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2021 Volume 1
Article Contents
Abstract
Introduction
Types of functional rice
High resistant starch rice
Colored rice
Giant embryo rice
Low glutelin rice
Golden rice
Fe and zn fortified rice
Breeding technology of functional rice
Traditional polymerization breeding
Transgenic polymerization breeding
Molecular marker-assisted selection polymerization breeding
Cultivation and regulation technology of functional rice
Effect of light on the accumulation of active components in rice
Effect of temperature on the accumulation of active components in rice
Effect of water management on the accumulation of active components in rice
Effect of fertilizer application on the accumulation of active components in rice
Effect of harvesting time on the accumulation of active components in rice
Prevention and control of pests and diseases for functional rice
Conclusions
Acknowledgments
Conflict of interest
Supplementary information
Rights and permissions
References
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ARTICLE   Open Access    

Comparative analysis of the TCP gene family in celery, coriander and carrot (family Apiaceae)

  • 1.

    College of Life Sciences/Center for Genomics and Bio-computing, North China University of Science and Technology, Tangshan 063210, China

  • 2.

    School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China

  • 3.

    Food Science and Technology Department, University of Nebraska-Lincoln, Lincoln, NE 68588, USA

  • 4.

    Faculty of Life Science, Tangshan Normal University, Tangshan 063000, China

  • These authors contributed equally: Qiaoying Pei, Nan Li
More Information
  • Apiaceae is one of the most important families in Apiales and includes many economically important vegetables and medicinal plants. The TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR 1/2 (TCP) gene family plays an important role in regulating plant growth and development, but it has not been widely studied in Apiaceae. In the present study, we identified 215 TCP family genes in six species of plant, of which 122 genes were present in three Apiaceae including 29 in celery (Apium graveolens), 43 in coriander (Coriandrum sativum), and 50 in carrot (Daucus carota). Whole-genome duplication likely contributed to TCP gene family expansion in Apiaceae. There were more paralogs in carrot than in coriander and celery, which was attributable to the greater number of tandem and proximal duplicated genes on chromosome 1. Nine microRNAs were found to regulate 20 TCP genes in the three Apiaceae species, with miR-319 having the most target genes. Several TCP genes showed high expression in the root, petiole and leaf of celery and coriander. These results provide a basis for comparative and functional genomic analyses of TCP genes in Apiaceae and other plants.

  • Introduction

    With the development of the world economy, people's lifestyles have changed dramatically, and long-term high-intensity work has put many people's bodies in a sub-healthy state. The increasing incidence of various chronic diseases has not only put enormous pressure on society's healthcare systems but also caused endless suffering to people[1]. Therefore, people's demands on the functionality and safety of food are increasing, and it has become the consensus of people that 'not just eating enough, but more importantly eating well'.

    Rice is the staple food for more than half of the world's population and the main economic source for a large number of rural people[2]. However, due to the rising cost of rice cultivation, farmers are gaining less and less economic benefits from growing rice, which seriously undermines their incentive to grow rice and poses a serious threat to world food security. Increasing the added value of rice not only helps to increase farmers' income but also helps to ensure world food security. The presence of a large number of functional ingredients in rice makes it possible to increase the added value of rice, and functional rice has therefore been widely noticed.

    Functional rice refers to rice containing certain specific components that play a regulatory and balancing role in human physiological functions in addition to the nutrients necessary for human growth and development in the endosperm, embryo, and rice bran. They can increase human physiological defense mechanisms, prevent certain diseases, help recovery, delay aging, and boost physical strength and energy levels[3]. Rice is a staple food for more than half of the world's population[4], and its functional components have a great potential to be exploited for human welfare. Using functional rice as a carrier to address health problems and realize 'medicine-food homology' is an excellent motivation for promoting functional rice. The current typical functional rice is introduced in this paper. It also summarizes the breeding and cultivation technologies of functional rice.

    Types of functional rice

    High resistant starch rice

    Rice has a high glycemic index. Its long-term consumption leads to obesity, diabetes, and colon disease in many people[5]. However, the consumption of rice rich in resistant starch (RS) can greatly reduce the risk of these diseases[6]. Therefore, breeding rice varieties with high RS content has attracted considerable attention from breeders in various countries. However, the variability of RS content between different rice varieties is low, and there are few germplasm resources available for selection, thus making it challenging to breed rice varieties with high RS content using traditional breeding methods. Combining traditional and modern molecular breeding techniques can greatly improve the successful production of high RS rice breeds. Nishi et al.[7] selected a high RS rice variety EM10 by treating fertilized egg cells of Kinmaze with N-methyl-N-nitrosourea. However, its yield was very low, and it was not suitable for commercial production. Wada et al.[8] crossed 'Fukei 2032' and 'EM129' as parents and selected Chikushi-kona 85, a high RS rice variety with a higher yield than EM10. Miura et al.[9] bred ultra-high RS BeI-BEIIB double mutant rice by crossing the Abe I and Abe IIB mutant strains, and the content of RS in the endosperm reached 35.1%. Wei et al.[10] found that the simultaneous inhibition of starch branching enzyme (SBE) genes SBEIIb and SBEI in Teqing by antisense RNA could increase the RS content in rice to 14.9%. Zhu et al.[11] used RNAi technology to inhibit the expression of SBEI and SBEII genes in rice, which increased the content of RS in rice endosperm from 0 to 14.6 %. Zhou et al.[6] found that rice RS formation is mainly controlled by soluble starch synthase (SSIIA). However, its regulation is dependent on the granule-bound starch synthase Waxy (Wx), and SSIIA deficiency combined with high expression of Wxa facilitates the substantial accumulation of RS in the rice. The results of Tsuiki et al.[12] showed that BEIB deficiency was the main reason for the increased accumulation of RS in rice. Itoh et al.[13] developed new mutant rice lines with significantly higher levels of RS in rice by introducing genes encoding starch synthase and granule-bound starch synthase in the rice into the BEIB-deficient mutant line be2b.

    Colored rice

    The accumulation of anthocyanins/proanthocyanidins in the seed coat of the rice grain gives brown rice a distinct color[14]. Most common rice varieties lack anthocyanins in the seed coat, and so far, no rice variety with colored endosperm in its natural state has been identified. However, Zhu et al.[15] bred rice with purple endosperm using transgenic technology. Red rice contains only proanthocyanidins, while black and purple rice contain anthocyanidins and proanthocyanidins[16]. Red seed coat of rice was found to be controlled by the complementary effects of two central effect genes Rc and Rd. The loss of function of the Rc gene prevented the synthesis of proanthocyanidins, while the Rd gene could enhance the effect of the Rc gene in promoting proanthocyanidins synthesis[17]. Purple seed coat color is controlled by two dominant complementary genes Pb and Pp. Pb determines the presence or absence of seed coat color, and Pp determines the depth of seed coat color[18]. In addition, phycocyanin synthesis is also regulated by transcription factors such as MYB, bHLH, HY5, and WD40[14], but the exact regulatory mechanism is not clear. Colored rice is rich in bioactive components, such as flavonoids, phenolic acids, vitamin E (VE), glutelin, phytosterols, and phytic acid (PA). It also contains large amounts of micronutrients such as Ca, Fe, Zn, and Se[19], and has a much higher nutritional and health value than ordinary white rice. In addition, Zhu et al.[20] successfully developed rice with enriched astaxanthin in the endosperm by introducing the genes sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY. This achievement has laid a solid foundation for the further development of functional rice industry.

