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Genome-wide discovery of CBL genes in Nitraria tangutorum Bobr. and functional analysis of NtCBL1-1 under drought and salt stress

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  • Calcineurin B-like (CBL) proteins are a class of important Ca2+ receptors that play key roles in plant stress response. CBLs have been shown to participate in responses to abiotic stresses such as drought, salt, and cold in many plant species, including Arabidopsis and rice. However, little is known about their potential functions in the desert halophyte Nitraria tangutorum. Here, we have identified 11 CBL genes distributed across six chromosomes of N. tangutorum and categorized them into four groups through phylogenetic analysis. Synteny analysis showed that they have strong collinear relationships and have undergone purifying selection during their evolution. NtCBL promoter regions contain a variety of cis-acting elements related to hormone and environmental stress responses. Real-time quantitative PCR showed that the expression of NtCBLs differed significantly among various tissues and was upregulated by salt and drought stress. We chose NtCBL1-1 for an in-depth functional characterization and observed that transgenic Arabidopsis plants expressing NtCBL1-1 exhibited increased tolerance to both drought and salt stress. Compared to wild-type Arabidopsis, transgenic lines showed higher germination rates, slower chlorophyll degradation, more soluble proteins, and reduced accumulation of the oxidative stress marker malondialdehyde. These findings indicate that NtCBL1-1 plays a significant role in responding to drought and salt stress, laying the foundation for further investigations into the functional mechanisms of NtCBL genes in N. tangutorum.
  • 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.

    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.

    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 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.

    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).

    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 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].

    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.

    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 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 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.

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

    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.

    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.

    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.

    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
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    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
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    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.

    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.

    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.

    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 Primers for Quantitative RT-PCR in N.tangutorum.
    Supplemental Table S2 Primers for isolation of NtCBL1-1 fragment and construction of overexpression vector.
    Supplemental Table S3 CBL genes used to construct phylogenetic trees from 12 species except N.tangutorum.
    Supplemental Table S4 KaKs analysis in N.tangutorum.
    Supplemental Table S5 Identity between CBLs gene in Nitraria tangutorum.
    Supplemental Fig. S1 Cis-regulatory elements analysis of NtCBLs. (A) A heat map showing the number of cis-acting elements related to stress response in NtCBLs. (B) The distribution of cis-acting elements in the promoter region (ATG upstream) of NtCBLs, where different boxes represent different cis-acting elements.
    Supplemental Fig. S2 Positive identification of partial NtCBL1-1 transgenic T3 generation; different lanes represent different transgenic lines.
    Supplemental Fig. S3 Relative expression levels in partial transgenic T3 generations, with wild-type Arabidopsis as a reference.
    Supplemental Fig. S4 Phenotypic changes in overexpression lines 1 and 3 following exposure to 200 mM NaCl for 0−7 days.
    Supplemental Fig. S5 Phenotypic changes in overexpression lines 1 and 3 following exposure to 300 mM mannitol for 0−7 days.
    Supplemental file S1 Genome annotations.
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  • Cite this article

    Zhu L, Wu J, Li M, Fang H, Zhang J, et al. 2023. Genome-wide discovery of CBL genes in Nitraria tangutorum Bobr. and functional analysis of NtCBL1-1 under drought and salt stress. Forestry Research 3:28 doi: 10.48130/FR-2023-0028
    Zhu L, Wu J, Li M, Fang H, Zhang J, et al. 2023. Genome-wide discovery of CBL genes in Nitraria tangutorum Bobr. and functional analysis of NtCBL1-1 under drought and salt stress. Forestry Research 3:28 doi: 10.48130/FR-2023-0028

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ARTICLE   Open Access    

Genome-wide discovery of CBL genes in Nitraria tangutorum Bobr. and functional analysis of NtCBL1-1 under drought and salt stress

Forestry Research  3 Article number: 28  (2023)  |  Cite this article

Abstract: Calcineurin B-like (CBL) proteins are a class of important Ca2+ receptors that play key roles in plant stress response. CBLs have been shown to participate in responses to abiotic stresses such as drought, salt, and cold in many plant species, including Arabidopsis and rice. However, little is known about their potential functions in the desert halophyte Nitraria tangutorum. Here, we have identified 11 CBL genes distributed across six chromosomes of N. tangutorum and categorized them into four groups through phylogenetic analysis. Synteny analysis showed that they have strong collinear relationships and have undergone purifying selection during their evolution. NtCBL promoter regions contain a variety of cis-acting elements related to hormone and environmental stress responses. Real-time quantitative PCR showed that the expression of NtCBLs differed significantly among various tissues and was upregulated by salt and drought stress. We chose NtCBL1-1 for an in-depth functional characterization and observed that transgenic Arabidopsis plants expressing NtCBL1-1 exhibited increased tolerance to both drought and salt stress. Compared to wild-type Arabidopsis, transgenic lines showed higher germination rates, slower chlorophyll degradation, more soluble proteins, and reduced accumulation of the oxidative stress marker malondialdehyde. These findings indicate that NtCBL1-1 plays a significant role in responding to drought and salt stress, laying the foundation for further investigations into the functional mechanisms of NtCBL genes in N. tangutorum.

