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On a global scale (Vose et al., 2005; IPCC, 2007) and at the farm level (Peng et al., 2004), minimum night temperatures are increasing at a much faster pace than maximum day temperatures, and this trend is projected to continue into the future (Christensen et al., 2007). Controlled environment studies (Cheng et al., 2009; Mohammed & Tarpley, 2009a,b; Kanno & Makino, 2010), as well as field experiments (Peng et al., 2004; Nagarajan et al., 2010), have recorded a significant negative impact of higher minimum night temperature on rice yield. Hence, efforts must be intensified to address this emerging phenomenon in synchrony with the progress being achieved in breeding for high day temperature tolerance in rice mega varieties (Jagadish et al., 2010; Ye et al., 2012) to induce diurnal temperature tolerance in rice. To achieve this target, a diverse set of entries must be tested for their response to high night temperatures (HNTs), which is a prerequisite to the identification of contrasting entries in order to better understand and explore the physiological and molecular mechanisms that induce tolerance.
The yield penalty under HNT has been attributed to a reduction in the number of panicles per square meter (Peng et al., 2004), final grain weight (Morita et al., 2005; Kanno & Makino, 2010) and spikelet fertility (Cheng et al., 2009; Mohammed & Tarpley, 2009a, 2010), which are partly explained by increased respiration rates, membrane leakage (Mohammed & Tarpley, 2009b) and reduced pollen germination (Mohammed & Tarpley, 2009a). However, the majority of the conclusions drawn above are based on individual genotype performance – for example, IR72 (Cheng et al., 2009), Cocodrie (Mohammed & Tarpley, 2009a,b, 2010) and Akita-63 (Kanno & Makino, 2010); almost all of these studies were conducted under controlled environments. Therefore, there is a significant gap in the identification of contrasting rice genotypes and their physiological and molecular responses on exposure to HNTs under realistic field conditions.
Temperature at night has been speculated to have an impact on the flowering dynamics on the following morning (Kobayashi et al., 2010), but it has not been studied systematically. Photoassimilates generated either during grain filling (post-anthesis) or redistributed from the reserve pool of the vegetative tissues (pre-anthesis) determine successful grain filling in rice (Yang & Zhang, 2006). Limited information is available on the effect of HNT on dry matter production, carbohydrate (sugars and starch) and nitrogen (N) partitioning, and grain filling, which are critical determinants of final grain yield. Final grain weight is determined by the rate and duration of grain filling in rice. However, the magnitude of change with HNT on the rate and duration of grain filling in contrasting rice genotypes has not been estimated. The above-mentioned sequence of yield-influencing processes could have a major influence on grain quality, which is increasingly becoming an essential determinant of the market price, and thus warrants detailed investigation.
To capture the impact of extreme temperatures and other abiotic stresses at the molecular level in rice, the proteomic (two-dimensional gel electrophoresis) approach has been effectively employed (Cui et al., 2005; Jagadish et al., 2010, 2011). However, in the majority of the studies, either vegetative (Salekdeh et al., 2002; Yan et al., 2005) or reproductive (Imin et al., 2004; Liu & Bennett, 2011) tissues, and generally at a single time point, have been used to study proteome changes. Yan et al. (2005), using salt stress-affected rice seedling roots, and Kerim et al. (2003), using anthers at different developmental stages, applied the two-dimensional proteomic approach and demonstrated the proteome dynamics at different time points. To our knowledge, no reports have addressed the proteome changes with HNT using both vegetative and reproductive tissues at economically relevant time points, such as flowering and early grain filling (EGF).
Unlike all the above-mentioned studies, our trial was carried out using temperature-controlled chambers under field conditions. Preliminary wide genetic diversity screening for HNT among 36 different rice accessions using the above-mentioned field chambers formed the basis of this experiment, from which the most contrasting entries were selected for physiological and molecular characterization. Both field and laboratory analyses were undertaken as follows: to estimate the impact of HNT on grain yield and yield components between two contrasting rice genotypes under field conditions; to quantify N, nonstructural carbohydrate (NSC) and biomass partitioning at key developmental stages in response to HNT; to determine the impact of HNT on flowering pattern, rate and duration of grain filling along different sections of the panicle, and grain quality; and to unravel the temporal reprogramming of the flag leaf and spikelet proteome exposed to HNT at flowering and EGF, and to establish their relevance to physiological responses.
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- Materials and Methods
- Supporting Information
Conclusions drawn from controlled environment experiments have documented an HNT-induced increase in respiration rate and decrease in pollen germination (Mohammed & Tarpley, 2010) and poor assimilate translocation to grains (Morita et al., 2005; Cheng et al., 2009; Kanno & Makino, 2010), with a subsequent reduction in seed set and/or grain weight. These conclusions are based on individual genotype performance, whereas our study builds on the outcome of a wide genetic diversity screening (36 accessions) and tests the most contrasting entries from both studies using the same chambers established under field conditions.
