Nucleus-Encoded Thylakoid Protein,OsY3IP1,Confers Enhanced Tolerance to Saline and Alkaline Stresses in Rice

Hyeran MOON,Young-Ah KIM,Ryoung SHIN,Chang-Jin PARK

(1Department of Molecular Biology,Sejong University,Seoul 05006,South Korea;2Department of Bioresources Engineering,Sejong University,Seoul 05006,South Korea;3RIKEN Center for Sustainable Resource Science,Yokohama 230-0045,Japan;4Plant Engineering Research Institute,Sejong University,Seoul 05006,South Korea;#These authors contributed equally to this work)

Abstract:Abiotic stress confers serious damage to the photosynthetic machinery,often resulting in plant growth inhibition.Hypothetical chloroplast open reading frame 3 (Ycf3)-interacting protein 1 (Y3IP1) is a nucleus-encoded thylakoid protein and plays an essential role in the assembly of photosystem I.The full-length cDNA over-expresser (FOX) gene-hunting system is an approach using systemically generated gain-of-function mutants.Among the FOX-rice lines,a line CE175 overexpressing rice Y3IP1 gene (OsY3IP1) displayed less inhibition of root growth under saline (NaCl) stress.The expression of OsY3IP1 was up-regulated under saline and alkaline (Na2CO3) stresses in the rice variety Kitaake.After saline and alkaline treatments,transgenic Kitaake overexpressing OsY3IP1-GFP (OsY3IP1-GFPox/Kit) displayed higher levels of chlorophyll content compared to Kitaake.Under the stress conditions,the maximum quantum yield of photosystem II photochemistry levels was higher in OsY3IP1-GFPox/Kit than in Kitaake.The increased tolerance conferred by OsY3IP1 overexpression correlated with reduced reactive oxygen species accumulation.Our data provide new insights into the possible role of OsY3IP1 in the pathway suppressing photooxidative damage under stress conditions.These features can be further exploited to improve saline and alkaline tolerances of rice plants in future.

Key words:alkaline;reactive oxygen species;rice;saline;stress tolerance

Rice is one of the most important food crops in the world,feeding more people than any other crops.The enhanced growth of rice plants with the same amounts of nutrients and water contributes to improved yield potential.Plant growth depends on the photosynthetic activity derived from cooperating photosystem I (PSI) and photosystem II (PSII) complexes (Peltier et al,2006;Yu et al,2018).PSI is a large pigment-protein complex in the thylakoid membrane of cyanobacteria,green algae and plants (Jordan et al,2001;Nelson and Ben-Shem,2004;Amunts and Nelson,2009;Nellaepalli et al,2021).PSI consists of two subcomplexes,the PSI core complex and the light-harvesting complex I,and catalyzes the sunlight-driven transmembrane electron transport (Yang et al,2015).In higher plants,it is made up of at least 15 core subunits,4 different light-harvesting complex proteins,and many numbers of cofactors (Albus et al,2010;Qin et al,2015;Yang et al,2015).

Although the PSI structure and composition have been studied for decades,little is known about the PSI complex assembly at the membrane (Nelson and Junge,2015;Yu et al,2018).Several assembly factors have been reported,and one of them is hypothetical chloroplast reading frame number 3 (Ycf3) encoded by a chloroplast gene (Boudreau et al,1997;Ruf et al,1997).In tobacco (Nicotianatabacum) and green alga (Chlamydomonas reinhardtii),ycf3knockout mutants completely lack PSI complexes,demonstrating that it is essential for the PSI complex biogenesis (Boudreau et al,1997;Ruf et al,1997).Now it is believed that Ycf3 helps the proper folding of the newly synthesized PSI subunits and mediates the PSI complex biogenesis and assembly in the thylakoid membrane (Naver et al,2001;Albus et al,2010;Krech et al,2012;Yang et al,2015).The Ycf3 protein interacts with a nucleus-encoded thylakoid protein,Ycf3-interacting protein (Y3IP1),inArabidopsisand tobacco (Albus et al,2010).Recently,in vivofunction of Y3IP1 for the assembly process of the PSI complex was elucidated (Nellaepalli et al,2018,2021).Ycf3 and another chloroplast-encoded protein,hypothetical chloroplast reading frame number 4 (Ycf4),form the module mediating PSI assembly.The initial module consists of Ycf3 and Y3IP1,and mainly facilitates the assembly of reaction center subunits.

