山内 大輔

To study plant organs, it is necessary to investigate the three-dimensional (3D) structures of plants. In recent years, non-destructive measurements through computed tomography (CT) have been used to understand the 3D structures of plants. In this study, we use the Chrysanthemum seticuspe capitulum inflorescence as an example and focus on contact points between the receptacles and florets within the 3D capitulum inflorescence bud structure to investigate the 3D arrangement of the florets on the receptacle. To determine the 3D order of the contact points, we constructed slice images from the CT volume data and detected the receptacles and florets in the image. However, because each CT sample comprises hundreds of slice images to be processed and each C. seticuspe capitulum inflorescence comprises several florets, manually detecting the receptacles and florets is labor-intensive. Therefore, we propose an automatic contact point detection method based on CT slice images using image recognition techniques. The proposed method improves the accuracy of contact point detection using prior knowledge that contact points exist only around the receptacle. In addition, the integration of the detection results enables the estimation of the 3D position of the contact points. According to the experimental results, we confirmed that the proposed method can detect contacts on slice images with high accuracy and estimate their 3D positions through clustering. Additionally, the sample-independent experiments showed that the proposed method achieved the same detection accuracy as sample-dependent experiments.

Studies visualizing plant tissues and organs in three-dimension (3D) using micro-computed tomography (CT) published since approximately 2015 are reviewed. In this period, the number of publications in the field of plant sciences dealing with micro-CT has increased along with the development of high-performance lab-based micro-CT systems as well as the continuous development of cutting-edge technologies at synchrotron radiation facilities. The widespread use of commercially available lab-based micro-CT systems enabling phase-contrast imaging technique, which is suitable for the visualization of biological specimens composed of light elements, appears to have facilitated these studies. Unique features of the plant body, which are particularly utilized for the imaging of plant organs and tissues by micro-CT, are having functional air spaces and specialized cell walls, such as lignified ones. In this review, we briefly describe the basis of micro-CT technology first and then get down into details of its application in 3D visualization in plant sciences, which are categorized as follows: imaging of various organs, caryopses, seeds, other organs (reproductive organs, leaves, stems and petioles), various tissues (leaf venations, xylems, air-filled tissues, cell boundaries, cell walls), embolisms and root systems, hoping that wide users of microscopes and other imaging technologies will be interested also in micro-CT and obtain some hints for a deeper understanding of the structure of plant tissues and organs in 3D. Majority of the current morphological studies using micro-CT still appear to be at a qualitative level. Development of methodology for accurate 3D segmentation is needed for the transition of the studies from a qualitative level to a quantitative level in the future.

Land plants have two types of shoot-supporting systems, root system and rhizoid system, in vascular plants and bryophytes. However, since the evolutionary origin of the systems are different, how much they exploit common systems or distinct systems to architect their structures are largely unknown. To understand the regulatory mechanism how bryophytes architect rhizoid system responding to environmental factors, we have developed the methodology to visualize and quantitatively analyze the rhizoid system of the moss, Physcomitrium patens in 3D. The rhizoids having the diameter of 21.3 µm on the average were visualized by refraction-contrast X-ray micro-CT using coherent X-ray optics available at synchrotron radiation facility SPring-8. Three types of shape (ring-shape, line, black circle) observed in tomographic slices of specimens embedded in paraffin were confirmed to be the rhizoids by optical and electron microscopy. Comprehensive automatic segmentation of the rhizoids which appeared in different three form types in tomograms was tested by a method using Canny edge detector or machine learning. Accuracy of output images was evaluated by comparing with the manually-segmented ground truth images using measures such as F1 score and IoU, revealing that the automatic segmentation using the machine learning was more effective than that using Canny edge detector. Thus, machine learning-based skeletonized 3D model revealed quite dense distribution of rhizoids. We successfully visualized the moss rhizoid system in 3D for the first time. High resolution refraction-contrast X-ray micro-CT using coherent X-ray optics successfully visualized 3D architecture of rhizoid system of moss, Physcomitrium patens, which is composed of cellular filaments having the diameter of 21.3 µm on the average, for the first time by using machine learning for segmentation.

Plant roots change their morphological traits in order to adapt themselves to different environmental conditions, resulting in alteration of the root system architecture. To understand this mechanism, it is essential to visualize morphology of the entire root system. To reveal effects of long-term alteration of gravity environment on root system development, we have performed an experiment in the International Space Station using Arabidopsis plants and obtained dried root systems grown in rockwool slabs. X-ray computed tomography (CT) technique using industrial X-ray scanners has been introduced to visualize root system architecture of crop species grown in soil in 3D non-invasively. In the case of the present study, however, root system of Arabidopsis is composed of finer roots compared with typical crop plants and rockwool is also composed of fibers having similar dimension to that of the roots. A higher spatial resolution imaging method is required for distinguishing roots from rockwool. Therefore, in the present study, we tested refraction-contrast X-ray micro-CT using coherent X-ray optics available at the beamline of the synchrotron radiation facility SPring-8 for bio-imaging. We have found that wide field of view but with low resolution obtained at the experimental Hutch 3 of this beamline provided an overview map of the root systems, while narrow field of view but with high resolution obtained at the experimental Hutch 1 provided extended architecture of the secondary roots, by clear distinction between roots and individual rockwool fibers, resulting in successful tracing of these roots from their basal regions.

© The Author(s) 2018. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. X-ray micro-CT is one of the most useful techniques to examine 3D cellular architecture inside dry seeds. However, the examination of imbibed seeds is difficult because immersion in water causes a decline in the image quality. Here, we examined the use of ionic liquids for specimen preparation of chemically fixed imbibed seeds of Arabidopsis. We found that treatment with high concentrations of ionic liquids after osmium tetroxide fixation helped not only to prevent the structural damage caused by seed shrinkage, but also to preserve the image quality. Under these conditions, the cellular architecture of seeds was also well maintained.

<p>Insertion of a sheet of cover glass in the lid of a glass-based dish is a useful technique for observation by differential interference contrast microscopy. However, microscopic observations of algal cultures using this glass-based dish are often interfered with by dew condensation on the glass surface of the lid when the culture is incubated for a long time. To prevent dew development on the lid surface during incubation, we employed a glass-based dish whose inner surface of the glass-lid is coated with 2-methacryloyloxyethyl phosphorylcholine polymer. Using this modified dish, we could observe thalli development of Coleochaete scutata successively for four weeks without dew condensation on the lid.</p>

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 mu m. Treatments with cytoskeletal inhibitors for 15min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.

The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1.

A clinical X-ray CT scanner is an instrument that is used to observe our bodies non-invasively, using the distribution of the linear absorption coefficient. However, X-ray irradiation energy and its spatial resolution are not suitable for the visualization of small objects. Further development and optimization of measurement techniques have led to the development of an X-ray micro-CT scanner for the visualization of small specimens with diameters of a few mm. A technique called refraction-contrast imaging was developed to facilitate the visualization of soft tissue structures, and has become more widely used over time. In this review, the history of X-ray imaging is briefly recounted, and the applicability of these techniques to plant biological research is summarized. In addition, we introduce the authors works related to these techniques.

The cotyledon of legume seeds is a storage organ that provides nutrients for seed germination and seedling growth. The spatial and temporal control of the degradation processes within cotyledons has not been elucidated. Calcium oxalate (CaOx) crystals, a common calcium deposit in plants, have often been reported to be present in legume seeds. In this study, micro-computed tomography (micro-CT) was employed at the SPring-8 facility to examine the three-dimensional distribution of crystals inside cotyledons during seed maturation and germination of Lotus miyakojimae (previously Lotus japonicus accession Miyakojima MG-20). Using this technique, we could detect the outline of the embryo, void spaces in seeds and the cotyledon venation pattern. We found several sites that strongly inhibited X-ray transmission within the cotyledons. Light and polarizing microscopy confirmed that these areas corresponded to CaOx crystals. Three-dimensional observations of dry seeds indicated that the CaOx crystals in the L. miyakojimae cotyledons were distributed along lateral veins however, their distribution was limited to the abaxial side of the procambium. The CaOx crystals appeared at stage II (seed-filling stage) of seed development, and their number increased in dry seeds. The number of crystals in cotyledons was high during germination, suggesting that CaOx crystals are not degraded for their calcium supply. Evidence for the conservation of CaOx crystals in cotyledons during the L. miyakojimae germination process was also supported by the biochemical measurement of oxalic acid levels. © The Author 2012. Published by Oxford University Press [on behalf of Japanese Society of Microscopy]. All rights reserved.

