Stomata actively regulate internal aeration of the sacred lotus Nelumbo nucifera

The sacred lotus Nelumbo nucifera (Gaertn.) possesses a complex system of gas canals that channel pressurized air from its leaves, down through its petioles and rhizomes, before venting this air back to the atmosphere through large stomata found in the centre of every lotus leaf. These central plate stomata (CPS) lie over a gas canal junction that connects with two‐thirds of the gas canals within the leaf blade and with the larger of two discrete pairs of gas canals within the petiole that join with those in the rhizome. It is hypothesized that the lotus actively regulates the pressure, direction and rate of airflow within its gas canals by opening and closing these stomata. Impression casting the CPS reveal that they are open in the morning, close at midday and reopen in the afternoon. The periodic closure of the CPS during the day coincides with a temporary reversal in airflow direction within the petiolar gas canals. Experiments show that the conductance of the CPS decreases in response to increasing light level. This behaviour ventilates the rhizome and possibly directs benthic CO₂ towards photosynthesis in the leaves. These results demonstrate a novel function for stomata: the active regulation of convective airflow.     

Anatomy of the gas canal system of Nelumbo nucifera

Nelumbo nucifera (Gaertn.) grows by extending a creeping rhizome through anaerobic sediments. Nodes form at intervals along the rhizome, each producing a single leaf, and gas canals channel air from the leaves throughout the petioles and rhizomes. The gas flow pathway was mapped by casting the canals in growing shoots with silicone and by blowing air through complexes of rhizomes and petioles. Air from a leaf flows to a rhizome through one of two petiolar canal pairs, joining with the lowermost of three canal pairs in the rhizome through a chamber in the node. The lowermost canal pair links these nodal chambers along the length of a rhizome, allowing air from a node to flow both forward, toward a growing shoot, and backward, toward preceding leaves. These linked chambers also connect with the middle pair of canals on their proximal side, enabling flow to proceed backward along the rhizome to an adjacent node. A chamber in the next node then diverts the flow into the upper canal pair. This pair leads to a third node and chamber from which the air vents to the atmosphere through the second petiolar canal pair. Thus, pressurised air from one leaf must flow backward through two nodes before it returns to the atmosphere. Forward flow also ventilates a shoot's growing tip, with air from the lowermost canal pair entering a chamber in the developing node which, as described above, connects with the middle canal. This allows the air to reverse direction at the tip and enter the vent flow pathway.     

Knudsen-transitional flow and gas pressurization in leaves of nelumbo

Pressures in gas spaces of leaves of the lotus Nelumbo are higher than ambient pressure. The pressurization capacity of leaves was studied as a function of leaf temperature, and the composition of air entering evacuated leaves was used to calibrate the pore sizes which determine flow in these leaves. The adaxial side of the leaf of Nelumbo has two distinct regions in terms of gas exchange characteristics. There is a region of relatively high mean pore diameter in the center of the leaf opposite the point of petiole insertion. Gas exchange between the remainder of the leaf (>99% by area) and the atmosphere is restricted by “pores” with an effective mean diameter less than 0.03 micrometer. As a result, a flowthrough ventilation operates within each leaf. Air enters the leaf across the expanse of the lamina, and escapes back to the atmosphere through the highly porous region at the center of the lamina.     

The Importance of Water Vapour for the Circulating Air Flow through Nelumbo nucifera

Aquatic vascular plants depend on an adequate oxygen supplyin order to maintain growth and reproduction in anaerobic environments.Nelumbo nucifera is able to survive with a gas transport systemwhich supplies oxygen to the roots and rhizomes submerged inthe anaerobic sediment. It was possible to demonstrate thatthis gas transport system is based on a purely physical phenomenonThermo-osmotic oxygen transport was first demonstrated on freshleaves with the help of an oxygen-sensitive electrode. A definiteenhancement of oxygen flow was obtained through excised leaveswhen a temperature difference between the ambient and lacunarair was present in light. These leaves were then dried to brittlenessand the enhanced oxygen flow was still detectable. This showsthat not only photosynthetic oxygen, but also atmospheric oxygencan be transported to the buried organs. The absolute flow ofoxygen through dry leaves was much lower than through freshleaves, but the thermo-osmotic transport of oxygen still functioned.Furthermore, the process of thermo-osmosis need not rely ona difference in humidity between the two sides of a porous partition,but may be linked causally to the temperature difference andthe pore size. Key words: Nelumbo nucifera, oxygen transport, thermo-osmosis of gases     

