Boron deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of

Journal of Plant Physiology 165(2008)

1331—1341

Boron de?ciency decreases growth

and photosynthesis,and increases starch and hexoses in leaves of citrus seedlings

Shuang Han a ,Li-Song Chen a,?,Huan-Xin Jiang b ,Brandon R.Smith c ,Lin-T ong Yang a ,Cheng-Yu Xie a

College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,China b

College of Life Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China c

Organic and Alternative Crop Production,Department of Plant Sciences,University of Tennessee,2431Joe Johnson Drive,Knoxville,TN 37996,USA

Received 3July 2007;received in revised form 16November 2007;accepted 16November 2007

KEYWORDS Antioxidant;

Boron de?ciency;Citrus;

Photosynthesis

Summary

Seedlings of sweet orange (Citrus sinensis )were fertilized for 14weeks with boron (B)-free or B-suf?cient (2.5or 10m M H 3BO 3)nutrient solution every other day.Boron de?ciency resulted in an overall inhibition of plant growth,with a reduction in root,stem and leaf dry weight (DW).Boron-starved leaves showed decreased CO 2assimilation and stomatal conductance,but increased intercellular CO 2concentra-tions.Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco),NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH)and stromal fruc-tose-1,6-bisphosphatase (FBPase)were lower in B-de?cient leaves than in controls.Contents of glucose,fructose and starch were increased in B-de?cient leaves while sucrose was decreased.Boron-de?cient leaves displayed higher or similar superoxide dismutase (SOD),ascorbate peroxidase (APX),monodehydroascorbate reductase (MDAR)and glutathione reductase (GR)activities,while dehydroascorbate reductase (DHAR)and catalase (CAT)activities were lower .Expressed on a leaf area or protein basis,B-de?cient leaves showed a higher ascorbate (AsA)concentration,but a similar AsA concentration on a DW basis.For reduced glutathione (GSH),we found a similar GSH concentration on a leaf area or protein basis and an even lower content

on a DW basis.Superoxide anion (O 2d à

)generation,malondialdehyde (MDA)

www.elsevier .de/jplph

Abbreviations:APX,ascorbate peroxidase;AsA,ascorbate;B,boron;Car ,carotenoids;CAT ,catalase;Chl,chlorophyll;DHAR,

dehydroascorbate reductase;DW,dry weight;FBPase,fructose-1,6-bisphosphatase;FW,fresh weight;GR,glutathione reductase;GSH,reduced glutathione;MDA,malondialdehyde;MDAR,monodehydroascorbate reductase;NADP-GAPDH,NADP-glyceraldehyde-3-phosphate dehydrogenase;O 2d à,superoxide anion;ROS,reactive oxygen species;Rubisco,ribulose-1,5-bisphosphate carboxylase/oxygenase;SOD,superoxide dismutase;TNC,total nonstructural carbohydrates.?Corresponding author .Tel.:+8659187645506;fax:+8659183727618.E-mail address:lisongchen2002@https://www.360docs.net/doc/4c287269.html, (L.-S.Chen).

concentration and electrolyte leakage were higher in B-de?cient than in control leaves.In conclusion,CO2assimilation may be feedback-regulated by the excessive accumulation of starch and hexoses in B-de?cient leaves via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes.Although B-de?cient leaves remain high in activity of antioxidant enzymes,their antioxidant system as a whole does not provide suf?cient protection from oxidative damage. &2007Elsevier GmbH.All rights reserved.

Introduction

Boron(B)is an essential element required for the normal growth of higher plants.Boron de?ciency is a widespread problem in many agricultural crops, including citrus(Shorrocks,1997).The role of B in plant nutrition is little understood,which is surprising,since on a molar basis the requirement for B is,at least for dicotyledons,higher than that of any other micronutrient(Marschner,1995;Alves et al.,2006).

