Mobilization of stored nitrate in broccoli (Brass ica oleracea var. italical Liangxue Liu and Barry J. ShelP Department of Horticuftural Science, lJniversity ol Guelph, Guelph, Ontario, Canada NIG 2W1. Received 22 November 1994, accepted 10 February 1995.

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Liu, L. and Shelp, B. J. 1995. Mobilization of stored nitrate in broccoli (Brassica oleracea var. italica). Can. J. Plant

Sci.

75: 7@-715. The study tested the hypothesis that substitution of nitrate supply with other anions induces the mobilization of stored nitrate in broccoli (Brassica oleraceavar. italica) plants. Broccoli plants, grown under greenhouse conditions in soilless culture, were provided with either a continuous supply of nitrate in the nutrient solution until commercial maturity, or with a nitrate supply only up to inflorescence emergence. At inflorescence emergence, nitrate was also substituted with various chloride or sulphate salts of sodium, potassium and ammonium (15 and 5 mmol L-'). During the period of inflorescence development studied, plant size increased by two- to threefold. Nitrate removal or its substitution with chloride or sulphate salts of sodium and potassium (15 mmol L-r) resulted in 13% less dry-matter accumulation afi 63-97% less xylem-sap nitrate than continuous nitrate supply , arrd 57 -62% decline in the plant-nitrate content found at inflorescence emergen@ . In contrast, chloride or sulphate salts of ammonium (5 mmol L-r) sustained plant growth, while also decreasing xylem-sap nitrate by 83-9OVo and plant-nitrate content by 58-62% . Phloem-exudate nitrate of plants receiving nitrate continuously was only 7 Vo of that of xylem sap, and was decreased by 63%by mintemrpted nitrate supply. Therefore, the removal of nitrate and substitution with a low supply of ammonium, rather than other anions, is recommended for decreasing plant-nitrate content.

Key words: Broccoli, chloride, mobilization, nitrate, Liu, L. etShelp, B. 1995. Mobilisationdesnitratesdestockagechezlebrocoli

sulphate

(Brassicaoleraceavar.ilalba). Can. J. Plant

Sci. 75: 7W-715. Nous avons test6 I'hypothdse selon laquelle le remplacement des nitrates de fumure par d'autres anions provoquerait la mobilisation des nitrates stock6s dans les plants de brocoli (Brassica oleracea var. italica). Des plants de brocoli cultiv€s en serre sur substrat sans sol 6taient alimentds, soit par un apport continu de nitrates jusqu'i maturit6 de r6colte, soit par un aPPort de nitrates seulement jusqu'i la sortie de I'inflorescnece. A. ce stade, le nitrate 6tait remplac6 par divers chlorures et sulfates de sodium, de potassium it d'ammonium (15 et 5 mmol L-l). Durant la phase de grossissement de I'inflorescence, la taille de la plante augmentait de2d3 fois. Le retrait des nitrates ou leur remplacement par les sels de sodium et de potassium (15 mmol L-f) reduisait de l3Vo I'accumulation de matidres sbches. En outre, on observait une baisse de 63 97% de la teneur en nitrate Oani ta sbve de xyldme par rapport avec I'alimentation continue en nitrates, ainsi qu'une baisse de 57 d62Vo dans la teneur de la plante en nitrates observ6e ir la sortie de I'inflorescence. Par constrate, le chlorure ou le sulfate d'ammonium (5 mmol maintenait la croissance normale de la plante, tout en abaissant 6galement les concentrations de nitrate dans la slve du xyldme (83 a 90%) et la teneur en nitrates de la plante (58 d 62%). I€s concentrations de nitrates dans I'exsudat du phlodme chez les plantes fertilis6es aux nitrates sans intemrption n'6taient qredeT%o de celles de la sbve du xylbme. Par aillsurs, quand I'apport de nitrates dans I'alimentation 6tait interrompu, elles accusaient une rdduction de 63%. Il appert que le retrait des nitrates dans I'alimentation des plantes et leur remplacement par de faibles concentrations de sels d'ammonium, de pr6fdrence aux autres anions est ir recommander pour abaisser les concentrations en nitrates du brocoli.

i

L-')

