Shu Yng,Guizhi Ling,Qiuyue Li,Ke Yi,Xinlin Tng,Muqing Zhng,Xiofeng Li,*

a State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangxi Key Laboratory for Sugarcane Biology,College of Agriculture,Guangxi University,Nanning 530004,Guangxi,China

b Institute for New Rural Development,Guangxi University,Nanning 530004,Guangxi,China

Keywords:Sugarcane Manganese toxicity Chlorosis Chlorophyll biosynthesis Gene expression

ABSTRACT Manganese(Mn)toxicity-induced leaf chlorosis limits crop production in acidic soils,but its underlying mechanisms remain unknown.The effects of excessive Mn on chlorophyll(Chl)biosynthesis in sugarcane(Saccharum officinarum L.)leaves were investigated.Under Mn treatment,Chl concentration decreased with Mn accumulation and chlorosis appeared in expanding leaves.Before that,levels of the initial Chl precursor 5-aminolevulinic acid(ALA)and its downstream intermediates decreased,whereas magnesium-protoporphyrin IX monomethyl ester(MgPME)accumulated.Overaccumulation of Mn in leaves downregulated the ALA biosynthetic gene GluTR(encoding glutamyl-tRNA reductase)and MgPME conversion gene MgPMEC(encoding MgPME cyclase),upregulated the ALA biosynthesis inhibitor FLU(encoding FLUORESCENT),but had no significant effect on the expression of other Chl biosynthetic genes.The above Mn-induced changes of Chl precursors and expression of corresponding genes commenced before the Chl decline and leaf chlorosis,and were reversed by ALA supplementation.Thus,excessive Mn-induced chlorosis in sugarcane is mediated by a Chl-biosynthesis disorder resulting from the inhibition of ALA synthesis and MgPME conversion.

1.Introduction

Manganese(Mn)toxicity is the second-most important factor limiting crop production in acid soils[1,2].Excessive Mn can interfere with the absorption,translocation,and utilization of other essential elements,inhibit enzyme activity,induce oxidative damage,and ultimately inhibit photosynthesis and plant growth[3,4].Mn tends to accumulate in plant shoots and induces visible symptoms of Mn toxicity in the leaves,such as chlorosis in maize(Zea mays L.)[5],bush bean(Phaseolus vulgaris L.)[6],and barley(Hordeum vulgare L.)[7].We previously showed[8]that sugarcane(Saccharum officinarum L.)grown in acidic soils of southern China suffered from severe leaf chlorosis resulting from excessive soil Mn.The planting area of sugarcane in regions exhibiting leaf chlorosis accounts for approximately 40% of the total sugarcane acreage in China.The chlorosis has resulted in a 23%-40% reduction in cane production[8].

It has been hypothesized[9]that excessive Mn-induced chlorosis is mediated by chlorophyll(Chl)breakdown resulting from Chl photobleaching and/or chloroplast photooxidative disruption.Previous studies[6,10]indicated that excessive Mn-induced chlorosis was caused not by insufficient production of Chl but by photooxidation or Chl bleaching.In common bean(Phaseolus vulgaris L.)[11],sugar maple(Acer saccharum Marsh.)and red maple(Acer rubrum L.)[12],light-dependent Chl destruction or photobleaching is observed in excessive Mn-induced leaf chlorosis.These studies emphasize the irreplaceable role of high light exposure,particularly sunlight,in Mn-induced chlorosis,because high levels of light not only exacerbate foliar Mn overaccumulation but also provoke oxidative stress[9,12].However,accumulating evidence[8,13]indicates that photooxidative bleaching cannot well explain Mninduced chlorosis under low light exposure.In fact,Mn-induced porphyrin accumulation has been reported to inhibit Chl biosynthesis in tobacco(Nicotiana tabacum L.)callus[14],blue-green algae(Anacystis nidulans)[15],and detached leaves of barley[16].Thus,whether excessive Mn directly influences Chl biosynthesis in higher plants remains unknown.

