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Above-optimal temperatures reduce yield in many crops, including tomato, largely because of the heat-sensitivity of their reproduction process. A full understanding of heat-stress (HS) response and thermotolerance of tomato reproduction is still lacking. Recently, using external application of the plant hormone ethylene, it was demonstrated that ethylene plays a role in heat-tolerance of tomato pollen (the male reproductive cells). In order to expand our understanding on involvement of ethylene in tomato pollen thermotolerance, we analyzed the response of wild type and ethylene-related tomato mutant plants to HS, at physiological and molecular levels. We report that mild chronic HS conditions highly reduce the number of viable and germinating pollen grains as well as the production of seeded fruits in wild type tomato plants, while no significant reduction was detected/observed in pollen quality, number of seeded fruits and seeds per fruit in plants of the ethylene over-producer mutant epinastic. Our findings suggest that ethylene is involved in thermotolerance of tomato reproduction, pointing to an effect on pollen viability and germination potential, highlighting candidate genes involved in pollen response to HS (like SlHSP17, SlHSP101, SlMBF1) and suggesting directions for further studies.

Sweetpotato is an important food crop globally, serving as a rich source of carbohydrates, vitamins, fiber, and micronutrients. Sweetpotato yield depends on the modification of adventitious roots into storage roots. The underlying mechanism of this developmental switch is not fully understood. Interestingly, storage-root formation is manifested by formation of starch-accumulating parenchyma cells and bulking of the distal part of the root, while the proximal part does not show bulking. This system, where two parts of the same adventitious root display different developmental fates, was used by us in order to better characterize the anatomical, physiological, and molecular mechanisms involved in sweetpotato storage-root formation. We show that, as early as 1 and 2 weeks after planting, the proximal part of the root exhibited enhanced xylem development together with increased/massive lignin deposition, while, at the same time, the distal root part exhibited significantly elevated starch accumulation. In accordance with these developmental differences, the proximal root part exhibited up-regulated transcript levels of sweetpotato orthologs of Arabidopsis vascular-development regulators and key genes of lignin biosynthesis, while the distal part showed up-regulation of genes encoding enzymes of starch biosynthesis. All these recorded differences between proximal and distal root parts were further enhanced at 5 weeks after planting, when storage roots were formed at the distal part. Our results point to down-regulation of fiber formation and lignification, together with up-regulation of starch biosynthesis, as the main events underlying storage-root formation, marking/highlighting several genes as potential regulators, providing a valuable database of genes for further research.

Tomato pollen grains have the capacity for ethylene production, possessing specific components of the ethylene-biosynthesis and -signaling pathways, being affected/responsive to high-temperature conditions. Exposure of plants to heat stress (HS) conditions reduces crop yield and quality, mainly due to sensitivity of pollen grains. Recently, it was demonstrated that ethylene, a gaseous plant hormone, plays a significant role in tomato pollen heat-tolerance. It is not clear, however, whether, or to what extent, pollen grains are dependent on the capacity of the surrounding anther tissues for ethylene synthesis and signaling, or can synthesize this hormone and possess an active signaling pathway. The aim of this work was (1) to investigate if isolated, maturing and mature, tomato pollen grains have the capacity for ethylene production, (2) to find out whether pollen grains possess an active ethylene-biosynthesis and -signaling pathway and characterize the respective tomato pollen components at the transcript level, (3) to look into the effect of short-term HS conditions. Results from accumulation studies showed that pollen, anthers, and flowers produced ethylene and HS affected differentially ethylene production by (rehydrated) mature pollen, compared to anthers and flowers, causing elevated ethylene levels. Furthermore, several ethylene synthesis genes were expressed, with SlACS3 and SlACS11 standing out as highly HS-induced genes of the pollen ethylene biosynthesis pathway. Specific components of the ethylene-signaling pathway as well as several ethylene-responsive factors were expressed in pollen, with SlETR3 (ethylene receptor; named also NR, for never ripe) and SlCTR2 (constitutive triple response2) being HS responsive. This work shows that tomato pollen grains have the capacity for ethylene production, possessing active ethylene-biosynthesis and -signaling pathways, highlighting specific pollen components that serve as a valuable resource for future research on the role of ethylene in pollen thermotolerance.

Pollen development at high temperature depends on a fine balance between acclimation and injury.

Sweet potato yield depends on a change in the developmental fate of adventitious roots into storage-roots. The mechanisms underlying this developmental switch are still unclear. We examined the hypothesis claiming that regulation of root lignification determines storage root formation. We show that application of the plant hormone gibberellin increased stem elongation and root gibberellin levels, while having inhibitory effects on root system parameters, decreasing lateral root number and length, and significantly reducing storage root number and diameter. Furthermore, gibberellin enhanced root xylem development,caused increased lignin deposition, and, at the same time, decreased root starch accumulation. In accordance with these developmental effects, gibberellin application upregulated expression levels of sweet potato orthologues of Arabidopsis vascular development regulators (IbNA075, IbVND7, and IbSND2) and of lignin biosynthesis genes(IbPAL, IbC4H, Ib4CL, IbCCoAOMT, and IbCAD), while down regulating starch biosynthesis genes (IbAGPase and IbGBSS) in the roots. Interestingly, gibberellin down regulated root expression levels of orthologues of the Arabidopsis BREVIPEDICELLUS transcription factor (IbKN2 and IbKN3), regulator of meristem maintenance. The results substantiate our hypothesis and mark gibberellin as an important player in regulation of sweet potato root development, suggesting that increased fiber formation and lignification inhibit storage root formation and yield. Taken together, our findings provide insight into the mechanisms underlying sweet potato storage-root formation and provide a valuable database of genes for further research.

Phytohormones control the development and growth of plants, as well as their response to biotic and abiotic stress. The seven most well-studied phytohormone classes defined today are as follows: auxins, ethylene, cytokinin, abscisic acid, jasmonic acid, gibberellins, and brassinosteroids. The basic principle of hormone regulation is conserved in all plants, but recent results suggest adaptations of synthesis, transport, or signaling pathways to the architecture and growth environment of different plant species. Thus, we aimed to define the extent to which information from the model plant Arabidopsis thaliana is transferable to other plants such as Solanum lycopersicum. We extracted the co-orthologues of genes coding for major pathway enzymes in A. thaliana from the translated genomes of 12 species from the clade Viridiplantae. Based on predicted domain architecture and localization of the identified proteins from all 13 species, we inspected the conservation of phytohormone pathways. The comparison was complemented by expression analysis of (co-) orthologous genes in S. lycopersicum. Altogether, this information allowed the assignment of putative functional equivalents between A. thaliana and S. lycopersicum but also pointed to some variations between the pathways in eudicots, monocots, mosses, and green algae. These results provide first insights into the conservation of the various phytohormone pathways between the model system A. thaliana and crop plants such as tomato. We conclude that orthologue prediction in combination with analysis of functional domain architecture and intracellular localization and expression studies are sufficient tools to transfer information from model plants to other plant species. Our results support the notion that hormone synthesis, transport, and response for most part of the pathways are conserved, and species-specific variations can be found.

