ABA-Induced Vegetative Diaspore Formation in Physcomitrella patens

Abstract

The phytohormone abscisic acid (ABA) is a pivotal regulator of gene expression in response to various environmental stresses such as desiccation, salt and cold causing major changes in plant development and physiology. Here we show that in the moss Physcomitrella patens exogenous application of ABA triggers the formation of vegetative diaspores (brachycytes or brood cells) that enable plant survival in unfavorable environmental conditions. Such diaspores are round-shaped cells characterized by the loss of the central vacuole, due to an increased starch and lipid storage preparing these cells for growth upon suitable environmental conditions. To gain insights into the gene regulation underlying these developmental and physiological changes, we analyzed early transcriptome changes after 30, 60, and 180 min of ABA application and identified 1,030 differentially expressed genes. Among these, several groups can be linked to specific morphological and physiological changes during diaspore formation, such as genes involved in cell wall modifications. Furthermore, almost all members of ABA-dependent signaling and regulation were transcriptionally induced. Network analysis of transcription-associated genes revealed a large overlap of our study with ABA-dependent regulation in response to dehydration, cold stress, and UV-B light, indicating a fundamental function of ABA in diverse stress responses in moss. We also studied the evolutionary conservation of ABA-dependent regulation between moss and the seed plant Arabidopsis thaliana pointing to an early evolution of ABA-mediated stress adaptation during the conquest of the terrestrial habitat by plants.

Keywords: ABA; brachycyte; cell wall; diaspore; gene expression; moss.

FIGURE 1

Developmental changes of protonema upon exogenous ABA application. Comparison of protonema before and after ABA application observed via (A) light microscopy, (B) cryo scanning electron microscopy (SEM), and (C) confocal microscopy after propidium iodide staining. The left column shows chloronema (ch) and caulonema (ca) cells prior to ABA application, while the middle and right columns show brachycytes (b, rounded brood cells) and tmema cells (t, preformed filament breaking points) 2 weeks after exogenous application of 100 μm ABA. (D) The bar chart shows average cell wall thickness in μm (measured from TEM sections) after 2 weeks of culture without (WT) and with (WT + 100 μm ABA) treatment. Error bars show standard deviation (n = 11); the difference is significant (two-sided t-test, p < 0.01). (E–H) Ultrastructural changes upon exogenous ABA application, cryo-SEM. Images are false colored: nucleus (pink), chloroplasts (green), and central vacuole (blue). Cryo-SEM after freeze breaking and etching; (E,G) 2 weeks 100 μm ABA, (F,H) mock control. In (E,F) the cell is broken perpendicular, in (G,H) the cell wall (CW) has been removed, allowing to see the plasma membranes’ (PM) outer surface. The central vacuole (blue) clearly visible in (F,H) is not recognizable in (E,G). ABA-treated cells exhibit more furrows and a denser patterns of depressions (marked by arrows) that most probably represent negatives (relief) of cellulose synthase complexes. (I,J) Ultrastructural changes upon exogenous ABA application, transmission electron microscopy (TEM) images of epon embedded, Osmium-stained cells. (I) Five weeks 100 μm ABA, (J) without ABA. Intracellular structures are labeled as follows: vacuole (V), starch granules (S), oleosomes (O), plasma membrane (PM), cell wall (CW).

FIGURE 2

Analysis of ABA-responsive genes and microarray data validation by quantitative real-time PCR. Differentially expressed genes from P. patens protonema in response to exogenously applied 10 μM ABA. (A) Differentially expressed genes after 30, 60, and 180 min of ABA treatment. (B) Venn diagram of ABA-responsive genes after 30, 60, and 180 min of ABA treatment. (C) Validation of microarray data by qRT-PCR; expression values were normalized to the reference gene PpEF1α (Le Bail et al., 2013). Error bars indicate standard deviation calculated from three biological replicates. The values from the microarray experiments were scaled down by the indicated factors to fit to the qRT-PCR scale: Phypa_141045, Phypa_172697, and Phypa_163821 by 10⁴; Phypa_72483, Phypa_28324 by 10³.

FIGURE 3

Comparison of DEGs with previously published studies. (A) Venn diagram depicting the overlap of DEGs from our study and two previously performed studies by Stevenson et al. (2016) and Komatsu et al. (2013). (B) Comparison of all our DEGs with both studies. (C) Unique identification of early induced genes by our study.

FIGURE 4

Identification of conserved ABA-regulated genes in P. patens and A. thaliana. UpSet plot of ABA-responsive genes from four A. thaliana studies (Matsui et al., 2008; Wang et al., 2011; Liu et al., 2013; Zhan et al., 2015) and their comparison with putative A. thaliana orthologs that are regulated by ABA in P. patens.

FIGURE 5

Principal component analysis (PCA) of the P. patens transcriptome time series. Replicates have been linked by a convex hull to visualize the sample distances.

FIGURE 6

Global transcriptome analysis. Euclidean distance between the global and ordered gene subset response for subsets of different sizes. The red line is a polynomial fit to all data points. The light and dark rectangles depict the significantly regulated genes from the F-test for the FDR corrected p-values < 0.01 and <0.05, respectively. Genes were ranked according to the p-value from the F-test analysis.

FIGURE 7

Overlap of transcription associated proteins (TAPs) in our and other stress related studies. (A) Identification of differentially expressed TAPs after 100 μM ABA application for 30, 60, and 180 min. (B) Overlap of ABA-regulated TAPs found in this study with Richardt et al. (2010) and Timmerhaus et al. (2011) (C) All TAPs differentially expressed during cold stress, dehydration and UV-B light are also differentially regulated in response to ABA. (D) Comparison of differentially expressed genes in response to cold and ABA.