Boron homeostasis affects Longan yield: a study of NIP and BOR boron transporter of two cultivars

Wang, Jing; Wei
, Junbin; Guo, Dongliang; Lv, Xinmin; Wang, Boyun; Wang, Yiyun; Li, Jianguang

doi:10.1186/s12870-023-04689-8

Research
Open access
Published: 02 January 2024

Boron homeostasis affects Longan yield: a study of NIP and BOR boron transporter of two cultivars

Jing Wang¹,
Junbin Wei ¹,
Dongliang Guo¹,
Xinmin Lv¹,
Boyun Wang¹,
Yiyun Wang¹ &
…
Jianguang Li¹

BMC Plant Biology volume 24, Article number: 9 (2024) Cite this article

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Abstract

Background

Essential micronutrient Boron (B) plays crucial roles in plant survival and reproduction but becomes toxic in higher quantities. Although plant cells have different B transport systems, B homeostasis is mainly maintained by two transporter protein families: B exporters (BOR) and nodulin-26-like intrinsic proteins (NIP). Their diversity and differential expression are responsible for varied B tolerance among plant varieties and species. Longan is a highly admired subtropical fruit with a rising market in China and beyond. In the present study, we cultured Shixia (SX) and Yiduo (YD), two differently characterized Longan cultivars, with foliar B spray. We analyzed their leaf physiology, fruit setting, B content, and boron transporter gene expression of various tissue samples. We also traced some of these genes' subcellular localization and overexpression effects.

Results

YD and SX foliage share similar microstructures, except the mesophyll cell wall thickness is double in YD. The B spray differently influenced their cellular constituents and growth regulators. Gene expression analysis showed reduced BOR genes expression and NIP genes differential spatiotemporal expression. Using green fluorescent protein, two high-expressing NIPs, NIP1 and NIP19, were found to translocate in the transformed tobacco leaves' cell membrane. NIPs transformation of SX pollen was confirmed using magnetic beads and quantified using a fluorescence microscope and polymerase chain reaction. An increased seed-setting rate was observed when YD was pollinated using these pollens. Between the DlNIP1 and DlNIP19 transformed SX pollen, the former germinated better with increasing B concentrations and, compared to naturally pollinated plants, had a better seed-setting rate in YD♀ × SX♂.

Conclusion

SX and YD Longan have different cell wall structures and react differently to foliar B spray, indicating distinct B tolerance and management. Two B transporter NIP genes were traced to localize in the plasma membrane. However, under high B concentrations, their differential expression resulted in differences in Jasmonic acid content, leading to differences in germination rate. Pollination of YD using these NIPs transformed SX pollen also showed NIP1 overexpression might overcome the unilateral cross incompatibility between YD♀ × SX♂ and can be used to increase Longan production.

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Background

Boron (B) is one of the essential micronutrients that play multiple crucial physiological functions in higher plants, such as cell wall formation, cell division, cell elongation, flowering, fruiting, rooting, nutrient uptake, ion flux, carbohydrate metabolism, nitrogen metabolism, and hormone regulation [1]. However, high B levels are hazardous to plant development [2]. Different parts of a plant have unique requirements and functions of B [1]. Furthermore, various plant species, varieties, and plants of various ages respond differently to B shortage or toxicity [2]. The primary source of B in plants is from the soil, which can be found as borate or boric acid (B(OH)₃) in soil solutions. Plants absorb boric acid using three different methods, depending on its accessibility: diffusion, major intrinsic proteins (MIP), and B exporters (BOR) transporters [3]. Passive diffusion and MIP transport are used when B is adequately available, whereas BOR transporters are used when B is absent. Previous studies revealed B transport in Arabidopsis thaliana plant occurs not just by passive diffusion but is also regulated by transport proteins such as boric acid channels (nodulin-26-like intrinsic proteins or NIPs) and B exporters (BORs) [4]. NIPs and BORs work together to keep plant development regular in high and low B environments.

