Differential Morpho-Physiological and Transcriptomic Responses to Heat Stress in Two Blueberry Species

Abstract

Blueberries (Vaccinium spp.) are highly vulnerable to changing climatic conditions, especially increasing temperatures. To gain insight into mechanisms underpinning the response to heat stress, two blueberry species were subjected to heat stress for 6 and 9 h at 45 °C, and leaf samples were used to study the morpho-physiological and transcriptomic changes. As compared with Vaccinium corymbosum, Vaccinium darrowii exhibited thermal stress adaptation features such as small leaf size, parallel leaf orientation, waxy leaf coating, increased stomatal surface area, and stomatal closure. RNAseq analysis yielded ~135 million reads and identified 8305 differentially expressed genes (DEGs) during heat stress against the control samples. In V. corymbosum, 2861 and 4565 genes were differentially expressed at 6 and 9 h of heat stress, whereas in V. darrowii, 2516 and 3072 DEGs were differentially expressed at 6 and 9 h, respectively. Among the pathways, the protein processing in the endoplasmic reticulum (ER) was the highly enriched pathway in both the species: however, certain metabolic, fatty acid, photosynthesis-related, peroxisomal, and circadian rhythm pathways were enriched differently among the species. KEGG enrichment analysis of the DEGs revealed important biosynthesis and metabolic pathways crucial in response to heat stress. The GO terms enriched in both the species under heat stress were similar, but more DEGs were enriched for GO terms in V. darrowii than the V. corymbosum. Together, these results elucidate the differential response of morpho-physiological and molecular mechanisms used by both the blueberry species under heat stress, and help in understanding the complex mechanisms involved in heat stress tolerance.

Keywords: RNAseq; blueberry; differentially expressed genes; heat stress; pathway analysis.

Figure 1

Laser scanning confocal microscopy (LSCM) imaging of stomata internal organelles and cell surface features of two blueberry species. (A) Stomata internal organelle surface area at different heat stress imposition periods (0, 6, and 9-h heat stress): Maximum projection of z stack with staining shows the wide opened structure of stoma organelle surface area that exhibits an intense red signal in V. darrowii compared to V. corymbosum. (B) LSCM micrographs showing the surface structure of stomata cell opening (guard cells).

Figure 2

Venn diagram comparing the number of genes with statistically significant differential expression in response to heat stress at 6 and 9-h in V. darrowii and V. corymbosum species.

Figure 3

Protein processing in the endoplasmic reticulum pathway: (A) Map of alignment of V. corymbosum differentially expressed genes (DEGs) to Vitis vinifera genome at 6-h heat stress. (B) Map of alignment of V. corymbosum DEGs to V. vinifera genome at 9-h heat stress. The colored box represents DEGs, and their degree of expression is depicted by color variations, with red the most upregulated and green the most downregulated.

Figure 4

Protein processing in endoplasmic reticulum pathway: (A) Map of alignment of V. darrowii DEGs to V. vinifera genome at 6-h heat stress. (B) Map of alignment of V. darrowii DEGs to V. vinifera genome at 9-h heat stress. The colored box represents DEGs, and their degree of expression is depicted by color variations, with red the most upregulated (p < 0.05) and green the most downregulated (p < 0.05), white box represents no significant differences in gene expression.

Figure 5

DEGs enriched in the top 20 metabolic pathways. Highly enriched pathways among the down- and upregulated DEGs represented based on the counts. Counts indicate the number of DEGs annotated in each pathway, and the circle colour blue indicates minimal downregulated gene count and red color shows the maximum number of downregulated gene count. The circle size depicts upregulated gene counts in each pathway, and the position of the circle depicts the total number of DEGs. (A) DEGs in metabolic pathways of V. corymbosum at 6-h heat stress, (B) DEGs in metabolic pathways of V. corymbosum at 9-h heat stress, (C) DEGs in metabolic pathways of V. darrowii at 6-h heat stress, (D) DEGs in metabolic pathways of V. darrowii at 9-h heat stress.

Figure 6

Gene ontology enrichment analysis of DEGs from V. corymbosum. Highly enriched Gene Ontology (GO) terms among the DEGs from V. corymbosum under 6-h heat stress (A) and 9-h heat stress (B).

Figure 7

Gene ontology enrichment analysis of DEGs from V. darrowii. Highly enriched Gene Ontology (GO) terms among the DEGs from V. darrowii under 6-h heat stress (A) and 9-h heat stress (B).

Figure 8

RT-qPCR validation of DEGs: Gene expression patterns of randomly selected genes in both blueberry species (V. darrowii and V. corymbosum) at 6- and 9-h heat stress.

Figure 9

Schematic representation of differential morpho-physiological and molecular mechanisms involved in response to heat stress in the blueberry species.