Advances in Crop Science and Technology
Open Access

Triazoles; Strigolactone Inhibitors; Phytohormones; MOFs

Introduction

Paclobutrazol (PBZ) [(2RS, 3RS)-1-(4-chlorophenyl)-4, 4-dimethyl- 2-(1H-1, 2, 4-trizol-1-yl)-pentan-3-ol] belongs to the triazole family. Paclobutrazol (PBZ) [(2RS, 3RS)-1-(4-chlorophenyl)-4, 4-dimethyl- 2-(1H-1, 2, 4-trizol-1-yl)-pentan-3-ol] belongs to the triazole family. This compound regulates plant growth by influencing the isoprenoid pathway, inhibiting GA synthesis, decreasing ethylene production, and enhancing the content of both CKs and ABA. Coolbaugh et al. showed that ancymidol blocks with high specificity the oxidative steps leading from ent-kaurene to ent-kaurenoic acid in the pathway of GA’ biosynthesis. The same oxidative steps are thought to be inhibited by the active triazol derivatives. Paclobutrazol has been reported to inhibit GA biosynthesis in plants by inhibiting kaurene oxidase, a Cyt P-450 oxidase, thus, blocking the oxidation of kaurene to kaurenoic acid. The objectives of this study were to determine ‘Abbreviations: GA, gibberellin; El, electron impact; TMSi, trimethylsilyl ether; amu, atomic mass unit, the translocation and distribution pattern of paclobutrazol from root system of apple seedlings at various time intervals by GC and to confirm the presence of paclobutrazol in apple seedling tissues by GC-MS. Plant growth retardants are compounds which are used to reduce plant growth without changing developmental patterns or being phytotoxic [1]. PBZ, a member of triazole plant growth regulator group, is used widely in agriculture. It is a cell elongation and internode extension inhibitor that retards plant growth by inhibition of gibberellins biosynthesis. Gibberellins stimulate cell elongation. When gibberellin production is inhibited, cell division still occurs, but the new cells do not elongate. The result is shoots with the same numbers of leaves and internodes compressed into a shorter length. Reduced growth in the diameter of the trunk and branches has also been observed. Another response of trees to treatment with PBZ is increased production of the hormone abscisic acid and the chlorophyll component phytol, both beneficial to tree growth and health. PBZ may also induce morphological modifications of leaves, such as smaller stomatal pores, thicker leaves, and increased number and size of surface appendages, and increased root density that may provide improved environmental stress tolerance and disease resistance. PBZ also has some fungicidal activity due to its capacity as a triazole to inhibit sterol biosynthesis.

Chemistry of Paclobutrazol

PBZ ([(2R, 3R + 2S, 3S)-1-(4-chloro-phenyl) 4,4-dimethyl-2- (1,2,4-triazol-1-yl)-pentan-3-ol]) has been developed as a plant growth regulator and is registered with trade names such as Bonzi, Clipper, Cultar, and Parsley. It belongs to the triazole compounds that are characterized by a ring structure containing three nitrogen atoms, chlorophenyl and carbon side chains. Structurally, PBZ is a substituted triazole with two asymmetric carbon atoms and is produced as a mixture of 2R, 3R, and 2R, 3R, and 2S, 3S enantiomers. Paclobutrazol (PBZ) is a plant growth retardant and triazole fungicide [2]. It is a known antagonist of the plant hormone gibberellin. It acts by inhibiting gibberellin biosynthesis, reducing internodial growth to give stouter stems, increasing root growth, causing early fruitset and increasing seedset in plants such as tomato and pepper. PBZ has also been shown to reduce frost sensitivity in plants.

The structure of Paclobutrazol (Table 1)

Table 1:Structure of Paclobutrazol.

Chemistry of Paclobutrazol (PBZ)

PBZ was first announced in 1986 as a new bioregulator, which was introduced to the market by ICI Agrochemicals (now part of Syngenta). It is a synthetic compound [(2 RS, 3 RS) -1- (4-chlorophenyl) - 4,4-dimethyl-2- (1 H-1,2,4-triazol-1-yl) -pentan-3 ol] that inhibits vegetative growth , belonging to the triazole group. Chemical properties of PBZ include: molecular weight 293.8, molecular formula C15H20ClN3O, melting point 165°C–166°C, density 1.22 g ml–1 and water solubility 35 mg L–1. PBZ is a hydrophobic and slightly polar molecule, with hydrophilic parts. It has two chiral centres (two asymmetric carbons), hence the existence of two pairs of enantiomers [(2R, 3R)- and (2S, 3S)-] and [(2S, 3R)- and (2R, 3S)-]. However, among the stereoisomers, 2S and 3S show a higher inhibition efficiency in gibberellin biosynthesis, but 2R and 3R are more easily degraded.

Paclobutrazol (PBZ) is the ISO common name for an organic compound that is used as a plant growth retardant and triazole fungicide. It is a known antagonist of the plant hormone gibberellin, acting by inhibiting gibberellin biosynthesis, reducing internodal growth to give stouter stems, increasing root growth, causing early fruitset and increasing seedset in plants such as tomato and pepper. PBZ has also been shown to reduce frost sensitivity in plants. Moreover, paclobutrazol can be used as a chemical approach for reducing the risk of lodging in cereal crops. PBZ has been used by arborists to reduce shoot growth and shown to have additional positive effects on trees and shrubs. Among those are improved resistance to drought stress, darker green leaves, higher resistance against fungi and bacteria, and enhanced development of roots. Cambial growth, as well as shoot growth, has been shown to be reduced in some tree species.

Structure and synthesis

The first synthesis of paclobutrazol was disclosed in patents filed by an ICI group working at Jealott’s Hill. 4-Chlorobenzaldehyde and pinacolone are combined in an aldol condensation to form a chalcone which is hydrogenated using Raney nickel as catalyst to give a substituted ketone. This material is brominated and the resulting compound treated with the sodium salt of 1, 2, 4-triazole in a nucleophilic substitution reaction. The final reduction reaction uses sodium borohydride, which in cold methanol gives almost exclusively the diastereomer pair having the absolute configuration (2R,3R) and its enantiomer (2S,3S), with only about 2% of the alternative (2R,3S) and (2S,3R) isomers. However, this pair of isomers can be produced when the reduction is carried out using butylmagnesium bromide.

In a 1984 study, ICI workers separated the individual enantiomers by chiral resolution and were able to demonstrate that only the (2R,3R) isomer displays substantial fungicidal activity, whereas the (2S,3S) isomer is responsible for the growth regulating properties [3]. However, the commercial product (developed under the code number PP333) was the racemic material, since separation of the isomers was unnecessary when both components had utility in agriculture.

Mechanism of action

Paclobutrazol is an inhibitor of enzymes which use cytochrome P450 as a co-factor. Their active site contains a heme center which activates oxygen from the air to oxidise their substrates. The (2S,3S) isomer inhibits the enzyme ent-kaurene oxidase which is on the main biosynthetic pathway to gibberellins, which are important plant hormones. A secondary effect arising from the inhibition of entkaurene oxidase is that its precursor, geranylgeranyl pyrophosphate accumulates in the plant and some of this is diverted into additional production of the phytol group of chlorophyll and the hormone abscisic acid. The latter is responsible for controlling transpiration of water through the leaves and hence PBZ treatment can lead to better tolerance of drought conditions. The (2R,3R) isomer is a better fit to the active site of the fungal cytochrome P450 14α-demethylase. This inhibits the conversion of lanosterol to ergosterol, a component of the fungal cell membrane, which is lethal for many species Many other azole derivatives including propiconazole and tebuconazole show this type of activity, so the main commercial opportunity for paclobutrazol was as a plant growth retardant and it was first marketed by ICI in 1985 under the trade names Bonzi, Clipper, Cultar and Parlay. PBZ is found as an active ingredient in several commercial products such as: “Cultar® 25 SC” and “Bonzi®” (Syngenta, USA), “Regalis® Plus” (BASF, USA) and “AuStar®” (Chemicals Direct Pty, Ltd., Australia). It is a non-polar compound with a broad-spectrum nature that is mainly translocated via xylem. However, it will depend on the application route, as it can also be transported via phloem.

The mode of action of PBZ is framed as part of the terpene pathway [4]. This is, it inhibits the biosynthesis of gibberellins by inactivating the enzyme ent-kaurene oxidase, which catalyses their oxidation to ent-kaurenoic acid. This favours the activation of the enzymes geranylgeranyl reductase and phytoene synthase for chlorophyll and abscisic acid biosynthesis, respectively. As a result, it decreases vigour and promotes floral induction and development.

Plant growth and development is associated with cell division and expansion induced by gibberellin activity. PBZ applications inhibit its synthesis; consequently, cell elongation does not occur. In the tree you can see a greater number of leaves, shoots and shorter internodes. Likewise, it increases the thickness of the leaves and reduces the size of stomatal pores through transpiration. Improvement of water relations in treated plants takes place because of enhancement in ABA content that decreases stomatal aperture, decreases shoot growth and causing less surface area for transpiration, more roots for uptake of water, and anatomical alterations in leaves that impart barriers to water loss.

Mode of action

Although the precise features of the molecular structure which confer plant growth regulatory activities are not well understood, it appears to be related to the stereochemical arrangement of the substituents on the carbon chain. There are indications that enantiomers having S configuration at the chiral carbon bearing the hydroxyl group are inhibitors of GA biosynthesis. One of the inhibitor of GA biosynthesis, paclobutrazol, is mainly used as growth retardant and stress protectant. This retardation of growth is due to the interference of PBZ with gibberellin biosynthesis by inhibiting the oxidation of ent-kaurene to ent-kauronoic acid through inactivating cytochrome P450-dependent oxygenase [5]. In addition, it tends to be much more effective than various other plant growth regulators at relatively low rate of applications.

PBZ is also known to affect the synthesis of the hormone abscisic acid and phytol. Abscisic acid is also synthesized via the terpenoid pathway. When gibberellins synthesis is blocked, more precursors in the terpenoid pathway are accumulated and shunted to promote the genesis of abscisic acid. It has also been reported to inhibit normal catabolism of ABA. The effect of PBZ on both the synthesis and catabolism processes leads to enhanced concentrations of ABA in leaves. One of the major roles of ABA is to cause closing of stomatal aperture and decreasing loss of water from leaves through transpiration. Improvement of water relations in treated plants takes place because of enhancement in ABA content that decreases stomatal aperture, decreases shoot growth and causing less surface area for transpiration, more roots for uptake of water, and anatomical alterations in leaves that impart barriers to water loss.

Terpenoid pathway for biosynthesis of gibberellins, abscisic acid, phytol, and steroids, and path for degradation of abscisic acid. Steps blocked by paclobutrazol indicated with Geranyl diphosphate synthase (GPS), Farnesyl diphosphate synthase (FPS), Geranyl geranyl diphosphate synthase (GGPS), ent-copalyl-diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), Geranyl geranyl reductase (GGRS), Chlorophyll synthase (CHL) and Phytoene synthase (PSY) are the enzymes involved in the terpenoid pathway. ABA 8′-hydroxylase (ABA 8′OH) involved in the enzymatic degradation of ABA into Phaseic acid. KO, KAO and ABA 8′OH are the enzymes inhibited upon PBZ application.

Plant growth regulators are widely used in contemporary agriculture to promote plant growth, yield and grain quality. Both beneficial and adverse effects of plant growth regulators on growth and development as well as plant metabolism have been documented. The term growth retardants is used for all chemicals that retard cell division and cell elongation in shoot tissues and regulate plant height physiologically without formative effects. Paclobutrazol is a member of the triazole family of plant growth regulators and has been found to protect several crops from various environmental stresses, including drought, chilling, heat and UV radiation.

Paclobutrazol (PBZ) is a triazole derivative that inhibits sterol and gibberellin biosynthesis. This compound can markedly affect plant growth and development by altering the photosynthetic rate and modifying the phytohormone levels. Paclobutrazol inhibits the activity of ent-kaurene oxidase, which is an enzyme in the GA biosynthetic pathway that catalyzes the oxidation of ent-kaurene to ent-kaurenoic acid. PBZ application has reduced plant height, improved stem diameter and leaf number, altered root architecture directly contributed to yield increase, and indirectly reduced the event of lodging. It was also reported that application of paclobutrazol effectively reduced vegetative growth of rice plants and increased chlorophyll content. Rice seedlings treated with paclobutrazol allocated less photosynthates for vegetative growth; allocated more photosynthates for seed development compared to control plants or those plants treated with gibberellin. In corn (Zea mays L.) under drought stress, application of 50 ppm paclobutrazol increased yield and average weight of 1,000 seeds [5]. Moreover, the possible hypotheses on drought tolerance regulation by PBZ have been proposed, which state that it maintains the endogenous cytokinin levels and stabilizes leaf water potential and causing increased leaf and epidermal thickness. Alternatively, regulation of free proline and glycine betaine as major osmoprotectants and promotion of enzymatic and non-enzymatic antioxidant activities, reduce the toxicity derived from drought stress.In a view of this, the objective of this article is to review the effect of paclobutrazol on morphological, biochemical, yield and stress responses of crop.

