RPBF induced by HT, while RISBZ1 was possibly mainly responsible for regulating the response of glutelin fam-ily genes to HT at the earlier filling stage. In addition, HT accelerated expressions of PDI and BiP to assist with the proper folding and assembly of proglutelin.
Keywords Rice (Oryza sativa L.) · High temperature · Storage protein · Gene expression
Introduction
Temperature is one of the most important environmental factors affecting plant growth and development. It is well known that high temperature (HT) above certain growth-optimal ranges can negatively affect the yield and quality of crop products by impairing dry matter production and alter-ing grain chemical components (Peng et al. 2004; Geigen-berger 2011). As an important crop, rice (Oryza sativa L.) provides staple food for more than half of the world popu-lation; however, rice yield and grain quality are particularly susceptible to HT at the reproductive stage. Average daily temperatures exceeding 26–28 °C during the grain-filling period notably deteriorate grain quality, and make rice unsatisfactory for human consumption, thus air temperature at grain-filling stage is the dominant environmental factor influencing rice grain quality, with higher chalky appear-ance and lower amylose content typically caused by HT during this stage, in addition to decreased grain weight and filling duration for the HT-ripening rice grain (Krishnan et al. 2011; Geigenberger 2011; Jagadish et al. 2015).
Storage proteins are the second major abundant com-ponent next to starch in rice grain, contributing approxi-mately 8–10 % of grain weight, and play crucial role in determining nutritional quality and the pasting and textural
Abstract High temperature (HT) at the filling stage is a major environmental constraint to rice (Oryza sativa L.) grain quality. However, the effects of HT on the accumu-lation and composition of storage proteins are not well understood. In this study, the transcriptional expressions of genes related to storage protein synthesis responsive to HT, and their relationship with storage protein compo-sition, were comprehensively analyzed under controlled temperature using two non-waxy indica rice cultivars, 9311 and II-7954. HT reduced grain weight and increased total protein content (TPC) irrespective of rice genotype, and HT-inducible increase in TPC was attributable to the relative increase in proportion of the aleurone fraction to whole grain, as well as increased absolute amount of TPC in rice grain; HT resulted in a relatively higher ratio of glu-telin to prolamin compared with the control temperature, as reflected by a significant decrease in amount of 13-kDa prolamin polypeptide and an enhanced accumulation of proglutelin, α-glutelin and β-glutelin subunits throughout the whole grain filling; The remarkably lowering of tran-scripts of glutelin and/or prolamin family genes in HT-ripening grain at the middle and late filling stage possibly were closely associated with the repressing regulation of
Electronic supplementary material The online version of this article (doi:10.1007/s10725-016-0225-4) contains supplementary material, which is available to authorized users.
properties of cooking rice (Tan and Corke 2002; Kawakatsu et al. 2010). Traditionally, storage proteins in cereal plants are classified into albumin, globulin, prolamin and glute-lin based on solubility (Shewry and Halford 2002). In rice, glutelin is the major storage protein accounting for 80 % of the total endosperm protein, and consists mainly of 37-kDa acid subunit and 22-kDa basic subunit linked by both intra- and inter-molecular disulfide bonds; whereas prolamin is regarded as a minor rice storage protein only accounting for about 5 % of the total endosperm protein, with three distinct polypeptide subgroups (10, 13 and 16 kDa) being distinguished by their molecular mass on SDS-PAGE (Yamagata et al. 1982; Ogawa et al. 1987). Previous stud-ies suggested that storage protein-related polypeptides in cereal endosperms are synthesized by the membrane-bound polysomes derived from the rough endoplasmic reticulum (RER), and then deposited mostly in special storage orga-nelles called protein bodies (PBs), which are grouped into two different types: PB-I and PB-II. The polypeptides of prolamin are directly formed within the lumen of the RER and preferentially accumulated in PB-I, while glutelin appears to be preferentially deposited in the PB-II derived from the protein body surrounding vacuoles (PSV), and the two smaller glutelin subunits are derived from a precursor polypeptide and packaged in protein bodies by the cleavage of 57-kDa polypeptide (Yamagata and Tanaka 1986; Kum-amaru et al. 2010; Kawakatsu et al. 2010).
The storage protein content and its different fractions in rice endosperms depend primarily on genotype, but are also substantially affected by environmental factors. It is widely accepted that HT-ripening rice grain has relatively higher levels of storage proteins compared with that grown under low temperature (Yamakawa et al. 2007; Cooper et al. 2008; Ashida et al. 2013). Nevertheless, considering the altering extent of storage protein in rice endosperm influenced by HT exposure, some previous reports seem controversial. For instance, Cooper et al. (2008) and Lin et al. (2010) concluded that HT exposure could lead to a significant increase in crude protein con-tent of rice endosperm, but non-significant differences in storage protein content were also found among different temperature regimes in many cases. In contrast, accord-ing to Yamakawa et al. (2007), HT impaired the storage protein deposition in rice endosperm, and HT-inducible increase in storage protein content in rice endosperm was likely due to severely repressed starch accumulation for rice plants exposed to HT during grain filling, thereby enhancing the dry-weight percentage of storage protein to total starch. Recently, Ashida et al. (2013) revealed that HT could affect the balance of different protein fractions in rice endosperm, and lead to a marked increase in the ratio of glutelin to prolamin. Up to date, the influence of HT exposure on accumulation of different classes of rice
storage protein and the relationship to the storage protein biosynthesis in filling grain are not clearly understood.