    • Plants encounter various abiotic stresses throughout their life. Salt, drought, cold, and other abiotic stresses adversely affect plant growth and development, sometimes even leading to plant death[1]. Over the course of evolution, plants have gradually developed various mechanisms to mitigate the damage caused by environmental stress[2]. These pathways encompass the calcium ion response mechanism, where external stimuli induce alterations in the concentration of free calcium ions within the cytoplasm. This shift is subsequently transduced into downstream signals, instigating a sequence of responses that empower plants to adapt to or resist changes in their external environment. Calcineurin B-like proteins (CBLs) are a family of Ca2+ receptors. The CBL protein was first identified in the model plant Arabidopsis and was named for its high homology to animal neuronal calcium sensors (NCS) and yeast calcineurin B (CNB)[3].

      As Ca2+ receptors, CBLs have a typical helix-loop-helix elongation factor hand (EF-hand) domain that binds calcium ions. Various numbers of CBL genes have been identified in plants, including 10 in Arabidopsis[4], six in Brassica napus[5], and eight in grape[6]. Plant CBLs typically interact with CBL-interacting protein kinases (CIPKs) to form CBL–CIPK complexes, which transmit the Ca2+ signal[7]. The CBL–CIPK signaling network mediates plant responses to abiotic stresses such as high salt, drought, ABA, and low temperature, and it has an important role in the maintenance of normal plant physiological activities[8]. For example, AtCBL4–AtCIPK24 phosphorylates the Na+/H+ antiporter AtNHX7 on the cell membrane under high-salt stress[9], promoting efflux of excess Na+ to maintain an appropriate balance of Na+ inside and outside the cell. Both CBL1 and CBL9 target CIPK23, which plays a role in the regulation of potassium uptake and stomatal movement[10,11]. Overexpression of AtCBL9 and AtCIPK3 increased the tolerance of transgenic plants to exogenous ABA[12]. CaCIPK3 and CaCBL2 interact to improve drought tolerance in pepper[13].

      Nitraria tangutorum Bobr. (N. tangutorum) is a deciduous shrub from the Nitrariaceae that is endemic to China and widely distributed in arid, semi-arid, and saline desert areas of northwest China[14]. N. tangutorum shows strong resistance to salt and alkali drought and has a highly developed root network, enabling it to stabilize sand and act as an effective windbreak. It therefore has an important role in protecting and maintaining the balance of the ecological environment, particularly in desert, semi-desert, and salinized areas of China.

      Currently, research on N. tangutorum predominantly centers on physiological and ecological aspects, with relatively few studies addressing gene function analysis, especially for stress-resistant gene families. This has hindered the progress of molecular biology research on N. tangutorum. The CBL gene family has been extensively demonstrated to play a role in responding to abiotic stresses in other plant species. Nevertheless, their distribution and functionality in the halophyte N. tangutorum have yet to be documented.

      In this study, CBL family members in N. tangutorum were identified, and their basic physicochemical properties, phylogeny, and stress responses were characterized. Subsequently, a representative CBL gene, NtCBL1-1, was cloned, and the effects of its overexpression in Arabidopsis were analyzed. These findings offer insights into the evolution and biological functions of the CBL gene family in N. tangutorum, thus establishing a theoretical basis for further investigations into the mechanisms of abiotic stress resistance in this desert halophyte. Also, it provides valuable insights for improving the stress tolerance of other agricultural and forestry plants.

    • N. tangutorum was collected in Dengkou County, Inner Mongolia in 2020. The fruit was subsequently extracted, dried, and stored in sandy soil at 4 °C for a vernalization period of 3 months. After vernalization, the seeds were germinated in a seedling tray, and the resulting seedlings were transplanted into nutrient-rich soil. They were then cultivated in a greenhouse with a temperature of 23 °C, under a light-dark cycle of 16 h of light and 8 h of darkness.

      Stress experiments were conducted using 2-month-old seedlings with multiple replicating seedlings employed for each treatment. One group was exposed to salt stress at 500 mM NaCl, while another group was exposed to drought stress at 20% PEG 6000. During stress treatment, the corresponding salt or PEG solution was carefully added to the seedling culture medium several times until the new solution began to slowly permeate. Subsequently, seedlings were immersed in an equal concentration response solution to maintain consistent stress levels. For each stress condition (0, 1, 4, 8, or 24 h), whole seedlings were exposed, and three seedlings were sampled from each treatment at each time point. Following sampling, the seedlings were rapidly frozen in liquid nitrogen and stored at −80 °C for RNA extraction.

    • Arabidopsis AtCBL protein sequences and rice (Oryza sativa) OsCBL protein sequences were downloaded from TAIR (www.arabidopsis.org/) and Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) databases. CBL sequences from rice and Arabidopsis were used as BLASTP search queries to identify potential CBL genes in the unpublished genome sequence of N. tangutorum. The candidate sequences were submitted to the SMART database for further confirmation (http://smart.embl-heidelberg.de/). The identified CBL genes were named according to their homologous to AtCBL genes. Cell-PLoc2.0 (www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) was used to predict their subcellular localization, and ExPASy (https://web.expasy.org/) was used to calculate their isoelectric points (PIs) and molecular weights (MWs).

    • MAFFT was used with default parameters to construct a multiple alignment of CBL nucleotide sequences[15]. IQ-TREE[16] was used to build a phylogenetic tree (iqtree -s -m MFP -b 1000 -nt auto), which was then visualized with iTOL[17]. Inter-chromosomal relationships among N. tangutorum CBLs were determined using TBtools, and inter-species synteny analysis was performed with the MCscan pipeline of JCVI[18]. Ka/Ks ratios between NtCBL gene pairs were calculated with KaKs_Calculator 2.0[19].

