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

    Physiological and Molecular Studies of Stay Green Caused by Pod Removal and Seed Injury in Soybean Xinxin Zhang, Min Wang, Tingting Wu, Cunxiang Wu, Bingjun Jiang, Changhong Guo, Tianfu Han PII: DOI: Reference:

S2214-5141(16)30027-7 doi: 10.1016/j.cj.2016.04.002 CJ 161

To appear in:

The Crop Journal

Received date: Revised date: Accepted date:

19 February 2016 28 March 2016 3 April 2016

Please cite this article as: Xinxin Zhang, Min Wang, Tingting Wu, Cunxiang Wu, Bingjun Jiang, Changhong Guo, Tianfu Han, Physiological and Molecular Studies of Stay Green Caused by Pod Removal and Seed Injury in Soybean, The Crop Journal (2016), doi: 10.1016/j.cj.2016.04.002

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Physiological and Molecular Studies of Stay Green Caused by Pod

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Removal and Seed Injury in Soybean Xinxin Zhanga, b, Min Wangb, Tingting Wub, Cunxiang Wub, Bingjun Jiangb,

Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang,

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a

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Changhong Guoa, *, Tianfu Hanb, *

College of Life Science and Technology, Harbin Normal University, Harbin 150025, b

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China;

MOA Key Laboratory of Soybean Biology (Beijing), Institute of Crop Science, the

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Chinese Academy of Agricultural Sciences, Beijing100081, China *

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Corresponding Authors

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E-mail address: [email protected] (T. Han), [email protected] (C. Guo)

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Phone: +86-10-82105875 (T. Han) Fax: +86-10-82108784 (T. Han)

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ACCEPTED MANUSCRIPT Abstract Leaves provide substances and signals for pod and seed development in soybean.

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However, the regulatory feedbacks of pod and seed on leaf development remain

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unclear. Here, we investigated the effects of pod/seed on leaf senescence by

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conducting pod removal and seed injury experiments. Our results showed that pod removal and seed injury delayed leaf senescence and caused the stay green phenotype

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of leaves. There were dosage effects of pod number on the extent of stay green in the depodded plants. The concentrations of chlorophyll (SPAD value, the index

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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

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content of IAA decreased while that of ABA increased. This trend was more

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pronounced in the intact plants compared with the depodded and seed-injured ones.

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The ratio of GA3/ABA decreased gradually in all treatments. The content of GA3 was relatively stable, and it was higher in the intact plants than in the depodded plants. The expression levels of the four senescence-related genes, GmSARK, GmSGR1, GmCYN1 and GmNAC, declined in the depodded or seed-injured treatments and were positively correlated with the number of leaves retained in the 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 plants than in intact plants. We postulated that pod or seed can regulate leaf development. When the seed is aborted due to disease infection or pest attack, the leaves stay green because of the absence of the seed signals for senescence. Key words: Soybean; Seed injury; Stay green; Source-sink relation

1. Introduction 1

ACCEPTED MANUSCRIPT Leaf not only plays a major role in photosynthesis but is also the organ for perceiving the environmental signals during plant development. For soybean, the dry matter

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derived from leaf photosynthesis constitutes over 90% of the overall dry matter accumulation and is considered as a determinant of yield [1]. The role of leaf in signal

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acceptance is performed measuring the day-length changes and producing flowering

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substances that transfer to shoot apical meristem, resulting in adaptive changes of plant growth and development [2]. In soybean, a typical short-day (SD) plant, SD

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promotes and long day (LD) inhibits flowering and maturity [3,4]. For some

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photoperiod-sensitive varieties of soybean, continuous SD is required for the maintenance of the post-flowering reproductive status; the plants could revert to vegetative growth from reproductive growth if the plants were moved from SD to LD.

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New branches and leaves in the reverted plants stay green without SD conditions [5]. The floral stimuli are transmissible in soybean, and it was evidenced in previous study

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that the late maturing scion could be induced to flowering when grafted on an early-maturing stock with enough leaves [6]. Leaf produces and exports photosynthates 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, nucleic acids, and the remobilization and transportation of nutrients to sink organs [7]. Leaf senescence is controlled by an intricate network, which is programmed and regulated by growth stage and internal and external stimuli [7]. Several genes in metabolic and signaling pathways have been found to be involved in the senescence process [8-12]. Among them, GmSARK plays specific roles in senescence-inducing hormonal

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ACCEPTED MANUSCRIPT pathways [8-9]; SGR1and CYN1 are crucial in chlorophyll degradation, and NAC is a transcriptional factor for ABA synthesis [10-12].

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Abnormal senescence, including premature death and stayg reen, is caused by both genetic variations and environmental factors, such as drought, nutrition

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deficiency and organ damage [13-15]. Stay green is an abnormal developmental

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phenotype, and exhibits delayed leaf senescence in crops [13]. It has been shown that disruption of chloroplast degradation and related metabolic pathways leads to

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stay-green leaves[13,16-17]. In recent years, a soybean stay green syndrome called “Zhengqing”, characterized by senescence-delayed leaves, aborted pods, dead

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seeds, became a wide-spread problem in the Yellow-Huai-Hai River Valley of China and caused great losses in soybean yield [18]. Compared with the stay green

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phenomena in other crops, “Zhengqing” in soybean is a special type of stay-green which is caused by disease or insect attack.

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The interactions between multiple organs were also related to leaf senescence.

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Wittenbach proposed that pod removal might exert an important influence on the leaf senescence progress in soybean [19]. Previous studies also showed that some physiological parameters in soybean were influenced by pod removal [20-21], while the molecular changes of leaves by seed regulation remains rarely reported. In the current study, we tracked leaf development process and measured the physiological parameters and expression of senescence-related and flowering-timing genes under the treatments of pod removal and seed injury to evaluate the effects of pod/seed status on leaf development, to understand the relationship between source and sink in soybean and to know the cause for the outbreak of “Zhengqing”.

2. Materials and Methods 2.1 Plant Materials

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ACCEPTED MANUSCRIPT Zhonghuang30, 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

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Institute of Crop Science, the Chinese Academy of Agricultural Sciences, Beijing, China (39º54′N, 116º46′E). Seeds were sown on the 28th of June 2014 and the 1st of

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July 2015 in plastic pots with 26 cm height × 30 cm diameter on the top and 22 cm at

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the bottom. Each pot contained 4 kilogram of soil (turf: loam: vermiculite = 4:2:1, V/V/V). Seeds were thinned to five healthy plants in each pot at V2 (second-node

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stage) [22]. Plants were placed outdoor and were irrigated as needed to avoid any

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water stress. Other environmental conditions were controlled at the optimum level to minimize environmental effects on the results.

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2.2 Experiment design

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The experiment was arranged in a randomized complete block design with three

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replications. At the stage of R4 (full pod) [22], the pots were randomly divided into five groups for five treatments. In Treatment 1, 0 podswere retained (0-podded) in each plant (all pods were removed) after R4; In Treatment 2-3, 10 (10-podded) and 20 pods (20-podded) were retained in each plant after R4 stage, respectively. The pod removal was made by cutting the pods at the carpopodium by scissors, and the remaining pods were evenly distributed on 10 nodes of the main stem. In Treatment 4, all pods were (approximately 30) retained, but the seeds were destroyed by an injection syringe at the pod cavity. The intact plants (fully podded) (Treatment 5) were used as the control. After R4, we checked the plants and continuously depodded (Treatments 1-3) or punctured the new pods (Treatment 4) every other day to meet the designed pod numbers or condition.

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ACCEPTED MANUSCRIPT 2.3 Measurement of physiological parameters Trifoliate leaves on the seventh nodes (from bottom) on the main stem were sampled

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at the interval of 5 days for the analysis of physiological parameters and the expression of senescence-related genes (Table 1). The leaves on the same node of the

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0-podded and intact plants were sampled daily in the first week after R4 for the

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expression analysis of the flowering genes of GmFT2a [23, 24] and E1 [25]. The samples were taken from each treatment and each replication, frozen in liquid

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nitrogen, stored at 80 °C until processing. Each sample was extracted separately and

2.3.1 Chlorophyll concentration

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measured three times.

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SPAD value, the index of relative chlorophyll content, was measured by SPAD-502

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chlorophyll meter (Konica Minolta Inc., Tokyo, Japan), as described by Li et al. [26].

