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Horticultural commodities suffer chilling injury following exposure to extremely low temperatures, which results in visible symptoms and considerable quality loss. Therefore, it is of significance to understand the mechanism of this physiological disorder and to develop effective strategies to control it. Chilling stress causes alteration in structure and function of the plasma membrane, which is assumed to be the primary event in response to cold stress. During this process, the membrane lipid metabolism plays a pivotal role in membrane fluidity and stability. In this review, we summarized the possible roles of membrane lipid metabolism in the development of chilling injury, having the potential for developing effective strategies to alleviate chilling injury in horticultural products under refrigerated storage in practice.

Carotenoids are a class of bioactive compounds that exhibit health-promoting properties for humans, but their regulation in bananas during fruit ripening remains largely unclear. Here, we found that the total carotenoid content continued to be elevated along the course of banana ripening and peaked at the ripening stage followed by a decrease, which is presumably caused by the transcript abundances of carotenoid biosynthetic genes MaLCYB1.1 and MaLCYB1.2. Moreover, a ripening-inducible transcription factor MaSPL16 was characterized, which was a nuclear protein with transactivation activity. Transient transformation of MaSPL16 in banana fruits led to enhanced transcript levels of MaLCYB1.1 and MaLCYB1.2 and hence the total carotenoid accumulation. Importantly, MaSPL16 stimulated the transcription of MaLCYB1.1 and MaLCYB1.2 through directly binding to their promoters. Collectively, our findings indicate that MaSPL16 behaves as an activator to modulate banana carotenoid biosynthesis, which may provide a new target for molecular improvement of the nutritional and bioactive qualities of agricultural crops that accumulate carotenoids.

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.

Starch degradation is a necessary process determining banana fruit quality during ripening. Many starch degradation-related genes are well studied. However, the transcriptional regulation of starch degradation during banana fruit ripening remains poorly understood. In this study, we identified a MYB transcription factor (TF) termed MaMYB3, as a putative protein binding the promoter of MaGWD1, a member of glucan water dikinase (GWD) family which has been demonstrated as an important enzyme of starch degradation. MaMYB3 was ripening- and ethylene-repressible, and its expression was negatively correlated with starch degradation. Acting as a nucleus-localized transcriptional repressor, MaMYB3 repressed the transcription of 10 starch degradation-related genes, including MaGWD1, MaSEX4, MaBAM7-MaBAM8, MaAMY2B, MaAMY3, MaAMY3A, MaAMY3C, MaMEX1, and MapGlcT2-1, by directly binding to their promoters. Interestingly, a previously identified activator of starch degradation-related genes, MabHLH6, was also suppressed by MaMYB3. The ectopic overexpression of MaMYB3 in tomato down-regulated the expression of starch degradation-related genes, inhibited starch degradation and delayed fruit ripening. Based on these findings, we conclude that MaMYB3 negatively impacts starch degradation by directly repressing starch degradation-related genes and MabHLH6, and thereby delays banana fruit ripening. Collectively, our study expands our understanding of the complex transcriptional regulatory hierarchy modulating starch degradation during fruit ripening.

SlARF10 plays an important role in the chlorophyll accumulation and photosynthesis of tomato plants, and regulation of its expression affects the starch, fructose, and sucrose content of fruit., The photosynthesis of green tomatoes contributes to fruit growth and carbon economy. The tomato auxin response factor 10 (SlARF10) belongs to the ARF family and is located in nucleus. In this study, we found that SlARF10 was highly expressed in green fruit. Overexpression of SlARF10 in fruit produced a dark-green phenotype whilst knock-down by RNAi produced a light-green phenotype. Autofluorescence and chlorophyll content analyses confirmed the phenotypes, which indicated that SlARF10 plays an important role in chlorophyll accumulation. Overexpression of SlARF10 positively affected photosynthesis in both leaves and fruit. Furthermore, SlARF10-overexpression lines displayed improved accumulation of starch, fructose, and sucrose in fruit, whilst SlARF10-RNAi lines showed decreased accumulation of starch and sucrose. Regulation of SlARF10 expression altered the expression of AGPase starch biosynthesis genes. SlARF10 positively regulated the expression of SlGLK1, POR, CBP1, and CBP2, which are related to chlorophyll metabolism and regulation. Electrophoretic mobility shift assays confirmed that SlARF10 directly targets to the SlGLK1 promoter. Our results thus indicate that SlARF10 is involved in chlorophyll accumulation by transcriptional activation of SlGLK1 expression in tomato fruit, and provide insights into the link between auxin signaling, chloroplast activity, and sugar metabolism during tomato fruit development.

The transcriptional regulation of aroma formation genes remains poorly understood in the banana. In this work, we found that the expressions of a subset of aroma biosynthetic genes including MaOMT1, MaMT1, MaGT1, MaBCAT1, MaACY1, MaAGT1, and BanAAT, as well as two bZIP genes, MabZIP4 and MabZIP5, were down-regulated when prestored at 7 °C compared to those prestored at 22 °C during the ripening process of banana. Furthermore, MabZIP4 and MabZIP5 were shown to be able to activate the transcription of these aroma biosynthetic genes. Importantly, MabZIP4 directly binds to BanAAT promoter, while MabZIP5 binds to the promoters of MaMT1, MaACY1, MaAGT1, and BanAAT via the G-box motif, implicating the diverse functional significances of MabZIPs in controlling aroma biosynthesis in banana. Overall, this work sheds new insights on the understanding of transcriptional regulatory mechanisms associated with aroma formation during banana ripening.

