Genome-wide characterization of plant CTP:phosphocholine cytidylyltransferases through evolutionary, biochemical, and structural analyses.

Phosphatidylcholine has essential functions in many eukaryotic cells, and its de novo biosynthesis is rate-limited by cytidine triphosphate:phosphocholine cytidylyltransferase (CCT). Although the biological and biochemical functions of CCT have been reported in mammals and several plants, this key enzyme has yet to be examined at a genome-wide level. As such, certain fundamental questions remain unanswered, including the evolutionary history, genetic and functional relationships, and structural variations among CCTs in the green lineage. In the current study, in-depth phylogenetic analysis, as well as the conservation and diversification in CCT gene structure and motif patterns, indicated that CCTs exist broadly in chlorophytes, bryophytes, lycophytes, monilophytes, gymnosperms, early-diverging angiosperms, monocots, and eudicots, and form eight relatively conserved clades. To further explore the potential function of selection pressure, we conducted extensive selection pressure analysis with a representative CCT gene, CCT1 from the model plant Arabidopsis thaliana (AthCCT1), and identified two positive selection sites, L59 and Q156. Site-directed mutagenesis and in vitro enzyme assays demonstrated that these positively selected sites were indeed important for the activity and substrate affinity of AthCCT1, and subsequent 3D structure analyses explained the potential biochemical mechanism. Taken together, our results unraveled the evolution and diversity of CCTs in the green lineage, as well as their association with the enzyme's biochemical and structural properties, and expanded our understanding of this important enzyme at the genome-wide level.

Microalgal glycerol-3-phosphate acyltransferase role in galactolipids and high-value storage lipid biosynthesis.

Glycerolipids are the most abundant lipids in microalgae, and glycerol-3-phosphate:acyl-CoA acyltransferase (GPAT) plays an important role in their biosynthesis. However, the biochemical and biological functions of algal GPAT remain poorly characterized. Here, we characterized the endoplasmic reticulum (ER)-associated GPAT of the model unicellular green alga Chlamydomonas reinhardtii (CrGPATer). Enzymatic assays indicated that CrGPATer is an sn-1 acyltransferase using a variety of acyl-CoAs as the acyl donor. Subcellular localization revealed that CrGPATer was associated with ER membranes and lipid droplets. We constructed overexpression (OE) and knockdown (KD) transgenic C. reinhardtii lines to investigate the in vivo function of CrGPATer. Lipidomic analysis indicated that CrGPATer OE enhanced the cellular content of galactolipids, especially monogalactosyldiacylglycerol, under nitrogen deficiency stress. Correspondingly, CrGPATer KD lines contained lower contents of galactolipids than the control. Feeding experiments with labeled phosphatidic acid revealed that the intermediate of the eukaryotic Kennedy pathway could be used for galactolipid biosynthesis in the chloroplasts. These results provided multiple lines of evidence that the eukaryotic Kennedy pathway mediated by CrGPATer may be involved in galactolipid biosynthesis in C. reinhardtii. OE of CrGPATer significantly increased the content of triacylglycerol and the yield of biomass. Moreover, the content and yield of 1, 3-olein-2-palmitin, a high-value lipid that can be used as an alternative for human milk fat in infant formula, were significantly enhanced in the OE transgenic lines. Taken together, this study provided insights into the biochemical and biological functions of CrGPATer and its potential as a genetic engineering target in functional lipid manufacturing.

CRISPR/Cas-Mediated Gene Editing in Plant Immunity and Its Potential for the Future Development of Fungal, Oomycete, and Bacterial Pathogen-Resistant Pulse Crops.

Pulses provide myriad health benefits and are advantageous in an environmental context as a result of their leguminous nature. However, phytopathogenic fungi, oomycetes and bacteria pose a substantial threat to pulse production, at times leading to crop failure. Unfortunately, existing disease management strategies often provide insufficient control, and there is a clear need for the development of new pulse cultivars with durable and broad-spectrum disease resistance. CRISPR/Cas-mediated gene editing has proven its potential for rapidly enhancing disease resistance in many plant species. However, this tool has only very recently been applied in pulse species, and never in the context of plant immunity. In this review, we examine the recent successful utilization of this technology in pulse species for proof-of-concept or the improvement of other traits. In addition, we consider various genes that have been edited in other plant species to reduce susceptibility to pathogens, and discuss current knowledge regarding their roles in pulses. Given the functional conservation of the selected genes across diverse plant species, there is a high likelihood that their editing would elicit similar effects in non-oilseed grain legumes, thus providing a suite of potential targets for CRISPR/Cas-mediated gene editing to promote pulse crop productivity in coming years.

