INFLUENCE OF PARTIAL ROOT DRYING ON MORPHO-ANATOMICAL AND PHYSIOLOGICAL CHARACTERISTICS OF TOMATO FRUITS (

Partial root drying (PRD) is a new irrigation and plant growing technique which improves water use efficiency without significant yield reduction. The aim of the presented thesis was to investigate the effect of this technique on the growth of vegetative and generative tomato organs, and especially fruits, its influence on the activity of enzyme cell wall peroxidase, morpho-anatomical characteristics of flower and fruit pedicels, influence on fruits yield and quality and ions distribution in tomato organs. Tomato line L-4 (Lycopersicon esculentum L.) was grown in commercial compost with the root system divided equally between two plastic pots. During the vegetative and generative phases of development, one half of the root system of PRD was maintained in a dry state, while the other half was optimally watered. After 10 days an inversion was made in such a way that previously wet side was exposed to drought and dried side was watered. Obtained results showed that as a consequence of PRD treatment the growth of whole plants was reduced, with fruit size and yield of PRD plants being similar to control, as well as morphoanatomical characteristics of pedicels, ions and lycopen contents, although sugar content was higher in PRD than in control plants. These results suggest that with PRD technique we can halve the demand for irrigation water and increase the nutritional value of the tomato fruit.


I n t r o d u c t i o n
Nowadays, in agriculture, great emphasis is placed on the area of crop physiology and crop management for dry conditions with the aim to make plants more efficient in water use.Recent results demonstrated that regulated deficit irrigation (RDI) and partial root drying (PRD) are the irrigation methods that tend to decrease agricultural use of water.
The PRD technique was developed on the basis of knowledge of root-toshoot chemical signalling in drying soil and, therefore, understanding of this process is essential for successful application of the PRD technique (G o w i n g et al ., 1990).The conventional view of drought is that soil drying induces restriction of water supply and this results in a reduction of tissue water content, growth and stomatal conductance.Recent results, however, demonstrated that drought might induce synthesis of some metabolites in roots and their transfer from root to shoots via xylem.These so-called root chemical signals may have an influence on different physiological processes (D o d d et al., 1997).In the past decade a large number of field studies have validated the existence of chemical signalling in the field.L o v e y s (1991) was the first to apply the split-root technique for inducing chemical signals in the root system of grapevine grown in field conditions, and these results showed that PRD reduced vine vigour and increased the quality and yield of fruit.Excessive plant vigour is a major problem for many horticultural crops, since the use of assimilates in leaf growth might restricts fruit development.The PRD field results, mainly from the vineyards of Southern Australia, demonstrated that in addition to the benefit in terms of reduced canopy density and consequent improvement of fruit quality, the benefit comes also in the form of improved water-use efficiency (D r y et al. 2000).
The aim of the presented master´s thesis was to investigate the effect of PRD treatment on tomato plant growth, yield and its quality.Investigations also included the acivity of the enzyme cell wall peroxidase since recent results demonstrated significant role of this enzyme in the growth processes of fruit (T h o m p s o n et al., 1998 ).In addition to investigate hydraulic implications for transport to fruit, our aim was also to test a hypothesis that PRD induces hydraulic and chemical isolation of fruits from the rest of plant (D a v i e s et al., 2000).Therefore, investigations of pedicel vasculature area (xylem and phloem) and ions accumulation and distribution were done.

Material and Methods
Tomato seeds, Lycopersicon esculentum L., line L-4 (provided by the Center for vegetable ″Palanka″ , Serbia) were germinated in commercial compost in a growth cabinet until the emergence of the fifth leaf.In a growth cabinet photoperiod was 12 h ; light intensity at the level of the plants 300 μmolm -2 s -1 , temperature 28/18 o C and relative humidity 70%.In the phase of the fifth leaf the plants were removed from their pots and the root system of each plant was divided in to two parts and placed into two separate plastic bags (volume 3.0 dm -3 each).Thereby, the root system of each plant was split into two hydraulically separate compartments.For PRD treatment we repeated the D a v i e s et al. (2000) experiment in such a way that during the vegetative and generative stages of tomato development, one half of the root system of PRD was maintained in a dry state, while the remainder of the root system was watered.One PRD treatment took c.10 days and during this period the soil water content in the dry root side was reduced to 30%.After this, the treatment was reversed, allowing the previously dry compartment to be well-watered and the well-watered compartment to dry down.In control plants both compartments were watered daily throughout the experimental period.
During the experimental period measurements of several parameters were done including: plant height, number of flowers and fruits, fruit diameter, biomass, fruit and petiole anatomy, fruit yield and quality (sugars, lycopene and minerals) and ion content in roots, stems, leaves and fruits.

Morpho-anatomical measurements
Measurements were done in three stages of fruit development, namely -in stage I (early stage of green fruit) and 18 days after PRD treatment, then in stage II (stage of green fruit) and 39 days after PRD treatment, and finally in stage III (stage of ripe fruit) and 57 days after PRD treatment.Fruit pedicels in control and PRD plants were cut in three different positions (proximally, abscission zone, and distally).Hystological procedure was done by the method of B l a ž e n č i ć (1988).Investigated tissue samples were infiltrated in paraffin, dehydrated, sectioned on a microtome and finally stained with safranine-aniline blue.The microscopic examination (by OLYMPUS A041) included several parameters: 1.The total area of a pedicel; 2. The area of the individual elements of the vascular elements (xylem and phloem); 3. The number and density of xylem elements, and 4. The area of other tissues of the fruit pedicel.

Biochemical analysis
Within the biochemical analysis, dry weight, total minerals, total sugars and lycopene in ripe fruit were measured (S p a n y a r , 1954; D ž a m i ć , 1989).Ions content (P, K, Ca, Mg, Zn, Cu) measurements were done by the method of atomic absorption spectrophotometry, although for N content Kjeldahl method was used (D ž a m i ć et al., 1999 ).Cell wall peroxidase activity was measured in the samples of tomato fruit epidermis.Enzyme activity was measured by guaicol test with spectrophotometric method (T h o m p s o n et al., 1998).

Plant growth
The obtained results show that PRD treatment reduced the growth of vegetative organs (Tab.1).At the end of the investigated period, the average height of PRD plants was 66.01, and of the control ones 88.92 cm.Similar results were obtained for biomass (results not shown).The effect of PRD treatment on generative organs was different.The number of flower trusses was, in statistical terms, significantly reduced (P<0.01),being in PRD plants about 30% less compared to the control plants.However, this effect was not manifested in number and growth of fruits, so that at the end of the investigated period the fruits number and diameter were about the same in the control and PRD plants, ranging from 35.15 mm to 37.19 mm, respectively.A smaller number of flower trusses in PRD plants, compared to their number in the control plants, did not cause formation of less number of fruits, which means that PRD treatment also influenced the flowers abortivity.D a v i e s et al. (2000) also obtained a significant PRD effect on shoot and root growth as well as flower formation.Therefore, our results confirmed that applied partial root drying of the root system was sufficient to trigger a shoot response.
T a b.On the basis of the biochemical analysis (Tab.2), it was found that PRD and control plants fruit did not differ significantly in statistical terms regarding dry weight (10.05% and 10.73%), lycopene (3.18 mg% and 3.43 mg%), and total minerals (1.09% and 1.03%).Statistically significant differences (P<0.05) were found in total sugars content, namely -fruits of PRD plants had more total sugars (4.20%) compared to the fruits of control plants (3.70%).These results show that PRD growing system, in spite of its strong effect on reduction in growth of vegetative organs, does not reduce fruit yield (based on dry weight and fruit diameter), but -by the rising sugars content, it improves the fruits quality.PRD treatment also significantly increased the activity of cell wall peroxidase at the stage of complete maturity.The results in other phases (early green and green) have not being shown but these results confirmed for both treatment (PRD and control), similarly to T h o m p s o n et al. (1998), an increase in the activity of investigated enzyme during fruit development.
However, the physiological basis of PRD effect on fruits has not been explained yet.D r y et al. (1996) and D a v i e s et al. (2000) pointed out that PRD treatment influenced transport of assimilates in a such a way that reduction of shoot growth redirected carbohydrates towards the fruit.According to D a v i e s et al. too (2000), another of the explanations may be a partial hydraulic and chemical isolation of fruits from the rest of the plant.

