Single-cell RNA sequencing of batch Chlamydomonas cultures reveals heterogeneity in their diurnal cycle phase.

The photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of its ease of culture in the laboratory. Genomic and systems biology approaches have previously described transcriptome responses to environmental changes using bulk data, thus representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing by the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample natural algal communities. Second, scRNA-seq successfully separated single cells into nonoverlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest photosynthesis and cell division to economize resources. Notably, these groups of cells not only recapitulated known patterns observed with bulk RNA-seq but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We document experimentally that bulk RNA-seq data represent an average of typically hidden heterogeneity in the population.

Sorting out small RNAs.

Small RNAs carry out their functions by guiding Argonaute (AGO) proteins to their targets. Diverse types of small RNAs and multiple AGO proteins exist in most eukaryotic species, but how small RNAs are sorted into specific AGO complexes remains unclear. Two papers in this issue (Mi et al., 2008; Montgomery et al., 2008) now reveal the importance of the 5' terminal nucleotide of the small RNA in the sorting process in Arabidopsis.

Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide.

Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.

A low-bias and sensitive small RNA library preparation method using randomized splint ligation.

Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2'-O-methyl modifications (2'OMe) on the 3' terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2'OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2'OMe modified RNA with new levels of detail.

A universal method for the rapid isolation of all known classes of functional silencing small RNAs.

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.

Genome-Wide Analysis Identified a Set of Conserved lncRNAs Associated with Domestication-Related Traits in Rice.

Crop domestication, which gives rise to a number of desirable agronomic traits, represents a typical model system of plant evolution. Numerous genomic evidence has proven that noncoding RNAs such as microRNAs and phasiRNAs, as well as protein-coding genes, are selected during crop domestication. However, limited data shows plant long noncoding RNAs (lncRNAs) are also involved in this biological process. In this study, we performed strand-specific RNA sequencing of cultivated rice Oryza sativa ssp. japonica and O. sativa ssp. indica, and their wild progenitor O. rufipogon. We identified a total of 8528 lncRNAs, including 4072 lncRNAs in O. rufipogon, 2091 lncRNAs in japonica rice, and 2365 lncRNAs in indica rice. The lncRNAs expressed in wild rice were revealed to be shorter in length and had fewer exon numbers when compared with lncRNAs from cultivated rice. We also identified a number of conserved lncRNAs in the wild and cultivated rice. The functional study demonstrated that several of these conserved lncRNAs are associated with domestication-related traits in rice. Our findings revealed the feature and conservation of lncRNAs during rice domestication and will further promote functional studies of lncRNAs in rice.

Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess Their Role in RNA Spray-Based Crop Protection.

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5'-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.

Effect of small coding genes on the circadian rhythms under elevated CO2 conditions in plants.

Increase in atmospheric carbon dioxide (CO2) has a significant effect on plant growth and development. To explore the elevated-CO2 response, we generated transcriptional profiles over a time course (2 h-14 days) of exposure to elevated CO2 in Arabidopsis thaliana. Genes related to photosynthesis were down-regulated and circadian rhythm-related genes were abnormally regulated in the early to middle phase of elevated CO2 exposure. To understand the novel mechanism of elevated CO2 signaling, we focused on 42 unknown small coding genes that showed differential expression patterns under elevated CO2 conditions. Four transgenic plants overexpressing the small coding gene exhibited a growth-defective phenotype under elevated CO2 but not under current CO2. Transcriptome analysis showed that circadian rhythm-related genes were commonly regulated in four transgenic plants. These circadian rhythm-related genes were transcribed in the dark when CO2 concentrations in the leaf was high. Taken together, our identified four small coding genes are likely to participate in elevated CO2 signaling to the circadian rhythm.

GSM2, a transaldolase, contributes to reactive oxygen species homeostasis in Arabidopsis.

