Differential expression of <Emphasis Type="Italic">Histone H3</Emphasis> gene in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze) suggests its role in growing tissue

Histone proteins are integral part of chromatin and their expression is typically linked to DNA replication in the S phase of cell cycle. Histone H3 is one of the four histones, along with H2A, H2B and H4, which forms the eukaryotic nucleosome octomer core. Using differential display of mRNA and rapid amplification of cDNA ends (RACE), a full-length Histone H3.1 cDNA (CsH3) was isolated from tea leaves. The open reading frame consisted of 411 nucleotides and deduced amino acid sequence comprised of 136 amino acid residues. CsH3 shared 79-82% and 98% identity at nucleotide and amino acid sequences, respectively with Histone H3 isolated from other plant species. During active-growth period of tea, higher expression was observed in apical buds that decreased gradually with increasing age of the leaf. During dormancy season, the expression of CsH3 was severely down-regulated in all the leaves studied. CsH3 was found to be down regulated in response to drought stress and ABA treatment and up-regulated by GA(3) treatment. A positive association of CsH3 abundance with active cellular growth suggested its role in plant growth and development.     

茶树CPP转录因子家族的全基因组鉴定及分析

CPP(cysteine-rich polycomb-like protein)转录因子广泛存在于动植物中,是一类成员数目较少的转录因子家族,在调控植物生长发育和响应非生物胁迫中起重要作用。该研究通过对茶树基因组系统的鉴定,获得10个CsCPP转录因子均具有典型的CXC结构域。系统发育分析将CsCPP家族成员分为4类(A^D),且大部分成员与葡萄在进化关系上更为接近。A类和C类成员的CXC结构域分布在蛋白序列的N端,而B类和D类成员分布在C端。茶树组织表达分析表明,CsCPP转录因子在生长活跃的顶芽和嫩叶中普遍高表达,不同组织的表达水平排序为:顶芽和嫩叶>根和茎>成熟叶和果>老叶和花。启动子分析发现了CsCPP家族成员的启动子区域存在大量的ABA和干旱响应元件;干旱处理下,6个CsCPP成员的表达水平均有不同程度上调,其中4个成员在ABA处理后表达迅速上调,表明CsCPP转录因子可能在ABA介导的干旱胁迫响应中起正调控作用;低温处理下,大部分CsCPP成员的表达均有轻微下调,而CsCPP 2和CsCPP 6的表达水平在6 h达到顶峰,表达量均超过2倍。研究结果为进一步发掘茶树CPP转录因子家族的功能奠定了基础。     

Agrobacterium-mediated transformation of Lupinus mutabilis L. using shoot apical explants

A procedure for regenerating plants of Lupinus mutabilis from shoot apices, from which the leaf primordia and initial cell layer(s) of the apical meristem were removed, has been used to generate transgenic plants following Agrobacterium tumefaciens-mediated gene delivery. Transformation competent cells, from which buds developed, were located at the periphery of the apical meristem. Kanamycin resistant plants were obtained which expressed β-glucuronidase activity. Integration of the neomycin phosphotransferase II and β-glucuronidase genes into the genomes of transgenic plants was confirmed by non-radioactive DNA-DNA hybridisation. This is the first report of the generation of transgenic plants in L. mutabilis.     

Photoregulation of growth and branching of plum shoots: Physiological action of two photosystems

Summary Plum shoot proliferation was investigated in terms of two distinct processes: axillary bud differentiation and axillary shoot development. Results showed that light quality influenced bud differentiation and interacted with apical dominance in determining shoot outgrowth, resulting in a differentiated structure of shoot clusters and type of branching. Results suggested that blue light, acting through its photoreceptor, increased the number of axillary buds differentiated from apical meristem, but did not remove the apical dominance. Red light removed apical dominance, while reducing the formation of axillary buds; both events appeared to be dependent on the putative amount of phytochrome active form, and independent of light photon fluence rate. On the contrary, blue light action appeared to be dependent on photon fluence rate. In addition, apparent blue-red interactions related to photomorphogenic events fit an antagonistic model for branching regulated by light via cryptochrome and phytochrome photoreceptors. Our results show that the dynamics of shoot cluster development is the product of two events: the formation of new axillary buds and their release from apical dominance.     

Cryopreservation of in vitro-grown apical meristems of lily by vitrification

Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Lilium japonicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25°C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25°C for 20 min or at 0°C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40°C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0°C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige & Skoog (1962) medium - PVS2 vitrification solution     

Accumulation of 32P and [14C]sucrose in decapitated and intact shoots of the fern Davallia trichomanoides blume

The transport and accumulation of ³²P and [¹⁴C] sucrose in decapitated and intact shoot segments of the fern Davallia were studied. The apical buds of intact shoots and the expanding buds of shoots decapitated 4 weeks before application are major sinks for these nutrients. Decapitation results in a shift of ¹⁴C accumulation from the apex to the lateral buds within 36 h. This shift can be reversed in shoots decapitated for 12 h by replacing the apex. Increased ¹⁴C accumulation into the stump region occurs when decapitated shoot segments are treated with indole-3-acetic acid, and decreased label accumulation into the apical region results when intact shoots are treated with 2,3,5-triiodobenzoic acid.     

