Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.)

The identification of microspores or pollen grains at particular developmental stages during microsporogenesis or microgamentogenesis is an important step for different basic and applied purposes. Among them, the most relevant example from a biotechnological perspective is the production of androgenic doubled haploids. For this and other techniques, precise, fast, easy and reliable criteria to identify flower buds carrying microspores or pollen at particular stages are essential. In anthocyanin-producing pepper types, the particularities of flower development allow for the identification of several morphological markers potentially useful as criteria for such an identification. In this work, our aim was to determine the easiest and more accurate criterion to correlate visible, measurable traits of bud and anther development with each of the individual stages of microsporogenesis and microgametogenesis. For this, we used three Spanish sweet pepper F1 hybrids (‘Herminio’, ‘Gacela’ and ‘Águila’). We analyzed and discussed the accuracy and practical usefulness of using anther length, bud length, anther purple pigmentation and the ratio between calyx length and bud length (calyx/bud ratio) as predictors of individual microspore/pollen developmental stages. According to our results, we propose a combination of calyx/bud ratio and anther pigmentation as an easy, fast and accurate criterion potentially applicable to anthocyanin-producing pepper cultivars to determine their particular markers.We acknowledge Dr. Rosa Peiro, Mrs. Nuria Palacios and Mrs. Patricia Corral for their valuable help, as well as the staff of the COMAV greenhouses. VPV is a predoctoral fellow of the FPU program of the Spanish Ministry of Education. This work was supported by grant from Spanish Ministry of Science and Innovation (MICINN) AGL2010-17895 to JMSS.Parra Vega, V.; Gonzalez Garcia, B.; Seguí Simarro, JM. (2013). Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.). Acta Physiologiae Plantarum. 35(2):627-633. https://doi.org/10.1007/s11738-012-1104-xS627633352Barany I, Gonzalez-Melendi P, Fadón B, Mityko J, Risueño MC, Testillano PS (2005) Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development. Biol Cell 97:709–722Barany I, Fadon B, Risueno MC, Testillano PS (2010) Cell wall components and pectin esterification levels as markers of proliferation and differentiation events during pollen development and pollen embryogenesis in Capsicum annuum L. J Exp Bot 61:1159–1175. doi: 10.1093/jxb/erp392Buyukalaca S, Comlekcioglu N, Abak K, Ekbic E, Kilic N (2004) Effects of silver nitrate and donor plant growing conditions on production of pepper (Capsicum annuum L.) haploid embryos via anther culture. Eur J Hortic Sci 69(5):206–209Dumas de Vaulx R, Chambonnet D, Pochard E (1981) Culture in vitro d’anthères de piment (Capsicum annuum L.): amèlioration des taux d’obtenction de plantes chez différents génotypes par des traitments à +35 °C. Agronomie 1 (10):859-864Dunwell JM (2010) Haploids in flowering plants: origins and exploitation. Plant Biotechnol J 8(4):377–424. doi: 10.1111/j.1467-7652.2009.00498.xErcan N, Sensoy FA, Sirri Sensoy A (2006) Influence of growing season and donor plant age on anther culture response of some pepper cultivars (Capsicum annuum L.). Sci Hort 110(1):16–20Irikova T, Grozeva S, Rodeva V (2011) Anther culture in pepper (Capsicum annuum L.) in vitro. Acta Physiol Plant 33(5):1559–1570. doi: 10.1007/s11738-011-0736-6Kim M, Kim J, Yoon M, Choi DI, Lee KM (2004) Origin of multicellular pollen and pollen embryos in cultured anthers of pepper (Capsicum annuum). Plant Cell, Tissue Organ Cult 77:63–72Kim M, Jang IC, Kim JA, Park EJ, Yoon M, Lee Y (2008) Embryogenesis and plant regeneration of hot pepper (Capsicum annuum L.) through isolated microspore culture. Plant Cell Rep 27(3):425–434Koleva-Gudeva LR, Spasenoski M, Trajkova F (2007) Somatic embryogenesis in pepper anther culture: the effect of incubation treatments and different media. Sci Hort 111(2):114–119Lantos C, Juhász A, Somogyi G, Ötvös K, Vági P, Mihály R, Kristóf Z, Somogyi N, Pauk J (2009) Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat. Plant Cell Tiss Org Cult 97(3):285–293. doi: 10.1007/s11240-009-9527-9Ltifi A, Wenzel G (1994) Anther culture of hot and sweet pepper (Capsicum annuum L.): influence of genotype and plant growth temperature. Capsicum Eggplant Newsl 13:74–77Mityko J, Andrasfalvy A, Csillery G, Fari M (1995) Anther culture response in different genotypes and F1 hybrids of pepper (Capsicum Annuum L). Plant Breed 114(1):78–80Nowaczyk P, Kisiala A (2006) Effect of selected factors on the effectiveness of Capsicum annuum L. anther culture. J Appl Genet 47(2):113–117Regner F (1996) Anther and microspore culture in Capsicum. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol 3. Kluwer, Dordrecht, pp 77–89Regnet F (1994) Microspore culture of Capsicum annuum. Capsicum Eggplant Newsl 13(1114):69–70Salas P, Rivas-Sendra A, Prohens J, Seguí-Simarro JM (2012) Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica 184(2):235–250. doi: 10.1007/s10681-011-0569-9Seguí-Simarro JM (2010) Androgenesis revisited. Bot Rev 76(3):377–404. doi: 10.1007/s12229-010-9056-6Seguí-Simarro JM, Nuez F (2005) Meiotic metaphase I to telophase II is the most responsive stage of microspore development for induction of androgenesis in tomato (Solanum lycopersicum). Acta Physiol Plant 27(4B):675–685Seguí-Simarro JM, Corral-Martínez P, Parra-Vega V, González-García B (2011) Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep 30(5):765–778. doi: 10.1007/s00299-010-0984-8Shivanna KR (2003) Pollen biology and biotechnology. Science Publishers Inc., EnfieldSupena EDJ, Muswita W, Suharsono S, Custers JBM (2006a) Evaluation of crucial factors for implementing shed-microspore culture of Indonesian hot pepper (Capsicum annuum L.) cultivars. Sci Hort 107(3):226–232Supena EDJ, Suharsono S, Jacobsen E, Custers JBM (2006b) Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.). Plant Cell Rep 25(1):1–1

