Selection of transcripts related to low-temperature tolerance using RNA sequencing from F2 plants between japonica and indica rice (Oryza sativa L.) cultivars.

At low temperatures (18°C), seedlings of an indica rice (Oryza sativa L.) cultivar Kasalath showed symptoms of chlorosis, although the leaves of a japonica cultivar Arroz da Terra remained green. In this study, transcripts related to the chlorophyll content of rice seedlings grown at 18°C were investigated using RNA-sequencing (RNA-Seq) data for F2 crosses between cultivars Arroz da Terra and Kasalath, as well as their parental cultivars. Differential expression analysis revealed that gene ontology terms related to 'photosynthesis' were significantly enriched in lowly expressed genes at 18°C than at 25°C in Kasalath. However, the gene ontology terms related to 'response to stress' were significantly enriched in highly expressed genes at 18°C than at 25°C in Kasalath. When the F2 plants were grown at 18°C, their chlorophyll contents varied. Transcripts with expression levels related to chlorophyll content were statistically selected using RNA-Seq data from 21 F2 plants. In regression models, frequently selected genes included four photosynthetic and two stress-responsive genes. The expression values of four photosynthetic and two stress-responsive genes in high-frequency selected genes were significantly correlated with chlorophyll content not only in plants analysed using RNA-Seq but also in 95 F2 plants.

Characterisation of a rice vacuolar invertase isoform, OsINV2, for growth and yield-related traits.

OsINV2, a rice vacuolar invertase isoform, was assessed for its functional roles in plant growth and development with key focus on its agronomic traits such as grain weight, grain filling percentage, grain number and dry weights at various stages until harvest. Lack of differences between the wild-type and the mutants with respect to any of the aforementioned traits tested revealed a possibility of functional compensation of OsINV2 in the mutants conceivably by its isoform. This was confirmed by OsINV2 promoter::GUS studies, where its spatial and temporal expression in the panicle elongation stages showed that although OsINV2 expression was observed from the stage with young panicles ~1 cm in length to the flag leaf stage, significant differences with respect to panicle and spikelet phenotypes between the wild-type and the mutant were not present. However, complement lines displaying an overexpression phenotype of OsINV2 possessed a higher stem non-structural carbohydrate content under both monoculm and normal tillering conditions. A trade-off between the spikelet number and grain weight in the complement lines grown under monoculm conditions was also observed, pointing towards the necessity of OsINV2 regulation for grain yield-related traits.

Genetic Evidence for the Role of a Rice Vacuolar Invertase as a Molecular Sink Strength Determinant.

BACKGROUND: Rice is a major crop feeding the majority of the global population, and increasing its sink strength is one of the modes to alleviate the declining availability of food for the rapidly growing world population. We demonstrate a role for an important rice vacuolar invertase isoform, OsINV3, in sink strength determination. RESULTS: OsINV3 mutants showed shorter panicles with lighter and smaller grains, owing to a smaller cell size on the outer and inner surfaces of the palea and lemma as observed by scanning electron microscopy. Further, strong promoter::GUS expression was observed in the palea, lemma and the rachis branches in the young elongating panicles, which supported the role of OsINV3 in cell expansion and thus, in spikelet size and panicle length determination. Size of the spikelet was found to directly influence the grain weight, which was confirmed by the lack of differences in weights of hulled grain for differently segregated alleles in the heterozygous lines. Assessment of field grown mutants not only revealed a drastic reduction in the percentage of ripened grain, 1000-grain weight and final yield, but also significantly reduced partitioning of assimilates to the panicles, whereby the total dry weight remained unaffected. Determination of the non-structural carbohydrate contents revealed a lower hexose-to-sucrose ratio in the panicles of the mutants from panicle initiation to 10 days after heading, a stage that identifies as the critical pre-storage phase of grain filling, whereas the starch contents were not affected. In addition, strong promoter::GUS expression was observed in the dorsal end of ovary during the pre-storage phase until 6 days after flowering, highlighting a function for OsINV3 in monitoring the initial grain filling stage. CONCLUSIONS: OsINV3 was found to regulate spikelet size by playing a key role in cell expansion, driving the movement of assimilates for grain filling by modulating the hexose-to-sucrose ratio, contributing in grain weight determination and thus, the grain yield.

