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2022 Volume 2
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    

A mutation in CsKTN1 for the Katanin p60 protein results in miniature plant in cucumber, Cucumis sativus L.

  • State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

More Information
  • Katanin, a microtubule shearing protein, plays an important role in plant architecture formation. However, little is known about its mechanisms in regulating plant architecture in cucumber. In the present study, through EMS mutagenesis, we identified a novel micro-plant (mp) mutant in the North China type inbred line CCMC that may be of value for cucumber breeding. The size and number of stem cells were altered in the mp mutant. Through bulked segregant analysis (BSA) sequencing approach combined with genetic mapping, the mp locus was delimited to an interval of 130.9-kb. Multiple lines of evidence suggested that the mp mutation is due to a single nucleotide polymorphism in Csa7G435510 that is predicted to encode the Katanin p60 subunit protein (CsKTN1). The expression levels of CsKTN1 decreased significantly in all tissues except the tendril of mp mutant. Subcellular localization showed that both wild-type and mutant CsKTN1 proteins were located in cell membrane, cytoplasm and nucleus of tobacco leaf cells. The mutant protein lost part of its ability to bind and shear microtubule in vitro. These findings provide new insight into the regulatory function of microtubule-shearing protein, Katanin p60, in plant architecture of cucumber.

  • 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 Sequences of primers used in the study.
    Supplemental Table S2 Photosynthetic and fluorescence parameters of mp mutant and wild type.
    Supplemental Table S3 The whole genome sequencing reports of mp and CCMC pools.
    Supplemental Table S4 Gene annotation in candidate intervals.
    Supplemental Table S5 Annotation of mutation sites in candidate intervals.
    Supplemental Fig. S1 Phenotypic data of mp mutant and CCMC. Values are mean±SD. **P<0.01(Student′s t-test).
    Supplemental Fig. S2 Stereoscope images of ovaries of mutant and CCMC by paraffin section. Micrographs of transverse section of ovaries of mp mutant (a) and CCMC (b).
    Supplemental Fig. S3 cDNA sequence alignment of Csa7G435510 originated from 3 cucumbers as follow: 9930,CCMC and mp.
    Supplemental Fig. S4 Sequence alignment of Katanin p60 homologs. Green line, the AAA+type ATPase domain; red rectangle, amino acid substitution in CsKTN1 protein of mp mutant.
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  • Cite this article

    Song M, Fu W, Wang Y, Cheng F, Zhang M, et al. 2022. A mutation in CsKTN1 for the Katanin p60 protein results in miniature plant in cucumber, Cucumis sativus L.. Vegetable Research 2:3 doi: 10.48130/VR-2022-0003
    Song M, Fu W, Wang Y, Cheng F, Zhang M, et al. 2022. A mutation in CsKTN1 for the Katanin p60 protein results in miniature plant in cucumber, Cucumis sativus L.. Vegetable Research 2:3 doi: 10.48130/VR-2022-0003
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A mutation in CsKTN1 for the Katanin p60 protein results in miniature plant in cucumber, Cucumis sativus L.

  • State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

  • Received: 22 October 2021
  • Accepted: 11 February 2022
  • Published online: 24 February 2022
Vegetable Research  2,  Article number: 3  (2022)  |  Cite this article

Abstract: Katanin, a microtubule shearing protein, plays an important role in plant architecture formation. However, little is known about its mechanisms in regulating plant architecture in cucumber. In the present study, through EMS mutagenesis, we identified a novel micro-plant (mp) mutant in the North China type inbred line CCMC that may be of value for cucumber breeding. The size and number of stem cells were altered in the mp mutant. Through bulked segregant analysis (BSA) sequencing approach combined with genetic mapping, the mp locus was delimited to an interval of 130.9-kb. Multiple lines of evidence suggested that the mp mutation is due to a single nucleotide polymorphism in Csa7G435510 that is predicted to encode the Katanin p60 subunit protein (CsKTN1). The expression levels of CsKTN1 decreased significantly in all tissues except the tendril of mp mutant. Subcellular localization showed that both wild-type and mutant CsKTN1 proteins were located in cell membrane, cytoplasm and nucleus of tobacco leaf cells. The mutant protein lost part of its ability to bind and shear microtubule in vitro. These findings provide new insight into the regulatory function of microtubule-shearing protein, Katanin p60, in plant architecture of cucumber.

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    INTRODUCTION

    • Plant architecture refers to the morphological characteristics and spatial distribution of plants. Distinct plant architecture may change the distribution of solar radiation in the population, affecting carbon assimilation and dry matter accumulation of photosynthetic organs, ultimately resulting in different yields. It also directly determines the amount of labor input in cultivation management. Plants with ideal architecture can have beneficial characteristics including reduced area required for cultivation, reduced labor needs, enhanced light utilization, and increased yield. Crops such as rice[1], wheat[2] and maize[3] have all benefited from genetic modification of plant architecture. The green revolution was primarily due to the finding and use of semi-dwarf genes in rice and wheat. Many of the genes underlying plant architecture related traits have been cloned[4,5].

