JI Dongling, XIAO Wenhui, SUN Zhiwei, LIU Lijun, GU Junfei, ZHANG Hao,Matthew Tom HARRISON, LIU Ke, WANG Zhiqin, WANG Weilu,2, YANG Jianchang

(1Jiangsu Key Laboratory of Crop Genetics and Physiology / Jiangsu Key Laboratory of Crop Cultivation and Physiology, Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China; 2Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou 225009, China; 3Tasmanian Institute of Agriculture, University of Tasmania, Newnham Drive, Launceston, Tasmania 7248, Australia)

Abstract: Due to climate change, extreme heat stress events have become more frequent, adversely affecting rice yield and grain quality. The accumulation and translocation of dry matter and nitrogen substances are essential for rice yield and grain quality. To assess the impact of high temperature stress(HTS) at the early panicle initiation (EPI) stage on the accumulation, transportation, and distribution of dry matter and nitrogen substances in various organs of rice, as well as the resulting effects on rice yield and grain quality, pot experiments were conducted using an indica rice cultivar Yangdao 6 (YD6) and a japonica rice cultivar Jinxiangyu 1 (JXY1) under both normal temperature (32 °C / 26 °C) and high temperature (38 °C / 29 °C) conditions. The results indicated that exposure to HTS at the EPI stage significantly decreased rice yield by reducing spikelet number per panicle, grain-filling rate, and grain weight. However, it improved the nutritional quality of rice grains by increasing protein and amylose contents. The reduction in nitrogen and dry matter accumulation accounted for the changes in spikelet number per panicle, grain-filling rate, and grain size. Under HTS, the decrease in nitrogen accumulation accompanied by the reduction in dry matter may be due to the down-regulation of leaf net photosynthesis and senescence, as evidenced by the decrease in nitrogen content. Furthermore, the decrease in sink size limited the translocation of dry matter and nitrogen substances to grains, which was closely related to the reduction in grain weight and the deterioration of grain quality. These findings significantly contribute to our understanding of the mechanisms of HTS on grain yield and quality formation from the perspective of dry matter and nitrogen accumulation and translocation. Further efforts are needed to improve the adaptability of rice varieties to climate change in the near future.

Key words: rice; early panicle initiation stage; high temperature stress; carbon-nitrogen translocation;grain yield; grain quality

Rice serves as the primary source of caloric sustenance for a significant proportion of the global population,surpassing the 50% threshold (Guo et al, 2022). High temperature conditions substantially constrain the growth and development of this crucial crop. Looking ahead,the predicted rise in global temperatures by 1.5 °C between 2021 and 2040 may amplify the existing challenges confronting rice production (IPCC, 2022).The increase in average temperature or the emergence of extreme high temperature occurrences exceeding the threshold levels at vulnerable growth stages can substantially impede rice productivity (Wang W L et al,2020; Shi et al, 2023). The escalating impacts of climate change are exacerbating the frequency and severity of heat stress events during the rice cultivation season (Shi et al, 2017; Gaupp et al, 2020). Of particular note,predictions indicate that by the year 2030, roughly 16% of the global area for rice cultivation may be exposed to over 5 d of heat stress during the reproductive phase (Gourdji et al, 2013). The looming threat of high temperature stress (HTS) to rice production has attracted considerable attention (Wang Y L et al, 2020; Zhen et al, 2020b; Hu et al, 2021;Schaarschmidt et al, 2021).

The reproductive phase of rice is highly vulnerable to heat stress (Jagadish et al, 2007; Shi et al, 2017; Wu et al, 2017). Numerous investigations have established that temperatures surpassing critical thresholds of 33 °C or 35 °C have deleterious effects on rice yield by impeding spikelet fertility, shortening grain-filling duration, diminishing grain weight, and compromising grain quality (Shi W J et al, 2016; Zhen et al, 2020a, b;Chen et al, 2021; Schaarschmidt et al, 2021; Wu et al,2023). At the flowering or grain-filling stages, even short periods of heat stress (＞ 33 °C) can negatively impact rice yield (Shi P H et al, 2016; Sun et al, 2018).Despite this, previous studies have primarily focused on heat stress at these stages, overlooking the potential effects of HTS before the heading stage, which can also significantly impact yield formation and grain quality to a comparable or greater extent than the postheading stage (Wu et al, 2017, 2023; Zhen et al, 2019,2020b; Wang Y L et al, 2020). HTS affects yield formation and grain quality differently between these crucial growth stages. For instance, heat stress results in a more significant reduction in grain number and grain-filling rate at the booting stage compared with the post-heading stage at similar temperature treatments(Shi et al, 2017; Zhen et al, 2020b). Additionally, heat stress at the early panicle initiation (EPI) stage has contrasting effects on grain quality, particularly for head rice rate and chalkiness degree, compared with the booting and post-heading stages (Shi et al, 2017;Zhen et al, 2019; Wu et al, 2023). The above results suggest that further experiments are needed to improve our understanding of the effects of HTS at the EPI stage on grain yield and quality.

