Burkholderia phytofirmans PsJN acclimates grapevine to cold by modulating carbohydrate metabolism

Low temperatures damage many temperate crops, including grapevine, which, when exposed to chilling, can be affected by symptoms ranging from reduced yield up to complete infertility. We have previously demonstrated that Burkholderia phytofirmans PsJN, a plant growth-promoting rhizobacteria (PGPR) that colonizes grapevine, is able to reduce chilling-induced damage. We hypothesized that the induced tolerance may be explained at least partly by the impact of bacteria on grapevine photosynthesis or carbohydrate metabolism during cold acclimation. To investigate this hypothesis, we monitored herein the fluctuations of photosynthesis parameters (net photosynthesis [P(n)], intercellular CO(2) concentration, stomatal conductances, ΦPSII, and total chlorophyll concentration), starch, soluble sugars (glucose, fructose, saccharose, mannose, raffinose, and maltose), and their precursors during 5 days of chilling exposure (4°C) on grapevine plantlets. Bacterization affects photosynthesis in a non-stomatal dependent pattern and reduced long-term impact of chilling on P(n). Furthermore, all studied carbohydrates known to be involved in cold stress tolerance accumulate in non-chilled bacterized plantlets, although some of them remained more concentrated in the latter after chilling exposure. Overall, our results suggest that modification of carbohydrate metabolism in bacterized grapevine plantlets may be one of the major effects by which this PGPR reduces chilling-induced damage.     

Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26 degrees C) and low (4 degrees C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26 degrees C and 4 degrees C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26 degrees C and 4 degrees C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO(2) fixation and O(2) evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.     

Enhancement of Chilling Resistance of Inoculated Grapevine Plantlets with a Plant Growth-Promoting Rhizobacterium, Burkholderia phytofirmans Strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26°C) and low (4°C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26°C and 4°C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26°C and 4°C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO₂ fixation and O₂ evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.     

Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera co-cultured with plant growth-promoting rhizobacteria

The potential of a plant growth-promoting rhizobacterium, Pseudomonas sp. (strain PsJN), to stimulate the growth and enhancement of the resistance of grapevine (Vitis vinifera L.) transplants to gray mould caused by Botrytis cinerea has been investigated. In vitro inoculation of grapevine plantlets induced a significant plant growth promotion which made them more hardy and vigorous when compared to non-inoculated plantlets. This ability increased upon transplanting. When grown together with B. cinerea, the causal agent of gray mould, significant differences of aggressiveness were observed between the inoculated and non-inoculated plants. The presence of bacteria was accompanied by an induction of plant resistance to the pathogen. The beneficial effect from this plant-microbe association is being postulated.     

Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.     

Endophytic Colonization of Vitis vinifera L. by Plant Growth-Promoting Bacterium Burkholderia sp. Strain PsJN

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.     

Biochemical and physiological mechanisms related to cold acclimation and enhanced freezing tolerance in poplar plantlets

Temperature is one of the abiotic factors limiting growth and productivity of plants. In the present work, the effect of low non-freezing temperature, as inducer of 'cold acclimation', was studied in poplar. Actively growing plantlets of Populus tremula  ×  Populus tremuloides cv. Muhs 1 were used, and cold treatment consisted in whole plants exposure to 4°C in controlled conditions. Leaves of cold-treated poplars were shown to be acclimated, as an increase of their freezing tolerance was measured using electrolyte leakage. Chlorophyll fluorescence measurements revealed a decrease in photosystem II efficiency while the pigment contents of leaves did not vary. In contrast, after 1 week of cold exposure, an accumulation of pigments was noted in the stems near the apex of the stressed plants as confirmed by chromatographic analyses. Simultaneously, a rapid accumulation of osmoprotectants, i.e. carbohydrates (measured by spectrometry), and of stress indicators (e.g. putrescine) occurred; changes in protein patterns also arose. Indeed, Western blot studies revealed that the expression of three families of stress-related proteins, i.e. dehydrins, stress protein 1 and heat-shock protein 70, was activated or induced by low temperatures. This study complements a previous work on proteomic and individual carbohydrates and provides insight in the ability of poplar plantlets to cold acclimate and to cope with low temperatures by diverse mechanisms (growth cessation, carbohydrate, pigment, polyamine and protein accumulations) related to stress response or involved in acclimation process.     

Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues

The colonization pattern of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN was determined using grapevine fruiting cuttings with emphasis on putative inflorescence colonization under nonsterile conditions. Two-week-old rooted plants harbouring flower bud initials, grown in nonsterile soil, were inoculated with PsJN:gfp2x. Plant colonization was subsequently monitored at various times after inoculation with plate counts and epifluorescence and/or confocal microscopy. Strain PsJN was chronologically detected on the root surfaces, in the endorhiza, inside grape inflorescence stalks, not inside preflower buds and flowers but rather as an endophyte inside young berries. Data demonstrated low endophytic populations of strain PsJN in inflorescence organs, i.e. grape stalks and immature berries with inconsistency among plants for bacterial colonization of inflorescences. Nevertheless, endophytic colonization of inflorescences by strain PsJN was substantial for some plants. Microscopic analysis revealed PsJN as a thriving endophyte in inflorescence organs after the colonization process. Strain PsJN was visualized colonizing the root surface, entering the endorhiza and spreading to grape inflorescence stalks, pedicels and then to immature berries through xylem vessels. In parallel to these observations, a natural microbial communities was also detected on and inside plants, demonstrating the colonization of grapevine by strain PsJN in the presence of other microorganisms.     

Comparative analysis of defence responses induced by the endophytic plant growth-promoting rhizobacterium Burkholderia phytofirmans strain PsJN and the non-host bacterium Pseudomonas syringae pv. pisi in grapevine cell suspensions

Plant growth-promoting rhizobacteria (PGPR) are beneficial microorganisms that colonize the rhizosphere of many plant species and confer beneficial effects, such as an increase in plant growth. PGPR are also well known as inducers of systemic resistance to pathogens in plants. However, the molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain largely unknown. Burkholderia phytofirmans strain PsJN is an endophytic PGPR that colonizes grapevine and protects the plant against the grey mould disease caused by Botrytis cinerea. This report focuses on local defence events induced by B. phytofirmans PsJN after perception by the grapevine cells. It is demonstrated that, after addition to cell suspension cultures, the bacteria were tightly attaching to plant cells in a way similar to the grapevine non-host bacteria Pseudomonas syringae pv. pisi. B. phytofirmans PsJN perception led to a transient and monophasic extracellular alkalinization but no accumulation of reactive oxygen species or cell death were detected. By contrast, challenge with P. syringae pv. pisi induced a sustained and biphasic extracellular alkalinization, a two phases oxidative burst, and a HR-like response. Perception of the PGPR also led to the production of salicylic acid (SA) and the expression of a battery of defence genes that was, however, weaker in intensity compared with defence gene expression triggered by the non-host bacteria. Some defence genes up-regulated after B. phytofirmans PsJN challenge are specifically induced by exogenous treatment with SA or jasmonic acid, suggesting that both signalling pathways are activated by the PGPR in grapevine.     

Differential responses of Arabidopsis ecotypes to cold, chilling and freezing temperatures

Arabidopsis plants show an increase in freezing tolerance in response to exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. In the present study, we evaluated the physiological and morphological responses of various Arabidopsis ecotypes to continuous growth under chilling (14°C) and cold (6°C) temperatures and evaluated their basal freezing tolerance levels. Seedlings of Arabidopsis plants were extremely sensitive to low growth temperatures: the hypocotyls and petioles were much longer and the angles of the second pair of true leaves were much greater in plants grown at 14°C than in those grown at 22°C, whereas just intermediate responses were observed under the cold temperature of 6°C. Flowering time was also markedly delayed at low growth temperatures and, interestingly, lower growth temperatures were accompanied by longer inflorescences. Other marked responses to low temperatures were changes in pigmentation, which appeared to be both ecotype specific and temperature dependent and resulted in various visual phenotypes such as chlorosis, necrosis or enhanced accumulation of anthocyanins. The observed decreases in chlorophyll contents and accumulation of anthocyanins were much more prominent in plants grown at 6°C than in those grown at 14°C. Among the various ecotypes tested, Mt‐0 plants markedly accumulated the highest levels of anthocyanins upon growth at 6°C. Freezing tolerance examination revealed that among 10 ecotypes tested, only C24 plants were significantly more sensitive to subzero temperatures. In conclusion, Arabidopsis ecotypes responded differentially to cold (6°C), chilling (14°C) and freezing temperatures, with specific ecotypes being more sensitive in particular traits to each low temperature.     

Effect of inoculation with the endophyte Clavibacter sp. strain Enf12 on chilling tolerance in Chorispora bungeana

Endophytic bacteria have been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. The objective of this study was to evaluate the effect of an endophytic bacterium, Clavibacter sp. strain Enf12, in regenerated plantlets of Chorispora bungeana subjected to chilling stress (0°C). Aerial biomass and physiological markers for chilling stress, such as electrolyte leakage, lipid peroxidation, reactive oxygen species (ROS) accumulation, proline content and activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), guaiacol peroxidase (EC 1.11.1.7) and ascorbate peroxidase (EC 1.11.1.11), were assessed. We demonstrated that Clavibacter sp. strain Enf12 was capable of colonizing internal tissues of regenerated plantlets of C. bungeana and maintained stable population densities under both normal (20°C) and chilling (0°C) conditions. Inoculation enhanced plantlet growth under both conditions and significantly attenuated the chilling-induced electrolyte leakage, lipid peroxidation and ROS accumulation. The endophyte significantly increased the activities of antioxidant enzymes and proline content in C. bungeana plantlets under chilling stress. These findings suggest that Clavibacter sp. strain Enf12 inoculation stimulates the growth of C. bungeana plantlets and improves their tolerance to chilling stress through enhancing the antioxidant defense system.     

Preparing plants for improved cold tolerance by priming

Cold is a major stressor, which limits plant growth and development in many parts of the world, especially in the temperate climate zones. A large number of experimental studies has demonstrated that not only acclimation and entrainment but also the experience of single short stress events of various abiotic or biotic kinds (priming stress) can improve the tolerance of plants to chilling temperatures. This process, called priming, depends on a stress “memory”. It does not change cold sensitivity per se but beneficially modifies the response to cold and can last for days, months, or even longer. Elicitor factors and antagonists accumulate due to increased biosynthesis or decreased degradation either during or after the priming stimulus. Comparison of priming studies investigating improved tolerance to chilling temperatures highlighted key regulatory functions of ROS/RNS and antioxidant enzymes, plant hormones, especially jasmonates, salicylates, and abscisic acid, and signalling metabolites, such as β‐ and γ‐aminobutyric acid (BABA and GABA) and melatonin. We conclude that these elicitors and antagonists modify local and systemic cold tolerance by integration into cold‐induced signalling cascades.