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2.3.2 Soluble sugar and protein

We measured the soluble protein content of leaves using Coomassie Brilliant Blue G250 [27] and the soluble sugar content by Anthronecolorimetry method [27]. 2.3.3 Plant hormone content The IAA, GA3 and ABA contents were measured by Huakong Center, College of Agronomy and Biotechnology, China Agricultural University, using enzyme-linked immunosorbentassay (ELISA) methods [28]. 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 SuperMix (TransGenBiotech, Beijing, China) was used to obtain single-stranded cDNA. The RT-PCR primers (Table 1) were 5

ACCEPTED MANUSCRIPT designed according to target genes. GmCYP2 was used as the reference gene for normalization [29]. RT-PCR was conducted by ABI7900 Sequence Detection System

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(Applied Biosystems, CA, USA) using KAPA SYBR FAST qPCR Kit (KAPA BIOSYSTEMS, Boston, Massachusetts, United States) for 40 cycles (95 °C for 15 s

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denaturation; 60 °C for 1 min annealing). All reactions were repeated at least three

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times.

2.5 Data analysis

treatments (pod removal or seed injury) were

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The variations among years and

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[Insert Table 1 here]

determined by the analysis of variance using Microsoft Excel 2007 (Microsoft

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Corporation, WA, USA). A Levene’s test was conducted to analyse the homogeneity

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of variance to ensure the appropriateness of combining analyses across two years. The

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homogenous variance was found among two years. The Duncan’s multiple range tests were performed by using the software SPASS.

3. Results

3.1 Effect of Pod removal and seed injury on leaf senescence and plant development Under the outdoor condition of this study in Beijing, the 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 from R4. The results demonstrated that the trifoliolate leaf on the 7th node of the intact plants became yellow (Fig. 1a), and more than 60% (Fig. 2a) of the leaves abscised attheR7 6

ACCEPTED MANUSCRIPT stage (Fig. 1b). At the same time, the trifoliolate leaf on the 7th node of the 20-podded plants also turned yellow, and approximately 50% (Fig. 2a) of the leaves abscised.

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Leaves in the 10-podded plants became yellowish, but the abscised leaves were less than 30% (Fig. 2a). 0-podded and seed-injured plants behaved similarly in foliar color

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(Fig. 1a), and most leaves stayed green in the 0-podded and seed-injured treatments

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(Fig. 1b). When the intact plants reached R8, approximately 50% and 70% (Fig. 2b) leaves of the 10-podded and 20-podded plants abscised, respectively. The pods

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showed mature color, and the remaining leaves became yellow. However, the

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0-podded and seed-injured plants still kept most of the leaves, and the remaining leaves and punctured pods stayed green (Fig. 1c), indicating that pod removal and seed injury resulted in the delay of leaf senescence and stay green of soybean plants.

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pods (seeds).

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The extent of the stay green was inversely correlated with the number of remaining

[Insert Figs. 1 and 2 here]

3.2 Effect of pod removal and seed injury on physiological parameters The SPAD value, the index of relative chlorophyll content, of the intact plants began to decrease at day 15 after R4, while that of the SPAD value under other treatments kept 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 had the highest chlorophyll content at day35 after R4 (Fig. 3a and Table S1). The contents of soluble protein in leaves decreased gradually after R4 stage in all treatments, but the rate was obviously lower in pod removal and seed injury treatments compared with that in the leaves of the intact plants (Fig. 3b and Table S2). The soluble sugar content increased in the first 10 days after R4 in all treatments then 7

ACCEPTED MANUSCRIPT gradually decreased in the leaves of the intact plants but showed an increasing tendency in other treatments (Fig. 3c and Table S3). The differences of soluble sugar

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content between the depodded and seed-injured treatments were not statistically

[Insert Fig. 3 here]

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significant.

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

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The GA3 content of the intact plants showed an increasing tendency in the first 15 days after R4. After that stage, it began to decline at a slow rate (Fig. 4a and Table S4).

