Aquaporins as a novel role to enhance salinity tolerance in wheat

Literature Review

Salinity as an Issue in Agriculture:

It has been estimated that nearly 950 million hectares on earth were affected due to salinity and over half of the arable irrigated land in the world suffers from secondary- induced salinity. Due to globalization and rapid climatic changes the annual rainfall is expected to decrease in subtropical regions. The shortage of rainfall and water must be replaced with brackish and saltwater for better agriculture production. By the time 2050 global food production need to be increased to 50% to reach the demands of a growing population(Shabala 2013). In Australia, by the year 2020, an expectation of 25% of land may be out of production due to Secondary salinization. The threats imposed by salinity were causing billions of dollars lost to the agriculture industry. In the past few years at Murray-Darling Basin, an estimated range of 260 million dollars of the economy got affected.

Major Constraints impose by salinity:

Osmotic and ionic stresses were the major consequences of salinity. The extra osmotic gradient generates by excess levels of Na+ that drives water out of the cell. In order to undergo normal cellular metabolism water from vacuole moves to the cytosol and compensates the water depletion. As a result, the cell turgor get reduces and elongation of roots takes place.

The second limitation due to salt stress was Na+ toxicity. In general, the physiochemical property of Na+ and K+ were similar in nature, due to their similarity, Na+ compensate with K+ ions in the key metabolic processes like ribosome functions, protein synthesis and enzymatic reactions(Marschner 1995).

The third important constraint induced by salinity is the K+ deficiency and other nutritional disorders (Shabala 2013).Na+ competes with K+ at specific sites of a plasma membrane. When Na+ crosses along the plasma membrane, a momentous membrane depolarization is observed and such kind of depolarization disturbs the K+ uptake. The Final result will be an impaired metabolism in the cell.

The other constraint imposed by Salinity is reducing of Hydraulic conductance in roots and minimize the absorption of H2O2 (Aroca et al. 2005; Ye & Steudle 2006).

Few adaptive mechanisms of the plant under Salinity: Salinity Tolerance is a combination of three important basic mechanisms:

• Osmotic Tolerance: It refers to the capability of the plant to resist toxic salinity components under saline conditions by maintaining both stomatal conductance and normal shoot growth.

• Na+ exclusion: It refers to the plant ability to minimize the amount of Na+ entry into shoots was the major toxicity occurs. This is achieved by controlling Na+ entry into roots from the soil and developing of Na+ efflux mechanism that excludes sodium ions back to the external environment. HKT family genes were more highlighted in the breeding world, whereas these genes were responsible for root adaptive mechanism seen in Arabidopsis, barley, eucalyptus, wheat etc.HKT family genes were usually divided into two subclasses based on their transport selectivity. Transporters of one family are selective to Na+ ions rather than K+, whereas the other family is selective to both Na+ and K+ absorption. K+ selectivity is strictly due to the deposition of glycine in specific positions of HKT subfamilies. (Shabala 2017)In recent times an important concept of water and solute transporters came into existence and role played by Aquaporins in this relation. So far More than 30 MIPs were identified in plants like Rice, Arabidopsis and maize (Maurel 2007) There was no clear information about the mechanism behind the role of Aquaporins played in the increase of salinity tolerance. Still, more research is needed to be done.
• Na+ Tissue tolerance:  It refers to plant's ability to compartmentalise the Na+ ions into vacuoles that minimize the toxic effect in cellular metabolisms that occurs in the cytoplasm of the cells. Plants that are capable of intracellular compare.

International progress in developing Saline Tolerant Plants: 

Development of Saline tolerant transgenic crops were not fruitful approach because the fundamental mechanism of Ion fluxes in plants was not completely known. Although many attempts were made to develop transgenic crops the complete success was not achieved yet. Failure of these experiments was due to the trails of experiments was carried mostly in greenhouse basis, when the scenario applied in on-field the problems arises again. This failure is due to the availability of different salt levels in the field (Richards 1983). Shoot salt exclusion was considered as a special character to control adverse effects of salinity. Plants membranes-localised with high K transporters called as (HKT) genes had given a limited success (Golldack et al. 2003). Whereas in conventional approach of breeding the expression and elevation of SOS1 and SOS4 genes responsible for salinity tolerance in halophytes were failed to perform in transgenic agricultural crops (Talaat 2018) Genes like NHX ( anti-transporters’)  of Na and protons have been failed whereas the enzymes responsible for detoxification of ROS has differing levels of success(Roy, Negrão & Tester 2014).

