A simple procedure for the isolation of high quality RNA from ripening banana fruit

Isolation of high quality RNA from ripening banana fruit tissue is difficult due to high levels of polysaccharides and other substances that interfere when using conventional procedures for RNA isolation. These substances not only decrease the yield but the quality of RNA is almost unusable. We describe here a simple RNA procedure that effectively removes these contaminating substances without affecting the yield. Following this procedure, we routinely obtained 80–150 μg of total RNA per g fresh tissue. The RNA is of good quality and suitable for northern analysis, RT-PCR and cDNA library construction. NBRI publication No. 488(NS).     

Upsurge of particulate peroxidase in ripening banana fruit

Peroxidase was isolated from the pulp of ripening banana fruit and assayed with o-dianisidine as hydrogen-donor. Cell macerates contained soluble and particle-bound peroxidase. Soluble peroxidase levels did not appreciably differ in pre-climacteric, climacteric and post-climacteric fruit. Particulate peroxidase levels increased 3-fold with the initiation of the respiration climacteric and gradually declined with the onset of senescence. Bound peroxidase was released from cell wall and membrane fractions with washing in 0–8 M CaCl₂.     

Expression of multiple forms of polygalacturonase gene during ripening in banana fruit.

The activity of polygalacturonase (PG, E.C 3.2.1.15) during ripening in climacteric fruits has been positively correlated with softening of the fruit tissue and differential expression of its gene is suspected to be regulated by the plant hormone ethylene. We have cloned four partial cDNAs, MAPG1 (acc. no. AF311881), MAPG2 (acc. no. AF311882), MAPG3 (acc. no. AF542382) and MAPG4 (acc. no. AY603341) for PG genes and studied their differential expression during ripening in banana. MAPG3 and MAPG4 are believed to be ripening related and regulated by ethylene whereas MAPG2 is associated more with senescence. MAPG1 shows constitutive expression and is not significantly expressed in fruit tissue. The genomic clone MAGPG (acc. No. AY603340) includes the complete MAPG3 gene, which consists of four exons and three introns. The structure of the gene has more similarity to tomato abscission PG rather than tomato fruit PG. It is concluded that softening during ripening in banana fruit results from the concerted action of at least four PG genes, which are differentially expressed during ripening.     

果实成熟过程相关调控基因研究进展

果实成熟过程中,多聚半乳糖醛酸酶（ＰＧ）参与果胶的分解,从而在果实软化中起作用,新近发现,果实软化过程中,协同展蛋白具有一定的作用：ＡＣＣ合成酶（ＡＣＳ）、ＡＣＣ氧化酶（ＡＣＯ）和ＡＣＣ脱氨酶与乙烯合成直接有关,ＡＣＳ是乙烯形成的关键酶,由多基因家族编码,各个基因协同表达,每一基因都有自己的转录特性,新近不断发现果实中ＡＣＳ基因家族中的新成员;ＡＣＯ是一种与膜结合的酶,这种酶具有结构上的立体专一性     

Molecular and genetic regulation of fruit ripening

Fleshy fruit undergo a novel developmental program that ends in the irreversible process of ripening and eventual tissue senescence. During this maturation process, fruit undergo numerous physiological, biochemical and structural alterations, making them more attractive to seed dispersal organisms. In addition, advanced or over-ripening and senescence, especially through tissue softening and eventual decay, render fruit susceptible to invasion by opportunistic pathogens. While ripening and senescence are often used interchangeably, the specific metabolic activities of each would suggest that ripening is a distinct process of fleshy fruits that precedes and may predispose the fruit to subsequent senescence.     

Changes in gene expression during strawberry fruit ripening and their regulation by auxin

Changes in messenger RNA during the development of the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, were analysed by extracting total RNA and separating the in-vitro translated products by two-dimensional polyacrylamide gel electrophoresis. Alterations in numerous messenger RNAs accompanied fruit development between the immature green stage and the overripe stage, with prominent changes detected at or before the onset of ripening. A number of messenger RNAs undetectable in immature green fruit increased as the fruit matured and ripened. Others showed a marked decrease in advance of the ripening phase. A further group of messenger RNAs was prominent in immature and ripe fruit but absent just prior to the turning stage. Removing the achenes from a segment of the fruit accelerated anthocyanin accumulation in the de-achened portion and produced a pattern of translated polypeptides similar to normal ripe fruit. Application of the synthetic auxin 1-naphthaleneacetic acid to the de-achened receptacle produced a translation pattern similar to that in mature green fruit. These findings indicate that ripening in strawberry is associated with the expression of specific genes.     

