Next Article in Journal
Improved Method for Apple Fruit Target Detection Based on YOLOv5s
Next Article in Special Issue
Traditional Olive Tree Varieties in Alto Aragón (NE Spain): Molecular Characterization, Single-Varietal Oils, and Monumental Trees
Previous Article in Journal
A Review: Development of Plant Protection Methods and Advances in Pesticide Application Technology in Agro-Forestry Production
Previous Article in Special Issue
Pollen Development and Stainability in Vicia faba L. and Lupinus angustifolius L.
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agriculture
Volume 13
Issue 11
10.3390/agriculture13112166
agriculture-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Deletion of the OsLA1 Gene Leads to Multi-Tillering and Lazy Phenotypes in Rice

by
Zhanglun Sun
1,
Tianrun Mei
1,
Tingting Feng
1,
Hao Ai
1,
Yafeng Ye
2,
Sumei Duan
1,
Binmei Liu
2,* and
Xianzhong Huang
1,*
1
Center for Crop Biotechnology, College of Agriculture, Anhui Science and Technology University, Chuzhou 239000, China
2
Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230001, China
*
Authors to whom correspondence should be addressed.
Agriculture 2023, 13(11), 2166; https://doi.org/10.3390/agriculture13112166
Submission received: 9 October 2023 / Revised: 3 November 2023 / Accepted: 16 November 2023 / Published: 17 November 2023
(This article belongs to the Special Issue Genetic Identification, Exploration, Evaluation and Breeding of Plant Genetic Resources)
Browse Figures
Review Reports Versions Notes

Abstract

:
Plant architecture, one of the key factors that determine grain yield in rice, is mainly affected by components such as plant height, tiller number, and panicle morphology. For this paper, we obtained a multi-tillering and lazy mutant from a japonica rice cultivar, Wuyunjing 7 (WYJ7), via treatment with a heavy ion beam. Compared to WYJ7, the mutant showed a significant increase in tiller angle, tiller number, number of primary and secondary branches, and number of grains; however, the plant height and grain thickness of the mutant was significantly decreased. Phenotypic analysis of the F1 hybrids revealed that the multi-tillering and lazy mutant phenotypes were regulated by a recessive gene. The segregation ratio of 1׃3 of the mutant phenotype and the wild-type plant in the F2 population indicated that the former was controlled by a single gene named Multi-Tillering and Lazy 1 (MTL1). Bulked segregant analysis was performed using the individual plants with extremely typical tiller angles in the F2 population. The MTL1 gene was initially mapped within a region of 5.58–17.64 Mb on chromosome 11. By using the F2 segregated population for fine mapping, the MTL1 gene was ultimately fine mapped within the range of 66.67 kb on chromosome 11. The analysis of genes in this region revealed the presence of the previously identified LAZY1 (LA1) gene. Genomic PCR amplification and semi-quantitative RT-PCR assays showed that the LA1 gene could not be amplified and was not expressed, thus indicating that the MTL1 gene might be identical to the LA1 gene. This study suggests that the multi-tillering and lazy mutant phenotypes might be caused by the deletion of LA1 function. This finding can guide further investigations on the functional mechanisms of the LA1 gene, thus enriching the theoretical knowledge of plant architecture in relation to rice.

