plants

Article Characterization of Atypical Protein (PTK) and Their Role in Abiotic Stress Response in Rice

Allimuthu Elangovan 1, Monika Dalal 2,*, Gopinathan Kumar Krishna 1 , Sellathdurai Devika 1, Ranjeet Ranjan Kumar 3, Lekshmy Sathee 1 and Viswanathan Chinnusamy 1,* 1 Division of Plant Physiology, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India; [email protected] (A.E.); [email protected] (G.K.K.); [email protected] (S.D.); [email protected] (L.S.) 2 ICAR-National Institute for Plant Biotechnology, New Delhi 110012, India 3 Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India; [email protected] * Correspondence: [email protected] (M.D.); [email protected] or [email protected] (V.C.); Tel.: +11-91-258-42-815 (V.C.)

 Received: 4 April 2020; Accepted: 14 May 2020; Published: 23 May 2020 

Abstract: Tyrosine phosphorylation constitutes up to 5% of the total phophoproteome. However, only limited studies are available on protein tyrosine kinases (PTKs) that catalyze protein tyrosine phosphorylation in plants. In this study, domain analysis of the 27 annotated PTK genes in rice genome led to the identification of 18 PTKs with tyrosine kinase domain. The kinase domain of rice PTKs shared high homology with that of dual specificity kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1) of Arabidopsis. In phylogenetic analysis, rice PTKs clustered with receptor-like cytoplasmic kinases-VII (RLCKs-VII) of Arabidopsis. mRNAseq analysis using Genevestigator revealed that rice PTKs except PTK9 and PTK16 express at moderate to high level in most tissues. PTK16 expression was highly abundant in panicle at flowering stage. mRNAseq data analysis led to the identification of drought, heat, salt, and submergence stress regulated PTK genes in rice. PTK14 was upregulated under all stresses. qRT-PCR analysis also showed that all PTKs except PTK10 were significantly upregulated in root under osmotic stress. Tissue specificity and abiotic stress mediated differential regulation of PTKs suggest their potential role in development and stress response of rice. The candidate dual specificity PTKs identified in this study paves way for molecular analysis of tyrosine phosphorylation in rice.

Keywords: BRI1; drought; dual specificity; osmotic stress; receptor-like protein kinases

1. Introduction Protein kinases catalyze phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residue of target proteins by transferring the γ-phosphate from ATP. Based on their substrate specificity to phosphorylate Ser/Thr and Tyr residues, protein kinases were initially classified into Ser/Thr kinases and Tyr kinases, respectively [1]. Later dual-specificity protein tyrosine kinases (DsPTKs) which phosphorylate all three residues viz. Ser, Thr, and Tyr were also identified [2]. Among these, serine phosphorylation (pS) is most common followed by threonine (pT) and tyrosine phosphorylation (pY). In plants such as rice and Arabidopsis, the relative abundance of pS and pT is about 80–85% and 10–15%, respectively, while pY ranges between 0–5% [3–5]. The lower representation of pY in phosphoproteome has been attributed to occurrence of Tyr phosphorylation on less abundant proteins, methods of detection and sample type, i.e., subcellular fractions or whole-cell extracts [3,6]. In spite of lower

Plants 2020, 9, 664; doi:10.3390/plants9050664 www.mdpi.com/journal/plants Plants 2020, 9, 664 2 of 17 abundance, pY has been shown to plays an important role in cell division, growth, and differentiation in humans. Impairment of PTK activity has been associated with diabetes, while hyperactivity leads to oncogenesis [7]. In plants, Ser/Thr kinases are the most studied kinases, while Tyrosine kinases are relatively less known. Tyr phosphorylation was first detected in Pisum sativum by immunoblotting with anti-phospho-Tyr antibodies [8]. Later pharmacological studies using inhibitors demonstrated the role of Tyr phosphorylation in different plant processes, for example, organization of microtubules [9], Abscisic acid (ABA) pathway in seeds [10], and leaf bending in Mimosa pudica L. [11]. The involvement of Tyr phosphorylation in different stages of plant development starting from embryo to leaf senescence and abiotic stress response reiterates its significance in plants [12–15]. Furthermore, inhibitors of protein tyrosine phosphatases (PTPs) have been shown to inhibit ABA-mediated stomatal closure [16,17]. A loss-of function mutation in a dual-specificity PTP (DsPTP) PHS1 conferred hypersensitivity to the plant stress hormone ABA and thus enhanced expression of ABA-dependent RAB18 [18]. Recently, poplar PTP1 was found to be a negative regulation of salt tolerance [19]. These evidences suggest that tyrosine phosphorylation and dephosphorylation play important role in stress response of plants. Structurally, protein kinases share a conserved catalytic domain of ~250 to 300 amino acids with eleven conserved sub-domains (I to XI). A conserved lysine residue in the subdomain II helps in binding to ATP, and is essential for maximal activity. The presence of aspartic acid residue in the subdomain VII is important for the kinase activity and subdomain VI helps in recognition of specific hydroxyl amino acid [20,21]. The presence of DLRAAN or DLAARN in sub-domain VI indicates Tyr specificity, while the presence of DLKPEN indicates Ser/Thr specificity. Subdomain VIII helps in recognition of peptide substrates, where, Tyr specificity is defined by the highly conserved consensus sequence PI/VK/RWT/MAPE and the relatively less conserved GT/SXXY/FXAPE sequence confers Ser/Thr specificity. The subdomain XI consensus motif CW(X)6RPXF is highly specific to tyrosine kinase and it was found to be conserved in tyrosine kinases from mammals, fruit fly, and Dictyostelium [21]. Hence, based on CW(X)6RPXF motif, 57 PTKs were identified in Arabidopsis. However, all these Arabidopsis PTKs have KXXN in sub-domain VIB, which is a Ser/Thr specific motif. Based on this observation, it was inferred that plants do not encode bona fide PTKs, and hence these kinases were inferred as dual specificity protein kinases (DsPTKs) [21]. Though typical PTKs were predicted in rice, yet the number was far less (3 in O. sativa ssp. indica and four in O. sativa ssp. japonica)[22]. Moreover these were predicted based on the residues known to be essential for catalytic activity, hence their role as typical tyrosine kinases still remains to be verified. Therefore, to characterize the PTKs in rice genome, we carried out the in silico analysis of annotated PTK genes in rice genome and their expression analysis under osmotic and drought stress at seedling and reproductive stages respectively.

2. Results and Discussion

2.1. Analysis of PTK Genes in Rice Twenty seven genes annotated as “tyrosine domain containing protein” were identified from rice genome RGAP Release 7. Based on pfam domain analysis, 19 of these 27 annotated proteins were identified with protein tyrosine kinase domain (PF07714.16). Further, HMMER analysis also identified 19 proteins with protein tyrosine kinase domain (PF07714.16) and remaining 8 with (PF00069.24) based on the lowest E-value. Hence, these 19 putative PTKs were further analyzed for presence of conserved motifs in 11 (I-XI) subdomains characteristic to protein kinases. The subdomains for ATP binding and catalysis were conserved among the 19 putative PTKs of rice. The subdomain I of protein kinases is characterized by GxGxφGXV, where φ can be F or Y. Among the putative PTK proteins, LOC_Os02g39560 lacked sub domain I as well as many of the conserved residues in other sub domains. Hence it was not considered for further analysis. The remaining 18 candidate PTK proteins of rice showed highly conserved GxGxFGXV motif in subdomain I, invariant Plants 2020, 9, 664 3 of 17

