pcg proteins msi1 and bmi1 function upstream of mir156 to ... · pcg proteins msi1 and bmi1...

PcG Proteins MSI1 and BMI1 Function Upstream ofmiR156 to Regulate Aerial Tuber Formationin Potato1[OPEN]

Amit Kumar,2 Kirtikumar Ramesh Kondhare ,2 Pallavi Vijay Vetal, and Anjan Kumar Banerjee3,4

Biology Division, Indian Institute of Science Education and Research, Pune 411008, Maharashtra, India

ORCID IDs: 0000-0002-4398-4455 (A.K.); 0000-0002-0762-3767 (K.R.K.); 0000-0002-9001-7943 (P.V.V.); 0000-0002-1825-7926 (A.K.B.).

Polycomb Repressive Complexes (PRC1 and PRC2) regulate developmental transitions in plants. AtBMI1, a PRC1 member,represses micro RNA156 (miR156) to trigger the onset of adult phase in Arabidopsis (Arabidopsis thaliana). miR156overexpression (OE) reduces below-ground tuber yield, but stimulates aerial tubers in potato (Solanum tuberosum sspandigena) under short-day (SD) photoperiodic conditions. Whether PRC members could govern tuber development throughphotoperiod-mediated regulation of miR156 is unknown. Here, we investigated the role of two PRC proteins, StMSI1 (PRC2member) and StBMI1-1, in potato development. In wild-type andigena plants, StMSI1 and miR156 levels increased in stolon,whereas StBMI1-1 decreased under SD conditions. StMSI1-OE and StBMI1-1-antisense (AS) lines produced pleiotropic effects,including altered leaf architecture/compounding and reduced below-ground tuber yield. Notably, these lines showed enhancedmiR156 accumulation accompanied by aerial stolons and tubers from axillary nodes, similar to miR156-OE lines. Further,grafting of StMSI1-OE or StBMI1-1-AS on wild-type stock resulted in reduced root biomass and showed increasedaccumulation of miR156a/b and -c precursors in the roots of wild-type stocks. RNA-sequencing of axillary nodes fromStMSI1-OE and StBMI1-1-AS lines revealed downregulation of auxin and brassinosteroid genes, and upregulation ofcytokinin transport/signaling genes, from 1,023 differentially expressed genes shared between the two lines. Moreover,we observed downregulation of genes encoding H2A-ubiquitin ligase and StBMI1-1/3, and upregulation of Trithorax groupH3K4-methyl-transferases in StMSI1-OE. Chromatin immunoprecipitation-quantitative PCR confirmed H3K27me3-mediatedsuppression of StBMI1-1/3, and H3K4me3-mediated activation of miR156 in StMSI1-OE plants. In summary, we show thatcross talk between histone modifiers regulates miR156 and alters hormonal response during aerial tuber formation in potatounder SD conditions.

Plants sense multiple environmental cues, such astemperature, light, and nutrient availability, and syn-chronize developmental programs accordingly. Pho-toperiod is one such environmental cue that playsan important role during tuber development (tuber-ization) in potato (Solanum tuberosum ssp andigena).During tuberization, the stolon (a modified below-ground stem) passes through various developmentalstages andmatures into a potato under short-day (SD)

condition. Apart from phytohormones (auxin, cytokinin[CK], and gibberellin [GA]; Xu et al., 1998), phyto-chromes, flowering genes (CONSTANS [CO]; Martínez-García et al., 2002), a number ofmobile signals, includingmRNAs (StBEL5, -11, -29, and POTH1; Banerjee et al.,2006a, Mahajan et al., 2012, Ghate et al., 2017), microRNAs (miR172 and miR156; Martin et al., 2009, Bhogaleet al., 2014), and a Flowering Locus T (FT) orthologousprotein StSP6A (Navarro et al., 2011) are now known toregulate tuberization. Earlier, we showed that miR156levels increase in stolon under tuber-inducing SD pho-toperiodic conditions and its overexpression (OE) led toaerial tuber formation in potato (Bhogale et al., 2014).However, the basis for aerial tuber formation and whatregulates miR156 under SD condition is not known.Previous studies in Arabidopsis (Arabidopsis thaliana)revealed that Polycomb Group (PcG) proteins mediatethe repression of several miRNAs (Lafos et al., 2011),including miR156 and miR172 (Picó et al., 2015).PcG proteins are important regulators of growth

and development across eukaryotic lineages. Theywere first identified in Drosophila as multiproteincomplexes, termed as Polycomb Repressive Com-plex1 (PRC1) and PRC2. The PRC1 complex in Dro-sophila contains four members, namely the Polycomb(Pc), Polyhomeotic (Ph), Posterior sex comb (Psc), and

1This work was supported by the Indian Institute of Science Edu-cation and Research Pune (IISER Pune) (a grant to A.K.B. and a fel-lowship to K.R.K.) and the Council of Scientific & Industrial Research(research fellowship to A.K.).

2Authors contributed equally to this article.3Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Anjan Kumar Banerjee ([email protected]).

A.K., K.R.K., and A.K.B. designed the research; A.K., K.R.K., andP.V.V. performed experiments; A.K., K.R.K., and A.K.B. analyzed thedata and wrote the article; all authors approved the final article.

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http://orcid.org/0000-0002-4398-4455

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http://orcid.org/0000-0002-0762-3767

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http://orcid.org/0000-0002-9001-7943

http://orcid.org/0000-0002-9001-7943

http://orcid.org/0000-0002-1825-7926

http://orcid.org/0000-0002-1825-7926

http://orcid.org/0000-0002-4398-4455

http://orcid.org/0000-0002-0762-3767

http://orcid.org/0000-0002-9001-7943

http://orcid.org/0000-0002-1825-7926

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dRING1 proteins (Shao et al., 1999; Peterson et al.,2004). They repress target chromatin by H2A mon-oubiquitination (Cao et al., 2005). Arabidopsis hasthree homologs of Psc (AtBMI1A, AtBMI1B, andAtBMI1C) and two homologs of dRING1 (AtRING1A,and AtRING1B; Calonje, 2014). BMI assists in activityof E3 ubiquitin ligases that monoubiquitylate histoneH2A at Lys-119 position leading to the repression oftarget genes. A recent study in Arabidopsis has shownthat BMI1 regulates meristem maintenance and celldifferentiation by repressing PLETHORA (PLT) andWUS homeobox-containing (WOX) genes (Merini et al.,2017). Further, BMI1 mutants show downregula-tion of important flowering genes, like SQUAMOSAPROMOTER BINDING PROTEIN-LIKE (SPL) and FT,indicating an important role in flowering response. Toavoid precocious flowering, SPLs are suppressed bymiR156 during the juvenile phase of plants. However,during adult and reproductive phases, miR156 ex-pression is suppressed by BMI1 to allow the expressionof SPLs (Merini et al., 2017). The core PRC2 complex inDrosophila consists of four subunits, namely Enhancerof Zeste [E(z)], Suppressor of Zeste 12 [Su(z)12], Extrasex combs (Esc), and p55. The [E(z)] protein repressestarget genes by catalyzing H3K27me3 modificationof these genes (Müller et al., 2002), whereas p55 helpsin recruitment of PRC2 complex to target chromatin.Arabidopsis has five p55 homologs named as MSI1–5(Hennig et al., 2005). They belong to the Trp Asp (WD-40) or b-transducin repeat-containing protein familyand have seven WD repeats with four antiparallelb-sheets at the C-terminal end that assist in its interac-tion with other proteins. A previous report on MSI1in Arabidopsis showed that it regulates overall plantarchitecture and ovule development (Hennig et al.,2003). Subsequent studies also revealed that MSI1 is acomponent of several histone modifier complexes thatregulates different phases of plant development. It isa part of three PRC2 complexes, known as FERTILI-ZATION INDEPENDENT SEED (FIS) complex thatregulates seed development (Köhler et al., 2003),EMBRYONIC FLOWER(EMF) complex that sup-presses flowering during juvenile stage (Yoshidaet al., 2001), and VERNALIZATION (VRN) com-plex, which is essential for the onset of floweringafter vernalization (De Lucia et al., 2008). Addition-ally, MSI1 is also a part of CHROMATIN ASSEMBLYFACTOR1 (CAF-1; Exner et al., 2006), nucleosome-remodeling factor (Martínez-Balbás, 1998), and his-tone deacetylase (Mehdi et al., 2016), indicating itsdiverse role in plant development. MSI1 also pro-motes flowering in Arabidopsis in a photoperiod-dependent manner by assisting in expression of COand SUPPRESSOR OF CO (SOC1) through H3K4methylation and H3K9 acetylation over SOC1 locus(Bouveret et al., 2006; Steinbach and Hennig, 2014).

Tuberization and flowering are two reproductivephenomena that share commonmolecular players andenvironmental cues (Martínez-García et al., 2002).ConsideringPcGproteins’ role inflowering,wehypothesize

that they might govern tuber development in potato. In anexperiment,weobserved thatOEofStMSI1produced aerialstolons and tubers under SD photoperiodic conditions fromaxillary nodes, a phenotype that was demonstrated earlierfor miR156 OE in potato (Bhogale et al., 2014). This raised anumber of interesting questions with respect to the functionof PcG proteins in potato, such as: (1) What is the cause ofaerial stolonand tuberdevelopment fromaxillarynodes? (2)Do PcG proteins have any role in photoperiod-mediatedcontrol of tuberization? (3) Is miR156 directly regulatedby StMSI1 or there are other epigenetic modifiers thatcould regulate miR156? In this study, using severalapproaches, such as OE or knockdown of two PcGproteins StMSI1 and StBMI1-1, RNA-sequencing (RNA-seq) analysis of axillary nodes of StMSI1OE and StBMI1-1 knockdown lines, homo- and hetero-grafting and thechromatin immunoprecipitation-quantitative PCR(ChIP-qPCR) method, we established that StMSI1and StBMI1-1 function upstream of miR156 to regu-late aerial tubers in potato under SD photoperiodicconditions.