Plants are exposed to various environmental cues that lead to reactive oxygen species (ROS) accumulation. ROS production and detoxification are tightly regulated to maintain balance. Although studies of glucose (Glc) are always accompanied by ROS in animals, the role of Glc in respect of ROS in plants is unclear. We isolated gsm2 (Glc-hypersensitive mutant 2), a mutant with a notably chlorotic-cotyledon phenotype. The chloroplast-localized GSM2 was characterized as a transaldolase in the pentose phosphate pathway. With 3% Glc treatment, fewer or no thylakoids were observed in gsm2 cotyledon chloroplasts than in wild-type cotyledon chloroplasts, suggesting that GSM2 is required for chloroplast protection under stress. gsm2 also showed evaluated accumulation of ROS with 3% Glc treatment and was more sensitive to exogenous H2O2 than the wild type. Gene expression analysis of the antioxidant enzymes in gsm2 revealed that chloroplast damage to gsm2 cotyledons results from the accumulation of excessive ROS in response to Glc. Moreover, the addition of diphenyleneiodonium chloride or phenylalanine can rescue Glc-induced chlorosis in gsm2 cotyledons. This work suggests that GSM2 functions to maintain ROS balance in response to Glc during early seedling growth and sheds light on the relationship between Glc, the pentose phosphate pathway and ROS.

Plasma Membrane Aquaporin Members PIPs Act in Concert to Regulate Cold Acclimation and Freezing Tolerance Responses in Arabidopsis thaliana.

Aquaporins play a major role in plant water uptake at both optimal and environmentally stressed conditions. However, the functional specificity of aquaporins under cold remains obscure. To get a better insight to the role of aquaporins in cold acclimation and freezing tolerance, we took an integrated approach of physiology, transcript profiling and cell biology in Arabidopsis thaliana. Cold acclimation resulted in specific upregulation of PIP1;4 and PIP2;5 aquaporin (plasma membrane intrinsic proteins) expression, and immunoblotting analysis confirmed the increase in amount of PIP2;5 protein and total amount of PIPs during cold acclimation, suggesting that PIP2;5 plays a major role in tackling the cold milieu. Although single mutants of pip1;4 and pip2;5 or their double mutant showed no phenotypic changes in freezing tolerance, they were more sensitive in root elongation and cell survival response under freezing stress conditions compared with the wild type. Consistently, a single mutation in either PIP1;4 or PIP2;5 altered the expression of a number of aquaporins both at the transcriptional and translational levels. Collectively, our results suggest that aquaporin members including PIP1;4 and PIP2;5 function in concert to regulate cold acclimation and freezing tolerance responses.

Transcriptomic Analysis of the Grapevine LEA Gene Family in Response to Osmotic and Cold Stress Reveals a Key Role for VamDHN3.

Late embryogenesis abundant (LEA) proteins comprise a large family that plays important roles in the regulation of abiotic stress, however, no in-depth analysis of LEA genes has been performed in grapevine to date. In this study, we analyzed a total of 52 putative LEA genes in grapevine at the genomic and transcriptomic level, compiled expression profiles of four selected (V. amurensis) VamLEA genes under cold and osmotic stresses, and studied the potential function of the V. amurensis DEHYDRIN3 (VamDHN3) gene in grapevine callus. The 52 LEA proteins were classified into seven phylogenetic groups. RNA-seq and quantitative real-time PCR results demonstrated that a total of 16 and 23 VamLEA genes were upregulated under cold and osmotic stresses, respectively. In addition, overexpression of VamDHN3 enhanced the stability of the cell membrane in grapevine callus, suggesting that VamDHN3 is involved in osmotic regulation. These results provide fundamental knowledge for the further analysis of the biological roles of grapevine LEA genes in adaption to abiotic stress.

Salt-Induced Stability of SR1/CAMTA3 mRNA Is Mediated by Reactive Oxygen Species and Requires the 3' End of Its Open Reading Frame.

Soil salinity, a prevalent abiotic stress, causes enormous losses in global crop yields annually. Previous studies have shown that salt stress-induced reprogramming of gene expression contributes to the survival of plants under this stress. However, mechanisms regulating gene expression in response to salt stress at the posttranscriptional level are not well understood. In this study, we show that salt stress increases the level of Signal Responsive 1 (SR1) mRNA, a member of signal-responsive Ca2+/calmodulin-regulated transcription factors, by enhancing its stability. We present multiple lines of evidence indicating that reactive oxygen species generated by NADPH oxidase activity mediate salt-induced SR1 transcript stability. Using mutants impaired in either nonsense-mediated decay, XRN4 or mRNA decapping pathways, we show that neither the nonsense-mediated mRNA decay pathway, XRN4 nor the decapping of SR1 mRNA is required for its decay. We analyzed the salt-induced accumulation of eight truncated versions of the SR1 coding region (∼3 kb) in the sr1 mutant background. This analysis identified a 500-nt region at the 3' end of the SR1 coding region to be required for the salt-induced stability of SR1 mRNA. Potential mechanisms by which this region confers SR1 transcript stability in response to salt are discussed.

