Endogenous cytokinin in developing kiwifruit is implicated in maintaining fruit flesh chlorophyll levels

Background and Aims

Green kiwifruit (Actinidia deliciosa) retain high concentrations of chlorophyll in the fruit flesh, whereas in gold-fleshed kiwifruit (A. chinensis) chlorophyll is degraded to colourless catabolites during fruit development, leaving yellow carotenoids visible. The plant hormone group the cytokinins has been implicated in the delay of senescence, and so the aim of this work was to investigate the link between cytokinin levels in ripening fruit and chlorophyll de-greening.

Methods

The expression of genes related to cytokinin metabolism and signal transduction and the concentration of cytokinin metabolites were measured. The regulation of gene expression was assayed using transient activation of the promoter of STAY-GREEN2 (SGR2) by cytokinin response regulators.

Key Results

While the total amount of cytokinin increased in fruit of both species during maturation and ripening, a high level of expression of two cytokinin biosynthetic gene family members, adenylate isopentenyltransferases, was only detected in green kiwifruit fruit during ripening. Additionally, high levels of O-glucosylated cytokinins were detected only in green kiwifruit, as was the expression of the gene for zeatin O-glucosyltransferase, the enzyme responsible for glucosylating cytokinin into a storage form. Season to season variation in gene expression was seen, and some de-greening of the green kiwifruit fruit occurred in the second season, suggesting environmental effects on the chlorophyll degradation pathway. Two cytokinin-related response regulators, RRA17 and RRB120, showed activity against the promoter of kiwifruit SGR2.

Conclusions

The results show that in kiwifruit, levels of cytokinin increase markedly during fruit ripening, and that cytokinin metabolism is differentially regulated in the fruit of the green and gold species. However, the causal factor(s) associated with the maintenance or loss of chlorophyll in kiwifruit during ripening remains obscure.     

Induced polyploidy dramatically increases the size and alters the shape of fruit in Actinidia chinensis

Background and Aims

Some otherwise promising selections of Actinidia chinensis (kiwifruit) have fruit that are too small for successful commercialization. We have therefore made the first detailed study in diploid kiwifruit of the effects of chromosome doubling induced by colchicine on fruit size, shape and crop loading.

Methods

Flow cytometric analysis of young leaves and chromosome analysis of flower buds and root tips was used to confirm the stability of induced autotetraploids. Fruit weight, size and crop load were measured in the third year after planting in the field and for three consecutive years. DNA fingerprinting was used to confirm the origin of the material.

Key Results

There was a very significant increase in fruit size in induced autotetraploids of different genotypes of A. chinensis. With the commercially important diploid cultivar ‘Hort16A’, most regenerants, Type A plants, had fruit which were much the same shape as fruit of the diploid but, at the same fruit load, were much larger and heavier. Some regenerants, Type B plants, produced fruit similar to ‘fasciated’ fruit. Fruit of the autotetraploids induced from three female red-fleshed A. chinensis selections were also 50–60 % larger than fruit of their diploid progenitors. The main increase in fruit dimensions was in their diameters. These improved fruit characteristics were stable over several seasons.

Conclusions

Chromosome doubling has been shown to increase significantly fruit size in autotetraploid A. chinensis, highlighting the considerable potential of this technique to produce new cultivars with fruit of adequate size. Other variants with differently shaped fruit were also produced but the genetic basis of this variation remains to be elucidated. Autoploids of other Actinidia species with commercial potential may also show improved fruit characteristics, opening up many new possibilities for commercial development.     

Hydraulic connections of leaves and fruit to the parent plant in Capsicum frutescens (hot pepper) during fruit ripening

Background and Aims

The hydraulic architecture and water relations of fruits and leaves of Capsicum frutescens were measured before and during the fruiting phase in order to estimate the eventual impact of xylem cavitation and embolism on the hydraulic isolation of fruits and leaves before maturation/abscission.

Methods

Measurements were performed at three different growth stages: (1) actively growing plants with some flowers before anthesis (GS1), (2) plants with about 50 % fully expanded leaves and immature fruits (GS2) and (3) plants with mature fruits and senescing basal leaves (GS3). Leaf conductance to water vapour as well as leaf and fruit water potential were measured. Hydraulic measurements were made using both the high-pressure flow meter (HPFM) and the vacuum chamber (VC) technique.

