Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene

Strawberry (Fragaria x 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.     

Abscisic acid plays an important role in the regulation of strawberry fruit ripening

The plant hormone abscisic acid (ABA) has been suggested to play a role in fruit development, but supporting genetic evidence has been lacking. Here, we report that ABA promotes strawberry (Fragaria ananassa) fruit ripening. Using a newly established Tobacco rattle virus-induced gene silencing technique in strawberry fruit, the expression of a 9-cis-epoxycarotenoid dioxygenase gene (FaNCED1), which is key to ABA biosynthesis, was down-regulated, resulting in a significant decrease in ABA levels and uncolored fruits. Interestingly, a similar uncolored phenotype was observed in the transgenic RNA interference (RNAi) fruits, in which the expression of a putative ABA receptor gene encoding the magnesium chelatase H subunit (FaCHLH/ABAR) was down-regulated by virus-induced gene silencing. More importantly, the uncolored phenotype of the FaNCED1-down-regulated RNAi fruits could be rescued by exogenous ABA, but the ABA treatment could not reverse the uncolored phenotype of the FaCHLH/ABAR-down-regulated RNAi fruits. We observed that down-regulation of the FaCHLH/ABAR gene in the RNAi fruit altered both ABA levels and sugar content as well as a set of ABA- and/or sugar-responsive genes. Additionally, we showed that exogenous sugars, particularly sucrose, can significantly promote ripening while stimulating ABA accumulation. These data provide evidence that ABA is a signal molecule that promotes strawberry ripening and that the putative ABA receptor, FaCHLH/ABAR, is a positive regulator of ripening in response to ABA.     

Phospholipase D1 plays a key role in TNF-alpha signaling

The primary characteristic features of any inflammatory or infectious lesions are immune cell infiltration, cellular proliferation, and the generation of proinflammatory mediators. TNF-alpha is a potent proinflammatory and immuno-regulatory cytokine. Decades of research have been focused on the physiological/pathophysiological events triggered by TNF-alpha. However, the signaling network initiated by TNF-alpha in human leukocytes is still poorly understood. In this study, we report that TNF-alpha activates phospholipase D1 (PLD1), in a dose-dependent manner, and PLD1 is required for the activation of sphingosine kinase and cytosolic calcium signals. PLD1 is also required for NFkappaB and ERK1/2 activation in human monocytic cells. Using antisense oligonucleotides to reduce specifically the expression of PLD isozymes showed PLD1, but not PLD2, to be coupled to TNF-alpha signaling and that PLD1 is required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, the coupling of TNF-alpha to activation of the phosphorylation of ERK1/2 and the activation of NFkappaB were inhibited by pretreating cells with antisense to PLD1, but not to PLD2; thus, demonstrating a specific requirement for PLD1. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required for TNF-alpha-induced production of several important cytokines, such as IL-1beta, IL-5, IL-6, and IL-13, in human monocytes. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by TNF-alpha and its functional role for coordinating the signals to inflammatory responses.     

Brassinosteroid plays a role on pink stage for receptor and transcription factors involved in strawberry fruit ripening

In contrast to climacteric fruits, the ripening regulation of non-climacteric fruits is not well understood. Strawberry is a representative example of this kind of fruit, so it has been used as a model system for this category. In this study, the effect of exogenous brassinosteroid (BR) on the expression of the receptor (FaBRI1) and two components of the signaling pathway (FaBIN2 and FaBRZ1) was analyzed in Fragaria × ananassa cultivar Camino Real by quantitative real-time polymerase chain reaction (RT-qPCR). The physicochemical and phytochemical characteristics of fruits were evaluated in the field and postharvest trials. Perception and signal transduction pathway show little gene action mainly when elicited by epibrassinolide, having treatment differences due mainly the pink stage. This leads us to suggest that BR is involved in strawberry fruit ripening, where the threshold to action seems to be very low and act in the pink stage according to perception and transduction signals. However, owing to the physicochemical and phytochemical characteristics, the BR influence mainly starts in the white stage for total sugar and soluble solid in field assay and for total sugar in the postharvest assays. In addition, there is a positive effect on vitamin C content and total anthocyanins for the treated red fruits in the postharvest assay. All results show that BR is involved in strawberry fruit ripening, in different stages, mainly in a phenylpropanoid pathway. However, new assays to confirm the real BR importance on strawberry maturation and fruit quality.     

Sts1 plays a key role in targeting proteasomes to the nucleus

The evidence that nuclear proteins can be degraded by cytosolic proteasomes has received considerable experimental support. However, the presence of proteasome subunits in the nucleus also suggests that protein degradation could occur within this organelle. We determined that Sts1 can target proteasomes to the nucleus and facilitate the degradation of a nuclear protein. Specific sts1 mutants showed reduced nuclear proteasomes at the nonpermissive temperature. In contrast, high expression of Sts1 increased the levels of nuclear proteasomes. Sts1 targets proteasomes to the nucleus by interacting with Srp1, a nuclear import factor that binds nuclear localization signals. Deletion of the NLS in Sts1 prevented its interaction with Srp1 and caused proteasome mislocalization. In agreement with this observation, a mutation in Srp1 that weakened its interaction with Sts1 also reduced nuclear targeting of proteasomes. We reported that Sts1 could suppress growth and proteolytic defects of rad23Δ rpn10Δ. We show here that Sts1 suppresses a previously undetected proteasome localization defect in this mutant. Taken together, these findings explain the suppression of rad23Δ rpn10Δ by Sts1 and suggest that the degradation of nuclear substrates requires efficient proteasome localization.     

The Phe105 loop of Alix Bro1 domain plays a key role in HIV-1 release

Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects that result from interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical boomerang folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently, mutation of Phe105 and surrounding residues at the tip of the loop compromise the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix.     

An expansin gene expressed in ripening strawberry fruit

Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria x ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.     

Grp1 plays a key role in linking insulin signaling to glut4 recycling

The glucose transporter type 4 (glut4) is critical for metabolic homeostasis. Insulin regulates glut4 by modulating its expression on the cell surface. This regulation is mainly achieved by targeting the endocytic recycling of glut4. We identify general receptor for 3-phosphoinositides 1 (Grp1) as a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (ARF6) that promotes glut4 vesicle formation. Grp1 also promotes the later steps of glut4 recycling through ARF6. Insulin signaling regulates Grp1 through phosphorylation by Akt. We also find that mutations that mimic constitutive phosphorylation of Grp1 can bypass upstream insulin signaling to induce glut4 recycling. Thus, we have uncovered a major mechanism by which insulin regulates glut4 recycling. Our findings also reveal the complexity by which a single small GTPase in vesicular transport can coordinate its multiple steps to accomplish a round of transport.     

Notch signaling plays a key role in cardiac cell differentiation

Results from lineage tracing studies indicate that precursor cells in the ventricles give rise to both cardiac muscle and conduction cells. Cardiac conduction cells are specialized cells responsible for orchestrating the rhythmic contractions of the heart. Here, we show that Notch signaling plays an important role in the differentiation of cardiac muscle and conduction cell lineages in the ventricles. Notch1 expression coincides with a conduction marker, HNK-1, at early stages. Misexpression of constitutively active Notch1 (NIC) in early heart tubes in chick exhibited multiple effects on cardiac cell differentiation. Cells expressing NIC had a significant decrease in expression of cardiac muscle markers, but an increase in expression of conduction cell markers, HNK-1, and SNAP-25. However, the expression of the conduction marker connexin 40 was inhibited. Loss-of-function study, using a dominant-negative form of Suppressor-of-Hairless, further supports that Notch1 signaling is important for the differentiation of these cardiac cell types. Functional studies show that the expression of constitutively active Notch1 resulted in abnormalities in ventricular conduction pathway patterns.     

Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening

Despite the fact that the organic acid content of a fruit is regarded as one of its most commercially important quality traits when assessed by the consumer, relatively little is known concerning the physiological importance of organic acid metabolism for the fruit itself. Here, we evaluate the effect of modifying malate metabolism in a fruit-specific manner, by reduction of the activities of either mitochondrial malate dehydrogenase or fumarase, via targeted antisense approaches in tomato (Solanum lycopersicum). While these genetic perturbations had relatively little effect on the total fruit yield, they had dramatic consequences for fruit metabolism, as well as unanticipated changes in postharvest shelf life and susceptibility to bacterial infection. Detailed characterization suggested that the rate of ripening was essentially unaltered but that lines containing higher malate were characterized by lower levels of transitory starch and a lower soluble sugars content at harvest, whereas those with lower malate contained higher levels of these carbohydrates. Analysis of the activation state of ADP-glucose pyrophosphorylase revealed that it correlated with the accumulation of transitory starch. Taken together with the altered activation state of the plastidial malate dehydrogenase and the modified pigment biosynthesis of the transgenic lines, these results suggest that the phenotypes are due to an altered cellular redox status. The combined data reveal the importance of malate metabolism in tomato fruit metabolism and development and confirm the importance of transitory starch in the determination of agronomic yield in this species.     

