Search
2023 Volume 3
Article Contents
ARTICLE   Open Access    

Overexpression of a pear B-class MADS-box gene in tomato causes male sterility

  • # These authors contributed equally: Haiqi Zhang, Wei Han

More Information
  • B-class MADS-box genes are sufficient for the specification of petals and stamens; however, the role of Tomato MADS-box protein 6 (TM6) in seed formation in pear remains largely unknown. In this study, PbTM6a and PbTM6b, characterized as negative regulators of the response to GA4+7, were identified as classic B-class MADS-box genes. Additionally, both of the genes encoding proteins carried the highly conserved MADS-box domain, and showed high expression levels in anther, petal and filament of 'Dangshansu'. Overexpression of PbTM6a in tomato reduced the number of seeds per fruit. Analysis of the anatomical structure of floral organs revealed that the reduction in seed number in transgenic fruits might be attributed to an obstacle of pollen release due to strongly formed stomium and limited ovary space of ovule development. Moreover, triphenyl tetrazolium chloride (TTC) staining and in vitro germination tests of pollen grains indicated that PbTM6a overexpression reduced pollen viability and germination rates. Reciprocal crosses showed that the reduction in seed number in transgenic fruits was dominantly caused by the decreased fertility of pollen grains. Subsequent scanning electron microscopy showed that sterile pollen grains were caused by abnormal pollen grains. Additionally, the reduced levels of jasmonic acid (JA), abscisic acid (ABA), indole-3-acetic acid (IAA) and gibberellin A3 (GA3) in transgenic stamens contributed to the development of sterile pollen. Collectively, our results reveal the role of PbTM6a genes in controlling male infertility and broaden our understanding of the mechanism underlying the function of B-class MADS-box genes in pear.
  • 加载中
  • Supplemental Table S1 List of qRT-PCR primers.
    Supplemental Table S2 List of primers cloning PbDEFs.
    Supplemental Fig. S1 Expression levels of PbTM6a and PbTM6a in control and GA4+7-treated ovaries. DAA, Days After Anthesis. Data represents mean (± standard deviation (SD). Asterisk represents significant differences (P< 0.05) as determined by one-way ANOVA.
    Supplemental Fig. S2 Floral organs and PbTM6a subcellular localization. (a) Different floral organs of pear. (b) Subcellular localization of PbTM6a.
    Supplemental Fig. S3 Detection and observations of reproductive and vegetative growth of PbTM6a-OE lines (a) DNA amplify of PbTM6a in transgenic tomato (b) Observations of vegetative growth of wild type and transgenic tomato. (c) Observations of flowers of wild type and transgenic tomato.
    Supplemental Table S3  Seeds numbers of tomato with overexpressing PbTM6a gene in mutual pollination experiment.
  • [1]

    Zeng X, Liu H, Du H, Wang S, Yang W, et al. 2018. Soybean MADS-box gene GmAGL1 promotes flowering via the photoperiod pathway. BMC Genomics 19:51

    doi: 10.1186/s12864-017-4402-2

    CrossRef   Google Scholar

    [2]

    Mandel MA, Yanofsky MF. 1995. A gene triggering flower formation in Arabidopsis. Nature 377:522−24

    doi: 10.1038/377522a0

    CrossRef   Google Scholar

    [3]

    Adamczyk BJ, Fernandez DE. 2009. MIKC* MADS domain heterodimers are required for pollen maturation and tube growth in Arabidopsis. Plant Physiology 149:1713−23

    doi: 10.1104/pp.109.135806

    CrossRef   Google Scholar

    [4]

    Busi MV, Bustamante C, D'Angelo C, Hidalgo-Cuevas M, Boggio SB, et al. 2003. MADS-box genes expressed during tomato seed and fruit development. Plant Molecular Biology 52:801−15

    doi: 10.1023/A:1025001402838

    CrossRef   Google Scholar

    [5]

    Dreni L, Zhang D. 2016. Flower development: the evolutionary history and functions of the AGL6 subfamily MADS-box genes. Journal of Experimental Botany 67:1625−38

    doi: 10.1093/jxb/erw046

    CrossRef   Google Scholar

    [6]

    Zik M, Irish VF. 2003. Flower development: initiation, differentiation, and diversification. Annual Review of Cell and Developmental Biology 19:119−40

    doi: 10.1146/annurev.cellbio.19.111301.134635

    CrossRef   Google Scholar

    [7]

    Causier B, Schwarz-Sommer Z, Davies B. 2010. Floral organ identity: 20 years of ABCs. Seminars In Cell & Developmental Biology 21:73−79

    doi: 10.1016/j.semcdb.2009.10.005

    CrossRef   Google Scholar

    [8]

    Burko Y, Shleizer-Burko S, Yanai O, Shwartz I, Zelnik ID, et al. 2013. A role for APETALA1/fruitfull transcription factors in tomato leaf development. The Plant Cell 25:2070−83

    doi: 10.1105/tpc.113.113035

    CrossRef   Google Scholar

    [9]

    Krizek BA, Meyerowitz EM. 1996. The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity function. Development 122:11−22

    doi: 10.1242/dev.122.1.11

    CrossRef   Google Scholar

    [10]

    Theißen G. 2001. Development of floral organ identity: Stories from the MADS house. Current Opinion in Plant Biology 4:75−85

    doi: 10.1016/S1369-5266(00)00139-4

    CrossRef   Google Scholar

    [11]

    Angenent GC, Franken J, Busscher M, van Dijken A, van Went JL, et al. 1995. A novel class of MADS box genes lnvolved in ovule development in Petunia. The Plant Cell 7:1569−82

    doi: 10.2307/3870020

    CrossRef   Google Scholar

    [12]

    Melzer R, Verelst W, Theißen G. 2009. The class E floral homeotic protein SEPALLATA3 is sufficient to loop DNA in 'floral quartet'-like complexes in vitro. Nucleic Acids Research 37:144−57

    doi: 10.1093/nar/gkn900

    CrossRef   Google Scholar

    [13]

    Jack T, Fox GL, Meyerowitz EM. 1994. Arabidopsis homeotic gene APETALA3 ectopic expression: transcriptional and posttranscriptional regulation determine floral organ identity. Cell 76:703−16

    doi: 10.1016/0092-8674(94)90509-6

    CrossRef   Google Scholar

    [14]

    Jack T, Brockman LL, Meyerowitz EM. 1992. The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell 68:683−97

    doi: 10.1016/0092-8674(92)90144-2

    CrossRef   Google Scholar

    [15]

    Schwarz-Sommer Z, Hue I, Huijser P, Flor PJ, Hansen R, et al. 1992. Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development. The EMBO Journal 11:251−63

    doi: 10.1002/j.1460-2075.1992.tb05048.x

    CrossRef   Google Scholar

    [16]

    Tröbner W, Ramirez L, Motte P, Hue I, Huijser P, et al. 1992. GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis. The EMBO Journal 11:4693−704

    doi: 10.1002/j.1460-2075.1992.tb05574.x

    CrossRef   Google Scholar

    [17]

    Zahn LM, Leebens-Mack J, DePamphilis CW, Ma H, Theissen G. 2005. To B or Not to B a flower: the role of DEFICIENS and GLOBOSA orthologs in the evolution of the angiosperms. Journal of heredity 96:225−40

    doi: 10.1093/jhered/esi033

    CrossRef   Google Scholar

    [18]

    Hernández-Hernández T, Martínez-Castilla LP, Alvarez-Buylla ER. 2007. Functional diversification of B MADS-box homeotic regulators of flower development: Adaptive evolution in protein-protein interaction domains after major gene duplication events. Molecularl Biology and Evolution 24:465−81

    doi: 10.1093/molbev/msl182

    CrossRef   Google Scholar

    [19]

    Kramer EM, Dorit RL, Irish VF. 1998. Molecular evolution of genes controlling petal and stamen development: Duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages. Genetics 149:765−83

    doi: 10.1093/genetics/149.2.765

    CrossRef   Google Scholar

    [20]

    Kramer EM, Su HJ, Wu CC, Hu JM. 2006. A simplified explanation for the frameshift mutation that created a novel C-terminal motif in the APETALA3 gene lineage. BMC Evolutionary Biology 6:30

    doi: 10.1186/1471-2148-6-30

    CrossRef   Google Scholar

    [21]

    Okabe Y, Yamaoka T, Ariizumi T, Ushijima K, Kojima M, et al. 2019. Aberrant Stamen Development is Associated with Parthenocarpic Fruit Set Through Up-Regulation of Gibberellin Biosynthesis in Tomato. Plant and Cell Physiology 60:38−51

    doi: 10.1093/pcp/pcy184

    CrossRef   Google Scholar

    [22]

    Tanaka N, Tanaka-Moriya Y, Mimida N, Honda C, Iwanami H, et al. 2016. The analysis of transgenic apples with down-regulated expression of MdPISTILLATA. Plant Biotechnology 33:395−401

    doi: 10.5511/plantbiotechnology.16.1109a

    CrossRef   Google Scholar

    [23]

    Yao JL, Dong YH, Morris BAM. 2001. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor. PNAS 98:1306−11

    doi: 10.1073/pnas.98.3.1306

    CrossRef   Google Scholar

    [24]

    Mazzucato A, Olimpieri I, Siligato F, Picarella ME, Soressi GP. 2008. Characterization of genes controlling stamen identity and development in a parthenocarpic tomato mutant indicates a role for the DEFICIENS ortholog in the control of fruit set. Physiologia Plantarum 132:526−37

    doi: 10.1111/j.1399-3054.2007.01035.x

    CrossRef   Google Scholar

    [25]

    de Martino G, Pan I, Emmanuel E, Levy A, Irish VF. 2006. Functional analyses of two tomato APETALA3 genes demonstrate diversification in their roles in regulating floral development. The Plant Cell 18:1833−45

    doi: 10.1105/tpc.106.042978

    CrossRef   Google Scholar

    [26]

    Cao X, Liu X, Wang X, Yang M, van Giang T, et al. 2019. B-class MADS-box TM6 is a candidate gene for tomato male sterile-1526. Theoretical and Applied Genetics 132:2125−35

    doi: 10.1007/s00122-019-03342-z

    CrossRef   Google Scholar

    [27]

    Yao JL, Xu J, Tomes S, Cui W, Luo Z, et al. 2018. Ectopic expression of the PISTILLATA homologous MdPI inhibits fruit tissue growth and changes fruit shape in apple. Plant Direct 2:e00051

    doi: 10.1002/pld3.51

    CrossRef   Google Scholar

    [28]

    Zouine M, Maza E, Djari A, Lauvernier M, Frasse P, et al. 2017. TomExpress, a unified tomato RNA-Seq platform for visualization of expression data, clustering and correlation networks. The Plant Journal 92:727−35

    doi: 10.1111/tpj.13711

    CrossRef   Google Scholar

    [29]

    Liu L, Wang Z, Liu J, Liu F, Zhai R, et al. 2018. Histological, hormonal and transcriptomic reveal the changes upon gibberellin-induced parthenocarpy in pear fruit. Horticulture Research 5:1

    doi: 10.1038/s41438-017-0012-z

    CrossRef   Google Scholar

    [30]

    Roque E, Serwatowska J, Cruz Rochina M, Wen J, Mysore KS, et al. 2013. Functional specialization of duplicated AP3-like genes in Medicago truncatula. The Plant Journal 73:663−75

    doi: 10.1111/tpj.12068

    CrossRef   Google Scholar

    [31]

    Martín-Pizarro C, Triviño JC, Posé D. 2019. Functional analysis of the TM6 MADS-box gene in the octoploid strawberry by CRISPR/Cas9-directed mutagenesis. Journal of Experimental Botany 70:885−95

    doi: 10.1093/jxb/ery400

    CrossRef   Google Scholar

    [32]

    Zhai R, Wang Z, Zhang S, Meng G, Song L, et al. 2016. Two MYB transcription factors regulate flavonoid biosynthesis in pear fruit (Pyrus bretschneideri Rehd.). Journal of Experimental Botany 67:1275−84

    doi: 10.1093/jxb/erv524

    CrossRef   Google Scholar

    [33]

    van der Linden CG, Vosman B, Smulders MJM. 2002. Cloning and characterization of four apple MADS box genes isolated from vegetative tissue. Journal of Experimental Botany 53:1025−36

    doi: 10.1093/jexbot/53.371.1025

    CrossRef   Google Scholar

    [34]

    Suog SK, Yu GH, An G. 1999. Characterization of MdMADS2, a member of the SQUAMOSA subfamily of genes, in apple. Plant physiology 120:969−78

    doi: 10.1104/pp.120.4.969

    CrossRef   Google Scholar

    [35]

    Wada M, Oshino H, Tanaka N, Mimida N, Moriya-Tanaka Y, et al. 2018. Expression and functional analysis of apple MdMADS13 on flower and fruit formation. Plant Biotechnology 35:207−13

    doi: 10.5511/plantbiotechnology.18.0510a

    CrossRef   Google Scholar

    [36]

    Pnueli L, Hareven D, Rounsley SD, Yanofsky MF, Lifschitz E. 1994. Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants. The Plant Cell 6:163−73

    doi: 10.1105/tpc.6.2.163

    CrossRef   Google Scholar

    [37]

    Vrebalov J, Pan IL, Arroyo AJM, McQuinn R, Chung M, et al. 2009. Fleshy fruit expansion and ripening are regulated by the Tomato SHATTERPROOF gene TAGL1. The Plant Cell 21:3041−62

    doi: 10.1105/tpc.109.066936

    CrossRef   Google Scholar

    [38]

    Ampomah-Dwamena C, Morris BA, Sutherland P, Veit B, Yao JL. 2002. Down-regulation of TM29, a tomato SEPALLATA homolog, causes parthenocarpic fruit development and floral reversion. Plant Physiology 130:605−17

    doi: 10.1104/pp.005223

    CrossRef   Google Scholar

    [39]

    Poupin MJ, Federici F, Medina C, Matus JT, Timmermann T, et al. 2007. Isolation of the three grape sub-lineages of B-class MADS-box TM6, PISTILLATA and APETALA3 genes which are differentially expressed during flower and fruit development. Gene 404:10−24

    doi: 10.1016/j.gene.2007.08.005

    CrossRef   Google Scholar

    [40]

    Boss PK, Vivier M, Matsumoto S, Dry IB, Thomas MR. 2001. A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF, is not only expressed in flowers but also throughout berry development. Plant Molecular Biology 45:541−53

    doi: 10.1023/A:1010634132156

    CrossRef   Google Scholar

    [41]

