Elongating Leaf Bases Research Articles

Sucrose-Phosphate Synthase and Sucrose Synthase contribute to refoliation in ryegrass, a grassland fructan-accumulating species.

The perennity of grassland species such as Lolium perenne greatly depends on their ability to regrow after cutting or grazing. Refoliation largely relies on the mobilization of fructans in the remaining tissues and on the associated sucrose synthesis and transport towards the basal leaf meristems. However, nothing is known yet about the sucrose synthesis pathway. Sucrose Phosphate Synthase (SPS) and Sucrose Synthase (SuS) activities, together with their transcripts, were monitored during the first hours after defoliation along the leaf axis of mature leaf sheaths and elongating leaf bases (ELB) where the leaf meristems are located. In leaf sheaths, which undergo a sink-source transition, fructan and sucrose contents declined while SPS and SuS activities increased, along with the expression of LpSPSA, LpSPSD.2, LpSuS1, LpSuS2, and LpSuS4. In ELB, which continue to act as a strong carbon sink, SPS and SuS activities increased to varying degrees while the expression of all the LpSPS and LpSuS genes decreased after defoliation. SPS and SuS both contribute to refoliation but are regulated differently depending on the source or sink status of the tissues. Together with fructan metabolism, they represent key determinants of ryegrass perennity and, more generally, of grassland sustainability.

Defoliation, wounding, and methyl jasmonate induce expression of the sucrose lateral transporter LpSUT1 in ryegrass (Lolium perenne L.)

Ryegrass (Lolium perenne L.) regrowth after defoliation results from the mobilization of sugar reserves (mainly fructans) and, simultaneously, the efficient lateral transport of sucrose towards growing tissues. However, as for grasses overall, it is not yet known if the induction of this transport is solely linked to the sugar demand of growing tissues via the modification of sugar content at the tissue or cellular level or if it could be triggered by a wounding signal due to the defoliation itself. Ryegrass plants were therefore submitted to total or partial defoliation, pinning of the leaf blades to simulate wounding, or to leaf spraying with 100 μM methyl jasmonate (MeJA), a phytohormone related to wounding. As a response to total or partial defoliation, fructans were mobilized, and the expression of the sucrose lateral transporter LpSUT1 was induced. This highlights an efficient intra-plant compensatory partitioning of sugar resources between defoliated and intact tillers, resulting in the adaptation to regrow after moderate to severe defoliation. The MeJA treatment strongly decreased fructan content. Pinning and especially MeJA largely and quickly increased sucrose content and LpSUT1 transcript levels in leaf sheaths and elongating leaf bases, suggesting a direct effect of wounding on the up-regulation of the sucrose lateral transporter. The overall results suggest that sucrose transport capacity and fructan degradation are induced by defoliation through the modification of source-sink relationships for sugars at the plant level and are mediated by phytohormones associated with wounding, such as jasmonates. This article is protected by copyright. All rights reserved.

Short-term effects of defoliation intensity on sugar remobilization and N fluxes in ryegrass.

In grassland plant communities, the ability of individual plants to regrow after defoliation is of crucial importance since it allows the restoration of active photosynthesis and plant growth. The aim of this study was to evaluate the effects of increasing defoliation intensity (0, 25, 65, 84, and 100% of removed leaf area) on sugar remobilization and N uptake, remobilization, and allocation in roots, adult leaves, and growing leaves of ryegrass over 2 days, using a 15N tracer technique. Increasing defoliation intensity decreased plant N uptake in a correlative way and increased plant N remobilization, but independently. The relative contribution of N stored before defoliation to leaf growth increased when defoliation intensity was severe. In most conditions, root N reserves also contributed to leaf regrowth, but much less than adult leaves and irrespective of defoliation intensity. A threshold of defoliation intensity (65% leaf area removal) was identified below which C (glucose, fructose, sucrose, fructans), and N (amino acids, soluble proteins) storage compounds were not recruited for regrowth. By contrast, nitrate content increased in elongating leaf bases above this threshold. Wounding associated with defoliation is thus not the predominant signal that triggers storage remobilization and controls the priority of resource allocation to leaf meristems. A framework integrating the sequential events leading to the refoliation of grasses is proposed on the basis of current knowledge and on the findings of the present work.

