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).