The protein kinases AtMAP3Kε1 and BnMAP3Kε1 are functional homologues of S. pombe cdc7p and may be involved in cell division

We identified an Arabidopsis thaliana gene, AtMAP3Kε1, and a Brassica napus cDNA, BnMAP3Kε1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT–PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kε1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.This work was mainly supported by a EU grant (SIME project BIOTEC-RTD-CEE PL 960275). The authors also acknowledge the financial support of the MERS and CNRS to UMR 8618, and DGESIG PB98–0678

PGI1-mediated vascular pentose phosphate pathway activity determines growth, photosynthesis and metabolism through 2-C-methyl-D-erythritol 4-P pathway action in Arabidopsis

Resumen del trabajo presentado en el XVI Meeting of Plant Molecular Biology, celebrado en Sevilla (España), del 14 al 16 de septiembre de 2022Phosphoglucose isomerase is involved in the early steps of glycolysis and regeneration of glucose-6-phosphate pools in the pentose phosphate pathway (PPP). In Arabidopsis, plastidial phosphoglucose isomerase (PGI1) is an important determinant of growth, metabolism and photosynthesis, probably due to its involvement in the synthesis of 2-C-methyl-D-erythritol 4-P (MEP)-derived hormones in root tips and vascular tissues (Bahaji et al., 2015; Bahaji et al., 2018). To test this hypothesis, we conducted proteomic and metabolic characterization of PGI1-null pgi1-2 plants. We also characterized pgi1-2 plants ectopically expressing PGI1 under the control of a root tip- and vascular tissue-specific promoter. Furthermore, we characterized pfk4/pfk5 knockout plants impaired in the early steps of plastidial glycolysis, and pgl3-1 plants with reduced activity of the plastidial PPP enzyme 6-phosphogluconolactonase 3. The overall data obtained in this work provide strong evidence that root tip and vascular PGI1-mediated plastidial PPP determines growth, development and photosynthesis through MEP pathway action.This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI) / 10.13039/501100011033/ (grants BIO2016-78747-P, PID2019-104685GB-100) and the Ministry of Education, Youth and Sport of the Czech Republic and ERDF project entitled “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827)