Previous studies have shown that B de?ciency decreases plant photosynthetic capacity(Kastori et al.,1995;Zhao and Oosterhuis,2002,2003).In mustard(Brassica campestris),decreased rate of CO2assimilation appears to result from both decreased Hill reaction activity and low intercel-lular CO2concentration(Sharma and Ramchandra, 1990).Plesnicˇar et al.(1997)suggested that the decreased rate of photosynthetic O2evolution in B-de?cient sun?ower(Helianthus annuus)leaves could be correlated with reduced chlorophyll(Chl) content,photosynthetic electron transport rate and photophosphorylation.El-Shintinawy(1999) investigated the structural and functional damage caused by B de?ciency in sun?ower leaves and suggested that B de?ciency affected photosynthesis directly or indirectly.Accumulation of starch and hexoses occurs despite decreased photosynthesis (Kastori et al.,1995;Zhao and Oosterhuis,2002; Camacho-Cristo′bal et al.,2004)because growth is more affected than photosynthesis,and they are not used.This leads us to hypothesize that CO2 assimilation is feedback-regulated by excessive accumulation of starch and hexoses in B-de?cient leaves via direct interference with chloroplast function and/or indirect repression of photosyn-thetic enzymes.

A consequence of the imbalance between photo-synthetic production of carbohydrates and their use in growth is that less of the absorbed photon-energy captured by the light harvesting system is used in CO2assimilation,so the photosystem electron transport chain becomes over-reduced. This leads to accelerated production of reactive oxygen species(ROS)such as single oxygen(1O2),superoxide anion(O2dà),hydrogen peroxide(H2O2) and hydroxyl radicals(d OH).To minimize cellular damage caused by ROS,plants have evolved a scavenging system composed of antioxidants such as ascorbate(AsA)and reduced glutathione(GSH) and antioxidant enzymes such as superoxide dis-mutase(SOD,EC1.15.1.1),ascorbate peroxidase (APX,EC1.11.1.11),glutathione reductase(GR,EC 1.6.4.2),monodehydroascorbate reductase(MDAR, EC 1.6.5.4),and dehydroascorbate reductase (DHAR,EC 1.8.5.1)involved in the water–water cycle,as well as catalase(CAT,EC 1.11.1.16) involved in scavenging the bulk H2O2generated by photorespiration(Chen et al.,2005).Although there have been several reports investigating the antioxidant responses in leaves of plants treated with excess B(Keles et al.,2004;Molassiotis et al., 2006),data are limited on the effects of

B de?ciency.Liu and Yang(2000)reported that low B decreased SOD,APX,and CAT activities,and AsA content of soybean(Glycine max)leaves.In sun-?ower leaves,B de?ciency increased APX activity, decreased GR and CAT activities,decreased AsA and non-protein SH-compound contents,but did not affect SOD activity(Cakmak and Ro¨mheld,1997; Dube et al.,2000).Thus,it is not well known how B de?ciency affects antioxidant system in plants.

In China,B de?ciency is frequently observed in citrus orchards,and is responsible for considerable loss of productivity and poor fruit quality.To our knowledge,there is hardly any information on gas exchange,photosynthetic enzymes,carbohydrates, and antioxidant system of citrus leaves in response to B de?ciency.The objectives of this study were to test the hypothesis that CO2assimilation is feed-back-regulated by excessive accumulation of starch and hexoses in B-de?cient leaves,and to determine how B de?ciency affects antioxidant system in B-de?cient leaves.

Materials and methods

Plant culture and B treatments

This study was conducted outdoors from March to November2005at Fujian Agriculture and Forestry

S.Han et al.

1332

University,Fuzhou.Seeds of citrus(Citrus sinensis(L.) Osbeck cv.Xuegan)were germinated in plastic trays containing sand,and fertilized when necessary with1/4 strength nutrient solution until dripping.Full-strength nutrient solution contained6mM KNO3,4mM Ca(NO3)2, 2mM NH4H2PO4,1mM MgSO4,50m M KCl,10m M H3BO3, 2m M MnSO4,2m M ZnSO4,0.5m M CuSO4,0.065m M (NH4)6Mo7O24,and40m M Fe-EDTA(Ferna′ndez-Ballester et al.,2003).In mid-June(5weeks after germination), uniform seedlings with a single stem were selected, transplanted into6L pots containing sand.Each pot contained three seedlings,and was supplied with500mL of1/3strength nutrient solution every other day.Ten weeks after transplanting,the treatment was applied for14weeks:until the end of the experiment,each pot was supplied with500mL B-de?cient(0m M H3BO3)or B-suf?cient(2.5or10m M H3BO3)nutrient solution every other day.At the end of the experiment,fully expanded (about7weeks old)leaves from different replicates and treatments were used for all the measurements.Leaf disks(0.61cm2in size)were collected at noon under full sun and immediately frozen in liquid N2.Samples were stored atà801C until they were used for the determina-tion of photosynthetic enzymes,carbohydrates,malon-dialdehyde(MDA),antioxidant enzymes and metabolites, pigments,and soluble protein.

Measurements of root,stem and leaf dry weight(DW), and speci?c leaf weight

At the end of the experiment,12plants per treatment from different pots were harvested.The plants were divided into their separate parts(roots,stems,and leaves).The plant material was then dried at801C for48h and the DW measured.Speci?c leaf weight was measured according to Syvertsen et al.(1980). There were?ve replicates per treatment(one leaf per replicate).

Determination of pigments,total soluble protein,

and B

Chl,Chl a,Chl b,and carotenoids(Car)were assayed according to Lichtenthaler(1987).There were?ve replicates per treatment(two disks from the same leaf per replicate).Soluble protein was determined according to Bradford(1976).There were?ve replicates per treatment(two disks from the same leaf per replicate). Boron was determined using the modi?ed curcumin method(Kowalenko and Lavkulich,1976)after leaf samples were ashed at5001C for5h,and dissolved in 0.1N HCl.There were?ve replicates per treatment(two leaves from the same plant per replicate).

Gas exchange measurements

Measurements were made with a CI-301PS portable photosynthesis system(CID,WA,USA)at ambient CO2 concentration with a natural photosynthetic photon?ux density of1300735m mol mà2sà1between10:30and 12:00on a clear day.During measurements,leaf temperature and ambient vapor pressure were26.07 1.01C and1.970.1kPa,respectively.There were eight replicates per treatment(one leaf per replicate). Photosynthetic enzymes

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco,EC 4.1.1.39),NADP-glyceraldehyde-3-phos-phate dehydrogenase(NADP-GAPDH,EC 1.2.1.13), and stromal fructose-1,6-bisphosphatase(FBPase,EC 3.1.3.11)were extracted according to Chen and Cheng (2004).There were?ve replicates per treatment(two disks from the same leaf per replicate).Total Rubisco activity was assayed according to Cheng and Fuchigami (2000).NADP-GAPDH and stromal FBPase activities were determined according to Chen and Cheng(2004). Nonstructural carbohydrates

Sucrose,fructose,glucose,and starch were extracted three times with80%(v/v)ethanol at801C(3mL each, 30min per extraction),and determined according to Jones et al.(1977).There were four or?ve replicates per treatment(three disks from the same leaf per replicate). O2dàgeneration

O2dàgeneration was determined by the reduction of nitroblue tetrazolium(Doke,1983).There were four or ?ve replicates per treatment(six fresh disks from the same leaf per replicate).

Lipid peroxidation

Lipid peroxidation was determined by measuring MDA concentration(Hodges et al.,1999).There were?ve replicates per treatment(six disks from the same leaf per replicate).

Electrolyte leakage

Fifteen fresh leaf disks from the same leaf taken at mid-day under full sun were immediately incubated in 15mL distilled water at room temperature.T wenty-four hours after incubation,the?rst electrical conductance (C1)was measured.Then,the sample was incubated in boiling water bath for15min and the second electrical conductance(C2)was measured after the sample was cooled.The electrolyte leakage was calculated according to the formula:(C1/C2)?100.There were four replicates per treatment(one leaf per replicate).

