System-wide analysis of short-term response to high temperature in Pinus radiata

Abstract

Pinus radiata seedlings, the most widely planted pine species in the world, were exposed to temperatures within a range mimicking future scenarios based on current models of heat increase. The short-term heat response in P. radiata was studied in detail by exploring the metabolome, proteome and targeted transcriptome. The use of complementary mass spectrometry techniques, GC-MS and LC-Orbitrap-MS, together with novel bioinformatics tools allowed the reliable quantification of 2,075 metabolites and 901 protein groups. Integrative analyses of different functional levels and plant physiological status revealed a complex molecular interaction network of positive and negative correlations between proteins and metabolites involved in short-term heat response, including three main physiological functions as: 1) A hormone subnetwork, where fatty acids, flavonoids and hormones presented a key role; 2) An oxidoreductase subnetwork, including several dehydrogenase and peroxidase proteins; and 3) A heat shock protein subnetwork, with numerous proteins that contain a HSP20 domain, all of which were overexpressed at the transcriptional level. Integrated analysis pinpointed the basic mechanisms underlying the short-term physiological reaction of P. radiata during heat response. This approach was feasible in forest species and unmasked two novel candidate biomarkers of heat resistance, PHO1 and TRANSCRIPTION FACTOR APFI, and a MITOCHONDRIAL SMALL HEAT SHOCK PROTEIN, for use in future breeding programs.

Metabolome Integrated Analysis of High-Temperature Response in Pinus radiata

“…In-depth analysis of metabolite accumulation in relation to KEGG pathways ( Figure 2 and Supplementary Figure S1 ) allowed to confirm the high dynamism of the metabolome in relation to high-temperature response ( Escandón et al, 2017 ). Although two main trends in the KEGG pathways have been identified by heatmap-clustering ( Figure 2 and Supplementary Figure S1 ), each pathway seem to have a specific role in a particular moment of the stress, seemingly following an orchestrated succession, particularly, in the case of flavonoids related pathways.…”

“…Recovered plants (R) represent an intermediate exposure time (T3 recovered plants) because there was no significant difference (at the morpho-physiological level) between recovered plants exposed to the different heat exposures. This experimental design aimed to cover the entire stress sensing–response–adaption process, increasing the density of analysis at short-term (T1/2, T1, T2), longer exposures (T5), and recovered plants (R), complementing previous short-term response (C, T1, and T3) analysis ( Escandón et al, 2017 ).…”

“…The polar fraction of each sample was analyzed twice on an LC-Orbitrap-MS, first in positive ion mode and then in negative. A Dionex Ultimate 3000 (Thermo Fisher Scientific, United States) UHPLC was used and a LC-Orbitrap LTQ XL-MS system (controlled by Xcalibur version 2.2, Thermo Fisher Corporation) was run according to the procedure described in Escandón et al (2017) . The resolution and sensitivity of the Orbitrap were controlled by the injection of a mixed standard after the analysis of each batch, and the resolution was also checked with the aid of lock masses (phthalates).…”

“…The individual peaks were identified following different approaches; the first step was performed against an in-house library (>100 compounds) and manual annotation considering m/z and retention times. In the second step, masses were assigned using the KEGG, PubChem, METLIN, MassBank, HMDB, and Plantcyc databases as reported by Escandón et al (2017) with built-in MZmine plugins with a 5 ppm threshold and considering as “identified” beyond doubt those metabolites that were defined after the comparison to our standard compound library or by a matching of MS/ MS to the small number of plant compounds for which their MS/MS is available in public databases ( Meijón et al, 2016 ); and as “tentatively assigned” those with molecular ions with exact masses corresponding to identified metabolites in databases. Metabolite identification against our library was confirmed by retention time (RT), mass, isotopic pattern, and ring double bound parameters.…”

Hot drought reduces the effects of elevated CO2 on tree water‐use efficiency and carbon metabolism

“…It is noteworthy that e[CO 2 ] trees, which had a lower protein content at 25°C, exhibited a relatively greater increase in protein content with heat stress. This increase in soluble protein might be due to an upregulation of heat‐shock proteins (Aspinwall et al , 2019) to prevent failure of the photosynthetic apparatus (Escandón et al , 2017) or could be caused by N remobilization for Rubisco, which can explain up to 30% of changes in total protein (Warren & Adams, 2001). In summary, we found the stress response of the primary metabolome to be highly tissue specific and to be largely independent of growth [CO 2 ] in contrast to our third hypothesis.…”