1. Introduction

As one of the most important food crops in the world, rice (Oryza sativa L.) is a staple food for more than half of the world’s population. Rice yield is an important factor that can be adapted to meet the increasing global demand for food among the world’s population. The plant height, tiller number, tiller angle, and panicle morphology of rice determine the plant’s architecture, a crucial factor for yield formation [1]. In the 1960s, based on Semidwarf 1 (SD1) mutation, the identification and utilization of semi-dwarf varieties greatly increased the yield of rice [2,3,4]. Therefore, achieving the ideal plant structure plays an important role in improving rice yield.
Tiller is one of the important factors that determine rice plant architecture and yield. Tillering mainly includes tiller number, tiller angle, and tiller ability. Previous studies have shown that rice tillers are affected by both environmental and genetic factors, among which genetic factors are crucial [5]. In recent years, several genes that regulate tiller-related traits in rice have been identified. The first gene cloned in rice to control rice tillers was MONOCULM 1 (MOC1), which is a homologous gene of LATERAL SUPPRESSOR (LAS) in tomato and Arabidopsis; this gene encodes a GRAS family nucleoprotein [6,7,8]. The mutant moc1 does not have any tillers; it has only one main stem, and the inflorescence axis and panicles are also smaller than those of the wild-type plant [8]. The overexpression of the MOC1 gene increased tillers and led to a high level of tillering [8]. The MOC2 gene encodes the enzyme fructose-1,6-diphosphatase. Although the moc2 mutant produces tiller buds, the growth of these buds is suppressed or they cannot further develop into tillers; this condition manifests via single tillers, dwarfing, light green leaves, short and narrow leaves, small panicles, and reduced grain number per panicle [9]. The MOC3 gene encodes a WUSCHEL-related homeobox protein family member that functions as a transcription suppressor. The moc3 mutant cannot form axillary buds and has no tillers; during the reproductive stage, the mutant produces only one inflorescence, and the development of spikelets is affected, resulting in a reduced number of spikelets and several morphological defects [10,11]. The rice MOC3/WUS dysfunction mutant decreased culm number 1 (dc1) showed increased apical dominance of the main stem, and the growth of tillers was inhibited by the main stem [12]. Removal of the top of the main stem of the dc1 mutant or knockout of the auxin-related gene ABERRANT SPIKELET AND PANICLE 1 (ASP1) during the tillering stage can prevent the inhibitory effect of the main stem on tiller buds, thereby promoting their growth [12]. The critical genes involved in rice tiller differentiation include DWARF3 (D3), D10, D14, D27, and D53. The proteins encoded by these genes carry out their functions through strigolactones (SLs), and mutants with function deletion of these genes exhibit stunted plant height and increased tiller numbers [13,14,15,16,17].
Tiller angle is another important agronomic trait of rice plant architecture [5]. The PROSTRATE GROWTH 1 (PROG1) gene is a critical gene that controls the growth of wild rice (Oryza rufipogon L.) from prostrate growth to erect growth; this gene encodes a zinc finger transcription factor composed of 167 amino acids, and it is mainly expressed in axillary shoot meristems [18,19]. The loss function of PROG1 not only improves plant structure but also increases the number of grains per panicles, thereby leading to a significant increase in the yield (manifested as “pleiotropism”) [18,19]. TILLER ANGLE CONTROLLING 1 (TAC1) is a major gene that regulates the tiller angle in rice. In one specific study, TAC1 overexpression increased the tiller angle in rice, and plants with TAC1-RNAi showed a compact structure [20]. The genes TAC1, TAC3, and D2 play important roles in the regulation of the tiller angle in rice; however, the molecular mechanisms underlying their roles in plant architecture remain unclear [21]. TAC4 controls the tiller angle in rice by regulating indoleacetic acid content and affecting auxin distribution, thereby regulating the gravitational response of rice [22]. The LAZY1 (LA1) gene is a negative regulator of auxin polar transport and regulates the tiller angle in rice [23,24,25,26]. LA1 encodes a Cys(2)-His(2) zinc finger protein that functions as a transcription factor and binds to the cis-acting elements of its downstream genes to regulate their expression, thereby controlling tillers [23]. PROG1 acts upstream of the LA1 gene as a repressor protein of rice and controls the tiller angle by regulating the geotropism of seedlings and the asymmetric distribution of auxin mediated by LA1 [27]. LA1 gene overexpression partially rescues the defects of a larger tiller angle and stem orientation in PROG1 complementary transgenic plants [27].
To summarize, many related studies on the tiller angle and tiller number in rice have been conducted in recent years. The identification of genes related to controlling these traits has facilitated the development of the ideal plant type and molecular design breeding in rice. However, the genetic regulatory network of many genes and their molecular mechanisms for regulating the tiller angle have not yet been fully understood, and further studies on this topic are required from different perspectives. We obtained a multi-tillering and lazy 1 (mtl1) mutant from a japonica rice cultivar Wuyunjing 7 (WYJ7) via mutagenesis treatment with a heavy ion beam. In the present study, the mtl1 mutant was used as the research material to conduct agronomic trait investigations, genetic law analysis, and fine mapping to (1) identify the regulation of rice tiller number and the tiller angle gene MTL1, (2) explore the genetic network of plant architecture development, and (3) strengthen the theory of ideal plant type breeding in rice.

2. Materials and Methods

2.1. Material Planting and Investigation of Agronomic Traits

An mtl1 mutant was obtained from the japonica cultivar WYJ7 via mutagenesis treatment with a heavy ion beam in 2015. The methods used for mutagenesis have been described by Xu et al. (2012) [28]. The parameters used in the present study were as follows: an ion energy of 25 keV; N+ ion fluence of 2.5 × 1016 ions/cm2; an irradiation time of 10 min [28]. In May 2021, the seeds of WYJ7 and mtl1 were germinated under dark conditions in an oven at 37 °C. After sprouting, the seeds were spread on the seedling bed at the Anhui Science and Technology University, Fengyang Campus, Anhui Province (32°52′30″ N, 117°33′15″ E). Once the seedlings reached the growth stage of four leaves and one heart, they were transplanted into rice fields consisting of rice plants with equal fertility and similar textures. The phenotypes of WYJ7 and mtl1 were observed throughout the growth period. During the tillering stage, 15 plants of WYJ7 and mtl1 with consistent growth stature were selected to determine their tiller angle and tiller number. During the maturity stage of rice, the plant height, panicle length, number of primary and secondary branches, and number of grains per panicle of WYJ7 and mtl1 were counted. Grain length, grain width, and grain thickness were measured using a vernier caliper, and the 1000-grain weight and yield per plant were quantified. The obtained data were statistically analyzed via Student’s t-test using SPSS software (Version 21.0, IBM SPSS), and graphs were created using GraphPad Prism software (FreeImage Public License—Version 1.0).

2.2. Construction and Genetic Analysis of the F2 Segregated Population

The mtl1 mutant was crossed with the indica rice cultivar Huajingxian 74 (HJX74) to obtain F1 generation seeds, and the F1 generation plants were self-crossed to obtain the F2 generation segregated population. During the maturity stage, the number of individual plants with wild-type and multi-tillering and lazy phenotypes in the F2 generation segregated population was determined, and the data were analyzed via the chi-square test using SPSS software (Version 21.0, IBM SPSS).

2.3. Mixed-Pool Sequencing and Single-Nucleotide Polymorphism Analysis

Twenty-five young leaves of each individual were collected from the HJX74 phenotypes and the extreme multi-tillering and lazy phenotypes (large tiller angle and unable to grow upright, like lazy 1 mutant [23]) in the F2 population of HJX74 and mtl1. The samples were sent to Wuhan Shuangluyuan Chuangxin Science and Technology Research Institute Co., Ltd. for bulked segregation analysis (BSA). First, two DNA mixing pools were constructed from the individuals of mtl1 and wild-type phenotypes in the segregated population. By using the Japanese version 7.0 genome as a reference genome, the mixing pools were scanned using Green Super Rice 40K high-density rice gene chips containing 44,263 single-nucleotide polymorphism (SNP) checkpoints in 4726 cultivated rice varieties, and the allelic differences between the two mixed pools and their parents were analyzed. The gene segments related to the phenotypes were mapped to preliminarily locate the MTL1 gene.

2.4. Fine Mapping of the MTL1 Gene

To perform fine mapping of the MTL1 gene, we collected young leaves of each individual that exhibited mtl1 traits from the F2 segregated population hybridized between mtl1 and HJX74, and a modified cetyltrimethylammonium bromide method was used to extract the DNA from the leaves [29].
Based on the nucleotide differences between Nipponbare and 93-11 reference genomes, insertion and deletion (InDel) primers (Table S1) were designed using the Primer 3 online website (https://primer3.ut.ee/, accessed on 10 December 2021) and synthesized by GENERAL Biosystems Co., Ltd. (Chuzhou, China).
PCR amplification was performed on LongGene (T20, LongGene Scientific Instrument Co., Ltd., Hangzhou, China) in a 20 μL reaction volume containing 2 μL genomic DNA template (25 ng/μL), 10 μL 2× Santaq PCR mix (Vazyme Biotech Co., Ltd., Nanjing, China) and 200 nmol/L of each primer. The reaction procedure was as follows: denaturation at 95 °C for 3 min, followed by 35 cycles at 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 5 s, and a final extension for 10 min at 72 °C. The PCR amplification products were analyzed via electrophoresis with 4% agarose gel.

2.5. Analysis of the Candidate Genes

Based on our fine mapping results, the genes in the localization interval were annotated using the Rice Genome Annotation Project Database website (http://rice.plantbiology.msu.edu/, accessed on 10 October 2022), and the gene functions in the target interval were preliminarily determined. Gene-specific primers (Table S1) were designed according to the reference genes in the target region annotated on the website. The expression period of the candidate genes was predicted based on information from the Rice Annotation Project Database website (https://rapdb.dna.affrc.go.jp/, accessed on 10 March 2023). RNA was extracted and reverse-transcribed, and semi-quantitative RT-PCR primers were used to analyze the difference in the expression levels between candidate genes in mtl1 and WYJ7.
To determine the expression profiles of the candidate genes, RNA of WYJ7 and mtl1 seedlings was extracted using TRIzol (Vazyme Biotech Co., Ltd., Nanjing, China), and cDNA was synthesized using MonScript™ RTIII All-in-One Mix with dsDNase (Monad Biotech Co., Ltd., Suzhou, China) in accordance with the manufacturer’s instructions.
Semi-quantitative RT-PCR was performed using the gene-specific primers (Table S1). The PCR amplification profile was as follows: 94 °C for 2 min, followed by 22–28 cycles of 95 °C for 15 s, 55 °C for 15 s, 72 °C for 5 s, and a final extension at 72 °C for 5 min. The rice OsActin gene was used as the reference gene for the internal control.

3. Results

3.1. Phenotypic Analysis of the mtl1 Mutant

3.1.1. Plant Architecture Analysis of mtl1

Under field cultivation conditions, the tiller angle of mtl1 significantly increased during the vegetative growth period of rice (Figure 1a,b; P = 2.06 × 10−19), with an average tiller angle of approximately 110°, whereas the tiller of WYJ7 exhibited near-erect growth (Figure 1c). With the transition from the vegetative growth to reproductive growth stage, WYJ7 gradually stopped tillering, while mtl1 continued to produce a high level of tillers. Before heading, the number of tillers in mtl1 was significantly higher than that in WYJ7 (Figure 1c,d; P = 4.02 × 10−10). At the mature stage of rice, the plant height in mtl1 was significantly lower than that in WYJ7 (Figure 1e,f; P = 2.64 × 10−8).

3.1.2. Panicle Type Analysis of mtl1

The investigation of panicle morphology (Figure 2a) and yield (Figure 2b) indicated that, compared to WYJ7, the average panicle length of mtl1 increased by 1 cm (Figure 2c). Moreover, the number of primary and secondary branches per panicle in mtl1 significantly increased (Figure 2d,e; P = 1.72 × 10−8 and 1.80 × 10−11, respectively), The average number of grains per panicle in mtl1 also significantly increased (P = 2.07 × 10−8), with WYJ7 and mtl1 having an average of 161 and 214 grains per panicle, respectively (Figure 2f). The evaluation of yield per plant showed that mtl1 had a significantly higher yield than WYJ7 (P = 1.27 × 10−4); the average yield per plant of WYJ7 and mtl1 was approximately 54 g and 100 g, respectively (Figure 2g).

3.1.3. Grain Type Analysis of mtl1

We further compared the grain phenotypes, including grain length (Figure 3a), grain width (Figure 3b), and grain thickness (Figure 3c), between WYJ7 and mtl1. Compared to WYJ7, mtl1 showed a moderate decrease in grain length (P = 1.13 × 10−2), which was approximately 98.79% of that of WYJ7 (Figure 3d). Grain width did not show significant differences between the two genotypes (Figure 3e; P = 8.66 × 10−1). However, the grain thickness of mtl1 was significantly reduced (P = 6.42 × 10−3), which was approximately 98.04% of that of WYJ7 (Figure 3f). Additionally, the 1000-grain weight of mtl1 was only 85.09% of that of WYJ7 (Figure 3g).

3.2. Genetic Analysis of the mtl1 Mutant Phenotypes in Rice

To determine the genetic characteristics of the multi-tillering and lazy traits, we performed a genetic analysis of the mtl1 mutant phenotypes. The observations revealed that the mtl1 mutant phenotypes may be controlled by a recessive gene, as the F1 generation plants resulting from crossing mtl1 with HJX74 exhibited wild-type phenotypes. To clarify the genetic law of the MTL1 gene, we performed a statistical analysis on the F2 segregated population. The results showed a 3:1 segregation model between the wild-type and mutant phenotypes (χ2 < χ20.05, 1 = 3.84) (Table 1), thus suggesting that the mtl1 mutant phenotypes was controlled by a recessive gene.

3.3. Preliminary Mapping of the MTL1 Gene

To mine the MTL1 gene that controls the multi-tillering and lazy phenotypes, we used gene chips to preliminarily map the MTL1 gene by using the BSA method. According to the SNP chip scanning results, the genotype of the parent P1--mtl1 was almost of the same color (gray); the genotype of P2-HJX74 was also almost of the same color (red), thus indicating that the parents had homozygous genotypes (Figure 4a). Moreover, in the F2 generation segregated population, most of the genotypes in the WT mixed pool F2-HJX74 and the mtl1 mutant phenotypes mixed pool F2-mtl1 were almost heterozygous (blue); however, some segments on chromosome 11 showed differences between the two mixed pools.
A comparison of genomic differences between the two samples in the preliminary localization segment found that the genotype of the F2-mtl1 mixed pool was consistent with the mtl1 mutant genotype (AA) (Figure 4b), while the genotype of the F2-HJX74 mixed pool was predominantly heterozygous (AB) (Figure 4b,c). Based on the principle of BSA and the SNP loci of this differential segment, the results indicated that the gene related to the MTL phenotypes was preliminarily mapped between 5.58 and 17.64 Mb on chromosome 11.

3.4. Fine Mapping of the MTL1 Gene

According to the preliminary mapping, eight pairs of InDel primers (M1–M8) were first developed (Table S1), and the 230 individual plants of the mtl1 mutant phenotypes from the F2 population were used for MTL1 gene mapping. Twenty-six crossing-over individual plants were found within the preliminary interval. Based on these 26 individual plants, the MTL1 gene was then narrowed down to be between markers M6 and M8 (Figure 5a). Four polymorphic primers (M9–M12) were developed between M6 and M8, and six crossing-over individual plants between M6 and M8 were used to narrow down the MTL1 gene between M12 and M8 (Figure 5b). According to the Rice Genome Annotation Project database website, the target interval was 66.67 kb and contained five putative genes (Figure 5c).

3.5. Analysis of the Candidate Genes

Based on the annotation results of the Rice Genome Annotation Project database, the target interval contains five genes (Table S2), among which three genes (LOC_Os11g29810, LOC_Os11g29820, and LOC_Os11g29830) encode predictive proteins of unknown functions; LOC_Os11g29850 encodes an ATP-binding cassette transporter, and the LA1 gene (LOC_Os11g29840) controls the rice tiller angle (Figure 5, Table S2).
To analyze the nucleotide differences of these five genes between WYJ7 and mtl1, we performed genome PCR assays using gene-specific primers (Table S1). The results showed that all five candidate genes were amplified normally in WYJ7. However, LA1 could not be amplified normally in mtl1. Sequence comparisons involving these genes showed that LOC_Os11g29840 was polymorphic in mapping parents and Nipponbare. Next, five exons of LA1 were PCR amplified using WYJ7 and mtl1 genomic DNA as templates. The five exons were easily amplified from WYJ7; however, no amplification products were detected for each exon from mtl1, thus suggesting that the MTL1 gene was deleted in mtl1 (Figure 5d). Semi-quantitative RT-PCR also confirmed that the LA1 gene was not expressed in mtl1 (Figure 5e).