A moderate increase in night temperature during the entire reproductive period led to a significant decline in grain yield and total dry matter at physiological maturity with the highly sensitive Gharib. This decline in yield was mainly attributed to a substantial reduction in 1000-grain weight, a phenomenon observed by Morita et al. (2005) and Kanno & Makino (2010). However, the percentage seed set was unaffected in Gharib, which contrasted with the finding of Mohammed & Tarpley (2009a), who noted a 90% reduction in fertility at HNT of 32°C using cultivar Cocodrie (which could be highly susceptible). Our ongoing controlled environment work indicates a similar response from very sensitive varieties exposed to temperatures > 30°C, but the tolerant N22, even under 35°C HNT, recorded a < 5% reduction in sterility (O. Coast et al., unpublished; University of Reading, UK). Hence, preliminary diversity analysis is essential to avoid an overestimation of the temperature effects. In addition, the grain weight of tolerant N22 was unaffected in our field study. However, the number of spikelets per panicle was reduced significantly in N22 with HNT, accompanied by a higher seed set, demonstrating the plastic response of maintaining yield under HNT. Competition for assimilates between the spikelets and the stem during panicle formation has been documented, with spikelets being poorer competitors than the stem for available assimilates (Fischer & Stockman, 1980). In this competition for assimilates between panicle and stem, the stem in N22 appears to have prevailed over the panicle, as evidenced by a 6.5% increase in height and a simultaneous decrease (14.6%) in spikelet number under HNT (Table 1). Moreover, a similar quantitative impact of HNT on spikelet degradation (9.6%) in N22 was observed in an independent experiment using the same chambers, but such plastic responses were not observed with the sensitive Gharib.
A steady supply of assimilates in the 0–10 and 10–20 d following heading is a crucial determining factor for endosperm expansion and grain filling, respectively (Nagata et al., 2001). Carbohydrates for grain filling could either be assimilated during the ripening period or translocated from assimilates accumulated in the leaf sheath and culms before heading (Nagata et al., 2001; Lafarge & Bueno, 2009). In our study, a significant decline in N and NSC content in the sensitive Gharib throughout the ripening period until physiological maturity resulted in assimilate shortage and, with reduced 1000-grain weight and grain yield, indicated a greater limitation with source, although sink strength reduction could not be ruled out. After accounting for the accumulated N after flowering from the initial content + the translocation from the leaves and stem, unaccounted values of 11 mg per hill and 26.2 mg per hill N were recorded in N22 and Gharib panicles, respectively, at 22°C, and 14 mg per hill in both entries at 28°C, indicating the contribution of direct N uptake or active translocation of N stored in the roots during the active grain-filling stage (Fig. 3). Compared with N, NSC translocation to the panicle was more pronounced, with a higher contribution from stem NSC than from leaf NSC (data not shown), as documented earlier (Fu et al., 2011).Comparison of N22 and Gharib across both temperatures independently showed a smaller decrease in NSC translocation in Gharib, which could be equated to the HNT effect only, whereas a larger decrease in N22 could be caused by a combination of the HNT effect and reduced sink size (Fig. 3).
Figure 3. Remobilization of nitrogen (N; mg per hill) from rice (Oryza sativa) leaves (N content in leaves at flowering – N content in leaves at physiological maturity (PM)), stem (N content in stems at flowering – N content in stems at PM) and possibly roots into panicles, that is, N increase in panicles (N content in panicles at PM – N content in panicles at flowering), and nonstructural carbohydrate (NSC) translocation ratio from stem to panicles (amount of NSC transferred from stem to grains/NSC in stems at flowering × 100) of N22 (a) and Gharib (b); white circles represent 28°C and black circles represent 22°C. Horizontal bars indicate ± SE.
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Grain filling, the final stage of growth in cereals, is determined by the product of the rate and duration of grain growth. A negative relationship between the rate and duration of grain filling has been established (Yang et al., 2008). N22, which has considerably higher initial, maximum and mean grain-filling rates across the whole panicle, was able to compensate for a significant reduction in active grain-filling duration and maintained grain yield (Table 3). Interestingly, the plastic behavior of N22 to the deliberate reduction in the number of spikelets per square meter probably allowed the remaining spikelets to receive sufficient assimilates within the shortened grain-filling duration, a response that was absent in the susceptible Gharib. In addition, this response would allow assimilate saving, which otherwise would have been utilized for the production of additional nonproductive spikelets. Gharib, however, showed a higher initial grain-filling rate, but the maximum and mean grain-filling rates were decreased greatly, in both middle and bottom portions of the panicles, together with the grain-filling duration in the top and middle parts of the panicle, thereby reducing the final grain weight. Our results confirm the conclusions of Kobata & Uemuki (2004) that a lower yield caused by high temperature during grain filling may be a result of the failure of assimilate supply to meet the accelerated grain-filling rate. This was the case with Gharib. Further, a significant synergistic correlation between the grain-filling rate and grain weight (but not between the grain-filling duration and grain weight) in bread and durum wheat under high temperature has been recorded (Dias & Lidon, 2009). Ideally, rice varieties with sufficient biomass, equipped with efficient translocation efficiency (high grain-filling rates) to compensate for the reduced grain-filling period, could potentially overcome the impact of HNT on grain yield.
