José A. Mercado

Manipulation of Strawberry Fruit Softening by Antisense Expression of a Pectate Lyase Gene

Strawberry (Fragaria ×ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.

Pollen sporopollenin: degradation and structural elucidation

Abstract We report the isolation of purified sporopollenin from pollen grains of different species and its complete solubilization. Exine from Pinus pinaster, Betula alba, Ambrosia elatior and Capsicum annuum was extracted by treatment with hydrogen fluoride in pyridine. These exines were purified from their aromatic moieties and from fatty acids linked by ester bonds using acidolysis and saponification treatments. The biopolymer obtained retains almost completely the shape of the original pollen grain. Fourier-transform infrared spectroscopy analysis of the isolated sporopollenin showed the absence of polysaccharide and phenolic material and the presence of carboxylic acid groups joined to unsaturations and ether linkages. Sporopollenin samples were successfully degraded by exhaustive 24-h ozonolysis at room temperature. Gentle ozonolysis (3 h at 0°C) did not completely degrade the biopolymer. The compounds obtained after exhaustive ozonolysis were analysed by gas chromatography-mass spectrometry. Dicarboxylic acids with a low number of carbon atoms were identified as major components of sporopollenin from P. pinaster, A. elatior and C. annuum, representing 28.8%, 63.2% and 88.5%, respectively, of the total compounds obtained. Fatty acids and n-alkanes also were identified in P. pinaster, A. elatior and B. alba sporopollenin. From the data obtained, an hypothesis about the chemical nature and structural arrangement of the sporopollenin is proposed.

Antisense Down-Regulation of the FaPG1 Gene Reveals an Unexpected Central Role for Polygalacturonase in Strawberry Fruit Softening

The strawberry (Fragaria × ananassa ‘Chandler’) fruit undergoes a fast softening during ripening. Polygalacturonase (PG) activity is low during this process, but two ripening-related PG genes, FaPG1 and FaPG2, have been cloned. Both genes were up-regulated during fruit ripening and were also negatively regulated by auxin. To further assess the role of FaPG1 on strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the 35S promoter (APG lines) were obtained. Sixteen out of 30 independent transgenic lines showed fruit yields similar to those of the control. Several quality parameters were measured in ripe fruits from these 16 lines. Fruit weight was slightly reduced in four lines, and most of them showed an increase in soluble solid content. Half of these lines yielded fruits significantly firmer than did the control. Four APG lines were selected, their ripened fruits being on average 163% firmer than the control. The postharvest softening of APG fruits was also diminished. Ripened fruits from the four selected lines showed a 90% to 95% decrease in FaPG1 transcript abundance, whereas the level of FaPG2 was not significantly altered. Total PG activity was reduced in three of these lines when compared with control fruits. Cell wall extracts from APG fruits showed a reduction in pectin solubilization and an increase in pectins covalently bound to the cell wall. A comparative transcriptomic analysis of gene expression between the ripened receptacle of the control and those of the APG fruits (comprising 1,250 receptacle expressed sequence tags) did not show any statistically significant change. These results indicate that FaPG1 plays a central role in strawberry softening.The loss of firm texture is one of the most characteristic physiological processes that occur during the ripening of fleshy fruits. It is generally accepted that the disassembly of primary cell wall and middle lamella is the main factor involved in fruit softening. In this process, polygalacturonase (PG) has been implicated in the degradation of the polyuronide network in several fruits. However, the minor effect of PG downregulation on tomato softening, reported during the nineties, minimized the role of this enzyme in softening. Further works in other fruits are challenging this general assumption, as is occurring in strawberry. The strawberry (Fragaria x ananassa) fruit undergoes an extensive and fast softening that limit its shelf life and postharvest. Traditionally, it has also been considered that PG plays a minor role on this process, due to the low PG activity found in ripened strawberry fruits. Transgenic strawberry plants expressing an antisense sequence of the ripening-specific PG gene FaPG1 have been generated to get an insight into the role of this gene in softening. Half of the transgenic lines analyzed yielded fruits significantly firmer than control, without being affected other fruit parameters such as weight, color or soluble solids. The increase on firmness was maintained after several days of posharvest. In these firmer lines, FaPG1 was silenced to 95%, but total PG activity was only minor reduced. At the cell wall level, transgenic fruits contained a higher amount of covalently bound pectins whereas the soluble fraction was diminished. A microarray analysis of genes expressed in ripened receptacle did not show any significant change between control and transgenic fruits. Thus, contrary to the most accepted view, it is concluded that PG plays a key role on pectin metabolism and softening of strawberry fruit.

Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler

The effects of growth regulator balance and culture conditions on the morphogenetic response of leaf disks from greenhouse grown plants of the strawberry cultivar Chandler, have been studied. Best results were obtained in the presence of 2.46 μM IBA and 8.88 μM BA, where 47% of the cultures regenerated after 16 weeks with 2.9 shoot colonies per regenerating leaf disk. Optimum incubation conditions included two weeks in the dark with subsequent transfer to light (40 μmol m-2 s-1, 16 h). The regeneration protocol was also valuable when leaf disks from in vitro grown plants were used as explants. Transformation was attempted using Agrobacterium tumefaciens carrying the plasmid pBI121. Leaf disks from in vitro cultures proliferating in the presence of 2.21 μM kinetin were best explants for transformation. A 4.22% of inoculated explants showed kanamycin resistance after 16 weeks in a medium containing 25 mg l-1 of this antibiotic. The transgenic nature of several shoots was also confirmed by the GUS assay and PCR analysis.

Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening.

Cell wall disassembly in softening fruits is a complex process involving the cumulative action of many families of wall-modifying proteins on interconnected polysaccharide matrices. One strategy to elucidate the in vivo substrates of specific enzymes and their relative importance and contribution to wall modification is to suppress their expression in transgenic fruit. It has been reported previously that inhibiting the expression of pectate lyase genes by antisense technology in strawberry (Fragaria×ananassa Duch.) fruit resulted in prolonged fruit firmness. This suggested that pectin depolymerization might make a more important contribution to strawberry fruit softening than is often stated. In this present study, three independent transgenic lines were identified exhibiting a greater than 90% reduction in pectate lyase transcript abundance. Analyses of sequential cell wall extracts from the transgenic and control fruit collectively showed clear quantitative and qualitative differences in the extractability and molecular masses of populations of pectin polymers. Wall extracts from transgenic fruits showed a reduction in pectin solubility and decreased depolymerization of more tightly bound polyuronides. Additional patterns of differential extraction of other wall-associated pectin subclasses were apparent, particularly in the sodium carbonate- and chelator-soluble polymers. In addition, microscopic studies revealed that the typical ripening-associated loss of cell–cell adhesion was substantially reduced in the transgenic fruits. These results indicate that pectate lyase plays an important degradative role in the primary wall and middle lamella in ripening strawberry fruit, and should be included in synergistic models of cell wall disassembly.

Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L.

SummaryAn efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg/l) and indole-3-butyric acid (0.25 mg/l) induced the maximum percentage of shoot regeneration (98%) and the highest number of shoot colonies per explant (4.6) after 8 weeks of culture. Isolated shoots would elongate and proliferate when the benzyladenine concentration was lowered to 0.5 mg/l. The established protocol for shoot regeneration was employed to transform leaf disks using Agrobacterium tumefaciens carrying the plasmid pBI121. A 7.7% of the inoculated explants showed kanamycin resistance after 10 weeks of selection in a medium containing 25 mg/l of this antibiotic. The transgenic shoots obtained were rooted in the presence of 25 mg/ kanamycin and successfully acclimatized. The final percentage of transformation obtained based on beta-glucuronidase expression was 6.9%.

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy

BACKGROUND One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening. SCOPE In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.

The strawberry (Fragaria×ananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae

Pectins are essential components of primary plant cell walls and middle lamellae, and are related to the consistency of the fruit and its textural changes during ripening. In fact, strawberries become soft as the middle lamellae of cortical parenchyma cells are extensively degraded during ripening, leading to the observed short post-harvest shelf life. Using a custom-made oligonucleotide-based strawberry microarray platform, a putative rhamnogalacturonate lyase gene (FaRGlyase1) was identified. Bioinformatic analysis of the FaRGlyase1 sequence allowed the identification of a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the databases. Expression of FaRGlyase1 occurred mainly in the receptacle, concurrently with ripening, and it was positively regulated by abscisic acid and negatively by auxins. FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harbouring RNA interference constructs into fruit receptacles. Light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells. In addition, genetic linkage association analyses in a strawberry-segregating population showed that FaRGLyase1 is linked to a quantitative trait loci linkage group related to fruit hardness and firmness. The results showed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.

Effects of low temperature on pepper pollen morphology and fertility: Evidence of cold induced exine alterations

SummaryThe effects of low night temperatures on pollen viability and pollen grain morphology were investigated in bell pepper (Capsicum annuum, L.) commercial hybrids ‘Latino’ and ‘Novi’. Night temperatures of 10 and 15°C decreased pollen viability, estimated by an in vitro assay and acetocarmine staining, and by decreased seed number per fruit. At anthesis, in a separate experiment, pollen grains that developed at low temperature (25/14°C, day/night) were smaller and appeared aggregated and shrunken with a differently sculptured exine which was thinner than the pollen exine developed at the 30/20°C temperature regime. The results obtained when a 10°C night-temperature treatment was applied to plants grown at 30/20°C with flower buds of different diameters, indicated that meiosis and the young microspore stage were affected. However, the later phases of microspore development and pollen maturation were not affected by the same temperature treatments.

Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits

Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell–cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit.