Exploiting DNA methylation in cassava under water deficit for crop improvement.

DNA methylation plays a key role in the development and plant responses to biotic and abiotic stresses. This work aimed to evaluate the DNA methylation in contrasting cassava genotypes for water deficit tolerance. The varieties BRS Formosa (bitter) and BRS Dourada (sweet) were grown under greenhouse conditions for 50 days, and afterwards, irrigation was suspended. The stressed (water deficit) and non-stressed plants (negative control) consisted the treatments with five plants per variety. The DNA samples of each variety and treatment provided 12 MethylRAD-Seq libraries (two cassava varieties, two treatments, and three replicates). The sequenced data revealed methylated sites covering 18 to 21% of the Manihot esculenta Crantz genome, depending on the variety and the treatment. The CCGG methylated sites mapped mostly in intergenic regions, exons, and introns, while the CCNGG sites mapped mostly intergenic, upstream, introns, and exons regions. In both cases, methylated sites in UTRs were less detected. The differentially methylated sites analysis indicated distinct methylation profiles since only 12% of the sites (CCGG and CCNGG) were methylated in both varieties. Enriched gene ontology terms highlighted the immediate response of the bitter variety to stress, while the sweet variety appears to suffer more potential stress-damages. The predicted protein-protein interaction networks reinforced such profiles. Additionally, the genomes of the BRS varieties uncovered SNPs/INDELs events covering genes stood out by the interactomes. Our data can be useful in deciphering the roles of DNA methylation in cassava drought-tolerance responses and adaptation to abiotic stresses.

Propagation of yellow passion fruit seedlings by cutting, grafting and seeds under salt stress

Salinity is one of the most limiting abiotic stresses in the global agricultural sector. The objective of this study was to evaluate the effect of irrigation water salinity on the initial development of P. edulis Sims. seedlings propagated by seeds, grafting and cuttings. Treatments were distributed in a completely randomized design, in a 5 x 3 factorial arrangement, corresponding to 5 levels of irrigation water electrical conductivity (0.3 - control, 1.8, 3.3, 4.8, and 6.3 dS m-1) and 3 propagation methods (seeds, cuttings and grafting), with four replicates and one plant per plot. Fifty days after the imposition of treatments with saline water, plant height, number of leaves, stem diameter, total chlorophyll, stomatal conductance, relative leaf water content, total water consumption, dry matter of root, shoot and total, root length and root volume were evaluated. The increase in water salinity affected with greater intensity the growth and development variables (height, total dry matter and root volume of the plant) and the physiological characteristics (stomatal conductance) of the species Passiflora edulis, regardless of the method of propagation. Seedlings propagated by grafting showed better development compared to the other propagation methods (seeds and cuttings). The interaction between the propagation methods and water salinity affected seedlings propagated by seeds and cuttings with greater intensity.

Epigenetic responses to Phytophthora citrophthora gummosis in citrus.

Studies show that DNA methylation is associated with plant immunity but little is known as to how this epigenetic mechanism assists plants in adjusting their responses to biotic stress, especially when interacting with an hemibiotrophic pathogen such as citrus Phytophthora. The aim of the present study was to assess the effects of scion-rootstock interaction on plant resistance to P. citrophthora infection and DNA methylation patterns in 'Pera' sweet orange and 'Tahiti' acid lime grafted onto 'Rangpur' lime and 'Tropical' sunki rootstocks reinoculated with P. citrophthora. Results showed that reinoculated plants of the 'Pera' sweet orange/'Rangpur' lime and 'Tahiti' acid lime/'Tropical' sunki combinations with more and less sensitive varieties to Phytophthora, presented smaller stem lesions and increased frequency of full methylation and hemimethylation rates, compared to inoculated plants. In contrast, 'Tahiti' acid lime/'Rangpur' lime, two highly sensitive varieties, and 'Pera'/'Tropical' sunki, two much less sensitive varieties, showed high increases in the frequency of hemimethylation and non-methylation levels. Results suggest that in citrus, both the scion-rootstock interaction and DNA methylation affect the response to P. citrophthora infection. Reinoculated plants, depending on the combination, showed changes in intracellular hyphae growth through the formation of sets of fibers and crystal accumulation in the periderm, cortex, and phloem. In addition, starch grain concentration was higher in reinoculated plants in comparison to inoculated plants. These findings support the assumption that DNA methylation is a plant defense mechanism and therefore may be exploited to improve the response of plants to the gummosis of P. citrophthora in citrus.

