1. Growth rate is a fundamental property of organisms. In trees, growth is indeterminate and varies in space and time depending on resource availability, genetic constraints, competition and stress from biotic and abiotic environmental factors.
2. The ratio between the abundance of RNA and DNA in the tissue has been used to indicate recent growth rates in many systems, but not for trees. We assessed the applicability of using RNA : DNA ratios for assessing intraspecific and interspecific differences in growth rates in two species of mangrove trees under field conditions. We manipulated growth by fertilizing mangrove trees over a period of 4 years and measured tree growth as increments in linear extension of tagged shoots. The C, N and P contents per unit biomass were measured to test the hypothesis that faster growing organisms require more P per unit biomass (the Growth Rate Hypothesis).
3. We found that interspecific differences in the RNA : DNA ratio clearly reflected the difference in shoot elongation rates between the species. Intraspecific differences in RNA : DNA were significantly correlated with growth rates only for Avicennia marina and not for Ceriops australis. C : N and C : P ratios were lower in trees with higher growth rates and exhibited a negative correlation with RNA : DNA ratios.
4. Our results indicate that RNA : DNA ratios can reliably predict interspecific differences in growth rates between the two mangrove species and that RNA : DNA ratios can be used as an indication of variation in growth rate for Avicennia marina. The interaction between C : N : P ratios, RNA : DNA ratios and growth rates supported the Growth Rate Hypothesis on an interspecific level, but not on an intraspecific level.
Individual growth rate is a fundamental property of the life history of an organism and has vast implications for community structure and population ecology. In trees, growth is an important variable in understanding responses to disturbance and environmental conditions such as resource availability and competition (Givnish 1988). Trees are often large and long-lived, having complex forms and varying phenologies, with components (stem, roots and canopy) that may grow at different rates. Growth is slow compared to herbaceous plants and thus changes in growth over short periods of time are often difficult to measure, with growth often estimated as annual increments in stem circumference (Clark et al. 2007) or shoot extension (McGraw & Garbutt 1990). In this study, we tested the application of the ratio between ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) abundance for determining growth rates in trees by testing for a correlation between the ratio and growth rates. The relative abundance of RNA compared with DNA in the cell has been used to indicate recent growth in a wide-range of organisms for over 60 years [see reviews by (Neidhardt & Magasanik, 1960, Elser et al. 2000, Chìcharo & Chìcharo, 2008)]. RNA : DNA ratios are believed to provide estimates of growth because protein synthesis is intensive during active growth and cell enlargement. RNA is directly involved in protein synthesis and therefore increases in RNA content are observed during periods of rapid growth, whereas DNA content is usually stable (Bulow 1970) making the RNA : DNA ratio an indicator of protein synthesis capacity per cell. The RNA : DNA ratio is thus a frequently measured indicator of growth rate.
While rarely assessed, interspecific differences in RNA abundance relative to DNA content or to dry mass have been shown to reflect differences in growth rates between species, with faster growing organisms (such as bacteria and unicellular eukaryotes) having higher RNA contents than slower growing insects or molluscs (Vrede et al. 2004). An interspecific pattern of the relationship between RNA content and mean growth rate was demonstrated among organisms from within lower taxonomic groups. For example, a comparison between five species of the genus Drosophila revealed that the average RNA content was positively correlated with average growth rates for each species (Elser et al. 2006).
While the RNA : DNA ratio has been used extensively as an indicator of growth rates in other systems, few studies have attempted to test its validity for plants. Kato & Asakura (1981) showed that RNA : DNA ratios increased linearly with specific growth rate in tobacco cell cultures. When cell elongation in peas was enhanced with addition of gibberellic acid (Broughton 1969), an initial doubling of the RNA : DNA ratio was observed in those cells. RNA : DNA ratios of shoot tips were shown to be four times higher during periods of shoot growth than during the rest period (winter) for giant sequoia (Monteuuis & Gendraud 1987) and a similar pattern was found in leaves of orange trees (Monselise, Cohen & Kessler 1962). However, the applicability of using RNA : DNA ratios to elucidate interspecific variation in growth has not yet been assessed for plants.