Selection of Transcripts Affecting Initial Growth Rate of Rice Backcrossed Inbred Lines Using RNA Sequencing Data.

Seedling growth is an important factor for direct seeding of rice. However, the genetic and transcriptomic factors involved in this process are largely unknown. In this study, transcripts affecting shoot weight were identified in rice (Oryza sativa L.) using RNA sequencing (RNA-Seq) data from 20 backcrossed inbred lines (BILs) and their parental cultivars. The selection frequency of the genes for the regression model was determined using repeated analysis of random subsets of the transcriptome. The qLTG3-1gene, controlling low-temperature germinability, and short grain 1 gene (SG1), known to decrease organ elongation, showed high frequency. The quantitative trait loci (QTLs) analysis performed for BILs revealed that qLTG3-1 was included in the QTLs for shoot weight but SG1 was not. No nucleotide polymorphisms were found in the coding region of SG1 in either of the parental cultivars. Quantitative real-time PCR showed that SG1 expression was negatively correlated with shoot weight for all 104 BILs analyzed in this study. Expression QTL (eQTLs) analysis showed an eQTL for SG1 expression located in the same region as the QTL for shoot weight. However, no eQTLs were detected on the same chromosome as SG1, suggesting that nucleotide polymorphisms around the gene do not affect its expression in analyzed growth stage. Overall, these results indicate that RNA-Seq is a useful tool for identifying transcripts that can be related to seedling growth rate.

A reduction of sucrose phosphate synthase (SPS) activity affects sucrose/starch ratio in leaves but does not inhibit normal plant growth in rice.

Sucrose phosphate synthase (SPS) has been shown to mediate sucrose/starch ratio in plant leaves through studies of 'starch leaf' species that mainly accumulate starch in their leaves. However, the contribution of SPS to sucrose/starch ratio in rice leaves, which mainly accumulate sucrose (i.e., 'sugar leaf'), has not been confirmed due to inconsistencies in the results of previous studies. In this study, we analyzed mutant lines with reduced SPS activity, which were generated using Tos17 insertion, RNAi, and the CRISPR/Cas9 system. The knockdown and knockout mutants of OsSPS1 showed a 29-46% reduction in SPS activity in the leaves, but the carbohydrate content in the leaves and plant growth were not significantly different from those of wild-type plants. In a double knockout mutant of OsSPS1 and OsSPS11 (sps1/sps11), an 84% reduction in leaf SPS activity resulted in higher starch accumulation in the leaves than in the wild-type leaves. However, the sps1/sps11 plants grew normally, which is in contrast to the inhibited growth of SPS mutants of Arabidopsis thaliana, a typical starch leaf plant. These results suggest that SPS has a smaller effect on the sucrose/starch ratio in leaves and growth of rice than on starch leaf species.

Control of grain protein contents through SEMIDWARF1 mutant alleles: sd1 increases the grain protein content in Dee-geo-woo-gen but not in Reimei.

A new possibility for genetic control of the protein content of rice grains was suggested by the allele differences of the SEMIDWARF1 (SD1) mutation. Two quantitative trait loci-qPROT1 and qPROT12-were found on chromosomes 1 and 12, respectively, using backcrossed inbred lines of Sasanishiki/Habataki//Sasanishiki///Sasanishiki. One of them, qPROT1, increased almost all grain proteins instead of only certain proteins in the recessive Habataki allele. Fine mapping of qPROT1 revealed that two gene candidates-Os01g0883800 and Os01g0883900-were included in this region. Os01g0883800 encoded Gibberellin 20 oxidase 2 as well as SD1, the dwarf gene used in the so-called 'Green Revolution'. Mutant analyses as well as sequencing analysis using the semi-dwarf mutant cultivars Dee-geo-woo-gen and Calrose 76 revealed that the sd1 mutant showed significantly higher grain protein contents than their corresponding wild-type cultivars, strongly suggesting that the high protein contents were caused by sd1 mutation. However, the sd1 mutant Reimei did not have high grain protein contents. It is possible to control the grain protein content and column length separately by selecting for sd1 alleles. From this finding, the genetic control of grain protein content, as well as the column length of rice cultivars, might be possible. This ability might be useful to improve rice nutrition, particularly in areas where the introduction of semi-dwarf cultivars is not advanced.