In addition to the involvement of in the very early stage of PSI complex assembly,functions of Y3IP1 in stress tolerance have been reported.For example,Arabidopsisy3ip1mutant is characterized by retarded growth,delayed development,light-green leaf color,and reduced PSI accumulation (Albus et al,2010).A rice FOXArabidopsisline R07303 overexpressing a rice full-length cDNA AK068219 (OsY3IP1) has been isolated by screening theArabidopsislibrary under salinity stress (Kondou et al,2009;Yokotani et al,2011).TransgenicArabidopsisplants overexpressing rice andArabidopsisY3IP1genes display increased tolerance to multiple environmental stresses,leading the gene to be named chloroplast protein-enhancing stress tolerance (CEST) (Yokotani et al,2011).Recently,apple Y3IP1 protein (MdY3IP1) was isolated,and its ectopic expression inArabidopsistriggers early-flowering and enhances saline-tolerance (Yu et al,2018).However,little is known about the molecular mechanism explaining the stress tolerance phenotypes observed in its ectopic expression plants.Furthermore,the stress tolerance has not been examined on important monocotyledon crops such as rice plants.

Salinity and alkalinity cause serious damage in plants,inducing remarkable reactive oxygen species (ROS) accumulation,photooxidative damage and chlorophyll loss (Kura-Hotta et al,1987;Mittler,2002;Takahashi and Murata,2008;Baxter et al,2014;Zhang et al,2017;Yu et al,2018;Sun et al,2019).Therefore,the stresses are two major constraints inhibiting crop production worldwide.It is estimated that approximately 950 million hectares of the global land surface are affected by salt and alkaline (Guo et al,2017).Because elevated soil pH is much more destructive to plants,negative effect of soil alkalinization is greater than that of soil salinization (Fan et al,2013;Liu et al,2017).In order to effectively improve crop saline and alkaline tolerance,it is essential to first understand how plants react and adapt to the stress effects.In this study,we aimed to understand how possible alteration at the early stage of PSI complex formation by Y3IP1 overexpression affects in a broad range of abiotic stress responses,particularly saline (NaCl) and alkaline (Na2CO3) stresses,in rice.

RESULTS

Salt-tolerant phenotype of FOX-rice line CE175 overexpressing rice Y3IP1 gene

To examine whether the full-length cDNA over-expresser (FOX) rice line,CE175,with integrated AK068219 cDNA displays improvement in the salt tolerance conferred byOsY3IP1,the rice cDNA introduced into CE175 was determined by nucleotide sequence analysis of the DNA fragment amplified with specific primers for the Ubi promoter and Nos terminator according to Ichikawa et al (2006).As expected,the characterized sequence corresponded to a rice full-length cDNA clone AK068219 (OsY3IP1) (Fig.S1-A).The transcript levels of theOsY3IP1in CE175 were significantly enhanced compared to its background variety Nipponbare (Fig.S1-B).We tested the effects of salinity stress on seedling growth of CE175 and Nipponbare.When grown in standard 1/2 MS (Murashige and Skoog) agar medium including 150 mmol/L NaCl for 4 d,the inhibition of shoot growth was not significantly reduced in CE175 (Fig.1-A and -C).However,on the same agar medium,the root growth inhibition in CE175 was less severe than that in Nipponbare (Fig.1-A and -B),suggesting that the salt tolerance in root growth of CE175 could be caused byOsY3IP1overexpression.All the putative CE175 seedlings analyzed in Fig.1 were genotyped with primers,HygB_For and HygB_Rev,and turned out to be transgenic rice carryingOsY3IP1(Fig.S1-C).

Isolation and sequence analysis of OsY3IP1 gene

To further characterizeOsY3IP1,we identified and cloned theOsY3IP1gene from Kitaake,a model rice variety with a short life cycle (Li et al,2017).OsY3IP1amplified from Kitaake displayed 100% identity with that from Nipponbare (Fig.S2-A).OsY3IP1 encodes predicted protein of 292 amino acids with estimated molecular masses of 31.7 kDa.The OsY3IP1 protein shared significant homology with various Y3IP1 family members fromSetaria italic,Brachypodium distachyon,Panicum miliaceum,Zea mays,Sorghum bicolor,Ananas cosmosus,Vitis vinifera,Malus domestica,Arabidopsis thaliana,andNicotiana tabacum(Fig.2).The TargetP-2.0 and ChloroP 1.1 predicted that OsY3IP1 carries chloroplast transfer peptides (red solid box in Fig.2) but not a cleavable signal peptide in its N-terminals.Conserved domain analysis using the SOSUI 1.11 revealed that OsY3IP1 contains a transmembrane domain,which is highly conserved in the C-terminal region (red dashed box in Fig.2).A phylogenetic tree of Y3IP1 family members displayed that OsY3IP1 is clustered into a subgroup of monocot Y3IP1s fromZ.mays,S.bicolor,P.miliaceum,B.distacgyonandA.cosmosus(Fig.S2-B).

