Os07g0622900

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Annotated Information

Function

Please input function information here. DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1) encodes for an endoplasmic reticulum (ER) It is a membrane protein that is present only in cereals. It can controls the early stages of that development It is plays important roles in the ER membrane during early tapetum development, functioning after MSP1 and before UDT1, and also in meiocyte development, after MEL1 and before PAIR1.

mutation and phenotype

Please input mutation information here. Mutant generated T-DNA insertion in japonica rice. And isolated two another T-DNA-tagged line，dtm1-2, in which T-DNA was inserted into the first intron, 284 bp upstream of the mutant dtm1-1 insertion site . These dtm1-2 mutant plants exhibited the same male-sterile phenotype as dtm1-1.

Mutant anthers were white and smaller than those from segregating wild-type (WT) siblings (Figure 1a,b). And no grains existed in the mutant anthers (Figure1c). Nonetheless, mutant spikelets pollinated with WT pollen grains produced fertile seeds. When their heteroprogeny was crossed with WT pollen, two types of offspring resulted: the WT and one that carried T-DNA at an approximately 1:1 ratio. These results indicated that the female organs were normal. When the heterozygous progeny was selfed, normal and sterile plants were produced at a 3:1 ratio, thereby demonstrating that this sterile phenotype was the result of a male defect caused by a single recessive mutation. Plants of this mutant, dtm1, had no other developmental abnormalities.

In WT, the primordium was formed via cell division in the L1, L2 and L3 layers of the floral meristem at stage 1. At the four corners of the hypodermis, archespores began to differentiate at stage 2, before developing to sporogenous cells and parietal cells during stages 3 and 4. At stage 5, the parietal layers developed endothecium, middle and tapetal layers, creating four layers of anther walls (Figure 4a). The sporogenous cells generated PMCs within the locule at stage 6 (Figure 4b). During stages 7 and 8, The PMCs then underwent meiosis and formed dyads and tetrads (Figure 4c,d). By that time, the tapetal-cell layers showed considerable development and differentiation, whereas cells from the middle layer had started to shrink. Afterward, the cytoplasm of the tapetal cells pulled away from the peritapetal region and condensed toward the middle layer (Figure 4d). The dtm1 mutant anthers did not differ from the WT at stages 5–7 (Figure 4e,f). That is, the four layers of the anther wall were normally formed and PMCs had developed as expected. However, differences between the WT and mutant started to emerge at stage 8a (Figure 4g). That is, whereas the middle cell layer in the WT was almost entirely diminished, no change was seen for that layer in dtm1, remaining, instead, in the condition associated with stage 7. During stages 6–8a, OsGSL5(encodes an anther-specific callose synthase )and Osg1 (required for callose degradation). Expression of OsGSL5 was not altered in the mutants compared with the WT, whereas Osg1 transcripts were reduced in the mutants. Such a decrease in Osg1 expression would have caused a hyper-accumulation of callose in dtm1. At stage 8b, the dtm1 tapetum, unlike that of the WT, did not differentiate, but instead became enlarged (Figure 4h). Meiocytes were degenerated and crushed meiotic products were present in the locule. The middle layer appeared to remain the same as at the previous stage, without degeneration. Stage 9 for the WT began with tapetal cells being condensed and young microspores detaching from the tetrads (Figure 4i). In stage 10, microspores enlarged and had expanded vacuoles (Figure 4j). By stage 11, the tapetal cells had almost disappeared and young microspores became bi-nucleus pollen grains, with both a generative nucleus and a vegetative nucleus (Figure 4k). The vacuole shrank and cytoplasm contents increased. At stage 12, the tapetal cell layer was completely empty and vacuoles in the grains had disappeared, being filled instead with starch granules and other substances (Figure 4l). In contrast, dtm1 anthers at stage 9 had more swollen tapetal cells (Figure 4m). At the later stages, those swollen cells and remnants of degenerated meiotic cells were still evident (Figure 4n–p).

The transmission electron microscopy (TEM) analysis show the mutant tapetal cell cytoplasm was filled with globular structures lacking any obvious organelle development (Figure5f); these phenotypes appeared from late stage 7. Also observed spiral forms that appeared to be undeveloped ER stacks (Figure 5g). The WT cytoplasm began to detach from the peritapetal regions and radial cell wall, making spaces obvious between tapetal cells arrowheads in (Figure 5b). In the mutant, however, the tapetum cytoplasm was not so detached (Figure 5f). At this stage, a callose layer was deposited over the cell walls of meiocytes (Figure 5d). However, the cell wall and callose wall of dtm1 meiocytes were irregular (Figure 5h). At stage 8b, these morphological differences were more evident. The WT tapetal cell cytoplasm was shrunken, with the tapetum cell wall toward the peritapetal region remaining, but the radial cell walls starting to disintegrate (Figure5i). In contrast, the tapetal cell cytoplasm from dtm1 did not shrink (Figure 5j). Instead, mutants retained the original cell walls, which contained irregularly shaped, small, dense bodies (arrows in Figure 5j) and expanded vesicles (arrowheads in Figure 5j). Some vesicles also had spiral deposits (Figure 5k). Cells were occasionally occupied by numerous small vesicles and acuoles (Figure 5l), whereas others had large vacuoles (Figure 5m). For the WT, tetrads formed at this stage and were enveloped by newly produced primexine walls (Figure 5n). However, mutant meiocytes did not develop to tetrads. These degenerated meiocytes contained numerous vesicles and, instead of primexine, a callose wall was present around them (Figure 5o).

One aspect of the dtm1 phenotype is the failure to form tetrads. Using anther length as a criterion for assessing stage progression show meiosis in the dtm1 mutant anthers was slow, and was eventually arrested at prophase I. Further experiment suggested that the loss of function of DTM1 affects PMCs entrance to meiosis, and hinders further progress in the early stages of prophase I.

Expression

Please input expression information here. DTM1 was expressed in all organs examined, transcripts were more abundant in young panicles . In developing anthers, expression was higher during meiosis. Laser microdissection transcriptome analyses also demonstrated its high expression in tapetal-layer cells and meiocytes. RNAin-situ hybridization analyses of the anthers at stages 6–8 show the transcripts of DTM1 were most abundant in the tapetal cells at stages 6 and 7, and moderately in the PMCs and meiocytes. The transcripts were also weakly detected in most anther walls, including the middle layer, endothecial cells, and epidermal cell layer. The level of transcript was significantly reduced at stage 8.

Real-time PCR analysis showed that expression of MSP1 was not altered in dtm1. But its expression was decreased in the UDT1 mutant. Therefore, these results suggest that DTM1 functions later than MSP1, but prior to UDT1. GAMYB and TDR, which are related to tapetal programmed cell death (PCD) transcript levels of these genes were not significantly, but OsC4, OsC6 and CP1 were significantly reduced in the dtm1 anthers. OsC6 and CP1 are direct targets of TDR: the former is related to pollen wall formation and the latter functions in tapetal PCD.

OsMEL1, which acts in premeiotic germ cell division, was not significantly changed in dtm1. Among PAIR1, PAIR2, PAIR3, DMC1 and ZEP1, which are known to function in chromosome pairing or synapsis the transcript level of PAIR1 was diminished in the mutants, whereas the other genes were not affected. In addition, the expression of PSS1, which encodes microtubule-stimulated ATPase activity, was reduced.

DTM1 levels were lower in msp1 anthers, but unchanged in the udt1, tdr and pair1 mutants.

Evolution

Please input evolution information here. DTM1 is unique to cereals: no homologous gene occurs in dicot species, including Arabidopsis. BLASTP search indicated that DTM1 has 17% identity with yeast SPC1p, a subunit of the signal peptidase complex (SPC), which is located in the ER.

Labs working on this gene

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1、Crop Biotech Institute, Kyung Hee University

2、Department of Life Science, Pohang University of Science and Technology (POSTECH)

3、Department of Plant Molecular Systems Biotechnology, Kyung Hee University

References

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Jakyung Yi, et al. The rice gene DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1) is required for early tapetum development and meiosis. The Plant Journal, (2012), 70, 256–270