Phenotypic analysis of a dwarf and deformed flower3 (ddf3) mutant in rice (Oryza sativa L.) and characterization of candidate genes

1. Introduction

Rice is an important food crop that feeds over half of the world’s population, and it is also one of the most important monocot model systems because of characteristics such as a small sequenced genome and a mature Agrobacteriummediated genetic transformation system. Rice production is correlated with multiple factors, such as tiller number, grain number per panicle, grain size, and plant height. Plant height,which is established during the vegetative growth periods,plays a crucial role in plant architecture, photosynthetic efficiency, nutrient absorption, and lodging resistance (Krag and Nielsen 1989; Wang et al. 2016). Generally, taller plants are advantageous for maintaining a higher photosynthetic efficiency, but have reduced lodging tolerance; dwarf plants have increased lodging tolerance, but severe dwarfism affects the development of other organs, which is not conducive to the improvement of rice yield (Ding et al. 2015; Zhao et al. 2015;Zhang et al. 2016). The appropriate plant height, therefore,is a critical factor in improving rice yield.

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一要更好促进短期绩效与长期发展之间的综合平衡。农业产业扶贫资金多是在财政体制常规分配渠道之外，按照专项资金和项目制方式进行资源配置。农业生产的周期性特征决定了农业产业发展的特殊性，往往需要三五年甚至更长时间的持续投入，才能形成比较成熟的产业链。当前，个别贫困地区在产业扶贫项目资金的投入使用上，更偏向于追求财政绩效考核意义上的短、平、快，希望收到立竿见影的效果，这容易导致产业扶贫出现重短期效应、轻长效机制、组织化程度低、同质化严重等问题。解决这些问题，必须有“功成不必在我”的精神境界和“功成必定有我”的责任担当，真正以长远眼光，厚植产业发展的长久根基。

During the last 20 years, considerable progress has been made in elucidating the regulatory mechanisms controlling plant height in rice. Previous studies have shown that plant hormones such as gibberellin (GAs) (Daviere and Achard 2013), brassinolide (BRs) (Ye et al. 2011), and strigolactone(SLs) (Screpanti et al. 2016), and genes associated with cell wall synthesis, cellular differentiation, and cell elongation(Yang et al. 2011), are the main factors controlling plant height. Mutations in these genes inhibit the development of stem internodes, leading to a dwarf phenotype. So far, the genes known to be related to plant dwarfism are mainly involved in GA, BR, and SLs biosynthesis and signal transduction. Additionally, some genes that are not associated with hormone metabolic pathways, such as OsKinesin-13A (Deng et al. 2014), OSH15 (Sato et al.1999), OsGLP1 (Banerjee and Maiti 2010), and DTH8 (Wei et al. 2010), have been found to in fluence plant height.

Although determined during the vegetative growth stage,plant height has a significant effect on reproductive traits,such as heading date, flower development, and rice quality(Song et al. 2008; Duan et al. 2012a, b). Many dwarf mutants are associated with the abnormal flower development. For example, a mutation in DDF1, which encodes an F-box protein, leads to much shorter plant height compared with wild type and significant structural abnormalities in florets (Duan et al. 2012b). The ddf2 mutant has a severe dwarf phenotype and defects in spikelet and floral organs (Zhang et al. 2015).DEFORMED FLORAL ORGAN1 (DFO1) was identified as a rice epigenetic repressor. The dfo1 mutant has a small stature and defects in palea identity (Zheng et al. 2015). Although a few genes have been reported to control both plant height and floral organ development, flower-deformation/dwarf genes remain largely undiscovered.

To gain further insight into the relationship between plant height and flower development and the molecular basis for these phenotypes, we identified and characterized the dwarf and deformed flower 3 (ddf3) mutant, which in addition to a severe dwarf phenotype, exhibits pollen abortion and pistils with multiple stigmas. The mutant gene was mapped between insertion-deletion (InDel) markers M15 and M16 on the long arm of chromosome 7, which corresponds to an approximate physical distance of 45.21 kb. Sequencing revealed a 13.98-kb deletion in the ddf3 mutant genome,which caused the deletion of three genes. Our work has laid the foundation for further gene cloning and functional analysis of DDF3.

2. Materials and methods

2.1. Plant materials and growth conditions

The functions of ZF and EP are not known, but FH2 is an allele of BENT UPPERMOST INTERNODE1(BUI1)/RICE MORPHOLOGY DETERMINANT(RMD) (Yang et al. 2011;Zhang et al. 2011), which has been reported to be involved in the regulation of cell development. To gain insight into the roles of these genes in rice plant development, we investigated their expression patterns in different organs.We found that the expression of ZF was the highest in panicles, but ZF expression was very low in stems, leaves,and roots (Fig. 6-B). EP was not expressed in any of these organs, whereas the expression of FH2 was relatively high in all four organs (Fig. 6-C). These results indicated that ZF and FH2 could potentially be involved in the regulation of rice morphogenesis or flower organ development, while EP might not function at the heading stage.

2.2. Phenotypic characterization

At the heading stage, plant height, leaf length, leaf width,and internode length were measured in the mutant and WT.Yield-related traits, including panicle length, no. of grains per panicle, seed-setting rate, grain size, and 1 000-grain weight, were measured at harvest. All trait measurements are presented as the average from 10 plants.

2.3. Histological analysis of internodes

To identify the DDF3 locus, we evaluated the phenotypes of F1 plants and an F2 mapping population derived from a cross between ddf3 and 9311. All F1 plants displayed thick and tall stems, broad leaves, and low seed-setting rates.Furthermore, the segregation ratio of WT to mutant plants in the F2 population was 3:1 (normal:mutants=529:150;χc

2.4. Analysis of pollen activity and stigma development

Pollen abortion is divided into three types, typical abortion,spherical abortion, and stained abortion, based on the stage when abortion occurs. Typical abortion occurs during the early stages of pollen development and pollen generally only develop to the single nucleus pollen stage; spherical abortion usually occurs during the dual core pollen stage;stained abortion type mainly happens during the trinucleate pollen stage (Li 1980; Xiang et al. 2011). The pollen abortion types observed in the ddf3 mutant were mainly the spherical abortion and stained abortion types (Fig. 3-C), indicating that the mutated gene(s) mainly affect the dual core and trinucleate stages of pollen development. In addition to pollen defects, many pistils in the mutant showed a multistigma phenotype (Fig. 3-D and E), and in some pistils the stigma didn’t elongate. Therefore, it is very likely that the DDF3 gene(s) affects the seed-setting rate by regulating the development both of pollen and stigma.

2.5. Map-based cloning of the mutant gene

At harvest, the segregation of mutant traits was determined by counting the rate of plants with WT and mutant phonotypes in the F2 mapping population. Rice leaf genomic DNA was extracted using the CTAB method(Murray and Thompson 1980). Among 512 simple sequence repeat (SSR) markers, 242 were found to be polymorphic between the mutant and 9311. Bulked segregant analysis (BSA) was used for rough mapping of the mutant locus (Zhang et al. 1994). New InDel markers were developed using primer 5.0 based on the gaps in sequence between the japonica Nipponbare variety and the indica variety 9311, which were found on the gramene website (http://www.gramene.org/). Gene sequences and gene models were obtained from RGAP7 (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/). The candidate genes were identified by PCR ampli fication and sequence analysis.

情况 6.5 若f3(v)=4,由引理1(6)知此时最坏的情况是v点关联4个6-面,两个(3,3,8)-面,两个(3,4,8)-面且3-面的邻面均为6-面。由R1,R2.1,R3.5及最坏3-面8-点情形得

2.6. qRT-PCR

The relative expression levels of the candidate genes in 20-day-old mutant and WT seedlings were compared.The relative expression levels of the candidate genes in different tissues were determined in WT plants at the heading stage. Total RNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan).Then, cDNA was synthesized from 1 μg RNA with PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa)according to manufacturer’s instructions. qRT-PCR was carried out in a Bio-Rad CFX96 Touch with a 25-μL reaction volume, which included 2 μL cDNA, 0.2 μmol L–1 gene-specific primers, ddH2O, and SYBR® Premix Ex Taq™ (TaKaRa) reagent. The rice ubiquitin gene was used as an internal control. The 2–ΔΔCT method was used to calculate the relative expression (Yu et al. 2007).

3. Results

3.1. Phenotypic characterization of the ddf3 mutant

To identify the gene responsible for the ddf3 mutant phenotype, we amplified all six genes located between markers M15 and M16 using gene-specific primers.Interestingly, only three genes could be successfully amplified from both WT and ddf3. Sequence analysis revealed that the sequences of these three genes were the same in WT and ddf3. However, the other three genes,which encode a zinc finger, DHHC-type domain containing protein (ZF), an expressed protein (EP), and an actinbinding FH2 domain containing protein (FH2), respectively,could be amplified from WT but could not be completely amplified from ddf3 (Fig. 5-A and B). We next evaluated the expression of these genes at seedling stage in ddf3 and WT using qRT-PCR. This assay suggested that compared with WT, the expression of ZF in ddf3 was increased about 15-fold, while the expression of FH2 could not be detected in ddf3 (Fig. 5-C). Considering the direction and states of these three genes, we inferred that there was a large deletion in this region. Then we amplified genomic DNA fragment deletion by designing primers (P0) at both ends of the region that couldn’t be amplified in ddf3. Consistent with our inference, this region contained a gene large deletion in ddf3, compared with WT (Fig. 5-D). Sequence analysis revealed a 13.98-kb deletion in the ddf3 genomic DNA.These results suggest that the ddf3 mutant is caused by a multi-gene deletion, and the three genes in the deleted region are DDF3 candidate genes.

3.2. The ddf3 mutant exhibits severe defects in cell elongation

2=3.06, P＞0.05). These results indicated that the mutant traits of ddf3 are controlled by a single or tightly linked nuclear gene. The DDF3 locus was roughly mapped to a 3.3-Mb region between two SSR markers, RM6403 and RM234, on the long arm of chromosome 7 (Fig. 4-A and B). Using 663 mutant plants in the F2 population, the DDF3 locus was fine mapped to a 45.21-kb region between the InDel markers M15 and M16 (Fig. 4-C). According to the RGAP7 gene predictions, there are six genes in this region (Fig. 4-D).

3.3. Mutation of DDF3 affects pollen activity and stigma development

In order to determine why the seed-setting rates of ddf3 were lower than those in WT, we compared pollen activity in ddf3 and WT. We found that pollen activity was significantly lower in the ddf3 mutant than that in WT and that there was an abundance of abnormal pollen in ddf3 (Fig. 3-A and B).Analysis of pollen types revealed that ddf3 had a great deal of abortive pollen. The aborted pollen grains were mainly spherical and stained abortion types, although some were typical abortion types (Fig. 3-C). The pistil is an important reproductive organ, and the normal development of pistils is critical for obtaining good seed-setting rates. Therefore, we wanted to know whether pistil development was normal in the ddf3 mutant. Interestingly, we found that many pistils in ddf3 showed a multi-stigma phenotype (Fig. 3-D and E), and in some pistils, it appeared that the stigma didn’t elongate(data not shown). These results suggest that mutation of DDF3 affects pollen activity and stigma development.

Fig. 1 Comparison of phenotype characteristics between ddf3 and wild-type (WT) plants. A, WT and ddf3 plants during the heading stage. B and C, plant heights (B) and tiller numbers (C) statistical analysis. D, panicle lengths of WT and ddf3. E, leaf characteristics of WT and ddf3. F and G, comparison of leaf lengths (F) and leaf widths (G) between ddf3 and WT. H, phenotypes of grain shapes in WT and ddf3. I, grain lengths, grain widths, and grain thicknesses of WT and ddf3. Values are mean±SD (B,C, F, and G, n=10 plants; I, n=30 replicates). The Student’s t-test analysis indicates a significant difference (compared with WT,＊＊, P＜0.01). Scale bars, 10 cm (A, D, and E) and 1 cm (H).

3.4. There is a multi-gene deletion in the ddf3 mutant

At the heading stage, the middle sections of the second internodes were collected from the ddf3 mutant and WT and then fixed in FAA (formalin:acetic acid:50% ethanol, 2:1:17(v/v)) overnight. After a series of dehydration and in filtration steps, the tissues were embedded in paraffin (Paraplast Plus; Sigma-Aldrich). The embedded tissues were cut into 8 μm sections with a microtome (Leica RM2265, Germany).Then the paraffin was removed from the sections with xylene. This was followed by a dehydration through an ethanol gradient, and toluidine blue staining. The stained sections were observed and photographed with a Nikon 50i microscope (Nikon, Japan).

To investigate the reason why ddf3 plants show a severely dwarfed phenotype, we measured the lengths of each internode in WT and ddf3. We found that all internodes were significantly shorter in ddf3 compared with WT (Fig. 2-A and B). Analysis of longitudinal sections revealed that the lengths of the cells in ddf3 internodes were significantly shorter than those in WT, whereas there was no significant difference in internode cell width (Fig. 2-C–F). Taken together, these results suggest that the DDF3 mutation inhibits cell elongation, but might not affect the transverse development of internode cells.

Compared with WT, the ddf3 mutant displayed reduced height and increased tillering; the plant height of ddf3 was 69.23% shorter, and tiller number was 56.58% higher(Fig. 1-B and C). In addition, panicle length, leaf length,and grain shape, were shorter in ddf3 (Fig. 1-D, E, and H). Interestingly, the lengths of the flag leaf, the 2nd leaf,the 3rd leaf, and the grain in ddf3 were significantly shorter compared with those in WT, but there was no significant difference in leaf width, grain width, and grain thickness(Fig. 1-F, G, and I). These results indicate that DDF3 may not influence the transverse development of organs. We further investigated yield-related traits. We found that panicle length, number of grains per panicle, seed-setting rate, and 1 000-grain weight were reduced by 40.96, 53.87,61.78, and 27.49%, respectively, in ddf3 compared with WT(Appendix A).

此外，还通过数值计算进行了检验。例如，Cupillard和Capdeville（2010）揭示出：在考虑球状地壳构造的情况下，当噪声源在地表呈空间均匀分布时，就可以正确推定其衰减，但当噪声源为局部存在时，就无法进行正确推定了。相同的数值验证也经Weaver（2011）得出了结论。

Fig. 2 Morphological characteristics and histological characterization of internodes of wild type (WT) and ddf3. A, internodes of WT and ddf3. B, comparison of internode lengths between WT and ddf3 plants. C and D, longitudinal section analysis of WT (C)and ddf3 (D) in second internode (internode II). E and F, the statistical analysis of parenchyma (PC) cell length (E) and width (F)in internode II. Scale bars, 20 cm (A) and 100 μm (C and D). Values are mean±SD (B, n=10 plants; E and F, n=30 replicates).The Student’s t-test analysis indicates a significant difference (compared with WT, ＊＊, P＜0.01). NS, not significant.

Fig. 3 The pollen activity and stigma development state of wild type (WT) and ddf3. A and B, the pollen activity of WT (A) and ddf3 (B). C, the abortive pollen types of WT and ddf3. The Student’s t-test analysis indicates a significant difference (compared with WT, ＊＊, P＜0.01). Values are mean±SD (n=5 plants). D and E, stigma development state of WT and ddf3. Scale bar, 2 mm.

Fig. 4 The molecular mapping of the DDF3 gene. A and B, rough mapping of DDF3. C, fine mapping of ddf3. D, gene predication in fine mapping region of DDF3.

Fig. 5 Identification of the mutant site of DDF3. A and B, the amplified fragments of ZF, EP, and FH2 genes in wild type (WT,A) and ddf3 (B). ZF-1 and ZF-2, the 1st and 2nd fragments of ZF; FH2-1 and FH2-2, the 1st and 2nd fragments of FH2. C, the relative expression of ZF, EP, and FH2 between WT and ddf3. D, the amplified fragment around the deletion region in WT and ddf3. Values are mean±SD (B, n=3 plants). The Student’s t-test analysis indicates a significant difference (compared with WT,＊＊, P＜0.01). Red arrows indicate the DNA fragments which can be successfully amplified in WT genome but can’t be amplified in ddf3 genome.

3.5. ZF and FH2 could be involved in the regulation of rice morphogenesis or flower organ development

To further understand the effects of mutating each DDF3 candidate gene, we first analyzed which parts of these genes have been deleted. According to the RGAP7 gene prediction models, WT ZF has nine exons and eight introns, but the promoter, two exons and some parts of introns have been deleted in ddf3. In WT, EP only has two exons and one intron, and this gene was completely deleted in ddf3. WT FH2 has 15 exons and 14 introns, but in ddf3 10 exons, 9 introns, and the 3´ untranslated region have been completely deleted (Fig. 6-A). These results indicate these three genes are completely or partly nonfunctional in ddf3.

A dwarf mutant, which was named dwarf and deformed flower3 (ddf3), was identified in the progeny of an Oryza sativa cultivar Dongjin plant derived from tissue culture transformation. We con firmed that the mutant phenotypes were stably inherited after five years of cultivation in natural condition. To map the mutant locus, we generated an F2 mapping population derived from a cross between the ddf3 mutant and the cultivar Yangdao 6 (9311). For phenotypic characterization, microscopic observation, analysis of floral organ development, mapping, and qRT-PCR assays, ddf3,wild-type (WT) Dongjin and the F2 mapping population were cultivated in an experimental field at Nanchang (28°41´N,115°55´E) during the normal growing season.

4. Discussion

4.1. ddf3 is a novel flower-deformation dwarf mutant

In this present study, we found a novel flower-deformation dwarf mutant, ddf3. The gene(s) responsible for the ddf3 mutant phenotype is(are) located in an interval of 45.21 kb on the long arm of chromosome 7. There was a 13.98-kb mutation in the ddf3 mutant genome in this region, in which three genes are deleted. This indicates that the ddf3 mutant phenotype is due to the loss of function of one or more of these deleted genes. Further transgenic complementation of the mutant and gene knockout experiments in WT will allow the responsible gene(s) to be identified.

带宽矢量h可以通过质点群的方差依比例进行设定，这主要是通过对协方差矩阵进行Cholesky分解计算得到（Bickel and Levina，2008）。

4.2. DDF3 gene(s) affects the seed-setting rate by regulating the development of pollen and stigma

The I2-KI staining method was used to detect pollen activity as described previously (Jiang et al. 2007). Briefly, the anthers of the mutant and WT were crushed on a slide in a drop water and then stained with I2-KI solution. In this study, pollen type includes normal and abortion, and abortion type is divided into typical abortion, spherical abortion, and stained abortion type (Li 1980; Xiang et al. 2011). Typical aborted pollen grains are shrunken and unstained. Spherical aborted pollen grains appear circular but cannot be stained.Stained aborted pollen grains appear circular and weakly stained. A Nikon 50i microscope (Nikon) was used to observe the stained pollen grains and count the number of different pollen types. To analyze stigma development, the pistils of the mutant and WT were observed under a stereo microscope (Nikon SMZ800, Japan).

