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ABSTRACT MYC2, a basic helix-loop-helix (bHLH) transcription factor, is a key regulator in the activation of jasmonate (JA) response. However, the molecular details of MYC2 involving in methyl jasmonate (MeJA)-induced chilling tolerance of fruit remain largely unclear. In the present work, two MYC2 genes, MaMYC2a and MaMYC2b, and one homolog of the inducer of the C-repeat-binding factor (CBF) gene, MaICE1 were isolated and characterized from banana fruit. MaMYC2s and MaICE1 were found to be all localized in the nucleus. In addition, the proline-rich domain (PRD) and the acidic domain (AD) in the N-terminus were important for the transcriptional activation of MaMYC2 in yeast cells. Unlike MaICE1's constitutive expression, MaMYC2a and MaMYC2b were induced rapidly following MeJA treatment during cold storage. Moreover, protein-protein interaction analysis confirmed that MaMYC2s interacted with MaICE1. The expression of ICE-CBF cold-responsive pathway genes including MaCBF1, MaCBF2, MaCOR1, MaKIN2, MaRD2 and MaRD5 was also significantly induced by MeJA. Taken together, our work provides strong evidence that MaMYC2 is involved in MeJA-induced chilling tolerance in banana fruit through physically interacting and likely functionally coordinating with MaICE1, revealing a novel mechanism for ICE1 in response to cold stress as well as during development of induced chilling tolerance. [ABSTRACT FROM AUTHOR]