      As a climbing crop, cucumber (Cucumis sativus L.) has the indeterminate growth habit under optimum greenhouse management. Reducing plant height (vine length) and organ size can increase plant density and productivity. To address this trait, eight associated loci/genes have been reported and cloned, including CsCLAVATA1 (dwarf, dw), CsVFB1 (short internode, si), CsCullin1 (compact, cp), CsCKX (compact-1, cp-1), CsCYP85A1 (super compact-1, scp-1) and CsDET2 (super compact-2, scp-2)[6]. These mutants exhibited reduced internode length or compact growth habit and were reported to be mainly involved in CLAVATA signaling pathway and phytohormones biosynthesis pathway including brassinosteroid (BR), auxin, cytokinin, and jasmonic acid[7−11]. A tendril-less mutant (td-1) obtained from the EMS-induced mutagenesis population was found to have significantly reduced vine length although the regulatory mechanism remains unknown[12]. Three loci related to leaf size were mapped for the study of small organs in cucumber: little leaf (ll), little leaf 2 (ll-2) and small and cordate leaf 1 (scl-1)[13,14]. Among them, scl-1 showed significant reduction in leaf length and width, whereas ll and ll-2 mutants showed shrunken phenotypes in more organs such as leaves, flowers, seeds, and fruits. Although a number of mutants have been reported, the currently available mutants or genotypes have not been applied to cucumber breeding with modified plant architecture, which is perhaps due to their extreme phenotypes or the vague understanding of their regulatory mechanisms

      The size and shape of cells could be determined by the de-polymerization state of microtubules, which would affect the plant architecture[15]. As one of the microtubule shearing proteins, Katanin protein is a heterodimer composed of two subunits, p60 and p80[16]. Katanin p60 subunit protein which belongs to the AAA protein family, could catalyze the hydrolysis of ATP and shear microtubules under the action of ROP6-RIC1 signaling pathway[17]. Its cleavage activity is mainly regulated by several factors including Katanin p80 subunit, intracellular ATP and Ca2+ content, light signal induction, phytohormones, and microtubule morphology[18]. Katanin heterodimer was directed by Katanin p80 microtubule cleavage site for specific microtubule recognition to complete the cleavage[19]. In addition, the Katanin-mediated severing could be antagonized by augmin protein at microtubule crossovers[20]. In post-translational regulation, Katanin p60 was ubiquitinated by MAB1 (MATH-BTB1) through decoupling with E3 ubiquitin ligase complex containing CLU-3[21].

      Mutations in Katanin p60 coding genes may disrupt the morphogenesis during plant growth by affecting cell division plane orientation[15,22]. A total of seven defective loci in Katanin p60 protein were reported including BOT1, DGL1, ERH3, FRA2/AtKTN1, FRC2, KTN1, and LUE1/AtKSS, which were identified in Arabidopsis (six mutations allelic of the same locus) and rice (one locus)[23−28]. These mutants had varying degrees of microtubule and cell growth defects such as reduced cell length or impaired cell elongation, abnormal cell wall location or reduced cellulose content, and cell division plane orientation defects[22]. As a result, both vegetative and reproductive organs showed irregular growth patterns, such as shorter internodes, compact organs, root radial swelling, abnormally shaped hypophysis, sterile flowers and malformed ovule development[22]. In conclusion, microtubule severing by Katanin p60 displayed a broad range of association with cellular and developmental processes in plants.

      Previous studies indicated that there is a link between plant architecture and Katanin protein. In the present study, we identified a micro-plant cucumber mutant mp with small organ phenotype caused by defects in Katanin protein. This mutant is a novel material to study its function and application in plant architecture breeding in horticulture plants, especially for climbing plants such as cucurbit crops. To increase the potential value of cucumber plant architecture breeding, we investigated the molecular mechanism and biological function of microtubule-shearing protein Katanin p60 involved in cell morphogenesis and plant architecture.

    MATERIALS AND METHODS

      Plant materials

    • Micro-plant mutant (mp) displaying reduced organ size was obtained from the M2 generation of ethyl methanesulfonate (EMS)-induced mutagenesis population. The donor line for mutagenesis was CCMC (Changchunmici), a North China fresh market type with normal plant architecture (Fig. 1). Another normal line 'Hazerd', a European greenhouse inbred line with wild type plant architecture, was used for developing a segregation population with mp mutant. The following traits were measured to determine the accurate phenotype, hypocotyl length (one true-leaf stage), plant height, internode length (10 true-leaf stage), leaf length, leaf width, petiole length (10th true-leaf), ovary length (flowering stage), commercial fruit length (7 d after pollination) and mature fruit length (40 d after pollination).

