Shahid Aziz

Ascorbic acid (AsA) is an essential plant metabolite that acts primarily as an antioxidant, regulates cell division and elongation, and enhances stress tolerance. Despite its crucial physiological role, the biosynthesis of AsA in G. max, a major crop of significant commercial importance, remains largely unexplored. This gap highlights the need for a thorough investigation of AsA biosynthesis pathways and their role in optimizing the nutritional value and stress tolerance of soybeans. This study identified 41 key genes linked to four AsA biosynthesis pathways in G. max, highlighting specific gene duplications compared to Arabidopsis. Their expression levels were assessed by analyzing a diverse set of RNA-Seq data from the NCBI database. Additionally, to cross-validate the expression levels of genes and the accumulation levels of AsA in the principal tissues, G. max plants were grown under controlled conditions following the protocols from selected RNA-seq experiments. Genes associated with the D-mannose/L-galactose pathway exhibited ubiquitous expression, and the expression patterns of genes from alternative pathways reflected their responsiveness to specific tissues or environmental conditions. Germination and leaf development were accompanied by strong expression of gene members from all pathways, whereas leaf aging was characterized by downregulation. Specific gene members, such as GMP_2a (D-mannose/L-galactose pathway), GulLO_1f (L-gulose pathway), and MIOX_3a (Myo-inositol pathway) were highly stress-responsive and linked to stress-resistant genotypes and cultivars. Consistent with gene expression analyses, the quantification of AsA revealed the highest mass fractions in young leaves and germinating seeds. However, AsA mass fractions were significantly reduced or unchanged under stress conditions, depending on the type of stress and the duration of exposure. Overall, this study validated the relevance of AsA biosynthesis pathways in soybeans, highlighting key genes that could be targeted to enhance stress tolerance and improve ascorbate production, thereby boosting the nutritional value of soybeans.

Xylella fastidiosa (Xf) is a global bacterial threat for a diversity of plants, including olive trees. However, current understanding of host responses upon Xf-infection is limited to allow early disease prediction, diagnosis, and sustainable strategies for breeding on plant tolerance. Recently, we identified a major complex trait for early de novo programming, named CoV-MAC-TED, by comparing early transcriptome data during plant cell survival with SARS-CoV-2-infected human cells. This trait linked ROS/RNS balancing during first hours of stress perception with increased aerobic fermentation connected to alpha-tubulin-based cell restructuration and control of cell cycle progression. Furthermore, our group had advanced concepts and strategies for breeding on plant holobionts. Here, we studied tolerance against Xf-infection by applying a CoV-MAC-TED-related gene set to (1) progress proof-of-principles, (2) highlight the importance of individual host responses for knowledge gain, (3) benefit sustainable production of Xf-threatened olive, (4) stimulate new thinking on principle roles of secondary metabolite synthesis and microbiota for system equilibration and, (5) advance functional marker development for resilience prediction including tolerance to Xf-infections. We performed hypothesis-driven complex analyses in an open access transcriptome of primary target xylem tissues of naturally Xf-infected olive trees of the Xf-tolerant cv. Leccino and the Xf-susceptible cv. Ogliarola. The results indicated that cyanide-mediated equilibration of oxygen-dependent respiration and carbon-stress alleviation by the help of increased glycolysis-driven aerobic fermentation paths and phenolic metabolism associate to tolerance against Xf. Furthermore, enhanced alternative oxidase (AOX) transcript levels through transcription Gleichschaltung linked to quinic acid synthesis appeared as promising trait for functional marker development. Moreover, the results support the idea that fungal endophytes strengthen Xf-susceptible genotypes, which lack efficient AOX functionality. Overall, this proof-of-principles approach supports the idea that efficient regulation of the multi-functional AOX gene family can assist selection on multiple-resilience, which integrates Xf-tolerance, and stimulates future validation across diverse systems.

Plants subjected to stress need to respond rapidly and efficiently to acclimatize and survive. In this paper, we investigated a selected gene set potentially involved in early cell reprogramming in two rice genotypes with contrasting salinity tolerance (Pokkali tolerant and IR29 susceptible) in order to advance knowledge of early molecular mechanisms of rice in dealing with salt stress. Selected genes were evaluated in available transcriptomic data over a short period of 24 h and involved enzymes that avoid ROS formation (AOX, UCP and PTOX), impact ATP production (PFK, ADH and COX) or relate to the antioxidant system. Higher transcript accumulation of AOX (ROS balancing), PFK and ADH (alcohol fermentation) was detected in the tolerant genotype, while the sensitive genotype revealed higher UCP and PTOX transcript levels, indicating a predominant role for early transcription of AOX and fermentation in conferring salt stress tolerance to rice. Antioxidant gene analyses supported higher oxidative stress in IR29, with transcript increases of cytosolic CAT and SOD from all cell compartments (cytoplasm, peroxisome, chloroplast and mitochondria). In contrast, Pokkali increased mRNA levels from the AsA-GSH cycle as cytosolic/mitochondrial DHAR was involved in ascorbate recovery. In addition, these responses occurred from 2 h in IR29 and 10 h in Pokkali, indicating early but ineffective antioxidant activity in the susceptible genotype. Overall, our data suggest that AOX and ADH can play a critical role during early cell reprogramming for improving salt stress tolerance by efficiently controlling ROS formation in mitochondria. We discuss our results in relation to gene engineering and editing approaches to develop salinity-tolerant crops.

Key message We found 34 and 71 key genes potentially involved in flavonoid biosynthesis and cell wall disassembly, respectively, which could be associated with specific peel coloration and softening of each genotype. Abstract Cashew apple (Anacardium occidentale) has a great economic importance worldwide due to its high nutritional value, peculiar flavor and aroma. During ripening, the peduncle develops different peel color and becomes quickly fragile due to its oversoftening, impacting its consumers’ acceptance. In view of this, the understanding about its transcriptional dynamics throughout ripening is imperative. In this study, we performed a transcriptome sequencing of two cashew apple genotypes (CCP 76 and BRS 265), presenting different firmness and color peel, in the immature and ripe stages. Comparative transcriptome analysis between immature and ripe cashew apple revealed 4374 and 3266 differentially expressed genes (DEGs) to CCP 76 and BRS 265 genotypes, respectively. These genes included 71 and 34 GDEs involved in the cell wall disassembly and flavonoid biosynthesis, respectively, which could be associated with firmness loss and anthocyanin accumulation during cashew apple development. Then, softer peduncle of CCP 76 could be justified by down-regulated EXP and up-regulation of genes involved in pectin degradation (PG, PL and PAE) and in cell wall biosynthesis. Moreover, genes related to flavonoid biosynthesis (PAL, C4H and CHS) could be associated with early high accumulation of anthocyanin in red-peel peduncle of BRS 265. Finally, expression patterns of the selected genes were tested by real-time quantitative PCR (qRT-PCR), and the qRT-PCR results were consistent with transcriptome data. The information generated in this work will provide insights into transcriptome responses to cashew apple ripening and hence, it will be helpful for cashew breeding programs aimed at developing genotypes with improved quality traits.

Northern Pakistan is home to many diverse ethnicities and languages. The region acted as a prime corridor for ancient invasions and population migrations between Western Eurasia and South Asia. Kho, one of the major ethnic groups living in this region, resides in the remote and isolated mountainous region in the Chitral Valley of the Hindu Kush Mountain range. They are culturally and linguistically distinct from the rest of the Pakistani population groups and their genetic ancestry is still unknown. In this study, we generated genome-wide genotype data of ~1 M loci (Illumina WeGene array) for 116 unrelated Kho individuals and carried out comprehensive analyses in the context of worldwide extant and ancient anatomically modern human populations across Eurasia. The results inferred that the Kho can trace a large proportion of their ancestry to the population who migrated south from the Southern Siberian steppes during the second millennium BCE ~110 generations ago. An additional wave of gene flow from a population carrying East Asian ancestry was also identified in the Kho that occurred ~60 generations ago and may possibly be linked to the expansion of the Tibetan Empire during 7th to 9th centuries CE (current era) in the northwestern regions of the Indian sub-continent. We identified several candidate regions suggestive of positive selection in the Kho, that included genes mainly involved in pigmentation, immune responses, muscular development, DNA repair, and tumor suppression.

