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Title: N-deficiency damps out circadian rhythmic changes of stem 1

diameter dynamics in tomato plant. 2

Syunsuke Kanai1 a, Joseph Adu-Gymfi2, Kei Lei1, Junki Ito3, Katsumi Ohkura4, 3

Reda E.A. Moghaieb5, Hany El-Shemy5, Rashmi Mohapatra6, Pravat K. 4

Mohapatra6, Hirofumi Saneoka1, Kounosuke Fujita1 5

Running title: N deficiency on stem diameter in tomato 6

1Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, 7

Higashi-hiroshima, 739-8528, Japan. 8

2International Atomic Energy Agency (IAEA), Wagramer Strasse 5, A-1400 Vienna Austria. 9

3Hiroshima Prefectural Agricultural Center, Hara, Hachihonmatsu, Higashi-hiroshima, 739-0151, 10

Japan. 11

4Faculty of Environmental and Information Sciences, Yokkaichi University, 1200 Kayoucho, 12

Yokkaichi, 512-8512, Japan. 13

5Faculty of Agriculture, Cairo University, Cairo University Street, Giza, 12613, Egypt. 14

6School of Life Science, Sambalpur University, Jyoti vihar, Sambalpur 768019, India. 15

aAuthor for correspondence. E-mail: skanai@hiroshima-u.ac.jp 16

Tell: +81-(0)82-424-7963, Fax: +81-(0)82-424-7963. 17

Number of pages of manuscripts: 40, Number of Table: 1, Number of Figure: 11. 18

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Abstract 1

Tomato (Lycopersicon esculentum) plants were grown in hydroponics. At the fruiting stage, N 2

was withdrawn from the growing medium for a period of 19 days and its effects were studied on 3

plant biomass production, photosynthesis, partitioning of 13C and 15N, and changes in the stem 4

and fruit diameters etc, in order to monitor the mechanism of resource management on the plant 5

parts at low N and prevent excess use of the fertilizer. N-deficiency treatment decreased leaf 6

photosynthesis immediately and affected biomass accumulation of tomato. Conversely, 7

N-deficiency increased stem diameter for a period of two weeks before reducing it below the 8

control. During this period, these results suggest that N deficiency more suppress source activity 9

than sink activity. N-deficiency reduced the amplitude of the circadian pattern of daytime 10

shrinkage and nocturnal expansion of the stem diameter by decreasing the magnitude of the 11

former. Circadian pattern of contraction and expansion of diameter was less evident in the fruit. 12

Under N-deficiency, distribution of 13C and 15N decreased and increased to fruits respectively. 13

Restricted partitioning of carbon to fruits could be responsible for accumulation of unused 14

assimilates and consequential osmotic adjustment for maintenance of stem water potential. This 15

effect might have precluded contraction of stem diameter of N-deficient plants until the 16

production of assimilates became limiting on account of depression of leaf photosynthesis. 17

18

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Key words: Nitrogen, source-sink relationship, stem and fruit diameter, tomato, 1

micro-morphometry 2

3

Introduction 4

Glasshouse tomatoes grown hydroponically, is provided with high levels of inorganic nutrients. 5

Nitrogen fertilizers are most essential for growth and development of tomato plant [1,2] and 6

always given to the plant in excess of requirement. While this practice ensures unhindered 7

growth of the plant, it can exacerbate the release of extra nutrition to the environment. The 8

ecological price for nitrogen waste of aqua culture tomato is very high to pay since large amount 9

of fertilizers are leached from this agrosystem to ground water [3]. Siddiqi et al. [2] reported that 10

reduction of nitrogen to 50% of the normal levels had no adverse effect on fruit yield and quality. 11

The storage capacity of the plant for the element is in excess of that required for growth. 12

Moreover, N in one organ can be reused for the growth another [4]. Therefore, it is important to 13

grow aqua culture tomato at reduced N-concentrations and restrict eutrophying nitrogen waste. 14

But, it is not easy to define the critical N-concentration, which allows maximum growth rate [5] 15

in order to avoid deficiency symptoms. Under the circumstances, a quick and reliable technique 16

is necessary to identify the N-deficiency symptoms and replenish to stock solution in exigency. 17

Nitrogen is always a part of the carbon compounds in plant cells [6]. Assimilatory processes of 18

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carbon and nitrogen are closely interlinked and their dynamics are of key importance for crop 1

production [7]; deficiency of either of the nutrients can result in marked changes in the 2

assimilation and metabolic activities of the other [8]. Many studies have shown strong positive 3

correlations between nitrogen content and photosynthetic capacities of leaves [9,10]. 4