RESULTS

Phylogenetic Analysis Revealed Conservation ofMSI1- and BMI1-like Proteins in Potato

BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE5Proteins) results revealed ;91% identity be-tween potatoMSI1-like protein (StMSI1; XP_006349413.1)and Arabidopsis MSI1 (AtMSI1; NP_200631.1;Supplemental Fig. S1A). From the Potato GenomeSequencing Consortium (PGSC) database, we ob-served that the StMSI1 gene (;5.20 kb) resides onchromosome 1 (Supplemental Table S1). Its longestopen reading frame spans 1,368 bp and encodesfor 425 amino acid residues (a 48.36-kD protein).Using WD repeat protein structure predictor tool,we could identify seven WD repeats in StMSI1protein, positioned between amino acids 33 and 403(Supplemental Fig. S1, B and C). Further analysisrevealed the presence of 14 hotspot residues in theStMSI1 protein sequence that are likely to be involvedin protein–protein interactions (Supplemental Fig.S1B). Arabidopsis MSI2 and MSI3 proteins matchwith potato nucleosome/caf (StMSI2) and share;68%identity (Supplemental Fig. S1A). Like StMSI1 protein,StMSI2 also has seven WD repeats and it shares 57%sequence similarity with StMSI1. In contrast, other MSI-like proteins from Arabidopsis showed less conservationwith potato proteins (Supplemental Fig. S2A). STRINGtool (https://string-db.org/) analysis predicted thatStMSI1 could interact with a range of proteins includingother PRC proteins, CAF, histone acetylases, and deace-tylases (Supplemental Fig. S2B).

In potato, four BMI1 proteins (StBMI1-1, StBMI1-2,StBMI1-3, and StBMI1-4) have been identified and theyshare;55%, 50%, 45%, and 32% sequence identity withArabidopsis BMI1a, respectively. When potato BMI1

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https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins








https://string-db.org/



proteins were analyzed for conserved domains,we found that a Cys-rich RING domain involved inzinc binding and the ubiquitination process is pre-sent in StBMI1-1, StBMI1-2, and StBMI1-4 similar toArabidopsis BMI1 proteins (AtBMI1a, AtBMI 1b, andAtBMI 1c), but this domain was absent in StBMI1-3(Supplemental Fig. S3). Arabidopsis BMI1 proteinsalso have a RAWUL domain (ubiquitin-like domainlikely to be involved in protein–protein interac-tions), but this domain is absent in potato or tomato(Solanum lycopersicum) BMI1 homologous proteins.StBMI1–4 has an additional RAD18 domain, which isa putative nucleic acid binding domain (SupplementalFig. S3). From the PGSC database, we found thatthe StBMI1-1 gene was located on chromosome 9, theStBMI1-2 and StBMI1-3 genes on chromosome 6, andthe StBMI1-4 gene was on chromosome 1 (SupplementalTable S1). Phylogenetic analysis indicated that StBMI1-1, StBMI1-2, and StBMI1-3 displayed close conservationto respective tomato BMI1 proteins, whereas StBMI1-4had close conservation to AtBMI1c (SupplementalFig. S4).

SD Photoperiod Influences StMSI1 Expression in Stolonand Root Tissues

qPCR analysis showed a significant increase ofStMSI1 transcript abundance in stolons under SD thanlong-day (LD) photoperiodic conditions (Fig. 1A).However, its transcript level was significantly lowerin roots (Fig. 1A) and mature tubers (SupplementalFig. S5A) under SD compared to LD. The expressionof StMSI1 remain unchanged in shoot tip, leaf, andstem under SD compared to LD conditions (Fig. 1A).From the RNA-seq data available in the PGSC

database (Xu et al., 2011), it was further evident thatthree StMSI genes (StMSI1, StMSI2, and StMSI4) arehighly expressed in the stolons, but their expressionis reduced in mature tubers and roots (SupplementalFig. S5B). To characterize StMSI1 promoter activity, wegenerated promStMSI1::GUS-pBI121 potato transgeniclines. GUS assay on in vitro grown plantlets showeda ubiquitous StMSI1 expression pattern. Promoter ac-tivity was observed in shoot tip, stem, leaf, shoot–rootjunction, and root (Fig. 1, B–H), with strong activityin meristematic regions (axillary nodes and root tips;Fig. 1, C and E). When promoter activity was assayedfrom soil-grown plants induced under LD/SD condi-tions for 14 d, it was observed that swollen stolonsamples from the SD condition had strong GUS activity(Fig. 1F, right) compared to stolons from LD conditions(Fig. 1F, left). GUS activity was also noticed in tuberpeel and pith of SD-induced promoter transgenic plants(Fig. 1D).

OE of StMSI1 Results in Pleotropic Effects in Potato,Including Aerial Stolons/Tubers

Several constitutive OE lines of StMSI1 (StMSI1-OE)driven by 35S Cauliflower Mosaic Virus (CaMV) pro-moter were generated to characterize its role in potatodevelopment (Supplemental Fig. S6A). Of them, twoindependent OE lines (OE1 and OE3) with moder-ate levels of StMSI1 OE were used for further anal-ysis (Fig. 2A). OE lines showed drastic changes inoverall plant phenotype compared to wild-type plants(Fig. 2B). OE plants exhibited decreased plant height(Supplemental Fig. S6B) and internodal distance(Supplemental Fig. S6C); they had a lower numberof leaflets per leaf (Fig. 2, C and D) and leaf length

Figure 1. StMSI1 promoter has ubiquitous expression, but was induced in stolon under the SD photoperiod. A, Effect of LD andSD photoperiod on transcript accumulation of StMSI1 in different tissues (shoot tip, leaf, stem, root, and stolon) of wild-typeandigena (7540) potato plants grown under LD/SD conditions for 14 d, post 8 weeks of LD induction in soil. Fold-change ofStMSI1 across different tissues is compared between SD versus LD in a tissue-specific manner. Data are mean 6 SD for threebiological and three technical replicates. EIF3e was used as a reference gene for expression analysis. Student’s t test was per-formed to check the level of significance. The asterisk represents statistical significance (*P , 0.05). Promoter activity of StMSI1in promStMSI1::GUS transgenic lines (B). B to H, GUS activity in 3-weeks–old entire plant grown in vitro (B), stem and nodes (C),tuber pith (D), root tip (E), LD stolon (F, left), SD swollen stolon (F, right), leaf (G), and the shoot–root junction (H). Stolon and tubersamples are from soil-grown plants incubated under LD/SD conditions for 14 d. Scale bars5 2 cm (B), and 2 mm (C–H). ns, notsignificant.

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PcG Proteins and miR156 Regulate Potato Development

















was reduced (Fig. 2E), although leaf thickness wasincreased (Fig. 2F) compared to wild-type plants. OElines also showed altered epidermal cells, biggertrichomes, increased stomatal number, and alteredvascular bundle arrangement in stem compared towild-type plants (Fig. 2, G– N). Moreover, the rootlength (Supplemental Fig. S6D) and root biomass(Fig. 2O) were decreased in OE lines compared towild-type plants. To evaluate tuber yield potential,soil-grown StMSI1-OE lines maintained under LDconditions were subjected to SD inductions for 6 weeks.Interestingly, these lines produced numerous aerialstolons from axillary nodes post 3 weeks of induction(Fig. 3, A and B). On further incubation of 2–3 weeks,the aerial stolons were noticed to branch profuselyand to develop into mini-tubers in ;70% to 80% ofthe plants (Fig. 3, C and D). The mini-tubers werepurple in color and had characteristic tuber-eyeswith 100% sprouting efficiency when attemptedfor germination. Neither StMSI1-OE (this study) normiR156-OE (Bhogale et al., 2014) showed aerial tuberphenotype in potato under LD conditions. Throughout

our experiments, vector control (VC) plants behaved likewild-type plants.

StMSI1-OE Line Showed an Altered Expression of miR156and StBMI1

To analyze if miR156 levels were affected in StMSI1-OE lines, miR156a/b/c expression was measured inleaf tissues of SD-inducedplants. Interestingly,miR156a/b/c expression was nearly 5-fold higher in the OE line(OE3) compared to the VC (Fig. 3E). Earlier, a PRC1member, AtBMI1, has been shown to repress miR156during reproductive phase maintenance in Arabidopsis(Picó et al., 2015). Anticipating cross talk amongStMSI1, StBMI1, and miR156 in potato, the relativetranscript levels of all four StBMI1 genes (StBMI1-1,-2, -3, and -4) were quantified in the StMSI1-OE line(OE3) using primers from nonconserved regions ofeach variant. The transcript levels of StBMI1-1, -3,and -4 were low in the StMSI1-OE line comparedto VC (Fig. 3F). Due to the close conservation of mRNA

Figure 2. StMSI1 OE affects plant architecture in potato. A, Transcript levels of StMSI1 in leaves of OE lines (OE1 and OE3)compared towild type (WT) . Data aremean6 SD for three biological replicates. EIF3ewas used as a reference gene for expressionanalysis. B, Plant architecture of StMSI1OE potato lines (OE1 and OE3) along with wild-type and VC plants. C to F, The leaf size(C), the number of leaflets per leaf (D), the leaf length (E), and thickness (F) in StMSI1 OE potato lines (OE1 and OE3) are shownalong with wild-type and VC plants. Six individual plants per line were considered for phenotypic data analysis. Student’s t testwas performed to check significancewith *P values, 0.05, **P, 0.01, ***P, 0.001, and ****P, 0.0001. Error bars represent6SD. G andH, Transverse cross section of the stem of wild-type plant (G) and StMSI1OE line OE3 (H). K and L, Magnified images ofvascular bundles in wild type and OE line, respectively. I, J, M, and N, Scanning electron microscopy images showing the leafepidermis cells, number of stomata (J), and trichomes (N) in OE lines compared to wild type (I and M), respectively. O, Rootbiomass in StMSI1OE potato lines (OE1 and OE3) are shown along with wild-type and VC plants. Student’s t test was performedto check significance at P, 0.05. Error bars represent6 SD from six independent plant per line. Scale bars5 10 cm (B), 5 cm (C),300 mm (G, H, K, and L), and 50 mm (I, J, M, and N). gfw, grams fresh weight; ns, not significant.


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sequences between StBMI1-1 and -2 transcript variants,we could not validate the StBMI1-2 variant. The tran-script levels of a CK biosynthesis gene, StLOG3, and aGA catabolism gene, StGA2ox1, were significantlyhigher in leaves of the StMSI1-OE line comparedto VC plants (Fig. 3, G and H). Moreover, StMSI1and miR156 levels in leaves were high in the juvenilephase of wild-type andigena plants, whereas theirlevels were significantly lower in the adult phase ofthe plant (Fig. 3I).

