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Additional nitrogen fertilization at heading time of rice down-regulates cellulose synthesis in seed endosperm.

Midorikawa K, Kuroda M, Terauchi K, Hoshi M, Ikenaga S, Ishimaru Y, Abe K, Asakura T - PLoS ONE (2014)

Bottom Line: As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed.Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression.These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
The balance between carbon and nitrogen is a key determinant of seed storage components, and thus, is of great importance to rice and other seed-based food crops. To clarify the influence of the rhizosphere carbon/nitrogen balance during the maturation stage of several seed components, transcriptome analysis was performed on the seeds from rice plants that were provided additional nitrogen fertilization at heading time. As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed. Moreover, cellulose and sucrose synthases, which are involved in cellulose synthesis, were down-regulated. Therefore, we compared cellulose content of mature seeds that were treated with additional nitrogen fertilization with those from control plants using calcofluor staining. In these experiments, cellulose content in endosperm from plants receiving additional nitrogen fertilization was less than that in control endosperm. Other starch synthesis-related genes such as starch synthase 1, starch phosphorylase 2, and branching enzyme 3 were also down-regulated, whereas some α-amylase and β-amylase genes were up-regulated. On the other hand, mRNA expression of amino acid biosynthesis-related molecules was up-regulated. Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression. These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm.

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β-Glucan content of rice grains.(A) Histochemical staining of mature rice seed. Cross-sections of grains from the control plot (left) and from the N-fertilized plot (+NH4Cl; right) are shown. Sections were stained for β-glucan using calcofluor white. The white-dotted line indicates the area analyzed for fluorescence intensity. The fluorescence intensity of the endosperm was calculated by adjusting the background intensity. Scale bars, 1 mm. (B) Endosperm fluorescence intensity was analyzed using ImageJ. Sections were cut from 30 randomly selected grains from each plot. The horizontal line inside the box plot indicates the median value. The inner box indicates the interquartile range and runs between 25th and 75th percentiles. The upper line extending from the box indicates the largest value between the 75th percentile and the point at 1.5 times the interquartile range. The lower line extending from the box indicates the smallest point between the 25th percentile and the point at 1.5 times the interquartile range; *P<0.05.
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pone-0098738-g006: β-Glucan content of rice grains.(A) Histochemical staining of mature rice seed. Cross-sections of grains from the control plot (left) and from the N-fertilized plot (+NH4Cl; right) are shown. Sections were stained for β-glucan using calcofluor white. The white-dotted line indicates the area analyzed for fluorescence intensity. The fluorescence intensity of the endosperm was calculated by adjusting the background intensity. Scale bars, 1 mm. (B) Endosperm fluorescence intensity was analyzed using ImageJ. Sections were cut from 30 randomly selected grains from each plot. The horizontal line inside the box plot indicates the median value. The inner box indicates the interquartile range and runs between 25th and 75th percentiles. The upper line extending from the box indicates the largest value between the 75th percentile and the point at 1.5 times the interquartile range. The lower line extending from the box indicates the smallest point between the 25th percentile and the point at 1.5 times the interquartile range; *P<0.05.

Mentions: Cell wall-related genes such as those encoding cellulose synthase A catalytic subunit (CESA) and xyloglucan endotransglucosylase/hydrolase, were down-regulated following additional fertilization (Table 2). Genes encoding OsCESA2, OsCESA5, OsCESA6, OsCESA8, CESA-like protein A5 (OsCslA5), and CESA-like protein F6 (OsCslF6) are all involved in cell wall synthesis, and were down-regulated in the presence of additional fertilization. Thus, seeds were examined to determine whether β-glucan constituents of cellulose and (1, 3; 1, 4)-β-D-glucan actually decreased. Staining with calcofluor white demonstrated weaker fluorescence intensity in seed endosperms from the N-fertilized plot than from the control plot (Fig. 6). Cell walls of rice endosperm comprise cellulose and hemicellulose [21]. Rice hemicellulose comprises arabinoxylans and (1, 3; 1, 4)-β-D-glucan, also known as mixed-linkage glucan (MLG). Although calcofluor white stains both cellulose and MLG, rice does not accumulate significant amount of MLG in its grains [21], [22]. Thus the present data suggest decreased cellulose content of endosperm. In general, cellulose is synthesized from UDP-glucose by CESA, which comprises a cellulose synthase complex of six subunits [23]–[25]. Although at least 11 genes have been annotated as CESA in the MSU rice genome annotation project, the functions of only three genes, OsCESA4, OsCESA7, and OsCESA9, have been examined [26]. Sucrose synthase (SUS), which is involved in UDP-glucose metabolism, was down-regulated by additional fertilization, and plays a direct role in cell wall biosynthesis by forming a complex with CESA [27]–[30]. Six SUS homologs are present in the rice genome [31]. Among these, SUS2 and SUS3 were extracted from DEGs as genes that were down-regulated by additional fertilization (Table 1). In particular, SUS3 is reportedly localized primarily in the endosperm and in the aleurone layer [31], [32], indicating that CESA2, CESA5, CESA6, and CESA8 may form a complex with SUS3 to synthesize cellulose in the endosperm. The relationship between rice quality and cellulose content remains poorly understood. However, it is accepted that rice with a high nitrogen content has less stickiness, greater hardness after cooking, less palatability, and less processing quality [33]–[35]. Accordingly, cell walls may become harder with increased cellulose content. However, in the present experiments, rice with high nitrogen content contained less cellulose. Thus, the complexities of relationship between cooked-rice properties and rice-seed components require further detailed study.