Carbohydrate partitioning is a fundamental process in plants that involves carbon assimilation, transport,and distribution from source organs such as leaves to sink organs like stems, roots, and grains. This process plays a crucial role in plant growth and enhances the ability of plants to tolerate various biotic and abiotic stresses (Zhang et al, 2017; Wang W L et al, 2020).Before reaching the heading stage, non-structural carbohydrates accumulate within the stems. These reserves are subsequently translocated from the stems to the developing grains, serving as a significant carbohydrate source for grain filling (Shi et al, 2013).The differentiation and formation of spikelets depend on the energy provided by carbohydrate metabolism and dry matter accumulation at the pre-anthesis stage.Insufficient carbohydrate and nitrogen supply results in reduced differentiation and increased degradation of spikelets (Wang W L et al, 2018). Heat stress can lead to a decrease in spikelet density, primarily due to a reduction in dry matter weight and nitrogen uptake at the pre-anthesis stage (Wang W L et al, 2018).Approximately 9.1%-42.2% of the grain matter at maturity derives from assimilate transport at the preanthesis stage (Ntanos and Koutroubas, 2002). However,many factors, such as genetics and environments,affect the accumulation and transport of carbohydrates(Yang et al, 2006; Wang W L et al, 2020). Heat stress decreases the dry matter weight of the panicle (Chen et al, 2021), resulting in a significant reduction in assimilation allocation to grains, a decrease in grain weight, and a deterioration in rice product quality,including increased chalkiness, and poorer cooking and eating quality (Tu et al, 2022). The appropriate buildup and satisfactory transfer of stem carbohydrates are of significant importance to rice breeders in the context of securing stable yields in unpredictable environments, as they have been demonstrated to be crucial factors for enhancing grain yield and quality under HTS conditions (Shi et al, 2017; Zhen et al,2020a).

Nitrogen is a fundamental nutrient element for rice plants, as it plays a role in numerous physiological processes, including carbon and nitrogen storage and metabolism, leaf photosynthesis, and senescence (Chen et al, 2014; Cai et al, 2016; Wang et al, 2022). The acquisition and partitioning of nitrogen in various plant organs are critical in determining grain yield and quality (Yang et al, 2006; Kim et al, 2011). Grain nitrogen content and its accumulation are closely related to plant nitrogen uptake, and excessive grain nitrogen accumulation increases protein content butsignificantly reduces cooking and eating quality (Tu et al,2022). Before heading, rice plants store nitrogen acquired from the soil in their vegetative organs, while grains become the primary nitrogen sink following heading (Peng et al, 2010). Approximately 80% of the nitrogen accumulated in the harvested grain is derived from nitrogen remobilization in senescing tissues (Mae and Ohira, 1981). Studies indicate that exposure to HTS during the entire rice cultivation period and after flowering diminishes nitrogen allocation to grains but enhances nitrogen allocation to vegetative organs (Kim et al, 2011; Wang W L et al, 2020). Furthermore, elevated temperatures increase grain nitrogen concentration because the effect of heat stress on carbon accumulation in rice grains surpasses its effect on nitrogen (Kim et al,2011; Wang W L et al, 2020).

Table 1. Effects of high temperature stress at early panicle initiation stage on grain yield and its components.

Several studies have indicated that elevated levels of stem carbohydrates and nitrogen compounds at the booting and post-heading stages can serve as potential markers for heat-tolerant rice cultivars. This discovery has proven useful in developing new breeding strategies aimed at mitigating the adverse effects of heat stress on grain quality and yield (Xiong et al, 2017; Zhen et al,2020b; Liu et al, 2022; Sun et al, 2023). Nonetheless,there is limited research on the repercussions of shortterm heat stress at the EPI stage on the accumulation,translocation, and distribution of carbohydrates and nitrogen in rice plants and their relationships with rice yield formation and grain quality. In the present study,two representative rice cultivars, anindicarice cultivar Yangdao 6 (YD6), and ajaponicarice cultivar Jinxiangyu 1 (JXY1), were exposed to HTS at the EPI stage. The objectives were to examine the influence of EPI heat stress on rice yield formation, grain quality,and the dynamics of dry matter and nitrogen accumulation in different plant parts. Additionally, we aimed to assess the effects of EPI heat stress on the accumulation and translocation of dry matter and nitrogen, and their associations with grain yield and quality.