Behavioral Responses of Asian Citrus Psyllid (Hemiptera: Liviidae) to Salinity-Stressed Citrus.

Most commercial citrus varieties are intolerant of salinity stress, but some rootstocks, such as Rangpur lime, tolerate moderately saline irrigation water. Development of salinity-tolerant citrus may allow for citriculture in semiarid and arid regions where salinity stress is problematic. Because salinity stress influences shoot growth in citrus, we compared the behavioral responses of Asian citrus psyllid, Diaphorina citri Kuwayama, to salinity-stressed versus nonstressed Rangpur lime seedlings. The effects of salinity stress on key physiological processes in the seedlings were also examined. Seedlings in the control group were fertilized with a solution having a salinity of 1.7 dS m-1 while seedlings in the salinity-stressed group were fertilized with a solution having a salinity of 10 dS m-1. The seedlings were exposed to salinity stress for increasing durations (15, 20, or 60 d). Seedlings presented differential physiological responses 15 d after the imposition of salinity stress, and differences in psyllid settling rate on control versus salinity-stressed seedlings were discernable within 1 h following the imposition of salinity stress. The levels of settling, oviposition, and egg survivorship were significantly lower on salinity-stressed versus control seedlings. Olfactometer tests showed that female psyllids preferred the odor from control seedlings, suggesting that the odors of control and salinity-stressed seedlings were different. The results showed that D. citri avoids salinity-stressed seedlings; this suggests the possibility of using moderate salinity stress as a management strategy to minimize psyllid settlement and reproduction and to reduce the spread of huanglongbing, especially in citrus grown in semiarid and arid areas.

Salt stress on physiology, biometry and fruit quality of grafted Passiflora edulis / Estresse salino na fisiologia, biometria e qualidade dos frutos de Passiflora edulis enxertada

The production of grafted passion fruit is an alternative for plant adaptation to saline environments. The objective of this study was to evaluate the effect of salt stress on physiology, biometry and fruit quality of P. edulis grafted on Passiflora spp. The experiment was conducted in completely randomized design, in a 3 x 2 factorial scheme, corresponding to three species of Passiflora (P. edulis, P. gibertii and P. cincinnata) with P. edulis scion and two levels of irrigation water salinity (0.5 - control and 4.5 dS m-1), with four repetitions. Water salinity compromises gas exchanges (CO2 assimilation raste and transpiration) and physiological variables (total chlorophyll and total water consumption) in grafted P. edulis. The interaction between the factors (water salinity x species) compromised only the growth in plant height and number of leaves. In relation to the species, auto-grafted P. edulis stood out from the other species, with higher internal CO2 concentration, number of leaves, stem dry mass, peel thickness, total soluble solids (TSS) of the pulp and TSS/TA ratio (titratable acidity). Auto-grafted P. edulis under saline conditions develops vital mechanisms (TSS and TSS/TA), which attenuates the effects of salt stress on the physico-chemical quality of the fruits.

A produção de maracujazeiro enxertado é uma alternativa para adaptação das plantas a ambientes salinos. Objetivou-se avaliar o efeito do estresse salino na fisiologia, biometria e qualidade de frutos de P. edulis enxertado em espécies de Passiflora spp. O delineamento utilizado foi inteiramente casualizado, em esquema fatorial 3 x 2, sendo três espécies de Passiflora (P. edulis, P. gibertii e P. cincinnata) tendo como copa P. edulis e dois níveis de salinidade de água de irrigação (0,5 ­ testemunha e 4,5 dS m-1), com quatro repetições. A salinidade da água compromete as trocas gasosas (taxa de assimilação de CO2 e transpiração) e variáveis fisiológicas (clorofila total e consumo hídrico total) em P. edulis enxertado. A interação entre os fatores (salinidade da água x espécie) compromete apenas o crescimento em altura de plantas e número de folhas. Em relação às espécies, o P. edulis auto enxertado se destaca em relação as demais espécies apresentando maior concentração interna de CO2, número de folhas, massa seca de caule, espessura da casca do fruto, sólidos solúveis totais (SST) da polpa e razão sólidos solúveis totais por acidez titulável (SST/AT). O P. edulis auto enxertado sob condições de salinidade, desenvolve mecanismos vitais (SST e SST/AT), que atenuam os efeitos do estresse salino na qualidade físico-química dos frutos.

