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Abstract <p>Alkaline stress represents a major abiotic factor that adversely affects, whereas proline has been widely reported to alleviate its detrimental effects. To investigate the molecular mechanisms underlying proline-mediated mitigation of alkaline stress, one-year-old <i>Cinnamomum bodinieri</i> Levl. seedlings were subjected to alkaline stress and subsequently foliar-sprayed with either 0 μmol/L (CK) or 500 μmol/L proline (PRO). Transcriptome sequencing was performed on the DNBSEQ-T7 platform to identify differentially expressed genes (DEGs). The results indicated that exogenous proline significantly increased endogenous proline content and enhanced peroxidase (POD) and catalase (CAT) activities, while reducing malondialdehyde (MDA) accumulation in <i>C. bodinieri</i> seedlings. A total of 353 DEGs were identified, comprising 170 up-regulated and 183 down-regulated genes. KEGG pathway analysis revealed that upregulated DEGs were mainly enriched in pathways related to starch and sucrose metabolism (ko00500), alanine, aspartate, and glutamate metabolism (ko00250), carbon fixation in photosynthetic organisms (ko00710), calcium signaling (ko04020), arginine biosynthesis (ko00220), tropane, piperidine and pyridine alkaloid biosynthesis (ko00960), and plant MAPK signaling (ko04016). Conversely, downregulated DEGs were enriched in pathways related to flavone and flavonol biosynthesis (ko00944), Drug metabolism-other enzymes (ko00983), MAPK signaling (ko04010), α-linolenic acid metabolism (ko00592), NF-κB signalling (ko04064), and Toll and Imd signaling (ko04624). Notably, no significant differences were observed in the expression levels of proline metabolism-related genes such as <i>CbP</i>5<i>CS</i>, <i>CbOAT</i>, <i>CbP</i>5<i>CR</i>, and <i>CbProT</i> between the PRO and CK treatments. However, <i>CbProDH</i> expression was significantly lower than that observed in CK conditions while <i>CbAAT</i> exhibited a significantly higher expression level compared to CK. Six DEGs were randomly selected for validation using quantitative real-time PCR (qRT-PCR), and the results were largely consistent with the RNA-Seq data, confirming the reliability of the sequencing results. In conclusion, exogenous proline enhanced the tolerance of <i>C. bodinieri</i> seedlings to alkaline stress by increasing endogenous proline levels, sustaining photosynthetic carbon assimilation, and promoting sucrose synthesis. Proline treatment altered the proline metabolic pathway, primarily relying on the aspartate aminotransferase (AAT) route to maintain continuous proline biosynthesis and normal tricarboxylic acid (TCA) cycle activity. These findings provide new insights into the molecular mechanism of proline metabolism relieving the tolerance of <i>C. bodinieri</i> to alkaline stress.</p>

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Transcriptome Analysis provides new Insights into the Molecular Mechanisms of Enhancing Alkali-Tolerance of Cinnamomum bodinieri by Exogenous Proline

  • 期刊论文

  • 发表日期：

    2026年5月7日

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  • L. Y. Gu,
  • Z. X. Liu,
  • Y. Q. He,
  • Y. X. Cao,
  • R. Dai,
  • H. Z. Han,
  • L.H. Zhang

摘要

Abstract

Alkaline stress represents a major abiotic factor that adversely affects, whereas proline has been widely reported to alleviate its detrimental effects. To investigate the molecular mechanisms underlying proline-mediated mitigation of alkaline stress, one-year-old Cinnamomum bodinieri Levl. seedlings were subjected to alkaline stress and subsequently foliar-sprayed with either 0 μmol/L (CK) or 500 μmol/L proline (PRO). Transcriptome sequencing was performed on the DNBSEQ-T7 platform to identify differentially expressed genes (DEGs). The results indicated that exogenous proline significantly increased endogenous proline content and enhanced peroxidase (POD) and catalase (CAT) activities, while reducing malondialdehyde (MDA) accumulation in C. bodinieri seedlings. A total of 353 DEGs were identified, comprising 170 up-regulated and 183 down-regulated genes. KEGG pathway analysis revealed that upregulated DEGs were mainly enriched in pathways related to starch and sucrose metabolism (ko00500), alanine, aspartate, and glutamate metabolism (ko00250), carbon fixation in photosynthetic organisms (ko00710), calcium signaling (ko04020), arginine biosynthesis (ko00220), tropane, piperidine and pyridine alkaloid biosynthesis (ko00960), and plant MAPK signaling (ko04016). Conversely, downregulated DEGs were enriched in pathways related to flavone and flavonol biosynthesis (ko00944), Drug metabolism-other enzymes (ko00983), MAPK signaling (ko04010), α-linolenic acid metabolism (ko00592), NF-κB signalling (ko04064), and Toll and Imd signaling (ko04624). Notably, no significant differences were observed in the expression levels of proline metabolism-related genes such as CbP5CS, CbOAT, CbP5CR, and CbProT between the PRO and CK treatments. However, CbProDH expression was significantly lower than that observed in CK conditions while CbAAT exhibited a significantly higher expression level compared to CK. Six DEGs were randomly selected for validation using quantitative real-time PCR (qRT-PCR), and the results were largely consistent with the RNA-Seq data, confirming the reliability of the sequencing results. In conclusion, exogenous proline enhanced the tolerance of C. bodinieri seedlings to alkaline stress by increasing endogenous proline levels, sustaining photosynthetic carbon assimilation, and promoting sucrose synthesis. Proline treatment altered the proline metabolic pathway, primarily relying on the aspartate aminotransferase (AAT) route to maintain continuous proline biosynthesis and normal tricarboxylic acid (TCA) cycle activity. These findings provide new insights into the molecular mechanism of proline metabolism relieving the tolerance of C. bodinieri to alkaline stress.