Functional Analysis Of An Ethylene-responsive HD-Zip Family Member RhHB2 In Rose Flower Opening

Rose is one of the most important cut flowers in the world with a wide range of aesthetic demand all over the global market. The economic value of rose flowers primarily depends on the quality of flower opening, which is mainly driven by cell expansion. In the post-harvest storage and transportation process, the exogenous and endogenous ethylene can inhibit petal expansion and lead to abnormal opening phenomena, such as accelerated flower opening and abnormal petal shape, which ultimately decreases the flower quality and causes serious loss. To date, however, the molecular mechanism in this process remains unknown. Therefore, it is necessary to screen and identify the important regulators which can respond to ethylene and impact the flower opening.In this work, cut rose cv. 'Samantha' was used as material to investigate the potential regulator in rose flower opening. Based on transcriptomics database and microarray data, we isolated an ethylene-responsive transcription factor gene, RU00731. The full length sequence of RU00731 was cloned by RACE. Its ORF is 651 bp in length,and encodes a protein with 216 amino acids. According to sequence analysis, RU00731 contains the conserve Homeodomain and leucie zipper domains, and belongs to the HD-Zip Class I sub-family in HB superfamily. The RU00731 was named as RhHB2. The phylogenetic analysis showed that RhHB2 is a homolog of AtHB7 and AtHB12 in Arabidopsis. RhHB2 protein is localized in the nucleus, and possesses transactivation activity in C-ternimus. RhHB2 could be homodimerized in yeast cells. The expression of RhHB2 was relative lower in all floral organs of unopened flower, while it increased substantially in stamen, pistils and petals in fully opened ones. In petals, the expression level of RhHB2 was enhanced associated with the opening process and droped down in aged flowers. Furthermore, RhHB2 gene was significantly induced by ethylene and dehydration treatments.Silencing of RhHB2 by VIGS, mildly delayed the flower opening and decrased the flower diameter,but didn't change the flower height. In petals, Silencing of RhHB2 did not significantly accelerate or delay the senescence process. On the other hand, the length and width of petals were substantially decreased in RhHB2-silenced petals compared with the control TRV, indicating that RhHB2 is a positive regulator for regulating petal expansion. According to qRT-PCRs, expression level of RU08526(sugar transporter), RU22926 (Cellulase) and RU05224 (expansin) were up-regulated RhHB2-silenced petals,, while that of RU06454 (Endoxyloglucan transferase) and RU02382 (Extension) was drastically down-regulated.Overexpression of RhHB2 in Arabidopsis produced bigger leaves, flowers and petals, and thicker main stem as well. Moreover, the silique numbers was dramatically increased. Anatomical analysis showed that the size of pavement cells of leaves was not obviously different from wild type and transgenic plants harboring empty vector. However, the cell numbers largely increased in the tissues surrounding main vascular bundle of leaves.We tested the expression of 11 GA synthesis and degradation-related genes and 15 cell division and expansion-related genes in transgenic Arabidopsis by RT-PCR. The results showed that the expression of GA2ox2, GA2ox6, KLUH/CYP78A5, AN3, CYCD3;2 and CYCA2;3 was higher in transgenic plants than WT, while that of SIAMESE(SIM) and CYP79B2 was significantly lower than WT plants. These genes are involved in the GA degradation and synthesis, auxin synthesis, cell division and cell endoreduplication. Collectively, we argued that RhHB2 gene may be an key player in regulating cell division and expansion in plants.