    • Protein motifs in the CBLs were analyzed using MEME website tools (http://meme-suite.org/tools/meme); the motif length ranged from 10 to 50 amino acid residues, the maximum number of motifs identified was 50, and other parameters were set to default values. Gene structures were analyzed using TBtools[20]. The promoter sequence was derived from the genome annotations file (Supplemental File S1), covering a 3,000 bp region upstream of the CBL gene's initiation codon. This extracted promoter sequence was then submitted to the PlantCARE platform[21] for the prediction of cis-acting elements. The results of this prediction were subsequently visualized using Adobe Illustrator.

    • Plant total RNA was extracted using the Easte Super Total RNA Extraction Kit (Promega, Shanghai, China). Subsequently, cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Quantitative real-time PCR primers were designed using the NCBI Primer-BLAST tools (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and the primer details can be found in Supplemental Table S1. The quantitative PCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). For each stress condition and time point, three biological replicates were analyzed. The relative expression levels were determined using the 2−ΔΔCᴛ method as described by Livak & Schmi[22].

    • Based on the gene family identification results, specific primers were designed using the reference CDS sequence of NtCBL1-1 (Supplemental Table S2) for the cloning process. Following successful cloning, the NtCBL1-1 fragment was incorporated into the pBI121 overexpression vector utilizing XmaI and SacI restriction sites. The resultant overexpression vector was then introduced into wild-type Arabidopsis (Col-0) plants using the Agrobacterium tumefaciens infection transgenic method, as previously reported by Clough & Bent[23]. Positive transgenic plants were selected and subsequently propagated to generate homozygous strains of the T3 generation, which were employed for stress resistance experiments.

    • To measure germination rate, seeds of wild-type and T3-generation transgenic Arabidopsis were sterilized and placed onto ½ Murashige and Skoog (MS) medium containing 0 or 150 mM NaCl or 300 mM mannitol. Fifty to 70 seeds of each transgenic line were arrayed on each petri dish, and there were three biological replicate plates. On day 7, the plates were photographed and the germination percentage calculated.

      For phenotypic and physiological assessments, transgenic Arabidopsis plants were transferred from petri dishes to nutrient-rich soil after 10 d of initial growth and then cultivated for an additional 10 d. Plants with roughly uniform growth were selected and irrigated with 200 mM NaCl or 300 mM mannitol to simulate salt or drought stress. The amount of solution added to each pot was the same. Phenotypic changes were observed continuously; leaves were collected, snap-frozen in liquid nitrogen, and stored in a −80 °C freezer for physiological and biochemical measurements. Soluble protein and MDA were detected using commercial kits (Jiancheng Bioengineering, Nanjing, China). Chlorophyll content was determined by the method of Lichtenthaler & Wellburn[24]. In brief, the main veins were removed from fresh Arabidopsis leaves, and samples of leaf material (0.1 g) were weighed and placed into 10 mL centrifuge tubes. Extraction solution (1:1 absolute ethanol:acetone) was added to each centrifuge tube, and extraction was performed under dark conditions for 24 h, during which time the chlorophyll was completely dissolved by shaking 3–5 times. Each assay was performed with three biological replicates of each treatment and time point.

    • By performing blastp analysis using candidate sequences from Arabidopsis and rice CBL proteins, combined with redundancy elimination and domain analysis, we successfully pinpointed 11 members of the CBL gene family in the entire genome of N. tangutorum. These genes were designated NtCBL1-1 to NtCBL10-2, primarily based on their homology with AtCBLs.

      Subsequently, basic information on these identified CBL genes was compiled. It was observed that these genes were unevenly distributed across six chromosomes. The amino acid lengths ranged from 213 to 321, with isoelectric points (pI) between 4.66 and 5.22. Their molecular weight (Mw) sizes ranged from 24.65 kDa to 36.84 kDa (Table 1). Furthermore, predictions of subcellular localization indicated a likelihood of expression on the plasma membrane for all identified members.

      Table 1.  Physicochemical properties of CBLs of N.tangutorum

      Gene IDOriginal IDLocusLength (aa)MW (kDa)PISubcellular localization prediction
      NtCBL1-1NITAA04G1075Chr4A21324.484.66Plasma membrane
      NtCBL1-2NITAB04G1197Chr4B21324.484.66Plasma membrane
      NtCBL3-1NITAB02G1010Chr2B25229.065.05Plasma membrane
      NtCBL3-2NITAA02G0832Chr2A22626.104.82Plasma membrane
      NtCBL4-1NITAA02G2027Chr2A21324.655.25Plasma membrane
      NtCBL4-2NITAA02G2024Chr2A21324.725.17Plasma membrane
      NtCBL4-3NITAB02G2334Chr2B21324.695.24Plasma membrane
      NtCBL8-1NITAB04G1638Chr4B32136.845.22Plasma membrane
      NtCBL8-2NITAA04G1516Chr4A22626.195.01Plasma membrane
      NtCBL10-1NITAB05G0933Chr5B26730.714.88Plasma membrane
      NtCBL10-2NITAA05G0858Chr5A27531.594.94Plasma membrane
    • To explore the phylogenetic relationships among CBLs, we constructed a maximum likelihood phylogenetic tree using 120 CBL sequences from 13 species (Supplemental Table S3), including N. tangutorum. Our analysis revealed that these CBL genes fall mainly into five distinct categories, with most genes clustered together based on their respective gene names. Clade 5 stands out with only three CBL genes found in Selaginella moellendorffii, while the other four subgroups (1 to 4) contain 30, 19, 43, and 25 genes, respectively. NtCBL genes were identified in Clades 1, 2, 3, and 4, with Clade 3 being particularly abundant, housing up to five NtCBL genes (Fig. 1).

