Developmental role of the tomato Mediator complex subunit MED18 in pollen ontogeny

[EN] Pollen development is a crucial step in higher plants, which not only makes possible plant fertilization and seed formation, but also determines fruit quality and yield in crop species. Here, we reported a tomato T-DNA mutant, pollen deficient1 (pod1), characterized by an abnormal anther development and the lack of viable pollen formation, which led to the production of parthenocarpic fruits. Genomic analyses and the characterization of silencing lines proved that pod1 mutant phenotype relies on the tomato SlMED18 gene encoding the subunit 18 of Mediator multi-protein complex involved in RNA polymerase II transcription machinery. The loss of SlMED18 function delayed tapetum degeneration, which resulted in deficient microspore development and scarce production of viable pollen. A detailed histological characterization of anther development proved that changes during microgametogenesis and a significant delay in tapetum degeneration are associated with a high proportion of degenerated cells and, hence, should be responsible for the low production of functional pollen grains. Expression of pollen marker genes indicated that SlMED18 is essential for the proper transcription of a subset of genes specifically required to pollen formation and fruit development, revealing a key role of SlMED18 in male gametogenesis of tomato. Additionally, SlMED18 is able to rescue developmental abnormalities of the Arabidopsis med18 mutant, indicating that most biological functions have been conserved in both species. Significance Statement Pollination is a key development process in the life cycle of flowering plants. Genetic and molecular characterization of a tomato mutant have led to the identification of POD1 gene encoding the Mediator complex subunit MED18 whose function is required for tapetum tissue degeneration, a crucial step for pollen development. Furthermore, we show that MED18 fulfils an essential role in tomato, ensuring proper gene regulation during pollen ontogeny.This research was supported by the Spanish Ministry of Economy and Competitiveness (grants AGL2015-64991-C3-1-R, AGL2015-64991-C3-2-R, AGL2015-64991-C3-3-R, BIO2013-43098-R, BFU2016-77243-P and BIO2016-77559-R) and Junta de Andalucia (grant P12-AGR-1482).Pérez Martín, F.; Juan Yuste-Lisbona, F.; Pineda, B.; García Sogo, B.; Del Olmo, I.; Alché, JDD.; Egea, I.... (2018). Developmental role of the tomato Mediator complex subunit MED18 in pollen ontogeny. The Plant Journal. 96(2):300-315. https://doi.org/10.1111/tpj.14031S300315962Allen, B. L., & Taatjes, D. J. (2015). The Mediator complex: a central integrator of transcription. Nature Reviews Molecular Cell Biology, 16(3), 155-166. doi:10.1038/nrm3951Atarés, A., Moyano, E., Morales, B., Schleicher, P., García-Abellán, J. O., Antón, T., … Pineda, B. (2011). An insertional mutagenesis programme with an enhancer trap for the identification and tagging of genes involved in abiotic stress tolerance in the tomato wild-related species Solanum pennellii. Plant Cell Reports, 30(10), 1865-1879. doi:10.1007/s00299-011-1094-yBaulcombe, D. C. (1996). Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants. The Plant Cell, 1833-1844. doi:10.1105/tpc.8.10.1833Bourbon, H.-M. (2008). Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex. Nucleic Acids Research, 36(12), 3993-4008. doi:10.1093/nar/gkn349Buendía-Monreal, M., & Gillmor, C. S. (2016). Mediator: A key regulator of plant development. Developmental Biology, 419(1), 7-18. doi:10.1016/j.ydbio.2016.06.009Canales, C., Bhatt, A. M., Scott, R., & Dickinson, H. (2002). EXS, a Putative LRR Receptor Kinase, Regulates Male Germline Cell Number and Tapetal Identity and Promotes Seed Development in Arabidopsis. Current Biology, 12(20), 1718-1727. doi:10.1016/s0960-9822(02)01151-xCarbonell-Bejerano, P., Urbez, C., Carbonell, J., Granell, A., & Perez-Amador, M. A. (2010). A Fertilization-Independent Developmental Program Triggers Partial Fruit Development and Senescence Processes in Pistils of Arabidopsis. Plant Physiology, 154(1), 163-172. doi:10.1104/pp.110.160044Chadick, J. Z., & Asturias, F. J. (2005). Structure of eukaryotic Mediator complexes. Trends in Biochemical Sciences, 30(5), 264-271. doi:10.1016/j.tibs.2005.03.001Chuang, C.