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. 2023 Oct 26;43(11):77. doi: 10.1007/s11032-023-01424-x

The molybdenum cofactor biosynthesis gene, OsCNX1, is essential for seedling development and seed germination in rice

Xiaoguang Lu 1,#, Di Zhang 1,#, Yi Zhang 1, Xing Liu 1, Sheng Wang 1, Xin Liu 1,✉
PMCID: PMC10616024  PMID: 37916037

Abstract

Pre-harvest sprouting (PHS) frequently occurs in rice due to the long spells of rainy weather, and causes severe yield loss and grain quality decrease. Here, we identified one PHS-related gene OsCNX1 cloned from rice PHS mutant, which encoded a molybdenum cofactor (MoCo) biosynthesis enzyme. Genetic complementation indicated OsCNX1 could rescue the PHS and seedling lethal phenotype of the mutant. Expression pattern showed that OsCNX1 was expressed in rice tissue including seedling shoot, culm, blade, and sheath of flag leaf, young panicle, and the seeds at different development stages. Overexpression of OsCNX1 significantly decreased the plant height, and the seed germination of the dormant seeds harvested from fresh panicles, comparing to the wild type (WT). In addition, 1492 differentially expressed genes (DEGs) were identified between OsCNX1-overexpressed line and WT by RNA-sequencing, which were mainly classified in plant-pathogen interaction, plant hormone signal transduction, and starch/sucrose metabolism. These results showed that OsCNX1 was not only necessary for rice seed germination, but also participated in plant development, indicating that OsCNX1 may be useful in rice breeding of PHS resistance and plant height.

Supplementary information

The online version contains supplementary material available at 10.1007/s11032-023-01424-x.

Keywords: Pre-harvest sprouting (PHS), OsCNX1, Molybdenum cofactor (MoCo), Seed germination, Seedling development

Introduction

Pre-harvest sprouting (PHS) or vivipary, which refers to the phenomenon that the seeds germinate on the spike before harvest, led to a significant decline in the yield and quality of rice (Wang et al. 2020b; Nakamura 2018; Vetch et al. 2019; Moullet et al. 2022). In 2023, many provinces such as Henan in China experienced prolonged rainfall during the wheat harvest period, leading to extensive sprouting of wheat, greatly affecting the yield and quality of wheat and posing a serious threat to the food security. Many factors affect pre-harvest sprouting, including seed coat color, grain structure, seed dormancy characteristics, endogenous phytohormones, and environmental factors (Finkelstein et al. 2008; Zhao et al. 2022a). More than 140 quantitative trait loci (QTL) or genes, which were associated with PHS or seed dormancy, have been identified in rice (Sohn et al. 2021; Zhao et al. 2022a). Previous studies showed that ABA played an important role in plant growth and development, especially in primary dormancy and seed germination , and the genes involving ABA biosynthesis and ABA metabolic signal pathway, which affected crop pre-harvest sprouting, have been most and deeply studied (Koornneef et al. 2002; Gubler et al. 2005; Leng et al. 2014; Verma et al. 2016). Generally, increased expression of ABA biosynthesis genes has been shown to enhance seed dormancy, whereas increased ABA catabolism releases seed dormancy in rice.

