Mannans and endo-β-mannanase transcripts are located in different seed compartments during Brassicaceae germination

Abstract

Mannans but not endo-β-mannanases are mainly found in the mucilage layer of two Brassicaceae seeds. Nonetheless, mannanase mobilization from inner to outer seed layers cannot be ruled out. The contribution of endo-β-mannanase (MAN) genes to the germination of the wild-type Sisymbrium officinale and cultivated Brassica rapa (Brassicaceae) species has been explored. In both species, mannans have been localized to the imbibed external seed coat layer (mucilage) by fluorescence immunolocalization and MAN enzymatic activity increases in seeds as imbibition progresses, reaching a peak before 100% germination is achieved. The MAN gene families have been annotated and the expression of their members analyzed in vegetative and reproductive organs. In S. officinale and B. rapa, MAN2, MAN5, MAN6, and MAN7 transcripts accumulate upon seed imbibition. SoMAN7 is the most expressed MAN gene in S. officinale germinating seeds, as occurs with its ortholog in Arabidopsis thaliana, but in B. rapa, the most abundant transcripts are BrMAN2 and BrMAN5. These genes (MAN2, MAN5, MAN6, and MAN7) are localized, by mRNA in situ hybridization, to the micropylar at the endosperm layer and to the radicle in S. officinale, but in B. rapa, these mRNAs are faintly found to the micropylar living seed coat layer and are mainly present at the radicle tip and the vascular bundles. If the domestication process undergone by B. rapa is responsible for these different MAN expression patterns, upon germination remains to be elucidated. Since mannans and MAN genes are not spatially distributed in the same seed tissues, a movement of MAN enzymes that are synthesized with typical signal peptides from the embryo tissues to the mucilage layer (via apoplastic space) is necessary for the mannans to be hydrolyzed.

A Possible Role of the Aleurone Expressed Gene HvMAN1 in the Hydrolysis of the Cell Wall Mannans of the Starchy Endosperm in Germinating Hordeum vulgare L. Seeds

“…Interestingly, HvMAN1 is the most expressed gene throughout zygotic embryogenesis, clearly decreasing from phase W1 (high mitotic activity) to LG in which the caryopsis is preparing for desiccation and for the establishment of dormancy (Nonogaki et al, 2018). In other crop and wild species, several MAN paralogs have also been described as functionally relevant in grains: Brachypodium distachyon (BdMAN1, BdMAN2, BdMAN4, and BdMAN6), Oryza sativa (OsMAN1, OsMAN2, and OsMAN6), Arabidopsis thaliana (AtMAN2, AtMAN5, AtMAN6, and AtMAN7), Brassica rapa (BrMAN2, BrMAN5, BrMAN6, and BrMAN7), Sisymbrium officinale (SoMAN2, SoMAN5, SoMAN6, and SoMAN7), and Solanum lycopersicum (LeMAN1 and LeMAN2; Nonogaki et al, 2000;Nonogaki et al,2007;Yuan et al, 2007;Ren et al, 2008;Iglesias-Fernández et al, 2011a;Iglesias-Fernández et al, 2011b;González-Calle et al, 2015;Carrillo-Barral et al, 2018).…”

“…In Hordeum vulgare, it has also been proposed that starch mobilization requires a previous CW degradation in order to allow a rapid diffusion of amylolytic enzymes (Andriotis et al, 2016). In the Brassicaceae Sisymbrium officinale and Brassica rapa, mannans and MAN genes have been localized to different seed compartments, again suggesting the movement of MAN enzymes from the embryo tissues to the mucilage layer where mannans are localized and hydrolyzed (Carrillo-Barral et al, 2018). Taking into account the MAN enzymatic activity in embryos and endosperms of germinating grains (Figure 5B), the highest expression levels of HvMAN1 and the mannan polymers disappearance upon germination, a possible role for HvMAN1 can be anticipated, without excluding the activity of other MANs (HvMAN3, HvMAN5, and HvMAN6).…”

“…The supernatant (400 ml) was mixed with 200 ml of 0.25% AZCL galactomannan (Megazyme, Chicago, IL, USA). Enzymatic activity was determined as previously described (Iglesias-Fernández et al, 2011a;Carrillo-Barral et al, 2018). For quantification of total protein, the Bradford reagent was utilized (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and Bovine Serum albumin (BSA) was the standard.…”

“…In Arabidopsis, the importance of MAN enzymes in the germination sensu stricto has been further supported by genetic experiments with T-DNA insertion mutants in genes encoding MANs (AtMAN5, AtMAN6, and AtMAN7;Iglesias-Fernández et al, 2011a, 2011b. Interestingly, the presence of mannan polymers has been shown in the mucilage layer of imbibed A. thaliana, B. rapa and S. officinale seeds by fluorescence immunolocalization assays (Lee et al, 2012;Carrillo-Barral et al, 2018). In the monocotyledonous Brachypodium distachyon, a model species for temperate cereals, BdMAN2, BdMAN4, and BdMAN6 genes have been demonstrated to be relevant during grain germination, and mannans have been immunolocalized to the coleorhiza, disappearing as germination progresses (González-Calle et al, 2015).…”

“…In dicotyledonous seeds, such as those from Arabidopsis thaliana, Brassica rapa, and Sisymbrium officinale. MAN enzymes have been involved in the weakening of the seedcovering layers to facilitate radicle emergence during germination sensu stricto (Iglesias-Fernández et al, 2011a;Iglesias-Fernández et al, 2011b;Rodríguez-Gacio et al, 2012;Bewley et al, 2013;Zhang et al, 2013;Nonogaki, 2014;Steinbrecher and Leubner-Metzger, 2017;Carrillo-Barral et al, 2018). In Arabidopsis, the importance of MAN enzymes in the germination sensu stricto has been further supported by genetic experiments with T-DNA insertion mutants in genes encoding MANs (AtMAN5, AtMAN6, and AtMAN7;Iglesias-Fernández et al, 2011a, 2011b.…”