Xinxin Zhang,Min Wang,Tingting Wu,Cunxiang Wu,Bingjun Jiang, Changhong Guo*,Tianfu Han,**

aKey Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang,College of Life Science and Technology, Harbin Normal University,Harbin 150025,China

bMOA Key Laboratory of Soybean Biology(Beijing),Institute of Crop Science,the Chinese Academy of Agricultural Sciences,Beijing 100081,China

Physiological and molecular studies of staygreen caused by pod removal and seed injury in soybean

Xinxin Zhanga,b,Min Wangb,Tingting Wub,Cunxiang Wub,Bingjun Jiangb, Changhong Guoa,*,Tianfu Hanb,**

aKey Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang,College of Life Science and Technology, Harbin Normal University,Harbin 150025,China

bMOA Key Laboratory of Soybean Biology(Beijing),Institute of Crop Science,the Chinese Academy of Agricultural Sciences,Beijing 100081,China

ARTICLEINFO

Article history:

Received 19 February 2016

Received in revised form 3 April 2016

Accepted 6 June 2016

Available online 21 April 2016

Soybean

Seed injury

Staygreen

Source–sink relationship

Leaves provide substances and signals for pod and seed development in soybean.However, the regulatory feedbacks of pod and seed to leaf development remain unclear.We investigated the effects of pod and seed on leaf senescence by conducting pod removal and seed injury experiments.Pod removal and seed injury delayed leaf senescence and caused the staygreen phenotype of leaves.There were dosage effects of pod number on the extent of staygreen in depodded plants.The concentrations of chlorophyll(SPAD value,an index of relative chlorophyll content),soluble protein,and soluble sugar in the leaves of depodded plants were higher than those of intact plants.During seed development,the content of IAA decreased,while that of ABA increased.This trend was more pronounced in intact than in depodded and seed-injured plants.The GA3/ABA ratio decreased gradually in all treatments. The content of GA3was relatively stable and was higher in intact than in depodded plants.The expression levels of four senescence-related genes,GmSARK,GmSGR1,GmCYN1,and GmNAC, declined in depodded or seed-injured treatments and were positively correlated with the number of leaves retained on plants.GmFT2a,the major flowering-promoting gene,was expressed at a higher level while E1,a key flowering inhibitory gene,was expressed at a lower level in depodded than in intact plants.We propose that the pod or seed can regulate leaf development.When the seed is aborted owing to disease infection or pest attack,the leaves stay green because of the absence of the seed signals for senescence.

?2016 Crop Science Society of China and Institute of Crop Science,CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

The leaf not only plays a major role in photosynthesis but also is an organ perceiving environmental signals during plant development.For soybean,dry matter derived from leaf photosynthesis constitutes over 90%of overall dry matter accumulation and is considered a determinant of yield[1].The role of the leaf in signal reception comprises the measurementof day-length changes and production of florigen that is transferred to the shoot apical meristem,resulting in adaptive changes in plant growth and development[2].In soybean,a typical short-day(SD)plant,SD promotes and long day(LD) inhibits flowering and maturation [3,4]. For some photo period-sensitive varieties of soybean,continuous SD is required for the maintenance of post-flowering reproductive status;plants can revert to vegetative from reproductive growth if moved from SD to LD.New branches and leaves in the reverted plants stay green without SD conditions[5].The floral stimuli are transmissible in soybean,and a late-maturing scion could be induced to flower when grafted onto an early-maturing stock with enough leaves[6].

The leaf produces photosynthates and exports them to seeds during the seed-filling period of crops.However,in the last stage of leaf development and seed filling,the function of leaves weakens,accompanied by the degradation of chlorophyll, protein,and nucleic acids and the remobilization and transportation of nutrients to sink organs[7].Leaf senescence is controlled by an intricate genetic network that is programmed and regulated by growth stage and internal and external stimuli [7].Several genes in metabolic and signaling pathways are involved in the senescence process[8–12].Among them,GmSARK plays specific roles in senescence-inducing hormonal pathways [8,9],SGR1 and CYN1 are crucial in chlorophyll degradation,and NAC is a transcription factor for ABA synthesis[10–12].

