Tolerance analysis of chloroplast OsCu/Zn-SOD overexpressing rice under NaCl and NaHCO3 stress

The 636-bp-long cDNA sequence of OsCu/Zn-SOD (AK059841) was cloned from Oryza sativa var. Longjing11 via reverse transcription polymerase chain reaction (RT-PCR). The encoded protein comprised of 211 amino acids is highly homologous to Cu/Zn-SOD proteins from tuscacera rice and millet. Quantitative RT-PCR revealed that in rice, the level of OsCu/Zn-SOD gene expression was lowest in roots and was highest in petals and during the S5 leaf stage. Moreover, the expression level of OsCu/Zn-SOD gene expression decreased during the L5 leaf stage to maturity. The level of OsCu/Zn-SOD gene expression, however, was increased under saline–sodic stress and NaHCO3 stress. Germination tests under 125, 150, and 175 mM NaCl revealed that OsCu/Zn-SOD-overexpressing lines performed better than the non-transgenic (NT) Longjing11 lines in terms of germination rate and height. Subjecting seedlings to NaHCO3 and water stress revealed that OsCu/Zn-SOD-overexpressing lines performed better than NT in terms of SOD activity, fresh weight, root length, and height. Under simulated NaHCO3 stress, OsCu/Zn-SOD-overexpressing lines performed better than NT in terms of survival rate (25.19% > 6.67%) and yield traits (average grain weight 20.6 > 18.15 g). This study showed that OsCu/Zn-SOD gene overexpression increases the detoxification capacity of reactive oxygen species in O. sativa and reduces salt-induced oxidative damage. We also revealed the regulatory mechanism of OsCu/Zn-SOD enzyme in saline–sodic stress resistance in O. sativa. Moreover, we provided an experimental foundation for studying the mechanism of OsCu/Zn-SOD enzymes in the chloroplast.


Introduction
Plants adapt to various environmental conditions by actively regulating their internal metabolism. The enzyme superoxide dismutase (SOD, EC 1.15.1.1) determines the reaction rate in any metabolic pathway in plants. SOD is the most important enzyme that responds to reactive oxygen species (ROS) and is specifically produced by the biological PSI-PSП system. SOD a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Premier 5.0 software was used to design RT-PCR primers based on the nucleotide sequence for Cu/Zn-SOD of rice (AK059841, LOC_Os08g44770 OsCu/Zn-SOD,) that were retrieved from the GenBank gene registry. Takara Ex-Taq high fidelity enzyme was used to synthesize the following primer sequences: OsCu/Zn-SOD-P1 F1: 5'-tatcatcgtcaggtcaggca-3'; OsCu/Zn-SOD-R1: 5'-acacttcagctgcaacttgc-3'. Total RNA was extracted from seedling leaves (four leaves and a bud) for the reverse transcription of the cDNA template. The OsCu/Zn-SOD gene fragment was amplified and cloned via PCR.
PCR conditions consisted of pre-denaturation at 95˚C for 5min; followed by 35 cycles of: 95˚C for 30 s, 55˚C for 30 s, 72˚C for 45s, and and an extension of 72˚C for 10 min. The PCR products were resolved by 1% agarose gel electrophoresis. After the target DNA fragment was then retrieved, they were linked with pMD18-T vector. Subsequently, they were transformed to JM109, and then sent to Beijing Genomics Institute (BGI) for restriction enzyme digestion.
Quantitative RT-PCR analysis of the tissue-specific expression of OsCu/ Zn-SOD gene and expression characteristics under saline alkali stress Tissue samples were collected from Longjing 11 plants that were pot-cultured in a greenhouse. The tissue samples were frozen in liquid nitrogen at −80˚C. To simulate salt and sodic stress, plants were cultured in Hough nutrient solution with 150 mM NaCl and 60 mM NaHCO 3 for 2, 4, 8, 16, and 24 h. Water was used as the control treatment (0 h stress time). Three leaves and a bud were harvested from each plant. Roots and leaves were also collected and frozen in liquid nitrogen at −80˚C.
