Leaf relative water content at 50% stomatal conductance measured by noninvasive NMR is linked to climate of origin in nine species of eucalypt.

Stomata are the gatekeepers of plant water use and must quickly respond to changes in plant water status to ensure plant survival under fluctuating environmental conditions. The mechanism for their closure is highly sensitive to disturbances in leaf water status, which makes isolating their response to declining water content difficult to characterise and to compare responses among species. Using a small-scale non-destructive nuclear magnetic resonance spectrometer as a leaf water content sensor, we measure the stomatal response to rapid induction of water deficit in the leaves of nine species of eucalypt from contrasting climates. We found a strong linear correlation between relative water content at 50% stomatal conductance (RWCgs50 ) and mean annual temperature at the climate of origin of each species. We also show evidence for stomata to maintain control over water loss well below turgor loss point in species adapted to warmer climates and secondary increases in stomatal conductance despite declining water content. We propose that RWCgs50 is a promising trait to guide future investigations comparing stomatal responses to water deficit. It may provide a useful phenotyping trait to delineate tolerance and adaption to hot temperatures and high leaf-to-air vapour pressure deficits.

In situ pod growth rate reveals contrasting diurnal sensitivity to water deficit in Phaseolus vulgaris.

The development of reproductive tissues determines plant fecundity and yield. Loading of resources into the developing reproductive tissue is thought to be under the co-limiting effects of source and sink strength. The dynamics of this co-limitation are unknown, largely due to an inability to measure the flux of resources into a developing sink. Here we use nuclear magnetic resonance (NMR) sensors to measure sink strength by quantifying rates of pod dry matter accumulation (pod loading) in Phaseolus vulgaris at 13-min intervals across the diel period. Rates of pod loading showed contrasting variation across light and dark periods during the onset of water deficit. In addition, rates of pod loading appeared decoupled from net photosynthetic rates when adjusted to the plant scale. Combined, these observations illustrate that the rate of pod development varies under water limitation and that continuous, non-invasive methodologies to measure sink strength provide insight into the governing processes that determine the development of reproductive tissues.

Proline-Mediated Drought Tolerance in the Barley (Hordeum vulgare L.) Isogenic Line Is Associated with Lateral Root Growth at the Early Seedling Stage.

A vigorous root system in barley promotes water uptake from the soil under water-limited conditions. We investigated three spring barley genotypes with varying water stress responses using rhizoboxes at the seedling stage. The genotypes comprised two elite German cultivars, Barke and Scarlett, and a near-isogenic line, NIL 143. The isogenic line harbors the wild allele pyrroline-5-carboxylate synthase1-P5cs1. Root growth in rhizoboxes under reduced water availability conditions caused a significant reduction in total root length, rooting depth, root maximum width, and root length density. On average, root growth was reduced by more than 20% due to water stress. Differences in organ proline concentrations were observed for all genotypes, with shoots grown under water stress exhibiting at least a 30% higher concentration than the roots. Drought induced higher leaf and root proline concentrations in NIL 143 compared with any of the other genotypes. Under reduced water availability conditions, NIL 143 showed less severe symptoms of drought, higher lateral root length, rooting depth, maximum root width, root length density, and convex hull area compared with Barke and Scarlett. Within the same comparison, under water stress, NIL 143 had a higher proportion of lateral roots (+30%), which were also placed at deeper substrate horizons. NIL 143 had a less negative plant water potential and higher relative leaf water content and stomatal conductance compared with the other genotypes under water stress. Under these conditions, this genotype also maintained an enhanced net photosynthetic rate and exhibited considerable fine root growth (diameter class 0.05-0.35 mm). These results show that water stress induces increased shoot and root proline accumulation in the NIL 143 barley genotype at the seedling stage and that this effect is associated with increased lateral root growth.

Reduced spatial resolution MRI suffices to image and quantify drought induced embolism formation in trees.

