Alternative 3'-untranslated regions regulate high-salt tolerance of Spartina alterniflora.

High-salt stress continues to challenge the growth and survival of many plants. Alternative polyadenylation (APA) produces mRNAs with different 3'-untranslated regions (3' UTRs) to regulate gene expression at the post-transcriptional level. However, the roles of alternative 3' UTRs in response to salt stress remain elusive. Here, we report the function of alternative 3' UTRs in response to high-salt stress in S. alterniflora (Spartina alterniflora), a monocotyledonous halophyte tolerant of high-salt environments. We found that high-salt stress induced global APA dynamics, and ∼42% of APA genes responded to salt stress. High-salt stress led to 3' UTR lengthening of 207 transcripts through increasing the usage of distal poly(A) sites. Transcripts with alternative 3' UTRs were mainly enriched in salt stress-related ion transporters. Alternative 3' UTRs of HIGH-AFFINITY K+ TRANSPORTER 1 (SaHKT1) increased RNA stability and protein synthesis in vivo. Regulatory AU-rich elements were identified in alternative 3' UTRs, boosting the protein level of SaHKT1. RNAi-knock-down experiments revealed that the biogenesis of 3' UTR lengthening in SaHKT1 was controlled by the poly(A) factor CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 30 (SaCPSF30). Over-expression of SaHKT1 with an alternative 3' UTR in rice (Oryza sativa) protoplasts increased mRNA accumulation of salt-tolerance genes in an AU-rich element-dependent manner. These results suggest that mRNA 3' UTR lengthening is a potential mechanism in response to high-salt stress. These results also reveal complex regulatory roles of alternative 3' UTRs coupling APA and regulatory elements at the post-transcriptional level in plants.

Sulfate-TOR signaling controls transcriptional reprogramming for shoot apex activation.

Plants play a primary role for the global sulfur cycle in the earth ecosystems by reduction of inorganic sulfate from the soil to organic sulfur-containing compounds. How plants sense and transduce the sulfate availability to mediate their growth remains largely unclear. The target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator of nutrient sensing and metabolic signaling to control cell proliferation and growth in all eukaryotes. By tissue-specific Western blotting and RNA-sequencing analysis, we investigated sulfate-TOR signal pathway in regulating shoot apex development. Here, we report that inorganic sulfate exhibits high potency activating TOR and cell proliferation to promote true leaf development in Arabidopsis in a glucose-energy parallel pathway. Genetic and metabolite analyses suggest that this sulfate activation of TOR is independent from the sulfate-assimilation process and glucose-energy signaling. Significantly, tissue specific transcriptome analyses uncover previously unknown sulfate-orchestrating genes involved in DNA replication, cell proliferation and various secondary metabolism pathways, which largely depends on TOR signaling. Systematic comparison between the sulfate- and glucose-TOR controlled transcriptome further reveals that TOR kinase, as the central growth integrator, responds to different nutrient signals to control both shared and unique transcriptome networks, therefore, precisely modulates plant proliferation, growth and stress responses.

Metabolite-mediated TOR signaling regulates the circadian clock in Arabidopsis.

Circadian clocks usually run with a period close to 24 h, but are also plastic and can be entrained by external environmental conditions and internal physiological cues. Two key nutrient metabolites, glucose and vitamin B3 (nicotinamide), can influence the circadian period in both mammals and plants; however, the underlying molecular mechanism is still largely unclear. We reveal that the target of rapamycin (TOR) kinase, a conserved central growth regulator, is essential for glucose- and nicotinamide-mediated control of the circadian period in Arabidopsis Nicotinamide affects the cytosolic adenosine triphosphate concentration, and blocks the effect of glucose-TOR energy signaling on period length adjustment, meristem activation, and root growth. Together, our results uncover a missing link between cellular metabolites, energy status, and circadian period adjustment, and identify TOR kinase as an essential energy sensor to coordinate circadian clock and plant growth.

The Full-Length Transcriptome of Spartina alterniflora Reveals the Complexity of High Salt Tolerance in Monocotyledonous Halophyte.

