Metabolite Profiling Reveals the Dynamic Changes in Non-Volatiles and Volatiles during the Enzymatic-Catalyzed Processing of Aijiao Oolong Tea.

The enzymatic reaction stage (ECS) of oolong tea processing plays an important role in the formation of the flavor quality of the oolong tea. To investigate the dynamic changes in the volatile and non-volatile components in the leaves of oolong tea during the ECS, metabolomic studies were carried out using the leaf samples collected at different stages of the ECS of Aijiao oolong tea. Out of the identified 306 non-volatile metabolites and 85 volatile metabolites, 159 non-volatile metabolites and 42 volatile metabolites were screened out as key differential metabolites for dynamic changes during the ECS. A multivariate statistical analysis on the key differential metabolites showed that the accumulations of most metabolites exhibited dynamic changes, while some amino acids, nucleosides, and organic acids accumulated significantly after turning-over treatment. The evolution characteristics of 27 key precursors or transformed VOCs during the ECS of Aijiao oolong tea were clarified, and it was found that the synthesis of aroma substances was mainly concentrated in lipids as precursors and glycosides as precursor pathways. The results revealed the dynamic changes in the flavor metabolites in the ECS during the processing of Aijiao oolong tea, which provided valuable information for the formation of the characteristic flavor of Aijiao oolong tea.

Transcriptomic Analysis for Diurnal Temperature Differences Reveals Gene-Regulation-Network Response to Accumulation of Bioactive Ingredients of Protocorm-like Bodies in Dendrobium officinale.

Dendrobium officinale Kimura et Migo (D. officinale) is one of the most important traditional Chinese medicinal herbs, celebrated for its abundant bioactive ingredients. This study demonstrated that the diurnal temperature difference (DIF) (T1: 13/13 °C, T2: 25/13 °C, and T3: 25/25 °C) was more favorable for high chlorophyll, increased polysaccharide, and total flavonoid contents compared to constant temperature treatments in D. officinale PLBs. The transcriptome analysis revealed 4251, 4404, and 4536 differentially expressed genes (DEGs) in three different comparisons (A: 25/13 °C vs. 13/13 °C, B: 13/13 °C vs. 25/25 °C, and C: 25/13 °C vs. 25/25 °C, respectively). The corresponding up-/down-regulated DEGs were 1562/2689, 2825/1579, and 2310/2226, respectively. GO and KEGG enrichment analyses of DEGs showed that the pathways of biosynthesis of secondary metabolites, carotenoid biosynthesis, and flavonoid biosynthesis were enriched in the top 20; further analysis of the sugar- and flavonol-metabolism pathways in D. officinale PLBs revealed that the DIF led to a differential gene expression in the enzymes linked to sugar metabolism, as well as to flavonol metabolism. Certain key metabolic genes related to ingredient accumulation were identified, including those involved in polysaccharide metabolism (SUS, SUT, HKL1, HGL, AMY1, and SS3) and flavonol (UGT73C and UGT73D) metabolism. Therefore, these findings indicated that these genes may play an important role in the regulatory network of the DIF in the functional metabolites of D. officinale PLBs. In a MapMan annotation of abiotic stress pathways, the DEGs with significant changes in their expression levels were mainly concentrated in the heat-stress pathways, including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs). In particular, the expression levels of HSP18.2, HSP70, and HSF1 were significantly increased under DIF treatment, which suggested that HSF1, HSP70 and HSP18.2 may respond to the DIF. In addition, they can be used as candidate genes to study the effect of the DIF on the PLBs of D. officinale. The results of our qPCR analysis are consistent with those of the transcriptome-expression analysis, indicating the reliability of the sequencing. The results of this study revealed the transcriptome mechanism of the DIF on the accumulation of the functional metabolic components of D. officinale. Furthermore, they also provide an important theoretical basis for improving the quality of D. officinale via the DIF in production.

Functional and Transcriptome Analysis Reveal Specific Roles of Dimocarpus longan DlRan3A and DlRan3B in Root Hair Development, Reproductive Growth, and Stress Tolerance.

