Exploring Thrips Preference and Resistance in Flowers, Leaves, and Whole Plants of Ten Capsicum Accessions.

Capsicum species grown for pepper production suffer severely from thrips damage, urging the identification of natural resistance. Resistance levels are commonly assessed on leaves. However, Capsicum plants are flower-bearing during most of the production season, and thrips also feed on pollen and flower tissues. In order to obtain a comprehensive estimate of elements contributing to thrips resistance, flower tissues should be considered as well. Therefore, we assessed resistance to Frankliniella occidentalis in flowers, leaves, and whole plants of ten Capsicum accessions. Using choice assays, we found that thrips prefer flowers of certain accessions over others. The preference of adult thrips for flowers was positively correlated to trehalose and fructose concentration in anthers as well as to pollen quantity. Resistance measured on leaf discs and thrips population development on whole plants was significantly and positively correlated. Leaf-based resistance thus translates to reduced thrips population development. Results of the flower assays were not significantly correlated with resistance in leaves or on whole plants. This suggests that both leaves and flowers represent a different part of the resistance spectrum and should both be considered for understanding whole plant resistance and the identification of resistant Capsicum varieties.

Low Salicylic Acid Level Improves Pollen Development Under Long-Term Mild Heat Conditions in Tomato.

Exposure to high temperatures leads to failure in pollen development, which may have significant implications for food security with ongoing climate change. We hypothesized that the stress response-associated hormone salicylic acid (SA) affects pollen tolerance to long-term mild heat (LTMH) (≥14 days exposure to day-/nighttime temperature of 30-34/24-28°C, depending on the genotype), either positively, by inducing acclimation, or negatively, by reducing investment in reproductive development. Here, we investigated these hypotheses assessing the pollen thermotolerance of a 35S:nahG tomato line, which has low SA levels. We found that reducing the SA level resulted in increased pollen viability of plants grown in LTMH and further characterized this line by transcriptome, carbohydrate, and hormone analyses. Low expression of JAZ genes in 35S:nahG and LTMH hypersensitivity of low-jasmonic acid (JA) genotypes together suggest that the increased pollen thermotolerance in the low-SA line involves enhanced JA signal in developing anthers in LTMH. These findings have potential application in the development of more thermotolerant crops.

A temperature regime that disrupts clock-controlled starch mobilization induces transient carbohydrate starvation, resulting in compact growth.

In nature plants are usually subjected to a light/temperature regime of warm day and cold night (referred to as +DIF). Compared to growth under +DIF, Arabidopsis plants show compact growth under the same photoperiod, but with an inverse temperature regime (cold day and warm night: -DIF). Here we show that -DIF differentially affects the phase and amplitude of core clock gene expression. Under -DIF the phase of the morning clock gene CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) is delayed, similar to that of plants grown on low sucrose. Indeed, under -DIF carbohydrate (CHO) starvation marker genes are specifically upregulated at the End of the Night (EN) in Arabidopsis rosettes. However, only in inner-rosette tissue (small sink leaves and petioles of older leaves) sucrose levels are lower under -DIF compared to under +DIF, suggesting that sucrose in source leaf blades is not sensed for CHO status and that sucrose transport from source to sink may be impaired at EN. CHO-starvation under -DIF correlated with increased starch breakdown during the night and decreased starch accumulation during the day. Moreover, we demonstrate that different ways of inducing CHO-starvation all link to reduced growth of sink leaves. Practical implications for control of plant growth in horticulture are discussed.

Increased arbuscular mycorrhizal fungal colonization reduces yield loss of rice (Oryza sativa L.) under drought.

