Selection of Optimal Reference Genes and Transcriptional Regulation of Betalamic Pigments in Two Congeneric Species of Alternanthera Elicited with Methyl Jasmonate

Plant pigments, including betalains, have drawn both scientific and economic interest. However, the molecular basis behind its synthesis and catabolism is not entirely understood. In this sense, transcription analysis largely improves our understanding, but it is essential to select optimal reference genes. Thus, the present study aimed (i) to investigate the stability of a set (10) of candidate reference genes, including ABCG18 ( ABC-2 transporter ), ACS ( ACT domain ), ACTIN ( Actin ), AUX1 ( transmembrane protein) , DDF2 ( DNA interase) , GAPCP-1 ( glyceraldehyde-3-phosphate dehydrogenase) , UBC4 ( Ubiquitin 4) , RP ( Ribosomal protein ), TH7 ( Thioredoxin 7 ), and UBQ ( Ubiquitin ) genes in Alternanthera sessilis and Alternanthera philoxeroides and (ii) to evaluate the expression of key genes that code for enzymes involved in the biosynthesis pathway of betalamic pigments in plants elicited with methyl jasmonate (MeJA). For this, these plants were first established and multiplied in vitro and then transfer to the continuous flow hydroponic system with Hoagland solution (control plants) or Hoagland solution plus 100 µM MeJA. Comprehensive analysis showed that the most stable genes for RT-qPCR studies were GAPCP-1 and ACS for A . sessilis and TH7 and AUX1 for A . philoxeroides . Conversely, TH7 and DDF2 were not suitable reference genes in A. sessilis and UBC4 and RP for A. philoxeroides . Additionally, the regulation of genes involved in betalamic pigment biosynthesis pathway responds differently in the two congeneric species when treated with MeJA (100 µM) for 48 h, with a pronounced increase in the expression of the Corismate mutase , Cytochrome CYP76AD1 , and DOPA 4,5- dioxygenase genes in A . phyloxeroids.