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PcG Proteins MSI1 and BMI1 Function Upstream of miR156 to Regulate Aerial Tuber Formation in Potato

Amit Kumar, Kirtikumar Ramesh Kondhare, Pallavi Vijay Vetal, Anjan Kumar Banerjee

Published January 2020. DOI: https://doi.org/10.1104/pp.19.00416

Abstract

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). miR156 overexpression (OE) reduces below-ground tuber yield, but stimulates aerial tubers in potato (Solanum tuberosum ssp andigena) under short-day (SD) photoperiodic conditions. Whether PRC members could govern tuber development through photoperiod-mediated regulation of miR156 is unknown. Here, we investigated the role of two PRC proteins, StMSI1 (PRC2 member) 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 enhanced miR156 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 increased accumulation of miR156a/b and -c precursors in the roots of wild-type stocks. RNA-sequencing of axillary nodes from StMSI1-OE and StBMI1-1-AS lines revealed downregulation of auxin and brassinosteroid genes, and upregulation of cytokinin 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 group H3K4-methyl-transferases in StMSI1-OE. Chromatin immunoprecipitation-quantitative PCR confirmed H3K27me3-mediated suppression of StBMI1-1/3, and H3K4me3-mediated activation of miR156 in StMSI1-OE plants. In summary, we show that cross talk between histone modifiers regulates miR156 and alters hormonal response during aerial tuber formation in potato under SD conditions.

Plants sense multiple environmental cues, such as temperature, light, and nutrient availability, and synchronize developmental programs accordingly. Photoperiod is one such environmental cue that plays an important role during tuber development (tuberization) in potato (Solanum tuberosum ssp andigena). During tuberization, the stolon (a modified below-ground stem) passes through various developmental stages and matures into a potato under short-day (SD) condition. Apart from phytohormones (auxin, cytokinin [CK], and gibberellin [GA]; Xu et al., 1998), phytochromes, flowering genes (CONSTANS [CO]; Martínez-García et al., 2002), a number of mobile signals, including mRNAs (StBEL5, -11, -29, and POTH1; Banerjee et al., 2006a, Mahajan et al., 2012, Ghate et al., 2017), micro RNAs (miR172 and miR156; Martin et al., 2009, Bhogale et al., 2014), and a Flowering Locus T (FT) orthologous protein StSP6A (Navarro et al., 2011) are now known to regulate tuberization. Earlier, we showed that miR156 levels increase in stolon under tuber-inducing SD photoperiodic conditions and its overexpression (OE) led to aerial tuber formation in potato (Bhogale et al., 2014). However, the basis for aerial tuber formation and what regulates miR156 under SD condition is not known. Previous studies in Arabidopsis (Arabidopsis thaliana) revealed that Polycomb Group (PcG) proteins mediate the 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. They were first identified in Drosophila as multiprotein complexes, termed as Polycomb Repressive Complex1 (PRC1) and PRC2. The PRC1 complex in Drosophila contains four members, namely the Polycomb (Pc), Polyhomeotic (Ph), Posterior sex comb (Psc), and dRING1 proteins (Shao et al., 1999; Peterson et al., 2004). They repress target chromatin by H2A monoubiquitination (Cao et al., 2005). Arabidopsis has three homologs of Psc (AtBMI1A, AtBMI1B, and AtBMI1C) and two homologs of dRING1 (AtRING1A, and AtRING1B; Calonje, 2014). BMI assists in activity of E3 ubiquitin ligases that monoubiquitylate histone H2A at Lys-119 position leading to the repression of target genes. A recent study in Arabidopsis has shown that BMI1 regulates meristem maintenance and cell differentiation by repressing PLETHORA (PLT) and WUS homeobox-containing (WOX) genes (Merini et al., 2017). Further, BMI1 mutants show downregulation of important flowering genes, like SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) and FT, indicating an important role in flowering response. To avoid precocious flowering, SPLs are suppressed by miR156 during the juvenile phase of plants. However, during adult and reproductive phases, miR156 expression is suppressed by BMI1 to allow the expression of SPLs (Merini et al., 2017). The core PRC2 complex in Drosophila consists of four subunits, namely Enhancer of Zeste [E(z)], Suppressor of Zeste 12 [Su(z)12], Extra sex combs (Esc), and p55. The [E(z)] protein represses target genes by catalyzing H3K27me3 modification of these genes (Müller et al., 2002), whereas p55 helps in 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 β-transducin repeat-containing protein family and have seven WD repeats with four antiparallel β-sheets at the C-terminal end that assist in its interaction with other proteins. A previous report on MSI1 in Arabidopsis showed that it regulates overall plant architecture and ovule development (Hennig et al., 2003). Subsequent studies also revealed that MSI1 is a component of several histone modifier complexes that regulates different phases of plant development. It is a part of three PRC2 complexes, known as FERTILIZATION INDEPENDENT SEED (FIS) complex that regulates seed development (Köhler et al., 2003), EMBRYONIC FLOWER(EMF) complex that suppresses flowering during juvenile stage (Yoshida et al., 2001), and VERNALIZATION (VRN) complex, which is essential for the onset of flowering after vernalization (De Lucia et al., 2008). Additionally, MSI1 is also a part of CHROMATIN ASSEMBLY FACTOR1 (CAF-1; Exner et al., 2006), nucleosome-remodeling factor (Martínez-Balbás, 1998), and histone deacetylase (Mehdi et al., 2016), indicating its diverse role in plant development. MSI1 also promotes flowering in Arabidopsis in a photoperiod-dependent manner by assisting in expression of CO and SUPPRESSOR OF CO (SOC1) through H3K4 methylation and H3K9 acetylation over SOC1 locus (Bouveret et al., 2006; Steinbach and Hennig, 2014).

Tuberization and flowering are two reproductive phenomena that share common molecular players and environmental cues (Martínez-García et al., 2002). Considering PcG proteins’ role in flowering, we hypothesize that they might govern tuber development in potato. In an experiment, we observed that OE of StMSI1 produced aerial stolons and tubers under SD photoperiodic conditions from axillary nodes, a phenotype that was demonstrated earlier for miR156 OE in potato (Bhogale et al., 2014). This raised a number of interesting questions with respect to the function of PcG proteins in potato, such as: (1) What is the cause of aerial stolon and tuber development from axillary nodes? (2) Do PcG proteins have any role in photoperiod-mediated control of tuberization? (3) Is miR156 directly regulated by StMSI1 or there are other epigenetic modifiers that could regulate miR156? In this study, using several approaches, such as OE or knockdown of two PcG proteins StMSI1 and StBMI1-1, RNA-sequencing (RNA-seq) analysis of axillary nodes of StMSI1 OE and StBMI1-1 knockdown lines, homo- and hetero-grafting and the chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) method, we established that StMSI1 and StBMI1-1 function upstream of miR156 to regulate aerial tubers in potato under SD photoperiodic conditions.

RESULTS

Phylogenetic Analysis Revealed Conservation of MSI1- and BMI1-like Proteins in Potato

BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) results revealed ∼91% identity between potato MSI1-like protein (StMSI1; XP_006349413.1) and Arabidopsis MSI1 (AtMSI1; NP_200631.1; Supplemental Fig. S1A). From the Potato Genome Sequencing Consortium (PGSC) database, we observed that the StMSI1 gene (∼5.20 kb) resides on chromosome 1 (Supplemental Table S1). Its longest open reading frame spans 1,368 bp and encodes for 425 amino acid residues (a 48.36-kD protein). Using WD repeat protein structure predictor tool, we could identify seven WD repeats in StMSI1 protein, positioned between amino acids 33 and 403 (Supplemental Fig. S1, B and C). Further analysis revealed the presence of 14 hotspot residues in the StMSI1 protein sequence that are likely to be involved in protein–protein interactions (Supplemental Fig. S1B). Arabidopsis MSI2 and MSI3 proteins match with 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 conservation with potato proteins (Supplemental Fig. S2A). STRING tool (https://string-db.org/) analysis predicted that StMSI1 could interact with a range of proteins including other PRC proteins, CAF, histone acetylases, and deacetylases (Supplemental Fig. S2B).