RESULTS

Yield, yield components, grain morphology, and panicle architecture

Compared with nature temperature (NT, 32 °C / 26 °C),HTS (38 °C / 29 °C) significantly reduced grain yield in both YD6 (by 28.7%) and JXY1 (by 24.5%) (Table 1). Among the yield components, the number of panicles per pot was not affected by HTS, while the number of spikelets per panicle, grain-filling rate, and 1000-grain weight were significantly decreased by HTS in both cultivars. Under HTS, the number of spikelets per panicle, grain-filling rate, and 1000-grain weight decreased by 15.1%, 12.0%, and 2.6% for YD6,and 6.8%, 14.8%, and 3.7% for JXY1, respectively.Comparatively, YD6 experienced a more substantial yield decrease than JXY1, mainly due to the higher reduction in the number of spikelet per panicle. HTS also resulted in a significant reduction in the harvest index of YD6 and JXY1. HTS induced a significantreduction in grain length for YD6 and JXY1 (Table 2 and Fig. S1). Additionally, grain length had a higher reduction for YD6 (5.9%) than JXY1 (4.7%). Thus,HTS had no apparent effects on the length-width ratio of JXY1, while significant effects were observed on YD6 (Table 2).

Table 2. Effects of high temperature stress at early panicle initiation stage on grain morphology.

Under HTS, the number of differentiated primary and secondary branches per panicle in YD6 and JXY1 was not affected, while the number of degenerated primary branches per panicle significantly increased,and the number of surviving secondary branches per panicle decreased in JXY1 and YD6, respectively(Table 3). HTS significantly reduced panicle length for YD6 and JXY1 by 8.5% and 10.6%, respectively.Compared with NT, the total number of differentiated spikelets per panicle for YD6 and JXY1 decreased significantly under HTS, with a greater decrease observed in YD6 than in JXY1 (Table 4). This was primarily due to a reduction in the number of differentiated spikelets on secondary branch per panicle and an increase in the number of degenerated secondary spikelets per panicle. The numbers of differentiated and surviving spikelets on secondary branch per panicle decreased by 21.8% and 28.0% in YD6, and by 17.5% and 25.9% in JXY1, respectively.The number of degenerated spikelets on secondary branch per panicle in YD6 and JXY1 significantly increased by 225.2% and 82.0% relative to NT,respectively (Table 4).

Processing, steaming palatability, and nutritional quality

At the EPI stage, HTS significantly reduced the milled rice rate, head rice rate, and gel consistency, but significantly increased the protein and amylose contents of both YD6 and JXY1, compared with NT (Table 5).The responses of total essential and non-essential amino acid contents to HTS significantly differed between the two cultivars. Compared with NT, HTS significantly increased the total essential amino acid content of YD6, while having no significant effect on JXY1. HTS significantly increased the total nonessential amino acid content in both rice cultivars(Tables S1 and S2).

Dry matter and nitrogen accumulation and its distribution rate

At the meiosis stage, aboveground dry matteraccumulation in YD6 significantly decreased under HTS (Fig. 1). At the heading and maturity stages,aboveground dry matter accumulation was significantly reduced by 27.7% and 20.8% in YD6, and 27.2% and 19.1% in JXY1 under HTS, respectively, compared with NT. Regarding dry matter distribution, significant decreases in the dry matter distribution rate in the stem at the heading stage were observed for both YD6 and JXY1 under HTS (Table 6). The dry matter distribution rates in the panicles of YD6 and JXY1 at the maturity stage were significantly reduced by HTS.The dry matter distribution rates in the leaves of YD6 and JXY were increased by 27.1% and 16.4% under HTS, respectively (Table 6).

Table 3. Effects of high temperature stress at early panicle initiation stage on differentiation and degeneration of branches and panicle length.

Table 4. Effects of high temperature stress at early panicle initiation stage on differentiation and degeneration of spikelets.

Table 5. Effects of high temperature stress at early panicle initiation stage on milling, cooking, eating, and nutrition qualities.

Fig. 1. Effects of high temperature stress at early panicle initiation stage on aboveground dry matter accumulation at meiosis (ME),heading (HD), and maturity (MA) stages.

Fig. 2. Effects of high temperature stress at early panicle initiation stage on nitrogen accumulation at meiosis (ME), heading (HD),and maturity (MA) stages.

Table 6. Effects of high temperature stress at early panicle initiation stage on dry matter distribution rate and nitrogen distribution rate at meiosis, heading, and maturity stages. %

The nitrogen content in the stem, leaf, and panicle showed significant differences among treatments(Table S3). At the meiosis and heading stages, the nitrogen contents in the stems and leaves of both cultivars were significantly decreased compared with NT. At maturity, the nitrogen contents in the leaves were significantly increased in both cultivars under HTS relative to NT. HTS apparently reduced the nitrogen accumulation in the leaves, stems, and roots of YD6 and JXY1 at the meiosis and heading stages and in the panicles at the heading and maturity stages(Fig. 2). Panicle nitrogen accumulation of both cultivars declined significantly at maturity, with YD6 decreasing more than JXY1. The panicle nitrogen distribution rates were significantly affected by HTS (Table 6).Under HTS, the nitrogen distribution rate in the stem of YD6 was significantly reduced by 12.8% and 19.8%at the meiosis and heading stages, respectively, while that of JXY1 was significantly reduced by 11.9% only at the heading stage. At maturity, the nitrogen allocation rates to leaves of YD6 and JXY1 were significantly increased by 34.4% and 29.1%, respectively.

