Overexpression of OsMed16 inhibits rice growth and causes spontaneous cell death

The Mediator complex transduces information from the DNA-bound transcription factors to the RNA polymerase II transcriptional machinery. Research on plant Mediator subunits was mainly performed in Arabidopsis, while very few of them have been functionally characterized in rice. Here the rice Mediator subunit 16, OsMed16, was studied. OsMed16 encoded a putative protein of 1301 amino acids, which is longer than the reported version. It was expressed in various rice organs, and localized in nucleus. Knockout of OsMed16 caused rice seedling lethality. Its overexpression led to rice growth retardation, low yield, and spontaneous cell death in leaf blade and leaf sheath. RNA sequencing suggested that overexpression of OsMed16 altered the expression of a large number of genes. Among them, the up-regulation of some defense-related genes was veried.

. Biochemical identi cation of Mediator complex in plants was very late. The rst plant Mediator complex was puri ed from Arabidopsis cell suspension culture in 2007 (Bäckström et al. 2007).
In addition to multiple Mediator subunits, Pol II subunits were also isolated in the puri ed Arabidopsis Mediator fraction, but the Kinase module subunits (Med12, Med13, CDK8, and CycC) were not isolated together with the bulk complex. Furthermore, the Kinase module subunits were identi ed by bioinformatics approaches (Bäckström et al. 2007). Now it is usually thought that the Arabidopsis Mediator complex comprises 33 subunits, among which 29 subunits are conserved with yeast or animal counterparts, and 4 subunits are unique to plants (Yang et al. 2016; Zhai and Li 2019). To date, Mediator subunits in other plants have not yet been biochemically identi ed, but were characterized by bioinformatics analysis. Mathur et al. (2011) in silico identi ed Mediator subunits in 16 plant species from algae to higher angiosperms. It is found that at least one homolog for all the animal/fungal Mediator subunits is present in the plant kingdom (Mathur et al. 2011). In addition to in silico analysis, the biological functions of some Arabidopsis Mediator subunits have been studied through genetic and molecular analysis. It is found that these Mediator subunits participate in multiple biological processes, including plant growth, development, owering, pathogen defense and stress tolerance (Elfving et  Rice is an important staple crop, and also used as a monocot model plant. 55 Mediator genes, including paralogs of some main module subunits and Kinase module subunits, have been identi ed in the whole rice genome by in silico approaches (Mathur et al. 2011). However, unlike that in Arabidopsis, very few rice Mediator subunits have been functionally characterized. OsMed15a and OsMed14-1 are the two wellstudied Mediator subunits in rice. OsMED15a is implicated in rice seed development through linking rice grain size/weight-regulating TFs to their target genes. Reduction in OsMed15a expression (RNAi plants) down-regulated the expression of genes associated with grain size/weight, GW2, GW5 and DR11, and reduced grain length, weight, and yield (Dwivedi et al. 2019). OsMed14-1 plays an important role in rice development. RNAi-mediated repression of OsMed14-1 expression led to growth inhibition and slender organs, which was caused by defective cell-cycle progression and reduced auxin level in OsMed14-1 knockdown plants (Malik et al. 2020).
OsMed16 (OsSFR6) is a homolog of AtSFR6, and its function was preliminarily studied in Arabidopsis (Wathugala et al. 2011). The atsfr6 mutant showed freezing sensitivity, pale cotyledons and leaves. Overexpression of OsMed16 in atsfr6 mutant could restore the wild-type phenotype and elevate freezing and osmotic tolerance (Wathugala et al. 2011). Moreover, the expression of COLD-ON REGULATED (COR) genes could also be restored in atsfr6 mutant overexpressing OsMed16, so OsMed16 is thought to act as a regulator of COR gene expression, osmotic stress and freezing tolerance in Arabidopsis (Wathugala et al. 2011). However, the biological function of OsMed16 remains unclear in rice. In this study, the expression pattern and function of OsMed16 was investigated in rice. The results revealed that the knockout mutant osmed16 exhibited severe growth inhibition, and were unable to complete the life cycle. Overexpression of OsMed16 also led to growth inhibition, low yield and spontaneous cell death. RNA-seq data indicated that overexpression of OsMed16 altered the expression of a large number of genes involved in multiple biological processes. In particular, alterations of some defense related genes were further examined.