Drought reduces the availability of soil water and the mobility of nutrients, thereby limiting the growth and productivity of rice. Under drought, arbuscular mycorrhizal fungi (AMF) increase P uptake and sustain rice growth. However, we lack knowledge of how the AMF symbiosis contributes to drought tolerance of rice. In the greenhouse, we investigated mechanisms of AMF symbiosis that confer drought tolerance, such as enhanced nutrient uptake, stomatal conductance, chlorophyll fluorescence, and hormonal balance (abscisic acid (ABA) and indole acetic acid (IAA)). Two greenhouse pot experiments comprised three factors in a full factorial design with two AMF treatments (low- and high-AMF colonization), two water treatments (well-watered and drought), and three rice varieties. Soil water potential was maintained at 0 kPa in the well-watered treatment. In the drought treatment, we reduced soil water potential to - 40 kPa in experiment 1 (Expt 1) and to - 80 kPa in experiment 2 (Expt 2). Drought reduced shoot and root dry biomass and grain yield of rice in both experiments. The reduction of grain yield was less with higher AMF colonization. Plants with higher AMF colonization showed higher leaf P concentrations than plants with lower colonization in Expt 1, but not in Expt 2. Plants with higher AMF colonization exhibited higher stomatal conductance and chlorophyll fluorescence than plants with lower colonization, especially under drought. Drought increased the levels of ABA and IAA, and AMF colonization also resulted in higher levels of IAA. The results suggest both nutrient-driven and plant hormone-driven pathways through which AMF confer drought tolerance to rice.

Proximal and Distal Parts of Sweetpotato Adventitious Roots Display Differences in Root Architecture, Lignin, and Starch Metabolism and Their Developmental Fates.

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.

Gibberellin Promotes Sweetpotato Root Vascular Lignification and Reduces Storage-Root Formation.

Sweetpotato 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 sweetpotato orthologues of Arabidopsis vascular development regulators (IbNA075, IbVND7, and IbSND2) and of lignin biosynthesis genes (IbPAL, IbC4H, Ib4CL, IbCCoAOMT, and IbCAD), while downregulating starch biosynthesis genes (IbAGPase and IbGBSS) in the roots. Interestingly, gibberellin downregulated 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 sweetpotato 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 sweetpotato storage-root formation and provide a valuable database of genes for further research.

Quantitative trait loci analysis of hormone levels in Arabidopsis roots.

Quantitative trait loci (QTL) analyses for five groups of hormones, including cytokinins in Arabidopsis roots were performed using recombinant inbred lines (Ler×Cvi). Significant QTLs were detected for cytokinins, jasmonic acid and salicylic acid. Separate analysis of two sub-populations, viz., vegetative and flowering plants revealed that many of the QTLs were development-specific. Using near-isogenic lines, several significant QTLs were confirmed; three co-localized QTL regions were responsible for determining several cytokinin metabolites. Using a knock-out plant, a functional role of zeatin N-glucosyltransferase gene (UGT76C2) underlying a large-effect QTL for levels of tZ-N-glucosides and tZRMP was evaluated in the metabolism of cytokinins. Pleotropic effects of this gene were found for cytokinin levels in both roots and leaves, but significant changes of morphological traits were observed only in roots. Hormone QTL analysis reveals development-specific and organ-dependent aspects of the regulation of plant hormone content and metabolism.

NADP-MALIC ENZYME 1 Affects Germination after Seed Storage in Arabidopsis thaliana.

Aging decreases the quality of seeds and results in agricultural and economic losses. The damage that occurs at the biochemical level can alter the seed physiological status. Although loss of viability has been investigated frequently, little information exists on the molecular and biochemical factors involved in seed deterioration and loss of viability. Oxidative stress has been implicated as a major contributor to seed deterioration, and several pathways are involved in protection against this. In this study, we show that seeds of Arabidopsis thaliana lacking a functional NADP-MALIC ENZYME 1 (NADP-ME1) have reduced seed viability relative to the wild type. Seeds of the NADP-ME1 loss-of-function mutant display higher levels of protein carbonylation than those of the wild type. NADP-ME1 catalyzes the oxidative decarboxylation of malate to pyruvate with the simultaneous production of CO2 and NADPH. Upon seed imbibition, malate and amino acids accumulate in embryos of aged seeds of the NADP-ME1 loss-of-function mutant compared with those of the wild type. NADP-ME1 expression is increased in imbibed aged as compared with non-aged seeds. NADP-ME1 activity at testa rupture promotes normal germination of aged seeds. In seedlings of aged seeds, NADP-ME1 is specifically active in the root meristematic zone. We propose that NADP-ME1 activity is required for protecting seeds against oxidation during seed dry storage.

A disturbed auxin signaling affects adventitious root outgrowth in Solanum dulcamara under complete submergence.

