Plant Physiology Preview. Published on February 21, 2017, as DOI: /pp

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1 Plnt Physiology Preview. Pulished on Ferury 21, 217, s DOI:1.114/pp Running title: Spliing regultor STA1 in het stress dpttion Corresponding Author: Sng-Dong Yoo Deprtment of Life Sienes Division of Life Sienes KOREA University 145 Anmro, Seonguk-gu Seoul, Kore, 2841 Tel: Fx: E-mil: sngdong@kore..kr Artile type: Reserh Artile Reserh re: Signling nd Response 1 Copyright 217 y the Amerin Soiety of Plnt Biologists

2 STABILIZED1 Modultes pre-mrna Spliing for Thermotolerne Geun-Don Kim 1, Young-Hee Cho 1, Byeong-H Lee 2, Sng-Dong Yoo 1* 1 Division of Life Siene, College of Life Siene nd Biotehnology, KOREA University, Seoul, Kore 2 Deprtment of Life Siene, Sogng University, Seoul, Kore One-Sentene Summry: Het induile STA1 tivity ws found to e involved in the pre-mrna spliing of het stress response genes nd ontriutes to the estlishment of het stress tolerne in Aridopsis. Author ontriution S.-D. Yoo initited nd designed experiments; G.-D. Kim nd Y.-H. Cho performed nd nlyzed most of experiments; S.-D. Yoo nd Y.-H. Cho wrote the mnusript. Footnotes 1 This study ws supported y the Koren Ntionl Reserh Foundtion (NRF-213R1A1A2 5714; 216R1A2B49167), funding y the Koren Rurl Development Administrtion Woo Jng Chun s Speil Projet (PJ ) nd the grnt from the KOREA University (KU- FRG-NARS327Q) to S.-D. Yoo, Koren NRF (NRF-216R1A2B111338) nd KOREA University grnt to Y.-H. Cho, nd Koren NRF (214R1A1A258769) to B.-H. Lee. These uthors ontriuted eqully to the work * Address orrespondene to sngdong@koe..kr 2

3 Astrt High temperture stress often leds to differentil RNA spliing, thus umulting different types nd/or mounts of mture mrnas in eukryoti ells. However, regultory mehnisms underlying plnt pre-mrna spliing in the environmentl stress onditions remin elusive. Herein, we desrie tht U5-snRNP-interting protein homolog STABILIZED1 (STA1) hs pre-mrna spliing tivity for het-induile trnsripts inluding HEAT STRESS TRANSCRIPTION FACTORS nd vrious HEAT SHOCK PROTEINS for the estlishment of het stress tolerne in Aridopsis thlin. Our ell-sed spliing reporter ssy demonstrted STA1 ts on pre-mrna spliing for speifi susets of stress-relted genes. Cellulr reonstitution of het-induile trnsription sdes supported the view tht STA1-dependent pre-mrna spliing plys role in DREB2A-dependent HSFA3 expression for het responsive gene expression. Further geneti nlysis with loss-of-funtion mutnt st1-1, STA1-expressing trnsgeni plnts in Col kground nd STA1-expressing trnsgeni plnts in st1-1 kground verified tht STA1 is essentil in expression of neessry genes inluding HSFA3 for two-step het stress tolerne in plnts. However, onstitutive overexpression of DNA version of HSFA3 in st1-1 kground is unle to exeute plnt het stress tolerne in st1-1. Consistently our glol trget nlysis of STA1 showed tht its spliing tivity modultes rther rod rnge of gene expression in response to het tretment. The findings of this study revel tht hetinduile STA1 tivity for pre-mrna spliing serves s moleulr regultory mehnism underlying the plnt stress tolerne to high temperture stress. 3

4 INTRODUCTION The spliing of preursor messenger RNA (pre-mrna) is neessry step for intron-ontining gene expression in eukryoti ells in order to produe mture trnsripts for protein trnsltion (Whl et l., 29). This proess is highly ordered nd tightly ontrolled y multi-suunit splieosome tivity to mix nd mth introns nd exons of pre-mrnas. The high-moleulrweight splieosome omplex omprises smll nuler rionuleoprotein prtiles (snrnps) lled U1, U2, U4/U6, nd U5 snrnps. For spliing of pre-mrna introns, U1 snrnp reognizes the 5 -spliing site (SS), nd U2 snrnp inds to the denosine t the rnh point of introns with the ssistne of U2 uxiliry ftors (U2AFs). U4/U6 nd U5 trimeri snrnps ssoite with eh other nd undergo step-wise 3 -SS levge proess. Eventully, U5 snrnp dissoites from the omplex long with lrit form of the intron. In this proess, U5 snrnp urtely nd dynmilly swps interting prtners with other snrnp suunits (Whl et l., 29). The funtions of rionuleoprotein prtiles re evolutionrily onserved in most eukryotes (Knowler nd Wilks, 198). Reent dvnes in funtionl genomi nlysis pltforms enled the inferene tht lterntive spliing of pre-mrnas is entrl regultory module in extending gene reservoir with limited numer of struturl genes enoded in genomes for protein informtion (Reddy et l., 213). Consequently, fewer genomi resoures ddress physiologil nd developmentl omplexities through trnsript vrints produed in ellulr nd environmentl ontexts (Reddy et l., 213; Stiger nd Brown, 213). The sessile lifestyle of plnts filittes the evolution of diverse dpttion proesses t the multidimensionl lyers from ells to orgnisms under vrious environmentl stress onditions (Godfry nd Grnett, 214; MClung, 214). For exmple, in the ell utonomous mnner, low temperture tivtes the ICE-CBF-COR pthwy nd estlishes old stress tolerne in Aridopsis (Zhu et l., 27). Suh ellulr dpttion proesses lso require posttrnsriptionl regultory steps in gene expression. For exmple, COR15A expression ws originlly reported to e trnsriptionlly upregulted nd further modulted posttrnsriptionlly upon old tretment (Artus et l., 1996; Lee et l., 26). In reessive loss-offuntion mutnt st1-1, pre-mrna of COR15A umulted in response to the old stress nd thus STA1 is implited in ontrolling pre-mrna spliing of COR15A (Lee et l., 26). 4

5 STA1 is stress-induile U5 snrnp-interting prtner, of whih ellulr funtions involve in the estlishment of old stress nd ABA tolerne (Lee et l., 26). Reently STA1 is lso doumented for het stress responses without mehnisti detils (Yu et l., 216). High temperture (het) is detrimentl environmentl stress ondition ffeting plnt iomss yields nd its ourrene eomes more prevlent in the ropping fields under tody s limte hnge (Godfry nd Grnett, 214; MClung, 214). Evolutionrily onserved het stress trnsription ftors (HSFs) nd het shok proteins (HSPs) ply key roles in estlishing plnt sl nd/or quired het stress tolerne (Kotk et l., 27; Shrf et l., 212). For exmple, the gene regultory modules of HSF nd HSP expression hve een well hrterized in Aridopsis. The key stress-relted trnsription ftors DEHYDRATION-RESPONSIVE ELEMENT- BINDING PROTEIN (DREB) 2A nd DREB2C, indue expression of het-induile trnsription ftor HEAT SHOCK TRANSCRIPTION FACTOR A3 (HSFA3), nd its trnsription tivity indue downstrem HSPs expression (Skum et l., 26; Shrmm et l., 28; Chen et l., 21). A similr trnsription sde is lso found in the mize, nd thus this trnsriptionl regultory module is most likely onserved through the evolution of diverse plnt speies (Qin et l., 27). Nevertheless, post-trnsriptionl regultory steps of het-induile gene expression remin lrgely unknown in plnts. In this study, we utilized novel spliing reporter in Aridopsis lef mesophyll protoplsts to exmine STA1 tivity on pre-mrna spliing of stress-indued genes. STA1 ws involved in the pre-mrna spliing of het-induile HSFA3 s well s old-induile COR15A nd IDD14. Clerly, STA1 spliing tivity ws indispensle for mture mrna expression of HSFA3 nd its downstrem HSPs in reonstituted DREB2A-dependent gene regultory module. Further geneti nlysis verified tht STA1 involved in pre-mrna spliing of essentil genes, inluding HSFA3 nd its trget gene HSA32, neessry for in the estlishment of plnt het stress tolerne. However, single het stress trnsription ftor HSFA3 ws unle to repitulte the stress tolerne in the sene of STA1 tivity. Our glol trget serh of STA1 tivity using differentil disply of RNA-followed y sequening (DDR-seq) reveled tht STA1 plyed entrl roles in roder rnge of het induile gene expression. Our findings unrveled tht het-induile STA1 tivity seures pproprite gene expression under het stress onditions, of whih gene produts ontriute to plnt tolerne. 5

6 RESULTS AND DISCUSSION STA1 Funtion in Pre-mRNA Spliing To evlute the iohemil tivity of STA1 in pre-mrna spliing t ellulr system, spliing reporter onstrut ws generted using the trnsltionl fusion of the GUS reporter gene to the 3 - end of genomi version of the struture gene under the regultion of onstitutive 35S promoter (Fig. 1A). This GUS reporter tivity inreses only when the intron of pre-mrna is splied out orretly. Otherwise, premture termintion odon (PTC) of the intron retined in the pre-mrna would interrupt the omplete trnsltion of the GUS reporter protein. Therefore, in priniple, n inrese or derese in the ellulr GUS tivity my primrily reflet the mount of mture mrnas serving s protein trnsltion templtes. The design priniple of the spliing reporter ws vlidted y the spliing tivity of STA1 with gcor15a-gus reporter onstrut using the well-estlished Aridopsis lef mesophyll protoplsts. The GUS-sed spliing reporter nd UBQ1 promoter-driven renill luiferse (rluc) ontrol reporter onstruts were o-trnsfeted to either wild type (Col) or st1-1 protoplsts with or without STA1-HA effetor onstrut nd then inuted for 6 h under light (Yoo et l., 27). The spliing reporter tivity of intron-ontining gcor15a-gus onstrut ws lerly indued with STA1 expression when ompred to the sl tivity otined without the effetor expression (Fig. 1, B nd C). In the ssy the ontrol reporter tivity of intron-free rluc onstrut ws not ltered in the presene or sene of STA1 expression, inditing tht STA1 did not modulte trnsription tivity in this system. To further verify this notion, ontrol experiment ws independently rried out with 35S-driven intron-free fluc reporter onstrut in st1-1 LMPs. Agin, STA1 did not ffet the LUC reporter tivity t ll (Supplementl Fig. S1). Then, to re-exmine whether the differene in the GUS reporter tivity origintes from the spliing effiieny depending on STA1 tivity, oth the intronretined nd intron-free forms of COR15A-GUS were deteted using semi-quntittive reverse trnsriptse-dependent PCR (RT-PCR) using the RNA extrted from the trnsfeted protoplsts of Col nd st1-1 (Supplementl Fig. S2). The mture mrna of gcor15a-gus ws sustntilly enrihed in Col, wheres its pre-mrna ws rther enrihed in st1-1, inditing tht ler shift of pre-mrna to mture mrna ws mde for gcor15a-gus y STA1. Tken together, our ell-sed ssy demonstrted pre-mrna spliing tivity of STA1. 6

7 Then, to investigte whether STA1 tivity hs ny preferene in pre-mrna spliing trgets, two more spliing reporters were onstruted with genomi versions of INDETERMINATE DOMAIN 14 (gidd14) nd MITOGEN-ACTIVATED PROTEIN KINASE 1 (gmpk1). IDD14 is nother old-induile gene tht produes splied vrint nd ontrols its own gene produt tivity in response to old (Seo et l., 211). The stress-relted MPK1 enodes gene for signling potent kinse, the funtion of whih is lrgely unknown (Mo et l., 211). The reporter tivity of gidd14-gus, ut not gmpk1-gus, ws inresed in st1-1 protoplsts y STA1 expression (Fig. 1, D nd E). gidd14 ontins single intron s gcor15 ut gmpk1 hs multiple introns. However, the numer of introns is seemingly irrelevnt to the trget preferene of STA1 for pre-mrna spliing sed on the glol nlysis of STA1- dependent het induile gene expression in this study (see elow). To hrterize STA1 funtions in ioti stress responsive gene regultion other thn old stress nd ABA responses reported in the previous study (Lee et l., 26), STA1 expression ptterns were serhed through Aridopsis efp dtse, whih integrtes high-throughput trnsriptome nlyses (Winter et l., 27). High-temperture stress indues STA1 expression in ddition to old stress (Supplementl Fig. S3). To verify the gene expression of STA1 in response to het, semi-quntittive RT-PCR ws performed fter mild het tretment (37 C). STA1 expression inresed within 1 h nd mintined up to 3 h, nd then deresed in 12 h fter het tretment (Fig. 2A). Sine STA1 expression is under the regultion of het stress, its gene produt tivity most likely plys role in the stress-responsive gene expression. To investigte STA1 funtions in het stress relted gene expression, the gene expression of severl het stress ftors (HSF) inluding HSFA3, HSFB1, nd HSFB2 were exmined upon het tretment. These HSFs re trnsription ftors tht hve importnt funtions in plnt het stress dpttion (Shrmm et l., 28; Shrf et l., 212). To preisely hrterize the regultory funtions of STA1 on spliing of het induile trnsripts, het induile expression of the HSFs ws speifilly mesured for the totl mrna (primer omintion of f1 nd r1), pre-mrna (primer omintion of f1 nd r2) nd mture mrna (primer omintion of f1 nd r3) in Col nd st1-1 (Fig. 2B). Totl mrna expression of HSFA3 ws indued nd mintined in oth Col nd st1-1 seedlings t 37 C, inditing tht totl trnsript levels of HSFA3 inresed with nd without STA1 tivity y het stress (Fig. 2, C nd D). Notly, premrna expression ws mrginl in Col; however, it ws rther signifintly indued in st1-1 7

8 inditing tht pre-mrna trnsripts of HSFA3 were not splied ppropritely in st1-1 ompred to those in Col. Mture HSFA3 mrna expression ws then indued in Col, ut not in st1-1. The expression ptterns of HSFB1 nd HSFB2 were similr to those of HSFA3 (Supplementl Fig. S4). To exmine STA1 tivity for pre-mrna spliing of het stress responsive genes new spliing reporter ws onstruted with the genomi version of HSFA3 (ghsfa3). This time the reporter ws generted with Nno-luiferse (nluc) tht is smller in size nd thus more sensitive in ellulr responses ompred to firefly-luiferse (Hll et l., 212). To sustntite this spliing ssy, evolutionrily onserved spliing sites of the intron in ghsfa3 (GT-intron- AG) were mutted to e un-splied t splie donor (Do) or eptor (A) site (Fig. 2E) nd sujeted to the ell-sed ssy with nd without STA1 in st1-1 protoplsts. The reporter tivity from wild type ghsfa3-nluc inresed with STA1 expression, ut those from two mutted forms of ghsfa3-nluc did not (Fig. 2F), onfirming tht the indution of spliing reporter tivity resulted from uthenti pre-mrna spliing of the reporter genes y STA1. In the loss-of-funtion llele st1-1, STA1 trnsript is deleted with six nuleotides infrme nd it produes st1-1 protein omitting two mino ids (Lee et l., 26). To investigte whether st1-1 protein hs ny spliing tivity, the reporter ssy using ghsfa3-nluc ws rried out in omintion of STA1 nd st1-1 expression. Protein lot nlysis first showed tht STA1 nd st1-1 umulted to similr level in Col protoplsts (Fig. 2G). The reporter tivity ws indued y STA1, ut not y st1-1, nd its tivity y oth STA1 nd st1-1ws similr to the tivity otined y STA1 lone (Fig. 2G), inditing st1-1 did not rry ny notiele spliing tivity of ghsfa3. However, st1-1 lone redued the reporter tivity to some extent, nd this might indite tht st1-1 hs little dominnt negtive (DN) funtion, lthough suh tivity is ompetitively weker thn STA1. This null spliing tivity of st1-1 is oherent with the ft tht st1-1 is reessive loss-of-funtion mutnt (Lee et l., 26). Reonstitution of Het Induile Trnsription Csdes y DREB2A nd STA1 To sustntite this finding, we reonstituted trnsription regultory iruits of het-response gene expression in Aridopsis protoplsts. Nmely, DREB2A responsile for HSFA3 trnsription under het stress onditions ws trnsiently expressed in Col or st1-1 protoplsts with nd without STA1 (Fig. 3A; Skum et l., 26; Shrmm et l., 28). Prior to the 8

9 mesurement of HSFA3 pre-mrna, DREB2A nd STA1 expressions were verified y RT-qPCR with the RNA extrted from the trnsfeted protoplsts (Supplementl Fig. S5). With forwrd nd reverse primers speifilly designed to reognize the pre-mrna trnsripts of HSFA3 (Fig. 3B), the endogenous expression of pre-mrna of HSFA3 exhiited sl level nd it ws not indued y STA1 (Figs. 3, C nd D). In ontrst the pre-mrna expression ws indued y DREB2A nd its umultion ws rther pronouned in st1-1 protoplsts when ompred to tht in Col protoplsts. Furthermore the pre-mrna of HSFA3 deresed in the presene of STA1, impliting its role in the spliing of HSFA3 pre-mrna. This ws onsistent with our oservtion tht pre-mrna spliing of HSFA3 ws dependent on the STA1 tivity (Figs. 2, C nd D). To exmine whether DREB2A-driven HSFA3 eomes funtionl trnsription tivtor in the presene of STA1 tivity, mture mrna expression of HSP23.6, HEAT STRESS-ASSOCIATED PROTEIN 32 (HSA32), nd HSP7T-2 ws mesured y RT-qPCR s potentil trget genes of HSFA3. Either STA1 or DREB2A lone ould not indue mture mrna of HSP23.6, HSA32 nd HSP7T-2 in st1-1 protoplsts tht re disonneted in the proess from pre-mrna to mture-mrna (Fig. 3E). The o-expression of STA1 nd DREB2A did indue mture mrna umultion of the HSPs. Evidently, pre-mrna of HSFA3 indued y DREB2A ws proessed y STA1, nd then its mture mrna produts ould indue the HSP gene expression ppropritely. These results suggested tht STA1 tivity served n importnt regultory step in mture mrna expression of HSFA3, whose trnsription ws driven y DREB2A. Pre-mRNA Spliing Funtion of STA1 for Plnt Thermotolerne The STA1 spliing tivity ws then exmined for plnt het stress tolerne with wild type (Col), loss-of-funtion st1-1, STA1-expressing trnsgeni plnts in Col kground (STA1/Col) nd STA1-expressing trnsgeni plnts in st1-1 kground (STA1/st1-1). Before het induile gene expression nlysis endogenous nd trnsgene expression of STA1 ws onfirmed y using semi-quntittive RT-PCR (Supplementl Fig. S6). For two-step quired het stress tolerne ssy, Aridopsis seedlings were grown for 5 d t room temperture nd exposed to het t 38 C for 2 h nd then t 45 C for nother 2 h. Seedling viility ws exmined t 7 d fter het tretment. Col seedlings limted nd survived from the step-wise het tretment, wheres 9

10 st1-1 seedlings grew slowly, hd shorter primry roots, displyed ompletely lehed shoots, nd eventully died (Fig. 4A, upper pnel). Both STA1/Col nd STA1/st1-1 seedlings showed their tolerne t level similr to Col in response to the step-wise het tretment, onfirming tht geneti defet of STA1 led to the lk of het stress tolerne in st1-1. These seedling phenotypes were exmined repetedly with multiple independent STA1-expressing trnsgeni Col nd trnsgeni st1-1 lines (Supplementl Fig. S7). In ontrst, fter one-step sl het tolerne ssy onduted with 5-d-old seedlings exposed to 45 C for 2 h, none of these genotypes inluding Col exeuted het stress tolerne (Fig. 4A, lower pnel). Seedling survivl nd lethl phenotypes of Col displyed respetively for one-step sl nd two-step quired het stress tretments were onsistent with previous report (Silv-Correi et l., 214). In summry het induile STA1 hs regultory role in the estlishment of the two-step quired het stress tolerne, ut not tht of the one-step sl het stress, perhps through its spliing funtion of het responsive gene expression. To further understnd STA1 funtion in het responsive gene expression, totl RNA ws extrted from whole seedlings efore nd t 1 d fter the step-wise het tretment, nd gene expression ws mesured y RT-qPCR. To speifilly mesure the expression of wild-type STA1 nd mutnt st1 trnsripts we designed primers sed on the 6-nuleotide-in-frme deletion in st1-1 (Lee et l., 26). Totl STA1 (tsta1) expression omining STA1 nd st1 expression ws omprtively high in STA1/st1-1 seedlings efore het tretment (Fig. 4B) refleting the ft tht trnsgene expression ws under the regultion of 35S onstitutive promoter. In response to het tretment, tsta1 expression ws highly indued in Col, st1-1 nd STA1/st1-1. However, STA1 ws highly indued only in Col y het tretment, ut not in st1-1 nd STA1/st1-1 (Fig. 4C). The results suggested tht the het-indued tsta1 expression resulted from the indution of st1 expression in STA1/st1-1, nd further implited tht STA1 nd/or STA1-dependent spliing produts do not seem to involve in the intron-free STA1 nd/or st1 expression. Then, expression of our model trnsript HSFA3 ws monitored speifilly for totl mrna (primer omintion of f1 nd r1), pre-mrna (primer omintion of f1 nd r2) nd mture mrna (primer omintion of f1 nd r3) under het stress onditions. HSFA3 expression in ny type of mrna ws low in ll genotypes efore het tretment (Fig. 4D). Totl mrna expression of HSFA3 ws highly indued in ll genotypes y het tretment (Fig. 4D, upper 1

11 pnel). Pre-mRNA of HSFA3 ws umulted to high level in st1-1 y het tretment, ut not to the sme level in Col nd STA1/st1-1 (Fig. 4D, middle pnel). On the ontrry mture HSFA3 mrna expression level ws high in Col nd STA1/st1-1 y het tretment, ut suh high expression level ws not rehed in st1-1 (Fig. 4D, lower pnel). These results indited tht HSFA3 trnsription ws driven y het tretment regrdless of STA1 tivity, resulting in the synthesis nd umultion of its pre-mrnas. However, HSFA3 spliing ws rried out ppropritely only in the presene of STA1 tivity. To verify the RT-qPCR dt t tehnil point, STA1 nd HSFA3 expression ws monitored with totl RNA in the sene of reversetrnsription s negtive ontrol, resulting in null mplifition of trget genes (Supplementl Fig. S8). Tken together, mture mrna expression of HSFA3 resulted from STA1-independent trnsription nd STA1-dependent pre-mrna spliing in response to het stress. The higher indution HSFA3 expression in Col nd STA1/st1-1 orrelted well with their seedling viility fter the two-step het tretment (Fig. 4A, upper pnel). All these experiments thus lerly indited tht STA1-dependent spliing plyed n importnt regultory step for HSFA3 expression in the estlishment of Aridopsis het stress tolerne. To exmine whether STA1-dependent spliing of HSFA3 pre-mrna led to the expression of its downstrem genes, HSA32 expression ws monitored using RT-qPCR. Totl mrna expression of HSA32 ws highly indued in Col- nd STA1/st1-1 y het tretment, ut unlike HSFA3, it ws never indued to the sme level in st1-1 (Fig. 4E, upper pnel) suggesting full indution of HSA32 expression requires STA1 tivity. Pre-mRNA of HSA32 umulted in st1-1 to ertin level tht ws higher thn those in Col nd STA1/st1-1 under the stress onditions (Fig. 4E, middle pnel) nd thus pre-mrna spliing of HSA32 gin required STA1 tivity s HSFA3. Consequently, mture HSA32 mrna expression level ws higher in Col nd STA1/st1-1 thn st1-1 under the stress onditions (Fig. 4E, lower pnel). These results indited tht hetindued HSA32 expression is seemingly under the trnsriptionl regultion of HSFA3, spliing of whih is lso dependent on STA1 tivity. In ddition its mture mrna expression is lso under the post-trnsriptionl regultion of STA1 tivity. In se of intron-free HSP18.2 tht is nother trget gene of HSFA3 (Shrmm et l., 28) the gene expression ws highly indued in Col nd STA1/st1-1 in response to het tretment, ut suh indution ws lerly ompromised in st1-1 (Supplementl Fig. S9) gin suggesting tht HSP18.2 indution requires funtionl HSFA3. Tken ll together, these results suggest tht STA1 plys neessry regultory step in 11

12 het response gene expression, nd its gene produts hve importnt roles in estlishing plnt two-step quired het stress tolerne. The higher umultion of HSFA3 pre-mrna in st1-1 (Fig. 4D, middle pnel) ould reflet the possiility tht nonsense-medited mrna dey (NMD) tivity is ompromised in the sene of STA1 tivity (Mqut, 24). To ddress whether STA1 funtions in NMD, the seedling survivl ssy in response to two-step het tretment ws rried out with wellhrterized loss-of-funtion NMD mutnts, upf1 nd upf3 (Jeong et l., 211) together with Col, st1-1, nd STA1/st1-1. Consistent with the previous growth phenotype responses to het stress tretment (Fig. 4A), Col nd STA1/st1-1 showed het stress tolerne, ut st1-1 did not (Supplementl Fig, S1). upf1 nd upf3 lso showed phenotype similr to Col efore nd fter the step-wise het tretment inditing tht STA1 most unlikely funtions in NMD t lest for the estlishment of het stress tolerne. HSFA3 plys key role in expression of mny HSP genes, gene produts of whih ontriute to either positive or negtive feedk of het stress responses nd eventully provides effiient plnt het stress dpttion (Chrng et l., 26). Sine mture mrna expression of HSFA3 orrelted well with plnt het tolerne in our two-step quired het ssy, we further exmined whether HSFA3 ould drive plnt het stress tolerne for itself downstrem of STA1. Two trnsgeni Aridopsis lines in st1-1 kground were generted to onstitutively express DNA version of HSFA3 tht does not require spliing tivity for its mture mrna expression (HSFA3/st1-1). The HSFA3/st1-1 lines with single homozygous trnsgene insertion were seleted nd used for further nlysis. The expression of HSFA3 ws monitored in two trnsgeni HSFA3/st1-1 lines y RT-qPCR (Supplementl Fig. S11). There ws no drsti phenotypi differene mong Col, st1-1, STA1/st1-1, nd HSFA3/st1-1, exept seedlings in the st1-1 kground showed reltively slower growth nd simpler root rhiteture thn Col (Supplementl Fig. S12). These trnsgeni lines were tested for het stress tolerne under oth mild nd two-step quired het stress onditions together with Col nd st1-1. For mild het stress tolerne ssy, seedlings were exposed t 38 C for 1 d nd seedling viility ws monitored t 7 d fter the het tretment. Approximtely two-thirds of seedlings of Col nd trnsgeni STA1/st1-1 omplementtion lines were survived under mild het stress onditions, ut trnsgeni HSFA3/st1-1 lines were unle to keep their viility s like Col nd trnsgeni STA1/st1-1 omplementtion lines (Fig. 5, A nd B). In onsistent with previous dt (Fig. 4A), 12

