An in Vivo Imaging Assay Detects Spatial Variability in Glucose Release from Plant Roots.

Plants secrete a plethora of metabolites into the rhizosphere that allow them to obtain nutrients necessary for growth and modify microbial communities around the roots. Plants release considerable amounts of photosynthetically fixed carbon into the rhizosphere; hence, it is important to understand how carbon moves from the roots into the rhizosphere. Approaches used previously to address this question involved radioactive tracers, fluorescent probes, and biosensors to study sugar movement in the roots and into the rhizosphere. Although quite effective for studying sugar movement, it has been challenging to obtain data on spatial and temporal variability in sugar exudation using these techniques. In this study, we developed a gel-based enzyme-coupled colorimetric and fluorometric assay to image glucose (Glc) in vivo and used this assay to show that there is spatial variability in Glc release from plant roots. We found that the primary roots of maize (Zea mays) released more Glc from the base of the root than from the root tip and that the Glc release rate is reduced in response to water stress. These findings were confirmed independently by quantifying Glc release in well-watered and water-stressed maize primary roots using high-performance anion-exchange chromatography. Additionally, we demonstrated differential patterns of Glc exudation in different monocot and eudicot plant species. These findings and their implications on root-rhizosphere interactions are discussed.

Plasma membrane proteomics in the maize primary root growth zone: novel insights into root growth adaptation to water stress.

Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress.

Overexpression of AtDREB1D transcription factor improves drought tolerance in soybean.

Drought is one of the major abiotic stresses that affect productivity in soybean (Glycine max L.) Several genes induced by drought stress include functional genes and regulatory transcription factors. The Arabidopsis thaliana DREB1D transcription factor driven by the constitutive and ABA-inducible promoters was introduced into soybean through Agrobacterium tumefaciens-mediated gene transfer. Several transgenic lines were generated and molecular analysis was performed to confirm transgene integration. Transgenic plants with an ABA-inducible promoter showed a 1.5- to two-fold increase of transgene expression under severe stress conditions. Under well-watered conditions, transgenic plants with constitutive and ABA-inducible promoters showed reduced total leaf area and shoot biomass compared to non-transgenic plants. No significant differences in root length or root biomass were observed between transgenic and non-transgenic plants under non-stress conditions. When subjected to gradual water deficit, transgenic plants maintained higher relative water content because the transgenic lines used water more slowly as a result of reduced total leaf area. This caused them to wilt slower than non-transgenic plants. Transgenic plants showed differential drought tolerance responses with a significantly higher survival rate compared to non-transgenic plants when subjected to comparable severe water-deficit conditions. Moreover, the transgenic plants also showed improved drought tolerance by maintaining 17-24 % greater leaf cell membrane stability compared to non-transgenic plants. The results demonstrate the feasibility of engineering soybean for enhanced drought tolerance by expressing stress-responsive genes.

Apoplastic hydrogen peroxide in the growth zone of the maize primary root under water stress. I. Increased levels are specific to the apical region of growth maintenance.

Previous work on the adaptation of maize (Zea mays L.) primary root growth to water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. Cell wall proteomic analysis suggested that levels of apoplastic reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), may be modified in a region-specific manner within the growth zone of water-stressed roots. Apoplastic ROS may have wall loosening or tightening effects and may also have other growth regulatory functions. To gain an understanding of how apoplastic ROS levels change under water stress, cerium chloride staining was used in conjunction with transmission electron microscopy to examine the spatial distribution of apoplastic H2O2. The results revealed that apoplastic H2O2 levels increased specifically in the apical region of the growth zone under water stress, correlating spatially with the maintenance of cell elongation. The basal regions of the growth zone of water-stressed roots and the entire growth zone of well-watered roots exhibited relatively low levels of apoplastic H2O2. The increase in apoplastic H2O2 in the apical region under water stress probably resulted, at least in part, from a pronounced increase in oxalate oxidase activity in this region. By contrast, well-watered roots showed negligible oxalate oxidase activity throughout the growth zone. The results show that changes in apoplastic ROS levels in the root growth zone under water-deficit conditions are regulated in a spatially-specific manner, suggesting that this response may play an important role in maize root adaptation to water stress.

Antioxidant Metabolism Underlies Different Metabolic Strategies for Primary Root Growth Maintenance under Water Stress in Cotton and Maize.