Paclobutrazol induced responses in plants

Morphological response

Paclobutrazol is used in high input crop management to shorten the stem, thereby reducing the risk of lodging. There are several reports describing the various effects of paclobutrazol on plant morphology of crops. For example, reported PBZ application significantly decreased plant height of Camelina sativa when compared to control and induced dwarfing effect and with highest concentration of PBZ in which maximum reduction (47.5% decrease) in plant height with respect to control was obtained. Similarly, paclobutrazol concentrations of 200 mg/L to 600 mg/L decreased gibberellin content in the leaves compared to that of control when applied to rice plant during preanthesis]. Paclobutrazol application reduced plant height and the greater concentration of paclobutrazol caused severe dwarfism as indicated in (Figure 1). Reduction in plant height is considered as the most imperative morphological outcome of paclobutrazol application. According to Tesfahun and Menzir 2018, plant height reduction strongly associated with reduced elongation of the internodes, rather than lowering the number of internodes and they found uppermost internodes to be shortened under paclobutrazol application [6]. reported that foliar application of paclobutrazol at 12.5 g a.i ha−1, under a single-application scheme reduced plant height of sunflowers without adverse effects on achene and oil yields, thus providing a basis for reducing the risk of plant lodging.

Figure 1: Isoprenoid biosynthetic pathways in plants. Metabolites discussed in this review are shown in bold. The mevalonate (MVA) and methylerythritol phosphate (MEP) pathways both generate isopentenyl diphosphate (IPP) in parallel and contribute to particular isoprenoids (Swiezewska and Danikiewicz, 2005). Thick grey arrows show the exchange of intermediates between the MVA and MEP pathways. Abbreviations: DMAPP: dimethylallyl diphosphate; FPP: farnesyl diphosphate; GPP: geranyl diphosphate; GGPP: geranylgeranyl diphosphate; IPP: isopentenyl diphosphate.

Yield response

The positive effects of paclobutrazol on yield components such as greater fertile tillers, spike, fertile panicle or spikelet and in some cases mean grain weight has been shown in studies evaluating the production potential of cereals; however, numerous studies have revealed that the increased fertile tiller, altered phenology and better canopy have been the main important components that significantly associated with enhanced grain yield in response to paclobutrazol application. One of the possible increments in grain yield is (i) the change in canopy coverage, in which the plant developed broader canopy this in turn facilitated improved light interception for better photosynthesis in leaves and stems of PBZ treated plants. Further, (ii) the leaves in PBZ treated plants were closely packed, dark green and remained on plants for a larger period than controls. This may explain increased dry matter accumulation in stem and root and simultaneous yield increments despite reduced plant height due to PBZ treatments. linked the grain yield increment (iii) with slow senescence in leaves which prolong the phase of seed development and maturation and as a consequence, the yield can be increased, but the harvest time delayed. The other possible grain yield increment is closely related to (iv) the spread of roots, which determines the uptake and utilization of water and nutrients. In similar way, reported that greater root biomass is significantly and positively correlated with ear characteristics and enhanced biomass and grain yields. The increased in the grain yield is attributed partly to (v) decreased investment in above ground parts, due to a relatively stouter canopy of paclobutrazol treated plants, (vi) as well as enhanced grain filling in the treated plants due to the improved rooting system, which possibly increased the nutrients and water uptake.

Physiological response

Chlorophyllis a critical component of the primary photosynthetic reaction has a dual function in photosynthesis. It captures light, and also serves as a medium for the light-driven charge separation and transport of electrons. The biosynthesis of chloroplast pigments was significantly affected by paclobutrazol as indicated in (Table 2). Several studies on tef and camilena showed that chlorophyll was higher on plants treated with paclobutrazol compared to control. The increased chlorophyll content treated with paclobutrazol might be from minimized damage caused by reactive oxygen and changes in the levels of carotenoids, ascorbate and the ascorbate peroxidase. The report of Nivedithadevi, Somasundaram and Pannerselvam 2015 also showed that plants treated with paclobutrazol synthesized more cytokinin, which in turn enhanced chloroplast differentiation and chlorophyll biosynthesis, and prevented chlorophyll degradation [7]. Furthermore, paclobutrazol appears to have delayed the onset of senescence, represented by the rate of chlorophyll degradation in attached mung bean leaves, which was probably due to the enhanced endogenous level of cytokinins through their secondary effect on plants. Paclobutrazol application in Camelina sativa L. Crantz also increased chlorophyll content which led to greater rate in photosynthesis and higher yield. The results of showed that black rice plants treated with either 25 or 50 ppm paclobutrazol have greener leaves compared to control and the leaves also experienced late senescence. This could be due to an increase in the activity of oxidative enzymes that prevented cell maturation.

Table 2: Growth retardents.

Stress response

Since early migration from aquatic to terrestrial environments, plants have had to cope with periodic and unpredictable environmental stresses, such as drought and salinity. Crop production in arid or semiarid regions is usually restricted by soil moisture deficit as well as soil salinity. Water deficit coupled with salinity in irrigation water is the major limiting factor in most regions where cereals are subjected to extreme water deficit during dry seasons. Enhanced stress tolerance in cereals can be achieved by exogenous application of some plant growth regulators, including paclobutrazol. Exogenous application of paclobutrazol can reduce some of the harmful effects of drought and salt stress and in some cases, compensate losses or damages caused by these stresses. Paclobutrazol increased stress tolerance of plants through the following methods.

Increasing root activity

Paclobutrazol are often referred as multi-stress protectants due to their innate potential of mitigating the negative effects of abiotic stresses had on plant growth and development, by regulating hormones level, enzymatic and non-enzymatic antioxidants and osmolytes. The 2-year results showed that root activity and root-bleeding sap flow were significantly higher in paclobutrazol treatments than compared to control. As root-bleeding sap is the indicator of root pressure, therefore, the improved root-bleeding sap is attributed to higher root growth and root vigor in response to the paclobutrazol application. Also the study of Morita, Okamoto, Abe and Yamagishi 2008 showed the presence of a close relationship between the bleeding rate and the root traits in maize. The rate of root bleeding sap is correlated to active water absorption of the root system and reflects the physiological root activity. Yan et al. 2013 also observed that uniconazole, a triazole with a function similar to paclobutrazol promoted root activity, root bleeding sap and improved root growth in soybean. Previously, Zhao, Fang and Gao 2006 also observed a higher root activity in rice and wheat treated with plant growth regulators. Thus the application of paclobutrazol may improve plant performance under stressful condition through stimulating root activity of the plant [8].

