3.1. Influence of As(V) with or without Si and Se on Krebs cycle intermediates
3.1.1. Pyruvic acid content
Pyruvic acid contents was increased in 3 weeks old rice seedlings under As(V) stress. In root, the said contents was increased by about 35, 44 and 55% while in shoot it was increased by about 25, 42 and 52% under 25, 50 and 75 µM As(V) treatments respectively, over control. During co-application of 25, 50 and 75 µM As(V) and Si, the said contents were increased further by about 69, 83, 90% in root and 35, 69, 77% in shoot respectively, over control [Table 1]. Under co-application of Se along with said concentrations of As(V), the pyruvic acid contents were increased more than As(V) treatments alone but less than Si + As(V) treatments, that were about 46, 51, 67% in root and 27, 47, 59% in shoot respectively, over control [Table 1].
3.1.2. Citric acid content
Citric acid contents was significantly increased in the test seedlings under As(V) stress. In root, the citrate contents was increased by about 30, 44 and 47% while in shoot it was increased by about 15, 37 and 49% under 25, 50 and 75 µM As(V) treatments respectively, over control. During co-application of 25, 50 and 75 µM As(V) and Si, the said contents were increased further by about 58, 85, 94% in root and 49, 61, 73% in shoot respectively, over control [Table 1]. Under co-application of Se along with said concentrations of As(V), the citric acid contents were increased more than As(V) treatments alone but less than Si + As(V) treatments, that were about 36, 47, 66% in root and 31, 43, 52% in shoot respectively, over control [Table 1].
3.1.3. Succinic acid content
Succinic acid contents was significantly increased in both root and shoot of 3 weeks old rice seedlings under As(V) stress. In root, said contents was increased by about 54, 67 and 76% while in shoot it was increased by about 52, 61 and 66% under 25, 50 and 75 µM As(V) treatments respectively, over control. During co-application of 25, 50 and 75 µM As(V) and Si, the said contents were increased more by about 71, 90, 96% in root and 67, 77, 81% in shoot respectively, over control [Table 1]. Under co-application of Se along with said concentrations of As(V), the succinate contents were increased more than As(V) treatments alone but less than Si + As(V) treatments, that were about 60, 71 and 81% in root and 56, 66 and 71% in shoot respectively, over control [Table 1].
3.1.4. Malic acid content
Malic acid contents was significantly increased in the test seedlings under As(V) stress. In root, the malate contents was increased by about 33, 43 and 50% while in shoot it was increased by about 22, 31 and 43% under 25, 50 and 75 µM As(V) treatments respectively, over control. Joint application of Si and said concentrations of As(V), said contents was increased more by about 58, 71, 94% in root and 51, 62, 74% in shoot respectively, over control [Table 1]. Under co-application of Se along with said concentrations of As(V), the malate contents were increased more than As(V) treatments alone but less than Si + As(V) treatments, that were about 42, 46 and 61% in root and 34, 40 and 47% in shoot respectively, over control [Table 1].
3.2. Influence of As(V) with or without Si and Se on the activities of respiratory enzymes
3.2.1. Pyruvate dehydrogenase (PDH; EC 1.2.4.1) activity
In the test seedlings, PDH activity was decreased under As(V) stress. The enzyme activity was decreased in root by about 48, 58 and 83% while in shoot by about 38, 51 and 78% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was applied along with same concentrations of As(V), relatively less decrease in PDH activity was recorded compared to As(V) treatment alone that were about 12, 21 and 26% in root and 5, 12 and 17% in shoot respectively, over control [Fig. 1(a)]. In rice seedlings, Se application did not show any notable decrease than Si application in As(V) treated media. During Se + As(V) application, the said activity was decreased by about 27, 39 and 43% in root and 15, 27 and 44% in shoot respectively, over control [Fig. 1(b)].
3.2.2. Isocitrate dehydrogenase (ICDH; EC 1.1.1.41) activity
In rice seedlings, ICDH activity was decreased under As(V) application. In root, the enzyme activity was decreased by about 64, 74 and 89% while in shoot it was decreased by about 51, 66 and 71% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was applied along with As(V) there was relatively less decrease in ICDH activity that were about 15, 24, 30% in root and 13, 19, 21% in shoot under Si + 25, Si + 50 and Si + 75 µM As(V) treatments respectively, over control [Fig. 2(a)]. During Se + As(V) application in the test seedlings, the enzyme activity was decreased by about 25, 48 and 60% in root and 23, 30 and 49% in shoot respectively, over control [Fig. 2(b)].
