1. Introduction
The rice (
Oryza sativa L.) is an important widely adapted food crop and 20% of the world’s dietary energy supply which is feeding more than half of the world’s population and 3 billion people uptake rice daily [
1,
2]. Due to the fast-growing population, the global rice consumption is projected to increase from 450 million tons in 2011 to about 490 million tons in 2020 and 40% more rice is needed to be produced by 2050 to meet people’s demand for food [
3,
4]. The cytoplasmic male sterility (CMS) is the foundation to exploit the heterosis of hybrid rice which uses a three-line system consisting of a cytoplasmic male sterile (CMS) line (A line), a maintainer (B), and a restorer (R line) for hybrid seed production [
5]. China is the pioneer of hybrid rice production and with the development of latest breeding tools the yield of rice has been increased more than 20% and newly developed genotypes performing better than conventional verities and now accounts more than half of the annual rice planting area in China [
6]. The development of new CMS has become the main interest of breeders because very few genotypes exhibit a strong restoration ability as effective restorer for CMS in the development of hybrid rice [
7,
8]. Yield and quality are typical quantitative traits governed by multiple genomic loci, while yield is directly depends on grain weight (GW) which is mainly determined by the synthesis and accumulation of starch in the endosperm of the grain [
9,
10]. To solve this problem, we must resort to new technologies and new genetic improvement strategies. Starch is one of the important indicators for evaluating rice quality and 90% of rice endosperm is starch [
11].
Rice waxy gene
Wx-encoded granular bound starch synthase I (GBSSI), also known as Waxy protein is the major gene controlling amylose synthesis in endosperm [
12].
Wx gene differentiates into alleles
Wx a and
Wx b, indica rice is dominated by
Wx a which confers higher amylose content by producing 10-fold higher mRNA and protein level than
Wx b while japonica rice is dominated by
Wx b with lower amylose content [
13,
14].
Wx exon or intron structural change would affect
Wx expression by affecting messenger RNA (mRNA) stability [
12,
15]. Several studies have reported that mutations in the functional site of the
Wx gene led to 14.6 to 2.6% reduced amylose content (AC) in rice transgenic lines and hybrids obtained with mutant lines [
15,
16,
17,
18,
19,
20,
21,
22,
23], while
Wx overexpression lines showed increased AC by 6–11% [
24].
At present, the GW related genes that have been cloned including
qSW5/GW5 [
25,
26],
TGW6 [
27],
GS3 [
28],
GS5 [
29],
GW2 [
30],
GW8/OsSPL16 [
31],
qGL3/
qGL3-1/
GL3.1 [
32,
33,
34],
GW7 [
35], and
OsSPL13 [
36]. Among them,
TGW6 is one of the most important genes regulating rice GW traits, which encodes a purine acetic acid-glucose hydrolase. Its loss-of-function mutation causes a decrease in the content of indoleacetic acid in the endosperm resulted in increased cell numbers which finally resulted with increased grain length and GW with 15% enhanced production of rice [
27]. Rice genes including
DEP1, GS3,
GW2,
GS5,
Gn1a, and
TGW6, that are negative regulators of grain size and number and grain weight has been knocked-out to improve yield [
37,
38], and CRISPR/Cas9 based simultaneous mutations of
GW2,
GW5, and
TGW6 resulted in 29.3% increase in GW [
39]. This suggests that generation of mutation in major yield related genes in a single cultivar would be helpful to increase large scale production of rice.
With the development of some new molecular biology techniques such as CRISPR/Cas9 (clustered regulatory interspersed short palindromic repeat/CRISPR associated proteins) a lot of achievements has been made in plants and animals. CRISPR/Cas9 technology is widely used to study the gene function and regarded as the third-generation genome-editing tool established after zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALEN), based on guided RNA (gRNA) engineered nucleases, which is most applicable due to their simplicity, efficiency, and versatility [
40,
41]. CRISPR/Cas9 make a double-stranded break (DSB) in the target DNA which is subsequently repaired by natural repair mechanism of homologous recombination (HR) precise pathway or non-homologous end joining (NHEJ) [
42], which creates random insertions and deletions and results in targeted gene knockouts or gene replacement [
40,
43,
44]. CRISPR/Cas9 is the most advanced genome editing tool in plant biology [
45,
46] and has been widely used in animals, yeast, human non-human cell lines [
42,
47,
48], as well as in the model species
A. thaliana and
N. benthamiana [
43,
49], as well as crops such as rice [
50,
51,
52], wheat [
53], maize [
54], potato [
55], and tomato [
56].
Conventional plant breeding techniques are effective but laborious and time consuming, therefore we used CRISPR/Cas9-mediated gene editing to introduce a loss-of- function mutations into the Wx and TGW6 genes associated with lower AC and increase yield in rice maintainer line 209B. Our results show that mutations in the Wx and TGW6 gene produce decreased AC and enhanced yield in rice CMS line offering an effective strategy of accelerating the hybrid rice breeding program. Through one generation of hybridization and two generations of backcrossing with mutant maintainer lines as the male parent and 209A as female parent, the glutinous cytoplasmic male sterile lines (CMS) were successfully achieved. The protein of CMS line pollen and mutant maintainer line were separated by two-dimensional electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and differentially expressed spots were analyzed. This study gave new insights into the mechanism of CMS and maintainer lines and demonstrated the power of proteomic in plant biology. Present study showed that the CRISPR/Cas9 technology provides the tool set to fasten the rice breeding program to achieve desired agronomic characters and improved yield.
4. Discussion
CRISPR/Cas9 is an emerging genome editing technology developed in past few years with high specificity and editing efficiency. Relative to ZFNs [
68] and TALENs [
69,
70], CRISPR/Cas9 is simple and flexible and only one gRNA and one nuclease (Cas9) are needed to achieve the mutations in the DNA sequence of the target gene. Current research focuses on the development of CRISPR/Cas9 technology and specific gene knockouts.
Breeding for consumer-preferred grain yield and quality have thus become a major goal for breeding programs and in the last few decades, the classical, mutational, and molecular breeding approaches have brought about tremendous increase in rice productivity with the development of novel rice varieties for food security considerations. The improved living standards and fast economic growth are shifting public attention toward quality characteristics such as, nutrition, flavor, appearance, and cooking which are linked to starch physical properties. With the development of latest gene editing technologies such as CRISPR/Cas9, many yield related quantitative trait loci (QTLs) has been edited and their functions have been explored in different verities [
37]. In the rice grain endosperm, starch is the major component consisting of a linear polysaccharide amylose which determines the cooking and eating quality of rice. Cooking of high AC (25–33%) verities results in separated, dry and firm rice grains, becoming hard after cooling while glutinous rice with low AC (5–20%) is especially sticky and soft when cooked [
71]. The improvement of maintainer line in hybrid rice breeding system is most inevitable to achieve target traits. In China, the
indica hybrids considered low quality owing to high AC that makes them hard and dry when cooked. The breeding for low AC and improved grain quality and yield is a major objective of breeders.
In this study the CRISPR/Cas9 construct with 20-nt target sequence for the sgRNA was carefully designed with high GC content and low off-target score and the
Wx and
TGW6 gene with expectation to produce a null mutation were edited. The schematic representation of whole procedure of generation and analysis of targeted mutated plants was described in
Figure S6. The goal of this study was to develop a high yielding CMS rice line with low amylose content to facilitate the hybrid rice breeding program and proteins from anthers of maintainer and CMS line were also identified. We sequenced the Wx and TGW6 gene and confirmed that 209B contains both genes (
Figure S7). Four targets were designed in the exon regions of
Wx and
TGW6 gene, the corresponding promoters were OsU6a, OsU6b, OsU6c, and OsU3m and
Agrobacterium tumefaciens based transformations was successfully achieved with the CRISPR/Cas9 cassette and mutations in the target regions were analyzed by sanger sequencing by decoding it using online DSDecodeM tool. The results of this study indicate that the CRISPR/Cas9 gene editing technology can successfully edit rice targeting DNA sequences with high efficiency and multiple mutations can be generated at the same target site, and base deletion or insertion occurs before the target site PAM.