Longan (Dimocarpus longan Lour.) is a prominent subtropical fruit tree native to South China, holding 70 and 60% of the world's Longan cultivation field and yield in 2010 [5]. There are over 400 varieties of Longan cultivars in China [6], and the majority (68%) of them grow during the mid-season (August-September) [5]. This season is presided by the rainy season of southeast Asia that depletes relatively soluble B in the soil by leaching it with the rainfall [2]. B effect on Longan's fruit quality is well studied and practised [5]. A foliar spray of boric acid and applying B fertilizer have increased fruit size, quality, and soluble sugar [3] and reduced flower and fruit drops [7]. For optimal Longan harvesting, it is necessary to understand and employ the proper dose of B. However, the molecular mechanism for B uptake and translocation in various Longan species are not fully understood. We studied two commonly cultivated Longan varieties, the traditional cultivar Shixia (SX), with 140 years of cultivation history in China [5], and the newer Yiduo (YD) cultivar, with better fruit quality [6, 7]. YD was introduced to China from Thailand, making up 90% of the Thai Longan yield [7].

In this study, we observed cellular structural differences, the sugar content of their leaf cells, the flower's Jasmonic acid (JA) content, the fruit-setting rate, and the B content of different plant organs after foliar B treatment. We also identified the genes of the BOR and NIP gene families and studied their evolutionary relation, cis-acting elements, and level of expression in different tissues. Depending on this analysis, genes that expressed most in various organs of SX and YD were selected for further study. Subcellular localization of this gene product was studied by creating versatile green fluorescent protein (GFP)-gene expression vectors and transforming SX pollen. B tolerance of the GFP-gene transformed pollens was studied, and Longan plants were pollinated using this pollen to check the germination rate. The unilateral cross incompatibility (UCI) between SX and YD was reported to hinder the attempt to generate successful crossbreeding [6]. So, we established a crossing of YD♀ × SX♂ using hybrid pollen to study the fruit-setting rate of the hybrid panicle.

Result

Leaf phenotyping, structural and ultrastructural differences

Mature YD and SX, both cultivars of Longan, have oblong, blunt-tipped leaves with 6 to 9 pairs of leaflets per leaf in pinnately compounded and alternate formation. SX leaves were smaller (10–12 cm) (Fig. 1a) than the YD leaves (18–20 cm) (Fig. 1b). Under the light microscope, toluidine blue stained longitudinal sections of both leaves showed the diameter of the vascular bundles is more prominent in the YD leaf (Fig. 1d) than in the SX leaf (Fig. 1c, g). Transmission electron microscopy (TEM) of mesophyll cells of both foliage showed similar cell size (Fig. 1h, i), but the cell wall of the YD (Fig. 1f) is significantly thicker than the SX (Fig. 1e, j).

B influences various vascular plant functions, including root extension, indole-3-acetic acid oxidase, sugar translocation, carbohydrate metabolism, nucleic acid synthesis, and pollen tube formation, in different manners [8]. We also observed varied effects of the foliar B spray on some SX and YD leaf constituents. The sucrose content of the SX leaves significantly increased (15.01 mg/g to 15.90 mg/g). In contrast, the YD leaves substantially decreased (16.41 mg/g to 14.99 mg/g) (Fig. 2a). The concentration of glucose (8.85 mg/g to 7.76 mg/g) (Fig. 2b), Quina cortisol (16.49 mg/g to 14.77 mg/g) (Fig. 2c), and total chlorophyll (19.71 mg/L to 15.60 mg/L) (Fig. 2j) in SX leaves significantly decreased but didn’t change much in YD (8.82 mg/g to 8.79 mg/g, 16.88 mg/g to 16.93 mg/g, and 20.79 mg/L to 20.75 mg/L respectively). The fructose content decreased in both SX (5.80 mg/g to 5.16 mg/g) and YD (5.53 mg/g to g.30 mg/g) (Fig. 2d) but was more notable in SX. SX's cellulose (112.07 mg/g to 170.90 mg/g) (Fig. 2e) and lignin (63.80 mg/g to 179.58 mg/g) (Fig. 2f) content significantly increased, but the YD (159.46 mg/g to 160.49 mg/g, and 116.49 mg/g to 113.67 mg/g) remained relatively unchanged. The starch (Fig. 2g) content of both SX (32.22 mg/g to 38.01 mg/g) and YD (20.16 mg/g to 29.67 mg/g) significantly increased. The SX chlorophyll-a (Fig. 2h) and chlorophyll-b (Fig. 2i) content slightly decreased (15.72 mg/L to 12.43 mg/L and 3.99 mg/L to 3.17 mg/L, respectively) but was relatively unchanged in YD (16.32 mg/L to 16.02 mg/L and 4.46 mg/L to 4.73 mg/L respectively). The carotenoid (Fig. 2k) content increased in SX (3.24 mg/L to 3.75 mg/L) and decreased in YD (6.36 mg/L to 4.91 mg/L), but both were insignificant.