Recurrent water deficit causes alterations in the profile of redox proteins in citrus plants.

Plant acclimation to recurrent stress involves profound alterations in multiple genetic, metabolic and physiological processes. Stressful conditions usually implicate imbalance in reactive oxygen species (ROS) production and removal rates, which may lead to oxidative stress. However, the primary cellular targets of oxidative stress and their relevance in plant acclimation to abiotic stresses remains poorly characterized. By comparing redox proteomic and sugar profiles in citrus Valencia (VO) scions grafted onto two rootstocks with different soil water extraction capacities - Rangpur Lime (RL) and Sunki Maravilha (SM) - here we demonstrate that both ROS-mediated post-translational protein modification and changes in sugar composition are associated with acclimation to recurrent drought in citrus. The redox proteomic analysis of the distinct scion/rootstock combinations exposed to one (WD1), two (WD2) or three (WD3) water deficit episodes revealed a total of 32 and 55 redox protein spots present in VO/RL and VO/SM plants, respectively. Mass spectrometry analysis of these protein spots revealed essential targets of ROS-mediated posttranslational protein modification in citrus plants challenged by recurrent drought. The oxidation of cysteine thiol groups into glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was shown to increase in WD3 samples of the VO/RL combination, whereas the opposite was observed for the VO/SM combination. Similarly, recurrent drought promoted the oxidation of catalase thiol groups in VO/SM, but not in VO/RL. Carbohydrate profiling revealed that glucose, fructose and galactose may also contribute to the phenotypic differences observed between the citrus genotypes exposed to drought. These findings reveal for the first time that recurrent drought differentially affects the profile of redox proteomics of citrus, suggesting that this alteration may be part of the stress memory in perennial plants.

Overcoming dormancy in stored and recently harvested Passiflora cincinnata Mast. seeds / Superação de dormência em sementes de Passiflora cincinnata Mast. armazenadas e recém-colhidas

The treatment of seeds using growth regulators can overcome seed dormancy and increase the emergence and development of seedlings. The objective of this research was to evaluate the effect of different concentrations of GA4+7 + N-(phenyl methyl)-aminopurine on the dormancy breaking of Passiflora cincinnata Mast. seeds recently harvested and stored for six years. The study was conducted in a greenhouse at Embrapa Cassava and Tropical Fruits, in completely randomized design, in a 2 x 4 factorial scheme, with the following factors: year of seed harvest (2010 and 2016) and the emergence promoter GA4+7 + N-(phenyl methyl)-aminopurine at four concentrations (0%; 0.03%; 0.45% and 0.90%), with 4 replicates of 25 seeds. Emergence percentage, first count, emergence speed index, number of leaves, root length, stem diameter, dry matter of shoots, roots and total, and root/shoot ratio were evaluated. The results showed that GA4+7 + N-(phenyl methyl)-aminopurine is recommended to overcome dormancy of Passiflora cincinnata in recently harvested seeds (2016) and increase the emergence of seedlings from seeds stored for six years in cold chamber (2010). The emergence promoter concentration of 0.03% can be used to overcome dormancy in recently harvested seeds and maintains the vigor of seeds stored for six years. Passiflora cincinnata seeds remain physiologically viable for six years and their emergence is potentiated by the use of GA4+7 + N-(phenyl methyl)-aminopurine.

