DcGST1, encoding a glutathione S-transferase activated by DcMYB7, is the main contributor to anthocyanin pigmentation in purple carrot.

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.

Overexpressing CsPSY1 Gene of Tea Plant, Encoding a Phytoene Synthase, Improves α-Carotene and ß-Carotene Contents in Carrot.

Tea plants (Camellia sinensis (L.) O. Kuntze) belong to Theaceae family, in the section Thea. Tea plants are widely distributed in subtropical and tropical regions in the word. α-carotene and ß-carotene in the tea leaves belong to carotenoids, which are associated with the aroma and color of the tea. Phytoene synthase (PSY) is a rate-limiting enzyme in carotenoids biosynthesis. We identified three CsPSY genes in 'Shuchazao', named CsPSY1, CsPSY2, and CsPSY3. Structural analysis of three CsPSY genes showed that CsPSY1 had a longer intro structure. The cis-acting elements of CsPSYs promoter were mainly associated with light-responsiveness, abiotic stress-responsiveness, and hormone-responsiveness. CsPSY1 exhibited expression in all tissues of the tea plants, whereas CsPSY2 and CsPSY3 were trace expression levels in all tissues. The positive expression of CsPSY1 under hormonal and abiotic stresses suggested its role in plant development and defense responses. The amino acid sequence of CsPSY1 was highly conserved in eight tea cultivars. The recombinant vector pCAMBIA1301-CsPSY1 was constructed to stabilize the overexpression of CsPSY1 in carrot. The contents of α-carotene and ß-carotene in transgenic carrot callus were significantly increased. This study provides a foundational basis for further research on the function of CsPSYs and carotenoids accumulation in tea plants.

Generating colorful carrot germplasm through metabolic engineering of betalains pigments.

Betalains are tyrosine-derived plant pigments exclusively found in the Caryophyllales order and some higher fungi and generally classified into two groups: red-violet betacyanins and yellow-orange betaxanthins. Betalains attract great scientific and economic interest because of their relatively simple biosynthesis pathway, attractive colors and health-promoting properties. Co-expressing two core genes BvCYP76AD1 and BvDODA1 with or without a glycosyltransferase gene MjcDOPA5GT allowed the engineering of carrot (an important taproot vegetable) to produce a palette of unique colors. The highest total betalains content, 943.2 µg·g-1 DW, was obtained in carrot taproot transformed with p35S:RUBY which produces all of the necessary enzymes for betalains synthesis. Root-specific production of betalains slightly relieved tyrosine consumption revealing the possible bottleneck in betalains production. Furthermore, a unique volcano-like phenotype in carrot taproot cross-section was created by vascular cambium-specific production of betalains. The betalains-fortified carrot in this study is thus anticipated to be used as functional vegetable and colorful carrot germplasm in breeding to promote health.

Effect of betanin synthesis on photosynthesis and tyrosine metabolism in transgenic carrot.

BACKGROUND: Betalain is a natural pigment with important nutritional value and broad application prospects. Previously, we produced betanin biosynthesis transgenic carrots via expressing optimized genes CYP76AD1S, cDOPA5GTS and DODA1S. Betanin can accumulate throughout the whole transgenic carrots. But the effects of betanin accumulation on the metabolism of transgenic plants and whether it produces unexpected effects are still unclear. RESULTS: The accumulation of betanin in leaves can significantly improve its antioxidant capacity and induce a decrease of chlorophyll content. Transcriptome and metabolomics analysis showed that 14.0% of genes and 33.1% of metabolites were significantly different, and metabolic pathways related to photosynthesis and tyrosine metabolism were markedly altered. Combined analysis showed that phenylpropane biosynthesis pathway significantly enriched the differentially expressed genes and significantly altered metabolites. CONCLUSIONS: Results showed that the metabolic status was significantly altered between transgenic and non-transgenic carrots, especially the photosynthesis and tyrosine metabolism. The extra consumption of tyrosine and accumulation of betanin might be the leading causes.

A MYB activator, DcMYB11c, regulates carrot anthocyanins accumulation in petiole but not taproot.

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.

Differential hydroxylation efficiency of the two non-heme carotene hydroxylases: DcBCH1, rather than DcBCH2, plays a major role in carrot taproot.