4. Discussion

Tiller is one of the key factors for developing an ideal rice plant type. The tiller angle determines the planting density of rice and affects the yield by influencing photosynthetic efficiency [30,31]. During the domestication process of rice, the rice plant underwent a process from creeping growth to erect growth. The creeping growth pattern of wild rice is beneficial, allowing rice plants to compete with weeds for growth space in the wild environment, while the erect growth pattern of cultivated rice is beneficial for improving plant density and yield under agricultural conditions [32]. The results of domestication and artificial selection are mainly driven by C2H2-type zinc finger transcription factors, including PROG1, PROG7, and RICE PLANT ARCHITECTURE DOMESTICATION (RPAD) [18,19,33,34]. The change in the growth pattern of the Asian cultivated rice from creeping to erect is mainly caused by allelic mutations in the PROG1 gene and a deletion of 110 kb in the RPAD gene [18,19,34], while the change in the African cultivated rice is mainly due to allelic mutations in the PROG7 gene and a deletion of 113 kb in the RPAD gene [33,34]. The TAC1 gene shows a low expression level in japonica rice, which exhibits a more compact plant architecture than indica rice and has been widely planted in high-latitude temperate regions and high-altitude regions [20,35]. Although many genes controlling rice tillers have been identified in recent years, only a few genes have been studied for their application in rice production. Therefore, there is an urgent requirement to explore new plant type control genes and enrich the theory of ideal plant types.
In the present study, the investigation of the agronomic traits of mtl1 showed that while the tiller angle of mtl1 increased significantly, the number of effective panicles did not continue to increase with the increase in tiller number (Figure 1a–e). The grain thickness and 1000-grain weight in mtl1 decreased significantly (Figure 2 and Figure 3). However, the panicle length, number of primary and secondary branches, and number of grains in mtl1 increased significantly; therefore, yield per plant increased significantly. Although the yield per plant of mtl1 increased significantly, the large tiller angle presented a disadvantage in the form of an inability to increase plant density. Therefore, balancing the relationships between tiller angle, tiller number, and planting density and the relationship between grain number and grain size are urgent issues that need to be addressed in the design of an appropriate breeding approach and development of an ideal plant type.
We narrowed down the MTL1 gene to the range of 17.27–17.33 Mb (66.67 kb) on chromosome 11 by using map-based cloning (Figure 5a–c). Our analysis of the candidate genes in the mapping interval revealed that the cloned gene LA1 was missing (Figure 5d,e). Previous studies have shown that LA1 regulates the polar transport of auxin and controls the gravitropism of the aboveground part of rice and ultimately the tiller angle. Exon 4 of the lazy1-ZF802 mutant in rice has a deletion of 8 bp, leading to the premature termination of protein translation, and the entire gene of the lazy1-Shiokari mutant is missing; both these plants exhibit the same dispersed tillering phenotypes [23]. By carrying out the appropriate knockdown of LA1 gene expression by using RNAi, the tiller angle can be moderately increased, and the RNAi plant phenotypes of the LA1 gene are consistent with the la1 mutant phenotypes [23]. Brevis Radix Like 4 (OsBRXL4) interacts with LA1 and affects its nuclear localization, thereby influencing the gravity response and tiller angle of rice. The overexpression of OsBRXL4 can reduce LA1 localization in the nucleus, thereby increasing the tiller angle and leading to semi-scattered phenotypes in rice [36]. These results showed that the tiller angle of rice is associated with the expression level of the endogenous LA1 gene. In rice, the loss of LA1 function increased the tiller angle; however, it did not cause any significant changes in panicle morphology. In maize, ZmLA1 gene mutation not only formed a creeping main stem but also affected the morphology of corn tassels and ears, as noted by the authors of [36]. In another study, the insertion of a transposon MuDR at 25 bp of the fourth exon of ZmLA1 significantly decreased the expression level of LA1, thereby resulting in a reduced number of tassel branches, a decrease in pollen shedding and ear silk, irregular axis arrangement, and reduced seed setting rate [37]. In the present study, compared to the la1 mutant, the deletion of the MTL1 gene also produced a prostrate growth pattern. However, mtl1 showed a larger panicle and an increase in grain yield per plant, which have not been reported for the existing la1 mutant. Considering our results in tandem with the findings of previous studies, we speculate that the mtl1 mutant phenotypes are caused by the deletion of the LA1 gene. A recently published study revealed that in Arabidopsis, gravistimulation triggers and regulates the MAPK-mediated phosphorylation of LA proteins [38]. Phosphorylated LA proteins enhance their interaction with the TOC proteins on the surface of amyloplasts, leading to the enrichment of LA proteins on the surface of amyloplasts, asymmetrical auxin distribution, and root differential growth; the shuttling of LA proteins from amyloplasts to the adjacent plasma membrane triggers gravity sensing in plants [38]. So, how does the complete deletion of LA proteins in rice affect auxin polar transport? The mtl1 mutant could be a potentially good genetic material to understand this aspect.
Thus far, the regulatory networks and signaling pathways associated with LA1 controlling the tiller angle of rice are largely unknown. Moreover, the limited progress made in identifying new genes hinders their applications in the structural improvement and elucidation of the molecular mechanisms underlying tiller angle regulation.
To summarize, in the present study, we found that the different mutation types of LA1 play a very important role in controlling the tiller angle of rice. In the future, CRISPR-Cas9 technology could be used to conduct in-depth studies on the different mutations of LA1, along with the aggregation of other genes, to create a suitable tiller angle and ideal plant type for rice plants.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture13112166/s1, Table S1: Primers used in the experiments; Table S2: Gene annotations in the fine mapping interval.

Author Contributions

X.H. and B.L. designed the work; Z.S. and T.M. performed experiments and wrote the original manuscript. T.F., H.A. and Y.Y. contributed to genetic hybridizations and the analysis of experimental data; S.D. helped with plant materials in the fields; X.H. and B.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Excellent Scientific Research and Innovation team of the Education Department of Anhui Province (2022AH010087), the Anhui Province Key Research and Development Program (202204c06020070), the Talent Introduction Start-up Fund Project of Anhui Science and Technology University (NXYJ202001), and the Industry University Research Cooperation Project Shouxian Branch Center for the Transformation of Scientific and Technological Achievements of Anhui University of Science and Technology (kycg881215).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data reported in this study are available in the article and supplementary materials.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Jiao, Y.Q.; Wang, Y.H.; Xue, D.W.; Wang, J.; Yan, M.X.; Liu, G.F.; Dong, G.J.; Zeng, D.L.; Lu, Z.F.; Zhu, X.D.; et al. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nat. Genet. 2010, 42, 541–544. [Google Scholar] [CrossRef]
  2. Peng, J.; Richards, D.E.; Hartley, N.M.; Murphy, G.P.; Devos, K.M.; Flintham, J.E.; Beales, J.; Fish, L.J.; Worland, A.J.; Pelica, F.; et al. ‘Green revolution’ genes encode mutant gibberellin response modulators. Nature 1999, 400, 256–261. [Google Scholar] [CrossRef]
  3. Sasaki, A.; Ashikari, M.; Ueguchi-Tanaka, M.; Itoh, H.; Nishimura, A.; Swapan, D.; Ishiyama, K.; Saito, T.; Kobayashi, M.; Khush, G.S.; et al. Green revolution: A mutant gibberellin-synthesis gene in rice. Nature 2002, 416, 701–702. [Google Scholar] [CrossRef]
  4. Qian, Q.; Guo, L.B.; Smith, S.M.; Li, J.Y. Breeding high-yield superior quality hybrid super rice by rational design. Natl. Sci. Rev. 2016, 3, 283–294. [Google Scholar] [CrossRef]
  5. Wang, B.; Smith, S.M.; Li, J.Y. Genetic Regulation of Shoot Architecture. Annu. Rev. Plant Biol. 2018, 69, 437–468. [Google Scholar] [CrossRef]
  6. Schumacher, K.; Schmitt, T.; Rossberg, M.; Schmitz, G.; Theres, K. The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family. Proc. Natl. Acad. Sci. USA 1999, 96, 290–295. [Google Scholar] [CrossRef]
  7. Greb, T.; Clarenz, O.; Schafer, E.; Muller, D.; Herrero, R.; Schmitz, G.; Theres, K. Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation. Genes Dev. 2003, 17, 1175–1187. [Google Scholar] [CrossRef] [PubMed]
  8. Li, X.Y.; Qian, Q.; Fu, Z.M.; Wang, Y.H.; Xiong, G.S.; Zeng, D.L.; Wang, X.Q.; Liu, X.F.; Teng, S.; Hiroshi, F.; et al. Control of tillering in rice. Nature 2003, 422, 618–621. [Google Scholar] [CrossRef] [PubMed]
  9. Koumoto, T.; Shimada, H.; Kusano, H.; She, K.C.; Iwamoto, M.; Tanako, M. Rice monoculm mutation moc2, which inhibits outgrowth of the second tillers, is ascribed to lack of a fructose-1,6-bisphosphatase. Plant Biotechnol. J. 2013, 30, 47–56. [Google Scholar] [CrossRef]
  10. Lu, Z.F.; Shao, G.N.; Xiong, J.S.; Jiao, Y.Q.; Wang, J.; Liu, G.F.; Meng, X.B.; Liang, Y.; Xiong, G.S.; Wang, Y.H.; et al. MONOCULM 3, an ortholog of WUSCHEL in rice, is required for tiller bud formation. J. Genet. Genomics 2015, 42, 71–78. [Google Scholar] [CrossRef]
  11. Tanaka, W.; Ohmori, Y.; Ushijima, T.; Matsusaka, H.; Matsushita, T.; Kumamaru, T.; Kawano, S.; Hirano, H.Y. Axillary Meristem Formation in Rice Requires the WUSCHEL Ortholog TILLERS ABSENT1. Plant Cell 2015, 27, 1173–1184. [Google Scholar] [CrossRef]
  12. Xia, T.Y.; Chen, H.Q.; Dong, S.J.; Ma, Z.Y.; Ren, H.B.; Zhu, X.D.; Fang, X.H.; Chen, F. OsWUS promotes tiller bud growth by establishing weak apical dominance in rice. Plant J. 2020, 104, 1635–1647. [Google Scholar] [CrossRef]
  13. Ishikawa, S.; Maekawa, M.; Arite, T.; Onishi, K.; Takamure, I.; Kyozuka, J. Suppression of tiller bud activity in tillering dwarf mutants of rice. Plant Cell Physiol. 2005, 46, 79–86. [Google Scholar] [CrossRef]
  14. Lin, H.; Wang, R.X.; Qian, Q.; Yan, M.X.; Meng, X.B.; Fu, Z.M.; Yan, C.Y.; Jiang, B.; Su, Z.; Li, J.Y.; et al. DWARF27, an iron-containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth. Plant Cell 2009, 21, 1512–1525. [Google Scholar] [CrossRef]
  15. Ito, S.; Kitahata, N.; Umehara, M.; Hanada, A.; Kato, A.; Ueno, K.; Mashiguchi, K.; Kyozuka, J.; Yoneyama, K.; Yamaguchi, S.; et al. A new lead chemical for strigolactone biosynthesis inhibitors. Plant Cell Physiol. 2010, 51, 1143–1150. [Google Scholar] [CrossRef]
  16. Jiang, L.; Liu, X.; Xiong, G.S.; Liu, H.H.; Chen, F.L.; Wang, L.; Meng, X.B.; Liu, G.F.; Yu, H.; Yuan, Y.D.; et al. DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature 2013, 504, 401–405. [Google Scholar] [CrossRef] [PubMed]