13 seedlings of Col nd STA1/st1-1 showed stress tolerne to the step-wise het tretment, ut trnsgeni HSFA3/st1-1 lines gin filed to survive under the stress onditions (Fig. 5C). To monitor protein funtions of trnsgene HSFA3 in gene regultion, its trget gene expression ws mesured y RT-qPCR. Expression of HSFA3 trget genes HSP18.2 nd HSA32 ws highly indued in Col nd STA1/st1-1 under two-step quired het stress onditions (Fig. 5, D nd E). Although HSFA3 expression ws deteted in trnsgeni HSFA3/st1-1 lines efore het tretment euse of the nture of 35S promoter used in the trnsgeni line onstrution (Supplementl Fig. S11), HSP18.2 nd HSA32 expression ws not ny further indued in HSFA3/st1-1 ompred to st1-1 fter het tretment. Furthermore, mture mrna trnsripts of two well hrterized het stress responsive genes HSP23.6 nd HSC7-5 were filed to umulte in HSFA3/st1-1 lines fter het tretment (Fig. 5, F nd G). These results indited tht HSFA3 expresses in response to het stress nd its gene produts indue HSP expression, ut its sole tivity is not suffiient to drive plnt het tolerne. This implited tht STA1 involves in pre-mrna spliing of roder rnge of genome responses to het stress. Glol Trget Anlysis for STA1-dependent Spliing Ativity To serh for genome-wide glol trgets of STA1-depdnednet spliing tivity in het induile trnsriptomes, differentil disply of RNA-followed y sequening (DDR-seq) ws onduted with Col nd st1-1. Aridopsis seedlings were first grown on hlf strength MS gr ontining.5% surose under yle of 16 h light nd 8 h drk t C for 5 dys. For het-indued DDR-seq nlysis, seedlings were exposed to het tretment t 38 C for 2 h nd then immeditely t 45 C for 2 h, nd hrvested 24 h fter het tretment. Beuse two genotypes showed some differenes in growth phenotypes, we identified het induile genes within eh genotype nd ompred these identified sets of genes to lern similrities nd differenes in het responsive gene expression in Col nd st1-1. DDR-seq nlysis ws thus rried out with Col (driver) vs. het-treted Col (tester) nd st1-1 (driver) vs. het-treted st1-1 (tester) nd sequene reds were normlized nd nlyzed using CLC Genomis Workenh. After mpping sequene reds to genome informtion 7762 nd 8529 genes were uniquely identified from 398,37 nd 436,311 se pirs from Col nd st1-1, respetively (Supplementl Tle S1, Supplementl Fig. S6A). These orresponded to pproximtely 3 35% of protein oding genes in Aridopsis genome. In n ordinry proess of differentilly expressed 13

14 gene nlysis, trnsripts of whih expression ws ffeted in opposite diretion in wild type nd loss-of funtion mutnt would e reognized s primry responsive genes under the regultion of geneti ftors of interests. However, we rther tried to nlyze 5745 genes tht were ommonly sequened in Col nd st1-1 (Supplementl Fig. S13A), sine genes under STA1 regultion seem to umulte their pre-mrnas in st1-1 under indution onditions. Consistently twie more sequene reds mthed to introns in the rw dt of st1-1 ompred to Col (Supplementl Tle S1). In results roughly two thirds of het-induile genes in Col- overlpped with those in st1-1 suggesting tht STA1 onveys pre-mrna spliing tivity for signifint portion of trnsriptome responses to het stress. In the ommonly identified DDR-seq dt 268 genes overlpped with those found in mirorry-sed dt for het stress responses to 38 C for 3 h (Supplementl Dtset; Sine oth DDR-seq nd mirorry experiments were rried out with different het stress onditions, only ertin level of het responses presented ommonly in oth dtsets. Despite the limittion, gene ontology (GO) term nlysis ws rried out for this set of genes to understnd representtive ellulr pthwys under het stress onditions. First genes for responses to het (GO:948, 2.9e-19) nd protein folding (GO:6457, 6.2e-15) were reognized eing enrihed signifintly (Fig. 6A). In ddition genes for responses to high light intensity, hydrogen peroxide, old nd regultion of progrmmed ell deth were enrihed with equl or less thn 1% of flse disovery rtes (FDR), whih perhps implited tht retive oxygen speies ould ply role in het responsive gene expression. Furthermore 14 HSP genes were notied in the tegory of genes for response to het nd those for protein folding tht re known to e the mjor funtion of HSPs in stressed ells (Supplementl Fig. S13B). Twelve of them hve more thn one intron, of whih spliing is most likely under STA1 regultion. HSP9-2 expression ould not e mesured y RT-qPCR with limited nlysis nd thus it ws exluded from further nlysis. We foused on these 11 intronontining het stress-relted genes in the eginning. When these intron-ontining HSP genes were nlyzed for their gene expression ptterns under het stress onditions, mture mrna expression of HSP23.6, HSC7-5, BOB1, CPHSC7-1 nd HSP6-3B ws highly indued in Col, ut muh less in st1-1 (Fig. 6B nd Supplementl Fig. S14A). This pttern of mture mrna umultion ws similr to tht of HSFA3 (Fig. 4D). 14

15 However, mture mrna expression of HSP7-3, HSP7-4, HSP7-16 nd HSP89.1 ws indued similrly in oth Col nd st1-1 or even higher in st1-1 (Supplementl Fig. S14A), inditing tht mture trnsript umultion of these genes ws likely independent of STA1 tivity. Tken together, mture mrna expression of HSP23.6, HSC7-5, BOB1, CPHSC7-1 nd HSP6-3B is most likely under the ontrol of STA1-dependent pre-mrna spliing. To verify this notion, umultion of totl mrna nd pre-mrna of HSP23.6, HSC7-5, BOB1, CPHSC7-1 nd HSP6-3B ws mesured seprtely y RT-qPCR. Totl mrna expression of HSP23.6, HSC7-5 nd CPHSC7-1 ws indued in Col nd st1-1 y het tretment (Fig. 6C nd Supplementl Fig. S14B). However, their pre-mrna trnsripts were higher in st1-1 thn Col (Fig. 6D nd Supplementl Fig. S14C). These results indited tht indution of HSP23.6, HSC7-5 nd CPHSC7-1 expression y het tretment ws minly regulted t post-trnsriptionl level y STA1 spliing tivity s shown with HSFA3 expression (Fig. 4D). In ses of BOB1 nd HSP6-3B, totl mrna umultion ws reltively less indued in st1-1 ompred to Col y het tretment (Fig. 6C nd Supplementl Fig. S14B), ut their pre-mrna levels were still higher in st1-1 thn Col (Fig. 6D nd Supplementl Fig. S14C). These results implited tht STA1 not only modulted spliing of BOB1 nd HSP6-3B t posttrnsriptionl level, ut lso influened their trnsription perhps y the funtion of STA1 spliing produts s shown with HSA32 expression (Fig. 4E). Tken ll together STA1 spliing tivity is indispensle for mture mrna umultion of suset of HSPs under het stress onditions. In this study, STA1, puttive omponent of the U5 snrnp omplex, ws hrterized s n essentil regultory ftor involved in the spliing of speifi suset of pre-mrnas inluding HSFA3 nd its trget gene HSA32 in response to high temperture stresses. Phenome nd moleulr nlyses in response to het tretment demonstrted tht STA1 funtions in spliing, ut not in NMD, s regultor in het stress tolerne. HSFA3-dependent trnsript sdes tht were reonstituted using trnsient expression of the stress-relted DREB2A nd STA1 unmiguously linked to the STA1 tivity with pre-mrna spliing of HSFA3, lthough it is not suffiient ftor for the estlishment of plnt het stress tolerne. Consistently STA1 ontrols pre-mrna spliing of roder rnge of het induile trnsriptomes. In onlusion, het-induile STA1-dependent post-trnsriptionl regultion of the stress responsive gene expression plys role in the estlishment of the quired het stress tolerne in Aridopsis. 15