      Figure 1. 

      Morphological characterization of CCMC and mp mutant. Representative photographs of mp mutant cucumber plants showing miniaturized development in (a) seed, (b) hypocotyl and root, (c) the 1st true leaf, (d) male flower, (e) the whole plant expanded 10 true-leaf stage, (f) female flower and ovary, and (g) mature fruit. Scale bar = 10 mm.

      Several F2 populations were developed to investigate the inheritance of mp including F2 populations obtained separately from the crosses between CCMC and mp mutant, 'Hazerd' and mp mutant. All cucumber germplasm resources used in this research were obtain from the Laboratory of Cucurbitae Genetic and Germplasm Enhancement in the College of Horticulture, Nanjing Agricultural University. The plant materials were grown and phenotyped in greenhouses at Baima Cucumber Research Station of Nanjing Agricultural University, Nanjing, China.

      Scanning electron microscopy

    • Scanning electron microscopy was performed according to the protocol as described previously[29] with minor modification. The main stems of the second internode of CCMC and mp mutant plants at 10 true-leaf stage were investigated under a scanning electronic microscope (SEM). Firstly, the tissue samples were flushed with 1.0% formaldehyde to remove the surface wax powder and cut longitudinally into 2 mm × 5 mm size, then quickly fixed in 4% glutaraldehyde phosphate buffer (pH = 6.8) for 24 h. The fixed samples were dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, and 100%) and critical-point drying. A Hitachi JSM-6360LV scanning electron microscope was used for sample observation. The cell length, width and number were calculated by ImageJ software.

      Paraffin sectioning

    • Paraffin sectioning was performed according to the protocol as described previously[30] with minor modification. Ovaries of WT and mp mutant plants grown under the same conditions were sampled separately, and then fixed in 50% FAA fixation solution (50% ethanol, 10% formalin, 5% acetic acid) for at least 16 h and embedded in paraffin. Transverse sections of 3 mm thickness were mounted in neutral balsam. Neutral balsam was melted, and tissue was incubated three times in balsam bath. Melted balsam was poured into a block mold together with the tissue and allowed to cool. Longitudinal sections were cut with 8 μm thickness and then fixed on micro slides and stained with 1% hematoxylin for 3−5 min. Finally, haematoxylin eosin (HE) staining results were examined and photographed using an OLYMPUS BX53 fluorescence microscope.

      Measurement of photosynthetic parameters

    • Photosynthetic parameters including net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (E) and chlorophyll fluorescence parameters Fv/Fm of CCMC and mp mutant plants were measured using the US-made LI-6800 portable photosynthesis measurement system. All plants were grown in a plant culture incubator at 28 °C day / 24 °C night temperature and 10,000 Lux light intensity. The first fully expanded true leaf was selected to measure photosynthetic parameters at 8−10 in the morning. Ten leaves for each material were selected as biological repeats.

      Genetic mapping of mp candidate gene

    • Among 485 F2 individuals obtained from the cross between CCMC and mp mutant, equal amounts of DNA from 21 and 24 individuals with mutant and normal plant types were bulked, respectively. The DNA was extracted by CTAB method. A modified BSA-seq method[31] was used to map the mp locus. The two bulks were subjected to whole genome resequencing using the Illumina HiSeq 2500 (500 bp) platform. CCMC was re-sequenced in our previous study[32]. Reads from both bulks were independently aligned to the CCMC consensus sequence reads for variants calling. SNP indix, Δ (SNP index), and the p-values were calculated to determine the candidate region. The average SNP index of SNPs in a genomic interval was calculated using sliding window analysis with 1 Mb window size and 10 kb increment.

      Due to the low level of polymorphism between mp and CCMC, a linkage group for target chromosome was constructed using the 273 F2 population from mp x Hazerd. To identify more recombinants in the target region, additional 708 F2 individuals from the same cross were employed. Plant genotypes were determined by identification of molecular markers. 'Hazerd' was re-sequenced in our previous study[32]. Indel (Insertion/Deletion) and SNP markers were developed within the preliminary interval using re-sequenced data of 'Hazerd' and CCMC. The candidate SNPs in refined region were annotated using the Cucumber Genome Database (http://cucurbitgenomics.org)[33]. All primer sequences are listed in Supplemental Table S1.

