Correction

PLANT BIOLOGYCorrection for “Sugar starvation-regulated MYBS2 and 14-3-3protein interactions enhance plant growth, stress tolerance, andgrain weight in rice,” by Yi-Shih Chen, Tuan-Hua David Ho,Lihong Liu, Ding Hua Lee, Chun-Hua Lee, Yi-Ru Chen,Shu-Yu Lin, Chung-An Lu, and Su-May Yu, which was firstpublished October 8, 2019; 10.1073/pnas.1904818116 (Proc. Natl.Acad. Sci. U.S.A. 116, 21925–21935).The authors note that an additional affiliation should be listed

for Tuan-Hua David Ho and Su-May Yu. The new affiliationshould appear as Biotechnology Center, National Chung HsingUniversity, Taichung, 402 Taiwan, Republic of China. Thecorrected author and affiliation lines appear below. The onlineversion has been corrected.

Yi-Shih Chena,b, Tuan-Hua David Hoc,d, Lihong Liua, DingHua Leec, Chun-Hua Leeb, Yi-Ru Chena, Shu-Yu Line,Chung-An Lub, and Su-May Yua,d

aInstitute of Molecular Biology, Academia Sinica, Nankang, 115 Taipei,Taiwan, Republic of China; bDepartment of Life Sciences, National CentralUniversity, Jhongli City, 320 Taoyuan County, Taiwan, Republic of China;cInstitute of Plant and Microbial Biology, Academia Sinica, Nankang, 115Taipei, Taiwan, Republic of China; dBiotechnology Center, National ChungHsing University, Taichung, 402 Taiwan, Republic of China; and eInstitute ofBiological Chemistry, Academia Sinica, Nankang, 115 Taipei, Taiwan,Republic of China

Published under the PNAS license.

First published November 11, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1918414116

www.pnas.org PNAS | November 19, 2019 | vol. 116 | no. 47 | 23861

CORR

ECTION

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0 D

ownl

oade

d by

gue

st o

n O

ctob

er 2

3, 2

020

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0 D

ownl

oade

d by

gue

st o

n O

ctob

er 2

3, 2

020

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0 D

ownl

oade

d by

gue

st o

n O

ctob

er 2

3, 2

020

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0 D

ownl

oade

d by

gue

st o

n O

ctob

er 2

3, 2

020

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0 D

ownl

oade

d by

gue

st o

n O

ctob

er 2

3, 2

020

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0 D

ownl

oade

d by

gue

st o

n O

ctob

er 2

3, 2

020

Dow

nloa

ded

by g

uest

on

Oct

ober

23,

202

0

Sugar starvation-regulated MYBS2 and 14-3-3 proteininteractions enhance plant growth, stress tolerance,and grain weight in riceYi-Shih Chena,b, Tuan-Hua David Hoc,d, Lihong Liua, Ding Hua Leec, Chun-Hua Leeb, Yi-Ru Chena, Shu-Yu Line,Chung-An Lub,1, and Su-May Yua,d,1

aInstitute of Molecular Biology, Academia Sinica, Nankang, 115 Taipei, Taiwan, Republic of China; bDepartment of Life Sciences, National CentralUniversity, Jhongli City, 320 Taoyuan County, Taiwan, Republic of China; cInstitute of Plant and Microbial Biology, Academia Sinica, Nankang, 115 Taipei,Taiwan, Republic of China; dBiotechnology Center, National Chung Hsing University, Taichung, 402 Taiwan, Republic of China; and eInstitute of BiologicalChemistry, Academia Sinica, Nankang, 115 Taipei, Taiwan, Republic of China

Edited by Julia Bailey-Serres, University of California, Riverside, CA, and approved September 16, 2019 (received for review April 3, 2019)

Autotrophic plants have evolved distinctive mechanisms formaintaining a range of homeostatic states for sugars. The on/offswitch of reversible gene expression by sugar starvation/provisionrepresents one of the major mechanisms by which sugar levels aremaintained, but the details remain unclear. α-Amylase (αAmy) is thekey enzyme for hydrolyzing starch into sugars for plant growth, andit is induced by sugar starvation and repressed by sugar provision.αAmy can also be induced by various other stresses, but the physio-logical significance is unclear. Here, we reveal that the on/off switchof αAmy expression is regulated by 2 MYB transcription factors com-peting for the same promoter element. MYBS1 promotes αAmy ex-pression under sugar starvation, whereas MYBS2 represses it. Sugarstarvation promotes nuclear import of MYBS1 and nuclear export ofMYBS2, whereas sugar provision has the opposite effects. Phosphor-ylation of MYBS2 at distinct serine residues plays important roles inregulating its sugar-dependent nucleocytoplasmic shuttling andmaintenance in cytoplasm by 14-3-3 proteins. Moreover, dehydra-tion, heat, and osmotic stress repress MYBS2 expression, therebyinducing αAmy3. Importantly, activation of αAmy3 and suppressionof MYBS2 enhances plant growth, stress tolerance, and total grainweight per plant in rice. Our findings reveal insights into a uniqueregulatory mechanism for an on/off switch of reversible gene expres-sion in maintaining sugar homeostatic states, which tightly regulatesplant growth and development, and also highlight MYBS2 andαAmy3 as potential targets for crop improvement.

rice | MYB transcription factor | sugar repression | abiotic stress tolerance |grain weight

In plants, as autotrophic organisms, a range of sugar homeostaticstates is crucial for growth regulation, environmental stress toler-

ance, and productivity, meaning that sugar status must be continu-ously monitored and elicit an appropriate reaction. An integratedsignaling network coordinates sugar status—reflecting sugar pro-duction in source tissues to its utilization or storage in sink tissues—that involves cross-talk among sugars, hormones, and environmentalcues and that regulates developmental and stress-adaptive processes(1, 2). Nearly all fundamental processes throughout the lifecycle ofplants are modulated by sugars. In general, sugar provision up-regulates genes involved in biosynthesis, transport, and storage ofreserves as well as cell growth, and it down-regulates those asso-ciated with photosynthesis, reserve mobilization, and stress re-sponses, whereas sugar starvation has the opposite effects (3–5).Upon assimilation in photosynthetic source leaves, newly fixed

carbon is utilized for cellular respiration and metabolism, tran-siently stored in vacuoles as sucrose or in plastids as starch, andtransported as sucrose to sink tissues, such as growing tissues (togenerate energy) or developing organs (for long-term storage)(1, 6). Despite sugars being of central importance to plant growth,too much of them can be detrimental. For example, ectopic ex-pression of a yeast invertase, which converts sucrose to glucose

and fructose in the apoplast, leads to decreased sucrose export andaccumulation of carbohydrates in leaves, with subsequent inhibi-tion of photosynthesis, stunted growth, impaired root formation,and necrosis in tobacco leaves (7). Rice and maize mutant linesdefective in a tonoplast sucrose transporter, SUT2, accumulatehigher concentrations of sugars in leaves but exhibit growth re-tardation and reduced biomass and grain yield, presumably due toreduced transport of sucrose out of vacuoles in source leaves tosink tissues/organs where sugar is in high demand (8, 9).Starch, which constitutes ∼75% of cereal grain dry weight (10),

acts as the major carbon source for generating energy and me-tabolites during germination and seedling growth. α-Amylase(αAmy) is the most abundant hydrolase and plays a centralrole in starch mobilization and, thus, in the rate of seedlinggrowth. Our previous studies in rice revealed that sugar starva-tion up-regulates αAmy expression by controlling its transcriptionrate and mRNA stability (3, 11, 12). All αAmy isolated fromcereals contain a TATCCA element (the TA box) or variant atpositions ∼90 to ∼150 base pairs upstream of the transcriptionstart sites (13). αAmy transcriptional regulation is mediated througha sugar response complex (SRC) in αAmy promoters, in which the

Significance

As autotrophic organisms, sugar status in plants must be con-stantly monitored and reacted to in order to maintain sugarhomeostatic states crucial for growth regulation, environmentalstress tolerance, and productivity. α-Amylase (αAmy) is the keyenzyme hydrolyzing starch into sugars and is regulated by sugarlevels; it is induced by sugar starvation but repressed by sugarprovision. Two MYBs compete for binding to the same αAmypromoter element to regulate this process, with MYBS1 pro-moting and MYBS2 repressing αAmy expression. Induction ofαAmy expression by suppressing MYBS2 enhances stress toler-ance and productivity. Phosphorylation of MYBS2 is critical forregulating its sugar-dependent nucleocytoplasmic shuttling andinteractions with 14-3-3 proteins, representing a regulatorymechanism for reversible gene expression by sugar status.

Author contributions: Y.-S.C., T.-H.D.H., C.-A.L., and S.-M.Y. designed research; Y.-S.C.performed research; L.L. and Y.-R.C. contributed new reagents/analytic tools; Y.-S.C.,T.-H.D.H., L.L., D.H.L., C.-H.L., S.-Y.L., C.-A.L., and S.-M.Y. analyzed data; and Y.-S.C., T.-H.D.H.,and S.-M.Y. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: chungan@cc.ncu.edu.tw or sumay@imb.sinica.edu.tw.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1904818116/-/DCSupplemental.