N-deficiency decreases the amount of thylakoids, carboxylation activity [11], photochemical 5

efficiency [12,13], chlorophyll contents [14] and stomatal conductance of leaves [15] and these 6

effects are ultimately reflected on biomass production. Glasshouse tomato grown in hydroponics 7

is no exception to the adverse effects of N-deficiency on the source activities of the plant as 8

illustrated above [16, 17]. Effect of N-deficiency on sink and source has already well studied, 9

respectively. However, it has not been elucidated that effect of N-deficiency on source organ, 10

sink organ and translocation of photosynthate at the same time in terms of source-sink 11

relationship. Recently, it has been reported that separation of effects of source and sink on 12

biomass production is possible by simultaneous monitoring of the amplitude of rhythmic changes 13

of fruit and stem diameter dynamics and photosynthetic activities of the source leaf [18-20]. 14

There are many reports in the literature that major sink organs like stem and fruits of plants 15

fluctuated diurnally to coincide with the changes in water status of the plant [21-24]. It is possible 16

that sub-optimal nitrogen supply decreases hydraulic conductance of root, therefore growth of 17

sink organs is inhibited by deficient of turgor. [25-28]. The manner in which the nutrient stress is 18

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transduced into a hydraulic response is not known. But, monitoring changes in the amplitude of 1

the circadian rhythm in shrinkage and expansion of the diameter of the plant organ by 2

micromorhometric technique [29] can be a reliable method to measure the changes in water 3

status of the plant resulting from adverse effects of the stress. In the present study, the objective 4

was to identify the process of resource management on the part of the plant at low N by 5

monitoring changes in stem and fruit diameter dynamics as well as primary production. 6

7

Materials and methods 8

Plant material and culture 9

Tomato (Lycopersicon esculentum L.cv. Momotarou) plants were grown in pots (70L) inside the 10

glass house of Graduate School of Biosphere Science, Hiroshima University, Japan. Each pot was 11

filled with nutrient solutions, consisting of N (Ca(NO3)2.4H2O) 3.57 mM, P (NaH2PO4.2H2O) 12

0.32 mM, K (K2SO4/KCl 1:1) 1.02 mM, Ca (CaCl2.H2O) 0.75 mM, Mg (MgSO4.7H2O) 0.82 mM, 13

Fe (Fe+3-EDTA) 0.02 mM, Mn (MnSO4.4H2O) 3.64 μM, B (H3BO3) 0.05 mM, Zn (ZnSO4.7H2O) 14

0.15 μM, Cu (CuSO4.5H2O) 0.16 μM, Mo (NaMoO4.2H2O) 0.1 μM, Co (CoSO4.7H2O) 0.17 μM. 15

There were six pots, each having three plants. At the first fruiting stage (65 days old), N was 16

withdrawn from the nutrient medium in three pots and this treatment was continued for 20 days. 17

The plants of the other pots received full nutrition. The plants were grown under natural light 18

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(3200 E m-2 s-1). The maximum and minimum temperatures were 32 and 23oC respectively. The 1

relative humidity of the glass house varied between 45 to 65%. 2

3

Measurement of biomass production 4

Plants from both control and N-deficiency treatment were sampled at 1, 9 and 19 days after 5

treatment in three replicates. Each plant was separated into roots, leaves, fruits and stem. The 6

plant organs were dried in an open air draught oven at 70oC for 72 h before the estimation of dry 7

weight. The dry materials were ground to powder with a vibrating sample mill (Model T1-100, 8

Heiko Co Ltd., Fukushima, Japan) and aliquots were taken for analysis of nitrogen. 9

Leaf blade immediately below the second truss was harvested at 0, 3, 6, 12 and 20 day after 10

treatment at 4 replicates. The leaf blades were lyophilized, and then estimated for dry weight. 11

These blades were ground to powder for measurements of N and sugar concentration in leaf 12

blade to employ examination of relationship between photosynthetic rate and leaf nitrogen and 13

sugar content. 14

15

Measurement of photosynthesis, transpiration and stomatal conductance 16

Photosynthetic rate, transpiration and stomatal conductance of the 1st and 2nd leaves below the 17

fruiting truss were measured with a portable infra red gas analyser (Model L1 6400, Licor Co. 18

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Ltd., Lincoln, Nebraska, USA) as described in our earlier report [19]. The leaf chamber was open 1

type and measurements were taken once at 11.00 am on each day in both control and 2

