Laser microdissection transcriptome data derived gene regulatory networks of developing rice endosperm revealed tissue- and stage-specific regulators modulating starch metabolism.

KEY MESSAGE: Laser microdissection applied on the developing rice endosperm revealed tissue- and stage-specific regulators modulating programmed cell death and desiccation tolerance mechanisms in the central starchy endosperm following starch metabolism. Rice (Oryza sativa L.) filial seed tissues are heterozygous in its function, which accumulate distinct storage compounds spatially in starchy endosperm and aleurone. In this study, we identified the 18 tissue- and stage-specific gene co-regulons in the developing endosperm by isolating four fine tissues dorsal aleurone layer (AL), central starchy endosperm (CSE), dorsal starchy endosperm (DSE), and lateral starchy endosperm (LSE) at two developmental stages (7 days after flowering, DAF and 12DAF) using laser microdissection (LM) coupled with gene expression analysis of a 44 K microarray. The derived co-expression regulatory networks depict that distinct set of starch biosynthesis genes expressed preferentially at first in CSE at 7 DAF and extend its spatial expression to LSE and DSE by 12 DAF. Interestingly, along with the peak of starch metabolism we noticed accumulation of transcripts related to phospholipid and glycolipid metabolism in CSE during 12 DAF. The spatial distribution of starch accumulation in distinct zones of starchy endosperm contains specific transcriptional factors and hormonal-regulated genes. Genes related to programmed cell death (PCD) were specifically expressed in CSE at 12DAF, when starch accumulation was already completed in that tissue. The aleurone layer present in the outermost endosperm accumulates transcripts of lipid, tricarboxylic acid metabolism, several transporters, while starch metabolism and PCD is not pronounced. These regulatory cascades are likely to play a critical role in determining the positional fate of cells and offer novel insights into the molecular physiological mechanisms of endosperm development from early to middle storage phase.

Effects of single ingestion of rice cracker and cooked rice with high resistant starch on postprandial glucose and insulin responses in healthy adults: two randomized, single-blind, cross-over trials.

Rice with double mutation of starch synthase IIIa and branching enzyme IIb (ss3a/be2b) has much higher amounts of apparent amylose and resistant starch (RS) than usual varieties. In this study, we conducted two randomized, single-blind, crossover trials to investigate the effect of single ingestion of two processed foods composed of ss3a/be2b mutant rice on postprandial blood glucose and insulin response in healthy adults, compared to those of usual cultivar. In trial 1, of ingestion of rice crackers, the incremental area under the curves of glucose (IAUCglc) and insulin (IAUCins) in RS group was significantly lower than in the control group. In trial 2, of the ingestion of cooked rice, IAUCglc in the RS group was significantly lower than in the control group. These results showed that the ss3a/be2b-mutant rice cracker and cooked rice having high RS can attenuate postprandial blood glucose and insulin response.

Laser Microdissection-Based Tissue-Specific Transcriptome Analysis Reveals a Novel Regulatory Network of Genes Involved in Heat-Induced Grain Chalk in Rice Endosperm.

Heat stress occurrence during seed filling leads to the formation of a chalky portion in the limited zone of the starchy endosperm of rice grains. In this study, isolation of aleurone, dorsal, central and lateral tissues of developing endosperm by laser-microdissection (LM) coupled with gene expression analysis of a 44 K microarray was performed to identify key regulatory genes involved in the formation of milky-white (MW) and white-back (WB) grains during heat stress. Gene regulatory network analysis classified the genes changed under heat stress into five modules. The most distinct expression pattern was observed in modules where most of the small heat shock proteins and cellular organization genes were changed under heat stress in dorsal aleurone cells and dorsal starchy endosperm zones. The histological observation supported the significant increase in cell number and size of dorsal aleurone cells in WB grains. With regard to the central starchy endosperm zone, preferential down-regulation of high molecular weight heat shock proteins (HMW HSPs), including a prominent member encoding endoplasmic reticulum (ER) chaperones, by heat stress was observed, while changes in expression of starch biosynthesis genes were minimal. Characterization of transgenic plants suppressing endosperm lumenal binding protein gene (BiP1), an ER chaperone preferentially down-regulated at the MW zone under heat stress, showed evidence of forming the chalky grains without disturbing the expression of starch biosynthesis genes. The present LM-based comprehensive expression analysis provides novel inferences that HMW HSPs play an important role in controlling redox, nitrogen and amino acid metabolism in endosperm leading to the formation of MW and WB chalky grains under heat stress.