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ABA content kept rising after R4 (Fig. 4b and Table S5), and IAA content decreased rapidly in the first 10 days and became stable thereafter (Fig. 4c and Table S6). The

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GA3, ABA and IAA contents in the leaves of depodded or seed-injured plants showed

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a changing trend that was similar with the intact plant, but the change in the value of

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the hormonal content was smaller than the intact plants. Regarding the GA3 to ABA ratio, all depodded and seed-injured treatments showed a decreasing tendency in accordance with the intact control (Fig. 4d). [Insert Fig. 4 here]

3.4 Effect of pod removal and seed injury on the expression of senescence-related genes GmSARK, GmNAC, GmCYN1 and GmSGR1 were involved in the pathways of phytohormone biosynthesis or chlorophyll degradation [8-12]. The results of this study showed that the GmSARK transcriptions in the leaves of the intact plants increased rapidly at day 5 after R4. The GmSARK expression in the 20-podded and 10-podded treatments was also elevated, and the increase of GmSARK expression was higher than that in 0-podded and seed-injured plants. There was a positive correlation 8

ACCEPTED MANUSCRIPT between the number of seed pod development in the plants and GmSARK expression level in all treatments (Fig. 5a and Table S7). The GmSARK expression in leaves of

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the 0-podded and seed-injured plants retained at a low level and did not show significant differences between the two treatments (Fig. 5a). The expression of

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GmNAC, GmCYN1 and GmSGR1kept low in the first 25 days after R4 in all

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treatments, and a rapid increase occurred first in the intact plant, followed by that of the 20-podded treatment. In contrast, the expression levels of GmNAC, GmCYN1 and

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GmSGR1 kept at low levels all the time in other treatments (Fig. 5b, 5c, 5d and Tables

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S8–S10). Furthermore, the gene expression patterns in seed-injured plants showed similar results compared with the 0-podded treatment, in accordance with the changes

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of leaf senescence and physiological parameters in relative treatments.

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[Insert Fig. 5 here]

3.5 Effect of pod removal and seed injury on the expression of the flowering time

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genes of GmFT2a and E1

The expression of GmFT2a in the leaves of the intact plants was quite stable after R4, but the expression in the fully depodded plants started to increase 5 days after R4. The difference of the GmFT2a expression level between the two treatments was not apparent in the first 4 days (Fig. 6a). In contrast, E1 expression showed a marked decline after pod removal (Fig. 6b). During the experiment, we found that the depodded plants produced many new flowers, showing great potential to resume reproductive process. That could be attributed by the enhancement of the flowering promoting effects of GmFT2aand/or the alleviation of the inhibitory effect of E1 in the leaves after pod removal. [Insert Fig. 6 here] 9

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

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At the late reproductive development stage of plants under normal conditions, certain

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metabolic pathways functioned and their related genes were expressed, resulting in

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the remobilization and transportation of dry matters from leaves to sink organs [8, 10, 12]. The leaves, as a source organ, turned yellow and abscised after seeds were filled.

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However, the senescence progress could be interrupted when the plants were depodded. Our results showed that in depodded plants, the senescence was delayed,

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and the leaves stayed green, accompanied with an inhibition of chlorophyll and

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protein degradation and an accumulation of carbohydrates (Fig. 3). In contrast, the ABA content decreased (Fig. 4), and the expression of genes related to chlorophyll

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degradation and ABA synthesis was down-regulated (Fig. 5) in the depodded plants. It is interesting that the increasing expression of the senescence-related genes showed a certain timeline in the intact plants. In the leaves of the intact plants, GmSARK expression increased 5 days after R4, but GmNAC,GmCYN1,and GmSGR1 expression remained low until day25 after R4 (Fig. 5), indicating that hormones should be regulated earlier than chlorophyll degradation during seed development. 4.2 “Zhengqing” is a stay green phenomenon caused by disease and pest attack The stay green phenomenon has been reported in many crops [3, 13, 15-18], and it mainly happens at the late stage of crop development, characterized by the delay of both senescence and foliar yellowing with a rather high photosynthetic capability [13]. Alteration of the genetic progress in the chlorophyll degradation, phytohormonal 10

ACCEPTED MANUSCRIPT biosynthesis and even flowering pathways could cause stay green in crops [13, 15, 17, 30, 31]. The current study showed that change of the sink products were caused by the

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external factors, such as pod removal or seed injury, could also result in stay green (Fig. 1). Similarities were found when comparing the developmental rate,

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physiological parameters and gene expression of the depodded plants with the

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seed-injured plants, indicating that the seed itself, instead of the pod as a whole, is the

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source of signal inducing leaf senescence in the late stage of soybean development. In recent years, the “Zhengqing” syndrome of summer-planting soybean

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occurred in a large range in the Yellow-Huai-Hai River Valley in China. It is a special type of stay green in soybean, and is caused by disease or insect attack. The leaves of

