Making Epidermal Bladder Cells Bigger: Developmental- and Salinity-Induced Endopolyploidy in a Model Halophyte

Abstract

Endopolyploidy occurs when DNA replication takes place without subsequent mitotic nuclear division, resulting in cell-specific ploidy levels within tissues. In plants, endopolyploidy plays an important role in sustaining growth and development, but only a few studies have demonstrated a role in abiotic stress response. In this study, we investigated the function of ploidy level and nuclear and cell size in leaf expansion throughout development and tracked cell type-specific ploidy in the halophyte Mesembryanthemum crystallinum In addition to developmental endopolyploidy, we examined the effects of salinity stress on ploidy level. We focused specifically on epidermal bladder cells (EBC), which are modified balloon-like trichomes, due to their large size and role in salt accumulation. Our results demonstrate that ploidy increases as the leaves expand in a similar manner for each leaf type, and ploidy levels up to 512C were recorded for nuclei in EBC of leaves of adult plants. Salt treatment led to a significant increase in ploidy levels in the EBC, and these cells showed spatially related differences in their ploidy and nuclear and cell size depending on the positions on the leaf and stem surface. Transcriptome analysis highlighted salinity-induced changes in genes involved in DNA replication, cell cycle, endoreduplication, and trichome development in EBC. The increase in cell size and ploidy observed in M. crystallinum under salinity stress may contribute to salt tolerance by increasing the storage capacity for sodium sequestration brought about by higher metabolic activity driving rapid cell enlargement in the leaf tissue and EBC.

Figure 1.

Average normalized nuclear count for each ploidy level detected in M. crystallinum juvenile plants. A, Seed. B, Cotyledon. C, First leaf pair. D, Second leaf pair. E, Third leaf pair. F, Fourth leaf pair. G, Fifth leaf pair. H, Roots. Leaves were measured over a 3-week period, as follows: week 1 (light gray bars), week 2 (medium gray bars), and week 3 (dark gray bars). Data are means ± se of three independent biological replicates.

Figure 2.

Mean ploidy levels in cotyledons, leaves, and roots from M. crystallinum. A, Mean ploidy was calculated as shown in “Materials and Methods” from cotyledons, leaf pair 1, leaf pair 2, leaf pair 3, leaf pair 4, and leaf pair 5 over a 3- or 2-week period, as follows: week 1 (light gray bars), week 2 (medium gray bars), and week 3 (dark gray bars). Data are means ± se of three independent biological replicates. Columns with the same letter are not significantly different in Duncan’s multiple range test (P < 0.05), with white uppercase letters denoting the comparison with developmental stages with each week and black lowercase letters comparing values for the weeks within each development stage. B, Mean ploidy levels in young and old roots from control or salt-treated plants. Significance in Duncan’s multiple range test (P < 0.05) is indicated by the asterisk.

Figure 3.

Flow cytometry analysis of endopolyploidy in M. crystallinum adult plants. Propidium iodide (PI) was used to stain nuclei in the chopped plant material from branch leaf of 9-week-old plants (A), branch leaf of 10-week-old plants (B), and bract leaf (C). Plants were untreated (left) or salt treated (right). Acquisition was performed on a BD FACS Canto II flow cytometer, and data were analyzed using Flowing Software version 2.5.1. The histogram is representative of three independent experiments with biological replicates from three different plants. Mean ploidy levels in the samples shown were calculated as described in “Materials and Methods.”

Figure 4.

Bar graphs showing average normalized nuclear counts for each ploidy level in the branch or bract leaves from adult plants. Samples were as follows: peeled leaf (dark gray bars), upper epidermal peels (medium gray bars), and lower epidermal peels (light gray bars). Insets show expansion of the y axis to visualize high-ploidy nuclear counts. Data are means ± se of three independent biological replicates.

Figure 5.

Mean ploidy levels calculated in a peeled leaf, the upper epidermal peel, and the lower epidermal peel of bract and branch leaves taken from untreated (light gray bars) or salt-treated (dark gray bars) M. crystallinum plants. Mean ploidy was calculated as shown in “Materials and Methods” from peeled leaves, upper leaf peels, and lower leaf peels from both bract leaves (A) and branch leaves (B). Data are means ± se of three independent biological replicates. Columns with the same letter are not significantly different in Duncan’s multiple range test (P < 0.05), with uppercase letters denoting the effects of NaCl treatment and lowercase letters referring to the comparison of different tissues.

Figure 6.

Fluorescence microscopy of nuclei in different M. crystallinum cell types from salt-treated plants. A, Lower epidermal peels were stained in PI solution for 5 min in the dark, and a representative fluorescence microscopy image is shown. The white arrow indicates an EBC nucleus, the yellow arrow indicates the nucleus in a guard cell, and the blue arrow indicates a pavement cell nucleus. B, Table showing calculated nuclear diameters for selected cell types. Data are means ± se of 50 measurements per cell type. C, Composed figure showing the relative nuclear size of representative nuclei from different cell types.

Figure 7.

Fold change (log₂) of significantly differentially expressed genes associated with endoreplication in the M. crystallinum bladder cell transcriptome in response to 200 mm NaCl. Significantly different expression was evaluated at an adjusted P < 0.05 and false discovery rate = 0.01.

Figure 8.

Differential responses of selected genes to salt stress in root and EBC transcriptomes. A, Normalized expression values within a category scaled from 0 to 1 on the y axis. B, Fold change (FC) between salt and control treatments.