Quantitative RT-PCR analysis of 20 transcription factor genes of MADS, ARF, HAP2, MBF and HB families in moisture stressed shoot and root tissues of sorghum

Abstract

Transcription factors (TFs) are an important target in understanding the regulation of plant responses to environmental stress including moisture stress. Members of the same TF family may differ in their response to moisture stress. The expression pattern could vary between shoot and root tissues depending on level of moisture stress. A set of five rarely studied TF families viz., MADS-box (MCM1, AGAMOUS, DEFICIENS and SRF), Auxin Responsive Factor (ARF), Heme Activator Protein 2 (HAP2), Multiprotein Bridging Factor (MBF) and Homeobox (HB) together having 20 members in sorghum, were expression analyzed through quantitative real-time PCR (qRT-PCR) in well watered and moisture stressed shoot and root tissues of sorghum using SYBR Green® to quantify dsDNA synthesis. Fluorescence values were used to calculate PCR efficiency by using LinRegPCR. The PTSb00029.1 and PTSb00033.1 of ARF family and PTSb00174.1 and PTSb00175.1 of HB family recorded 2 to 5, PTSb00221.1 and PTSb00208.1 of MADS family and PTSb00128.1 of HAP2 family recorded 5 to 10 fold up-regulation under moisture stress regimes. The PTSb00128.1, a HAP2 family member, recorded 15 fold up-regulation in mild moisture stressed root tissues. TF genes such as PTSb00218.1, PTSb00220.1, PTSb00031.1, PTSb00032.1, PTSb00034.1 and PTSb00223.1 were found down regulating in both tissues types under moisture stress condition. However, the PTSb00128.1, PTSb00221.1, PTSb00029.1, PTSb00033.1 and PTSb00174.1 TFs were found up-regulating to varied levels in mild and severe moisture stressed root tissues only. Verification of qRT-PCR results was done by in situ hybridization (ISH) of randomly selected two TF genes in shoot and root tissues of sorghum. Taken together, moisture stress triggered up-regulation of more genes in root tissue compared to shoot tissue in sorghum.

Keywords: Moisture stress; Quantitative real-time PCR; Sorghum; Transcription factor genes; cDNA.

Fig. 1

Integrity of total RNA isolated from sorghum: two bright bands corresponding to ribosomal 28S rRNA and 18S rRNA with a ratio of intensities of ~ 2:1 are visible in gel photograph

Fig. 2

Specificity of qRT-PCR reaction. a Separation of qRT-PCR products of 22 genes on 3 % agarose gel gives single product of the expected size for most of the reactions with few reactions yielding no product (arrow). b Typical real-time PCR amplification plot of 22 genes for two types of cDNA samples of shoot and root tissues of sorghum. As number of PCR cycle increases there is increase in fluorescence. Some curves do not cross threshold line in such reactions there is no detectable PCR product when loaded on 3 % agrose gel

Fig. 3

Technical precision of qRT-PCR reaction. a Technical precision of real-time PCR reflected as correlation coefficient between the duplicate measurements of cDNA levels of TF genes from the separate reverse transcription reaction (biological replicates). The correlation coefficient values for biological replicate is 0.97. b Technical precision of real-time PCR reflected as correlation coefficient between the duplicate measurements of cDNA levels of TF genes from the same reverse transcription reaction (technical replicates). The correlation coefficient values for technical replicate is 0.96

Fig. 4

Trends of regulated expression of TFs during moisture stress in sorghum shoot and root tissues. Relative change in up and down-regulation of transcription factor genes in mild and severe moisture stressed shoot and root tissues compared to respective well watered control. The x-axis showes the TF gene ID, and the y-axis shows the diffrence between Ct values of moisture stressed samples and respective well watered control. MS = Mild Root; MR = Mild Root; SS = Severe stressed Shoot; SR = Severe stressed Root

Fig. 5

Sorghum leaf sections hybridized with DIG-labeled RNA probe: presence of targeted mRNA is indicated by pinkish purple colour. a, b leaf sections; negative (without probe) control c Well watered leaf section hybridized with PSTb00220.1 DIG-labeled RNA probe d Mild moisture stressed leaf section hybridized with PSTb00220.1 DIG-labeled RNA probe e Severe moisture stressed leaf section hybridized with PSTb00220.1 DIG-labeled RNA probe. f Well watered leaf section hybridized with PSTb00223.1 DIG-labeled RNA probe g Mild moisture stressed leaf section hybridized with PSTb00223.1 DIG-labeled RNA probe h Severe moisture stressed leaf section hybridized with PSTb00223.1 DIG-labeled RNA probe

Fig. 6

Sorghum leaf sections hybridized with DIG-labeled RNA probe: The presence of targeted mRNA is indicated by pinkish purple colour. a, b root sections; negative (without probe) control c Well watered root section hybridized with PSTb00220.1 DIG-labeled RNA probe d Mild moisture stressed root section hybridized with PSTb00220.1 DIG-labeled RNA probe e Severe moisture stressed root section hybridized with PSTb00220.1 DIG-labeled RNA probe. f Well watered root section hybridized with PSTb00223.1 DIG-labeled RNA probe g Mild moisture stressed root section hybridized with PSTb00223.1 DIG-labeled RNA probe h Severe moisture stressed root section hybridized with PSTb00223.1 DIG-labeled RNA probe