-F., & Meyerowitz, E. M. (2000). Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, 97(9), 4985-4990. doi:10.1073/pnas.060034297Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. The Plant Journal, 16(6), 735-743. doi:10.1046/j.1365-313x.1998.00343.xColeman, A. W., & Goff, L. J. (1985). Applications of Fluorochromes to Pollen Biology. I. Mithramycin and 4′,6-Diamidino-2-Phenylindole (Dapi) as Vital Stains and for Quantitation of Nuclear Dna. Stain Technology, 60(3), 145-154. doi:10.3109/10520298509113905Conaway, R. C., Sato, S., Tomomori-Sato, C., Yao, T., & Conaway, J. W. (2005). The mammalian Mediator complex and its role in transcriptional regulation. Trends in Biochemical Sciences, 30(5), 250-255. doi:10.1016/j.tibs.2005.03.002Cottrell, H. J. (1948). Tetrazolium Salt as a Seed Germination Indicator. Annals of Applied Biology, 35(1), 123-131. doi:10.1111/j.1744-7348.1948.tb07355.xCrane, M. B. (1915). Heredity of types of inflorescence and fruits in tomato. Journal of Genetics, 5(1), 1-11. doi:10.1007/bf02982149Davoine, C., Abreu, I. N., Khajeh, K., Blomberg, J., Kidd, B. N., Kazan, K., … Björklund, S. (2017). Functional metabolomics as a tool to analyze Mediator function and structure in plants. PLOS ONE, 12(6), e0179640. doi:10.1371/journal.pone.0179640Ellul, P., Garcia-Sogo, B., Pineda, B., Ríos, G., Roig, L., & Moreno, V. (2003). The ploidy level of transgenic plants in Agrobacterium-mediated transformation of tomato cotyledons (Lycopersicon esculentum L.Mill.) is genotype and procedure dependent. Theoretical and Applied Genetics, 106(2), 231-238. doi:10.1007/s00122-002-0928-yFallath, T., Kidd, B. N., Stiller, J., Davoine, C., Björklund, S., Manners, J. M., … Schenk, P. M. (2017). MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana. PLOS ONE, 12(4), e0176022. doi:10.1371/journal.pone.0176022Feng, B., Lu, D., Ma, X., Peng, Y., Sun, Y., Ning, G., & Ma, H. (2012). Regulation of the Arabidopsis anther transcriptome by DYT1 for pollen development. The Plant Journal, 72(4), 612-624. doi:10.1111/j.1365-313x.2012.05104.xGillaspy, G., Ben-David, H., & Gruissem, W. (1993). Fruits: A Developmental Perspective. The Plant Cell, 1439-1451. doi:10.1105/tpc.5.10.1439Gleave, A. P. (1992). A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Molecular Biology, 20(6), 1203-1207. doi:10.1007/bf00028910Gómez, J. F., Talle, B., & Wilson, Z. A. (2015). Anther and pollen development: A conserved developmental pathway. Journal of Integrative Plant Biology, 57(11), 876-891. doi:10.1111/jipb.12425Gorman, S. W., McCormick, S., & Rick, C. (1997). Male Sterility in Tomato. Critical Reviews in Plant Sciences, 16(1), 31-53. doi:10.1080/07352689709701945Helliwell, C. (2003). Constructs and methods for high-throughput gene silencing in plants. Methods, 30(4), 289-295. doi:10.1016/s1046-2023(03)00036-7Honys, D., & Twell, D. (2004). Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biology, 5(11). doi:10.1186/gb-2004-5-11-r85Jeong, H.-J., Kang, J.-H., Zhao, M., Kwon, J.-K., Choi, H.-S., Bae, J. H., … Kang, B.-C. (2014). Tomato Male sterile 1035 is essential for pollen development and meiosis in anthers. Journal of Experimental Botany, 65(22), 6693-6709. doi:10.1093/jxb/eru389Jimenez-Lopez, J. C., Zienkiewicz, A., Zienkiewicz, K., Alché, J. D., & Rodríguez-García, M. I. (2015). Biogenesis of protein bodies during legumin accumulation in developing olive (Olea europaea L.) seed. Protoplasma, 253(2), 517-530. doi:10.1007/s00709-015-0830-5Kornberg, R. D. (2005). Mediator and the mechanism of transcriptional activation. Trends in Biochemical Sciences, 30(5), 235-239. doi:10.1016/j.tibs.2005.03.011Lai, Z., Schluttenhofer, C. M., Bhide, K., Shreve, J., Thimmapuram, J., Lee, S. Y., … Mengiste, T. (2014). MED18 interaction with distinct transcription factors regulates multiple plant functions. Nature Communications, 5(1). doi:10.1038/ncomms4064Larivière, L., Geiger, S., Hoeppner, S., Röther, S., Sträßer, K., & Cramer, P. (2006). Structure and TBP binding of the Mediator head subcomplex Med8–Med18–Med20. Nature Structural & Molecular Biology, 13(10), 895-901. doi:10.1038/nsmb1143Lee, S. K., Chen, X., Huang, L., & Stargell, L. A. (2013). The head module of Mediator directs activation of preloaded RNAPII in vivo. Nucleic Acids Research, 41(22), 10124-10134. doi:10.1093/nar/gkt796Li, D.