Floret Initiation and Development in Spring Wheat (Triticum aestivum L.)

The number and developmental stages of florets were determinedin each spikelet of the spike in the main shoots of spring wheat.Samples were taken frequently from plants grown in a phytotronand in a nitrogen application field-test. Ten stages of development,from floret initiation until anthesis, were recognized and described. Inter-spikelet variation in the total number of initiated floretswas rather small. However, the number of florets at advancedstages of development, as well as the number of grains, washighest in the central spikelets in which florets initiatedfirst. Floret initiation did not proceed beyond spike emergence,whereafter the distal florets and the spikelet apex degenerated.Grain-set was restricted to florets which had developed at leastto the stage of visible anther lobes at spike emergence. Thenumber of these florets was increased significantly by nitrogenapplication. Wheat, Triticum aestivum L., spikelet, floret, grain set, nitrogen     

Free Polyamines in Senescing Wheat(Triticum aestivum L.)

The free polyamine content of flag leaves, peduncles, rachis,glumes, and grains of wheat (Triticum aestivum L., cv. Castell)plants, ripening under field conditions, has been investigatedduring three consecutive growing seasons. Putrescine was quantitativelythe most important of all polyamines detected in these organs.Concentrations were highest in the grains, glumes and flag leaves.No correlation was found between polyamine content and the onsetof senescence of flag leaves and other organs. Excised primaryleaves, however, showed a decrease in polyamine content in thedark and also in light/dark cycles, but in the latter case onlyafter an initial increase. Sink removal of otherwise intactwheat plants caused an accumulation of putrescine in flag leavesat the later stages of senescence, whereas removal of all otherleaves was without any significant effect. Putrescine was alsorecovered in phloem-exudate samples collected throughout theperiod of grain development. In both grains and glumes, peakconcentrations of polyamines were found early during seed development. Key words: Triticum aestivum, polyamines, ripening, senescence     

Effects of Boron Deficiency on Anther Development and Floret Fertility in Wheat (Triticum aestivum L. 'Wilgoyne')

Boron (B) deficiency limits reproductive growth more than vegetativegrowth in cereals such as wheat. The purpose of the presentstudy was to identify the critical stages of anther developmentof wheat (‘Wilgoyne’) during which B deficiencycauses a significant and irreversible decrease of floret fertilityin order to formulate timely measures for correcting or preventingthis problem. Withdrawing B from the rooting medium for 3 dbetween premeiotic interphase through meiosis to late tetrad,limited anther elongation and resulted in the loss of pollenviability. The negative effects of B withdrawal on anther lengthsuggest that the role of B in reproductive cell walls is similarto that in vegetative ones. The results indicated that as moreflorets reached meiosis within the period of B withdrawal, thelower was floret fertility and the number of grains set in awhole ear. For the whole ear, the critical period during whichB deficiency causes maximal and irreversible damage to floretfertility was about 7 d, extending from the early emergenceof the flag leaf to 2–3 d after its full emergence. Theresults suggest that there are two phases of pollen developmentsensitive to boron deficiency: the period from premeiotic interphasethrough meiosis to late tetrad was the most sensitive stageof microsporogenesis in wheat while the period from mitosis-Ito II during which starch accumulation occurred in pollen grainswas less sensitive. Copyright 2000 Annals of Botany Company Anther, boron (B) deficiency, floret fertility, Grain Set Index (GSI), pollen, wheat (‘Wilgoyne’)     

Colchicine-induced Paracrystals in Root Cells of Wheat (Triticum aestivum L.)