Morpho-anatomical measurements
Graph 1 shows the results of measuring of anatomical characteristics in the zone of flower pedicels abscission (xylem area, area of inner and outer phloem) in PRD and the control plants.The results show that in the examined plants -the measured phloem area, especially the inner one, was significantly larger (0.0899 mm 2 for the control, and 0.1020 mm 2 for PRD), compared to the xylem area (0.0179 mm 2 for the control, and 0.0180 mm 2 for PRD), but without statistically significant differences between the applied procedures of plants growing.The relation of areas of the total phloem and xylem amounted to 8.48 for the control, and 8.66 for PRD.These results confirmed that in both treatments in the zone of pedicels abscission was a decrease in xylem area compared to phloem.
Table 3 presents the results of measuring of xylem area of tomato fruit pedicel in control and PRD plants in three different positions and in three stages of fruit development.Calculation was done in relative units in order to eliminate differences in pedicel areas in PRD and control plants, which might later affect the calculation of final results.These results show that in fruit pedicels, similar to the flower pedicels, the xylem area in PRD plants did not differ significantly from that in control plants.Differences in xylem areas appeared between the observed stages of fruit development, and the increase in xylem area was particularly pronounced between stage I and stage II, when the xylem area in all investigated zones was almost doubled.Differences in areas were also expressed between the investigated zones, and in both PRD and control plants -the measured relative area of xylem was less in the abscission zone related to the proximal and distal positions.Obtained results indicate that there were no significant differences in the area of inner and outer phloem between PRD and control plants.Differences that appeared between the stages of development were in consequence of growth, where, during the fruits development, the areas of inner and outer phloem were enlarged.In both treatments, the measured area of the inner phloem was significantly larger compared to the outer one, and also there was a noticable enlargement of the inner phloem area in the zone of abscission related to distal and proximal position.The ratio of the total phloem area/pedicel xylem area in the investigated stages of fruit development was increased in the zone of abscission, relative to the proximal and distal position.The results of our researches confirmed that in both treatments in the zone of pedicels abscission there was a decrease in xylem area compared to phloem.Xylem relative area in the zone of abscission (ca 20%) was, however, much smaller compared to the results presented by L e e (1988).He found that xylem in the zone of abscission was reduced to 3.2 -5.7% of the total pedicel area.H o et al. (1987), researching into the anatomy of tomato fruit pedicel, found that, in later stages of fruit growth, in the zone of abscission of fruit pedicel, due to reduction in xylem area related to phloem, the mechanism of water and assimilates transport was changed, so that even 90% of water was transported through phloem.The hydraulic isolation of tomato fruit from the rest of plant was also observed by M a l o n e and A n d r e w s (2001).V a n I e p e r e n et al. ( 2003) measured the distribution of hydraulic resistance along the pedicel and found that it was almost in full caused by high resistance located in relatively thin abscission zone in the node of pedicel.However, our results did not confirmed significant effect of PRD on the vasculature of the abscission zone in the pedicel of flower and fruit.Therefore, they did not confirm the hypothesis that higher hydraulic isolation of PRD fruit compared to well-watered fruit might explain PRD effects on fruit and shoot growth and sugar accumulation in fruits.
The above is also supported by the results of Ca 2+ distribution, where no differences were noted between PRD and the control.E h r e t and H o (1986) were the first to point out that the isolation of tomato fruit from the xylem flow (might lead to the certain extent to Ca 2+ deficiency).They showed that in the early stages of fruit development Ca 2+ was transported into fruit by xylem, and that the total Ca 2+ content was imported into the fruit very early during fruit development.If this early Ca 2+ deposit is insufficient, it will result in Ca 2+ deficiency in later development.The similar was found by H o et al. (1987).This reduction in xylem cross-section area reduced then xylem transport capacity, and accordingly -it reduced delivery of apoplast mobile ions, such as Ca 2+ .
Ion Ca 2+ is the element transported by xylem, and therefore low concentration of Ca 2+ in fruits also indirectly confirms that during ripening tomato fruits become hydraulically isolated from stem.Results for other measured ions (N, P, K, Ca, Mg, Zn, Cu) are not presented but they also confirmed that PRD treatment did not significantly change ion transport and distribution.

C o n c l u s i o n
Partial root drying (PRD) caused a significant reduction in shoot but not fruit growth, although total fruit sugars content was increased compared to control plants.These results confirmed that application of PRD techniques might improve crop water use efficiency and nutritional values of tomato fruit.Obtained results also confirmed that activity of enzyme cell wall peroxidase is correlated with growth cessation during fruit ripening.Differences in enzyme activity between PRD and control mature fruit may be due to the appearance of new isoforms.There was no significant PRD effect on the vasculature of the abscission zone in the pedicel of flowers and fruits.The hydraulic architecture of the pedicel's abscission zone showed that in both treatments the resistance to water movement through the xylem was higher in flowers than in developed fruits.Ca 2+ and other ions data (N, P, K, Mg, Zn, Cu) indirectly and similarly in both treatments pointed out to an increased resistance to xylem water flow in fruits compared to other organs.However, our results did not confirm the hypothesis that higher hydraulic isolation of PRD fruit compared to well-watered fruit might explain PRD effects on fruit and shoot growth and sugar accumulation in fruits.
Obtained results pointed out that PRD effects on tomato fruit yield and quality are based on the changed assimilates partitioning between source organs (mature leaves) and sink organs (flowers and fruits).Increased relative sink strength of fruits, comparing to flowers (reduced truss numbers), redirected carbohydrates (produced by source tissues) to fruits, and as a consequence sugar content in PRD plants was increased.Further investigations of this hypothesis for understanding of fruit growth and yield in both well-watered and PRD conditions will be worthwhile.

Graph. 1 .
-Influence of PRD on morpho-anatomical parameters of pedicel of tomato flowers in the abscission zone (KCA -control and TCA-PRD treated plants) T a b. 3. -The influence of partial root drying on absolute and relative xylem cross-sectional area through different zones (proximal, abscission and distal) of tomato fruit pedicel

Graph. 2 .
-The influence of partial root drying on Ca content (%) in different organs (root, stem with leaves and fruits) in phase I (A) and phase III (B).
1. -Investigated traits of tomato plants grown under PRD regime