Introduction Untargeted metabolomics is a powerful tool to detect hundreds of metabolites within a given tissue and to compare the metabolite composition of samples in a comprehensive manner. However, with regard to pollen research such comprehensive metabolomics approaches are yet not well developed. To enable isolation of pollen that is tightly enclosed within the anthers of the flower, such as immature pollen, the current pollen isolation protocols require the use of a watery solution. These protocols raise a number of concerns for their suitability in metabolomics analyses, in view of possible metabolic activities in the pollen and contamination with anther metabolites. Objectives We assessed the effect of different sample preparation procedures currently used for pollen isolation for their suitability to perform metabolomics of tomato pollen. Methods Pollen were isolated using different methods and the metabolic profiles were analysed by liquid chromatography–mass spectrometry (LC–MS). Results Our results demonstrated that pollen isolation in a watery solution led to (i) rehydration of the pollen grains, inducing marked metabolic changes in flavonoids, phenylpropanoids and amino acids and thus resulting in a metabolite profile that did not reflect the one of mature dry pollen, (ii) hydrolysis of sucrose into glucose and fructose during subsequent metabolite extraction, unless the isolated and rehydrated pollen were lyophilized prior to extraction, and (iii) contamination with anther-specific metabolites, such as alkaloids, thus compromising the metabolic purity of the pollen fraction. Conclusion We conclude that the current practices used to isolate pollen are suboptimal for metabolomics analyses and provide recommendations on how to improve the pollen isolation protocol, in order to obtain the most reliable metabolic profile from pollen tissue. Electronic supplementary material The online version of this article (10.1007/s11306-018-1471-4) contains supplementary material, which is available to authorized users.

Recently, we have developed a quantitative shotgun proteomics strategy called mass accuracy precursor alignment (MAPA). The MAPA algorithm uses high mass accuracy to bin mass-to-charge (m/z) ratios of precursor ions from LC-MS analyses, determines their intensities, and extracts a quantitative sample versus m/z ratio data alignment matrix from a multitude of samples. Here, we introduce a novel feature of this algorithm that allows the extraction and alignment of proteotypic peptide precursor ions or any other target peptide from complex shotgun proteomics data for accurate quantification of unique proteins. This strategy circumvents the problem of confusing the quantification of proteins due to indistinguishable protein isoforms by a typical shotgun proteomics approach. We applied this strategy to a comparison of control and heat-treated tomato pollen grains at two developmental stages, post-meiotic and mature. Pollen is a temperature-sensitive tissue involved in the reproductive cycle of plants and plays a major role in fruit setting and yield. By LC-MS-based shotgun proteomics, we identified more than 2000 proteins in total for all different tissues. By applying the targeted MAPA data-processing strategy, 51 unique proteins were identified as heat-treatment-responsive protein candidates. The potential function of the identified candidates in a specific developmental stage is discussed.

UNLABELLED • PREMISE OF THE STUDY Yield in sweetpotato is determined by the number of storage roots produced per plant. Storage roots develop from adventitious roots (ARs) present in stem cuttings that serve as propagation material. Data on the origin of sweetpotato ARs and the effect of nodal position on AR establishment and further development are limited.• METHODS We anatomically described root primordium initiation using stem sections and measured number of root primordia formed at different nodal positions using light microscopy and correlated nodal positions with AR number and length 14 d after planting (DAP).• KEY RESULTS Primordia for ARs initiate at the junction of the stem pith ray and the cambium, on both sides of the leaf gap, and they are well developed before emerging from the stem. The number of ARs that develop from isolated stem nodes 14 DAP corresponded to the number of AR primordia detected inside the stem. The total length of established roots at nodes 9-13 from the apex is about 2-fold longer than at nodes 5-8.• CONCLUSIONS Nodal position (age) has a significant effect on the developmental status and number of root primordia inside the stem, determining the number and length of ARs that have developed by 14 DAP. Adventitious roots originating from nodes 9-13 possess similar AR systems and develop better than those originating from younger nodes 3-8. The mechanism regulating AR initiation in nodes is discussed. This system can serve for studying the effect of environmental conditions on AR initiation, development, and capacity to form storage roots.

It is becoming increasingly evident that optimization of root architecture for resource capture is vital for enabling the next green revolution. Although cereals provide half of the calories consumed by humans, root and tuber crops are the second major source of carbohydrates globally. Yet, knowledge of root architecture in root and tuber species is limited. In this opinion article, we highlight what is known about the root system in root and tuber crops, and mark new research directions towards a better understanding of the relation between root architecture and yield. We believe that unraveling the role of root architecture in root and tuber crop productivity will improve global food security, especially in regions with marginal soil fertility and low-input agricultural systems.

Greenhouse and field culture systems were used to study the effect of drought conditions on the storage root (SR) formation in ‘Beauregard’ sweetpotato (Ipomoea batatas). In the greenhouse culture system, drought was simulated by withholding water for 5 and 10 days after transplanting (DAT) cuttings in dry sand. Control plants received water at planting and every 3 days thereafter. In the field studies, natural drought conditions and selective irrigation were used to impose water deprivation during the critical SR formation period. Greenhouse drought for 5 and 10 DAT reduced the number of SRs by 42% and 66%, respectively, compared with the controls. Field drought resulted in a 49% reduction in U.S. #1 SR yield compared with the irrigated condition. Quantitative real-time polymerase chain reaction (PCR) analysis showed differential expression of a set of sweetpotato transcription factors and protein kinases among greenhouse-grown plants subjected to well-watered conditions and water deficit during 5 DAT. A significant enhancement of expression was observed for known drought stress-associated genes such as an abscisic acid-responsive elements-binding factor, dehydration-responsive element-binding factor, and homeo-domain-zip proteins. Members of calcium-binding proteins showed differential expression under drought stress. For the first time it is reported that knotted1-like homeobox and BEL1-like genes showed altered expression in response to drought stress under a greenhouse condition. In summary, the results suggest that water deprivation during the SR formation period influences root development and expression patterns of stress-responsive genes and those previously found associated with SR formation in sweetpotato.

