The damage caused by Cd toxicity to photosynthesis, cellular ultrastructure, antioxidant metabolism, and gene expression in young cacao plants are mitigated by high Mn doses in soil.

Manganese (Mn) is one of the essential mineral micronutrients most demanded by cacao. Cadmium (Cd) is highly toxic to plants and other living beings. There are indications that Mn can interact with Cd and mitigate its toxicity. The objective of this study was to evaluate the action of Mn on the toxic effect of Cd in young plants of the CCN 51 cacao genotype, subjected to different doses of Mn, Cd, and Mn+Cd in soil, through physiological, biochemical, molecular, and micromorphological and ultrastructural changes. High soil Mn doses favored the maintenance and performance of adequate photosynthetic processes in cacao. However, high doses of Cd and Mn+Cd in soil promoted damage to photosynthesis, alterations in oxidative metabolism, and the uptake, transport, and accumulation of Cd in roots and leaves. In addition, high Cd concentrations in roots and leaf tissues caused irreversible damage to the cell ultrastructure, compromising cell function and leading to programmed cell death. However, there was a mitigation of Cd toxicity when cacao was grown in soils with low Cd doses and in the presence of Mn. Thus, damage to the root and leaf tissues of cacao caused by Cd uptake from contaminated soils can be attenuated or mitigated by the presence of high Mn doses in soil.

Mitigation of Pb toxicity by Mn in seedling of the cacao clonal CCN 51 genotype grown in soil: physiological, biochemical, nutritional and molecular responses.

Lead (Pb) is a highly toxic metal for humans, animals and plants even at low concentrations in the soil. The ingestion of chocolate produced from contaminated beans can contribute to consumer exposure to Pb. While, Mn is an element essential for plants and participates as enzymatic cofactors in several metabolic pathways. The objective of this study was to evaluate the influence of Mn on mitigation of Pb toxicity in seedling of the cacao clonal CCN 51 genotype grown in soils with different doses of Pb, Mn and Mn+Pb, through physiological, biochemical, molecular and nutritional responses. It was found that the seedling of the cacao clonal CCN 51 genotype grown in soils with high Pb, Mn and Mn+Pb contents accumulated these heavy metals in the roots and leaves. Mn doses reduced the Pb uptake by root system and prevented that the Pb accumulated at toxic levels in the roots and leaves of the plants. High doses of Pb applied in soil were highly toxic to the plants, leading, in some cases, them to death. However, no Mn toxicity was observed in cocoa plants, even at high doses in the soil. Uptake of Pb and Mn by the roots and its transport into the aerial part of the plant promoted changes in photosynthesis, leaf gas exchange, respiration, carboxylation and in the instantaneous efficiency of carboxylation, reducing in the treatments with the highest concentrations of Pb, and the emission of chlorophyll fluorescence, affecting the efficiency of photosystem 2 and the production of photoassimilates. Besides that, Pb, Mn and Mn+Pb toxicities activated defense mechanisms in plants that alter the gene expression of met, psbA and psbO, increasing in plants subjected to high concentrations of Pb and the activity of the enzymes involved in the cellular detoxification of excess ROS at the leaf level. In addition, high uptake of Mn by root system was found to reduced Pb uptake in plants grown with Mn+Pb in the soil. Therefore, application of Mn in the soil can be used to mitigate the Pb toxicity in seedling of the cacao clonal CCN 51 genotype grown in contaminated soils.

Impact of Ambient and Elevated [CO2] in Low Light Levels on Growth, Physiology and Nutrient Uptake of Tropical Perennial Legume Cover Crops.