Fig. 6 Diagram of deletion segment and analysis of mutant genes relative expression. A, diagram of deletion segment in genome.B and C, the analysis of ZF (B) and FH2 (C) relative expression in different organs of wild type (WT). Values are mean±SD (n=3).–, means the site of the primer;or mean the direction of the primer.

4.3. FH2 (BUI1/RMD) may be an important factor regulating morphological and flower development

BUI1/RMD, which encodes the Class II Formin Homology5(FH5), controls the rearrangements of the actin cytoskeleton and the spatial organization of actin filaments and plays a crucial role in proper cell expansion and rice morphogenesis(Yang et al. 2011; Li et al. 2014). The bui1/rmd mutant, as well the ddf3 mutant reported in this study, display dwarfism,short leaves, a decrescent grain shape, and blocked cell elongation. Additionally, Arabidopsis FH5 was reported to facilitate pollen tube growth, and RNAi suppression of FH5 expression in Nicotiana tabacum led to diminished subapical actin structure and compromised tip-focused growth of pollen tubes (Cheung et al. 2010). In this study,there was a complete deletion of 10 exons, 9 introns, and the 3´ untranslated region of FH2 (BUI1/RMD) in the ddf3 mutant (Fig. 6-A), resulting in the deletion of most of the conserved FH2 functional domain. FH2 (BUI1/RMD) was relatively highly expressed in the panicle, stem, leaf, and root(Fig. 6-C). Therefore, FH2 (BUI1/RMD) may be an important factor regulating morphological and flower development,and loss of FH2 may explain the morphological and floral defects in the ddf3 mutant.

4.4. ZF may be responsible for the morphogenic defects and deformed floral organs observed in ddf3

ZF encodes an S-acyltransferases (PATs) with a conserved Asp-His-His-Cys (DHHC) motif. The promoter, two exons and some parts of introns are deleted in the ddf3 mutant(Fig. 6-A). Evidence from previous studies suggests that S-acyltransferases are involved in cell expansion, pollen tube growth, ovule fertilization, and release of pollen in Arabidopsis (Hemsley et al. 2005; Chae et al. 2009; Qi et al.2013). Several OsDHHCs have also been reported to play important roles in plant growth and development, and stress responses in rice (Li et al. 2016; Zhou et al. 2017). We found that ZF was expressed in the stem, leaf, and root and was especially highly expressed in the panicle (Fig. 6-B).Therefore, loss of ZF function may also be responsible for the morphogenic defects and deformed floral organs observed in the mutant.

不同区域不同地区具有不同的优势和劣势，对当地产业结构的研究有利于指导正确的政策制定，有利于对具体问题的具体解决，有利于充分发挥地区优势，有利于发挥部分促进整体的作用。对当地环境分析可以更好地吸收国内外各地区的优秀理论成果，结合当地的现实情况对当前的经济状况做出正确的分析，从而制定合理有效解决问题的方法。

英格曼神甫这时在阅览室打盹儿——他已经搬到阅览室住了，为了不额外消耗一份柴火去烧他居处的壁炉，也为了能听见法比·阿多那多上楼下楼、进门出门的声音，这声音使他心里踏实，觉得得到了法比的间接陪伴，法比也在间接给他壮胆。

4.5. EP may not be involved in the regulation of plant development during the heading stage

EP encodes an expressed protein, and its function is unclear.It was not expressed in any tissue, which indicates that EP may not be involved in the regulation of plant development during the heading stage or there is the possibility of gene annotation wrong. However, future studies should also focus on investigating the role of EP at other stages of plant development.

5. Conclusion

The mutant gene, DDF3, is involved in the regulation of cell elongation and pollen and stigma development in rice.A 13.98-kb deletion in the ddf3 resulted in the complete or partial deletion of three genes. Disruption of the function of one or more of these genes is the ultimate cause of the ddf3 mutant phenotype.

Acknowledgements

The research was supported by the National Natural Science Foundation of China (31560350 and 31760350)and the Science and Technology Program of Jiangxi, China(20171ACF60018).

Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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