Introduction
Zinc (Zn) deficiency is one of the most important micronutrient constraints affecting wheat growth, yield, and grain nutritional quality worldwide. Wheat is a major source of carbohydrates and a staple food for a large proportion of the global population; therefore, improving Zn content of bread wheat grains is of great importance. Previous studies suggest that genetic biofortification is a fundamental and sustainable approach for increasing Zn concentration in wheat grains.
Materials and Methods
This study was conducted as a factorial experiment arranged in a completely randomized design with three replications under greenhouse conditions at the Faculty of Agriculture, Urmia University. Two bread wheat cultivars differing in Zn-efficiency, the Zn-efficient cultivar (Niknejad) and the Zn-inefficient cultivar (Farin), were grown under Zn-deficient (0 mg Zn kg⁻¹ soil) and Zn-sufficient (5 mg Zn kg⁻¹ soil) conditions. Root and leaf samples were collected at two growth stages, 30% heading and grain filling. Total RNA was extracted from root and leaf tissues using RNX-Plus solution, and the relative expression of genes encoding elongation factor Tu (Ef-Tu), the beta subunit of the pyrophosphate-dependent phosphofructokinase (PFP) enzyme complex (PFP β-subunit), and the snowy cotyledon3 (Sco3) protein was analyzed using the real-time PCR technique. Gene sequences were retrieved from the GenBank database, and gene-specific primers were designed using Gene Runner and Fast PCR softwares. Real-time PCR reactions were performed using a Rotor-Gene Q system, with the actin gene used as the reference gene for normalization. Relative gene expression levels were calculated using the 2⁻ΔΔCt method, and statistical analyses were performed using SAS software (version 9.2).
Results and Discussion
Analysis of variance revealed that the three-way interaction effect of cultivar × tissue × sampling stage was significant for the relative expression of all three genes (P ≤ 0.01). Under Zn-deficient conditions, Ef-Tu expression decreased in the roots of both cultivars at both the 30% heading and grain filling stages. In contrast, leaf expression of Ef-Tu increased in the Niknejad cultivar at the 30% heading stage, showing a markedly greater increase compared to the Farin cultivar. However, expression levels declined in both cultivars during the grain filling stage. Regarding the PFP β-subunit gene, higher expression was observed in the roots of the Farin cultivar at the 30% heading stage, whereas the Niknejad cultivar exhibited lower expression at this stage. In both cultivars, root expression decreased during the grain filling stage. In leaf tissues, PFP β-subunit expression increased in both cultivars at the 30% heading stage, with more than a twofold higher expression in the Niknejad cultivar compared to Farin. A similar trend was observed at the grain filling stage, although the magnitude of increase remained greater in the Zn-efficient cultivar. Expression of the Sco3 gene decreased in the roots of both cultivars at both growth stages, whereas its expression increased in leaf tissues at the 30% heading stage in both cultivars. At the grain filling stage, the highest Sco3 expression was recorded in the leaves of the Niknejad cultivar.
Conclusion
The findings of the present study demonstrated that, under zinc-deficient conditions, the highest upregulation of the genes encoding EF-Tu and the β-subunit of the pyrophosphate-dependent phosphofructokinase complex was observed in the leaves of the Niknejad cultivar at the 30% heading stage. Similarly, the greatest increase in the expression of the Sco3 gene was detected in the leaves of the Niknejad cultivar, although this peak occurred at the grain filling stage. In contrast, enhanced root expression of the PFP β-subunit gene was observed exclusively in the Farin cultivar at the 30% heading stage. However, similar to Ef-Tu and Sco3, the expression of this gene decreased in the Zn-efficient cultivar at the 30% heading stage and in both cultivars during the grain filling stage. Collectively, these results indicate that Zn deficiency elicits distinct tissue-specific and developmental stage-dependent gene expression responses in bread wheat cultivars differing in Zn efficiency. The stronger induction of Ef-Tu, PFP β-subunit, and Sco3 genes in the leaves of the Zn-efficient cultivar suggests their potential involvement in mechanisms underlying enhanced Zn efficiency. Therefore, these genes may represent promising molecular targets for improving Zn efficiency and advancing wheat biofortification strategies.