16 More reently STA1 hs een reported to ply role in mirna iogenesis nd RNAdireted DNA methyltion sed on nlyses of relevnt moleulr nd ellulr phenotypes of st1-1 (Chne et l., 213; Dou et l., 213). Moreover, het stress seemingly lters spliing of mirna, perhps linking STA1 tivity to mirna spliing (Yn et l., 212). This new STA1 funtions in smll RNA expression deserve more ttentions to eluidte speifi STA1 funtions in gene regultion. Our findings unrvel tht STA1 hs speifi trgets in pre-mrna spliing for the posttrnsriptionl regultion of gene expression response to het stress. Even so, how STA1 modultes het stress tolerne remins unler. Identifition of geneti omponents nd eluidtion of their moleulr mehnisms in the STA1-ontining splieosome omplex tht is responsile for its spliing speifiity will enlighten the moleulr sis underlying stressspeifi gene expression, nd eventully stress dptive responses in plnts. 16

17 MATERIALS AND METHODS Plnt Mterils nd Growth Conditions Plnts were grown in soil for 22 to 24 d under photoperiod of 13 h light/11 h drk (6 μmol/m 2 /s) t 25 C. Aridopsis thlin Columi- (Col) plnts were used s the wild-type, st1-1 (Lee et l., 26), upf1-5 (Jeong et l., 211) nd upf3-1 (Jeong et l., 211) mutnts were used for experiments. Plsmid onstruts for trnsgeni plnts were generted y inserting the DNA of STA1 or HSFA3 etween the 35SC4PPDK promoter (designted s HBT) nd the NOS termintor in mini-inry vetor, pcb32 (Cho et l., 212). The onstruts were expressed in Col or st1-1 plnts. Trnsgeni lines with similr trnsgene expression levels were seleted nd used for further nlyses. Multiple independent trnsgeni lines were generted nd nlyzed to identify onsistent geneti effets. Trnsgeni plnt phenotypes from t lest two independent lines of the T 3 genertion were nlyzed. Het Stress Survivl Assy To exmine plnt het stress tolerne, surfe-sterilized seeds were strtified for 4 d t 4 C in the drk, nd plnts were vertilly grown on hlf-strength Murshige nd Skoog (MS) gr medium ontining.5 % surose for 5 d under photoperiod of 16 h light/8 h drk (6 μmol/m 2 /s) t 25 C. For the quired het stress tolerne ssy, seedlings were sujeted to step-wise het tretment of 38 C for 2 h followed y 45 C for 2 h. For the sl het stress tolerne ssy, seedlings were sujeted to 45 C for 2 h. For the mild het stress tolerne ssy, horizontlly grown 3-dy-old seedlings were sujeted to 38 C for 24 h. Plnt survivl ws oserved 7 d fter the het tretment nd sored sed on the retention of green shoots. Aridopsis Mesophyll Protoplst Trnsient Expression Assy Protoplst isoltion nd trnsient expression ssys were rried out s previously desried y Yoo et l. (27) nd Cho nd Yoo (21). The effetor onstrut (STA1) ws generted y inserting DNA etween the HBT promoter nd the NOS termintor in plnt expression vetor. All the reporter onstruts (gcor15a, ghsfa3, gidd14, nd gmpk1) were generted y inserting genomi DNA fused with GUS etween the HBT (modified 35S) promoter nd the NOS termintor in plnt expression vetor. All the onstruts were verified y DNA sequening. The renill luiferse driven y UBQ1 promoter (UBQ1-rLUC) ws inluded s n internl ontrol 17

18 in the protoplst trnsient expression ssy. The experiments were repeted, inditing onsistent results mong the replites. The primers used for loning re listed in Supplementl Tle S2. In the funtionl spliing ssy, reporter tivities were lulted sed on the GUS/renill-LUC rtio nd normlized to the vlues otined without the effetor expression. To mesure the GUS tivity, the trnsfeted protoplsts with designted onstruts were lysed using pssive lysis uffer (Promeg) ontining 1% Triton-X (USB) nd riefly mixed y vortexing. The lyste ws inuted t room temperture for 1 min nd entrifuged t 13, rpm for 3 s. The protoplst lyste ws mixed with 1 mm MUG (Gold Biotehnology) nd inuted t 37 C for 9 min. The retion mixture ws frozen t -8 C to quenh the retion. The mixture ws then diluted with.2 M N 2 CO 3, nd the GUS tivity ws mesured using the Glomx (Promeg) single tue system with UV module, following the mnufturer s instrutions. RNA Isoltion nd Trnsript Mesurement For STA1 expression test, Col-RD29A-LUC ws used s the WT (Lee et l., 26). Seeds were sown on 1 MS medium (2% surose nd.3% gelrite) fter surfe steriliztion with sodium hypohlorite (4%). The seeds were strtified t 4 C for 2 d nd grown t 22 C under ontinuous light. For het tretment, the plnts were pled in 37 C inutor. For gene expression nlysis, totl RNA ws isolted y the Trizol method (Invitrogen), nd 1 μg of totl RNA ws used for DNA synthesis using M-MLV reverse trnsriptse (Promeg). Gene expression ws quntittively mesured using rel-time PCR (Bio-Rd) with the SYBR Green dye-dded PCR mix (Bio-Rd). PROTEIN PHOSPHATASE 2A (PP2A, AT1G1332), TUBULIN4 (TUB4, At1g482), nd ELONGATION INITIATION FACTOR 4 (ELF4, At3g1392) trnsripts were used s the ontrols with gene-speifi primers. Detiled primer sequenes re listed in Supplementl Tle S2 of the Supplementry mteril. Eh primer set ws pre-tested y PCR for single gene produt. The experiments were repeted thrie, nd onsistent results were otined. Differentil disply of RNA-followed y sequening (DDR-seq) For smple preprtion, plnts were grown on hlf-strength MS gr medium ontining.5 % surose for 5 d nd sujeted to n quired het stress s desried in mteril nd method. Het treted (tester) nd ontrol (driver) seedlings were hrvested 1 d fter het tretment. 18

19 Totl RNA ws extrted sed on the Trizol method (Invitrogen) nd poly-(a)-rna ws isolted using Dyned mrna DIRECT TM Kit (Thermo Sientifi). Fifty ng of poly-(a)-rna ws used to synthesize doule strnded DNA. After ethnol preipittion, doule strnded DNA ws digested with Dpn II enzyme (NEB) for 2 h, phenol/chcl 3 extrted, ethnol preipitted nd resuspended with H 2 O. DNA ws ligted with pre-nneled R-24/12 dptor (2 mg/ml) t 16 C for 12 h. R dptor ligted DNA ws phenol/chcl 3 extrted, ethnol preipitted nd resuspended with H 2 O. To generte tester nd driver mplions, DNA ws mplified with multiple PCR retions using high-fidelity DNA polymerse (Phusion Highfidelity DNA Polymerse, NEB) nd R-24 primer (72 C 5 min, 2 yles of 94 C 1 min; 72 C 3 min, 72 C 1 min). The PCR produts were phenol/chcl 3 extrted nd isopropnol preipitted. To remove R-24/12 dptor, the PCR produts were digested with Dpn II for 2 h, phenol/chcl 3 extrted twie, ethnol preipitted nd resuspended with H 2 O. The onentrtions of the tester nd driver produts were quntified using Quiit 2. Fluorometer (Invitrogen). Five hundred ng of the tester ws further ligted with pre-nneled J-24/12 dptor (2 mg/ml) t 14 C or 12 h. For the sutrtive hyridiztion, 25 μg of driver nd 25 ng of J-dptor ligted tester DNA were omined nd phenol/chcl 3 extrted, CHCl 3 extrted nd ethnol preipitted. The pellet ws resuspended with 4 μl of 3x uffer solution ontining 3 mm HEPES (USB) ph 8. t 2 C nd 3 mm EDTA (USB). The solution ws inuted t 98 C for 5 min for DNA denturtion. One μl of 5 M NCl ws dded into the solution nd the mixture ws further inuted t 67 C for 2 h to hyridize tester nd driver. The hyridiztion solution ws mixed well with 8 μl of 5 mg/ml of yest RNA nd further diluted with 367 μl H 2 O. For eh sutrtion, eight 1 μl PCR retions were prepred inluding 2 μl of hyridiztion mix without the primer. The PCR mixtures were inuted t 72 C for 5 min nd.8 μl of J-24 primer (1 mg/ml) ws dded. Ten yles of PCR retion were performed (94 C 1 min, 7 C 3 min) nd inuted t 72 C for 1 min. PCR produts were omined, phenol/chcl 3 extrted twie, ethnol preipitted nd resuspended with 4 μl H 2 O. 2 μl PCR produts were digested with Mung Ben Nulese (NEB) t room temperture for 3 min. To stop the retion, 16 μl of 5 mm Tris ph 8.9 ws dded nd inuted t 7 C for 1 min. For mplifition, eight 1 μl PCR retions were prepred inluding 2 μl of Mung Ben Nulese-treted produts nd.8 μl of J-24 primer (1 mg/ml) without polymerse. The PCR mixtures were inuted t 95 C for 1 19

20 min nd ooled down to 8 C. 2.5 U of polymerse ws dded nd eighteen yles of PCR retion were performed (94 C 1 min, 7 C 3 min). After further inution t 7 C for 1 min, PCR produts were omined, phenol/chcl 3 extrted twie, CHCl 3 extrted, ethnol preipitted nd resuspended with 5 μl H 2 O, generting differene produt (DP). For next genertion sequening, lirry ws prepred ording to Ion Xpress TM Plus Frgment Lirry Kit (Thermo Sientifi) nd sequening were onduted with the mnufture s instrution with Ion PGM TM (Thermo Sientifi). Rw reds were normlized nd nlyzed using tril version of CLC Genomis Workenh (QIAGEN, Vleni, CA, USA) pltform. Gene Ontology Anlysis Gene ontology (GO) nlysis ws rried out using grigo we-sed tool (Du et l., 21). Complete_GO ws used for identifition of enrihed gene ontology with Fisher s test. For multiple signifine tests, Benjmini nd Yekutieli FDR orretion ws used nd GO terms with FDR<.1 were visulized with grphil view. ACCESSION NUMBERS Sequene dt from this study n e found in the Aridopsis Genome Inititive or GenBnk/EMBL dtses under the following ession numers: STA1, AT4g343; COR15A, AT2G4254; IDD14, AT1G6813; MPK1, AT3G5979; DREB2A, AT5G541; HSFA3, AT5G541; HSFB1, AT4G3699; HSFB2, AT5G622; UPF1, AT5G471; UPF3, AT1G3398; HSA32, AT4G2132; HSP23.6, AT4G252; HSP7T-2, AT2G3212; HSP7-16, AT1G1166; HSP7-4, AT3G1258; HSP6-3B, AT3G2399; HSP7-1, AT5G25; BOB1, AT5G534; HSP7-3, AT3G944; CPHSC7-1, AT4G2428; HSP7-7, AT5G4991; HSC7-5, AT5G959; HSP89.1 nd AT3G777. ACKNOWLEDGEMENTS We thnk for Jungwoo Hong for initil tehnil support. SUPPLEMENTAL MATERIALS 2

21 Supplementl Figure S1. Spliing tivity ws mesured in the presene nd sene of STA1 for 35S-fLUC tivity in lef mesophyll protoplsts (LMPs) of st1-1. Supplementl Figure S2. Intron-retined pre-mrna nd exon-only mture mrna of gcor15a- GUS were diserned using semi-quntittive RT-PCR with RNA extrted from trnsfeted LMPs of Col nd st1-1. Supplementl Figure S3. STA1 expression upon old nd het tretment. Supplementl Figure S4. Different types of HSF trnsripts were mesured in Col nd st1-1 in response to het tretment (37 C) using semi-quntittive RT-PCR. Supplementl Figure S5. Anlysis of STA1 nd DREB2A trnsripts. Supplementl Figure S6. Anlysis of the endogenous nd trnsgene expression of STA1 in trnsgeni plnts. Supplementl Figure S7. Seedling survivl ssy ws rried out with Col, st1-1, two lines of STA1-expressing Col (STA1/Col), nd two lines of STA1-expressing st1-1 (STA1/st1-1) for two-step quired nd one-step sl het tretments. Supplementl Figure S8. The expression levels of STA1 nd HSFA3 mrna were monitored using RT-qPCR without reverse trnsription s ontrol. Supplementl Figure S9. The expression levels of HSP18.2 in Col, st1-1 nd STA1-expressing st1-1 (STA1/st1-1) were monitored using RT-qPCR. Supplementl Figure S1. STA1 involves in pre-mrna spliing ut not in nonsense-medited mrna dey for the estlishment of plnt het stress tolerne. Supplementl Figure S11. Anlysis of HSFA3 expression in two HSFA3/st1-1 trnsgeni plnts. Supplementl Figure S12.. Non-het treted twelve-dy-old seedlings of Col, st1-1, two lines of STA1/st1-1 nd two lines of HSFA3/st1-1 re shown. Supplementl Figure S13. Anlysis of DDR-seq of Col nd st1-1 in response to het stress. Supplementl Figure S14. Anlysis of het induile genes enrihed in DDR-seq nlysis. Supplementl Tle S1. DDR-seq informtion. Supplementl Tle S2. Primers used in the study. Supplementl Dtset. GO nlysis of ommonly enrihed 268 genes in DDR-seq dt of Col nd st

22 FIGURE LEGENDS Figure 1. STA1 indues spliing tivity of speifi pre-mrna. A, A shemti digrm of the funtionl spliing ssy is shown. The genomi version of spliing trget gene ws loned into the GUS reporter onstrut with trnsltionl fusion. B, C, Spliing tivity ws mesured in the presene nd sene of STA1 for gcor15a-gus in lef mesophyll protoplsts of Col (B) nd st1-1 (C). UBQ1-rLUC tivity served s n internl ontrol. STA1 protein expression ws shown using protein lot nlysis with nti-epitope speifi ntiody. Ruiso smll suunit proteins (RBC) served s protein loding ontrol using oomssie lue stining. The mens of three replites re shown with stndrd error rs. Asterisks represent Pired t-test signifine etween smples (***P <.1, **P <.1, nd *P <.5). D, E, Spliing tivity ws mesured in the presene nd sene of STA1 for gidd14- GUS (D) nd gmpk1-gus (E) in lef mesophyll protoplsts of st1-1. UBQ1-rLUC tivity served s n internl ontrol. The mens of three replites re shown with stndrd error rs. Figure 2. Het-induile STA1 involves in pre-mrna spliing of HSFA3. A, STA1 expression ws mesured in response to het tretment (37 C) using semi-quntittive RT-PCR. PP2A served s RNA ontrol. B, A shemti digrm of genomi DNA of HSFA3 is shown with primer positions. C, D, Different types of HSFA3 trnsripts were mesured in Col (C) nd st1-1 (D) in response to het tretment (37 C) using semi-quntittive RT-PCR. Het induile totl mrna (f1/r1), pre-mrna (f1/f2) nd mture mrna (f1/f3) of HSFA3 were mesured. Experiments were triplited with onsistent results. Representtive dt re shown. E, Shemti digrms WT (wild type), mdo (muttion on splie donor) nd ma (muttion on splie eptor) of ghsfa3-nluc re shown. F, Spliing reporter tivities from WT ghsfa3- nluc or mdo nd mac forms of ghsfa3-nluc were mesured in the presene nd sene of STA1. Expression of HA-tgged STA1 vrints ws shown y protein lot nlysis using nti- HA ntiody. RBC used s loding ontrol. G, Spliing reporter tivity from ghsfa3-nluc ws mesured in omintion of STA1 nd st1-1 expression. Expression of HA-tgged STA1 nd Flg-tgged st1-1 ws shown y protein lot nlysis using with nti-epitope speifi ntiody. Ruiso smll suunit proteins (RBC) served s protein loding ontrol using oomssie lue stining. 35S-fLUC tivity served s n internl ontrol. All of the experiments were repeted three times with onsistent results. The mens of three replites re shown with 22

23 stndrd error rs. Different letters indite signifine differene y Tukey-Krmer test (p<.5). Figure 3. STA1 indues pre-mrna spliing of HSFA3 driven y DREB2A. A, A working model is proposed for DREB2A-dependent het-induile gene expression. B, A shemti digrm of HSFA3 is shown with set of primers used for deteting intron-retined pre-mrna. C, D, Intron-retined HSFA3 pre-mrna, gene expression of whih ws driven y DREBA2, ws diserned in LMPs of Col nd st1-1 with/without STA1. The pre-mrna umultion ws monitored using semi-quntittive RT-PCR (C) nd RT-qPCR (D). E, Expression of HSP23.6, HSA32, nd HSP7T-2 in st1-1 ws monitored in omintion of DREB2A nd STA1 expression using RT-qPCR. Quntittive vlues were normlized with n internl ontrol EIF4 nd presented in omprison to those vlues in Col without effetor trnsfetion. All of the experiments were repeted three times nd produed onsistent results. The mens of triplites re shown with stndrd error rs. Asterisks represent Pired t-test signifine etween smples (***P <.1, **P <.1, nd *P <.5). Figure 4. STA1 involves in the estlishment of stress tolerne in response to two-step het tretment in Aridopsis. A, Seedling survivl ssy ws rried out with Col, st1-1, STA1-expressing Col (STA1/Col), nd STA1-expressing st1-1 (STA1/st1-1) for two-step quired nd one-step sl het tretments. B,C, The expression levels of tsta1 (B) nd STA1 (C) were quntittively monitored efore (Cont) nd t 1d fter het tretment (Het) using RT-qPCR. D, E, The expression levels of totl mrna, pre-mrna nd mture-mrna of HSFA3 (D) nd HSA32 (E) were quntittively monitored using RT-qPCR in Col, st1-1 nd STA1/st1-1 efore (Cont) nd t 1 d fter het tretment (Het). Primer sets for totl mrna (f1/r1), pre-mrna (f1/r2) nd mture-mrna (f1/r3) were desried with shemti digrms. Quntittive vlues were normlized with internl ontrols ELF4. The mens of triplites re shown with stndrd error rs. Different letters indite signifine differene y Tukey-Krmer test (p<.5). Figure 5. STA1, ut not HSFA3, estlishes plnt stress tolerne for mild het nd twostep quired het stresses. 23

24 A, Seedling survivl ssy ws rried out with Col, st1-1, two lines of STA1-expressing st1-1 (STA1/st1-1), two lines of HSFA3-expressing st1-1 (HSFA3/st1-1) for mild het tretment. B, Vile seedlings ws mesured to lulte survivl rte. The mens of triplites re shown with stndrd error rs. C, Seedling survivl ssy ws rried out for two-step quired het tretment. D-G, The expression levels of mture mrna of HSP18.2 (D), HSA32 (E), HSP23.6 (F) nd HSC7-5 (G) were monitored in Col, st1-1, two lines of STA1/st1-1 nd two lines of HSFA3/st1-1 efore nd 1 d fter the step-wise het tretment using RT-qPCR. The mens of triplites re shown with stndrd error rs. Different letters indite signifine differene y Tukey-Krmer test (p<.5). Figure 6. Het induile genes enrihed in DDR-seq nlysis. A, Grphil view of gene ontology (GO) term nlysis for gene (see Supplementl Dtset). B, Mture mrna expression of HSP23.6, HSC7-5 nd BOB1 ws mesured in Col nd st1-1 using RT-qPCR. C, D, Totl mrna (C) nd premture mrna (D) of HSP23.6, HSC7-5 nd BOB1 were mesured using RT-qPCR in Col nd st1-1. The mens of three replites re shown with stndrd error rs. Different letters indite signifine differene y Tukey-Krmer test (p<.5). 24

25 LITERATURE CITED Artus NN, Uemur M, Steponkus PL, Gilmour SJ, Lin C, Thomshow MF (1996) Constitutive expression of the old-regulted Aridopsis thlin COR15 gene ffets oth hloroplst nd protoplst freezing tolerne. Pro Ntl Ad Si USA 93: Chne BS, Liu R, Chinnusmy V, Kwon Y, Prk J, Kim S, Zhu JK, Yng S, Lee B (213) STA1, n Aridopsis pre-mrna proessing ftor 6 homolog, is new plyer involved in mirna iogenesis. Nulei Aids Res 41: Chrng YY, Liu HC, Liu, NY, Hsu, FC, Ko SS (26) Aridopsis Hs32, novel het shok protein, is essentil for quired thermotolerne during long reovery fter limtion. Plnt Physiol 14: Chen H, Hwng JE, Lim CJ, Kim DY, Lee SY, Lim CO (21) Aridopsis DREB2C funtions s trnsriptionl tivtor of HsfA3 during the het stress response. Biohemil nd Biophysil Reserh Comm 41: Cho YH, Hong JW, Kim EC, Yoo SD (212) Regultory funtions of SnRK1 in stressresponsive gene expression nd in plnt growth nd development. Plnt Physiol 158: Cho YH, Yoo SD (21) Expression of epitope-tgged proteins in Aridopsis lef mesophyll protoplsts. Methods Mol Biol 657: Dou K, Hung CF, M ZY, Zhng CJ, Zhou JX, Hung HW, Ci T, Tng K, Zhu JK, He XJ (213) The PRP6-like spliing ftor STA1 is involved in RNA-direted DNA methyltion y filitting the prodution of Pol V-dependent sffold RNAs. Nulei Aids Res 41: Du Z, Zhou X, Ling Y, Zhng Z, Su Z (21) grigo: GO nlysis toolkit for the griulturl ommunity. Nulei Aids Res 38: W64-7 Godfry HCJ, Grnett T (214) Food seurity nd sustinle intensifition. Philosophil Trnstions of the Royl So B 369: Hll MP, Unh J, Binkowski BF, Vlley MP, Butler BL, Wood MG, Otto P, Zimmermn K, Vidugiris G, Mhleidt T, Roers MB, Benink HA, Eggers CT, Slter MR, Meisenheimer PL, Kluert DH, Fn F, Enell LP, Wood KV (212) Engineered 25

26 luiferse reporter from deep se shrimp utilizing novel imidzopyrzinone sustrte. ACS Chem Biol 7: Jeong H, Kim Y, Kim S, Kim Y, Lee I, Kim Y, Shin J (211) Nonsense-medited mrna dey ftors, UPF1 nd UPF3, ontriute to plnt defense. Plnt Cell Physiol 52: Knowler JT nd Wilks AF (198) Rionuleoprotein prtiles nd the mturtion of eukryote mrna. Trends Biohem Si 5: Kotk S, Lrkindle J, Lee U, von Koskull-Döring P, Vierling E, Shrf KD (27) Complexity of the het stress response in plnts. Curr Opin Plnt Biol 1: Lee B, Kpoor A, Zhu J, Zhu J (26) Stilized1, stress-upregulted nuler protein, is required for pre-mrna spliing, mrna turnover, nd stress tolerne in Aridopsis. Plnt Cell 18: Mo G, Meng X, Liu Y, Zheng Z, Chen Z, Zhng S (211) Phosphoryltion of WRKY trnsription ftor y two pthogen-responsive MAPKs drives phytolexin iosynthesis in Aridopsis. Plnt Cell 23: Mqut LE (24) Nonsense-medited mrna dey: spliing, trnsltion, nd mrnp dynmis. Nt Rev Mol Cell Biol 5: MClung CR (214) Mking hunger yield. Siene 344: Qin F, Kkimoto M, Skum Y, Mruym K, Oske Y, Trn LS, Shinozki K, Ymguhi-Shinozki K (27) Regultion nd funtionl nlysis of ZmDREB2A in response to drought nd het stresses in Ze mys L. Plnt J 5: Reddy ASN, Mrquez Y, Klyn M, Brt A (213) Complexity of the lterntive spliing lndspe in plnts. Plnt Cell 25: Skum Y, Mruym K, Qin F, Oske Y, Shinozki K, Ymguhi-Shinozki K (26) Dul funtion of n Aridopsis trnsription ftor DREB2A in wter-stress-responsive nd het-stress-responsive gene expression. Pro Ntl Ad Si USA 13: Shrf KD, Bererih T, Eerserger I, Nover L (212) The plnt het stress trnsription ftor (Hsf) fmily: struture, funtion nd evolution. Biohim Biophys At 1819:

27 Shrmm F, Lrkindle J, Kiehlmnn E, Gnguli A, Englih G, Vierling E, von Koskull- Döring P (28) A sde of trnsription ftor DREB2A nd het stress trnsription ftor HsfA3 regultes the het stress response of Aridopsis. Plnt J 53: Seo P, Kim M, Ryu J, Jeong E, Prk C (211) Two splie vrints of the IDD14 trnsription ftor ompetitively form nonfuntionl heterodimers whih my regulte strh metolism. Nt Commun 2: 33 Silv-Correi J, Freits S, Tvres RM, Lino-Neto T, Azevedo H (214) Phenotypi nlysis of the Aridopsis het stress response during germintion nd erly seedling development. Plnt Methods 1: 7 Stiger D, Brown JWS (213) Alterntive spliing t the intersetion of iologil timing, development, nd stress responses. Plnt Cell 25: Whl MC, Will CL, Luhrmnn R (29) The splieosome: Design priniples of dynmi RNP mhine. Cell 136: Winter D, Vinegr B, Nhl H, Ammr R, Wilson GV, Provrt NJ (27) An eletroni fluoresent pitogrph rowser for exploring nd nlyzing lrge-sle iologil dt sets. PLoS ONE 2: e718 Yn K, Liu P, Wu CA, Xu R, Guo QH, Hung JG, Zheng CC (212) Stress-indued lterntive spliing provides mehnism for the regultion of mirorna proessing in Aridopsis thlin. Mol Cell 48: Yoo S, Cho Y, Sheen J (27) Aridopsis mesophyll protoplsts: A verstile ell system for trnsient gene expression nlysis. Nt Proto 2: Yu S, Hn J, Chhoeun C, Lee B. (216) Geneti Sreening for Aridopsis Mutnts Defetive in STA1 Regultion under Therml Stress Implites the Existene of Regultors of Its Speifi Expression, nd the Geneti Intertions in the Stress Signling Pthwys. Front Plnt Si 7: 618 Zhu J, Dong CH, Zhu JK (27) Interply etween old-responsive gene regultion, metolism nd RNA proessing during plnt old limtion. Curr Opin Plnt Biol 1:

