Genomic dissection and expression analysis of stress-responsive genes in C₄ panicoid models, Setaria italica and Setaria viridis

Highlights

• Eighty three C₄ pathway-specific genes identified in Setaria italica and Setaria viridis.

• Segmental and tandem duplications resulted in expansion of these genes.

• Expression profiling was performed in response to stress and hormone treatments.

• Potential candidate genes were identified for further functional characterization.

Abstract

The study reports the identification and expression profiling of five major classes of C₄ pathway-specific genes, namely, carbonic anhydrase (CaH), phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), NADP-dependent malate dehydrogenase (MDH) and NADP-dependent malic enzyme (NADP-ME), in the model species, Setaria italica and Setaria viridis. A total of 42 and 41 genes were identified in S. italica and S. viridis, respectively. Further analysis revealed that segmental and tandem duplications have contributed to the expansion of these gene families. RNA-Seq derived expression profiles of the gene family members showed their differential expression pattern in tissues and dehydration stress. Comparative genome mapping and Ks dating provided insights into their duplication and divergence in the course of evolution. Expression profiling of candidate genes in contrasting S. italica cultivars subjected to abiotic stresses and hormone treatments showed distinct stress-specific upregulation of SiαCaH1, SiβCaH5, SiPEPC2, SiPPDK2, SiMDH8, and SiNADP-ME5 in the tolerant cultivar. Overexpression of SiNADP-ME5 in heterologous yeast system enabled the transgenic cells to survive and grow in dehydration stress conditions, which highlights the putative role of SiNADP-ME5 in conferring tolerance to dehydration stress. Altogether, the study highlights key genes that could be potential candidates for elucidating their functional roles in abiotic stress response.

Introduction

Photosynthesis converts light energy into chemical energy to fix atmospheric carbon dioxide (CO₂) for synthesizing reduced carbon compounds. Three distinct mechanisms of carbon fixation, namely, C₃, C₄ and crassulacean acid metabolism (CAM) have been reported in terrestrial plants (Furbank and Taylor, 1995). In C₃ photosynthesis, plants assimilate CO₂ through the Calvin cycle that occurs inside of the chloroplast in the mesophyll cells (Furbank and Taylor, 1995). In C₄ plants, CO₂ is converted into bicarbonate followed by its carboxylation to form a four-carbon compound, oxaloacetate (OAA) in the mesophyll. OAA undergoes several possible modifications and diffuses into bundle sheath, where decarboxylation and refixation of CO₂ by Rubisco in the Calvin cycle takes place (Furbank and Taylor, 1995). The mesophyll-located C₄ cycle serves as a biochemical CO₂ pump to elevate the CO₂ levels in the bundle sheath and ensures an uninterrupted supply of CO₂ to Rubisco, thus enhancing the photosynthetic water use efficiency of C₄ plants (Schuler et al., 2016). Also, Nitrogen requirement in the C₄ leaves is lower due to a higher Rubisco catalytic turnover rate, which confers better photosynthetic nitrogen use efficiency in these plants (Schuler et al., 2016). C₄ photosynthesis is common among monocots, but not very common among dicots (Furbank and Taylor, 1995; Schuler et al., 2016). Particularly, the grass family, Poaceae constitutes C₃, C₃–C₄ intermediate and C₄ species (Grass Phylogeny Working Group II, 2012). Among C₃ grasses, rice (Erhartoideae) and wheat (Pooideae) are considered as staple crops, whereas barley and Brachypodium (Pooideae) are equally important as bioenergy feedstocks. The PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae and Danthonioideae) clade of Poaceae is C₄-rich (Grass Phylogeny Working Group II, 2012), where the important C₄ grasses including sorghum, maize, millets and sugarcane are present in the Panicoid tribe.

