Mycorrhiza of Brazil Pine (Araucaria angustifolia [Bert. O. Ktze.])

Abstract: Tropical and subtropical forests once covered large areas of Central and South America. An important member of forests of the southern hemisphere is the genus Araucaria. Because of clear cutting only small remnants of Araucaria angustifolia forests still exist in Southern Brazil. Attempts at reforestation have had only limited success because of lack of knowledge about the environmental requirements of this species. This is especially true with respect to the root/fungus symbiosis (mycorrhiza) which is necessary for enhanced water and nutrient uptake and present in more than 90 % of land plants. Analysis of the root systems of Araucaria trees from forest and grassland (campo) sites revealed mycorrhizal structures (appressoria, penetration and coiled hyphae, vesicles, arbuscules, spores) which are characteristic for the arbuscular mycorrhiza (AM) type. The spores of AM fungi at both sites - forest and campo - were identified. The biodiversity at the forest site was much higher, with 13 species, whereas only 6 different species could be identified at the campo site. Glomus and Acaulospora were the only genera present at the campo. The forest, however, also contained spores of Entrophospora and Scutellospora. In addition to the greater biodiversity, the spore number in soil as well as the percent mycorrhizal colonization in roots were significantly higher at the forest site than at the campo site. Because of the low frequency of hyphal coils and the dominating intercellular growth of hyphae, these mycorrhizas can be classified as an Arum -type, which is the first report of this kind in gymnosperms.     

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.     

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.     

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.     

Changes in IAA, tryptophan and activity of soluble peroxidase associated with zygotic embryogenesis in Araucaria angustifolia (Brazilian pine)

In A. angustifolia seeds the highest values of freeIAA occurred in the embryonic axis, at the initial phases of development. Thesevalues decreased sharply coincident with the increase of IAA with thedifferentiation of cotyledons and seed elongation. During seed development,tryptophan concentrations varied inversely with free IAA and directly withconjugated IAA. An increase in peroxidase activity was followed by a decreaseinfree IAA in the embryo axis, and in conjugated IAA in the megagametophyte.Megagametophyte tissues did not exhibit significant variation in free IAAduringseed development. Following the stage where cotyledons arise, tryptophandecreased in the megagametophyte and increased in the cotyledons and embryonicaxis.     

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.     

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.     

Slow development of leaf photosynthesis in an evergreen broad-leaved tree, Castanopsis sieboldii: relationships between leaf anatomical characteristics and photosynthetic rate

Changes in net photosynthetic rate on a leaf area basis and anatomical properties during leaf development were studied in an evergreen broad‐leaved tree, Castanopsis sieboldii and an annual herb, Phaseolus vulgaris. In C. sieboldii, surface area of mesophyll cells facing the intercellular air spaces on a leaf area basis (S_mes) was already considerable at the time of full leaf area expansion (FLE). However, surface area of chloroplasts facing the intercellular air spaces on a leaf area basis (S_c), and chlorophyll and Rubisco contents on a leaf area basis increased to attain their maximal values 15–40 d after FLE. In contrast, in P. vulgaris, chloroplast number on a leaf area basis, S_c and S_mes at 10 d before FLE were two to three times greater than the steady‐state levels attained at around FLE. In C. sieboldii, the internal CO₂ transfer conductance (g_i) slightly increased for 10 d after FLE but then decreased toward the later stages. Limitation of photosynthesis by g_i was only about 10% at FLE, but then increased to about 30% at around 40 d after FLE. The large limitation after FLE by g_i was probably due to the decrease in CO₂ concentration in the chloroplast caused by the increases in thickness of mesophyll cell walls and in Rubisco content per chloroplast surface area. These results clearly showed that: (1) in C. sieboldii, chloroplast development proceeded more slowly than mesophyll cell expansion and continued well after FLE, whereas in P. vulgaris these processes proceeded synchronously and were completed by FLE; (2) after FLE, photosynthesis in leaves of C. sieboldii was markedly limited by g_i. From these results, it is suggested that, in the evergreen broad‐leaved trees, mechanical protection of mesophyll cells has priority over the efficient CO₂ transfer and quick construction of the chloroplasts.     

Seasonal development of the photosynthetic performance of Norway spruce (Picea abies [L.] Karst.) under magnesium deficiency

In order to investigate the influence of different magnesium nutrition on photosynthesis, one hundred 6-year-old spruce trees derived from one clone were planted in October 1990 into a special out-door experimental construction, where they were cultivated in sand culture with an optimal supply of nutrients, except magnesium, via circulating nutrient solutions. Magnesium was added to the nutrient solutions in three different concentrations, varying from optimal to severe deficient supplies. During the first vegetative period in 1991, photosynthetic performance and carboxylation efficiency were measured under saturating light, controlled CO₂ conditions, optimal temperature and humidity, using a minicuvette system.During summer, the trees under moderate magnesium deficiency developed tip yellowing symptoms on older needles, while the youngest needles remained green with unchanged chlorophyll contents. Trees under severe magnesium deficiency showed yellowing symptoms on all needle age classes combined with decreased chlorophyll contents in the youngest needles as well. In comparison with the controls, the photosynthetic performance of the 1-year-old needles was significantly lower in both deficiency treatments. The same was observed in the youngest needles of the trees under severe deficiency. Trees under moderate deficiency treatment decreased in photosynthetic performance during the summer without reduction of chlorophyll contents. The reduction of photosynthetic rates corresponded to a decrease in carboxylation efficiency, which is taken as a measure of the activity of the enzyme ribulose-1,5-bisphosphate carboxylase. This reduction, together with the observed increase of carbohydrate contents in needles of trees growing under magnesium deficiency, led to the assumption that the photosynthetic carbonfixation is reduced as a consequence of the accumulation of carbohydrates.     

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.     

The Multiple Forms of alpha-Amylase Enzyme of the Araucaria Species of South America: A. araucana (Mol.) Koch and A. angustifolia (Bert.) O. Kutz : A Comparative Study

α-Amylase is one of the major enzymes present in the seeds of both Araucaria species of South America and it initiates starch hydrolysis during germination and early seedling growth. The pattern of the multiple forms of α-amylase of the two Araucaria species was investigated by electrophoresis and isoelectrofocusing of the native enzyme in polyacrylamide gels. The enzyme forms were compared in the embryo and megagametophyte of quiescent seeds and of seeds imbibed for 18, 48, and 90 hours. Specific α-amylase enzyme forms appear and disappear during these imbibition periods showing both similarities and differences between tissues and species. Before imbibition, there are five α-amylase forms identical in both tissues, but different between species. After 18 hours of imbibition, there are two enzyme forms in both tissues of Araucaria araucana seeds, only one form in the embryo of Araucaria angustifolia but two forms in the megagametophyte of this specie. After 48 hours of seed imbibition, most of the enzyme forms present in quiescent seeds reappear. At 90 hours of imbibition different enzyme forms are detected in the embryo with respect to the gametophyte. The changes in form patterns of α-amylase are discussed according to a possible regulation of gene expression by endogenous gibberellins.     

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.)

Stimulation of leaf expansion by an exogenous cytokinin was studied in isolated leaf discs of sweet pepper with emphasis on the assimilate utilization of the tissue. Leaf discs were floated on solutions containing sucrose and plant growth regulators. Benzyladenine (BA) promoted the area expansion rate of the leaf discs. Sucrose at 100 mM resulted in increased area expansion rate compared with 10 mM sucrose. However, the increased sucrose concentration had no influence on the effect of BA. Over a period of 24 h, treatment with BA did not result in any change of sucrose uptake nor of the partitioning of assimilated carbon in the leaf discs. Neither did BA treatment affect the activity of acid invertase (EC 3.2.1.26) or pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) in the leaf discs. We conclude that the observed promotion of leaf area expansion by exogenous BA is not mediated through the uptake of sucrose or the carbohydrate metabolism of the leaf tissue.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid - PPi-PFK pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) This study was supported by grants from the Danish Research Counsil (SJVF 13-4148 and 13-4547 to P.U. SJVF 13-4146 and 13-4494 to T.H.N.) and from The Research Center for Plant Biotechnology to P.U.     

Local Knowledge,Use and Management of Ethnovarieties of <Emphasis Type="Italic">Araucaria angustifolia</Emphasis> (Bert.) Ktze. in the Plateau of Santa Catarina,Brazil

This study examines the human use and management of Araucaria angustifolia ethnovarieties from Santa Catarina, Brazil, and contributes to what is known about the ethnobotany of Araucaria species. The available literature on varietal differences of A. angustifolia is somewhat divergent, and there are currently no ethnobotanical studies on the intraspecific variation and management of this species. The study examined local knowledge and sociocultural and economic values of A. angustifolia varieties to understand how the varieties are managed and how management practices are influencing the conservation of the species. Semi-structured interviews were conducted with 33 informants (identified using the snowball method) in the Painel and Urubici municipalities. Participants identified 12 local varieties, four of which were cited by more than one informant. Characteristic differences include size, color and flavor of the nut-like seeds (pinhão), and most importantly, season of maturation of the cone. The “Caiová” variety was preferred for its bigger, firmer, and sweeter seeds that are considered easier to peel and last longer in storage. Even though there is some interest in developing management practices that favor some varieties in order to guarantee year-round production, seedlings are commonly removed. This management practice is most likely a response to current regulations that prohibit cutting down adult trees. The results of this study have important implications for the relationship between the knowledge of A. angustifolia practices and the current legal framework that protects this species. A more detailed understanding of the relevant ethnobotanical knowledge is required in order to establish the best practices for sustainable use of A. angustifolia and its varietal diversity and to support the communities that depend on this species as a resource.     

Influence of UV-B radiation on photosynthetic activity and chlorophyll fluorescence kinetics in Norway spruce [Picea abies (L.) Karst.] seedlings

Norway spruce [Picea abies (L.) Karst.] seedlings were grown in greenhouses with two supplemental levels of ultraviolet-B (UV-B) radiation. Photochemical efficiency of photosystem II and vitality index were determined monthly. At the end of the experiment, growth, chlorophyll content and photosynthetic rates were measured. The data indicate that low temperature in winter affected light dependent processes in experimental plants including control, while the rise of ambient temperatures, moderate this effect. The synergistic effects of UV-B radiation and low temperatures could only be observed in the second winter period. Measurements of net photosynthetic activity in the second winter period showed significant differences between treated and untreated plants.     