Key Results

The hydraulic architecture of hot pepper plants during the fruiting phase was clearly addressed to favour water supply to growing fruits. Hydraulic measurements revealed that leaves of GS1 plants as well as leaves and fruit peduncles of GS2 plants were free from significant xylem embolism. Substantial increases in leaf petiole and fruit peduncle resistivity were recorded in GS3 plants irrespective of the hydraulic technique used. The higher fraction of resistivity measured using the VC technique compared with the HPFM technique was apparently due to conduit embolism.

Conclusions

The present study is the first to look at the hydraulics of leaves and fruits during growth and maturation through direct, simultaneous measurements of water status and xylem efficiency of both plant regions at different hours of the day.     

Light enhances differentiation of the vascular system in the fruit of Actinidia deliciosa

Light is recognized as crucial in determining high quality of fleshy fruits, for example, kiwifruit [Actinidia deliciosa var. deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson]. Among the possible mechanisms through which light improves the quality of kiwifruit berry, there may be a direct morphogenic role on the differentiation of the fruit's vascular system, though this has not yet been investigated. The present study's aim was to determine (1) whether light positively affects the differentiation of the vascular system of the fruit and/or the pedicel, and, if so, (2) which component (xylem, phloem, or both) is more affected, and (3) in which period of the berry's development the improvement of the vascular differentiation (if any) occurs. To this end, fruit morphogenesis of kiwifruit was studied in two developmental environments (i.e., in full sunlight and in paper bags that reduced the full sunlight to 10%), and in two phases of fruit development (i.e., 1 and 5 months [harvest] after anthesis). During the growth period, the type of environment did not affect the differentiation pattern of the vascular system in the three types of bundles present in the fruit. However, in comparison with shade, light improved the vasculature in the fruit pericarp and pedicel, inducing a consistently higher extent of the xylary component in the main bundles of the fruit and pedicel, principally due to an increase in the number of xylem elements. The phloic component was also increased by light, but to a much lesser extent than that of the xylary. During the entire period of development, light-grown fruits contained higher concentrations of calcium and magnesium, as compared with shade-grown fruits. In conclusion, in the berry of Actinidia deliciosa, light enhances the differentiation of the vascular system, in particular the xylary component. The hypothesis that fruit quality is improved through a more efficient translocation of specific mineral nutrients (e.g., calcium) via the xylem is presented.     

Changes in vascular and transpiration flows affect the seasonal and daily growth of kiwifruit (Actinidia deliciosa) berry

Background and Aims

The kiwifruit berry is characterized by an early stage of rapid growth, followed by a relatively long stage of slow increase in size. Vascular and transpiration flows are the main processes through which water and carbon enter/exit the fruit, determining the daily and seasonal changes in fruit size. This work investigates the biophysical mechanisms underpinning the change in fruit growth rate during the season.

Methods

The daily patterns of phloem, xylem and transpiration in/outflows have been determined at several stages of kiwifruit development, during two seasons. The different flows were quantified by comparing the diurnal patterns of diameter change of fruit, which were then girdled and subsequently detached while measurements continued. The diurnal courses of leaf and stem water potential and of fruit pressure potential were also monitored at different times during the season.

Key Results

Xylem and transpiration flows were high during the first period of rapid volume growth and sharply decreased with fruit development. Specific phloem import was lower and gradually decreased during the season, whereas it remained constant at whole-fruit level, in accordance with fruit dry matter gain. On a daily basis, transpiration always responded to vapour pressure deficit and contributed to the daily reduction of fruit hydrostatic pressure. Xylem flow was positively related to stem-to-fruit pressure potential gradient during the first but not the last part of the season, when xylem conductivity appeared to be reduced.

Conclusions

The fruit growth model adopted by this species changes during the season due to anatomical modifications in the fruit features.     