In past decades, many efforts have been made to elu-cidate the genetic and physiological mechanisms of cereal storage protein accumulation. In rice, many differ-ent classes of genes encoding storage proteins have been explicitly identified, and their metabolic functions in the regulation of storage protein biosynthesis have been basi-cally recognized (Kawakatsu and Takaiwa 2010). Rice glutelin is encoded by a multigene family, which can be classified into two classes of gene subfamilies: GluA1-A3 and GluB1-B5 (Kawakatsu and Takaiwa 2010), while prolamin multigene family members for rice mainly com-prise Pro13, Pro14 and Pro 17 (Saito et al. 2012). All these genes play unique or overlapping roles in the poly-peptide synthesis of precursor glutelin and prolamin. Fur-thermore, an ER luminal binding protein (BiP) facilitates the folding and assembly of prolamin in the formation of PB-I, and protein disulfide isomerase (PDI) may help in the sorting process for both glutelin and prolamin, which play crucial roles in the post-translational folding/sort-ing and processing of polypeptide biosynthesis (Li et al. 1993; Takahashi et al. 2005; Satoh et al. 2010; Kim et al. 2012). Recently, two types of transcription factor, basic leucine zipper factor (RISBZ1) and rice prolamin box binding factor (RPBF), are identified as the cis-regula-tory elements that bind to the promoter region of storage proteins genes and are involved in the endosperm-specific expression of storage protein genes in rice and other higher plants (Kawakatsu et al. 2009). However, so far the influence of HT exposure on comprehensive expres-sions of these genes and its relationship to storage protein accumulation has been little investigated in rice.
In this study, two non-waxy indica rice cultivars, 9311 and II-7954, were applied to examine the effect of HT exposure at the filling stage on storage protein accumu-lation and composition in brown and milled rice grain. Aiming to clarify the metabolic alterations in storage protein accumulation caused by HT exposure, we further investigated the temporal and spatial patterns of tran-scriptional expression of several gene families related to storage protein synthesis. We comprehensively compared the mRNA transcriptional levels in response to HT expo-sure for these genes and their various isoforms involved in rice storage protein accumulation. Our candidate genes covered a wide range of multigene families controlling some key metabolic steps relevant to storage protein accumulation, such as protein polypeptide biosynthesis, protein subunit assembly, protein disulfide-bond folding and other regulatory factors. Such results will be helpful for understanding the HT-inducible alteration in the tran-script expressions of multiple family genes participating in storage protein biosynthesis.
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Materials and methods
Plant materials and growth conditions
Two indica rice genotypes (O. sativa L.) with similar growth period and plant morphology, namely, 9311 and II-7954 were used for this study. The two genotypes dif-fer in their tolerances of grain weight to HT, in terms of a previous screening survey of grain-yield loss under HT exposure (Su et al. 2014). Seeds were sown on 30th April and transplanted on 28th May, 2013 in the experimental field of Zhejiang University, Hangzhou, China. Plants were managed normally in paddy field until heading stage; then 50 plants with the same size were selected and transplanted into 10-L plastic pots (2 plants per pot) filled with paddy soil. The pots were placed in a greenhouse under natural light conditions and moderate growth temperatures (28 °C daytime/22 °C nighttime) until rice plants were imposed to different temperature treatments. At full heading stage, 34–40 panicles with uniformity anthesis day were randomly selected and tagged. The plastic pots were moved into phy-totrons (Model PGV-36, Conviron, Canada) set at differ-ent temperatures. The tagged panicles were sampled at a 7-days interval. Grain samples were immediately frozen in liquid nitrogen and stored at −80 °C until further analysis. At maturity, the other tagged panicles were harvested for determining storage protein content and composition.
Two temperature treatments were imposed in two phy-totrons. The daily mean temperatures were 33 °C (high temperature treatment, HT) and 23 °C (normal temperature treatment, NT), respectively. The diurnal change of tem-perature was designed by a simulation of daily temperature fluctuation based on natural climate. The daily maximum and minimum temperatures were set up at 2:00 p.m. and 5:00 a.m., with 38 and 28 °C for HT, 26 and 20 °C for NT, respectively. Two phytotrons were kept without distinction in other climate conditions except for temperature treat-ment, the photoperiod was from 5:30 a.m. to 7:00 p.m. with 150–180 J m−2 s−1 of light intensity, and the relative humidity was maintained around 80–85 % with a wind speed of 0.5 m s−1.
