Identification of banana ADA1 gene family members and their expression profiles under biotic and abiotic stresses / 生物工程学报

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

Efficient regeneration and genetic transformation platform applicable to five Musa varieties

Background: Banana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics. Results: In this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 µM benzylaminopurine and 9.1 µM thidiazuron. One immature male flower could regenerate 380­456, 310­372, 200­240, 130­156, and 100­130 well-developed shoots in only 240­270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless ß-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot. Conclusions: Our robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.

Molecular cloning and expression analysis of the MaASR1 gene in banana and functional characterization under salt stress

Background Abscisic acid (ABA)-, stress- and ripening-induced protein (ASR) is plant-specific hydrophilic transcriptional regulators involved in sucrose stress and wounding in banana. However, it is not known whether banana ASR genes confer salt stress tolerance. The contexts of the study was to analysis the sequence characterization of banana ASR1, and identify its expression patterns and function under salt stress using quantitative real-time PCR (qPCR) and overexpression in Arabidopsis. The purpose was to evaluate the role of banana ASR1 to salt stress tolerance employed by plants. Results A full-length cDNA isolated from banana fruit was named MaASR1, and it had a 432 bp open reading frame (ORF) encoding 143 amino acids. MaASR1 was preferential expression in roots and leaves compared to low expression in fruits, rhizomes and flowers. Under salt stress, the expression of MaASR1 quickly increased and highest expression level was detected in roots and leaves at 4 h, and then gradually decreased. These results suggested that MaASR1 expression was induced under salt stress. MaASR1 protein was localized in the nucleus and plasma membrane. MaASR1 was transformed to Arabidopsis and verified by southern and northern analysis, transgenic lines L14 and L38 integrated one and two copies of MaASR1, respectively, while overexpression in transgenic lines provided evidence for the role of MaASR1 to salt stress tolerance. Conclusions This study demonstrated that overexpression of MaASR1 in Arabidopsis confers salt stress tolerance by reducing the expression of ABA/stress-responsive genes, but does not affect the expression of the ABA-independent pathway and biosynthesis pathway genes.

Phenylpropanoid enzymes, phenolic polymers and metabolites as chemical defenses to infection of Pratylenchus coffeae in roots of resistant and susceptible bananas (Musa spp.).

Activity differences of the first (phenylalanine ammonia lyase, PAL) and the last (cinnamyl alcohol dehydrogenase, CAD) enzymes of phenylpropanoid pathway in the roots of resistant (Yangambi Km5 and Anaikomban) and susceptible (Nendran and Robusta) banana cultivars caused by root lesion nematode, Pratylenchus coffeae, were investigated. Also, the accumulation of phenolics and deposition of lignin polymers in cell walls in relation to resistance of the banana cultivars to the nematode were analyzed. Compared to the susceptible cultivars, the resistant cultivars had constitutively significantly higher PAL activity and total soluble and cell wall-bound phenolics than in susceptible cultivars. The resistant cultivars responded strongly to the infection of the nematode by induction of several-time higher PAL and CAD enzymes activities, soluble and wall-bound phenolics and enrichment of lignin polymers in cell wall and these biochemical parameters reached maximum at 7th day postinoculation. In addition, profiles of phenolic acid metabolites in roots of Yangambi Km5 and Nendran were analyzed by HPLC to ascertain the underlying biochemical mechanism of bananas resistance to the nematode. Identification and quantification of soluble and cell wall-bound phenolic acids showed six metabolites and only quantitative, no qualitative, differences occurred between the resistant and susceptible cvs. and between constitutive and induced contents. A very prominent increase of p-coumaric, ferulic and sinapic acids, which are precursors of monolignols of lignin, in resistant cv. was found. These constitutive and induced biochemical alterations are definitely the chemical defenses of resistant cvs. to the nematode infection.

Biochemical markers assisted screening of Fusarium wilt resistant Musa paradisiaca (L.) cv. Puttabale micropropagated clones.

