Physiological and comparative transcriptome analysis of leaf response and physiological adaption to saline alkali stress across pH values in alfalfa (Medicago sativa)

Soil salinization is a critical factor limiting growth and causing physiological dysfunction in plants. The damage from alkaline salt in most plants is significantly greater than that from neutral salt. However, there is still a lack of research on the action mechanism by which saline alkali stress on plants under the same salt concentration across different pH values. The present study examined the effects of different pH values (7.0, 8.0, 9.0, and 10.0) under the same salt concentration (200 mmolL-1) on photosynthetic function, photoprotective mechanism, nitrogen metabolism, and osmotic regulation in alfalfa (Medicago sativa) leaves, including a transcriptomic analysis of changes in gene expression related to the above metabolic processes. The results showed that low pH saline alkali stress (pH 7.0 and 8.0) promoted chlorophyll synthesis in alfalfa leaves, and non-photochemical quenching (NPQ) and cyclic electron transfer (CEF) were promoted. There was no significant effect on plant growth or photochemical activity. The soluble sugar, proline, and soluble protein contents did not change significantly, and there was no obvious oxidative damage in alfalfa leaves. However, when pH increased to 9.0 and 10.0, KEGG enrichment analysis showed that photosynthesis (map00195) and nitrogen metabolism (map00910) were significantly enriched (P < 0.05), and PSII antenna protein coding genes were down-regulated under pH 9.0 and 10.0 treatments. The activities of PSII and PSI were decreased under high pH saline alkali stress, and the expression levels of the photosynthetic electron transporter-related genes PetA, PetB, petE, and petF were also significantly down-regulated. PSII was more sensitive to high pH saline alkali stress than PSI, and the PSII receptor side was more sensitive to high pH saline alkali stress than the PSII donor side. The activities of the oxygen-evolving complex (OEC) and PSI were significantly damaged only at pH 10.0. The activities of nitrate reductase (NR) and nitrite reductase (NiR), the expression levels of their genes, and the content of soluble protein were also decreased under pH 9.0 and 10.0 treatments. The inhibition of plant growth and oxidative damage to alfalfa leaves caused by high pH saline alkali stress were mainly related to the inhibition of photosynthesis (light energy absorption, electron transfer) and nitrogen metabolism (NO3- reduction). Under high pH saline alkali stress (pH 10.0), the photoprotection mechanisms such as CEF and NPQ were inhibited, which was also one of the important reasons for photoinhibition in alfalfa leaves. The accumulation of osmotic adjustment substances, such as soluble sugar and proline, was an important mechanism by which alfalfa physiologically adapted to high pH alkaline salt stress.