Abstract
The induction of microspore embryogenesis produces dramatic changes in different aspects of the cell physiology and structure. Changes at the cell wall level are among the most intriguing and poorly understood. In this work, we used high pressure freezing and freeze substitution, immunolocalization, confocal, and electron microscopy to analyze the structure and composition of the first cell walls formed during conventional Brassica napus microspore embryogenesis, and in cultures treated to alter the intracellular Ca2+ levels. Our results revealed that one of the first signs of embryogenic commitment is the formation of a callose-rich, cellulose-deficient layer beneath the intine (the subintinal layer), and of irregular, incomplete cell walls. In these events, Ca2+ may have a role. We propose that abnormal cell walls are due to a massive callose synthesis and deposition of excreted cytoplasmic material, and the parallel inhibition of cellulose synthesis. These features were absent in pollen-like structures and in microspore-derived embryos, few days after the end of the heat shock, where abnormal cell walls were no longer produced. Together, our results provide an explanation to a series of relevant aspects of microspore embryogenesis including the role of Ca2+ and the occurrence of abnormal cell walls. In addition, our discovery may be the explanation to why nuclear fusions take place during microspore embryogenesis.
Highlights
Microspore embryogenesis is a fascinating experimental process whereby a haploid microspore is reprogrammed to become a haploid or doubled haploid (DH) embryo (Seguí-Simarro and Nuez, 2008a)
Equivalent suspensor-bearing microspore-derived embryos (MDEs) presented a similar cellulose pattern at the embryo proper domain, whereas suspensor cell walls were clearly delineated by cellulose staining (Supplementary Figure Scarlet 4B (S4B)). This pattern, changed from globular MDEs onward (Supplementary Figure S4C), where staining consistently decorated all the cell walls of the embryo proper in a pattern similar to a conventional mature cell wall. These results indicated that the first embryogenic divisions of the induced microspore are defined by abnormal patterns of callose and cellulose deposition, establishing a clear correlation between the morphological features that define a microspore as embryogenic, the presence of callose at both the subintinal layer and the cell walls, and the total absence of cellulose
Figure S6), which represented 240% of the yield of control cultures (∼250 MDEs/ml). These results indicated that alteration of Ca2+ levels by caffeine and benzyl alcohol (BA) affected the amount of callose deposited in the cell walls of embryogenic the first stages of the switch, the mechanisms of synthesis and/or deposition of cell wall components are altered, giving rise to the transient formation of atypical walls
Summary
Microspore embryogenesis is a fascinating experimental process whereby a haploid microspore is reprogrammed to become a haploid or doubled haploid (DH) embryo (Seguí-Simarro and Nuez, 2008a) This inducible pathway has a great biotechnological potential, since among many other advantages, it allows for faster and cheaper ways to obtain pure lines for hybrid seed production (Forster et al, 2007). Abnormal Cell Walls During Microspore Embryogenesis a developmental switch toward embryogenesis, and with the cessation of the old gametophytic program (Malik et al, 2007; Seguí-Simarro and Nuez, 2008a). All these changes must imply a profound remodeling at the genetic and molecular levels, and in cell architecture. Among all the changes undergone by the embryogenic microspore, one of the aspects that attracted the attention of the first cell biologists that studied this process was how induced cells are divided (Zaki and Dickinson, 1991; Simmonds and Keller, 1999)
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