Modifications of Phytohormone Metabolism Aimed at Stimulation of Plant Growth, Improving Their Productivity and Tolerance to Abiotic and Biotic Stress Factors

Due to the growing human population, the increase in crop yield is an important challenge for modern agriculture. As abiotic and biotic stresses cause severe losses in agriculture, it is also crucial to obtain varieties that are more tolerant to these factors. In the past, traditional breeding methods were used to obtain new varieties displaying demanded traits. Nowadays, genetic engineering is another available tool. An important direction of the research on genetically modified plants concerns the modification of phytohormone metabolism. This review summarizes the state-of-the-art research concerning the modulation of phytohormone content aimed at the stimulation of plant growth and the improvement of stress tolerance. It aims to provide a useful basis for developing new strategies for crop yield improvement by genetic engineering of phytohormone metabolism.


Introduction
Due to the constantly growing human population, ensuring high crop productivity is an important challenge for 21st century agriculture. The research aimed at obtaining highyielding varieties is being carried out [1]. Another important issue is to obtain varieties displaying enhanced tolerance to biotic and abiotic stresses that cause significant loss of yield. Among the abiotic stresses, the most important are drought, thermal stress (too high or too low temperature), light stress, salt stress, and stress caused by environmental pollution, e.g., by heavy metal ions. Due to anthropogenic climate change, an increase in abiotic-stress-evoked losses of crop yield is expected in the near future [2]. Apart from abiotic factors, the biotic ones, such as pathogens, competing plants, parasites, and herbivores, also limit plant growth and productivity.
The application of mineral fertilizers, herbicides, and pesticides, as well as growing high-yielding varieties obtained via traditional breeding methods, enabled a significant increase in crop productivity during the second half of the 20th century [3]. For example, the average cereal yield in 1951 was 1.2 t/ha, while in 1993 it was 2.3 t/ha [4]. However, this yield increase has slowed down in the 21st century. It is currently believed that for the most important crop species, further increases in their productivity obtained by traditional breeding methods are possible only to a small extent. For this reason, research based on genetic engineering became crucial for the future of agriculture [1]. In addition to experiments on transgenic organisms, extensive genome analyses of major crop species are also being carried out. Their goal is to identify quantitative trait loci (QTLs), which are genes determining quantitative traits [5].
Research on transgenic plants conducted over the past decades resulted in the development of various strategies of genetic modification aimed at obtaining lines with increased yield or improved tolerance to stress [6,7]. One of the promising research directions is associated with the modulation of phytohormone levels [2]. Phytohormones participate in the regulation of plant growth and development. They also play a role in response to environmental factors. These compounds include auxins, cytokinins, gibberellins, abscisic acid (ABA), ethylene, jasmonic acid (JA) and its derivatives, and brassinosteroids. Auxins, cytokinins, gibberellins, and brassinosteroids are considered particularly important for the regulation of plant growth and development, while JA, ABA, and ethylene play crucial roles in stress response. However, one needs to remember that growth-stimulating hormones participate in stress responses, while those primarily associated with the stress response are also involved in the regulation of various plant developmental processes, such as dormancy, fruit maturation, or senescence [8]. Plant hormones have pleiotropic effects. Furthermore, the result of their action often depends on cross-talk between various phytohormones and signaling molecules [9]. Phytohormones occur in plants at very low concentrations; their biosynthesis and degradation are strictly regulated. In some cases, reversible inactivation by conjugate formation is also possible [10].
This review presents the current state of research on the modulation of phytohormone content aimed at obtaining transgenic plants with traits favorable for the breeder, such as increased yield and improved tolerance to abiotic and biotic stress factors.

Strategies Applied in Phytohormone-Targeted Genetic Engineering
The research aimed at improving crop performance by modification of phytohormone metabolism and signaling starts with identification of the crucial genes. This is possible mainly due to the studies carried out on mutants or by comparing crop varieties displaying desirable traits with the other ones [10]. Gene and genome sequencing enables the identification of loci crucial for the observed effects. Analyses of phenotypes and detailed analyses at the biochemical level, i.e., determination of phytohormone content, enable scientists to discover gene functions. When the sequence and function of its product are known, bioinformatics provides tools to find homologues in other species. At this point, the plant transformation can be carried out to increase or decrease the level of a certain phytohormone. The increase in hormone level can be achieved by the overexpression of the gene encoding enzyme participating in the phytohormone biosynthetic pathway or silencing of the gene whose product catalyzes hormone degradation. The decrease can be achieved by silencing of the gene crucial for phytohormone biosynthesis or by overexpression of the gene whose product is involved in hormone degradation. The manipulation of the genes encoding enzymes carrying out phytohormone conjugation was also carried out [10]. Sometimes, the increase in phytohormone level may be achieved by the enhanced production of an enzyme catalyzing the formation of a metabolite that serves as a phytohormone precursor (for example, xanthophyll precursors of ABA biosynthesis) or a cofactor needed by the hormone-synthetizing enzyme (for example, molybdenum cofactor required for abscisic aldehyde oxidase activity) (see subchapter concerning ABA). The research on the engineering of phytohormone transport was also carried out (see subchapter about auxins). The significant progress in our understanding of phytohormone signaling opens a wide range of possibilities, as various elements of signaling cascades, transcription factors, and miRNAs are emerging targets for potential modifications. These strategies have been mentioned, but their detailed description is beyond the scope of the present review. In the early research, scientists used strong, constitutive promoters to provide the overexpression of desired genes. The discovery of tissue-specific, developmental-stagespecific, and stress-responsive promoters enabled the improved control of the time and site of transgene expression [10]. Furthermore, artificial promoters have been developed. Considering gene silencing, various constructs may be applied, including antisense sequences, 3 -untranslated regions, and hairpin constructs. The recent development of the CRISPR/Cas9 system paved the way for extensive genome editing (see subchapter about future perspectives).

