Isotopically nonstationary metabolic flux analysis of plants: recent progress and future opportunities.

Metabolic flux analysis (MFA) is a valuable tool for quantifying cellular phenotypes and to guide plant metabolic engineering. By introducing stable isotopic tracers and employing mathematical models, MFA can quantify the rates of metabolic reactions through biochemical pathways. Recent applications of isotopically nonstationary MFA (INST-MFA) to plants have elucidated nonintuitive metabolism in leaves under optimal and stress conditions, described coupled fluxes for fast-growing algae, and produced a synergistic multi-organ flux map that is a first in MFA for any biological system. These insights could not be elucidated through other approaches and show the potential of INST-MFA to correct an oversimplified understanding of plant metabolism.

Phytochemical rich Himalayan Rhododendron arboreum petals inhibit SARS-CoV-2 infection in vitro.

Phytochemicals with potential to competitively bind to the host receptors or inhibit SARS-CoV-2 replication, may prove to be useful as adjunct therapeutics for COVID-19. We profiled and investigated the phytochemicals of Rhododendron arboreum petals sourced from Himalayan flora, undertook in vitro studies and found it as a promising candidate against SARS-CoV-2. The phytochemicals were reported in various scientific investigations to act against a range of virus in vitro and in vivo, which prompted us to test against SARS-CoV-2. In vitro assays of R. arboreum petals hot aqueous extract confirmed dose dependent reduction in SARS-CoV-2 viral load in infected Vero E6 cells (80% inhibition at 1 mg/ml; IC50 = 173 µg/ml) and phytochemicals profiled were subjected to molecular docking studies against SARS CoV-2 target proteins. The molecules 5-O-Feruloyl-quinic acid, 3-Caffeoyl-quinic acid, 5-O-Coumaroyl-D-quinic acid, Epicatechin and Catechin showed promising binding affinity with SARS-CoV-2 Main protease (MPro; PDB ID: 6LU7; responsible for viral replication) and Human Angiotensin Converting Enzyme-2 (ACE2; PDB ID: 1R4L; mediate viral entry in the host). Molecular dynamics (MD) simulation of 5-O-Feruloyl-quinic acid, an abundant molecule in the extract complexed with the target proteins showed stable interactions. Taken together, the phytochemical profiling, in silico analysis and in vitro anti-viral assay revealed that the petals extract act upon MPro and may be inhibiting SARS-CoV-2 replication. This is the first report highlighting R. arboreum petals as a reservoir of antiviral phytochemicals with potential anti-SARS-CoV-2 activity using an in vitro system.

Plant microProteins: Small but powerful modulators of plant development.

MicroProteins (miPs) are small and single-domain containing proteins of less than 20 kDa. This domain allows microProteins to interact with compatible domains of evolutionary-related proteins and fine-tuning the key physiological pathways in several organisms. Since the first report of a microProtein in mice, numerous microProteins have been identified in plants by computational approaches. However, only a few candidates have been functionally characterized, primarily in Arabidopsis. The recent success of synthetic microProteins in modulating physiological activities in crops makes these proteins interesting candidates for crop engineering. Here, we comprehensively summarise the synthesis, mode of action, and functional roles of microProteins in plants. We also discuss different approaches used to identify plant microProteins. Additionally, we discuss novel approaches to design synthetic microProteins that can be used to target proteins regulating plant growth and development. We finally highlight the prospects and challenges of utilizing microProteins in future crop improvement programs.

Transcription factors BBX11 and HY5 interdependently regulate the molecular and metabolic responses to UV-B.

UV-B radiation acts as a developmental cue and a stress factor for plants, depending on dose. Activation of the transcription factor ELONGATED HYPOCOTYL 5 (HY5) in a UV RESISTANCE LOCUS 8 (UVR8)-dependent manner leads to the induction of a broad set of genes under UV-B. However, the underlying molecular mechanisms regulating this process are less understood. Here, we use molecular, biochemical, genetic, and metabolomic tools to identify the B-BOX transcription factor B-BOX PROTEIN 11 (BBX11) as a component of the molecular response to UV-B in Arabidopsis (Arabidopsis thaliana). BBX11 expression is induced by UV-B in a dose-dependent manner. Under low UV-B, BBX11 regulates hypocotyl growth suppression, whereas it protects plants exposed to high UV-B radiation by promoting the accumulation of photo-protective phenolics and antioxidants, and inducing DNA repair genes. Our genetic studies indicate that BBX11 regulates hypocotyl elongation under UV-B partially dependent on HY5. Overexpression of BBX11 can partially rescue the high UV-B sensitivity of hy5, suggesting that HY5-mediated UV-B stress tolerance is partially dependent on BBX11. HY5 regulates the UV-B-mediated induction of BBX11 by directly binding to its promoter. BBX11 reciprocally regulates the mRNA and protein levels of HY5. We report here the role of a BBX11-HY5 feedback loop in regulating photomorphogenesis and stress tolerance under UV-B.

