Water relations, growth and development, leaf gas exchange, nutrient uptake, and transport are just a few of the developmental or biochemical processes that may be affected by boron toxicity in plants. B toxicity in grapevines is usually evident by characteristic symptoms on above-ground parts, including decreased shoot growth, smaller leaves, and also marginal chlorosis and necrosis.
4.1. Vine Growth and Nutrient Concentrations in Plant Tissues
For both ungrafted and grafted vines, the boron concentrations in each plant organ increased with excess boron treatment (
Table 2). Among the different vine organs, leaves showed the highest boron concentration, followed by roots and other parts. It has been reported that in most plant species, boron is absorbed by roots, loaded into the xylem, and translocated to shoots via the transpiration stream and accumulated in leaves without undergoing redistribution [
29]. However, in relatively lower concentrations, boron can also be found accumulated in roots. Dannel et al., 1998 [
30] reported that after long-term experiments with excess B, the concentration in sunflower leaves was about 10-fold higher than in the roots, indicating that roots do not retain B to prevent the above-ground from accumulating excess B. These tissues typically contain between 40 and 100 mg B kg
−1 d.w. in species that accumulate B in their leaves. However, when B levels in the soil approach toxic levels, the leaves may contain 250 mg kg
−1 d.w. B. Under extremely toxic B conditions, leaf B concentrations may exceed 700 to 1000 mg kg
−1 d.w. [
31]. In grapevines, similar symptoms and decreased shoot vigor have been reported in field conditions when leaf B concentrations were greater than 140 mg kg
−1 d.w. [
22].
Boron is absorbed by plants primarily passively as boric acid but also in small amounts actively as borate ions [
3]. A system of active transportation using B transporters has been suggested in the scenario of restricted B availability. In Arabidopsis, BOR1 was reported to be the initially identified transporter involved in xylem loading [
32]. In B-limiting conditions, a mechanism involving various protein channels and transporters is established to supply this component to aerial tissues [
33]. Transcriptional analysis of a boron transporter gene (VvBOR1) in the grapevine revealed it is preferentially expressed in flowers at anthesis, and there is a direct correlation between the expression pattern and the amount of boron in grapes [
34,
35].
The results of the present research are in accordance with those presented in citrus [
36] and mandarin [
37], where B accumulated primarily in the leaves as opposed to other plant tissues. The increased boron concentrations in leaves as a result of an excess of B application is a common and expected response. However, in our experiment, significant differences were found between the different rootstocks. Both the own-rooted treated vines and vines grafted onto 101-14 Mgt rootstock concentrated increased boron in leaves, resulting in the earlier appearance of leaf toxicity symptoms in vines grafted onto 101-14 Mgt rootstock, in comparison with 1103P.
Macronutrient levels in leaves were adequate for vine growth since N, P, K, Mg and Ca concentrations in leaves during this experiment ranged between 1.68 and 2.44%, 0.20 and 0.32%, 0.97 and 3.35%, 0.42 and 0.72%, 1.96 and 2.88% in d.w., respectively. However, excess boron significantly decreased macronutrients in various parts of the vine in most cases. These results are consistent with those of other researchers who reported decreased leaf nutrients because of a high B supply [
22,
38]. Moreover, it has been reported that P concentration was decreased by high B in grapevines [
22] as well as in kiwi fruit [
39] and tomato [
40].
Ca and Mg are crucial for the development and growth of plants. Ca crosslinks the carboxyl groups of the pectic polymers to enhance the cell wall, whereas Mg is a crucial component of proteins and chlorophyll [
41]. The distribution of Mg was generally more concentrated in leaves, indicating its high mobility and transport efficiency in plants.