Evaluation of the transpiration rate of lotus using the stem heat-balance method

To reveal the mechanism of transpiration by hydrophytes in the field, it is necessary to evaluate the transpiration rate without the effect of the evaporation from the water surface. In order to test the suitability for evaluating the transpiration rate of lotus (Nelumbo nucifera Gaertn.) leaves in the field, stem heat-balance method was applied and the obtained sap-flow rate was compared with the transpiration rate measured by weighing and with the overall canopy evapotranspiration rate by means of the eddy covariance technique. The transpiration rate estimated with the sap-flow measurements showed good agreement with that obtained from the weighing method. Lotus has many air canals in its petiole to carry oxygen-rich air to the rhizome and methane- and carbon dioxide-rich air back to the atmosphere, but there was little effect of the mass flow of air through these canals on the sap-flow rates. In the field observations, the canopy evapotranspiration rate (0.28 mm h⁻¹ at maximum) was nearly equal to the sum of the transpiration rate from all sunlit leaves (0.30 mm h⁻¹), and the contribution of the transpiration from shaded leaves and evaporation from the water surface was considered to be minor in the seasons when the leaves were fully developed. Evaluation of bulk leaf conductance revealed that the conductance in the leaf boundary layer of lotus could be low (ca. 0.23 mol m⁻² s⁻¹) because of its large leaf area. The low conductance in the leaf boundary layer would increase leaf temperature, which, in turn, would generate air circulation within the plant's ventilation system. Because there was a linear relationship between transpiration rate and the leaf-to-air vapor-pressure deficit, with no apparent maximum, high vapor-pressure deficits (3.4 kPa at maximum) did not appear to cause significant stomatal closure in lotus plants. The stomata of lotus leaves play a role as air inlets to carry oxygen-rich air to the rhizome, so their low sensitivity would help to increase air intake.     

A BENEFICIAL GAS TRANSPORT SYSTEM IN NYMPHOIDES PELTATA

The aquatic vascular plant Nymphoides peltata (Gmel.) O. Kunze which inhabits anaerobic environments depends highly on the availability of oxygen for its submerged organs buried in the sediment of the lake. In tracer gas studies, carried out with shaded plants, it is shown that ethane is taken up by one of the youngest leaves in a whorl and transported down the petiole to the axis, returning to the surface via the older leaves. In sunlight, this gas diffusion through the plant is replaced by an effect which enhances the gas movement up to 1,200% due to the increased difference between leaf temperature and the surrounding air (ΔT = 1.7 K). The temperature difference is accompanied by a pressurization 50 Pascal above ambient inside the aerenchyma of the young leaves. These findings confirm that a pressurized flow-through system is established by N. peltata, whereby the oxygen supply to the rhizomes is improved. The temperature difference derived from irradiation energy initiates a circulating air stream, which transports air from the young leaves through the plant. Enhanced transport of a tracer gas as well as oxygen can be demonstrated by warming an excised young leaf with red-filtered light or warm water. A similar increase in gas transport is not detected through older leaves. The light energy needed to create a temperature difference can be substituted with warm water. Evidence is thus given, which shows that the increased oxygen emission from the petiole of young leaves is independent of photosynthesis. This gas transport is a result of thermo-osmosis under slip-flow conditions (Knudsen diffusion), limiting this effect to a temperature gradient between the surrounding air and the lacunar air of young leaves.     