Dry matter, nitrogen translocation, and contribution rate

Individual effects of cultivars and temperature treatments were observed on translocation, translocation rate, and their contribution to grain dry matter and nitrogen substances, but no interaction effects between these two factors were found (Table 7). Under HTS,pre-anthesis aboveground dry matter translocation,translocation rate, and their contribution rate to grains significantly decreased by 38.7%, 25.8%, and 14.1%in YD6, and 39.8%, 30.5%, and 17.7% in JXY1 compared with NT, respectively. Similarly, pre-anthesis nitrogen translocation, translocation rate, and contribution rate showed significant differences among treatments,with pre-anthesis aboveground nitrogen translocation,translocation rate, and their contribution rate to grains being significantly reduced by 50.2%, 24.2%, and 37.3% in YD6, and 39.3%, 16.6%, and 29.3% in JXY1 under HTS, respectively.

Table 7. Effects of high temperature stress at early panicle initiation stage on pre-anthesis aboveground dry matter and nitrogen translocation amounts, and their translocation efficiencies, and contribution rates of translocation amount to grain yield.

Hormone content

HTS significantly reduced the levels of zeatin + zeatin riboside (Z + ZR) and auxin (IAA) in the panicles of YD6 and JXY1 (Fig. 3). The Z + ZR and IAA contents in the roots of both cultivars also decreased under HTS, with the IAA content in the roots of YD6 being more affected than that of JXY1. Additionally,HTS significantly increased abscisic acid (ABA)content in the panicles and roots of both cultivars,with the ABA response to HTS in the panicle of YD6 being significantly higher than that of JXY1.

Correlation analysis

Correlation analysis revealed that IAA and Z + ZR contents in the panicle, Z + ZR content in the root, and aboveground dry matter and nitrogen accumulation at the meiosis stage were significantly and positively correlated with the number of spikelets per panicle,grain-filling rate, panicle length, grain length, and the numbers of differentiated and surviving secondary branches and spikelets per panicle (Fig. 4). Conversely,they were significantly and negatively correlated with the number of degenerated primary branches per panicle and the numbers of degenerated secondary branches and their spikelets per panicle. ABA contents in the panicles and roots at the meiosis stage were significantly and positively correlated with the number of degenerated spikelets on secondary branch per panicle.

Fig. 3. Effects of high temperature stress at early panicle initiation stage on plant hormones.

Fig. 4. Correlation analysis of hormone, dry matter and nitrogen accumulation and translocation with yield and spikelet traits.

According to the correlation analysis in Fig. S2,grain length displayed a significant and positive correlation with various traits, including 1000-grain weight, protein content, amylose content, and the total essential and non-essential amino acid contents.Moreover, pre-anthesis aboveground dry matter and nitrogen translocation amount, as well as their translocation rate, had a positive impact on grain length. Furthermore, pre-anthesis aboveground dry matter and nitrogen translocation amounts and their translocation rate, contributions rate of pre-anthesis aboveground dry matter and nitrogen to the grains demonstrated a similar pattern of positive correlation with head milled rice rate and gel consistency, and a negative correlation with protein and amylose contents.

DISCUSSION

Effects of HTS at EPI stage on yield formation

Recent research has confirmed that rising temperatures have adverse effect on the yield of rice plants. The extent of this impact varies depending on the frequency and intensity of extreme temperature events.An increase in temperature of 1.5 °C during the entire rice growth period has resulted in yield reduction ranging from 8% to 40%, with the degree of decrease being linked to the natural background temperature (Cai et al, 2016; Wang W L et al,2018). Temperature stress at the anthesis and grain-filling stages, lasting for 6 d with temperatures of 38 °C during the day and 28 °C at night, leads to reductions in rice yield of 26.4% and 19.9%, respectively(Shi P H et al, 2016). At the booting stage,temperature stress (40 °C / 30 °C) causes a significant decrease in yield, ranging from 63.5% to 90.3%, surpassing the effects of HTS at the post-heading stage (Shi P H et al, 2016; Zhen et al, 2020b). Wu et al(2021) found that the yield of temperaturesensitive rice cultivars is reduced by approximately 60% due to HTS at the EPI stage. In this study, HTS at the EPI stage led to an average decrease of 26.7% in rice yield for both cultivars.