The Chl biosynthetic pathway has been deciphered in plants[17].The initial Chl biosynthetic precursor 5-aminolevulinic acid(ALA)is synthesized from glutamate by glutamyl-tRNA reductase(GluTR).ALA is condensed by ALA dehydratase(ALAD)to form porphobilinogen(PBG)and finally oxidized by protoporphyrinogen IX oxidase(PPOX)to form protoporphyrinogen IX(Proto).At a branch point,Proto is converted into Mg-protoporphyrin IX(MgProto)by magnesium chelatase(MgCh)or to heme by ferrous chelatase(FeCh).Then Mg-protoporphyrin IX methyltransferase(MgMT)transfers MgProto to Mg-protoporphyrin IX monomethyl ester(MgPME),which is oxidized by Mg-protoporphyrin IX monomethyl ester cyclase(MgPMEC)to form divinyl protochlorophyllide(Pchlide).Finally,Pchlide is consecutively catalyzed by NADPHprotochlorophyllide oxidoreductase(POR),3,8-divinyl-protochlorophyllide a 8-vinyl reductase(DVR),and Chl synthase(ChlS)to form the Chl pool.Because Chl biosynthesis is a complex and tightly coordinated process in plants,any metabolic disorder in intermediates,such as inhibited synthesis and/or blocked downstream metabolism,will interfere with the normal metabolic flow.ALA synthesis as well as the branch point between Chl and heme are major regulatory points for Chl biosynthesis[17].Given that all Chl biosynthetic precursors from the final steps are phototoxic when present in excessive,plants have evolved heme-and FLUmediated feedback control of ALA synthesis to regulate Chl biosynthesis[17,18].Identifying the effects of excessive Mn on Chl biosynthesis will thus shed light on Mn-induced chlorosis.

Sugarcane,a tropical crop,is used as a raw material in sugar,fiber,and commercial biofuel production worldwide[19,20].In recent years,sugarcane grown in acidic soils has suffered severe Mn toxicity-induced chlorosis,resulting in large losses in productivity[8].However,the mechanism underlying Mn-induced chlorosis in sugarcane is unknown.In this study,the effects of Mn on accumulation of the Chl biosynthetic precursor ALA and its downstream intermediates,and Chl biosynthesis-associated gene expression were investigated.The mechanisms underlying the effects of Mn on Chl biosynthesis are discussed.

2.Materials and methods

2.1.Plant cultures and treatments

Surface-sterilized single-bud sets of the Mn-sensitive sugarcane genotype YC58-21,were germinated at 25°C for a week[21].Seedlings of similar size were precultured in 5-L plastic pots(4 seedlings per pot)filled with aerated 1/5-strength Hoagland nutrient solution.The pH of the solution was adjusted frequently to 5.5 with 0.1 mol L-1HCl/KOH and the solution was renewed every two days.Plants were grown in an environmental chamber with 16 h(30°C)/8 h(25°C)day/night regime,75%-85%relative humidity and about 800μmol m-2s-1photosynthetically active radiation at canopy level.After another 15-20 days,seedlings of uniform size at third-leaf stage(with three fully expanded leaves)were used for subsequent experiments.

To monitor the dynamic processes of Mn accumulation and chlorosis,the seedlings were grown in the above nutrient solution containing 0.5 mmol L-1MnCl2until sixth-leaf stage.Young leaves(YL,expanding leaves),mature leaves(ML,the last fully expanded leaves),and older leaves(OL,leaves fully expanded before the ML)were harvested after Mn treatment for 0,4,8,16,and 24 days(corresponding to third-,fourth-,fifth-,and sixth-leaf stages,respectively).Mn and Chl concentrations in the leaf blades were determined as described below.The means of Mn and Chl concentrations in leaves were visualized by electronic fluorescent pictograph(eFP)and heat maps using open-source application TBtools(v 1.0987)[22].

To investigate the effects of excessive Mn on Chl synthesis and corresponding processes,seedlings were cultured in 1/5 Hoagland nutrient solution(pH 5.5)with 0(control,Con)or 0.5(Mn)mmol L-1MnCl2for 0,2,4,8,16,and 24 days.After photographing,the concentrations of Mn,Chl,and Chl biosynthetic precursors as well as the expression of associated genes in expanding leaves were determined as described below.

To investigate the significance of Chl biosynthetic precursors in leaf chlorosis and Mn-induced alteration of Chl biosynthesis,roots of seedlings were exposed to ALA and Mn-free nutrient solution(Con),nutrient solution with 120μmol L-1ALA(ALA),1.0 mmol L-1MnCl2(Mn),or 1.0 mmol L-1MnCl2plus 120μmol L-1ALA(ALA+Mn).The concentrations of Chl and the endogenous Chl precursors and associated gene expression in the expanding leaves were measured after treatment for 8 days.

