Next Article in Journal
Response of Nicandra physalodes (Linn.) Gaertn. and Its Rhizospheric Organisms to the Selective Pressures of High-Concentration Oxytetracycline, Ciprofloxacin, and Tobramycin
Previous Article in Journal
Precise Short-Term Small-Area Sunshine Forecasting for Optimal Seedbed Scheduling in Plant Factories
 
 
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 9
10.3390/agriculture13091791
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

RNA-Seq Revealed the Molecular Mechanism of Nutritional Quality Improvement in o16-wx Double-Mutation Maize

by
Zhoujie Ma
1,†,
Peizhen Wu
1,†,
Lei Deng
2,3,
Kaiwu Zhang
2,3,
Wenpeng Yang
2,3,
Hong Ren
2,3,
Li Song
1,* and
Wei Wang
2,3,*
1
Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), College of Life Sciences/Institute of Agro-Bioengineering, Guizhou University, Guiyang 550025, China
2
Guizhou Institute of Upland Food Crops, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China
3
Key Laboratory of Maize Biology and Genetic Breeding in Karst Mountainous Region (Ministry of Agriculture and Rural Affairs), Guiyang 550006, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2023, 13(9), 1791; https://doi.org/10.3390/agriculture13091791
Submission received: 24 July 2023 / Revised: 15 August 2023 / Accepted: 6 September 2023 / Published: 11 September 2023
(This article belongs to the Section Genotype Evaluation and Breeding)
Browse Figures
Review Reports Versions Notes

Abstract

:
The enhancement of the nutritional composition of maize grains could be achieved by the introduction of a mutation with a heightened lysine content. To obtain double-recessive mutant lines for the o16 and wx genes, a molecular-marker-assisted selection technique was used to backcross them into conventional maize lines. The resultant maize was subsequently utilized to explore the molecular mechanism responsible for the maize’s nutritional quality. Based on this, an RNA-seq investigation was conducted using the employing kernels during the development period of maize kernel (18, 28, and 38 DAP) to examine the gene expression involved in amino metabolism. The results revealed that a total of 27 and 34 differentially expressed genes (DEGs) were identified in tryptophan metabolism and lysine metabolism, respectively, across three time periods. In the lysine synthesis pathway, the genes encoding AK, ASD, and DapF were found to be up-regulated at various stages, encouraging lysine synthesis. Conversely, in the lysine degradation pathway, the genes encoding ALDH7A1 and LKR/SDH were down-regulated, suggesting an increase in lysine content. In the process of tryptophan metabolism, the down-regulation of genes encoding TAA and ALDH led to an increase in tryptophan content. In addition, the down-regulation of genes encoding α-zein resulted in a decrease in zein content, thereby enhancing the nutritional quality of maize. These findings hold substantial significance for elucidating the transcriptional-level molecular mechanism, underlying the accumulation of o16 and wx genes to improve maize grain quality, as well as offering valuable insight for the development of biomarkers and gene editing.

1. Introduction

Maize (Zea mays L.) is the main food and feed crop in the world, possessing high economic value and application prospects. The nutritional components of its grains, including protein, fat, starch, vitamins, and mineral elements, are important indicators for measuring the nutritional quality of corn, which directly affects the taste and healthy development of humans and livestock. Among them, lysine, which is contained in proteins, is an essential amino acid for both humans and animals. Ordinary corn has a low lysine content and is unable to satisfy the needs of human consumption or livestock and poultry feed. Therefore, high-quality protein maize (QPM), also known as high-lysine corn, has received widespread attention. In 1935, the maize mutations opaque1(o1) and opaque2(o2) were initially found [1]. Mertz et al. and Hartings et al. [2,3] found that o2 mutation can significantly increase the lysine level in maize. Subsequently, researchers found numerous similar mutations, including floury endosperm1(fl1) (opaque8, opaque4), floury endosperm2(fl2), floury endosperm3(fl3), opaque5, proline responding1(pro1) (opaque6), opaque7, shrunken4(sh4) (opaque9), opaque10, opaque13, and opaque18 [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. However, these mutations were difficult to apply in breeding procedures and production due to various reasons, such as complex genetic patterns. Yang et al. used the Mu transposon technique to produce the high-lysine mutant opaque16 (o16), whose lysine content reached 0.36% and whose gene was placed on chromosome 8, in order to increase the germplasm resources of high-lysine maize. The distance between the umc1141 and umc1121 molecular markers was 3 cm from the umc1141 molecular marker [19].
In order to boost maize’s nutritional value even more. Yang et al. [20] used MAS (marker-assisted selection) to conduct a prospect selection of target genes (o2 and o16) and obtained a high-lysine innovative germplasm of o2 and o16 gene accumulation in maize. The tryptophan and lysine contents were more than 20% higher than those of the o2 parent, Qi205. In 2010, Zhang et al. [21] used a new high-lysine mutation, QCL3021 (o16), as a donor and Taixi19 (o2) as a receptor to obtain 17 BC2F4 families that contained o2 and o16 double genes. The lysine content obtained was 0.469–0.599%, which was about 122.63% higher than that of common maize, 22.33% higher than that of the parent Taixi19 (o2o2), and 65.86% higher than that of the parent QCL3021 (o16o16). Hartings et al. [22] studied the o2o7 mutant and found that its lysine level was more than 3.5 times higher compared to the wild type. In 2018, Konsam Sarika et al. [23] backcrossed o16 and o2 genes into different parents through MAS. On the basis of the parent, the tryptophan and lysine content increased by 91% and 76%, respectively. In 2022, Gulab Chand et al. [24] analyzed 21 different maize hybrids of the o2o16 genotype in two locations using a half-diallel cross design. Compared with o2 genotype QPM hybrids (tryptophan: 0.086%; lysine: 0.346%), the o2o2/o16o16 genotype hybrids had significantly higher tryptophan (0.126%) and lysine (0.506%) contents. Therefore, the accumulation of high-lysine mutant genes can increase the content of lysine and tryptophan in maize grains, thereby improving their nutritional quality.
Waxy corn, also known as cake corn or sticky corn, is popular with consumers because of its excellent taste and edible digestibility. It was found that waxy cornstarch is completely composed of amylopectin, which is characterized by a small molecular weight and multiple branches, and its wx(waxy) gene is located on chromosome 9 [25,26]. Although waxy corn has a good edible taste, its nutritional value is low. In order to improve the nutritional quality of waxy maize, Zhou et al. [27] introduced the o2 allele into the waxy maize line Zhao OP-6/O2O2 by using labeled MAS technology, and the lysine content increased by 51.6% compared with that of the parent. Yang et al. [28] used MAS to obtain seven materials that polymerized the wx and o16 genes, and compared with the waxy parents, the lysine content increased by 16-28%. Wang et al. [29] inserted the o2 and o16 genes into waxy corn to obtain two materials, QCL8006_1 and QCL8006_2, which had a lysine content of 0.49% and 0.54%, respectively. Thus, through the accumulation of the high-lysine and waxy genes, a new high-lysine maize germplasm with unchanged waxy properties could be obtained.
The introduction of the o2 gene into waxy maize not only reduced the synthesis of various zeins but also affected the accumulation of other correlative endosperm proteins [27]. However, whether o16 and wx genes can affect the key development process of maize the kernel when they are simultaneously infiltrated into common maize and how it is different from the effect of o2 in the wx background. have not been reported. To this end, we used MAS to backcross the o16 and wx genes into common maize and obtained the o16 and wx double-recessive mutant accumulation line QCL8012_2. A transcriptome sequencing analysis was performed on the kernels during their development period of the kernel to compare the differences in transcriptional expression between the o16wx mutant line and its parents in order to understand the molecular mechanism of the o16 and wx genes in improving the nutritional quality of the kernels after their insertion into common maize.