BACKGROUND: Magnetic resonance imaging (MRI) is uniquely suited to non-invasively and continuously monitor embolism formation in trees. Depending on the MRI method used, quantitative parameter maps of water content and MRI signal relaxation behavior can be generated. The ability to measure dynamic differences in water content and relaxation behavior can be used to detect xylem embolism formation, even if xylem conduits are too small to be spatially resolved. This is especially advantageous when using affordable small-scale low-field MRI scanners. The amount of signal that can be obtained from an object strongly depends on the strength of the magnetic field of the imager's magnet. Imaging at lower resolutions thus would allow to reduce the cost, size and weight of the MRI scanner and to shorten image acquisition times. RESULTS: We investigated how much spatial resolution can be sacrificed without losing the ability to monitor embolism formation in coniferous softwood (spruce, Picea abies) and diffuse porous beech (Fagus sylvatica). Saplings of both species were bench dehydrated, while they were continuously imaged at stepwise decreasing spatial resolutions. Imaging was done by means of a small-scale MRI device, utilizing image matrix sizes of 128 × 128, 64 × 64 and 32 × 32 pixels at a constant FOV of 19 and 23 mm, respectively. While images at the lowest resolutions (pixel sizes 0.59 × 0.59 mm and 0.72 × 0.72 mm) were no longer sufficient to resolve finer details of the stem anatomy, they did permit an approximate localization of embolism formation and the generation of accurate vulnerability curves. CONCLUSIONS: When using MRI, spatial resolution can be sacrificed without losing the ability to visualize and quantify embolism formation. Imaging at lower spatial resolution to monitor embolism formation has two advantages. Firstly, the acquisition time per image can be reduced dramatically. This enables continuous imaging at high time resolution, which may be beneficial to monitor rapid dynamics of embolism formation. Secondly, if the requirements for spatial resolution are relaxed, much simpler MRI devices can be used. This has the potential to make non-invasive MR imaging of embolism formation much more affordable and more widely available.

A Wild Allele of Pyrroline-5-Carboxylate Synthase1 Leads to Proline Accumulation in Spikes and Leaves of Barley Contributing to Improved Performance Under Reduced Water Availability.

Water stress (WS) during spike development strongly affects final grain yield and grain quality in cereals. Proline, an osmoprotectant amino-acid, may contribute to alleviating the effects of cell and tissue dehydration. We studied five spring barley genotypes contrasting in their drought response, including two introgression lines, S42IL-143 and S42IL-141, harboring a Pyrroline-5-carboxylate synthase1- P5cs1 allele originating from the wild barley accession ISR42-8. We tested the hypothesis that barley genotypes harboring a wild allele at P5cs1 locus are comparatively more drought-tolerant at the reproductive stage by inducing proline accumulation in their immature spikes. At the booting stage, we subjected plants to well-watered and WS treatments until physiological maturity. Several morpho-physiological traits had significant genotype by treatment interaction and reduction under WS. Varying levels of genotypic proline accumulation and differences in WS tolerance were observed. Spike proline accumulation was higher than leaf proline accumulation for all genotypes under WS. Also, introgression lines carrying a wild allele at P5cs1 locus had a markedly higher spike and leaf proline content compared with the other genotypes. These introgression lines showed milder drought symptoms compared with elite genotypes, remained photosynthetically active under WS, and maintained their intrinsic water use efficiency. These combined responses contributed to the achievement of higher final seed productivity. Magnetic resonance imaging (MRI) of whole spikes at the soft dough stage showed an increase in seed abortion among the elite genotypes compared with the introgression lines 15 days after WS treatment. Our results suggest that proline accumulation at the reproductive stage contributes to the maintenance of grain formation under water shortage.

Low field, time domain NMR in the agriculture and agrifood sectors: An overview of applications in plants, foods and biofuels.

In this contribution, a selective overview of low field, time-domain NMR (TD-NMR) applications in the agriculture and agrifood sectors is presented. The first applications of commercial TD-NMR instruments were in food and agriculture domains. Many of these earlier methods have now been recognized as standard methods by several international agencies. Since 2000, several new applications have been developed, using state of the art instruments, new pulse sequences and new signal processing methods. TD-NMR is expected, in the coming years, to become even more important in quality control of fresh food and agricultural products, as well as for a wide range of food-processed products. TD-NMR systems provide excellent means to collect data relevant for use in the agricultural environment and the bioenergy industry. Data and information collected by TD-NMR systems thus may support decision makers in business and public organizations.

A Mobile NMR Sensor and Relaxometric Method to Non-destructively Monitor Water and Dry Matter Content in Plants.