OsY3IP1 gene is induced by saline and alkaline treatments

To investigate the possible involvement of OsY3IP1 in saline and alkaline stresses,its transcript levels were examined in Kitaake after saline and alkaline treatments.In the salinity treatment (250 mmol/L NaCl),ascorbateperoxidase 8(APX8) used as a positive control was induced,indicating that saline treatment was successful (Fig.3-A).Expression ofOsY3IP1was examined with specific primers,CEST_RT_F and CEST_RT_R.The transcriptional level ofOsY3IP1was also graduallyincreased at 12 and 24 h after NaCl treatment.In the alkaline treatment (15 mmol/L Na2CO3),thecysteine protease 1(OsCP1) gene was used as a positive control for Na2CO3treatment (Fig.3-B).OsY3IP1was induced at 24 h after Na2CO3treatment.These results indicated that the expression ofOsY3IP1is clearly up-regulated by saline and alkaline stresses in rice plants.

OsY3IP1 is specific to photosynthetic tissues and localized in chloroplasts

We analyzed the expression ofOsY3IP1in various tissues (Fig.S3) of both 10-and 3-week-old Kitaake plants by reverse transcripts polymerase chain reaction (RT-PCR) using specific primers,CEST_RT_F/R (Fig.4-A).The transcriptswere detected at high levels in leaves from the main tiller,subtiller and stem tissues,but at significantly low levels in the roots and anthers.The photo-synthetic tissue-specific expression ofOsY3IP1was also observed in 3-week-old plants.Web-based prediction tools (TargetP-2.0,ChloroP 1.1 and SOSUI 1.11) suggested that OsY3IP1 carries a chloroplast transfer peptide in its N-terminal region (Fig.2) and is located at the thylakoid of chloroplasts.To experimentally determine the OsY3IP1 location,its subcellular localization was examined after PEG-mediated transient expression of OsY3IP1 proteins fused with a C-terminal GFP tag (OsY3IP1-GFP) in rice protoplasts (Fig.4-B).Under fluorescence microscopy,the fluorescent signal from control GFP co-localized homogeneously throughout the cytoplasm and nucleus,but not in chloroplasts (lower panel of Fig.4-B).In contrast,most of the green fluorescentsignal from OsY3IP1-GFP was overlapped with multiple dot-like structures associated with the chlorophyll auto-fluorescence,indicating that the OsY3IP1 protein was targeted to the chloroplasts (upper panel of Fig.4-B).These results suggested that OsY3IP1 plays a role in the chlorophylls of photosynthetic tissues.

Transgenic rice plants overexpressing OsY3IP1 are generated

To confirm whether salt-tolerance phenotype of CE175 could be ascribed to overexpression of OsY3IP1 and further investigate its biological relevance,we generated transgenic rice plants (OsY3IP1-GFPox/Kit) overexpressingOsY3IP1under the control of the maize Ubi promoter (Ubi::OsY3IP1-GFP/pC1300,Fig.S4-A) in Kitaake.The putative independent transgenic lines (OsY3IP1-GFPox/Kit T0,lines 1A to 11A) were subjected to PCR-based selection using the specific primers,Ubi_pro_junction_F and CEST_RT_R,and 10 out of the 11 lines,except line 11A,generatedOsY3IP1-GFP-specific 971-bp amplicon (Fig.S4-B).Of the 10 transgenic lines,the segregation ratio in the T1progenies of the self-pollinated T0transgenic plants,1A,5A and 9A,suggested single copy integration of the transgene,Ubi::OsY3IP1-GFP/pC1300 (Fig.S4-C).Overexpression ofOsY3IP1-GFPwas assessed in OsY3IP1-GFPox/Kit T1progeny from 1A,5A and 9A using qRT-PCR withOsY3IP1specific primers,CEST_q1 and CEST_RT_R and western blot analysis with an anti-GFP antibody (Fig.5),respectively.In qRT-PCR analysis,significant up-regulation ofOsY3IP1-GFPwas observed in all the tested transgenic lines (T1),1A-4,5A-6 and 9A-13 (Fig.5-A).The T2progenies (1A-4-12,5A-6-12 and 9A-13-18) of these lines displayed high accumulation level of the recombinant protein,OsY3IP1-GFP (Fig.5-B).These results indicated the successful generation of transgenic Kitaake plants overexpressing OsY3IP1-GFP (OsY3IP1-GFPox/Kit).

