Acetoacetyl-CoA thiolase (EC 2.3.1.9), also called thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA. This is the first enzymatic step in the biosynthesis of isoprenoids via mevalonate (MVA). In this work, thiolase II from alfalfa (MsAACT1) was identified and cloned. The enzymatic activity was experimentally demonstrated in planta and in heterologous systems. The condensation reaction by MsAACT1 was proved to be inhibited by CoA suggesting a negative feedback regulation of isoprenoid production. Real-time RT-PCR analysis indicated that MsAACT1 expression is highly increased in roots and leaves under cold and salinity stress. Treatment with mevastatin, a specific inhibitor of the MVA pathway, resulted in a decrease in squalene production, antioxidant activity, and the survival of stressed plants. As expected, the presence of mevastatin did not change chlorophyll and carotenoid levels, isoprenoids synthesized via the plastidial MVA-independent pathway. The addition of vitamin C suppressed the sensitive phenotype of plants challenged with mevastatin, suggesting a critical function of the MVA pathway in abiotic stress-inducible antioxidant defence. MsAACT1 over-expressing transgenic plants showed salinity tolerance comparable with empty vector transformed plants and enhanced production of squalene without altering the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity in salt-stress conditions. Thus, acetoacetyl-CoA thiolase is a regulatory enzyme in isoprenoid biosynthesis involved in abiotic stress adaptation.

Potato (Solanum tuberosum L.) tuberization is regulated by many signals, such as abscisic acid (ABA), sucrose and gibberellic acid (GA). ABA and sucrose are positive modulators, while GA is an inhibitor of the process. ABF (ABRE-binding factor) proteins are transcription factors involved in ABA and stress signaling. Previously, we reported that S. tuberosum StABF1 could mediate the ABA effects on tuberization. The aim of the present study was to evaluate the potential use of ABF genes to enhance tuberization and to determine the molecular mechanism involved. For this purpose, transgenic potato plants expressing the Arabidopsis ABF4 or ABF2 genes were generated, and their tuberization capacity and response to tuberization-related signals were analyzed in vitro. The results indicate that both ABF4 and ABF2 proteins positively regulate potato tuber induction; however, only ABF4 expression significantly increases the number and weight of the tubers obtained, without stunting growth. ABF4 and ABF2 transgenic plants exhibit ABA hypersensitivity during tuberization, accompanied by a GA-deficient phenotype. ABF4 expression triggers a significant rise in ABA levels in stolons under tuber-inducing conditions as compared with wild-type plants and a transcriptional deregulation of GA metabolism genes. Our results demonstrate that Arabidopsis ABF4 functions in potato ABA-GA signaling crosstalk during tuberization by regulating the expression of ABA- and GA-metabolism genes. ABF4 gene might be a potential tool to increase tuber production, since its heterologous expression in potato enhances tuber induction without affecting plant growth.

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Genetic evidence is provided for the role of potato PM H+-ATPases in tuberization, demonstrating that the isoform PHA1 promotes stolon elongation and tuber growth, and is involved in sugar–starch metabolism.