Cram-Trees.doc

22salt allocation during leaf development and leaf fall in mangroves w. john cram * () peter g. torr *derek a. rose +* department of biological and nutritional sciences,university of newcastle, newcastle upon tyne, ne1 7ru, uk.+ department of agricultural and environmental sciences,university of newcastle, newcastle upon tyne, ne1 7ru, ukw.j.cramncl.ac.ukfax: +44 191 222 8684phone: +44 191 222 7886presented at the international symposium on mangroves: evolution, physiology and conservation. the university of tokyo, tokyo, japan 10-12 july 2001.abstractby taking samples along individual branches and measuring leaf size, thickness and na+ and k+ concentrations, we have shown in bruguiera cylindrica, avicennia rumphiana and avicennia marina that there are two phases of salt accumulation by leaves. this is confirmed by re-analysis of published data for other species. the first phase is a rapid increase in leaf content as it grows from bud to maturity, the second is a slower but continuous change in quantity in the leaf, via changes in ion concentration and/or in leaf thickening. leaf thickening most not be overlooked in estimating changes in leaf contents with age. generally, leaf na+ content increases significantly, and k+ content falls slightly. mangrove leaves thus continue as sinks for na+ throughout their lifetime.at the end of a leafs life, just before abscission, no burst of salt accumulation has been found. yellow, senescent leaves do not have higher na+ than old green ones. we point out that leaf drop involves losing both salt and biomass, not just salt, and hence does not reduce the salt concentration in the plant. we conclude that leaf drop is not a salt excretion mechanism, but is simply the point in time at which the leaf ceases to accumulate salt.using a simple model, the contribution to salt accumulation of slowly accumulated na+ by mature leaves has been calculated. for bruguiera cylindrica, the most extensively studied species, 60% of the salt in the leaf is accumulated slowly in the mature phase, thus more than during the initial phase of rapid expansion growth.the limited data suggest that gland-bearing species show smaller changes in mature leaf na+ content than do gland-less species.key words. mangrovemature leafsalinity tolerancesalt disposalleaf dropintroductionwhen mangroves grow in sea water, as they normally do, salt inevitably enters the roots in large quantities. this is not because the cell membranes are permeable to salt they are not but because the channels which facilitate the flow of essential k+ into plant incidentally let in large quantities of na+. the inwards k+ channels are about 100-fold selective for k+ over na+, but in sea water the na+ concentration is about 45x that of k+, and so, as elegantly shown by amtmann & sanders (1999), na+ will enter root cells in significant quantities via k+channels.mangroves and other halophytes use na+ as an energetically cheap osmoticum (raven, 1997), and control its accumulation in the vacuole to maintain turgor pressure. at the same time, plant cells scavenge na+ from the cytoplasm to avoid its toxic effects on enzymes, keeping na+ below 50 mm by pumping the ions into the vacuole to 500 mm or more, and maintaining the concentration difference between the two compartments (eg raven, 1997; hasegawa et al 2000).in this paper we are considering not the mechanism of na+ entry to the root, but rather the way in which the plant allocates the na+ that does enter. three processes are agreed as being involved (eg tomlinson, 1994; hogarth, 1999): 1. re-secretion from root cells, 2. accumulation in expanding vacuoles of growing tissues, and 3. excretion by leaf salt glands in species which have them. salt arriving in the shoot is accumulated in cells in a controlled manner and if glands are present excess above that utilised is excreted. this paper concerns accumulation in leaf cells, but will first consider another process, viz.4. leaf drop.the fourth process, the shedding of old leaves, is widely quoted as a salt tolerance mechanism (eg munns, 1993; hasegawa et al, 2000). it is described as a mechanism for “the elimination of salt” (tomlinson, 1994) or to “remove salt from metabolic tissues” (hogarth, 1999). zheng et al. (1999) write explicitly eliminating excessive salt by loss of old leaves that are rich in salt is one of the characteristics by which salt-nonsecretors adapt themselves to saline environments. leaf drop as a means of secreting or eliminating salt?in discussing this question, words must be used with precision. no-one would argue with the statement that after a leaf falls, the parent mangrove holds less salt than before. however, the burning question for the mangrove is, does leaf fall separate salt from the plant, as do root secretion and gland excretion? does salt move from inside to outside the plant? the answer, we suggest, is “no”. this can be justified in several ways. first, cutting leaves off a plant is not a horticultural means of conferring salinity tolerance, and abscission is the natural equivalent. by analogy, surgical removal of a limb is never prescribed as a treatment for high blood cholesterol in humans second, the essential difference between salt excretion by leaf glands and salt loss by abscission is that glands separate salt from the plant, they excrete salt only, whereas abscission removes salt and its associated biomass from the tree. third, it is critical to distinguish between concentration and quantity. root secretion, leaf expansion and gland excretion all reduce the concentration of salt in the tree; but losing a leaf does not.we therefore suggest that leaf shedding by itself is not an adaptation that removes salt from other mangrove tissues; it is not a process to which salt is allocated. it is simply the loss of part of the plant at the end of its life-span. accumulation of ions in