Total RNA was extracted via Trizol one-step method. First, 1 μg of total RNA template was reverse-transcribed into cDNA and then diluted ten-fold for quantitative PCR. OsCu/Zn-SOD gene transcripts were identified via quantitative RT-PCR (qRT-PCR) with the following specific primers for OsCu/Zn-SOD-RT-P4: qRT-F: 5 0 -tatcatcgtcaggtcaggca-3 0 ; qRT-R: 5 0 -acacttcagctgcaacttgc-3 0 , Amplification was conducted with the following protocol: 95˚C for 5min; followed by 40 cycles of: 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s. PCR was conducted with 2× Brilliant III SYBR Green QPCR Master Mix (Agilent). Data were acquired on the MxPro-Mx3000P system. All gene expression levels were normalized to that of the internal reference actin1 gene (Actin1-F: 5 0 -cttcatag gaatggaagct gcgggta-3 0 ; Actin1-R: 5 0 -cgacca ccttgatcttcatgctgcta-3 0 ). No template control (NTC) was set at the same time. Each reaction was repeated thrice. After PCR, relative gene expression was calculated by the system based on the relative quantitative method with the following formula: Rel Exp = 2 −ΔΔCt , where ΔCt = Ct(OsCu/Zn-SOD) − Ct(Actin1); ΔΔCt = (Under stress treatment ΔCt) − (Not under treatment ΔCt). A chart was generated based on fold changes in expression. Excel format was used for the output.
Detection of the subcellular localization of OsCu/Zn-SOD pBI121-OsCu/Zn-SOD::GFP and the chloroplast marker mcherry were transformed into epidermal cells of onion via particle bombardment [24]. Then the onion epidermal cells were placed onto a tablet and the transformants were incubated for 18-24 h. The GFP fluorescence was observed under a confocal microscope (Olympus).

Tolerance analysis of OsCu/Zn-SOD-overexpressing transgenic rice
Longjing 11 was genetically transformed with the 35S::OsCu/Zn-SOD construct. After rice Longjing 11 seeds were sterilized, 2 mg/L 2, 4-dichlorophenoxyacetic acid was added to the culture medium for callus induction. The calluses were cultured in an illuminated incubator at 30˚C for 3 weeks. Naturally split embryogenic calluses were screened on a clean workbench with tweezers. The selected calluses were then co-cultured after 1 week. Agrobacterium was treated with electroporation. Then, pCXSN::OsCu/Zn-SOD plasmid DNA monoclone was placed into 4 ml YEP culture medium with 50 mg/L Kana and 0 mg/L Hyg and then shaking culture at 28˚C and 250 rpm for approximately 20-36 h until reaching an OD λ = 600 of approximately 0.5. The culture supplemented with acetosyringone (AS, its final concentration is 100 m mol/L) were used to infect the calluses [26,27].
Total RNA was extracted from transgenic, NT, and T3-#1, #2, #3 lines via Trizol one-step method and the 5 μg of the total sample was used for denaturation at 65˚C for 10 min. After separation by the 1.5% formaldehyde-agarose gel and then the sample was transferred to the Hybond-N + cellulose nitrate membrane. RNA was cross-linked to the membrane using ultraviolet irradiation. The membrane was crossed by DIG-chloroplast precursor OsCu/Zn-SOD (AK059841) DNA specific probes at 50˚C for 12 h using CDP-Star TM reagent (Roche company). Signals were detected using LAS 4000 imaging analyzer [29].
NT and T3-#1, #2, #3 seeds were disinfected with 3% H 2 O 2 for 30 min. The seeds were first cultivated at 30˚C to investigate resistance to 0, 125, 150, 175 mM NaCl stress during germination. Germination rate, fresh weight, plant height, and MDA content were determined after 7 days. MDA content under stress treatment was determined via thiobarbituric acid-reactivesubstance assay [30].
The growth characteristics of NT and T3-#1, #2, #3 under NaHCO 3 stress were detected. Germination was accelerated at 30˚C in an incubator for 5 days. The uniform-sized seedlings were transferred to Hough stress treatment solutions with 0, 5, 7.5, 10 m mol/L NaHCO 3 (each treatment replicated three times). Plants were grown in a culture room at 28˚C. The nutrient solution was replaced once every 2 days to its original volume. Growth characteristics, plant height, fresh weight, SOD activity, and chlorophyll changes were investigated after 21 days. NBT staining detected the content of superoxide anion in three leaves from seedlings after stress treatment.