Enhancing cryopreservation survival in Petunia × Calibrachoa 'Light Yellow' callus: Insights into material characteristics and genetic integrity.

Petunia × Calibrachoa 'Light Yellow' (× Petchoa 'Light Yellow') is a kind of perennial herbaceous flower obtained through intergeneric hybridization of Petunia and Calibrachoa with high ornamental value and wide application, facing challenges in seed acquisition. Expanding propagation through tissue culture is an economically efficient means. Hence, establishing an effective procedure for the storage of callus is essential for × Petchoa 'Light Yellow'. Cryopreservation is an effective method for the in vitro propagation and long-term preservation of × Petchoa 'Light Yellow' germplasms. For formulating the optimization of the vitrification procedure, first, an orthogonal experimental design was employed to pinpoint critical steps in the vitrification protocol (pre-culture, osmoprotection, dehydration, and dilution) for Petunia × Calibrachoa callus tissues and then five additional factors (pre-culture, osmoprotection I and II, dehydration, and dilution) were optimized to further reduce the sample water content and enhance cell viability levels. The vitrification procedure was described as follows: callus tissues were precultured in MS solid medium with 0.3 M sucrose for 5 d, incubated with osmoprotection solution I and II for 15 min at 25 °C, respectively, cryoprotected with PVS2 for 30 min at 0 °C, and rapidly immersed in liquid nitrogen. Cryopreserved callus tissues were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25 °C and recovered on MS solid medium with 0.5 mg/L 6-BA and 0.1 mg/L NAA, and sucrose. The cell viability measured by TTC staining was approximately 16 %-18 % after 72 h-recovery. Following 45 days, the relative survival of callus reached up to 49.48 %. Furthermore, EST-SSR analysis showed no significant difference in the genetic stability of cryopreserved callus compared to the control. Based on the cryopreservation of × Petchoa 'Light Yellow' callus, we further evaluated the response of callus water contents to the osmotic stress in the optimized and original protocols (CK) for a higher cryopreservation survival. A comparative analysis of water content demonstrated that the procedure of gradual and gentle dehydration significantly improved water content and cell survival. Ultrastructural changes between cryopreserved and non-cryopreserved callus were examined and high vacuolation emerged as a key determinant, indicating its substantial impact on the low survival of cryopreserved cells, which should help us to understand the effectiveness of osmotic protectants in dehydration.

Amyloid and SCD jointly predict cognitive decline across Chinese and German cohorts.

INTRODUCTION: Subjective cognitive decline (SCD) in amyloid-positive (Aß+) individuals was proposed as a clinical indicator of Stage 2 in the Alzheimer's disease (AD) continuum, but this requires further validation across cultures, measures, and recruitment strategies. METHODS: Eight hundred twenty-one participants from SILCODE and DELCODE cohorts, including normal controls (NC) and individuals with SCD recruited from the community or from memory clinics, underwent neuropsychological assessments over up to 6 years. Amyloid positivity was derived from positron emission tomography or plasma biomarkers. Global cognitive change was analyzed using linear mixed-effects models. RESULTS: In the combined and stratified cohorts, Aß+ participants with SCD showed steeper cognitive decline or diminished practice effects compared with NC or Aß- participants with SCD. These findings were confirmed using different operationalizations of SCD and amyloid positivity, and across different SCD recruitment settings. DISCUSSION: Aß+ individuals with SCD in German and Chinese populations showed greater global cognitive decline and could be targeted for interventional trials. HIGHLIGHTS: SCD in amyloid-positive (Aß+) participants predicts a steeper cognitive decline. This finding does not rely on specific SCD or amyloid operationalization. This finding is not specific to SCD patients recruited from memory clinics. This finding is valid in both German and Chinese populations. Aß+ older adults with SCD could be a target population for interventional trials.

