Abstract: Gas exchange and leaf water relations were studied on attached and excised oil palm fronds. Excised palm fronds experienced water stress and responded by closing their stomata in an attempt to avoid water loss through transpiration. This inhibited the diffusion of CO2 into the leaf, decreased the intercellular CO2 level and resulted in a reduction in photosynthetic rate. Water deficit develops in the excised tissue as the demand by transpiration exceeds the supply of water. The leaf water potential (ψl), leaf osmotic potential (ψs) and leaf turgor potential (ψp) were reduced in response to the disruption in water supply. Results show that gas exchange measurements on excised fronds must be carried out immediately after excision in order to avoid water stress effects.
INTRODUCTION
The frond leaflets are the main site of photosynthesis and gas exchange processes in oil palm (Elaeis guineensis). They represent the main interface between the palm and the atmosphere, for exchange of gases (e.g., carbon dioxide (CO2), water vapour and oxygen) with the environment. During daytime, photosynthesis increases the oxygen concentration in the sub-stomatal air space and decreases the intercellular CO2 concentration (Ci). This affects the concentration gradients of these gases and hence, their diffusion rates. Palm leaflets have stomata on their surface that regulate the entry and exit of gases including water vapour. Mature oil palms are hypostomatous, having stomata only on the abaxial leaf surface[1]. This could be an adaptation to alterations in environmental conditions as the palm matures, since juvenile palms have stomata on both leaf surfaces.
Stomata are small pores on the surfaces of leaves that control the exchange of gases between the interior of the leaf and the atmosphere. Each stoma has a pair of guard cells that can swell up or shrink to control its pore size. In this capacity they make major contributions to the ability of the plant to control its water relations and to gain carbon. It is commonly assumed that stomata provide the control of both transpiration and photosynthesis. However, gas exchange is regulated by controlling the aperture of the stomatal pore and the number of stomata that form on the epidermis.
Environmental factors such as light intensity, temperature, Vapour Pressure Deficit (VPD), carbon dioxide concentration and endogenous plant hormones control stomatal aperture and development[2-4]. Stomata open when the light strikes the leaf and close in the dark. The mechanism involves a change in the turgor of the guard cells. When turgor develops within the two guard cells next to each stoma, their thin outer walls bulge out and forces the inner walls into a crescent shape, which opens the stoma. When the guard cells lose turgor, the inner walls regain their original shape and the stoma closes. The increase in turgor or osmotic pressure in the guard cells is caused by an uptake of potassium ions (K+), such that their concentrations in open guard cells are higher than that in the surrounding cells. Plant hormone such as abscisic acid can also trigger stomata closure when soil water is insufficient to keep up with transpiration[5-9]. However, in leaves excised from well-watered plants, their stomatal conductance decreases as the leaves dry, so a signal from dry roots is not required for stomatal closure[10]. Stomatal conductance (gs) is a numerical measure of the rate of passage of either water vapour or carbon dioxide through the stomata pores.
Carbon dioxide concentration can control both stomatal opening and their number. Plants grown in an artificial atmosphere with a high level of CO2 have fewer stomata than normal[11]. High CO2 levels can also reduce gs. The difference in water vapour pressure between the inside and outside of leaves can significantly influence gs. Stomata normally close with increasing VPD, a survival response to prevent excessive dehydration and hydraulic failure[2,12,13]. Leaves of plants developing under high levels of light have increased stomatal densities[14].
In addition to opening and closing the stomata, plants may exert control over their gas exchange rates by varying stomata density in new leaves when they are produced. The more stomata per unit area (stomata density) the more CO2 can be taken up and the more water can be released, which will lower the leaf temperature.
Gas exchange measurements are useful for comparing and understanding palm productivity (or biomass accumulation) at the leaflet, palm or community level as well as their response to environmental stresses. Since CO2 intake and H2O release share the same pathway via the stomata, gas exchange measurements commonly include the estimation of CO2 uptake, stomatal conductance and transpiration. Portable open-path gas exchange systems are available for measuring leaf photosynthesis, with options for controlling CO2, humidity, temperature and light. They provide real-time measurements of CO2 uptake, transpiration, leaf conductance and the intercellular CO2 level.