BIOLOGÍA Y BIOTECNOLOGÍA REPRODUCTIVA DE LAS PLANTAS

La biología reproductiva de las plantas engloba todos los procesos que permiten a un organismo vegetal tener descendencia, sea por vía sexual o asexual. El conocimiento de estos procesos es esencial para poder sacar provecho de ellos mediante aproximaciones biotecnológicas. Esta obra, dividida en dos grandes bloques, dedica el primero de ellos a la exposición secuencial de todos estos procesos (inducción y desarrollo floral, gametogénesis, polinización, fecundación, embriogénesis, formación, maduración y dispersión de frutos y semillas, etc.). El segundo bloque se centra en las distintas aplicaciones biotecnológicas derivadas de los procesos reproductivos, de interés práctico en muy distintos ámbitos de la sociedad.Seguí Simarro, JM. (2011). BIOLOGÍA Y BIOTECNOLOGÍA REPRODUCTIVA DE LAS PLANTAS. Editorial Universitat Politècnica de València. http://hdl.handle.net/10251/72437EDITORIA

Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L.

A stress treatment of 32°C for at least 8h was able to change the gametophytic program of the microspore, switching it to embryogenesis in Brassica napus, an interesting model for studying this process in vitro. After induction, some microspores started symmetric divisions and became haploid embryos after a few days, whereas other microspores, not sensitive to induction, followed their original gametophytic development. In this work the distribution and ultrastructural localization of two heat-shock proteins (Hsp70 and Hsp90) throughout key stages before and after embryogenesis induction were studied. Both Hsp proteins are rapidly induced, localizing in the nucleus and the cytoplasm. Immunogold labeling showed changes in the distribution patterns of these proteins, these changes being assessed by a quantitative analysis. Inside the nucleus, Hsp70 was found in association with RNP structures in the interchromatin region and in the nucleolus, whereas nuclear Hsp90 was mostly found in the interchromatin region. For Hsp70, the accumulation after the inductive treatment was accompanied by a reversible translocation from the cytoplasm to the nucleus, in both induced (embryogenic) and noninduced (gametophytic) microspores. However, the translocation was higher in embryogenic microspores, suggesting a possible additional role for Hsp70 in the switch to embryogenesis. In contrast, Hsp90 increase was similar in all microspores, occurring faster than for Hsp70 and suggesting a more specific role for Hsp90 in the stress response. Hsp70 and Hsp90 colocalized in clusters in the cytoplasm and the nucleus, but not in the nucleolus. Results indicated that stress proteins are involved in the process of microspore embryogenesis induction. The differential appearance and distribution of the two proteins and their association at specific stages have been determined between the two systems coexisting in the same culture: embryogenic development (induced cells) and development of gametes (noninduced cells). © 2003 Elsevier Science (USA). All rights reserved.This work was supported by research projects funded by EU BIO4-CT-0275, Spanish CICYT PB98-0678, BOS2002-03572, and Comunidad de Madrid 07G/0054/2000.Peer reviewe

Cell architecture during gametophytic and embryogenic microspore development in Brassica napus L.

