AaZFP3, a Novel CCCH-Type Zinc Finger Protein from Adonis amurensis, Promotes Early Flowering in Arabidopsis by Regulating the Expression of Flowering-Related Genes.

CCCH-type zinc finger proteins (ZFP) are a large family of proteins that play various important roles in plant growth and development; however, the functions of most proteins in this family are uncharacterized. In this study, a CCCH-type ZFP, AaZFP3, was identified in the floral organ of Adonis amurensis. Quantitative real-time PCR (qPCR) analysis revealed that AaZFP3 was widely expressed in the flowers of A.amurensis. Subcellular localization analysis showed that the AaZFP3 protein was mainly localized to the cytoplasm in tobacco and Arabidopsis. Furthermore, the overexpression of AaZFP3 promoted early flowering in Arabidopsis under both normal and relatively low-temperature conditions. RNA-sequencing and qPCR analyses revealed that the expression of multiple key flowering-time genes was altered in transgenic Arabidopsis overexpressing AaZFP3 compared to wild-type. Of these genes, FLOWERING LOCUS T (AtFT) expression was most significantly up-regulated, whereas FLOWERING LOCUS C (AtFLC) was significantly down-regulated. These results suggest that the overexpression of AaZFP3 promotes early flowering in Arabidopsis by affecting the expression of flowering-time genes. Overall, our study indicates that AaZFP3 may be involved in flowering regulation in A.amurensis and may represent an important genetic resource for improving flowering-time control in other ornamental plants or crops.

Identification and Characterization of MIKCc-Type MADS-Box Genes in the Flower Organs of Adonis amurensis.

Adonis amurensis is a perennial herbaceous flower that blooms in early spring in northeast China, where the night temperature can drop to -15 °C. To understand flowering time regulation and floral organogenesis of A. amurensis, the MIKCc-type MADS (Mcm1/Agamous/ Deficiens/Srf)-box genes were identified and characterized from the transcriptomes of the flower organs. In this study, 43 non-redundant MADS-box genes (38 MIKCc, 3 MIKC*, and 2 Mα) were identified. Phylogenetic and conserved motif analysis divided the 38 MIKCc-type genes into three major classes: ABCDE model (including AP1/FUL, AP3/PI, AG, STK, and SEPs/AGL6), suppressor of overexpression of constans1 (SOC1), and short vegetative phase (SVP). qPCR analysis showed that the ABCDE model genes were highly expressed mainly in flowers and differentially expressed in the different tissues of flower organs, suggesting that they may be involved in the flower organ identity of A. amurensis. Subcellular localization revealed that 17 full-length MADSs were mainly localized in the nucleus: in Arabidopsis, the heterologous expression of three full-length SOC1-type genes caused early flowering and altered the expression of endogenous flowering time genes. Our analyses provide an overall insight into MIKCc genes in A. amurensis and their potential roles in floral organogenesis and flowering time regulation.

The N-terminal and third transmembrane domain of PsCor413im1 are essential for targeting to chloroplast envelope membrane.

Cold-regulated (COR) genes, located downstream of the C-repeat binding factors (CBFs) in cold signaling pathways, play a central role in plant response to cold stress. In our previous studies, a Cor413 chloroplast envelope membrane protein, PsCor413im1, was identified from the cold-tolerant plant Phlox subulata. Its overexpression enhanced cold tolerance and altered AtCor15 expression in Arabidopsis. In the present study, the function of PsCor413im1 was further investigated. Transmission electron microscope observation showed that the chloroplast envelope membrane of cold-treated transgenic Arabidopsis seedlings was more stable than that of cold-treated wild-type seedlings. Subcellular localization of green fluorescent protein as a marker revealed that the N-terminal and putative third transmembrane domain (TMD) of PsCor413im1 were essential for its targeting of the chloroplast envelope membrane. Furthermore, overexpression of PsCor413im1 fragments containing N-terminal and third TMD also altered the expression of AtCor15 genes in Arabidopsis. Overall, our results suggest that PsCor413im1 may stabilize the chloroplast envelope membrane under cold stress, and its N-terminal and third TMD are important for its targeting capability and function.

AabHLH48, a novel basic helix-loop-helix transcription factor from Adonis amurensis, promotes early flowering in Arabidopsis by activating FRUITFULL expression.

Basic helix-loop-helix (bHLH) transcription factors play various important roles in plant growth and development. In this study, a AabHLH48 was identified in the floral organ of Adonis amurensis, a perennial herb that can naturally complete flowering at extreme low temperatures. AabHLH48 was widely expressed in various tissues or organs of A. amurensis and was localized in the nucleus. Overexpression of AabHLH48 promotes early flowering in Arabidopsis under both photoperiod (12 h light/12 h dark and 16 h light/8 h dark) and temperature (22 and 18 °C) conditions. Transcriptome sequencing combined with quantitative real-time PCR analysis showed that overexpression of AabHLH48 caused a general upregulation of genes involved in floral development in Arabidopsis, especially for AtAGAMOUS-LIKE 8/FRUITFULL (AtAGL8/FUL). The yeast one-hybrid assay revealed that AabHLH48 has transcriptional activating activity and can directly bind to the promoter region of AtAGL8/FUL. These results suggest that the overexpression of AabHLH48 promoting early flowering in Arabidopsis is associated with the upregulated expression of AtAGL8/FUL activated by AabHLH48. This indicates that AabHLH48 can serve as an important genetic resource for improving flowering-time control in other ornamental plants or crops.

Identification of Transcription Factors Involved in the Regulation of Flowering in Adonis Amurensis Through Combined RNA-seq Transcriptomics and iTRAQ Proteomics.

Temperature is one of the most important environmental factors affecting flowering in plants. Adonis amurensis, a perennial herbaceous flower that blooms in early spring in northeast China where the temperature can drop to -15 °C, is an ideal model for studying the molecular mechanisms of flowering at extremely low temperatures. This study first investigated global gene expression profiles at different developmental stages of flowering in A. amurensis by RNA-seq transcriptome and iTRAQ proteomics. Finally, 123 transcription factors (TFs) were detected in both the transcriptome and the proteome. Of these, 66 TFs belonging to 14 families may play a key role in multiple signaling pathways of flowering in A. amurensis. The TFs FAR1, PHD, and B3 may be involved in responses to light and temperature, while SCL, SWI/SNF, ARF, and ERF may be involved in the regulation of hormone balance. SPL may regulate the age pathway. Some members of the TCP, ZFP, MYB, WRKY, and bHLH families may be involved in the transcriptional regulation of flowering genes. The MADS-box TFs are the key regulators of flowering in A. amurensis. Our results provide a direction for understanding the molecular mechanisms of flowering in A. amurensis at low temperatures.