The efficacy of melatonin on postharvest quality of Chinese flowering cabbage was investigated in this study. Treatment of Chinese cabbage with melatonin significantly delayed leaf yellowing and maintained higher contents of nutrients such as soluble sugars, protein, vitamin C, total flavonoids and phenols during a 7-day storage. Also, melatonin enhanced the activities and expression level of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH). Melatonin treatment decreased tissue weight loss, respiration rate, activities of phosphohexose isomerase (PHI), succinate dehydrogenase (SDH), cytochrome C oxidase (CCO), and ascorbic acid oxidase (AAO) and the expression of their corresponding genes. Additionally, melatonin-treated leaves maintained high energy status, as evidenced by higher total ATPase and nicotinamide adenine dinucleotide kinase (NADK) activities, higher energy charge level, adenosine triphosphate (ATP) and adenosine diphosphate (ADP) contents, lower level of adenosine monophosphate (AMP) and transcript abundance of respiratory enzymes. These findings collectively suggest that melatonin maintains leaf quality of postharvest Chinese flowering cabbage might be, at least in part, due to the reduced ratio of Embden-Meyerhof-Parnas pathway (EMP), tricarboxylic acid (TCA) cycle, and cytochrome pathway (CCP), a relatively higher level of pentose phosphate pathway (PPP) and energy status.

Summary Although starch degradation has been well studied in model systems such as Arabidopsis leaves and cereal seeds, this process in starchy fruits during ripening, especially in bananas, is largely unknown. In this study, 38 genes encoding starch degradation‐related proteins were identified and characterized from banana fruit. Expression analysis revealed that 27 candidate genes were significantly induced during banana fruit ripening, with concomitant conversion of starch‐to‐sugars. Furthermore, iTRAQ‐based proteomics experiments identified 18 starch degradation‐associated enzymes bound to the surface of starch granules, of which 10 were markedly up‐regulated during ripening. More importantly, a novel bHLH transcription factor, MabHLH6, was identified based on a yeast one‐hybrid screening using MaGWD1 promoter as a bait. Transcript and protein levels of MabHLH6 were also increased during fruit ripening. Electrophoretic mobility shift assays, chromatin immunoprecipitation and transient expression experiments confirmed that MabHLH6 activates the promoters of 11 starch degradation‐related genes, including Ma GWD 1, Ma LSF 2, Ma BAM 1, Ma BAM 2, Ma BAM 8, Ma BAM 10, Ma AMY 3, Ma AMY3 C, Ma ISA 2, Ma ISA 3 and MapGlcT2‐2 by recognizing their E‐box (CANNTG) motifs present in the promoters. Collectively, these findings suggest that starch degradation during banana fruit ripening may be attributed to the complex actions of numerous enzymes related to starch breakdown at transcriptional and translational levels, and that MabHLH6 may act as a positive regulator of this process via direct activation of a series of starch degradation‐related genes.

Low-temperature storage is a common strategy for preserving and transporting vegetables and fruits. However, many fruits are hypersensitive to chilling injury, including bananas. In the present study, storage conditions of 11 &deg, C delayed the ripening of Fenjiao (Musa ABB Pisang Awak) banana, and the pulp could be softened after ethephon treatment. Storage conditions of 7 &deg, C prevented fruit from fully softening, and fruit contained a significantly higher starch content and lower soluble sugar content. MaEBF1, a critical gene component in the ethylene signaling pathway, was repressed during ripening after fruit had been stored for 12 days at 7 &deg, C. The expression of a series of starch degradation-related genes and a MaNAC67-like gene were also severely repressed. Both MaEBF1 and MaNAC67-like genes were ethylene-inducible and localized in the nucleus. MaNAC67-like protein was able to physically bind to the promoter of genes associated with starch degradation, including MaBAM6, MaSEX4, and MaMEX1. Yeast two-hybrid, GST-pull down, and BiFC assays showed that MaEBF1 interacted with the MaNAC67-like protein, and their interaction further activated the promoters of MaBAM6 and MaSEX4. The current study indicates that MaNAC67-like is a direct regulator of starch degradation and potential for involvement in regulating chilling-inhibited starch degradation by interacting with the ethylene signaling components in banana fruit. The present work paves the way for further functional analysis of MaEBF1 and MaNAC67-like in banana, which will be useful for understanding the regulation of banana starch metabolism and fruit ripening.

Auxin response factors (ARFs) are involved in auxin-mediated transcriptional regulation in plants. In this study, we performed functional characterization of SlARF6A in tomato. SlARF6A is located in the nucleus and exhibits transcriptional activator activity. Overexpression of SlARF6A increased chlorophyll contents in the fruits and leaves of tomato plants, whereas downregulation of SlARF6A decreased chlorophyll contents compared with those of wild-type (WT) plants. Analysis of chloroplasts using transmission electron microscopy indicated increased sizes of chloroplasts in SlARF6A-overexpressing plants and decreased numbers of chloroplasts in SlARF6A-downregulated plants. Overexpression of SlARF6A increased the photosynthesis rate and accumulation of starch and soluble sugars, whereas knockdown of SlARF6A resulted in opposite phenotypes in tomato leaves and fruits. RNA-sequence analysis showed that regulation of SlARF6A expression altered the expression of genes involved in chlorophyll metabolism, photosynthesis and sugar metabolism. SlARF6A directly bound to the promoters of SlGLK1, CAB, and RbcS genes and positively regulated the expression of these genes. Overexpression of SlARF6A also inhibited fruit ripening and ethylene production, whereas downregulation of SlARF6A increased fruit ripening and ethylene production. SlARF6A directly bound to the SAMS1 promoter and negatively regulated SAMS1 expression. Taken together, these results expand our understanding of ARFs with regard to photosynthesis, sugar accumulation and fruit development and provide a potential target for genetic engineering to improve fruit nutrition in horticulture crops.

Papaya fruits accumulate carotenoids during fruit ripening. Although many papaya carotenoid biosynthesis pathway genes have been identified, the transcriptional regulators of these genes have not been characterized. In this study, a NAC transcription factor, designated as CpNAC1, was characterized from papaya fruit. CpNAC1 was localized exclusively in nucleus and possessed transcriptional activation activity. Expression of carotenoid biosynthesis genes phytoene desaturases (CpPDSs) and CpNAC1 was increased during fruit ripening and by propylene treatment, which correlates well with the elevated carotenoid content in papaya. The gel mobility shift assays and transient expression analyses demonstrated that CpNAC1 directly binds to the NAC binding site (NACBS) motifs in CpPDS2/4 promoters and activates them. Collectively, these data suggest that CpNAC1 may act as a positive regulator of carotenoid biosynthesis during papaya fruit ripening possibly via transcriptional activation of CpPDSs such as CpPDS2/4.

