Transcriptome dynamics uncovers long non-coding RNAs response to salinity stress in Chenopodium quinoa.

Chenopodium quinoa is a crop with outstanding tolerance to saline soil, but long non-coding RNAs (LncRNAs) expression profile driven by salt stress in quinoa has rarely been observed yet. Based on the high-quality quinoa reference genome and high-throughput RNA sequencing (RNA-seq), genome-wide identification of LncRNAs was performed, and their dynamic response under salt stress was then investigated. In total, 153,751 high-confidence LncRNAs were discovered and dispersed intensively in chromosomes. Expression profile analysis demonstrated significant differences between LncRNAs and coding RNAs. Under salt stress conditions, 4,460 differentially expressed LncRNAs were discovered, of which only 54 were differentially expressed at all the stress time points. Besides, strongly significantly correlation was observed between salt-responsive LncRNAs and their closest neighboring genes (r = 0.346, p-value < 2.2e-16). Furthermore, a weighted co-expression network was then constructed to infer the potential biological functions of LncRNAs. Seven modules were significantly correlated with salt treatments, resulting in 210 hub genes, including 22 transcription factors and 70 LncRNAs. These results indicated that LncRNAs might interact with transcription factors to respond to salinity stress. Gene ontology enrichment of the coding genes of these modules showed that they were highly related to regulating metabolic processes, biological regulation and response to stress. This study is the genome-wide analysis of the LncRNAs responding to salt stress in quinoa. The findings will provide a solid framework for further functional research of salt responsive LncRNAs, contributing to quinoa genetic improvement.

Genome-Wide Analysis and Expression Profiles of the VOZ Gene Family in Quinoa (Chenopodium quinoa).

Vascular plant one zinc-finger (VOZ) proteins are a plant-specific transcription factor family and play important roles in plant development and stress responses. However, little is known about the VOZ genes in quinoa. In the present study, a genome-wide investigation of the VOZ gene family in quinoa was performed, including gene structures, conserved motifs, phylogeny, and expression profiles. A total of four quinoa VOZ genes distributed on three chromosomes were identified. Based on phylogenetic analysis, CqVOZ1 and CqVOZ3 belong to subfamily II, and CqVOZ2 and CqVOZ4 belong to subfamily III. Furthermore, the VOZ transcription factors of quinoa and sugarbeet were more closely related than other species. Except for CqVOZ3, all the other three CqVOZs have four exons and four introns. Analysis of conserved motifs indicated that each CqVOZ member contained seven common motifs. Multiple sequence alignment showed that the CqVOZ genes were highly conserved with consensus sequences, which might be plausibly significant for the preservation of structural integrity of the family proteins. Tissue expression analysis revealed that four CqVOZ genes were highly expressed in inflorescence and relatively low in leaves and stems, suggesting that these genes had obvious tissue expression specificity. The expression profiles of the quinoa CqVOZs under various abiotic stresses demonstrated that these genes were differentially induced by cold stress, salt stress, and drought stress. The transcript level of CqVOZ1 and CqVOZ4 were down-regulated by salt stress and drought stress, while CqVOZ2 and CqVOZ3 were up-regulated by cold, salt, and drought stress, which could be used as abiotic stress resistance candidate genes. This study systematically identifies the CqVOZ genes at the genome-wide level, contributing to a better understanding of the quinoa VOZ transcription factor family and laying a foundation for further exploring the molecular mechanism of development and stress resistance of quinoa.

Evaluating the fermentation quality and bacterial community of high-moisture whole-plant quinoa silage ensiled with different additives.

AIM: To explore the potential of whole-plant quinoa (WPQ) as a high-protein source for livestock feed, this study evaluated the effects of additives on the fermentation quality and bacterial community of high-moisture WPQ silage. METHODS AND RESULTS: High-moisture WPQ was ensiled with one of the following additives: untreated control (C), fibrolytic enzyme (E), molasses (M), LAB inoculant (L), a combination of fibrolytic enzyme and LAB inoculant (EL) and a combination of molasses and LAB inoculant (ML). The fermentation quality and bacterial community after 60 days of ensiling were analysed. Naturally fermented WPQ exhibited acetic acid-type fermentation dominated by enterobacteria, with low lactic acid content (37.0 g/kg DM), and high pH value (5.65), acetic acid (70.8 g/kg DM) and NH3 -N production (229 g/kg TN). Adding molasses alone or combined with LAB inoculant shifted the fermentation pattern towards increased intensity of lactic acid fermentation, lowering the pH value (<4.56), contents of acetic acid (<46.7 g/kg DM) and NH3 -N (<140 g/kg TN) and total abundance of enterobacteria (<16.0%), and increasing the lactic acid content (>60.5 g/kg DM), lactic/acetic acid ratio (>1.40) and the relative abundance of Lactobacillus (>83.0%). CONCLUSIONS: The results suggested that the lack of fermentable sugar could be the main factor of restricting extensive lactic acid fermentation in WPQ silage. Supplementing fermentable sugar or co-ensiling with materials with high WSC content and low moisture content are expected to be beneficial strategies for producing high-quality WPQ silage. SIGNIFICANCE AND IMPACT OF STUDY: High biomass production and high protein content make WPQ to be an ideal forage source for livestock feed. Results of this study revealed the restricting factor for extensive lactic acid fermentation in WPQ silage, which could be helpful in producing high-quality WPQ silage.

The Novel Cucurbitaceae miRNA ClmiR86 Is Involved in Grafting-Enhanced Phosphate Utilization and Phosphate Starvation Tolerance in Watermelon.

Watermelon (Citrullus lanatus) is a globally important Cucurbitaceae crop in which grafting is commonly used to improve stress tolerance and enhance nutrient utilization. However, the mechanism underlying grafting-enhanced nutrient assimilation remains unclear. Here, we demonstrate the possible involvement of a novel Cucurbitaceae miRNA, ClmiR86, in grafting-enhanced phosphate-starvation tolerance via CALCINEURIN B-LIKE INTERACTING PROTEIN KINASE 5 (ClCIPK5) suppression in watermelon. Transcript analyses revealed that the induction of ClmiR86 expression was correlated with the downregulation of ClCIPK5 in squash-grafted watermelon under phosphate starvation. In addition, the differential expression of ClmiR86 in various watermelon genotypes was consistent with their phosphate utilization efficiency. Furthermore, ClmiR86 overexpression in Arabidopsis enhanced root growth and phosphate uptake under phosphate starvation and promoted inflorescence elongation under normal conditions. These results suggest that the ClmiR86-ClCIPK5 axis is involved in phosphate starvation response as well as grafting-enhanced growth vigor and phosphate assimilation. The present study provides valuable insights for investigating long-distance signaling and nutrient utilization in plants.

Brassicaceae transcriptomes reveal convergent evolution of super-accumulation of sinigrin.

Wasabi, horseradish and mustard are popular pungent crops in which the characteristic bioactive hydrolysis of specialized glucosinolates (GSLs) occurs. Although the metabolic pathways of GSLs are well elucidated, how plants have evolved convergent mechanisms to accumulate identical GSL components remains largely unknown. In this study, we discovered that sinigrin is predominantly synthesized in wasabi, horseradish and mustard in Brassicaceae. We de novo assembled the transcriptomes of the three species, revealing the expression patterns of gene clusters associated with chain elongation, side chain modification and transport. Our analysis further revealed that several gene clusters were convergently selected during evolution, exhibiting convergent shifts in amino acid preferences in mustard, wasabi and horseradish. Collectively, our findings provide insights into how unrelated crop species evolve the capacity for sinigrin super-accumulation and thus promise a potent strategy for engineering metabolic pathways at multiple checkpoints to fortify bioactive compounds for condiment or pharmaceutical purposes.

Transcriptome analysis and differential gene expression profiling of two contrasting quinoa genotypes in response to salt stress.

BACKGROUND: Soil salinity is one of the major abiotic stress factors that affect crop growth and yield, which seriously restricts the sustainable development of agriculture. Quinoa is considered as one of the most promising crops in the future for its high nutrition value and strong adaptability to extreme weather and soil conditions. However, the molecular mechanisms underlying the adaptive response to salinity stress of quinoa remain poorly understood. To identify candidate genes related to salt tolerance, we performed reference-guided assembly and compared the gene expression in roots treated with 300 mM NaCl for 0, 0.5, 2, and 24 h of two contrasting quinoa genotypes differing in salt tolerance. RESULTS: The salt-tolerant (ST) genotype displayed higher seed germination rate and plant survival rate, and stronger seedling growth potential as well than the salt-sensitive (SS) genotype under salt stress. An average of 38,510,203 high-quality clean reads were generated. Significant Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified to deeper understand the differential response. Transcriptome analysis indicated that salt-responsive genes in quinoa were mainly related to biosynthesis of secondary metabolites, alpha-Linolenic acid metabolism, plant hormone signal transduction, and metabolic pathways. Moreover, several pathways were significantly enriched amongst the differentially expressed genes (DEGs) in ST genotypes, such as phenylpropanoid biosynthesis, plant-pathogen interaction, isoquinoline alkaloid biosynthesis, and tyrosine metabolism. One hundred seventeen DEGs were common to various stages of both genotypes, identified as core salt-responsive genes, including some transcription factor members, like MYB, WRKY and NAC, and some plant hormone signal transduction related genes, like PYL, PP2C and TIFY10A, which play an important role in the adaptation to salt conditions of this species. The expression patterns of 21 DEGs were detected by quantitative real-time PCR (qRT-PCR) and confirmed the reliability of the RNA-Seq results. CONCLUSIONS: We identified candidate genes involved in salt tolerance in quinoa, as well as some DEGs exclusively expressed in ST genotype. The DEGs common to both genotypes under salt stress may be the key genes for quinoa to adapt to salinity environment. These candidate genes regulate salt tolerance primarily by participating in reactive oxygen species (ROS) scavenging system, protein kinases biosynthesis, plant hormone signal transduction and other important biological processes. These findings provide theoretical basis for further understanding the regulation mechanism underlying salt tolerance network of quinoa, as well establish foundation for improving its tolerance to salinity in future breeding programs.

Subcellular distribution of aluminum associated with differential cell ultra-structure, mineral uptake, and antioxidant enzymes in root of two different Al+3-resistance watermelon cultivars.

Crop plants, such as watermelon, suffer from severe Aluminum (Al3+)-toxicity in acidic soils with their primary root elongation being first arrested. However, the significance of apoplastic or symplastic Al3+-toxicity in watermelon root is scarcely reported. In this work, we identified a medium fruit type (ZJ) and a small fruit type (NBT) as Al+3-tolerant and sensitive based on their differential primary root elongation rate respectively, and used them to show the effects of symplastic besides apoplastic Al distribution in the watermelon's root. Although the Al content was higher in the root of NBT than ZJ, Al+3 allocated in their apoplast, vacuole and plastid fractions were not significantly different between the two cultivars. Thus, only a few proportion of Al+3 differentially distributed in the nucleus and mitochondria corresponded to interesting differential morphological and physiological disorders recorded in the root under Al+3-stress. The symplastic amount of Al+3 substantially induced the energy efficient catalase pathway in ZJ, and the energy consuming ascorbate peroxidase pathway in NBT. These findings coincided with obvious starch granule visibility in the root ultra-structure of ZJ than NBT, suggesting a differential energy was used in supporting the root elongation and nutrient uptake for Al+3-tolerance in the two cultivars. This work provides clues that could be further investigated in the identification of genetic components and molecular mechanisms associated with Al+3-tolerance in watermelon.

Gene coexpression network analysis reveals the role of SRS genes in senescence leaf of maize (Zea mays L.).

Shi-related sequence (SRS) proteins are plant-specific transcription factors that play important roles in developmental processes, including regulating hormone biosynthesis, response or signal transduction. However, systematical analysis of the SRS gene family in maize has not yet been conducted. In this study, 11 SRS genes with 13 transcripts were identified and characterized. The characteristics of the gene family were analysed in terms of phylogenetic relationships, chromosome distribution and gene structure. RNA-sequencing data analysis showed that the expression patterns of SRS genes were quite different from each other in maize, indicating their divergence in function. Interestingly, the GRMZM2G077752 gene is highly expressed in senescent leaves. Using further coexpression network analysis, we determined that the module containing GRMZM2G077752 were over-represented by genes related to abscisic acid (ABA) stimulus and carbohydrate metabolic process. This result indicated that GRMZM2G077752 might perceive ABA signal and cause the activation of carbohydrate remobilization during leaf ageing. This study provides valuable information for understanding the functions of the SRS genes in maize.

Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses.

Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants.

Genome-wide identification and expression analysis of the ClTCP transcription factors in Citrullus lanatus.

BACKGROUND: The plant-specific TCP transcription factor family, which is involved in the regulation of cell growth and proliferation, performs diverse functions in multiple aspects of plant growth and development. However, no comprehensive analysis of the TCP family in watermelon (Citrullus lanatus) has been undertaken previously. RESULTS: A total of 27 watermelon TCP encoding genes distributed on nine chromosomes were identified. Phylogenetic analysis clustered the genes into 11 distinct subgroups. Furthermore, phylogenetic and structural analyses distinguished two homology classes within the ClTCP family, designated Class I and Class II. The Class II genes were differentiated into two subclasses, the CIN subclass and the CYC/TB1 subclass. The expression patterns of all members were determined by semi-quantitative PCR. The functions of two ClTCP genes, ClTCP14a and ClTCP15, in regulating plant height were confirmed by ectopic expression in Arabidopsis wild-type and ortholog mutants. CONCLUSIONS: This study represents the first genome-wide analysis of the watermelon TCP gene family, which provides valuable information for understanding the classification and functions of the TCP genes in watermelon.