Determination of TPC and protein composition
The TPC was measured by the Kjeldahl method using an Auto-analyzer (Kjeltec 8400, FOSS, Sweden). Prior to TPC analysis, the matured grains were sampled from the two rice cultivars with different temperature regimes. After air-drying, the aleurone fraction and milled rice grain were separated from the husked rice grain using a rice polisher (Model JNMJ3, Taizhou Grain Industry Instrument Crop, China) based on the different degree of milling as described by Wang et al. (2011). The samples of brown and milled
rice grain were ground using a portable grinder (Model JFSJ100, Taizhou Grain Industry Instrument Crop, China) fitted with a 0.25 mm screen. The aleurone fraction was ground by hand to obtain fine powdered flour using a mor-tar and pestle. Subsequently, the flour samples from brown rice grain, milled rice grain and aleurone fraction were separately digested with H2SO4 at 360 °C for 150 min in a digestion apparatus (Tecator Digestor Auto, FOSS, Swe-den). After cooling, the sample solutions were injected into a Kjeldahl analyzer (Kjeltec 8400 Analyzer Unit, FOSS, Sweden). Distillation and titration were carried out auto-matically. After the total N concentration was determined, TPC was calculated by multiplying 5.95 as the protein conversion factor, and the results were reported on a dry weight basis. Triplicate measurements were performed for each sample.
The flour samples from brown and milled rice grain were further used to measure their storage protein composi-tions. Four protein fractions (Albumin, globulin, prolamin and glutelin) were sequentially extracted in the order given below by stirring the flour (0.5 g flour/25 mL solvent) for 3 h at room temperature in the following solvents: albumin, 10 mM Tris–HCl, pH 7.5; globulin, 1 M NaCl, 10 mM Tris–HCl, pH 7.5; prolamin, 55 % (v/v) n-propanol, 10 mM Tris–HCl, pH 7.5; glutelin, 0.24 % CuSO4, 1.68 % KOH, 0.5 % potassium sodium tartrate, and 50 % (v/v) iso-pro-panol. After centrifugation at 4000×g for 10 min at room temperature, the contents of the former 3 protein fractions were determined using the Bradford reagent according to Bollag and Edelstein (1990), and the glutelin content was determined by using a calibration curve established by the Kjeldahl method (Holme and Peck 1998).
SDS‑PAGE analyses of storage proteins
SDS-PAGE was run according to the method described by Yamagata et al. (1982) using a Bio-Rad Mini-Protean II cell (Bio-Rad Laboratories; USA). The immature grain and mature grain of 9311 were sampled at 14 DAA (day after anthesis) and the physiological maturity period (28 DAA), respectively. After being dried, the brown and milled rice grain were separately ground into powder, and then the powdered samples were used to extract the glutelin plus prolamin by SDS-urea method (Yamagata et al. 1982). The extracting SDS-urea solution contained 100 mM Tris–HCl (pH 6.8), 4 M urea, 2 % SDS, 5 % β-mercaptoethanol, 20 % glycerol. An amount of 30 mg powder was placed into a 2.0 mL Eppendorf tube contained 0.8 mL SDS-urea solu-tion, the mixture was shaken overnight at room tempera-ture, and subsequently were centrifuged at 12,000×g for 10 min. Then the supernatant fluid was moved into a 0.2 mL Eppendorf tube, and 20 μL of supernatant fluid was used for the electrophoresis. Electrophoresis was conducted
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with 15 % SDS-PAGE gel, followed by Coomassie Brilliant Blue R-250 staining. The molecular weight of each band was determined by using pre-stained full-range rainbow molecular weight markers (PM0032, Dingguo Biotech, China) as indicated.
The amount of protein was determined by densitom-etry analysis (Lin et al. 2010). Gel images were taken by scanner (UVP EC3 Imaging System, USA), and signal intensities of target polypeptide bands were measured by Image software (Vision Works LS) and estimated as the area × density of band. Net signal intensities of the target bands were averaged from at least two biological replicates.
Quantitative real‑time PCR and semi‑quantitative RT‑PCR
Total RNA was extracted from 20 grains of different fill-ing stages using the RNeasy plant mini kit (Qiagen, Ger-many). First-strand cDNA was prepared from 1 μg total RNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas, Lithuania) and Oligo-dT18 primer. Aliquots of the first-stand cDNA mixtures served as the templates for quantitative Real-time PCR analysis by using the SYBR Green Real-time PCR Master Mix reagent Kit (Toyobo, Japan). Reactions were performed on CFX96 (Bio-Rad, USA) according to the manufactures protocols. The gene-specific primer pairs were designed by Primer Premier 5.0 (Premier, Canada) as listed in Supplement 1. The ampli-fication of Actin-1 gene was performed as a control and a standard curve was checked. The average value and stand-ard error were calculated from three independent biological replicates.
Semi-quantitative RT-PCR was also performed to exam-ine the expressions of candidate genes by using the same primer pairs of each gene (Supplement 1). The reactions were performed using EasyTaq™ DNAPolymerase reagent Kit (Invitrogen, USA). The amplification products were checked on 2 % (w/v) electrophoresis agarose gel.
Data analyses
Statistical analyses were performed with statistical software SPSS 17.0 (SPSS Inc., Chicago). Data were submitted to one way-ANOVA and the means were tested by least sig-nificant difference (LSD) at 5 % probability.
Results
Influence of HT exposure on total protein content and protein composition in brown and milled rice grain
Table 1 showed that temperature treatments during grain filling strongly influenced grain weight and total protein content (TPC) in rice grain. HT-ripened grain had lower grain weight and higher TPC than NT-ripened one irre-spective of genotype, and significantly elevated TPC was observed for both brown and milled rice grain, as well as aleurone fraction of HT-ripened grain (P < 0.01).