First published October 8, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1904818116 PNAS | October 22, 2019 | vol. 116 | no. 43 | 21925–21935

PLANTBIOLO

GY

TA box is a key cis-acting element (14–16). MYBS1 is a singleDNA binding repeat (R1) MYB transcription factor that interactswith the TA box and induces the αAmy promoters under sugarstarvation (17, 18). MYBS1 expression and its nuclear import ispromoted by sugar starvation, whereas sugar provision has theopposite effects (17, 19).In cereals, the stored reserves in the endosperm are degraded

and mobilized by a battery of enzymes and transporters acting inconcert, and gibberellin (GA) is the major hormone that initiatesthese processes (20). GA activates αAmy promoters through theGA response complex (GARC), in which the adjacent GA re-sponse element (GARE) and TA box are key elements that actsynergistically (14, 21). The MYBS1–TA box interaction is es-sential for GARC and SRC functions (14, 17), demonstratingthat MYBS1 is an essential node in GA and sugar starvationcross-signaling. In rice and barley, MYBGA is a GA-inducibleR2R3 MYB transcriptional factor that binds the GARE andactivates αAmy and hydrolase gene promoters in aleurone cellssurrounding the starchy endosperm (19, 22). GA antagonizessugar-mediated repression of αAmy expression by enhancingconuclear transport of MYBGA and MYBS1 and formation of astable bipartite MYB–DNA complex to activate αAmy and hy-drolase gene promoters (19).The 14-3-3 protein family is a highly conserved group of di-

meric proteins that dock onto phosphorylated serine (Ser) andthreonine (Thr) residues in their target proteins (23, 24). Inplants, target proteins of 14-3-3 proteins are involved in signaltransduction and gene regulation of various biological processes,and binding of client proteins by 14-3-3 proteins may lead toalteration in conformation, activity, stability, or intracellular lo-calization (25, 26). That 14-3-3 proteins are involved in sugarregulation has been reported for yeast cells. In Saccharomycescerevisiae, a 14-3-3 protein (Bmh1) is required for interactionwith heat-shock protein HSP70 (Ssb) to recruit a phosphatase(Glc7) that dephosphorylates and inactivates the protein kinaseSNF1, a process necessary for glucose repression (27). However,14-3-3 protein-mediated sugar regulation of gene expression hasnot been explored in plants.We previously identified another R1 MYB (MYBS2) that also

specifically binds to the TA box of αAmy promoters (17). Thefunction of MYBS2 had been unknown prior to this study. Here,by using αAmy as a biochemical marker, we show that sugarprovision and starvation counteract each other by regulating thecompetition between MYBS1 and MYBS2 for binding to the TAbox as a transcriptional activator and repressor of αAmy pro-moters, respectively. Notably, phosphorylation regulates thesugar-dependent nucleocytoplasmic shuttling of MYBS2 and itscytoplasmic interaction with 14-3-3 proteins, which plays an es-sential role in regulating the on/off switch of reversible geneexpression in response to sugar status. We also observed thatmanipulation of MYBS2 and αAmy can lead to beneficial effectson plant growth, stress tolerance, and grain productivity, repre-senting a unique approach to crop improvement.

ResultsMYBS2 Is a Negative Regulator of Germination and Plant Growth, andSuppresses αAmy Expression. We first investigated the physiolog-ical function of MYBS2 in plant growth by gain- and loss-of-function analyses in transgenic rice overexpressing either MYBS2cDNA or MYBS2 RNA interference (Ri) constructs under thecontrol of the Ubi promoter. Levels of recombinant MYBS2mRNAs increased by 52- to 60-fold in transgenic seedlings of2 overexpressing (Ox) lines, whereas endogenousMYBS2mRNAdecreased by 50 to 70% in 2 silencing (Ri) lines, relative to thesegregated WT (sWT) (Fig. 1A). We compared the phenotypesof these transgenic lines to sWT and found that germinationrates in 2 Ox lines were reduced by 34 to 59%, but were un-changed in the Ri lines (Fig. 1B). Moreover, seedling growth up

to 14 d was delayed in Ox lines but remained unchanged in theRi lines (Fig. 1 C and D). Plant height at 90 d (i.e., directly beforeheading) was also shorter in Ox lines, but similar to sWT in Rilines (Fig. 1 E and F). The seed size of Ox lines is not differentfrom that of the sWT.To determine whether αAmy expression is regulated by

MYBS2, we first cultured embryo calli of MYBS2 Ox and Ri linesin a medium without sugar (−S), and then shifted cells to amedium with sugar (+S). Levels of αAmy3 and aAmy8 mRNAsgradually decreased with time in sWT, and were rapidly reducedin an Ox line, but not significantly altered in an Ri line (SI Ap-pendix, Fig. S1). We also observed that αAmy3 expression in riceseedlings was suppressed in Ox lines (by 40 to 60%), but wasactivated in RNAi lines (by 5- to 6-fold) (Fig. 2A).

MYBS2 Competes against MYBS1 for Binding to the TA Box andRepresses αAmy Promoter under Sugar Starvation. To understandthe mechanism of MYBS2-mediated sugar repression of αAmyexpression, we assessed the effect of MYBS1, MYBS2, andMYBS2(Ri) (expression driven by the Ubi promoter) (Fig. 2B) onthe activity of promoters containing the αAmy3 SRC and 6 tandemrepeats of the TA box (6xTA) individually fused to the CaMV35Sminimal promoter using a rice embryo transient expression sys-tem. Our results showed that the activity of the 2 promoters wasenhanced byMYBS1, and was even more significantly enhanced bythe MYBS2 Ri construct, but was repressed by MYBS2 in −Smedium (Fig. 2C). These gain- and loss-of-function analyses sug-gest that MYBS2 is a transcriptional repressor and that it offsetsthe transcriptional transactivation activity of MYBS1 on the TAbox of αAmy3 SRC promoters under sugar starvation.We investigated the effect of different MYBS1:MYBS2 ratios

on the activity of the 6xTA promoter using effector and reporterconstructs and the rice embryo transient expression system (SIAppendix, Fig. S2A). We found that promoter activity was con-siderably enhanced upon transfection of a fixed amount of MYBS2

Fig. 1. MYBS2 is a negative regulator of germination and plant growth.sWT and transgenic rice lines overexpressing (Ox) or underexpressing (Ri)MYBS2 were used in the experiment. (A) Total RNA were extracted fromleaves of 7-d-old seedlings and subjected to qRT-PCR analysis. The Insetshows comparison of MYBS2 mRNA levels between sWT and Ri lines. (B)Transgenic seeds were germinated in −S medium at 28 °C for 5 d, beforedetermining germination rates. Error bars represent SD. Asterisks indicatesignificant differences (Student’s t test, ***P < 0.001). (C and D) Two-day-oldseedlings of sWT and transgenic lines with similar shoot lengths were grownin medium for up to 14 d. Seedling growth was determined by measuringshoot length. (E and F) Plants in C and D were transferred to a greenhousefor continuous growth and the morphology of 90-d-old plants was assessed.n = 30 for all experiments.

21926 | www.pnas.org/cgi/doi/10.1073/pnas.1904818116 Chen et al.

but increasing amounts of MYBS1 in both +S and −S media (SIAppendix, Fig. S2B), and the same was true for fixed amounts ofMYBS1 but increasing amounts of MYBS2(Ri) (SI Appendix, Fig.S2C). Together, these data indicate that MYBS1 and MYBS2compete against each other for binding to the 6xTA promoter.

Nuclear Import of MYBS2 Is Promoted by Sugar Provision. To in-vestigate if MYBS2 function is regulated at the subcellular level,we expressed CaMV35S:MYBS2-GFP and CaMV35S:GFP intransgenic rice and examined images of GFP signal in root tips.GFP by itself localized to both the nucleus and cytoplasm, re-gardless of whether +S or −S medium was used, whereas MYBS2-GFP was found in the nuclei of root cells incubated in +S mediumbut in the cytoplasm of those cultured in −S medium (Fig. 3A andSI Appendix, Fig. S3).We also investigated sugar-mediated regulation of the sub-

cellular localization of MYBS2 based on the spatial dynamics ofMYBS2-GFP using a barley aleurone transient expression sys-tem (19, 28). As MYBS2-GFP signal was distributed throughoutdifferent focal planes of the aleurone cells, we prepared 30 op-tical sections for each cell (SI Appendix, Fig. S4), but only 5regularly spaced sections representing each cell are shown in Fig.3B. MYBS2 was mainly localized (∼70%) in the nucleus in +Smedium, but predominantly (∼84%) in the cytoplasm in −Smedium (Fig. 3B and SI Appendix, Fig. S4 A and B). Localizationof MYBS2-GFP shifted from the nucleus to the cytoplasm whenthe same cell was transferred from +S to −S medium (Fig. 3Cand SI Appendix, Fig. S4C).Collectively, these data demonstrate that although MYBS2

shuttles between the nucleus and cytoplasm, it is preferentiallyimported into the nucleus when sugar is provided.

