Screening of freezing stress-responsive genes related to photosynthesis in in vitro seedlings of Malus sieversii via RNA-seq

Abstract:【Objective】In order to explore the response of photosynthesis of Malus sieversii seedlings to freezing stress, we screened the responsive genes related to photosynthesis through transcriptomic analysis.【Methods】In vitro seedlings of a single plant line were used as materials. Under simulated freezing stress at -3 ℃, the changes in morphology, chlorophyll content, PSII maximum photochemical efficiency (Fv/Fm) and the performance index (PIabs) of photosystem II of the in vitro seedlings of M. sieversii exposed to the freezing stress for 0, 3, 6, 12 and 24 h (seedlings under 25 ℃ as control) were analyzed, and the RNA- seq was performed on the leaves of the seedlings under the above five treatments.【Results】Under the freezing stress, the edge and top of the leaves began to shrink at 12 h. Compared with CK, the content of chlorophyll a decreased significantly by 16.2% within 24 h, and had no significant difference among the other four time points. Compared with CK, the content of chlorophyll b increased significantly by 52% at 0 h, but did not change significantly at 3 h, while at 6 h, 12 h and 24 h, it decreased significantly by 12.3%, 28.6% and 36.7% respectively. The change trend of total chlorophyll content was completely consistent with chlorophyll b. Carotenoid content was significantly lower than that of the control, and decreased by 23.5%, 23.5%, 25.5%, 19.6% and 29.4% at 0, 3, 6, 12 and 24 h, respectively. Compared with CK, chlorophyll a / b ratio decreased significantly at 0 h and 3 h but increased significantly at 12 h and 24 h by 14.9% and 14.1%, respectively. Under simulated freezing stress, the fluorescence images of the leaves gradually changed from dark blue to light blue and to blue-green from CK to 24 h treatment, indicating that the photochemical efficiency gradually decreased. Fv / Fm decreased significantly by 7.9%, 12.3%, 12.8%, 19.9% and 29.4% at 0, 3, 6, 12 and 24 h, respectively. Compared with CK, the PIabs decreased significantly by 61.2%, 63.7%, 78.3%, 77.2% and 87.7% at 0, 3, 6, 12 and 24 h, respectively. RNA-seq analysis of samples of CK and 0, 3, 6 and 12 h, showed that total clean reads of each sample ranged from 42.48 M to 43.31 M, accounting for 95.33% to 97.35% of the total clean reads. The total clean bases for each sample averaged between 6.37 Gb- 6.50 Gb, of which the number of bases with a quality greater than 30 in total clean reads accounted for 90.97%-91.67%, and the total mapping was between 86.75%-87.50%. Through KEGG enrichment analysis with a critical Q-value of ≤ 0.05, 58 genes related to photosynthesis were screened out, including 18 genes related to photosystem Ⅱ, mainly involved in oxygen evolution enhancer proteins Psb27, PsbQ, PsbY, etc. There were 12 genes involved in photosystem Ⅰ, including photosystem Ⅰ subunits PsaG, PsaN and PsaK genes. There were 13 genes related to photosynthetic electron transfer chain, including the genes of photosystem Ⅰ receptor- side ferredoxin petE, petH and petF. Three ATP synthase- related genes were atpF and atpG genes, and 12 cytochrome b6f- related genes (mainly petC genes). The expression of PsbQ (Msi_13A005910) and PsbY (BGI_novel_G011171) was significantly up-regulated by freezing stress.【Conclusion】Compared with CK, Fv/Fm and PIabs of the in vitro seedlings of M. sieversii were significantly decreased under -3 ℃. Leaves began to shrink at 12 h of freezing exposure, and chlorophylls were decreased and chlorophyll a/b ratio increased at 24 h of treatment. 58 significantly differentially expressed genes related to photosynthetic regulation were screened by RNA-seq analysis, and PsbQ and PsbY were found to be significantly up-regulated. The results provide a reference for indepth study on the molecular mechanism of the response of M. sieversii to freezing stress.