Analysis of green kiwi fruit (<i>Actinidia deliciosa</i> cv. Hayward) proteinases by two‐dimensional zymography and direct identification of zymographic spots by mass spectrometry

Larocca, Marilena; Rossano, Rocco; Riccio, P.

doi:10.1002/jsfa.4100

Cited by 19 publications

(11 citation statements)

References 25 publications

(35 reference statements)

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“…Bromelain is a cysteine-type protease unique to pineapple. Cysteine-type proteases are also found in other fruits, such as kiwifruit [44], papaya and fig [45]. Pineapple EST library analyses identified an abundance of a bromelain inhibitor (Ac124) and a fruit bromelain (Ac122) from green mature fruit [4].…”

Section: Resultsmentioning

confidence: 99%

Microarray analysis of gene expression profiles in ripening pineapple fruits

2012

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BackgroundPineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening.ResultsMicroarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested.ConclusionsThis is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general.

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Section: Resultsmentioning

confidence: 99%

Microarray analysis of gene expression profiles in ripening pineapple fruits

2012

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“…The reason for the difference (approximately 3 kD) in molecular mass is unclear but appears to be due to a change in protein folding, as LC-MS analysis of the recombinant S68G mutant did not reveal any differences in protein processing (data not shown). Actinidin contains a number of Cys bonds, and even under fully denaturing conditions, it is difficult to get actinidin to resolve at the predicted size (Larocca et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit

et al. 2011

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Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.

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“…Proteolytic enzymes, their isoforms and post-translational variants, present in the extracts are detected as clear, unstained and translucent spots on a dark or blue background. Therefore, 2-DZ represents a useful technique to detect the whole pattern of proteolytic enzymes present in the sample under study [18]–[20].This study describes for the first time the proteolytic enzymes present in honey and their effects on honey proteins.…”

Section: Introductionmentioning

confidence: 99%

What Are the Proteolytic Enzymes of Honey and What They Do Tell Us? A Fingerprint Analysis by 2-D Zymography of Unifloral Honeys

et al. 2012

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Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.

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Analysis of green kiwi fruit (Actinidia deliciosa cv. Hayward) proteinases by two‐dimensional zymography and direct identification of zymographic spots by mass spectrometry

Abstract: The innovative achievements of the present study are the: (1) two-dimensional zymographic map of kiwi gelatinases without the need for extensive purification; and (2) direct identification of proteinase isotypes by means of direct MALDI-ToF MS analysis of the zymographic spots.

Cited by 19 publications

References 25 publications

Microarray analysis of gene expression profiles in ripening pineapple fruits

Microarray analysis of gene expression profiles in ripening pineapple fruits

Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit

What Are the Proteolytic Enzymes of Honey and What They Do Tell Us? A Fingerprint Analysis by 2-D Zymography of Unifloral Honeys

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