MYBS2 Is a Phosphoprotein.Bioinformatics analysis using the NetPhos3.1 server (http://www.cbs.dtu.dk/services/NetPhos/) revealed

several potential phosphorylation sites in MYBS2. To determine ifMYBS2 is phosphorylated, total proteins extracted from the em-bryo calli of transgenic rice carrying CaMV35S:MYBS2-GFP weresubjected to regular and Phos-Tag immunoblot assays using anti-GFP antibodies. Two protein bands were detected in most sam-ples with the Phos-Tag immunoblot assay (SI Appendix, Fig. S5 Aand B). To confirm that MYBS2 is phosphorylated, we treatedtotal proteins with λ protein phosphatase prior to Phos-Tag im-munoblotting. We detected 2 protein bands with molecular massof ∼70 kDa in cells cultured in +S and −S media, but their mo-lecular weights shifted to ∼56 kDa (the predicted molecularweights of MYBS2 plus GFP) upon treatment with λ proteinphosphatase (SI Appendix, Fig. S5C), indicating that the 2 highermolecular mass proteins were phosphorylated forms of MYBS2.Thus, our analysis reveals that MYBS2 is phosphorylated in both+S and −S media.Immunoprecipitation (IP) coupled with mass spectrometry

can identify phosphorylation sites in a target protein with highsensitivity (29). Accordingly, we extracted total protein fromembryo calli of the CaMV35S:MYBS2-GFP transgenic line andsubjected it to IP using a GFP-trapping method followed by massspectrometry-based analysis. We unequivocally identified 2 phos-phorylation sites at Ser53 and Ser75 in MYBS2 (SI Appendix,Fig. S6).

Phosphorylation at Ser75 Promotes the Sugar-Dependent NuclearLocalization of MYBS2. MYBS2 contains 2 putative nuclear locali-zation signals (NLSs) rich in Arg (R) and Lys (K) amino acids—86KRRRRK91 (designated as NLS1) and 153KKKRR157 (NLS2)—flanking the MYB domain (Fig. 4A). To determine if these 2putative NLSs play a role in regulating nucleocytoplasmic shut-tling of MYBS2, we generated constructs with mutations in the2 putative NLSs by substituting Arg/Lys with Ala (A) and an-alyzed their activity in barley aleurones. As depicted in Fig. 4A,WT and both single NLS-mutated MYBS2 constructs were pri-marily distributed in the cytoplasm in −S medium. In +S me-dium, WTMYBS2 was mainly localized in the nucleus, but bothsingle NLS-mutated MYBS2 constructs were predominantly dis-tributed in the cytoplasm. However, mutation of both NLSsresulted in exclusively cytoplasmic localization of MYBS2, re-gardless of whether medium included sugar (+S) or lacked it (−S).These results indicate that mutation of either NLS1 or NLS2impairs the nuclear import of MYBS2 and that mutation of bothNLSs completely prevents nuclear import of MYBS2, regardlessof the presence or absence of sugar in the medium.Phosphorylation plays an important role in regulating the

nucleocytoplasmic trafficking of cargo proteins, and phosphory-lation upstream of the NLS has been shown to enhance the nu-clear import of the large tumor antigen of simian-virus 40 (SV40T-antigen) (30). We investigated if phosphorylation at Ser75 (SIAppendix, Fig. S6B) impacts MYBS2 nuclear import by generatingconstructs in which Ser75 was substituted with an amino acid thatcannot be phosphorylated (Ala) or mimics constitutive phos-phorylation (Asp). We found that relatively more MYBS2(S75A)-GFP accumulated in the cytoplasm and more MYBS2(S75D)-GFP accumulated in the nucleus of both +S and −S media (Fig.4B and SI Appendix, Fig. S7), demonstrating that phosphorylationat Ser75 promotes nuclear import of MYBS2 and that it may beresponsible for the nuclear localization of MYBS2 under condi-tions of sugar provision.During the course of identifying functional domains of MYBS2,

we found that deletion of amino acids 1 to 53 conferred greaterrepression on 6xTA promoter activity (SI Appendix, Fig. S8 A,Left), resulting in exclusively nuclear localization of MYBS2-GFP in both +S and −S media (SI Appendix, Fig. S8 A, Right).This observation indicates that this N-terminal domain may con-tain a nuclear export signal (NES) or amino acid sequences re-quired for retention of MYBS2 in the cytoplasm. This supposition

Fig. 2. MYBS2 represses αAmy expression and promoter activities throughthe TA box. (A) Seedlings of sWT, MYBS2 (full-length or truncated) Ox and Rilines were cultured in −S medium for 10 d. Total RNAs were extracted fromleaves and used for qRT-PCR analysis using αAmy3-specific primers. (B and C)Rice embryos were cotransfected with effector and reporter plasmids, in-cubated in −S medium for 24 h, before assaying for luciferase activity. Thevalue for luciferase activity of the reporter construct in the absence of theeffector was set to 1×, and all other values were calculated relative to thisvalue. Error bar indicates the SE for 3 replicate experiments. (B) Effectorconstructs. (C) Luciferase activities of in rice embryos carrying reporterconstructs aAmy3-35Smp:Luc and 6xTA-35Smp:Luc in the presence of ef-fector constructs. Asterisks indicate significant differences (Student’s t test,*P < 0.05, **P < 0.01).

Chen et al. PNAS | October 22, 2019 | vol. 116 | no. 43 | 21927

PLANTBIOLO

GY

was confirmed by MYBS2(54-265)-GFP with a functional NLS1or NLS2 being exclusively localized in the nucleus, or strictly in thecytoplasm upon both NLSs being mutated, in both +S and −Smedia (SI Appendix, Fig. S8B). MYBS2(54-265)-GFP was alsoexclusively localized in the nucleus in transgenic rice roots in-cubated in both +S and −S media (Fig. 3A). Bioinformatic analysispredicted a putative NES that is located at amino acids 46 to 65 ofMYBS2 (Fig. 4C). Mutation of hydrophobic amino acids at theN-terminal end of this putative NES did not affect the nuclearlocalization of MYBS2 in +S or −S medium, but changes of hy-drophobic amino acids at the C-terminal region (amino acids57 to 65) led to increased nuclear localization of MYBS2 in −Smedium (Fig. 4C). This result indicates that amino acids 57 to 65,which match well the canonical NES consensus sequence (Φ-X2-Φ-X2-Φ-X-Φ, Φ = L, I, or V) (31), is indeed the core of afunctional NES.

MYBS2 Interacts with Specific 14-3-3 Protein. To identify proteinsthat may interact with MYBS2 and regulate its nucleocytoplas-mic shuttling, we extracted total proteins from embryo calli ofthe CaMV35S:MYBS2-GFP and CaMV35S:GFP (as a control)transgenic lines and subjected them to coimmunoprecipitation(co-IP) using a GFP-trapping method followed by linear-trapquadrupole mass spectrometry-based proteomic analysis. MYBS2-interacting proteins were identified from 2 MYBS2-GFP over-expressing transgenic lines, but not from the control, underhighly stringent criteria with a false-discovery rate of <1% forpeptides identification. Proteins with top Mascot scores >400 areshown in SI Appendix, Table S1. Among them, 7 rice GF14 (14-3-3)proteins were identified as being highly abundant MYBS2-interacting partners. Most of these GF14 proteins exhibitedhigh sequence coverage and a large number of significant uniquepeptides in datasets from 2 transgenic lines, indicative of rela-tively high-confidence candidate interacting proteins. Expressionof several GF14 is activated by sugar starvation (SI Appendix,Fig. S9).We selected 4 of the GF14 candidate proteins with high

Mascot scores (GF14B, GF14C, GF14D, and GF14E) as rep-

resentatives for interaction with MYBS2 and 1 with a lower score(GF14G) for further analysis. For in vivo interaction assays, wecotransfected rice embryo calli with Ubi:MYBS2-mCherry andindividual Ubi:GFP-GF14, Ubi:GF14-GFP or Ubi:GFP (as anegative control) constructs. Total proteins were extracted andthe MYBS2-GF14 protein complex was isolated using the GFP-trapping method before conducting immunoblot analysis usinganti-GFP and anti-RFP antibodies. The anti-GFP antibodiescould detect the GFP− control and all GFP-tagged GF14 proteins,whereas the anti-RFP antibodies could only detect MYBS2-mCherry in the elution of GFP-tagged GF14B, GF14C, GF14D,and GF14E, but not GF14G or the GFP-negative control (Fig.5A). Similar results were obtained with the in vitro interactionassays (SI Appendix, Fig. S10).

Sugar-Mediated Repression of the αAmy3 Promoter Is Released byCytoplasmic 14-3-3 Proteins Restricting MYBS2. We further investi-gated the role of 14-3-3 proteins in regulating MYBS2 func-tion. Rice embryo calli were cotransfected with the effectorsUbi:MYBS2 and Ubi:GF14 and the reporter 6xTA-35S mp:Luc.Individually, GF14B, GF14C, and GF14D significantly enhanced6xTA promoter activity in both +S and −S medium, whereascotransfection of GF14 with MYBS2 only partially did so (Fig.5B). Since MYBS2 is a phosphoprotein, we further investigatedif 14-3-3 proteins regulate the subcellular localization of MYBS2in response to sugar levels. Barley aleurones were transfectedwith Ubi:MYBS2-mCherry or Ubi:GFP-GF14 alone and incu-bated in +S or −S medium. MYBS2-mCherry was localized inthe nucleus in +S medium, whereas 3 GFP-GF14s were foundin the cytoplasm regardless of whether +S or −S medium wasemployed (Fig. 5C and SI Appendix, Fig. S11). We then cotrans-fected Ubi:MYBS2-mCherry and individual Ubi:GFP-GF14 intobarley aleurones and incubated them in +S medium. Interestingly,MYBS2-mCherry became mostly localized in the cytoplasm uponinteracting with individual GFP-GF14 proteins (Fig. 5D), dem-onstrating that retention of MYBS2 in the cytoplasm throughinteraction with 14-3-3 proteins is responsible for relieving sugarrepression of the TA box under sugar starvation.