Emerging role of Silica in regulating Aquaporins under Salinity

Silicon is the 2^nd most abundant mineral that is present in the earth crust, and it is reported that it played a key role in elevating salinity stress in barley and rice by minimising the ion accumulation in leaves.(Gong, Randall & Flowers 2006) The underlying mechanism behind the concept is Si elevates plant roots is by minimising the absorption of sodium uptake and this is achieved by accumulating the Si content in roots. In a general view, salinity reduces plant growth in two phases: ion toxicity and making difficulty in water uptake by reducing osmotic potential (Munns & Tester 2008). One of the immediate effects on plant due to osmotic stress is reduced in root hydraulic conductivity. Water uptake in plants occurs in three ways: Transcellular, Apoplastic and Symplastic. Among them, Symplastic and Apoplastic moments were elaborated as cell-to-cell pathways that are further regulated by Aquaporins (Sutka et al. 2011) Aquaporins are major intrinsic proteins that covey water and solutes to pass over them (Maurel 2007). These integral membrane proteins are divided into subclasses like Tonoplast intrinsic proteins, plasma membrane intrinsic proteins, Nodulin 26-like proteins, x intrinsic proteins, SIPs (small intrinsic proteins). In addition to the transportation of water few  reports suggest that MIPs transport some non-charged molecules along with water and solutes (Ma et al. 2008). MIPs were observed to transport many important substrates like urea, boron, lactic acid, arsenate, Formamide and glycerol(Hove & Bhave 2011).

Gene Cloning: It refers to the cloning of those genes of wheat which owns the ability of tolerating salinity maximum. It can be said that by using gene cloning the researcher can enhance level crop production as wheat is a mandaory crop which is required by human beings. The procedure will directly contribute in developing growth level of crop production. In this procedure, farmers can take help of scientific methodologies in order to make their crop successful by increasing their level of tolerence for salinity.

Phenotyping glasshouse experiements: Phenotyping refers to the quantification of plant's quality, productivity level, development, architecture and photosynthesis. In this sensors are placed in the laboratory where crops are irrigated. These sensors specify actual feature of plant which will be seen in future after its completion, sensors also sets the level of salinity tolerence of wheat in order to protect the plant for longer duration.

Research gap:

1. Plant aquaporins are the membrane intrinsic proteins that usually regulate water and solutes in plant roots. As we know aquaporins are different types, some channels transport water and some transport, metalloids, ROS, solutes etc (Yool & Campbell 2012), but there is no clear evidence that the aquaporins transports ions like sodium in plant roots (Holm et al. 2005). Then there may be an important role of aquaporins in plant osmotic adjustment and nutrient transport.
2. NIP Subclass of Aquaporins directly influences plant responses by influencing the Si transport and SI uptake under saline conditions. For rice varieties, the OSLSIL gene (NIP2 HOMOLOG) expression was elevated in the salt tolerant varieties by inducing greater Silicon intake. The expression of NIP2 homolog in addition to Si application only was the opposite which was observed in salinity only. So the combinational study of these two Principles may open the gates for understanding a clear principle behind the role of Aquaporins in controlling salinity  (Senadheera, Singh & Maathuis 2009; Shi et al. 2016).NIP subclass Aquaporins enhance the Si entrance into the cell that further promotes the expression of PIP subfamily that enhances the effect of root hydraulic conductance, Optimal water moment, Reduced levels of Na+. Hence this concept lack in clear evidence that states the right mechanism of Aquaporins and the direct involvement of silicon in the regulation of NIPs function. This concept needs further experiments and validations.

Aims and Objectives of research

1. My principle aim was to investigate and conform that Aquaporins regulate sodium entry in plant roots.
2. Investigating the role of silicon in Osmotic adjustment, Na accumulation, and root water uptake via aquaporins.

Research Plan 

Methodology:  10 Seedlings of wheat variety will be imbibed in de-ionised water for at least two days and transferred to an incubator at a temperature of 30C in dark environment. The seeds are then transferred to trays that are filled with vermiculite. After 5 days the seed will be transferred to a container that contains Hoagland nutrient solution. The relative humidity will be maintained at 60% during days and 80% at nights. After 15 days of plant growth the plantlets will be transferred to a new container where plants treated with 50mM of NaCl and five plantlets will be treated with both NaCl and 10Mm of CaCl2.Here CaCl2 acts as an aquaporin blocker. (Martínez-Ballesta et al. 2008)

Achieving the 1^st objective:

Measurement of Sodium Content in Roots: The oven dried roots must be digested in HNO3 solution. In the next step the digested root material is filtered and subjected to dilute with distilled water and Na+ analysis will be done by using atomic absorption spectrophotometer. An analysis was performed in all the treatments and the results will be compared between both aquaporin blocked and unblocked plants.

