CLAIRO Zoom-in 2: Plant hormones help planted trees withstand unfavourable environmental conditions in Ostrava

Plant hormones, also known as phytohormones, are naturally occurring signalling molecules that influence physiological processes, such as plant growth, differentiation and development [1,2,3]. They include a wide variety of organic compounds, which regulate physiological processes at extremely low concentrations [4,3].

The first class of plant hormones to be identified were auxins already in the late 1920s [5]. Later investigations resulted in the discovery of other classes of hormones, ethylene, gibberellins, cytokinins, and abscisic acid. More recently further groups of compounds were identified as plant hormones, such as brassinosteroids, jasmonates and salicylic acid [3], and most recently strigolactones were discovered.

Plant hormones can cause a lot of change in plants and each hormone has its distinct job. Auxins positively influence cell enlargement, bud formation, and root initiation [6]; gibberellins are responsible for stem elongation [3], ethylene stimulates the ripening of fruit, the opening of flowers, and the shedding of leaves [7]; cytokinins regulate a number of important developmental processes [8]; abscisic acid is an important plant growth inhibitor [9]; jasminates have an important role in plant defence [3], by deterring insect feeding; and salicylic acid plays an essential role in the defence against pathogens [10]. In most of the cases various plant hormones effect not only one, but a wide range of physiological processes in plants.

Phytohormones interact with each other and act together either in unison or in opposition to each other. The responses given by the plant to various environmental changes is the result of the effect of the hormonal balance. [3,11]

Although the term ‘hormone’ used in medicine has begun to carry the meaning, ‘a chemical transported message’, long distance transport has not been demonstrated for all plant hormones. By way of example the production of the gas ethylene can act at very short distances triggering changes within the same tissue, or even within the same cell, where it has been synthesized. [3]

The accurate identification and quantification of plant hormones is essential for determining their biological activity, as it depends on the concentration of these compounds in the plant [12]. The development of sensitive analytical methods is critical for gaining a better understanding of the biosynthesis of hormones in the plants, of how chemical signals are transmitted through a cell, and about the effects of various compounds [3].

However, the extremely low concentration of these compounds makes their effective extraction and analysis difficult [12] calling for the application of highly sensitive techniques. For the identification and quantification of plant hormones the plant tissue must be first homogenized and extracted and then purified, during which a pure sample is prepared for the analysis through removing interfering substances from the crude extract [13].

The advancement of the technology has made analysis much easier. A couple of decades ago it was not uncommon that hundreds of grams of plant tissues were needed for purification. In contrast, nowadays only a few milligrams of tissue are sufficient for the determination of hormone levels in certain organs of the plant [3]. Furthermore, major progress in mass spectrometry has made the analytical methodology highly sensitive and accurate [13].

Among the different classes of plant hormones cytokinins are the ones that are applied on the new greenery during the treatment of the plants under CLAIRO, because they are very active growth regulators and have a key role in abiotic stress responses. Growth is an essential factor as quickly growing trees with large canopies are more efficient in removing pollutants. On the other hand, the various abiotic stressors present in the target sites of CLAIRO can significantly hinder the healthy development of the newly planted greenery.

Cytokinins are purine-based plant hormones that are involved in and numerous physiological processes in plants. They promote cell division, control cell growth and differentiation, delay the biological aging of the leaves, increase antioxidant activity, regulate photosynthesis, participate in shoot and root growth, bud formation and flowering, and are also involved in plant defence against biotic and abiotic stress. Cytokinins include a large array of natural and synthetic compounds and their various derivatives. [8,11]

Although the roots are the primary site of cytokinin production, they are also synthetized in the leaves. These small compounds act both short and long distances. The cytokinins that are produced in roots are transported to the shoots through the xylem, that is a tissue in plants to transport water and minerals. However, some cytokinins are transported from shoots to roots through the phloem (a tissue, which transfers organic compounds produced during photosynthesis) [11,14].

Perhaps the most studied plant hormones are cytokinins due to the potential economic benefits linked to their critical role in influencing the biological aging of leaves (senescence) [14]. They regulate the last phase of leaf development, slowing down the disintegration of the photosynthetic apparatus and chlorophyll breakdown, which are linked to the process of aging [8]. In case of most plants, senescence causes leaf yellowing, that is the result of the degradation of the green pigment chlorophyll [8, 14].

The aging of leaves, including yellowing, can be triggered by age, but also by environmental stressors, such as drought, extreme temperatures, or nutrient deficiency, as well as mechanical damage or biotic stress [14,15]. While ethylene and abscisic acid promote biological aging, cytokinins and auxin are delaying it [14].