Antioxidant enzymes and metabolites

SOD,APX,MDAR,DHAR,GR,and CAT were extracted according to Chen and Cheng(2003).There were four or ?ve replicates per treatment(two disks from the same leaf per replicate).SOD activity was assayed according to

Citrus seedlings under boron de?ciency1333

Giannoppolitis and Rice (1977).APX,CAT ,MDAR,DHAR,and GR activities were measured according to Chen and Cheng (2003).

GSH was determined according to Grif?th (1980).There were four or ?ve replicates per treatment (two disks from the same leaf per replicate).

AsA was determined according to Chen and Cheng (2003).There were four or ?ve replicates per treatment (one disk per replicate).

Experimental design and statistical analysis

There were 25pots seedlings per tretment in a completely randomized design.Experiments were per-formed with 4–12replicates (one plant from different pots per replicate).Results represented the mean-s 7standard errors.Differences among treatments were separated by the least signi?cant difference test at P o 0.05level.

Results

Plant growth

Plants grown in the absence of B ?rstly developed B-de?cient symptoms at the apex and in the actively growing leaves.In the young leaves B-de?cient symptoms included dieback of terminal growth,yellow,water-soaked spots in the lamina,and general deformation.Symptoms in the mature leaves were characterized by yellowing,enlarge-ment,splitting,and corking of leaf veins.No

symptoms were observed in plants supplied with 2.5or 10m M B,and foliar B concentration was in the normal range for the two treatments (Table 2;Chapman,1968).Based on these results,plants that did not receive B are considered B de?cient,and those treated with 2.5or 10m M B are considered B suf?cient.

Boron de?ciency decreased leaf,stem and root DW,and increased speci?c leaf weight.Leaf and stem DW decreased to a larger extent than root DW in response to B de?ciency,and resulted in a greater root DW/shoot DW ratio (Table 1).

Chl,Car,total soluble protein and B

Contents of Chl,Chl a ,Chl b ,Car ,soluble protein and B,and ratio of Chl a /b decreased in B de?ciency,but the ratio of Car/Chl increased compared with B suf?ciency (Table 2).

Gas exchange and photosynthetic enzymes

Both CO 2assimilation and stomatal conductance decreased signi?cantly,but intercellular CO 2concentration increased in B-de?cient compared B-suf?cient leaves (Table 3).

On an area,protein or DW basis,activities of key enzymes in the Calvin cycle,including Rubisco,NADP-GAPDH,and stromal FBPase were lower in B-de?cient than in control leaves (Table 4;acti-vities on a DW basis not shown).

Table 1.Effects of boron (B)supply on leaf,stem and root dry weight (DW),and speci?c leaf weight in citrus

seedlings

B treatments (m M)Root DW (g plant à1)Stem DW (g plant à1)Leaf DW (g plant à1)Root DW/shoot DW

Speci?c leaf weight (g FW m à2)

(g DW m à2)

0 3.4570.32b 2.8970.30b 3.4170.30b 0.5770.05b 336.8732.8a 138.173.9a 2.5 4.9270.48a 4.5770.40a 6.3570.55a 0.4570.02a 252.0711.0b 99.575.0b 10

4.1670.21ab

4.4970.33a

6.1570.37a

0.4070.02a

250.179.1b 101.875.2b

Data are means 7standard errors (n ?5or 12).Within a column,values followed by different letters are signi?cantly different at P o 0.05.

Table 2.Effects of B supply on chlorophyll (Chl),carotenoid (Car),B,and soluble protein concentrations in citrus

leaves

B treatments (m M)Chl

(mg m à2)Chl a (mg m à2)

Chl b (mg m à2)

Chl a /b

Car

(mg m à2)

Car/Chl

B (m g g à1DW)

Soluble protein (g m à2)

0388764b 283750b 104713b 2.6370.16b 94715b 0.24670.012a 6.070.7b 6.870.4b 2.5719734a 553727a 16477a 3.3770.03a 16278a 0.22570.002b 37.673.9a 9.570.6a 10

725716a 555711a 16875a 3.3070.05a 15474a 0.21270.007b 43.471.9a 9.070.2a

Data are means 7standard errors (n ?5).Within a column,values followed by different letters are signi?cantly different at P o 0.05.