Auxins
Auxins play a key role in the regulation of plant growth and development, therefore, modulation of their biosynthesis and signaling has been a subject of intensive research [11].
The most important auxin is indole-3-acetic acid (IAA). The substrate for its synthesis is tryptophan, but this amino acid can be transformed in various ways, all of them leading to the same final product. One of the known pathways of IAA synthesis involves two steps: tryptophan oxidation to indole-3-pyruvate, followed by oxidation of indole-3-pyruvate to IAA. The second reaction is catalyzed by monooxygenase encoded by YUCCA genes. Alternatively, indole-3-pyruvate can be converted to indole-3-acetaldehyde and then to IAA. Another known IAA biosynthetic pathway leads through indole-3-acetamide. There is also a pathway specific to the Brassicaceae family, for which the intermediate is indole-3acetonitrile [12]. The concentration of auxins depends on the synthesis, degradation, and transport of this phytohormone; it is also regulated by conjugation. In the latter case, the important role is fulfilled by enzymes encoded by the GH3 family of genes [13].
The results of the experiments carried out on transgenic lines with changed auxin content are summarized in Table 1. In terms of practical application, the modification of auxin levels in developing flowers seems to be the most promising direction. This effect is achieved by the expression of bacterial gene encoding tryptophan monooxygenase (e.g., iaaM from Pseudomonas syringae pv. savastanoi) under the control of an ovule-specific promoter. Increased auxin content stimulates the growth of generative shoots and fruits of transgenic plants. The application of tissue-specific promoters is better than the use of constitutive ones because the enhanced auxin synthesis occurring in whole plants often leads to undesirable developmental disorders (Table 1). Apart from the genes directly involved in auxin biosynthesis and degradation, the genes whose products play regulatory roles can also be targets of manipulation. It was observed that the expression of OsIAA6 was highly induced by drought stress. Transgenic rice with overexpression of this gene under the control of a constitutive promoter displayed enhanced expression of YUCCA genes and was more tolerant to drought [14].
The research carried out on mutants is also important for better understanding of auxin roles. Arabidopsis thaliana mutant yuc7-1D, with the altered gene YUCCA7, displayed a phenotype characteristic for plants with auxin overproduction: tall stems and curled, narrow leaves. It was also more tolerant to drought when compared to control plants [15]. A. thaliana mutant arf2 producing inactive Auxin Response Factor protein (ARF) developed longer and thicker flower shoots, larger and darker leaves, and larger seeds [16]. The brachytic2 (br2) maize mutant with impaired auxin transport in the stem had shorter internodes. This observation may be of practical significance because dwarfism is a desirable trait of cereals as it provides a more favorable ratio of grain biomass to shoot biomass and increases lodging resistance [17]. The modification of soybean GmPIN1 using CRISPR/Cas9 method resulted in plants displaying changed architecture [18]. The knowledge concerning auxin-initiated signaling pathways has intensively broadened during the last decades. The modification of these pathways allows us to obtain plants with desired traits. For example, overexpression of the gene encoding auxin-induced protein ARGOS in A. thaliana resulted in the stimulation of cell proliferation and an increase in organ size [29]. Overexpression of maize ARGOS1 (ZAR1) stimulated organ growth, enhanced grain yield, and drought stress tolerance in transgenic maize [30]. Overexpression of Auxin Response Factor 19 (ARF19) homolog from Jatropha curcas in A. thaliana and J. curcas increased seed size and yield [31]. Expression of gene IbARF5 from sweet potato under the 35S promoter in A. thaliana resulted in enhanced tolerance to drought and salinity in transgenic plants [32]. On the other hand, downregulation of Auxin Response Factor 4 (ARF4) in tomato increased tolerance to salinity and drought stress [33]. Similar results were obtained by Chen et al. [34]. Overexpression of the gene OsAFB6, encoding an auxin receptor, in rice resulted in increased grain yield per plant both in short day and long day conditions [35]. Another auxin receptor, AFB3, when overexpressed in Arabidopsis caused the increase in salt stress tolerance [36]. Transgenic maize overexpressing Auxin Binding Protein 1 (ABP1) was more resistant to sugarcane mosaic virus [37].

Cytokinins
Cytokinins are another class of phytohormones necessary for plant growth stimulation and controlling many developmental processes. They also participate in the regulation of plant senescence. Prolonging organ longevity due to cytokinin action enables longer biomass production by the plant [38]. Among the enzymes participating in cytokinin biosynthesis, the main targets of genetic engineering are as follows: isopentenyl transferase (IPT) catalyzing condensation of isoprenoid residue with adenine nucleotide, cytokinin dehydrogenase (CKX) involved in the degradation of these phytohormones, and glycosyl transferases converting cytokinins into their conjugates [38][39][40][41]. It was observed that mutations in ckx genes led to the increase in cytokinin content. Arabidopsis ckx3 ckx5 double mutant formed larger inflorescences, floral meristems, and flowers and displayed increased seed yield per plant [42]. Similarly, ckx3 ckx5 mutants of oilseed rape showed an increased cytokinin concentration that resulted in larger and more active inflorescence meristems, increased amounts of flowers and ovules and slightly increased seed yield [43]. Natural variations in soybean GmCKX7-1 were linked to altered cytokinin profiles and yield characteristics [44].