-D., Xue, J.-S., Zhu, J., & Yang, Z.-N. (2017). Gene Regulatory Network for Tapetum Development in Arabidopsis thaliana. Frontiers in Plant Science, 8. doi:10.3389/fpls.2017.01559Liu, X., Huang, J., Parameswaran, S., Ito, T., Seubert, B., Auer, M., … Zhao, D. (2009). The SPOROCYTELESS/NOZZLE Gene Is Involved in Controlling Stamen Identity in Arabidopsis. Plant Physiology, 151(3), 1401-1411. doi:10.1104/pp.109.145896Lora, J., Hormaza, J. I., Herrero, M., & Gasser, C. S. (2011). Seedless fruits and the disruption of a conserved genetic pathway in angiosperm ovule development. Proceedings of the National Academy of Sciences, 108(13), 5461-5465. doi:10.1073/pnas.1014514108Lozano, R., Angosto, T., Gómez, P., Payán, C., Capel, J., Huijser, P., … Martı́nez-Zapater, J. M. (1998). Tomato Flower Abnormalities Induced by Low Temperatures Are Associated with Changes of Expression of MADS-Box Genes. Plant Physiology, 117(1), 91-100. doi:10.1104/pp.117.1.91Ma, H. (2005). MOLECULAR GENETIC ANALYSES OF MICROSPOROGENESIS AND MICROGAMETOGENESIS IN FLOWERING PLANTS. Annual Review of Plant Biology, 56(1), 393-434. doi:10.1146/annurev.arplant.55.031903.141717McNeil, K. J., & Smith, A. G. (2009). A glycine-rich protein that facilitates exine formation during tomato pollen development. Planta, 231(4), 793-808. doi:10.1007/s00425-009-1089-xMercier, R. (2003). The meiotic protein SWI1 is required for axial element formation and recombination initiation in Arabidopsis. Development, 130(>14), 3309-3318. doi:10.1242/dev.00550Mukundan, B., & Ansari, A. (2011). Novel Role for Mediator Complex Subunit Srb5/Med18 in Termination of Transcription. Journal of Biological Chemistry, 286(43), 37053-37057. doi:10.1074/jbc.c111.295915Muschietti, J., Dircks, L., Vancanneyt, G., & McCormick, S. (1994). LAT52 protein is essential for tomato pollen development: pollen expressing antisense LAT52 RNA hydrates and germinates abnormally and cannot achieve fertilization. The Plant Journal, 6(3), 321-338. doi:10.1046/j.1365-313x.1994.06030321.xOzga, J. A., & Reinecke, D. M. (2003). Hormonal Interactions in Fruit Development. Journal of Plant Growth Regulation, 22(1), 73-81. doi:10.1007/s00344-003-0024-9Pacini, E. (2010). Relationships between Tapetum, Loculus, and Pollen during Development. International Journal of Plant Sciences, 171(1), 1-11. doi:10.1086/647923Pérez-Martín, F., Yuste-Lisbona, F. J., Pineda, B., Angarita-Díaz, M. P., García-Sogo, B., Antón, T., … Lozano, R. (2017). A collection of enhancer trap insertional mutants for functional genomics in tomato. Plant Biotechnology Journal, 15(11), 1439-1452. doi:10.1111/pbi.12728Pina, C., Pinto, F., Feijó, J. A., & Becker, J. D. (2005). Gene Family Analysis of the Arabidopsis Pollen Transcriptome Reveals Biological Implications for Cell Growth, Division Control, and Gene Expression Regulation. Plant Physiology, 138(2), 744-756. doi:10.1104/pp.104.057935Polowick, P. L., & Sawhney, V. K. (1993). An ultrastructural study of pollen development in tomato (Lycopersicon esculentum). I. Tetrad to early binucleate microspore stage. Canadian Journal of Botany, 71(8), 1039-1047. doi:10.1139/b93-120Polowick, P. L., & Sawhney, V. K. (1993). An ultrastructural study of pollen development in tomato (Lycopersicon esculentum). II. Pollen maturation. Canadian Journal of Botany, 71(8), 1048-1055. doi:10.1139/b93-121Rutley, N., & Twell, D. (2015). A decade of pollen transcriptomics. Plant Reproduction, 28(2), 73-89. doi:10.1007/s00497-015-0261-7Samanta, S., & Thakur, J. K. (2015). Importance of Mediator complex in the regulation and integration of diverse signaling pathways in plants. Frontiers in Plant Science, 6. doi:10.3389/fpls.2015.00757Schiefthaler, U., Balasubramanian, S., Sieber, P., Chevalier, D., Wisman, E., & Schneitz, K. (1999). Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, 96(20), 11664-11669. doi:10.1073/pnas.96.20.11664Scott, R. J. (2004). Stamen Structure and Function. THE PLANT CELL ONLINE, 16(suppl_1), S46-S60. doi:10.1105/tpc.017012Smirnova, A., Leide, J., & Riederer, M. (2012). Deficiency in a Very-Long-Chain Fatty Acid β-Ketoacyl-Coenzyme A Synthase of Tomato Impairs Microgametogenesis and Causes Floral Organ Fusion. Plant Physiology, 161(1), 196-209. doi:10.1104/pp.112.206656Sorensen, A.-M., Kröber, S., Unte, U. S., Huijser, P., Dekker, K., & Saedler, H. (2003). TheArabidopsis ABORTED MICROSPORES(AMS) gene encodes a MYC class transcription factor. The Plant Journal, 33(2), 413-423. doi:10.1046/j.1365-313x.2003.01644.xWang, Y., Hu, Z., Zhang, J., Yu, X., Guo, J.-E., Liang, H., … Chen, G. (2018). Silencing SlMED18, tomato Mediator subunit 18 gene, restricts internode elongation and leaf expansion. Scientific Reports, 8(1). doi:10.1038/s41598-018-21679-1Wesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M., Rouse, D. T., Liu, Q., … Waterhouse, P. M. (2001). Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal, 27(6), 581-590. doi:10.1046/j.1365-313x.2001.01105.xWilson, Z. A., & Zhang, D.-B. (2009). From Arabidopsis to rice: pathways in pollen development. Journal of Experimental Botany, 60(5), 1479-1492. doi:10.1093/jxb/erp095Wilson, Z. A., Morroll, S. M., Dawson, J., Swarup, R., & Tighe, P. J. (2001). The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors. The Plant Journal, 28(1), 27-39. doi:10.1046/j.1365-313x.2001.01125.xWiner, J., Jung, C. K. S., Shackel, I., & Williams, P. M. (1999). Development and Validation of Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction for Monitoring Gene Expression in Cardiac Myocytesin Vitro. Analytical Biochemistry, 270(1), 41-49. doi:10.1006/abio.1999.4085Yang, W.-C., Ye, D., Xu, J., & Sundaresan, V. (1999). The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein. Genes & Development, 13(16), 2108-2117. doi:10.1101/gad.13.16.2108Yang, C.-Y., Spielman, M., Coles, J. P., Li, Y., Ghelani, S., Bourdon, V., … Dickinson, H. G. (2003). TETRASPORE encodes a kinesin required for male meiotic cytokinesis in Arabidopsis. The Plant Journal, 34(2), 229-240. doi:10.1046/j.1365-313x.2003.01713.xYang, C., Vizcay-Barrena, G., Conner, K., & Wilson, Z. A. (2007). MALE STERILITY1 Is Required for Tapetal Development and Pollen Wall Biosynthesis. The Plant Cell, 19(11), 3530-3548. doi:10.1105/tpc.107.054981Yuan, W., Li, X., Chang, Y., Wen, R., Chen, G., Zhang, Q., & Wu, C. (2009). Mutation of the rice genePAIR3results in lack of bivalent formation in meiosis. The Plant Journal, 59(2), 303-315. doi:10.1111/j.1365-313x.2009.03870.xYuste-Lisbona, F. J., Quinet, M., Fernández-Lozano, A., Pineda, B., Moreno, V., Angosto, T., & Lozano, R. (2016). Characterization of vegetative inflorescence (mc-vin) mutant provides new insight into the role of MACROCALYX in regulating inflorescence development of tomato. Scientific Reports, 6(1). doi:10.1038/srep18796Zhao, D.-Z. (2002). The EXCESS MICROSPOROCYTES1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther. Genes & Development, 16(15), 2021-2031. doi:10.1101/gad.997902Zheng, Z., Guan, H., Leal, F., Grey, P. H., & Oppenheimer, D. G. (2013). Mediator Subunit18 Controls Flowering Time and Floral Organ Identity in Arabidopsis. PLoS ONE, 8(1), e53924. doi:10.1371/journal.pone.0053924Zhou, S., Wang, Y., Li, W., Zhao, Z., Ren, Y., Wang, Y., … Wan, J. (2011). Pollen Semi-Sterility1 Encodes a Kinesin-1–Like Protein Important for Male Meiosis, Anther Dehiscence, and Fertility in Rice. The Plant Cell, 23(1), 111-129. doi:10.1105/tpc.109.07369