The regulatory role of cytokinins (CTKs) in leaf senescence has been documented in different species, including Chinese flowering cabbage. However, its physiological and molecular basis relatively remains unknown. In this study, exogenous application of a CTK analogue 6-benzylaminopurine (6-BA) significantly retarded leaf senescence of Chinese flowering cabbage, with less chlorophyll degradation and lower accumulation of reactive oxygen species (ROS) and malondialdehyde compared with the control group. Meanwhile, higher levels of soluble sugars and proteins, flavonoids, cellulose, amino acids, total phenols, procanthocyanins, and vitamin C were retained in 6-BA-treated leaves. 6-BA treatment also prevented the decline in endogenous CTK content and the increase in ethylene, abscisic acid, and jasmonic acid contents. Moreover, the comparative transcriptome analysis revealed that a total of 21,895 differently expressed genes (DEGs) were identified from four comparisons of 6-BA treatment versus the control during senescence. Further analysis showed that most of the identified DEGs were enriched in ROS, respiratory metabolism, and phytohormone pathways, and a total of 50 classes of transcription factors that were possibly involved in modulating these DEGs were obtained. The transcriptional levels of 18 DEGs were verified by Quantitative real-time PCR (qRT-PCR), which confirmed the accuracy of the transcriptomic data. Overall, these findings and data provide a comprehensive view of physiological and molecular events concerning with the CTK-mediated leaf senescence and -maintained quality in economical leafy vegetables., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Banana fruit is prone to chilling injury (CI) during cold storage, resulting in quality deterioration and commodity reduction. The hot water treatment (HWT), dipping banana fruit in hot water (52 °C) for 3 min, reduced CI symptom at 7 °C storage. The purpose of this study was to investigate the potential molecular mechanism of HWT on the alleviation of CI of postharvest banana fruit. It was found that HWT treatment obviously inhibited the increases in CI index, relative electrolytic leakage, and the contents of malonaldehyde (MDA) and O 2 •- , while enhanced proline accumulation. Further transcriptome analysis in the pericarp of banana fruit was evaluated during storage. The results showed that differentially expressed genes (DEGs) in the comparison between control and HWT group were mainly enriched in photosynthesis, chlorophyll metabolism, lipid metabolism, glutathione metabolism, and brassinosteroid and carotenoid biosynthesis. Moreover, transcriptome expression profiles and RT-qPCR analyses exhibited that the corresponding genes involved in these metabolism pathways and heat shock proteins (HSPs) were upregulated by HWT during cold storage. In general, our findings clearly reveal the potential pathways by which HWT alleviates CI in banana fruit, enriching the theoretical basis for the application of hot water to reduce CI in fruits., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Fruit ripening is a complex developmental process, which is modulated by both transcriptional and post-translational events. Control of fruit ripening is important in maintaining moderate quality traits and minimizing postharvest deterioration. In this study, we discovered that the transcription factor MaMYB4 acts as a negative regulator of fruit ripening in banana. The protein levels of MaMYB4 decreased gradually with banana fruit ripening, paralleling ethylene production, and decline in firmness. DNA affinity purification sequencing combined with RNA-sequencing analyses showed that MaMYB4 preferentially binds to the promoters of various ripening-associated genes including ethylene biosynthetic and cell wall modifying genes. Furthermore, ectopic expression of MaMYB4 in tomato delayed tomato fruit ripening, which was accompanied by downregulation of ethylene biosynthetic and cell wall modifying genes. Importantly, two RING finger E3 ligases MaBRG2/3, whose protein accumulation increased progressively with fruit ripening, were found to interact with and ubiquitinate MaMYB4, contributing to decreased accumulation of MaMYB4 during fruit ripening. Transient overexpression of MaMYB4 and MaBRG2/3 in banana fruit ripening delayed or promoted fruit ripening by inhibiting or stimulating ethylene biosynthesis, respectively. Taken together, we demonstrate that MaMYB4 negatively modulates banana fruit ripening, and that MaMYB4 abundance could be regulated by protein ubiquitination, thus providing insights into the role of MaMYB4 in controlling fruit ripening at both transcriptional and post-translational levels., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Ripening of fleshy fruits involves both diverse post-translational modifications (PTMs) and dynamic transcriptional reprogramming, but the interconnection between PTMs, such as protein phosphorylation and transcriptional regulation, in fruit ripening remains to be deciphered. Here, we conducted a phosphoproteomic analysis during banana (Musa acuminata) ripening and identified 63 unique phosphopeptides corresponding to 49 proteins. Among them, a Musa acuminata basic leucine zipper transcription factor21 (MabZIP21) displayed elevated phosphorylation level in the ripening stage. MabZIP21 transcript and phosphorylation abundance increased during banana ripening. Genome-wide MabZIP21 DNA binding assays revealed MabZIP21-regulated functional genes contributing to banana ripening, and electrophoretic mobility shift assay, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction, and dual-luciferase reporter analyses demonstrated that MabZIP21 stimulates the transcription of a subset of ripening-related genes via directly binding to their promoters. Moreover, MabZIP21 can be phosphorylated by MaMPK6-3, which plays a role in banana ripening, and T318 and S436 are important phosphorylation sites. Protein phosphorylation enhanced MabZIP21-mediated transcriptional activation ability, and transient overexpression of the phosphomimetic form of MabZIP21 accelerated banana fruit ripening. Additionally, MabZIP21 enlarges its role in transcriptional regulation by activating the transcription of both MaMPK6-3 and itself. Taken together, this study reveals an important machinery of protein phosphorylation in banana fruit ripening in which MabZIP21 is a component of the complex phosphorylation pathway linking the upstream signal mediated by MaMPK6-3 with transcriptional controlling of a subset of ripening-associated genes., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Fruit ripening in tomato (Solanum lycopersicum) is the result of selective expression of ripening-related genes, which are regulated by transcription factors (TFs). The NAC (NAM, ATAF1/2, and CUC2) TF family is one of the largest families of plant-specific TFs and members are involved in a variety of plant physiological activities, including fruit ripening. Fruit ripening-associated NAC TFs studied in tomato to date include NAC-NOR (non-ripening), SlNOR-like1 (non-ripening like1), SlNAC1, and SlNAC4. Considering the large number of NAC genes in the tomato genome, there is little information about the possible roles of other NAC members in fruit ripening, and research on their target genes is lacking. In this study, we characterize SlNAM1, a NAC TF, which positively regulates the initiation of tomato fruit ripening via its regulation of ethylene biosynthesis. The onset of fruit ripening in slnam1-deficient mutants created by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology was delayed, whereas fruit ripening in OE-SlNAM1 lines was accelerated compared with the wild type. The results of RNA-sequencing (RNA-seq) and promoter analysis suggested that SlNAM1 directly binds to the promoters of two key ethylene biosynthesis genes (1-aminocyclopropane-1-carboxylate synthase: SlACS2 and SlACS4) and activates their expression. This hypothesis was confirmed by electrophoretic mobility shift assays and dual-luciferase reporter assay. Our findings provide insights into the mechanisms of ethylene production and enrich understanding of the tomato fruit ripening regulatory network., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Pitaya (Hylocereus) is the most economically important fleshy-fruited tree of the Cactaceae family that is grown worldwide, and it has attracted significant attention because of its betalain-abundant fruits. Nonetheless, the lack of a pitaya reference genome significantly hinders studies focused on its evolution, as well as the potential for genetic improvement of this crop. Herein, we employed various sequencing approaches, namely, PacBio-SMRT, Illumina HiSeq paired-end, 10× Genomics, and Hi-C (high-throughput chromosome conformation capture) to provide a chromosome-level genomic assembly of 'GHB' pitaya (H. undatus, 2n = 2x = 22 chromosomes). The size of the assembled pitaya genome was 1.41 Gb, with a scaffold N50 of ~127.15 Mb. In total, 27,753 protein-coding genes and 896.31 Mb of repetitive sequences in the H. undatus genome were annotated. Pitaya has undergone a WGT (whole-genome triplication), and a recent WGD (whole-genome duplication) occurred after the gamma event, which is common to the other species in Cactaceae. A total of 29,328 intact LTR-RTs (~696.45 Mb) were obtained in H. undatus, of which two significantly expanded lineages, Ty1/copia and Ty3/gypsy, were the main drivers of the expanded genome. A high-density genetic map of F1 hybrid populations of 'GHB' × 'Dahong' pitayas (H. monacanthus) and their parents were constructed, and a total of 20,872 bin markers were identified (56,380 SNPs) for 11 linkage groups. More importantly, through transcriptomic and WGCNA (weighted gene coexpression network analysis), a global view of the gene regulatory network, including structural genes and the transcription factors involved in pitaya fruit betalain biosynthesis, was presented. Our data present a valuable resource for facilitating molecular breeding programs of pitaya and shed novel light on its genomic evolution, as well as the modulation of betalain biosynthesis in edible fruits.