Recently, we identified early reactive oxygen species (ROS) and reactive nitrogen species (RNS) rebalancing as part of a major complex trait, named CoV-MAC-TED, that characterized host cell response on SARS-CoV-2 infections from two genetic virus variants.[1–3] Transcript level changes of the enzymes acetylserotonin O-methyltransferase-like (ASMTL) and alcohol dehydrogenase 5 (ADH5) were hypothesized to mark relevant adaptive ROS/RNS equilibration early during respiratory infections and preliminary validation in cultured primary target cells supported this view.[2] ASMTL is a paralog of ASMT, which is involved in final melatonin biosynthesis. ADH5, also known as S-nitrosoglutathione reductase, is involved in nitric oxide (NO) homeostasis.[1,2] In parallel to these studies, we released our results on ASMTL and ADH5 transcriptome accumulation from influenza H3N2-infected human nasal epithelial cells (NECs) that originated from seven healthy cell donator origins (D01–D07).[4] We observed that variability of ASMTL and ADH5 transcript levels linked to the timing of virus replication and that this connected to the initiation of the classical immune system response, when marked by interferon regulatory factor 9 (IRF9) transcript level changes. Cells of the donator D02 showed the highest ASMTL transcript levels among all cell origins at 8 hours post-infection (hpi), indicating rapidly unbalanced ROS/RNS that linked to the earliest immune response. On the contrary, transcript levels of ADH5 from D05 indicated early and over times highest NO stress among all seven origins, suggesting unbalanced ROS/RNS in favor of RNS. This response connected to the highest immune response at 24 hpi among all cell origins. In contrast, cells originating from D01 indicated a stably balanced relationship between ROS and RNS marked by ASMTL and ADH5 that associated to the lowest IRF9 response over all time points among the seven cell origins. This equilibrated response of D01 was in accordance with postponed influenza virus replication in relation to all other cell origins.[4] Of note, also under infection by two SARS-CoV-2 variants NECs from one of three donator origins displayed contrasting response to other donator cells as follows: lower transcript levels for enolase (representing glycolysis) and lactate dehydrogenase (representing aerobic fermentation) were observed at 24 hpi for cell cultures from origin D1 that linked to delayed replication of SARS-CoV-2.[3]These overall observations made us optimistic that our approach and marker systems in combination with appropriate primary target human cells could be promising, in general, to early identify differential individual host cell responses on viral infections. Concurrently, we hypothesized that this tool could also help to discriminate individuals through genetic polymorphisms in the relevant functional genes that relate to CoV-MAC-TED (see ReprogVirus gene set in References 1–4).ROS signaling is one of the earliest and relevant cell responses upon abiotic and biotic stresses that transmits any change in environment, including viral attacks, to structures and molecules (e.g., membranes, ion channels [Ca2+], enzymes, microtubulines), which then are crucially determining cell performance. Therefore, we studied polymorphisms in both highlighted marker genes for ROS/RNS balancing, ASMTL, and ADH5. As a first step, we checked the genetic material of the public transcriptome data of these seven donators for NECs.[5] The coding sequence of ADH5 was highly conservative. No sequence nucleotide polymorphisms or other major differences among the seven donor cell origins were encountered. To the contrary, we found several polymorphic sites (sequence nucleotide polymorphisms) in the coding sequence of the ASMTL gene (unpublished data). This might indicate a higher potential for acclimation acquired during evolution.In this brief report, we want to point to transcript level profiles that we observed for one of the polymorphic sites during early virus-induced cell reprogramming. In the upper part of Table 1, we demonstrate the crucial nucleotide for polymorphism in this site (G or A) for influenza H3N2-infected cells from seven donator origins. On the one hand, we observed the following cell origins with the same preferential nucleotide profile over times in this site: cells of D02, D03, and D07 show continuously G at all time points (0, 8, and 24 hpi); D04 and D06 display stably A, such as in the reference gene. On the other hand, we discovered for D01 and D05 unique differential transcript level profiles. Cells from D05 changed at 8 hpi to the preferential transcription of an allele, which shows A instead of G in this polymorphic site. However, at 24 hpi a change in preferential allelic transcription became visible through the reappearance of G instead of A. In contrast, at 8 hpi D01 changed in the same way as observed for D05 to preferential transcription of an allele that shows A instead of the initial G. However, in cells of D01 this allelic site continued longer to be preferentially transcribed and A is still the more frequent nucleotide seen at 24 hpi (boldface at 8 and 24 hpi). Post-infection time-dependent allelic genotyping profiles of the same polymorphic site was also found on infection by two SARS-CoV-2 variants that demonstrated diverse effects on disease severity.[3] ASMTL genotype profiles are shown for NECs from three donators (Table 1, lower part). Overall, these results demonstrate virus- and variant-specific allelic genotype profiles over times when both virus types (influenza H3N2 and SARS-CoV-2) and both SARS-CoV-2 variants are compared within each cell origin.In summary, and despite the restricted number of studied individual cell origins, these observations highlight to our view that efficient allelic genotyping for specific or general virus tolerance and immunophenotyping requires integrative studies on time-dependent individual...