Additional nitrogen fertilization at heading time of rice down-regulates cellulose synthesis in seed endosperm.

Midorikawa K, Kuroda M, Terauchi K, Hoshi M, Ikenaga S, Ishimaru Y, Abe K, Asakura T - PLoS ONE (2014)

β-Glucan content of rice grains.(A) Histochemical staining of mature rice seed. Cross-sections of grains from the control plot (left) and from the N-fertilized plot (+NH4Cl; right) are shown. Sections were stained for β-glucan using calcofluor white. The white-dotted line indicates the area analyzed for fluorescence intensity. The fluorescence intensity of the endosperm was calculated by adjusting the background intensity. Scale bars, 1 mm. (B) Endosperm fluorescence intensity was analyzed using ImageJ. Sections were cut from 30 randomly selected grains from each plot. The horizontal line inside the box plot indicates the median value. The inner box indicates the interquartile range and runs between 25th and 75th percentiles. The upper line extending from the box indicates the largest value between the 75th percentile and the point at 1.5 times the interquartile range. The lower line extending from the box indicates the smallest point between the 25th percentile and the point at 1.5 times the interquartile range; *P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048278&req=5

pone-0098738-g006: β-Glucan content of rice grains.(A) Histochemical staining of mature rice seed. Cross-sections of grains from the control plot (left) and from the N-fertilized plot (+NH4Cl; right) are shown. Sections were stained for β-glucan using calcofluor white. The white-dotted line indicates the area analyzed for fluorescence intensity. The fluorescence intensity of the endosperm was calculated by adjusting the background intensity. Scale bars, 1 mm. (B) Endosperm fluorescence intensity was analyzed using ImageJ. Sections were cut from 30 randomly selected grains from each plot. The horizontal line inside the box plot indicates the median value. The inner box indicates the interquartile range and runs between 25th and 75th percentiles. The upper line extending from the box indicates the largest value between the 75th percentile and the point at 1.5 times the interquartile range. The lower line extending from the box indicates the smallest point between the 25th percentile and the point at 1.5 times the interquartile range; *P<0.05.
Mentions: Cell wall-related genes such as those encoding cellulose synthase A catalytic subunit (CESA) and xyloglucan endotransglucosylase/hydrolase, were down-regulated following additional fertilization (Table 2). Genes encoding OsCESA2, OsCESA5, OsCESA6, OsCESA8, CESA-like protein A5 (OsCslA5), and CESA-like protein F6 (OsCslF6) are all involved in cell wall synthesis, and were down-regulated in the presence of additional fertilization. Thus, seeds were examined to determine whether β-glucan constituents of cellulose and (1, 3; 1, 4)-β-D-glucan actually decreased. Staining with calcofluor white demonstrated weaker fluorescence intensity in seed endosperms from the N-fertilized plot than from the control plot (Fig. 6). Cell walls of rice endosperm comprise cellulose and hemicellulose [21]. Rice hemicellulose comprises arabinoxylans and (1, 3; 1, 4)-β-D-glucan, also known as mixed-linkage glucan (MLG). Although calcofluor white stains both cellulose and MLG, rice does not accumulate significant amount of MLG in its grains [21], [22]. Thus the present data suggest decreased cellulose content of endosperm. In general, cellulose is synthesized from UDP-glucose by CESA, which comprises a cellulose synthase complex of six subunits [23]–[25]. Although at least 11 genes have been annotated as CESA in the MSU rice genome annotation project, the functions of only three genes, OsCESA4, OsCESA7, and OsCESA9, have been examined [26]. Sucrose synthase (SUS), which is involved in UDP-glucose metabolism, was down-regulated by additional fertilization, and plays a direct role in cell wall biosynthesis by forming a complex with CESA [27]–[30]. Six SUS homologs are present in the rice genome [31]. Among these, SUS2 and SUS3 were extracted from DEGs as genes that were down-regulated by additional fertilization (Table 1). In particular, SUS3 is reportedly localized primarily in the endosperm and in the aleurone layer [31], [32], indicating that CESA2, CESA5, CESA6, and CESA8 may form a complex with SUS3 to synthesize cellulose in the endosperm. The relationship between rice quality and cellulose content remains poorly understood. However, it is accepted that rice with a high nitrogen content has less stickiness, greater hardness after cooking, less palatability, and less processing quality [33]–[35]. Accordingly, cell walls may become harder with increased cellulose content. However, in the present experiments, rice with high nitrogen content contained less cellulose. Thus, the complexities of relationship between cooked-rice properties and rice-seed components require further detailed study.

Bottom Line: As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed.Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression.These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
The balance between carbon and nitrogen is a key determinant of seed storage components, and thus, is of great importance to rice and other seed-based food crops. To clarify the influence of the rhizosphere carbon/nitrogen balance during the maturation stage of several seed components, transcriptome analysis was performed on the seeds from rice plants that were provided additional nitrogen fertilization at heading time. As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed. Moreover, cellulose and sucrose synthases, which are involved in cellulose synthesis, were down-regulated. Therefore, we compared cellulose content of mature seeds that were treated with additional nitrogen fertilization with those from control plants using calcofluor staining. In these experiments, cellulose content in endosperm from plants receiving additional nitrogen fertilization was less than that in control endosperm. Other starch synthesis-related genes such as starch synthase 1, starch phosphorylase 2, and branching enzyme 3 were also down-regulated, whereas some α-amylase and β-amylase genes were up-regulated. On the other hand, mRNA expression of amino acid biosynthesis-related molecules was up-regulated. Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression. These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm.

Show MeSH