2. Materials and Methods

2.1. Plant Materials

In order to obtain an F1 generation, QCL8007_5 (o16wx) was used as the donor parent, and the common maize inbred line CML530 was used as the recipient parent. Through molecular-marker-assisted selection, combined with a genetic background analysis, a lysine content analysis, and plant field performance, the o16wx gene’s pyramiding line was finally screened. After several generations of self-crossing to achieve stability inheritance, it was named QCL8012_2. An SNP (55K) chip analysis showed that the recovery rate of the inherited background of QCL8012_2 based on molecular markers was 94.69%. QCL8012_2 and CML530 were planted in the experimental base of the Guizhou Academy of Agricultural Sciences (Guiyang, Guizhou, China) in the summer of 2017.

2.2. Kernel Phenotype and Electron Microscope Observation

The mature ear and dry grain of QCL8012_2 and CML530 were observed under natural light and were photographed using a Canon camera. After removing the peel of the mature and dry kernels, a piece of endosperm was cut from the middle using a knife blade, and platinum was embedded by ion sputtering. The observation was performed using a scanning electron microscope (SEM, Munich, Germany; S-3400N).
A light box test for opaqueness was performed. One hundred randomly selected seeds in CML530 and QCL8012-2 were used for analyses of endosperm opaqueness. The degree of opaqueness of seeds was analyzed by using a standard ‘light box’ with the formula: Degree of opaqueness = [(N100 × 100) + (N75 × 75) + (N50 × 50) + (N25 × 25) +(N0 × 0)]/100, where N100, N75, N50, N25 and N0 are the numbers of seeds with 100%, 75%, 50%,25% and 0% opacity, respectively [30].

2.3. Determination of Amino Acids

The amino acid content (17 amino acids) in the mature maize kernels was analyzed using an automatic amino acid analyzer (S-433D; Sykam, Eresing, Germany). After drying the corn kernels, they were crushed using a universal pulverizer and passed through an 80-mesh sieve. Subsequently, they were weighed (120–150 mg) and placed in a 15 mL hydrolysis tube (with a screw). Next, 5 mL of 6 M HCl was added to the tube, and the kernels were placed in a water bath (110 °C) for 20–24 h. They were then cooled down to room temperature, and the pH was adjusted to 2.0 using a 6 M NaOH solution. The volume of the solution was kept constant at 100 mL, and a syringe was used to absorb the reaction liquid, which was filtered using a 0.45 μm water filter into a 2 mL chromatography vial and tested for 17 free amino acids. Each sample procedure was repeated three times.
Tryptophan (Trp) content was determined, according to the national standard (GB7650-87 of China) [31]. Each sample was repeated three times. The specific steps are as follows: After drying the corn kernels, they were crushed using a universal pulverizer and passed through an 80-mesh sieve. After drying, the corn kernels were crushed by a universal pulverizer and passed through an 80-mesh sieve. After degreasing, two portions of a 40 mg sample were weighed and placed in a 20 mL glass test tube, added with 1 mL of 10% KOH, and placed in a 40 °C incubator for hydrolysis for 16–18 h. The test tube was taken out, and 0.2 mL of 5% p-dimethylaminobenzaldehyde was added after cooling and shaken well. Then, 0.2 mL of 1% NaNO3 was added. The test tube was placed in ice water, and then 5 mL of concentrated hydrochloric acid was added. The test tube was taken out and placed in a 40° incubator for 45 min (the temperature rose to 40 °C). After the test tube was taken out, it was diluted with water to the scale, and centrifuged at 4000 r/min for 10 min. The supernatant reagent blank was taken as the control, and the absorbance was read at 590 nm wavelength. The tryptophan content was obtained by comparison with the standard curve.

2.4. RNA-Seq

During the development period of the maize kernel, the total RNA of the whole grain was extracted using a Plant RNA Kit (OMEGA, Beijing, China) at 18 days, 28 days, and 38 days after pollination (DAP). The first step in the workflow involved purifying poly-A-containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations at elevated temperatures. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers, which was followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. These cDNA fragments then underwent the addition of a single ‘A’ base and subsequent ligation of the adapter. Subsequently, the products were purified and enriched using PCR amplification. We then quantified the PCR yield using Qubit and pooled the samples together to produce a single-strand DNA circle (ssDNA circle), which provided the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling-circle replication (RCR) to enlarge the fluorescent signals during the sequencing process. The DNBs were loaded into the patterned nanoarrays, and single-end reads of 50 bp were read through on the BGISEQ-500 platform for the data analysis study. For this step, the BGISEQ-500 platform combined DNA nanoball-based nanoarrays and stepwise sequencing using a combinational probe–anchor synthesis sequencing method. Transcriptome sequencing was performed using the BGISEQ-500 sequencing platform (completed by Shenzhen Huada Gene Technology Service Co., Ltd., Shenzhen, China), and each sampling procedure was repeated twice.

2.5. Identification of Differentially Expressed Genes (DEGs)

DEG detection was performed using the SOAPnuke software [32], HISAT software [33], Bowtie2 software [34], and RSEM [35] and NOISeq2 [36] algorithms, and the differentially expressed genes between the two groups were screened according to a difference fold value of ≥2 and a corrected p-value of ≤0.05.

2.6. GO and KEGG Enrichment Analysis of DEGs

The selected DEGs were compared to the GO database. Based on the GO annotation results, the differential genes were functionally classified and enrichment analyzed. A pathway analysis of the differential genes was performed using the KEGG (2008) [37] public database. A p-value calculation was performed along with a GO functional significance enrichment analysis. Additionally, false discovery rate (FDR) correction was performed. Pathways with FDR values of ≤0.01 were considered to have significant enrichment.

2.7. qRT-PCR Analysis

Twenty-five key DEGs were selected for qRT-PCR verification during the three periods, and primers were designed online, as shown in Table S1 (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi (accessed on 11 May 2022)). The total RNA for the RNA-seq analysis was reverse transcribed into cDNA using the Thermo Scientific RevertAid First Strand cDNA Synthesis (BIO-RAD, Hercules, CA, USA). The reverse transcription system (20 μL) included the addition of the following: 10 μL of RNA, 1 uL of random hexamer primer, 4 μL of 5× reaction buffer, 1 μL of RiboLock RNase inhibitor (20 U/μL), 2 μL of 10 mM dNTP mix, 1 μL of RevertAid M-MuLV RT (200 U/μL), and 1 uL of water (nuclease-free). The reverse transcription program was performed under the following conditions: 25 °C for 5 min; 42 °C for 60 min; 70 °C for 5 min; and 4 °C for preservation. The cDNA was diluted 10–20 times according to the following system (10 μL): 2.0 μL of cDNA, 0.5 μL of primer F (10 mM), 0.5 μL of primer R (10 mM), 5 μL of SYBR Select Master MIX, and 2.0 μL of H2O. The program used was performed under the following conditions: 50 °C for 2 min; 95 °C for 10 min; 95 °C for 15 s; 60 °C for 1 min; for a total of 40 cycles. Subsequently, a qRT-PCR procedure was performed using the FX Connect Real-Time PCR System (BIO-RAD, Hercules, CA, USA), with actin used as the internal reference, and each sampling procedure was repeated three times.

2.8. Accession Numbers

The transcriptome data uploaded to https://ngdc.cncb.ac.cn/: PRJCA017345.
BioSample accessions were as follows: SAMC1272017, SAMC1272018, SAMC1272019, SAMC1272020, SAMC1272021, SAMC1272022, SAMC1272023, SAMC1272024, SAMC1272025, SAMC1272026, SAMC1272027, and SAMC1272028; PRJCA017345 records will be accessible after the specified release date is published.

3. Results

3.1. Kernel Phenotype and Electron Microscope Observation

The grain phenotypes and ears of QCL8012_2 and CML530 were observed under natural light. The o16wx mutant line exhibited the following characteristics: it had a cylindrical-type ear, its grain was dull, its endosperm was waxy, and its grains were primarily opaque. However, the recurrent parent, CML530, exhibited the following characteristics: its seed coat was smooth and glossy, its grain was full, its endosperm was keratinous, and its grains were transparent (Figure 1A–C). A histochemical test for starch staining showed that CML530 was blue and QCL8012_2 was purplish red.
By scanning electron microscopy, it was found the endosperm of the o16wx mutant’s starch granules were primarily ellipsoidal or spherical, and their volume was slightly smaller than that of the recurrent parent, CML530. In addition, the density of their matrix protein scattered between the gaps of starch granules was large. The endosperm starch granules of CML530 were irregular in shape, and their matrix protein density was slightly smaller (Figure 1E).
One hundred randomly selected CML530 and QCL8012_2 seeds were grouped into five classes with the scores in degree of opaqueness of 100%, 75%, 50%, 25%, and 0% [28]. In CML530 and QCL8012_2, the opaqueness was found to be 0% and 75.5%, respectively (Figure 2, Table 1)

3.2. Changes in Free Amino Acid (FAA) Composition of o16wx Endosperm

Compared with the recurrent parent, CML530, the 18 amino acids in the o16wx mutant line had different degrees of change (Figure 3; Table S2). Among them, the amino acid content decreased for Asp, Leu, Glu, Cys, Ile, Phe, Met, ALa, Ser, and Val, and the amino acid content increased for Pro, Thr, Gly, Tyr, His, Arg, Lys, and Trp. The amino acid content of Lys and Trp increased by 50.58% and 11.39%, respectively.

3.3. Correlation between RNA-Seq Quality and Biological Repeats

In order to analyze the regulatory network of the o16wx lines, we used an RNA-seq analysis of the QCL8012_2 and CML530 seeds (18, 28, and 38 DAP). In this study, we used two biological replicates. In each sample, an average of 23,985,593 clean reads were obtained (Table S3). The average alignment rate with the reference gene and reference genome was 81.31% (Table S4) and 87.11% (Table S5). The average Q20 was 97.565%, and the average Q30 was 90.143% (Table S6), indicating that the result of the sequencing data was better preferable. According to the results of quantitative analysis (Table S7), more than 81% of the total genes in maize were detected in each sample (Figure 1), showing that the sequencing saturation was in line with expectations. The two repeated samples’ correlation coefficients were both greater than 0.86 (Figure S2), indicating that the RNA-seq data for all samples with two biological replicates were highly reliable, which was conducive to the analysis of DEGs.

3.4. Differentially Expressed Genes between o16wx Double-Recessive Mutant Line and Recurrent Parent

To identify significant DEGs, the NOISeq software was used to analyze the differences between QCL8012_2 and CML530. The results indicated that compared with CML530, QCL8012_2 had 10388 DEGs at 18 DAP, of which 5329 were down-regulated and 5059 were up-regulated. At 28 DAP, QCL8012_2 had 4553 DEGs, and among them, 2213 were down-regulated and 2340 were up-regulated. At 38 DAP, QCL8012_2 had 3958 DEGs, and among them, 1645 were down-regulated and 2313 were up-regulated (Figure 4).

3.5. Functional Annotation of Significantly DEGs

The GO function classification statistics of the differential genes in the three periods were performed using the WEGO software. The results involved the following three items: cellular components, biological processes, and molecular functions (Figure 5). At 18 DAP, 6467 DEGs were involved in 42 GO terms, 11 molecular functionals, 19 biological processes, and 12 cellular component pathways. At 28 DAP, 2905 DEGs were involved in 38 GO terms, 10 molecular functionals, 17 biological processes, and 11 cellular component pathways. At 38 DAP, 2419 DEGs were involved in 38 GO terms, 10 molecular functionals, 18 biological processes, and 11 cellular component pathways.
In order to understand the molecular function of DEGs more clearly, the KEGG public database was used to perform Pathway enrichment analysis of differential genes (Figure 6). As a result, at 18 DAP, 3984 DEGs were annotated for 19 pathways, of which 349 DEGs were annotated for amino acid metabolism and 599 DEGs were annotated for carbohydrate metabolism. At 28 DAP, 1857 DEGs were annotated for 19 pathways, of which 137 DEGs were annotated for amino acid metabolism and 290 DEGs were annotated for carbohydrate metabolism. In 38 DAP, 1566 DEGs were annotated for 19 pathways, of which 147 DEGs were annotated for amino acid metabolism and 241 DEGs were annotated for carbohydrate metabolism.

3.6. DEGs in Lysine Metabolism

DEGs were annotated into the KEGG database, and the results showed that, in the lysine biosynthesis pathway, 15, 8 and 12 DEGs were detected in the three periods, where 21 were up-regulated and 14 down-regulated; in the lysine degradation pathway, 9, 4 and 2 DEGs were detected in the three periods, where 7 were up-regulated and 8 down-regulated (Figure 7).
In the first step of lysine synthesis, from L-aspartate to L-4-aspartyl phosphate, Zm00001d042264 and Zm00001d005151 encoding AK (EC: 2.7.2.4) were up-regulated at 28 DAP. In the process of converting L-aspartyl-4-phosphate to L-aspartate-semialdehyde, nine DEGs encoding ASD (EC: 1.2.1.11) were detected. Among them, Zm00001d042275 was up-regulated at 18 and 28 DAP; Zm00001d034765 and Zm00001d009968 were up-regulated at 28 and 38 DAP. Zm00001d002602 encoding DapA (EC: 4.3.3.7) was down-regulated in the process of converting L-aspartate-semialdehyde to (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate. During the process of (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate to (S)-2,3,4,5-tetrahydrodipicolinate, Zm00001d047935 and Zm00001d049956, which encode DapB (EC: 1.17.1.8), were down-regulated at 18 DAP. Three DEGs encoding DapF (EC: 5.1.1.7) were detected in the process of converting (S)-2,3,4,5-tetrahydrodipicolinate to meso-diaminopimetlate. Among them, Zm00001d023842 and Zm00001d030677 were up-regulated at 18 DAP, and Zm00001d046371 was up-regulated at 18 and 38 DAP. In the process of converting meso-diaminopimetlate to L-lysine, Zm00001d000153 and Zm00001d023429 encoding DD (EC: 4.1.1.20) were up-regulated at 38 DAP.
In the lysine degradation pathway, the lkrsdh1 (Zm00001d052079) which encodes SDH (EC: 1.5.1.8, 1.5.1.9), was down-regulated at 18 DAP and up-regulated at 28 DAP during the conversion from L-lysine to (S)-2-aminoe-6-oxohexanoate. Zm00001d021241, which encodes L-PO (EC: 1.5.3.7), was down-regulated in the three periods and inhibited lysine degradation. During the conversion of (S)-2-aminoe-6-oxohexanoate to L-2-aminoadipate, Zm00001d050495, which encodes ALDH7A1 (EC: 1.2.1.31), was down-regulated at 18 DAP and inhibited lysine degradation. During the conversion of L-2-aminoadipate to glutaryl-CoA, Zm00001d021889, which encodes OGDH (EC: 1.2.4.2), was down-regulated at 18 DAP. Zm00001d031212, which encodes ACAT (EC: 2.3.1.9), was down-regulated at 28 DAP during the process of glutaryl-CoA to acetyl-CoA.

3.7. DEGs in Tryptophan Metabolism

DEGs were annotated into the KEGG database, and the results showed that, in the tryptophan biosynthesis pathway, 7, 5, and 2 DEGs were detected in the three periods, where 9 were up-regulated and 5 were down-regulated; in the lysine degradation pathway, 21, 5, and 6 DEGs were detected in the three periods, where 19 were up-regulated and 13 down-regulated (Figure 8).
Zm00001d034713, which encodes AS (EC: 4.1.3.27), was up-regulated in the three periods during the conversion process chorismiteate to anthranilate in the tryptophan synthesis pathway. In the conversion process of anthranilate to N-(5-Phosphoribosyl)-anthranilate, Zm00001d030185 and Zm00001d021606, which encode APT (EC: 2.4.2.18), were down-regulated at 18 DAP and up-regulated at 38 DAP, respectively. In the bidirectional process of converting N-(5-Phosphoribosyl)-anthranilate to 1-(o-Carboxyp henylamino)-1′-deoxyribulose-5′-phosphate, Zm00001d053347, which encodes PAI (EC: 5.3.1.24), was down-regulated at 18 DAP. Five DEGs encoding TS (EC: 4.2.1.20) were detected during the conversion of (3-indoyl)-glycerolphosphate to indole and the co-synthesis of tryptophan with L-serine. Among them, Zm00001d046676 was up-regulated at 18 and 28 DAP, Zm00001d024702 and Zm00001d049610 were up-regulated at 18 DAP, and Zm00001d018969 and Zm00001d024702 were down-regulated at 18 DAP. During the process of tryptophan to indolepyruvate in the tryptophan degradation pathway, seven DEGs encoding TAA (EC: 2.6.1.99) were detected. Among them, Zm00001d008708 and Zm00001d012728 were down-regulated at 18 DAP, Zm00001d037674 was down-regulated at 28 DAP, and Zm00001d043651 was down-regulated at 38 DAP and Zm00001d037498 was up-regulated at 18 and 38 DAP. In the conversion process of indole-3-ethidium to (indol-3-yl) acetate, eight DEGs encoding ALDH (EC: 1.2.1.3) were detected. Among them, Zm00001d050495 and Zm00001d022554 were down-regulated at 18 DAP.

3.8. qRT-PCR Validation

Twenty-five key DEGs were selected for qRT-PCR verification during the three periods. The results indicated that the qRT-PCR was consistent with RNA-seq results (Figure 9A–C). At the three periods, the determination coefficient r2 values between the RNA-seq and qRT-PCR analyses’ relative expression levels were 0.8580, 0.7431, and 0.8193, respectively (Figure 9D–F), indicating the reliability of RNA-seq.