SD Photoperiod Affects StBMI1-1 and miR156 Expressionin Shoot Tip and Stolons

StBMI1-1 level was quantified by qPCR in differenttissues and the stages of stolon-to-tuber transitions inandigena plants grown under LD/SD conditions for14 d (Fig. 4, A and B). Our analysis demonstrated that

StBMI1-1 transcript levels were significantly low underSD photoperiod in shoot tip, stem, and stolon tissuescompared to LD conditions (Fig. 4A). However, thetranscript levels remained unchanged in leaf and roottissues under LD and SD photoperiodic conditions(Fig. 4A). Further, StBMI1-1 transcript levels weresignificantly low in stolon, swollen stolon, and mini-tuber, but high in tubers under SD conditions com-pared to the stolons from LD (Fig. 4B). Moreover,miR156 levels were quantified in shoot tip and stolontissues under LD/SD conditions. We noticed ;2.5-and 2-fold increase of miR156 expression in shoottip and stolon (respectively) under SD conditionscompared to LD (Fig. 4C). From the PGSC resources(Xu et al., 2011), it was evident that StBMI1-4 isexpressed only in floral organs and not in any othertissue types (i.e. shoot apex, leaf, stem, root, stolon,young/mature tuber, sprouted tuber) in potato. Be-cause StBMI1-1 is expressed more abundantly than

Figure 3. StMSI1 OE lines produce aerial tubers accompanied by reduced expression of StBMI1 and increased expression ofmiR156a/b/c. Aerial stolons (A and B; white arrows) and tubers from axillary nodes under SD induction (C and D) in StMSI1OElines, OE1 andOE3, respectively. Relative miR156a/b/c levels in leaves of StMSI1OE line OE3 compared to wild-type (WT) plant(E). As miR156a, miR156b, and miR156c sequences in potato cannot be distinguished at the mature miRNA level, we havereferred them as “miR156a/b/c” throughout the text. Relative transcript levels of StBMI1-1, StBMI1-3, and StBMI1-4 in leaves ofStMSI1-OE line (OE3) compared to VC line (F). Relative levels of StLOG3 (G) and StGA2ox1 (H) in StMSI1-OE line (OE3) areshown in comparison to VC plants. Relative levels of StMSI1 andmiR156a/b/c in leaves during juvenile versus adult phase inwild-type potato plants (I). For (E), (F), (H), and (I), data are mean of three biological and three technical replicates with6 SD. U6 wasused as a reference gene for miRNAs, whereas EIF3e was used for gene expression analysis. Relative level in wild type wasconsidered as “1” with 6 SD for (E), (F), (H), and (I), whereas relative levels in juvenile phase was considered as “1” with 6 SD.Student’s t test was performed to check significance with *P , 0.05, **P , 0.01, and ***P , 0.001. ns, not significant. Scalebars 5 7 cm (A) and 5 cm (B–D).





StBMI1-2 (Supplemental Fig. S7), and the absence ofthe RING domain in the StBMI1-3 protein, we choseStBMI1-1 for its functional characterization in thisstudy.

StBMI1-1 Knockdown Affects Leaf and Root DevelopmentBut Induces Aerial Tuber Formation

To investigate if StBMI1-1 functions upstream ofmiR156 in potato, its antisense (AS; StBMI1-1-AS)lines were generated (Supplemental Fig. S8, A and B).Of the two lines (G9 and G12), G9 had ;35% down-regulation, whereas G12 had ;30% downregulationof StBMI1-1 (Fig. 4D). The overall architecture of theplant was weak in StBMI1-AS line (G9) when com-pared to wild-type or VC plants (Fig. 4E). Shoot bio-mass was significantly lower in StBMI1-1-AS lines G9

and G12 (Supplemental Fig. S8C). The expression ofStLOG3 remained unchanged, whereas that of StGA2ox1was upregulated in the StBMI1-1-AS line (SupplementalFig. S8D). The StBMI1-1-AS line (G9) showed a reduc-tion in leaf size as well as leaf compounding post2–3 weeks of incubation under LD conditions insoil (Fig. 4F). The leaf phenotypes were similar tomiR156-OE lines K1 and K6 (Fig. 4G) as well asStMSI1-OE lines (Fig. 2C). On an average, wild-typeor VC plants had seven leaflets per leaf in matureplants, whereas the StBMI1-1-AS lines (G9 and G12)always had fewer than five leaflets per leaf (Fig. 4H).Root biomass was also significantly lower in theStBMI1-1-AS line (G9) compared to wild-type plants(Fig. 4I). qPCR analysis demonstrated that the StBMI1-1-AS line (G9) had .2-fold increase of miR156a/b/clevels in leaves compared to wild-type or VC plants(Fig. 4J). Similar to the StMSI1-OE lines, an extended

Figure 4. StBMI1-1 and miR156 show antagonistic expression pattern in stolon under LD/SD conditions and AS lines of StBMI1-1 develop aerial tubers under SD. A and B, Effect of LD and SD photoperiod on the expression of StBMI1-1 in different tissuetypes—shoot tip, leaf, stem, and root (A) and in various stages of stolon-to-tuber transitions (B).Wild-type (WT) potato plants weregrown under LD/SD conditions for 14 d, after 8 weeks of growth in soil under LD conditions. C, The relative levels of miR156a/b/cin shoot-tip and stolon tissues at 14 d under LD/SD conditions. The relative transcript levels of StBMI1-1 or miR156a/b/c indifferent tissues under SD conditions is calculated considering its levels under LD condition as “1” with6 SD. EIF3e andU6wereused as reference genes for StBMI1 and miR156a/b/c expression analysis, respectively. D, The transcript levels of StBMI1-1 inleaves of AS transgenic lines G9 and G12 compared to wild type and VC lines. Analysis was performed with three biologicalreplicates per line. qPCR was performed using StBMI1-1 specific primers. E, StBMI1-1-AS transgenic lines along with wild typeand VC. F and G, Leaf phenotype of StBMI1-1-AS line G9 along with wild-type and VC plants after the second and third week insoil (F) and the leaf phenotype of miR156-OE lines (K1 and K6) after the week in soil (G). H and I, Number of leaflets per leaf (H)and root biomass (I) in StBMI1-1-AS transgenic line (#G9) and VC are presented with respect to wild type. Data are representedfrom nine independent plants per line. J, Relative miR156a/b/c levels in AS line #G9 and VC with respect to wild type. K, For-mation of aerial tubers in StBMI1-1 AS line (#G9) after 4 weeks of SD incubation. White arrows indicate aerial tubers. U6 wasused as a reference gene. Student’s t test was performed to check significance with *P , 0.05, **P , 0.01, ***P , 0.001, and****P , 0.0001. gfw, grams fresh weight; ns, not significant. Error bars represent 6 SD. Scale bars 5 1 cm (E–G and K).


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incubation of the StBMI1-1-AS line (G9) under SDconditions resulted in formation of aerial tubers in;50% of the plants (Fig. 4K); however, no such phe-notype was observed in G9 line under LD conditions(Fig. 4E).

Knockdown of StMSI1 and OE of StBMI1-1 AffectsLeaf Development

To assess the effect of StMSI1 knockdown on potatophenotype, two independent StMSI1-AS lines (AS8and AS9) displaying up to 50% reduction of StMSI1transcript levels were selected for phenotypic analysis(Supplemental Fig. S9A). StMSI1-AS lines exhibitedreduced plant height (Supplemental Fig. S9B), inter-nodal distance (Supplemental Fig. S9C), and leaf length(Supplemental Fig. S9, D and E) compared to wild-typeor VC plants. Unlike StMSI1-OE lines, there wasno effect on the root biomass of the StMSI1-AS lines(Supplemental Fig. S9, F andG). The levels ofmiR156a/b/c were significantly decreased in the StMSI1-AS line(AS8) compared to the VC plants (Supplemental Fig.

S9H). Two independent StBMI1-1-OE lines (II-9 andII-10; Supplemental Fig. S10, A and B) showed an in-crease in leaf size and number of leaflets per leaf com-pared to wild-type plants (Supplemental Fig. S10C). Inmature plants, the average number of leaflets per leafincreased to nine or more in OE lines in comparison toseven in the wild type or the VC (Supplemental Fig.S10D). However, root biomass was not affected in theStBMI1-1-OE lines (Fig. 5I). Shoot and root biomass didnot show any changes in the StBMI1-1-OE lines (II-9and II-10) compared to the VC plants (SupplementalFig. S10, E and F).

OE or Knockdown of StMSI1 or StBMI1-1Influences Tuberization

Tuber yield potential was assessed both in in vitroand in soil-grown plants of the StMSI1-OE/AS,miR156-OE, and StBMI1-1-OE/AS lines. Under tuber-inducing in vitro conditions, the StMSI1-OE (OE3),StMSI1-AS (AS8), StBMI1-1-AS (G9), and miR156-OElines exhibited delayed tuberization and reduced

Figure 5. OE or knockdown of StMSI1 or StBMI1-1 influences tuberization. The number of tubers produced byOE and AS lines ofStMSI1 and StBMI1-1 on 8% (w/v) Suc in dark under in vitro conditions over a period of 28 d. Data are plotted at 7-d interval alongwith miR156a/b/c OE, wild-type, and VC lines. A, For better representation, one representative line per transgenic construct wasplotted. B, The relative transcript levels of StBEL5, StSP6A, miR172, StSP5G, StCO2, and StCO-LIKE9 in leaves of StMSI1-OE(OE3) and StBMI1-1-AS (#G9) lines incubated under SD for 20 d are shown with respect to VC plants. Three biological and threetechnical replicates were used for the analysis. EIF3e was used as a reference for genes, and U6 for miR172. C to F, Number oftubers and tuber yield was calculated from soil-grown plants after 1 month of SD induction in all four types of transgenic lines—StMSI1-OE (OE1 and OE3), StMSI1-AS (AS8 and AS9), StBMI1-1-OE (II-9 and II-10), and StBMI1-1-AS (G9 and G12) lines,compared to wild-type (WT) and VC plants. Data are plotted from six individual plants per line for StMSI1-OE/AS lines, andnine individual plants per line for the StBMI1-1-OE/AS lines. Student’s t test was performed to check significance with *P, 0.05,**P , 0.01, ***P , 0.001, and ****P , 0.0001. gfw, grams fresh weight; ns, not significant. Error bars represent 6 SD.


















tuber yield compared to wild type, whereas theStBMI1-1-OE line (II-9) showed an earliness forin vitro tuberization as well as increased tuber yield(Fig. 5A). The expression of tuber marker genes, suchas miR172 (Martin et al., 2009), StBEL5 (Banerjee et al.,2006a), and StSP6A (Navarro et al., 2011) were sig-nificantly reduced in leaves of the StMSI1-OE andStBMI1-1-AS lines (Fig. 5B). In contrast, the expres-sion of StSP6A repressors, such as StCO2, StCO-like 9,and StSP5G were significantly higher in leaves of theStMSI1-OE and StBMI1-1-AS lines (Fig. 5B), which isconsistent with the reduced tuber yield phenotype inthese lines. Although there was no effect on tubernumbers in the StMSI1-OE or -AS lines in soil-grownplants (Fig. 5C), both showed ;3- to 5-fold reductionin tuber yield compared to wild type (Fig. 5D;Supplemental Fig. S11, A and B). The StBMI1-1-OElines showed no difference in tuber numbers, but theyhad increased tuber yield (Fig. 5, E and F). However,the StBMI1-1-AS lines showed a reduction in tubernumbers as well as in tuber yield (Fig. 5, E and F).

