## 4 Discussion
### NO Is Essential for mVOCs-Induced Plant Fe Acquisition  
Considerable attention has been paid to plant Fe deficiency due to the low solubility of soil Fe [30]. The bioavailability of Fe is extremely low in alkaline soils, and Fe forms insoluble Fe\({}^{3+}\) oxy-hydroxides [29]. In this study, we assessed the ability of VOCs-exposed plants to take up Fe under alkaline conditions. When plants were cultured on an alkaline medium containing different Fe levels, the VOCs-exposed plants exhibited better growth traits with a high level of chlorophyll content. Previous studies have indicated that chlorophyll is the major component of the chloroplasts, which is closely associated with the Fe content of leaves [28]. Fe deficiency results in a significant decrease in chlorophyll content and massive degradation of chloroplast proteins, thereby damaging the structural and functional integrity of chloroplasts [14]. In this study, the control plants exhibited chloride leaves under Fe-limited conditions, and fewer photosynthetic lamellae occurred in the chloroplasts. Nevertheless, BF06 VOCs exposure apparently promoted normal grana stacking and increased the number of grana lamellae in the chloroplasts, and thus alleviated chlorotic symptoms. Consistently, total Fe content was significantly higher in the VOCs-exposed plants than in the controls, but this was not observed in plants grown at 0 \(\upmu\)M Fe(III)-EDTA. Interestingly, the VOCs-exposed plants grown with no Fe also displayed greener leaves with better chloroplast development under alkaline stress. Thus, BF06 VOCs exposure could promote reutilization of cell wall Fe in plants under Fe-limited conditions. To verify the hypothesis, the content of soluble and cell wall Fe was examined in both the control and VOCs-exposed plants. Our results showed that VOCs exposure led to higher levels of soluble Fe in plants, and a significant decrease of root cell wall Fe. This raised a question as to how BF06 VOCs regulated Fe assimilation and reutilization of cell wall Fe in plants.  
NO is an essential regulator that participates in Fe deficiency responses in plants [58]. NO not only activates FER or FIT1-dependent signaling pathways [55], but also functions as an Fe chelator to increase the mobilization of cell wall Fe [60]. Intriguingly, the emission of VOCs by some bacterial strains, such as 2R or 3R-butanediol, induces NO synthesis in plants [65]. In our experiments, BF06 could produce VOCs, containing 2R or 3R-butanediol. Thus, we further investigated whether the VOCs-induced increase of total or soluble Fe content contributed to enhancing NO synthesis. Our data reveled that VOCs exposure resulted in a greater increase of root NO accumulation, but this induction was notably curtailed by the application of L-NAME or c-PTIO. It is well demonstrated that NO regulates Fe deficiency responses via the induction of _FRO2_ and _IRT1_ expression regulated by _FIT1_[59]. Here, we found that treatment with L-NAME largely abrogated the expression of VOCs-induced _FIT1_, _IRT1_, and _FRO2_. It was also observed that total or soluble Fe content was not pronouncedly increased in the VOCs-exposed plants when treated with L-NAME or c-PTIO under alkaline stress. Consistently, the VOCs-induced reduction of cell wall Fe was not found in the NO-deficient plants. Therefore, the enhanced reutilization of cell wall Fe and Fe uptake was dependent on the VOCs-induced NO production in roots.