3.2.3. α-ketoglutarate dehydrogenase (α-KGDH; EC 1.2.4.2) activity
In the test seedlings, the α-KGDH activity was decreased under As(V) application. The enzyme activity was decreased in root by about 74, 86 and 92% while in shoot by about 64, 75 and 84% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was supplemented along with 25, 50 and 75 µM As(V) there was relatively less decrease in α-KGDH activity by about 23, 28, 38% in root and 20, 25, 32% in shoot respectively, over control [Fig. 3(a)]. During Se + said concentrations of As(V) application in the test seedlings, the enzyme activity was reduced further than Si + As(V) application but reduced less, than As(V) treatments alone that were by about 47, 57, 61% in root and 46, 52, 55% in shoot respectively, over control [Fig. 3(b)].
3.2.4. Succinate dehydrogenase (SDH; EC 1.3.5.1) activity
SDH activity was decreased in the test seedlings under As(V) application. In root, the enzyme activity was decreased by about 69, 72 and 77% while in shoot it was decreased by about 54, 60 and 65% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was supplemented along with 25, 50 and 75 µM As(V) there was relatively less decrease in SDH activity by about 18, 22, 32% in root and 10, 18, 21% in shoot respectively, over control [Fig. 4(a)]. Se supplementation in As(V) treated test seedlings reduced the said activity more than Si + As(V) treated test seedlings. During Se application along with As(V), the rate of reductions in enzyme activity were 37, 56, 62% in root and 33, 49, 56% in shoot respectively, over control [Fig. 4(b)].
3.2.5. Fumarase (EC 4.2.1.2) activity
Fumarase activity in the test seedlings increased under As(V) treatments. In root, the enzyme activity was increased by about 70, 77 and 88% while in shoot it was increased by about 66, 69 and 72% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was supplemented along with 25, 50 and 75 µM As(V) there was relatively less increase in enzyme activity that was by about 25, 33, 36% in root and 10, 21, 26% in shoot respectively, over control [Fig. 5(a)]. Se supplementation in As(V) treated test seedlings enhanced the said activity more than Si supplementation in As(V) treated medium. During joint application of As(V) and Se, the rate of increments in enzyme activity were 35, 46, 58% in root and 22, 40, 41% in shoot respectively, over control [Fig. 5(b)].
3.2.6. Malate dehydrogenase (MDH; EC 1.1.1.37) activity
The MDH activity was decreased under As(V) stress in the test seedlings. In root, the enzyme activity was decreased by about 79, 84 and 92% while in shoot it was decreased by about 61, 72 and 88% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was applied along with 25, 50 and 75 µM As(V) there was relatively less decrease in MDH activity that was by about 20, 27 and 36% in root and 19, 24 and 29% in shoot respectively, over control [Fig. 6(a)]. During joint application of As(V) and Se, the rate of reductions were narrowed down to about 47, 57, 64% in root and 31, 48, 65% in shoot respectively, over control [Fig. 6(b)].
3.2.7. Citrate synthase (CS; EC 2.3.3.1) activity
The CS activity was decreased under As(V) stress in 3 weeks old rice seedlings. In root, the enzyme activity was decreased by about 76, 86 and 91% while in shoot it was decreased by about 62, 69 and 76% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was applied along with 25, 50 and 75 µM As(V) there was relatively less decrease in CS activity by about 22, 30 and 34% in root and 10, 23 and 27% in shoot respectively, over control [Fig. 7(a)]. During joint application with As(V) and Se, the rate of reductions were more than Si + As(V) but less than As(V) treatments only that were by about 34, 48, 51% in root and 26, 29, 37% in shoot respectively, over control [Fig. 7(b)].
3.3. Influence of arsenate with or without silicate and selenate on GABA accumulation and activities of its regulatory enzymes
3.3.1. γ-aminobutyric acid (GABA) content
In rice seedlings, GABA contents increased in As(V) treatments. The enhancement in GABA contents were about 54, 72, 97% in root and in shoot by about 31, 49, 75% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was applied along with said concentrations of As(V) there was relatively less increase in the said contents that were about 18, 28, 30% in root and 6, 14, 21% in shoot respectively, over control [Fig. 8(a)]. The rate of enhancement was more during Se application along with 25, 50 and 75 µM As(V) than the jointly treated test seedlings with As(V) and Si. During joint application with As(V) and Se, the rate of increments in GABA contents were by about 31, 43, 51% in root and 18, 29, 43% in shoot respectively, over control [Fig. 8(b)].