Among the Panicoids, Setaria italica (foxtail millet) and its wild progenitor, Setaria viridis (green foxtail) are considered as model species for studying C₄ photosynthesis (Brutnell et al., 2010; Li and Brutnell, 2011), abiotic stress tolerance (Diao et al., 2014; Muthamilarasan and Prasad, 2015; Huang et al., 2016), biofuel (Lata et al., 2013) and nutritional traits (Muthamilarasan et al., 2016; Pant et al., 2016). Both S. italica and S. viridis (collectively termed Setaria), possess small diploid genome (∼500 Mb), self-pollination and inbreeding nature, shorter life-span (∼90 days; seed-to-seed), short stature (∼100−150 cm average) and prolific seed production (several thousand seeds per panicle; Lata et al., 2013). The genomes of S. italica and S. viridis have also been sequenced (Bennetzen et al., 2012; Zhang et al., 2012), which has enabled the development of several genetic and genomic resources in Setaria (Muthamilarasan and Prasad, 2015). In structural genomics perspective, large-scale, genome-wide molecular markers were developed (Pandey et al., 2013; Kumari et al., 2013; Muthamilarasan et al., 2014a; Zhang et al., 2014a, 2014b) and high-density genetic linkage maps were constructed for mapping the genomic regions controlling agronomic traits (Jia et al., 2013; Fang et al., 2016). In functional genomics, several important gene families including NAC transcription factors (Puranik et al., 2013), WD40 proteins (Mishra et al., 2014), MYB transcription factors (Muthamilarasan et al., 2014b), AP2/ERF transcription factors (Lata et al., 2014), C₂H₂-type zinc finger proteins (Muthamilarasan et al., 2014c), Nuclear Factor Y (Feng et al., 2015), 14-3-3 proteins (Kumar et al., 2015), WRKY transcription factors (Muthamilarasan et al., 2015), heat-shock proteins (Singh et al., 2016), SET domain-containing proteins (Yadav et al., 2016), autophagy-associated genes (Li et al., 2016), pentatricopeptide repeat proteins (Liu et al., 2016), DOF containing proteins (Zhang et al., 2017) and HD-Zip transcription factors (Chai et al., 2018) were identified and characterized. However, no elaborate study on C₄ photosynthetic/stress-responsive genes has been performed in any Panicoid species including Setaria to understand the structure, organization, evolution and expression profiles in response to environmental perturbations.

In the present study, five major classes of enzymes that play pivotal roles in C₄ biochemical carbon-concentrating mechanism were identified in sequenced Poaceae genomes with emphasis on the Setaria. Carbonic anhydrase (CaH) converts CO₂ to bicarbonate (HCO₃⁻) which is then pre-assimilated by phosphoenolpyruvate carboxylase (PEPC) to form OAA in the mesophyll (Furbank and Taylor, 1995; Schuler et al., 2016). The conversion of OAA to malate is catalyzed by malate dehydrogenase (MDH) and following the diffusion of malate through plasmodesmata to the bundle sheath, decarboxylation of malate is catalyzed by one of three different decarboxylating enzymes to form pyruvate and CO₂ (Hatch and Burnell, 1990). Finally, PEP is regenerated in the mesophyll by pyruvate orthophosphate dikinase (PPDK) (Aubry et al., 2011). Based on the decarboxylating enzymes, three biochemical subtypes of C₄ photosynthesis were defined. These include NADP-dependent malic enzyme (NADP-ME), NAD-dependent ME (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK) (Gutierrez et al., 1974). The present study involves two subtypes, namely NADP-ME (S. italica, S. viridis, Zea mays and Sorghum bicolor) and NAD-ME (Panicum hallii and P. virgatum). Also, Oryza sativa and Brachypodium distachyon from C₃ clade have been included. Since no genome of a PEPCK subtype has been sequenced, it has been excluded from the present study.

All five gene family members were identified using computational approaches in these eight species. However, importance has been given to analyze the genic, genomic and physicochemical protein properties of these gene family members in S. italica and S. viridis. The different classes of enzymes were further categorized according to their phylogeny, domain architecture, subcellular localization and sequence conservation. The RNA-seq data of different tissues and stress treatment were systematically analyzed to derive the expression profiles of these five gene family members. Based on this, candidate genes were chosen for expression profiling in response to different abiotic stresses (dehydration, salt and heat) and hormone treatments (abscisic acid, jasmonic acid and salicylic acid) in two contrasting S. italica cultivars. Comparative mapping of these five gene families between the sequenced grass genomes and Ks dating of orthologous gene pairs were performed to deduce the evolutionary aspects of these gene families.