There is no direct evidence of the effect of lignin metabolism on early storage root development in sweet potato. In this study, we found that heterologous expression of the maize leaf color (Lc) gene in sweet potato increased anthocyanin pigment accumulation in the whole plant and resulted in reduced size with an increased length/width ratio, low yield and less starch content in the early storage roots. RT-PCR analysis revealed dramatic up-regulation of the genes involved in the lignin biosynthesis pathway in developing storage roots, leading to greater lignin content in the Lc transgenic lines, compared to the wild type. This was also evidenced by the enhanced lignification of vascular cells in the early storage roots. Furthermore, increased expression of the β-amylase gene in leaves and storage roots also accelerated starch degradation and increased the sugar use efficiency, providing more energy and carbohydrate sources for lignin biosynthesis in the Lc transgenic sweet potato. Lesser starch accumulation was observed in the developing storage roots at the initiation stage in the Lc plants. Our study provides experimental evidence of the basic carbohydrate metabolism underlying the development of storage roots, which is the transformation of lignin biosynthesis to starch biosynthesis.

This study aimed to investigate the effect of 1-methylcyclopropene (1-MCP) on adventitious rooting in two sweetpotato cultivars. Experiments with ‘Beauregard’ and ‘Evangeline’ sweetpotato cuttings revealed differential adventitious root (AR) emergence responses to 1-MCP application. ‘Beauregard’ AR count and length decreased with 1-MCP application in two of four experiments. In contrast, 1-MCP did not influence ‘Evangeline’ root count. However, ‘Evangeline’ root length decreased in three of four experiments. Trypan blue staining of ‘Beauregard’ nodal tissue with delayed AR primordia emergence showed localized dead tissue in the general area where ARs emerge. The degree of staining appeared to correspond with the stage of AR emergence with the staining becoming more intense around the time an AR primordium eventually emerged through a crack in the epidermis. This response agrees with reported results of ethylene-mediated AR emergence in other plant species. These results also appear to suggest that ‘Beauregard’ and ‘Evangeline’ cuttings differ in ethylene sensitivity. This represents the first evidence of genotype-specific ethylene involvement in adventitious rooting of sweetpotato cuttings.

This study characterized lateral root (LR) development attributes during the onset of storage root (SR) initiation stage in ‘Beauregard’ sweetpotato. SR initiation has been defined as the appearance of cambia around the protoxylem and secondary xylem elements. Our results showed that 20-day-old adventitious roots (ARs) classified as SRs had 53% and 85% greater mean LR count than pencil roots (PRs) and lignified roots (LGs), respectively. SRs had 53% and 78% greater mean LR density relative to PRs and LGs, respectively. SRs had 66% and 130% greater mean total LR length than PRs and LGs, respectively. SRs had lower mean main root (MR)/LR length ratio compared with PRs (–38%) and LGs (–60%). SRs had 70% and 134% greater mean surface area than PRs and LGs, respectively. SRs had lower mean MR/LR surface area ratio compared with PRs (–42%) and LGs (–62%). The plot of the first and second principal components revealed the presence of a gradient between extreme LG and SR clusters, suggesting a developmental transition between LGs and SRs with PRs representing an intermediate developmental stage. Although AR architecture is not the sole determinant of SR formation, our data help provide a basis for integrating AR architecture attributes with other factors that are known to influence SR initiation. Growth substrate moisture variability influenced LR development during the critical SR initiation period. Relative to the control treatments, water deprivation 10 to 20 days after transplanting (DAT) reduced mean LR count, length, and surface area by 49%, 103%, and 94%, respectively. Saturated conditions 10 to 20 DAT reduced mean LR count, length, and surface area by 75%, 81%, and 77%, respectively. These results represent the first evidence for the association between anatomical cues of SR initiation and root architecture and provide corroborating data that soil moisture variability 10 to 20 DAT directly influences SR yield potential through AR architecture modifications that are associated with diminished SR formation. This information can be used to further optimize SR yield by identifying agroclimatic and management variables that are associated with desirable LR development during the critical SR initiation stage.

The formation of mature and fertile pollen grains, taking place inside the anther, depends on supply of assimilates, in the form of sucrose, provided mainly by the leaves. Data is limited, however, with respect to the understanding of sucrose metabolism in microspores and the supporting tissues. The aims of the present work were to 1) follow the changes in total and relative concentrations of sucrose, glucose, fructose and starch in the stamen parts and microspores up until anthesis, 2) follow the activities of sucrose-metabolism-related enzymes, in the anther walls fraction and microspores of the crop plant tomato. Sucrose was found to be partially cleaved in the filament, decreasing by more than twofold in the anther wall layers and the locular fluid, and to accumulate in the mature pollen grains, constituting 80% of total soluble sugars. Thus, sucrose was both the starting sugar, supporting microspore development, and the main carbohydrate accumulated at the end of the pollen-development program. The major invertase found to be active in both the anther wall layers and in maturing microspores was cell-wall-bound invertase. High fructokinase 2 and sucrose phosphate synthase activities during pollen maturation coincided with sucrose accumulation. The potential importance of sucrose accumulation during pollen dehydration phase and germination is discussed.

Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.

A thermal time-based approach using growing degree days was used to describe the onset of morpho-anatomical features associated with storage root developmental stages in the sweetpotato cultivar ‘Beauregard.’ Initially, we calculated accumulated growing degree days corresponding to adventitious root initiation, the onset of storage root formation (appearance of anomalous cambium), and appearance of visible storage roots with localized thickening and pigmentation in sweetpotatoes grown under greenhouse conditions. Subsequently, we performed field calibration of this approach to verify the timing of these storage root developmental stages under natural growing conditions. Under field conditions, transplants required relatively fewer calendar days to achieve the thermal time corresponding to each developmental stage in comparison to greenhouse-grown plants. Further calibration of this approach for ‘Beauregard’ and other sweetpotato varieties grown in diverse environments will help enhance our understanding of the effects of management and agroclimatic variables on sweetpotato yield determination. This study demonstrates the advantage of using thermal time vs. calendar days in estimating storage root development in sweetpotatoes.

Additional index words. storage root development, storage root anatomy, Ipomoea batatas Abstract. Adventitious roots of 'Beauregard' and 'Georgia Jet' sweetpotato were observed and anatomically characterized over a period of 60 days of storage root development. The majority of 'Beauregard' and 'Georgia Jet' adventitious roots sampled at 5 to 7 days after transplanting (DAT) possessed anatomical features (five or more protoxylem elements) associated with storage root development. The majority of 'Beauregard'(86%)and'GeorgiaJet'(89%)storagerootssampledat60to65DATwere traced directly to adventitious roots extant at 5 to 7 DAT. The two varieties, however, differed in the timing in which regular and anomalous cambia were formed. Regular vascular cambium development, i.e., initiation and completion, was observed in both varieties at 19 to 21 DAT. Formation of complete regular vascular cambium was negligible for 'Beauregard' (4%) in comparison with 'Georgia Jet' (32%) at 26 to 28 DAT. However, anomalous cambia development adjacent to xylem elements was greater in 'Beauregard' (30%) in comparison with 'Georgia Jet' (13%). Nearly 40% to 50% of samples in both varieties showed extensive lignification in the stele region. At 32 to 35 DAT, 62% to 70% of the adventitious roots for both varieties had either been initiated (developed anomalous cambium) or were lignified. The remaining adventitious roots showed intermediate stages of vascular cambium development. The adventitious root count increased up to 19 to 21 DAT and then remained constant up to 32 to 35 DAT. These accumulatedresults suggestthatthe initialstages ofadventitiousrootdevelopment are critical in determining storage root set in sweetpotato.

Pollen thermotolerance. Global warming is predicted to increase the frequency and severity of extreme weather phenomena such as heat waves thereby posing a major threat for crop productivity and food security. The yield in case of most crop species is dependent on the success of reproductive development. Pollen development has been shown to be highly sensitive to elevated temperatures while the development of the female gametophyte as well as sporophytic tissues might also be disturbed under mild or severe heat stress conditions. Therefore, assessing pollen thermotolerance is currently of high interest for geneticists, plant biologists and breeders. A key aspect in pollen thermotolerance studies is the selection of the appropriate heat stress regime, the developmental stage that the stress is applied to, as well as the method of application. Literature search reveals a rather high variability in heat stress treatments mainly due to the lack of standardized protocols for different plant species. In this review, we summarize and discuss experimental approaches that have been used in various crops, with special focus on tomato, rice and wheat, as the best studied crops regarding pollen thermotolerance. The overview of stress treatments and the major outcomes of each study aim to provide guidelines for similar research in other crops.

Sequencing costs are falling, but the cost of data analysis remains high, often because unforeseen problems arise, such as insufficient depth of sequencing or batch effects. Experimenting with data analysis methods during the planning phase of an experiment can reveal unanticipated problems and build valuable bioinformatics expertise in the organism or process being studied. This protocol describes using R Markdown and RStudio, user-friendly tools for statistical analysis and reproducible research in bioinformatics, to analyze and document the analysis of an example RNA-Seq data set from tomato pollen undergoing chronic heat stress. Also, we show how to use Integrated Genome Browser to visualize read coverage graphs for differentially expressed genes. Applying the protocol described here and using the provided data sets represent a useful first step toward building RNA-Seq data analysis expertise in a research group.

Exposure to high temperatures (heat stress) causes reduced yield in tomatoes (Lycopersicon esculentum), mainly by affecting male gametophyte development. Two experiments were conducted where several tomato cultivars were grown under heat stress, in growth chambers (day/night temperatures of 31/25 °C) or in greenhouses (day/night temperatures of 32/26 °C), or under control (day/night temperatures of 28/22 °C) conditions. In heat-sensitive cultivars, heat stress caused a reduction in the number of pollen grains, impaired their viability and germinability, caused reduced fruit set and markedly reduced the numbers of seeds per fruit. In the heat-tolerant cultivars, however, the number and quality of pollen grains, the number of fruits and the number of seeds per fruit were less affected by high temperatures. In all the heat-sensitive cultivars, the heat-stress conditions caused a marked reduction in starch concentration in the developing pollen grains at 3 days before anthesis, and a parallel decrease in the total soluble sugar concentration in the mature pollen, whereas in the four heat-tolerant cultivars tested, starch accumulation at 3 days before anthesis and soluble sugar concentration at anthesis were not affected by heat stress. These results indicate that the carbohydrate content of developing and mature tomato pollen grains may be an important factor in determining pollen quality, and suggest that heat-tolerant cultivars have a mechanism for maintaining the appropriate carbohydrate content under heat stress.

In petunia, a mitochondrial (mt) locus, S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). The S-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF, Pcf, contains parts of the atp9 and coxII genes and an unidentified reading frame, urf-s. The second and third ORFs contain NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) sequences, respectively. The nad3 and rps12 sequences included in the S-Pcf locus are identical to the corresponding sequences on the mt genome of fertile petunia. In both CMS and fertile petunia, only a single copy of nad3 and rps12 had been detected on the physical map of the main mt genome. The origin of the urf-s sequence and the molecular events leading to the formation of the chimeric S-Pcf locus are not known. This paper presents evidence indicating that two different mt sequences, related to urf-s and found in fertile petunia lines (orf-h and Rf-1), might have been involved in the molecular evolution of the S-Pcf locus. Southern analysis of mtDNA derived from both fertile and sterile petunia plants suggests that one of these urf-s related sequences (showing 100% homology to urf-s and termed orf-h) is located on a sublimon. An additional, low-homology urf-s related sequence (Rf-1) is shown to be located on the main mt genome 5' to the nad3 gene. It is, thus, suggested that the sequence of events leading to the generation of the S-Pcf locus might have involved introduction of the orf-h sequence, via homologous recombination, into the main mt genome 5' to nad3 at the region where the Rf-1 sequence is located.

The paper supports the view that ethylene plays a significant role in maintaining tomato pollen thermotolerance. Interfering with the ethylene signalling pathway or reducing ethylene levels and increased tomato pollen sensitivity to heat stress. On the other hand, increasing ethylene levels before heat-stress improved pollen quality., Background and aims Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due to sensitivity of developing pollen grains. The mechanisms maintaining high pollen quality under heat-stress conditions are poorly understood. Our recently published data indicate high heat-stress-induced expression of ethylene-responsive genes in tomato pollen, indicating ethylene involvement in the pollen heat-stress response. Here we elucidated ethylene's involvement in pollen heat-stress response and thermotolerance by assessing the effects of interfering with the ethylene signalling pathway and altering ethylene levels on tomato pollen functioning under heat stress. Methodology Plants of the ethylene-insensitive mutant Never ripe (Nr)—defective in an ethylene response sensor (ERS)-like ethylene receptor—and the corresponding wild type were exposed to control or heat-stress growing conditions, and pollen quality was determined. Starch and carbohydrates were measured in isolated pollen grains from these plants. The effect of pretreating cv. Micro-Tom tomato plants, prior to heat-stress exposure, with an ethylene releaser or inhibitor of ethylene biosynthesis on pollen quality was assessed. Principal results Never ripe pollen grains exhibited higher heat-stress sensitivity, manifested by a significant reduction in the total number of pollen grains, reduction in the number of viable pollen and elevation of the number of non-viable pollen, compared with wild-type plants. Mature Nr pollen grains accumulated only 40 % of the sucrose level accumulated by the wild type. Pretreatment of tomato plants with an ethylene releaser increased pollen quality under heat stress, with an over 5-fold increase in the number of germinating pollen grains per flower. Pretreatment with an ethylene biosynthesis inhibitor reduced the number of germinating pollen grains following heat-stress exposure over 5-fold compared with non-treated controls. Conclusions Ethylene plays a significant role in tomato pollen thermotolerance. Interfering with the ethylene signalling pathway or reducing ethylene levels increased tomato pollen sensitivity to heat stress, whereas increasing ethylene levels prior to heat-stress exposure increased pollen quality.