FvbHLH9 Functions as a Positive Regulator of Anthocyanin Biosynthesis by Forming a HY5-bHLH9 Transcription Complex in Strawberry Fruits.

Anthocyanin accumulation is transcriptionally regulated by the MYB-bHLH-WD40 complex. Light is indispensable for anthocyanin accumulation, and light-inducible MYB and HY5 were considered to promote anthocyanin accumulation in many fruits. Whether and how light-inducible bHLH transcription factor and HY5 regulate anthocyanin synthesis in strawberry is unknown. In this study, we identified a bHLH transcription factor, FvbHLH9, which was induced by light as well as FvHY5, and found that, similar to FvHY5, the transient overexpression and interference FvbHLH9 in strawberry fruits can promote and decrease anthocyanin accumulation, respectively, indicating FvbHLH9 functions as a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both FvHY5 and FvbHLH9 specifically bind to the promoter region of some key enzyme genes, including FvDFR, and the expression of FvDFR was activated through the heterodimer formation between FvHY5 and FvbHLH9. Finally, we confirmed that FvbHLH9-promoted anthocyanin accumulation is dependent on HY5-bHLH heterodimerisation in Arabidopsis. Our findings provide insights into a mechanism involving the synergistic regulation of light-dependent coloration and anthocyanin biosynthesis via a HY5-bHLH heterodimer formed by the interaction of FvHY5 and FvbHLH9 in strawberry fruits.

Functional Characterization of Three Novel Genes Encoding Diacylglycerol Acyltransferase (DGAT) from Oil-Rich Tubers of Cyperus esculentus.

Cyperus esculentus is probably the only plant that is known to accumulate large amounts of oil in its tubers. However, the underlying metabolic mechanism and regulatory factors involved in oil synthesis of tubers are still largely unclear. In this study, one gene encoding type I diacylglycerol acyltransferase (DGAT) (CeDGAT1) and two genes encoding type II DGAT (CeDGAT2a and CeDGAT2b) from C. esculentus were identified and functionally analyzed. All three DGAT genes were found to be expressed in tuber, root and leaf tissues. CeDGAT1 is highly expressed in roots and leaves, whereas CeDGAT2b is dominantly expressed in tubers. Furthermore, the temporal expression pattern of CeDGAT2b is well coordinated with the oil accumulation in developing tubers. When each CeDGAT was heterologously expressed in triacylglycerol (TAG)-deficient mutant of Saccharomyces cerevisiae, Arabidopsis thaliana wild type or its TAG1 mutant with AtDGAT1 disruption, only CeDGAT2b showed the ability to restore TAG biosynthesis with lipid body formation in yeast mutant, enhance seed oil production of Arabidopsis wild type and rescue multiple seed phenotypes of TAG1 mutant. In addition, CeDGAT2b was shown to have a substrate preference for unsaturated fatty acids toward TAG synthesis. Taken together, our results indicated that CeDGAT2b from C. esculentus is an actively functional protein and is most likely the major contributor to tuber oil biosynthesis containing common fatty acids, in contrast to oil-rich seeds and fruits where DGAT1 plays a more central role than DGAT2 in oil production accumulating normal fatty acids, whereas DGAT2 is a primary regulator for oil synthesis rich in unusual fatty acids.

Dynamic changes in the expression pattern of miRNAs and associated target genes during coconut somatic embryogenesis.