Spartina alterniflora (Spartina) is the only halophyte in the salt marsh. However, the molecular basis of its high salt tolerance remains elusive. In this study, we used Pacific Biosciences (PacBio) full-length single-molecule long-read sequencing and RNA-seq to elucidate the transcriptome dynamics of high salt tolerance in Spartina by salt gradient experiments. High-quality unigenes, transcription factors, non-coding RNA and Spartina-specific transcripts were identified. Co-expression network analysis found that protein kinase-encoding genes (SaOST1, SaCIPK10 and SaLRRs) are hub genes in the salt tolerance regulatory network. High salt stress induced the expression of transcription factors but repressed the expression of long non-coding RNAs. The Spartina transcriptome is closer to rice than Arabidopsis, and a higher proportion of transporter and transcription factor-encoding transcripts have been found in Spartina. Transcriptome analysis showed that high salt stress induced the expression of carbohydrate metabolism, especially cell-wall biosynthesis-related genes in Spartina, and repressed its expression in rice. Compared with rice, high salt stress highly induced the expression of stress response, protein modification and redox-related gene expression and greatly inhibited translation in Spartina. High salt stress also induced alternative splicing in Spartina, while differentially expressed alternative splicing events associated with photosynthesis were overrepresented in Spartina but not in rice. Finally, we built the SAPacBio website for visualizing full-length transcriptome sequences, transcription factors, ncRNAs, salt-tolerant genes and alternative splicing events in Spartina. Overall, this study suggests that the salt tolerance mechanism in Spartina is different from rice in many aspects and is far more complex than expected.

Engineering Herbicide-Tolerance Rice Expressing an Acetohydroxyacid Synthase with a Single Amino Acid Deletion.

The acetohydroxyacid synthase (AHAS) is an essential enzyme involved in branched amino acids. Several herbicides wither weeds via inhibiting AHAS activity, and the AHAS mutants show tolerance to these herbicides. However, most AHAS mutations are residue substitutions but not residue deletion. Here, residue deletion was used to engineering the AHAS gene and herbicide-tolerant rice. Molecular docking analysis predicted that the W548 of the AHAS was a residue deletion to generate herbicide tolerance. The AHAS-ΔW548 protein was generated in vitro to remove the W548 residue. Interestingly, the deletion led to the tetramer dissociation of the AHAS, while this dissociation did not reduce the activity of the AHAS. Moreover, the W548 deletion contributed to multi-family herbicides tolerance. Specially, it conferred more tolerance to sulfometuron-methyl and bispyribac-sodium than the W548L substitution. Further analysis revealed that AHAS-ΔW548 had the best performance on the sulfometuron-methyl tolerance compared to the wild-type control. Over-expression of the AHAS-ΔW548 gene into rice led to the tolerance of multiple herbicides in the transgenic line. The T-DNA insertion and the herbicide treatment did not affect the agronomic traits and yields, while more branched-chain amino acids were detected in transgenic rice seeds. Residue deletion of W548 in the AHAS could be a useful strategy for engineering herbicide tolerant rice. The increase of branched-chain amino acids might improve the umami tastes of the rice.

Identification and expression analyses of the NAC transcription factor family in Spartina alterniflora.

As a coastal halophyte, Spartina alterniflora has high salt tolerance. However, the mechanism at the molecular level has not been widely studied due to the absence of a reference genome. The proteins of NAC families are plant-specific transcription factors that regulate the growth, development and stress response in plants. To identify the NAC family and explore the relationship between NAC proteins and the growth, development and stress response of Spatina alterniflora, full-length transcriptome data of Spartina alterniflora by the third generation sequencing technology was used as reference sequences in this study to blast with the NAC protein sequences from Oryza sativa, Arabidopsis thaliana and Zea mays. Finally, 62 SaNAC proteins were found in Spartina alterniflora by deep analysis on conserved domains. Then we analyzed sequence alignment, evolution, motif prediction, homology comparison, subcellular localization, tissue and abiotic stress-induced gene differential expression profile on the NAC family members in Spartina alterniflora. As a result, all SaNAC proteins were found containing a conserved NAM domain and having certain evolutionary similarity with rice; two family proteins, SaNAC9 and SaNAC49, were expressed in the nucleus; moreover, SaNAC genes were identified to have distinct expressional profiles in different tissues and stress response of Spartina alterniflora. These results indicated the SaNAC transcription factor family not only had conserved functional domains but also played important role in the regulation of growth, development and abiotic stress response.