According to our results, all measured trace elements accumulated in various tissues decreased after boron application. Fe content in vine tissues was significantly higher compared to the other elements, which may be attributed to the plant’s absorption mechanism and functions. Iron participates in redox reactions, electron transport, and maintaining the chloroplast’s structural integrity in cells to ensure that plants can absorb and distribute sufficient Fe to leaves in various environments [
42]. Furthermore, we noticed a significant decline in the uptake of Fe in grapevine plants exposed to excess boron. The results clearly demonstrate that B treatment reduces Cu, Mn, and Zn concentrations in various vine parts. Although Cu and Zn are contaminants at certain concentrations, they are also essential micronutrients for plants. For instance, Zn plays a role as an activator of enzymes, and Cu plays an important role in regulating protein composition, photosynthesis, mitochondrial respiration, and cell wall metabolism [
43]. In the current study, Cu and Zn were equally distributed throughout all tissues, while Cu was more concentrated in roots. The decrease in C uptake by roots could be due to the toxic effect of B on root cells, leading to an impaired absorption process. The low uptake of Cu and Zn by plants was attributed to excess boron, according to Singh et al., 1990 [
44]. However, previous studies did not show consistent effects of excess boron on nutrient elements of different plant species [
45,
46,
47]. These inconsistencies between various studies could be caused by differences in the mobility of nutrients in different species or variations in the demands of these crops for nutrients throughout the growing period [
45].
In our experiment, the first visible symptoms of B toxicity developed on leaf blades of B-treated vines 25 days after the beginning of the experimentation (
Figure 1).
It has been reported that after being absorbed, boron is transported to the shoot via transpiration flux, and tends to accumulate radially at the leaf edges where leaf veins end. These tissues indeed show the most severe symptoms of B toxicity [
48]. In grapevines, which have leaf reticulate venation, B toxicity is observed around the leaf margins, whereas in grasses, such as wheat and barley, with parallel-veined leaves, the toxic effect develops black patches in leaf tips where the veins terminate [
49]. The disorder was also characterized by the downward or upward cupping of leaves. Occasionally, leaf cupping has been reported as a symptom of B toxicity [
50,
51]. According to Loomis and Durst 1992 [
52], the disorder may be induced by the inhibition of cell growth, possibly due to an abnormal high level of cross-links in the cell wall. B toxicity symptoms do not appear in roots, which suggests that B distribution is related to the transpiration stream. B commonly accumulated in the leaves, but it remained in the root system when B was in relatively lower concentrations. Although there were no visible symptoms in roots under excess B, the overall root volume significantly decreased (
Table 3). A phenotypic effect of B toxicity was related to the inhibition of root growth, which was accompanied by a reduction in plant dry weight and an increase in B levels in root tissues. The reduction of root growth has been previously observed in grapevines by Gunes et al., 2006 [
21], and also by others in different plant species [
53,
54]. Additionally, Aquea et al., 2012 [
55] revealed the molecular basis of root growth inhibition caused by B toxicity in
Arabidopsis. They revealed that B toxicity induced the expression of genes associated with cell wall modifications, abscisic acid response, and abscisic acid signaling. In this study, the decreased leaf chlorophyll content because of increased boron levels led to decreased dry weights of shoots and roots. Regarding the rootstock cultivar effect, it was found that vines grafted onto 1103P showed increased root and shoot weights compared to those grafted onto 101-14 Mgt rootstock.
4.2. Photosynthetic Activity, Water Status, and Chlorophyll Pigments
Excess boron treatments gradually decreased photosynthesis, simultaneously with a decrease in leaf chlorophyll. The damage to the photosynthetic apparatus constitutes the most significant consequence of excess boron. In our experiment, leaf chlorophyll (Chl) content and CCM-200 index dropped thirty days after the beginning of boron treatment (
Figure 2). It was reported that plants under boron stress conditions presented a decreased amount of Chl and, therefore, decreases in photosynthetic rate occurred [
56,
57]. The strong decline of Chl induced by B toxicity in grapevines, as reported in other species [
56,
58], can indicate a decrease in the synthesis of these molecules, as well as an enhancement of their oxidative processes. It is also well-known that during the late stages of the experiment, plants regulate the construction and destruction of a specific subset of light-harvesting complexes through the formation and degradation of light-reduced total leaf Chl concentrations. According to Kiani-Pouya and Rasouli 2014 [
59], the relative Chl measurement (CCM-200 index) could be used for a cost-effective and rapid chlorophyll assessment because of a close correlation between the CCM-200 index and leaf Chl content. The vines exposed to excess boron during a period of thirty or sixty days showed a decreased photosynthetic activity. At the beginning of the experimental cycle, there were no statistically significant differences for the control plants. To better comprehend the impact of excess B on PSII machinery, we measured the chlorophyll fluorescence parameter F
v/F
m (
Table 6). The most frequently used fluorescence parameter is F
v/F
m (maximum quantum yield of PSII). In our study, the maximum quantum yield of PSII was affected by B (
Table 6), indicating serious damage to PSII machinery. Similar findings were obtained by Papadakis et al., 2004 [
37], who reported a significant decrease in F
v/F
m in leaves of Navelina orange plants grown with excess B concentrations. Our results indicated that both control and stressed vines had relatively high F
v/F
m ratios after 30 days of boron treatment (0.729–0.828). Strong and significant differences between stressed and control vines were observed during the following period, especially at the end of the experimental cycle (60 d). At this stage, Cabernet Franc grafted onto 101-14 Mgt rootstock recorded the lowest value of F
v/F
m ratio (0.489).
The significant decrease in the F
v/F
m ratio indicated that leaves were photoinhibited, a condition that molecular oxygen can represent an alternative electron acceptor for unused electrons, leading to Reactive Oxygen Species (ROS) generation [
60]. Additionally, due to the inhibition in the electron transport rate, reduced activity of some CO
2 assimilation enzymes (carboxylase/oxygenase, ribulose-1,5-bisphosphate, and fructose-1,6-bisphosphate phosphatase) has been reported [
56]. As shown in
Figure 2 and
Figure 5, the observed decrease in F
v/F
m ratio was coincident with a decline in leaf chlorophyll content and CO
2 assimilation rate. It is well-known that the lowest rate of CO
2 assimilation may be induced by several different factors, including water potential, limitations on CO
2 gas exchange, degradation of photosynthetic pigments, ion toxicity, and nutritional imbalance.
Over the experimental period, a gradual decrease in stem water potential was observed in the treated vines throughout the duration of the experiment (
Figure 4). Aquea et al., 2012 [
55], reported that boron toxicity induces the expression of genes involved in abscisic acid (ABA) signaling and represses genes that code for water transporters. The global changes in gene expression suggest that boron principally triggers a molecular response associated with a water-stress-related response.
In addition, significantly reduced values of CO
2 assimilation rate were observed in Boron-treated plots, whereas stomatal conductance was not affected. On the contrary, the period of the experimentation had a significant effect on stomatal conductance, which from day thirty to day sixty of the experimentation was reduced by a range of 30%. Additionally, the net CO
2 assimilation rate was reduced by 19.64 and 48.54% in control and treated vines, respectively. In addition, both the scion and rootstock cultivars had a significant effect on the net CO
2 assimilation rate. In general, the Merlot cultivar and 1103P rootstock showed an increased net CO
2 assimilation rate, whereas the Cabernet Franc cultivar and 101-14 Mgt rootstock rated the lowest values (2.76 μmol CO
2 m
−2s
−1). Even though it is known that excess boron inhibits photosynthesis, information on the effects of B on the photosynthetic process is still scarce [
57,
61]. According to certain authors, stomatal conductance did not decrease along with the decline in photosynthetic rate in plants exposed to excess B [
62]. In contrast, other authors observed a reduction in stomatal conductance [
37]. The structural damage of thylakoids, according to Pereira et al., 2000 [
63], was one of the potential causes of the reduction in photosynthesis caused by excess B. This, in turn, altered the rate of electron transport and the CO2 photosynthetic rate, which can also be restricted by stomatal reduction. Following B toxicity, a significant decline in the F
v/F
m ratio (maximum quantum yield of chlorophyll fluorescence) was observed in many species [
37,
61]. Our results showed that the reduction of CO
2 photosynthetic rate was mainly related to a downregulation of photosystem II photochemical efficiency and partly to the stomatal conductance.
In the case of photoinhibition, because of the decrease in F
v/F
m ratio, molecular oxygen can serve as an alternative electron acceptor for unused electrons and light [
60], which occurs in the generation of ROS. The ROS, which are by-products of various abiotic stress, lead to dysfunction of membranes and cell death. Plants have developed a powerful scavenging system composed of antioxidant molecules and antioxidant enzymes to prevent the negative effects of these reactive molecules. Among those, phenolic substances represent a large class of secondary metabolites with strong antioxidative properties. As shown in
Figure 3, sixty days from the beginning of boron treatment, leaf phenolic substances were increased by boron stress. In addition, no significant differences were found between stressed scion and rootstock cultivars.
Phenolic compounds protect plants from physiological stresses, such as oxidative stress, by preventing breakdown of macromolecules and cellular membranes. It has been reported that the antioxidant role of these compounds is due to their molecular structure [
64].