The Mediator subunit OsMED15a is a transcriptional co-regulator of seed size/weight–modulating genes in rice

Highlights

• This study establishes OsMed15a as a novel grain size/weight QTL in rice.

• OsMed15a governs seed size by regulating the expression of GW2, GW5 and D11.

• KIX domain of OsMed15a interacts with the activation domains of OsNAC024 and OsNAC025 transcription factors.

• OsNAC024 binds to the NAC-binding elements present in the promoter of grain size/weight-associated genes GW2, GW5 and D11.

• OsMed15a is important for the transcriptional activity of OsNAC024 transcription factor.

Abstract

Although several transcription factors (TFs) that regulate seed size/weight in plants are known, the molecular landscape regulating this important trait is unclear. Here, we report that a Mediator subunit, OsMED15a, links rice grain size/weight-regulating TFs to their target genes. Expression analysis and high-resolution quantitative trait loci (QTL) mapping suggested that OsMED15a is involved in rice seed development. OsMED15a has an N-terminal, three-helical KIX domain. Two of these helices, α1 and α3, and three amino acids, 76LRC78, within OsMED15a helix α3 were important for its interaction with several proteins, including interactions with the transactivation domains of two NAC-type TFs, OsNAC024 and OsNAC025. Moreover, OsMED15a, OsNAC024, and OsNAC025 all exhibited increased expression during seed development, and we identified several grain size/weight-associated SNPs in these genes in 509 low- and high-grain-weight rice genotypes. RNAi-mediated repression of OsMED15a expression down-regulated the expression of the grain size/weight regulating genes GW2, GW5 and DR11 and reduced grain length, weight, and yield. Of note, both OsNAC024 and OsNAC025 bound to the promoters of these three genes. We conclude that the transactivation domains of OsNAC024 and OsNAC025 target the KIX domain of OsMED15a in the regulation of grain size/weight–associated genes such as GW2, GW5, and D11. We propose that the integrated molecular-genetics approach used here could help identify networks of functional alleles of other regulator and co-regulator genes and thereby inform efforts for marker-assisted introgression of useful alleles in rice crop improvement.

Introduction

Rice is a staple food for more than half of the world population and nearly 3.5 billion people depend on it for their daily calorific intake. According to few surveys, by 2030, rice production should be almost doubled to fulfil the demand of growing human population [1]. Total yield in rice is mainly determined by different traits like number of panicles per plant, number of grains per panicle and grain size/weight [2]. The grain weight trait is influenced by the size (length and width) of the grain. Among these traits, grain size/weight has been the preferred target for different breeding programs for enhancing the productivity in rice [3,4]. Different approaches including genome mapping, sequencing and functional genomics have delineated many genes that are important for grain size/weight trait [5]. However, at the molecular level, the mechanistic details of regulation of this complex trait of grain size/weight is not yet known.

The NAC (named after NAM (No Apical Meristem) ATAF1/2 (Arabidopsis thaliana ACTIVATION FACTOR 1/2) and CUC (CUP-SHAPED COTYLEDON 2) proteins) proteins form one of the largest plant-specific TF super families that expanded during the evolution of land plants. Till date, 117 NAC TFs have been identified in Arabidopsis whereas rice genome codes for at least 151 NAC TFs [[6], [7], [8]]. NAC TFs contain a conserved NAC domain, which is around 150 amino acids long, towards N-terminus and a transcription regulatory region (TRR) towards C-terminus [6,9]. Within NAC domain, there are five different subdomains for DNA binding activity and protein-protein interactions [6]. Presence of these domains makes NAC TFs versatile in terms of their function. They regulate various biological processes affecting growth and development of plants. For instance, NAC TFs have been implicated in Shoot Apical Meristem (SAM) formation and development, secondary cell wall biosynthesis, xylem vessel element formation and leaf senescence [[10], [11], [12], [13], [14], [15]]. Recently, on the basis of single nucleotide polymorphism (SNP) analysis and expression profile, three NAC TFs have been predicted to be associated with seed size of rice [16]. However, the mechanistic details of recruitment of transcriptional machinery by NAC TFs are largely unexplored.

Mediator is an important component of transcriptional machinery and is well conserved in all the eukaryotes. It is a large complex made up of 25 subunits in yeast. In metazoans and plants, the number of Mediator subunits is >30 [17,18]. These subunits are arranged into different modules; Head, Middle, Tail and Kinase [19,20]. Recently, we reported interaction map of Arabidopsis Mediator complex revealing the arrangement of subunits in it [21]. Position of few subunits within the complex is yet to be assigned. Head, Middle and Tail modules form the core part of the complex, and the Kinase module reversibly associates with the core part as and when required [19,20]. Usually, Head and Middle modules establish contact with RNA polymerase II and its associated proteins, whereas, the Tail module interacts with different transcription factors enabling Mediator complex function as a bridge to relay transcriptional signal from transcription factor to transcriptional machinery [22]. However, many reports have established important roles of Mediator in several other aspects of transcription including initiation, elongation, splicing, gene looping, and termination of transcription [17,23,24].