Induction of AGAMOUS gene expression plays a key role in ripening of tomato sepals in vitro

In vitro culture of VFNT Cherry tomato sepals (calyx) at 16–21 °C results in developmental changes that are similar to those that occur in fruit tissue [10]. Sepals become swollen, red, and succulent, produce ethylene, and have increased levels of polygalacturonase RNA. They also produce many flavor volatiles characteristic of ripe tomato fruit and undergo similar changes in sugar content [11]. We examined the expression of the tomato AGAMOUS gene, TAG1, in ripening, in vitro sepal cultures and other tissues from the plant and found that TAG1 RNA accumulates to higher levels than expected from data from other plants. Contrary to reports on the absence of AGAMOUS in sepals, TAG1 RNA levels in green sepals from greenhouse-grown plants is detectable, its concentration increasing with in vitro ripening to levels that were even higher than in red, ripe fruit. Sepals of fruit on transgenic tomato plants that expressed TAG1 ectopically were induced by low temperature to ripen in vivo, producing lycopene and undergoing cell wall softening as is characteristic of pericarpic tissue. We therefore propose that the induction of elevated TAG1 gene expression plays a key role in developmental changes that result in sepal ripening.     

The alpha 1 beta 1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen.

When the alpha and beta chains were separated from human oxyhemoglobin (HbO(2)), each individual chain was oxidized easily to the ferric form, their rates being almost the same with a very strong acid-catalysis. In the HbO(2) tetramer, on the other hand, both chains become considerably resistant to autoxidation over a wide range of pH values (pH 5-11). Moreover, HbA showed a biphasic autoxidation curve containing the two rate constants, i.e. k(f) for the fast oxidation due to the alpha chains, and k(s) for the slow oxidation to the beta chains. The k(f)/k(s) ratio increased from 3.2 at pH 7.5-7.3 at pH 5.8, but became 1 : 1 at pH values higher than 8.5. In the present work, we used the valency hybrid tetramers such as (alpha(3+))2(beta O(2))(2) and (alpha O(2)(2)(beta(3+))(2), and demonstrated that the autoxidation rate of either the alpha or beta chains (when O2- ligated) is independent of the valency state of the corresponding counterpart chains. From these results, we have concluded that the formation of the alpha 1 beta 1 or alpha 2 beta 2 contact suppresses remarkably the autoxidation rate of the beta chain and thus plays a key role in stabilizing the HbO(2) tetramer. Its mechanistic details were also given in terms of a nucleophilic displacement of O(2)(-) from the FeO(2) center, and the emphasis was placed on the proton-catalyzed process performed by the distal histidine residue.     

Pectin esterase gene family in strawberry fruit: study of FaPE1, a ripening-specific isoform

Pectin esterases (PE, EC 3.1.1.11) catalyse the demethylation of pectin. As a result of its activity, structural interactions among cell wall components during cell wall turnover and loosening are affected. In plants, PEs are typically encoded by a gene family. This family has been studied in strawberry (Fragaria x ananassa Duch.) in order to investigate the role of distinct PE genes during fruit ripening and senescence. By a combination of a PCR-based library screening and RT-PCR four different strawberry PE cDNAs, termed FaPE1 to FaPE4, have been isolated. Differential expression of each FaPE gene in various organs and during fruit development was revealed by northern blot. FaPE1 is specifically expressed in fruit, showing an increasing expression during the ripening process up to a maximum in the turning stage. Concerning hormone regulation, auxin treatment increased FaPE1 mRNA levels in green fruit, whereas exogenous ethylene decreased FaPE1 mRNA levels in ripe and senescing fruits. It is proposed that this repression of FaPE1 expression could be involved in textural changes occurring during fruit senescence.