Two splice forms of OsbZIP1, a homolog of AtHY5, function to regulate skotomorphogenesis and photomorphogenesis in rice.

Plants possess well-developed light sensing mechanisms and signal transduction systems for regulating photomorphogenesis. ELONGATED HYPOCOTYL5 (HY5), a basic leucine zipper (bZIP) transcription factor, has been extensively characterized in dicots. In this study, we show that OsbZIP1 is a functional homolog of Arabidopsis (Arabidopsis thaliana) HY5 (AtHY5) and is important for light-mediated regulation of seedling and mature plant development in rice (Oryza sativa). Ectopic expression of OsbZIP1 in rice reduced plant height and leaf length without affecting plant fertility, which contrasts with OsbZIP48, a previously characterized HY5 homolog. OsbZIP1 is alternatively spliced, and the OsbZIP1.2 isoform lacking the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-binding domain regulated seedling development in the dark. Rice seedlings overexpressing OsbZIP1 were shorter than the vector control under white and monochromatic light conditions, whereas RNAi knockdown seedlings displayed the opposite phenotype. While OsbZIP1.1 was light-regulated, OsbZIP1.2 showed a similar expression profile in both light and dark conditions. Due to its interaction with OsCOP1, OsbZIP1.1 undergoes 26S proteasome-mediated degradation under dark conditions. Also, OsbZIP1.1 interacted with and was phosphorylated by CASEIN KINASE2 (OsCK2α3). In contrast, OsbZIP1.2 did not show any interaction with OsCOP1 or OsCK2α3. We propose that OsbZIP1.1 likely regulates seedling development in the light, while OsbZIP1.2 is the dominant player under dark conditions. The data presented in this study reveal that AtHY5 homologs in rice have undergone neofunctionalization, and alternative splicing of OsbZIP1 has increased the repertoire of its functions.

Stress-induced F-Box protein-coding gene OsFBX257 modulates drought stress adaptations and ABA responses in rice.

F-box (FB) proteins that form part of SKP1-CUL1-F-box (SCF) type of E3 ubiquitin ligases are important components of plant growth and development. Here we characterized OsFBX257, a rice FB protein-coding gene that is differentially expressed under drought conditions and other abiotic stresses. Population genomics analysis suggest that OsFBX257 shows high allelic diversity in aus accessions and has been under positive selection in some japonica, aromatic and indica cultivars. Interestingly, allelic variation at OsFBX257 in aus cultivar Nagina22 is associated with an alternatively spliced transcript. Conserved among land plants, OsFBX257 is a component of the SCF complex, can form homomers and interact molecularly with the 14-3-3 rice proteins GF14b and GF14c. OsFBX257 is co-expressed in a network involving protein kinases and phosphatases. We show that OsFBX257 can bind the kinases OsCDPK1 and OsSAPK2, and that its phosphorylation can be reversed by phosphatase OsPP2C08. OsFBX257 expression level modulates root architecture and drought stress tolerance in rice. OsFBX257 knockdown (OsFBX257KD ) lines show reduced total root length and depth, crown root number, panicle size and survival under stress. In contrast, its overexpression (OsFBX257OE ) increases root depth, leaf and grain length, number of panicles, and grain yield in rice. OsFBX257 is a promising breeding target for alleviating drought stress-induced damage in rice.

Seed germination variability: why do genetically identical seeds not germinate at the same time?

For survival in the natural environment, plants have evolved a 'bet-hedging' strategy where individual variation is high and a range of phenotypes is produced. When faced with unpredictable environmental conditions, fluctuation in seed behaviour is a beneficial trait that allows plant species to survive, particularly if seedlings from early-germinated seeds die. However, this is not a desired trait from an agricultural perspective, where a set of uniformly growing seedlings is required. Whilst variability in seed behaviour is unavoidable, over the centuries humans have attempted to select seeds with minimum variability for agricultural use. In the model plant Arabidopsis, even non-stratified seeds in the same silique germinate variably, and it remains elusive how this variability is manifested from genes to a physiological outcome and what molecular mechanisms of bet-hedging facilitate this diversity. Will the re-introduction of valuable wild alleles into domesticated crops contribute to this variability between individual seeds by promoting bet-hedging? Recent advances have shed light on possible molecular pathways of germination that are affected at the level of single seeds and single cells. Here, we review the hormonal, molecular, and cellular mechanisms that might affect the germination outcome of individual genetically identical seeds.

A comprehensive transcriptome analysis of contrasting rice cultivars highlights the role of auxin and ABA responsive genes in heat stress response.

Sensing a change in ambient temperature is key to survival among all living organisms. Temperature fluctuations due to climate change are a matter of grave concern since it adversely affects growth and eventually the yield of crop plants, including two of the major cereals, i.e., rice and wheat. Thus, to understand the response of rice seedlings to elevated temperatures, we performed microarray-based transcriptome analysis of two contrasting rice cultivars, Annapurna (heat tolerant) and IR64 (heat susceptible), by subjecting their seedlings to 37 °C and 42 °C, sequentially. The transcriptome analyses revealed a set of uniquely regulated genes and related pathways in red rice cultivar Annapurna, particularly associated with auxin and ABA as a part of heat stress response in rice. The changes in expression of few auxin and ABA associated genes, such as OsIAA13, OsIAA20, ILL8, OsbZIP12, OsPP2C51, OsDi19-1 and OsHOX24, among others, were validated under high-temperature conditions using RT-qPCR. In particular, the expression of auxin-inducible SAUR genes was enhanced considerably at both elevated temperatures. Further, using genes that expressed inversely under heat vs. cold temperature conditions, we built a regulatory network between transcription factors (TF) such as HSFs, NAC, WRKYs, bHLHs or bZIPs and their target gene pairs and determined regulatory coordination in their expression under varying temperature conditions. Our work thus provides useful insights into temperature-responsive genes, particularly under elevated temperature conditions, and could serve as a resource of candidate genes associated with thermotolerance or downstream components of temperature sensors in rice.

Differential quantitative regulation of specific gene groups and pathways under drought stress in rice.

Abiotic stresses like drought are detrimental for growth and development and lead to loss in crop production. To be able to adapt and survive under such adverse conditions, synchronous regulation of a rather large number of genes is required. Here, we have used a bioinformatics approach to identify gene groups and associated pathways from microarray and RNA-seq experiments that are restricted in their gene expression amplitude within fold change intervals (FCI) under drought stress conditions. We find that the expression of genes as functional groups is coordinated quantitatively, in a fold change specific manner, and differs among three rice cultivars distinct in their drought stress response. By networking these groups and further categorization into components like ubiquitin proteasome system, we identify relatively less studied E2 ubiquitin conjugating enzyme coding genes as an important constituent of differential drought stress response in rice. By extending this approach to find hexamer DNA motifs in the upstream promoter regions of genes within the FCIs under stress, we find that genes with strong to very strong or a moderate expression under stress are coordinated through cis-regulatory motifs. Few of these, such as TSO1, L-Box, PE1, GT binding site, ABRE/G-box or AP2/ERF binding site can be candidate cis-regulatory motifs to coordinate fold change limited gene expression under drought stress. This work thus provides an insight into a quantitative regulation of gene expression under drought stress in rice and a useful resource for designing approaches towards coordinating the expression of identified candidate genes under stress in order to achieve drought tolerance in rice.

Gut microbiome contributes to impairment of immunity in pulmonary tuberculosis patients by alteration of butyrate and propionate producers.

Tuberculosis (TB) is primarily associated with decline in immune health status. As gut microbiome (GM) is implicated in the regulation of host immunity and metabolism, here we investigate GM alteration in TB patients by 16S rRNA gene and whole-genome shotgun sequencing. The study group constituted of patients with pulmonary TB and their healthy household contacts as controls (HCs). Significant alteration of microbial taxonomic and functional capacity was observed in patients with active TB as compared to the HCs. We observed that Prevotella and Bifidobacterium abundance were associated with HCs, whereas butyrate and propionate-producing bacteria like Faecalibacterium, Roseburia, Eubacterium and Phascolarctobacterium were significantly enriched in TB patients. Functional analysis showed reduced biosynthesis of vitamins and amino acids in favour of enriched metabolism of butyrate and propionate in TB subjects. The TB subjects were also investigated during the course of treatment, to analyse the variation of GM. Although perturbation in microbial composition was still evident after a month's administration of anti-TB drugs, significant changes were observed in metagenome gene pool that pointed towards recovery in functional capacity. Therefore, the findings from this pilot study suggest that microbial dysbiosis may contribute to pathophysiology of TB by enhancing the anti-inflammatory milieu in the host.

Impact of Tocilizumab on Inflammatory Markers and Oxygen Status in Severe COVID-19 Patients: A Single Centre Retrospective Study.

