Fengxian Zhen, Junjie Zhou, Aqib Mahmood,Wei Wang, Xini Chang, Bing Liu, Leilei Liu,Weixing Cao, Yan Zhu*, Liang Tang*

National Engineering and Technology Center for Information Agriculture, Key Laboratory for Crop System Analysis and Decision Making,Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Jiangsu Key Laboratory for Information Agriculture, Jiangsu Collaborative Innovation Center for Modern Crop Production,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China

A B S T R A C T

Keywords:Rice (Oryza sativa L.)Heat stress Yield components Nonstructural carbohydrates Translocation Extreme heat stress events are becoming more frequent under anticipated climate change,which can have devastating impacts on rice growth and yield. To quantify the effects of short-term heat stress at booting stage on nonstructural carbohydrates (NSC) remobilization in rice, two varieties (Nanjing 41 and Wuyunjing 24) were subjected to 32/22/27 °C(maximum/minimum/mean),36/26/31 °C,40/30/35 °C,and 44/34/39 °C for 2,4 and 6 days in phytotrons at booting stage during 2014 and 2015.Yield and yield components, dry matter partitioning index (DMPI), NSC accumulation and translocation were measured and calculated. The results showed that the increase of high-temperature level and duration significantly reduced grain yield by suppressing spikelet number per panicle, seed-setting rate,and grain weight.Heat stress at booting decreased DMPI in panicles,increased DMPI in stems, but had no significant effect on photosynthetic rate. Stem NSC concentration increased whereas panicles NSC concentration, stem NSC translocation efficiency, and contribution of stem NSC to grain yield decreased.Severe heat stress even transformed the stem into a carbohydrate sink during grain filling.The heat-tolerant Wuyunjing 24 showed a higher NSC transport capacity under heat stress than the heat-sensitive Nanjing 41.Heat degree-days(HDD),which combines the effects of the intensity and duration of heat stress,used for quantifying the impacts of heat stress indicates the threshold HDD for the termination of NSC translocation is 9.82 °C day.Grain yield was negatively correlated with stem NSC concentration and accumulation at maturity, and yield reduction was tightly related to NSC translocation reduction. The results suggest that heat stress at booting inhibits NSC translocation due to sink size reduction.Therefore,genotypes with higher NSC transport capacity under heat stress could be beneficial for rice yield formation.

1. Introduction

Rice (Oryza sativa L.) is one of the world's major staple crops that feeds ＞50%of the world's population[1].Rice growth and development is heavily limited by high temperatures. Currently, rice is mainly grown in areas where temperatures are already close to optimum rice production temperatures (28/22 °C), and any further increase in mean temperature or episodes of high temperatures during sensitive stages may adversely affect rice yield [2]. However, as climate change intensifies, the frequency and intensity of heat stress events during the rice growing season are increasing[3,4].By 2030,it is expected that 16% of the rice-growing area will be exposed to temperatures above the critical threshold for at least 5 days during the reproductive period [4]. Therefore, heat stress poses a great threat to the production stability and yield of rice,which has been well documented thus far[5,6].

Rice is susceptible to heat stress, particularly during its reproductive period[2,6-9].The response of rice plants to heat stress varies with growth stage and variety [2,8,10].The most sensitive stages to heat stress are flowering and booting[11,12]. Booting stage, the early stage of rice reproductive phase, is considered to be the second most sensitive stage after flowering,and heat stress during microsporogenesis can induce spikelet sterility and significantly reduce the yield of heat susceptible varieties [12,13]. A large number of studies have shown that temperatures above the threshold temperature of 33 °C [11,14] or 35 °C [12,15] are detrimental to rice yield and quality due to the reduced spikelet fertility [2,9],shortened grain filling duration[8,16],decreased grain weight[7,12] and deteriorated grain quality [17,18] caused by high temperatures. During flowering or grain filling, even if the heat stress(＞33 °C)lasted for only a short period,such as 4 or 6 days, it would have adverse effects on rice yield [7-9]. In these studies, the effects varied with the intensity and duration of heat stress.However,most previous studies have focused on heat stress during flowering or grain-filling stages.Rarely have studies investigated the effects of heat stress at booting stage or examined if heat stress can significantly reduce the spikelet number,spikelet fertility and grain weight during booting stage [12,13]. The effects of short-term heat stress at booting stage on rice physiological processes and yield formation are still not well understood.

Carbohydrate partitioning,the process of carbon assimilation,transport,and distribution from source organs(leaves)to sink organs (stems, roots, grains), is crucial for plant growth and its tolerance to abiotic and biotic stresses [19,20]. Before heading, non-structural carbohydrates are accumulated in the stem primarily in the form of soluble sugars and starch.These pre-heading reserves are subsequently remobilized from stems to grains and are considered an important source of carbohydrates for grain-filling, supplementing the newly assimilated carbohydrates [21-23]. Normally, the rapid translocation of NSC from stems to grains occurs 10-20 days after heading [24], contributing 10%-40% of the final grain weight[23]. However, many factors, such as genetics and the environment, affect the accumulation and transport of carbohydrates [25]. Adequate accumulation and sufficient remobilization of stem NSC are of interest to rice breeders in the context of yield stability in variable environments,as they have been shown to be important contributors to grain yield under adverse climatic conditions such as water deficit and low radiation [26,27]. Recently, a few studies have suggested that higher stem NSC concentration at heading may be one of the indicators of heat-tolerant rice varieties, which could be used in rice breeding to reduce the yield loss caused by heat stress [28,29]. However, few studies have investigated the effects of short-term heat stress at booting stage on the accumulation and translocation of carbohydrates. Therefore,it is necessary to improve our understanding of the impact of short-term heat stress at booting stage on rice yield formation from the perspective of NSC remobilization.

In the present study, in order to evaluate the effects of short-term heat stress at booting stage on the remobilization of nonstructural carbohydrates,two rice varieties with different heat tolerances were exposed to heat stress of various temperature levels and durations.The objectives of this study were: (i) to investigate the effects of high-temperature levels and durations at booting stage on rice yield, yield components, and NSC dynamics in rice leaves, stems and panicles;and (ii) to evaluate and quantify the impacts of short-term heat stress at booting on NSC accumulation and translocation and their relationships with grain yield.

2. Materials and methods

2.1. Experimental site description

Temperature-controlled experiments were conducted during the rice-growing seasons of 2014 and 2015 at the Experimental Station of National Engineering and Technology Center for Information Agriculture (NETCIA), Rugao City, Jiangsu Province, China (32°16′N, 120°45′E; altitude 6 m). The climate in this region is subtropical humid monsoon, with average annual temperature of 14.6 °C, average annual precipitation of 1059.7 mm, and average annual solar radiation of 5023 MJ m-2. The soil in this region is clay loam containing an organic matter of 15.8 g kg-1, available nitrogen (N) of 1.1 g kg-1, available phosphorus (P) of 13.5 mg kg-1, available potassium (K) of 73.9 mg kg-1in the 0-30 cm soil layer, and the pH value was 8.26. The heat stress treatment was carried out in four independent phytotron rooms situated in the experimental station.

2.2. Experimental design and treatments

Two main japonica varieties in Jiangsu province, the heatsensitive Nanjing 41 and the heat-tolerant Wuyunjing 24,were used in the present study.The growth period was about 148 days for Nanjing 41 and 147 days for Wuyunjing 24. In mid-May of 2014 and 2015, rice seeds were sown in a nearby seedbed.One month later,3-leaf seedlings were transplanted into plastic pots with two seedlings per hill and three hills for each pot.The pots measured 30 cm in diameter and 35 cm in height.Each pot contained 20 kg local clay loam soil.One day before transplanting, 1.5 g N, 1.5 g K2O, and 2 g P2O5were applied as basal fertilizer in each pot.At the mid-tillering and panicle initiation stages, 0.3 g N and 1.2 g N were applied as top dressing, respectively. Rice plants were grown under natural conditions outside the phytotrons before and after the heat stress treatment and were regularly irrigated to maintain a 3 cm water-layer until 10 days before harvest.Weeds were controlled by herbicides and pests or diseases were controlled by chemicals. Other managements were performed according to local standard rice management practices.

Once the rice plants developed into the booting stage,they were transferred into phytotron rooms to be exposed to various heat stress treatments. The booting stage was determined by manually dissecting the stem and visually observing it. The booting stage was defined as when 50% of the plants had the panicle became visible to the naked eye as a tiny and transparent growth ＜2 mm in length buried within the leaf sheaths near the base of the plant,approximately two weeks before heading. The treatments involved four maximum/minimum/mean temperature levels of 32/22/27 °C (T1),36/26/31 °C (T2), 40/30/35 °C (T3), and 44/34/39 °C (T4), and three heat stress durations of 2 days (D1), 4 days (D2), and 6 days (D3). T1 (which was considered to be the optimal temperature for rice growth during the booting stage) was treated for only 4 days and was considered as the control treatment[8,18].Thirty pots were selected for each treatment and were randomly arranged in the phytotrons. After the treatment, the plants were transferred back to the initial environment and grown to harvest. Experimental details are shown in Table 1.

The size of each phytotron room was 2.8 m high by 4.2 m wide by 4.4 m long. The phytotrons were built of highly transparent glass (75% optical transparency). Photosynthetically active radiation (PAR) inside the phytotrons was 70% of ambient PAR on sunny days and 50% on rainy days,respectively, which was sufficient for the photosynthesis and growth of rice plant. The daily sunshine hours during the treatment were from 5:30 to 18:30. The gradual temperature fluctuations in the phytotrons were controlled to simulate the daily temperature dynamics of the local ambient environment (Fig. 1). The relative humidity inside the phytotrons was controlled at 70% ± 5%. Differences in CO2concentration inside and outside the phytotrons were minimized by using fans that exchanged air with the ambient air. The air temperature and relative humidity inside the phytotrons were monitored using VP-3 sensors (Decagon Devices, Pullman, Wash, USA), and the PAR was monitored using PYR solar radiation sensors(Decagon Devices,Pullman,Wash, USA). The air temperature and relative humidity, and PAR in phytotrons were recorded at a 5-minute interval in EM50 digital data loggers (Decagon Devices, Pullman, Wash,USA).