Nucleic acids are molecules rich in phosphorus (P), with a carbon (C) : nitrogen (N) : P stoichiometry of 12 : 4 : 1. Some of the major cellular pools of organic P are associated with nucleic acids, with RNA usually being the most abundant P-containing molecule in the cell (Geider & Roche 2002). The Growth Rate Hypothesis (GRH; Elser et al. 1996, Elser & Hamilton 2007) proposes that as increase in growth rate requires an additional allocation of resources (mainly P) to ribosomal RNA, more rapidly growing organisms should have an overall greater P content per unit biomass. The hypothesis that variation in P content is associated with growth rate has been tested and supported for a variety of organisms (reviewed in Elser et al. 2000, 2003), including recently for mangroves (Lovelock et al. 2007). Fewer studies have investigated the association between P content and RNA content, with some studies reporting correlations between growth rates, RNA content and P content (Vrede, Persson & Aronsen 2002; Acharya, Kyle & Elser 2004; Gillooly et al. 2005; Elser et al. 2006) and some studies not supporting the GRH (e.g. Rhee 1978; Arnold et al. 2004).
In the present study, we assessed the applicability of using RNA : DNA ratio for assessing intraspecific and interspecific differences in growth rates in trees under field conditions. We selected two common coexisting mangrove species: Avicennia marina (Forssk.) Vierh. and the slower growing Ceriops australis (White) Ballment, Smith & Stoddart (Fig. 1). We manipulated intraspecific growth rates by fertilizing the trees over 4 years. RNA : DNA ratios were measured for two tissue types (vascular cambium and leaves). C, N and P contents per unit biomass were also measured to test the Growth Rate Hypothesis in trees by measuring the interaction among growth rate, the RNA : DNA ratio and the C : N : P ratio.
Materials and Methods
Site and species description
Bald Hills creek (27°17′17′S, 153°02′50′E) is a tidal creek in Moreton Bay, Queensland. The climate is subtropical with a dry winter (64 mm of rainfall between June and August) and a hot wet summer (mean rainfall between December and February 597 mm). The mean annual rainfall is 1186 mm and the mean annual temperature is 25·4 °C (Australian Bureau of Meteorology, averages for 1951–2000). The high-intertidal mangrove scrub forest at this site is dominated by Avicennia marina and Ceriops australis. The latter (Ceriops australis) is a slow growing, relatively small tree that usually occurs in high intertidal mangroves on well-drained soil (Ball 2002). Avicennia marina is a fast growing species (Clarke, Kerrigan & Westphal 2001), occupying diverse mangrove habitats. Both species are tolerant of high salinities (Clarke, Kerrigan & Westphal 2001). The mean tree height in the scrub zone was 1·4 m for A. marina and 1·1 m for C. australis. Salinity of the soil porewater varied over the site from 31 to 73 practical salinity units (PSU).
Growth was manipulated by fertilization with either N or P. Nine replicate trees of co-occurring A. marina and C. australis were fertilized annually since September 2005 by inserting 300 g of N (urea) or P (triple superphosphate) into 30 cm deep holes cored on each side of the main stem of the target trees. Holes were then sealed with the soil core that was extracted. For control trees, holes were cored and sealed in the same manner, but no nutrients were added. Salinity of the soil porewater was assessed for each tree in the experiment. Porewater was extracted from 30 cm depth using a suction device (McKee 1993) and analysed using a handheld refractometer (model 300011, Sper Scientific, Scottsdale, AZ, USA).
Net CO2 assimilation and growth
Net CO2 assimilation was measured using a Li-Cor 6400 photosynthesis system (Li-Cor Corp, Lincoln, NE, USA) in May 2009 between 08.30 am and 10.00 am local time using a 6400-40 leaf chamber with illumination set at 1000 μE and ambient air as reference. Photosynthesis measurements were made on three leaves, all exposed to full sunlight, from each of three trees per treatment for each species.
Demographic absolute growth rates (DAGR; McGraw & Garbutt 1990) were calculated for monthly increases in mean shoot extension of five shoots tagged per tree for 1 year (October 2006–November 2007) as described in (Feller et al. 2003). Tree heights were measured with a telescopic pole in September 2005 and then again in November 2007.