Result 1 Sequence and Phylogenic Analysis of OsMed16
Owing to its high homology to AtSRF6 (AtMed16), the rice gene LOC_Os10g35560 was previously named OsSRF6 (Wathugala et al. 2011). However, as a subunit of the Mediator complex, LOC_Os10g35560 should be named OsMed16 according to the common uni ed nomenclature for Mediator subunits (Bourbon et al. 2004). In Wathugala's studies, OsSFR6 (OsMed16) was predicted to encode a protein of 1170 amino acids. When searching in GenBank (National Center for Biotechnology Information, NCBI) and Rice Genome Annotation Project database, we found the ORF of OsMed16 was 3906 bp in length, and thus encoded a putative protein with 1301 amino acid residues, which is 131-aa longer than OsSRF6 reported by Wathugala et al. (2011). To test this, the full-length ORF of OsMed16 (3906 bp) was ampli ed from the model japonica rice variety Nipponbare by high-delity PCR, and veri ed by sequencing. Subsequently, the gene structure of OsMed16 was analyzed, which contains 16 exons and 15 introns (Fig. 1a).
To understand the evolutionary relationship of OsMed16, its counterparts were obtained from different plant species, including algae, mosses, ferns, gymnosperms and angiosperms. Then sequence alignment and phylogenetic analysis were performed. On the whole, the phylogenetic tree is organized into two major clades. The Med16 subunits from unicellular algae (CrMed16, VcMed16, GpMed16) were grouped in one clade and shared less than 15% identity with OsMed16 (Fig. 1b); The Med16 subunits from other plant species were grouped in another clade and shared higher identity with OsMed16 (Fig. 1b). Among the sequences retrieved from NCBI database, OsMed16 displays the highest percentage of identity with ObMed16 from Oryza brachyantha (96%), and has 69% identity with AtMed16.

OsMed16 mRNA Expression Pattern and Protein Subcellular Localization
Quantitative real-time PCR (qRT-PCR) assays were performed with total RNA isolated from rice root, leaf, stem, leaf sheath and young panicle. The results showed that OsMed16 mRNA was expressed in all the examined organs, which have similar expression level except a little lower in leaf sheath (Fig. 2a). Furthermore, public microarray databases (eFP browser) indicated that OsMed16 was also expressed in in orescence and seed (Additional le 1 Fig. S1) (Winter et al. 2007). The wide expression pattern of OsMed16 is consistent with its function as a basic transcriptional regulator.
To determine the subcellular localization of OsMed16, a p35S-OsMed16-GFP construct was generated and transiently expressed in rice protoplasts with a red uorescent protein (RFP) fused to OsGhd7, a nucleus-localized protein (Xue et al. 2008). The p35S-GFP empty vector was used as a control. As a result, the green uorescence signal in the control was observed in cytoplasm, while OsMed16-GFP uorescence was present in the nucleus, co-localized with the OsGhd7-RFP protein (Fig. 2b). These results indicated that OsMed16 is localized in the nucleus, which was in agreement with its role as a Mediator subunit.

