Transcriptional insights into sugarcane aquaporin genes under water decit conditions

Water decit in soil during formative growth stage adversely hinders the crop productivity. Plant develop a key chain of mechanisms to cope these strains. Characterization of genotypes under water decit will provide the basis for breeding new germplasm for ecient utilization of water and nutrients and adaptation to water stress. To achieve this, two tolerant (Co 98014 and Co 0118) and two sensitive (CoJ 85 and Co 89003) sugarcane genotypes were assessed for antioxidant response followed by differential expression of three aquaporin genes (ShPIP2-1, ShPIP-5 and ShPIP2-6) under two water decit conditions. The MDA and H 2 O 2 contents were signicantly higher (p < 0.05) in sensitive genotypes as compared to tolerant ones, whereas SOD activity was higher (p < 0.05) in tolerant than sensitive genotypes. The transcript analysis of AQPs reveals upregulation of ShPIP2;5, whereas down-regulation of ShPIP2;1 and ShPIP2;6 when plants were imposed to water decit conditions. The ndings under study suggested the role of PIP2 AQPs in regulation of plant water status under water decit conditions.


Introduction
Sugarcane (Saccharum o cinarum L.), an economically important and the largest area occupying Indian crop suffers due to various biotic and abiotic constraints. Among various abiotic constraints, drought or water de cit is the most drastic factor in regions suffering with water scarcity, that adversely affects this crop from initial growth to development phases (Kumar et al. 2019). Production of sugarcane is worsening with the rapid expansion of drought affected areas of the world. As a major part of sugarcane growing area in India is directly affected by drought that alters plant functioning at very early stages.
Sugarcane production can still be improved further if we can put a check on a major agricultural problem i.e. water stress. Thus, the varieties having tolerance towards drought is prior need to sustain sugarcane productivity during this changing climate scenario.
Drought stress is known to decreased chlorophyll and relative water contents, water potential and affects other various metabolic and physiological processes (Singh et al. 2016, Kumar et al. 2019. Water de cit induce reactive oxygen species (ROS) accumulation and lipid peroxidation in plants that could have an effect on membrane lipids, proteins and nucleic acids, resulting in increased membrane leakage of solutes under drought stress (Liu 2011). Thus, cellular damage is caused in plants due to oxidative stress (Miller et al. 2010). To reduce such stress, plants have developed a complex multitude of antioxidant defense systems to limit ROS and maintain redox homeostasis such as superoxide dismutase, catalase and peroxidase etc. (Kumar et al. 2019).

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In apart to the physio-biochemical changes, a coordinated set of signalling networks are involved to control stress consequences by regulating numerous of genes that encode protein for plant survival (Chaves et al. 2003). The genes having role in stress acclimation encode for chaperones (HSPs), aquaporins (water channel proteins), proteases, detoxifying enzymes and free radical scavengers.
Importance of AQPs in water transport suggest role of these proteins in various physiological responses to abiotic stresses like drought (Heinen et al. 2009). Amino acid identity of plant AQPs classify aquaporins into ve subfamilies: plasma membrane intrinsic protein (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like proteins (NIPs), small basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs) (Sakurai et al. 2005). Similarly, phylogenetic study based on nucleotides sequence revealed 33 isoforms of AQPs consisting four subfamilies i.e. 13 PIPs, 11 TIPs, 6 NIPs and 3 SIPs (de- Andrade et al. 2016). The subfamily PIP can be further subdivided into PIP1 and PIP2 (Chaumont et al. 2000), consists ve and eight proteins, respectively. Three PIP2 isoforms involved in drought stress in higher plants. The transcript study of three PIPs (ShPIP2;1, ShPIP2;5 and ShPIP2;6) evaluated in two sugarcane genotypes (IACSP94-2094 and IACSP97-7065) under water de cit through qPCR showed these isoforms were responsive to drought and their expression pattern were dependent of genotype, experimental conditions (de- Andrade et al. 2016). The isoform PIP2 exhibit more e cient water channel activity than PIP1 (Chaumont et al. 2000, Kaldenhoff andFischer 2006). The abundance of PIP2 transcripts has been established by qPCR studies suggest their presence in different tissues and organs under water stress conditions (Zhang et al. 2008, de Andrade et al.2016. Drought tolerance in sugarcane is not same in all species, besides in certain species, mechanisms can operate concomitantly producing tolerance through their amalgamated effects. No particular studies of tolerance mechanisms in response to water stress has been done in the sugarcane genotypes of semiarid region of India. Even the type of tolerance mechanism for drought tolerance are not fully understood. Thereby, breeding for drought tolerance in sugarcane require a depth understanding of divergent resistance mechanisms. Molecular techniques, particularly gene expression pro les has been proved as a reliable tool to identify transcript involved in drought tolerance (Rodrigues et al. 2009). Therefore, present study was designed on evaluation of sugarcane genotypes of semi-arid region of India through antioxidant activity and differential expression pattern analysis of aquaporin genes under different water de cit conditions.