Identification of a new sucrose transporter in rye-grass (LpSUT2): Effect of defoliation and putative fructose sensing

Rye-grass fast regrowth after defoliation results from an efficient mobilization of C reserves which are transported as sucrose towards regrowing leaves, and which can be supported by one or several sucrose transporters (SUTs) like LpSUT1. Therefore, our objectives were to isolate, identify, characterize and immunolocalize such sucrose transporters.A protein (LpSUT2) showing a twelve spanning trans-membrane domain, extended N terminal and internal cytoplasmic loop, and kinetic properties consistent with well-known sucrose transporters, was isolated and successfully characterized. Along with LpSUT1, it was mainly localized in mesophyll cells of leaf sheaths and elongating leaf bases. These transporters were also found in parenchyma bundle sheath (PBS) cells but they were not detected in the sieve element/companion cell complex of the phloem. Unlike LpSUT1 transcript levels which increased as a response to defoliation in source and sink tissues, LpSUT2 transcript levels were unaffected by defoliation and weakly expressed. Interestingly, sucrose transport by LpSUT2 was inhibited by fructose.LpSUT1 and LpSUT2 appeared to have different functions. LpSUT1 is proposed to play a key role in C storage and mobilization by allowing sucrose transport between PBS and mesophyll cells, depending on the plant C status. LpSUT2 could be involved in sucrose/fructose sensing at sub-cellular level.

Cloning and characterization of a novel fructan 6-exohydrolase strongly inhibited by sucrose in Lolium perenne.

The first 6-fructan exohydrolase (6-FEH) cDNA from Lolium perenne was cloned and characterized. Following defoliation, Lp6 - FEHa transcript level unexpectedly decreased together with an increase in total FEH activity. Lolium perenne is a major forage grass species that accumulates fructans, mainly composed of β(2,6)-linked fructose units. Fructans are mobilized through strongly increased activities of fructan exohydrolases (FEHs), sustaining regrowth following defoliation. To understand the complex regulation of fructan breakdown in defoliated grassland species, the objective was to clone and characterize new FEH genes in L. perenne. To find FEH genes related to refoliation, a defoliated tiller base cDNA library was screened. Characterization of the recombinant protein was performed in Pichia pastoris. In this report, the cloning and enzymatic characterization of the first 6-FEH from L. perenne is described. Following defoliation, during fructan breakdown, Lp6-FEHa transcript level unexpectedly decreased in elongating leaf bases (ELB) and in mature leaf sheaths (tiller base) in parallel to increased total FEH activities. In comparison, transcript levels of genes coding for fructosyltransferases (FTs) involved in fructan biosynthesis also decreased after defoliation but much faster than FEH transcript levels. Since Lp6-FEHa was strongly inhibited by sucrose, mechanisms modulating FEH activities are discussed. It is proposed that differences in the regulation of FEH activity among forage grasses influence their tolerance to defoliation.

Differential regulation of two sucrose transporters by defoliation and light conditions in perennial ryegrass

Sucrose transport between source and sink tissues is supposed to be a key-step for an efficient regrowth of perennial rye-grass after defoliation and might be altered by light conditions. We assessed the effect of different light regimes (high vs low light applied before or after defoliation) on growth, fructans and sucrose mobilization, as well as on sucrose transporter expression during 14 days of regrowth. Our results reported that defoliation led to a mobilization of C reserves (first sucrose and then fructans), which was parallel to an induction of LpSUT1 sucrose transporter expression in source and sink tissues (i.e. leaf sheaths and elongating leaf bases, respectively) irrespective to light conditions. Light regime (high or low light) had little effects on regrowth and on C reserves mobilization during the first 48 h of regrowth after defoliation. Thereafter, low light conditions, delaying the recovery of photosynthetic capacities, had a negative effect on C reserves re-accumulation (especially sucrose). Surprisingly, high light did not enhance sucrose transporter expression. Indeed, while light conditions had no effect on LpSUT1 expression, LpSUT2 transcripts levels were enhanced for low light grown plants. These results indicate that two sucrose transporter currently identified in Lolium perenne L. are differentially regulated by light and sucrose.

Impact of defoliation frequency on regrowth and carbohydrate metabolism in contrasting varieties of Lolium perenne.