Plant Mediator complex was first purified from Arabidopsis cell suspension culture in 2007 [25]. Since then functions of several Mediator subunits have been studied mostly in Arabidopsis. Med25, which was identified as Phytochrome and Flowering Time 1 (PFT1), was found to be important for flowering [25]. MED25 is also involved in pathogen defense [26], drought and salt stress [27] and jasmonic acid signalling [28]. Till date, MED25 has been reported to interact with at least eleven transcription factors [27,28]. MED14 which is also known as STRUWWELPETER (SWP) was found to be important for shoot meristem development [29], cold response [30] and plant immunity [31]. Kinase module subunits, MED12, MED13 and CDK8 were found to regulate cell differentiation leading to proper development of Arabidopsis [[32], [33], [34]]. Floral transition and flower development were found to be regulated by different Mediator subunits including MED8, MED12, MED13, MED18, MED20, MED25 and CDK8 [26,[35], [36], [37], [38]]. On the other hand, Mediator subunits like MED8, MED12, MED13, MED25, MED31, and CDK8 could regulate root development [[39], [40], [41]]. Overall, Mediator complex functions as the key conversing point of multiple signalling pathways regulating expression of almost all the class II genes (the genes that are transcribed by RNA Pol II) affecting all the aspects of plant growth and development and immunity [18,42].

MED15, a subunit in the Tail module, has emerged as a very important subunit involved in transcriptional regulation of several processes in different organisms. MED15 was first discovered in yeast as Gal11p required for galactose metabolism [43]. The master regulator of galactose metabolism, Gal4p, interacts with Gal11p/MED15 to recruit transcriptional machinery on the target promoters [44]. In yeast, mammals and worms, MED15 has been found to be important for fatty acid beta-oxidation and lipid homeostasis [[45], [46], [47]]. In C. elegans, MED15 was shown to be involved in the maintenance of phospholipid saturation [48]. Inactivation of MED15 caused constitutive activation of unfolded protein response of endoplasmic reticulum (ER), suggesting its importance in ER homeostasis [48]. We and others have found critical and essential role played by MED15 in transcriptional regulation of multidrug resistance in fungi and animals [46,49,50]. Thus, MED15 is very important transcriptional co-regulator in fungi and metazoans.

MED15 harbors a KIX domain at its amino-terminal region [51]. KIX was first discovered in mouse cAMP-response element-binding protein (CREB)-binding protein (CBP) as kinase-inducible domain (KID)-interacting domain [52]. Because of its ability to interact with KID, it was called KID-interacting domain or KIX domain. KIX domain has three α helices arranged to form a hydrophobic groove that functions as a docking site for different activation domains including KID and transactivation domains (TADs) [51]. In most cases, nine amino acid transactivation domain (9AA TAD) of transcription factors has been found to interact with the KIX domain of MED15 to recruit Mediator and RNA Pol II transcriptional machinery on the promoter of target genes [47,49,53,54]. 9AA TAD is a domain found in a large superfamily of transcription factors represented by zinc-cluster transcription factors in yeast and by p53, NF-kB and VP16 in animals [55]. 9AA TADs are not well studied in plants.

In last five years, few reports have explained the importance of MED15 in plants. The role of MED15 was discovered in salicylic acid-mediated defense response in Arabidopsis but the mechanism of its involvement in it could not be deciphered [56]. In a very recent study, the role of AtMED15 was explained in lipid homeostasis [57]. The N-terminal region of AtMED15 was found to interact with the N-terminal part of plant-specific transcription factor Wrinkled 1 (WRI1), which activates glycolysis-related and fatty acid biosynthesis genes during embryogenesis [57]. Earlier, we reported the presence of two paralogs of MED15 in rice and found an increase in the expression level of OsMED15_1 (hereafter referred to as OsMED15a) in different stages of seed development indicating its possible role in the process [58]. In this study, we scanned 509 rice genotypes and found one G/A single nucleotide polymorphism (SNP) in OsMED15a gene that showed strong association with grain weight trait. We validated the SNP in a RIL mapping population generated by crossing of short grain ‘Sonasal’ and long grain ‘IR 64’. Indeed, reduction in OsMED15a expression (but not OsMED15b) in transgenic plants affected seed development, seed size and total yield, confirming the importance of OsMED15a in rice. To dissect the molecular mechanism behind this phenotype, we identified the proteins that interact with OsMED15a. A number of proteins including a couple of NAC TFs (OsNAC024 and OsNAC025) were found to interact with OsMED15a. Interestingly, all the interactions that we found involved KIX domain of OsMED15a. In-silico structure analysis of KIX domain of OsMED15a followed by experimental validation revealed the importance of three amino acids of α3 helix in protein-protein interactions. Further, SNP analysis of genes coding for OsMED15a-interacting proteins revealed strong association of OsNAC024 and OsNAC025 with rice grain weight trait highlighting the relevance of MED15-NAC interaction in rice seed development. This study reveals that activation domains of OsNAC024 and OsNAC025 TFs interact with the KIX domain of OsMED15a to regulate expression of seed size-regulating genes. We suggest that OsMED15a could be one of the potential candidates for the modification of size/weight trait of rice grain. However, further in-depth analysis is required to characterize all the genes and traits regulated and affected by this particular Mediator subunit.