4. Discussion

In this study, the o16 and wx genes of maize were backcrossed into common maize using MAS technology, and the o16 and wx double-recessive gene mutation line QCL8012_2 was obtained. The levels of tryptophan and lysine increased by 11.39% and 50.58%, respectively, compared with their levels in common maize. An SNP (55K) chip analysis showed that the recovery rate of the inherited background of QCL8012_2 based on molecular markers was 94.69%. A transcriptome sequencing analysis indicated 27 and 34 DEGs were detected in tryptophan metabolism and lysine metabolism, respectively, during the development period of maize kernel (18, 28, and 38 DAP). This provides a reference for elucidating the mechanism of the o16 and wx gene insertions in common maize to increase the levels of tryptophan and lysine in maize grains. In maize, the protein content of the grains is mainly divided into gliadin, glutelin, globulin, and albumin, and the content of gliadin is the highest. Additionally, zein is rich in glutamic acid and proline, and the content of lysine and tryptophan is quite low, as well as the amino acid composition is unbalanced. The total amount of protein in maize grains is fixed. When the content of gliadin decreases, the content of other proteins increases, and the nutritional quality of maize is improved. Therefore, reducing the content of zein in maize grains through genetic regulation represents the main way for breeders to improve protein quality. Hartings et al. [22] found that the transcription levels of 19 kDa and 22 kDa α-zein and 10, 27, and 50 kDa γ-zein in the endosperm of maize, an o2o7 mutant, were decreased. Wang et al. [38] detected 16, 15, and 14 genes encoding α-zein at 18, 28, and 38 DAP of a maize o2o16 grain after pollination, respectively. All these genes were down-regulated, reducing zein synthesis and increasing lysine levels. Zhou et al. found that the introduction of an o2 gene into waxy maize lines reduced the accumulation of various zein types, except for 27 kDa γ-zein. In this study, a total of 18 DEGs encoding α-zein were detected in the three periods (Table S8). Except for Zm00001d019160 and Zm00001d019162, which were up-regulated in the three periods, the rest were down-regulated at different periods, reducing the synthesis of α-zein and resulting in an increase in lysine content. In addition, 16 genes encoding α-zein, which were initially detected by Wang et al., were all detected in this study.
In the lysine metabolism process, AK (EC: 2.7.2.4) is the first key enzyme [39,40]. Brennecke et al. found that AK was up-regulated in maize’s o2 mutant and promoted the synthesis of lysine [41]. In this study, Zm00001d042264 and Zm00001d005151 encoding AK were up-regulated at 28 DAP, and Zm00001d050133, Zm00001d050134, and Zm00001d021142 genes were up-regulated at 38 DAP, which promoted the process of L-aspartate to L-4-aspartyl phosphate. ASD (EC: 1.2.1.11) is the second key enzyme in the aspartate metabolic pathway, which catalyzes the process of L-4-aspartyl phosphate to L-asparte-4-semialdehyde. A total of nine DEGs encoding ASD were detected in this study. Among them, Zm00001d042275 was up-regulated at 18, 28, and 38 DAP. In addition, Zm00001d046371, Zm00001d023842, and Zm00001d030677, encoding DapF (EC: 5.1.1.7) were up-regulated at 18 DAP during the conversion process of L,L-2,6-diaminopimelate to meso-diaminopimetlate. These changes promoted the synthesis of lysine. LKR/SDH is a bifunctional enzyme that is regulated by the o2 gene and involved in lysine catabolism [42]. Lkrsdh1 (Zm00001d052079), which encodes LKR/SDH, was down-regulated in the o2 mutant’s endosperm [43]. In this study, Zm00001d052079, which encodes LKR/SDH, was down-regulated at 18 DAP during lysine’s conversion to 2-aminoadipate-6-semialdehyde. Simultaneously, Zm00001d050495, which encodes ALDH7A1 (EC: 1.2.1.31), was down-regulated at 18 DAP.
In the tryptophan synthesis process, AS is the first key regulatory enzyme for tryptophan synthesis. In this study, Zm00001d034713, which encodes AS, was up-regulated in the three periods for the conversion process chorismiteate to anthranilate. Wang et al. showed that after the o2o16 gene was inserted into waxy maize, the gene Zm00001d030185 encoding APT was significantly up-regulated at 18 DAP during endosperm development [29], which promoted tryptophan synthesis. In this study, five DEGs encoding TS were detected in the conversion process of (3-indoyl)-glycerophosphate to tryptophan. Among them, Zm00001d046676 was up-regulated at 18 DAP and 28 DAP, and Zm00001d024702 and Zm00001d049610 were up-regulated at 18 DAP. These changes promoted the synthesis of tryptophan. In the tryptophan degradation pathway, the TAA of the aminotransferases family is first converted to indole-3-pyruvate (IPA), and then indole-3-acetic acid (IAA) is synthesized by IPA through the IPMO flavonoid monooxygenases family. The primary mechanism of auxin synthesis, which is the conversion of tryptophan to IAA, is crucial for several developmental processes [44,45]. In this study, seven DEGs encoding TAA were detected, which catalyzed the conversion of tryptophan to indole pyruvic acid. Zm00001d037674 was down-regulated at 28 DAP, Zm00001d012728, and Zm00001d008708 were down-regulated at 18 DAP, and Zm00001d043651 was down-regulated at 38 DAP. Several enzymes involved in the metabolism of aldehydes and responsible for converting aldehydes into their corresponding carboxylic acids make up the aldehyde dehydrogenase (ALDH) super-family [46]. Eight DEGs encoding ALDH (EC: 1.2.1.3) were detected in the process of indole-3-ethidium to (indol-3-yl) acetate. Among them, Zm00001d050495 and Zm00001d022554 were down-regulated at 18 DAP. It is worth noting that Zm00001d050495 was down-regulated during both lysine degradation and tryptophan degradation, which increased the contents of lysine and tryptophan.
In this study, in addition to significant increases in lysine and tryptophan of the o16wx line, significant decreases in glutamic acid, cysteine, leucine, and isoleucine were also found. Further analysis of the metabolic pathways of these four amino acids revealed that 1 and 2 were down-regulated in glutamate anabolism. Further analysis of the metabolic pathways of these four amino acids revealed that: Zm00001d038948 and Zm00001d038948 were down-regulated in the glutamate synthesis pathway; Zm00001d040235 and Zm00001d040235 were down-regulated in cysteine anabolism; Zm00001d040477 and Zm00001d043156 were up-regulated in leucine degradation metabolism; Zm00001d051600 and Zm00001d009564 were down-regulated in isoleucine anabolism.

5. Conclusions

This study used MAS to introduce the o16 and wx alleles into CML530 and obtained a double-recessive mutation, QCL8012_2 (o16wx). Compared with the recurrent parent, the lysine and tryptophan contents of QCL8012_2 were increased by 50.58% and 11.39%. An RNA-seq analysis of QCL8012_2 and CML530 grains (18, 28, and 38 DAP) showed 34 and 27 DEGs were detected in lysine metabolism and tryptophan metabolism, respectively. Further analysis showed that the genes encoding ASD, AK, and Dap F were up-regulated, and the genes encoding ALDH7A1 and LKR/SDH were down-regulated in the lysine metabolism pathway. These changes increased lysine content. At the same time, the genes encoding AS and TS were up-regulated, and the genes encoding TAA and ALDH were down-regulated in the tryptophan metabolism pathway. These changes increased the tryptophan content. In addition to changes in the amino acid metabolic pathway, we also found that the down-regulated expression of 16 genes related to α-zein synthesis inhibited the synthesis of zein in the endosperm, thereby improving the nutritional quality of maize. This is of great significance to explain the molecular mechanism of o16 and wx gene accumulation to improve the nutritional quality of maize at the transcriptional level, as well as for the development of biomarkers and gene editing.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture13091791/s1, Figure S1: The number of identified genes in each sample; Figure S2: The coefficient of repeated correlation between each sample; Table S1: Twenty-five DEGs validated by quantitative real-time PCR analysis; Table S2: Amino acid content of CML530 and QCL8012-2 endosperm; Table S3: Amino acid content of CML530 and QCL8012-2 endosperm; Table S4: Alignment statistics of reads aligned to the reference gene; Table S5: Alignment statistics of reads aligned to the reference genome B73v4; Table S6: QC20 and QC30 of RNA sequencing data for all samples; Table S7: FPKM of all gene for all samples; Table S8: Eighteen distinct zein genes in QCL8012_2 compared to CML530 at 18 DAP, 28 DAP, and 38 DAP.