The first attempts of genetic engineering of cytokinin metabolism were carried out in the 1990s. Gan and Amasino [45] transformed tobacco with the IPT gene under the control of SAG12 promoter from A. thaliana, responsible for triggering gene expression in senescing leaves. The obtained transgenic lines displayed a greater number of flowers and seeds, delayed leaf senescence, and enhanced biomass production [45]. Due to the success of this strategy, promoters from the SAG family have been often used for plant genetic engineering. However, it has to be mentioned that in some cases, the delayed senescence of older leaves delayed nutrient allocation to seeds and storage organs. As a result, no yield increase and sometimes even yield reduction were observed. Furthermore, in the situation of nitrogen shortage, it was observed that old non-senescing leaves started to compete with younger leaves, which disturbed nitrogen recycling in plants. In rice, early senescing cultivars have a higher yield than those which undergo senescence later [46]. Positive effects were obtained by crossing the transgenic line of A. thaliana overexpressing CKX3 under the control of root-specific PYK10 promoter and displaying enhanced root growth with the lines displaying enhanced leaf growth [47].
Another promoter involved in the regulation of gene expression during senescence but also during stress response is senescence associated receptor protein kinase promoter (SARK). Expression of the IPT gene under the control of this promoter allowed for obtaining transgenic rice and tobacco with increased drought tolerance [48,49]. The examples of the experiments concerning the modulation of cytokinin metabolism are shown in Table 2.
The observed effects vary depending on the species and method used; not all of them are beneficial [50]. The application of inducible promoters responding to specific conditions allows better control of cytokinin content in transgenic plants. This allows us to avoid adverse effects occurring when too many of these phytohormones are synthesized in the plant [9]. Interestingly, the overexpression of AGO2, encoding protein belonging to the ARGONAUTE family and playing a role in the regulation of gene expression, led to the enhanced expression of cytokinin transporter BG3 and changed the pattern of cytokinin distribution in transgenic rice. This, in turn, resulted in an increase in grain length and salt tolerance [51]. Interesting results were obtained by Wang et al. [52], who used CRISPR/Cas gene editing to introduce changes into the cytokinin biosynthetic gene OsLOG5. The researchers managed to obtain rice lines with improved yield properties under drought stress when compared to stressed control [52].
It is noteworthy that in many cases researchers managed to obtain transgenic lines with increased biomass production or seed yield, but also more tolerant to abiotic stresses, such as drought and salinity (Table 2). However, it needs to be emphasized that there are some inconsistencies between the literature data, because the increased tolerance to some abiotic stresses was reported for plants with both increased and decreased cytokinin content. It needs to be remembered that in the experiments on transgenic plants, various species and promoters were used; there were also differences in the stress conditions applied [53].
Furthermore, cytokinins are a group of compounds, including trans-zeatin; cis-zeatin; N 6 -isopentenyladenine; dihydrozeatin; N 6 -benzylaminopurine; kinetin; ortho-, meta-, paratopolins; and ribosides of above-mentioned compounds [54]. It is already known that particular cytokinins vary in sites and timing of their production and degradation, transport routes, signaling pathways, and activity [54,55]. In A. thaliana, trans-zeatin and isopentenyladenine are the most active forms, present in higher concentrations than other cytokinins [54]. Trans-hydroxylated cytokinins, namely, the trans-zeatin-type, are synthesized in the roots and transported to the shoots in xylem sap. They are thought to play an important role as a nitrogen-supply signal in stimulation of the shoot growth. On the other hand, N 6 -isopentenyladenine and cis-zeatin-types are predominant in the phloem sap of A. thaliana. These species are thought to participate in systemic shoot-to-root signaling in cooperation with other signaling molecules [56]. The understanding of the specificity of certain cytokinin types synthesis, transport, and signaling is crucial for the successful genetic engineering of these phytohormones.
Plant responses aimed at restoring the homeostasis of cytokinin levels and signaling were also observed in plants with changed biosynthesis or degradation of these hormones. In some of the experiments, the cytokinin content in transgenic lines was not assessed, while in some others, the methods of cytokinin measurements were questioned by other scientists [55]. The intensive research on cytokinin synthesis, degradation, transport, and signaling is being carried out, which should enable us to explain these effects in the future.   increased drought tolerance: delayed wilting, increased total dry biomass and seed yield per plant in stress-exposed plants (plants not watered for 6-10 days before flowering phase or 2 weeks after flowering) [49] SARK::IPT rice (Oryza sativa) increased tolerance to drought: less pronounced stress symptoms, increased RWC and maximum quantum efficiency of PSII, no decrease in carbon and nitrogen assimilation and protein content (plants not watered for 3 days at pre anthesis) -increased sucrose and starch content in flag leaf, enhanced nitrate content, higher nitrate and nitrite reductase activity, and sustained ammonium content in drought-exposed plants [72] SARK::IPT cotton (Gossypium hirsutum) -delayed leaf senescence (detached leaf assay) -increased drought tolerance: increased root and shoot biomass, Chl content, and photosynthetic rate under water deficit in the greenhouse; increased root and shoot biomass and cotton yield under water deficit in growth chamber (66% less watering) [73] SARK::IPT maize (Zea mays) -increased drought tolerance: delayed wilting and leaf senescence, increased water content in stress-exposed plants, 30  increase in seed dry weight and protein content faster growth of seedlings [77] TP12::IPT flower-specific promoter narrow-leafed lupin (Lupinus angustifolius) increased branching increased total number of fruits (pods) in some lines [78] AtMYB32xs::IPT developmentalprocess-related promoter canola (Brassica napus) -delayed leaf senescence both under controlled conditions and in the field more flowers and siliques increased yield (field trials) [79] AtMYB32xs-p::IPT wheat (Triticum aestivum) -delayed leaf senescence increased yield improved drought tolerance: improved canopy green cover, lower canopy temperatures, higher leaf water potential (field trials) [80] rd29A::IPT stress-induced promoter tobacco (Nicotiana tabacum) -increased tolerance to salt stress: delayed leaf senescence and decreased MDA content in stressed plants (plants exposed to 150 