Overexpression of OsMed16 Caused Rice Growth Inhibition and Spontaneous Cell Death
To investigate the function of OsMed16 in planta, it was disrupted using CRISPR/Cas9 genome-editing technology (Additional le 1 Fig. S2a). The osmed16 mutants exhibited a stunted growth phenotype, failed to head, and died prematurely (Additional le 1 Fig. S2b), indicating that disruption of OsMed16 caused rice seedling lethality.
We further employed gain-of-function approach to investigate the roles of OsMed16. OsMed16 overexpression vector driven by CaMV 35S promoter was constructed and transformed into Nipponbare via an Agrobacterium-mediated method. The expression level of OsMed16 in transgenic plants was detected using qRT-PCR assay, and two representative homozygous transgenic lines with high expression level of OsMed16 (named OsMed16-OE) were used for further investigation (Additional le 1 Fig. S3). Unexpectedly, overexpression of OsMed16 also inhibited rice growth. Compared with the wild-type, OsMed16-OE lines had a dwarf phenotype with fewer tillers (Fig. 3d). Another distinct visible phenotype observed was spontaneous cell death in OsMed16-OE lines. At three leaves stage, small necrotic spots rst appeared on the leaf sheath of OsMed16-OE seedlings (Fig. 4a), and then were also observed on leaves (spotted leaf, Fig. 4b-c). As plants grew, the brown spots gradually became large irregular lesions ( Fig. 4b-c). The cell death was further con rmed by Trypan Blue staining. The OsMed16-OE leaves showed increased staining intensity compared to wild-type leaves ( Fig. 4d-f). Accumulation of reactive oxygen species may cause cell damage and even death (Khanna-Chopra. 2012). By DAB (3,3diaminobenzidine) staining, the over accumulation of H 2 O 2 was observed in the leaves of OsMed16-OE plants ( Fig. 4g-i). We also used NBT (nitroblue tetrazolium) staining and observed the increase of superoxide anion in OsMed16-OE plants ( Fig. 4j-l). With the increasing of number and size of lesions, the old leaves of OsMed16-OE lines withered prematurely, and the whole plants exhibited early senescence (Fig. 3b).

Overexpression of OsMed16 Reduced Rice Grain Yields
In addition to the growth inhibition, plants overexpressing OsMed16 exhibited signi cant yield reduction. Compared to wild-type plants, grain yield per plant was reduced by 91.8% and 91.3% in the two overexpression lines (Fig. 5a-b). The yield components were further analyzed. The panicle number per plant, panicle length, and 1,000-grain weight of OsMed16-OE plants decreased signi cantly compared to the wild-type (Fig. 5c-e). Additionally, the seed length and width were also compared between the OsMed16-OE lines and the wild-type. The results showed that the seed length was unchanged (Additional le 1 Fig. S4a-b), but seed width decreased slightly in OsMed16-OE lines (Additional le 1 Fig. S4c-d).

Transcriptome Changes in OsMed16-OE Plants
To assess the in uence of OsMed16 overexpression on gene expression, OsMed16-OE plants exhibiting necrotic lesions were harvested, and RNA sequencing (RNA-Seq) was performed on wild-type and OsMed16-OE plants. Overall, we obtained 6 transcriptome data sets, each of which contains an average of about 50 million paired-end (PE) reads (Additional le 1 Fig. S5). The raw sequencing reads were rst trimmed and mapped to the rice reference genome using HISAT2. More than 96% reads could map to unique loci per sample (Additional le 1 Fig. S5). Differentially expressed genes (DEGs) were determined with stringent criteria: log 2 fold change ≥ 1 and P-value (false discovery rate, FDR) ≤ 0.05. Compared with the wild-type, 2402 DEGs were detected in OsMed16-OE plant leaves, of which 1419 were up-regulated (Additional le 2 Table.S1), whereas 983 were down-regulated (Additional le 2 Table. (39), and oxidoreductase activity (34) (Fig. 6a, b). Among these genes, OsAPX2 (LOC_Os07g49400) is an ascorbic acid peroxidase gene that plays an important role in the growth and development of rice by clearing reactive oxygen species to protect seedlings from abiotic stress (Zhang et al. 2013). CYP93G2 (LOC_Os06g01250) encodes the avanone 2-hydroxylase, which is not only a member of the cytochrome P450 gene but also the rst enzyme in its biosynthetic pathway (Du et al. 2010). Our results con rmed that the up-regulated genes and the down-regulated genes were indeed associated with multiple biological pathways in rice.
Overexpression of OsMed16 led to spontaneous cell death in rice, which resembled the hypersensitive response (HR) caused by pathogenic infection. This led us to speculate that overexpression of OsMed16 might trigger the expression of defense-related genes. Thus, we examined these genes in the RNA-seq data. Indeed, some defense-related genes, including PR1a and PR1b, were up-regulated in OsMed16-OE compared with the wild-type. To con rm these results, we further performed qRT-PCR to check the expression levels of eight defense-related genes in the OsMed16-OE and wild-type plants. The transcript levels of all these genes were elevated in OsMed16-OE plants (Fig. 7), suggesting that overexpression of OsMed16 did activate the expression of defense-related genes. OsMed16 may exist in a signaling pathway which was usually activated in the absence of pathogen infection, leading to hypersensitivity in OsMed16-overexpressing plants. The cell death phenotype and the activation of defense-related genes in OsMed16-overexpressing plants indicated that OsMed16 may play a positive regulatory role in programmed cell death (PCD) and resistance-related signaling pathways in plants. However, the mechanism of OsMed16 gene's positive regulation of PCD and defense signals is still unclear. The results of this study will provide a new perspective for the molecular regulation mechanism of Mediator complex in plant cell death and disease resistance signaling, especially in monocotyledons. The results of agronomic traits showed that the overexpression of OsMed16 seriously affected the growth and development of plants. Therefore, it is speculated that OsMed16 may be related to the overall physiology and morphology of plants. Further elucidating the mechanism of OsMed16 and its downstream genes will contribute to the understanding of the role of OsMed16 in regulating the plant cell death and defense mechanisms as well as the rice plant growth and development. . Meanwhile, in Arabidopsis thaliana, Med16 was not only proved to regulate the immune response, but also proved to be an important subunit in the tail module, because the whole tail module was missing after extraction of the mediator complex in atsrf6 mutants. Our experimental results showed that the OsMed16-overexpressing plants affected the various periods of rice growth and development, that is to say OsMed16 plays a crucial role in different developmental stages. However, the mechanisms by which OsMed16 regulates rice growth and development are still largely unknown. Future studies will be required to dissect these regulatory mechanisms.