The male gametophyte developmental programme can be divided into five phases which differ in relation to the environment and pollen hydration state: (1) pollen develops inside the anther immersed in locular fluid, which conveys substances from the mother plant--the microsporogenesis phase; (2) locular fluid disappears by reabsorption and/or evaporation before the anther opens and the maturing pollen grains undergo dehydration--the dehydration phase; (3) the anther opens and pollen may be dispersed immediately, or be held by, for example, pollenkitt (as occurs in almost all entomophilous species) for later dispersion--the presentation phase; (4) pollen is dispersed by different agents, remaining exposed to the environment for different periods--the dispersal phase; and (5) pollen lands on a stigma and, in the case of a compatible stigma and suitable conditions, undergoes rehydration and starts germination--the pollen-stigma interaction phase.This review highlights the issue of pollen water status and indicates the various mechanisms used by pollen grains during their five developmental phases to adjust to changes in water content and maintain internal stability.Pollen water status is co-ordinated through structural, physiological and molecular mechanisms. The structural components participating in regulation of the pollen water level, during both dehydration and rehydration, include the exine (the outer wall of the pollen grain) and the vacuole. Recent data suggest the involvement of water channels in pollen water transport and the existence of several molecular mechanisms for pollen osmoregulation and to protect cellular components (proteins and membranes) under water stress. It is suggested that pollen grains will use these mechanisms, which have a developmental role, to cope with environmental stress conditions.

Accurate and rapid cultivar identification is important for breeders'-rights protection, especially for vegetatively propagated plants. The objective of this study was to test the feasibility of developing cultivar-specific RAPD markers in commercial strawberries (Fragaria ananassa Dutch). Efforts were focused on distinguishing between two newly developed Volcani cultivars, ‘Ofra’ and ‘Dorit’, and six other cultivars, ‘Douglas’, ‘Chandler’, ‘Oso Grande’, ‘Dover’, ‘Nurit’ and ‘Parker’. Reproducible RAPD fingerprints were generated, each containing at least one polymorphic DNA product. A combination of 10 polymorphic DNA products exhibited cultivar-specific patterns enabling the distinction between closely related varieties, such as ‘Ofra’ (which is the progeny of ‘Dorit’ and ‘Parker’) and ‘Dorit’ (which is the progeny of ‘Nurit’ and ‘Dover’). This study shows that RAPD markers can help in the protection of breeders’ rights to strawberry cultivars.

Comparative histological and ultrastructural studies were made on the gametogenesis and microsporogenesis in isonuclear fertile and cytoplasmic male sterile petunia lines. Using electron microscopy, changes in the mitochondrial double membrane were observed in the tapetal and sporogenic cells of cytoplasmic male sterile anthers as the first cytological sign of breakdown of the process of microsporogenesis at early prophase I. These changes were manifested by a larger space between the outer and the inner membrane and fewer sites of close proximity (“contact sites”) in the sterile mitochondria, as compared to fertile mitochondria. The mitochondrial membrane of the parietal tissue in the cytoplasmic male sterile anthers was not affected in the same way. Even when total breakdown was already obvious in the tapetal and sporogenic cells, the mitochondria of the parietal layer remained intact.

Original objectives: Anatomical study of storage root initiation and formation. Induction of storage root formation. Isolation and characterization of genes involved in storage root formation. During the normal course of storage root development. Following stress-induced storage root formation. Background:Sweetpotato is a high value vegetable crop in Israel and the U.S. and acreage is expanding in both countries and the research herein represents an important backstop to improving quality, consistency, and yield. This research has two broad objectives, both relating to sweetpotato storage root formation. The first objective is to understand storage root inductive conditions and describe the anatomical and physiological stages of storage root development. Sweetpotato is propagated through vine cuttings. These vine cuttings form adventitious roots, from pre-formed primordiae, at each node underground and it is these small adventitious roots which serve as initials for storage and fibrous (non-storage) “feeder” roots. What perplexes producers is the tremendous variability in storage roots produced from plant to plant. The marketable root number may vary from none to five per plant. What has intrigued us is the dearth of research on sweetpotato during the early growth period which we hypothesize has a tremendous impact on ultimate consistency and yield. The second objective is to identify genes that change the root physiology towards either a fleshy storage root or a fibrous “feeder” root. Understanding which genes affect the ultimate outcome is central to our research. Major conclusions: For objective one, we have determined that the majority of adventitious roots that are initiated within 5-7 days after transplanting possess the anatomical features associated with storage root initiation and account for 86 % of storage root count at 65 days after transplanting. These data underscore the importance of optimizing the growing environment during the critical storage root initiation period. Water deprivation during this phenological stage led to substantial reduction in storage root number and yield as determined through growth chamber, greenhouse, and field experiments. Morphological characterization of adventitious roots showed adjustments in root system architecture, expressed as lateral root count and density, in response to water deprivation. For objective two, we generated a transcriptome of storage and lignified (non-storage) adventitious roots. This transcriptome database consists of 55,296 contigs and contains data as regards to differential expression between initiating and lignified adventitious roots. The molecular data provide evidence that a key regulatory mechanism in storage root initiation involves the switch between lignin biosynthesis and cell division and starch accumulation. We extended this research to identify genes upregulated in adventitious roots under drought stress. A subset of these genes was expressed in salt stressed plants.

Seven members of an Ashkenazi Jewish family with Gaucher disease in 3 successive generations were tested for the presence of the 2 common mutations known to occur in the glucocerebrosidase gene. Genomic DNA from blood or skin fibroblasts of relatives was amplified by using the PCR technique and individual mutations identified by oligonucleotides specific to the mutated sequences. Four individuals were homozygous for a mutation at amino acid 370 (370 mutation) known to occur only in type 1 disease. The other 3 affected relatives were compound heterozygotes for this mutation and for a mutation at amino acid 444 (NciI mutation) which, in the homozygous state, is associated with neurological disease. Clinical severity was more marked in the compound heterozygotes than in the homozygotes. Since the mutation is present in Ashkenazim, molecular diagnosis in families which carry the NciI mutation should prove useful in assessing their risk of the neurologic forms of Gaucher disease.

Exposure to higher than optimal temperatures - heat-stress (HS) - is becoming increasingly common to all crop plants worldwide. Heat stress coinciding with microgametogenesis, especially during the post-meiotic phase that is marked by starch biosynthesis, is often associated with starch-deficient pollen and male sterility and ultimately, greatly reduced crop yields. The molecular basis for the high sensitivity of developing pollen grains, on one hand, and factors involved in pollen heat-tolerance, on the other, is poorly understood. The long-term goal of this project is to provide a better understanding of the genes that control pollen quality under heat-stress conditions. The specific objectives of this project were: (1) Determination of the threshold heat stress temperature(s) that affects tomato and sorghum pollen quality whether: a) Chronic mild heat stress conditions (CMHS), or b) Acute heat stress (AHS). (2) Isolation of heat-responsive, microgametogenesis-specific sequences. During our one-year feasibility project, we have accomplished the proposed objectives as follows: Objectrive 1: We have determined the threshold HS conditions in tomato and sorghum. This was essential for achieving the 2nd objective, since our accumulated experience (both Israeli and US labs) indicate that when temperature is raised too high above "threshold HS levels" it may cause massive death of the developing pollen grains. Above-threshold conditions have additional major disadvantages including the "noise" caused by induced expression of genes involved in cell death and masking of the differences between heatsensitive and heat-tolerant pollen grains. Two different types of HS conditions were determined: a) Season-long CMHS conditions: 32/26°C day/night temperatures confirmed in tomato and 36/26°C day maximum/night minimum temperatures in sorghum. b) Short-term AHS: In tomato, 2 hour exposure to 42-45°C (at 7 to 3 days before anthesis) followed by transfer to 28/22±2oC day/night temperatures until flower opening and pollen maturation, caused 50% reduced germinating pollen in the heat-sensitive 3017 cv.. In sorghum, 36/26°C day/night temperatures 10 to 5 days prior to panicle emergence, occurring at 35 days after sowing (DAS) in cv. DeKalb28E, produced starch-deficient and sterile pollen. Objective 2: We have established protocols for the high throughput transcriptomic approach, cDNA-AFLP, for identifying and isolating genes exhibiting differential expression in developing microspores exposed to either ambient or HS conditions and created a databank of HS-responsivemicrogametogenesis-expressed genes. A subset of differentially displayed Transcript-Derived Fragments (TDFs) that were cloned and sequenced (35 & 23 TDFs in tomato and sorghum, respectively) show close sequence similarities with metabolic genes, genes involved in regulation of carbohydrate metabolism, genes implicated in thermotolerance (heat shock proteins), genes involved in long chain fatty acids elongation, genes involved in proteolysis, in oxidation-reduction, vesicle-mediated transport, cell division and transcription factors. T-DNA-tagged Arabidopsis mutants for part of these genes were obtained to be used for their functional analysis. These studies are planned for a continuation project. Following functional analyses of these genes under HS – a valuable resource of genes, engaged in the HS-response of developing pollen grains, that could be modulated for the improvement of pollen quality under HS in both dicots and monocots and/or used to look for natural variability of such genes for selecting heat-tolerant germplasm - is expected.

Mitochondria (mt), which are specialized organelles whose primary function is the conservation of oxidatively derived energy and its utilization for ATP synthesis, produce a large proportion of the energy that cells require and contain their own DNA. Plant mt genomes are large in size and complex in sequence organization. The rapid structural mt DNA changes observed in plants (Gray 1992) may cause duplication of genes and generation of pseudogenes and novel open reading frames (orfs) as well as re-shuffling of genes and their regulatory sequences. These phenomena cause diversity that may have evolutionary advantages during stress conditions. The study of plant mt genome evolution and genome expression may be facilitated by the analysis of mt mutants. One of the few mt mutations characterized in plants so far is the cytoplasmic male sterility (CMS) mutation (Hanson, Conde 1985), that is usually manifested by the abortion of normal pollen development and is commercially used for the efficient production of hybrid seed.

The gene copies for the alpha-subunit of the mitochondrial F1ATPase (atpA) were isolated and characterized in both male-fertile and cytoplasmic male sterile (CMS) petunia. Two copies, an intact gene and a truncated gene, were detected in both cytoplasms. The accumulated data, based upon a comparison of the sequences (the open reading frames as well as the 5' and 3' flanking regions) of the two atpA copies, both in male-fertile and CMS Petunia, indicate that: (1) they differ in their genomic organization and (2) a common progenitor cytoplasm, containing two copies of an intact atpA sequence, served as the origin for the atpA copies of the fertility and CMS-inducing cytoplasms. Homologous recombination through the progenitor intact atpA sequences is assumed to have caused the rearrangement in the 3' portion of the atpA open reading frame and the generation of the truncated atpA gene. It is thus suggested that the atpA pseudogenes, in both male-fertile and CMS cytoplasms, originated from a common progenitor atpA pseudogene sequence.

The main goal of the proposed research was to continue the mutual investigations into the molecular basis of CMS and male fertility restoration [MRF], with the ultimate goal of understanding these phenomena in higher plants. The experiments focused on: (1) dissecting apart the complex CMS - specific mitochondrial S-Pcf locus, in order to distinguish its essential parts which cause sterility from other parts and study its molecular evolution. (2) Studying the expression of the various regions of the S-Pcf locus in fertile and sterile lines and comparing the structure and ultrastructure of sterile and fertile tissues. (3) Determine whether alteration in respiration is genetically associated with CMS. Our mutual investigations further substantiated the association between the S-Pcf locus and CMS by the findings that the fertile phenotype of a population of unstable petunia somatic hybrids which contain the S-Pcf locus, is due to the presence of multiple muclear fertility restoration genes in this group of progenies. The information obtained by our studies indicate that homologous recombination played a major role in the molecular evolution of the S-Pcf locus and the CMS trait and in the generation of mitochondrial mutations in general. Our data suggest that the CMS cytoplasm evolved by introduction of a urs-s containing sublimon into the main mitochondrial genome via homologous recombination. We have also found that the first mutation detected so far in S-Pcf is a consequence of a homologous recombination mechanism involving part of the cox2 coding sequence. In all the cases studied by us, at the molecular level, we found that fusion of two different cells caused mitochondrial DNA recombination followed by sorting out of a specific mtDNA population or sequences. This sequence of events suggested as a mechanism for the generation of novel mitochondrial genomes and the creation of new traits. The present research also provides data concerning the expression of the recombined and complex CMS-specific S-Pcf locus as compared with the expression of additional mitochondrial proteins as well as comparative histological and ultrastructural studies of CMS and fertile Petunia. Evidence is provided for differential localization of mitochondrially encoded proteins in situ at the tissue level. The similar localization patterns of Pcf and atpA may indicate that Pcf product could interfere with the functioning of the mitochondrial ATPase in a tissue undergoing meiosis and microsporogenesis. Studies of respiration in CMS and fertile Petunia lines indicate that they differe in the partitioning of electron transport through the cytochrome oxidase and alternative oxidase pathways. The data indicate that the electron flux through the two oxidase pathways differs between mitochondria from fertile and sterile Petunia lines at certain redox states of the ubiquinone pool. In summary, extensive data concerning the CMS-specific S-Pcf locus of Petunia at the DNA and protein levels as well as information concerning different biochemical activity in CMS as compared to male fertile lines have been accumulated during the three years of this project. In addition, the involvement of the homologous recombination mechanism in the evolution of mt encoded traits is emphasized.

