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First published online August 18, 2006; 10.1104/pp.106.080135 Plant Physiology 142:710-721 (2006) © 2006 American Society of Plant Biologists Suppression of LX Ribonuclease in Tomato Results in a Delay of Leaf Senescence and Abscission1,[W]Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel (A.L., L.S., S.B.); and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711 (P.J.G.)
Although present in different organisms and conserved in their protein sequence, the biological functions of T2 ribonucleases (RNase) are generally unknown. Tomato (Lycopersicon esculentum) LX is a T2/S-like RNase and its expression is known to be associated with phosphate starvation, ethylene responses, and senescence and programmed cell death. In this study, LX function was investigated using antisense tomato plants in which the LX protein level was reduced. LX protein levels normally become elevated when leaves senesce and antisense inhibition of LX retarded the progression of senescence. Moreover, we observed a marked delay of leaf abscission in LX-deficient plants. This correlated with specific induction of LX protein in the tomato mature abscission zone tissue. LX RNase gene regulation and the consequences of antisense inhibition indicate that LX has an important functional role in both abscission and senescence.
The T2 RNases (EC 3.1.27.1) are secreted endoribonucleases that are found outside cells or in compartments of the endomembrane system with no absolute substrate base specificity (Irie and Ohgi, 2001  ; Deshpande and Shankar, 2002). First isolated from fungi, proteins in this family have subsequently been identified in a wide variety of organisms, ranging from viruses and bacteria to mammals; thus, it is the most broadly distributed family of RNA-degrading enzymes known (Irie, 1997, 1999; Deshpande and Shankar, 2002). Although biological functions of various T2 RNases have been postulated on the basis of enzyme location or regulation of gene expression, the physiological roles of these enzymes are generally unknown. RNases have been hypothesized to have roles in RNA turnover and metabolism and also to be involved in nutrition and viral pathogenicity. A novel function in regulation of cell membrane permeability was suggested for the yeast (Saccharomyces cerevisiae) Rny1 T2 RNase (MacIntosh et al., 2001).
The only plant enzymes in the T2 family for which the in vivo role is known are the S-RNases, which are involved in gametophytic self incompatibility in plants (Lee et al., 1994
S-like RNase genes are often expressed in diverse organs and in response to different environmental conditions (Green, 1994
The presence of vacuole-localized RNases and RNA (Boller and Kende, 1979
The expression of genes encoding for nucleic acid-degrading enzymes was described for several types of programmed cell death (PCD) processes in plants (Mittler and Lam, 1997
The tomato LX S-like RNase was originally identified as a Pi-induced RNase (Loffler et al., 1992
LX RNase Protein Is Induced during Senescence and Its Level Is Reduced in the LX Antisense Lines
To detect and measure LX protein levels during development and following modulation of LX gene expression, we generated polyclonal antibodies specific for the LX protein that was overexpressed in bacterial cells. The specificity of the antibodies was demonstrated by lack of cross-reaction with any induced protein following western-blot analysis performed with tomato leaf proteins extracted from wounded leaves (data not shown). The LE RNase, which shares the highest degree of amino acid sequence similarity with LX among known tomato proteins, is induced by wounding (Lers et al., 1998
The LX antibodies were further used to determine whether induction of the LX gene by ethylene in nonsenescing leaves occurred at the protein level, as predicted from our previous analysis showing induction at the mRNA level (Lers et al., 1998 ). Intact soil-grown young tomato plants, with six developed leaves, were exposed to 10 µL L–1 ethylene on air in a continuous gas flow-through system. Proteins extracted from four leaves at different positions on the stem, from position 1 (the first leaf at the lower position that was fully expanded and green with no signs of yellowing) up to position 4, were used in western-blot analysis. Proteins extracted from stem, cotyledon, and root tissues were also used. The analysis revealed induction of LX protein within 24 h into the ethylene treatment (Fig. 1B). The LX level increased in a leaf age-dependent manner, but, after an additional 24 h under air, high levels of LX protein were observed in leaves in all positions. Antisense plants were generated to investigate the consequences of inhibiting the expression of the LX gene. Tomato VF36 plants were transformed with an LX antisense construct. The transforming vector, pLXAS9-11, was based on the pBI121 plasmid in which the full cDNA of LX was cloned in inverse orientation under the direction of a doubly enhanced cauliflower mosaic virus (CaMV) 35S promoter and the E9 terminator. Plants homozygous for the antisense transgene were identified by PCR analysis and further screened for the effects on LX gene expression. The LX protein level was measured with western-blot analysis and the LX antibodies we generated. Initially, the level of LX was compared between senescing, same-age leaves of wild-type and antisense plants. In some of the antisense lines, the LX protein level was found to be reduced (data not shown). To more accurately quantify and compare the LX protein level between wild-type and LX antisense plants, the LX level was measured following its induction by ethylene in young green leaves. Young tomato plants, about 6 weeks old and harboring seven or eight leaves, were treated by streaming ethylene in air at 10 µL L–1 for 18 h. The plants were grown on perlite with a complete artificial nutrient supply. Total proteins were extracted from the second and third leaves and used for measurement of LX protein by western-blot analysis. In three independent LX antisense lines—A2, H9, and T2—the LX level was reduced markedly compared with that observed in wild-type plants (Fig. 1C). LX gene expression was also measured by reverse transcription-PCR, which revealed a reduced LX transcript level in the antisense lines compared with wild-type plants (data not shown). These three LX antisense lines, A2, H9, and T2, were used for the remainder of this study.
