Due to global climate changes, mainly caused by the increase in global average temperature, the weather is becoming more unpredictable, with unusual precipitation and extreme temperatures becoming widespread phenomena. According to recent studies, by 2050 the global average temperature will rise and probably exceed by 2◦C under the current high emission scenario. This escalation will significantly amplify the consequences of climate change, affecting billions of people worldwide.
Abiotic stresses represent key challenges for agriculture, as crop production is the sector most affected by global climate change. Among the various types of abiotic stresses, drought stress is the most detrimental one to plants, because it causes transpiration rates to exceed the water uptake, resulting in disruption of water potential gradients, changes in cell volume, denaturation of proteins and reactive oxygen species (ROS) production and disruption of membrane integrity. These processes as a whole lead to serious damage to plant organisms and yield reductions or even losses. Maize has high yield potential, which requires sufficient water supply: according to the transpiration coefficient, it needs 250 g of water to synthesize 1 g of dry matter.
In most countries, maize is grown in rainfed areas with a rainfall of 300–500 mm, which is below the critical value to obtain a decent yield. Depending on the intensity or duration of drought stress and the harvest stage, the maize yield losses vary between 30 and 90%, which has a strong impact on the flowering and grain filling phase. To combat the negative effects of water stress, plants have involved a wide range of responses including physiological and biochemical mechanisms. Most of them are the result of the regulating the expression of stress-associated genes, leading to shifts in phytohormone balance, stomatal closure, osmolytes and antioxidants production, cuticle thickness augmentation and root growth stimulation, making plants more resilient to harsh environmental conditions. Drought stress-related genes are of great interest for genomics studies as they represent a key to crop resistance improvement, ensuring sustainable crop production under unpredictable conditions.
Our study aimed to evaluate the effects of microbiologicals, based on plant growth-promoting bacteria (PGPB) and their metabolites, particularly exopolysaccharides known for their water stress mitigation ability, on drought resistance and metabolite profiles of maize.
The experiment was conducted during the 2023 growing season at the trial station of the Northeast Institute of Agriculture of the National Academy of Agrarian Sciences of Ukraine. We cultivated Tristan “FAO 270” maize under field conditions. The experimental plot was exposed to water stress due to limited precipitation during critical growth stages in May and June. Maize plants were treated with commercially available biologicals from BTU Biotech Company, including AZOTOHELP, LIPOSAM and ORGANIC-BALANCE.
AZOTOHELP® contains living Agrobacterium pusense cells and growth-promoting biologically active metabolites. It enhances stress resistance and boosts crop yields. LIPOSAM® is made of oligo/polysaccharides of microbial origin, it is an effective sticking agent, which enhances plants' drought tolerance. ORGANIC-BALANCE® contains nitrogen-fixing, phosphorus- and potassium-mobilizing plant growth-promoting bacteria, biologically active microbial compounds of microbial origin.
To assess the drought resistance of maize, we analyzed the expression of key water stress marker genes (ZmNHL1, ZmVPP1, ZmNAC111) using qRT-PCR. Relative Water Content (RWC) of leaves was measured to evaluate overall plant well-being and the severity of water stress. It recognizes other gene expression and encodes a transcription factor of the NAM, ATAF, and CUC (NAC) family. ZmNAC111 expression is induced by drought, high temperatures and salinity and it regulates the expression of genes involved in the response to abiotic stresses.
Why did we decide to analyze gene expression profiles? While investigating abiotic stress reactions, it is very important to take into account not only yields and morphological characteristics, but also molecular mechanisms underlying stress resistance processes. Stress-associated genes lead to activation of different biochemical and physiological mechanisms of stress adaptation and mitigation. According to experimental data from extensive studies of the role of the late embryogenesis abundant (LEA) proteins, they are involved in protecting higher plants from damage caused by environmental stresses, especially osmotic stress. Bioinformatics analysis of ZmNHL1 revealed that the protein encoded by ZmNHL1 belongs to the LEA-2 protein family. Tissue specific expression analysis showed that ZmNHL1 is relatively abundant in stems and leaves, highly expressed in tassels and only slightly expressed in roots, pollens and ears. Under favorable conditions, no significant differences in relative water content (RWC) and antioxidant enzymes activity were found between transgenic and wild-type (WT) plants, but under the drought stress, the RWC, the activity of superoxide dismutase and peroxidase of plants from the three 35S::ZmNHL1 transgenic lines were significantly higher than that of the WT plant. It is thought that ZmNHL1 promotes maize tolerance to drought stress in transgenic plants by improving ROS scavenging and maintaining the cell membrane permeability.
ZmNAC111 was chosen as a drought resistance marker in maize plants. It is a regulatory gene that controls activity and influences other genes expression, which code for a transcription factor of different types, regulating numerous reactions in plant organisms. ZmVPP1 was also analyzed as another marker of plant drought resistance.