Author Contributions

Z.M. and P.W. conducted most of the experiments, analyzed the data, and prepared the manuscript; W.Y., H.R. and W.W. provided materials and financial support; W.W. conceived and designed this study; L.D. and K.Z. contributed analytical tools and provided technical support; L.S. provided revisions to the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32060461), the National Maize Industry Technology System (No. CARS-02-091), Guizhou Provincial Financial Seeds Industry Development Project (2023) and the Guizhou Provincial Science and Technology Plan Project (Nos. Qianke Zhongyindi (2022) 4011; Qiankehe Service Enterprise (2022) 007; Qiankehe Support (2020) 1Y050).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The datasets supporting the conclusions of this article are included within the article and its additional files. The datasets used and/or analyzed during the current study are available from the authors on reasonable request (Zhoujie Ma, [email protected]; Wei Wang, [email protected]).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Beadle, G.W.; Fraser, A.C. A Summary of Linkage Studies in Maize; Memoir Cornell University: Ithaca, NY, USA, 1935. [Google Scholar]
  2. Mertz, E.T.; Bates, L.S.; Nelson, O.E. Mutant Gene That Changes Protein Composition and Increases Lysine Content of Maize Endosperm. Science 1964, 145, 279–280. [Google Scholar] [CrossRef]
  3. Hartings, H.; Lazzaroni, N.; Rossi, V.; Riboldi, G.R.; Thompson, R.D.; Salamini, F.; Motto, M. Molecular Analysis of Opaque-2 Alleles from Zea Mays L. Reveals the Nature of Mutational Events and the Presence of a Hypervariable Region in the 5′ Part of the Gene. Genet. Res. 1995, 65, 11–19. [Google Scholar] [CrossRef] [PubMed]
  4. Mains, E.B. Heritable Characters in Maize; Linkage of a Factor for Shrunken Endosperm with the A1 Factor for Aleurone Color. J. Hered. 1949, 40, 21–24. [Google Scholar] [CrossRef] [PubMed]
  5. Nelson, O.E.; Mertz, E.T.; Bates, L.S. Second Mutant Gene Affecting the Amino Acid Pattern of Maize Endosperm Proteins. Science 1965, 150, 1469–1470. [Google Scholar] [CrossRef]
  6. Ma, Y.; Nelson, O. Aminoacid Composition and Storage Proteins in Two New High-Lysine Mutants in Maize. Cereal Chem. 1975, 52, 412–419. [Google Scholar]
  7. Robertson, D.S. A New Opaque Gene Located on Chromosome 7. Maize Genet. Coop. Newsl. 1967, 41, 94–95. [Google Scholar]
  8. Ashman, R.B. Gene Linkages in Translocation T9-10a Heterozygotes. Maize Genet. Coop. Newsl. 1968, 42, 155–156. [Google Scholar]
  9. McWhirter, K.S. A Floury Endosperm, High Lysine Locus on Chromosome 10. Maize Genet. Coop. Newsl. 1971, 45, 184. [Google Scholar]
  10. Pirona, R.; Hartings, H.; Lauria, M.; Rossi, V.; Motto, M. Genetic Control of Endosperm Development and of Storage Products Accumulation in Maize Seeds. Maydica 2005, 50, 515. [Google Scholar]
  11. Nelson, O. The Mutations Opaque-9 through Opaque-13. Corn Genet. Coop. Newsl. 1981, 55, 68. [Google Scholar]
  12. Soave, C.; Tardani, L.; Di Fonzo, N.; Salamini, F. Zein Level in Maize Endosperm Depends on a Protein under Control of the Opaque-2 and Opaque-6 Loci. Cell 1981, 27, 403–410. [Google Scholar] [CrossRef] [PubMed]
  13. Tian, Q.; Wang, G.; Ma, X.; Shen, Q.; Ding, M.; Yang, X.; Luo, X.; Li, R.; Wang, Z.; Wang, X.; et al. Riboflavin Integrates Cellular Energetics and Cell Cycle to Regulate Maize Seed Development. Plant Biotechnol. J. 2022, 20, 1487–1501. [Google Scholar] [CrossRef] [PubMed]
  14. Holding, D.R.; Otegui, M.S.; Li, B.; Meeley, R.B.; Dam, T.; Hunter, B.G.; Jung, R.; Larkins, B.A. The Maize Floury1 Gene Encodes a Novel Endoplasmic Reticulum Protein Involved in Zein Protein Body Formation. Plant Cell 2007, 19, 2569–2582. [Google Scholar] [CrossRef]
  15. Li, Q.; Wang, J.; Ye, J.; Zheng, X.; Xiang, X.; Li, C.; Fu, M.; Wang, Q.; Zhang, Z.; Wu, Y. The Maize Imprinted Gene Floury3 Encodes a Platz Protein Required for tRNA and 5S rRNA Transcription through Interaction with RNA Polymerase III. Plant Cell 2017, 29, 2661–2675. [Google Scholar] [CrossRef] [PubMed]
  16. Myers, A.M.; James, M.G.; Lin, Q.; Yi, G.; Stinard, P.S.; Hennen-Bierwagen, T.A.; Becraft, P.W. Maize Opaque5 Encodes Monogalactosyldiacylglycerol Synthase and Specifically Affects Galactolipids Necessary for Amyloplast and Chloroplast Function. Plant Cell 2011, 23, 2331–2347. [Google Scholar] [CrossRef]
  17. Wang, G.; Zhang, J.; Wang, G.; Fan, X.; Sun, X.; Qin, H.; Xu, N.; Zhong, M.; Qiao, Z.; Tang, Y. Proline Responding1 Plays a Critical Role in Regulating General Protein Synthesis and the Cell Cycle in Maize. Plant Cell 2014, 26, 2582–2600. [Google Scholar] [CrossRef]
  18. PLOS Genetics Editors. Retraction: Maize Opaque10 Encodes a Cereal-Specific Protein that Is Essential for the Proper Distribution of Zeins in Endosperm Protein Bodies; Public Library of Science: San Francisco, CA, USA, 2022.
  19. Yang, W. Mapping of a High Lysine Mutant Gene in Maize and Analysis of O2 Endosperm Hetero and Rogenesis and O2 Locus Allelic Variation. Ph.D. Thesis, University of Huazhong Agricultural, Wuhan, China, 2005. [Google Scholar]
  20. Yang, W.; Zheng, Y.; Zheng, W.; Feng, R. Molecular Genetic Mapping of a High-Lysine Mutant Gene (Opaque-16) and the Double Recessive Effect with Opaque-2 in Maize. Mol. Breed. 2005, 15, 257–269. [Google Scholar] [CrossRef]
  21. Zhang, W.-L.; Yang, W.-P.; Chen, Z.-W.; Wang, M.-C.; Yang, L.-Q.; Cai, Y.-L. Molecular Marker-Assisted Selection for O2 Introgression Lines with O16 Gene in Corn. Acta Agron. Sin. 2010, 36, 1302–1309. [Google Scholar] [CrossRef]
  22. Hartings, H.; Lauria, M.; Lazzaroni, N.; Pirona, R.; Motto, M. The Zea Mays Mutants Opaque-2 and Opaque-7 Disclose Extensive Changes in Endosperm Metabolism as Revealed by Protein, Amino Acid, and Transcriptome-Wide Analyses. BMC Genom. 2011, 12, 1–18. [Google Scholar] [CrossRef]
  23. Sarika, K.; Hossain, F.; Muthusamy, V.; Zunjare, R.U.; Baveja, A.; Goswami, R.; Bhat, J.S.; Saha, S.; Gupta, H.S. Marker-Assisted Pyramiding of Opaque2 and Novel Opaque16 Genes for Further Enrichment of Lysine and Tryptophan in Sub-Tropical Maize. Plant Sci. 2018, 272, 142–152. [Google Scholar] [CrossRef]