Developmental study onHypolepis punctata (Thunb.) Mett.

The origins of the first and second petiolar buds ofHypolepis punctata were clarified in relation to the early development of the leaf primordium, which arises from a group of superficial cells of the shoot apical meristem. One of these superficial cells produces a two-sided leaf apical cell which subsequently cuts off segments to make a well-defined cell group, called here the leaf apical cell complex, on the distal part of the leaf primordium. Meanwhile, cells surrounding the leaf apical cell complex also divide frequently to form the basal part of the leaf primordium. Two groups of basal cells of the leaf primordium located on the abaxial and the adaxial sides initiate the first and the second petiolar buds, respectively. The initial cells are usually contiguous to the leaf apical cell complex, constructing the abaxial and adaxial flanks of the very young leaf primordium. However, the first petiolar bud sometimes develops from cells located farther from the leaf apical cell complex. These cells are derived from those originally situated in the peripheral region of the shoot apical meristem. This study was supported by a Grant-in-Aid for Encouragement of Young Scientists by the Ministry of Education, Science and Culture, of Japan No. 474322 in 1979.     

Poly(a)+ rna during vegetative development ofacetabularia peniculus

Summary In the juvenile stage, the diploid giant-celled green algae Acetabularia spp. are differentiated into an upright stalk and an irregularly branched rhizoid. Early amputation and grafting experiments as well as biochemical and molecular analyses have shown that mRNA (as poly(A)⁺ RNA) is continuously supplied from the primary nucleus in the rhizoid and accumulates in the stalk apex. In the present study, localization of poly(A)⁺ RNA in the juvenile stage of theAcetabularia peniculus was investigated by fluorescent in situ hybridization using oligo(dT) as a probe. The signal was localized in the apical cytoplasm and, in addition, multiple longitudinal striations throughout the stalk and rhizoid cytoplasm. A large portion of the poly(A)⁺ RNA striations exhibited structural polarity, broadened at one end and gradually thinned toward the other end. Some of the striations in the rhizoid cytoplasm were continuous with a zone of signal in the area of the perinuclear rim. The poly(A)⁺ RNA striations were associated with thick bands of longitudinal actin bundles which run through the entire length of the stalk. Cytochalasin D caused fragmentation of the actin bundles and irregular distribution of the fluorescent signal. We suggest that the poly(A)⁺ RNA striations constitute a hitherto unknown form of packaged mRNA that is transported over large distances along the actin cytoskeleton to be stored and expressed in the growing apex.     

In nature dormant buds and in vitro dormant-like buds ofFraxinus excelsior L.

Summary Microcuttings ofFraxinus excelsior, sampled from adult trees during the period of cell cycle blockage of bud in G_0–1, developed long rejuvenated sprouts on the Woody Plant Medium (WPM) with benzylaminopurine (BAP) (4.0 mg/l) and indolebutyric acid (IBA) (0.03 mg/l). These sprouts had the ability to enter a resting period, building dormant-like buds when maintained on the original WPM. Sprouts developed from subcultures also entered a resting period without any transfer. Comparison of in nature buds in active growth and dormancy with buds of growing sprouts and in vitro dormant-like buds revealed similarity in behaviour at the shoot apical level. In particular, in dormant-like buds in a constant environment, shoot apical functioning was suppressed while the cell cycle of the shoot apex was blocked at the G_0–1 phase, like in nature dormant buds.Abbreviations a.u. arbitrary unit - BAP benzylaminopurine - CF cumulative frequency - d₁, d₂ diameters of the shoot apex - IBA indolebutyric acid - P_n last opposite primordia of range n - WPM woody plant medium     

Floral-stimulus activity in tobacco stem pieces

The growth patterns of axillary buds of dayneutral tobacco (Nicotiana tabacum L. cv. Wisconsin 38) plants were assessed by using expiants of single buds attached to leafless stem cuttings and allowing the buds to grow to flowering without additional manipulation. Buds located 5, 10 and 15 nodes below the inflorescence were employed. For a given bud position, when a cutting had few internodes the growth pattern of a bud tended to fall into one of two groups: buds that produced few-noded shoots and buds that produced many-noded shoots. For example, in a group of 13 cuttings composed of bud 5 with 2 associated internodes, 11 buds produced 14.2 nodes (range, 11–17) and 2 buds produced 32.0 nodes (range, 30–34). As the number of internodes on the cutting increased, the number of buds producing few-noded shoots increased and the number of nodes produced decreased (e.g. in contrast to the data above, all 5th buds with 6 internodes produced 12.8 nodes; range 11–15). When cuttings from the 3 positions had the same number of internodes, the more apical cuttings had buds that produced fewer nodes (e.g. for cuttings with 6 internodes all 5th buds produced 12.8 nodes, all 10th buds produced 15.5 nodes and 85% of 15th buds produced few-noded shoots with 19.3 nodes). The number of nodes produced by a bud was a function of the original position of the stem piece and not the original position of the bud. That is, bud 5 associated with the 6 internodes below it produced 12.8 nodes and bud 10 associated with essentially the same 6 internodes (i.e. the 6 above it) produced 12.9 nodes while bud 10 associated with the 6 internodes below it produced 15.5 nodes. Thus, the number of nodes produced by a bud was dependent upon the original main-axis position of the cutting as well as the number of internodes on the cutting. Buds forced to grow out in situ on main axes devoid of leaves produced substantially more nodes than similar buds on cuttings. Buds isolated without associated internodes produced many-noded plants with a number of nodes similar to that of plants grown from seed. The simplest interpretation of these data is that stem pieces contain floral-stimulus activity and that this activity is present in a gradient with the highest activity being located in the apical part of the stem.We thank Susan Smith and Harry Roy (Rensselaer) for comments, and the National Science Foundation for financial support (IBN-9003739 to C.N.M.).     

Effect of phytoregulators and physiological characteristics of the explants on micropropagation of Maytenus ilicifolia

Micropropagated shoots of Maytenus ilicifolia Mart. were obtained from axillary buds cultured in Murashige & Skoog medium supplemented with 13.3 M 6-benzyladenine (BA). Addition of 1.1 M 1-indole-3-acetic acid (IAA) to the medium increased shoot elongation. The number of shoots formed was influenced by BA concentration, degree of juvenility of the explant, and by bud explant position on the stem. Cultures of buds taken from stem parts located close to the shoot tip yielded more callus than shoots, whereas axillary buds at distant positions from the apical bud yielded more shoots.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic-acid     

Changes in adenine-nucleotide content in the apical bud of Sinapis alba L. during floral transition

The total adenylate pool of the apical buds of vegetative plants of Sinapis alba L. continuously grown in short days fluctuates over a 24-h cycle with the minimum occurring at the end of the dark period. In the buds of plants induced to flower by a single long-day treatment, total adenylate pool increases above the control level 16 h after the start of the long day, resulting mainly from a rise in ATP and ADP contents. This occurs 6 h after the increase in the soluble carbohydrate content previously shown to occur in the apical buds of plants induced to flower (Bodson 1977, Planta 135, 19–23). A transient rise of the energy charge occurs 22 h after the start of the inductive long day.Abbreviations LD long day - SD short day     

Partial purification and characterization of mRNAs encoding glycollate oxidase and catalase

Polyadenylated mRNA was prepared from etiolated and greening leaves of Lens culinaris and cotyledons of Cucumis sativus during the transition from etiolated to photoautotrophic stage. These mRNA preparations were used to identify, by translation in vitro, the precursor forms of glycollate oxidase and catalase, both enzymes being markers of microbodies. The level (per fresh weight) of translatable RNA coding for glycollate oxidase was found to increase ten fold during the first 3 d of illumination of etiolated leaves. For catalase mRNA activity, this increase was less pronounced. Characterizing the products of in-vitro translation directed by the mRNA prepared, we observed a 43-kDa species of glycollate oxidase and a 56-kDa species of apo-catalase. Limited proteolysis of the in-vitro-formed proteins and comparison with the respective mature enzymes present in vivo revealed differences between the cucumber and the lens protein but not between the monomeric precursor and the subunit of mature glycollate oxidase from Lens culinaris. Messenger RNA coding for glycollate oxidase was highly purified by electrophoresis on low-melting-point agarose in the presence of methylmercuric hydroxide. The size of the mRNA was determined to be 1.47 kb. By this procedure, the mRNA for glycollate oxidase in the subfraction could be enriched in such a way that the activity, assayed by translation in a reticulocyte lysate, amounted to 30% of the total translation activity.Abbreviations PAGE polyacrylamide gel electrophoresis - poly(A)⁺ RNA polyadenylated RNA - SDS sodium dodecyl sulfate     

Chromosome doubling of 2x Solanum species by oryzalin: method development and comparison with spontaneous chromosome doubling in vitro

A potato breeding scheme implies the possibility of ploidy level manipulation either by reducing the chromosome number of cultivars from 48 to 24 to be able to cross them with diploid related species or by doubling diploid material to reach the generally optimal tetraploid level. In vitro spontaneous chromosome doubling is widely used but can lead to somaclonal variation. Since oryzalin has proven to be efficient as a chromosome doubling agent on potato cell suspension cultures, we tried this herbicide on various Solanum species and interspecific diploid hybrids. A 24 h dip in a 28.8 M aqueous oryzalin solution applied on apical buds was the most efficient treatment in terms of tetraploid plant production (mean = 4.1 tetraploid plants for 10 treated buds over 4 genotypes). However 50–100% of the regenerated tetraploid plants acclimatized after in vitro treatment proved to be chimaeric. Consequently, a selection procedure in the progeny was necessary to obtain real and stable doubled clones and final yields were low. This technique is easy to apply and could be a good alternative to chromosome doubling by spontaneous in vitro regeneration in the case of refractory genotypes especially where somaclonal variation is problematic. Percentage of tetraploids among the regenerated plants varied from 6 to 29% with the oryzalin doubling technique while it varied from 20 to 78% by in vitro spontaneous doubling for five diploid genotypes. An observation of the progeny indicated that chimaeras were more frequent using oryzalin (50–100% of the initially supposed tetraploid plants) than when chromosomes doubled spontaneously (4–67% of the initially supposed tetraploid plants).     

Does ice crystal formation in buds explain growth disturbances in boron-deficient Norway spruce?

Loss of apical dominance in boron-deficient trees has been suggested to be due to frost damage of terminal buds and leaders. Excessive nitrogen (N) supply can exacerbate boron (B) deficiency by the dilution-effect. N may also have direct effects on winter hardiness. We studied frost hardening of buds of Norway spruce (Picea abies L. Karst.) in healthy-looking trees and in trees with growth disturbances. The effect of B and N on frost hardiness was studied in a factorial fertilisation experiment during cold acclimation. Frost hardiness was determined by differential temperature analysis (DTA) and scoring of visual damage. In a DTA profile of apical buds with a piece of stem, low-temperature exotherm (LTE) predicted bud injury, while two of the observed high-temperature exotherms and two of the observed intermediate-temperature exotherms were non injurious. Appearance of LTE followed changes in air temperature. The risk of frost damage was not affected by fertilisation treatments or previously observed growth disturbances. However, when the bud structure was deformed by severe B deficiency, the supercooling ability disappeared. Such buds are probably killed by freezing in nature and therefore, frost damage may play a secondary role in the development of growth disturbances.     

Development of shoot inHydrangea macrophylla II. sequence and timing

The development of axillary buds, terminal buds, and the shoots extended from them was studied inHydrangea macrophylla. The upper and lower parts in a nonflower-bearing shoot are discernible; the preformed part of a shoot develops into the lower part and the neoformed part into the upper part (Zhou and Hare, 1988). These two part are formed by the different degrees of internode elongation at early and late phases during a growth season, respectively. Leaf pairs in the neoformed part of the shoot are initiated successively with a plastochron of 5–20 days after the bud burst in spring. The upper axillary buds are initiated at approximately the same intervals as those of leaf pairs, but 10–30 days later than their subtending leaves. Changes in numbers of leaf pairs and in lengths of successive axillary buds show a pattern similar to the changes in internode lengths of the shoot at the mature stage. The uppermost axillary buds of the flower-bearing shoot often begin extending into new lateral shoots when the flowering phase has ended. The secondary buds in terminal and lower axillary buds are initiated and developed in succession during the late phase of the growth season. Internode elongation seems to be important in determining the degrees of development of the axillary buds. Pattern of shoot elongation is suggested to be relatively primitive. Significances of apical dominance and environmental conditions to shoot development are discussed.     

Automated sampling and RNA isolation at room temperature for measurements of circadian rhythms in Chlamydomonas reinhardtii

The development of techniques allowing the unattended collection of RNA from cell samples at room temperature makes practical accurate and facile monitoring of circadian rhythms in Chlamydomonas reinhardtii. The utility of these methods was demonstrated by collecting RNA samples for three days from cells maintained in continuous darkness. Every hour, cells were automatically collected and lysed with buffer containing SDS and proteinase K. Samples were maintained at room temperature with little or no evidence of degradation of RNA. Strong, non-damping circadian rhythms of cab mRNA abundance were measured. Free-running rhythms of about 24 h were measured from cultures maintained at 16, 20, 25 and 30 °C, thus demonstrating temperature compensation of circadian period. Simultaneous collections from cultures previously entrained to 12 h light/12 h dark cycles of opposite phase displayed circadian rhythms of cab mRNA abundance that were in phase with their previous entraining light cycles. Thus, this result suggests that the measured circadian rhythms of cab mRNA abundance was not an artifact of the collection procedure.