An enhanced percentage of glutelin to TPC was observed for HT-ripened grain, but the ratio of prolamin to TPC was reduced by HT, although the altering extent was genotype-dependent (Table 2). In contrast, the storage pro-tein composition of 9311 was more susceptible to HT expo-sure than that of II-7954, as reflected by their differential values in the percentage of prolamin and/or glutelin to TPC between the two temperature regimes. However, HT did not significantly affect the percentage of albumin and globulin to TPC in brown and milled rice grain, with a significant difference in albumin only found for milled rice grain of 9311 between different temperature regimes. Furthermore, milled rice grain tended to have a relatively higher ratio of glutelin to prolamin than brown rice grain irrespective of temperature.
HT-ripened grain contained significantly higher amount of 57-kDa proglutelin at different sampling stages (14 and 28 DAA) compared with NT grain, while they accumulated almost similar amounts of β-glutelin at 28 DAA (Fig. 1a,
Table 1 Differences in grain weight and total protein contents in 9311 and 7954 genotypes in response to temperature treatments
HT high temperature treatment, NT normal temperature treatment, difference = value at HT − value at NT; Data are means ± standard errors of three biological replicates**P < 0.01 between HT and NT
Cultivars Temperature treatment Grain weight (mg/grain) Total protein content (%)
b). In addition, HT exposure tend to increase the amount of α-glutelin in rice grain (brown and milled), although there was no significant difference between the two temperature regimes for brown rice grain at 28 DAA. Interestingly, the amounts of 57-kDa proglutelin accumulated in rice grain (brown and milled) at 28 DAA were somewhat lower than that at 14 DAA; however, there was a reverse tendency for β-glutelin, indicating that the accumulation of β-glutelin in rice grain lags behind the bulk of 57-kDa proglutelin.
The evident band of the prolamin fraction on SDS-PAGE gels was 13-kDa polypeptide, and other prolamin types (16 and 10 kDa) were detectable at very low levels, independence of different temperature regimes (Fig. 1a, b). In response to HT, relatively lower amounts of 13-kDa pol-ypeptide were detected for HT-ripened grain at 14 and 28 DAA, except for HT-ripened milled rice grain at 14 DAA, implying that HT exposure represses accumulation of 13- kDa prolamin in rice grain at middle and late filling stage.
HT‑inducible change in transcriptional expressions of various genes relevant to storage protein synthesis in filling grain
The genes of three GluA, four GluB and Pro13 were expressed exclusively in developing rice grain and little of their mRNAs was detected in leaf (Fig. 2a). Moreover, most proglutelin biosynthesis-related genes (three GluA and four GluB genes) displayed almost similar changes in their temporal patterns of transcript abundance under NT, with the relatively higher transcript levels at the early and/or middle filling stages compared with the later filling stage (28 DAA). Interestingly, HT exposure generally enhanced the transcript levels of the seven genes encoding proglutelin
biosynthesis at the early filling stage (7 and/or 14 DAA), although the enhancing extent was greatly variable depend-ing on different genes, however, their transcript levels in HT-ripening grain significantly decreased and were barely detectable at the middle and late stage (21 and 28 DAA). Further detection using quantitative real-time reverse tran-scription PCR (Fig. 2c) was similar to those obtained by semi-quantitative reverse transcription (RT)-PCR analy-sis (Fig. 2b). These results suggest that HT accelerates the transcriptional expressions of glutelin family genes at the early filling stage, but it has an inhibitory impact on the bio-synthesis and accumulation of glutelin composition in rice grain at the middle and late filling stage. In contrast, the transcripts of Pro13, Pro14 and Pro17 encoding 13-kDa prolamin increased and peaked at 14–21 DAA under NT, however, their transcript abundances in HT-ripening grain were strongly repressed by HT exposure throughout the entire filling stage (Fig. 2b, c), indicating that the prolamin family genes are somewhat difference from the glutelin related genes in their responses of transcriptional expres-sion to HT exposure.
Similar to genes of GluA, GluB and Pro families, RISBZ1 and RPBF were expressed preferentially in devel-oping rice grain and weakly in leaf (Fig. 3a). However, RISBZ1 differed from RPBF in the temporal pattern of transcriptional expression in filling rice grain. Under NT, the transcript amounts of RISBZ1 decreased gradually with grain filling; however, it was not this case for RPBF, with relatively enhanced transcript level at the later filling stage (Fig. 3b, c). In response to HT exposure, there was a slight change in RPBF transcript in the earlier filling stage, but the expression of RPBF in HT-ripening grain was strongly repressed at the later filling stage (21 and 28 DAA).
Table 2 Differences in four storage protein composition in 9311 and 7954 genotypes in response to temperature treatments
HT high temperature treatment, NT normal temperature treatment; difference = value at HT − value at NT; Data are means ± standard errors of three biological replicates*P < 0.05 between HT and NT, **P < 0.01 between HT and NT
Cultivars Grain samples Temperature treatment Albumin (%) Globulin (%) Prolamin (%) Glutelin (%) Glutelin/prolamin
Comparatively, transcript level of RISBZ1 was substan-tially enhanced by HT exposure at the initial filling stage (7 DAA), and then the RISBZ1 expression in HT-ripening grain decreased rapidly to an extremely low level, and remained significantly lower than that for NT grain there-after (14 and 21 DAA) (Fig. 3b, c). These results suggest
that the remarkably lowering transcripts of glutelin and/or prolamin family genes in HT-ripening grain at the mid-dle and late stage were possibly closely associated with the repressing regulation of RISBZ1 and RPBF induced by HT.