Throughout the entire growth period with HTS, we observed reductions in panicle number per unit area, spikelet number per panicle, and grain-filling rate, but there were only marginal effects on grain weight(Cai et al, 2016; Wang W L et al, 2018).When HTS occurred during anthesis or post-anthesis, both grain-filling rate and grain weight are reduced (Shi P H et al,2016). Studies by Zhen et al (2020b) and Wang Y L et al (2020) showed that spikelet number per panicle,grain-filling rate, and grain weight are concurrently reduced by HTS at the booting and panicle differentiation stages, which aligns with our findings.Additionally, HTS significantly altered the morphology of the panicles, leading to slower growth and development at all stages of panicle differentiation,resulting in smaller panicle sizes compared with the control treatment (Chen et al, 2021). The number of spikelets per panicle is determined by the numbers of differentiated and degenerated spikelets. This study found that HTS significantly impacted the differentiation and degeneration of secondary branches per panicle,resulting in decreased spikelet density. This finding was consistent with previous studies (Wu et al, 2017;Wang W L et al, 2018, 2020). HTS at the EPI stage reduces spikelet fertility due to reduced pollen viability, poor anther dehiscence, and abnormal tapetum cell function (Hu et al, 2021). Additionally, it has been observed that HTS at the grain-filling stage reduced grain weight by an average of 2.9% (Xiong et al, 2017),a value consistent with the effects of HTS at the EPI stage on grain weight. This reduction in grain weight is partly attributed to alterations in grain morphology.Heat stress has been found to significantly affect the development and morphology of the glume, lemma,and palea, ultimately determining the shape of the grain (Li et al, 2021). In line with these findings, our study also observed that exposure to HTS at the EPI stage significantly reduced grain length.

Dry matter accumulation, translocation, and distribution are crucial for plant organ differentiation and yield formation (Wang W L et al, 2018; Zhen et al, 2020a,b). Previous research has indicated that increased temperatures throughout the growth cycle and grainfilling stage can decrease dry matter accumulation and distribution to panicles, despite an increase in leaf net photosynthesis with improved nitrogen levels (Wang W L et al, 2018). However, heat stress (40 °C / 35 °C)at the booting stage has marginal effects on leaf photosynthetic rate and dry matter production but significantly decreases them in grains (Zhen et al,2020b). Our study revealed that HTS at the EPI stage not only decreased dry matter accumulation at different growth stages, but also reduced its distribution and contribution rate to the harvested grains. As previous literature has addressed, leaf nitrogen is closely related to leaf net photosynthesis, which can prolong leaf senescence and provide more carbohydrates for plant growth (Chen et al, 2014). In this study, HTS at the EPI stage decreased leaf nitrogen at the meiosis and heading stages, which would affect the production of carbohydrate substances. Even if heat stress did not affect leaf net photosynthetic rate as reported, the leaf area might have decreased, still adversely affecting dry matter production and accumulation (Wang W L et al,2018). An adequate supply of carbohydrates is required to form rice branches, spikelets, and floret skeletons(Wang Y L et al, 2020; Chen et al, 2021). Heat stress at the EPI stage significantly reduced aboveground dry matter and nitrogen accumulation, except for a slight increase in panicle distribution in YD6 at the heading stage. This reduction hindered branch and spikelet development, resulting in a significant decrease in spikelet differentiation and grain length. Assimilates produced during grain filling (post-anthesis) or redistributed from the vegetative tissues sink (preanthesis) determine grain-filling quality (Shi et al,2013). Dry matter distribution to panicles was reduced at maturity under HTS compared with NT, possibly due to the disrupted transport of sucrose from leaves to spikelets and the inhibition of the activities of sucrose hydrolysis, glycolysis, and tricarboxylic acid cycle enzymes (Wang Y L et al, 2020; Chen et al,2021). It has been found that nitrogen fertilizer usage enhances root morphology and oxidative activity,resulting in increased nitrogen uptake, as well as carbon and nitrogen accumulation and translocation to grains (Zhu et al, 2020, 2022). Regulating critical enzyme activity responsible for the conversion of sucrose to starch through alternating wet and dry irrigation or moderate drying promotes the transfer and distribution of non-structural carbohydrates and free amino acids from the source organs (stems and leaves) to the grain reservoir, thereby facilitating grain filling (Yang and Zhang, 2010; Zhang et al, 2012;Wang et al, 2016). This study observed a significant decrease in grain length at maturity, which was closely related to a decrease in carbon and nitrogen accumulation,transportation, and distribution. The decrease in grain weight caused by HTS at the EPI stage can be attributed to the decrease in non-structural carbohydrates,underdeveloped vascular bundles, and reduced grain length and width (Wu et al, 2019). These results indicated that the decreases of pre-anthesis ‘flow’,‘source’ restriction, and ‘sink’ allocation directly affect the development of spikelets, including glume development and grain length, and indirectly reduce grain weight and yield.

In rice, more than 70% to 80% of nitrogen accumulation occurs during the pre-anthesis period,and low nitrogen accumulation during the postanthesis period promotes the transition from nitrogen metabolism to carbon metabolism, which leads to high yield (Liu et al, 2022). The increase in temperature enhances nitrogen uptake to a certain extent but does not increase dry matter accumulation, leading to a decrease in nitrogen accumulation from the jointing to the booting stage, resulting in a decrease in branch and spikelet differentiation (Wang W L et al, 2018).Additionally, HTS leads to a reduction in leaf and stem nitrogen accumulation accompanied by a reduction in dry matter, decreased activities of nitrate reductase and glutamine synthetase in plants, and hindered nitrogen metabolism (Sun et al, 2023), which may also contribute to the stagnation of nitrogen at maturity, resulting in a lower nitrogen distribution rate in panicles at maturity. We found that HTS at the EPI stage decreased nitrogen accumulation, transportation,and its contribution rate to grains, which was in line with previous studies conducted at the booting or post-heading stages (Shi et al, 2017; Zhen et al,2020a). The decreases in the dynamics of nitrogen substances might be related to the reduction in the requirements of the limited sink size (Sun et al, 2023).The above findings suggested that HTS reduces spikelet differentiation, increases degenerated spikelet,and decreases grain weight by impairing source-sink transport and disturbing nitrogen distribution.