N-deficiency treatment plants during the period of treatment. The photosynthetically active 3

radiation was above 1000 µmol m-2 s-1 and observations were recorded after the plant reached a 4

steady photosynthesis state. All measurements were recorded five times and differences between 5

the observations were negligible. 6

7

Measurement of stem and fruit diameter 8

Changes in stem and fruit diameter were continuously recorded in both control and N-deficient 9

plants during the period of treatment with a shrinkage type micro-displacement detector [19]. The 10

sensors were connected to a computerized data acquisition system (NEC, Sanei Kogyo Co. Ltd., 11

Tokyo). The sensors were fastened to the stem or a growing fruit and connected to the power 12

system and data logger. The control run was carried out by placing a glass rod of 12 mm diameter. 13

The fluctuation of the measuring system was within ±2µm. Measurement of stem and fruit 14

diameter were recorded three times and the pattern of response was similar in all stem and fruit. 15

16

Measurement of nitrogen 17

An aliquot of the plant organs and leaf blade were used for N content determination by the 18

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Kjerdahl method [30]. 1

2

Measurement of sugar concentration 3

Aliquots of the powdered leaf blade were boiled with 80% (v/v) aqueous ethanol three times for 4

the extraction of sugars. The extracts were pooled in a volumetric flask and the flask was filled 5

up to the mark with distilled water. The sugar content in the ethanol-soluble extract was 6

determined using the anthrone reagent, according to the method of Suwa et al. [31]. 7

8

13CO2 feeding 9

13CO2 feedings were given to the leaf immediately above the first fruiting node on days 7 and 17 10

after N-deficiency treatment in both control and treated conditions. The leaf was enclosed in a 11

transparent plastic bag and 200 mL of gas mixture comprising of 80% nitrogen, 19% oxygen and 12

350 µ L L-1 of 13CO2 (99 13C atom % excess) was introduced from a cylinder. The leaf was 13

allowed to assimilate 13CO2 for one hour. The PAR was above 1700 µ mol m-2 s-1 during the 14

assimilation process. The plants were harvested 48 h after feeding by separating into 13CO2 fed 15

leaf, other leaves, fruits, stem and other plant parts. The plant parts were ground to powder for 16

measurement of 13C abundance. 17

18

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15N-NO3 feeding 1

The roots were dipped into a solution of 15N-NO3 (10 atom %, 100 ppm N) in a 20 L container 2

for 24 h before the initiation of N-deficiency treatment. The plants were harvested 19 days after 3

imposition of N-deficiency treatment. The plants were separated into leaves, stem, fruits and 4

roots. The samples were dried in an oven for 72 h before estimation of dry weight. The materials 5

were ground to powder for determination of 15N abundance. 6

7

13C and 15N analyses 8

The 13C or 15N abundance in the powdered plant sample was determined with a mass 9

spectrometer (model Delta plus, Finnigan Co., San Jose, CA, USA). The 13C atom % excess in 10

the plant sample was calculated as the difference in 13C atom % between the sample and standard 11

pure chemical glycine. The amount of labeled C or N in the plant sample was calculated using the 12

equation mentioned below [19]. 13

14

The element analyzer facilitated in the mass spectrometer determined the amount of total C. 15

Similarly; the amount of labeled N in plant sample was calculated. The total N of the plant 16

material was determined by micro-kjeldahl method [30]. 17

13C or 15N amount in the plant part A

100

Amount of C or N in the sample = ×

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1

Statistical analysis 2

The experimental data were analysed for the effect of N-deficiency treatment according to the 3

expected mean squares given by [32]. For mean separation, treatment sum squares was 4

partitioned by method of orthogonal contrasts. The coefficients of variation for all response 5

variables were below 10%. 6

7

8

Results 9

Dry mass accumulation 10

Increase in dry mass accumulation was slower in the N-deficient plants compared to the control 11

during the 19 d period of treatment (Fig.1). N-deficiency affected fruit and leaf growth more than 12

the other organs during this period. The effect of N-deficiency on root growth was not significant. 13

14

Stem and fruit diameter 15

Diameter of the stem in the control exhibited daytime shrinkage and nighttime expansion and 16

increased temporally up to 19 days after treatment (Fig. 2). The circadian pattern of change in 17

diameter of stem was similar for the plants subjected to N-deficiency. During the first two weeks 18

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of the treatment period, the diameter of the N-deficient plant was larger than the control. But, the 1

stem diameter did not expand in size thereafter in N-deficient plants and became lower than the 2

control plants. The diameter started to decrease early in the morning with sunrise (above 140 E 3

m-2 s-1) and the shrinkage was high throughout the day (Fig.3). It recovered in the evening 4