In potato, four BMI1 proteins (StBMI1-1, StBMI1-2, StBMI1-3, and StBMI1-4) have been identified and they share ∼55%, 50%, 45%, and 32% sequence identity with Arabidopsis BMI1a, respectively. When potato BMI1 proteins were analyzed for conserved domains, we found that a Cys-rich RING domain involved in zinc binding and the ubiquitination process is present in StBMI1-1, StBMI1-2, and StBMI1-4 similar to Arabidopsis BMI1 proteins (AtBMI1a, AtBMI 1b, and AtBMI 1c), but this domain was absent in StBMI1-3 (Supplemental Fig. S3). Arabidopsis BMI1 proteins also have a RAWUL domain (ubiquitin-like domain likely to be involved in protein–protein interactions), but this domain is absent in potato or tomato (Solanum lycopersicum) BMI1 homologous proteins. StBMI1–4 has an additional RAD18 domain, which is a putative nucleic acid binding domain (Supplemental Fig. S3). From the PGSC database, we found that the StBMI1-1 gene was located on chromosome 9, the StBMI1-2 and StBMI1-3 genes on chromosome 6, and the StBMI1-4 gene was on chromosome 1 (Supplemental Table S1). Phylogenetic analysis indicated that StBMI1-1, StBMI1-2, and StBMI1-3 displayed close conservation to respective tomato BMI1 proteins, whereas StBMI1-4 had close conservation to AtBMI1c (Supplemental Fig. S4).

SD Photoperiod Influences StMSI1 Expression in Stolon and Root Tissues

qPCR analysis showed a significant increase of StMSI1 transcript abundance in stolons under SD than long-day (LD) photoperiodic conditions (Fig. 1A). However, its transcript level was significantly lower in roots (Fig. 1A) and mature tubers (Supplemental Fig. S5A) under SD compared to LD. The expression of StMSI1 remain unchanged in shoot tip, leaf, and stem 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 that three StMSI genes (StMSI1, StMSI2, and StMSI4) are highly expressed in the stolons, but their expression is reduced in mature tubers and roots (Supplemental Fig. S5B). To characterize StMSI1 promoter activity, we generated promStMSI1::GUS-pBI121 potato transgenic lines. GUS assay on in vitro grown plantlets showed a ubiquitous StMSI1 expression pattern. Promoter activity was observed in shoot tip, stem, leaf, shoot–root junction, and root (Fig. 1, B–H), with strong activity in meristematic regions (axillary nodes and root tips; Fig. 1, C and E). When promoter activity was assayed from soil-grown plants induced under LD/SD conditions for 14 d, it was observed that swollen stolon samples 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 tuber peel and pith of SD-induced promoter transgenic plants (Fig. 1D).

Figure 1.
Figure 1.

StMSI1 promoter has ubiquitous expression, but was induced in stolon under the SD photoperiod. A, Effect of LD and SD photoperiod on transcript accumulation of StMSI1 in different tissues (shoot tip, leaf, stem, root, and stolon) of wild-type andigena (7540) potato plants grown under LD/SD conditions for 14 d, post 8 weeks of LD induction in soil. Fold-change of StMSI1 across different tissues is compared between SD versus LD in a tissue-specific manner. Data are mean ± sd for three biological and three technical replicates. EIF3e was used as a reference gene for expression analysis. Student’s t test was performed to check the level of significance. The asterisk represents statistical significance (*P < 0.05). Promoter activity of StMSI1 in 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 tuber samples are from soil-grown plants incubated under LD/SD conditions for 14 d. Scale bars = 2 cm (B), and 2 mm (C–H). ns, not significant.

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) promoter were generated to characterize its role in potato development (Supplemental Fig. S6A). Of them, two independent OE lines (OE1 and OE3) with moderate levels of StMSI1 OE were used for further analysis (Fig. 2A). OE lines showed drastic changes in overall 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 number of leaflets per leaf (Fig. 2, C and D) and leaf length was reduced (Fig. 2E), although leaf thickness was increased (Fig. 2F) compared to wild-type plants. OE lines also showed altered epidermal cells, bigger trichomes, increased stomatal number, and altered vascular bundle arrangement in stem compared to wild-type plants (Fig. 2, G– N). Moreover, the root length (Supplemental Fig. S6D) and root biomass (Fig. 2O) were decreased in OE lines compared to wild-type plants. To evaluate tuber yield potential, soil-grown StMSI1-OE lines maintained under LD conditions were subjected to SD inductions for 6 weeks. Interestingly, these lines produced numerous aerial stolons 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 profusely and to develop into mini-tubers in ∼70% to 80% of the plants (Fig. 3, C and D). The mini-tubers were purple in color and had characteristic tuber-eyes with 100% sprouting efficiency when attempted for germination. Neither StMSI1-OE (this study) nor miR156-OE (Bhogale et al., 2014) showed aerial tuber phenotype in potato under LD conditions. Throughout our experiments, vector control (VC) plants behaved like wild-type plants.

Figure 2.
Figure 2.

StMSI1 OE affects plant architecture in potato. A, Transcript levels of StMSI1 in leaves of OE lines (OE1 and OE3) compared to wild type (WT) . Data are mean ± sd for three biological replicates. EIF3e was used as a reference gene for expression analysis. B, Plant architecture of StMSI1 OE 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 shown along with wild-type and VC plants. Six individual plants per line were considered for phenotypic data analysis. Student’s t test was performed to check significance with *P values < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars represent ± sd. G and H, Transverse cross section of the stem of wild-type plant (G) and StMSI1 OE line OE3 (H). K and L, Magnified images of vascular bundles in wild type and OE line, respectively. I, J, M, and N, Scanning electron microscopy images showing the leaf epidermis cells, number of stomata (J), and trichomes (N) in OE lines compared to wild type (I and M), respectively. O, Root biomass in StMSI1 OE potato lines (OE1 and OE3) are shown along with wild-type and VC plants. Student’s t test was performed to check significance at P < 0.05. Error bars represent ± sd from six independent plant per line. Scale bars = 10 cm (B), 5 cm (C), 300 μm (G, H, K, and L), and 50 μm (I, J, M, and N). gfw, grams fresh weight; ns, not significant.

Figure 3.
Figure 3.

StMSI1 OE lines produce aerial tubers accompanied by reduced expression of StBMI1 and increased expression of miR156a/b/c. Aerial stolons (A and B; white arrows) and tubers from axillary nodes under SD induction (C and D) in StMSI1 OE lines, OE1 and OE3, respectively. Relative miR156a/b/c levels in leaves of StMSI1 OE 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 have referred them as “miR156a/b/c” throughout the text. Relative transcript levels of StBMI1-1, StBMI1-3, and StBMI1-4 in leaves of StMSI1-OE line (OE3) compared to VC line (F). Relative levels of StLOG3 (G) and StGA2ox1 (H) in StMSI1-OE line (OE3) are shown in comparison to VC plants. Relative levels of StMSI1 and miR156a/b/c in leaves during juvenile versus adult phase in wild-type potato plants (I). For (E), (F), (H), and (I), data are mean of three biological and three technical replicates with ± sd. U6 was used as a reference gene for miRNAs, whereas EIF3e was used for gene expression analysis. Relative level in wild type was considered as “1” with ± sd for (E), (F), (H), and (I), whereas relative levels in juvenile phase was considered as “1” with ± sd. Student’s t test was performed to check significance with *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant. Scale bars = 7 cm (A) and 5 cm (B–D).

