Effects of Manganese on Some Biochemical Indices of Rice (Oryza sativa L.) Crop in Acid Soil Condition

It’s known that Manganese (Mn) is toxic when its concentration goes beyond a critical limit of 23ppm in plants. In Assam soil, available Mn concentration is 3-52ppm. There is paucity of information how the excess Mn brings about biochemical changes in upland rice crop grown in acid soil condition of Assam. A dose response study of Mn (0, 10,20,30 ppm Mn as MnSO4H2O) on ten upland rice genotypes (Kanaklata, Mulagabharu, Kapilee, Disang, Kolong, Joymoti, Jyoti Prasad, Luit, Lachit and Chilarai) was accomplished applying Mn as foliar spray (each 1000cm 3 ) through panicle initiation to heading stage (60-70 days after sowing:DAS). At lower dose of Mn treatment, there were higher increments in total Chlorophyll (27.17%), Chlorophylla (26.72%) and Chlorophyll-b (27.77%), NR activity (35.39%), Carbohydrate content in grain (9.04%), and Cell membrane stability (37.06%). The highest dose of Mn treatment (30ppm) reduced all these physiological attributes in the study. However, Mn content in shoots (87%) and grain (87.9%), distribution of Mn contents in intercellular (84.21%), exchangeable locations (74.84%) increased significantly by 30ppm Mn treatment. Based on the overall, Kanaklata followed by Chilarai was superior in terms of the biochemical traits deviated by Mn treatments in the present studies.


INTRODUCTION
Manganese (Mn) being an essential micronutrient imparts metabolic roles within different plant cell compartments. However, Mn beyond a critical limit interfere biochemical processes, and Mn becomes toxic to all susceptible plants on acid soils in contrast to calcareous soils, and in organic soils (Alejandro et al., 2020). The manifestation of both interveinal and marginal leaf chlorosis, necrotic leaf spots are plausible due to the biochemical changes caused by excess Mn in plants ( Mora et al., 2009). with NPK @60:40:20 Kgha -1 as Urea (23.25g=half dose of N as), SSP(89.25g) & MoP (11.857g) as basal; and rest half of N (11.625g urea) while the crop was at the maximum tillering stage. A regular irrigation (2-3cm water) was supplied from transplanting to harvesting time of the crop. The experimental site was kept weed free and prophylactic measures were followed as and when required. The abaxial leaves were misted with Mn (@0,10,20 and 30ppm) as MnSO 4 .H 2 O (MW:159.08g) solution (1000cm 3 ) in three splits during tillering to heading stage (60-70 DAS) using a hand sprayer. All the treatments were given in an isolation manner to avoid any contamination. Chlorophyll contents in leaf were estimated by non-maceration, Dimethyl Sulphoxide (DMSO) method (Arnon, 1949). Mn content in shoot at Heading stage (70DAS) and in grain at harvest was solubilised by digestion with a mixture of sulphuric and nitric acids, and its contents were estimated using the new extraction spectrophotometric method with Methylene Blue (MB) referred by Beck et al. (2006) In vivo nitrate reductase activity was estimated experimentally at 540nm (Thimmaiah, 1999). Total carbohydrate content in grain was estimated following Anthrone method (Hedge et al., 1962). Mn within the leaf cells were separated into intercellular and exchangeable fractions using a sequential elution procedure (Bates, 1992;Badacsonyi et al., 2000;& Bharali & Bates, 2002). Samples of hydrated apices were placed in 100 X 25 mm glass specimen tubes ("master tubes") paired with identical ("slave") tubes to which extracts could be added, and linked to them via fine plastic tubes. Solutions were added and withdrawn from the master tubes using a manifold system attached to a reversible pump, and during incubation, the samples were agitated by bubble streams. Freely soluble ions on the samples were removed with three serial incubations (each 10 min, 20 cm 3 ) with double distilled water (DDW). Exchangeable Mn held on the fixed negative charges of the cell walls were eluted by two treatments (each 1h, 20 cm 3 ) with 25 Mm SrCl 2 (Wells and Brown, 1987). After extraction of Mn, the leaf samples were oven dried (at 80 0 C) to a constant weight. Cell Membrane stability (CMS) was measured for cellular injury caused by lethal Mn, if any, as suggested by Sullivan and Rose (1972). The CMS was determined using the same sample extracts for inter and exchangeable cellular ions. The electrical conductivity readings of these extracts for the samples collected from the experimental treatments were used to compute CMS.

RESULTS AND DISCUSSION
The soil used in the pots was acidic in nature as the pH (4.92-5.62) was consistent throughout the experiment. Of course, there was 20.1% increase in soil pH at harvest stage of the crop over the initial soil pH. The exchangeable Mn content in soil varied (27.426 ppm to 32.2-ppm) during the crop growth stages. So, the Mn status of the soil was medium (Basumatary et al., 2014). Despite the congenial climatic conditions during the growing season, biochemical deviations were brought about by the supra optimal concentration of Mn in the upland rice genotypes.