Overexpression of OsY3IP1 delays chlorophyll loss under saline and alkaline stresses

To compare the chlorophyll loss of OsY3IP1-GFPox/Kit and Kitaake control under saline and alkaline stresses,chlorophyll retention assay using floating leaf disk was performed,and total chlorophyll content was quantified.Before NaCl treatment,there were no significant difference in chlorophyll content of leaf disks from OsY3IP1-GFPox/Kit and Kitaake (Fig.6-A).After incubation on 300 mmol/L NaCl for 8 d,however,the chlorophyll contents in Kitaake decreased to 20.5% of those before treatment,whereas the chlorophyll contents in OsY3IP1-GFPox/ Kit (T2progenies from lines 1A and 9A) were significantly less decreased to 62.6% and 50.1%,respectively.The chlorophyll contents were also analyzed after alkaline treatment (Fig.6-B).After incubated in distilled water (0 mmol/L Na2CO3) for 3 d,no significant difference was observed in the chlorophyll contents from OsY3IP1-GFPox/Kit (T1progenies from lines 1A and 9A) and Kitaake.However,when 30 mmol/L Na2CO3was treated for 3 d,more chlorophyll was conserved in the OsY3IP1-GFPox/Kit compared with Kitaake.The chlorophyll content in Kitaake treated with Na2CO3decreased to 12.9% of those from non-treated Kitaake after 3 d,whereas the chlorophyll contents in OsY3IP1-GFPox/Kit were decreased to 46.4% and 57.0%,respectively.These results indicated thatOsY3IP1overexpression exhibited greater capacity for maintaining chlorophyll under saline and alkaline stresses.

OsY3IP1-overexpressing rice plants display increased photosynthetic activity

Damage to PSI would lead to further inhibition of electron transfer and aggravate the extent of damage on PSII (Sonoike,2011;Zhang et al,2012).Because Y3IP1 is involved in the PSI complex assembly (Nellaepalli et al,2018),we investigated whether stress tolerance observed in OsY3IP1-overexpressing plants was related with photosynthetic activity within photosystems.After 300 mmol/L NaCl treatments for 3 d,the ratio of maximum quantum yield of PSII photochemistry (Fv/Fm) in Kitaake plants was significantly decreased,reflecting a decline in PSII function.By comparison,the salinity-induced decrease ofFv/Fm was suppressed in all OsY3IP1-GFPox/Kit T2lines (1A-21-5,5A-6-7 and 9A-15-5) (Fig.7-A).Treated with high concentration of Na2CO3(30 mmol/L),the photosynthetic efficiency (Fv/Fm) of the Kitaake plants also dropped within 1 d and remained low for 3 d.However,the decrease ofFv/Fm caused by alkalinity stress was suppressed in all the tested transgenic lines (T2,1A-21-5,5A-6-7 and 9A-15-5) (Fig.7-B).These data showed that OsY3IP1-overexpressing Kitaake plants displays increased photosynthetic activity.

OsY3IP1 expression reduces ROS accumulation

To explore possible changes of ROS accumulation in OsY3IP1-GFPox/Kit transgenic lines,we compared hydrogen peroxide (H2O2) levels in Kitaake and transgenic plants by histochemically staining of leaves with 3,3′-diaminobenzidine (DAB) after saline (Fig.8-A and -B) and alkaline treatments (Fig.8-C and -D).After treatment of 700 mmol/L NaCl,a majority of the area on the leaf fragments from Kitaake was stained dark brown,indicating excessive accumulation of H2O2(Fig.8-A).However,leaf fragments from OsY3IP1-GFPox/Kit T1lines (1A-4,5A-6 and 9A-15) displayed much less accumulation of H2O2compared with Kitaake controls.Quantitative image analysis showed significantly lower H2O2accumulation compared to Kitaake plants under NaCl treatment (Fig.8-B).Treatment of 50 mmol/L Na2CO3also induced much less H2O2in OsY3IP1-GFPox/Kit T1lines compared to Kitaake plants (Fig.8-C and -D).These results suggested that ROS accumulation caused by saline and alkaline stresses is reduced in OsY3IP1-overexpressing transgenic plants,resulting in the enhanced saline and alkaline tolerance.

