Protein expression plasticity contributes to heat and drought tolerance of date palm

Climate change is increasing the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding the resistance and resilience of plants to climate change is, therefore, urgently needed. As date palm (Phoenix dactylifera) evolved adaptation mechanisms to a xeric environment and can tolerate large diurnal and seasonal temperature fluctuations, we studied the protein expression changes in leaves, volatile organic compound emissions, and photosynthesis in response to variable growth temperatures and soil water deprivation. Plants were grown under controlled environmental conditions of simulated Saudi Arabian summer and winter climates challenged with drought stress. We show that date palm is able to counteract the harsh conditions of the Arabian Peninsula by adjusting the abundances of proteins related to the photosynthetic machinery, abiotic stress and secondary metabolism. Under summer climate and water deprivation, these adjustments included efficient protein expression response mediated by heat shock proteins and the antioxidant system to counteract reactive oxygen species formation. Proteins related to secondary metabolism were downregulated, except for the P. dactylifera isoprene synthase (PdIspS), which was strongly upregulated in response to summer climate and drought. This study reports, for the first time, the identification and functional characterization of the gene encoding for PdIspS, allowing future analysis of isoprene functions in date palm under extreme environments. Overall, the current study shows that reprogramming of the leaf protein profiles confers the date palm heat- and drought tolerance. We conclude that the protein plasticity of date palm is an important mechanism of molecular adaptation to environmental fluctuations. Supplementary Information The online version contains supplementary material available at 10.1007/s00442-021-04907-w.

. Daily environmental conditions Figure S2. Effects of climate and drought on fresh weight and dry to fresh weight ratio of date palm leaves Figure S3. Comparison of consensus PdIspS protein sequence to functionally characterized plant isoprene synthases Figure S4. Consensus PdIspS CDS Figure S5. PdIspS protein sequence Table S1. VOC emitted from date palm leaves and log2 of fold changes between treatments Table S2. Proteomic data (see separate excel file)

Material and Methods MM1: Reconstruction of the PdIspS CDS sequence
To reconstruct the N-and C-termini of the partial PdIspS sequence XP_008779509.1, we identified close orthologs of this gene from other monocots from the NCBInr database (ASF20076.1 and XP_010923912.1 from Arundo donax and Elaeis guineensis, respectively) and searched genome sequence data for two P. dactylifera varieties (NCBI; Bioprojects PRJNA396270 and PRJNA322046) using terminal parts of these genes. Three contigs from the Khanizi cultivar (PEFZ01192174.1, PEFZ01199267.1, and PEFZ01103933.1) and two unplaced scaffolds from the Khalas cultivar (NW_008252718.1 and NW_008252718.1) resulting from this search were assembled in a single pseudo-scaffold. A complete CDS of the P. dactylifera putative IspS gene was reconstructed based on the RNA-seq reads aligned to this pseudoscaffold. Briefly, to identify samples in which XP_008779509.1 is expressed, RNA-seq data for the P. dactilyfera leaf tissue from the Bioprojects PRJEB18007 and PRJEB22923 were obtained from NCBI. All transcript models predicted for the Khalas genome assembly have been indexed and transcript abundance in the aforementioned RNA-seq libraries were quantified using Kallisto v0.44.0 (Bray et al. 2016) using default parameters. Libraries characterized by a high abundance of the XP_008779509.1 transcripts were selected for further analysis. RNA-seq reads were filtered, quality trimmed using Trimmomatic v.0.35 (Bolger et al. 2014), and mapped to the reference consisting of the P. dactilyfera genome assembly GCA_000413155.1 and the IspS pseudo-scaffold using STAR v2.5.2a (Dobin et al. 2013). The IspS pseudo-scaffold alignment region was extracted from the bam files using SAMtools (Li et al. 2009) and visualized and processed using Integrative Genomics Viewer v2.4.19 (Robinson et al. 2011). The IspS coding sequence (CDS) encoded by this pseudo-scaffold was reconstructed based on the consensus of RNA-seq sequences aligned to this region. The IspS intron-exon splice sites were defined manually based on the alignments. Nucleotide and amino acid CDS sequences of PdIspS are provided in the Supplementary Figs S3-5.

Material and Methods MM2: In vitro enzymatic activity assay of P. dactylifera IspS following expression in E. coli and protein purification
Date palm IspS activities were determined at 5mM of the substrate dimethylallyl diphosphate (DMADP) for isoprene biosynthesis or by 5 mM geranyl diphosphate (GDP) for monoterpenes.
Each sample contained 50 µL protein extract, corresponding to 169 µg in the empty vector (EV) control and 174 µg in PdIspS samples, respectively, as determined by the Bradford assay. The incubation time was 30 min, and the standard incubation temperature was 40°C. For the temperature dependence analysis of PdIspS, samples were incubated between 20 and 60° with 5°C steps and the respective data collected from EV were used for background corrections.
After incubation, the reaction was stopped by removal of the aqueous solution and rinsing the vial with 100 µL buffer solution. Finally, the headspace was injected by a self-made autosampler in the inlet of PTR-QMS using N2 as carrier gas (170 ml min -1 ) for VOC analysis.
Supplementary Table:   Table S1. VOC emitted from date palm leaves and log2 of fold changes between treatments.
No indicate compounds shown in Figure 2 (biplot of PCA). Increased and decreased emissions due to treatments are indicated by the intensity of red and blue colors, respectively. Significant differences between treatments at p <0.05 as calculated by non-parametric t-test are indicated in bold.