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the stay-greened plants can neither become yellow nor abscise, even after the frost

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injury, and then seriously decreased the yield [32]. Li et al. [33] proposed that the abscission of pods and flowers should be the main reason of stay green. Guo et al. [34]

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and Boethel et al. [35] suggested that insect pest might be the main reason of stay green. Our results in the stimulated experiments showed that pod removal and seed injury can result in stay green, similar with the “Zhengqing” syndrome, indicating that “Zhengqing” is mainly caused by the halt of seed development. When seeds abort, they cannot produce signal substances to regulate leaf senescence and plant development. Meanwhile, leaves cannot export photosynthates and receive senescence signals from the seeds, resulting in stay green. It can be concluded that the seed injury caused by insect attack, disease infection or other external injury are the inducing factors of the soybean “Zhengqing” syndrome. Strategies to prevent the soybean from “Zhengqing” should be focused on the control of pest insects and diseases on the pods and seeds. 11

ACCEPTED MANUSCRIPT 4.3 Dual regulation of leaf (source) and seed (sink) in soybean development During plant development, the leaf perceives external signals, such as photoperiod

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change, and produces flowering stimuli or florigen to initiate the reproductive process.

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FT and its homologues have proved to be the genes encoding florigen [36, 37]. In soybean, GmFT2a is a major flowering promoting gene and one of the integrators in

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the flowering pathway [23, 24]. In this study, it was found that pod removal enhanced

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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

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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

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sensitive communication mechanism between the leaves and seeds resulted in the

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highly-regulated expression of specific leaf genes. A previous study showed that ribulosebisphosphate carboxylase (Rubisco) levels and leaf photosynthesis declined with

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soybean pod removal. A possible mechanism is that the leaf changes from a photosynthesizing source organ to a sink organ in the depoded plants [19].

Many substances are involved in leaf senescence. In addition to ethylene, a major plant senescence hormone [7, 9], ABA also induces leaf senescence [12]. ABA is synthesized in leaves but could be transported to other organs under normal conditions [38]. Previously, we found that seeds accumulated more ABA under SD than under LD, accompanied with the earlier senescence of leaves under SD [39], indicating that the senescence of SD-treated plants was related to the ABA signal from the seeds. In this study, ABA was found to decline in the leaves of the depodded plants in which the senescence of leaves was delayed. Because seeds accumulated ABA under normal condition and the depodded plants contained less ABA in leaves 12

ACCEPTED MANUSCRIPT compared with the intact plants, we proposed that the seeds can both withdraw ABA from leaves and accumulate it as a sink in the early- and mid-stage of seed

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development and export (resend) ABA to leaves as a source in late stage of plant development. The accumulation of ABA in young seeds can enhanced the sink

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strength, and the ABA exported to leaves after full seed may promote leaf senescence

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and remobilize the metabolites.

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4.4 Depodded soybean plant is an ideal system to study the source-sink relation of crops

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Delaying leaf senescence is particularly advantageous under stress conditions such as drought, high temperature. Stress conditions tend to accelerate senescence and

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decrease the supply of assimilates to the seeds [15]. In soybean, premature senescence

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is a limiting factor for yield enhancement [1]. To breed varieties that can extend the

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duration of active photosynthesis, the balance between leaves, seeds and other organs should be emphasized [1]. Depodding is a simple and easy way to control the organ balance quantitatively. Taken together, these results provided a ideal basis for further elucidating the balance between sink and source, and also facilitating breeding for optimizing organ and yield components. 4.5 Solutions for “Zhengqing” in agricultural practice in the fields Given the significant yield loss caused by the “Zhengqing” syndrome, figuring out a solution for “Zhengqing” syndrome in agricultural practice is necessary and urgent. Here we put forward two steps to find the solutions. First, to identify the causal bacteria, fugi, virus or pests. Second, to find the best way to prevent the

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ACCEPTED MANUSCRIPT prevailing of diseases and pests by chemical, biological, agronomic and integrated ways.

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5. Conclusion

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Pod removal and seed injury delayed leaf senescence, retarded leaf abscission and

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kept plants in vegetative status or stayed green. Compared with the leaves of intact plants, the stay-green leaves contained higher levels of chlorophyll, soluble protein,

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soluble sugar and IAA but lower levels of ABA. The expression of four genes, GmSARK, GmNAC, GmCYN1 and GmSGR1, which are involved in chlorophyll

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degradation or hormonal metabolism, and E1, a key flowering inhibitory gene, was decreased in depodded or seed-injured plants. This decrease indicated that pod

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removal and seed injury play an important role in regulating leaf senescence and plant

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development. “Zhengqing”, or the stay green syndrome of soybean, results from the

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injury of seeds by insect attack or disease infection. This study provided the basis for understanding the “Zhengqing” syndrome and could facilitate the solution of “Zhengqing” in agricultural practices in the fields.

Acknowledgments

This study was supported by the China Agriculture Research System (CARS-04) and the Chinese Academy of Agricultural Sciences Innovation Project to T. F. Han, and the National Major Project for Breeding of Transgenic Crops (2016ZX08004002).

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[23] F. Kong, B. Liu, Z. Xia, S. Sato, B.M. Kim, S. Watanabe, T. Yamada, S. Tabata, A. Kanazawa, K. Harada, J. Abe. Two coordinately regulated homologs of FLOWERING LOCUS T are involved in the control of photoperiodic flowering in soybean. Plant Physiology 154(2010) 1220-1231. [24] H. Sun, Z. Jia, D. Cao, B. Jiang, C. Wu, W. Hou, Y. Liu, Z. Fei, D. Zhao, T. Han, GmFT2a, a soybean homolog of FLOWERING LOCUS T, is involved in flowering transition and maintenance. PLoS ONE 6(2011) e29238. [25] Z. Xia, S. Watanabe, T. Yamada, Y. Tsubokura, H. Nakashima, H. Zhai, T. Anai, S. Sato, T. Yamazaki, S. Lü, H. Wu, S. Tabata, and K. Harada, Positional cloning and 1

ACCEPTED MANUSCRIPT characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering. Proc. Natl. Acad. Sci. USA 109(2012)E2155 -E2164. [26] X.H. Li, Y.H. Xie, X.F. Yang, Y.J. Wang, S.M. Ma, Dynamic changes of SPAD value of soybean leaves under different nitrogen concentrations, Hunan Agric. Sci. 10 (2013)

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39-42 (in Chinese with English abstract).

[27] X.K. Wang, Principle and Technology of Plant Physiological and Biochemical

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Experiments, Higher Education Press (2006) pp. 190-191 (in Chinese).

[28] J. Zhao, G. Li, G.X. Yi, B.M. Wang, A.X. Deng, T.G. Nan, Z.H. Li, Q.X. Li,

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Comparison between conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA for small molecules, Analytica Chimica.

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Acta.571 (2006) 79-85.

[29] B. Jian, B. Liu,, Y.R. Bi,, W.S. Hou , C.X. Wu, T.F. Han, Validation of internal control

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[30] Y.Y. Meng, H.Y. Li, Q. Wang, B. Liu, C.T. Lin, Blue light–dependent interaction between cryptochrome2 and CIB1 regulates transcription and leaf senescence in soybean,

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different types of stay- green wheat and relevant physiological mechanisms, Acta Agron. Sin. 39 (2013) 1096-1104 (in Chinese with English abstract). [32] L.D. Chang, W. Ma, J.Q. Guo, S.F. Fang, Control efficiency of spraying frequency on “Zhengqing” phenomenon of soybean, Heilongjiang Agric. Sci. 2 (2015) 53-55 (in Chinese with English abstract). [33] X.Z. Li, Discussion the causes and treating measurements of Zhengqing disease of soybean, J. Hebei Agric. Sci. 11 (2007) 64-65 (in Chinese). [34] J.Q. Guo, W. Ma, Q.K. Lei, X.L. Yang, Y.X. Li, Tentative analysis of “Zhengqing” phenomena of soybean in the Huanghuai Valleys, J. Henan Agric. Sci. 41 (2012) 45-48 (in Chinese with English abstract). [35] D.J. Boethel, J.S. Russin, A.T. Wier, M. Blake Layton, J.S. Mink, M.L. Boyd, Delayed maturity associated with southern green stink bug (Heteroptera: Pentatomidae) injury at various soybean phenological stages. J. Econ. Entomol. 93 (2000) 707-712. [36] L. Corbesier, C. Vincent, S. Jang, F. Fornara, Q. Fan, I. Searle, A. Giakountis, S. Farrona, L. Gissot, C. Turnbull, G. Coupland, FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis, Science 316 (2007) 1030–1033. 2

ACCEPTED MANUSCRIPT [37] S. Tamaki, S. Matsuo, H.L. Wong, S. Yokoi, K. Shimamoto, Hd3a protein is a mobile flowering signal in rice, Science 316 (2007) 1033–1036. [38] T.L. Setter, W.A. Brun, Abscisic acid translocation and metabolism in soybeans following depodding and petiole girdling treatments, Plant Physiol. 67 (1981) 774-779.

