Involvement of calcium-mediated effects on ROS metabolism in the regulation of growth improvement under salinity.

Salinity reduces Ca(2+) availability, transport, and mobility to growing regions of the plant and supplemental Ca(2+) is known to reduce salinity damages. This study was undertaken to unravel some of the ameliorative mechanisms of Ca(2+) on salt stress at the cellular and tissue levels. Zea mays L. plants were grown in nutrient solution containing 1 or 80 mM NaCl with various Ca(2+) levels. Measurements of growth and physiological parameters, such as ion imbalance, indicated that the Ca(2+)-induced alleviation mechanisms differed between plant organs. Under salinity, H(2)O(2) levels increased in the leaf-growing tissue with increasing levels of supplemental Ca(2+) and reached the levels of control plants, whereas superoxide levels remained low at all Ca(2+) levels, indicating that Ca(2+) affected growth by increasing H(2)O(2) but not superoxide levels. Salinity completely abolished apoplastic peroxidase activity. Supplemental Ca(2+) increased its activity only slightly. However, under salinity, polyamine oxidase (PAO) activity was shifted toward the leaf base probably as an adaptive mechanism aimed at restoring normal levels of reactive oxygen species (ROS) at the expansion zone where NADPH oxidase could no longer provide the required ROS for growth. Interestingly, addition of Ca(2+) shifted the PAO-activity peak back to its original location in addition to its enhancement. The increase in PAO activity in conjunction with low levels of apoplastic peroxidase is supportive of cellular growth via nonenzymatic wall loosening derived by the increase in H(2)O(2) and less supportive of the peroxidase-mediated cross-linking of wall material. Thus extracellular Ca(2+) can modulate ROS levels at specific tissue localization and developmental stages thereby affecting cellular extension.

Involvement of the plant antioxidative response in the differential growth sensitivity to salinity of leaves vs roots during cell development.

Sensitivity to salinity varies between plant organs and between cells of different developmental stages within a single organ. The physiological and molecular bases for the differential responses are not known. Exposure of plants to salinity is known to induce formation of reactive oxygen species (ROS), which are involved in damage mechanisms but also in cell growth processes. The objective of this study was to elucidate developmental-stage-specific and organ-specific involvement of oxidative defense in the plant response to salinity in maize (Zea mays L.). Plants were grown in nutrient solution containing 1mM NaCl (control) or 80mM NaCl. The oxidative stress response and damage symptoms along the cell developmental gradient in growing and mature tissue of leaves and roots were examined. Unlike leaves, roots did not suffer oxidative damage in either growing or mature cells and demonstrated reduced antioxidant response. This may reflect different requirements of ROS for growth mechanisms of leaf and root cells. In leaves, growing tissue demonstrated higher stimulation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity under salinity than mature tissue, whereas mature tissue demonstrated higher stimulation of catalase. These results indicate differential roles for these ROS-scavenging enzymes at different cell developmental stages. Because ROS are required for cell expansion, the higher increase in SOD and APX activities in the growing leaf cells that resulted in reduction of ROS content under salinity could lead to the inhibition of cell growth under salinity.

Differential expression of maize chitinases in the presence or absence of Trichoderma harzianum strain T22 and indications of a novel exo- endo-heterodimeric chitinase activity.

BACKGROUND: The interaction of plants with endophytic symbiotic fungi in the genus Trichoderma alters the plant proteome and transcriptome and results in enhanced plant growth and resistance to diseases. In a previous study, we identified the numerous chitinolytic enzyme families and individual enzymes in maize which are implicated in plant disease resistance and other plant responses. RESULTS: We examined the differential expression of the entire suite of chitinolytic enzymes in maize plants in the presence and absence of T. harzianum. Expression of these enzymes revealed a band of chitinolytic enzyme activity that had greater mass than any known chitinase. This study reports the characterization of this large protein. It was found to be a heretofore undiscovered heterodimer between an exo- and an endo-enzyme, and the endo portion differed between plants colonized with T. harzianum and those grown in its absence and between shoots and roots. The heterodimeric enzymes from shoots in the presence and absence of T. harzianum were purified and characterized. The dimeric enzyme from Trichoderma-inoculated plants had higher specific activity and greater ability to inhibit fungal growth than those from control plants. The activity of specific chitinolytic enzymes was higher in plants grown from Trichoderma treated seeds than in control plants. CONCLUSIONS: This is the first report of a dimer between endo- and exochitinase. The endochitinase component of the dimer changed post Trichoderma inoculation. The dimer originating from Trichoderma inoculated plants had a higher antifungal activity than the comparable enzyme from control plants.