The cotyledon of legume seeds is a storage organ that provides nutrients for seed germination and seedling growth. The spatial and temporal control of the degradation processes within cotyledons has not been elucidated. Calcium oxalate (CaOx) crystals, a common calcium deposit in plants, have often been reported to be present in legume seeds. In this study, micro-computed tomography (micro-CT) was employed at the SPring-8 facility to examine the three-dimensional distribution of crystals inside cotyledons during seed maturation and germination of Lotus miyakojimae (previously Lotus japonicus accession Miyakojima MG-20). Using this technique, we could detect the outline of the embryo, void spaces in seeds and the cotyledon venation pattern. We found several sites that strongly inhibited X-ray transmission within the cotyledons. Light and polarizing microscopy confirmed that these areas corresponded to CaOx crystals. Three-dimensional observations of dry seeds indicated that the CaOx crystals in the L. miyakojimae cotyledons were distributed along lateral veins; however, their distribution was limited to the abaxial side of the procambium. The CaOx crystals appeared at stage II (seed-filling stage) of seed development, and their number increased in dry seeds. The number of crystals in cotyledons was high during germination, suggesting that CaOx crystals are not degraded for their calcium supply. Evidence for the conservation of CaOx crystals in cotyledons during the L. miyakojimae germination process was also supported by the biochemical measurement of oxalic acid levels.

Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state C-13 nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.

The effect of environmental factors on the regulation of aerenchyma formation in rice roots has been discussed for a long time, because aerenchyma is constitutively formed under aerated conditions. To elucidate this problem, a unique method has been developed that enables sensitive detection of differences in the development of aerenchyma under two different environmental conditions. The method is tested to determine whether aerenchyma development in rice roots is affected by osmotic stress.To examine aerenchyma formation both with and without mannitol treatment in the same root, germinating rice (Oryza sativa) caryopses were sandwiched between two agar slabs, one of which contained 270 mm of mannitol. The roots were grown touching both slabs and were thereby exposed unilaterally to osmotic stress. As a non-invasive approach, refraction contrast X-ray computed tomography (CT) using a third-generation synchrotron facility, SPring-8 (Super photon ring 8 GeV, Japan Synchrotron Radiation Research Institute), was used to visualize the three-dimensional (3-D) intact structure of aerenchyma and its formation in situ in rice roots. The effects of unilateral mannitol treatment on the development of aerenchyma were quantitatively examined using conventional light microscopy.Structural continuity of aerenchyma was clearly visualized in 3-D in the primary root of rice and in situ using X-ray CT. Light microscopy and X-ray CT showed that the development of aerenchyma was promoted on the mannitol-treated side of the root. Detailed light microscopic analysis of cross-sections cut along the root axis from the tip to the basal region demonstrated that aerenchyma developed significantly closer to the root tip on the mannitol-treated side of the root.Continuity of the aerenchyma along the rice root axis was morphologically demonstrated using X-ray CT. By using this osandwich' method it was shown that mannitol promoted aerenchyma formation in the primary roots of rice.

