RESUMEN
Multienzyme associations localized to specific subcellular sites are involved in several critical functions in cellular metabolism, such as plant survival and reproduction. To date, few multienzyme complexes involved in male fertility have been examined in Brassica napus Here, we reported that in B. napus, the members of a multienzyme complex work in an interaction pattern different from that in Arabidopsis thaliana for sporopollenin biosynthesis. 7365A, a male-sterile mutant with a relatively smooth anther cuticle, was found to have a dramatic reduction in both cutin monomers and wax composition. Proteomic comparison between the mutant 7365A and wild-type 7365B showed down-regulation of three sporopollenin biosynthetic enzymes, namely BnPKSA, BnPKSB and BnTKPR; these enzymes were tightly co-expressed with BnACOS5. BnPKSA and BnPKSB showed similar expression patterns but distinct accumulation levels, suggesting that they had partially distinct functions during sporopollenin biosynthesis. In vitro and in vivo analyses demonstrated that BnPKSB directly interacted with BnPKSA and BnACOS5, but no such interactions were found in the present investigation for BnTKPR1. Interestingly, the interaction between PKSA and PKSB has not been discovered in Arabidopsis, which may indicate a new interaction representing an additional efficient regulation method in B. napus Taken together, we propose that BnPKSA and BnPKSB may comprise a heterodimer combined with BnACOS5, constituting a sporopollenin metabolon in tapetal cells that is related to male reproductive development in B. napus.
Asunto(s)
Brassica napus/enzimología , Regulación de la Expresión Génica de las Plantas , Complejos Multienzimáticos , Proteómica , Biopolímeros/biosíntesis , Biopolímeros/genética , Brassica napus/genética , Brassica napus/crecimiento & desarrollo , Brassica napus/ultraestructura , Carotenoides/biosíntesis , Carotenoides/genética , Regulación hacia Abajo , Flores/enzimología , Flores/genética , Flores/crecimiento & desarrollo , Flores/ultraestructura , Lípidos de la Membrana/metabolismo , Modelos Biológicos , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Multimerización de ProteínaRESUMEN
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.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Brassica napus/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Glucanos/metabolismo , Proteínas de Plantas/metabolismo , Polen/crecimiento & desarrollo , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/fisiología , Biopolímeros/metabolismo , Brassica napus/citología , Brassica napus/genética , Brassica napus/crecimiento & desarrollo , Carotenoides/metabolismo , Diferenciación Celular , Análisis por Conglomerados , Regulación hacia Abajo/genética , Flores/citología , Flores/genética , Flores/crecimiento & desarrollo , Flores/fisiología , Perfilación de la Expresión Génica , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Infertilidad Vegetal , Proteínas de Plantas/genética , Polen/citología , Polen/genética , Polen/fisiología , Alineación de Secuencia , Factores de TiempoRESUMEN
In this study, total 19 straw samples from four Brassica species were determined with a diverse cell wall composition and varied biomass enzymatic digestibility under sulfuric acid or lime pretreatment. Correlation analysis was then performed to detect effects of cell wall compositions and wall polymer features (cellulose crystallinity, hemicellulosic monosaccharides and lignin monomers) on rapeseeds biomass digestibility. As a result, coniferyl alcohol (G-lignin) showed a strongly negative effect on biomass saccharification, whereas hemicellulosic monosaccharides (fucose, galactose, arabinose and rhamnose) were positive factors on lignocellulose digestions. Notably, chemical analyses of four typical pairs of samples indicated that hemicellulosic monosaccharides and G-lignin may coordinately influence biomass digestibility in rapeseeds. In addition, Brassica napus with lower lignin content exhibited more efficiency on both biomass enzymatic saccharification and ethanol production, compared with Brassica junjea. Hence, this study has at first time provided a genetic strategy on cell wall modification towards bioenergy rapeseed breeding.