Double knockout of OsWRKY36 and OsWRKY102 boosts lignification with altering culm morphology of rice
Introduction
Lignocellulose, which constitutes the secondary cell walls of vascular plants, is the most abundant renewable source of energy and materials on Earth. Lignocellulosic biomass has long been utilized primarily as fuels, woody materials, and paper feedstocks. Fuel use comprises approximately half of the world’s annual wood consumption [1]. However, a large part of wood biomass used as fuels is currently being exploited from natural forests mainly in developing countries, whereas utilization of lignocelluloses from planted forests remains limited for production of pulp and woody materials [2]. Breeding to increase the calorific value of plant biomass obtained from artificial plantation and agricultural lands may be a promising strategy for reducing deforestation and increasing the sustainable energy production.
Lignin, a major lignocellulosic component, contributes primarily to the increased heating value of biomass because of its higher carbon content compared with those of cell-wall polysaccharides [2]. Therefore, plant biomass rich in lignin may be suitable for producing energy-rich solid biofuels. In addition, increasing the lignin content of plants may be beneficial for their applications into valuable aromatic chemicals [[3], [4], [5], [6]], and may contribute to improved resistances against various biotic and abiotic stresses [[7], [8], [9], [10], [11]]. Taken together, plant breeding toward lignin enrichment is strategic for improving utilization characteristics of grass biomass for biorefinery applications.
Grass crops such as maize (Zea mays), wheat (Triticum aestivum), rice (Oryza sativa), sugarcane (Saccharum spp.) and Sorghum (Sorghum spp.) annually provide more than 2500 M tons of lignocelluloses as agricultural residue [12], which surpasses the world consumption of wood lignocelluloses (approximately 2000 M tons) [2]. In addition to these crops, grass biomass plants such as Erianthus spp., Miscanthus spp., Napier grass (Pennisetum purpureum), and switchgrass (Panicum virgatum) have also attracted attention as potent biomass feedstocks, especially because of their superior biomass productivity over tree species [[13], [14], [15]].
Recently, because of the importance of grass lignocelluloses, the structure and biosynthesis of grass lignins have been actively investigated. In angiosperms, i.e., in both eudicots and monocots including grasses, lignin incorporates mainly guaiacyl (G) and syringyl (S) aromatic units derived from the radical coupling of coniferyl and sinapyl alcohols, respectively, with much lower numbers of p-hydroxyphenyl (H) units from p-coumaryl alcohol [[16], [17], [18], [19]]. Although sharing the three typical lignin aromatic units with eudicots, grasses possess several unique lignin units such as γ-p-coumaroylated G and S units derived from polymerization of γ-p-coumaroylated monolignols [[20], [21], [22], [23]] and tricin (T) units derived from polymerization of a flavone tricin [24,25]. In addition, a minor amount of γ-feruloylated units derived from γ-feruloylated monolignols is present in grass lignins [26], although a large part of ferulate (FA) exists on arabinoxylans (typical grass hemicelluloses) [20,27].
Transcription factors involved in secondary cell wall formation have been extensively studied, mainly using model eudicots [[28], [29], [30], [31], [32]]. On the other hand, although fewer studies have been conducted on grass species than eudicots, information about transcriptional regulation on grass cell wall biosynthesis has been collected [33,34]. Lignin-enriched grass cell walls have been achieved using bioengineering techniques to manipulate the transcriptional regulation. For example, heterologous expression of the transcriptional activator gene Arabidopsis thaliana AtMYB61 enriched lignins, especially grass-specific lignins such as γ-p-coumaroylated and tricin lignins, in rice culm biomass [35]. In addition, overexpression of native MYB transcriptional activators in grasses, such as SbMYB60 in Sorghum bicolor [36,37] and PvMYB58/63 and PvMYB42/85 in switchgrass [38], augmented the lignin content of biomass. Furthermore, our recent work has demonstrated that targeted knockout of the transcriptional repressor gene OsMYB108 using CRISPR/Cas9 [Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated 9 (Cas9)] [39,40] significantly enriched grass-specific γ-p-coumaroylated and tricin lignins in rice biomass [41]. Such a knockout mutation may be achieved by non-transgenic approaches using chemical- or irradiation-induced mutagenesis.