Effects of heat-killed Lactobacillus casei subsp. casei 327 intake on defecation in healthy volunteers: a randomized, double-blind, placebo-controlled, parallel-group study.

Lactobacillus casei subsp. casei 327 (L. casei 327) was isolated from brown rice. A preliminary study showed that intake of 50 mg of heat-killed L. casei 327 is effective in improving defecation. In this study, we conducted a randomized, double-blind, placebo-controlled, parallel-group trial to investigate the effect of intake of heat-killed L. casei 327 (25 mg: approximately 5 × 1010 bacteria) on defecation in healthy volunteers with relatively low defecation frequencies. We selected 104 healthy Japanese adults with relatively low defecation frequencies (approximately 3-5 times a week) by screening and pretrial tests. Subjects (n=52 in each group) were randomly given a tablet containing L. casei 327 (group A) or a placebo tablet (group P) daily for 2 weeks. After eliminating data for 9 subjects who met the exclusion criteria for efficacy analysis, data for 95 subjects were analyzed. The defecation frequency and number of defecation days during the intake period and their changes from the pretrial period were significantly higher in group A than in group P. The fecal volume during the intake period was higher in group A than group P, but the difference was not statistically significant. However, the change from the pretrial period was significantly higher in group A than in group P. There were no significant differences between groups in the values of fecal shape, color, odor, and feeling after defecation. These results suggested that intake of L. casei 327 improves defecation in healthy adults who have relatively low defecation frequencies.

Starch Synthase IIa-Deficient Mutant Rice Line Produces Endosperm Starch With Lower Gelatinization Temperature Than Japonica Rice Cultivars.

The gelatinization temperature of endosperm starch in most japonica rice cultivars is significantly lower than that in most indica rice cultivars. This is because three single nucleotide polymorphisms in the Starch synthase (SS) IIa gene in japonica rice cultivars (SSIIaJ ) significantly reduce SSIIa activity, resulting in an increase in amylopectin short chains with degree of polymerization (DP) ≤ 12 compared to indica rice cultivars (SSIIaI ). SSIIa forms a trimeric complex with SSI and starch branching enzyme (BE) IIb in maize and japonica rice, which is likely important for the biosynthesis of short and intermediate amylopectin chains (DP ≤ 24) within the amylopectin cluster. It was unknown whether the complete absence of SSIIa further increases amylopectin short chains and reduces gelatinization temperature and/or forms altered protein complexes due to the lack of a suitable mutant. Here, we identify the SSIIa-deficient mutant rice line EM204 (ss2a) from a screen of ca. 1,500 plants of the rice cultivar Kinmaze (japonica) that were subjected to N-methyl-N-nitrosourea mutagenesis. The SSIIa gene in EM204 was mutated at the boundary between intron 5 and exon 6, which generated a guanine to adenine mutation and resulted in deletion of exon 6 in the mRNA transcript. SSIIa activity and SSIIa protein in developing endosperm of EM204 were not detected by native-PAGE/SS activity staining and native-PAGE/immunoblotting, respectively. SSIIa protein was completely absent in mature seeds. Gel filtration chromatography of soluble protein extracted from developing seeds showed that the SSI elution pattern in EM204 was altered and more SSI was eluted around 300 kDa, which corresponds with the molecular weight of trimeric complexes in wild type. The apparent amylose content of EM204 rice grains was higher than that in its parent Kinmaze. EM204 also had higher content of amylopectin short chains (DP ≤ 12) than Kinmaze, which reduced the gelatinization temperature of EM204 starch by 5.6°C compared to Kinmaze. These results indicate that EM204 starch will be suitable for making foods and food additives that easily gelatinize and slowly retrograde.

Starch synthase IIIa and starch branching enzyme IIb-deficient mutant rice line ameliorates pancreatic insulin secretion in rats: screening and evaluating mutant rice lines with antidiabetic functionalities.

Diabetes mellitus is a metabolic disease spreading worldwide that has been reported to worsen the development and progression of other diseases (cancer, vascular diseases and dementia). To establish functional rice lines with anti-postprandial hyperglycaemic effects, we developed mutant rice lines, which lack one or two gene(s) related to starch synthesis, and evaluated their effects. Powder of mutant rice lines or other grains was loaded to rats fasted overnight (oral grain powder loading test). Incremental area under time-concentration curves (iAUC) were calculated with monitored blood glucose levels. Rice lines with anti-postprandial hyperglycaemic effects were separated by cluster analysis with calculated iAUC. A double mutant rice #4019 (starch synthase IIIa (ss3a)/branching enzyme IIb (be2b)), one of the screened mutant rice lines, was fed to Goto-Kakizaki (GK) rats, an animal model for type 2 diabetes, for 5 weeks. Plasma levels of C-peptide, a marker of pancreatic insulin secretion, were measured with ELISA. For in vitro study, a rat pancreatic cell line was cultured with a medium containing rat serum which was sampled from rats fed #4019 diet for 2 d. After 24-h of incubation, an insulin secretion test was performed. Through the oral rice powder loading test, seven rice lines were identified as antidiabetic rice lines. The intake of #4019 diet increased plasma C-peptide levels of GK rats. This result was also observed in vitro. In rat serum added to cell medium, ornithine was significantly increased by the intake of #4019. In conclusion, the mutant rice #4019 promoted pancreatic insulin secretion via elevation of serum ornithine levels.

Effects of intake of Lactobacillus casei subsp. casei 327 on skin conditions: a randomized, double-blind, placebo-controlled, parallel-group study in women.

Lactic acid bacteria are gut flora that play key roles in intestinal homeostasis, which may affect a variety of physiological functions. Our preliminary double-blind, placebo-controlled, parallel-group trials have suggested that intake of heat-killed Lactobacillus casei subsp. casei 327 (designated L. K-1) is effective for improving skin conditions. The aim of this study was to confirm the effect of L. K-1 intake in a randomized, double-blind, placebo-controlled, parallel-group study in healthy female volunteers. Sixty-four subjects were allocated to either the placebo food group (group P, n=32) or active food group (group A, n=32), in which subjects consumed lactobacillus K-1 50 mg (approximately 1 × 1011 bacteria) daily for 8 weeks. After excluding subjects who declined to participate (n=1), violated restrictions (n=4), or were judged ineligible by the principal investigators (n=1), data obtained with 58 subjects (30 in group A and 28 in group P) were analyzed for efficacy by comparing differences from pretrial levels between the two groups. When the level of transepidermal water loss (TEWL) was measured at the arm, the level of TEWL at week 4 of the intake period was significantly lower in group A than group P (p=0.021), suggesting an improvement of skin barrier function. Analysis of skin condition questionnaire data revealed a significant reduction in skin flakiness on the face (week 4). No adverse events were associated with intake of the test foods. The safety of L. K-1 was also confirmed in an independent open-label trial in 11 healthy subjects who consumed excessive amounts of L. K-1 250 mg (approximately 5 × 1011 bacteria). Intake of L. K-1 may therefore be beneficial to skin condition improvement.

Randomized, double-blind, placebo-controlled, parallel-group study of the effect of Lactobacillus paracasei K71 intake on salivary release of secretory immunoglobulin A.