Genome-wide dynamics of alternative polyadenylation in rice.

Alternative polyadenylation (APA), in which a transcript uses one of the poly(A) sites to define its 3'-end, is a common regulatory mechanism in eukaryotic gene expression. However, the potential of APA in determining crop agronomic traits remains elusive. This study systematically tallied poly(A) sites of 14 different rice tissues and developmental stages using the poly(A) tag sequencing (PAT-seq) approach. The results indicate significant involvement of APA in developmental and quantitative trait loci (QTL) gene expression. About 48% of all expressed genes use APA to generate transcriptomic and proteomic diversity. Some genes switch APA sites, allowing differentially expressed genes to use alternate 3' UTRs. Interestingly, APA in mature pollen is distinct where differential expression levels of a set of poly(A) factors and different distributions of APA sites are found, indicating a unique mRNA 3'-end formation regulation during gametophyte development. Equally interesting, statistical analyses showed that QTL tends to use APA for regulation of gene expression of many agronomic traits, suggesting a potential important role of APA in rice production. These results provide thus far the most comprehensive and high-resolution resource for advanced analysis of APA in crops and shed light on how APA is associated with trait formation in eukaryotes.

Genome-Wide Characterization and Gene Expression Analyses of GATA Transcription Factors in Moso Bamboo (Phyllostachys edulis).

Moso bamboo is well-known for its rapid-growth shoots and widespread rhizomes. However, the regulatory genes of these two processes are largely unexplored. GATA transcription factors regulate many developmental processes, but their roles in moso bamboo height control and rhizome development remains unexplored. Here, thirty-one bamboo GATA factors (PeGATAs) were identified, which are evolutionarily closer to rice than Arabidopsis, and their gene expression patterns were analyzed in bamboo development and phytohormone response with bioinformatics and molecular methods. Interestingly, PeGATAs could only be classified into three groups. Phytohormone responsive cis-elements were found in PeGATA promoters and the expression profiles showed that PeGATA genes might respond to gibberellin acid and abscisic acid but not to auxin at the transcriptional level. Furthermore, PeGATA genes have a tissue-specific expression pattern in bamboo rhizomes. Interestingly, most PeGATA genes were down-regulated during the rapid-growth of bamboo shoots. In addition, over-expressing one of the PeGATA genes, PeGATA26, significantly repressed the primary root length and plant height of transgenic Arabidopsis plants, which may be achieved by promoting the gibberellin acid turnover. Overall, our results provide insight into the function of GATA transcription factors in bamboo, and into genetic resources for engineering plant height.

Comprehensive profiling of rhizome-associated alternative splicing and alternative polyadenylation in moso bamboo (Phyllostachys edulis).

Moso bamboo (Phyllostachys edulis) represents one of the fastest-spreading plants in the world, due in part to its well-developed rhizome system. However, the post-transcriptional mechanism for the development of the rhizome system in bamboo has not been comprehensively studied. We therefore used a combination of single-molecule long-read sequencing technology and polyadenylation site sequencing (PAS-seq) to re-annotate the bamboo genome, and identify genome-wide alternative splicing (AS) and alternative polyadenylation (APA) in the rhizome system. In total, 145 522 mapped full-length non-chimeric (FLNC) reads were analyzed, resulting in the correction of 2241 mis-annotated genes and the identification of 8091 previously unannotated loci. Notably, more than 42 280 distinct splicing isoforms were derived from 128 667 intron-containing full-length FLNC reads, including a large number of AS events associated with rhizome systems. In addition, we characterized 25 069 polyadenylation sites from 11 450 genes, 6311 of which have APA sites. Further analysis of intronic polyadenylation revealed that LTR/Gypsy and LTR/Copia were two major transposable elements within the intronic polyadenylation region. Furthermore, this study provided a quantitative atlas of poly(A) usage. Several hundred differential poly(A) sites in the rhizome-root system were identified. Taken together, these results suggest that post-transcriptional regulation may potentially have a vital role in the underground rhizome-root system.