    Giant embryo rice

    Giant embryo rice refers to rice varieties whose embryo volume is more than twice that of ordinary rice[21]. Rice embryo contains more nutrients than the endosperm; therefore, the nutritional value of giant embryo rice greatly exceeds that of ordinary rice. Studies have found that the levels of γ-aminobutyric acid (GABA), essential amino acids, VE, γ-oryzanol, phenols, and trace elements in giant embryo rice are considerably higher than that in ordinary rice[21]. Satoh & Omura[22] used the chemical mutagen N-methyl-N-nitrosourea to treat the fertilized egg cells of the rice variety Kinmaze to obtain a 'giant embryo' mutant. The mutants’ embryo occupied 1/4–1/3 of the rice grain volume and was 3–4 times larger than normal rice embryo[23]. Its GABA content increased dramatically after the rice was soaked in water. Maeda et al.[24] crossed the giant embryo mutant EM40 of Kinmaze with the high-yielding variety Akenohoshi to produce the giant embryo rice variety 'Haiminori'. The embryo size of 'Haiminori' is 3–4 times that of ordinary rice, and the GABA content of its brown rice is 3–4 times higher than that of 'Nipponbare' and 'Koshihikari' after soaking for four hours in water. A few genes that can regulate the size of rice embryos have been identified, and GE is the first identified rice giant embryo gene[25]. Nagasawa et al.[26] found that the loss of GE gene function resulted in enlarged embryos and smaller endosperm in rice. Lee et al.[27] found that the inhibition of LE gene expression by RNAi technology could lead to embryo enlargement in rice, but the regulatory mechanism remains to be investigated.

    Low glutelin rice

    Protein is the second most crucial nutrient in rice, accounting for 7–10% of the grain weight, and glutenin accounts for 60%–80% of the total protein content in rice grains[28]. Compared to other proteins, glutenin is more easily digested and absorbed by the body[29]. Therefore, higher glutenin content in rice can improve its nutritional value. However, people with renal disease (a common complication of diabetes) have impaired protein metabolism, and consumption of rice with lower glutelin content can help reduce their protein intake and metabolic burden[30]. Japanese breeders treated Nihonmasari with the chemical mutagen ethyleneimine and selected the low-glutelin rice mutant NM67[31]. Iida et al.[31] developed a new rice variety LGC-1 (Low glutelin content-1) with a glutelin content of less than 4% by backcrossing the NM67 mutant with the original variety 'Nihonmasari'. According to Miyahara[32], the low glutelin trait in LGC-1 is controlled by a single dominant gene Lgc-1 located on chromosome 2. Subsequently, Nishimura et al.[33] produced two rice varieties, 'LGC Katsu' and 'LGC Jun' with lower glutelin content by crossing LGC1 with a mutant line Koshikari (γ-ray induction) lacking 26 kDa globulin (another easily digestible protein).

    Golden rice

    Vitamin A (VA) is one of the essential nutrients for the human body[34]. However, rice, a staple food, lacks VA, leading to a VA deficiency in many people. β-carotene is a precursor for VA synthesis and can be effectively converted into VA in the human body[35]. Therefore, breeding rice varieties rich in β-carotene has attracted the attention of breeders in various countries. Ye et al.[36] simultaneously transferred phytoene synthase (psy), phytoene desaturase (crt I), and lycopene β-cyclase (lcy) genes into rice using the Agrobacterium-mediated method and produced the first generation of golden rice with a β-carotene content of 1.6 µg·g−1 in the endosperm. However, due to the low content of β-carotene in rice, it is difficult to meet the human body's demand for VA. To increase β-carotene content in rice, Paine et al.[37] introduced the phytoene synthase (psy) gene from maize and the phytoene desaturase (crt I) gene from Erwinia into rice. They obtained the second generation of golden rice with 37 µg g−1 of β-carotene in the endosperm, with nearly 23-fold increase in β-carotene content compared to the first generation of golden rice.

    Fe and Zn fortified rice

    Fe and Zn are essential trace elements for human beings. The contents of Fe and Zn in common rice are about 2 μg·g−1 and 16 μg·g−1, respectively[38], which are far from meeting human needs. In 2004, to alleviate micronutrient deficiencies among underprivileged people in developing countries, the Consultative Group on International Agricultural Research launched the HarvestPlus international collaborative program for improving Fe, Zn, and β-carotene levels in staple crops, with breeding targets of 13 μg·g−1 and 28 μg·g−1 for Fe and Zn in rice, respectively. Masuda et al.[39] found that expression of the nicotianamine synthase (NAS) gene HvNAS in rice resulted in a 3-fold increase in Fe and a 2-fold increase in Zn content in polished rice. Trijatmiko et al.[38] overexpressed rice OsNAS2 gene and soybean ferritin gene SferH-1 in rice, and the Fe and Zn content in polished rice of rice variety NASFer-274 reached 15 μg·g−1 and 45.7 μg·g−1, respectively. In addition, it has been found that increasing Fe intake alone does not eliminate Fe deficiency but also decreases the amount of Fe absorption inhibitors in the diet or increases the amount of Fe absorption enhancers[40]. The negatively charged phosphate in PA strongly binds metal cations, thus reducing the bioavailability of Fe and Zn in rice[41], while the sulfhydryl group in cysteine binds Fe, thereby increasing the absorption of non-heme Fe by the body[42]. To improve the bioavailability of Fe and Zn, Lucca et al.[40] introduced a heat-tolerant phytase (phyA) gene from Aspergillus fumigatus into rice and overexpressed the cysteine-rich protein gene (rgMT), which increased the content of phytase and cysteine residues in rice by 130-fold and 7-fold, respectively[40].

    Breeding technology of functional rice

    The functional quality of rice is highly dependent on germplasm resources. Current functional rice breeding mainly adopts transgenic and mutagenic technologies, and the cultivated rice varieties are mainly enriched with only one functional substance and cannot meet the urgent demand by consumers for rice enriched with multiple active components. The diversity of rice active components determines the complexity of multifunctional rice breeding. In order to cultivate multifunctional rice, it is necessary to strengthen the application of different breeding technologies. Gene polymerization breeding is a crop breeding technology that can polymerize multiple superior traits that have emerged in recent years, mainly including traditional polymerization breeding, transgenic polymerization breeding, and molecular marker-assisted selection polymerization breeding.

    Traditional polymerization breeding

    The transfer of beneficial genes in different species during traditional polymeric breeding is largely limited by interspecific reproductive isolation, and it is challenging to utilize beneficial genes between different species effectively. Gene transfer through sexual crosses does not allow accurate manipulation and selection of a gene and is susceptible to undesirable gene linkage, and in the process of breed selection, multiple backcrosses are required[43]. Thus, the period of selecting target plants is long, the breeding cost is high, and the human resources and material resources are costly[44]. Besides, it is often difficult to continue the breakthrough after a few generations of backcrossing due to linkage drag. Thus, there are significant limitations in aggregating genes by traditional breeding methods[45].

    Transgenic polymerization breeding

    Transgenic technology is an effective means of gene polymerization breeding. Multi-gene transformation makes it possible to assemble multiple beneficial genes in transgenic rice breeding rapidly and can greatly reduce the time and workload of breeding[46]. The traditional multi-gene transformation uses a single gene transformation and hybridization polymerization method[47], in which the vector construction and transformation process is relatively simple. However, it is time-consuming, laborious, and requires extensive hybridization and screening efforts. Multi-gene-based vector transformation methods can be divided into two major categories: multi-vector co-transformation and multi-gene single vector transformation[47]. Multi-vector co-transformation is the simultaneous transfer of multiple target genes into the same recipient plant through different vectors. The efficiency of multi-vector co-transformation is uncertain, and the increase in the number of transforming vectors will increase the difficulty of genetic screening, resulting in a reduced probability of obtaining multi-gene co-transformed plants. Multi-gene single vector transformation constructs multiple genes into the T-DNA region of a vector and then transfers them into the same recipient plant as a single event. This method eliminates the tedious hybridization and backcrossing process and solves the challenges of low co-transformation frequency and complex integration patterns. It can also avoid gene loss caused by multi-gene separation and recombination in future generations[47]. The transgenic method can break through the limitations of conventional breeding, disrupt reproductive isolation, transfer beneficial genes from entirely unrelated crops to rice, and shorten the cycle of polymerizing target genes significantly. However, there are concerns that when genes are manipulated, unforeseen side effects may occur, and, therefore, there are ongoing concerns about the safety of transgenic crops[48]. Marker-free transgenic technology through which selective marker genes in transgenic plants can be removed has been developed. This improves the safety of transgenic crops, is beneficial to multiple operations of the same transgenic crop, and improves the acceptance by people[49].

