Abstract
Microspore embryogenesis is a manifestation of plant cell totipotency whereby new cell walls are formed as a consequence of the embryogenic switch. In particular, the callose-rich subintinal layer created immediately upon induction of embryogenesis was recently related to protection against stress. However, little is currently known about the functional significance of other compositional changes undergone by the walls of embryogenic microspores. We characterized these changes in Brassica napus at different stages during induction of embryogenic microspores and development of microspore-derived embryos (MDEs) by using a series of monoclonal antibodies specific for cell wall components, including arabinogalactan-proteins (AGPs), pectins, xyloglucan and xylan. We used JIM13, JIM8, JIM14 and JIM16 for AGPs, CCRC-M13, LM5, LM6, JIM7, JIM5 and LM7 for pectins, CCRC-M1 and LM15 for xyloglucan, and LM11 for xylan. By transmission electron microscopy and quantification of immunogold labeling on high-pressure frozen, freeze-substituted samples, we profiled the changes in cell wall ultrastructure and composition at the different stages of microspore embryogenesis. As a reference to compare with, we also studied in vivo microspores and maturing pollen grains. We showed that the cell wall of embryogenic microspores is a highly dynamic structure whose architecture, arrangement and composition changes dramatically as microspores undergo embryogenesis and then transform into MDEs. Upon induction, the composition of the preexisting microspore intine walls is remodeled, and unusual walls with a unique structure and composition are formed. Changes in AGP composition were related to developmental fate. In particular, AGPs containing the JIM13 epitope were massively excreted into the cell apoplast, and appeared associated to cell totipotency. According to the ultrastructure and the pectin and xyloglucan composition of these walls, we deduced that commitment to embryogenesis induces the formation of fragile, plastic and deformable cell walls, which allow for cell expansion and microspore growth. We also showed that these special walls are transient, since cell wall composition in microspore-derived embryos was completely different. Thus, once adopted the embryogenic developmental pathway and far from the effects of heat shock exposure, cell wall biosynthesis would approach the structure, composition and properties of conventional cell walls.
Highlights
The plant kingdom is characterized by a high level of developmental plasticity and totipotency
Anticallose immunogold labeling revealed that the subintinal layer was rich in callose (Figure 1E)
JIM14 epitopes presented detectable levels in the intine of in vivo pollen and in vitro pollen-like grains (Figure 2G), the general pattern was similar to that obtained with JIM8, with higher particle densities only in the inner cell walls and epidermis of torpedo and cotyledonar embryos (Figure 2H). These results demonstrated that the arabinogalactan proteins (AGPs) composition of the cell walls produced during microspore embryogenesis is unique, remarkably different from in vivo microspore/pollen development, and highly dynamic, changing dramatically as the microspore becomes embryogenic and develops as an microspore-derived embryos (MDEs)
Summary
The plant kingdom is characterized by a high level of developmental plasticity and totipotency. In the model species Brassica napus, the androgenic switch is induced by a heat stress treatment of at least one day at 32◦C (Lichter, 1982; Supena et al, 2008), after which some microspores/pollen change their developmental fate and form MDEs. MDEs will simultaneously undergo considerable molecular and cellular modifications related to both heat stress exposure (the so-called heat stress response) and the onset of the embryogenic program. The newly formed inner walls are irregular, incomplete, and present large deposits of cytoplasmic material secreted to the apoplast (Corral-Martínez et al, 2013; Parra-Vega et al, 2015a,b) Beneath their original intine, embryogenic microspores develop an additional layer, the subintinal layer, which is absent in non-embryogenic cultured microspores (Parra-Vega et al, 2015b; Rivas-Sendra et al, 2019). Both subintinal layers and inner walls are callose-rich and cellulose-deficient during the first embryogenic stages (Parra-Vega et al, 2015b; Rivas-Sendra et al, 2019)
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