Fig. 3. Nuclear import of MYBS2 is promoted by sugarprovision. (A) Roots of 5-d-old transgenic rice seedlingsoverexpressing CaMV35S:GFP and CaMV35S:MYBS2-GFP and CaMV35S:MYBS2(54-264)-GFP were incubatedin +S or −S medium for 24 h under darkness, and theGFP signal in root tips was examined by confocal mi-croscopy. The red-colored cell walls were stained withpropidium iodide that is a membrane-impermeable dyefor staining the extracellular space and its constituents(including cell walls and secreted polysaccharides).(Scale bar, 20 μm.) (B and C) Barley aleurones weretransfected with Ubi:MYBS2-GFP and incubated in +Sor −S medium for 24 h. Thirty optical sections, each of0.9 to 1.1 μm thickness, were prepared for each cell (SIAppendix, Fig. S4), but only 5 regularly spaced sections(sections 4, 10, 16, 22, and 28; from top to bottom ofcell) are shown here. “N” and “C” indicate higher GFPsignals in the nucleus and cytoplasm, respectively,whereas “n” and “c” represent lower respective signals.Percentage indicates the number of cells with GFP dis-tribution in the nucleus or cytoplasm divided by thetotal number of cells examined. n > 200. (B) Aftertransfection, barley aleurones were incubated in +Sor −S medium for 24 h. (C) After transfection, barleyaleurones were incubated in +S medium for 24 h, andtransferred (indicated by the arrow) to −Smedium for afurther 24 h.

21928 | www.pnas.org/cgi/doi/10.1073/pnas.1904818116 Chen et al.

Phosphorylation of MYBS2 at Ser53 Is Necessary for Interaction with14-3-3 Proteins in the Cytoplasm under Sugar Starvation. MYBS2contains a putative 14-3-3 binding domain 49KKSSSMP55 (con-sensus sequence underlined) that matches well with the consensus14-3-3 binding motif (R/K)XX(pS/pT)XP or (R/K)XXX(pS/pT)XP (32, 33). The Ser53 residue in the consensus 14-3-3 bindingmotif of MYBS2 (Fig. 6A) was predicted as a phosphorylation sitebased on our mass spectrometry analysis (SI Appendix, Fig. S6A).To investigate whether phosphorylation in the consensus 14-3-3binding motif regulates its interaction with MYBS2, we substitutedeach Ser residue in the Ser51-53 amino acid cluster to Ala. Sin-gle or triple Ser mutation variants of MYBS2—MYBS2(S51A),MYBS2(S52A), MYBS2(S53A), and MYBS2(S51-53A)—werefused to mCherry, and rice embryo calli were cotransfected withUbi:GFP-GF14D (a representative of GF14) and Ubi:MYBS2(WT or mutant). The MYBS2-GF14D protein complex was iso-lated from total cell extract using the GFP-trapping method andsubjected to immunoblot analysis using anti-GFP and anti-RFPantibodies. The anti-GFP antibodies could detect GFP-GF14Dpresent in all protein complex samples, but the anti-RFP an-tibodies could only detect MYBS2-mCherry in protein samplescontaining WT MYBS2, MYBS2(S51A), or MYBS2(S52A), butnot MYBS2(S53A) or MYBS2(S51-53A) (Fig. 6B). This analysisdemonstrates that phosphorylation at Ser53 is necessary for theinteraction between MYBS2 and 14-3-3 proteins.To further investigate whether phosphorylation at Ser53

is necessary for sequestering MYBS2 in the cytoplasm un-der sugar starvation, we transfected barley aleurones withUbi:MYBS2(mutant)-GFP constructs. The MYBS2-mCherry pro-tein had already been shown to localize in the nucleus in +S me-dium (Fig. 5C). We used GFP as the reporter here to demonstratethat the subcellular localization of mutant MYBS2 is not affected byfusion with different reporters. In +S medium, MYBS2(mutant)-GFP alone localized in the nucleus (Fig. 6C). We then cotrans-

fected barley aleurones with Ubi:MYBS2(mutant)-mCherry andUbi:GFP-GF14D constructs. In +S medium, GF14D localized inthe cytoplasm, as did MYBS2(S51A) and MYBS2(S52A), whereasMYBS2(S53A) remained in the nucleus (Fig. 6D). In −S medium,a large proportion of MYBS2(S51A) and MYBS2(S52A) becamelocalized in the cytoplasm, probably due to sequestration by en-dogenous GF14 proteins, but MYBS2(S53A) remained in thenucleus (Fig. 6E). We then cotransfected barley aleurones withUbi:MYBS2(mutant)-mCherry and Ubi:GFP-GF14D and incu-bated in −S medium, and found that GF14D, MYBS2(S51A)and MYBS2(S52A) were all localized in the cytoplasm, whereasMYBS2(S53A) remained in the nucleus (Fig. 6F). We also per-formed a control transfection experiment to show that theMYBS2(mutant)-mCherry signal was not a bleed-over due tostrong GFP signal (SI Appendix, Fig. S12). Together, these findingsdemonstrate that phosphorylation of MYBS2 at Ser53 is necessaryfor this protein to be retained in the cytoplasm by 14-3-3 proteinsunder sugar starvation.

Both MYBS1 and MYBS2 Are Present in the Nucleus Early in the Transitionfrom Normal Metabolic to Sugar-Depleted Conditions. Nuclear ex-port of MYBS1 occurs concomitantly with nuclear import ofMYBS2 under conditions of sugar provision (19) (Fig. 3). To de-termine whether MYBS1 and MYBS2 can both be present in thenucleus and compete for binding to the TA box in αAmy promotersduring the transition from normal metabolic (high sugar level) tosugar-depleted (low sugar level) conditions, we investigated thekinetics of nucleus-cytoplasm shuttling of MYBS1 and MYBS2.We cotransfected barley aleurones with Ubi:MYBS1-mCherry andUbi:MYBS2-GFP. Transfected cells were incubated in −S or +Smedia for 16 h, and then transferred to +S or −S media for an-other 20 h, respectively (SI Appendix, Fig. S13 A and C). A shift influorescence intensity between the nucleus and cytoplasm could bedetected for both MYBSs upon transferring cells between the +S

Fig. 4. MYBS2 contains NLS and NES and phos-phorylation at Ser75 regulates its sugar-dependentnuclear localization. (Left) Barley aleurones weretransfected with Ubi:MYBS2 (full-length or mu-tated)-GFP and incubated in +S or −S medium for24 h. (Center) Dark green indicates higher GFP sig-nal, light green indicates lower GFP signal, and whiteindicates no GFP signal in the nucleus (N) or cyto-plasm (C). (Right) nucleocytoplasmic partitioning (inpercent, %) of different forms of MYBS2-GFP. “N”and “C” indicate higher GFP signals in the nucleusand cytoplasm, respectively, whereas “n” and “c”represent lower respective signals. n > 200. (A) Con-served amino acid residues Lys (K) and Arg (R) of thebipartite NLS (NLS1 and NLS2) flanking the MYB do-main in MYBS2 were substituted with Ala (A). (B)Ser75 of MYBS2 was substituted with Ala or Asp (D). (C)Conserved hydrophobic amino acid residues (under-lined) in NES of MYBS2 were substituted with Ala.

Chen et al. PNAS | October 22, 2019 | vol. 116 | no. 43 | 21929

PLANTBIOLO

GY

and −S conditions (SI Appendix, Fig. S13 B and D). Althoughincrease in nuclear export of MYBS1 occurred, MYBS2 wasrapidly imported into the nucleus upon transferring cells from −Sto +S conditions (SI Appendix, Fig. S13B). In contrast, althoughMYBS1 was rapidly imported to the nucleus and MYBS2 wasexported to the cytoplasm upon transferring cells from +S to −Sconditions, MYBS1 and MYBS2 could still be colocalized in thenucleus prior to transferring to −S conditions (SI Appendix, Fig.S13D). These results indicate that relatively high levels of MYBS1 arecolocalized with MYBS2 in the nucleus during the transition betweenhigh sugar level and sugar-depleted conditions.

