R2R3-type MYB transcription factors are implicated in drought stress, which is a primary factor limiting the growth and development of woody plants. The identification of R2R3-MYB genes in the Populus trichocarpa genome has been previously reported. Nevertheless, the diversity and complexity of the conserved domain of the MYB gene caused inconsistencies in these identification results. There is still a lack of drought-responsive expression patterns and functional studies of R2R3-MYB transcription factors in Populus species. In this study, we identified a total of 210 R2R3-MYB genes in the P. trichocarpa genome, of which 207 genes were unevenly distributed across all 19 chromosomes. These poplar R2R3-MYB genes were phylogenetically divided into 23 subgroups. Collinear analysis demonstrated that the poplar R2R3-MYB genes underwent rapid expansion and that whole-genome duplication events were a dominant factor in the process of rapid gene expansion. Subcellular localization assays indicated that poplar R2R3-MYB TFs mainly played a transcriptional regulatory role in the nucleus. Ten R2R3-MYB genes were cloned from P. deltoides × P. euramericana cv. Nanlin895, and their expression patterns were tissue-specific. A majority of the genes showed similar drought-responsive expression patterns in two out of three tissues. This study provides a valid cue for further functional characterization of drought-responsive R2R3-MYB genes in poplar and provides support for the development of new poplar genotypes with elevated drought tolerance.

The R2R3-MYB genes in plants play an essential role in the drought-responsive signaling pathway. Plenty of R2R3-MYB S21 and S22 subgroup genes in Arabidopsis have been implicated in dehydration conditions, yet few have been covered in terms of the role of the S21 and S22 subgroup genes in poplar under drought. PdMYB2R089 and PdMYB2R151 genes, respectively belonging to the S21 and S22 subgroups of NL895 (Populus deltoides × P. euramericana cv. ‘Nanlin895′), were selected based on the previous expression analysis of poplar R2R3-MYB genes that are responsive to dehydration. The regulatory functions of two target genes in plant responses to drought stress were studied and speculated through the genetic transformation of Arabidopsis thaliana. PdMYB2R089 and PdMYB2R151 could promote the closure of stomata in leaves, lessen the production of malondialdehyde (MDA), enhance the activity of the peroxidase (POD) enzyme, and shorten the life cycle of transgenic plants, in part owing to their similar conserved domains. Moreover, PdMYB2R089 could strengthen root length and lateral root growth. These results suggest that PdMYB2R089 and PdMYB2R151 genes might have the potential to improve drought adaptability in plants. In addition, PdMYB2R151 could significantly improve the seed germination rate of transgenic Arabidopsis, but PdMYB2R089 could not. This finding provides a clue for the subsequent functional dissection of S21 and S22 subgroup genes in poplar that is responsive to drought.

Centipedegrass (Eremochloa ophiuroides (Munro.) Hack.) is a species originating in China and is an excellent warm-season turfgrass. As a native species in southern China, it is naturally distributed in the phosphorus-deficient and aluminum-toxic acid soil areas. It is important to research the molecular mechanism of centipedegrass responses to phosphorus-deficiency and/or aluminum-toxicity stress. Quantitative Real-Time PCR (qRT-PCR) is a common method for gene expression analysis, and the accuracy of qRT-PCR results depends heavily on the stability of internal reference genes. However, there are still no reported stable and effective reference genes for qRT-PCR analysis of target genes under the acid-soil-related stresses in different organs of centipedegrass. For scientific rigor, the gene used as a reference for any plant species and/or any stress conditions should be first systematically screened and evaluated. This study is the first to provide a group of reliable reference genes to quantify the expression levels of functional genes of Eremochloa ophiuroides under multiple stresses of P deficiency and/or aluminum toxicity. In this study, centipedegrass seedlings of the acid-soil-resistant strain ‘E041’ and acid-soil-sensitive strain ‘E089’ were used for qRT-PCR analysis. A total of 11 candidate reference genes (ACT, TUB, GAPDH, TIP41, CACS, HNR, EP, EF1α, EIF4α, PP2A and actin) were detected by qRT-PCR technology, and the stability of candidate genes was evaluated with the combination of four internal stability analysis software programs. The candidate reference genes exhibited differential stability of expression in roots, stems and leaves under phosphorus-deficiency and/or aluminum-toxicity stress. On the whole, the results showed that GAPDH, TIP41 and HNR were the most stable in the total of samples. In addition, for different tissues under various stresses, the selected reference genes were also different. CACS and PP2A were identified as two stable reference genes in roots through all three stress treatments (phosphate deficiency, aluminum toxicity, and the multiple stress treatment of aluminum toxicity and phosphate deficiency). Moreover, CACS was also stable as a reference gene in roots under each treatment (phosphate deficiency, aluminum toxicity, or multiple stresses of aluminum toxicity and phosphate deficiency). In stems under all three stress treatments, GAPDH and EIF4α were the most stable reference genes; for leaves, PP2A and TIP41 showed the two highest rankings in all three stress treatments. Finally, qRT-PCR analysis of the expression patterns of the target gene ALMT1 was performed to verify the selected reference genes. The application of the reference genes identified as internal controls for qRT-PCR analysis will enable accurate analysis of the target gene expression levels and expression patterns in centipedegrass under acid-soil-related stresses.