Developmental synchronization of male and female gametophytes in Ginkgo biloba and its neck mother cell division prior to fertilization.

This study investigated male and female gametophytes in Ginkgo biloba, while a droplet of fluid was present in the fertilization chamber and found that the central cell, the generative cell and the neck mother cell divided simultaneously prior to fertilization. In male gametophytes, the generative cell divided to yield two sperm cells. Concomitantly, the two neck mother cells of the archegonium increased in size then divided asymmetrically resulting in two big cover cells and two small base cells. Each cell had a fixed end in direct contact with an adjacent jacket cell and a free end overlapping its counterpart. This unique arrangement could allow for their free ends to swing into the fertilization chamber as a result of the force from the interior of the archegonium where a polar periclinal division had occurred to produce a canal cell and an egg. The subsequent withdrawal of the content of the archegonium may facilitate the entry of sperm into the archegonium. The neck apparatus closed after the fertilization occurred. The concurrence of the above divisions and the delicate structure of neck apparatus suggest that the gametophytes undergo a synchronization process to become receptive at the time of fertilization. However, the formation of neck cells and the opening time of neck apparatus of the archegonia within the same ovule were slightly different, which could lead to the formation of zygotes at a temporally distinct interval. The earlier formed zygote may progress as the only mature embryo in the ovule.

The proteasome is responsible for caspase-3-like activity during xylem development.

Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase-3, which displays Asp-Glu-Val-Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase-3 is absent in plants, caspase-3-like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase-3-like activity exists in xylem cell death. In this study, we showed that caspase-3-like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase-3-like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase-3-like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven ß subunits encoded by 12 genes. Pharmacological assays showed that Ac-DEVD-CHO, a caspase-3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto-lactacystin ß-lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6-induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase-3-like activity and is involved in xylem development.

The Ca2+ -dependent DNases are involved in secondary xylem development in Eucommia ulmoides.

Secondary xylem development has long been recognized as a typical case of programmed cell death (PCD) in plants. During PCD, the degradation of genomic DNA is catalyzed by endonucleases. However, to date, no endonuclease has been shown to participate in secondary xylem development. Two novel Ca(2+) -dependent DNase genes, EuCaN1 and EuCaN2, were identified from the differentiating secondary xylem of the tree Eucommia ulmoides Oliv., their functions were studied by DNase activity assay, in situ hybridization, protein immunolocalization and virus-induced gene silencing experiments. Full-length cDNAs of EuCaN1 and EuCaN2 contained an open reading frame of 987 bp, encoding two proteins of 328 amino acids with SNase-like functional domains. The genomic DNA sequence for EuCaN1 had no introns, while EuCaN2 had 8 introns. EuCaN1 and EuCaN2 digested ssDNA and dsDNA with Ca(2+) -dependence at neutral pH. Their expression was confined to differentiating secondary xylem cells and the proteins were localized in the nucleus. Their activity dynamics was closely correlated with secondary xylem development. Secondary xylem cell differentiation is influenced by RNAi of endonuclease genes. The results provide evidence that the Ca(2+) -dependent DNases are involved in secondary xylem development.

Molecular features of secondary vascular tissue regeneration after bark girdling in Populus.

Regeneration is a common strategy for plants to repair damage to their tissue after attacks from other organisms or physical assaults. However, how differentiating cells acquire regenerative competence and rebuild the pattern of new tissues remains largely unknown. Using anatomical observation and microarray analysis, we investigated the morphological process and molecular features of secondary vascular tissue regeneration after bark girdling in trees. After bark girdling, new phloem and cambium regenerate from differentiating xylem cells and rebuild secondary vascular tissue pattern within 1 month. Differentiating xylem cells acquire regenerative competence through epigenetic regulation and cell cycle re-entry. The xylem developmental program was blocked, whereas the phloem or cambium program was activated, resulting in the secondary vascular tissue pattern re-establishment. Phytohormones play important roles in vascular tissue regeneration. We propose a model describing the molecular features of secondary vascular tissue regeneration after bark girdling in trees. It provides information for understanding mechanisms of tissue regeneration and pattern formation of the secondary vascular tissues in plants.