RNA-Seq Analysis Revealed Common DE Genes betweenthe StMSI1-OE and StBMI1-1-AS Lines

To understand the cause for aerial tuber formation,we performed paired-end RNA-seq from axillarynodes of the SD-induced StMSI1-OE and StBMI1-1-ASlines along with the VC plants. Overall, 172 millionfinal clean reads were obtained from 181 million rawreads after quality-filtering and adapter trimming.Of them, 88.86% read pairs uniquely mapped to thepotato genome (Table 1). Downstream processing ofRNA-seq data revealed that ;7,386 and 1,690 geneswere differentially expressed (DE) in the StMSI1-OEand StBMI1-1-AS lines, respectively, compared tothe VC plants (Fig. 6A). Among the DE genes in theStMSI1-OE line,;3,360 and 4,026 genes were up- anddownregulated, respectively, whereas 921 genes wereup- and 769 were downregulated in the StBMI1-1-ASline. Approximately 6,363 DE genes were unique onlyto the StMSI1-OE line, whereas ;667 genes werespecific to the StBMI1-1-AS line (Fig. 6A). Interest-ingly, we observed that out of 1,690 DE genes iden-tified in the StBMI1-1-AS line, 1,023 genes (;60%)

were common between both of the lines (StMSI1-OEand StBMI1-1-AS). When common DE genes were an-alyzed further, we found that 345 genes were upregu-lated and 371 were downregulated in both lines.However, 307 DE genes from the common pool showedopposite expression patterns in the StMSI1-OE andStBMI1-1-AS lines; here, 123 genes were upregulated inthe StMSI1-OE, but downregulated in the StBMI1-1-ASline, and 184 genes were downregulated in the StMSI1-OE, but upregulated in the StBMI1-1-AS line (Fig. 6A).Among the common DE genes, a large number relatedto auxin and brassinosteroid (BR) biosynthesis, trans-port, and signaling were downregulated, whereas thegenes involved in CK transport and signaling wereupregulated.

RNA-Seq and qPCR Analysis Revealed Altered Expressionof Histone Modifiers, Tuber Markers, andDevelopment-Related Genes in the StMSI1-OE andStBMI1-1-AS Lines

RNA-seq analysis also revealed downregulation ofStBMIs (StBMI1-1 and StBMI1-3), LHP1 (which re-cruits PRC1 complex over H3K27me3-modified targetgenes; Turck et al., 2007), and HDA19 (that cata-lyzes the removal of acetylation marks on targetgenes), in the StMSI1-OE line compared to the VC(Supplemental Table S2). Further, we could identifythat Trithorax group members, such as SDG4 and amember of SET7/9 family (SET7/9) having histoneH3K4 methyltransferase activity, were significantlyupregulated in these lines (Supplemental Table S2).

The expression of SDG4 and SET7/9 genes wereupregulated, whereas that of LHP1was downregulatedin the StMSI1-OE line (Fig. 6B). The abscisic acid sig-naling gene PYL4, and auxin-responsive genes such asSAUR and ARF16 and epidermal patterning factor (EPF),were downregulated in the StMSI1-OE line (Fig. 6B;Supplemental Table S2). The transcript levels of a genethat induces tuber formation, StSP6A (Navarro et al.,2011) and a member of the tuberigen activation com-plex, St 14-3-3 (Teo et al., 2017), were significantly re-duced in the StMSI1-OE line (Fig. 6B; SupplementalTable S2). On the other hand, genes involved incell division (cyclin A2, CycA2), shoot-apical meristem

Table 1. Summary of read counts and alignment statistics for axillary node samples of potato after RNA-seq

Sample Name Raw Reads Cleaned Reads% Read Pairs Uniquely

Mapped to the Genome

VC line replicate 1 26,452,575 25,414,994 89.06%VC line replicate 2 21,156,895 20,288,178 86.84%VC line replicate 3 19,426,628 18,589,972 85.46%StMSI1 OE line OE3 replicate 1 23,727,153 22,661,717 90.78%StMSI1 OE line OE3 replicate 2 19,741,392 18,514,718 92.89%StMSI1 OE line OE3 replicate 3 28,251,381 26,807,328 86.92%StBMI1-1 AS line G9 replicate 1 25,269,133 24,046,116 87.22%StBMI1-1 AS line G9 replicate 2 16,943,329 16,239,229 91.72%Total 180,968,486 172,562,252 –


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formation and maintenance (CLAVATA1, CLV andERECTA, ERC), and leaf development (TEOSINTEBRANCHED1/CYCLOIDEA/PCF TF and SPL9)were altered in the StBMI1-1-AS line compared to theVC (Fig. 6C; Supplemental Table S3). Moreover, thetranscript levels of tuberization repressors, such asPHYB2 and CO1 and CO2, were significantly in-creased in the StBMI1-1-AS lines (Fig. 6C; SupplementalTable S3). Validation of common DE genes betweenthe StMSI1-OE and StBMI1-1-AS lines showed thatthree genes, such as pseudo-response regulator (governscircadian rhythm and plant fitness), protease (associatedwith leaf senescence), and chalcone synthase (involvedin flavonoid biosynthesis) were upregulated, whereasseveral other genes, such as HD-ZIP TF (required forvascular development), longifolia (involved in leafmorphology), and glabra (associated with trichomebranching), were downregulated in the StMSI1-OEand StBMI1-1-AS lines (Fig. 7A; Supplemental TableS4). Among the common DE genes between theStMSI1-OE and StBMI1-1-AS lines, we found 22 genes(of 1,023) were associated with sugar transport orsugar/starch metabolism (Supplemental Table S4).

Phytohormone-Related Genes Were Affected inStMSI1-OE and StBMI1-1-AS Lines

Analysis of common DE genes between the StMSI1-OE and StBMI1-1-AS lines showed that the genesencoding auxin transport proteins (auxin efflux1 and 2)

and auxin response proteins (ARP, expansin,AUX/IAA,andAUX-IAA3) were downregulated in the StMSI1-OEand StBMI1-1-AS lines compared to the VC (Fig. 7B;Supplemental Table S4). Additionally, genes relatedto BR biosynthesis (cytoP450) and signaling (BR kinase,thesasus, and Phi-1 protein) were downregulated (Fig. 7B;Supplemental Table S4). The transcript of a gene (sigmafactor sigb regulation protein rsbq), which acts as a neg-ative regulator of strigolactone (STL) signaling, was high,whereasanSTL-responsivegene (PGSC0003DMT400043632)was downregulated in the axillary nodes of the StMSI1-OE and StBMI1-1-AS lines (Supplemental Table S4).Further, the StMSI1-OE lines had reduced levels ofGA biosynthesis genes, GA20ox and GA3ox, and in-creased expression of a GA catabolic gene, GA2ox, inaxillary nodes (Supplemental Table S2). Genes encod-ing CK transporters, such as purine transporter2 and 3, aswell as a responsive gene (zeatin riboside), were upre-gulated in StMSI1-OE and StBMI1-1-AS lines in com-parison to the VC (Fig. 7B). Further, heat map (Fig. 7C;Supplemental Table S4) showed that among the 28 auxin-related genes, 23 were downregulated in the StMSI1-OE line and 19were downregulated in the StBMI1-1-ASline. The remaining five genes were upregulated in theStMSI1-OE line and nine were downregulated in StBMI1-1-AS line compared to the VC (Fig. 7C; SupplementalTable S4). Of the seven BR-related genes, six weredownregulated in the StMSI1-OE line, whereas all sevenwere downregulated in the StBMI1-1-AS line comparedto the VC (Fig. 7C; Supplemental Table S4). Gene ontol-ogy (GO) analysis forDEgenes categorizedGO terms into

Figure 6. RNA-seq analysis and validation of DE genes specific to StMSI1-OE or StBMI1-1-AS line. A, Venn diagram shows thesummary of DE genes in StMSI1-OE and StBMI1-AS lines compared to VC line. B and C, Validation of selective StMSI1-specific (B)and StBMI1-1-specific genes (C) compared to VC. The relative fold-change of respective gene expression in StMSI1-OE or StBMI1-1-AS lines was calculated with respect to its transcript level in VC plant. Student’s t test was performed to check significance with*P, 0.05, **P, 0.01, ***P, 0.001, and ****P, 0.0001. ns, not significant. Error bars represent6 SD from three biological andthree technical replicates. EIF3e was used as a reference gene.



















different biological processes, molecular functions,and cellular components (Supplemental Tables S5–S7;Supplemental Fig. S12).

Grafting of StMSI1-OE or StBMI1-1-AS on Wild TypeInfluenced miR156 Accumulation and Reduced RootBiomass In Wild-Type Stock

Considering the mobile nature of tuberization sig-nals, to investigate if OE of StMSI1-OE or StBMI1-1 knockdown has any effect on miR156 expression,different combinations of homo- and hetero-graftswere made under in vitro conditions (Fig. 8A). Over-all, we produced ;70% to 80% successful grafts and3 weeks after grafting, several root growth parametersweremeasured. As expected, homo-grafts of StMSI1-OEor StBMI1-1-AS showed a reduction in number of roots,root length, and biomass compared to wild-type homo-grafts. Interestingly, we noticed that root growth wasaffected in hetero-grafts containing StMSI1-OE orStBMI1-1-AS as scion and wild type as stock as well asin reverse grafts (Fig. 8, A–D). To analyze the cause ofreduced root growth, the expression of auxin and CK

transport/signaling genes was quantified in roots of allhomo- and hetero-grafts (Fig. 8E). The expression levels ofauxin efflux carrier1 and expansin were reduced, whereasthose of CK transporters (purine transporter2 and 3) wereincreased in all homo- and hetero-grafts compared towild-type homo-grafts (Fig. 8E), Additionally, we foundthat the precursor levels ofmiR156a/b andmiR156cwerealso high in roots of all homo- andhetero-grafts comparedto wild-type homo-grafts (Fig. 8E).