      Sequence analysis, annotation, and identification of the mp candidate gene

    • Total RNA was extracted from leaves of CCMC and mp mutant plants using Trizol method. cDNA synthesis was performed using a Prime ScriptTM RT Reagent Kit (TaKaRa). The coding sequences of the candidate gene from CCMC and mp mutant were cloned and sequenced by Sanger sequencing. Functional annotations of candidate genes were acquired from Cucumber Genome Database (http://cucurbitgenomics.org) and NCBI (National Center for Biotechnology Information) databases (www.ncbi.nlm.nih.gov). The homologous amino acid sequences of predicted proteins from nine plant species were downloaded from NCBI database (www.ncbi.nlm.nih.gov) (NCBI GenBank No: Cucumis melo: XP_016902686.1; Gossypium hirsutum: XP_016739756.1; Arabidopsis thaliana: BAB87822.1; Solanum lycopersicum: XP_004241721.1; Nicotiana tabacum: XP_016456600.1; Oryza sativa: XP_015622511.1; Sorghum bicolor: XP_002456154.1; Brachypodium distachyon: XP_003569573.1; Hordeum vulgare: KAE8818098.1). MEGA 7.0.21 software was used to construct a neighbor-joining tree based on 1,000 bootstrap replications[34].

      Subcellular localization of CsKTN1

    • Full-length CDSs (coding sequences) of CsKTN1 from CCMC and mp mutant alleles were fused with GFP (green fluorescent protein) in pGreen vector (the modified vector contains 35S and GFP fragments) to generate plant binary expression vector p35S::CsKTN1S(CCMC)-GFP and p35S::CsKTN1F(mp)-GFP. Restriction sites were included in primers to facilitate cloning and the corresponding fragments were cloned into SmaI-cut pGreen plasmid to produce p35S::Gene-GFP recombinant plasmids. Two recombinant plant expression plasmids and one free GFP plasmid p35S::GFP used as a positive control, were used for transient expression in tobacco leaf cells (4 weeks old) via agrobacterium (C58) infection. Agrobacterium was cultured overnight until OD600 value = 1.0 and suspended with binding buffer (MgCl2 10 mmol·l−1, MES 10 mmol·l−1, Acetosyringone 200 umol·l−1, pH 5.6) after centrifugation. Agrobacterium was injected into tobacco leaves. Infected tobacco plants were kept in darkness at 27 °C for 2−3 d to allow transient expression of transgenes. Fluorescent signals were detected using a Zeiss LSM800 ultra high-resolution confocal microscope. The primer sequences are listed in Supplemental Table S1.

      Quantitative real-time PCR (qRT-PCR) analysis

    • Quantitative real-time PCR (qRT-PCR) was performed on tissue samples from the first fully expanded true leaf, root, main stem of second internode, male and female flower at anthesis, tendril at 10-true-leaf stage from CCMC and mp plants. Roots were dug out of the soil and quickly frozen in liquid nitrogen. Total RNA was extracted with RNAprep Plant Mini Kit (Tiangen) following the manufacturer's instructions. First strand cDNA was synthesized using Prime Script 1st Strand cDNA Synthesis Kit (TaKaRa). qRT-PCR was performed with a TB-GREEN Premix Ex TaqTM Kit (TaKaRa) in Bio-Rad CFX96 quantitative real-time PCR system, and the values of triplicate reactions were averaged. The threshold cycle (Ct) value of each gene was investigated and normalized to Ct value of CsActin. Relative mRNA expression data was analyzed using the 2−ΔΔCᴛ method. The primer sequences are listed in Supplemental Table S1.

      Protein expression and purification

    • Full length CDS of CsKTN1C (CCMC allele) and CsKTN1T (mp allele) were cloned into pCZN1 vector (His-tag) and expressed in Arctic ExpressTM bacteria (DE3). The IPTG (isopropyl β-D-1-thiogalactopyranoside) was used to induce protein expression. The expressed proteins were purified by Ni-IDA-Sefinose (TM) Resin (Novagen) following the manufacturer's instructions. Purified proteins were analyzed by 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

      Microtubule binding assay

    • Microtubule coprecipitation test was performed to verify the binding relationship between target protein (CsKTN1) and microtubule in vitro as previously described[35]. Purified tubulin protein (Cytoskeleton, Porcine Brain, Cat. #T240) and target proteins, CsKTN1S (CCMC allele)-His and CsKTN1F (mp allele)-His, were centrifuged at 200,000 g at 4 °C for 20 min before use. Two target proteins (PBS buffer dissolved) at a concentration of 2 μM were mixed with a 2 μM tubulin solution in binding buffer (100 mM PIPES, 1 mM MgCl2, 1 mM EGTA, 20 mM paclitaxel and 1 mM GTP, pH = 6.9), at room temperature for 30 min. After centrifugation at 100,000 g for 30 min, the pellets (resuspended by binding buffer) and supernatant were analyzed by SDS-PAGE.

      Microtubule shearing and observation

    • Rhodamine-labeled tubulin (Cytoskeleton, Porcine Brain, Cat. #TL590M) was polymerized in PEM binding buffer (100 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH = 6.9) containing 1 mM ATP and 20 mM taxol at 37 °C. Recombinant proteins CsKTN1S-His and CsKTN1F-His were diluted to the final concentration of 2μM in PEM binding buffer and mixed with the polymerized tubulin (6 μM) at 37 °C for 30 min. The mixed proteins were fixed with 1% glutaraldehyde for observation by fluorescence microscopy.