An efficient protocol was standardized for screening of panama wilt resistant Musa paradisiaca cv. Puttabale clones, an endemic cultivar of Karnataka, India. The synergistic effect of 6-benzyleaminopurine (2 to 6 mg/L) and thidiazuron (0.1 to 0.5 mg/L) on MS medium provoked multiple shoot induction from the excised meristem. An average of 30.10 ± 5.95 shoots was produced per propagule at 4 mg/L 6-benzyleaminopurine and 0.3 mg/L thidiazuron concentrations. Elongation of shoots observed on 5 mg/L BAP augmented medium with a mean length of 8.38 ± 0.30 shoots per propagule. For screening of disease resistant clones, multiple shoot buds were mutated with 0.4% ethyl-methane-sulfonate and cultured on MS medium supplemented with Fusarium oxysporum f. sp. cubense (FOC) culture filtrate (5–15%). Two month old co-cultivated secondary hardened plants were used for screening of disease resistance against FOC by the determination of biochemical markers such as total phenol, phenylalanine ammonia lyase, oxidative enzymes like peroxidase, polyphenol oxidase, catalase and PR-proteins like chitinase, β-1-3 glucanase activities. The mutated clones of M. paradisiaca cv. Puttabale cultured on FOC culture filtrate showed significant increase in the levels of biochemical markers as an indicative of acquiring disease resistant characteristics to FOC wilt.

Applicability of the use of waste from different banana cultivars for the cultivation of the oyster mushroom

The objective of this research was to evaluate the oyster mushroom Pleurotus ostreatus- (Jacq.: Fr.) Kumm. cultivation in substrates based on different combinations of wastes (leaf, pseudo-stem and pseudo-stem + leaf) and banana cultivars - Musa spp. (Thap Maeo, Prata Anã, Pelipita and Caipira) during 49 days. Organic matter loss in the substrate by action of the fungus was also evaluated during that period. It was verified that the pseudo-stem waste provided the best averages of biological efficiency among all cultivars tested and best rates were obtained by Thap Maeo (61.5%). The highest organic matter loss (OML) was obtained from pseudo-stem + leaf wastes (Prata Anã - 78.6%; Thap Maeo - 67.6%; Pelipita - 64.8%; Caipira - 60.6%). Therefore, the use of those wastes showed itself viable for P. ostreatus cultivation due to its availability and low cost, besides decreasing discards to environment.

Ocorrência e biossíntese de frutooligossacarídeos em banana / Occurrence and biosynthesis of fructooligosaccharides in banana

A banana tem sido comumente indicada como uma boa fonte de frutooligossacarídeos (FOS), que são considerados componentes funcionais de alimentos. Contudo, diferenças significantes em suas quantidades têm sido referidas na literatura. Portanto, uma parte do trabalho foi destinada à identificação e quantificação de FOS durante o amadurecimento de cultivares de bananas pertencentes aos grupos genômicos mais comumente cultivados no Brasil. Considerando as diferenças de cultivar, estágio do amadurecimento e metodologia usada para análise de FOS, os conteúdos dos açúcares foram analisados por cromatografia líquida de alta performance (HPAEC-PAD) e cromatografia a gás (CG-MS). Uma pesquisa inicial entre oito cultivares no estágio maduro, mostrou acúmulo de 1-cestose, primeiro membro da série de FOS, em todas elas (quantidades entre 297 e 1600 µg/g M.S). A nistose, o segundo membro, foi detectado somente na cultivar Prata. Com bases nestes dados, foram escolhidas cinco cultivares, para que fossem analisadas durante todo o amadurecimento. Os resultados mostraram uma forte correlação entre a chegada a um nível específico de sacarose (~200 mg/g M.S) e a síntese de 1-cestose. Em uma segunda fase, os níveis de sacarose e FOS total foram quantificados em diferentes fases de amadurecimento de banana Prata, armazenada em temperatura ambiente e em baixa temperatura. As supostas enzimas envolvidas em sua síntese também foram avaliadas. Para explorar a possibilidade da invertase ser responsável pela atividade de frutosiltransferase em banana, foi medido o efeito do inibidor Piridoxal HCl, os níveis de concentração do substrato e as atividades de hidrólise e transglicosilação, e o efeito do tempo no estudo cinético da enzima. A baixa temperatura atrasou todos os eventos analisados por 15 dias e os níveis de sacarose tiveram um pequeno aumento, porém constante, enquanto a banana estava armazenada ao frio, e uma rápida elevação no final do amadurecimento. Foi detectado FOS total desde o primeiro dia pós-colheita, enquanto que a 1-cestose permaneceu indetectável até os níveis de sacarose atingirem aproximadamente 200 mg/g M.S., em ambos os grupos. Os níveis de sacarose e FOS total foram ligeiramente maiores em bananas armazenadas em baixas temperaturas do que em frutos controle. Em ambas as amostras os níveis de FOS total foram maiores que de 1-cestose. Os perfis de carboidratos por HPLC e TLC sugeriram a presença de neocestose, 6-cestose e bifurcose. A enzima supostamente responsável pela atividade de transglicosilação em banana parece ser a invertase. Contudo, os altos níveis de sacarose encontrados em banana armazenadas em baixa temperatura, poderiam ser resultado de várias mudanças de enzimas degradativas e biossíntéticas, como sacarose-sintase (SuSy), sacarose-fosfato-sintase (SPS), invertase e outras, uma vez que a sacarose possui um papel central, direta ou indiretamente, em diversas vias do metabolismo de carboidrato em banana. Assim, na última parte do trabalho foram analisados o acúmulo de sacarose e a síntese e atividade de enzimas sintéticas, hidrolíticas e fosforolíticas, importantes no metabolismo de amido-sacarose, durante o amadurecimento de banana Prata nos dois tratamentos. A baixa temperatura não danificou os frutos, aumentando a vida de prateleira deles. As amostras do frio apresentaram pequeno aumento no nível de degradação de amido e um acréscimo de 20 % na sacarose acumulada durante o amadurecimento. Foi verificado o atraso na produção de etileno, CO2, e no início de degradação de amido durante o acondicionamento ao frio, concomitante ao atraso no pico de atividade de α-amilase. O atraso no climatério também manteve alta a atividade e síntese protéica de SuSy durante o armazenamento a frio, que declinaram após a retirada do frio, como no controle. As enzimas ß-amilase, fosforilase (forma citosólica e plastidial) e SPS reagiram positivamente, sofrendo uma indução positiva na síntese e atividade enzimática durante o armazenamento ao frio, que poderia ser parte do mecanismo necessário para os maiores níveis de açúcares e para o processo de tolerância do fruto à baixa temperatura

Banana has been currently indicated as a good source of fructooligosaccharides (FOS), which are considered to be functional components of foods. However, significant differences in their amounts in bananas have been observed in the literature. So, a part of this work aims to identify and quantify FOS during ripening in different banana cultivars belonging to the most common genomic groups cultivated in Brazil. Considering that these differences can be due to cultivar, stage of ripening, and the methodologies used for FOS analyses, sugar contents were analyzed by high performance anion exchange chromatography pulsed amperiometric detection (HPAEC-PAD) and gas chromatography- mass spectrometry (GC-MS). An initial screening of eight cultivars in a full-ripe stage showed that 1-Kestose, the first member of the FOS series (amounts between 297 and 1600 µg/g of D.M), was accumulated in all of them. Nystose, the second member, was detected only in Prata cultivar. Five of the cultivars were analyzed during ripening, and a strong correlation could be established with a specific sucrose level (~200 mg/g of D.M.), which seems to trigger the synthesis of 1-Kestose. In a second part of this work, the levels of sucrose and total-FOS were quantified in different phases of banana Prata ripening stored at ambient and low temperature. The supposed enzymes involved in their synthesis were also evaluated. To explore the possibility that invertase could be responsible for the fructosyltransferase activity in banana, we measured the effect of the inhibitor Pyridoxal HCl, the level of substrate concentration on both hydrolyze and transglycosylase activity in the same protein extract and the effect of time on kinetic study of the enzyme. The cold temperature delayed all the analyzed events for 15 days and sucrose levels increased low, but constantly, while banana were stored at low temperature and had a burst when it increased. Total-FOS were detected in the first days after harvest, while 1-kestose remained undetectable until the sucrose levels were around 200 mg.g (dry weight), in both groups. Total-FOS and sucrose levels were higher in banana stored at low temperature than in control. In both samples total-FOS levels were higher than 1-kestose. The carbohydrate profiles by HPLC and TLC suggest the presence of neokestose, 6-kestose and bifurcose. The enzyme supposed to be responsible for the transglycosilation activity in banana, seems to be an invertase. However, the higher sucrose levels found in banana stored at low temperature could be result of several changes in biosynthetic and degradative enzymes, such sucrose-synthase, sucrose-phosphate-synthase, invertase and others, once that sucrose plays a central role in a lot of direct and indirect carbohydrate pathways in banana fruits. So, in the last part of this work, we analyzed the sucrose accumulation and synthesis and activity of synthetic, hydrolytic and phosphorolytic enzymes that are important in the starch-sucrose metabolism during ripening of banana Prata stored at ambient and low temperature. The levels of starch degradation and sucrose accumulation (around 20% over) showed high levels in cold fruits as compared with control, during the ripening. The cold temperature delayed the ethylene and CO2 production, and the beginning of the starch degradation, concomitantly with a delay in the profile of α-amylase synthesis and activity. The late climateric also maintained the high synthesis and activity of SuSy during the cold storage that decreased just after ending the cold exposure. The ß-amylase, phosphorylase (plastidial and citossolic forms) and the SPS enzymes showed a positive induction in the both activity and synthesis of protein during the cold storage. It could be important to the higher sugars levels showed at low temperature and that could contribute to the process of cold resistance in banana fruit

Genetic diversity of carotenoid-rich bananas evaluated by Diversity Arrays Technology (DArT)

The aim of this work was to evaluate the carotenoid content and genetic variability of banana accessions from the Musa germplasm collection held at Embrapa Cassava and Tropical Fruits, Brazil. Forty-two samples were analyzed, including 21 diploids, 19 triploids and two tetraploids. The carotenoid content was analyzed spectrophotometrically and genetic variability was estimated using 653 DArT markers. The average carotenoid content was 4.73 µg.g-1, and ranged from 1.06 µg.g-1 for the triploid Nanica (Cavendish group) to 19.24 µg.g-1 for the triploid Saney. The diploids Modok Gier and NBA-14 and the triploid Saney had a carotenoid content that was, respectively, 7-fold, 6-fold and 9-fold greater than that of cultivars from the Cavendish group (2.19 µg.g-1). The mean similarity among the 42 accessions was 0.63 (range: 0.24 to 1.00). DArT analysis revealed extensive genetic variability in accessions from the Embrapa Musa germplasm bank.

Obtainment of embryogenic cell suspensions from scalps of the banana CIEN-BTA-03 (Musa sp., AAAA) and regeneration of the plants

The purposes of this work were to obtain embryogenic cell suspensions (ECS) from scalps and to regenerate plants of the banana CIEN-BTA-03. Shoot apexes were grown in the scalp-induction medium of Murashige and Skoog plus BA and IAA, following four diverse treatments. The first two, ME22 and ME25, were solid media supplemented with (mg L-1) 22.7 BA plus 0.192 IAA, and 25 BA plus 0.217 IAA, respectively, all containing 1.8 g L-1 of phytagel, and subcultures were performed monthly and bimonthly over 16 months. The other two treatments, IT22 and IT25, resembled ME22 and ME25 but consisted in temporary immersion for four months without subcultures, followed by two months in solid media. The scalps were grown in callus-induction medium and embryogenic calluses were obtained with abundant somatic embryos, especially in scalps from IT25. About 10 to 15 embryos from each were transferred to 5 ml of multiplication medium to initiate the ECS. The scalps obtained from the IT25 treatment were the most successful as they led to ECS with high embryogenic capability. In addition, IT25 decreased the timespan required for the production of scalps. The obtained ECS gave rise to secondary somatic embryos. It showed a high multiplication index, as well as numerous mature somatic embryos, and good conversion of embryos and plant regeneration.

In vitro cloning and homestead cultivation of primitive Musa cultivars.

Two primitive diploid Musa cultivars, Matti and Chemmatti from the extreme southern part of the Western Ghats were multiplied by in vitro culture of sucker-derived shoot apices. Decontaminated corm explants (1 cm x 1 cm) having shoot apex (approximately 0.3 cm) cultured for 1 month in Murashige and Skoog basal agar medium was cut vertically into eight segments and each segment having a part of shoot meristem was cultured in presence of 6-benzylaminopurine (BAP) and combinations of BAP and indole-3-acetic acid (IAA) or indole-3-butyricacid (IBA) to produce multiple shoots. After 12 weeks of culture, maximum number of shoots (32) in both the cultivars were produced in approximate 60% of the explants in presence of BAP and IAA each at 1.5 mg/l(-1) (Matti) and 40% of the explants in 2.5 mg/l(-1) of BAP and 1.5 mg/l(-1) of IAA (Chemmatti). Buds were formed from the base of the subcultured shoots and somewhat more number (34) of shoots were obtained in Matti than in Chemmatti (31) after 8 weeks. Difference in the concentration of cytokinin required for shoot initiation and multiplication, persistence of exudation through the subculture and red colouration of the early formed sheathing leaf bases in the shoots in Chemmatti indicated possible genotypic differences between the two cultivars. Multiple shoot proliferation achieved through five subcultures of the isolated shoots without any decline. Transfer of shoots (4-5 cm) into MS basal medium favoured rooting in 4 weeks and rooted plants (9 cm) were hardened and established (80-95%). Mericlones of Matti cultivated in homesteads produced bunches of uniform characters in 13 months.