This document summarizes research on engineering agricultural plants for improved salt tolerance through genetic engineering. It discusses genes from model organisms like Arabidopsis and yeast that improve salt tolerance when transferred to crop plants. Genes from halophytic plants, which are naturally salt tolerant, are also discussed. The document outlines past research and calls for future work, including combining multiple salt tolerance genes, comparative analysis of similar genes, and further research on halophytes and the biological costs of salt tolerance.

1. Review: Engineering agricultural plant species for improved salt tolerance
John Kern
Plant Development & Biotechnology w/ Marina Tourlakis
16/10/2015
Introduction
The vast majority of the water on earth is found in the ocean, which is 3.5% salt (599 mM). Not only is
the largest reserve of water high is salt, the available reserves of freshwater relative to human demands
are steadily decreasing. It has been estimated that barring drought and other abiotic factors, salinity
hampers the efficiency of 20% to 50% of land that is presently used for agriculture (1). This effect
includes semi-arid areas that have been used in long term agriculture. The very process itself causes
buildup of mineral salts in soil when drainage or rainfall are limited. As such, a means to deal with
salinity is a factor even in landlocked areas.
Aside from land that is already used for agriculture, there is a glut of arid land that had been deemed
feasible to use should irrigation using sea water become an option. Studies have estimated that as much as
20% of coastal arid land could potentially be irrigated in this manner (1). Should this become an option,
the ability to greatly increase both food production and carbon fixation would be greatly increased. If salt
water agriculture becomes viable, benefits should be felt in both short and long term scales.
With the advent of genetic engineering, the ability to mix the beneficial traits between salt tolerant
(halophytic) and agriculturally useful plant species is close to within reach. There are however several
approaches that scientists can take. Methods are greatly varied but by no means incompatible. Some
experiments work to isolate genes in well-known species (like Arabidopsis and Yeast) that will improve
salinity response when overexpressed. Other studies work to compare freshwater plants’ and halophytes’
salt tolerance in order to identify genomic differences that convey the resistance. Other researchers
examine plants that grow in salt that have simply never been examined in detail as to whether they could
be cultivated and/or upgraded for utilitarian purposes.
Between 2001 and 2005, four science reviews discussing transgenic saline tolerance improvement have
been published. In 2001, work on Arabidopsis and yeast was acknowledged and more work on halophytes
was called for (2). In 2002, it was predicted that future plants would be engineered to sequester sodium in
leaves, away from fruit (3), a hypothesis which I will argue has since been disproven. In 2005, reviews
called for attempts at combining several slat tolerance improving genes in the same plant (4), and for
more precise work to be done to quantify the productive effects of transgenic approaches (5). In this

2. review, the possible approaches to improving salt tolerance using transgenes will be outlined, and future
directions for improving on the work will be contrasted.
Stress Response Genes in Model Organisms
A popular approach by groups that study salt tolerance is to compare gene expression in individuals of the
same species before and after stress exposure. One such study was able to isolate the BADH gene
complex from spinach, which express it under stress. When the gene complex transferred to and
overexpressed in carrots and potatoes, the resulting specimens had higher tolerance to stresses including
both drought and saline exposure (6, 7).
The HAL1 gene complex is another stress tolerance candidate. I can be moved from yeast to plants like
tomatoes and watermelons to improve their salt tolerances (8, 9). Teams that have produced transgenic
plants with this gene have observed that under non-stressed circumstances, the plants behave
indistinguishably from wild types. In this gene’s case, there remains further work to be completed, as the
exact mechanism by which it improves stress tolerance remains unverified. However, there has been some
work suggesting that it plays a role in a larger gene complex that helps keep cells from losing K+ ions
while subjected to salt stress (10). The exact mechanism used to accomplish this is as of yet unidentified
though.
The GmDREB2 gene, isolated from soybean and moved to Arabidopsis and Tobacco has been found to
play a role in abiotic stress response (11). Salinity was one such abiotic stress, however, the gene played a
role in protecting from other stresses, like droughts. Further research in genes like this show potential in
producing hardier crops, as opposed to ones that are tailored to specific environments. Similar works have
included study of the SNAC1 gene complex, which codes for several transcription factors, has been found
to play a role in abiotic stress response in rice, producing hardier (to salt and drought) plants when
overexpressed (12).
Beyond these genes, several studies have experimented with the roles of other genes in salt tolerance, and
are working to understand the mechanisms by which they assist. Genes that help with salinity tolerance
generally fall into three categories; vacuolar antiporters, membrane antiporters, and stress response genes.
Of these, the first two categories generally play roles in the mechanics behind sodium evacuation, while
the latter play roles in plants’ response to stress after being otherwise unprepared. Each method
contributes the plant’s resilience by a unique mechanism.

3. Genes Promoting Intrinsic Salt Tolerance in Model Organisms
The other genes that have been researched each play roles in increasing plants’ normal levels of salt
expulsion. As opposed to genes that are primarily activated in response to abiotic stress, these constantly
maintain levels of proteins that contribute to their evacuative function. One such gene which is in the
early phases of research is the OsNHX1 gene, which is naturally present in rice and codes for a vacuolar
Na+/H+ antiporter (13). Researcher have found a positive correlation between the gene’s expression and
Oryza Sativa’s tolerance to saline water.
Another such gene is AtHKT1 plays a role in salt response and is also in the early phase of research.
While naturally found in Arabidopsis, the gene has been found to play a role in moving sodium ions from
tissue into the phloem, so that it can be moved back down to the roots (14). A separate study has found
that the gene also plays a role in limiting Na+ ion entry at the roots (15). This gene may indeed play roles
in both evacuating and limiting the entry of sodium ions, in which case it would be a major component of
further transgenic work. It is worth noting that this mechanism actually evacuates sodium from the entire
plant, leaving it in groundwater.
Another such gene is the AtNHX1 complex, which is also naturally present in yeast, and has been studied
extensively. By examination of both the protein structure, and ion contents in plants with and without the
gene, researchers have identified the role of the protein it codes for. The AtNHX1 gene codes for a protein
that moves Na+ and H+ ions out of vacuoles, and into the cytosol. As such, it plays a role in moving the
ions out of the cells. By moving this gene to other species, researchers have found that when
overexpressed, it can increase salt tolerance in wheat, tomatoes, and maize (16, 17, and 18).

4. Sodium Transport in Stem Tissue
Figure 1: Cross Section of Plant stem tissue, arrows indicate net transport of Na+ ions as a result of
increased expression of associated genes. Arrow A: Vacuole to cytosol transport that genes such as
OsNHX1 & AtNHX1 contribute to. Arrow B: Cytosol to intercellular space by genes such as SOS1 and
subsidiaries of the HAL1 complex. Arrow C: Transport of NA+ to the phloem promoted by genes such as
SOS1 & AtHKT1. Arrow D: Transport of Na+ to the xylem promoted by the SOS1 gene that was
specifically derived from the halophyte Salicornia brachiate.
Study of Halophytes
Despite that extensive genomic research has been performed on the species that are popular to work on,
relatively little has been done to investigate the plants that are naturally tolerant of high salt levels. Plants
referred to as halophytes are a category of species that are able to thrive in highly saline environments.
Several have been investigated, but little extensive or genomic research has been done on most. However,
the field of halophyte study is in development.
Some of the earliest lines of research performed on halophytes has been to delve into their potential to be
used as crops unaltered. Studies have indeed found that Salicornia herbacea L. and Panicum Turgidum
could both be used as saline irrigated cattle feed crops (19, 20). They are however, inedible to people.
Recently, halophytes have been studied at the genome level significantly more extensively.
An investigation where halophyte gene expressions were compared to Arabidopsis under salt stressed
conditions has identified a number of candidate genes for further investigation. These included Fe-SOD,
Central Vacuole Parenchyma cell PhloemXylem

