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Extensive cultivation of ginger for years caused ecological environment worse off and ginger productivity decrease due to wilt diseases, which also happens in nearly all crops in continuous cropping, especially in Ralstonia solanacearum caused wilt farmlands. In the present work, based on analysis of Ralstonia solanacearum species complex (RSSC) host plant phylogeny and RSSC control factors, an organic ecological method to combat the wilt is proposed that is expected to guarantee continuous cropping of ginger (Zingiber officinale Rosc.) achieving double harvest in economy and environment.

Plant somatic embryogenesis (SE) is an in vitro biological process wherein bipolar structures are induced to form somatic cells and regenerate into whole plants. MicroRNA (miRNA) is an essential player in plant SE. However, the mechanism of microRNA408 (miR408) in SE remains elusive. Here, we used stable transgenic technology in longan (Dimocarpus longan) embryogenic calli to verify the mechanism by which miR408 promotes cell division and differentiation of longan early SE. dlo-miR408-3p regulated riboflavin biosynthesis by targeting nudix hydrolase 23 (DlNUDT23), a previously unidentified gene mediating N6-methyladenosine (m6A) modification and influencing RNA homeostasis and cell cycle gene expression during longan early SE. We showed that DlMIR408 overexpression (DlMIR408-OE) promoted 21-nt miRNA biosynthesis. In DlMIR408-OE cell lines, dlo-miR408-3p targeted and downregulated DlNUDT23, promoted riboflavin biosynthesis, decreased flavin mononucleotide (FMN) accumulation, promoted m6A level, and influenced miRNA homeostasis. DNA replication, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, the pentose phosphate pathway, and taurine and hypotaurine metabolism were also closely associated with riboflavin metabolism. In a riboflavin feeding assay, dlo-miR408-3p and pre-miR408 were upregulated and DlNUDT23 was downregulated, increasing the m6A level and cell division and differentiation in longan globular embryos. When riboflavin biosynthesis was inhibited, dlo-miR408-3p was downregulated and DlNUDT23 was upregulated, which decreased m6A modification and inhibited cell division but did not inhibit cell differentiation. FMN artificial demethylated m6A modification affected the homeostasis of precursor miRNA and miRNA. Our results revealed a mechanism underlying dlo-miR408-3p-activated riboflavin biosynthesis in which DlNUDT23 is targeted, m6A modification is dynamically mediated, and cell division is affected, promoting early SE in plants.

Circular RNAs (circRNAs) are widely involved in plant growth and development. However, the function of circRNAs in plant somatic embryogenesis (SE) remains elusive. Here, by using high-throughput sequencing, a total of 5029 circRNAs were identified in the three stages of longan (Dimocarpus longan Lour.) early SE. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that differentially expressed (DE) circRNA host genes were enriched in the ‘non-homologous end-joining’ (NHEJ) and ‘butanoate metabolism’ pathways. In addition, the reactive oxygen species (ROS) content during longan early SE was determined. The results indicated that ROS-induced DNA double-strand breaks may not depend on the NHEJ repair pathway. Correlation analyses of the levels of related metabolites (glutamate, γ-aminobutyrate and pyruvate) and the expression levels of circRNAs and their host genes involved in butanoate metabolism were performed. The results suggested that circRNAs may act as regulators of the expression of cognate mRNAs, thereby affecting the accumulation of related compounds. A competing endogenous RNA (ceRNA) network of DE circRNAs, DE mRNAs, DE long noncoding RNAs (lncRNAs) and DE microRNAs (miRNAs) was constructed. The results showed that the putative targets of the noncoding RNA (ncRNAs) were significantly enriched in the KEGG pathways ‘mitogen-activated protein kinase signaling’ and ‘nitrogen metabolism’. Furthermore, the expression patterns of the candidate circRNAs, lncRNAs, miRNAs and mRNAs confirmed the negative correlation between miRNAs and ceRNAs. In addition, two circRNA overexpression vectors were constructed to further verify the ceRNA network correlations in longan early SE. Our study revealed the potential role of circRNAs in longan early SE, providing new insights into the intricate regulatory mechanism underlying plant SE.

The NAM, ATAF1/2, and CUC2 form a huge plant-specific gene family of NAC TFs that are involved in the growth, development, and regulation of biotic and abiotic stress responses. Although the draft genome of longan (Dimocarpus longan Lour.) has been published, however the comprehensive data regarding the functions, evolution, and expression patterns of the NAC family are still unavailable. In this study, a comprehensive analysis of the NAC transcription factor family in longan was performed, and a total of 114 NAC genes were found. We investigated the NAC gene family exploring the phylogeny, domain conservation, intron/exon, motifs, cis-regulatory elements, protein-protein interaction, and expression profiles of RNA-seq samples in different tissues and early somatic embryogenesis of longan. Phylogenetic analysis showed that the genes with similar gene structure and motif distribution were clustered in the same group. Cis-element identification indicates the possible role of NAC genes in biological and physiological processes. Protein-protein interaction identified the DlNACs homologous with Arabidopsis proteins. We further investigated the expression pattern of DlNAC genes in different tissues (pulp, stem, large fruit, young fruit, and flower) during somatic embryogenesis at embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC), and globular embryos (GE) stages. The qRT-PCR results showed that the DlNAC genes were expressed higher at EC and GE stage compared with ICpEC stage. In conclusion, our results provide insight into the evolution, diversity, and characterization of NAC genes in the longan and provide a base for understanding their biological roles and molecular mechanisms in plants.

Amaranth plants are rich in betalains. Additionally, the molecular structure and metabolic pathway of betalains are different in amaranth than in beet. To date, only a few studies have examined the...

The dynamic alterations in cell wall (CW) biosynthesis play an essential role in physiological isolation during the plant somatic embryogenesis (SE). However, the mechanisms underlying the functions of cell wall-associated miRNAs (CW-miRNA) remain poorly understood in plant SE. Here, we have identified 36 distinct candidate miRNAs associated with CW biosynthesis from longan third-generation genome as well as miRNA transcriptome, and modified RLM-RACE validated four distinct miRNA, which specifically targeted four CW-related genes. More importantly, we found that the dlo-miR397a-antagomir significantly enhanced DlLAC7 expression and improved laccase activity. Interestingly, inhibition of dlo-miR397a increased CW lignin deposition and promoted the tightening of protodermal cell by miRNA-mimic technology during early SE. Moreover, overexpression of dlo-miR408-3p (dlo-miR408-3p-agomir) markedly decreased DlLAC12 expression. dlo-miR408-3p-agomir activated rapid cell division, thus promoting the globular embryo (GE) development, which might be due to high DNA synthesis activity in protoepidermal cells, rather than affecting lignin synthesis. The subcellular location also indicated that both DlLAC7 and DlLAC12 proteins were primarily localized in CW and regulated CW biosynthesis. Overall, our findings provided new insight on the molecular regulatory networks comprising various miRNAs associated with cell wall, and established that dlo-miR397a and dlo-miR408-3p played differential roles during early SE in longan. The findings also shed some light on the potential role of miRNA target DlLAC regulating in vivo embryonic development of plant.

Cytochrome P450 (CYP), a multi-gene superfamily, is involved in a broad range of physiological processes, including hormone responses and secondary metabolism throughout the plant life cycle. Longan (Dimocarpus longan), a subtropical and tropical evergreen fruit tree, its embryonic development is closely related to the yield and quality of fruits. And a large number of secondary metabolites, such as flavonoids and carotenoids, are also produced during the longan somatic embryogenesis (SE). It is important, therefore, to study potential functions of CYPs in longan. However, the knowledge of longan CYPs is still very limited. Here, a total of 327 DlCYPs were identified using the genome-search method, which could be classified into nine clans. The expansion of the DlCYP family was mainly caused by tandem duplication (TD) events. Promoter cis-acting elements analysis elucidated that DlCYPs played important roles in hormonal responses. A total of 246 DlCYPs exhibited six different expression patterns during the early SE based on longan transcriptomic data. Eight DlCYPs underwent alternative splicing (AS) events, and they might produce one to six isoforms. And the AS transcript of DlCYP97C1 might act as an alternative to the full-length transcript in ICpEC and GE stages. Finally, protein-protein interaction (PPI) networks and miRNA target prediction elucidated that DlCYPs might be involved in the phenylpropanoid metabolic pathway and primarily regulated and targeted by miR413. In summary, our results provided valuable inventory for understanding the classification and biological functions of DlCYPs and provided insight into further functional verification of DlCYPs during the longan early SE.

