Retarded Root Growth Research Articles

Arabidopsis Pentatricopeptide Repeat Protein GEND2 Participates in Mitochondrial RNA Editing

Abstract In Arabidopsis, RNA editing alters more than 500 cytidines (C) to uridines (U) in mitochondrial transcripts, a process involving the family of pentatricopeptide repeat (PPR) proteins. Here, we report a previously uncharacterized mitochondrial PLS-type PPR protein, GEND2, which functions in the mitochondrial RNA editing. The T-DNA insertion in the 5′-untranslated region of GEND2, referred to as gend2-1, results in defective root development compared to wild-type (WT) plants. A comprehensive examination of mitochondrial RNA-editing sites revealed a significant reduction in the gend2-1 mutant compared to WT plants, affecting six specific mitochondrial RNA editing sites, notably within the mitochondrial genes CcmFn-1, RPSL2 and ORFX. These genes encode critical components of cytochrome protein maturation pathway, mitochondrial ribosomal subunit and twin arginine translocation subunits, respectively. Further analysis of the transcriptional profile of the gend2-1 mutant and WT revealed a striking induction of expression in a cluster of genes associated with mitochondrial dysfunction and regulated by ANAC017, a key regulator coordinating organelle functions and stress responses. Intriguingly, the gend2-1 mutation activated an ANAC017-dependent signaling aimed at countering cell wall damage induced by cellulose synthase inhibitors, as well as an ANAC017-independent pathway that retarded root growth under normal condition. Collectively, our findings identify a novel mitochondrial PLS-type PPR protein GEND2, which participates in the editing of six specific mitochondrial RNA editing sites. Furthermore, the gend2-1 mutation triggers two distinct pathways in plants: an ANAC017-dependent pathway and ANAC017-independent pathway.

Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5–7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

Ethylene and ROS mediate root growth inhibition induced by the endocrine disruptor bisphenol A (BPA)

Bisphenol A (BPA) functions as a detrimental substance that disrupts the endocrine system in animals while also impeding the growth and development of plants. In our previous study, we demonstrated that BPA hinders the growth of roots in Arabidopsis by diminishing cell division and elongation, which is ascribed to the increased accumulation and redistribution of auxin. Here, we examined the mediation of ROS and ethylene in BPA-induced auxin accumulation and root growth inhibition. BPA enhanced ROS levels, and ROS increased auxin contents but reduced cell division activity and the expression of EXPA8 involved in root elongation. ROS scavenger treatment reversed BPA-triggered root growth retardation, auxin accumulation, and cell division inhibition. In addition, BPA induced ethylene, and ethylene synthesis inhibitor treatment reversed BPA-triggered root growth retardation and auxin accumulation. Taken together, ROS and ethylene are involved in BPA-inhibited cell elongation and cell division by mediating auxin accumulation and redistribution.

Jasmonic Acid Effect on Cucumis sativus L. Growth Is Related to Inhibition of Plasma Membrane Proton Pump and the Uptake and Assimilation of Nitrates.

When plants are exposed to environmental stress, their growth is inhibited. Under such conditions, controlled inhibition of growth is beneficial for plant survival. Jasmonic acid (JA) is a well-known phytohormone that limits plant growth, which has been confirmed in several species. However, its role in cucumber seedlings has not yet been comprehensively investigated. For this reason, we aimed to determine the involvement of JA in the regulation of proteins crucial for growth including plasma membrane proton pump (PM H+-ATPase), PM nitrate transporters, and nitrate reductase (NR). Treatment of cucumber seedlings with JA not only limited their growth but also increased the H2O2 content in their roots. The main sources of ROS generated for signalling purposes are PM NADPH oxidase (RBOH) and superoxide dismutase (SOD). Exposure of seedlings to JA induced the expression of some CsRBOH and SOD encoding genes, suggesting that ROS signalling can be activated by JA. As a consequence of JA exposure, the activity of all analysed proteins was inhibited and the expression of their genes was modified. The results indicate that reduction of PM H+-ATPase activity and the related decrease in nitrate uptake and assimilation are responsible for the root growth retardation of JA-treated plants.