Itlots cl6s: Brocoli, chlorure, mobilisation, nitrate, The intake of NO3 from vegetables poses potential health hazards to humans, such as methaemoglobinaemia in infants

and the formation

of

carcinogenic N-nitrosamines

(Committee on Nitrate Accumulation 1972'). Nitrate, the major N source for most higher plants, is accumulated when its uptake exceeds reduction and subsequent assimilation. The extent of accumulation depends upon many internal and external factors, such as light int€nsity, length of photoperiod, temperature, amount and form of N application, species and cultivar (Maynard et al. 1976). Because NO3 is primarily translocated in xylem, and fruits and seeds are phloem fed (Pate 1980), leafy vegetables, such as spinach and lettuce,

contain much higher NO3 concentrations than fruiting vegetables such as peppers and tomatoes (Corrd and Breimer 1979). Excess NO3 is stored in the vacuole (Martinoia

",

^. ,0,

sulfate

1981; Granstedt and Huffaker 1982) where it serves as an osmoticum (Mott and Steward 1972; MacRobbie 1976). It has been suggested that this role can be replaced by other anions such as Cl and SOa (Blom-Zandstra and Lampe

1983; Schipper 1985; Wehrmann and Hiihndel 1985), thereby making this stored NO3 available for growth and metabolism. Thus, it may be possible to decrease NO3 accumulation in vegetables without decreasing yield.

When NO3

is

substituted with either

decrease in NO3 concentrations

Cl or

SOa, a

of lettuce (Blom-Zandstra

and Lampe 1983) and spinach plants (Schipper 1985) is observed. In those studies, a minus-NO3 treatrnent was not included; therefore, it is not known whether the decrease in NO3 concentration is attributable to substitution, or to a decrease in NO" availabiliW in the nutrient solution.

7IO

CANADIAN JOURNAL OF PLANT SC'E'VCE

A decline in NO3 concentration of the nutrient solution markedly decreases the NO3 contents of plants (Clement a al. 1979; Koch et al. 1988; Gojon et al. l9l).

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Other research reports that Cl substitution decreases the NO3 concentration of lettuce only when NII+ is present in the nutrient solution (van der Boon and Steenhuizen 1986; van der Boon et al. 1988, 1990). Furttrermore, the vacuolar NO3 pool of Chenopodium rubrurn suspension culture cells is mobilizerl when NlIa is also present @eck and Renner 1989). This study tested the hypothesis that substitution of the NO3 supply with other enions induces the mobilization of stored NO3 in broccoli plants. Broccoli was chosen because of the possibility of collecting both xylem sap and phloem exudate from commercially mature plants.

iIATERIALS AND iIETHODS Plant Material Broccoli (Brassica oleracea var. inlica'Emperor') seeds were germinated in seedling trays containing vermiculite, and supplied once a week with one-quarter-strength Hoagland-type Cl-free nutrient solution (Shelp et al. 1992). After 4 wk, the seedlings were transplanted individually into 6-L pots containing a mixture of three parS vermiculite:one part perlite (by volume) and grown under greenhouse conditions. Two separate experiments were conducted, one from January to April 1992, and the other from October to December lg3. Natural light was supplemented by high intensity sodium vapour lamps yielding a quantum flux density at pot level

of 60 mol m-' s-'. The plans were maintained on a lGh light/8-h dark period, an average day and night temperature of 25'Cl18'C, and fertilized every second day until inflorescence emergence with I full-strength Hoagland-type nutrient solution (Shelp et al. 19912) containing 15 mmol L-r NO3, 3 mmol SO4 and no Cl. At inflorescence emergence, all pos were thoroughly flushed with deionized water. In the first experiment, which was conducted

L

L-l

nlW\ control plants continued to receive 15 mmol L-l NO3,

whereas in the experimental treatments, NOr was substituted with various Cl -or SOa salts of Na, K and NHa at either 15 or 5 mmol L-1. In the second experiment, which was conducted in 1993, control plants continued to receive 15 mmol L-t NO3, whereas in the experimental treaEnents, NO3 was simply removed or was substituted with Cl or SOa salts of Na at 15 mmol L-1. Previously, it was shown that the growth characteristics of greenhouse-grown broccoli plants appeared optimal with NO3 nutrition when it was supplied at 14-19 mmol L-r in the nutrient solution (Shelp 1987a).