Submergence tolerance

Also paclobutrazol has a role on submergence stress. The longtime submergence is also detrimental to rice crop, and where this cannot be avoided some corrective measures are to be taken to exploit yield potential of rice crop. Under submerged conditions, 200 ppm paclobutrazol spray to rice seedlings resulted in 50% increase in percent survival over control. The increased seedling survival is presumably due to low energy use in elongation, while, the same was available for maintenance processes, for synthesis of anaerobic proteins and maintenance of membrane integrity essential for submergence tolerance.

Increasing antioxidant enzyme

Increased the levels of antioxidant enzyme activities in plants under stress conditions are natural responses, which can help plants better tolerate the stress. Exogenous application of paclobutrazol enlarged these traits and enhanced stress tolerance in plants. Additionally, the enhanced antioxidant enzyme activities in response to paclobutrazol application may also protect their photosynthetic machineries against damages caused by Reactive oxygen species during water-deficit conditions.

Among these SOD and CAT are well-known antioxidative enzymes in cells, which can catalyze the poorly reactive oxygen species converting them to non-toxic substances. SOD constitutes the first line of defence against active oxygen species (AOS). This enzyme removes O2 ⁻ by catalyzing its dismutation, wherein one O2 ⁻Mis reduced to hydrogenperoxide (H2O2) and another is oxidized to oxygen. CAT is an enzyme that can convert H2O2 directly into water and oxygen. This enzyme is present in every cell and in particular on peroxisome. SOD and CAT plays a significant role in defending against oxidative stress induced by abiotic stress in plant tissues. Similarly, Rady and Gaballah 2012 also found that the application of paclobutrazol on barley crop had a significant role in increasing CAT and SOD concentration. This compound reduced damage in plants grown under water stress conditions by enhancing the activity of these antioxidative enzymes. A number of studies showed that paclobutrazol minimizes the adverse effects of water-deficit stress by increasing the levels of the activities of antioxidative enzymes in many plants such as groundnuts, sesame seeds, mangos and tomatoes.

Proline content

Proline is well-known as an osmotic regulator that can reduce osmotic damage. It was reported that under non-water-stressed condition paclobutrazol does not have any significant effect; however, under water stress conditions, paclobutrazol (40 mg l−1) treatment resulted in a significant increase in proline content of barley plant as indicated in. Recent studies showed that paclobutrazol has effect in increasing free proline content of crops to protect from drought stress. However, the effect of paclobutrazol on proline content is still unclear. Supporting this idea Mohamed et al. 2011 reported that free proline content in 50 mg L−1 paclobutrazol-treated tomato plants grown under 60% field capacity peaked at 54.56 mg g−1, which is 1.52-fold compared to control. In contrast, free proline content in 10 mgL−1paclobutrazol pretreated peanut under water deficit conditions (1.04-folds over control) was lower than non-treated plants (1.49-folds over control) [9]. The accumulation of proline in leaves could possibly play a protection role apart from osmoregulation during drought stress. In sight of this sense we understand that paclobutrazol might act as a stress ameliorating agent crops, as this plant does not need to accumulate the proline content in the leaves. Previous studies have proved that proline accumulation was lower in tolerant plants when compared to sensitive plants during periods of drought stress. However, further study is needed in order to reach conclusive agreement on the effect of paclobutrazol on free proline content of crop leaves.

Translocation and chemical stability

It was previously believed that triazoles were primarily transported acropetally in the xylem. However, PBZ has been detected in xylem and phloem sap of castor bean and pear indicating that triazoles can be transported acropetally and basipetally. Although the metabolic fate of applied has not been investigated in detail most of them have a high chemical stability and depending on the site of application tend to be metabolized slowly. Early and Martin observed more rapid PBZ metabolism in apple leaves than other plant parts, while Sterrett found little evidence for PBZ metabolism in apple seedlings. PBZ is comparatively more resistant to degradation than BAS 111.

Methods of application

The most common application methods of PBZ are foliar sprays and media drench. PBZ shows good results for both methods; however, drenches act longer and provide uniform control of plant height with lower doses. When PBZ is applied by foliar spray, the compound is poorly soluble in water and consequently little translocated in the phloem. Thus, when applied by spray to the plant canopy, its action is restricted to the wet contact area. On the other hand, the application of PBZ by drench is uniform and increases the product efficiency in lower concentrations compared to foliar spray. Moreover, drench application of PBZ may directly inhibit GA synthesis as roots synthesize large quantities of GA. Similarly, Banon et al. and AlKhassawneh et al.

demonstrated that drench applications were more effective, allowing to use lower quantity of PBZ, which is desirable for both ecological and economic reasons. This effectiveness may be directly related to its high persistence in the soil drench and in plant organs. Gent and McAvoy also indicated that PBZ persists in annuals, herbaceous perennials and, especially, woody ornamentals. PBZ is considered a phloem immobile chemical evidence exists that it is partially mobile in phloem. Studies indicate that PBZ and uniconazole-P move in plants acropetally via the xylem, accumulate in leaves, and have very low mobility in phloem. This results in a low level of PBZ residues in seeds and fruits as they are supplied with nutrients via the phloem However, low phloem mobility of PBZ further reduces the effectiveness of foliar spraying, since PBZ action on plant growth would be restricted to the site of application.

Application rates

A lot has been done to identify the best application rate of PBZ in different places. Factors like age of the trees, extent of vegetative growth and method of application should be considered when determining the rate of PBZ to be applied. The rates also affect the different tree parameters variously. In general, the amount of PBZ required to promote flowering and fruiting in fruit crops is very low.