OsPEX1, an extensin-like protein, negatively regulates root growth in a gibberellin-mediated manner in rice

Leucine-rich repeat extensins (LRXs) are required for plant growth and development through affecting cell growth and cell wall formation. LRX gene family can be classified into two categories: predominantly vegetative-expressed LRX and reproductive-expressed PEX. In contrast to the tissue specificity of Arabidopsis PEX genes in reproductive organs, rice OsPEX1 is also highly expressed in roots in addition to reproductive tissue. However, whether and how OsPEX1 affects root growth is unclear. Here, we found that overexpression of OsPEX1 retarded root growth by reducing cell elongation likely caused by an increase of lignin deposition, whereas knockdown of OsPEX1 had an opposite effect on root growth, indicating that OsPEX1 negatively regulated root growth in rice. Further investigation uncovered the existence of a feedback loop between OsPEX1 expression level and GA biosynthesis for proper root growth. This was supported by the facts that exogenous GA3 application downregulated transcript levels of OsPEX1 and lignin-related genes and rescued the root developmental defects of the OsPEX1 overexpression mutant, whereas OsPEX1 overexpression reduced GA level and the expression of GA biosynthesis genes. Moreover, OsPEX1 and GA showed antagonistic action on the lignin biosynthesis in root. OsPEX1 overexpression upregulated transcript levels of lignin-related genes, whereas exogenous GA3 application downregulated their expression. Taken together, this study reveals a possible molecular pathway of OsPEX1mediated regulation of root growth through coordinate modulation of lignin deposition via a negative feedback regulation between OsPEX1 expression and GA biosynthesis.

Cytogenotoxic effects of 3-epicaryoptin in Allium cepa L. root apical meristem cells.

Diterpenoid 3-epicaryoptin (C26H36O9) is abundant in the leaves of Clerodendrum inerme, a traditionally used medicinal plant, and has insect antifeedant activities. Here, we aim to explore the cytogenotoxic effects of compound 3-epicaryoptin in Allium cepa root apical meristem cells. 3-epicaryoptin (concentrations of 100, 150, and 200µgmL-1) and the standard compound colchicine (200µgmL-1) were applied to A. cepa roots for 2, 4, and 4 + 16h (4-h treatment followed by 16-h recovery). Cytogenotoxicity was analyzed by studying the root growth retardation (RGR), mitotic index (MI), and chromosomal aberrations. The result showed statistically significant (p < 0.01), concentration-dependent RGR effects of 3-epicaryoptin treatment compared with the negative control. A study of cell frequency in different phases of cell division observed a significant (p < 0.001) increase in the metaphase cell percentage (66.2 ± 0.58%, 150µgmL-1), which subsequently caused an increase in the frequency of MI (12.29 ± 0.34%, 150µgmL-1) at 4h of 3-epicaryoptin treatment and that was comparable with the colchicine action. The cytological study revealed that the 3-epicaryoptin treatment could induce different types of chromosomal abnormalities, such as colchicine-like metaphase, vagrant chromosomes, sticky chromosomes, anaphase bridge, lagging chromosomes, multipolar anaphase-telophase, and an increased frequency of micronuclei and polyploid cells. These findings indicate that 3-epicaryoptin is cytogenotoxic, and thus, C. inerme should be used with caution in traditional medicine.

Tea plant roots respond to aluminum-induced mineral nutrient imbalances by transcriptional regulation of multiple cation and anion transporters

BackgroundTea is one of the most popular non-alcoholic beverages in the world for its flavors and numerous health benefits. The tea tree (Camellia sinensis L.) is a well-known aluminum (Al) hyperaccumulator. However, it is not fully understood how tea plants have adapted to tolerate high concentrations of Al, which causes an imbalance of mineral nutrition in the roots.ResultsHere, we combined ionomic and transcriptomic profiling alongside biochemical characterization, to probe the changes of metal nutrients and Al responsive genes in tea roots grown under increasing concentrations of Al. It was found that a low level of Al (~ 0.4 mM) maintains proper nutrient balance, whereas a higher Al concentration (2.5 mM) compromised tea plants by altering micro- and macro-nutrient accumulation into roots, including a decrease in calcium (Ca), manganese (Mn), and magnesium (Mg) and an increase in iron (Fe), which corresponded with oxidative stress, cellular damage, and retarded root growth. Transcriptome analysis revealed more than 1000 transporter genes that were significantly changed in expression upon Al exposure compared to control (no Al) treatments. These included transporters related to Ca and Fe uptake and translocation, while genes required for N, P, and S nutrition in roots did not significantly alter. Transporters related to organic acid secretion, together with other putative Al-tolerance genes also significantly changed in response to Al. Two of these transporters, CsALMT1 and CsALS8, were functionally tested by yeast heterologous expression and confirmed to provide Al tolerance.ConclusionThis study shows that tea plant roots respond to high Al-induced mineral nutrient imbalances by transcriptional regulation of both cation and anion transporters, and therefore provides new insights into Al tolerance mechanism of tea plants. The altered transporter gene expression profiles partly explain the imbalanced metal ion accumulation that occurred in the Al-stressed roots, while increases to organic acid and Al tolerance gene expression partly explains the ability of tea plants to be able to grow in high Al containing soils. The improved transcriptomic understanding of Al exposure gained here has highlighted potential gene targets for breeding or genetic engineering approaches to develop safer tea products.