MAIN CONCLUSION: Genome-wide analysis of small RNAs identifies somatic embryogenesis- specific miRNAs and their targets and provides novel insights into the mechanisms governing somatic embryogenesis in coconut, a highly in vitro recalcitrant species. Coconut, a major plantation crop of the tropics is recalcitrant to in vitro culture with a very low rate of somatic embryo turnover. Clonal propagation to enhance the production of high yielding, disease-free planting material in coconut has remained a distant reality. To better understand the molecular basis of this recalcitrance and to throw light on the complex regulatory network involved in the transition of coconut somatic cells to embryogenic calli, genome-wide profiling of small RNAs from embryogenic (EC) and non-embryogenic calli (NEC) was undertaken using Illumina Hiseq 2000 platform. We have identified a total of 110 conserved miRNAs (representing 46 known miRNA families) in both types of calli. In addition, 97 novel miRNAs (48 specific to EC, 21 specific to NEC and 28 common to both the libraries) were also identified. Among the conserved miRNAs, 10 were found to be differentially expressed between NEC and EC libraries with a log2 fold change > 2 following RPM-based normalization. miR156f, miR167c, miR169a, miR319a, miR535a, and miR5179 are upregulated and miR160a, miR166a, miR171a, and miR319b are down-regulated in NEC. To confirm the differential expression pattern and their regulatory role in SE, the expression patterns of miRNAs and their putative targets were analyzed using qRT- PCR and most of the analyzed miRNA-target pairs showed inverse correlation during somatic embryogenesis. Selected targets were further validated by RNA ligase mediated rapid amplification of 5' cDNA ends (5'RLM-RACE). Our data suggest that a few conserved miRNAs and species-specific miRNAs act in concert to regulate the process of somatic embryogenesis in coconut. The results of this study provide the first overview into the regulatory landscape of somatic embryogenesis in coconut and possible strategies for fine-tuning or reprogramming to enhance somatic embryo turn over in coconut.

Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance.

Element content and expression of genes of interest on single cell types, such as stomata, provide valuable insights into their specific physiology, improving our understanding of leaf gas exchange regulation. We investigated how far differences in stomatal conductance (gs ) can be ascribed to changes in guard cells functioning in amphistomateous leaves. gs was measured during the day on both leaf sides, on well-watered and drought-stressed trees (two Populus euramericana Moench and two Populus nigra L. genotypes). In parallel, guard cells were dissected for element content and gene expressions analyses. Both were strongly arranged according to genotype, and drought had the lowest impact overall. Normalizing the data by genotype highlighted a structure on the basis of leaf sides and time of day both for element content and gene expression. Guard cells magnesium, phosphorus, and chlorine were the most abundant on the abaxial side in the morning, where gs was at the highest. In contrast, genes encoding H+ -ATPase and aquaporins were usually more abundant in the afternoon, whereas genes encoding Ca2+ -vacuolar antiporters, K+ channels, and ABA-related genes were in general more abundant on the adaxial side. Our work highlights the unique physiology of each leaf side and their analogous rhythmicity through the day.

Identification of drought resistant miRNA in Macleaya cordata by high-throughput sequencing.

Drought is one of the most serious factors affecting crop yields in the world. Macleaya cordata (Willd.) is a draught-tolerant medicinal plant that has been proposed as a pioneer crop to be cultivated in arid areas. However, the exact molecular mechanisms through which M. cordata responds to draught stress remain elusive. In recent years, microRNA (miRNAs) in plants have been associated with stress response. Based on these findings, the current study aimed to shed light on the potential regulatory roles of miRNAs in the draught tolerance of M. cordata by employing high-throughput RNA sequencing and degradation sequencing. Six M. cordata plants were randomly divided into two equal experiment groups, including one draught group and one control group. High-throughput sequencing of the M. cordata samples led to the identification of 895 miRNAs, of which 18 showed significantly different expression levels between the two groups. PsRobot analysis and degradation sequencing predicted the differential miRNAs to target 59 and 36 genes, respectively. Functional analysis showed that 38 of the predicted genes could be implicated in the modulation of stress response. Four miRNAs and eight target genes were selected for quantitative real-time polymerase chain reaction (qRT-PCR) validation. The expression trend of each miRNA analyzed by qRT-PCR was consistent with that determined by sequencing, and was negatively correlated with those of its target genes. The results of our current study supported the involvement of miRNAs in the draught tolerance of M. cordata and could pave the way for further investigation into the related regulatory mechanisms.

Drought-induced disturbance of carbohydrate metabolism in anthers and male abortion of two Gossypium hirsutum cultivars differing in drought tolerance.