Regulation of rhizosphere acidification by photosynthetic activity in cowpea (Vigna unguiculata L. walp.) seedlings

In contrast to cereals or other crops, legumes are known to acidify the rhizosphere even when supplied with nitrates. This phenomenon has been attributed to N2 fixation allowing excess uptake of cations over anions; however, as we have found previously, the exposure of the shoot to illumination can cause rhizosphere acidification in the absence of N2 fixation in cowpea (Vigna unguiculata L. Walp). In this study, we examined whether the light-induced acidification can relate to photosynthetic activity and corresponding alterations in cation-anion uptake ratios. The changes of rhizosphere pH along the root axis were visualized using a pH indicator agar gel. The intensity of pH changes (alkalization/acidification) in the rhizosphere was expressed in proton fluxes, which were obtained by processing the images of the pH indicator agar gel. The uptake of cations and anions was measured in nutrient solution. The rhizosphere was alkalinized in the dark but acidified with exposure of the shoots to light. The extent of light-induced acidification was increased with leaf size and intensity of illumination on the shoot, and completely stopped with the application of photosynthesis inhibitor. Although the uptake of cations was significantly lower than that of anions, the rhizosphere was acidified by light exposure. Proton pump inhibitors N,N'-dicyclohexyl carbodimide and vanadate could not stop the light-induced acidification. The results indicate that light-induced acidification in cowpea seedlings is regulated by photosynthetic activity, but is not due to excess uptake of cations.     

Contribution of corticular photosynthesis to bud development in African baobab (Adansonia digitata L.) and Castor bean (Ricinus communis L.) seedlings

The African baobab (Adansonia digitata L.) and castor bean (Ricinus communis L.) are drought resistant green-stemmed succulent plants which grow in the arid and semi-arid regions of Africa. Photosynthesis in the stems of green-stemmed plants is known to contribute to plant carbon gain especially during leafless periods. To study the contribution of stem photosynthesis in stem succulent plants, the height and stem diameter of baobab and castor bean plants grown in the greenhouse were measured. The plants were completely defoliated and subjected to different treatments: Watered with open stems (WO), watered and stems covered with aluminium foil (WC) to achieve 100% light exclusion, drought and open (DO) and drought and covered (DC). Stem coverage with aluminium foil resulted in a higher stem height and diameter during drought for baobab with similar trends seen in castor bean. Light exclusion resulted in a significantly lower bud DW production and enrichment in ¹³C in bud dry matter of castor bean and in stem dry matter of baobab. These show that corticular photosynthesis contributes in carbon gain in these species.     

Diversity of Regenerating Plants in Reforestations with Araucaria angustifolia (Bertol.) O. Kuntze of 12, 22, 35, and 43 Years of Age in Paraná State, Brazil

In the current context of intense forest fragmentation and consequent loss of biodiversity, commercial reforestations have gained special attention in conservation biology because the colonization of the understory in these wooded areas may result in a considerable number of species. A record was made of the regenerating species in the understory of reforestations with Araucaria angustifolia at planting ages of 12, 22, 35, and 43 years, located in the central east region of Paraná State, Brazil. In total, an area of 1,200 m² was sampled, with 231 recorded species belonging to 60 botanical families. Species diversity and richness increased with planting age up to 35 years. Canopy cover was the variable that best explained the colonization of the understory in some stages. Maintenance activities in the reforestations over the years selected species capable of germinating or sprouting and growing rapidly. However, our results show that A. angustifolia commercial reforestations are home to many native plant species. Thus, the objectives of commercial reforestation can be harmonized with the interests of conservation biology, applying management alternatives with the aim of changing current forestry models.     

Light effects on leaf development and photosynthetic capacity of Hydrocotyle bonariensis Lam.

Rates of net CO₂ uptake were examined in developing leaves of Hydrocotyle bonariensis. Leaves that developed under high photosynthetically active radiation (48 mol m^-2 day^-1 PAR) were smaller, thicker, and reached maximum size sooner than did leaves that developed under low PAR (4.8 mol m^-2 day^-1). Maximum net CO₂ uptake rates were reached after 5 to 6 days expansion for both the low and the high PAR leaves. Leaves grown at high PAR had higher maximum photosynthetic rates and a higher PAR required for light saturation but showed a more rapid decline in rate with age than did low PAR leaves. To assess the basis for the difference observed in photosynthetic rates, CO₂ diffusion conductances and the mesophyll surface available for CO₂ absorption were examined for mature leaves. Stomatal conductance was the largest conductance in all treatments and did not vary appreciably with growth PAR. Mesophyll conductance progressively increased with growth PAR (up to 48 mol m^-2 day^-1) as did the mesophyll surface area per unit leaf area, but the cellular conductance exhibited most of its increase at low PAR (up to 4.8 mol m^-2 day^-1).