Regulation of α-expansins genes in Arabidopsis thaliana seeds during post-osmopriming germination.

Seed osmopriming is a pre-sowing treatment that involves limitation of the seed water imbibition, so that pre-germinative metabolic activities proceed without radicular protrusion. This technique is used for improving germination rate, uniformity of seedling growth and hastening the time to start germination. In Arabidopsis thaliana, seed germination has been associated with the induction of enzymes involved in cell wall modifications, such as expansins. The α-expansins (EXPAs) are involved in cell wall relaxation and extension during seed germination. We used online tools to identify AtEXPA genes with preferential expression during seed germination and RT-qPCR to study the expression of five EXPA genes at different germination stages of non-primed and osmoprimed seeds. In silico promoter analysis of these genes showed that motifs similar to cis-acting elements related to abiotic stress, light and phytohormone responses are the most overrepresented in promoters of these AtEXPA genes, showing that their expression is likely be regulated by intrinsic developmental and environmental signals during Arabidopsis seed germination. The osmopriming conditioning had a decreased time and mean to 50% germination without affecting the percentage of final seed germination. The dried PEG-treated seeds showed noticeable high mRNA levels earlier at the beginning of water imbibition (18 h), showing that transcripts of all five EXPA isoforms were significantly produced during the osmopriming process. The strong up-regulation of these AtEXPA genes, mainly AtEXPA2, were associated with the earlier germination of the osmoprimed seeds, which qualifies them to monitor osmopriming procedures and the advancement of germination.

Identification of Endogenous Reference Genes for RT-qPCR Expression Analysis in Urochloa brizantha Under Abiotic Stresses.

Urochloa brizantha is one of the most important warm season forage grasses in tropical countries. Despite its importance, there are few studies on gene expression in this species under stressful conditions. Real-time (RT-qPCR) is an accurate technique for gene quantification analysis, but reference genes must be validated under the same conditions used to assess the expression of the target genes. Here, we evaluated the stability of nine reference genes: Actin 12, Eukaryotic initiation factor 4 A, Elongation factor-1 alpha, FTSH protease 4, U2 auxiliary fator, Succinol Co-enzyme A, Tubulin alfa-5, Tubulin beta-6, Ubiquitin conjugating enzyme. Total RNA was extract from leaf tissues of U. brizantha subjected to 6, 12 and 24 h of cold and heat stresses (10 and 45 °C, respectively), and drought, including moderate (-0.5 to -0.7 MPa), severe (-1.1 to -1.8 MPa) and recovery after re-watering. The RefFinder web-based tool was used to rank the most stable reference genes for each stress. Elongation factor-1 alpha, Elongation factor-1 alpha or Ubiquitin conjugating enzyme, and Eukaryotic initiation factor 4 A were the most stable genes for heat, cold and drought stress, respectively. The expression of Rubisco large subunit gene was normalized against the most stable gene selected by ReFfinder for each stress.