Overlapping transcriptional expression response of wheat zinc-induced facilitator-like transporters emphasize important role during Fe and Zn stress

Abstract

Background: Hexaploid wheat is an important cereal crop that has been targeted to enhance grain micronutrient content including zinc (Zn) and iron (Fe). In this direction, modulating the expression of plant transporters involved in Fe and Zn homeostasis has proven to be one of the promising approaches. The present work was undertaken to identify wheat zinc-induced facilitator-like (ZIFL) family of transporters. The wheat ZIFL genes were characterized for their transcriptional expression response during micronutrient fluctuations and exposure to multiple heavy metals.

Results: The genome-wide analyses resulted in identification of fifteen putative TaZIFL-like genes, which were distributed only on Chromosome 3, 4 and 5. Wheat ZIFL proteins subjected to the phylogenetic analysis showed the uniform distribution along with rice, Arabidopsis and maize. In-silico analysis of the promoters of the wheat ZIFL genes demonstrated the presence of multiple metal binding sites including those which are involved in Fe and heavy metal homeostasis. Quantitative real-time PCR analysis of wheat ZIFL genes suggested the differential regulation of the transcripts in both roots and shoots under Zn surplus and also during Fe deficiency. Specifically, in roots, TaZIFL2.3, TaZIFL4.1, TaZIFL4.2, TaZIFL5, TaZIFL6.1 and TaZIFL6.2 were significantly up-regulated by both Zn and Fe. This suggested that ZIFL could possibly be regulated by both the nutrient stress in a tissue specific manner. When exposed to heavy metals, TaZIFL4.2 and TaZIFL7.1 show significant up-regulation, whereas TaZIFL5 and TaZIFL6.2 remained almost unaffected.

Conclusion: This is the first report for detailed analysis of wheat ZIFL genes. ZIFL genes also encode for transporter of mugineic acid (TOM) proteins, that are involved in the release of phytosiderophores to enhance Fe/Zn uptake. The detailed expression analysis suggests the varying expression patterns during development of wheat seedlings and also against abiotic/biotic stresses. Overall, this study will lay foundation to prioritize functional assessment of the candidate ZIFL as a putative TOM protein in wheat.

Keywords: Biofortification; Iron deficiency; Micronutrient uptake; Triticum aestivum L.; Zinc transport.

Fig. 1

Phylogenetic analysis of wheat ZIFL protein sequences. The analysis was performed by using 35 ZIFL protein sequences from wheat, thirteen from Oryza sativa, seven from Zea mays and three from Arabidopsis. Rooted phylogenetic tree was constructed by using Neighbour-joining method using MEGA7 software with 1000 bootstrap replicates

Fig. 2

Genomic and chromosomal distribution of wheat ZIFL genes on wheat genome. Distribution of 35 TaZIFL across: a A, B and D sub genomes. b Wheat chromosomal distribution. c Chromosomal distribution shared on different Chromosomes and sub-genomes

Fig. 3

Intron exon arrangement and protein conservation. The intron-exon structure was obtained using Gene Structure Display Server (GSDS 2.0:) Pink boxes and black lines depict the introns and exons, respectively

Fig. 4

Protein sequence analysis for conserved and unique motifs in wheat ZIFL. a Conserved motifs across 15 TaZIFL proteins, as obtained from MEME. Different colors represent distinct motifs. b Unique motifs found through MEME for group 2, group 4, as well as TaZIFL6.2

Fig. 5

Relative gene expression levels of wheat ZIFL transcripts under +Zn condition. Gene expression profiles of wheat ZIFL genes were studied in a roots and b shoots. Total RNA was extracted from the wheat seedlings subjected to three and 6 days of treatments +Zn. The roots and shoots samples were collected, and qRT-PCR was performed on the DNA free RNAs. A total of 2 µg of RNA was used for cDNA synthesis. C_t values were normalized against wheat ARF1 as an internal control. Data represents mean of three biological replicates each treatment containing 12–15 seedlings. Vertical bars represent the standard deviation. # on the bar indicates that the mean is significantly different at p < 0.05 with respect to their respective control samples

Fig. 6

Relative gene expression levels of wheat ZIFL genes under −Fe condition. Gene expression profiles of wheat ZIFL genes were studied in a roots and b shoots. Total RNA was extracted from the wheat seedlings subjected to 3 and 6 days of treatments. The roots and shoots samples were collected, and qRT-PCR was performed on the DNA free RNAs. A 2 µg of total RNA was used for cDNA synthesis. C_t values were normalized against wheat ARF1 as an internal control. Data represents mean of three biological replicates with each treatment containing 12–15 seedlings. Vertical bars represent the standard deviation. # on the bar indicates that the mean is significantly different at p < 0.05 with respect to their respective control treatments

Fig. 7

qRT-PCR expression analysis of shoots and roots of wheat seedlings exposed to multiple heavy metals (Ni, Co and Cd). Five days old wheat seedlings were exposed to the mentioned heavy metals for the period of 14 days. Total RNA was extracted from the treated and control samples and 2 µg of RNA was used to construct the cDNA. C_t values were normalized against wheat ARF1 as an internal control. Fold expression values were calculated relative to the control tissue of the mentioned wheat ZIFL. Vertical bars represent the standard deviation. # represent the significantly difference at p < 0.05 with respect to their respective control treatments

Fig. 8

Heat map for relative expression of putative TaZIFL genes in different tissues and at multiple developmental stages. Heatmaps were generated using expression values from expVIP database for grain, leaf, root, spike and shoot tissues. Green to red color change depicts increase in transcript expression, as shown by the color bar