Measuring Root Hydraulic Conductivity: Plant stem will be sliced just above the root collar and the root plugs are transferred to a pressure bomb in a way that protrudes the excised stem from the chamber. In the next step, the pressure inside the chamber should be increased from 0.7 Mpa to 0.69 Mpa and flow should measure after thirty minutes equilibration to constant flow. The same procedure will be repeated for having 5 readings by introducing a pre-weighed capsule that is attached to a sponge in contact with the stem portion and weight is determined by using a digital balance. The same procedure should be followed by manipulating different pressure levels. In the final step root, hydraulic conductance should be calculated by using the slope of linear regression between the variables.(Trubat, Cortina & Vilagrosa 2012) The results will be compared between aquaporin blocked and normal saline treated plants.

Measurement of Root Xylem Osmotic Potential: Roots from all the treatments must be thoroughly rinsed in deionised water and should be placed on a filter paper and subjected to frozen in nitrogen solution. In the next step with the help of syringe is used to extract the root sap. Osmotic potential of the extracted sap is determined by using Cryoscopic Osmometer at 25 C. The osmotic potential can be calculated by the equation: áµ s = -RTC, where T is a thermodynamic temperature, R is molar gas constant and C is recorded by using micro voltmeter(Yin et al. 2013). Same process of comparison should be done between CaCl2 treated and normal saline treated plants.

Achieving the 2^nd objective:

RNA extraction and identification of wheat NIP Aquaporin Genes: Total RNA from wheat roots can be extracted by using TRIZOL reagent. In the next step to find out the RNA concentration, the Spectrophotometrical measurement will be performed and the integrity can be checked by using Agarose gels. The first stand of wheat DNA sequence can be extracted from total RNA and amplified. Multiple DNA sequences were then deducted by using Clustalx.

Heterogeneous expression of wheat NIPs and SI transport essay in Xenopus Oocytes. To find the heterogeneous expression DNA NIPs should be amplified with the help of PCR by using isoform-specific primers that contains uracil-specific excision reagent and cloned into PacL- containing the PSP64T vector that carries the non-translated b-globin gene from Xenopus Laevis. The exact orientation of clones is then identified by a sequencing process and restriction mapping technique. The oocyte isolation, In vitro RNA synthesis and microinjection of RNA, can be performed as described by (Fetter et al. 2004). In the process, the pool oocytes will be treated with Si and intracellular Si measurements were taken.

Dosing of Silicon in Oocytes: The concentrated nitric acid solution will be added to oocytes and further dried for 2 hours. In the next step, an ultra-trace elemental analyser (plasma- grade water) will be added and incubated for 2 hours at normal room temperature. These samples will be Vortexed and subjected to centrifugation for 10 minutes and a Zeeman atomic spectrometer was used to trace the concentrations of Si. In the final step, a standard curve can be plotted by using Ammonium Hexafluorosilicate solution and data will be analysed by using Spectra software.

Water permeability: After 24 hours of injecting CRNA oocytes will be transferred to the Hypo-Osmotic solution. The Osmolality can be estimated by using Osmometer. The changes in the volume of oocytes can be captured by using a victim colour camera. The osmotic adjustment can be calculated by using an equation POS=VO (d (V/Vo) / dt) / S ×Vw (Osmin=Osmout).

Electrophysiology:  Electrode voltage clamps will be performed in oocytes after 48 hours of water injection.  The voltage electrodes were removed to find the resistance in anND96 solution that is filled with KCl. The both solution will be continually perfused in the experimental process. TEVC experiments will be performed on oocytes with high membrane potential and the hyperpolarised Oocytes were eliminated. In the solutions with low levels of Na concentration Ethane Sulfonic Acid is used as anions. The voltage readings will be recorded using gene CLAMPS software and Gene clamp Amplifier.

