Seeds from 20 HGP and 10 LGP mutants along with the wild type N22 were harvested from low soil P and normal soil P field plots. Before sowing these seeds, the initial grain P content was estimated in seeds obtained from low P (LP) and normal P (NP) conditions. The difference in grain P content was insignificant between NP-HGP and NP-LGP mutants. Notably, the grain P content was 27% more in LP-HGP mutants than LP-LGP mutants (Fig. 1). The seeds obtained from LP and NP conditions were germinated in normal P soil, and the seedlings were designated as LP-HGPS (low P plot seeds-HGP seedlings), LP-LGPS (low P plot seeds-LGP seedlings), NP-HGPS (normal P plot seeds-HGP seedlings), and NP-LGPS (normal P plot seeds-LGP seedlings). Various morphological and physiological parameters of these seedlings were analyzed to assess the differential response of seed P content in germinability and seedling growth.
Initial grain P concentration in seeds collected from low P (shown in red dots) and normal P (shown in blue dots) filed plots.
LP-HGPS show better seedling growth than LP-LGPS
The root and shoot length of LP-HGPS was significantly more than LP-LGPS and N22. The mean root length of LP-HGPS and LP-LGPS was 11.04 cm and 10.26 cm at 12 days and 15.76 cm and 14.58 cm at 24 days, respectively. The mean root dry weight of LP-HGPS and LP-LGPS was 0.017 and 0.009 g at 12 days, and 0.024 and 0.022 g at 24 days, respectively. Notably, LP-HGPS, LP-LGPS and LP-N22 showed significant increase in root length as compared to NP-HGPS, NP-LGPS, and NP-N22 (Fig. 2). Shoot length was also recorded at 12 and 24 days of germination. NP-HGPS, NP-LGPS, and NP-N22 did not show any significant variation in shoot length, however, LP-HGPS showed higher shoot length than LP-LGPS at both the time points. The mean shoot length of LP-HGPS and LP-LGPS was 8.4 cm and 6.7 cm at 12 days, and 23 cm and 21.6 cm at 24 days, respectively (Fig. 3). Interestingly, one of the HGP mutants (NH4614) showed similar shoot length under LP and NP at 24 days of germination. In contrast, LGP mutants such as NH4777, NH4684, and NH4725 showed severe impact on shoot length under LP when compared with NP.
Root length of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) Root length at 12 days of germination. (B) Root length at 24 days of germination. The values represent the mean of 100 plants.
Shoot length of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) Shoot length at 12 days of germination. (B) Shoot length at 24 days of germination. The values represent the mean of 100 plants.
NP-HGPS, NP-LGPS, and NP-N22 did not show significant variation in root and shoot dry weight, however, LP-HGPS showed higher root and shoot dry weight than LP-LGPS at both the time points (Figs. 4, 5). The average root dry weight of LP-HGPS and LP-LGPS was 0.017 and 0.009 g at 12 days, and 0.024 and 0.022 g at 24 days, respectively. LP-HGPS exhibited 89% and 10% more root dry weight than LP-LGPS at 12 and 24 days (Fig. 4). The average shoot dry weight of LP-HGPS and LP-LGPS was 0.033 and 0.027 g at 12 days, and 0.054 and 0.053 g at 24 days, respectively. LP-HGPS exhibited 22% and 2% more shoot dry weight than LP-LGPS at 12 and 24d, respectively (Fig. 5). The difference between LP-HGPS and LP-LGPS was more pronounced at 12 days than 24 days old seedlings.
Root dry weight of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) Root dry weight at 12 days of germination. (B) Root dry weight at 24 days of germination. The values represent the mean of 20 plants.
Shoot dry weight of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) Shoot dry weight at 12 days of germination. (B) Shoot dry weight at 24 days of germination. The values represent the mean of 20 plants.
Seedling vigour
SVI was comparable in NP-HGPS, NP-LGPS, and NP-N22 at both the time points, but it was more severely affected in LP-LGPS than LP-HGPS (Fig. 6). The average mean SVI of LP-HGPS and LP-LGPS was 1651.8 and 1256.8 at 12 days and 3290.7 and 2680.1 at 24 days, respectively. Germination percentage of LP-HGPS and LP-LGPS was 85 and 74 percent, respectively. Notably, mutants NH4785 and NH4791 showed similar SVI under NP and LP conditions at 24 days of germination. Both of these mutants showed lesser effect of LP on SVI at 12 days of germination also. Among all the mutants, NH4688 showed highest SVI under LP and NP at 12 days of germination.
Seedling vigour index (SVI) of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) SVI at 12 days of germination. (B) SVI at 24 days of germination. The values represent the mean of 100 plants.
Chlorophyll (Chl) and fluorescence parameters
Reciprocal trend of Chl a content was observed at 12 and 24 days. At 12 days, all the mutants showed reduced Chl a under LP in comparison to NP, though the degree of reduction was more severe in LP-LGPS than LP-HGPS. On the other hand, at 24 days, all the mutants showed increased Chl a under LP in comparison to NP, and there was not much difference between LP-LGPS and LP-HGPS. Chl a did not show significant difference in NP-HGPS, NP-LGPS, and NP-N22 at both the time points (Fig. 7). LP-NH4663 showed maximum gain of Chl a when compared with NP-NH4663, while NH4618 did not show difference under LP and NP conditions at 24 days. Chl b was reduced in all the mutants under LP condition at both the time points, however, the degree of reduction was more severe in LP-LGPS than LP-HGPS (Fig. 8). Mutants NH4785 and NH4791 showed similar Chl b content at 12 days under LP and NP conditions. Similarly, fluorescence parameters such as Fv/Fm and ETR were reduced in all the mutants under LP condition at 24 days. LP-LGPS showed more reduction of Fv/Fm and ETR than LP-HGPS (Fig. 9). Fv/Fm ranged from 0.64 to 0.71 in LP-HGPS and 0.63 to 0.65 in LP-LGPS, while ETR ranged from 20.28 to 22.50 in LP-HGPS and 15.62 to 18.46 in LP-LGPS. Fv/Fm and ETR were comparable in NP-HGPS, NP-LGPS, and NP-N22.
Chlorophyll a (Chl a) content of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) Chl a at 12 days of germination. (B) Chl a at 24 days of germination. The values represent the mean of 6 plants.
Chlorophyll b (Chl b) content of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) Chl b at 12 days of germination. (B) Chl b at 24 days of germination. The values represent the mean of 6 plants.
Fv/Fm and ETR of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) Fv/Fm at 24 days of germination. (B) ETR at 24 days of germination. The leaf samples at 24th day were collected and kept in dark for 30 min before measuring the Fv/Fm and ETR. The values represent the mean of 6 plants.
P content in root and shoot tissues
The differences in root and shoot P content were significant between LP and NP seedlings and also between HGPS and LGPS at 12 and 24 days (Figs. 10, 11). Compared to NP, the P content was significantly less in LP seedlings in all the mutants but the degree of reduction was more in LP-LGPS than LP-HGPS. The root and shoot P content was 13% and 27% more in LP-HGPS than LP-LGPS at 12d. Similarly, at 24 days, LP-HGPS exhibited 10% and 14% more root and shoot P content than LP-LGPS. At 12 days, LP-HGPS showed 29% and 14% lesser P content than NP-HGPS in root and shoot while LP-LGPS showed 40% and 36% lesser root and shoot P content than NP-LGPS. At 24 days, LP-HGPS showed 29% and 2% lesser root and shoot P content than NP-HGPS while LP-LGPS showed 34% and 14% lesser root and shoot P content than NP-LGPS.
Root P content of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) Root P content at 12 days of germination. (B) Root P content at 24 days of germination. The values represent the mean of 6 plants.