HSPs are functionally involved in the repair and renaturation of stress-damaged proteins, in addition to protecting the cells against the effects of stress (Wang et al., 2004; Sarkar et al., 2009; Jagadish et al., 2011). Peptidyl-prolyl cis–trans isomerase (FKBP-type) was particularly up-regulated in early grain-filling spikelets in N22 and was down-regulated in the case of Gharib, with PPIase (peptidylprolyl isomerase) possibly having a positive role in maintaining protein synthesis and trafficking proteins during the active grain-filling stage. This protein is known to be induced in floral tissues under heat stress in wheat (Kurek et al., 1999) and works in tandem with HSP90 to ensure the correct folding of proteins in Arabidopsis thaliana (Hagai et al., 2007). Late embryogenesis abundant protein, which behaves like HSP12 in Saccharomyces cerevisiae, was up-regulated in the early grain-filling panicle of both varieties, showing its role in grain filling under heat stress and preventing other proteins from heat-induced desiccation. Calcium, a universal signaling molecule under heat stress, triggers cytosolic Ca2+ bursts, which are transduced by several Ca2+-binding proteins (CBPs), such as calmodulin (CaM), CaM-related proteins, Ca2+-dependent protein kinases (CDPKs), etc., that further up-regulate the expression of HSPs (Liu et al., 2003; Yang & Poovaiah, 2003). In our study, CBPs, such as CaM-dependent protein kinases, CaM-binding protein and IQ CaM-binding motif family protein, were more strongly up-regulated in tolerant N22, whereas the first two proteins were unchanged and down-regulated, respectively, in the susceptible Gharib panicles. Phosphatidylinositol 3- and 4-kinase family protein, which is involved in phosphate signaling in animals, was up-regulated at the 100% flowering stage, but more strongly at 12 DAF, indicating its role in high temperature stress signaling in N22, whereas the same protein was undetected in susceptible Gharib. Among the proteins involved in sugar metabolism, β-mannosidase/glucosidase homolog was highly up-regulated only in N22, whereas the three other proteins were equally up-regulated in both entries. The CUE (coupling of ubiquitin to ER degradation) domain-containing protein, which is involved in the degradation of misfolded proteins in the endoplasmic reticulum and protein sorting, was up-regulated in both varieties, with a higher level of expression in N22 at the EGF stage. In addition, histone acylation by GCN5 (general control non-repressed protein 5) and HAC (histone acetyl transferase) helps in the transcriptional regulation of HSP70 and HSP17 genes, which are actively involved in correct protein folding and sequestration under high temperature stress (Bharti et al., 2004; Han et al., 2008). Maturase K could assist in splicing its own and other chloroplast group II introns, showing more active transcription of heat stress-responsive gene up-regulation in N22 (but down-regulation in Gharib). Proteins involved in the biosynthesis of RuBISCo were down-regulated in both genotypes, which could result in reduced photosynthetic rate with a pre-exposure to HNT, a phenomenon documented in wheat (Prasad et al., 2008). The majority of the significantly changing proteins at the 100% flowering stage were detected at 12 DAF in both flag leaves and spikelets, whereas those that were sequenced from tissues at 12 DAF were undetected at 100% flowering. This indicated dynamic proteome programming with different tissues at key developmental stages in rice when exposed to HNT. The combined increase in HSPs and Ca signaling proteins, and the better nucleic acid/protein modification and repair in tolerant N22 at the EGF stage, could have allowed for better enzymatic activity in the conversion of sucrose to starch.
Rice market prices are largely determined by milling quality outcomes and appearance, that is, higher chalk or brokens reduce rice prices dramatically. The significant reduction in milled rice yield and the increase in chalk content (with the highest chalk category in Gharib) are proxy for the negative impact of HNT on grain weight (reduced grain width), leading to reduced yield and total milled rice. The decrease in grain width could be associated with a reduction in average endosperm cell area observed under HNT (Morita et al., 2005), or with abnormal amyloplast packaging, resulting in white core chalk formation (Ishimaru et al., 2009). From source–sink manipulation studies, a close relationship between assimilate supply and milky white chalk formation has been established (Tsukaguchi & Iida, 2008), with increasing assimilate supply overcoming chalk formation even under high temperatures (Kobata & Uemuki, 2004). In addition, higher maintenance respiration with increasing night temperatures could partly be responsible for reduced assimilate supply, as documented by Cheng et al. (2009) and Mohammed & Tarpley (2010). Chalkiness was not a problem with N22, mainly because of the increased grain-filling rates and little influence on overall biomass, even under HNT. Interestingly, chalkiness under the 50–75% category was reduced significantly in Gharib with HNT, a feature that could be attributed to better assimilate transfer at the initial grain-filling stages, but, with a lack of sustained supply of assimilates, this resulted in a 36.4% increase in the > 75% chalkiness category. Moreover, Gharib with a comparatively higher biomass than N22 could have a relatively higher demand for maintenance respiration, depriving a larger share of assimilates over the 2-month-long HNT exposure.