28 A strt stop 35S promoter Intron-retined signture GUS NOS termintor Pre-mRNA spliing GUS GUS Pre-mture Termintion Codon (PTC) B Fold indution (GUS/rLUC) gcor15a -GUS * C gcor15a -GUS ** STA STA1-HA (-HA) RBC D 3 Col gidd14-gus E 3 st1-1 gmpk1-gus Fold indution (GUS/rLUC) STA1 - + STA1 - +

29 Fig. 1. STA1 indues spliing tivity of speifi pre-mrna. A, A shemti digrm of the funtionl spliing ssy is shown. The genomi version of spliing trget gene ws loned into the GUS reporter onstrut with trnsltionl fusion. B, C, Spliing tivity ws mesured in the presene nd sene of STA1 for gcor15a-gus in lef mesophyll protoplsts of Col (B) nd st1-1 (C). UBQ1-rLUC tivity served s n internl ontrol. STA1 protein expression ws shown using protein lot nlysis with nti-epitope speifi ntiody. Ruiso smll suunit proteins (RBC) served s protein loding ontrol using oomssie lue stining. The mens of three replites re shown with stndrd error rs. Asterisks represent Pired t-test signifine etween smples (***P <.1, **P <.1, nd *P <.5). D, E, Spliing tivity ws mesured in the presene nd sene of STA1 for gidd14-gus (D) nd gmpk1- GUS (E) in lef mesophyll protoplsts of st1-1. UBQ1-rLUC tivity served s n internl ontrol. The mens of three replites re shown with stndrd error rs.

30 A STA1 PP2A (h/37 C) B f1 HSFA3 5 3 r2 r1 r3 C (h/37 C) D (h/37 C) f1/r1 f1/r1 f1/r2 f1/r2 f1/r3 f1/r3 UBQ1 UBQ1 Col st1-1 E WT mdo ma Splie Splie donor eptor GT AG TT AG GT GG ghsfa3-nluc Fold indution (nluc/fluc) F STA WT mdo ma STA1-HA (-HA) RBC ghsfa3-nluc G Fold indution (nluc/fluc) STA1 st1-1 STA1-HA (-HA) st1-1-flg (-Flg) RBC ghsfa3-nluc

31 Fig. 2. Het-induile STA1 involves in pre-mrna spliing of HSFA3. A, STA1 expression ws mesured in response to het tretment (37 C) using semiquntittive RT-PCR. PP2A served s RNA ontrol. B, A shemti digrm of genomi DNA of HSFA3 is shown with primer positions. C, D, Different types of HSFA3 trnsripts were mesured in Col (C) nd st1-1 (D) in response to het tretment (37 C) using semi-quntittive RT-PCR. Het induile totl mrna (f1/r1), pre-mrna (f1/f2) nd mture mrna (f1/f3) of HSFA3 were mesured. Experiments were triplited with onsistent results. Representtive dt re shown. E, Shemti digrms WT (wild type), mdo (muttion on splie donor) nd ma (muttion on splie eptor) of ghsfa3- nluc re shown. F, Spliing reporter tivities from WT ghsfa3-nluc or mdo nd mac forms of ghsfa3-nluc were mesured in the presene nd sene of STA1. Expression of HA-tgged STA1 vrints ws shown y protein lot nlysis using nti-ha ntiody. RBC used s loding ontrol. G, Spliing reporter tivity from ghsfa3-nluc ws mesured in omintion of STA1 nd st1-1 expression. Expression of HA-tgged STA1 nd Flg-tgged st1-1 ws shown y protein lot nlysis using with nti-epitope speifi ntiody. Ruiso smll suunit proteins (RBC) served s protein loding ontrol using oomssie lue stining. 35S-fLUC tivity served s n internl ontrol. All of the experiments were repeted three times with onsistent results. The mens of three replites re shown with stndrd error rs. Different letters indite signifine differene y Tukey-Krmer test (p<.5).

32 A DREB 2A HSFA3 Chromtin B C intron_f2 5 3 r2 HSFA3 Intron retined HSFA3 U1 U6 U4 U2 U5 STA TUB4 STA1 DREB2A HSFA3 pre-mrna Col Intron retined HSFA3 HSFA3 mrna TUB4 STA1 DREB2A D Fold indution HSFA3 _Intron * Col HSFA3 _Intron st1-1 ** st1-1 STA1 DREB2A

33 E Fold indution HSP23.6 * HSA32 ** HSP7T-2 * STA DREB2A Fig. 3. STA1 indues pre-mrna spliing of HSFA3 driven y DREB2A. A, A working model is proposed for DREB2A-dependent het-induile gene expression. B, A shemti digrm of HSFA3 is shown with set of primers used for deteting intronretined pre-mrna. C, D, Intron-retined HSFA3 pre-mrna, gene expression of whih ws driven y DREBA2, ws diserned in LMPs of Col nd st1-1 with/without STA1. The pre-mrna umultion ws monitored using semi-quntittive RT-PCR (C) nd RT-qPCR (D). E, Expression of HSP23.6, HSA32, nd HSP7T-2 in st1-1 ws monitored in omintion of DREB2A nd STA1 expression using RT-qPCR. Quntittive vlues were normlized with n internl ontrol EIF4 nd presented in omprison to those vlues in Col without effetor trnsfetion. All of the experiments were repeted three times nd produed onsistent results. The mens of triplites re shown with stndrd error rs. Asterisks represent Pired t-test signifine etween smples (***P <.1, **P <.1, nd *P <.5).

34 A Aquired het resistne Bsl het resistne B Reltive gene expression tsta1 Col st1-1 STA1/ STA1/ Col st1-1 Col st1-1 STA1/st1-1 C Reltive gene expression 1 STA1 d st1-1 d Cont Het Cont Het

35 D f1 HSFA3 E f1 HSA32 Reltive gene expression Reltive gene expression Reltive gene expression r1 f1/r1 Col st1-1 STA1/st1-1 f1/r2 f1/r3 Cont r2 r3 Cont Het Het Reltive gene expression Reltive gene expression Reltive gene expression Cont Het Cont Het r1 x1 2 r2 r3 f1/r1 f1/r2 f1/r3 Cont d d d Cont Het Het

36 Fig. 4. STA1 involves in the estlishment of stress tolerne in response to two-step het tretment in Aridopsis. A, Seedling survivl ssy ws rried out with Col, st1-1, STA1-expressing Col (STA1/Col), nd STA1-expressing st1-1 (STA1/st1-1) for two-step quired nd one-step sl het tretments. B,C, The expression levels of tsta1 (B) nd STA1 (C) were quntittively monitored efore (Cont) nd t 1d fter het tretment (Het) using RT-qPCR. D, E, The expression levels of totl mrna, pre-mrna nd mture-mrna of HSFA3 (D) nd HSA32 (E) were quntittively monitored using RT-qPCR in Col, st1-1 nd STA1/st1-1 efore (Cont) nd t 1 d fter het tretment (Het). Primer sets for totl mrna (f1/r1), pre-mrna (f1/r2) nd mture-mrna (f1/r3) were desried with shemti digrms. Quntittive vlues were normlized with internl ontrols ELF4. The mens of triplites re shown with stndrd error rs. Different letters indite signifine differene y Tukey-Krmer test (p<.5).

37 A Col st1-1 HSFA3/ st1-1 #4-4 STA1/st1-1 #1-7 HSFA3/st1-1 #2-2 STA1/ st1-1 #3-3 Cont Mild het resistne B C 1.8 Aquired het resistne Survivl rte D x1 4 E Reltive gene expression HSP18.2 Col st1-1 STA1/st1-1 #1-7 STA1/st1-1 #3-3 HSFA3/st1-1 #2-2 HSFA3/st1-1 #4-4 e e e e e e Cont Het d Reltive gene expression HSA32 _f1/r3 Cont Het

38 F Reltive gene expression x1 4 5 HSP _f1/r e e e e e e Cont d d Het G Reltive gene expression HSC7-5 _f1/r3 e e e e e e Cont d d Het d Fig. 5. STA1, ut not HSFA3, estlishes plnt stress tolerne for mild het nd twostep quired het stresses. A, Seedling survivl ssy ws rried out with Col, st1-1, two lines of STA1-expressing st1-1 (STA1/st1-1), two lines of HSFA3-expressing st1-1 (HSFA3/st1-1) for mild het tretment. B, Vile seedlings ws mesured to lulte survivl rte. The mens of triplites re shown with stndrd error rs. C, Seedling survivl ssy ws rried out for two-step quired het tretment. D-G, The expression levels of mture mrna of HSP18.2 (D), HSA32 (E), HSP23.6 (F) nd HSC7-5 (G) were monitored in Col, st1-1, two lines of STA1/st1-1 nd two lines of HSFA3/st1-1 efore nd 1 d fter the step-wise het tretment using RT-qPCR. The mens of triplites re shown with stndrd error rs. Different letters indite signifine differene y Tukey-Krmer test (p<.5).

39 A