ChIP-qPCR Shows Enrichment of H3K27me3 andH3K4me3 Over StBMI1-1 and miR156 Genes, Respectively

We noted the upregulation of miR156a/b/c (Figs.3E and 4J) and suppression of StBMI1-1 and StBMI1-3(Fig. 3F) in the StMSI1-OE line. To understand thepossible cross talk between these regulators, ChIP-qPCR analysis was performed to quantify the levelof repressive mark (H3K27me3) at the first intron ofStBMI1-1 and StBMI1-3 genes. Our analysis foundthat the levels of H3K27me3 on StBMI1-1 and StBMI1-3 genes were significantly increased in the StMSI1-OEline (Fig. 9A). Apart from increased levels of miR156a/

Figure 7. Auxin- and BR-related genes were downregulated, whereas CK-related genes were upregulated in StMSI1-OE andStBMI1-1-AS lines. A and B, Validation of selective DE genes common between StMSI1-OE and StBMI1-1-AS lines compared toVC line. The relative fold-change of respective gene expression in StMSI1-OE or StBMI1-1-AS line was calculated with respect toits transcript level in VC plant. Student’s t test was performed to check significance with * P, 0.05, **P, 0.01, and ***P, 0.001.Error bars represent 6 SD from three biological and three technical replicates. EIF3e was used as a reference gene. In (A), redunderlines represent the genes related to auxin, CK and BR metabolism and/or transport. C, Heat map was plotted for all auxin-and BR transport/signaling-related genes from a pool of DE genes common between StMSI1-OE and StBMI1-AS lines. Pseudo-Res. Reg., Pseudo Response Regulator; HD-ZIP TF, Homeodomain-ZIP Transcription Factor; ARP, Auxin Response Protein;Theseus, Receptor-like protein kinase THESEUS 1.


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b/c (Figs. 3E and 4J), the expression ofmiR156ewas alsohigh in leaves of StMSI1-OE and StBMI1-1-AS lines(Fig. 9B). Moreover, miR156f level was high in theStMSI1-OE line; however, it was surprisingly low inthe StBMI1-1-AS line (Fig. 9B). The transcript level ofTrithorax group members having histone H3K4 meth-yltransferase activity (SET7/9 and SDG4) was signifi-cantly higher in both StMSI1-OE and StBMI1-1-AS linescompared to VC (Fig. 9B). Hence, we tested the possi-bility of miR156 activation by quantifying the levels ofH3K4me3 marks made by Trithorax group members.We observed that H3K4me3 marks were increased atthe upstream regions of different miR156 family mem-bers (miR156b, miR156e, miR156f, and miR156g) in theStMSI1-OE and StBMI1-1-AS lines (Fig. 9C).

RLM-RACE Assay Confirms miR156-Mediated Cleavageof StSPL13

FromRNA-seq data, three SPL genes, including StSPL8,StSPL9, and StSPL13, were differentially downregulated in

StMSI1-OE or StBMI1-1-AS lines (Supplemental Tables S2and S3). Of these, StSPL9 and StSPL13 are predicted tobe cleaved by different miRNA156 members. Througha degradome analysis, Seo et al. (2018) recently showedthat StSPL13 is cleaved by miR156 in potato. In ouranalysis, we observed that the transcript levels ofStSPL6 and 13 were significantly reduced in bothStMSI1-OE and StBMI1-1-AS lines compared to theVC (Fig. 9D). However, StSPL3 and 8 transcriptlevels remain unchanged in the StMSI1-OE line, butboth were significantly reduced in the StBMI1-1-AS line(Fig. 9D). Further, psRNATarget analysis (http://plantgrn.noble.org/psRNATarget/) predicted that miR156e/f-5p/g-5p can also cleave StSPL13 with an expectancy value ofE 5 1.0, followed by miR156a/b/c members. Through amodified 59 RNALigase-Mediated RapidAmplification ofcDNA Ends (RLM-RACE) assay, we confirmed thatStSPL13 transcript is cleaved by miR156 members with100% cleavage efficiency (7 out of 7) at the 11th/12th nu-cleotide position (Fig. 9E). However, it may be noted thatthe cleavage site confirmed here on StSPL13 transcript isdifferent than that reported in Bhogale et al. (2014),

Figure 8. Hetero-grafts of StMSI1-OE or StBMI1-AS line with wild type showed altered root growth compared to homo-grafts ofwild type (WT). A, Two representative images of in vitro grown plants are shown for each combination of homo- and hetero-graftsafter 3 weeks of graft initiation. Scale bar5 1 cm. B to D, Average number of roots (B), root length (C), and root biomass (D) perhomo- or hetero-graft are presented. Statistical analysis was performed using Student’s t test, assuming unequal variances.Number of biological replicates (n) per each homo- or hetero-graft combination are shown below graphs. Statistical significanceindicatedwith *P, 0.05, **P, 0.01, ***P, 0.001, and ****P, 0.0001. gfw, grams freshweight; ns, not significant. Comparisonfor each homo- or hetero-graft was performed with respect to wild-type/wild-type homograft. Arrows (white, A) indicate the graftunions. E, Relative transcript levels of auxin and CK transport/signaling genes as well as premiR156a/b and premiR156c levels inroots of different homo- and hetero-grafts after 21 d. In (E), mean values of two biological replicates per graft combination wereplotted. EIF3ewas used as the reference gene for qPCR analysis. Error bars represent6 SD. AEC1, Auxin efflux carrier 1; PT, Purinetransporter; EXP, Expansin; miR156a/b, precursor of miR156a/b; miR156c, precursor of miR156c.






http://plantgrn.noble.org/psRNATarget/

http://plantgrn.noble.org/psRNATarget/


suggesting a different member of the miR156 family iscleaving StSPL13 transcript rather than miR156a/b/c.Additionally, psRNATarget analysis (Dai and Zhao, 2011)of 1,023 common DE genes unveiled that many of thesegenes could be targets of different miRNAs (SupplementalTable S8).

Promoters of StMSI1, StBMI1, and miR156 Members HaveNumerous Light Regulatory Motifs

When the promoter sequence of StMSI1 (;1.5 kb)was analyzed by the PlantCARE tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/),we could identify several light regulatory motifs (LREs),e.g. two Box4motifs, fourGT1motifs, oneGAmotif, twoTCT motifs, and one ATCT motif (Supplemental TableS9). In addition, twoCAT-boxmotifs related tomeristemexpression were identified in its promoter sequence(Supplemental Table S9). Similarly, several LREs were

found in the 1.5-kb promoters of all 11 miR156 mem-bers in potato (miR156a-k) and three StBMI genes(StBMI1-2, StBMI1-3, and StBMI1-4; SupplementalTable S9) Additionally, numerous Polycomb re-sponse elements (PREs) were identified in the pro-moters of miR156 members and StBMI1 genes(StBMI1-2, StBMI1-3, and StBMI1-4; SupplementalTable S10).

DISCUSSION

PcG proteins are important regulators of plant de-velopment and control the expression of homeoticgenes involved in meristematic activity and organ dif-ferentiation (Goodrich et al., 1997). Previous studiesin Arabidopsis have shown that a large number ofmiRNAs involved in phytohormone regulation andother key developmental processes are also regulatedby PcG proteins (Lafos et al., 2011; Teotia and Tang,

Figure 9. ChIP-qPCR validatesH3K27me3-mediated repression of StBMI1 andH3K4me3-mediated activation ofmiR156. A, Theenrichment of H3K27me3 repressive marks on the promoters of StBMI1-1 and StBMI1-3 in StMSI1-OE line (OE3) compared toVC. B, The relative levels of miR156e and miR156f in StMSI1-OE and StBMI1-AS lines compared to VC. C, The enrichment ofH3K4me3 activation marks over the promoters of miR156 members in the transgenic lines StMSI1-OE and StBMI1-AS comparedto VC. D, The relative levels of StSPLs (StSPL3, StSPL6, StSPL8, and StSPL13) in StMSI1-OE and StBMI1-AS lines compared to VC.E, Alignment of StSPL13 transcript and miR156f-5p is shown with predicted efficiency of cleavage (E 5 2). “7/7” represents theactual cleavage frequency after RLM-RACE analysis. The relative enrichment of respectivemarks in the StMSI1-OE and StBMI1-ASlines was calculatedwith respect to the VC sample. Student’s t test was performed to check significance indicatedwith *P, 0.05,**P , 0.01, ***P , 0.001, and ****P , 0.0001. ns, not significant. Error bars represent 6 SD from three biological and threetechnical replicates. EIF3e orU6was used as a reference gene. F, The proposed model considering the cross talk among StMSI1,StBMI1-1, andmiR156 during aerial and below-ground tuber formation in potato under SD photoperiodic conditions. (Note: Thenomenclature of potato miR156 members is as per the miRbase [http://www.mirbase.org/]. The genomic locations of miR156precursors and promoters used for ChIP-qPCR analysis are given in Supplemental Table S13.


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http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

http://bioinformatics.psb.ugent.be/webtools/plantcare/html/








http://www.mirbase.org/



2015). Although much progress has been made to un-derstand the role of PcG proteins in Arabidopsis, theirrole in potato development remains to be explored.Here, we have shown that two PRC members, StMSI1and StBMI1-1, regulate tuber development in potato bycontrolling the expression of miR156 and hormonalresponse in a photoperiod-dependent manner.

SD Photoperiod Influences Expression of StMSI1,StBMI1-1, and miR156 in Stolon

MSI1 is a crucial component of several histonemodifier complexes that are involved in meristemmaintenance, branching, flowering, and leaf andovule development (Hennig et al., 2003, 2005). Pre-vious studies (Hennig et al., 2003; Liu et al., 2016)showed ubiquitous expression ofMSI1 in Arabidopsisand tomato. Similarly, we noticed a ubiquitous expres-sion pattern of GUS in the promStMSI1::GUS-pBI121potato lines, with highest expression in axillary nodesand root tips (Fig. 1, C and F). Additionally, when thesepromoter lineswere subjected to LD/SD induction, GUSexpression was higher in swollen stolon under SD pho-toperiod compared to stolons from LD (Fig. 1, G and H).qPCR analysis further validated the higher expressionof StMSI1 in stolon under SD conditions (Fig. 1A).These observations indicate that photoperiod couldregulate StMSI1 expression in potato. In potato,miR156 has been shown to play an important role incontrolling tuber development and its expressionincreases in stolons under SD photoperiod (Bhogaleet al., 2014). Additionally, both in potato and Ara-bidopsis, miR156 has been shown to control juvenile-to-adult phase transition by targeting SPL proteins;miR156 is highly expressed during juvenile phase,but remain suppressed during the adult phase of theplant (Wu et al., 2009; Bhogale et al., 2014). Inter-estingly, in our study, the expression pattern of bothStMSI1 and miR156 was similar in stolons during SDinduction as well as in leaves during juvenile andadult phases in the potato plant (Fig. 3I). Apart fromthe factors reported in several studies (Hsieh et al.,2009; Lee et al., 2010; Xing et al., 2010; Yu et al., 2012,2013; Yang et al., 2013), recently, a PRC1 member(AtBMI1) has been shown to control miR156 ex-pression in Arabidopsis (Picó et al., 2015). We foundthat StBMI1-1 expression was significantly low, whereasthat ofmiR156was high in shoot tip and stolon under SDphotoperiod compared to LD conditions (Fig. 4A). Fur-ther, the presence of numerous LREs in the promoters ofStMSI1 and StBMI1 genes and 11 miR156 members inpotato (miR156a–j; Supplemental Table S9) suggeststhat photoperiod could regulate the expression of thesegenes. The identification of PREs in the promoters of allmiR156 members (Supplemental Table S10), suggests apossible role of PcG proteins in photoperiod-mediatedregulation of miR156 during the stolon-to-tuber tran-sition in potato. In this report, StMSI1 OE and StBMI1knockdown lines had an increased level of miR156(Figs. 3E and 4J) and both showed phenotypes similar

to miR156 OE, including aerial tubers (Figs. 3, C and D,and 4K) and reduced below-ground tuber yield (Fig. 5,D and F), suggesting that StMSI1 and StBMI1-1 func-tion upstream of miR156 in potato.

StMSI1-OE and StBMI1-1-AS Lines Exhibit Altered PlantArchitecture and Reduced Tuber Yield

Both StMSI1-OE and StBMI1-AS lines had drasticchanges in overall plant architecture (Figs. 2B and 4E)including reduced leaf compounding, lamina size,petiole length (Figs. 2, B and C; 4, E and F), root biomass(Figs. 3G and 4I), and tuber yield (Fig. 5, D and F). TheStMSI1-OE line also showed increased numbers of leafstomata, trichome length, and altered stem vascularbundles (Fig. 2, G–N). We demonstrated earlier thatmiR156 OE leads to reduced leaf size, compounding,and tuber yield in potato (Bhogale et al., 2014). Weobserved that StMSI1-OE and StBMI1-AS lines had 5-and 2-fold increase of miR156 expression, respectively(Figs. 3E and 4J). SPLs, the targets of miR156, workantagonistically to TEOSINTE BRANCHED1/CYCLO-IDEA/PCF transcription factors belonging to the class-IICINCINNATA subgroup during leaf development(Rubio-Somoza et al., 2014). The reduced transcriptlevels of several SPLs, including StSPL6 and -13 inboth lines (StMSI1-OE and StBMI1-AS; Fig. 9D) andRLM-RACE–mediated validation of StSPL13 cleavageby miR156 (Fig. 9E), further justifies the reduced leafsize and compounding phenotype in both transgeniclines. A number of reports (Uchida et al., 2007; Hayand Tsiantis, 2010, Mahajan et al., 2016) have dem-onstrated that class-I KNOX genes regulate meristemactivity, leaf architecture, and compounding. Thehigh level of POTH15 (a class-I KNOX gene in potato)transcript in the StMSI1-OE line (Supplemental Table S2)and the presence of ;800 common DE genes betweenthe POTH15-OE and StMSI1-OE lines (SupplementalTable S4) could also be the cause of altered leaf ar-chitecture. In StMSI1-OE and StBMI1-AS lines, wenoticed downregulation of several genes coding forauxin efflux carriers (PIN proteins), HD-ZIP TFs, andBR signaling pathway genes (Fig. 7, A and B), whichhave been shown to affect vascular bundle formation(Mattsson et al., 1999; Sieburth, 1999; Lee et al., 2018).PcG proteins control the development of primary

and lateral roots through regulating stem cell activity(Aichinger et al., 2011) and auxin transporter PIN1expression (Gu et al., 2014). Auxin and BR stimulate,whereas CK inhibits, lateral root development (Müssiget al., 2003; Aloni et al., 2005). Our RNA-seq datashowed high expression of CK and low expressionof auxin- and BR-signaling–related genes (Fig. 7B;Supplemental Tables S2–S4). A number of genes, suchas PINs, PLETHORA (PLT), SCARECROW, and ARFsthat are involved in root development (Sabatini et al.,2003; Aida et al., 2004; Blilou et al., 2005; Wang et al.,2005), were affected in the StMSI1-OE or StBMI1-1-ASlines, possibly explaining the reduced root growth












phenotype. Similar to miR156-OE lines (Bhogale et al.,2014), StMSI1-OE and StBMI1-1-AS lines showed asignificant reduction in below-ground tuber yieldcompared to wild-type plants (Fig. 5, D and F). Incontrast, the StBMI1-1-OE lines showed increasedtuber yield (Fig. 5F). We further noticed that theStMSI1-AS lines had comparatively reduced plantarchitecture (Supplemental Fig. S9) and tuber yield(Fig. 5D). ConsideringMSI1 functions as a componentof both activator and repressor complexes, we assumethat its moderate levels are essential for tuber devel-opment. Similar results were also observed in thein vitro tuberization experiment (Fig. 5A). One ofthe reasons for reduction in below-ground tuber yieldcould be due to the weaker plant architecture of theselines than that found in wild type. Further, the down-regulation of crucial tuber marker genes downstreamof miR156 in the tuberization pathway, for examplemiR172 (Martin et al., 2009), StBEL5 (Banerjee et al.,2006a), and StSP6A (Navarro et al., 2011), and theupregulation of tuber growth repressors (StPHYB,StCO, and StSP5G; Jackson et al., 1996; Navarro et al.,2011; Kloosterman et al., 2013), could be another reasonfor reduced tuber yield in these lines (Figs. 5B and 6C;Supplemental Tables S2 and S3). Moreover, we ob-served an increase in miR156 (Figs. 3E and 4J), but areduction in miR172 expression (Fig. 5B) in the StMSI1-OE and StBMI1-1-AS lines. psRNATarget analysis un-veiled numerous common DE genes as targets ofdifferent miRNAs, including miR156 and miR172.Approximately 247 common DE genes (of 1,023) re-lated to plant growth and development were pre-dicted to be cleaved by miR156 and miR172 familymembers (Supplemental Table S8), suggesting thataltered levels of miR156 and miR172 and their po-tential downstream target genes could have alsocontributed to the low tuber yield phenotype. Inter-estingly, grafting of StMSI1-OE or StBMI1-1-AS ontowild-type stock resulted in reduced root biomass(Fig. 8, A–D) and showed increased accumulation ofmiRNA156a/b and -c precursors in the roots of wild-type stocks (Fig. 8E), suggesting that PRC proteinscould have influenced the accumulation of miR156 inroots. The reduced root biomass in these hetero-graftscould possibly be due to altered expression of genesencoding auxin and CK transport/signaling proteinsin roots of these hetero-grafts (Fig. 8E). These findingsare consistent with the earlier report of Bhogale et al.(2014), where the authors demonstrated that miR156functions as a potential mobile signal in potato.

Cross Talk of Histone Modifiers Regulates miR156 andAlters Hormonal Response during Aerial Tuber Formationin StMSI1-OE and StBMI1-1-AS Lines under SD Photoperiod

In potato plant, every axillarymeristem possesses theability to form a stolon/tuber; however, this potentialremains suppressed in all meristems except the below-ground one (Ewing and Struik, 1992). In this study, the

StMSI1-OE and StBMI1-1-AS lines produced aerialstolons/tubers from axillary nodes (Figs. 3, A–D, and4K), a phenotype that matched with our previousdemonstration of miR156 OE in potato (Bhogale et al.,2014). Both of these lines showed high levels of miR156expression (Figs. 3E and 4J), indicating a possible reg-ulation of miR156 either through StMSI1 or StBMI1-1.The StBMI1-1-AS lines show a weaker phenotype ofaerial tuber development than either the StMSI1-OE ormiR156-OE lines. It could be because of a lower level ofRNA suppression (which was ;35%) in the StBMI1-1-AS line (G9). Also, it is possible that the function of fourpotato BMI proteins could be redundant. Hence, si-lencing only StBMI1-1 might not result in a more ro-bust phenotype. Recent studies have demonstratedthat BMI-mediated suppression of miR156 (Picó et al.,2015) triggers onset of the adult phase in Arabidopsis,which is consistent to our observation of increasedmiR156 levels in the StBMI1-1-AS line in potato(Fig. 4J). Moreover, the presence of multiple BMI1-binding motifs (Merini et al., 2017) in the promoterand precursor sequences of miR156 further supportsthe notion. However, the reason behind the increasedlevel of miR156 in the StMSI1-OE line (Fig. 3E) and theaerial tuber phenotype (Fig. 3, C and D) was not clear.RNA-seq of axillary nodes from both of these linesprovided crucial insights of StMSI1-mediated regulationof miR156.