Ran GTPases play essential roles in plant growth and development. Our previous studies revealed the nuclear localization of DlRan3A and DlRan3B proteins and proposed their functional redundancy and distinction in Dimocarpus longan somatic embryogenesis, hormone, and abiotic stress responses. To further explore the possible roles of DlRan3A and DlRan3B, gene expression analysis by qPCR showed that their transcripts were both more abundant in the early embryo and pulp in longan. Heterologous expression of DlRan3A driven by its own previously cloned promoter led to stunted growth, increased root hair density, abnormal fruits, bigger seeds, and enhanced abiotic stress tolerance. Conversely, constitutive promoter CaMV 35S (35S)-driven expression of DlRan3A, 35S, or DlRan3B promoter-controlled expression of DlRan3B did not induce the alterations in growth phenotype, while they rendered different hypersensitivities to abiotic stresses. Based on the transcriptome profiling of longan Ran overexpression in tobacco plants, we propose new mechanisms of the Ran-mediated regulation of genes associated with cell wall biosynthesis and expansion. Also, the transgenic plants expressing DlRan3A or DlRan3B genes controlled by 35S or by their own promoter all exhibited altered mRNA levels of stress-related and transcription factor genes. Moreover, DlRan3A overexpressors were more tolerant to salinity, osmotic, and heat stresses, accompanied by upregulation of oxidation-related genes, possibly involving the Ran-RBOH-CIPK network. Analysis of a subset of selected genes from the Ran transcriptome identified possible cold stress-related roles of brassinosteroid (BR)-responsive genes. The marked presence of genes related to cell wall biosynthesis and expansion, hormone, and defense responses highlighted their close regulatory association with Ran.

Genome-Wide Identification and Expression Analysis Reveal bZIP Transcription Factors Mediated Hormones That Functions during Early Somatic Embryogenesis in Dimocarpus longan.

The basic leucine zip (bZIP) transcription factors (TFs) are a group of highly conserved gene families that play important roles in plant growth and resistance to adversity stress. However, studies on hormonal regulatory pathways and functional analysis during somatic embryogenesis (SE) in Dimocarpus longan is still unavailable. In this study, a total of 51 bZIP family members were systematically identified in the whole genome of longan, a comprehensive bioinformatics analysis of DlbZIP (bZIP family members of D. longan) was performed, and subcellular localization and profiles patterns after transiently transformed DlbZIP60 were analyzed. The combined analysis of RNA-seq, ATAC-seq and ChIP-seq showed that four members have different H3K4me1 binding peaks in early SE and differentially expressed with increased chromatin accessibility. Comparative transcriptome analysis of bZIPs expression in early SE, different tissues and under 2,4-D treatment revealed that DlbZIP family might involved in growth and development during longan early SE. The qRT-PCR results implied that DlbZIP family were subjected to multiple hormonal responses and showed different degrees of up-regulated expression under indole-3-acetic acid (IAA), abscisic acid (ABA) and methyl jasmonate (MeJA) treatments, which indicated that they played an important role in the hormone synthesis pathways associated with the early SE of longan. Subcellular localization showed that DlbZIP60 was located in the nucleus, and the contents of endogenous IAA, MeJA and ABA were up-regulated in transiently DlbZIP60 overexpressed cell lines. These results suggest that DlbZIP60 may mediate hormones pathways that functions the development during early SE in longan.

The miR166 targets CsHDZ3 genes to negatively regulate drought tolerance in tea plant (Camellia sinensis).

Drought is the stressor with a significant adverse impact on the yield stability of tea plants. HD-ZIP III transcription factors (TFs) play important regulatory roles in plant growth, development, and stress responses. However, whether and how HD-ZIP III TFs are involved in drought response and tolerance in tea plants remains unclear. Here, we identified seven HD-ZIP III genes (CsHDZ3-1 to CsHDZ3-7) in tea plant genome. The evolutionary analysis demonstrated that CsHDZ3 members were subjected to purify selection. Subcellular localization analysis revealed that all seven CsHDZ3s located in the nucleus. Yeast self-activation and dual-luciferase reporter assays demonstrated that CsHDZ3-1 to CsHDZ3-4 have trans-activation ability whereas CsHDZ3-5 to CsHDZ3-7 served as transcriptional inhibitors. The qRT-PCR assay showed that all seven CsHDZ3 genes could respond to simulated natural drought stress and polyethylene glycol treatment. Further assays verified that all CsHDZ3 genes can be cleaved by csn-miR166. Overexpression of csn-miR166 inhibited the expression of seven CsHDZ3 genes and weakened drought tolerance of tea leaves. In contrast, suppression of csn-miR166 promoted the expression of seven CsHDZ3 genes and enhanced drought tolerance of tea leaves. These findings established the foundation for further understanding the mechanism of CsHDZ3-miR166 modules' participation in drought responses and tolerance.