Magnesium deficiency of kiwifruit (Actinidia deliciosa)

Magnesium deficiency was associated with large yield reductions in a five-year-old commercial kiwifruit (Actinidia deliciosa) orchard. The effect on yield resulted primarily from a reduction in fruit numbers, there being no difference in mean fruit weight between fruit harvested from affected and unaffected vines. Magnesium deficiency had no deleterious effect on postharvest storage characteristics of fruit stored at 0.5–1°C for 18 weeks; fruit from deficient vines were firmer but had slightly lower soluble solids than fruit from control vines. Although deficiency symptoms were first observed on the basal leaves of the non-fruiting shoots mid season, indications of the impending deficiency could be established very early in the season using foliar analysis. Magnesium concentrations in youngest fully expanded leaves (YFEL) on the affected vines were less than 2.0 g kg⁻¹ DM four weeks after budbreak and remained below this value for the rest of the season; concentrations in YFEL on unaffected vines did not decrease below this value and gradually increased after fruitset to 4.5 g kg⁻¹ DM at harvest. To avert potential production losses, it is suggested that soluble magnesium fertilizers (containing at least 200 kg ha⁻¹ Mg) should be broadcast early in the season if foliar magnesium concentrations less than 2.0 gkg⁻¹ DM are measured four–six weeks after budbreak.     

Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var.deliciosa)

Five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var.deliciosa cv. Hayward) were isolated from a library made from young fruit, 8–10 days after anthesis. One gene (pKIWI503) has low levels of expression in young fruit but is induced late in fruit development and during fruit ripening, and has some homology to plant metallothionein-like proteins. The other four genes are highly expressed in young fruit with reduced expression in the later stages of fruit development. pKIWI504 has strong homology to plant metallothionein-like proteins and pKIWI505 exhibits homology to the -subunit of the mitochondrial ATP synthase gene. The two other genes (pKIWI501 and 502) encode proteins with no significant homology to other known sequences.     

Temporal expression of effects of varying nitrogen supply on canopy growth, photosynthesis and fruit production for Actinidia deliciosa vines in the field

The effects of varying nitrogen supply on canopy leaf area, response of leaf net photosynthesis (A_n) to quantum flux density (Q), and fruit yields of kiwifruit vines (Actinidia deliciosa var. deliciosa) were examined in a two-year field experiment. Vines were grown with 0, 250 or 750 kg N ha^?1 year^?1. The responses to nitrogen supply were compared with responses to shade, to examine the impact of reduced carbon assimilation on canopy leaf area and fruit yields. Nitrogen supply did not affect significantly any of the measured variables during the first season of the experiment. In the second season, canopy leaf area was reduced significantly where nitrogen supply was limited. The quantum efficiency of photosynthesis (φ_q) increased from 0. 03 mol CO₂ mol^?1 Q soon after leaf emergence to more than 0. 05 mol CO₂ mol^?1 Q during the middle of the growing season. The quantum saturated rate of A_n (A_sat) also increased during the season, from 7–10 μmol CO₂ m^?2 s^?1 soon after leaf emergence, to 15–20 (μmol CO₂ m^?2 s^?1 during the middle of the growing season. φ_q and A_sat increased significantly with nitrogen supply at all measurement times during the second season. For vines with high nitrogen, fruit yields in both seasons were similar, averaging 3. 05 kg m^?2. Fruit yields in the second season were reduced significantly where nitrogen supply was limited, due to reduced fruit numbers. The relative effects of reduced leaf area and reduced leaf photosynthesis for carbon assimilation by nitrogen deficient vines were examined using a mathematical model of canopy photosynthesis for kiwifruit vines. Simulations of canopy photosynthesis indicated that effects on leaf area and on leaf photosynthesis were of similar importance in the overall effects of nitrogen deficiency on carbon assimilation. The effects of nitrogen supply on fruit numbers (i. e. flower development) preceded the measured effects on carbon assimilation, indicating that the nitrogen supply affected carbon partitioning to reserves in the first season.     

A functional�Cstructural kiwifruit vine model integrating architecture, carbon dynamics and effects of the environment

Background and Aims

Functional–structural modelling can be used to increase our understanding of how different aspects of plant structure and function interact, identify knowledge gaps and guide priorities for future experimentation. By integrating existing knowledge of the different aspects of the kiwifruit (Actinidia deliciosa) vine''s architecture and physiology, our aim is to develop conceptual and mathematical hypotheses on several of the vine''s features: (a) plasticity of the vine''s architecture; (b) effects of organ position within the canopy on its size; (c) effects of environment and horticultural management on shoot growth, light distribution and organ size; and (d) role of carbon reserves in early shoot growth.

