Seasonal shifts in dormancy status, carbohydrate metabolism, and related gene expression in crown buds of leafy spurge

Crown buds of field-grown leafy spurge (Euphorbia esula L.) were examined to determine relationships between carbohydrate metabolism and gene expression throughout para-, endo-, and eco-dormancy during the transition from summer, autumn, and winter, respectively. The data indicates that endo-dormancy plays a role in preventing new shoot growth during the transition from autumn to winter. Cold temperature was involved in breaking endo-dormancy, inducing flowering competence, and inhibiting shoot growth. An inverse relationship developed between starch and soluble sugar (mainly sucrose) content in buds during the shift from para- to endo-dormancy, which continued through eco-dormancy. Unlike starch content, soluble sugars were lowest in crown buds during para-dormancy but increased over two- to three-fold during the transition to endo-dormancy. Several genes (AGPase, HK, SPS, SuSy, and UGPase) coding for proteins involved in sugar metabolism were differentially regulated in conjunction with well-defined phases of dormancy in crown buds. Marker genes for S-phase progression, cell wall biochemistry, or responsive to auxin were also differentially regulated during transition from para-, endo-, and eco-dormancy. The results were used to develop a model showing potential signalling pathways involved in regulating seasonal dormancy status in leafy spurge crown buds.     

Changes in the expression of carbohydrate metabolism genes during three phases of bud dormancy in leafy spurge

Underground adventitious buds of leafy spurge (Euphorbia esula) undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy. In this study, relationships among genes involved in carbohydrate metabolism and bud dormancy were examined after paradormancy release (growth induction) by decapitation and in response to seasonal signals. Real-time PCR was used to determine the expression levels of carbohydrate metabolism genes at different phases of bud dormancy. Among differentially-regulated genes, expression of a specific Euphorbia esula β-amylase gene (Ee-BAM1) increased 100-fold after growth induction and 16,000-fold from July (paradormancy) to December (ecodormancy). Sequence data analysis indicated that two genes, Ee-BAM1 and Ee-BAM2, could encode this β-amylase. However, real-time PCR using gene-specific primer pairs only amplified Ee-BAM1, indicating that Ee-BAM2 is either specific to other organs or not abundant. The deduced amino acid sequences of these two genes are very similar at the N-terminal but differ at the C-terminal. Both contain a nearly identical, predicted 48-amino acid plastid transit peptide. Immunoblot analyses identified a 29 kD (mature Ee-BAM1 after cleavage of the transit peptide) and a 35 kD (unprocessed EeBAM1) protein. Both 35 and 29 kD proteins were constitutively expressed in growth-induced and seasonal samples. Immunolocalization indicated that Ee-BAM1 is in the cytosol of cells at the shoot tip of the bud. Ee-BAM1 also surrounds the amyloplasts in mature cells toward the base of the bud. These observations suggests that Ee-BAM1 may have dual functions; serving as reserve protein in the cytosol and as a degrading enzyme at the surface of amyloplasts.     

Dormancy and Flowering Are Regulated by the Reciprocal Interaction Between Cytokinin and Gibberellin in <Emphasis Type="Italic">Zantedeschia</Emphasis>

Floral productivity of Zantedeschia is dependent on the conversion rate of buds to shoots, which is controlled by varying intensities of para- (apical dominance), endo- (dormancy), and ecodormancy. We present evidence of cross-talk between cytokinin and gibberellin in their complementary roles to alleviate bud dormancy and enhance flowering in a perennial geophyte. We assessed the impact of cytokinin and gibberellin, applied alone and in sequential combinations, on bud fate during three phases along the ontogeny of growth, which coincide with the progressive transition of buds from apical dominance to dormancy. Given that cytokinin can stimulate branching and gibberellin can induce flowering in Zantedeschia, we measured these phenotypic responses as parameters of bud commitment. The efficacy of cytokinin alone to stimulate branching declined with the transition to dormancy (phase 1 = 3.8 ± 0.2 shoots; phase 3 = 1.0 ± 0.3 shoots). To sustain branching during this transition, a sequential application of gibberellin was necessary. Gibberellin alone failed to stimulate branching. The efficacy of gibberellin alone to stimulate flowering diminished with the transition to dormancy. Any flowering during this transition occurred only after the sequential application of cytokinin. Cytokinin alone failed to stimulate flowering. Alleviating bud dormancy and enhancing flowering in Zantedeschia, achieved by the reciprocal cross-talk between cytokinin and gibberellin, contributes to the pool of evidence drawing common mechanisms between dormancy and flowering and may have commercial implications.     