Genome-Wide Identification and Expression Analysis of DWARF53 Gene in Response to GA and SL Related to Plant Height in Banana.

Dwarfing is one of the common phenotypic variations in asexually reproduced progeny of banana, and dwarfed banana is not only windproof and anti-fallout but also effective in increasing acreage yield. As a key gene in the strigolactone signalling pathway, DWARF53 (D53) plays an important role in the regulation of the height of plants. In order to gain insight into the function of the banana D53 gene, this study conducted genome-wide identification of banana D53 gene based on the M. acuminata, M. balbisiana and M. itinerans genome database. Analysis of MaD53 gene expression under high temperature, low temperature and osmotic stress based on transcriptome data and RT-qPCR was used to analyse MaD53 gene expression in different tissues as well as in different concentrations of GA and SL treatments. In this study, we identified three MaD53, three MbD53 and two MiD53 genes in banana. Phylogenetic tree analysis showed that D53 Musa are equally related to D53 Asparagales and Poales. Both high and low-temperature stresses substantially reduced the expression of the MaD53 gene, but osmotic stress treatments had less effect on the expression of the MaD53 gene. GR24 treatment did not significantly promote the height of the banana, but the expression of the MaD53 gene was significantly reduced in roots and leaves. GA treatment at 100 mg/L significantly promoted the expression of the MaD53 gene in roots, but the expression of this gene was significantly reduced in leaves. In this study, we concluded that MaD53 responds to GA and SL treatments, but "Yinniaijiao" dwarf banana may not be sensitive to GA and SL.

[Identification of banana ADA1 gene family members and their expression profiles under biotic and abiotic stresses].

The Spt-Ada-Gcn5-acetyltransferase (SAGA) is an ancillary transcription initiation complex which is highly conserved. The ADA1 (alteration/deficiency in activation 1, also called histone H2A functional interactor 1, HFI1) is a subunit in the core module of the SAGA protein complex. ADA1 plays an important role in plant growth and development as well as stress resistance. In this paper, we performed genome-wide identification of banana ADA1 gene family members based on banana genomic data, and analyzed the basic physicochemical properties, evolutionary relationships, selection pressure, promoter cis-acting elements, and its expression profiles under biotic and abiotic stresses. The results showed that there were 10, 6, and 7 family members in Musa acuminata, Musa balbisiana and Musa itinerans. The members were all unstable and hydrophilic proteins, and only contained the conservative SAGA-Tad1 domain. Both MaADA1 and MbADA1 have interactive relationship with Sgf11 (SAGA-associated factor 11) of core module in SAGA. Phylogenetic analysis revealed that banana ADA1 gene family members could be divided into 3 classes. The evolution of ADA1 gene family members was mostly influenced by purifying selection. There were large differences among the gene structure of banana ADA1 gene family members. ADA1 gene family members contained plenty of hormonal elements. MaADA1-1 may play a prominent role in the resistance of banana to cold stress, while MaADA1 may respond to the Panama disease of banana. In conclusion, this study suggested ADA1 gene family members are highly conserved in banana, and may respond to biotic and abiotic stress.

Identification and Validation of the miR156 Family Involved in Drought Responses and Tolerance in Tea Plants (Camellia sinensis (L.) O. Kuntze).

The microRNA156 (miR156) family, one of the first miRNA families discovered in plants, plays various important roles in plant growth and resistance to various abiotic stresses. Previously, miR156s were shown to respond to drought stress, but miR156s in tea plants (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. Herein, we identify 47 mature sequences and 28 precursor sequences in tea plants. Our evolutionary analysis and multiple sequence alignment revealed that csn-miR156s were highly conserved during evolution and that the rates of the csn-miR156 members' evolution were different. The precursor sequences formed typical and stable stem-loop structures. The prediction of cis-acting elements in the CsMIR156s promoter region showed that the CsMIR156s had diverse cis-acting elements; of these, 12 CsMIR156s were found to be drought-responsive elements. The results of reverse transcription quantitative PCR (RT-qPCR) testing showed that csn-miR156 family members respond to drought and demonstrate different expression patterns under the conditions of drought stress. This suggests that csn-miR156 family members may be significantly involved in the response of tea plants to drought stress. Csn-miR156f-2-5p knockdown significantly reduced the Fv/Fm value and chlorophyll content and led to the accumulation of more-reactive oxygen species and proline compared with the control. The results of target gene prediction showed that csn-miR156f-2-5p targeted SQUAMOSA promoter binding protein-like (SPL) genes. Further analyses showed that CsSPL14 was targeted by csn-miR156f-2-5p, as confirmed through RT-qPCR, 5' RLM-RACE, and antisense oligonucleotide validation. Our results demonstrate that csn-miR156f-2-5p and CsSPL14 are involved in drought response and represent a new strategy for increasing drought tolerance via the breeding of tea plants.

Analysis of Characteristics in the Macro-Composition and Volatile Compounds of Understory Xiaobai White Tea.

Understory planting affects the growth environment of tea plants, regulating the tea plant growth and the formation of secondary metabolites, which in turn affects the flavor of Xiaobai white tea. The present research adopted biochemical composition determination, widely targeted volatilities (WTV) analysis, multivariate statistical analysis, and odor activity value (OAV) analysis to analyze the characteristics in the macro-composition and volatile compounds of understory white tea. The sensory evaluation results indicated that understory Xiaobai white tea (LWTs) was stronger than ordinary Xiaobai white tea (PWTs) in terms of the taste of smoothness, sweetness, and thickness as well as the aromas of the flower and sweet. Understory planting reduced light intensity and air temperature, increased air humidity, organic matter, total nitrogen, and available nitrogen contents, which improved the growth environment of tea plants. The phytochemical analysis showed that the water-extractable substances, caffeine, flavonoids, and soluble sugar contents of understory tea fresh-leaf (LF) were higher than those of ordinary fresh-leaf (PF). The phytochemical analysis showed that the free amino acids, theaflavins, thearubigins, water-extractable substances, and tea polyphenols contents of LWTs were significantly higher than those of PWTs, which may explain the higher smoothness, sweetness, and thickness scores of LWTs than those of PWTs. The 2-heptanol, 2-decane, damasone, and cedar alcohol contents were significantly higher in LWTs than in PWTs, which may result in stronger flowery and sweet aromas in LWTs than in PWTs. These results provide a firm experimental basis for the observed differences in the flavor of LWTs and PWTs.

Identification of WRKY gene family members in amaranth based on a transcriptome database and functional analysis of AtrWRKY42-2 in betalain metabolism.

Introduction: WRKY TFs (WRKY transcription factors) contribute to the synthesis of secondary metabolites in plants. Betalains are natural pigments that do not coexist with anthocyanins within the same plant. Amaranthus tricolor ('Suxian No.1') is an important leaf vegetable rich in betalains. However, the WRKY family members in amaranth and their roles in betalain synthesis and metabolism are still unclear. Methods: To elucidate the molecular characteristics of the amaranth WRKY gene family and its role in betalain synthesis, WRKY gene family members were screened and identified using amaranth transcriptome data, and their physicochemical properties, conserved domains, phylogenetic relationships, and conserved motifs were analyzed using bioinformatics methods. Results: In total, 72 WRKY family members were identified from the amaranth transcriptome. Three WRKY genes involved in betalain synthesis were screened in the phylogenetic analysis of WRKY TFs. RT-qPCR showed that the expression levels of these three genes in red amaranth 'Suxian No.1' were higher than those in green amaranth 'Suxian No.2' and also showed that the expression level of AtrWRKY42 gene short-spliced transcript AtrWRKY42-2 in Amaranth 'Suxian No.1' was higher than that of the complete sequence AtrWRKY42-1, so the short-spliced transcript AtrWRKY42-2 was mainly expressed in 'Suxian No.2' amaranth. Moreover, the total expression levels of AtrWRKY42-1 and AtrWRKY42-2 were down-regulated after GA3 treatment, so AtrWRKY42-2 was identified as a candidate gene. Therefore, the short splice variant AtrWRKY42-2 cDNA sequence, gDNA sequence, and promoter sequence of AtrWRKY42 were cloned, and the PRI 101-AN-AtrWRKY42-2-EGFP vector was constructed to evaluate subcellular localization, revealing that AtrWRKY42-2 is located in the nucleus. The overexpression vector pRI 101-AN-AtrWRKY42-2-EGFP and VIGS (virus-induced gene silencing) vector pTRV2-AtrWRKY42-2 were transferred into leaves of 'Suxian No.1' by an Agrobacterium-mediated method. The results showed that AtrWRKY42-2 overexpression could promote the expression of AtrCYP76AD1 and increase betalain synthesis. A yeast one-hybrid assay demonstrated that AtrWRKY42-2 could bind to the AtrCYP76AD1 promoter to regulate betalain synthesis. Discussion: This study lays a foundation for further exploring the function of AtrWRKY42-2 in betalain metabolism.