    Molecular marker-assisted selection polymerization breeding

    Molecular marker-assisted selection is one of the most widely used rice breeding techniques at present. It uses the close linkage between molecular markers and target genes to select multiple genes directly and aggregates genes from different sources into one variety. This has multiple advantages, including a focused purpose, high accuracy, short breeding cycle, no interference from environmental conditions, and applicability to complex traits[50]. However, few genes have been targeted for the main effect of important agronomic traits in rice, and they are mainly focused on the regulation of rice plant type and the prevention and control of pests and diseases, and very few genes related to the synthesis of active components, which can be used for molecular marker-assisted selection are very limited. Furthermore, the current technical requirements and costs for analyzing and identifying DNA molecular markers are high, and the identification efficiency is low. This greatly limits the popularization and application of functional rice polymerization breeding. Therefore, to better apply molecular marker-assisted selection technology to breed rice varieties rich in multiple active components, it is necessary to construct a richer molecular marker linkage map to enhance the localization of genes related to functional substance synthesis in rice[51]. Additionally, it is important to explore new molecular marker technologies to improve efficiency while reducing cost.

    It is worth noting that the effects of gene aggregation are not simply additive. There are cumulative additive effects, greater than cumulative epistatic effects, and less than cumulative epistatic effects among the polymerization genes, and the effects are often smaller than the individual effect. Only with a clearer understanding of the interaction between different QTLs or genes can functional rice pyramiding breeding be carried out reasonably and efficiently. Except for RS and Se, other active components of rice mainly exist in the rice bran layer, and the content of active components in the endosperm, the main edible part, is extremely low. Therefore, cultivating rice varieties with endosperm-enriched active components have broad development prospects. In addition, because crops with high quality are more susceptible to pests and diseases[52], the improvement of rice resistance to pests and diseases should be considered during the polymerization breeding of functional rice.

    Cultivation and regulation technology of functional rice

    The biosynthesis of active components in rice is influenced by rice varieties but also depends on cultivation management practices and their growth environment.

    Effect of light on the accumulation of active components in rice

    Environmental conditions have a greater effect on protein content than genetic forces[53]. Both light intensity and light duration affect the synthesis and accumulation of active components in rice. Low light intensity in the early stage of rice growth is not conducive to the accumulation of glutelin in rice grains but favors the accumulation of amylose, while the opposite is true in the late stage of rice growth[54]. Low light intensity during the grain-filling period reduces the accumulation of total flavonoids in rice[55] and decreases Fe ions' movement in the transpiration stream and thereby the transport of Fe ions to rice grains[56]. An appropriate increase in light intensity is beneficial to the accumulation of flavonoids, anthocyanins, and Fe in rice, but the photostability of anthocyanins is poor, and too much light will cause oxidative degradation of anthocyanins[57]. Therefore, functional rice is best cultivated as mid-late rice, which would be conducive to accumulating active components in rice.

    Effect of temperature on the accumulation of active components in rice

    The temperature has a great influence on the synthesis of active components in rice. An appropriate increase in the temperature is beneficial to the accumulation of γ-oryzanol[58] and flavonoids[59] in rice. A high temperature during the grain-filling period leads to an increase in glutelin content in rice[60], but an increase in temperature decreases the total phenolic content[61]. The results regarding the effect of temperature on the content of PA in rice were inconsistent. Su et al.[62] showed that high temperatures during the filling period would increase the PA content, while Goufo & Trindade[61] reported that the increase in temperature would reduce the PA content. This may be due to the different growth periods and durations of temperature stress on rice in the two studies. The synthesis of anthocyanins/proanthocyanidins in colored rice requires a suitable temperature. Within a certain range, lower temperatures favor the accumulation of anthocyanins/proanthocyanidins in rice[63]. Higher temperatures will lead to degradation, and the thermal stability of proanthocyanidins being higher than that of anthocyanins[64]. In addition, cold or heat stress facilitates GABA accumulation in rice grains[65]. Therefore, in actual production, colored rice and low-glutelin rice are best planted as late rice, and the planting time of other functional rice should be determined according to the response of its enriched active components to temperature changes.

    Effect of water management on the accumulation of active components in rice

    Moderate water stress can significantly increase the content of glutelin[66] and GABA[67] in rice grains and promote the rapid transfer of assimilation into the grains, shorten the grain filling period, and reduce the RS content[68]. Drought stress can also induce the expression of the phytoene synthase (psy) gene and increase the carotenoid content in rice[69]. Soil moisture is an important medium in Zn diffusion to plant roots. In soil with low moisture content, rice roots have low available Zn, which is not conducive to enriching rice grains with Zn[70]. Results from studies on the effect of soil water content on Se accumulation in rice grains have been inconsistent. Li et al.[71] concluded that flooded cultivation could significantly increase the Se content in rice grains compared to dry cultivation. However, the results of Zhou et al.[72] showed that the selenium content in rice grains under aerobic and dry-wet alternative irrigation was 2.44 and 1.84 times higher than that under flood irrigation, respectively. This may be due to the forms of selenium contained in the soil and the degree of drought stress to the rice that differed between experiments[73]. In addition, it has been found that too much or too little water impacts the expression of genes related to anthocyanin synthesis in rice, which affects the accumulation of anthocyanins in rice[74]. Therefore, it is recommended to establish different irrigation systems for different functional rice during cultivation.

    Effect of fertilizer application on the accumulation of active components in rice

    Both the amount and method of nitrogen application affect the accumulation of glutelin. Numerous studies have shown that both increased and delayed application of nitrogen fertilizer can increase the accumulation of lysine-rich glutelin to improve the nutritional quality of rice (Table 1). However, this improvement is not beneficial for kidney disease patients who cannot consume high glutelin rice. Nitrogen stress can down-regulate the expression of ANDs genes related to the anthocyanins biosynthesis pathway in grains, resulting in a decrease in anthocyanins synthesis[55]. Increased nitrogen fertilizer application can also increase the Fe, Zn, and Se content in rice[75,76]. However, some studies have found that increased nitrogen fertilizer application has no significant effect on the Fe content of rice[77], while other studies have shown that increased nitrogen fertilizer application will reduce the Fe content of rice[78]. This may be influenced by soil pH and the form of the applied nitrogen fertilizer. The lower the soil pH, the more favorable the reduction of Fe3+ to Fe2+, thus promoting the uptake of Fe by rice. Otherwise, the application of ammonium fertilizer can improve the availability of soil Fe and promote the absorption and utilization of Fe by rice. In contrast, nitrate fertilizer can inhibit the reduction of Fe3+ and reduce the absorption of Fe by rice[79].

    Table 1.  Effect of nitrogen fertilizer application on glutelin content of rice.
    SampleN level
    (kg ha−1)    		Application timeGlutelin content
    (g 100 g−1)    		References
    Rough rice05.67[66]
    270Pre-transplanting : mid tillering : panicle initiation : spikelet differentiation = 2:1:1:16.92
    300Pre-transplanting : mid tillering : panicle initiation : spikelet differentiation = 5:2:2:16.88
    Brown rice05.35[83]
    90Pre-transplanting : after transplanting = 4:16.01
    Pre-transplanting : after transplanting = 1:16.60
    180Pre-transplanting : after transplanting = 4:16.53
    Pre-transplanting : after transplanting = 1:17.29
    270Pre-transplanting : after transplanting = 4:17.00
    Pre-transplanting : after transplanting = 1:17.66
    Rough rice05.59[84]
    187.5Pre-transplanting : after transplanting = 4:16.47
    Pre-transplanting : after transplanting = 1:16.64
    300Pre-transplanting : after transplanting = 4:17.02
    Pre-transplanting : after transplanting = 1:17.14
    Polished rice03.88[85]
    90Pre-transplanting : tillering : booting = 2:2:14.21
    180Pre-transplanting : tillering : booting = 2:2:14.43
    270Pre-transplanting : tillering : booting = 2:2:16.42
    360Pre-transplanting : tillering : booting = 2:2:14.87
    Brown rice09.05[86]
    120Flowering22.14
     | Show Table
    DownLoad: CSV

    Appropriate application of phosphorus fertilizer is beneficial in promoting the translocation of Fe and Zn from leaves to rice grains, thus increasing the content in rice grains[80]. However, the excessive application of phosphate fertilizer will reduce the availability of Fe and Zn in soil, resulting in less uptake by the roots and a lower content in the rice grains[81]. The content of PA in rice increased with a higher phosphorus fertilizer application rate[80]. Increasing the phosphorus fertilizer application rate would increase the values of [PA]/[Fe] and [PA]/[Zn] and reduce the effectiveness of Fe and Zn in rice[80]. Currently, there are few studies on the effect of potassium fertilization on the synthesis of active components in rice. Available studies report that increased application of nitrogen fertilizer can increase the Zn content in rice[82]. Therefore, the research in this area needs to be strengthened.

    Because the iron in soil mainly exists in the insoluble form Fe3+, the application of iron fertilizer has little effect on rice biofortification[87]. There are different opinions about the effect of Zn fertilizer application methods. Phattarakul et al.[88] believed that foliar spraying of Zn fertilizer could significantly improve the Zn content in rice grains. Jiang et al.[89] concluded that most of the Zn accumulated in rice grains were absorbed by the roots rather than from the reactivation of Zn in leaves. In contrast, Yuan et al.[90] suggested that soil application of Zn fertilizer had no significant effect on Zn content in rice grains. The different results may be affected by the form of zinc fertilizer applied and the soil conditions in the experimental sites. Studies have found that compared with the application of ZnEDTA and ZnO, zinc fertilizer in the form of ZnSO4 is most effective for increasing rice's Zn[70]. In addition, the application of zinc fertilizer reduces the concentration of PA in rice grains[70].

    The form of selenium fertilizer and the method and time of application will affect the accumulation of Se in rice grains. Regarding selenium, rice is a non-hyperaccumulative plant. A moderate application of selenium fertilizer can improve rice yield. However, the excessive application can be toxic to rice, and the difference between beneficial and harmful supply levels is slight[91]. Selenite is readily adsorbed by iron oxide or hydroxide in soil, and its effectiveness in the soil is much lower than selenite[92]. In addition, selenate can migrate to the roots and transfer to rice shoots through high-affinity sulfate transporters. In contrast, selenite is mainly assimilated into organic selenium in the roots and transferred to the shoots in smaller amounts[93]. Therefore, the biological effectiveness of Se is higher in selenate-applied soil than in selenite application[94] (Table 2). Zhang et al.[95] found that the concentration of Se in rice with soil application of 100 g Se ha-1 was only 76.8 μg·kg-1, while the concentration of Se in rice with foliar spray of 75 g Se ha-1 was as high as 410 μg·kg-1[73]. However, the level of organic selenium was lower in rough rice with foliar application of selenium fertilizer compared to soil application[96], while the bioavailability of organic selenium in humans was higher than inorganic selenium[97]. Deng et al.[73] found that the concentrations of total selenium and organic selenium in brown rice with selenium fertilizer applied at the full heading stage were 2-fold higher than those in brown rice with selenium fertilizer applied at the late tillering stage (Table 2). Although the application of exogenous selenium fertilizer can rapidly and effectively increase the Se content of rice (Table 2), it can easily lead to excessive Se content in rice and soil, which can have adverse effects on humans and the environment. Therefore, breeding Se-rich rice varieties is a safer and more reliable way to produce Se-rich rice. In summary, functional rice production should include the moderate application of nitrogen and phosphorus fertilizer and higher levels of potassium fertilizer, with consideration to the use of trace element fertilizers.

    Table 2.  Effect of selenium fertilizer application on the selenium content of rice.
    SampleSe level (g Se ha−1)Selenium fertilizer formsApplication methodSe content (μg·g−1)References
    Rough rice00.002[98]
    18SeleniteFoliar spray at full heading0.411
    Polished rice00.071[99]
    20SeleniteFoliar spray at full heading0.471
    20SelenateFoliar spray at full heading0.640
    Rough rice75SeleniteFoliar spray at late tillering0.440[73]
    75SeleniteFoliar spray at full heading1.290
    75SelenateFoliar spray at late tillering0.780
    75SelenateFoliar spray at full heading2.710
    Polished rice00.027[100]
    15SeleniteFoliar spray at full heading0.435
    45SeleniteFoliar spray at full heading0.890
    60SeleniteFoliar spray at full heading1.275
     | Show Table
    DownLoad: CSV

    Effect of harvesting time on the accumulation of active components in rice

    The content of many active components in rough rice is constantly changing during the development of rice. It was found that the content of total flavonoids in brown rice increased continuously from flowering stage to dough stage and then decreased gradually[101]. The γ-oryzanol content in rice decreased by 13% from milk stage to dough stage, and then gradually increased to 60% higher than milk stage at full maturity[101]. The results of Shao et al.[102] showed that the anthocyanin content in rice reached its highest level at two weeks after flowering and then gradually decreased. At full ripeness, and the anthocyanins content in brown rice was only about 50% of the maximum level. The content of total phenolics in rice decreased with maturity from one week after flowering to the fully ripe stage, and the loss of total phenolics reached more than 47% by the fully ripe stage. In contrast, the content of total phenolics in black rice increased with maturity[102]. Moreover, RS content in rough rice decreases during rice maturation[68]. Therefore, the production process of functional rice should be timely and early harvested to obtain higher economic value.

    Prevention and control of pests and diseases for functional rice

    Pests and diseases seriously impact the yield and quality of rice[103]. At present, the two most effective methods to control pests and diseases are the use of chemical pesticides and the planting of pest and disease-resistant rice varieties. The use of chemical pesticides has greatly reduced the yield loss of rice. However, excessive use of chemical pesticides decreases soil quality, pollutes the environment, reduces soil biodiversity[104], increases pest resistance, and aggravates the adverse effects of pests and diseases on rice production[105]. It also increases residual pesticide levels in rice, reduces rice quality, and poses a severe threat to human health[106].

    Breeding pest and disease-resistant rice varieties are among the safest and effective ways to control rice pests and diseases[107]. In recent years, many pest and disease resistance genes from rice and microorganisms have been cloned[47]. Researchers have used these genes to breed rice varieties resistant to multiple pests and diseases through gene polymerization breeding techniques. Application in production practices delivered good ecological and economic benefits[108].

