Introduction

Teosinte branched1, Cycloidea, Proliferating cell factor(TCP)-domain proteins are plant specific regulators of growth and organ patterning.These are basic helix-loop-helix (bHLH) transcription factors (TFs) but do not bind toE-Box DNA sequence. Sequence divergence in the TCP-domain of these non-conventional bHLHproteins further divides them into Class-I and -II TCP TFs, manifests position specificpreferences for certain bases in their otherwise similar DNA-binding sequence and allowsdimerization more freely between members of the same class1,2. Theabundance of Class-I and -II TCP DNA-binding element in promoter of contrasting groupsof genes creates functional antagonism between these two groups of proteins. WhileClass-I TCP TFs generally promote cell division and proliferation and support thegrowth of organs and tissues, Class-II TCP proteins are known to functionoppositely3. Also, owing to overlapping expression pattern andfunction of various Class-I TCP TFs, the phenotypes of their overexpression as well asmutant lines are mostly feeble or undetectable4,5.

In a wide variety of plants, TCP TFs regulate different developmental aspects throughtheir effect on similar molecular pathways that include cytokinin, auxin, jasmonic acid(JA) and strigolactone6. These proteins also function by interacting withother TFs5,7 and regulate gene expression by recruiting chromatinmodifiers like BRAHMA (BRM)8. TCP-regulated phenotypes include leafshape, branch pattern, epidermal cell differentiation and floral structure andpatterning6. TCP proteins have also been shown to integrate externalsignals into developmental pathways as exemplified by dark-responsive mesocotylelongation in rice9.

The intrinsic developmental program of plants always remains knotted to external cues andis severely affected by abiotic stress conditions. Plants have developed mechanisms towithstand such harsh conditions by activating enzymes, transcription regulators andother factors that operate in pathways governed by hormones like abscisic acid (ABA) andsecond messengers like Ca2+. Interestingly, knockdown of a subset ofClass-II TCP TFs by overexpression of mir319 increases tolerance to dehydrationand salinity stress in bentgrass10. Moreover,Ca2+-triggered signaling in Arabidopsis is known to activate genesthrough CAMTA-, DREB-, ABRE- and Class-I TCP-like factor binding sites in their promoterregions11. Mutation disrupting the function of MSI1 (atranscriptional repressor), not only induces stress and ABA-responsive genes but alsoupregulates two Class-I TCP and a subset of Class-I TCP-regulated genes12. These reports do indicate a possible relation between pathways regulated by abioticstress and ABA and those governed by Class-I TCP TFs.

In a previous study from our laboratory, based on microarray data, upregulation ofOsTCP19, a Class-I TCP TF, in response to dehydration, salinity and cold wasinferred13. The present work was undertaken to explore any possiblerole of Class-I TCP TFs in stress signaling network in rice. The results of the presentwork provide evidence about the possible mechanism by which OsTCP19 may confer salt andwater-deficit tolerance.

Results

Abiotic stress-responsiveness of OsTCP19

A previous microarray analysis from our laboratory pointed out an increase inexpression of a Class-I TCP TF gene, OsTCP19, within a few hours exposureof rice seedlings to salt, drought and cold stress13 (GSE6901;Supplementary Fig. S1a,b online). To substantiate thisobservation and elucidate the role of this gene in stress tolerance, a detailedqRT-PCR analysis was conducted and the expression profile of OsTCP19 fromstress-sensitive indica rice variety Pusa Basmati 1(PB1) was compared with thatfrom salt-tolerant Pokkali and drought-tolerant Nagina 22 (N22) rice genotypesunder salt and drought stress, respectively. Compared to the untreated controlsamples (0 h), qRT-PCR analysis for shoots of 0, 0.5, 3, 6 and 24 h saltstressed PB1 and Pokkali rice seedlings confirmed 5 to 6-fold upregulation ofthis gene within 6 h of stress (Figure 1a,b). About 2-foldupregulation of this gene within 3 h of salt stress was also observed for rootsof salt stressed PB1 and Pokkali seedlings (Figure 1d,e).While this expression increases up to 9-fold (by 24 h) and 5-fold (by 5 and 6 h)under water-deficit stress in shoots of PB1 and N22, respectively,water-deprived roots of both these varieties only show marginal fluctuation intranscript abundance (Figure 1g,h,j,k). Interestingly, acomparison of the relative transcript level with respect to the reference gene(UBQ5) expression (2−ΔCtplot) indicated higher abundance of OsTCP19 in the tissues ofstress-tolerant varieties than the sensitive PB1 variety at least during earlyhours of stress exposure (Figure 1c,f,i,l). These resultssuggest a probable role of OsTCP19 in early response to abioticstresses.

Figure 1
figure 1

OsTCP19 is upregulated under salt and water-deficit stress.

(a,b,d,e,g,h,j,k) qRT PCR analysis indicating fold-change inexpression (2−ΔΔCt plot) ofOsTCP19 in shoots and roots of PB1, Pokkali and N22 rice undersalt (200 mM NaCl) or water-deficit (air-drying) stress over theirrespective controls (unstressed tissue, 0 h sample). Value of control sample(not shown in the histogram) is equivalent to 1 and‘*’ indicates data significantly different fromcontrol sample (t-test, two tailed p-value ≤ 0.05).(c,f,i,l) Comparison of the OsTCP19 transcript level underwater-deficit or salt stress in PB1, Pokkali and N22 rice varieties relativeto the reference gene (UBQ5) expression(2–ΔCt plot).‘*’ indicates data significantly different fromPB1 rice (t-test, two-tailed p-value ≤ 0.05). In allcases, X-axis indicates different time points after stress subjection andthe error bars represent SD and the p-value is mentioned over therespective bars. All data were simulated from three independent set ofexperiments (biological replicates). For each set of experiment, allvarieties of rice were grown and subjected to stress simultaneously.

