Total ceramide analysis.

<p>6A) Total ceramide analysis in leafs of RNAi rice lines and WT rice plants. 6B) Total ceramide analysis in panicles of tms2 mutant and WT rice plants. Ceramide levels were determined from the leaves of <i>LOC_Os07g26940</i> RNAi (3ES-41 and 7ES-3) or <i>ORMDL</i> RNAi (29KS38 and 29KS-40) transgenic rice plants, and compared with equivalent tissue from WT. Results were expressed as absolute levels of ceramides (nmol/g dw). Similar analysis was performed on panicles of either WT or tms2 mutant rice, and data is presented as absolute levels of ceramides (nmol/g dw). Absolute levels (A, B) represent the mean of 6 technical replicates for each independent line. Bar indicates the standard error (n = 6). * indicates statistical significance at p-value of 0.05 as determined by t-test. 3ES-41 and 7ES-3 or 29KS38 and 29KS-40 are two independent transgenic lines from the same construct.</p

Expression analysis and pollen staining of RNAi transgenic rice plants.

<p>A) qRT-PCR analysis of <i>ORMDL LOC_Os07g26940</i> and other <i>ORMDL</i> gene expression in leaves of <i>LOC_Os07g26940</i> RNAi (3ES-41 and 7ES-3). B) qRT-PCR analysis of <i>ORMDL LOC_Os07g26940</i> and other <i>ORMDL</i> gene expression in leaves of <i>ORMDL</i> RNAi (29KS38 and 29KS-40) transgenic rice plants. 3ES-41 and 7ES-3 or 29KS38 and 29KS-40 are two independent transgenic lines from the same construct. The expression level of <i>EF-1α</i> was used as internal control. All data are representative from at least two biological repeats, each based on three technical replicates; similar results were obtained in the repeated experiments. Bars indicate the standard error (n = 3). which was calculated form technical replicates. Each biological sample was a mixture of 3 plants. C) Pollen staining of wild type, <i>LOC_Os07g26940</i> RNAi, and <i>ORMDL</i> RNAi transgenic rice plants. Similar results were obtained from the two tested transgenic lines form each construct. The pictures are a representative from each construct.</p

Sphingolipid long-chain base composition ng LCB mg⁻¹ dry weight in wild type and <i>tms2</i> mutant rice plants in leaf tissue(A) and in panicles(B).

<p>Sphingolipid long-chain base composition ng LCB mg⁻¹ dry weight in wild type and <i>tms2</i> mutant rice plants in leaf tissue(A) and in panicles(B).</p

Expression analysis by quantitative RT-PCR.

<p>(A) qRT-PCR analysis of <i>LOC_Os07g26930</i> and all three splicing forms of <i>ORMDL LOC_Os07g26940</i> in panicles and anthers of wild type rice plants grown in control growth room at 26°C and 32°C. The expression level of <i>Actin1</i> (<i>Os05g36290</i>) was used as internal control. (B) qRT-PCR analysis of all three splicing forms of <i>LOC_Os07g26940</i> in wild type rice tissues, including stems, leaves, roots, small panicles (1–2 cm), medium panicles (3–14 cm), large panicles (15–25 cm) and anthers. The expression level of <i>EF-1α</i> (<i>Os03g08020</i>) was used as internal control. All data are representative from at least two biological repeats, each based on three technical replicates; similar results were obtained in the repeated experiments. Bars indicate the standard error (n = 3), which was calculated from technical replicates. Each biological sample was a mixture of 3 plants.</p

Ceramide composition in leaves of <i>Japonica</i> WT and RNAi rice lines.

<p>Unit: nmol g dw⁻¹, N: a minimum of 3± SE.</p><p>Ceramide composition in leaves of <i>Japonica</i> WT and RNAi rice lines.</p

Rice <i>ORMDL</i> Controls Sphingolipid Homeostasis Affecting Fertility Resulting from Abnormal Pollen Development

<div><p>The orosomucoids (ORM) are ER-resisdent polypeptides encoded by <i>ORM</i> and <i>ORMDL</i> (ORM-like) genes. In humans, ORMDL3 was reported as genetic risk factor associated to asthma. In yeast, ORM proteins act as negative regulators of sphingolipid synthesis. Sphingolipids are important molecules regulating several processes including stress responses and apoptosis. However, the function of <i>ORM</i>/<i>ORMDL</i> genes in plants has not yet been reported. Previously, we found that temperature sensitive genetic male sterility (TGMS) rice lines controlled by <i>tms2</i> contain a deletion of about 70 kb in chromosome 7. We identified four genes expressed in panicles, including an <i>ORMDL</i> ortholog, as candidates for <i>tms2</i>. In this report, we quantified expression of the only two candidate genes normally expressed in anthers of wild type plants grown in controlled growth rooms for fertile and sterile conditions. We found that only the <i>ORMDL</i> gene (<i>LOC_Os07g26940</i>) showed differential expression under these conditions. To better understand the function of rice <i>ORMDL</i> genes, we generated RNAi transgenic rice plants suppressing either <i>LOC_Os07g26940</i>, or all three <i>ORMDL</i> genes present in rice. We found that the RNAi transgenic plants with low expression of either <i>LOC_Os07g26940</i> alone or all three <i>ORMDL</i> genes were sterile, having abnormal pollen morphology and staining. In addition, we found that both sphingolipid metabolism and expression of genes involved in sphingolipid synthesis were perturbed in the <i>tms2</i> mutant, analogous to the role of ORMs in yeast. Our results indicated that plant ORMDL proteins influence sphingolipid homeostasis, and deletion of this gene affected fertility resulting from abnormal pollen development.</p></div

Phylogenetic relationship of rice ORMDL and other ORMDL from different species across kingdom.

<p>An un-rooted neighbour joining phylogenetic tree was constructed from the protein sequences of rice ORMDL obtained from GRAMENE rice database and ORMDL from other species selected from NCBI Reference Sequence database. Multiple sequence alignment was performed using the ClustalW in MEGA5 and the tree was generated using MEGA5. The numbers for interior branches indicate the bootstrap values (%) for 1000 replications.</p

Identification of genes involved in early steps of sphingolipid biosynthesis in rice using homologous genes from <i>A. thaliana</i> and <i>S. cerevisiae</i>.

<p>Identification of genes involved in early steps of sphingolipid biosynthesis in rice using homologous genes from <i>A. thaliana</i> and <i>S. cerevisiae</i>.</p

Ceramide composition in panicles of <i>Indica</i> WT and <i>tms</i>2 rice lines.

<p>Unit: nmol g dw⁻¹, N: a minimum of 3± SE.</p><p>Ceramide composition in panicles of <i>Indica</i> WT and <i>tms</i>2 rice lines.</p