Histomorphological changes in shoot apices of <Emphasis Type="Italic">Lactuca sativa</Emphasis> treated with gibberellic acid

Lettuce plants were treated with gibberellic acid (GA₃) and uniconazole (UZ; a gibberellin synthesis inhibitor) to investigate the influence of GA₃ on cell division frequency in the shoot apical meristem (SAM) during stem elongation and flower initiation in lettuce (Lactuca sativa) grown in a greenhouse. GA₃ (0.1 mM) was sprayed on the surface of outer leaves and uniconazole solution (0.86 mM) was applied to the soil. GA₃ increased cell division frequency in the peripheral zone and the rib meristem of shoot apices, and this was associated with the stimulation of stem elongation. UZ treatment decreased cell division frequency in the peripheral zone, rib meristem and subapical pith, and this was associated with restricted stem elongation. Treatment with UZ and GA₃ together induced minor stem elongation. Flower induction occurred 3 d earlier in the GA₃ and UZ+GA₃ treatments than in the control, while the UZ treatment delayed flower initiation for more than 9 d relative to the control.     

Putative dual pathway of auxin transport in organogenesis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, damage to the superficial acropetal polar auxin transport (PAT) inhibits generative but not vegetative organ initiation. In order to verify whether in a vegetative phase auxin can be transported to the meristem in a different way, the research on wild-type and plants with defective PAT was performed. Distance from the differentiated vascular elements to the shoot apical meristem (SAM) was measured for Arabidopsis cultured in different experimental systems. The influence of this distance on the ability to induce organogenesis as well as transport of the fluorescent dye to the SAM, and the LEAFY gene expression were analyzed. The youngest protoxylem elements were used as a marker of the vascular tissues. The distance of protoxylem to the SAM and organogenesis were interrelated. Organ initiation occurred only when protoxylem was localized near to the SAM. Experimental elongation of internodes in a vegetative rosette caused an increase in the distance between protoxylem and the SAM organogenic zone. Thus, the inhibition of organ initiation took place already during the vegetative phase. The results suggest the presence of at least two pathways of acropetal transport of auxin inducing organogenesis: one superficial way through PAT, and the second, putative one, internal through the vascular system. Possibly, organogenesis is completely blocked only when both these pathways are dysfunctional.     

Effect of methylglyoxal bis-(guanylhydrazone) on polyamine and ethylene biosynthesis of apple fruit after harvest

Polyamine and ethylene both play important roles in fruit ripening, whose biosynthetic pathways share a common substrate, S-adenosylmethionine (SAM). To unravel the interrelationship between polyamine and ethylene, their metabolism and expression of relevant genes were investigated in apple fruit (Malus domestica Borkh.) treated with methylglyoxal bis-(guanylhydrazone) (MGBG). The MGBG-treated fruit had higher ethylene production until 16 days after treatment (DAT) with preceding accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) than control fruit and then decreased to nearly the same level as control. Ethylene promotion at the early stage by MGBG was accompanied by increased expression of apple ACC synthase (Md-ACS1) and ACC oxidase (MdACO). The expression of apple SAM synthase (MdSAMS) in MGBG-treated fruit was slightly higher than that in control. On the other hand, significant changes in free polyamine titers were observed at some stages, but the changes did not show consistent trends. Based on these observations, possible relationship between polyamine and ethylene pathways was discussed.     

Genetic control of α‐farnesene production in apple fruit and its role in fungal pathogenesis

Terpenes are important compounds in plant trophic interactions. A meta‐analysis of GC‐MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)‐α‐farnesene. Four quantitative trait loci (QTLs) for α‐farnesene production in ripe fruit were identified in a segregating ‘Royal Gala’ (RG) × ‘Granny Smith’ (GS) population with one major QTL on linkage group 10 co‐locating with the MdAFS1 (α‐farnesene synthase‐1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC‐MS analysis of headspace and solvent‐extracted terpenes showing that cold‐treated GS apples produced higher levels of (E,E)‐α‐farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)‐α‐farnesene. To evaluate the role of (E,E)‐α‐farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post‐harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)‐α‐farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post‐inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)‐α‐farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.     