Cytokinins also promote the process of photosynthesis at different levels. At the level of tissues, they change the structure of the leaves by increasing the number of cells per leaf area, as well as the number vascular bundles that are responsible for transporting water, minerals and organic compounds within the plant; regulate leaf gas exchange to increase the availability of carbon dioxide for photosynthesis. At the level of cells, cytokinins maintain the photosynthetic apparatus of plants by increasing the number of chloroplasts, which are specialized cellular parts that serve as the site of photosynthesis, and by delaying the breakdown chlorophylls. [8]

What is particularly relevant in the context of the CLAIRO project, cytokinins have an essential role in plant defence against abiotic stress [8]. The increase of cytokinin can improve both heat tolerance and low-temperature resistance, as well as drought resistance. Plants give common responses under different types of stress, which make cytokinins fit to alleviate the adverse effects of various abiotic stressors [16]. Cytokinins were found to increase immunity to biotic stress, such as pathogen attacks [8].

As extremely low abundance of plant hormones makes the extraction of these compounds very difficult, chemical synthesis has been gradually gaining ground in the field of organic chemistry [17]. The discovery of new cytokinin compounds has brought about a boom in synthesis and testing a range of cytokinin derivatives. Modern biotechnology makes possible the preparation of new derivatives with more robust biological activity linked to a range of physiological processes, such as biological aging, environmental stress tolerance, mass production, cell division, shoot and root growth [11], as compared to naturally occurring cytokinins. In addition, within the same class of compounds some artificially prepared cytokinins has been found to be more stable than naturally occurring ones [18]. Karel Dolezal, the Head of the Department of Chemical Biology at Palacky University Olomouc, who is also the leader of the research team responsible for plant treatment under CLAIRO has pointed out that

Naturally occurring cytokinins are easily inactivated, while artificial ones can be more persistent.

Scientific achievements in the field led to the application of synthetic cytokinins in agriculture, horticulture and medicine [11]. Some cytokinin compounds are used in the micropropagation of endangered plant species or medicinal plants, during which small pieces of tissue taken from plants are grown under laboratory conditions to produce new plants [11,8].

External application of cytokinins and their derivatives can slow down changes linked to biological aging [8], and can even regreen an already yellowing leaf [19].

Today a huge variety of new cytokinin derivatives can be synthetized and tested with the help of modern biotechnology. As it was explained by Karel Dolezal,

A really small change in the molecular structure can cause a major change in the biological activity of the compound.

These changes in biological activity can be explained by differences in the perception and transmission of chemical signals [20].

Genetic engineering linked to plant hormones also opens new prospects for the application of cytokinins. With current genetic modification methods, the endogenous levels of plant hormones, or their perception by receptors can be increased significantly [8].

Specific preparations, biostimulants will have a key role in the soil and plant treatment applied by the Palacky University Olomouc at the target sites of CLAIRO.

The use of biostimulants holds great potential and offers an innovative and environmental-friendly opportunity to reduce the negative impacts of chemical fertilizers and pesticides in global food production [21]. Plant biostimulants are substances and microorganisms applied to soils and plants to promote plant growth and increase nutrient efficiency and abiotic stress tolerance [22].

They differ from fertilizers and pesticides as they are not synthetic, do not provide nutrients directly to the plants and they neither protect them directly from pathogens and pests [23]. Nevertheless, biostimulants improve plant growth by acting on plant metabolism and facilitating the uptake and assimilation of nutrients [22]. Even though biostimulants do not interact in reality with pathogens or pests, they still strengthen the self-defence mechanism of plants [23]. There is evidence that plants treated with biostimulants are better equipped to cope with both abiotic and biotic stresses [23].

In addition, plant biostimulants can increase the microbial and enzymatic activity of the soil [22], and through this affect positively the structure of the roots and help the transport of nutrients [24, 25].

Biostimulants include organic and nonorganic substances, inorganic compounds and microorganisms. They are commonly applied in small amounts as compared to nutrients which are applied in significantly higher quantities. They can be composed of a range of different components. These include among others the following categories:

• humic substances, which are organic materials from microbial materials and the decomposition of dead organic matter, and the main component of which is humus; they increase soil fertility;
• protein hydrolysates that are available in powder form or as liquid extracts and are applied to the leaves or near the roots;
• extracts of seaweeds that stimulate flowering and the development of plants
• vermicompost, that is a product of the decomposition process using various types of worms, and which improve soil structure;
• plant-derived smoke and smoke-water (snoke contains biologically active compounds); and
• beneficial fungi or bacteria, that can be applied to seeds, seedlings or plants, to improve nutrient efficiency and tolerance to various stressors. [23]

Biostimulants are most commonly applied to the soil, but in certain cases application can also be beneficial on the foliage or on seeds. Biostimulants are generally used for promoting the growth and development of high-value crops such as fruits and vegetables, but their application for cereals and oil seeds has also started to gain popularity. [23]

The Palacky University Olomouc, the project partner responsible for the innovative soil and plant treatment in CLAIRO, is one of the pioneers in Europe in the analysis and synthesis of plant hormones.

The work of Department of Chemical Biology at the Palacky University on plant hormones is undertaken in close collaboration with the Laboratory Growth Regulators (LGR) as well as the Haná Regional Center for Biotechnological and Agricultural Research (CRH), that deals with plant biotechnology.