Plant tissue harvest
Tissue was harvested in July 2009 from the fertilized and control trees for RNA : DNA ratio determination. For RNA : DNA ratio determination in the leaves (primary growth), the youngest fully developed leaf pair was collected without the petioles and frozen by plunging in liquid N2. The leaf pair was selected from an unshaded twig at the top of the canopy. Two leaf pairs were selected per tree and the average RNA : DNA ratio of the four leaves was defined as the RNA : DNA ratio for the tree. For RNA : DNA ratio determination in the vascular cambium (secondary growth) two ∼3 cm2 scrapes (∼0·5 g) of vascular cambium tissue (∼1 mm thick) were collected from each tree trunk using a razor blade and frozen in liquid N2. The vascular cambium samples were taken near the base of the tree in areas without lichen growth. The average RNA : DNA ratio of the two samples was defined as the RNA : DNA ratio of the tree. Only trees that yielded consistent RNA : DNA ratios (replicate values that were within two standard deviations from the mean) were used for this analysis.
DNA and RNA determination
The relative quantities of RNA and DNA were determined by a fluorescence assay using 2,7-diamino-10-ethyl-9-phenylphenanthridinium bromide (ethidium bromide, EtBr). EtBr binds specifically to double stranded polynucleotides and, while doing so, emits a yellow fluorescence when irradiated with UVR. Hydrolysis of the nucleic acid strands results in loss of fluorescence. We used the sequential enzymatic method (Bentle, Dutta & Metcoff 1981) to quantify the contribution of the RNA and DNA fractions to the EtBr-polynucleotide complex by the sequential addition of RNase and DNase in excess to specifically hydrolyse first RNA and then DNA.
Vascular cambium or leaf tissue was ground in liquid N2 and homogenized (Tissue-Tearor, BioSpec Products Inc. Bartlesville, OK, USA) in homogenizing buffer (1 m NaCl, 2% Sarkosyl) and left on ice for 30 min. Homogenized samples were then spun at 5000 g and supernatant was transferred to a tube with extraction buffer: 20 mm Tris-acetate (pH 7·5), 1 mm MgCl2, 0·8 mm CaCl2, 6·5 μm EtBr, 0·01% Proteinase-K. All reagents were molecular grade and certified DNase and RNase free. Extraction was then carried out at 37 °C for 90 min in the dark, mixing occasionally. Following incubation, each sample was aliquoted to three triplicate wells in a 96 well plate (μClear black, Greiner Bio-One GmbH, Frickenhausen, Germany). Fluorescence was measured using a plate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA) with excitation set at 365 nm and emission measured at 592 nm. This measurement was designated F1. Samples were then incubated with DNase free RNase A (Fermentas Inc., ON, Canada) for 45 min at 37 °C. Fluorescence was measured again (designated F2), after which samples were incubated with RNase free DNase 1 (Fermentas Inc.) for 45 min at 37 °C. A final fluorescence measurement was then made (F3). The contribution of RNA to the total fluorescence was measured as F1-F2 and that of DNA as F2-F3. Remaining fluorescence at F3 was attributed to background fluorescence of the sample.
Alongside the samples, each plate included standards consisting of blank aliquots (extraction buffer: homogenizing buffer 1 : 10 v/v with no plant tissue) as well as blanks spiked with 5, 10 and 20 μg of pure DNA or RNA (DNA from calf thymus, and RNA from torula yeast, Sigma-Aldrich Corp., St Louis, MO, USA) to control for inter-plate variability and to suppress slight changes in background fluorescence because of weak fluorescence by the nucleases. In all plates, both RNA and DNA standards exceeded the amount of DNA and RNA in the samples and were reduced to zero following incubation with the respective nuclease. To test our analytical accuracy and to determine the fluorescence ratio between RNA and DNA for each species and tissue type, we spiked a series of tissue samples with known quantities of nucleic acids and measured the fluorescence contributed by the spikes. The DNA-EtBr complex has a higher fluorescence yield than the RNA-EtBr complex and fluorescence data were normalized to account for this difference. In this experimental system, the fluorescence yield of RNA was 0·42 that of DNA for A. marina and 0·33 that of DNA for C. australis respectively (Fig. 2), which is within the range of (Le Pecq & Paoletti 1966) or slightly higher than (Bentle, Dutta & Metcoff 1981; Buckley & Szmant 2004) fluorescence ratios achieved using similar techniques. Spike recovery was unsuccessful for Avicennia marina leaves and thus RNA : DNA ratios were not calculated for this tissue. In A. marina vascular cambium tissue, the slope for DNA standard fluorescence was 0·12 and the slope for RNA for 0·05 (fluorescence ratio of 0·42, Fig. 2a). For C. australis vascular cambium, the slope for DNA was 1·48 and for RNA the slope was 0·49 (ratio of 0·33, Fig. 2b). The difference in the RNA : DNA fluorescence between the cambium tissue of the two mangrove species is probably because of a different amount of tannins or different levels of salts (especially NaCl). Tannins can precipitate proteins and chelate metal ions in the solution, as well as alter the pH (tannins are slightly acidic polyphenolic compounds) (Kraus, Dahlgren & Zasoski 2003) thus affecting the EtBr-nucleotide complex fluorescence. In the same manner, differences in NaCl concentrations in the tissue can affect the EtBr-nucleotide complex fluorescence ratio as it suppresses RNA-EtBr fluorescence more than it does DNA-EtBr fluorescence (Bentle, Dutta & Metcoff 1981).
