Functional analysis of ARF1 from Cymbidium goeringii in IAA response during leaf development

Cymbidium is an economically important genus of flowering orchids cultivated in China because of showing graceful leaf shapes and elegant flower coloration. However, the deterioration of the ecological environment and the difficulty of conservation management have become increasing challenges for maintaining its germplasm resources. ARFs are critical transcription factors in the auxin signaling pathway and have been found to play pivotal roles in leaf growth and development in previous studies. However, their functions and mechanisms in Cymbidium goeringii remain to be clarified.

The sequence of the CgARF1 gene was analyzed by bioinformatics. The expression of this gene in different tissues and under IAA treatment was detected by quantitative real-time PCR analysis. The CgARF1 gene was overexpressed in wild-type Arabidopsis and Nicotiana benthamiana via the Agrobacterium infection method. Acetone-ethanol solvent extraction was applied for the determination of chlorophyll contents, and the contents of endogenous hormones were determined using the enzyme-linked immunosorbent assay technique.

CgARF1 cloned from C. goeringii 'Songmei' was 2,049 bp in length and encoded 682 amino acids containing three typical domains: a B3 DNA binding domain, an Aux_resp domain and an AUX/IXX family domain. The expression pattern of CgARF1 in different tissues of C. goeringii showed that its expression was highest in the leaves and changed greatly under IAA treatment. Subcellular localization studies showed that the protein was mainly localized in the cell nucleus. CgARF1-overexpressing lines exhibited leaf senescence and a decreased chlorophyll content. Under IAA treatment, CgARF1 regulates the rooting length, rooting number and rooting rate from detached leaves. The levels of endogenous hormones in transgenic leaves were also significantly changed.

These results indicated that CgARF1 overexpression is responsive to IAA treatment during leaf development. This study provides a foundation for future research on the function of the ARF gene family in C. goeringii.