Whole-genome resequencing of Sorghum bicolor and S. bicolor \xd7 S. halepense lines provides new insights for improving plant agroecological characteristics

Sorghum (Sorghum bicolor L. (Moench)) is the world’s fifth economically most important cereal and is a staple particularly in the semi-arid tropics of Africa and Asia. Genetic gains in this crop can benefit from wild relatives such as Sorghum halepense. Genome sequences including those from this wild species can boost the study of genome-wide and intraspecific variation for dissecting the genetic basis and improving important traits in sorghum. The whole-genome resequencing carried out in this work on a panel of 172 populations of S. bicolor and S. bicolor × S. halepense (SbxSh) advanced lines generated a total of 567,046,841 SNPs, 91,825,474 indels, 1,532,171 SVs, and 4,973,961 CNVs. Clearly, SbxSh accumulated more variants and mutations with powerful effects on genetic differentiation. A total of 5,548 genes private to SbxSh mapped to biological process GO enrichment terms; 34 of these genes mapped to root system development (GO: 0022622). Two of the root specific genes i.e., ROOT PRIMORDIUM DEFECTIVE 1 (RPD1; GeneID: 8054879) and RETARDED ROOT GROWTH (RRG, GeneID: 8072111), were found to exert direct effect on root growth and development. This is the first report on whole-genome resequencing of a sorghum panel that includes S. halepense genome. Mining the private variants and genes of this wild species can provide insights capable of boosting sorghum genetic improvement, particularly the perenniality trait that is compliant with agroecological practices, sustainable agriculture, and climate change resilience.

Exogenous proanthocyanidins improve tolerance of Cu-toxicity by amelioration of oxidative damage and re-programming of gene expression in Medicago sativa.

Excess copper (Cu) in soil due to industrial and agricultural practices can result in reduced plant growth. Excess Cu resulted in severely retarded root growth with severe discoloration of Alfalfa (Medicago sativa) and Medicago truncatula. Growth in the presence of hydrogen peroxide resulted in similar symptoms that could be partially recovered by the addition of the reductant ascorbic acid revealing damage was likely due to oxidative stress. The addition of proanthocyanidins (PAs) in the presence of Cu prevented much of the damage, including plant growth and restoration of lignin synthesis which was inhibited in the presence of excess Cu. Transcriptome analyses of the impact of excess Cu and the amelioration after PAs treatment revealed that changes were enriched in functions associated with the cell wall and extracellular processes, indicating that inhibition of cell wall synthesis was likely the reason for retarded growth. Excess Cu appeared to induce a strong defense response, along with alterations in the expression of a number of genes encoding transcription factors, notably related to ethylene signaling. The addition of PAs greatly reduced this response, and also induced novel genes that likely help ameliorate the effects of excess Cu. These included induction of genes involved in the last step of ascorbic acid biosynthesis and of enzymes involved in cell wall synthesis. Combined, these results show that excess Cu causes severe oxidative stress damage and inhibition of cell wall synthesis, which can be relieved by the addition of PAs.

In vitro and in vivo activity analysis of poplar CLE dodecapeptides that are most divergent from Arabidopsis counterparts

Intercellular communication mediated by the plant-specific CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) family members is one of the fundamental mechanisms coordinating the development of complex bodies of plants. In this work, we chose 8 out of 38 putative CLE dodecapeptides encoded in the genome of P. trichocarpa based on their lowest sequence similarity with Arabidopsis CLE peptides, and investigated how such sequence variations affect their functional characteristics. In group 1, PtCLE16p faithfully retained the AtCLE1-7p activity, while PtCLE49p reversed the root-enhancing effect to an inhibitory one with two extra amino acid substitutions, which might have disrupted the capacity of PtCLE49p to recognize the corresponding receptors. In group 2, PtCLE9p conferred Arabidopsis with retarded root growth and suppressed phloem differentiation in a negative dominant manner just like AtCLE25G6T did. PtCLE9p enhanced the vegetative growth in both basal and aerial rosettes by regulating the expression of AERIAL ROSETTE 1 (ART1) and FRIGIDA (FRI) as well as the downstream FLOWERING LOCUS C (FLC) genes. In group 3, PtCLE34p and PtCLE5p slightly promoted primary root growth, while PtCLE40p revealed CLV3p-like and TDIF activity in root and hypocotyls, respectively. The remaining PtCLE18p in group 4 dramatically disturbed the expression of WOX5 and promoted the development of root hairs by repressing the expression of GLABRA2 (GL2) gene, which encoded a negative regulator of epidermal cells differentiation towards root hairs. In summary, our data indicated that with significant functional conservation and common signaling machinery existing for CLE families of land plants, unique and diverse activities of CLE peptides have evolved to perform specific functions in different plant species.

Involvement of plasma membrane H+-ATPase in diamide-induced extracellular alkalization by roots from pea seedlings.

The plasma membrane H+-ATPase can be considered as a redox-dependent enzyme, because diamide-mediated inhibition of its hydrolytic and transport activities is accompanied by alkalization of the rhizosphere and retardation of root growth. Plasma membranes were isolated from roots of etiolated pea seedlings treated in the presence of an oxidant-diamide and an inhibitor of redox-sensitive protein phosphatase-phenylarsine oxide. Hydrolytic and proton transport activities of H+-ATPase were determined. The effects of diamide appeared in inhibition of both ATP hydrolysis and the proton transport. However, root treatment with phenylarsine oxide only slightly reduced Vmax, but did not affect ATP-dependent proton transport. The thiol groups of cysteines in the proteins can act as molecular targets for both compounds. However, treatment of isolated membranes with diamide or dithiothreitol did not have any effect on the H+ transport. It can be assumed that water-soluble diamide acts indirectly and its effects are not associated with oxidation of H+-ATPase cysteines. Therefore, plasmalemma was subjected to PEGylation-process where reduced cysteines available for PEG maleimide (5kDa) were alkylated. Detection of such cysteines was carried out by Western blot analysis with anti-ATPase antibodies. It was found that shifts in the apparent molecular weight were detected only for denaturated proteins. These data suggest that available thiols are not localized on the enzyme surfaces. BN-PAGE analysis showed that the molecular weights of the ATPase complexes are almost identical in all samples. Therefore, oligomerization is probably not the reason for the inhibition of ATPase activity. Roots treated with these inhibitors in vivo exhibited stunted growth; however, a strong alkaline zone around the roots was formed only in the presence of diamide. Involvement of H+-ATPase redox regulation in this process is discussed.

Host sunflower-induced silencing of parasitism-related genes confers resistance to invading Orobanche cumana

Orobanche cumana is a holoparasitic plant that attaches to host-plant roots and seriously reduces the yield of sunflower (Helianthus annuus L.). Effective control methods are lacking with only a few known sources of genetic resistance. In this study, a seed-soak agroinoculation (SSA) method was established, and recombinant tobacco rattle virus vectors were constructed to express RNA interference (RNAi) inducers to cause virus-induced gene silencing (VIGS) in sunflower. A host target gene HaTubulin was systemically silenced in both leaf and root tissues by the SSA-VIGS approach. Trans-species silencing of O. cumana genes were confirmed for 10 out of 11 target genes with silencing efficiency of 23.43%-92.67%. Knockdown of target OcQR1, OcCKX5, and OcWRI1 genes reduced the haustoria number, and silencing of OcEXPA6 caused further phenotypic abnormalities such as shorter tubercles and necrosis. Overexpression of OcEXPA6 caused retarded root growth in alfalfa (Medicago sativa). The results demonstrate that these genes play an important role in the processes of O. cumana parasitism. High-throughput small RNA (sRNA) sequencing and bioinformatics analyses unveiled the distinct features of target gene-derived siRNAs in O. cumana such as siRNA transitivity, strand polarity, hotspot region, and 21/22-nt siRNA predominance, the latter of which was confirmed by Northern blot experiments. The possible RNAi mechanism is also discussed by analyzing RNAi machinery genes in O. cumana. Taken together, we established an efficient host-induced gene silencing technology for both functional genetics studies and potential control of O. cumana. The ease and effectiveness of this strategy could potentially be useful for other species provided they are amenable to SSA.

The pre-mRNA splicing factor RDM16 regulates root stem cell maintenance in Arabidopsis.

Pre-mRNA (messenger RNA) splicing participates in the regulation of numerous biological processes in plants. For example, alternative splicing shapes transcriptomic responses to abiotic and biotic stress, and controls developmental programs. However, no study has revealed a role for splicing in maintaining the root stem cell niche. Here, a screen for defects in root growth in Arabidopsis thaliana identified an ethyl methane sulfonate mutant defective in pre-mRNA splicing (rdm16-4). The rdm16-4 mutant displays a short-root phenotype resulting from fewer cells in the root apical meristem. The PLETHORA1 (PLT1) and PLT2 transcription factor genes are important for root development and were alternatively spliced in rdm16-4 mutants, resulting in a disordered root stem cell niche and retarded root growth. The root cap of rdm16-4 contained reduced levels of cytokinins, which promote differentiation in the developing root. This reduction was associated with the alternative splicing of genes encoding cytokinin signaling factors, such as ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5 and ARABIDOPSIS RESPONSE REGULATORS (ARR1, ARR2, and ARR11). Furthermore, expression of the full-length coding sequence of ARR1 or exogenous cytokinin application partially rescued the short-root phenotype of rdm16-4. This reveals that the RDM16-mediated alternative splicing of cytokinin signaling components contributes to root growth.

Ginsenosides regulate adventitious root formation in Panax ginseng via a CLE45–WOX11 regulatory module

Adventitious root branching is vital to plant growth and regeneration, but the regulation of this process remains unclear. We therefore investigated how ginsenosides regulate adventitious root branching in Panax ginseng. Cell proliferation and adventitious root branching were decreased in the presence of ginsenoside Rb1 and a high concentration of ginsenoside Re, but increased when treating with a low concentration of Re. Moreover, the exogenous application of a synthetic dodeca-amino acid peptide that has a CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) motif corresponding to PgCLE45 retarded root growth in both ginseng and Arabidopsis. The root Re levels and the expression of the DDS, CYP716A47, and CYP716A53 genes that encode enzymes involved in ginsenoside synthesis were decreased in the presence of PgCLE45. The expression profiles of PgWOX and PgCLE genes were determined to further investigate the CLE-WOX signaling pathway. The levels of PgWOX11 transcripts showed an inverse pattern to PgCLE45 transcripts. Using yeast one-hybrid assay, EMSA, and ChIP assay, we showed that PgWOX11 bound to the PgCLE45 promoter, which contained the HD motif. Transient expression assay showed that PgWOX11 induced the expression of PgCLE45 in adventitious roots, while PgCLE45 suppressed the expression of PgWOX11. These results suggest that there is a negative feedback regulation between PgCLE45 and PgWOX11. Taken together, these data show that ginsenosides regulate adventitious root branching via a novel PgCLE45-PgWOX11 regulatory loop, providing a potential mechanism for the regulation of adventitious root branching.

Application of cytogenetic model Allium cepa for screening potential cytogenotoxicity of herbal-based hair dyes

Plant models may be useful as test organisms for initial screening of potential toxicity of personal care products. The objective of the present study was to assess the efficacy of the Allium cepa (common onion) test system as a bioanalytical tool for screening potential cytotoxicity and genotoxicity of herbal-based hair dye formulations. Exposure of black hair dye formulations for 48 hours resulted in root growth retardation and mitosis suppression in the root meristems of A. cepa bulbs indicating concentration dependent cytotoxicity. At the 72 hour post exposure, cytotoxic effects on the roots were reduced but not recovered completely signifying prolong toxic action of the hair dyes. The condensed nuclei was the most frequent nuclear abnormality found in the dye exposed root meristematic cells indicating the cell death process. Induction of micronuclei and chromosomal aberrations in the root meristematic cells even at the post exposure stage indicates persistent genotoxicity of the hair dyes which may be attributed to the interactive effects of chemical mixtures present in the commercial hair dye formulations. The results revealed that A. cepa test system is an effective bioanalytical tool for screening cytogenotoxicity of commercial hair dye formulations.

Toxic hazards of industrial waste receiving canal system in the lower catchment of Kelani River basin, Sri Lanka

Assessing toxic hazards associated with polluted riverine ecosystems is essential for the development of effective strategies for their management. The present study explored the combined utility of bioassay responses of the plant, Allium cepa and surface water physico-chemical characteristics to assess the toxic hazards of an industrial waste receiving canal system located in the lower catchment of the Kelani River basin, Sri Lanka. Surface water samples from seven sites, viz. Maha Ela upstream (Site A), Manikagara Ela (Site B), Manikagara Ela - Maha Ela confluence (Site C), Maha Ela downstream (Site D), Maha Ela - Kelani River confluence (Site E), River downreach (Site F) and upper-reach (Site R) were analysed on three occasions in 2015 covering dry and wet periods. Irrespective of the sampling periods, exposure of A. cepa bulbs to water from the Sites B, C, D and E resulted in root growth retardation and mitosis depression (p

Heavy metals analysis and toxicity evaluation of some textile and dyeing effluents in Kano, Nigeria using &lt;I&gt;Allium cepa&lt;/I&gt; bioassay

Insufficiencies in dyeing and textile processing coupled with incomplete sewage treatment system lead to indiscriminate discharge of large amount of dyestuff into water bodies mostly used for irrigation and fishing. These wastes are potentially mutagenic and some are even carcinogenic. In this research, cytotoxicity of three industrial effluents viz; African Textile Manufacturer (ATM), Kofar Naisa and Kofar Mata dyeing centres were evaluated using Allium cepa bioassay. The concentrations of some heavy metals (Cr, Cd, Zn, Fe, Cu, Co, Pb, Mn and Ni) were also determined. Three sets of small onion bulbs were cultivated in 25%, 50% 75% and 100% effluent concentrations (v/v) and a control for each site. Root tips from each bulb were harvested and processed for cytological studies using squashing technique. After 48hrs of exposure, cytotoxic effects of the effluents on root tips (compared to control) showed root growth retardation which was more apparent at higher concentrations. This indicated that, the root growth inhibition was concentration dependent. Number of dividing cells observed and the Mitotic Index (MI) were also concentration dependent. There were decrease in number of dividing cells and MI with increase in concentration of the treatment. The effective concentrations that caused 50% effect (EC50) was 95% for ATM and K/Naisa dyeing points while it was 100% in K/mata dyeing point. Diverse structural aberrations and abnormalities were observed ranging from chromosomal bridge, which was apparent even at low concentrations of the treatment to cytokinesis failure, Micronucleus (MN) and nuclear buds or vacuolated nuclei. Analysis of Variance (ANOVA) showed no significant difference (P&gt;0.05) in mean concentrations of Cr, Fe, Cu and Mn for K/Naisa and K/Mata dyeing centres, but there was statistical difference (P&lt;0.05) for these parameters in ATM. Likewise, there was significant difference (P&lt;0.05) in the root growth of A. cepa exposed to different concentrations of the effluent in ATM and K/Naisa sites but no statistical difference existed in root growth of A. cepa exposed to K/Mata effluents.&#x0D; Keywords: Allium cepa, Bioassay, Effluents, Cytotoxicity, Mitotic Index

Enhancing Abiotic Stress Response via a Kalanchoe fedtschenkoi NF‐Y Transcription Factor in C3 Plants

