A Bacterial Indole-3-acetyl-L-aspartic Acid Hydrolase Inhibits Mung Bean (Vigna radiata L.) Seed Germination Through Arginine-rich Intracellular Delivery

Indole-3-acetyl-L-aspartic acid (IAA-Asp) is a natural product in many plant species and plays many important roles in auxin metabolism and plant physiology. IAA-Asp hydrolysis activity is, therefore, believed to affect plant physiology through changes in IAA metabolism in plants. We applied a newly discovered technique, arginine-rich intracellular delivery (AID), to deliver a bacterial IAA-Asp hydrolase into cells of mung bean (Vigna radiata) seeds and measured its effects on mung bean seed germination. IAA-Asp hydrolase inhibited seed germination about 12 h after the enzyme was delivered into cells of mung bean seeds both covalently and noncovalently. Mung bean seed germination was delayed by 36 h when the enzyme protein was noncovalently attached to the AID peptide and longer than 60 h when the enzyme protein was covalently attached to the AID peptide. Root elongation of mung bean plants was inhibited as much as 90% or 80%, respectively, when the IAA-Asp hydrolase was delivered with the AID peptide by covalent or noncovalent association. Further thin-layer chromatography analysis of plant extracts indicated that the levels of IAA increased about 12 h after treatment and reached their peak at 24 h. This result suggests that IAA-Asp hydrolase may increase IAA levels and inhibit seed germination of mung bean plants and that the AID peptide is a new, rapid, and efficient experimental tool to study the in vivo activity of enzymes of interest in plant cells.     

Free and conjugated indole-3-acetic Acid in developing bean seeds

The changes in conjugated indole-3-acetic acid (IAA) levels compared to the levels of free IAA have been analyzed during the development of bean (Phaseolus vulgaris L.) seed using quantitative mass spectrometry. Free and ester-linked IAA levels are both relatively high in the early stages of seed development but drop during seed maturation. Concomitantly, the amide-linked IAA becomes the major form of IAA present as the seed matures. In fully mature seed, amide IAA accounts for 80% of the total IAA. The total IAA pool in the seed is maintained at approximately the same level (150-170 nanograms/seed) once the level of free IAA has attained its maximum. Thus, the amount of amide IAA conjugates that accumulate in mature seed is closely related to the amounts of free and ester-linked IAA that disappeared from the rapidly growing seed. Analysis of developing bean pods, from which the seeds were taken for analysis, showed very low levels of both ester and amide-linked IAA conjugates. The pattern of changes seen in the levels of free and conjugated IAA in developing bean seed supports our prior hypothesis suggesting a role of IAA conjugates in the storage of the phytohormone in the seed.     

Indoleacetic Acid Levels in Phaseolus, Zea, and Pincus during Seed Germination

The content of indoleacetic acid (IAA) was determined in dry and germinating seeds of French bean (Phaseolus vulgaris), sugar maize (Zea mays), and Scots pine (Pinus silvestris). IAA was found in both the dry and the soaked seeds of the three species examined. The amount of IAA per gram fresh weight was extremely different in the three species whereas the variation between different harvests of the same species was small. Maize contained the highest level of IAA and bean the smallest. The time of imbibition was of decisive importance for the level of IAA. In all three species the content of IAA increased considerably during the initial 4 hours of swelling. The highest level of IAA was found in seeds that had swelled for 24 to 48 hours, during which period the radicles began to emerge from the seed coat. Later, during the period of rapid root growth, the content of IAA declined.     

Auxin Biosynthesis during Seed Germination in Phaseolus vulgaris

The relative roles of de novo biosynthesis of indoleacetic acid (IAA) and IAA conjugates stored in mature seeds (Phaseolus vulgaris L.) in supplying auxin to germinating bean seedlings were studied. Using ²H oxide and 2,4,5,6,7-[²H]l-tryptophan as tracers of IAA synthesis, we have shown that de novo biosynthesis of IAA, primarily from tryptophan, is an important source of auxin for young bean seedlings. New synthesis of IAA was detected as early as the second day of germination, at which time the seedlings began to accumulate fresh weight intensively and the total content of free IAA began to increase steadily. IAA conjugates that accumulate in large amounts in cotyledons of mature seeds may thus be considered to be only one of the possible sources of IAA required for the growth of bean seedlings.     

Heterologous expression of IAP1, a seed protein from bean modified by indole-3-acetic acid,in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The seed protein IAP1 from bean (PvIAP1; Phaseolus vulgaris L.) that is modified by the phytohormone indole-3-acetic acid (IAA) was heterologously expressed in the two reference plant species Arabidopsis thaliana and Medicago truncatula. For the transformation of Medicago we devised a novel protocol using seedling infiltration. When PvIAP1 was overexpressed under the control of the constitutive 35SCaMV promoter in Arabidopsis, the plants showed signs of earlier bolting and enhanced branching. Expression of a fusion protein of PvIAP1 with both a green fluorescence protein (GFP) as reporter and 6× histidine (His) tag under the control of the native bean IAP1 promoter resulted in the accumulation of the protein in both plant species exclusively in seeds as shown by immunoblotting and by fluorescence microscopy. During seed development, PvIAP1 was first expressed in the vascular bundle of Arabidopsis, whereas in later stages GFP fluorescence was visible essentially in all tissues of the seed. Fluorescence decreased rapidly after imbibition in the seeds for both Arabidopsis and Medicago, although the fluorescence persisted longer in Arabidopsis. GFP fluorescence was distributed evenly between an organelle fraction, the microsomal membrane fraction, and the cytosol. This was also confirmed by immunoblot analysis. Clusters of higher GFP fluorescence were observed by confocal microscopy. Although PvIAP1 protein accumulated in seeds of both Arabidopsis and Medicago, neither species post-translationally modified the protein with an indoleacyl moiety as shown by quantitative GC–MS analysis after alkaline hydrolysis. These results indicate an apparent specificity for IAA attachment in different plant species. Alexander Walz and Claudia Seidel contributed equally to the paper.     

Germination dimorphism in Suaeda acuminata: A new combination of dormancy types for heteromorphic seeds

Desert annual Suaeda acuminata produces two morphologically distinct types of seeds on the same plant. The main aims of our study were to compare germination characteristics of the dimorphic seeds, ascertain their dormancy types and give the hormonal explanation. The two seed types of S. acuminata absorbed water at different rates with brown seeds imbibing water faster. Germination percentages of brown seeds were significantly higher than those of black seeds in all temperature and light regimes tested. Eight weeks of cold stratification did not break dormancy of black seeds, whereas exogenous GA₃ promoted germination. Excised embryos of untreated black seeds produced normal seedlings. Contents of ZR, GA₃ and ABA of brown seeds were significantly higher than that of black seeds; while contents of IAA of black seeds were significantly higher than that of brown seeds. Brown seeds of S. acuminata are non-dormant, whereas black seeds have intermediate physiological dormancy (PD). Interaction among ZR, ABA and GA₃ may play an important role in dormancy status of both seed types. This is the first report of non-dormancy and intermediate PD in a heteromorphic species.     

Role of Pseudomonas putida Indoleacetic Acid in Development of the Host Plant Root System

Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.     

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : I. The Use of Steady-State Labeling

The pathway and kinetics of photosynthate unloading in developing seeds of bean (Phaseolus vulgaris L.) were investigated using steady-state labeling with ¹⁴CO₂. The continuous assimilation of ¹⁴CO₂ at constant specific activity produced stable tracer fluxes that facilitated straightforward analyses of photosynthate import and unloading in developing seeds. The kinetics of tracer equilibration within intact seeds were compatible with a symplastic route of photosynthate unloading in the seed coat. The import and partitioning of tracer within seeds were partially disrupted by the surgical excision of the distal halves of seeds as practiced during the preparation of “empty” seed coats for perfusion.     

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims

Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination.

Methods

Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC.

Key Results

During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied.

Conclusions

The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.     

Pyruvate orthophosphate dikinase of c(3) seeds and leaves as compared to the enzyme from maize

Pyruvate orthophosphate dikinase (PPDK) was found in various immature seeds of C₃ plants (wheat, pea, green bean, plum, and castor bean), in some C₃ leaves (tobacco, spinach, sunflower, and wheat), and in C₄ (maize) kernels. The enzyme in the C₃ plants cross-reacts with rabbit antiserum against maize PPDK. Based on protein blot analysis, the apparent subunit size of PPDK from wheat seeds and leaves and from sunflower leaves is about 94 kdaltons, the same as that of the enzyme from maize, but is slightly less (about 90 kdaltons) for the enzyme from spinach and tobacco leaves. The amount of this enzyme per mg of soluble protein in C₃ seeds and leaves is much less than in C₄ leaves. PPDK is present in kernels of the C₄ plant, Zea mays in amounts comparable to those in C₄ leaves.

Regulatory properties of the enzyme from C₃ tissues (wheat) are similar to those of the enzyme from C₄ leaves with respect to in vivo light activation and dark inactivation (in leaves) and in vivo cold lability (seeds and leaves).

Following incorporation of ¹⁴CO₂ by illuminated wheat pericarp and adjoining tissue for a few seconds, the labeled metabolites were predominantly products resulting from carboxylation of phosphoenolpyruvate, with lesser labeling of compounds formed by carboxylation of ribulose 1,5-bisphosphate and operation of the reductive pentose phosphate cycle of photosynthesis. PPDK may be involved in mechanisms of amino acid interconversions during seed development.

     

Concentration of Indole-3-acetic Acid and Its Derivatives in Plants

Seeds of oat, coconut, soybean, sunflower, rice, millet, kidney bean, buckwheat, wheat, and corn and vegetative tissue of oat, pea, and corn were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. Three conclusions were drawn: (a) all plant tissues examined contained most of their IAA as derivatives, either esterified or as a peptide; (b) the cereal grains examined contained mainly ester IAA; (c) the legume seeds examined contained mainly peptidyl IAA. Errors in analysis of free and bound IAA are discussed.     

Myo-Inositol Esters of Indole-3-acetic Acid as Seed Auxin Precursors of Zea mays L

Indole-3-acetyl-myo-inositol esters constitute 30% of the low molecular weight derivatives of indole-3-acetic acid (IAA) in seeds of Zea mays. [¹⁴C]Indole-3-acetyl-myo-inositol was applied to a cut in the endosperm of the seed and found to be transported from endosperm to shoot at 400 times the rate of transport of free IAA. The rate of transport of indole-3-acetyl-myo-inositol from endosperm to shoot was 6.3 picomoles per shoot per hour and thus adequate to serve as the seed auxin precursor for the free IAA diffusing downward from the shoot tip. Indole-3-acetyl-myo-inositol is the first seed auxin precursor to be identified.     

An abundant, highly conserved tonoplast protein in seeds

We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with M_r 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds.     

The influence of kinetin on the endogenous content of indoleacetic acid in swelling seeds of Phaseolus, Zea and Pinus and young plants of Phaseolus

Treatment of different plant materials, seeds of Phaseolus vulgaris, Zea mays and Pinus silvestris and young plants of Phaseolus, with kinetin increased the level of extractable IAA. For seeds this increase was most pronounced in bean seeds, which contained the lowest amount of endogenous IAA and cytokinins, and lower in maize seeds with high endogenous content of IAA and cytokinins. – For young bean plants the kinetin treatment significantly increased the extractable amounts of IAA from all parts of the plant, hypocotyls, cotyledons, epicotyls and primary leaves, when the cut plants were placed for 24 h in kinetin solution. For plants sprayed with kinetin solution only the primary leaves showed a significantly higher level of extractable IAA, which could be explained by the fact that the plants were growing very close together, so that the primary leaves received most of the kinetin during spraying.     

The Glycoproteins of Plant Seeds : ANALYSIS BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS AND BY THEIR LECTIN-BINDING PROPERTIES

Protein from the jack bean, peanut, soybean and kidney bean seeds were extracted with a solution containing 9.3 molar urea, 5 millimolar K₂CO₃, 0.5% dithiothreitol and 2% Nonidet P-40 and then subjected to two-dimensional gel electrophoresis. After electrophoresis, the slab gels were stained with a variety of ¹²⁵I-labeled lectins and the lectin-binding proteins were identified after autoradiography. Incubation of slab gels of jack bean with concanavalin A, peanut with peanut agglutinin, soybean with soybean agglutinin, and kidney bean with phytohemagglutinin showed that the majority of the polypeptides in each seed type were able to bind to their homologous lectins. Control slab gels in which incubations were carried out with identical amounts of proteins, ¹²⁵I-lectin and an appropriate sugar inhibitor showed little or no lectin binding to the polypeptides. Additionally, incubation of slab gels of peanut proteins with ¹²⁵I-ricin, ¹²⁵I-wheat germ agglutinin, ¹²⁵I-concanavalin A, and ¹²⁵I-soybean agglutinin each revealed a clearly distinct binding pattern compared to the one observed with the peanut agglutinin. The results demonstrate that a large number of legume seed polypeptides are glycoproteins and that the carbohydrate groups within a seed species are heterogeneous in structure, thus indicating the existence of complex glycosylating enzyme systems in legume seeds. It is suggested that the high degree of binding between seed proteins and their homologous lectins might have some functional significance in maintaining large aggregates of protein in compact, insoluble form.     

Symbiotic N2 fixation in pea and field bean estimated by15N fertilizer dilution in field experiments with barley as a reference crop

Summary The total amount of nitrogen derived from symbiotic nitrogen fixation in two pea and one field bean cultivar, supplied with 50 kg N ha⁻¹ at sowing (‘starter’-N), was estimated to 165, 136, and 186 kg N ha⁻¹, respectively (three-year means). However, estimates varied considerably between the three years. At the full bloom/flat pod growth stage from 30 to 59 per cent of total N₂ fixation had taken place. The proportion of total N derived from N₂ fixation at maturity was higher in seeds than in vegetative plant parts and amounted to 59.5, 51.3 and 66.3 per cent of total above-ground plant N in the two pea cultivars and field bean, respectively (three-year means). The recovery of fertilizer N was 62.2, 70.2, 52.1, and 69.5 per cent in the two pea cultivars, field bean and barley, respectively. Growth analysis indicated that barley did not meet the claims for an ideal reference crop in the¹⁵N fertilizer dilution technique for estimating N₂ fixation in pea and field bean. ‘Starter’-N neither increased the seed yield nor the N content of the grain legumes.     

农田开垦对三江平原湿地土壤种子库影响及湿地恢复潜力

种子库是湿地植被恢复的重要途径之一,不同时期的耕作土壤中残留的种子对开垦湿地恢复具有重要的作用.本文采用温室萌发法在两种水分条件下对三江平原天然湿地、不同开垦年限湿地种子库结构和规模进行了研究,以了解不同开垦年限湿地种子库特征及其在湿地植被恢复中的潜力.本次实验共萌发物种50种,随着开垦年限增加,萌发物种逐渐减少,天然湿地、开垦1年、3年、10年、20年的湿地分别为34种、31种、21种、21种和8种,萌发物种数与种子库规模均表现出极显著差异(F1=8.32,F2=5.946,P＜0.001).种子库密度以天然湿地和开垦1年湿地最大,分别为7624粒/m2,9836粒/m2.随着开垦年限增加,种子库规模逐渐减小,开垦3年、10年种子库密度为4336粒/m2,4872粒/m2.开垦20 a后,显著减少为432粒/m2.湿润条件下萌发物种数及种子密度显著高于淹水处理,种子库具有明显的分层现象,0-5 cm土层种子库规模显著高于5-10 cm.小叶章(Calamagrostis angustifolia)作为该地区优势物种,由最初的1192粒/m2,经过20 a开垦后在种子库中消失.研究表明,在一定的开垦年限范围内,开垦湿地土壤中仍然保留大量的湿地物种种子,在湿地恢复中具有重要的作用.     

Bean arcelin

Summary SDS-PAGE of seed proteins from the seeds of a nondomesticated bean of Mexican origin (Phaseolus vulgaris L., PI 325690) revealed the presence of a novel 38 kd protein which appeared to be neither an altered phaseolin nor a lectin fraction. The protein was named arcelin, after Arcelia, the town in the state of Guerrero near which PI 325690 had been collected. The pure line, UW 325, was derived by self fertilization of the plant from a single arcelin-containing seed of PI 325690. Despite a low percentage seed phaseolin (14.6%), seed phenotype, seed germination, plant growth, pollen fertility, and percentage seed protein of UW 325 were normal. Analyses of F₂ and F₃ seeds from a single F₁ plant of the cross SanilacXPI 325690-3 revealed that arcelin expression was inherited as a single gene and that presence was dominant to absence of arcelin. The mean percentage phaseolin in the seeds of homozygous dominant Arc/Arc F₃ families (14.0%) was significantly lower than that of the homozygous recessive arc/arc seeds (44.7%). The distribution of percentage phaseolin values for seeds within segregating families was bimodal and nonoverlapping. Without exception, seeds containing arcelin (Arc+phenotype) contained a lower percentage phaseolin than seeds lacking arcelin (Arc-phenotype). Although arcelin presence was associated with low percentage phaseolin, the Arc/Arc and Arc/arc genotypes were similar for seed weight and percentage total seed protein.     

Direct observation of cell wall structure in living plant tissues by solid-state C NMR spectroscopy

Solid-state ¹³C nuclear magnetic resonance (NMR) spectra of the following intact plant tissues were recorded by the crosspolarization magic-angle spinning technique: celery (Apium graveolens L.) collenchyma; carob bean (Ceratonia siliqua L.), fenugreek (Trigonella foenum-graecum L.), and nasturtium (Tropaeolum majus L.) endosperm; and lupin (Lupinus polyphyllus Lindl.) seed cotyledons. All these tissues had thickened cell walls which allowed them to withstand the centrifugal forces of magic angle spinning and which, except in the case of lupin seeds, dominated the NMR spectra. The celery collenchyma cell walls gave spectra typical of dicot primary cell walls. The carob bean and fenugreek seed spectra were dominated by resonances from galactomannans, which showed little sign of crystalline order. Resonances from β(1,4′)-d galactan were visible in the lupin seed spectrum, but there was much interference from protein. The nasturtium seed spectrum was largely derived from a xyloglucan, in which the conformation of the glucan core chain appeared to be intermediate between the solution form and solid forms of cellulose.