SUMMARY
Carotenoids are important natural pigments that give bright colors to plants. The difference in the accumulation of carotenoids is one of the key factors in the formation of various colors in ...carrot taproots. Carotenoid cleavage dioxygenases (CCDs), including CCD and 9‐cis epoxycarotenoid dioxygenase, are the main enzymes involved in the cleavage of carotenoids in plants. Seven CCD genes have been annotated from the carrot genome. In this study, through expression analysis, we found that the expression level of DcCCD4 was significantly higher in the taproot of white carrot (low carotenoid content) than orange carrot (high carotenoid content). The overexpression of DcCCD4 in orange carrots caused the taproot color to be pale yellow, and the contents of α‐ and β‐carotene decreased sharply. Mutant carrot with loss of DcCCD4 function exhibited yellow color (the taproot of the control carrot was white). The accumulation of β‐carotene was also detected in taproot. Functional analysis of the DcCCD4 enzyme in vitro showed that it was able to cleave α‐ and β‐carotene at the 9, 10 (9′, 10′) double bonds. In addition, the number of colored chromoplasts in the taproot cells of transgenic carrots overexpressing DcCCD4 was significantly reduced compared with that in normal orange carrots. Results showed that DcCCD4 affects the accumulation of carotenoids through cleavage of α‐ and β‐carotene in carrot taproot.
Significance Statement
We analyzed seven carotenoid cleavage dioxygenase genes annotated from the carrot genome. Functional analysis revealed that DcCCD4 catalyzes the degradation of α‐ and β‐carotene to affect carotenoid accumulation. This work may provide a reference for generating new plant varieties with ideal color.
The first domesticated carrots were thought to be purple carrots rich in anthocyanins. The anthocyanins biosynthesis in solid purple carrot taproot was regulated by DcMYB7 within P3 region containing ...a gene cluster of six DcMYBs. Here, we described a MYB gene within the same region, DcMYB11c, which was highly expressed in the purple pigmented petioles. Overexpression of DcMYB11c in ‘Kurodagosun’ (KRDG, orange taproot carrot with green petioles) and ‘Qitouhuang’ (QTHG, yellow taproot carrot with green petioles) resulted in deep purple phenotype in the whole carrot plants indicating anthocyanins accumulation. Knockout of DcMYB11c in ‘Deep Purple’ (DPPP, purple taproot carrot with purple petioles) through CRISPR/Cas9‐based genome editing resulted in pale purple phenotype due to the dramatic decrease of anthocyanins content. DcMYB11c could induce the expression of DcbHLH3 and anthocyanins biosynthesis genes to jointly promote anthocyanins biosynthesis. Yeast one‐hybrid assay (Y1H) and dual‐luciferase reporter assay (LUC) revealed that DcMYB11c bound to the promoters of DcUCGXT1 and DcSAT1 and directly activated the expression of DcUCGXT1 and DcSAT1 responsible for anthocyanins glycosylation and acylation, respectively. Three transposons were present in the carrot cultivars with purple petioles but not in the carrot cultivars with green petioles. We revealed the core factor, DcMYB11c, involved in anthocyanins pigmentation in carrot purple petioles. This study provides new insights into precise regulation mechanism underlying anthocyanins biosynthesis in carrot. The orchestrated regulation mechanism in carrot might be conserved across the plant kingdom and useful for other researchers working on anthocyanins accumulation in different tissues.
Summary statement
Here, we described a MYB gene, DcMYB11c, which was highly expressed in the purple pigmented petioles. The orchestrated regulation mechanism in carrot might be conserved across the plant kingdom and useful for other researchers working on anthocyanins accumulation in different tissues.
SUMMARY
The color of purple carrot taproots mainly depends on the anthocyanins sequestered in the vacuoles. Glutathione S‐transferases (GSTs) are key enzymes involved in anthocyanin transport. ...However, the precise mechanism of anthocyanin transport from the cytosolic surface of the endoplasmic reticulum (ER) to the vacuoles in carrots remains unclear. In this study, we conducted a comprehensive analysis of the carrot genome, leading to the identification of a total of 41 DcGST genes. Among these, DcGST1 emerged as a prominent candidate, displaying a strong positive correlation with anthocyanin pigmentation in carrot taproots. It was highly expressed in the purple taproot tissues of purple carrot cultivars, while it was virtually inactive in the non‐purple taproot tissues of purple and non‐purple carrot cultivars. DcGST1, a homolog of Arabidopsis thaliana TRANSPARENT TESTA 19 (TT19), belongs to the GSTF clade and plays a crucial role in anthocyanin transport. Using the CRISPR/Cas9 system, we successfully knocked out DcGST1 in the solid purple carrot cultivar ‘Deep Purple’ (‘DPP’), resulting in carrots with orange taproots. Additionally, DcMYB7, an anthocyanin activator, binds to the DcGST1 promoter, activating its expression. Compared with the expression DcMYB7 alone, co‐expression of DcGST1 and DcMYB7 significantly increased anthocyanin accumulation in carrot calli. However, overexpression of DcGST1 in the two purple carrot cultivars did not change the anthocyanin accumulation pattern or significantly increase the anthocyanin content. These findings improve our understanding of anthocyanin transport mechanisms in plants, providing a molecular foundation for improving and enhancing carrot germplasm.
Significance Statement
These findings improve our understanding of anthocyanin transport mechanisms in plants, providing a molecular foundation for improving and enhancing carrot germplasm.
In the whole life process, many factors including external and internal factors affect plant growth and development. The morphogenesis, growth, and development of plants are controlled by genetic ...elements and are influenced by environmental stress. Transcription factors contain one or more specific DNA-binding domains, which are essential in the whole life cycle of higher plants. The AP2/ERF (APETALA2/ethylene-responsive element binding factors) transcription factors are a large group of factors that are mainly found in plants. The transcription factors of this family serve as important regulators in many biological and physiological processes, such as plant morphogenesis, responsive mechanisms to various stresses, hormone signal transduction, and metabolite regulation. In this review, we summarized the advances in identification, classification, function, regulatory mechanisms, and the evolution of AP2/ERF transcription factors in plants. AP2/ERF family factors are mainly classified into four major subfamilies: DREB (Dehydration Responsive Element-Binding), ERF (Ethylene-Responsive-Element-Binding protein), AP2 (APETALA2) and RAV (Related to ABI3/VP), and Soloists (few unclassified factors). The review summarized the reports about multiple regulatory functions of AP2/ERF transcription factors in plants. In addition to growth regulation and stress responses, the regulatory functions of AP2/ERF in plant metabolite biosynthesis have been described. We also discussed the roles of AP2/ERF transcription factors in different phytohormone-mediated signaling pathways in plants. Genomic-wide analysis indicated that AP2/ERF transcription factors were highly conserved during plant evolution. Some public databases containing the information of AP2/ERF have been introduced. The studies of AP2/ERF factors will provide important bases for plant regulatory mechanisms and molecular breeding.
Celery is a widely cultivated vegetable abundant in ascorbate (AsA), a natural plant antioxidant capable of scavenging free radicals generated by abiotic stress in plants. Ascorbate peroxidase (APX) ...is a plant antioxidant enzyme that is important in the synthesis of AsA and scavenging of excess hydrogen peroxide. However, the characteristics and functions of APX in celery remain unclear to date.
In this study, a gene encoding APX was cloned from celery and named AgAPX1. The transcription level of the AgAPX1 gene was significantly upregulated under drought stress. AgAPX1 was expressed in Escherichia coli BL21 (DE3) and purified. The predicted molecular mass of rAgAPX1 was 33.16 kDa, which was verified by SDS-PAGE assay. The optimum pH and temperature for rAgAPX1 were 7.0 and 55 °C, respectively. Transgenic Arabidopsis hosting the AgAPX1 gene showed elevated AsA content, antioxidant capacity and drought resistance. Less decrease in net photosynthetic rate, chlorophyll content, and relative water content contributed to the high survival rate of transgenic Arabidopsis lines after drought.
The characteristics of APX in celery were different from that in other species. The enhanced drought resistance of overexpressing AgAPX1 in Arabidopsis may be achieved by increasing the accumulation of AsA, enhancing the activities of various antioxidant enzymes, and promoting stomatal closure. Our work provides new evidence to understand APX and its response mechanisms to drought stress in celery.
Melatonin (MT) is a bioactive molecule that can regulate various developmental processes. Changes in lignin content play important roles in plant growth and development. Herein, quantitative analysis ...and histochemical staining showed that lignin content significantly increased over time, and melatonin treatment triggered the lignification at 8 and 16 d in tea leaves. The POD activity participated in lignin formation had also been significantly improved. The effect of melatonin on the increase of lignin content was attenuation over time. Sequencing results based on transcriptome at 8 and 16 d showed that 5273 and 3019 differentially expressed genes (DEGs) were identified in CK1 vs. MT1 and CK2 vs. MT2, respectively. A total of 67 DEGs were annotated to lignin biosynthesis, and 38 and 9 genes were significantly up-regulated under melatonin treatment, respectively. Some transcription factor genes such as MYB were also identified among the two pairwise comparisons, which might relate to lignin metabolism. Melatonin increased the degree of lignification in tea leaves by modifying the enzyme genes expression involved in lignin synthesis pathway. These results provide a reference for further study on the molecular mechanism of the dynamic changes of lignin content induced by melatonin treatment in tea plants.
The AP2/ERF family is a large family of transcription factors (TFs) in higher plants that plays a central role in plant growth, development, and response to environmental stress. Here, 209 AP2/ERF ...family members were identified in celery based on genomic and transcriptomic data. The TFs were classified into four subfamilies (i.e., DREB, ERF, RAV, and AP2) and Soloist. Evolution analysis indicated that the AP2/ERF TFs are ancient molecules and have expanded in the long-term evolution process of plants and whole-genome duplication events. AgAP2/ERF proteins may be associated with multiple biological processes as predicted by the interaction network. The expression profiles and sequence alignment analysis of the TFs in the DREB-A1 group showed that eight genes could be divided into four branches. Two genes, AgDREB1 and AgDREB2, from the DREB-A1 group were selected for further analysis. Subcellular localization assay suggested that the two proteins are nuclear proteins. Yeast one hybrid assay demonstrated that the two proteins could bind to the dehydration-responsive element (DRE). The overexpression of AgDREB1 and AgDREB2 in Arabidopsis induced the increased tolerance to cold treatment and the up-regulation of the COR genes expression. AgDREB1 and AgDREB2 might function as transcriptional activators in regulating the downstream genes by binding to corresponding DRE to enhance stress tolerance in celery.
