Sugar-Controlled Ca2+ Uptake and {alpha}-Amylase Secretion in Cultured Cells of Rice (Oryza sativa L.)

Sugar starvation-induced synthesis and extracellular liberationof -amylase molecules in suspension-cultured cells of rice (Oryzasativa L.) required Ca²⁺, although the level of translatable-amylase mRNA was not affected in the presence of Ca²⁺. Sugardepletion markedly stimulated Ca²⁺ uptake by rice cells andsucrose supplementation reduced it. Immunohistochemical andelectron probe microanalyzer studies indicated an apparent resemblancebetween the distribution pattern of Ca²⁺ and that of -amylasemolecules induced in the sugar-depleted cells. Ca²⁺ uptake wasreduced by sucrose, maltose, fructose, and glucose similarlyat more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose(10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effectiveCa²⁺ channel blocker, La³⁺ significantly inhibited the Ca²⁺uptake and the synthesis and extracellular liberation of -amylasemolecules in the absence of sucrose, while a general P-typeATPase inhibitor, vanadate greatly stimulated both in the presenceof sucrose. We concluded that, by controlling the Ca²⁺ uptake,metabolic sugars regulate the protein synthesis and posttranslationalsecretory processes of -amylase molecules in rice cells. ⁴ Invited research fellow of the Japan Society for the Promotionof Science. Present address: Plant Physiology Department, WarsawAgricultural University, Rakowiecka Str. 26/30 02-528 Warsaw,Poland.     

Changes in {alpha} and {beta}-Amylase Activities during Germination of Seeds of Alfalfa (Medicago sativa L.)

We examined the methods available for the assay of -amylasein alfalfa (Medicago sativa) and found the Phadebas test mostsuitable. The Phadebas assay and activity staining on ampholinegels after isoelectrofocusing revealed that an amylase is presentin the dry seeds of alfalfa and that its activity decreasesrapidly after the second day of seed germination. An amylasewas purified by affinity chromatography and gel filtration.The kinds of sugar generated from soluble starch by the purifiedamylase resembled those generated by other -amylases from plants,in particular those from mung bean (Vigna radiata). These resultsindicate that the amylase in alfalfa seeds belongs to the familyof -amylases. The molecular weight and isoelectric point ofthe -amylase were determined to be 43 kDa and 4.92, respectively. The Pantrac assay and activity staining on immobiline gels afterisoelectrofocusing revealed that the activities of ß-amylasesincreased during the initial 4 to 5 days of germination. Furthermore,treatment of whole seedlings with cycloheximide or actinomycinD inhibited the increase in activity of ß-amylasesbut did not affect the reduction in activity of -amylase. During germination of alfalfa seeds, -amylase activity decreaseswhile, in contrast, ß-amylase activity increases (inthe cotyledons of germinating seeds), changes that are specificto the germinating seeds of alfalfa. (Received September 8, 1990; Accepted February 20, 1991)     

Metabolism of Abscisic Acid in Developing Seeds of Phaseolus vulgaris L. and its Correlation to Germination and {alpha}-Amylase Activity

Bean seeds were unable to germinate during their developmentwhich was characterized either by an increasing ABA content(expressed as ng ABA/seed) or by a fairly high and constantABA concentration (expressed as ng ABA g^–1 fr. wt). During seed maturation the mother plant induces a dormant stateby depressing the ABA catabolism and keeping the endogenousABA at a high level to prevent both the premature hydrolysisof starch (-amylase activity) and the germination of the morphogeneticallyadult yet immature seeds. The depth of this induced dormancyis positively correlated with endogenous ABA concentration. Application of exogenous ABA to fully mature seeds, which containno endogenous ABA and show a very active ABA catabolism, onlyprolonged the lag phase but had no influence on the furtherdevelopment of the -amylase activity.     

Ethylene-Induced Inhibition of Root Growth Requires Abscisic Acid Function in Rice (Oryza sativa L.) Seedlings