    RESULTS

      Changes in cell morphology and size as determinants of the micro-plant mutant phenotype

    • From an EMS cucumber mutant library, a micro-plant (mp) mutant was identified showing miniaturized phenotypes in all organs (Fig. 1a−g). The plant height of mp mutant shrank significantly by ~80% of normal cucumber plant (CCMC) at the expanded 10 true-leaf stage. Other organs such as hypocotyl, internode length, leaf size, ovary size and fruit length decreased by about 50% to 80% compared with that of CCMC (Supplemental Fig. S1). The mp mutant was female sterile hence unable to produce viable seeds. Specifically, there were fewer seeds in mature fruit of mp mutant, and all of them were empty, deflated and unable to germinate. The hybrid F1 of CCMC and mp mutant displayed an intermediate phenotype, and its growth state was between CCMC and mp mutant (Fig. 1). In addition, the mutant also exhibited significant reduction of several key photosynthetic parameters (Pn, Gs, E and Fv/Fm) in mp mutant than CCMC (Supplemental Table S2).

      Micro-structure of stem epidermal cells in mp mutant displayed irregular cell size and deformed cell shape compared with normal cucumber (Fig. 2). Triangle, trapezoid, or sub-circular cells were observed in mp mutant, while only long rhombus cells were observed in the normal plants (Fig. 2a, 2b). The length of epidermal cells in mp stem was significantly shorter than that of CCMC stem, while the width did not change significantly (Fig. 2c, 2d). In addition, the cell number of epidermal cells in mp stem was significantly increased (Fig. 2e). Paraffin sections of the ovary cells of mp mutant and CCMC were also examined, and the result showed that the ovary cells of mutant were smaller and irregularly arranged compared with CCMC (Supplemental Fig. S2). According to these findings, the emergence of micro plant phenotype was caused by changes in cell development and proliferation.

      Figure 2. 

      Scanning electron microscopy (SEM) observation of stem epidermal cells of CCMC and mp mutant. Micrographs of stem epidermal cells in (a) mp mutant and (b) CCMC. Scale bar = 100 μm. Boxplot indicating the (c) length, (d) width and (e) cell number of epidermal cells on stems of CCMC and mp mutant.

      Genetic mapping placed the mp locus to a 130.9 kb interval

    • To explore the inheritance of mp phenotype, we developed F2 population consisting of 485 plants derived from the cross between CCMC and mp mutant. 117, 255 and 113 plant individuals exhibited micro-plant type, intermediate type and normal plant phenotype in the F2 population respectively, which fits the segregation ratio of 1:2:1 (P = 0.51). This suggested that the micro-plant mutant phenotype is controlled by a single gene with incomplete dominance.

      From the F2 plants, we bulked plants with extreme micro-plant and normal-plant phenotypes for re-sequencing. Each pool has a sequencing depth of over 82.26 and coverage of over 93.43% (Supplemental Table S3). The potential polymorphisms were found by aligning the short reads of two bulks to CCMC consensus assembly. The calculated (SNP index) throughout the majority of the genome areas was 0.5, indicating that there was no substantial genetic variation between the two groups ( Fig. 3a ). A single genomic region harboring a cluster of SNPs with a high SNP index was identified in the 15.5−17.6 Mb interval of chromosome 7, suggesting that this candidate genomic region probably harbors the causative mutation ( Fig. 3a).

      Figure 3. 

      BSA-seq and linkage analysis of the mp locus. (a) BSA-seq analysis identified the candidate interval for mp locus to a 2.1 Mb genomic region harboring a high ΔSNP index (subtracting the SNP-index value of the mutant-pool from the CCMC-pool) on chromosome 7 (15.5−17.6 Mb). (b) A genetic map based on a F2 segregation population containing 273 individual plants delimited the mp locus to a 1.1 cM region. (c) Seven polymorphic markers and nine recombinants were applied to narrow down the mp locus to a 130.9 kb region. The numbers in parentheses indicate the number of recombinant plants of each marker. The white box indicates the mutant genotype, the black box for the CCMC genotype, and the striped box for the heterozygous genotype. (d) Genes (black boxes) and candidate mutation site in the mapping region.

      For the low level of polymorphism between mp and CCMC at the candidate region, we developed another F2 population (n = 273) from the cross between 'Hazerd' variety and mp mutant to narrow the potential region of mp locus. Ten polymorphic InDel markers and one SNP marker were developed between 'Hazerd' and mp mutant. After the marker genotypes of all 273 individuals were identified, an initial linkage map was constructed and mp locus was mapped to a 0.6 cM region flanking by two Indel markers (MIndel-23 and MIndel-10) (Fig. 3b). To identify more recombinants in this region, a larger F2 population with 708 plants was examined, and two additional SNP markers were developed. Nine more recombinants were obtained, delimiting the mp locus into a 130.9 kb area flanked on Chr7 by markers MSNP-3 (17,480,086 bp) and MIndel-30 (17,611,033 bp) (Fig. 3c).

      A gene that encodes the Katanin p60 subunit protein was identified for mp locus

    • Nineteen genes were annotated in the 130.9 kb region (Fig. 3d, Supplemental Table S4). Based on the re-sequencing data of CCMC and two bulked pools, five SNPs were found in this region (Supplemental Table S5). Among these SNPs, SNP (SNP-7G17507331) causes a non-synonymous homozygous mutation (Supplemental Table S5). The other four SNPs were located in the intergenic region and had a low SNP index (below 0.6) in mp pool (Supplemental Table S5). There were no other base differences (like Indel or SV) in this region between two bulked pools. As a result, the SNP-7G17507331 was considered as the causal SNP for the mp mutant.

      According to the Cucumber Genome Database, SNP-7G17507331 is located inside the gene Csa7G435510 (Fig. 3d). We performed Sanger sequencing to clone the genomic and cDNA sequences of Csa7G435510 from CCMC and mp mutant (Supplemental Fig. S3). The gene Csa7G435510 consists of seven exons and encodes a Katanin p60 protein containing an AAA+-type ATPase domain of amino acid residues 225 to 514 (Fig. 4a, Supplemental Fig. S4). We named it as CsKTN1. The causal SNP at the exon 5 of CsKTN1 resulted in an amino acid substitution from serine (S353) in CCMC to phenylalanine (F353) in mp mutant, which was located at the conserved AAA+-type ATPase domain, suggesting the potential modification in protein function (Fig. 4a, Supplemental Fig. S4).

      Figure 4. 

      Identification of CsKTN1 gene. (a) Diagram of the gene structure of Csa7G435510. A single-nucleotide mutation occurs in the fifth exon of Csa7G435510 resulted in S (Serine) to F (Phenylalanine) substitution in mp mutant. (b) A neighbor-joining tree for cucumber Katanin p60 protein and its homologs with other selected plant species, constructed by MEGA 7. The numbers at the branch points represent bootstrap values (%) of 1000 replications. (c) Relative expression of CsKTN1 in different tissues of mp and CCMC plants measured by qRT-PCR. Values are mean±SD. ** P < 0.01 (Student's t-test). (d) Subcellular localization of CsKTN1 protein (CCMC and mp mutant) in tobacco epidermal cell. GFP signal was observed by confocal fluorescence microscopy. GFP, Bright field and Merged represent the images observed under different light fields. Free-GFP represents the free GFP plasmid p35S::GFP, while CsKTN1F(mp)-GFP and CsKTN1S(CCMC)-GFP represent two KNT1-GFP protein.

      To understand the structural and functional relationship among Katanin p60 homologs, we performed protein alignment and constructed a phylogenetic tree for CsKTN1 with 9 Katanin p60 protein sequences from other crops (Supplemental Fig. S4). The alignment showed that AAA+-type ATPase domain is highly conserved across dicot and monocot plants. The similarity of CsKTN1 to other Katanin p60 protein varied from 78% (Arabidopsis homolog) to 93.99% (Melon homolog). The phylogenetic tree showed that CsKTN1 was clustered together with Katanin p60 protein from dicot species and far away from monocot crops (Fig. 4b).

      Furthermore, we investigated the gene expression patterns of CsKTN1 in various organs of mp and CCMC, including root, stem, leaf, male flower, ovary and tendril. The result showed that the expression level of CsKTN1 was the highest in stem, followed by root, leaf, flower and tendril, and the lowest in ovary of CCMC. The expression level of CsKTN1 gene was the highest in root, followed by tendril, leaf, stem and flower, and the lowest in ovary of mp mutant. Except for the tendril, the expression of CsKTN1 was significantly lower in all organs of mp mutant compared to the CCMC (Fig. 4c). To characterize the gene function, we examined the subcellular localizations of the protein encoded by two CsKTN1 alleles (CCMC and mp mutant) in tobacco leaf epidermal cells. As a result, both CsKTN1-GFP fusion proteins (CsKTN1S(CCMC)-GFP and CsKTN1F(mp)-GFP) were localized in cell membrane, cytoplasm, and nucleus, similar to free GFP protein (Fig. 4d).

      The mutated CsKTN1 protein lost part of its ability to bind and shear microtubule in vitro

    • Given that Katanin p60 protein is required for microtubule-shearing in cell[17], the molecular basis for CsKTN1 in regulation of microtubule morphology was investigated in vitro. We applied the coprecipitation assay for two CsKTN1-His fusion proteins (CCMC and mp) to determine whether CsKTN1 could bind to microtubule. As shown in Fig. 5a, in the absence of microtubules, none of the CsKTN1-His proteins was sedimented and retained in the supernatant. After incubation with microtubules, the amount of CsKTN1S (CCMC)-His in the pellets was found more than CsKTN1F (mp mutant)-His, indicating that CsKTN1S (CCMC)-His has a stronger ability to bind and coprecipitate with microtubules (Fig. 5a).

      Figure 5. 

      Microtubule binding and shearing capacity of CsKTN1. (a) Binding of CsKTN1 to microtubules in vitro. The proteins of supernatants (S) and the pellets (P) were analyzed on a coomassie-blue-stained polyacrylamide gel. C: CsKTN1S (CCMC)-His, M: CsKTN1F (mp mutant)-His, +: present, −: absent. (b) Microtubule-shearing activity of CsKTN1 in vitro. The shearing situation of microtubules was observed by fluorescence microscopy. Bar = 10 μm.

      To investigate whether the microtubule-shearing function of CsKTN1 was affected by amino substitution in mp mutant, we examined the shearing activity of CsKTN1S-His and CsKTN1F-His proteins. The taxol-stabilized and rhodamine-labeled microtubules were observed under fluorescence microscopy after perfusion with two recombinant proteins, CsKTN1S-His and CsKTN1F-His. As a result, CsKTN1S-His mediated complete microtubule shearing, while CsKTN1F-His mediated less rate of microtubule shearing in vitro (Fig. 5b). These results suggested that the amino acid substitution in the conserved domain of CsKTN1 gene may affect the microtubules binding and shearing ability of target protein.

      DISCUSSION

    • Plant architecture is a key factor affecting photosynthetic efficiency and cultivation pattern of crops. Optimal plant architecture enables plant to have a superior form for light capture, which could boost planting density and increase the light energy utilization during the growth and development phase, hence improving the economic output[4,36]. Miniature or semi-dwarf wheat and rice varieties have been developed and cultivated worldwide, initiating a 'green revolution' in crop breeding and production[2,5]. In this study, a novel cucumber plant architecture mutant was identified from an EMS-induced mutagenesis population, which was regulated by Katanin p60 protein. The mutant with homologs mp locus exhibited extremely miniaturized phenotypes in various organs (Fig. 1). It also possesses both female sterile and male fertile characteristics, and the resultant offspring following the cross with normal cucumber plants in producing heterozygous mp locus showed potentially useful downsizing phenotypes (Fig. 1). Thus, it could be used not only as a good resource for cucumber hybrid production, but also as a good variety resource for vine length reduction, which might provide impetus for a new direction of breeding varieties with useful downsizing plant architecture to obtain better economic benefits.

      Using BSA-seq and map-based cloning methods, we identifiedand cloned the causal gene (Csa7G435510) underlying cucumber micro-plant mutant (Fig. 3). Csa7G435510 encodes a Katanin p60 protein that contains an AAA+-type ATPase domain which was highly conserved among homologous proteins from other species. Thus, it was designated as CsKTN1 (Fig. 4a, 4b). A single nucleotide substitution (C1058T) in the coding region of CsKTN1 leading to an amino acid substitution (S353F) was responsible for the plant miniatures (Fig. 4a, Supplemental Fig. S2). The mutation caused the down-regulation of CsKTN in various tissues in the mutant except for the tendril (Fig. 4c). This may be due to the feedback effect of protein inactivation on gene transcription. This phenomenon of mutation in the coding sequence of gene rather than promoter region causing changes in gene expression has been reported in several mutants of cucumber[9−10, 14].

      CsKTN1 gene has been reported to control cucumber fruit length. A non-synonymous mutation of CsKTN1 resulted in short fruit3 (sf3) mutant[37]. The mutation sites of CsKTN1 gene in sf3 mutant and mp mutant are different, but the phenotypes of these two mutants are similar, such as shorter fruit, which confirmed the accuracy of CsKTN1 as a candidate gene for mp mutant. The ability of Katanin p60 protein to regulate plant morphology has great potential in the cultivation of diverse plant architecture varieties of cucumber.

      Plant architectural modifications could be caused by the Katanin p60 mutation in other plants. In Arabidopsis, the Katanin p60 mutations, fra2, Bot1 and lue1, showed the micro-plant phenotype[23,24,26]. In rice, the dg1 mutant, a Katanin p60 mutation, exhibited dwarf, reduced organ size and short root phenotype[27]. In cotton, the Katanin-silenced plant showed dwarf phenotype with shorter internodes, and produced dark green and smaller leaf blades with shorter petioles[38]. Phylogenetic analysis showed that Katanin p60 proteins of cucumber had highly conserved domain and close evolutionary relationship with homolog proteins in other plants, implying that Katanin p60 homologs may share a regulatory function. However, phenotypic differences in ktn1 mutations still exist in different species. For example, in Arabidopsis and rice, ktn1 decreased fertility and produced less fertile seeds compared to wild type[26,27], while in cucumber, it resulted in female sterility with no active seeds. In addition, F1 hybrids from the crosses of ktn1 and wild type in cucumber and rice showed intermediate phenotypes (Fig. 1), indicating that mutant trait was regulated by an incomplete dominant gene[27], while in Arabidopsis, the trait was regulated by a recessive gene[24]. These discrepancies could be caused by species differences or the different mutation sites in KTN1 gene.

