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Radish, Raphanus sativus L., is an important root crop that is cultivated worldwide. Owing to its evolutionary proximity to Arabidopsis thaliana, radish can be used as a model root crop in research on the molecular basis of agronomic traits. Pithiness is a significant defect that reduces the production of radish with commercial value; however, traditional breeding to eliminate this trait has thus far been unsuccessful. Here, we performed transcriptomics and genotype-by-sequencing (GBS)-based quantitative trait locus (QTL) analyses of radish inbred lines to understand the molecular basis of pithiness in radish roots. The transcriptome data indicated that pithiness likely stems from the response to oxidative stress, leading to cell death of the xylem parenchyma during the root-thickening process. Subsequently, we narrowed down a list of candidates responsible for pithiness near a major QTL and found polymorphisms in a radish homologue of Arabidopsis ANAC013 (RsNAC013), an endoplasmic reticulum bound NAC transcription factor that is targeted to the nucleus to mediate the mitochondrial retrograde signal. We analysed the effects of polymorphisms in RsNAC013 using Arabidopsis transgenic lines overexpressing RsNAC013 alleles as well as in radish inbred lines bearing these alleles. This analysis indicated that non-synonymous variations within the coding sequence result in different levels of RsNAC013 activities, thereby providing a genetic condition for root pithiness. The elevated oxidative stress or hypoxia that activates RsNAC013 for mitochondrial signalling enhances this process. Collectively, this study serves as an exemplary case of translational research taking advantage of the extensive information available from a model organism.

Gynandropsis gynandra (Cleomaceae) is a cosmopolitan leafy vegetable and medicinal plant, which has also been used as a model to study C4 photosynthesis due to its evolutionary proximity to Arabidopsis. Here, we present a high-quality genome sequence of G. gynandra, anchored onto 17 main super- scaffolds with a total length of 740 Mb, an N50 of 42 Mb and 30,933 well-supported gene models. The G. gynandra genome and previously released genomes of C3 relatives in the Cleomaceae and Brassicaceae make an excellent model for studying the role of genome evolution in the transition from C3 to C4 photosynthesis. We revealed that G. gynandra and its C3 relative Tarenaya hassleriana shared a whole-genome duplication event (Gg-α), then an addition of a third genome (Th-α, +1x) took place in T. hassleriana but not in G. gynandra. Analysis of syntenic copy number of C4 photosynthesis-related gene families indicates that G. gynandra generally retained more duplicated copies of these genes than C3 T. hassleriana, and also that the G. gynandra C4 genes might have been under positive selection pressure. Both whole-genome and single-gene duplication were found to contribute to the expansion of the aforementioned gene families in G. gynandra. Collectively, this study enhances our understanding of the impact of gene duplication and gene retention on the evolution of C4 photosynthesis in Cleomaceae.

Background The importance of uridine 5′-diphosphate glucose (UDP-G) synthesis and degradation on carbon (C) partitioning has been indicated in several studies of plant systems, whereby the kinetic properties and abundance of involved enzymes had a significant effect upon the volume of C moving into the hemicellulose, cellulose and sucrose pools. In this study, the expression of 136 genes belonging to 32 gene families related to UDP-G metabolism was studied in 3 major sugarcane organs (including leaf, internode and root) at 6 different developmental stages in 2 commercial genotypes. Results Analysis of the genes associated with UDP-G metabolism in leaves indicated low expression of sucrose synthase, but relatively high expression of invertase genes, specifically cell-wall invertase 4 and neutral acid invertase 1–1 and 3 genes. Further, organs that are primarily responsible for sucrose synthesis or bioaccumulation, i.e., in source organs (mature leaves) and storage sink organs (mature internodes), had very low expression of sucrose, cellulose and hemicellulose synthesis genes, specifically sucrose synthase 1 and 2, UDP-G dehydrogenase 5 and several cellulose synthase subunit genes. Gene expression was mostly very low in both leaf and mature internode samples; however, leaves did have a comparatively heightened invertase and sucrose phosphate synthase expression. Major differences were observed in the transcription of several genes between immature sink organs (roots and immature internodes). Gene transcription favoured utilisation of UDP-G toward insoluble and respiratory pools in roots. Whereas, there was comparatively higher expression of sucrose synthetic genes, sucrose phosphate synthase 1 and 4, and comparatively lower expression of many genes associated with C flow to insoluble and respiratory pools including myo-Inositol oxygenase, UDP-G dehydrogenase 4, vacuolar invertase 1, and several cell-wall invertases in immature internodes. Conclusion This study represents the first effort to quantify the expression of gene families associated with UDP-G metabolism in sugarcane. Transcriptional analysis displayed the likelihood that C partitioning in sugarcane is closely related to the transcription of genes associated with the UDP-G metabolism. The data presented may provide an accurate genetic reference for future efforts in altering UDP-G metabolism and in turn C partitioning in sugarcane.