S.Han et al.

1334

Nonstructural carbohydrates

On an area basis,B-de?cient leaves had higher concentrations of glucose,fructose,soluble sugars (glucose+fructose+sucrose),starch,and total non-structural carbohydrates(TNC),but a lower con-tent of sucrose(Figure1A–F).When expressed on a DW basis,nonstructural carbohydrates followed the same trend as when expressed on an area basis (Figure1G–I and K–L),except there was no dif-ference in the content of soluble sugars(Figure1J). The increase in TNC content was mainly due to the increased starch level(Figure1E and K).

O2dàgeneration,MDA,and electrolyte leakage

O2dàgeneration,MDA content,and electrolyte leakage were higher in B-de?cient leaves than in controls(Table5).

Antioxidant enzymes and metabolites

Boron-de?cient leaves had higher or similar SOD, APX,MDAR,and GR activities depending on whether the results were expressed on an area, protein,or DW basis.The activities of DHAR and CAT were always lower under B de?ciency,regard-less of how the data were expressed(Figure2). AsA content was elevated in response to B de?ciency when expressed on a leaf area or protein basis,but did not change on a DW basis.Conversely, B de?ciency decreased GSH concentration only when expressed on a DW basis,and did not change on an area or protein basis(Table6). Discussion

Our?nding that B-de?cient symptoms started at the apex and in the actively growing leaves is consistent with the result that B is phloem immobile in lime(Citrus aurantifolia)(Konsaeng et al.,2005).Evidence suggests that the predomi-nant role of B is in the formation of primary cell walls,where it cross-links the pectic polysacchar-ide rhamnogalacturonan II(RG-II)(Brown and Hu, 1997;O’Neill et al.,2004).The borate cross-linked RG-II has been shown to be essential for normal plant growth using the Arabidopsis thaliana mur1 mutant,in which the amount of borate cross-linked RG-II is reduced(O’Neill et al.,2001).Boron de?ciency in pumpkin(Cucurbia moschata)plants results in a decrease in growth and is accompanied by cell wall thickening and a decrease in borate cross-linking of RG-II.Supplying borate to the B-de?cient pumpkin plants restores normal growth, reduces wall thickening,and increase the amount of RG-II cross-linking to normal levels(Ishii et al., 2001).Our?nding that B de?ciency resulted in a decrease in growth and an increase in speci?c leaf weight(Table1)also can be explained by the role of B in the formation of primary cell walls.

Table3.Effects of B supply on CO2assimilation,stomatal conductance,and intercellular CO2concentration in citrus leaves

B treatments(m M)CO2assimilation

(m mol mà2sà1)Stomatal conductance

(mmol mà2sà1)

Intercellular CO2

concentration(m mol molà1)

0 1.7070.26b44.975.6b353.878.9a

2.58.3970.52a86.675.5a258.6716.1b

108.7670.65a96.1712.5a266.7713.4b

Data are means7standard errors(n?8).Within a column,values followed by different letters are signi?cantly different at P o0.05. Table4.Effects of B supply on activities of ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco),NADP-glyceraldehyde-3-phosphate dehydrogenase(NADP-GAPDH),and stromal fructose-1,6-bisphosphatase(FBPase)in citrus leaves

B treatments(m M)Activities on an area basis(m mol mà2sà1)Activities on a protein basis(m mol gà1protein sà1)

Rubisco NADP-GAPDH FBPase Rubisco NADP-GAPDH FBPase

0 6.070.7b9.170.5b 2.370.4b0.970.1b 1.370.1b0.3370.06b 2.541.871.8a112.474.2a8.070.6a 4.470.2a11.870.4a0.8470.06a 1040.373.9a100.9713.5a8.671.2a 4.570.4a11.271.5a0.9570.14a Data are means7standard errors(n?5).Within a column,values followed by different letters are signi?cantly different at P o0.05. Citrus seedlings under boron de?ciency1335

Boron de?ciency decreased the root/shoot ratio of spinach (Spinacea oleracea )(Bottrill et al.,1970),tobacco (Nicotiana tabacum )(Camacho-Cristo ′bal and Gonza ′lez-Fontes,1999),and lupin (Lupinus albus )(Alves et al.,2006).Cakmak et al.(1995)reported that B de?ciency decreased sun?ower root/shoot ratio,especially under high light.Withholding of B for 3or 5weeks also decreased cotton (Gossypium hirsutum )root/shoot ratio (Zhao and Oosterhuis,2003).In contrast,a 15-day B-de?cient treatment increased cotton root/shoot ratio (Rosolem and Costa,2000).Our results

G l u (m m o l m -2)

(m m o l m -2)

G l u (μm o l g -1 D W )02550

100

F r u 051015

F r u (μm o l g -1 D W )

050

100

150S u c 0102030

S u c (μm o l g -1 D W )

010*******

400G l u + F r u + S u c 02040

G l u + F r u + S u c (μm o l g -1 D W )0100200

300

400

S t a r c h (m m o l h e x o s e m -2)

(m m o l h e x o s e m -2)

010*******

400

G l u + F r u + S u c (μm o l g -1 D W )

500100015002000

2500B treatments (μM)

0.5

10T N C 0

100200300

400

0 2.5

T N C (μm o l g -1 D W )

5001000

15002000

2500Figure 1.Effects of boron (B)supply on contents of glucose (Glu,A and G),fructose (Fru,B and H),sucrose (Suc,C and I),Glu+Fru+Suc (D and J),starch (E+K),total nonstructural carbohydrates (TNC,F and L)of citrus leaves on a leaf area (A –F)or DW (G –L)basis.Bars represent means 7standard errors (n ?4or 5).Different letters above standard error bars indicate signi?cant difference at P o 0.05.

S.Han et al.

1336

showed that B-de?cient treatment increased citrus root/shoot ratio(Table1).Similar results have been obtained in Dittrichia viscosa(Stavrianakou et al.,2006)and sun?ower(Kastori et al.,1995). Thus,it appears that the in?uence of B de?ciency on the root/shoot ratio depends on the duration of B deprivation,light intensity,and plant species or cultivar.

Our?nding that starch content was higher in B-de?cient than in control leaves is similar to those observed in cotton(Zhao and Oosterhuis,2002)and tobacco leaves(Camacho-Cristo′bal et al.,2004). The elevated starch level in B-de?cient leaves in this study may result from the decreased demand for reduced C in growing sink tissues due to growth inhibition.Because starch is the major storage carbohydrate in citrus leaves(Iglesias et al.,2002), the corresponding increases in speci?c leaf weight and starch in B-de?cient leaves(Table1,Figure1E and K)suggest that starch accumulation may contribute to speci?c leaf weight.

Our results showed that B de?ciency increased the concentration of soluble sugars on an area basis (Figure1D),but had no effect on the content of soluble sugars on a DW basis(Figure1J).This indicates that the higher content of soluble sugars on an area basis is probably associated with the increase in speci?c leaf weight(Table1).In tobacco,both glucose and fructose contents were 4.8and 2.6times higher in B-de?cient than in control leaves,respectively,whereas sucrose con-centration did not change(Camacho-Cristo′bal et al.,2004).In this study,B de?ciency increased the contents of glucose and fructose(Figure1A,B, G and H),but decreased the concentration of sucrose(Figure1C and I).In B-de?cient mustard leaves,there was a greater increase in reducing sugars compared with non-reducing sugars,and this was attributed to an increase in invertase activity (Ramchandra et al.,1987).Similarly,we found that B de?ciency decreased sucrose content on a leaf area basis,and increased acid invertase activity and hexose concentration on an area basis in Citrus grandis(Chen et al.,unpublished data).Based on these results,we suggest that the increase in glucose and fructose,and the decrease in sucrose in B-de?cient leaves are likely caused by increased invertase activity.