The total mutation frequency was up to 90%, wherein homozygous mutations were about 28%, which indicate that the CRISPR/Cas9 editing facilitates homozygous mutations in the T
0 generation (
Table 1). The previous studies showed that the CRISPR/Cas9 induced the homozygous mutations in T
0 generation and mutations mainly take place in transformed calli cells [
72]. The expression level of targeted genes was lower in mutant lines than WT (
Figure 6). The off-target mutations were not detected for all targets (
Table S3). The comparison of T
0 and T
1 generations showed that the mutation frequency of homozygotes was stably inherited regardless of whether T-DNA is present. The conserved amino acid sequence was totally changed in mutant plants and mutant plants showed divergence to WT in amino acid sequence alignment (
Figure 5).
The glutinous rice lines were obtained, and all mutant lines seeds showed low AC decreased from 18.2% to 1.7 % and homozygous mutant lines showed less percentage of AC than heterozygous and bi-allelic mutants (
Table 2). It is reported that the
Wx gene also affect the GC and GT of rice [
22,
73] and our results showed an increase in GC from 58.65 mm to 138.62 mm and decrease in GT from 5.67 ASV to 3.12 ASV, while there was no effect on total SC (
Table 2). The GW was increased from 21.1 g to 240.8 g (
Table 3), while there was no effect on PH, NOP, FLL, FLW, PL, GPS, and SSR (
Table 4). The cross-section analysis by electron microscope showed that endosperm of mutant grains was shrunken corresponding to their WT. The T-DNA free lines were obtained to address the social values of laws about genetically modified (GM) foods by selecting the transgene free lines by self-pollination in the T
1 and T
2 generations (
Figure 7). Our results showed that the T
1 mutant lines were re-edited while mutations were inherited and stable in T
2 generation (
Figure 8). The T
0 lines are frequently difficult to predict which suggests that the mutations in T
0 generations are not stable but the mutations in T
1 generations transmitted stably to later generations. These results are consistent with previous reports that the editing site of the T
1 generation mutant plant target sequence may also have a sequence recognized by the gRNA target, resulting in re-editing, which makes the T
1 generation unpredicted which can stabilize in later generations [
74]. Together, these results clearly demonstrate that CRISPR/Cas9-induced gene mutations can be stably transmitted to subsequent generations.
The shape of pollen grains and staining patterns in male sterility inducing cytoplasm and sterility maintaining nuclear genes are influenced by the pollen abortion stage related to nuclear stage [
75]. Mutant maintainer lines were assessed for pollen fertility status and results showed that pollen fertility rate was randomly distributed and six genotypes were found sterile having pollen fertility 0–9%, two genotypes PS with 10–29% pollen fertility, two genotypes were recorded PF which is 12.5% of total and four genotypes were identified as CF and two were F as these genotypes had above 80% pollen and spikelet fertility which is 37.50% of the total genotypes (
Table 5). The developed maintainer lines were crossed with CMS line to develop F
1 and after subsequent backcrossing glutinous CMS line was achieved.
The CMS lines has been widely used in hybrid rice production, but the molecular mechanism of CMS remains poor understood. The protein identification tool is a powerful tool to study anther development and pollen production in plants [
76,
77,
78,
79]. The CMS is different plant species are cause by a specific ORF containing chimeric genes in mitochondrial genome [
80] with rare similarity but sharing same male gamete abortion phenomenon [
81]. The mitochondrial amplification events suggest an increased demand for energy during pollen development [
82] but lowered ATP production was also observed in some CMS flowers [
81,
83]. In this study the proteins identified in maintainer and CMS line helped to understand the molecular mechanism of rice male sterility. Sixteen proteins were identified between sterile and maintainer anthers (
Table 6). The identified proteins have potential roles in anther and pollen development and may help to clarify the mechanism of male sterility in rice.
