The health of plants is paramount for farmers, gardeners, and scientists alike. They not only provide the food we need to survive, but their overall well-being is a marker for the strength of the environmental conditions around them. And there are many things, much like with us humans, that can be a cause for alarm. Plants not only have to deal with pathogens of every variety assaulting them and not only the amount of water and sunlight they get for nutrition, but the state of the very soil beyond that is also critical.

If the soil has too high a pH, too low of a mineral content, or more commonly a concern, too high of a salinity level, then that can negatively impact the survivability of any plant and those around it. Salt buildup is something that can happen gradually, but inexorably over time, and the only way to remove it is either through the water table (which brings its own problems) or invasive methods that often require digging up the soil structure itself.

It is estimated that around 6% of the total land area in the world suffers from excess salt concentrations and this heavily restricts the kind of life that can survive on this land. Unlike some other forms of abiotic stress, salinity affects all stages of plant growth, from germination to adulthood, and reduces stalk height and root length both. Exposure to high salt levels begins a simple, physical process known as osmosis, where molecules in a solution try to balance themselves depending on certain ions within the solvent. Salt molecules, specifically the sodium in the molecule of sodium chloride when using the term, are one of those ions to consider.

When in high saline soil, osmosis results in a restriction on water transfer between the soil and the plant roots. The plants themselves often have cell walls rigid enough to prevent water loss from occurring, but the high water transfer force into the soil due to osmosis reduces the capability of roots to take up water itself. Additionally, any water take up has a likelihood of introducing salt ions into the plant vascular system as well, leading to other untoward effects. The ions travel from the roots into the long-distance transport system, ultimately reaching the leaves through transpiration.

The stress response this creates in the plants reduces their ability to photosynthesize, since water is a part of the equation needed to convert carbon dioxide into glucose with the addition of light energy. It also leaves the plants open to harm from other ions, like reactive oxygen species (ROS) with the type humans are more familiar with being hydrogen peroxide. These radical molecules can build up in plant cells while the plant is under salt stress and have a high possibility of causing death to any individual cell affected. This is showcased physically on the plants by a browning and blackening of leaf ends and other parts of the plant structure.

Attempts by farmers to irrigate their land to provide additional water resources in order to offset the stress usually only worsen the problem, as irrigation water often has some amount of dissolved salts in them that adds to the soil salinity after evaporation. They may be options to leach the salts into groundwater, but as just mentioned, it is a costly process.

However, not all plants are ultimately susceptible to salt-heavy soils. Many have, as salinity has increased slowly over the centuries, millennia, and eons, developed mechanisms to deal with or at least abate the effects of high saline surroundings. And it is these biochemical methods that must be discussed first before we can go into the modern research being done with that knowledge. Since the study of plant response to abiotic stresses is an ongoing field of research, there remains plenty to be uncovered about plants around the world and how they individually deal with stresses like salt buildup.

Plant Salt Stress Responses

The Stresses of Osmosis

The first step to dealing with osmotic pressures is to alter the differences between the inner area and the outside environment. This is referred to as osmotic adjustment. These alterations allow plant cells to remain functioning in the face of extreme soil differentials around them and continue metabolic processing that the rest of the plant desperately needs. The primary method of pulling this off is the synthesis of specific molecules that can counter the differential while not being toxic to the cell itself.

Proline is an amino acid routinely used in the biosynthesis of proteins. It also serves as a critical osmotic adjustment device, helped by its characteristics that include a low molecular weight, a high solubility in water, and a neutral charge overall under normal pH conditions. The best way to check for whether a plant is currently under osmotic stress is to see if the genes responsible for proline production have been activated. These genes have already, in the past, been used transgenically in order to confer better osmotic stress resistance in plants that normally lack proper salinity tolerance.

Soluble sugars like glucose and sucrose can also mitigate the impact of osmotic stress by stabilizing the structure of the cell wall and membrane. They also prevent the salt ions from interfering with enzyme activity. Of course, sugars can be difficult to come by, since they are the direct result of photosynthesis. Meaning that resistance to salinity levels requires a reduction in energy stores within the plant cells. The levels of these sugars in a plant under such stress can be measured in order to determine whether a plant has strong salt tolerance capabilities.

Lastly among the molecular options is the amino acid derivative betaine, also known as trimethylglycine. Previous research has shown that it is used by plants to block the transport of ions like salt from the roots to the rest of the plant when osmotic stress is occurring. Using betaine as a spray has showed some amount of positive benefit for the plants it is applied to and their tolerance of salt.

