Desiccation of Phaseolus vulgaris Seeds During and Following Germination, and its Effect Upon the Translatable mRNA Population of the Seed Axes

Misra, S. and Bewley, J. D. 1986. Desiccation of Phaseolus vulgansseeds during and following germination, and its effect uponthe translatable mRNA population of the seed axes.—J.exp. BoL 37: 364–374. After imbibition and germination, seeds of P. vulgaris passfrom a stage where they are insensitive to desiccation to astage where they are sensitive. Desiccation of seeds duringthe sensitive stage results in an almost total impairment ofprotein synthesis upon subsequent rehydration. Seeds desiccatedduring the desiccation-tolerant stage, however, resume proteinsynthesis at almost control levels. The protein patterns obtained following in Vitro translationof bulk RNA from fresh imbibed, desiccated, and desiccated-rehydratedseed axes were qualitatively similar at 5 HAI (the desiccation-tolerant stage). The drying treatment resulted in increasedintensity of extant proteins at 5 and 12 HAI. At 12 HAI (thetransition stage between the desiccation-tolerant and desiccation-intolerantphases) desiccation and subsequent rehydration triggered synthesisof a unique set of proteins-the rehydration proteins. At 20HAI (the desiccation-intolerant stage) desiccation resultedin an overall decline in the intensity of proteins synthesizedin vitro. Also the rehydration proteins were not synthesizedin response to a drying and rehydration treatment at this time. Key words: Seed germination, desiccation, mRNA, in vitro translation, Phaseolus vulgaris     

l-Phenylalanine Ammonia-Lyase Activity During Germination of Phaseolus vulgaris

l-Phenylalanine ammonia-lyase (PAL) activity develops in excised bean axes after approximately 5 hours of incubation and reaches a maximum level after 14 hours of incubation. Light does not affect the development of activity, but puromycin, cycloheximide, actinomycin D, and 5-fluorouracil inhibit.     

Gene Expression and Synthesis of Phytohemagglutinin in the Embryonic Axes of Developing Phaseolus vulgaris Seeds

Phytohemagglutinin (PHA), the major seed lectin of the common bean (Phaseolus vulgaris), is found largely in the cotyledons, but is also present in the embryonic axis. At mid-maturation, the percentage of total protein synthesis which is directed towards making PHA is 5 to 10 times greater in the cotyledons than in the axes. This lower rate of synthesis in the axes is correlated with a lower abundance of mRNA for PHA, as determined by dot blot hybridization using a cDNA clone for PHA. Manen and Pusztai (Planta 1982 155: 328-334) have claimed on the basis of immunocytochemical evidence that, in the axis, PHA is found in the cytosol although it is present in protein bodies in the cotyledons. In the cotyledons, PHA is synthesized on rough endoplasmic reticulum, and its transport to the protein bodies via the Golgi complex is associated with specific posttranslational processing steps (Vitale and Chrispeels, J Cell Biol 1984 In press). A cytosolic localization of axis PHA would be an indication of a different site of synthesis and transport pathway. The results presented here indicate that the site of synthesis of PHA and the posttranslational modifications of PHA are the same in the axes as in the cotyledons. Since in the cotyledons these modifications take place in the endoplasmic reticulum, the Golgi, and the protein bodies, it appears that the transport pathway and the site of accumulation of PHA in the axes is similar to that in the cotyledons. On the basis of our evidence, we suggest that the subcellular localization of PHA in the axes should be reexamined.     

Germination of Phaseolus vulgaris: IV. Patterns of Protein Synthesis in Excised Axes

Soluble proteins from excised Phaseolus vulgaris axes incubated for 1 hour in ³H or ¹⁴C- amino acid mixtures at different times during the period leading up to initiation of cell elongation were compared by acrylamide gel electrophoresis. Differences in electrophoretic patterns were found when proteins from axes incubated during the 1st hour of imbibition were compared with proteins from axes incubated during the hour when cell elongation was initiated. These differences greatly diminished by the 2nd hour of imbibition which suggests that they were due primarily to incomplete axis imbibition. A 5-hour actinomycin D treatment which reduced amino acid incorporation by 40% in the 5th hour had no apparent effect on the electrophoretic pattern during that hour.     

Desiccation of Axes of Phaseolus vulgaris during Development of a Switch from a Development Pattern of Protein Synthesis to a Germination Pattern

Immature seeds of Phaseolus vulgaris removed from the pod at 32 days of development do not germinate unless first subjected to a desiccation treatment. This change from development to germination caused by premature drying is mirrored in the pattern of protein synthesis by the axes. Rehydrated axes from 32-day-developed seeds cease to synthesize proteins that are uniquely associated with development, but instead synthesize some proteins that are similar to those made in the germinating axes from mature dry seeds. Desiccation of 22-day-developed seeds does not lead to their germination, nor does it cause a switch from a developmental to a germination mode of protein synthesis by the axes. It is proposed that desiccation plays a role in permanently suppressing developmental protein synthesis and in inducing germination protein synthesis.     

Desiccation-Tolerant and Desiccation-Intolerant Stages during the Development and Germination of Phaseolus vulgaris Seeds

Embryonic axes of Phaseolus vulgaris undergo a transition fromdesiccation-intolerance to desiccation-tolerance in the courseof their development. Biochemical and ultrastructural observationsindicate that desiccation and rehydration during the early intolerantdevelopmental stages drastically reduces the metabolic and cellularintegrity of the axis. During the desiccation-tolerant stagesuch perturbations do not occur. Coincident with the acquisitionof desiccation-tolerance during development the seeds gain thecapacity to germinate upon subsequent rehydration. Drying presumablyacts to terminate developmental processes and to initiate themetabolic processes essential for the completion of germination.During germination of the mature axis, desiccation and rehydration,up to 12 h from the start of imbibition, does not affect subsequentseedling growth and development. But gradually desiccation-tolerancedecreases and metabolism is severely and irreversibly reducedby drying.     

Cellular double-stranded RNA in Phaseolus vulgaris

Summary High molecular weight double-stranded (ds) RNAs have been detected in apparently virus-free French (common) bean Phaseolus vulgaris cv. Black Turtle Soup (BTS). Several other bean cultivars were free of detectable high molecular weight dsRNAs. The dsRNAs have been partially characterized and have homology to the BTS genome as well as to the genomes of other bean cultivars. The T_m of hybrids formed between BTS DNA and denatured dsRNA have been estimated.     

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.     

The Effects of Germination on the Subcellular Distribution of Cholinesterase in Cotyledons of Phaseolus vulgaris

Homogenates of Phaseolus vulgaris cotyledons have been found capable of hydrolyzing acetylthiocholine. The hydrolysis occurs optimally at pH 8.0, and is inhibited by neostigmine but not eserine. Total activity of the enzyme increases about three-fold between the second and third days of germination, and remains high until day 6 before dropping coincident with the appearance of visible morphological symptoms of senescence in the tissue. Fractionation studies have revealed that the enzyme is enriched in preparations of purified cell wall and plasma membrane and is also present in a soluble fraction. The soluble enzyme accounts for more than 70% of the total cholinesterase activity two days after planting but by the fourth day of germination only about 30% of the total activity in the tissue is soluble. During the same period there is a large increase in the specific activities of both the cell wall and plasma membrane enzymes. By the seventh day of germination the particulate and soluble forms of the enzyme both show much reduced activities, but the specific activities of the cell wall and plasma membrane enzymes subsequently increase again. This is thought to reflect breakdown of protein other than cholinesterase in these structures as they in turn become subject to the increasing pressures of senescence. Cholinesterase in plant tissue presumably serves to regulate the endogenous titre of acetylcholine. The behaviour of this enzyme in bean cotyledons has been interpreted in terms of patterns of physiological and ultrastructural change known to characterize this tissue during germination.     

The Effects of Photoperiod on Flower-bud Development in Phaseolus vulgaris

For a variety of Phaseolus vulgaris of Peruvian origin, extendingthe daylength from 11 to 15 or 16 h with low-intensity incandescentor natural light, while not affecting the rate of initiationof flower primordia, inhibited the development of the flowerbuds and caused many, and sometimes all of them, to senesceand abscise. The inhibition of the oldest flower bud was firstdetected at the stage of pollen formation. The long photoperiodsalso inhibited the elongation and over-all development of theterminal inflorescence but, in contrast, promoted the elongationof those internodes below the first trifoliate leaf and alsothe elongation of the petioles.     

Changes in Cell Wall Polysaccharides During the Growth of Phaseolus vulgaris Leaves

The changes in pectic and hemicellulosic polysaccharides, and-cellulose during the expansion growth of the primary leavesof Phaseolus vulgaris L. var. Pinta have been studied. -Celluloseincreased continuously with age, while pectic and water-solublehemicellulose extracted with 4% KOH fractions slightly decreased.The water-soluble hemicelluloses extracted with 24% KOH showedthe most conspicuous changes, increasing until the 8th day,when the absolute growth rate was maximal, and thereafter decreasing.Furthermore, the study of the molecular mass distribution ofpectin, and water-soluble polysaccharides extracted with 4%and 24% KOH, showed an increase in the degree of polymerizationof polyuronic acid and xylan, and an important depolymerizationof galactan and xyloglucan. Accordingly, the mechanism of cellwall loosening in the leaf cell walls is similar to that describedfor plant axes. Key words: Cell wall, growth, leaf     

Long-lived Messenger RNA and its Relationship to Protein Synthesis During Germination of Pea (Pisum sativum L.) Seeds

Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Synthesis of both protein and RNA is initiated very early ingermination in the embryo axes of pea seeds. The early RNA synthesisinvolves all three types, although there is some evidence forpreferential synthesis of mRNA in the first few hours afterthe onset of imbibition. In addition to newly synthesized mRNA,the embryo axis also contains long-lived mRNA. The amount ofthis long-lived mRNA declines markedly during the first 20 hof germination. Results from in vitro and in vivo protein synthesis experimentsand from studies of polysome formation suggest that much ofthe long-lived mRNA present in the embryo axis does not directprotein synthesis. The increase in the rate of protein synthesisduring germination is thus dependent on recruitment of newlysynthesized mRNA molecules. Pea, Pisum sativum L., germination, mRNA, protein synthesis     

Seed Development in Phaseolus vulgaris L. cv Seminole: II. Precocious Germination in Late Maturation

Seeds of Phaseolus vulgaris L. cv Seminole in late maturation phase germinated precociously in vitro. Germination occurred in the absence of free water after 5 days but within 24 to 48 hours in contact with water. Excised axes germinated within 12 hours and embryos by 48 hours only if supplied with water. Ethylene accelerated the germination of seeds and embryos irrespective of water availability. There was no effect of ethylene on the rate of axis germination. Ethylene was equally effective within the range 0.5 to 1000 parts per million and 1 hour exposure was fully effective. Induction of precocious germination in vivo was observed by manipulating water content inside pods or by ethylene injection, whether pods were attached to the parent plant or not. These results demonstrate the importance of endogenous regulation of water supply in suppressing precocious germination. Ethylene is identified as a powerful antagonist to the natural control.     

Structural Development and Respiratory Activity of the Funiculus During Bean Seed (Phaseolus vulgaris L.) Maturation

Changes in the structural organization of the funiculus of Phaseolusvulgaris were correlated with mitochondrial respiration rates,including both cytochrome and alternative pathway activitiesand seed weight during development of the seed. After fertilization,vascular elements are still differentiating within the funiculus.The central core of the funiculus consists mainly of procambialcells together with a few mature xylem and phloem elements.As the seed gradually matures, more vascular elements beginto appear. Procambial cells in the upper region of the funiculusadjacent to the pod differentiate and result in xylem and phloemappearing as a convoluted, intertwining network of strands.In the lower part of the funiculus adjacent to the seed, fewervascular elements are present and they organize into a smallbundle prior to entering the seed. The funiculus is fully developedat the cotyledon stage judging from the size of the funiculusand the organization of the vascular tissues. At the early maturationstage, the seed begins to enlarge in both size and weight. Correlatedwith development of the funiculus tissue is a gradual decreasein total rates of respiration. Inhibitor studies using potassiumcyanide and/or salicylhydroxamic acid show that the CN-insensitive,or alternative pathway is the predominant route of electrontransport in funiculus mitochondria during the early stagesof development. This pathway declines in activity with age whereuponcytochrome pathway activity accounts for all of the respirationby the time vascular tissues are mature and the seed is rapidlyexpanding.Copyright 1994, 1999 Academic Press Funiculus, vascular tissue, cytochrome, respiratory pathway, alternative respiratory pathway, Phaseolus vulgaris     

Alterations in Endogenous Levels of Gibberellin-like Substances during Germination of Phaseolus vulgaris Seeds

The endogenous gibberellin-like substances were determined in mature dry and germinating bean seeds, Phaseolus vulgaris L. cv. Alabaster. Methanol extracts were partitioned against ethyl acetate and butanol at neutral and acid pH. Each phase was individually chromatographed on a silica gel column. The gibberellin activity was measured with the Tan-ginbozu dwarf rice micro-drop bioassay. Each extract was tested in two dilutions. Extracts from dry seeds showed the highest gibberellin activity, largely attributable to ethyl acetate-soluble substances. The activity was considerably reduced in extracts from seeds imbibed for 1 day. Gibberellin-like substances soluble in butanol appeared in extracts from seeds soaked for 1 or more days.