The aims of the study were to gain a better understanding of fructan metabolism regulation during regrowth of Lolium perenne, and to evaluate the role of fructans of remaining tissues as well as carbon assimilation of new leaf tissues in refoliation. Two varieties that contrast for carbohydrate metabolism, Aurora and Perma, were subject to severe and frequent or infrequent defoliations before regrowth. Aurora, which had a greater content of fructans in leaf sheaths than Perma before defoliation, produced more leaf biomass within the 4 days following the first cut. At the end of the regrowth period, Aurora produced more leaf biomass than Perma. Photosynthetic parameters, which were barely affected by defoliation frequency, could not explain these differences. Fructan synthesising activities [sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 6G-fructosyltransferase (6G-FFT)], declined after defoliation. In elongating leaf bases, corresponding transcript levels did not decline concomitantly, suggesting a post-transcriptional regulation of expression, while in leaf sheaths the gene expression pattern mostly followed the time-course of the enzyme activities. Regulation of Lp1-SST and Lp6G-FFT gene expression depends, therefore, on the sink-source status of the tissue after defoliation. During the phase of reserve accumulation, fructosyltransferase activities together with corresponding transcripts increased more in frequently defoliated plants than in infrequently defoliated plants.

Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.)

Fructans are the main storage compound in Lolium perenne. To account for the prevailing neokestose-based fructan synthesis in this species, a cDNA library of L. perenne was screened by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) as a probe. A full length Lp6G-FFT clone was isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNA was tested by heterologous expression in Pichia pastoris. The recombinant protein demonstrated both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1-FFT) with a maximum 6G-FFT/1-FFT ratio of two. The activity of 6G-FFT was investigated with respect to developmental stage, tissue distribution, and alterations in carbohydrate status expression and compared to sucrose:sucrose 1-fructosyltransferase (1-SST). Lp6G-FFT and Lp1-SST were predominantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of both genes declined along the leaf axis, in parallel with the spatial occurrence of fructan and fructosyltransferase activities. Surprisingly, Lp6G-FFT was highly expressed in photosynthetically active tissues where very low extractable fructosyltransferase activity and fructan amounts were detected, suggesting a post-transcriptional regulation of expression. Lp6G-FFT gene expression increased only in elongating leaves following similar increases of sucrose content in blades, sheaths, and elongating leaf bases. Regulation of Lp6G-FFT gene expression depends on the tissue according to its sink-source status.

Fructans, but not the sucrosyl-galactosides, raffinose and loliose, are affected by drought stress in perennial ryegrass.

The aim of this study was to evaluate the putative role of the sucrosyl-galactosides, loliose [alpha-D-Gal (1,3) alpha-D-Glc (1,2) beta-D-Fru] and raffinose [alpha-D-Gal (1,6) alpha-D-Glc (1,2) beta-D-Fru], in drought tolerance of perennial ryegrass and to compare it with that of fructans. To that end, the loliose biosynthetic pathway was first established and shown to operate by a UDP-Gal: sucrose (Suc) 3-galactosyltransferase, tentatively termed loliose synthase. Drought stress increased neither the concentrations of loliose and raffinose nor the activities of loliose synthase and raffinose synthase (EC 2.4.1.82). Moreover, the concentrations of the raffinose precursors, myoinositol and galactinol, as well as the gene expressions of myoinositol 1-phosphate synthase (EC 5.5.1.4) and galactinol synthase (EC 2.4.1.123) were either decreased or unaffected by drought stress. Taken together, these data are not in favor of an obvious role of sucrosyl-galactosides in drought tolerance of perennial ryegrass at the vegetative stage. By contrast, drought stress caused fructans to accumulate in leaf tissues, mainly in leaf sheaths and elongating leaf bases. This increase was mainly due to the accumulation of long-chain fructans (degree of polymerization > 8) and was not accompanied by a Suc increase. Interestingly, Suc but not fructan concentrations greatly increased in drought-stressed roots. Putative roles of fructans and sucrosyl-galactosides are discussed in relation to the acquisition of stress tolerance.

Ascorbate‐glutathione cycle and H2O2 detoxification in elongating leaf bases of ryegrass: effect of inhibition of glutathione reductase activity on foliar regrowth

The role of the ascorbate‐glutathione cycle and AOS detoxification was investigated during leaf growth of defoliated and undefoliated plants of ryegrass (Lolium perenne L. cv. Bravo). Antioxidants and related enzymatic activities were located in elongating leaf bases (ELBs) of undefoliated plants, following a decreasing gradient from basal (meristem) to distal segments, inverse to H2O2 levels. In the meristematic zone, the intense activity of the ascorbate‐glutathione cycle and the supply of reducing power by the oxidative pentose phosphate pathway allowed the maintenance of both antioxidant reduction and H2O2 detoxification. BCNU (1–3 bis(2‐chloroethyl)‐N‐nitrosourea), a glutathione reductase inhibitor, induced an increase in the meristematic zone in both H2O2 and antioxidant levels and a decrease in reduced/oxidized ratios of glutathione and ascorbate. These changes were associated with a reduced foliar regrowth activity. In the absence of BCNU, defoliation did not modify the ratios of reduced/oxidized antioxidants, although it triggered a temporary increase in H2O2 level. The results are discussed on the basis of a possible control of leaf growth by glutathione and ascorbate.