Growth and developmental characteristics of the OsCu/Zn-SODoverexpressing rice lines under saline-sodic soils NT and T3-#1, #2, #3 seeds were planted in pots to simulate saline-sodic stress in a field experiment during spring. Field soil and saline-sodic soil patches were obtained from Songneng. Plain and mixed in a basin at ratios of 1:0, 2:1, and 1:1. Groundwater was used for irrigation. Each pot contained three transgenic or NT plants. Ten pots were used for per treatment. The transplant survival rate after 1 month was investigated and plant characteristics under treatment were detected.

Results
OsCu/Zn-SOD gene cloning, sequence alignment, and phylogenetic tree construction A 726-bp-long target gene fragment was cloned via RT-PCR. The cloned fragment was then sent to BGI for sequencing via restriction enzyme digestion. As expected, the cloned nucleotide sequence was identical to OsCu/Zn-SOD (AK059841). The sequences included an ORF that was 636 bp in length and that encoded 211 amino acids. Sequence alignment revealed that Cu/Zn-SOD protein sequences from different species, such as sorghum, wheat, wild rice, millet, and A. thaliana, shared 69%-96% homology ( Fig 1A). NCBI analysis showed that the sequences contained one Cu 2+ (His-103,105,120,177) and Zn 2+ (His-120,128,137,140) binding site and SOD active domain (His-103105120137140177). The cluster analysis and phylogenetic tree of OsCu/Zn-SOD protein and 12 other Cu/Zn-SOD proteins revealed that Cu/ Zn-SOD proteins from Longjing 11 rice, wild rice, and millet clustered in the same branch, thus indicating that these three proteins shared the closest evolutionary relationship ( Fig 1B).

qRT-PCR analysis of OsCu/Zn-SOD gene expression
The specific expression of the OsCu/Zn-SOD gene in different rice tissues and organs was analyzed via qRT-PCR. Moreover, the level of transcriptional expression under NaCl and NaHCO 3 stress was detected. Results showed (Fig 2A) that the level of OsCu/Zn-SOD gene expression was in all stages of rice growth and development, and was lowest in roots and highest in flowers and during S5. However, it decreased during the L5 stage of growth. In addition, the level of OsCu/Zn-SOD gene expression increased significantly with the increasing duration of exposure to NaCl and NaHCO 3 stress (Fig 2B and 2C). The level of OsCu/Zn-SOD gene expression in leaves was significantly higher than that in the root. Under NaCl stress, the level of OsCu/Zn-SOD gene expression increased first and then decreased. The maximum peak expression in roots occurred at 8 h of stress. The maximum peak expression in leaves occurred at 16 h of stress. The relative expression under stress was 9.797-fold higher than that in the control. As a whole, the level of OsCu/Zn-SOD gene expression in leaves increased more significantly than in roots. The level of OsCu/Zn-SOD gene expression was significantly induced by NaHCO 3 stress. The maximum peak expression in leaves occurred at 8 h of NaHCO 3 stress and was 31.362-fold that of the control. The results showed the level of OsCu/Zn-SOD gene expression was significantly higher under NaHCO 3 stress than under NaCl stress. ROS were produced during salt-alkali-induced oxidative stress. We speculated that OsCu/Zn-SOD plays a major role in improving the growth of rice when it is subjected to carbonate stress.