The proteins of CMS line and GX4-2 maintainer mutant line anthers were separated by two-dimensional electrophoresis and SDS-PAGE as the second. The silver stained proteins were analyzed using Image Master 2D software. The identified proteins were, 20S proteasome beta 4 subunit, putative RNA-binding protein, Putative berberine bridge enzyme (BBE), putative mitochondrial NAD
+-dependent malic enzyme, Putative calcium-binding protein annexin, UDP-glucuronic acid decarboxylase, putative phosphoribosyl pyrophosphate synthase, putative RNA binding protein (RBP), H
+ -transporting two-sector ATPase alpha chain–rice mitochondria, glucose-1-phosphate adenylyltransferase large subunit 3, putative membrane-associated salt-inducible protein, putative leucine-rich repeat protein, putative acetyl-CoA synthetase (ACOS), putative lipoamide dehydrogenase, Isoamylase (fragrant), and DNA binding protein (
Table 6). These proteins are closely associated with metabolism, protein biosynthesis, transcription, signal transduction and many other activities which are important in cell activities and essential to pollen development.
Dysfunctions of mitochondria in the pollen caused CMS in plants and several other mitochondrion regions have been identified associated with CMS [
84]. ATP synthase β-subunit helps to fulfill the demand of energy for respiratory function and cellular energy to develop male gametophyte also observed in mitochondria [
85], and defective β-subunit resulted non-functional pollens and abnormal anther development [
84]. The 20S proteasome is the proteolytic complex actively involved in removing abnormal proteins with several biological functions [
86], while RBP is involved to regulate transcriptional and post-transcriptional levels to control the gene expression. Plants respond to pathogen infection with rapid reprogramming of gene expression and loss of function of RBP showed enhanced resistance to pathogens [
77]. Biochemical and biological function of BBE are unexplored [
87]. Plant annexins regulate diverse aspects of plant development, stress responses and growth [
88]. ACOS played role in plastids and in several metabolic pathways [
89] and has significant role in anther development [
90,
91]. The ACOS in anther prevent the conversion of pyruvate into acetyl-CoA which leads to pollen sterility. The degeneration and formation of various tissues during pollen development needs high energy for key biosynthetic intermediates. Isoamylase in combination with pullulanase plays a predominant role in amylopectin synthesis and also essential for the construction of the amylopectin multiple-cluster structure by removing the excessive branches to avoid interference with the formation of double helices of the cluster chains of amylopectin and crystallization of starch in the endosperm. These proteins or enzymes are involved in multiple physiological and biochemical reactions such as carbon metabolism and starch synthesis, as well as signal transduction and protein expression regulation [
92].
In short, the increase yield and reduction of AC are valuable parameters in crop breeding and CRISPR/Cas9 is excellent technology to achieve targeted mutations in genes. In this study the rice maintainer line and new CMS lines were developed with increased yield and improved quality while maintaining all agronomic traits. We also took precautionary approach and produced T-DNA-free plants to avoid foreign bacterial DNA integration and bypass GMO rules. The most likely off-target effects were analyzed and Cas9 free plants were selected for food safety assessments and it was ensured that the other plant traits were not affected. In our work, we improved existing traits by directly rewriting the plant genetic code without any cutting and pasting genes from animals or bacteria into rice plants. Our study provides some insights to study the gene functions and generation of new rice CMS lines with increased yield and improved quality without compromising on nutritional value to facilitate the hybrid breeding programs of rice to develop elite crop verities. This study is the first example to develop rice CMS lines with increased yield and low AC and the protein identification in mutant rice maintainer and CMS line which will be the source material for further breeding of hybrid glutinous rice verities in short period. The identified proteins in anther of maintainer and CMS lines provide the insights to the actual mechanism underlying in sterility of rice lines. The study showed the genetic mutations are not only helpful to improve the plant characteristics, they also help in understanding the mechanisms underlying the biochemical behavior changes in cell of the plants.