Key Message: Four MaHDZs are possibly involved in banana fruit ripening by activating the transcription of genes related to ethylene biosynthesis and cell wall degradation, such as MaACO5, MaEXP2, MaEXPA10, MaPG4 and MaPL4. The homeodomain-leucine zipper (HD-ZIP) proteins represent plant-specific transcription factors, which contribute to various plant physiological processes. However, little information is available regarding the association of HD-ZIPs with banana fruit ripening. In this study, we identified a total of 96 HD-ZIP genes in banana genome, which were divided into four different groups consisting of 35, 31, 9 and 21 members in the I, II, III and IV subfamilies, respectively. The expression patterns of MaHDZ genes during fruit ripening showed that MaHDZI.19, MaHDZI.26, MaHDZII.4 and MaHDZII.7 were significantly up-regulated in the ripening stage and thus suggested to be potential regulators of banana fruit ripening. Furthermore, MaHDZI.19, MaHDZI.26, MaHDZII.4 and MaHDZII.7 were found to localize exclusively in the nucleus and exhibit transcriptional activation capacities. Importantly, MaHDZI.19, MaHDZI.26, MaHDZII.4 and MaHDZII.7 stimulated the transcription of several ripening-related genes including MaACO5 related to ethylene biosynthesis, MaEXP2, MaEXPA10, MaPG4 and MaPL4 were associated with cell wall degradation, through directly binding to their promoters. Taken together, our findings expand the functions of HD-ZIP transcription factors and identify four MaHDZs likely involved in regulating banana fruit ripening by activating the expression of genes related to ethylene biosynthesis and cell wall modification, which may have potential application in banana molecular breeding.

Key Message: Banana MaBZR1/2 interact with MaMPK14 to enhance the transcriptional inhibition of cell wall modifying genes including MaEXP2, MaPL2 and MaXET5. Fruit ripening and softening, the major attributes to perishability in fleshy fruits, are modulated by various plant hormones and gene expression. Banana MaBZR1/2, the central transcription factors of brassinosteroid (BR) signaling, mediate fruit ripening through regulation of ethylene biosynthesis, but their possible roles in fruit softening as well as the underlying mechanisms remain to be determined. In this work, we found that MaBZR1/2 directly bound to and repressed the promoters of several cell wall modifying genes such as MaEXP2, MaPL2 and MaXET5, whose transcripts were elevated concomitant with fruit ripening. Moreover, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that MaBZR1/2 physically interacted with a mitogen-activated protein kinase MaMPK14, and this interaction strengthened the MaBZR1/2-mediated transcriptional inhibitory abilities. Collectively, our study provides insight into the mechanism of MaBZR1/2 contributing to fruit ripening and softening, which may have potential for banana molecular improvement.