Summary Ascorbic acid-glutathione (AsA-GSH) cycle represents important antioxidant defense system in planta. Here we utilized Oncidium cytosolic ascorbate peroxidase (OgCytAPX) as a model to demonstrate that CytAPX of several plants possess dual catalytic activity of both AsA and GSH, compared with the monocatalytic activity of Arabidopsis APX (AtCytAPX). Structural modeling and site-directed mutagenesis identified that three amino acid residues, Pro63, Asp75, and Tyr97, are required for oxidization of GSH in dual substrate catalytic type. Enzyme kinetic study suggested that AsA and GSH active sites are distinctly located in cytosolic APX structure. Isothermal titration calorimetric and UV-visible analysis confirmed that cytosolic APX is a heme-containing protein, which catalyzes glutathione in addition to ascorbate. Biochemical and physiological evidences of transgenic Arabidopsis overexpressing OgCytAPX1 exhibits efficient reactive oxygen species-scavenging activity, salt and heat tolerances, and early flowering, compared with Arabidopsis overexpressing AtCytAPX. Thus results on dual activity CytAPX impose significant advantage on evolutionary adaptive mechanism in planta., Graphical Abstract, Highlights • Cytosolic ascorbate peroxidase in several plants possess two substrate oxidation • Amino acid residues Pro63, Asp75, and Tyr97 were identified as GSH oxidation • OgcytAPX1-catalyzing GSH is an independent biochemical step in AsA-GSH cycle • CytAPXs with two substrate-binding activities enhance stress tolerance and flowering, Plant Biochemistry; Plant Biology; Plant Evolution

Radish root formation under short photoperiod is a common sense, however, cherry radish is a kind of four-season radish and the relation of photoperiod and cherry radish root formation is not clear until now. Effect of different photoperiods (8 h/16 h, 12 h/12 h, and 16 h/8 h) on the development and antioxidant ability of cherry radish as well as expression of relative genes was investigated in this study. Surprisingly, the most robust radish plant and largest biomass were found at the long photoperiod regime (16h/8 h), even though both the polyphenol content and antioxidant ability in terms of DPPH and FRAP were lower than those in 12/12 h photoperiod. Moreover, under 16h/8h photoperiod it can form the radish product organs in 30 d but not for the others. Gene expression analysis showed that eight non-coding miRNAs and coding RNAs were both involved in the regulation of radish root formation. MiR160, miR165, and miR396 were up-regulated by 16h/8 h photoperiod treatment while miR156 and miR157 were down-regulated. The up-regulation of miR160 and the opposite expression trend of ARF16 indicated that miR160 may mediate the light-respond root formation by targeting ARF16. An auxin response factor ARF6 and a cell wall structure related WRKY2 were also significantly up-regulated by the longer illumination treatment compared with the control. Thus, unlike other radish roots that form in short-day condition, the cherry radish roots grow fastest in long photoperiod and miR160-ARF16 are involved in the quick formation of cherry radish root.

DNA methylation plays essential roles in gene regulation, chromatin structure stability, gene imprinting, X chromosome inactivation and embryonic development. However, the dynamics and functions of DNA methylation during the early stage of longan (Dimocarpus longan) somatic embryogenesis (SE) are still unclear. In this study, we carried out whole genome bisulphite sequencing and transcriptome sequencing analyses for embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC) and globular embryos (GE) in an early SE system. At a global level, the DNA 5-methylcytosine content in EC, ICpEC and GE was 24.59, 19.65 and 19.74%, respectively, suggesting a global decrease in DNA methylation from EC to ICpEC and then a slight increase from ICpEC to GE. Differentially methylated region (DMR) analysis showed that hypomethylation mainly occurred in CHH contexts. Gene ontology and Kyoto encyclopedia of genes and genomes analysis of hypomethylated-CHH-DMR-associated genes revealed that zein biosynthesis, fatty acid biosynthesis, circadian rhythm and mitophagy pathways were involved in longan early SE. Expression patterns of DNA methyltransferase and demethylase genes during longan early SE suggested that the decrease in DNA methylation was probably regulated by DNA methyltransferase genes and the DNA demethylase gene REPRESSOR OF SILENCING 1 (ROS1). The correlation between DNA hypomethylation and gene expression revealed that decreased DNA methylation did not cause extensive changes in gene expression during early longan SE and that gene expression may be affected by methylation changes in gene and downstream regions. Inhibiting DNA methylation with 5-azacytidine treatment in EC promoted the formation of GE and enhanced the capability of longan SE. Our results suggest that DNA demethylation has important roles in longan SE development.