Plants 2020, 9, x FOR PEER REVIEW 3 of 17 Lysine (K) residue in subdomain II, Glutamic acid (E) in subdomain III and motif DFG in subdomain 95VII whichacid (E) are in subdomain required for III anchoringand motif DFG of ATP in subdomain and enzyme VII catalysis,which are required respectively for anchoring [23,24]. of ATP 96 andProtein enzyme kinases catalysis, are respectively differentiated [23,24]. as Ser/Thr or Tyr protein kinases based on subdomain VI, 97VIII, andProtein XI [16 kinases,18]. Theare differentiated tyrosine kinases as Ser/Thr are characterized or Tyr protein bykinases presence based of on consensus subdomain sequenceVI, 98 of DL(RVIII,/ A)A(Aand XI /[16,18].R)N, XP(I The/ V)(Ktyrosine/R)W(T kinases/M)APE, are char andacterized CW(X)6RPXF by presence in subdomainsof consensus sequence VI, VIII, of and XI, 99 DL(R/A)A(A/R)N, XP(I/V)(K/R)W(T/M)APE, and CW(X)6RPXF in subdomains VI, VIII, and XI, respectively. Of the 18 candidate PTK proteins of rice, 17 showed motifs (Y/H)RDFK(A/T)SN, 100 respectively. Of the 18 candidate PTK proteins of rice, 17 showed motifs (Y/H)RDFK(A/T)SN, TXXYXAPE, and CL(X) RPXM at subdomain VI, VIII and XI, respectively (Figure1) instead of 101 TXXYXAPE, and CL(X)6 6RPXM at subdomain VI, VIII and XI, respectively (Figure 1) instead of 102typical typical tyrosine tyrosine kinase kinase specific specific motifs motifs foundfound in in animal animal system system viz., viz., HRDLXARN, HRDLXARN, PXXWXAPE, PXXWXAPE, and and 103CW(X) CW(X)6RPXF6RPXF [24 [24].]. Of Of thethe 1818 candidatecandidate PTK PTK loci loci identified identified from from rice, rice, LOC_Os07g31290 LOC_Os07g31290 has all has all 104subdomains subdomains except except subdomain subdomain XI. XI. SinceSince LOC_Os07g31290 showed showed expres expressionsion in both in bothin silico in silicoand and 105qRT-PCR qRT-PCR analysis, analysis, it was it alsowas includedalso included in this in analysis. this analysis. Notably Notably the consensus the consensus sequence sequence for subdomain for 106 6 XI showedsubdomain a conserved XI showed motif a conserved CL(X)6RPxM motifamong CL(X) RPxM the rest among of the the 17 candidaterest of the PTK17 candidate protein sequences.PTK 107These protein results sequences. prompted These us to results analyze prompted already us validated to analyze dual already specificity validated protein dual kinases specificity from proteinArabidopsis . 108 kinases from Arabidopsis.

109

110 FigureFigure 1. Sequence 1. Sequence logo logo of of conserved conserved motifsmotifs in in protein protein tyrosine tyrosine kinases kinases of rice. of rice. The Thesequence sequence logos logos 111 are basedare based on multipleon multiple alignment alignment analysis analysis of 18 ri ricece PTK PTK proteins. proteins. The The bit bitscore score indicates indicates the residue the residue 112 contentcontent for for each each position position in in the the sequence. sequence. * cons conservederved residues residues in ineach each sub-domain. sub-domain. Roman Roman numbers numbers 113 I-XI denote sub-domains in kinase domain. I-XI denote sub-domains in kinase domain.

114 In Arabidopsis, a classical example of tyrosine phosphorylation is presented by brassinosteroid In Arabidopsis, a classical example of tyrosine phosphorylation is presented by brassinosteroid 115 receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and its co-receptor BRI1-ASSOCIATED receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and its co-receptor BRI1-ASSOCIATED KINASE 116 KINASE 1 (BAK1). BRI1 and BAK1 are leucine-rich repeat receptor-like kinases (LRR-RLKs) which 1171 (BAK1).were initially BRI1 and classified BAK1 are as leucine-richserine/threonine repeat prot receptor-likeein kinases. kinases Later it (LRR-RLKs) was found whichthat both were the initially 118classified cytoplasmic as serine domains/threonine of BRI1 protein and kinases.BAK1 also Later autophosphorylate it was found that on bothtyrosine the residues, cytoplasmic thereby domains 119of BRI1proved and that BAK1 these also two autophosphorylate are dual-specificity kinases on tyrosine [25]. Sequence residues, analysis thereby revealed proved that that these these two two are 120dual-specificity kinases do not kinases have the [25 typical]. Sequence tyrosine analysis kinase specific revealed CW(X) that6 theseRPXF twomotif kinases of subdomain do not XI have reported the typical 121tyrosine for animal kinase PTKs. specific Furthermore, CW(X)6RPXF sequence motif alignment of subdomain of BRI1 XI with reported the 18 forPTK animal proteins PTKs. identified Furthermore, in 122sequence rice revealed alignment >80% of identity BRI1 within consensus the 18 PTKsequence proteins of subdomains identified VI, in VIII, rice and revealed XI (Figure>80% 2). identity This in 123consensus indicates sequence that the of18 subdomainsPTKs identified VI, in VIII, rice andmight XI fu (Figurenction 2as). dual This specificit indicatesy BRI-like that the kinases. 18 PTKs There identified in rice might function as dual specificity BRI-like kinases. There is a high probability that in the absence of bona fide tyrosine kinases, these members of kinase family might be carrying out tyrosine Plants 2020 9 Plants, , 2020 664, 9, x FOR PEER REVIEW 4 of 174 of 17