Previously, loss-of-function of eudicot WRKY-family transcriptional repressors MtSTP and AtWRKY12 induced ectopic lignification in usually unlignified pith tissues of Medicago truncatula and Arabidopsis stems, respectively [42]. AtWRKY12 directly repressed genes encoding transcriptional activators for secondary cell wall formation [42]. Eudicot poplar (Populus trichocarpa) PtrWRKY19 [43] and grass Miscanthus lutarioriparius MlWRKY12 [44] have been isolated as putative orthologs of AtWRKY12 in earlier studies, where heterologous expressions of PtrWRKY19 and MlWRKY12 restored the ectopic lignification of Arabidopsis atwrky12 mutant plants. AtWRKY12 [42], PtrWRKY19 [43] and PvWRKY12 [38] were expressed particularly in usually unlignified tissues of Arabidopsis, poplar and switchgrass stems, respectively. However, transcripts of MlWRKY12 were detected mainly in sclerenchyma fibers of stem tissues [44]. Suppression of AtWRKY12 homologs with a dominant repression (WRKY-DR) technique induced ectopic lignification in eudicot alfalfa (Medicago sativa) and grass maize and switchgrass stems [45]. The WRKY-DR significantly increased cell wall thickness in pith tissues and decreased the relative thickness of culm relative to the central cavity of switchgrass [38].
However, loss-of-function of AtWRKY13, which belongs to the same WRKY subgroup as AtWRKY12, inhibited sclerenchyma development and decreased stem diameter of Arabidopsis [46]. AtWRKY13 acted as a transcriptional activator of secondary cell wall biosynthesis [46]. Thus, AtWRKY12 and AtWRKY13 oppositely regulate lignification and flowering time under short-day conditions [47] in Arabidopsis.
In the present study, we generated and characterized rice mutants deficient in OsWRKY36 and OsWRKY102, which encode protein homologs of AtWRKY12 and AtWRKY13, respectively, in the same WRKY subgroup. Interestingly, we found that lignin was relatively enriched in rice oswrky36 and oswrky102 cell walls, where ectopic lignification apparently did not occur. These phenotypes differed from those of Arabidopsis atwrky12 and atwrky13 mutant lines [42,46]. OsWRKY36/OsWRKY102-double-mutant lines, which were generated by crossing the OsWRKY36- and OsWRKY102-single-mutant lines, displayed increased lignin content in cell walls with altered culm morphology, like switchgrass with WRKY-DR [38]. Our data suggest that OsWRKY36 and OsWRKY102 are associated with repression of lignification in rice culm tissues. Our results provide new insight into distinct transcriptional regulation of grass cell wall formation and beneficial information for breeding grass biomass toward improved biorefinery.
Section snippets
Bioinformatics
Protein homologs that have the most recent shared families (MRSFs) with AtWRKY12 (At2g44745) [42] and AtWRKY13 (At4g39410) [46,47] were retrieved from Phytozome v12 [48] and multiple sequence alignment of the proteins was performed using ClustalW [49] with default parameters (Fig. S1). Miscanthus MlWRKY12 [44] was also included in the analysis. A phylogenetic tree of the proteins was constructed using the neighbor-joining method [50] and evolutionary analyses were conducted using MEGA7 [51].
Generation of transgenic rice deficient in OsWRKY36 and OsWRKY102
At the onset of this study, we performed phylogenetic analysis of amino acid sequences of protein homologs of AtWRKY12 [42] and AtWRKY13 [46,47] (Fig. 1). Grass WRKY clusters (A, B, and D) were clearly separated from the eudicot clusters (C and E). ZmWRKY (GRMZM2G123387_T01) and PvWRKY12, which have been reported as negative regulators for secondary cell wall formation in maize and switchgrass [38,45], and OsWRKY36 was included in the grass cluster B. This supports our notion that OsWRKY36 acts
Conclusions
Rice mutant lines deficient in OsWRKY36 and OsWRKY102 generated using CRISPR/Cas9 displayed distinct phenotypes from those of dicot mutants lack in MtSTP, AtWRKY12 and AtWRKY13. Although ectopic lignification was not observed, lignin content based on the thioglycolic acid assay was relatively increased in the OsWRKY36 and OsWRKY102 mutant cell walls, suggesting that OsWRKY36 and OsWRKY102 are involved in repression of rice lignification. The enriched lignins in the WRKY mutant lines displayed
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgements
We thank Dr. Seiichi Toki, Dr. Masaki Endo and Mr. Masafumi Mikami (National Agricultural and Food Organization) for providing the pZH_OsU6gRNA_MMCas9 vector. We also thank Ms. Kaori Kanazawa, Ms. Keiko Tsuchida, Ms. Kumiko Murata, Ms. Mayumi Inutsuka, Ms. Mayumi Shichi, Ms. Megumi Ozaki and Ms. Naoko Tsue (RISH, Kyoto University) for their assistance in rice cultivation and chemical analyses, and Dr. Hironori Kaji, Ms. Ayaka Maeno and Ms. Kyoko Yamada (ICR, Kyoto University) for their
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- 1
These authors contributed equally to this article.
- 2
Present address: Graduate School of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan.