Amnon Lers

Senescence-induced RNases in tomato

A main feature of leaf senescence is the hydrolysis of macromolecules by hydrolases of various types, and redistribution of released materials. We have initiated a study for the characterization of RNases involved in nucleic acid catabolism during senescence. Using a PCR-based cloning approach we isolated from tomato two senescence-induced RNase cDNA clones. Each of these cDNAs hybridized to a senescence-induced transcript in northern analysis. One RNase cDNA was identical to the tomato LX RNase while the second corresponded to the LE RNase. Both LX and LE RNase genes had originally been demonstrated to be induced after phosphate starvation of tomato cell culture but nothing was known about their expression or function in plants. We observed that the expression of the LX and LE genes is induced in leaves during an advanced stage of senescence with the LX transcript level being much more induced than that of LE. Low-level expression of the RNase genes was observed in flowers and artificially senescing detached leaves while no expression could be detected in stems, roots, or fruits at different ripening stages. Ethylene activated the LX gene expression in detached young leaves while LE gene expression, which could be transiently induced by wounding, appeared to be activated by abscisic acid. We suggest that the LX RNase has a role in RNA catabolism in the final stage of senescence, and LE may function during wounding as a plant defense protein.

Delayed storage and controlled atmosphere storage of nectarines: two strategies to prevent woolliness

Both delayed storage (DS) of nectarine (Prunus persica cv. Flavortop) fruits held for 48 h at 20°C before storage, and controlled atmosphere (CA) storage, fruits stored at 10% CO2 ,3 % O 2, alleviated or prevented chilling injury manifested as woolliness in nectarine fruits stored for 4 or 6 weeks at 0°C. Control fruits showed 80 and 100% woolliness during ripening after 4 or 6 weeks at 0°C, respectively. DS and CA were similar in their beneficial effect after 4 weeks and CA was better after 6 weeks storage. The two storage processes appeared to prevent woolliness by different mechanisms. DS initiated ripening so that at removal from storage polygalacturonase (PG) activity was higher and pectin esterase (PE) activity lower than in control fruits. The PG activity increased further during ripening, and normal softening occurred in DS fruits. There was no difference in mRNA abundance of PG and PE between DS and control fruits. CA repressed both mRNA levels and activity of PG during storage, but allowed recovery of activity during ripening. Endoglucanase (EGase) activity declined during ripening in all fruits, but control fruits retained more activity than DS or CA fruits. The EGase mRNA level was high in control fruits during ripening after storage, and almost undetectable in all treatments at all other times. We postulate that the ratio between PG:PE either at removal (DS) or during ripening (CA) will determine whether woolliness develops or not.

Ethylene involvement in the cold storage disorder of ‘Flavortop’ nectarine

Abstract ‘Flavortop’ nectarine was either held at 20°C for ripening or stored at 0°C for 30 days after treatment with 0.1 ppm 1-methylcyclopropene (MCP) at harvest. Half of the untreated fruits were exposed to 15 ppm ethylene during storage. Fruit softening was retarded by MCP both without storage or after storage but not affected by ethylene treatment during storage. Fruits treated with MCP developed severe flesh woolliness and reddening and had lower expressible juice compared to the ethylene treatment. Ethylene production of the fruits following treatment was not affected by MCP but was inhibited after storage while it was enhanced by exogenous ethylene. mRNA abundance of ACC oxidase (ACO), polygalacturonase (PG) and pectin esterase (PE) during storage was also inhibited by MCP, and ACO and PG expression was furthermore inhibited after post-storage ripening. Ethylene-treated fruits had higher message levels of ACC synthase (ACS) and PE than control fruit following storage while ACO levels were higher in control fruit and PG was similar in both. The accumulation of endogluconase (EGase) mRNA was enhanced by MCP at all stages while inhibited by ethylene after post-storage ripening. The data suggest that a certain level of ethylene production is essential for normal ripening of nectarines after cold storage. Exogenous ethylene during storage enhanced ethylene production after storage, thereby promoting the sequence of cell wall hydrolysis necessary for normal ripening. MCP blocked the ethylene action and inhibited its synthesis after cold storage, subsequently leading to abnormal softening and the occurrence of severe disorders.

Microarray Analysis of the Abscission-Related Transcriptome in the Tomato Flower Abscission Zone in Response to Auxin Depletion

The abscission process is initiated by changes in the auxin gradient across the abscission zone (AZ) and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the molecular and biochemical basis of the increased AZ sensitivity to ethylene. We examined transcriptome changes in the tomato (Solanum lycopersicum ‘Shiran 1335’) flower AZ during the rapid acquisition of ethylene sensitivity following flower removal, which depletes the AZ from auxin, with or without preexposure to 1-methylcyclopropene or application of indole-3-acetic acid after flower removal. Microarray analysis using the Affymetrix Tomato GeneChip revealed changes in expression, occurring prior to and during pedicel abscission, of many genes with possible regulatory functions. They included a range of auxin- and ethylene-related transcription factors, other transcription factors and regulatory genes that are transiently induced early, 2 h after flower removal, and a set of novel AZ-specific genes. All gene expressions initiated by flower removal and leading to pedicel abscission were inhibited by indole-3-acetic acid application, while 1-methylcyclopropene pretreatment inhibited only the ethylene-induced expressions, including those induced by wound-associated ethylene signals. These results confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes resulting from auxin depletion. Our results shed light on the regulatory control of abscission at the molecular level and further expand our knowledge of auxin-ethylene cross talk during the initial controlling stages of the process.

Overexpression of the CBF2 transcriptional activator in Arabidopsis delays leaf senescence and extends plant longevity

Leaf senescence is a programmed developmental process governed by various endogenous and exogenous factors, such as the plant developmental stage, leaf age, phytohormone levels, darkness, and exposure to stresses. It was found that, in addition to its well-documented role in the enhancement of plant frost tolerance, overexpression of the C-repeat/dehydration responsive element binding factor 2 (CBF2) gene in Arabidopsis delayed the onset of leaf senescence and extended the life span of the plants by approximately 2 weeks. This phenomenon was exhibited both during developmental leaf senescence and during senescence of detached leaves artificially induced by either darkness or phytohormones. Transcriptome analysis using the Affymetrix ATH1 genome array revealed that overexpression of CBF2 significantly influenced the expression of 286 genes in mature leaf tissue. In addition to 30 stress-related genes, overexpression of CBF2 also affected the expression of 24 transcription factor (TF) genes, and 20 genes involved in protein metabolism, degradation, and post-translational modification. These results indicate that overexpression of CBF2 not only increases frost tolerance, but also affects other developmental processes, most likely through interactions with additional TFs and protein modification genes. The present findings shed new light on the crucial relationship between plant stress tolerance and longevity, as reported for other eukaryotic organisms.

Expression analysis of the BFN1 nuclease gene promoter during senescence, abscission, and programmed cell death-related processes

Little is known about the biological role of nucleases induced during plant senescence and programmed cell death (PCD). Arabidopsis BFN1 has been identified as a senescence-associated type I nuclease, whose protein sequence shares high homology with some other senescence- or PCD-associated plant nucleases. To learn about BFN1 regulation, its expression pattern was analysed. A 2.3 kb portion of the 5′ promoter sequence of BFN1 was cloned and its ability to activate the GUS reporter gene was examined. Transgenic Arabidopsis and tomato plants harbouring this chimeric construct were analysed for GUS expression. In both, the BFN1 promoter was able specifically to direct GUS expression in senescent leaves, differentiating xylem and the abscission zone of flowers. Thus, at least part of the regulation of BFN1 is mediated at the transcriptional level, and the regulatory elements are recognized in the two different plants. In tomato, specific expression was observed in the leaf and the fruit abscission zones. The BFN1 promoter was also active in other tissues, including developing anthers and seeds, and in floral organs after fertilization. PCD has been implicated in all of these processes, suggesting that in addition to senescence, BFN1 is involved in PCD associated with different development processes in Arabidopsis.

NAC transcription factor ORE1 and senescence-induced BIFUNCTIONAL NUCLEASE1 (BFN1) constitute a regulatory cascade in Arabidopsis

Senescence is a highly regulated process that involves the action of a large number of transcription factors. The NAC transcription factor ORE1 (ANAC092) has recently been shown to play a critical role in positively controlling senescence in Arabidopsis thaliana; however, no direct target gene through which it exerts its molecular function has been identified previously. Here, we report that BIFUNCTIONAL NUCLEASE1 (BFN1), a well-known senescence-enhanced gene, is directly regulated by ORE1. We detected elevated expression of BFN1 already 2 h after induction of ORE1 in estradiol-inducible ORE1 overexpression lines and 6 h after transfection of Arabidopsis mesophyll cell protoplasts with a 35S:ORE1 construct. ORE1 and BFN1 expression patterns largely overlap, as shown by promoter-reporter gene (GUS) fusions, while BFN1 expression in senescent leaves and the abscission zones of maturing flower organs was virtually absent in ore1 mutant background. In vitro binding site assays revealed a bipartite ORE1 binding site, similar to that of ORS1, a paralog of ORE1. A bipartite ORE1 binding site was identified in the BFN1 promoter; mutating the cis-element within the context of the full-length BFN1 promoter drastically reduced ORE1-mediated transactivation capacity in transiently transfected Arabidopsis mesophyll cell protoplasts. Furthermore, chromatin immunoprecipitation (ChIP) demonstrates in vivo binding of ORE1 to the BFN1 promoter. We also demonstrate binding of ORE1 in vivo to the promoters of two other senescence-associated genes, namely SAG29/SWEET15 and SINA1, supporting the central role of ORE1 during senescence.

The expression of a grapefruit gene encoding an isoflavone reductase-like protein is induced in response to UV irradiation

Exposure of harvested grapefruit to UV-C (254 nm) irradiation was previously found to induce resistance against the green mold decay caused by Penicillium digitatum. In order to gain insight into the mechanism of this UV-induced resistance we initiated a study for isolation of genes induced during this process. Using the differential display method we cloned cDNA representing an mRNA which is accumulated in grapefruit peel upon UV irradiation. Sequence analysis revealed that this cDNA represents a gene encoding for an isoflavone reductase-like protein and was termed IRL (isoflavone reductase-like). The grapefruit IRL protein sequence has high homology also to a novel family of other isoflavone reductase-like proteins present in few non-legume plants and whose function is not clear yet. The UV dose, and time following it, which lead to maximal accumulation of the IRL transcript were found to be similar to those leading to maximal induced resistance. The expression of the IRL gene was demonstrated to be induced also by wounding and pathogen infection.

Ripening of 'Red Rosa' plums: effect of ethylene and 1-methylcyclopropene

‘Red Rosa’, a Japanese type plum, was treated with 0.1 ppm 1-methylcyclopropene (1-MCP) for 20 h at 20˚C at harvest and then held at 20˚C for ripening together with untreated fruit, or placed at 0˚C storage for 5 weeks. Another batch of plums were exposed to 15 ppm ethylene during storage at 0˚C. Fruits ripening directly after 1-MCP treatment had lower ethylene production and softened more slowly than untreated fruits. Following storage, both the ethylene-treated and 1-MCP-treated fruits were low in ethylene production and softened more slowly than untreated fruits. They also showed lower exo-polygalacturonase (exo-PG) and endo-glucanase (EGase) activities. Respiration rates, pectin esterase (PE) and endo-PG activities were similar in 1-MCP-treated and control fruits. Cell wall enzyme activities were different amongst the treatments, but did not correlate with the differences in fruit softening. 1-Aminocyclopropane–1-carboxylic acid (ACC) oxidase (ACO) activity was highest in ethylene treated fruit during ripening after storage, while the mRNA abundance was highest in control fruit at removal from storage. ACC synthase (ACS) activity was higher in control and 1-MCP-treated fruit than in ethylene-treated during ripening after storage, and message abundance was highest at removal from storage in control fruit. ACC content was highest in fruits at removal from storage. The results are discussed in terms of the role of ethylene in ripening of ‘Red Rosa’ plums.

Programmed Cell Death Occurs Asymmetrically during Abscission in Tomato

This work examines abscission in tomato and finds that it is associated with programmed cell death and expression of the RNase LX, both of which occur on the distal side of the abscission zone, indicating asymmetric processes occurring during abscission. Abscission occurs specifically in the abscission zone (AZ) tissue as a natural stage of plant development. Previously, we observed delay of tomato (Solanum lycopersicum) leaf abscission when the LX ribonuclease (LX) was inhibited. The known association between LX expression and programmed cell death (PCD) suggested involvement of PCD in abscission. In this study, hallmarks of PCD were identified in the tomato leaf and flower AZs during the late stage of abscission. These included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species and enzymatic activities, and expression of PCD-associated genes. Overexpression of antiapoptotic proteins resulted in retarded abscission, indicating PCD requirement. PCD, LX, and nuclease gene expression were visualized primarily in the AZ distal tissue, demonstrating an asymmetry between the two AZ sides. Asymmetric expression was observed for genes associated with cell wall hydrolysis, leading to AZ, or associated with ethylene biosynthesis, which induces abscission. These results suggest that different abscission-related processes occur asymmetrically between the AZ proximal and distal sides. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during normal progression of abscission and suggest an important role for LX in this PCD process.