124 Plantsis a 2020 high, 9 ,probability x FOR PEER REVIEWthat in the absence of bona fide tyrosine kinases, these members of kinase4 family of 17 125phosphorylation. might be carrying This out is furthertyrosine supported phosphorylation. by fact Th thatis is membersfurther supported of the plantby fact calcium-dependent that members of 124126protein is thea kinasehigh plant probability (CDPK calcium-dependent/ CPK)-relatedthat in the absence protein PKs of (CRK) bonakinase fi superfamily,de (C tyrosineDPK/CPK)-related kinases, in addition these PKs members to Ser(CRK)/Thr of superfamily, kinase phosphorylation, family in 125127can alsomightaddition catalyze be carrying to trans-Ser/Thr out and tyrosinephosphorylation, autophosphorylation phosphorylation. can also ofTh catalyze Tyris is residuesfurther trans- supported in andArabidopsis autophosphorylation by fact[26 that]. The members identification of ofTyr 126128and characterizationtheresidues plant calcium-dependentin Arabidopsis of kinases [26]. capable protein The of identificationkinase phosphorylating (CDPK/CPK)-related and characterization tyrosine residuesPKs (CRK) of wouldkinases superfamily, be capable an important in of 127129aspect additionphosphorylating of understanding to Ser/Thr tyrosine phosphorylation, the signaling residues pathwayswould can alsobe anincatalyze plants.important trans- aspect and ofautophosphorylation understanding the signalingof Tyr 128130 residuespathways in inArabidopsis plants. [26]. The identification and characterization of kinases capable of 129 phosphorylating tyrosineIIIIIIIVV residues would be an important aspect of understanding the VIsignaling 130 pathwaysBRI in plants. IGSGGFGDVYKA --- AIKKL --- QGDREFMAEME --- KHRNLVPLLGYCKVG ---LVYEFMKYGSLEDVLH --- HIIHRDMKSSNVLLD LOC_Os08g04420 LGQGAFGPVYKA --- AVKVL --- QGEKEFQTEVL --- HHRNLVNLVGYCAEK ---LLYAFMPNGSLASHLY --- PVVHRDIKSPNILLD LOC_Os04g52840 IGEGGFGSVFRGIIIIIIIVV --- AVKVL --- QGVREFLTELT --- KHENLVTLIGCCAEG ---LVYNYLENNSLAQTLL --- PIIHRDIKASNILLD VI LOC_Os09g39930 LGEGGFGPVYRG --- AVKQL --- QGNREFLVEVL --- EHPNLVTLLGYCTDG ---LVYEYMARGSLEDHLL --- PVIYRDFKASNILLD BRILOC_Os07g31290 IGSGGFGDVYKA IGEGGFGSVYKG ------AIKKL AVKKL ------QGDREFMAEME QGHKQWLAEVQ ------KHRNLVPLLGYCKVG DHPNLVKLIGYCGTD ---LVYEFMKYGSLEDVLH ---LVYEFMPNKTLEYHLF ------HIIHRDMKSSNVLLD QVIYRDLKSSNVLLD LOC_Os08g04420LOC_Os09g36320 LGQGAFGPVYKA LGEGGFGSVYKG ------AVKVL AVKCL ------QGEKEFQTEVL QGHKQWLAEVQ ------HHRNLVNLVGYCAEK EHPNLVKLLGYCAV- ---LLYAFMPNGSLASHLY ---LVYEYMPNKSLEDHLF ------PVVHRDIKSPNILLD QVIYRDFKASNILLD LOC_Os04g52840LOC_Os07g48730 IGEGGFGSVFRG LGEGGFGCVYKG ------AVKVL AIKKL ------QGVREFLTELT QGHKEWLAEVT ------KHENLVTLIGCCAEG HHENLVKLVGYCSDS ---LVYNYLENNSLAQTLL ---LVYEYMLRGSLENHLF ------PIIHRDIKASNILLD PIIFRDLKSSNVLLA LOC_Os09g39930LOC_Os03g06330 LGEGGFGPVYRG LGGGGFGRVYKG ------AVKQL AVKVH ------QGNREFLVEVL QGHREWLAEVI ------EHPNLVTLLGYCTDG SHPNLVKLIGYCCED ---LVYEYMARGSLEDHLL ---LVYEFMPLGSVESHLF ------PVIYRDFKASNILLD PVIYRDFKTSNILLD LOC_Os07g31290LOC_Os10g29620 IGEGGFGSVYKG IGGGRFGRVYKG ------AVKKL AVKVH ------QGHKQWLAEVQ QGHREWLAEVI ------DHPNLVKLIGYCGTD SHPNLVRLVGYCCEG ---LVYEFMPNKTLEYHLF ---LVYEYMPRGSVESHLF ------QVIYRDLKSSNVLLD PVIYRDFKTSNILLD LOC_Os09g36320LOC_Os01g40590 LGEGGFGSVYKG LGEGGFGCVFKG ------AVKCL AVKTL ------QGHKQWLAEVQ QGHKEWVAEVD ------EHPNLVKLLGYCAV- QHPHLVKLVGYCIED ---LVYEYMPNKSLEDHLF ---LVYEFMPRGSLENHLF ------QVIYRDFKASNILLD PVIYRDFKTSNILLD LOC_Os07g48730LOC_Os06g07230 LGEGGFGCVYKG LGEGGFGYVFKG ------AIKKL AVKSL ------QGHKEWLAEVT QGHREWVAEVD ------HHENLVKLVGYCSDS HHKHLVKLIGYCIED ---LVYEYMLRGSLENHLF ---LVYEFMARGSLENHLF ------PIIFRDLKSSNVLLA PVIYRDFKTSNILLD LOC_Os03g06330LOC_Os03g24930 LGGGGFGRVYKG LGEGGFGAVHKG ------AVKVH AVKQL ------QGHREWLAEVI QGHREWLAEVI ------SHPNLVKLIGYCCED RHPHLVKLLGYCCED ---LVYEFMPLGSVESHLF ---LVYEFMPRGSLENHLF ------PVIYRDFKTSNILLD PVIYRDFKASNILLD LOC_Os10g29620LOC_Os08g35600 IGGGRFGRVYKG LGSGGFGPVYKG ------AVKVH AVKYL ------QGHREWLAEVI QGHREWLAEVF ------SHPNLVRLVGYCCEG RHKNLVKLIGYCYED ---LVYEYMPRGSVESHLF ---LVYEYMSNGSLEKHLF ------PVIYRDFKTSNILLD PVIYRDFKASNILLD LOC_Os01g40590LOC_Os09g27010 LGEGGFGCVFKG LGCGGFGPVYKG ------AVKTL AVKYL ------QGHKEWVAEVD QGHKEWLAEVF ------QHPHLVKLVGYCIED RHKNLVKLIGYCYEA ---LVYEFMPRGSLENHLF ---LVYEYMSGESLEKHLF ------PVIYRDFKTSNILLD PVIYRDFKASNILLD LOC_Os06g07230LOC_Os02g53750 LGEGGFGYVFKG IGEGGFGPVYKG ------AVKSL AVKYL ------QGHREWVAEVD QGHREWLAEVV ------HHKHLVKLIGYCIED SHPHLVKLIGYCCQD ---LVYEFMARGSLENHLF ---LVYEYMARGSLEHHLF ------PVIYRDFKTSNILLD PVIYRDFKASNILLD LOC_Os03g24930LOC_Os06g10160 LGEGGFGAVHKG LGEGGFGPVYKG ------AVKQL AVKYL ------QGHREWLAEVI QGHREWLAEVV ------RHPHLVKLLGYCCED SHPHLVKLVGFCNQD ---LVYEFMPRGSLENHLF ---LVYEYMPRGSLENHLF ------PVIYRDFKASNILLD PVIYRDFKASNILLD LOC_Os08g35600LOC_Os10g25550 LGSGGFGPVYKG VGEGGFGPVYKG ------AVKYL AVKLL ------QGHREWLAEVF QGHKEWLAEVI ------RHKNLVKLIGYCYED RHHHLVKLIGYCYED ---LVYEYMSNGSLEKHLF ---LVYEFMARGSLEKHLF ------PVIYRDFKASNILLD PVIYRDFKTSNILLN LOC_Os09g27010LOC_Os03g29410 LGCGGFGPVYKG LGEGGFGPVYKG ------AVKYL AVKLW ------QGHKEWLAEVF QGHKEWLSEVI ------RHKNLVKLIGYCYEA RHPNLVKLIGYCCED ---LVYEYMSGESLEKHLF ---LVYEYMAKGSLENHLF ------PVIYRDFKASNILLD PVIYRDFKTSNILLD LOC_Os02g53750LOC_Os07g42200 IGEGGFGPVYKG LGEGGFGPVYKG ------AVKYL AVKLW ------QGHREWLAEVV QGHKEWLAEVI ------SHPHLVKLIGYCCQD RHPNLVKLVGYCCED ---LVYEYMARGSLEHHLF ---LVYEYMEHGSLENHLF ------PVIYRDFKASNILLD PVIYRDFKASNILLD LOC_Os06g10160 LGEGGFGPVYKG --- AVKYL --- QGHREWLAEVV --- SHPHLVKLVGFCNQD ---LVYEYMPRGSLENHLF --- PVIYRDFKASNILLD