Water content (WC) and dry matter content (DMC) are some of the most basic parameters to describe plant growth and yield, but are exceptionally difficult to measure non-invasively. Nuclear Magnetic Resonance (NMR) relaxometry may fill this methodological gap. It allows non-invasive detection of protons in liquids and solids, and on the basis of these measures, can be used to quantify liquid and dry matter contents of seeds and plants. Unfortunately, most existing NMR relaxometers are large, unwieldy and not suitable to measure intact plants or to be used under field conditions. In addition, currently the appropriate NMR relaxometric methods are poorly suited for non-expert use. We here present a novel approach to overcome these drawbacks. We demonstrate that a basic NMR relaxometer with the capability to accept intact plants, in combination with straightforward NMR and data processing methods, can be used as an NMR plant sensor to continuously, quantitatively and non-invasively monitor changes in WC and DMC. This can be done in vivo, in situ, and with high temporal resolution. The method is validated by showing that measured liquid and solid proton densities accurately reflect WC and DMC of reference samples. The NMR plant sensor is demonstrated in an experimental context by monitoring WC of rice leaves under osmotic stress, and by measuring the dynamics of water and dry matter accumulation during seed filling in a developing wheat ear. It is further demonstrated how the method can be used to estimate leaf water potential on the basis of changes in leaf water content.

An integrated magnetic resonance plant imager for mobile use in greenhouse and field.

In this contribution we demonstrate a mobile, integrated MR plant imager that can be handled by one single person and used in the field. Key to the construction of it was a small and lightweight gradient amplifier, specifically tailored to our combination of magnet, gradient coils and the requirements of the desired pulse sequences. To allow imaging of branches and stems, an open C-shaped permanent magnet was used. In the design of the magnet, pole gap width, low weight and robustness were prioritized over homogeneity and field strength. To overcome the adverse effects of short T2*, multi-spin echo imaging was employed, using short echo times and high spectral widths. To achieve microscopic resolution under these constraints requires fast switching field gradients, driven by strong and fast gradient amplifiers. While small-scale spectrometers and RF amplifiers are readily available, appropriate small-scale gradient amplifiers or designs thereof currently are not. We thus constructed a small, 3-channel gradient amplifier on the basis of a conventional current-controlled AB amplifier design, using cheap and well-known parts. The finished device weighs 5 kg and is capable of delivering 40 A gradient pulses of >6 ms in duration. With all components built onto an aluminum hand trolley, the imaging setup weighs 45 kg and is small enough to fit into a car. We demonstrate the mobility and utility of the device imaging quantitative water content and T2, first of an apple tree in an orchard; second, of a beech tree during spring leaf flushing in a greenhouse. The latter experiment ran for a continuous period of 62 days, acquiring more than 6000 images.

A small-scale MRI scanner and complementary imaging method to visualize and quantify xylem embolism formation.

Magnetic resonance imaging (MRI) is a useful tool to image xylem embolism formation in plants. MRI scanners configured to accept intact plants are rare and expensive. Here, we investigate if affordable small-scale, custom-built low-field MRI scanners would suffice for the purpose. A small-scale, C-shaped permanent magnet was paired with open, plane parallel imaging gradients. The setup was small enough to fit between leaves or branches and offered open access for plant stems of arbitrary length. To counter the two main drawbacks of the system, low signal to noise and reduced magnetic field homogeneity, a multi-spin echo (MSE) pulse sequence was implemented, allowing efficient signal acquisition and quantitative imaging of water content and T2 signal relaxation. The system was tested visualizing embolism formation in Fagus sylvatica during bench dehydration. High-quality images of water content and T2 were readily obtained, which could be utilized to detect the cavitation of vessels smaller than could be spatially resolved. A multiplication of both map types yielded images in which filled xylem appeared with even greater contrast. T2 imaging with small-scale MRI devices allows straightforward visualization of the spatial and temporal dynamics of embolism formation and the derivation of vulnerability curves.

In vivo pressure gradient heterogeneity increases flow contribution of small diameter vessels in grapevine.

Leaves lose approximately 400 H2O molecules for every 1 CO2 gained during photosynthesis. Most long-distance water transport in plants, or xylem sap flow, serves to replace this water to prevent desiccation. Theory predicts that the largest vessels contribute disproportionately to overall sap flow because flow in pipe-like systems scales with the fourth power of radius. Here, we confront these theoretical flow predictions for a vessel network reconstructed from X-ray µCT imagery with in vivo flow MRI observations from the same sample of a first-year grapevine stem. Theoretical flow rate predictions based on vessel diameters are not supported. The heterogeneity of the vessel network gives rise to transverse pressure gradients that redirect flow from wide to narrow vessels, reducing the contribution of wide vessels to sap flow by 15% of the total. Our results call for an update of the current working model of the xylem to account for its heterogeneity.

Magnetic resonance imaging suggests functional role of previous year vessels and fibres in ring-porous sap flow resumption.

Reactivation of axial water flow in ring-porous species is a complex process related to stem water content and developmental stage of both earlywood-vessel and leaf formation. Yet empirical evidence with non-destructive methods on the dynamics of water flow resumption in relation to these mechanisms is lacking. Here we combined in vivo magnetic resonance imaging and wood-anatomical observations to monitor the dynamic changes in stem water content and flow during spring reactivation in 4-year-old pedunculate oaks (Quercus robur L.) saplings. We found that previous year latewood vessels and current year developing earlywood vessels form a functional unit for water flow during growth resumption. During spring reactivation, water flow shifted from latewood towards the new earlywood, paralleling the formation of earlywood vessels and leaves. At leaves' full expansion, volumetric water content of previous rings drastically decreased due to the near-absence of water in fibre tissue. We conclude (i) that in ring-porous oak, latewood vessels play an important hydraulic role for bridging the transition between old and new water-conducting vessels and (ii) that fibre and parenchyma provides a place for water storage.

Heat girdling does not affect xylem integrity: an in vivo magnetic resonance imaging study in the tomato peduncle.

Heat girdling is a method to estimate the relative contribution of phloem vs xylem water flow to fruit growth. The heat girdling process is assumed to destroy all living tissues, including the phloem, without affecting xylem conductivity. However, to date, the assumption that xylem is not affected by heat girdling remains unproven. In this study, we used in vivo magnetic resonance imaging (MRI) velocimetry to test if heat girdling can cause xylem vessels to embolize or affect xylem water flow characteristics in the peduncle of tomato (Solanum lycopersicum cv Dirk). Anatomical and MRI data indicated that, at the site of girdling, all living tissues were disrupted, but that the functionality of the xylem remained unchanged. MRI velocimetry showed that the volume flow through the secondary xylem was not impeded by heat girdling in either the short or the long term (up to 91 h after girdling). This study provides support for the hypothesis that in the tomato peduncle the integrity and functionality of the xylem remain unaffected by heat girdling. It therefore confirms the validity of the heat girdling technique as a means to estimate relative contributions of xylem and phloem water flow to fruit growth.

Stomatal Closure, Basal Leaf Embolism, and Shedding Protect the Hydraulic Integrity of Grape Stems.

The time scale of stomatal closure and xylem cavitation during plant dehydration, as well as the fate of embolized organs, are under debate, largely due to methodological limitations in the evaluation of embolism. While some argue that complete stomatal closure precedes the occurrence of embolism, others believe that the two are contemporaneous processes that are accompanied by daily xylem refilling. Here, we utilize an optical light transmission method to continuously monitor xylem cavitation in leaves of dehydrating grapevine (Vitis vinifera) in concert with stomatal conductance and stem and petiole hydraulic measurements. Magnetic resonance imaging was used to continuously monitor xylem cavitation and flow rates in the stem of an intact vine during 10 d of dehydration. The results showed that complete stomatal closure preceded the appearance of embolism in the leaves and the stem by several days. Basal leaves were more vulnerable to xylem embolism than apical leaves and, once embolized, were shed, thereby preventing further water loss and protecting the hydraulic integrity of younger leaves and the stem. As a result, embolism in the stem was minimal even when drought led to complete leaf shedding. These findings suggest that grapevine avoids xylem embolism rather than tolerates it.

Rhizophoraceae Mangrove Saplings Use Hypocotyl and Leaf Water Storage Capacity to Cope with Soil Water Salinity Changes.

Some of the most striking features of Rhizophoraceae mangrove saplings are their voluminous cylinder-shaped hypocotyls and thickened leaves. The hypocotyls are known to serve as floats during seed dispersal (hydrochory) and store nutrients that allow the seedling to root and settle. In this study we investigate to what degree the hypocotyls and leaves can serve as water reservoirs once seedlings have settled, helping the plant to buffer the rapid water potential changes that are typical for the mangrove environment. We exposed saplings of two Rhizophoraceae species to three levels of salinity (15, 30, and 0-5‰, in that sequence) while non-invasively monitoring changes in hypocotyl and leaf water content by means of mobile NMR sensors. As a proxy for water content, changes in hypocotyl diameter and leaf thickness were monitored by means of dendrometers. Hypocotyl diameter variations were also monitored in the field on a Rhizophora species. The saplings were able to buffer rapid rhizosphere salinity changes using water stored in hypocotyls and leaves, but the largest water storage capacity was found in the leaves. We conclude that in Rhizophora and Bruguiera the hypocotyl offers the bulk of water buffering capacity during the dispersal phase and directly after settlement when only few leaves are present. As saplings develop more leaves, the significance of the leaves as a water storage organ becomes larger than that of the hypocotyl.

Grapevine petioles are more sensitive to drought induced embolism than stems: evidence from in vivo MRI and microcomputed tomography observations of hydraulic vulnerability segmentation.

The 'hydraulic vulnerability segmentation' hypothesis predicts that expendable distal organs are more susceptible to water stress-induced embolism than the main stem of the plant. In the current work, we present the first in vivo visualization of this phenomenon. In two separate experiments, using magnetic resonance imaging or synchrotron-based microcomputed tomography, grapevines (Vitis vinifera) were dehydrated while simultaneously scanning the main stems and petioles for the occurrence of emboli at different xylem pressures (Ψx ). Magnetic resonance imaging revealed that 50% of the conductive xylem area of the petioles was embolized at a Ψx of -1.54 MPa, whereas the stems did not reach similar losses until -1.9 MPa. Microcomputed tomography confirmed these findings, showing that approximately half the vessels in the petioles were embolized at a Ψx of -1.6 MPa, whereas only few were embolized in the stems. Petioles were shown to be more resistant to water stress-induced embolism than previously measured with invasive hydraulic methods. The results provide the first direct evidence for the hydraulic vulnerability segmentation hypothesis and highlight its importance in grapevine responses to severe water stress. Additionally, these data suggest that air entry through the petiole into the stem is unlikely in grapevines during drought.

A portable NMR sensor to measure dynamic changes in the amount of water in living stems or fruit and its potential to measure sap flow.

Nuclear magnetic resonance (NMR) and NMR imaging (magnetic resonance imaging) offer the possibility to quantitatively and non-invasively measure the presence and movement of water. Unfortunately, traditional NMR hardware is expensive, poorly suited for plants, and because of its bulk and complexity, not suitable for use in the field. But does it need to be? We here explore how novel, small-scale portable NMR devices can be used as a flow sensor to directly measure xylem sap flow in a poplar tree (Populus nigra L.), or in a dendrometer-like fashion to measure dynamic changes in the absolute water content of fruit or stems. For the latter purpose we monitored the diurnal pattern of growth, expansion and shrinkage in a model fruit (bean pod, Phaseolus vulgaris L.) and in the stem of an oak tree (Quercus robur L.). We compared changes in absolute stem water content, as measured by the NMR sensor, against stem diameter variations as measured by a set of conventional point dendrometers, to test how well the sensitivities of the two methods compare and to investigate how well diurnal changes in trunk absolute water content correlate with the concomitant diurnal variations in stem diameter. Our results confirm the existence of a strong correlation between the two parameters, but also suggest that dynamic changes in oak stem water content could be larger than is apparent on the basis of the stem diameter variation alone.

Phloem flow and sugar transport in Ricinus communis†L. is inhibited under anoxic conditions of shoot or roots.

Anoxic conditions should hamper the transport of sugar in the phloem, as this is an active process. The canopy is a carbohydrate source and the roots are carbohydrate sinks. By fumigating the shoot with N2 or flooding the rhizosphere, anoxic conditions in the source or sink, respectively, were induced. Volume flow, velocity, conducting area and stationary water of the phloem were assessed by non-invasive magnetic resonance imaging (MRI) flowmetry. Carbohydrates and δ(13) C in leaves, roots and phloem saps were determined. Following flooding, volume flow and conducting area of the phloem declined and sugar concentrations in leaves and in phloem saps slightly increased. Oligosaccharides appeared in phloem saps and after 3 d, carbon transport was reduced to 77%. Additionally, the xylem flow declined and showed finally no daily rhythm. Anoxia of the shoot resulted within minutes in a reduction of volume flow, conductive area and sucrose in the phloem sap decreased. Sugar transport dropped to below 40% by the end of the N2 treatment. However, volume flow and phloem sap sugar tended to recover during the N2 treatment. Both anoxia treatments hampered sugar transport. The flow velocity remained about constant, although phloem sap sugar concentration changed during treatments. Apparently, stored starch was remobilized under anoxia.