Longan (Dimocarpus longan Lour.) is an important commercial fruit tree in southern China. The embryogenesis of longan affects the quality and yield of fruit. A large number of alternative splicing events occurs during somatic embryogenesis (SE), which is regulated by serine/arginine-rich (SR) proteins. However, the functions of SR proteins in longan are poorly understood. In this study, 21 Dlo-SR gene family members belonging to six subfamilies were identified, among which Dlo-RSZ20a, Dlo-SR30, Dlo-SR17, Dlo-SR53 and Dlo-SR32 were localized in the nucleus, Dlo-RSZ20b, Dlo-RSZ20c, Dlo-RSZ20d, Dlo-SC18, Dlo-RS2Z29, Dlo-SCL41, and Dlo-SR33 were localized in chloroplasts, and Dlo-RS43, Dlo-SC33, Dlo-SC37, Dlo-RS2Z33, Dlo-RS2Z16, Dlo-RS2Z24, Dlo-SCL43, Dlo-SR112, and Dlo-SR59 were localized in the nucleus and chloroplasts. The Dlo-SR genes exhibited differential expression patterns in different tissues of longan. The transcript levels of Dlo-RSZ20a, Dlo-SC18, Dlo-RS2Z29, DLo-SR59, Dlo-SR53, and Dlo-SR17 were low in all analyzed tissues, whereas Dlo-RS43, Dlo-RS2Z16, Dlo-RS2Z24, and Dlo-SR30 were highly expressed in all tissues. To clarify their function during SE, the transcript levels of Dlo-SR genes were analyzed at different four stages of SE, comprising non-embryonic callus (NEC), friable-embryogenic callus (EC), incomplete compact pro-embryogenic culture (ICpEC) and globular embryo (GE). Interestingly, the transcript levels of Dlo-RS2Z29 and Dlo-SR112 were increased in embryogenic cells compared with the NEC stage, whereas transcript levels of Dlo-RSZ20a, Dlo-RS43, Dlo-SC37, and Dlo-RS2Z16 were especially increased at the GE stage compared with the other stages. Alternative splicing events of Dlo-SR mRNA precursors (pre-mRNAs) was detected during SE, with totals of 41, 29, 35, and 44 events detected during NEC, EC, ICpEC, and GE respectively. Protein-protein interaction analysis showed that SR proteins were capable of interaction with each other. The results indicate that the alternative splicing of Dlo-SR pre-mRNAs occurs during SE and that Dlo-SR proteins may interact to regulate embryogenesis of longan.

BackgroundSomatic embryogenesis (SE) is a process of somatic cells that dedifferentiate to totipotent embryonic stem cells and generate embryos in vitro. Longan SE has been established and wildly used as model system for studying embryogenesis in woody plants, SE-related genes had been characterized. In spite of that, a comprehensive overview of SE at a molecular level is still absent. To understand the molecular mechanisms during longan SE, we examined the transcriptome changes by using Illumina HiSeq from the four distinct developmental stages, including non-embryogenic callus (NEC), embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC), globular embryos (GE).ResultsRNA-seq of the four samples generated a total of 243.78 million high quality reads, approximately 81.5% of the data were mapped to longan genome. The cDNA libraries of NEC, EC, ICpEC and GE, generated 22,743, 19,745, 21,144, 21,102 expressed transcripts, 1935, 1710, 1816, 1732 novel transcripts, 2645, 366, 505, 588 unique genes, respectively. Comparative transcriptome analysis showed that a total of 10,642, 4180, 5846 and 1785 genes were differentially expressed in the pairwise comparisons of NEC_vs_EC, EC_vs_ICpEC, EC_vs_GE, ICpEC_vs_GE, respectively. Among them, plant hormones signalling related genes were significantly enriched, especially the auxin and cytokinin signalling components. The transcripts of flavonoid biosynthesis related genes were mainly expressed in NEC, while fatty acid biosynthesis related genes mainly accumulated in early SE. In addition, the extracelluar protein encoding genesLTP,CHI,GLP,AGP,EP1were related to longan SE. Combined with the FPKM value of longan nine tissues transcription, 27 SE specific or preferential genes (LEC1,LEC1-like,PDF1.3,GH3.6,AGL80,PIN1,BBM,WOX9,WOX2,ABI3, et al.) and 28 NEC preferential genes (LEA5,CNOT3,DC2.15,PR1–1,NsLTP2,DIR1,PIP1,PIP2.1,TIP2–1,POD-P7andPOD5et al.) were characterized as molecular markers for longan early SE. qRT-PCR validation of SE-related genes showed a high correlation between RNA-seq and qRT-PCR data.ConclusionThis study provides new insights into the role of the transcriptome during early SE in longan. Differentially expressed genes reveal that plant hormones signalling, flavonoid and fatty acid biosynthesis, and extracelluar protein related genes were involved in longan early SE. It could serve as a valuable platform resource for further functional studies addressing embryogenesis in woody plants.

Background Long non-coding RNAs (lncRNAs) are involved in variable cleavage, transcriptional interference, regulation of DNA methylation and protein modification. However, the regulation of lncRNAs in plant somatic embryos remains unclear. The longan (Dimocarpus longan) somatic embryogenesis (SE) system is a good system for research on longan embryo development. Results In this study, 7643 lncRNAs obtained during early SE in D. longan were identified by high-throughput sequencing, among which 6005 lncRNAs were expressed. Of the expressed lncRNAs, 4790 were found in all samples and 160 were specifically expressed in embryogenic callus (EC), 154 in incomplete embryogenic compact structures (ICpECs), and 376 in globular embryos (GEs). We annotated the 6005 expressed lncRNAs, and 1404 lncRNAs belonged to 506 noncoding RNA (ncRNA) families and 4682 lncRNAs were predicted to target protein-coding genes. The target genes included 5051 cis-regulated target genes (5712 pairs) and 1605 trans-regulated target genes (3618 pairs). KEGG analysis revealed that most of the differentially expressed target genes (mRNAs) of the lncRNAs were enriched in the “plant-pathogen interaction” and “plant hormone signaling” pathways during early longan SE. Real-time quantitative PCR confirmed that 20 selected lncRNAs showed significant differences in expression and that five lncRNAs were related to auxin response factors. Compared with the FPKM expression trends, 16 lncRNA expression trends were the same in qPCR. In lncRNA-miRNA-mRNA relationship prediction, 40 lncRNAs were predicted to function as eTMs for 15 miRNAs and 7 lncRNAs were identified as potential miRNA precursors. In addition, we verified the lncRNA-miRNA-mRNA regulatory relationships by transient expression of miRNAs (miR172a, miR159a.1 and miR398a). Conclusion Analyses of lncRNAs during early longan SE showed that differentially expressed lncRNAs were involved in expression regulation at each SE stage, and may form a regulatory network with miRNAs and mRNAs. These findings provide new insights into lncRNAs and lay a foundation for future functional analysis of lncRNAs during early longan SE. Electronic supplementary material The online version of this article (10.1186/s12864-018-5158-z) contains supplementary material, which is available to authorized users.

Amaranth plants contain large amounts of betalains, including betaxanthins and betacyanins. Amaranthin is a betacyanin, and its molecular structure and associated metabolic pathway differ from those of betanin in beet plants. The chlorophyll, carotenoid, betalain, and flavonoid contents in amaranth leaves were analyzed. The abundance of betalain, betacyanin, and betaxanthin was 2-5-fold higher in the red leaf sectors than in the green leaf sectors. Moreover, a transcriptome database was constructed for the red and green sectors of amaranth leaves harvested from 30-day-old seedlings. 22 unigenes were selected to analyze the expression profiles in the two leaf sectors. The RNA-sequencing data indicated that many unigenes are involved in betalain metabolic pathways. The potential relationships between diverse metabolic pathways and betalain metabolism were analyzed. The validation of the expression of 22 selected unigenes in a qRT-PCR assay revealed the genes that were differentially expressed in the two leaf sectors. Betalains were biosynthesized in specific tissues of the red sectors of amaranth leaves. Almost all of the genes related to betalain metabolism were identified in the transcriptome database, and the expression profiles were different between the red sectors and green sectors in the leaf. Amaranth plants consist of diverse metabolic pathways, and the betalain metabolic pathway is linked to a group of other metabolic pathways.