Effects of HTS at EPI stage on grain quality

Heat stress at the post-heading stage increases chalky rate and degree while decreasing head rice rate (Tang et al, 2019). In contrast, heat stress at the booting or EPI stage decreases chalkiness but has contradictory effects on head rice rate (Zhen et al, 2019; Wu et al,2023). Our research suggested that HTS at the EPI stage may decrease head rice rate by increasing chalkiness. High temperature reduces head rice yield by increasing the occurrence of chalky and cracked/notched grains, which are easily broken during milling(Fitzgerald and Resurreccion, 2009). Even though HTS occurred at the early heading stage in our study,the arrangement of starch granules may have been affected due to side effects resulting from a reduction in dry matter in panicles. The lack of assimilated supply to grains under heat stress has also been suggested to contribute to the occurrence of chalky grains (Chen et al, 2013; Sreenivasulu et al, 2015).

Grain quality can be impacted by variations in grain length, width, thickness, and the degree of grain filling(Huang et al, 2013). Glume cell proliferation and expansion influence grain development, with the endosperm occupying most of the volume in mature grains. Grain morphology affects rice processing quality, with protein content and grain sink capacity showing a positive correlation (Sreenivasulu et al,2015). Short-term heat stress at the booting and grainfilling stages has increased grain protein content while decreasing grain size and weight. Our study observed a correlation between decreased grain length and increased grain protein content. Sufficient early-stage nitrogen accumulation and reduced spikelet number per panicle can increase grain protein content by affecting the source-sink balance (Liu et al, 2022; Tu et al, 2022). The increased protein content might be attributed to the more adverse effects of heat stress on non-carbohydrate accumulation than the nitrogen in grains. HTS at the grain-filling stage can alter starch metabolism, decreasing amylopectin and amylose contents and increasing starch particle size and crystal structure (Xiong et al, 2017; Tang et al, 2019).Similarly, short-term HTS treatment at the booting stage decreases amylose content (Zhen et al, 2019).However, Zhong et al (2005) suggested that amylose content decreases in low-amylose cultivars, while increases or remains stable in high-amylose cultivars under heat stress during grain filling. In this study,HTS significantly increased the amylose content in low-amylose cultivars. This might be related to a reconstructed balance between the sink and source, as the decrease in sink size is much higher than that in the source size, which can deliver much more substrate for the synthesis of amylose at the grainfilling stage. However, the underlying mechanisms of HTS effects on grain quality at the EPI stage require further investigation. From a nutritional perspective,increasing protein content is beneficial as it provides more nutrition per unit grain, which can partly mitigate the hunger facing future climate change (Loladze et al,2019; Wei et al, 2021), and high amylose content in rice is also beneficial for human health as it can lower blood glucose response in diabetic control (Zhu et al,2012). However, the taste of cooked rice might be down-regulated by excessive protein and amylose contents (Madan et al, 2012). One solution to this trade-off between nutrition and taste is to select and breed heat tolerant rice cultivars.

Endogenous hormone response

The differentiation of rice spikelets is controlled by various hormones, including IAA, cytokinin (CTK),and ABA (Wang Z Q et al, 2018). IAA regulates the generation of lateral meristem during spikelet differentiation by controlling cell polarity determination and elongation(Zhao, 2010). An increase in IAA content or signal transduction leads to an increase in the numbers of branches and spikelets (Tabuchi et al, 2011). Increasing the content of CTK in inflorescence tissue can effectively increase the number of differentiated branches and improve the numbers of branches and spikelets per panicle (Wu et al, 2017, Zhang et al,2022). HTS can disrupt hormone levels, explicitly reducing CTK content and activity, which leads to a decrease in the number of spikelets per panicle (Wang Z Q et al, 2018). This effect can be alleviated by the exogenous application of 6-benzylaminopurine. Under HTS, CTK and ABA regulate the number of spikelets per panicle (Wu et al, 2019). In this study, HTS reduced the contents of growth hormone (IAA and Z +ZR) and increased the content of inhibitory hormone(ABA) in panicles and roots at the meiosis stage. This led to a reduction in the number of differentiated and existing branches and spikelets per panicle, and an increase in the numbers of degenerated branches and spikelets per panicle. Plant hormones play a role in regulating grain development. For example, IAA promotes the growth and development of glume or endosperm cells and affects the shape of rice grains(Hu et al, 2018). The role of glume and lemma in grain development has been observed in studies where the removal of these structures resulted in smaller grains with smaller aleurone cells, and an exacerbated glume degradation upon the application of ABA (Radley,1981). Furthermore,OsGRF4has been found to have a positive impact on grain shape and panicle length by regulating two CTK dehydrogenase precursor genes(Sun et al, 2016).