Aims: Mutant EGFR Non-small cell lung cancer has benefit from gefitinib, but it has limited effect for wild-type EGFR tumors. Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicine, the plant Lithospermum erythrorhizon (zicao), not only can inhibit the tumor growth, but also overcome cancer drug resistance. Our aim is to investigate whether shikonin can enhance antitumor effect of gefitinib in EGFR wild-type lung cancer cells in vitro and in vivo., Materials and Methods: CCK-8 was used to determine the proliferation of EGFR wild-type non-small cell lung cancer. Apoptosis and cell cycle were detected by flow cytometry. PKM2, STAT3, p-STAT3 and cyclinD1 were detected by Western blot. A549 tumor model was established to observe the antitumor effect of shikonin combination with gefitinib in vivo., Key Findings: The results showed that combination of shikonin with gefitinib exhibited synergistic antitumor effect in vitro and in vivo. Its potential molecular mechanisms may be associated with inhibition of PKM2/STAT3/cyclinD1., Significance: These results provide a promising therapeutic approach for the treatment of wild-type EGFR non-small cell lung cancer., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The biological processes involved in banana fruit ripening are extremely complex and modulated by a number of genes such as transcription factors (TFs). Although TFs like MADS, ERF and NAC are implicated in controlling banana ripening, little is known about other TFs such as TCP in this process. In this work, 25 MaTCPs named MaTCP1 to MaTCP25 were characterized from our previously reported transcriptomes related to banana ripening. Expression analysis revealed that these MaTCPs displayed differential expression patterns during the progression of banana ripening. Particularly, MaTCP5, MaTCP19 and MaTCP20 were ethylene-inducible and nuclear-localized, with MaTCP5 and MaTCP20 acting as transcriptional activators while MaTCP19 being a transcriptional inhibitor. Moreover, MaTCP5 and MaTCP20 promoted the transcription of MaXTH10/11 that may play a role in fruit softening during banana ripening, whereas MaTCP19 repressed their transcription, by directly binding to their promoters. Importantly, protein-protein interaction assays demonstrated that MaTCP20 physically interacts with MaTCP5 and MaTCP19 to form heterodimers in vitro and in vivo, and these protein complexes affects their transcriptional activities in regulating the target genes. Taken together, our results provide an overview of the interactions between MaTCPs in controlling the ripening-associated genes and lay a foundation for further investigation of MaTCP gene family in regulating banana fruit ripening., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The ethylene response factor (ERF) and phytohormone jasmonate (JA) are reported to function in leaf senescence. The involvement of ERF in JA-mediated leaf senescence, however, needs to be elucidated. In the present work, we demonstrate a Chinese flowering cabbage ERF transcription factor (TF), BrERF72, that is associated with JA-promoted leaf senescence. Exogenous application of methyl jasmonate (MeJA)-accelerated leaf senescence of Chinese flowering cabbage, evidenced by the data that MeJA treatment led to the stronger reduction in the maximum quantum yield (Fv/Fm), photosynthetic electron transport rate (ETR), and total chlorophyll content, while significant induction in the expression of several senescence-associated genes ( SAGs ) including BrSAG12, BrSAG19 , and chlorophyll catabolic genes ( CCGs ) BrPAO1 , BrNYC1 , BrPPH1 , and BrSGR1 . Increases in levels of endogenous JA and transcripts of JA biosynthetic genes BrLOX4 , BrAOC3 , and BrOPR3 were also found after MeJA treatment. BrERF72 was a MeJA-inducible, nucleus-localized protein, and possessed trans-activation ability. Transient overexpression of BrERF72 in tobacco leaves also promoted leaf senescence. More importantly, further experiments revealed that BrERF72 directly activated expression of BrLOX4 , BrAOC3 , and BrOPR3 through binding to their promoters via the GCC or DRE/CRT cis -element. Together, the novel JA-ERF association reported in our study uncovers a new insight into the transcriptional regulation of JA production mediated by ERF during JA-promoted leaf senescence in Chinese flowering cabbage., Competing Interests: The authors declare that they have no conflict of interest.