Analysis of gene-disruption mutants of a sucrose phosphate synthase gene in rice, OsSPS1, shows the importance of sucrose synthesis in pollen germination.

The molecular function of an isoform of sucrose phosphate synthase (SPS) in rice, OsSPS1, was investigated using gene-disruption mutant lines generated by retrotransposon insertion. The progeny of the heterozygote of disrupted OsSPS1 (SPS1(+/-)) segregated into SPS1(+/+), SPS1(+/-), and SPS1(-/-) at a ratio of 1:1:0. This distorted segregation ratio, together with the expression of OsSPS1 in the developing pollen revealed by quantitative RT-PCR analysis and promoter-beta-glucuronidase (GUS) fusion assay, suggested that the disruption of OsSPS1 results in sterile pollen. This hypothesis was reinforced by reciprocal crosses of SPS1(+/-) plants with wild-type plants in which the disrupted OsSPS1 was not paternally transmitted to the progeny. While the pollen grains of SPS(+/-) plants normally accumulated starch during their development, pollen germination on the artificial media was reduced to half of that observed in the wild-type control. Overall, our data suggests that sucrose synthesis via OsSPS1 is essential in pollen germination in rice.

Phenotypic analyses of rice lse2 and lse3 mutants that exhibit hyperaccumulation of starch in the leaf blades.

BACKGROUND: To identify genes that potentially regulate the accumulation, mobilization, and transport of photoassimilates in rice (Oryza sativa L.) leaves, we recently screened a mutant collection of rice by iodine staining to visualize leaf starch contents. From this screening, we isolated a rice mutant that exhibits hyperaccumulation of starch in leaves and designated it as the Leaf Starch Excess 1 (LSE1) mutant. Here, we report two other rice LSE mutants, LSE2 and LSE3. RESULTS: Unlike lse1 plants, lse2 and lse3 plants displayed retarded growth; lse2 showed an extremely dwarf phenotype and rarely survived in paddy fields; lse3 showed inhibited growth with pale green leaf blades, low tiller numbers, reduced height, and low grain yield. In lse2 and lse3 plants, the mature source leaves contained larger amounts of starch and sucrose than those in wild-type and lse1 plants. Furthermore, microscopic observations of leaf transverse sections indicated that hyperaccumulation of starch in chloroplasts of mesophyll and bundle sheath cells occurred in lse2 and lse3 plants, while that in vascular cells was noticeable only in lse3 leaves. CONCLUSIONS: The distinct phenotypes of these three LSE mutants suggest that the LSE2 and LSE3 mutations occur because of disruption of novel genes that might be involved in the path of sucrose transport from mesophyll cells to phloem sieve elements in rice leaves, the mechanism for which has not yet been elucidated.

Suppression of starch synthesis in rice stems splays tiller angle due to gravitropic insensitivity but does not affect yield.

In rice (Oryza sativa L.), tiller angle - defined as the angle between the main culm and its side tillers - is one of the important factors involved in light use efficiency. To clarify the relationship between tiller angle, gravitropism and stem-starch accumulation, we investigated the shoot gravitropic response of a low stem-starch rice mutant which lacks a large subunit of ADP-glucose pyrophosphorylase (AGP), called OsAGPL1 and exhibits relatively spread tiller angle. The insensitive gravitropic response exhibited by the mutant led us to the conclusion that insensitivity of gravitropism caused by stem-starch reduction splayed the tiller angle. Furthermore, since another AGP gene called OsAGPL3 was expressed at considerable levels in graviresponding sites, we generated a double mutant lacking both OsAGPL1 and OsAGPL3. The double mutant exhibited still lower stem-starch content, less sensitive gravitropic response and greater tiller angle spread than the single mutants. This indicated that the expansion of the tiller angle caused by the reduction in starch level was intense according to the extent of the reduction. We found there were no significant differences between the double mutant and wild-type plants in terms of dry matter production. These results provided new insight into the importance of stem-starch accumulation and ideal plant architecture.

Disruption of a rice gene for α-glucan water dikinase, OsGWD1, leads to hyperaccumulation of starch in leaves but exhibits limited effects on growth.

To identify potential regulators of photoassimilate partitioning, we screened for rice mutant plants that accumulate high levels of starch in the leaf blades, and a mutant line leaf starch excess 1 (LSE1) was obtained and characterized. The starch content in the leaf blades of LSE1 was more than 10-fold higher than that in wild-type plants throughout the day, while the sucrose content was unaffected. The gene responsible for the LSE1 phenotype was identified by gene mapping to be a gene encoding α-glucan water dikinase, OsGWD1 (Os06g0498400), and a 3.4-kb deletion of the gene was found in the mutant plant. Despite the hyperaccumulation of starch in their leaf blades, LSE1 plants exhibited no significant change in vegetative growth, presenting a clear contrast to the reported mutants of Arabidopsis thaliana and Lotus japonicus in which disruption of the genes for α-glucan water dikinase leads to marked inhibition of vegetative growth. In reproductive growth, however, LSE1 exhibited fewer panicles per plant, lower percentage of ripened grains and smaller grains; consequently, the grain yield was lower in LSE1 plants than in wild-type plants by 20~40%. Collectively, although α-glucan water dikinase was suggested to have universal importance in leaf starch degradation in higher plants, the physiological priority of leaf starch in photoassimilate allocation may vary among plant species.

The promoter activities of sucrose phosphate synthase genes in rice, OsSPS1 and OsSPS11, are controlled by light and circadian clock, but not by sucrose.

Although sucrose plays a role in sugar sensing and its signaling pathway, little is known about the regulatory mechanisms of the expressions of plant sucrose-related genes. Our previous study on the expression of the sucrose phosphate synthase gene family in rice (OsSPSs) suggested the involvement of sucrose sensing and/or circadian rhythm in the transcriptional regulation of OsSPS. To examine whether the promoters of OsSPSs can be controlled by sugars and circadian clock, we produced transgenic rice plants harboring a promoter-luciferase construct for OsSPS1 or OsSPS11 and analyzed the changes in the promoter activities by monitoring bioluminescence from intact transgenic plants in real-time. Transgenic plants fed sucrose, glucose, or mannitol under continuous light conditions showed no changes in bioluminescence intensity; meanwhile, the addition of sucrose increased the concentration of sucrose in the plants, and the mRNA levels of OsSPS remained constant. These results suggest that these OsSPS promoters may not be regulated by sucrose levels in the tissues. Next, we investigated the changes in the promoter activities under 12-h light/12-h dark cycles and continuous light conditions. Under the light-dark cycle, both OsSPS1 and OsSPS11 promoter activities were low in the dark and increased rapidly after the beginning of the light period. When the transgenic rice plants were moved to the continuous light condition, both P OsSPS1 ::LUC and P OsSPS11 ::LUC reporter plants exhibited circadian bioluminescence rhythms; bioluminescence peaked during the subjective day with a 27-h period: in the early morning as for OsSPS1 promoter and midday for OsSPS11 promoter. These results indicate that these OsSPS promoters are controlled by both light illumination and circadian clock and that the regulatory mechanism of promoter activity differs between the two OsSPS genes.

Starch reduction in rice stems due to a lack of OsAGPL1 or OsAPL3 decreases grain yield under low irradiance during ripening and modifies plant architecture.