High throughput characterizations of poly(A) site choice in plants.

The polyadenylation of mRNA in eukaryotes is an important biological process. In recent years, significant progress has been made in the field of mRNA polyadenylation owing to the advent of the next generation DNA sequencing technologies. The high-throughput sequencing capabilities have resulted in the direct experimental determinations of large numbers of polyadenylation sites, analysis of which has revealed a vast potential for the regulation of gene expression in eukaryotes. These collections have been generated using specialized sequencing methods that are targeted to the junction of 3'-UTR and the poly(A) tail. Here we present three variations of such a protocol that has been used for the analysis of alternative polyadenylation in plants. While all these methods use oligo-dT as an anchor to the 3'-end, they differ in the means of generating an anchor for the 5'-end in order to produce PCR products suitable for effective Illumina sequencing; the use of different methods to append 5' adapters expands the possible utility of these approaches. These methods are versatile, reproducible, and may be used for gene expression analysis as well as global determinations of poly(A) site choice.

A comparative metabolomics analysis of domestic yak (Bos grunniens) milk with human breast milk.

Yaks are tough animals living in Tibet's hypoxic stress environment. However, the metabolite composition of yak milk and its role in hypoxic stress tolerance remains largely unexplored. The similarities and differences between yak and human milk in hypoxic stress tolerance are also unclear. This study explored yak colostrum (YC) and yak mature milk (YMM) using GC-MS, and 354 metabolites were identified in yak milk. A comparative metabolomic analysis of yak and human milk metabolites showed that over 70% of metabolites were species-specific. Yak milk relies mainly on essential amino acids- arginine and essential branched-chain amino acids (BCAAs): L-isoleucine, L-leucine, and L-valine tolerate hypoxic stress. To slow hypoxic stress, human breast milk relies primarily on the neuroprotective effects of non-essential amino acids or derivates, such as citrulline, sarcosine, and creatine. In addition, metabolites related to hypoxic stress were significantly enriched in YC than in YMM. These results reveal the unique metabolite composition of yak and human milk and provide practical information for applying yak and human milk to hypoxic stress tolerance.

High-Altitude Stress Orchestrates mRNA Expression and Alternative Splicing of Ovarian Follicle Development Genes in Tibetan Sheep.

High-altitude stress threatens the survival rate of Tibetan sheep and reduces their fertility. However, the molecular basis of this phenomenon remains elusive. Here, we used RNA-seq to elucidate the transcriptome dynamics of high-altitude stress in Tibetan sheep ovaries. In total, 104 genes were characterized as high-altitude stress-related differentially expressed genes (DEGs). In addition, 36 DEGs contributed to ovarian follicle development, and 28 of them were downregulated under high-altitude stress. In particular, high-altitude stress significantly suppressed the expression of two ovarian lymphatic system marker genes: LYVE1 and ADAMTS-1. Network analysis revealed that luteinizing hormone (LH)/follicle-stimulating hormone (FSH) signaling-related genes, such as EGR1, FKBP5, DUSP1, and FOS, were central regulators in the DEG network, and these genes were also suppressed under high-altitude stress. As a post-transcriptional regulation mechanism, alternative splicing (AS) is ubiquitous in Tibetan sheep. High-altitude stress induced 917 differentially alternative splicing (DAS) events. High-altitude stress modulated DAS in an AS-type-specific manner: suppressing skipped exon events but increasing retained intron events. C2H2-type zinc finger transcription factors and RNA processing factors were mainly enriched in DAS. These findings revealed high-altitude stress repressed ovarian development by suppressing the gene expression of LH/FSH hormone signaling genes and inducing intron retention of C2H2-type zinc finger transcription factors.

Functional analyses unveil the involvement of moso bamboo (Phyllostachys edulis) group I and II NIN-LIKE PROTEINS in nitrate signaling regulation.