2.3. Data measurement

The net photosynthetic rate (Pn) of flag leaves was measured with a portable photosynthetic LI-6400 (Li-Cor Inc., Lincoln,NE,USA)on sunny days between 9:00 AM and 11:00 AM every 5-7 days after treatment, and three flag leaves were selected each time. Rice plants were sampled every 6-7 days from heading to physiological maturity. At each sampling time,three pots were sampled for each treatment to comprise three replicates. Rice plants were separated into leaves, stems(including leaf sheaths and culms), and panicles. The dry weights of the plant parts were determined after drying in a forced-air oven at 85 °C for 72 h. The partitioning index of plant organs was determined by the ratio of organ biomass to aboveground biomass at physiological maturity.The samples were then grounded into a fine powder using a steel grinder to determine the NSC concentration.

NSC concentration (sugars and starch) in different plant parts was determined following the methods of Yoshida et al.[30]. NSC accumulation in each plant part was calculated by multiplying the NSC concentration by the corresponding dry matter. The NSC translocation was determined by the difference in NSC accumulation in stems at heading and physiological maturity. The NSC translocation efficiency was the ratio of NSC translocation to stem NSC accumulation at heading. The contribution of stem NSC to grain yield was the ratio of NSC translocation to grain yield.

At physiological maturity, plants in three pots were harvested for each treatment.The total number of spikelets per panicle was counted. The seed-setting rate was calculated as the ratio of the number of filled spikelets to the total number of spikelets. The 1000-grain weight of fertilegrains and the total grain yield for each pot were also determined.

Table 1-Experimental information details.

Fig.1- The daily temperature, relative humidity and photosynthetically active radiation(PAR) profiles inside the phytotrons during treatments in growing season 2015.

2.4. Heat stress indices

Three indices of mean temperature (T), heat stress duration(D), and heat degree-days (HDD) were used to quantify the dose/severity of the heat stress treatment. HDD is the accumulative index that considers both the hightemperature level and the heat stress duration according to Shi et al.[16].HDD is calculated by the following equations:

where HDD(°C day)is the sum of daily heat degree-days(HDi)from the first day of treatment to the mth day after treatment;HHDj(°C day) is the hourly heat degree-days; Tj(°C) is the air temperature at the jth hour of a day, which was recorded by EM50 data loggers;and Tc(°C)is the threshold temperature for heat stress.In the present study,Tcwas set to 33 °C according to previous studies[11,14].

2.5. Statistical analysis

Data were analyzed using SPSS 19.0 (SPSS, Chicago, IL, USA)software. Analysis of variance (ANOVA) was performed to determine the main and interactive effects of multiple factors on grain yield,yield components, and NSC accumulation and translocation in the two rice-growing seasons.Additionally,a two-way ANOVA in each growing season was conducted to determine the main and interaction effects between treatment temperature and heat stress duration. Means of different treatments were compared using least significant difference tests with P ＜0.05 (LSD0.05). Pearson's correlation was used to determine the relationships between grain yield and NSC translocation with heat stress indices. Simple linear regression was used to fit the relationships between NSC translocation and HDD, and the relationships between NSC accumulation at maturity and yield components.Graphs were produced with OriginPro 9.0 software (OriginLab, Wellesley Hills,MA, USA).

3. Results

3.1. Effects of heat stress on grain yield and yield components

Short-term heat stress at booting stage significantly decreased grain yield and yield components (Table 2). The effects of high-temperature level and duration and their interaction are statistically significant. Grain yield decreased with increasing high-temperature level and duration, but yield losses differed. Under the treatment duration D1, the differences among different temperatures were minimal,except for T4. But under the treatment duration D2 and D3,the yield declined significantly with increasing temperature.For example, at the treatment duration D2, the grain yield of Nanjing 41 and Wuyunjing 24 under temperature treatment T4 decreased by 69.4%and 70.6%compared with temperature treatment T1. The effects of heat stress duration also varied with the temperature level, and the yield difference at different durations was smaller for temperature treatment T2 than for T3 and T4. The yield loss was due to the reduction in spikelet number per panicle,seed-setting rate and 1000-grain weight.For example, following treatment duration D2, compared with T1,T4 reduced spikelet number per panicle, seed-setting rate and 1000-grain weight by 33.9%,39.6%,and 21.4%for Nanjing 41,and by 26.3%, 45.8%, and 15.4% for Wuyunjing 24, respectively.Generally, Wuyunjing 24 had a higher 1000-grain weight than Nanjing 41. Averaged across all treatments and years,Wuyunjing 24 had higher yield and yield components compared

with Nanjing 41. However, under severe heat stress treatment T3D3 and T4D3, the yield performance of Wuyunjing 24 was worse than Nanjing 41.

Table 2-Effects of short-term heat stress during the booting stage on grain yield and yield components of the two varieties during 2014 and 2015.

ulation and translocation under short-term heat stress at the booting stage during 2014 Contribution of stem NSC to grain yield 0.35 0.06 1.73 4.74*4.48**9.50**0.56 2.47 2.45 6.00**0.86 NSC translocation efficiency 0.01 542.57**149.95**575.10**151.71**116.98**86.03**1.16 0.80 17.39**0.67 2.47 10.33**1.67 NSC translocation 0.21 133.78**107.03**1.07 1.50 0.17 8.77**3.72 1.59 6.78**1.95 4.87*0.71 ulation at maturity Leaves Stems Leaves Stems Grains 1.96 3.98*709.21** 634.84** 657.24**3.46*1.13 802.33** 980.26** 787.99**1.39 16.05** 6.82**21.24** 6.91**1.81 NSC accum 0.61 1.19 1.52 17.16** 61.72** 39.59**52.44**5.31**123.19** 6.65**1.24 7.24**1.92 4.86*2.30 1.08 ponents and NSC accum 5.81*68.66**5.59**150.20** 83.33** 166.58**1.43 0.10 10.79**0.52 1.08 0.34 2.94*NSC accumulation at heading 7.27**0.35 58.82** 2.78*79.01**40.73** 0.02 10.07** 4.90**790.30** 167.48** 141.2**0.28 1.40 11.74** 5.70**0.85 7.69**8.73**8.84**21.04** 0.06 14.86** 20.16** 5.75**5.01**0.66 7.75**0.38 1.69 2.99*3.85*25.01** 5.16**2.37 Table 3-Analysis of variance for grain yield and yield com Grain weight 3.64 13.72**4.57*3.80*5.76*0.20 5.22**14.48** 0.75 1.67 0.65 1.06 Seed-settingrate 130.68**51.46**43.74**0.09 8.03**2.02 11.58**4.74*0.40 0.24 5.16**1.69 1.59 12.44**2.56 5.91**1.35 Spikeletnumber 0.02 0.34 23.04**1.64 14.03**2.17 11.45**1.37 0.61 Yield 14.95** 17.29**28.08**208.87** 46.99**133.09**59.77**241.73** 143.33**32.31** 6.52**0.51 11.37** 5.47**2.33 1.13 2.74 0.24 7.15**0.58 0.89 1.18 3.29*2.54 and 2015.Source of variation Year (Y)Temperature(T)Duration (D)T × D Variety (V)Y × T Y × D Y × V V × T V × D Y × T × D Y × V × T Y × V × D V × T × D Y × T × D × V Numbers in the table indicate the F-values. *and **indicate significant difference at P ＜0.05 and P ＜0.01,respectively.

The main effects and interaction effects of treatment factors are shown in Table 3. Significant effects of main treatment factors such as temperature level and heat stress duration were observed among the yield indices. Additionally, the interaction between treatment temperature and duration also had significant effects on all yield indices. The inter-annual differences between years existed mainly because the rice plant suffered heat stress (＞35 °C) under ambient environment during the panicle initiation stage in 2014, and thus the spikelet number per panicle in 2014 was lower than that in 2015. Other factors, such as lower solar radiation in 2014 than in 2015, may also have affected the two-year results.

3.2. Effects of heat stress on dry matter production

3.2.1.Effects of heat stress on photosynthetic efficiency

After the heat stress treatment, Pnof flag leaves decreased progressively with the aging process of rice plants. Following the treatment duration D2, Pnof T3 and T4 was gradually higher than those of T1 and T2 (Fig. 2-A, B). Under T3 and T4 treatments, Pneven increased with the increase of the heat stress duration in the late grain-filling stage (Fig. 2-C-F). In general, the differences in Pnamong different treatments were relatively small.

3.2.2. Effects of heat stress on dry matter accumulation and partitioning

Fig.2- Effects of short-term heat stress at booting stage on flag leaf net photosynthetic rate(Pn)during growing season 2015.T1,32/22 °C(27 °C); T2,36/26 °C (31 °C); T3,40/30 °C (35 °C);T4,44/34 °C (39 °C);D1,2 days;D2,4 days;D3,6 days.

Short-term heat stress exhibited no substantial effect on the total aboveground dry matter accumulation but caused a marked change in the dry matter partitioning within rice plant at maturity (Fig. 3). The increase of high-temperature level and duration significantly increased stem dry matter and stem dry matter partitioning index (DMPI), but reduced panicle dry matter and panicle DMPI, especially at T3 and T4 for 4 and 6 days. For example, following the treatment duration D2, T4 increased the stem DMPI by 104.8% and 63.8% for Nanjing 41 and Wuyunjing 24, respectively, compared with T1.

3.3. Dynamics of NSC concentration in leaves, stems, and panicles

Leaf NSC was relatively stable during grain filling(Fig.4).Heat stress at booting had no significant effect on leaf NSC concentration. Following the T4 temperature treatment, the leaf NSC concentration of T4D3 was slightly higher than that of T4D2 and T4D1.