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[39] T.F. Han, J.Y. Gai, Phytohormonal analysis of some photoperiod effects in soybean,

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Acta Agron. Sin. 25 (1999) 349-355 (in Chinese with English abstract).

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ACCEPTED MANUSCRIPT Figure Captions Figure 1.Senescent phenotype of soybean leaves and plants in different treatments.

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a. The leaves at the seventh node on main stem when the intact control reached the R7

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stage (beginning maturity). b and c. Soybean plants when the intact control reached

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the R7 and R8 (full maturity) stages, respectively. 0, 10 and 20 represented the 0-podded, 10-podded and 20-podded treatments, respectively; Punctured, the

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seed-injured treatment; Intact, the intact or fully podded control.

Figure 2.The number of retained leaves when the intact control reached the R7

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(a) and R8 (b)stages.

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0, 10 and 20, respectively represented the 0-podded, 10-podded and 20-podded treatments; Punctured, the seed-injured treatment; Intact, the intact or fully podded

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control. Error bars indicated the standard deviation (SD) of three biological replicates. Means were not significantly different at P< 0.01 when they were labeled with the same letter.

Figure 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. 0, 10 and 20 represented the 0-podded, 10-podded and 20-pod treatments, respectively; Punctured, the seed-injured treatment; Intact, the intact or fully podded control.

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Error bars indicated the standard deviation (SD) of three biological replicates. Figure 4.The levels of GA3, ABA and IAA and the ratio of GA3/ABA in leaves

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under different treatments.

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a-c. The levels of GA3(a), ABA (b) and IAA (c), respectively, in leaves under

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different treatments. d. The ratio of GA3/ABA in leaves under different treatments. 0, 10 and 20 represented the 0-podded, 10-podded and 20-podded treatments,

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respectively; Punctured, the seed-injured treatment; Intact, the intact or fully podded control.

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Means were not significantly different at P< 0.01 when they were labeled with the

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same letter.

Error bars indicated the standard deviation (SD) of three biological replicates.

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Figure 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. 0, 10 and 20 represented the 0-podded, 10-podded and 20-podded treatments, respectively; Punctured, the seed-injured treatment; Intact, the intact or fully podded control. GmCYP2 was used as a normalized control. Error bars indicated the standard deviation (SD) of three biological replicates.

Figure 6. Relative expression levels of flowering time genes of GmFT2a (a) and E1 (b) under different treatments.

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deviation (SD) of three biological replicates.

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ACCEPTED MANUSCRIPT Table 1-Sequence, annealing temperature and predicted product size of PCR Sequence (5’-3’)

TTCAACAAAGAGGAGGCGCT

GmSARK-R

TTCTAGCATGCTGACCAVVG

GmNAC-F

TCCACCAACTTTGCCATTACCT

GmNAC-R

AGCAACGTCCATTGGAACAA

GmCYN1-F

GGACAGGTAATTGGTGCCTGA

GmCYN1-R

TGTCTGAGCTAAGGGTGTCA

GmSGR1-F

CCGCTTACGTTGAGCCCTAT

GmSGR1-R

AATTTGGCAGCATCCCCGTA

GmFT2a-F

GCTGACATCTCTGTTATTGTAGGTA

GmFT2a-R

TAATTCATAACAAAGCAAACGAGTA

E1-F

TGCACCAACTCGTTCTAAAGG

temperature

size (bp)

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105

56

244

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134

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139

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190

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112

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154

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Product

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GmSARK-F

E1-R

Annealing

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RT-Primer

CCGATCTCATCACCTTTCCTGA

GmCYP2-F

CGGGACCAGTGTGCTTCTTCA

GmCYP2-R

CCCCTCCACTACAAAGGCTCG

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