Fruit-setting rate

Fruit-setting was observed for SX and YD Longan varieties with and without foliar B spray (Fig. 3). Although the fruit-setting rate differed without B spray in SX and YD, the fruit-setting rate increased in both varieties after spraying but was significant in SX. The fruit-setting of B unsprayed and B sprayed SX were 30% and 47%, whereas in YD were 0% and 11%.

Total boron content

With foliar B spray, the B content of the root, stem, leaf (Fig. 4a), and flowers (Fig. 4b) significantly increases in both YD and SX Longan varieties. The root B content increased in SX from 12.43 mg/Kg to 17.44 mg/Kg and from 14.56 mg/Kg to 20.82 mg/Kg in YD (Fig. 4a). The stem B content significantly increased in SX from 10.92 mg/Kg to 11.74 mg/Kg and from 12.99 mg/Kg to 25.58 mg/Kg in YD (Fig. 4a). The leaf B content increased significantly in SX from 30.84 mg/Kg to 97.21 mg/Kg and from 66.83 mg/Kg to 136.04 mg/Kg in YD (Fig. 4a). Among these, the amount of B was highest in the YD leaves (136.04 mg/Kg). Although to start with, SX had higher B in the root, but with B spray, YD had more B in its root. Also, before treatment, the B content of YD leaves (66.83 mg/Kg) (Fig. 4a) than SX (30.84 mg/Kg). Before treatment, the B content of the SX female flower (16.62 mg/Kg) SX was higher than the male (13.30 mg/Kg) (Fig. 4b), but after B spray, both male and female flowers had similar B content (20.95 mg/Kg and, 21.01 mg/Kg respectively). Before treatment, the B content of YD male (20.44 mg/Kg) and female (20.38 mg/Kg) flower was higher than the SX, and after treatment, the female YD flower (22.64 mg/Kg) had a higher B content than the YD male flower (21.07 mg/Kg) and both of the SX flowers (Fig. 4b). Above results indicates YD has higher B translocation than SX and is more prominent in foliage and female reproductive organs.

Jasmonic acid content

The YD and the SX Longan female flower’s JA concentrations significantly rose following foliar B spray, with the former showing a more noticeable change. After foliar B spray, the JA content of the SX increased from 30.2 μg/g to 36.7 μg/g, whereas in the YD, it increased from 37.9 μg/g to 78.1 μg/g (Fig. 5).

Gene expression analysis

We found five candidate genes of the BOR gene family (Additional file 1: Table 1) and thirty-three candidate genes of the NIP gene family (Additional file 2: Table 2). The cis-acting element of the genes was identified using Gene Structure Display Server (GSDS) and visualized in Additional file 3: Fig. 1. Eight different cis-acting elements including salicylic acid responsiveness, core promoter element, stress responsiveness, light responsive element, MeJA-responsiveness, regulatory element, zein metabolism regulation, flavonoid biosynthetic genes regulator, promoter and enhancer regions, anaerobic induction, salicylic acid responsiveness, etc. were identified in the BOR genes promoters (Additional file 3: Fig. 1a). Fourteen different cis-acting elements such as meristem expression, light responsive element, conserved DNA module array, drought-inducibility, MeJA-responsiveness, abscisic acid responsiveness, anaerobic induction, gibberellin-responsiveness cell cycle regulation, core promoter element, etc. were found in the NIP genes promoters (Additional file 3: Fig. 1b). The gene extraction and alignment using the clustalW revealed six BOR and nine NIP domains, respectively (Additional file 4: Fig. 2).

Side-by-side comparison of the phylogenetic analysis (Additional file 5: Fig. 3a and d), the conserved motif (Additional file 5: Fig. 3b and e) and the exon/intron organization (Additional file 5: Fig. 3c and f) of both the BOR and the NIP genes revealed sequence similarity among members of the same phylogenetic domain; however, the distribution of their coding sequences was unique. Ten conserved BOR (Additional file 5: Fig. 3b) and NIP (Additional file 5: Fig. 3e) gene family regions were identified, of which motifs 1, 2, and 3 were shared among all five BOR genes of the gene family, and only motif one was shared among all members of the NIP family (Additional file 6: Table 3).