Regulation of cell cycle regulators by environmental signals during growth-dormancy cycle of trees.

Climate change such as changing temperature and increasing concentrations of atmospheric CO(2), are likely to drive significant modifications in forests. While many studies have demonstrated the responses and adoptions of tree to fluctuations in climatic and environmental conditions, the impact of environmental signals on trees is complex and poorly understood with respect to the molecular biology in context of the seasonal change of environmental signals. This addendum is focused on the impact of environmental signals on growth-dormancy cycle of trees growing in temperate regions, especially the regulation of cell cycle regulators by temperature and photoperiod. In addition, the plant hormone control of growth-dormancy cycle of trees and cell cycle regulators in the cambium is also discussed.

Induction of PtoCDKB and PtoCYCB transcription by temperature during cambium reactivation in Populus tomentosa Carr.

Cell cycle progression requires interaction between cyclin-dependent kinase B (CDKB) and cyclin B (CYCB). The seasonal expression patterns of the CDKB and CYCB homologues from Populus tomentosa Carr. were investigated, and effects of temperature and exogenous indole-3-acetic acid (IAA) on their expression were further studied in water culture experiments. Based on the differential responses of dormant cambium cells to exogenous IAA, four stages of cambium dormancy were confirmed for P. tomentosa: quiescence 1 (Q1), rest, quiescence 2-1 (Q2-1), and quiescence 2-2 (Q2-2). PtoCDKB and PtoCYCB transcripts were strongly expressed in the active phases, weakly in Q1, and almost undetectable from rest until late Q2-2. Climatic data analysis showed a correlation between daily air temperature and PtoCDKB and PtoCYCB expression patterns. Water culture experiments with temperature treatment further showed that a low temperature (4 degrees C) kept PtoCDKB and PtoCYCB transcripts at undetectable levels, while a warm temperature (25 degrees C) induced their expression in the cambium region. Meanwhile, water culture experiments with exogenous IAA treatment showed that induction of PtoCDKB and PtoCYCB transcription was independent of exogenous IAA. The results suggest that, in deciduous hardwood P. tomentosa growing in a temperate zone, the temperature in early spring is a vital environmental factor for cambium reactivation. The increasing temperature in early spring may induce CDKB and CYCB homologue transcription in the cambium region, which is necessary for cambium cell division.

Occurrence of the transition of apical architecture and expression patterns of related genes during conversion of apical meristem identity in G2 pea.

G2 pea exhibits an apical senescence delaying phenotype under short-day (SD) conditions; however, the structural basis for its apical development is still largely unknown. In the present study, the apical meristem of SD-grown G2 pea plants underwent a transition from vegetative to indeterminate inflorescence meristem, but the apical meristem of long-day (LD)-grown G2 pea plants would be further converted to determinate floral meristem. Both SD signal and GA(3) treatment enhanced expression of the putative calcium transporter PPF1, and pea homologs of TFL1 (LF and DET), whereas LD signal suppressed their expression at 60 d post-flowering compared with those at 40 d post-flowering. Both PPF1 and LF expressed at the vegetative and reproductive phases in SD-grown apical buds, but floral initiation obviously increased the expression level of PPF1 compared with the unchanged expression level of LF from 40 to 60 d post-flowering. In addition, although the floral initiation significantly enhanced the expression levels of PPF1 and DET, DET was mainly expressed after floral initiation in SD-grown apical buds. Therefore, the main structural difference between LD- and SD-grown apical meristem in G2 pea lies in whether their apical indeterminate inflorescence meristem could be converted to the determinate structure.

PPF1 may suppress plant senescence via activating TFL1 in transgenic Arabidopsis plants.

Senescence, a sequence of biochemical and physiological events, constitutes the final stage of development in higher plants and is modulated by a variety of environmental factors and internal factors. PPF1 possesses an important biological function in plant development by controlling the Ca2+ storage capacity within chloroplasts. Here we show that the expression of PPF1 might play a pivotal role in negatively regulating plant senescence as revealed by the regulation of overexpression and suppression of PPF1 on plant development. Moreover, TFL1, a key regulator in the floral repression pathway, was screened out as one of the downstream targets for PPF1 in the senescence-signaling pathway. Investigation of the senescence-related phenotypes in PPF1(-) tfl1-1 and PPF1(+) tfl1-1 double mutants confirmed and further highlighted the relation of PPF1 with TFL1 in transgenic plants. The activation of TFL1 expression by PPF1 defines an important pathway possibly essential for the negative regulation of plant senescence in transgenic Arabidopsis.

Phloem transdifferentiation from immature xylem cells during bark regeneration after girdling in Eucommia ulmoides Oliv.

Eucommia ulmoides Oliv. (Eucommiaceae), a traditional Chinese medicinal plant, was used to study phloem cell differentiation during bark regeneration after girdling on a large scale. Here it is shown that new sieve elements (SEs) appeared in the regenerated tissues before the formation of wound cambium during bark regeneration after girdling, and they could originate from the transdifferentiation of immature/differentiating axial xylem cells left on the trunk. Assays of water-cultured twigs revealed that girdling blocked sucrose transport until the formation of new SEs, and the regeneration of the functional SEs was not dependent on the substance provided by the axis system outside the girdled areas, while exogenous indole acetic acid (IAA) applied on the wound surface accelerated SE differentiation. The experiments suggest that the immature xylem cells can transdifferentiate into phloem cells under certain conditions, which means xylem and phloem cells might share some identical features at the beginning of their differentiation pathway. This study also showed that the bark regeneration system could provide a novel method for studying xylem and phloem cell differentiation.

Formation of archegonium chamber is associated with nucellar-cell programmed cell death in Ginkgo biloba.

The archegonium chamber in Ginkgo biloba L. is a pathway for spermatozoids swimming towards the archegonium for fertilization. The objective of this study was to elucidate the mechanism of archegonium chamber formation. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and DNA ladder demonstrated that the nucellar cell death, coordinated with the archegonium chamber formation, was a process of programmed cell death. Cytochemical localization of Ca(2+) in these nucellar cells was determined by means of in situ precipitation with potassium pyroantimonate and electron microscopic visualization, in order to study the relation between Ca(2+) and programmed cell death. The results showed an early uptake of the mitochondrial calcium particles in the nucellar cells undergoing programmed cell death. Together with other dynamic changes in Ca(2+) subcellular distribution, this indicates that Ca(2+) may play a role in the regulation of mitochondria-mediated programmed events in the nucellar cells.

ABP1 expression regulated by IAA and ABA is associated with the cambium periodicity in Eucommia ulmoides Oliv.

A cDNA clone of Eucommia ulmoides Oliv. encoding auxin binding protein 1 (ABP1), one of the putative receptors of auxin, was isolated, and the seasonal expression of ABP1 in relation to IAA and ABA annual variation was investigated by different technical approaches including RT-PCR, real-time PCR, northern blotting, western blotting, and immunolocalization. In the cambial region, ABP1 expression at both the protein and the mRNA level was found to be high, low, and remarkably scarce in the active, quiescent, and resting stages, respectively, during cambium periodicity. The signal abundance of ABP1 follows the opposite pattern to ABA accumulation and correlates with auxin responsiveness of the cambial tissues, suggesting a role for ABP1 in mediating auxin-dependent regulation of cambial activation in the activity-dormancy cycle. This paper attempts to explain why IAA would 'boost' the reactivation of a quiescent cambium, and not that of a resting cambium. Results also show that ABP1 expression is improved by IAA, while inhibited by ABA.

Regeneration of the secondary vascular system in poplar as a novel system to investigate gene expression by a proteomic approach.

Wood formation is a complex process composing many biological events. To access its key developmental stages, we have established a regeneration system that can mimic the initiation and differentiation of cambium cells for Chinese white poplar. Anatomical studies showed that new cambium and xylem re-appeared in sequence within a few weeks after being debarked. This provides the opportunity to follow key stages of wood formation by sampling clonal trees at different regeneration times. We used this system in combination with a proteomic approach to analyze proteins expressed in different regeneration stages. PMFs for 244 proteins differentially displayed were obtained and queried against public databases. Putative functions of 199 of these proteins were assigned and classified. Regulatory genes for cell cycle progression, differentiation and cell fate were expressed in the formation of cambial tissue, while 27 genes involved in secondary wall formation were predominantly found in the xylem developing stage. This indicates that the change of gene expression pattern is corresponding to the progression of second vascular system regeneration when and where the key events of wood development occur. Further exploration of these interesting genes may provide insight into the molecular mechanisms of wood formation.

Opposite patterns in the annual distribution and time-course of endogenous abscisic acid and indole-3-acetic acid in relation to the periodicity of cambial activity in Eucommia ulmoides Oliv.

The seasonal change of free abscisic acid (ABA) and indole-3-acetic acid (IAA) and their relationship with the cambial activity in Eucommia ulmoides trees were investigated by ABA and IAA immunolocalization using primary polyclonal and rhodamine-red fluorescing secondary antibodies, ABA and IAA quantification using high performance liquid chromatography (HPLC), and systematic monitoring of vascular cell layers production. ABA and IAA clearly displayed opposite annual distribution patterns. In the active period (AP), both immunolocalization and HPLC detected an abrupt decrease of ABA, reaching its lowest level in the summer. During dormancy, ABA started increasing in the first quiescence (Q1) (autumn), peaked in the rest (winter), and gradually decreased from the onset of the second quiescence (Q2) (the end of winter). IAA showed a reverse pattern to that of ABA: it sharply increased in AP, but noticeably decreased from the commencement of Q1. Longitudinally, the ABA distribution increased apico-basally, contrasting with IAA. Laterally, most of the ABA was located in mature vascular tissues, whereas the IAA essentially occurred in the cambial region. The concomitant IAA-ABA distribution and seasonal changes in vascular tissues greatly correlated with xylem and phloem cell production, and late wood differentiation and maturation. Interestingly, the application of exogenous ABA to quiescent E. ulmoides branches, in a water-culture system, inhibited external IAA action on cambial activity reactivation. These results suggest that, in E. ulmoides, ABA and IAA might probably interact in the cambial region. The annual cambial activity could be influenced by an IAA:ABA ratio; and ABA might play a key role in vascular cambium dormancy in higher plants. The relationship between hormonal changes and the (particular) annual life cycle of E. ulmoides is also discussed.

PPF1 inhibits programmed cell death in apical meristems of both G2 pea and transgenic Arabidopsis plants possibly by delaying cytosolic Ca2+ elevation.

PPF1 encodes a putative calcium ion carrier that affects the flowering time of transgenic Arabidopsis by modulating Ca(2+) storage capacities in chloroplasts of a plant cell. In the current work, we found that differential expression of PPF1 might affect processes of programmed cell death (PCD) since DNA fragmentation was detected in senescencing apical buds of long day-grown G2 pea (Pisum sativum L.) plants, but was not in non-senescencing short day-grown counterparts at all growth stages. An animal inhibitor of caspase-activated DNase (ICAD) homologue was detected in short day-grown plant continuously throughout the whole experiment and only in early stages of long day-grown pre-floral G2 pea apical buds. DNA fragmentation was significantly inhibited in apical meristems of transgenic Arabidopsis that over-expressed the PPF1 gene when compared to that of either wild-type control or to PPF1 (-) plants. The expression of ICAD-like protein decreased to undetectable level at 45 dpg in apical tissues of PPF1 (-) Arabidopsis, which was much earlier than that found in PPF1 (+) or wild-type controls. In epidermal cells of PPF1 (-) plants, we recorded significantly earlier calcium transient prior to PCD. We suggest that the expression of PPF1, a chloroplast localized Ca(2+) ion channel may inhibit programmed cell death in apical meristems of flowering plants by keeping a low cytoplasmic calcium content that might inhibit DNA fragmentation in plant cells.

Relationship between endogenous indole-3-acetic acid and abscisic acid changes and bark recovery in Eucommia ulmoides Oliv. after girdling.

Eucommia ulmoides (Eucommiaceae), a traditional Chinese medicinal plant, is often subjected to severe manual peeling of its bark. If the girdled trunk is well protected from desiccation, new bark forms within 1 month. It has been proposed that phytohormones play a key role in this process. Research has been conducted to determine the distribution of endogenous indole-3-acetic acid (IAA) and abscisic acid (ABA) during the bark recovery, using high-performance liquid-chromatography (HPLC) and fluoro-immuno-localization techniques. Results showed that, from 2 d after girdling, the IAA content in the recovering bark (RB) increased markedly while that of ABA decreased. The opposite pattern was observed during progressive re-establishment of the tissues. Immuno-localization showed that most of the IAA was located in the RB tissue layers undergoing cell division, dedifferentiation and (re)differentiation, such as xylary rays, immature xylem, phellogen and cambial regions. This study also provides evidence that IAA and ABA are involved in the bark reconstitution.

Time-course of programmed cell death during leaf senescence in Eucommia ulmoides.

Leaves of Eucommia ulmoidesOliv. harvested between April to November were examined for programmed cell death (PCD) during growth and senescence. Leaves developed in April, becoming fully expanded in late May, remaining unchanged until November when they started to dehisce. Falling leaves retained a green color. Our results showed that (1) mesophyll cells gradually reduced their nuclei from September to November, (2) positive TUNEL signals appeared on the nuclei from August, (3) ladder-like DNA fragmentation occurred in September and October, and (4) a 20-kDa Ca(2+)-dependent DNase appeared in these same months. In fallen leaves, intact mesophyll cell nuclei could not be detected, but a few cells around the vascular bundle had nuclei. Therefore, (1) programmed cell death (PCD) of leaf cells occurred in the leaves of E. ulmoides, (2) the progress of mesophyll cell PCD lasted for more than 2 months, and (3) PCD of leaf cells was asynchronous in natural senescing leaves.

Transgenic expression of a putative calcium transporter affects the time of Arabidopsis flowering.

PPF1 is a gibberellin-induced, vegetative growth-specific gene, first isolated from short-day (SD)-grown G2 pea plants. In the current work, we found that transgenic Arabidopsis plants overexpressing the PPF1 gene (PPF1 (+)) flowered much later and had a significantly longer lifespan compared to control plants, whereas suppression of this gene (PPF1 (-)) resulted in a very rapid reproductive cycle. Western blotting analyses of PPF1 (+) and (-) plant lines revealed a positive correlation between the amount of antibody-reactive protein and the time of flowering. Green fluorescent protein (GFP) co-expression assays showed that the PPF1 protein is likely localized in chloroplast membranes. Transgenic expression of PPF1 affected the calcium storage capacities since chloroplasts isolated from PPF1 (+) plants contained high Ca2+ levels while chloroplasts of PPF1 (-) plants contained very low amounts of calcium ion. Using Novikoff human hepatoma cells, we demonstrated that expression of PPF1 leads to a significant inward calcium ion current that was absent in untransformed cells. We conclude that, as a putative calcium ion carrier, PPF1 affects the flowering time of higher plants by modulating Ca2+ storage capacity within chloroplasts.

Toward understanding the different function of two types of parenchyma cells in bamboo culms.

The bamboo, woody monocot, has two types of parenchyma cells in the ground tissues of its culm, in contrast to a single type of parenchyma cell in rice, maize and other major crop species. The distribution of cell wall components, including lignin, (1-->3), (1-->4)-beta-D-glucans (MGs), the highly-substituted glucuronoarabinoxylans (hsGAXs) and low-branched xylans (lbXs) in ground parenchyma tissue of Phyllostachys heterocycla var. pubescens culms was studied at various developmental stages using light microscopy (LM), UV-microscopy, transmission electron microscopy (TEM) and immunolabeling techniques. The short parenchyma cell walls were lignified in 2-month-old bamboo culms just as the long parenchyma cell walls were. The lignified regions were confined to the portions in contact with the long parenchyma cell walls, while the walls at the cell corner region never lignified, even in 7-year-old culms. Significant differences were also found in the hemicellulose distribution between the short and long parenchyma cell walls. In bamboo parenchyma tissue, MGs were localized in short parenchyma cell walls and few were found in long parenchyma cell walls in both young and 7-year-old culms. The distribution of hsGAXs was similar to that of MGs in young culms, but they only appeared in the cell corner region of short parenchyma cells in old culms. Low-branched xylans were distributed in the lignified, but not in unlignified parenchyma cell walls. Based on this evidence, the differences of function in both short and long parenchyma cells in a bamboo culm are discussed.