Seasonal and micro-environmental factors controlling clonal propagation of mature trees of marwar teak [<Emphasis Type="Italic">Tecomella undulata</Emphasis> (Sm.) Seem]

A simple method has been developed for clonal propagation of mature trees of Tecomella undulata (Sm.) Seem, a medicinally important deciduous timber tree of hot arid regions, via multiple shoot proliferation from axillary buds after examining the role of season influences and physico–chemical conditions on micropropagation. Spring season (March–April) was the best period for contamination free establishment of explants and maximum sprouting of healthy axillary buds. Shoots proliferated directly from the explant nodes cultured on Murashige and Skoog’s medium containing cytokinins, BAP supporting better growth compared to kinetin during shoot induction as well as multiplication phase. Cytokinin concentration influenced the bud induction frequency and optimal response of 2.6 buds per explant was achieved in 86.66% explants on media supplemented with 10 µM BAP. Stunted shoot buds with excessive callus were observed when cytokinin concentration was increased beyond optimal levels. Ascorbic acid (50 mg/l), arginine and citric acid (25 mg/l each) were added to proliferation and multiplication media for reducing callus proliferation and better shoot growth. Among the media (B5, MS, NN, WPM and SH) tested, SH was best for shoot multiplication. Shoot cultures were multiplied by regular subculture of axillary shoots on SH medium containing 5.0 µM each of BAP and kinetin. Shoots produced roots when cultured on ½× SH medium + 10 μM IBA. Regenerated plantlets were successfully transferred to field after hardening and acclimatization. Genetic homogeneity of tissue culture raised plants was confirmed by generation of monomorphic DNA fragments with Start codon targeted and intersimple sequence repeat (ISSR) markers.     

Bud dormancy breaking affects respiration and energy balance of bilberry shoots in the initial stage of growth

Respiration and heat production in the shoots of bilberry (Vaccinium myrtillus L.) were studied at the beginning of growth after breaking bud dormancy by means of transfer of the shoots to indoor conditions (November–April) and upon natural sprouting in spring (May). The buds released from dormancy at the beginning of winter sprouted slower and showed lower respiratory activity than the buds that started growing in May. In May, cytochrome respiratory pathway in sprouting buds was 1.3 times more active than energetically ineffective alternative pathway, whereas activity of cytochrome pathway in December was 1.4 times lower as compared with the alternative. In November–December, the rate of heat evolution by the buds was 3–5 times lower than in April–May. In case of early breaking of bud dormancy, the share of respiration energy dissipated as heat was 30% on average. In the buds whose growth was induced later, the value of this parameter was twice as much. The ratio between heat evolution and respiration depended on temperature. High temperature more intensely activated heat evolution than respiration, which caused a decrease in the level of metabolic energy available for growth. In the temperature range of 5–15°C characteristic of the beginning of vegetation, the share of respiration energy dissipated as heat was 2–3 times lower than at 20–30°C, which reflects a great adaptability of V. myrtillus to climatic conditions of the region. Our data suggest that progression through a full cycle of winter dormancy is physiologically important for shoot growth. Early dormancy release brought about changes in respiration and energy balance of the shoots in the initial stage of extra-bud growth.     

Characterization, expression and function of DORMANCY ASSOCIATED MADS-BOX genes from leafy spurge

DORMANCY ASSOCIATED MADS-BOX (DAM) genes are related to AGAMOUS-LIKE 24 and SHORT VEGETATIVE PHASE genes of arabidopsis and are differentially regulated coordinately with endodormancy induction and release in buds of several perennial plant species. DAM genes were first shown to directly impact endodormancy in peach where a deletion of a series of DAM resulted in loss of endodormancy induction. We have cloned and characterized several MADS box genes from the model perennial weed leafy spurge. Leafy spurge DAM genes are preferentially expressed in shoot tips and buds in response to cold temperatures and day length in a manner that is relative to the level of endodormancy induced by various environmental conditions. Over-expression of one DAM gene in arabidopsis delays flowering. Additionally, we show that at least one DAM gene is differentially regulated by chromatin remodeling. Comparisons of the DAM gene promoters between poplar and leafy spurge have identified several conserved sequences that may be important for their expression patterns in response to dormancy-inducing stimuli.     

Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog’s (MS) medium supplemented with 2.0 mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25–0.5 mg/l NAA (1-napthalene acetic acid) and 3 % (w/v) sucrose within 4 weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0 mg/l BA, 0.5 mg/l NAA, 40 mg/l Ads (adenine sulphate) within 4–6 weeks of subculture. Inclusion of 0.25–0.50 mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25–1.0 mg/l NAA or indole-3-butyric acid (IBA) and 2 % (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5 mg/l NAA with 3 % (w/v) sucrose. About 80 % of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.     

Encapsulation of internode regenerated adventitious shoot buds of Indian Siris in alginate beads for temporary storage and twofold clonal plant production

This study for the first time demonstrates single bead alginate encapsulation and conversion (multiple shootlets rejuvenation) from adventitious shoot buds (AB) of Albizia lebbeck (L.) Benth. Internodal (IN) segments isolated from field grown 1-year-old plant of A. lebbeck were used for AB induction under in vitro conditions. IN segments incubated on MS medium augmented with 10.0 µM BA exhibited highest adventitious shoot bud induction frequencies (76 %) on all over the surface after 10 weeks of culture. Induced AB were detached from in vitro proliferating cultures and used for encapsulation as an explant to produce large number of synseeds (07–08) from a single IN explant. Four to five AB were encapsulated in a single calcium alginate bead to manage mass propagation, interim storage and germplasm sharing. The finer gel matrix for encapsulation was attained using 3.0 % sodium alginate and 100 mM calcium chloride. Highest percentage of shoot emergence and multiplication (75 %) from synseed was obtained on MS + 10.0 µM BA + 1.0 µM NAA (RM) after 10 weeks of culture. Encapsulated adventitious buds stored at 4 °C for 1–8 weeks (2 months) too showed thriving shoot emergence (68 %) and multiplication in encapsulated AB and development into complete plantlets when returned to RM. Hence, 4–5 encapsulated AB stored at 4 °C, when cultured back to RM also showed shoot induction resulting in up to 10 shoots per synseeds after 10 weeks of culture. Healthy root formation (½ MS + 2.0 µM IBA) and acclimatization were optimized by using previously standardized protocol (Perveen et al. in J For Res 22:47–52, 2011). Genetic stability of synseed-derived plantlets acclimatized under ex vitro was assessed and compared with mother plant using inter-simple sequence repeats (ISSR) analysis. The synthetic seeds have the achievability of being a substitute planting material for the forestry sector in future, especially for the multipurpose plant species.     

Label-free quantitative proteomics analysis of dormant terminal buds of poplar

Induction and break of bud dormancy are important features for perennial plants surviving extreme seasonal variations in climate. However, the molecular mechanism of the dormancy regulation, still remain poorly understood. To better understand the molecular basis of poplar bud dormancy, we used a label-free quantitative proteomics method based on nanoscale ultra performance liquid chromatography-ESI-MS^E for investigation of differential protein expression during dormancy induction, dormancy, and dormancy break in apical buds of poplar (Populus simonii × P. nigra). Among these identified over 300 proteins during poplar bud dormancy, there are 74 significantly altered proteins, most of which involved in carbohydrate metabolism (22 %), redox regulation (19 %), amino acid transport and metabolism (10 %), and stress response (8 %). Thirty-one of these proteins were up-regulated, five were down-regulated during three phase, and thirty-eight were expressed specifically under different conditions. Pathway analysis suggests that there are still the presence of various physiological activities and a particular influence on photosynthesis and energy metabolism during poplar bud dormancy. Differential expression patterns were identified for key enzymes involved in major metabolic pathways such as glycolysis and the pentose phosphate pathway, thus manifesting the interplay of intricate molecular events in energy generation for new protein synthesis in the dormant buds. Furthermore, there are significant changes present in redox regulation and defense response proteins, for instance in peroxidase and ascorbate peroxidase. Overall, this study provides a better understanding of the possible regulation mechanisms during poplar bud dormancy.     

Exogenous dormancy-breaking substances positively change endogenous phytohormones and amino acids during dormancy release in ‘Anna’ apple trees

A 2-season trial was conducted to verify the effects of foliar applications of some dormancy-breaking substances (DBS) on dormancy release in buds of ‘Anna’ apple (Malus sylvestris, Mill) trees, as well as on metabolic changes in the contents of phytohormones, proline and arginine in buds during their release from dormancy. The efficiency of early bud break induced by Dormex?, potassium nitrate, mineral oil, calcium nitrate and thiourea was noticed in varying degrees. Although Dormex? was distinguished, all DBS hastened bud break, shortened flowering duration, improved bud break% and fruit-set%, increased the contents of gibberellic acid, indole-3-acetic acid, proline and arginine, but reduced abscisic acid content in buds as compared to the control. These results were positively reflected in the final tree yield. Accordingly, it is concluded that the use of Dormex?, at a rate of 4 %, could be recommended for reaching bud break as early as possible and improving ‘Anna’ apple tree yield under the short winters in Egypt and similar regions by regulating the contents of proline, arginine and phytohormones in buds.     

Gibberellin mediates daylength-controlled differentiation of vegetative meristems in strawberry (Fragaria × ananassa Duch)

Background  

Differentiation of long and short shoots is an important developmental trait in several species of the Rosaceae family. However, the physiological mechanisms controlling this differentiation are largely unknown. We have studied the role of gibberellin (GA) in regulation of shoot differentiation in strawberry (Fragaria × ananassa Duch.) cv. Korona. In strawberry, differentiation of axillary buds to runners (long shoot) or to crown branches (short shoot) is promoted by long-day and short-day conditions, respectively. Formation of crown branches is a prerequisite for satisfactory flowering because inflorescences are formed from the apical meristems of the crown.     

The effect of LAB 173711 and ethephon on time of flowering and cold hardiness of peach flower buds

Peach flowers are often killed during bloom by spring frosts. LAB 173711, a compound with abscisic (ABA)-like activity, and ethephon delayed flowering in peach trees. In greenhouse experiments, LAB 173711, at concentrations of 10^?3–10^?2 M, was most effective in delaying bloom when applied after a 5°C cold storage period, rather than before the dormancy breaking treatment. In contrast, ethephon delayed bloom most effectively when applied before 5°C cold storage; ethephon caused flower bud abscission when treatments were made after the chilling requirement had been satisfied. In field experiments, ethephon delayed flowering by 6–7 days, which reduced bud injury after a spring frost during bloom. No flower bud injury was found on ethephon-treated trees after temperatures of ?4.3°C; whereas without ethephon 25% of the flower buds were frost damaged. LAB 173711 delayed the time to 50% bloom by 2–3 days. However, this was not long enough to avoid low-temperature injury to the flower buds.     

In vitro Protein Synthesis by Polyribosomes of Peach Flower Buds at Different Physiological Stages

The in vitro phenylalanine incorporation by polyribosomes of peach flower buds (Prunus persica Stokes) during dormancy, dormancy break and flowering was investigated. Protein synthesis was measured using as catalyst either calf liver soluble factors or the ribosomal supernatant from the peach flower buds in the presence or the absence of the synthetic mRNA, polyuridylic acid. In the presence of polyuridylic acid, the activity of protein synthesis of dormant ribosomes is the same as that of ribosomes during dormancy break and flowering. The absence of synthetic messenger did not cause a change in activity. The ribosomal supernatant of dormant buds, but not of flowering buds, reduces the phenylalanine incorporation by polyribosomes from buds harvested at dormancy break.     

Disbudding treatments on pistachio trees cv. mateur: dry matter accumulation and distribution within fruiting and non-fruiting branches under dry climate

Key message

After applying disbudding treatments, removal of fifty percent of flower bud each year improves dry matter accumulation in fruiting and non-fruiting branches of pistachio trees, which could minimize alternate bearing.

Abstract

Dry matter accumulation and distribution within branches of pistachio trees were investigated during 2005-2008 to determine the effects of fruiting on shoot growth under rain-fed conditions in arid climate. Four treatments were applied: T ₀ normal alternation cycle, T ₁ trees disbudded for 1 year, T ₂ trees disbudded for two successive years, and T ₃ removal of 50 % of all floral buds for each year. Consecutive disbudded treatment (T ₂) allowed a higher growth potential of pistachio trees with reference to normal biennial cycle (T ₀). Individual current shoot of T ₂ accumulated 44 % as much dry matter cm^?1 as those of ‘On’ trees, which have the highest yield. Removal of 50 % of floral buds (T ₃) significantly increased the dry matter accumulation in fruiting branch to reach 57 g in postharvest compared to 42.6 g for the control T ₀. Trees disbudded for 2 years (T ₂) had increased dry matter accumulation in the non-fruiting branch from 3.3 to 16.3 g. Leaves, current shoot, 1-year-old wood and inflorescence buds represented, respectively, 87, 5.3, 5 and 2.7 % of the total dry matter of individual branch of T ₂. In fruiting branches, nuts consisted of 83–87 %, leaves 7–10 %, rachises 4 %, 1-year-old wood 1.6–2 % and current shoot 0.8–1.3 % of the total dry matter. One-year-old wood played a major role as sources and sinks for developing current year shoot, leaves, inflorescence buds and nuts. Removal of 50 % of flower bud (T ₃) improves the dry matter accumulation in fruiting and non-fruiting branches. Thus, under rain-fed conditions, annual pruning could be used to minimize alternate bearing of pistachio.