  24. Chand, G.; Zunjare, R.U.; Allen, T.; Muthusamy, V.; Mishra, S.J.; Kasana, R.K.; Bhatt, V.; Ismail, M.R.; Sarika, K.; Guleria, S.K. Genetic Analysis of Maize Genotypes Possessing Novel Combination of Opaque2 and Opaque16 Genes Affecting Higher Accumulation of Lysine and Tryptophan in Kernels. J. Cereal Sci. 2022, 107, 103534. [Google Scholar] [CrossRef]
  25. Weatherwax, P. A Rare Carbohydrate in Waxy Maize. Genetics 1922, 7, 568. [Google Scholar] [CrossRef] [PubMed]
  26. Lu, D.; Lu, W. Effects of Protein Removal on the Physicochemical Properties of Waxy Maize Flours. Starch-Stärke 2012, 64, 874–881. [Google Scholar] [CrossRef]
  27. Zhou, Z.; Song, L.; Zhang, X.; Li, X.; Yan, N.; Xia, R.; Zhu, H.; Weng, J.; Hao, Z.; Zhang, D. Introgression of Opaque2 into Waxy Maize Causes Extensive Biochemical and Proteomic Changes in Endosperm. PLoS ONE 2016, 11, e0158971. [Google Scholar] [CrossRef]
  28. Yang, L.; Wang, W.; Yang, W.; Wang, M. Marker-Assisted Selection for Pyramiding the Waxy and Opaque-16 Genes in Maize Using Cross and Backcross Schemes. Mol. Breed. 2013, 31, 767–775. [Google Scholar] [CrossRef]
  29. Wang, W.; Niu, S.; Dai, Y.; Wang, M.; Li, Y.; Yang, W.; Zhao, D. The Zea Mays Mutants Opaque2 and Opaque 16 Disclose Lysine Change in Waxy Maize as Revealed by RNA-Seq. Sci. Rep. 2019, 9, 12265. [Google Scholar] [CrossRef]
  30. Hossain, F.; Prasanna, B.M.; Kumar, R.; Singh, B.B. The Effect of Genotype X Pollination Mode Interaction on Kernel Modification in Quality Protein Maize (QPM) Genotypes. Indian J. Genet. Plant Breed. 2008, 68, 132–138. [Google Scholar]
  31. GB 7650-1987; Determination of Tryptophan in Cereal Grains. National Standards of the People’s Republic of China: Beijing, China, 1987.
  32. Chen, Y.; Chen, Y.; Shi, C.; Huang, Z.; Zhang, Y.; Li, S.; Li, Y.; Ye, J.; Yu, C.; Li, Z.; et al. Soapnuke: A Mapreduce Acceleration-Supported Software for Integrated Quality Control and Preprocessing of High-Throughput Sequencing Data. Gigascience 2018, 7, 1–6. [Google Scholar] [CrossRef]
  33. Kim, D.; Langmead, B.; Salzberg, S.L. Hisat: A Fast Spliced Aligner with Low Memory Requirements. Nat. Methods 2015, 12, 357–360. [Google Scholar] [CrossRef]
  34. Langmead, B.; Salzberg, S.L. Fast Gapped-Read Alignment with Bowtie 2. Nat. Methods 2012, 9, 357–359. [Google Scholar] [CrossRef]
  35. Li, B.; Dewey, C.N. Rsem: Accurate Transcript Quantification from RNA-Seq Data with or without a Reference Genome. BMC Bioinform. 2011, 12, 323. [Google Scholar] [CrossRef] [PubMed]
  36. Love, M.I.; Huber, W.; Anders, S. Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with Deseq2. Genome Biol. 2014, 15, 1–21. [Google Scholar] [CrossRef]
  37. Kanehisa, M.; Araki, M.; Goto, S.; Hattori, M.; Hirakawa, M.; Itoh, M.; Katayama, T.; Kawashima, S.; Okuda, S.; Tokimatsu, T.; et al. Kegg for Linking Genomes to Life and the Environment. Nucleic Acids Res. 2007, 36, D480–D484. [Google Scholar] [CrossRef]
  38. Wang, W.; Dai, Y.; Wang, M.; Yang, W.; Zhao, D. Transcriptome Dynamics of Double Recessive Mutant, o2o2o16o16, Reveals the Transcriptional Mechanisms in the Increase of Its Lysine and Tryptophan Content in Maize. Genes 2019, 10, 316. [Google Scholar] [CrossRef] [PubMed]
  39. Rinder, J.; Casazza, A.P.; Hoefgen, R.; Hesse, H. Regulation of Aspartate-Derived Amino Acid Homeostasis in Potato Plants (Solanum Tuberosum L.) by Expression of E. Coli Homoserine Kinase. Amino Acids 2008, 34, 213–222. [Google Scholar] [CrossRef] [PubMed]
  40. Lo, C.C.; Bonner, C.A.; Xie, G.; D’Souza, M.; Jensen, R.A. Cohesion Group Approach for Evolutionary Analysis of Aspartokinase, an Enzyme That Feeds a Branched Network of Many Biochemical Pathways. Microbiol. Mol. Biol. Rev. 2009, 73, 594–651. [Google Scholar] [CrossRef]
  41. Brennecke, K.; Neto, A.J.S.; Lugli, J.; Lea, P.J.; Azevedo, R.A. Aspartate Kinase in the Maize Mutants Ask1-Lt19 and Opaque-2. Phytochemistry 1996, 41, 707–712. [Google Scholar] [CrossRef]
  42. Kemper, E.L.; Neto, G.C.; Papes, F.; Moraes, K.C.M.; Leite, A.; Arruda, P. The Role of Opaque2 in the Control of Lysine-Degrading Activities in Developing Maize Endosperm. Plant Cell 1999, 11, 1981–1993. [Google Scholar] [CrossRef]
  43. Li, C.; Qiao, Z.; Qi, W.; Wang, Q.; Yuan, Y.; Yang, X.; Tang, Y.; Mei, B.; Lv, Y.; Zhao, H.; et al. Genome-Wide Characterization of Cis-Acting DNA Targets Reveals the Transcriptional Regulatory Framework of Opaque2 in Maize. Plant Cell 2015, 27, 532–545. [Google Scholar] [CrossRef]
  44. Zhao, Y. Auxin Biosynthesis: A Simple Two-Step Pathway Converts Tryptophan to Indole-3-Acetic Acid in Plants. Mol. Plant 2012, 5, 334–338. [Google Scholar] [CrossRef]
  45. Tao, Y.; Ferrer, J.L.; Ljung, K.; Pojer, F.; Hong, F.; Long, J.A.; Li, L.; Moreno, J.E.; Bowman, M.E.; Ivans, L.J.; et al. Rapid Synthesis of Auxin Via a New Tryptophan-Dependent Pathway Is Required for Shade Avoidance in Plants. Cell 2008, 133, 164–176. [Google Scholar] [CrossRef] [PubMed]
  46. Brocker, C.; Vasiliou, M.; Carpenter, S.; Carpenter, C.; Zhang, Y.; Wang, X.; Kotchoni, S.O.; Wood, A.J.; Kirch, H.H.; Kopečný, D.; et al. Aldehyde Dehydrogenase (ALDH) Superfamily in Plants: Gene Nomenclature and Comparative Genomics. Planta 2013, 237, 189–210. [Google Scholar] [CrossRef] [PubMed]
Agriculture 13 01791 g001
Figure 1. (A) Photographs of intact ears; (B) Photographs of mature kernels in natural light; (C) Photographs of mature kernels in transmitted light; (D) Histochemical test for starch staining photos of mature kernels; (E) Scanning electron micrograph for endosperms at 1500× magnification, Bars = 30 μm. Note: SG is starch granules, and the blue arrow indicates a matrix protein.
Figure 1. (A) Photographs of intact ears; (B) Photographs of mature kernels in natural light; (C) Photographs of mature kernels in transmitted light; (D) Histochemical test for starch staining photos of mature kernels; (E) Scanning electron micrograph for endosperms at 1500× magnification, Bars = 30 μm. Note: SG is starch granules, and the blue arrow indicates a matrix protein.
Agriculture 13 01791 g001
Agriculture 13 01791 g002
Figure 2. Light box testing of CML530 and QCL8012-2 seeds.
Figure 2. Light box testing of CML530 and QCL8012-2 seeds.