Accumulation of excessive amounts of starch may disrupt chloroplast structure,leading to lower CO2 assimilation and Chl content(Cave et al.,1981). There is often a more pronounced down-regulation of photosynthesis in starch-accumulating species when sink capacity is limiting(Goldschmidt and Huber,1992).Previous work on citrus has shown that girdling and defruiting may increase carbohy-drates,particularly starch,and decrease photo-synthesis(Iglesias et al.,2002).In this study,the starch level observed in B-de?cient leaves may be high enough to affect chloroplast structure and function,as indicated by the decreased Chl a/b ratio(Table2).

It has been suggested that accumulation of hexoses through invertase activity may exceed the capacity of the hexokinase(EC 2.7.1.1)to phosphorylate hexoses and thus trigger down-regulation of the Calvin cycle(Jeannette et al., 2000).Herbers et al.(1996)reported that photo-synthetic gene transcripts(Chl a/b binding protein) in tobacco expressing a yeast invertase gene were down-regulated when the levels of glucose and fructose reached about4.5mmol mà2.In this study, the hexose level observed was far higher than the reported threshold level for hexose regulation of gene expression in tobacco(Herbers et al.,1996). Our?nding that B-de?cient leaves had higher hexose(Figure1A,B,G and H)and lower sucrose (Figure1C and I)contents,and lower activities of photosynthetic enzymes(Table4)is consistent with the feedback repression of photosynthetic genes by excess hexoses.Our data are also similar to the results of Goldschmidt and Huber(1992),who investigated the relationship between feedback inhibition of photosynthesis caused by girdling and

Table5.Effects of B supply on superoxide anion(O2dà)generation,malondialdehyde(MDA)concentration,and electrolyte leakage in citrus leaves

B treatments (m M)O2dàgeneration MDA Electrolyte

leakage(%) On an area basis

(OD580mà2sà1)

On a DW basis

(OD580

kgà1DW sà1)

On an area basis

(m mol mà2)

On a DW basis

(m mol kgà1DW)

00.3170.07a 2.2770.05a17.172.0a124.0714.8a18.372.7a

2.50.1170.01b 1.2070.08b10.171.0b101.8710.2a1

3.370.9b 100.1470.03b 1.3970.27b8.970.9b92.079.3a1

4.270.5b

Data are means7standard errors(n?4or5).Within a column,values followed by different letters are signi?cantly different at P o0.05.

Citrus seedlings under boron de?ciency1337

the type of carbohydrates accumulated in a wide range of plant species.They observed that high-invertase-type species tended to show a marked decrease in maximum photosynthetic capacity,and that all of the strongly inhibited plants did not accumulate high-level sucrose.It was suggested that the appearance of free hexoses can trigger down-regulation of the Calvin cycle and,hence,inhibition of maximum photosynthetic ca-pacity.Since CO 2assimilation was decreased in B-de?cient leaves (Table 3),less of the absorbed light energy was used in electron transport.As a result,there was more excess excitation energy in B-de?cient leaves than in controls,particularly under high light.The excess absorbed photon ?ux can potentially lead to the production of 1O 2and reduced ROS (Chen and Cheng,2003).As expected,O 2d àgeneration increased in B de?ciency compared with B suf?ciency (Table 5).Our ?nding that

S O D (105 u n i t s m -2)

510

15S O D (105 u n i t s g -1 P r o t e i n )0.0.51.01.5

2.0A P X (μm o l m -2 s -1

)

050100150200

250M D A R (μm o l m -2 s -1)0204060

80D H A R (μm o l m -2 s -1)01020

30G R (μm o l m -2 s -1)

05101520

25C A T (μm o l m -2 s -1)