Reduced MYBS2 Expression Up-Regulates αAmy under Abiotic Stress,and Ectopic Expression of αAmy3 Enhances Abiotic Stress Tolerance.Since αAmy is activated by various biotic and abiotic stressessuch as water stress, viral/bacterial infection, wounding, heat, orabscisic acid (ABA) in different plant species (34–37), we inves-tigated whether MYBS2 regulates αAmy3 expression in responseto abiotic stresses. We found that expression of MYBS2 in riceseedlings was suppressed by dehydration (air drying; by 70%) andheat (42 °C; by 80%), with concomitant dramatic activation ofαAmy3 expression under conditions of dehydration (by 95-fold)and heat stress (by 2,478-fold) (Fig. 7A).To determine whether suppression of MYBS2 and activation

of αAmy is correlated with stress tolerance, we subjected MYBS2Ox and Ri lines to osmotic stress treatment (i.e., sortibol treat-ment to mimic osmotic stress). We found that aAmy3 and aAmy7expression in both control and sorbitol-treated seedlings wasreduced inMYBS2-Ox andMYBS2(54-265)-Ox lines but increasedin Ri lines (Fig. 7B). Seedling shoot length of MYBS2-Ox lines wasshorter than that of the sWT under both control and sorbitol-treated

conditions (Fig. 7 C and D), and significantly greater than that of thesWT in Ri lines under sorbitol-treated conditions (Fig. 7D). Thesedata indicate that reduced MYBS2 expression not only up-regulatesaAmy expression, particularly under osmotic stress, but it also en-hances osmotic stress tolerance in rice.We also generated transgenic rice carrying Ubi:αAmy3 or

αAmy3:αAmy3, and first showed that accumulation of αAmy3 wassignificantly increased in transgenic lines under sorbitol treatment(Fig. 7E). The significantly greater expression of αAmy3 driven bythe Ubi promoter under sorbitol treatment could be due to en-hanced stability of αAmy3mRNA, a posttranscriptional regulationsimilar to sugar starvation (3, 11, 12). The germination rate andseedling shoot length of αAmy3-Ox lines were similar to sWTwithout sorbitol treatment (Fig. 7 F and G) but they were signif-icantly greater than sWT under sorbitol treatment (Fig. 7H and I),indicating that ectopic expression of αAmy3 confers osmotic stresstolerance on rice.To further investigate whether down-regulation of MYBS2

improves plant stress tolerance, we subjected the MYBS2 Ox andRi lines to various abiotic stress treatments. Using PEG treat-ment to mimic osmotic stress, we observed that the survival rateof the Ri lines (100%) was twice that of the Ox lines (40 to 50%)and higher than that for the sWT (90%) (Fig. 8A). Upon droughttreatment in soil, the survival rate of the Ri lines (100%) wassignificantly greater than that of the sWT line (70%), and dra-matically greater than that of the MYBS2-Ox lines (0–17%) andMYBS2(54-265)-Ox lines (0–10%) (Fig. 8B). Deletion of the N-terminal amino acid residues 1 to 53 caused exclusively nuclearlocalization of MYBS2 (Fig. 3A and SI Appendix, Fig. S8B),which is consistent with the low expression of αAmy3 in riceseedlings (Figs. 2A and 7B) and the reduced survival rate of

Fig. 5. MYBS2 specifically interacts with certain 14-3-3 proteins (GF14). (A) In vivo co-IP and immunoblotanalysis: Rice embryo calli were cotransfected withUbi:MYBS2-mCherry and Ubi:GFP-GF14 or Ubi:GFP(negative control) constructs by particle bombard-ment and incubated in +S medium for 16 h. Totalproteins were extracted and GFP/GFP-fused proteinswere isolated using GFP trap. Anti-GFP and anti-RFPantibodies were then used to detect coprecipitatedGFP-GF14 or GF14-GFP and MYBS2-mCherry pro-teins, respectively, by means of immunoblotting. (B)Rice embryo calli were cotransfected with the ef-fectors Ubi:MYBS2 and Ubi:GF14 and the reporter6xTA-35Smp:Luc, then incubated in +S or −S me-dium for 24 h, before assaying for luciferase activity.Values for luciferase activity of the reporter con-struct in the absence of the effector were set to 1×and other values were calculated relative to thisvalue. (C) Barley aleurones were cotransfected withUbi:MYBS2-mCherry or Ubi:GFP-GF14 alone, in-cubated in +S medium for 24 h, and then examinedunder confocal microscopy. (Scale bars, 50 μm.) (D) Barleyaleurones were cotransfected with Ubi:MYBS2-mCherryand Ubi:GFP-GF14, incubated in +S medium for 24 h,and then examined under confocal microscopy. (Scalebars, 50 μm.)

21930 | www.pnas.org/cgi/doi/10.1073/pnas.1904818116 Chen et al.

seedlings under drought stress (Fig. 8B). The growth of seedlingswas delayed slightly under aerobic conditions but significantlyunder submerged conditions in the Ox lines relative to the sWT.However, the performance of the Ri lines was similar to the sWT(Fig. 8C). These studies demonstrated that reduced MYBS2expression or enhanced αAmy3 expression promotes osmotic anddrought stress tolerance in rice. Although overexpression ofMYBS2 results in lower tolerance to submergence, reducedMYBS2 expression does not enhance submergence tolerance.We also found that the total grain weight per plant was re-

duced in 3MYBS2Ox lines, yet increased in 2MYBS2 Ri lines, ingreenhouse and field conditions (SI Appendix, Fig. S14). Theincrease in grain weight per plant in the Ri-1 line is consistentlygreater than that in the Ri-2 line, which correlates with the lowerMYBS2 mRNA level in the Ri-1 line than in the Ri-2 line (Fig.1A). In a recent field test with T4 generation seeds, grain weightsper plant were reduced by 36 to 45% for the Ox lines, butincreased by 26 to 54% in the Ri lines, relative to the sWT(Fig. 8D). Together, these findings demonstrate that MYBS2 isa negative regulator of abiotic stress tolerance and seeddevelopment.

DiscussionMechanisms Underlying Reciprocal Regulation of αAmy byMYBS1 andMYBS2 in Relation to Plant Growth and Stress Tolerance. Our in-vestigations have not only demonstrated that reversible sugarhomeostatic states play important roles in plant growth and de-velopment, but we have also deciphered the molecular mechanismby which sugar levels switch on and off gene expression. As il-lustrated in Fig. 9, when sugar is supplied, Ser75-phosphorylatedMYBS2 is imported into the nucleus to prevent MYBS1 fromactivation of αAmy. Under sugar starvation, Ser53-phosphorylatedMYBS2 is exported from the nucleus and interacts with 14-3-3proteins in the cytoplasm, leading to de-repression of αAmy. Thesugar-mediated nucleocytoplasmic shuttling and competition be-tween MYBS2 and MYBS1 for reversible gene expression con-stitutes an essential regulatory mechanism of sugar status on plantgrowth, stress tolerance and, ultimately, productivity. This regu-latory system confers adaptive plasticity and secures optimal carbonsupplies for plant growth under an ever-changing environment, andhighlightsMYBS2 and αAmy3 as targets for breeding stress-tolerantcrops. Moreover, the significance of this work is beyond plant bi-ology as reversible regulation of gene expression is a key to anyorganism in need to maintaining any sugar homeostatic states.Sugar homeostatic states represent a balance between de-

mand for sugars and their production, and in plants, sugarhomeostasis appears to cover a far broader range than in hu-mans. In rice, hexose concentrations are increased from 30 to60 mM in the embryo/seedling and from 80 to 500 mM in theendosperm within 7 d after germination (38). Together, MYBS1and MYBGA are responsible for the rapid increase in sugarconcentrations in the endosperm after germination (19). Sugarsare transported from the endosperm to the embryo to supportseedling growth, reflecting sugar status dynamics between theendosperm and the developing seedlings. While sugar con-centration is increasing after germination, MYBS2 expressionis also enhanced up to day 6 after germination (SI Appendix,Fig. S15A), when a balance between the action of MYBS1 andMYBS2 is likely achieved and sugar concentrations in theembryo/seedling stabilize by day 7 (38). As we have shown inthis work, the balance between MYBS1 (to enhance αAmy ex-pression) and MYBS2 (to lower αAmy expression) does notfollow an all-or-none pattern, but reflects a more gradual shiftin cellular localizations (SI Appendix, Fig. S13). Thus, MYBS1and MYBS2 work together to maintain sugar levels within anacceptable range during rice seed germination and seedlinggrowth in rice.

Fig. 6. Phosphorylation of MYBS2 at Ser53 is necessary for interactions with14-3-3 (GF14) proteins in the cytoplasm. (A) Consensus 14-3-3 binding motif(underlined) in MYBS2. Potential phosphorylation residues Ser51-53 (redfont) in MYBS2 were identified by LC/MA/MS (SI Appendix, Fig. S6A). (B) Riceembryo calli were cotransfected with Ubi:MYBS2 (point mutated)-mCherryand Ubi:GFP-GF14D constructs and incubated in +S medium for 24 h. Totalproteins were extracted and GFP-GF14D was isolated using GFP trap. Anti-GFP and anti-RFP antibodies were then used to detect coprecipitated GF14and MYBS2 proteins, respectively, by means of immunoblotting. (C–F) Barleyaleurones were transfected with point-mutated MYBS2 alone or with 14-3-3protein (GF14D), then incubated in +S (C and D) or −S (E and F) medium for24 h, before being examined under confocal microscopy. (Scale bars, 50 μm.)“N” and “C” indicate higher GFP or mCherry signals in the nucleus and cy-toplasm, respectively, whereas “n” represents lower signals in the nucleus.(C and E ) Barley aleurones were transfected with Ubi:MYBS2(S51A)-GFP,Ubi:MYBS2(S52A)-GFP or Ubi:MYBS2(S53A)-GFP. (D and F) Barely aleuroneswere cotransfected with Ubi:MYBS2(mutant)-mCherry and Ubi:GFP-GF14D.

