Dr. Claudio Stasolla, Department of Plant Science, University of Manitoba — Bethany Asmundson and Dr. Mohamed Mira conducted this work under the supervision of Dr. Stasolla
IRON IS AN essential micronutrient, and despite its relatively high abundance in soils, it is often a limiting factor for plant growth and development that can cause crop yield loss. Due to its chemical properties, iron often exists as insoluble forms, especially in calcareous soils with high pH (common to Manitoba and comprising more than 30% of soils worldwide), and it can therefore be unavailable to plants. Even when taken up and acquired by plant cells, iron is often immobilized or rendered inactive.
Most (80%) of leaf iron is present in photosynthetic tissue, where it is needed for the development of chloroplasts, as well as for the synthesis of chlorophyll. A common iron deficit symptom is, in fact, iron deficiency chlorosis (IDC), characterized by the yellowing of leaves ascribed to impaired chlorophyll production and accompanied by stunted growth and reduced seed yield.
THE SCIENCE BEHIND IDC
Being a constituent of the electron transport chain, iron, when limited, can disrupt the electron flow leading to the accumulation of reactive oxygen species (ROS), which are deleterious to cell functionality. Over-production of ROS linked to iron depletion has been the cause of lipid peroxidation and depression of photosynthetic rate in several species. Several studies have demonstrated that accumulation of ROS in iron-deficient plants
co-localizes precisely with chlorophyll. A further consequence of iron deficit, favouring ROS accumulation, is the depression of the antioxidant redox system comprising the enzymes catalase, superoxide dismutase and ascorbate peroxidase, and the production of antioxidants such as ascorbic acid and glutathione, all of which are needed to counteract oxidative stress by reducing the levels of ROS.
SOYBEAN RESEARCH FINDINGS
Our work in soybean demonstrates that altering a single gene producing phytoglobin (Pgb) is sufficient to influence tolerance to iron stress. Phytoglobins are heme-containing proteins found in several plant tissues which are responsive to stress and control how plants respond to sub- optimal environmental conditions, including excess or limited moisture. Their role during iron deficiency has never been investigated before.
Our studies show that relative to susceptible cultivars, tolerant soybean plants, when exposed to iron deficiency, can retain a higher amount of chlorophyll and photosynthetic capacity and are characterized by lower levels of Pgb in leaf tissue. The negative correlation between resilience to iron deficit and Pgb level was also demonstrated using transgenic plants characterized by pronounced changes in the levels of Pgb in shoot tissue (80-fold increase or 8-fold decrease relative to the natural, untransformed plants). Transgenic soybean lines suppressing Pgb were able to tolerate iron deficiency, while lines overproducing Pgb showed the highest susceptibility to this condition (Figure 1).
NITRIC OXIDE HELPS SOYBEANS COPE WITH IDC
The main function of Pgbs during conditions of stress is to modulate the level of nitric oxide — an important molecule regulating plant response to stress conditions. Plants tolerant to iron deficiency characterized by a lower Pgb content, accumulated nitric oxide in their cells. We demonstrated that nitric oxide is required for the acquisition of tolerance to iron deficiency, as applications of nitric oxide to susceptible plants elevated their ability to cope with low iron. This observation demonstrated that soybean response to iron depletion is controlled by Pgb through nitric oxide.
THE ROLE OF ASCORBIC ACID (VITAMIN C)
We further elaborated a model whereby high levels of nitric oxide, associated with tolerance, are required to activate antioxidant responses limiting the accumulation of ROS, which are responsible for the damage of photosynthetic tissue during iron deficiency.
In lines suppressing Pgb (and exhibiting tolerance to iron deficit), we observed the activation of important ROS-removing enzymes such as catalase and superoxide dismutase. Induction of these enzymes removed ROS and elevated the ability of plants to cope with low iron conditions. One important antioxidant molecule that was produced in leaves of tolerant plants (suppressing Pgb) was ascorbic acid (vitamin C). Besides its function in removing ROS, ascorbic acid is required to convert iron from Fe(III) to Fe (II). This conversion is crucial for plant survival to iron stress.
ASCORBIC ACID IMPROVES PLANT TOLERANCE TO IRON STRESS
Within the plant tissue, iron can exist in different forms: Fe(III) is the less mobile and unavailable form while Fe(II) is more mobile and active, being able to cross cells and reach sites where iron is needed, such as the photosynthetic tissue. Under conditions of iron deficiency in the soil, plant survival is often dependent upon the ability to mobilize the internal pool of iron present in the cells by converting Fe(III) to its more mobile form Fe(II). This conversion was indeed observed in tolerant plants suppressing Pgb, as well as susceptible plants sprayed with ascorbic acid — a treatment that elevated tolerance to iron stress.
SUMMARY OF FINDINGS
Collectively, this work shows that tolerance to iron deficiency in soybeans can be enhanced by suppression of the protein Pgb. This occurs through 1) a rise in nitric oxide, which influences antioxidant responses that reduce the deleterious effects of ROS, and 2) an elevated level of ascorbic acid at the same, which is required to make iron more available to the plant by converting Fe(III) to Fe(II). Our data indicate that the level of Pgb, easily measurable in plant tissue, could be used as a reliable marker to predict plant response to iron stress and select/screen germplasm that is better able to cope with iron deficit. Furthermore, applications of ascorbic acid to leaf tissue could be used as an effective treatment to limit iron deficiency stress.