Shoot P content of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) Shoot P content at 12 days of germination. (B) Shoot P content at 24 days of germination. The values represent the mean of 6 plants.
Acid phosphatase activity
Low P stress responsive enzymes such as externally secreted acid phosphatase (ESApase), root acid phosphatase (RApase), and shoot acid phosphatase (SApase) play a key role in mobilizing the organic P to available P in plants. The low P condition triggers these P responsive enzymes to compensate the P deficiency/P availability upto some extent. The ES Apase, RApase and SApase enzymes activity was induced in all the LP seedlings (Figs. 12, 13, 14). Though the percent increase (from NP to LP) of ESApase, RApase, and SApase enzyme activity was more in LGPS than HGPS, the differences in the ESApase enzyme activity among LP-HGPS, LP-LGPS and LP-N22 were not significant at both 12 days and 24 days, respectively (Fig. 12). The mean RApase enzyme activity in LP-HGPS, LP-LGPS and LP-N22 at 12d was 4.47, 5.38 and 5.77, respectively (Fig. 13). The mean RApase enzyme activity in LP-HGPS, LP-LGPS, and LP-N22 at 24 days was 2.62, 2.48 and 2.27, respectively. The mean SApase enzyme activity in LP-HGPS, LP-LGPS and LP-N22 at 12d was 3.86, 4.53 and 4.31, respectively (Fig. 14). The mean SApase enzyme activity in LP-HGPS, LP-LGPS, and LP-N22 at 24 days was 2.24, 2.27 and 2.17, respectively. Compared to 12 days old LP seedlings, the ESApase, RApase, and SApase activity was 10%, 47%, and 45% lower in 24 days old LP seedlings.
Externally secreted acid phosphatase (ESApase) enzyme activity of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) ESApase enzyme activity at 12 days of germination. (B) ESApase enzyme activity at 24 days of germination. The values represent the mean of 6 plants.
Root acid phosphatase (RApase) enzyme activity of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) RApase enzyme activity at 12 days of germination. (B) RApase enzyme activity at 24 days of germination. The values represent the mean of 6 plants.
Shoot acid phosphatase (SApase) enzyme activity of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) SApase enzyme activity at 12 days of germination. (B) SApase enzyme activity at 24 days of germination. The values represent the mean of 6 plants.
Alpha-amylase activity
Alpha-amylase enzyme (AAE) plays a significant role during the germination and later throughout the growth period of rice. AAE activity was comparable in NP-HGPS, NP-LGPS, and NP-N22 at both the time points (4 days and 8 days), but it was significantly high in LP-HGPS than LP-LGPS, and LP-N22 (Fig. 15). The average mean AAE of LP-HGPS and LP-LGPS was 7.46 and 5.16 at 4 days and 5.49 and 4.65 at 8 days, respectively. The AAE activity was significantly more in NP than LP condition in all the mutants and N22.
Alpha amylase enzyme (AAE) activity of seedlings obtained from germination of seeds harvested from low P (shown as red dots) and normal P (shown as blue dots) fields. Wild type N22 (W), high grain P mutant seedlings (HGPS), and low grain P mutant seedlings (LGPS) were grown under normal P condition. (A) AAE activity at 4 days of germination. (B) AAE activity at 8 days of germination. The values represent the mean of 10 plants.
Correlation studies
Correlation analysis of various parameters taken from 12 days old seedlings was performed. In LP-HGPS, seedling vigour was positively and significantly correlated with germination percentage, root length, and shoot length (Fig. 16A). Root dry weight was positively correlated while shoot length was negatively correlated with grain P content. In LP-LGPS, root length was positively correlated with RApase but negatively correlated with SApase. Similarly, P content in root and root dry weight showed significant positive correlation with Chl b content. RApase showed significant negative correlation with Chl a content (Fig. 16B). These correlations were not noticed in NP-HGPS and NP-LGPS (Fig. 16C,D).
