Functional Characteristics Analysis of Dehydrins in Larix kaempferi under Osmotic Stress.

Dehydrins (DHN) belong to the late embryogenesis abundant II family and have been found to enhance plant tolerance to abiotic stress. In the present study, we reported four DHNs in Larix kaempferi (LkDHN) which were identified from the published transcriptome. Alignment analysis showed that these four LkDHNs shared close relationships and belonged to SK3-type DHNs. The electrophoretic mobility shift assay indicated that these four LkDHNs all possess sequence-independent binding capacity for double-strands DNAs. The subcellular localizations of the four LkDHNs were in both the nucleus and cytoplasm, indicating that these LkDHNs enter the nucleus to exert the ability to bind DNA. The preparation of tobacco protoplasts with different concentrations of mannitol showed that LkDHNs enhanced the tolerance of plant cells under osmotic stress. The overexpression of LkDHNs in yeasts enhanced their tolerance to osmotic stress and helped the yeasts to survive severe stress. In addition, LkDHNs in the nucleus of salt treated tobacco increased. All of these results indicated that the four LkDHNs help plants survive from heavy stress by participating in DNA protection. These four LKDHNs played similar roles in the response to osmotic stress and assisted in the adaptation of L. kaempferi to the arid and cold winter of northern China.

Homogalacturonan deesterification during pollen-ovule interaction in Larix decidua Mill.: an immunocytochemical study.

Studies on angiosperm plants have shown that homogalacturonan present in the extracellular matrix of pistils plays an important role in the interaction with the male gametophyte. However, in gymnosperms, knowledge on the participation of HG in the pollen-ovule interaction is limited, and only a few studies on male gametophytes have been reported. Thus, the aim of this study was to determine the distribution of HG in male gametophytes and ovules during their interaction in Larix decidua Mill. The distribution of HG in pollen grains and unpollinated and pollinated ovules was investigated by immunofluorescence techniques using monoclonal antibodies that recognise high methyl-esterified HG (JIM7), low methyl-esterified HG (JIM5) and calcium cross-linked HG (2F4). All studied categories of HG were detected in the ovule. Highly methyl-esterified HG was present in the cell walls of all cells throughout the interaction; however, the distribution of low methyl-esterified and calcium cross-linked HG changed during the course of interaction. Both of these categories of HG appeared only in the apoplast and the extracellular matrix of the ovule tissues, which interact with the male gametophyte. This finding suggests that in L. decidua, low methyl-esterified and calcium cross-linked HG play an important role in pollen-ovule interaction. The last category of HG is most likely involved in adhesion between the pollen and the ovule and might provide an optimal calcium environment for pollen grain germination and pollen tube growth.

Role of adventitious roots in water relations of tamarack (Larix laricina) seedlings exposed to flooding.

BACKGROUND: Flooding reduces supply of oxygen to the roots affecting plant water uptake. Some flooding-tolerant tree species including tamarack (Larix laricina (Du Roi) K. Koch) produce adventitious roots in response to flooding. These roots were reported to have higher hydraulic conductivity under flooding conditions compared with non-adventitious roots. In the present study, we examined structural and functional modifications in adventitious roots of tamarack seedlings to explain their flooding tolerance. RESULTS: Seedlings were subjected to the flooding treatment for six months, which resulted in an almost complete disintegration of the existing root system and its replacement with adventitious roots. We compared gas exchange parameters and water relations of flooded plants with the plants growing in well-drained soil and examined the root structures and root water transport properties. Although flooded seedlings had lower needle chlorophyll concentrations, their stomatal conductance, net photosynthesis rates and shoot water potentials were similar to non-flooded plants, indicative of flooding tolerance. Flooded adventitious roots had higher activation energy and a higher ratio of apoplastic to cell-to-cell water flow compared with non-flooded control roots as determined with the 1-hydroxypirene 3,6,8-trisulfonic acid apoplastic tracer dye. The adventitious roots in flooded plants also exhibited retarded xylem and endodermal development and accumulated numerous starch grains in the cortex. Microscopic examination of root sections treated with the PIP1 and PIP2 antibodies revealed high immunoreactivity in the cortex of non-flooded roots, as compared with flooded roots. CONCLUSIONS: Structural modifications of adventitious roots suggest increased contribution of apoplastic bypass to water flow. The reduced dependence of roots on the hypoxia-sensitive aquaporin-mediated water transport is likely among the main mechanisms allowing tamarack seedlings to maintain water balance and gas exchange under flooding conditions.

Periodic expression of Sm proteins parallels formation of nuclear Cajal bodies and cytoplasmic snRNP-rich bodies.

Small nuclear ribonucleoproteins (snRNPs) play a fundamental role in pre-mRNA processing in the nucleus. The biogenesis of snRNPs involves a sequence of events that occurs in both the nucleus and cytoplasm. Despite the wealth of biochemical information about the cytoplasmic assembly of snRNPs, little is known about the spatial organization of snRNPs in the cytoplasm. In the cytoplasm of larch microsporocytes, a cyclic appearance of bodies containing small nuclear RNA (snRNA) and Sm proteins was observed during anther meiosis. We observed a correlation between the occurrence of cytoplasmic snRNP bodies, the levels of Sm proteins, and the dynamic formation of Cajal bodies. Larch microsporocytes were used for these studies. This model is characterized by natural fluctuations in the level of RNA metabolism, in which periods of high transcriptional activity are separated from periods of low transcriptional activity. In designing experiments, the authors considered the differences between the nuclear and cytoplasmic phases of snRNP maturation and generated a hypothesis about the direct participation of Sm proteins in a molecular switch triggering the formation of Cajal bodies.

Over-Expression of the Cell-Cycle Gene LaCDKB1;2 Promotes Cell Proliferation and the Formation of Normal Cotyledonary Embryos during Larix kaempferi Somatic Embryogenesis.

Somatic embryogenesis is an effective tool for the production of forest tree seedlings with desirable characteristics; however, the low initiation frequency and productivity of high-quality mature somatic embryos are still limiting factors for Larix kaempferi (Japanese larch). Here, we analyzed the expression pattern of L. kaempferi cyclin-dependent kinase B 1;2 (LaCDKB1;2) during somatic embryogenesis in L. kaempferi and its relationship with the cell proliferation rate. We also analyzed the effect of LaCDKB1;2 over-expression on somatic embryo quality. The results revealed a positive correlation between LaCDKB1;2 expression and the cell proliferation rate during the proliferation stage. After LaCDKB1;2 over-expression, the proliferation rate of cultures increased, and the number of somatic embryos in transgenic cultures was 2.69 times that in non-transformed cultures. Notably, the number of normal cotyledonary embryos in transgenic cultures was 3 times that in non-transformed cultures, indicating that LaCDKB1;2 not only increases the proliferation of cultures and the number of somatic embryos but also improves the quality of somatic embryos. These results provide insight into the regulatory mechanisms of somatic embryogenesis as well as new Larix breeding material.

Timing and duration of European larch growing season along altitudinal gradients in the Swiss Alps.

The 2007 European larch (Larix decidua Mill.) growing season was monitored along two elevational transects in the Lötschental valley in the Swiss Alps. Phenological observations and weekly microcore sampling of 28 larch trees were conducted between April and October 2007 at seven study sites regularly spaced from 1350 to 2150 m a.s.l. on northwest- and southeast-facing slopes. The developmental stages of nearly 75,000 individual cells assessed on 1200 thin sections were used to investigate the links between the trees' thermal regimes and growth phases including the beginning and ending of cell enlargement, wall thickening and maturation of the stem wood. Needles appeared approximately 3-4 weeks earlier than stem growth. The duration of ring formation lasted from mid-May to the end of October, with the length of the growing season decreasing along elevation from 137 to 101 days. The onset of the different growing seasons changed by 3-4 days per 100 m elevation; the ending of the growing season, however, appeared minimally related to altitude. If associated with the monitored altitudinal lapse rate of -0.5 degrees C per 100 m, these results translate into a lengthening of the growing season by approximately 7 days per degree Celsius. This study provides new data on the timing and duration of basic growth processes and contributes to quantification of the impacts of global warming on tree growth and productivity.

Summer temperature dependency of larch budmoth outbreaks revealed by Alpine tree-ring isotope chronologies.

Larch budmoth (LBM, Zeiraphera diniana Gn.) outbreaks cause discernable physical alteration of cell growth in tree rings of host subalpine larch (Larix decidua Mill.) in the European Alps. However, it is not clear if these outbreaks also impact isotopic signatures in tree-ring cellulose, thereby masking climatic signals. We compared LBM outbreak events in stable carbon and oxygen isotope chronologies of larch and their corresponding tree-ring widths from two high-elevation sites (1800-2200 m a.s.l.) in the Swiss Alps for the period AD 1900-2004 against isotope data obtained from non-host spruce (Picea abies). At each site, two age classes of tree individuals (150-250 and 450-550 years old) were sampled. Inclusion of the latter age class enabled one chronology to be extended back to AD 1650, and a comparison with long-term monthly resolved temperature data. Within the constraints of this local study, we found that: (1) isotopic ratios in tree rings of larch provide a strong and consistent climatic signal of temperature; (2) at all sites the isotope signatures were not disturbed by LBM outbreaks, as shown, for example, by exceptionally high significant correlations between non-host spruce and host larch chronologies; (3) below-average July to August temperatures and LBM defoliation events have been coupled for more than three centuries. Dampening of Alps-wide LBM cyclicity since the 1980s and the coincidence of recently absent cool summers in the European Alps reinforce the assumption of a strong coherence between summer temperatures and LBM defoliation events. Our results demonstrate that stable isotopes in tree-ring cellulose of larch are an excellent climate proxy enabling the analysis of climate-driven changes of LBM cycles in the long term.

Prominent differences in leaf fatty acid composition in the F1 hybrid compared with parent trees Larix gmelinii var. japonica and L. kaempferi.

Fatty acid (FA) compositions in leaves were investigated for two families of F(1) hybrids of Larix gmelinii var. japonica x L. kaempferi (F(1)) and their parent clones. Twenty-one FAs, from C(12) to C(32), were found in the leaves of both adult trees and seedlings. The levels of 18:1/(18:2 + 18:3) increased in the order L. kaempferi, F(1), and L. gmelinii var. japonica, with significant differences between L. gmelinii var. japonica and F(1) in adult trees, but these differences were not found in the seedlings. Moreover, in the adult trees, the 18:1/(18:2 + 18:3) levels in the neutral phospholipid fraction and the SigmaC(18)/SigmaC(16), especially in the glycolipid fraction, showed significant differences among the three species. These characteristics are discussed from the viewpoint of lipid synthesis in the endoplasmic reticulum and chloroplasts, and of the activities and substrate specificities in sequential FA desaturation. Linear discriminant analysis suggested that the FA compositions are useable as an index in the identification of hybrid seedlings.

[Patterns of somatic embryo formation in Siberian larch: embryological aspects].

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5-1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5-10 days, such embryos formed the embryogenic tissue including two cell types--elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.

Gene expression associated with increased supercooling capability in xylem parenchyma cells of larch (Larix kaempferi).

Xylem parenchyma cells (XPCs) in larch adapt to subfreezing temperatures by deep supercooling, while cortical parenchyma cells (CPCs) undergo extracellular freezing. The temperature limits of supercooling in XPCs changed seasonally from -30 degrees C during summer to -60 degrees C during winter as measured by freezing resistance. Artificial deacclimation of larch twigs collected in winter reduced the supercooling capability from -60 degrees C to -30 degrees C. As an approach to clarify the mechanisms underlying the change in supercooling capability of larch XPCs, genes expressed in association with increased supercooling capability were examined. By differential screening and differential display analysis, 30 genes were found to be expressed in association with increased supercooling capability in XPCs. These 30 genes were categorized into several groups according to their functions: signal transduction factors, metabolic enzymes, late embryogenesis abundant proteins, heat shock proteins, protein synthesis and chromatin constructed proteins, defence response proteins, membrane transporters, metal-binding proteins, and functionally unknown proteins. All of these genes were expressed most abundantly during winter, and their expression was reduced or disappeared during summer. The expression of all of the genes was significantly reduced or disappeared with deacclimation of winter twigs. Interestingly, all but one of the genes were expressed more abundantly in the xylem than in the cortex. Eleven of the 30 genes were thought to be novel cold-induced genes. The results suggest that change in the supercooling capability of XPCs is associated with expression of genes, including genes whose functions have not been identified, and also indicate that gene products that have been thought to play a role in dehydration tolerance by extracellular freezing also have a function by deep supercooling.

Larch (Larix kaempferi) xylem parenchyma cells respond to subfreezing temperature by deep supercooling.

In previous studies, xylem parenchyma cells (XPCs) in the boreal softwood species larch, which has thick and rigid walls similar to those of XPCs in boreal hardwood species, were shown to respond to subfreezing temperature by deep supercooling during summer but change their freezing behavior to extracellular freezing during winter. In this study, we re-examined freezing behavior of XPCs in larch by observation of deep etching of frozen samples as well as observation of re-warmed samples after freezing using a cryo-scanning electron microscope. The results showed that XPCs in larch adapts to subfreezing temperature by deep supercooling throughout all seasons. Such freezing behavior is the same as that of XPCs in boreal hardwood species.

[Induction of androgenic cultures in Siberian larch].

Male generative buds of the Siberian larch have no organic quiescence during the autumn-winter period and are capable of completing the development of male generative structures under favorable conditions. When microsporophylls of the Siberian larch were cultivated on medium MS with 0.2 mg/l 2,4-D, embryoids of two types were obtained: directly from the microspore body and via formation of organogenic callus. This means that in the Siberian larch, direct and indirect androgenesis in vitro is possible. The Siberian larch pollen was first germinated in vitro, which was enhanced by pretreatment with hydrogen peroxide.

Transcriptional activity in diplotene larch microsporocytes, with emphasis on the diffuse stage.

Manuscript provides insights into the biology of long-lived plants, different from Arabidopsis, tomato or grass species that are widely studied. In the European larch the diplotene stage lasts approximately 5 months and it is possible to divide it into several substages and to observe each of them in details. The diplotene stage is a period of intensive microsporocyte growth associated with the synthesis and accumulation of different RNA and proteins. Larch microsporocytes display changes in chromatin morphology during this stage, alternating between 4 short stages of chromatin condensation (contraction) and 5 longer diffusion (relaxation) stages. The occurrence of a diplotene diffusion stage has been observed in many plant species. Interestingly, they have also been observed during spermiogenesis and oogenesis in animals. The aim of this study was to examine whether chromatin relaxation during the diplotene is accompanied by the synthesis and maturation of mRNA. The results reveal a correlation between the diffusion and chromatin decondensation, transcriptional activity. We also found decreasing amount of poly(A) mRNA synthesis in the consecutive diffusion stages. During the early diffusion stages, mRNA is intensively synthesized. In the nuclei large amounts of RNA polymerase II, and high levels of snRNPs were observed. In the late diffusion stages, the synthesized mRNA is not directly subjected to translation but it is stored in the nucleus, and later transported to the cytoplasm and translated. In the last diffusion stage, the level of poly(A) RNA is low, but that of splicing factors is still high. It appears that the mRNA synthesized in early stages is used during the diplotene stage and is not transmitted to dyad and tetrads. In contrast, splicing factors accumulate and are most likely transmitted to the dyad and tetrads, where they are used after the resumption of intense transcription. Similar meiotic process were observed during oogenesis in animals. This indicates the existence of an evolutionarily conserved mechanism of chromatin-based regulation of gene expression during meiotic prophase I.

Development of male gametophyte of Larix leptolepis Gord. with emphasis on diffuse stage of meiosis.

