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. 2019 Dec 6;9(1):18492.
doi: 10.1038/s41598-019-54898-1.

The Expression Pattern of OsDim1 in Rice and Its Proposed Function

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Free PMC article

The Expression Pattern of OsDim1 in Rice and Its Proposed Function

Henry Akrofi Doku et al. Sci Rep. .
Free PMC article

Abstract

Development of plant tissues is dependent on numerous factors, including hormone activity, signaling, cell division, and elongation. In plants, Defective Entry into Mitosis 1 (Dim1) homologs are recognized as pivotal in leaf senescence and progress of normal growth, but their role in rice has not been functionally characterized. The findings presented in this paper suggest that OsDim1 is important in early seedling development, pollen tube elongation, and impacts rice yield components. The gene is expressed in the scutellum, endosperm, embryonic root, shoot, pollen grains and tubes, as well as in several organs of the rice flower. According to the present study findings, RNAi mediated knockdown of OsDim1 resulted in phytohormonal imbalance, reduced amylase activity, affected differentiation of embryonic root elongation zone tissues, suppressed embryonic root and shoot growth, and impaired pollen tube elongation. In contrast, overexpression of OsDim1 showed significant growth in embryonic roots and shoots, while it increased culm length, total number of tillers per plant, seed setting rate, and total number of grains per panicle compared to its wild type line. In summary, we propose OsDim1 plays an important role in seedling growth and pollen tube elongation, and has pleiotropic effects on reproductive tissues.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Phylogenetic Tree and Sequence homology of OsDim1 Protein. (a) Phylogenetic relationship between OsDim1 protein and some members of the thioredoxin-like super family. OsDim1 protein is indicated in the phylogenetic tree as OsTRX-Dim1 (enclosed in a box) and forms a branch with thioredoxin-like Zea mays mitosis Dim1, Arabidopsis thaliana YLS8, Glycine max Dim1, and S. pombe Dim1 proteins at a bootstrap value of 100. The phylogenetic tree was constructed by means of MEGA software (version 5.0) using the neighbor-joining method. Bootstrap value = 0.2 and the text in parentheses denote the accession numbers or Unigene entry codes (https://www.ncbi.nlm.nih.gov/unigene) of the proteins. (b) Amino acid sequence alignment of the thioredoxin-like Dim1 proteins that formed a branch with OsDim1 protein at a bootstrap value of 100%, as shown in the phylogenetic tree. The boxes indicate the critical sites of the conserved domains of Dim1 the protein family. The alignment was performed with DNAMAN8.
Figure 2
Figure 2
Expression Pattern of OsDim1 in Different Tissues of OsDim1pro:GUS (GUS) Transgenic Rice Plant. (a) Histochemical staining assay showing the expression of OsDim1 in the endosperm (EN) as well as zones of cell elongation (EZ) and root hair (HZ) of the seminal root (SR) of a 5-day-old GUS transgenic rice seedling. Histochemical assay showing expression of OsDim1 in the nodal septal (NO) (b), auricles (AU), collar (CO), leaf sheath (LS) (c), leaf blade (LB) (d), immature panicle (PA) (e), style (SY) (f), pollen grains (PO) and stigma (ST) (g) at the early reproductive stage of the rice plant. (h) Expression of OsDim1 in the embryogenic callus of GUS transgenic rice. Pollen mean time transit test showing expression of OsDim1 in the stigma (SB), as well as the pollen tips or the entire pollen tube (PT) during its emergence and elongation at three minutes after pollination (3MAP) (i), 5MAP (j) and 30 MAP (k). (l) RT-PCR analysis of OsDim1 in comparison with the rice β-actin gene. OsDim1 expression was detected in the panicles, nodes and internodes, leaf blade, leaf shoot and root of the rice plant at the seedling stage (SS), tiller stage (TS), panicle initiation stage (PIS), meiotic division phase of the rice panicle (flower) development (MS), just before the flowering stage (JFS), flowering stage (FS), five days after pollination (5 D), and fifteen days after pollination (15 D) of the rice plant. Abbreviations: COL, coleoptile; LI, ligule; OV, ovary; OU, ovules, SH, shoot. TP, tip of pollen tube. Full length gels of the RT-PCR results are displayed in Supplementary Fig. S1.
Figure 3
Figure 3
Expression Pattern of OsDim1 by β-Glucuronidase and Enhanced Green Fluorescent Protein (EGFP). Histochemical staining showing expression of OsDim1 in the internal structure of the endosperm of two (a), three (b), five (c) and seven (d) days old OsDim1pro:GUS (GUS) transgenic rice seedlings respectively. (e) Histochemical staining showing OsDim1 expression in the shoot apex of a 5-day-old GUS transgenic rice seedling. Histochemical staining showing the expression of OsDim1 on the external structure/layer of the endosperm of 2 (f) and 7 (g) days old GUS transgenic rice seedlings, respectively. Histochemical staining showing OsDim1 expression in the seminal root DZ (h) and branch root (i) of 5-day-old GUS transgenic rice seedlings, respectively. (j) EZ cross-section of the