There were significant effects of Mn on total chlorophyll, Chlorophyll-a and Chlorophyll-b contents in the varieties (Table  1, 2, and 3). The highest total chlorophyll content in leaf tissues was found at 10ppm Mn (1.810 mg g -1 f.w.) followed by (>) control (1.473 mg g -1 f.w.)>20 ppm Mn (1.467 mg g -1 f.w.), and the least was at 30ppm Mn (1.410 mg g -1 f.w.). On an average, the highest total chlorophyll content in leaf tissues was recorded in Kanaklata (1.927 mg g -1 f.w.)> Luit (1.705 mg g -1 f.w.)>Chilarai (1.694 mg g -1 f.w.), while the lowest was recorded in Kolong (1.109 mg g -1 f.w.). The total chlorophyll content increased significantly by 10ppm Mn in Kanaklata (27.17%)>Disang (23.68%) as compared with control. There is disorganisation of the normal arrangement of the grana in chloroplast Ohki, 1988) in presence of critical Mn concentration. The oxidation of Mn in chloroplasts by light activated chlorophyll generates reactive oxygen species, and thereby it degrades chlorophyll (Balidisserotto et al., 2007). The highest chlorophyll-a content in leaf tissues was produced, too, by 10ppm Mn (0.900 mg g -1 f.w.)> control (0.732 mg g -1 f.w.)>20 ppm Mn (0.730 mg g -1 f.w.), and the least was at 30ppm Mn (0.710 mg g -1 f.w.). On an average, among the varieties, the highest chlorophyll-a content in leaf tissues was recorded in Kanaklata (0.955 mg g -1 f.w.)> Luit (0.848 mg g -1 f.w.)>Chilarai (0.842 mg g -1 f.w.), while the lowest was recorded in Kolong (0.541 mg g -1 f.w.). So, Chlorophyll-a content increased significantly from 3.17% (Disang) at 10ppm Mn to 26.72% (Kanaklata) up to 20ppm Mn, but at 30 ppm Mn, all the rice varieties showed significant reductions (1.56 to 10.79%) in chlorophyll-a content as compared to the control. In case of Chlorophyll-b, the highest chlorophyll-b content in leaf tissues was recorded in Kanaklata (0.963 mg g -1 f.w.) > Luit (0.856 mg g -1 f.w.) > Chilarai (0.850 mg g -1 f.w.), while the lowest was recorded in Kolong (0.559 mg g -1 f.w.). There was significant increase in Chlorophyll-b from 0.44% (Kapili) to 27.77% (Kanaklata) up to 20ppm Mn. However, a drastic reduction of Chlorophyll-b (0.27 to 13.40%) was found in the genotypes at 30 ppm Mn as compared to the control. At an excess of Mn, older leaves of rice developed brown spots and mild interveinal chlorosis in addition to depression in growth. Both deficiency and excess of manganese resulted in low concentration of Chlorophyll-a and Chlorophyll-b as well as reduced Hill reaction activity in leaves (Lidon et al., 2004;& Rezai & Farboodnia 2008). A significant variation of Mn content of shoot was found among the rice genotypes due to the Mn treatments (  (Marcar & Graham, 1987). There were significant reductions of NR activity commensuration with the Mn concentrations in the treatment (Fig.1) Leidi and Gomez (1985) studied the role of Mn in the regulation of soybean NR activity under light and dark conditions. An indirect role of Mn on activation of NR was reported where Mn deficient plants had lower activation of NR than the plants supplied with Mn irrespective of light regimes. Carbohydrate contents in grains at harvest reduced significantly due to Mn treatments (Fig.2). The highest carbohydrate content in grain was found at 10ppm Mn (7.69 mgg -1 d.w)> 20ppm (7.26 mgg -1 d.w)>control (7.24 mgg -1 d.w), and the lowest was at 30ppm Mn (6.55 mgg -1 d.w). On an average, among the genotypes, the highest carbohydrate content was in Kanaklata (9.06 mgg -1 d.w)> Chilarai (8.68 mgg -1 d.w)>Jyoti prasad (8.45 mgg -1 d.w), while the lowest carbohydrate content in grain was recorded in Kolong (4.91 mgg -1 d.w). The carbohydrate content in grain was increased significantly by 10ppm Mn in Kanaklata (9.04%)> Disang (6.89%). In case of treatment 20 ppm Mn, Lachit (5.27%) showed significant increase in the carbohydrate content in grain> Joymoti (4.22%) and Disang (6.04%), However, at 30 ppm Mn treatment, all the rice varieties showed significant reductions (1.49 to 15.72%) in the carbohydrate content in grain as compared to the control. As per the report of Kumar et al. (2017), grain quality including carbohydrate viz., amylase contents in grains can be enhanced with application of Mn @5kgha -1 .

