Combined transcriptomic and metabolomic analyses identifies CsERF003, a citrus ERF transcription factor, as flavonoid activator.

Transcription factor (TF) modulation is a promising strategy for plant flavonoid improvement. Here, we observed evident decreases in some major flavones and flavonols and the expression of some key related genes in a 'Newhall' navel orange mutant (MT) relative to the wild type (WT). A consistently downregulated ERF TF CsERF003 in MT could increase the contents of major flavonoids and the precursor phenylalanine when transiently overexpressed in citrus fruit. Overexpression of CsERF003 in 'Micro-Tom' tomato (OE) resulted in a darker and redder fruit color than wild type 'Micro-Tom' (WTm). Two major flavonoids, naringeninchalcone and kaempferolrutinoside, were averagely induced by 7.99- and 36.83-fold in OEs, respectively, while little change was observed in other polyphenols, such as caffeic acid, ferulic acid, and gallic acid. Key genes involved in the initiation of phenylpropanoid (PAL, 4CH, and 4CL) and flavonoid (CHS and CHI) biosynthesis were up-regulated, while most genes participating in the biosynthesis of other polyphenols, such as HCT and CCR, were down-regulated in OEs. Therefore, it could be concluded that carbon flux floods into the phenylpropanoid biosynthetic pathway and is then specifically directed for flavonoid biosynthesis. CsERF003 may be a potentially promising gene for fruit quality improvement and engineering of natural flavonoid components.

Heavy metal contamination and risk assessment in winter jujube (Ziziphus jujuba Mill. cv. Dongzao).

Winter jujube (Ziziphus jujuba Mill. cv. Dongzao) is a major fresh-eating jujube fruit with various important nutrients for humans. It can absorb heavy metals from polluted air, water and soils and applied pesticides, which may pose potential threats to consumers. Here, to evaluate the content of heavy metals in winter jujube and systematically evaluate the potential risks, we collected 212 winter jujube samples from four main producing areas in China and determined the contents of eight heavy metals (Cd, Cr, Pb, Ni, Cu, Zn, As, and Mn) using inductively coupled plasma mass spectrometer (ICP-MS). Based on the integrated pollution index (IPI) evaluation standard, more than 99.06% of samples were at safe levels. Moreover, clustering analysis divided the eight heavy metals into four groups, namely Cr/Ni, Cd/Pb, Cu/Mn/Zn, and As. Importantly, none of the analyzed heavy metals posed risks to adults as indicted by the average carcinogenic and non-carcinogenic risks. Notably, Cr and Cd could pose low carcinogenic risks to children (≤12 age group) when their concentration reached the 90th percentile. This study systematically assessed the health risks associated with heavy metal intake through winter jujube consumption and highlighted the necessity of constant heavy metal monitoring in winter jujube.

Transcription factor CsESE3 positively modulates both jasmonic acid and wax biosynthesis in citrus.

PLIP lipases can initiate jasmonic acid (JA) biosynthesis. However, little is known about the transcriptional regulation of this process. In this study, an ERF transcription factor (CsESE3) was found to be co-expressed with all necessary genes for JA biosynthesis and several key genes for wax biosynthesis in transcriptomes of 'Newhall' navel orange. CsESE3 shows partial sequence similarity to the well-known wax regulator SHINEs (SHNs), but lacks a complete MM protein domain. Ectopic overexpression of CsESE3 in tomato (OE) resulted in reduction of fruit surface brightness and dwarf phenotype compared to the wild type. The OE tomato lines also showed significant increases in the content of wax and JA and the expression of key genes related to their biosynthesis. Overexpression of CsESE3 in citrus callus and fruit enhanced the JA content and the expression of JA biosynthetic genes. Furthermore, CsESE3 could bind to and activate the promoters of two phospholipases from the PLIP gene family to initiate JA biosynthesis. Overall, this study indicated that CsESE3 could mediate JA biosynthesis by activating PLIP genes and positively modulate wax biosynthesis. The findings provide important insights into the coordinated control of two defense strategies of plants represented by wax and JA biosynthesis. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-022-00085-2.

C24 and C26 aldehydes are potential natural additives of coating for citrus water retention.

Water loss is a key factor for the postharvest senescence of fruit. It has been reported that natural cuticular wax at high concentrations has better performance than commercial coating in water retention of fruit, which can prevent postharvest water loss without the accumulation of off-flavor. Here, we analyzed the correlation between epicuticular wax and postharvest water loss with 75 citrus varieties from a natural population. The water loss rate of the fruit was little influenced by the wax microstructure (stomata and wax crystal morphology), but strongly affected by epicuticular wax components. Further, C24 and C26 aliphatic aldehydes showed the greatest impact on fruit water loss rate, whose correlation coefficients reached -0.63 and -0.67, respectively. These two substances could significantly reduce the fruit water loss rate, indicating that they are potential natural additives to be used in the coating for citrus fruit water retention.

Comprehensive analysis of KCS gene family in Citrinae reveals the involvement of CsKCS2 and CsKCS11 in fruit cuticular wax synthesis at ripening.

Cuticular wax covers the surface of fleshy fruit and plays a protective role in fruit development and postharvest storage, including reducing fruit water loss, resisting biotic and abiotic stress and affecting fruit glossiness. The ß-ketoacyl-CoA synthase (KCS) is the rate-limiting enzyme of very long chain fatty acids (VLCFAs) synthesis, which provides precursors for the synthesis of cuticular wax. In this study, a total of 96 KCS genes were identified in six Citrinae species, including 13, 16, 21, 14, 16 and 16 KCS genes in the primitive species (Atalantia buxifolia), the wild species (Citrus ichangensis), and four cultivated species (Citrus medica, Citrus grandis, Citrus sinensis and Citrus clementina), respectively. Compared with primitive species, wild and cultivated species showed expansion of KCS gene family. Evolutionary analysis of KCS gene family indicated that uneven gain and loss of genes resulted in variable numbers of KCS genes in Citrinae, and KCS genes have undergone purifying selection. Expression profiles in C. sinensis revealed that the KCS genes had diverse expression patterns among various tissues. Furthermore, CsKCS2 and CsKCS11 were predominantly expressed in the flavedo and their expression increased sharply with ripening. Subcellular localization analysis indicated that CsKCS2 and CsKCS11 were located in the endoplasmic reticulum. Further, heterologous expression of CsKCS2 and CsKCS11 in Arabidopsis significantly increased the content of cuticular wax in leaves. Thus, CsKCS2 and CsKCS11 are involved in the accumulation of fruit cuticular wax at ripening. This work will facilitate further functional verification and understanding of the evolution of KCS genes in Citrinae.

Genome assembly of wild tea tree DASZ reveals pedigree and selection history of tea varieties.

Wild teas are valuable genetic resources for studying domestication and breeding. Here we report the assembly of a high-quality chromosome-scale reference genome for an ancient tea tree. The further RNA sequencing of 217 diverse tea accessions clarifies the pedigree of tea cultivars and reveals key contributors in the breeding of Chinese tea. Candidate genes associated with flavonoid biosynthesis are identified by genome-wide association study. Specifically, diverse allelic function of CsANR, CsF3'5'H and CsMYB5 is verified by transient overexpression and enzymatic assays, providing comprehensive insights into the biosynthesis of catechins, the most important bioactive compounds in tea plants. The inconspicuous differentiation between ancient trees and cultivars at both genetic and metabolic levels implies that tea may not have undergone long-term artificial directional selection in terms of flavor-related metabolites. These genomic resources provide evolutionary insight into tea plants and lay the foundation for better understanding the biosynthesis of beneficial natural compounds.

Parallel Metabolomic and Transcriptomic Analysis Reveals Key Factors for Quality Improvement of Tea Plants.

As one of the most popular beverages globally, tea has enormous economic, cultural, and medicinal importance that necessitates a comprehensive metabolomics study of this species. In this study, a large-scale targeted metabolomics analysis on two types of leaf tissues of nine tea cultivars from five representative geographical origins within China was carried out using the liquid chromatography-mass spectrometry technique. RNA-seq-based transcriptomic analysis was in parallel conducted on the same samples, and gene expression and metabolic differentiation between tissues as well as between the multiple tea cultivars were investigated. The data obtained provide an accessible resource for further studies of naturally occurring metabolic variation of tea plants, which will aid in thoroughly interpreting the underlying genetic and molecular mechanisms of biosynthesis of specialized metabolites in this critical species. Candidate genes including a transcription factor (CsMYB5-like), which were highly correlated with both the content of flavonoids and the expression level of genes participating in the phenylpropanoid and flavonoid biosynthesis pathway, were identified as potential targets for quality improvement of tea.

Lipidomic and transcriptomic analysis reveals reallocation of carbon flux from cuticular wax into plastid membrane lipids in a glossy "Newhall" navel orange mutant.

Both cuticle and membrane lipids play essential roles in quality maintenance and disease resistance in fresh fruits. Many reports have indicated the modification of alternative branch pathways in epicuticular wax mutants; however, the specific alterations concerning lipids have not been clarified thus far. Here, we conducted a comprehensive, time-resolved lipidomic, and transcriptomic analysis on the "Newhall" navel orange (WT) and its glossy mutant (MT) "Gannan No. 1". The results revealed severely suppressed wax formation accompanied by significantly elevated production of 36-carbon plastid lipids with increasing fruit maturation in MT. Transcriptomics analysis further identified a series of key functional enzymes and transcription factors putatively involved in the biosynthesis pathways of wax and membrane lipids. Moreover, the high accumulation of jasmonic acid (JA) in MT was possibly due to the need to maintain plastid lipid homeostasis, as the expression levels of two significantly upregulated lipases (CsDAD1 and CsDALL2) were positively correlated with plastid lipids and characterized to hydrolyze plastid lipids to increase the JA content. Our results will provide new insights into the molecular mechanisms underlying the natural variation of plant lipids to lay a foundation for the quality improvement of citrus fruit.

Identification of Key Residues Required for RNA Silencing Suppressor Activity of p23 Protein from a Mild Strain of Citrus Tristeza Virus.

The severe strain of citrus tristeza virus (CTV) causes quick decline of citrus trees. However, the CTV mild strain causes no symptoms and commonly presents in citrus trees. Viral suppressor of RNA silencing (VSR) plays an important role in the successful invasion of viruses into plants. For CTV, VSR has mostly been studied in severe strains. In this study, the N4 mild strain in China was sequenced and found to have high sequence identity with the T30 strain. Furthermore, we verified the functions of three VSRs in the N4 strain, and p23 was found to be the most effective in terms of local silencing suppressor activity among the three CTV VSRs and localized to both nucleus and plasmodesmata, which is similar to CTV T36 strain. Several conserved amino acids were identified in p23. Mutation of E95A/V96A and M99A/L100AA impaired p23 protein stability. Consequently, these two mutants lost most of its suppressor activity and their protein levels could not be rescued by co-expressing p19. Q93A and R143A/E144A abolished p23 suppressor activity only and their protein levels increased to wild type level when co-expressed with p19. This work may facilitate a better understanding of the pathogenic mechanism of CTV mild strains.

Fatty acid metabolic flux and lipid peroxidation homeostasis maintain the biomembrane stability to improve citrus fruit storage performance.

Little is known about the variations of fresh fruit biomembrane and its physiological and biochemical characteristics during storage. A navel orange mutant 'Gannan No.1' (Citrus sinensis Osbeck) showed higher membrane stability and titratable acid while lower calyx senescence compared with wild-type 'Newhall'. The membrane damage was significantly reduced in 'Gannan No.1' under 10% polyethylene-glycol (41.16% vs. 8.77%) and 30% polyethylene-glycol (52.59% vs.16.11%) treatments on day 45 after harvest. Consistently, membrane electrolyte leakage and malondialdehyde were significantly decreased in 'Gannan No.1', and superoxide dismutase and glutathione reductase were activated. A metabolic analysis was performed to evaluate membrane fatty acid unsaturation and peroxidation. Linolenic acid and hexadecylenic acid contributed to the higher degree of unsaturated fatty acids in 'Gannan No.1'. Furthermore, 'Gannan No.1' accumulated stress-resistant metabolites such as proline, α-tocopherol and glutathione. Correlation analysis of membrane homeostasis indexes with quality parameters showed the importance of biomembrane stability in maintaining citrus fruit quality.