Plant materials and experimental conditions
Two tolerant (Co 98014, Co 0118), and two sensitive (CoJ 85, Co 89003) sugarcane genotypes of semiarid region of India that have contradictory response to water stress (Kumar et al. 2019) were chosen under this study. Experimental trial was conducted under rain out shelter, eld laboratory, Department of Biotechnology, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, U.P., India and qRT-PCR was performed at DUVASU, Mathura, India. Each genotype was planted in plastic pots (39 × 33 cm 2 ) containing 25.0 kg of soil and 5.0 kg farm yard manure (FYM) in completely randomized design. Healthy and infection free sugarcane setts (2 budded) were prepared and treated prior to planting with 0.2 percent mixture of mancozeb + carbendazim for 10 min followed by chlorpyriphos 25 EC for 5 min. The soil pH, electrical conductivity and organic carbon were 7.2, 1.3 dSm -1 and 4.3 g/kg. Particle size distribution (%) was 58 (sand), 25 (silt) and 22 (clay). Three setts were planted in each pot with three biological replications. After 60 days of planting, thinning was done by leaving only three plants in each pot. Moisture of soil contents was determined as per gravimetric method. Soil moisture content obtained was 23.9% (0-20 cm soil depth) at the time of sowing and it was 18.1%, 5.9% (0-20 cm soil depth) on 135 th and 150 th day, respectively. The similar management inputs like fertilizer and insecticides for proper growth and disease control were provided. On 120 th day, samples were collected and marked as control, after that plants were imposed to water withholding. Next sampling was done on 135 th and 150 th day after imposing water de cit. The third expanded leaf from top was collected as sample in aseptic manner by minimizing activity of ribonucleases at 09:00 h, frozen in LN 2 and immediately stored at -80 °C. The samples were collected in two replications, one for analysis of antioxidant activity and another one for AQPs expression.

Climatic variables
The meteorological observations during experimental period were recorded by an automatic weather station of Indian Institute of Farming System and Research (IIFSR), Modipuram, Meerut, India. The observation recorded were min and max temp, percent relative humidity in morning and evening, average rainfall and bright sunshine for Jan-2018 to June-2018 ( Fig. 1).

Antioxidant analysis
Malondialdehyde (MDA) assay was done by the measuring thio-barbituric acid reactive substance (Heath and Packer 1968) and expressed as μM MDA/g FW using extinction coe cient of 155 mM −1 cm −1 . H 2 O 2 content was measured by the peroxidase coupled assay according to Veljovic-Jovanovic et al. (2002). Superoxide dismutase (SOD) measurement was performed by nitroblue tetrazolium (NBT) method of Beyer and Fridovich (1987) and expressed as units/mg protein.

RNA extraction and cDNA synthesis
Total RNA was isolated using TRIZOL reagent (Invitrogen, USA) with some modi cations. The 200 mg of leaf sample was crushed in LN 2 and 1.5 ml of TRIZOL was added and transferred to RNase free tube.
Then, 30 µl β-mercaptoethanol (BME) and 40 µl dithiothreitol (DTT) was added and mixed vigorously by pipetting by passing several times through tip of pipette. Samples were incubated for 5 min at room temp by adding chloroform (300 µl) and centrifuged (12000g, 15 min at 4C). Upper aqueous phase was separated carefully without disturbing interphase and precipitated by adding chilled isopropanol (250 µl), 1.5 M NaCl (125 µl), 0.8 M potassium acetate (125 µl) and incubated for 10 min. The samples were centrifuged (12000g, 10 min, 4C) and supernatant was decanted. RNA pellet was washed twice with 1 ml of 75% ethanol and pellet was air dried and dissolved in 75 µl nuclease free water (NFW). Residual genomic DNA contamination was removed using DNase I (GeNei, Bangalore) as per the manufacturer instructions. The concentration of RNA was determined using Biophotometer (Eppendorf, Germany). The integrity of RNA was analyzed using 0.08% agarose gel electrophoresis. A total of 36 samples of RNA were isolated and reverse transcribed to cDNA in a nal volume of 20 µl using m-mulv RT-PCR kit (GeNei, Bangalore) by taking 1µl oligo dT, 1µl random Hexamer primers and 2 µg of total RNA. The volume was nalized to 12 µl by NFW and incubated at 65 °C for 10min. The tubes were ice chilled and master mix was prepared by taking 10 µl of 5X reaction buffer, 1 µl m-mulv RT, 0.5µl each dNTPs and 1 µl RNase inhibitor. To minimize pipetting error, the master mix was prepared in one PCR tube (0.2 ml) by taking multiple values of required reagents as per the number of samples and mixed. The 8 μl of master mix was mixed to each tube and incubated with 1 µl oligo dT and 1 µl of random hexamer primers. Reaction was carried out in thermal cycler at 37 °C for 1 h followed by 95 °C for 5 minutes. The cDNA was stored at -20 °C.

Primer sequences
Primer for sugarcane AQPs used under study were taken from already published primer sequences (de- Andrade et al. 2016). These sequences were further aligned using PRIMER-BLAST at NCBI for speci city. β-tubulin (Andrade et al. 2015) was ampli ed as internal reference gene (Table 1).

Meteorological variables
The meteorological observations during experimental period are presented in Fig. 1 Fig. 2b).

Superoxide dismutase (SOD)
During water de cit conditions, a dramatic increase in SOD activity was showed in tolerant genotypes as compared to sensitive ones. The genotype Co 98014 was shown with the highest and Co 89003 with the lowest SOD activity (Table 2, Fig 2c).
A dramatic increase in ShPIP2;5 transcript was observed in water de cit level-II (150 th day) in comparison to both normal (120 th day) and water de cit level-I (135 th day) (Fig. 3b)

Discussion
Unfavorable circumstances like water de cit induce ROS production that causes oxidative injuries to plant cell. In response to oxidative stress, plant develop its antioxidant defense system to maintain cellular homeostasis (Liu et al. 2011, Kumar et al. 2019. It is known that water de cit is detected early in roots, and subsequently its effects trigger modi cations in leaves (Locy et al. 2002).
Malondialdehyde ( (Patade et al. 2011, Kumar et al. 2019. Under current study, SOD was signi cantly increased under water de cit conditions (Fig. 2c). The SOD activity was higher in tolerant genotypes than sensitive ones responding to water de cit stress. It indicated that the genotypes induced oxidative stress and their antioxidant enzymes caused to detoxify cells. The results under study are similar to previous reports in sugarcane (dos-Santos and Silva 2015), wheat (Ekmekci and Terzioglu 2005).
Aquaporins are integral membrane proteins, present in all plant organs/tissues and participate in transfer of water and solutes. Plasma membrane integral proteins (PIPs) are highly in uenced by environmental constraints like water de cit or drought (Maurel et al. 2008). Gene expression study through real-time quantitative PCR had been successfully used to identify stress-induced genes in barley (Maraschin et al. 2006), transposable elements in sugarcane and regulatory pathways of stress tolerance in M. truncatula (Merchan et al. 2007). The response to water de cit is detected early in roots and leaves (Locy et al. 2002). Drought stress affected the expression pattern of PIP2 proteins in plants leaves of rice (Li et al. 2008, Sakurai et al. 2005 and maize (Zelazny et al. 2009). Difference in AQPs expression was also showed in tolerant and sensitive rice (Lian et al. 2006). Expression pattern of PIP Aquaporins vary with the level of stress, species and isoform (Galmes et al. 2007). The AQPs expression pattern under study revealed upregulation of ShPIP2;5, and down-regulation of ShPIP2;1 and ShPIP2;6 when plants were imposed to water de cit conditions. Findings of study are in harmony with earlier researchers reports (Silva et al. 2013, de Andrade et al. 2016. HT-SuperSAGE libraries analysis of two bulks under drought showed divergent AQPs expression in tolerant and sensitive sugarcane genotypes indicated AQPs expression is genotype speci c (Silva et al. 2013). Cultivars of same species also respond differently to drought as per their tolerance ability (Heinen et al. 2009). Jang et al. (2004) also reported AtPIP2;1 and AtPIP2;5 were up-regulated whereas AtPIP2;6 was down-regulated in leaves of Arabidopsis thaliana under 250 mM mannitol stress. The tolerant genotypes showed higher antioxidant enzyme activity were comparatively more upregulated the ShPIP2;5 expression during water de cit genotypes than sensitive ones suggesting ShPIP2;5 gene may possess a possible mechanism for plant to direct the water ux for speci c tissues, organs or cells that are vital for plant survival during water de cit. On the other hand, down regulation of some AQPs might be necessary to stop the aquaporin synthesis at low moisture availability in soil to minimize plant water loss as well as maintain turgor pressure in leaves (Almeida-Rodriguez et al. 2010, Afzal et al. 2016).

Conclusion
The study discriminated wide variations in antioxidant responses. Differential expression pattern of AQPs reveals that PIP2 was responsive to water de cit conditions. The ShPIP2;5 upregulation suggested its role in maintenance of cell water status under water de cit as these proteins affects water transport. However, further protein pro ling-based studies need to be employed in other crops to explore the role of these proteins.

Declarations
Author contribution Conceptualization and Data curation, D.K.; Formal analysis and Investigation, D.K. and N.M.; Writingoriginal draft, D.K. All authors read and approved the manuscript.
Veljovic-Jovanovic S, Noctor G and Foyer CH (2002) The potential in uence of artefactual interference by tissue phenolics and ascorbate.