mM NaCl for 2 weeks) [81] AtCOR15a::IPT cold-stress-induced promoter sugarcane (Saccharum officinarum and S. spontaneum hybrids) increased tolerance to cold stress: less pronounced symptoms of leaf senescence (detached leaves exposed to 27 • C, 4 • C or 4 • C and then 0 • C), -increased Chl content, decreased MDA content, and electrolyte leakage in cold-stressed plants (plants were exposed to decreasing temperatures for acclimation, then incubated in 0 • C for 8 h and recovered for 24 h) [82] AtMT::IPT stress-induced promoter   RCc3::OsCKX4 root-specific promoter rice (Oryza sativa) -enhanced root development [96] RCc3::OsCKX4 rice (Oryza sativa) -increased Zn concentration in roots, shoots, and grains increased grain yield per plot [97] bGLU::AtCKX1 root-specific promoter barley (Hordeum vulgare) -stimulated lateral root growth improved drought tolerance: higher RWC, less pronounced decrease in yield (plants exposed to water deficit) -faster recovery and higher RWC in some drought-exposed transgenic lines (hydroponically grown plants were deprived of the growth medium for 24 h), improved growth after stress recovery (observed 2 weeks after severe stress application to hydroponically grown plants and 4 weeks after 3 days of watering withdrawal in soil-grown plants) [98] EPP::CKX1 EPP::CKX2 root-specific promoter barley (Hordeum vulgare) -stimulation of root growth increased concentration of various micro-and macro-elements in leaves increased tolerance to drought: higher CO 2 assimilation rate in stressed plants (plants were not watered until the soil moisture level dropped to 10%, this level was maintained for the next 2 weeks)

Gibberellins
Gibberellins are a large group of tetracyclic diterpenoids. Among them, only a few compounds participate in the regulation of growth and development of higher plantsprimarily GA 1 and GA 4 [2]. Gibberellin deficiency causes dwarfism [123]. For arable crops, especially cereals, dwarfism can be an advantage, because it improves lodging resistance and changes assimilate partitioning so that more assimilates are allocated to flowers and grains. Breeding of semi-dwarf cereal varieties has been proven to be enormously successful in increasing grain yield since the advent of the "green revolution" [124]. The rice semidwarf-1 (sd-1) gene, encoding gibberellin 20 oxidase, is well known as the "green revolution gene" and is considered to be the one of the most important genes deployed in modern rice breeding. It has contributed to the significant increase in crop production that occurred in the 1960s and 1970s, especially in Asia [125,126]. The genes responsible for the "green revolution" in wheat are semi-dwarfing genes Reduced height (Rht). The most important and widely used are the alleles Rht-B1b and Rht-D1b that are found in >70% of current commercial wheat cultivars. They are known to reduce stem extension by causing partial insensitivity to gibberellins due to the changed signaling process [127,128].
The crucial enzymes associated with gibberellin metabolism are GA20ox and GA3ox gibberellin oxidases, catalyzing the last two steps of the synthesis of biologically active gibberellins, as well as GA2ox oxidase, catalyzing the oxidation of these phytohormones to inactive catabolites. The three types of enzymes mentioned above are encoded by small gene families, GA20ox, GA3ox, and GA2ox, respectively [129]. All of them were targets of genetic engineering. The introduction of GA20ox and GA3ox or GA2ox genes enables us to obtain plants with either increased or decreased active gibberellin content (Table 3). Increased gibberellin content stimulates elongation growth and lignin synthesis, while a reduced level of these phytohormones results in dwarfism, stimulation of lateral shoot formation, and reduction of lignin content (Table 3). Change in gibberellin content also allows us to obtain other useful traits. Tomato fruits with GA20ox1 gene overexpression remained firm for a longer time, which prolonged their shelf life [130]. The formation of longer xylem fibers in transgenic poplars overexpressing AtGA20ox1 is beneficial for paper production [131]. On the other hand, reduced lignin content in transgenic switchgrass with decreased gibberellin content facilitates the bioethanol production process. However, the GA2ox-overexpressing lines were semi-dwarf, which is not a desired trait in plants grown for biomass production [132]. Enhanced activity of gibberellin biosynthetic enzymes may be not beneficial in terms of resistance to certain pathogens. Transgenic rise overexpressing OsGA20ox3 was more susceptible to Xanthomonas oryzae pv. oryzae (causing bacterial blight) and Magnaporthe oryzae (causing rice blast), while OsGA20ox3 knockdown lines displayed enhanced resistance to these pathogens [133].
Modification of gibberellin signaling was also taken into consideration. DELLA protein was identified as a repressor in the gibberellin signaling pathway. The above-mentioned Rht alleles encode DELLA proteins [128]. Canola mutant ds-3 bearing a mutation in the gene encoding DELLA protein is semi-dwarf [134]. The overexpression of SLR1 encoding rice DELLA protein enhanced cold tolerance in this species, while plants with overexpression of GA20ox1 were more sensitive to this kind of stress. These results suggest that weakening of gibberellin signaling leads to the improvement in chilling tolerance [135]. Overexpression of GoGID1 encoding gibberellin receptor in alfalfa allowed to obtain transgenic plants with increased growth rates, heights, and biomass production when compared to the control [136].  dwarfism, delayed onset of generative phase increased starch granule accumulation and gravity responses increased tolerance to salt stress: improved survival rate and less reduced seedling growth (seeds exposed to 100 mM or 140 mM NaCl for 7 days); significantly improved survival rate (seedlings exposed to 170 mM NaCl) [142] rbcs::PtGA2ox1 leaf-specific promoter

Brassinosteroids
Similar to other phytohormones, brassinosteroids have a pleiotropic effect and they participate both in the regulation of development and stress response [149]. Considering their chemical structure, these compounds belong to polyhydroxy steroids and are similar to animal steroid hormones [150]. Many experiments concerning the impact of brassinosteroids on plant development and stress tolerance were carried out by spraying the plants with solutions of these phytohormones. There are also data in the literature on genetic engineering of the pathway of their biosynthesis [151]. Important determinants of yield that are regulated by brassinosteroids are plant height, leaf angle, and inflorescence architecture [152]. The results of the experiments on transgenic plants with increased brassinosteroid content are shown in Table 4. There were also experiments aiming at decreasing the level of these phytohormones. Overexpression of AtBAT1 encoding brassinosteroidinactivating acyltransferase in bentgrass resulted in decreased growth rate, erect leaves, prolonged leaf longevity, and improved drought resistance [153].