Anthocyanins are involved in tissue coloration and stress response in plants. Foods containing high anthocyanin content are also beneficial to human health. Purple celery accumulated amounts of ...anthocyanins in the petioles. The biosynthesis of anthocyanin in plants is mainly regulated by the R2R3-MYB transcription factor (TF). However, the R2R3-MYB TF that controls anthocyanin accumulation in purple celery remains unclear. In this study, an R2R3-MYB TF gene, AgMYB2, was cloned from purple celery and characterized as anthocyanin biosynthetic regulator. Sequence analysis indicated that AgMYB2 contained highly conserved R2R3 domain and two anthocyanin characteristic motifs, ANDV motif and KPRPR S/T F motif. The relative expression level of AgMYB2 in purple celery was significantly higher than that in non-purple celery at three developmental stages. Heterologous expression of AgMYB2 in Arabidopsis generated more anthocyanins and resulted in dark-purple leaves and flowers. The expression levels of anthocyanin biosynthetic genes and the antioxidant activity of transgenic Arabidopsis carrying AgMYB2 were up-regulated. The determination of anthocyanin glycosylation activity of Arabidopsis crude enzyme verified the anthocyanin biosynthesis regulatory function of AgMYB2 at the protein level. The interaction between AgMYB2 and bHLH proteins was shown by yeast two-hybrid assay. The results will help to elucidate the molecular mechanism of anthocyanin biosynthesis in purple celery and provide an approach for cultivating plants with high anthocyanin content.
Key message
Overexpression of
AgMYB12
in celery improved the accumulation of apigenin by interacting with the
AgFNS
gene.
Celery is a common vegetable, and its essential characteristic is ...medicine food homology. A natural flavonoid and a major pharmacological component in celery, apigenin plays an important role in human health. In this study, we isolated a novel R2R3-MYB transcription factor that regulates apigenin accumulation from the celery cultivar ‘Jinnan Shiqin’ through yeast one-hybrid screening and designated it as AgMYB12. The AgMYB12 protein was located in the nucleus. It showed transcriptional activation activity and bound specifically to the promoter of
AgFNS
, a gene involved in apigenin biosynthesis. Phylogenetic tree analysis demonstrated that AgMYB12 belongs to the flavonoid branch. It contains two flavonoid-related motifs, SG7 and SG7-2, and shared a highly conserved R2R3 domain with flavonoid-related MYBs. The homologous overexpression of
AgMYB12
induced the up-regulation of
AgFNS
gene expression and accumulation of apigenin and luteolin in celery. Additionally, the expression levels of apigenin biosynthesis-related genes, including
AgPAL
,
AgCHI
,
AgCHS
,
Ag4CL
, and
AgC4H
, increased in transgenic celery plants. These results indicated that AgMYB12 acted as a positive regulator of apigenin biosynthesis and activated the expression of
AgFNS
gene. The current study provides new information about the regulation mechanism of apigenin metabolism in celery and offers a strategy for cultivating the plants with high apigenin content.
Abstract
Water dropwort (Liyang Baiqin,
Oenanthe javanica
(BI.) DC.) is an aquatic perennial plant from the Apiaceae family with abundant protein, dietary fiber, vitamins, and minerals. It usually ...grows in wet soils and can even grow in water. Here, whole-genome sequencing of
O. javanica
via HiSeq 2000 sequencing technology was reported for the first time. The genome size was 1.28 Gb, including 42,270 genes, of which 93.92% could be functionally annotated. An online database of the whole-genome sequences of water dropwort, Water dropwortDB, was established to share the results and facilitate further research on
O. javanica
(database homepage:
http://apiaceae.njau.edu.cn/waterdropwortdb
). Water dropwortDB offers whole-genome and transcriptome sequences and a Basic Local Alignment Search Tool. Comparative analysis with other species showed that the evolutionary relationship between
O. javanica
and
Daucus carota
was the closest. Twenty-five gene families of
O. javanica
were found to be expanded, and some genetic factors (such as genes and miRNAs) related to phenotypic and anatomic differentiation in
O. javanica
under different water conditions were further investigated. Two miRNA and target gene pairs (miR408 and
Oja15472
, miR171 and
Oja47040
) were remarkably regulated by water stress. The obtained reference genome of
O. javanica
provides important information for future work, thus making in-depth genetic breeding and gene editing possible. The present study also provides a foundation for the understanding of the
O. javanica
response to water stress, including morphological, anatomical, and genetic differentiation.
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