Abnormal senescence,including premature death and staygreen,is caused by both genetic variation and environmental factors[13–15].Staygreen is an abnormal crop developmental phenotype involving delayed leaf senescence [13].Disruption of chloroplast degradation and related metabolic pathways leads to staygreen leaves[13,16,17].In recent years,a soybean staygreen syndrome called“Zhengqing”, characterized by senescence-delayed leaves,aborted pods, and dead seeds has become a widespread problem in the Yellow-Huai-Hai river valley of China and has caused large soybean yield losses[18].Compared with the staygreen phenomena in other crops,“Zhengqing”in soybean is a special type of staygreen caused by disease or insect attack.

Interactions between multiple organs are also involved in leaf senescence.Wittenbach[19]proposed that pod removal might exert an important influence on leaf senescence progress in soybean.Some physiological parameters in soybean were influenced by pod removal[20,21],although molecular changes in leaves caused by seed regulation have remained rarely reported.In the present study,we followed the leaf development process and measured the physiological parameters and expression of senescence-related and flowering-timing genes in response to pod removal and seed injury treatments.Our aim was to evaluate the effects of pod/seed status on leaf development,to characterize the relationship between source and sink in soybean,and to identify the cause of“Zhengqing”outbreaks.

2.Materials and methods

2.1.Plant materials

Zhonghuang 30,a mid-maturing(maturity group III)variety of soybean[Glycine max(L.)Merr.],was used in a two-year pot experiment conducted in 2014 and 2015 at the Institute of Crop Science,the Chinese Academy of Agricultural Sciences,Beijing,China(39°54′N,116°46′E).Seeds were sown on June 28,2014 and July 1,2015 in plastic pots of 26 cm height×30 cm diameter at the top and 22 cm at the bottom. Each pot contained 4 kg of soil (turf:loam:vermiculite 4:2:1,v/v/v).Seeds were thinned to five healthy plants in each pot at V2(the second-node stage) [22].Plants were placed outdoors and were irrigated as needed to avoid water stress.Other environmental conditions were controlled at the optimum level to minimize environmental effects on the results.

2.2.Experiment design

The experiment was arranged in a randomized complete block design with three replications.At the R4(full pod)[22] stage,the pots were randomly divided into five groups for five treatments.In treatment 1,0 pods were retained(0-pod)in each plant(all pods were removed)after R4;in treatments 2 and 3,10(10-pod)and 20 pods(20-pod),respectively,were retained in each plant after R4.Pod removal was performed by excision of the pods at the carpopodium with scissors, with the remaining pods evenly distributed on 10 nodes of the main stem.In treatment 4,all(approximately 30)pods were retained,but the seeds were destroyed by puncturing with a syringe in the pod cavity.Intact(fully podded)plants (treatment 5)were used as controls.After R4,plants were checked and continuously depodded(treatments 1–3)or new pods were punctured(treatment 4)every other day to meet the designed pod numbers or conditions.

2.3.Measurement of physiological parameters

Trifoliate leaves at the seventh node(from bottom)on the main stem were sampled at intervals of 5 days for analysis of physiological parameters and of expression of senescence-related genes(Table 1).The leaves on the same node of the 0-pod and intact plants were sampled daily in the first week after R4 for expression analysis of the flowering genes GmFT2a [23,24]and E1[25].Samples were taken from each treatment and replication,frozen in liquid nitrogen,and stored at?80°C until processing.Each sample was extracted separately and measured three times.

2.3.1.Chlorophyll concentration

SPAD value,an index of relative chlorophyll content,was measured with a SPAD-502 chlorophyll meter(Konica Minolta Inc.,Tokyo,Japan),as described by Li et al.[26].

2.3.2.Soluble sugar and protein

The soluble protein content of leaves was measured using Coomassie Brilliant Blue G250[27]and soluble sugar content by anthrone colorimetry[27].