Mother of FT and TFL1 (MFT) belongs to phosphatidylethanolamine-binding protein (PEBP) family, which plays an important role in flowering time regulation, seed development, and germination. To gain insight into the molecular function of DlMFT in Dimocarpus longan Lour., we isolated DlMFT and its promoter sequence from longan embryogenic callus (EC). Bioinformatic analysis indicated that the promoter contained multiphytohormones and light responsive regulatory elements. Subcellular localization showed that the given the DlMFT signal localized in the nucleus, expression profiling implied that DlMFT showed significant upregulation during somatic embryogenesis (SE) and zygotic embryogenesis (ZE), and particular highly expressed in late or maturation stages. The accumulation of DlMFT was mainly detected in mature fruit and seed, while it was undetected in abortive seeds, and notably decreased during seed germination. DlMFT responded differentially to exogenous hormones in longan EC. Auxins, salicylic acid (SA) and methyl jasmonate (MeJa) suppressed its expression, however, abscisic acid (ABA), brassinosteroids (BR) showed the opposite function. Meanwhile, DlMFT differentially responded to various abiotic stresses. Our study revealed that DlMFT might be a key regulator of longan somatic and zygotic embryo development, and in seed germination, it is involved in complex plant hormones and abiotic stress signaling pathways.

Betalains are abundant in amaranth plants. Additionally, the betalain molecular structure and metabolic pathway differ from those of betanin in beet plants. To date, only a few studies have examined the regulatory roles of miRNAs in betalain biosynthesis in plants. Thus, we constructed small RNA libraries for the red and green sectors of amaranth leaves to identify miRNAs associated with betalain biosynthesis. We identified 198 known and 41 novel miRNAs. Moreover, 216 miRNAs were distributed in 44 miRNA families, including miR156, miR159, miR160, miR166, miR172, miR319, miR167, miR396, and miR398. An analysis of all unigene sequences in an amaranth transcriptome database resulted in the detection of 493 target genes for the 239 screened miRNAs. The targets included SPL2, ARF18, ARF6, and NAC. A quantitative real-time polymerase chain reaction validation of 20 miRNAs and nine target genes revealed expression-level differences between the red and green sectors of amaranth leaves. This study involved the application of an Illumina sequencing platform to identify miRNAs regulating betalain metabolism in amaranth plants. The data presented herein may provide insights into the molecular mechanisms underlying the regulation of betalain biosynthesis in amaranth and other plant species.

Longan (Dimocarpus longan Lour.) is an important tropical and subtropical fruit tree in Southeast Asia and China. We investigated flower type, morphology, and development, pollen morphology, and pollen vigor of a popular Chinese cultivar �Honghezi�. Results showed that the cultivar developed three types of flower, i.e. two types of male flowers (M1 and M2) and female flower (F).

Piriformospora indica is a plant growth- and disease resistance-promoting fungus in the Sebacinales family. In this study, the effect of this symbiotic fungus on the growth of tissue-cultured banana seedlings was investigated to explore its potential applications in the banana tissue culture industry. P. indica plugs (P-1) and a P. indica chlamydospore suspension (P-2) were co-cultured with banana seedlings in rooting medium to investigate their growth-promoting efficiency. Both the P-1 and P-2 treatments improved the plant biomass and chlorophyll contents of banana seedlings. The P-2 treatment had stronger promoting effects than the P-1 treatment, especially on plant height and root length in the first 3 weeks. At 4 weeks after inoculation, seedlings in the P-1 and P-2 treatments showed increased plant height, more and longer roots, increased shoot fresh weight, leaf width and length, and stem circumference compared with controls. The P. indica treatments also significantly enhanced the chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents in banana leaves. The results of this study verified that the fungus can facilitate the growth of banana seedlings in vitro and shorten the rooting time of tissue-cultured banana seedlings. Therefore, it has the potential to improve the growth and quality of tissue-cultured banana plants.