The rice mutant ebisu dwarf (d2) with impaired brassinosteroid biosynthesis had erect leaves, which improves light penetration into the canopy. On the other hand, it produced smaller seeds [154]. However, another rice mutant, osdwarf4-1, had a stature similar to the d2 mutant, while the morphology of its flowers and seeds remained unchanged [155,156]. Barley accessions carrying a single recessive gene uzu, encoding the brassinosteroid receptor, are semi-dwarf. This gene has been introduced in almost all Japanese hull-less barley cultivars [157,158]. Brassinosteroid insensitive semi-dwarf barley mutants were more tolerant to drought [159].
Transgenic tomatoes overexpressing SIBRI1 encoding brassinosteroid receptor displayed increased height, yield, and fruit number per plant. Transgene overexpression also caused an increase in the levels of carotenoids, ascorbic acid, soluble solids, and soluble sugars during fruit ripening [160]. On the other hand, it led to a decrease in drought tolerance [161]. Rice line overexpressing SERK2, encoding membrane protein interacting with brassinosteroid receptor, produced larger grains and was more tolerant to salt stress [162]. Overexpression of kinases participating in brassinosteroid-induced signaling, e.g., membrane localized ZmBSK1 and downstream acting ZmCCaMK, in corn, led to the improved drought tolerance [163]. Overexpression of the gene TaBRI1 from wheat, encoding transmembrane receptor kinase, in Arabidopsis resulted in increased sensitivity to brassinosteroids, earlier flowering, and increased silique size and seed yield [164]. While the modification of signaling through BRI receptors is linked to growth arrest, the overexpression of BRL3, a vascular-enriched member of the brassinosteroid receptor family, in A. thaliana enhanced tolerance to drought without penalizing plant growth [165]. In recent years, there has been significant progress in the deciphering of brassinosteroid signaling, which opens the way to successful modification of crop plants [152].  •− formation, decreased MDA content, increased levels of soluble proteins and proline, increased SOD activity (plants exposed to 50 mM NaCl for 3 days and then to 100 mM NaCl for 12 days) [174]  increased tolerance to chilling stress: lesser amount of oxidized proteins, lower level of lipid peroxidation and electrolyte leakage, increased maximum quantum efficiency of PSII, increased activity of APX and enzymes participating in ascorbate and glutathione recycling (plants exposed to 4 • C for 3 days) [176] enzyme catalyzing the conversion of 6deoxocathasterone and 3-dehydroteasterone to 6-deoxoty phasterol and typhasterol, respectively Ubi::TaD11-2A rice (Oryza sativa) -increased grain length and 1000-grain weight increased starch content and decreased amylose content [177]

Abscisic Acid
The most important functions of ABA include regulation of dormancy, stomata opening, as well as maturation and germination of seeds. This phytohormone also participates in the response to abiotic stress, primarily drought [2]. In higher plants, carotenoids, specifically violaxanthin or neoxanthin, are substrates for ABA biosynthesis. An important enzyme necessary for the synthesis of violaxanthin (and indirectly neoxanthin) is zeaxanthin epoxidase (ZEP). In the ABA biosynthetic pathway, both xanthophylls are converted to the conformation 9-cis, and then 9-cis-epoxycarotenoid dioxygenase (NCED) catalyzes xanthoxin formation. Later, xanthoxin undergoes two-step oxidation-first to abscisic aldehyde and then to ABA. These reactions are catalyzed by short-chain alcohol dehydrogenase/reductase (SDR) and abscisic aldehyde oxidase (AAO), respectively [178]. Seo et al. [179] observed that under stress conditions AAO expression does not change, while the expression of the LOS5/ABA3 (LOS5) gene is enhanced. This gene encodes an enzyme responsible for sulphation of AAO molybdenum cofactor [180]. Therefore, experiments on plants overexpressing the LOS5 gene were also conducted.
The results of the experiments on transgenic plants with increased ABA content are collected in Table 5. They indicate that the modifications associated with this phytohormone are a very promising direction of research aimed at obtaining varieties with increased drought tolerance. On the other hand, it was reported that ABA overproducing transgenic tomato was significantly more vulnerable to xylem embolism [181]. Apart from the synthesis and degradation of this phytohormone, ABA transport also has an impact on stress tolerance. The Lr34res gene conferring durable resistance to multiple fungal pathogens in rice was reported to be an ABA transporter [182].