      Figure 1. 

      The phylogenetic tree shows the relationships between CBL protein sequences from N. tangutorum and 12 other species. Phylogenetic analysis was performed using IQ-TREE software with maximum likelihood (ML) method and subjected to 1,000 bootstrap replicates. The modules are color coded to represent the five subclades of CBLs. NtCBLs are indicated by dark red stars. Green dots of different sizes indicate Bootstraps confidence levels above 80.

      To investigate the structural relationships among the 11 NtCBLs, we performed a genomic collinearity analysis using JCVI. Seven collinear CBL pairs were identified in the N. tangutorum genome: NtCBL1-1 and NtCBL1-2, NtCBL3-1 and NtCBL3-2, NtCBL8-1 and NtCBL8-2, NtCBL10-1 and NtCBL10-2, NtCBL4-1 and NtCBL4-2, NtCBL4-1 and NtCBL4-3, and NtCBL4-2 and NtCBL4-3 (Fig. 2a). Their chromosomal distribution and sequence similarity suggest that most NtCBL gene pairs were generated through whole genome duplication/polyploidization, while some NtCBLs, such as NtCBL4-1 and NtCBL4-2, appear to have undergone tandem duplication.

      Figure 2. 

      Genome-wide synteny analysis of CBL gene family among N. tangutorum and three other species. (a) inter-chromosomal relationships of NtCBLs (the links on the green curve indicate synteny relationships between genes). (b) Synteny analyses between the CBLs of N. tangutorum, Arabidopsis, Vitis vinifera, and Oryza sativa, the links between species indicate homologous relationships between genes.

      We conducted Ka/Ks calculations for the gene pairs, uncovering that, aside from NtCBL4-1 and NtCBL4-2, which displayed a Ks value of zero, the Ka/Ks values for the remaining gene pairs were notably below 1 (Supplemental Table S4). This observation suggests that these gene pairs underwent evolutionary purifying selection, emphasizing a constrained evolutionary process. Collinearity between genes from different species often indicates functional similarities. In light of this, we performed a collinear analysis of CBL genes across Arabidopsis, rice, grape, and N. tangutorum. The outcome indicated the existence of 12, 6, and 10 collinear relationships between NtCBLs and Arabidopsis, rice, and grape, respectively (Fig. 2b). This suggests that NtCBLs may share a close evolutionary relationship with Arabidopsis and grape, while showing a relatively distant relationship with rice.

    • To obtain a more comprehensive understanding of the gene structure of CBL genes in N. tangutorum, we conducted an analysis of their gene structures. We observed that all CBL genes exhibited intron structures, with gene lengths spanning from 3,667 to 6,098 base pairs and coding sequence (CDS) lengths ranging from 8 to 11 exons. Notably, a lack of a 5' UTR region was observed in NtCBL8-1, while NtCBL10-2 lacked a 3' UTR region. Conversely, typical 5' UTR and 3' UTR structures were present in the remaining genes (Fig. 3).

      Figure 3. 

      Gene structure of the CBL gene family in N. tangutorum. The dark green boxes represent the UTR (Untranslated Region), the light green boxes represent the CDS (gene coding region), and the black lines represent the intron region.

      Simultaneously, the pattern distribution within NtCBL proteins was investigated. As shown in Fig. 4, eight major motifs were identified among NtCBLs. Notably, motifs 1, 2, and 6 exhibited the highest degree of conservation of all CBLs. However, it is worth noting that motif 3 was absent in NtCBL4-1. In addition, pattern alterations were observed in four gene pairs that underwent fragment duplication, such as NtCBL8-1 and NtCBL8-2, as well as NtCBL10-1 and NtCBL10-2. Collectively, these findings suggest that functional differentiation may have occurred in NtCBLs during evolution.

      Figure 4. 

      Conservative motifs distribution in N. tangutorum. (a) Distribution of different motifs on NtCBL genes. (b) Specific sequence information of different motifs, with larger letters indicating higher conservation.

    • Promoter cis-acting elements constitute a critical region for transcription initiation, as highlighted by Hernandez-Garcia & Finer[25]. Analysis of these elements holds significant value in unraveling the potential functions of genes. To investigate plausible biological roles of the N. tangutorum CBL gene family. We examined the sequence located 3 kb upstream of the NtCBL gene initiation codon for cis-acting element analysis.

      This analysis unveiled the existence of a multitude of cis-regulatory elements intricately associated with hormone and stress responses. Specifically, cis-acting elements associated with abscisic acid responsiveness, auxin-responsive elements, methyl jasmonate (MeJA) responsiveness, gibberellin responsiveness, and salicylic acid responsiveness were identified within hormone signaling pathways. In addition, a range of abiotic stress-related elements were identified, including anaerobic induction, defense and stress responsiveness, drought inducibility, and low-temperature responsiveness (Supplemental Fig. S1). Collectively, these findings strongly suggest that CBL genes may indeed participate in biological functions related to these hormone and stress response pathways.

    • Relevant studies have shown that CBLs play a role in drought and salt stress processes in plants[26]. Meanwhile, previous analysis revealed cis-acting elements associated with drought or salt stress response in the NtCBL promoter region. All these findings point to the potential involvement of NtCBLs in drought or salt stress responses in N. tangutorum.

      Therefore, we have devised a set of experiments using qRT-PCR to investigate the expression patterns of NtCBL genes in different tissues and under abiotic salt stress conditions. First, we conducted an analysis of sequence similarity among NtCBLs (Supplemental Table S5). Due to the tetraploid nature of N. tangutorum, the genes within the NtCBL gene family exhibit extremely high similarity (nearly exceeding 95%). This high similarity made it nearly impossible to design specific primers. Therefore, NtCBL1-1 and NtCBL1-2 were combined as NtCBL1 for qRT-PCR, and the same approach was applied to the other genes.

      The expression patterns of the NtCBL gene family in the roots, stems, and leaves of N. tangutorum were initially examined through a comparison of expression results across different tissues. It was observed that the relative expression of all NtCBLs was highest in the stem. NtCBLs could be roughly divided into two groups based on their expression patterns. In the first group, which included NtCBL-1 and NtCBL-10, gene expression was ranked stem > leaf > root. In the second group, which included NtCBL-3, NtCBL-4, and NtCBL-8, gene expression was ranked stem > root > leaf. These different expression patterns of NtCBLs suggest that they may have distinct, tissue-specific functions (Fig. 5).

      Figure 5. 

      The gene expression characteristics of N. tangutorum CBL genes in root, stem, and leaf tissues were analyzed by fluorescence quantitative PCR. **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey’s HSD).

      Subsequently, we examined the expression patterns of NtCBL genes under salt and drought stress. The majority of NtCBLs demonstrated distinct responses to these stresses, although the timing and magnitude of the response differed among individual genes (Fig. 6). Under drought stress, the expression of NtCBL1 and NtCBL3 peaked at 8 h, whereas NtCBL4 and NtCBL10 reached their highest expression levels at 1 h, and NtCBL8 showed no significant change throughout the experiment. All NtCBL genes were significantly upregulated under salt stress, with their expression peaking at 1 h of salt stress and subsequently declining. These findings unequivocally illustrate that the expression of NtCBL genes is highly influenced by drought and salt stress, implying that NtCBL proteins likely contribute to N. tangutorum's response to these challenging environmental conditions.

      Figure 6. 

      Expression patterns of NtCBL genes during drought (upper panels) and salt stress (lower panels) in N. tangutorum. 'ck' stands for untreated control, while '1h', '4h', '8h', and '24h' respectively represent different time points of salt or stress treatment, which are 1, 4, 8, and 24 h. **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey’s HSD).

    • With reference to research in other plant species[2729] and response to drought and salt stress in N.tangutorum, we chose NtCBL1 as the representative gene for NtCBL and examined its potential role in enhancing stress resistance through overexpression in Arabidopsis. We designed specific primers only for NtCBL1 (Supplemental Table S5), and confirmed that the cloned gene was NtCBL1-1 by first-generation sequencing comparison, then constructed the 35S:NtCBL1-1 overexpression vector and transformed it into wild-type Arabidopsis via Agrobacterium-mediated transformation to obtain positive transgenic plants. We obtained multiple transgenic positive lines (Supplemental Fig. S2) and randomly selected eight of these lines for analysis of their relative expression levels. We observed variations in the expression levels among these lines (Supplemental Fig. S3). Based on their expression levels, we specifically chose lines 1, 4, and 6 and renamed them as lines 1, 2, and 3, respectively, for subsequent functional validation of NtCBL1-1.

      Previous studies have shown that stress can significantly affect seed germination rates[30]. To determine whether heterologous overexpression of NtCBL1-1 affected Arabidopsis germination, we sowed seeds of wild-type Arabidopsis and three independent transgenic lines on ½ MS medium containing 0 or 150 mM NaCl or 300 mM mannitol, and we observed their germination rate after 7 d. Under normal growing conditions, wild-type and transgenic lines germinated rapidly, and their germination rates were similar (Fig. 7). Under 150 mM salt stress, the germination rate of wild-type Arabidopsis was reduced, and the three overexpression lines showed higher germination than wild Arabidopsis. Under drought stress, both wild-type and transgenic lines showed reduced germination compared to control conditions, but the germination rate of transgenic lines was significantly higher than that of wild-type lines. Thus, overexpression of NtCBL1-1 in Arabidopsis ameliorated—at least in part—the inhibition of germination caused by salt and drought stress.

      Figure 7. 

      Heterologous expression of NtCBL1-1 increases Arabidopsis germination rates under salt and drought stress. (a)−(c) Phenotypic charts of germination rates under normal growth conditions, 150 mM NaCl treatment, and 300 mM mannitol treatment, with about 70 seeds per dish and three replicates for each experiment. (d) Germination rate statistics of Arabidopsis overexpressing NtCBL1-1 under normal growth conditions, 150 mM NaCl treatment, and 300 mM mannitol treatment. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey’s HSD).