OBJECTIVE: This retrospective study aims to investigate the impact of tocilizumab on inflammatory markers in patients with severe COVID-19. The effect on oxygenation was also assessed. METHODS: This study is a single-centre, retrospective cohort study conducted at NIMS hospital. Data of the eligible patients with severe COVID-19 pneumonia who received injection tocilizumab (max 800 mg) were charted and analysed. Oxygenation and inflammatory markers were compared before and after (day 3 and day 7) tocilizumab injection. Effect of dysglycemia on the efficacy of tocilizumab was assessed. Outcomes were analysed in the form of discharge without oxygen, discharge with oxygen, and death. Data were analysed by SPSS v22. RESULTS: The mean age of the subjects was 57.8 ± 12.2 years, and 78.57% were male. Forty-four percent of the patient had type 2 diabetes. Tocilizumab treatment was associated with reduction in the oxygen requirement [median:10 L/min (IQR6- 14)] v/s 4 L/min (IQR 3-7, p-0.005]. Peripheral oxygen saturation also improved after tocilizumab [92 % (IQR 90-96)] v/s [95 % (IQR 94-96), p-0.01)], respectively. Serum CRP level decreased significantly when evaluated after three days (44±5 v/s 20 ±3 mg/dl, p=< 0.001). Out of the 42, 12 (29%) patients died due to severe COVID-19 or its complications. When compared with the patients who survived, patients who died had a higher level of D-dimer (1.2 ± 0.51 v/s 3.1 ±1.2 ng/dl, p-value- 0.04), and LDH: (845 ±55 v/s 1364 ±198 U/L, p - 0.01). At day seven of the tocilizumab injection, diabetic patients (n-13) had higher IL-6 serum level than nondiabetic patients (n-16) [(median- 311(IQR-1245.5) v/s (209 (IQR-546.2), p-value- 0.048]. CONCLUSION: In this retrospective pre-post analysis, tocilizumab injection was associated with reduced inflammation and improved oxygenation in severe COVID-19. Despite high IL-6 levels, diabetes had no impact on the efficacy of the tocilizumab.

OsRR6, a type-A response regulator in rice, mediates cytokinin, light and stress responses when over-expressed in Arabidopsis.

Plants have evolved a complex network of components that sense and respond to diverse signals. In the present study, we have characterized OsRR6, a type-A response regulator, which is part of the two-component sensor-regulator machinery in rice. The expression of OsRR6 is induced by exogenous cytokinin and various abiotic stress treatments, including drought, cold and salinity stress. Organ-specific expression analysis revealed that its expression is high in anther and low in shoot apical meristem. The Arabidopsis plants constitutively expressing OsRR6 (OsRR6OX) exhibited reduced cytokinin sensitivity, adventitious root formation and enhanced anthocyanin accumulation in seeds. OsRR6OX plants were more tolerant to drought and salinity conditions when compared to wild-type. The hypocotyl growth in OsRR6OX seedlings was significantly inhibited under red, far-red and blue-light conditions and also a decline in transcript levels of OsRR6 was observed in rice under the above monochromatic as well as white light treatments. Transcriptome profiling revealed that the genes associated with defense responses and anthocyanin metabolism are up-regulated in OsRR6OX seedlings. Comparative transcriptome analysis showed that the genes associated with phenylpropanoid and triterpenoid biosynthesis are enriched among differentially expressed genes in OsRR6OX seedlings of Arabidopsis, which is in conformity with reanalysis of the transcriptome data performed in rice transgenics for OsRR6. Further, genes like DREB1A/CBF3, COR15A, KIN1, ERD10 and RD29A are significantly upregulated in OsRR6OX seedlings when subjected to ABA and abiotic stress treatments. Thus, a negative regulator of cytokinin signaling, OsRR6, plays a positive role in imparting abiotic stress tolerance.

Prevalence of Symptoms in Patients Discharged from COVID Care Facility of NIMS Hospital: Is RT PCR Negativity Truly Reflecting Recovery? A Single-Centre Observational Study.