2.3.3.Plant hormone content

IAA,GA3,and ABA contents were measured by Huakong Center, College of Agronomy and Biotechnology,China Agricultural University,using enzyme-linked immunosorbent assay(ELISA) methods[28].

Table 1–Sequence,annealing temperature,and predicted product size of PCR.

2.4.RNA extraction and RT-PCR

Total RNA was extracted using TransZOL Up(TransGen Biotech,Beijing,China).One-Step gDNA Removal and cDNA Synthesis Super Mix(Trans Gen Biotech,Beijing,China)was used to obtain single-stranded cDNA.The RT-PCR primers (Table 1)were designed according to target genes.GmCYP2 was used as the reference gene for normalization[29].RT-PCR was performed with an ABI7900 Sequence Detection System (Applied Biosystems,CA,USA)using KAPA SYBR FAST qPCR Kit(KAPA BIOSYSTEMS,Boston,Massachusetts,United States) for 40 cycles(95°C for 15 s denaturation;60°C for 1 min annealing).All reactions were repeated at least three times.

2.5.Data analysis

Variations among years and treatments(pod removal or seed injury)were tested by analysis of variance using Microsoft Excel 2007(Microsoft Corporation,WA,USA).Levene's test was performed to assess the homogeneity of variance to ensure the appropriateness of combining analysis across two years. Homogeneous variance among the two years was confirmed. Duncan's multiple range tests were performed with IBM-SPSS Statistics 21(SPSS,an IBM Company,Chicago,IL,USA).

3.Results

3.1.Effect of pod removal and seed injury on leaf senescence and plant development

Under the outdoor conditions of this study in Beijing,intact plants of soybean cv.Zhonghuang 30 began to flower(R1) 27 days after emergence(VE).The days to R2(full bloom),R3 (beginning pod),R4(full pod),R5(beginning seed),R6(full seed), R7(beginning maturity),and R8(full maturity)[22]were 30,48, 51,61,71,85,and 95 after VE,respectively.The pod removal and seed injury experiments started at R4.The results showed that the trifoliolate leaves on the seventh nodes of the intact plants became yellow(Fig.1-a),and more than 60%(Fig.2-a)of the leaves abscised at the R7 stage(Fig.1-b).The trifoliolate leaves on the seventh node of the 20-pod plants also turned yellow and approximately 50%(Fig.2-a)abscised.Leaves on the 10-pod plants became yellowish,but abscised leaves were fewer than 30%(Fig.2-a).0-pod and seed-injured plants behaved similarly with respect to foliar color(Fig.1-a),and most leaves stayed green in the 0-pod and seed-injured treatments(Fig.1-b).When the intact plants reached R8,approximately 50%and 70% (Fig.2-b)of the leaves of the 10-pod and 20-pod plants, respectively,abscised.The pods showed mature color and the remaining leaves became yellow.However,the 0-pod and seed-injured plants retained most of their leaves,and the remaining leaves and punctured pods stayed green(Fig.1-c), indicating that pod removal and seed injury resulted in the delay of leaf senescence and staygreen of soybean plants.The extent of staygreen was inversely correlated with the number of remaining pods(seeds).

3.2.Effect of pod removal and seed injury on physiological parameters

SPAD values of intact plants began to decrease at day 15 after R4,whereas those under other treatments remained stable until day 30 after R4.The SPAD value was inversely correlated with the number of intact pods remaining on the plants.The leaves of the pod-punctured plants showed the highest chlorophyll content at day 35 after R4(Fig.3-a;Table S1).The contents of soluble protein in leaves decreased gradually after R4 in all treatments,but the rate was markedly lower in the pod removal and seed injury treatments compared with that in leaves of intact plants(Fig.3-b;Table S2).Soluble sugar content increased in the first 10 days after R4 in all treatments,then gradually decreased in leaves of intact plants,but showed an increasing tendency in other treatments(Fig.3-c; Table S3).Differences in soluble sugar content between the depodded and seed-injured treatments were not statistically significant.