    • To assess whether the heterologous expression of NtCBL1-1 impacts the salt and drought tolerance of Arabidopsis, we subjected soil-grown seedlings to irrigation with 200 mM NaCl or 300 mM mannitol for a duration of 7 d. On the first day of salt stress, leaves of wild-type Arabidopsis began to show slight wilting, but transgenic plants overexpressing NtCBL1-1 showed no visible changes (Fig. 8a, Supplemental Fig. S4). On day 3, leaf yellowing became visible on wild-type Arabidopsis, and the transgenic plants began to wilt. On day 5, leaves of wild-type Arabidopsis were severely wilted and showed large areas of yellowing, whereas leaves of the transgenic plants had begun to turn yellow. On day 7, wild-type plants were completely withered, but only some leaves of the transgenic plants were withered and yellow.

      Figure 8. 

      Heterologous overexpression of NtCBL1-1 increased salt tolerance in Arabidopsis. (a) Wild-type Arabidopsis phenotypes and overexpression lines exposed to 200 mM NaCl for 0–7 d. (b) Chlorophyll content, soluble protein content, and MDA content after 0–5 d of exposure to 200 mM NaCl. Statistical significance denoted as *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Tukey’s HSD).

      Since the wild-type plants had nearly died by the 7th day of the salt stress treatment, we conducted physiological measurements exclusively on plants harvested on days 0, 3, and 5. Before salt stress treatment, there were no significant differences in chlorophyll content between wild-type and transgenic lines. Chlorophyll content decreased as the duration of salt stress increased in both wild-type and transgenic Arabidopsis, but chlorophyll content was significantly higher in transgenic lines than in the wild type. This difference was most striking on day 5 (Fig. 8b). Soluble protein content showed a trend similar to that of chlorophyll content; it was lower in the wild type than in the transgenic lines under salt stress. Although all genotypes showed accumulation of MDA during the stress treatment, MDA content was significantly lower in the transgenic lines.

      The transgenic lines also showed less severe stress symptoms than the wild type in response to simulated drought (Fig. 9a, Supplemental Fig. S5). In contrast to the salt stress results, there were no significant differences in MDA or soluble protein content on day 3 of drought stress. However, on day 5, the MDA content was significantly higher in the wild type than in the transgenic lines, and the soluble protein content was significantly lower (Fig. 9b). Thus, overexpression of NtCBL1-1 also improved drought stress tolerance of transgenic Arabidopsis.

      Figure 9. 

      Heterologous overexpression of NtCBL1-1 in Arabidopsis increased drought stress tolerance. (a) Phenotypic comparison between wild-type Arabidopsis and overexpression lines under 300 mM mannitol exposure for 0–7 d. (b) Chlorophyll content, soluble protein content, and MDA content of wild-type Arabidopsis and overexpression lines exposed to 300 mM mannitol for 0–5 d. Statistical significance denoted as *p < 0.05, **p < 0.01 (ANOVA followed by Tukey’s HSD).

    • Deterioration of the soil environment significantly limits plant growth and development, and soil salinization is becoming an increasingly serious threat to forestry and agricultural production. As of 2021, about one billion hectares of land worldwide have been affected by soil salinization, accounting for about 7% of the Earth's total land area[31]. Excessive salinization impairs the normal growth of most plants, but halophytes can typically complete their entire life cycle under such conditions. Studying the adaptive mechanisms that enable halophytes to thrive under saline-alkali conditions is therefore important for the utilization and restoration of saline-alkali land.

      As important Ca2+ sensors, CBLs have key roles in plant perception and response to a variety of abiotic stresses, including drought[32], salinity[33], and cold[34], as well as the stress hormone ABA[35]. CBLs have been studied in a number plants, and 10, 9, and 7 CBL genes have been identified and characterized in Arabidopsis[4], Saccharum spontaneum[36], and Triticum aestivum[37], respectively. However, the number of CBL genes and their functions in the halophyte N. tangutorum have not previously been reported. In this study, we identified 11 CBL genes in the N. tangutorum genome and classified them into four clades on the basis of their phylogenetic relationships. Each clade showed general conservation of gene structure and protein domain composition, although there were some differences (Fig. 4) that may reflect functional differentiation of the NtCBLs.

      Plant polyploidization refers to the increase in the number of chromosomes within plant cells, often manifested as the duplication of entire sets of chromosomes[38]. Polyploidization leads to the presence of multiple identical or highly similar gene copies within the genome. These duplicated copies of genes undergo mutations during evolution and accumulate new features, driving the expansion of gene families. For instance, in the polyploid crop Brassica napus, the number of FBA gene family members is higher than in diploid plants such as Arabidopsis and rice[39]. Additionally, the MADS-box gene count in tetraploid Gossypium hirsutum was significantly higher than diploid Gossypium hirsutum[40].

      Plant polyploidization also leads to gene redundancy and enhanced stability[41]. In N. tangutorum, all CBL genes have homologs in their corresponding subgenomes. That is, except for the NtCBL4 gene, all NtCBL genes exhibit two highly similar gene duplications on corresponding chromosomes in their respective subgenomes. For example, NtCBL1-1 and NtCBL1-2 occur on CHR4A and CHR4B, while NtCBL10-1 and NtCBL10-2 are located on CHR5A and CHR5B, respectively. This duplication probably results from the tetraploidization process in N. tangutorum. In general, polyploidization offers significant benefits to plants, especially in adapting to environmental changes. This may be one reason N. tangutorum adapts to extreme saline-alkaline and drought conditions. Through polyploidization, plants may produce more copies of genes, and these copies could mutate, leading to new traits or functions. This diversity helps plants better adapt to various pressures or environmental conditions and helps them maintain a competitive advantage during their evolutionary process.