When wild-type tomato and the three antisense lines were grown under optimal growth conditions, either on soil or on perlite fully supplemented with nutrients, no obvious phenotypic differences were detected. Next, we compared the growth of the wild-type and the LX-deficient lines under conditions of Pi deficiency. Tomato seeds were germinated on perlite and irrigated with full nutrient solution. Once cotyledons had emerged, irrigation was continued with Pi-deficient nutrient solution. Leaves and cotyledons of the antisense lines appeared more purple than those of the wild type, especially in the veins, which suggests that anthocyanin was elevated in the antisense plants. To quantify anthocyanin levels, leaf disc samples were harvested from the first true leaf of each plant and anthocyanin and chlorophyll levels were measured. The results shown in Figure 2 demonstrate significantly higher anthocyanin levels in the three LX-deficient lines than in the wild type, whereas the chlorophyll content showed no significant difference. When Pi starvation was imposed at a later stage of plant development, anthocyanin induction was observed mainly in the new leaves, which had developed after the application of Pi starvation (data not shown).
Senescence Is Delayed in LX-Deficient Plants To determine whether inhibition of LX gene expression affected senescence, the progress of senescence was compared between the wild type and three antisense lines. Tomato plants were grown in the greenhouse in propagation trays with fertilizer included in the soil and irrigated with tap water. Following about 4 weeks of growth under these conditions, the plants developed four true leaves, but cotyledons in some of them had entered senescence, as indicated by the initiation of yellowing. At this stage, the cotyledons from all the plants were collected for analysis. Following 3 weeks of further growth, when the first true leaves in some of the plants had begun to senesce and significant yellowing was apparent, the first true leaves of all the plants were collected for analysis. Leaf or cotyledon tissue was homogenized and used for quantifying the levels of chlorophyll and total soluble protein as senescence markers and for measuring the LX protein level by western-blot analysis. Samples for all three measurements were taken from the same tissue homogenate. These analyses revealed higher chlorophyll and protein content relative to fresh weight in both the cotyledons (Fig. 3A ) and first leaves (Fig. 3B) in the three antisense lines than in the wild type, indicating a delay in senescence in the antisense lines. The delay in senescence in the three antisense lines was also manifested in a slower decline, compared to wild type, in the levels of the large and small subunits of the Rubisco enzyme (data not shown). Western-blot analysis of the protein extracts confirmed the higher level of the LX RNase protein in the wild-type plants than in the three LX-deficient antisense lines. To examine the effect of LX inhibition on senescence that was induced under different growth conditions, we have compared the initiation of leaf senescence between wild-type and LX-deficient plants under Pi starvation. Tomato plants were grown in perlite and watered with full nutrient solution for 5 weeks and then with Pi-deficient solution. Following a further 3 weeks, there was a visible difference between wild-type and LX antisense transgenic plants in the progress of senescence. Fixed-area leaf discs were sampled from the first true leaves of the plants and chlorophyll and protein were extracted and quantified. Both chlorophyll and protein content were found to be much lower in the leaves of the wild type than in the three lines of LX-deficient antisense plants (Fig. 3C).
Leaf Abscission Is Delayed in LX-Deficient Plants We observed that when LX-deficient and wild-type plants were germinated and grown in perlite under Pi-limiting conditions or were irrigated with water with no nutrient supply, the cotyledons and leaves of the young plants abscised much earlier in the wild-type plants than in the antisense ones (Fig. 4A ). Abscission occurred before the appearance of any signs of senescence in the abscised cotyledons or leaves so that early abscission seemed to be unrelated to the effect of LX inhibition on senescence. Furthermore, in many cases, signs of epinasty were observed in the nutrient-limited plants, which suggest that high levels of ethylene were synthesized in response to stress conditions. Ethylene is known to have a key role in the induction of abscission processes in plants and we hypothesized that the observed delay in cotyledon/leaf abscission in the LX-deficient antisense plants may have resulted from lack of a sufficiently high LX level. To establish and quantify the difference between LX-deficient antisense and wild-type plants in the occurrence of abscission, we have measured the difference in the time needed for cotyledon and leaf detachment following induction of the process. The wild-type and three LX-deficient antisense lines were grown on soil under optimal growth conditions for about 6 weeks, until five or six leaves had developed. To induce abscission, we initially removed the leaf blades (deblading) from the three lower leaves in each plant, but not from the cotyledons. Deblading is known to accelerate petiole abscission because it reduces the auxin supply. After 36 h, plants were treated with ethylene at 2 µL L–1 in flowing air for another 36 h and then returned to the greenhouse. Immediately following ethylene treatment (designated as time zero of the experiment) and on each subsequent day, the numbers of abscising cotyledons and petioles were counted and the results were presented as percentages of the initial total numbers in each line (Fig. 4, B and C).
The cotyledons were more sensitive than the leaves to the abscission-inducing ethylene treatment. Differences in the percentage of abscising cotyledons were observed immediately following ethylene treatment: Whereas more than 50% of the cotyledons abscised from the wild-type plant, less than 10% of the cotyledons abscised in each of the three antisense LX-deficient lines (Fig. 4B). After 1 d, the differences were less pronounced, but cotyledon abscission was still lower in transgenics than in wild-type plants. After 3 d, cotyledon abscission reached 90% to 100% in all lines (Fig. 4B). Abscission of the petioles occurred at a much reduced rate compared to that observed for the cotyledons: Immediately following ethylene treatment, about 10% petiole abscission was observed in wild-type plants, but none in any of the LX-deficient lines (Fig. 4C) and, after 3 d, petiole abscission was more than 80% in the wild-type plants compared with 10% to 40% in the three antisense lines (Fig. 4C). Even after 10 d, a difference in the progress of abscission was maintained between the wild-type and the antisense lines and only after 19 d following the treatment were there comparable levels of petiole abscission in the various tomato lines (Fig. 4C). The duration of the abscission delay varied among the three LX-deficient lines, with the A2 and T2 lines showing a more inhibited phenotype than the H9 line for both cotyledon and leaf abscission (Fig. 4, B and C). In additional experiments aimed at comparing abscission between wild-type and LX-deficient plants, more mature tomato plants were used and abscission was induced only by deblading. The plants were grown on soil in the greenhouse for about 3 months, by which time they had initiated flowering and had developed about 10 mature leaves. Deblading was applied to the leaves in positions 5 to 8, counting from the base of the plant upward, leaving 5-cm-long petioles. The total number of petioles that abscised from each position was counted separately for each line and the abscission levels are presented as percentages that abscised out of the total number of debladed petioles (Fig. 5 ). The three LX-deficient lines exhibited pronounced delay in the abscission of petioles from all positions compared with that of wild-type plants (Fig. 5). The difference in the progress of the petiole abscission process was more evident in the higher positions, where the induction of the process was slower than in the more mature, lower positions. For example, among the petioles in the eighth position, by 20 d after leaf deblading, wild-type abscission had reached 100%, compared with about 10% for the H9 line, and no abscission in the A2 and H2 LX-deficient lines (Fig. 5). Interestingly, in this experiment, as well as in the previous one (Fig. 4, B and C), the H9 line seemed to be less inhibited in abscission progress than the two other lines, A2 and T2 (Fig. 5).
