Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development

Abstract

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants.Microspore embryogenesis describes the process in which the immature male gametophyte is induced to form a haploid embryo in vitro. Microspore embryo culture is a valuable tool for plant breeders because the haploid embryos that are produced (microspore-derived embryos [MDEs]) can be converted to homozygous doubled haploids using chromosome-doubling agents.The ability to obtain a homozygous population of plants in a single generation not only significantly reduces the time needed to develop inbred lines, but also facilitates selection for recessive and polygenic traits and speeds up the development of mapping populations and molecular markers for marker-assisted breeding (Forster and Thomas, 2005). Microspore embryogenesis has been described for more than 250 plant species and efficient protocols are available for about 20 of these (Maluszinsky et al., 2003). As with other regeneration processes, many species still remain recalcitrant for microspore embryogenesis and, even within a successful species, there are usually recalcitrant genotypes. There is, therefore, considerable interest in identifying the molecular genetic factors that define and control this process...

Identification of proteins related to microspore embryogenesis responsiveness in anther cultures of winter triticale (×Triticosecale Wittm.)

“…The hypothesis has been put forward ( _ Zur et al 2014), claiming that the effectiveness of ME in triticale anther cultures depends on both cell tolerance to/defence ability against oxidative stress and ROS-induced signal transduction associated with the stress treatment used for microspore reprogramming. Similarly to our study, one of the antioxidative enzyme-L-ascorbate peroxidase 1 (spot no 9) was accumulated after ME-inducing treatment in maize and rapeseed (Joosen et al 2007;Cordewener et al 2009;Uváčková et al 2012). Its role can be more complex as in various plant cell culture systems high ascorbate levels stimulate cell division (de Pinto et al 1999).…”

“…Microspore reprogramming not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program. Numerous studies focused on searching for the gene(s) responsible for efficient ME induction with the use of model plant species: rapeseed (Joosen et al 2007), wheat (Sánchez-Díaz et al 2013) and barley (Muñoz-Amatriaín et al 2009). The first report for molecular regulation of ME in triticale was published recently ( _ Zur et al 2013).…”

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation

“…Due to these advantages, it is not surprising that the B. napus MDE culture system has been extensively utilized to investigate anatomical, physiological, and molecular events occurring during embryo development in vitro (reviewed in Yeung 2002;Boutilier et al 2005). Recent transcriptome and proteome analyses have focused on the molecular events delineating the induction of microspore embryogenesis, that is the initial transition from gametophytic to embryonic cell state (Joosen et al 2007;Malik et al 2007). Both studies have revealed the existence of robust markers and pathways associated with the early phases of embryo development.…”

“…Gene expression proWling is a powerful tool for dissecting the molecular mechanisms of relevant developmental processes, including embryogenesis. Over the past few years cDNA microarray studies have revealed the existence of transcript patterns associated with embryo development in both angiosperms (Girke et al 2000;Thibaud-Nissen et al 2003;Joosen et al 2007;Malik et al 2007) and gymnosperms (Stasolla et al 2003(Stasolla et al , 2004b. Most importantly, these studies have revealed the existence of key genes able to aVect the embryogenic process if their expression level is experimentally altered (Tahir et al 2006;).…”

Expression Profiles in Barley Microspore Embryogenesis

“…Recently, studies using macro-and microarray technology have been used to further understanding of the molecular processes that take place in embryogenic microspores and microspore-derived embryos in Brassica (Pauls et al 2006, Joosen et al 2007, Malik et al 2007. Although microspore embryogenesis is highly dependent on the species, genotype, stress pretreatment and culture media, utilization of microarray techniques is providing new insights into the molecular mechanisms that control microspore embryogenesis.…”