Subcellular localization of OsCu/Zn-SOD
Onion epidermal cells were co-transformed via particle bombardment. The levels of OsCu/ Zn-SOD::GFP fusion green fluorescent protein and mCherry red fluorescent protein expression were observed under confocal microscopy. The subcellular localization of OsCu/Zn-SOD in the chloroplast is shown in Fig 3A. In this image, OsCu/Zn-SOD::GFP fusion protein appears as green fluorescent dots and mcherry appears as red fluorescent dots. PCR detection was used to analyze mesophyll protoplast cells from the T1 generation of Arabidopsis that was integrated with OsCu/Zn-SOD::GFP. Cells were observed under confocal microscopy ( Fig  3B). The green fluorescent channel displayed the green fluorescence of GFP. The spontaneous  Fig 4A. The callus of Longjing 11 were genetically transformed via Agrobacterium EHA105-mediated infection. T0-generation transgenic lines were obtained and the T1 generation seeds were then harvested. Germination tests were then conducted in 50 mg/L Hyg solution. Although seeds from both NT and T1-#1, #2, #3 were able to germinate, the plumule color and radicle growth of the lines showed drastic differences after 9 days. The plumule of the transgenic lines was green, whereas that of the NT was white. In addition, transgenic lines showed normal radicle growth (S1), whereas NT showed arrested radicle growth. Unlike NT plants, transgenic Cu/Zn-SOD lines (T1-#1, #2, #3) were able to normally grow, develop, and produce seeds. Moreover, the OsCu/Zn-SOD-overexpressing T1-#1, #2, #3, #4, #5 lines are capable of growth in Hyg solution. PCR products were resolved via agarose gel electrophoresis. As shown in Fig 4B, the transgenic lines had 700 bp bands, which is the same as the template plasmid DNA. This result indicated that the 35S::OsCu/Zn-SOD gene was integrated into the genome of the transgenic lines. Northern blot detected the expression of OsCu/Zn-SOD gene in T3-#1, #2, #3. Fig 4C shows the expression of OsCu/Zn-SOD in #1, #2, #3. In this image, the hybrid signal is clear and that the target gene is overexpressed in T3-#1, #2, #3. Therefore, we harvested the seeds from these plants for subsequent experiments.

Resistance of OsCu/Zn-SOD-overexpressing transgenic rice lines
Resistance to NaCl stress during germination NT and OsCu/Zn-SOD (T3-#1, #2) lines showed different seed germination rates after 9 days. Although the root and leaf growth of NT and T3- #1, #2 were the same under water treatment (Fig 5A), those of T3-#1, #2, and #3, were superior to that of NT under 125, 150, 175 mM NaCl treatment. Moreover, although germination rate was inhibited under treatment compared with that under the control, the germination rate of the overexpressing lines was higher than that of NT (Fig 5B). In addition, although leaf growth was inhibited by NaCl stress, the overexpressing lines showed better resistance to NaCl treatment and higher fresh weight than NT (Fig 5C). MDA damage to the cell membrane system occurred as a result of salt and oxidative stress. The MDA content in NT was higher than those in the overexpressing lines. (Fig 5D). NBT staining detected that: (1) there was no difference in the color of 5-day rice seedlings of NT and T3 (#1, #2, #3) which germinated under 0mmol/L NaCl stress. (2) Additionally, there was slight difference in color of 5-day rice seedlings between NT and T3 (#1, #2, #3) which germinated under 125mmol/L NaCl stress. (3) Especially, there was a big difference in color of 5-day rice seedlings between NT and T3 (#1, #2, #3) which germinated under 150mmol/L and 175 mmol/L NaCl stress, in mesocotyl (S2). This result indicated that OsCu/Zn-SOD gene overexpression decreases oxidative stress damage and the T3 #1, #2, #3 lines had a certain tolerance to salt stress during germination stage, which were validated by the SOD scavenging activity of these lines.

Growth characteristics of seedlings under NaCHO 3 stress
To detect the effect of OsCu/Zn-SOD gene overexpression on the growth characteristics of rice, we investigated the effect of NaHCO 3 stress solution on seedlings at 7 d and 21 d after germination. As NaHCO 3 concentration increased, the growth of NT and T3-#1, #2, #3 lines gradually increased. Although the roots of transgenic lines were much better than those of NT, the leaves of transgenic lines were less green relative to those of NT (Fig 6A, 6B, 6C and 6D). Plant height was not significantly different between NT and transgenic lines. In the stress solution containing 5, 7.5, 10 mM NaHCO3, T3-#1, #2, #3 lines were significantly taller than NT ( Fig  6E). The fresh weights per plant of T3-#1, #2, #3 were significantly greater than those of NT ( Fig  6F). The SOD activity of transgenic lines was significantly higher than that of NT (Fig 6G). The chlorophyll content of plant leaves from T3-#1, #2, #3 lines (ρ<0.05) were significantly higher Tolerance analysis of chloroplast OsCu/Zn-SOD overexpressing rice under NaCl and NaHCO 3 stress than those from NT (Fig 6H). The phenotypic and physiological indicators revealed that the lines of OsCu/Zn-SOD gene overexpression were resistant to NaHCO 3 stress, as well as confirmed that the OsCu/Zn-SOD gene increases the resistance of rice to saline-sodic stress during the late stage of germination by increasing ROS-scavenging activity.