This study investigates the gas exchange response of oil palm leaflets before and after frond excision, in order to develop a suitable technique of measuring gas exchange in tall palms, where access to attached fronds is often difficult.
MATERIALS AND METHODS
Gas exchange measurements were carried out in January 2005 on 6-year old DxP palms grown under standard estate practice on Rengam Series soil at an inland site in Peninsular Malaysia. Five palms with uniform canopy growth were selected. Measurements were taken from fronds 1, 9 and 17 while still attached to the palm, immediately after frond excision and 2 h later. Excised fronds were placed on the ground under shade to minimize water loss.
Leaf gas exchange: An infrared gas analyzer system (LI-6400, LI-COR Inc., USA) was used to measure leaf gas exchange at optimal cuvette condition, i.e. photon flux density of 1000 μmol photosynthetically active radiation (PAR) m2 s-1, 400 ppm CO2, 30oC and 60% relative humidity. Measurements were taken from six upper rank leaflets while still attached to the frond rachis from the middle of each chosen frond i.e., before and after frond excision.
Leaf water relations: Leaf water potential (ψl) was determined using a pressure chamber (PMS Instruments, USA) according to Scholander and Hamme[15]. The leaflet to be measured was enclosed in a plastic bag before being excised from the frond and partly sealed in a pressure chamber and pressurized with compressed nitrogen gas until the distribution of water between the living cells and the xylem conduits was returned to its initial pre-excision state. This was detected visually by observing the appearance of sap at the cut leaf surface, which becomes wet and shiny and the reading indicated on the pressure gauge was recorded[16].
The leaf osmotic potential (ψs) of expressed leaflet sap was estimated using a Roebling Automatic Freezing-Point Osmometer (Hermann Roebling, Berlin). Excised leaflets were harvested, immediately frozen using liquid nitrogen and stored at –30°C. They were then thawed for 30-60 min at room temperature and the sap was extracted for immediate osmotic potential determination according to Hawa[17]. Leaf turgor potential (ψp) was estimated as the difference between ψl and ψs.
Frond moisture content: Frond moisture content was determined on frond 17 taken from another five palms at the same location. Immediately after excision, the frond was separated into rachis and leaflets. Their fresh weights were recorded before drying in an oven at 70°C until constant weight. Subsequently, the dried frond components were weighed and the moisture content calculated.
RESULTS AND DISCUSSION
Gas exchange: In general, reductions of photosynthetic rate, transpiration rate and stomatal conductance from the same leaflets were small, but significant (p<0.05) immediately after frond excision, with reductions of about 14% in photosynthetic rate, 28% in stomatal conductance, 25% in transpiration rate and 14% in intercellular CO2 level (Table 1). This result contradicts those of Smith[18,19] who reported that excision of leaflets had no significant effect on stomatal conductance or photosynthetic rate within 1 to 4 min after removal from tall palms.
The parameters were further reduced at two hours after excision, when photosynthetic rate was reduced by about 77%, stomatal conductance by 96% and transpiration rate by 93% as compared to before excision. The decline in transpiration rate immediately after frond excision can be assailed to stomatal closure. With the onset of desiccation, stomatal closure proceeded until it reached a very low level. This also greatly reduced the intercellular CO2 level by about 73% as compared to before frond excision, because of the low CO2 diffusion from outside. Frond number had no significant effect on the gas exchange parameters.
Photosynthetic was significantly correlated with stomatal conductance and transpiration rates as shown in Fig. 1 and 2. A similar observation on the relationship between photosynthetic rate and stomatal conductance was reported by Dufrene[20], Smith[21] and Lamade[22]. It demonstrates the regulatory role of stomata for both the loss of water from plants and the exchange of CO2, providing a short-term control of both transpiration and photosynthesis in plants[23-25].