A robust method of extraction and GC-MS analysis of Monophenols exhibited UV-B mediated accumulation in Arabidopsis.

Studies on specialized metabolites like phenolics are of immense interest owing to their significance to agriculture, nutrition and health. In plants, phenolics accumulate and exhibit spatial and temporal regulations in response to growth conditions. Robust methodologies aimed at efficient extraction of plant phenolics, their qualitative and quantitative analysis is desired. We optimized the analytical and experimental bottlenecks that captured free, ester, glycoside and wall-bound phenolics after acid or alkali treatments of the tissue extracts and subsequent GC-MS analysis. Higher recovery of phenolics from the methanolic extracts was achieved through (a) Ultrasonication assisted extraction along with Methyl tert-butyl ether (MTBE) enrichment (b) nitrogen gas drying and (c) their derivatization using MSTFA for GC-MS analysis. The optimized protocol was tested on Arabidopsis rosette exposed to UV-B radiation (280-315 nm) which triggered enhanced levels of 11 monophenols and might be attributed to photoprotection and other physiological roles. Interestingly, coumaric acid (308 m/z) and caffeic acid (396 m/z) levels were enhanced by 12-14 folds under UV-B. Other phenolics such as cinnamic acid (220 m/z), hydroxybenzoic acid (282 m/z), vanillic acid (312 m/z, gallic acid (458 m/z), ferulic acid (338 m/z), benzoic acid (194 m/z), sinapinic acid (368 m/z) and protocatechuic acid (370 m/z) also showed elevated levels by about 1 to 4 folds. The protocol also comprehensively captured the variations in the levels of ester, glycoside and wall-bounded phenolics with high reproducibility and sensitivity. The robust method of extraction and GC-MS analysis can readily be adopted for studying phenolics in plant systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01150-2.

The unique bryophyte-specific repeat-containing protein SHORT-LEAF regulates gametophore development in moss.

Convergent evolution of shoot development across plant lineages has prompted numerous comparative genetic studies. Though functional conservation of gene networks governing flowering plant shoot development has been explored in bryophyte gametophore development, the role of bryophyte-specific genes remains unknown. Previously, we have reported Tnt1 insertional mutants of moss defective in gametophore development. Here, we report a mutant (short-leaf; shlf) having two-fold shorter leaves, reduced apical dominance, and low plasmodesmata frequency. UHPLC-MS/MS-based auxin quantification and analysis of soybean (Glycine max) auxin-responsive promoter (GH3:GUS) lines exhibited a striking differential auxin distribution pattern in the mutant gametophore. Whole-genome sequencing and functional characterization of candidate genes revealed that a novel bryophyte-specific gene (SHORT-LEAF; SHLF) is responsible for the shlf phenotype. SHLF represents a unique family of near-perfect tandem direct repeat (TDR)-containing proteins conserved only among mosses and liverworts, as evident from our phylogenetic analysis. Cross-complementation with a Marchantia homolog partially recovered the shlf phenotype, indicating possible functional specialization. The distinctive structure (longest known TDRs), absence of any known conserved domain, localization in the endoplasmic reticulum, and proteolytic cleavage pattern of SHLF imply its function in bryophyte-specific cellular mechanisms. This makes SHLF a potential candidate to study gametophore development and evolutionary adaptations of early land plants.

ELONGATED HYPOCOTYL5 Negatively Regulates DECREASE WAX BIOSYNTHESIS to Increase Survival during UV-B Stress.