The rate of soil application is a function of tree size and cultivar. The rate is determined by multiplying the diameter of tree canopy in meters by 1–1.5 g of active ingredients of PBZ. They indicated that other factors including soil type, irrigation system, etc. may affect PBZ activity and, thus, may be necessary to improve the effectiveness of the chemical. As to them, overdose may cause undesirable effects such as restricted growth, panicle malformation (too compact), and shoot deformity. They also asserted that to insure uniform flowering and reduce the detrimental side effects, the search for better application methods were investigated and one approach is to apply high volume of low PBZ concentration to improve better coverage. Optimizing PBZ dose is a prerequisite for any yield improvement programmes. Severe and undesirable loss in seed and oil yield of Camelina was observed when the plants were treated with higher PBZ concentration (125 mg L−1), while PBZ dose between 75 mg L−1 and 100 mg L−1 can effectively improve the economic traits, including higher seed and oil yields in Camelina. Severe retardation of Camelina growth was also reflected in plant height, branch and canopy size when the plants were sprayed with higher PBZ concentration (125 mg L−1). He also reported that Camelina seed yield increased by 74.23% when compared to the control with the applications of 100 g L−1. Similarly, reduced yields were recorded in peanut and Jatropha associated with higher PBZ concentrations. Kamran et al. described that soaking of seeds under 300 mg L−1 PBZ increased the average maize grain yield by 61.3% as compared to the control. Patil and Talathi also reported that application of 5 g of PBZ through soil enabled to induce early and regular fruiting with 2.8 times increase in yield in mango var. Alphonso. In addition, PBZ at a rate of 150 mg L−1 in bottle gourd, 100 mg L−1 in bitter gourd, 150 mg L−1 in French bean, 125 mg L−1 in cucumber and 40 mg L−1 in tomato increased the yield and quality of fruits.

Morphological and physio-biochemical responses of plants to PBZ

Effect of PBZ on relative water content

Relative water content (RWC), directly related to the content of soil water is a significant indicator of water stress in leaves. Plant exposure to water stress results in an immediate reduction of RWC. PBZ accelerated the stomatal closure, improved water retention, and increased drought tolerance in jack pine and oak. PBZ-treated plants maintained higher RWC than the non-treated ones’, stated that the application of PBZ (30 mg/l) in wheat under control and water-stressed plants resulted in an increase of 5% and 11% respectively in the mean RWC. The reduced rate of evapotranspiration helps plants maintain a higher RWC, and overcome stress, and developed tolerance to various environmental stresses. RWC increased in PBZ-treated triticale (Triticale hexaploide) plants during water stress, Under water stress, PBZ treatment assists plants in retaining water for 30-40 days (, observed that application of PBZ (90 mg/l) under drought in rice genotypes was responsible for about a 15% increase in RWC as compared to drought without PBZ treatment., found that in Curcuma alismatifolia leaves, PBZ (1500 mg/l) increased RWC by 5% under drought., reported that in okra (Abelmoschus esculentu) cultivar Nutec, application of PBZ (80 mg/l) along with drought increased RWC (60.1%) compared to drought without PBZ treatment (57.2%) although the result was not statistically significant. Similarly, in Safflower (Carthamus tinctorius L.) application of PBZ under drought enhances the RWC. Overall PBZ enhances the RWC of plants under drought conditions by a reduction in evapotranspiration.

Effect of PBZ on membrane stability index

Membrane stability is a common criterion for determining drought tolerance because water deficit induces water loss from plant tissues, which severely impairs membrane structure and function. The stability of the cell membrane was used as a drought tolerance indicator and leakage of electrolytes showed an increase in water deficit, reported that PBZ (90 mg/l) in rice genotypes led to an 11% increase in mean MSI as compared to drought-stressed plants without PBZ treatment. PBZ (20 mg/l) minimized the leakage of electrolytes in carrots Reported that the application of PBZ (30 mg/l) in wheat under control and water-stressed plants resulted in an increase of 1-2% and 4-5% respectively in the mean MSI. Similarly, reported that PBZ (1500 mg/l) decreased electrolyte leakage by 60% under water deficit stress in Curcuma alismatifolia., observed that the application of PBZ (150 mg/l) in mungbean under drought decreased electrolyte leakage from 52.6% (drought without PBZ) to 47.1%. Similarly, in Safflower (Carthamus tinctorius L.) application of PBZ under drought enhances the cell membrane stability. Collectively, these findings suggest that PBZ improves MSI by minimizing electrolyte and ion leakage under stress conditions.

Effect of PBZ on plant growth

The most striking growth response observed in PBZ-treated plants is a reduction in shoot growth. This response is mainly attributed to internode length reduction., reported that canola plant height was reduced by 27% when PBZ was applied at 10 cm stalk height as compared to without PBZ., reported that red firespike plants treated with PBZ (.24 mg/pot) under drought were 11 cm taller than untreated plants. Under water deficit stress, found that applying PBZ (1500 mg/l) decreased the plant height of Curcuma alismatifolia by 50% relative to non-treated plants. In Amorpha fruticosa, found that PBZ treatment (150 mg/l) under extreme drought (RWC 35-40%) resulted in a 61% increase in height relative growth rate compared to drought without PBZ, observed that in Patumma after 40 days of withholding water, the plant height was 1.2 times lower in PBZ (1500 mg/l) treated plants compared to water-stressed without PBZ. When PBZ (3750 mg/L) was applied to Patumma, shoot height was reduced by 48.93% relative to untreated plants. In comparison to non-treated plants, soil drenching with PBZ (1500 mg/l) under water stress for 20- and 30-days periodsmaintained shoot length. However, in sunflower and zinnia shoot height was reduced by 26.3 and 42.1%, respectively, after soil drenching with PBZ (2.0 mg/pot), Syzygium myrtifolium (Roxb.) Walp Plant height was reduced by 19.93% when treated with PBZ (3750 mg/L). According to PBZ (500ppm) increased panicle number, resulting in higher grain yield while reducing water demand, hence increasing rice water use efficiency under drought conditions.

Reported that PBZ (50 mg/l) increased wheat seedling length, fresh and dry weight of shoots, under low-temperature stress as compared to control (low-temperature stress without PBZ) PBZ has been shown to increase both the fresh and dry weight of shoots and roots in cucumber seedlings that have been exposed to high temperatures., reported that seed soaking of maize with PBZ (300 mg/l) under a semi-arid region increased root dry weight by 102.1% at the seventh leaf stage, 65.1% at the ninth leaf stage, 47.9% at the twelfth leaf stage, compared to drought without PBZ treatment., reported that in peanut plants at 80 days after sowing (DAS) application of PBZ (10 mg/l) under drought increased root length from 18.17 to 28.15 cm/plant, total leaf area from 96.38 to 117.31 cm2/plant, whole plant fresh weight from 33.72 to 39.16 g/ plant, whole plant dry weight from 3.49 to 4.12 g/plant as compared to drought-stressed plants without PBZ treatment. A similar pattern of results was also obtained by in Sesamum indicum by application of PBZ (5 mg/l) during drought. observed that in sweet potatoes, PBZ (34 μM) under drought increased vine fresh weight, root fresh weight, vine dry weight, and root dry weight by 40.10, 65.47, 66.91, and 67.86% respectively, compared to water-stressed plants.

