Transcriptome Profiling of the Green Alga Spirogyra pratensis (Charophyta) Suggests an Ancestral Role for Ethylene in Cell Wall Metabolism, Photosynthesis, and Abiotic Stress Responses

Abstract

It is well known that ethylene regulates a diverse set of developmental and stress-related processes in angiosperms, yet its roles in early-diverging embryophytes and algae are poorly understood. Recently, it was shown that ethylene functions as a hormone in the charophyte green alga Spirogyra pratensis Since land plants evolved from charophytes, this implies conservation of ethylene as a hormone in green plants for at least 450 million years. However, the physiological role of ethylene in charophyte algae has remained unknown. To gain insight into ethylene responses in Spirogyra, we used mRNA sequencing to measure changes in gene expression over time in Spirogyra filaments in response to an ethylene treatment. Our analyses show that at the transcriptional level, ethylene predominantly regulates three processes in Spirogyra: (1) modification of the cell wall matrix by expansins and xyloglucan endotransglucosylases/hydrolases, (2) down-regulation of chlorophyll biosynthesis and photosynthesis, and (3) activation of abiotic stress responses. We confirmed that the photosynthetic capacity and chlorophyll content were reduced by an ethylene treatment and that several abiotic stress conditions could stimulate cell elongation in an ethylene-dependent manner. We also found that the Spirogyra transcriptome harbors only 10 ethylene-responsive transcription factor (ERF) homologs, several of which are regulated by ethylene. These results provide an initial understanding of the hormonal responses induced by ethylene in Spirogyra and help to reconstruct the role of ethylene in ancestral charophytes prior to the origin of land plants.

Figure 1.

The effect of 24-h ethylene exposure (10 ppm) on the average cell length (µm) of S. pratensis. A, Cell length distribution was obtained by measuring 200 to 300 individual cells for each treatment. All boxes bound the 25th and 75th percentiles, whiskers show the outermost data points <1.5 times the interquartile range beyond the box. Blue diamonds indicate the mean; horizontal lines indicate the median; white circles indicate outliers. Significant differences (P < 0.05) are indicated by letters above the bars. B, Representative microscopic image of Spirogyra filaments showing a subtle cell length increase after a 24-h ethylene treatment. Arrows indicate the ends of individual cells in the filaments. Scale bar is 50 µm.

Figure 2.

Volcano plots showing the relation between the FDR and the FC for all transcripts. The transcripts that are differentially expressed (the P value of the FDR < 0.05 and the FC > 4) are indicated in red for the different time points: A, 3-h control versus 3-h ethylene; B, 6-h control versus 6-h ethylene; C, 12-h control versus 12-h ethylene; and D, 24-h control versus 24-h ethylene. The differentially expressed transcripts for each time point are listed in Supplemental File S1.

Figure 3.

Overview of the differential expression analysis statistics (with a FDR value of P < 0.05 and an expression level change of at least 4-fold). A, Venn diagram showing the number of differentially expressed genes that are regulated by ethylene over time (by comparing the ethylene versus control samples for each time point). The Venn diagram was created online using Venny (http://bioinfogp.cnb.csic.es/tools/venny/). B, Number of transcripts that are differentially regulated by ethylene for each time point (ethylene-treated compared to untreated samples for each time point). Light gray bars are up-regulated genes and dark gray bars are down-regulated genes.

Figure 4.

Expression profiles of S. pratensis genes that show significant expression differences after 3 h of treatment. Relative normalized gene expression (TMM-FPKM) profiles are shown for a PROTEIN KINASE SUPERFAMILY homolog (At3g19300; comp14026_c4) (A), the CHLOROPLAST MEMBRANE TRANSLOCON TIC214 (or YCF1) homolog (AtCg01130; comp11735_c0) (B), a basic helix-loop-helix DNA binding superfamily protein homolog of TT8 (At4g09820; comp6246_c0) (C), granulin repeat Cys protease homolog (RESPONSIVE TO DEHYDRATION21B [RD21B]; At5g43060; comp38750_c0) (D), LATE EMBRYOGENESIS ABUNDANT (LEA) related homolog (At3g19430; comp14490_c0) (E), 20S PROTEASOME ALPHA SUBUNIT C1 homolog (PAC1; At3g22110; comp54358_c0) (F), TRANSDUCING/WD40 REPEAT PROTEIN homolog (WD40; At5g15550; comp13964_c1) (G), and protein of unknown function homolog (At1g47980; comp11757_c0) (H). Values represent the average of three biological replicates with sd of the mean. Significant differences between treatments (P < 0.05) are indicated with an asterisk.

Figure 5.

GO enrichment analysis of biological processes regulated by ethylene (24 h untreated versus 24 h ethylene treated) on a subset of genes that are up-regulated (A) and a subset of genes that are down-regulated (B) by ethylene. To identify similar GO terms among the enriched terms, this set of GO terms was categorized using semantic clustering (REVIGO). Each ball represents a cluster of GO terms related to a similar process, and the size of the ball represents the number of GO terms grouped in that cluster. The color of the balls indicates the P value of the GO enrichment analysis; red indicates the highest P value and blue the lowest (least likely to occur by chance). The cutoff P value for the GO enrichment analysis was set to 0.01. The background used for the GO enrichment analysis was all the annotated Spirogyra transcripts of the assembly. Clusters of GO terms related to abiotic stress are indicated by the green plane.

Figure 6.

Ethylene-regulated cell wall modifying enzymes in Spirogyra. Relative normalized gene expression (TMM-FPKM) profiles are shown for EXPANSIN A2 homolog (EXPA2; At5g39270; comp46631_c0; A), EXPANSIN A8 homolog (EXPA8; At2g40610; comp37004_c0; B), EXPANSIN A12 homolog (EXPA12; At3g15370; comp14442_c0; C), EXPANSIN A14 homolog (EXPA14; At5g56320; comp13155_c0; D), EXPANSIN A18 homolog (EXPA18; At1g62980; comp42456_c0; E), EXPANSIN A22 homolog (EXPA22; At5g39270; comp46631_c0; F), EXPANSIN B3 homolog (EXPB3; At4g28250; comp10991_c1; G), XTH1 (At4g13080; comp26605_c0; H), XTH3 (At3g25050; comp14616_c0; I), XTH6 (At5g65730; comp46154_c0; J), XTH15 (At4g14130; comp6908_c0; K), XTH27 (At2g01850; comp6450_c0; L), XTH30 (At1g32170; comp6861_c0; M), XYLOGLUCAN XYLOSYLTRANSFERASE5 homolog (XXT5; At1g74380; comp6335_c0; N), CELLULOSE SYNTHASE8 homolog (CESA8; At4g18780; comp14512_c9; O), and CELLULOSE SYNTHASE-LIKE C4 homolog (CSLC4; At3g28180; comp51653_c0; P). Values represent the average of three biological replicates with sd of the mean. Significant differences between treatments (P < 0.05) are indicated with an asterisk.

Figure 7.

Ethylene reduces chlorophyll content (mg⋅gFW⁻¹; A) and net photosynthetic capacity (CO₂ exchange rate [CER]; µmol⋅h⁻¹⋅gFW⁻¹; B) after 10 d of treatment with 10 ppm ethylene gas. This ethylene effect was reduced by a pretreatment of 10 ppm 1-MCP. Bars represent the average of three biological replicates with sd of the mean. The values of individual replicates are superimposed (white circles) on the bars. Significant differences (P < 0.05) are indicated by letters above the bars.

Figure 8.

Effect of abiotic stress on cell length of Spirogyra after 10 d. A, Different stress treatments (WC medium at low pH 4.4), osmolarity/nutrient (deionized H₂O), constant light (24 h), and salinity (WC medium with 50 mm NaCl) significantly increased the average cell length compared to the no-stress control (WC medium). This cell elongation response was completely inhibited when treated with 1-MCP (10 ppm; gray boxes). Heat stress (25°C) and low light conditions (10 µmol⋅m⁻²⋅s⁻¹) do not significantly increase cell length. The two positive controls, ACC (500 µm in WC medium) and ethylene (10 ppm) treatment, also induced the cell elongation response. All boxes bound the 25th and 75th percentiles, whiskers show the outermost data points <1.5 times the interquartile range beyond the box. Blue diamonds indicate the mean; horizontal lines indicate the median; white circles indicate outliers. Significant differences (P < 0.05) are indicated by letters above the bars. B, Microscopic images of filaments after 10 d of stress with deionized H₂O compared to the no-stress control (WC medium). The 1-MCP treatment (10 ppm) can inhibit the stress-induced elongation response. Cell lengths were quantified in A. Arrows indicate the ends of individual cells in the filaments. The scale bar is 50 µm.