Understanding how the distinct cell types of the shoot apical meristem (SAM) withstand ultraviolet radiation (UVR) stress can improve cultivation of plants in high-UVR environments. Here, we show that UV-B irradiation selectively kills epidermal and niche cells in the shoot apex. Plants harboring a mutation in DECREASE WAX BIOSYNTHESIS (DEWAX) are tolerant to UV-B. Our data show that DEWAX negatively regulates genes involved in anthocyanin biosynthesis. ELONGATED HYPOCOTYL5 (HY5) binds to the DEWAX promoter elements and represses its expression to promote the anthocyanin biosynthesis. The HY5-DEWAX regulatory network regulates anthocyanin content in Arabidopsis (Arabidopsis thaliana) and influences the survivability of plants under UV-B irradiation stress. Our cell sorting-based study of the epidermal cell layer transcriptome confirms that core UV-B stress signaling pathway genes are conserved and upregulated in response to UV-B irradiation of the SAM. Furthermore, we show that UV-B induces genes involved in shoot development and organ patterning. We propose that the HY5-DEWAX regulatory relationship is conserved; however, changes in the expression levels of these genes can determine anthocyanin content in planta and, hence, fitness under UV-B irradiation stress.

Light signaling and UV-B-mediated plant growth regulation.

Light plays an important role in plants' growth and development throughout their life cycle. Plants alter their morphological features in response to light cues of varying intensity and quality. Dedicated photoreceptors help plants to perceive light signals of different wavelengths. Activated photoreceptors stimulate the downstream signaling cascades that lead to extensive gene expression changes responsible for physiological and developmental responses. Proteins such as ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) act as important factors which modulate light-regulated gene expression, especially during seedling development. These factors function as central regulatory intermediates not only in red, far-red, and blue light pathways but also in the UV-B signaling pathway. UV-B radiation makes up only a minor fraction of sunlight, yet it imparts many positive and negative effects on plant growth. Studies on UV-B perception, signaling, and response in plants has considerably surged in recent times. Plants have developed different strategies to use UV-B as a developmental cue as well as to withstand high doses of UV-B radiation. Plants' responses to UV-B are an integration of its cross-talks with both environmental factors and phytohormones. This review outlines the current developments in light signaling with a major focus on UV-B-mediated plant growth regulation.

BBX31 promotes hypocotyl growth, primary root elongation and UV-B tolerance in Arabidopsis.

Photomorphogenesis is an important developmental process that helps the seedlings adapt to external light conditions. B-Box proteins are a family of transcription factors that regulate photomorphogenic responses. BBX31 negatively regulates photomorphogenesis under visible light. In contrast, it promotes photomorphogenesis under UV-B and enhances tolerance to high doses of UV-B radiation. BBX31 and HY5 independently and oppositely regulate the ability of seedlings to adapt to varying light intensities. BBX31 also regulates primary root elongation under low intensities of white light. GC-MS and HPLC-based metabolite profiling identified differential accumulation of multiple primary and secondary metabolites in 35S:BBX31 that might enhance tolerance to UV-B.

The B-Box-Containing MicroProtein miP1a/BBX31 Regulates Photomorphogenesis and UV-B Protection.

The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) represents a major hub in the light-signaling cascade both under visible and UV-B light. The mode of transcriptional regulation of HY5, especially under UV-B light, is not well characterized. B-BOX (BBX) transcription factors regulate HY5 transcription and also posttranscriptionally modulate HY5 to control photomorphogenesis under white light. Here, we identify BBX31 as a key signaling intermediate in visible and UV-B light signal transduction in Arabidopsis (Arabidopsis thaliana). BBX31 expression is induced by UV-B radiation in a fluence-dependent manner. HY5 directly binds to the promoter of BBX31 and regulates its transcript levels. Loss- and gain-of-function mutants of BBX31 indicate that it acts as a negative regulator of photomorphogenesis under white light but is a positive regulator of UV-B signaling. Genetic interaction studies suggest that BBX31 regulates photomorphogenesis independent of HY5 We found no evidence for a direct BBX31-HY5 interaction, and they primarily regulate different sets of genes in white light. Under high doses of UV-B radiation, BBX31 promotes the accumulation of UV-protective flavonoids and phenolic compounds. It enhances tolerance to UV-B radiation by regulating genes involved in photoprotection and DNA repair in a HY5-dependent manner. Under UV-B radiation, overexpression of BBX31 enhances HY5 transcriptional levels in a UV RESISTANCE LOCUS8-dependent manner, suggesting that BBX31 might regulate HY5 transcription.