Lactobacillus paracasei K71 was shown to be effective in alleviating the severity of atopic dermatitis in a randomized controlled trial, and a preliminary open-label trial suggested that strain K71 intake enhanced secretory immunoglobulin A (sIgA) release in the saliva. This study investigated the effect of K71 on sIgA release in a randomized, double-blind, placebo-controlled, parallel-group trial. The trial included 62 Japanese subjects aged 20-64 years with relatively low rates of salivary sIgA release. Subjects (n=31 in each group) were randomly given a tablet containing 100 mg (approximately 2 × 1011 bacteria) of K71 or a placebo tablet daily for 12 weeks. After eliminating data for eight subjects (four in each group) who met the exclusion criteria for efficacy analysis, data for 54 subjects were analyzed. The change in the rate of salivary sIgA release 8 weeks after initiation of the study compared with baseline was significantly higher in the K71 tablet group (105.5 ± 119.0 µg/min) than in the placebo group (52.7 ± 62.6 µg/min; p=0.047). There were no adverse events associated with intake of tablets containing K71. The safety of intake of L. paracasei K71 was also confirmed in an independent open-label trial with 20 healthy subjects who consumed excessive amounts of K71-containing food. L. paracasei K71 intake may therefore have some benefits in promoting mucosal immune function.

Control of foreign polypeptide localization in specific layers of protein body type I in rice seed.

KEY MESSAGE: Prolamin-GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin-GFP fusion proteins were gradually digested from outside by pepsin digestion. In rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin-GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin-GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin-GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin-GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin-GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion.

In vivo digestibility of rice prolamin/protein body-I particle is decreased by cooking.

Rice has storage proteins, e.g., glutelin, globulin and prolamin, in the seeds, which are used as nitrogen sources during germination. Rice prolamin has been reported to be an indigestible protein that decreases the nutritional value of rice. However, the causes for the indigestibility of prolamin are currently not clear. The objective of this study was to determine if prolamin is naturally indigestible or if cooking affects its digestibility. The gastrointestinal (GI) transit of rice 23 kDa glutelin (23G) and 13 kDa prolamin (13P) in Wistar/ST rats fed raw rice (RR) and cooked rice (CR) diets was assessed using Western blot analysis. We also measured the excretion of these proteins in the feces of these rats. Additionally, morphological observation of the structure of type-I protein bodies in the feces was performed using electron microscopy. Assessment of GI transit revealed that 23G rapidly disappeared from the GI contents of both the RR and CR groups, but 13P accumulated in the cecum of the CR group. In the CR group, prolamin, maintaining the structure of PB-I, was fully excreted in the feces. These results indicate that rice prolamin is not indigestible by nature, but is rendered indigestible by cooking.

Separation and identification of rice prolamins by two-dimensional gel electrophoresis and amino acid sequencing.

There are difficulties in detecting and separating rice prolamin polypeptides by 2D-PAGE analysis because prolamin polypeptides are insoluble, and the amino acid sequences show high homology among them. In this study, we improved the prolamin extraction method and the 2D-PAGE procedure, and succeeded in separating prolamin polypeptide species by 2D-PAGE and in identifying major prolamin polypeptide sequences.

Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes.

Rice prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.

Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins.

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 µg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.

Ultrastructure of mature protein body in the starchy endosperm of dry cereal grain.

The development of the protein body in the late stage of seed maturation is poorly understood, because electron-microscopy of mature cereal endosperm is technically difficult. In this study, we attempted to modify the existing method of embedding rice grain in resin. The modified method revealed the ultrastructures of the mature protein body in dry cereal grains.

Improvement in the in vivo digestibility of rice protein by alkali extraction is due to structural changes in prolamin/protein body-I particle.

Rice prolamin, constituting type-I protein body (PB-I), is indigestible and causes deterioration of rice protein nutritional quality. In this study, the in vivo digestibility of rice protein isolates was investigated by tracing their intraluminal transit in the gastrointestinal (GI) tract of rats by western blotting and by observing the structures excreted in the feces by electron microscopy. Two types of rice protein isolates, produced by alkali extraction (AE-RP) and by starch degradation (SD-RP), were compared. The protein patterns in the isolates were similar, but their digestion in the GI-tract showed striking differences. In the AE-RP group, 13-kDa prolamin (13P) quickly disappeared in the lower GI tract and was not excreted in the feces. By contrast, in the SD-RP group, 13P accumulated massively and nearly intact PB-Is were excreted. These results indicate that the in vivo digestibility of prolamin can be improved by alkali extraction through structural changes to it.