    Green pest and disease control technologies must consider the synergies between rice and water, fertilizer, and pest and disease management. In this regard, the rice-frog, rice-duck, and other comprehensive rice production models that have been widely used in recent years are the most representative. These rice production models significantly reduced chemical pesticide usage and effectively controlled rice pests and diseases[109]. The nutritional imbalance will reduce the resistance of rice to pests and diseases[110]. Excessive application of nitrogen fertilizer stimulates rice overgrowth, protein synthesis, and the release of hormones, increasing its attractiveness to pests[111]. Increased soluble protein content in rice leaves is more conducive to virus replication and increases the risk of viral infection[112]. Increasing the available phosphorus content in the soil will increase crop damage by pests[113], while insufficient potassium supply will reduce crop resistance to pests and diseases[114]. The application of silica fertilizer can boost the defense against pests and diseases by increasing silicon deposition in rice tissue, inducing the expression of genes associated with rice defense mechanisms[115] and the accumulation of antifungal compounds in rice tissue[116]. The application of silica fertilizer increases the release of rice volatiles, thereby attracting natural enemies of pests and reducing pest damage[117]. Organic farming increases the resistance of rice to pests and diseases[118]. In addition, rice intercropping with different genotypes can reduce pests and diseases through dilution and allelopathy and changing field microclimate[119].

    In conclusion, the prevention and control of rice pests and diseases should be based on chemical and biological control and supplemented by fertilizer management methods such as low nitrogen, less phosphorus, high potassium and more silicon, as well as agronomic measures such as rice-aquaculture integrated cultivation, organic cultivation and intercropping of different rice varieties, etc. The combined use of multiple prevention and control measures can improve the yield and quality of functional rice.

    Conclusions

    Functional rice contains many active components which are beneficial to maintaining human health and have high economic and social value with broad market prospects. However, the current development level of the functional rice industry is low. The development of the functional rice requires extensive use of traditional and modern polymerization breeding techniques to cultivate new functional rice varieties with endosperm that can be enriched with multiple active components and have broad-spectrum resistance to pests and diseases. It is also important to select suitable planting locations and times according to the response characteristics of different functional rice active components to environmental conditions.

    Acknowledgments

    This work is supported by the National Natural Science Foundation of China (Project No. 32060430 and 31971840), and Research Initiation Fund of Hainan University (Project No. KYQD(ZR)19104).

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1 The TCP gene family members and their abbreviation name in celery, coriander, carrot, lettuce, grape, and Arabidopsis.
    Supplemental Table S2 The summary of TCP gene family members in  A.  graveolens , C. sativum, D. carota and compared with  A. thaliana .
    Supplemental Table S3 The list of orthologous TCP gene pairs between A. graveolens,  C. sativum, and  D. carota.
    Supplemental Table S4 The list of paralogous TCP gene pairs in each of other examined species.
    Supplemental Table S5 Ka/Ks calculation and divergent time of the orthologous  gene pairs between A. graveolens, C. sativum, and  D. carota.
    Supplemental Table S6 The duplicated type of TCP genes in A. graveolens,C. sativum and D. carota. The 0 to 4 indicate the singleton, dispersed, proximal, tandem, WGD duplication type, respectively.
    Supplemental Table S7 The prediction of target TCP family genes of miRNA in carrot, coriander, and celery.
    Supplemental Table S8 The expression level of the TCP genes in root, leaf and petiole for A. graveolens and C. savitum.  The gene expression was determined by the RNA-Seq data (RPKM).
    Supplemental Fig. S1 The gene structure of TCP gene family in three Apiaceae species.
    Supplemental Fig. S2 The circle plot of paralogous TCP gene pairs among three Apiaceae species.
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  • Cite this article

    Pei Q, Li N, Bai Y, Wu T, Yang Q, et al. 2021. Comparative analysis of the TCP gene family in celery, coriander and carrot (family Apiaceae). Vegetable Research 1: 5 doi: 10.48130/VR-2021-0005
    Pei Q, Li N, Bai Y, Wu T, Yang Q, et al. 2021. Comparative analysis of the TCP gene family in celery, coriander and carrot (family Apiaceae). Vegetable Research 1: 5 doi: 10.48130/VR-2021-0005
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Comparative analysis of the TCP gene family in celery, coriander and carrot (family Apiaceae)

  • 1.

    College of Life Sciences/Center for Genomics and Bio-computing, North China University of Science and Technology, Tangshan 063210, China

  • 2.

    School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China

  • 3.

    Food Science and Technology Department, University of Nebraska-Lincoln, Lincoln, NE 68588, USA

  • 4.

    Faculty of Life Science, Tangshan Normal University, Tangshan 063000, China

  • Received: 05 January 2021
  • Accepted: 11 June 2021
  • Published online: 22 June 2021
Vegetable Research  1,  Article number: 5  (2021)  |  Cite this article

Abstract: Apiaceae is one of the most important families in Apiales and includes many economically important vegetables and medicinal plants. The TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR 1/2 (TCP) gene family plays an important role in regulating plant growth and development, but it has not been widely studied in Apiaceae. In the present study, we identified 215 TCP family genes in six species of plant, of which 122 genes were present in three Apiaceae including 29 in celery (Apium graveolens), 43 in coriander (Coriandrum sativum), and 50 in carrot (Daucus carota). Whole-genome duplication likely contributed to TCP gene family expansion in Apiaceae. There were more paralogs in carrot than in coriander and celery, which was attributable to the greater number of tandem and proximal duplicated genes on chromosome 1. Nine microRNAs were found to regulate 20 TCP genes in the three Apiaceae species, with miR-319 having the most target genes. Several TCP genes showed high expression in the root, petiole and leaf of celery and coriander. These results provide a basis for comparative and functional genomic analyses of TCP genes in Apiaceae and other plants.

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    INTRODUCTION

    • The Apiaceae family of plants includes more than 400 genera and 3,000 species[1,2]. Several Apiaceae species such as carrot (Daucus carota), celery (Apium graveolens), and coriander (Coriandrum sativum) are cultivated as a vegetable or for medicinal purposes worldwide[3,4].

      Celery is an annual or biennial herbage species originating from the Mediterranean and Middle East[5,6]. Besides being a vegetable, celery is also used as a medicinal plant[7]. Coriander, which is also known as cilantro, is a popular herb and a major ingredient of curry powder[8]. Carrot is an economically important vegetable with a high nutritional value[9]. All three of these plants are diploid species although their chromosome number and genome size differ: celery and coriander each have 22 chromosomes (2n = 2x = 22) whereas carrot has 18 (2n = 2x = 18)[3,10,11], and the assembled genome size of celery is 3.33 Gb, which is larger than coriander (2.11 Gb) and carrot (421.5 Mb)[3,11,12].

      The TCP gene family in plants is named after the first identified members—ie, TEOSINTE BRANCHED 1 (TB1) in maize (Zea mays)[13], CYCLOIDEA (CYC) in snapdragon (Antirrhinum majus)[14], and PROLIFERATING CELL FACTOR 1 (PCF1) and PCF2 in rice (Oryza sativa)[15]. TCP genes regulate multiple processes in plant growth and development such as shoot branching[16], seed germination[17,18], gametophyte development[19,20], leaf development[21−25], leaf senescence[26−28], mitochondrial biogenesis[29], flower development[30−32] and cell cycle[29,33]. There are 24 TCP genes in Arabidopsis thaliana[34], 22 in rice[34], 24 in tomato[35], 19 in plum[26], 42 in switchgrass[36], 36 in carrot[37], and 32 in celery[38]. However, to date, no TCP genes have been identified in coriander. High-quality genome sequences of celery, carrot and coriander were recently released[3,11,12], which can facilitate comparative analyses of specific genes in Apiaceae.

      In this study, we identified and characterized TCP genes in celery, coriander and carrot and performed comparisons with genes in Lactuca sativa (lettuce), Vitis vinifera (grape), and Arabidopsis. We mapped the TCP genes to chromosomes, identified orthologs and paralogs, detected collinearity and gene expansion or loss, and analyzed their expression patterns in plant tissues. Our results provide a basis for comparative studies on the function and evolution of TCP genes in plants.

    MATERIALS AND METHODS

      Identification of TCP family genes in celery, coriander and carrot

    • The genome sequences of coriander and celery were downloaded from the coriander genome database (http://cgdb.bio2db.com) and celery genome database (http://celerydb.bio2db.com), respectively[3,10]. The Arabidopsis genome sequence was retrieved from The Arabidopsis Information Resource (TAIR10; http://www.arabidopsis.org). The sequences of carrot (version 2), lettuce (version 5), and grape (Genoscope.12X) were downloaded from Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html)[11,39,40]. The Pfam database (https://pfam.sanger.ac.uk) was used to identify TCP family genes with the identifier PF03634 (E value < 1e−4)[41]. The Simple Modular Architecture Research Tool (SMART v9.0) database and Conserved Domains Database (CDD) were used for domain validation[42,43].

      Phylogenetic analysis of TCP gene family

    • To analyze the evolutionary relationships of TCP genes in Apiaceae, multiple sequence alignment of the TCP amino acid sequences of Arabidopsis, grape, lettuce, carrot, coriander and celery was performed with ClustalW software (v2.0)[44] and a phylogenetic tree was constructed with the neighborhood-joining method (bootstrap = 1,000) using MEGA X[45]. The reconstructed TCP gene tree was compared to the actual species tree using Notung v2.9 software with default parameters[46,47].

      Chromosome location, gene structure and conserved motif analysis

    • The location of coriander, celery and carrot TCP genes on chromosomes was drawn using Tbtools software and the files were saved in general feature format (gff)[48]. Gene structure was determined using Gene Structure Display Server 2.0 (https://gsds.cbi.pku.edu.cn)[49]. Conserved motifs were analyzed using Multiple Expectation maximizations for Motif Elicitation suite 5.2.0 (http://meme-suite.org)[50].

      Analysis of orthologs and paralogs

    • Orthologous and paralogous TCP gene pairs in celery, coriander and carrot were analyzed using OrthoMCL software v2.0 (https://orthomcl.org/orthomcl)[51]. The relationships of the genes among the three species was depicted using Circos software (v0.69)[52].

      Identification of collinear blocks and duplication types

    • MCScanX was used to identify collinear blocks and duplication types of the TCP genes[53]. Whole-genome sequences were searched against themselves using BLASTp (E value < 1e−5). We extracted TCP genes located in the collinear blocks using Perl scripts. The duplication type of TCP genes was estimated using the subprogram duplicate_gene_classifier.

      Calculation of Ka/Ks and estimation of divergence time

    • The nonsynonymous rate (Ka), synonymous rate (Ks), their ratio (Ka/Ks) and divergence time among orthologous gene pairs of the three species were calculated using KaKs_Calculator 2.0[54]. The coding sequence of orthologous gene pairs were aligned using ClustalW (v2.0)[44]; AXTconvertor software (v1.0) was then used to convert the alignment file to axt format. Lastly, the Ka value, Ks value and their ratio were calculated based on the Nei–Gojobori method[54]. Ks was used to estimate the divergence time based on the formula T = Ks/2r, where r indicates neutral substitutions (5.2 × 10−9 for Apiaceae)[12].

      Analysis of selective pressures

    • We used the maximum likelihood method and codon substitution models to determine the likelihood ratio of positive selection. We analyzed each branch of the phylogenetic tree to infer ω (the ratio of nonsynonymous to synonymous distances) using CodeML implemented in PAML4.9[47,55]. We adopted a complete deletion method for analyzing alignments with gaps and eliminated sequences with gaps in over 40% of their length. The likelihood ratio test between M0 and M1 and between M7 and M8 models were used to determine variation sites.

      Identification of micro (mi)RNA target genes in the TCP gene family

    • Mature miRNA sequences of A. thaliana were downloaded from miRBase (release 22.1; http://www.mirbase.org)[56]. TCP genes that are miRNA targets were predicted using psRNATarget Schema v2 (2017 release)[57] with maximum expectation ≤ 3 and other default parameters. A miRNA–TCP gene network was constructed using Cytoscape v3.7.2 software[58].

      Expression of TCP genes

    • TCP gene expression data in celery and coriander were extracted from RNA sequencing (RNA-seq) datasets previously published by our group[3,10] using Perl script; the values were normalized as reads per kilobase per million reads (RKPM). An expression heatmap was created using TBtools software (v1.0)[48].

    RESULTS

      Identification of TCP genes in three Apiaceae species

    • We identified 29 TCP genes in the genome of celery, 43 in coriander, and 50 in carrot (Supplemental Table S1 and S2). Additionally, 24 TCP genes were identified in Arabidopsis along with 20 in grape and 49 in lettuce (Supplemental Table S2). Thus, a total of 215 TCP genes were identified in the six species for further analysis.

      Phylogenetic and functional analyses of TCP gene family

    • To classify the TCP gene family in plants, we constructed a phylogenetic tree of all 215 amino acid sequences from the six abovementioned species using MEGA X (Fig. 1). Consistent with the phylogenetic relationships described in Arabidopsis and grape, the phylogenetic tree had three groups according to the type of TCP protein domain including the PCF, CINCINNATA (CIN), and CYC/TB1 classes.

      Figure 1.  Phylogenetic tree of TCP family genes in three Apiaceae species (carrot, celery and coriander) and lettuce, grape and Arabidopsis. The topology of the phylogenetic tree was determined using IQ-TREE with maximum likelihood (ML) based on the JTT+F+R8 model. The bootstrap was set to 1,000 replicates, and values > 40% are shown. The three classes were identified based on bootstrap values and phylogenetic topology.

      In class PCF, there were ten AgTCP, 26 CsTCP, and 36 DcTCP genes; in class CYC/TB1, there were eight AgTCP, seven CsTCP, and seven DcTCP genes; and in class CIN, there were 11 AgTCP, ten CsTCP and seven DcTCP genes (Fig. 1 and Supplemental Table S2). Notably, in coriander and carrot there were more TCP genes in class PCF than in the other two classes.

      The functions of most TCP family genes have been well studied in the model plant Arabidopsis. We inferred the function of homologous genes within the same taxonomic group in the phylogenetic tree in order to clarify the function of TCP genes in Apiaceae. For example, AT1G53230.1 (AtTCP3) is known to suppress the expression of CUP-SHAPED COTYLEDON (CUC), resulting in cotyledon fusion[24]. We identified three TCP genes—namely, DcTCP34, AgTCP2, and CsTCP6—that clustered together with AtTCP3 (Fig. 1), suggesting that they are also related to cotyledon fusion. It may also be possible to deduce the function of other Apiaceae TCP genes based on the function of the homologous genes in Arabidopsis.

      TCP gene family structure and conserved motifs

    • We carried out a gene structure analysis of TCP family genes to identify exons, introns and untranslated regions (Supplemental Fig. S1). Of the 122 TCP genes in Apiaceae, 94—including all 50 DcTCP genes—lacked introns. Most TCP genes had a single exon, although there were some exceptions. For example, CsTCP15 (class CIN) had four exons and DcTCP11 (class PCF) had three. In general, genes in the same class or subclass had similar gene structure and size.

      As gene structure varied among genes, we performed a motif analysis to examine the structure in greater detail. We compared five motifs in Apiaceae species and found that motif 3 was found at the beginning of most genes, followed by motif 1 and motif 2 (Fig. 2). However, motif 2 was located at the start of the CsTCP7, DcTCP15, CsTCP23, CsTCP36, and CsTCP2 and motif 5 was present at the beginning of DcTCP11. Almost all TCP genes had motif 1 except for DcTCP and CsTCP in class PCF, indicating that this motif is highly conserved and plays an important role in Apiaceae. Motif 3 was also present in most TCP genes and is likely conserved in Apiaceae.

      Figure 2.  Conversed motifs in TCP family genes of three Apiaceae species.