      Apart from chromosomal duplication, gene self-replication and differentiation also play significant roles in the expansion and evolution of gene families. Systematic phylogenetic analysis in this study reveals the presence of multiple copies of the CBL4 gene in several non-polyploid plants. For instance, there are three copies in Populus euphratica, five copies in E. grandis, two copies in C. sinensis, and three copies in M. domestica (Fig. 1). Multiple copies of CBL4 have also been observed in other plants, such as two copies in canola[5], and three copies in M. sativa [42]. Generally, gene redundancy is critical to maintaining essential biological functions in certain plants and may play a pivotal role in their adaptability and survival. Expansion of the CBL4 subfamily may be closely related to this phenomenon. In N. tangutorum, despite being a polyploid plant, the CBL4 gene had undergone replication prior to polyploidization, resulting in the formation of NtCBL4-1 and NtCBL4-2, suggesting a potentially significant role for CBL4 in the life processes of N. tangutorum.

      The cis-acting elements in the promoter region can regulate gene expression, thereby revealing internal information about gene function[43]. Analysis of the NtCBL promoter reveals various cis-acting elements associated with hormone regulation and defense responses. For instance, within the NtCBL gene promoter region, there are abscisic acid (ABA) responsive elements known as ABREs (Fig. 5). In the promoter regions of CBL genes in other species such as pepper, quinoa, and Vitis vinifera, we have found ABRE elements, indicating the relative conservation of ABRE elements in the CBL gene promoter regions. When plants face stress like drought, their water levels decrease, leading to the production of ABA. The ABA signaling pathway is activated, triggering a series of physiological and biochemical responses to help plants adapt to drought stress[44]. The varying responses of CBL genes to drought in pepper[45], quinoa[46], Vitis vinifera [6], and N. tangutorum might be associated with ABRE elements.

      Genes typically display patterns of tissue-specific expression. In our study, we observed predominant expression of CBL genes in stem and root, similar to the expression pattern of CBL in rice. However, in rice, CBL4 exhibits major expression in leaves[47], while in N. tangutorum, NtCBL4 exhibits the least expression in leaves. In cotton research, the expression of GhCBL4-5 was found to be higher in stem and root compared to leaves[48], which is somewhat similar to the expression pattern of NtCBL4 in this study. Despite differences in the expression patterns of CBL genes across species and tissues, they exhibit notable responses to stressors such as drought, salinity, and other adverse conditions. This high degree of conservancy in their response to environmental stressors might indicate the pivotal role CBL genes play in physiological regulation and stress responses in plants. Wide adaptability suggests the universality and significance of CBL genes and their regulated signaling pathways across different species.

      Currently, multiple genes within the CBL family have been cloned and their function under non-biological stressors has been validated[4951]. Among these genes, CBL1 is one of the most extensively studied representatives within the CBL gene family. CBL1 has been studied in many plant species, including Arabidopsis[33] and Brassica napus[27], but its function in N. tangutorum remains to be fully characterized. Here, we selected NtCBL1-1 as a representative of the NtCBL gene family and performed a preliminary functional characterization by overexpression in Arabidopsis. Although the sequences of NtCBL1-1 and NtCBL1-2 were highly similar, we were able to design specific primers for cloning NtCBL1-1, and we confirmed the identity of the cloned gene by Sanger sequencing. We then used this gene to construct an overexpression vector for Arabidopsis transformation.

      Previous reports on CBL1 in other species, as well as our own qRT-PCR results, led us to speculate that NtCBL1-1 might participate in responses to salt and drought stress. Although both stresses impaired seed germination, seeds of the NtCBL1-1 overexpression lines had a higher germination rate than those of the wild type under stressed conditions (Fig. 8). Likewise, the transgenic plants showed less severe symptoms in response to salt stress and simulated drought. Leaves of the wild type showed severe wilting and yellowing under both stresses, whereas those of transgenic plants were only slightly yellow, and this difference became more obvious with increasing stress duration.

      Studies have shown that plant stress can lead to chlorophyll degradation[52], and plants with greater stress resistance tend to have higher soluble protein content[53]. MDA is a lipid peroxidation product whose content can serve as a measure of oxidative damage[54].

      Overexpression of stress-resistant genes may affect plant physiological characteristics, such as soluble protein content, malondialdehyde (MDA) levels, and chlorophyll content. These physiological traits are often associated with a plant's ability to withstand stress and its mechanisms for coping with environmental pressures. Over-expression of stress-resistant genes typically triggers the activation of signaling pathways, leading to a series of biochemical responses in plants. This results in the production of more stress-resistant proteins and metabolites, including soluble proteins and antioxidant enzymes. This process helps alleviate oxidative stress, enhances the plant's resistance to free radicals and oxidative damage, thereby reducing the accumulation of oxidative byproducts such as soluble proteins and MDA. Chlorophyll, a key molecule in photosynthesis, plays an important role in protecting plants from environmental stressors. Overexpression of stress-resistant genes can reduce the damage to chlorophyll caused by stress factors, helping maintain photosynthetic efficiency and normal plant growth.

      For example, researchers have found that overexpression of the stress-resistant gene HvPIP2;5 in transgenic Arabidopsis leads to retention of more chlorophyll and a reduction in MDA accumulation[55]. In another study, overexpression of the superoxide dismutase gene reduced the accumulation of soluble proteins in alfalfa leaf tissues[56].