The plans were harvested at inflorescence emergence (about 22 d, t9y2 and 16 d in 1993 before commercial maturity) and commercial maturity (i.e. just prior to bud opening at the time of maximum expansion of the inflorescence, about 10-12 crn diameter under our greenhouse conditions). At inflorescence emergence, the shoots of plants from six pots were harvested and separated from thebottom to the top into several stata fleaves 1-5, leaves G14, leaves 15-19 and stem (including petioles*midribs)1. Xylem sap

n

was collected from the base of the stem remaining attached to the root system in the pot as described previously (Shelp 1987b). At commercial maturity, the shoots were further divided into leaves?-E24, florets and the inflorescence stalks. The inflorescence was considered to extend 15 cm from the top of the inflorescence along the stem. Prior to harvest of each shoot, phloem exudate was collected from shallow incisions in stalks of the infloresoenoe (Shelp 1987b); after harvest, the xylem sap was collected from the stem base. All plant parts were dried in a forced air oven at 70'C and ground to a fine powder. The fresh and dry weights of the total aboveground parts and inflorescence were determined.

Plant Analyses Preparation of plant material, collection of xylem saps and phloem exudates, and analyses of NO3, NlIa (Tel and Heseltine l90a), Cl (Tel and Heseltine 1990b) and toal N (Tel et al. 1992) were described previously (Liu and Shelp 1993a). The concentration of SOa in xylem sap and phloem exudate was determined using a Dionex series 40(Xli HPLC-

ion chromatography system @ionex Corp.

Sunnyvale,

California) equippd with an lonPac AS4A separator column and an AG4A guard column. Sulphate was eluted isocratically at a flow rate of ll4 mL h-' from the column with alo% (voltvol) solution containing 1.8 mmol L-r Na2CO3 and 1.7 mmol L-r NaHCO3.

Erperimental Design and Statistical Analysis The experiment was a completely randomized block design with six replicates of one plant per pot. Analyses of variance were conducted on the yield data and elemental content in plant tissues and hanslocation fluids (SAS Institute, Inc. 1988). The difference between treatments was compared using Duncan's multiple range test at a 5% sigrificance level.

RESULTS

Yield Response With continuous NO3 supply, two- to threefold increases in fresh and dry werghts were evident during the period from inflorescence emergence to final harvest (Table 1). Substitution of NO3 with Cl or SOa salts of Na and K at inflorescence emergenoe slightly reduced the fresh and dry weights shoot and inflorescence at final harvest (although the

ofboth

shoot dry weight was not significantly affected). In both years, the mean shoot dry weight of these treatments was less than 13% lower than when broccoli was grown with a continuous NO3 supply. With the exception of the response of inflorescence dry weight to NI{4CI at 15 mmol L-', sutr stitution of NO3 with NI{4CI or (NIIa)2SOa had no effect on yield. Plants supplied with NII4 salts at 15 mmol L-r had significantly higher yields than those supplied with Na or K salts, whereas plants receiving 5 mmol L-' had similar yields. At find harvest, plants supplied with Cl or SOa salts of Na and K were generally shorter, with thinner stems and fewer lower leaves (the fallen leaves were included in the yield determination) than plants receiving an intemrpted supply of NO3 (daa not shown).

L'U AND SHEIP - N'TMTE ITOB|/UZIT'O'V

Nitrogen, Chloride and Sulphate Goncentrations of Xylem Sap and Phloem Exudate When NO3 in the nutrient solution was substituted with various Cl or SOa salts, NO3 concentrations of xylem sap Table

l.

,,V

BFOCCOU

711

tlnt found for plants receiving NOr continuously (Table 2). With NII+ salts of Cl or SOa at 15 mmol L-1, xylem-sap NI{4 increased by 220-2ffi%. decreased to less than 37% of

The concentrations of total N found with Cl or SOa salts

of

yield characteristics G plant-t) at commercial maturity of greenhusegrown Emperor broccoli in response to nitrate removd or nitrate substitution wlth chloride or sulphat€ salts at inlloresc€oce enerEence lnflorescence

Shoot

Dry weight

Fresh weight

Fresh weight

Dry weight

269.U 7ll.Zab

33.3c

99.lab

ri.at

21.5b

532.8c

91.80

I13.0c

16.&

5O.5c

94.0b

124.7c

17.6c

Na2SOa (15)

519.5c

m.7b

l18.2c

16.8c

(NHd2SO4/K2SO4r (15)

7l5.sab

la

17E.la r57.9b

24.Mb 2r.5b

lEl.5a

E.3a

r70.tub

22.9ab

ll.3a

Year/treatnent

tvn NaNO3 GE)t NaNO3 (15)Y Nacl (15)

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KCr (15)

(NHd2SO4/K2SO4 (5)

6E5.4b

NH4CI (15) NH4CI (5)

763.5a

105.