leaves during development.expansion growth, maturity, senescence and abscission are internally programmed stages of leaf development (gan and amasino, 1997). leaves will, of course, maintain their value as photosynthetic organs during the period of maturity, including the import and reduction of nitrate and sulphate. during senescence they will retranslocate nitrogen, phosphorous, potassium and other solutes to the rest of the plant (eg. slim et al. 1996). here we are asking, in contrast to the metabolites like nitrate, how is nacl in mangroves (and by implication other halophytes) accumulated during these stages? is nacl only accumulated during expansion growth? a priori one might expect that insofar as nacl is accumulated by leaves to serve an osmotic purpose, it would be accumulated to a set concentration during expansion growth, and maintained at that concentration during maturity. the quantity would then increase with leaf area during expansion growth, and with leaf thickness then and during maturity. in this paper we show that, in contrast to the simplest supposition, na accumulation is not restricted to the initial expansion phase, but continues during maturity and into senescence. fig 1mangrove leaves grow to full size in a few weeks, accumulating ions in their vacuoles as they do so, and then remain on the tree for months until senescence and abscission (eg tomlinson, 1994). each leaf must accumulate salt arriving in the shoot from the root system during expansion growth (phase 1). in the mature state (phase 2) a leaf might (a) take in no more salt, or (b) accumulate further ions continuously or (c) take up further salt only in the last stage before abscission (fig. 1). if leaves accumulate more salt during maturity, this could be via increased concentration and/or increased thickness. which of these alternatives applies to mangrove leaves?to answer this question one needs to follow the nacl content of individual leaves, measuring it regularly during its life. in practice this cannot be done, but it is possible to collect leaves of increasing age from single branches of mangroves, new leaves at the tip and progressively older ones further back. leaves have somewhat variable maximum surface areas (fig 2a), so their contents cannot be directly compared to see whether there is change after reaching maturity. however, contents per unit area can easily be calculated and allow one to compare thickness and concentration changes, which are in fact the two variables we want to follow in the mature leaf. the time scale of the changes can be established from the position of leaves along the branch, and demographic data in the literature for bruguiera cylindrica and avicennia marina in thailand, with a climate not dissimilar from that of singapore (wium-andersen and christensen, 1978; table 2). previous studies of changes in leaf ion contents with age include those of biebl and kinzel (1965), atkinson et al (1967), slim et al (1996) and zheng et al (1999). some of their results will be used in drawing some general conclusions about the role of mature leaves in salt disposal in mangroves. materials and methodssamples were collected from mature trees of bruguiera cylindrica (l.) bl. and avicennia rumphiana hallier f. growing on the seawards edge of the mangrove forest in the kranji estuary, singapore. samples of avicennia marina (forssk.) vierh. subsp. australasica (walp.) j. everett were collected from the board walk at kurnell bay, sydney, australia. both collections were made in january at about 11:00. bruguiera spp have no salt glands, whereas avicennia species do (tomlinson, 1994) and this was the reason for selecting the two species.terminal branches with leaves attached were placed in individual plastic bags and taken immediately to the laboratory. each leaf was identified while on the plant with a water resistant marker. leaves develop in pairs in all three species, and those of a pair were numbered a and b. in the laboratory, individual leaves were broken off, separating at the abscission zone. surface salt from glands in avicennia was washed off using the procedure of boon and allaway (1982), and other surface contamination was also washed off all the leaves, which were then gently dried with absorbent paper. the fresh weight and area of each leaf was measured and the leaves immediately put into a drying oven for several days. dried material was subsequently redried, weighed and analysed in newcastle upon tyne, uk. na+ and k+ were extracted from 0.1 g of mid-leaf samples in 100 mm nitric acid in a boiling water bath for 1 hr, made up to a suitable volume and analysed using a flame photometer. the extraction procedure removed over 96% of both na+ and k+ from the leaves, since a second lengthy extraction removed only an additional 4.1 0.2 % (sd, 3). leaf densities were measured in the related species rhizophora mangle and avicennia germinans. in r. mangle the density was 0.99 g cm-3 in leaves from node 1, falling to 0.91 0.02 g cm-3 in older leaves. in a. germinans the density was 0.97 0.03 g cm-3 independent of age. for the purposes of calculation the densities of leaves investigated in this paper were treated as 1.0 g cm-3. this introduces an error of a few percent, but is insignificant in comparison with the changes being observed and cannot affect the conclusions.from these data the leaf area and thickness, the ion concentrations in the leaf (mol (gram fresh weight of leaf)-1 ) and the quantities per unit area of leaf (mol cm-2) were calculated.resultsbruguiera cylindrica fig 2changes in leaf area, thickness, cation contents per unit surface area and cation concentrations are shown in fig 2. during the development of its leaves b. cylindrica approximately doubles the quantity of na+ (fig 2b) via a 30-40% increase in leaf thickness (fig 2c) plus a 30-40% increase in na+ concentration in the leaf