ABA-induced signaling pathways are a subject of intensive research. The expression of tomato genes encoding ABA receptors belonging to PYR/PYL/RCAR family in A. thaliana improved drought tolerance of transgenic plants [183]. Similarly, overexpression of native genes of subfamily III of PYR/PYL/RCAR family in A. thaliana resulted in increased ABA-sensitivity and enhanced drought resistance [184]. Overexpression of OsPYL3 and OsPYL9 in rice enhanced tolerance to cold stress and drought; and similarly, overexpression of TaPYL4 in wheat improved drought tolerance [185]. Transgenic poplars overexpressing PtPYRL1 or PtPYRL5 were more tolerant to drought, cold, and osmotic stress [186]. Membrane-bound kinase OsPKR15 was shown to interact with Os-PYL11, an orthologue of AtPYL9. Ectopic expression of OsPKR15 in A. thaliana increased its sensitivity to ABA and resulted in the enhancement of drought tolerance [187]. The potential of ABA receptors overexpression for the improvement of water-use efficiency (WUE) in crops was proposed by Mega et al. [188]. Rice overexpressing OsPYL6 under the control of Arabidopsis thaliana Responsive to Dehydration 29A (AtRD29A) promoter displayed enhanced tolerance to dehydration. On the other hand, the reduced grain yield under non-stress conditions due to reduction in height, biomass, panicle branching, and spikelet fertility was also observed in transgenic plants [189]. The role of SNF 1-RELATED PROTEIN KINASE 2 (SnRK2), comprising a subfamily of plant-specific protein kinases, in ABA signaling and stress tolerance is being investigated [190]. Overexpression of wheat genes of TaSnRK2s in A. thaliana resulted in improved tolerance to drought, salt, and cold stress [185]. Overexpression of ARR5, encoding one of the SnRK2 targets, in A. thaliana resulted in ABA hypersensitivity and enhanced drought tolerance [191]. Transgenic A. thaliana expressing TaCIPK27, encoding a wheat kinase involved in stress response, displayed enhanced ABA-sensitivity and improved drought tolerance [192]. increased tolerance to salt stress: increased fresh weight of stressed plants (seedlings exposed to 0-160 mM NaCl for 10 days) -increased tolerance to osmotic stress: increased fresh weight of stressed plants (seedlings exposed to 0-400 mM mannitol for 10 days) -increased drought tolerance: drought survival (plants not watered for 3 weeks, control plants died); reduced water loss from detached shoots [193] 35S::MsZEP tobacco (Nicotiana tabacum) -increased tolerance to salt stress: increased content of soluble sugars and proline, increased activity of SOD, and decreased content of MDA (plants watered with 200 mM NaCl for 2 weeks) -increased drought tolerance: less pronounced wilting symptoms, increased content of soluble sugars and proline, increased activity of SOD and decreased content of MDA (plants not watered for 2 weeks) [194] EsABA1 under the control of artificial superpromoter tobacco (Nicotiana tabacum) -increased tolerance to salt stress: increased shoot dry weight and total root length in stressed plants (seedlings exposed to 250 mM NaCl for 4 weeks) -reduced Chl degradation in leaf discs incubated in 400 and 600 mM NaCl solutions for 3 days [195] [196] 35S::VuNCED1 creeping bentgrass (Agrostis stolonifera) increased tolerance to salt stress: increased fresh and dry biomass and less pronounced wilting symptoms in stressed plants, increased survival rate (plants watered with 0.2-0.8% NaCl for 10 weeks) -increased drought tolerance: increased fresh and dry biomass, less pronounced wilting symptoms in plants exposed to water deficit, increased survival rate (reduced watering for 10 weeks) [197] 35S::OsNCED3 thale cress (Arabidopsis thaliana) -delayed seed germination, slower growth, changed leaf morphology sugar oversensitivity increased drought tolerance: less severe stress symptoms (plants not watered for 9-13 days) [198]  delayed seed germination, slower growth, changed morphology sugar oversensitivity increased drought tolerance: less severe stress symptoms (plants not watered for 9-13 days) [200] 35S::OsNCED3 rice (Oryza sativa) -promotion of leaf senescence (darkness induction protocol) -increased drought tolerance: increased survival rate of stressed plants (seedlings not watered for 18 days) -increased tolerance to salt stress: increased survival rate of stressed plants (seedlings exposed to 150 NaCl) [201] 35S::VaNCED1 grapevine (Vitis vinifera) changed leaf morphology increased drought tolerance: less severe stress symptoms (plants not watered for 50 days) [202]  increased drought tolerance: reduced water loss from detached leaves; less pronounced wilting symptoms, increased activity of CAT and APX and increased proline content in stressed plants (plants not watered for 6 days) [206] AtLOS5 under the control of artificial superpromoter cotton (Gossypium hirsutum) increased drought tolerance: reduced water loss from detached shoots; less pronounced wilting symptoms in stressed plants (plants not watered for 5 days) -increased fresh weight; SOD, POX, and APX activities; and proline content; decreased MDA content in plants exposed to reduced watering for 8 weeks [207] AtLOS5 under the control of artificial superpromoter maize (Zea mays) -increased drought tolerance: reduced water loss from stressed plants and increased survival rate (plants not watered for 2 weeks) -increased SOD, CAT, and POX activities and proline content, decreased content of H 2 O 2 and MDA in plants exposed to reduced watering for 5 days Many of the ABA-responsive transcription factors have been identified to date, among them, those belonging to NAC, bZIP, AP2/ERF, and WRKY families [210]. Stress-responsive grapevine transcription factor VvNAC17 was shown to increase sensitivity to ABA and drought tolerance when its gene was overexpressed in Arabidopsis [211]. Similarly, the overexpression of soybean GmNAC019 in A. thaliana led to the hypersensitivity to ABA and higher survival rate in a soil-drying assay [212]. The overexpression of droughtinduced maize ZmWRKY26 in A. thaliana improved its tolerance to drought and heat [213]. The positive role of MaWRKY80 from banana in drought stress resistance was shown in the experiment with transgenic Arabidopsis. Among other effects, this transcription factor modulated the expression of genes encoding ABA biosynthetic enzymes [214]. Capsicum annuum ABA Induced ERF (CaAIEF1) expressed in A. thaliana enhanced drought tolerance of transgenic plants [215]. The overexpression of VlbZIP30, encoding a transcription factor belonging to the bZIP family in grapevine, in transgenic A. thaliana improved dehydration tolerance [216]. It was shown that corn transcription factor ZmbZIP33 interacts with core components of ABA signaling. Its overexpression in Arabidopsis led to the increase in ABA content and drought tolerance [217]. Arabidopsis plants overexpressing TabZIP14-B from wheat exhibited enhanced tolerance to salt and cold, as well as increased ABA sensitivity [218]. A maize gene ZmMYB3R, encoding MYB transcription factor, is known to be induced by ABA. Its overexpression in A. thaliana caused increased sensitivity to ABA and enhanced tolerance to drought and salt stress [219]. Increased sensitivity to ABA resulting in the enhanced tolerance to drought, salt, and osmotic stress was also observed in A. thaliana with overexpression of another transcription factor from maize, ZmHDZIV14 [220]. Dehydration responsive element binding factors (DREB) belong to the AP2/ERF family. The expression of ABA-induced AhDREB1 from peanuts in A. thaliana resulted in increased ABA levels and increased sensitivity to this phytohormone, as well as in improved drought tolerance [221]. ZmPTF1 transcription factor, belonging to the bHLH family, is known to be a positive regulator of ABA synthesis. Its overexpression in maize caused an increase in ABA content and enhanced drought tolerance [222]. The other examples of genetic modification of ABA receptors, ABA signaling components, and ABA-responsible transcription factors can be found in the reviews [223][224][225].