Starch accumulated in rice (Oryza sativa L.) stems before heading as nonstructural carbohydrates (NSCs) is reported to be important for improving and stabilising grain yield. To evaluate the importance of stem starch, we investigated a retrotransposon (Tos17) insertion rice mutant lacking a gene encoding a large subunit of ADP-glucose pyrophosphorylase (AGP) called OsAGPL1 or OsAPL3. The AGP activity and starch contents of the mutant were drastically reduced in the stem (i.e. leaf sheath and culm) but not in the leaf blade or endosperm. This starch reduction in the leaf sheaths of the mutant was complemented by the introduction of wild-type OsAGPL1. These results strongly suggest that OsAGPL1 plays a principal role in stem starch accumulation. Field experimentations spanning 2 years revealed that the mutant plants were shorter than the wild-type plants. Moreover, the tiller number and angle were larger in the mutant plants than the wild-type plants, but the dry weight at heading stage was not different. The grain yield was slightly lower in control plots without shading treatment. However, this difference increased substantially with shading. Therefore, stem starch is indispensable for normal ripening under low irradiance conditions and probably contributes to the maintenance of appropriate plant architecture.

Overexpression of a rice TIFY gene increases grain size through enhanced accumulation of carbohydrates in the stem.

Screening of rice full-length cDNA overexpressing (FOX) lines allowed the identification of a TIFY gene, TIFY11b, as a growth-promoting gene whose overexpression increased plant height and seed size. The grains of TIFY11b-overexpressing plants exceeded those of non-transformants in length, width and thickness, resulting in 9-21% increases in grain weight. The increase was achieved by overexpressing the gene in the whole plant body, but not by seed-restricted expression, indicating that seed enlargement is attributable to overexpression in vegetative organs such as the leaf. The whole-body overexpressing plants developed longer leaves along with higher levels of starch and sucrose in the leaf sheath and culm at the heading stage than the non-transformants. Although overexpression of TIFY11b did not alter the photosynthetic rate per leaf area before and after heading, it caused an accumulation of higher levels of the carbohydrate assimilate, probably due to increased photosynthesis per plant, suggesting that the increase in grain size and weight is attained by enhanced accumulation and translocation of the carbohydrate in the culms and leaf sheaths of the transgenic plants. Thus, TIFY11b is a novel grain-size increasing gene.

Tissue specificity and diurnal change in gene expression of the sucrose phosphate synthase gene family in rice.

The rice genome contains 5 isogenes for sucrose phosphate synthase (SPS), the key enzyme in sucrose synthesis; however, little is known about their transcriptional regulation. In order to determine the expression patterns of the SPS gene family in rice plants, we conducted an expression analysis in various tissues and developmental stages by real-time quantitative RT-PCR. At the transcript level, the rice SPS genes, particularly SPS1, were preferentially expressed in source tissues, whereas SPS2, SPS6, and SPS8 were expressed equally in source and sink tissues. We also investigated diurnal changes in SPS gene expression, SPS activity, and soluble sugar content in leaf blades. Interestingly, the expression of all the SPS genes, particularly that of SPS1 and SPS11, tended to be higher at night when the activation state of the SPS proteins was low, and the mRNA levels of SPS1 and SPS6 were negatively correlated with sucrose content. Furthermore, the temporal patterns of SPS gene expression and sugar content under continuous light conditions suggested the involvement of endogenous rhythm and/or sucrose sensing in the transcriptional regulation of SPS genes. Our data revealed differential expression patterns in the rice SPS gene family and part of the complex mechanisms of their transcriptional control.

Disruption of a gene for rice sucrose transporter, OsSUT1, impairs pollen function but pollen maturation is unaffected.

Sucrose transporters (SUTs) are known to play critical roles in the uptake of sucrose from the apoplast in various steps of sugar translocation. Because developing pollen is symplastically isolated from anther tissues, it is hypothesized that SUTs are active in the uptake of apoplastic sucrose into pollen. To investigate this possibility, a comprehensive expression analysis was performed for members of the SUT gene family in the developing pollen of rice (Oryza sativa L.) using real-time RT-PCR combined with a laser microdissection technique. Among the five SUT genes, OsSUT1 and OsSUT3 were found to be preferentially expressed and had temporal expression patterns that were distinct from each other. Expression of OsSUT1 in pollen was confirmed by a promoter-GUS fusion assay. The physiological function of OsSUT1 in pollen was further investigated using retrotransposon insertion mutant lines. While the homozygote of disrupted OsSUT1 (SUT1-/-) could not be obtained, heterozygote plants (SUT1+/-) showed normal grain filling. Their progeny segregated into SUT1+/- and SUT1+/+ with the ratio of 1:1, suggesting that the pollen disrupted for OsSUT1 is dysfunctional. This hypothesis was reinforced in vivo by a backcross of SUT1+/- plants with wild-type plants and also by in vitro pollen germination on the artificial media. However, starch accumulation during pollen development was not affected by disruption of OsSUT1, suggesting that the sugar(s) required for starch biosynthesis is supplied by other sugar transporters.

Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese.

Rice (Oryza sativa) is indispensable in the diet of most of the world's population. Thus, it is an important target in which to alter iron (Fe) uptake and homeostasis, so as to increase Fe accumulation in the grain. We previously isolated OsYSL2, a functional iron [Fe(II)]- and manganese [Mn(II)]-nicotianamine complex transporter that is expressed in phloem cells and developing seeds. We produced RNAi (OsYSL2i) and overexpression lines (OXOsYSL2) of OsYSL2. At the vegetative stage in an OsYSL2i line, the Fe and Mn concentrations were decreased in the shoots, and the Fe concentration was increased in the roots. At the reproductive stage, positron-emitting tracer imaging system analysis revealed that Fe translocation to the shoots and seeds was suppressed in OsYSL2i. The Fe and Mn concentrations were decreased in the seeds of OsYSL2i, especially in the endosperm. Moreover, the Fe concentration in OXOsYSL2 was lower in the seeds and shoots, but higher in the roots, compared with the wild type. Furthermore, when OsYSL2 expression was driven by the sucrose transporter promoter, the Fe concentration in the polished rice was up to 4.4-fold higher compared with the wild type. These results indicate that the altered expression of OsYSL2 changes the localization of Fe, and that OsYSL2 is a critical Fe-nicotianamine transporter important for Fe translocation, especially in the shoots and endosperm.

A gene controlling the number of primary rachis branches also controls the vascular bundle formation and hence is responsible to increase the harvest index and grain yield in rice.

The quantitative trait locus controlling the number of primary rachis branches (PRBs) in rice was identified using backcrossed inbred lines of Sasanishiki/Habataki//Sasanishiki///Sasanishiki. The resultant gene was ABERRANT PANICLE ORGANIZATION 1 (APO1). Habataki-genotype segregated reciprocal recombinant lines for the APO1 locus increased both the number of PRB (12-13%) and the number of grains per panicle (9-12%), which increased the grain yield per plant (5-7%). Further recombination dividing this region revealed that different alleles regulated the number of PRB and the number of grains per panicle. The PRB1 allele, which includes the APO1 open reading frame (ORF) and the proximal promoter region, controlled only the number of PRB but not the number of grains per panicle. In contrast, the HI1 allele, which includes only the distal promoter region, increased the grain yield and harvest index in Habataki-genotype plants, nevertheless, the ORF expressed was Sasanishiki type. It also increased the number of large vascular bundles in the peduncle. APO1 expression occurred not only in developing panicles but also in the developing vascular bundle systems. In addition, Habataki plants displayed increased APO1 expression in comparison to Sasanishiki plants. It suggests that APO1 enhances the formation of vascular bundle systems which, consequently, promote carbohydrate translocation to panicles. The HI1 allele is suggested to regulate the amount of APO1 expression, and thereby control the development of vascular bundle systems. These findings may be useful to improve grain yield as well as quality through the improvement of translocation efficiency.

Elevation of myostatin and FOXOs in prolonged muscular impairment induced by eccentric contractions in rat medial gastrocnemius muscle.

This study aimed to investigate torque deficit and activation of protein synthesis and/or protein degradation signaling pathways during the early and recovery phase after high- and low-velocity eccentric contractions (ECs). Male Wistar rats (n = 36) were randomly divided into fast angular velocity ECs group (FAST; 180 degrees/s; n = 12), slow ECs group (SLOW; 30 degrees/s; n = 12), and control group (control; n = 12). ECs comprised four sets of five forced dorsiflexions combined with electrical stimulation of the plantar flexors. Isometric tetanic torque was measured before and after ECs. Tissue contents of Akt(P) (P, phosphorylated), mammalian target of rapamycin (mTOR)(P), 70-kDa ribosomal protein S6 kinase (P70S6k), P70S6k(P), forkhead transcription factor 1 of the O class (FOXO1), FOXO1(P), FOXO3, FOXO3(P), myostatin, and activin receptor type IIB (ActRIIB) were measured. The isometric tetanic torque after ECs was significantly lower in FAST than in SLOW (days 1, 3, and 5, P < 0.05; day 2, P < 0.01). The ratio of P70S6k(P) against total P70S6k on days 2 and 7 was significantly higher in SLOW than in the control. The ratio of FOXO1 against total FOXO1, the ratio of FOXO3a against total FOXO3a, and myostatin on days 2 and 7 were significantly higher in FAST than in the control, while that of ActRIIB on day 7 was significantly lower in SLOW than in the other two groups. These results suggest that EC intensity plays a key role in impairment of muscular function and activation of protein synthesis and/or protein degradation signaling pathways.

TGF-beta1 and TNF-alpha are involved in the transcription of type I collagen alpha2 gene in soleus muscle atrophied by mechanical unloading.

The aim of this study was to examine the effect of hindlimb suspension (HS) on the expressions of COL1A2 (type I collagen alpha(2) chain) mRNA and its regulatory factors, transforming growth factors (TGF)-beta(1), -beta(2), and -beta(3), phosphorylated Smad3, and tumor necrosis factor-alpha (TNF-alpha) in rat hindlimb muscles. Forty-eight male Wistar rats (age, 5 wk) were randomly assigned to HS for 1, 3, 7, and 14 days and control (n = 6 for each). During the exposure to HS, COL1A2 mRNA expression decreased in the soleus muscle at day 3 and recovered to control level at day 7. The content of TNF-alpha, one of the negative regulatory factors for COL1A2, increased from day 3 until day 14. On the other hand, the contents of TGF-beta(1), TGF-beta(3), and Smad3, positive regulatory factors for COL1A2, increased at day 7. The in situ hybridization for COL1A2 and the immunohistochemistry of TGF-beta(1) and TNF-alpha revealed their expressions around nerve-related tissues, including muscle spindles and connective tissue sheath. The results indicate that the transcriptional activity of COL1A2 in the soleus muscle initially decreases in response to unloading through an increase in TNF-alpha production; thereafter, it returns toward normal level through the activated TGF-beta/Smad pathway.

Involvement of the sucrose transporter, OsSUT1, in the long-distance pathway for assimilate transport in rice.

The roles of the rice sucrose transporter, OsSUT1, have previously been examined in filling grain, germination, and early seedling growth. In the current work, the role that OsSUT1 plays in the transport of assimilate along the entire long-distance pathway, from the flag leaf blade to the base of the filling grain, was investigated. OsSUT1 promoter::GUS (beta-glucuronidase) reporter gene analysis and immunolocalization revealed that both OsSUT1 promoter::GUS activity and OsSUT protein were present in the mature phloem of all the vegetative tissues involved in the long-distance assimilate transport pathway during grain filling. In addition, expression was observed in the flag leaf blade and sheath prior to heading. The OsSUT1 promoter::GUS activity appeared to be largely confined to the companion cells within the phloem, whereas the protein localized to both the sieve tubes and the companion cells. RT-PCR analysis confirmed that the OsSUT1 transcript is expressed in the uppermost internode of the rice plant (internode-1). These OsSUT localization data were related to measurements of starch and soluble sugar content of these tissues, and localization of the carbohydrate reserves stored in the stem. Results from dye feeding experiments, to examine cellular connections, revealed a symplastic continuity between the phloem and surrounding parenchyma in the flag leaf blade, sheath, and internode-1 tissues. It is proposed that OsSUT1 may primarily play a role in phloem loading of sucrose retrieved from the apoplasm along the transport pathway.