Carotene hydroxylase plays an important role in catalyzing the hydroxylation of carotene to xanthopylls, including two types: non-heme carotene hydroxylase (BCH type) and heme-containing cytochrome P450 hydroxylase (P450 type). Two BCH-encoding genes were annotated in the carrot genome. However, the role of BCHs and whether there are functional interactions between the duplicated BCHs in carrot remains unclear. In this study, two BCH encoding genes, DcBCH1 and DcBCH2, were cloned from carrot. The relative expression level of DcBCH1 was much higher than that of DcBCH2 in carrot taproots with different carotene accumulation levels. Overexpression of DcBCH1 in 'KRD' (high carotene accumulated) carrot changed the taproot color from orange to yellow, accompanied by substantial reductions in α-carotene and ß-carotene. There was no obvious change in taproot color between transgenic 'KRD' carrot overexpressing DcBCH2 and control carrot. Simultaneously, the content of α-carotene in the taproot of DcBCH2-overexpressing carrot decreased, but the content of ß-carotene did not change significantly in comparison with control carrot. Using the CRISPR/Cas9 system to knock out DcBCH1 in 'KRD' carrot lightened the taproot color from orange to pink-orange; the content of α-carotene in the taproot increased slightly, while the ß-carotene content was still significantly decreased, compared with control carrot. In DcBCH1-knockout carrot, the transcript level of DcBCH2 was significantly increased. These results indicated that in carrot taproot, DcBCH1 played the main function of BCH enzyme, which could hydroxylate α-carotene and ß-carotene; DcBCH1 and DcBCH2 had functional redundancy, and these two DcBCHs could partially compensate for each other.

Genome-Wide Identification and Evolution Analysis of R2R3-MYB Gene Family Reveals S6 Subfamily R2R3-MYB Transcription Factors Involved in Anthocyanin Biosynthesis in Carrot.

The taproot of purple carrot accumulated rich anthocyanin, but non-purple carrot did not. MYB transcription factors (TFs) condition anthocyanin biosynthesis in many plants. Currently, genome-wide identification and evolution analysis of R2R3-MYB gene family and their roles involved in conditioning anthocyanin biosynthesis in carrot is still limited. In this study, a total of 146 carrot R2R3-MYB TFs were identified based on the carrot transcriptome and genome database and were classified into 19 subfamilies on the basis of R2R3-MYB domain. These R2R3-MYB genes were unevenly distributed among nine chromosomes, and Ka/Ks analysis suggested that they evolved under a purified selection. The anthocyanin-related S6 subfamily, which contains 7 MYB TFs, was isolated from R2R3-MYB TFs. The anthocyanin content of rhizodermis, cortex, and secondary phloem in 'Black nebula' cultivar reached the highest among the 3 solid purple carrot cultivars at 110 days after sowing, which was approximately 4.20- and 3.72-fold higher than that in the 'Deep purple' and 'Ziwei' cultivars, respectively. The expression level of 7 MYB genes in purple carrot was higher than that in non-purple carrot. Among them, DcMYB113 (DCAR_008994) was specifically expressed in rhizodermis, cortex, and secondary phloem tissues of 'Purple haze' cultivar, with the highest expression level of 10,223.77 compared with the control 'DPP' cultivar at 70 days after sowing. DcMYB7 (DCAR_010745) was detected in purple root tissue of 'DPP' cultivar and its expression level in rhizodermis, cortex, and secondary phloem was 3.23-fold higher than that of secondary xylem at 110 days after sowing. Our results should be useful for determining the precise role of S6 subfamily R2R3-MYB TFs participating in anthocyanin biosynthesis in carrot.

[Ecological compensation strategy of the old revolutionary base areas along the route of Long March based on ecosystem service value evaluation]. / 基于生态系统服务价值评估的长征沿线革命老区生态补偿策略.

The conflict between ecological protection and socio-economic development in the old revolutionary base areas along the route of Long March can be addressed by ecological compensation. In this study, we collected 2018 data of land use, net primary productivity of vegetation, climate factors and so on. The ecosystem service value of 310 counties in the old revolutionary base areas was calculated using the data and modified equivalent factor. Then, the regional differentiated ecological compensation estimation method was applied to estimate the priority and amount of ecological compensation within the area. We further proposed the ecological compensation strategy adopted for the old revolutionary base areas along the route of Long March. The results showed that the total value of ecosystem services in the old revolutionary base areas along the route of Long March amounted to 2593.74 billion yuan in 2018. Forest services took the highest proportion (58.8%), followed by grassland and waters. The total amount of ecological compensation made for the old revolutionary base areas along the route of Long March reached 183.405 billion yuan. Compared with this amount, there was a large gap in the actual compensation funds available to the region. In addition, the total amount of ecological compensation accounted for only 0.04% of the aggregate GDP in this area, which put some strain on local fiscal resources and showed certain viability in ecological compensation. In 2018, totally 100 old revolutionary base areas along the route of Long March were designated as the priority areas for ecological compensation, consistent with the national key eco-function zone policy. However, there were deviations in the allocation of ecological compensation funds, with some priority areas receiving no compensation. As for the compensation strategy, it is necessary to put in place a diversified, market-oriented and comprehensive ecological compensation mechanism to increase the sources of ecological compensation funds at first. Then, efforts should be continued to optimize the spatial selection of ecological compensation in the old revolutionary base areas along the route of Long March and to improve the efficiency of regional ecological compensation mechanism.