After PBZ (500 mg/l) application, the root dry weight of Aesculus hippocastanum was improved (18.4% reduction) after water deficit stress. Under drought conditions, the dry weight of PBZ (60 mg/l) treated tomato shoots (37.17% reduction) and root dry weight (13.04% reduction) were higher as compared to the control. Similarly, the dry weight of PBZ (50 mg/l) treated plants decreased by 20.45%, compared to 36.77% for non-treated plants. In turf grass, shoot dry weight was extremely responsive to water deficit conditions (25% FC), resulting in 95 to 97% reduction, respectively, while treatment with PBZ (30 mg/l) reduced the shoot dry weight by 3.14% only. The leaf area of P. angustifolia plants treated with PBZ (30 mg/l) and grown under wellwatered conditions was reduced by 83.25%. However, when exposed to mild water deficit conditions, the growth of PBZ-treated plants improved but declined when exposed to severe water deficit stress. When exposed to drought, shoot height, leaf area, and root length of PBZ (10 mg/l) pre-treated peanut plants improved compared to the control, reported that the diameter of Vetiveria Zizanioides increased in stressed plants due to 12% PBZ application. According to, PBZ (1.6 mg/l) reduced leaf area (LA) in tomato plants by 24% under water deficit conditions. Overall, PBZ enhanced plant development under stressful circumstances by increasing shoot and root biomass. Although some research implies that PBZ reduces plant height, others report that PBZ increases plant height, hence a greater knowledge of the influence of PBZ application on plant development is required before future application.

Effect of PBZ on photosynthetic pigments

Water stress alters the total chlorophyll content and stability within thylakoid membrane protein-pigment complexes which are the first structures to be weakened under stress conditions. Chlorophyll reduction under water deficit stress is mainly due to chloroplast damage caused by ROS. PBZ (3 g a.i./tree) increased Chlorophyll a (27.35%), Chlorophyll b (54.54%), total chlorophyll (30.98%) and carotenoids (13.55%) compared to control without PBZ in cashew. According to applying PBZ (30 mg/l) to wheat plants under water deficit stress resulted in a 25.7% increase in chlorophyll content as compared to stressed plants without PBZ., reported that in maize PBZ (300 mg/l) increased the chlorophyll content by 48.2%, 54.3%, 51.2%, and 79.0%, at 0, 15, 30, and 45 DAS respectively Similarly carotenoid contents increased by 15.7%, 17.3%, 27.9% and 36.7% at 0, 15, 30 and 45 DAS in water deficit stress as compared to control (drought without PBZ application) observed that PBZ treatment was 15–18% more effective than the control at preventing chlorophyll loss in wheat during low-temperature stress. PBZ (10 mg/l) increased total chlorophyll, carotenoid, xanthophyll, and anthocyanin content in 80 days old Arachis hypogaea by 120.22%, 112.66%, 116.48%, 111.26%, 114.44%, and 112.24% respectively over control under drought reported that PBZ (2 mg/l) increased chlorophyll content by 62% as compared to control in maize., observed that treatment with 25 or 50 mg/l PBZ in black rice plants had greener leaves and encountered late senescence than control plants. Similarly, in Safflower (Carthamus tinctorius L.) application of PBZ under drought enhances the photosynthetic pigments., reported that net photosynthesis was 51% higher in red firespike plants treated with PBZ (0.24 mg/pot) under drought than in those without PBZ. In Zoysia japonica, PBZ (50 mg/l) during water deficit stress increased leaf chlorophyll content by 0.6 mg/g FW compared to water-stressed without PBZ. Similarly, , recorded that PBZ in both irrigated and deficitirrigated plants increased Chlorophyll content as compared to control plants (without PBZ). PBZ increased the photosynthetic pigment content in Festuca arundinacea and Lolium perenne under water stress. Under water deficit stress, PBZ significantly increased chlorophyll a, chlorophyll b, and carotenoids in wheat cultivars reported that PBZ (150 mg/l) treatment in mungbean under drought increased SPAD value from 34 (drought without PBZ) to 37.7. All prior investigations have concluded that PBZ improves photosynthesis by increasing chlorophyll and other photosynthetic pigments under stressful circumstances.

Effect of PBZ on grain yield and dry matter partitioning

Drought primarily affects production by reducing the number of seeds by either influencing the quantity of dry matter produced at the time of flowering or by directly affecting pollen or ovules, leading to a decrease in seed collection. PBZ has been shown to modify sink efficiency, prompting assimilates to be redistributed to meristematic regions other than shoot apices and improving assimilate flow to reproductive structures in plants. Under drought, the use of PBZ (50 mg/l) increased the average weight of 1,000 seeds and yield in maize (Zea mays L.). According, average maize grain yields increased by 61.3% after seed soaking with 300 mg/l PBZ, while seed dressing with PBZ at 2.5 g/kg increased yield by 33.3% compared to control without PBZ in semi-arid regions.