10 pages.In this work, the cell architecture of the microspore following both gametophytic and embryogenic developmental pathways in vitro was compared with the gametophytic development in vivo in Brassica napus, at both light and electron microscopy level. The microspore reprogramming to embryogenesis involves defined changes affecting cell activities and structural organization which can be considered as markers of the microspore embryogenic pathway, but less is known about others developmental programmes followed by the microspore in vitro after both, inductive and non-inductive conditions. Low-temperature processing of the samples, cytochemical and immunocytochemical approaches to identify various cell components were performed. Differences in specific cellular features such as cellular size and shape, nuclear architecture, starch accumulation, presence of vacuoles and ribosomal population were studied to characterize sequential stages of microspore embryogenesis and other pathways occurring in vitro. The presence of abundant starch grains in a defined cytoplasmic region appeared as a specific feature of the in vitro gametophytic development, as well as of the non-induced microspores of in vitro cultures under embryogenic-inductive conditions.Peer reviewe

The Mitochondrial Cycle of Arabidopsis Shoot Apical Meristem and Leaf Primordium Meristematic Cells Is Defined by a Perinuclear Tentaculate/Cage-Like Mitochondrion1[W][OA]

Plant cells exhibit a high rate of mitochondrial DNA (mtDNA) recombination. This implies that before cytokinesis, the different mitochondrial compartments must fuse to allow for mtDNA intermixing. When and how the conditions for mtDNA intermixing are established are largely unknown. We have investigated the cell cycle-dependent changes in mitochondrial architecture in different Arabidopsis (Arabidopsis thaliana) cell types using confocal microscopy, conventional, and three-dimensional electron microscopy techniques. Whereas mitochondria of cells from most plant organs are always small and dispersed, shoot apical and leaf primordial meristematic cells contain small, discrete mitochondria in the cell periphery and one large, mitochondrial mass in the perinuclear region. Serial thin-section reconstructions of high-pressure-frozen shoot apical meristem cells demonstrate that during G1 through S phase, the large, central mitochondrion has a tentaculate morphology and wraps around one nuclear pole. In G2, both types of mitochondria double their volume, and the large mitochondrion extends around the nucleus to establish a second sheet-like domain at the opposite nuclear pole. During mitosis, approximately 60% of the smaller mitochondria fuse with the large mitochondrion, whose volume increases to 80% of the total mitochondrial volume, and reorganizes into a cage-like structure encompassing first the mitotic spindle and then the entire cytokinetic apparatus. During cytokinesis, the cage-like mitochondrion divides into two independent tentacular mitochondria from which new, small mitochondria arise by fission. These cell cycle-dependent changes in mitochondrial architecture explain how these meristematic cells can achieve a high rate of mtDNA recombination and ensure the even partitioning of mitochondria between daughter cells

Nuclear bodies domain changes with microspore reprogramming to embryogenesis

10 pages, 5 figures.-- PMID: 16584983 [PubMed].We analysed the presence of nuclear bodies and particularly Cajal bodies during representative stages of gametophytic and haploid embryogenic development in isolated microspore and anther cultures of a model system (Brassica napus cv. Topas) and a recalcitrant species (Capsicum annuum L. var. Yolo Wonder B). The nuclear bodies domain is involved on several important roles on nuclear metabolism, and Cajal bodies are specifically involved on the storage and maturation of both snRNPs and snoRNPs, as well as other splicing factors, necessary for mRNA and pre-rRNA processing, but not directly on the transcription. In this study, immunofluorescence and immunogold labelling with anti-trimethylguanosine antibodies against the specific cap of snRNAs, ultrastructural and cytochemical analysis were performed on cryoprocessed samples at confocal and electron microscopy respectively. Results showed that Cajal bodies increase during the early stages of microspore embryogenic development (young pro-embryos), compared to microspore and pollen development. Our results suggest that Cajal bodies may have a role in the transcriptionally active, proliferative stages that characterise early microspore embryogenic development.This work was supported by projects granted by Spanish Ministry of Education and Science (MEC) BOS2002-03572, AGL2005-05104, BFU2005-01094, and Comunidad de Madrid, CM 07G/0026/2003.Peer reviewe