LOC_Os10g25550 VGEGGFGPVYKGVII --- AVKLL --- VIII QGHKEWLAEVI --- RHHHLVKLIGYCYED IX ---LVYEFMARGSLEKHLF X --- PVIYRDFKTSNILLN XI LOC_Os03g29410 LGEGGFGPVYKG --- AVKLW --- QGHKEWLSEVI --- RHPNLVKLIGYCCED ---LVYEYMAKGSLENHLF --- PVIYRDFKTSNILLD LOC_Os07g42200BRI LGEGGFGPVYKG VSDFGMARL ------STLAGTPGYVPPEYYQSFRCAVKLW --- QGHKEWLAEVI ------KGDVYSYGVVLLELLTGKRPTD RHPNLVKLVGYCCED ---LVYEYMEHGSLENHLF --- VFDPELMK ------AVACLDDRAWRRPTMVQVM PVIYRDFKASNILLD LOC_Os08g04420 VADFGLSRE --- ANIRGTYGYLDPEYVSSRSF --- KSDVYSYGVLLFEMIAGRNPQQ --- IADSRLEG --- AYRCVSRVSRKRPAMRDVV LOC_Os04g52840 ISDFGLARL --- TRVAGTLGYLAPEYAIRGQV --- KSDIYSFGVLLLEIVSGRCNTN --- IIDADLGN --- GLLCTQDAMARRPNMSTVV VII VIII IX X XI LOC_Os09g39930 LSDFGLAKV --- TRVMGTYGYCAPEYALTGKL --- CSDVYSFGVVFLEIITGRRAID --- MADPLLRG --- AAMCLQEDATMRPAISDVV BRILOC_Os07g31290 VSDFGMARL LSDFGLARE ------STLAGTPGYVPPEYYQSFRC TAVMGTYGYAAPDYVETGRL ------KGDVYSYGVVLLELLTGKRPTD RSDVWSFGVVLLELLTGHRAFD ------VFDPELMK ------AVACLDDRAWRRPTMVQVM ------LOC_Os08g04420LOC_Os09g36320 VADFGLSRE LSDFGLARE ------ANIRGTYGYLDPEYVSSRSF TAVVGTHGYAAPDYIETGHL ------KSDVYSYGVLLFEMIAGRNPQQ KSDVWSFGVVLYEILTGRRTLD ------IADSRLEG IMDPRLRG ------AYRCVSRVSRKRPAMRDVV AESCLLKNAKERPTMSEVV LOC_Os04g52840LOC_Os07g48730 ISDFGLARL LSDFGLARN ------TRVAGTLGYLAPEYAIRGQV TRVVGTRGYAAPEYVATGHL ------KSDIYSFGVLLLEIVSGRCNTN KSDVYSFGVVLLELLTGRRALD ------IIDADLGN IMDTRLGG ------GLLCTQDAMARRPNMSTVV ALRCLHHDPKLRPAM---- LOC_Os09g39930LOC_Os03g06330 LSDFGLAKV LSDFGLAKD ------TRVMGTYGYCAPEYALTGKL TRIMGTYGYAAPEYIMTGHL ------CSDVYSFGVVFLEIITGRRAID MSDVYSYGVVLLELLTGRKSLD ------MADPLLRG IVDPRLAE ------AAMCLQEDATMRPAISDVV AYHCLNRNPKARPLMRDIV LOC_Os07g31290LOC_Os10g29620 LSDFGLARE LSDFGLAKD ------TAVMGTYGYAAPDYVETGRL TRIMGTYGYAAPEYVMTGHL ------RSDVWSFGVVLLELLTGHRAFD MSDVYSYGVVLLELLTGRKSLD ------IVDPRLSA ------AYHCLNRNPKARPLMRDIV LOC_Os09g36320LOC_Os01g40590 LSDFGLARE LSDFGLAKD ------TAVVGTHGYAAPDYIETGHL TRVMGTYGYAAPEYVMTGHL ------KSDVWSFGVVLYEILTGRRTLD KSDVYSFGVVLLEMMSGRRSMD ------IMDPRLRG LVDPRLEG ------AESCLLKNAKERPTMSEVV ACACLNRDPKARPLMSQVV LOC_Os07g48730LOC_Os06g07230 LSDFGLARN LSDFGLAKA ------TRVVGTRGYAAPEYVATGHL TRVVGTYGYAAPEYVMTGHL ------KSDVYSFGVVLLELLTGRRALD KSDVYSFGVVLLEMLTGRRSMD ------IMDTRLGG LVDPRLGL ------ALRCLHHDPKLRPAM---- CYHCLSRDTKSRPTMDEVV LOC_Os03g06330LOC_Os03g24930 LSDFGLAKD LSDFGLAKM ------TRIMGTYGYAAPEYIMTGHL TRVMGTHGYAAPEYVMTGHL ------MSDVYSYGVVLLELLTGRKSLD KSDVYSYGVVLLELLTGRRAME ------IVDPRLAE IMDPRLAG ------AYHCLNRNPKARPLMRDIV AVQCTSPQPRDRPRMAAVV LOC_Os10g29620LOC_Os08g35600 LSDFGLAKD LSDFGLAKD ------TRIMGTYGYAAPEYVMTGHL TRVMGTNGYAAPEYIMTGHL ------MSDVYSYGVVLLELLTGRKSLD KSDVYSFGVVLLELLSGRHSVD ------IVDPRLSA VMDPAMEG ------AYHCLNRNPKARPLMRDIV AYKCLSPSPKSRPSMREVV LOC_Os01g40590LOC_Os09g27010 LSDFGLAKD LSDFGLAKD ------TRVMGTYGYAAPEYVMTGHL TRVMGTHGYAAPEYIMTGHL ------KSDVYSFGVVLLEMMSGRRSMD KSDVYSFGVVLLELLSGRKSVD ------LVDPRLEG VMDPALEC ------ACACLNRDPKARPLMSQVV AYKCLSENPKSRPTMREVV LOC_Os06g07230LOC_Os02g53750 LSDFGLAKA LSDFGLAKE ------TRVVGTYGYAAPEYVMTGHL TRVMGTHGYAAPEYILTGHL ------KSDVYSFGVVLLEMLTGRRSMD KSDVYSFGVVLLELLTGRRSVD ------LVDPRLGL VMDPSLEG ------CYHCLSRDTKSRPTMDEVV AYHCLHSVPKSRPHMRDVV LOC_Os03g24930LOC_Os06g10160 LSDFGLAKM LSDFGLAKE ------TRVMGTHGYAAPEYVMTGHL TRVMGTHGYAAPEYILTGHL ------KSDVYSYGVVLLELLTGRRAME RSDVYSFGVVLLELLTGRRSVD ------IMDPRLAG IMDPSLEL ------AVQCTSPQPRDRPRMAAVV AHQCLQSVPKSRPCMRDVV LOC_Os08g35600LOC_Os10g25550 LSDFGLAKD LSDFGLAKD ------TRVMGTNGYAAPEYIMTGHL TRVMGTQGYAAPEYIMTGHL ------KSDVYSFGVVLLELLSGRHSVD KSDVYSYGVVLLELLTGRKAVD ------VMDPAMEG VIDKSLNG ------AYKCLSPSPKSRPSMREVV AYQCLSVSPKSRPRMSAVV LOC_Os09g27010LOC_Os03g29410 LSDFGLAKD LSDFGLAKD ------TRVMGTHGYAAPEYIMTGHL TRVMGTHGYAAPEYILTGHL ------KSDVYSFGVVLLELLSGRKSVD KSDVYSFGVVLLEILSGRRAVD ------VMDPALEC VMDPALEG ------AYKCLSENPKSRPTMREVV AYKCLSGNPKNRPDMCQVV 131 LOC_Os02g53750LOC_Os07g42200 LSDFGLAKE LSDFGLAKD ------TRVMGTHGYAAPEYILTGHL TRVMGTHGYAAPEYIMTGHL ------KSDVYSFGVVLLELLTGRRSVD KSDVYSFGVVLLEILTGRRAVD ------VMDPSLEG IMDPALEG ------AYHCLHSVPKSRPHMRDVV AYRCLSGSPKNRPDMSAVV LOC_Os06g10160 LSDFGLAKE --- TRVMGTHGYAAPEYILTGHL --- RSDVYSFGVVLLELLTGRRSVD --- IMDPSLEL --- AHQCLQSVPKSRPCMRDVV LOC_Os10g25550 LSDFGLAKD --- TRVMGTQGYAAPEYIMTGHL --- KSDVYSYGVVLLELLTGRKAVD --- VIDKSLNG --- AYQCLSVSPKSRPRMSAVV 132 FigureLOC_Os03g29410Figure 2. Multiple 2. LSDFGLAKDMultiple sequence --- sequence TRVMGTHGYAAPEYILTGHL alignment alignment --- ofof KSDVYSFGVVLLEILSGRRAVD ricerice PTKPTK proteinsproteins --- with VMDPALEG BRI BRI ---(BRASSINOSTEROID (BRASSINOSTEROID AYKCLSGNPKNRPDMCQVV 131 LOC_Os07g42200 LSDFGLAKD --- TRVMGTHGYAAPEYIMTGHL --- KSDVYSFGVVLLEILTGRRAVD --- IMDPALEG --- AYRCLSGSPKNRPDMSAVV 133 INSENSITIVEINSENSITIVE 1). Roman1). Roman numbers numbers denote denote sub-domainssub-domains of of protein protein kinase. kinase. The codes The codesfor shades for shadesare: 134 Green, conserved and essential for kinase activity; Cyan, conserved; Grey, >80% conservation with 132 are:Figure Green, 2. conserved Multiple andsequence essential alignment for kinase of rice activity; PTK proteins Cyan, conserved; with BRI Grey,(BRASSINOSTEROID>80% conservation 133135 INSENSITIVEBRI. 1). Roman numbers denote sub-domains of protein kinase. The codes for shades are: with BRI. 134 Green, conserved and essential for kinase activity; Cyan, conserved; Grey, >80% conservation with 135136 BRI.We named the rice candidate PTKs identified in this study as PTK1 to PTK18 based on their We named the rice candidate PTKs identified in this study as PTK1 to PTK18 based on their 137 chromosomal localization starting from 1 to 12 in an ascending order (Table S1). These 136138chromosomal PTKWe genes named localization were the distributed rice startingcandidate across from PTKs nine chromosome identified in 1this to with 12study in 1 an asto ascendingPTK13 to perPTK18 order chromosome based (Table onS1). theirexcept These 137139PTK chromosomalgeneschromosome were localization distributed 5, 11, and 12starting across which from do nine not chromosome chromosomes harbour any 1 toPTK 12 with ingene an 1 (Figureascending to 3 locus S1). order Gene per (Table chromosome structure S1). analysisThese except 138140chromosome PTKrevealed genes 5, thatwere 11, the and distributed PTK 12 genes which across have do not1–6 nine introns harbour chromosomes (Figure any PTK 3). with gene 1 to (Figure 3 locus S1).per chromosome Gene structure except analysis 139revealed chromosome that the 5,PTK 11, andgenes 12 havewhich 1–6 do intronsnot harbour (Figure any3 PTK). gene (Figure S1). Gene structure analysis 140 revealed that the PTK genes have 1–6 introns (Figure 3).

141

141 Figure 3. Gene structure of PTKs genes in rice. Coding sequence (CDS) is shown as green color box, intron as Block line and 0, 1, 2 on the introns depics intron phase. Plants 2020, 9, 664 5 of 17

The protein length of PTKs varied from 376 to 500 amino acids, while the molecular weight ranged from 41.43 to 55.05 kDa. The pI (isoelectric point) of the PTKs varied from 5.69 to 10.24 (Table S1). Based on TargetP 1.1 prediction, there were four PTKs viz. PTK11, PTK14, PTK15, and PTK17, with high probability of chloroplast localization, PTK12 contained a signal peptide for secretory pathway, while rest of the PTKs were predicted to be localized in other locations. LOCALIZER1.0 predicted nuclear localization signal (NLS) in PTK1, PTK2, PTK5, PTK7, PTK8, PTK9, PTK10, PTK13, and PTK17, while NLS, cTP (chloroplast transit peptide) and mTP (mitochondrial transit peptide) in PTK12, NLS and cTP in PTK14, and mTP and NLS in PTK15 (Table S2). To understand the phylogenetic relationship with Arabidopsis PTKs and to identify the subfamily of kinases, rice PTK sequences were used as query and 18 Arabidopsis homologs were identified. The analysis of homologs in the phylogenetic tree revealed that most of the Arabidopsis homologs of rice PTKs belonged to group VII of receptor-like cytoplasmic kinases (RLCKs), which are mainly AVRPPHB SUSCEPTIBLE 1 (PBS1)-like 1 (PBL) proteins (Figure4). The orthologs of OsPTK1, OsPTK4 and OsPTK15 in Arabidopsis encode PBL proteins. The PBL proteins are involved in plant innate immunity [27–30]. Besides their role in innate immunity, OsPTK1 orthologs of Arabidopsis PBL34 (AT3G01300) and PBL35 (AT3G01300) have also been shown to regulate vitamin E biosynthesis in seeds [31]. Orthologs of OsPTK6 and OsPTK12 in Arabidopsis COLD-RESPONSIVE PROTEIN KINASE 1 (CRPK1, AT1G16670) [32] and Calcium/Calmodulin-Regulated Receptor-Like Kinase 1 (CRLK1, AT5G54590) [33], respectively, have been shown to regulate cold tolerance. RLKs contain a ligand binding ectodomain, a transmembrane domain, and a cytoplasmic kinase domain [34]. RLCKs are a subfamily of RLKs which lack transmembrane domains and ligand binding domain but retain the intracellular domains with kinase activity. There are about 162, 160, and 402 RLCK genes in maize, rice, and Arabidopsis, respectively [35–37]. Based on phylogenetic analysis these RLCKs have been divided into 15–17 subgroups in Arabidopsis, rice and maize [34,37]. However, these RLCKs were not further classified based on substrate specificity. To understand the regulation of PTK genes, in silico analysis of 2kb promoter region of PTK genes was carried out. The analysis revealed presence of several elements such as light responsive element, wound/elicitor response elements, abiotic stress response elements related to drought and salt stresses, and hormone responsive element associated with plant hormones such as ABA, IAA, GA and Ethylene (Table S3). Among cis-elements there was high propensity of recognition site for dehydration, salt, pathogenesis and wound response. Cis-elements related to root, mesophyl cell, grain and pollen specific expression were also highly presented in these promoters. Plants 2020, 9, 664 6 of 17 Plants 2020, 9, x FOR PEER REVIEW 6 of 17

177 Figure 4. Phylogenetic analysis of OsPTK and AtPTK protein sequences. The unrooted phylogenetic 178 Figure 4. Phylogenetic analysis of OsPTK and AtPTK protein sequences. The unrooted phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis software version 7.0 (MEGA7). 179 tree was constructed using Molecular Evolutionary Genetics Analysis software version 7.0 (MEGA7). The amino acid sequences were aligned using the Clastalw and Neighbor joining analysis was performed 180 The amino acid sequences were aligned using the Clastalw and Neighbor joining analysis was with the pairwise deletion option with Poisson correction, bootstrap analysis was conducted with 181 performed with the pairwise deletion option with Poisson correction, bootstrap analysis was 1000 replicates. The digits on branches indicate boot strap values. 182 conducted with 1000 replicates. The digits on branches indicate boot strap values. 2.2. mRNAseq Expression Analysis of PTK Genes 183 2.2. mRNAseq Expression Analysis of PTK Genes The expression potential of PTKs in different tissues, developmental stages and abiotic stresses was 184 analyzedThe byexpression using GENEVESTIGATOR. potential of PTKs in The different cluster analysistissues, development based on the expressional stages and levels abiotic in di stressesfferent 185 tissueswas analyzed and developmental by using GENEVESTIGATOR. stages revealed that The expression cluster analysis levels of basedPTK16 onand thePTK9 expressionwere very levels low in 186 exceptdifferent at flowering tissues and stage developmental where PTK16 stagesshowed revealed high level that of expression expression levels in panicles of PTK16 (Figure and5 ).PTK9 Most were of 187 very low except at flowering stage where PTK16 showed high level of expression in panicles (Figure 188 5). Most of the PTK genes were highly expressed in root. The cis-regulatory element (CRE)

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189 ROOTMOTIFTAPOX1the PTK genes were highly, which expressed is known in root.to induce The cis high-regulatory expression element in roots (CRE) [38,39],ROOTMOTIFTAPOX1 was found in all , 190 PTKswhich (Table is known S3). to induce high expression in roots [38,39], was found in all PTKs (Table S3). 191 PTK1PTK1 waswas ubiquitously ubiquitously expressed expressed in in all all the the tissues tissues wi withth high high levels levels of of expr expressionession at at booting booting and and 192 headingheading stage stage in in spikelets, spikelets, pistil, pistil, and and anther. anther. PTK3,PTK3, PTK4,PTK4, PTK7,PTK7, PTK8,PTK8 ,PTK10,PTK10 ,PTK17,PTK17 ,and and PTK18PTK18 193 werewere highlyhighly expressedexpressed in vegetativein vegetative (coleoptiles, (coleoptil rootes,/root root/root tips, shoot tips,/leaf, shoot flag/leaf, leaf) andflag reproductive leaf) and 194 reproductivetissues (panicle), tissues and (panicle), hence expressed and hence during expre allssed developmental during all developmental stages of rice. stagesPTK1 ,ofPTK6 rice., PTK13PTK1, , 195 PTK6PTK14, PTK13, and PTK15, PTK14,showed and PTK15 highlevel showed of expression high level in of anther expression and pistil in anther but their and expression pistil but levelstheir 196 expressionwere lower levels in leaf were/shoot lower and in flag leaf/shoot leaf. Expression and flag leaf. levels Expression of PTK2, PTK5,levels andof PTK2PTK11, PTK5,were and highest PTK11 in 197 wereroot followedhighest in by root leaf followed (Figure5 by). In leaf consistent (Figure 5). with In expressionconsistent with of PTKs expressionat flowering of PTKs stage at flowering in anthers, 198 stagethe CREs in anthers,POLLEN1LELAT52 the CREs POLLEN1LELAT52and GTGANTG10 and[40], GTGANTG10 which regulate [40], expression which regulate in pollens expression in rice, were in 199 pollensfound toin berice, highly were representedfound to be inhighly the promoter represented of allin thethe PTKspromoter. At milkof all stage the PTKs of grain. At milk development stage of 200 grainall PTKs developmentwere downregulated all PTKs were except downregulatedPTK1, PTK3 ,except and PTK10 PTK1., Interestingly,PTK3, and PTK10 during. Interestingly, dough stage 201 duringand mature dough grain stage stage, andPTK4 mature, PTK7 grain, PTK10 stage,, and PTK4PTK18, PTK7were, PTK10, upregulated and PTK18 which were suggests upregulated a role for 202 whichthese genessuggests in graina role maturation. for these genesPTK12 in, grainPTK13 maturation., and PTK14 PTK12were, highly PTK13, expressed and PTK14 up were to flowering highly 203 expressedstage but wereup to downregulatedflowering stage during but were grain downregulated development during (Figure grain5b). The development overrepresentation (Figure 5b). of CREThe 204 overrepresentationEBOXBNNAPA for of seed CRE specific EBOXBNNAPA expression for [41 ]seed was specific found in expression promoter of[41]PTKs was(Table found S3). in promoter 205 of PTKs (Table S3).

206

207 FigureFigure 5. 5. ExpressionExpression analysis analysis of of PTKPTK genes of rice. a (a) )Different Different tissues; tissues; b (b) )developmental developmental stages. stages. 208 ExpressionExpression analysis waswas carried carried out out with with mRNAseq mRNAse datasetsq datasets using using Genevestigator. Genevestigator. Genes Genes in same in clustersame 209 clusterare given are samegiven color same to color facilitate to facilitate easy comparison easy comparison between between (a) tissues a) andtissues (b) developmentaland b) developmental stages. 210 stages. Stress responsive expression pattern of PTKs were analyzed using mRNAseq data from different 211 abioticStress stress responsive experiments. expression The analysis pattern revealed of PTKs that werePTK11 analyzedand PTK16 usingwere mRNAseq upregulated data infrom root 212 differenttissues on abiotic 2nd and stress 3rd day,experiments. while PTK14 Thewas analysis upregulated revealed only that on PTK11 2nd day and of droughtPTK16 were stress. upregulated Expression 213 inlevels root oftissuesPTK2 on, PTK4 2nd, andPTK6 3rd, PTK8 day,, PTK13while ,PTK14PTK17, wasand upregulatedPTK18 were only significantly on 2nd day downregulated of drought stress. in root 214 Expressionon 2nd and levels 3rd days of ofPTK2 drought, PTK4 stress, PTK6 (OS-00230;, PTK8, PTK13 Figure,6 ).PTK17,PTK2 and PTK3PTK18showed were significantly 1.5 to 2 folds 215 downregulatedincrease in leaf tissuesin root afteron 2nd 13 and to 26 3rd days days of droughtof drought stress stress (OS-00369; (OS-00230; Figure Figure6). 6). PTK2 and PTK3 216 showedIn another1.5 to 2 droughtfolds increase stress in experiment leaf tissues (OS-00143), after 13 to drought26 days tolerantof drought (DT) stress donor (OS-00369; parent PSBRC28 Figure 217 6).(P28), DT introgression line H471 and drought sensitive (DS) recurrent parent Huang-Hua-Zhan (HHZ) 218 wereIn grown another in drought control (20%stress soil experiment moisture) (OS-00143) and drought, drought stress tolerant (soil moisture (DT) donor 15%, parent 10%) atPSBRC28 tillering 219 (P28),stage [DT42]. introgressionPTK3, PTK7, andline PTK14H471 andshowed drought upregulation sensitive in (DS) leaf tissuesrecurrent at least parent in oneHuang-Hua-Zhan genotype under 220 (HHZ)drought were stress. grown It is interestingin control (20% to note soil that moisture)PTK3 was and upregulated drought stress in leaves (soil atmoisture 15% not 15%, at 10% 10%) AWC, at 221 tilleringwhile PTK7 stagewas [42]. upregulated PTK3, PTK7, in leaves and PTK14 at 10% notshowed at 15% upregulation AWC in all threein leaf di fftissueserent genotypes. at least inPTK4 one , 222 genotypePTK5, PTK11 under, PTK13 drought, and stress.PTK17 It isshowed interesting significant to note downregulationthat PTK3 was upregulated under drought in leaves stress at in 15% leaf 223 not at 10% AWC, while PTK7 was upregulated in leaves at 10% not at 15% AWC in all three different