Activity and expression of banana starch phosphorylases during fruit development and ripening

Two main forms of starch phosphorylase (EC 2.4.1.1) were identified and purified from banana (Musa acuminata Colla. cv. Nanic?o) fruit. One of them, designated phosphorylase I, had a native molecular weight of 155 kDa and subunit of 90 kDa, a high affinity towards branched glucans and an isoelectric point around 5.0. The other, phosphorylase II, eluted at a higher salt concentration from the anion exchanger, had a low affinity towards branched glucans, a native molecular weight of 290 kDa and subunit of 112 kDa. Kinetic studies showed that both forms had typical hyperbolic curves for orthophosphate (Pi) and glucose-1-phosphate, and that they could not react with substrates with a blocked reducing end or alpha-1,6 glucosidic bonds. Antibodies prepared against the purified type-II form and cross-reacting with the type-I form showed that there was an increase in protein content during development and ripening of the fruit. The changes in protein level were parallel to those of phosphorylase activity, in both the phosphorolytic and synthetic directions. Considering the kinetics, indicating that starch phosphorylases are not under allosteric control, it can be argued that protein synthesis makes a contribution to regulating phosphorylase activity in banana fruit and that hormones, like gibberellic acid and indole-3-acetic acid, may play a regulating role. For the first time, starch phosphorylases isoforms were detected as starch-granule-associated proteins by immunostaining of SDS-PAGE gels.     

Post-harvest physiology of tetraploid banana fruit: response to storage and ripening

Aspects of the post-harvest physiology relating to storage and ripening of the fruit of tetraploid banana clones resistant to Sigatoka disease, have been compared with fruit of Valery, an important commercial triploid cultivar. Significant differences in susceptibility to low temperature injury, duration of the preclimacteric period, the texture of pulp and peel and ethylene evolution have been found between tetraploid and Valery fruit and also between tetraploid fruit of different clones. Fruit of Valery and one tetraploid clone developed serious chilling injury during storage at 12 °C whereas that of five other tetraploid clones showed only slight damage. The preclimacteric period for fruit of two tetraploid clones was 30–45% less than for Valery fruit at an equivalent stage of physical development. Pulp firmness of preclimacteric tetraploid fruit was 20–30% less than that of Valery fruit and the differences persisted through ripening. The softening response to applied ethylene was up to 15 h earlier in fruit of tetraploid clones than of Valery but respiratory patterns, colour development and starch-to-sugar conversion were similar. Unlike Valery fruit, ripe tetraploid fruit did not develop senescent spotting, and shelf life was terminated by rapid deterioration of peel strength to a state of severe finger drop. Temporal and quantitative differences occurred between fruit of tetraploid clones and Valery in production of ethylene and these may relate to the observed differences in control of softening in both pulp and peel.     

Flower development schedule and AGAMOUS‐like gene expression patterns in two morphs of Nigella damascena (Ranunculaceae) differing in floral architecture

Love‐in‐a‐mist (Nigella damascena) is an annual species of Ranunculaceae native to the Mediterranean Basin, characterized by delicate flowers lying on long lacy bracts. Two floral morphs of N. damascena, designated [P] and [T], differ in the identity and number of perianth organs and in the position of the perianth–androecium boundary on the meristem. They both occur in the wild. Here we describe a precise comparative schedule of floral development in the two morphs. We divided the sequence of developmental events affecting the floral meristem into six stages and related them to the height of the elongating stem and to the time elapsed after the beginning of stem elongation. In addition, we characterized the expression pattern of C‐class genes in floral organs of both morphs in an attempt to better characterize the differences between the two floral groundplans. In the [T] morph an expansion of the expression domain of AGAMOUS (AG) paralogues outside the fertile organs was observed, correlating with the change in identity of the inner perianth organs. Expression of AG‐like genes in the sepal‐like organs suggests these are not identical to true sepals at the molecular level. The morpho‐temporal framework we have defined will allow us to compare various gene expression profiles at targeted developmental stages in both morphs, providing further insight into the molecular control of the floral dimorphism in N. damascena and into the processes underlying the transition from a differentiated (bipartite) to an undifferentiated (unipartite) perianth. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 608–619.     

Chlorophyll catabolism and gene expression in the peel of ripening banana fruits

Vacuolar malate transport in Catharanthus roseus is probably mediated by a 37 kDa intrinsic tonoplast protein identified with a photolyzable malate analog. Antibodies raised against the protein inhibit malate uptake in isolated vacuoles. We report here the native molecular mass and the oligomeric state of the putative malate transporter which were determined from two-dimensional native electrophoresis. In its first dimension, the electrophoresis used a charge shift method developed for isolating native membrane protein complexes from purified tonoplast vesicles. In combination with a second dimension of sodium dodecylsulfate electrophoresis, it enables the determination of the oligomeric state and subunit composition of non-dissociated complexes. In such analyses, most of the tonoplast proteins of Catharanthus roseus appear to have a complex structure. In native gels (first dimension), both the photoprobe and the antibodies recognized a 160 kDa protein. The photolabelling characteristics correlate well with the main features of malate transport activity. The 160 kDa protein, when analyzed in the second dimension, contained the 37 kDa polypeptide as a subunit. In addition, cross-linking with dimethyl suberimidate (DMS) in the intact tonoplast vesicles resulted in the disappearance of the 37 kDa monomer protein band with concomitant appearance of additional bands of molecular masses higher than the monomer, i.e. 73 and 160 kDa. These results, taken together, suggest that the putative malate transporter exists in the tonoplast as a tetramer.