| Table 1: | Mean gas exchange parameters of three frond ages before and after excision |
| n.b. Mean±SE followed by the same letter are not significant at p<0.05 (small letter for frond ages and capital letter for excision treatments, n = 30) | |
| Table 2: | Mean leaf water potential, osmotic potential and turgor of three frond ages before and after excision |
| n.b. Mean±S.E. followed by the same letter are not significant at p<0.05 (small letter for frond ages and capital letter for excision treatments, n = 30). | |
| Table 3: | Moisture content of frond 17 components (n = 5) |
| Fig. 1: | Relationship between stomatal conductance and photosynthesis rates of 6-year old palms in response to frond excision |
Intercellular CO2 level is a function of stomatal aperture, also of ambient CO2 and photosynthetic rate, which in turn is a function of the plant metabolic system, water availability, leaf transpiration and carbon utilization in the plant.
| Fig. 2: | Relationship between transpiration and photosynthesis rates of 6-year old palms in response to frond excission |
The intercellular CO2 level is a very important limiting resource in plant growth and development since photosynthesis proceeds on the basis of CO2 availability to the chloroplast. A decrease in stomatal conductance will limit CO2 uptake into intercellular spaces and result in a decrease in intercellular CO2 level. However, increased stomatal closure can also produce high intercellular CO2 level due to mitochondrial respiration, but this occurs only at high stress levels.
Leaf water relations: As water evaporated from the leaf, ψl became more negative (Table 2). Excision stopped the supply of water to the fronds and induced water stress. The ψl was not significantly reduced immediately after excision, although it was decreased by about 24% as compared to before excision. It was significantly reduced by about 56% of the prior-to-excision value at two hours after excision, when its mean value was less than –2.0 MPa. Frond number had no significant effect on the leaf water potential parameters.
Leaf osmotic potential (ψs) fell by 13% immediately after excision and showed a significant 35% reduction two hours after excision (Table 2). Plants have the ability to accumulate solutes in cell vacuoles that lower the osmotic potential of the cell contents and subsequently lower the water potential[26,27]. The leaf cells contain various organic and inorganic solutes, which determine the ψs that is generally lower or more negative than ψl. Thus, ψs could regulates ψl when the leaf continues to lose water through accumulation of cellular solutes, such as potassium (K+) ions[28]. Guard cells can become hypertonic to the mesophyll cells by increasing their sugar concentration through active photosynthesis, or by pumping K+ ions into their cytoplasm. When mesophyll cells loose excessive amounts of water, they become hypertonic to guard cells and water leaves guard cells. Plants under stress produce abscissic acid that results in opening of K+ channels in guard cell plasma membrane. The loss of solutes then causes water loss and stomatal closure.
Leaf turgor potential (ψp) fell 24% immediately after excision and by 56% two hours after excision (significant at p<0.05). ψp is associated with cellular growth and function, e.g. stomata closure response to low turgor to reduce transpiration[29,30]. The palm leaf water potential relations were therefore significantly reduced after two hours as a result of the water deficit induced by frond excision.
Frond moisture content: The initial frond moisture content was about 71% for the rachis and about 60% for leaflets (Table 3). This was not sufficient to sustain gas exchange after frond excision. Vascular vessels are cut open when the frond is excised, breaking the water columns in the xylem that would initially be under tension[31]. According to the Scholander assumptions[15,32], the water content of xylem conduits will be displaced by air when leaves or stems are cut in air, if the leaf is transpiring at the time of excision from the plant. Thus, the disruption to the supply of water induces water stress in the excised frond.
Excised palm fronds experience water stress and respond by closing their stomata in an attempt to avoid water loss through transpiration. This inhibits the diffusion of CO2 into the leaf, decreasing its intercellular CO2 level and reducing the photosynthetic rate. Water deficit develops in the excised tissue as the demand by transpiration exceeds the supply of water. This was demonstrated by the reductions in ψl, ψs and ψp values in response to the disruption in water supply. Gas exchange measurements on excised fronds must therefore be done immediately after excision in order to avoid water stress effects.
ACKNOWLEDGMENTS
I wish to thank the Director General of MPOB for permission to publish this work. The valuable technical assistance from members of the MPOB Crop Physiology Group was most appreciated. Comments from Dr. I.E. Henson, Hj. Mohd Tayeb Dolmat, Dr. Norman Kamarudin and Dr. Ahmad Kushairi Din were greatly acknowledged.