Global crop production is expected to decrease because of increasing abiotic stressors including extremes in temperature, water, light intensity, nutrients, and radiation due to climate change and anthropogenic activities. Increased abiotic stressors will alter the metabolism in C3 plants and thus negatively affect their growth and productivity. Developing C3 crop varieties with improved abiotic stress tolerances emerges as the most sustainable solution towards ensuring global food security under the predicted climate change scenario. Crassulacean Acid Metabolism (CAM) plants are considered well‐adapted to drought, heat, increased light intensities, higher UV irradiation, and are far more water‐use efficient compared to C3 photosynthesis plants. The use of abiotic stress‐responsive transcription factors (TFs) from CAM plants is one feasible approach to improve crop production under adverse abiotic stress conditions. We have identified a NF‐YB TF from the heat‐tolerant obligate CAM plant Kalanchoe fedtschenkoi as a possible regulator of abiotic stresses. This NF‐Y is a ubiquitous transcription factor that is expressed as one of three possible isoforms NF‐YA, NF‐YB, or NF‐YC. Their isoforms interact to form a heterotrimeric complex with each subunit playing key roles in the abiotic stress responses including drought, heat, freezing, and salinity. We are functionally characterizing this KfNF‐YB via overexpression in Arabidopsis thaliana to determine its role in abiotic stress responses. Preliminary results following in vitro salinity stress assay of T2 transgenic lines after 12 days in MS media with 200 mM NaCl indicate increased root growth, whereas the wild type had retarded root growth. In addition, transgenic lines had higher biomass dry weight compared to wild type after 12 days. T2 lines also have increased rosette size in two‐week old transgenic A. thaliana compared to wild type. We will discuss the implications of the preliminary results as a potential strategy to enhance multiple abiotic stress tolerance responses in C3 plants.Support or Funding InformationThis work is funded by South Dakota Board of Regents Grant #A18‐0015‐001 to BWM Wone and supported in part by U. S. Department of Energy, Office of Science, Genomic Science Program under award #DE‐SC0008834 to JC Cushman.

Defective Quiescent Center/AtTRS85 Encoding a TRAPPIII-specific Subunit Required for the Trans-golgi Network/Early Endosome Integrity is Essential for the Proper Root Development in Arabidopsis

Tethering factors mediate the initial contact between donor and acceptor membranes. Transport protein particle (TRAPP)III, a multisubunit tethering complex, controls both secretory pathway and autophagy in yeast. However, the roles of TRAPPIII is poorly understood in plants. We have isolated a recessive mutant displaying retarded root growth, defective root hair elongation, and compromised organization of quiescent center by introducing UAS tag in Arabidopsis thaliana and named defective quiescent center (dqc)-1. The tag localized in the 25th exon of At5g16280 encoding AtTRS85 specifying the TRAPPIII complex. Three additional T-DNA insertion mutants of dqc/attrs85 exhibited the defective gravitropic responses in addition to the weak dqc-1-like phenotypes. In dqc-2, the auxin-responsive DR5 expression in the columella was extended and an auxin efflux carrier PIN-FORMED1-GFP abnormally aggregated in the vacuole-like subcellular compartments. In the root epidermis of dqc-2, an early endosome (EE) marker was dispersed in the cytoplasm, a trans-Golgi network (TGN) marker localized in tonoplast, and a vacuole marker localized in the fragmented tonoplast. Furthermore, the endocytosis of FM4-64 in dqc-2 was compromised as evidenced by the decrease in the EE punctum formation, the rapid accumlation into the tonoplast, and the infrequent brefeldin A body formation. Together, DQC/AtTRS85 is essential for the integrity of TGN/EE and the vacuole fusion.

Multi-phased internalization of murine norovirus (MNV) in Arabidopsis seedlings and its potential correlation with plant defensive responses

Norovirus is a highly infectious human pathogen that causes acute foodborne diseases worldwide. As global diet patterns have begun to incorporate a higher consumption of fresh agricultural products, the internalization of norovirus into plants has emerged as a potential threat to human health. Here, we demonstrated that murine norovirus (MNV1) was internalized into Arabidopsis in multiple phases, and this internalization was correlated with Arabidopsis innate immunity responses. Under hydroponic conditions, continuous treatment of MNV1 retarded root growth and facilitated flower development of Arabidopsis without causing necrotic lesions. Examination of viral titers and RNA levels revealed that MNV1 was internalized into Arabidopsis in at least three different phases. In response to MNV1 treatment, the Arabidopsis defensive marker PR1 (a salicylic acid signaling marker) was transiently up-regulated at the early stage. PDF1.2, a jasmonic acid signaling marker, exhibited a gradual induction over time. Noticeably, Arabidopsis RNS1 (T2 ribonuclease) was rapidly induced by MNV1 and exhibited anti-correlation with the internalization of MNV1. Exposure to recombinant Arabidopsis RNS1 protein reduced the viral titers and degraded MNV1 RNA in vitro. In conclusion, the internalization of MNV1 into Arabidopsis was fluctuated by mutual interactions that were potentially regulated by Arabidopsis immune systems containing RNS1.