Expression pattern of BnPHT5;1b genes
We further focused on the expression pattern of BnPHT5;1b genes in B. napus . To this end, the expression levels ofBnA09PHT5;1b and BnCnPHT5;1b genes in the leaves and roots at 5 µM, 100 µM and 1000 µM Pi supply were measured (Figure 2a). Expression of BnA09PHT5;1b and BnCnPHT5;1b was greater in leaves than in roots (Figure 2a). BnA09PHT5;1b was induced by Pi supply in all tissues except in young leaves at 5 µM and 100 µM Pi supply (Figure 2a). The expression of BnCnPHT5;1b in roots was higher at 100 µM and 1,000 µM Pi supply than that of 5 µM Pi supply, and that in the old and mature leaves was similar between at 5 µM and 100 µM Pi supply but increased greatly at 1,000 µM Pi supply (Figure 2a). Interestingly, downregulation of BnCnPHT5;1b in young leaves was observed at 100 µM Pi supply compared with 5 µM and 1,000 µM Pi supply (Figure 2a). To understand the details of tissue expression, we established transgenic Arabidopsis lines expressingProBnA09PHT5;1b: β-glucuronidase (GUS ) andProBnCnPHT5;1b: GUS , respectively. GUS staining results indicated that BnA09PHT5;1b and BnCnPHT5;1b have similar tissue expression patterns with evident transcriptional activity in root vascular bundle and leaf vein (Figure 2b, c). These results suggest thatBnPHT5;1b genes may be involved in cellular Pi homeostasis in multiple tissues.
BnPHT5;1bsfunctionally complement Atpht5;1 growth defects
The high protein sequence identities of BnA09PHT5;1b and BnCnPHT5;1b and AtVPT1/AtPHT5;1 (Figure S2) imply that BnPHT5;1b proteins might have a tonoplast localization and Pi transport activity. A novel neural network-based model, Alphafold provides accurate protein 3-D structure prediction (Jumper et al., 2021). Alphafold was used to predict the 3-D structure of BnPHT5;1b proteins and to compare them with AtVPT1. As expected, both BnA09PHT5;1b or BnCnPHT5;1b showed a high degree of coincidence in 3-D structure with the AtVPT1 protein, especially in the domains composed of helices (Figure 3a, b). To validate the localization of BnA09PHT5;1b and BnCnPHT5;1b, green fluorescent protein (GFP) labeled BnA09PHT5;1b and BnCnPHT5;1b were constructed and their subcellular localization was analyzed in Arabidopsis protoplast. As shown in figure 3C, the GFP signals were observed on the tonoplast, indicating that BnA09PHT5;1b and BnCnPHT5;1b localized on the vacuole membrane. Such tonoplast localization of BnA09PHT5;1b and BnCnPHT5;1b proteins were further confirmed when 35s:GFP-BnA09PHT5;1b or 35s:GFP-BnCnPHT5;1b were co-expressed in tobacco (Nicotiana benthamiana ) leaves with γTIP:mCherry, a tonoplast marker (Figure S3).
To analyze the Pi transport activity of these two BnPHT5;1 proteins, a yeast mutant (YP100) was employed. The YP100 yeast mutant lacks the function of Pi transport, thus cannot survive (Popova et al., 2010). When induced in YP100 yeast, PHO84 (high-affinity Pi transporter) strain had much better growth than the PHO91 strain (low-affinity Pi transporter), suggesting that this yeast system can be used to assay Pi transport activity. The AtVPT1 strain, BnA09PHT5;1b strain and BnCnPHT5;1b strain had comparable growth at 30 mM Pi supply (Figure 3d), suggesting that BnPHT5;1b proteins and AtVPT1 protein may have similar Pi transport activity in yeast.
Homozygous lines of 35s:BnA09PHT5;1b-GFP/Atvpt1 and 35s:BnCnPHT5;1b/Atvpt1 were generated by Agrobacterium-mediated transformation of Arabidopsis thaliana (Clough and Bent, 1998). Col-0, Atvpt1 mutant and BnPHT5;1b transgenic lines were grown in Arabidopis nutrient solution (ANS solution) (Liu et al., 2016) for four weeks for phenotype comparison. The Atvpt1 mutant developed shorter roots and smaller shoots than Col-0 plants from 13 µM to 3,900 µM Pi supply (Figure 4a, Figure S4). However, bothBnA09PHT5;1b and BnCnPHT5;1b transgenic lines complemented the Atvpt1 growth defects at all Pi supplies though the plants were slightly smaller than the Col-0 wildtype (Figure 4a, b). Phosphorus concentrations were elevated in all BnPHT5;1b transgenic plants compared to the Atvpt1 mutant (Figure 4c). These results demonstrate that BnPHT5;1b genes can functionally complement theAtvpt1 growth defects. Moreover, overexpression ofBnPHT5;1b genes individually in Col-0 background enhanced plant adaptability to fluctuations in Pi supply (Figure S5).