Comprehensive Network Analysis of Anther-Expressed Genes in Rice by the Combination of 33 Laser Microdissection and 143 Spatiotemporal Microarrays

Co-expression networks systematically constructed from large-scale transcriptome data reflect the interactions and functions of genes with similar expression patterns and are a powerful tool for the comprehensive understanding of biological events and mining of novel genes. In Arabidopsis (a model dicot plant), high-resolution co-expression networks have been constructed from very large microarray datasets and these are publicly available as online information resources. However, the available transcriptome data of rice (a model monocot plant) have been limited so far, making it difficult for rice researchers to achieve reliable co-expression analysis. In this study, we performed co-expression network analysis by using combined 44 K agilent microarray datasets of rice, which consisted of 33 laser microdissection (LM)-microarray datasets of anthers, and 143 spatiotemporal transcriptome datasets deposited in RicexPro. The entire data of the rice co-expression network, which was generated from the 176 microarray datasets by the Pearson correlation coefficient (PCC) method with the mutual rank (MR)-based cut-off, contained 24,258 genes and 60,441 genes pairs. Using these datasets, we constructed high-resolution co-expression subnetworks of two specific biological events in the anther, “meiosis” and “pollen wall synthesis”. The meiosis network contained many known or putative meiotic genes, including genes related to meiosis initiation and recombination. In the pollen wall synthesis network, several candidate genes involved in the sporopollenin biosynthesis pathway were efficiently identified. Hence, these two subnetworks are important demonstrations of the efficiency of co-expression network analysis in rice. Our co-expression analysis included the separated transcriptomes of pollen and tapetum cells in the anther, which are able to provide precise information on transcriptional regulation during male gametophyte development in rice. The co-expression network data presented here is a useful resource for rice researchers to elucidate important and complex biological events

Abscisic Acid–Induced Transcription Is Mediated by Phosphorylation of an Abscisic Acid Response Element Binding Factor, TRAB1

The rice basic domain/Leu zipper factor TRAB1 binds to abscisic acid (ABA) response elements and mediates ABA signals to activate transcription. We show that TRAB1 is phosphorylated rapidly in an in vivo labeling experiment and by phosphatase-sensitive mobility shifts on SDS–polyacrylamide gels. We had shown previously that a chimeric promoter containing GAL4 binding sites became ABA inducible when a GAL4 binding domain–TRAB1 fusion protein was present. This expression system allowed us to assay the ABA response function of TRAB1. Using this system, we show that Ser-102 of TRAB1 is critical for this function. Because no ABA-induced mobility shift was observed when Ser-102 was replaced by Ala, we suggest that this Ser residue is phosphorylated in response to ABA. Cell fractionation experiments, as well as fluorescence microscopy observations of transiently expressed green fluorescent protein–TRAB1 fusion protein, indicated that TRAB1 was localized in the nucleus independently of ABA. Our results suggest that the terminal or nearly terminal event of the primary ABA signal transduction pathway is the phosphorylation in the nucleus of preexisting TRAB1

Culm morphology and culm strength of T65, <i>smos1</i>, ST-4, and LRC1.

<p>(A) Culm cross-sections from the uppermost (1st) to the fourth (4th) internodes of the main culms. Bar = 5 mm. (B) Cross-sections of the fourth internode showing the culm thickness of each plant. Bar = 1 mm. (C) Culm thickness of the fourth internodes of each plant (n = 3). (D) Culm diameter measured from the uppermost to the fourth internode of the main culms (n = 3). (E) Section modulus and (F) bending stress of each plant (n = 3). Tukey’s test was conducted for panels (C) to (F).</p

Rice lines with improved lodging resistance.

<p>(A) Bending-type lodging resistance of selected lines evaluated in terms of cLr value. Data are means ± SD (n> = 3). (B) Gross morphology of the selected lines. First up to the second panel of the third row show T65 (original cultivar) and T65 mutant lines. MN-1 and MN-2 at the third row are Nipponbare mutants and MK-1 at the fourth row is a Kinmaze mutant, respectively. Bar = 20 cm.</p

Breaking-type lodging resistance and culm morphologies of selected lines.

<p>(A) Breaking-type lodging resistance evaluated in terms of bending moment at breaking. Data are means ± SD (n> = 3). The fourth internodes of MT-6 and MT-8 could not be evaluated due to short internode lengths. (B) Magnified view of the third internode cross-section of 7 lines showing high bending-moment-at-breaking value in comparison with original cultivars (T65, Nipponbare, and Kinmaze). Bar = 500 µm. (C) Relative culm thickness of each line. Thickness of the original cultivars is set as 1. Data are means ± SD (n> = 3). The uppermost to fourth internodes were measured.</p

Selection of lines with high grain yield and improved lodging resistance from the F5 population of the <i>smos1</i> and ST-4 cross.

<p>(A) Procedure to select for high grain yield and improved lodging resistance. (B) Grain weight per plant of lines from the F5 population of the <i>smos1</i> and ST-4 cross. Data are means ± SD (n> = 3). (C) Bending-moment-at-breaking values of line #1 and line #5 (n> = 3).</p

Morphology, plant height, and tiller numbers of T65, <i>smos1</i>, ST-4, and LRC1.

<p>(A) Gross morphology of plants at 30 days after heading. Bar = 10 cm. (B) Diagram for plant height. (C) Diagram for tiller numbers. Plant height and tiller numbers (n> = 3) were measured at 40 days after heading. Tukey’s test was conducted for panels (B) and (C).</p