PHS9 which encoded a higher plant unique CC-type glutaredoxin could interact with OsGAP, which was an interaction partner of the abscisic acid (ABA) receptor OsRCAR1, and acted as a negative regulator in ABA signaling involving in rice seed dormancy (Xu et al. 2019). 9-cis-Epoxycarotenoid dioxygenase (NCED) was a crucial enzyme for ABA synthesis, and the mutation of the gene caused PHS in rice (Fang and Chu 2008; Chen et al. 2023). Mutation of OsNCED3, which was a member of 9-cis-epoxycarotenoid dioxygenase family, enhanced rice PHS due to the breaking of seed dormancy before seed maturity, and overexpression of OsNCED3 enhanced PHS resistance by regulating proper ABA/GA ratio in rice embryo (Chen et al. 2023). Seed Dormancy 6 (SD6) which encoded a basic helix-loop-helix transcription factor interacted with C-repeat binding factors expression 2 (ICE2) and antagonistically balanced the expression of the ABA catabolism gene ABA8OX3 and OsNCED2 to regulate rice seed dormancy (Xu et al. 2022). OsCNX6 encoded a molybdenum cofactor (MoCo) synthesis gene, which involved in ABA biosynthesis, played a significant role in rice seed development, and the OsCNX6 mutation caused twisting and slender leaves, and lethal phenotype (four or five leaves stage), and the plants with heterozygous genotype (OsCNX6/cnx6) caused PHS phenotype in the progeny seeds (Liu et al. 2019). qSD1-2 was proposed to control primary dormancy by a GA-regulated dehydration, and loss-of-function mutations of the gibberellin (GA) synthesis gene enhanced seed dormancy (Ye et al. 2015). The mutation of Viviparous1 (OsVP1), which was orthologs gene of ABA Insensitive3 (ABI3) in Arabidopsis, caused PHS, and OsVP1 could interact with Rc and OsC1 to increase ABA sensitivity and PHS resistance (Wang et al. 2020a). Seed Dormancy 4 (Sdr4), which acted as a downstream target of OsVP1, integrated the abscisic acid and gibberellic acid signaling pathways at the transcriptional level and functioned as central modulator of dormancy in the seed maturation program (Sugimoto et al. 2010; Chen et al. 2021; Zhao et al. 2022a). In addition, gene mutation in sugar metabolism also affected pre-harvest sprouting. Mutation in PHS8 which encoded a starch debranching enzyme resulted in the phytoglycogen breakdown and sugar accumulation in the endosperm and exhibited PHS phenotype (Du et al. 2018). Although these genes have been cloned and functionally validated, few of them were widely used in rice PHS resistance breeding practices. Therefore, identifying more genes related to PHS has great significance for rice breeding.

In the previous mutant screening, we obtained a rice pre-harvest sprouting mutant and located the candidate gene OsCNX1 through map-based cloning, which encodes the molybdenum cofactor synthesis gene (Liu et al. 2019). In this study, we proved that the mutation of OsCNX1 is the cause of PHS and lethal phenotype in the mutant through genetic complementary transformation. Furthermore, overexpression of OsCNX1 significantly reduced plant height and seed germination of dormant seeds. Thus, these results indicated that OsCNX1 involved the plant development and seed dormancy, and genetic engineering of OsCNX1 had potential application value in rice PHS-resistant and height breeding.

Materials and methods

Plant materials and growth conditions

Oryza sativa L. ssp. japonica cv Nipponbare plants were used as wild type (WT) and planted in the experimental field with shading outdoors (17:00–08:00 h) from elongation stage to the heading stage in Harbin (45°700N, 126°600E), China. For the phenotype study, WT, heterozygous mutant plants and transgenic plants were grown in a growth chamber with controlled short day (10-h light/14-h darkness) or controlled long day (16-h light/8-h darkness). The seedlings were cultured with Yoshida’s culture solution, and the osmotic and salt stress treatments were according to our previous studies (Liu et al. 2019). Fifty mature and dormant seeds collected from fresh panicles at 25 days after pollination (DAP) were placed on filter papers with water, and incubated at 27°C for 84 h. Germination was scored at the first germination stage (S1) when radicle or coleoptile was visually ≥ 1 mm (Counce et al. 2000). Days to heading of each plant were recorded when the first panicle emerged from main stem.

Vector constructions and rice transformation

To construct expression vector, the 1356-bp full length coding sequence of OsCNX1 (Os04g0661600) was cloned into pC1390, digesting with KpnI and BamHI restriction enzymes by ClonExpress® technology (Vazyme Biotech Co., Ltd), in which the target gene was driven by the Ubiquitin promoter. The construct was introduced into cnx1 heterozygous mutant plants (cv. Nipponbare background) to develop transgenic lines by agrobacterium (EHA105)-mediated transformation as described previously (Zhao et al. 2022b). All primers used for vector construction and selecting transgenic lines were listed in Supplementary Table S1.

