First Report of the Physiological Race (XXIV) of Hemileia vastatrix (Coffee Leaf Rust) in Hawaii.

Hawaii's coffee industry, produced commercially on six islands by over 1,470 growers on ~10,000 acres, is conservatively valued at $100M per year (USDA NASS 2023). Until late October 2020, Hawaii was the only major coffee producing region of the world that was free of Coffee Leaf Rust (CLR). Growers are currently facing their most formidable production challenge with the arrival of Hemileia vastatrix Berk. & Broome, the most economically devastating pathogen of coffee worldwide. Since its introduction (Keith et al. 2022), CLR has rapidly spread throughout the state and can be found on coffee farms and feral coffee throughout the six islands. Implementation of CLR control measures will be difficult in Hawaii, given the extreme environmental heterogeneity, differences in management practices, high production costs, and labor shortages. Compounding these challenges is that all coffee genotypes grown on a large scale in the state are susceptible to CLR. More than 55 different rust races from coffee growing countries worldwide have been identified (Silva et al. 2022). Since key control measures include developing and establishing resistant coffee cultivars, determining the rust race(s) present in Hawaii was imperative. In June 2021, nine spore samples from symptomatic cultivated and feral plants ('Typica') growing on three islands (Hawaii Island: 3, Maui: 5, Molokai: 1) were collected in gelatin capsules using a G-R Electric Manufacturing portable vacuum pump with a mini cyclone spore adapter. The samples were sent to the Coffee Rust Research Center (CIFC) in Portugal. At CIFC, the urediniospores were bulked on susceptible genotype 849/1 Matari and inoculated on a set of coffee differentials following a standard race-typing procedure (Várzea and Marques 2005). The genotype of virulence of rust samples was inferred according to Flor's gene-for-gene theory (Silva et al. 2022). The genes of virulence v2, v4, and v5 (Race XXIV) were identified in all rust samples from all islands in Hawaii, supporting the theory of a single introduction to the state, which subsequently spread (Ramírez-Camejo et al. 2022). Race XXIV was previously characterized at CIFC and is commonly found in the majority of coffee-growing countries in South and Central America, Africa and Asia (CIFC's data base). According to Figueiredo & Arruda (1974), race XXIV is considered highly aggressive with a high spore germination rate, medium germ tube length, and short incubation period required for infection. Race XXIV is pathogenic to all coffee Arabica genotypes with the resistance genes SH5 or SH2,5 like varieties Blue Mountain, Bourbon, Catuaí, Caturra, Kent's, Kona, K7, Mundo Novo, SL 28, SL 39, as well as Accession "Agaro" with resistance genes SH4,5 (CIFC's records). On the other hand, this race is not virulent to some other Arabica genotypes, such as Geisha (SH1,5), S.288 (SH3,5), and Dilla & Alghe (SH1). Race XXIV is unable to infect derivatives of interspecific tetraploid hybrids like the groups Catimor and Sarchimor (Bettencourt and Rodrigues 1988). This is the first report of race XXIV on Coffea arabica in Hawaii. This finding is essential to evaluate the potential resistance of coffee germplasm existing in Hawaii or to be introduced in this region to develop new varieties. Since the emergence of new H. vastatrix races occur preferentially at germplasm collections (Li et al. 2021), proper management is imperative where multiple genotypes/varieties are planted.

Genetic Diversity of Viral Populations Associated with Ananas Germplasm and Improvement of Virus Diagnostic Protocols.

Pineapple (Ananas comosus L. [Merr.]) accessions from the U.S. Tropical Plant Genetic Resources and Disease Research (TPGRDR) in Hilo, Hawaii were subjected to RNA-sequencing to study the occurrence of viral populations associated with this vegetatively propagated crop. Analysis of high-throughput sequencing data obtained from 24 germplasm accessions and public domain transcriptome shotgun assembly (TSA) data identified two novel sadwaviruses, putatively named "pineapple secovirus C" (PSV-C) and "pineapple secovirus D" (PSV-D). They shared low amino acid sequence identity (from 34.8 to 41.3%) compared with their homologs in the Pro-pol region of the previously reported PSV-A and PSV-B. The complete genome (7485 bp) corresponding to a previously reported partial sequence of the badnavirus, pineapple bacilliform ER virus (PBERV), was retrieved from one of the datasets. Overall, we discovered a total of 69 viral sequences representing ten members within the Ampelovirus, Sadwavirus, and Badnavirus genera. Genetic diversity and recombination events were found in members of the pineapple mealybug wilt-associated virus (PMWaV) complex as well as PSVs. PMWaV-1, -3, and -6 presented recombination events across the quintuple gene block, while no recombination events were found for PMWaV-2. High recombination frequency of the RNA1 and RNA2 molecules from PSV-A and PSV-B were congruent with the diversity found by phylogenetic analyses. Here, we also report the development and improvement of RT-PCR diagnostic protocols for the specific identification and detection of viruses infecting pineapple based on the diverse viral populations characterized in this study. Given the high occurrence of recombination events, diversity, and discovery of viruses found in Ananas germplasm, the reported and validated RT-PCR assays represent an important advance for surveillance of viral infections of pineapple.

Genome sequence of pineapple secovirus B, a second sadwavirus reported infecting Ananas comosus.

The complete genome sequence of pineapple secovirus B (PSV-B), a new virus infecting pineapple (Ananas comosus) on the island of Oahu, Hawaii, was determined by high-throughput sequencing (HTS). The genome comprises two RNAs that are 5,956 and 3,808 nt long, excluding the 3'-end poly-A tails, both coding for a single large polyprotein. The RNA1 polyprotein contains five conserved domains associated with replication, while the RNA2 polyprotein is cleaved into the movement protein and coat protein. PSV-B is representative of a new species in the subgenus Cholivirus (genus Sadwavirus; family Secoviridae), as the level of amino acid sequence identity to recognized members of this subgenus in the Pro-Pol and coat protein regions is below currently valid species demarcation thresholds.

Coffee Leaf Rust (Hemileia vastatrix) from the Recent Invasion into Hawaii Shares a Genotypic Relationship with Latin American Populations.

Hawaii has long been one of the last coffee-producing regions of the world free of coffee leaf rust (CLR) disease, which is caused by the biotrophic fungus Hemileia vastatrix. However, CLR was detected in coffee farms and feral coffee on the island of Maui in February 2020 and subsequently on other islands of the Hawaiian archipelago. The source of the outbreak in Hawaii is not known, and CLR could have entered Hawaii from more than 50 coffee-producing nations that harbor the pathogen. To determine the source(s) of the Hawaii inoculum, we analyzed a set of eleven simple sequence repeat markers (SSRs) generated from Hawaii isolates within a dataset of 434 CLR isolates collected from 17 countries spanning both old and new world populations, and then conducted a minimum spanning network (MSN) analysis to trace the most likely pathway that H. vastatrix could have taken to Hawaii. Forty-two multilocus genotypes (MLGs) of H. vastatrix were found in the global dataset, with all isolates from Hawaii assignable to MLG 10 or derived from it. MLG 10 is widespread in Central America and Jamaica, making this region the most probable source of inoculum for the outbreak in Hawaii. An examination of global weather patterns during the months preceding the introduction of CLR makes it unlikely that the pathogen was windborne to the islands. Likely scenarios for the introduction of CLR to Hawaii are the accidental introduction of spores or infected plant material by travelers or seasonal workers, or improperly fumigated coffee shipments originating from Central America or the Caribbean islands.

Signatures of selection in recently domesticated macadamia.

Macadamia is a high value nut crop that is recently domesticated, ideal for testing the effect of artificial selection. Here, we sequence the genome of Hawaiian cultivar 'Kau' and assemble into 794 Mb in 14 pseudo-chromosomes with 37,728 genes. Genome analysis reveals a whole-genome duplication event, occurred 46.8 million years ago. Gene expansions occurred in gene families involves in fatty acid biosynthesis. Gene duplication of MADS-Box transcription factors in proanthocyanidin biosynthesis are relevant for seed coat development. Genome re-sequencing of 112 accessions reveals the origin of Hawaiian cultivars from Mount Bauple in southeast Queensland in Australia. Selective sweeps are detected in macadamia cultivars, including genes involved in fatty acid biosynthesis, seed coat development, and heat stress response. Such strong effects of artificial selection in few generations reveals the genomic basis for 'one-step operation' for clonal crop domestication. The knowledge gained could accelerate domestication of new crops from wild species.

Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis.

The burrowing nematode, Radopholus similis, is an economically important plant-parasitic nematode that inflicts damage and yield loss to a wide range of crops. This migratory endoparasite is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops (e.g., citrus, banana). Despite the economic importance of this nematode, little is known about the repertoire of effectors owned by this species. Here we combined spatially and temporally resolved next-generation sequencing datasets of R. similis to select a list of candidates for the identification of effector genes for this species. We confirmed spatial expression of transcripts of 30 new candidate effectors within the esophageal glands of R. similis by in situ hybridization, revealing a large number of pioneer genes specific to this nematode. We identify a gland promoter motif specifically associated with the subventral glands (named Rs-SUG box), a putative hallmark of spatial and concerted regulation of these effectors. Nematode transcriptome analyses confirmed the expression of these effectors during the interaction with the host, with a large number of pioneer genes being especially abundant. Our data revealed that R. similis holds a diverse and emergent repertoire of effectors, which has been shaped by various evolutionary events, including neofunctionalization, horizontal gene transfer, and possibly by de novo gene birth. In addition, we also report the first GH62 gene so far discovered for any metazoan and putatively acquired by lateral gene transfer from a bacterial donor. Considering the economic damage caused by R. similis, this information provides valuable data to elucidate the mode of parasitism of this nematode.

Rambutan genome revealed gene networks for spine formation and aril development.

Rambutan is a popular tropical fruit known for its exotic appearance, has long flexible spines on shells, extraordinary aril growth, desirable nutrition, and a favorable taste. The genome of an elite rambutan cultivar Baoyan 7 was assembled into 328 Mb in 16 pseudo-chromosomes. Comparative genomics analysis between rambutan and lychee revealed that rambutan chromosomes 8 and 12 are collinear with lychee chromosome 1, which resulted in a chromosome fission event in rambutan (n = 16) or a fusion event in lychee (n = 15) after their divergence from a common ancestor 15.7 million years ago. Root development genes played a crucial role in spine development, such as endoplasmic reticulum pathway genes, jasmonic acid response genes, vascular bundle development genes, and K+ transport genes. Aril development was regulated by D-class genes (STK and SHP1), plant hormone and phenylpropanoid biosynthesis genes, and sugar metabolism genes. The lower rate of male sterility of hermaphroditic flowers appears to be regulated by MYB24. Population genomic analyses revealed genes in selective sweeps during domestication that are related to fruit morphology and environment stress response. These findings enhance our understanding of spine and aril development and provide genomic resources for rambutan improvement.

A novel ampelovirus associated with mealybug wilt of pineapple (Ananas comosus).

Mealybug wilt of pineapple (MWP) is the most important and complex viral disease affecting pineapple worldwide. High-throughput sequencing was conducted to characterize a new virus identified only in symptomatic pineapple plants and tentatively named pineapple mealybug wilt-associated virus 6 (PMWaV-6). Data analyses revealed a genome of 17,854 nucleotides with an organization resembling members of the genus Ampelovirus, family Closteroviridae. Encoded proteins shared sequence identity with the corresponding proteins of grapevine leafroll-associated virus 3, blackberry vein banding-associated virus, and PMWaV-2. The present study reports the discovery of PMWaV-6, a putative and distinct new member of the genus Ampelovirus, subgroup I, its potential involvement in MWP, and the development of PMWaV-6-specific RT-PCR assays to detect and monitor this virus in field samples.

Genetic diversity among naturally-occurring strains of Beauveria bassiana associated with the introduced coffee berry borer, Hypothenemus hampei, (Coleoptera: Curculionidae) on Hawai'i Island.

The coffee berry borer (CBB), Hypothenemus hampei, is considered the most important insect pest of coffee worldwide. CBB was discovered on Hawai'i Island in 2010 and soon thereafter on the islands of O'ahu (2014) and Maui (2016). As part of an areawide effort to manage CBB in Hawai'i, we conducted a survey of naturally-occurring Beauveria associated with the beetle to complement field efficacy studies of the commercial B. bassiana strain GHA. Sampling of CBB from coffee farms or unmanaged sites in various districts on the islands of Hawai'i and O'ahu, and also from Puerto Rico, resulted in >1800 Beauveria isolates. These were initially characterized using colony morphology to differentiate strain GHA, registered for use in Hawai'i, from indigenous congenerics. A total of 114 isolates representative of these indigenous morphotypes were selected for further characterization. Sequencing of the intergenic regions B locus and EFutr identified all as Beauveria bassiana sensu stricto. Sixteen haplotypes were observed, with one more common haplotype present in 12 of 16 sites sampled on Hawai'i Island. This B locus-EFutr haplotype, designated Bb1, was the only haplotype observed in 2016 epizootics on two high-elevation coffee farms on Hawai'i Island with no history of GHA application. Many of the haplotypes showed genetic similarity to those collected from CBB from other countries, including Brazil, Columbia, Nicaragua, and Kenya, but a few were identical to those from other insect species collected in Hawai'i before 2010. This diversity suggests a mixed lineage among B. bassiana strains associated with CBB in the three Hawaiian islands.

Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus).

The complete genomic sequence of a putative novel member of the family Secoviridae was determined by high-throughput sequencing of a pineapple accession obtained from the National Plant Germplasm Repository in Hilo, Hawaii. The predicted genome of the putative virus was composed of two RNA molecules of 6,128 and 4,161 nucleotides in length, excluding the poly-A tails. Each genome segment contained one large open reading frame (ORF) that shares homology and phylogenetic identity with members of the family Secoviridae. The presence of this new virus in pineapple was confirmed using RT-PCR and Sanger sequencing from six samples collected in Oahu, Hawaii. The name "pineapple secovirus A" (PSVA) is proposed for this putative new sadwavirus.

Using spatially explicit, time-dependent analysis to understand how social factors influence conservation outcomes.

Conservation across human-dominated landscapes requires an understanding of the social and ecological factors driving outcomes. Studies that link conservation outcomes to social and ecological factors have examined temporally static patterns. However, there may be different social and ecological processes driving increases and decreases in conservation outcomes that can only be revealed through temporal analyses. Through a case study of the invasion of Falcataria moluccana in Hawaii, we examined the association of social factors with increases and decreases in invader distributions over time and space. Over 7 years, rates of invader decrease varied substantially (66-100%) relative to social factors, such as building value, whether land was privately or publically owned, and primary residence by a homeowner, whereas rates of increase varied only slightly (<0.1-3.6%) relative to such factors. These findings suggest that links between social factors and invasion in the study system may be driven more by landowners controlling existing invasive species, rather than by landowners preventing the spread of invasive species. We suggest that spatially explicit, time-dependent analyses provide a more nuanced understanding of the way social factors influence conservation outcomes. Such an understanding can help managers develop outreach programs and policies targeted at different types of landowners in human-dominated landscapes.

Uso de un Análisis Espacialmente Explícito y Dependiente del Tiempo para Entender cómo Influyen los Factores Sociales sobre los Resultados de la Conservación Resumen La conservación dentro de los paisajes dominados por humanos requiere de un entendimiento de los factores sociales y ecológicos que afectan los resultados. Los estudios que conectan los resultados de la conservación con los factores sociales y ecológicos han examinado temporalmente los patrones estáticos. Sin embargo, puede haber diferentes procesos sociales y ecológicos que produzcan un incremento o una disminución en los resultados de la conservación, los cuales sólo pueden ser revelados por medio de los análisis temporales. Examinamos la asociación entre los factores sociales y el incremento y la disminución de la distribución de una invasión en el tiempo y el espacio con el estudio de caso de la invasión de Falcataria moluccana en Hawái. A lo largo de siete años las tasas de disminución de la invasión variaron considerablemente (66-100%) en relación con los factores sociales, como el valor de construcción, si la tierra era pública o privada, y si era la residencia principal del propietario, mientras que las tasas de incremento variaron solamente un poco (<0.1-3.6%) en relación con dichos factores. Estos hallazgos sugieren que las conexiones entre los factores sociales y la invasión en el sistema de estudio podrían ser causados más por los propietarios que controlan a las especies invasoras existentes en lugar de ser causados por los propietarios que previenen la expansión de las especies invasoras. Sugerimos que los análisis espacialmente explícitos y dependientes del tiempo proporcionan un entendimiento más matizado de cómo los factores sociales influyen sobre los resultados de la conservación. Dicho entendimiento puede ayudar a los administradores a desarrollar programas y políticas de compromiso con la comunidad enfocadas en diferentes tipos de propietarios en los paisajes dominados por el humano.

The bracteatus pineapple genome and domestication of clonally propagated crops.

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a 'one-step operation'. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513 Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars 'Smooth Cayenne' and 'Queen' exhibited ancient and recent admixture, while 'Singapore Spanish' supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated bromelain inhibitors. Four candidate genes for self-incompatibility were linked in F153, but were not functional in self-compatible CB5. Our findings support the coexistence of sexual recombination and a one-step operation in the domestication of clonally propagated crops. This work guides the exploration of sexual and asexual domestication trajectories in other clonally propagated crops.