(bellow 420 E m-2 s-1) and expanded at night (0 E m-2 s-1). During the initial period, N-deficiency 5

reduced daytime shrinkage and encouraged nocturnal expansion of stem diameter more than the 6

control. The amplitude of the rhythm damped out gradually two weeks after the initiation of the 7

treatment; the night-time expansion became very low in the treated plant in contrast to the control 8

by the end of the treatment period (Fig.3). 9

10

The diameter of the fruit increased with passage of time in both control and N-deficient plants 11

during the period of treatment (Fig.4). N-deficiency marginally decreased expansion during the 12

first two weeks, but this effect did not continue till the end of the treatment period. The amplitude 13

of the circadian rhythm in contraction and expansion of fruit diameter was not as distinct as that 14

of the stem (Figs.3, 5). In the early part of treatment period (Fig.5A), there was no shifting of the 15

rhythmic phase in the fruit diameter and expansion occurred all throughout the 24-hour daily 16

period in control plants. N-deficiency reduced daytime expansion of fruit diameter and effect 17

lasted till the night. The reduction of fruit diameter size was restored gradually 7 days after 18

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treatment (Fig.5B) due to faster expansion in N-deficient plants and the diameter became larger 1

than the control towards the end of the treatment period (Fig.5C). 2

3

Apparent photosynthetic rate, stomatal conductance and transpiration rate 4

N-deficiency depressed the apparent photosynthetic rate immediately after initiation of the 5

treatment and the impact continued to increase with passage of time (Fig. 6 A). However, the 6

adverse effect of N-deficiency could not be noticed on stomatal conductance and leaf 7

transpiration rate until 8 days after treatment (Fig.6B, C). The stomatal conductance and rate of 8

transpiration continued to decline thereafter till the end of the period. 9

10

N concentration 11

N-deficiency decreased significantly nitrogen concentration of all plant parts in spite of the 12

preferential partitioning (data not shown), which occurred in favour of the fruits and roots. 13

N concentration of leaf blade was lower in N-deficient plants than in the controls at 3 DAT 14

(control and N-deficiency; 31.4 and 25.5 mg kg-1 D.W., respectively) (Fig. 10A). This 15

phenomenon was increased with passage of time, but N concentration of the control was kept 16

almost constant during treatment periods. At 20 DAT, leaf N concentration was lowered from 17

31.8 mg kg-1 D.W. in control to 12.9 mg kg-1 D.W. in N-stressed plants. 18

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1

Leaf sugar concentration 2

Treatment of N stress increased leaf sugar concentration in comparison to the control after 3 DAT 3

(control: 98, -N: 134 mg kg-1 D.W.) (Fig. 10B). The phenomenon was continued throughout the 4

period of experiment. 5

6

13C partitioning 7

13C atom % accumulation was very high in the fed leaf and the next preferred target was the 8

fruit (Table 1). The other leaves, upper stem and roots gained very low percentage of the isotope 9

on day 9. On day 19, the concentration of the isotope did not change in the fed leaf, but receded 10

in the fruits and increased in the other leaves and upper parts of stem. N-deficiency did not 11

influence 13C atom % of the fruits; in case of roots, the treatment improved 13C atom % on day 19. 12

The export rate of 13C into other plant parts from the fed leaf in the control was lower at day 9 13

after treatment than at day 19 (Fig.7). N-deficiency decreased the export rate of 13C from the fed 14

leaf into other parts on day 9 after treatment and the effect increased with passage of time. In the 15

control, 13C mostly partitioned into the fruits and other plant parts received only a small part of 16

the isotope (Fig.8). N-deficiency had no influence on partitioning of 13C to fruits on day 9 after 17

treatment. But at day 19, it decreased partitioning to fruits and leaves and increased export in 18

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favour of roots and stem 1

2

15N partitioning 3

Compared to 13C, percentage of 15N partitioning into the fruits was low (Fig.9). In the control, 4

15N partitioning occurred mostly in favor of the leaves and fruits were the next favorite 5

destination. Unlike the situation in carbon partitioning, N-deficiency significantly increased 6

partitioning in favor of the fruits. This was done largely at the cost of the leaves. N-deficiency 7

also increased partitioning into the roots. 8

9

10

Discussion 11

We have previously evaluated quality of resource management of tomato [19, 20] and Japanese 12

persimmon [18] on the basis their capacity for partitioning of assimilates from source to sink in 13

response to P and K deficiency and salinity stress respectively. Nitrogen is another important 14

growth factor and its influence on source sink relationship of tomato was elucidated in the 15

present endeavor. The source activity of the leaves declined immediately after imposition of the 16

stress (Fig. 6), and diameter of stem in the sub-optimal N treatment expanded more than that of 17

the control (Fig. 2) due to accumulation of a lot of carbohydrate reserves (data not shown) 18