Conclusion
In the present study, the rice Mediator subunit 16, OsMed16, was functionally characterized. It was expressed in various rice organs, and localized in nucleus. Loss of function of OsMed16 causes rice seedling lethality. Its overexpression led to rice growth inhibition, low yield and spontaneous cell death in leaf blade and leaf sheath. RNA-seq data suggested that overexpression of OsMed16 altered the expression of a large number of genes, including the defense-related genes. These results demonstrated that OsMed16 regulates not only rice growth and development, but also defense response.

Plant Materials and Growth Conditions
The WT rice (Oryza sativa cv. Nipponbare), the two knockout lines of OsMed16, and the two OsMed16 overexpression lines were used in this study. The seeds were soaked in deionized water for 2 days in an incubator at 28℃ under dark conditions. After germination, the seeds were grown either hydroponically or in paddy eld. For hydroponic culture, the seeds were rst grown in 0.5 mM CaCl 2 solution for 5-7 days.
Then the seedlings were transferred to a 4 L plastic pot containing 1/2 Kimura B solution (pH 5.6) ( Yamaji and Ma. 2007). The nutrient solution was changed with fresh solution every two days. The plants were grown in a greenhouse under natural light at 25℃-30℃. The paddy eld is located in the rice planting base of Guangxi University, Nanning city, Guangxi Province. Each experiment had at least three biological replicates.