Agriculture 13 01791 g002
Agriculture 13 01791 g003
Figure 3. The contents of eighteen FAAs in mature grains of CML530 and QCL8012_2. The “*” indicates that the difference is significant.
Figure 3. The contents of eighteen FAAs in mature grains of CML530 and QCL8012_2. The “*” indicates that the difference is significant.
Agriculture 13 01791 g003
Agriculture 13 01791 g004
Figure 4. MA-plot distribution of DEGs in QCL8012_2 vs. CML530 at 18 (A), 28 (B), and 38 DAP (C); (D) Venn diagram of DEGs in CML530 vs. QCL8012_2 at 18, 28, and 38 DAP. Note: The X-axis represents the average expression level after log2 conversion, and the Y-axis represents the fold change after log2 conversion.
Figure 4. MA-plot distribution of DEGs in QCL8012_2 vs. CML530 at 18 (A), 28 (B), and 38 DAP (C); (D) Venn diagram of DEGs in CML530 vs. QCL8012_2 at 18, 28, and 38 DAP. Note: The X-axis represents the average expression level after log2 conversion, and the Y-axis represents the fold change after log2 conversion.
Agriculture 13 01791 g004
Agriculture 13 01791 g005
Figure 5. The Go functional annotation of DEGs in QCL8012_2 vs. CML530 at 18 (A), 28 (B), and 38 DAP (C); X-axis represents GO terms. Y-axis means number of DEGs.
Figure 5. The Go functional annotation of DEGs in QCL8012_2 vs. CML530 at 18 (A), 28 (B), and 38 DAP (C); X-axis represents GO terms. Y-axis means number of DEGs.
Agriculture 13 01791 g005
Agriculture 13 01791 g006
Figure 6. The KEGG pathway enrichment results of DEGs in QCL8012_2 vs. CML530 at 18 (A), 28 (B), and 38 DAP (C); X-axis means rich factor, and Y-axis represents second KEGG pathway terms.
Figure 6. The KEGG pathway enrichment results of DEGs in QCL8012_2 vs. CML530 at 18 (A), 28 (B), and 38 DAP (C); X-axis means rich factor, and Y-axis represents second KEGG pathway terms.
Agriculture 13 01791 g006
Agriculture 13 01791 g007
Figure 7. The DEGs in lysine metabolism. Notes: In the figure, red and blue represent up-regulated expression and down-regulated expression, respectively. Color depth represents RNA expression level, and rows and columns on the heat map represent genes and periods (18, 28, and 38 DAP). Identified enzymes include DapB−4-hydroxy tetrahydrodipicolinate reductase; DapA−4-hydroxy-tetrahydrodipicolinate synthase; DapF−diaminopimelate epimerase; AK−aspartate kinase; DD−diaminopimelate decarboxylase; ASD−aspartate-semialdehyde dehydrogenase; ALDH7A1−aldehyde dehydrogenase family 7 member A1; SDH−saccharopine dehydrogenase; L-PO−L-pipecolate oxidase; ACAT−acetyl-CoA C-acetyltransferase; OGDH−2-oxoglutarate dehydrogenase.
Figure 7. The DEGs in lysine metabolism. Notes: In the figure, red and blue represent up-regulated expression and down-regulated expression, respectively. Color depth represents RNA expression level, and rows and columns on the heat map represent genes and periods (18, 28, and 38 DAP). Identified enzymes include DapB−4-hydroxy tetrahydrodipicolinate reductase; DapA−4-hydroxy-tetrahydrodipicolinate synthase; DapF−diaminopimelate epimerase; AK−aspartate kinase; DD−diaminopimelate decarboxylase; ASD−aspartate-semialdehyde dehydrogenase; ALDH7A1−aldehyde dehydrogenase family 7 member A1; SDH−saccharopine dehydrogenase; L-PO−L-pipecolate oxidase; ACAT−acetyl-CoA C-acetyltransferase; OGDH−2-oxoglutarate dehydrogenase.
Agriculture 13 01791 g007
Agriculture 13 01791 g008
Figure 8. The DEGs in tryptophan metabolism. Notes: In the figure, red and blue represent up-regulated expression and down-regulated expression, respectively. Color depth represents RNA expression level, and rows and columns on the heat map represent genes and periods (18, 28, and 38 DAP). Identified enzymes include AS—anthranilate synthase; APT−anthranilate phosphoribosyltransferase; PAI−phosphoribosylanthranilate isomerase; TS−tryptophan synthase; ALDH−aldehyde dehydrogenase; IAO−indole-3-acetaldehyde oxidase; TAA−L-tryptophan-pyruvate aminotransferase.
Figure 8. The DEGs in tryptophan metabolism. Notes: In the figure, red and blue represent up-regulated expression and down-regulated expression, respectively. Color depth represents RNA expression level, and rows and columns on the heat map represent genes and periods (18, 28, and 38 DAP). Identified enzymes include AS—anthranilate synthase; APT−anthranilate phosphoribosyltransferase; PAI−phosphoribosylanthranilate isomerase; TS−tryptophan synthase; ALDH−aldehyde dehydrogenase; IAO−indole-3-acetaldehyde oxidase; TAA−L-tryptophan-pyruvate aminotransferase.
Agriculture 13 01791 g008
Agriculture 13 01791 g009
Figure 9. The qRT−PCR verification of DEGs. Histogram of DEGs in o16wx and wild-type endosperm at 18 (A), 28 (B), and 38 DAP (C). The scatter plot of DEGs in o16wx and wild-type endosperm at 18 (D), 28 (E), and 38 DAP (F).
Figure 9. The qRT−PCR verification of DEGs. Histogram of DEGs in o16wx and wild-type endosperm at 18 (A), 28 (B), and 38 DAP (C). The scatter plot of DEGs in o16wx and wild-type endosperm at 18 (D), 28 (E), and 38 DAP (F).
Agriculture 13 01791 g009
Table 1. Average degree of opaqueness (%) in CML530 and QCL8012-2 seeds.
Table 1. Average degree of opaqueness (%) in CML530 and QCL8012-2 seeds.
PopulationsGenotypesOpaqueness
0%25%50%75%100%Average (%)
CML530wt10000000
QCL8012_2o16o16wxwx0520433275.5
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

Ma, Z.; Wu, P.; Deng, L.; Zhang, K.; Yang, W.; Ren, H.; Song, L.; Wang, W. RNA-Seq Revealed the Molecular Mechanism of Nutritional Quality Improvement in o16-wx Double-Mutation Maize. Agriculture 2023, 13, 1791. https://doi.org/10.3390/agriculture13091791

AMA Style

Ma Z, Wu P, Deng L, Zhang K, Yang W, Ren H, Song L, Wang W. RNA-Seq Revealed the Molecular Mechanism of Nutritional Quality Improvement in o16-wx Double-Mutation Maize. Agriculture. 2023; 13(9):1791. https://doi.org/10.3390/agriculture13091791

Chicago/Turabian Style

Ma, Zhoujie, Peizhen Wu, Lei Deng, Kaiwu Zhang, Wenpeng Yang, Hong Ren, Li Song, and Wei Wang. 2023. "RNA-Seq Revealed the Molecular Mechanism of Nutritional Quality Improvement in o16-wx Double-Mutation Maize" Agriculture 13, no. 9: 1791. https://doi.org/10.3390/agriculture13091791

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