2.2.Determination of Mn

Leaf samples were oven-dried at 65 °C to constant weight and digested with concentrated HNO3at 140°C.The Mn concentrations in the digests were determined by atomic absorption spectrometry(AAS,PinAAcle 900 T,PerkinElmer,Waltham,MA,USA).

2.3.Determination of Chl

Chl of fresh leaf samples(0.10 g)was extr acted with 20 mL 80%(v/v)aqueous acetone solution in the dark until complete bleaching was achieved.The absorbances of the extracts were measured spectrophotometrically at 645 and 663 nm(UV2600,Shimadzu,Kyoto,Japan).Total Chl concentration was calculated following Arnon[23].

2.4.Determination of ALA and PBG

Measurement of ALA concentration followed Tewari and Tripathy[24].Fresh leaf samples(2.00 g each)were fully homogenized in a prechilled mortar and pestle with 5 mL of chilled 1 mol L-1sodium acetate buffer(pH 4.6).The homogenate was centrifuged at 12,000×g for 10 min(4°C)using a high-speed refrigerated centrifuge(H2050R-1,Cence,Changsha,Hunan,China).A 3-mL aliquot of the supernatant was then mixed with 150μL of acetylacetone and heated in a boiling water bath for 10 min.After cooling to room temperature,an equal volume of modified fresh Ehrlich’s reagent solution(consisting of 42 mL glacial acetic acid,8 mL 70%(v/v)perchloric acid,and 1.0 g dimethylaminobenzaldehyde)was added and the mixture was vortexed for 2 min.After 10 min of incubation,the absorbance of the mixture was measured spectrophotometrically at 555 nm.ALA concentration was calculated using a standard curve based on ALA reference standards.Similarly,PBG was extracted with 0.6 mol L-1Tris-HCl buffer(pH 8.2,with 0.1 mol L-1EDTA)and measured using Ehrlich’s reagent,and the concentration was calculated following Peng et al.[25].

2.5.Determination of heme

The concentrations of heme in the expanding leaves were determined following Wu et al.[26].Fresh leaf samples(2.00 g each)were ground in liquid nitrogen and washed with 8 mL of 90%alkaline acetone(v/v,alkalizing with 0.1 mol L-1NH4OH)and then centrifuged at 8000×g for 10 min(4 °C).The washing steps were repeated until the chlorophyll was completely removed.Then the final pellets were extracted in 5 mL of acidified acetone(acetone:dimethyl sulfoxide:concentrated hydrochloric acid 20:4:1,v/v/v)for 30 min and then centrifuged at 8000×g for 10 min(4 °C).The supernatant was mixed with 0.7 mL absolute ethanol and absorbance was determined at 386 nm by spectrophotometer.Heme concentrations were calculated using a standard curve of heme.

2.6.Determination of Proto,MgProto,and Pchlide

The concentrations of Proto,MgProto and Pchlide in the leaves were determined following Hodgins and Van Huystee[27].Fresh leaf samples(0.20 g each)were ground in liquid nitrogen and extracted in 25 mL of 80% alkaline acetone(v/v,alkalized with 0.1 mol L-1NH4OH).The extraction was incubated in the dark until the tissue was bleached.After centrifugation at 15,000×g for 10 min(4 °C),the absorbance of the supernatant was measured spectrophotometrically at 575,590,and 628 nm and the concentrations were calculated using the corresponding formulas described previously[28].

2.7.Determination of MgPME

The concentration of MgPME in the leaves was determined by the method of Rebeiz et al.[29].Fresh leaf samples(0.20 g each)were ground in liquid nitrogen and extracted three times in 5 mL of 90% alkaline acetone(v/v,alkalized with 0.1 mol L-1NH4OH).After centrifugation at 15,000×g for 10 min(4 °C),the supernatants were collected,combined,and brought to 25 mL.The supernatants were then extracted three times with an equal volume of n-hexane.MgPME in the hexane-extracted acetone residue solvent mixture(bottom phase)was quantified using a fluorescence spectrophotometer(LS-55,PerkinElmer).The excitation and emission spectra were 440 nm and 595 nm respectively,with a slit width of 4 nm.