3.3.Effect of pod removal and seed injury on the content of plant hormones

The GA3content of intact plants showed an increasing tendency in the first 15 days after R4.After that stage,it began to decline slowly(Fig.4-a;Table S4).ABA content continued to rise after R4(Fig.4-b;Table S5),and IAA content decreased rapidly in the first 10 days and became stablethereafter(Fig.4-c;Table S6).The GA3,ABA,and IAA contents in the leaves of depodded or seed-injured plants showed a trend similar to that of intact plants,but the change in the value of the hormonal content was smaller than in intact plants.For the GA3/ABA ratio,all depodded and seed-injured treatments showed a decreasing tendency similar to that in the intact control(Fig.4-d).

Fig.1–Senescent phenotype of soybean leaves and plants in different treatments.a.Leaves at the seventh node on the main stem when the intact control reached the R7 stage(beginning maturity).b and c.Soybean plants when the intact control reached the R7 and R8(full maturity)stages,respectively.Punctured,the seed-injured treatment;Intact,the intact control.

3.4.Effect of pod removal and seed injury on expression of senescence-related genes

GmSARK,GmNAC,GmCYN1,and GmSGR1 are involved in the pathways of phytohormone biosynthesis or chlorophyll degradation[8–12].GmSARK transcription in leaves of intact plants increased rapidly atday 5 after R4.The GmSARK expression in the 20-pod and 10-pod treatments was also elevated,and the increase of GmSARK expression was higher than that in 0-pod and seed-injured plants.There was a positive correlation between the number of seed pods developing in plants and GmSARK expression level in all treatments(Fig.5-a;Table S7). GmSARK expression in leaves of the 0-pod and seed-injured plants remained low and did not differ significantly between the two treatments(Fig.5-a).The expression of GmNAC,GmCYN1, and GmSGR1 remained low in the first 25 days after R4 in all treatments,and a rapid increase occurred first in intact plants, followed by the 20-pod treatment.In contrast,expression of GmNAC,GmCYN1,and GmSGR1 remained consistently low in other treatments(Fig.5-b–d;Tables S8–S10).Gene expressionpatterns in seed-injured plants were similar to those in the 0-pod treatment,in accord with the changes in leaf senescence and physiological parameters in the corresponding treatments.

Fig.2–The number of retained leaves when the intact control reached the R7(a)and R8(b)stages.Numbers 0,10,and 20, represent respectively the 0-pod,10-pod,and 20-pod treatments;Punctured,the seed-injured treatment;Intact,the intact control.Error bars indicate the standard deviation(SD)of three biological replicates.Means labeled with the same letter are not significantly different at P＜0.01.

Fig.3–Contents of chlorophyll,soluble protein and soluble sugar in leaves under different treatments.The contents of chlorophyll(a),soluble protein(b),and soluble sugar(c)in leaves under different treatments.Numbers 0,10,and 20 represent 0-pod,10-pod,and 20-pod treatments,respectively; Punctured,the seed-injured treatment;Intact,the intact. Means labeled with the same letter are not significantly different at P＜0.01.Error bars indicate the standard deviation (SD)of three biological replicates.

3.5.Effect of pod removal and seed injury on the expression of the flowering time genes GmFT2a and E1

The expression of GmFT2a in leaves of intact plants was stable after R4,but the expression in fully depodded plants started to increase 5 days after R4.No difference in GmFT2a expression level between the two treatments was apparent in the first 4 days(Fig.6-a).In contrast,E1 expression showed a marked decline after pod removal(Fig.6-b).During the experiment, depodded plants produced many new flowers,showing high potential to resume the reproductive process.This finding could be attributed to the enhancement of flowering promotion of GmFT2a and/or the alleviation of the inhibitory effect of E1 in leaves after pod removal.

