Decoding the functionality of plant transcription factors.

Transcription factors (TFs) intricately govern cellular processes and responses to external stimuli by modulating gene expression. TFs help plants to balance the trade-off between stress tolerance and growth, thus ensuring their long-term survival in challenging environments. Understanding the factors and mechanisms that define the functionality of plant TFs is of paramount importance for unravelling the intricate regulatory networks governing development, growth, and responses to environmental stimuli in plants. This review provides a comprehensive understanding of these factors and mechanisms defining the activity of TFs. Understanding the dynamic nature of TFs has practical implications for modern molecular breeding programmes, as it provides insights into how to manipulate gene expression to optimize desired traits in crops. Moreover, recent studies also report the functional duality of TFs, highlighting their ability to switch between activation and repression modes; this represents an important mechanism for attuning gene expression. Here we discuss what the possible reasons for the dual nature of TFs are and how this duality instructs the cell fate decision during development, and fine-tunes stress responses in plants, enabling them to adapt to various environmental challenges.

Effect of ACGT motif in spatiotemporal regulation of AtAVT6D, which improves tolerance to osmotic stress and nitrogen-starvation.

KEY MESSAGE: Plasma membrane-localized AtAVT6D importing aspartic acid can be targeted to develop plants with enhanced osmotic and nitrogen-starvation tolerance. AtAVT6D promoter can be exploited as a stress-inducible promoter for genetic improvements to raise stress-resilient crops. The AtAVT6 family of amino acid transporters in Arabidopsis thaliana has been predicted to export amino acids like aspartate and glutamate. However, the functional characterization of these amino acid transporters in plants remains unexplored. The present study investigates the expression patterns of AtAVT6 genes in different tissues and under various abiotic stress conditions using quantitative Real-time PCR. The expression analysis demonstrated that the member AtAVT6D was significantly induced in response to phytohormone ABA and stresses like osmotic and drought. The tissue-specific expression analysis showed that AtAVT6D was strongly expressed in the siliques. Taking together these results, we can speculate that AtAVT6D might play a vital role in silique development and abiotic stress tolerance. Further, subcellular localization study showed AtAVT6D was localized to the plasma membrane. The heterologous expression of AtAVT6D in yeast cells conferred significant tolerance to nitrogen-deficient and osmotic stress conditions. The Xenopus oocyte studies revealed that AtAVT6D is involved in the uptake of Aspartic acid. While overexpression of AtAVT6D resulted in smaller siliques in Arabidopsis thaliana. Additionally, transient expression studies were performed with the full-length AtAVT6D promoter and its deletion constructs to study the effect of ACGT-N24-ACGT motifs on the reporter gene expression in response to abiotic stresses and ABA treatment. The fluorometric GUS analyses revealed that the promoter deletion construct-2 (Pro.C2) possessing a single copy of ACGT-N24-ACGT motif directed the strongest GUS expression under all the abiotic conditions tested. These results suggest that Pro.C2 can be used as a stress-inducible promoter to drive a significant transgene expression.

Promoter profiling of Arabidopsis amino acid transporters: clues for improving crops.

KEY MESSAGE: The review describes the importance of amino acid transporters in plant growth, development, stress tolerance, and productivity. The promoter analysis provides valuable insights into their functionality leading to agricultural benefits. Arabidopsis thaliana genome is speculated to possess more than 100 amino acid transporter genes. This large number suggests the functional significance of amino acid transporters in plant growth and development. The current article summarizes the substrate specificity, cellular localization, tissue-specific expression, and expression of the amino acid transporter genes in response to environmental cues. However, till date functionality of a majority of amino acid transporter genes in plant development and stress tolerance is unexplored. Considering, that gene expression is mainly regulated by the regulatory motifs localized in their promoter regions at the transcriptional levels. The promoter regions ( ~ 1-kbp) of these amino acid transporter genes were analysed for the presence of cis-regulatory motifs responsive to developmental and external cues. This analysis can help predict the functionality of known and unexplored amino acid transporters in different tissues, organs, and various growth and development stages and responses to external stimuli. Furthermore, based on the promoter analysis and utilizing the microarray expression data we have attempted to identify plausible candidates (listed below) that might be targeted for agricultural benefits.

Primer Designing for Amplifying an AT-Rich Promoter from Arabidopsis thaliana.

The aim of the present study is to optimize the PCR conditions required to amplify the promoter sequence of an amino acid transporter having an AT-rich base composition with a high number of tandem repeats. The present study also covers the key parameters that need to be kept in mind while designing primers. Results show that successful can be achieved by performing a 2-step PCR reaction at a lower extension temperature of 65 ̊C for an increased extension period of 1.5 min/kb, with MgCl2 concentration ranging from 2.5 to 3.0mM. The results also suggest that the DNA concentration of around 25-30 ng/µl was essential to achieve this amplification.

Genome wide analysis of W-box element in Arabidopsis thaliana reveals TGAC motif with genes down regulated by heat and salinity.

To design, synthetic promoters leading to stress-specific induction of a transgene, the study of cis-regulatory elements is of great importance. Cis-regulatory elements play a major role in regulating the gene expression spatially and temporally at the transcriptional level. The present work focuses on one of the important cis-regulatory element, W-box having TGAC as a core motif which serves as a binding site for the members of the WRKY transcription factor family. In the present study, we have analyzed the occurrence frequency of TGAC core motifs for varying spacer lengths (ranging from 0 to 30 base pairs) across the Arabidopsis thaliana genome in order to determine the biological and functional significance of these conserved sequences. Further, the available microarray data was used to determine the role of TGAC motif in abiotic stresses namely salinity, osmolarity and heat. It was observed that TGAC motifs with spacer sequences like TGACCCATTTTGAC and TGACCCATGAATTTTGAC had a significant deviation in frequency and were thought to be favored for transcriptional bindings. The microarray data analysis revealed the involvement of TGAC motif mainly with genes down-regulated under abiotic stress conditions. These results were further confirmed by the transient expression studies with promoter-reporter cassettes carrying TGAC and TGAC-ACGT variant motifs with spacer lengths of 5 and 10.

Optimization of PCR conditions for amplifying an AT-rich amino acid transporter promoter sequence with high number of tandem repeats from Arabidopsis thaliana.

OBJECTIVE: The aim of the present study is to optimize the PCR conditions required to amplify the promoter sequence of an amino acid transporter having an AT-rich base composition with a high number of tandem repeats. RESULT: Results show that successful amplification can be achieved by performing a 2-step PCR at a lower extension temperature of 65 °C for an increased extension period of 1.5 min/kb, with MgCl2 concentration ranging from 2.5 to 3.0 mM. The results also suggest that the DNA concentration of about 25-30 ng/µl was essential to achieve this amplification.