Stem NSC concentration first reached its maximum near heading then decreased during grain filling under most treatments, whereas the stem NSC concentration of Nanjing 41 under T3D3, T4D2, and T4D3, and Wuyunjing 24 under T4D3 continued to increase (Fig. 5). At heading, stem NSC concentration did not differ significantly among treatments,but later on, stem NSC concentrations of both varieties increased significantly with increasing temperature level and heat stress duration. For example, at maturity, the NSC concentration in stems of T4D2 increased by 282.8% and 197.2% for Nanjing 41 and Wuyunjing 24, respectively,compared with the T1D2; and the stem NSC concentration of T4D3 increased by 91.1% and 232.0% for Nanjing 41 and Wuyunjing 24, respectively, compared with T4D1. At maturity, the stem NSC concentration of Wuyunjing 24 was lower than that of Nanjing 41.

Panicle NSC concentration increased gradually during grain filling (Fig. 6). Under the treatment duration D2, the panicle NSC concentrations of T3D2 and T2D2 increased more rapidly than that of T1D2 in the first 21 days of grain filling, but thereafter, the panicle NSC concentration was T1D2 ＞T2D2 ＞T3D2 (Fig. 6-A, B). The panicle NSC concentration of T4D2 remained the lowest throughout the grain-filling process.Similarly, at temperature level T2, panicle NSC concentrations were T2D3 ＞T2D2 ＞T2D1 in the first 21 days of grain filling,but the opposite was true at maturity(Fig.6-C,D).Under T3 and T4 treatments,panicle NSC concentrations decreased with the increase of heat stress duration(Fig.6-E,G,H).

3.4. Effects of heat stress on NSC accumulation and translocation

Fig.3- Effects of short-term heat stress at booting stage on dry matter and dry matter partitioning index at physiological maturity during growing seasons of 2014(A,B)and 2015(C,D).Numbers in the bars represent the dry matter partitioning index(DMPI,%)of each organ.T1,32/22 °C(27 °C);T2,36/26 °C(31 °C);T3,40/30 °C(35 °C);T4,44/34 °C(39 °C);D1,2 days;D2,4 days;D3,6 days.

In general, the NSC accumulation in leaves and stems at heading of Wuyunjing 24 was greater than that of Nanjing 41(Table 4). Averaged across all treatments and years, the NSC accumulation in leaves was 0.14 and 0.22 g tiller-1, and the NSC accumulation in stems was 0.75 and 0.91 g tiller-1for Nanjing 41 and Wuyunjing 24, respectively. NSC accumulation in leaves at heading increased with increasing temperature level and duration for Wuyunjing 24, while for Nanjing 41,the difference among treatments was not significant.NSC accumulation in stems at heading also showed an increasing trend with increasing temperature level for Wuyunjing 24,except for the treatment T4D3. However, for Nanjing 41, the difference among treatments was not significant.At maturity,the leaf and stem NSC accumulation of Wuyunjing 24 was smaller than that of Nanjing 41. Averaged across all treatments and years, the NSC accumulation in leaves was 0.19 and 0.15 g tiller-1, and the NSC accumulation in stems was 0.87 and 0.75 g tiller-1at maturity for Nanjing 41 and Wuyunjing 24,respectively.Moreover,an increase in temperature level and duration increased the leaf and stem NSC accumulation, but decreased the NSC accumulation in grains at maturity for both varieties. Heat stress at booting stage significantly increased the NSC accumulation in stems during grain filling(Fig.7).In severe heat stress treatments(T4D2 and T4D3 for Nanjing 41, and T4D3 for Wuyunjing 24), stem NSC accumulations continued to increase rather than decrease.These results indicate that under severe heat stress,no NSC is transported from stems into the developing grains and the newly assimilated NSC re-accumulates in stems.

Fig.4-Effects of short-term heat stress on the leaf NSC concentration at booting stage during growing season 2015.T1,32/22 °C(27 °C);T2,36/26 °C (31 °C);T3,40/30 °C (35 °C);T4,44/34 °C(39 °C);D1,2 days;D2,4 days;D3,6 days.

Fig.6-Effects of short-term heat stress on the panicle NSC concentration at booting stage during growing season 2015.T1,32/22 °C (27 °C);T2,36/26 °C (31 °C);T3,40/30 °C(35 °C);T4,44/34 °C(39 °C); D1,2 days;D2,4 days;D3,6 days.

Table 4-Effects of short-term heat stress at booting stage on NSC accumulation and translocation during 2014 and 2015.

Both the NSC translocation and NSC translocation efficiency in stems decreased with increasing temperature level and duration, and there is a strong interaction between treatment temperature and duration (Table 4). For example,following temperature duration D2, NSC translocation efficiency of T4D2 decreased by 368.6%and 164.7%for Nanjing 41 and Wuyunjing 24, respectively, compared with T1D2. The average NSC translocation efficiency was-18.46%and 17.81%for Nanjing 41 and Wuyunjing 24,respectively.Notably,under the severe heat stress treatments T3D3, T4D2 and T4D3, NSC translocations and NSC translocation efficiencies were negative,indicating that NSC was not remobilized from stems to panicles but re-accumulated in stems. Similarly, the contribution of pre-heading NSC in stems to grain yield decreased with increasing temperature and heat stress duration, while it increased slightly with increasing temperature at D1 (2 days) treatments in Wuyunjing 24. Averaged across years,the contribution of pre-heading NSC to the grain yield of Nanjing 41 and Wuyunjing 24 ranged from 19.2% to-263.6% and 19.8% to -248.8%, respectively. In general, NSC translocation, NSC translocation efficiency and contribution of stem NSC to grain yield of Wuyunjing 24 were higher than that of Nanjing 41 in most treatments, implying that Wuyunjing 24 had a relatively higher NSC transport capacity than Nanjing 41 under heat stress.

3.5. Quantification of the effects of heat stress on NSC translocation

There was a strong interaction between temperature level and heat stress duration (Table 3). In order to better quantify the effects of different heat stress treatments, correlation comparisons were performed for grain yield and NSC translocation in stems with the three heat stress indices (T, D, HDD) (Fig. 8).Averaged across varieties, the correlation coefficients between yield with T, D, and HDD were -0.66, -0.59, and -0.83,respectively, indicating that gain yield was more strongly correlated with the combined index of HDD than with the single factors of T or D(Fig.8-A).Likewise,NSC translocation in stems was more closely associated with HDD than with T and D(Fig.8-B).Besides,yield and stem NSC translocation of the two varieties varied in their relationships with the three different heat stress indices. Yield and NSC translocation of Nanjing 41 showed stronger correlation with T and HDD than that of Wuyunjing 24,while weaker correlation with D than that of Wuyunjing 24.

HDD considers both temperature level and heat stress duration,and had better relationships with grain yield and NSC translocation.Therefore,HDD was used to quantify the effects of heat stress on NSC translocation(Fig.9).Stem NSC translocation showed a significant negative correlation with HDD (R2of 0.89 and 0.81 for Nanjing 41 and Wuyunjing 24, respectively).Similarly,stem NSC translocation efficiency showed a significant negative correlation with HDD(R2of 0.88 and 0.84 for Nanjing 41 and Wuyunjing 24, respectively). When HDD exceeded 7.75 °C day and 11.89 °C day for Nanjing 41 and Wuyunjing 24,respectively,NSC translocation was negative.

The relationships between NSC concentration in leaves,stems and panicles at maturity with grain yield are presented in Fig.10.At maturity, grain yield was slightly negatively correlated with leaf NSC concentration (Fig. 10-A). Grain yield and NSC concentration in stems at maturity displayed a significant negative correlation (Fig. 10-B). However, grain yield was positively correlated with NSC concentration in panicles (Fig. 10-C). In addition,NSC accumulation in stems at maturity was negatively correlated with grain yield and yield components(Fig.11).As NSC accumulation in stems increased, the seed-setting rate, 1000-grain weight, and spikelet number per panicle all decreased.These results indicate that the accumulation of NSC in stems at maturity is not conductive to yield formation. Grain yield reduction under heat stress at booting stage was closely related to NSC translocation reduction and NSC translocation efficiency reduction(P ＜0.001,Fig.12).Grain yield reduction conforms well to the quadratic function of NSC translocation reduction and NSC translocation efficiency reduction.

4. Discussion

4.1. Effects of heat stress at booting on rice yield and yield components

Fig.7-Effects of short-term heat stress at booting stage on the dynamics of NSC accumulation in stems during growing season 2015.T1,32/22 °C(27 °C);T2,36/26 °C(31 °C); T3,40/30 °C (35 °C); T4,44/34 °C (39 °C);D1,2 days;D2,4 days;D3,6 days.

Studies have shown that heat stress reduced rice yield through increased spikelet sterility during flowering and reduced grain weight during grain-filling stage [8,9,11]. However, heat stress at booting stage caused a considerable yield loss by reducing spikelets per panicle, seed-setting rate, and 1000-grain weight. Temperature level and duration displayed a strong interaction.Studies on heat stress at early reproductive stages and meiosis also reported that heat stress reduced spikelet number per panicle, seed-setting rate, grain weight,and grain size [31,32]. Heat stress at booting could promote floret degradation, thereby reducing spikelet number per panicle [31]. Moreover, heat stress at booting could cause spikelet sterility by disrupting pollen development during microsporogenesis and reducing anther dehiscence[13,32].In rice caryopsis, ＞90% of grain weight comes from endosperm cells, and the grain size is largely depended on glume size[10,29]. Heat stress can disrupt ovule cell division at booting stage, thereby reducing the size of endosperm cells or the number of cells per grain, resulting in smaller grains and lower grain weight. On the other hand, the inhibited translocation of NSC from stems into developing grains during grain filling could also result in a decrease in grain weight and grain size [31]. The reduction in spikelet number per panicle,spikelet fertility rate, and the number of cells per grain could lead to a reduction in sink size.

Short-term heat stress at booting stage did not negatively affect the photosynthetic rate and total aboveground biomass. Under severe heat stress, the photosynthetic rate even increased slightly. Previous studies have also shown that heat stress has little impact on photosynthesis,because the strong transpiration rate in leaves can reduce the tissue temperature, thus avoiding the damage of hightemperature to photosynthetic tissue [10,33]. Therefore, it is not possible that photosynthetic capacity during grain filling should be responsible for the observed low grain yield in the present study. However, heat stress at booting resulted in a marked change in the dry matter partitioning within the rice plants. More dry matter was distributed to leaves and stems, whereas less dry matter was distributed to panicles, indicating a decrease in the proportion of sink to source. These results are consistent with previous studies on heat stress during grain filling, which showed that leaves maintained photosynthetic capacity and supplied assimilates into other plant tissues except grains,resulting in an increase in the dry matter partitioning to leaves and stems [10,21].

Fig.8- Person correlations of grain yield (A) and NSC translocation in stems (B) with high-temperature level, heat stress duration and heat degree-days in 2014-2015. T, high-temperature level; D, heat stress duration; HDD, heat degree-days.

Fig.9- Relationships of NSC translocation(A)and NSC translocation efficiency(B) in stems with heat degree-days(HDD)in 2014-2015.

4.2. Source-sink related NSC remobilization

Fig.10-Relationships of NSC concentrations at maturity in leaves(A),stems(B),and panicles(C)with grain yield in 2014-2015.

Fig.11-Relationships between NSC accumulation at maturity in stems and yield components in 2014-2015.

The remobilization of pre-stored assimilate reserves from vegetative tissues into grains can be a limiting factor for rice yield formation, especially under challenging environments[10,21,26]. Our results revealed that increasing temperature level and heat stress duration at booting could significantly inhibit the translocation of carbohydrates from stems into panicles, leading to an increase in NSC accumulation in vegetative organs. The negative contributions of pre-heading NSC to grain yield in treatments T3D3, T4D2 and T4D3 suggested that no pre-heading NSC was transported from stems into grains. Additionally, the negative contributions suggested that grain yield originated from the newly assimilated carbohydrates under severe heat stress. During the grain-filling period, many factors, such as source-sink relationships, sink activity, vascular bundle characters, and phloem loading and unloading, affect the translocation of stem NSC [21,34-36]. We observed that the photosynthetic rate, leaf dry matter,NSC concentration and accumulation in rice leaves and stems did not decrease, but even increased,suggesting that short-term heat stress at booting did not limit the availability of assimilates in both leaves and stems.Therefore, the availability of assimilates could not be the reason for the poor NSC translocation observed in our study.However, sink size (spikelet number, spikelet fertility, grain weight/size) was reduced by heat stress at booting. Sink capacity is the pulling force for carbohydrate transport [37].The reduction in sink size could lead to a reduction in carbohydrate demand and in the pulling force exerted on the carbohydrates in stems, thereby reducing the NSC translocation efficiency. Moreover, under severe heat stress, grain weight/size and fertilized grains were drastically reduced,whereas leaves still maintained photosynthetic capacity,resulting in an excess of assimilates, making the stem a new carbon pool.

In addition to sink size, sink activity also affect the translocation and partitioning of stem NSC during the grain filling. Several enzymes, such as invertase, sucrose synthase(SUS), and ADP-glucose pyrophosphorylase (AGPase), are considered as key enzymes associated with sucrose to starch conversion in developing grains [38,39]. Rapid conversion of sucrose to starch may facilitate the translocation of carbohydrates into grains [34]. Heat stress at booting stage may indirectly affect the activity of these enzymes, thereby inhibiting starch synthesis and metabolism and carbohydrate translocation. There are indications that heat stress during the early reproductive stage (just before our treatments) [31]and meiosis[32]can cause changes in some phytohormones,such as reducing active cytokinins, increasing abscisic acid(ABA) and ethylene in young panicles, thereby regulating grain filling/NSC translocation and final grain yield.Moreover,heat-induced ethylene may reduce the activity of key enzymes in the sucrose-starch metabolic pathway in pollen and grain,thereby inhibiting pollen development and grain filling[32].Other explanations for translocation efficiency reduction may involve physical blockage of vascular bundles or poor phloem unloading caused by heat stress [29,34,35]. Zhang et al. [29] reported that heat stress at anthesis induced callose accumulation onto the plasmodesmata of leaf and sheath cells,which would inhibit the sucrose transport in rice plants.The higher stem NSC concentration observed at maturity under heat stress may be due to NSC translocated to panicle rachis, which cannot be unloaded into grains. The case may feedback to inhibit the NSC transport in stems, resulting in the high NSC accumulation in stems [34]. The underlying physiological mechanism of heat stress at booting inhibiting carbohydrate transport is still not fully understood and requires further study.

4.3. Quantification of the effects of heat stress

In the past decade, many studies have involved quantitative methods to study the impact of heat stress on rice physiological performance and yield [9,16,22]. However, these quantitative algorithms require further improvement in regard to prediction accuracy. Furthermore, few studies have focused on rice physiological responses and yield formation under heat stress at the booting stage. We found that the relationships between the heat stress index HDD and grain yield and NSC translocation were closer than the mean temperature(Fig. 8), indicating that the index of HDD is superior than the mean temperature in quantifying the effects of heat stress,even though both are used by crop modelers[9,16].The strong negative relationship between NSC translocation and HDD allowed us to quantify the threshold HDD for NSC translocation termination (P ＜0.001, Fig. 9). The average value of the two varieties(9.82 °C day)could be used as the critical value of heat stress intensity, which can cause the pre-heading carbohydrates to completely stop remobilization to some extent. The established relationships between yield and NSC translocation, as well as the relationships between NSC translocation and HDD in the present study could be integrated to existing crop simulation models to improve predictions of grain yield and quality in the face of heat stress at the booting stage in future climate scenarios.