Correlogram of traits under LP and NP conditions in HGPS and LGPS at 12 days. The scale represents Pearson correlation values. Blue and red colours represent negative and positive correlation, respectively. root length (rl), shoot length (sl), root dry weight (rdwt), shoot dry weight (sdwt), P content in root (pr), P content in shoot (ps), P content in grain (pg), externally secreted acid phosphatase (esa), root acid phosphatase (ra), shoot acid phosphatase (sa), grain length (gl), grain width (gw), grain area (ga), chlorophyll A content (chla), chlorophyll B content (chlb).
At 24 days, LP-HGPS showed significant and positive correlation of germination%, root length, and shoot length with seedling vigour. FV/Fm and ETR showed significant positive correlation with root dry weight. Chl a showed significant positive correlation with SApase (Fig. 17A). In LP-LGPS, shoot length was negatively and significantly correlated with SApase, while Chl b was negatively correlated with Fv/Fm, RApase, ES Apase, and root P content. P content in root was positively and significantly correlated with ES Apase, RApase, and Fv/Fm. Shoot P content was negatively correlated with root P content, ES Apase, and Chl a (Fig. 17B). These correlations were not observed in NP-HGPS and NP-LGPS (Fig. 17C,D).
Correlogram of traits under LP and NP conditions in HGPS and LGPS at 24 days. The scale represents Pearson correlation values. Blue and red colours represent negative and positive correlation, respectively. root length (rl), shoot length (sl), root dry weight (rdwt), shoot dry weight (sdwt), P content in root (pr), P content in shoot (ps), P content in grain (pg), externally secreted acid phosphatase (esa), root acid phosphatase (ra), shoot acid phosphatase (sa), grain length (gl), grain width (gw), grain area (ga), (fvfm) electron transport rate (etr), chlorophyll A content (chla), chlorophyll B content (chlb).
Gene expression analysis
To decipher the relative expression of phosphate starvation responsive (PSR) genes, a set of seven P transporter genes was selected based on their critical roles in P metabolism28. The expression level of all the genes was similar in NP-HGPS, NP-LGPS, and NP-N22. While comparing the expression level of genes in LP with NP, all the seven genes showed a distinct expression pattern among LGPS and HGPS. In root of 12 days old seedling, these genes showed up-regulation in all the LGPS, while in root of 24 days old seedlings, OsPT6, OsPT8, and OsPT4 showed up-regulation in HGPS but OsPT2, and OsPT10, OsPT1, and OsPAP10a showed up-regulation in LGPS (Fig. 18). Similar analysis was performed in shoot tissue of 12- and 24-days old seedlings. At 12 days, shoot of HGPS showed up-regulation of OsPT4, OsPT2 while LGPS showed up-regulation of OsPT6. Other genes did not show a distinct expression between LGPS and HGPS. At 24 days, shoot of HGPS showed up-regulation of OsPT4 and OsPT10 while LGPS showed up-regulation of OsPT1, OsPT2, OsPT8, and OsPAP10a. OsPT6 did not show a distinct expression between LGPS and HGPS (Fig. 19).
Heatmaps of relative expression levels of the genes involved in the P uptake and transport. Expression of these genes in seedlings obtained from germination of seeds harvested from low P plot was compared with the seedlings obtained from germination of seeds harvested from normal P plot. (A) Relative expression levels of genes in roots of 12 days old seedlings. (B) Relative expression levels of genes in roots of 24 days old seedlings. LGPS mutants and WT are shown by red bar.
Heatmaps of relative expression levels of the genes involved in the P uptake and transport. Expression of these genes in seedlings obtained from germination of seeds harvested from low P plot was compared with the seedlings obtained from germination of seeds harvested from normal P plot. (A) Relative expression levels of genes in shoots of 12 days old seedlings. (B) Relative expression levels of genes in shoots of 24 days old seedlings. LGPS mutants and WT are shown by red bar.