Chen et al. PNAS | October 22, 2019 | vol. 116 | no. 43 | 21931

PLANTBIOLO

GY

The On/Off Switching of αAmy Expression by Sugar Is Regulated byNucleocytoplasmic Shuttling of 2 MYBSs That Compete for the SamePromoter cis-Element. Although MYBS1 and MYBS2 shuttle be-tween the nucleus and cytoplasm due to the presence of bothNLS and NES, their respective high and low sugar-regulated nu-clear export seems to follow different kinetics. The +S condition-induced nuclear export of MYBS1 is a relatively slow process (SIAppendix, Fig. S13B). This scenario is further supported by othercytoplasm-nucleus shuttling experiments. Under the +S conditionfor 24 h, 51% of MYBS1 is still present in the nucleus, yet 90% ofMYBS2 has already been imported into the nucleus (19) (SIAppendix, Table S2). In contrast, under the −S condition for 24 h,90% of MYBS1 has already been imported into the nucleus,whereas only 16% of MYBS2 is present in the nucleus, probablydue to being efficiently trapped by cytoplasmic 14-3-3 proteins (SIAppendix, Table S2). Since MYBS1 does not exit the nucleusquickly, there are occasions when both MYBS1 andMYBS2 are inthe nucleus soon (within hours) after shifting from −S to +Sconditions, by which time competition for binding to the TA box inthe aAmy promoter comes into play under normal metabolic (highsugar level) conditions. Therefore, our studies appear to supportthe model proposed in Fig. 9.

Here we show that MYBS2 is preferentially expressed in tis-sues (such as root hairs, companion cells and vascular bundles)where sugars are transported (SI Appendix, Fig. S15 B–E), and itsexpression is up-regulated by sugar transcriptionally and post-transcriptionally (SI Appendix, Figs. S9, S15B, and S16). Ourprevious study shows that MYBS2 represses the TA box promoteractivity but it may weakly activate the αAmy3 SRC promoter ac-tivity (17). In the present study, our gain- and loss-of-functionanalyses indicate that MYBS2 competes against MYBS1 forbinding to the TA box to repress αAmy promoters. Throughtransient expression assays, we found that αAmy3 SRC and the6xTA box promoters were suppressed (by 50%) by MYBS2overexpression, and activated (by 2.2- to 3.7-fold) by MYBS2(Ri)(Fig. 2C). These results are consistent with the outcome of ourtransgenic stable expression assays in which αAmy3 mRNA ac-cumulation was suppressed (by 40 to 60%) by MYBS2 over-expression and activated (by 5.4- to 5.7-fold) by MYBS2(Ri) (Fig.2A). Our studies consistently showed that MYBS1 is a muchstronger activator than MYBS2 (17). Even if MYBS2 has a weakactivator activity, it behaves like a repressor when competing withMYBS1. Since reduced expression of MYBS2 enhances plantgrowth, seed development and stress tolerance, plants would

Fig. 7. Reduced MYBS2 expression up-regulatesαAmy3 that enhances abiotic stress tolerance inrice. (A and B) Total RNAs were extracted from seed-lings for qRT-PCR analysis using αAmy3-, αAmy7- andMYBS2-specific primers. The value of mRNA level in thenontreated control (CK) was set to 1×, and all othervalues were calculated relative to this value. (A) Ten-day-old seedlings of sWT rice were treated with theindicated abiotic stress. (B) Seeds of sWT, MYBS2 (full-length or truncated) Ox and Ri lines were germinated inmedium with or without 400 mM sorbitol (S) for 8 d.n = 60. (C and D) Shoot length of seedlings in experi-ment (B) was determined and plant morphology wasphotographed on day 8. (E–I) Transgenic rice over-expressing Ubi:αAmy3 or αAmy3:αAmy3 were used inthe experiment. Seeds were germinated in mediumwithout sorbitol (CK) or with 400 mM sorbitol (S). (E)Total RNA were extracted from 8-d-old seedlings forqRT-PCR analysis using aAmy3-specific primers. (F andH) Germination rates were determined daily up to day5. (G and I) Shoot length was determined and plantmorphology was photographed at day 8. n = 30. Errorbars represent SD. Asterisks indicate significant differ-ences (Student’s t test, **P < 0.01, ***P < 0.001).

21932 | www.pnas.org/cgi/doi/10.1073/pnas.1904818116 Chen et al.

appear to be subject to sugar repression most of the time duringtheir life cycle.

Phosphorylation of MYBS2 Plays Critical Roles in Regulating Its Sugar-Dependent Nucleocytoplasmic Shuttling and Interaction with 14-3-3Proteins. We found that sugar-dependent regulation of nucleo-cytoplasmic shuttling of MYBS2 is the reverse of that of MYBS1.The NLS and NES are directly involved in the sugar-dependentsubcellular targeting of MYBS1 (19), whereas they do not seemto be directly involved in the sugar-dependent subcellular lo-calization of MYBS2 (Fig. 4 and SI Appendix, Fig. S8B). Instead,we show that phosphorylated Ser75 upstream of NLS1 promotesthe sugar-dependent nuclear localization of MYBS2 (Fig. 4B);this is a scenario somewhat similar to the phosphorylation at aSer residue upstream of the NLS of the SV40 T-antigen to fa-cilitate recognition of the NLS by importin α1 (an essentialadaptor protein for the nuclear import of cargo proteins) thatsubsequently interacts with the nuclear pore complex and passesthrough the channel (30).Our studies support the model that sugar-mediated repression

of the TA box promoter is caused by MYBS2 (localized in thenucleus when sugar is supplied) preventing MYBS1 from bindingto the TA box (Fig. 9A). Regulation of nucleocytoplasmic shut-tling of a transcription factor by a 14-3-3 protein has previouslybeen demonstrated in mammalian cells. The carbohydrate-responseelement-binding protein (ChREBP) is a transcriptional activatorof genes encoding enzymes that convert excess carbohydrates to

fat storage in the liver. Under glucose starvation, ChREBP isexported to the cytoplasm where it is sequestered by a 14-3-3protein, leading to inhibition of the expression of enzymes nec-essary for glycolysis and lipogenesis (39). In our study, MYBS2 is a

Fig. 8. Reduced MYBS2 expression enhances osmotic and drought stress tolerance and total grain weight per plant in rice. Segregated (sWT), MYBS2-Ox,MYBS2(54-265)-Ox, and MYBS2-Ri lines were used in these experiments. (A) Ten-day-old seedlings were treated with 15% PEG for 7 d and 20% PEG foranother 16 d before determining the survival rate. n = 10. (B) Ten-day-old seedlings were transferred to soil with regular water for 3 wk, before cessation ofwatering for the next 21 d, and then the survival rate was determined. n = 10. (C) Seeds were germinated in medium, and shoots grown under aerobic orsubmerged condition for 14 d, shoot length was then determined. n = 30. (D) Total grain weight per rice plant grown in irrigated field. The value of totalgrain weight per plant in the sWT was set to 100%, and all other values were calculated relative to this value. n = 12. Significance levels with the t test: *P <0.05, ***P < 0.001.

Fig. 9. Proposed mechanism underlying reciprocal regulation of αAmy byMYBS1 and MYBS2 during sugar provision (A) and sugar starvation (B).Details of the model are described in the text.

Chen et al. PNAS | October 22, 2019 | vol. 116 | no. 43 | 21933

PLANTBIOLO

GY

transcriptional repressor and its nuclear export and retention by14-3-3 proteins in the cytoplasm are important for activation ofαAmy under sugar starvation.Based on our IP-coupled mass spectrometry analysis, MYBS2

possesses 1 phosphorylation site at Ser53 that resides within theconsensus 14-3-3 protein-binding motif 49KKSSSMP55 (consensussequence underlined). We found that phosphorylation at Ser53alone is necessary for the interaction of MYBS2 with 14-3-3 pro-teins to retain MYBS2 in the cytoplasm under sugar starvation(Figs. 6 and 9B). This result is consistent with the predicted phos-phorylation site in the consensus 14-3-3 protein-binding motif(R/K)XX(pS/pT)XP or (R/K)XXX(pS/pT)XP (32, 33).MYBS2(S51A)-GFP and MYBS2(S52A)-GFP with normal Ser53 are still expor-ted to the cytoplasm, whereas MYBS2(S53A)-GFP without Ser53is retained in the nucleus (Fig. 6E), indicating that phosphoryla-tion of Ser53 is necessary for the localization of MYBS2 in thecytoplasm under sugar starvation.MYBS2 possesses functional NLSs and NES and may shuttle

dynamically between the nucleus and cytoplasm. However, 2different mechanisms may be responsible for the preferentialnuclear localization of MYBS2 when sugar is supplied and cy-toplasmic localization of MYBS2 under sugar starvation. First,when sugar is supplied, lower 14-3-3 protein expression (SI Ap-pendix, Fig. S9) in the cytoplasm facilitates the import of Ser75-phosphorylated MYBS2 to the nucleus (Fig. 9A). Second, higher14-3-3 protein expression (SI Appendix, Fig. S9) in the cytoplasmunder sugar starvation means greater opportunity for interactionwith Ser53-phosphorylated MYBS2 exported from the nucleusand retention in the cytoplasm (Fig. 9B). This notion is sup-ported by the observation that the localization of MYBS2 shiftsfrom the cytoplasm to the nucleus when the NES loses func-tionality under sugar starvation (Fig. 4C). However, the otherpossibility that additional unidentified factors interact with andsequester the MYBS2 and 14-3-3 protein complex in the cyto-plasm when sugar is scarce cannot be completely ruled out.Eight 14-3-3 genes have been identified in rice, and expression

of the 14-3-3 gene family is differentially or coordinately regu-lated by various abiotic stresses and hormones (40, 41), indi-cating that 14-3-3 proteins may act in a complex network toregulate gene expression. Increased levels of 14-3-3 proteins maysequester MYBS2, preventing it from entering the nucleus dur-ing certain plant growth stages or under abiotic stress conditions.This assumption is supported by our previous study showing thatGF14C is activated by both sugar starvation and drought stress,and that ectopic overexpression of GF14C confers drought tol-erance on transgenic rice seedlings (42). In the present study, wehave demonstrated that several GF14s interact with and restrictMYBS2 in the cytoplasm, allowing MYBS1 to activate the TAbox-containing promoters of αAmy in response to low sugarlevels or abiotic stress (Fig. 9B).

