Identification of novel genes potentially involved in somatic embryogenesis in chicory (Cichorium intybus L.)

Abstract

BackgroundIn our laboratory we use cultured chicory (Cichorium intybus) explants as a model to investigate cell reactivation and somatic embryogenesis and have produced 2 chicory genotypes (K59, C15) sharing a similar genetic background. K59 is a responsive genotype (embryogenic) capable of undergoing complete cell reactivation i.e. cell de- and re-differentiation leading to somatic embryogenesis (SE), whereas C15 is a non-responsive genotype (non-embryogenic) and is unable to undergo SE. Previous studies [1] showed that the use of the β-D-glucosyl Yariv reagent (β-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that β-GlcY is a useful tool for investigating somatic embryogenesis (SE) in chicory. In addition, a putative AGP (DT212818) encoding gene was previously found to be significantly up-regulated in the embryogenic K59 chicory genotype as compared to the non-embryogenic C15 genotype suggesting that this AGP could be involved in chicory re-differentiation [2]. In order to improve our understanding of the molecular and cellular regulation underlying SE in chicory, we undertook a detailed cytological study of cell reactivation events in K59 and C15 genotypes, and used microarray profiling to compare gene expression in these 2 genotypes. In addition we also used β-GlcY to block SE in order to identify genes potentially involved in this process.ResultsMicroscopy confirmed that only the K59, but not the C15 genotype underwent complete cell reactivation leading to SE formation. β-GlcY-treatment of explants blocked in vitro SE induction, but not cell reactivation, and induced cell wall modifications. Microarray analyses revealed that 78 genes were differentially expressed between induced K59 and C15 genotypes. The expression profiles of 19 genes were modified by β-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by β-GlcY-treatment: AGP (DT212818), 26 S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein-1 (MT1), two non-specific lipid transfer proteins genes (SDI-9 and DEA1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and snakin 2 (SN2). These results suggest that the 8 genes, including the previously-identified AGP gene (DT212818), could be involved in cell fate determination events leading to SE commitment in chicory.ConclusionThe use of two different chicory genotypes differing in their responsiveness to SE induction, together with β-GlcY-treatment represented an efficient tool to discriminate cell reactivation from the SE morphogenetic pathway. Such an approach, together with microarray analyses, permitted us to identify several putative key genes related to the SE morphogenetic pathway in chicory.

LEAFY COTYLEDON2 (LEC2) promotes embryogenic induction in somatic tissues of Arabidopsis, via YUCCA-mediated auxin biosynthesis

“…Only recently, the molecular events governing embryogenic induction in somatic plant cells begun to be decoded, and with the use of global transcriptome analysis many genes have been shown to be involved in the somatic embryogenesis (SE) that is induced in different plant species (Thibaud-Nissen et al 2003; Sharma et al 2008; Imin et al 2008; Lucau-Danila et al 2010). However, very few genes have been experimentally confirmed to be SE-specific and even fewer have been elucidated in terms of their mechanism of action during SE induction.…”

Ultrastructural changes and the distribution of arabinogalactan proteins during somatic embryogenesis of banana (Musa spp. AAA cv. ‘Yueyoukang 1’)

“…The protein moiety is rich in hydroxyproline and usually constitutes <10% (w/w) of the molecule. Although the mechanism of AGPs action remains unclear, they have been thought to play important roles in various aspects of plant growth and development such as cell–cell interactions (Kreuger and van Holst 1993, Motose et al 2004), cell division and/or cell elongation (Park et al 2003, Šamaj et al 1998, 1999b, Showalter 2001), as well as the regulation of somatic embryogenesis (Chapman et al 2000b, Legrand et al 2007, Lucau‐Danila et al 2010, Šamaj et al 2008, van Hengel et al 2001). For example, Kreuger and van Holst (1993) found that direct addition of mature carrot seed AGPs to a weakly embryogenic cell (EC) line caused reinitiation of EC formation.…”

“…JIM8‐binding AGP might play a principal role in proembryo stages, whereas both JIM8‐ and JIM13‐binding AGPs could be involved in embryo differentiation (Tang et al 2006). Recently, some putative AGP genes involved in cell fate determination events leading to somatic embryogenesis commitment in chicory have been identified (Legrand et al 2007, Lucau‐Danila et al 2010). Nevertheless, to our knowledge, there are no ultrastructural and developmental studies focussed on AGPs in banana somatic embryogenesis.…”

Pretreatments, conditioned medium and co-culture increase the incidence of somatic embryogenesis of different Cichorium species

“…77 In these genotypes, Lucau-Danila et al also showed that at least eight genes, including the previously identified AGP gene (DT212818), were differentially expressed in both genotypes. 29 They were probably involved in cell fate determination in chicory (i.e. acquisition of totipotency).…”

“…Moreover, stressed environment could modify AGPs gene-expression. 29,78,79 Finally, the set of released AGP could also reflect the regulation of gene expression that resulted from different stresses due to in vitro culture (collecting, fragments, liquid culture, orbital shaking…), but also resulted from the competence of each genotype to commit into SE. Legrand et al established that one AGP gene was upregulated in the high embryogenic chicory (C. intybus, Kospool Hungarian cultivar) compared with the low embryogenic one.…”