StMSI1-OE Line Showed an Altered Expression of miR156 and StBMI1

To analyze if miR156 levels were affected in StMSI1-OE lines, miR156a/b/c expression was measured in leaf tissues of SD-induced plants. Interestingly, miR156a/b/c expression was nearly 5-fold higher in the OE line (OE3) compared to the VC (Fig. 3E). Earlier, a PRC1 member, AtBMI1, has been shown to repress miR156 during reproductive phase maintenance in Arabidopsis (Picó et al., 2015). Anticipating cross talk among StMSI1, StBMI1, and miR156 in potato, the relative transcript 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 of each variant. The transcript levels of StBMI1-1, -3, and -4 were low in the StMSI1-OE line compared to VC (Fig. 3F). Due to the close conservation of mRNA sequences between StBMI1-1 and -2 transcript variants, we could not validate the StBMI1-2 variant. The transcript levels of a CK biosynthesis gene, StLOG3, and a GA catabolism gene, StGA2ox1, were significantly higher in leaves of the StMSI1-OE line compared to VC plants (Fig. 3, G and H). Moreover, StMSI1 and miR156 levels in leaves were high in the juvenile phase of wild-type andigena plants, whereas their levels were significantly lower in the adult phase of the plant (Fig. 3I).

SD Photoperiod Affects StBMI1-1 and miR156 Expression in Shoot Tip and Stolons

StBMI1-1 level was quantified by qPCR in different tissues and the stages of stolon-to-tuber transitions in andigena plants grown under LD/SD conditions for 14 d (Fig. 4, A and B). Our analysis demonstrated that StBMI1-1 transcript levels were significantly low under SD photoperiod in shoot tip, stem, and stolon tissues compared to LD conditions (Fig. 4A). However, the transcript levels remained unchanged in leaf and root tissues under LD and SD photoperiodic conditions (Fig. 4A). Further, StBMI1-1 transcript levels were significantly low in stolon, swollen stolon, and mini-tuber, but high in tubers under SD conditions compared to the stolons from LD (Fig. 4B). Moreover, miR156 levels were quantified in shoot tip and stolon tissues under LD/SD conditions. We noticed ∼2.5- and 2-fold increase of miR156 expression in shoot tip and stolon (respectively) under SD conditions compared to LD (Fig. 4C). From the PGSC resources (Xu et al., 2011), it was evident that StBMI1-4 is expressed only in floral organs and not in any other tissue types (i.e. shoot apex, leaf, stem, root, stolon, young/mature tuber, sprouted tuber) in potato. Because StBMI1-1 is expressed more abundantly than StBMI1-2 (Supplemental Fig. S7), and the absence of the RING domain in the StBMI1-3 protein, we chose StBMI1-1 for its functional characterization in this study.

Figure 4.
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 tissue types—shoot tip, leaf, stem, and root (A) and in various stages of stolon-to-tuber transitions (B). Wild-type (WT) potato plants were grown under LD/SD conditions for 14 d, after 8 weeks of growth in soil under LD conditions. C, The relative levels of miR156a/b/c in shoot-tip and stolon tissues at 14 d under LD/SD conditions. The relative transcript levels of StBMI1-1 or miR156a/b/c in different tissues under SD conditions is calculated considering its levels under LD condition as “1” with ± sd. EIF3e and U6 were used as reference genes for StBMI1 and miR156a/b/c expression analysis, respectively. D, The transcript levels of StBMI1-1 in leaves of AS transgenic lines G9 and G12 compared to wild type and VC lines. Analysis was performed with three biological replicates per line. qPCR was performed using StBMI1-1 specific primers. E, StBMI1-1-AS transgenic lines along with wild type and 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 in soil (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 represented from nine independent plants per line. J, Relative miR156a/b/c levels in AS line #G9 and VC with respect to wild type. K, Formation of aerial tubers in StBMI1-1 AS line (#G9) after 4 weeks of SD incubation. White arrows indicate aerial tubers. U6 was used 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 ± sd. Scale bars = 1 cm (E–G and K).

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

To investigate if StBMI1-1 functions upstream of miR156 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% downregulation, whereas G12 had ∼30% downregulation of StBMI1-1 (Fig. 4D). The overall architecture of the plant was weak in StBMI1-AS line (G9) when compared to wild-type or VC plants (Fig. 4E). Shoot biomass was significantly lower in StBMI1-1-AS lines G9 and G12 (Supplemental Fig. S8C). The expression of StLOG3 remained unchanged, whereas that of StGA2ox1 was upregulated in the StBMI1-1-AS line (Supplemental Fig. S8D). The StBMI1-1-AS line (G9) showed a reduction in leaf size as well as leaf compounding post 2–3 weeks of incubation under LD conditions in soil (Fig. 4F). The leaf phenotypes were similar to miR156-OE lines K1 and K6 (Fig. 4G) as well as StMSI1-OE lines (Fig. 2C). On an average, wild-type or VC plants had seven leaflets per leaf in mature plants, 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 the StBMI1-1-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/c levels in leaves compared to wild-type or VC plants (Fig. 4J). Similar to the StMSI1-OE lines, an extended incubation of the StBMI1-1-AS line (G9) under SD conditions resulted in formation of aerial tubers in ∼50% of the plants (Fig. 4K); however, no such phenotype was observed in G9 line under LD conditions (Fig. 4E).

Knockdown of StMSI1 and OE of StBMI1-1 Affects Leaf Development

To assess the effect of StMSI1 knockdown on potato phenotype, two independent StMSI1-AS lines (AS8 and AS9) displaying up to 50% reduction of StMSI1 transcript levels were selected for phenotypic analysis (Supplemental Fig. S9A). StMSI1-AS lines exhibited reduced plant height (Supplemental Fig. S9B), internodal distance (Supplemental Fig. S9C), and leaf length (Supplemental Fig. S9, D and E) compared to wild-type or VC plants. Unlike StMSI1-OE lines, there was no effect on the root biomass of the StMSI1-AS lines (Supplemental Fig. S9, F and G). The levels of miR156a/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 and II-10; Supplemental Fig. S10, A and B) showed an increase in leaf size and number of leaflets per leaf compared to wild-type plants (Supplemental Fig. S10C). In mature plants, the average number of leaflets per leaf increased to nine or more in OE lines in comparison to seven in the wild type or the VC (Supplemental Fig. S10D). However, root biomass was not affected in the StBMI1-1-OE lines (Fig. 5I). Shoot and root biomass did not show any changes in the StBMI1-1-OE lines (II-9 and II-10) compared to the VC plants (Supplemental Fig. S10, E and F).

Figure 5.
Figure 5.

OE or knockdown of StMSI1 or StBMI1-1 influences tuberization. The number of tubers produced by OE and AS lines of StMSI1 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 along with miR156a/b/c OE, wild-type, and VC lines. A, For better representation, one representative line per transgenic construct was plotted. 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 three technical replicates were used for the analysis. EIF3e was used as a reference for genes, and U6 for miR172. C to F, Number of tubers 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, and nine 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 ± sd.