In Petunia, a mitochondrial (mt) locus, S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). The S-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF, termed Pcf, contains an unidentified reading frame urf-s that has been detected so far only in sterile Petunia lines and sterile somatic hybrids. In the study described here, a urf-s-related sequence was detected in seven different normal fertile Petunia lines and species as well as in additional members of the Solanaceae family by means of the polymerase chain reaction. The urf-s-related sequence identified in the fertile lines was termed orf152. In Petunia the nucleotide sequence of orf152 was found to be identical to the corresponding part of urf-s. However, the genome organization around orf152 was found to be different from that of urf-s. These results indicate that: (1) at least part of the urf-s sequence is present in fertile lines and species of Petunia and in other Solanaceae species; (2) the orf152 sequence of Petunia is not part of the Pcf ORF. The relevance of these findings to a better understanding of the evolution of the S-pcf locus in (S) cytoplasm in Petunia is discussed.

Single-stranded cDNA was prepared from RNA obtained from a patient with type 1 Gaucher disease. The cDNA was amplified in vitro and analyzed by sequencing. Three base-pair changes were identified which included a G to C transversion at nucleotide 3119 of the active gene (Asp140----His), an A to C transversion at nucleotide 3170 (Lys157----Gln) and a G to A change at nucleotide 5309 (Glu326----Lys). To study the mode of inheritance of the three different base-pair changes, genomic DNA was prepared from blood or skin fibroblasts of several family members. Genomic glucocerebrosidase DNA sequences were amplified and subjected to hybridization with allele-specific oligonucleotides (ASOs). The hybridization profiles demonstrated that two of the base-pair changes originated from the mother and were transmitted to her two affected sons and to a grandchild, while the third base-pair change, originating from the father, was transmitted to his two affected sons, a carrier daughter and a second grandchild. Tests of other patients with Gaucher disease failed to disclose the presence of the three base-changes. This is a unique family with three base-pair changes tightly linked to Gaucher disease.

The system responsible for the oxidative burst (OB) activity in Paracentrotus lividus eggs is different from those described earlier for other sea urchin species. The OB in P. lividus is associated with a soluble fraction resulting from centrifugation at 150,000 g . A low-molecular-weight, -SH-containing molecule present in this supernatant is required for the OB activity. The sulfhydryl reagent N -ethylmaleimide completely inhibits the calcium stimulated OB activity of intact eggs in the presence of the calcium ionophore A23187, suggesting that this requirement of low-molecular-weight -SH-containing molecules for OB exists also in vivo .

Exposing pepper (Capsicum annuum) plants to extremely high day temperatures (HDT) (day/night temperatures of 36 ± 2/10 ± 2°C), obtained by keeping the greenhouse closed during the day to exploit solar heating, prevented the development of low night temperature (LNT) symptoms. Plants of cultivars Fiesta and Selica grown under LNTs (10 ± 2°C) and moderate day temperatures (25 ± 2°C) during winter exhibited retarded growth, reduced leaf numbers, and deformed fruits with few or no seeds. LNT caused a reduction in the number and quality of pollen grains: the reduction in pollen quality was associated with reduced starch accumulation in pollen grains at 3 days before anthesis (DBA) and a decrease of more than two-fold in total soluble sugars in the mature pollen grains. This inhibitory effect was associated with more than 50% reduction in the enzymatic activities of the cell wall-bound and soluble acid invertases that catalyze the hydrolysis of incoming sucrose molecules. All these symptoms were prevented by HDT treatment which matched the vegetative and reproductive performance of the plants to those of plants grown under optimal night temperature (ONT) conditions (day/night temperatures of 23 ± 2/18 ± 2°C). HDT also prevented the inhibitory effect of LNT on enzymatic activities of both invertases in pollen at 5 DBA and brought about the accumulation of high levels of starch in pollen at 3 DBA. The results presented could support the development of a novel procedure for producing greenhouse crops with minimum or even with no fuel consumption for heating during the winter nights in regions with bright and sunny days.

We measured the mannose-specific adherence of radiolabeled Escherichia coli, carrying type 1 fimbriae, to monolayers of wild-type baby hamster kidney (BHK) cells and to 3 ricin-resistant mutants defective in the synthesis of complex N-linked oligosaccharide units. RicR14, a mutant accumulating N-linked oligomannose units in its glycoproteins at the expense of complex (N-acetyllactosamine) units, bound the largest number of bacteria, about 4 times more than the wild-type cells. The mutant cells in suspension were also readily agglutinated by the bacteria, while no agglutination of wild-type cells occurred under the conditions used. RicR21, a mutant which accumulates hybrid structures, bound about twice as many bacteria as wild-type cells, and was agglutinated by the bacteria to a lesser extent than RicR14. Binding and agglutination of RicR19, also presumed to accumulate hybrid structures, were the same as those of RicR14. These results provide evidence that oligomannose and hybrid units of cell surface glycoproteins serve as preferred receptors for mannose-specific E. coli. Lectin-resistant mutants are therefore useful for the investigation of sugar-specific adherence.

The sugar specificity of Escherichia coli 346 and of the type-1 fimbriae isolated from this organism has been studied by quantitative inhibition of the agglutination of mannan-containing yeast cells. The best inhibitors of the agglutination by the bacteria were the oligosaccharides Manα1→6[Manα1→3]Manα1→6[Manα1→2Manα1→3]ManαOMe, Manα1→6[Manα1→3]Manα1→6[Manα1→3]ManαOMe and Manα1→3Manβ1→4GlcNAc, and the aromatic glycoside p-nitrophenyl α- d -mannoside, all of which were 20–30 times more inhibitory than methyl α- d -mannoside. The disaccharides Manα1→3Man, Manα1→2Man and Manα1→6Man, the tetrasaccharide Manα1→2Manα1→3Manβ1→4GlcNAc and the pentasaccharide Manα1→2Manα1→2Manα1→3Manβ1→4GlcNAc, were all poor inhibitors. A very good correlation was found between the relative inhibitory activity of the different sugars tested with intact bacteria and with the isolated fimbriae. Our findings show that the combining site of the E. coli lectin is an extended one, corresponding to the size of a trisaccharide, that it contains a hydrophobic region, and that it is in the form of a pocket on the surface of the lectin. The combining site fits best the structures found in short oli gomannosidic chains present in N-glycosidically linked glycoproteins.

A large number of linear and branched oligosaccharides and several glycosides of D-mannose were tested for their inhibitory activity on the agglutination of yeast cells or guinea pig erythrocytes by three D-mannose-specific enteric bacteria possessing type 1 fimbriae. With Escherichia coli 346, the best inhibitors found are the alpha glycosides of the branched oligosaccharides alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-alpha-D-Manp-(1 leads to 6)-alpha-D-Manp-(1 leads to 3)-D-Manp and alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-alpha-D-Manp- (1 leads to 6)-[alpha-D-Manp-(1 leads to 2)-alpha-D-Manp-(1 leads to 3) ]-D-Manp and the trisaccharide alpha-D-Manp-(1 leads to 3)-beta-D-Manp-(1 leads to 4)-D-GlcNAc, all of which are 21-30 times more inhibitory than methyl alpha-D-mannopyranoside. The aromatic glycoside p-nitrophenyl alpha-D-mannopyranoside was also a strong inhibitor (30 times more inhibitory than methyl alpha-D-mannopyranoside), whereas the corresponding beta-D-glycoside was only a weak inhibitor (approximately as methyl alpha-D-mannopyranoside). A nearly identical pattern of inhibitory activity was observed with the fimbriae. This suggests that the combining site of the E. coli fimbrial lectin is in the form of an extended pocket on the surface of the lectin corresponding to the size of a trisaccharide and fitting best the structure alpha-D-Manp-(1 leads to 3)-beta-D-Manp-(1 leads to 4)-D-GlcNAc. Since p-nitrophenyl alpha-D-mannopyranoside is a strong inhibitor, the existence of a hydrophobic region in the combining site or close to it was assumed. The combining site of the Klebsiella pneumoniae fimbrial lectin is probably similar to that of E. coli, but that of the Salmonella typhimurium fimbrial lectin differs considerably. It appears that the combining sites of the three bacterial lectins tested exhibit preference for structures found in N-glycosylic oligomannoside units of mammalian cell surface glycoproteins.

This study characterized the influence of nitrogen (N) rates and variation in local availability on root architecture as measured by lateral root (LR) development attributes during the onset of the storage root (SR) initiation stage in ‘Beauregard’ sweetpotato adventitious roots (ARs). In N rate experiments, plants grown without fertilizer N showed significantly lower values for all measured LR attributes compared with fertilized plants. Total first- (1LR) and second-order LR (2LR) length increased by 78% and 2873%, respectively, as N was increased from 0 to 50 kg·ha−1. Total 1LR and 2LR number increased by 32% and 1465%, respectively. Increasing the N rates to 100 and 200 kg·ha−1 did not result in further increases for all LR attributes measured. There were no differences in AR number between untreated controls and plants fertilized with 50 kg N/ha. However, the number of ARs increased by 65% when fertilizer N was increased from 50 to 100 kg·ha−1. Increasing the rate to 200 kg·ha−1 did not result in further increases in AR number. In split-root experiments, roots grown in the compartment with 50 kg N/ha had 135% and 2916% increase in total 1LR and 2LR length, respectively, compared with roots grown in the compartment without fertilizer N. Total 1LR and 2LR number increased by 110% and 2114%, respectively. There were 111% more ARs in the fertilized compartment relative to the unfertilized compartment. There were no differences in LR attributes and AR number between compartments that received similar fertilizer N rates. In fertilizer placement experiments, there were no differences in LR attributes between pre-mixing fertilizer N and placement of fertilizer ≈4 cm below the surface of the growth substrate. There were also no differences between the unfertilized control and placement of fertilizer ≈4 cm from the bottom of the pot. Plants grown in substrate with pre-mixed N showed 38% and 342% increase in 1LR and 2LR length, respectively, relative to the bottom placement of N. Total number of 1LR and 2LR in the growth substrate with pre-mixed N increased by 30% and 312%, respectively, relative to the bottom placement of N. These results represent the first evidence for the association between sweetpotato root architectural attributes and variation in N rate and localized availability. These results are also consistent with findings in model systems in which local N presence is necessary for LR development. This information can be used to further optimize SR yield by helping to ensure the availability of N at the optimum rate across time and space.

Background The number of fibrous roots that develop into storage roots determines sweetpotato yield. The aim of the present study was to identify the molecular mechanisms involved in the initiation of storage root formation, by performing a detailed transcriptomic analysis of initiating storage roots using next-generation sequencing platforms. A two-step approach was undertaken: (1) generating a database for the sweetpotato root transcriptome using 454-Roche sequencing of a cDNA library created from pooled samples of two root types: fibrous and initiating storage roots; (2) comparing the expression profiles of initiating storage roots and fibrous roots, using the Illumina Genome Analyzer to sequence cDNA libraries of the two root types and map the data onto the root transcriptome database. Results Use of the 454-Roche platform generated a total of 524,607 reads, 85.6% of which were clustered into 55,296 contigs that matched 40,278 known genes. The reads, generated by the Illumina Genome Analyzer, were found to map to 31,284 contigs out of the 55,296 contigs serving as the database. A total of 8,353 contigs were found to exhibit differential expression between the two root types (at least 2.5-fold change). The Illumina-based differential expression results were validated for nine putative genes using quantitative real-time PCR. The differential expression profiles indicated down-regulation of classical root functions, such as transport, as well as down-regulation of lignin biosynthesis in initiating storage roots, and up-regulation of carbohydrate metabolism and starch biosynthesis. In addition, data indicated delicate control of regulators of meristematic tissue identity and maintenance, associated with the initiation of storage root formation. Conclusions This study adds a valuable resource of sweetpotato root transcript sequences to available data, facilitating the identification of genes of interest. This resource enabled us to identify genes that are involved in the earliest stage of storage root formation, highlighting the reduction in carbon flow toward phenylpropanoid biosynthesis and its delivery into carbohydrate metabolism and starch biosynthesis, as major events involved in storage root initiation. The novel transcripts related to storage root initiation identified in this study provide a starting point for further investigation into the molecular mechanisms underlying this process.