Roles of the fructans from leaf sheaths and from the elongating leaf bases in the regrowth following defoliation of Lolium perenne L.

The study of carbohydrate metabolism in perennial ryegrass (Lolium perenne L. cv. Bravo) during the first 48 h of regrowth showed that fructans from elongating leaf bases were hydrolysed first whereas fructans in mature leaf sheaths were degraded only after a lag of 1.5 h. In elongating leaf bases, the decline in fructan content occurred not only in the differentiation zone (30-60 mm from the leaf base), but also in the growth zone. Unlike other soluble carbohydrates, the net deposition rate of fructose remained positive and even rose during the first day following defoliation. The activity of fructan exohydrolase (FEH; EC 3.2.1.80) was maximal in the differentiation zone before defoliation and increased in all segments, but peaked in the growth zone after defoliation. These data strongly indicate that fructans stored in the leaf growth zone were hydrolysed and recycled in that zone to sustain the refoliation immediately after defoliation. Despite the depletion of carbohydrates, leaves of defoliated plants elongated at a significantly higher rate than those of undefoliated plants, during the first 10 h of regrowth. This can be partly attributed to the transient increase in water and nitrate deposition rate. The results are discussed in relation to defoliation tolerance.

Endogenous gibberellins in Lolium perenne and influence of defoliation on their contents in elongating leaf bases and in leaf sheaths

Gibberellin (GA) levels in leaf sheaths and in elongating leaf bases of perennial ryegrass (Lolium perenne L. cv. Bravo) were monitored in undefoliated and defoliated plants. Nine C‐13‐hydroxylated GAs (GA8, GA97, GA29, GA1, GA20, GA44, GA19, GA17, GA53) and one C‐13‐non‐hydroxylated GA (GA9) were identified by combined gas chromatography‐mass spectrometry in leaf extracts. The total level of GA8, GA29, GA1, GA20, GA44, GA19 and GA53, determined by selected ion monitoring, was 7 times higher in elongating leaf bases than in mature leaf sheaths. In both leaf tissues, defoliation induced an increase in GA53 level, while GA20 and GA1 levels decreased, suggesting that the GA53→GA44, as well as GA19→GA20, conversions were slowed down. The roles of GA1 in the control of leaf elongation and fructan mobilization following defoliation are discussed.

Influence of initial levels of carbohydrates, fructans, nitrogen, and soluble proteins on regrowth of Lolium perenne L. cv. Bravo following defoliation

An experiment was designed to evaluate the role of N and C reserves on regrowth of Lolium perenne cv. Bravo following defoliation. By using two nitrogen fertilization levels together with three photoperiodic conditions, plants with variable contents of water-soluble carbohydrates (43–216 mg g−1 DW in stubble) and contrasting amounts of nitrogen (7–49 mg g−1 DW) were obtained. Plants were severely defoliated and regrowth was followed for 28 d under the same environmental conditions. The yield of leaf dry matter at the end of the regrowth period was not related to the initial level of carbohydrate reserves. However, levels of fructan in leaf sheaths and in elongating leaf bases strongly influenced the shoot yield during the first 2 d following defoliation. Fructan exohydrolase activity increased 2–3-fold in sheaths and 3.5–5-fold in elongating leaf bases, suggesting that not only fructans from sheaths but also fructans from immature cells may be used as substrates for growth. In contrast, no direct relationship was found between shoot production and nitrogen or soluble protein accumulation in source organs during early regrowth. A significant correlation existed with the initial amount of soluble proteins in sheaths and in elongating leaf bases after only 6 d of regrowth.