Under water stress, wheat genotypes treated with PBZ increased grain yield per plant by 6-7%, grain numbers per panicle by 24-33%, 1,000-grain mass by 3-6%, and harvest index by 2-4%. According to , under water stress, yield per plant was reduced. Stress effects, on the other hand, were found to be reduced when PBZ was applied (40 mg/l). reported that the application of PBZ (150 mg/l) in mungbean under drought increased seed yield from 622 (drought without PBZ) to 1921 kg/ha. Drought impaired flowering in red firespike plants, but PBZ treatment (0.24 mg/plant) promoted flowering and maintained the same number of flowers (6 flowers/plant) as the control. Tomato plants treated with PBZ (50 mg/l) produced 1.37 times more fruit than non-treated plants. The yield of pre-treated plants was reduced by 4.79% when they were subjected to drought at 60% field capacity. observed that PBZ (30 mg/l) pre-treated tomato plants retained their fruit yield (3.89 kg/plant) and fruits per plant (31 fruits/plant) when exposed to water deficit stress. Overall, past research indicates that the use of PBZ boosted grain yield/ fruit set under drought by improving sink efficiency. PBZ hampered the gibberellin biosynthesis. GAs are growth regulators which fall under a large family of tetracyclic diterpenoids. GAs are plant hormones that are required for a variety of developmental activities in plants such as pollen maturation, stem elongation, leaf expansion, trichome creation, seed germination, and flowering induction. Furthermore, the exogenous application of gibberellins can reverse PBZ-induced growth inhibition. These findings support the theory that PBZ-induced growth inhibition is due to a reduction in gibberellin biosynthesis. studied the effect of PBZ (200 mg/l) in rice varieties under submergence stress and found that gibberellic acid content was decreased by the application of PBZ compared to submergence stress without PBZ. found that PBZ (150 mg/l) under severe drought (RWC 35-40%) decreased GA content more than drought without PBZ in Amorpha fruticosa. PBZ-induced abscisic acid biosynthesiAbscisic acid (ABA) is classified as a stress phytohormone because it accumulates quickly in response to stress and mediates many stress responses that help plants survive. The effect of PBZ on ABA is of significant importance because ABA is synthesized through the isoprenoid pathway. Reported that PBZ (150 mg/l) under severe drought (RWC 35-40%) increased ABA (27.1%) than without PBZ in Amorpha fruticosa Similarly, , recorded that treatment with PBZ in wheat cultivars did not significantly affect ABA content, however, mean ABA content was significantly enhanced by 25% under water deficit stress., showed that DI (Deficit irrigated) + PBZ treated plants significantly increased ABA accumulation compared to DI control plants. PBZ application increased ABA and decreased gibberellins during the reproductive stage in the shoot of mango plants. Compared to untreated seedlings, PBZ treatment has been shown to minimize endogenous ABA by about one-third caused by water stress in apples and wheat, found that PBZ-induced stress tolerance in snap beans was due to increased endogenous ABA content. PBZ substantially enhanced endogenous ABA levels in hydroponically grown seedlings and detached leaves of oilseed rape, according to. According to, PBZ enhanced the endogenous level of ABA in wheat under water deficit stress., observed that PBZ (200 mg/l) increased ABA content in rice varieties under submergence stress compared to submergence stress without PBZ application. The effect of PBZ on ABA may be the source of stress defense.

PBZ elevated antioxidant enzymes activity

PBZ enhances the detoxification of ROS, antioxidant, and chlorophyll (Chl) content. As photosystem II (PSII) operation is reduced, an imbalance between electron generation and usage occurs, causing quantum yield shifts. These changes in chloroplastic photochemistry cause excess light energy to be dissipated in the PSII core and antenna under drought, resulting in the development of potentially harmful active oxygen species (O2-1, 1O2, H2O2, OH).

ROS detoxification pathways can be found in all plant species and are classified as enzymatic which include ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and non-enzymatic which include reduced glutathione (GSH), ascorbic acid and tocopherol.

showed that PBZ (5 mg/l) application to Sesamum indicum resulted in 464.74%, 267.49%, and 359.08% increase in SOD, APX, and POX activity respectively in leaf tissue under drought conditions as compared to without PBZ. Different PBZ treatments increased SOD activity in maize grown in the semi-arid environment to varying degrees. From 0 to 15 days after silking (DAS), SOD activity increased, then decreased until it reached 45 DAS. The APX activity of PBZ-treated ryegrasses was found to be 25% higher than that of untreated under drought. No considerable difference in CAT activity was observed in PBZ-treated plants under drought. PBZ increased POX activity considerably under drought.

PBZ enhanced proline content

Proline is a key amino acid in protein and membrane structures, as well as a ROS scavenger under drought. PBZ treatment enhanced proline content and improved drought tolerance. However, further research is needed to determine the actual molecular mechanism underlying the effect of PBZ on mobile proline concentration in plants. PBZ treatment (75 mg/L) significantly reduced proline content (0.030 μmol/g FW) in pomegranate leaves by 59.22% to control (0.067 μmol/g FW) found that free proline concentration increased by 54.56 mg g-1 in PBZ (50 mg/l) treated tomato plants grown at 60% field capacity, which was 1.52-fold greater than the control. However, in water-stressed conditions, the free proline level in PBZ (10 mg/l) in pre-treated peanuts was lower (1.04-fold over control) than in untreated plants (1.49-fold over control), showed that the wheat plants treated with PBZ under water stress had a 40% decrease in proline content as compared to the stressed plants without PBZ. These findings suggested that the wheat genotypes experienced less stress (as indicated by the proline content) and improved drought tolerance as a result of PBZ application. Another study showed a considerable increase in free proline content after Mannitol+PBZ treatment in wheat cultivar Sakha 8 (3.342 mg g-1 f.w) as compared to control (without PBZ+Mannitol) and the same pattern was observed in all the wheat cultivars. Endogenous proline level increased by 17% in mango leaves treated with PBZ (1500 mg/L) under salt stress when compared to salinized plants without PBZ treatment, showed a significant increase in proline content in drought-sensitive and drought tolerant rice genotypes after priming with PBZ under drought as compared to their unprimed samples. reported that the application of PBZ (150 mg/l) in mungbean under drought increased proline content from 7.28 (drought without PBZ) to 7.87 μmol/g f.wt. Similarly, in Safflower (Carthamus tinctorius L.) application of PBZ under drought enhances the proline content.

PBZ reduced malondialdehyde content

Usually, membrane lipid peroxidation in plants is detected by measuring malondialdehyde (MDA). MDA is a widely used marker of oxidative lipid injury caused by environmental stress., showed that the MDA content was significantly lower in the PBZ-treated maize plants over the control under drought. PBZ treatment under drought considerably reduced the MDA content in maize leaf by 31.5% at 0 DAS, 31.4% at 15 DAS, 32.2% at 30 DAS, and 20.2% at 45 DAS compared with drought without PBZ. Other studies carried out on PBZ-primed rice samples indicated that PBZ showed insignificant change in MDA content in the sensitive genotype under drought while a 55% decrease in MDA content was found in the tolerant genotype as compared to PBZ treated under control conditions. Similar findings were documented by, who observed that plants raised from PBZ-primed seeds had lower MDA levels under control and drought conditions than plants raised from unprimed seeds. The amount of MDA decreased as the amount of PBZ increased. PBZ (80 mg/l) decreased MDA content (51.15 mol/g f.wt.) under water deficit stress relative to drought alone (61.92 mol/g f.wt.) reported that PBZ (300 mg/l) in the semi-arid region reduced MDA content by 44.1%, 50.4%, 66.3%, 40.5%, at 0, 15, 30, and 45 DAS respectively compared with the water-stressed plants without PBZ treatment.