Generation of Transgenic Plants
To create the knockout lines of OsMed16, the CRISPR/Cas9 genome targeting system was used. The pCRISPR-OsMed16 plasmids with OsMed16 speci c target sites were constructed as described previously (Ma et al. 2015). Brie y, speci c target sequences (ATGCCCTCGTGCATTACTGG and GTTGCTTTTGATCCCACTCG) within the OsMed16 gene were selected by a Blast search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the rice genome sequence. Then the two speci c sequences of OsMed16 gene were respectively introduced into the sgRNA expression box by overlapping PCR to produce pU6a-OsMed16-SgRNA and pU6b-OsMed16-SgRNA.
These fragments were cloned into pYLCRISPR/Cas9 Pubi to construct pCRISPR-OsMed16 by using the restricted connection reactions containing BsaI and T4 DNA ligase. The constructed plasmids were introduced into Agrobacterium tumefaciens EHA101 and transformed into wild-type Nipponbare rice. The mutants were screened by PCR using primer pairs anking the OsMed16-speci c target site, and the homozygous mutants were selected for further study and analysis.
Transgenic plants overexpressing OsMed16 gene (named OsMed16-OE) was obtained by Agrobacteriummediated transformation. Total RNA was extracted from Nipponbare using the TRIzol reagent kit (Life Technologies) and reverse transcribed with a Hiscript II Q RT SuperMix Kit (Vazyme). The resulting cDNA was used as template for PCR-ampli cation of the OsMed16 full length cDNA with 5'-AATTGGTACCATGACCTCTTCCTCCGCCCC-3' and 5'-AATTACGCGTTCAAACGACTTTCACCCATG-3' as primers. The full-length cDNA of OsMed16 was inserted into the pCAMBIA1300-Ubi vector carrying the maize Ubiquitin promoter and the terminator of the nopaline synthase gene. OsMed16 gene speci c primers (5'-CGATGGCAATTACACTGTGC-3' and 5'-TAGAAGGCCAGCAGCATCA-3') and were used to identify the positive transgenic plants. The relative expression levels of OsMed16 in transgenic plant leaves were determined by quantitative reverse transcription-PCR (qRT-PCR) as described below.

RNA Isolation and Gene Expression Analysis
To examine the expression pattern of OsMed16 gene, the root, leaf blade, leaf sheath and spike were sampled at the heading stage for extraction of total RNA. Total RNA (1 μg Table 1).

Subcellular Localization of OsMed16
To detect the subcellular localization of OsMed16, plasmid to express the OsMed16-GFP fusion protein was constructed. OsMed16 cDNA was ampli ed from the Nipponbare cDNA by PCR using the OsMed16 speci c primers 5'-CCGGAATTCATGACCTCTTCCTCCGCCCC-3' (EcoRI site in italic text) and 5'-CGGGGTACCCAACGACTTTCACCCATGTCC-3' (KpnI site in italic text). The ampli ed cDNA was cloned downstream of the green uorescent protein coding region in PYL322-GFP vector (Ma et al. 2018) to produce OsMed16-GFP vector.
The vectors expressing the nuclear marker OsGhd7-mcherry, the OsMed16-GFP fusion protein and GFP alone were all transduced into the protoplast of rice. Preparation of rice protoplasts and plasmid transformation have been described previously (Chang et al. 2015). After transformation, the cells were incubated at 28 °C in dark for 12-15 h, and images were taken using a confocal laser scanning microscope (TCS SP8; Leica Microsystems).

Histochemical Stain
Leaves from the OsMed16 overexpressing plants with obvious lesions mimics and the WT at the same growth stage were harvested for histochemical analysis. Dead cells were detected by trypan blue staining (Yin et al. 2000). H 2 O 2 accumulation was determined using DAB staining (Thordal-Christensen et al. 1997). The amount of reactive oxygen species in cells were determined using NBT staining (Qiao et al. 2010).

RNA-seq Data Analysis
Leaves from OsMed16-overexpressing plants showing spontaneous lesions and WT plants at the same developmental stage were collected for RNA-seq analysis. Puri cation and construction of the cDNA library were as described (Shen et al. 2014). The library was sequenced using the Illumina NovaSeq platform to generate raw reads, then low quality and adaptor reads were ltered to obtain clean reads for further research.
To identify statistically signi cant differentially expressed genes, the standard of log2 fold change ≥1 and false discovery rate (FDR) ≤0.05 were adopted. In order to obtain the GO term with signi cant gene enrichment, GO gene function annotation analysis was performed to obtain functional annotations, biological functions, and metabolic pathways of screened differential genes. GO (Gene Ontology; http://geneontology.org/) analysis of DEGs was conducted by hypergeometric tests, and each p-value indicates the enrichment of the corresponding category.

Availability of data and materials
All data supporting the conclusions of this article are provided within the article (and its additional les).
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Not applicable.  Asterisks indicate signi cant differences from the wild type (*P < 0.05; **P < 0.01 by Student's t-test).