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Expression of OsTCP19 was also positively influenced by exogenous application ofstress-related hormones, namely, ABA, salicylic acid and methyl jasmonate (Supplementary Fig. S1c online). However, ABA caused mostconsistent and intensified expression (about 5-fold) of this gene, indicating astrong association of OsTCP19 with ABA-mediated abiotic stress-signalingpathways. The untreated PB1 seedlings, incubated simply in fresh Yoshida mediumfor the same duration as mentioned for stress or hormone treatments, did notshow any significant difference in OsTCP19 expression (Supplementary Fig. 1d online). This indicated that the alteration inOsTCP19 expression is specific to stress and hormone treatments.

OsTCP19 from indica rice contains an alternatively splicedintron

OsTCP19 was found to share more similarity with homologous protein sequences frommonocots than other plant groups (Supplementary Fig. S2aonline; Supplementary Table S1 online). AmongArabidopsis sequences, TCP15 and TCP14 were found closest(49–52% similarity) to OsTCP19 (Supplementary Fig.S2b,c online). RGAP database annotates this gene as intronless.However, its cloning using PB1 rice genomic DNA revealed the presence of anin-frame 36 bp insertion just before the designated TCP-domain. Owing to broadconservation in TCP-regulated pathways across plant species, this clonedfragment was overexpressed in Arabidopsis thaliana (Col-0) under thecontrol of CaMV 35S promoter (p35S:OsTCP19) for evaluating itsrole in stress tolerance (Supplementary Fig. S4a online).The 36 bp insertion and the flanking regions bear little similarity toArabidopsis sequences. Hence, primers designed from the flankingregions (36-i primers) were used in a RT-PCR analysis meant for recording thelevel of expression of the transgene. This, however, resulted in amplificationof about 136 bp DNA fragment instead of 172 bp suggesting this insertion, whichbegins and ends with GC and AG dinucleotides, is spliced and represents anintron.

Further RT-PCR analyses for studying the splicing of this gene using 36-i primersdetected higher abundance of the spliced form (OsTCP19s; 136 bp amplicon)than the unspliced form (OsTCP19i; 172 bp amplicon) of OsTCP19 inall tested samples of PB1 rice except 24 h salt stressed shoots and 3 h or morewater-deficit stressed roots (Figure 2a). As amino acidstretch similar to that encoded by the 36 bp intron is present in homologousproteins from other monocots (Supplementary Fig. S2donline), higher abundance of OsTCP19i in other rice varietiesappeared possible. On further analysis, OsTCP19i was observed as themajor transcript form in all the stressed tissues of N22 whereas OsTCP19stranscript seems scarcely detectable (Figure 2b). Whileboth forms were detected in unstressed Pokkali shoots, OsTCP19i wassignificantly enriched under salt stress (Figure 2b).Expression of OsTCP19s remained rather low in Pokkali roots. Anypossibility of genomic DNA contamination in these assays was ruled out by acontrol RT-PCR using primers flanking an intron of OsEF1α(LOC_Os03g08020) which only amplified DNA fragment (103 bp) of sizeexpected from cDNA. A graphical representation of this analysis is shown in‘Supplementary Fig. S3 online’.Thus, it appears that OsTCP19 from indica rice bears an alternativelyspliced GC-AG intron and its splicing is dependent on plant type, variety,tissue and stress condition.

Figure 2
figure 2

Splicing of OsTCP19 and subcellular localization of the encodedproteins.

(a,b) RT-PCR analysis using primers flanking an intron of OsTCP19 andOsEF1α for unstressed (0 h) and stressed (0.5-24 h)tissues of indica rice seedlings (as indicated). M indicates the markerlane. The black and grey arrows correspond to band size of 172 bp and 136bp, respectively. (c) ClustalW alignment showing region of mutation (blackarrows) at the intron boundaries of mOsTCP19i relative toOsTCP19s and OsTCP19i and the corresponding change inprotein sequence (grey arrow). The numbers correspond to the respectivenucleotide or amino acid position. (d) RT-PCR analysis of tobacco leavesinfiltrated with Agrobacterium cells bearing constructp35S:OsTCP19s (lane s) and p35S:mOsTCP19i (lane m) usingprimers flanking OsTCP19 intron. (e,f) Fluorescence microscopy of onionepidermal cells expressing either YFP, YFP-mOsTCP19i orYFP-OsTCP19s as indicated (scale bar = 50 µm). (g)Higher resolution images of nuclear YFP fluorescence (scale bar = 10µm).

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For further characterization, OsTCP19s was cloned from PB1 rice seedlings.The 5’ and 3’ intron boundaries of OsTCP19i were alsomutated (5’GC≫GG, 3’AG≫AA) torestrict its splicing (mOsTCP19i) which, however, resulted in an Ala toGly transition in the protein sequence (Figure 2c). Thismutant construct was validated experimentally and only unspliced transcriptscould be detected in tobacco leaf cells transiently expressing mOsTCP19i underthe regulation of CaMV 35S promoter (Figure 2d). Particlebombardment of constructs bearing these ORFs fused to C-terminus of YFP(p35S:YFP-mOsTCP19i and p35S:YFP-OsTCP19s) on onion epidermalcells revealed the nuclear enrichment for both the proteins (Figure 2e). Control construct, p35S:YFP, generatesfluorescence dispersed throughout the cell (Figure 2f). Inaddition to their presence in the whole nucleus, both these proteins were alsodetected in the form of multiple nuclear bodies (NB; Figure2g) indicating the role of OsTCP19 in transcription as well as othernuclear phenomena.