Leaf C, N and P
Three green leaves were harvested from each tree, dried at 60 °C and subsequently weighed. N and C concentrations (presented as % mass) of dried leaves were determined using a CNS combustion analyser (Analytical Services, School of Land Crop and Food Sciences, The University of Queensland, Australia). The P concentration (% mass) in finely ground leaves was determined using an acidified persulfate autoclave digestion of the organic compounds (Menzel & Corwin 1965) followed by quantification of the released orthophosphate in a colourimetric assay with ammonium molybdate and malachite green (Van Veldhoven & Mannaerts 1987).
Data were analysed in a linear model (ancova) with nutrient treatment and species as factors, RNA : DNA ratio and salinity of the porewater as cofactors and DAGR as the variable. We verified the assumptions of normality and homoscedasticity by visually inspecting quantile-normal plots. Data were log transformed, if necessary, to improve homoscedasticity and linearity. The relationship between RNA : DNA ratio and DAGR growth rates was further analysed using Pearson’s correlation. Statistical analysis was performed using R version 2.9.2 (Team 2009).
Growth manipulation and interspecific variation in growth rates
The nutrient addition created significant differences in DAGR growth rates both between species and among treatment (ancova, F(1,36) = 34·15, P < 0·001 and F(2,36) = 8·6, P < 0·001 respectively; Fig. 3a). Both C. australis and A. marina exhibited N limitation to growth. Shoot extension of N-fertilized plants was three times faster than control trees for both A. marina and C. australis. Phosphate addition did not significantly affect DAGR (Tukey HSD, P = 0·92; Fig. 3a) for either species, indicating that P did not limit tree growth at this site. For all treatments, A. marina shoot extension rates were, on average, almost three times higher than those of C. australis indicating A. marina is a faster growing species at this site. Porewater salinity was variable 57·5 ± 10·7 (Mean ± SD), but salinity was not found to have a significant effect on DAGR and was removed from the statistical model.
Photosynthetic carbon assimilation rates were significantly higher for A. marina leaves than for C. australis (F(1,12) = 283·8, P < 0·001, Fig. 3b). Carbon assimilation rates had a mean of 8·2 (±0·7) mol CO2 m−2 s−1 for A. marina and 3·7 (±0·44) mol CO2 m−2 s−1 for C. australis. Nutrient addition did not affect photosynthetic rates for either species (F(2,12) = 2·19, P = 0·15).
RNA : DNA ratio as an indicator for growth rate
RNA : DNA ratios of the vascular cambium tissue ranged between 1·8 and 17·5 for A marina and between 0·6 and 5·9 for C. australis (Fig. 4). RNA : DNA ratios of leaves ranged between 5·3 and 14·3 for C. australis (Fig. 5), but could not be quantified from A. marina leaves. The RNA : DNA ratios of A. marina vascular cambium were significantly higher than those of C. australis (Fig. 4, F(1,37) = 41·27, P < 0·001). Nutrient treatment did not significantly affect RNA : DNA ratios in the vascular cambium tissue of either species (F(2,37) = 0·21 P = 0·8) or the RNA : DNA ratio in C. australis leaves (F(2,21) = 0·06, P = 0·94).