Papaya is an important tropical fruit with a rich source of carotenoids. The ripening of papaya is a physiological and metabolic process with remarkable changes including accumulation of carotenoids, which depends primarily on the action of ethylene. Ethylene response is mediated by a transcriptional cascade involving the transcription factor families of EIN3/EILs and ERFs. Although ERF members have been reported to control carotenoid production in Arabidopsis and tomato, whether EIN3/EILs are also involved in carotenoid biosynthesis during fruit ripening remains unclear. In this work, two EIN3 genes from papaya fruit, namely CpEIN3a and CpEIN3b, were studied, of which CpEIN3a was increased during fruit ripening, concomitant with the increase of transcripts of carotenoid biosynthesis-related genes including CpPDS2/4, CpZDS, CpLCY-e and CpCHY-b, and carotenoid content. Electrophoretic mobility shift assays (EMSAs) and transient expression analyses revealed that CpEIN3a was able to bind to the promoters of CpPDS4 and CpCHY-b, and promoted their transcription. Protein-protein interaction assays indicated that CpEIN3a physically interacted with another transcription factor CpNAC2, which acted as a transcriptional activator of CpPDS2/4, CpZDS, CpLCY-e and CpCHY-b by directly binding to their promoters. More importantly, the transcriptional activation abilities of CpPDS2/4, CpLCY-e and CpCHY-b were more pronounced following their interaction. Collectively, our findings suggest that CpEIN3a interacts with CpNAC2 and, individually or co-operatively, activates the transcription of a subset of carotenoid biosynthesis-related genes, providing new insights into the regulatory networks of carotenoid biosynthesis during papaya fruit ripening., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Key Message: CpERF9 controls papaya fruit ripening through transcriptional repression of cell-wall-modifying genes CpPME1/2 and CpPG5 by directly binding to their promoters. Papaya fruit ripening is an intricate and highly coordinated developmental process which is controlled by the action of ethylene and expression of numerous ethylene-responsive genes. Ethylene response factors (ERFs) representing the last regulators of ethylene-signaling pathway determine the specificities of ethylene response. However, knowledge concerning the transcriptional controlling mechanism of ERF-mediated papaya fruit ripening is limited. In the present work, a gene-encoding AP2/ERF protein with two ERF-associated amphiphilic repression (EAR) motifs, named CpERF9, was characterized from papaya fruit. CpERF9 was found to localize in nucleus, and possess transcriptional repression ability. CpERF9 expression steadily decreased during papaya fruit ripening, while several genes encoding pectin methylesterases (PMEs) and polygalacturonases (PGs), such as CpPME1/2 and CpPG5, were gradually increased, paralleling the decline of fruit firmness. Electrophoretic mobility shift assay (EMSA) demonstrated a specific binding of CpERF9 to promoters of CpPME1/2 and CpPG5, via the GCC-box motif. Transient expression of CpERF9 in tobacco repressed CpPME1/2 and CpPG5 promoter activities, which was depended on two EAR motifs of CpERF9 protein. Taken together, these findings suggest that papaya CpERF9 may act as a transcriptional repressor of several cell-wall modifying genes, such as CpPME1/2 and CpPG5, via directly binding to their promoters.

Phytohormone ethylene controls diverse developmental and physiological processes such as fruit ripening via modulation of ethylene signaling pathway. Our previous study identified that ETHYLENE RESPONSE FACTOR11 (MaERF11), a transcription factor in the ethylene signaling pathway, negatively regulates the ripening of banana, but the mechanism for the MaERF11-mediated transcriptional regulation remains largely unknown. Here we showed that MaERF11 has intrinsic transcriptional repression activity in planta. Electrophoretic mobility shift assay and chromatin immunoprecipitation analyses demonstrated that MaERF11 binds to promoters of three ripening-related Expansin genes, MaEXP2, MaEXP7 and MaEXP8, as well as an ethylene biosynthetic gene MaACO1, via the GCC-box motif. Furthermore, expression patterns of MaACO1, MaEXP2, MaEXP7, and MaEXP8 genes are correlated with the changes of histone H3 and H4 acetylation level during fruit ripening. Moreover, we found that MaERF11 physically interacts with a histone deacetylase, MaHDA1, which has histone deacetylase activity, and the interaction significantly strengthens the MaERF11-mediated transcriptional repression of MaACO1 and Expansins Taken together, these findings suggest that MaERF11 may recruit MaHDA1 to its target genes and repress their expression via histone deacetylation., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The DNA binding with one finger (Dof) proteins, a family of plant-specific transcription factors, are involved in a variety of plant biological processes. However, little information is available on their involvement in fruit ripening. We have characterized 25 MaDof genes from banana fruit (Musa acuminata), designated as MaDof1-MaDof25 Gene expression analysis in fruit subjected to different ripening conditions revealed that MaDofs were differentially expressed during different stages of ripening. MaDof10, 23, 24, and 25 were ethylene-inducible and nuclear-localized, and their transcript levels increased during fruit ripening. Moreover, yeast two-hybrid and bimolecular fluorescence complementation analyses demonstrated a physical interaction between MaDof23 and MaERF9, a potential regulator of fruit ripening reported in a previous study. We determined that MaDof23 is a transcriptional repressor, whereas MaERF9 is a transcriptional activator. We suggest that they might act antagonistically in regulating 10 ripening-related genes, including MaEXP1/2/3/5, MaXET7, MaPG1, MaPME3, MaPL2, MaCAT, and MaPDC, which are associated with cell wall degradation and aroma formation. Taken together, our findings provide new insight into the transcriptional regulation network controlling banana fruit ripening., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Key Message: The ripening-induced MaBSD1 acts as a transcriptional activator, and might be involved in banana fruit ripening partly through directly activating the expression of two ripening-associated genes, MaEXP1/2. BSD (BTF2-like transcription factors, synapse-associated proteins and DOS2-like proteins) transcription factors are characterized by a typical BSD domain. However, little information is available concerning their possible roles in plant growth and development, especially in fruit ripening. In the present study, one BSD gene, designated as MaBSD1, was isolated from banana fruit. MaBSD1 has an open reading frame (ORF) of 921 bp which encodes a polypeptide of 306 amino acid residues with molecular weight of 34.80 kDa, and isoelectric point (pI) of 4.54. Subcellular localization and transcriptional activation assays showed that MaBSD1 was localized in both the nucleus and cytoplasm and possessed transcriptional activity. RT-qPCR and promoter activity analysis indicated that MaBSD1 was ethylene and ripening inducible, and the accumulation of MaBSD1 transcript was correlated well with the evolution of ethylene production and ripening process. Moreover, transient assay showed that MaBSD1 could activate the expression of two cell wall modification-related genes, MaEXP1/2, via directly interacting with their promoters. Together, these data suggest that ripening-induced MaBSD1 acts as a transcriptional activator and might be associated with banana fruit ripening, at least partially through directly activating the expression of MaEXP1/2, expanding the limited information concerning the BSD transcription factor in relation to fruit ripening.