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis

Total RNA was extracted using RNeasy Plant Mini Kit (QIAGEN, Germany) according to the manufacturer’s protocol. First-strand cDNA was synthesized using HiScript III RT SuperMix for qPCR (Vazyme, China) following the kit manual. qRT-PCR was performed using a Bio-Rad CFX Connect (USA) system with three technical replicates, as previously described (Zhang et al. 2012). The rice Actin gene (Os03g0718100) was used as internal control to calculate the relative expression of genes via 2-ΔΔCT method (Livak and Schmittgen 2001). The gene-specific primers used for qRT-PCR were listed in Supplementary Table S1.

RNA-sequencing

Seedling shoots of OsCNX1OE-01 and WT at the four-leaf stage were collected with three biological replications for performing transcriptome sequencing. The library preparation, transcriptome sequencing, and raw data analysis were conducted by Biomarker Technologies Corporation. The raw datasets were aligned to the rice Nipponbare reference genome (https://rapdb.dna.affrc.go.jp/download/irgsp1.html) using BMKCloud online platform (http://www.biocloud.net). Differentially expressed genes (DEGs) (FDR < 0.01 and fold change ≥ 2) were analyzed using the DESeq2 (Love et al. 2014). DEGs were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) datasets (Kanehisa et al. 2012). KOBAS software was used to assay the statistical enrichment in KEGG pathways (Mao et al. 2005). Gene Ontology (GO) enrichment analysis was implemented by GOseq R package–based Wallenius non-central hyper-geometric distribution (Young et al. 2010).

Statistical analysis

All numerical data were presented as mean ± SE calculated from three biological replicates. All P values were based on a two-tailed t-test. P < 0.05 (*) and P < 0.01 (**) were considered as statistically significant using SPSS software (https://www.ibm.com/cn-zh/spss).

Results

Functional complementation of PHS mutant

By previous large-scale screening for PHS mutants, we isolated one PHS mutant (Fig. 1a), and approximately 25% seedling plants of the mutant progenies showed twisting or slender leaves (Fig. 1b) and then presented lethal phenotypes when the seedling developed four to five leaves. Further study showed that a candidate gene OsCNX1 (Os04g0661600), which was disrupted by a retrotransposon Karma DNA insertion in the PHS mutant, was identified underlying the PHS locus by map-based cloning method (Liu et al. 2019). For further identification, an expression vector carrying CDS of OsCNX1 under the control of constitutive the Ubiquitin promoter and HYGROMYCIN B PHOSPHOTRANSFERASE (HPT) selectable marker gene was transformed into cnx1 seeds (Fig. 2a). In addition, specific selection primers, Primer-F and Primer-R, were designed for screening resultant plants with a homozygous cnx1 mutant locus (Fig. 2a). As shown in Fig. 2b, eight positive transgenic lines were obtained by HPT selection, and three candidate complementary lines (OsCNX1/cnx1-04, OsCNX1/cnx1-06, and OsCNX1/cnx1-07) were screened in the T0 generation. The three lines presented normal development and seed setting rate compared with WT (Fig. 2c). To confirm the results, we observed progeny plants of three selected lines and found that all the HPT positive seedling progeny plants developed well and showed little differences with WT (Fig. 2d), indicating that the introduced OsCNX1 could complement the twisting leaf and seedling lethal phenotypes of the cnx1 mutant.

Fig. 1.

Fig. 1

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Phenotype of pre-harvest sprouting (PHS) mutant. a Sprouting phenotype of freshly collected panicles (35 days after pollination) after 4-day seed imbibition. b The seedling phenotype of PHS mutant. Scale bars, 5 cm

Fig. 2.