From Fig. 3, the transcript level of PDI in rice grain was significantly enhanced by HT exposure. However, the
Fig. 1 SDS-PAGE analysis of glutelin and prolamin in response to temperature treatments for 9311 milled and brown rice grain. a Coomassie brilliant blue staining of SDS-PAGE gel containing glu-telin and prolamin extracts. b Signal intensities of target polypeptide bands of glutelin and prolamin. M-HT milled rice-HT, M-NT milled
rice-NT, B-HT brown rice-HT, B-NT brown rice-NT, 14 and 28 indi-cate different days after anthesis (DAA), respectively; MW the protein marker of standard molecular weight, NS no significant difference between HT and NT, *P < 0.05 and **P < 0.01 between HT and NT; data are means ± standard errors of two biological replicates
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Fig. 2 Organ specificity (a) and RT-PCR, qRT-PCR (b, c) analysis of temporal expressions of genes related to storage protein polypep-tide biosynthesis in response to temperature treatments. HT high temperature treatment, NT normal temperature treatment, GHT rice
grain-HT, GNT rice grain-NT, LHT rice leaf-HT, LNT rice leaf-NT, 7–28 indicate different days after anthesis (DAA), respectively; data are means ± standard errors of three biological replicates
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response of BiP expression to HT exposure was phase-dependent, the elevated level of BiP transcript in HT-rip-ening grain occurred at the earlier filling stage (7 and 14 DAA), but HT exposure resulted in greatly lowering lev-els thereafter (21 and 28 DAA) (Fig. 3b, c). Furthermore,
PDI was consistently expressed both in developing grain and in leaf, while BiP was predominantly expressed in rice grains (Fig. 3a), indicating a difference in tissue-specificity between PDI and BiP expressions in response to HT exposure.
Fig. 3 Organ specificity (a) and RT-PCR, qRT-PCR (b, c) analysis of temporal expressions of genes related to storage protein transcrip-tional regulation and subunit assembly in response to temperature treatments. HT high temperature treatment, NT normal temperature
treatment, GHT rice grain-HT, GNT rice grain-NT, LHT rice leaf-HT, LNT rice leaf-NT, 7–28 indicate different days after anthesis (DAA), respectively; data are means ± standard errors of three biological rep-licates
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Discussion
Effect of HT exposure at grain filling stage on storage protein accumulation and composition in brown and milled rice grain
Changes in environmental temperature can change the chemical ingredients of rice grain such as starch and stor-age proteins during grain filling, thus affecting rice nutri-tional quality (Krishnan et al. 2011; Jagadish et al. 2015). In the past decades, most research has focused on the effects of HT on starch, and it is well recognized that higher environmental temperature during the grain-filling period causes decrease in starch deposition in rice grain, thereby leading to reduced grain weight (Geigenberger 2011). Compared with the effect of HT on starch in rice grain, the effect on protein has been little studied. Some reports have demonstrated that HT-ripening rice grain has relatively higher level of storage protein compared with that grown under low temperatures (Yamakawa et al. 2007; Cooper et al. 2008; Lin et al. 2010). Nevertheless, the extent of HT-inducible increase in storage protein content has been con-troversial. For instance, Yamakawa et al. (2007) observed that HT had more negative effect on the level of starch than it did on protein for rice plants exposed to HT during grain filling, thereby leading to smaller developing grain and a relatively high percentage of protein for HT-ripening rice. In contrast, Cooper et al. (2008) and Lin et al. (2010) observed that rice grain weight and starch accumulation were significantly lower under HT, whereas crude protein did not vary among temperature treatments for all culti-vars. In the present study, HT caused significant decrease in grain weight and increase in TPC in HT-ripened grain compared with NT-ripened one irrespective of genotype (Table 1). Moreover, significantly increased TPC was not only observed for brown rice grain and the aleurone frac-tion of HT-ripened grains, but also observed for milled rice grain (P < 0.01), suggesting that the increase of TPC in HT-ripened grains is not only attributed to the decrease in grain weight and the relatively increase in proportion of the aleu-rone fraction to whole grain, but also due to the increase absolute amount of TPC in rice grain, because the statisti-cal significant difference in TPC (P < 0.01) was also shown for milled rice grain between the two temperature regimes (Table 1). The increased TPC in milled rice grain was pos-sibly due to the fact that HT exposure accelerated stor-age protein biosynthesis during grain filling, leading to an increased accumulation of storage protein for HT-ripened grain; One the other hand, relative to the storage protein, starch accumulation was more susceptible to HT exposure, resulting in reduced grain weight and relatively enhanced percentage of storage protein to whole grain when rice were exposed to HT during grain filling.