Plants 2020, 9, x FOR PEER REVIEW 8 of 17 Plants 2020, 9, 664 8 of 17 224 genotypes. PTK4, PTK5, PTK11, PTK13, and PTK17 showed significant downregulation under 225 drought stress in leaf tissues in all three genotypes (OS-00143; Figure 6). PTKs were found to be tissues in all three genotypes (OS-00143; Figure6). PTKs were found to be upregulated in seeds under 226 upregulated in seeds under heat stress with 1.5 to 2.5 fold increase in expression as compared with heat stress with 1.5 to 2.5 fold increase in expression as compared with control except PTK3, PTK9, and 227 control except PTK3, PTK9, and PTK12 (OS-00155; Figure 6). Expression levels of PTKs were largely PTK12 (OS-00155; Figure6). Expression levels of PTKs were largely unaltered under salt stress except 228 unaltered under salt stress except PTK14 which showed about 1.5 fold increase at 24 h of salt stress PTK14 which showed about 1.5 fold increase at 24 h of salt stress (OS-00148; Figure6). 229 (OS-00148; Figure 6).

230 Figure 6. Expression analysis of PTK genes under different abiotic stress conditions in rice. Expression 231 Figure 6. Expression analysis of PTK genes under different abiotic stress conditions in rice. analysis was carried out with mRNAseq datasets using Genevestigator. 232 Expression analysis was carried out with mRNAseq datasets using Genevestigator. The anther transcriptome analysis of photo thermo sensitive genic male sterile lines (PTGMS) rice 233 The anther transcriptome analysis of photo thermo sensitive genic male sterile lines (PTGMS) Y58S and Pei’ai64S (P64S) under cold stress [43] revealed that 1.25 to 2 fold upregulation of PTK2, PTK5, 234 rice Y58S and Pei’ai64S (P64S) under cold stress [43] revealed that 1.25 to 2 fold upregulation of PTK8, PTK14, PTK16, and PTK18 in P64S, a genotype relatively tolerant to cold, while these genes 235 PTK2, PTK5, PTK8, PTK14, PTK16, and PTK18 in P64S, a genotype relatively tolerant to cold, while were downregulated by 1.5 to 2 fold in cold susceptible Y58S. Only PTK13 showed 1.5 fold increase in 236 these genes were downregulated by 1.5 to 2 fold in cold susceptible Y58S. Only PTK13 showed 1.5 expression levels in both genotypes (OS-00153; Figure6). Interestingly PTK2, PTK5 and PTK6, PTK14, 237 fold increase in expression levels in both genotypes (OS-00153; Figure 6). Interestingly PTK2, PTK5 PTK16, and PTK18 showed upregulation in relatively cold tolerant genotype but were downregulated 238 and PTK6, PTK14, PTK16, and PTK18 showed upregulation in relatively cold tolerant genotype but in susceptible genotype. This suggests their potential role in spikelet fertility under cold stress. 239 were downregulated in susceptible genotype. This suggests their potential role in spikelet fertility In submergence stress experiment with deepwater rice cv. C9285 and non-deepwater rice cv. 240 under cold stress. Taichung 65 (T65) [44], several PTKs were upregulated (OS-00362; Figure6). PTK6, PTK13 and PTK14 241 In submergence stress experiment with deepwater rice cv. C9285 and non-deepwater rice cv. appears to be early submergence regulated genes as these were upregulated even at 1 h of submergence 242 Taichung 65 (T65) [44], several PTKs were upregulated (OS-00362; Figure 6). PTK6, PTK13 and stress in deepwater rice cv. C9285, while only PTK13 showed about 1.5 fold increase in expression 243 PTK14 appears to be early submergence regulated genes as these were upregulated even at 1 h of in T65. PTK6 expression was upregulated throughout the stress period in deepwater rice cv. C9285. 244 submergence stress in deepwater rice cv. C9285, while only PTK13 showed about 1.5 fold increase in PTK8 and PTK18 showed upregulation in expression (1.5 to 2 fold) even at 12-24 h in both deepwater 245 expression in T65. PTK6 expression was upregulated throughout the stress period in deepwater rice

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rice andPlants non-deepwater 2020, 9, x FOR PEER REVIEW rice genotypes. PTK1, PTK2, and PTK4 were preferentially upregulated9 of 17 246in non-deepwatercv. C9285. PTK8 rice and T65, PTK18 while showedPTK6 upregulationand PTK14 in wereexpression upregulated (1.5 to 2 fold) in deepwater even at 12-24 rice h in cv. both C9285. 247Interestingly deepwaterPTK14 rice showedand non-deepwater highest increase rice genotypes. in expression PTK1 after, PTK2, 1 h submergence and PTK4 were in C9285, preferentially while it was 248significantly upregulated downregulated in non-deepwater in non-deepwater rice T65, while rice PTK6 cv. and T65 PTK14 (Figure were6). PTK8upregulated, PTK13 in, anddeepwaterPTK18 ricemay be 249involved cv. C9285. in general Interestingly submergence PTK14 tolerance showed highest responses increase as these in expression were upregulated after 1 h insubmergence both tolerant in and 250susceptible C9285, genotypes,while it was while significantPTK14ly appearsdownregulated to be associated in non-deepwater with specific rice cv. mechanism T65 (Figure of 6). submergence PTK8, 251tolerance PTK13, in deep-waterand PTK18 may rice. be involved in general submergence tolerance responses as these were 252 upregulated in both tolerant and susceptible genotypes, while PTK14 appears to be associated with 2532.3. Real-Timespecific mechanism RT-PCR Analysis of submergence of Expression tolerance of PTK in deep-water Genes rice.

254 The2.3. stressReal-Time responsive RT-PCR expressionAnalysis of Expression of PTK genes of PTK was Genes further analyzed by quantitative RT-PCR analysis at seedling stage and anthesis stage in drought tolerance rice cv. Sahbhagi Dhan (IR74371-70-1-1). It is a 255 The stress responsive expression of PTK genes was further analyzed by quantitative RT-PCR 256drought analysis tolerant at seedling rice cultivar stage with and wider anthesis adaptability. stage in drought It was also tolerance released rice in cv. Nepal Sahbhagi and Bangladesh Dhan 257in the(IR74371-70-1-1). name of Sukha It Dhan is a drought 3, and tolerant BRRI Dhan rice cultivar 56, respectively. with wider Atadaptability. seedling stage,It was osmoticalso released stress in was 258imposed Nepal for and two Bangladesh weeks. PTK1 in theand namePTK10 of Sukhawere Dhan upregulated 3, and BRRI by Dhan 2 and 56, 2.5 respectively. folds in shoots At seedling at seedling 259stage stage, under osmotic osmotic stress stress was (Figure imposed7). for two weeks. PTK1 and PTK10 were upregulated by 2 and 2.5 260 folds in shoots at seedling stage under osmotic stress (Figure 7).