LUb Itl.9a

66.9.3b

99.zab

rgln NaNO3 GE)

253.3c

22.5c

NaNO3 (15)

547.9a

-NaNO3

54.Ia 48.&b

ll9.5a

rc.9b

9.9b

ro.tu

NaCl (15)

4./.3.2b

47.3b

94.&

10.0d

Na2SOa (15)

450.8'

49.Mb

99.2b

ll.3a

zIE indicates harvest at inflorescence emergenc€. vNumbers in parentheses indicate concentration (mmol L-l). I(NH4)2SO4 -a XrSOo were supplied in a ratio of l:l to give both 15 mmol L-l NH,4 and lS.mmol Li SO1.^a-dun&r each cotrinn ina for eaci year, mgans not folowJ by the same letter are significantly different at P s 0.05 using Duncan's multiple range test. Table

2. Xylem-sap conpmition (mmol L-5 at commenclal mrturtty of greenhouse-grown Emperor brwcoli in

Year/treatment

response to nttrate rernoval

or

nitrate subctitution with chloride or sulphate salts at lnflonescence e[refgpme NO3-N

NH4-N

Total N

so4-s

cl

38.47a

2.29b

71.3tub

l.7lbc

85.50a

NDg ND ND ND ND ND ND ND ND

0.E7c

38.79a

rvln NaNO3 flE)z NaNO3 (15)Y

NaCl (15)

1.36c

O.79bc

3l.E6cde

KCI (15)

O.7lbc

26.93e

Na2SOa (15)

6.43b 0.79c

O.29c

45.Acd

(NHj6SO4/K2SO4I (15)

6.ffi

5.5tu

76.79ab

0.43c 6.21a 0.36c

47.36c

ND ND ND ND

ND

(NH4J2SO4/K2SO4 (5)

4.g7bc

NH4CI (15) NH4CI (5)

6.Ulb

tv'8 NaNO3 (15)

-NaNO3 NaCl (15) Na2SOa (15)

6.29b

l9.Ma 2.23c S.llbc 6.89b

zIE indicates hanest at inflorescence emergenc€. vNumben in parentheses indicarc concentration (mmol L-l). x(NHr2SO4 frsOn were supplied in a ratio of l:l to give both 15 mmol wND, not determined. "tta

67.21b 30.43dc

ND

ND ND

L-l

NlI4 and

15 mmol

a-e Under each column and for each year, means not followed by the same letter are significantly different at

8.59b

l.tlc 1.89c

2.31c 35.10c 5.55bc

2.42c 6.33b

0.04b

4.9tb

3.63a 0.45b

9.35a

L-r

l. l3c 9.5?b

o.4tb

SO4.

P5

0.05 using Duncan's multiple ratrge test.

712

CANAD'AN JOURNAL OF PLANr SC'Et'CE

L-t NlI4 were decreasedto 32-55yo of that found for broccoli receiving NO3 continuously, whereas these were decreased to 79-g% with 15 mmol L-r NH4. When NO3 was the N source about 43-53% of xylem N was organic N. However, when NO3 was sub-

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Na, K and 5 mmol

stituted with various Cl or SOa salts,73-ggyo of xylem N was organic N. Chloride substitution for NO3 resulted in much higher Cl concentrations in xylem sap than the remaining treaunents; this was particularly evident with the higher concentration of NHa. Removal of NOr or its substitution with SOa increased the SOa conce-ntrations in xylem sap by 103-286Vo compared to that found with plants receiving NO3 continuously. Phloem exudate con0ained less NO3 and more NIIa, total N, Cl and SOa (table 3) than xylem sap. ln !9y2, we were unable to collect phloem exudate from plants grown with NO3. However, in the other treatrnents, the phloem-Cl concentrations were much higher rrn1992 than in l9fl3.ln1993., broccoli receiving an intemrpted NO3 supply had a phloemNO3 concentration that was only 37Vo of that found for plants receiving an unintemrpted supply. In contrast to rylem sap, the concentration of SOa in phloem exudate was highest with NO3. In each treaffnent, at least 94Vo of the phloem N

was organic N.