Combating Ion Toxicity

Other than the direct stresses produced from salinity, there are also the salt ions themselves that cause toxic metabolic impacts over time, especially upon photosynthesis capabilities. It is the sodium ions, the Na+, that are of highest concern. The tolerance that plants form is not only against these ions, but also on the secondary effects including depletion of water uptake.

When a plant is under sodium toxicity, they will automatically be forced to reduce the amount of potassium (K+) in the cytosol in order to keep ion balance. The ability for a plant to maintain high potassium levels even while under such stress is an example of salt tolerance that is desired by farmers and scientists. Metabolomic tests can be run to observe the flow of both kinds of ions in real time and as a measure of how a plant is responding to salinity in the surrounding soil.

In addition to keeping a high potassium to sodium ratio within its cells, another option for a plant if it is unable to evacuate the sodium from its tissues is to move all the excess salt ions to a metabolically inactive region. Normally, this involves moving the ions from the cytosol in the cell to the vacuole storage. A transporter protein called an antiporter that is able to move ions across a membrane is used for this purpose and helps to maintain not only ion levels in the cytosol, but also overall pH levels resulting from positive or negatively charged molecules like the ions, but often in the form of H+ cations.

The Na+/H+ antiporter is a prime source for salt tolerance in plants and has been extensively studied because of that. Simple overexpression of the gene responsible for the antiporter protein has been shown to increase tolerance responses. The antiporter genes in different plants have slightly different mechanisms and can be combined transgenically in order to have the benefits from both or even more varieties. These transgenic plants can also be crossbred with any number of cultivars in order to confer the wanted tolerance genes.

Reducing ROS Activity

The third and final prime area for pursuing salt tolerance is dealing with the tertiary side molecules that become an issue while a plant is combating a saline environment. Oxygen is, as with most organisms, a critical part of metabolic processes in plants. It is needed in energy production systems and in mitochondrial respiration, but the use of oxygen leaves behind some much less desired byproducts. Reactive oxygen species are, as their name implies, highly reactive and highly oxidative toward other molecules.

They are able to damage the cytoplasmic membrane of the cell, interfere or even outright break metabolic interactions, and generally result in cell death if enough of them builds up. One of the most damaging actions they can take is binding with individual amino acids in active proteins. In total, this sort of activity is referred to as oxidative stress.

The counter to all of this and the first goto for plants is superoxide dismutase (SOD), a part of the antioxidant enzyme defense system. It converts oxide anions into other forms in order to reduce their toxicity before other enzymes expel them from the cell. Largely this is through altering O2 into H2O2. Transformation of SOD production genes into other plants has shown success in the past.

Ascorbate peroxidase (APX) is another enzyme that acts as a counterpart to SOD. It is responsible for doing the H2O2 expulsion and has strong activity when the plant is responding to ozone in the cell. Thus, ozone tolerance is conferred when a plant has strong APX genes. Another enzyme with the same job is catalase (GAT), but it works slightly differently due to requiring a substrate to reduce in order to continue its active state. Its heightened response under light respiration has made it a key component in C3 plants that are most efficient at photosynthesis.

An additional area that ROS cause damage is through the degradation of lipids via peroxidation. When this occurs to the structures that make up the phospholipid bilayer of the cell, a product of this is malondialdehyde (MDA), which can act as an indicator for how strongly a plant is capable of dealing with reactive oxygen species. Certain genes can reduce the speed of ROS peroxidation and in turn reduce the amount of MDA that is seen in measurements.

With that breakdown of how the overall plant response happens, now we can move onto recent research to improve those defenses through biotechnology solutions.

A Biotechnology Focus on Salt Tolerance

Halophytes: A Genetic Goldmine

When testing plants for their individual resistance to saline conditions, they can commonly be split into two separate categories. The majority of plants fall into the glycophyte grouping, the plants that lack any meaningful salt tolerance and can be easily damaged by high salinity soils. The other class of plants are the more biologically intriguing ones. Halophytes are the rare kind of plant that does have an elevated tolerance to salinity and can be found growing in salt-rich environments like marshes or other locations close to the ocean.