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probavly along the phloem transport pathway. However, there was no reflection on the expansion 1

of the sinks like fruit (Figs.4). The responses of fruit to N-deficiency were similar to that of 2

P-deficiency, but influences on source activity differed considerably between the stresses. Both 3

the stresses affected 13C export from the source leaf to other organs (Fig.7), but the preferential 4

partitioning carbon and nitrogen elements in favour of the fruit ensured partial homeostasis in 5

growth. However, adverse effects of N-deficiency were more steadfast on carbon translocation to 6

the sink organs of tomato compared to P-deficiency; the export of 13C from source leaf (Fig.7) 7

declined severely in N-deficient plant compared to P-deficient plant within the 19 days period of 8

treatment. Unlike phosphorus, nitrogen always moves with carbon compounds in the phloem 9

solutes and deficiency of one element is bound to have a consequential influence on the other. 10

Severe reduction of the nitrogen concentration of the N-deficient plant fruit, stem, root (data not 11

shown) and leaf blade (Fig. 10A) corroborates decrease of 13C export rate from leaves (Fig.7) by 12

diminished utilizable N in leaves (Fig 10A). 13

14

Further, De Groot et al. [33] distinguished the contrasting effects of N and P deprivation on leaf 15

photosynthesis in tomato plants; N-stress affects photosynthesis by reduced utilization of 16

assimilates and absorption of light while P-stress produces a decreased rate of CO2 fixation. 17

Therefore, influence of N-deprivation on leaf photosynthesis could be more instantaneous 18

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compared to that of P-deprivation and our results support this proposition. 1

2

Proseus et al. [34] proposed that rapid changes in turgor pressure of plant organs could provide 3

new information on the mechanism of growth. Johnson et al. [23] observed a strong correlation 4

between circadian rhythmic fluctuation in diameter and the water potential of fruit and stem of 5

tomato plants. Low water potential can decrease phloem turgor reducing the driving force for 6

entry of sap to the fruit. In our study, withdrawal of N from the growth medium, encouraged stem 7

diameter expansion more than that of the control during the first two weeks of treatment period 8

before reducing it subsequently. In plants nitrogen is a part of carbon compounds [6]. Carbon 9

assimilates provide the skeleton for synthesis of various amino acids. Excess soluble 10

carbohydrates that cannot be used in amino acid synthesis accumulate in plant parts [6]. It is 11

plausible that abrupt limitation of exogenous N could increase organic acids used in NO3 12

reduction and sucrose concentration, all of which may be involved in turgor maintenance of the 13

plant cells [8,35,36]. Accumulation of sucrose, an osmotic solute, can increase the gradient for 14

water flux and maintain cell turgor [37,38]. In our study, N-deficient tomato might have 15

accumulated such carbon osmolytes for retention of high phloem turgor, and the stem diameter 16

increased temporarily as long as the reduced hydraulic conductance of roots [25] did not depress 17

water potential of plant below the control level. 18

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1

However, a high concentration of sugar could not be sustained for long and the stem diameter 2

decreased below the control level two weeks after imposition of the stress (Fig.2). Photosynthesis 3

of N-deficient leaves decrease on account of end product accumulation [39] and loss of Rubisco 4

activity [10]. In our experiment, apparent photosynthetic rate of leaf increased and decreased 5

with increase of nitrogen (Fig. 10A) and sugar concentrations of the leaves (Fig. 10B), 6

respectively (Fig. 11 A, B). These observations provide support for change in source sink balance 7

among plant organs, which became necessary for reallocation of resources under sub-optimal N 8

[40]. Since photosynthetic assimilate production was low in N-deficient plants (Fig.6A), the stem 9

could not conserve water for osmotic adjustment and the diameter decreased below the control 10

level. N-deficiency reduced stomatal conductance and transpiration rate from 8 DAT 11

(Fig.6B,C)）; and the effect on stomatal conductance [15, 41] possibly led to a concomitant 12

increase of water potential of the plant organs. 13

14

Presence of high concentration of solutes in the phloem might have reduced daytime shrinkage of 15

stem diameter. The resulting decrease of amplitude might have contributed to the damping out of 16

the circadian rhythm of stem diameter dynamics (Fig.2) Similar to the stem, the fruit maintained 17

its water potential and did not exhibit any change of diameter under N-deficiency, although the 18

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diameter declined marginally during the first few days of treatment. Unlike the stem, fruit 1

diameter did not change in the later part of the treatment period, because of the preference 2

received in partitioning of assimilates (Fig.8). Similar preference in partitioning to heterotrophic 3

organs at the cost of photosynthetic organs was noticed under P-deficiency stress [19, 42]. But 4

unlike phosphorus, N-deficiency effects are more explicit on the source activity and also on the 5

sink activity. In our study, N-deprivation did not decrease fruit diameter in spite of reduction in 6

13C assimilate partitioning (Fig.8) and 13C export rate from leaves (Fig.7). Whereas N-deficiency 7

decrease dray weight of fruits at 19 DAT, but not influence at 9 DAT (Fig. 1). These results 8

suggest that N-deficiency do not affect water content of fruit before 9 DAT and then depress dry 9

weight production of fruit and diameter of fruit is maintained by water content. In fact, water 10

content percentage of fruit became higher than control by N deficiency treatment (data not 11

shown). 12

13

14

Simultaneous monitoring of both source and sink activities has distinguished the effects of the 15

stress on these organs. N-deficiency effects were instantaneous and comparable to that of 16

K-deficiency [20]. However, the mechanisms of these effects are not similar, the former regulates 17

water potential, because unused sugar accumulate in the plant [33], whereas, K becomes a direct 18

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osmolyte for cell water. Conversely, P limitation down regulates carbohydrate synthesis [33] and 1

soluble carbohydrates concentration cannot be a factor for regulation of plant water potential in 2

the deficient plants. The water potential of P-deficient plants might have increased for sometime 3

on account of reduced stomatal conductance [19]. 4

5

6

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Physiol. 46 (1970) 419-422. 12

13

14

15

16

17

18

Table

1. T

he effect of N deficiency on

13C atom

% excess in various parts of tomato plants at different tim

es after treatment

Plan

t parts9 D

AT

19 D

AT

Contro

lN

defic

iency

Contrl

N de

ficie

ncy

Fed le

af0.5

57

±0.0

14

1.0

83

±0.1

41

0.5

83

±0.0

34

0.9

38

±0.1

18

Oth

er le

aves

0.0

06

±0.0

01

0.0

02

±0.0

01

0.0

38

±0.0

02

0.0

07

±0.0

00

Fru

its0.1

15

±0.0

21

0.1

13

±0.0

32

0.0

48

±0.0

05

0.0

45

±0.0

03

Ste

m, u

pper

0.0

06

±0.0

02

0.0

06

±0.0

01

0.0

34

±0.0

08

0.0

18

±0.0

04

Ste

m, lo

wer

0.0

16

±0.0

01

0.0

28

±0.0

03

0.0

22

±0.0

05

0.0

22

±0.0

03

Roots

0.0

06

±0.0

01

0.0

06

±0.0

02

0.0

04

±0.0

01

0.0

13

±0.0

01

Ave

rage±

SEM

-26-

- 27 -

Figure legends 1

Fig. 1. The effect of N-deficiency on dry weight of tomato plant organs. 0, Initial of treatment; 2

Cont.: control; -N: nitrogen deficiency. Vertical bars denote SEM for whole plant weight and 3

each organ weight. Fruit (black and white checks), root (white), stem (diagonal stripe), leaves 4

(spotted). 5

6

Fig. 2.The effect of N-deficiency on diurnal changes of stem diameter of tomato plant. Changes 7

in stem diameter were monitored by shrinkage type micro-displacement detector. Data represent 8

the means of three plants. Control (fine line), -N treatment (thick line) and PAR (most bottom 9

line). 10

11

Fig. 3. The effect of N deficiency on diurnal changes of stem diameter of tomato plants. On each 12

occasion, the diameter changes in both control and N-deficient plants were recorded with 13

reference to 'Zero change' at midnight (00.00 h). Control (fine line), -N treatment (thick line) and 14

PAR (most bottom line). 15

16

Fig. 4. The effect of N deficiency on changes in fruit diameter in tomato. Changes in fruit 17

diameter was monitored as described in Fig. 2. Control (fine line), -N (thick line) and PAR (most 18

- 28 -

bottom line). 1

2

Fig. 5. The effect of N deficiency on diurnal changes of fruit diameter of tomato plants. On each 3

occasion, the diameter changes in both control and N-deficient plants were recorded with 4

reference to 'Zero change' at midnight (00.00 h). Control (fine line), -N treatment (thick line). 5