LX Protein Level Is Specifically Induced in the Abscission Zone The observed influence of LX inhibition on the progress of abscission raised the possibility that LX RNase is involved in this process. Experiments were performed to examine the possibility that LX is expressed in the abscission zone (AZ) in association with the progress of abscission. We used LX-specific antibodies to measure the level of LX protein in mature and young AZs and in adjacent tissue that served as a control. Tissue slices of 2- to 3-mm thickness that included the AZ cells were sampled from leaf petioles that were approaching abscission and from young leaf petioles. As a control tissue, an adjacent slice of the petiole a few millimeters away from the AZ was sampled as well. Proteins were extracted from pooled tissue slices from different plants and, following electrophoresis, LX levels were measured by western-blot analysis. The results shown in Figure 6A indicated high LX protein levels in the tissue that included the mature AZ, whereas much less of the protein was observed in the nearby petiole control tissue (Fig. 6A). In the young AZ, as well as in the nearby tissue, LX protein levels were very low (Fig. 6A). Additional experiments in which LX protein level was measured in the AZ at different stages during its development revealed that LX protein accumulation is not a late event that occurs just a few days before actual abscission, but that it is initiated as much as 2 weeks before abscission takes place and is induced to a higher level as abscission approaches (data not shown). We compared the levels of LX protein accumulation in the AZs of wild-type and of LX-deficient plants: Slices of tissue that included the AZs and nearby petiole tissue were sampled from petioles of leaves at the same stage of advanced senescence, and western-blot analysis of the extracted proteins revealed higher levels of LX protein in the AZs of wild-type plants than in those of the antisense plants (Fig. 6B). Similar analysis of LX protein levels in the AZs of ripe tomato fruit demonstrated induction of LX in the fruit pedicle AZ similar to that in the leaf AZ, as well as reduced levels of LX in antisense plants (Fig. 6C). The reduction in the LX protein level was observed mainly in the A2 and T2 lines; it was less in the H9 line in which, as mentioned above, there was less retardation of abscission than in the other two lines.
To localize better the site of LX protein expression, we performed immunoblot analyses by tissue printing tomato petiole AZs. The joint containing the AZ, including the main stem and part of the relevant petiole, was vertically cross sectioned and used for blotting on a membrane. LX antibodies showed the presence of the protein in the AZ and in the surrounding tissue only in mature AZs, but not in young ones, which serves to confirm that the LX gene was expressed specifically around the AZ layer (Supplemental Fig. S2). To examine whether AZ-related expression of an RNase occurs in a different plant, tree tobacco (Nicotiana glauca) plants were examined for the presence of a related RNase associated with both leaf senescence and abscission. When tomato LX antibodies were used in western-blot analysis of proteins extracted from senescing or young tobacco leaves, a protein with a similar molecular weight to that of LX was detected specifically in senescing leaves (Fig. 7A ). This tobacco LX-related protein was found to be induced in the tobacco leaf AZ compared to its level in the nearby petiole tissue (Fig. 7B).
Although widespread among a variety of different organisms, the in vivo functions of the T2 RNases are largely unknown. In only one case has a function been attributed to T2 RNases in plants, that of the S-RNases involved in self incompatibility. Our results demonstrate the involvement of a T2/S-like RNase, the LX, in plant abscission, as well as provide evidence for a function in senescence. Previously, a role for RNases in senescence was only implied on the basis of correlations between activation of gene expression or enzyme activity and senescence. Retardation of leaf senescence was observed in LX-deficient antisense plants, suggesting a link between RNA degradation and senescence progress. The involvement of LX RNase in abscission is supported by both its specific expression in the AZ and the marked delay of leaf abscission observed in the LX-deficient antisense plants. The finding of LX involvement in abscission suggests the association of a new group of hydrolytic enzymes, namely, nucleic acid-degrading enzymes, in abscission.
The tomato LX RNase was identified originally as a Pi starvation-induced RNase (Loffler et al., 1992 Overall, the observed expression pattern of LX suggests a complex regulation of this gene in response to tissue identity, developmental stage, hormone action, and environmental conditions.
The expression pattern of LX suggests that it has a function in the response to Pi-limiting conditions, PCD-related processes, and senescence. The antisense tomato lines developed in this study to examine the consequences of inhibiting LX expression had strongly reduced levels of LX protein, but residual LX protein could be detected. Tomato plants with reduced LX protein levels developed without major phenotypic alteration under optimal growth conditions and only when they were exposed to Pi-limiting stress conditions did the limiting level of LX result in elevated anthocyanin levels compared to that measured in the wild type, indicating stress while chlorophyll levels remained similar (Fig. 2). Elevated anthocyanin levels were also reported for RNS1- or RNS2-deficient Arabidopsis plants made using antisense constructs (Bariola et al., 1999
The negative influence of reduced LX RNase levels on the progression of leaf senescence that we observed seems unlikely to be a consequence of a possible function of LX in Pi reutilization. If, in the antisense plants, the reduced level of LX results in elevated stress due to Pi limitation, we would expect senescence to be accelerated because nutrient limitation is known to induce senescence. However, we observed delayed senescence in LX-deficient plants compared with the wild type whether the process was induced either by general nutrient limitation caused by reduced soil volume or by reduced Pi supply. Retardation of leaf senescence was manifested in higher levels of both chlorophyll and protein content. This senescence delay effect of LX deficiency was fairly unexpected. Because the LX gene was found to be induced at a relatively late stage of senescence, we did not anticipate that reducing its level would result in retardation of leaf senescence at such an early stage. One possible explanation is that there is cross talk (i.e. coordination) between the efficient recycling of macromolecules and the progress of senescence. Another possibility is that activity of the LX RNase is required for reducing the influence of negative regulators of senescence, which are turned off as part of the senescence process. We are not aware of any reports of a functional relationship between senescence and nucleic acid-degrading enzymes, but few reports support a relationship between senescence regulation and expression of genes involved in processes related to protein (Woo et al., 2001
A clear delay in leaf abscission was observed in the LX-deficient antisense plants relative to wild-type plants. The involvement of LX RNase in the abscission process is an unexpected finding. To our knowledge, no relationship between RNases and abscission was reported before. Abscission is a natural part of plant development in which leaves, flowers, or fruits separate from the plant (Bleecker and Patterson, 1997 In this study, the effects of reduced LX levels on abscission were demonstrated during various stages of plant development and they do not seem to be related to a function of LX in senescence. Retarded abscission of cotyledons was observed in very young antisense plants when abscission was accelerated by nutrient limitation. It is likely that enhanced production of stress ethylene, which led to detectable leaf epinasty, resulted in induction of abscission in the nonsenescing cotyledons. Retardation of cotyledon and petiole abscission was observed in 6-week-old antisense plants following deblading and ethylene application, a treatment that did not result in senescence in the abscising cotyledons or petioles. As the kinetics of abscission became more moderate, the differences in abscission rates between LX-deficient and wild-type plants increased. Comparison between the effects of LX inhibition on abscission of the eighth petiole and on that of the more mature fifth petiole (Fig. 5) revealed a greater difference between antisense and wild-type plants in the younger eighth petiole. These differences might result from elevated expression of the endogenous LX gene in mature AZs possibly due to increased ethylene sensitivity. This elevated expression could then diminish differences in the level of the LX protein between antisense and wild-type plants. The antisense H9 line exhibited less inhibition of abscission in most of the experiments in accordance with less suppression of the LX gene observed in this line.
Specific induction of LX in the AZs of both leaves and fruits further supports its role in the abscission process. The tissue print analysis indicated expression in an area that seems to include more than the few cell layers known to be included in the AZ. However, such spreading of the signal could result from the pressure imposed on the tissue during the printing process. More accurate experiments, such as immunocytochemistry, will be carried out to determine the specific cell localization of the LX protein in the AZs. Induction of LX-related, senescence-associated RNase in the tobacco leaf AZ suggests that this enzyme is involved in abscission in tobacco as well.
Both our functional and expression studies indicate that LX RNase has a role in the abscission process; however, the specific function of LX is not clear yet. On the basis of gene expression analysis, LX was hypothesized to function in PCD-related processes (Lehmann et al., 2001
The possible involvement of PCD in the abscission process has been discussed in several reviews (Sexton and Roberts, 1982 The demonstrated involvement of an RNase in abscission opens up a new avenue in the study of the abscission process, which so far was mostly associated with cell wall-degrading enzymes. We believe this finding may lead to new insight on both the general function of RNases in plants and the mechanism of the abscission process and further studies of LX and other nucleases is required.