Protein interactomes for plant lipid biosynthesis and their biotechnological applications.

Plant lipids have essential biological roles in plant development and stress responses through their functions in cell membrane formation, energy storage and signalling. Vegetable oil, which is composed mainly of the storage lipid triacylglycerol, also has important applications in food, biofuel and oleochemical industries. Lipid biosynthesis occurs in multiple subcellular compartments and involves the coordinated action of various pathways. Although biochemical and molecular biology research over the last few decades has identified many proteins associated with lipid metabolism, our current understanding of the dynamic protein interactomes involved in lipid biosynthesis, modification and channelling is limited. This review examines advances in the identification and characterization of protein interactomes involved in plant lipid biosynthesis, with a focus on protein complexes consisting of different subunits for sequential reactions such as those in fatty acid biosynthesis and modification, as well as transient or dynamic interactomes formed from enzymes in cooperative pathways such as assemblies of membrane-bound enzymes for triacylglycerol biosynthesis. We also showcase a selection of representative protein interactome structures predicted using AlphaFold2, and discuss current and prospective strategies involving the use of interactome knowledge in plant lipid biotechnology. Finally, unresolved questions in this research area and possible approaches to address them are also discussed.

Genetic architecture of seed glycerolipids in Asian cultivated rice.

Glycerolipids are essential for rice development and grain quality but its genetic regulation remains unknown. Here we report its genetic base using metabolite-based genome-wide association study and metabolite-based quantitative traits locus (QTL) analyses based on lipidomic profiles of seeds from 587 Asian cultivated rice accessions and 103 chromosomal segment substitution lines, respectively. We found that two genes encoding phosphatidylcholine (PC):diacylglycerol cholinephosphotransferase (OsLP1) and granule-bound starch synthase I (Waxy) contribute to variations in saturated triacylglycerol (TAG) and lyso-PC contents, respectively. We demonstrated that allelic variation in OsLP1 sequence between indica and japonica results in different enzymatic preference for substrate PC-16:0/16:0 and different saturated TAG levels. Further evidence demonstrated that OsLP1 also affects heading date, and that co-selection of OsLP1 and a flooding-tolerant QTL in Aus results in the abundance of saturated TAGs associated with flooding tolerance. Moreover, we revealed that the sequence polymorphisms in Waxy has pleiotropic effects on lyso-PC and amylose content. We proposed that rice seed glycerolipids have been unintentionally shaped during natural and artificial selection for adaptive or import seed quality traits. Collectively, our findings provide valuable genetic resources for rice improvement and evolutionary insights into seed glycerolipid variations in rice.

Physaria fendleri and Ricinus communis lecithin:cholesterol acyltransferase-like phospholipases selectively cleave hydroxy acyl chains from phosphatidylcholine.

Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12-HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn-1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over-expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase-like PLAs (LCAT-PLAs) in HFA biosynthesis were characterized. The LCAT-PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2 , and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT-PLAs from the HFA-accumulating plant species Physaria fendleri (PfLCAT-PLA) and castor (RcLCAT-PLA) could cleave acyl chains at both the sn-1 and sn-2 positions of PC, and displayed substrate selectivity towards sn-2-ricinoleoyl-PC over sn-2-oleoyl-PC. Furthermore, co-expression of RcFAH12 with PfLCAT-PLA or RcLCAT-PLA, but not Arabidopsis AtLCAT-PLA, resulted in increased occupation of HFA at the sn-1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT-PLA and RcLCAT-PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops.

A Positive Feedback Loop Mediated by CsERF31 Initiates Female Cucumber Flower Development: ETHYLENE RESPONSE FACTOR31 mediates a positive feedback loop that initiates female cucumber flower development.

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid (ACC) synthases. However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein-DNA interaction assays and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signalling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female-male determination and female-bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.

A positive feedback loop mediated by CsERF31 initiates female cucumber flower development.

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid synthases (CsACSs). However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein-DNA interaction assays, and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signaling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female-male determination and female-bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination.