Transcriptional response of Arabidopsis thaliana's root-tip to spaceflight.

Plants are expected to play a critical role in the biological life support systems of crewed spaceflight missions, including in the context of upcoming missions targeting the Moon and Mars. Therefore, understanding the response of plants to spaceflight is essential for improving the selection and engineering of plants and spaceflight conditions. In particular, understanding the root-tip's response to spaceflight is of importance as it is the center of orchestrating the development of the root, the primary organ for the absorption of nutrients and anchorage. GLDS-120 is a pioneering study by Paul et al. that used transcriptomics to evaluate the spaceflight response of the root-tip of the model plant Arabidopsis thaliana in dark and light through separate analyses of three genotype groups (Wassilewskija, Columbia-0, and Columbia-0 PhyD) and comparison of genotype responses. Here, we provide a complementary analysis of this dataset through a combined analysis of all samples while controlling for the genotypes in a paired analysis. We identified a robust transcriptional response to spaceflight with 622 DEGs in light and 200 DEGs in dark conditions. Gene enrichment analysis identified 37 and 13 significantly enriched terms from biological processes in light and dark conditions, respectively. Prominent enrichment for hypoxia-related terms in both conditions suggests hypoxia is a key stressor for root development during spaceflight. Additional enriched terms in light conditions include the circadian cycle, light response, and terms for the metabolism of flavonoid and indole-containing compounds. These results further our understanding of plants' responses to the spaceflight environment.

Tissue growth constrains root organ outlines into an isometrically scalable shape.

Organ morphologies are diverse but also conserved under shared developmental constraints among species. Any geometrical similarities in the shape behind diversity and the underlying developmental constraints remain unclear. Plant root tip outlines commonly exhibit a dome shape, which likely performs physiological functions, despite the diversity in size and cellular organization among distinct root classes and/or species. We carried out morphometric analysis of the primary roots of ten angiosperm species and of the lateral roots (LRs) of Arabidopsis, and found that each root outline was isometrically scaled onto a parameter-free catenary curve, a stable structure adopted for arch bridges. Using the physical model for bridges, we analogized that localized and spatially uniform occurrence of oriented cell division and expansion force the LR primordia (LRP) tip to form a catenary curve. These growth rules for the catenary curve were verified by tissue growth simulation of developing LRP development based on time-lapse imaging. Consistently, LRP outlines of mutants compromised in these rules were found to deviate from catenary curves. Our analyses demonstrate that physics-inspired growth rules constrain plant root tips to form isometrically scalable catenary curves.

Substrate heterogeneities cause root growth trajectories to switch from vertical to oblique.

Hard pans, soil compaction, soil aggregation and stones create physical barriers that can affect the development of a root system. Roots are known to exploit paths of least resistance to avoid such obstacles, but the mechanism through which this is achieved is not well understood. Here, we combined 3D-printed substrates with a high-throughput live imaging platform to study the responses of plant roots to a range of physical barriers. Using image analysis algorithms, we determined the properties of growth trajectories and identified how the presence of rigid circular obstacles affects the ability of a primary root to maintain its vertical trajectory. Results showed the types of growth responses were limited, both vertical and oblique trajectories were found to be stable and influenced by the size of the obstacles. When obstacles were of intermediate sizes, trajectories were unstable and changed in nature through time. We formalised the conditions for root trajectory to change from vertical to oblique, linking the angle at which the root detaches from the obstacle to the root curvature due to gravitropism. Exploitation of paths of least resistance by a root may therefore be constrained by the ability of the root to curve and respond to gravitropic signals.

Evaluation of the probable synergistic toxicity of selected potentiated antiretroviral and antibiotics on some aquatic biomarker organisms.

Environmental effects of active pharmaceutical compounds (APCs) in the environment are not well characterized, hence the need for comprehensive evaluation. This study employed three bioassays using three organisms, namely, Allium cepa, Daphnia magna, and Salmonella typhimurium, in the ecotoxicity study of lone and a mixture of selected APCs, namely, lamivudine (L), an antiretroviral, and ciprofloxacin (C) and sulfamethoxazole (S), antibiotics, at a concentration range between 10 and 100 ppb, in order to evaluate the potential of the lone and ternary mixture to exert synergistic toxicity. Study results from exposure to lone APCs showed that the L, C, and S trio individually had fatal impacts on daphnids, with mortality rates of 100, 75, and 95%, respectively, after 48 h. Sulfamethoxazole showed a mutagenic tendency, with a mutation ratio (background/sample ratio) of 2.0. Lamivudine showed a lethal impact on the root length of A. cepa (p > 0.05, p = 3.60E-3). Further microscopic examination of the A. cepa root tip revealed chromosomal aberrations on exposure to each compound. The LCS-mix ecotoxicology bioassays indicated a synergistic effect on the daphnids, probably due to potentiation. Although the LCS mix had a cytotoxic effect (evidenced by the absence of bacteria colonies) on exposed TA 98 P450 Salmonella typhimurium strain, this effect was not observed in other bacterial strains. Microscopic examination of A. cepa exposed to the LCS-mix revealed an aberration in the mitotic stage of the cell. The impact of combination of the pharmaceuticals in aqueous ecosystems was greater than when exposed to the tested individual pharmaceutical compounds. Study result showed that these compounds have tendencies to pose a higher risk to exposed living entities when in combined/potentiated forms, and this could lead to distortion of the regular functioning of the ecosystem, particularly bacterial and other microbial populations that are listed among primary producers of the aquatic food web.

Proteomic analysis of the meristematic root zone in contrasting genotypes reveals new insights in drought tolerance in rice.

Drought is responsible for major losses in rice production. Root tips contain meristematic and elongation zones that play major roles in determination of root traits and adaptive strategies to drought. In this study we analysed two contrasting genotypes of rice: IR64, a lowland, drought-susceptible, and shallow-rooting genotype; and Azucena, an upland, drought-tolerant, and deep-rooting genotype. Samples were collected of root tips of plants grown under control and water deficit stress conditions. Quantitative proteomics analysis resulted in the identification of 7294 proteins from the root tips of IR64 and 6307 proteins from Azucena. Data are available via ProteomeXchange with identifier PXD033343. Using a Partial Least Square Discriminant Analysis on 4170 differentially abundant proteins, 1138 statistically significant proteins across genotypes and conditions were detected. Twenty two enriched biological processes showing contrasting patterns between two genotypes in response to stress were detected through gene ontology enrichment analysis. This included identification of novel proteins involved in root elongation with specific expression patterns in Azucena, including four Expansins and seven Class III Peroxidases. We also detected an antioxidant network and a metallo-sulfur cluster assembly machinery in Azucena, with roles in reactive oxygen species and iron homeostasis, and positive effects on root cell cycle, growth and elongation.

AtNUF2 modulates spindle microtubule organization and chromosome segregation during mitosis.

The NDC80 complex is a conserved eukaryotic complex composed of four subunits (NUF2, SPC25, NDC80, and SPC24). In yeast and animal cells, the complex is located at the outer layer of the kinetochore, connecting the inner layer of the kinetochore and spindle microtubules (MTs) during cell division. In higher plants, the relationship of the NDC80 complex with MTs is still unclear. In this study, we characterized the biological function of AtNUF2, a subunit of the Arabidopsis NDC80 complex. We found that AtNUF2 is widely expressed in various organs, especially in different stages of embryonic development. It was verified that AtNUF2 co-localized with α-tubulin on MTs during mitosis by immunohistochemical assays. Mutation of AtNUF2 led to severe mitotic defects, not only in the embryo and endosperm, but also in seedlings, resulting in seed abortion and stagnating seedling growth. Furthermore, the biological function of AtNUF2 was studied using partially complemented nuf2-3/-DD45;ABI3pro::AtNUF2 (nuf2-3/-DA ) seedlings. The chromosome bridge and lagging chromatids occurred in nuf2-3/-DA root apical meristem cells, along with aberration of spindle MTs, resulting in blocked root growth. Meanwhile, the direct binding of AtNUF2 and AtSPC25 to MTs was determined by an MT co-sedimentation assay in vitro. This study revealed the function of AtNUF2 in mitosis and the underlying mechanisms, modulating spindle MT organization and ensuring chromosome segregation during embryo, endosperm, and root development, laying the foundation for subsequent research of the NDC80 complex.

Effects of Elevated Aluminum Concentration and Distribution on Root Damage, Cell Wall Polysaccharides, and Nutrient Uptake in Different Tolerant Eucalyptus Clones.

Aluminized acidic soil can damage Eucalyptus roots and limit tree growth, hindering the productivity of Eucalyptus plantations. At present, the negative impacts of elevated aluminum (Al) on the cell morphology and cell wall properties of Eucalyptus root tip are still unclear. In order to investigate the responses of two different tolerant clones, Eucalyptus urophylla (G4) and Eucalyptus grandis × Eucalyptus urophylla (G9), to Al toxicity, seedling roots were treated hydroponically with an Al solution, and the polysaccharide content in the root tip cell wall and the characteristics of programmed cell death were studied. The results show that the distribution of Al was similar in both clones, although G9 was found to be more tolerant to Al toxicity than G4. The Al3+ uptake of pectin in root tip cell walls was significantly higher in G4 than in G9. The root tip in G4 was obviously damaged, enlarged, thickened, and shorter; the root crown cells were cracked and fluffy; and the cell elongation area was squeezed. The lower cell wall polysaccharide content and PME activity may result in fewer carboxylic groups in the root tip cell wall to serve as Al-binding sites, which may explain the stronger Al resistance of G9 than G4. The uptake of nitrogen and potassium in G4 was significantly reduced after aluminum application and was lower than in G9. Al-resistant Eucalyptus clones may have synergistic pleiotropic effects in resisting high aluminum-low phosphorus stress, and maintaining higher nitrogen and potassium levels in roots may be an important mechanism for effectively alleviating Al toxicity.

Selenium restores mitochondrial dysfunction to reduce Cr-induced cell apoptosis in Chinese cabbage (Brassica campestris L. ssp. Pekinensis) root tips.

Chromium (Cr) disrupts the growth and physiology of plants. Selenium (Se) is considered as a promising option to help plants ameliorate Cr toxicity. To investigate the effects of exogenous Se on reactive oxygen species (ROS) burst and programmed cell death (PCD) in root tip cells under Cr stress, hydroponic experiments were carried out with Chinese cabbage seedlings grown in Hoagland solution containing 1 mg L-1 Cr and 0.1 mg L-1 Se. Results showed that Se scavenged the overproduction of H2O2 and O2－·, and alleviated the level of lipid peroxidation in root tips stressed by Cr. Moreover, Se effectively prevented DNA degradation and reduced the number of apoptotic cells in root tips. Compared with Cr treatment, Se supplementation reduced the content of ROS and malondialdehyde in mitochondria by 38.23% and 17.52%, respectively. Se application decreased the opening degree of mitochondrial permeability transition pores by 32.30%, increased mitochondrial membrane potential by 40.91%, alleviated the release of cyt c from mitochondria into cytosol by 18.42% and caused 57.40% decrease of caspase 3-like protease activity, and thus restored mitochondrial dysfunction caused by Cr stress. In addition, the alteration of Se on mitochondrial physiological properties maintained calcium homeostasis between mitochondria and cytosol, which further contributed to reducing the appearance of Cr-induced PCD. Findings suggested that Se restored mitochondrial dysfunction, which further rescued root tip cells from PCD, consequently activating defense strategies to protect plants from Cr toxicity and maintaining plant growth.

Whole-Tissue Three-Dimensional Imaging of Rice at Single-Cell Resolution.

The three-dimensional (3D) arrangement of cells in tissues provides an anatomical basis for analyzing physiological and biochemical aspects of plant and animal cellular development and function. In this study, we established a protocol for tissue clearing and 3D imaging in rice. Our protocol is based on three improvements: clearing with iTOMEI (clearing solution suitable for plants), developing microscopic conditions in which the Z step is optimized for 3D reconstruction, and optimizing cell-wall staining. Our protocol successfully 3D imaged rice shoot apical meristems, florets, and root apical meristems at cellular resolution throughout whole tissues. Using fluorescent reporters of auxin signaling in rice root tips, we also revealed the 3D distribution of auxin signaling events that are activated in the columella, quiescent center, and multiple rows of cells in the stele of the root apical meristem. Examination of cells with higher levels of auxin signaling revealed that only the central row of cells was connected to the quiescent center. Our method provides opportunities to observe the 3D arrangement of cells in rice tissues.

Processes controlling programmed cell death of root velamen radicum in an epiphytic orchid.

BACKGROUND AND AIMS: Development of the velamen radicum on the outer surface of the root epidermis is an important characteristic for water uptake and retention in some plant families, particularly epiphytic orchids, for survival under water-limited environments. Velamen radicum cells derive from the primary root meristem; however, following this development, velamen radicum cells die by incompletely understood processes of programmed cell death (PCD). METHODS: We combined the use of transmission electron microscopy, X-ray micro-tomography and transcriptome methods to characterize the major anatomical and molecular changes that occur during the development and death of velamen radicum cells of Cymbidium tracyanum, a typical epiphytic orchid, to determine how PCD occurs. KEY RESULTS: Typical changes of PCD in anatomy and gene expression were observed in the development of velamen radicum cells. During the initiation of PCD, we found that both cell and vacuole size increased, and several genes involved in brassinosteroid and ethylene pathways were upregulated. In the stage of secondary cell wall formation, significant anatomical changes included DNA degradation, cytoplasm thinning, organelle decrease, vacuole rupture and cell wall thickening. Changes were found in the expression of genes related to the biosynthesis of cellulose and lignin, which are instrumental in the formation of secondary cell walls, and are regulated by cytoskeleton-related factors and phenylalanine ammonia-lyase. In the final stage of PCD, cell autolysis was terminated from the outside to the inside of the velamen radicum. The regulation of genes related to autophagy, vacuolar processing enzyme, cysteine proteases and metacaspase was involved in the final execution of cell death and autolysis. CONCLUSIONS: Our results showed that the development of the root velamen radicum in an epiphytic orchid was controlled by the process of PCD, which included initiation of PCD, followed by formation of the secondary cell wall, and execution of autolysis following cell death.

Genotoxicity response of Vicia faba seedlings to cadmium in soils as characterized by direct soil exposure and micronucleus test.

To overcome the drawbacks of the Vicia faba root tip micronucleus test in soil using the solution extract method, we conducted a potting experiment by direct soil exposure. Cadmium was spiked into 3 typical soils (brown soil, red soil, and black soil) to simulate environmental concentrations (0.625, 1.25, 2.5, 5, and 10 mg kg-1). Multiple Vicia faba tissues (primary root tips, secondary root tips, and leaf tips) were sampled, and mitotic index (MI), chromosome aberration frequency (CA), and micronucleus frequency (MN) were used as endpoints after a seedling period of 5 days. The results showed a response between Cd concentrations and multiple sampling tissues of Vicia faba, and the secondary root tips responded to Cd stress the most, followed by primary root tips and leaf tips. Soil physicochemical properties (e.g., pH, total phosphorus, total organic carbon, etc.) influenced the genotoxicity of Cd, and pH was the dominant factor, which resulted in the genetic toxicity response of Cd in soils in the order: red soil > brown soil > black soil. The lowest observable effect concentration (LOEC) of Cd was 1.25 mg kg-1 for both brown soil and red soil and 2.5 mg kg-1 for black soil. In view of this, we suggested that soil properties should be considered in evaluating genotoxicity risk of Cd in soil, especially with soil pH range, and the secondary root tips should be taken as suitable test tissues in the MN test due to its more sensible response feature to Cd stress in soil.

The Class III Peroxidase Encoding Gene AtPrx62 Positively and Spatiotemporally Regulates the Low pH-Induced Cell Death in Arabidopsis thaliana Roots.

Exogenous low pH stress causes cell death in root cells, limiting root development, and agricultural production. Different lines of evidence suggested a relationship with cell wall (CW) remodeling players. We investigated whether class III peroxidase (CIII Prx) total activity, CIII Prx candidate gene expression, and reactive oxygen species (ROS) could modify CW structure during low pH-induced cell death in Arabidopsis thaliana roots. Wild-type roots displayed a good spatio-temporal correlation between the low pH-induced cell death and total CIII Prx activity in the early elongation (EZs), transition (TZs), and meristematic (MZs) zones. In situ mRNA hybridization showed that AtPrx62 transcripts accumulated only in roots treated at pH 4.6 in the same zones where cell death was induced. Furthermore, roots of the atprx62-1 knockout mutant showed decreased cell mortality under low pH compared to wild-type roots. Among the ROS, there was a drastic decrease in O2·- levels in the MZs of wild-type and atprx62-1 roots upon low pH stress. Together, our data demonstrate that AtPrx62 expression is induced by low pH and that the produced protein could positively regulate cell death. Whether the decrease in O2·- level is related to cell death induced upon low pH treatment remains to be elucidated.

Photoinducible DRONPA-s: a new tool for investigating cell-cell connectivity.

The development of multicellular plants relies on the ability of their cells to exchange solutes, proteins and signalling compounds through plasmodesmata, symplasmic pores in the plant cell wall. The aperture of plasmodesmata is regulated in response to developmental cues or external factors such as pathogen attack. This regulation enables tight control of symplasmic cell-to-cell transport. Here we report on an elegant non-invasive method to quantify the passive movement of protein between selected cells even in deeper tissue layers. The system is based on the fluorescent protein DRONPA-s, which can be switched on and off repeatedly by illumination with different light qualities. Using transgenic 35S::DRONPA-s Arabidopsis thaliana and a confocal microscope it was possible to activate DRONPA-s fluorescence in selected cells of the root meristem. This enabled us to compare movement of DRONPA-s from the activated cells into the respective neighbouring cells. Our analyses showed that pericycle cells display the highest efflux capacity with a good lateral connectivity. In contrast, root cap cells showed the lowest efflux of DRONPA-s. Plasmodesmata of quiescent centre cells mediated a stronger efflux into columella cells than into stele initials. To simplify measurements of fluorescence intensity in a complex tissue we developed software that allows simultaneous analyses of fluorescence intensities of several neighbouring cells. Our DRONPA-s system generates reproducible data and is a valuable tool for studying symplasmic connectivity.

Excess iron stress reduces root tip zone growth through nitric oxide-mediated repression of potassium homeostasis in Arabidopsis.

The root tip zone is regarded as the principal action site for iron (Fe) toxicity and is more sensitive than other root zones, but the mechanism underpinning this remains largely unknown. We explored the mechanism underpinning the higher sensitivity at the Arabidopsis root tip and elucidated the role of nitric oxide (NO) using NO-related mutants and pharmacological methods. Higher Fe sensitivity of the root tip is associated with reduced potassium (K+ ) retention. NO in root tips is increased significantly above levels elsewhere in the root and is involved in the arrest of primary root tip zone growth under excess Fe, at least in part related to NO-induced K+ loss via SNO1 (sensitive to nitric oxide 1)/SOS4 (salt overly sensitive 4) and reduced root tip zone cell viability. Moreover, ethylene can antagonize excess Fe-inhibited root growth and K+ efflux, in part by the control of root tip NO levels. We conclude that excess Fe attenuates root growth by effecting an increase in root tip zone NO, and that this attenuation is related to NO-mediated alterations in K+ homeostasis, partly via SNO1/SOS4.

Allium cepa root tip assay in assessment of toxicity of magnesium oxide nanoparticles and microparticles.

Allium cepa bioassay had been used from decades for the assessment of toxicants and their harmful effects on environment as well as human health. Magnesium oxide (MgO) particles are being utilized in different fields. However, reports on the adverse effects of MgO nanoparticles on the environment and mankind are scarce. Hence, the toxicity of MgO particles is of concern because of their increased utilization. In the current study, A. cepa was used as an indicator to assess the toxicological efficiency of MgO nano- and microparticles (NPs and MPs) at a range of exposure concentrations (12.5, 25, 50, and 100µg/mL). The toxicity was evaluated by using various bioassays on A. cepa root tip cells such as comet assay, oxidative stress and their uptake/internalization profile. Results indicated a dose dependent increase in chromosomal aberrations and decrease in mitotic index (MI) when compared to control cells and the effect was more significant for NPs than MPs (at p<0.05). Comet analysis revealed that the Deoxyribonucleic acid (DNA) damage in terms of percent tail DNA ranged from 6.8-30.1 over 12.5-100µg/mL concentrations of MgO NPs and was found to be significant at the exposed concentrations. A significant increase in generation of hydrogen peroxide and superoxide radicals was observed in accordance with the lipid peroxidation profile in both MgO NPs and MPs treated plants when compared with control. In conclusion, this investigation revealed that MgO NPs exposure exhibited greater toxicity on A. cepa than MPs.

An Al-inducible expansin gene, OsEXPA10 is involved in root cell elongation of rice.

Expansins are cell wall loosening proteins, which are encoded by multigene families. However, the physiological role of most expansin genes is still poorly understood. Here, we functionally characterized an Al-inducible expansin gene, OsEXPA10, which is regulated by a C2H2-type zinc-finger transcription factor, ART1 in rice. A detailed expression analysis showed that OsEXPA10 was expressed in both the roots and shoots at a similar level, but only the expression in the roots was rapidly upregulated in response to Al. Furthermore, spatial expression analysis showed that the Al-induced expression was only found in the root tips (0-3 mm), but not in the mature root zones. The expression was neither induced by other metals including Cd and La nor by low pH. Immunostaining showed that OsEXPA10 was localized at all cells of the root tips. Knockout of OsEXPA10 resulted in a significant decrease in the cell elongation of the roots in the absence of Al. In the presence of Al, knockout of OsEXPA10 did not alter the Al sensitivity evaluated by relative root elongation, but the root cell wall of knockout lines accumulated less Al compared to those of the wild-type rice. Collectively, our results indicate that OsEXPA10 expressed in the root tips is required for the root cell elongation, but that the contribution of this gene to high Al tolerance in rice is small.

Proteomic Analysis of Calcium Effects on Soybean Root Tip under Flooding and Drought Stresses.

Flooding and drought are disadvantageous environmental conditions that induce cytosolic calcium in soybean. To explore the effects of flooding- and drought-induced increases in calcium, a gel-free/label-free proteomic analysis was performed. Cytosolic calcium was decreased by blocking calcium channels in the endoplasmic reticulum (ER) and plasma membrane under both stresses. Calnexin, protein disulfide isomerase, heat shock proteins and thioredoxin were predominantly affected as the ER proteins in response to calcium, and ER-associated degradation-related proteins of HCP-like superfamily protein were up-regulated under stress exposure and then down-regulated. Glycolysis, fermentation, the tricarboxylic acid cycle and amino acid metabolism were mainly induced as the types of cellular metabolism in response to calcium under both stresses. Pyruvate decarboxylase was increased and decreased under flooding and drought, respectively, and was further decreased by the reduction of cytosolic calcium; however, it was recovered by exogenous calcium under both stresses. Furthermore, pyruvate decarboxylase activity was increased under flooding, but decreased under drought. These results suggest that calcium is involved in protein folding in the ER, and ER-associated degradation might alleviate ER stress during the early stage of both stresses. Furthermore, calcium appears to modify energy metabolism, and pyruvate decarboxylase may be a key enzyme in this process under flooding and drought.

Rapid root responses of seedlings exposed to a postdrought water pulse.

PREMISE OF THE STUDY: Mediterranean-type climate ecosystems experience significant variability in precipitation within and across years and may be characterized by periods of extreme drought followed by a brief, high-intensity precipitation pulse. Rapid root growth could be a key factor in effective utilization of precipitation pulses, leading to higher rates of seedling establishment. Changes in root growth rate are rarely studied, however, and patterns in seedling root traits are not well explored. We investigated the influence of an extreme postdrought precipitation event on seedlings that occur in southern California coastal sage scrub. METHODS: We measured root elongation rate, root tip appearance rate, new leaf appearance rate, and canopy growth rate on 18 mediterranean species from three growth forms. KEY RESULTS: Root elongation rate responded more strongly to the precipitation pulse than did root tip appearance rate and either metric of aboveground growth. The majority of species exhibited a significant change in root growth rate within 1 week of the pulse. Responses varied in rapidity and magnitude across species, however, and were not generally predictable based on growth form. CONCLUSIONS: While the majority of species exhibited shifts in belowground growth following the pulse, the direction and magnitude of these morphological responses were highly variable within growth form. Understanding the implications of these different response strategies for plant fitness is a crucial next step to forecasting community dynamics within ecosystems characterized by resource pulses.

Expression patterns of the native Shrunken-2 promoter in Sorghum bicolor visualised through use of the GFP reporter gene.

KEY MESSAGE: The AGPase large subunit (shrunken-2) promoter was demonstrated to be active in the placentochalaza and endosperm of developing grain as well as the root tips in transgenic sorghum. The temporal and spatial expression patterns of the Sorghum bicolor Shrunken-2 (Sh2) promoter were evaluated using the green fluorescence protein reporter gene (gfp) in transgenic sorghum, within the context of upregulating starch biosynthesis in the developing grain. GFP fluorescence was analysed throughout development in various tissue types using confocal laser scanning microscopy techniques. Sh2 promoter activity was first detected in the placentochalaza region of the developing caryopsis and apoplasm adjacent to the nucellar epidermis at 7 days post anthesis (dpa) where fluorescence remained relatively constant until 17 dpa. Fluorescence in this region weakened by 20 dpa and disappeared by 25 dpa. Expression was also detected in the developing endosperm, but not until 12 dpa, continuing until 25 dpa. Whilst the endosperm expression was expected, the fluorescence detected in the placentochalaza was completely unexpected. Although transcript presence does not mean the resulting biochemistry is also present, these preliminary findings may suggest alternate spatial activity of ADP-glucose pyrophosphorylase prior to uptake by the developing grain. Sh2 promoter activity was also unexpectedly detected in the root tips at all developmental time points. Sh2 promoter activity was not detected in any reproductive floral tissue (both pre and post anthesis) or in pollen. Similarly, no expression was detected in leaf tissue at any stage.

Gel-Free/Label-Free Proteomic Analysis of Endoplasmic Reticulum Proteins in Soybean Root Tips under Flooding and Drought Stresses.

Soybean is a widely cultivated crop; however, it is sensitive to flooding and drought stresses. The adverse environmental cues cause the endoplasmic reticulum (ER) stress due to accumulation of unfolded or misfolded proteins. To investigate the mechanisms in response to flooding and drought stresses, ER proteomics was performed in soybean root tips. The enzyme activity of NADH cytochrome c reductase was two-fold higher in the ER than other fractions, indicating that the ER was isolated with high purity. Protein abundance of ribosomal proteins was decreased under both stresses compared to control condition; however, the percentage of increased ribosomes was two-fold higher in flooding compared to drought. The ER proteins related to protein glycosylation and signaling were in response to both stresses. Compared to control condition, calnexin was decreased under both stresses; however, protein disulfide isomerase-like proteins and heat shock proteins were markedly decreased under flooding and drought conditions, respectively. Furthermore, fewer glycoproteins and higher levels of cytosolic calcium were identified under both stresses compared to control condition. These results suggest that reduced accumulation of glycoproteins in response to both stresses might be due to dysfunction of protein folding through calnexin/calreticulin cycle. Additionally, the increased cytosolic calcium levels induced by flooding and drought stresses might disturb the ER environment for proper protein folding in soybean root tips.