Comparative study of leaf architecture and cuticles of Nelumbo changchangensis from the Eocene of Hainan Island,China, and the two extant species of Nelumbo (Nelumbonaceae)

Fossil leaves of Nelumbo changchangensis, collected from the Eocene of Hainan Island, China, were studied and compared with those of the extant species of Nelumbo, N. nucifera Gaertn. and N. lutea Willd. The fossil leaves have all the specialized features of extant Nelumbo in leaf architecture, except that the organization of the areolae looks much more irregular than that of extant Nelumbo. Comparisons of the cuticle and epicuticular ultrastructure indicate that: (1) N. changchangensis resembles N. nucifera in that anticlinal cell walls of the lower epidermis are straight along the major veins and near leaf bases and are shallowly undulate with U‐ to V‐shaped undulations inside the areolae; (2) N. changchangensis differs from N. lutea in that anticlinal cell walls of the lower epidermis of the latter are deeply undulate with U‐, V‐ to reversed Ω‐shaped undulations inside the areolae; and (3) epicuticular wax crystals are more densely distributed on the leaves of N. changchangensis and N. nucifera than they are in N. lutea. These findings shed significant light on the cuticle differentiation of fossil and extant Nelumbo species. The morphometric comparisons indicate that almost all the synapomorphies of extant Nelumbo were already present by the Eocene, © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 123–137.     

Growth characteristics ofNelumbo nucifera Gaertn. in response to water depth and flooding

Three experiments on the effects of water depth and flooding onNelumbo nucifera Gaertn. were made in the artificial environment of concrete ponds. First, plants were harvested in autumn after growing under seven different water levels ranging from 0.2–3 m The number of floating leaves, the total number of leaves and the leaf area index of emergent leaves were greatest in the tanks at 0.5 m depth. The petiole dry weight per unit length of emergent leaves and the ratio of aboveground to belowground biomass rose with increasing water depth up to 2 m. In contrast, that of floating leaves was constant at about 10 mg dry weight cm⁻¹. The proportion of biomass in tubers fell from 20% at 0.2 m to 6% at 2 m. Second, petiole elongation responses to the amplitude of flooding were investigated in early summer. The maximum rate of petiole elongation was 25 cm per day at 2.4 m water depth. This was the maximum depth at whichN. nucifera could grow. No petioles could elongate from 3 m to 5 m depth. Finally, the effects of timing of flooding on growth were investigated. At the end of growing season, the belowground biomass of plants in the flooding treatment in late summer was smallest among the flooding treatment plants (P<0.05), and was most severe when flooding occurred in this season. Based on the results of these experiments, the growth characteristics ofN. nucifera in relation to petiole elongation, biomass allocation, and flooding tolerance were discussed.     

Interrelationships of petiolar air canal architecture,water depth,and convective air flow in Nymphaea odorata (Nymphaeaceae)

• Premise of the study: Nymphaea odorata grows in water up to 2 m deep, producing fewer larger leaves in deeper water. This species has a convective flow system that moves gases from younger leaves through submerged parts to older leaves, aerating submerged parts. Petiolar air canals are the convective flow pathways. This study describes the structure of these canals, how this structure varies with water depth, and models how convective flow varies with depth. • Methods: Nymphaea odorata plants were grown at water depths from 30 to 90 cm. Lamina area, petiolar cross-sectional area, and number and area of air canals were measured. Field-collected leaves and leaves from juvenile plants were analyzed similarly. Using these data and data from the literature, we modeled how convective flow changes with water depth. • Key results: Petioles of N. odorata produce two central pairs of air canals; additional pairs are added peripherally, and succeeding pairs are smaller. The first three pairs account for 96% of air canal area. Air canals form 24% of petiolar cross-sectional area. Petiolar and air canal cross-sectional areas increase with water depth. Petiolar area scales with lamina area, but the slope of this relationship is lower in 90 cm water than at shallower depths. In our model, the rate of convective flow varied with depth and with the balance of influx to efflux leaves. • Conclusions: Air canals in N. odorata petioles increase in size and number in deeper water but at a decreasing amount in relation to lamina area. Convective flow also depends on the number of influx to efflux laminae.     