Trans-acting short-interfering RNAs (tasiRNAs) originate from TAS3 families through microRNA (miRNA) 390-guided cleavage of primary transcripts and target auxin response factors (ARF3/-4), which are involved in the normal development of lateral roots and flowers in plants. However, their roles in embryo development are still unclear. Here, the pathway miR390-TAS3-ARF3/-4 was identified systematically for the first time during somatic embryo development in Dimocarpus longan. We identified the miR390 primary transcript and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, salicylic acid, anaerobic induction, fungal elicitor, circadian control, and heat stress. The longan TAS3 transcript, containing two miR390-binding sites, was isolated; the miR390- guided cleavage site located near the 3′ end of the TAS3 transcript was verified. Eight TAS3-tasiRNAs with the 21-nucleotides phase were found among longan small RNA data, further confirming that miR390-directed TAS3 cleavage leads to the production of tasiRNA in longan. Among them, TAS3_5′D5+ and 5′D6+ tasiRNAs were highly abundant, and verified to target ARF3 and -4, implying that miR390-guided TAS3 cleavage with 21-nucleotides phase leading to the production of tasiRNA-ARF is conserved in plants. Pri-miR390 was highly expressed in friable-embryogenic callus (EC), and less expressed in incomplete compact pro-embryogenic cultures, while miR390 showed its lowest expression in EC and highest expression in torpedo-shaped embryos (TEs). DlTAS3 and DlARF4 both exhibited their lowest expressions in EC, and reached their peaks in the globular embryos stage, which were mainly inversely proportional to the expression of miR390, especially at the globular embryos to cotyledonary embryos (CEs) stages. While DlARF3 showed little variation from the EC to TEs stages, and exhibited its lowest expression in the CEs stage. There was a general lack of correlation between the expressions of DlARF3 and miR390. In addition, pri-miR390, DlTAS3, DlARF3 and -4 were up-regulated by 2,4-D in a concentration-dependent manner. They were also preferentially expressed in roots, pulp, and seeds of ‘Sijimi’ longan, implying their extended roles in the development of longan roots and fruit. This study provided insights into a possible role of miR390-tasiRNAs-ARF in plant somatic embryo development.

Cold stress seriously affects banana growth, yield and fruit quality. Long noncoding RNAs (lncRNAs) have been demonstrated as key regulators of biotic and abiotic stress in plants, but the identification and prediction of cold responsive mRNAs and lncRNAs in wild banana remains unexplored. In present study, a cold resistant wild banana line from China was used to profile the cold-responsive mRNAs and lncRNAs by RNA-seq under cold stress conditions, i.e. 13°C (critical growth temperature), 4°C (chilling temperature), 0°C (freezing temperature) and normal growing condition, i.e. 28°C (control group). A total of 12,462 lncRNAs were identified in cold-stressed wild banana. In mRNA, much more alternative splicing events occurred in wild banana under the cold stress conditions compared with that in the normal growing condition. The GO analysis of differential expression genes (DEGs) showed the biochemical processes and membrane related genes responded positively to the cold stress. The KEGG pathway enrichment analysis of the DEGs showed that the pathways of photosynthesis, photosynthesis-antenna proteins, circadian rhythm-plant, glutathione metabolism, starch and sucrose metabolism, cutin/suberine/biosynthesis were altered or affected by the cold stress conditions. Our analyses of the generated transcriptome and lncRNAs provide new insights into regulating expression of genes and lncRNAs that respond to cold stress in the wild banana.

We investigated immunocytochemical staining of microtubular cytoskeleton of free nuclear endosperm, a tissue which is particularly difficult to fix. This tissue requires fixation for 45 hr to preserve the integrity of the microtubular network after paraformaldehyde based fixation. Low glutaraldehyde concentration in the fixative and the ethanol dehydration retains beta-tubulin antigenicity and the former improves preservation of tissue structure. An ethanol-free embedding method is recommended for immunocytochemical studies of ethanol sensitive target proteins.