O tratamento de sementes com reguladores de crescimento pode superar a dormência das sementes, aumentar o vigor e o desenvolvimento das mudas. O objetivo desta pesquisa foi avaliar o efeito de diferentes concentrações de GA4 + 7 + N- (fenilmetil) -aminopurina na quebra de dormência de sementes de Passiflora cincinnata recentemente colhidas e armazenadas por seis anos. O estudo foi conduzido em uma estufa na Embrapa Mandioca e Fruticultura, em delineamento inteiramente casualizado, em esquema fatorial 2 x 4, com os seguintes fatores: ano de colheita de sementes (2010 e 2016) e promotor de emergência GA4 + 7 + N - (fenilmetil) - aminopurina em quatro concentrações (0%; 0,03%; 0,45% e 0,90%), com 4 repetições de 25 sementes. Foram avaliados a porcentagem de emergência, a primeira contagem, o índice de velocidade de emergência, o número de folhas, o comprimento da raiz, o diâmetro do caule, a matéria seca da parte aérea, das raízes, total e a relação raiz/parte aérea. O GA4 + 7 + N - (fenilmetil) - aminopurina é recomendado para superar a dormência de Passiflora cincinnata em sementes recentemente colhidas (2016) e aumenta a emergência de sementes armazenadas por seis anos em câmara fria. A concentração do promotor de emergência de 0,03% pode ser usada para superar a dormência em sementes recentemente colhidas e potencializa o vigor das sementes armazenadas durante seis anos. As sementes de Passiflora cincinnata permanecem fisiologicamente viáveis por seis anos e seu vigor é potencializado pelo uso de GA4 + 7 + N - (fenilmetil) - aminopurina.

Salinity reduces nutrients absorption and efficiency of their utilization in cassava plants / Salinidade reduz a absorção e a eficiência de utilização de nutrientes em plantas de mandioca

ABSTRACT: The objective of this research was to evaluate the influence of salinity on the absorption and utilization of nutrients by cassava. For the study, cassava was submitted to four saline concentrations: 0, 20, 40, and 60mM NaCl. Results showed that the absorption of all nutrients, except nitrogen (N), was reduced by salinity, with highest reduction for potassium (K). However, all nutrients were maintained at concentrations which did not indicate mineral deficiency problem. The abnormal concentration of calcium in the tuberous roots may have been one of the factors that contributed to the lower growth of this organ and of the plant as a whole. Transports of nitrogen, potassium, phosphorus and sulfur from root to the aerial part was higher under salinity treatment. Efficiency in the use of all the nutrients, mainly N, was reduced due to salinity. Given that: (i) the absorption of K was the most impaired, (ii) there was abnormal accumulation of Ca in tuberous roots, and (iii) the efficiency in the use of N was the most affected, it is suggested to prioritize studies on these three issues, as a way to better understand the aspects related to the tolerance/sensitivity of cassava plants to salinity.

RESUMO: O objetivo deste trabalho foi o de avaliar a influência da salinidade sobre a absorção e utilização de nutrientes pela mandioca. Para o estudo, as plantas foram submetidas a quatro concentrações salinas: 0, 20, 40 e 60mM de NaCl. Os resultados mostraram que a absorção de todos os nutrientes, exceto o nitrogênio (N), foi reduzida pela salinidade, com maior redução para o potássio (K). No entanto, todos os nutrientes foram mantidos em concentrações que não indicaram problema de deficiência mineral. A concentração anormal de cálcio nas raízes tuberosas pode ter sido um dos fatores que contribuíram para o menor crescimento desse órgão e da planta como um todo. Os transportes de nitrogênio, potássio, fósforo e enxofre da raiz para a parte aérea foram maiores sob tratamento com salinidade. A eficiência no uso de todos os nutrientes, principalmente N, foi reduzida devido à salinidade. Considerando que: (i) a absorção de K foi a mais prejudicada, (ii) houve acúmulo anormal de Ca nas raízes tuberosas e (iii) a eficiência no uso de N foi a mais afetada, sugere-se priorizar estudos sobre estas três questões, como forma de melhor entender os aspectos relacionados à tolerância/sensibilidade das plantas de mandioca à salinidade.

Recurrent water deficit causes epigenetic and hormonal changes in citrus plants.