Widely targeted metabolomics analysis reveals the formation of nonvolatile flavor qualities during oolong tea manufacturing: a case study of Jinguanyin.

Background: The manufacturing processes of oolong tea significantly impact its nonvolatile components, leading to the emergence of distinct flavor attributes. Understanding the dynamic changes in nonvolatile components during the manufacturing stages of the Jinguanyin (JGY) cultivar is crucial for unraveling the potential mechanism behind flavor formation. Methods: Comprehensive metabolomics and sensomics analyses were conducted to investigate the dynamic changes in nonvolatile components throughout various phases of oolong tea processing, focusing on the JGY cultivar. Results: A total of 1,005 nonvolatile metabolites were detected, with 562 recognized as significant differential metabolites during various phases of oolong tea processing. Notably, the third turning-over, third setting, and high-temperature treatments exhibited the most significant effects on the nonvolatile metabolites of oolong tea. JGY finished tea demonstrated a characteristic flavor profile, marked by mellowness, sweetness in aftertaste, and a significant Yin rhyme. This flavor profile was collectively promoted by the accumulation of amino acids and organic acids, the decrease in flavonols (3-O-glycosides) and sugar substances, the alteration of phenolic acids, and the stabilization of caffeine. Conclusion: This study contribute to the understanding of the formation of oolong tea flavor qualities. The dynamic changes observed in various types of nonvolatile compounds during oolong tea processing shed light on the intricate interplay of metabolites and their influence on the final flavor characteristics.

Integrated Metabolomic and Transcriptomic Analyses Reveal the Potential Regulation of Flavonoids in the Production of Embryogenic Cultures during Early Somatic Embryogenesis of Longan (Dimocarpus longan Lour.).

Embryogenic cultures of longan (Dimocarpus longan Lour.) contain various metabolites with pharmacological properties that may function in the regulation of somatic embryogenesis (SE). In this study, based on widely targeted metabolomics, 501 metabolites were obtained from the embryogenic calli, incomplete compact proembryogenic cultures, and globular embryos during early SE of longan, among which 41 flavonoids were differentially accumulated during the SE. Using RNA sequencing, 36 flavonoid-biosynthesis-related genes and 43 MYB and 52 bHLH transcription factors were identified as differentially expressed genes. Furthermore, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the flavonoid metabolism-related pathways were significantly enriched during the early SE. These results suggested that the changes in flavonoid levels in the embryogenic cultures of longan were mediated by MYBs and bHLHs via regulating flavonoid-biosynthesis-related genes, thus potentially regulating early SE. The identified metabolites in the embryogenic cultures of longan can be used to develop pharmaceutical ingredients.

Multiomics Analysis Reveals the Involvement of JsLHY in Controlling Aroma Production in Jasmine Flowers.

The Jasminum sambac flower is famous for its rich fragrance. However, our knowledge of the regulatory network for its aroma formation remains largely unknown and therefore needs further study. To this end, an integrated analysis of the volatilomics and transcriptomics of jasmine flowers at different flowering stages was performed. The results revealed many candidate transcription factors (TFs) may be involved in regulating the aroma formation of jasmine, among which the MYB-related TF LATE ELONGATED HYPOCOTYL (JsLHY) was identified as a hub gene. Using the DNA affinity purification sequencing method, dual-luciferase reporter, and yeast one-hybrid assays, we demonstrate that JsLHY can bind the gene promoter regions of six aroma-related structural genes (JsBEAT1, JsTPS34, JsCNL6, JsBPBT, JsAAAT5, and Js4CL7) and directly promote their expression. In addition, suppressing JsLHY expression decreased both the expression of JsLHY-bound genes and the content of related VOCs. The present study reveals how JsLHY participates in jasmine aroma formation.

