Early growth of Brazilian pine (Araucaria angustifolia[Bertol.] Kuntze) in response to soil compaction and drought

Soil compaction leads to changes in soil physical properties such as density, penetration resistance and porosity, and, by consequence, affects root and plant growth. The initial growth of Brazilian pine is considered as being more affected by soil physical than chemical conditions, and the presence of a well-developed tap root system has been associated with this fact. A greenhouse experiment was conducted in order to evaluate the impact of soil compaction on the growth of Brazilian pine seedlings and on their susceptibility to a simulated drought period. In the first phase of the experiment, the effects of three levels of soil compaction on root morphology and plant growth were examined. Soil cylinders were artificially compacted in PVC tubes. Pre-germinated seeds were planted, and 147 days later 10 plants from each treatment were harvested for analysis. Higher values of soil density were associated with a shorter and thicker tap root. Growth of lateral roots and shoots remained unaffected at this stage. In the second phase, half of the plants (12) in each compaction treatment were drought-stressed by withholding water for a period of 77 days. Increased soil compaction again resulted in reduced length and increased diameter of the main tap root. This time, the effects were also extended to the lateral roots. Shoot extension growth and overall plant mass, however, increased with soil compaction. This greater mass accumulation in plants growing under increased soil compaction may be attributed to a more intimate contact between roots and soil particles. Drought stress reduced both root and shoot growth, but root mass was more negatively affected by drought stress in plants growing under high levels of soil compaction. Future investigations on the effects of soil compaction on the initial growth of Brazilian pine should include a wider range of compaction levels to better establish the relationship between soil physical parameters and plant growth.     

High-efficiency cryopreservation of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures: ultrastructural characterization and morpho-physiological features

Here we evaluated and characterized the growth dynamics of A. angustifolia embryogenic cultures (EC) submitted to different cryotreatment incubation times through morphological and time-lapse cell tracking analyzes. The EC submitted to cryopreservation protocol were evaluated by regrowth rates, and ultrastructural characterization by transmission electron microscopy (TEM). The results indicated that A. angustifolia EC support all the cryoprotection times evaluated, without cell proliferation inhibition, but with noticeable genotype-dependent response in all tested cell lines. The use of 1M DMSO showed non-inhibitory effects to EC regrowth independent of cell line or cryotreatment incubation time. However, after cryopreservation, Cr01 cell line regrowth was 100 % for all cryotreatments incubation times evaluated (30, 60, 120, 240 min), while Cr02 cell line only showed 100 % regrowth in 240 min of cryotreatment. The 100 % cell regrowth obtained in both cell lines indicates that the proposed protocol can be successful applied to A. angustifolia EC cryopreservation. Cell tracking analysis showed a survival and initial proliferation of embryogenic cells, with the first cell regrowth signs after 30 days in culture. TEM analysis revealed a conspicuous cell wall thickening in embryogenic cells after cryotreatment and after thawing, which may be related to osmotic stress response caused by the cryopreservation process. An increased heterochromatin presence was also observed in cryotreated or after thawing cells, may possibly be acting as a cell defense mechanism, decreasing the DNA vulnerability to cleavage and preserving the cell integrity.

     

Araucaria angustifolia (Bertol.) Kuntze has neuroprotective action through mitochondrial modulation in dopaminergic SH-SY5Y cells

Molecular Biology Reports - Brain disorders (BD) including neuropsychiatric and neurodegenerative diseases, are often associated with impairments in mitochondrial function and oxidative damage that...     

Patterns of pollen and seed dispersal in a small, fragmented population of the wind-pollinated tree Araucaria angustifolia in southern Brazil

Paternity analysis based on eight microsatellite loci was used to investigate pollen and seed dispersal patterns of the dioecious wind-pollinated tree, Araucaria angustifolia. The study sites were a 5.4 ha isolated forest fragment and a small tree group situated 1.7 km away, located in Paranalpha State, Brazil. In the forest fragment, 121 males, 99 females, 66 seedlings and 92 juveniles were mapped and genotyped, together with 210 seeds. In the tree group, nine male and two female adults were mapped and genotyped, together with 20 seeds. Paternity analysis within the forest fragment indicated that at least 4% of the seeds, 3% of the seedlings and 7% of the juveniles were fertilized by pollen from trees in the adjacent group, and 6% of the seeds were fertilized by pollen from trees outside these stands. The average pollination distance within the forest fragment was 83 m; when the tree group was included the pollination distance was 2006 m. The average number of effective pollen donors was estimated as 12.6. Mother-trees within the fragment could be assigned to all seedlings and juveniles, suggesting an absence of seed immigration. The distance of seedlings and juveniles from their assigned mother-trees ranged from 0.35 to 291 m (with an average of 83 m). Significant spatial genetic structure among adult trees, seedlings, and juveniles was detected up to 50 m, indicating seed dispersal over a short distance. The effective pollination neighborhood ranged from 0.4 to 3.3 ha. The results suggest that seed dispersal is restricted but that there is long-distance pollen dispersal between the forest fragment and the tree group; thus, the two stands of trees are not isolated.     

Impact of monoecy in the genetic structure of a predominately dioecious conifer species, Araucaria angustifolia (Bert.) O. Kuntze

Understanding the mating system of a tree species is important in genetic conservation and tree breeding strategies because it affects the inbreeding and genetic diversity of the descendant populations. Araucaria angustifolia (Bert.) O. Kuntze is a mainly dioecious species that reproduces through outcrossing. However, some monoecious trees have been identified and they may reproduce through self-fertilization. The objective of this study was to confirm the expected relatedness of full-sibs in outcrossed hand-pollinated progenies of female seed trees, self-sibs of hand self-pollinated monoecious seed trees, and to investigate the mating system of open-pollinated progenies of female and monoecious A. angustifolia trees. To do this, eight microsatellite loci were used to genotype hand- and open-pollinated progenies. Our results show that the relatedness of outcrossed hand-pollinated progenies are true full-sibs and progenies from a selfed monoecious seed tree are self-sibs, which confirms the hand-pollination method used. Open-pollinated female seed trees reproduced only by outcrossing, generating progenies with a mixture of full- and half-sibs. Monoecious seed trees reproduced mainly by xenogamy, generating progenies with mixtures of self-sibs, full-sibs, half-sibs and self-half-sibs. We also found that an increase in the effective number of pollen donors ( $ N_{\text{ep}} $ ) would lead to an increase in the total number of alleles ( $ K $ ) within progenies. Our results also suggest that monecious trees have limited potential to modify the genetic structure through selfed seed production due to the very low estimated selfing rate in these trees and the rare occurrence of these trees in natural populations.     

Intrapopulational genetic diversity of Araucaria angustifolia (Bertol.) Kuntze is different when assessed on the basis of chloroplast or nuclear markers

A natural population of Araucaria angustifolia (Bertol.) Kuntze was evaluated to quantify genetic diversity, using nucleotide sequences of chloroplast maturase K genes (matK) and six specific nuclear polymorphic microsatellite loci (nuSSR). Estimates of nucleotide diversity assessed from matK sequences were low (π = 0.001), but they indicated the potential of this gene for evaluating divergence among widespread populations. The matK sequence (1,565 bp) was used for analysis of phylogenetic relationships, enabling clustering of the South American species (A. angustifolia and A. araucana). For analysis of intraspecific genetic diversity, nuSSR were more informative than matK, resulting in a polymorphic loci percentage of 69, a number average of alleles per locus of 11.6, expected genetic diversity 0.741, and significant spatial genetic structure up to 25 m. Our data can help in the selection of genetic sources for conservation of this species, which is at risk of extinction.     

Cloning and expression of embryogenesis-regulating genes in Araucaria angustifolia (Bert.) O. Kuntze (Brazilian Pine)

Angiosperm and gymnosperm plants evolved from a common ancestor about 300 million years ago. Apart from morphological and structural differences in embryogenesis and seed origin, a set of embryogenesis-regulating genes and the molecular mechanisms involved in embryo development seem to have been conserved alike in both taxa. Few studies have covered molecular aspects of embryogenesis in the Brazilian pine, the only economically important native conifer in Brazil. Thus eight embryogenesis-regulating genes, viz., ARGONAUTE 1, CUP-SHAPED COTYLEDON 1, WUSCHEL-related WOX, S-LOCUS LECTIN PROTEIN KINASE, SCARECROW-like, VICILIN 7S, LEAFY COTYLEDON 1, and REVERSIBLE GLYCOSYLATED POLYPEPTIDE 1, were analyzed through semi-quantitative RT-PCR during embryo development and germination. All the eight were found to be differentially expressed in the various developmental stages of zygotic embryos, seeds and seedling tissues. To our knowledge, this is the first report on embryogenesis-regulating gene expression in members of the Araucariaceae family, as well as in plants with recalcitrant seeds.     

Internal genetic structure and outcrossing rate in a natural population of Araucaria angustifolia (Bert.) O. Kuntze

The internal genetic structure and outcrossing rate of a population of Araucaria angustifolia (Bert.) O. Kuntze were investigated using 16 allozyme loci. Estimates of the mean number of alleles per loci (1.6), percentage of polymorphic loci (43.8%), and expected genetic diversity (0.170) were similar to those obtained for other gymnosperms. The analysis of spatial autocorrelation demonstrated the presence of internal structure in the first distance classes (up to 70 m), suggesting the presence of family structure. The outcrossing rate was high (0.956), as expected for a dioecious species. However, it was different from unity, indicating outcrossings between related individuals and corroborating the presence of internal genetic structure. The results of this study have implications for the methodologies used in conservation collections and for the use or analysis of this forest species.     

The transition of proembryogenic masses to somatic embryos in Araucaria angustifolia (Bertol.) Kuntze is related to the endogenous contents of IAA,ABA and polyamines

In somatic embryogenesis (SE) of conifers, the inability of many embryogenic cell lines to form well-developed somatic embryos may results from failure and constraints during the transition of proembryogenic masses (PEMs) to early somatic embryos. In the present work, we propose the inclusion of a preculture and prematuration steps looking at enhancing PEM III-to-early somatic embryos transition. It was further hypothesized that these results would correlate with the contents of endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and polyamines (PA). To test these hypotheses, the embryogenic culture was subjected to preculture with fluridone (FLD) and prematuration treatments with different combinations of carbon source and polyethylene glycol (PEG). The frequency of PEM III was increased after FLD preculture and the contents of IAA and ABA decreased, while the contents of PA increased. Putrescine (Put) was the most abundant PA present at this stage, followed by spermidine (Spd) and spermine (Spm). In early embryogenesis, prematuration treatments supplemented with maltose or lactose plus PEG enhanced the PEM III-to-early somatic embryos transition. IAA and ABA contents increased at this stage, while a decrease of the total free PA levels was observed. Put was the most abundant PA, followed by Spd and Spm, mainly in the treatment supplemented with PEG. This resulted in a decrease of PA ratio (Put/Spd + Spm) and, hence, PEM III-to-early somatic embryos transition. It was concluded that the preculture with FLD and prematuration treatments promote the PEM III-to-early somatic embryos transition throughout the whole early developmental process in Araucaria angustifolia.     