Organ-specific transcriptome profiling of metabolic and pigment biosynthesis pathways in the floral ornamental progenitor species Anthurium amnicola Dressler.

Anthurium amnicola Dressler possesses a number of desirable and novel ornamental traits such as a purple-colored upright spathe, profuse flowering, and floral scent, some of which have been introgressed into modern Anthurium cultivars. As a first step in identifying genes associated with these traits, the transcriptome from root, leaf, spathe, and spadix from an accession of A. amnicola was assembled, resulting in 28,019 putative transcripts representing 19,458 unigenes. Genes involved in pigmentation, including those for the metabolism of chlorophyll and the biosynthesis of carotenoids, phenylpropanoids, and flavonoids were identified. The expression levels of one MYB transcription factor was highly correlated with naringenin 3-dioxygenase (F3H) and dihydroflavonol-4-reductase (DFR) in leaves, whereas a bHLH transcription factor was highly correlated with flavonoid 3'-monooxygenase (F3'H) and a DFR in spathes, suggesting that these two transcription factors might regulate flavonoid and anthocyanin synthesis in A. amnicola. Gene sequence and expression data from four major organs of A. amnicola provide novel basal information for understanding the genetic bases of ornamental traits and the determinants and evolution of form and function in the Araceae.

Developing single nucleotide polymorphism markers for the identification of pineapple (Ananas comosus) germplasm.

Pineapple (Ananas comosus [L.] Merr.) is the third most important tropical fruit in the world after banana and mango. As a crop with vegetative propagation, genetic redundancy is a major challenge for efficient genebank management and in breeding. Using expressed sequence tag and nucleotide sequences from public databases, we developed 213 single nucleotide polymorphism (SNP) markers and validated 96 SNPs by genotyping the United States Department of Agriculture - Agricultural Research Service pineapple germplasm collection, maintained in Hilo, Hawaii. The validation resulted in designation of a set of 57 polymorphic SNP markers that revealed a high rate of duplicates in this pineapple collection. Twenty-four groups of duplicates were detected, encompassing 130 of the total 170 A cosmos accessions. The results show that somatic mutation has been the main source of intra-cultivar variations in pineapple. Multivariate clustering and a model-based population stratification suggest that the modern pineapple cultivars are comprised of progenies that are derived from different wild Ananas botanical varieties. Parentage analysis further revealed that both A. comosus var. bracteatus and A. comosus var. ananassoides are likely progenitors of pineapple cultivars. However, the traditional classification of cultivated pineapple into horticultural groups (e.g. 'Cayenne', 'Spanish', 'Queen') was not well supported by the present study. These SNP markers provide robust and universally comparable DNA fingerprints; thus, they can serve as an efficient genotyping tool to assist pineapple germplasm management, propagation of planting material, and pineapple cultivar protection. The high rate of genetic redundancy detected in this pineapple collection suggests the potential impact of applying this technology on other clonally propagated perennial crops.

Developing single nucleotide polymorphism (SNP) markers from transcriptome sequences for identification of longan (Dimocarpus longan) germplasm.

Longan (Dimocarpus longan Lour.) is an important tropical fruit tree crop. Accurate varietal identification is essential for germplasm management and breeding. Using longan transcriptome sequences from public databases, we developed single nucleotide polymorphism (SNP) markers; validated 60 SNPs in 50 longan germplasm accessions, including cultivated varieties and wild germplasm; and designated 25 SNP markers that unambiguously identified all tested longan varieties with high statistical rigor (P<0.0001). Multiple trees from the same clone were verified and off-type trees were identified. Diversity analysis revealed genetic relationships among analyzed accessions. Cultivated varieties differed significantly from wild populations (F st=0.300; P<0.001), demonstrating untapped genetic diversity for germplasm conservation and utilization. Within cultivated varieties, apparent differences between varieties from China and those from Thailand and Hawaii indicated geographic patterns of genetic differentiation. These SNP markers provide a powerful tool to manage longan genetic resources and breeding, with accurate and efficient genotype identification.

Biogas energy production from tropical biomass wastes by anaerobic digestion.

Anaerobic digestion (AD) is an attractive technology in tropical regions for converting locally abundant biomass wastes into biogas which can be used to produce heat, electricity, and transportation fuels. However, investigations on AD of tropical forestry wastes, such as albizia biomass and food wastes, such as taro, papaya, and sweet potato, are limited. In this study, these tropical biomass wastes were evaluated for biogas production by liquid AD (L-AD) and/or solid-state AD (SS-AD), depending on feedstock characteristics. When albizia leaves and chips were used as feedstocks, L-AD had greater methane yields (161 and 113 L kg(-1)VS, respectively) than SS-AD (156.8 and 59.6 L kg(-1)VS, respectively), while SS-AD achieved 5-fold higher volumetric methane productivity than L-AD. Mono-digestion and co-digestion of taro skin, taro flesh, papaya, and sweet potato achieved methane yields from 345 to 411 L kg(-1)VS, indicating the robustness of AD technology.

Screening promoters for Anthurium transformation using transient expression.

KEY MESSAGE : There are multiple publications on Anthurium transformation, yet a commercial product has not been achieved. This may be due to use of non-optimum promoters here we address this problem. Different promoters and tissue types were evaluated for transient ß-glucuronidase (GUS) expression in Anthurium andraeanum Hort. 'Marian Seefurth' following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35S promoter from Cauliflower Mosaic Virus fused to a GUS reporter gene were bombarded into in vitro grown anthurium lamina, somatic embryos and roots. The number of GUS foci and the intensity of GUS expression were evaluated for each construct. Ubiquitin promoters from rice and maize resulted in the highest number of expressing cells in all tissues examined. Due to the slow growth of anthurium plants, development of transgenic anthurium plants takes years. This research has rapidly identified multiple promoters that express in various anthurium tissues facilitating the development of transformation vectors for the expression of desirable traits in anthurium plants.

Preliminary study of genetic variation in Hawaiian isolates of Beauveria bassiana [Hypocreales, Cordycipitaceae].

There have been no previous surveys documenting genetic diversity in Beauveria bassiana (Balsamo) Vuillemin in Hawaii. We used PCR primers and DNA sequencing to genetically characterize 14 isolates of B. bassiana collected from insects in east Hawaii island (the largest Hawaiian island, known as the 'Big Island') and compared these with the 'GHA' strain found in the commercial product BotaniGard®. Twelve of the 14 Hawaiian isolates were unique and the GHA strain was not among those isolated from the wild. Our data provides evidence that genetic diversity of B. bassiana in Hawaii is high over small spatial scales.

The externally derived portion of the hyperosmotic shock-activated cytosolic calcium pulse mediates adaptation to ionic stress in suspension-cultured tobacco cells.

The influx of Ca(2+) into the cytosol has long been suggested to serve as a signaling intermediate in the acquisition of tolerance to hyperosmotic and/or salinity stresses. Here we use aequorin-transformed suspension-cultured tobacco cells to directly assess the role of cytosolic calcium (Ca(2+)(cyt)) signaling in salinity tolerance acquisition. Aequorin luminescence recordings and (45)Ca influx measurements using inhibitors of Ca(2+) influx (Gd(3+) and the Ca(2+)-selective chelator EGTA), and modulators of organellar Ca(2+) release (phospholipase C inhibitors U73122 or neomycin) demonstrate that hyperosmolarity, whether imposed by NaCl or by a non-ionic molecule sorbitol, induces a rapid (returning to baseline levels of Ca(2+) within 10 min) and complex Ca(2+)(cyt) pulse in tobacco cells, deriving both from Gd(3+)-sensitive externally derived Ca(2+) influx and from U73122- and neomycin-sensitive Ca(2+) release from an organelle. To determine whether each of the two components of this brief Ca(2+) signal regulate adaptation to hyperosmotic shock, the Ca(2+) pulse was modified by the addition of Gd(3+), U73122, neomycin, or excess Ca(2+), and then cells were treated with salt or sorbitol. After 10 min the cell culture medias were diluted with additional hyperosmotic media to reduce the toxic affects of the modulators, and the growth of cells was measured after 1 week. Gd(3+) treatment reduced growth in salt relative to control cells but not in sorbitol, and exposure to excess Ca(2+) increased growth in salt but not in sorbitol. In contrast, exposure to inhibitors of IP(3) formation had no effect on growth in salt or sorbitol. Therefore, although hyperosmotic treatment stimulates both Ca(2+) influx and Ca(2+) release from an internal Ca(2+) depot, only Ca(2+) influx has a measurable impact on ionic stress tolerance acquisition in tobacco cell suspensions. In contrast, osmoadaptation in these cells appears to occur independent of Ca(2+) signaling.