Flooding negatively affects the growth and even survival of most terrestrial plants. Upon flooding, the excess water quickly decreases the gas exchange between atmosphere and the submerged plant tissues, which leads to oxygen deficiency resulting in a plant cell energy crisis, and eventually plant death. Solanum dulcamara survives flooding by producing aerenchymatous adventitious roots (ARs) from pre-formed primordia on the stem, which replace the original flood-sensitive root system. However, we found that under complete submergence, AR outgrowth was impaired in S. dulcamara. In the present work, we tried to elucidate the mechanisms behind this phenomenon in particular the involvement of the phytohormones auxin, abscisic acid and jasmonic acid. Abscisic acid (ABA) is a negative regulator of AR outgrowth, but surprisingly the ABA content and signaling were decreased to a similar extent under both partial and complete submergence, suggesting that ABA might not be responsible for the difference in AR outgrowth. Auxin, which is necessary for AR outgrowth, was at similar concentrations in either partially or completely submerged primordia, but complete submergence resulted in a decrease of auxin signaling in the primordia. Application of 1-naphthaleneacetic acid (NAA) to completely submerged plants restored AR outgrowth, implying that auxin response in the rooting tissues of completely submerged plants was reduced. Furthermore, jasmonic acid (JA) concentrations did not differ between partial and complete submergence. To conclude, a disruption in the auxin signaling within S. dulcamara AR primordia may result in the abortion of AR outgrowth under complete submergence.

Role of Tulipa gesneriana TEOSINTE BRANCHED1 (TgTB1) in the control of axillary bud outgrowth in bulbs.

KEY MESSAGE: Tulip vegetative reproduction. Tulips reproduce asexually by the outgrowth of their axillary meristems located in the axil of each bulb scale. The number of axillary meristems in one bulb is low, and not all of them grow out during the yearly growth cycle of the bulb. Since the degree of axillary bud outgrowth in tulip determines the success of their vegetative propagation, this study aimed at understanding the mechanism controlling the differential axillary bud activity. We used a combined physiological and "bottom-up" molecular approach to shed light on this process and found that first two inner located buds do not seem to experience dormancy during the growth cycle, while mid-located buds enter dormancy by the end of the growing season. Dormancy was assessed by weight increase and TgTB1 expression levels, a conserved TCP transcription factor and well-known master integrator of environmental and endogenous signals influencing axillary meristem outgrowth in plants. We showed that TgTB1 expression in tulip bulbs can be modulated by sucrose, cytokinin and strigolactone, just as it has been reported for other species. However, the limited growth of mid-located buds, even when their TgTB1 expression is downregulated, points at other factors, probably physical, inhibiting their growth. We conclude that the time of axillary bud initiation determines the degree of dormancy and the sink strength of the bud. Thus, development, apical dominance, sink strength, hormonal cross-talk, expression of TgTB1 and other possibly physical but unidentified players, all converge to determine the growth capacity of tulip axillary buds.

Natural variation of hormone levels in Arabidopsis roots and correlations with complex root architecture.

Studies on natural variation are an important tool to unravel the genetic basis of quantitative traits in plants. Despite the significant roles of phytohormones in plant development, including root architecture, hardly any studies have been done to investigate natural variation in endogenous hormone levels in plants. Therefore, in the present study a range of hormones were quantified in root extracts of thirteen Arabidopsis thaliana accessions using a ultra performance liquid chromatography triple quadrupole mass spectrometer. Root system architecture of the set of accessions was quantified, using a new parameter (mature root unit) for complex root systems, and correlated with the phytohormone data. Significant variations in phytohormone levels among the accessions were detected, but were remarkably small, namely less than three-fold difference between extremes. For cytokinins, relatively larger variations were found for ribosides and glucosides, as compared to the free bases. For root phenotyping, length-related traits-lateral root length and total root length-showed larger variations than lateral root number-related ones. For root architecture, antagonistic interactions between hormones, for example, indole-3-acetic acid to trans-zeatin were detected in correlation analysis. These findings provide conclusive evidence for the presence of natural variation in phytohormone levels in Arabidopsis roots, suggesting that quantitative genetic analyses are feasible.

Auxin synthesis gene tms1 driven by tuber-specific promoter alters hormonal status of transgenic potato plants and their responses to exogenous phytohormones.

KEY MESSAGE: Ectopic auxin overproduction in transgenic potato leads to enhanced productivity accompanied with concerted and occasional changes in hormonal status, and causing altered response of transformants to exogenous auxin or cytokinin. Previously, we generated potato transformants expressing Agrobacterium-derived auxin synthesis gene tms1 driven by tuber-specific patatin gene promoter (B33-promoter). Here, we studied the endogenous hormonal status and the response to exogenous phytohormones in tms1 transformants cultured in vitro. Adding indole-3-acetic acid (IAA) or kinetin to culture medium affected differently tuberization of tms1-transformed and control plants, depending also on sucrose content in the medium. Exogenous phytohormones ceased to stimulate the tuber initiation in transformants at high (5-8%) sucrose concentration, while in control plants the stimulation was observed in all experimental settings. Furthermore, exogenous auxin partly inhibited the tuber initiation, and exogenous cytokinin reduced the average tuber weight in most transformants at high sucrose content. The elevated auxin level in tubers of the transformants was accompanied with a decrease in content of cytokinin bases and their ribosides in tubers and most shoots. No concerted changes in contents of abscisic, jasmonic, salicylic acids and gibberellins in tubers were detected. The data on hormonal status indicated that the enhanced productivity of tms1 transformants was due to auxin and not mediated by other phytohormones. In addition, exogenous cytokinin was shown to upregulate the expression of genes encoding orthologs of auxin receptors. Overall, the results showed that tms1 expression and local increase in IAA level in transformants affect both the balance of endogenous cytokinins and the dynamics of tuberization in response to exogenous hormones (auxin, cytokinin), the latter reaction depending also on the carbohydrate supply. We introduce a basic model for the hormonal network controlling tuberization.

Thermodynamic and structural properties of tuber starches from transgenic potato plants grown in vitro and in vivo.

Potato plants harboring Phytochrome B (PHYB) gene from Arabidopsis thaliana or rol genes from Agrobacterium rhizogenes were used to study the effect of transgene expression on structure and properties of starch in tubers. Thermodynamic characteristics of starch (melting temperature, enthalpy of melting, thickness of crystalline lamellae) were shown to be variable depending on the transgene expression and plant culturing mode: in vitro or in soil. The expression of rolB or rolC genes in in vitro cultured plants evoked opposite effects on starch melting temperature and crystalline lamellae thickness. AtPHYB or rolB expression in the soil-grown potato led to the formation of more defective or more ordered starch structures, respectively, in comparison with starches of the same lines grown in vitro. On the whole, our study revealed genotype-dependent differences between starches extracted from tubers of in vitro or in vivo grown plants.

Expression of auxin synthesis gene tms1 under control of tuber-specific promoter enhances potato tuberization in vitro.

Phytohormones, auxins in particular, play an important role in plant development and productivity. Earlier data showed positive impact of exogenous auxin on potato (Solanum tuberosum L.) tuberization. The aim of this study was to generate potato plants with increased auxin level predominantly in tubers. To this end, a pBinB33-tms1 vector was constructed harboring the Agrobacterium auxin biosynthesis gene tms1 fused to tuber-specific promoter of the class I patatin gene (B33-promoter) of potato. Among numerous independently generated B33:tms1 lines, those without visible differences from control were selected for detailed studies. In the majority of transgenic lines, tms1 gene transcription was detected, mostly in tubers rather than in shoots. Indoleacetic acid (IAA) content in tubers and the auxin tuber-to-shoot ratio were increased in tms1-expressing transformants. The organ-specific increase in auxin synthesis in B33:tms1-transformants accelerated and intensified the process of tuber formation, reduced the dose of carbohydrate supply required for in vitro tuberization, and decreased the photoperiodic dependence of tuber initiation. Overall, a positive correlation was observed between tms1 expression, IAA content in tubers, and stimulation of tuber formation. The revealed properties of B33:tms1 transformants imply an important role for auxin in potato tuberization and offer prospects to magnify potato productivity by a moderate organ-specific enhancement of auxin content.

Vacuolar invertase regulates elongation of Arabidopsis thaliana roots as revealed by QTL and mutant analysis.

The possible role of the sucrose-splitting enzymes sucrose synthase and invertase in elongating roots and hypocotyls of Arabidopsis was tested by using a combination of histochemical methods and quantitative trait locus (QTL) analysis. Lengths of roots and hypocotyls correlated better with invertase activities than with sucrose synthase activities. The highest correlations were observed with activities in the elongating zones of roots. The genetic basis of these correlations was studied by using QTL analysis. Several loci, affecting invertase activity, colocated with loci that had an effect on root or hypocotyl length. Further fine mapping of a major locus for root length, but not for hypocotyl length (top chromosome 1), consistently showed colocation with the locus for invertase activity containing a gene coding for a vacuolar invertase. The analysis of a functional knockout line confirmed the role of this invertase in root elongation, whereas other invertase genes might play a role in hypocotyl elongation. Thus, we show the power of QTL analysis, combined for morphological and biochemical traits, followed by fine-mapping and mutant analysis, in unraveling the function of genes and their role in growth and development.

Histochemical analysis reveals organ-specific quantitative trait loci for enzyme activities in Arabidopsis.

To identify genetic loci involved in the regulation of organ-specific enzyme activities, a specific histochemical staining protocol was used in combination with quantitative trait locus (QTL) analysis. Using phosphoglucomutase (PGM) as an example, it is shown that enzyme activity can specifically, and with high resolution, be visualized in non-sectioned seedlings of Arabidopsis. The intensities of staining were converted to quantitative data and used as trait for QTL analysis using Landsberg erecta x Cape Verde Islands recombinant inbred lines. Independently, PGM activities were quantified in whole-seedling extracts, and these data were also used for QTL analysis. On the basis of extract data, six significant (P < 0.05) loci affecting PGM activity were found. From the histochemical data, one or more specific QTLs were found for each organ analyzed (cotyledons, shoot apex, hypocotyl, root, root neck, root tip, and root hairs). Loci detected for PGM activity in extracts colocated with loci for histochemical staining. QTLs were found coinciding with positions of (putative) PGM genes but also at other positions, the latter ones supposedly pointing toward regulatory genes. Some of this type of loci were also organ specific. It is concluded that QTL analysis based on histochemical data is feasible and may reveal organ-specific loci involved in the regulation of metabolic pathways.

In situ analysis of enzymes involved in sucrose to hexose-phosphate conversion during stolon-to-tuber transition of potato.

An in situ study of enzymes involved in sucrose to hexose-phosphate conversion during in vitro stolon-to-tuber transition of potato (Solanum tuberosum L. cv. Bintje) was employed to follow developmental changes in spatial patterns. In situ activity of the respective enzymes was visualized by specific activity-staining techniques and they revealed distinct spatially and developmentally regulated patterns. Two of the enzymes studied were also subject to in situ investigations at the transcriptional level. During the stages of stolon formation high hexokinase (EC 2.7.1.1) and acid (cell wall-bound) invertase (EC 3.2.1.26) activities were restricted to the mitotically active (sub)apical region, suggesting a possible importance of these enzymes for cell division. At the onset of tuberization sucrose synthase (EC 2.4.1.13) and fructokinase (EC 2.7.1.4) were strongly induced (visualized at transcriptional and translational level) and the acid invertase activities disappeared from the swelling subapical region as expected. The high degree of similarity in the spatial pattern and the temporal induction of sucrose synthase and fructokinase suggests a tightly co-ordinated coarse (up)regulation, which may be subject to a sugar-modulated mechanism(s) by which genes involved in the metabolic sucrose-starch converting potential are co-ordinately regulated during tuber growth. The overall activity of uridine-5-diphosphoglucose pyrophosphorylase (EC 2.7.7.9) was present in all tissues during stolon and tuber development, implying that its coarse control is not subject to (in)direct developmental regulation.

In situ staining of activities of enzymes involved in carbohydrate metabolism in plant tissues.

A powerful technique is described to localize the activities of a range of enzymes in a wide variety of plant tissues. The method is based on the coupling of the enzymatic reaction to the reduction of NAD and subsequent reduction and precipitation of nitroblue tetrazolium. Enzymes that did not reduce NAD could be visualized by coupling their activities to glucose-6-phosphate dehydrogenase activity via one or more intermediary 'coupling' enzymes. The method is shown to be applicable for the detection of the activities of hexokinase, fructokinase, sucrose synthase, uridine 5'-diphospho-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase, phosphoglucomutase, and phosphoglucose isomerase. It could be used for all tissues tested, including green leaves, stems, roots, fruits, and seeds. The method is specific, very sensitive, and has a high spatial resolution, giving information at the cellular and the subcellular level. The localization of sucrose synthase, invertase, and uridine 5'-diphospho-glucose pyrophosphorylase in transgenic potato plants, carrying a cytokinin biosynthesis gene, is studied and compared with wild-type plants.