Background: Early metabolic reorganization was only recently recognized as an essentially integrated part of immunology. In this context, unbalanced ROS/RNS levels connected to increased aerobic fermentation, which is linked to alpha-tubulin-based cell restructuring and control of cell cycle progression, were identified as a major complex trait for early de novo programming (‘CoV-MAC-TED’) during SARS-CoV-2 infection. This trait was highlighted as a critical target for developing early anti-viral/anti-SARS-CoV-2 strategies. To obtain this result, analyses had been performed on transcriptome data from diverse experimental cell systems. A call was released for wide data collection of the defined set of genes for transcriptome analyses, named ‘ReprogVirus’, which should be based on strictly standardized protocols and data entry from diverse virus types and variants into the ‘ReprogVirus Platform’. This platform is currently under development. However, so far, an in vitro cell system from primary target cells for virus attacks that could ideally serve for standardizing the data collection of early SARS-CoV-2 infection responses has not been defined. Results: Here, we demonstrate transcriptome-level profiles of the most critical ‘ReprogVirus’ gene sets for identifying ‘CoV-MAC-TED’ in cultured human nasal epithelial cells infected by two SARS-CoV-2 variants differing in disease severity. Our results (a) validate ‘Cov-MAC-TED’ as a crucial trait for early SARS-CoV-2 reprogramming for the tested virus variants and (b) demonstrate its relevance in cultured human nasal epithelial cells. Conclusion: In vitro-cultured human nasal epithelial cells proved to be appropriate for standardized transcriptome data collection in the ‘ReprogVirus Platform’. Thus, this cell system is highly promising to advance integrative data analyses with the help of artificial intelligence methodologies for designing anti-SARS-CoV-2 strategies.

Background: Transcriptome studies of a selected gene set ( ReprogVirus ) had identified unbalanced ROS/RNS levels, which connected to increased aerobic fermentation that linked to alpha-tubulin-based cell restructuration and cell cycle control, as a ma jor c omplex t rait for e arly d e novo programming ( CoV-MAC-TED ) upon SARS-CoV-2 infection. Recently, CoV-MAC-TED was confirmed as promising marker by using primary target human nasal epithelial cells (NECs) infected by two SARS-CoV-2 variants with different effects on disease severity. To further explore this marker/cell system as a standardized tool for identifying anti-viral targets in general, testing of further virus types is required. Results: Transcriptome level profiles of H3N2 influenza-infected NECs indicated ROS/RNS level changes and increased transcript accumulation of genes related to glycolysis, lactic fermentation and α-tubulin at 8 hours post infection. These early changes linked to energy-dependent, IRF9-marked rapid immunization. However, ReprogVirus -marker genes indicated the absence of initial cell cycle progress, which contrasted our findings during infections with two SARS-CoV-2 variants, where cell cycle progress was linked to delayed IRF9 response. Our results point to the possibility of CoV-MAC-TED-assisted, rapid individual host cell response identification upon virus infections. Conclusion: The complex trait CoV-MAC-TED can identify similar and differential early responses of SARS-CoV-2 and influenza H3N2 viruses. This indicates its appropriateness to search for anti-viral targets in view of therapeutic design strategies. For standardization, human NECs can be used. This marker/cell system is promising to identify differential early cell responses upon viral infections also depending on cell origins.