Additional Nitrogen Fertilization at Heading Time of Rice Down-Regulates Cellulose Synthesis in Seed Endosperm

<div><p>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.</p></div

Rice cultivation schedule.

<p>Samples were grown in a growth chamber at 28°C/22°C over a 12-h light/12-h dark cycle. Fertilizer was supplied at planting and 37 days after germination. In addition, 400 mg ammonium chloride (NH₄Cl) was supplied at heading time to the “+NH₄Cl” group.</p

Significantly enriched categories were identified using QuickGO.

<p>In response to additional fertilization, 1,365 genes were up- or down-regulated; FDR-corrected <i>P</i>-values of categories at the deepest hierarchical level are shaded; *<i>P</i><0.05.</p

Cluster dendrogram.

<p>A cluster dendrogram was generated using rice seed gene expression data from six samples and the “pvclust” function. Each sample was prepared at 15-DAF seeds from 24 control or 24 N-fertilized plants and were pooled. Three sets of five seeds were picked from each seed pool. C, control samples; N, N-fertilized samples.</p

Genes related to synthesis of storage substances in seeds with altered mRNA expression in response to additional fertilizer.

<p>Genes related to synthesis of storage substances in seeds with altered mRNA expression in response to additional fertilizer.</p

Analysis of proteins in polished rice from incubator cultivated plants.

<p>Three independent plants were selected from the N-fertilized and control plots. (A) Nitrogen content of polished rice was determined using a Rapid-N analyzer. Error bars represent standard deviation (SD; *<i>P</i><0.001) (B) Protein composition of polished rice was analyzed using SDS-PAGE. Soluble proteins and insoluble proteins were extracted sequentially from the same sample. Major storage proteins in rice seed were extracted as insoluble proteins, as indicated at the right side of the gel.</p

Amino acid and protein contents of mature polished rice cultivated in a field.

<p>Field trials were conducted for three years and data are expressed as means of the three trials. Filled bars correspond with samples from the N-fertilized plot and unfilled bars correspond with samples from the control plot. Error bars represent standard deviation (SD; *<i>P</i><0.01) (A) Total amino acid content was analyzed after hydrolysis using hydrogen chloride. (B) Protein content was estimated using the Kjeldahl method.</p

β-Glucan content of rice grains.

<p>(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 25^th and 75^th percentiles. The upper line extending from the box indicates the largest value between the 75^th percentile and the point at 1.5 times the interquartile range. The lower line extending from the box indicates the smallest point between the 25^th percentile and the point at 1.5 times the interquartile range; *<i>P</i><0.05.</p

Changes in gene expression and rice seed compounds with additional fertilization.

<p>Notable change in mRNA expression and seed compounds are summarized. Molecules in the open oval are DEGs from DNA microarray experiments. Rectangles show the compound whose content was measured in this study. Red and blue arrows indicate up- and down-regulation by additional nitrogen fertilization, respectively. SUS3, sucrose synthase 3; CESA, cellulose synthase A catalytic subunit; SBE3, starch-branching enzyme 3; SS1, starch synthase 1; SP2, starch phosphorylase 2; αAmy, α-amylase; βAmy, β-amylase.</p