Ethylene and abscisic acid (ABA) have a complicated interplay in many developmental processes. Their interaction in rice is largely unclear. Here, we characterized a rice ethylene-response mutant mhz4, which exhibited reduced ethylene-response in roots but enhanced ethylene-response in coleoptiles of etiolated seedlings. MHZ4 was identified through map-based cloning and encoded a chloroplast-localized membrane protein homologous to Arabidopsis thaliana (Arabidopsis) ABA4, which is responsible for a branch of ABA biosynthesis. MHZ4 mutation reduced ABA level, but promoted ethylene production. Ethylene induced MHZ4 expression and promoted ABA accumulation in roots. MHZ4 overexpression resulted in enhanced and reduced ethylene response in roots and coleoptiles, respectively. In root, MHZ4-dependent ABA pathway acts at or downstream of ethylene receptors and positively regulates root ethylene response. This ethylene-ABA interaction mode is different from that reported in Arabidopsis, where ethylene-mediated root inhibition is independent of ABA function. In coleoptile, MHZ4-dependent ABA pathway acts at or upstream of OsEIN2 to negatively regulate coleoptile ethylene response, possibly by affecting OsEIN2 expression. At mature stage, mhz4 mutation affects branching and adventitious root formation on stem nodes of higher positions, as well as yield-related traits. Together, our findings reveal a novel mode of interplay between ethylene and ABA in control of rice growth and development.     

Effect of Gibberellic Acid on the Ultrastructure and {alpha}-Amylase Activity of Aleurone Cells of Avena sativa L.

The -amylase activity and ultrastructure of aleurone cells inseeds of Avena sativa L. were studied using seed halves withembryo (embryo seeds) which had imbibed water and seed halveswithout embryo (embryo-less seeds) which had imbibed water withor without GA₃. -Amylase activity was detected in the aleurone layers of embryoseeds that had imbibed water and embryo-less seeds that hadimbibed GA₃-water. The ultrastructure of aleurone cells withdetectable -amylase activity showed marked changes in the roughsurfaced endoplasmic reticulum (rER), in the flattened sacculesforming stacks and in the aleurone grains. The progressive changesin the rER were as follows: first, the number of slender rERincreased; then, the inner space became wider and showed roundor oval profile; and finally, the rER became slender again witha reduced number of adhering ribosomes. The flattened sacculesforming stacks were appressed to the surface of aleurone grains.With time, they decreased in number and finally disappeared.In parallel with the decrease of flattened saccules, digestionof proteinaceous material inside the aleurone grains proceeded. (Received February 24, 1987; Accepted September 3, 1987)     

{alpha}-Amylase Production by Pre-mature Wheat (Triticum aestivum L.) Embryos

Embryo/scutellar tissue of pre-mature wheat grains usually containslittle -amylase but readily produces the enzyme upon removalfrom the caryopsis. Enzyme production is accompanied by cytologicalchanges in the scutellar epithelium cells characteristic ofgerminating mature embryos, although -amylase production bypre-mature tissue is not always associated with germinativegrowth of the embryonic axis. Production of -amylase is influencedby embryo age, stimulated by GA₃ and overall is inhibited byABA. Examination by isoelectric focussing and rocket-line immuno-electrophoresisreveals the presence of both -AMYl and -AMY2 isoenzymes, thelatter being the major constituent. In the presence of ABA certain-AMY2 isoenzymes not detected previously are observed. Key words: a-Amylase, wheat, embryo     

Effects of Atmospheric Humidity on Abscisic Acid Accumulation and Water Status in Leaves of Rice (Oryza sativa L.)

Rice (Oryza sativa L ) plants were grown in controlled environmentcabinets with either low (c 0 4 kPa) or high (c 1 6 kPa) atmosphericwater vapour pressure deficit (v p d) The capacity of detachedleaves to accumulate ABA in response to rapidly induced waterstress was increased when plants were grown at high v p d Highv p d significantly lowered solute potential (     

Abscisic Acid and Water Relations of Rice (Oryza sativa L.): Effects of Drought Conditioning on Abscisic Acid Accumulation in the Leaf and Stomatal Response

The effects of a period of water stress (drought conditioning)on responses to a second (challenge) stress were examined inyoung vegetative rice (Oryza sativa L.) plants. Drought conditioningdid not affect the rate of subsequent stress development, nor,in a first experiment, did it influence relations between turgor(_p) and total () leaf water potential. However, conditioningdid extend the range of _p over which stomata remained open andsignificantly reduced the amount of ABA which accumulated inthe leaf at a given _p. The change in stomatal behaviour (stomataladjustment) was quantitatively accounted for by the change inleaf ABA accumulation. The reduction in ABA accumulation due to conditioning did notinvolve a change in the potential capacity to produce ABA, asABA accumulation in partially dehydrated detached leaves wasnot reduced by conditioning. It is suggested that effects ofconditioning on leaf ABA content in the intact plant involvechanges in the rate of ABA export from the leaf. Oryza sativa L, rice, drought conditioning, stomata, water stress, abscisic acid     

Effects of Gibberellins and Prohexadione on the Activities of Oryzain and {alpha}-Amylase in Rice Seeds

Oryzains, cysteine proteinases of rice seeds, are induced byGA₃ in germinating rice seeds [Abe et al. (1987) Agric. Biol.Chem. 51: 1509]. The effects of GA₁, GA₃, GA₄, GA₉, and GA₂₀on the production of oryzain and -amylase were investigatedin embryoless half- and whole-seeds of rice (cv. Nipponbare).When gibberellins (GAs) were incubated with embryoless half-seeds,GA₁, GA₃ and GA₄ induced oryzain and -amylase, but GA₉, andGA₂₀ did not. GA₉ and GAM induced oryzain and -amylase productionin whole seeds, but this production was inhibited by the simultaneousapplication of prohexadione, an inhibitor of 2ß- and3ß-hydroxylation of GAs. Prohexadione did not inhibitthe activities of oryzain and -amylase induced by GA₁. Theseresults suggest that GAs possessing the 3ß-hydroxylgroup induce activities of oryzain and -amylase in rice seedsand that GA₉ and GA₂₀ have activity only after they are convertedmetabolically to active GAs, probably GA₄ and GA₁, respectively.GA₁, was more active than GA₄ in both half seeds and wholeseeds incubation. Oryzain and -amylase activities induced byGA₄ were significantly inhibited in the presence of 10^–4M prohexadione. This suggests that the conversion of GA₁, toGA₄ (13-hydroxylation) might be inhibited at a high dose ofprohexadione in whole seeds. ⁴Present address: Institute of Food Development, Kyung Hee University,Suwon 449-701, Korea     

Modification of Leaf Size in Rice (Oryza sativa L.) and its Effects on Water Stress-induced Abscisic Acid Accumulation

The ability of detached leaves of the rice cultivars IR20 and63–83 and their F₂ progeny to accumulate ABA in responseto water stress is negatively correlated with leaf size. Itwas shown that this association was not an artifact of incubationconditions following the imposition of stress. Also, it waspossible to break the correlation by selecting plants in segregatingpopulations which differed in ABA yet had similar leaf size. In further experiments, leaf size was altered phenotypicallyby various treatments; either being increased by gibberellinapplication or periodic removal of tillers, or reduced by priorexposure to water stress or ABA. Although responses to thesetreatments were complex, the results demonstrated that leafsize and accumulation capacity were at least partially independent.It is suggested that the correlations observed previously inF₂ populations from the cross IR20 x 63–83 were a resultof genetic linkage. Oryza saliva L., rice, leaf size, abscisic acid, water stress     

Abscisic Acid and Water Relations of Rice (Oryza sativa L.): Sequential Responses to Water Stress in the Leaf

Water stress was imposed by withholding water at an early vegetativestage from plants of two rice cultivars (IR20 and 63–83)grown in pots. As stress intensified the following sequenceof responses of the leaves was observed: (i) rise in abscisicacid (ABA) content, (ii) closure of stomata, (iii) initiationof leaf rolling. In both cultivars, turgor (_p) declined linearly with total waterpotential () of the leaf. Bulk leaf ABA content increased linearlyas _p declined, and attained twice the control (unstressed) levelfollowing a reduction in _p of about 0.12 MPa. Stomatal conductance exhibited a sigmoidal relationship to _p,declining abruptly when a particular ‘critical’_p was reached (threshold response). The critical potentialsvaried considerably between experiments, but were closely correlatedwith control potentials and with the potentials at which ABAconcentration doubled relative to controls. Leaf rolling was initiated at s near to zero _p. Increases inthe ratio of adaxial to abaxial conductance were associatedwith rolling. Variations in the above responses could be accounted for byvariations in the rate of stress development, which in termsof reduction ranged from 0.38 to 0.86 MPa day^–1. Fastdrying rates resulted in: (a) reduced osmotic adjustment, (b)increased amounts of ABA in the leaf at a given level of or_p, (c) an increase in the ABA concentration present at 50 percent stomatal closure, and (d) initiation of leaf rolling ata higher . Oryza sativa L., rice, water stress, stomata, leaf rolling, abscisic acid     

Rhizobium-Mediated Induction of Phenolics and Plant Growth Promotion in Rice (Oryza sativa L.)

Qualitative and quantitative estimation of phenolic compounds was done through reverse phase–high performance liquid chromatography (RP-HPLC) from different parts (leaf, stem, and root) of rice plants after inoculation with two rhizobial strains, RRE6 (Rhizobium leguminosarum bv. phaseoli) and ANU 843 (R. leguminosarum bv. trifolii) and infection by Rhizoctonia solani. On the basis of their retention time, the major phenolic acids detected in HPLC analysis were gallic, tannic, ferulic, and cinnamic acids. Furthermore, in all Rhizobium-inoculated rice plants, synthesis of phenolic compounds was more consistently enhanced than in control (uninoculated plants), where the maximum accumulation of phenolic compounds was observed in plants inoculated with RRE6 and infection with R. solani. Under pathogenic stress, RRE6 performed better because a relatively higher amount of phenolics was induced as compared with plants treated with ANU 843. Phenolic acids mediate induced systemic resistance and provide bioprotection to plants during pathogenic stresses. In addition, both rhizobial strains promote growth and productivity of rice plants in greenhouse conditions. This report on Rhizobium-mediated defense responses and growth promotion of nonlegume (such as rice) provides a novel paradigm of symbiotic plant–microbe interaction.     

Purification and Properties of a Basic Endo-1,3-r{beta}-Glucanase from Rice (Oryza sativa L.)

A 1,3-rß-glucanase purified from rice grain is a 33kDa monomer with a pI of     

Desiccation Tolerance and Potential Longevity of Developing Seeds of Rice (Oryza sativa L.)

The longevity and desiccation tolerance of samples of seedsof a japonica rice (Oryza sativa L.) harvested serially duringdevelopment from plants grown in two temperature regimes, viz28/20 °C and 32/24 °C (12/12 h) were determined. Massmaturity (defined as the end of the seed-filling phase) occurred19·7 and 18·3 d after 50% anthesis, respectively.Longevity (determined at 40 °C with 15% moisture contentand quantified by the value of the constant K_i of the seed viabilityequation) improved during seed development and maturation until17 and 14 d after mass maturity in the cooler and warmer regimes,respectively, but declined thereafter. Changes in K_i with timewere similar in the two environments until mass maturity, butthe increase in K_i values after mass maturity was much greaterin the cooler regime. Tolerance of desiccation to low (4%) moisturecontents improved until 22 and 14 d after mass maturity in thecooler and warmer regimes, respectively, when maturation dryinghad reduced seed moisture contents naturally to 24 and 32% moisturecontent, respectively. Further delays to seed harvest reduceddesiccation tolerance, particularly in the warmer environment.Comparison among 15 samples of seeds harvested at differenttimes in the two environments showed a strong correlation (r= 0·947, P < 0·01) between longevity (K_i) anddesiccation tolerance (to 4% moisture content). Hence, it issuggested that the regulation of desiccation tolerance to lowmoisture contents and potential air-dry longevity during seeddevelopment and maturation determined here may have a commoncause.Copyright 1994, 1999 Academic Press Oryza sativa L., rice, desiccation tolerance, genebanks, seed development, seed longevity, temperature     

Coleoptile Senescence in Rice (Oryza sativa L.)

We investigated the cellular events associated with cell deathin the coleoptile of rice plants (Oryza sativa L.). Seeds germinatedunder submergence produced coleoptiles that were more elongatedthan those grown under aerobic conditions. Transfer of seedlingsto aerobic conditions was associated with coleoptile opening(i.e. splitting) due to death of specific cells in the sideof the organ. Another type of cell death occurred in the formationof lysigenous aerenchyma. Senescence of the coleoptile was alsonoted, during which discolouration of the chlorophyll and tissuebrowning were apparent. DNA fragmentation was observed by deoxynucleotidyltransferase-mediateddUTP nick end labelling (TUNEL) assay, and further confirmedby the appearance of oligonucleosomal DNA ladders in senescentcoleoptile cells. Two nucleases (Nuc-a and Nuc-b) were detectedby in-gel-assay from proteins isolated from coleoptiles. Nuc-a,commonly observed in three cell death phases required eitherCa²⁺or Mg²⁺, whereas Nuc-b which appeared during senescencerequired both Ca²⁺and Mg²⁺. Both nucleases were strongly inhibitedby Zn²⁺. Copyright 2000 Annals of Botany Company Aerenchyma, rice, cell death, coleoptile, fragmentation, nuclease, Oryza sativa, senescence, split, submergence, TUNEL     

水稻体细胞无性系变异

水稻体细胞无性系变异研究取得了很大进展 ,获得了大量抗病、抗逆、优质、矮杆等突变体。对这些突变体遗传分析表明 ,大多数突变性状由 1对或 2对基因控制。水稻体细胞无性系变异的发生与基因型、性状、继代时间、培养方式等有关 ,并具有内在的机制 ,点突变和反转录转座子插入可能是引起水稻无性系变异的两个重要原因。     

Changes in Cytokinin Activity during Seed Germination in Rice (Oryza sativa L.)

Cytokinin activity was determined in dry mature rice seeds,in endosperm and embryo tissues 24, 48 and 72 over imbibitionand in radicles 96 h after germination. Cytokinins with chromatographicproperties similar to zeatin, zeatin riboside, zeatin glucosideand zeatin riboside glucoside were datected in embryo and endosperm,but only the latter two were detected in mature seeds. Cytokininactivity was low during early toges of germination. Qualitativeand quantitative changes in cytokinins were observed in bothembryo and endosperm. The presence of higher cytokinin activityin the endosperm than in the embryo during the first 24 h aftergermination suggests that the endosperm may supply cytokininsuntil the embryo is able to synthaize its own cytokinins. Thepossible significance of high cytokinin glucoside activity inthe embryo early during germination and high cytokinin activityin the radicle during the later stages is discussed. Oryza sativa L., rice, cytokinin, germination, seed     

Inhibition of Tonoplast and Plasma Membrane H$-ATPase Activity in Rice (Oryza sativa L.) Culture Cells by Local Anesthetics

Tonoplast and plasma membrane vesicles were prepared from rice(Oryza sativa L. var. Yuukara) culture cells with step sucrosegradient (30% and 42.9%, w/v) and/or step dextran T-70 gradient(1% and 8%, w/w) to determine the inhibition of tonoplast andplasma membrane AT-Pases by local anesthetics. The degree towhich the anesthetics inhibited these ATPases was of the followingorder: dibucaine>lidocainetetracaine>procaineGABA. Dibucaineranging in concentration from 0.2 nui to 2 mM inhibited tonoplastATPase activity more than plasma membrane ATPase, the half inhibitionsbeing 0.8 and 1.1 mM, respectively. The K_m values of tonoplastand plasma membrane ATPases were not affected by dibucaine,but various values were noted for V_max. Dibucaine inhibitedtonoplast and plasma membrane ATPases solubilized from 0.1%DOC pellet by n-octylglucoside and zwittergent 3–14, respectively.The addition of a phospholipid mixture (asolectin) to solubilizedboth ATPases had no effect on the inhibition by dibucaine. Thus,local anesthetics may act directly on the ATPase moiety withoutlipid mediation. (Received June 15, 1987; Accepted November 13, 1987)     

Penicillin Induction of Gibberellin and {alpha}-Amylase Biosynthesis in Rice Endosperm

Penicillin induces the synthesis of -amylase in embryoless riceendosperm and enhances the gibberellin-induced response. Penicillininduction of -amylase can be prevented by inhibitors of nucleicacid and protein synthesis, CCC and 2,4-DNP. A characteristic gibberellin-like activity in the extracts frompenicillin-treated endosperms becomes detectable after 12 hfrom the addition of penicillin. This gibberellin-like activityis located on paper chromatograms at the R_F typical for GA₃and its formation is blocked by CCC, an inhibitor of GA biogenesis.Glucose has no effect on the biosynthesis of either gibberellinor -amylase induced by penicillin. The time-course study of the levels of different constituentsshows that penicillin probably induces RNA and DNA synthesisin the first place, which results in gibberellin biosynthesis,which in turn stimulates the synthesis of -amylase. The possiblemode of action of penicillin in higher plants is discussed.     

Blue Light Mediated Regulation of {beta}-Amylase Activity in Mustard (Sinapis alba L.) Cotyledons

In the cotyledons of mustard (Sinapis alba L.) seedlings grownunder continuous blue light, ß-amylase activity increasedbetween 42–96 h from sowing and thereafter the ß-amylaseactivity abruptly declined. Preirradiation with blue light didnot increase the responsivity of the subsequent phytochrome-mediatedß-amylase increase in the cotyledons. The run-offkinetics of ß-amylase increase in seedlings transferredfrom blue light to darkness indicated that the components ofthe blue light-triggered signal chain are kinetically identicalto those of the phytochrome-mediated signal chain. Far-red reversibilityexperiments showed that the above blue light response is eithermediated by phytochrome directly or the blue light photoreceptorrequires the coaction of phytochrome. (Received November 11, 1987; Accepted March 23, 1988)