Varieties
There were significant changes of intercellular manganese contents due to Mn treatments (Fig.3). The highest intercellular manganese content was detected at 30ppm Mn (0.042 mgg -1 d.w.)>20ppm Mn (0.030 mgg -1 d.w.)>10ppm Mn (0.021 mgg -1 d.w.), and the lowest of intercellular Mn was at controlled plants (0.014 mgg -1 d.w.). Among the genotypes, the highest intercellular manganese content was recorded in Chilarai (0.038 mgg -1 d.w.)>Joymoti (0.037 mgg -1 d.w.)>Kanaklata (0.030 mgg -1 d.w.), while the lowest was recorded in Mulagabharu>Kolong (0.021 mgg -1 d.w.). The intercellular Mn content increased significantly in variety Joymoti (84.21%) at 30ppm Mn treatment>Lachit (78.18%). In case of 20 ppm Mn, Joymoti (80.85%) showed significant increase the intercellular Mn>Disang (70.27%). Of course, 10 ppm Mn also enhanced intercellular Mn significantly (16.66 to 75.67%) in the varieties as compared with control. Similarly, the highest exchangeable manganese content was estimated at 30ppm Mn (0.140 mgg -1 d.w.) > 20ppm Mn (0.109 mgg -1 d.w.)>10ppm Mn (0.081 mgg -1 d.w.), and the lowest at the control 0ppm Mn (0.057). On an average, the highest exchangeable manganese content was recorded in Joymoti (0.149 mgg -1 d.w.)> Chilarai (0.147 mgg -1 d.w.)>Kanaklata (0.122 mgg -1 d.w.), while the lowest of it was recorded in the Jyoti prasad (0.064 mgg -1 d.w.). The exchangeable Mn content increased significantly commensuration to the concentration of Mn in the treatments (Fig. 4). So, at 30ppm Mn, variety Lachit (74.84%) followed by (>) Chilarai (73.33%) showed the highest increment of Mn. In case of 20 ppm Mn also, the variety Lachit (72.48%) showed significant increase in the exchangeable Mn > Chilarai (64.86%). Even, at10 ppm Mn treatment, all the rice varieties showed significant increases in the exchangeable Mn (13.91% to 56.98%) as compared to the control. The concentrations of Mn in cellular locations are highly dependent on plant species and genotypes (Husted et al., 2009;Broadley et al., 2012;& Fernando & Lynch, 2015). It had been established that excess Mn may be stored in vacuoles (Duˇci´c & Polle, 2007;& Dou et al., 2009), cell walls (Führs et al., 2010), and distributed to different leaf tissues (Fernando et al., 2006a,b). In the present study, higher concentrations of Mn were detected in the intercellular and exchangeable fractions of tissues in case of 30ppm Mn treatment irrespective of the genotypes. Plausibility is that cell membrane permeability increased gradually (with lowering CMS) in commensuration with the increases in Mn concentration in the treatments compared to the control as described below. Plant cells treated with higher Mn concentration had higher alteration of membrane permeability. So, higher leakage of Mn from the protoplast was evidenced by the recovery of more amounts of intercellular and exchangeable Mn contents in the plant tissues as compared to the controlled tissues in the experiment.
There were significant variations of cell membrane stability (CMS) among the genotypes due to Mn treatments (Fig.5) (Lynch & St. Clair, 2004;& Doncheva et al., 2009). The ROS contributes to the peroxidation of lipids present in plasma membrane leading to fracture of membrane, and causing alteration of permeability properties of the membrane. High concentration of Mn in plant cell causes swelling of thylakoids and changes membrane stability of the internal organelles like chloroplast (Hauck et al., 2003). In the current study, ROS might have caused lipid breakdown in membrane, induced cellular plasmolysis and lowered CMS (Pryor & Lightsey, 1981 in the plants treated with 30ppm Mn. Plants treated with the supra optimal concentration of Mn reduced inter cellular and exchangeable cations viz., calcium, magnesium, and potassium etc in plants (Horst & Marchehner,1978). Calcium being integral component of membrane helps maintain CMS (Legge et al., 1982, & Bharali & Bates 2004). The plant cell may become vulnerable to solute leakage due to the ROS attack on the membrane. Calcium ions bind with modulator proteins e.g. Calmodulin (Dieter, 1984), and serves as chemical signalling that in some cases equips the plant to resist external stresses (Bharali & Bates, 2004). These possibilities have not been explored meticulously in the present studies.