      Most genes in classes CYC/TB1 and CIN lacked motif 2 except for DcTCP17 and CsTCP79. Only five genes in class CIN had motif 4, which was present in all class CYC/TB1 genes. Motif 5 was only found in class PCF and not in other classes. Interestingly, DcTCP14 (class PCF) did not have any of the 5 motifs, suggesting that they were lost during the course of the evolution of carrot. Thus, genes in the same class had similar motif composition, indicating that they are functionally similar.

      Chromosomal distribution of TCP genes in Apiaceae species

    • In celery, 27/29 TCP genes mapped to nine chromosomes (Fig. 3a and Supplemental Table S1). Two celery TCP genes did not map to any chromosomes, and no TCP family gene was found on chromosomes Agr6 and Agr10. There were five genes that mapped to chromosomes Agr3, Agr5, and Agr11 while only one was located on chromosomes Agr1 and Agr2.

      Figure 3.  Distribution of TCP transcription factors on each chromosome in three Apiaceae species. (a) Celery, (b) Coriander, (c) Carrot.

      In coriander, 35/43 TCP genes mapped to nine chromosomes (Fig. 3b and Supplemental Table S1); none were found on chromosome Csa8. Chromosome Csa9 had the most TCP genes (10), followed by chromosomes Csa7 (6) and Csa1 (5). Chromosomes Csa2 and Csa3 each had just one TCP gene.

      The 50 carrot TCP genes were unevenly distributed across nine chromosomes (Dca1–9) (Fig. 3c and Supplemental Table S1). Interestingly, TCP gene expansion was observed on chromosome 1, which had 16 genes. Additionally, 15 genes (DcTCP1–15) were clustered together, mainly through tandem and proximal duplication.

      Identification of orthologous and paralogous TCP genes in Apiaceae

    • We examined orthologous and paralogous gene pairs in Apiaceae and found that there were 22 orthologous gene pairs between any two of celery, coriander and carrot (Fig. 4 and Supplemental Table S3), indicating a close phylogenetic relationship between these species. There were only three paralogous gene pairs in celery and coriander (Supplemental Fig. S2 and Supplemental Table S4) but 29 were found in carrot.

      Figure 4.  Circle plot of orthologous TCP gene pairs among three Apiaceae species.

      The Ks value was calculated to estimate the divergence time of orthologous TCP gene pairs among celery, coriander and carrot (Fig. 5 and Supplemental Table S5). The divergence time ranged from 14.03 to 116.15 million years between celery and coriander TCP genes, 24.94 to 81.44 million years between celery and carrot, and 22.59 to 94.72 million years between coriander and carrot orthologous TCP gene pairs. Therefore, the divergence time of most TCP genes was earlier than that of any two species (celery vs coriander, 11–13 Mya; carrot vs celery or coriander, 20–23 Mya)[4].

      Figure 5.  Ks and divergence time of TCP orthologs. Ks values (a) and divergence time estimation (b) of orthologous gene pairs between any two of three Apiaceae species.

      Detection of duplication type for TCP family genes in Apiaceae

    • Various types of gene duplication can lead to the expansion of a gene family. We examined five types of gene duplication in celery, coriander and carrot (Fig. 6a, Table 1 and Supplemental Table S6)—namely, singleton, dispersed, proximal, tandem and whole-genome duplication (WGD). There were no singleton TCP gene in the three species of Apiaceae (Table 1). Dispersed and tandem duplications were the predominant types in celery and coriander. In celery, the percentage of genes showing dispersed duplication and WGD was 48.1% and 40.7%, respectively; in coriander, the percentages were 40.0% and 45.7%, respectively. WGD was also the predominant type in carrot (32.0%), which had a lower percentage of dispersed duplication (24.0%) than celery and coriander. Moreover, the percentage of the tandem type was higher in carrot (30.0%) than in celery (11.1%) and coriander (11.4%). These results demonstrate that WGD played an important role in TCP gene expansion in celery, coriander and carrot, which is supported by the previous suggestion that they underwent two WGD events since their divergence from lettuce[3].

      Figure 6.  Duplication and loss of TCP family genes. (a) Percentage of duplication types for TCP family genes and genes in the whole genome of three Apiaceae species. (b) Duplication (+) or loss (−) of TCP family genes in three Apiaceae species and three other representative species. Numbers after '+' and '−' are the number of genes. Agr, Apium graveolens (celery); Ath, Arabidopsis thaliana (Arabidopsis); Csa, Coriandrum sativum (coriander); Dca, Daucus carota (carrot); Lsa, Lactuca sativa (lettuce); Vvi, Vitis vinifera (grape).

      Table 1.  The identification of duplicated type for TCP family genes and all genes in A. graveolens, C. sativum and D. carota.

      Duplication typea Categoryb A. graveolens C. sativum D. carota
      Singleton All genes 3,028 3,577 3,543
      TCP 0 0 0
      Percentage (%) 0 0 0
      Dispersed All genes 15,258 14,963 13,378
      TCP 13 14 12
      Percentage (%) 48.15 40 24
      Proximal All genes 1,167 2,161 1,428
      TCP 0 1 7
      Percentage (%) 0 2.86 14
      Tandem All genes 2,426 3,032 3,501
      TCP 3 4 15
      Percentage (%) 11.11 11.43 30
      WGD/segmental All genes 7,787 10,200 8,974
      TCP 11 16 16
      Percentage (%) 40.74 45.71 32
      Total All genes 29,666 33,933 30,824
      TCP 27 35 50
      Note: a the classification of duplicate genes was conducted using the MCScanX program. WGD/segmental duplicates were inferred by the anchor genes in collinear blocks. Tandem duplicates were defined as paralogs that were adjacent to each other on chromosomes. Proximal duplicates were paralogs near each other, while interrupted by several other genes. Dispersed duplicates were paralogs that were neither near each other on chromosomes, nor do they showed conserved synteny. b TCP indicated the TCP family genes. Percentage (%) indicated the percentage of TCP family gene number among all genes.

      TCP gene loss and duplication during the evolution of Apiaceae

    • We compared species and gene trees in celery, coriander and carrot to identify gene losses and duplications during the evolution of the TCP gene family. There were more gene losses in celery (20) than in coriander (7) and carrot (17) but more gene duplications in carrot (18) than in coriander (4) and celery (3). In the common ancestor of coriander, celery and carrot, there were 20 gene duplications and 14 gene losses (Fig. 6b).

      Positive selection of TCP family genes in Apiaceae

    • We analyzed natural selection in the evolution of TCP genes in Apiaceae (Fig. 7). Strong positive selection was observed at the major nodes of the phylogenetic tree, which may have contributed to the functional divergence of Apiaceae species. We detected 35 positive selection sites overall; most were in class PCF (29), followed by class CYC/TB1 (5) and class CIN (1), indicating that TCP genes in class PCF underwent greater positive selection in the evolution of Apiaceae.

      Figure 7.  Positive selection of TCP family genes in celery, coriander and carrot. Red stars represent branches in which positive selection occurred. The maximum likelihood (ML) phylogenetic tree was constructed using PhyML software.

      miRNA target TCP genes in Apiaceae

    • We next sought to identify TCP family genes in Apiaceae that are regulated by miRNAs. We found nine miRNAs that regulated 20 TCP family genes including five genes in celery, nine in coriander and six in carrot (Supplemental Table S7 and Fig. 8). Of the nine miRNAs, miR-319 had the most target genes (11), followed by miR-172 (3) and miR-181 (3) (Fig. 8). Specifically, miR-319 regulated four TCP family genes in celery, four in coriander, and three in carrot. Our results are supported by other studies demonstrating that miR-319 regulates TCP family genes[31,59−61]. We also found that four genes were regulated by more than one miRNA: DcTCP20 was regulated by miR-319 and miR-837, CsTCP33 was regulated by miR-319 and miR-8181, and AgTCP26 and AgTCP27 were regulated by miR-319 and miR-8181 (Supplemental Table S7 and Fig. 8).

      Figure 8.  Interaction network of miRNAs and target TCP family genes in carrot, celery and coriander.

      Expression of TCP family genes in celery and coriander

    • We analyzed the expression patterns of TCP genes in root, petiole and leaf tissues of celery and coriander. In celery, all TCP family genes in class CYC/TB1 had relatively low expression in the three tissues (Fig. 9a and Supplemental Table S8) whereas AgTCP22 had the highest expression level (RPKM > 60), suggesting a key role in celery growth and development. In coriander and celery, all TCP family genes in class CYC/TB1 were expressed at a relatively low level in the three tissues (Fig. 9b and Supplemental Table S8); meanwhile, several genes including CsTCP39, CsTCP14, CsTCP12, CsTCP31, and CsTCP40 had high expression. CsTCP6 and CsTCP30 were more highly expressed in leaf than in the other two tissues.

      Figure 9.  Expression of TCP family genes in three replicates of plant tissues including root (R1, R2 and R3), petiole (P1, P2 and P3), and leaf (L1, L2 and L3). Hierarchical gene expression clustering of TCP genes in celery (a) and coriander (b). Expression levels were calculated based on RPKM.

    DISCUSSION

    • Celery, coriander and carrot are typical members of the Apiaceae family. The draft genomes of these three species were recently released[3,11,12] and there have been several studies on TCP family genes in carrot and celery based on the sequences[37,38,62,63]. The latest versions of the celery, coriander and carrot genomes are of high quality with chromosomal-level assembly, allowing us to accurately and comprehensively analyze the TCP gene family in Apiaceae. To date there have been no reports on TCP genes in coriander. In this study, we identified 43 TCP genes in coriander as well as 29 in celery and 50 in carrot. Our results provide a resource for future studies on the TCP gene family in Apiaceae or related species.

      TCP transcription factors have a 59-amino acid basic helix-loop-helix (bHLH) motif that is involved in DNA binding and protein–protein interaction[64]. The bHLH-like domain of TCP differs from the canonical bHLH in its basic region[34,65]. PCF1 and PCF2 interact with DNA-binding proteins that specifically bind to the PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) promoter[15]. Our analyses of gene structure and motifs of TCP genes in Apiaceae revealed similarities within the same class or subclass.

      Gene duplication is the main mechanism underlying the evolution of complex phenotypes[66]. Many duplicated genes in plants were produced by WGD or whole-genome triplication[67−70]. Most duplicated genes were functionally redundant and had one of four fates during the course of evolution namely: conservation, neofunctionalization, subfunctionalization and specialization[67,71]. In conservation, the ancestral function was maintained in both copies, thus preserving gene dosage[71]. In neofunctionalization, one copy retained the original function while the other acquired a novel function[71,72]. In subfunctionalization, both copies were required to preserve the ancestral gene function[71,73,74]. In specialization, subfunctionalization and neofunctionalization acted cooperatively, producing two gene copies that were functionally distinct from each other and from the ancestral gene[71,75]. Functional redundancies of TCP genes have been reported in Arabidopsis[76]. In carrot, 15 TCP genes were clustered on chromosome 1, and the number of paralogous gene pairs was greater in carrot (29) than in celery (3) and coriander (3). Although there were more gene losses than duplications in the evolution of celery, coriander and carrot, we found that WGD made a major contribution to TCP gene family expansion in Apiaceae, which is similar to what has been reported in most other gene families in higher plants[47,77−80].

      The broad range of functions of TCP family genes in plants can be attributed to the diverse structures of different members. Most TCP genes are highly expressed in meristematic tissues, suggesting that their main function is to promote plant proliferation and growth[81]. However, some TCP genes, such as CIN and CYC/TB1, are known to negatively regulate plant proliferation and development[82] (lateral organ development for CIN genes and flower and lateral shoot development for CYC/TB1 genes)[26]. Our gene expression analysis showed that TCP gene expression in celery (AgTCP22) and coriander (CsTCP12) was nearly 2x higher in root and petiole than in leaf, suggesting roles in plant growth and development.

      In conclusion, we identified and characterized TCP genes in three Apiaceae species. We described their chromosomal location, exon–intron structure, motifs, collinearity, positive selection and expression patterns in plant tissues. These results provide a basis for investigations on the molecular networks regulating growth and development in Apiaceae and other plants.

      Acknowledgments
      • This work was supported by the National Natural Science Foundation of China (31801856), China Postdoctoral Science Foundation (2020M673188), Hebei Province Higher Education Youth Talents Program (BJ2018016), and Key Science Research and Development Program of Tangshan (19150206E).
      Conflict of interest
      • The authors declare that they have no conflict of interest.
      Supplementary information
      • Supplemental Table S1 The TCP gene family members and their abbreviation name in celery, coriander, carrot, lettuce, grape, and Arabidopsis.
      • Supplemental Table S2 The summary of TCP gene family members in  A.  graveolens , C. sativum, D. carota and compared with  A. thaliana .
      • Supplemental Table S3 The list of orthologous TCP gene pairs between A. graveolens,  C. sativum, and  D. carota.
      • Supplemental Table S4 The list of paralogous TCP gene pairs in each of other examined species.
      • Supplemental Table S5 Ka/Ks calculation and divergent time of the orthologous  gene pairs between A. graveolens, C. sativum, and  D. carota.
      • Supplemental Table S6 The duplicated type of TCP genes in A. graveolens,C. sativum and D. carota. The 0 to 4 indicate the singleton, dispersed, proximal, tandem, WGD duplication type, respectively.
      • Supplemental Table S7 The prediction of target TCP family genes of miRNA in carrot, coriander, and celery.
      • Supplemental Table S8 The expression level of the TCP genes in root, leaf and petiole for A. graveolens and C. savitum.  The gene expression was determined by the RNA-Seq data (RPKM).
      • Supplemental Fig. S1 The gene structure of TCP gene family in three Apiaceae species.
      • Supplemental Fig. S2 The circle plot of paralogous TCP gene pairs among three Apiaceae species.
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      • Copyright: © 2021 by the author(s). Exclusive Licensee Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
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    Cite this article
    Pei Q, Li N, Bai Y, Wu T, Yang Q, et al. 2021. Comparative analysis of the TCP gene family in celery, coriander and carrot (family Apiaceae). Vegetable Research 1: 5 doi: 10.48130/VR-2021-0005
    Pei Q, Li N, Bai Y, Wu T, Yang Q, et al. 2021. Comparative analysis of the TCP gene family in celery, coriander and carrot (family Apiaceae). Vegetable Research 1: 5 doi: 10.48130/VR-2021-0005
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  • Introduction
  • Materials and methods

  • Identification of tcp family genes in celery, coriander and carrot

  • Phylogenetic analysis of tcp gene family

  • Chromosome location, gene structure and conserved motif analysis

  • Analysis of orthologs and paralogs

  • Identification of collinear blocks and duplication types

  • Calculation of ka/ks and estimation of divergence time

  • Analysis of selective pressures

  • Identification of micro (mi)rna target genes in the tcp gene family

  • Expression of tcp genes

  • Results

  • Identification of tcp genes in three apiaceae species

  • Phylogenetic and functional analyses of tcp gene family

  • tcp gene family structure and conserved motifs

  • Chromosomal distribution of tcp genes in apiaceae species

  • Identification of orthologous and paralogous tcp genes in apiaceae

  • Detection of duplication type for tcp family genes in apiaceae

  • tcp gene loss and duplication during the evolution of apiaceae

  • Positive selection of tcp family genes in apiaceae

  • Mirna target tcp genes in apiaceae

  • Expression of tcp family genes in celery and coriander

  • Discussion
  • Acknowledgments
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
  • About this article
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