Past century changes in Araucaria angustifolia (Bertol.) Kuntze water use efficiency and growth in forest and grassland ecosystems of southern Brazil: implications for forest expansion

Araucaria angustifolia (Bertol.) Kuntze is an indigenous conifer tree restricted to the southern region of South America that plays a key role in the dynamics of regional ecosystems where forest expansion over grasslands has been observed. Here, we evaluate the changes in intrinsic water use efficiency (iWUE) and basal area increment (BAI) of this species in response to atmospheric CO₂, temperature and precipitation over the last century. Our investigation is based on tree-rings taken from trees located in forest and grassland sites in southern Brazil. Differences in carbon isotopic composition ( δ ¹³C), ¹³CO₂ discrimination (Δ¹³C) and intracellular carbon concentration ( C _i ) are also reported. Our results indicate an age effect on Δ¹³C in forest trees during the first decades of growth. This age effect is not linked to an initial BAI suppression, suggesting the previous existence of nonforested vegetation in the forest sites. After maturity all trees show similar temporal trends in carbon isotope-derived variables and increasing iWUE, however, absolute values are significantly different between forest and grassland sites. The iWUE is higher in forest trees, indicating greater water competition or nutritional availability, relative to grassland, or both. BAI is also higher in forest trees, but it is not linked with iWUE or atmospheric CO₂. Nevertheless, in both forest and grassland sites A. angustifolia has had growth limitations corresponding to low precipitation and high temperatures observed in the 1940s.     

AaMps1 protein inhibition regulates the protein profile,nitric oxide,carbohydrate and polyamine contents in embryogenic suspension cultures of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)

Plant Cell, Tissue and Organ Culture (PCTOC) - The Monopolar Spindle 1 (Mps1) protein is a dual-specificity kinase that plays a critical role in the progression of the cell cycle. Studies on...     

Reversal of height dimorphism promotes pollen and seed dispersal in a wind-pollinated dioecious plant

Variation in the timing of reproductive functions in dioecious organisms may result in adaptive changes in the direction of sexual dimorphism during the breeding season. For plants in which both pollen and seeds are wind-dispersed, it may be advantageous for male plants to be taller when pollen is dispersed and female plants to be taller when seeds are dispersed. We examined the dynamics of height dimorphism in Rumex hastatulus, an annual, wind-pollinated, dioecious plant from the southern USA. A field survey of seven populations indicated that females were significantly taller than males at seed maturity. However, a glasshouse experiment revealed a more complex pattern of height growth during the life cycle. No dimorphism was evident prior to reproduction for six of seven populations, but at flowering, males were significantly taller than females in all populations. This pattern was reversed at reproductive maturity, consistent with field observations. Males flowered later than females and the degree of height dimorphism was greater in populations with a later onset of male flowering. We discuss the potential adaptive significance of temporal changes in height dimorphism for pollen and seed dispersal, and how this may be optimized for the contrasting reproductive functions of the sexes.     

Patterns of genetic diversity in southern and southeastern Araucaria angustifolia (Bert.) O. Kuntze relict populations

Habitat fragmentation and a decrease in population size may lead to a loss in population genetic diversity. For the first time, the reduction in genetic diversity in the northernmost limit of natural occurence (southeastern Brazil) of Araucaria angustifolia in comparison with populations in the main area of the species continuous natural distribution (southern Brazil), was tested. The 673 AFLPs markers revealed a high level of genetic diversity for the species (Ht = 0.27), despite anthropogenic influence throughout the last century, and a decrease of H in isolated populations of southeastern Brazil (H = 0.16), thereby indicating the tendency for higher genetic diversity in remnant populations of continuous forests in southern Brazil, when compared to natural isolated populations in the southeastern region. A strong differentiation among southern and southeastern populations was detected (AMOVA variance ranged from 10%-15%). From Bayesian analysis, it is suggested that the nine populations tested form five "genetic clusters" (K = 5). Five of these populations, located in the northernmost limit of distribution of the species, represent three "genetic clusters". These results are in agreement with the pattern of geographic distribution of the studied populations.     