For rapid growth, moso bamboo (Phyllostachys edulis) requires large amounts of nutrients. Nitrate is an indispensable molecular signal to regulate nitrogen absorption and assimilation, which are regulated by group III NIN-LIKE PROTEINs (NLPs). However, no Phyllostachys edulis NLP (PeNLP) has been characterized. Here, eight PeNLPs were identified, which showed dynamic expression patterns in bamboo tissues. Nitrate did not affect PeNLP mRNA levels, and PeNLP1, -2, -5, -6, -7, and -8 successfully restored nitrate signaling in Arabidopsis atnlp7-1 protoplasts through recovering AtNiR and AtNRT2.1 expression. Four group I and II PeNLPs (PeNLP1, -2, -5, and -8) interacted with the nitrate-responsive cis-element of PeNiR. Moreover, nitrate triggered the nuclear retention of PeNLP8. PeNLP8 overexpression in Arabidopsis significantly increased the primary root length, lateral root number, leaf area, and dry and wet weight of the transgenic plants, and PeNLP8 expression rescued the root architectural defect phenotype of atnlp7-1 mutants. Interestingly, PeNLP8 overexpression dramatically reduced nitrate content but elevated total amino acid content in Arabidopsis. Overall, the present study unveiled the potential involvement of group I and II NLPs in nitrate signaling regulation and provided genetic resources for engineering plants with high nitrogen use efficiency.

GSK3/shaggy-like kinase 1 ubiquitously regulates cell growth from Arabidopsis to Moso bamboo (Phyllostachys edulis).

Moso bamboo (Phyllostachys edulis) is one of the fastest growing species with a maximum growth rate of 1 m/day. However, the regulator genes for this explosive growth phenomenon have not been functionally studied. Here, we found that Moso bamboo GSK3/shaggy-like kinase 1 (PeGSK1) acts as a negative regulator of cell growth. Over-expression of PeGSK1 in Arabidopsis showed significant growth arrest phenotypes, including dwarfism, small leaves, reduced cell length, and disturbed cell elongation of petiole. Furthermore, Overexpression of PeGSK1 fully inhibited the longer hypocotyl phenotype of Arabidopsis atgsk1 mutants. In addition, PeGSK1-overexpressing lines were resistant to exogenous BR treatment and PeGSK1 interacted with the brassinosteroid signal transduction key regulator BZR1. The BZR1-dependent cell growth genes were down-regulated in PeGSK1-overexpressing lines. These results indicated that PeGSK1 is functionally similar to AtGSK1 and inhibited cell growth via the brassinosteroid signaling pathway. Importantly, PeGSK1 also interacted with PeBZR1, and the expression pattern of PeGSK1 was negatively correlated with the internode elongation of bamboo, indicating that PeGSK1 is involved in the cell growth of bamboo. In summary, our results provide insight into the role of brassinosteroids in the rapid-growth of bamboo culms and identifying target genes for the genetic manipulation of plant height.

A 3' RACE protocol to confirm polyadenylation sites.

A routine procedure in the study of polyadenylation involves the determinations of the junctions of the mRNA body and the poly(A) tail (the poly(A) site). This is typically accomplished by selectively amplifying the 3'-ends of cDNAs (3'-Rapid Amplification of cDNA Ends, or 3'-RACE), followed by sequencing of individual clones. We have developed a modification of the standard 3'-RACE protocol that couples high specificity with the ability to characterize thousands (or more) of individual sequences. This protocol may be used for numerous purposes, including the confirmation of results obtained using high throughput sequencing technologies.

Genome-wide determination of poly(A) site choice in plants.

Second generation DNA sequencing technologies have been a great boon for the study of mRNA polyadenylation. The experimental determination of large numbers of polyadenylation sites using high-throughput sequencing strategies has provided the necessary platform for deeper understanding the regulation of gene expression in eukaryotes. For generating large sets of data to map poly-A sites, specialized sample preparations that target the junction of 3'-UTR and the poly(A) tail are usually employed. Here, we describe three different protocols that are effectively used for global determinations of poly(A) site choice in plants.