Fig. 2

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Complementation of cnx1 mutant. a DNA fragment used for complementation. LB, left border; RB, Right border. b Screening for OsCNX1 complementary plants in T0 generation (line 01–08). c Seedling phenotypes of complementary lines in T0 generation. d Seedling phenotypes of complementary lines in T1 generation. HPT, HYGROMYCIN B PHOSPHOTRANSFERASE. Scale bars, 5 cm

Phenotypes of OsCNX1-overexpressed lines

Except the three complementary lines, the other five transgenic positive lines (line 01, 02, 03, 05, and 08) were obtained (Fig. 2b). We further examined the transcript of OsCNX1 in these transgenic positive lines. The results showed that the transcript abundance of OsCNX1 in all five lines was significantly higher than WT, and the line-01 and line-03 were selected for further study, in which the transcript levels were increased more than 80-fold and 40-fold than WT, respectively (Fig. 3a). Then, we observed that the seedling plants of OsCNX1-overexpressed lines (OsCNX1OE-01 and OsCNX1OE-03) were significantly shorter than WT (Fig. 3b). As OsCNX1 mutant caused PHS, we also examined the seed germination of two OsCNX1-overexpressed lines and found that the seed germination rates of OsCNX1OE-01 and OsCNX1OE-03 were 32% and 23%, respectively, which were significantly lower than 75% in WT (Fig. 3c, d), indicating that OsCNX1 participated in seed development, and may be useful in crop breeding of PHS resistance.

Fig. 3.

Fig. 3

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Phenotypes of OsCNX1-overexpressed plants. a Expression analysis of OsCNX1-overexpressed lines (OsCNX1OE-01 and OsCNX1OE-03) detected by qRT-PCR. b Seedling height of OsCNX1-overexpressed lines. c d Seed germination of OsCNX1-overexpressed lines. Values are means ± SEM with three replicates. Scale bars, 5 cm

Expression pattern of OsCNX1 in rice

To verify the expression pattern of OsCNX1 in rice, the OsCNX1 transcripts were monitored by qRT-PCR in rice samples including seedling shoot, culm, blade, and sheath of flag leaf, young panicle, and the seeds at different development stages. The results showed that OsCNX1 transcripts were detected in all surveyed tissues (Fig. 4) and were abundantly expressed in blade, but weakly expressed in sheath. In addition, OsCNX1 was also expressed during the seed development stages, especially in the seeds of 5, 15, and 20 DAP, and the expression level increased with seed filling (Fig. 4). These results indicated that OsCNX1 probably participated in many plant development pathways, including plant growth and seed development.

Fig. 4.

Fig. 4

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qRT-PCR analysis of OsCNX1 in different tissues of wild type

Transcriptome profiling of OsCNX1-overexpressed plants

To survey the regulation pathway of OsCNX1, the RNA-sequencing experiment was performed to profile the transcriptome changes between OsCNX1-overespressed plant and WT. The data quality assessment of RNA-seq was shown in Supplementary Fig. S1a. In addition, eight genes randomly selected from transcriptome data were assayed using qRT-PCR to determine the reliability of RNA-seq and showed that the expression mode of the genes was consistent with transcriptome results (Supplementary Fig. S1b), indicating that the RNA-seq data was reliable. Finally, 1492 DEGs were identified between OsCNX1OE-01 and WT in 20-day-old seedling shoots, including 894 up-regulated and 598 down-regulated genes. Several metabolite pathways in KEGG terms were identified including plant-pathogen interaction, plant hormone signal transduction, and starch/sucrose metabolism (Fig. 5a). Plant hormone was closely related to plant development (Santner et al. 2009; Oliva et al. 2013). Given the differences of seedling phenotype between OsCNX1-overexpressed plants and WT, we screened transcriptome data and found 14 obvious DEGs involving in plant hormone signal transduction pathway (Supplementary Table S2). qRT-PCR results further verified the expression trends of these DEGs (Fig. 5b). The above results indicated that OsCNX1 overexpression probably affected plant growth and development during the seedling stage.

Fig. 5.

Fig. 5

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Identification of the differentially expressed genes (DEGs). a KEGG enrichment analysis of DEGs. b Expression analysis of DEGs in plant hormone signal transduction by qRT-PCR. Gene annotations within parentheses were predicted by The Rice Annotation Project (RAP) (https://rapdb.dna.affrc.go.jp/)

Discussion

In previous studies, MoCo was identified as a sort of essential factor for plant and seed development (Schwarz and Mendel 2006; Mendel and Hänsch 2002; Liu et al. 2019). Our previous study proved that OsCNX6, which encoded a MoCo synthesis gene, was crucial for seed development and plant development, and OsCNX6-overexpressed plants showed no obvious phenotype differences including heading date and plant height (Liu et al. 2019). In this study, the mutation of OsCNX1, which also involved in the MoCo biosynthesis, caused PHS and severe growth defect (Fig. 1b). Furthermore, OsCNX1 overexpression in rice decreased the plant height during the seedling and adult stage (Fig. 3b; Supplementary Fig. S2), indicating that OsCNX1 may have potential value in rice height breeding, though its possibility remains to be explored by further effort.