OE or Knockdown of StMSI1 or StBMI1-1 Influences Tuberization

Tuber yield potential was assessed both in in vitro and 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-OE lines exhibited delayed tuberization and reduced tuber yield compared to wild type, whereas the StBMI1-1-OE line (II-9) showed an earliness for in vitro tuberization as well as increased tuber yield (Fig. 5A). The expression of tuber marker genes, such as miR172 (Martin et al., 2009), StBEL5 (Banerjee et al., 2006a), and StSP6A (Navarro et al., 2011) were significantly reduced in leaves of the StMSI1-OE and StBMI1-1-AS lines (Fig. 5B). In contrast, the expression of StSP6A repressors, such as StCO2, StCO-like 9, and StSP5G were significantly higher in leaves of the StMSI1-OE and StBMI1-1-AS lines (Fig. 5B), which is consistent with the reduced tuber yield phenotype in these lines. Although there was no effect on tuber numbers in the StMSI1-OE or -AS lines in soil-grown plants (Fig. 5C), both showed ∼3- to 5-fold reduction in tuber yield compared to wild type (Fig. 5D; Supplemental Fig. S11, A and B). The StBMI1-1-OE lines showed no difference in tuber numbers, but they had increased tuber yield (Fig. 5, E and F). However, the StBMI1-1-AS lines showed a reduction in tuber numbers as well as in tuber yield (Fig. 5, E and F).

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

To understand the cause for aerial tuber formation, we performed paired-end RNA-seq from axillary nodes of the SD-induced StMSI1-OE and StBMI1-1-AS lines along with the VC plants. Overall, 172 million final clean reads were obtained from 181 million raw reads after quality-filtering and adapter trimming. Of them, 88.86% read pairs uniquely mapped to the potato genome (Table 1). Downstream processing of RNA-seq data revealed that ∼7,386 and 1,690 genes were differentially expressed (DE) in the StMSI1-OE and StBMI1-1-AS lines, respectively, compared to the VC plants (Fig. 6A). Among the DE genes in the StMSI1-OE line, ∼3,360 and 4,026 genes were up- and downregulated, respectively, whereas 921 genes were up- and 769 were downregulated in the StBMI1-1-AS line. Approximately 6,363 DE genes were unique only to the StMSI1-OE line, whereas ∼667 genes were specific to the StBMI1-1-AS line (Fig. 6A). Interestingly, we observed that out of 1,690 DE genes identified in the StBMI1-1-AS line, 1,023 genes (∼60%) were common between both of the lines (StMSI1-OE and StBMI1-1-AS). When common DE genes were analyzed further, we found that 345 genes were upregulated and 371 were downregulated in both lines. However, 307 DE genes from the common pool showed opposite expression patterns in the StMSI1-OE and StBMI1-1-AS lines; here, 123 genes were upregulated in the StMSI1-OE, but downregulated in the StBMI1-1-AS line, 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 related to auxin and brassinosteroid (BR) biosynthesis, transport, and signaling were downregulated, whereas the genes involved in CK transport and signaling were upregulated.

View this table:
Table 1. Summary of read counts and alignment statistics for axillary node samples of potato after RNA-seq
Figure 6.
Figure 6.

RNA-seq analysis and validation of DE genes specific to StMSI1-OE or StBMI1-1-AS line. A, Venn diagram shows the summary 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 represent ± sd from three biological and three technical replicates. EIF3e was used as a reference gene.

RNA-Seq and qPCR Analysis Revealed Altered Expression of Histone Modifiers, Tuber Markers, and Development-Related Genes in the StMSI1-OE and StBMI1-1-AS Lines

RNA-seq analysis also revealed downregulation of StBMIs (StBMI1-1 and StBMI1-3), LHP1 (which recruits PRC1 complex over H3K27me3-modified target genes; Turck et al., 2007), and HDA19 (that catalyzes the removal of acetylation marks on target genes), in the StMSI1-OE line compared to the VC (Supplemental Table S2). Further, we could identify that Trithorax group members, such as SDG4 and a member of SET7/9 family (SET7/9) having histone H3K4 methyltransferase activity, were significantly upregulated in these lines (Supplemental Table S2).

The expression of SDG4 and SET7/9 genes were upregulated, whereas that of LHP1 was downregulated in the StMSI1-OE line (Fig. 6B). The abscisic acid signaling gene PYL4, and auxin-responsive genes such as SAUR and ARF16 and epidermal patterning factor (EPF), were downregulated in the StMSI1-OE line (Fig. 6B; Supplemental Table S2). The transcript levels of a gene that induces tuber formation, StSP6A (Navarro et al., 2011) and a member of the tuberigen activation complex, St 14-3-3 (Teo et al., 2017), were significantly reduced in the StMSI1-OE line (Fig. 6B; Supplemental Table S2). On the other hand, genes involved in cell division (cyclin A2, CycA2), shoot-apical meristem formation and maintenance (CLAVATA1, CLV and ERECTA, ERC), and leaf development (TEOSINTE BRANCHED1/CYCLOIDEA/PCF TF and SPL9) were altered in the StBMI1-1-AS line compared to the VC (Fig. 6C; Supplemental Table S3). Moreover, the transcript levels of tuberization repressors, such as PHYB2 and CO1 and CO2, were significantly increased in the StBMI1-1-AS lines (Fig. 6C; Supplemental Table S3). Validation of common DE genes between the StMSI1-OE and StBMI1-1-AS lines showed that three genes, such as pseudo-response regulator (governs circadian rhythm and plant fitness), protease (associated with leaf senescence), and chalcone synthase (involved in flavonoid biosynthesis) were upregulated, whereas several other genes, such as HD-ZIP TF (required for vascular development), longifolia (involved in leaf morphology), and glabra (associated with trichome branching), were downregulated in the StMSI1-OE and StBMI1-1-AS lines (Fig. 7A; Supplemental Table S4). Among the common DE genes between the StMSI1-OE and StBMI1-1-AS lines, we found 22 genes (of 1,023) were associated with sugar transport or sugar/starch metabolism (Supplemental Table S4).

Figure 7.
Figure 7.

Auxin- and BR-related genes were downregulated, whereas CK-related genes were upregulated in StMSI1-OE and StBMI1-1-AS lines. A and B, Validation of selective DE genes common between StMSI1-OE and StBMI1-1-AS lines compared to VC line. The relative fold-change of respective gene expression in StMSI1-OE or StBMI1-1-AS line 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, and ***P < 0.001. Error bars represent ± sd from three biological and three technical replicates. EIF3e was used as a reference gene. In (A), red underlines 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.

Phytohormone-Related Genes Were Affected in StMSI1-OE and StBMI1-1-AS Lines

Analysis of common DE genes between the StMSI1-OE and StBMI1-1-AS lines showed that the genes encoding auxin transport proteins (auxin efflux1 and 2) and auxin response proteins (ARP, expansin, AUX/IAA, and AUX-IAA3) were downregulated in the StMSI1-OE and StBMI1-1-AS lines compared to the VC (Fig. 7B; Supplemental Table S4). Additionally, genes related to BR biosynthesis (cytoP450) and signaling (BR kinase, thesasus, and Phi-1 protein) were downregulated (Fig. 7B; Supplemental Table S4). The transcript of a gene (sigma factor sigb regulation protein rsbq), which acts as a negative regulator of strigolactone (STL) signaling, was high, whereas an STL-responsive gene (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 of GA biosynthesis genes, GA20ox and GA3ox, and increased expression of a GA catabolic gene, GA2ox, in axillary nodes (Supplemental Table S2). Genes encoding CK transporters, such as purine transporter2 and 3, as well as a responsive gene (zeatin riboside), were upregulated in StMSI1-OE and StBMI1-1-AS lines in comparison 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 19 were downregulated in the StBMI1-1-AS line. The remaining five genes were upregulated in the StMSI1-OE line and nine were downregulated in StBMI1-1-AS line compared to the VC (Fig. 7C; Supplemental Table S4). Of the seven BR-related genes, six were downregulated in the StMSI1-OE line, whereas all seven were downregulated in the StBMI1-1-AS line compared to the VC (Fig. 7C; Supplemental Table S4). Gene ontology (GO) analysis for DE genes categorized GO terms into 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 Type Influenced miR156 Accumulation and Reduced Root Biomass In Wild-Type Stock

Considering the mobile nature of tuberization signals, to investigate if OE of StMSI1-OE or StBMI1-1 knockdown has any effect on miR156 expression, different combinations of homo- and hetero-grafts were made under in vitro conditions (Fig. 8A). Overall, we produced ∼70% to 80% successful grafts and 3 weeks after grafting, several root growth parameters were measured. As expected, homo-grafts of StMSI1-OE or 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 was affected in hetero-grafts containing StMSI1-OE or StBMI1-1-AS as scion and wild type as stock as well as in reverse grafts (Fig. 8, A–D). To analyze the cause of reduced root growth, the expression of auxin and CK transport/signaling genes was quantified in roots of all homo- and hetero-grafts (Fig. 8E). The expression levels of auxin efflux carrier1 and expansin were reduced, whereas those of CK transporters (purine transporter2 and 3) were increased in all homo- and hetero-grafts compared to wild-type homo-grafts (Fig. 8E), Additionally, we found that the precursor levels of miR156a/b and miR156c were also high in roots of all homo- and hetero-grafts compared to wild-type homo-grafts (Fig. 8E).

Figure 8.
Figure 8.

Hetero-grafts of StMSI1-OE or StBMI1-AS line with wild type showed altered root growth compared to homo-grafts of wild type (WT). A, Two representative images of in vitro grown plants are shown for each combination of homo- and hetero-grafts after 3 weeks of graft initiation. Scale bar = 1 cm. B to D, Average number of roots (B), root length (C), and root biomass (D) per homo- 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 significance indicated with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. gfw, grams fresh weight; ns, not significant. Comparison for each homo- or hetero-graft was performed with respect to wild-type/wild-type homograft. Arrows (white, A) indicate the graft unions. E, Relative transcript levels of auxin and CK transport/signaling genes as well as premiR156a/b and premiR156c levels in roots of different homo- and hetero-grafts after 21 d. In (E), mean values of two biological replicates per graft combination were plotted. EIF3e was used as the reference gene for qPCR analysis. Error bars represent ± sd. AEC1, Auxin efflux carrier 1; PT, Purine transporter; EXP, Expansin; miR156a/b, precursor of miR156a/b; miR156c, precursor of miR156c.

ChIP-qPCR Shows Enrichment of H3K27me3 and H3K4me3 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 the possible cross talk between these regulators, ChIP-qPCR analysis was performed to quantify the level of repressive mark (H3K27me3) at the first intron of StBMI1-1 and StBMI1-3 genes. Our analysis found that the levels of H3K27me3 on StBMI1-1 and StBMI1-3 genes were significantly increased in the StMSI1-OE line (Fig. 9A). Apart from increased levels of miR156a/b/c (Figs. 3E and 4J), the expression of miR156e was also high in leaves of StMSI1-OE and StBMI1-1-AS lines (Fig. 9B). Moreover, miR156f level was high in the StMSI1-OE line; however, it was surprisingly low in the StBMI1-1-AS line (Fig. 9B). The transcript level of Trithorax group members having histone H3K4 methyltransferase activity (SET7/9 and SDG4) was significantly higher in both StMSI1-OE and StBMI1-1-AS lines compared to VC (Fig. 9B). Hence, we tested the possibility of miR156 activation by quantifying the levels of H3K4me3 marks made by Trithorax group members. We observed that H3K4me3 marks were increased at the upstream regions of different miR156 family members (miR156b, miR156e, miR156f, and miR156g) in the StMSI1-OE and StBMI1-1-AS lines (Fig. 9C).

Figure 9.
Figure 9.

ChIP-qPCR validates H3K27me3-mediated repression of StBMI1 and H3K4me3-mediated activation of miR156. A, The enrichment of H3K27me3 repressive marks on the promoters of StBMI1-1 and StBMI1-3 in StMSI1-OE line (OE3) compared to VC. B, The relative levels of miR156e and miR156f in StMSI1-OE and StBMI1-AS lines compared to VC. C, The enrichment of H3K4me3 activation marks over the promoters of miR156 members in the transgenic lines StMSI1-OE and StBMI1-AS compared to 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 = 2). “7/7” represents the actual cleavage frequency after RLM-RACE analysis. The relative enrichment of respective marks in the StMSI1-OE and StBMI1-AS lines was calculated with respect to the VC sample. Student’s t test was performed to check significance indicated with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant. Error bars represent ± sd from three biological and three technical replicates. EIF3e or U6 was used as a reference gene. F, The proposed model considering the cross talk among StMSI1, StBMI1-1, and miR156 during aerial and below-ground tuber formation in potato under SD photoperiodic conditions. (Note: The nomenclature of potato miR156 members is as per the miRbase [http://www.mirbase.org/]. The genomic locations of miR156 precursors and promoters used for ChIP-qPCR analysis are given in Supplemental Table S13.

RLM-RACE Assay Confirms miR156-Mediated Cleavage of StSPL13

From RNA-seq data, three SPL genes, including StSPL8, StSPL9, and StSPL13, were differentially downregulated in StMSI1-OE or StBMI1-1-AS lines (Supplemental Tables S2 and S3). Of these, StSPL9 and StSPL13 are predicted to be cleaved by different miRNA156 members. Through a degradome analysis, Seo et al. (2018) recently showed that StSPL13 is cleaved by miR156 in potato. In our analysis, we observed that the transcript levels of StSPL6 and 13 were significantly reduced in both StMSI1-OE and StBMI1-1-AS lines compared to the VC (Fig. 9D). However, StSPL3 and 8 transcript levels remain unchanged in the StMSI1-OE line, but both 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 of E = 1.0, followed by miR156a/b/c members. Through a modified 5′ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) assay, we confirmed that StSPL13 transcript is cleaved by miR156 members with 100% cleavage efficiency (7 out of 7) at the 11th/12th nucleotide position (Fig. 9E). However, it may be noted that the cleavage site confirmed here on StSPL13 transcript is different than that reported in Bhogale et al. (2014), suggesting a different member of the miR156 family is cleaving StSPL13 transcript rather than miR156a/b/c. Additionally, psRNATarget analysis (Dai and Zhao, 2011) of 1,023 common DE genes unveiled that many of these genes could be targets of different miRNAs (Supplemental Table S8).

Promoters of StMSI1, StBMI1, and miR156 Members Have Numerous 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 Box4 motifs, four GT1 motifs, one GA motif, two TCT motifs, and one ATCT motif (Supplemental Table S9). In addition, two CAT-box motifs related to meristem expression were identified in its promoter sequence (Supplemental Table S9). Similarly, several LREs were found in the 1.5-kb promoters of all 11 miR156 members in potato (miR156a-k) and three StBMI genes (StBMI1-2, StBMI1-3, and StBMI1-4; Supplemental Table S9) Additionally, numerous Polycomb response elements (PREs) were identified in the promoters of miR156 members and StBMI1 genes (StBMI1-2, StBMI1-3, and StBMI1-4; Supplemental Table S10).

DISCUSSION

PcG proteins are important regulators of plant development and control the expression of homeotic genes involved in meristematic activity and organ differentiation (Goodrich et al., 1997). Previous studies in Arabidopsis have shown that a large number of miRNAs involved in phytohormone regulation and other key developmental processes are also regulated by PcG proteins (Lafos et al., 2011; Teotia and Tang, 2015). Although much progress has been made to understand the role of PcG proteins in Arabidopsis, their role in potato development remains to be explored. Here, we have shown that two PRC members, StMSI1 and StBMI1-1, regulate tuber development in potato by controlling the expression of miR156 and hormonal response in a photoperiod-dependent manner.

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

MSI1 is a crucial component of several histone modifier complexes that are involved in meristem maintenance, branching, flowering, and leaf and ovule development (Hennig et al., 2003, 2005). Previous studies (Hennig et al., 2003; Liu et al., 2016) showed ubiquitous expression of MSI1 in Arabidopsis and tomato. Similarly, we noticed a ubiquitous expression pattern of GUS in the promStMSI1::GUS-pBI121 potato lines, with highest expression in axillary nodes and root tips (Fig. 1, C and F). Additionally, when these promoter lines were subjected to LD/SD induction, GUS expression was higher in swollen stolon under SD photoperiod compared to stolons from LD (Fig. 1, G and H). qPCR analysis further validated the higher expression of StMSI1 in stolon under SD conditions (Fig. 1A). These observations indicate that photoperiod could regulate StMSI1 expression in potato. In potato, miR156 has been shown to play an important role in controlling tuber development and its expression increases in stolons under SD photoperiod (Bhogale et al., 2014). Additionally, both in potato and Arabidopsis, 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 the plant (Wu et al., 2009; Bhogale et al., 2014). Interestingly, in our study, the expression pattern of both StMSI1 and miR156 was similar in stolons during SD induction as well as in leaves during juvenile and adult phases in the potato plant (Fig. 3I). Apart from the 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 expression in Arabidopsis (Picó et al., 2015). We found that StBMI1-1 expression was significantly low, whereas that of miR156 was high in shoot tip and stolon under SD photoperiod compared to LD conditions (Fig. 4A). Further, the presence of numerous LREs in the promoters of StMSI1 and StBMI1 genes and 11 miR156 members in potato (miR156a–j; Supplemental Table S9) suggests that photoperiod could regulate the expression of these genes. The identification of PREs in the promoters of all miR156 members (Supplemental Table S10), suggests a possible role of PcG proteins in photoperiod-mediated regulation of miR156 during the stolon-to-tuber transition in potato. In this report, StMSI1 OE and StBMI1 knockdown 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 function upstream of miR156 in potato.

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

Both StMSI1-OE and StBMI1-AS lines had drastic changes 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). The StMSI1-OE line also showed increased numbers of leaf stomata, trichome length, and altered stem vascular bundles (Fig. 2, G–N). We demonstrated earlier that miR156 OE leads to reduced leaf size, compounding, and tuber yield in potato (Bhogale et al., 2014). We observed 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, work antagonistically to TEOSINTE BRANCHED1/CYCLOIDEA/PCF transcription factors belonging to the class-II CINCINNATA subgroup during leaf development (Rubio-Somoza et al., 2014). The reduced transcript levels of several SPLs, including StSPL6 and -13 in both lines (StMSI1-OE and StBMI1-AS; Fig. 9D) and RLM-RACE–mediated validation of StSPL13 cleavage by miR156 (Fig. 9E), further justifies the reduced leaf size and compounding phenotype in both transgenic lines. A number of reports (Uchida et al., 2007; Hay and Tsiantis, 2010, Mahajan et al., 2016) have demonstrated that class-I KNOX genes regulate meristem activity, leaf architecture, and compounding. The high 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 between the POTH15-OE and StMSI1-OE lines (Supplemental Table S4) could also be the cause of altered leaf architecture. In StMSI1-OE and StBMI1-AS lines, we noticed downregulation of several genes coding for auxin efflux carriers (PIN proteins), HD-ZIP TFs, and BR signaling pathway genes (Fig. 7, A and B), which have 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 PIN1 expression (Gu et al., 2014). Auxin and BR stimulate, whereas CK inhibits, lateral root development (Müssig et al., 2003; Aloni et al., 2005). Our RNA-seq data showed high expression of CK and low expression of auxin- and BR-signaling–related genes (Fig. 7B; Supplemental Tables S2–S4). A number of genes, such as PINs, PLETHORA (PLT), SCARECROW, and ARFs that 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-AS lines, possibly explaining the reduced root growth phenotype. Similar to miR156-OE lines (Bhogale et al., 2014), StMSI1-OE and StBMI1-1-AS lines showed a significant reduction in below-ground tuber yield compared to wild-type plants (Fig. 5, D and F). In contrast, the StBMI1-1-OE lines showed increased tuber yield (Fig. 5F). We further noticed that the StMSI1-AS lines had comparatively reduced plant architecture (Supplemental Fig. S9) and tuber yield (Fig. 5D). Considering MSI1 functions as a component of both activator and repressor complexes, we assume that its moderate levels are essential for tuber development. Similar results were also observed in the in vitro tuberization experiment (Fig. 5A). One of the reasons for reduction in below-ground tuber yield could be due to the weaker plant architecture of these lines than that found in wild type. Further, the downregulation of crucial tuber marker genes downstream of miR156 in the tuberization pathway, for example miR172 (Martin et al., 2009), StBEL5 (Banerjee et al., 2006a), and StSP6A (Navarro et al., 2011), and the upregulation of tuber growth repressors (StPHYB, StCO, and StSP5G; Jackson et al., 1996; Navarro et al., 2011; Kloosterman et al., 2013), could be another reason for reduced tuber yield in these lines (Figs. 5B and 6C; Supplemental Tables S2 and S3). Moreover, we observed an increase in miR156 (Figs. 3E and 4J), but a reduction in miR172 expression (Fig. 5B) in the StMSI1-OE and StBMI1-1-AS lines. psRNATarget analysis unveiled numerous common DE genes as targets of different miRNAs, including miR156 and miR172. Approximately 247 common DE genes (of 1,023) related to plant growth and development were predicted to be cleaved by miR156 and miR172 family members (Supplemental Table S8), suggesting that altered levels of miR156 and miR172 and their potential downstream target genes could have also contributed to the low tuber yield phenotype. Interestingly, grafting of StMSI1-OE or StBMI1-1-AS onto wild-type stock resulted in reduced root biomass (Fig. 8, A–D) and showed increased accumulation of miRNA156a/b and -c precursors in the roots of wild-type stocks (Fig. 8E), suggesting that PRC proteins could have influenced the accumulation of miR156 in roots. The reduced root biomass in these hetero-grafts could possibly be due to altered expression of genes encoding auxin and CK transport/signaling proteins in roots of these hetero-grafts (Fig. 8E). These findings are consistent with the earlier report of Bhogale et al. (2014), where the authors demonstrated that miR156 functions as a potential mobile signal in potato.

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

In potato plant, every axillary meristem possesses the ability to form a stolon/tuber; however, this potential remains 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 aerial stolons/tubers from axillary nodes (Figs. 3, A–D, and 4K), a phenotype that matched with our previous demonstration of miR156 OE in potato (Bhogale et al., 2014). Both of these lines showed high levels of miR156 expression (Figs. 3E and 4J), indicating a possible regulation of miR156 either through StMSI1 or StBMI1-1. The StBMI1-1-AS lines show a weaker phenotype of aerial tuber development than either the StMSI1-OE or miR156-OE lines. It could be because of a lower level of RNA suppression (which was ∼35%) in the StBMI1-1-AS line (G9). Also, it is possible that the function of four potato BMI proteins could be redundant. Hence, silencing only StBMI1-1 might not result in a more robust phenotype. Recent studies have demonstrated that BMI-mediated suppression of miR156 (Picó et al., 2015) triggers onset of the adult phase in Arabidopsis, which is consistent to our observation of increased miR156 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 promoter and precursor sequences of miR156 further supports the notion. However, the reason behind the increased level of miR156 in the StMSI1-OE line (Fig. 3E) and the aerial tuber phenotype (Fig. 3, C and D) was not clear. RNA-seq of axillary nodes from both of these lines provided crucial insights of StMSI1-mediated regulation of miR156.