The present study evaluated the physiological, molecular and hormonal parameters from scion/rootstock interaction of citrus plants during recurrent water deficit. Responses of the Valencia (VO) scion variety grafted on two rootstocks with different soil water extraction capacities, Rangpur Lime (RL) and Sunki Maravilha (SM), during three successive periods of water deficit: plants exposed to a single episode of water deficit (WD1) and plants exposed to two (WD2) and three (WD3) recurrent periods of WD were compared. The combinations VO/RL and VO/SM presented polymorphic alterations of epigenetic marks and hormonal (i.e. abscisic acid, auxins and salicylicacid) profiles, which were particularly prominent when VO/SM plantswere exposed toWD3 treatment. Upon successive drought events, the VO/SM combination presented acclimatization characteristics that enable higher tolerance to water deficit by increasing transpiration (E), stomatal conductance (g s ) and photosynthetic rate (A), which in turn may have facilitated the whole plant survival. Besides providing comprehensive data on the scion/rootstock interactions upon successive stress events, this study brings the first dataset suggesting that epigenetic alterations in citrus plants triggered by recurrent water deficit lead to improved drought tolerance in this crop species.

Differential accumulation of flavonoids and phytohormones resulting from the canopy/rootstock interaction of citrus plants subjected to dehydration/rehydration.

Water scarcity can elicit drastic changes in plant metabolic and hormonal regulation, which may be of fundamental importance to stress tolerance. The study of plant the metabolic alterations in response to water deficit, especially the effects of the rootstocks level, is important to elucidate the mechanisms associated to drought tolerance. To verify the influence of rootstock and grafting on the tolerance to drought in citrus plants, we analyzed the growth, phytohormone levels and flavonoid profiles in grafted and ungrafted citrus plants subjected to different soil water regimes on plant status (well-watered, moderate drought and severe drought and rehydrated) under field conditions. The experiments were conducted under field conditions in the Brazilian Agricultural Research Corporation (EMBRAPA), Cruz das Almas, BA, Brazil. Water deficit reduced the total leaf area per plant in all canopy/rootstock combinations. Self-grafting reduce root volume, area and length when compared to ungrafted plants. Drought-induced increases in salicylic acid and abscisic acid associated with concomitant reductions in indoleacetic acid were observed in most canopy/rootstock combinations. However, plants with 'Sunki Maravilha' rootstocks exhibited the most pronounced changes in hormonal levels upon drought stress. Associated to these hormonal changes, drought also significantly affected flavonoid content and profile in both leaves and roots of the distinct citrus combinations. Glycosylated (GFs) and polimethoxylated flavonoids were predominantly found in leaves, whereas prenylated coumarins were found in the roots. Leaf levels of GFs (vicenin, F11, rutin and rhoifolin) were particularly modulated by drought in plants with 'Rangpur Santa Cruz' lime rootstock, whereas root levels of prenylated coumarins were most regulated by drought in plants with the 'Sunki Maravilha' root system. Taken together, these data indicate that the impacts of water deficit restriction on growth, hormonal balance and flavonoid profiles significantly varies depending on the canopy/rootstock combinations.

Formation of seedlings of species from the genus passiflora under saline stress / Formação de mudas de espécies do gênero passiflora sob estresse salino

The genus Passiflora of the passion fruit crop is the most important from economic point of view. However, its cultivation in the semi-arid region is at risk due to salinity problems, requiring the identification of tolerant species, which develop better in environments with salinity problems. The objective of this study was to evaluate the effects of water salinity on growth and formation of seedlings of three species from the genus Passiflora. The treatments were distributed in a completely randomized design, adopting a 5 x 3 factorial arrangement, with five levels of irrigation water salinity (ECw) of 0.3; 1.4; 2.5; 3.6 and 4.7 dS m-1 and three species of Passiflora: gibertii; cincinnata and edulis 'BRS Gigante Amarelo', with four replicates. P. edulis surpassed the others in leaf area, shoot, root and total dry matter and Dickson index of seedling quality. Irrigation water salinity inhibits the formation of seedlings evaluated by growth in height, stem diameter and shoot, root and total dry matter of the studied species of Passiflora. The interaction between ECw and the Passiflora species interfered significantly in height, root, shoot and total dry matter and Dickson quality index. ECw above 0.3 dS m-1 affects the Dickson quality index of P. gibertii more,compared with P. edulis and P. cincinnata.

