Introduction

Strigolactones (SLs) are a new class of plant hormones regulating shoot branching1,2 and symbiotic interactions with arbuscular mycorrhizal fungi3,4. In addition, SLs regulate many other processes in plant growth and development including primary root growth, lateral root formation, adventitious root formation, root hair development, seed germination, photomorphogenesis and nodulation (reviewed in references5,6,7,8,9,10,11), protonema branching in moss12, as well as responses to stresses13 and nutrient deficiency (reviewed in reference8). In the last decade, great progresses have been made to identify genes regulating the biosynthesis and signaling of SLs, in particular, by analyzing branching mutants in Arabidopsis, pea, rice and petunia (reviewed in references14,58. T1 transformants were selected using 20 μg/L hygromycin B. A minimum of 20 independent transgenic lines were selected for each transformation. Six independent transgenic lines were used for further studies. When T1 plants reached maturity, the number of primary rosette-leaf branches was counted.

RT-PCR analysis

To examine the expression of PtD14 genes in the Arabidopsis transgenic lines, total RNA was extracted from the rosette leaves of four-week-old plants using the Invisorb Spin Plant Mini Kit (Stratec Molecular). Two μg of total RNA were reversely transcribed to cDNA using Fermentas RevertAid Reverse Transcriptase (Thermo Scientific). PtD14-specific primers were used in PCR reactions. PCR amplification of Arabidopsis ACTIN2 served as a control in the analysis of Arabidopsis transgenic lines.

Quantitative RT-PCR

To examine the expression patterns of PtD14 genes, total RNA was extracted from various tissues and organs of Populus plants using the Spectrum™ Plant Total RNA isolation kit (Sigma). Two μg of total RNA were reversely transcribed to cDNA using Fermentas RevertAid Reverse Transcriptase. Quantitative RT-PCR was performed using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific). Thermal cycling consisted of 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 15 s at 95 °C and 60 s at 60 °C. PtD14-specific primers (PtD14a: TTAGCCGAACGCTTTTCAACA and TTCCACAGTAGCTTTGCCACC; PtD14b: CTAAGAGGGATACTGGGCCT and TTCCACGGTATTTTCGCCAC) were used in the quantitative RT-PCR reactions. PCR amplification of Populus UBIQUITIN C served as a control for normalizing the relative transcript level. All PCR reactions were done with three technical replicates.

Yeast two-hybrid assay

The full-length open-reading frame of PtD14a, PtD14b and PtMax2a45 was each cloned into pENTR vector (Life technologies, CA). For the bait construct, the pENTR vector containing PtD14a or PtD14b was transferred into the pDEST32 destination vector by LR clonase-mediated reactions (Life technologies). For the prey construct, the pENTR vector containing PtMAX2a was transferred into the pDEST22 destination vector. One hundred ng of each plasmid of bait and prey construct was added into 100 μl of MaV203 competent yeast cells (Life technologies). For negative control, 100 ng of pDEST22 and pDEST32 empty vector was co-transformed with each other or with the counterpart of pDEST plasmid DNA. Co-transformation was performed by adding 600 μl of 40% PEG/1× LiAc to yeast cell and plasmid mixture followed by incubation at 30 °C for 30 min. After incubation, 35.5 μl of DMSO was added into the cell mixture to improve transformation efficiency. Then, the yeast cells were incubated for 20 min in a 42 °C water bath. Co-transformed yeast cell was centrifuged and the pellet was diluted in 1 ml of 0.9% NaCl. A total of 100 μl of diluted yeast cells was spread on SD plate deficient of Trytophane and Leucine (SD/-Trp/-Leu). Correctly co-transformed yeast cells were cultured in 2 ml of SD/-Trp/-Leu solution overnight at 28 °C. Cultured yeast cells were diluted up to 100 times with 0.9% NaCl. Fifteen μl of diluted yeast cells were dropped on SD plate deficient of Tryptophan, Leucine and Histidine (SD/-Trp/-Leu/-His) supplemented with 5 mM 3-Amino-1,2,4-triazole (3AT; Sigma-Aldrich, MO) or 5 mM of 3AT plus 5 μM GR24. The plates were incubated for 3 days at 28 °C. Yeast cells grown on SD plate were imaged with Canon power shot SX210 IS digital camera (Canon USA Inc., NY).

Protein degradation assay

Populus mesophyll protoplasts were isolated from P. tremula × alba clone 717-1B4 leaves as described previously46. A total of 30 μg of plasmid expressing 10× Myc-tagged PtD14 proteins was purified with Qiagen Plasmid Midi Kit and transfected into 200 μl of protoplasts (~2 × 105) using PEG-calcium mediated transfection method59. After 12 hr incubation at room temperature, GR24 was added into the transfected protoplast suspension to a final concentration of 5 μM and incubated for another 5 hr. Protoplasts were collected by centrifuging. Total protein was then extracted from protoplasts using 50 mM Tris-HCl (pH8.0), 100 mM NaCl, 10 mM EDTA (pH 8.0), 1% SDS, 1 mM PMSF and protease inhibitor (Sigma). After centrifuging, the supernatant was collected and protein concentration was determined by Bradford method. Protein extracts were mixed with SDS loading buffer (60 mM Tris-HCl pH 8.0, 1% SDS, 10% glycerol, 20 mM DTT) and denatured by boiling for 8 min. To detect 10× Myc-tagged PtD14 proteins with western blotting, 1 μg of total protein was separated in 10% SDS-PAGE gel and transferred to PVDF membrane. Membranes were then probed with anti-Myc antibody (1:4000; Abgent), detected with ECL reagent (Thermo) and imaged with CCD imager (Bio-rad). In parallel, same amount of protein extracts were separated in 10% SDS-PAGE gel and stained with ProteoSilver Silver Stain Kit (Sigma). One protein band existing in all samples (~80 kDa) was selected to demonstrate equal protein loading.

Additional Information

How to cite this article: Zheng, K. et al. Characterization of DWARF14 Genes in Populus. Sci. Rep. 6, 21593; doi: 10.1038/srep21593 (2016).