Plant Growth Conditions and Treatments
Tomato (Lycopersicon esculentum) line VF36 was used throughout the study. Tomato seeds were germinated on perlite support at 28°C in the dark and, after 3 d, were transferred to light. About a week later, when cotyledons had fully developed, the seedlings were transferred to 12-cm containers filled with either perlite or HR1 artificial soil (Hagarin Ltd). The plants were grown in a greenhouse under a controlled temperature of 25°C and natural daylight. The plants grown on perlite were watered with nutrient solution, with or without Pi, as described by Bosse and Köck (1998) Abscission was induced by removal of the leaf blade with a sharp razor, leaving most of the petiole intact. For ethylene application, intact tomato plants in their containers were placed for the required time in sealed transparent Perspex jars fitted with inlet and outlet ports and connected to a flow-through system. Ethylene mixed with air was bubbled through sterile water to maintain humidity in the jars, and the flow rate was maintained at 100 mL min–1.
Chlorophyll and protein were quantified in the same tissue extract. One to three discs, 7 mm in diameter, were sampled from the leaves, placed in a microtube in 150 µL of extraction buffer (50 mM Tris-HCl, pH 7.5, 0.1% [w/v] SDS, and 10% [w/v] polyvinylpyrrolidone), and ground with a fitting pestle and a motorized drill. Chlorophyll was extracted from a sample of the solution with 80% acetone and its content was measured spectroscopically according to Porra et al. (1989)
To generate the LX antisense plant transformation vector p9-11, the entire LX cDNA (accession no. X79338) was first fused in the antisense orientation between the doubly enhanced CaMV 35S promoter and the E9 terminator in plasmid p1185 (Diehn et al., 1998
LX sequences corresponding to amino acids 25 to 211 of the protein (accession no. X79338) were fused to a His tag, overexpressed in bacteria, and used for rabbit immunization. The appropriate 562-bp-long DNA was synthesized by PCR and cloned in the EcoRI site of the pET-22b(+) vector (Novagen) by means of specific primers extended by the EcoRI recognition site: LXRIA primer (5'-CGAATTCACAAGACTTTGATTTCTTCTACTTTGTTC-3') and LXRIB primer (5'-GACGTAGTTTTAAGTAACTAACGGGCTTAAGC-3'). The LX expression vector was transformed into Escherichia coli ER2566 strain and protein synthesis was induced by 1 mM isoprylthio-
Protein for immunoblot analysis was extracted from one to three leaf discs (7-mm diameter) or from stem segments. The tissue was homogenized in the presence of 2-fold (v/w) extraction buffer (50 mM Tris-HCl, pH 7.5, 0.1% [w/v] SDS, 10% [w/v] polyvinylpyrrolidone, and 1 mM phenylmethylsulfonyl fluoride) in a microtube by means of a fitting pestle and a motorized drill. Following 15-min centrifugation in a microfuge at 4°C, the soluble protein extract was assayed for protein content with the Bradford assay (Bio-Rad) and stored at –80°C. For analysis of proteins in experiments that involved senescing tissue, gel loading was based on fixed volumes of samples that contained proteins extracted from equal fresh weights of tissues because protein content markedly decreases during senescence. Flowers were harvested at three different developmental stages: Young flowers were those from when the bud had begun to open, with petals already yellow, until just before the flowers were fully opened (between stages 2 and 3; as described by Barry et al., 1996
Protein extracts were mixed with sample buffer and boiled for 3 min before being separated on 12.5% or 15% SDS-PAGE gels (Laemmli, 1970 For tissue printing, the joints, including part of the primary stems and the relevant petioles, were halved longitudinally and the exposed tissue was pressed gently on a dry nitrocellulose membrane. The membrane was then used for LX protein detection by immunoblot analysis as described above.
The following materials are available in the online version of this article.
We are grateful to Dr. Jay De Rocher and Beth Kasiborski for the construction of plasmid p9-11. Received March 8, 2006; accepted August 10, 2006; published August 18, 2006.
1 This work was supported by the U.S.-Israel Binational Agricultural Research and Development Fund (research grant no. IS–2982–98C).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Amnon Lers (alers{at}volcani.agri.gov.il).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.080135 * Corresponding author; e-mail alers{at}volcani.agri.gov.il; fax 972–3–9683608.
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-1,4-glucanases (cellulases), have been hypothesized to have a central role in the process (Greenberg et al., 1975