DISCUSSION

Sophisticated mechanisms have evolved in plants to adapt to the various environmental stresses.Understanding these mechanisms increases the yield potential of crop plants.Previous studies in dicotyledon plants,Arabidopsisand tobacco,have shown that Y3IP1 protein plays a pivotal role in plant responses to a number of abiotic stresses (Albus et al,2010;Yokotani et al,2011;Nellaepalli et al,2018;Yu et al,2018).However,most of the studies have been done on model systems,not on important monocotyledon crops such as rice plants.Although transgenicArabidopsisoverexpressingOsY3IP1displays improved tolerance to salinity stress (Yokotani et al,2011),the salt tolerance has not been examined in rice plants.In addition,whether transgenic rice plants overexpressingOsY3IP1display tolerance to multiple stresses has not been investigated.

Y3IP1 is a nucleus-encoded chloroplast protein that is post-translationally translocated into chloroplasts to assemble PSI complex (Albus et al,2010;Yu et al,2018;Nellaepalli et al,2021).Consistent with previous reports,OsY3IP1was specifically expressed in primary photosynthetic tissue and localized to the chloroplasts in rice protoplasts.Yokotani et al (2011) reporte thatY3IP1genes from rice andArabidopsisare not drastically affected by any stress treatment.However,in our study,OsY3IP1gene was apparently induced under salinity and alkaline stresses in rice plants,which was confirmed with stress marker genes,APX8andOsCP1.This suggested thatOsY3IP1should be transcriptionally regulated and involved in responses to abiotic stresses.Saline and alkaline stresses often result in the breakdown of chlorophyll in chloroplasts (Xing et al,2013;Srivastava et al,2016).Transgenic rice plants overexpressingOsY3IP1,OsY3IP1-GFPox/Kit,retained more chlorophyll than Kitaake under high saline and alkaline stresses.Therefore,it is obvious thatOsY3IP1overexpression suppresses the reduction of the chlorophyll content in chloroplasts damaged by saline and alkaline stresses in rice leaves.

Chlorophyll is an important part of two photosystem,PSI and PSII,on the thylakoid membrane and its content directly reflects the photosynthetic efficiency and assimilation capacity (Xing et al,2013).Therefore,it is reasonable to assume thatOsY3IP1-overexpressing plants maintain more photosynthetic activity due to less damaged chlorophyll.Several parameters representing photosynthetic activity are less decreased in transgenicArabidopsisplants overexpressingY3IP1s after saline treatment (Yokotani et al,2011).In addition,appleY3IP1-overexpression inArabidopsisstrongly and specifically increases PSI accumulation (Yu et al,2018).Consistent with the reports,a photosynthetic parameterFv/Fm in OsY3IP1-GFPox/Kit plants mitigates the decrease of the parameter under salinity and alkalinity stresses.Because reduction ofFv/Fm reveals a defect in energy transfer within the photosystem and often indicates the extension of damage to PSII (Baker,2008),these results suggested that OsY3IP1 protein can confer saline and alkaline tolerance by changing the molecular structure and/or function of the photosynthetic apparatus.

The accumulation of ROS is connected to physiological balance,and any fluctuations in ROS level can disturb the normal function of cellular machinery (Miller et al,2010;Suzuki et al,2012).In the histochemical detection of ROS accumulation with DAB staining,OsY3IP1-overexpressing transgenics maintained relatively lower ROS accumulation when Kitaake displayed an enormous increase in ROS level in response to the saline and alkaline stresses.This suggested that OsY3IP1 might directly or indirectly inhibit detrimental ROS accumulation caused by abiotic stress.The reduction of ROS accumulation is also observed in transgenicArabidopsisoverexpressing appleY3IP1(Yu et al,2018).Therefore,it seems thatY3IP1overexpression enhanced stress tolerance by reducing ROS production.However,the molecular mechanism how Y3IP1 involves in the photosystem and reduces ROS accumulation should be more elucidated further.

Difficult questions remain as to why and how alteration at the early stage of PSI complex formation by Y3IP1 affects a broad range of environmental stresses.Although stress-tolerant plants display complex molecular responses,the processes generally consume lots of adenosine triphosphate (ATP) (He et al,2015).The ATP is the most important product of photosynthesis composed of PSI and PSII within plant chloroplasts.One of the most important events around PSI is linear and cyclic electron transport in the light reactions of photosynthesis (Yamori and Shikanai,2016).Linear electron transport of photosynthesis generates both ATP and NADPH,whereas PSI cyclic electron transport produces ATP without NADPH.When plant cells suffer from stress,PSI cyclic electron transport needs to be upregulated to optimize the redox state of the cell and satisfy the high ATP requirement (Shikanai,2007;Yamori and Shikanai,2016).Supporting these results,extensive additional investigations elucidated thatin vivofunction of PSI cyclic electron transport is involved in the response to various environmental stresses such as strong light (Takabayashi et al,2002;DalCorso et al,2008;Nishikawa et al,2012),low humidity (Horvath et al,2000),drought (Munne-Bosch et al,2005;Long et al,2008),rapid temperature changes (Wang and Portis,2007),and low and high temperatures (Wang et al,2006;Yamori et al,2011).Therefore,further study will be focused on a possible connection between PSI assembly promoted by accumulated OsY3IP1 and PSI cyclic electron transport.

In this study,a FOX-rice line CE175 integrated with rice full-length cDNA clone AK068219 displayed salinity tolerance.From rice variety Kitaake,OsY3IP1corresponding to the AK068219 was isolated and characterized.We showed that overexpression ofOsY3IP1in Kitaake plants conferred salinity and alkalinity tolerance phenotype without apparent abnormal growth,suggesting its usefulness for molecular breeding.

METHODS

Rice materials

Twojaponicarice varieties,Kitaake and Nipponbare,were used as the wild type.Rice seeds sterilized with 70% ethanol and 40% sodium hypochlorite solution were germinated in sterile water at 23 °C for 7 d and then transferred to the greenhouse facility at Sejong University in Korea.Healthy and well-expanded leaves from 8-week-old plants were used for all experiments,unless otherwise indicated.

Vector construction

Full-length complementary DNA (cDNA) ofOsY3IP1was amplified from Kitaake cDNA by PCR usingOsY3IP1-specific primers,Os01g0798500_FL_F (5′-ATGGCGCTGCTCTCACC ACC-3′) and CESTw/o stop_FL_R (5′-CCCCTGTAGAGAAT TGTAGAAAAGA-3′).The PCR amplified product was cloned into pCR8TM/GW/TOPO?vector (Invitrogen,CA,USA) to create an OsY3IP1/pCR8 vector.The overexpression construct,Ubi::OsY3IP1-GFP/pC1300,was constructed by recombining the OsY3IP1/pCR8 construct into the gateway vector Ubi::GFP/CAMBIA1300 (Rohila et al,2006;Park et al,2017) through a Gateway?LR clonase (Invitrogen,CA,USA) reaction.

Rice transformations

Rice transformations were carried out as previously described using Kitaake (Chern et al,2005).Agrobacterium tumefaciensstrain LBA4404 was used to infect callus tissue induced from Kitaake seeds.Transformants carrying Ubi::OsY3IP1-GFP/ pC1300 constructs were selected onhygromycin(50 mg/L) and confirmed by PCR using specific primers,Ubi_pro_junction_F (5′-ACATCTTCATAGTTACGAGTTT-3′) and CEST_RT_R (5′-TCTCCTTCGCAAGCAACTGA-3′).

Seedling growth assay

Seedling growth assay was carried out after saline treatment on the seedlings of FOX-rice line CE175 as previously reported (Zeng et al,2016).For saline treatment,putative transgenic CE175 seeds were selected on 1/2 MS agar medium (Duchefa Biochemie,Haarlem,Netherlands) containing 50 mg/Lhygromycin,and Nipponbare seeds were germinated on 1/2 MS agar medium at 23 °C for 3 d and then transferred to vertically standing 1/2 MS agar medium containing 150 mmol/L NaCl.At 4 d after treatment,shoot and root lengths were measured.

Sequence alignment and gene accession numbers

Deduced amino acid sequence of OsY3IP1 was aligned with amino acid sequences of Y3IP1 homologues obtained from NCBI/EMBL databases:Z.mays(GRMZM2G002165),S.bicolor(Sb03g037100),P.miliaceum(PM08G28210),S.italic(5G348300v2),O.sativa(LOC_Os01g58470),B.distachyon(Bradi2g52400),A.cosmosus(Aco002105.1),V.vinifera(GSV IVT01037088001),M.domestica(MDP0000930948),N.tabacum(TC52264),andA.thaliana(At5g44650).Sequence alignment was performed using DNASTAR MegAlign by ClustalW program (Ver.5.01).Chloroplast transfer peptide was carried by the TargetP-2.0 (http://www.cbs.dtu.dk/services/TargetP/) and ChloroP 1.1 (http://www.cbs.dtu.dk/services/ChloroP/),and the conserved domain analysis was test by the SOSUI 1.11 (http://harrier.nagahama-i-bio.ac.jp/sosui/).Other sequences in this study can be found in the GenBank/EMBL database under the following accession numbers:rEF1α(LOC_Os03g08020),APX8(LOC_Os02g34810),andOsCP1(LOC_Os04g57490).