Quantitative real-time PCR analyses:

After the treatment of plants with NACL, the roots must be subjected to frozen in a liquid nitrogen solution. And must be stored -80áµ’ C. In the next stem a prime script RT reagent kit was used to extract the First-strand DNA sequence which is further used for QPCR analyses. Quantitative PCR experiments should be conducted on a CFX 96 PCR analysing system using SYBR green master mix. To perform the real-time PCR the temperatures should be maintained at 95 C for nearly 10 min and followed for 40 cycles at 94 C for a period of 15 s and 60 C for 1 min. All the above-mentioned reaction should be performed on a reaction plate using the iQ5 machine. In the final step quantification analysis will be carried by using comparative method a standard curve transform the threshold cycle to express the level of genes. Standard deviations and prime efficiencies are then calculated by using q-base software(Hellemans et al. 2007). The standard curve should be plotted by using a 5-fold dilution series for 6 dilution points. The expression levels of these genes are next transformed to analysis tools, Normfinder(Andersen, Jensen & Ørntoft 2004), Bestkeeper (Pfaffl et al. 2004), which are needed to be performed according to the guidelines given in the respective manuals.

• Gantts Chart and timeline

Outputs of the Projects

The first objective of the project delivers evidences about how aquaporins transport sodium and how their function takes place under saline conditions. The second objective of the project highlights the osmotic adjustment under salinity and studies the role of Silicon on water uptake and sodium uptake and delivers the expression of aquaporins. Moreover the project delivers the role of silicon in salinity tolerance, enhanced water permeability under saline conditions and the underlying mechanisms in wheat roots. Once the mechanism was clearly understood then it will be easy to exploit the breeding processors that control the aquaporin expression and new saline varieties can be generated. In conventional agronomic practice silicon usage can be increased and can be recommended as a supplement to control sodium threat under saline conditions. The expected outputs of the project are expected to be published in six high quality publications at various national and international research conferences on salinity stress in plants results will be available to wider community and industry via ACROSS annual reports as well as through media reports.

Outcomes from this Project

With a most important experiment in the direction of world agriculture that requires production of 70 percent additional food yield for an added 2.3 billion individuals by the year 2050. Salinity is a most important stress preventive the upsurge in the ultimatum for the food.  The national land and water resources audit estimates that approximately 5.8 million hectares of land has a potential to convert into salinity this figure may be estimated to increase up to 17 million hectares by the year 2050 if the effective control is not implemented this is expected to cost 1 billion a year. My project is going to contribute to overcome these problems. Salinity tolerance includes a compound of rejoinder at molecular, metabolic, biological and entire plant level. With the aid of most recent knowledge and the techniques described above can help the healthy growth of the plant that enhances salinity tolerance in crop production. Also the Genetic manufacturing has been demonstrated to be a methodology to the expansion of Salinity Tolerant plant and this attitude will turn out to be more commanding as additional applicant genes related to aquaporin over expression can have a clear solution that can enhance salinity tolerant. Also the time to time checking of the plant growth after supplementing the needed silicon fertilizers to the plant shall help it to grow healthy and tolerate the salinity. As the root is the important part of the plant methodologies that avoid the sodium absorption can restrict the accumulation of sodium concentrations in roots. Prevention is better than cure so the above concept of aquaporins can prevent the consequences of salinity stress in plants.

Academic outcomes: The new approach and methodologies in studying the functions and Sodium transport by aquaporins are highly original and there is only limited instances used before. It allows answering large number of questions related to aquaporins and salinity tolerance and can result in a series of presentations and publications at major international conferences in the area, promoting the image of Australian science worldwide.

Social outcomes: understanding the molecular and ionic transport through aquaporins and effect of Si in salinity tolerance is more vital for sustainable agricultural practices, with long-term benefits for Australian rural communities.

Project feasibility and risk management:

The feasibility of this project will be high and there will be no expected risks. But a partial risk from the project is, as we get the results of the project according to the aims then further there will be a chance to develop many transgenic wheat varieties that have capability of over expressing the aquaporin genes. These transgenic plants can be saline tolerant but in the process of genetic modification there is a chance of losing several desired characters that may further reduce the productivity. So it is better to opt for conventional agronomic practices by applying silicon fertilizers that increase the activity of aquaporins and controls sodium accumulation in plants.

REFERENCES

• Andersen, CL, Jensen, JL & Ørntoft, TF 2004, 'Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets', Cancer research, vol. 64, no. 15, pp. 5245-5250.
• Aroca, R, Amodeo, G, Fernández-Illescas, S, Herman, EM, Chaumont, F & Chrispeels, MJ 2005, 'The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots', Plant physiology, vol. 137, no. 1, pp. 341-353.
• Fetter, K, Van Wilder, V, Moshelion, M & Chaumont, F 2004, 'Interactions between plasma membrane aquaporins modulate their water channel activity', The Plant Cell, vol. 16, no. 1, pp. 215-228.

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