Induced systemic resistance and plant responses to fungal biocontrol agents.

Biocontrol fungi (BCF) are agents that control plant diseases. These include the well-known Trichoderma spp. and the recently described Sebacinales spp. They have the ability to control numerous foliar, root, and fruit pathogens and even invertebrates such as nematodes. However, this is only a subset of their abilities. We now know that they also have the ability to ameliorate a wide range of abiotic stresses, and some of them can also alleviate physiological stresses such as seed aging. They can also enhance nutrient uptake in plants and can substantially increase nitrogen use efficiency in crops. These abilities may be more important to agriculture than disease control. Some strains also have abilities to improve photosynthetic efficiency and probably respiratory activities of plants. All of these capabilities are a consequence of their abilities to reprogram plant gene expression, probably through activation of a limited number of general plant pathways.

Genome-wide identification, expression and chromosomal location of the genes encoding chitinolytic enzymes in Zea mays.

Chitinolytic enzymes are important pathogenesis and stress related proteins. We identified 27 putative genes encoding endochitinases in the maize genome via in silico techniques and four exochitinases. Only seven of the endochitinases and segments of the exochitinases were heretofore known. The endochitinases included members of family 19 chitinases (classes I-IV of PR3, II of PR4) and members of family 18 chitinases (class III of PR8). Some similar enzymes were detected on adjacent regions of the same chromosome, and seem to result from duplication events. Most of the genes expressed were identified from EST libraries from plants exposed to biotic or abiotic stresses but also from libraries from tissues not exposed to stresses. We isolated proteins from seedlings of maize in the presence or absence of the symbiotic root colonizing fungus Trichoderma harzianum strain T22, and analyzed the activity of chitinolytic enzymes using an in-gel activity assay. The activity bands were identified by LC/MS/MS using the database from our in silico study. The identities of the enzymes changed depending on whether or not T22 was present. One activity band of about 95 kDa appeared to be a heterodimer between an exochitinase and any of several different endochitinases. The identity of the endochitinase component appeared to be dependent upon treatment.

The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach.

Trichoderma spp. are effective biocontrol agents for several soil-borne plant pathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases and overall plant growth. Root colonization with Trichoderma harzianum Rifai strain 22 (T22) induces large changes in the proteome of shoots of maize (Zea mays) seedlings, even though T22 is present only on roots. We chose a proteomic approach to analyze those changes and identify pathways and genes that are involved in these processes. We used two-dimensional gel electrophoresis to identify proteins that are differentially expressed in response to colonization of maize plants with T22. Up- or down-regulated spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry and nanospray ion-trap tandem mass spectrometry. We identified 91 out of 114 up-regulated and 30 out of 50 down-regulated proteins in the shoots. Classification of these revealed that a large portion of the up-regulated proteins are involved in carbohydrate metabolism and some were photosynthesis or stress related. Increased photosynthesis should have resulted in increased starch accumulation in seedlings and did indeed occur. In addition, numerous proteins induced in response to Trichoderma were those involved in stress and defense responses. Other processes that were up-regulated were amino acid metabolism, cell wall metabolism, and genetic information processing. Conversely, while the proteins involved in the pathways noted above were generally up-regulated, proteins involved in other processes such as secondary metabolism and protein biosynthesis were generally not affected. Up-regulation of carbohydrate metabolism and resistance responses may correspond to the enhanced growth response and induced resistance, respectively, conferred by the Trichoderma inoculation.

The relationship between increased growth and resistance induced in plants by root colonizing microbes.

Trichoderma spp. are effective biocontrol agents for numerous foliar and root phytopathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases as well as overall plant growth. Root colonization with T. harzianum strain T22 induces large changes in the proteome of shoots of maize seedlings (Zea mays) even though T22 is present only on roots; changes also were recorded in the roots. In shoots, we identified 91 of 114 upregulated and 30 of 50 downregulated proteins. In roots, 20 upregulated and 11 downregulated proteins were found and 17 and eight, respectively, were identified. Classification of proteins differentially expressed in both shoots and roots revealed that the largest number of upregulated proteins was involved in carbohydrate metabolism; in shoots, some upregulated proteins were involved in photosynthesis. Increases in these protein classifications suggest enhanced respiratory and photosynthetic rates. These changes may be required for the enhanced growth response induced by colonization of Trichoderma following seed or soil treatments. Stress and defense related proteins that were upregulated probably are related to the enhanced resistance conferred by the Trichoderma inoculation. We suggest that Trichoderma induces both increased growth, which is mediated by an increase in photosynthetic and respiratory rates, and systemic induced resistance. These two general effects may be mediated by different elicitors.

Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance.

The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.

Involvement of Jasmonic Acid/Ethylene Signaling Pathway in the Systemic Resistance Induced in Cucumber by Trichoderma asperellum T203.

ABSTRACT Trichoderma spp. are effective biocontrol agents for a number of soilborne plant pathogens, and some are also known for their ability to enhance plant growth. It was recently suggested that Trichoderma also affects induced systemic resistance (ISR) mechanism in plants. Analysis of signal molecules involved in defense mechanisms and application of specific inhibitors indicated the involvement of jasmonic acid and ethylene in the protective effect conferred by Trichoderma spp. against the leaf pathogen Pseudomonas syringae pv. lachrymans. Moreover, examination of local and systemic gene expression by real-time reverse transcription-polymerase chain reaction analysis revealed that T. asperellum (T203) modulates the expression of genes involved in the jasmonate/ethylene signaling pathways of ISR (Lox1, Pal1, ETR1, and CTR1) in cucumber plants. We further showed that a subsequent challenge of Trichoderma-preinoculated plants with the leaf pathogen P. syringae pv. lachrymans resulted in higher systemic expression of the pathogenesisrelated genes encoding for chitinase 1, beta-1,3-glucanase, and peroxidase relative to noninoculated, challenged plants. This indicates that Trichoderma induced a potentiated state in the plant enabling it to be more resistant to subsequent pathogen infection.

Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins.

Most studies on the reduction of disease incidence in soil treated with Trichoderma asperellum have focused on microbial interactions rather than on plant responses. This study presents conclusive evidence for the induction of a systemic response against angular leaf spot of cucumber (Pseudomonas syringae pv. lachrymans) following application of T. asperellum to the root system. To ascertain that T. asperellum was the only microorganism present in the root milieu, plants were grown in an aseptic hydroponic growth system. Disease symptoms were reduced by as much as 80%, corresponding to a reduction of 2 orders of magnitude in bacterial cell densities in leaves of plants pretreated with T. asperellum. As revealed by electron microscopy, bacterial cell proliferation in these plants was halted. The protection afforded by the biocontrol agent was associated with the accumulation of mRNA of two defense genes: the phenylpropanoid pathway gene encoding phenylalanine ammonia lyase (PAL) and the lipoxygenase pathway gene encoding hydroxyperoxide lyase (HPL). This was further supported by the accumulation of secondary metabolites of a phenolic nature that showed an increase of up to sixfold in inhibition capacity of bacterial growth in vitro. The bulk of the antimicrobial activity was found in the acid-hydrolyzed extract containing the phenolics in their aglycone form. High-performance liquid chromatography analysis of phenolic compounds showed a marked change in their profile in the challenged, preelicited plants relative to that in challenged controls. The results suggest that similar to beneficial rhizobacteria, T. asperellum may activate separate metabolic pathways in cucumber that are involved in plant signaling and biosynthesis, eventually leading to the systemic accumulation of phytoalexins.