RT-PCR analysis

Total RNA was isolated from fully expended leaves of Kitaake using the TRIzol reagent (Invitrogen,CA,USA) as per manufacturer instructions.We quantified RNA using a NanoDrop?ND-1000 spectrophotometer (Thermo Fisher Scientific,MA,USA).For the first-strand cDNA synthesis,total 2 μg RNA was reverse-transcribed in a total volume of 25 μL reaction using M-MLV reverse transcriptase (Promega,MA,USA).For RT-PCR,the expression ofOsY3IP1was confirmed using specific primers,CEST_RT_F (5′-GAGGGAGAAGGG GATTCCGA-3′) and CEST_RT_R (5′-TCTCCTTCGCAAGC AACTGA-3′).The rice elongation factor 1α (rEF1α)cDNA fragment was amplified as a constitutive internal control using specific primers,rEF1a1048F (5′-ACTGCCACACCTCCCAC ATTG-3′) and rEF1a1552R (5′-CAACAGTCGAAGGGCAAT AATAAGTC-3′).qRT-PCR analysis was performed using SYBR Green I dye (NEXpro,South Korea) in a LightCycler?96 real-time PCR machine (Roche,Basel,Switzerland) with the following conditions:95 °C for 10 s,55 °C for 10 s,45 cycles.Amplicon specificity was confirmed by checking the melting curve at the completion of 45 cycles.TherEF1αwas used as an internal standard using specific primers,rEF-1a_F (5′-TTTCACTCTTGGTGTGAAGCAGAT-3′) and rEF-1a_R (5′-GACTTCCTTCACGATTTCATCGTAA-3′).RelativeOsY3IP1gene expression was examined with specific primers,CEST_q1 (5′-CCAGGTCAAAAGGGTGCTTG-3′) and CEST_RT_R (5′-TCTCCTTCGCAAGCAACTGA-3′) and normalized torEF1αexpression.Saline treatment was confirmed with positive controlAPX8with specific primers,APX8_F (5′-TG GTCTGATGACCTCCTCTGA-3′) and APX8_R (5′-CATGAG CCATGACAACTAGA-3′) (Hong et al,2007;Shafi et al,2015).Alkaline treatment was confirmed with positive controlOsCP1by RT-PCR using specific primers OsCP1_RT_F (5′-CGATC AAGAAGGGGCCCAAC-3′) and OsCP1_RT_R (5′-CAGCAG GTGGAGTGATCCTT-3′) (Zhang et al,2017).

Expression analysis

OsY3IP1expression analyses were carried out after saline and alkaline treatments as previously described (Srivastava et al,2016;Zhang et al,2017).For saline stress,leaves from 4-week-old Kitaake were cut into approximately 2.5 cm pieces and treated with 250 mmol/L NaCl solution.For alkaline stress,4-week-old Kitaake leaves were cut into 2.5 cm pieces and treated with 15 mmol/L Na2CO3solution (pH 10.8).To determine theOsY3IP1gene expression,we sampled leaf pieces at 0,6,12 and 24 h after treatment with NaCl and Na2CO3.For RNA extraction,samples were collected immediately,fixed in liquid nitrogen,and stored in a deep-freezer at -80 °C.

Rice protoplast isolation and polyethylene glycol (PEG)-mediated transformation

To observe the cellular localization of OsY3IP1,we used the Ubi::OsY3IP1-GFP/pC1300 construct which had been generated for the overexpression of OsY3IP1-GFP in rice plants.Vector 326-GFP was used as free GFP control (Lee et al,2001).To transiently express OsY3IP1-GFP in rice protoplasts,the plasmid was transformed into protoplasts of 10-day-old rice seedlings through PEG-mediated transformation method as previously described (Bart et al,2006).The expression of the OsY3IP1-GFP construct was visualized 16-20 h after transformation,and images were captured using an inverted fluorescence microscope Eclipse Ti (Nikon,Tokyo,Japan) fitted with an objective (400×).The filter sets used were C-FL-C FITC (excitation 465 nm to 495 nm) and C-FL-C DAPI (excitation 361 nm to 389 nm) (Nikon,Tokyo,Japan) to detect GFP and the chlorophyll autofluorescence,respectively.