  17. Kagiyama, M.; Hirano, Y.; Mori, T.; Kim, S.Y.; Kyozuka, J.; Seto, Y.; Yamaguchi, S.; Hakoshima, T. Structures of D14 and D14L in the strigolactone and karrikin signaling pathways. Genes Cells 2013, 18, 147–160. [Google Scholar] [CrossRef]
  18. Jin, J.; Huang, W.; Gao, J.P.; Yang, J.; Shi, M.; Zhu, M.Z.; Luo, D.; Lin, H.X. Genetic control of rice plant architecture under domestication. Nat. Genet. 2008, 40, 1365–1369. [Google Scholar] [CrossRef]
  19. Tan, L.B.; Li, X.R.; Liu, F.X.; Sun, X.Y.; Li, C.G.; Zhu, Z.F.; Fu, Y.C.; Cai, H.W.; Wang, X.K.; Xie, D.X.; et al. Control of a key transition from prostrate to erect growth in rice domestication. Nat. Genet. 2008, 40, 1360–1364. [Google Scholar] [CrossRef] [PubMed]
  20. Yu, B.S.; Lin, Z.W.; Li, H.X.; Li, X.J.; Li, J.Y.; Wang, Y.H.; Zhang, X.; Zhu, Z.F.; Zhai, W.X.; Wang, X.K.; et al. TAC1, a major quantitative trait locus controlling tiller angle in rice. Plant J. 2007, 52, 891–898. [Google Scholar] [CrossRef] [PubMed]
  21. Dong, H.J.; Zhao, H.; Xie, W.B.; Han, Z.M.; Li, G.W.; Yao, W.; Bai, X.F.; Hu, Y.; Guo, Z.L.; Lu, K.; et al. A Novel Tiller Angle Gene, TAC3, together with TAC1 and D2 Largely Determine the Natural Variation of Tiller Angle in Rice Cultivars. PLoS Genet. 2016, 12, e1006412. [Google Scholar] [CrossRef] [PubMed]
  22. Li, H.; Sun, H.Y.; Jiang, J.H.; Sun, X.Y.; Tan, L.B.; Sun, C.Q. TAC4 controls tiller angle by regulating the endogenous auxin content and distribution in rice. Plant Biotechnol. J. 2021, 19, 64–73. [Google Scholar] [CrossRef]
  23. Li, P.J.; Wang, Y.H.; Qian, Q.; Fu, Z.M.; Wang, M.; Zeng, D.L.; Li, B.H.; Wang, X.J.; Li, J.Y. LAZY1 controls rice shoot gravitropism through regulating polar auxin transport. Cell Res. 2007, 17, 402–410. [Google Scholar] [CrossRef]
  24. Yoshihara, T.; Iino, M. Identification of the gravitropism-related rice gene LAZY1 and elucidation of LAZY1-dependent and -independent gravity signaling pathways. Plant Cell Physiol. 2007, 48, 678–688. [Google Scholar] [CrossRef] [PubMed]
  25. Sang, D.J.; Chen, D.Q.; Liu, G.F.; Liang, Y.; Huang, L.Z.; Meng, X.B.; Chu, J.F.; Sun, X.H.; Dong, G.J.; Yuan, Y.D.; et al. Strigolactones regulate rice tiller angle by attenuating shoot gravitropism through inhibiting auxin biosynthesis. Proc. Natl. Acad. Sci. USA 2014, 111, 11199–11204. [Google Scholar] [CrossRef]
  26. Zhang, N.; Yu, H.; Yu, H.; Cai, Y.Y.; Huang, L.Z.; Xu, C.; Xiong, G.S.; Meng, X.B.; Wang, J.Y.; Chen, H.F.; et al. A Core Regulatory Pathway Controlling Rice Tiller Angle Mediated by the LAZY1-Dependent Asymmetric Distribution of Auxin. Plant Cell 2018, 30, 1461–1475. [Google Scholar] [CrossRef]
  27. Zhang, H.; Li, X.; Sang, D.J.; Huang, L.Z.; Song, Y.Q.; Du, M.C.; Cao, J.J.; Wang, W.G. PROG1 acts upstream of LAZY1 to regulate rice tiller angle as a repressor. Crop J. 2023, 11, 386–393. [Google Scholar] [CrossRef]
  28. Xu, X.; Liu, B.M.; Zhang, L.L.; Wu, Y.J. Mutagenic effects of heavy ion irradiation on rice seeds. Nucl. Instrum. Methods Phys. Res. 2012, 290, 19–25. [Google Scholar] [CrossRef]
  29. Edwards, K.; Johnstone, C.; Thompson, C. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 1991, 19, 1349. [Google Scholar] [CrossRef]
  30. Wang, Y.H.; Li, J.Y. Molecular basis of plant architecture. Annu. Rev. Plant Bio. 2008, 59, 253–279. [Google Scholar] [CrossRef] [PubMed]
  31. Wang, Y.H.; Li, J.Y. Rice, rising. Nat. Genet. 2008, 40, 1273–1275. [Google Scholar] [CrossRef] [PubMed]
  32. Wang, W.G.; Gao, H.B.; Liang, Y.; Li, J.Y.; Wang, Y.H. Molecular basis underlying rice tiller angle: Current progress and future perspectives. Mol. Plant. 2022, 15, 125–137. [Google Scholar] [CrossRef] [PubMed]
  33. Hu, M.; Lv, S.W.; Wu, W.G.; Fu, Y.C.; Liu, F.X.; Wang, B.B.; Li, W.G.; Gu, P.; Cai, H.W.; Sun, C.Q.; et al. The domestication of plant architecture in African rice. Plant J. 2018, 94, 661–669. [Google Scholar] [CrossRef] [PubMed]
  34. Wu, Y.Z.; Zhao, S.S.; Li, X.R.; Zhang, B.S.; Jiang, L.Y.; Tang, Y.Y.; Zhao, J.; Ma, X.; Cai, H.W.; Sun, C.Q.; et al. Deletions linked to PROG1 gene participate in plant architecture domestication in Asian and African rice. Nat. Commun. 2018, 9, 4157. [Google Scholar] [CrossRef]
  35. Jiang, J.H.; Tan, L.B.; Zhu, Z.F.; Fu, Y.C.; Liu, F.X.; Cai, H.W.; Sun, C.Q. Molecular evolution of the TAC1 gene from rice (Oryza sativa L.). J. Genet. Genomics 2012, 39, 551–560. [Google Scholar] [CrossRef]
  36. Li, Z.; Liang, Y.; Yuan, Y.D.; Wang, L.; Meng, X.B.; Xiong, G.S.; Zhou, J.; Cai, Y.Y.; Han, N.P.; Hua, L.K.; et al. OsBRXL4 Regulates Shoot Gravitropism and Rice Tiller Angle through Affecting LAZY1 Nuclear Localization. Mol. Plant 2019, 12, 1143–1156. [Google Scholar] [CrossRef]
  37. Dong, Z.B.; Jiang, C.; Chen, X.Y.; Zhang, T.; Ding, L.; Song, W.B.; Luo, H.B.; Lai, J.S.; Chen, H.B.; Liu, R.Y.; et al. Maize LAZY1 mediates shoot gravitropism and inflorescence development through regulating auxin transport, auxin signaling, and light response. Plant Physiol. 2013, 163, 1306–1322. [Google Scholar] [CrossRef]
  38. Chen, J.Y.; Yu, R.B.; Li, N.; Deng, Z.G.; Zhang, X.X.; Zhao, Y.R.; Qu, C.F.; Yuan, Y.F.; Pan, Z.X.; Zhou, Y.Y.; et al. Amyloplast sedimentation repolarizes LAZYs to achieve gravity sensing in plants. Cell 2023, 186, 4788–4802.e15. [Google Scholar] [CrossRef]
Agriculture 13 02166 g001
Figure 1. Comparison of plant types between WYJ7 and mtl1 rice under field growth conditions. (a) Plant phenotypes grown under field conditions; (b) tiller angle; (c,d) tiller number; (e,f) plant height. Scale bar: 15 cm.
Figure 1. Comparison of plant types between WYJ7 and mtl1 rice under field growth conditions. (a) Plant phenotypes grown under field conditions; (b) tiller angle; (c,d) tiller number; (e,f) plant height. Scale bar: 15 cm.
Agriculture 13 02166 g001
Agriculture 13 02166 g002
Figure 2. Comparison of panicle patterns between WYJ7 and mtl1 rice. (a) Panicle type; (b) grains of an individual plant; (c) panicle length; (d) number of primary branches; (e) number of secondary branches; (f) number of grains per panicle; (g) grain yield per plant. Scale bar: 5 cm.
Figure 2. Comparison of panicle patterns between WYJ7 and mtl1 rice. (a) Panicle type; (b) grains of an individual plant; (c) panicle length; (d) number of primary branches; (e) number of secondary branches; (f) number of grains per panicle; (g) grain yield per plant. Scale bar: 5 cm.
Agriculture 13 02166 g002
Agriculture 13 02166 g003
Figure 3. Grain type comparison between WYJ7 and mtl1 rice. (a,b) Grain length; (c,d) grain width; (e,f) grain thickness; (g) 1000-grain weight. Scale bar: 1 cm.
Figure 3. Grain type comparison between WYJ7 and mtl1 rice. (a,b) Grain length; (c,d) grain width; (e,f) grain thickness; (g) 1000-grain weight. Scale bar: 1 cm.
Agriculture 13 02166 g003
Agriculture 13 02166 g004
Figure 4. Preliminarily mapping of the MTL1 gene using the BSA method. (a) Genotype analysis of parents and the mixed pools of F2; (b,c) SNP analysis of the differential loci. Homozygous gene loci in the female parent P1-mtl1 are shown in gray (AA); homozygous loci in the male parent P2-HJX74 are shown in red (BB); heterozygous gene loci in the segregated populations are shown in blue (AB).
Figure 4. Preliminarily mapping of the MTL1 gene using the BSA method. (a) Genotype analysis of parents and the mixed pools of F2; (b,c) SNP analysis of the differential loci. Homozygous gene loci in the female parent P1-mtl1 are shown in gray (AA); homozygous loci in the male parent P2-HJX74 are shown in red (BB); heterozygous gene loci in the segregated populations are shown in blue (AB).
Agriculture 13 02166 g004
Agriculture 13 02166 g005
Figure 5. Fine mapping and candidate gene analysis. (a) Preliminary mapping of MTL1; (b) fine mapping of MTL1 by using newly designed InDels; (c) candidate genes in the mapping interval. (d) PCR amplification of the exons of LA1; (e) semi-quantitative RT-PCR analysis of LA1 expression level. Ms represent polymorphic markers; the numbers below the lines represent the number of recombinants. The arrow indicates the site of the predicted genes in the M8 to M12 interval.
Figure 5. Fine mapping and candidate gene analysis. (a) Preliminary mapping of MTL1; (b) fine mapping of MTL1 by using newly designed InDels; (c) candidate genes in the mapping interval. (d) PCR amplification of the exons of LA1; (e) semi-quantitative RT-PCR analysis of LA1 expression level. Ms represent polymorphic markers; the numbers below the lines represent the number of recombinants. The arrow indicates the site of the predicted genes in the M8 to M12 interval.
Agriculture 13 02166 g005
Table 1. Genetic analysis of the mtl1 mutant rice.
Table 1. Genetic analysis of the mtl1 mutant rice.
Hybrid Crosses.F1 PhenotypesF2χ23∶1
HJX 74 Phenotypesmtl1 PhenotypesTotal Number of Plants
mtl1 × HJX74HJX745051616660.20
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Sun, Z.; Mei, T.; Feng, T.; Ai, H.; Ye, Y.; Duan, S.; Liu, B.; Huang, X. Deletion of the OsLA1 Gene Leads to Multi-Tillering and Lazy Phenotypes in Rice. Agriculture 2023, 13, 2166. https://doi.org/10.3390/agriculture13112166

AMA Style

Sun Z, Mei T, Feng T, Ai H, Ye Y, Duan S, Liu B, Huang X. Deletion of the OsLA1 Gene Leads to Multi-Tillering and Lazy Phenotypes in Rice. Agriculture. 2023; 13(11):2166. https://doi.org/10.3390/agriculture13112166

Chicago/Turabian Style

Sun, Zhanglun, Tianrun Mei, Tingting Feng, Hao Ai, Yafeng Ye, Sumei Duan, Binmei Liu, and Xianzhong Huang. 2023. "Deletion of the OsLA1 Gene Leads to Multi-Tillering and Lazy Phenotypes in Rice" Agriculture 13, no. 11: 2166. https://doi.org/10.3390/agriculture13112166

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop