The role and proteomic analysis of ethylene in hydrogen gas-induced adventitious rooting development in cucumber (Cucumis sativus L.) explants

Previous studies have shown that both hydrogen gas (H2) and ethylene (ETH) play positive roles in plant adventitious rooting. However, the relationship between H2and ETH during this process has not been explored and remains insufficiently understood. In this study, cucumber (Cucumis sativus L.) was used to explore the proteomic changes in ETH-H2-induced rooting. Our results show that hydrogen-rich water (HRW) and ethylene-releasing compound (ethephon) at proper concentrations promote adventitious rooting, with maximal biological responses occurring at 50% HRW or 0.5 µM ethephon. ETH inhibitors aminoethoxyvinylglycine (AVG) and AgNO3 cause partial inhibition of adventitious rooting induced by H2, suggesting that ETH might be involved in H2-induced adventitious rooting. According to two-dimensional electrophoresis (2-DE) and mass spectrometric analyses, compared with the control, 9 proteins were up-regulated while 15 proteins were down-regulated in HRW treatment; four proteins were up-regulated while 10 proteins were down-regulated in ethephon treatment; and one protein was up-regulated while nine proteins were down-regulated in HRW+AVG treatment. Six of these differentially accumulated proteins were further analyzed, including photosynthesis -related proteins (ribulose-1,5-bisphosphate carall boxylase smsubunit (Rubisco), sedoheptulose-1,7-bisphosphatase (SBPase), oxygen-evolving enhancer protein (OEE1)), amino and metabolism-related protein (threonine dehydratase (TDH)), stress response-related protein (cytosolic ascorbate peroxidase (CAPX)), and folding, modification and degradation-related protein (protein disulfide-isomerase (PDI)). Moreover, the results of real-time PCR about the mRNA levels of these genes in various treatments were consistent with the 2-DE results. Therefore, ETH may be the downstream signaling molecule during H2- induced adventitious rooting and proteins Rubisco, SBPase, OEE1, TDH, CAPX and PDI may play important roles during the process.

IDENTIFICATION AND VALIDATION OF REFERENCE GENES FOR THE NORMALIZATION IN REAL-TIME RT-QPCR ON RICE AND RED RICE IN COMPETITION, UNDER DIFFERENT NITROGEN DOSES

ABSTRACT Real time reverse transcription polymerase chain reaction (RT-qPCR) is an important technique to analyze differences in gene expression due to its sensitivity, accuracy, and specificity. However, before analyzing the expression of the target gene, it is necessary to identify and evaluate the stability of candidate reference genes for the proper normalization. This study aimed at evaluating the stability of candidate reference genes in order to identify the most appropriate genes for the normalization of the transcription in rice and red rice in competition under different nitrogen levels, as well as to demonstrate the effectiveness of the reference gene selected for the expression of the cytosolic ascorbate peroxidase (OsAPX2). Eleven candidate reference genes were assessed using the RefFinder which integrates the four leading software: geNorm, NormFinder, Bestkeeper, and the comparative delta-Ct method in addition to the analysis of variance to identify genes with lower standard deviation and coefficient of variation values. Eight out of the eleven genes have shown the desired effectiveness and, among them, the gene UBC-E2 has the highest stability according to RefFinder and the analysis of variance. The expression of the gene OsAPX2 has proven to be effective in validating the candidate reference gene. This study is the first survey on the stability of candidate reference genes in rice and red rice in competition, providing information to obtain more accurate results in RT-qPCR.