Tubulin conformations other than microtubules in the meristematiccells of wheat roots grown in the presence of 2 mM colchicinesolution were investigated by immunofluorescence and electronmicroscopy. In the affected cells microtubules disappeared andwere replaced by tubulin fluorescent strands that occurred inthe cortical cytoplasm. With increasing time of exposure tocolchicine the tubulin strands became better organized and occurredalso in the subcortical cytoplasm and finally they were restrictedto the area around the nucleus. In prophase and preprophasecells thick strands occupied the cortical cytoplasmic zone wherein normal cells a preprophase microtubule band (PMB) was expectedto be assembled. In the colchicine-treated cells electron microscopy revealedan accumulation of paracrystalline aggregates, which initiallyoccurred along the cell wall and later deeper in the cytoplasm,in the perinuclear regions and the cytoplasmic invaginationsof the nucleus. In transverse planes the paracrystalline strandsappear to consist of hexagonal subunits in a 'honeycomb' arrangement,while in longitudinal and oblique sections they exhibit variableimages. Since their distribution coincides with that of thetubulin strands visualized by immunofluorescence, they are consideredto be the same structure. Therefore, the paracrystals consistof, or at least contain, tubulin. They are most likely to bepolymers of tubulin-colchicine complexes.Copyright 1995, 1999Academic Press Wheat roots, colchicine, immunofluorescence, electron microscopy, tubulin paracrystals, Triticum aestivum L     

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane-associated 3′-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products.     

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, α-mannosidase, and β-N-acetylglucosamidase were found in the vacuole to varying degrees, but no β-glucosidase was localized in the vacuole.     

Gibberellic Acid Regulates Cell Wall Extensibility in Wheat (Triticum aestivum L.)

Mutations (Rht genes) blocking sensitivity to gibberellic acid (GA) were used to examine phytohormone mediated cell wall expansion in wheat (Triticum aestivum L.). Irreversible extensibility of immature leaf segments, as determined by stress/strain (instron) measurements, declined with Rht gene dose. Exogenous GA₃ significantly increased wall extensibility in the nonmutant controls but had no effect on the near-isogenic GA-insensitive genotypes. Furthermore, ancymidol, an inhibitor of gibberellin biosynthesis, diminished wall extensibility in the nonmutant control. Extensibility of immature segments was highly correlated with mature leaf sheath length (R = +0.95). The results indicate that wall yielding properties of expanding wheat leaves are associated with leaf cell expansion potential and that GA is involved in the determination of those properties.     

Effect of Seed Damage on Growth and Development in Pea (Pisum sativum L.) and Wheat (Triticum aestivum L.)

The effect of damage to the food storage of the seed on theensuing plant was compared in cultivars of two species differingin seed structure, the Greenfeast pea with cotyledons and theGabo wheat with endosperm. Partial removal of storage tissue slightly retarded growth ratein both species and slowed development rate in wheat. Completeremoval lowered the germination rate, drastically slowed thegrowth rate of the survivors for the first 20 d after sowingand lowered the development rate throughout the life cycle. This treatment doubled the time to flower initiation (20 d later)compared with the control, thus indicating the promotive roleof both cotyledon and endosperm in the progress of the shoottowards the reproductive state. The number of vegetative nodes in the pea was lowered by twonodes whereas it was raised by one in the wheat.     

Osmotic Stress-Induced Growth Suppression of Dark-Grown Wheat (Triticum aestivum L.) Coleoptiles

Application of 60 mM polyethylene glycol (PEG) to dark-grownwheat (Triticum aestivum L.) roots substantially reduced growthof coleoptiles. However, when PEG was removed, the growth rateof these coleoptiles greatly increased. Cell walls of stressedcoleoptiles remained loosened as compared with those of unstressedones. The osmotic potential of the stressed coleoptiles decreasedto that of the 60 mM PEG solution. On the other hand, the extentof decrease in the osmotic potential of stressed roots was smallerthan that of stressed coleoptiles. The osmotic potential difference() between the cell sap and the incubation medium of stressedroots was substantially higher than that of unstressed ones.The amount of ink moved from roots, where it was applied, tothe apical region of coleoptiles was significantly reduced underosmotic stress conditions. When water was exogenously appliedto abraded coleoptiles, the growth of these stressed coleoptileswas greatly promoted. These results suggest that inhibitionof coleop-tile growth under osmotic stress conditions is notdirectly related to a decrease in cell wall extensibility orto loss of the capacity to maintain osmotic potential gradients,but is caused by the reduction of the water supply from theroots to the coleoptiles. (Received August 26, 1996; Accepted December 18, 1996)     

抗除草剂草甘膦EPSPs基因在小麦中的转化

通过基因枪法,用抗除草剂草甘膦的ＥＰＳＰｓ基因转化小麦京花１号的幼穗约１０００个,及幼胚约８００个,经草甘膦选择后分别获得３８株和４株再生植株。这些再生植株经ＰＣＲ和（或）Ｓｏｕｔｈｅｒｎ杂交证明,其中有部分再生植株的基因组中稳定整合了外源ＥＰＳＰｓ基因,并且转化体中部分是可育的。首次用实验证明,抗除草剂草甘膦的ＥＰＳＰｓ基因作为单子叶禾谷类作物小麦基因转化的选择标记是行之有效的。     

Light-dependent Proton Excretion of Wheat (Triticum aestivum L.) and Maize (Zea mays L.) Roots

We investigated the change of root net proton excretion of seedlings of Triticum aestivum L. and Zea mays L. with daily variation of illumination using a multi-channel pH-stat system. We found an increase of net proton excretion during darkness and a drop after the beginning of illumination. Inhibition of carotenoid biosynthesis by norflurazone and photooxidation of chlorophylls did not change the periodicity or its induction. The induction of diurnal periodicity was possible with blue, green and red light. After induction the oscillation of net proton excretion continued for at least two cycles under constant light. We conclude that net H⁺ excretion of wheat and maize roots may be regulated by an endogenous clock or by a signal from the leaves. The nature of such a hypothetical signal remains unknown.     

Movement of C-labeled Sugars into Kernels of Wheat (Triticum aestivum L.)

An anatomical study of wheat (Triticum aestivum L.) kernels 14 days after anthesis revealed that the tracheary elements of the pericarp vascular bundle are not in direct continuity with those of the rachilla. The phloem was continuous from the rachilla into the crease of the pericarp.     

High Frequency Divisions in Leaf Base Protoplasts of Wheat (Triticum aestivum L.)

Protoplasts were isolated from the basal meristematic region of leaves from 6-day-old seedlings of wheat (Triticum aestivum). Protoplasts divided when cultured on MS medium (as agarose beads) in presence of nurse tissue. Donor seedlings when grown on BAP-supplemented MS medium were found to be considerably superior for protoplast isolation and culture than when grown on MS basal medium, in terms of protoplast viability, cell wall formation and cell division frequency. In addition, reduction of ammonium content of the culture medium, together with a dark Incubation, led to a high protoplast division frequency of about 70%. Microcolonies of 10-to 12-celled stages were obtained in Triticum aestivum, varieties HD 2329, HD 2285, Kalyan Sona, Arjun and CPAN 1676.     

Nitrogen and Photosynthesis in the Flag Leaf of Wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L. cv Yecora 70) plants were grown with various concentrations of nitrate nitrogen available to the roots. Sampling of flag leaves began after they had reached full expansion and continued throughout senescence. Rates of gas exchange, ribulose-1,5-bisphosphate (RuP₂) carboxylase activity, and the amounts of chlorophyll, soluble protein, nitrogen, and phosphorus were determined for each flag leaf. Rate of CO₂ assimilation was uniquely related to total leaf nitrogen irrespective of nutrient treatment, season, and leaf age. Assimilation rate increased with leaf nitrogen, but the slope of the relationship declined markedly when leaf nitrogen exceeded 125 millimoles nitrogen per square meter. Chlorophyll content and RuP₂ carboxylase activity were approximately proportional to leaf nitrogen content. As leaves aged, RuP₂ carboxylase activity and calculated Hill activity declined in parallel. With normal ambient partial pressure of CO₂, the intercellular partial pressure of CO₂ was always such that rate of assimilation appeared colimited by RuP₂ carboxylation and RuP₂ regeneration capacity.

The initial slope of rate of CO₂ assimilation against intercellular partial pressure of CO₂ varied nonlinearly with carboxylase activity. It is suggested that this was due to a finite conductance to CO₂ diffusion in the wall and liquid phase which causes a drop in CO₂ partial pressure between the intercellular spaces and the site of carboxylation. A double reciprocal plot was used to obtain an estimate of the transfer conductance.

     

Elicitor-Induced Cinnamyl Alcohol Dehydrogenase Activity in Lignifying Wheat (Triticum aestivum L.) Leaves

The substrate-specific induction of wheat (Triticum aestivum L. cv Fenman) leaf cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was examined in relation to its role in regulating the composition of defensive lignin induced at wound margins. Treatment of wounds with a partially acetylated chitosan hydrolysate or spores of the nonpathogen Botrytis cinerea elicited lignification at wound margins and invoked significant increases in phenylalanine ammonia-lyase (EC 4.3.1.5), peroxidase (EC 1.11.1.7), and CAD activities. The substrate-specific induction of CAD with time was determined in elicitor-treated leaves and in excised lignifying wounds. In whole leaf extracts no significant increases in p-cou-maryl and coniferyl alcohol dehydrogenase activities were detectable, but a significant 5-fold increase in sinapyl alcohol dehydrogenase activity was evident 32 h after elicitor treatment. Similarly, fungal challenge resulted in elevated levels of only sinapyl alcohol dehydrogenase in whole-leaf extracts. In excised lignifying tissues p-coumaryl alcohol dehydrogenase levels were similar to those observed in healthy tissue. A small yet significant increase in coniferyl alcohol dehydrogenase was apparent, but the most dramatic increase occurred in sinapyl alcohol dehydrogenase activity, which increased to values approximately 10 times higher than the untreated controls. Our results show for the first time that CAD induction in lignifying tissues of wheat is predominantly attributable to highly localized increases in sinapyl alcohol dehydrogenase activity.     

Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)

Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M(2) mutants in a common wheat cultivar 'Jinmai 47'. Numerous phenotypes with altered morphological and agronomic traits were observed from the M(2) and M(3) lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.     

小麦幼苗期水分胁迫所诱导基因表达谱的初步分析

利用抑制差减杂交(Suppression Subtractive Hybridization,SSH)和高密度点阵膜技术研究小麦2叶幼苗期水分胁迫诱导表达基因。通过筛选具有1530个克隆的SSH文库,获得181个阳性克隆。序列同源性比较和功能查询结果发现,83．2％的水分胁迫诱导表达基因分别与不同逆境胁迫条件下表达的基因具有较高的同源性,这些基因在生物体内的功能都是直接或间接对细胞遭受逆境胁迫起保护作用。其中17个EST未找到同源性较高的匹配序列,已经在GenBank注册。用反向Northern、RT-PCR和Northern进一步检验所获得的功能已知EST,初步建立了小麦幼苗期水分胁迫诱导的基因表达谱。     

The Morphology and Development of Cross Veins in the Leaves of Bread Wheat (Triticum aestivum L.)

In the leaves of bread wheat Triticum aestivum L. the longitudinalvascular bundles are linked by small transverse bundles Pairsof similar small vascular bundles also link the upper ends ofminor longitudinal bundles to their neighbours in a Y-shapedarrangement The cross-vein procambial strands arise from unexpanded cellsof one layer of the mesophyll tissue. Lines of these cells connectone longitudinal procambial strand to the next The procambialcells subsequently undergo two tangential divisions to producecells which differentiate to form the conducting and parenchymatouselements of the mature cross veins. Anomalous cross veins are sometimes found. possible modes oforigin of these anomalous cross veins are considered.