Phenotype and stress response of p35S:OsTCP19Arabidopsis transgenics

For functional analysis, four T3 generation p35S:OsTCP19 transgenic lines,L1, L5, L6 and L8, homozygous for single insertion were selected. Duringscreening of T0 seeds, a line negative for hygromycin selection marker was alsopicked. This line, NT, was used as a negative control besides wild-type, WT,plants in various analyses. As mentioned before, OsTCP19s but notOsTCP19i transcripts could be detected by RT-PCR analysis in thetransgenic seedlings grown on only MS medium (control) or supplemented with 125mM NaCl or 350 mM mannitol (Figure 3a). Moreover, theexpression was rather low in L5 compared to other transgenic lines, whereas nospecific amplification was obtained for WT and NT plants.

Figure 3
figure 3

Phenotypes of p35S:OsTCP19 Arabidopsis transgenic plants.

(a) Semi-qRT-PCR using 36-i primers depicting amplification of 136 bpfragments in transgenics (L1, L5, L6, L8) but not in NT and WT plants undercontrol (C; unstressed), salt stress (N; 125 mM NaCl) and water-deficitstress (M; 350 mM mannitol). ACT2 was used as endogenous control. (b)Root growth in 10-day-old transgenic, NT and WT seedlings. The analysis wasperformed with a total of 250–300 plants grown in threeindependent batches. (c) LR formation in 15-day-old transgenic and WTseedlings grown in glass vials containing 0.2% phytagel or Petri platescontaining 0.8% agar as solidification base. Histogram was plotted from theanalysis of at least 100 plants grown in three independent batches in Petriplates. (d) RH in 15-day-old seedlings of transgenic and WT plants. (e)Trichomes in ~11 cm long inflorescence stem of transgenic and WT plants.Error bars in the histograms indicate SD. ‘*’indicates data significantly different from WT (t-test, two-tailedp-value ≤ 0.05). In all histograms, the p-value is mentioned overthe respective bars.

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Although both transgenic and non-transgenic plants displayed similar efficiencyand rate of germination (Supplementary Fig. S4b online),slower initial growth of the transgenic lines was clearly inferred and wasevident by reduced rate of root elongation till 15 days after germination (DAG;Figure 3b, Supplementary Fig. S4eonline). Strikingly, by 15 DAG, the transgenic plants displayedsignificantly fewer numbers of lateral roots (LRs) as compared to WT plants(Figure 3c). The transgenic plants visually appearedto have higher number of trichomes on the inflorescence stem and more root hairs(RHs; Figure 3d,e), indicating a possible role ofOsTCP19 in epidermal cell differentiation as well. In addition, earlyflowering was observed in the transgenic lines grown on MS-agar medium insidevertically oriented Petri plates (Supplementary Fig. S4conline). However, this phenotype could not be inferred convincingly inplants grown in pots containing Soilrite mix. Therefore, constitutiveoverexpression of OsTCP19 in Arabidopsis affects initial seedlinggrowth, LR development, trichome and RH formation and condition-dependent earlyflowering.

The seeds of WT, NT and transgenic plants had no major difference in germinationresponse under salt and water-deficit stress (Supplementary Fig.S4f online). However, following germination, seedling establishment,growth and biomass accumulation were strikingly better in transgenic lines thanWT or NT plants under higher (125 mM NaCl or 350 mM mannitol) but not lower (100mM NaCl or 200 mM mannitol) degree of stress (Figure4a–d; Supplementary Fig. S4g,honline). These results suggest a role for OsTCP19 instress-responsive post-germination growth, seedling establishment and biomassaccumulation but not in germination per se. In a different experiment,when 12-day-old unstressed plants were transferred and observed for further 33days in vertically oriented Petri plates containing MS-agar medium supplementedwith 100 mM NaCl, better efficiency of flowering was found in transgenic than WTplants (Supplementary Fig. S4d online). This probably isrelated to mechanisms that caused early flowering in unstressed plants (Supplementary Fig. S4c online).

Figure 4
figure 4

Abiotic stress tolerance of p35S:OsTCP19 Arabidopsis transgenicplants.

(a–c) Post-germination growth and seedling establishment of WT, NTand transgenic plants (15 DAG) in response to salt and water-deficit stressunder horizontal and vertical growth conditions. (d) Ratio of total biomassaccumulated (per 100 seeds sown) by different transgenic, WT or NT linesunder abiotic stresses (as indicated) to that under control condition. (e)Analysis of water-deficit (water withholding) and salt (200 mM NaCl)tolerance level in 2-week stresses plants (24-day-old) that were allowed torecover for 1 week. (f,g) Percentage of plant recovered or survived at theend of recovery phase. Error bars in the histograms represent SD.‘*’ indicates data significantly different from WT(t-test, two-tailed p-value ≤ 0.05). The p-value ismentioned over the respective bars of the histograms. All analyses wereperformed with plants grown in three independent batches (biologicalreplicates). For each independent set of experiment, the data was eithersimulated from six Petri plates (for a–d) or 50 plants (fore–g).

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Compared to WT, detached leaves of 22-day-old plants of transgenic lines L1 andL8 suffered less cell death after 15 h incubation in salt solution which wasevident by weaker staining using Evans blue (Supplementary Fig.S5a online). Decrease in the rate of water loss in detached leaves oftransgenic plants was also clearly revealed by monitoring the time-dependentloss in fresh weight (Supplementary Fig. S5b online). Whenstressed with 200 mM NaCl or by water-withholding for two weeks, the 24-day-oldL1 and L8 transgenic lines displayed better survival and appeared healthier thanWT plants (Figure 4e). Not only better relative watercontent (RWC) but also reduced reactive oxygen species (ROS) accumulation, asrevealed by H2DCFDA staining, was observed in the leaves of 12-daystressed transgenic plants (Supplementary Fig. S5c-eonline). Within 1-week of irrigation with RO water, while nearly 80% L8and 60% L1 transgenics recovered from water-deficit stress, this recovery wasconfined only to 25–30% WT plants (Figure4e,f). None of the tested plant lines showed recovery from salt stressduring 1-week of irrigation with RO water. Nonetheless, the transgenicsdisplayed a slower rate of death since significantly more number of transgenicsbearing green and expanded leaves was observed towards the end of this recoveryphase than the WT plants (Figure 4e,g). Plants grown onlyunder control condition do not display any distinct difference between them(Figure 4e).

Overexpression of OsTCP19 affects ABA, auxin and JA signaling inArabidopsis

Since OsTCP19 overexpression transgenics were compromised in LRdevelopment, it was decided to explore the underlying changes in gene expressionto gain knowledge about the role of OsTCP19 in the signalingnetwork. Moreover, as TCP TFs influence the expression of genes ofvarious hormonal pathways, important regulators of LR development belonging tosuch pathways were picked for expression analysis in the transgenics. Inhibitionof various auxin responsive factors (ARFs) by many AUX/IAAs and modulation ofPIN transporters are known to cause reduction in LR formation14.Elevation of endogenous cytokinin level and inhibition of jasmonate signalingalso attenuates LR formation15,16. Consistent with thesereports, genes for four AUX/IAAs (IAA3, IAA12, IAA14,IAA28), two PIN transporters (PIN1 and PIN2), threeisopentenyltransferases (IPT1, IPT2 and IPT5; involved incytokinin biosynthesis) and two lipoxygenases (LOX1 and LOX2;involved in JA biosynthesis) were selected for expression analysis. ABA alsomediates LR inhibition through ABI417. Although ABI3 (a B3 domaincontaining TF) fine tunes the auxin-mediated LR formation, it works in closeassociation with ABI4 (an AP2 TF) along with ABI5 in many ABA-dependentsignaling pathways18,19. Hence, these three genes were alsoconsidered. As ethylene also inhibits lateral root formation20,five well characterized ERFs (RAP2.2, RAP2.3, RAP2.12,HRE1 and TINY2) were also added to the list of genes fortranscript analysis.

To ensure robustness of the data, transcript level of these genes were monitoredin three different transgenic lines (L1, L6 and L8) and results were consideredsignificant only if all these lines exhibited similar trend compared to WT.ABI3, ABI4 and IAA3 transcripts were enriched by 2 ormore folds in the transgenic plants compared to WT (Figure5). About 3-fold downregulation of LOX2 was also observed inthese transgenic lines. Coexpression analysis (using ‘Abioticstress’ option; correlation coefficient cut off 0.5) in RiceOligonucleotide array database (ROAD) also shows that expression ofOsTCP19 positively correlates with nine IAAs, thirteenAP2 (like ABI4) and one B3 domain protein (like ABI3). Inaddition, a negative correlation between OsTCP19 and a LOX gene(similar to ArabidopsisLOX2) was observed (Supplementary Table S2 online).This further suggests that OsTCP19 negatively influences auxin and JAsignaling and affects expression of similar class of genes inArabidopsis and rice. Presence of Class-I TCP binding sites (site-IIelements) in the promoter of many of these genes points to a possibility ofdirect regulation by OsTCP19 (Supplementary Fig. S6aonline).

Figure 5
figure 5

Fold-change in expression of different genes in transgenics (L8, L6 and L1)over WT plants.

The analyzed genes and the respective hormonal pathways are mentioned foreach histogram. The error bars represent the SD. ‘*’indicates data significantly different from WT (t-test, two-tailedp-value ≤ 0.05). The p-value is mentioned over the respectivebars of the histograms. The analysis was performed with 15-day oldseedlings grown on MS medium in three independent batches (biologicalreplicates). For each independent set of experiment, the sampling was donefrom a single Petri plate supporting the growth all transgenic and WTplants.

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OsTCP19 influences lipid droplet synthesis andmetabolism

Under abiotic stress, upregulation of triacylglycerol (TAG) biosynthesis genediacylglycerolacetyl transferase (DGAT1) by ABI4 leads toaccumulation of lipid droplets (LDs) in vegetative tissue ofArabidopsis21. Incidentally, dgat1 Arabidopsismutants are hypersensitive to abiotic stresses during seedling establishmentwhich is in contrast to that observed in case of p35S:OsTCP19Arabidopsis transgenics22. By qRT-PCR analysis, about 1.5times higher expression of DGAT1 in the transgenic (L1 and L8) than WTplants was observed (Figure 6a) and appears to beconsistent with the increase in ABI4 expression. Nile red staining ofleaf protoplasts revealed hyperaccumulation of LDs in transgenic line L8(relative to WT; Figure 6b). Further analysis revealedincreased expression of two other stress-responsive TAG biosynthesis genes21, DGAT2 and phospholipid:diacylglycerol acyltransferase1 (PDAT1), in transgenic lines by ca.1.5-fold and 2-fold, respectively (Figure 6a,c). ALD-associated Arabidopsis protein Caleosin 3 (CLO3 or RD20; aperoxygenase) is known to play an important role in abiotic stresssignaling23. Analysis in ROAD suggests positive correlationbetween expression of OsTCP19 and two caleosin genes, one of them(LOC_Os03g12230) being highly similar to ArabidopsisCLO3 (Supplementary Table S2 online). Higherexpression of this gene was also observed in p35S:OsTCP19 transgenicscompared to WT Arabidopsis plants (Figure 6c).

Figure 6
figure 6

OsTCP19 influences LD accumulation.

(a,c) Fold-change in expression of AtDGAT1,AtDGAT2, PDAT1 and CLO3 in p35S:OsTCP19 (L1 and L8) over WTArabidopsis plants. (b) Fluorescence microscopy images of Nilered stained LDs in leaf protoplasts of transgenic (L8-D) and wildtype (WT-D) plants. The image of the same protoplasts under bright field isalso shown (L8-B and WT-B). (d) Expression analysis of uidA gene intobacco leaves transiently transformed with constructs as indicated. Errorbar in the histograms represents SD and ‘*’ indicatesdata significantly different from the respective controls (t-test,two-tailed p-value ≤ 0.05). The p-value is mentioned over therespective bars of the histograms. All analyses were performed with 15-dayold seedlings grown on MS medium in three independent batches (biologicalreplicates). For each independent set of experiment, the sampling was donefrom a single Petri plate supporting the growth all transgenic and WTplants.

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The OsDGAT gene (LOC_Os02g48350) was observed to coexpress withOsTCP19 in ROAD (Supplementary Table S2 online).Its promoter also contains three distinct Class-I TCP TF binding sites (Supplementary Fig. S6b online). Using promoter:GUSconstruct bearing 1097 bp DNA region upstream of start codon of this gene(pOsDGAT:uidA) and effector constructs prepared using ORF encodingOsTCP19s activation of OsDGAT expression by OsTCP19 wasdemonstrated by agroinfiltration of tobacco leaves. Construct bearing a ricegene encoding a member of secretory phosphatases (p35S:OsPHOS;LOC_Os01g57240) and unrelated to nuclear activities was used ascontrol effector. Nearly 2-fold upregulation of uidA was inferred byqRT-PCR analysis for leaf zones co-expressing OsTCP19s compared to thoseco-expressing the control effector (Figure 6d). In thisanalysis, the plant selection marker of these vectors, hptII, was used asreference gene. OsTCP19i and mOsTCP19i also caused activation of OsDGATto essentially similar levels (Figure 6d). All these dataindicate that OsTCP19 plays an important role in stress signaling byinfluencing LD biosynthesis as well as its metabolism.

OsTCP19 interacts with OsABI4 and OsULT1

p35S:OsTCP19Arabidopsis transgenics showed better seedling establishment and survivalunder abiotic stresses and conditional early flowering which contradict theestablished activities of the upregulated genes ABI3 and ABI421,24,25. This led to hypothesize a model involvingcondition-dependent regulation of ABI3 and ABI4 beyondtranscriptional level in the transgenic plants and might involve physicalinteraction of these proteins with OsTCP19. A bimolecular fluorescencecomplementation (BiFC) assay was carried out to test this hypothesis in riceusing constructs bearing N-terminus of YFP fused to N-terminus ofOsABI4 (LOC_Os05g28350; p35S:YFPn-OsABI4) andC-terminus of YFP fused to C-terminus of OsTCP19s(p35S:OsTCP19s-YFPc). On particle bombardment, YFP fluorescence wasdetected exclusively in nucleus of the transformed onion epidermal cells. Thus,it indicated a nucleus-specific interaction between OsTCP19 and OsABI4 (Figure 7a). This also substantiates the hypothesis thatOsTCP19 can modulate the activity of OsABI4 and control pathways in rice asobserved in Arabidopsis.

Figure 7
figure 7

BiFC analysis showing nucleus specific interaction of OsTCP19 with OsABI4 andOsULT1.

(a) Fluorescence microscopy images of onion epidermal cells cotransfectedwith p35S:YFPn-OsABI4 and p35S:OsTCP19s-YFPc constructs (scalebar = 100 µm). (b) Similar analysis for cellscotransfected with p35S:YFPn-OsULT1 and p35S:YFPc-OsTCP19s(scale bar = 50 µm).

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A subset of NB localizing proteins in metazoans is known to containSAND-domain26. OsTCP19 was envisaged to interact with fewsuch proteins based on the presence SAND-domain containing proteins in plantsand localization of OsTCP19 to NBs. Two functionally redundant SAND-domaincontaining transcriptional regulators, ULT1 and ULT2, regulate set of genesincluding those belonging to KNOX1 group which, interestingly, are also thetargets of many Class-I TCP proteins5,27. This further suggestsa possibility of interaction between OsTCP19 and ULT-like genes from rice. BiFCanalysis in onion epidermal cells using constructs bearing N-terminus YFPfused to N-terminus of OsULT1 (LOC_Os01g57240;p35S:YFPn-OsULT1) and C-terminus YFP fused to N-terminus ofOsTCP19s (p35S:YFPc-OsTCP19s) revealed strong YFPfluorescence in the nucleus (Figure 7b). Thus, OsTCP19 caninteract with ULT-like proteins which are known coregulators of transcription.Similar analyses also indicate interaction of OsABI4 and OsULT1 with the proteinencoded by the unspliced form of OsTCP19 (Supplementary Fig. S7,S8 online). No true fluorescence was observed when BiFC assays weredone for various negative controls (Supplementary Fig. S9online). Interaction of OsABI4 and OsULT1 with OsTCP19s was alsoobserved in yeast two-hybrid assays, thus substantiating BiFC data (Supplementary Fig. S10 online).

Discussion

Nuclear localization of TCP proteins is expected as they belong to TF family. Besideswhole nucleus, OsTCP19 was also localized in NBs. Apart from general eukaryotic NBs(like speckles, histone locus body, stress granules etc.)28, thosecontaining cyclophillin, HYL1, phytochrome and AKIP1 are known to exist inplants29. However, the nature of NBs where OsTCP19s and OsTCP19ilocalize remains to be determined.

ULT1 and ULT2 are trithorax group (trxG) factors30 and function byrecruiting trxG proteins like ATX1, which is a histone H3 lysine 4tri-methyltransferase and regulator of dehydration response inArabidopsis31. TrxG proteins antagonise the activity ofpolycomb group (PcG) gene repression complexes which include MSI1, a negativeregulator of stress signaling and Class-I TCP regulated pathways12.Hence, the interaction between OsTCP19 and OsULT1 might suggest the existence of aregulatory module comprised of few Class-I TCP TFs, ULT1- and ATX1-like proteinsthat can configure the abiotic stress signal network.

The decrease in LR number in p35S:OsTCP19Arabidopsis transgenic plants were attributed to upregulation of ABI4and IAA3 and downregulation of LOX2. These expression changes couldeven explain slow initial root growth and increased formation of trichomes and RHsin the transgenics32,33,34. Reduction in LR number is considered asan adaptive response towards drought stress tolerance35. RHs bearmembrane integrated H+-ATPases which mediate root-to-shoot signalingand maintain osmoregulation and water content under drought stress36.Trichomes aid in reducing transpiration and behave as a sink for glutathione whichcontributes in combating oxidative stress and water loss under harsh environmentalconditions37. Thus, the phenotypes displayed byp35S:OsTCP19 transgenics also contribute to abiotic stress tolerance.Earlier, it has been found that TCP14/15 regulate trichome formationand disturbances in TCP20 activity severely affect roots elongation inArabidopsis38,39.

Earlier studies reveal better drought tolerance for mutants of jasmonate signaling inplants40. Jasmonate signaling promotes ROS production41 which though aids in generating stress responses but is deleteriousto plants at higher concentration42. Hence, by regulating LOX2expression, OsTCP19 might assist in keeping a partial check over ROSconcentration. This in turn provides an optimum chance for survival underenvironmental stresses. Upregulation of IAA3 in the transgenic plants mighthave negated the effect of increased expression of ABI3 which usually has arole in auxin-mediated LR formation14,19. Interestingly,OsTCP19 upregulates under cold stress (as per microarray data) andABI3 also provides freezing tolerance in Arabidopsis43. Few Class-I TCPs are known to regulate the expression ofIAA3 (e.g. AtTCP15) positively and LOX2 negatively (e.g. AtTCP20)by binding to their promoter sequences in Arabidopsis3,44.Presence of Class-I TCP binding sites in upstream region of similar genes which arepositively or negatively coexpressed with OsTCP19 imply the conservation ofsimilar regulatory pathways in rice.

Knowledge about the role of LDs in vegetative tissues is limited. Recent studies haveshown hyperaccumulation of LDs in vegetative tissues of Arabidopsis inresponse to various abiotic stresses and hormones treatments21.Similar accumulation of TAG has also been reported for monocots under abioticstress45. Despite a positive correlation between the increase inLDs and inhibition of seedling establishment, LDs are still considered to have arole in seed germination and seedling establishment under various stresses. This isevident from Arabidopsisdgat1 mutant plants which are compromised in these traits22.Elevated expression of DGAT1 during both cell division (in shoot and root apicalmeristem) and senescence (of leaves) suggests a contrasting role for LD in theseprocesses46,47. Thus, it is being hypothesized that by servingas a rich source of energy and nutrients to sustain cell division, increased levelsof TAG in p35S:OsTCP19Arabidopsis plants might support seedling establishment under conditionsseverely affecting normal metabolic pathways, like abiotic stresses.

OsTCP19 caused hyperaccumulation of LDs by increasing the expression ofmultiple abiotic stress-upregulated TAG biosynthesis genes like DGAT1,DGAT2 and PDAT1 and this is partly dependent on ABI4 as itdirectly activates DGAT1 in association with ABI521. In thepresent study, two-fold increase in the activity of OsDGAT promoter due todirect activation by OsTCP19 was also observed. A higher activation might depend onrelative abundance of other endogenous factors which probably were limiting duringthe transient assays in tobacco leaves. An earlier study also reported the failureof many Class-I TCP proteins from rice to cause any transactivation in culturedtobacco cells or mesophyll protoplasts by co-transfection assays1. Inanother case, AtTCP20-EAR (AtTCP20 fused to EAR repression domain) failed to repressPCNA in Arabidopsis transgenics, although binding of AtTCP20 to PCNA promoterhas been shown by in vitro and in vivo assays38.

Recent studies revealed that LDs per se could be of little importance and, infact, the associated proteins and the process of lipid metabolism decide theirfunction48. Expression of CLO3 was increased inp35S:OsTCP19Arabidopsis plants. This Ca2+-binding LD-associated caleosinprotein upregulates in vegetative tissue under abiotic stresses and plays a role indrought tolerance by reducing transpiration23. The expression of asimilar gene from rice also correlates (as per ROAD) with that of OsTCP19.Similar CLO3-regulated pathways in rice may get affected byOsTCP19.

In the present study, early flowering of the transgenics in vertically oriented Petriplates revealed the condition-dependent activity of OsTCP19. Earlier studiesreported condition-dependent contrasting functions for TCP14 and TCP15 inArabidopsis49. This study postulates a conditionalregulation of ABI4 activity by OsTCP19 and was substantiated by the interactionbetween these proteins. This hypothesis seems to fit in a model that will allowincreased tolerance to dehydration and salinity and phenotype like early floweringto occur despite higher expression of ABI4 and ABI3. Interestingly,mutants of TCP14 show hypersensitivity to ABA during germination andexpression of a dominant repressor form of TCP15 affects seedling establishment inArabidopsis44,50. Although, genes of similar classes werefound to coexpress with OsTCP19 in rice, none were direct homologue ofABI3 or ABI4. However, a conditional upregulation of these genesby OsTCP19 remains possible due to disturbances in other hormone (like auxin)pathways17.

Based on many reports, ABI4 was chosen as a target of direct regulation by OsTCP19.First, ABI4 is known to regulate ABI3 expression in many signalingpathways18. Second, ABI4 is a dynamic transcription factor whichcan switch its activity from activator to repressor in a conditional manner51. Third, it can serve as a link between cytokinin and ABAsignaling17. Coincidently, Class-I TCP TFs also play a role incytokinin-dependent pathways and, in the present study too, OsTCP19transcript levels were upregulated by exogenous ABA application. Moreover, ABI4 frommonocots are functionally similar to that from Arabidopsis52,53,54. These reports and the present results together suggest arole for OsTCP19 in fine-tuning ABA signaling by regulating the expression andactivity of key proteins like ABI4.

In short, the present study assigns a role for OsTCP19 in calibrating andcrosslinking the developmental and stress-response pathways by interfering withauxin and JA acid pathways and manipulation of the ABA-signaling network. It couldpartly mediate this by recruiting trxG factor for activation of the target genes andby interacting with key regulators like ABI4. Its role in stress tolerance ismediated by the accumulation of LDs and associated proteins besides reduction incell death, water loss and ROS production. The phenotypes displayed by thetransgenics, stress tolerance assays and expression analysis together indicate anextensive role of OsTCP19 in water-deficit stress signaling. However, itmight be involved in shaping the early signaling pathways in response to variousabiotic stresses. In conclusion, this study unravels the role of a rice gene,OsTCP19 and extends the role of Class-I TCP TFs in abiotic stressresponse and ABA signaling.

Methods

Plant material and growth conditions

Sterilized (70% ethanol, 1 min; 3.5% NaOCl, 40 min) seeds of PB1, Pokkali and N22indica rice were grown in liquid Yoshida medium55 under 12 hlight condition. 10-day-old seedlings were subjected to different treatments (asmentioned in results) in hydroponic culture system in the presence of Yoshidamedium. Sterilized (70% ethanol, 30 sec; 0.6% NaOCl and 0.001% Tween-20, 10 min)Arabidopsis thaliana Col-0 seeds were germinated and grown onMurashige and Skoog (MS; Duchefa) medium containing 1% sucrose and 0.8% purifiedagar under continuous illumination at 21oC followingstratification. For salt and water-deficit treatments, NaCl (100 mM or 125 mM)or mannitol (200 mM or 350 mM) were provided as additives to Arabidopsisgrowing medium. When required, 6-8 leaf stage, healthy plants were transferredto pots containing Soilrite (a combination of Vermiculture, Perlite and Spagnummoss; 1:1:1 ratio) and irrigated with RO water. During assessment of stresstolerance in pots, either irrigation was stopped or was done with 200 mM NaClsolution every four days. For recovery from these stresses, plant were irrigatedwith normal RO water and observed for one week. Samples were either processedfor further analysis or stored under frozen condition till use. All experimentswere done with at least three biological replicates.

Insilico analysis

Details about the databases and software used for doing various in silicoanalyses are mentioned in ‘Supplementary Table S3online’.

Gene expression analysis

RNA was extracted from different samples using Trizol (Sigma). cDNA wassynthesized using ‘Applied biosystems High capacity cDNA synthesiskit’ (Life technologies). Expression analysis of genes or theirsplice forms was achieved either by qRT-PCR following manufacturer’sprotocol (‘Applied biosystems 7500 fast real time machine’and ‘Applied biosystems fast SYBR green mix’, Lifetechnologies) or semi-qRT-PCR under standard PCR conditions followed by gelelectrophoresis. Sequences of all primers used for the analyses are mentioned in‘Supplementary Table S4 online’.For qRT-PCR analysis either rice Ubiquitin5 (UBQ5; for ricesamples) or ArabidopsisActin2 (ACT2; for Arabidopsis samples) were used asreference genes. The Ct values obtained for various samples were firstnormalized with that for the respective reference gene (ΔCt). Toobtain fold change in expression (as per the case), the ΔCt values ofvarious genes for different samples were again normalized to that for unstressedtissue (0 h samples) or the wild type plants (ΔΔCt). Thefinal values for fold change in expression were derived by calculating2–ΔΔCt. To compare theabundance of OsTCP19 transcripts across various rice varieties,2–ΔCt were calculated which representthe relative expression level of the gene with respect to the referencegene.