      As the cytoskeleton, microtubules participate in a series of important life activities in cells, such as maintenance of cell structure, intracellular material transport, and mitosis[15]. The microtubules are in a state of controllable instability and dynamics, and are precisely regulated by the microtubule-associated protein (MAP) to maintain normal physiological functions of cells[39]. Several MAPs have been identified in plants and animals that are involved in promoting microtubule stabilization or destabilization[40]. The ability of Katanin p60 protein to bind and shear microtubules in vitro has also been found in Arabidopsis[16]. We confirmed that the CsKTN1 protein is a MAP by co-precipitation and microtubule shearing experiments (Fig. 5). In addition, the ability of CsKTN1 mutant protein to bind and shear microtubules decreased, indicating that the mutated amino acid has a negative impact on the function of CsKTN1 (Fig. 5). We speculate that the change of dynamic state of microtubules was the direct reason for the change of cell structure and eventually led to the change of plant architecture of the mp mutant.

      In conclusion, we propose that a mutation in the CsKTN1 gene encoding Katanin p60 protein caused the changes in cucumber plant architecture. The results obtained in this paper will be useful for elucidating the mechanisms of Katanin p60 protein regulating cucumber plant architecture.

      Acknowledgments

      • This research was supported by the National Natural Science Foundation of China (31772318), the Fund for Independent Innovation of Agricultural Science and Technology of Jiangsu Province [CX(20)2019], the Key Research and Development Program (BE2021357 and 2021YFD1200201-04), Jiangsu Belt and Road innovation cooperation project (BZ2019012), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. And we thank Obel Hesbon and Marzieh Davoudi for polishing the language of the manuscript.

      Conflict of interest

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

      Supplementary information
      • Supplemental Table S1 Sequences of primers used in the study.
      • Supplemental Table S2 Photosynthetic and fluorescence parameters of mp mutant and wild type.
      • Supplemental Table S3 The whole genome sequencing reports of mp and CCMC pools.
      • Supplemental Table S4 Gene annotation in candidate intervals.
      • Supplemental Table S5 Annotation of mutation sites in candidate intervals.
      • Supplemental Fig. S1 Phenotypic data of mp mutant and CCMC. Values are mean±SD. **P<0.01(Student′s t-test).
      • Supplemental Fig. S2 Stereoscope images of ovaries of mutant and CCMC by paraffin section. Micrographs of transverse section of ovaries of mp mutant (a) and CCMC (b).
      • Supplemental Fig. S3 cDNA sequence alignment of Csa7G435510 originated from 3 cucumbers as follow: 9930,CCMC and mp.
      • Supplemental Fig. S4 Sequence alignment of Katanin p60 homologs. Green line, the AAA+type ATPase domain; red rectangle, amino acid substitution in CsKTN1 protein of mp mutant.
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      • 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/.
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    Cite this article
    Song M, Fu W, Wang Y, Cheng F, Zhang M, et al. 2022. A mutation in CsKTN1 for the Katanin p60 protein results in miniature plant in cucumber, Cucumis sativus L.. Vegetable Research 2:3 doi: 10.48130/VR-2022-0003
    Song M, Fu W, Wang Y, Cheng F, Zhang M, et al. 2022. A mutation in CsKTN1 for the Katanin p60 protein results in miniature plant in cucumber, Cucumis sativus L.. Vegetable Research 2:3 doi: 10.48130/VR-2022-0003
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  • Html

  • Introduction
  • Materials and methods

  • Plant materials

  • Scanning electron microscopy

  • Paraffin sectioning

  • Measurement of photosynthetic parameters

  • Genetic mapping of mp candidate gene

  • Sequence analysis, annotation, and identification of the mp candidate gene

  • Subcellular localization of csktn1

  • Quantitative real-time pcr (qrt-pcr) analysis

  • Protein expression and purification

  • Microtubule binding assay

  • Microtubule shearing and observation

  • Results

  • Changes in cell morphology and size as determinants of the micro-plant mutant phenotype

  • Genetic mapping placed the mp locus to a 130.9 kb interval

  • A gene that encodes the katanin p60 subunit protein was identified for mp locus

  • The mutated csktn1 protein lost part of its ability to bind and shear microtubule in vitro

  • Discussion

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