With a combined species data set, we found a positive correlation between RNA : DNA ratio and growth rate (DAGR). The Pearson’s correlation coefficient was r = 0·648 (P < 0·001, Fig. 4). When considering the species individually, we found that RNA : DNA ratios correlated with growth rates for A. marina (r = 0·536, P = 0·02), but not for C. australis (r = −0·069, NS).
Leaf RNA : DNA ratio in C. australis was significantly higher than that of the vascular cambium (paired t-test, P < 0·0001, Fig. 5). No significant linear relationship between leaf and vascular cambium RNA : DNA ratios in Ceriops australis was detected (r = 0·016, NS).
C : N : P, RNA : DNA ratios and the growth rate hypothesis
Carbon : phosphorus mass ratios ranged between 250 and 526 for A. marina and between 184 and 843 for C. australis. C : N ranged between 11 and 30 for A. marina and between 21 and 124 for C. australis. Both C : P and C : N mass ratios explained a significant proportion of the variation in RNA : DNA ratios observed between the two species (Fig. 6a,b). However, we did not detect a significant interaction between either C : P or C : N and RNA : DNA ratios from trees within each species (Table 1). RNA : DNA ratios were higher in trees with lower C : N and C : P mass ratios.
Table 1. Pearson’s product-moment correlation coefficients (r) for the relationships between log RNA : DNA or log DAGR and log C : N mass ratio (first column) or log C : P mass ratio (second column) for each species separately and for both species combined
C : N
C : P
Significant correlations are given in bold.
RNA : DNA
r = 0·035, P = 0·87
r = 0·007, P = 0·97
r = −0·03, P = 0·91
r = 0·07, P = 0·81
r = −0·53, P = 0·0007
r = −0·48, P = 0·003
r = −0·687, P = 0·0002
r = −0·21, P = 0·32
r = −0·62, P = 0·02
r = 0·1, P = 0·73
r = −0·82, P < 0·0001
r = −0·54, P = 0·0005
Intraspecific variations in growth (measured as DAGR) were negatively correlated with the C : N mass ratio of the leaves for both A. marina and C. australis (Fig. 7a, Table 1). Intraspecific variations in C : P were not significantly correlated with variation in growth rate for either species (Fig. 7b, Table 1). However, both C : P and C : N mass ratios showed a negative correlation with growth rate when both species were combined in one data set (Fig. 7, Table 1).
Tree growth is indeterminate and varies in space and time depending on resource availability, genetic constraints, competition and stress from biotic and abiotic environmental factors. Being able to predict tree growth is key to understanding the ecology of species and communities. Here, we show that RNA : DNA ratios can reflect the growth rate for A. marina and that they reliably predict interspecific differences in growth rates between the two mangrove species. RNA : DNA ratios in A. marina were significantly higher than those of C. australis, and A. marina also exhibited higher DAGR and CO2 assimilation rates than C. australis. RNA : DNA ratios obtained for A. marina this study were within the same range of values calculated for orange trees (Monselise, Cohen & Kessler 1962), and four times higher than those calculated for growing shoots of the slow growing giant sequoia (Monteuuis & Gendraud 1987). In comparison, C. australis RNA : DNA ratios were only slightly higher than those found in giant sequoia.
Growth rates of C. australis were very low and in many of the trees, shoot extension was not detected. Low growth rates of this species and other species within the genus have been observed by others (e.g. Smith 1988; Ball 2002). Low growth rates are consistent with the high level of salt tolerance (Smith 1988; McGuinness 1997; Aziz & Khan 2001) and tolerance of low soil water content (Ball 1998) observed in C. australis and congenitor C. tagal.
Variation in growth rate was notably higher for A. marina than for C. australis, (SD = 0·9 vs. 0·25 respectively). This could imply that the ability of the RNA : DNA ratio to assess changes in growth rates within species may depend on large variation in growth rates and that RNA : DNA ratios might not be able to detect small variations in growth rates, such as those observed in C. australis in response to nutrient enrichment in this study. The lack of correlation between RNA : DNA ratios and DAGR in both tissue types of C. australis might arise from uncoupling of the growth rate from the need for protein synthesis in non-growing organs. The slow growing (and even the non-growing) C. australis trees in this study were not necessarily allocating carbon to shoot extension (Chapin et al. 1987), but may have been translocating fixed carbon to other organs and tissues, for example, to roots (reviewed in Wilson 1988) and other storage pools (Kobe 1997), to support recycling of nutrients and futile cycling of nitrogen (Britto et al. 2001) and for investment in secondary metabolism of antiherbivore defences (Coley, Bryant & Chapin 1985). Thus, it is possible that RNA : DNA ratios reflect metabolic activity not apparent in observations of shoot extension.