Rice was a short-day (SD) model plant, of which flowering was promoted under SD condition, and suppressed under long-day (LD) condition (Tsuji et al. 2011; Li et al. 2022). Intriguingly, we observed that two OsCNX1-overexpressed lines (Nipponbare background) flowered under LD treatment, though Nipponbare (WT), a typical SD variety, was still in vegetative growth stage under LD treatment with solution culture (Supplementary Fig. S2a). In addition, the heading date of OsCNX1-overexpressed plants was also reduced comparing to WT under SD conditions (Supplementary Fig. S2b). Then, we screened the DEGs in RNA-Seq data and found that seven genes which were annotated as flowering-regulated genes by Swiss-Prot (http://www.uniprot.org/) differentially expressed between OsCNX1-overexpressed line and WT (Supplementary Table S2). These results indicated that that the OsCNX1 not only participated in plant development, but also involved in rice flowering pathway under both SD and LD conditions. However, additional effort is required to elucidate the phenomenon and detailed interactions between OsCNX1 and these genes involving in the process of rice flowering.

OsCNX6 overexpression in rice increased the osmotic and salt stress compared with WT (Liu et al. 2019). Given the similar biosynthesis pathway between OsCNX1 and OsCNX6, we also performed the stress treatment to OsCNX1-overexpressed plants，including 15% polyethylene glycol and 150 mmol NaCl (Supplementary Fig. S3). Unexpectedly, no significant differences were observed between OsCNX1-overexpressed plants and WT. The above results indicated that OsCNX1 was essential for plant growth and seed development, but probably involved in the different pathways of regulating plant development compared with OsCNX6. We also found that OsCNX1 was reliably expressed in the developing seeds (Fig. 4), and OsCNX1-overexpressed plants significantly decreased the seed germination rate compared with WT (Fig. 3c, d), indicating that OsCNX1 was necessary for seed development, and probably involved in seed germination. The results also showed that the regulation of OsCNX1 expression and further evaluation of natural variations in OsCNX1 may be helpful in breeding PHS-resistant varieties.

To explore the phenotypic correlation with genes expression which involved in plant growth and development, we comprehensively analyzed transcriptome data and screened 1492 DEGs including several metabolite pathways (Fig. 5a). Unexpectedly, the significant network with the most DEGs was plant-pathogen interaction by KEGG analysis, though a serious of DEGs were screened in both Plant hormone signal transduction, and Starch/sucrose metabolism, which involved in plant growth and development (Fig. 5a, b). The result indicated that it would be valuable to further confirm interactions between OsCNX1 and these genes involving in plant-pathogen interaction and explore the detailed plant-pathogen regulation networks for possible disease resistance breeding in the future.

Supplementary information

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Acknowledgements

We are grateful to Prof. Jun Fang (Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences) for supporting the early research and to Dr. Guangxin Zhao (Jilin Agricultural University) for providing helpful discussions and advisement.

Author contribution

XL and DZ performed most of the experiments; YZ, XL, and SW provided technical assistance. XL designed the research, performed parts of the experiments, and wrote the manuscript. All authors approved the final manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (31901524) and by the Heilongjiang Provincial Natural Science Foundation of China (YQ2022C008).

Data availability

All data are included in this published article and its supplementary information files (Supplementary Table S1, S2; Fig. S1, S2, S3).

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xiaoguang Lu and Di Zhang contributed equally to this work.

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Associated Data

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Supplementary Materials

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Data Availability Statement

All data are included in this published article and its supplementary information files (Supplementary Table S1, S2; Fig. S1, S2, S3).

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