Transcriptomic sequencing and expression verification of identified genes modulating the alkali stress tolerance and endogenous photosynthetic activities of industrial hemp plant

Abstract

Hemp (Cannabis sativa L.) has a long cultivation history around the world. In northeast part of China, the alkaline soil geology severely reduces crop production. In this study, we tried to evaluate the impacts of alkali-induced stress on the photosynthetic status and physiological indices of hemp plants. The microscopic evaluation of endogenous ultrastructure clearly demonstrated significant oxidative damage to the structure of the photosynthetic tissues associated with the membrane, resulting from an increase in the levels of MGDG and DGDG. The deformed photosynthetic apparatus induced by alkali-stress significantly inhibited the biosynthesis process of photosynthetic pigments, causing 49.25%, 52.72%, 65.31%, and 28.13% loss in total Chl, Chl a, Chl b, and carotenoids, respectively. Meanwhile, the reduction in chlorophyll fluorescence parameters (Pn (74.62%), Gs (39.69%), and Tr (83.77%)) along with the obviously increased MDA (28.57%) and H2O2 (35.18%) content exhibited that the inhibitory effect of alkali-stress not only decreased the photosynthetic efficiency by intercepting the nutrient supply but also generated excessive ROS, resulting in oxidative stress. Transcriptomic analysis (RNA-sequencing) revealed the considerably enriched GO terms as well as KEGG pathways that exposed the significant DEGs. The qPCR expression evaluation of down-regulated chlorophyll biosynthesis-related major genes (GOGAT (LOC115699366) and HEMA (LOC133032634)) and photosystem-related major genes (PSB (LOC115701338) and HCF (LOC115707994)) exhibited important molecular evidence for modulating the photosynthesis activity of hemp plant under devastating mechanism of alkali-stress. However, the transcript patterns of photorespiration-related genes (GOX (LOC115697365) and GDC (LOC115707082)) showed a slower decreasing trend at late stress stage (at 24 ~ 48 h), and the transcription of SGAT gene (LOC115699360) was even enhanced by stress treatment at 48 h, probably in an attempt to adjust cellular carbon balance and elevate the antioxidant properties induced by alkali-stress.

Fig 1. A pictorial view of hemp seedling and leaves grown under normal condition (CK, control without stress) and alkali-stress (NaHCO₃ (250 mmol·L⁻¹)) during three different time intervals (h, hours), respectively.

Fig 2. Microscopic observation of photosynthesis related endogenous ultrastructure in leaves of hemp seedling grown under control (CK, control without stress) and alkali stress (NaHCO₃ (250 mmol·L⁻¹)) treatment at 48 h.

Chl, chloroplast; T, thylakoid; GL, granal lamellae; SG, starch granule.

Fig 3. Effect of alkali-stress on photosynthetic pigments of hemp seedlings compared to control treatment.

(A) Total chlorophyll content, (B) Chlorophyll a content, (C) Chlorophyll b content, (D) Carotenoid content. The statistical error bars indicate mean+SDs and dissimilar letters are denoting the significant change from each other (p < 0.05). Control (CK, without stress treatment).

Fig 4. Effect of alkali-stress to chlorophyll fluorescence parameters compared to control treatment.

(A) Net photosynthetic rate, (B) Stomatal conductance, (C) Transpiration rate, (D) Intercellular CO₂ concentration. The statistical error bars indicate mean+SDs and dissimilar letters are denoting the significant change from each other (p < 0.05).

Fig 5. Effect of alkali-stress on physiological indices of antioxidant system compared to control treatment.

(A) Malondialdehyde (MDA) content, (B) Hydrogen peroxide (H₂O₂) content, (C) Glutathione content, (D) Peroxidase (POD) activity. The statistical error bars indicate mean+SDs and dissimilar letters are denoting the significant change from each other (p < 0.05).

Fig 6. Analysis of categorized DEGs related to hemp leaves under comparative groups of alkali-stress and control treatments.

(A) Venn diagram of DEGs in comparative treatment groups at different time intervals, (B) Volcano plot of DEGs between alkali-stress and control at 6 h, (C) at 24 h, (D) at 48 h, (E) Hierarchical clustering analysis of DEGs, (F) Pathway information of clustered gene involved in the T48 group of alkali-stress.

Fig 7. A schematic overview of genetic regulatory mechanisms against effect of alkali-stress on hemp seedling leaves.

(A) Chlorophyll metabolism, (B) Photorespiration metabolism. The expression profiles of putative DEGs contributed in the pathway are shown by heat map, respectively.

Fig 8. qRT-PCR analysis of identified hub DEGs.

The expression verification of 13 DEGs of photosynthesis linked chlorophyll synthesis and photorespiratory metabolism. Statistical letters exhibit the significant differences among expression levels in comparative groups of control (CK) and alkali-stress (NaHCO₃ (250 mmol·L⁻¹)) treatments at different time intervals, respectively.