Structural and component characterization of meristem cells in Araucaria angustifolia (Bert.) O. Kuntze zygotic embryo

Araucaria angustifolia, the Brazilian pine, is an endangered native conifer with economic and ecological importance. The female cone develops seeds containing the zygotic embryo, which, at cotyledonary stage, shows well-developed meristems. Little is known about the structure of gymnosperm meristems. In the present work, the composition and morphological organization of Araucaria angustifolia shoot and root apical meristems were studied during embryo development, using histochemical and microscope analyses. Histochemical evaluation revealed the presence of cellulose within the cell wall, cells with the presence of total proteins that react with Coomassie Brilliant Blue, starch grains, and large nuclei with evident nucleoli in the cytoplasm. Scanning electron microscopy showed apical meristem surface morphology, and both scanning and transmission microscopy revealed a thin and irregular cell wall with plasmodesmata and within the cells, mitochondria, many vacuoles, lipid bodies, Golgi bodies, and many amyloplasts with endoplasmic reticulum surrounding them and large nuclei. Similar to angiosperm cells, A. angustifolia meristem cells exhibit pluripotent characteristics, such as apparatus for intercellular communication and differentiation.     

Cell-to-cell trafficking patterns in cell lines of Araucaria angustifolia (Brazilian pine) with contrasting embryogenic potential

Plant Cell, Tissue and Organ Culture (PCTOC) - The acquisition of embryogenic competence involves aspects at a cellular level related to cell communication and signaling. For Araucaria...     

Initial growth of Brazilian pine (Araucaria angustifolia) under equal soil volumes but contrasting rooting depths

Araucaria angustifolia is a critically endangered tall tree species of valuable wood, and field observations led to the suggestion that limitations imposed to the vertical growth of its tap root system greatly restrict the height of mature individuals. However, experimental studies dealing with the effects of soil depth on the species growth are mostly lacking. This study evaluated and compared the growth responses of young plants of A. angustifolia to distinct rooting depths but same soil volumes. Seeds were planted in pots of different heights and diameters, all containing 3 liters of soil mixture. Plants were submitted to four available rooting depths: 65 (T1), 35 (T2), 20 (T3), and 10 (T4) cm. There were eight experimental units in each treatment, arranged in a randomized complete block design, each block containing two units per treatment. Contrary to what was expected, the T3 and T4 plants had accumulated more mass and attained the same height as the other two groups, after a 10-month growth period in a green house. Those plants also had thicker stems, longer shoot branches, and thicker and longer lateral roots, which were interpreted as compensatory responses to increase plant anchorage and stability. The inverse relationship between rooting depth and plant mass was attributed to a down-regulation of shoot growth because or restricted lateral space and/or poor soil aeration of the longer and narrower pots. This experiment allowed us to demonstrate that is not the possibility of the tap root to grow deep into the soil that ensures a better growth to plants of A. angustifolia: provided that the offer of soil volume and resources are the same, the vertical extension of the tap root does not result in greater growth of the plants. In fact, much greater growth impairment is expected from lateral than from vertical restriction to root growth.     

Genetic analysis of 50-year old Brazilian pine (Araucaria angustifolia) plantations: implications for conservation planning

In order to determine the implications of including planted populations in conservation planning, we investigate the structure and genetic diversity, mating system, and effective population size within families in three planted and one natural Araucaria angustifolia populations. The study area is a Brazilian National Forest in southern Brazil, established in the 1950s. Genetic analysis was investigated using nine allozyme loci. Significant and positive fixation index was detected for the adults of all studied populations. Although we observed lower levels of genetic diversity and biparental inbreeding in some populations, their pattern of mating, similar to that expected in panmixia, indicates that all populations can be used in conservation planning. Furthermore, the presence of unique alleles and the significant genetic differentiation among all adult populations (F _ST = 0.404) reinforces that all populations are important conservation resources. The average paternity correlation was high in almost all populations, showing that open-pollinated families are composed of mixtures of half- and full-sibs. With regards to conservation strategies, our results suggest that seed collection from at least 133 seed trees is necessary to retain a reference effective population size of 500. Our results confirm that the planted Araucaria forests in southern Brazil can be useful as a genetic resource for conservation, breeding and reforestation strategies for this threatened species.