Basic helix-loop-helix (bHLH) transcription factors (TFs) are ubiquitously involved in the response of higher plants to various abiotic stresses. However, little is known about bHLH TFs involved in the cold stress response in economically important fruits. Here, five novel full-length bHLH genes, designated as MabHLH1-MabHLH5, were isolated and characterized from banana fruit. Gene expression profiles revealed that MabHLH1/2/4 were induced by cold stress and methyl jasmonate (MeJA) treatment. Transient assays in tobacco BY2 protoplasts showed that MabHLH1/2/4 promoters were activated by cold stress and MeJA treatments. Moreover, protein-protein interaction analysis demonstrated that MabHLH1/2/4 not only physically interacted with each other to form hetero-dimers in the nucleus, but also interacted with an important upstream component of cold signaling MaICE1, with different interaction domains at their N-terminus. These results indicate that banana fruit cold-responsive MabHLHs may form a big protein complex in the nucleus with MaICE1. Taken together, our findings advance our understanding of the possible involvement of bHLH TFs in the regulatory network of ICE-CBF cold signaling pathway.

The involvement of ethylene response factor (ERF) transcription factor (TF) in the transcriptional regulation of ethylene biosynthesis genes during fruit ripening remains largely unclear. In this study, 15 ERF genes, designated as MaERF1-MaERF15, were isolated and characterized from banana fruit. These MaERFs were classified into seven of the 12 known ERF families. Subcellular localization showed that MaERF proteins of five different subfamilies preferentially localized to the nucleus. The 15 MaERF genes displayed differential expression patterns and levels in peel and pulp of banana fruit, in association with four different ripening treatments caused by natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and combined 1-MCP and ethylene treatments. MaERF9 was upregulated while MaERF11 was downregulated in peel and pulp of banana fruit during ripening or after treatment with ethylene. Furthermore, yeast-one hybrid (Y1H) and transient expression assays showed that the potential repressor MaERF11 bound to MaACS1 and MaACO1 promoters to suppress their activities and that MaERF9 activated MaACO1 promoter activity. Interestingly, protein-protein interaction analysis revealed that MaERF9 and -11 physically interacted with MaACO1. Taken together, these results suggest that MaERFs are involved in banana fruit ripening via transcriptional regulation of or interaction with ethylene biosynthesis genes.