How biological form is determined is one of the important questions in developmental biology. Physical forces are thought to be the primary determinants of the biological forms, and several theories for this were proposed nearly a century ago. To evaluate how physical forces can influence biological forms, precise determination of cell and tissue shapes and their geometries is necessary. Computed tomography (CT) is useful for visualizing three-dimensional structures without destroying a sample. Because recent progress in micro-CT has enabled visualizing cells and tissues at the sub-micron level, we investigated if we could extract cell and tissue outlines of seeds using refraction contrast X-ray CT available at the SPring-8 synchrotron radiation facility. We used Arabidopsis seeds because Arabidopsis is a well-known model plant and its seed size is small enough to obtain whole images using the X-ray CT experimental system. We could trace the outlines of tissues in dry seeds using beamline BL20B2 (10 keV, 2.4 mu m.pixel(-1)). Although we could also detect the outlines of some cell types, the image resolution was not adequate to extract whole cell edges. To detect the edges of cells in the epidermis and cortex, we obtained CT images using beamline BL20XU (8 keV, 0.5 mu m.pixel(-1)). With these CT images, we could extract the facets and edges of each cell and determine cell vertices. This method enabled us to compare the numbers of cell facets among various cell types. We could also describe cell geometry as a set of points that showed these cell vertices.

Gibberellins (GAs) are involved in the expression of cysteine proteinase genes in germinated cotyledons of common bean seeds. Because DELLA proteins are known to be transcriptional repressors mediating GA signaling, we isolated two cDNA clones encoding DELLA proteins (PvGAI1 and PvGAI2) from common bean seedlings to examine the mechanism of GA signaling involved in the expression of the proteinase genes. RT-PCR and RNA blot analyses indicated that the level of mRNA in germinated cotyledons was higher for PvGAI2 than for PvGAI1. We also found that transient expression of PvGAI2, but not that of PvGAI1, repressed the promoter activities of GA-inducible cysteine proteinase genes, EP-C1 and CP2, in germinated cotyledons. These findings suggest that PvGAI2 is mainly responsible for regulating the expression of proteinase genes in germinated cotyledons. Application of a GA-biosynthesis inhibitor, prohexadione calcium to common bean seeds had little effect on the RNA level of PvGAI2, although the inhibitor repressed genes for EP-C1 and CP2. Because it is known that GA induces degradation of DELLA proteins, our findings suggest that the level of GA, but not the mRNA expression of PvGAI2, regulates the protein level of PvGAI2 suppressing the proteinases genes in germinated cotyledons.

The analysis of expressed sequences from a diverse set of plant species has fueled the increase in understanding of the complex molecular mechanisms underlying plant growth regulation. While representative data sets can be found for the major branches of plant evolution, fern species data are lacking. To further the availability of genetic information in pteridophytes, a normalized cDNA library of Adiantum capillus-veneris was constructed front prothallia grown under white light. A total of 10,420 expressed sequence tags (ESTs) were obtained and clustering of these sequences resulted in 7,100 nonredundant clusters. Of these, 1.608 EST clusters were found to be similar to sequences of known function and 1,092 EST clusters showed similarity to sequences of unknown function. Given the usefulness of Adiantum for developmental studies, the sequence data represented in this report stand to make a significant contribution to the understanding of plant growth regulation, particularly for pteridophytes.

Bacillus brevis (Brevibacillus parabrevis) ATCC 8185 synthesizes two kinds of antibiotic peptides, cyclopeptide tyrocidine and linear gramicidin. The production of linear gramicidin can be induced by the standard method (using a skim milk medium for pre-culture and beef broth for the main culture) employed for the induction of tyrocidine. In this study, we tried to determine the optimal growth medium for B. brevis ATCC 8185 for synthesizing linear gramicidin. The yield of linear gramicidin produced by the standard method was 3.11 μg/ml. When beef broth was used both as the pre-medium and the main medium, the yield of the antibiotic was only 0.59 μg/ml. To confirm the influence of skim milk, the strain was grown in a 1% skim milk medium. As a result, the amount of linear gramicidin produced reached 20.3 μg/ml. These findings show the importance of skim milk in the production of linear gramicidin. In the skim milk medium, the cells produced an extracellular protease 2h before the linear gramicidin was expressed. The 1% skim milk medium pretreated by this protease did not allow the induction of linear gramicidin into the cells, and protease activity was not detected in the supernatant of the culture. When the cells were cultivated in a 1% egg albumin medium, protease activity from the supernatant of the culture was detected, but production of linear gramicidin was not observed. Therefore, a 1% casein medium was used for production of linear gramicidin. As a result, the yield of linear gramicidin produced in the medium reached 6.69 μg/ml. We concluded that a digested product of the extracellular protease from casein enhances linear gramicidin production.

Pod storage protein (PSP) accumulates in both developing pods and wounded leaves of common bean (Phaseolus vulgaris). Since jasmonic acid (JA) is known to be involved in wound responses, we examined hormonal regulation of PSP expression. Methyl jasmonate (MeJA)-induced expression of PSP was blocked by both salicylic acid (SA) and ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The PSP promoter fused to β-glucuronidase (GUS) coding region was introduced into Arabidoposis thaliana using Agrobacterium infection. No MeJA-inducible GUS expression was found in the transformants. The chimeric gene was then introduced into stems of common bean using particle bombardment. Exogenously applied MeJA enhanced GUS activity, and this was prevented by both SA and ACC. Deletion analysis of the promoter indicated that the region between positions-747 and-555 included cis-regulatory elements for JA induction and ethylene suppression, and that cis-elements for SA-suppression were located in the region downstream of position-86.

alpha-Amylase (EC 3.2.1.1) expression was found in calli of French bean (Phaseolus vulgaris L. cv Goldstar). We examined enzyme activity in the calli to investigate influence of gibberellin and sugars on enzyme expression. After subculture of the calli, alpha-amylase activity decreased, and then increased at a stationary phase of callus growth. Exogenous application of gibberellin and an inhibitor of gibberellin synthesis, uniconazole, did not have any significant effects on the enzyme expression, Sugar starvation increased the activity, while addition of metabolizable sugars, such as sucrose, glucose and maltose, to the medium repressed expression, Addition of 6% mannitol, a non-metabolizable sugar, to the medium induced higher alpha-amylase expression as compared to addition of 3% mannitol, This result suggests that osmotic stress enhances alpha-amylase activity in the calli, Furthermore, high concentrations of agar in the medium increased alpha-amylase activity in the calli, It is probable that high concentrations of agar prevented incorporation of nutrient into the calli and induced the alpha-amylase activity in the calli.

The development of endopeptidase activity in cotyledons of Vigna mungo seedlings was examined after application of exogenous amino acids, sugars and plant hormones. The endopeptidase activity in the cotyledons fell when germinating seeds were allowed to absorb a solution of amino acids at high concentrations, and it was postulated that this effect might have been caused in part by osmotic stress and in part by end-product repression. Protein immunoblotting with an antiserum against SH-EP, the major cysteine endopeptidase occurring in the cotyledons, showed that sugars and amino acids at high concentrations also delayed the post-translational processing of SH-EP intermediates, Endopeptidase activity equivalent to nearly twice that in controls was observed when GA(3) was applied at 10 to 100 mu M to cotyledons that had been detached from the embryonic axis. In addition, naphthaleneacetic acid at 1 to 100 mu M, kinetin at 1 to 10 mu M and jasmonic acid at 1 to 10 mu M also increased the activity to a limited extent. Results of pulse-chase experiments suggested that the effect of GA(3) on the endopeptidase activity in the detached cotyledons was attributable to suppression of the degradation of the enzyme. Protein immunoblotting revealed the presence of 34-kDa and 35-kDa intermediates of SH-EP in addition to previously reported 36-kDa and 43-kDa intermediates.

Pulse-chase experiments indicated that the higher levels of a-amylase in detached and incubated cotyledons of Vigna mungo than those in cotyledons attached to the embryonic axis were due to both faster synthesis and slower degradation of the enzyme in the detached cotyledons than in the attached cotyledons. Levels of a-amylase in the cotyledons were examined in terms of possible effects of end-products and the effects of exogenously applied plant hormones and growth regulators. Levels of a-amylase activity and content were reduced by high concentrations of glucose and sucrose, and it is suggested that this effect was caused mostly by osmotic stress and partly by end-product repression. The level of a-amylase was nearly twice that in controls after 1 to 10 mu M GA(3) had been applied to the cotyledons. In addition, 0.1 mM kinetin, 0.1 mM 2,4-D and 0.1 to 0.5 mM naphthaleneacetic acid also increased the level by 34% to 66% as compared to the control. ABA and uniconazole both prevented the synthesis of a-amylase.

Endopeptidase activity increases continually in pods of maturing fruits of French bean (Phaseolus vulgaris L. cv Goldstar) plants and is thought to participate in the protein mobilization in pods during the development of seeds (M. Endo, T. Minamikawa, D. Yamauchi, W. Mitsuhashi [1987] J Exp Bot 38: 1988-1995). In the present studies, one of the major endopeptidase forms, designated EP-C1, was purified as a 34-kD polypeptide from pods of maturing French bean fruits. EP-C1 was found to be immunologically distinguished from other forms in extracts from pods, but homologous to SH-EP, the major cysteine endopeptidase expressed in cotyledons of germinating Vigna mungo seeds (W. Mitsuhashi, T. Minamikawa [1989] Plant Physiol 89: 274-279). The level of endopeptidase that reacted with the antiserum to EP-C1 increased in pods as the fruit maturation proceeded. EP-C1 was also immunologically detected in stems of French bean plants bearing fruits of later maturation stages. Protein immunoblotting showed that a 34-kD polypeptide corresponding to EP-C1 in molecular mass occurred in extracts from 7- to 9-d cotyledons of germinating French bean seeds. In addition, two other polypeptides with slightly higher molecular masses were observed in extracts from 3- to 5-d cotyledons. We suggest that these two polypeptides are intermediates involved in posttranslational processing of EP-C1. RNA blot hybridization with EP-C1 cDNA as a probe showed that EP-C1 mRNA occurred in pods of fruits at later maturing stages and also in cotyledons of 3-to 7-d germinating seeds.

A cysteine endopeptidase (SH-EP) is involved in the degradation of storage globulin in cotyledons of germinating seeds of Vigna mungo. Using SH-EP cDNA as a probe, we isolated two overlapping genomic clones for SH-EP from a V. mungo genomic library. The results of Southern blot analysis of the nuclear DNA suggested that there is a single gene for SH-EP. The site of initiation of transcription was located 31 bp upstream from the initiation codon. Protein immunoblotting showed that, in addition to the presence of the protein in cotyledons of germinating seeds, levels of a protein that reacted with an antiserum against SH-EP increased in pods of maturing fruits of V. mungo, with very low levels being present in other organs of the seedlings. The change in levels of SH-EP mRNA in cotyledons of germinating seeds followed a time course similar to that of alpha-amylase mRNA. Levels of both enzymes, as well as levels of their mRNAs, were also examined in cotyledons of seedlings of three other legumes by protein immunoblotting and RNA blot hybridization, respectively.

Levels of starch and soluble sugar in pods of Phaseolus vulgaris and Vigna mungo plants were analyzed during the course of maturation of fruits. The results suggest that the immature pods of P. vulgaris function to some extent as temporary reservoirs of carbohydrates for growth of seeds. A less clear pattern of accumulation of starch was observed in pods of maturing fruits of Vigna mungo. Measurements of a-amylase activities in pods of maturing fruits and immunoblotting with an antiserum against a-amylase from V. mungo cotyledons suggested that the enzyme participates in the mobilization of starch that is stored in pods of both species. High levels of expression of a-amylase of the same molecular size as that found in pods were observed in cotyledons of germinating seeds of both species. The results indicate that only one form of a-amylase, with respect to molecular size, is expressed in pods of the maturing fruits, as it is in the cotyledons and other organs of seedlings of P. vulgaris and V. mungo.