To detect the relative concentration of superoxide anion in the leaf tissue of OsCu/Zn-SOD -overexpressing lines, we detected the difference in superoxide anion concentration between transgenic and NT via NBT staining method. The color depth of the stain reflects the relative content of superoxide anions (Fig 7). The microscopic observation of the OsCu/Zn-SOD-overexpressing lines was different from that of NT: as NaHCO 3 concentration increased, the blue color of the stain gradually deepened. Therefore, the relative concentration of superoxide anion in OsCu/Zn-SOD-overexpressing lines was lower than that in NT. This result indicated that SOD activity significantly was increased via OsCu/Zn-SOD overexpression, thus enhancing superoxide anion scavenging. This phenomenon improved the growth of transgenic lines as well as enhanced photosynthesis, which was consistent with the high chlorophyll content under NaHCO 3 treatment.

Growth characteristics OsCu/Zn-SOD-overexpressing and NT lines under saline-sodic stress in the field
To simulate rice cultivation in saline-sodic soils during spring, we irrigated transgenic and NT seedlings with groundwater in saline-sodic soil. The different survival characteristics of the two lines after one month (Fig 8). Seedling survival rate was not significantly different (96.667% vs 93.334%) in field soil. However, when grown in field soil with a 2:1 ratio of salinity to sodicity, seedling survival rate was significantly different. The survival rate of OsCu/Zn-SOD-overexpressing lines was 73.33%, whereas that of NT was 40%. When grown in field soil with a 1:1 ratio of salinity to sodicity, the seedling survival rate of OsCu/Zn-SOD-overexpressing lines was 25.19%, whereas that of NT was 6.67%. Effective tillering rates were investigated in field experiments during autumn. When grown in field soil with a 2:1 ratio of salinity to sodicity, OsCu/Zn-SOD-overexpressing lines had 13 tillers/plant and a seed setting rate of 52.5%; NT plants had 8.012 tillers/plant and a seed setting rate of 49.3%. When grown in field soil with a 1:1 ratio of salinity to sodicity, OsCu/Zn-SOD-overexpressing lines had more tillers (7.122/plant) than NT (4.318/plant). Furthermore, thousand-seed weight analysis showed that OsCu/Zn-SOD-overexpressing lines (average 20.6 g/thousand seed) performed better than NT (average 18.15 g/thousand seed) in terms of yield. Although OsCu/Zn-SOD-overexpressing lines were also affected by saline-sodic damage, the lines continued growing and developing, with certain rate of seed setting. These growth responses indicated that the transgenic OsCu/ Zn-SOD rice has enhanced tolerance to saline-sodic soils.

Discussion and conclusion
Rice is a staple cereal that is widely consumed all over the world [31; 32; 33]. Regions with saline-sodic soils account for 5% of the earth's arable areas. Salinity-sodicity damage mainly occurs as a result of high Na+ ions and high pH value. Salinity-sodicity damage can slow down the growth and development of plant vegetative organs; decrease the activity of antioxidant enzymes in cells and cause the excessive oxidation of cell membranes, thus leading to metabolic disorders even acceleration of cell senescence and death [34; 35]. The lifespan of Tolerance analysis of chloroplast OsCu/Zn-SOD overexpressing rice under NaCl and NaHCO 3 stress Tolerance analysis of chloroplast OsCu/Zn-SOD overexpressing rice under NaCl and NaHCO 3 stress organisms is intrinsically associated with the level of SOD enzyme activity and the ability to resist oxidation [36]. However, plants have developed efficient strategies to alleviate the harmful effects of ROS during evolution. The enzymatic system such as superoxide dismutase (SOD) provides a dynamic balance of ROS concentration in plants [37; 38]. The OsCu/Zn-SOD gene was cloned from Longjing 11 rice in this study. The cloned OsCu/Zn-SOD gene encodes an amino acid sequence that contains Cu 2+ and Zn 2+ binding sites and a SOD active domain (Fig 1). qRT-PCR detection showed that saline-sodic stress upregulated the level of OsCu/Zn-SOD expression in transcripts, particularly in leaves (Fig 2). This result illustrated that the level of the OsCu/Zn-SOD gene expression in rice during growth and development under salt stress is similar to that of the cytoplasmic copper/zinc superoxide dismutase (Cu/ Zn-SOD) gene in the rotifer Brachionus calyciflorus under temperature