Relative concentration was used to express the levels of Chlbiosynthetic precursors and was calculated as the ratio of the concentrations in Mn(and/or ALA)-treated plants to those in controls.

2.8.Total RNA extraction and gene expression analysis

After termination of the experiments,the expanding leaves were collected,frozen immediately in liquid nitrogen,and stored at-80 °C until use.Total RNA was extracted using the FastPure Plant Total RNA Isolation Kit(Vazyme,Nanjing,Jiangsu,China).cDNA was synthesized using the HiScript III RT SuperMix for qPCR(+gDNA wiper)Kit(Vazyme).Quantitative real time polymerase chain reaction(qRT-PCR)assays were performed using ChamQ Universal SYBR qPCR Master Mix(Vazyme)in an automated real-time PCR thermal cycler(qTOWER2.2,Analytik Jena AG,Jena,Germany).The amplification program was initiated with an initial incubation at 95 °C(10 min),followed by 35 cycles of 95 °C(15 s)and 60 °C(60 s)plus a melting curve protocol.All of the experiments were repeated in triplicate.Gene-specific primers(Table 1)were designed based on full-length transcriptome sequencing data(unpublished data in our laboratory)and synthesized by Sangon Biotech(Shanghai,China).Theβ-actin gene was used as a reference gene for gene expression data normalization.The 2-ΔΔCTmethod[30]was used to quantify relative fold changes in the expression patterns of selected genes by comparing the expression levels between different times(2,4,or 8 days versus 0 day)in the time-course experiment,or different treatments(Mn,ALA,or ALA+Mn versus Con)in the short-term experiment.

Table 1Sequences of gene-specific primers used for qRT-PCR assays.

2.9.Statistical analysis

All experiments were arranged in a randomized,complete block design with 3 replicates.Treatment means were compared by analysis of variance(ANOVA)and Duncan’s multiple-range test.

3.Results

3.1.Mn-induced reduction of Chl and subsequent chlorosis in sugarcane

Compared to controls,a significant increase in leaf Mn concentration started at 2 days of Mn treatment and further increased over the treatment time(Fig.1A).The Chl concentrations in expanding leaves of Mn-treated plants were comparable to those of controls within 4 days.However,when the treatment duration was prolonged to 8,16,and 24 days,the Chl concentrations in Mn-treated plants decreased to 1.98,1.63,and 0.69 mg g-1FW,with respective decreases of 15%,29%,and 70%(Fig.1B).Expanding leaves and even the last fully expanded leaf showed chlorosis after 24 days of Mn treatment(Fig.1C).

The dynamical process of Mn accumulation and Mn-induced chlorosis in sugarcane were further visualized in Fig.2.Although Mn accumulated gradually in all leaves with increasing Mnexposure duration,leaves of different ages(LY,ML,and OL)showed comparable levels of Mn accumulation during the third-leaf stage after Mn-treatment for 0 or 4 days.When the Mn-exposure period extended to 8,16,24 days,and when the seedlings grew to the fourth-,fifth-,and sixth-leaf stages respectively,the level of Mn accumulation increased with leaf age:YL＜ML＜OL(Fig.2A).The leaf blades of LY,ML,and OL showed similar Chl levels with no symptom of chlorosis during the third-leaf stage.Mild chlorosis appeared in the seedlings at the fourth-leaf stage(8 days)and was aggravated to marked chlorosis at the fifth-leaf age(16 days),and to severe,irreversible chlorosis at the sixth-leaf stage(24 days).Chlorosis developed only in newly emerging leaves,such as YL of seedlings exposed to Mn for 8-24 days and ML(L6)at the sixthleaf stage(Fig.2B).

3.2.Effect of Mn on Chl biosynthetic precursors

The steady-state levels of ALA,the initial precursor of Chl biosynthesis,and its downstream intermediates were significantly influenced by Mn treatment(Fig.3).The relative concentration(%of control,same as below)of ALA decreased significantly over the course of treatment,with a reduction of 37%after 24 days(Fig.3A).The ALA downstream intermediates including PBG,heme,Proto,MgProto,and Pchlide showed similar time-dependent decreases of respectively 35%,45%,57%,58%,and 58% by the end of the experiment(Fig.3A-C).By contrast,the relative concentrations of MgPME increased by respectively 32%,52%,70%,and 117%after Mn-treatment for 4,8,16,and 24 days(Fig.3D).The changes in the levels of ALA and its downstream intermediates(except heme)began after 2 or 4 days of Mn treatment,preceding the Mninduced decline in leaf Chl concentration(starting at 8 days)(Figs.1,3).

Fig.1.Mn-induced suppression of chlorophyll and chlorosis in sugarcane.Seedlings were treated with 0(Con,Control)or 0.5 mmol L-1(Mn)MnCl2 for 0-24 days.Mn(A)and Chl(B)concentrations in expanding leaves were determined.Mn-induced chlorosis was also photographed after treatment for 24 days(C).Values are means±standard deviation(SD,n=3).Different letters indicate that values are significantly different at P＜0.05 by Duncan’s multiple-range test.

Fig.2.Electronic fluorescent pictograph and heat maps of leaf Mn and Chl concentrations among sugarcane leaves.Seedings were treated with 0.5 mmol L-1 MnCl2 for 0,4,8,16,and 24 days,Mn and Chl concentrations in the blades of young leaves(YL,expanding leaves),mature leaves(ML,last fully expanded leaves),and older leaves(OL,leaves fully expanded before the ML)were measured.Images show the means(n=3)of Mn(A)and Chl(B)in leaves at the leaf stages.L1-L6 are labels for fully expanded leaves ordered from old to young.

3.3.Effect of Mn on the transformations of Chl biosynthetic precursors

Transformations of ALA and its downstream intermediates under excessive Mn stress were evaluated(Fig.4).Mn treatment showed no effect on the transformation of ALA to its downstream product PBG,because the concentration ratios of PBG/ALA did not change during Mn treatment for 0-24 days(Fig.4A).The allocation of metabolic flow at the branch point between Chl and heme biosynthesis was also unchanged by Mn-stress,given that the concentration ratios of heme/Proto and MgProto/Proto were unaffected(Fig.4A,B).By contrast,the concentration ratios of Pchlide/MgProto,and correspondingly the conversion ratio of MgProto to Pchlide,decreased significantly throughout the Mntreatment process(Fig.4B).Simultaneously,the Mn-induced repression of the conversion of MgProto to Pchlide started at 2 days of Mn-treatment,preceding the Mn-induced decline in leaf Chl(Figs.2,4B).

Fig.3.Effects of Mn on relative concentrations of Chl biosynthetic precursors.Seedings were treated with 0(Con)or 0.5 mmol L-1(Mn)MnCl2 for 0-24 days,after which the relative concentrations(%of control)of ALA and PBG(A),Heme and Proto(B),MgProto and Pchlide(C),and MgPME(D)in the expanding leaves were measured.Values are means±SD(n=3).Different letters on the same line indicate that the values are different at P＜0.05 by Duncan’s multiple-range test.

3.4.Effect of excessive Mn on the expression of genes encoding Chl biosynthetic enzymes

The expression of representative genes during Mn-induced chlorosis in newly emerging leaves of sugarcane seedlings was measured.The expression of GluTR decreased rapidly and markedly in response to Mn treatment,by 54% at 2 days and 87% at 8 days(Fig.5).The decline in GluTR expression preceded the changes in the levels of Chl,ALA,and its downstream intermediates,as well as Mn-induced chlorosis(Figs.1,3,and 5).By contrast,the expression of ALAD was not affected after Mn-treatment for 8 days(Table 2).Thus,the effects of excessive Mn on Chl biosynthesis were initiated by inhibition of GluTR-catalyzed ALA biosynthesis,followed by a reduction in leaf concentrations of ALA and its downstream intermediates.

The expression of PPOX,FeCh,MgCh,and MgMT after treatment with 0.5 mmol L-1MnCl2for 0,2,4,and 8 days was shown in Table 2.Excessive Mn did not affect the transcription of these genes(relative expression about 1.0),during Mn treatment for 0-8 days.By contrast,MgPMEC showed pronounced timedependent downregulation(Fig.5).After Mn-exposure for 0,2,4,and 8 days,MgPMEC was downregulated from 1.01 to 0.93,0.37 and 0.11,with respective decreases of 8%,63%,and 89%.The significant decrease in MgPMEC expression induced by Mn occurred simultaneously with the accumulation of MgPME and decrease in Pchlide at 4 days,but preceded the significant decrease in Chl concentration(8 days)(Figs.1,3,and 5).