Preparation of different constructs

OsTCP19 was cloned in TA-cloning vector (pGEMT-easy, Promega) followingits amplification by PCR using PB1 rice genomic DNA and primers flanking the ORF(Supplementary Table S5 online, S.No. 1-2). Byincorporating NcoI and SpeI sites as overhangs in the concernedprimers (Supplementary Table S5 online, S.No. 3-4), the ORFwith its stop codon was PCR amplified from OsTCP19_PGEMT plasmid andmobilized into pCAMBIA1302 vector (www.cambia.org) between CaMV 35S promoter and mGFP usingthe facility of these restriction sites to create p35S:OsTCP19 construct.Applying ‘Phusion site directed mutagenesis kit’ (Thermoscientific) and primers as described in ‘SupplementaryTable S5 online (S.No. 7-8)’, a mutated version ofOsTCP19 (mOsTCP19i) was created by replacing the first GC andlast AG dinucleotides of the intron to GG and AA, respectively. OsABI4(LOC_Os05g28350; homologous to ABI4 from Zea mays andArabidopsis), OsULT1 (LOC_Os01g57240; homologous toArabidopsisULT1 and ULT2) and spliced OsTCP19 form were amplified fromPB1 cDNA using primer as mentioned in Supplementary Table S5online (S.No. 1-2, 9-10, 13-14). As per manufacturer’sinstructions, primers were designed (Supplementary Table S5online, S.No. 5-6, 11-12, 15-16) and following PCR the ORFs ofOsULT1, OsABI4 and OsTCP19 (spliced, unspliced andmutated forms) were first cloned in pENTR-D-Topo entry vector (Invitrogen, LifeTechnologies) and then mobilized into various destination vectors (Supplementary Table S6 online, S.No. 1-13) by recombination using‘Invitrogen LR clonase II mix’ (Life technologies).Similarly, 1097 bp genomic fragment upstream of OsDGAT(LOC_Os02g48350) was also amplified and cloned in Gateway vector forthe preparation of pOsDGAT:uidA construct (SupplementaryTable S5 online, S.No 17-20; Supplementary Table S6online, S.No. 17).

Yeast Two-Hybrid analysis

OsABI4 and OsULT1 were cloned in PGBKT7-DEST (bait vector) andOsTCP19s in PGADT7-DEST (prey vector) by gateway cloning to createOsABI4-BD, OsULT1-BD and OsTCP19-AD, respectively (Supplementary Table S6 online, S.No 14-16). Pairwiseco-transformation of these constructs into Saccharomyces cerevisiae AH109cells was conducted using EZ-transformation kit (MP biomedical) and were thenselected and grown on appropriate medium to check their interactions as per BDMatchmaker protocol (Clontech).

Subcellular localization and BiFC analysis

For subcellular localization, construct p35S:YFP-OsTCP19s orp35S:YFP-mOsTCP19i was bombarded on onion epidermal cells asdescribed56 and imaged by fluorescence microscopy (Eclipse80i, Nikon). For BiFC, any of the p35S:OsTCP19s-YFPc,p35S:OsTCP19i-YFPc, p35S-mOsYFP19i-YFPc,p35S:YFPc-OsTCP19s, p35S:YFPc-OsTCP19i orp35S:YFPc-mOsTCP19i construct was co-expressed in onion epidermalcells by particle bombardment and visualized by fluorescence or confocalmicroscopy (AOBS TCS-SP2, Leica). Experiments were validated from at least threeseparate sets of bombardment, each done with four different onion peels.

Agrobacterium-mediated Arabidopsis transformation

Arabidopsis plants transformation was done using Agrobacteriumtumefaciens strain GV3101 bearing p35S:OsTCP19 construct byfloral dip method as described by Giri et al56.Transgenic selection was done on hygromycin (15 mg/ml) containing medium. Duringselection of T1 plants, a plant line negative for hygromycinresistance was selected and maintained as a negative control plant (NT).

Visualization of oil bodies

Protoplasts isolated from leaves of 15-day-old plants57 werestained for 10 min with 0.1% Nile red (stock solution in acetone) in MMG bufferfollowed by two brief washings. The protoplasts were then visualized byfluorescence microscopy (Nikon 80i) using FITC filter.

Analysis of abiotic stress-related parameters

To examine post-germination biomass accumulation of seedlings, the ratio for thetotal weight of seedlings developed from hundred seeds under stress and controlcondition was calculated. Measurements of relative water content (RWC) and theanalysis of cell death by Evan’s blue staining in the leaves ofArabidopsis plants were done according to Ji et al.58. Thepercentage water loss measurements from excised leaves were done according toSaez et al.59. ROS accumulation, was studied by stainingleaves with 100 µM2′,7′-dichlorodihydrofluorescein diacetate(H2DCFDA) in 10 mM Tris-HCl, pH 7.2 and imaging the fluorescingstomata using a fluorescence microscope. Fluorescence signal from more thanfifty stomata per leaf were quantified in ImageJ software for preparing agraphic representation of the data. Data presented are average of threereplicates in each case.

Agroinfiltration of tobacco leaves

This was achieved by injecting a mix of equal proportion of Agrobacteriumtumefaciens strain LBA4404 bearing pDGAT:uidA construct and thosebearing either p35S:OsPHOS, p35S:OsTCP19s, p35S:OsTCP19i orp35S:OsmTCP19i into tobacco leaves as described by Pandey etal60. Each analysis was performed in leaves fromthree different plants.