C2H2 zinc finger proteins (C2H2-ZFPs), as one of the largest TF families in eukaryotes, play an essential role in plant development and stress responses, but their involvement in the ripening of economically important fruits, as well as their transcriptional regulatory mechanisms, remain largely unclear. In this study, two C2H2-ZFPs, named as MaC2H2-1/2, were identified and characterized from banana fruit. MaC2H2-1/2 contain a typical ERF-associated amphiphilic repression (EAR) motif at their C-terminus. Subcellular localization demonstrated that MaC2H2-1/2 proteins were both localized to the nucleus. Dual-luciferase reporter assay (DLR) showed that MaC2H2-1/2 were transcriptional repressors in vivo. Gene expression analysis revealed a significant induction of MaC2H2-1/2 transcripts during the ripening of banana fruit with three different ripening characteristics caused by natural, ethylene-induced, and 1-methylcyclopropene (1-MCP)-delayed treatments, which correlated well with ethylene production. More importantly, electrophoretic mobility shift assay (EMSA) and transient expression showed that MaC2H2-1/2 bound to the promoters of the key ethylene biosynthetic genes MaACS1 and MaACO1, and repressed their activities. Collectively, these results suggest that MaC2H2-1/2 are transcriptional repressors and may mediate a finely tuned regulation of ethylene production during banana fruit ripening, possibly via transcriptional repression of ethylene biosynthetic genes, expanding the knowledgement of the involvement of C2H2 zinc finger proteins in fruit ripening.

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., Crop genetics: the secrets of aging in leaves Researchers in China have uncovered a gene involved leaf aging in response to the plant hormone jasmonate in Chinese flowering cabbage. A team led by Jian-ye Chen of the South China Agricultural University treated Chinese flowering cabbage with jasmonate, which is known to promote leaf senescence in other species. In addition to accelerating senescence, the treatment caused the cabbage to produce more jasmonate. The team also used RNA sequencing to discover a new transcription factor, BrERF72, linking jasmonate and leaf senescence. BrERF72 is active during leaf senescence and was more strongly expressed in jasmonate-treated leaves. The team found that BrERF72 activates jasmonate biosynthesis genes, potentially creating a feedback loop reinforcing senescence. These findings extend our understanding of how leaves age, which will help improve breeding, treatment, and storage of leafy crops.

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.

Many reports indicate that RING finger proteins play key roles in regulating plant defense responses against abiotic and biotic stresses, but few have thus far been investigated in economic fruit, such as bananas. In this study, a RING finger gene, designated MaRING1, was isolated and characterized from banana fruit. MaRING1 belonged to the ATL RING-H2 finger protein family, and localized preferentially to the nucleus. Gene expression profiles revealed that MaRING1 was cold-responsive and induced by methyl jasmonate (MeJA) treatment during cold storage. Moreover, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that MaRING1 physically interacted with MaMYC2a, a transcription factor (TF) related to MeJA-induced chilling tolerance of banana fruit. Taken together, our results suggest that MaRING1 might functionally coordinate with MaMYC2a in response to cold stress of banana fruit, expanding our understanding of the possible involvement of RING finger proteins in the regulatory network of MeJA-induced chilling tolerance.

Red pitaya (Hylocereus polyrhizus) fruit is a high-value, functional food, containing a high level of betalains. Several genes potentially related to betalain biosynthesis, such as cytochrome P450-like (CytP450-like), have been identified in pitaya fruit, while their transcriptional regulation remains unclear. In this work, the potential involvement of a WRKY transcription factor, HpWRKY44, in regulating CytP450-like1 expression in pitaya fruit was examined. HpWRKY44, a member of the Group 1 WRKY family, contains two conserved WRKY motifs and is localized in the nucleus. HpWRKY44 also exhibits trans-activation ability. Gene expression analysis showed that the expression of HpCytP450-like1 and HpWRKY44 increased steadily during pitaya fruit coloration, which corresponded with the production of elevated betalain levels in the fruit. HpWRKY44 was also demonstrated to directly bind to and activate the HpCytP450-like1 promoter via the recognition of the W-box element present in the promoter. Collectively, our findings indicate that HpWRKY44 transcriptionally activates HpCytP450-like1, which perhaps, at least in part, contributes to betalain biosynthesis in pitaya fruit. The information provided in the current study provides novel insights into the regulatory network associated with betalain biosynthesis during pitaya fruit coloration.

The regulation of ICE1 protein stability is important to ensure effective cold stress response, and is extensively studied in Arabidopsis. Currently, how ICE1 stability in fruits under cold stress is controlled remains largely unknown. Here, we reported the possible involvement of a SEVEN IN ABSENTIA (SINA) ubiquitin ligase MaSINA1 from banana fruit in affecting MaICE1 stability. MaSINA1 was identified based on a yeast two-hybrid screening using MaICE1 as bait. Further yeast two-hybrid, pull-down, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays confirmed that MaSINA1 interacted with MaICE1. The expression of MaSINA1 was repressed by cold stress. Subcellular localization analysis in tobacco leaves showed that MaSINA1 was localized predominantly in the nucleus. In vitro ubiquitination assay showed that MaSINA1 possessed E3 ubiquitin ligase activity. More importantly, in vitro and semi-in vivo experiments indicated that MaSINA1 can ubiquitinate MaICE1 for the 26S proteasome-dependent degradation, and therefore suppressed the transcriptional activation of MaICE1 to MaNAC1, an important regulator of cold stress response of banana fruit. Collectively, our data reveal a mechanism in banana fruit for control of the stability of ICE1 and for the negative regulation of cold stress response by a SINA E3 ligase via the ubiquitin proteasome system.

The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) proteins are plant-specific transcriptional factors (TFs) functioning in the growth and development of Arabidopsis and other plant species. However, the involvement of LBD TFs in regulating the ripening of economically important fruits is largely unknown. In the present study, four full-length LBD genes, designated as MaLBD1–MaLBD4, were isolated and characterized from banana fruit. Expressions of MaLBD1–MaLBD4 in fruit with three ripening characteristics revealed that MaLBD1/2/3 were ethylene-inducible, and their transcript levels obviously increased during fruit ripening, while MaLBD4 changed slightly. Moreover, the activities of MaLBD1/2/3 promoters were activated after ethylene treatment, further supporting their involvement in fruit ripening. Subcellular localization showed that MaLBD1/2/3 were nuclear proteins, and a transactivation assay in protoplasts demonstrated that MaLBD1/2/3 had transactivation activity. More importantly, a transient expression assay further indicated that MaLBD1/2/3 were transcriptional activators that regulated ripening-related MaEXP1/2 expressions by directly binding to MaEXP1/2 promoters. These results suggest that MaLBDs are involved in regulating fruit ripening, in part by transcriptional activation of the EXPANSIN expression related to cell wall modification. Taken together, our findings provide some new information about the functions of LBD TFs involved in the regulation of fruit ripening.

Our previous studies have indicated that the banana ripening-induced MaNAC1, a NAC (NAM, ATAF1/2 and CUC2) transcription factor (TF) gene, is regulated by ethylene during fruit ripening, and propylene, a functional ethylene analogue, induces cold tolerance of banana fruits. However, the involvement of MaNAC1 in propylene-induced cold tolerance of banana fruits is not understood. In the present work, the possible involvement of MaNAC1 in cold tolerance of banana fruits was investigated. MaNAC1 was noticeably induced by cold stress or following propylene treatment during cold storage. Transient protoplast assays showed that MaNAC1 promoter was activated by cold stress and ethylene treatment. Yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA) and transient expression assays demonstrated MaNAC1 as a novel direct target of MaICE1, and that the ability of MaICE1 binding to MaNAC1 promoter might be enhanced by MaICE1 phosphorylation and cold stress. Moreover, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses revealed physical interaction between MaNAC1 and MaCBF1, a downstream component of inducer of C-repeat binding factor (CBF) expression 1 (ICE1) in cold signalling. Taken together, these results suggest that the cold-responsive MaNAC1 may be involved in cold tolerance of banana fruits through its interaction with ICE1-CBF cold signalling pathway, providing new insights into the regulatory activity of NAC TF.

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.

Fruit softening is an ethylene-dependent ripening event. 1-Methylcyclopropene (1-MCP), a synthetic plant growth regulator structurally related to the natural plant hormone ethylene, is used to slow down the fruit ripening. However, inappropriate 1-MCP treatment tends to cause the elastic texture in papaya fruit. Constitutive triple response 1 (CTR1), a downstream protein of the ethylene receptors, acts as a negative regulator of ethylene signaling. To elucidate the signal transduction pathway involved in the ethylene regulation during papaya ripening and softening, four genes homologous to Arabidopsis CTR1 were isolated from papaya fruit and designated as CpCTR1–4. Their molecular and biochemical properties were characterized, and their expression patterns in papaya fruit after treatments with 1-MCP were investigated. Four CTR1-like genes differ in sequence length and molecular size, and have a relative distance relationship in the evolutionary tree analysis. However, sequences analysis showed that the C-terminus of CpCTR1–4 proteins contained the highly conserved kinase domains, including a protein kinase ATP-binding signature (IGAGSFGTVH) and a serine/threonine protein kinase active site signature (IVHRDLKSPNLLV). Their N-terminus contains the conserved motifs (CN box) that exist in all CTR1-like proteins. CpCTR1–4 proteins were predicted to be located differently in either chloroplast or nuclei or both. Gene expression analysis showed that 1-MCP treatment significantly repressed the expression of CpCTRs and high concentration of 1-MCP treatment had a more significant effect. These results suggested that CpCTR1–4 genes may play different roles in papaya fruit ripening and softening and that the rubbery fruit might be relate to the expression of CpCTRs genes.

EIN3 Binding F-box protein (EBF) is an essential signaling component necessary for ethylene response. However, little information is available on EBF genes during banana fruit ripening. Two EBF genes designated MaEBF1 and MaEBF2 were isolated and characterized from banana fruit. Subcellular localization analysis showed that MaEBF1 and MaEBF2 were both nuclear proteins. Expression of MaEBF1 and MaEBF2 in fruit with four ripening characteristics revealed that MaEBF2 was enhanced by ethylene during fruit ripening, while MaEBF1 changed only slightly. Moreover, the MaEBF2 promoter was activated after ethylene treatment, further supporting its involvement in fruit ripening. More importantly, MaEBF2 was shown to physically interact with MaEIL5 , using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Together, these results suggest that MaEBF may be involved in banana fruit ripening, at least partly via interaction with MaEIL5 . Our findings expand our understanding of the regulatory network of ethylene signaling cascade in banana fruit ripening.

Polygalacturonase (PG), which degrades pectin in cell wall, was proposed to participate in the process of plant organ abscission. In an effort to understand the molecular mechanisms of lychee fruitlet abscission, a full-length cDNA encoding a polygalacturonase, termed as LcPG1, was isolated from lychee by using RT-PCR and RACEs. The LcPG1 cDNA was 1666 bp in length with an open reading frame (ORF) of 1443 bp, encoding a polypeptide of 481 amino acid residues. Homologous and phylogenetic analysis showed that the LcPG1 included three of the four typical domains conserved in PG proteins, and was clustered into the clade E of PG genes. Expression analysis in the fruitlet abscission zone (AZ) indicated that the expression of LcPG1 was positively correlated with the lychee fruitlet abscission, either induced by ethephon or inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the carbohydrate stress caused by the girdling plus defoliation was found to stimulate the fruitlet abscission, which was paralleled with an increase in LcPG1 expression level. All these results suggested that the LcPG1 played an essential role in the process of lychee fruitlet abscission. To our knowledge, it is the first case indicating that a PG gene, belonging to the clade E, is involved in organ shedding.

Δ1-Pyrroline-5-carboxylate synthetase (P5CS) catalyzes the rate-limiting step in proline biosynthesis in plants. In the present study, a full-length cDNA, denominated as CpP5CS2 for Δ1-pyrroline-5-carboxylate synthetase (P5CS), was cloned from papaya using in silico cloning and 3′- or 5′-rapid amplification of cDNA ends (RACE). The full-length cDNA of CpP5CS2 was 2583 bp, with a 2151 bp open reading frame (ORF) encoding a 717 amino acid polypeptide. Sequence analysis showed that CpP5CS2 contained several substrate-binding and catalytic domains and had high homology to other plant P5CSs. The expression pattern of CpP5CS2 in papaya under low (7 °C) and high temperature (35 °C) stresses was examined using real-time quantitative PCR. The results showed that both stresses induced CpP5CS2 expression during the storage period, and the increased expression of CpP5CS2 preceded proline accumulation. In addition, the high temperature caused a more significant induction of CpP5CS2 expression and a higher level of accumulated proline than low temperature.

Auxin, a vital plant hormone, regulates a variety of physiological and developmental processes. It is involved in fruit abscission through transcriptional regulation of many auxin-related genes, including early auxin responsive genes (i.e., auxin/indole-3-acetic acid (AUX/IAA), Gretchen Hagen3 (GH3) and small auxin upregulated (SAUR)) and auxin response factors (ARF), which have been well characterized in many plants. In this study, totally five auxin-related genes, including one AUX/IAA (LcAUX/IAA1), one GH3 (LcGH3.1), one SAUR (LcSAUR1) and two ARFs (LcARF1 and LcARF2), were isolated and characterized from litchi fruit. LcAUX/IAA1, LcGH3.1, LcSAUR1, LcARF1 and LcARF2 contain open reading frames (ORFs) encoding polypeptides of 203, 613, 142, 792 and 832 amino acids, respectively, with their corresponding molecular weights of 22.67, 69.20, 11.40, 88.20 and 93.16 kDa. Expression of these genes was investigated under the treatment of girdling plus defoliation which aggravated litchi fruitlet abscission due to the blockage of carbohydrates transport and the reduction of endogenous IAA content. Results showed that transcript levels of LcAUX/IAA1, LcGH3.1 and LcSAUR1 mRNAs were increased after the treatment in abscission zone (AZ) and other tissues, in contrast to the decreasing accumulation of LcARF1 mRNA, suggesting that LcAUX/IAA1, LcSAUR1 and LcARF1 may play more important roles in abscission. Our results provide new insight into the process of fruitlet abscission induced by carbohydrate stress and broaden our understanding of the auxin signal transduction pathway in this process at the molecular level.

Ethylene plays an essential role in many biological processes including fruit ripening via modulation of ethylene signaling pathway. Ethylene Response Factors (ERFs) are key transcription factors (TFs) involved in ethylene perception and are divided into AP2, RAV, ERF, and DREB sub-families. Although a number of studies have implicated the involvement of DREB sub-family genes in stress responses, little is known about their roles in fruit ripening. In this study, we identified a DREB TF with a EAR motif, designated as MaDEAR1, which is a nucleus-localized transcriptional repressor. Expression analysis indicated that MaDEAR1 expression was repressed by ethylene, with reduced levels of histone H3 and H4 acetylation at its regulatory regions during fruit ripening. In addition, MaDEAR1 promoter activity was also suppressed in response to ethylene treatment. More importantly, MaDEAR1 directly binds to the DRE/CRT motifs in promoters of several cell wall-modifying genes including MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 associated with fruit softening during ripening and represses their activities. These data suggest that MaDEAR1 acts as a transcriptional repressor of cell wall-modifying genes, and may be negatively involved in ethylene-mediated ripening of banana fruit. Our findings provide new insights into the involvement of DREB TFs in the regulation of fruit ripening.

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.

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.

Chromatin remodeling-related proteins, such as linker histone H1, involving in fruit ripening and stress responses are poorly understood. In the present study, a novel cDNA encoding linker histone H1 gene, designated as MaHIS1 was isolated and characterized from banana fruit. The full-length cDNA sequence was 1,253 bp with an open-reading frame (ORF) of 948 bp, encoding 315 amino acids with a molecular weight of 31.98 kDa and a theoretical isoelectric point of 10.67. Subcellular localization analysis showed that MaHIS1 was a nucleus-localized protein. Real-time PCR analysis indicated that expression of MaHIS1 gene is induced by external and internal ethylene during fruit postharvest ripening. Accumulation of MaHIS1 transcript was also obviously enhanced by exogenous hormones, including methyl jasmonate, abscisic acid, and hydrogen peroxide (H2O2), as well as stresses, such as chilling and pathogen Colletotrichum musae infection. Moreover, yeast two-hybrid and bimolecular fluorescence complementation assays showed that MaHIS1 could interact with a transcription factor (TF) MaWRKY1. Taken together, our results suggest that MaHIS1 may be related to ripening and stress responses of banana fruit, and be likely functionally coordinating with MaWRKY1 in these physiological processes. Key message MaHIS1 may be related to ripening and stress responses of banana fruit, and it also could interact with WRKY TF, which expands the very limited information regarding the functions of linker histone H1 in fruits.