3.3.2. Glutamate dehydrogenase (GDH; EC 1.4.1.2) activity
In the test seedlings, GDH activity was increased more under As(V) stress. In root, the enzyme activity was increased by about 43, 68 and 77% while in shoot it was increased by about 33, 44 and 60% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was applied along with 25, 50 and 75 µM As(V), there was relatively less increase in GDH activity by about 18, 24 and 32% in root and 10, 14 and 24% in shoot respectively, over control [Fig. 9(a)]. During Se + said concentrations of As(V) application in the test seedlings, the rate of increments were by about 26, 37, 48% in root and 18, 32, 40% in shoot respectively, over control [Fig. 9(b)].
3.3.3. Glutamate decarboxylase (GAD; EC 4.1.1.15) activity
In rice seedlings, GAD activity was increased more under As(V) stress. In root, the enzyme activity was increased by about 51, 64 and 90% while in shoot it was increased by about 39, 47 and 59% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was applied along with As(V), there was relatively less increase in GAD activity than As(V) treatment alone that were about 24, 26, 29% in root and 11, 17, 19% in shoot under Si + 25, Si + 50 and Si + 75 µM As(V) treatments respectively, over control [Fig. 10(a)]. The rate of enhancement was more during application of Se along with As(V) in the test seedlings than joint application of As(V) and Si. In As(V) + Se treated seedlings, the increments in the said activity were by about 47, 48, 53% in root and 26, 32, 42% in shoot respectively, over control [Fig. 10(b)].
3.3.4. GABA transaminase (GABA-T; EC 2.6.1.19) activity
In rice seedlings, GABA-T activity was increased more under As(V) stress. In root, the enzyme activity was increased by about 66, 87 and 90% while in shoot it was increased by about 41, 63 and 73% under 25, 50 and 75 µM As(V) treatments respectively, over control. When Si was applied along with said concentrations of As(V) there was relatively less increment in GABA-T activity that were about 20, 26 and 30% in root and 11, 13 and 23% in shoot respectively, over control [Fig. 11(a)]. During joint application of As(V) with Se the rate of increments in the enzyme activity were about 42, 53, 68% in root and 26, 30, 45% in shoot respectively, over control [Fig. 11(b)].
3.4. Influence of arsenate with or without silicate and selenate on polyamines content
Estimation of polyamines content by using thin layer chromatography
The polyamines viz. putrescine (Put), spermidine (Spd), and spermine (Spm) contents were detected from As(V), As(V) + Si and As(V) + Se treated rice seedlings. In both root and shoot obtained from As(V) treated seedlings, the levels of Put was increased while Spd and Spm were decreased (Fig. 12–14).
The Put contents was increased in root by about 30, 37, 45% and in shoot by about 24, 28, 36% under 25, 50 and 75 µM As(V) treatments respectively, over control. During co-application of stated concentrations of As(V) with Si, the Put contents had been detected more than As(V) treatment alone. The rate of increments were by about 52, 88, 98% in root and 38, 59 and 89% in shoot respectively, over control [Fig. 12(a)]. Se application along with As(V) could not cause more increment in Put contents than Si + As(V) treatments and increased by about 26, 39, 50% in root and 28, 44, 66% in shoot respectively, over control [Fig. 12(b)].
The Spd contents was decreased by about 45, 57, 66% in root and 44, 47, 54% in shoot under 25, 50 and 75 µM As(V) treatments respectively, over control. During co-application of stated concentrations of As(V) with Si, the level of Spd was also decreased but less than As(V) treatments alone. The reduction levels were about 7, 11, 16% in root and 5, 8 and 10% in shoot respectively, over control [Fig. 13(a)]. During application of As(V) with Se, the said contents was decreased more than Si + As(V) but less than only As(V) treated test seedlings that were by about 12, 32, 39% in root and 29, 31, 33% in shoot respectively, over control [Fig. 13(b)].
The Spm contents was decreased by about 41, 48, 60% in root and 32, 43, 53% in shoot under 25, 50 and 75 µM As(V) treatments respectively, over control. During As(V) + Si application, the Spm level was decreased less than As(V) treatment alone. The reduction levels were about 15, 23, 26% in root and 17, 21 and 28% in shoot under Si + 25, Si + 50 and Si + 75 µM As(V) treatments respectively, over control [Fig. 14(a)]. During joint application of As(V) with Se, the said contents were decreased more than Si + As(V) treatments but less than only As(V) treated test seedlings that were about 29, 34, 40% in root and 26, 34, 41% in shoot respectively, over control [Fig. 14(b)].