Reduced Photoinhibition under Low Irradiance Enhanced Kacip Fatimah (Labisia pumila Benth) Secondary Metabolites, Phenyl Alanine Lyase and Antioxidant Activity

A randomized complete block design experiment was designed to characterize the relationship between production of total flavonoids and phenolics, anthocyanin, photosynthesis, maximum efficiency of photosystem II (Fv/Fm), electron transfer rate (Fm/Fo), phenyl alanine lyase activity (PAL) and antioxidant (DPPH) in Labisia pumila var. alata, under four levels of irradiance (225, 500, 625 and 900 μmol/m2/s) for 16 weeks. As irradiance levels increased from 225 to 900 μmol/m2/s, the production of plant secondary metabolites (total flavonoids, phenolics and antocyanin) was found to decrease steadily. Production of total flavonoids and phenolics reached their peaks under 225 followed by 500, 625 and 900 μmol/m2/s irradiances. Significant positive correlation of production of total phenolics, flavonoids and antocyanin content with Fv/Fm, Fm/Fo and photosynthesis indicated up-regulation of carbon-based secondary metabolites (CBSM) under reduced photoinhibition on the under low light levels condition. At the lowest irradiance levels, Labisia pumila extracts also exhibited a significantly higher antioxidant activity (DPPH) than under high irradiance. The improved antioxidative activity under low light levels might be due to high availability of total flavonoids, phenolics and anthocyanin content in the plant extract. It was also found that an increase in the production of CBSM was due to high PAL activity under low light, probably signifying more availability of phenylalanine (Phe) under this condition