KEY MESSAGE: Cotton pollen abortion, under drought stress, was closely associated with changes in anther carbohydrate metabolism, and pollen abortion rate due to drought was higher in drought-sensitive cultivars than drought-tolerant cultivars. Cotton reproductive failure under drought stress is intrinsically connected with altered male fertility, however, studies investigating the effect of drought stress on cotton male fertility are nonexistent. Thus, a drought stress experiment was conducted with two cotton cultivars, differing in drought tolerance, to study pollen fertility and anthers' physiology. Results indicated that drought stress reduced pollen fertility of both cultivars due to decreases in anther starch and adenosine triphosphate (ATP) synthesis. Lower assimilate supply capacity in conjunction with impaired activities of ADP-glucose pyrophosphorylase and soluble starch synthase were the main reasons for the decreased starch levels in drought-stressed anthers. The decreased activities of sucrose synthetase and acid invertase were responsible for the higher sucrose level in drought-stressed anthers than well-watered anthers and the changing trend of sucrose was intensified by the decreased expressions of sucrose synthase genes (GhSusA, GhSusB, GhSusD) and acid invertase genes (GhINV1, GhINV2). However, despite sucrose degradation being limited in drought-stressed anthers, glucose level was higher in droughted anthers than well-watered ones, and that might be attributed to the down-regulated respiration since decreased anther ATP levels were detected in drought-stressed plants. Furthermore, compared to the drought-tolerant cultivar, pollen fertility was more suppressed by drought stress for the drought-sensitive cultivar, and that was attributed to the larger decrease in starch and ATP contents.

ABA signaling is negatively regulated by GbWRKY1 through JAZ1 and ABI1 to affect salt and drought tolerance.

KEY MESSAGE: GbWRKY1 can function as a negative regulator of ABA signaling via JAZ1 and ABI1, with effects on salt and drought tolerance. WRKY transcription factors play important roles in plant development and stress responses. GbWRKY1 was initially identified as a defense-related gene in cotton and negatively regulates the response to fungal pathogens by activating the expression of JAZ1. Here, we characterized the role of GbWRKY1, an orthologue of the Arabidopsis gene AtWRKY75, in abiotic stress (salt and drought) and established novel connection between JAZ1 and ABA signaling in Arabidopsis. GbWRKY1 is nucleus localized and its expression is significantly induced by treatment with ABA and osmotic stresses NaCl and PEG. Increased levels of expression of GbWRKY1 in transgenic Arabidopsis enhance sensitivity to salt and drought as revealed by seed germination tests and soil stress experiments. Similarly, GbWRKY1 overexpression cotton plants also display increased sensitivity to PEG treatment and drought. Expression analysis shows that the induction of two ABA responsive genes, RAB18 and RD29A by NaCl, mannitol, and ABA treatment is significantly impaired in GbWRKY1 overexpression Arabidopsis lines. GbWRKY1 overexpression Arabidopsis displays a strong ABA-insensitive phenotype at both germination and early stages of seedling development. Further genetic evidence suggested that the ABA-insensitive phenotype of GbWRKY1 overexpression Arabidopsis was dependent on JAZ1, and overexpression of JAZ1 also displayed an ABA-insensitive phenotype. In addition, yeast two hybrid and bimolecular fluorescence complementation assays showed that JAZ1 directly interacts with ABI1, a key negative regulator of ABA signaling. We, therefore, demonstrate that GbWRKY1 acts as a negative regulator of ABA signaling, through an interaction network involving JAZ1 and ABI1, to regulate salt and drought tolerance.

Transcriptome analysis of two soybean cultivars identifies an aluminum respon-sive antioxidant enzyme GmCAT1.

This study investigated the antioxidant defense system involved in the tolerance of soybean (Glycine max) to aluminum (Al) stress. Physiological assays showed that the amount of superoxide free radicals (O2 -), hydrogen peroxide (H2O2), and malondialdehyde (MDA) were significantly lower in an Al-resistant soybean cultivar (cv. PI416937) than in an Al-sensitive soybean cultivar (cv. Huachun18). Comparative analysis of microarray data from both genotypes following Al-stress treatment revealed that the expression of a series of antioxidant enzymes genes was induced in the Al-resistant cultivar. The quantitative real time-PCR (qRT-PCR) assay showed that the transcript levels of genes encoding antioxidant enzymes, including GmCAT1, GmPOD1, GmGST1, GmAPX, GmGSH1, and GmSOD, were higher in the Al-resistant cultivar than in the Al-sensitive cultivar in Al-stress conditions. Furthermore, GmCAT1-overexpressing Arabidopsis plants had improved tolerance to Al-stress and lower O2 -, H2O2, and MDA contents than wild-type plants. Therefore, providing evidence that the antioxidant defense system is essential for Al tolerance in soybean. ABBREVIATIONS: Al: aluminum; O2 -: superoxide free radicals; ROS: reactive oxygen species; H2O2: hydrogen peroxide; MDA: malondialdehyde; qRT-PCR: quantitative reverse transcription polymerase chain reaction; GO: gene ontology; WT: wild type; MS medium: Murashige and Skoog medium.