Thermal-Stable Proteins of Fruit of Long-Living Sacred Lotus Nelumbo nucifera Gaertn var. China Antique

Single-seeded fruit of the sacred lotus Nelumbo nucifera Gaertn var. China Antique from NE China have been shown to remain viable for as long as ~1,300 years, determined by direct radiocarbon-dating, and to have a germination rate of 84 %. The pericarp, a fruit tissue that encloses the single seeds of Nelumbo, is one of the major factors contributing to fruit longevity. Proteins that are heat stable and have a protective function are equally important to such centuries-long seed viability. We document proteins of Nelumbo fruit that are able to withstand heating, 32 % of which remained soluble in the 110 °C-treated embryo axis of a 549-year-old fruit and 76 % retained fluidity in its cotyledons. The genome of Nelumbo has recently been published and annotated. The amino-acid sequences of 11 “thermal proteins” (soluble at 100 °C) of modern Nelumbo embryo axes and cotyledons, identified by mass spectrometry, Western blot and bioassay, are assembled and aligned with those of an archaeal hyperthermophile Methancaldococcus jannaschii (“Mj,” an anaerobic methanogen having a growth optimum of 85 °C) and with those of five mesophile angiosperms. These thermal proteins have roles in protection and repair under stress. More than half (55 %) of the durable Nelumbo thermal proteins are present in the archaean Mj, indicating their ancient history. One Nelumbo protein-repair enzyme exhibits activity at 100 °C, having a heat-tolerance higher than the comparable enzyme of Arabidopsis. A list of 30 sequenced but unassembled thermal proteins of Nelumbo is appended.     

Rhizome transition to storage organ is under phytochrome control in lotus <Emphasis Type="Italic">(Nelumbo nucifera)</Emphasis>

We examined photoperiodic response of lotus (Nelumbo nucifera) rhizome morphogenesis (its transition to a storage organ) by using seed-derived plants. Rhizome enlargement (increase in girth) was brought about under 8, 10 and 12 h photoperiods, whereas the rhizomes elongated under 13 and 14 h photoperiods. Rhizomes elongated under 14 h light regimes supplied as 8 h of natural light plus 6 h supplemental hours of white, yellow or red light, but similar treatments with supplemental blue, green or far red light, caused enlargement in girth of the rhizomes. A 2 h interruption of the night with white, yellow or red light, in plants entrained to 8 h photoperiod brought rhizome elongation, whereas 2 h-blue, green or far red light night breaks still resulted in rhizome increase in girth. The inhibitory effect of a red (R) light night break on rhizome increase in girth was reversed by a far-red (FR) light given immediately afterwards. Irradiation with R/FR/R inhibited the rhizome increase in girth. FR light irradiation following R/FR/R irradiation cancelled the effect of the last R light irradiation. It was demonstrated that the critical photoperiod for rhizome transition to storage organ is between 12 and 13 h photoperiod. It was also evident that the optimal light quality range for interruption of dark period (night break) is between yellow and red light and that a R/FR reversible reaction is observed. From these results, we propose that phytochrome plays an important role in photoperiodic response of rhizome increase in girth in lotus. This is the first report on phytochrome-dependent morphogenesis of storage organs in rhizomous plants.     

Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract

Introduction – Betulinic acid (pentacyclic triterpenoid) is an important marker component present in Nelumbo nucifera Gaertn. rhizome. N. nucifera rhizome has several medicinal uses including hypoglycaemic, antidiarrhoeal, antimicrobial, diuretic, antipyretic, psychopharmacological activities. Objective – To establish a simple, sensitive, reliable, rapid and validated high‐performance thin‐layer chromatography method for estimation of betulinic acid in hydro‐alcoholic extract of N. nucifera Gaertn. rhizome. Materials and methods – The separation was carried out on a thin‐layer chromatography aluminium plate pre‐coated with silica gel 60F₂₅₄, eluted with chloroform, methanol and formic acid (49 : 1 : 1 v/v). Post chromatographic derivatisation was done with anisaldehyde–sulphuric acid reagent and densitometric scanning was performed using a Camag TLC scanner III, at 420 nm. Results – The system was found to produce a compact spot for betulinic acid (R_f = 0.30). A good linear precision relationship between the concentrations (2–10 µg) and peak areas were obtained with the correlation coefficient (r) of 0.99698. The limit of detection and limit of quantification of betulinic acid were detected to be 0.4 and 2.30 µg per spot. The percentage of recovery was found to be 98.36%. The percentage relative standard deviations of intra‐day and inter‐day precisions were 0.82–0.394 and 0.85–0.341, respectively. Conclusion – This validated HPTLC method provides a new and powerful approach to estimate betulinic acid as phytomarker in the extract. Copyright © 2010 John Wiley & Sons, Ltd.     