Partitioning of reserve and newly assimilated carbon in roots and leaf tissues of Lolium perenne during regrowth after defoliation: assessment by 13C steady‐state labelling and carbohydrate analysis

ABSTRACTThe relative significance of the use of stored or currently absorbed C for the growth of leaves or roots of Lolium perenne L. after defoliation was assessed by steady‐state labelling of atmospheric CO2. Leaf growth for the first two days after defoliation was to a large extent dependent on the use of C reserves. The basal part of the elongating leaves was mainly new tissue and 91% of the C in this part of the leaf was derived from reserves assimilated prior to defoliation. However, half of the sucrose in the growth zone was produced from photosynthesis by the emerged leaves. Fructans that were initially present in elongating leaf bases were hydrolysed (loss of 93 to 100%) and the resulting fructose was found in the new leaf bases, suggesting that this pool may be used to support cell division and elongation. Despite a negative C balance at the whole‐plant level, fructans were synthesized from sucrose that was translocated to the new leaf bases. After a regrowth period of 28 d, 45% of the C fixed before defoliation was still present in the root and leaf tissue and only 1% was incorporated in entirely new tissue.

Rise of fructan exohydrolase activity in stubble of Lolium perenne after defoliation is decreased by uniconazole, an inhibitor of the biosynthesis of gibberellins

SUMMARYThe objectives of this study were (i) to evaluate the relative involvement of fructans from leaf sheaths and from elongating leaf bases to regrowth after defoliation of Lolium perenne L. by following fructan exohydrolase (FEH), sucrose‐sucrose fructosyl transferase (SST) and invertase (INV) activities and (ii) to examine whether gibberellins could be implicated in regulation of FEH activity. In stubble, 36 % of fructans are located in leaf bases and 64 % in leaf sheaths. During the first phase of regrowth, the depletion of carbohydrates was mainly due to the decline of fructans, which represented 76% and 50 % of the carbohydrates mobilized from leaf sheaths and elongating leaf bases respectively. Despite a decrease in protein content, FEH activity increased 2‐5‐fold in leaf sheaths and sixfold in elongating leaf bases, so that the fructan‐hydrolysing activity was four times higher in growing leaves than in leaf sheaths, 2 d after defoliation. INV activity also increased, whereas the pattern of SST activity was inversely related to the variations of both hydrolysing activities. SST activity, which is highest in growing leaves, declined approximately threefold in leaf sheaths and elongating leaf bases at the beginning of regrowth.The increase in activity of FEH in stubble was strongly inhibited by an inhibitor of the biosynthesis of gibberellin, uniconazole. FEH activity was decreased to c. 67 %, 45 % and 33 % of the level in nontreated plants 24, 48 and 72 h following defoliation, respectively. The inhibition could be overcome by a subsequent treatment with gibberellic acids (GAs). For the first time, data are provided to support the view that GAs might play a role in the regulation of FEH activity, and the implication of this result is discussed.

Rise of fructan exohydrolase activity in stubble of Lolium perenne after defoliation is decreased by uniconazole, an inhibitor of the biosynthesis of gibberellins

The objectives of this study were (i) to evaluate the relative involvement of fructans from leaf sheaths and from elongating leaf bases to regrowth after defoliation of Lolium perenne L. by following fructan exohydrolase (FEH), sucrose‐sucrose fructosyl transferase (SST) and invertase (INV) activities and (ii) to examine whether gibberellins could be implicated in regulation of FEH activity. In stubble, 36% of fructans are located in leaf bases and 64% in leaf sheaths. During the first phase of regrowth, the depletion of carbohydrates was mainly due to the decline of fructans, which represented 76% and 50% of the carbohydrates mobilized from leaf sheaths and elongating leaf bases respectively. Despite a decrease in protein content, FEH activity increased 2·5‐fold in leaf sheaths and six‐fold in elongating leaf bases, so that the fructan‐hydrolysing activity was four times higher in growing leaves than in leaf sheaths, 2 d after defoliation. INV activity also increased, whereas the pattern of SST activity was inversely related to the variations of both hydrolysing activities. SST activity, which is highest in growing leaves, declined approximately threefold in leaf sheaths and elongating leaf bases at the beginning of regrowth.The increase in activity of FEH in stubble was strongly inhibited by an inhibitor of the biosynthesis of gibberellin, uniconazole. FEH activity was decreased to c. 67%, 45% and 33% of the level in nontreated plants 24, 48 and 72 h following defoliation, respectively. The inhibition could be overcome by a subsequent treatment with gibberellic acids (GAs). For the first time, data are provided to support the view that GAs might play a role in the regulation of FEH activity, and the implication of this result is discussed.