6

Fig. 6. The effect of N deficiency on (A) apparent photosynthetic rate, (B) stomatal conductance 7

and (C) transpiration rate of the leaf immediately below the second truss of tomato. Control 8

(white circle), -N treatment (black circle). Vertical bars denote SEM for each value. 9

10

Fig. 7. The effect of N deficiency on 13C export rate from the leaf immediately below the first 11

truss of tomato at 9 d and 19 d after treatment (DAT). Cont., control; -N, N deficiency. 12

13

Fig. 8. The effect of N deficiency on 13C partitioning among various part of tomato plant at 9 d 14

and 19 d after treatment (DAT). Cont., control; -N, N deficiency. 1st fruits (black and white 15

checks), root (white), stem (diagonal stripe), leaves (spotted), other (black). 16

17

Fig. 9. The effect of N deficiency on 15N partitioning among various part of tomato plant at 19 d 18

- 29 -

after treatment (DAT). Cont., control; -N, N deficiency. 1st fruits (black and white checks), root 1

(white), stem (diagonal stripe), leaves (spotted), other (black). 2

3

Fig. 10. The effect of N deficiency on (A) N concentration and (B) sugar concentration of the 4

leaf immediately below the second truss of tomato. Control (white circle), -N treatment (black 5

circle). Vertical bars denote SEM for each value. 6

7

Fig. 11. Relationship between (A) leaf N concentration and apparent photosynthetic rate, (B) leaf 8

sugar concentration and apparent photosynthetic rate. R2 is the linear regression coefficient 9

squared. P is level of significance. 10

0

10

20

30

40

50

60

70

Dry weight (g plant-1)

0

Cont.

Cont.

-N

-N

919

Days afte

r treatm

ent

Fig. 1

. The e

ffect o

f N-de

ficie

ncy o

n dry w

eigh

t of to

mato

plant o

rgans. 0

, Initial o

f treatm

ent; C

ont.: c

ontro

l; -N

: nitro

gen de

ficie

ncy.

Vertic

al bars denote

SEM

for w

hole

plant w

eigh

t and e

ach o

rgan w

eigh

t. Fru

it (black an

d white

checks), ro

ot (w

hite

), stem

(diago

nal

stripe), le

aves (spo

tted).

-30-

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

03

69

12

15

18

0 1600

3200

4800

6400

8000

9600

11200

12800

Changes of stem diameter (mm)

Days afte

r treatm

ent

Fig. 2

.The e

ffect o

f N-de

ficie

ncy o

n diu

rnal c

han

ges o

f stem

diamete

r of to

mato

plant. C

han

ges in

stem

diamete

r were

monito

red by

shrin

kage type

mic

ro-displac

em

ent de

tecto

r. Data re

prese

nt th

e m

ean

s of th

ree plan

ts. Contro

l (fine lin

e), -

N tre

atment (th

ick lin

e) an

d PA

R(m

ost bo

ttom

line).

-31-

PAR (E m-2

s-1

)

PAR (E m-2

s-1

)

-0.4

-0.2

5

-0.1

0.0

5

0.2

0.3

50:0

06:0

012:0

018:0

024:0

0 0 1600

3200

4800

6400

8000

9600

-0.5

0

-0.4

0

-0.3

0

-0.2

0

-0.1

0

0.0

0

0.1

0

0 1600

3200

4800

6400

8000

9600

-0.1

8

-0.1

2

-0.0

6

0.0

0

0.0

6

0.1

2

0 1600

3200

4800

6400

8000

9600

-0.2

2

-0.1

7

-0.1

2

-0.0

7

-0.0

2

0.0

3

0.0

80:0

06:0

012:0

018:0

024:0

0 0 1600

3200

4800

6400

8000

9600

Changes of stem diameter (mm)

Tim

e o

f dayFig. 3

. The e

ffect o

f N de

ficie

ncy o

n diu

rnal c

han

ges o

f stem

diamete

r of to

mato

plants. O

n e

ach o

ccasio

n, th

e diam

ete

r chan

ges in

both

contro

l and N

-de

ficie

nt plan

tsw

ere

recorde

d with

refe

rence to

'Zero

chan

ge' at m

idnigh

t (00.0

0 h

). Contro

l (fine lin

e), -

N tre

atment (th

ick lin

e) an

d PA

R (m

ost bo

ttom

line).