Nitrogen and Ghloride Contents of plant Organs The NO3 contents of different strata from the broccoli plant are shown in Table 4. For all treafinents, the foliage showed a decreasing gradient up the shoot. Compared with broccoli receiving NO3 continuously, plants receiving an intemrpted supply of NO3 possessed lower NO3 contents in all strata, except leaves l-5 n 1992 where NOr contents were similar for all treatnents. The NO3 contentsbf the stem + petioles

*

midribs decreased by 54-84Vo during inflorescence

development with Cl or SOa salts of Na, K and NlIc at final Table

3.

Phloem-exudate composition

Year/treaEnent

(nmol

L-)

at

6-7IVo with NO3 removal or with substiotion with Cl or SOa salts in 1993. The overall NO3 contents of the shoot were similarly decreased by NO3 removal alone and by substitution with Cl or SOa salts; 15 mmol L-' NH4 was less effective than 5 mmol L-t NIIa. Regardless of these treafrnents, 70-N% of the harvest in 1992, and by

*

substantially lower than when broccoli received NO3 continuously. Use of NII4 salts, rather than Na and K, increased the total-N contents. Compared to the continuous NO3 control, total-shoot N decreased to 52-55Vo with the Na or K salts of Cl and SOa, and to 78-827o with 5 mmol L-r NII4, and increased to 124-l3O% with 15 mmol L-r NI{4.

Chloride substitution for NO3 during inflorescence development generally increased the Cl contents of the shoot at commercial maturity (Table 6). NH4CI at 15 mmol L-r was particularly effective in increasing the Cl contents of the shoot. Because shoot-NO3 contents were not specifically lowered by substitution with either Cl or SOa (Table 4), the SOa contents were not dercrmined.

During the period

DtscussroN of inflorescpnce

development studied

(1G22 d after inflo'rescence emergence), NO3 removal or its substiurtion witr Cl or SOa salts of Na and K resulted n I3Vo less dry-matter accumulation than an unintemrpted supply

NO3 (Iable 1).

In

of

contrast, a low supply of NHaCI or (NII4}SO4 zustained plant growtr. Other shrdies have shown that the growttr of perennial ryegrass (Clement et aL

commercialffmrnfl**1ftx;;g3ffH"mt

NO3-N

*

decrease in shoot NO3 was from stem petioles midribs, a sfratum representing 43Vo of the shoot dry weight. In contast to NO3, the total-N contents were lowest in the bottom stratum (Table 5). When NO3 was removed or substituted with Cl or SOa salts of Na and K, the total-N contents of all plant strata, including the inflorescence, were

lng)

in response ro nibate removal

NH4-N

Total N

so4-s

zejclt

tgln NaNO, (15)z NaCl (15)

o.3sd

452.3c

ND9

KCI (15)

o.4td

25.39ab

584..lbc

ND

25.72.b

Na2SOa (15)

0.51c

29.97a

532.9a

ND

29.72b

(NHd2SO4/K2SO4v (15) (NHd2SO4/K2SO4 (5)

0.&a

2t.79b

892.Ia

ND

O.57abc

27.rkb

ffi.4bc

27.61b 25.54b

q.82a

15.30a

NH4CI (r5) NH4CI (5)

-I

?0.45b

O.52bc

25.43ab

634.rb

0.59ab

22.79b

5ll.&c

ND ND ND

l.3la o.4u

ND ND ND ND

ND

14.tu

ND

ro.20b

8.10,

ND ND

E.88c

8.41b 13.7M

24.llb

lEr3 NaNO3 (15)

-NaNO3 NaCl (15)

0.29c 0.29c

KCI (15) zNumbers

in

parentheses indicate concentration (mmol

KfOa ry9re supplM in a ratio of l$!lhSO..a."na tNot possible to collect phloem

l:l

L-l). to give both 15 mmol

exudate from these plants.

L-l

NH4 and 15 mmol

9.32bc

L-l

'ND, not determined. a-dUnder each column and for each year, means not followed by the same letter are significantly different at P

SO4.