HTS significantly decreased dry matter production by impairing the nitrogen uptake capacity. Additionally,the decreased sink size restricted the transportation and distribution of dry matter and nitrogen substances to grains, further reducing the number of spikelets per panicle and the quality of grain filling. HTS also had a negative impact on milling by decreasing milled rice and head rice rates, while improving nutritional quality by increasing protein, amino acid, and amylose contents. Different rice cultivars responded differently to HTS. YD6 was more sensitive to HTS than JXY1 in terms of protein and amylose contents. Further research is needed to understand the underlying mechanisms of the effects of HTS on dry matter and nitrogen accumulation, transportation, and distribution,in order to develop strategies to enhance rice adaptability to rising temperatures. Additionally, including more rice cultivars would refine the findings.

METHODS

Experimental design

Pot experiments were conducted at the research farm of Yangzhou University, Jiangsu Province, China (32° 30′ N, 119° 25′E), during the rice growing season from May to October in 2021, using two inbred rice cultivars YD6 (indica) and JXY1(japonica). Both cultivars were sown on 20 May, and seedlings were transplanted into pots on 18 June. Each pot (30 cm high ×25 cm top diameter) was filled with 14 kg of sandy loam soil and contained 3 holes with 2 seedlings per hole. The base fertilizer consisted of 2.2 g of urea, with an additional top dressing 1.1 g urea at 7 d after transplanting and 0.5 g urea at the tillering stage, respectively. Measures were taken to control pests, diseases, birds, and weeds. Calcium superphosphate,equivalent to 0.3 g of phosphorus, and potassium chloride,equivalent to 0.5 g of potassium, were used as base fertilizers for a one-time application. The potted rice plants were arranged randomly with three replications and grown under natural ambient conditions.

The development of young panicles was assessed using the leaf age index or residue method (Ling et al, 1983). Rice plants were grown under natural conditions until the onset of the EPI stage, marked by the visibility of the first row of floral primordia on the shoot apex. Subsequently, the rice pots were exposed to high temperature stress conditions in an artificial climate chamber. After 14 d of treatment, the pots were returned to a natural environment until harvest. The heat stress treatments were conducted in two separate artificial climate chambers. The rice plants were grown under controlled conditions, including 60% relative humidity, day/night cycles consisting of 13 h of light from 6:00 am to 19:00 pm, and 11 h of darkness from 19:00 pm to 6:00 am. Two treatments were applied in this experiment: the control treatment exposed to normal temperatures (NT, day/night: 32 °C / 26 °C) and the high temperature stress treatment (HTS, day/night: 38 °C / 29 °C).The temperature settings were based on historical weather data records, indicating that the temperatures in 2017 exceeded the average levels of 1981-2010 (Fig. S3). Daily temperature profiles inside the artificial growth chamber are shown in Fig. S3.

Dry matter and nitrogen content

At the meiosis, heading, and maturity stages, three specimens of rice plants exhibiting consistent growth were harvested,oven-dried at 105 °C for 30 min in kraft paper bags, and separated into four parts: roots, stems, leaves, and panicles(when present). The samples were cooled to 75 °C, dried to a constant weight, then crushed, passed through 100 mesh sieves,and weighed at 0.50 g. Subsequently, the crushed samples were digested using a combination of a catalyst and concentrated sulfuric acid (H2SO4) at 420 °C for 1.5 h. The solution was clarified, cooled to room temperature, and analyzed for nitrogen content using the automatic Kjeldahl nitrogen analysis method.

Panicle architecture

Three pots of rice plants were selected when 50% of the panicles had emerged from the flag leaf sheath. Fifteen uniform panicles per pot (a total of 45 panicles in each treatment) were selected and analyzed according to Wang W L et al (2018, 2019).Degenerated spikelets, observed under a stereomicroscope (SZH10,Olympus Co, Ltd, Japan) were light white in color, covered in white bract hair, and easily distinguishable from surviving spikelets, which were green. The numbers of surviving and degenerated spikelets were counted, and their respective positions on primary and secondary rachis branches were recorded.Spikelets that developed to a normal size while retaining a green color were considered surviving spikelets, while spikelets that were deformed and small were classified as degenerated spikelets.

Yield and yield components

Three pots of uniformly grown rice plants were selected per treatment at maturity to assess the number of panicles per pot,the number of spikelets per panicle, grain-filling rate, and 1000-grain weight. The number of panicles per pot was manually counted. After manually threshing the panicles, filled,half-filled, and empty spikelets were separated using water and an 80% ethanol solution, respectively. The grain-filling rate was calculated as the ratio of filled spikelet number to the total spikelet number. A subsample of 30 g of filled, 5 g of half-filled and 2 g of empty spikelets were collected to determine the number of spikelets. The grain yield was adjusted for a standard moisture content of 14%. Another five pots of rice per treatment were selected for grain quality measurement,including the manual measurement of grain length and width,and the determination of grain nitrogen content using the abovementioned method, with subsequent multiplication by 5.95 to calculate the protein content.