The StMSI1-OE line exhibited altered expression ofseveral genes encoding histone modifiers (SupplementalTable S2). For example, the expression of PRC1 mem-bers, such as StBMIs (StBMI1-1, 1-3, and 1-4) andStLHP1, was reduced in the StMSI1-OE line. BMI1 andLHP1 maintain repressed states of target genes throughassistingH2Aubiquitination andmaintainingH3K27me3modification, respectively (Derkacheva et al., 2013).Additionally, genes encoding Histone Deacetylase19(Supplemental Table S2), ring finger proteins, and E3ubiquitin ligase PUB14, involved in suppression oftarget genes, were downregulated (SupplementalTable S4; common down sheet). Enrichment ofthe repressive mark (H3K27me3) on the first intronsof StBMI1-1 and -3 genes in the StMSI1-OE line(Fig. 9A), as well as the presence of several PREsin StBMI (StBMI1-2, StBMI1-3, and StBMI1-4) pro-moters (Supplemental Table S9), further supports theregulation of StBMI1 genes by the PRC2 complex. Inthe StMSI1-OE line, we further observed upregula-tion of genes encoding JMJC domain-containing H3K9demethylase (Sun and Zhou, 2008; Supplemental TableS2) and Trithorax group members (SDG4 and SET7/9;Fig. 6B), which are involved in catalyzing H3K4 meth-ylation of target genes (Cartagena et al., 2008). More-over, ChIP assay confirmed the increased enrichmentof H3K4me3 modification of the miR156 promoter(Fig. 9C). On the basis of these findings, we assume thatthe combined effect of reduction in repressive histoneubiquitination and the increase in expressive methylmodification of the miR156 locus might have resultedin its enhanced expression in both StMSI1-OE and


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StBMI1-1-AS lines. Additionally, the presence of PREsand BMI1-binding motifs in the promoters of miR156members (Supplemental Table S10) suggests that PcGproteins can regulate miR156 expression in potato.From the RNA-seq analysis, we observed that 1,023

DE genes were common between the StMSI1-OE andStBMI1-1-AS lines (Supplemental Table S4). Subse-quent analysis of these common DE genes hinted at thecause of aerial stolon/tuber formation from axillarynodes of these lines. Bhogale et al. (2014) showed thatthe miR156-OE lines had higher levels of CK as wellas increased expression of a CK biosynthesis gene(LONELY GUY1; LOG1) and a responsive gene(StCyclin D3.1). In tomato, Eviatar-Ribak et al. (2013)demonstrated that OE of SlLOG1 causes developmentof mini-tubers from axillary nodes in tomato. Inter-estingly, in both the StMSI1-OE and StBMI-1-AS lines,we also found increased expression of CK transportand response genes in axillary nodes (Fig. 7B). Al-though we could not find CK biosynthesis genes to bedifferentially expressed in StMSI1-OE and StBMI1-ASlines in the RNA-seq data (Supplemental TablesS2–S4), we observed that the expression of StLOG3was high in leaves of the StMSI1-OE line (Fig. 3H).Besides this, both of the transgenic lines shared anumber of common DE genes with that of the SlLOG1OE lines as described in Eviatar-Ribak et al. (2013),indicating that common downstream effectors of PcGand/or LOG genes could be involved in aerial tuberdevelopment. These results are consistent with the roleof CK as a branching stimulator (Domagalska andLeyser, 2011) and a tuber inducer (Palmer and Smith,1969). Two previous studies (Eviatar-Ribak et al., 2013;Bhogale et al., 2014) emphasized the potential role ofCK during aerial tuber development. Although therole of auxin in developmental phase transition ofstolon-to-tuber (Roumeliotis et al., 2012) is wellestablished, its role in aerial stolon/tuber develop-ment was not known. In our RNA-seq analysis, wenoted a reduced expression of several auxin transportand signaling genes in StMSI1-OE and StBMI1-1-ASlines (Fig. 7, B and C), suggesting the involvement ofauxin in aerial stolon formation from axillary nodes.In this scenario, what possible explanations could

describe the development of aerial stolons/tubersphenotype from the above-ground axillary nodes? Anumber of interesting observations lead us to specu-late that formation of aerial stolons/tubers could be asynergistic or cumulative effect of multiple factors: (1)It appears that the development of stolons/tubers(below- and above-ground) are physiologically twodistinct phenomena (light versus dark condition); (2)The aerial tuber phenotype in our transgenic lines ischaracteristic of the above-ground axillary nodes andunder the strict control of SD photoperiod; (3) miR156levels increase in below-ground stolon, but decreasein leaves under SD conditions (Bhogale et al., 2014);(4) The altered expression of StMSI1 and StBMI1-1 instolon matched with miR156 expression and presenceof high levels of miR156 in the StMSI1-OE and

StBMI1-1-AS lines; (5) Downregulation of auxin andSTL, but increase of CK response genes in both lines,might have caused axillary-bud break; and (6) Thereduced level of GA biosynthesis and response genesin axillary nodes of the StMSI1-OE line could havepromoted aerial tubers from stolons.To summarize, we propose a model to explain PcG-

protein–mediated regulation of tuber developmentin potato. StBMI1-1 suppresses miR156 expression,whereas StMSI1-OE induces miR156 expression bydownregulating StBMI1-1. Further, StMSI1-OE in-creases the expression of miR156 through Trithoraxgroup members involved in H3K4me3 modification.Increased miR156 causes downregulation of keytuberization genes (miR172, StBEL5, StCO, StSP5G,and StSP6A), which results in reduced below-groundtuber yield in the StMSI1-OE and StBMI1-1-AS lines.Additionally, reduced expression of auxin-, BR-, andSTL- (Pasare et al., 2013) related genes and increasedexpression of CK transport/signaling genes in theaxillary nodes of both transgenic lines inhibit theapical dominance effect and stimulate the inductionof axillary stolons. Finally, the reduced expression ofGA biosynthesis and signaling genes could supportthe development of aerial tubers from axillary sto-lons under SD photoperiod (Fig. 9F).

MATERIALS AND METHODS

Plant Material and Growth Conditions

Potato cultivar (Solanum tuberosum ssp andigena 7540), which tuberizesunder SD conditions (16-h dark/8-h light), but not under LD conditions(16-h light/8-h dark), was used throughout this study. Wild-type andigenaplants were propagated by subculturing nodal stem explants in Murashigeand Skoog’s basal medium (MS; Murashige and Skoog, 1962) supplementedwith 2% (w/v) Suc. In vitro plants were maintained in a plant growth in-cubator (Percival Scientific) at 22°C and light intensity of 300 mmol m22 s21

under LD conditions unless mentioned otherwise.

Phylogenetic Analysis

A phylogenetic tree was constructed for putative MSI-like protein se-quences from Arabidopsis (Arabidopsis thaliana), potato, tomato (Solanumlycopersicum), rice (Oryza sativa), Selaginella, Physcomitrella patens, andChlamydomonas using the software “T-COFFEE” (hRp://www.ch.embnet.org/soaware/TCoffee.html) and graphical representation was performedwith the software “TreeDyn” (v198.3; http://www.phylogeny.fr/one_task.cgi?task_type5treedyn; Dereeper et al., 2008). Similarly, a phylogenetic treewas also prepared for putative BMI1 orthologs from potato, tomato, andArabidopsis. For both gene families, the full-length amino acid sequenceswere used to build the phylogenetic trees.

MSI1 and BMI1-like Proteins in Potato

StMSI1 protein structure, as well as the position of WD repeats in theprotein sequence, were predicted using the WD repeat protein StructurePredictor tool developed by Wu et al. (2012). The binding partners of potatoStMSI1 were predicted using the “STRING” database (Szklarczyk et al.,2017).The Web CD Search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) was used to identify conserved domains in BMI1 proteinsfrom Arabidopsis, tomato, and potato. Domain schematics were drawn usingthe software “DOG2.0” (Ren et al., 2009) and edited manually. Genomic loca-tion of putativeMSI and BMI1 orthologs in potato were retrieved from the SpudDB Genome Browser in the PGSC database (http://solanaceae.plantbiology.









http://www.ch.embnet.org/soaware/TCoffee.html

http://www.ch.embnet.org/soaware/TCoffee.html

http://www.phylogeny.fr/one_task.cgi?task_type=treedyn

http://www.phylogeny.fr/one_task.cgi?task_type=treedyn

https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi

https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi

http://solanaceae.plantbiology.msu.edu/cgi-bin/gbrowse/potato


msu.edu/cgi-bin/gbrowse/potato). The Gene Structure Display Server(http://gsds.cbi.pku.edu.cn) was used for visualization of gene features, e.g.position of introns, exons, and conserved domains in four potato BMI1 proteins.

Tissue-Specific Transcript Abundance under SD andLD Conditions

For investigating the influence of photoperiod on the tissue-specific ex-pression of StMSI1 and StBMI1-1, in vitro grown wild-type andigena plantswere transferred to soil and maintained under LD photoperiod with 300 mmolm22 s21 light intensity for a period of 10 weeks (until they attained 10–12 leafstages) in a growth chamber (Percival Scientific). Later, half of the plants weretransferred to tuber-inducing SD photoperiodic conditions for 14 d, while theremaining plants were maintained under LD conditions. Different tissues(shoot tip, leaf, stem, root, and the stages of stolon-to-tuber transitions) wereharvested at 14-d post LD/SD induction in triplicates between Zeitgeber time(ZT)5 2 and ZT5 4. Total RNA was isolated using RNAiso Plus (DSS Takara)as per the manufacturer’s instructions. Complementary DNA synthesis wascarried out using 2 mg of total RNA, Superscript IV Reverse Transcriptase(Invitrogen) and Oligo dT primers. qPCR reactions were performed on aCFX96 Real-Time System (Bio-Rad) with gene-specific primers (SupplementalTable S11). The reactions were carried out using SYBR green master mix(Takara-Clontech) and incubated at 95°C for 30 s, followed by 40 cycles at 95°Cfor 5 s, gene-specific annealing temperature for 15 s, and extension for 72°C for15 s. PCR specificity was checked by melting curve analysis, and data wereanalyzed using the 2–DDCt method (Livak and Schmittgen, 2001).

Generation of Constructs and Potato Transgenic Lines

To generate constitutive OE constructs, full-length coding sequences ofStMSI1 (1,368 bp) and StBMI1-1 (1,292 bp) were amplified by reversetranscription-PCR from in vitro grown andigena plants using primers listedin Supplemental Table S11. PCR-amplified sequences were mobilized intobinary vectors, pBI121 and pCB201, respectively, downstream of the CaMV35S promoter (Xiang et al., 1999). A respective nonconserved sequence fromthe sense strand was used to design AS constructs for both StMSI1 andStBMI1-1 genes. PCR-amplified fragments (584 bp for StMSI1, whereas357 bp for StBMI1-1) were cloned in AS directions into the binary vectorspBI121 and pCB201, respectively, driven by the CaMV 35S promoter.StMSI1 and StBMI1-1 OE constructs were referred to as 35S::StMSI1-pBI121(StMSI1-OE) and 35S::StBMI1-1-pCB201 (StBMI1-1-OE), respectively, andtheir AS constructs were referred to as 35S::StMSI1-AS-pBI121 (StMSI1-AS)and 35S::StBMI1-1-AS-pCB201 (StBMI1-1-AS), respectively. The StMSI1promoter sequence (1,544 bp) was amplified from andigena genomic DNA(Supplemental Table S11) and cloned into a binary vector pBI121 upstreamof the GUS gene (uidA) to generate the promStMSI1::GUS-pBI121 construct.The microRNA156 OE construct (miR156-OE) is from the previous studyfrom our lab (Bhogale et al., 2014). All six types of binary constructs weretransformed into Agrobacterium tumefaciens strain GV2260 and transgenicpotato lines were generated as per the method described in Banerjee et al.(2006b). Transgenic andigena line containing 35S::GUS construct was used asa VC in the study. Several phenotypic characters (plant height, internodaldistance, leaf length, leaflet number per leaf, root length, tuber numbers, androot and tuber biomass yields) were recorded after 4 weeks of LD/SDinductions.