3060

90A P X (μm o l g

-1 P r o t e i n s -1)(μm o l g -1 P r o t e i n s -1)(μm o l g -1 P r o t e i n s -1)(μm o l g -1 P r o t e i n s -1)(μm o l g -1 P r o t e i n s -1)0102030

40S O D

(10

2 u n i t s g -1 D W )

3060

90A P X (n m o l g -1 D W s -1)

(n m o l g -1 D W s -1)

(n m o l g -1 D W s -1)(n m o l g -1 D W s -1)

(n m o l g -1 D W s -1)

04008001200

1600M D A R 048

12M D A R 0200400

600G R 012

3D H A R 0123

4D H A R 0

100200

300

G R 050100150

200B treatments (μM)

0 2.5100

2.5

C A T 0

200400600

800Figure 2.Effects of B supply on activities of superoxide dismutase (SOD,A,G and M),ascorbate peroxidase (APX,B,H and N),monodehydroascorbate reductase (MDAR,C,I and O),dehydroascorbate reductase (DHAR,D,J and P),glutathione reductase (GR,E,K and Q),and catalase (CAT ,F ,L and R)of citrus leaves on a leaf area (A –F),protein (G –L)or DW (M –R)basis.Bars represent means 7standard errors (n ?4or 5).Different letters above standard error bars indicate signi?cant difference at P o 0.05.

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B-de?cient leaves showed higher SOD,APX,MDAR and GR activities(Figure2A–C,E,G–I and K),and AsA content(Table6)on an area or protein basis is consistent with the increased requirement for scavenging ROS.Foyer et al.(1995)reported that overexpression of GR in the chloroplasts of poplar (Populus tremula?Populus alba)caused an ap-proximately doubling of leaf GSH and AsA,and an increase in the reduction state of the GSH pool. Transgenic tobacco plants overexpressing A.thali-ana MDAR gene(AtMDAR1)in the cytosol exhibited a2.1-fold higher MDAR activity and2.2-fold higher AsA level compared with non-transformed ones (Eltayeb et al.,2007).Therefore,higher MDAR (Figure2C,I and O)and GR(Figure2E,K and Q) activities in B-de?cient leaves might explain why B-de?cient leaves displayed higher AsA content on an area or protein basis(Table6).Although B-de?cient leaves remain high in activity of antioxi-dant enzymes,MDA concentration and electrolyte leakage were higher in B-de?cient leaves than in controls(Table5).This indicates that B-de?cient leaves are damaged by oxidative stress,which is possibly the consequence of an over-reduction of the photosystem due to the slow-down of dark reactions resulting from a decrease in Rubisco and other photosynthetic enzymes.Our data are con-sistent with the results of El-Shintinawy(1999) and Cakmak et al.(1995),who observed that B de?ciency in sun?ower resulted in increases in both MDA level of chloroplasts and membrane perme-ability of leaf cells.

In conclusion,B de?ciency leads to an accumula-tion of starch,glucose,and fructose,but not sucrose,in citrus leaves.Assimilation of CO2may be feedback-regulated by the excessive accumula-tion of starch and hexoses via direct interference with chloroplast function and/or indirect repres-sion of photosynthetic enzymes.Although B-de?-cient leaves remain high in activity of antioxidant enzymes,their antioxidant system as a whole does not provide suf?cient protection from oxidative damage.Acknowledgments

This work was supported by the Agricultural Commonweal Industrial Special Fund Program of Department of Agriculture,China(nyhyzx07-023). The authors wish to thank Dr.David https://www.360docs.net/doc/4c287269.html,wor, Crop Performance and Improvement Division, Rothamsted Research,Harpenden,Herts.AL5 2JQ,UK,for language correction and constructive comments on this manuscript.

Appendix A.Supplementary materials Supplementary data associated with this article can be found in the online version at doi:10.1016/ j.jplph.2007.11.002.

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3.5070.28a140.772.9b301.0737.6a 3.0270.38a31.573.9a

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