Although FLU expression in Mn-treated seedlings was comparable to that in controls after 2 days,FLU was upregulated respectively 2.4-and 7.7-fold after 4 and 8 days(Fig.5).As expected,the upregulation of FLU occurred simultaneously with the accumulation of MgPME but before the decrease in Chl.qRT-PCR analysis showed that the expression of POR,DVR,and ChlS in the later steps of Chl biosynthesis was not affected by Mn treatment for 0-8 days(Table 2).

3.5.Effect of ALA supplementation on Mn-induced chlorosis

As shown in Fig.6,leaf ALA concentration in ALA-treated seedlings was comparable to that in control plants.However,in the presence of 1.0 mmol L-1Mn,leaf ALA level increased significantly after ALA treatment for 8 days,suggesting that exogenous ALA could compensate for the Mn-induced depletion of endogenous ALA in leaves(Fig.6A).The increase in endogenous ALA in leaves resulted in a significant increase in the Chl concentration(Fig.6B).

Fig.4.Effects of Mn on the concentration ratios of chlorophyll precursors.The concentration ratios of PBG/ALA and Heme/Proto(A),MgProto/Proto and Pchlide/MgPME(B)in expanding leaves after seedlings were treated with 0.5 mmol L-1 MnCl2 for 0-24 days.Values are means±SD(n=3).Different letters on the same line indicate that the values are different at P＜0.05 by Duncan’s multiple-range test.

Fig.5.Effects of Mn on expression of genes encoding chlorophyll biosynthetic enzymes.Seedlings were treated with 0.5 mmol L--1 MnCl2 for 0,2,4,and 8 days.qRT-PCR assays were used to measure the relative gene expression of GluTR,FLU,and MgPMEC in expanding sugarcane leaves.Values are means±SD(n=3).Different letters on the same line indicate that the values are different at P＜0.05 by Duncan’s multiple-range test.

Together with the level of leaf ALA,those of ALA downstream intermediates also increased(Fig.6A).ALA supplemented seedlings(ALA+Mn)showed higher levels of Proto,MgProto and Pchlide than Mn-treated plants(Mn).By contrast,the Mn-induced accumulation of MgPME declined after ALA supplementation.Similarly,the Mn-induced upregulation of FLU and downregulation of MgPMEC were suppressed by ALA supplementation(Fig.7).However,the Mn-induced downregulation of GluTR was unaffected by ALA supplementation.These effects of ALA on Chl precursors and the expression of the corresponding genes were accompanied by elevation of Chl level in leaves(Figs.6,7).Thus,Mn-induced changes in the expression of genes encoding enzymes implicated in MgPME conversion and its FLU-depended feedback control of ALA synthesis were reversed by exogenous ALA.

4.Discussion

Previous studies[6,9]found that chlorosis was the result of excessive Mn-induced photooxidation and subsequent Chl photobleaching and/or chloroplast disruption.Thus,leaves with greater Mn accumulation are at higher risk for oxidative stress and chlorosis[31].In the present study,the increase of leaf Mn accumulation with age(Fig.2A).This may have occurred because plants tend to distribute Mn into expanded,older leaves in parallel with their transpiration rate in the presence of excessive Mn[32].By contrast,Mn-induced chlorosis in sugarcane developed in a different pattern from Mn accumulation and occurred only in newly emerging leaves,rather than in previously expanded older leaves with higher Mn accumulation(Fig.2B).This observation cannot be explained by Mn-induced photooxidation alone,although leaves of different ages may have different detoxification capacities[4].Ratoon sugarcane seedlings emerge with severe Mn-induced chlorosis in naturally Mn-contaminated acid soil,and artificial cultured plantlets that emerged from cane stalks with a high Mn content also showed Mn-induced chlorosis even under weak illumination at a light intensity of 45μmol m-2s-1[8].In addition,in agreement with a previous report[16],surplus Mn inherited from paternal stalks delayed the greening of etiolated sugarcane seedlings during de-etiolation(unpublished result).The possible deleterious effects of Mn on Chl biosynthesis should be considered in evaluating the mechanisms underlying Mn-induced chlorosis.

Table 2Effect of Mn on the expression of genes encoding the chlorophyll biosynthetic enzymes in leaves.

Fig.6.Effects of ALA supplementation on the relative concentrations of Chl biosynthetic precursors(A)and Chl(B)in the leaves.Seedlings were treated with 0(Con),120μmol L-1 ALA(ALA),1 mmol L-1 MnCl2(Mn)or 1 mmol L-1 MnCl2 plus 120μmol L-1 ALA(ALA+Mn)for 8 days.Values are means±SD(n=3).Different letters on columns indicate that the values are different between the treatments at P＜0.05 by Duncan’s multiple-range test.

Fig.7.Effects of ALA supplementation on Mn-induced gene expression of the enzymes involved in the Chl biosynthesis.Seedlings were treated as described in Fig.6.The relative expression of GluTR,MgPMEC,and FLU in the expanding leaves was quantified.Values are means±SD(n=3).Different letters on columns indicate that the values are different between the treatments at P＜0.05 by Duncan’s multiple-range test.

Excessive Mn reduced the Chl level in sugarcane and caused chlorosis in newly emerging leaves(Figs.1,2).Leaf Mn overaccumulation reduced the level of the initial precursor of Chl biosynthesis ALA and its downstream intermediates(PBG,Proto,heme,MgProto,and Pchlide),but promoted accumulation of MgPME(Fig.3).Excessive Mn blocked the conversion of MgPME to protochlorophyllide:the concentration ratios of Pchlide/MgProto decreased significantly but did not affect the transformations of other intermediates(Fig.4).On the other hand,excessive Mn downregulated the ALA biosynthetic gene GluTR and the MgPMEconversion gene MgPMEC and upregulated the expression of the ALA biosynthesis feedback inhibitory regulator FLU(Fig.5).The changes in Chl biosynthetic precursor concentration and gene expression levels occurred before the decrease in Chl concentration(Figs.1-5).Moreover,the Mn overaccumulation-mediated effects were reversed by addition of ALA to the culture medium(Figs.6,7).These findings demonstrate that Mn-induced deprivation of Chl might be a result of the inhibition of ALA and its downstream intermediate biosynthesis and the excessive accumulation of MgPME(Fig.8).

As the initial precursor,ALA synthesis is a major regulatory point for Chl biosynthesis to adapt to stress[17].In this study,ALA level as well as the GluTR transcript level decreased rapidly with increasing Mn accumulation in leaves(Figs.3,5).The Mninduced decrease in leaf ALA was not caused by the accelerated conversion of ALA into PBG,given that the concentration ratio of PBG/ALA and ALAD expression were unchanged prior to the decrease in the Chl level(Fig.4;Table 2).Similar results have been reported in oilseed rape(Brassica napus L.)[33]:salt stress reduced Chl levels with a reduction of ALA,its net synthesis,and the expression of the key ALA biosynthesis genes GluTR and the ALA catabolic gene ALAD.In rice(Oryza sativa),reduced Chl biosynthesis in water-stressed seedlings was a result of downregulation of ALA,GluTR,and ALAD[34].These results suggest that ALAsynthesis reaction is an important Mn-influenced step in Chl biosynthesis.

Fig.8.Schematic diagram of excessive Mn-inhibited Chl biosynthesis in sugarcane seedlings.ALA synthesis and MgPME conversion in the Chl biosynthesis pathway are the steps inhibited by excessive Mn.Excessive Mn initially suppresses GluTR expression and induces a subsequent reduction in the levels of ALA and its downstream intermediates(PBG,Proto,heme,and MgProto).Mn-induced suppression of MgPMEC expression blocks the conversion of MgPME to Pchlide and leads to overaccumulation of MgPME,which results in a reduction in the levels of Pchlide and Chl.The accumulation of MgPME further aggravates the inadequate formation of ALA via the FLU-dependent feedback inhibition of ALA synthesis.Thus,the concentration of Chl in leaves decreases and the sugarcane seedlings show macroscopic chlorosis due to the overaccumulation of Mn.

Following ALA synthesis,the branch point between Chl and heme synthesis is another adjustment of Chl biosynthesis in response to changing environmental conditions[17].Salinityinduced chlorosis in cucumber(Cucumis sativus L.)results from heme accumulation,upregulation of FeCh,and down-regulation of genes involved in Chl biosynthesis,as well as redirection from Chl branch to heme branch[26].Cold-induced chlorosis in wheat(Triticum aestivum)line FA85 was associated with redirection of Proto from heme to Chl synthesis[35].However,we ruled out the involvement of metabolic redirection in Mn-induced chlorosis,given that the allocation of metabolic flow at the branch point and the expression of PPOX,FeCh,CHLD,CHLH,CHLI,and MgMT were unaffected by Mn stress(Fig.4;Table 2).

In the present study,MgPMEC expression decreased gradually as the Mn-treatment duration increased(Fig.5).Parallel to the downregulation of MgPMEC,the conversion of MgPME was blocked,markedly increasing MgPME accumulation and reducing the Pchlide level(Figs.3-5).In Arabidopsis,MgPMEC-deletion mutant display severe chlorosis and significant accumulation of MgPME in young leaves[36].The effects of Mn on the conversion of MgPME preceded the reduction of Chl concentration(Fig.1).This finding is inconsistent with a previous report[16]that the conversion of Proto into MgProto was blocked by excessive Mn.Our results are supported by other findings that downregulation of MgPMEC led to inhibition of Chl synthesis in rice under salt[37]and water stress[34],and resulted in excessive accumulation of MgPME and severe chlorosis in tobacco[38].These results indicate that MgPME conversion is another important Mn-influenced step in Chl biosynthesis.

FLU-dependent feedback control of ALA synthesis is reportedly[39,40]activated by the accumulation of Pchlide and MgPME.In the present study,Mn exposure induced overaccumulation of MgPME in leaves,upregulated FLU,and decreased GluTR expression and ALA level(Figs.3,5).Mn-induced expression of FLU and GluTR,and the decrease in the ALA level,were reversed by suppression of MgPME accumulation by addition of ALA to the culture medium(Figs.6,7).These results suggest that in sugarcane,Mn-induced overaccumulation of MgPME aggravates inadequate ALA formation caused by FLU-dependent feedback inhibition of ALA synthesis.

The finding in the present study that Mn toxicity-induced chlorosis involves the inhibition of chlorophyll biosynthesis may guide new sugarcane cultivar breeding.Improving Chl biosynthesis,especially ALA synthesis and MgPME conversion,will increase sugarcane production potential in Mn-toxic acidic soils.ALA is also recognized as a natural growth regulator that could improve plant adaptation to environmental stresses[26,41].The reversal in this study of Mn-induced chlorosis in sugarcane by ALA supplementation suggests the potential of ALA application to alleviate Mn toxicity-induced chlorosis in sugarcane grown in acidic soils.

5.Conclusions

We have identified in sugarcane seedlings a novel mechanism of Chl biosynthesis inhibition caused by excessive Mn,which involves the inhibition of ALA synthesis and MgPME conversion.Excessive Mn downregulates GluTR and reduces the levels of ALA and its downstream intermediates(including PBG,heme,Proto,MgProto,Pchlide).Simultaneously,excessive Mn blocks the conversion of MgPME by downregulating MgPMEC and causes an overaccumulation of MgPME,which in turn further aggravates the inadequate ALA synthesis by FLU-dependent feedback control.Finally,excessive Mn reduces the Chl level and induces visible chlorosis in leaves.

CRediT authorship contribution statement

Shu Yang:Conceptualization,Investigation,Methodology,Validation,Formal analysis,Writing-Original Draft,Writing-Review &Editing,Funding acquisition.Guizhi Ling:Formal analysis,Writing-Review & Editing,Funding acquisition.Qiuyue Li:Formal analysis,Writing-Review & Editing.Ke Yi:Validation,Methodology,Formal analysis,Writing-Review & Editing.Xinlian Tang:Methodology,Formal analysis,Writing-Review & Editing.Muqing Zhang:Resources,Supervision,Writing-Review & Editing.Xiaofeng Li:Conceptualization,Formal analysis,Project administration,Writing-Review & Editing,Funding acquisition,Writing-Original Draft.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by the National Natural Science Foundation of China(31660593),China Postdoctoral Science Foundation(2020M683620XB),Guangxi Natural Science Foundation(2021GXNSFAA075017,2021GXNSFAA220008),and Science and Technology Major Project of Guangxi(GK2018-266-Z01).