A2 D

ayB

11 D

ay

C14 D

ayD

19 D

ay

-32-

-33-

-5 0 5

10

15

20

03

69

12

15

18

0 1600

3200

4800

6400

8000

9600

11200

12800

14400

16000

17600

Dyas afte

r treatm

ent

Changes of fruit diameter (mm)

PAR (E m-2

s-1

)

Fig. 4

. The e

ffect o

f N de

ficie

ncy o

n c

han

ges in

fruit diam

ete

r in to

mato

. Chan

ges in

fruit diam

ete

r was m

onito

red as

desc

ribed in

Fig. 2

. Contro

l (fine lin

e), -

N (th

ick lin

e) an

d PA

R (m

ost bo

ttom

line).

-0.6

-0.3

0

0.3

0.6

0.9

0:00 6:00 12:00 18:00 24:00

0

1600

3200

4800

6400

8000

-0.8

-0.4

0.0

0.4

0.8

1.2

0

1600

3200

4800

6400

8000

-0.8

-0.4

0.0

0.4

0.8

1.2

0

1600

3200

4800

6400

8000

Chan

ges

of

fruit d

iam

ete

r (m

m)

Time of day

Fig. 5. The effect of N deficiency on diurnal changes of fruit diameter of tomato plants. On eachoccasion, the diameter changes in both control and N-deficient plants were recorded with referenceto 'Zero change' at midnight (00.00 h). Control (fine line), -N treatment (thick line).

A2 Day

B7 Day

C17 Day

-34-

PA

R (E

m-2

s-1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0

4

8

12

16

20

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Sto

mat

al c

onducta

nce

（m

ol H

2O

m-2 s

-1）

Days after treatment

Fig. 6. The effect of N deficiency on (A) apparent photosynthetic rate, (B) stomatalconductance and (C) transpiration rate of the leaf immediately below the second truss oftomato. Control (white circle), -N treatment (black circle). Vertical bars denote SEM for eachvalue.

Appar

ent

photo

synth

etic r

ate

（μ

mol C

O2 m

-2 s-

1）

A

B

C

Tra

nsp

irat

ion r

ate

（m

ol H

2O

m-2 s

-1）

-35-

0

10

20

30

40

50

60

70

80

90

13C

exp

ort

rat

e (

%)

Cont. -N

9 DAT

Cont. -N

19 DAT

Fig. 7. The effect of N deficiency on 13C export rate from the leaf immediatelybelow the first truss of tomato　at 9 d and 19 d after treatment (DAT). Cont.,control; -N, N deficiency.

-36-

0%

20%

40%

60%

80%

100%

13C

par

titionin

g ra

te

Cont. -N

9 DAT

Cont. -N

19 DAT

Fig. 8. The effect of N deficiency on 13C partitioning among various part of tomato plant at 9 dand 19 d after treatment (DAT). Cont., control; -N, N deficiency. 1st fruits (black and whitechecks), root (white), stem (diagonal stripe), leaves (spotted), other (black).

-37-

0%

20%

40%

60%

80%

100%15N

par

titionin

g ra

te

Cont. -N

19 DAT

Fig. 9. The effect of N deficiency on 15N partitioning among various part of tomato plant at 19d after treatment (DAT). Cont., control; -N, N deficiency. 1st fruits (black and white checks),root (white), stem (diagonal stripe), leaves (spotted), other (black).

-38-

10

15

20

25

30

35

40

0 3 6 12 20

Leaf

N c

oncentr

atio

n

（m

g kg

-1 D

.W.）

A

60

80

100

120

140

160

0 3 6 12 20

Leaf

suga

r concentr

atio

n

（m

g kg

-1 D

.W.）

B

Fig. 10. The effect of N deficiency on (A) N concentration and (B) sugarconcentration of the leaf immediately below the second truss of tomato. Control(white circle), -N treatment (black circle). Vertical bars denote SEM for each value.

Days after treatment

-39-

0

3

6

9

12

15

18

5 10 15 20 25 30 35 40

0

3

6

9

12

15

18

50 70 90 110 130 150 170

Fig. 11. Relationship between (A) leaf N concentration and apparentphotosynthetic rate, (B) leaf sugar concentration and apparent photosynthetic

rate. R 2 is the linear regression coefficient squared. P is level of significance.

Appar

ent

photo

synth

etic r

ate

（μ

mol C

O2 m

-2 s

-1）

Leaf N concentration (g mg-1

D.W.)

Leaf sugar concentration (g mg-1

D.W.)

A

B

y = 0.5086x - 1.4689

R 2 = 0.8022P < 0.001

y = -0.0914x + 21.331

R 2 = 0.4328P > 0.001

-40-