The StMSI1-OE line exhibited altered expression of several genes encoding histone modifiers (Supplemental Table S2). For example, the expression of PRC1 members, such as StBMIs (StBMI1-1, 1-3, and 1-4) and StLHP1, was reduced in the StMSI1-OE line. BMI1 and LHP1 maintain repressed states of target genes through assisting H2A ubiquitination and maintaining H3K27me3 modification, respectively (Derkacheva et al., 2013). Additionally, genes encoding Histone Deacetylase19 (Supplemental Table S2), ring finger proteins, and E3 ubiquitin ligase PUB14, involved in suppression of target genes, were downregulated (Supplemental Table S4; common down sheet). Enrichment of the repressive mark (H3K27me3) on the first introns of StBMI1-1 and -3 genes in the StMSI1-OE line (Fig. 9A), as well as the presence of several PREs in StBMI (StBMI1-2, StBMI1-3, and StBMI1-4) promoters (Supplemental Table S9), further supports the regulation of StBMI1 genes by the PRC2 complex. In the StMSI1-OE line, we further observed upregulation of genes encoding JMJC domain-containing H3K9 demethylase (Sun and Zhou, 2008; Supplemental Table S2) and Trithorax group members (SDG4 and SET7/9; Fig. 6B), which are involved in catalyzing H3K4 methylation of target genes (Cartagena et al., 2008). Moreover, ChIP assay confirmed the increased enrichment of H3K4me3 modification of the miR156 promoter (Fig. 9C). On the basis of these findings, we assume that the combined effect of reduction in repressive histone ubiquitination and the increase in expressive methyl modification of the miR156 locus might have resulted in its enhanced expression in both StMSI1-OE and StBMI1-1-AS lines. Additionally, the presence of PREs and BMI1-binding motifs in the promoters of miR156 members (Supplemental Table S10) suggests that PcG proteins can regulate miR156 expression in potato.

From the RNA-seq analysis, we observed that 1,023 DE genes were common between the StMSI1-OE and StBMI1-1-AS lines (Supplemental Table S4). Subsequent analysis of these common DE genes hinted at the cause of aerial stolon/tuber formation from axillary nodes of these lines. Bhogale et al. (2014) showed that the miR156-OE lines had higher levels of CK as well as 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 development of mini-tubers from axillary nodes in tomato. Interestingly, in both the StMSI1-OE and StBMI-1-AS lines, we also found increased expression of CK transport and response genes in axillary nodes (Fig. 7B). Although we could not find CK biosynthesis genes to be differentially expressed in StMSI1-OE and StBMI1-AS lines in the RNA-seq data (Supplemental Tables S2–S4), we observed that the expression of StLOG3 was high in leaves of the StMSI1-OE line (Fig. 3H). Besides this, both of the transgenic lines shared a number of common DE genes with that of the SlLOG1 OE lines as described in Eviatar-Ribak et al. (2013), indicating that common downstream effectors of PcG and/or LOG genes could be involved in aerial tuber development. These results are consistent with the role of CK as a branching stimulator (Domagalska and Leyser, 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 of CK during aerial tuber development. Although the role of auxin in developmental phase transition of stolon-to-tuber (Roumeliotis et al., 2012) is well established, its role in aerial stolon/tuber development was not known. In our RNA-seq analysis, we noted a reduced expression of several auxin transport and signaling genes in StMSI1-OE and StBMI1-1-AS lines (Fig. 7, B and C), suggesting the involvement of auxin in aerial stolon formation from axillary nodes.

In this scenario, what possible explanations could describe the development of aerial stolons/tubers phenotype from the above-ground axillary nodes? A number of interesting observations lead us to speculate that formation of aerial stolons/tubers could be a synergistic or cumulative effect of multiple factors: (1) It appears that the development of stolons/tubers (below- and above-ground) are physiologically two distinct phenomena (light versus dark condition); (2) The aerial tuber phenotype in our transgenic lines is characteristic of the above-ground axillary nodes and under the strict control of SD photoperiod; (3) miR156 levels increase in below-ground stolon, but decrease in leaves under SD conditions (Bhogale et al., 2014); (4) The altered expression of StMSI1 and StBMI1-1 in stolon matched with miR156 expression and presence of high levels of miR156 in the StMSI1-OE and StBMI1-1-AS lines; (5) Downregulation of auxin and STL, but increase of CK response genes in both lines, might have caused axillary-bud break; and (6) The reduced level of GA biosynthesis and response genes in axillary nodes of the StMSI1-OE line could have promoted aerial tubers from stolons.

To summarize, we propose a model to explain PcG-protein–mediated regulation of tuber development in potato. StBMI1-1 suppresses miR156 expression, whereas StMSI1-OE induces miR156 expression by downregulating StBMI1-1. Further, StMSI1-OE increases the expression of miR156 through Trithorax group members involved in H3K4me3 modification. Increased miR156 causes downregulation of key tuberization genes (miR172, StBEL5, StCO, StSP5G, and StSP6A), which results in reduced below-ground tuber yield in the StMSI1-OE and StBMI1-1-AS lines. Additionally, reduced expression of auxin-, BR-, and STL- (Pasare et al., 2013) related genes and increased expression of CK transport/signaling genes in the axillary nodes of both transgenic lines inhibit the apical dominance effect and stimulate the induction of axillary stolons. Finally, the reduced expression of GA biosynthesis and signaling genes could support the development of aerial tubers from axillary stolons under SD photoperiod (Fig. 9F).

MATERIALS AND METHODS

Plant Material and Growth Conditions

Potato cultivar (Solanum tuberosum ssp andigena 7540), which tuberizes under 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 andigena plants were propagated by subculturing nodal stem explants in Murashige and Skoog’s basal medium (MS; Murashige and Skoog, 1962) supplemented with 2% (w/v) Suc. In vitro plants were maintained in a plant growth incubator (Percival Scientific) at 22°C and light intensity of 300 μmol m−2 s−1 under LD conditions unless mentioned otherwise.

Phylogenetic Analysis

A phylogenetic tree was constructed for putative MSI-like protein sequences from Arabidopsis (Arabidopsis thaliana), potato, tomato (Solanum lycopersicum), rice (Oryza sativa), Selaginella, Physcomitrella patens, and Chlamydomonas using the software “T-COFFEE” (hRp://www.ch.embnet.org/soaware/TCoffee.html) and graphical representation was performed with the software “TreeDyn” (v198.3; http://www.phylogeny.fr/one_task.cgi?task_type=treedyn; Dereeper et al., 2008). Similarly, a phylogenetic tree was also prepared for putative BMI1 orthologs from potato, tomato, and Arabidopsis. For both gene families, the full-length amino acid sequences were 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 the protein sequence, were predicted using the WD repeat protein Structure Predictor tool developed by Wu et al. (2012). The binding partners of potato StMSI1 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 proteins from Arabidopsis, tomato, and potato. Domain schematics were drawn using the software “DOG2.0” (Ren et al., 2009) and edited manually. Genomic location of putative MSI and BMI1 orthologs in potato were retrieved from the Spud DB Genome Browser in the PGSC database (http://solanaceae.plantbiology.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 and LD Conditions

For investigating the influence of photoperiod on the tissue-specific expression of StMSI1 and StBMI1-1, in vitro grown wild-type andigena plants were transferred to soil and maintained under LD photoperiod with 300 μmol m−2 s−1 light intensity for a period of 10 weeks (until they attained 10–12 leaf stages) in a growth chamber (Percival Scientific). Later, half of the plants were transferred to tuber-inducing SD photoperiodic conditions for 14 d, while the remaining plants were maintained under LD conditions. Different tissues (shoot tip, leaf, stem, root, and the stages of stolon-to-tuber transitions) were harvested at 14-d post LD/SD induction in triplicates between Zeitgeber time (ZT) = 2 and ZT = 4. Total RNA was isolated using RNAiso Plus (DSS Takara) as per the manufacturer’s instructions. Complementary DNA synthesis was carried out using 2 μg of total RNA, Superscript IV Reverse Transcriptase (Invitrogen) and Oligo dT primers. qPCR reactions were performed on a CFX96 Real-Time System (Bio-Rad) with gene-specific primers (Supplemental Table 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°C for 5 s, gene-specific annealing temperature for 15 s, and extension for 72°C for 15 s. PCR specificity was checked by melting curve analysis, and data were analyzed using the 2–ΔΔCt method (Livak and Schmittgen, 2001).