A basic developmental framework of the Larix leptolepis Gord male gametophyte is presented in detail by squashing technique. The duration of the meiosis stage was more than 6 months, and included a long diffuse stage during winter. This long duration of the diffuse appearance of the diplotene stage makes L. leptolepis a unique suitable experimental material for studying the structure and function of the diffuse stage of meiosis. In particular, the processes of desynapsing and unpairing, which so far have received little attention, can be examined in detail. In L. leptolepis, the chromosomes undergo a dramatic structural reorganization during the diffuse diplotene stage. Based on the clearly visible differences in chromosome morphology, the diffuse diplotene stage was divided into four periods with suggested nomenclature as follows: schizonema, pre-diffuse diplotene, diffuse diplotene and post-diffuse diplotene. Both simultaneous and successive microsporogenesis were observed within L. leptolepis, and there was no strict relationship between the microsporogenesis types and the tetrad configurations, which are strongly influenced by spindle orientation, especially during meiosis II. The mature pollen grain at pollination consists of five cells aligned in an axial row. The prothallial cells cannot be regarded as senescent cells because they remain capable of division.

Additional nucleoli and NOR activity during meiotic prophase I in larch (Larix decidua Mill.).

Transcriptional activity was investigated in successive stages of prophase I (male meiosis) of larch meiocytes. Br-UTP incorporated into RNA was detected by light and electron microscopy. Two peaks of RNA synthesis were identified in the nucleolus. The first occurred during the zygotene-pachytene stage and the second (not previously described in plant meiocytes) in the diplotene. These processes correlated with a considerable increase in nucleolus volume during these periods. At the end of the zygotene, several perinucleolar structures lying close to each other and containing rRNA, argyrophilic proteins, U3 small nucleolar RNA, and fibrillarin were observed. The occurrence of newly formed RNA was also observed in these structures. This suggests that the observed perinucleolar structures correspond to the additional nucleoli known from animals.

13CO2 pulse-labelling of photoassimilates reveals carbon allocation within and between tree rings.

Post-photosynthetic fractionation processes during translocation, storage and remobilization of photoassimilate are closely related to intra-annual sigma13C of tree rings, and understanding how these processes affect tree-ring sigma13C is therefore indispensable for improving the quality of climate reconstruction. Our first objective was to study the relationship between translocation path and phloem grain. We pulse-labelled a branch of Larix gmelinii (Rupr.) Rupr. and later analysed the sigma13C distribution in the stem. A 13C spiral translocation path closely related to the spiral grain was observed. Our second objective was to study the use of remobilized storage material for earlywood formation in spring, which is a suspected cause of the autocorrelation (correlation of ring parameters to the climate in the previous year) observed in (isotope) dendroclimatology. We pulse-labelled whole trees to study how spring, summer and autumn photoassimilate is later used for both earlywood and latewood formation. Analysis of intra-annual sigma13C of the tree rings formed after the labelling revealed that earlywood contained photoassimilate from the previous summer and autumn as well as from the current spring. Latewood was mainly composed of photoassimilate from the current year's summer/autumn, although it also relied on stored material in some cases. These results emphasize the need for separating earlywood and latewood for climate reconstruction work with narrow boreal tree rings.

New type of snRNP containing nuclear bodies in plant cells.

In larch (Larix decidua Mill.) microspores a new type of nuclear bodies has been found which are an element of the spatial organization of the splicing system in plant cell. These are bizonal bodies, ultrastructurally differentiated into a coiled part and a dense part. Using immunocytochemistry and in situ hybridization at the EM level, the coiled part of the bizonal body was found to contain snRNA including U2 snRNA, Sm proteins and nucleolar proteins of the agyrophilic type and fibrillarin. The dense part contains Sm proteins but lacks snRNA. Such a separation of macromolecules related to splicing occurring within the bizonal bodies microspore is striking by the similarity of these bodies to amphibian oocyte snurposomes. The occurrence in plant cells, beside widely known coiled bodies (CBs), also of other nuclear bodies related to splicing proves that in plants similarly as for animals the differentiation among domains containing elements of the splicing system occurs.