Grafting in Hylocereus (Cactaceae) as a tool for strengthening tolerance to high temperature stress.

The Hylocereus species that are grown as exotic fruit crops are very often farmed under marginal agronomic conditions, which may include exposure to high temperatures. Here we present a pioneering investigation of grafting as an agro-technique to improve heat tolerance in Hylocereus. To this end, we studied the diploid species H. undatus, the tetraploid H. megalanthus and its di-haploid gamete-derived line 2719, and the interspecific-interploid tetraploid Z-10, all grafted onto H. undatus as the rootstock. Self-grafted, grafted and non-grafted plants were acclimated for one week (to obtain baseline values) and then exposed to heat stress (45/35 °C day/night) for three days, followed by a one-week recovery period under optimal temperatures (30/22 °C). A comparison of the physiological, biochemical and molecular performances of the grafted and self-grafted plants under heat stress and during the recovery period vs those of non-stressed plants (control; 30/22 °C) showed that the grafted and self-grafted plants performed better in most of the assessments: grafted and self-grafted plants recovered more rapidly from the heat stress and suffered far less stem damage. An unexpected - but important - finding that may have implications for other crop was that the self-grafted plants showed better performance than non-grafted plants throughout the trial. Our findings provide support for grafting as a strategy for coping with the stress induced by extremely high temperatures. This study thus paves the way for further investigations of grafting in Hylocereus as a valuable technique that will maintain crop productivity in the face of increasing worldwide temperatures.

Performance of Hylocereus (Cactaceae) species and interspecific hybrids under high-temperature stress.

High temperatures limit the successful cultivation of the Hylocereus species on a global basis. We aimed to investigate the degree of heat tolerance in three species, namely, the diploids Hylocereus undatus and H. monacanthus, and the tetraploid H. megalanthus, and nine of their interspecific-interploid hybrids. Rooted cuttings were exposed to heat stress (45/35 °C) or control conditions (25/20 °C) for eight days. Initially, the plants were screened for their tolerance to heat stress and ranked into four heat tolerance categories: good tolerance, moderate tolerance, low tolerance, or sensitive, according to the decrease in the maximum quantum efficiency of photosystem II (Fv/Fm) and visual stem damage. The physiological and biochemical performances of the parental species and of three hybrids representing three different heat-tolerance categories were further analyzed in depth. H. megalanthus (classified as heat sensitive) showed a 65% decrease in Fv/Fm and severe visual stem damage, along with a marked reduction in total chlorophyll content, a large increase in malondialdehyde, and inhibition of catalase activity. H. undatus and H. monacanthus, (classified as low-tolerance species) exhibited slight stem "liquification." The good-tolerance hybrid Z-16 exhibited the best performance under heat stress (21% decrease in Fv/Fm) and the absence of stem damage, coupled with a small decrease in total chlorophyll content, a slight increase in malondialdehyde, high antioxidant activity, and proline accumulation progressing with time. Our findings revealed that most of the hybrids performed better than their parental species, indicating that our breeding programs can provide Hylocereus cultivars suitable for cultivation in heat-challenging regions.

Expression of Hexokinase in Stomata of Citrus Fruit Reduces Fruit Transpiration and Affects Seed Development.

The temporal formation and spatial distribution of stomata on the surface of citrus floral organs and, specifically, on the ovule from which the fruit develops, were analyzed using citrus plants that express green fluorescent protein (GFP) under the guard cell-specific KST1 promoter. Stomata are found on the style, sepal, and anther of the closed flower and on ovules from the stage of anthesis. It has previously been shown that hexokinase (HXK) mediates sugar-sensing in leaf guard cells and stimulates stomatal closure. The activity and response of citrus fruit stomata to sugar-sensing by HXK was examined using plants that express HXK under the KST1 promoter. Those plants are referred to as GCHXK plants. The transpiration of young green GCHXK citrus fruits was significantly reduced, indicating that their stomata respond to sugar similar to leaf stomata. Toward fruit maturation, fruit stomata are plugged and stop functioning, which explains why WT and GCHXK mature yellow fruits exhibited similar water loss. Seeds of the GCHXK plants were smaller and germinated more slowly than the WT seeds. We suggest that the stomata of young green citrus fruits, but not mature yellow fruits, respond to sugar levels via HXK and that fruit stomata are important for proper seed development.

Impact of scion/rootstock reciprocal effects on metabolomics of fruit juice and phloem sap in grafted Citrus reticulata.