Transcription factors NF-YB involved in embryogenesis and hormones responses in Dimocarpus Longan Lour.

Introduction: NF-YB transcription factor is an important regulatory factor in plant embryonic development. Results: In this study, 15 longan NF-YB (DlNF-YB) family genes were systematically identified in the whole genome of longan, and a comprehensive bioinformatics analysis of DlNF-YB family was performed. Comparative transcriptome analysis of DlNF-YBs expression in different tissues, early somatic embryogenesis (SE), and under different light and temperature treatments revealed its specific expression profiles and potential biological functions in longan SE. The qRT-PCR results implied that the expression patterns of DlNF-YBs were different during SE and the zygotic embryo development of longan. Supplementary 2,4-D, NPA, and PP333 in longan EC notably inhibited the expression of DlNF-YBs; ABA, IAA, and GA3 suppressed the expressions of DlNF-YB6 and DlNF-YB9, but IAA and GA3 induced the other DlNF-YBs. Subcellular localization indicated that DlNF-YB6 and DlNF-YB9 were located in the nucleus. Furthermore, verification by the modified 5'RNA Ligase Mediated Rapid Amplification of cDNA Ends (5' RLM-RACE) method demonstrated that DlNF-YB6 was targeted by dlo-miR2118e, and dlo-miR2118e regulated longan somatic embryogenesis (SE) by targeting DlNF-YB6. Compared with CaMV35S- actuated GUS expression, DlNF-YB6 and DlNF-YB9 promoters significantly drove GUS expression. Meanwhile, promoter activities were induced to the highest by GA3 but suppressed by IAA. ABA induced the activities of the promoter of DlNF-YB9, whereas it inhibited the promoter of DlNF-YB6. Discussion: Hence, DlNF-YB might play a prominent role in longan somatic and zygotic embryo development, and it is involved in complex plant hormones signaling pathways.

Characterization of MicroRNAs and Gene Expression in ACC Oxidase RNA Interference-Based Transgenic Bananas.

Banana (Musa acuminata, AAA group) is a typically respiratory climacteric fruit. Previously, genes encoding ACC oxidase, one of the key enzymes in ethylene biosynthesis, Mh-ACO1 and Mh-ACO2 in bananas were silenced individually using RNAi interference technology, and fruit ripening of transgenic bananas was postponed. Here, the differential expression of miRNAs and their targeted mRNAs were analyzed in the transcriptomes of fruits at the third ripening stage, peel color more green than yellow, from the untransformed and RNAi transgenic bananas. Five significantly differentially expressed miRNAs (mac-miR169a, mac-miR319c-3p, mac-miR171a, mac-miR156e-5p, and mac-miR164a-5p) were identified. The predicted miRNA target genes were mainly enriched in six KEGG pathways, including 'sulfur relay system', 'protein digestion and absorption', 'histidine metabolism', 'pathogenic E. coli infection', 'sulfur metabolism', and 'starch and sucrose metabolism'. After ethylene treatment, the expression of ACC oxidase silencing-associated miRNAs was down-regulated, and that of their target genes was up-regulated along with fruit ripening. The evolutionary clustering relationships of miRNA precursors among 12 gene families related to fruit ripening were analyzed. The corresponding expression patterns of mature bodies were mainly concentrated in flowers, fruits, and leaves. Our results indicated that ethylene biosynthesis is associated with miRNAs regulating the expression of sulfur metabolism-related genes in bananas.

Transcriptome analysis provides insight into the regulatory mechanisms underlying pollen germination recovery at normal high ambient temperature in wild banana (Musa itinerans).

Introduction: Cultivated banana are polyploid, with low pollen fertility, and most cultivars are male sterile, which leads to difficulties in banana breeding research. The selection of male parent with excellent resistance and pollen fertility is therefore essential for banana breeding. Wild banana (Musa itinerans) have developed many good characteristics during natural selection and constitute an excellent gene pool for breeding. Therefore, research on wild banana breeding is very important for banana breeding. Results: In the current analysis, we examined the changes in viability of wild banana pollens at different temperatures by in vitro germination, and found that the germination ability of wild banana pollens cultured at 28°C for 2 days was higher than that of pollens cultured at 23°C (pollens that could not germinate normally under low temperature stress), 24°C (cultured at a constant temperature for 2 days) and 32°C (cultured at a constant temperature for 2 days). To elucidate the molecular mechanisms underlying the germination restoration process in wild banana pollens, we selected the wild banana pollens that had lost its germination ability under low temperature stress (23°C) as the control group (CK) and the wild banana pollens that had recovered its germination ability under constant temperature incubation of 28°C for 2 days as the treatment group (T) for transcriptome sequencing. A total of 921 differentially expressed genes (DEGs) were detected in CK vs T, of which 265 were up-regulated and 656 were down-regulated. The combined analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the activation, metabolism of various substances (lipids, sugars, amino acids) play a major role in restoring pollen germination capacity. TCA cycle and the sesquiterpenoid and triterpenoid biosynthetic pathways were also significantly enriched in the KEGG pathway. And we found that some DEGs may be associated with pollen wall formation, DNA methylation and DNA repair. The cysteine content, free fatty acid (FFA) content, H2O2 content, fructose content, and sucrose content of pollen were increased at treatment of 28°C, while D-Golactose content was decreased. Finally, the GO pathway was enriched for a total of 24 DEGs related to pollen germination, of which 16 DEGs received targeted regulation by 14 MYBs. Discussions: Our study suggests that the balance between various metabolic processes, pollen wall remodelling, DNA methylation, DNA repairs and regulation of MYBs are essential for germination of wild banana pollens.

Genome-wide identification and expression analysis of the GRAS family under low-temperature stress in bananas.

Introduction: GRAS, named after GAI, RGA, and SCR, is a class of plant-specific transcription factors family that plays a crucial role in growth and development, signal transduction, and various stress responses. Methods: To understand the biological functions of the banana GRAS gene family, a genome-wide identification and bioinformatics analysis of the banana GRAS gene family was performed based on information from the M. acuminata, M. balbisiana, and M. itinerans genomic databases. Result: In the present study, we identified 73 MaGRAS, 59 MbGRAS, and 58 MiGRAS genes in bananas at the whole-genome scale, and 56 homologous genes were identified in the three banana genomes. Banana GRASs can be classified into 10 subfamilies, and their gene structures revealed that most banana GRAS gDNAs lack introns. The promoter sequences of GRASs had a large number of cis-acting elements related to plant growth and development, phytohormone, and adversity stress responsiveness. The expression pattern of seven key members of MaGRAS response to low-temperature stress and different tissues was also examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The microRNAs-MaGRASs target prediction showed perfect complementarity of seven GRAS genes with the five mac-miRNAs. The expression of all seven genes was lowest in roots, and the expression of five genes was highest in leaves during low-temperature stress. The expression of MaSCL27-2, MaSCL27-3, and MaSCL6-1 was significantly lower under low-temperature stress compared to the control, except for MaSCL27-2, which was slightly higher than the 28°C control at 4 h. The expression of MaSCL27-2, MaSCL27-3, and MaSCL6-1 dropped to the lowest levels at 24 h, 12 h, and 4 h, respectively. The MaSCL27-4 and MaSCL6-2 expression was intermittently upregulated, rising to the highest expression at 24h, while the expression of MaSCL22 was less variable, remaining at the control level with small changes. Discussion: In summary, it is tentatively hypothesized that the GRAS family has an important function in low-temperature stress in bananas. This study provides a theoretical basis for further analyzing the function of the banana GRAS gene and the resistance of bananas to cold temperatures.

Genome-Wide Identification of the MPK Gene Family and Expression Analysis under Low-Temperature Stress in the Banana.

Mitogen-activated protein kinases (MAPKs and MPKs) are important in the process of resisting plant stress. In this study, 21, 12, 18, 16, and 10 MPKs were identified from Musa acuminata, Musa balbisiana, Musa itinerans, Musa schizocarpa, and Musa textilis, respectively. These MPKs were divided into Group A, B, C, and D. Phylogenetic analysis revealed that this difference in number was due to the gene shrinkage of the Group B subfamily of Musa balbisiana and Musa textilis. KEGG annotations revealed that K14512, which is involved in plant hormone signal transduction and the plant-pathogen interaction, was the most conserved pathway of the MPKs. The results of promoter cis-acting element prediction and focTR4 (Fusarium oxysporum f. sp. cubense tropical race 4) transcriptome expression analysis preliminarily confirmed that MPKs were relevant to plant hormone and biotic stress, respectively. The expression of MPKs in Group A was significantly upregulated at 4 °C, and dramatically, the MPKs in the root were affected by low temperature. miR172, miR319, miR395, miR398, and miR399 may be the miRNAs that regulate MPKs during low-temperature stress, with miR172 being the most critical. miRNA prediction and qRT-PCR results indicated that miR172 may negatively regulate MPKs. Therefore, we deduced that MPKs might coordinate with miR172 to participate in the process of the resistance to low-temperature stress in the roots of the banana. This study will provide a theoretical basis for further analysis of the mechanism of MPKs under low-temperature stress of bananas, and this study could be applied to molecular breeding of bananas in the future.

Structural and Functional Analysis of the MADS-Box Genes Reveals Their Functions in Cold Stress Responses and Flower Development in Tea Plant (Camellia sinensis).

MADS-box genes comprise a large family of transcription factors that play crucial roles in all aspects of plant growth and development. However, no detailed information on the evolutionary relationship and functional characterization of MADS-box genes is currently available for some representative lineages, such as the Camellia plant. In this study, 136 MADS-box genes were detected from a reference genome of the tea plant (Camellia sinensis) by employing a 569 bp HMM (Hidden Markov Model) developed using nucleotide sequencing including 73 type I and 63 type II genes. An additional twenty-seven genes were identified, with five MIKC-type genes. Truncated and/or inaccurate gene models were manually verified and curated to improve their functional characterization. Subsequently, phylogenetic relationships, chromosome locations, conserved motifs, gene structures, and gene expression profiles were systematically investigated. Tea plant MIKC genes were divided into all 14 major eudicot subfamilies, and no gene was found in Mß. The expansion of MADS-box genes in the tea plant was mainly contributed by WGD/fragment and tandem duplications. The expression profiles of tea plant MADS-box genes in different tissues and seasons were analyzed, revealing widespread evolutionary conservation and genetic redundancy. The expression profiles linked to cold stress treatments suggested the wide involvement of MADS-box genes from the tea plant in response to low temperatures. Moreover, a floral 'ABCE' model was proposed in the tea plant and proved to be both conserved and ancient. Our analyses offer a detailed overview of MADS-box genes in the tea plant, allowing us to hypothesize the potential functions of unknown genes and providing a foundation for further functional characterizations.

[Genome-wide identification of the banana GLR gene family and its expression analysis in response to low temperature and abscisic acid/methyl jasmonate].

Glutamate receptor-like (GLR) is an important class of Ca2+ channel proteins, playing important roles in plant growth and development as well as in response to biotic and abiotic stresses. In this paper, we performed genome-wide identification of banana GLR gene family based on banana genomic data. Moreover, we analyzed the basic physicochemical properties, gene structure, conserved motifs, promoter cis-acting elements, evolutionary relationships, and used real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) to verify the expression patterns of some GLR family members under low temperature of 4 ℃ and different hormone treatments. The results showed that there were 19 MaGLR family members in Musa acuminata, 16 MbGLR family members in Musa balbisiana and 14 MiGLR family members in Musa itinerans. Most of the members were stable proteins and had signal peptides, all of them had 3-6 transmembrane structures. Prediction of subcellular localization indicated that all of them were localized on the plasma membrane and irregularly distributed on the chromosome. Phylogenetic analysis revealed that banana GLRs could be divided into 3 subclades. The results of promoter cis-acting elements and transcription factor binding site prediction showed that there were multiple hormone- and stress-related response elements and 18 TFBS in banana GLR. RT-qPCR analysis showed that MaGLR1.1 and MaGLR3.5 responded positively to low temperature stress and were significantly expressed in abscisic acid/methyl jasmonate treatments. In conclusion, the results of this study suggest that GLR, a highly conserved family of ion channels, may play an important role in the growth and development process and stress resistance of banana.