261

262 FigureFigure 7. Expression 7. Expression analysis analysis of PTK of PTKgenes genes under under osmotic osmotic stress stress at at seedling seedling stage stage inin hydroponics,hydroponics, and 263 underand drought under stressdrought at anthesisstress at anthesis stage in stage field conditionsin field conditions in rice. in The rice. relative The relative expression expression was analysedwas ∆∆ct ΔΔ 264 by 2−analysedmethod. by 2− Ctct method values. Ct of controlvalues of served control as served calibrator. as calibrator. Bars in Bars blue in and blue red and colors red colors represent control and stress treatments, respectively. Small letters a, b, and c represents statistical difference,

different letters indicate significant differences; Error bars indicate SE; n = 3 biological replicates. Plants 2020, 9, 664 10 of 17

In contrast, all the PTKs except PTK10 were upregulated in roots at seedling stage under osmotic stress. The level of expression in root tissues was in the range of 2.6 to 6.5 fold except PTK14 with the highest (46.75 folds) induction under osmotic stress in the drought tolerant rice cultivar Sahbhagi Dhan (Figure7). Although most PTKs showed high level of expression in root tissues during normal development, only PTK11, PTK14, and PTK16 were found to be upregulated under drought stress in roots in Genevestigator datasets (Figures5 and6). PTK4 showed significant upregulation (6 fold) in the roots of drought tolerant rice cv. Sahbhagi Dhan in our study (Figure7). In an earlier study, bbs1 (bilateral blade senescence 1) rice mutant with early leaf senescence character was identified from EMS mutagenesis. Map-based cloning revealed that bbs1 is a single base insertion in LOC_Os03g24930 (i.e., PTK4 in our study) which led to loss-of function. BBS1 expression was upregulated up to 8 h and later down regulated under salt stress. The bbs1 mutant also showed hypersensitivity to salt stress [45]. In our study PTK4/BBS1 expression was upregulated under osmotic stress. Our results suggest that in addition to salt stress [45], PTK4/BBS1 may also confer osmotic stress tolerance to rice. To analyze the expression of PTK genes at anthesis stage, drought stress was imposed to the field grown plants of rice cv. Sahbhagi Dhan at anthesis stage by withholding irrigation. Flag leaf and panicle tissues were sampled for analysis when the soil moisture decreased to about 6% in the top 15 cm soil layer (Figure S2a) and the leaf relative water content was 64.3 3.1% in the ± drought stressed plants (Figure S2b). None of the PTKs were upregulated in flag-leaf and panicle. PTK6 was downregulated in flag leaf, while in panicle PTK4 and PTK18 were downregulated under drought stress. Although osmotic stress upregulated most of the PTKs in roots, only 1–2 PTKs were upregulated in leaf tissues under osmotic stress caused by PEG, drought and salt. In contrast, under temperature (cold and heat) stresses, many PTKs were upregulated (Figures6 and7). The reasons for upregulation of fewer genes under drought and salt stress as compared to that under temperature stresses may due to the target tissues and genotypes analyzed. For example, drought and salt stress studies were done in vegetative tissues (root and leaf), while the temperature stress studies were done in reproductive tissues (anther and seed). As ABA-dependent signaling plays a major role in stress inducible gene expression [46], mining of microarray data from RiceExPro was carried out. The results showed that ABA-mediated expression of PTKs in roots of rice seedlings is highly dynamic over durations of the treatment. ABA treatment moderately upregulated PTK3 and PTK13 at 1 h, but were downregulated at 6 h in roots. PTK2, PTK11, PTK14, PTK15, and PTK16 were upregulated by ABA at 3–6 h, especially PTK11, while PTK5 and PTK12 were also upregulated at 6h in roots of rice seedlings. However, PTK7, PTK8, and PTK18 were downregulated by ABA at 3–6 h in roots (Figure8). In the shoots of rice seedlings, PTK11 was highly upregulated at 6 h of ABA treatment, while PTK5, PTK6, and PTK13 were moderately upregulated (Figure8). Hence these 13 PTK genes may be regulated through ABA-dependent pathway under stress. Of these ABA-regulated PTKs, PTK6, PTK11, and PTK13 were regulated in both root and shoot, and rest 10 PTKs were regulated only in root. Jasmonic acid (JA) cross-talks with ABA and regulate abiotic stress tolerance. Some of the OsPTK orthologs in Arabidopsis have been shown to regulate plant immunity against hemibiotrophic pathogens [30,47]. Hence, we examined the expression of OsPTKs to understand whether they are also regulated by JA-dependent pathway. PTK5, PTK8, PTK11, PTK13, and PTK15 were upregulated at 1 h in roots by JA, but PTK13 and PTK15 were downregulated at 3–6 h. By 3 or 6 h of treatment, PTK3, PTK5, PTK7, PTK11, PTK17, and PTK18 were also upregulated in roots by JA (Figure8). As regards shoots, PTK11, PTK13, and PTK17 were upregulated by JA at 3 and/or 6 h of treatment (Figure8). Interestingly, PTK11 and to a lesser extent, PTK5 and PTK13 were upregulated both in roots and shoots by both ABA and JA (Figure8). All the PTKs examined showed upregulation in at least one of abiotic stress condition. PTK2, PTK8, PTK13, and PTK18 were upregulated under osmotic, heat, cold and submergence stresses, while PTK14 Plants 2020, 9, x FOR PEER REVIEW 11 of 17 Plants 2020, 9, 664 11 of 17 316 All the PTKs examined showed upregulation in at least one of abiotic stress condition. PTK2, 317 PTK8, PTK13, and PTK18 were upregulated under osmotic, heat, cold and submergence stresses, was upregulated under all abiotic stresses examined (Figures6 and7). Promoter analysis also revealed 318 while PTK14 was upregulated under all abiotic stresses examined (Figures 6 and 7). Promoter abundance of stress and ABA regulated CREs such as MYBCORE and MYCCONSENSUSAT in PTKs 319 analysis also revealed abundance of stress and ABA regulated CREs such as MYBCORE and (Table S3). 320 MYCCONSENSUSAT in PTKs (Table S3).

321 322 Figure 8.8. ExpressionExpression analysis analysis of PTKof PTKgenes genes under under ABA andABA Jasmonic and Jasmonic acid treatment acid treatment in rice. Expression in rice. 323 Expressionanalysis was analysis carried outwas with carried Rice out Expression with Rice Profile Expression Database Profile (RiceXPro) Database version (RiceXPro) 3.0 (https: version//ricexpro. 3.0 324 (https://ricexpro.dna.affrc.go.jp/).dna.affrc.go.jp/). Seven days old seedlings Seven days of japonica old seedlings rice cv. of Nipponbare japonica rice was cv. treated Nipponbare with Abscisic was µ µ 325 treatedacid (50 withM) Abscisic or Jasmonic acid (50 acid μM) (100 or JasmonicM) in Yoshida’s acid (100 nutrient μM) in solution.Yoshida’s Cy3nutrient (control) solution. and Cy3 Cy5 326 (control)(hormone and treatment). Cy5 (hormone treatment). Some of the Arabidopsis homologs of rice PTKs have been shown to play important roles in biotic 327 Some of the Arabidopsis homologs of rice PTKs have been shown to play important roles in and abiotic stress tolerance, and development. For example, At4G35600 (CAST AWAY), a homolog of 328 biotic and abiotic stress tolerance, and development. For example, At4G35600 (CAST AWAY), a OsPTK11, has been shown to inhibit organ abscission in Arabidopsis [48]. At1G61590 (SCHENGEN1), 329 homolog of OsPTK11, has been shown to inhibit organ abscission in Arabidopsis [48]. At1G61590 an ortholog of OsPTK4, is required for the positioning and correct formation of the centrally located 330 (SCHENGEN1), an ortholog of OsPTK4, is required for the positioning and correct formation of the Casparian strip membrane domain proteins (CASPs) domain endodermal cells in Arabidopsis [49]. 331 centrally located Casparian strip membrane domain proteins (CASPs) domain endodermal cells in In our study PTK4 was specifically found to be upregulated in root under drought (Figure6). Casparian 332 Arabidopsis [49]. In our study PTK4 was specifically found to be upregulated in root under drought

Plants 2020, 9, 664 12 of 17 strip formation is an important adaptive trait for drought tolerance. Hence, it will be worth examining the role of this kinase in Casparian strip formation in rice under drought. AT5G54590, an ortholog of OsPTK12, codes for CALCIUM/CALMODULIN-REGULATED RECEPTOR-LIKE KINASE 1 (CRLK1), plays important role in cold tolerance of Arabidopsis [50]. At2G05940 (RIPK/ACIK1A/PBL14) and At5G35580 (AvrPphB SUSCEPTIBLE1-LIKE13, PBL13), homologs of OsPTK4 and, At2G02800 (PBL3) and At5G01020 (PBL8), homologs of OsPTK11 and OsPTK18, respectively, have been shown to confer immunity against pathogenic bacteria in Arabidopsis [27,51]. AtPBL13 knockout mutation resulted in enhanced reactive oxygen species (ROS) burst in response to bacterial attack [27]. Since ROS signaling and ROS management are key for abiotic stress tolerance, and abiotic stresses differentially regulate the expression of OsPTK4, OsPTK11 and OsPTK18 in rice (Figure6), it is tempting to speculate the that these PTKs may play a role in abiotic stress tolerance through ROS signaling and management.

3. Materials and Methods

3.1. Plant Growth Conditions and Treatments Rice (O. sativa ssp. indica) variety Sahbhagi Dhan (IR74371-70-1-1) was used for expression analysis in the present study. Two experiments were conducted one each at seedling and reproductive stage. In experiment with seedlings, seeds were germinated on germination paper under culture room conditions. Five-day old uniform rice seedlings were transferred to Yoshida solution. For imposing osmotic stress, Yoshida’s solution was supplemented with PEG6000 i.e., 100 g PEG6000 was dissolved in 1000 mL Yoshida’s solution. Seedlings grown in Yoshida solution without PEG served as control. The seedlings were grown for 21 days in hydroponics at 25 ◦C under 16 h/8 h light/dark conditions. For imposition of drought stress at reproductive stage, 21 days old seedling were transplanted in 1 m2 puddled plots and grown under natural field conditions. Drought stress was imposed by with-holding irrigation at booting stage till soil matric potential (SMP) of ~ 70 KPa was reached. − The flag leaf and panicle tissues were collected upon attainment of SMP ~ 70 kPa stress, which − coincided with 50% anthesis. Plots maintained at < 10 kPa SMP served as control. The flag leaf − relative water content (RWC) was measured following Barrs and Weatherley (1962) [52]. Soil moisture content (SMC) was measured by gravimetric method.

3.2. Identification of PTK Gene Family Members in Rice To identify members of rice PTK gene family, a keyword search was performed using “Tyrosine” in the “Putative Function Search Tool” in Rice Genome Annotation Project Release 7 (RGAP, http://rice.plantbiology.msu.edu/). Twenty seven genes annotated as “tyrosine protein kinase domain containing protein” were identified from the rice genome. The nucleotide sequences and protein sequences were downloaded for further use. Protein sequences of the identified PTK homologs were subjected to domain analysis using PFAM (https://pfam.xfam.org/) and HMMER scan (https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan). The sequence logos were generated using web logo tool (https://weblogo.berkeley.edu/logo.cgi). The physical location of all identified PTK genes on chromosome was mapped using Chromosome Map Tool (http://viewer.shigen.info/oryzavw/maptool/MapTool.do). Gene structure of PTK gene, showing their exon-intron boundaries and inter phase, was generated using GSDS server (http://gsds.cbi.pku.edu.cn/). The protein sequences of putative OsPTKs were used for BLAST search of Arabidopsis thaliana genome at Ensembl Plants (http://plants.ensembl.org/index.html) and homologs were identified. After removing the redundant genes, a domain analysis was carried out. The protein sequences of rice and Arabidopsis were used for phylogenetic analysis using Molecular Evolutionary Genetics Analysis software version 7.0 (MEGA7) [53]. The amino acid sequences were aligned using the Clastalw program and Neighbor joining analysis was performed with the pairwise deletion option with Poisson Plants 2020, 9, 664 13 of 17 correction, bootstrap analysis was conducted with 1000 replicates. Subcellular localization of PTKs was predicted by using TargetP 1.1 [54] and LOCALIZER 1.0 [55].

3.3. Promoter Analysis For in silico analysis of conserved cis elements in promoters of PTK genes, 2 kb sequences upstream of translational start codon were analyzed using PLACE database (https://www.dna.affrc.go.jp/PLACE/)

3.4. In Silico Analysis of PTK Expression under Abiotic Stresses The in silico gene expression analysis was done using GENEVESTIGATOR software [56] (https://genevestigator.com/gv/index.jsp). The locus IDs of all the PTK genes were given as input in the development and perturbations tool and searched against experiments involving seedling root, shoot, panicle and flag leaf under drought and cold stresses. Expression potential represents the normalized expression value for a gene across all experiments available in the database [56] (https://genevestigator.com/gv/index.jsp). For analysis of Abscisic acid and Jasmonic acid mediated regulation of PTK genes, Rice Expression Profile Database (RiceXPro) version 3.0 (https://ricexpro.dna.affrc.go.jp/)[57] was used. In this data base, the data sets RXP_1001 (Root gene expression profile in response to abscisic acid), RXP_1006 (Shoot gene expression profile in response to abscisic acid), RXP_1011 (Root gene expression profile in response to jasmonic acid) and RXP_1012 (Shoot gene expression profile in response to jasmonic acid) were used. These data sets were generated by microarray analysis of 7-days old seedlings of japonica rice cultivar Nipponbare treated with Abscisic acid (50 µM) or Jasmonic acid (100 µM) in Yoshida’s nutrient solution under hydroponic condition in a growth chamber at 28 ◦C under continuous light. mRNA isolated from different time interval were labeled with Cy3 (control) and Cy5 (hormone treatment) for microarray hybridization.

3.5. RNA Isolation, cDNA Synthesis, and Quantitative RT-PCR Total RNA from seedling root and shoot, flag leaf and panicle was isolated using TRIzol reagent (Invitrogen). The quantitative and qualitative analysis carried out using spectrophotometer (Nano Drop Nd-1000 UV/Vis Spectrophotometer) and 1.2% agarose gel, respectively. Of the total RNA treated with DNase I (TURBO DNA-free ™ Kit (Ambion), 4.5 µg was reverse transcribed using Superscript III Reverse Transcription as per manufacturer’s instructions (Invtrogen). The sequence of primers used for qRT-PCR analysis as provided in Table S4. Quantitative PCR (qPCR) was performed with three biological and three technical replicates with appropriate primers using PoweUpSYBR®green Master Mix as per manufacturer’s instructions (Thermo Fisher, USA). qRT-PCR was carried out in StepOne real time PCR machine (Applied Biosystems). Expression data were normalized using endogenous control ∆∆Ct gene OsUBIQUITIN5 (UBQ5). Relative fold change was calculated using the 2− method [58]. The expression was represented in the form of relative fold change in expression of genes under stress conditions as compared with its expression under control conditions. The R package software (R studio) was used for analysis of variance for gene expression data and different letters denote significant variation (p < 0.05).

4. Conclusions Analysis of 27 annotated putative PTKs in rice genome led to the identification 18 PTKs with protein tyrosine kinase motif. The kinase domains of PTKs showed high homology with that of Arabidopsis BRI1, a well characterized dual specific kinase, suggesting that the 18 PTKs identified in this study are dual specific kinases. We identified Arabidopsis homologs of rice PTKs and showed that most of the PTKs are homologous to RLCK VII group of genes from Arabidopsis. mRNAseq analysis using Genevestigator and qRT-PCR analysis revealed developmental and abiotic stress responsive expression pattern of PTKs in rice. Expression analysis suggested that most of the PTKs are downregulated by drought stress, but were upregulated under temperature (heat and cold), and submergence stresses. Plants 2020, 9, 664 14 of 17

The upregulation of transcript levels under abiotic stresses mostly varied from 1.5 to 3 folds. Since regulatory function of kinases occurs through phosphorylation, even small increase in transcript levels might be sufficient for regulation stress response. Furthermore, RLCKs in association with receptor kinases (RKs) have been shown to play important role in signaling, and several plant processes and response to abiotic and biotic stresses. This study identified PTK2, PTK8, PTK13, PTK14, and PTK18 as multiple abiotic stress (osmotic, heat, cold and submergence) upregulated genes. PTK14 was preferentially upregulated in cold and submergence tolerant rice genotypes but was down regulated in susceptible genotypes. These PTKs may contribute to multiple stress tolerance in rice. The abiotic stress regulated OsPTKs identified in this study lays foundation for function validation of this enigmatic family of tyrosine kinases by using transgenic overexpression and CRISPR-Cas genome editing approaches.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/5/664/s1, Figure S1: PTKs Gene Location on Chromosomes of O. sativa, Figure S2: Soil moisture content (a) and leaf relative water content (b) of rice cv. Sahbhagi Dhan under well irrigated and drought stress in field conditions, Table S1: Protein characteristics of PTKs encoded by rice genome, Table S2: Prediction of cellular localization of PTKs, Table S3: Frequency of Cis-regulatory Elements (CRE) in the of promoter of PTK genes, Table S4: List of primers used for gene expression analysis. Author Contributions: V.C., M.D. and A.E. planned the study and designed the experiments; A.E., M.D. and V.C. carried out bioinformatic analyses and Genevestigator analysis; A.E., G.K.K. and S.D. conducted lab and field experiments; A.E., R.R.K. and L.S. prepared the manuscript; M.D. and V.C. edited the manuscripts. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by NAHEP, Indian Council of Agricultural Research (ICAR), New Delhi, grant No. NAHEP/CAAST/2018-19/07 and ICAR-IARI, New Delhi, grant no. CRSCIARISIL20144047279. Acknowledgments: A.E. acknowledges Junior Research Fellowship granted by ICAR-IARI, New Delhi for his M.Sc. study. Conflicts of Interest: The authors declare no conflict of interest.

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