Fig.12-Relationships between NSC translocation reduction(A)and NSC translocation efficiency reduction(B) in stems with yield reduction in 2014-2015.

4.4. Heat tolerance of varieties

Heat tolerance differences of varieties have been intensively studied from the perspective of seed-setting rate [2,9]. Recent studies have begun to discuss the heat tolerance of rice varieties from the perspective of carbohydrate utilization[10,21,28].In the present study, the seed-setting rates of the two varieties were more or less similar under most of the treatments at the booting stage. However, the results of the three NSC translocation parameters (NSC translocation, NSC translocation efficiency,contribution of NSC to grain yield)indicate that the heat-tolerant variety Wuyunjing 24 has a relatively higher NSC transport capacity than the heat-sensitive Nanjing 41 under heat stress,which is consistent with previous studies on heat stress during grain filling [10,21]. The two varieties also differed in their sensitivities to the high-temperature level and heat stress duration. Grain yield and NSC translocation correlation with T and D for the two varieties demonstrated that Nanjing 41 was more sensitive to the high-temperature level, whereas Wuyunjing 24 was more sensitive to heat stress duration. The results also showed strong interactions between temperature level and heat stress duration. Therefore, rice breeders and farmers should be very careful in identifying and selecting heattolerant genotypes because different temperatures and heat stress durations may produce different results. In the present study, the higher NSC translocation capacity of Wuyunjing 24 was related to its higher NSC accumulation in stems at heading,and lower NSC concentration and accumulation in stems at maturity. The ability to accumulate more NSC in stems before heading is thought to be conducive to sink activity in the early grain-filling stage and grain filling after heading[32,36].We also found strong negative relationships between NSC accumulation at maturity and yield components (P ＜0.001, Fig. 11). These results indicate that a high level of NSC in stems at maturity are likely due to poor unloading into grains,which is not conducive to yield formation. Therefore, high NSC translocation capacity(high NSC accumulation at heading and low NSC accumulation at maturity) under heat stress could be a valuable indicator in selecting heat-tolerant varieties in addition to seed-setting rate,especially when seed-setting rates of some varieties are similar.The heat tolerance of genotypes needs to be further studied and applied to adapt to climate change.

4.5. Remaining challenges

This study was carried out in phytotrons to ensure precise control of temperature and other environmental factors. Even though the light, humidity and other conditions are carefully controlled to minimize the differences with the ambient environment, there may still be some differences such as wind and precipitation [8,9]. Besides, the highest temperature treatment in our study was 44/34 °C(39 °C). The different translocation directions between T3D3 and T4D1 observed in our study were due to the fact that the HDD of T3D3 was higher than that of T4D1. However, if rice plants were exposed to higher (lethal)temperatures (＞44 °C), the results might differ from current results, although it is seldom observed in natural conditions.Therefore, the results of this study need to be validated in a broader range of environmental conditions in the near future.Moreover,further research is required to explore the physiological, biochemical, and molecular mechanisms of heat stress at booting on rice carbohydrate remobilization and yield formation.Mitigation strategies, such as integrated practices of nitrogen management to modulate source-sink balance [22], and the breeding of varieties with high NSC transport capacity under heat stress,should be developed to obtain yield stability in future climate scenarios.

5. Conclusions

Short-term heat stress at booting stage caused significant yield loss by reducing spikelet number per panicle, seed-setting rate,grain weight, and inhibiting NSC remobilization rather than limiting photosynthetic rate and aboveground biomass. The increase of high-temperature level and duration significantly decreased the pre-heading NSC translocation and its contribution to grain yield. Heat stress at booting significantly reduced sink size, which in turn inhibited the transport of NSC from stems to panicles, resulting in an increase in carbohydrate accumulation in vegetative organs. Severe heat stress even completely stopped the transport of NSC,transforming the stem into a sink organ to re-accumulate carbohydrates,rather than a source organ for grain filling. Heat degree-days (HDD), which combines the effects of the intensity and duration of heat stress,used for quantifying the impacts of heat stress, indicates the threshold HDD for the termination of NSC translocation is 9.82 °C day.The established relationships between NSC translocation, grain yield, and HDD could be used to improve the prediction of grain yield in future climate scenarios. This study also indicates that high NSC transport capacity under heat stress is beneficial for better yield formation of rice. Varieties with higher NSC translocation ability under heat stress could be used for rice breeding to mitigate the yield loss caused by heat stress.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2016YFD0300110), the National Natural Science Foundation of China (31571566), the National Science Fund for Distinguished Young Scholars(31725020), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We would like to thank Arielle Biro at Yale University for her assistance with English language and grammatical editing.