Ethylene
Ethylene is another phytohormone important for the regulation of the stress response. Among its other functions, the one important for farmers is the stimulation of fruit ripening [226]. Due to the simple structure of the molecule and ethylene occurrence in the gas phase, this compound is often applied exogenously. Treatment with the ethylene precursor, 1-amino-3-cyclopropane-1-carboxylic acid (ACC), was also applied [227].
As ethylene is known to be a plant growth inhibitor, many of the experiments aimed to decrease the synthesis of this phytohormone ( Table 6). The main target of genetic engineering is 1-amino-3-cyclopropane-1-carboxylic acid synthase (ACS), responsible for the synthesis of the direct precursor of this phytohormone. This enzyme was discovered to be crucial for the regulation of ethylene biosynthesis. Partial silencing of the expression of ACS encoding genes in maize resulted in higher yields of transgenic lines compared to control when plants were exposed to drought [228]. Apple and melon fruits with decreased ACS activity ripened more slowly and were firmer than the fruits of non-transformed plants, which is a desirable trait if there is a need for longer storage [229,230]. Interestingly, inoculation of pea with the soil bacterium Variovorax paradoxus synthesizing ACC deaminase (ACC decomposing enzyme) resulted in improved growth and seed yield under drought conditions when compared to plants inoculated with the V. paradoxus mutant, in which ACC deaminase activity was significantly lower [231].  Inactivation of ZmACO2 encoding ACC oxidase2 catalyzing the final step of ethylene biosynthesis via genome editing using CRISPR/Cas9 method led to the reduction of ethylene production in developing ears and increased grain yield per ear [239].
There were also attempts to modify ethylene-induced signaling. The ethylene response factor superfamily is known to participate in response to various environmental stresses, such as drought, salt, heat, and cold. An elegant summary of the research on ERFs, their participation in stress response, and their genetic engineering using CRISPR/Cas9 genome editing tool was written by Debbarma et al. [240]. The modification of ERF-dependent signaling turned out to be promising also in the case of improving biotic stress response. Transgenic rice overexpressing OsERF83 was more resistant to Magnaporthe orizae, causing one of the most destructive diseases in rice [241]. Overexpression of GmERF3 in tobacco resulted in increased tolerance to drought and salt stress but also enhanced resistance to the bacteria Ralstonia solanacearum, fungus Alternaria alternata, and tobacco mosaic virus [242]. Transgenic A. thaliana with overexpression of MbERF12 from Malus baccata displayed enhanced antioxidant response and increased tolerance to low temperature and salt stress [243]. Transgenic lines of A. thaliana overexpressing ERF1 were more tolerant to drought, salt, and heat stress [244]. It was shown that ectopic constitutive expression of ERF95 and ERF97 led to the increase in tolerance to the heat stress in A. thaliana [245]. Overexpression of native ERF in rubber tree resulted in the stimulation of root growth, increased dry biomass, and increased tolerance to salt stress [246]. Overexpression of TdSHN1, encoding cDNA of SHINE-type ERF transcription factor from durum wheat, in tobacco improved tolerance to Cd, Cu, and Zn [247]. Overexpression of MdERF1B from the apple tree significantly enhanced cold tolerance of Arabidopsis thaliana seedlings, and transgenic apple seedlings and calli [243]. Transgenic tomato overexpressing SlERF5 was more tolerant to drought and salt stress [248]. Other examples of modulation of ethylene signaling resulting in the enhancement of the tolerance to salt stress were reviewed by Riyazuddin et al. [249]. Members of the ARGOS family are known to be negative regulators of ethylene responses. Genetic engineering targeted at ARGOS8, including both overexpression and modification by the CRISPR/Cas 9 method, was used to obtain maize with improved grain yield under drought stress conditions [250]. The regulatory role of miRNA in stress response and its connection with ethylene signaling were also elucidated. For example, salinity-induced miR319 was reported to positively regulate ethylene synthesis and increase tolerance to salt stress in switchgrass [251].

Jasmonic Acid and Its Derivatives
Jasmonates play a role in plant response to various stress factors, including biotic ones [252]. They also participate in the regulation of plant development. The exogenous application of methyl jasmonate increased the yield of soybean [253]. Application of JA or JA together with gibberellin GA 3 resulted in an increase in ginseng yield [254]. The administration of JA alleviated the adverse effects of salt stress on rice and barley [255,256]. However, there is inconsistency in the results reported in the literature, as improved salt tolerance was also observed in transgenic plants with enhanced JA degradation [257]. JA is also used as an elicitor in the production of various secondary metabolites [258].