4.Discussion

4.1.Effects of pod removal and seed injury on soybean development

At the late reproductive development stage of plants under normal conditions,some metabolic pathways function and their associated genes are expressed,resulting in the remobilization and transportation of dry matter from leaves to sink organs [8,10,12].The leaves,as a source organ,turn yellow and abscise after seeds are filled.However,senescence progress can be interrupted when plants are depodded.Our results showed that in depodded plants,senescence was delayed,and the leaves stayed green,accompanied by an inhibition of chlorophyll and protein degradation and an accumulation of carbohydrates (Fig.3).In contrast,ABA content decreased(Fig.4),and the expression of genes involved in chlorophyll degradation and ABA synthesis was down regulated(Fig.5)in depodded plants. The increasing expression of senescence-related genes showed a clear time line in the intact plants.In the leaves of intact plants, GmSARK expression increased 5 days after R4,but GmNAC, GmCYN1,and GmSGR1 expression remained low until day 25 after R4(Fig.5),suggesting that hormones are regulated earlier than chlorophyll degradation during seed development.

4.2.“Zhengqing”is a staygreen phenomenon caused by disease and pest attack

The staygreen phenomenon has been reported in many crops [3,13,15–18],and occurs mainly at the late stage of crop development,characterized by a delay in both senescence and foliar yellowing with relatively high photosynthetic capability[13].Alteration of the genetic progress in chlorophyll degradation,phytohormonal biosynthesis and even flowering pathways can cause staygreen in crops[13,15,17,30,31].The present study showed that change in sink products caused by external factors,such as pod removal or seed injury,could also result in staygreen(Fig.1).Similarities were found between depodded and seed-injured plants with respect to developmental rate,physiological parameters,and gene expression,indicating that the seed itself,rather than the pod as a whole,is the source of the signal inducing leaf senescence in the late stage of soybean development.

In recent years,the“Zhengqing”syndrome of summer planted soybean has occurred over a large area in the Yellow-Huai-Hai river valley in China.It is a special type of staygreen in soybean,caused by disease or insect attack.The leaves of staygreen plants neither become yellow nor abscise, even after frost injury,and yield is severely reduced[32].Li et al.[33]proposed that abscission of pods and flowers is the main cause of staygreen.Guo et al.[34]and Boethel et al.[35] suggested insect pests as the main cause.Our results showed that pod removal and seed injury can result in staygreen similar to the“Zhengqing”syndrome,indicating that“Zhengqing”is caused mainly by a halt in seed development.When seeds abort,they cannot produce signal substances that regulate leaf senescence and plant development,and leaves cannot export photosynthates or receive senescence signals from the seeds, resulting in staygreen.It can be concluded that insect attack, disease infection,or other external injuries are the inducing factors of the soybean“Zhengqing”syndrome.Strategies to protect soybean from“Zhengqing”should be focused on the control of pest insects and disease on the pods and seeds.

Fig.4–Levels of GA3,ABA,and IAA and GA3/ABA ratio in leaves under different treatments.a–c:Levels of GA3(a),ABA(b),and IAA(c),respectively,in leaves under different treatments.d:GA3/ABA ratio in leaves under different treatments.Numbers 0, 10,and 20 represent 0-,10-,and 20-pod treatments,respectively;Punctured,the seed-injured treatment;Intact,the intact control.Means labeled with the same letter are not significantly different at P＜0.01.Error bars indicate the standard deviation (SD)of three biological replicates.

Fig.5–Relative expression levels of senescence-related genes under different treatments.a–d:Relative expression levels of senescence-related genes of GmSARK(a),GmNAC(b),GmCYN1(c),and GmSGR1(d)under different treatments.Numbers 0,10, and 20 represent 0-,10-,and 20-pod treatments,respectively;Punctured,the seed-injured treatment;Intact,the intact control. GmCYP2 was used as a normalized control.Error bars indicate the standard deviation(SD)of three biological replicates.

Fig.6–Relative expression levels of flowering-time genes of GmFT2a(a)and E1(b)under different treatments.The number 0 represents 0-pod treatments;Intact,the intact control.GmCYP2 was used as a normalized control.Error bars indicate the standard deviation(SD)of three biological replicates.