Methods

Using the L-system modelling platform, a functional–structural plant model of a kiwifruit vine was created that integrates architectural development, mechanistic modelling of carbon transport and allocation, and environmental and management effects on vine and fruit growth. The branching pattern was captured at the individual shoot level by modelling axillary shoot development using a discrete-time Markov chain. An existing carbon transport resistance model was extended to account for several source/sink components of individual plant elements. A quasi-Monte Carlo path-tracing algorithm was used to estimate the absorbed irradiance of each leaf.

Key Results

Several simulations were performed to illustrate the model''s potential to reproduce the major features of the vine''s behaviour. The model simulated vine growth responses that were qualitatively similar to those observed in experiments, including the plastic response of shoot growth to local carbon supply, the branching patterns of two Actinidia species, the effect of carbon limitation and topological distance on fruit size and the complex behaviour of sink competition for carbon.

Conclusions

The model is able to reproduce differences in vine and fruit growth arising from various experimental treatments. This implies it will be a valuable tool for refining our understanding of kiwifruit growth and for identifying strategies to improve production.     

Actinidia arguta: volatile compounds in fruit and flowers

More than 240 compounds were detected when the volatile components of the flowers and the fruit from several Actinidia arguta genotypes were investigated. Around 60-70 different compounds were extracted from individual tissues of each genotype. Two different methods of volatile sampling (headspace and solvent) favoured different classes of compounds, dependent upon their volatilities and solubilities in the flower or fruit matrices. The compounds extracted from flowers largely comprised linalool derivatives including the lilac aldehydes (12a-d) and alcohols (13a-d), 2,6-dimethyl-6-hydroxyocta-2,7-dienal (8), 8-hydroxylinalool (9), sesquiterpenes, and benzene compounds that are presumed metabolites of phenylalanine and tyrosine. Extracts of fruit samples contained some monoterpenes, but were dominated by esters such as ethyl butanoate, hexanoate, 2-methylbutanoate and 2-methylpropanoate, and by the aldehydes hexanal and hex-E2-enal. A number of unidentified compounds were also detected, including 8 from flowers that are so closely related that they are either isomers of one compound or two or more closely related compounds. This is the first report of the presence of a range of linalool derivatives in Actinidia.     

Tapetum and middle layer control male fertility in Actinidia deliciosa

Background and Aims

Dioecism characterizes many crop species of economic value, including kiwifruit (Actinidia deliciosa). Kiwifruit male sterility occurs at the microspore stage. The cell walls of the microspores and the pollen of the male-sterile and male-fertile flowers, respectively, differ in glucose and galactose levels. In numerous plants, pollen formation involves normal functioning and degeneration timing of the tapetum, with calcium and carbohydrates provided by the tapetum essential for male fertility. The aim of this study was to determine whether the anther wall controls male fertility in kiwifruit, providing calcium and carbohydrates to the microspores.

Methods

The events occurring in the anther wall and microspores of male-fertile and male-sterile anthers were investigated by analyses of light microscopy, epifluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and transmission electron microscopy coupled with electron spectroscopy. The possibility that male sterility was related to anther tissue malfunctioning with regard to calcium/glucose/galactose provision to the microspores was also investigated by in vitro anther culture.

Key Results

Both tapetum and the middle layer showed secretory activity and both degenerated by programmed cell death (PCD), but PCD was later in male-sterile than in male-fertile anthers. Calcium accumulated in cell walls of the middle layer and tapetum and in the exine of microspores and pollen, reaching higher levels in anther wall tissues and dead microspores of male-sterile anthers. A specific supply of glucose and calcium induced normal pollen formation in in vitro-cultured anthers of the male-sterile genotype.

Conclusions

The results show that male sterility in kiwifruit is induced by anther wall tissues through prolonged secretory activity caused by a delay in PCD, in the middle layer in particular. In vitro culture results support the sporophytic control of male fertility in kiwifruit and open the way to applications to overcome dioecism and optimize kiwifruit production.     