The ASH1 HOMOLOG 2 (ASHH2) Histone H3 Methyltransferase Is Required for Ovule and Anther Development in Arabidopsis

BACKGROUND:SET-domain proteins are histone lysine (K) methyltransferases (HMTase) implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein (also called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction. METHODOLOGY/PRINCIPAL FINDINGS:A slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to approximately 90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99) we observed a reduction of H3K36 trimethylation (me3), but not H3K4me3 or H3K36me2. CONCLUSIONS/SIGNIFICANCE:The severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to the observed defects, implicates ASHH2 in regulation of gene expression via H3K36 trimethylation in chromatin of Arabidopsis inflorescences

BnMs3 is required for tapetal differentiation and degradation, microspore separation, and pollen-wall biosynthesis in Brassica napus

7365AB, a recessive genetic male sterility system, is controlled by BnMs3 in Brassica napus, which encodes a Tic40 protein required for tapetum development. However, the role of BnMs3 in rapeseed anther development is still largely unclear. In this research, cytological analysis revealed that anther development of a Bnms3 mutant has defects in the transition of the tapetum to the secretory type, callose degradation, and pollen-wall formation. A total of 76 down-regulated unigenes in the Bnms3 mutant, several of which are associated with tapetum development, callose degeneration, and pollen development, were isolated by suppression subtractive hybridization combined with a macroarray analysis. Reverse genetics was applied by means of Arabidopsis insertional mutant lines to characterize the function of these unigenes and revealed that MSR02 is only required for transport of sporopollenin precursors through the plasma membrane of the tapetum. The real-time PCR data have further verified that BnMs3 plays a primary role in tapetal differentiation by affecting the expression of a few key transcription factors, participates in tapetal degradation by modulating the expression of cysteine protease genes, and influences microspore separation by manipulating the expression of BnA6 and BnMSR66 related to callose degradation and of BnQRT1 and BnQRT3 required for the primary cell-wall degradation of the pollen mother cell. Moreover, BnMs3 takes part in pollen-wall formation by affecting the expression of a series of genes involved in biosynthesis and transport of sporopollenin precursors. All of the above results suggest that BnMs3 participates in tapetum development, microspore release, and pollen-wall formation in B. napus

Transcriptome Analysis of the Arabidopsis Megaspore Mother Cell Uncovers the Importance of RNA Helicases for Plant Germline Development

Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant “germline” lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as illustrated by the identification of MEM, a novel gene involved in the restriction of germline fate

Leaf anatomy and photosynthetic acclimation in Valeriana jatamansi L. grown under high and low irradiance

Relationship of leaf anatomy with photosynthetic acclimation of Valeriana jatamansi was studied under full irradiance [FI, 1 600 μmol(PPFD) m-2 s-1] and net-shade [NS, 650 μmol(PPFD) m-2 s-1]. FI plants had thicker leaves with higher respiration rate (RD), nitrogen content per unit leaf area, chlorophyll a/b ratio, high leaf mass per leaf area unit (LMA), and surface area of mesophyll cell (Smes) and chloroplasts (Sc) facing intercellular space than NS plants. The difference between leaf thickness of FI and NS leaves was about 28 % but difference in photon-saturated rate of photosynthesis per unit leaf area (PNmax) was 50 %. This indicates that PNmax can increase to a larger extent than the leaf thickness with increasing irradiance in V. jatamansi. Anatomical studies showed that the mesophyll cells of FI plants had no open spaces along the mesophyll cell walls (higher Sc), but in NS plants wide open spaces along the mesophyll cell wall (lower Sc) were found. Positive correlation between Sc and PNmax explained the higher PNmax in FI plants. Increase in mesophyll thickness increased the availability of space along the mesophyll cell wall for chloroplasts (increased Sc) and hence PNmax was higher in FI plants. Thus this Himalayan species can acclimate to full sunlight by altering leaf anatomy and therefore may be cultivated in open fields