Drought stress significantly affected the expression of stress-responsive genes, which were positively altered in plants due to the action of biologicals. Investigating gene expression is a powerful tool for understanding and comparing the comprehensive responses of plants to both abiotic and biotic stresses. To analyze differences in gene expression levels between control and treated maize plants, a qRT-PCR analysis was conducted. This technique allows us to evaluate the level of particular gene expression. Increased levels of expression of stress-associated genes are associated with higher degree of stress.
Fig. 1. Relative gene expression
qRT-PCR analysis revealed increased regulation of some stress-related genes in control plant variants that experienced drought stress. The molecular genetics analysis results showed that the most substantial differences between control maize plants and treated with biologicals were observed following treatment with AZOTOHELP® individually, LIPOSAM® individually, and the complex (ORGANIC-BALANCE® 0.5 l/ha + AZOTOHELP® 0.3 l/ha + LIPOSAM® 0.25 l/ha). Speaking of ZmNHL1, the differences reached 5 and 4 times, respectively, and for ZmVPP1 - 7 times. The expression level of the transcription factor (ZmNAC111) decreased by 5.5 times in plants treated with AZOTOHELP® (Figure 1). This means that treated plants experienced lower levels of drought stress, compared to control ones.
Oxidative stress is an integral component of both abiotic and biotic stresses. To mitigate the consequences of massive reactive oxygen species formation, which are extremely harmful to biomolecules, living organisms have developed antioxidant systems. Antioxidants are various compounds, capable of scavenging reactive oxygen species and neutralizing them. The plant ability to synthesize a sufficient amount of antioxidant molecules during oxidative stress is a marker of its ability to overcome stress event with as minimal losses of productivity as possible.
Fig.2. DPPH radical scavenging activity
Antioxidant properties of maize leaves extracts were evaluated via the DPPH radical scavenging assay. 1,1-diphenyl-2-picrylhydrazyl (DPPH) molecule is a well-known stable free radical, widely used for antioxidant activity assessment. To assess the antioxidant potential through the scavenging of free radicals by the experimental samples, the change in optical density of DPPH radicals is measured. The degree of discoloration indicates the scavenging potential of the antioxidant extract. The higher the ability of plant extract to neutralize the DPPH solution, the higher antioxidant activity they have (Fig.2).
The accumulation of secondary metabolites and changes induced by PGPB represent an initial mechanism for priming the antioxidant system in plants, leading to a more robust defense system following stress signals. This phenomenon, known as priming, stimulates plant growth and underlies PGPB-induced metabolic reprogramming and oxidative stress mitigation during drought through non-enzymatic mechanisms.
The ability of crops to maintain adequate water status and efficiently utilize available resources is vital for their growth and survival in water-deficient environments. The water content of plant organs reflects their metabolic activity. The distribution of water among roots, stems, leaves, and fruits is a crucial aspect of resource allocation in crops, directly reflecting their pattern of water acquisition and utilization. During drought stress, the relative water content of leaf tissues is adversely affected, dropping from nearly 98% in fully turgid leaves to about 30-40% in severely desiccated and dying leaves.
Fig. 3. Relative water content
In our trial, the RWC of control plants was 86.7%, while in plants treated with microbiologicals, it ranged from 91% to 97% (Fig.3). This indicates significantly higher water stress resistance and the ability of plant organisms to maintain normal metabolism under harsh environmental conditions.
Fig. 4. Yield increase
The increased ability to sustain essential physiological processes under stress conditions translates into higher yields. Control plants yielded an average of 8.4 t/ha, whereas plants treated with ORGANIC-BALANCE, AZOTOHELP, LIPOSAM and a mixture of all three yielded 8.63 t/ha, 9.22 t/ha, 9.94 t/ha, and 10.0 t/ha, respectively (Fig.4).
In conclusion, our comprehensive investigation into the effects of microbiological products, incorporating PGPB and water stress-mitigating exopolysaccharides, on maize's drought resistance has yielded promising results. This study provides strong evidence of the significant benefits of biologicals. Treated maize plants exhibited enhanced drought tolerance, as indicated by decreased expression of key water stress marker genes and higher yields. Notably, the RWC measurements demonstrated the capacity of treated plants to maintain vital metabolic functions even in adverse environmental conditions.
These findings are of great significance in the context of sustainable agriculture, offering a potential solution to the challenges posed by water stress in crop production. By utilizing microbiological approaches and harnessing the beneficial properties of PGPB and their metabolites, we can enhance maize's ability to withstand drought. This not only leads to increased yields but also holds promise for more resilient and sustainable agricultural practices. As global concerns over water stress and climate change continue to grow, these insights pave the way for innovative and eco-friendly strategies that can contribute to food security and crop productivity in a changing world.
Author: Yaroslava Bukhonska, Plant Physiologist, M.Sc. in Plant Biology