Molecular study of drought response in the Mediterranean conifer Pinus pinaster Ait.: Differential transcriptomic profiling reveals constitutive water deficit-independent drought tolerance mechanisms

Abstract

Adaptation of long-living forest trees to respond to environmental changes is essential to secure their performance under adverse conditions. Water deficit is one of the most significant stress factors determining tree growth and survival. Maritime pine (Pinus pinaster Ait.), the main source of softwood in southwestern Europe, is subjected to recurrent drought periods which, according to climate change predictions for the years to come, will progressively increase in the Mediterranean region. The mechanisms regulating pine adaptive responses to environment are still largely unknown. The aim of this work was to go a step further in understanding the molecular mechanisms underlying maritime pine response to water stress and drought tolerance at the whole plant level. A global transcriptomic profiling of roots, stems, and needles was conducted to analyze the performance of siblings showing contrasted responses to water deficit from an ad hoc designed full-sib family. Although P. pinaster is considered a recalcitrant species for vegetative propagation in adult phase, the analysis was conducted using vegetatively propagated trees exposed to two treatments: well-watered and moderate water stress. The comparative analyses led us to identify organ-specific genes, constitutively expressed as well as differentially expressed when comparing control versus water stress conditions, in drought-sensitive and drought-tolerant genotypes. Different response strategies can point out, with tolerant individuals being pre-adapted for coping with drought by constitutively expressing stress-related genes that are detected only in latter stages on sensitive individuals subjected to drought.

Keywords: Mediterranean conifer; differential transcript profiles; pre‐adapted genotypes; response strategies; water stress.

FIGURE 1

Experimental design and data analysis. (a) Experimental design. Twelve cDNA libraries were constructed and sequenced using as template RNAs extracted from different organs (roots, stems, needles) from ramets of two sensitive genotypes (4 and 147) and two tolerant genotypes (132 and 144). These genotypes were vegetatively propagated and grown under well water (WW, pools 1, 2, 3, 7, 8, and 9) and water deficit conditions (WD, pools 4, 5, 6, 10, 11, and 12). (b) Pipeline used for data analysis. Schematic overview of the pipeline used to process raw sequence files to identify constitutive and differentially accumulated transcripts. The software and files used or generated in each step are indicated

FIGURE 2

Validation of sequencing data by qRT‐PCR. (a) Primer sequences for qRT‐PCR. (b) Relative quantification (Rq) by qRT‐PCR (black and gray bars) and RNA‐seq normalized expression values (hatched and dotted bars) of five DEGs identified: alpha‐dioxygenase 1 (ALPHA‐DOX1), disease resistance protein at5g63020 (DRP), chaperone protein dnaj chloroplastic (DNAJ), dehydration‐responsive protein rd22 (RD22), and glutathione S‐transferase (GST). T: tolerant genotypes (132, 144); S: sensitive genotypes (4, 147)

FIGURE 3

Gene expression analysis of Pinus pinaster genotypes under different water regimes. (a) Venn diagrams of 6.215 differentially expressed genes at the organ level. WW‐S: well‐watered sensitive plants. WD‐S: water deficit sensitive plants. WW‐T: well‐watered tolerant plants. WD‐T: water deficit tolerant plants. (b). Single enrichment analysis of differentially expressed genes in sensitive and tolerant genotypes, classified according to each GO terms. Red and green bars represent the percentage of GO biological process terms with upregulated and downregulated genes showing significant enrichment (p < .05), respectively

FIGURE 4

Drought‐responsive genes out of the 30 highest expressed genes in roots, stems, and needles of sensitive and tolerant genotypes. Differentially and constitutively expressed genes have been included. The colors indicate the different functionalities of the genes: structural and metabolic protection (dark blue), transport (light blue), signaling (light yellow) transcription factor and other regulator proteins (orange), detoxification (violet), cell wall component (light green), photosynthesis (dark green), plant growth regulators (light red), and hormone response (dark red)