Plant protein extraction and western blotting

Total protein was extracted from rice plants using a protein extraction buffer (6.8 mmol/L Na2HPO4,3.2 mmol/L NaH2PO4,150 mmol/L NaCl,2 mmol/L EDTA,20 mmol/L NaF,1.0% Triton X-100,and 1 mmol/L phenylmethylsulfonyl fluoride).Proteins (100 μg) were separated by 8.0% sodium dodecyl sulfate polyacrylamide gel electrophoresis.For OsY3IP1-GFP detection,anti-GFP mouse antibody (B-2) SC-9996 (SantaCruz biotecnology,CA,USA) and anti-mouse IgG,horseradish peroxidase linked whole antibody SC-2031 (SantaCruz biotecnology,CA,USA) were used as a primary and a secondary antibody at a final dilution of 1:1 000 and 1:10 000 for 2 h,respectively.GFP bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific,MA,USA) and Azure BiosystemsTMc280 (Azure Biosystems,CA,USA) according to the standard protocols.

Chlorophyll retention assay and chlorophyll fluorescence measurements

Chlorophyll retention was calculated using floating rice leaf disks according to Srivastava et al (2016) with minor modification.Rice leaf segments were excised and floated on 1/2 MS containing 300 mmol/L NaCl and 3 mmol/L 2-(N-morpholino)-ethanesulfonic acid (Biobasic,MA,USA),and 30 mmol/L Na2CO3for 8 and 3 d,respectively.After NaCl treatment,the leaf segments were immersed in extract solution (pure acetone) at 4 °C until the leaves were completely bleached (Gerona et al,2019).The total amount leaf chlorophyll content (chlorophyll a and chlorophyll b) was calculated by measuring the absorbance at 645 and 662 nm.Total Chl (μg/mL,pure acetone)=18.09A645+7.05A662,whereApresents absorbance in 1.00 cm cuvettes.The leaf segments from Na2CO3treatment were immersed in extract solution (95% ethanol) at room temperature until the leaves were completely bleached (Inskeep and Bloom,1985;Gerona et al,2019).The total amount leaf chlorophyll content was calculated by measuring the absorbance 647 and 665 nm.Total Chl (μg/mL,95% ethanol)=22.24A647+5.24A665.

Chlorophyll fluorescence of rice leaves was determined by a hand-held PAM-fluorometer (FluorPen P100,Czech Republic) (Baker,2008;?iv?ák et al,2008;Yokotani et al,2011).Eight-week-old rice leaves were cut into 1.5 cm-length and treated with 300 mmol/L NaCl and 30 mmol/L Na2CO3solution (pH 10.8),respectively.The leaf segments were sampled at 0,1,2 and 3 d after each treatment.Each segment was placed in leaf clip holder and dark-adapted for 15 min before determiningFv/Fm.

DAB staining

DAB staining for H2O2detection was performed according to Kaur et al (2016).To examine the accumulation of H2O2in rice leaves,approximately 2.5 cm leaf segments were cut and floated on distilled water containing 700 mmol/L NaCl or 50 mmol/L Na2CO3for 3 d,respectively.After each treatment,leaf segments were soaked in a 1% solution of DAB in 10 mmol/L sodium phosphate diabasic buffer (pH 7.0).After performing vacuum infiltrating twice for 10 min,the immersed leaves were incubated in the dark for 20 h at 28 °C.The leaves were destained in 2 mL of boiling destaining solution (1.5 mL of 95% ethanol and 0.5 mL of 10% glycerol) until the chlorophyll disappeared clearly.

Statistical analysis

All assays described above were repeated at least three times.All data are presented as mean and standard error.A one-way analysis of variance (ANOVA) was used to compare the treatment means.Means were compared at the 0.05 or 0.01 levels according to the Tukey’s test.SigmaPlot 11.0 (Systat Software Inc,United States) was used for graphical presentation of the data.

ACKNOWLEDGEMENTS

This study was supported by the National Research Foundation of South Korea (Grant Nos.NRF-2020R1A2C1007778 and 2015K2A2A4000129).We thank Professor Pamela RONALD (University of California Davis,USA),Dr.Hiroaki ICHIKAWA (National Agriculture and Food Research Organization Institute of Agrobiological Science,Japan) for providing Kitaake and FOX-rice lines,respectively,and Professor David HINDMAN (Sejong University,South Korea) for critical reviewing our manuscript.

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science;http://www.ricescience.org.

Fig.S1.FOX-rice line,CE175,integrated withOsY3IP1.

Fig.S2.Sequence comparison ofOsY3IP1from Nipponbare and Kitaake,and phylogenetic relationships of Y3IP1 homologues from selected plant species.

Fig.S3.Expression ofOsY3IP1in various tissues harvested from 10-and 3-week-old Kitaake plants.

Fig.S4.Generation and molecular analysis of transgenic lines overexpressingOsY3IP1.