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play important roles in plant growth, development, and stress responses. However, the precise role of NAC TFs in relation to fruit ripening is poorly understood. In this study, six NAC genes, designated MaNAC1-MaNAC6, were isolated and characterized from banana fruit. Subcellular localization showed that MaNAC1-MaNAC5 proteins localized preferentially to the nucleus, while MaNAC6 was distributed throughout the entire cell. A transactivation assay in yeast demonstrated that MaNAC4 and MaNAC6, as well as their C-terminal regions, possessed trans-activation activity. Gene expression profiles in fruit with four different ripening characteristics, including natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and a combination of 1-MCP with ethylene treatment, revealed that the MaNAC genes were differentially expressed in peel and pulp during post-harvest ripening. MaNAC1 and MaNAC2 were apparently upregulated by ethylene in peel and pulp, consistent with the increase in ethylene production. In contrast, MaNAC3 in peel and pulp and MaNAC5 in peel were constitutively expressed, and transcripts of MaNAC4 in peel and pulp and MaNAC6 in peel decreased, while MaNAC5 or MaNAC6 in pulp increased slightly during fruit ripening. Furthermore, the MaNAC2 promoter was activated after ethylene application, further enhancing the involvement of MaNAC2 in fruit ripening. More importantly, yeast two-hybrid and bimolecular fluorescence complementation analyses confirmed that MaNAC1/2 physically interacted with a downstream component of ethylene signalling, ethylene insensitive 3 (EIN3)-like protein, termed MaEIL5, which was downregulated during ripening. Taken together, these results suggest that MaNACs such as MaNAC1/MaNAC2, may be involved in banana fruit ripening via interaction with ethylene signalling components.

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors.

Histone deacetylation plays an important role in epigenetic control of gene expression. HD2 is a plant-specific histone deacetylase that is able to mediate transcriptional repression in many biological processes. To investigate the epigenetic and transcriptional mechanisms of longan fruit senescence, one histone deacetylase 2-like gene, DlHD2, and two ethylene-responsive factor-like genes, DlERF1 and DlERF2, were cloned and characterized from longan fruit. Expression of these genes was examined during fruit senescence under different storage conditions. The accumulation of DlHD2 reached a peak at 2 d and 30 d in the fruit stored at 25 °C (room temperature) and 4 °C (low temperature), respectively, or 6 h after the fruit was transferred from 4 °C to 25 °C, when fruit senescence was initiated. However, the DlERF1 transcript accumulated mostly at the later stage of fruit senescence, reaching a peak at 5 d and 35 d in the fruit stored at 25 °C and 4 °C, respectively, or 36 h after the fruit was transferred from low temperature to room temperature. Moreover, application of nitric oxide (NO) delayed fruit senescence, enhanced the expression of DlHD2, but suppressed the expression of DlERF1 and DlERF2. These results indicated a possible interaction between DlHD2 and DlERFs in regulating longan fruit senescence, and the direct interaction between DlHD2 and DlERF1 was confirmed by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Taken together, the results suggested that DlHD2 may act with DlERF1 to regulate gene expression involved in longan fruit senescence.

Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.

Background: Little attention has been paid to characterising the ethylene-signalling pathway genes in relation to abnormal ripening of harvested banana fruit during storage at high temperature. The aim of the present study was to investigate banana fruit abnormal ripening and the expression of ten genes associated with the ethylene-signalling pathway, namely MaACS1, MaACO1, MaERS1-4 and MaEIL1-4, at high temperature. Changes in these parameters of banana fruit at high temperature in response to 1-MCP pretreatment were also investigated., Results: High temperature accelerated the decline in fruit firmness, increased ethylene production and inhibited degreening in banana fruit, resulting in fruit abnormal ripening. In addition, the expression of MaACS1, MaACO1, MaERS2, MaERS3, MaERS4, MaEIL1, MaEIL3 and MaEIL4 was enhanced in banana fruit stored at high temperature. However, application of 1-MCP prior to high temperature storage delayed fruit abnormal ripening and simultaneously suppressed the expression of MaACS1, MaERS2, MaERS3, MaEIL1, MaEIL3 and MaEIL4., Conclusion: The findings of this study suggested that the expression of genes associated with the ethylene-signalling pathway might be involved in banana fruit abnormal ripening at high temperature. Application of 1-MCP suppressed the expression of genes associated with the ethylene-signalling pathway, which may be attributed at least partially to 1-MCP delaying fruit abnormal ripening at high temperature., (Copyright © 2010 Society of Chemical Industry.)

To investigate whether phospholipase D (PLD, EC 3.1.4.4) plays a role in adaptive response of post-harvest fruit to environment, a PLD gene was firstly cloned from grape berry (Vitis Vinifera L. cv. Chardonnay) using RT-PCR and 3'- and 5'-RACE. The deduced amino acid sequence (809 residues) showed 84.7% identity with that of PLD from Ricinus communis. The secondary structures of this protein showed the characteristic C2 domain and two active sites of a phospholipid-metabolizing enzyme. The PLD activity and its expression in response to heat acclimation were then assayed. The results indicated PLD was significantly activated at enzyme activity, as well as accumulation of PLD mRNA and synthesis of new PLD protein during the early of heat acclimation, primary suggesting that the grape berry PLD may be involved in the heat response in post-harvest grape berry. This work offers an important basis for further investigating the mechanism of post-harvest fruit adaptation to environmental stresses.

