In this step, we identified the sequence of the NCED3 gene through the analysis of the plant genome. We selected a 2000bp region upstream of the ATG start codon as the NCED3 promoter sequence. Subsequently, we designed homologous recombination primers based on the SmaI-PstI restriction enzyme sites and plasmid fragments to amplify the NCED3 promoter. PCR reactions were performed, resulting in the generation of DNA fragments containing the NCED3 promoter with homologous arms.

In this step, the pDR5-RUBY plasmid was subjected to SmaI-PstI double digestion to obtain digested plasmid fragments. The NCED3 promoter was then ligated with the digested plasmid to create an expression vector harboring the NCED3 promoter. These plasmids were subsequently transformed into DH5α Escherichia coli and plated on LB agar supplemented with kanamycin for selection and cultivation. Single colonies were picked from the plates for PCR and agarose gel electrophoresis verification, followed by sequencing to ensure the correct construction of the plasmid.

In this step, validated single colonies were subjected to oscillation culture to amplify and extract the target vector plasmids. These target plasmids were then transformed into Agrobacterium GV3 101 in a competent state. The transformed Agrobacterium was plated on LB agar plates containing rifampicin and kanamycin for selection and cultivation. Single colonies were picked from the plates for PCR and agarose gel electrophoresis verification to confirm the successful transformation of the plasmid into Agrobacterium.

In this step, Agrobacterium cultures containing the target gene plasmids were oscillated, and the cells were centrifuged and resuspended to prepare a suspension. The suspension was introduced into tobacco leaves through injection. Both injected and non-injected leaves from the same tobacco plant were maintained to create conditions for drought stress and non-drought stress. Subsequently, time-lapse photography was conducted to observe whether leaf color changes occurred under drought conditions, thereby validating the effectiveness of the system.

1.Rapid Verification

In this step, the extracted target plasmids were sent to a company for stable transformation. The transformed Arabidopsis seeds with the warning system and wild-type Arabidopsis seeds were subjected to drought stress on 1/2MS agar plates. The growth status and leaf color of the seeds were observed to validate the system's effectiveness.

2.Formal Experiment

Transplanting seedlings into soil pots with consistent initial weights, a stable cultivation period of three weeks was allowed. Subsequently, a drought stress experiment was initiated, simulating natural drought conditions based on the duration of withholding water to emulate varying degrees of drought severity. Leaf color changes were observed and assessed during this drought stress experiment.

Through the analysis of the plant genome, we identified the sequence of the GLR2.9 gene. To improve the success rate of subsequent steps involving multi-fragment assembly, we selected the 1500bp sequence upstream of the ATG start codon as the GLR2.9 promoter. As we did not have the sequence of the Bx gene, we entrusted a company to synthesize it. Subsequently, we designed homologous recombination primers to amplify the GLR2.9 promoter and the Bx reporter gene sequences, using the linearized vector fragment after NcoI-PstI digestion. PCR reactions were conducted to obtain GLR2.9 promoter DNA fragments and Bx reporter gene fragments, both containing homologous arms.

We performed NcoI-PstI double digestion on the pCAMBIA1300-C-MCS-GFP plasmid to obtain a linearized vector fragment. Next, we carried out homologous recombination by ligating the GLR2.9 promoter DNA fragment, Bx reporter gene fragment, and the linearized vector fragment, resulting in the expression vector plasmid containing the GLR2.9-Bx reporter system. These plasmids were then transformed into DH5α Escherichia coli and spread on LB agar plates supplemented with kanamycin for selection. Single colonies were picked from the plates and subjected to PCR and agarose gel electrophoresis verification, followed by sequencing to ensure correct plasmid construction.

The verified single colonies were cultured with agitation, and the target vector plasmids were extracted. These target vector plasmids were subsequently transformed into GV3 101 Agrobacterium tumefaciens in a state of competence. The transformed Agrobacterium cultures were spread on LB agar plates containing rifampicin and kanamycin for selection. Finally, single colonies were selected from the plates and subjected to PCR and agarose gel electrophoresis verification to confirm the successful transformation of the plasmid into Agrobacterium.

The Agrobacterium cultures containing the target gene plasmids were agitated, and the cells were pelleted and resuspended to prepare a suspension. This suspension was injected into tobacco leaves using a syringe. On the same tobacco plant, both injected and non-injected leaves were present. To simulate biological stress, Pst DC3000 bacteria, commonly used in the laboratory for plant biotic stress studies, were used to treat the leaves, while the control group leaves were sprayed with water. Subsequently, time-lapse photography was conducted to observe any color changes in the leaves, confirming the system's effectiveness. Furthermore, based on the property of Bx emitting green fluorescence under blue light, treated leaf samples were collected for fluorescence microscopy to further validate the results.