A green fluorescent protein fused to rice prolamin forms protein body-like structures in transgenic rice.

Prolamins, a group of rice (Oryza sativa) seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and deposited in ER-derived type I protein bodies (PB-Is) in rice endosperm cells. The accumulation mechanism of prolamins, which do not possess the well-known ER retention signal, remains unclear. In order to elucidate whether the accumulation of prolamin in the ER requires seed-specific factors, the subcellular localization of the constitutively expressed green fluorescent protein fused to prolamin (prolamin-GFP) was examined in seeds, leaves, and roots of transgenic rice plants. The prolamin-GFP fusion proteins accumulated not only in the seeds but also in the leaves and roots. Microscopic observation of GFP fluorescence and immunocytochemical analysis revealed that prolamin-GFP fusion proteins specifically accumulated in PB-Is in the endosperm, whereas they were deposited in the electron-dense structures in the leaves and roots. The ER chaperone BiP was detected in the structures in the leaves and roots. The results show that the aggregation of prolamin-GFP fusion proteins does not depend on the tissues, suggesting that the prolamin-GFP fusion proteins accumulate in the ER by forming into aggregates. The findings bear out the importance of the assembly of prolamin molecules and the interaction of prolamin with BiP in the formation of ER-derived PBs.

Thin frozen film method for visualization of storage proteins in mature rice grains.

There are technical difficulties in obtaining intact sections of cereal grains in which mature cells and their subcellular structures are well preserved. Here we describe a simple method for sectioning hard mature rice grains. It makes possible accurate localization of storage proteins in high-quality histological sections of rice endosperm.

Production of rice protein by alkaline extraction improves its digestibility.

Rice seed endosperm has two types of protein bodies (PB). Type I protein body (PB-I) accumulates prolamin and is hard to digest, while type II protein body (PB-II) mainly consists of glutelin, an easily digestible protein. A simple method to process rice protein and improve its digestibility was tested from the viewpoint of its application to food manufacturing. Rice protein prepared by alkaline extraction followed by neutralization sedimentation (AE-RP) was compared with that prepared by starch degradation by alpha-amylase (SD-RP). The crude protein content of AE-RP and SD-RP was 84.7% and 78.2%, respectively. There were no major differences in protein composition among AE-RP, SD-RP and rice flour by SDS-PAGE, except 16 kDa polypeptide. With respect to amino acids, all the groups showed quite similar compositions, although cysteine and methionine were lower in AE-RP. In an in vitro digestion study with pepsin and pancreatin, both the SDS-PAGE analysis of protein pattern and the crude protein content of undigested residue clearly demonstrated that AE-RP has a higher digestibility than SD-RP. To find the cause of the difference in digestibility, the structural property of protein bodies by two production methods was compared using electron microscopy. PB-II of AE-RP was transformed into small, amorphous granules, while that of SD-RP was still kept partial protein body structures. PB-I of AE-RP kept its protein body structure, but produced double layers. From the finding that glutelin-gold was detected by immunochemistry not only in small, amorphous granules but also in PB-I, mainly the cortex layer, in AE-RP, it became clear that PB-I was swollen and fragile as a result of alkali treatment. These results strongly indicate that the improvement in digestibility of AE-RP is a result of the structural change of PB-I and -II caused by alkaline extraction.

A novel vesicle derived directly from endoplasmic reticulum is involved in the transport of vacuolar storage proteins in rice endosperm.

We found novel vesicles derived from rough endoplasmic reticulum (ER) in rice endosperm. The novel vesicles had characteristic structures different from that of the ER-derived protein body type I and the Golgi-derived dense vesicles. Immunocytochemical analysis revealed that the novel vesicles are derived directly from the aggregates of vacuolar storage proteins in the rough ER. In addition, BiP, an ER-resident molecular chaperone, was localized in the novel vesicles, but also in protein storage vacuoles (PSVs). These results suggest that the novel vesicles mediate transport of vacuolar storage proteins directly from the ER to PSVs in rice endosperm.