Analysis of StMSI1 Promoter Activity

StMSI1 promoter transgenic lines (promStMSI1::GUS-pBI121) were grownin vitro under LD conditions for 20 d. Promoter lines were also transferred tosoil and subjected to LD/SD induction for 15 d. Entire in vitro grown plantletsas well as stolon and tuber samples from LD/SD-induced soil-grown plantswere used for GUS assay. The protocol described in Jefferson (1987) was fol-lowed. After overnight incubation at 37°C, samples were bleached with a seriesof ethanol gradients (50% to 100%, v/v) and photographed under a stereomicroscope (model no. S8APO; Leica).

Histology and Scanning Electron Microscopy

For anatomical studies, amodified protocol of Cai and Lashbrook (2006)wasfollowed on leaf and stem tissues of 8-weeks–old LD grown (StMSI1-OE3 and

wild-type) plants. Ten-micrometer (10-mm) sections were obtained using amicrotome (Leica), cleared with xylene, and photographed under a compoundmicroscope (Zeiss). External leaf architecture of transgenic andwild-type plantswas documented using a Quanta 200 3D eSEM apparatus (FEI), under envi-ronmental mode (eSEM).

In Vitro Tuberization

In vitro tuberization experimentwas conducted as per the previous reportof Prematilake and Mendis (1999) with minor modifications. Shoot apex(2–3 cm) of wild-type, VC, and five types of transgenic andigena lines—StMSI1-OE (OE3); StMSI1-AS (AS8), StBMI1-1-OE (#II-9), StBMI1-1-AS(#G9), and miR156-OE—were subcultured on MS medium containing 2%(w/v) Suc and 0.2% (w/v) phytagel, and grown in vitro for 4 weeks underLD conditions. Single-node explants from the middle region of individualshoots were further cultured on MS medium with 8% (w/v) Suc (inductionmedium) and incubated for 4 weeks. Twelve independent plants for each linewere recorded for the number of tubers formed up to a period of 4 weeks.

RNA-Seq Analysis

For RNA-seq, 35S::StMSI1-OE (OE3), 35S::StBMI1-1-AS (#G9), and35S::GUS (VC) lines were grown in soil for 12 weeks under LD conditions andsubjected to SD induction for another 3 weeks (until the aerial stolon initiationstarts in OE3 and #G9 lines). Axillary nodes (5 mm in length) containing a partof the stem from either end of the node were harvested between ZT5 2 and ZT5 4 from six independent plants per line. All the nodes were harvested from theupper half of the plant. Samples were pooled from two to three plants formingeither two or three biological replicates per line. The total RNA was isolatedusing RNAiso Plus (DSS Takara). RNA concentration and purity wasmeasuredusing Qubit RNA Assay Kit in Qubit4.0 Fluorometer (Life Technologies) andRNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technolo-gies). Total RNA (3mg per sample) was used as an input material for the samplepreparations. Sequencing libraries were generated using NEBNext Ultra RNALibrary Prep Kit for Illumina (New England Biolabs) and index codes wereadded to attribute sequences to each sample. The library quality was assessedusing a Bioanalyzer 2100 system (Agilent Technologies). The clustering of theindex-coded samples was performed on a cBot Cluster Generation System us-ing a TruSeq PE Cluster Kit v3-cBot-HS (Illumina). After cluster generation, thelibrary preparations were sequenced on an Illumina Hiseq platform and 150-bppaired-end reads were generated.

The reads were aligned to the potato reference genome(PGSC_DM_v3.4_gene.fasta.zip) using the alignment software “STAR” (2.6.1c;Dobin et al., 2013). Downstream differential expression analysis of aligned readswas done using suite tools from “Tuxedo” (Trapnell et al., 2013), based on theprotocol of Mahajan et al. (2016). GO analysis was performed using the software“Blast2GO” v1.3.3 (https://www.blast2go.com/) for functional annotation ofDE genes (Conesa et al., 2005; Götz et al., 2008), as described in Mahajan et al.(2016). Validation of select target genes identified in the RNA-seq analysis wasdone using qPCR as described above in the “Tissue-Specific Transcript Abun-dance under SD and LD Conditions” section. The list of primers used are pro-vided in Supplemental Table S11.

RLM-RACE Assay

To map the cleavage site of miR156e/f-5p on the StSPL13 transcript, amodified 59 RLM-RACE assay was carried out using the First Choice RLM-RACE kit (Ambion) as described in Bhogale et al. (2014).

ChIP-qPCR Analysis

ChIPwas performed on potato leaves from 35S::GUS (VC), StMSI1-OE3, andStBMI1-1-AS#G9 plants using the reagents and protocol provided in a universalplant ChIP-Seq kit (Cat. No. C01010152; Diagenode) as per the manufacturer’sinstructions. The sheared chromatin was immunoprecipitated using theDiaMag protein A-coated magnetic beads (Diagenode) and 1 mg of eitheranti-H3K4me3 (Cat. No. C15410003; Diagenode), anti-H3K27me3 (Cat. Noab6002; Abcam), or anti-IgG antibody (Cat. No. C15410206; Diagenode), in eachreaction. Finally, eluted DNA was used for subsequent qPCR analysis withgene-specific primers (Supplemental Table S11).


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https://www.blast2go.com/




Identification of LREs, PREs, and BMI-Binding Sites

The 1.5-kb promoter sequences of StMSI1, all 11 miR156 members(miR156a-k), and three StBMI1 genes (StBMI1-2, -3, and -4) were searched forthe presence of LREs using the tool “PlantCARE” (Lescot et al., 2002). PREs(Xiao et al., 2017) and BMI-binding sites (Merini et al., 2017) were also searchedin the promoters of miR156 members using the tool “RSAT” (van Helden 2003;Nguyen et al., 2018). PREs were also identified in the promoters and genebodies of StBMI1. As the promoter sequence of StBMI1-1 gene is not annotatedin potato, LREs and PREs were identified from the 59 untranslated region (298bp) of its transcript sequence.

Grafting

Wild-type, StMSI1-OE, and StBMI1-1-AS lines were maintained in vitroon MS medium for 1 month under LD conditions. Three combinations ofhomo-grafts (wild-type/wild-type, StMSI1-OE/StMSI1-OE, and StBMI1-1-AS/StBMI1-1-AS) and four types of hetero-grafts (StMSI1-OE/wild-type,StBMI1-1-AS/wild-type, wild-type/StMSI1-OE, and wild-type/StBMI1-1-AS) were made under in vitro conditions as per the protocol described inBanerjee et al. (2006a), with modifications. After 1 week of in vitro incuba-tion, successful grafts were again transferred to MS medium containing 2%(w/v) phytagel and cefotaxime (250 mg/L) and allowed to grow for anadditional 2 weeks under LD conditions. Three weeks after grafting, theaverage number of roots, the root length (in centimeters), and the biomass(grams of fresh weight) were recorded and tissues were harvested for furtherevaluation.

Statistical Analysis

Throughout the experiments, Student’s t test was performed to checksignificance with one, two, three, and four asterisks indicating P values, 0.05, , 0.01, , 0 .001, and , 0.0001, respectively. Error bars represent 6SD (SD).

Availability of Data

Results described in this article are included as main figures/tables andsupplemental figures/tables. RNA-seq raw data FASTQ files (http://maq.sourceforge.net/fastq.shtml) generated from this study were depositedand available at the National Centre for Biotechnology Information SequenceRead Archive (https://www.ncbi.nlm.nih.gov/sra) under accession numberPRJNA546591.

Accession Numbers

Accession numbers used in this study are listed in Supplemental Table S12.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Conservation between Arabidopsis MSI1 andpotato MSI proteins.

Supplemental Figure S2. StMSI1 interacts with several other histone mod-ifiers in potato.

Supplemental Figure S3. Potato and tomato orthologs of AtBMI1 containthe RING finer domain.

Supplemental Figure S4. Phylogenetic relationship of BMI1 proteins.

Supplemental Figure S5. Expression profiles of StMSI gene familymembers.

Supplemental Figure S6. Phenotypes of StMSI1-OE (OE1 and OE3) lines.

Supplemental Figure S7. Expression profiles of StBMI1 gene familymembers.

Supplemental Figure S8. StBMI1-1-AS line screening and phenotype.

Supplemental Figure S9. Phenotypes of StMSI1-AS (AS8 and AS9) lines.

Supplemental Figure S10. Phenotypes of StBMI1-1-OE (II-9 and II-10) lines.

Supplemental Figure S11. Tuber yield (below-ground) in StMSI1-OE and-AS lines.

Supplemental Figure S12. GO classification for DE genes commonbetween StMSI1-OE and StBMI1-1-AS lines.

Supplemental Table S1. MSI and BMI-like genes in potato and theirgenomic locations.

Supplemental Table S2. List of StMSI1-OE DE analysis.

Supplemental Table S3. List of StBMI1-1-AS DE analysis.

Supplemental Table S4. List of StMSI1-OE and StBMI1-1-ASlines—common DE genes.

Supplemental Table S5. B2G analysis for StMSI1-OE DE genes.

Supplemental Table S6. B2G analysis for StBMI1-1-AS DE genes.

Supplemental Table S7. B2G analysis for StMSI1-OE and StBMI1-1-AScommon DE genes.

Supplemental Table S8. List of miRNAs targeting common DE genes.

Supplemental Table S9. LREs in promoters of StMSI1, StBMI, and miR156members.

Supplemental Table S10. PREs in promoters of miR156 and StBMImembers.

Supplemental Table S11. List of primers.

Supplemental Table S12. List of accessions for the genes described in thisstudy.

Supplemental Table S13. The genome annotation, precursor, and pro-moter sequences of miR156 members in potato.

ACKNOWLEDGMENTS

Authors are grateful to Mr. Nitish Lahigude (Indian Institute of Science Educa-tion and Research Pune) for his help in potato plant maintenance. Nucleome Pvt.Ltd, Hyderabad is thanked for providing RNA-seq raw data inputs.

Received April 2, 2019; accepted August 7, 2019; published August 19, 2019.

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