Microspore embryogenesis: assignment of genes to embryo formation and green vs. albino plant production

Abstract

Plant microspores can be reprogrammed from their normal pollen development to an embryogenic route in a process termed microspore embryogenesis or androgenesis. Stress treatment has a critical role in this process, inducing the dedifferentiation of microspores and conditioning the following androgenic response. In this study, we have used three barley doubled haploid lines with similar genetic background but different androgenic response. The Barley1 GeneChip was used for transcriptome comparison of these lines after mannitol stress treatment, allowing the identification of 213 differentially expressed genes. Most of these genes belong to the functional categories "cell rescue, defense, and virulence"; "metabolism"; "transcription"; and "transport". These genes were grouped into clusters according to their expression profiles among lines. A principal component analysis allowed us to associate specific gene expression clusters to phenotypic variables. Genes associated with the ability of microspores to divide and form embryos were mainly involved in changes in the structure and function of membranes, efficient use of available energy sources, and cell fate. Genes related to stress response, transcription and translation regulation, and degradation of pollen-specific proteins were associated with green plant production, while expression of genes related to plastid development was associated with albino plant regeneration.

Fig. 1

DAPI staining of barley isolated microspores (top) and semithin sections of anthers stained with toluidine blue (bottom). a Anthers before mannitol treatment. b, c, d Anthers after 4 days of mannitol stress treatment of DH46 (b), DH6188 (c), and DH6004 (d). (T tapetum, Black arrows indicate binucleate microspores)

Fig. 2

Hierarchical clustering and expression profiles of differentially expressed genes. a Hierarchical cluster analysis of the 213 genes in DH46 (a), DH6004 (b), and DH6188 (c). b Expression profiles for the genes in each cluster presented in a graph format

Fig. 3

Principal component analysis of the five androgenic variables (grey squares) and the genes representative of each cluster (white squares). The three lines DH46, DH6004, and DH6188 are also identified (black circles). Each gene was designated as p (probe set), preceded by its cluster number and followed by its position in the cluster, according to the classified genes of Supplemental Table S1. nDM number of dividing microspores, nEMB number of embryos, pGP percentage of green plants, nAP number of albino plants, nGP number of green plants

Fig. 4

Expression analyses by semiquantitative RT-PCR of seven genes belonging to cluster 2 (Cl. 2), cluster 3 (Cl. 3), and cluster 8 (Cl. 8). Four different stages were assayed in each of the three DH lines: uninucleated microspores before stress treatment (UM), microspores after 4 days of stress treatment (s-4d), microspores after 4 days of culture (c-4d), and young pollen grains (P). Contig3116_at was used as control