/3-CIPK3/9/26 and CPK5 is critical for manganese homeostasis in Arabidopsis

Manganese(Mn)is an essential micronutrient for all living organisms.However,excess Mn supply that can occur in acid or waterlogged soils has toxic effects on plant physiology and development.Although a variety of Mn transporter families have been characterized,we have only a rudimentary understanding of how these transporters are regulated to uphold and adjust Mn homeostasis in plants.Here,we demonstrate that two calcineurin-B-like proteins,CBL2/3,and their interacting kinases,CIPK3/9/26,are key regulators of plant Mn homeostasis.Arabidopsis mutants lacking CBL2 and 3 or their interacting protein kinases CIPK3/9/26 exhibit remarkably high Mn tolerance.Intriguingly,CIPK3/9/26 interact with and phosphorylate the tonoplast-localized Mn and iron(Fe)transporter MTP8 primarily at Ser35,which is conserved among MTP8 proteins from various species.Mn transport complementation assays in yeast combined with multiple physiological assays indicate that CBL-CIPK-mediated phosphorylation of MTP8 negatively regulates its transport activity from the cytoplasm to the vacuole.Moreover,we show that sequential phosphorylation of MTP8,initially at Ser31/32 by the calcium-dependent protein kinase CPK5 and subsequently at Ser35 by CIPK26,provides an activation/deactivation fine-tuning mechanism for differential regulation of Mn transport.Collectively,our findings define a two-tiered calcium-controlled mechanism for dynamic regulation of Mn homeostasis under conditions of fluctuating Mn supply.

Arabidopsis Synaptotagmin 2 Participates in Pollen Germination and Tube Growth and Is Delivered to Plasma Membrane via Conventional Secretion

Arabidopsis synaptotagmin 2 （SYT2） has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen ofArabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmManl-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.

The functional change of perivascular adipose tissue in hypertensive rats and its mechanisms

Background Recent studies have showed that perivascular adipose tissue （PVAT） may secrete the adventitial-derived relaxing factor （ADRF） to affect vascular function.However,the functional change of ADRF in hypertensive status is seldom studied;and the mechanisms of ADRF remain unclear.Our study examined the ADRF secreted by perivascular adipose tissue of control rats with normal blood pressure （Wistar Kyoto rats,WKY） and discussed the mechanisms of ADRF;We observed the functional change in ADRF of perivascular adipose tissue in spontaneously hypertensive rats （SHRs）.Method The two adjacent thoracic aorta rings of SHR and WKY rats were divided into naked vessel subgroup and PVAT subgroup.The differences of vascular contractility between the two subgroups induced by 10-6 mmol/L phenylephrine were compared.The effect of PVAT culture medium of WKY on the vascular tension of Fat （-） vessels was observed by liquid transfer measure.The mechanism of ADRF was determined by tool drugs.Results In WKY group,vascular contractility of Fat （＋） subgroup was lower than that of the Fat （-） subgroup （P 0.05）;while in SHR group,there was no difference between the two subgroups （P 0.05）.Transferring the incubation solution of WKY Fat （＋） subgroup to the matched Fat （-） subgroup induced rapid vasodilation.When incubating blood vessels in calcium free PSS solution,there was no significant difference of phenylephrine-induced vasoconstriction between Fat （-） and Fat （＋） subgroup.Both glibenclamide,the blocker of ATP-sensitive potassium （KATP） channel and Tetraethy-lammonium chloride （TEA）,the inhibitor of calcium-dependent potassium （KCa） channel,effectively inhibited vasodilation function of ADRF.Conclusions Perivascular adipose tissue in WKY releases ADRF which can cause vasodilation,while this function was inhibited in SHR.ADRF acts through the activation of KCa and KATP channels and calcium ion is involved.