    Wang Y, Liu Z, Wu J, Hong L, Liang J, et al. 2021. MADS-box protein complex VvAG2, VvSEP3 and VvAGL11 regulates the formation of ovules in Vitis vinifera L. cv. 'Xiangfei'. Genes 12:647

    doi: 10.3390/genes12050647

    CrossRef   Google Scholar

    [42]

    Cong L, Wu T, Liu H, Wang H, Zhang H, et al. 2020. CPPU may induce gibberellin-independent parthenocarpy associated with PbRR9 in 'Dangshansu' pear. Horticulture Research 7:7

    doi: 10.1038/s41438-020-0285-5

    CrossRef   Google Scholar

    [43]

    Zhang H, Han W, Wang H, Cong L, Zhai R, et al. 2021. Downstream of GA4, PbCYP78A6 participates in regulating cell cycle-related genes and parthenogenesis in pear (Pyrus bretshneideri Rehd.). BMC Plant Biology 21:292

    doi: 10.1186/s12870-021-03098-z

    CrossRef   Google Scholar

    [44]

    Otero AJ, Rodríguez I, Falero G. 1991. 2,3,5-Triphenyl tetrazolium chloride (TTC) reduction as exponential growth phase marker for mammalian cells in culture and for myeloma hybridization experiments. Cytotechnology 6:137−42

    doi: 10.1007/BF00373031

    CrossRef   Google Scholar

    [45]

    Tovar-Méndez A, Kumar A, Kondo K, Ashford A, Baek YS, et al. 2014. Restoring pistil-side self-incompatibility factors recapitulates an interspecific reproductive barrier between tomato species. The Plant Journal 77:727−36

    doi: 10.1111/tpj.12424

    CrossRef   Google Scholar

    [46]

    Wang H, Zhang H, Liang F, Cong L, Song L, et al. 2021. PbEIL1 acts upstream of PbCysp1 to regulate ovule senescence in seedless pear. Horticulture Research 8:59

    doi: 10.1038/s41438-021-00491-5

    CrossRef   Google Scholar

    [47]

    Song S, Qi T, Huang H, Xie D. 2013. Regulation of stamen development by coordinated actions of jasmonate, auxin and gibberellin in Arabidopsis. Molecular Plant 6:1065−73

    doi: 10.1093/mp/sst054

    CrossRef   Google Scholar

    [48]

    Wasternack C. 2007. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. Annals of Botany 100:681−97

    doi: 10.1093/aob/mcm079

    CrossRef   Google Scholar

    [49]

    Nagpal P, Ellis CM, Weber H, Ploense SE, Barkawi LS, et al. 2005. Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation. Development 132:4107−18

    doi: 10.1242/dev.01955

    CrossRef   Google Scholar

    [50]

    Cecchetti V, Altamura MM, Falasca G, Costantino P, Cardarelli M. 2008. Auxin regulates Arabidopsis anther dehiscence, pollen maturation, and filament elongation. The Plant Cell 20:1760−74

    doi: 10.1105/tpc.107.057570

    CrossRef   Google Scholar

    [51]

    Yao X, Tian L, Yang J, Zhao Y, Zhu Y, et al. 2018. Auxin production in diploid microsporocytes is necessary and sufficient for early stages of pollen development. PLoS Genetics 14:e1007397

    doi: 10.1371/journal.pgen.1007397

    CrossRef   Google Scholar

    [52]

    Kovaleva LV, Voronkov AS, Zakharova EV, Andreev IM. 2018. ABA and IAA control microsporogenesis in Petunia hybrida L. Protoplasma 255:751−59

    doi: 10.1007/s00709-017-1185-x

    CrossRef   Google Scholar

    [53]

    Wang M, Hoekstra S, van Bergen S, Lamers GEM, Oppedijk BJ, et al. 1999. Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L. Plant Molecular Biology 39:489−501

    doi: 10.1023/A:1006198431596

    CrossRef   Google Scholar

    [54]

    Eckardt NA. 2002. Abscisic acid biosynthesis gene underscores the complexity of sugar, stress, and hormone interactions. The Plant Cell 14:2645−49

    doi: 10.1105/tpc.141110

    CrossRef   Google Scholar

    [55]

    Chen R, Zhao X, Shao Z, Wei Z, Wang Y, et al. 2007. Rice UDP-glucose pyrophosphorylase1 is essential for pollen callose deposition and its cosuppression results in a new type of thermosensitive genic male sterility. The Plant Cell 19:847−61

    doi: 10.1105/tpc.106.044123

    CrossRef   Google Scholar

    [56]

    Zanor MI, Osorio S, Nunes-Nesi A, Carrari F, Lohse M, et al. 2009. RNA interference of LIN5 in tomato confirms its role in controlling brix content, uncovers the influence of sugars on the levels of fruit hormones, and demonstrates the importance of sucrose cleavage for normal fruit development and fertility. Plant Physiology 150:1204−18

    doi: 10.1104/pp.109.136598

    CrossRef   Google Scholar

    [57]

    Jacobsen SE, Olszewski NE. 1991. Characterization of the arrest in anther development associated with gibberellin deficiency of the gib-1 mutant of tomato. Plant Physiology 97:409−14

    doi: 10.1104/pp.97.1.409

    CrossRef   Google Scholar

    [58]

    Li P, Tian J, Guo C, Luo S, Li J. 2021. Interaction of gibberellin and other hormones in almond anthers: phenotypic and physiological changes and transcriptomic reprogramming. Horticulture Research 8:94

    doi: 10.1038/s41438-021-00527-w

    CrossRef   Google Scholar

  • Cite this article

    Zhang H, Han W, Linghu T, Zhao Z, Wang A, et al. 2023. Overexpression of a pear B-class MADS-box gene in tomato causes male sterility. Fruit Research 3:1 doi: 10.48130/FruRes-2023-0001
    Zhang H, Han W, Linghu T, Zhao Z, Wang A, et al. 2023. Overexpression of a pear B-class MADS-box gene in tomato causes male sterility. Fruit Research 3:1 doi: 10.48130/FruRes-2023-0001

Figures(8)

Article Metrics

Article views(4599) PDF downloads(685)

ARTICLE   Open Access    

Overexpression of a pear B-class MADS-box gene in tomato causes male sterility

Fruit Research  3 Article number: 1  (2023)  |  Cite this article

Abstract: B-class MADS-box genes are sufficient for the specification of petals and stamens; however, the role of Tomato MADS-box protein 6 (TM6) in seed formation in pear remains largely unknown. In this study, PbTM6a and PbTM6b, characterized as negative regulators of the response to GA4+7, were identified as classic B-class MADS-box genes. Additionally, both of the genes encoding proteins carried the highly conserved MADS-box domain, and showed high expression levels in anther, petal and filament of 'Dangshansu'. Overexpression of PbTM6a in tomato reduced the number of seeds per fruit. Analysis of the anatomical structure of floral organs revealed that the reduction in seed number in transgenic fruits might be attributed to an obstacle of pollen release due to strongly formed stomium and limited ovary space of ovule development. Moreover, triphenyl tetrazolium chloride (TTC) staining and in vitro germination tests of pollen grains indicated that PbTM6a overexpression reduced pollen viability and germination rates. Reciprocal crosses showed that the reduction in seed number in transgenic fruits was dominantly caused by the decreased fertility of pollen grains. Subsequent scanning electron microscopy showed that sterile pollen grains were caused by abnormal pollen grains. Additionally, the reduced levels of jasmonic acid (JA), abscisic acid (ABA), indole-3-acetic acid (IAA) and gibberellin A3 (GA3) in transgenic stamens contributed to the development of sterile pollen. Collectively, our results reveal the role of PbTM6a genes in controlling male infertility and broaden our understanding of the mechanism underlying the function of B-class MADS-box genes in pear.