      These findings suggest that by overexpressing stress-resistant genes, plants can better cope with environmental stress, reduce oxidative damage, and reduce the accumulation of oxidative byproducts, thus maintaining their normal growth and survival.

      In this study, transgenic Arabidopsis overexpressing NtCBL1-1 contained more chlorophyll and soluble protein and less MDA than the wild type under salt stress (Fig. 8), indicating that they had higher salt tolerance. Responses to simulated drought stress were similar: Arabidopsis overexpressing NtCBL1-1 showed greater drought tolerance than the wild type (Fig. 9). Thus, overexpression of CBL1-1 from N. tangutorum improved the abiotic stress tolerance of transgenic Arabidopsis. The calcium receptor encoded by NtCBL1-1 may therefore form part of the signal transduction pathways that underlie the high salt tolerance of this dryland halophyte. Overexpression of the NtCBL1-1 may have activated multiple physiological pathways in Arabidopsis, aiding the plant in better coping with environmental stress, alleviating oxidative stress, protecting chlorophyll, and maintaining water balance. These physiological changes enhance the stress resistance of the transgenic Arabidopsis.

      This study revealed that the overexpression of the NtCBL1-1 in Arabidopsis led to enhanced phenotypes, germination rates, and chlorophyll content under drought stress compared to wild-type Arabidopsis. However, this is just the tip of the iceberg, as we still need a more comprehensive understanding of the underlying regulatory networks. In parallel, we are actively engaged in the development of a transgenic system specifically tailored for N. tangutorum. We eagerly anticipate conducting in-depth mechanistic inquiries through self-overexpression analysis. Moreover, the translocation of CBL into N. tangutorum holds the promise of yielding more robust salt-tolerant halophytes. This prospective outcome could offer valuable insights for future biotechnology-driven advancements in forestry breeding and ecological restoration.

    • In this study, we identified CBL family members in N. tangutorum and characterized their basic physicochemical properties, phylogeny, and responses to stress. The results indicate that there are a total of 11 NtCBL genes distributed across six chromosomes in N. tangutorum. Expression analysis reveals that these genes are highly responsive to both salt and drought stress. Functional studies of NtCBL1-1 demonstrate that transgenic Arabidopsis plants overexpressing NtCBL1-1 exhibit enhanced tolerance to both drought and salt stress. Under drought and salt stress conditions, compared to wild-type Arabidopsis, transgenic plants show increased germination rates, slower chlorophyll degradation, higher accumulation of soluble proteins, and reduced levels of the oxidative stress marker malondialdehyde. These findings underscore the significant role of NtCBL1-1 in responding to drought and salt stress and provide insight into the evolution and biological functions of the CBL gene family in N. tangutorum, providing a theoretical foundation for further study of abiotic stress resistance mechanisms in this desert halophyte.

    • The authors confirm contribution to the paper as follows: study conception and design: Chen J, Cheng T; data collection: Wu J, Li M, Zhang J; analysis and interpretation of results: Zhu L, Fang H, Chen Y; draft manuscript preparation: Zhu L, Li M. All authors reviewed the results and approved the final version of the manuscript.

    • All data generated or analyzed during this study are included in this article and its supplementary table files. The Gene sequence, CDS sequence, and GFF annotation information of all NtCBL gene families are included in the supplementary files.

      • This work was supported by the Natural Science Foundation of China (No. 31770715) and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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

      • Supplemental Table S1 Primers for Quantitative RT-PCR in N.tangutorum.
      • Supplemental Table S2 Primers for isolation of NtCBL1-1 fragment and construction of overexpression vector.
      • Supplemental Table S3 CBL genes used to construct phylogenetic trees from 12 species except N.tangutorum.
      • Supplemental Table S4 KaKs analysis in N.tangutorum.
      • Supplemental Table S5 Identity between CBLs gene in Nitraria tangutorum.
      • Supplemental Fig. S1 Cis-regulatory elements analysis of NtCBLs. (A) A heat map showing the number of cis-acting elements related to stress response in NtCBLs. (B) The distribution of cis-acting elements in the promoter region (ATG upstream) of NtCBLs, where different boxes represent different cis-acting elements.
      • Supplemental Fig. S2 Positive identification of partial NtCBL1-1 transgenic T3 generation; different lanes represent different transgenic lines.
      • Supplemental Fig. S3 Relative expression levels in partial transgenic T3 generations, with wild-type Arabidopsis as a reference.
      • Supplemental Fig. S4 Phenotypic changes in overexpression lines 1 and 3 following exposure to 200 mM NaCl for 0−7 days.
      • Supplemental Fig. S5 Phenotypic changes in overexpression lines 1 and 3 following exposure to 300 mM mannitol for 0−7 days.
      • Supplemental file S1 Genome annotations.
      • Copyright: © 2023 by the author(s). Published by 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/.
    Figure (9)  Table (1) References (56)
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    Zhu L, Wu J, Li M, Fang H, Zhang J, et al. 2023. Genome-wide discovery of CBL genes in Nitraria tangutorum Bobr. and functional analysis of NtCBL1-1 under drought and salt stress. Forestry Research 3:28 doi: 10.48130/FR-2023-0028
    Zhu L, Wu J, Li M, Fang H, Zhang J, et al. 2023. Genome-wide discovery of CBL genes in Nitraria tangutorum Bobr. and functional analysis of NtCBL1-1 under drought and salt stress. Forestry Research 3:28 doi: 10.48130/FR-2023-0028

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