Saccharum spontaneum, a wild relative of sugarcane, is highly tolerant to drought and salinity. The exploitation of germplasm resources for salinity tolerance is a major thrust area in India. In this study, we utilized suppression subtractive hybridization (SSH) followed by sequencing for the identification of upregulated transcripts during salinity stress in S. spontaneum clones coming from different geographical regions of India. Our sequencing of the SSH library revealed that 95% of the transformants contained inserts of size 200–1500 bp. We have identified 314 differentially expressed transcripts in the salinity-treated samples after subtraction, which were subsequently validated by quantitative real-time polymerase chain reaction. Functional annotation and pathway analysis revealed that the upregulated transcripts were a result of protein modifications, stress, and hormone signaling along with cell wall development and lignification. The prominently upregulated transcripts included UDP glucose dehydrogenase, cellulose synthase, ribulose, cellulose synthase COBRA, leucine-rich protein, NAC domain protein, pectin esterase, ABA-responsive element binding factor 1, and heat stress protein. Our results is a step forward the understanding of the molecular response of S. spontaneum under salinity stress, which will lead to the identification of genes and transcription factors as novel targets for salinity tolerance in sugarcane.

Background The composition of biomass determines its suitability for different applications within a biorefinery system. The proportion of the major biomass fractions (sugar, cellulose, hemicellulose and lignin) may vary in different sugarcane genotypes and growth environments and different parts of the plant. This study investigated the composition of mature and immature internodes, roots and mature leaves of sugarcane. Results Internodes were found to have a significantly larger alcohol-soluble component than leaves and roots. The primary difference between the immature and mature internodes was the ratio of soluble sugars. In mature tissues, sucrose content was significantly higher, whereas in immature internodal tissues there was lower sucrose and heightened concentrations of reducing sugars. Carbon (C) partitioning in leaf tissues was characterised by low levels of soluble components and high “other” and cell wall fractions. Root tissue had low ratios of soluble fractions relative to their cell wall contents, indicating a lack of storage of soluble carbon. There was no significant difference in the ratio of the major cell wall fractions between the major organ types. Characterisation of individual non-cellulosic monomers indicated leaf and root tissues had significantly higher arabinose and galactose fractions. Significantly larger proportions of syringyl lignin compounds and the hydroxycinnamic compound, p-coumaric acid were observed in mature internodal tissues compared to the other tissue types. Tissue-specific differences in composition were shown to greatly affect the recalcitrance of the cell wall to enzymatic saccharification. Conclusions Overall, this study displayed clear evidence of the differential partitioning of C throughout the sugarcane plant in specific organs. These organ-specific differences have major implications in their utility as a bioproduct feedstock. For example, the inclusion of trash (leaves) with the culms (internodes) may alter processing efficiency.

Background Despite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms. Results The sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes. Conclusions The transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3757-8) contains supplementary material, which is available to authorized users.

Summary Cambium drives the lateral growth of stems and roots, contributing to diverse plant growth forms. The root crop is one of the outstanding examples of the cambium-driven growth. To understand its molecular basis, we used radish to generate a compendium of root-tissue- and stage-specific transcriptomes from two contrasting inbred lines during root growth. Expression patterns of key cambium regulators and hormone signaling components were validated. Clustering and gene ontology (GO) enrichment analyses of radish datasets followed by a comparative analysis against the newly established Arabidopsis early cambium data revealed evolutionary conserved stress-response transcription factors that may intimately control the cambium. Indeed, an in vivo network consisting of selected stress-response and cambium regulators indicated ERF-1 as a potential key checkpoint of cambial activities, explaining how cambium-driven growth is altered in response to environmental changes. The findings here provide valuable information about dynamic gene expression changes during cambium-driven root growth and have implications with regard to future engineering schemes, leading to better crop yields.

Cambium drives lateral growth of stems and roots, contributing to diverse plant growth forms. Root crop is one outstanding example of the cambium-driven growth. To understand its molecular basis, we used radish to generate a compendium of root tissue- and stage-specific transcriptomes from two contrasting inbred lines in root growth. Expression patterns of key cambium regulators and hormone signaling components were validated. Clustering and GO enrichment analyses of radish datasets followed by comparative analysis against the newly established Arabidopsis early cambium data revealed evolutionary conserved stress-response transcription factors that might intimately control the cambium. Indeed, in vivo network made of selected stress-response and cambium regulators indicated ERF-1 as a potential key checkpoint of cambial activities, explaining how the cambium-driven growth is altered in response to environmental changes. Together, this study provides rich information about dynamic gene expression changes along the cambium-driven root growth with future engineering schemes for crop yields.

Summary Laser capture microdissection (LCM) coupled with transcriptome profiling is a powerful technique that allows for tissue-specific gene expression analysis in a complex system. One major challenge in using this technique is to obtain RNA without compromising its integrity. Here, we present a protocol optimized for radish root tissue sections using Steedman's wax embedding to obtain high-quality RNA suitable for next-generation sequencing analysis. For complete details on the use and execution of this protocol, please refer to Hoang et al. (2020)., Graphical Abstract, Highlights • Laser capture microdissection (LCM) for capturing tissues in radish tap roots • Steedman’s wax embedding method for samples incompatible with cryo-sectioning • High-quality tissue-specific RNAs suitable for next-generation sequencing analysis, Choe, Hoang, and Lee describe the stepwise protocol for laser capture microdissections of tissues in radish tap roots, focusing on how to prepare target tissues for dissection while keeping RNAs intact for next-generation sequencing. This method enabled identifying both novel and annotated protein-coding genes from de-novo assembled transcripts as described in Hoang et al. (2020).

Lignocellulosic biomass from sugarcane (Saccharum spp. hybrids) could potentially be a major feedstock for second-generation biofuel production. Consequently, selecting sugarcane varieties with favorable biomass characteristics, typically less enzymatic recalcitrance and better saccharification yield without sugar-yield penalty, will be important in sugarcane breeding. Economical and high-throughput techniques for profiling the major biomass components of this complex system will facilitate selection of clones with ideal lignocellulosic composition from large numbers of genotypes in breeding programs. We used a combined high-throughput profiling approach to evaluate the biomass composition of samples from a sugarcane germplasm collection. This employed near-infrared (NIR) spectroscopy for fiber characterization and high-performance liquid chromatography (HPLC) for determining the sugar content in juice. The results for 331 samples, from a diverse sugarcane population of 186 genotypes, derived from 143 parents of different genetic backgrounds, showed that high-quality NIR spectroscopic predictions were feasible for cellulose, hemicellulose, lignin, and extractives values in fiber, and sugars in juice were suitably analyzed by HPLC. The analysis of total biomass indicated that this NIR- and HPLC-based high-throughput method allowed a robust phenotypic assessment of a large number of samples for the key biomass traits in the sugarcane system, including total dry biomass, fiber, sugar content, and theoretical ethanol yields, and could potentially become the method of choice for sugarcane germplasm screening in breeding programs targeting the support of biofuel production.

Low-temperature (LT) stress is known to restrain sugarcane productivity in sub-tropical regions significantly. Many researchers have prioritized their work towards developing climate-resilient sugarcane varieties incorporating genome of stress-tolerant sugarcane related wild-type species in the pre-breeding programs. However, the lack of genomic resources for wild-type sugarcane limits the identification and utilization of stress-related genes in molecular breeding. In this study, for the first time, we generated ∼182 million RNA-seq paired-end reads for Saccharum spontaneum roots grown under low temperature (10 °C) at different time intervals (0 h, 3 h, 6 h, 12 h, 24 h, and 48 h), to identify LT stress responsive genes and pathways. These data were assembled into 141,409 unigenes and subsequently used to identify 2715 upregulated and 1710 downregulated transcripts under LT stress. Combining evidences from GO enrichment, KEGG pathways, histological studies, biochemical assays, and physiological analysis, our results revealed several key genes and pathways involved in cold acclimatization in the S. spontaneum roots. Transcription profiling of roots during LT stress revealed cold stress sensors (i.e., proline, MDA, calcium-dependent kinase, G-coupled proteins, and histidine kinase) that trigger and activate signal transduction through transcription factors (i.e., MYB, ERF, ARF2, DREB, CAMTA, and C2H2) resulting in upregulation of LT stress responsive genes (i.e, annexin, LEA, germins, LT dehydrins, osmotins, and COR) thereby enhancing cold tolerance. Also, transcriptomic analysis envisaged cold responsive metabolic pathways such as phenylpropanoid and sugar metabolism stimulate the synthesis of flavonoid, sucrose, galactose, raffinose, and fructose, antioxidants, phytohormones, and secondary metabolites, and thus trigger cold-responsive transcriptional regulation. Together, this study provides insights into cold tolerance of wild sugarcane roots to LT stress, thus providing a foundation for developing climate-resilient sugarcane varieties.

Sugarcane (Saccharum spp. hybrids) is a potential lignocellulosic feedstock for biofuel production due to its exceptional biomass accumulation ability, high convertible carbohydrate content and a favorable energy input/output ratio. Genetic modification of biofuel traits to improve biomass conversion requires an understanding of the regulation of carbohydrate and lignin biosynthesis. RNA-Seq was used to investigate the transcripts differentially expressed between the immature and mature tissues of the sugarcane genotypes varying in fiber content. Most of the differentially expressed transcripts were found to be down-regulated during stem maturation, highlighting their roles in active secondary cell-wall development in the younger tissues of both high and low fiber genotypes. Several cellulose synthase genes (including CesA2, CesA4, CesA7 and COBRA-like protein), lignin biosynthesis-related genes (ρ-coumarate 3-hydroxylase, ferulate 5-hydroxylase, cinnamyl alcohol dehydrogenase and gentiobiase) and transcription regulators for the secondary cell-wall synthesis (including LIM, MYB, PLATZ, IAA24, C2H2 and C2C2 DOF zinc finger gene families) were exclusively differentially expressed between immature and mature tissues of high fiber genotypes. These findings reveal target genes for subsequent research on the regulation of cellulose and lignin metabolism.

Background The chloroplast genome of plants has been frequently sequenced using chloroplast DNA derived by techniques involving chloroplast isolation and or by PCR amplification using primer sequences targeted to amplify the chloroplast genome. Using these approaches, chloroplast heteroplasmy, described as variations in the chloroplast sequence within an individual plant, has been reported in many plant species. More recently, next generation sequencing (NGS) technologies have allowed chloroplast genome sequences to be extracted from shotgun sequences of total plant DNA. Results Here, we used DNA preparations varying in nuclear, mitochondrial and chloroplast enrichment to explore the potential to distinguish genuine chloroplast heteroplasmy from apparent heteroplasmy due to sequence-variant homologues of chloroplast genome sequences inserted in nuclear or mitochondrial genomes. Application of NGS to the whole sugarcane genome followed by read mapping analysis of the complex sugarcane system allowed the assembly of a complete chloroplast genome sequence of sugarcane cv. Q155. Variant analysis showed that they were present only at frequencies that could be attributed to homologues of chloroplast sequences inserted in the nucleus or mitochondria. Conclusions The result suggests that earlier reports of heteroplasmy in chloroplasts may have been due to contaminating sequences from other genomes (nuclear or mitochondrial) in chloroplast preparations or specific amplification of sequences from these genomes. This demonstrates that the ability to evaluate sequence abundance avoids the risks of attributing a chloroplast gene homologue from the nucleus or mitochondria to the chloroplast.

Sugarcane (Saccharum spp. hybrids) is a leading industrial crop in tropical and subtropical regions worldwide. More recently, sugarcane has been selected as a key feedstock for biofuels due to its rapid growth, high fiber content and favorable energy input/output ratio. Breeding sugarcane varieties with biomass for efficient conversion to biofuels can be optimized by understanding the genetic control of biomass composition. However, the genetic analysis of these traits is hindered by the genomic complexity, and the limited availability of a reference genome. The aims of this project were: the development of a high-throughput profiling method for rapid screening of the key biomass traits in a sugarcane population; the construction of a new full-length transcriptome reference database; and the identification of transcripts associated with sugar and fiber accumulation in sugarcane. For the screening of genotypes, newly developed predictive models employing near-infrared (NIR) spectral analysis, coupled with the high performance liquid chromatography (HPLC), were shown to allow high-throughput profiling of major components in the fiber and sugar fractions in sugarcane biomass. Contrasting genotypes of low fiber and high fiber (minimum of ~29% and maximum of 61% total dry biomass) were identified amongst 331 samples from 186 sugarcane genotypes. The population studied exhibited a wide range of fiber/sugar ratio, from 0.4 (as low as that of the typical commercial sugarcane variety) to 2.2 (similar to that of energy-cane). In addition, the lignin content (the central factor in the biomass recalcitrance) ranged from 6 to 14% of the total dry biomass. To aid genotyping, a new sugarcane transcriptome (termed as SUGIT database) was constructed using PacBio full-length isoform sequencing (Iso-Seq), and a cDNA library derived from 22 diverse sugarcane genotypes, of the key tissues (leaf, internode and root), at different developmental stages (from immature to mature). Comparative analysis showed that this new SUGIT database included more full-length transcripts, longer predicted transcripts, and higher average length of the largest 1,000 proteins, compared to a de novo assembly from Illumina RNA-Seq short-read data from the same sample set. The annotation suggested that the majority (~94%) of the SUGIT database was from coding RNAs, while a very small proportion (~2%) could be long non-coding RNAs. About 70-82% of the RNA-Seq reads from different tissues mapped back to the SUGIT database, suggesting that it represented well the targeted tissues, while about 69% of this database was aligned with the sorghum genome, confirming the high conservation of orthologs in the genic regions of the two genomes. Applying the SUGIT database to differential expression analysis (FDR, false discovery rate corrected p-value

The productivity of plants as biofuel or biomaterial crops is established by both the yield of plant biomass per unit area of land and the efficiency of conversion of the biomass to biofuel. Higher yielding biofuel crops with increased conversion efficiencies allow production on a smaller land footprint minimizing competition with agriculture for food production and biodiversity conservation. Plants have traditionally been domesticated for food, fibre and feed applications. However, utilization for biofuels may require the breeding of novel phenotypes, or new species entirely. Genomics approaches support genetic selection strategies to deliver significant genetic improvement of plants as sources of biomass for biofuel manufacture. Genetic modification of plants provides a further range of options for improving the composition of biomass and for plant modifications to assist the fabrication of biofuels. The relative carbohydrate and lignin content influences the deconstruction of plant cell walls to biofuels. Key options for facilitating the deconstruction leading to higher monomeric sugar release from plants include increasing cellulose content, reducing cellulose crystallinity, and/or altering the amount or composition of noncellulosic polysaccharides or lignin. Modification of chemical linkages within and between these biomass components may improve the ease of deconstruction. Expression of enzymes in the plant may provide a cost-effective option for biochemical conversion to biofuel.

About 64% of the total aboveground biomass in sugarcane production is from the culm, of which ~90% is present in fiber and sugars. Understanding the transcriptome in the sugarcane culm, and the transcripts that are associated with the accumulation of the sugar and fiber components would facilitate the modification of biomass composition for enhanced biofuel and biomaterial production. The Sugarcane Iso-Seq Transcriptome (SUGIT) database was used as a reference for RNA-Seq analysis of variation in gene expression between young and mature tissues, and between 10 genotypes with varying fiber content. Global expression analysis suggests that each genotype displayed a unique expression pattern, possibly due to different chromosome combinations and maturation amongst these genotypes. Apart from direct sugar- and fiber-related transcripts, the differentially expressed (DE) transcripts in this study belonged to various supporting pathways that are not obviously involved in the accumulation of these major biomass components. The analysis revealed 1,649 DE transcripts between the young and mature tissues, while 555 DE transcripts were found between the low and high fiber genotypes. Of these, 151 and 23 transcripts respectively, were directly involved in sugar and fiber accumulation. Most of the transcripts identified were up-regulated in the young tissues (2 to 22-fold, FDR adjusted p-value

Background Sugarcane accumulates very high levels of sucrose in the culm. Elucidation of the molecular mechanisms that allows such high sucrose synthesis and accumulation (up to 650 mM) is made difficult by the complexity of the highly polyploid genome. Here we report the use of RNA Seq data to characterize the sucrose synthase (SuSy) genes expressed in the transcriptome of the mature sugarcane plant. Results Four SuSy gene families were identified in the sugarcane Iso-Seq long read transcriptome (SUGIT) through gene annotation of transcripts that mapped to reference SuSy genes from sorghum and maize. In total, 38, 19, 14, and 2 transcripts were identified for the four corresponding SuSy genes 1, 2, 4 and 7, respectively. Comparative studies using available SuSy genes from sorghum (1, 2, 4, 6, 7) and maize (1–7) revealed that the sugarcane SuSy genes were interrupted by multiple introns and that they share a highly conserved gene structure. Spatial expression of the four SuSy genes in sugarcane genotypes and in the progenitor species, Saccharum spontaneum and Saccharum officinarum, was studied in the leaf and root tissues and also in three regions of the culm tissue; top, middle and bottom internodes. Expression profiles indicated that all SuSy transcripts were differentially expressed between the top and bottom tissues, with high expression in the top tissues, lower expression in the bottom and moderate expression in the middle, indicating a gradient of SuSy activity in the sugarcane culm. Further, the root tissue had similar expression levels to that of the top internodes while leaf tissues showed lower expression. In the progenitors, SuSy7 was found to be highly expressed in S. officinarum while the other three SuSy genes had moderate expression in both the progenitors. Conclusions The high expression of the SuSy genes in sink tissues, the top internodes and the roots suggests functional roles in sucrose utilization to support growth. The SuSy7 gene has not been previously reported in sugarcane. As sugarcane is unique in storing such high amounts of sucrose, it is possible that there are more SuSy genes/isoforms with specific expression patterns to be discovered in this complex system.