The divergence of metabolic responses to water stress in the elongation zone of cotton and maize primary roots was investigated by establishing water-deficit conditions that generated steady root elongation at equivalent tissue water potentials. In water-stressed cotton roots, cell elongation was maintained in the apical 3 mm but was progressively inhibited with further displacement from the apex. These responses are similar to previous findings in maize, providing the foundation for comparisons of metabolic responses in regions of growth maintenance and inhibition between the species. Metabolomics analyses showed region-specific and species-specific changes in metabolite abundance in response to water stress, revealing both conserved responses including osmolyte accumulation, and key differences in antioxidative and sulfur metabolism. Quantitative assessment showed contrasting glutathione responses in the root elongation zone between the species, with glutathione levels declining in cotton as stress duration progressed, whereas in maize, glutathione levels remained elevated. Despite the lesser glutathione response in cotton, hydrogen peroxide levels were low in water-stressed cotton compared with maize roots and were associated with higher catalase, ascorbate peroxidase, and superoxide dismutase activities in cotton. The results indicate alternative metabolic strategies underlying the responses of primary root growth to water stress between cotton and maize.

Resistance to Russian wheat aphid biotype 2 in CIMMYT synthetic hexaploid wheat lines.

The Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), devastates wheat and barley production on all continents except Australia. Although D. noxia-resistant cultivars exist, virulent D. noxia populations exist in Asia, North America, and South America that have the ability to overcome resistance. In this study, synthetic hexaploid wheat genotypes created at the International Maize and Wheat Improvement Center (CIMMYT) were evaluated and characterized for resistance to North American biotype 2 (RWA2). Fewer RWA2 were produced on several genotypes, and D. noxia-induced leaf rolling and chlorosis were reduced on these genotypes as well. Aphid numbers were positively correlated with chlorosis and with leaf rolling. However, some genotypes were highly resistant to leaf rolling and chlorosis while supporting large RWA2 populations. There were negative correlations between leaf chlorosis and leaf dry weight (r = -0.267, df = 106, P = 0.006) and between aphid numbers and leaf dry weight (r = -0.297, df = 105, P = 0.002). These results indicate that chlorosis and aphid number individually explain at least 27% of the changes observed in leaf dry weights. Interestingly, there was no correlation between leaf rolling and leaf dry weight. The RWA2-resistant lines identified, which are also resistant to D. noxia populations in Mexico and to greenbug (Schizaphis graminum Rondani) biotype G, are strong candidates for use in improving the genetic diversity in bread wheat for resistance to different biotypes of both S. graminum and D. noxia.

Categories and inheritance of resistance to Russian wheat aphid (Homoptera: Aphididae) biotype 2 in a selection from wheat cereal introduction 2401.

The Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), is one of the most devastating insect pests of wheat (Triticum spp.) and barley (Hordeum spp.) in the world. Yield losses and control costs are valued at several hundred million dollars each year. The use of D. noxia-resistant cultivars is an ecologically, economically, and biologically sound method of managing this pest. Several D. noxia resistance (Dn) genes from wheat have been used to develop cultivars resistant to D. noxia. However, a new U.S. D. noxia biotype (biotype 2) in Colorado is virulent to all known Dn genes except the Dn7 gene from rye (Secale spp.). Hence, there is an immediate need to identify and characterize unique sources of D. noxia resistance to biotypes. In this article, we report resistance to D. noxia biotype 2, identified in a selection from wheat cereal introduction (CItr) 2401, that is controlled by two dominant genes. CItr2401 has a strong antibiosis effect that is exhibited as a reduced intrinsic rate of increase of D. noxia biotype 2. CItr2401 plants also exhibit tolerance to leaf rolling and chlorosis. No antixenosis was detected in CItr2401.

Developmental distribution of the plasma membrane-enriched proteome in the maize primary root growth zone.

Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM) proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein implicates that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

Genetic variability of oxalate oxidase activity and elongation in water-stressed primary roots of diverse maize and rice lines.

A previous study of maize primary roots under water stress showed pronounced increases in oxalate oxidase activity and apoplastic hydrogen peroxide in the apical region of the growth zone where cell elongation is maintained. We examined whether increased oxalate oxidase activity in water-stressed roots is conserved across diverse lines of maize and rice. The maize lines exhibited varied patterns of activity, with some lines lacking activity in the apical region. Moreover, none of the rice lines showed activity in the apical region. Also, although the genotypic response of root elongation to water stress was variable in both maize and rice, this was not correlated with the pattern of oxalate oxidase activity. Implications of these findings for root growth regulation under water stress are discussed.