Spatial-temporal differentiation and its influencing factors of agricultural eco-efficiency in China based on geographic detector.

Exploring the spatial-temporal variations of agricultural eco-efficiency (AEE) and its driving factors is of vital importance to achieve high-quality agro-ecological development in China. In this study, we used the super efficiency slack-based measure (SBM) model to measure the inter-provincial AEE based on the relevant panel data of 30 provinces/regions/cities in China from 2000 to 2018. Based on the time series analysis, spatial visualization, and trend surface analysis, the geographical detector model was further used to identify the core factors driving the spatial-temporal variations of AEE. The results showed that China's AEE level maintained stable upward progress from 2000 to 2018, which was still at a low level with much room for improvement. The AEE in China exhibited a significant spatial-temporal variation, presenting higher levels in the eastern and western parts but lower in the central part. The spatial variation of AEE was influenced by many factors, including agricultural resource endowment, socioeconomic condition, and the natural ecological environment. There were obvious variations in the influence factors on the spatial-temporal variation of AEE. The interactions among factors would enhance the spatial variation of AEE. Therefore, due to the spatial-temporal variation of AEE, emphasis should be placed on its core driving factors as well as the inter-parts agricultural cooperation in order to achieve high-quality agro-ecological development in China.

Overexpression of a carrot BCH gene, DcBCH1, improves tolerance to drought in Arabidopsis thaliana.

BACKGROUND: Carrot (Daucus carota L.), an important root vegetable, is very popular among consumers as its taproot is rich in various nutrients. Abiotic stresses, such as drought, salt, and low temperature, are the main factors that restrict the growth and development of carrots. Non-heme carotene hydroxylase (BCH) is a key regulatory enzyme in the ß-branch of the carotenoid biosynthesis pathway, upstream of the abscisic acid (ABA) synthesis pathway. RESULTS: In this study, we characterized a carrot BCH encoding gene, DcBCH1. The expression of DcBCH1 was induced by drought treatment. The overexpression of DcBCH1 in Arabidopsis thaliana resulted in enhanced tolerance to drought, as demonstrated by higher antioxidant capacity and lower malondialdehyde content after drought treatment. Under drought stress, the endogenous ABA level in transgenic A. thaliana was higher than that in wild-type (WT) plants. Additionally, the contents of lutein and ß-carotene in transgenic A. thaliana were lower than those in WT, whereas the expression levels of most endogenous carotenogenic genes were significantly increased after drought treatment. CONCLUSIONS: DcBCH1 can increase the antioxidant capacity and promote endogenous ABA levels of plants by regulating the synthesis rate of carotenoids, thereby regulating the drought resistance of plants. These results will help to provide potential candidate genes for plant drought tolerance breeding.

DcCCD4 catalyzes the degradation of α-carotene and ß-carotene to affect carotenoid accumulation and taproot color in carrot.

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 ß-carotene decreased sharply. Mutant carrot with loss of DcCCD4 function exhibited yellow color (the taproot of the control carrot was white). The accumulation of ß-carotene was also detected in taproot. Functional analysis of the DcCCD4 enzyme in vitro showed that it was able to cleave α- and ß-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 ß-carotene in carrot taproot.

[Relationship Between Urban Industrialization and PM2.5 Concentration in China and the Internal Mechanism of EKC].

Based on the panel data of 255 cities in China, this study built a spatial Durbin model to study the impact of China's industrialization on the PM2.5 pollution level. Meanwhile, an environmental governance tool characterized by forest and grass coverage was introduced to explore the internal mechanism and exogenous driving force of EKC. The results show that ① the relationship between industrialization and PM2.5 concentration is obviously an inverted u-shaped, and the EKC hypothesis was verified. ② The formation of the EKC curve was caused by the external factors and not the endogenous mechanism of economic growth. Forest-grass coverage plays a regulating role in the relationship between industrialization and PM2.5 pollution level, namely the inverted u-shaped structure of environmental EKC results from environmental governance tool with forest and grass cover rather than the automatic adjustment of economic growth. ③ The influence of industrialization on PM2.5 concentration has a spatial spillover effect. Urban industrialization not only affects the PM2.5 concentration in local regions but also its neighboring region.