Generation of Constructs and Potato Transgenic Lines

To generate constitutive OE constructs, full-length coding sequences of StMSI1 (1,368 bp) and StBMI1-1 (1,292 bp) were amplified by reverse transcription-PCR from in vitro grown andigena plants using primers listed in Supplemental Table S11. PCR-amplified sequences were mobilized into binary vectors, pBI121 and pCB201, respectively, downstream of the CaMV 35S promoter (Xiang et al., 1999). A respective nonconserved sequence from the sense strand was used to design AS constructs for both StMSI1 and StBMI1-1 genes. PCR-amplified fragments (584 bp for StMSI1, whereas 357 bp for StBMI1-1) were cloned in AS directions into the binary vectors pBI121 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, and their AS constructs were referred to as 35S::StMSI1-AS-pBI121 (StMSI1-AS) and 35S::StBMI1-1-AS-pCB201 (StBMI1-1-AS), respectively. The StMSI1 promoter sequence (1,544 bp) was amplified from andigena genomic DNA (Supplemental Table S11) and cloned into a binary vector pBI121 upstream of the GUS gene (uidA) to generate the promStMSI1::GUS-pBI121 construct. The microRNA156 OE construct (miR156-OE) is from the previous study from our lab (Bhogale et al., 2014). All six types of binary constructs were transformed into Agrobacterium tumefaciens strain GV2260 and transgenic potato lines were generated as per the method described in Banerjee et al. (2006b). Transgenic andigena line containing 35S::GUS construct was used as a VC in the study. Several phenotypic characters (plant height, internodal distance, leaf length, leaflet number per leaf, root length, tuber numbers, and root and tuber biomass yields) were recorded after 4 weeks of LD/SD inductions.

Analysis of StMSI1 Promoter Activity

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

Histology and Scanning Electron Microscopy

For anatomical studies, a modified protocol of Cai and Lashbrook (2006) was followed on leaf and stem tissues of 8-weeks–old LD grown (StMSI1-OE3 and wild-type) plants. Ten-micrometer (10-μm) sections were obtained using a microtome (Leica), cleared with xylene, and photographed under a compound microscope (Zeiss). External leaf architecture of transgenic and wild-type plants was documented using a Quanta 200 3D eSEM apparatus (FEI), under environmental mode (eSEM).

In Vitro Tuberization

In vitro tuberization experiment was conducted as per the previous report of 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 under LD conditions. Single-node explants from the middle region of individual shoots were further cultured on MS medium with 8% (w/v) Suc (induction medium) and incubated for 4 weeks. Twelve independent plants for each line were 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), and 35S::GUS (VC) lines were grown in soil for 12 weeks under LD conditions and subjected to SD induction for another 3 weeks (until the aerial stolon initiation starts in OE3 and #G9 lines). Axillary nodes (5 mm in length) containing a part of the stem from either end of the node were harvested between ZT = 2 and ZT = 4 from six independent plants per line. All the nodes were harvested from the upper half of the plant. Samples were pooled from two to three plants forming either two or three biological replicates per line. The total RNA was isolated using RNAiso Plus (DSS Takara). RNA concentration and purity was measured using Qubit RNA Assay Kit in Qubit4.0 Fluorometer (Life Technologies) and RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). Total RNA (3 μg per sample) was used as an input material for the sample preparations. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) and index codes were added to attribute sequences to each sample. The library quality was assessed using a Bioanalyzer 2100 system (Agilent Technologies). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina). After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 150-bp paired-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 reads was done using suite tools from “Tuxedo” (Trapnell et al., 2013), based on the protocol of Mahajan et al. (2016). GO analysis was performed using the software “Blast2GO” v1.3.3 (https://www.blast2go.com/) for functional annotation of DE 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 was done using qPCR as described above in the “Tissue-Specific Transcript Abundance under SD and LD Conditions” section. The list of primers used are provided in Supplemental Table S11.

RLM-RACE Assay

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

ChIP-qPCR Analysis

ChIP was performed on potato leaves from 35S::GUS (VC), StMSI1-OE3, and StBMI1-1-AS#G9 plants using the reagents and protocol provided in a universal plant ChIP-Seq kit (Cat. No. C01010152; Diagenode) as per the manufacturer’s instructions. The sheared chromatin was immunoprecipitated using the DiaMag protein A-coated magnetic beads (Diagenode) and 1 μg of either anti-H3K4me3 (Cat. No. C15410003; Diagenode), anti-H3K27me3 (Cat. No ab6002; Abcam), or anti-IgG antibody (Cat. No. C15410206; Diagenode), in each reaction. Finally, eluted DNA was used for subsequent qPCR analysis with gene-specific primers (Supplemental Table S11).

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 for the 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 searched in the promoters of miR156 members using the tool “RSAT” (van Helden 2003; Nguyen et al., 2018). PREs were also identified in the promoters and gene bodies of StBMI1. As the promoter sequence of StBMI1-1 gene is not annotated in potato, LREs and PREs were identified from the 5′ untranslated region (298 bp) of its transcript sequence.

Grafting

Wild-type, StMSI1-OE, and StBMI1-1-AS lines were maintained in vitro on MS medium for 1 month under LD conditions. Three combinations of homo-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 in Banerjee et al. (2006a), with modifications. After 1 week of in vitro incubation, successful grafts were again transferred to MS medium containing 2% (w/v) phytagel and cefotaxime (250 mg/L) and allowed to grow for an additional 2 weeks under LD conditions. Three weeks after grafting, the average number of roots, the root length (in centimeters), and the biomass (grams of fresh weight) were recorded and tissues were harvested for further evaluation.

Statistical Analysis

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

Availability of Data

Results described in this article are included as main figures/tables and supplemental figures/tables. RNA-seq raw data FASTQ files (http://maq.sourceforge.net/fastq.shtml) generated from this study were deposited and available at the National Centre for Biotechnology Information Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA546591.

Accession Numbers

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

Supplemental Data

The following supplemental materials are available.

Acknowledgments

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

Footnotes

  • www.plantphysiol.org/cgi/doi/10.1104/pp.19.00416

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Anjan Kumar Banerjee (akb{at}iiserpune.ac.in).

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

  • ↵1 This work was supported by the Indian Institute of Science Education and Research Pune (IISER Pune) (a grant to A.K.B. and a fellowship to K.R.K.) and the Council of Scientific & Industrial Research (research fellowship to A.K.).

  • ↵2 Authors contributed equally to this article.

  • ↵4 Senior author.

  • ↵[OPEN] Articles can be viewed without a subscription.

  • Received April 2, 2019.
  • Accepted August 7, 2019.
  • Published August 19, 2019.

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PcG Proteins MSI1 and BMI1 Function Upstream of miR156 to Regulate Aerial Tuber Formation in Potato
Amit Kumar, Kirtikumar Ramesh Kondhare, Pallavi Vijay Vetal, Anjan Kumar Banerjee
Plant Physiology Jan 2020, 182 (1) 185-203; DOI: 10.1104/pp.19.00416
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