At early stages of establishment of tropical plantation crops, inclusion of legume cover crops could reduce soil degradation due to erosion and nutrient leaching. As understory plants these cover crops receive limited irradiance and can be subjected to elevated CO2 at ground level. A glasshouse experiment was undertaken to assess the effects of ambient (450 µmol mol-1) and elevated (700 µmol mol-1) levels of [CO2] on growth, physiological changes and nutrient uptake of six perennial legume cover crops (Perennial Peanut, Ea-Ea, Mucuna, Pigeon pea, Lab lab, Cowpea) under low levels of photosynthetic photon flux density (PPFD; 100, 200, and 400 µmol m-2 s-1). Overall, total and root dry biomass, total root length, specific leaf area, and relative growth rates were significantly influenced by levels of [CO2] and PPFD and cover crop species. With few exceptions, all the cover crops showed significant effects of [CO2], PPFD, and species on net photosynthesis (PN ) and its components, such as stomatal conductance (gs) internal CO2 conc. (Ci), and transpiration (E). Increasing [CO2], from 450 to 700 µmol mol-1 and increasing PPFD from 100 to 400 µmol Ö¼m-2 Ö¼s-1 increased PN . Overall, the levels of [CO2], PPFD and species significantly affected total water use efficiency (WUETOTAL ), instantaneous water use efficiency (WUEINST ) and intrinsic water use efficiency (WUEINTR ). With some exceptions, increasing levels of [CO2] and PPFD increased all the WUE parameters. Interspecific differences were observed with respect to macro-micro nutrient uptake and use efficiency. With a few exceptions, increasing levels of [CO2] from 450 to 700 µmol mol-1 and PPFD from 100 to 400 µmol m-2 s-1 increased nutrient use efficiency (NUE) of all nutrients by cover crop species.

Proteomic profiles of young and mature cocoa leaves subjected to mechanical stress caused by wind.

Cocoa is a perennial and arboreal species intolerant to strong and frequent winds and, for this reason, is usually grown with windbreaks of trees. The mechanical alterations caused by the wind in the field have a great impact on the growth, development and productivity of cocoa. The present work had a main objective to understand the molecular mechanisms of responses to mechanical stress, caused by the action of constant wind flow in young plants of cocoa through alterations of the proteomic profile in young (YL) and mature leaves (ML). Plants were exposed to constant wind (CW) at a speed of 4.5 m s-1 for 12 h. There was a reduction in the accumulation of proteins in YL and a significant increase in ML submitted to CW in relation to the control. Differentially accumulated proteins, identified in YL and ML, belong to a broad functional group, related to energy production and carbon metabolism. Besides that, there was a higher efficiency in the protein relative abundance associated to energy production and the assimilation of carbon in the ML exposed to CW, in relation to the control. It was observed the appearance of new isoforms and, or post-transitional changes, which represent an acclimatization and tolerance response of these leaves to the stressor factor. In contrast, in YL, the energy production and the synthesis of gene products essential for their growth and development were affected by the mechanical stress caused by the wind, making them more intolerant.

Mitigation of Cd toxicity by Mn in young plants of cacao, evaluated by the proteomic profiles of leaves and roots.

Cd is a non-essential metal and highly toxic to plants, animals and humans, even at very low concentrations. Cd has been found in cocoa beans and in their products, as in the case of chocolate. Mn plays an important role in photosynthetic and can interact with Cd and attenuate its toxic effects on plants. The objective of this work was to evaluate the mechanisms of Mn response in the mitigation of Cd toxicity in young plants of the CCN 51 cacao genotype submitted to 0.8 mmol Cd kg-1, 1.6 mmol Mn kg-1 or the combination of 0.4 mmol Cd kg-1 + 0.8 mmol Mn kg-1 soil, together with the control treatment (without addition of Cd and Mn in soil), by means of analysis of changes in the profile of exclusive proteins (EP) and differentially accumulated proteins (DAP). Leaf and root proteins were extracted and quantified from the different treatments, followed by proteomic analysis. About eight DAP and 38 EP were identified in leaves, whereas in roots 43 DAP and 21 EP were identified. Some important proteins induced in the presence of Cd and repressed in the presence of Cd + Mn or vice versa, were ATPases, isoflavone reductase, proteasome and chaperonin. It was concluded that proteins involved in oxidoreduction and defense and stress response processes, in addition to other processes, were induced in the presence of Cd and repressed in the presence of Cd + Mn. This demonstrated that Mn was able to mitigate the toxic effects of Cd on young plants of the CCN 51 cocoa genotype.

Mechanical stress caused by wind on leaves of Theobroma cacao: Photosynthetic, molecular, antioxidative and ultrastructural responses.

Theobroma cacao is cultivated in the shade, in a so-called 'Cabruca' system, in intercropped with Erithryna or other tree species of economic value, and in full sun as a monoculture in irrigated or chemically-irrigated systems. Since it is a species quite intolerant to wind, it is practically impossible to implant cacao crops under full exposure to the sun, or in areas of frequent winds, without the protection of windbreaks, using arboreal species around the area of culture in the form of box. Wind can cause mechanical stimuli in plants, affecting their growth and development. The objective of this work was to evaluate the photosynthetic changes in mature leaves and the molecular, biochemical and ultrastructural changes in young and mature leaves of the CCN 51 cloned genotype of T. cacao subjected to intermittent (IW) and constant (CW) wind, with velocities of 2.5, 3.5 and 4.5 m s-1, during 3, 6 and 12 h of exposure. It was verified that CW and IW, considering different exposure times, interfered directly in stomatal conductance (gs), transpiration (E) and water use efficiency (WUE), causing a reduction of the photosynthetic rate (A) in mature leaves. In addition, the pulvinus and blade of young and mature leaves, exposed to IW and CW with different exposure times (3 and 12 h), showed marked macroscopic and microscopic mechanical injuries resulting from the constant leaf movement. At both speeds, there was rupture of the cell nuclear membrane in pulvinus and the mesophyll tissues, mainly in the young leaves. On the other hand, in young and mature leaves exposed to CW and IW at different speeds and exposure times, there was lipid peroxidation, increased activity of guaiacol (GPX) and ascorbate (APX) peroxidases in most treatments; and altered expression of transcripts of psba and psbo genes related to the phothosynthetic apparatus and Cu-Zn-sod and per genes related to antioxidative enzymes at the rate of 4.5 m s-1. Younger leaves were more intolerant to mechanical stress caused by the wind, since presented greater macro and microscopic damages and, consequently, greater molecular, biochemical and ultrastructural changes. High wind speeds can seriously compromise the development of young leaves of T. cacao plants and affect their productivity.

Physiological, ultrastructural, biochemical and molecular responses of young cocoa plants to the toxicity of Cr (III) in soil.

The objective of this study was to evaluate Cr toxicity in young plants of the CCN 51 Theobroma cacao genotype at different concentrations of Cr3+ in the soil (0, 100, 200, 400 and 600 mg kg-1) through physiological, ultrastructural, antioxidant and molecular changes. Doses of 400 and 600 mg Cr3+ kg-1 soil severely affected foliar gas exchange, promoted by damages in photosynthetic machinery evidenced by the decrease in CO2 fixation. Decreased expression of psbA and psbO genes, changes in enzymatic activity and lipid peroxidation also affected leaf gas exchange. A hormesis effect was observed at 100 mg Cr3+ kg-1 soil for the photosynthetic activity. As a metal exclusion response, the roots of the cocoa plants immobilized, on average, 75% of the total Cr absorbed. Ultrastructural changes in leaf mesophyll and roots, with destruction of mitochondria, plasmolysis and formation of vesicles, were related to the oxidative stress promoted by excess ROS. The activity of the antioxidant enzymes SOD, APX, GPX and CAT and the amino acid proline coincided with the greater expression of the sod cyt gene demonstrating synchronicity in the elimination of ROS. It was concluded, therefore, that the tolerance of the cocoa plants to the toxicity of Cr3+ depends on the concentration and time of exposure to the metal. Higher doses of Cr3+ in the soil promoted irreversible damage to the photosynthetic machinery and the cellular ultrastructure, interfering in the enzymatic and non-enzymatic systems related to oxidative stress and gene expression. However, the low mobility of the metal to the leaf is presented as a strategy of tolerance to Cr3+.

Path analysis of phenotypic traits in young cacao plants under drought conditions.

Drought is worldwide considered one of the most limiting factors of Theobroma cacao production, which can be intensified by global climate changes. In this study, we aimed to investigate the phenotypic correlation among morphological characteristics of cacao progenies submitted to irrigation and drought conditions and their partitions into direct and indirect effects. Path analysis with phenotypic plasticity index was used as criteria for estimation of basic and explanatory variables. The experiment was conducted in a greenhouse at the Cacao Research Center (CEPEC), Ilhéus, Bahia, Brazil, in a randomized block 21 x 2 factorial arrangement [21 cacao progenies obtained from complete diallel crosses and two water regimes (control and drought)] and six replications. In general, drought conditions influenced biomass production in most progenies, causing significant reductions in total leaf area, leaf number, leaf biomass, fine-roots length (diameter <1 mm), root volume and root area for considered drought intolerant. All progenies showed alterations in growth due to drought. Phenotypic plasticity was most strongly pronounced in root volume. Stem and root diameters, as well as stem dry biomass were the growth variables with the greatest direct effects on root volume under drought conditions, these characters being indicated in screening of cacao progenies drought tolerant.

Photosynthetic, antioxidative, molecular and ultrastructural responses of young cacao plants to Cd toxicity in the soil.

Cadmium (Cd) is a highly toxic metal for plants, even at low concentrations in the soil. The annual production of world cocoa beans is approximately 4 million tons. Most of these fermented and dried beans are used in the manufacture of chocolate. Recent work has shown that the concentration of Cd in these beans has exceeded the critical level (0.6mgkg-1 DM). The objective of this study was to evaluate the toxicity of Cd in young plants of CCN 51 cacao genotype grown in soil with different concentrations of Cd (0, 0.05 and 0.1gkg-1 soil) through photosynthetic, antioxidative, molecular and ultrastructural changes. The increase of Cd concentration in the soil altered mineral nutrient absorption by competition or synergism, changed photosynthetic activity caused by reduction in chloroplastidic pigment content and damage to the photosynthetic machinery evidenced by the Fv/Fm ratio and expression of the psbA gene and increased GPX activity in the root and SOD in leaves. Additionally, ultrastructural alterations in roots and leaves were also evidenced with the increase of the concentration of Cd in the soil, whose toxicity caused rupture of biomembranes in root and leaf cells, reduction of the number of starch grains in foliar cells, increase of plastoglobules in chloroplasts and presence of multivesiculated bodies in root cells. It was concluded, therefore, that soil Cd toxicity caused damage to the photosynthetic machinery, antioxidative metabolism, gene expression and irreversible damage to root cells ultrastructure of CCN 51 cocoa plants, whose damage intensity depended on the exposure time to the metal.

Combining ability, heritability and genotypic relations of different physiological traits in cacao hybrids.

Selecting parents and evaluating progenies is a very important step in breeding programs and involves approaches such as understanding the initial stages of growth and characterizing the variability among genotypes for different parameters, such as physiological, growth, biomass partitioning and nutrient translocation to the aerial part. In these cases, facilitating tools can be used to understand the involved gene dynamics, such as diallel crosses and genetic and phenotypic correlations. Our main hypothesis is that the contrasting phenotypes of these parental genotypes of cocoa used are due to genetic factors, and progenies derived from crosses of these parental genotypes are useful for breeding programs related to plant architecture, physiological parameters and translocation of mineral nutrients. We aimed to evaluate the combining abilities in progenies of cacao (Theobroma cacao L) originating from contrasting parents for canopy vigor. Emphasis was given to the evaluation of morphological and physiological parameters and the phenotypic and genotypic correlations to understand the dynamics of the action of the genes involved, as well as in expression profile from genes of gibberellins biosynthesis pathway in the parents. Fifteen F1 progenies were obtained from crosses of six clones (IMC 67, P4B, PUCALA, SCA 6, SCA 24 and SJ 02) that were evaluated in a randomized complete block design with four replicates of 12 plants per progeny, in a balanced half table diallel scheme. It is possible to identify and select plants and progenies of low, medium and high height, as there is expressive genetic variability for the evaluated parameters, some of these on higher additive effects, others on larger nonadditive effects and others under a balance of these effects. Most physiological parameters evaluated show that for selection of plants with the desired performance, no complex breeding methods would be necessary due to the high and medium heritability observed. Strong genetic components were observed from many of the correlations, which indicate the possibility to formulate selection indices for multi-traits, such as dwarfism or semidwarfism, tolerance to increase of leaf sodium concentrations and maintenance of the photosynthetic apparatus integrity under these conditions. Additionally, plants with higher carbon fixation, better water use, higher carboxylation efficiency and greater magnesium accumulation in leaves can be selected.

Diallel Analysis and Growth Parameters as Selection Tools for Drought Tolerance in Young Theobroma cacao Plants.

