Characterization of metallothionein genes from Broussonetia papyrifera: metal binding and heavy metal tolerance mechanisms.

BACKGROUND: Broussonetia papyrifera is an economically significant tree with high utilization value, yet its cultivation is often constrained by soil contamination with heavy metals (HMs). Effective scientific cultivation management, which enhances the yield and quality of B. papyrifera, necessitates an understanding of its regulatory mechanisms in response to HM stress. RESULTS: Twelve Metallothionein (MT) genes were identified in B. papyrifera. Their open reading frames ranged from 186 to 372 bp, encoding proteins of 61 to 123 amino acids with molecular weights between 15,473.77 and 29,546.96 Da, and theoretical isoelectric points from 5.24 to 5.32. Phylogenetic analysis classified these BpMTs into three subclasses: MT1, MT2, and MT3, with MT2 containing seven members and MT3 only one. The expression of most BpMT genes was inducible by Cd, Mn, Cu, Zn, and abscisic acid (ABA) treatments, particularly BpMT2e, BpMT2d, BpMT2c, and BpMT1c, which showed significant responses and warrant further study. Yeast cells expressing these BpMT genes exhibited enhanced tolerance to Cd, Mn, Cu, and Zn stresses compared to control cells. Yeasts harboring BpMT1c, BpMT2e, and BpMT2d demonstrated higher accumulation of Cd, Cu, Mn, and Zn, suggesting a chelation and binding capacity of BpMTs towards HMs. Site-directed mutagenesis of cysteine (Cys) residues indicated that mutations in the C domain of type 1 BpMT led to increased sensitivity to HMs and reduced HM accumulation in yeast cells; While in type 2 BpMTs, the contribution of N and C domain to HMs' chelation possibly corelated to the quantity of Cys residues. CONCLUSION: The BpMT genes are crucial in responding to diverse HM stresses and are involved in ABA signaling. The Cys-rich domains of BpMTs are pivotal for HM tolerance and chelation. This study offers new insights into the structure-function relationships and metal-binding capabilities of type-1 and - 2 plant MTs, enhancing our understanding of their roles in plant adaptation to HM stresses.

Genome-wide identification and expression analysis of ethylene responsive factor family transcription factors in Juglans regia.

BACKGROUND: Walnut is an important economic tree species with prominent economic value and ecological functions. However, in recent years, walnuts have become susceptible to drought stress, resulting in a decline in comprehensive benefits. Therefore, it is necessary to identify the regulatory molecular mechanism associated with walnut response to drought. In many plants, ethylene responsive factor (ERF) gene family plays important roles in response to biotic and abiotic stress, especial drought. Therefore, the identification and characterisation of walnut ERF genes will benefit walnut with regard to the clarification of drought response mechanism as well as the management, production, and quality of plantations. METHODS: 'ERF' was compared against the walnut transcriptome, and the JrERFs with a complete open reading frame (ORF) were identified by ORF Finder. The molecular weights, amino acid residues, and theoretical isoelectric point (pI) were predicted by ExPASy. The distribution of JrERFs in chromosome locations was determined based on walnut genome data from NCBI. The intron-exon structures and conserved domains were analysed using Gene Structure Display Server 2.0 and CD-Search, accordingly. Multi-sequence alignment and a phylogenetic tree were constructed by ClustalX2.1 and MEGA7, respectively. The conserved motifs were acquired using MEME. Total RNA was isolated using the cetyltrimethylammonium ammonium bromide (CTAB) method (Yang et al., 2018). Gene expression was determined by using real-time quantitative polymerase chain reaction (qRT-PCR) analysis and calculated according to the 2-ΔΔCT method (Livak & Schmittgen, 2001). RESULTS: A total of 44 JrERFs were identified from the walnut transcriptome, whose ORFs were 450-1,239 bp in length. The molecular weights of the JrERF proteins (consisting 149-412 amino acids) were 16.81-43.71 kDa, with pI ranging from 4.8 (JrERF11) to 9.89 (JrERF03). The JrERFs can be divided into six groups (B1-B6), and among the groups, B6 contained the most number of members. Each JrERF contained 1-6 motifs and each motif comprised 9-50 amino acids. Among the motifs, motif1, motif2, and motif3 were the most abundant. More than 40% of JrERFs were up-regulated continuously when subjected to ethephon (ETH), PEG6000, and PEG6000+ETH treatments. Of all the JrERFs, JrERF11 showed the highest expression. Therefore, we conclude that walnut ERF genes are highly conserved and involved in the regulation of drought response in the presence of ETH. JrERFs are possibly important candidate genes for molecular breeding; hence, the findings of this study provides the theoretical basis for further investigation of ERF genes in walnut and other species.