Knowing the spatiotemporal expression of these genes was also essential to understand their role in B translocation. Heatmap analysis of the BOR (Fig. 6a) and NIP (Fig. 7a) genes expression pattern before B spray revealed that the expression of the NIP genes was more prominent than BOR. DlBOR5 expressed more in the SX leaf, and DlBOR4 expressed more in the YD stem (Fig. 6a).

We also observed high chlorophyll a (Fig. 2h), chlorophyll b (Fig. 2i), and total chlorophyll (Fig. 2j) concentration in the YD shoot. DlNIP8, DlNIP15, DlNIP16, DlNIP30, and DlNIP31 expressed more in the YD leaf; DlNIP23 expressed more in the YD shoot; and DlNIP1, DlNIP6, and DlNIP22 expressed more in the YD root. Whereas DlNIP11 expressed more in the SX leaf, DlNIP4, DlNIP7, and DlNIP13 expressed more in the SX stem, and DlNIP18 expressed more in the SX root (Fig. 7b). It is noteworthy that no two same BOR or NIP gene was upregulated in both SX and YP at the same time. After acclimatization, before B treatment, the appearance of DlBOR3 in the YD leaf, upregulation of DlBOR5 and DlBOR2 in the SX leaf and downregulation of the DlBOR4 in the YD stem were observed (Fig. 6b). DlNIP15 and DlNIP30 were highly expressed in YD bud; DlNIP8 and DlNIP19 were highly expressed in YD leaf; DlNIP4, DlNIP9, and DlNIP22 were highly expressed in SX leaf; DlNIP23 and DlNIP29 were highly expressed in YD stem; DlNIP6 and DlNIP31 were highly expressed in YD root; DlNIP18 and DlNIP32 were highly expressed in SX root (Fig. 7b). After B treatment, most of the BOR genes were absent except for the higher expression of DlBOR2 in the SX male flower, DlBOR1 in the YD leaf, and DlBOR4 in the YD stem (Fig. 6c). With higher B concentration, the expression of BOR family genes gets diminished. A similar pattern was observed for the NIP gene family as we only found higher expression of DlNIP1 in SX female flower; DlNIP9 in SX Leaf; DlNIP22 in SX root; and DlNIP10 was highly expressed in YD’s roots, stems, and leaves. We also observed higher B content in the vegetative organs B untreated YD (Fig. 4), and DlNIP19 might be the reason behind it (Fig. 7b). DlNIP1 is specifically and highly expressed in the female flowers of SX (Fig. 7c), which might be why the B content of female SX flower (Fig. 4b) increased with foliar B spray, but the JA content did not (Fig. 5).

Subcellular localization

The DlNIP19 and DlNIP1 linked pBE-GFP vectors were transfected into tobacco leaf using Agrobacterium. The DlNIP19-GFP (Fig. 8a) and the DlNIP1-GFP (Fig. 8b) were observed to express and localize in the extremity of the leaves mesophyll pavement cell, the cell membrane. We also used the sequence of these two DlNIPs to search the WolfPSORT [9] database and found their subcellular localization to be the membrane of the vacuole, Golgi, and endoplasmic reticulum.

Pollen germination experiment

DlNIPs linked to pBE-GFP were also used to transform SX pollen using magnetic nanocarriers MNPs, and the transformations were confirmed using confocal laser scanning microscopy. GFP expressions were observed in untransformed pollen, and pBE-GFP, DlNIP19-GFP, and DlNIP1-GFP transformed SX pollen (Fig. 9a-d), while the non-transformed pollen showed no fluorescence. Quantitative real-time polymerase chain reaction (RT-PCR) quantified the expression of transferred genes in transgenic pollens (Additional file 7: Fig. 4). We found that compared to untransformed pollen, the DlNIP19-GFP pollen germinates better (41.18%) at low B concentrations (0.01%) but decreased significantly at intermediate (0.02%) and higher (0.05%) concentrations (22.78%, 20.19% respectively) (Fig. 10). In contrast, compared to untransformed pollen, the DlNIP1-GFP pollen germinates better (40.70%) at higher (0.05%) B concentrations but significantly lowered at low (0.01%) and intermediate (0.02%) concentrations (24%, and 18.75% respectively) (Fig. 10). Untransformed and empty vector transformed pollen showed a steady increase in germination with increasing B concentration (Fig. 10). Transgenic DlNIP1-SX and normal SX pollens were used to artificially pollinate YD female flower from 5 panicles of each tree (Fig. 11a) and bagged to prevent contamination (Fig. 11b). DlNIP1-SX pollinated YD Longans (YD♀ × DlNIP1-SX♂) showed increased fruit setting rate (11%) of crossbred (Fig. 11d, e) over the untransformed SX pollinated plants (Fig. 11c).

Discussion

Cell wall biosynthesis is one of the major factors influencing plant reproduction and growth. The differential expression of related genes, hormones, and the availability of nutrients such as sucrose, starch, etc., affects it [6]. B is the most abundant component of the vascular plant's cell wall (80–90% dry weight), forming borate ester cross-linked rhamnogalacturonan II dimmer essential for cell wall structure and function [8]. We observed thicker cell walls in the mesophyll cells of YD Longan, which were probably due to the higher B content in their leaves (Fig. 4a). Stronger cell walls and thicker vascular bundles (Fig. 1g) allowed YD to attain more significant translocation resulting in increased cellulose (Fig. 2e) and decreased lignification (Fig. 2f). However, cellulose and lignification of SX increased with B treatment. This, along with other changes in the leaf constituents due to B treatment, indicated the presence of a more complex system other than simple diffusion. Shireenet al. [1] also reviewed the effect of B in maintaining various cellular processes and components. They reported decreased carbohydrate metabolism, sugar transport, and photosynthesis due to low B content.

The foliar B spray has increased the fruit-setting rate (Fig. 3) in the SX more than in the YD, probably due to higher JA content in the YD compared to the SX (Fig. 5). Indigenous JA is a plant growth regulator (PGR) associated with abiotic stresses [10]. It has a complex synergistic and antagonistic relationship with other PGRs in managing stress. Under low B conditions, the JA biosynthesis gene upregulates and inhibits the growth in A. thaliana [11]. B is also known to influence pollen tube growth and pollination in hybrid Longan [6], and applying JA inhibitors such as ibuprofen and B increases the fruit-setting rate [7].

B is present in the soil as boric acid (H₃BO₃) or borate; unlike the other vital elements, B percolates as uncharged molecules rather than ions [1]. The plant has three types of boric acid transport mechanisms. The first is the simple diffusion of uncharged boric acid across lipid bilayers. The second is facilitated diffusion of boric acid via MIP, such as NIP. The third is the export of borate via BOR [3]. Passive diffusion and NIP transport are used when B is present in sufficient amounts, but BOR transporters are used when B is deficient [4]. It is shown that to maintain tissue B homeostasis, the gene expression that codes for these transporters is carefully regulated in response to the level of B present in the environment [2]. Our search found five genes of the BOR gene family (Additional file 3: Fig. 1a) and thirty-three genes of the NIP gene family (Additional file 3: Fig. 1b) differentially expressed in Longan. Among the eight cis-acting elements in BOR and fourteen cis-acting elements in NIP, several core promoters, promoter enhancers, stress-related promoters, flavonoid, and plant growth regulators-related promoters were identified. This indicates the BOR and NIP gene’s association with different stress and their role in plant growth and other physiological processes. When aligned with Citrus, Arabidopsis thaliana, and Litchi, we discovered the BOR gene family had six major clusters (Additional file 4: Fig. 2a), and the NIP gene family had nine (Additional file 4: Fig. 2b). However, Ozyigit et al. [4] reported three major BOR1 clusters and three major NIP5;1 cluster. Ten motifs were selected from the five BOR and thirty-three NIP genes to show the evolutionary relationships among the family members (Additional file 5: Fig. 3). However, in a study of NIP5;1, NIP6;1 and BOR1 genes expression among 18 different plants, Ozyigit et al. [4] reported identifying five conserved regions for NIP and BOR gene family.

Heatmap analysis of gene expression at different stages of development showed that after acclimatization, the BOR gene expression increased but decreased with increasing B concentration (Fig. 6). In their review, Miwa et al. [2] emphasized the function of BOR1 in B acquisition by root and translocation into the shoot in Oryza sativa. The expression of BOR1 in A. thaliana is controlled post-transcriptionally, where it is accumulated in the plasma membrane under low B conditions. However, in high B conditions, it is incorporated into the endosome, transported to the vacuole and degraded [12]. According to Miwa et al. [13], plants that overexpress BOR4 are better able to extend their leaves and store more chlorophyll in the shoot tissues in environments with high B availability. This coincides with our observation of stable chlorophyll concentration (Fig. 2h, i, and j) and over-expressed BOR4 in the YD (Fig. 6b).