CONSTANS (CO) gene is a key transcription regulator that controls the long-day induction of flowering in Arabidopsis plant. However, CO gene involved in fruit ripening and stress responses is poorly understood. In the present study, a novel cDNA encoding CONSTANS-like gene, designated as MaCOL1 was isolated and characterized from banana fruit. The full length cDNA sequence was 1887bp with an open reading frame (ORF) of 1242bp, encoding 414 amino acids with a molecular weight of 46.20kDa and a theoretical isoelectric point of 5.40. Sequence alignment showed that MaCOL1 contained two B-box zinc finger motifs and a CCT domain. In addition, MaCOL1 showed transcriptional activity in yeast and was a nucleus-localized protein. Real-time PCR analysis showed that MaCOL1 was differentially expressed among various banana plant organs, with higher expression in flower. Expression of MaCOL1 in peel changed slightly, while accumulation of MaCOL1 transcripts in pulp obviously increased during natural or ethylene-induced fruit ripening, suggesting that MaCOL1 might be associated with the pulp ripening of banana fruit. Moreover, accumulation of MaCOL1 transcript was obviously enhanced by abiotic and biotic stresses, such as chilling and pathogen Colletotrichum musae infection. Taken together, our results suggest that MaCOL1 is a transcription activator and may be involved in fruit ripening and stress responses.

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.

Summary3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC: 1.1.1.34), encoded by multiple genes in eukaryotes, is known to catalyse the first committed step in the mevalonic acid (MVA) pathway for the biosynthesis of isoprenoids, and has been implicated in fruit size determination. To elucidate whether the genes for HMGR are involved in the regulation of fruit growth in longan (Dimocarpus longan), we cloned two sequence-distinct HMGR genes, designated DlHMG1 and DlHMG2, respectively. Sequence analyses revealed that they shared a high level of sequence and domain conservation, with homologues identified in other species at both the nucleotide and amino acid levels. Gene expression analysis showed that the two genes were differentially regulated in the pericarp, arils, and seed, as well as during fruit development. DlHMG1 was up-regulated particularly in Phase I [0 – 42 d after anthesis (DAA)] and at the early stage of Phase II (42 – 56 DAA), followed by down-regulation in Phase III (≥ 56 DAA); while...

The effects of nitric oxide (NO) treatment on the sensitivity of banana fruit slices to ethylene were investigated. Fruit slices were partial-pressure infiltrated for 3 min in a NO donor Solution of 5 mM sodium nitroprusside (SNP), followed by treatment with 100 mu l l(-1) ethylene for 24 h, then packed into polyethylene bags and stored for 5 d at 24 degrees C. Control slices were treated with distilled water instead of SNP, then treated with ethylene. Changes in fruit firmness, chlorophyll content, respiration rate, the activities of three cell wall modifying enzymes [polygalacturonase (PG), endo-beta-1,4-glucanase (EGase), and beta-galactosidase (beta-Gal)], and total amylase activity were measured. Expression of the expansin (MaExp1) and ethylene receptor (MaERS2) genes were also analysed. The application of NO, prior to ethylene exposure, retarded pulp softening and peel de-greening. Pre-treatment with NO also decreased the respiration rate of banana fruit slices during ripening. Delayed pulp softening by NO was associated with decreased activities of PG, EGase, beta-Gal, and total amylase, as well as reduced expression of the MaExp1 gene. Expression of the MaERS2 gene was also suppressed in NO-treated banana fruit slices. Collectively, these findings suggest that NO pre-treament decreased the sensitivity of banana fruit slices to ethylene, and thereby delayed ripening.

Endo-1,4-β-glucanase (EGase, EC 3.2.1.4) is proposed to be involved in the control of cell wall modification. However, the relationship between EGase and longan fruit growth remains obscure. In the present work, the expression profiles of three Dl-EGase genes in pericarp and aril tissues of longan fruit during growth and development were characterized; moreover, the effects of plant growth substances, naphthalene acetic acid (NAA) and thidiazuron (TDZ) on their expressions of fruits at two different developmental stages were also investigated. The results showed that high levels of EGase activities in aril were in accordance with rapid aril growth, and three Dl-EGase genes exhibited different expression patterns in pericarp and aril during fruit growth and development. Dl-EGase1 and Dl-EGase3 seemed to be associated with the growth of both pericarp and aril, while Dl-EGase2 was closely related to pericarp growth. In addition, treatment of NAA and TDZ at the stage of pericarp growth increased the EGase activities and induced the expressions of all the three Dl-EGases. However, when NAA or TDZ was applied at the rapid aril growth stage, the accumulations of Dl-EGase1 and Dl-EGase2 in the pericarp, and the accumulations of Dl-EGase1 and Dl-EGase3 in the aril were induced. Furthermore, the expression patterns of the three Dl-EGases showed different tissue specificity. It is thus speculated that Dl-EGase genes played a different role in longan fruit growth and were developmentally regulated, and more importantly, the responses of Dl-EGases to plant growth substances were different and dependent on fruit development stage and fruit tissue.

Wounding of plants is a widespread stress in nature. Physical damage frequently occurs during harvesting, handling and marketing of fresh produce. Phenylalanine ammonia-lyase (PAL) is a key enzyme involved in plant wound related phenylpropanoid metabolism while heat shock proteins (HSPs) are involved in abiotic, including wounding, and biotic stress responses in plants. However, little is known about postharvest expression at gene and protein levels of PAL and HSPs in wounding (fresh-cut) fruits. In the present study, expression patterns of PAL and HSPs in fresh-cut and intact banana fruit pulp were characterized by Northern and Western blot analyses. Cuting enhanced Ma-PAL2 mRNA accumulation, new PAL protein synthesis, and PAL activity; thereby resulting in accumulation of total phenolics. HSP70 protein and Ma-HSP70-2 transcript were constitutively expressed and were not affected by cutting. However, Ma-HSP70-1 transcript levels increased in fresh-cut pulp tissue at 2–18 h after cutting. Ma-sHSP-1-CI, Ma-sHSP-3-CII, Ma-HSP90, and Ma-HSP101 transcripts also accumulated in fresh-cut tissues at 2–18 h after cuting, but Ma-sHSP-2-CI did not accumulate. Accumulation of sHSP (HSP17.6), HSP90 and HSP101 proteins exhibited the same pattern of expression as their mRNA. There was no evidence of a possible suppressive effect of HSPs synthesis on PAL protein accumulation. Thus, PAL, sHSP, HSP90 and HSP101 were regulated by cutting both at translational and transcriptional levels while HSP70 (Ma-HSP70-1) was regulated by cutting only at transcription level.

SummaryA full-length phenylalanine ammonia-lyase gene (PAL) cDNA, named DlPAL1, was isolated and characterised from harvested longan fruit. The expression pattern of DlPAL1 and PAL activities in relation to aril breakdown in longan fruit stored at different temperatures were investigated. The results showed that both PAL activity and the accumulation of DlPAL1 mRNA increased during storage at 25oC and at 10oC, which coincided with an increase in the aril breakdown index (ABI). The increase in PAL activity or in the accumulation of DlPAL1 mRNA coincided with the increase in ABI in fruit stored at 4oC for 40 d, then transferred to 25oC for 2 d. No accumulation of DlPAL1 mRNA throughout storage at 4oC could be detected, although the increase in PAL activity was in accordance with the increase in ABI. Furthermore, changes in DlPAL1 mRNA accumulation were not fully in accordance with the changes observed in PAL activity, and a rapid increase in PAL activity in arils was observed as aril breakdown occurred rapi...

Increasing evidence suggests that phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is associated with low temperature stress in plant tissues. Banana fruit are highly susceptible to chilling injury. However, little is known about the role of PAL (i.e. gene expression, protein level and activity) in fruit chilling. In this work, the involvement of PAL induced by heat treatment (38 degrees C for 3 days) prior to storage (8 degrees C) in chilling tolerance was investigated. The PAL inhibitor 2-aminoindan-2-phosphonic acid (AIP) was also used to further study the role of PAL in the chilling tolerance. The results showed that mRNA transcripts (MaPAL1 and MaPAL2) and PAL protein levels increased during storage at chilling temperature. Heat treatment prior to storage alleviated chilling injury and enhanced PAL activity, protein amount and MaPAL1 and MaPAL2 transcript levels. The increases in parameters of PAL upon heat pretreatment were all inhibited by AIP treatment, which resulted in aggravation of chilling injury. Thus, these findings indicate that the induction of PAL by heat pretreatment was regulated at both the transcriptional and the translational levels and that PAL may play a role in heat pretreatment-induced chilling tolerance of banana fruit.

Fruit ripening is generally attributed to disassembly of cellular wall, particularly due to solubilisation and depolymerisation of pectin and hemicellulose. Experiments were conducted to test effects of hydroxyl radicals (·OH) on the scission of cellular wall polysaccharides from pulp tissues of banana fruit at different ripening stage. Cellular wall materials were isolated from pulp tissues of banana fruit at different ripening stages. Two pectic fractions, water soluble pectin (WSP) and acid soluble pectin (ASP), and two hemicellulosic fractions, 1 M KOH soluble hemicellulose (HC1) and 4 M KOH soluble hemicellulos (HC2), were obtained from the cellular wall materials from pulp tissues, respectively. Effects of ·OH induced by the Fenton reaction on the scission of pectin and hemicellulose in vitro were investigated. As fruit ripening progressed, the sugar components of the WSP, HC1 and HC2 attacked by ·OH showed obvious molecular-mass downshifts. Thus, ·OH caused the disassembly of polysaccharides (WSP, ASP, HC1 and HC2) from cellular walls of pulp tissues of banana fruit, demonstrated by the reduced molecular mass distribution. Moreover, ·OH production in pulp tissues increased significantly as banana fruit ripened, which further help account for the role of ·OH in accelerated fruit ripening.

Bananas are highly susceptible to chilling injury (CI) and phenylalanine ammonia-lyase (PAL), as a key enzyme involved in plant phenylpropanoid metabolism, has been associated with low temperature stress in plant tissues. However, little is known about the role of PAL (including PAL activity, gene and protein expression) in postharvest chilling tolerance of banana fruit. Two partial cDNAs sequences (MaPAL1 and MaPAL2) with about 760 bp were cloned from banana pulp by RT-PCR. Western and northern hybridizations were used to investigate expression of PAL protein and PAL genes in fruit stored for 10 days at 7 °C (chilling temperature) and then transferred to 22 °C (room temperature). The effects of propylene (a functional ethylene analog) on their expression in relation to CI were also examined. Northern and western blot analyses revealed that mRNA transcripts of MaPAL1 and MaPAL2 and PAL protein levels in banana fruit during storage increased, reaching a peak at about day 8, and finally decreased at chilling temperature. Prior to low temperature storage, pretreatment with propylene could alleviate CI and enhance PAL activity, protein amount and mRNA transcripts of MaPAL1 and MaPAL2. Moreover, changes in PAL activity, protein amount and accumulation of MaPAL1 and MaPAL2 exhibited almost the same patterns. The results suggest that the induction of PAL in banana fruit during low temperature storage is regulated at transcriptional and translational levels, and is related to reduction in CI symptoms.

The feedback regulation of ethylene biosynthesis in banana [Musa sp. (AAA group, Cavendish subgroup) cv. Grand Nain] fruit was investigated in an attempt to clarify the opposite effect of 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, before and after the onset of ripening. 1-MCP pre-treatment completely prevented the ripening-induced effect of propylene in pre-climacteric banana fruit, whereas treatment after the onset of ripening stimulated ethylene production. In pre-climacteric fruit, higher concentrations of propylene suppressed ethylene production more strongly, despite their earlier ethylene-inducing effect. Exposure of the fruit ripened by propylene to 1-MCP increased ethylene production concomitantly with an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC content, and prevented a transient decrease in MA-ACS1 transcripts in the pulp tissues. In contrast, in the peel of ripening fruit, 1-MCP prevented the increase in ethylene production and subsequently the ripening process by reduction of the increase in MA-ACS1 and MA-ACO1 transcripts and of ACC synthase and ACC oxidase activities. These results suggest that ethylene biosynthesis in ripening banana fruit may be controlled negatively in the pulp tissue and positively in the peel tissue. This differential regulation by ethylene in pulp and peel tissues was also observed for MA-PL, MA-Exp, and MA-MADS genes.

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.

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.

In the present work, two full length cDNAs of GH3 genes, named DlGH3.1 and DlGH3.2 were cloned from pericarp and aril tissues of the longan fruit, respectively. Three conserved motifs, SSGTSAGERK, YASSE and YRVGD, as a characteristic of the acyladenylate/thioester forming enzyme superfamily were observed in DlGH3.1 and DlGH3.2 proteins. DlGH3.1 mainly expressed in pericarp tissues while DlGH3.2 accumulated in both the pericarp and aril tissues during fruit growth and development. In addition, NAA treatment induced the expression of DlGH3.1 and DlGH3.2 in the pericarp tissues at 21 and 77days after anthesis (DAA), while only DlGH3.2 in the aril tissues could be induced by NAA at 77DAA. More importantly, ABA and ethrel treatments suppressed the accumulations of DlGH3.1 and DlGH3.2 in the pericarp tissues of longan fruit at 21DAA (a rapid growth stage of pericarp), but enhanced DlGH3.2 expression in the aril tissues at 77DAA (a fruit ripening stage). Furthermore, the expression patterns of DlGH3.1 and DlGH3.2 showed different tissue specificity. Thus, our results suggest that DlGH3.1 gene expression might be associated with pericarp growth, while DlGH3.2 accumulation is likely to be related to both pericarp growth and fruit ripening, and the responses of DlGH3s to plant growth hormones are different and dependent on fruit development stage and fruit tissue.

Bananas (Musa acuminata, AAA group) fail to develop a yellow peel and stay green when ripening at temperatures >24 degrees C. The identification of the mechanisms leading to the development of stay-green ripe bananas has practical value and is helpful in revealing pathways involved in the regulation of chlorophyll (Chl) degradation. In the present study, the Chl degradation pathway was characterized and the progress of ripening and senescence was assessed in banana peel at 30 degrees C versus 20 degrees C, by monitoring relevant gene expression and ripening and senescence parameters. A marked reduction in the expression levels of the genes for Chl b reductase, SGR (Stay-green protein), and pheophorbide a oxygenase was detected for the fruit ripening at 30 degrees C, when compared with fruit at 20 degrees C, indicating that Chl degradation was repressed at 30 degrees C at various steps along the Chl catabolic pathway. The repressed Chl degradation was not due to delayed ripening and senescence, since the fruit at 30 degrees C displayed faster onset of various ripening and senescence symptoms, suggesting that the stay-green ripe bananas are of similar phenotype to type C stay-green mutants. Faster accumulation of high levels of fructose and glucose in the peel at 30 degrees C prompted investigation of the roles of soluble sugars in Chl degradation. In vitro incubation of detached pieces of banana peel showed that the pieces of peel stayed green when incubated with 150 mM glucose or fructose, but turned completely yellow in the absence of sugars or with 150 mM mannitol, at either 20 degrees C or 30 degrees C. The results suggest that accumulation of sugars in the peel induced by a temperature of 30 degrees C may be a major factor regulating Chl degradation independently of fruit senescence.

The effects of nitric oxide (NO) on ethylene synthesis and softening of ripening-initiated banana slice were investigated. Fruit firmness, color, and contents of starch and acid-soluble pectin (ASP) were measured. In addition, ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) content, expression and activities of ACC synthase (ACS) and ACC oxidase (ACO), and activities of cell-wall-modifying enzymes, polygalacturonase (PG), pectin methylesterase (PME), and endo-beta-1,4-glucanase, were analyzed. Application of NO reduced ethylene production, inhibited degreening of the peel and delayed softening of the pulp. The decrease of ethylene production was associated with the reduction in the activity of ACO and the expression of the MA-ACO1 gene. Moreover, the NO-treated fruit showed a lower expression of the MA-ACS1 gene but higher ACS activity and ACC content. In addition, NO treatment decreased the activities of PG, PME, and endo-beta-1,4-glucanase and maintained higher contents of ASP and starch, which may account for the delay of softening. We proposed that the inhibition of ACO activity and transcription of gene MA-ACO1 by NO resulted in decreased ethylene synthesis and the delay of ripening of banana slice.

Background ABA-, stress- and ripening-induced (ASR) proteins have been reported to act as a downstream component involved in ABA signal transduction. Although much attention has been paid to the roles of ASR in plant development and stress responses, the mechanisms by which ABA regulate fruit ripening at the molecular level are not fully understood. In the present work, a strawberry ASR gene was isolated and characterized (FaASR), and a polyclonal antibody against FaASR protein was prepared. Furthermore, the effects of ABA, applied to two different developmental stages of strawberry, on fruit ripening and the expression of FaASR at transcriptional and translational levels were investigated. Methodology/Principal Findings FaASR, localized in the cytoplasm and nucleus, contained 193 amino acids and shared common features with other plant ASRs. It also functioned as a transcriptional activator in yeast with trans-activation activity in the N-terminus. During strawberry fruit development, endogenous ABA content, levels of FaASR mRNA and protein increased significantly at the initiation of ripening at a white (W) fruit developmental stage. More importantly, application of exogenous ABA to large green (LG) fruit and W fruit markedly increased endogenous ABA content, accelerated fruit ripening, and greatly enhanced the expression of FaASR transcripts and the accumulation of FaASR protein simultaneously. Conclusions These results indicate that FaASR may be involved in strawberry fruit ripening. The observed increase in endogenous ABA content, and enhanced FaASR expression at transcriptional and translational levels in response to ABA treatment might partially contribute to the acceleration of strawberry fruit ripening.