PURPOSE: To assess the prevalence of post-COVID symptoms in patients with recovered COVID-19 (nasopharyngeal RT PCR negative) who were discharged from an acute COVID care facility at a tertiary care teaching hospital in North India. METHODS: This study was an observational study with retrospective data collection, conducted in the COVID follow-up clinic, a combined clinic of medicine and endocrinology. Patients discharged from the acute COVID care facility were recruited after 14 days of discharge if they fulfilled inclusion and exclusion criteria. The retrospective data was collected from the hospital records/EMR and analysed by the SPSSv23. RESULTS: Fifty patients, who fulfilled the inclusion and exclusion criteria, were included in the study. The Mean age of patients was 53.4±13.8 years (range 28-77). Seventy six percent were male, and 38% had type 2 diabetes. Fever (94%), cough (78%) and breathlessness (68%), were the most common symptoms at presentation to acute care facility. Oxygen saturation at presentation had a negative correlation with D-Dimer, age, and C reactive protein. When patients were evaluated clinically, after 14 days (range 15 to 50 days) of the discharge, 82% of patients had at least one persistent symptom. Fatigue (74%) was the most common symptoms in follow-up followed by breathlessness (44%), and muscle weakness (36%). Two patients had persistent fever, even after negative RT PCR status. CONCLUSION: Patients discharged from the acute COVID care facility had a high prevalence of post-COVID symptoms even after 14 days.

Drought-induced protein (Di19-3) plays a role in auxin signaling by interacting with IAA14 in Arabidopsis.

The members of early auxin response gene family, Aux/IAA, encode negative regulators of auxin signaling but play a central role in auxin-mediated plant development. Here we report the interaction of an Aux/IAA protein, AtIAA14, with Drought-induced-19 (Di19-3) protein and its possible role in auxin signaling. The Atdi19-3 mutant seedlings develop short hypocotyl, both in light and dark, and are compromised in temperature-induced hypocotyl elongation. The mutant plants accumulate more IAA and also show altered expression of NIT2, ILL5, and YUCCA genes involved in auxin biosynthesis and homeostasis, along with many auxin responsive genes like AUX1 and MYB77. Atdi19-3 seedlings show enhanced root growth inhibition when grown in the medium supplemented with auxin. Nevertheless, number of lateral roots is low in Atdi19-3 seedlings grown on the basal medium. We have shown that AtIAA14 physically interacts with AtDi19-3 in yeast two-hybrid (Y2H), bimolecular fluorescence complementation, and in vitro pull-down assays. However, the auxin-induced degradation of AtIAA14 in the Atdi19-3 seedlings was delayed. By expressing pIAA14::mIAA14-GFP in Atdi19-3 mutant background, it became apparent that both Di19-3 and AtIAA14 work in the same pathway and influence lateral root development in Arabidopsis. Gain-of-function slr-1/iaa14 (slr) mutant, like Atdi19-3, showed tolerance to abiotic stress in seed germination and cotyledon greening assays. The Atdi19-3 seedlings showed enhanced sensitivity to ethylene in triple response assay and AgNO3, an ethylene inhibitor, caused profuse lateral root formation in the mutant seedlings. These observations suggest that AtDi19-3 interacting with AtIAA14, in all probability, serves as a positive regulator of auxin signaling and also plays a role in some ethylene-mediated responses in Arabidopsis. SIGNIFICANCE STATEMENT: This study has demonstrated interaction of auxin responsive Aux/IAA with Drought-induced 19 (Di19) protein and its possible implication in abiotic stress response.

Analysis of drought-responsive signalling network in two contrasting rice cultivars using transcriptome-based approach.

Traditional cultivars of rice in India exhibit tolerance to drought stress due to their inherent genetic variations. Here we present comparative physiological and transcriptome analyses of two contrasting cultivars, drought tolerant Dhagaddeshi (DD) and susceptible IR20. Microarray analysis revealed several differentially expressed genes (DEGs) exclusively in DD as compared to IR20 seedlings exposed to 3 h drought stress. Physiologically, DD seedlings showed higher cell membrane stability and differential ABA accumulation in response to dehydration, coupled with rapid changes in gene expression. Detailed analyses of metabolic pathways enriched in expression data suggest interplay of ABA dependent along with secondary and redox metabolic networks that activate osmotic and detoxification signalling in DD. By co-localization of DEGs with QTLs from databases or published literature for physiological traits of DD and IR20, candidate genes were identified including those underlying major QTL qDTY1.1 in DD. Further, we identified previously uncharacterized genes from both DD and IR20 under drought conditions including OsWRKY51, OsVP1 and confirmed their expression by qPCR in multiple rice cultivars. OsFBK1 was also functionally validated in susceptible PB1 rice cultivar and Arabidopsis for providing drought tolerance. Some of the DEGs mapped to the known QTLs could thus, be of potential significance for marker-assisted breeding.