O gênero Passiflora da cultura do maracujazeiro é o importante de ponto de vista econômico. No entanto, seu cultivo na região semiárida apresenta risco, devido aos problemas de salinidade, sendo necessária a identificação de espécies tolerantes, que se desenvolvam em ambientes com problemas de salinidade. Com isso, objetivouse avaliar os efeitos da salinidade das águas no crescimento e formação de mudas de três espécies do gênero Passiflora. Os tratamentos foram distribuídos em delineamento inteiramente casualizado, usando arranjo fatorial 5 x 3, com cinco níveis de salinidade da água de irrigação (CEa) de 0,3; 1,4; 2,5; 3,6 e 4,7 dS m-1 e três espécies de Passiflora: gibertii; cincinnata e edulis BRS Gigante Amarelo, com quatro repetições. Dentre as espécies, P. edulis superou as demais em área foliar, matéria seca da parte aérea, de raiz e total e índice de qualidade de mudas de Dickson. A salinidade da água de irrigação inibe a formação de mudas avaliadas pelo crescimento em altura, diâmetro do caule, massa seca das partes aérea, raiz e total, das espécies estudadas. A interação entre CEa e as espécies de Passiflora interfere significativamente na altura, massa seca das aízes, parte aérea, total e índice de qualidade de Dickson das mudas. A irrigação com CEa acima de 0,3 dS m-1 compromete mais o índice de qualidade de Dickson da espécie P. gibertii que das espécies P. edulis e P. cincinnata.

Different adaptation strategies of two citrus scion/rootstock combinations in response to drought stress.

Scion/rootstock interaction is important for plant development and for breeding programs. In this context, polyploid rootstocks presented several advantages, mainly in relation to biotic and abiotic stresses. Here we analyzed the response to drought of two different scion/rootstock combinations presenting different polyploidy: the diploid (2x) and autotetraploid (4x) Rangpur lime (Citrus limonia, Osbeck) rootstocks grafted with 2x Valencia Delta sweet orange (Citrus sinensis) scions, named V/2xRL and V/4xRL, respectively. Based on previous gene expression data, we developed an interactomic approach to identify proteins involved in V/2xRL and V/4xRL response to drought. A main interactomic network containing 3,830 nodes and 97,652 edges was built from V/2xRL and V/4xRL data. Exclusive proteins of the V/2xRL and V/4xRL networks (2,056 and 1,001, respectively), as well as common to both networks (773) were identified. Functional clusters were obtained and two models of drought stress response for the V/2xRL and V/4xRL genotypes were designed. Even if the V/2xRL plant implement some tolerance mechanisms, the global plant response to drought was rapid and quickly exhaustive resulting in a general tendency to dehydration avoidance, which presented some advantage in short and strong drought stress conditions, but which, in long terms, does not allow the plant survival. At the contrary, the V/4xRL plants presented a response which strong impacts on development but that present some advantages in case of prolonged drought. Finally, some specific proteins, which presented high centrality on interactomic analysis were identified as good candidates for subsequent functional analysis of citrus genes related to drought response, as well as be good markers of one or another physiological mechanism implemented by the plants.

Survival strategies of citrus rootstocks subjected to drought.

Two citrus rootstocks, Rangpur lime (RL) and Sunki Maravilha mandarin (SM), were analyzed either ungrafted or grafted with their reciprocal graft combinations or with shoot scions of two commercial citrus varieties: Valencia orange (VO) and Tahiti acid lime (TAL). All graft combinations were subjected to distinct watering regimes: well-watered, severe drought and rehydration. Growth and water relation parameters, gas exchange as well as sugar and hormone profiles were determined. Data indicated that RL adopted a dehydration avoidance strategy and maintained growth, whereas SM adopted a dehydration tolerance strategy focused on plant survival. Compared with RL, the leaves and roots of SM exhibited higher concentrations of abscisic acid and salicylic acid, which induced drought tolerance, and accumulation of carbohydrates such as trehalose and raffinose, which are important reactive oxygen species scavengers. SM rootstocks were able to transfer their survival strategy to the grafted shoot scions (RL, VO, TAL). Because of their contrasting survival strategies, RL reached the permanent wilting point more quickly than SM whereas SM recovered from prolonged droughts more efficiently than RL. This is one of the most complete studies of drought tolerance mechanisms in citrus crops and is the first to use reciprocal grafting to clarify scion/rootstock interactions.