    • MADS-box genes regulate diverse aspects of growth and development in flowering plants, including photoperiod response and flowering time control[1], flower meristem determination and floral organ identification[2], pollen fertility regulation[3], seed and fruit development[4], and endocarp development and vegetative development regulation[5]. Typically, MADS-box transcription factors are involved in specifying the identity of floral organs, including sepals, petals, stamens, and carpels[6], the genetic formation control of which was deciphered with a genetic model (ABC model) was recently advanced to the ABCDE model[7]. Briefly, class A genes including APETALA1 (AP1) in Arabidopsis thaliana[8], are needed for the formation of sepals. Represented by AtAPETALA3 (AtAP3), AtPISTILLATAb(AtPI), class B plus class A genes determine the development of petals[9]. AtAGAMOUS (AtAG), a representative class C gene, functions alone to control the formation of carpels, Class B plus class C genes are required for the identification of stamens[10]. Class D genes, such as AtSEEDSTICK (AtSTK), individually regulate the specification of ovules[11], while class E genes represented by AtSEPALLATA (AtSEP) genes such as AtSEP1, AtSEP2, AtSEP3, and AtSEP4, are necessary for establishing the identity of petals, stamens, and carpels[12].

      Different lineages from B-class MADS box genes can give rise to distinct phenotypes. AtAP3 and AtPI function as B-class MADS box genes in Arabidopsis[13,14], DEFICIENS (DEF) and GLOBOSA (GLO) constitute B class MADS box genes function activity in A. majus[15,16]. Mutations in either one of these genes leads to the transformation of petals into sepals and that of stamens into carpels[1318]. The AP3 lineage gives rise to two AP3-like lineages due to another major duplication at the base of the core eudicots. Two AP3-like lineages contain euAP3 that contains AP3 itself, and TM6, which lacks a representative in A. thaliana[19,20]. Silencing Tomato AP3 (TAP3) in the tomato ovary using a specific promoter resulted in male sterility and parthenocarpy[21]. In consideration of the differences between siliques in Arabidopsis and pome fruit derived from both ovary and hypanthium tissues in apple (Malus domestica), silencing of the MdPI gene confers parthenocarpy and results in the transformation of petals into sepals and of stamens into pistils, respectively[22,23]. In the parthenocarpic fruit (pat) mutant of tomato, autonomous development of ovary is associated with the altered expression of class B MADS box genes, including stamenless (sl), pistillate (pi) and SlDEFICIENS (DEF)[24]. Different from the phenotype of mutation in TAP3, RNA interference (RNAi)-induced reduction in TM6 expression results in different phenotypes with homeotic defects primarily in stamens and normal carpel tissue in appearance[25]. Tomato male sterility may be a consequence of reduced expression of SlTM6[26]. Overexpression of TM6 can partially restore the tap3 second whorl phenotype, but TAP3 and TM6 showed different expression patterns and distinct functions in flower development[25]. Different lineages from class B MADS-box genes have complex biological functions during flower development, these orthologues in pear remain largely unrevealed.

      Ectopic expression is sufficient for the identification of classic B function. Ectopic expression of AtAP3 results in the conversion of carpels into stamens, and fails to form a functional gynoecium, resulting in a female sterile flower[13]. Flowers of plants ectopically expressing AtPI exhibit different phenotypes, with partial replacement of sepals by petals[9]. Plants overexpressing AtAP3 and AtPI together produce flowers containing two outer whorls of petals and inner whorls of stamens and the early flowering and leaf curling[9]. Overexpression of MdPI results in the complete conversion of sepals into petals and in the production of distinctly flattened fruit as a consequence of restricted cell expansion[27]. Despite that, overexpression of TM6 in wild-type background leads to the production of normal floral identification[25], given that TM6 has more prominent expression in the stamens and carpels, the function of TM6 and its roles in fruit development require further understanding.

      The down-regulation of TAP3 leads to parthenocarpic fruit set in tomato[21] and the members of B class MADS-box were down-regulated during fruit set[28]. Exogenous application of gibberellic acid 4+7 (GA4+7) reduced the expression of PbTM6a and PbTM6b in parthenocarpic pear ovary[29]. It compelled us to figure out the function of PbTM6 on the fruit set process in pear. Silencing TM6 in Medicago truncatula[30] or in tomato[25] or mutating FaTM6 in in the octoploid Strawberry leads to defects in the anthers and low viability of pollen grains[31]. Because of the long breeding cycle and arduous genetic transformation of pear, investigation of the function of candidate genes in this plant species is difficult. Research related to PbTM6 genes has not been reported in pear to date.

      In this study, we explored the potential role of PbTM6 downstream GA4+7 induced in pear, by generating transgenic tomato lines overexpressing PbTM6a. Specifically, we examined the function of PbTM6a in fertility, and preliminarily explored the regulatory mechanism underlying its role in male sterility. Our results have shed light on the function of PbTM6a in pear, and broaden the function of class B MADS box genes.

    • Sixteen-year-old 'Dangshansu' pear trees grafted onto Pyrus betulifolia Bge rootstocks were used as materials for sampling flowers, which were collected from pear experimental base of Northwest A&F University located in MeiXian County, Shaanxi Province, China (34.28° N, 108.22° E; 562 m). Samples of all treatments were immediately frozen in liquid nitrogen after removal, and stored at −80 °C.

      Tomato Ailsa Craig (Solanum lycopersicum L.), received from Xiangqiang Zhan, from Northwest A&F University, Yangling, China, was used for genetic transformation. Tomato planting conditions: 25 °C during the day, 18 °C at night, and photoperiod 18/6 h.

    • The MADS-box proteins from ABCDE model were retrieved from NCBI database (www.ncbi.nlm.nih.gov) and a phylogenetic tree was constructed by the software MEGA X with the Maximum Likelihood method and bootstrap analysis. Boot-strap values were calculated from 1,000 replicate analyses. The constructive methods were as per our previous study[32]. The amino acid sequence alignment of AtAP3, SlTM6, PbTM6a and PbTM6b was carried out through DNAMAN version 9.

      The protein accessions used were as follows: AtAG (Arabidopsis thaliana, P17839.2); AtAGL11 (Arabidopsis thaliana, Q38836.1); AtAP1 (Arabidopsis thaliana, CAA78909.1); AtAP3 (Arabidopsis thaliana, AAD51902.1); AtCAL (Arabidopsis thaliana, BAH30315.1); AtAGL8 (Arabidopsis thaliana, Q38876.1); AtFUL (Arabidopsis thaliana, OAO94650.1); AtPI (Arabidopsis thaliana, P48007.1); AtSEP1 (Arabidopsis thaliana, OAO95853.1); AtSEP2 (Arabidopsis thaliana, OAP07944.1); AtSHP1 (Arabidopsis thaliana, OAP06129.1); AtSHP2 (Arabidopsis thaliana, NP_565986.1); MdAG = MdMADS15 (Malus domestica, NP_001315863.1)[33]; MdAGL1 = MdMADS14 (Malus domestica, XP_008342376.1)[33]; MdAGL11 = MdMADS10 (Malus domestica, NP_001280931.1)[23]; MdAP1 (Malus domestica, ACD69426.1); MdCAL = MdMADS2 (Malus domestica, XP_008393256.1)[34]; MdDEF = MdMADS23 (Malus domestica, XP_028962429.1; HM122607); MdSEP1 = MdMADS8 (Malus domestica, NP_001280893.1)[35]; MdPI (Malus domestica, CAC28022.1); PbTM6a (Pyrus bretschneideri, XP_009346295.1); PbTM6b (Pyrus bretschneideri, XP_009378222.1); SlAG = TAG1 (Solanum lycopersicum, NP_001266181.1)[36]; SlAGL11 = TAGL1 (Solanum lycopersicum, XP_004241906.1)[37]; SlDEF=TAP3 (Solanum lycopersicum, NP_001234077.2)[25]; SlFUL (Solanum lycopersicum, NP_001294867.1); TAP3 (Solanum lycopersicum, ABG73412.1)[25]; SlPI = TPI = SlFBP1 (Solanum lycopersicum, NP_001234075.2; ABG73411.1; XP_004245202.1)[25]; SlMADS6 = TM6 = SlTDR6 (Solanum lycopersicum, QHB49937.1; ABG48621.1; NP_001311309.1)[25]; SlSEP1 = TM29 (Solanum lycopersicum, NP_001233911.1)[38]; SlTAGL1 (Solanum lycopersicum, NP_001300859.1); SlAGL6 (Solanum lycopersicum, NP_001348459.1); VvAGL11 = VvMADS5 = VvAG3 = (Vitis vinifera, QSX80212.1; A0A217EJJ0.1); VvAP3 = VvAP3a = VvPMADS1 = VvDEF (Vitis vinifera, E0CPH4.1; RVW50380.1)[39]; VvMADS2 (Vitis vinifera, XP_019080194.1); VvMADS1 = VvAGAMOUS/SHATTERPROOF (Vitis vinifera, NP_001268105.1)[40]; VvAG (VvAGAMOUS) = AG1 or AG2 (Vitis vinifera, NP_001268097.1)[41]; AnDEFA (Antirrhinum majus, P23706.1); AnGLO (Antirrhinum majus, Q03378.1).

    • PbTM6a and PbTM6b specific quantitative primers were designed by their reference sequence on the NCBI website (www.ncbi.nlm.nih.gov) (Supplemental Table S1). The flowers were separated into different floral organs including sepals, hypanthium, petals, filament, anther, style, and ovary of two days before anthesis, anthesis and four days after anthesis and then were used for RNA extraction and subsequently tissue-specific expression analysis. Total RNA extraction and Real-time fluorescent Quantitative Polymerase Chain Reaction (qRT-PCR) was performed as previously described[42]. The pear actin-β gene was used as an internal standard for expression analysis (Supplemental Table S1).

    • The CDS of PbTM6a after the removal of termination codons was cloned into the pCAMBIA2300 vector fusing with the Green Fluorescent Protein (GFP) reporter under the Cauliflower mosaic virus (CaMV) 35S promoter using primers with adaptors (Supplemental Table S2). Agrobacterium mediated transient transformation was used for subcellular localization. Agrobacterium containing recombinant plasmid was revitalized twice and suspended with cell resuspension solution (10 μmol·L−1 MES, 10 μmol·L−1 MgCl2, 200 μmol·L−1 AS, pH = 5.6), then was transformed into tobacco (Nicotiana benthamiana) leaves by injection and the empty vector expressing GFP was used as a positive control. After culture in the dark for 1 d and in the light for 2 d, the back of the tobacco leaf was made into freehand section for GFP observation. The GFP signal was observed with a fluorescence microscope (BX63, OLYMPUS, Japan). Activate and adjust the concentration of the bacterial solution till OD600 = 0.6, then inject the bacterial solution from the back of the tobacco leaf using a syringe.

    • The full-length PbTM6a (LOC103938026) coding sequence (CDs) was isolated from 'Dangshansu' cDNA using primers with adaptors (Supplemental Table S2), and then cloned into the pBI121 vector to generate a recombinant plasmid using a Clone-Express One Step Cloning kit (Vazyme, Nanjing, China), then transformed into Agrobacterium tumefaciens strain LBA4404 by heat-shock method. Agrobacterium revitalized and suspensions prepared were manufactured following the methods as reported in our lab[43]. Tomato genetic transformation was carried out as previously reported[43].

    • Paraffin sectioning experiments were carried out as previously reported[29]. Samples collected were immediately fixed in formaldehyde–acetic acid–alcohol fixative and stored at 4 °C. The samples were dehydrated in ethanol/xylene and embedded in paraffin, sectioned into 8-µm-thick slices, dried, and stained with safranin and fast green. A microscopic imaging system (BX51 + PD72 + IX71, Olympus, Japan) was used to observe the anatomical images.

    • TTC (2,3,5-triphenyl tetrazolium chloride) solution was performed to determine pollen grain viability[44]. Fresh pollen of wild-type and transgenic tomato were evenly sprinkled on the glass slide, respectively. Dripped with a drop of TTC dye solution, and then covered with a cover glass. Prepared glass slides were observed under an optical microscope after incubating at 37 °C for 15−20 min. The reaction of TTC with succinate dehydrogenase in the mitochondria of living cells yielded red formazan, which are used to indicate cell viability. Living pollen were stained in light red and deeper red. Percentage of pollen viability was calculated by the stained pollen number including light red and deeper red pollen, divided by the total pollen number. Percentage of vigorous pollen was calculated by the deeper stained pollen number divided by the total pollen number.

    • Pollen germination in vitro medium was prepared as follows: 12 g sucrose and 0.03 g Ca (NO3)2·4H2O, 0.02 g MgSO4·7H2O, 0.01g KNO3 and 0.005 g H3BO3 were added to 100 ml ddH2O. Agar powder was added to make the final concentration of 0.1% (m/v) after adjusting the pH to 6.5. Next, the medium was heated until the agar completely melted, and then cooled to room temperature. A drop of unset culture medium was added onto the glass slide as the pollen germination bed. The pollen was evenly sprinkled on the culture bed, cultivated for 1 h under dark conditions at 28 °C. A drop of Sigma-aldrich (0.1%) was added onto the glass slide treated above, then covered with a cover glass. After staining for 5 min, the pollen germination situation was assessed under a fluorescent microscope in vitro.

    • Reciprocal crosses experiment was carried out as previously reported[45]. For the female parent, the stamens were removed two days before flowering to prevent self-pollination. Before and after pollination, the inflorescence was bagged for the prevention of natural hybridization. For the male parent, blooming flowers with bright yellow petals, stamens golden, un-scattered pollen were chosen to take anthers and made into pollen. A pencil's rubber-head was used as a pollinator.

    • Pollens of PbTM6a-OE and wild type from the day before flowering was collected and glued to an aluminum sample holder with conductive glue. Scanning electron microscope (S-3400N) was applied for the visualization of pollen morphology. Three visual fields of scanning electron microscope were used for the statistics of normal pollen grain.

    • The determination of hormone content was performed as previously described[46]. Briefly, tomato stamen samples of wild-type and PbTM6a-OE were collected 2 d before anthesis with at least three replicates per treatment. 0.5−1 g of the sample was added into a 2 mL centrifuge tube, and the weight of each sample was recorded for analysis, 1 mL ethyl acetate was used as the extract and added into centrifuge tube, subsequently with vortex and shaken for 10 min, centrifuged at 12,000 rpm for 10 min, and the supernatant was transferred into a new 2 mL tube. The liquid collected was evaporated to dryness with a nitrogen blower, and then dissolved with 200 μL methanol (50%) and filtered through a 0.22-µm filter membrane before testing. The hormones levels were determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (AB SCIEX Triple TOF 5600+, Darmstadt, IN, USA). UPLC-MS/MS was performed using an ACQUITY UPLC HSS T3 (1.8 mm, Waters, USA) column (2.1 mm × 100 mm). The specific parameters were as in our previous study[46].

    • To confirm which class-B MADS box genes the two PbTM6 genes were orthologues of, phylogenetic analysis of classical MADS box genes were performed. The results showed that both PbTM6 belong to the B-class MADS box genes and were more closely related to TM6 than to TAP3 and AtAP3 (Fig. 1).

      Figure 1. 

      Phylogenetic analysis of PbTM6 and other MADS box proteins involved in the classic ABCDE model.

      Alignment of the amino acid sequences of PbTM6a, PbTM6b, AtAP3 and TM6 showed that all of these proteins possess a highly conserved MADS domain (1-60 aa) at the N-terminus, and the amino acid sequences of PbTM6a and PbTM6b were highly similar (Fig. 2a). Expression levels of PbTM6a and PbTM6b in the flower organs of pear were analyzed by qRT-PCR at developmental stages (2 d before anthesis, at anthesis and 4 d after anthesis) (Supplemental Fig. S1). The results showed that PbTM6a and PbTM6b shared similar expression patterns. The expression levels of PbTM6a and PbTM6b were high in anthers, filaments and petals, which suggested that these genes play important roles in the development of male reproductive organs and petal development (Fig. 2b & c). The expression of PbTM6a and PbTM6b was down-regulated in pear fruitlets by treatments of GA4+7 (Supplemental Fig. S2). The down-regulation of PbTM6a and PbTM6b might participate in the pear fruit set process. Subcellular localization analysis via the heterologous expression of a PbTM6a-GFP fusion in Nicotiana benthamiana leaf epidermal cells showed a fluorescence signal exclusively located in the cell nucleus (Supplemental Fig. S2b), indicating that PbTM6a is a nuclear-localized protein. In summary, our results showed that PbTM6a and PbTM6b correlate with B-class MADS-box genes, and highly expressed in male reproductive organs and petals.

      Figure 2. 

      (a) Amino acid sequence alignment of AtAP3, PbTM6a, PbTM6b, and TM6. MADS-box conserved domains are outlined in black. Expression of (b) PbTM6a and (c) PbTM6b in different floral organs of pear at different developmental stages. −2 DAA, 2 d before anthesis; 0 DAA, anthesis; 4 DAA, 4 d after anthesis. Data represents mean (± standard deviation (SD). Significant differences (P < 0.05) among treatments are determined by one-way analysis of variance (ANOVA), indicated with different lowercase letters.

    • Since the expression patterns of PbTM6a and PbTM6b in floral organs and the amino acid sequences of the encoded proteins were highly similar, PbTM6a had higher abundance and more significantly decreased expression in parthenocarpic ovaries than PbTM6b[29], PbTM6a was chosen for further analysis.

      To explore the potential function of PbTM6a in floral organ identity and fruit development, three PbTM6a overexpression (PbTM6a-OE) lines were generated in tomato (Supplemental Fig. S3a). It appeared similar between the wild type and PbTM6a-OE lines during the vegetative growth phase. Plant height and growth as leaf size, shape and color of wild-type and PbTM6a-OE plants were nearly uniform (Supplemental Fig. S3b). Overexpression of PbTM6a seemed to not affect the formation of floral organs compared with the wild type in appearance (Supplemental Fig. S3c). Moreover, flower and fruits of the wild type and PbTM6a-OE lines appeared similar during fruit development (Fig. 3a). The number and size of seeds were significantly reduced in the mature fruits of PbTM6a-OE lines compared with those of the wild type (Fig. 3b), although the mature fruits of wild-type plant showed traces of retarded seed formation. The statistics of seed number per fruit and seed weight proved the reduced number and size of seeds produced by PbTM6a-OE lines (Fig. 3c & d). Thus, we showed that the overexpression of PbTM6a in tomato reduces seed size and number.

      Figure 3. 

      Phenotypic analysis of wild type and transgenic tomato plants. (a) Comparison of the growth and development of tomato fruits and the structure of floral organs between the wild type and PbTM6a overexpression (PbTM6a-OE) lines. (b) Heterologous overexpression of PbTM6a decreases the number and size of tomato seeds. (c) Number of wild-type tomato plants and PbTM6a-OE lines. (d) Thousand seed weight of wild-type plants and PbTM6a-OE lines. Data represents mean (± standard deviation (SD). Significant differences (P < 0.05) among treatments are determined by one-way analysis of variance (ANOVA), indicated with different lowercase letters.

    • To explore the cause of the reduction in seed size and number in transgenic tomato, stamens at the late stage of anther development were selected for preparing and analyzing paraffin sections (Fig. 4a). Both transgenic and wild type lines could produce anthers with similar structure and pollen grains (Fig. 4bg). In addition, the connective tissues between homolateral pollen sacs were broken in wild type, but the stomium bound to the connective tissue appears to be more strongly formed in PbTM6a-OE anthers, which would influence anther dehiscence.

      Figure 4. 

      Morphological and histological observations of floral organs collected from wild type plants and PbTM6a-OE lines. Red lines indicate the section position. (a) Stamens of wild type plants and PbTM6a-OE lines after sepals removed at the anther dehiscence stage. (b)−(d) Histological analysis of anthers of the wild type plants and PbTM6a-OE lines. (e)−(g) Further analysis of anthers shown in (b)−(d). Arrows indicate the position of stomium. (h) Morphology of wild type and PbTM6a-OE flowers at anthesis. (i)−(k) Histological analysis of the cross-section of flowers shown in (h). (l) Analysis of the small tomato fruit of wild-type plants and PbTM6a-OE lines after fruit set. (m)−( o) Histological analysis of the cross-section of ovary shown in (l). Scale bars: 2 cm in (a), (h),( l); 200 µm in (b), (c), (d), (i), (j), (k), (m), (n), (o); 100 µm in (e), (f), (g).

      To confirm whether the reduction in seed number was related to defects in the ovule, the ovary was sampled at anthesis and fruit setting for histological observation. Results showed that the number of maternal ovules seemed similar between PbTM6a-OE lines and the wild type (Fig. 4hk). The total number of ovules in transgenic tomato plants was similar to that in wild type plants even after successful pollination and fertilization (Fig. 4mo). Moreover, the placenta in transgenic ovaries was larger than that in wild type and occupied the most space of ovaries which might lead to the arrest of ovules development (Fig. 4ho). These results indicate that the strongly formed stomium and inflated placenta in transgenic tomatoes might contribute to the reduction in seeds size and number.

    • Because tomato is a self-pollinating crop, defects in pollen germination were speculated as the cause of poor pollination and fertilization. To determine pollen vigor, the pollen of just-opened tomato flowers were selected and used for TTC staining and pollen germination in vitro. Pollen with cell viability was stained red. Pollen with no cell viability was not stained. Most of the pollen of wild type plants was stained deep pink with TTC, while most pollen of PbTM6a-OE lines stained light pink or did not stain (Fig. 5ac). The results of pollen germination experiments indicated that the majority of wild-type pollens were capable of germination, whereas only a few transgenic pollens could germinate (Fig. 5df). The percentage of fertile pollen was approximately 95% in wild-type plants, which was significantly higher than that in PbTM6a-OE1 (8%) and PbTM6a-OE2 (11%) lines (Fig. 5g). Similarly, the percentage of vigorous pollen in wild-type plants (81%) was higher than that in PbTM6a-OE lines (Fig. 5h). The statistics of germinated pollen supported the results of the germination experiment (Fig. 5i). Overall, our results demonstrated that tomato plants overexpressing PbTM6a produced pollen with weak vigor and reduced germination.

      Figure 5. 

      Assessment of pollen vigor via the viability evaluated by TTC staining and germination in vitro. (a)−(c) Visualization of pollen viability of PbTM6a-OE lines and wild-type plants in vitro by TTC staining. (d)−(f) Evaluation of PbTM6a-OE and wild-type pollen germination in vitro under a fluorescence microscope. Statistics of the percentage of (g) pollen viability, (h) vigorous pollen and (i) pollen germination rate (i) in PbTM6a-OE lines and wild-type plants. Data represents mean (± standard deviation (SD). Significant differences (P < 0.05) among treatments as determined by one-way analysis of variance (ANOVA), are indicated with different lowercase letters.

      To determine whether the reduced seed number and size of PbTM6a-OE line were maternally inherited, we carried out reciprocal crosses. Emasculated wild-type tomato plants pollinated with PbTM6a-OE pollen produced lower seed number per fruit because of inadequate fertilization; however the seed number was partly restored in transgenic tomato pollinated with wild-type pollen (Fig. 6a). The statistics of seed number coincide with our observation (Fig. 6b, Supplemental Table S3). Therefore, low-vigor pollen produced by PbTM6a-OE lines appear to be the dominant factor responsible for unsuccessful fertilization, which in turn led to the production of fruits with less seeds.

      Figure 6. 

      Comparison of seed formation and seed number among the progeny of reciprocal crosses and self-pollinations. (a) Seed formation and (b) seed number per fruit produced by PbTM6a-OE (♀) × wild Type (♂) and wild Type (♀) × PbTM6a-OE (♂) reciprocal crosses and wild-type and PbTM6a-OE self-pollinationas. Data represents mean (± standard deviation (SD). Significant differences (P < 0.05) among treatments are determined by one-way analysis of variance (ANOVA), indicated with different lowercase letters.

    • To determine the cause of the low viability of pollen, we observed pollen morphology by scanning electron microscopy (S-3400N). The pollen grain displayed a smooth and plump surface in wild-type (Fig. 7a), while transgenic pollen showed a sunken surface which was plump in appearance (Fig. 7b, c). Statistics showed that PbTM6a overexpressing tomato lines produced much less normal pollen grains compared to that of wild-type (Fig. 7d). Collectively, these findings led us to conclude that PbTM6a overexpression in tomato led to defective pollen grains with low vitality and germinating capacity, causing poor pollination and fertilization, which contributed to the formation of fewer seeds per fruit compared with the wild type.

      Figure 7. 

      Analysis of the morphology of wild-type and PbTM6a-OE pollen by scanning electron microscopy. (a) Overview of plump pollen grains of wild type. (b) Overview of plump pollen grains of PbTM6a-OE1. (c) Overview of plump pollen grains of PbTM6a-OE2. (d) Statistics of the percentage of pollen grain with normal surface in wild-type and PbTM6a-OE lines. Data represents mean (± standard deviation (SD) and asterisks represents significant differences (P < 0.01) as determined by one-way ANOVA.

    • Plant hormones were tightly related to pollen development. Therefore, we assessed plant hormone levels in transgenic tomato stamens overexpressing PbTM6a (Fig. 8). Remarkably, the content of JA and ABA were reduced in the stamens of PbTM6a-OE lines, and the levels of IAA and GA3 were significantly decreased. Thus, we concluded that overexpression of PbTM6a in tomato led to the suppression of endogenous JA, ABA, IAA and GA3 levels in stamens, which may be related to male sterility.

      Figure 8. 

      Levels of IAA, JA, SA, ABA, SA, GA3, and GA4 in wild-type and PbTM6a-OE stamens. Data represents mean (± standard deviation (SD). Asterisks represents significant differences (P < 0.05) as determined by one-way ANOVA.

    • Although class-B MADS box genes, especially for PI and AP3, exhibit a highly conserved function in the formation of petals and stamens[22,25,27], mutations in these genes have been reported to cause distinct new phenotypes including parthenocarpy, male sterility[25,26,27]. Orthologous to AP3, TM6, plays a key role in anther development in strawberry[31] and tomato[26]. However, the role of TM6 gene in pollen and seed development remains unknown, and research related to TM6 genes has not yet been reported in pear. Here we identified PbTM6a and PbTM6b genes from the transcriptome of parthenocarpic ovaries induced by GA4+7, and their expression was suppressed (Supplemental Fig. S1). Here, we reported the response of PbTM6 genes to GA4+7 in pear.

      Based on phylogenetic analysis of typical members of the ABCDE model[7], PbTM6a and PbTM6b were classified into class B of the MADS-box gene family (Fig. 1). Like other MADS-box proteins, which contain a highly conserved DNA-binding MADS-box domain of 56–60 amino acids at the N-terminus[3]. The PbTM6a and PbTM6b proteins were also found to possess the conserved DNA-binding MADS-box domain (Fig. 2a). In view of the parthenocarpic capacity of MdPI[22,27], repressed expression of PbTM6a and PbTM6b induced by the GA4+7 treatment impelled us to determine the involvement of PbTM6 in floral organ identity. Our results showing higher expression of PbTM6 in anther, filament and petal were consistent with previous reports, according to which class-B MADS-box genes are highly expressed in stamen and petal[9,25]. The PbTM6a and PbTM6b genes are homoeologues which arose through whole genome duplication. PbTM6a had higher abundance and more significantly decreased expression in parthenocarpic ovaries than PbTM6b[29], PbTM6a was chosen for further analysis.

      Because pear has a long breeding cycle and is difficult to transform, tomato was chosen as the heterologous expression system for exploring the role of PbTM6a in flowers and fruit development. Mutant in TM6 mediated by CRISPR/Cas9 system in the octoploid cultivated strawberry leads to distinct petals with modest defects in overall size and color and anthers with a severe reduction in pollen content and viability[31]. However, overexpression of PbTM6a in tomato did not lead to the conversion of petals and stamens and did not affect the vegetative development of transgenic plants compared with the wild-type (Fig. 3a & Supplemental Fig. S3). Thus, we speculated that PbTM6a has distinctive features compared with classical B-class MADS-box genes such as AP3 and PI.

      Surprisingly, the number of seeds per fruit was significantly reduced in PbTM6a-OE lines compared with the wild type (Fig. 3bd). Through histological observations of floral organs, PbTM6a-OE lines have more strongly formed stomium bound to the connective tissue in the pollen sac and squeezed ovules in a narrow ovary chamber, which seems to contribute to the reduction in seed number and size (Fig. 4). Further reciprocal crosses and the determination of pollen vigor and germination rate demonstrated that the low vigor pollen produced by PbTM6a-OE lines leading to unsuccessful fertilization is the dominant cause for the production of fruits with fewer and smaller seeds (Figs 57). Meanwhile the effect of the development of transgenic ovary and ovule itself on the seed formation cannot be excluded in this study. Given that the PbTM6a and PbTM6b have much lower expression in the ovary compared to petals and anthers (Fig. 2b, c), the development of the ovary and ovule seems not to be the dominant factor affecting seed number and size. Consistent with the speculation, FaTM6 is not expressed in carpels, carpels of mutants in FaTM6 shows normal development in octoploid strawberry[31].

      Interestingly, SlTM6 has been characterized as a candidate gene for the tomato male sterile-1526 locus, where the promoter and first four exons of the TM6 gene were absent[26]. The RNAi of SlTM6 resulted in flowers with homeotic defects primarily in stamens[25]. It is possible that the heterogenous overexpression of PbTM6a leads to post-transcriptional regulation of other endogenous MADS-box genes in tomato. Mutation in FaTM6 leads to smaller and darker anthers with clear defects in the epidermal cell layer which produce less pollen grain with aberrant and collapsed structure and impaired viability[31]. Here ectopic expression of PbTM6a can produce abnormal pollen with reduced viability and low germination and sunken surface (Figs 5 & 7). Sophisticated regulation of TM6 expression is required for pollen development. It demonstrated that TM6 has a conserved role in the anther and pollen formation.

      The integrated regulatory network of JA and other plant hormones participates in male organ development[47]. Decreased levels of JA, IAA, ABA and GA3 were detected in the stamens of PbTM6a overexpression lines (Fig. 8). In Arabidopsis, the deficiency of JA is tightly associated with male organ development, mutations in genes encoding JA biosynthetic enzymes result in failure of filament elongation, delayed anther dehiscence, and unviable pollen[48]. Decreased pollen vigor and germination in PbTM6a-OE lines may be a consequence of the decreased JA level. It has been reported that the exogenous application on undehisced anthers can remove the block in pollen release[49]. The specific mechanism between PbTM6a and genes related to regulating JA content needs to be further demonstrated.

      Auxin and its signaling play a key role in male gametogenesis. Two auxin biosynthesis genes, YUC2 and YUC6, exhibit high expression in anther procambium, endothecium, tapetum, tetrads and microspores, and the yuc2yuc6 double mutant is unable to form viable pollen grains[50]. Microsporocyte- or microspore-specific, but not tapetum-specific, overexpression of YUC2 gene in the yuc2yuc6 double mutant is sufficient to rescue abortive pollen formation[51]. Similar to the male-sterile phenotype of the JA mutants, the arf6-2 arf8-3 double mutant produces unviable pollens[49]. Male sterility observed in our study could also be a consequence of insufficient IAA content. The production of auxin in anthers is necessary for the advancement of pollen from the microspore stage to the bicellular stage in both Arabidopsis and tomato[51]. Auxin seems to act upstream of JA in the development of pollen grains.

      A gradual elevation in ABA and IAA levels accompanies the formation of microsporocytes and microspores in the male-fertile lines of petunia (Petunia hybrida L.)[52]. Ultrahigh levels of ABA and IAA arise in reproductive cells of male-sterile lines. The addition of ABA enhances the viability and reduces the death of barley Hordeum vulgare L., which suggests that ABA acts as a potential signal of male sterility[53]. Although the involvement of ABA in male sterility lacks direct evidence, accumulating indirect evidence shows that ABA interacts with the sugar signaling pathway[54]. Carbohydrates are necessary for sustaining pollen development[55]. In tomato, silencing of Lycopersicum Invertase 5 (LIN5), which encodes a cell wall invertase, decreases seed number per fruit and reduces pollen germination and viability[56]. The role of ABA in male sterility control needs to be further studied in the future.

      In addition, the promotion of bioactive GAs to pollen viability has been determined widely. Bioactive GAs is involved in pollen exine formation and the programmed cell death of tapetal cells in Arabidopsis, rice and tomato[57]. Exogenous GA3 application promotes pollen viability and number under cold conditions in almond (Amygdalus communis L.)[58]. Although the effects of GA4+7, when applied to flowers, on pollen germination in pear were not assessed in this study, the expression of PbTM6a was repressed by the application of GA4+7 in pear, confirming that PbTM6a acts downstream of GA4+7 in pear. The low pollen viability production mediated by PbTM6a maybe takes in the process of the regulation of GAs to pollen development. Overexpression of PbTM6a in tomato showed that low GA level may be the cause of the weak vigor of pollen (Fig. 8).

    • In summary, heterologous overexpression of PbTM6a is insufficient for the classical function of class B MADS-box genes in the identification of floral organs, but instead of reduced plant fertility. The specific mitotic progression and the molecular mechanism underlying the role of PbTM6a in pollen development should be elucidated in the future. The results of this study provide insight into the conserved role of MADS-box genes in controlling male fertility, and enhance our understanding of the mechanism underlying the function of PbTM6 in pear.

      • This study was supported by the earmarked fund for CARS-28 (China Agriculture Research System).

      • The authors declare that they have no conflict of interest.

      • # These authors contributed equally: Haiqi Zhang, Wei Han

      • Supplemental Table S1 List of qRT-PCR primers.
      • Supplemental Table S2 List of primers cloning PbDEFs.
      • Supplemental Fig. S1 Expression levels of PbTM6a and PbTM6a in control and GA4+7-treated ovaries. DAA, Days After Anthesis. Data represents mean (± standard deviation (SD). Asterisk represents significant differences (P< 0.05) as determined by one-way ANOVA.
      • Supplemental Fig. S2 Floral organs and PbTM6a subcellular localization. (a) Different floral organs of pear. (b) Subcellular localization of PbTM6a.
      • Supplemental Fig. S3 Detection and observations of reproductive and vegetative growth of PbTM6a-OE lines (a) DNA amplify of PbTM6a in transgenic tomato (b) Observations of vegetative growth of wild type and transgenic tomato. (c) Observations of flowers of wild type and transgenic tomato.
      • Supplemental Table S3  Seeds numbers of tomato with overexpressing PbTM6a gene in mutual pollination experiment.
      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (8)  References (58)
  • About this article
    Cite this article
    Zhang H, Han W, Linghu T, Zhao Z, Wang A, et al. 2023. Overexpression of a pear B-class MADS-box gene in tomato causes male sterility. Fruit Research 3:1 doi: 10.48130/FruRes-2023-0001
    Zhang H, Han W, Linghu T, Zhao Z, Wang A, et al. 2023. Overexpression of a pear B-class MADS-box gene in tomato causes male sterility. Fruit Research 3:1 doi: 10.48130/FruRes-2023-0001

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return