Rice storage proteins are generally classified into glute-lin, prolamin, globulin, and albumin. Glutelin is the major storage protein accounting for 80 % of the total endosperm protein (Shewry and Halford 2002). Previous studies have described that HT exposure during grain filling could lead to altering percentages of the four storage protein compo-sitions, as reflected by a marked increase in the ratio of glutelin to prolamin in HT-ripening rice grain (Lin et al. 2010; Ashida et al. 2013). In the present study, there was a relatively enhanced percentage of glutelin to TPC for HT-ripened grain, but the ratio of prolamin to TPC was reduced by HT exposure, leading to a remarkable increased ratio of glutelin to prolamin in brown and milled rice grain, although the altering extent was genotype-dependent (Table 2). Our observation strongly supported several pre-vious findings that HT exposure during grain filling could lead to a marked increase in ratio of glutelin to prolamin in HT-ripening rice grain (Lin et al. 2010; Ashida et al. 2013). Furthermore, it was also found that a relatively higher ratio of glutelin to prolamin in milled rice grain compared to brown rice grain irrespective of temperature treatments (Table 2). These results indicate that the increased TPC in HT-ripening rice grain is mainly caused by enhanced accu-mulation of glutelin in milled rice grain when rice plants are exposed to HT during grain filling.
Effect of HT exposure on transcriptional expressions of genes relevant to storage protein accumulation and their association with storage protein accumulation in filling grain
Proglutelin is encoded by a multigene family consisting of at least two distinct classes: GluA and GluB. The sub-family GluA contains three active gene numbers (GluA1, GluA2 and GluA3), while subfamily GluB consists of at least four active numbers: GluB1, GluB2, GluB4 and GluB5 (Kawakatsu and Takaiwa 2010). Moreover, the glutelin mature subunits are not directly encoded by indi-vidual mRNAs but are the products of post-translational processing (Yamagata et al. 1982; Ogawa et al. 1987). The multigene family for rice 13-kDa prolamin mainly com-prises Pro13, Pro14 and Pro17 (Saito et al. 2012). Previ-ous studies have demonstrated distinct differences in tem-poral expressions of proglutelin and prolamin family genes in rice endosperm development and their responses to HT. According to Yamakawa et al. (2007), the expression of 13-kDa prolamin genes was steadily decreased under HT with developing caryopses. In contrast, Lin et al. (2010) found that HT enhanced transcription of genes for proglu-telin and prolamin at the early filling stage but decreased their expressions at the later stage. In the present study, HT exposure accelerated the expressions of proglutelin family genes at the early filling stage (7 and 14 DAA), but
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repressed the expressions of these genes at the middle and late filling stages (21 and 28 DAA); In contrast, the tran-scripts of 13-KDa prolamin family genes were repressed by HT throughout the entire filling stage (Fig. 2b, c). These results suggest that 13-kDa prolamin family genes are more susceptible to HT than proglutelin family genes, which results in relatively lower 13-kDa prolamin polypeptide in HT-ripening grain at 14 and 28 DAA compared with NT one (Fig. 1). Interestingly, although significantly repressed expressions of proglutelin family genes were observed in HT-ripening grain at the middle and late filling stages (21 and 28 DAA), enhanced accumulation of proglutelin was detected at these stages (Figs. 1, 2). The possible reason is that the cleavage of 57-kDa polypeptide lagged behind its biosynthesis at the early filling stage under HT, result-ing in correspondingly increased accumulation of 57-kDa polypeptide at the middle and late filling stages (21 and 28 DAA) in HT-ripening grain.
The differential temporal expressions of proglutelin and prolamin family genes under HT are primarily regulated at the transcriptional level by transcription factors Two impor-tant transcription factors, RISBZ1 and RPBF, have been identified as the cis-regulatory elements that bind to the promoter region of storage protein genes and are involved in the endosperm-specific expression of storage protein genes in rice and other higher plants (Onodera et al. 2001; Yamamoto et al. 2006; Kawakatsu et al. 2009). In the pre-sent study, RISBZ1 and RPBF were expressed preferentially in developing rice grain and weakly in leaf (Fig. 3a), which is similar to expressions of the genes of GluA, GluB and Pro families (Fig. 2a), confirming that RISBZ1 and RPBF play essential roles in the endosperm-specific expres-sion of storage protein genes. Moreover, RPBF transcrip-tion was remarkably repressed throughout the grain-filling phase, whereas RISBZ1 expression was transiently induced by HT at the initial filling stage (7 DAA), and then rap-idly decreased to an extremely low level thereafter (14–28 DAA) (Fig. 3b, c). These results imply that rapidly lower-ing transcripts of glutelin and/or prolamin family genes in HT-ripening grain at the middle and late filling stages (14–28 DAA) are possibly closely associated with the repress-ing regulation of RPBF induced by HT exposure; RISBZ1 is possibly mainly responsible for regulating the response of proglutelin family genes to HT exposure at the earlier grain-filling stage, due to its high expression at that stage regardless of different temperature treatments. HT- induc-ible decrease in prolamin content at the later grain filling stage might be poorly correlated with altered expressions of RISBZ1 affected by HT.
PDI and BiP play crucial roles in the correct folding and precise assembly of nascent polypeptides and stor-age protein, as well as the sorting of storage proteins to PBs (Li et al. 1993; Takemoto et al. 2002; Satoh-Cruz
et al. 2010; Kim et al. 2012). PDI is largely restricted to the cisternal ER and required for maturation of proglute-lin to a conformation that is competent for export from the ER to the Golgi (Kumamaru et al. 2010). The rice PDI-deficient mutant esp2 is identified by the accumulation of abnormally large quantities of proglutelin with corre-sponding reductions in mature glutelin subunits (Takemoto et al. 2002; Satoh-Cruz et al. 2010). In the present study, the transcript level of PDI in rice grain was significantly enhanced by HT throughout grain filling (Fig. 3b, c), which is consistent with significant increases in the amounts of 57-kDa proglutelin and mature subunits (acid and basic subunit) in HT-ripened grain (Fig. 1), suggesting that the increase in proglutelin accumulation at the grain-filling stage in HT-ripened grain possibly activated PDI transcrip-tional expressions, which would accelerated proper folding and assembly of proglutelin, leading to relatively enhanced amounts of glutelin mature subunits in HT-ripened grain, although the extent of activation varied with the stage of endosperm development (Figs. 1, 3). In consistent with this, Nuttall et al. (2002) suggested that ER chaperons (BiP and PDI) were implicated in not only assisting in the fold-ing and assembling of nascent proteins but also in post-translational regulation. When secretory protein genes were highly expressed in transgenic plants, the synthesis of ER chaperons (BiP and PDI) increased to assist with the fold-ing and assembly of proteins. In addition, it is noteworthy that significantly up-regulated expression of PDI induced by HT was not only observed in developing rice grain but also observed for leaf, confirming that PDI participates in a variety of physiological processes, such as serves as a molecular chaperone involving in defective reaction of rice plants to multiple abiotic stresses (Urade 2007). Besides PDI, other lumenal chaperones such as BiP are also likely to be involved in storage protein folding and intracellular transport. BiP is highly enriched on the periphery of PB-I (Li et al. 1993; Takahashi et al. 2005), which facilitates the transport of the nascent prolamin polypeptides across the ER membrane and their folding and assembly into an intracisternal inclusion granule. In the present study, the level of BiP transcript was elevated at the earlier filling stage (7 and 14 DAA), and strongly decreased thereafter (21 and 28 DAA). This cannot fully explain the alteration in 13-kDa polypeptide caused by HT, because relatively lower amounts of 13-kDa polypeptide were detected for the HT-ripened grain at different sampling stages (14 and 28 DAA), except for HT-ripened milled rice grain at 14 DAA. The possible reason for this is that BiP is not only involved in retaining and assembly of prolamins, but also responsi-ble for regulating response of folding and transport of pro-glutelin to HT exposure at the earlier grain filling due to its high expression at that stage. Actually, Satoh-Cruz et al. (2010) observed higher amount of BiP protein in cisternal
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ER membrane than that in PB-I fraction, indicating the possible role of BiP in regulating proglutelin folding and assembly, especially at the earlier filling stage.
Conclusion
HT reduced grain weight and increased total protein con-tent (TPC) regardless of rice genotype, and HT-inducible increase in TPC was not only attributable to the decrease in grain weight and the relatively increase in proportion of the aleurone fraction to whole grain, but also due to increased absolute amount of TPC in rice grain; HT increased the amounts of proglutelin, α-glutelin and β-glutelin subnuits but significantly decreased the accumulation of 13-kDa prolamin polypeptide throughout the whole grain filling, leading to a higher ratio of glutelin to prolamin compared with NT; The remarkably lowering transcripts of glutelin and/or prolamin family genes in HT-ripening grain at the middle and late filling stages were possibly closely associ-ated with the repressed regulation of RPBF induced by HT exposure; while RISBZ1 was possibly mainly responsible for regulating the response of glutelin family genes to HT exposure at the earlier grain filling stage. In addition, HT exposure accelerated expressions of PDI and BiP to assist with proper folding and assembly of proglutelin, leading to relatively enhanced glutelin mature subunits in HT-ripened grain.
Acknowledgments The authors are deeply indebted to National Natural Science Foundation of China (No. 31271655) for its financial support to this research project.
