AtbZIP69 Overexpression Enhances Low-Nitrogen and Drought Tolerance in Wheat: A Mechanistic Study

This study evaluated the overexpression of AtbZIP69 in wheat material and verified its ability to enhance low-nitrogen (LN) and drought tolerance. We observed a 20-fold upregulation of AtbZIP69 expression under LN stress and several-fold upregulation under drought stress, suggesting its responsiveness to both stresses. This finding is consistent with previous studies in Arabidopsis, indicating a conserved function of AtbZIP69 across species. Numerous studies have confirmed the crucial role of bZIP transcription factors (TFs) in the responses of various species to drought and other abiotic stresses (Liu et al., 2014; Yang et al., 2020).

Transgenic wheat exhibited significantly improved performance compared to the wild type (WT) under drought stress, as evidenced by enhanced biomass, survival rate, and plant height stress index (Fig. 4). While several studies have linked bZIP TFs to the nitrate transport pathway, the role of bZIP TFs in improving LN tolerance remains relatively underexplored. This study used a chlorate toxicity assay to investigate the effect of AtbZIP69 overexpression on nitrate uptake in wheat. Chlorate, a toxic nitrate analogue, is commonly used to assess nitrate absorption capacity in wheat. Our results revealed that AtbZIP69 overexpression significantly increased nitrate uptake in wheat.

Greenhouse LN stress experiments demonstrated significantly higher chlorophyll a and b levels in transgenic wheat compared to the WT. The increased chlorophyll content in transgenic wheat overexpressing AtbZIP69 suggests a potential enhancement of photosynthesis, leading to increased energy production and synthesis of essential compounds for survival under LN stress. Chlorophyll content in leaves is known to be positively correlated with nitrogen availability, directly influencing the formation of photosynthetic products. Maize, for instance, can adapt to nitrogen stress by increasing its Chl a/Chl b ratio, apparent quantum yield, and light saturation point (Ya-Wei, 2019). Therefore, we believe that transgenic wheat improves LN tolerance through increased nitrate uptake and chlorophyll content.

Field experiments conducted over two years revealed no significant differences in yield and number of grains per year between the normal nitrogen treatment, transgenic wheat, and the WT. However, under LN treatment, the grain yield and spike number of transgenic wheat were significantly higher than those of the WT (Fig. 5). The use of nitrogen fertilizers, while contributing to increased wheat yield, also has implications for plant breeding (Yan et al., 2022). The wheat Green Revolution gene Rht1 significantly reduced plant height, leading to a substantial increase in wheat yield (Hedden, 2003). Additionally, the dwarfing gene improved collapse resistance and allowed the plant to tolerate higher nitrogen fertilizer levels (Liu et al., 2022a). Therefore, transgenic or gene-editing techniques offer promising approaches for achieving increased resistance and higher yields in wheat.

To further investigate the impact of AtbZIP69 overexpression on nitrogen allocation, we selected the most resistant strain, OE-1, and analyzed the N content of stems, leaves, and grains. The nitrogen content in transgenic wheat grains was significantly higher than that in the WT, while it decreased in the stems and leaves. The increased yield observed in transgenic wheat was likely due to its ability to preferentially allocate nitrogen to grains, potentially facilitated by the overexpression of genes involved in nitrogen transport, remobilization, or utilization. This observation aligns with previous studies demonstrating that modifying nitrogen allocation in crops can significantly enhance grain yield. In a previous field experiment, GmTDN1 was found to alter nitrogen distribution by allocating more nitrogen to the grains (Zhou et al., 2022). The overexpression of TaGS2-2Ab in wheat increased nitrogen remobilization to grains (Hu et al., 2018). Our findings suggest that transgenic wheat increases yield under LN treatment by preferentially allocating nitrogen to the grains.

The upregulation of nitrate transporter-related genes NRT2.1, NRT2.5, and NR1 in transgenic wheat under LN stress (Fig. 6) provides a mechanistic explanation for the observed improved LN tolerance. These genes are responsible for the uptake and transport of nitrate from the soil into plant roots. By upregulating these genes, transgenic wheat enhances its ability to take up and utilize nitrate, which is essential for plant growth and development. The increased expression of these genes is likely a direct consequence of AtbZIP69 overexpression, as AtbZIP69 has been shown to regulate nitrate transport genes in other species. This suggests that AtbZIP69 acts as a transcription factor, directly binding to the promoters of these genes and activating their transcription under LN stress.

Furthermore, the enhanced nitrate uptake observed in transgenic wheat is associated with higher chlorophyll content and improved photosynthetic capacity. Nitrate is a key component of chlorophyll, the pigment responsible for capturing light energy during photosynthesis. Therefore, the enhanced nitrate uptake in transgenic wheat leads to higher chlorophyll levels, promoting more efficient photosynthesis and the production of energy and essential compounds necessary for survival under LN stress.

In addition to the upregulation of nitrate transporter-related genes, the preferential allocation of nitrogen to grains in transgenic wheat likely contributes to the increased yield observed under LN treatment. This allocation may be facilitated by the overexpression of certain genes involved in nitrogen transport, remobilization, or utilization. By directing more nitrogen towards grain development, transgenic wheat is able to produce more grains and ultimately increase its yield under LN stress.

Overall, our findings suggest that AtbZIP69 overexpression in wheat improves LN tolerance by enhancing nitrate uptake, increasing chlorophyll content, and preferentially allocating nitrogen to grains. These mechanistic explanations provide insights into the molecular mechanisms underlying the improved LN tolerance observed in transgenic wheat and have important implications for the development of crops with increased nitrogen use efficiency and yield under stressful environmental conditions. These findings have significant implications for agriculture, especially in regions with poor soil quality and limited access to fertilizers. By developing transgenic crops capable of allocating more nitrogen to grains, we can potentially achieve significant increases in crop yields, even under stressful environmental conditions like LN. This advancement can contribute to improving food security and promoting sustainable agricultural practices.