The breakdown of macrophytes in a reservoir wetland

Weight losses from leaf laminae and petioles of Nelumbo lutea (Wild.) Pers., and from leaves of Ludwigia leptocarpa (Nutt.) Hara. and Typha angustifolia L., were measured by exposing air-dried leaf material in nylon mesh bags at upper (exposed 25 days, inundated 40 days) and lower (inundated 154 days) wetland sites in a Texas reservoir. No significant differences (P < 0.05) were found between sites, so data were combined to yield estimates of plant litter breakdown for the entire wetland. Breakdown rates (in percentage of ash free dry weight lost per day) for the 4 litter types were: Nelumbo leaves — 0.0108 ± 0.0016; Ludwigia — 0.0050 ± 0.0007; Typha — 0.0047 ± 0.0006; Nelumbo — petioles 0.003 ± 0.0010. Times required for a 95% loss of litter, based on an exponential model, are 278 days, 600 days, 638 days and 909 days for these 4 litter types, respectively. These rates are comparable to those reported for emergent aquatic macrophytes in other lakes and wetlands. Colonization of decaying wetland plants was dominated by the gatherers, Chironominii (54%) and Caenis sp. (26%).     

Effects of water level fluctuation on the growth ofNelumbo nucifera Gaertn. in Lake Kasumigaura,Japan

Investigations were made of the growth ofNelumbo nucifera, an aquatic higher plant, in a natural stand in Lake Kasumigaura. A rise of 1.0 m in the water level after a typhoon in August 1986 caused a subsequent decrease in biomass ofN. nucifera from the maximum of 291 g d.w. m⁻² in July to a minimum of 75 g d.w. m⁻². The biomass recovered thereafter in shallower regions. The underground biomass in October tended to increase toward the shore. The total leaf area index (LAI) is the sum of LAI of floating leaves and emergent leaves. The maximum total LAI was 1.3 and 2.8 m² m⁻² in 1986 and 1987, respectively. LAI of floating leaves did not exceed 1 m² m⁻². The elongation rates of the petiole of floating and emergent leaves just after unrolling were 2.6 and 3.4 cm day⁻¹, respectively. The sudden rise in water level (25 cm day⁻¹) after the typhoon in August 1986 caused drowning and subsequent decomposition of the mature leaves. Only the young leaves were able to elongate, allowing their laminae to reach the water surface. The fluctuation in water level, characterized by the amplitude and duration of flooding and the time of flooding in the life cycle, is an important factor determining the growth and survival ofN. nucifera in Lake Kasumigaura.     

Development and characterization of simple sequence repeat (SSR) markers in the water lotus (Nelumbo nucifera)

Simple sequence repeat (SSR) markers were developed in the water lotus (Nelumbo nucifera Gaertn.) from an SSR-enriched genomic library. Of the SSR markers tested, 11 primer pairs produced clearly distinguishable DNA banding patterns. Forty-three alleles were detected with the 11 markers. The allele number per locus ranged from 2 to 5 with an average of 3.9. Polymorphism values ranged from 0.11 to 0.66 with an average of 0.51. These primers were also applicable to another Nelumbo species, Nelumbo lutea (Willd.) Pers. (American lotus) and hybrids between N. nucifera and N. lutea. These results indicate that the SSR markers developed in this study are informative and will be useful for genetic analysis in Nelumbo species.     

VENTILATION BY FLOATING LEAVES IN NUPHAR

The network of internal gas spaces in the yellow waterlily constitutes a pressurized flow-through system which forces oxygen to the rhizome buried in anaerobic sediment. This ventilation has been documented previously in a subspecies of Nuphar luteum with aerial leaves, and appeared to occur only during daylight when the leaves are warmed by the sun's radiation. In this study we have found the ventilation system operating in two different subspecies of N. luteum growing in Alaska and in Massachusetts. These plants have floating leaves so that during certain times of the year the leaves are warmed not only by the sun in daylight but also by lake water at night, allowing the ventilation to continue during darkness.     

Simultaneous measurement of water flow velocity and solute transport in xylem and phloem of adult plants of Ricinus communis over a daily time course by nuclear magnetic resonance spectrometry

A new method for simultaneously quantifying rates of flow in xylem and phloem using the FLASH imaging capabilities of nuclear magnetic resonance (NMR) spectrometry was applied in this study. The method has a time resolution of up to 4 min (for the xylem) and was used to measure the velocity of flows in phloem and xylem for periods of several hours to days. For the first time, diurnal time course measurements of flow velocities and apparent volume flows in phloem and xylem in the hypocotyl of 40‐d‐old Ricinus communis L were obtained. Additional data on gas exchange and the chemical composition of leaves, xylem and phloem sap were used to assess the role of leaves as sinks for xylem sap and sources for phloem. The velocity in the phloem (0·250 ± 0·004 mm s^?1) was constant over a full day and not notably affected by the light/dark cycle. Sucrose was loaded into the phloem and transported at night, owing to degradation of starch accumulated during the day. Concentrations of solutes in the phloem were generally less during the night than during the day but varied little within either the day or night. In contrast to the phloem, flow velocities in the xylem were about 1·6‐fold higher in the light (0·401 ± 0·004 mm s^?1) than in the dark (0·255 ± 0·003 mm s^?1) and volume flow varied commensurately. Larger delays were observed in changes to xylem flow velocity with variation in light than in gas exchange. The relative rates of solute transport during day and night were estimated on the basis of relative flow and solute concentrations in xylem and phloem. In general, changes in relative flow rates were compensated for by changes in solute concentration during the daily light/dark cycle. However, the major solutes (K⁺, NO₃^?) varied appreciably in relative concentrations. Hence the regulation of loading into transport systems seems to be more important to the overall process of solute transport than do changes in mass flow. Due to transport behaviour, the chemical composition of leaves varied during the day only with regard to starch and soluble carbohydrates.     

Centuries-Old Viable Fruit of Sacred Lotus Nelumbo nucifera Gaertn var. China Antique

During the Sino-Japanese conflict of the 1920s, Japanese botanist Ichiro Ohga was presented single-seeded fruit of Nelumbo nucifera var. China Antique collected by a local farmer from a dry lakebed in Northeast China (then, “Manchuria”). Ohga studied the fruit and published his findings. Years later, we tested the germination of Nelumbo fruit from the same locality. The oldest seed sprouted, having a germination time of ~3 days, was radiocarbon dated to be ~1300 years old. These cold- and drought-tolerant seeds exhibited shoot-before-root emergence and a primary green plumule capable of “dim-light” photosynthesis. Such traits and the notable long-term viability of the fruit spurred the interest of Ray Ming, University of Illinois that has now led to the sequencing of the Nelumbo genome. Analyses of this genome may provide insight into the biochemistry of Nelumbo on wax-biosynthesis genes, and application of aging-related thermostable proteins to the extension of seed-life and improvement of food quality of economic crops. Here, we review the history of these long-lived Nelumbo fruit, and their occurrence, discovery, collection, propagation, and methods of seedling care. The robust impermeable wax- and suberin-covered pericarp is a major factor contributing to their remarkable longevity. New findings are presented on the modern and 459- and 464-year-old pericarp anatomy, impermeability to water, and whole fruit and pericarp mechanical properties, and comparison of the mode of fruit weight-gain during imbibition and germination time relative to fruit maturity.     

Temperature dependence of seedling establishment of a perennial, Dioscorea tokoro

The temperature dependence of seed germination and seedling growth was analyzed in Dioscorea tokoro, an East Asian summer-green perennial. Seeds were able to germinate fully only at 11°–20°C. At around 17°–20°C the first leaf petiole of the seedling elongated and quickly set the first leaf blade at a position enabling photosynthesis. At temperatures higher than 20°C petiole elongation was retarded, and seedlings formed a rhizome and established as a perennial. The rhizome size increased with temperature up to 29°C. Thus, during growth immediately after germination, temperature appears to be a key factor in determining whether the plant establishes as a perennial or grows rapidly without rhizome thickening. Received: April 6, 2001 / Accepted: September 14, 2001