cells (fig 2d and table 1). by contrast the leaf accumulates only a little more k+: the k+ concentration falls by 8%, but the increase in thickness more than makes up for this to give a 20% increase in leaf k+ content (figs 2c, 2d, and table 1). the changes in na+ and k+ concentration, leaf thickness and quantity are approximately linear with time, fitting alternative b in fig 1. table 1to examine whether there might be a rush of na+ into leaves just before abscission, yellow leaves still attached to the plant were compared with green leaves attached to the same or to the adjoining node. the ratio of na+ concentrations in yellow/green leaves is 1.09 0.11 (s.d. n = 6) and for k+ the ration is 0.7 0.1 (n = 6). though there is some variability, it is not sufficient to hide a large na+ increase or k+ loss just before abscission. avicennia rumphiana.the same measurements were made as for b. cylindrica, and selected data plus summaries are given in fig 3a and table 1. as with b. cylindrica, the leaves of a. rumphiana vary in size along different branches, and so we again use quantity per cm2 as the measure of leaf content. in a. rumphiana the changes in cation content, concentration and leaf thickness are again linear with time, as in b. cylindrica. a. rumphiana shows a modest progressive increase in na+ content of the leaf during the mature phase, but in this case a 33% increase in leaf thickness contributes more than the 10% increase in concentration. the species differs from b. cylindrica in having a 30-40% fall in k+ concentration (fig 3a), which leads to a small loss of k+ from the leaf despite the increase in thickness (table 1).fig 3in this species also there was no significant difference between yellow and similarly aged green leaves in na+ content (ratio = 0.87 0.08; n = 3) or k+ content (ratio = 0.87 0.09; n = 3). to check whether there might be any changes in solutes in the very last stage of senescence just before the leaf falls, a further comparison was made with yellow leaves collected from beneath the tree. these must have fallen less than a few hours earlier, or they would have been removed by crabs or high tide. attached leaves from nodes 5-7 contained 283 39 mmol/g fresh weight na+ and 57 6 k+ (n=9), whereas abscised leaves contained 257 35 na+ and 70 26 k+ (n = 6), showing that there is no significant difference between mature attached and abscised leaves, and certainly no evidence of any late increase in na+ or decrease in k+ concentrations. avicennia marinathis widely distributed species is clearly interesting, as it grows in temperate as well as tropical regions (tomlinson, 1994). a full set of measurements was made on this species also. fig 3b shows the progressive changes in na+ and k+ leaf contents, and other data are summarised in table 1. there are generally fewer nodes occupied by leaves than in the two tropical species already described. it is also unlike them in showing no significant increase in leaf thickness with age, a 50% fall in na+, and a 20% rise in k+ concentration. overall, a. marina leaves lose na+ content and gain k+ with age, the opposite of a. rumphiana. quantitative analysis of published data for other mangrove speciesrhizophora mucronata and r. mangle rhizophora mucronata (atkinson et al 1967) is qualitatively similar to b. cylindrica (of the same family, rhizophoraceae) in increasing na+ and decreasing k+ in its leaves via concentration and leaf size changes (table 1). in addition, both r. mucronata and r. mangle (biebl and kinzel, 1965) increase cl- content and leaf size (possibly mainly in thickness) by the same proportion, with cl- concentration constant. other measurements on r. mucronata (slim et al., 1996) suggest that cl concentration may increase with age of leaf, and may show a sharper rise during senescence. the age of leaves was not precisely defined, and this species deserves re-examination with leaf area and thickness as well as salt concentration being measured, as it is the only species out of all those studied for which accelerated salt accumulation during senescence has been reported. avicennia nitidacl- values have been published by biebl and kinzel (1965). a. nitida shows no increase in leaf thickness, and a 20% rise in cl- concentration (table 1), a modest change comparable with those seen in the other two species of avicennia discussed above. laguncularia racemosabiebl and kinzel (1965) published extensive measurements on laguncularia racemosa, mainly of structural changes and cl- contents and concentrations in leaves. l. racemosa differs quantitatively from the other species considered here by increasing its leaf thickness four-fold during maturity (fig 2c), via development of three to four layers of large, elongated, colourless cells between the epidermal layers. in addition the cl- concentration increases by 50%, and, together with a small increase in leaf size, this gives a nearly seven-fold increase in leaf content (table 1). it should be noted that laguncularia is in the combretaceae, a different family from the more widespread rhizophoraceae and avicenniaceae. other mangrove speciesaegialitis annulata (atkinson et al, 1967) differs from those discussed above by showing no change in leaf thickness, and a modest fall in na+, k+ and cl- concentrations (table 1). ceriops tagal (rhizophoraceae) has been reported by slim et al. (1996) to show progressive increase in cl concentration with age, but leaf thickness was not reported and so the results are ambiguous. data for eight other species are presented by zheng et al (1999), who report leaf concentrations (g/100 g dry weight) for young, mature and yellow leaves. values for six species of rhizophoraceae are reported, bruguiera gymnorrhiza, b. sexangula, rhizophora stylosa, r. apiculata, ceriops tagal and kandelia kandel. all show some modest increase in cl- and na+, and decrease in k+ concent