Direct Detection of Transcription Factors in Cotyledons during Seedling Development Using Sensitive Silicon-Substrate Photonic Crystal Protein Arrays  

Abstract

Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/ NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts.

Elucidating and mining the Tulipa and Lilium transcriptomes

“…Transcription factors orchestrate several networks by controlling when and where certain genes will be expressed (Lee et al 2006 ) and, therefore, have been well studied and characterized in plants. However, even though they function as master regulators, transcription factors are often expressed at relatively low abundance (Jones et al 2015 ). This low level of expression makes transcription factors suitable markers to further assess the sequencing depth and coverage of our two generated transcriptomes.…”

Recent Advances in Biosensing With Photonic Crystal Surfaces: A Review

“…PCs have been extensively used to enhance the signal from small concentrations of biological materials [16, 21, 29, 30, 39, 43, 47, 71, 78]. As a way to both increase the quality factor of the resonator and to get additional signal enhancement, we have demonstrated coupling of a Fabry-Perot cavity with a photonic crystal resonator, as shown in Fig.…”

“…For several of the fabrication approaches, PC biosensors have been fabricated with costs that are compatible with single-use disposable applications in formats that include microtiter plates [8, 23, 27], microscope slides [12, 13, 28–30], and microfluidic devices [31–34]. A wide variety of biomolecular assay applications have been demonstrated in these platforms that include small molecule high throughput screening [27, 35, 36], protein-protein interaction detection [37], inhibition of protein-DNA interactions [38], small molecule aggregation [27], molecular diagnostics [16, 21, 39–43], and direct detection of virus particles [44–46] at physiologically relevant concentrations. One of the most important application areas for PC biosensors has been for cell-based assays.…”

“…PCEF has been applied to boost the signal-to-noise ratio for fluorescent dye molecules [65, 70–72], quantum dots [65, 67], and Raman scattering [73–75] for applications in biosensing and high efficiency lighting [76, 77]. Within the field of biosensors, PCEF has been used to reduce the limits of detection for gene expression microarrays [30, 71], detection of cancer biomarkers in serum [16, 21, 39], and low-concentration transcription factors expressed by plant cells [43]. Label-free PC biosensing and PCEF have been implemented on PC structures and detection instruments that are capable of supporting both sensing modalities for applications that include DNA microarrays [29, 78] and cell-surface attachment imaging [47].…”

Mechanisms of vegetative propagation in bulbs : a molecular approach

“…Transcription factors orchestrate several networks by controlling when and where certain genes will be expressed ) and, therefore, have been well studied and characterized in plants. However, even though they function as master regulators, transcription factors are often expressed at relatively low abundance (Jones et al 2015). This low level of expression makes transcription factors suitable markers to further assess the sequencing depth and coverage of our two generated transcriptomes.…”