The Laboratory of Growth Regulators, that is a joint facility of the Palacky University and the Institute of Experimental Botany of the Czech Academy of Sciences, deals mainly with cytokinins, but recently the focus shifted to include also other groups of plant hormones [26]. Karel Dolezal has highlighted that

The Laboratory of Growth Regulators in Olomouc is unique in the world, with a large analytical capacity. There are very few laboratories in the world, which are able to quantify and identify all the classes of plant hormones. There are two of these in Europe, the laboratory in Olomouc and another one in Umea, Sweden.

LRG is engaged in the qualitative and quantitative determination of plant hormones, in optimizing the extraction of phytohormones from really small pieces of plant tissue, and in high quality purification of the samples. The use of the ultra-high-performance liquid chromatography (UHPLC) technique coupled with very sensitive tandem mass spectrometry (MS/MS) makes LRG in Olomouc one of the two best equipped laboratories in Europe together with a mass spectrometry laboratory based in Umea, Sweden. Ultra-high-performance liquid chromatography makes possible 5 times faster samples analysis, than would be possible with conventional high-performance liquid chromatography instrument. The highly sensitive mass spectrometry used in the laboratory allows the identification of compounds in case of extremely small substance concentrations. [27]

An internationally recognized contribution of LRG in the field of experimental biology is linked to the development of a number of anti-tumor agents derived from cytokinins that are used in cancer treatment. The laboratory has scientific results also in the agricultural field, associated with the use of cytokinins to increase the yield and stress resistance of a number of agricultural crops. [26]

At Palacky University, the Department of Chemical Biology focuses on phytochemistry and chemical biology. It is mainly engaged in the preparation and isolation of new plant hormone derivatives and in studying their biological effects [28]. Apart from research in the field of agriculture and medicine, the team of Palacky University is also involved in international projects that are targeting the use of cytokinins for the protection of endangered plant species and medicinal plants.

The experiment led by Palacky University under CLAIRO aims to demonstrate the applicability of cytokinins for increasing the tolerance of newly planted trees to air pollutants.

Dealing with trees in the project is an interesting new area for Karel Dolezal and his colleagues from Palacky University, as they have been engaged so far in research linked to agriculture and medicine, but forestry is a new field for the research team. Furthermore, because of the extreme localities of CLAIRO the task at hand is quite challenging for Palacky University. The research team normally needs to focus on only one type of stressor at one time and not several ones simultaneously, as it is the case with the Ostrava sites.

Three different treatment types are applied by Palacky University in CLAIRO on the planted greenery. Only commercial inorganic fertilizer is used in Variant A (classical treatment); in Variant B the soil and plants are treated by biostimulants (modern treatment); and finally, in Variant C cytokinins are added to the biostimulants (innovative treatment).

So as to ensure that the effects of the various treatment types can be compared under very similar environmental conditions, the application of all three variants is repeated in the experiment across different soil types. In case of both locations there are two different types of soil conditions. The larger segment of the Radvanice site is covered by brown earth, while a smaller area is represented by transitions between soils with stagnating water and soils with clay-enriched subsoil. In the Bartovice site one segment has a significant humus content containing also ash and sludge, while the other includes mixtures of coarse-grained building debris, sand and dust particles. Consequently, in practice this means that both in Radvanice and in Bartovice two larger segments are delineated, which are divided further into three smaller sections for each of the three different variants (Maps 1 and 2).

A biostimulant preparation was selected for the experiment, which contains humic subtsances and seaweed extracts, and which was designed to support the development of field crops and forest crops during the entire growth period.

The biostimulant used under Variant B is the base of the preparation applied under Variant C, which is supplemented with a biologically active specific cytokinin derivative.

For the experiment a specific cytokinin derivative was selected that had been synthetized by the Palacky University Olomouc. Karel Dolezal highlighted that the compound that was finally chosen, needed to meet a number of criteria. It is very similar to a naturally occurring compound, but actually more active. It is very effective in delaying biological aging of the plant, has strong anti-stress properties and it is also stable. Furthermore, it is easy and cheap to synthetise this derivative.

The first treatment took place in June 2021 following the planting of the trees. The second one is undertaken at the end of September 2021. Later plants will be treated twice each year, in May and in September for at least five years until the trees will be established and do not need help anymore. The preparations are applied in a liquid format on the soil and the plants.

The physiological effects of the preparations on plants are monitored by the Palacky University regularly. Monitoring is performed one week, one month and three months after each treatment. Photosynthetic parameters and gas exchange characteristics are measured, which are good indicators of the health condition of the plants.

The team of Palacky University was surprized to see that trees were in very good condition following the first treatment regardless of the variants applied. In case of each treatment type, the photosynthetic parameters are at very high levels and no significant differences can be seen currently among the three variants. The fact that also control plants are also in good condition shows that the right species were selected, and well-functioning ecosystems were designed by the Silesian University in Opava, which are adapted to the harsh local environmental conditions. The cooler weather with above average precipitation during summer could also be one of the reasons for the healthy state of the plants. Over time however, because of the influences of the locality this can change and differences are expected to emerge depending on the type of the treatment.

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