Suppression of MYBS2 and Activation of αAmy3 Improves PlantGrowth, Abiotic Stress Tolerance, and Total Grain Weight per Plantin Rice. Amylolytic breakdown of stored starch in seeds duringgermination, or of transitory starch accumulated in the chloro-plasts of photosynthetic tissues at night, is a central biochemicalevent for providing reducing sugars as carbon sources duringgrowth of both dicot and monocot plants. In the endosperm ofcereal grains and the cotyledon of most dicots, such as legumes,αAmy is induced by GA and suppressed by ABA upon germi-nation (43, 44). However, in vegetative tissues of both monocotsand dicots, αAmy can be induced by various biotic and abioticstresses, such as by water stress in barley leaves (34), virus in-fection in tobacco leaves (35), wounding in mung bean cotyle-dons (36), heat, prolonged darkness, and senescence in pealeaves (45, 46), and ABA, heat, and bacterial pathogens inArabidopsis leaves (37). The physiological significance of stress-induced αAmy in vegetative tissues was recently addressed in

Arabidopsis. Impaired mobilization of starch to sugars in leavesof αAmy- and βAmy-defective Arabidopsis mutants reducescarbon export to roots, which dampens sugar osmolyte accu-mulation and reduces root growth during osmotic stress, and thisosmotic stress-induced starch hydrolysis is mediated by an ABA-dependent pathway (47).Stress-induced accumulation of osmolytes lowers the water

potential of cells and promotes water retention in the plant—aprocess known as “osmotic adjustment”—thereby maintainingcell turgor for plant growth and survival under stress conditions(48, 49). However, sugars can also stabilize proteins and cellstructures (50), and they provide protection against oxidation byremoving excess reactive oxygen species (51). Hence, adjustmentof sugar production, storage, and utilization in response tochanging environments may affect plant fitness, survival, andbiomass production.The TA box is present in the promoters of up to 1,300 genes

expressed during rice germination (19, 52), and is also a highlyconserved motif involved in the up-regulation of promoters of atleast 20 genes under sugar starvation in rice suspension cells(53). MYBS2 competes with MYBS1 for binding to the TA box,therefore it is very likely that MYBS2 has multiple target genes.In this study, we show that expression of MYBS2 is suppressedwhereas that of αAmy3 and aAmy7 is induced by dehydration,heat, and osmotic stress in rice seedlings (Fig. 7 A and B), in-dicating that aAmy genes could be 1of key intermediaries in theMYBS2-dependent negative regulation of tolerance to abioticstress (osmotic, drought, and submergence) (Figs. 7 and 8). First,MYBS2 overexpression suppresses, whereas MYBS2 knockdownenhances, αAmy3 expression in seedlings grown under normalconditions (Fig. 2A) and osmotic stress conditions (Fig. 7 B–D).Second, αAmy3 overexpression promotes the germination rateand seedling growth of rice under osmotic stress (Fig. 7 H and I).Sugar production via αAmy-mediated starch degradation offers amechanism for osmotic adjustment under abiotic stress condi-tions. Our studies demonstrated that αAmy3 is necessary andsufficient for promoting osmotic and drought tolerance in rice.Other mechanisms may also regulate the antistress process,

such as active starch degradation by αAmy meeting the highdemand for sugars in cells to fulfill energy and carbon require-ments for protection against stresses. This mechanism is impor-tant for submergence tolerance of rice seedlings (Fig. 8C).Reduced MYBS2 expression does not enhance submergencetolerance of seedlings, indicating that overexpression of αAmy3 isnot sufficient for enhancing submergence tolerance. However,this enzyme is essential for submergence tolerance as indicatedby the reduced seedling growth in MYBS2-Ox lines undersubmergence.We previously showed that CIPK15, a calcineurin B-like–

interacting protein kinase, regulates the expression of MYBS1and αAmy necessary for germination and seedling growth undersubmergence (54). Here, we determined the transcript levels ofMYBS2 in cipk15 knockout mutant seedlings (54) grown in air orin water. We observed that for WT seedlings grown in water, theMYBS2 mRNA level decreased by 34%. In contrast, for thecipk15 mutants grown in water, their level of MYBS2 mRNA wassimilar to that of WT (SI Appendix, Fig. S17). Our studies suggestthat CIPK15 up-regulates MYBS1 and down-regulates MYBS2during submergence.In wheat and rice, mild soil drying or ABA treatment can

trigger whole-plant senescence, leading to accelerated remobi-lization of carbon from stems, and senescing leaves to grains (55,56). We have shown here that total grain weight per plant is alsoincreased by reduced expression of MYBS2, whereas MYBS2overexpression decreases it (Fig. 8D), likely due to enhancedcarbon remobilization to grains by elevated αAmy expression inrice stems and senescing leaves during grain filling stages whensoils normally undergo mild drought stress.

21934 | www.pnas.org/cgi/doi/10.1073/pnas.1904818116 Chen et al.

In summary, our studies decipher the regulatory mechanismcontrolling an on/off switch of reversible sugar signaling andgene expression at the molecular and cellular levels. Our inves-tigation reveals a potential approach to manipulating MYBS2and αAmy at the whole-plant level for improvement of plantgrowth, stress tolerance and grain productivity.

Materials and MethodsDetails about plant materials, primers, plasmids, plasmid construction, ricetransformation, GUS activity assay, rice embryo and barley aleurone transientexpression assays, real-time quantitative RT-PCR analysis, subcellular locali-zation in rice root and barley aleurone, antibodies and immunoblot analysis,

phosphorylation analysis and phosphor-peptide mapping, IP, and field trialsare described in SI Appendix, Materials and Methods.

ACKNOWLEDGMENTS. We thank Dr. John O’Brien for critical review of thismanuscript; Dr. Shuen-Fang Lo for technical assistance in field tests; and theAcademia Sinica Common Mass Spectrometry Facilities for proteomics andprotein modification analysis. This work was supported by Ministry of Sci-ence and Technology Grants MOST 104-2321-B-001-054, MOST 105-2321-B-001-035, MOST 105-2321-B-001-003, andMOST 106-2321-B-001-046 (to S.-M.Y.),and MOST 106-2311-B-008-003-MY3 (to C.-A.L.), and in part by the AdvancedPlant Biotechnology Center from the Featured Areas Research Center Programwithin the framework of the Higher Education Sprout Project by the Ministryof Education in Taiwan.

1. Y. L. Ruan, Sucrose metabolism: Gateway to diverse carbon use and sugar signaling.Annu. Rev. Plant Biol. 65, 33–67 (2014).

2. S. M. Yu, S. F. Lo, T. D. Ho, Source-sink communication: Regulated by hormone, nu-trient, and stress cross-signaling. Trends Plant Sci. 20, 844–857 (2015).

3. J.-J. Sheu, S.-P. Jan, H.-T. Lee, S.-M. Yu, Control of transcription and mRNA turnover asmechanisms of metabolic repression of alpha-amylase gene expression. Plant J. 5,655–664 (1994).

4. K. E. Koch, Carbohydrate-modulated gene expression in plants. Annu. Rev. PlantPhysiol. Plant Mol. Biol. 47, 509–540 (1996).

5. S. M. Yu, Cellular and genetic responses of plants to sugar starvation. Plant Physiol.121, 687–693 (1999).

6. S. Bihmidine, C. T. Hunter, 3rd, C. E. Johns, K. E. Koch, D. M. Braun, Regulation ofassimilate import into sink organs: Update on molecular drivers of sink strength.Front. Plant Sci. 4, 177 (2013).

7. A. von Schaewen, M. Stitt, R. Schmidt, U. Sonnewald, L. Willmitzer, Expression of ayeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads toaccumulation of carbohydrate and inhibition of photosynthesis and strongly influ-ences growth and phenotype of transgenic tobacco plants. EMBO J. 9, 3033–3044(1990).

8. J. S. Eom et al., Impaired function of the tonoplast-localized sucrose transporter inrice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth.Plant Physiol. 157, 109–119 (2011).

9. K. A. Leach, T. M. Tran, T. L. Slewinski, R. B. Meeley, D. M. Braun, Sucrose transporter2contributes to maize growth, development, and crop yield. J. Integr. Plant Biol. 59, 390–408 (2017).

10. B. M. Kennedy, “Nutritional quality of rice endosperm” in Rice: Production and Utili-zation, B. S. Luh, Ed. (AVI Publishing Co., Westport, CT, 1980), Chap. 11, pp. 439–469.

11. M. T. Chan, S. M. Yu, The 3′ untranslated region of a rice alpha-amylase gene func-tions as a sugar-dependent mRNA stability determinant. Proc. Natl. Acad. Sci. U.S.A.95, 6543–6547 (1998).