and hydrogen peroxide (H 2 O 2 ) stress [39]. The high level of OsCu/Zn-SOD expression in leaves is related to its subcellular localization in the chloroplast. The subcellular localization of a protein provides indirect evidence for protein function, which could be further elucidated and verified in future experiments. Protein synthesis in the cytoplasm is guided by a protein screening signal. Proteins may be transported to a specific organelle, secreted outside the cell, or remain in the cytoplasm. Cell function will be profoundly affected if protein localization deviates from the norm [40]. To verify the localization of OsCu/Zn-SOD protein in the chloroplastid (Fig 3), we used Tolerance analysis of chloroplast OsCu/Zn-SOD overexpressing rice under NaCl and NaHCO 3 stress epidermal cells of onion via particle bombardment and the localization of OsCu/Zn-SOD protein in the chloroplastid indicates its function in active oxygen scavenging. Saline-alkali stress induces the excessive production of superoxide anion (O 2 − ), hydrogen peroxide (H 2 O 2 ), and other ROS. What's worse, under the saline alkali environment, the activity of the antioxidant enzyme protection system was decrased, which causes ROS overproduction and disturbs clearing balance in plants [ 34; 35; 41; 42]. This study revealed that the OsCuZn-SOD-overexpressing lines showed better resistance to NaCl stress during germination than NT (Fig 5). The transgenic lines had a significant advantage over NT in terms of root length, plant height, and fresh weight. These results are similar to those of At Cu/Zn-SOD-overexpressing alfalfa lines with enhanced low-temperature resistance [43]. With the increase of membrane lipid peroxidation since the increase in stress intensity, MDA content was increased as increased; therefore, MDA production is an important indicator of the degree of damage to the plant [44]. Although MDA content in transgenic plants and in NT increased after NaCl stress treatment, MDA content in transgenic lines was significantly lower than that in NT (Fig 6), illustrating that the overexpression of OsCuZn-SOD gene enhanced ROS scavenging, reduced oxidative stress damage and MDA production. These results are similar to those of overexpressing of the A. marina Cu/Zn-SOD gene under drought and NaCl stress treatment in rice [45].
Saline-sodic stress is a carbonate-dominated complex salt stress that is characterized by high Na + ions and high pH value. The present study found that the growth of OsCu/Zn-SOD-overexpressing lines and NT were all inhibited after NaHCO 3 stress treatment. Moreover, the degree of damage increased as stress intensity increased (Fig 6); this response is consistent with that under Na 2 CO 3 treatment [46]. Under 5, 7.5, or 10 mM NaHCO 3 stress treatment, the OsCu/Zn-SOD-overexpressing lines had higher values for height, fresh weight, and chlorophyll index than NT. The Cu/Zn-SOD-overexpressing tobacco plants possess resistance to NaCl, water logging, and PEG stress, as well an optimized chloroplast antioxidant system [47]. Moreover, transgenic tobacco plants that overexpress TaSOD1.1 and TaSOD1.2 have improved physiological functions under NaCl stress, as well as enhanced resistance to salt stress [48]. Under NaCl stress, Cu/Zn-SOD-overexpressing transgenic rice had higher SOD activity than NT, which was consistent with the significant increase in Cu/Zn-SOD activity under the low temperature of 3˚C [49]. The overexpression of Cu/Zn-SOD gene also improves excess superoxide radical scavenging and alleviates damage to the plant body. In addition, the content of oxygen free radicals in the leaves of overexpression Cu/Zn-SOD gene rice plants was lower than that in NT (Fig  7). Similarly, the accumulation of active oxygen free radicals decreased when Kandelia candel-derived Cu/Zn-SOD is overexpressed in tobacco [50]. The 35S promoter increased the transcription level of the Cu/Zn-SOD gene, thus increasing SOD activity. In turn, the tolerance to NaHCO 3 stress was increased via the improved ROS balance, which becomes a protective role in chloroplast photosynthesis. When cultivated under field conditions in soils with a 1:1 ratio of salinity to sodicity, the transgenic lines grew and developed better than the NT lines. Therefore, the results of the present study will establish the foundation for the cultivation of rice lines which can be grown on saline-sodic soil.