References
Ashida K, Araki E, Maruyama-Funatsuki W, Fujimoto H, Ikegami M (2013) Temperature during grain ripening affects the ratio of type-II/type-I protein body and starch pasting properties of rice (Oryza sativa L.) J Cereal Sci 57:153–159
Bollag DM, Edelstein SJ (1990) Protein methods. Wiley-Liss, New York, pp 50–56
Cooper NTW, Siebenmorgen TJ, Counce PA (2008) Effects of night-time temperature during kernel development on rice physico-chemical properties. Cereal Chem 85:276–282
Geigenberger P (2011) Regulation of starch biosynthesis in response to a fluctuating environment. Plant Physiol 155:1566–1577
Holme DJ, Peck H (1998) Peck analytical biotechnology, 3rd edn. Addison Wesley Longman Limited, New York, pp 388–390
Jagadish SVK, Murty MVR, Quick WP (2015) Rice responses to ris-ing temperatures—challenges, perspectives and future directions. Plant Cell Environ 38:1686–1698
Kawakatsu T, Takaiwa F (2010) Cereal seed storage protein synthe-sis: fundamental processes for recombinant protein production in cereal grains. Plant Biotechnol J 8:939–953
Kawakatsu T, Yamamoto MP, Touno SM, Yasuda H, Takaiwa F (2009) Compensation and interaction between RISBZ1 and RPBF during grain filling in rice. Plant J 59:908–920
Kawakatsu T, Hirose S, Yasuda H, Takaiwa F (2010) Reducing rice seed storage protein accumulation leads to changes in nutri-ent quality and storage organelle formation. Plant Physiol 154:1842–1854
Kim YJ, Yeu SY, Park BS, Koh HJ, Song JT, Seo HS (2012) Pro-tein disulfide isomerase-like protein 1–1 controls endosperm development through regulation of the amount and composi-tion of seed proteins in rice. Plos One 7(9):e44493
Krishnan P, Ramakrishnan B, Raja Reddy K, Reddy VR (2011) High-temperature effects on rice growth, yield, and grain qual-ity. Adv Agron 111:87
Kumamaru T, Uemura Y, Inoue Y, Takemoto Y, Siddiqui SU, Ogawa M, Hara-Nishimura I, Satoh H (2010) Vacuolar pro-cessing enzyme plays an essential role in the crystalline struc-ture of glutelin in rice seed. Plant Cell Physiol 51:38–46
Li X, Wu Y, Zhang DZ, Gillikin JW, Boston RS, Franceschi VR, Okita TW (1993) Rice prolamine protein body biogenesis: a BiP-mediated process. Science 262:1054–1056
Lin CJ, Li CY, Lin SK, Yang FH, Huang JJ, Liu YH, Lur HS (2010) Influence of high temperature during grain filling on the accu-mulation of storage proteins and grain quality in rice (Oryza sativa L.) J Agric Food Chem 58:10545–10552
Nuttall J, Vine N, Hadlington JL, Drake P, Frigerio L, Ma JKC (2002) ER-resident chaperone interactions with recombinant antibodies in transgenic plants. Eur J Biochem 269:6042–6051
Ogawa M, Kumamaru T, Satoh H, Iwata N, Omura T, Kasai Z, Tan-aka K (1987) Purification of protein body-I of rice seed and its polypeptide composition. Plant Cell Physiol 28:1517–1527
Onodera Y, Suzuki A, Wu CY, Washida H, Takaiwa F (2001) A rice functional transcriptional activator, RISBZ1, responsible for endosperm-specific expression of storage protein genes through GCN4 motif. J Biol Chem 276:14139–14152
Peng S, Huang J, Sheehy JE, Laza RC, Visperas RM, Zhong X, Centeno GS, Khush GS, Cassman KG (2004) Rice yields decline with higher night temperature from global warming. Proc Natl Acad Sci USA 101:9971–9975
Saito Y, Shigemitsu T, Yamasaki R, Sasou A, Goto F, Kishida K, Kuroda M, Tanaka K, Morita S, Satoh S, Masumura T (2012) Formation mechanism of the internal structure of type I pro-tein bodies in rice endosperm: relationship between the locali-zation of prolamin species and the expression of individual genes. Plant J 70:1043–1055
Satoh-Cruz M, Crofts AJ, Takemoto-Kuno Y, Sugino A, Washida H, Crofts N, Okita TW, Ogawa M, Satoh H, Kumamaru T (2010) Protein disulfide isomerase like 1–1 participates in the maturation of proglutelin within the endoplasmic reticulum in rice endosperm. Plant Cell Physiol 51:1581–1593
Shewry PR, Halford NG (2002) Cereal seed storage proteins: structures, properties and role in grain utilization. J Exp Bot 53:947–958
Su D, Lei BT, Li ZW, Cao ZZ, Huang FD, Pan G, Ding Y, Cheng FM (2014) Influence of high temperature during filling period on grain phytic acid and its relation to spikelet sterility and grain weight in non-lethal low phytic acid mutations in rice. J Cereal Sci 60:331–338
Takahashi H, Saito Y, Kitagawa T, Morita S, Masumura T, Tanaka K (2005) A novel vesicle derived directly from endoplasmic reticulum is involved in the transport of vacuolar storage pro-teins in rice endosperm. Plant Cell Physiol 46:245–249
Takemoto Y, Coughlan SJ, Okita TW, Satoh H, Ogawa M, Kum-amaru T (2002) The rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase. Plant Physiol 128:1212–1222
Tan Y, Corke H (2002) Factor analysis of physicochemical proper-ties of 63 rice varieties. J Sci Food Agric 82:745–752
488 Plant Growth Regul (2017) 81:477–488
1 3
Urade R (2007) Cellular response to unfolded proteins in the endo-plasmic reticulum of plants. Febs J 274:1152–1171
Wang KM, Wu JG, Li G, Yang ZW, Shi CH (2011) Distribution of phytic acid and mineral elements in three indica rice (Oryza sativa L.) cultivars. J Cereal Sci 54:116–121
Yamagata H, Tanaka K (1986) The site of synthesis and accumulation of rice storage proteins. Plant Cell Physiol 27:135–145
Yamagata H, Sugimoto T, Tanaka K, Kasai Z (1982) Biosynthe-sis of storage proteins in developing rice seeds. Plant Physiol 70:1094–1100
Yamakawa H, Hirose T, Kuroda M, Yamaguchi T (2007) Compre-hensive expression profiling of rice grain filling-related genes under high temperature using DNA microarray. Plant Physiol 144:258–277
Yamamoto MP, Onodera Y, Touno SM, Takaiwa F (2006) Synergism between RPBF Dof and RISBZ1 bZIP activators in the regula-tion of rice seed expression genes. Plant Physiol 141:1694–1707
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