Hormone content

The determination of the developmental stage and size of panicles followed the method described by Ling et al (1983)and was based on preliminary experiments. The onset of panicle meiosis was identified as the point when the ligule of the flag leaf was 10 cm below that of the penultimate leaf.Three pots of rice plants were selected, and the leaves, root tips(measuring 3-4 cm), and young panicles were harvested. The harvested plant tissues were immediately wrapped in tin foil and flash-frozen in liquid nitrogen for 1 min. They were then transferred to an ultra-low temperature refrigerator at -80 °C for storage until further analysis. The levels of plant hormones including IAA, ABA, and Z + ZR were measured in each tissue sample. To extract the hormones, approximately 0.5 g of each tissue sample was ground into a fine powder using a grinder(MM400, Retsch Crop, Haan, Germany). The samples were then subjected to extraction according to the method described by Pan et al (2010). Subsequently, the extracted samples were analyzed using liquid chromatography-tandem mass spectrometry with multiple reaction monitoring, as described by Pan et al (2010).

Milling and appearance qualities

The rice grains were stored for three months until physical and chemical characteristics stabilized. Then, they were used for grain quality measurement. Before measurement, each treatment used an NP4350 type air separator to carry out air separation volume, in order to eliminate empty grains and half-full grains.Brown rice rate, milled rice rate, head rice rate, gel consistency,and amylose content were determined according to the National Standard of the People’s Republic of China (GB/T 17891-1999).

Amino acid component content

A sample of 10 mg of rice flour was weighed into a centrifuge tube with a rubber gasket, mixed with 500 μL of 6 mol/L hydrochloric acid, centrifuged at 5 000 r/min for 10 min, and then placed in an oven at 110 °C for 1 h. The cap was tightened,and the tube was placed back in an oven for 24 h, with the oven being turned off at the end of each day. The tube was removed,and the cap was opened and placed in a 57 °C water bath for evaporative drying for about 2 d. Then, 1 mL of amino acid sample diluent was added to the centrifuge tube, mixed thoroughly, and centrifuged at 10 000 r/min for 10 min. The supernatant was aspirated through a 0.45 microporous membrane into water sample bottles for the determination of amino acid content by the Biochrom 30 Automatic Amino Acid Analyzer(Biochrom, UK).

Data processes

Pre-anthesis aboveground dry matter (nitrogen) translocation was pre-anthesis aboveground (leaves + stems) dry matter(nitrogen) accumulation minus mature aboveground (leaves +stems) dry matter (nitrogen) accumulation. Pre-anthesis aboveground dry matter (nitrogen) translocation rate (%) was pre-anthesis aboveground dry matter (nitrogen) translocation divided by pre-anthesis aboveground dry matter (nitrogen)accumulation × 100%. Contribution of pre-anthesis aboveground dry matter (nitrogen) translocation to grains (%) was pre-anthesis aboveground dry matter (nitrogen) translocation divided by dry matter (nitrogen) accumulation of grains at maturity × 100%.

Statistics analysis

The data collected from the experiment were processed and analyzed using Microsoft Excel 2016 and IBM SPSS Statistics 27 software. Mean differences were assessed using the least significant difference method at theP= 0.05 level. Data were plotted using Origin 2021 and R (Corrplot, version 4.2.1,https://cran.r-project.org) software tools, with appropriate correlation formulas applied as needed.

ACKNOWLEDGEMENTS

This study was supported by the Jiangsu Agriculture Science and Technology Innovation Fund, China (Grant No.CX(23)1035), the National Natural Science Foundation of China (Grant Nos. 32201888, 32071943, and 32272197), the Provincial Natural Science Foundation of Jiangsu, China(Grant No. BK20200923), the National Key Research and Development Program of China (Grant Nos. SQ 2022YFD1500402 and SQ2022YFD2300304), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China.

SUPPLEMENTAL DATA

The following materials are available in the online version of

this article at http://www.sciencedirect.com/journal/rice-science;http://www.ricescience.org.

Fig. S1. Effects of high-temperature stress at early panicle initiation stage on grain morphology.

Fig. S2. Correlation analysis of grain morphology, carbon-nitrogen accumulation and transportation with grain quality parameters.Fig. S3. Temperature changes over the years and diurnal temperature setting.

Table S1. Effects of high-temperature stress at early panicle initiation stage on essential amino acids contents.

Table S2. Effects of high-temperature stress at early panicle initiation stage on non-essential amino acids contents.

Table S3. Effects of high-temperature stress at early panicle initiation stage on nitrogen contents at meiosis (ME),heading (HD), and maturity (MA) stages.