Background & Objective: Human brain myelin basic protein (MBP) distributes in nervous system and other tissues extensively, and can be detected in many kinds of tumor cells, such as lung cancer, breast cancer, and neuroglioma. However, it has not been reported whether MBP is relevant to the activity of neural invasion of tumors and whether MBP plays a role in biological behaviors of human lung cancer cells. This study was to investigate the inhibitory effect of MBP on hydrogen peroxide (H2O2)-induced apoptosis of human lung cancer cell line YTLMC-90., Methods: YTLMC-90 cells were transfected with plasmid pSVCEPMBPCAT containing MBP cDNA minigene (test group), or empty vector pSVCEPCAT, or received no transfection (control group), and exposed to H2O2. The expression of MBP in YTLMC-90 cells was detected by Western blot. Cell proliferation was measured by MTT assay. The morphologic and ultra-structural changes of apoptotic cells were observed by microscopy with fluorescent staining of acridine orange (AO) and electron microscopy. The DNA fragmentation was examined by agarose gel electrophoresis., Results: After exposed to 200 micromol/L H2O2 for 24 h, the inhibitory rate of cell growth was significantly lower in test group than in empty vector group and control group (36.67% vs. 78.67% and 84.00%, P<0.001). The morphologic and biochemical changes of apoptotic cells, such as shrinkage of cytoplasm and nucleus, fragmentation of chromatin, and ladder pattern of DNA, were commonly observed in cells in control group, but these apoptotic features were not discovered in test group., Conclusion: MBP markedly inhibits H2O2 cytotoxicity to YTLMC-90 cells through promoting cell proliferation and antagonizing H2O2-induced apoptosis.

Background & Objective: It has been shown that neurotrophic factors such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin (NT-3/4) are synthesized in a variety of cells inside and outside the nervous system. These factors are not only able to promote neural survival, proliferation and apoptosis of neural cells but also relevant to the activity of neural invasion of tumors. It has not been reported to date whether BDNF may play roles in the biological behavior of human lung cancer cells. The aim of this experiment was to investigate the effect of BDNF on hydrogen peroxide (H2O2)-induced apoptosis in the human lung cancer cell line YTMLC-90., Methods: The minigene pSVCEPBFCAT containing the promoter and enhancer elements of the human a1(I) collagen gene(COLIA1) at its 3' terminus followed by hBDNF gene cDNA was transfected and derived BDNF ectopic expression in the human lung cancer cells. The cell proliferation was measured by MTT assay. The morphological and ultra-structural changes of apoptotic cells were observed by microscopy with fluorescent stain of acridine orange and electron microscopy. The DNA fragmentation was examined by agarose gel electrophoresis., Results: After exposure of growing cells to 200 micromol/L H2O2 for 24 hours, the inhibition rate of cell growth was 30% in the pSVCEPBFCAT-transfected YTMLC-90, 84.60% in controls of non-transfected YTMLC-90, and 80.00% in pSVCEPCAT-transfected YTMLC-90, respectively (P< 0.001). The morphological and biochemical changes of apoptotic cells such as shrinkage of cytoplasm and nucleus,fragmentation of the chromatin, and ladder pattern of DNA were commonly observed in the cell population of controls, but these apoptotic features were not discovered in the pSVCEPBFCAT-transfected YTMLC-90., Conclusion: BDNF markedly inhibits H2O2 cytotoxicity on human lung cancer cell YTMLC-90 by promoting YTMLC-90 proliferation and antagonizing H2O2-induced apoptosis.

The ciliary neurotrophic factor (CNTF) plays a very important role in the development and regeneration of the nervous system. In this study, the prediction of secondary structure and the hydrophobicity analysis of human CNTF were performed according to the amino acid sequence deduced from the nucleotide sequence of the cDNA. Based on the results of the prediction of structure, the human CNTF gene was modified by insertion and deletion mutagenesis. The various mutants were all highly expressed in E. coli. The recombinant proteins were purified from bacterial via DEAE A-50 and Sephacryl S-200 chromatography, and their survival-promoting activities were determined by using cultures of the dorsal root ganglion neurons of embryonic chick. The results showed that the alpha-helixes in CNTF were critical for the biological activity and the flexible C-terminus of human CNTF was not essential. Our data also indicated that the middle and the tail part of the D-helix might play crucial roles in the biological functions of CNTF.