This study aimed to estimate the combining ability, of T. cacao genotypes preselected for drought tolerance through diallel crosses. The experiment was conducted under greenhouse conditions at the Cacao Research Center (CEPEC), Ilhéus, Bahia, Brazil, in a completely randomized block design, in an experimental arrangement 21 x 2 [21 complete diallel crosses and two water regimes (control and stressed)]. In the control, soil moisture was kept close to field capacity, with predawn leaf water potential (ΨWL) ranging from -0.1 to -0.5 MPa. In the drought regime, the soil moisture was reduced gradually by decreasing the amount of water application until ΨWL reached -2.0 to -2.5 MPa. Significant differences (p < 0.05) were observed for most morphological attributes analyzed regarding progenies, water regime and their interactions. The results of the joint diallel analysis revealed significant effects between general combining ability (GCA) x water regimes and between specific combining ability (SCA) x water regimes. The SCA 6 genetic material showed high general combining ability for growth variables regardless of the water regime. In general, the water deficit influenced the production of biomass in most of the evaluated T. cacao crosses, except for SCA-6 x IMC-67, Catongo x SCA, MOC-01 x Catongo, Catongo x IMC-67 and RB-40 x Catongo. Multivariate analysis showed that stem diameter (CD), total leaf area (TLA), leaf dry biomass (LDB), stem dry biomass (SDB), root dry biomass (RDB), total dry biomass (TDB), root length (RL), root volume (RV), root diameter (RD) <1 mm and 1 <(RD) <2 mm were the most important growth parameters in the separation of T. cacao genotypes in to tolerant and intolerant to soil water deficit.

Genetic Structure and Molecular Diversity of Cacao Plants Established as Local Varieties for More than Two Centuries: The Genetic History of Cacao Plantations in Bahia, Brazil.

Bahia is the most important cacao-producing state in Brazil, which is currently the sixth-largest country worldwide to produce cacao seeds. In the eighteenth century, the Comum, Pará and Maranhão varieties of cacao were introduced into southern Bahia, and their descendants, which are called 'Bahian cacao' or local Bahian varieties, have been cultivated for over 200 years. Comum plants have been used to start plantations in African countries and extended as far as countries in South Asia and Oceania. In Brazil, two sets of clones selected from Bahian varieties and their mutants, the Agronomic Institute of East (SIAL) and Bahian Cacao Institute (SIC) series, represent the diversity of Bahian cacao in germplasm banks. Because the genetic diversity of Bahian varieties, which is essential for breeding programs, remains unknown, the objective of this work was to assess the genetic structure and diversity of local Bahian varieties collected from farms and germplasm banks. To this end, 30 simple sequence repeat (SSR) markers were used to genotype 279 cacao plants from germplasm and local farms. The results facilitated the identification of 219 cacao plants of Bahian origin, and 51 of these were SIAL or SIC clones. Bahian cacao showed low genetic diversity. It could be verified that SIC and SIAL clones do not represent the true diversity of Bahian cacao, with the greatest amount of diversity found in cacao trees on the farms. Thus, a core collection to aid in prioritizing the plants to be sampled for Bahian cacao diversity is suggested. These results provide information that can be used to conserve Bahian cacao plants and applied in breeding programs to obtain more productive Bahian cacao with superior quality and tolerance to major diseases in tropical cacao plantations worldwide.

Altered physiology, cell structure, and gene expression of Theobroma cacao seedlings subjected to Cu toxicity.

Seedlings of Theobroma cacao CCN 51 genotype were grown under greenhouse conditions and exposed to increasing concentrations of Cu (0.005, 1, 2, 4, 8, 16, and 32 mg Cu L(-1)) in nutrient solution. When doses were equal or higher than 8 mg Cu L(-1), after 24 h of treatment application, leaf gas exchange was highly affected and changes in chloroplasts thylakoids of leaf mesophyll cells and plasmolysis of cells from the root cortical region were observed. In addition, cell membranes of roots and leaves were damaged. In leaves, 96 h after treatments started, increases in the percentage of electrolyte leakage through membranes were observed with increases of Cu in the nutrient solution. Moreover, there was an increase in the concentration of thiobarbituric acid-reactive substances in roots due to lipid peroxidation of membranes. Chemical analysis showed that increases in Cu concentrations in vegetative organs of T. cacao increased with the increase of the metal in the nutrient solution, but there was a greater accumulation of Cu in roots than in shoots. The excess of Cu interfered in the levels of Mn, Zn, Fe, Mg, K, and Ca in different organs of T. cacao. Analysis of gene expression via RTq-PCR showed increased levels of MT2b, SODCyt, and PER-1 expression in roots and of MT2b, PSBA, PSBO, SODCyt, and SODChI in leaves. Hence, it was concluded that Cu in nutrient solution at doses equal or above 8 mg L(-1) significantly affected leaf gas exchange, cell ultrastructure, and transport of mineral nutrients in seedlings of this T. cacao genotype.