12. J.-J. Sheu, T.-S. Yu, W.-F. Tong, S.-M. Yu, Carbohydrate starvation stimulates differ-ential expression of rice alpha-amylase genes that is modulated through complicatedtranscriptional and posttranscriptional processes. J. Biol. Chem. 271, 26998–27004 (1996).

13. S. M. Yu, “Regulation of alpha-amylase gene expression” in Molecular Biology ofRice, K. Shimamoto, Ed. (Springer, Tokyo, 1999), pp. 161–178.

14. P. W. Chen, C. M. Chiang, T. H. Tseng, S. M. Yu, Interaction between rice MYBGA andthe gibberellin response element controls tissue-specific sugar sensitivity of alpha-amylase genes. Plant Cell 18, 2326–2340 (2006).

15. P.-W. Chen et al., Rice alpha-amylase transcriptional enhancers direct multiple moderegulation of promoters in transgenic rice. J. Biol. Chem. 277, 13641–13649 (2002).

16. C. A. Lu, E. K. Lim, S. M. Yu, Sugar response sequence in the promoter of a rice alpha-amylase gene serves as a transcriptional enhancer. J. Biol. Chem. 273, 10120–10131(1998).

17. C. A. Lu, T. H. Ho, S. L. Ho, S. M. Yu, Three novel MYB proteins with one DNA bindingrepeat mediate sugar and hormone regulation of alpha-amylase gene expression.Plant Cell 14, 1963–1980 (2002).

18. C. A. Lu et al., The SnRK1A protein kinase plays a key role in sugar signaling duringgermination and seedling growth of rice. Plant Cell 19, 2484–2499 (2007).

19. Y. F. Hong et al., Convergent starvation signals and hormone crosstalk in regulatingnutrient mobilization upon germination in cereals. Plant Cell 24, 2857–2873 (2012).

20. G. G. Fincher, Molecular and cellular biology associated with endosperm mobilisation ingerminating cereal grains. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 305–346 (1989).

21. M. B. Lanahan, T. H. Ho, S. W. Rogers, J. C. Rogers, A gibberellin response complex incereal alpha-amylase gene promoters. Plant Cell 4, 203–211 (1992).

22. F. Gubler, R. Kalla, J. K. Roberts, J. V. Jacobsen, Gibberellin-regulated expression of amyb gene in barley aleurone cells: Evidence for Myb transactivation of a high-pIalpha-amylase gene promoter. Plant Cell 7, 1879–1891 (1995).

23. F. Madeira et al., 14-3-3-Pred: Improved methods to predict 14-3-3-binding phos-phopeptides. Bioinformatics 31, 2276–2283 (2015).

24. D. K. Morrison, The 14-3-3 proteins: Integrators of diverse signaling cues that impactcell fate and cancer development. Trends Cell Biol. 19, 16–23 (2009).

25. T. Gökirmak, A. L. Paul, R. J. Ferl, Plant phosphopeptide-binding proteins as signalingmediators. Curr. Opin. Plant Biol. 13, 527–532 (2010).

26. F. C. Denison, A. L. Paul, A. K. Zupanska, R. J. Ferl, 14-3-3 proteins in plant physiology.Semin. Cell Dev. Biol. 22, 720–727 (2011).

27. V. Hübscher et al., The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointlyregulate transcription of glucose repressed genes in Saccharomyces cerevisiae. NucleicAcids Res. 44, 5629–5645 (2016).

28. C. R. Lin et al., SnRK1A-interacting negative regulators modulate the nutrient star-vation signaling sensor SnRK1 in source-sink communication in cereal seedlings underabiotic stress. Plant Cell 26, 808–827 (2014).

29. S. B. Breitkopf, J. M. Asara, Determining in vivo phosphorylation sites using massspectrometry Curr. Protoc. Mol. Biol. Chap. 18:Unit 18, 19.1–19.27.

30. J. D. Nardozzi, K. Lott, G. Cingolani, Phosphorylation meets nuclear import: A review.Cell Commun. Signal. 8, 32 (2010).

31. S. Kosugi, M. Hasebe, M. Tomita, H. Yanagawa, Nuclear export signal consensus se-quences defined using a localization-based yeast selection system. Traffic 9, 2053–2062 (2008).

32. A. J. Muslin, J. W. Tanner, P. M. Allen, A. S. Shaw, Interaction of 14-3-3 with signalingproteins is mediated by the recognition of phosphoserine. Cell 84, 889–897 (1996).

33. M. B. Yaffe et al., The structural basis for 14-3-3:phosphopeptide binding specificity.Cell 91, 961–971 (1997).

34. J. V. Jacobsen, A. D. Hanson, P. C. Chandler, Water stress enhances expression of analpha-amylase gene in barley leaves. Plant Physiol. 80, 350–359 (1986).

35. T. Heitz, P. Geoffroy, B. Fritig, M. Legrand, Two apoplastic alpha-amylases are inducedin tobacco by virus infection. Plant Physiol. 97, 651–656 (1991).

36. N. Koizuka, Y. Tanaka, Y. Morohashi, Expression of alpha-amylase in response towounding in mung bean. Planta 195, 530–534 (1995).

37. E. A. Doyle, A. M. Lane, J. M. Sides, M. B. Mudgett, J. D. Monroe, An alpha-amylase(At4g25000) in Arabidopsis leaves is secreted and induced by biotic and abiotic stress.Plant Cell Environ. 30, 388–398 (2007).

38. S. M. Yu et al., Sugars act as signal molecules and osmotica to regulate the expressionof alpha-amylase genes and metabolic activities in germinating cereal grains. PlantMol. Biol. 30, 1277–1289 (1996).

39. Q. Ge et al., Importin-alpha protein binding to a nuclear localization signal of car-bohydrate response element-binding protein (ChREBP). J. Biol. Chem. 286, 28119–28127 (2011).

40. N. Yashvardhini, S. Bhattacharya, S. Chaudhuri, D. N. Sengupta, Molecular charac-terization of the 14-3-3 gene family in rice and its expression studies under abioticstress. Planta 247, 229–253 (2018).

41. Y. Yao, Y. Du, L. Jiang, J. Y. Liu, Molecular analysis and expression patterns of the 14-3-3gene family from Oryza sativa. J. Biochem. Mol. Biol. 40, 349–357 (2007).

42. S. L. Ho et al., Sugar starvation- and GA-inducible calcium-dependent protein kinase1 feedback regulates GA biosynthesis and activates a 14-3-3 protein to confer droughttolerance in rice seedlings. Plant Mol. Biol. 81, 347–361 (2013).

43. J. V. Jacobsen, F. Gubler, P. M. Chandler, “Gibberellin action in germinated cerealgrains” in Plant Hormones: Physiology, Biochemistry, and Molecular Biology,P. J. Davies, Ed. (Kluwer Academic Publishers, Dordrecht, 1995), pp. 246–271.

44. M. Taneyama, D. Yamauchi, T. Minamikawa, Synthesis and turnover of alpha-amylasein cotyledons of germinating Vigna-Mungo seeds—Effects of exogenously appliedend-products and plant hormones. Plant Cell Physiol. 36, 139–146 (1995).

45. P. D. Commuri, S. H. Duke, Apoplastic alpha-amylase in pea is enhanced by heat stress.Plant Cell Physiol. 38, 625–630 (1997).

46. M. Saeed, S. H. Duke, Amylases in pea tissues with reduced chloroplast density and/orfunction. Plant Physiol. 94, 1813–1819 (1990).

47. M. Thalmann et al., Regulation of leaf starch degradation by abscisic acid is importantfor osmotic stress tolerance in plants. Plant Cell 28, 1860–1878 (2016).

48. D. Bartels, R. Sunkar, Drought and salt tolerance in plants. Crit. Rev. Plant Sci. 24,23–58 (2005).

49. J. Krasensky, C. Jonak, Drought, salt, and temperature stress-induced metabolic re-arrangements and regulatory networks. J. Exp. Bot. 63, 1593–1608 (2012).

50. F. A. Hoekstra, E. A. Golovina, J. Buitink, Mechanisms of plant desiccation tolerance.Trends Plant Sci. 6, 431–438 (2001).

51. G. Miller, N. Suzuki, S. Ciftci-Yilmaz, R. Mittler, Reactive oxygen species homeostasisand signalling during drought and salinity stresses. Plant Cell Environ. 33, 453–467 (2010).

52. H. Tsuji et al., GAMYB controls different sets of genes and is differentially regulatedby microRNA in aleurone cells and anthers. Plant J. 47, 427–444 (2006).

53. H. J. Wang et al., Transcriptomic adaptations in rice suspension cells under sucrosestarvation. Plant Mol. Biol. 63, 441–463 (2007).

54. K. W. Lee et al., Coordinated responses to oxygen and sugar deficiency allow riceseedlings to tolerate flooding. Sci. Signal. 2, ra61 (2009).

55. J. C. Yang, J. H. Zhang, Z. Q. Wang, Q. S. Zhu, L. J. Liu, Involvement of abscisic acid andcytokinins in the senescence and remobilization of carbon reserves in wheat sub-jected to water stress during grain filling. Plant Cell Environ. 26, 1621–1631 (2003).

56. J. Yang, J. Zhang, Grain filling of cereals under soil drying. New Phytol. 169, 223–236 (2006).

Chen et al. PNAS | October 22, 2019 | vol. 116 | no. 43 | 21935

PLANTBIOLO

GY