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

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.

Keywords: Borate; Boric acid; Crossbreed; Exporter; Spatiotemporal expression; Translocation.

Fig. 1

Leaf morphology of YD and SX Longan cultivars. Mature leaves of SX (a) are smaller than the YD (b) leaves. Under a light microscope, the semi-thin and longitudinal sections of SX (c) and YD (d) leaves show that the vascular bundle diameter is relatively more prominent in the YD leaves. Here, red straight lines represent vascular bundle diameters (g) measurements. TEM showed that SX (e) and YD (f) have similar cell lengths (h), and width (i), but the YD mesophile's cell wall is double in size from SX (j). The Y-axis in the graphs represents the measurements of the cell components in µm, the bars depict the mean ± standard deviation of SX and the YD biological replicates (n = 3), and the asterisks indicate significant differences between SX and YD samples at a significance of p ≤ 0.05 (*) in the student's t-test

Fig. 2

Effect of boron spray on the leaf constituents of YD and SX Longan cultivars. We measured the effect of foliar boron spray on leaf constituents such as a Sucrose, b Glucose, c Quina cortisol, d Fructose, e Cellulose, f Lignin, g Starch, h Chlorophyll a, i Chlorophyll b, j Total chlorophyll, and k Carotenoids. The Y-axis in these diagrams represents the concentration of respective leaf constituents, the bars depict the mean ± standard deviation of the biological replicates (n = 3) of SX and YD, with and without boron spray, and the asterisks indicate significant differences between boron treated and untreated SX or YD plants at a significance of p ≤ 0.05 (*), p ≤ 0.01 (**) in the student’s t-test

Fig. 3

Foliar boron spray increases the fruit-setting rate in YD and SX Longan cultivars. The Y-axis of the chart represents the fruit setting rate in percentage, the columns show mean ± standard deviation of the biological replicates (n = 3) of both SX and YD, with and without boron spray, and the asterisks indicate significant differences between boron unsprayed and sprayed plant at a significance of p ≤ 0.05 (*) in student’s t-test

Fig. 4

Effect of foliar B spray on the B content of Longan organs. The B content of SX and YD vegetative (a) and reproductive (b) organs increased after foliar B spray. The Y-axis of the chart represents the concentration of B, and the bars represent the mean ± standard deviation of biological replicates (n = 3) before B treatment, with B treatment and without B treatment of SX and YD Longan’s vegetative and reproductive organs (both male and female) and the asterisks indicate significant differences compared to before treatment replicates at p ≤ 0.005 (**), p ≤ 0.001 (***) in the 2-way ANOVA analysis

Fig. 5

Effect of B spray on JA content of Longan. Foliar B spray increases the JA content of female YD and SX Longan flowers. The Y-axis here shows JA concentrations in µg/g of SX and YD female flower with and without B spray, the bars depict mean ± standard deviation of the biological replicates (n = 3), and the asterisks indicate significant differences between the B unsprayed and sprayed plants of the same variety at p ≤ 0.05 (*) in the students t-test

Fig. 6

Heatmap-based hierarchical cluster analysis of BOR gene expression in Longan at different stages of B treatment. Longan tissue samples were collected at the beginning of the experiment (a), before starting B treatment (b), and after one month of B treatment (c). Z-score refers to high (red) and low (blue) gene expression using normalized values compared to the mean of total sequencing reads

Fig. 7

Heatmap-based hierarchical cluster analysis of NIP gene expression in Longan at different stages of B treatment. Longan tissue samples were collected at the beginning of the experiment (a), before starting B treatment (b), and after one month of B treatment (c). Z-score refers to high (red) and low (blue) gene expression using normalized values compared to the mean of total sequencing reads

Fig. 8

Subcellular localization of GFP tagged DlNIP19 and DlNIP1 in Nicotiana benthamiana. The inserted DlNIP19 (a) and DlNIP1 (b) genes are localized in the cell membrane of the tobacco leaves epidermal cells

Fig. 9

Expression of DlNIP genes. a Untransformed pollen. b Pollen transformed with empty pBE-GFP plasmid. c Pollen transformed with DlNIP19-pBE-GFP plasmid. d Pollen transformed with DlNIP1 pBE-GFP plasmid

Fig. 10

Effect of incrementing B on pollen germination rate. With increasing B concentration in the solid medium, untransformed SX pollen germination gradually increased; the SX DlNIP19-GFP pollen germination decreased while the SX DlNIP-GFP pollen increased. The Y-axis of the chart shows the percentage (%) of germination rate of 0.01%, 0.02%, 0.05% B treated non-transfected (Not-transfected), empty vector-transfected (VEC), DlNIP19-GFP transfected (DlNIP19-GFP), and DlNIP1-GFP transfected (DlNIP1-GFP) pollens. The bars represent the mean ± standard deviation of the biological replicate (n = 3) of each group, and the asterisks indicate significant differences between different transfected groups of the same B treatment at * = p ≤ 0.05, *** = p ≤ 0.001, **** = p ≤ 0.0001 in the 2-way ANOVA analysis

Fig. 11

Pollination of YD flowers with transformed SX pollens. a YD flowers are inoculated with DlNIP1-transformed SX pollen. b Flower panicles are bagged after pollination. c YD fruit setting after one month of natural pollination. d YD fruit setting after one month of artificial pollination. e The fruit setting rate of the DlNIP1 transformed SX pollen inoculated YD panicles. The chart’s Y-axis shows the fruit setting rate of the non-transfected and transfected pollen germinated YD flowers. The bars represent the mean ± standard deviation of the group's biological replicates (n = 3) with asterisks indicating significant differences between groups at * = p ≤ 0.05 in a student's t-test