Expression profiling of auxin biosynthesis genes during inflorescence development in black pepper(Piper nigrum L)

Abstract

The study entitled “Expression profiling of auxin biosynthesis genes during inflorescence development in black pepper (Piper nigrum L.)” was carried out during 2017-2019, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to compare the transcript profile of auxin biosynthesis genes such as Flavin monooxygenase and Tryptophan aminotransferase during inflorescence development in different cultivars of black pepper (Piper nigrum L.) using Reverse Transcription quantitative PCR (RT- qPCR). Black pepper exhibits wide variability in inflorescence architecture. Auxin is a major hormone involved in patterning and formation of lateral organs and regulation of branching. Auxin biosynthesis in plants occurs mainly through tryptophan dependent and independent pathways. Among the different known pathways, the most significant and well studied is the Indole-3-Pyruvic Acid (IPA) pathway. Tryptophan aminotransferase (TAA1) and Flavin monooxygenase (YUC2) are the consecutive enzymes of the pathway converting tryptophan to IPA and subsequently to Indole Acetic Acid (IAA). Spike samples of three cultivars of black pepper viz., Panniyur, Karimunda and Thekken were used for the study. Samples were collected at three different developmental stages viz., stage 1 (1-2cm), stage 2 (6-8cm) and stage 3 (9-12cm) from two different plants of each cultivar. Genomic DNA and RNA were extracted by modified Cetyl Trimethyl Ammonium Bromide (CTAB) method and Trizol method respectively. Primers were designed for auxin biosynthesis genes such as Tryptophan aminotransferase and Flavin monooxygenase using “Primer Express” software and absence of secondary structure at the primer binding site was confirmed by “mfold” web server. Specificity of the designed gene specific primers was checked by PCR using genomic DNA. Single amplicon was obtained for all the primers indicating specificity of the designed primers. Amplification efficiency of the designed primers was determined by standard curve analysis and “Lin Reg” software. All the primers exhibited cent per cent amplification efficiency. RNA isolated from the spike samples was converted to cDNA and the quality was confirmed by PCR and agarose gel electrophoresis. cDNA was used for RT-qPCR using SYBR Green dye-based assay. Thermal conditions for RT-qPCR were 95°C for 2 min followed by 40 cycles of 95°C for 15 sec, 55°C for 15 sec and 72°C for 45 sec. RT-qPCR for each gene was performed with three technical replicates for each sample. Cq values obtained were used for further analysis. Gene expression analysis was performed using “qbase plus” software using Actin as the reference gene and two as the amplification factor for all the genes. The expression pattern of TAA1 showed regulation during inflorescence development in all the three cultivars. Higher expression was noticed in all the three stages of Panniyur-1 compared to Karimunda and Thekken. In Karimunda and Thekken, the expression was low in the first stage and peaked at stage 2 and decreased in stage 3. Expression of TAA1 was very less in stage 2 and 3 of Thekken. The expression pattern of YUC2 also showed differential regulation during inflorescence development in all the three cultivars. The expression pattern was similar in all the three cultivars with a peak at stage 2. The expression of YUC2 was highest in Panniyur-1 and lowest in Thekken. To conclude, auxin biosynthetic genes TAA1 and YUC2 were differentially regulated during different stages of inflorescence development in all the three cultivars of black pepper. The expression levels of both the biosynthetic genes were least in the branching type Thekken. Low expression levels of these genes may contribute towards low auxin accumulation in Thekken. Analysis of other genes involved in auxin signalling might help in a better understanding of inflorescence development in black pepper.