The chromosome-level Stevia genome provides insights into steviol glycoside biosynthesis.

Stevia (Stevia rebaudiana Bertoni) is well known for its very sweet steviol glycosides (SGs) consisting of a common tetracyclic diterpenoid steviol backbone and a variable glycone. Steviol glycosides are 150-300 times sweeter than sucrose and are used as natural zero-calorie sweeteners. However, the most promising compounds are biosynthesized in small amounts. Based on Illumina, PacBio, and Hi-C sequencing, we constructed a chromosome-level assembly of Stevia covering 1416 Mb with a contig N50 value of 616.85 kb and a scaffold N50 value of 106.55 Mb. More than four-fifths of the Stevia genome consisted of repetitive elements. We annotated 44,143 high-confidence protein-coding genes in the high-quality genome. Genome evolution analysis suggested that Stevia and sunflower diverged ~29.4 million years ago (Mya), shortly after the whole-genome duplication (WGD) event (WGD-2, ~32.1 Mya) that occurred in their common ancestor. Comparative genomic analysis revealed that the expanded genes in Stevia were mainly enriched for biosynthesis of specialized metabolites, especially biosynthesis of terpenoid backbones, and for further oxidation and glycosylation of these compounds. We further identified all candidate genes involved in SG biosynthesis. Collectively, our current findings on the Stevia reference genome will be very helpful for dissecting the evolutionary history of Stevia and for discovering novel genes contributing to SG biosynthesis and other important agronomic traits in future breeding programs.

Identification of GH1 gene family fgt members in Stevia rebaudiana and their expression when grown in darkness.

Stevia rebaudiana Bertoni is an important economic crop that is well known for its secondary metabolites, steviol glycosides (SGs), found in leaves. Because the enzymes of deglycosylation (glycoside hydrolases) play important roles in SGs biosynthetic processes, our study is focused on the functions of ß-glucosidases in SGs catabolism in stevia. We cloned and characterized 19 stevia GH1 genes based on transcriptomic sequences. The 19 genes were divided into five putative subfamilies in Arabidopsis. Conserved motifs in the SrGH1 proteins were analysed using the online motif-based sequence analysis tool, MEME. Most of the identified proteins contain the conserved 'TFNEP' motif (contains the catalytic acid/base) and 'ITENG' motif (contains the catalytic nucleophile). Furthermore, the steviol glycoside content and expression of these 19 genes were characterized under constant darkness. The dark treatment lowered the steviol glycoside content significantly, while SrBGLU16 responded to darkness and was markedly upregulated. This study is the first transcriptome-wide analysis of the GH1 family in Stevia rebaudiana. The sequences of 19 SrGH1 members and their expression when grown in darkness were characterized. Among the 19 genes, SrBGLU16 was markedly upregulated by darkness. Thus, we identified SrBGLU16 for further investigation as a possible steviol glycoside beta-glucosidase.

A beta-glucosidase gene from Stevia rebaudiana may be involved in the steviol glycosides catabolic pathway.

We herein report the preparation of a full-length raucaffricine-O-beta-D-glucosidase gene of stevia rebaudiana Bertoni (named SrRG1, GenBank accession number MK920450). Sequence analysis indicated SrRG1 consists of a 1650 bp open reading frame encoding a protein of 549 amino acids. Its deduced amino acid sequence showed a high identity of 82% with a raucaffricine-O-beta-D-glucosidase from H. annuus of glycoside hydrolase family 1. The expression pattern analyzed by real-time quantitative PCR showed no significant difference among different tissues, developmental stages, and cultivars under normal growth conditions. Furthermore, the gene function of SrRG1 was preliminarily studied by agrobacterium-mediated transformation on instantaneous expression. In the test of agrobacterium-mediated transformation on instantaneous expression, it was observed that overexpression of SrRG1 increased the accumulation of steviol content and decreased the major components and total SGs contents. Such results demonstrated that SrRG1 may participate in the steviol glycosides catabolic pathway. However, the effect of silencing construct infiltration on steviol and SGs content was not significant and its expression pattern was constitutive, which most probably, attributed the hydrolysis of SGs to the secondary activity of SrRG1. This study firstly identified the bate-glucosidase in stevia and advances our understanding of steviol glycosides hydrolyzation.

The SrWRKY71 transcription factor negatively regulates SrUGT76G1 expression in Stevia rebaudiana.

SrUGT76G1 is vital for the biosynthesis of rebaudioside A, D and M in Stevia rebaudiana Bertoni; however, its transcriptional regulatory mechanism remains unknown. In this study, the 2050-bp promoter region of SrUGT76G1 was isolated by the TAIL-PCR method, and sequence analysis revealed the presence of several W-box cis-elements, which are the recognition motifs of WRKY transcription factors. Furthermore, SrWRKY71, characterized by a typical WRKY domain and a C2H2 zinc finger-like motif, was identified as a putative transcriptional regulator of SrUGT76G1. The transcript of SrWRKY71 predominantly accumulated in leaves and was present at a lower level in stems, roots and flowers. The SrWRKY71-GFP fusion protein was specifically localized to the nucleus in tobacco epidermal cells. In addition, the N and C terminal regions of SrWRKY71 contributed to its transactivation activity. Y1H and EMSA assays validated that SrWRKY71 binds directly to W-box1 and W-box2 in the proximal promoter region of SrUGT76G1. Moreover, SrWRKY71 represses the expression level of SrUGT76G1 in both tobacco leaves and stevia callus. Taken together, the data in this study represent the first identification of an essential upstream transcription factor of SrUGT76G1 and provides new insight into the regulatory network of steviol glycoside biosynthesis in Stevia rebaudiana.

The evolutionary history of the sucrose synthase gene family in higher plants.

BACKGROUND: Sucrose synthase (SUS) is widely considered a key enzyme participating in sucrose metabolism in higher plants and regarded as a biochemical marker for sink strength in crops. However, despite significant progress in characterizing the physiological functions of the SUS gene family, knowledge of the trajectory of evolutionary processes and significance of the family in higher plants remains incomplete. RESULTS: In this study, we identified over 100 SUS genes in 19 plant species and reconstructed their phylogenies, presenting a potential framework of SUS gene family evolution in higher plants. Three anciently diverged SUS gene subfamilies (SUS I, II and III) were distinguished based on their phylogenetic relationships and unique intron/exon structures in angiosperms, and they were found to have evolved independently in monocots and dicots. Each subfamily of SUS genes exhibited distinct expression patterns in a wide range of plants, implying that their functional differentiation occurred before the divergence of monocots and dicots. Furthermore, SUS III genes evolved under relaxed purifying selection in dicots and displayed narrowed expression profiles. In addition, for all three subfamilies of SUS genes, the GT-B domain was more conserved than the "regulatory" domain. CONCLUSIONS: The present study reveals the evolution of the SUS gene family in higher plants and provides new insights into the evolutionary conservation and functional divergence of angiosperm SUS genes.

Nitrogen drives plant growth to the detriment of leaf sugar and steviol glycosides metabolisms in Stevia (Stevia rebaudiana Bertoni).

Steviol glycosides (SGs) in Stevia (Stevia rebaudiana Bertoni) leaves are important due to their high sweetness and low calorific value. The yield of SGs is dependent on fertilization regimes, but the relationship between nitrogen (N) administration and SGs synthesis is still unclear. In this study, we investigate the effects of N rates on SGs production through hydroponic and plot experiments. The SGs yield was not significantly changed by N fertilization, but leaf SGs concentrations were significantly reduced due to the "dilution effect". Additionally, N addition decreased leaf carbon (C)/N ratio and soluble sugar concentration, accompanied with the inhibited phosphoenolpyruvate carboxylase and L-phenylalanine ammonia_lyase activities. A significant positive correlation between leaf SGs concentrations, C/N ratio and soluble sugar concentration was observed. Overall, we suggest that N-driven Stevia growth negatively affects SGs concentrations. The leaf C/N ratio and soluble sugar changes indicated the occurrence of metabolic reprogramming.