BACKGROUND: Rootstock has a significant impact on plant growth and development, including fruit maturation. However, the existence of mutual interaction between scion and rootstock is often neglected. To explore the origin of different fruit quality traits in citrus, we studied the effect of rootstock and the reciprocal interaction between scion and rootstock of nine combinations; three mandarin varieties grafted on three different rootstocks. We analyzed the metabolic profile of juice via gas and liquid chromatography-mass spectrometry (GC-MS and LC-MS, respectively). Additionally, we profiled phloem sap composition in the scion and the rootstock. Quality traits of fruit and their physio-chemical characteristics were also evaluated. RESULTS: For all three cultivars, rootstock was found to affect fruit yield and biochemical fruit quality parameters (sugar and acidity) in interactions with the scions. In mandarin juice, eight of 48 compounds (two primary and six secondary) were related directly to the rootstock, and another seven (one primary and six secondary) were interactively affected by scion and rootstock. In scion and rootstock sap, six and 14 of 53 and 55 primary metabolites, respectively, were directly affected by the rootstock, while 42 and 33 were affected by rootstock interactively with scion, respectively. CONCLUSION: In this work, we show for the first time a reciprocal effect between rootstock and scion. Based on our results, the scion and rootstock interaction might be organ, distance or time dependent.

Expression of Arabidopsis Hexokinase in Tobacco Guard Cells Increases Water-Use Efficiency and Confers Tolerance to Drought and Salt Stress.

Abiotic stresses such as drought and saline water impose major limitations on plant growth. Modulation of stomatal behavior may help plants cope with such stresses by reducing both water loss and salt uptake. Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in guard cells' sugar-sensing, mediating stomatal closure and coordinating photosynthesis with transpiration. We generated transgenic tobacco lines expressing the Arabidopsis hexokinase1 (AtHXK1) under the guard cell-specific promoter KST1 and examined those plants using growth room and greenhouse experiments. The expression of AtHXK1 in tobacco guard cells reduced stomatal conductance and transpiration by about 25% with no negative effects on photosynthesis or growth, leading to increased water-use efficiency. In addition, these plants exhibited tolerance to drought and salt stress due to their lower transpiration rate, indicating that improved stomatal function has the potential to improve plant performance under stress conditions.

Biochar alleviates phytotoxicity in Ficus elastica grown in Zn-contaminated soil.

Zinc (Zn) immobilization by two distinct biochars in soil, together with concomitant alleviation of phytotoxic responses in Ficus elastica Roxb. ex Hornem., were examined. Rooted cuttings of F. elastica were grown in 880mgkg-1 Zn-spiked sandy soil amended with grain husk (GH) or cattle manure (CM) biochar at 0, 10, 30 and 50gkg-1 soil for a period of 6months. Addition of both GH and CM biochars had significant positive impacts on physiological parameters such as plant growth, leaf relative water content, photosynthetic pigments and leaf gas exchange characteristics. The responses to addition of CM biochar were significantly better than to GH biochar. Lipid peroxidation declined in leaves of plants grown in Zn-contaminated, biochar-amended soil. This was confirmed by luminescence and Fourier transform infrared analysis of the leaf material. Biochar significantly reduced the availability of soil Zn, as evidenced by lower concentrations of Zn in leaves and leachates of biochar treated plants relative to control plants. These findings show that biochar can effectively immobilize soil Zn, and as a result, alleviate Zn phytotoxicity by reducing its uptake and accumulation in the plant. Adding biochar to soils contaminated with metals thus holds promise as a means of restoring blighted lands.

Dormancy release and flowering time in Ziziphus jujuba Mill., a "direct flowering" fruit tree, has a facultative requirement for chilling.

In deciduous fruit trees, the effect of chilling on flowering has mostly been investigated in the "indirect flowering" group, characterized by a period of rest between flower bud formation and blooming. In the present study, we explored the effects of chilling and chilling deprivation on the flowering of Ziziphus jujuba, a temperate deciduous fruit tree belonging to the "direct flowering" group, in which flower bud differentiation, blooming and fruit development occur after dormancy release, during a single growing season. Dormancy release, vegetative growth and flowering time in Z. jujuba cv. Ben-Li were assessed following several treatments of chilling. Chilling treatments quantitatively decreased the timing of vegetative bud dormancy release, thereby accelerating flowering, but had no effect on the time from dormancy release to flowering. Trees grown at a constant temperature of 25°C, without chilling, broke dormancy and flowered, indicating the facultative character of chilling in this species. We measured the expression of Z. jujuba LFY and AP1 homologues (ZjLFY and ZjAP1). Chilling decreased ZjLFY expression in dormant vegetative buds but had no effect on ZjAP1expression, which reached peak expression before dormancy release and at anthesis. In conclusion, chilling is not obligatory for dormancy release of Z. jujuba cv. Ben-Li vegetative buds. However, the exposure to chilling during dormancy does accelerate vegetative bud dormancy release and flowering.

Non-structural carbohydrates in woody plants compared among laboratories.

Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g(-1) for soluble sugars, 6-533 (mean = 94) mg g(-1) for starch and 53-649 (mean = 153) mg g(-1) for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R(2) = 0.05-0.12 for soluble sugars, 0.10-0.33 for starch and 0.01-0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g(-1) for total NSC, compared with the range of laboratory estimates of 596 mg g(-1). Laboratories were reasonably consistent in their ranks of estimates among tissues for starch (r = 0.41-0.91), but less so for total NSC (r = 0.45-0.84) and soluble sugars (r = 0.11-0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory may be comparable within and between laboratories, especially for starch. To obtain comparable NSC estimates, we suggest that users can either adopt the reference method given in this publication, or report estimates for a portion of samples using the reference method, and report estimates for a standard reference material. Researchers interested in NSC estimates should work to identify and adopt standard methods.

Expression of Arabidopsis Hexokinase in Citrus Guard Cells Controls Stomatal Aperture and Reduces Transpiration.

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species.

Climate and soil attributes determine plant species turnover in global drylands.

AIM: Geographic, climatic, and soil factors are major drivers of plant beta diversity, but their importance for dryland plant communities is poorly known. This study aims to: i) characterize patterns of beta diversity in global drylands, ii) detect common environmental drivers of beta diversity, and iii) test for thresholds in environmental conditions driving potential shifts in plant species composition. LOCATION: 224 sites in diverse dryland plant communities from 22 geographical regions in six continents. METHODS: Beta diversity was quantified with four complementary measures: the percentage of singletons (species occurring at only one site), Whittake's beta diversity (ß(W)), a directional beta diversity metric based on the correlation in species occurrences among spatially contiguous sites (ß(R2)), and a multivariate abundance-based metric (ß(MV)). We used linear modelling to quantify the relationships between these metrics of beta diversity and geographic, climatic, and soil variables. RESULTS: Soil fertility and variability in temperature and rainfall, and to a lesser extent latitude, were the most important environmental predictors of beta diversity. Metrics related to species identity (percentage of singletons and ß(W)) were most sensitive to soil fertility, whereas those metrics related to environmental gradients and abundance ((ß(R2)) and ß(MV)) were more associated with climate variability. Interactions among soil variables, climatic factors, and plant cover were not important determinants of beta diversity. Sites receiving less than 178 mm of annual rainfall differed sharply in species composition from more mesic sites (> 200 mm). MAIN CONCLUSIONS: Soil fertility and variability in temperature and rainfall are the most important environmental predictors of variation in plant beta diversity in global drylands. Our results suggest that those sites annually receiving ~ 178 mm of rainfall will be especially sensitive to future climate changes. These findings may help to define appropriate conservation strategies for mitigating effects of climate change on dryland vegetation.

Phenotypic plasticity and water flux rates of Citrus root orders under salinity.

Knowledge about the root system structure and the uptake efficiency of root orders is critical to understand the adaptive plasticity of plants towards salt stress. Thus, this study describes the phenological and physiological plasticity of Citrus volkameriana rootstocks under severe NaCl stress on the level of root orders. Phenotypic root traits known to influence uptake processes, for example frequency of root orders, specific root area, cortical thickness, and xylem traits, did not change homogeneously throughout the root system, but changes after 6 months under 90 mM NaCl stress were root order specific. Chloride accumulation significantly increased with decreasing root order, and the Cl(-) concentration in lower root orders exceeded those in leaves. Water flux densities of first-order roots decreased to <20% under salinity and did not recover after stress release. The water flux densities of higher root orders changed marginally under salinity and increased 2- to 6-fold in second and third root orders after short-term stress release. Changes in root order frequency, morphology, and anatomy indicate rapid and major modification of C. volkameriana root systems under salt stress. Reduced water uptake under salinity was related to changes of water flux densities among root orders and to reduced root surface areas. The importance of root orders for water uptake changed under salinity from root tips towards higher root orders. The root order-specific changes reflect differences in vulnerability (indicated by the salt accumulation) and ontogenetic status, and point to functional differences among root orders under high salinity.

Evidence that differential gene expression between the halophyte, Thellungiella halophila, and Arabidopsis thaliana is responsible for higher levels of the compatible osmolyte proline and tight control of Na+ uptake in T. halophila.

Salt-sensitive glycophytes and salt-tolerant halophytes employ common mechanisms to cope with salinity, and it is hypothesized that differences in salt tolerance arise because of changes in the regulation of a basic set of salt tolerance genes. We explored the expression of genes involved in two key salt tolerance mechanisms in Arabidopsis thaliana and the halophytic A. thaliana relative model system (ARMS), Thellungiella halophila. Salt overly sensitive 1 (SOS1) is a plasma membrane Na+/H+ antiporter that retrieves and loads Na+ ions from and into the xylem. Shoot SOS1 transcript was more strongly induced by salt in T. halophila while root SOS1 was constitutively higher in unstressed T. halophila. This is consistent with a lower salt-induced rise in T. halophila xylem sap Na+ concentration than in A. thaliana. Thellungiella halophila contained higher unstressed levels of the compatible osmolyte proline than A. thaliana, while under salt stress, T. halophila accumulated more proline mainly in shoots. Expression of the A. thaliana ortholog of proline dehydrogenase (PDH), involved in proline catabolism, was undetectable in T. halophila shoots. The PDH enzyme activity was lower and T. halophila seedlings were hypersensitive to exogenous proline, indicating repression of proline catabolism in T. halophila. Our results suggest that differential gene expression between glycophytes and halophytes contributes to the salt tolerance of halophytes.