Polygalacturonase gene expression in kiwifruit: relationship to fruit softening and ethylene production

In kiwifruit, much of the softening process occurs prior to the respiratory climacteric and production of ethylene. This fruit therefore represents an excellent model system for dissecting the process of softening in the absence of endogenous ethylene production. We have characterized the expression of three polygalacturonase (PG) cDNA clones (CkPGA, B and C) isolated from fruit of Actinidia chinensis. Expression of CkPGA and B was detected by northern analysis only in fruit producing endogenous ethylene, and by RT-PCR in other tissues including flower buds, petals at anthesis, and senescent petals. CkPGA promoter fragments of 1296, 860 and 467 bp fused to the -glucuronidase (uidA) reporter gene directed fruit-specific gene expression during the climacteric in transgenic tomato. CkPGC gene expression was observed in softening fruit, and reached maximum levels (50-fold higher than for CkPGA and B) as fruit passed through the climacteric. However, expression of this gene was also readily detected during fruit development and in fruit harvested prior to the onset of softening. Using RT-PCR, expression of CkPGC was also detected at low levels in root tips and in senescent petals. These results suggest that PG expression is required not only during periods of cell wall degeneration, but also during periods of cell wall turnover and expansion.     

Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). II. Effects of reduced pollination and CPPU application

The significance of changes in cytokinin content during early fruit growth was examined in the kiwifruit ( Actinidia deliciosa var. deliciosa cv. Hayward). Fruit growth was modified by the reduction of seed number or by the application of the synthetic phenylurea cytokinin N -(2-chloro-4-pyridyl)- N -phenylurea (CPPU). The influence of these treatments on cell division was monitored by flow cytometry and changes in the endogenous cytokinins were measured at days 10 and 20 after anthesis, using high-performance liquid chromatography and radioimmunoassay. Total cytokinin levels appeared not to be limiting growth since the highest total cytokinin concentration was detected in unpollinated fruit, which abscised by day 25 after anthesis. However, compared with control fruit which had the highest concentration of zeatin (Z) 10 days post anthesis, Z levels were low in unpollinated fruit. It is hypothesised that an increase in Z is the critical change in cytokinin metabolism required for the initiation of cell division and fruit growth. The synthetic cytokinin CPPU promoted fruit development, but there was a decrease in the endogenous cytokinin concentration. Zeatin was not detected in CPPU-treated fruit. Cell division was reduced in unpollinated fruitlets but there was no significant difference ( P > 0.05) between the other treatments. Differences in final fruit size appeared to be due to cell expansion.     

中华猕猴桃基因组可变剪接事件鉴定及分析

可变剪接使一个基因能产生多种m RNA成熟体,极大地增加蛋白多样性.采用中华猕猴桃基因组数据做参考数据,利用中华猕猴桃叶片和果实3个不同发育时期(未成熟、半成熟和成熟期)的转录组数据,从中华猕猴桃基因组(39040个基因)中共鉴定出11651个基因(占总基因数的29%)对应的32180个可变剪接事件.在可变剪接不同类型中,内含子保留类型的发生频率最高,占50%以上;3′可变位点类型频率约为5′端可变类型的2倍.GO富集分析结果表明,可变剪接的基因主要富集于酶调控及核苷酸结合相关功能的GO类别中,而组织特有可变剪接基因功能富集热点与组织的重要功能关联,叶片多为肌动蛋白及微管相关;未成熟果实与双组分信号系统相关;半成熟果实多与磷脂合成过程相关;成熟果实多与信号传递过程相关.另外,55.6%的维生素合成相关基因发生可变剪接事件,显著高于基因组水平的29.6%,暗示着可变剪接参与维生素合成相关基因代谢过程中的重要作用.通过对中华猕猴桃全基因组可变剪接的分析,为解析中华猕猴桃基因组及进一步开展相关分子育种工作提供依据.     