4.3.Dual regulation of leaf(source)and seed(sink)in soybean development

During plant development,the leaf perceives external signals, such as photoperiod change,and produces flowering stimuli or florigen to initiate the reproductive process.FT and its homologs have proven to be genes encoding florigen[36,37]. In soybean,GmFT2a is a major flowering-promoting gene and one of the integrators in the flowering pathway[23,24].In the present study,pod removal enhanced the expression of GmFT2a but reduced the expression of E1,a key inhibitory gene of soybean[25],indicating that pod removal enhanced the flower-promoting process and retarded the inhibitory process.Pod removal can also activate the potential to resume the reproductive status of the depodded plants.These results confirmed that a sensitive communication mechanism connecting leaves and seeds results in the highly regulated expression of specific leaf genes.A previous study showed that ribulose bisphosphate carboxylase(Rubisco)levels and leaf photosynthesis declined with soybean pod removal.A possible explanation is that the leaf changes from a photosynthesizing source organ to a sink organ in depodded plants[19].

Many substances are involved in leaf senescence.Like ethylene,a major plant senescence hormone[7,9],ABA also induces leaf senescence[12].ABA is synthesized in leaves but can be transported to other organs under normal conditions [38].Previously,we found that seeds accumulated more ABA under SD than under LD,accompanied with earlier senescence of leaves under SD[39],indicating that the senescence of SD-treated plants was associated with ABA signal from the seeds.In the present study,ABA declined in the leaves of depodded plants,in which the senescence of leaves was delayed.Because seeds accumulate ABA under normal conditions and depodded plants contained less ABA in leaves than did intact plants,we propose that seeds can both withdraw ABA from leaves and accumulate it as a sink in the early-and mid-stage of seed development and export (resend)ABA to leaves as a source in late stage of plant development.The accumulation of ABA in young seeds can enhance sink strength,and ABA exported to leaves after full seed formation may promote leaf senescence and remobilize metabolites.

4.4.The depodded soybean plant is an ideal system for studying source–sink relationships in crops

Delaying leaf senescence is particularly advantageous under stress conditions such as drought and high temperature. Stress conditions tend to accelerate senescence and decrease the supply of assimilates to the seeds[15].In soybean, premature senescence is a limiting factor in yield improvement[1].To breed varieties that can extend the duration of active photosynthesis,the balance between leaves,seeds,and other organs should be emphasized[1].Depodding is a simple way to control the organ balance quantitatively.Taken together,these results provide an ideal basis for further elucidating the balance between sink and source and also for facilitating breeding for optimizing organ and yield components.

5.Conclusions

Pod removal and seed injury delayed leaf senescence,retarded leafabscission,and keptplants in vegetative or staygreen status. Compared with the leaves of intact plants,staygreen leaves contained higher levels of chlorophyll,soluble protein,soluble sugar,and IAA but lower levels of ABA.The expression of four genes,GmSARK,GmNAC,GmCYN1,and GmSGR1,that are involved in chlorophyll degradation or hormonal metabolism, and E1,a key flowering-inhibitory gene,was decreased in depodded or seed-injured plants.This decrease indicated that pod removal and seed injury play an importantrole in regulating leaf senescence and plant development.“Zhengqing”,or the staygreen syndrome of soybean,results from seed injury by insect attack or disease infection.This study has provided the basis for understanding the“Zhengqing”syndrome and could facilitate a reduction of incidence of“Zhengqing”in field agricultural practices.

Acknowledgments

This study was supported by the China Agriculture Research System(No.CARS-04)and the Agricultural Science and Technology Innovation Program to T.F.Han and the National MajorProject for Breeding of Transgenic Crops(No.2016ZX08004002) to C.H.Guo.

Supplementary data

Supplementary data for this article can be found online at http://dx.doi.org/10.1016/j.cj.2016.04.002.

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*Corresponding author.

**Corresponding author.Tel.:+86 10 82105875;fax:+86 10 82108784.

E-mail addresses:kaku2008@hotmail.com(C.Guo),hantianfu@caas.cn(T.Han).

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science,CAAS.

http://dx.doi.org/10.1016/j.cj.2016.04.002

2214-5141/?2016 Crop Science Society of China and Institute of Crop Science,CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).