5. P5CS, PDF1.2, AtNCED and SOS1 (21). The SOS1 gene is the only that has been transgenically
investigated further. It has been found to code for a transmembrane Na+/H+ antiporter (22). It was found
to be expressed to a greater degree under normal conditions in halophytic plants than in regular plants
(23). It is worth noting that the SOS1 gene is present in several species, including both halophytes and
Arabidopsis, and are slightly varied in structure, though similar in function between them (24).
In other experiments, the SOS1 gene has successfully been moved from Salicornia brachiate (a succulent
halophyte) to tobacco in order to confer increased salt tolerance. This tolerance was achieved by loading
the xylem loading with sodium ions, an effect unique to that gene (25). This is one of the earliest attempts
at using halophyte genes using transgenes. Further work with similar plants has yet to be expanded upon.
Discussion
Further study could include a wider variety of cultivar plants
Other reviews have suggested further expansion beyond yeast and Arabidopsis. There are some (tobacco,
carrots, tomatoes, maize, wheat) studies that have been performed since those calls. However, this work is
still in beginning phases, as the general pattern is to take a gene that has already been shown to work on
yeast &/or Arabidopsis, and then move it to the cultivar species. One such category of plants that could
see huge demand in the near to long term future are legumes. Regions that are so arid that saline irrigation
even becomes a factor also generally have extreme shortages of fixed nitrogen. As such, before highly
productive agriculture even comes into play, it may be necessary for future focus to be geared towards
soil structure and nutrient improvement. As such, plants with nitrogen fixing root bacteria may be the first
thing to invest in researching.
Combining transgenes in the same specimens: antiporters for vacuoles and cells
One avenue for future research would be to add several theoretically compatible genes to the same
recipient plants. As it stands, most of the experiments to date have involved one gene at a time. Many of
the genes that have been studied separately work by separate mechanisms. For example, the AtNHX1,
SOS1, and AtHKT1 genes could likely all be combined to provide a compounded intrinsic tolerance
beyond even the 400mM capacity achieved in BADH treated carrots. From that landmark, increasing
productive tolerance a further 50% would allow irrigation with untreated sea water. Several such
pathways that are theoretically possible could provide highly productive species with successful future
experiments.
Comparative analysis of genes that work by similar mechanisms

6. Ongoing studies of genes responsible for salinity tolerance have found both a variety of mechanisms,
along with several of genes that produce the same mechanisms. At present, there are no side by side
essays of similar transgenic species with slightly different genes that code for similar antiporters. For
example, the SOS1 complexes sourced from yeast and a halophytic plant could be compared. Such
analysis for every gene identified in the capability may play a crucial role in engineering crops to be as
efficiently halophytic as possible. Logically, those genes will be found in plants that undergo selective
pressure on tolerance, but only comparative experimentation can confirm the hypothesis.
Further study on plants that are already salt tolerant
Currently, some experimentation has worked on halophytic plants, this has lots of room to be furthered.
At the moment, the majority of genetic study on halophytes has been concentrated on genome comparison
against Arabidopsis or agricultural species. Should researchers wish to continue via the method of
database wide gene analysis, the next step could be to compare taxonomically diverse halophytes in
search of their similarities. Researchers could then cross reference those similarities with common
differences from large numbers of glycophytes. In essence, this would mean using gene databases to ask,
what halophytes have in common that is distinct from freshwater plants. The genes they find could then
be isolated and used in further transgenic study. Such an approach would be a major divergence from an
analysis of solely the genes glycophytes express under stressful conditions.
With reference of study into the potential naturally halophytic plants have as agricultural crops, another
future avenue of research would be to begin investigations into improving their nutrition and energy
content. In such a case, an entirely separate line of genomic analysis would be necessary, as the stress
tolerance, but not the nutrition, is already available in those species.
Research into the biological energy costs of salt tolerance
With the idea of using salt water crops for productive means, an avenue for research that is as of yet
untouched is an analysis of the energy toll of salinity tolerance. As of yet, researchers have not observed
any major productivity reduction in the transgenic species that have been produced. However, the
processes that provide saline resistance require the construction of new machinery within tissues. As such,
there is reason to suspect that it would be costly.
Considering the raw productive capacity of oceanic algae, there is reason to believe that salinity is not a
major limitation to well adapted autotrophs. However, a detailed study would be required to verify
whether the ability is costly or simply lost in freshwater plants through genetic drift when no longer

7. selected for. Understanding this difference could help in decisions as to whether desalination by organic
or mechanical means or saline agriculture are more energy efficient options for future primary production.
Conclusion
By examining the work that has been completed thus far, several avenues for future work emerge, ranging
from more aggressive approaches to genomic alteration, to broader database wide comparative analysis.
The current state of studies indicates fairly strongly that saline agriculture is possible, and on the horizon.
However, the strategies taken in future research will likely influence the time frame, and effectiveness
with which it can be implemented in the relatively near future. With hopes of improving global food
supplies, development of halophytic agriculture could potentially neutralize water supply limitations. This
area of research hold vast promise, barring legal and social barriers, evidence suggests that with adequate
investment, saline agriculture can be developed in the near future.
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