PBZ influence on protein content

The protein content in plants decreases with the onset of water deficiency. PBZ treatment increased the protein content of the leaves and tubers in carrots. From 0 to 15 DAS, the soluble protein content of maize increased slightly, then steadily decreased from 15 to 45 DAS. Plants treated with a high concentration of PBZ under drought retained higher protein content from 0 to 15 DAS, but protein content was significantly inhibited from 30 to 45 DAS. Wheat seeds primed with PBZ had increased protein content. Also, there are other similar reports which showed that PBZ priming increased the protein content under abiotic stress and non-stress conditions. According to when PBZ was applied under drought to the okra cultivar Nutec, total soluble proteins increased as the amount of PBZ was increased. Total soluble proteins were 11.04, 11.29, 10.75, and 11.76 mg/g f.wt. at four different PBZ treatments of 0, 20, 40, and 80 mg/l, respectively under water stress conditions.

PBZ influence on sugar content

During drought, the accumulation of compatible solutes such as carbohydrates is claimed to be an effective stress tolerance mechanism. Sugar resulting from transitory starch degradation was noticed in PBZ-pretreated plants, which retains the leaf water potential under water deficit stress conditions. PBZ treatment in mango increased total sugar, sugar: acid ratio, reducing sugar, and titratable acidity reduction. In drought-stressed ryegrass, PBZ application significantly increased soluble sugar content compared to untreated plants. The impact of PBZ was mainly pronounced on 30 and 45 days of drought treatment in Iranian perennial ryegrass. According to, PBZ (150 mg/l) under extreme drought (RWC 35-40%) had 119% higher soluble sugar content than drought without PBZ in Amorpha fruticosa. In untreated and PBZ-treated (50 mg/l) tomato plants total soluble sugars increased by 1.16 and 1.52 times under water deficit (60% FC), respectively. Sugar content increased by 2 mg/l after foliar application of PBZ under 6% PEG-induced water deficit stress in S. rebaudiana Bertoni as compared to stressed plants. Total soluble sugar enrichment in PBZ-treated sweet potatoes may be required for cellular osmotic adjustment under water deficit stress situations.

Molecular responses of plants to PBZ

PBZ inhibits GA biosynthesis by inactivating cytochrome P 450-dependent oxygenase, which inhibits the oxidation of ent-kaurene to ent-kauronoic acid. PBZ inhibits ABA degradation into phaseic acid, resulting in ABA accumulation. In drought-stressed tomato plants, PBZ increased the expression of ABA biosynthesis genes (SlZEP, SlNCED, and SlAAO1). To gain a better understanding of the dwarfism mechanism, , analyzed gene transcripts of Lily leaves after PBZ treatment. 2704 genes were found to be differentially expressed by comparing PBZ-treated samples to untreated samples. PBZ increased the expression of nine genes encoding GA biosynthesis enzymes (one KAO and eight GA20ox genes) while decreasing the expression of a gene involved in GA deactivation (GA2ox gene). reported that the expression of ent-kaurene oxidase (ZmKO1-2), ent-kaurene synthase (ZmKS1,2,4), and ent-copalyl diphosphate synthase (ZmCPS) decreased, whereas the expression of GA 3-oxidase (ZmGA3ox1), GA20-oxidase (ZmGA20ox1,5) and ent-kaurenoic acid oxidase (ZmKAO) increased in maize seedlings treated with PBZ. PBZ has been shown to increase SLGA20ox-3 and SLGA3ox2 expression in tomato plants through feedback regulation. Upregulation of SLGA20ox-3 and SLGA3ox2 transcript accumulation was observed in response to PBZ-induced ent-kaurene oxidase inhibition, which was thought to be a feedback upregulation of GA biosynthesis in response to lower GA content.

Another study examined the expression profiles of GA biosynthesis genes (ent-kaurene oxidase; KO, gibberellin 20-oxidase1; GA20ox1 and gibberellin 3-oxidase; GA3ox) and floral transcription factor genes (UFO, WUSCHEL; WUS, and LFY) in response to 1,250 mg/l of PBZ treatment of Jatropha floral buds. Then, samples were selected at the different time points of 14 days (no sex organs observed), and 20 days after treatment (blooming and sex organs observed). The results showed that PBZ significantly reduced the expression level of GA20ox1, GA3ox, and LFY as compared to the control (P<0.05) at 14 days. On the other hand, the expression level of UFO and WUS1 were significantly higher than the control. At 20 days, there was no difference in the expression level of GA biosynthesis genes between the control and treatment. At the same time blooming time of PBZ-treated flowers was delayed which might be due to low expression levels of GA20ox1, GA3ox, and LFY in treated floral buds.

PBZ (200 mg/l) inhibited the GAs content in rice varieties under submergence stress compared to submergence stress without PBZ. QRT-PCR was used to analyze the expression of GAs biosynthetic genes such as OsCPS1, OsKS1, and OsGA2ox1. OsCPS1 mRNA was repressed in PBZ treatment, which was consistent with the GA content in leaves. PBZ application increased ABA content regardless of rice genotypes due to the upregulation of 9-cis-epoxycarotenoid dioxygenase (NCED), the main enzyme in ABA biosynthesis, encoded by OsNCED. In contrast to plants not treated with PBZ, Rubisco-small subunit expression was higher at the anthesis and post-anthesis stages in all wheat cultivars with PBZ. At the anthesis and post-anthesis stages of wheat growth, the PBZ-treated water-stressed plants showed downregulation of the stress marker pyrroline-5-carboxylate synthase (P5CS) expression in all genotypes studied. At various growth stages after the formation of the basal second internode of wheat, the complex changes in the activities of enzymes involved in lignin biosynthesis, such as phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), were assessed in response to PBZ (200 mg/l) application. The activity of PAL and 4CL were higher by 42% and 35.6% respectively as compared to the control.

PBZ (PBZ) at 0.8 and 1.6 mg/l significantly increased aquaporin (gene and protein) expression in tomato plants compared to controls, implying a coordinated increase in ABA and aquaporin levels in response to water stress. Treatment with PBZ during deficit irrigation increased SlTIP2 expression by 5.3-fold above the control and resulted in greater PIP2-7 protein levels (compared to PBZ-irrigated). The increased expression

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