A portable Halbach magnet that can be opened and closed without force: the NMR-CUFF.

Portable equipment for nuclear magnetic resonance (NMR) is becoming increasingly attractive for use in a variety of applications. One of the main scientific challenges in making NMR portable is the design of light-weight magnets that possess a strong and homogeneous field. Existing NMR magnets can provide such magnetic fields, but only for small samples or in small regions, or are rather heavy. Here we show a simple yet elegant concept for a Halbach-type permanent magnet ring, which can be opened and closed with minimal mechanical force. An analytical solution for an ideal Halbach magnet shows that the magnetic forces cancel if the structure is opened at an angle of 35.3° relative to its poles. A first prototype weighed only 3.1 kg, and provided a flux density of 0.57 T with a homogeneity better than 200 ppm over a spherical volume of 5mm in diameter without shimming. The force needed to close it was found to be about 20 N. As a demonstration, intact plants were imaged and water (xylem) flow measured. Magnets of this type (NMR-CUFF = Cut-open, Uniform, Force Free) are ideal for portable use and are eminently suited to investigate small or slender objects that are part of a larger or immobile whole, such as branches on a tree, growing fruit on a plant, or non-metallic tubing in industrial installations. This new concept in permanent-magnet design enables the construction of openable, yet strong and homogeneous magnets, which aside from use in NMR or MRI could also be of interest for applications in accelerators, motors, or magnetic bearings.

Non-invasive approaches for phenotyping of enhanced performance traits in bean.

Plant phenotyping is an emerging discipline in plant biology. Quantitative measurements of functional and structural traits help to better understand gene-environment interactions and support breeding for improved resource use efficiency of important crops such as bean (Phaseolus vulgaris L.). Here we provide an overview of state-of-the-art phenotyping approaches addressing three aspects of resource use efficiency in plants: belowground roots, aboveground shoots and transport/allocation processes. We demonstrate the capacity of high-precision methods to measure plant function or structural traits non-invasively, stating examples wherever possible. Ideally, high-precision methods are complemented by fast and high-throughput technologies. High-throughput phenotyping can be applied in the laboratory using automated data acquisition, as well as in the field, where imaging spectroscopy opens a new path to understand plant function non-invasively. For example, we demonstrate how magnetic resonance imaging (MRI) can resolve root structure and separate root systems under resource competition, how automated fluorescence imaging (PAM fluorometry) in combination with automated shape detection allows for high-throughput screening of photosynthetic traits and how imaging spectrometers can be used to quantify pigment concentration, sun-induced fluorescence and potentially photosynthetic quantum yield. We propose that these phenotyping techniques, combined with mechanistic knowledge on plant structure-function relationships, will open new research directions in whole-plant ecophysiology and may assist breeding for varieties with enhanced resource use efficiency varieties.

Sieve tube geometry in relation to phloem flow.

Sieve elements are one of the least understood cell types in plants. Translocation velocities and volume flow to supply sinks with photoassimilates greatly depend on the geometry of the microfluidic sieve tube system and especially on the anatomy of sieve plates and sieve plate pores. Several models for phloem translocation have been developed, but appropriate data on the geometry of pores, plates, sieve elements, and flow parameters are lacking. We developed a method to clear cells from cytoplasmic constituents to image cell walls by scanning electron microscopy. This method allows high-resolution measurements of sieve element and sieve plate geometries. Sieve tube-specific conductivity and its reduction by callose deposition after injury was calculated for green bean (Phaseolus vulgaris), bamboo (Phyllostachys nuda), squash (Cucurbita maxima), castor bean (Ricinus communis), and tomato (Solanum lycopersicum). Phloem sap velocity measurements by magnetic resonance imaging velocimetry indicate that higher conductivity is not accompanied by a higher velocity. Studies on the temporal development of callose show that small sieve plate pores might be occluded by callose within minutes, but plants containing sieve tubes with large pores need additional mechanisms.