After B spray, DlNIP19 expressed most in the YD vegetative organs, and DlNIP1 expressed most in SX females (Fig. 7c), and when injected in tobacco leaf using A. tumefacient, both were found to localize in the cell walls. Since DlNIP1 expressed more in female flowers, the DlNIP1 transformed SX pollen germinated better at increasing B concentration than the DlNIP19 transformed SX pollen (Fig. 10). In the NIP gene family tree, DlNIP1 belongs to the NIP7 cluster, and DlNIP19 belongs to the NIP4 cluster (Additional file 4: Fig. 2). Genes from the NIP4 cluster were reported to be specific to the pollen [14] and NIP7 to the anther [15]. This indicates they have a different role in B transport. In A. thaliana, the NIP5;1 facilitates the influx of B into root cells from the soil and the NIP6;1 promoter showed strong expression at nodes of the stem [2]. Ozyigit et al. [4], on the other hand, showed that, in B-deficient conditions, the protein NIP5;1 was localized to the plasma membrane of the root cap and epidermal cells and highly increased, whereas the protein NIP6;1 was implicated in B distribution but in the shoots. JA has been reported to influence pollen germination negatively [6], which could be the reason behind the lower seed-setting rate in the female YD-containing crossbreed. Higher germination of DlNIP1 in high B also indicates its role in female flowers is crucial for germination (Fig. 10). Conversely, a lower DlNIP19 germination rate suggests it is more suited for managing high B in the vegetative organs.

Due to UCI, the seed setting rate between the YD♀ × SX♂ and the SX♀ × YD♂ were 0% and 22.4% [6]. We found that the YD pollinated using DlNIP1 transformed SX pollen had a better seed setting rate than the normal YD♀ × SX♂. It demonstrates the possibility of overcoming the UCI and making YD♀ × SX♂ Longan capable of harbouring the benefiting traits of both breeds.

Conclusion

B transporter BOR and NIP gene family plays a vital role in regulating longan metabolism, pollination, and growth regulators during low, normal, and high B conditions. Our study suggests different genes of these two gene families have activated in SX and YD Longan cultivars at different stages and B stresses during their cultivation, affecting their constituents and growth regulators differently. We selected two high-expressing NIP genes to study their spatiotemporal expression and found their accumulation in the plasma membrane. This differential expression is one of the reasons for the UCI between SX and YD. However, using YD♀ × SX♂, we achieved a higher seed setting than the naturally pollinated YD. In-vitro germination experiment showed that DlNIP1 transformed pollen germinates more than the DlNIP19 transformed pollen in an increasing B level. We hope this study will inspire further research in understanding the role of these B transporters for the betterment of the development of high-yielding Longan hybrid crops.

Methodology

Plant growth, treatments, and tissue collection

The experiment was conducted in Longan Resource Nursery of Guangzhou City, Guangzhou, China. We selected ten YD and ten SX healthy, ten-year-old Longan trees with similar growth for this experiment. The foliar spray of liquid sugar alcohol boron (151 g/L) acquired from Qingdao Arthur Import and Export Trade Co., Ltd. was diluted to 150 mg/L. The solution was sprayed once every two weeks onto five trees from both varieties. Roots, stems (twigs), leaves (final mature leaves), and flowers (male and female) were collected from all the trees at the beginning and the end of the B treatment, with three replicates for each. They were partly dried to measure the total B content. The others were frozen in liquid nitrogen and stored at -70 °C to detect gene expression. We collected female flowers from B-treated and untreated plants to measure the JA content and reveal the difference in floral organ PGR content under different B conditions. We collected leaves for optical and electronic microscopic analysis to discover the cell wall structure under various B conditions.

Leaf phenotyping

Mature YD and SX leaves were collected 130 days after B treatment. The anatomical characteristics of cells and vessels were evaluated in samples of mature leaves. Tissue samples (20mm²) observed under light microscopy (LM) were collected from the middle third of the leaf between 8:00 and 9:00 am. Tissue samples selected for LM were then fixed, sliced, and stained for semithin section LM and TEM following the methodology described by Mesquita et al. [16]. The concentration of sucrose, glucose, Quina cortisol, fructose, cellulose, lignin, starch, chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids in the frozen leaf samples from B untreated and B treated plants were analyzed using tandem mass spectrometry method described by C Sousa [17].

Leaf phenotypes were visualized as mean ± standard in a bar diagram. The significant difference was calculated using the student's t-test between the two varieties.