Salt-heavy soils are a growing problem in the world today, with decreasing rainfall, ingression and expansion of saltwater, and poor management of soils all contributing to a spread in saline-contaminated regions. Groundwater can itself become a hazard due to salt-infected water spreading through underground systems to burden even far apart locations with increasing salt concentrations. In a short amount of time, fertile soils can become barren ones where normal crops are unable to grow at all on them.

Because of this, halophytes have been a major source of study for agricultural scientists hoping to find the secret to better salt tolerance. In some cases, halophytic plants themselves have been explored as possible culturable crops for food production. Though there is a further distinction to be made. Based on how their particular brand of tolerance works, halophytes can be further subdivided into two groups.

For some, at least a small amount of salt is required in order for their metabolic processes to function. They have intertwined their biological systems with the salt in their soil environment in order to survive. Without it, their growth ceases altogether. These types of plants are referred to as obligate halophytes. The other side of the coin is of course those who can grow just fine on salt-free soils as well, referred to as facultative halophytes. Though they will generally see less impressive growth rates overall as compared to glycophytes in salt-free soils.

Unfortunately, it is commonly difficult to study the direct mechanisms of how the salt tolerance genes in halophytes function, as they can be multi-gene affairs. When transformed into a model plant organism like Arabidopsis, the genes fail to activate properly and we learn nothing in attempting to study them. Thus, research has to be done with halophyte test subjects themselves. The saltwater cress species Thellungiella halophila has begun to become the standard model organism within the category of halophytes. Whether it emerges as a de facto model organism or another will rise to prominence within research labs is hard to tell at this point.

An example of a unique mechanism used by halophytes is their ability to secrete salt ions in a liquid form that then crystallizes outside the plant once it has contacted with air. The structures on the plants that produce this secretion are called salt hairs or glands. A secondary option other than secretion is to concentrate excess salts in particular leaves and then shed those leaves to grow new ones.

As can be seen, many capabilities of halophytes are anatomical in nature, which is one of the reasons why they are hard to reproduce and replicate in other plant species. But they aren’t all about physically removing salts on a macroscopic level. Halophytes also have the expected ROS detoxification genes and cytosolic transporter proteins and also the osmoprotectants like betaine. These base level responses are fairly ubiquitous throughout plants, though some certainly work better and at a higher activation level than others. Though that’s not entirely or even mostly the reason for their tolerance.

It has recently been surmised by scientists that, overall, plants have the same response options when it comes to salt tolerance. There are variations on the genes and proteins involved, but the mechanisms employed are practically identical. And yet, even with these same methods, halophytes are salt tolerant and glycophytes largely aren’t. Why is this? The answer is about quantitative differences, rather than qualitative ones.

Halophytic plants just have more salt response genes that work at a heightened activity level as compared to their less fortunate counterparts. The specific proteins halophytes produce last longer due to that higher expression level. Thus, while the genes might be similar, finding which ones halophytes use and their mechanistic options for expression is key to imparting this onto other plant species.

As epigenomic, transcriptomic, and proteomic analyses have improved over the years, scientists have better been able to determine how the changes in halophyte expression happen inside the plant cells. What they have found is that it is likely post-translational modifications of proteins that cause the differential activity between glycophytes and halophytes.

The same genes may be used, but environmental pressures and stresses cause additional regulatory conformational changes to the proteins. In some cases, the high salinity of the cytosol in the cell may cause a conformational alteration and altered function from the very presence of salt ions alone.

Overall, the complex network of genes and regulation in halophytes appears to be responsible for their high tolerance to salinity and the combinatorial interactions of this system have to be understood before there is any chance of transgenically expressing the same functions in glycophytic plants as well.

Transcriptional Improvement of Soybeans

The importance of soybeans as a critical food source worldwide is well known. It is a major source of protein for societies where meat consumption is scarce both in the cooking sense and in regards to availability. The ability for soybeans as a legume crop to fix nitrogen into the soil is also a necessity for farmers that wish to retain the health of their soil microbiome.

While soybeans have some amount of salt tolerance through their genetics, their amount of growth is highly dependent and correlated to salinity in the soil. So, though they might not die from a moderately high salinity, it will greatly stunt their germination rate and amount of seeds produced.

When dealing with stresses, plants normally enact stress-inducible transcription factors that are able to regulate and bind to genes in order to turn them on. These genes in turn produce proteins that protect the plant against damage or mitigate the damage that is already being done. The activation of defense systems wouldn’t be possible without the work of transcription factors.

For environmental stresses, the WRKY family of transcription factors are one of the immediate picks for responding. They work throughout the growth and development phase of a plant, turning on and off hormones needed for those metabolic processes.

Alfalfa is a forage legume that is planted in pastures for livestock to later graze upon. It is thanks to a number of evolutionary pressures over time that modern alfalfa has become a plant well suited to almost any kind of environmental stress, including salt-ridden soils. Up until now, only a small handful of WRKY transcription factor genes have been isolated from alfalfa, but those that have are being put to use.

The gene MsWRKY11 codes for a DNA-binding protein that is found active in every single tissue of the alfalfa plant: roots, stem, leaf, flower, and fruit. Its expression is particularly profound in the roots and leaves however. This transcription factor was found to be upregulated under most abiotic stresses, so researchers studying salt tolerance and soybeans decided to see if it’s magic would work from a simple transgenic transfer.

The scientists found that overexpression of MsWRKY11 in soybeans caused an increased salt tolerance in the seedling stage in specific and enabled better growth in regards to stem length. The photosynthetic capabilities of the soybeans and their subsequent byproducts were also enhanced, along with a reduction in ROS molecules. In general, the transgenic soybeans were observed to grow at a faster and more productive rate than the wildtype in all areas, showing how the WRKY transcription factor families are able to confer a generalized and all-around improvement through salt tolerance.

Osmoprotectants Protecting Tomatoes

A study in January of last year decided to focus on removing the negative metabolic effects that salt ions have on plant growth and photosynthesis. As previously, though briefly, discussed, there are several compatible solutes, also known as osmoprotectants, that plants utilize to counteract the effects of osmotically uptaken salt molecules. Betaine, specifically the form of glycinebetaine (GB), is one of these solutes.

This one osmoprotectant is so useful that it is found across the kingdoms of life, in animals and bacteria in addition to plants. Its low molecular weight and water solubility allows it to accumulate in the cytosol and both block the salts from being transported elsewhere or causing harm, but also fixing some of the damage. This is primarily done by stabilizing the structure of proteins and the cell membrane to keep metabolic activity up and to prevent the membrane from being breached and cell death occurring.

There were suspicions that betaine could be used to maintain the homeostasis balance between sodium ions and potassium ions. Since the activity of sodium ions produces reactive oxygen species, which in turn bind to and cause the removal of potassium from the cells. This is not a positive process, due to the paramount necessity of potassium in mediating photosynthesis and stress responses. The researchers therefore decided to test whether betaine could accomplish this stability when a plant is under salt stress.

The experiment was conducted in two ways. With a transgenic tomato plant that was capable of naturally accumulating GB thanks to the added gene and with a wild type tomato plant where GB was added exogenously to the surrounding soil to be taken up by the plant. Both methods were put into experimental groups to see which fared better. The transgenic gene used, codA, was obtained from a bacterial species with known salt tolerance.

The results were simple enough. The transgenic plants successfully accumulated GB, while absolutely none was found in the wild-type plants. In turn, the transgenic group was shown to have higher photosynthesis rates, better antioxidant production, and less ROS accumulation in the leaf tissues. Additionally, the expulsion of potassium was reduced, while the removal of sodium ions was increased, along with a higher formation of ion transporter proteins.

The scientists concluded that the tolerance that codA confers likely has to do with enhanced transporter creation for shuttling unwanted ions out of the cell. This allows the cell to maintain a high potassium to sodium ratio, as is wanted for metabolic function, even under salt stress conditions.

The Expanding Application

Depending on how you look at it, salt tolerance in plants can seem simple or overbearingly complicated. The physical manifestations of the tolerance in the plants and their measured metabolites is pretty straightforward. We can understand why and how the mechanisms work to do what they do. But the genetics and corresponding proteins are not such an easy matter.

Over time and a considerable amount of effort, the scientific community has taken tremendous strides toward fixing that. Modern genomic analysis and other sequencing and application technologies combined with transgenic tools have more and more opened up how salt tolerance works on the smallest level.

This overview of how plants deal with high salinity soils as discussed above and some of the experiments being attempted should hopefully increase understanding of how this field of agricultural and biological research is progressing, along with where it is likely to lead to in the future. With continued scientific expertise, it is expected that general plant cultivars capable of growing in saline-rich areas should be just around the corner.

References

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Photo CCs: Samphire salt marsh – St Kilda South Australia from Wikimedia Commons

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