Ticarcillin degradation product thiophene acetic acid is a novel auxin analog that promotes organogenesis in tomato

Abstract

Efficient regeneration of transgenic plants from explants after transformation is one of the crucial steps in developing genetically modified plants with desirable traits. Identification of novel plant growth regulators and developmental regulators will assist to enhance organogenesis in culture. In this study, we observed enhanced shoot regeneration from tomato cotyledon explants in culture media containing timentin, an antibiotic frequently used to prevent Agrobacterium overgrowth after transformation. Comparative transcriptome analysis of explants grown in the presence and absence of timentin revealed several genes previously reported to play important roles in plant growth and development, including Auxin Response Factors (ARFs), GRF Interacting Factors (GIFs), Flowering Locus T (SP5G), Small auxin up-regulated RNAs (SAUR) etc. Some of the differentially expressed genes were validated by quantitative real-time PCR. We showed that ticarcillin, the main component of timentin, degrades into thiophene acetic acid (TAA) over time. TAA was detected in plant tissue grown in media containing timentin. Our results showed that TAA is indeed a plant growth regulator that promotes root organogenesis from tomato cotyledons in a manner similar to the well-known auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). In combination with the cytokinin 6-benzylaminopurine (BAP), TAA was shown to promote shoot organogenesis from tomato cotyledon in a concentration-dependent manner. To the best of our knowledge, the present study reports for the first time demonstrating the function of TAA as a growth regulator in a plant species. Our work will pave the way for future studies involving different combinations of TAA with other plant hormones which may play an important role in in vitro organogenesis of recalcitrant species. Moreover, the differentially expressed genes and long noncoding RNAs identified in our transcriptome studies may serve as contender genes for studying molecular mechanisms of shoot organogenesis.

Advancing Antibiotic Residue Analysis: LC-MS/MS Methodology for Ticarcillin Degradation Products in Tomato Leaves

“…The structural characteristics of β-lactam antibiotics are comparable to those of auxins, and the products of their decomposition may behave similarly to auxins in the culture media [31,32]. In our previous study, we postulated that Thiophene-3-Acetic acid (T3AA) may be a possible breakdown product of ticarcillin, based on the structure of ticarcillin (Figure 1 [24]). TAA, an organosulfur chemical, has two isomers: Thiophene-2-Acetic acid (T2AA) and Thiophene-3-Acetic acid (T3AA).…”

“…In agrobacterium-mediated plant transformation research, the antibiotic timentin is frequently employed to inhibit the growth of Agrobacterium tumefaciens following co-cultivation [23]. Timentin is a formulation that provides stability to the β-lactam ring in the broad-spectrum semi-synthetic β-lactam antibiotic ticarcillin by combining it with clavulanic acid, a β-Lactamase competitive inhibitor [24,25]. Timentin has been demonstrated to have a favorable impact on shoot regeneration in several plant species, including London plane trees, tomato, and tobacco, along with a few other β-lactam ring-containing antibiotics like penicillin G and carbenicillin [23,[26][27][28].…”

“…Its molecular formula is C 6 H 6 O 2 S. media [31,32]. In our previous study, we postulated that Thiophene-3-Acetic acid (T3AA) may be a possible breakdown product of ticarcillin, based on the structure of ticarcillin (Figure 1 [24]). TAA, an organosulfur chemical, has two isomers: Thiophene-2-Acetic acid (T2AA) and Thiophene-3-Acetic acid (T3AA).…”

“…The regulation of organogenesis through the application of exogenous substances, such as auxin analogs, can have a profound impact on tomato in vitro regeneration [33,34]. Our prior research has shown that the presence of timentin increases the rate of shoot regeneration in tomato cotyledon explants [24]. To verify the degradation of timentin into Thiophene-2-Acetic acid and Thiophene-3-Acetic acid, we regenerated tomato cotyledon explants in culture media designed for shoot regeneration in the presence and absence of timentin.…”

“…As far as we are aware, there is currently no reported LC-MS/MS methodology available for detecting ticarcillin and its metabolites in tomato leaves. Given that TAA has been recently demonstrated as an auxin analog capable of regulating organogenesis in tomato [24], a standardized method for the detection and quantification of TAA isomers in plant tissues is essential in deciphering mechanisms of organogenesis mediated by TAA. In addition, standardization of such a protocol would enable the detection of ticarcillin degradation products in other plant species.…”