seminal root of a 5-day-old GUS transgenic rice seedling showing OsDim1 fluorescence signals in the exodermis (EXO), sclerencymatous cells (SCL), endodermis (END), pericycle (PER) and vascular cylinder (VC). (k) Vertical section of seminal root EZ of a 5-day-old GUS transgenic rice seedling showing OsDim1 fluorescence signals. (l) Fluorescence microscopy showing subcellular localization of OsDim1 in onion epidermal cells. Top panel: Control EGFP (35Spro:EGFP) fluorescence signals were identified in the nucleus (NU) and cytoplasm of onion epidermal cells by bright fields, EGFP, and merged images, respectively. Bottom panel: OsDim1 fused to EGFP (35Spro:OsDim1-EGFP) signals were observed only in the nucleus of onion cells by bright fields, EGFP, and merged images, respectively. Bar length = 200 µm. Abbreviation: COR − cortex.
Figure 4
Figure 4
Comparison of the Morphology and Internal Structure of WT and OsDim1 Transgenic Rice Seedlings. (a) Molecular cloning of RNAi constructs. The diagram depicts the OsDim1 ORF length (429 bp), and the fragment length or cloning position for RNAi 1 (155 bp) and RNAi 2 (331 bp) transgenic rice development. (b) RT-PCR analysis showing low levels of expression of RNAi mediated knock down lines compared to the OVE and WT lines, with the rice β-actin gene as an internal standard to normalize the expressions. Full length gels of the RT-PCR results are displayed in Supplementary Fig. S2. (c) Phenotypic difference of the root and shoot length between the WT and OsDim1 transgenic rice lines after ten (10) days of seeding. Estimation of the differences in shoot (d) and root (e) length between the OVE, WT and RNAi rice lines. Shoot and root length in the WT and RNAi lines were assessed over a period of 20 days, while the period was shortened to only 15 days for the OVE line. (f) Comparison of the internal structure (cross-section) of the seminal roots of 5-day-old WT and RNAi rice seedlings. Abbreviations: OVE, OsDim1 overexpression transgenic seedlings; RNAi1, OsDim1-RNA interference (RNAi) transgenic line 1 rice seedlings; RNAi2, RNAi transgenic line 2 rice seedlings; WT, LiyuB wild type seedlings.
Figure 5
Figure 5
Assessment of Morphological Traits of the WT, OVE, and RNAi Lines. Phenotypic discrepancies in panicle length (a,b), culm length (c), seed setting rate (d), tiller number (e) and total number of grains per panicle (f) between the WT, OVE and RNAi rice lines. No significant differences were observed in the pollen fertility (g) between the WT, OVE and RNAi rice lines. Asterisks indicate statistically significant differences (*P < 0.05) and statistically highly significant differences (**P < 0.01 and ***P < 0.001). Error bars indicate standard deviation (±SD). Statistical analysis was performed by one way- ANOVA using the program PRISM 6.0.
Figure 6
Figure 6
Evaluation of Pollen Fertility, Viability, Germination, and Elongation of Pollen Tube of WT and RNAi Transgenic Rice Lines. Comparison of pollen fertility between WT (a) and RNAi (b) by I2-KI assay. Comparison of pollen viability between WT (c) and RNAi (d) by Alexander’s staining method. (e) A diagram summarizing the findings of the pollen mean time transit test of WT pollen and the growth of its pollen tube. Self-pollinated WT flower (WT/WT) developed pollen tubes that reached the style in 20 mins and penetrated the embryo sac at 40 MAP. (f) A diagram summarizing the effect of reciprocal crosses between WT and RNAi flowers for 60 mins. A cross-pollination between 30 WT pollen and 30 RNAi female flowers (RNAi/WT) resulted in germination of all WT pollen (100%), which subsequently developed pollen tubes that elongated and penetrated the embryo sac at 60 MAP. A reciprocal cross between 30 RNAi pollen and 30 WT female flowers (WT/RNAi) resulted in germination of only 60% of the RNAi pollen, which subsequently produced pollen tubes that elongated slowly and reached the style at 60 MAP. (g) A demonstration of WT pollen that grew successfully on the RNAi stigma with the tip of its pollen tube in the embryo sac at 60 MAP. The section of (g) enclosed in a square is shown next to the image. (h) A demonstration of one of the 60% RNAi pollen tubes that reached the WT style at 60 MAP. The section of (h) enclosed in a square is shown next to the image. (i) A demonstration of one of the 40% RNAi pollen grains on the WT stigma that produced pollen tubes but did not elongate. Bar length = 100 μm.
Figure 7
Figure 7
Difference in IAA (a), GA (b), ABA (c), CTK (d) and α-Amylase (e) concentrations between 5 days old WT and RNAi rice seedlings. Error bars indicate standard deviation (±SD). Asterisks indicate statistically significant differences (*P < 0.05) and highly significant differences (**P < 0.01 and ***P < 0.001). Abbreviations: ABA, abscisic acids; CTK, cytokinin; D, days old; ES, embryo sac; GA, gibberellin; IAA, indole acetic acid, MI, micropyle; OV, ovary, OVE, OsDim1 overexpressed transgenic line; PG, pollen grain; PT, pollen tube; ST, stigma; SY, style; WT, wild type rice. Statistical analysis was performed by one way- ANOVA using the program PRISM 6.0.

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