The experiments on transgenic plants with changed content of JA or its methyl ester were also carried out (Table 7). Among them, interesting ones concern plants overexpressing jasmonic acid carboxyl methyltransferase (JMT), which converts JA into its methyl ester. Another strategy is obtaining plants with overexpression of 13-lipoxygenase or 12-oxophytodienoate reductase participating in the biosynthesis of this phytohormone. Such modifications resulted in an increased tolerance to selected biotic and abiotic stresses and stimulation of the growth of underground storage organs.  shorter roots increased activity of SOD improved tolerance to salt stress: less pronounced reduction in root growth of transgenic wheat (seedlings were treated with increasing concentrations of NaCl for 4 days then exposed to 200 mM NaCl for the next 4 days); increased survival rate of transgenic Arabidopsis (plants were treated with increasing concentrations of NaCl for 4 days, then exposed to 200 mM NaCl for the next 2 weeks) -improved tolerance to osmotic stress and oxidative stress in transgenic Arabidopsis: no or very small reduction in root length (seedlings exposed to 100, 200, 300 mM mannitol or 1, 2 mM H 2 O 2 for 10 d) [263] 35S::TaAOS

tobacco (Nicotiana benthamiana)
enhanced tolerance to Zn: lesser decrease in Chl content in leaf discs exposed to 10 and 20 mM ZnCl 2 for 6 days [264] Ubi::AhAOC rice (Oryza sativa) -increased plant height and root length improved tolerance to salt stress: less pronounced reduction in seedling root growth (seeds germinated in presence of 80 or 120 mM NaCl); less severe stress symptoms, less pronounced reduction in plant height, increased content of proline and soluble sugars (plants exposed to 120 mM NaCl for 2 weeks) [265] 35S::GhAOC1 increased tolerance to salt and osmotic stress: improved germination in the presence of NaCl or mannitol (seeds exposed to 100-200 mM NaCl or 100-500 mM mannitol), more seedlings remained green (observations made after 7 days) -no differences in survival rate when older seedlings were exposed to NaCl (300 mM NaCl for 10 days) [  root growth stimulation increased content of protopanaxadiol group of ginsenosides [273] Jasmonic acid signaling is an object of intensive research [274]. For example, overexpression of TdTIFY11a, a member of TIFY protein family participating in JA signaling, from Triticum durum in A. thaliana promoted germination under salt stress [275]. Expression of VaNAC17, encoding Vitis amurensis transcription factor, known to be induced by drought stress, in A. thaliana resulted in enhanced JA synthesis and drought tolerance [276]. Similarly, the overexpression of VaNAC26 improved tolerance to drought and salt stress in A. thaliana [277]. The overexpression of OsbHLH034 gene encoding transcription factor acting as positive regulator in JA signaling resulted in the increased resistance to rice bacterial blight, but it also increased sensitivity to salt stress [278]. The overexpression of OsbHLH148 improved drought tolerance in transgenic rice [279]. Heterologous overexpression of JA-responsive transcription factor from Ipomea batatas IbMYB116 in A. thaliana caused upregulation of the expression of JA biosynthetic genes, promoted JA accumulation and the JA response, and improved the tolerance to drought stress [280]. Enhanced proline accumulation and increased drought tolerance were also observed in soybean overexpressing another JA-responsive transcription factor, GmTGA15 [281]. The modulation of the expression of JAZ proteins that are negative regulators of JA signaling allow us to obtain plants more tolerant to salinity and drought [257,282].

Future Perspectives
The examples presented here indicate that modification of phytohormone metabolism and signaling is a promising direction of research aimed at the improvement of crop productivity and stress tolerance. The progress in this field is possible due to broadening of the knowledge concerning the regulation of plant growth, development, and stress response, as well as due to the improvement of the methodology used. Many genes that can be targets of genetic engineering have been identified up to date [7]. The extensive research aiming at deciphering phytohormone signaling pathways is being carried out. The modification of this signaling at various levels, from elements of signaling cascades, through transcription factors to miRNAs, is a very promising direction of genetic engineering of crop plants.
Considering the methods of genetic engineering, the most promising innovation is genome editing using the so-called CRISPR/Cas9 system [283]. The system is based on nucleases that can be relatively easily programmed to search for specific DNA sequences. Available variants of effector nucleases allow various modifications of the target region. This makes CRISPR/Cas9 a fast, effective, and precise genome editing tool [283]. It is used both to discover functions of certain genes and to obtain plants of potential application in agriculture. CRISPR/Cas9 genome editing seems especially promising in research aimed at modulation of cytokinin levels [284].
Intensive research on the regulation of gene expression led to the discovery of many promoters specific to certain tissues, organs, or stage of plant development. The application of these promoters allows better control of the time and site of transgene expression. Scientists also designed artificial promoters [285]. There are systems enabling us to combine and introduce multiple genes at once (such as the Golden Gate modular cloning box), as well as methods for the introduction of large DNA fragments into plant cells. New successful protocols of crop species transformation are being developed [7].
An important obstacle in obtaining transgenic plants with improved yield is the well-known trade-off between stress defense and plant growth. One of its reasons is the energetic cost of the development and maintenance of various protective mechanisms, both biochemical and morphological. However, the negative effect of defense induction on growth often results from antagonistic crosstalk between phytohormones rather than from an identified metabolic expenditure. Sometimes, it is caused by pleiotropic effects of certain resistance traits or is a consequence of genetic linkage [286]. Therefore, it is possible to reduce the costs of plant defense. The strategies aimed at such a reduction were summarized by Karasov et al. [286].
To date, the majority of studies on transgenic lines with altered phytohormone content or signaling have been conducted under laboratory conditions. To obtain improved varieties suitable for regular cultivation, it is necessary to carry out large-scale field tests to determine whether the modifications introduced allow us to obtain the desired phenotype under natural conditions. At the same time, care should be taken to minimize the risk of transgene leak, so that genetically modified varieties would not pose the threat of contamination to the genomes of closely related wild species.