Microcallus formation from leaf mesophyll protoplasts in the genus Actinidia Lindl

Summary Microcallus (more than 60 cells) formation was obtained from leaf mesophyll protoplasts of 6 species and varieties in the genus Actinidia Lindl. (kiwifruit). The best results were achieved by using liquid over agarose culture for A. arguta var. arguta, liquid and agarose disc type culture for A. arguta var. purpurea, agarose disc type culture for A. arguta cv. Issaï and A. deliciosa and liquid agarose bead type- and disc type culture for A. kolomikta and A. polygama. Several factors influencing purification, browning, survival and sustained division of the protoplasts are briefly discussed.Abbreviations BAP benzylaminopurine - CPW cell and protoplast washing solution - 2,4-D dichlorophenoxyacetic acid - GA3 gibberellic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino) ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PVP polyvinylpyrrolidone - BT agarose bead type culture - DT agarose disc type culture     

Towards aspect-oriented functional--structural plant modelling

Background and Aims

Functional–structural plant models (FSPMs) are used to integrate knowledge and test hypotheses of plant behaviour, and to aid in the development of decision support systems. A significant amount of effort is being put into providing a sound methodology for building them. Standard techniques, such as procedural or object-oriented programming, are not suited for clearly separating aspects of plant function that criss-cross between different components of plant structure, which makes it difficult to reuse and share their implementations. The aim of this paper is to present an aspect-oriented programming approach that helps to overcome this difficulty.

Methods

The L-system-based plant modelling language L+C was used to develop an aspect-oriented approach to plant modelling based on multi-modules. Each element of the plant structure was represented by a sequence of L-system modules (rather than a single module), with each module representing an aspect of the element''s function. Separate sets of productions were used for modelling each aspect, with context-sensitive rules facilitated by local lists of modules to consider/ignore. Aspect weaving or communication between aspects was made possible through the use of pseudo-L-systems, where the strict-predecessor of a production rule was specified as a multi-module.

Key Results

The new approach was used to integrate previously modelled aspects of carbon dynamics, apical dominance and biomechanics with a model of a developing kiwifruit shoot. These aspects were specified independently and their implementation was based on source code provided by the original authors without major changes.

Conclusions

This new aspect-oriented approach to plant modelling is well suited for studying complex phenomena in plant science, because it can be used to integrate separate models of individual aspects of plant development and function, both previously constructed and new, into clearly organized, comprehensive FSPMs. In a future work, this approach could be further extended into an aspect-oriented programming language for FSPMs.     

Learning decreases heterospecific courtship and mating in fruit flies

Recent theory and data suggest that adaptive use of learning in the context of sexual behaviour could contribute to assortative mating. Experiments examining this issue indicated that male Drosophila persimilis that experienced courtship and rejection by heterospecific females exhibited significantly lower levels of heterospecific courtship and mating compared with those of inexperienced males. These results indicate that experience in the context of sexual behaviour in fruit flies could reduce gene flow between diverging populations, which may contribute to incipient speciation.     

Immunolocalisation of arabinogalactan proteins and pectins in Actinidia deliciosa pollen

Summary. The cell wall composition of germinating pollen grains of Actinidia deliciosa was studied by immunolocalization with monoclonal antibodies against arabinogalactan proteins (AGPs) and pectins. In ungerminated pollen, the JIM8 epitope (against a subset of AGPs) was located in the intine and in the cytoplasm, while the MAC207 epitope (against AGPs) was only located in the exine. After germination, the JIM8 and MAC 207 epitopes were located in the cytoplasm and in the pollen tube wall. The Yariv reagent that binds to AGPs was added to the germination medium inducing a reduction or inhibition in pollen germination. This indicates that AGPs are present in the growing pollen tube and play an important role in pollen germination. To identify the nature of the pectins found in pollen grains and tubes, four monoclonal antibodies were used. The JIM5 epitope (against unesterified pectins) was located in the intine, more intensely in the pore region, and along the pollen tube wall, and the JIM7 epitope (against methyl-esterified pectins) was also observed in the cytoplasm. After germination, the JIM5 epitope was located in the pollen tube wall; although, the tube tip was not labelled. The JIM7 epitope was located in the entire pollen tube wall. LM5 (against galactans) showed a labelling pattern similar to that of JIM5 and the pattern of LM6 (against arabinans) was similar to that of JIM7. Pectins show different distribution patterns when the degree of esterification is considered. Pollen tube wall pectins are less esterified than those of the pollen tube tip. The association of AGPs with pectins in the cell wall of the pollen grain and the pollen tube may play an important role in the maintenance of cell shape during pollen growth and development.Correspondence and reprints: Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.