Our results support the Growth Rate Hypothesis (GRH; Elser et al. 1996) on an interspecific level. For both species combined, we found a strong negative correlation between RNA : DNA ratios and both C : P and C : N ratios (Fig. 6), as well as a significant positive relationship between RNA : DNA ratio and growth (Fig. 4). Both P and N are part of the nucleic acid molecules and even though the C : N : P stoichiometry of nucleic acids (12 : 4 : 1) is more heavily enriched in P than in N compared with the mean atomic C : N : P ratios we have found in mangrove leaves (1062 : 31 : 1), N significantly correlated with RNA : DNA ratios. The correlation between C : N ratios and RNA : DNA ratios has been shown in other studies where the quantity of RNA strongly correlated with total N in tobacco cells (Kato & Asakura 1981) and orange leaves (Monselise, Cohen & Kessler 1962). Additionally, in agreement with the GRH, growth rate correlated negatively with C : N and C : P for both species combined, with the faster growing species (A. marina) having higher N and P content than the slower growth species.
We did not find strong support for the GRH when comparing individuals within each species. Intraspecific differences in growth rate correlated negatively with C : N within both species, but not with C : P, probably because of N rather than P limitation to growth in this environment for both A. marina and C. australis (Fig. 3). The potential success of the GRH in explaining P variation is greatly compromised under N-limited conditions as autotrophs under N-limitation can uptake and store considerable amounts of P (Ågren 2004). Indeed, the ability of plants to store nutrients in vacuoles has been suggested to result in a decoupling of elemental stoichiometry from metabolism, protein synthesis and growth (Matzek & Vitousek 2009). Variation in the relationship between C : P and N : P ratios and growth rate has been observed in other studies to be dependent on the nature of the nutrient limitation at the site. For example, at a site where growth of pines was limited by both N and P, trees showed both higher N and higher P content (lower C : N and C : P) in tissues of faster growing plants in the field in California (Matzek & Vitousek 2009). Additionally, in the mangrove Avicennia germinans growing at sites with contrasting N and P limitation, variation in growth rate was reflected more in C : N ratio at the N limited sites and by C : P ratio at the P limited site (Feller, Lovelock & McKee 2007). Neither C : N nor C : P ratios correlated with RNA : DNA ratios within either species, and RNA : DNA ratios correlated with growth rate only for A. marina.
Leaf and vascular cambium tissues had considerably different RNA : DNA ratios, with leaf RNA : DNA ratio of C. australis being on average >4 times higher than the vascular cambium (Fig. 5). This is not surprising as different tissues often have different RNA : DNA ratios and protein turnover (Houlihan et al. 1988). The rate of protein turnover in plants depends on the plant organ under study. Protein turnover of photo-damaged photosynthetic proteins in the leaves can be much more rapid than the turnover of other polypeptides (Raven 1989). The need for high rates of protein synthesis and turnover in the leaves may explain the high levels of RNA in the leaves compared with the vascular cambium. Furthermore, C : N : P stoichiometry in vascular plants can be substantially different between plant organs (Niklas et al. 2005).
Determining growth rates is essential for most ecological or physiological studies and for devising effective management practices for conservation of biodiversity and forest productivity. Using RNA : DNA ratios can provide an additional tool to assess interspecific and potentially intraspecific differences in growth rates. RNA : DNA ratios in this study reflected growth rates independently of the nutrient availability in the soil, suggesting the RNA : DNA ratio might be a robust tool for the assessment of growth potential over a range of environments.
We thank N. Stromsoe and A. Main for their help with sample analysis and V. Benion, A. Grinham, A. Chamberlain and B. Clegg for assistance with fieldwork. U. Motro helped with statistical analysis. We thank M. Motro and the anonymous reviewers for their useful comments on this manuscript. Australian Research Council Discovery Projects DP0774491 and DP0986170 and the Smithsonian Marine Science Network funded this work.