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An efficient biolistic transformation system of banana combined with a liquid medium selection system was developed during this study. An embryogenic cell suspension (ECS) of Musa acuminata cv. Baxi (AAA) was bombarded with a particle delivery system. After 7 days of restoring culture in liquid M2 medium, embryogenic cells were transferred to a liquid selection M2 medium supplemented with 10 μg/mL hygromycin for resistance screening. The untransformed cell clusters were inhibited or killed, and a small number of transformants proliferated in the liquid selection medium. After the 0th, first, second, and third generation of antibiotic screening, there were 0, 65, 212, and 320, respectively, vitality-resistant buds obtained from a 0.5-mL packed cell volume (PCV) of embryogenic cell suspension. The β-glucuronidase (GUS) staining, polymerase chain reaction (PCR) analysis, and Southern blot hybridization results all demonstrated a 100% positive rate of regenerated resistant seedlings. Interestingly, the number of buds obtained through third-generation screening was almost equal to that obtained from the original ECS in M2 medium without antibiotics. These results suggested that the liquid medium selection system facilitated the proliferation of a positive transgenic ECS, which significantly improved the regeneration rate of transformants. This protocol is suitable for the genetic transformation of all banana genotypes and is highly advantageous to varieties with low callusing potential.

Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.

Banana (Musa spp.) is an important tropical crop. Banana industry is under biotic and abiotic stresses such as Fusarium wilt, typhoon, cold stress. Genetic engineering offers a powerful strategy to create germplasm of banana with enhanced resistance. The safety of genetically modified organisms has become a bottleneck restricting the popularization and application of genetically modified technology. In this study, a candidate promoter, LEAFY (LFY) for expression and flower initiation in Arabidopsis, was cloned and constructed to ‘Gene-deletor’ vector. Histochemical β-glucuronidase (GUS) staining results showed that the ‘Gene-deletor’ vector driven by LFY promoter could lead to 88.5% excision efficiency from Arabidopsis seeds based on more than 200 T3 progeny examined per event. GUS staining was found to be partially negative in transgenic bananas, however, polymerase chain reaction could still detect the presence of large fragments of the vector. These results suggest that although LFY promoter could not completely drive the ‘Gene-deletor’ vector to achieve the effect of complete elimination of exogenous gene in bananas, its efficiency of eliminating exogenous gene laid a theoretical foundation for cloning banana fruit-specific promoters, that is, ‘non-transgenic’ GM bananas. These results suggest that LFY promoter could not completely drive the ‘Gene-deletor’ vector to achieve the effect of complete elimination of exogenous gene in bananas. Nevertheless, a certain effect of exogenous gene elimination laid a theoretical foundation for the next step of screening banana fruit-specific promoters, removing all exogenous genes from banana fruits, and solving the food safety problem of genetically modified bananas.

Anthocyanins spatiotemporally accumulate in certain tissues of particular species in the banana plant, and MYB transcription factors (TFs) serve as their primary regulators. However, the precise regulatory mechanism in banana remains to be determined. Here, we report the identification and characterization of MaMYB4, an R2R3-MYB repressor TF, characterized by the presence of EAR (ethylene-responsive element binding factor–associated amphiphilic repression) and TLLLFR motifs. MaMYB4 expression was induced by the accumulation of anthocyanins. Transgenic banana plants overexpressing MaMYB4 displayed a significant reduction in anthocyanin compared to wild type. Consistent with the above results, metabolome results showed that there was a decrease in all three identified cyanidins and one delphinidin, the main anthocyanins that determine the color of banana leaves, whereas both transcriptome and reverse transcription–quantitative polymerase chain reaction analysis showed that many key anthocyanin synthesis structural genes and TF regulators were downregulated in MaMYB4 overexpressors. Furthermore, dual-luciferase assays showed that MaMYB4 was able to bind to the CHS, ANS, DFR, and bHLH promoters, leading to inhibition of their expression. Yeast two-hybrid analysis verified that MaMYB4 did not interact with bHLH, which ruled out the possibility that MaMYB4 could be incorporated into the MYB-bHLH-WD40 complex. Our results indicated that MaMYB4 acts as a repressor of anthocyanin biosynthesis in banana, likely due to a two-level repression mechanism that consists of reduced expression of anthocyanin synthesis structural genes and the parallel downregulation of bHLH to interfere with the proper assembly of the MYB-bHLH-WD40 activation complex. To the best of our knowledge, this is the first MYB TF that regulates anthocyanin synthesis that was identified by genetic methods in bananas, which will be helpful for manipulating anthocyanin coloration in banana programs in the future.

Ceramide sphingolipids are major components of membranes. C2 and C6 ceramides induce programmed cell death (PCD) in animals and plants, and we previously showed that C2 and C6 ceramides induce PCD in rice (Oryza sativa) protoplasts. However, the mechanistic link between sphingolipids and PCD in rice remains unclear. Here, we observed that calcium levels increased rapidly after ceramide treatment. Moreover, the calcium channel inhibitor LaCl3 and the intracellular calcium chelator acetoxymethyl-1, 2-bis (2-aminophenoxy) ethic acid (BAPTA-AM) inhibited this calcium increase and prevented ceramide-induced PCD. Moreover, caspase-3-like protease activity increased significantly in C6 ceramide-treated protoplasts, and a caspase-specific inhibitor prevented C6 ceramide-induced cell death. We also detected the other typical PCD events including ATP loss. DIDS (4, 49-diisothiocyanatostilbene- 2, 29-disulfonic acid), an inhibitor of voltage-dependent anion channels (VDACs), decreased C6 ceramide-induced cell death. Together, this evidence suggests that mitochondria played an important role in C6 ceramide-induced PCD.

Banana (Musa spp.) is an important tropical fruit with high economic value, however, Fusarium wilt disease is a critical factor affecting the sound development of the banana industry. Different cultivars of banana exhibit a high degree of genetic variability for resistance to Fusarium wilt. Compared with Cavendish banana (Musa spp. AAA group), Dajiao (Musa spp. ABB group) has superior disease resistance and provides an excellent test material for the isolation and characterization of disease resistance genes. In this study, a Dajiao candidate gene, MpbHLH (basic helix-loop-helix), an encoding ICE1-like protein transcription factor (TF), was cloned and characterized. Real-time quantitative PCR (qPCR) results revealed that MpbHLH transcript levels were strongly induced under cold treatment, salt, and dehydration. Overexpression of MpbHLH in Foc TR4-sensitive Cavendish banana showed that the transgenic lines had superior resistance to Fusarium wilt compared with the wild-type (WT). Proteomic analysis showed that phenylalanine ammonia lyase (PAL) and peroxidase (POD) were highly expressed in transgenic plants. Physiological experiments further revealed that transgenic plants, especially their roots, had high POD activity. Taken together, overexpression of MpbHLH in banana may extensively activate stress-related genes. In particular, the activity of POD increases the resistance of transgenic banana to Fusarium wilt by enhancing the reactive oxygen species (ROS) scavenging system of a transgenic plant.

Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low K(m) for the fluorescent lipid C(6)-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.

Ceramidases hydrolyze ceramide into sphingosine and fatty acids. In mammals, ceramidases function as key regulators of sphingolipid homeostasis, but little is known about their roles in plants. Here we characterize the Arabidopsis ceramidase AtACER, a homolog of human alkaline ceramidases. The acer-1 T-DNA insertion mutant has pleiotropic phenotypes, including reduction of leaf size, dwarfing and an irregular wax layer, compared with wild-type plants. Quantitative sphingolipid profiling showed that acer-1 mutants and the artificial microRNA-mediated silenced line amiR-ACER-1 have high ceramide levels and decreased long chain bases. AtACER localizes predominantly to the endoplasmic reticulum, and partially to the Golgi complex. Furthermore, we found that acer-1 mutants and AtACER RNAi lines showed increased sensitivity to salt stress, and lines overexpressing AtACER showed increased tolerance to salt stress. Reduction of AtACER also increased plant susceptibility to Pseudomonas syringae. Our data highlight the key biological functions of ceramidases in biotic and abiotic stresses in plants.

Banana is an important tropical fruit with high economic value. One of the main cultivars (‘Cavendish’) is susceptible to low temperatures, while another closely related specie (‘Dajiao’) has considerably higher cold tolerance. We previously reported that some membrane proteins appear to be involved in the cold tolerance of Dajiao bananas via an antioxidation mechanism. To investigate the early cold stress response of Dajiao, here we applied comparative membrane proteomics analysis for both cold-sensitive Cavendish and cold-tolerant Dajiao bananas subjected to cold stress at 10°C for 0, 3, and 6 h. A total of 2,333 and 1,834 proteins were identified in Cavendish and Dajiao, respectively. Subsequent bioinformatics analyses showed that 692 Cavendish proteins and 524 Dajiao proteins were predicted to be membrane proteins, of which 82 and 137 differentially abundant membrane proteins (DAMPs) were found in Cavendish and Dajiao, respectively. Interestingly, the number of DAMPs with increased abundance following 3 h of cold treatment in Dajiao (80) was seven times more than that in Cavendish (11). Gene ontology molecular function analysis of DAMPs for Cavendish and Dajiao indicated that they belong to eight categories including hydrolase activity, binding, transporter activity, antioxidant activity, etc., but the number in Dajiao is twice that in Cavendish. Strikingly, we found peroxidases (PODs) and aquaporins among the protein groups whose abundance was significantly increased after 3 h of cold treatment in Dajiao. Some of the PODs and aquaporins were verified by reverse-transcription PCR, multiple reaction monitoring, and green fluorescent protein-based subcellular localization analysis, demonstrating that the global membrane proteomics data are reliable. By combining the physiological and biochemical data, we found that membrane-bound Peroxidase 52 and Peroxidase P7, and aquaporins (MaPIP1;1, MaPIP1;2, MaPIP2;4, MaPIP2;6, MaTIP1;3) are mainly involved in decreased lipid peroxidation and maintaining leaf cell water potential, which appear to be the key cellular adaptations contributing to the cold tolerance of Dajiao. This membrane proteomics study provides new insights into cold stress tolerance mechanisms of banana, toward potential applications for ultimate genetic improvement of cold tolerance in banana.

Arabidopsis thaliana plants that lack ceramide kinase, encoded by ACCELERATED CELL DEATH5 (ACD5), display spontaneous programmed cell death late in development and accumulate substrates of ACD5. Here, we compared ceramide accumulation kinetics, defense responses, ultrastructural features, and sites of reactive oxygen species (ROS) production in wild-type and acd5 plants during development and/or Botrytis cinerea infection. Quantitative sphingolipid profiling indicated that ceramide accumulation in acd5 paralleled the appearance of spontaneous cell death, and it was accompanied by autophagy and mitochondrial ROS accumulation. Plants lacking ACD5 differed significantly from the wild type in their responses to B. cinerea, showing earlier and higher increases in ceramides, greater disease, smaller cell wall appositions (papillae), reduced callose deposition and apoplastic ROS, and increased mitochondrial ROS. Together, these data show that ceramide kinase greatly affects sphingolipid metabolism and the site of ROS accumulation during development and infection, which likely explains the developmental and infection-related cell death phenotypes. The acd5 plants also showed an early defect in restricting B. cinerea germination and growth, which occurred prior to the onset of cell death. This early defect in B. cinerea restriction in acd5 points to a role for ceramide phosphate and/or the balance of ceramides in mediating early antifungal responses that are independent of cell death.

UNLABELLED PREMISE OF THE STUDY Single-walled carbon nanotubes (SWCNTs) have many unique structural and mechanical properties. Their potential applications, especially in biomedical engineering and medical chemistry, have been increasing in recent years, but the toxicological impact of nanoparticles has rarely been studied in plants. • METHODS We exposed Arabidopsis and rice leaf protoplasts to SWCNTs and examined cell viability, DNA damage, reactive oxygen species generation, and related gene expression. We also tested the effects of nanoparticles on Arabidopsis leaves after injecting a SWCNT solution. EM-TUNEL (electron-microscopic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) and a cerium chloride staining method were used. • KEY RESULTS SWCNTs caused adverse cellular responses including cell aggregation, chromatin condensation along with a TUNEL-positive reaction, plasma membrane deposition, and H(2)O(2) accumulation. The effect of SWCNTs on the survival of cells was dose dependent, with 25 μg/mL inducing 25% cell death in 6 h. In contrast, activated carbon, which is not a nano-sized carbon particle, did not induce cell death even 24 h after treatments. The data indicated that the nano-size of the particle is a critical factor for toxicity. Moreover, endocytosis-like structures with cerium chloride deposits formed after SWCNT treatment, suggesting a possible pathway for nanoparticles to traverse the cell membrane. • CONCLUSIONS Consequently, SWCNTs have an adverse effect on protoplasts and leaves through oxidative stress, leading to a certain amount of programmed cell death. Although nanomaterials have great advantages in many respects, the benefits and side effects still need to be assessed carefully.

Background Ceramide kinase (CERK) is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare) and investigate the effects of ceramides on rice cell viability. Principal Findings OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg2+ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK's substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing. Conclusions OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.