Selection of housekeeping genes for gene expression studies on the development of fruit bearing shoots in Chinese jujube (<Emphasis Type="Italic">Ziziphus jujube</Emphasis> Mill.)

Chinese jujube (Ziziphus jujube Mill.) is important economically for its fruit and also bears attractive medical value. Its flower development concurs with the growth of current fruit bearing shoots (FBSs). However, events involved in current FBSs, including FBS differentiation, flower development, and reliable housekeeping gene are still unknown. In this study, the morphology of FBS development and floral formation were examined and expression profiles of several potential internal control genes was determined. These included genes encoding proteins involved in protein translation (translation elongation factor 1α, ZjEF1), protein folding (cyclophilin, ZjCyP) or degradation (ubiquitin extension protein, ZjUBQ), and proteins involved in the structure of the cytoskeleton (β-actin, ZjACT) or nucleosome (histone3, ZjH3). Our results showed that the floral development in early growing FBSs (less than 20 mm in length) or shoot apices was not complete. Among ZjACT, ZjEF1, ZjCyP, ZjUBQ, and ZjH3, ZjH3 was the most suitable housekeeping gene to evaluate FBS development, based on their expression in early growing FBSs, shoot apices, and different organs. These results will be useful for further molecular mechanism study about FBS development in Chinese jujube.     

Reduced generation time of apple seedlings to within a year by means of a plant virus vector: a new plant‐breeding technique with no transmission of genetic modification to the next generation

Fruit trees have a long juvenile phase. For example, the juvenile phase of apple (Malus × domestica) generally lasts for 5–12 years and is a serious constraint for genetic analysis and for creating new apple cultivars through cross‐breeding. If modification of the genes involved in the transition from the juvenile phase to the adult phase can enable apple to complete its life cycle within 1 year, as seen in herbaceous plants, a significant enhancement in apple breeding will be realized. Here, we report a novel technology that simultaneously promotes expression of Arabidopsis FLOWERING LOCUS T gene (AtFT) and silencing of apple TERMINAL FLOWER 1 gene (MdTFL1‐1) using an Apple latent spherical virus (ALSV) vector (ALSV‐AtFT/MdTFL1) to accelerate flowering time and life cycle in apple seedlings. When apple cotyledons were inoculated with ALSV‐AtFT/MdTFL1 immediately after germination, more than 90% of infected seedlings started flowering within 1.5–3 months, and almost all early‐flowering seedlings continuously produced flower buds on the lateral and axillary shoots. Cross‐pollination between early‐flowering apple plants produced fruits with seeds, indicating that ALSV‐AtFT/MdTFL1 inoculation successfully reduced the time required for completion of the apple life cycle to 1 year or less. Apple latent spherical virus was not transmitted via seeds to successive progenies in most cases, and thus, this method will serve as a new breeding technique that does not pass genetic modification to the next generation.     

Forced flowering of pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> cv. Tainon 17) in response to cold stress,ethephon and calcium carbide with or without activated charcoal

Ethylene, a gaseous plant hormone, is responsible for the initiation of reproductive development in pineapple. Reproductive development can be forced in pineapple (Ananas comosus var. comosus) throughout the year with ethylene. Inhibition of natural flowering initiation with aviglycine [(S)-trans-2-amino-4-(2-aminoethoxy)-3-butenoic acid hydrochloride], an inhibitor of ethylene biosynthesis, provides evidence that reproductive development in response to cold stress and short daylength is also in response to ethylene production. We studied the effect of cold treatment of pineapple on ethylene production and flower induction by applying a short-term cold stress to stem apices. Shoot apices of pineapple treated with ice crystals also produced twice as much ethylene as did those of control plants and significantly more than was produced by “D” leaf basal tissue. Moreover, pineapple plants treated four times with ice crystals or ice water were induced to flower under field conditions and the forcing efficiency, as evaluated by the percentages of inflorescence emergence and fruit harvest, was comparable to forcing with calcium carbide (CaC₂) and ethephon. In another field experiment two applications of a 1.0% solution of CaC₂ or 0.15% ethephon applied at 48 h intervals was sufficient to force reproductive development of ‘Tainon 17’. Furthermore, 0.5 or 1.0% solutions of CaC₂ supplemented with 0.5% activated charcoal (AC) significantly improved the forcing effectiveness of CaC₂. This could/would make it possible to reduce the number or concentration, or both, of CaC₂ required to effect forcing in pineapple.     

DRL1 regulates adaxial leaf patterning and shoot apical meristem activity in<Emphasis Type="Italic">Arabidopsis</Emphasis>

Leaf shape is controlled early on by initiation at the shoot apical meristem (SAM), as well as by changes in the rates and planes of cell division and the polarity-dependent differentiation of leaf cells. To elucidate the regulation of this differentiation by signal(s) from the SAM, we screened for mutations in genes that might be involved in these early processes. A novel recessive mutant, 356-2 [identified as a new allele of thedeformed root and leaf1 (drl1) mutant], was isolated from a collection ofDs transposon insertion lines. The356- 2/drl1- 101 mutant produces narrow, filamentous leaves and defective mer-istems. Its palisade cells have a spongy cell-like structure and are fewer in number, indicating that the leaves are abaxialized. Interestingly, some of those filament-like leaves have no vascular tissues inside their blades.DRL1 encodes a protein similar to the yeast elongator-associated protein (EAP) KTI12. The amino acid sequence of DRL1 is universally conserved in prokaryotes and eukaryotes. These facts suggest that DRL1 might positively regulate leaf polarity and SAM activity by controlling cell proliferation and differentiation.     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis> homologs are linked to the expression of lateral dominance in the acaulescent <Emphasis Type="Italic">Streptocarpus rexii</Emphasis> (Gesneriaceae)

Acaulescent species of Streptocarpus Lindl. show unusual patterns of growth, characterized by anisocotyly (i.e. the unequal growth of cotyledons after germination) and lack of a conventional embryonic shoot apical meristem (SAM). A SAM-like structure appears during post-embryonic development on the axis of the continuously growing cotyledon. Since we have shown previously that KNOX genes are involved in this unusual morphology of Streptocarpus rexii, here we investigated the expression pattern of WUSCHEL (WUS), which is also required for the indeterminacy of the SAM, but is expressed independently from KNOX in Arabidopsis thaliana. In A. thaliana WUSCHEL is involved in the maintenance of the stem cell fate in the organizing centre. The expression pattern of the WUS ortholog in S. rexii (SrWUS) strongly deviates from that of the model plant, suggesting a fundamentally different spatial and temporal regulation of signalling involved in meristem initiation and maintenance. In S. rexii, exogenous application of growth regulators, i.e. gibberellin (GA₃), cytokinin (CK) and a gibberellin biosynthesis inhibitor (PAC), prevents anisocotyly and relocates meristematic cells to a position of conventional SAMs; this coincides with a re-localization of the two main pathways controlling meristem formation, the SrWUS and the KNOX pathways. Our results suggest that the establishment of a hormone imbalance in the seedlings is the basis of anisocotyly, causing a lateral dominance of the macrocotyledon over the microcotyledon. The peculiar morphogenetic program in S. rexii is linked to this delicate hormone balance and is the result of crosstalk between endogenous hormones and regulatory genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

The shoot apical meristem of oil palm (Elaeis guineensis; Arecaceae): developmental progression and dynamics

Background and Aims

Oil palm, an unbranched perennial monocotyledon, possesses a single shoot apical meristem (SAM), which is responsible for the initiation of the entire above-ground structure of the plant. To compare the palm SAM structure with those of other monocots and to study variations in its structure throughout the life of the plant, its organization was characterized from the embryonic stage to that of the reproductive plant.

Methods

SAM structure was studied by a combination of stained histological sections, light and confocal microscopy, and serial section-based three-dimensional reconstructions.

Key Results

The oil palm SAM is characterized by two developmental phases: a juvenile phase with a single tunica-corpus structure displaying a gradual increase in size; and a mature phase characterized by a stable size, a modified shape and an established histological zonation pattern. In mature plants, fluctuations in SAM shape and volume occur, mainly as a consequence of changes in the central zone, possibly in relation to leaf initiation.

Conclusions

Development of the oil palm SAM is characterized by a juvenile to mature phase transition accompanied by establishment of a zonal pattern and modified shape. SAM zonation is dynamic during the plastochron period and displays distinct features compared with other monocots.     

O‐Methyltransferases involved in biphenyl and dibenzofuran biosynthesis

Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O‐methylation reactions. cDNAs encoding the O‐methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab‐causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5‐dihydroxybiphenyl, supplied by the first pathway‐specific enzyme, biphenyl synthase (BIS). 3,5‐Dihydroxybiphenyl underwent a single methylation reaction in the presence of S‐adenosyl‐l ‐methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5‐hydroxyferulic acid. Both substrates were only methylated at the meta‐positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor‐treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N‐ and C‐terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.     

<Emphasis Type="Italic">compact shoot and leafy head 1</Emphasis>, a mutation affects leaf initiation and developmental transition in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The shoot apical meristem (SAM) produces lateral organs in a regular spacing (phyllotaxy) and at a regular interval (phyllochron) during the vegetative phase. In a Dissociation (Ds) insertion rice population, we identified a mutant, compact shoot and leafy head 1 (csl1), which produced massive number of leaves (∼70) during the vegetative phase. In csl1, the transition from the vegetative to the reproductive phase was delayed by about 2 months under long-day conditions. With a reduced leaf size and severe dwarfism, csl1 failed to produce a normal panicle after the transition to reproductive growth. Instead, it produced a leafy panicle, in which all primary rachis-branches were converted to vegetative shoots. Phenotypically csl1 resembled pla mutants in short plastochron but was more severe in the conversion of the reproductive organs to vegetative organs. In addition, neither the expression nor the coding region of PLA1 or PLA2 was affected in csl1. csl1 is most likely a dominant mutation because no mutant segregant was observed in progeny of 67 siblings of the csl1 mutant. CSL1 may represent a novel gene, which functions downstream of PLA1 and/or PLA2, or alternatively functions in a separate pathway, involved in the regulation of leaf initiation and developmental transition via plant hormones or other mobile signals.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration from shoot apices and gene transfer by particle bombardment in <Emphasis Type="Italic">Jatropha curcas</Emphasis>

An efficient and reproducible in vitro plant regeneration system from shoot apices was developed in Jatropha curcas. Benzylaminopurine (BAP; 2.5 μM) was most effective in inducing an average of 6.2 shoots per shoot apex. Incorporation of gibberellic acid (GA3; 0.5 μM) to basal medium was found essential for elongation of shoots. The BAP-habituated mother explants continuously produced shoots during successive subculture without any loss of morphogenic potential. The shoots rooted efficiently on half-strength MS medium. The rooted plantlets were acclimatized with more than 98 % success and the plants transferred to soil:compost in nursery showed no sign of variation compared to the seed-grown plants. The whole process of culture initiation to plant establishment was accomplished within 5–6 weeks. A genetic transformation system in J. curcas was established for the first time, using bombardment of particles coated with plasmid pBI426 with a GUS-NPT II fusion protein under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. The β-glucuronidase (GUS) activity in J. curcas shoot apices was significantly affected by the gold particle size, bombardment pressure, target distance, macrocarrier travel distance, number of bombardments, and type and duration of osmotic pre-treatment. The proliferating bombarded shoot apices were screened on medium supplemented with 25 mg dm⁻³ kanamycin and surviving shoots were rooted on medium devoid of kanamycin. The integration of the transgene into genomic DNA of transgenic plants was confirmed by PCR and Southern blot hybridization. The transgenic plants showed insertion of single to multiple copies of the transgene.     

Hide and seek: uncloaking the vegetative shoot apex of Arabidopsis thaliana

Leaf primordia are iteratively formed on the flanks of the shoot apical meristem (SAM) at the vegetative shoot apex of Arabidopsis thaliana. The youngest leaf primordia and the SAM are extensively covered by older proliferating leaves, making it difficult to obtain accurate volumetric data from these structures. Combination of serial histological sections combined with 3D reconstruction software allowed us to acquire such data. Here, we compared the SAMs of wild‐type plants of the Columbia‐0 and Landsberg erecta ecotypes with those of clavata3‐2 (clv3‐2) mutants, which produce an enlarged SAM. In addition, the SAM size and morphology of plants over‐expressing the gibberellin‐20 oxidase (GA20OX) gene was examined, and the effect of mild osmotic stress on primordium size was measured. Efficient 3D visualization of gene expression patterns is also possible with this method, as illustrated by the analysis of SHOOTMERISTEMLESS:GUS and WUSCHEL:GUS reporter lines.     

STEREOLOGICAL STUDY OF ULTRASTRUCTURAL CHANGES IN THE SHOOT APICAL MERISTEM OF NICOTIANA TABACUM DURING FLORAL INDUCTION

Shoot apices of a short-day sensitive line of Nicotiana tabacum L. cv. NCTG-22 have been examined by electron microscopy for ultrastructural changes which occurred in the central zone over a 17-day period during the transition from vegetative to reproductive growth. Plants were grown in controlled-environment chambers of the NCSU Phytotron and exposed to an inductive photoperiod after a 6-wk juvenile phase. Ultrastructural changes were investigated from photomicrographs using a semi-automatic stereological procedure and a microcomputer. After exposure to only one inductive cycle cell and nuclear cross sectional areas in short-day plants were significantly larger than in long-day controls. Subsequently, under short days cross sectional areas of cells, nuclei, vacuoles and proplastids decreased, while mitochondrial cross sectional area and relative volume increased during the course of the induction period. In induced apices as cross sectional areas and relative volumes of vacuoles and proplastids decreased, their profile numbers increased. The reduction in cross sectional areas of cells and most organelles was associated with an increase in rate of leaf initiation and size of the apical dome. The demand for sufficient energy input to maintain the surge in growth and activity preceding floral initiation was reflected by the significant increases in cross sectional area, profile numbers and density of the mitochondria population. Even though the transition period is quite long for Nicotiana, cells and organelles in the central zone were observed to progress through similar changes in morphology that are known to occur in Sinapis and Xanthium which exhibit a more rapid and absolute response to photo-induction.     

Developmental morphology of branching flowers in Nymphaea prolifera

Nymphaea and Nuphar (Nymphaeaceae) share an extra-axillary mode of floral inception in the shoot apical meristem (SAM). Some leaf sites along the ontogenetic spiral are occupied by floral primordia lacking a subtending bract. This pattern of flower initiation in leaf sites is repeated inside branching flowers of Nymphaea prolifera (Central and South America). Instead of fertile flowers this species usually produces sterile tuberiferous flowers that act as vegetative propagules. N. prolifera changes the meristem identity from reproductive to vegetative or vice versa repeatedly. Each branching flower first produces some perianth-like leaves, then it switches back to the vegetative meristem identity of the SAM with the formation of foliage leaves and another set of branching flowers. This process is repeated up to three times giving rise to more than 100 vegetative propagules. The developmental morphology of the branching flowers of N. prolifera is described using both microtome sections and scanning electron microscopy.     

Endophytic fungi from rhizomes of <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

About 63 fungal endophytic isolates were separated from rhizomes of Paris polyphylla var. yunnanensis, which is a traditional medicinal plant mainly distributed in China. The isolates were characterized and grouped based on the culture characteristics and the morphology of colony growth and conidia. Eleven representative ones were selected for further taxonomical identification. Five genera namely Fusarium, Gliocladiopsis, Gliomastix, Aspergillus and Cylindrocarpon were identified on the basis of their morphological characterizations. Of them, the most frequent genus was Fusarium (i.e. Ppf1, Ppf3 and Ppf14). Their ITS-rDNA sequences were compared with those available in the GeneBank databases to obtain the closest related species by BLAST analysis as well as to analyze their phylogenetic affiliation. The isolates were identified as Gliocladiopsis irregularis (Ppf2), Plectosphaerella cucumerina (Ppf4), Padospora sp. (Ppf6), Gliomastix murorum var. murorum (Ppf7), Aspergillus fumigatus (Ppf9), Pichia guilliermondii (Ppf10), Neonectria radicicola (anamorph: Cylindrocarpon) (Ppf12) and one uncultured mycorrhizal ascomycete (Ppf13) separately based on their morphological and molecular features. The molecular characters of the endophytic fungi were basically coincident with their morphology. The broad diversity and taxonomic spectrum were exhibited by the endophytic fungi from P. polyphylla var. yunnanensis.     

Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.