Glucomannan in Dendrobium catenatum: Bioactivities, Biosynthesis and Perspective

Dendrobium catenatum is a classical and precious dual-use plant for both medicine and food in China. It was first recorded in Shen Nong’s Herbal Classic, and has the traditional functions of nourishing yin, antipyresis, tonifying the stomach, and promoting fluid production. The stem is its medicinal part and is rich in active polysaccharide glucomannan. As an excellent dietary fiber, glucomannan has been experimentally confirmed to be involved in anti-cancer, enhancing immunity, lowering blood sugar and blood lipids, etc. Here, the status quo of the D. catenatum industry, the structure, bioactivities, biosynthesis pathway and key genes of glucomannan are systematically described to provide a crucial foundation and theoretical basis for understanding the value of D. catenatum and the potential application of glucomannan in crop biofortification.


Introduction
Dendrobium catenatum (also named D. officinale) is a rare and precious Traditional Chinese Medicinal (TCM) plant, and it was first recorded in the earliest works of Chinese Medicine, Shen Nong's Herbal Classic, written in the Eastern Han Dynasty nearly 2300 years ago. The main medicinal part of D. catenatum is the stem, which has the effects of nourishing yin, antipyresis, tonifying the stomach, and promoting fluid [1]. Wild D. catenatum is on the verge of extinction, and it was listed as a third-class protected species in the

Feature and Structure of Glucomannan
Unlike other soluble fibers, glucomannan is characterized by high viscosity [11]. As a hemicellulose polysaccharide, glucomannan is ubiquitous in the plant cell wall. Moreover, glucomannan exists as energy storage substance in Araceae, Liliaceae, and Iridaceae and Orchidaceae [12]. It was reported that glucomannan contributes to plant tolerance to the lack of water as a compatible solute and the succulence in Aloe vera [13]. Storage glucomannans in distinct species display different structures. The mannose:glucose (Man:Glc)ratio is 1.6:1.0 in glucomannan from Amorphophallus konjac and 3.0:1.0 in glucomannan from Orchis mascula [14,15]. The Man:Glc ratios of three glucomannans (ASP-4N, ASP-6N and ASP-8N) in Aloe leaves are 19.13:1, 8.97:1, and 2.96:1, respectively [16]. In Hidden hunger refers to malnutrition due to nutritional imbalance in the human body, even having taken in enough energy. More than two billion people worldwide have suffered from hidden hunger, which is highly correlated with the status that three staple cereals (rice, maize, and wheat) provide 60% of the world's food intake [9]. Recently, rediscovery and utilization of a nutrient-rich variety of "forgotten crops" in Asia offers a viable and promising approach to eliminate hidden hunger [10]. Glucomannan belongs to soluble hemicellulose and is an excellent source of soluble dietary fiber as the seventh essential nutrient in the human body. Therefore, incorporating D. catenatum as a rich source of glucomannan into the daily diet contributes to promoting nutritional diversification and alleviating hidden hunger.

Feature and Structure of Glucomannan
Unlike other soluble fibers, glucomannan is characterized by high viscosity [11]. As a hemicellulose polysaccharide, glucomannan is ubiquitous in the plant cell wall. Moreover, glucomannan exists as energy storage substance in Araceae, Liliaceae, and Iridaceae and Orchidaceae [12]. It was reported that glucomannan contributes to plant tolerance to the lack of water as a compatible solute and the succulence in Aloe vera [13]. Storage glucomannans in distinct species display different structures. The mannose:glucose (Man:Glc)ratio is 1.6:1.0 in glucomannan from Amorphophallus konjac and 3.0:1.0 in glucomannan from Orchis mascula [14,15]. The Man:Glc ratios of three glucomannans (ASP-4N, ASP-6N and ASP-8N) in Aloe leaves are 19.13:1, 8.97:1, and 2.96:1, respectively [16]. In addition, glucomannan can also be obtained from microorganisms, such as the cell walls of bacteria, yeast, or fungus [17,18]. Natural glucomannan is composed of D-glucose and D-mannose linked by a β-1,4-glucopyranoside bond to form polymer heteropolysaccharides in a certain molar ratio [19]. Moreover, the C3 position of D-mannose on the main chain can be linked to polysaccharides in the form of β-1,3-glycosidic bond or β-1,6-glycosidic bond [20].

Intestinal Health Improvement
It has been reported that glucomannan can modulate mouse cecal and fecal microbiota with favorable prebiotic effects [38]. The unique properties of glucomannan hydrolysate make it valuable as a prebiotic in a wide range of food, feed, and pharmaceutical products [39]. Prebiotics promote specific changes in the gastrointestinal microbiota [40], promote the growth of probiotics such as Lactobacillus and Bifidobacterium, inhibit the proliferation of harmful bacteria, reduce inflammation, improve the integrity of the intestinal mucosa, promote nutrient absorption [41], and control the blood glucose level of patients with type 2 diabetes [42]. Glucomannan selectively stimulates the production of beneficial gut microflora such as probiotics and benefits the treatment of functional gastrointestinal disorders related to abdominal pain in mice [43][44][45]. The combined laxative of glucomannan-probiotic promotes defecation in constipated rats [46]. Mannoseoligosaccharides, the oxidative degradation product of glucomannan, are potential prebiotics, which can affect the growth and species abundance of fecal microbiota and regulate the balance of intestinal flora [47,48]. For instance, D. catenatum polysaccharide DOP can restore the diversity of intestinal flora and regulate the abundance of intestinal flora by inhibiting the overexpression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), restoring the level of short-chain fatty acids (SCFAs), activating G-protein-coupled receptors (GPRs), and regulating the intestinal flora to alleviate the symptoms of colitis in mice [49].
Glucomannan also plays a direct role in protecting the intestinal epithelium. D. catenatum polysaccharide DOP is not easily digested and absorbed in the human body but is degraded into SCFAs by the gut microbiota in the large intestine, therefore improving intestinal health [22,50]. In addition, D. catenatum glucomannan can reduce intestinal epithelial injury and regulate intestinal mucosal immunity by keeping a balanced ratio of pro-and antiinflammatory cytokines and regulating the expressions of toll-like receptors (i.e., TLR-2, TLR-4, TLR-6, and TLR-9) important for recognizing pathogen-associated molecular patterns derived from various microbes in mice [51].

Immunomodulatory Activity
Glucomannan, one of the natural bioactive ingredients, can be used as an ideal immunomodulatory agent. Glucomannan can reduce brain inflammation, improve hippocampal neuron damage, maintain hippocampal cognitive function, and play an anti-epileptic role in epileptic rats [52]. D. catenatum polysaccharide DOPW3-B can improve the intestinal mucosal immune activity by increasing at Peyer's patches the levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4), two key effector cytokines for the differentiation of T helper types 1 and 2 with a positive effect on mesenteric lymph nodes [31,53]. Glucomannan can increase the expression of several cytokines that are important for immune homeostasis (e.g., TNF-α, IL-β and IL-10) [54]. Meanwhile, glucomannan plays a pivotal role in regulating the activation and proliferation of macrophages [55], and promoting phagocytosis [29]. Finally, the pretreatment of D. officinale with organic solvents enhances the immunostimulatory activity of polysaccharides and affects the mannose/glucose ratio of polysaccharides, which plays an important role in immunostimulation [56].

Anti-Cancer Activity
In a zebrafish xenograft model, D. catenatum polysaccharide DopW-1 inhibits the proliferation of HT-29 cells by the apoptosis pathway and has an anti-tumor effect on colorectal cancer [57]. It is known that hyper-activation of the phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling pathway in human cancers can promote the proliferation and survival of tumor cells [58]. Glucomannan can block the PI3K/AKT pathway, thereby promoting the apoptotic rate and reducing the proliferation ability of tumor cells [59]. In parallel, glucomannan can also inhibit the expression of major chemokine receptors, chemokine receptor 4 (CXCR4) and CC chemokine receptor 7 (CCR7), found in a wide range of tumor cells, thus reducing the dissemination ability of tumor cells [60]. In addition to directly interfering with tumor cells, glucomannan also acts in an indirect manner. The D. catenatum polysaccharide DOPa-3 significantly inhibits the formation and growth of colon tumors and alleviates colon injuries [61]. In rats, DOPA-4 effectively inhibits precancerous lesions of gastric cancer induced by 150µg/mL MNNG [62]. D. catenatum polysaccharides can reduce oxidative stress level, inhibit stress-induced activation of adenosine monophosphate-activated protein kinase (AMPK)/UNC-51 like autophagy activating kinase 1 (ULK1) pathway and the expression of light chain 3 (LC3) I and LC3 II proteins, reduce Beclin1 expression, and then reduce hypoxia/reoxygenation induced astrocyte autophagy, reduce human astrocyte apoptosis, and promote astrocyte survival [63].

Hypoglycemic Activity
Glucomannan is suitable as a dietary fiber supplement for the treatment of being overweight, hyperlipidemia, and diabetes. D. catenatum glucomannan DOP can alleviate hyperglycemia in high fat diet (HFD)/streptozocin (STZ)-induced diabetic mice through promoting the synthesis of liver glycogen and inhibiting the degradation of liver glycogen [64]. DOP treatment can reduce the level of bile acid in diabetic rats, reduce the binding of bile acid to the nuclear receptor FXR or the membrane receptor TGR5, increase the level of glucagon-like peptide-1 (GLP-1), and improve glucose and lipid metabolism and insulin sensitivity [65]. DOP promotes glycogen synthesis by regulating the expression of glycogen synthase kinase 3β (GSK-3β) and glycogen synthase (GS) in the liver or glucose transporter 4 (GLUT4) in muscle. Glucose levels are reduced by regulating the activity of glucose metabolism enzymes in the liver, including pyruvate kinase (PK), hexokinase (HK), and phosphoenolpyruvate carboxykinase (PEPCK) [66]. On the other hand, DOP can delay diabetic cataract by decreasing the level of serum malondialdehyde (MDA), increasing the activity of superoxide dismutase (SOD) and enhancing its antioxidant capacity [49]. Glucomannan AABP-2B, isolated and purified from Anemarrhena asphodeloides, demonstrates its hypoglycemic effect by inhibiting α-glucosidase activity and activating irS-1/PI3K/Akt signaling pathway in insulin-resistant cells [67]. Effects of glucomannan on insulin sensitivity contribute to weight loss, and taking as little as 4 g of glucomannan per day can promote weight loss [68].

Cosmetics
D. catenatum extracts are used as cosmetic raw materials to effectively solve the skin problems caused by yin deficiency and fire hyperactivity due to their rich polysaccharides, flavonoids, and other nutrients that are absorbed through the skin and have good moisturizing, anti-aging, and anti-wrinkle effects [69]. The aqueous extract of D. catenatum can resist drying damage to epidermal cells, increase cell vitality, and improve skin moisture content [70]. D. catenatum polysaccharide DSP has strong antioxidant activity, can scavenge DPPH free radicals, and has a better inhibitory effect on lipid peroxidation and oxidative damage of red blood cells [71,72]. Macromolecular polysaccharide as the main moisturizing component in D. catenatum is a kind of polyhydroxyl polymer, whose polar groups can form hydrogen bonds with water molecules to bind water. Meanwhile, polysaccharide can form a uniform film on the skin surface to prevent water loss [73]. Dendrobium huoshanense polysaccharide has certain hygroscopic and moisturizing properties with a higher moisture retention rate than glycerin, is non-irritating to skin, and serves as a natural moisturizing agent [74]. So far, many related skin care products are popular in the market, such as moistening and skin brightening masks with D. catenatum (SENYU, Jinhua, China), Dendrobium emulsion (MISS QUEEN, Ningbo, China), and D. catenatum skin corset firming lotion (BOTANIERA, Hangzhou, China).

Food and Functional Food
Glucomannan has wide application prospects in the food industry and can be used as a food additive, meal substitute food, and health care products, such as Tiepifengdou capsules, oral liquid, decoction pieces, yogurt [75], teabags [76,77], beverages, and noodles [78], and has been applied to a number of patents [79]. Due to its large molecular weight and strong water binding ability, glucomannan has excellent hydrophilicity, gelation, emulsification, film formation, thickening, and other unique functional properties [80]. With alkali treatment, the acetyl group in glucomannan is removed to promote the formation of intramolecular and intermolecular hydrogen bonds and get a gel with excellent stability [81]. Glucomannan is a licensed food additive, which is used as a stabilizing, thickening, and gellating agent [82]. For example, in yogurt and other drinks, glucomannan can be used as a thickening agent to increase flavor and nutrition [82]. In sausages, hams, and other meat products, glucomannan can replace fat, reduce fat content, increase viscosity, and water retention, to improve the texture and flavor of meat [83]. In starch products, the addition of glucomannan affects the paste characteristics, rheological properties, and texture of the starch system [84]. In addition, glucomannan can be used for food preservation, such as glucomannan film, which has good stability and food applicability and can be used for fruit and vegetable coating preservation and flavor microcapsule production [85].

Glucomannan Biosynthesis Pathway in Plant
In the biosynthesis pathway of glucomannan (Figure 4), the photosynthates of leaves are transported to the D. catenatum stem in the form of sucrose, which is then decomposed into glucose (Glc), UDP glucose (UDP-Glc), and fructose (Fru) under the action of sucrose synthase (SUS) and invertase (INV), and then, under the action of hexokinase (HXK) and fructokinase (FRK), Glc-6-P is further catalyzed by phosphoglucomutase (PGM) to produce glucose-1-phosphate (Glc-1-P). At the same time, Fru-6-P is catalyzed by phosphate mannose isomerase (PMI) to produce mannose-6-phosphate (Man-6-P), which is converted to mannose-1-phosphate (Man-1-P) by phosphomannomutase (PMM). GDP mannose pyrophosphorylase (GMP) catalyzes the production of GDP mannose (GDP-Man). Glc-1-P is converted to GDP-glucose (GDP-Glc) by GDP-Glc pyrophosphorylase (GGP) and Glc-1-P is also converted to ADP-glucose (ADP-Glc) by ADP-Glc pyrophosphorylase (AGP). Subsequently, GDP-Man, GDP-Glu, or ADP-Glu are each transported into the Golgi apparatus by specific transporters, and they are used as substrates of cellulose-like synthases A/D (CSLA/D) to synthesize glucomannan. Additionally, GDP-Man is also used for the synthesis of vitamin C/ascorbic acid (AsA) [86]. Glucomannan synthesized in the Golgi matrix might have two roles. On the one hand, it is localized in the cell wall through vesicle transport and functions as a structural polysaccharide [87]. On the other hand, glucomannan also acts as a storage polysaccharide in some plants, such as D. catenatum, Konjac, and A. vera. In Konjac, glucomannan was discovered to accumulate in the egg-shaped idioblast within the parenchyma [12].

Research Progresses of Key Glucomannan Biosynthetic Genes in Plant
The pathway of glucomannan synthesis involves some specific key enzymes: phosphate mannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP), and cellulose-like synthase A/D (CSLA/D). PMI, PMM, and GMP can provide precursors for the synthesis of GDP-Man, which serves as not only the glycosyl donor and metabolic intermediate widely existing in various organisms but also the substrate of the biosynthesis of glucomannan and ascorbic acid (AsA) [86]. In D. catenatum, the expression levels of DcPMI, DcPMM, DcGMP2, and DcCSLA4/8/13 in the stem are significantly higher than those in other tissues ( Figure 5), indicating their important roles for glucomannan accumulation in the D. catenatum stem. phosphorylase (GMP), and cellulose-like synthase A/D (CSLA/D). PMI, PMM, and GMP can provide precursors for the synthesis of GDP-Man, which serves as not only the glycosyl donor and metabolic intermediate widely existing in various organisms but also the substrate of the biosynthesis of glucomannan and ascorbic acid (AsA) [86]. In D. catenatum, the expression levels of DcPMI, DcPMM, DcGMP2, and DcCSLA4/8/13 in the stem are significantly higher than those in other tissues ( Figure 5), indicating their important roles for glucomannan accumulation in the D. catenatum stem.  The raw RNA-seq reads of different tissues and organs in D. catenatum are derived from the NCBI database (www.ncbi.nlm.nih.gov, accessed on 16 September 2022), including the leaf (SRR4431601), the root (SRR5722140), the green root tip (SRR4431599), the white part of the root (SRR4431598), the stem (SRR4431600), the flower bud (SRR4431603), the sepal (SRR4431597), the labellum (SRR4431602), the pollinia (SRR5722145), and the gynostemium (SRR4431596). A heatmap has been generated via TBtools software [89]. The color scale represents log2 of FPKM expression values; green and red indicate a low and high level of gene expression, respectively.

Phosphate Mannose Isomerase (PMI): Fru-6-P ↔ Man-6-P
Phosphate mannose isomerase (PMI) catalyzes the reversible conversion between Fru-6-P to Man-6-P in eukaryotes and prokaryotes and is a key enzyme during GDP-Man production [90], required for the first step of the mannose/L-galactose pathway during AsA biosynthesis in plants [91]. In Arabidopsis, there are two PMI1 isozymes, but PMI1, rather than PMI2, is involved in AsA biosynthesis. PMI1 has constitutive expression in both vegetative and reproductive organs under normal growth conditions, whereas PMI2 has no expression in any organs under light. Continuous light can induce PMI1 expression and an increased AsA level in leaves, whereas long-term darkness can induce PMI2 expression and decrease the AsA level. The diurnal expression pattern of PMI1 is in parallel with the total PMI activity and the AsA content in leaves. Moreover, knockdown of PMI1 results in a substantial decrease in the total AsA content of leaves, whereas knockout of PMI2 does not affect the total AsA levels in leaves [92]. In addition, AsA plays an essential role in scavenging reactive oxygen species (ROS) produced during cell metabolism and stress, and increase in the AsA level contributes to an improvement in abiotic stress tolerance [93]. Overexpression of BcPMI2 from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) improves AsA content and tolerance to oxidative and salt stress under NaCl or H 2 O 2 treatment, although transgenic tobacco has lower contents of AsA and soluble sugar than WT under normal conditions [94].
To date, PMI-like genes are mainly used in the mannose selection-based plant transformation system (plant PMI/Man system), which is non-antibiotic and environmentally friendly [95]. Most plant cells are sensitive to mannose and fail to utilize mannose as a sole carbon source because mannose can be taken up and phosphorylated to Man-6-P by endogenous hexokinase, but Man-6-P is not further utilized in the absence of enough PMI activity, subsequently leading to the accumulation of Man-6-P, inhibition of phosphoglucose isomerase, glycolysis blocking, and cell growth arrest [96]. However, plant cells with high PMI activity through genetic modification can metabolize Man-6-P and enter into glycolysis for ATP production and normal growth [97]. As a new safe positive selectable marker gene, PMI has been successfully used in the genetic selection of many plants, such as rice, maize, wheat, sorghum, sugarcane, onion, tomato, potato, cassava, grape, apple, cucumber, sugar beet, papaya, Arabidopsis, cabbage, and so on [98][99][100]. The Escherichia coli PMI (EcPMI) gene was first used in the plant PMI/Man system, which is the most popular one [101]. In 2009, the first genetically modified maize MIR604 via the EcPMI/Man selection system was approved for food and feed use, import, and processing in the European Union (European Food Safety Authority, 2009). Saccharomyces cerevisiae PMI (ScPMI) can also be used as a selectable marker in rice transformation [98]. However, non-plant type PMIs can still raise public safety concerns, and plant type PMIs may offer a superior alternative [102]. Hu et al. first proved that plant PMI genes from Chlorella variabilis and Oryza sativa can also be used as selectable markers to obtain transgenic plants exhibiting an accumulation of PMI transcripts and enhancement of PMI activity [100]. Through the selection system based on the green microalga Chlorococcum sp. PMI (ChlPMI), a polycistronic gene cluster containing crtB, HpBHY, CrBKT, and SlLCYB is transformed into tomato, resulting in the production of high astaxanthin content [103].

Phosphomannomutase (PMM): Man-6-P ↔ Man-1-P
Phosphomannomutase (PMM) catalyzes the interconversion between Man-6-P and Man-1-P and provides precursors for the synthesis of GDP-Man. GDP-Man is not only used for the synthesis of glucomannan but is also an indispensable intermediate in the AsA biosynthesis pathway. The PMM enzyme isolated from the cinnamon seed has the activity of converting D-glucose produced by photosynthesis or glycolysis into a mannose component of plant storage polysaccharide [104]. At present, there is much research on the catalytic role of PMM in the AsA synthesis pathway. In tobacco, decreased expression of PMM via the VIGS technique resulted in a reduction of AsA content in leaves, while overexpression of PMM increases AsA content in leaves [105]. Transgenic tobacco plants overexpressing the acerola (Malpighia glabra) PMM gene show around a 2-fold increase in AsA content compared with WT, which correlates with the level of PMM transcripts and the corresponding enzymatic activities [106]. Overexpression of rice PMM in transgenic rice increased AsA content in seeds by 25-50% [107]. Overexpressing D. catenatum PMM gene in Arabidopsis, resulting in a significant increase in the contents of both AsA and polysaccharide [108].

GDP-Mannose Pyrophosphorylase (GMP): Man-1-P ↔ GDP-Man
GDP-mannose pyrophosphorylase (GMP) (also named Vitamin C Defective1, VTC1) catalyzes the conversion of Man-1-P and GTP to GDP-Man and pyrophosphate. GMP plays an important role in maintaining the AsA level and redox balance in plants. Compared with WT, the Arabidopsis vtc1-1 mutant accumulated only 25% of leaf AsA content, but had no effect on the AsA redox state [109]. Arabidopsis KONJAC1 (KJC1) and KJC2 interact with VTC1 to stimulate GMP activity to affect the accumulation of AsA and glucomannan, and VTC1 mutants cause severe dwarfism [110]. The acerola plant has very high AsA levels, consistent with that of the M. glabra MgGMP gene promoter, which has higher activity than 35S and Arabidopsis GMP promoters. Transgenic tobacco plants containing the MgGMP gene and its original promoter, which displayed about 2-fold increased levels of AsA [111]. Transgenic tomatoes overexpressing a yeast-derived GMP exhibit up to a 31 and 17-fold increase in GMP activity in leaves and green fruit, respectively. The AsA levels increase by up to 70% in leaves, 50% in green fruit, and 35% in red fruit, especially in photosynthesizing organs [112]. The overexpression of tomato SlGMP3 increases total AsA levels and tolerance to oxidative stress in tomatoes and high or low temperature stress in tobacco, whereas knockdown of SlGMP3 in tomatoes leads to substantially decreased AsA content and a defective phenotype with lesions and senescence due to failing to instantly detoxify ROS [113,114]. Overexpression of tomato GMP in potatoes can improve the content of AsA and dehydroascorbate (DHA) under low temperature stress and enhance the cold tolerance of potatoes [115]. Transgenic tobacco expressing Pogonatherum paniceum GMP has a high germination rate and high AsA content under drought and salt stress, and has strong salt tolerance and drought resistance [116]. Overexpression of D. catenatum GMP in Arabidopsis results in increased mannose content in water-soluble polysaccharides and enhances salt stress tolerance [117], consistent with salt hypersensitivity in the Arabidopsis vtc-1 mutant [118].
In addition, the GMPase activity level can regulate the sensitivity of Arabidopsis to ammonium [119]. The vtc1-1 mutant exhibits stunted root growth with an elevated of NH 4 + efflux at the elongation zone and inhibition of cell elongation in the presence of NH 4 + [120], but this NH 4 + -hypersensitive phenotype in mutant is independent of AsAdeficiency. In fact, the GMP gene disruption can also cause N-glycosylation disorder of proteins associated with the destruction of hormone homeostasis and the increase of nitric oxide content under high NH 4 + conditions, consequently leading to conditional sensitivity to ammonium ions [121]. However, impaired GDP-mannose biosynthesis and defective N-glycosylation are required for but are not the primary causes of conditional NH 4 + sensitivity in vtc1-1, whereas pH alterations in the presence of NH 4 + associated with lost N for assimilation and alkalinization of the cytosol account for the drastic root growth defect in WT and vtc1-1 [122].

Cellulose-like Synthase A/D (CSLA/D): GDP-Man + UDP/GDP-Glu → Glucomannan
The cellulose synthase gene superfamily consists of the CesA family and 10 Csl families: CslA~CslH, CslJ, and CslM [123,124]. The 30 Arabidopsis CSL proteins is clustered into six families: CslA, CslB, CslC, CslD, CslE, and CslG [125], and the 33 rice CSL proteins are also divided into six families: CslA, CslC, CslD, CslE, CslF, and CslH [126], but only the CslA, CslC, CslD, and CslE families are shared by both. CSL proteins are usually located in the Golgi apparatus, which mediates the synthesis of hemicellulose and then transports it to the cell wall [127]. The CSLA/D families are mainly responsible for the skeleton synthesis of mannan and glucomannan [128,129]. So far, nine CSLA and six CSLD genes have been identified in Arabidopsis, while 10 CSLA and four CSLD genes have been identified in rice [130,131], and 13 CSLA [132] and eight CSLD members have been identified in D. catenatum [21].
Heterologous expression assays showed CSLA proteins from a variety of species catalyze the biosynthesis of the β-1,4-mannan or glucomannan backbone in vitro, such as CtManS in guar (Cyamopsis tetragonoloba) [133], AtCSLA2/7/9 in Arabidopsis [128], OsCSLA1 in rice [134], PtCslA1 in Populus trichocarpa [135], AkCSLA3 in A. konjac [136], and TfManS in fenugreek (Trigonella foenum-graecum) [137]. In Arabidopsis, the csla9 mutant exhibits significantly decreased glucomannan, and the csla2 csla3 csla9 triple mutant is absent of detectable glucomannan in inflorescence stems, although these mutants have no alteration in stem development or strength [138]. The clsa7 mutant is embryo lethal due to defective embryogenesis with evidently delayed development, abnormal cell patterning, and an arrested globular stage, associated with reduced nuclei proliferation and failed cellularization in the endosperm [139]. Overexpression of AtCSLA2, AtCSLA7, and AtCSLA9 leads to elevated glucomannan content in stems; AtCSLA9 overexpression can rescue the embryo lethality of csla7, indicating their functional redundancy [138]. Therefore, AtCSLA2, AtCSLA3, and AtCSLA9 are necessary for glucomannan synthesis in the stem, while AtCSLA7 is necessary for glucomannan synthesis in the embryo [138]. Heterologous expression of the O-fucosyltransferase family member AtMSR1 can enhance the glucomannan synthesis capability of AtCSLA2 and AkCSLA3, possibly via affecting enzymatic activity by protein glycosylation [140,141]. The mannosyl level in stem glucomannans is decreased by around 40% in Arabidopsis msr1 single mutant and by more than 50% in msr1 msr2 double mutant [142]. Transcription factors ANAC041, bZIP1, and MYB46 can directly bind the promoter of AtCLSA9 and regulate its expression [143]. Furthermore, overexpression of MYB46 leads to a significant increase in mannan content. In D. catenatum, overexpression of DoCSLA6 in Arabidopsis promotes mannan biosynthesis [144].
CSLD family members are required for the growth of stem and root, tip growth of root hair and pollen tube, and female gametophyte development and fertility. In Arabidopsis, the csld2 and csld3 mutants exhibit root hair bursts [145][146][147][148]; the csld5 mutant has reduced stem growth [149]; the csld1 and csld4 mutants are defective in male transmission and pollen tube production; the csld2 csld5, csld3, and csld5 and csld2, csld3, and csld5 mutants display dwarfism and severely reduced viability [129]; and csld2 and csld3 show synergid cell degeneration during megagametogenesis and reduced pollen tube penetration during fertilization [150]. In rice, the oscsld4 (nd1) mutant is dwarfed and its culm possesses a decreased content of xylan and cellulose but an increased amount of homogalacturonan, whereas disruption of the Arabidopsis AtCSLD5 gene results in decreased xylan and homogalacturonan synthase activities in Arabidopsis [151]. It seems that CSLD proteins are not only limited in (gluco)mannan biosynthesis. At first, researchers tended to believe that CSLD proteins have major functions in mannan synthesis because microsomes isolated from tobacco (Nicotiana benthamiana) leaves heterologously expressing AtCSLD5 or co-expressing AtCSLD2/3 have elevated mannan synthase activity, specifically using GDP-Man as an activated nucleotide-sugar donor [152]. However, genetic rescue assays with CSLD-CESA chimeric proteins and in vitro biochemical reconstitution suggested that CSLD3 prefers to function as a UDP-Glu-dependent β-1,4-glucan synthase and forms a protein complex displaying a similar ultrastructural feature to the CESA6-forming complex [153,154]. Overexpression of cotton GhCSLD3 in Arabidopsis enhances primary cell wall synthesis and restores the defects of the atceas6 mutant, including significantly reduced cellulose content, a defected cell wall, and a lower dry mass, indicating that GhCSLD3 and AtCESA6 may play a similar role in cellulose or cellulose-like polysaccharide synthesis [155]. Furthermore, the CSLD family is the most similar of the CSL gene families to the CESA family at the amino acid level [125] and, together with the CSLF family (accounting for mixed-linkage glucan synthesis), displays the closest relationship with the CESA family at the phylogeny level [156]. Therefore, the CSLD family might function as both a (gluco)mannan synthase and a β-1,4-glucan synthase.

Summary and Perspective
Food production concerns a new hot topic from meeting the need of "eating fully" to "eating healthily" [7,157]. In modern life, dietary structure and habits have changed, and the source of staple food is mostly limited to several starch crops, including rice, wheat, maize, and potatoes, leading to nutrition imbalance and hidden hunger [158]. Furthermore, severe malnutrition will hinder human growth and development, accompanied by mental disorders and diseases [159]. More than 7% of chronic diseases in contemporary society are caused by hidden hunger. The FAO proposed Zero Hunger as the 2nd Sustainable Development Goal to eliminate all forms of hunger by 2030, and biodiversity for food and agriculture is indispensable to achieve the Zero Hunger program [160]. Dietary diversity and a higher intake of dietary fiber can reduce the risk of chronic diseases such as diabetes, obesity, and cardiovascular disease. Intriguingly, D. catenatum as a traditional dual-purpose plant for both food and medicine in China, is able to supple glucomannan as an excellent dietary fiber and has great potential to fight hidden hunger.
The efficiency of D. catenatum is largely dependent on the active polysaccharide glucomannan, which has multiple medicinal effects such as anti-tumor, lowering blood lipids, and preventing diabetes. To date, most of the studies on the glucomannan biosynthetic pathway have focused on its role as a cell wall structural component. PMI, PMM, and GMP sequentially catalyze the production of GDP-mannose, thus providing precursors for the synthesis of glucomannan, and CSLA/D is required for the synthesis of the glucomannan skeleton. However, little is known about the subcellular localization, transportation, and regulation of storage glucomannan, which may be clarified by the comprehensive application of newly developed technologies including immunolocalization, high throughput omics, and gene editing, consequently contributing to creating a glucomannan biofortified crop through synthetic biology ( Figure 6). For instance, a stack of four identified committed genes, including manC and manB from Escherichia coli BL21(DE3), manA and pgi from Bacillus subtilis, are introduced into food-grade B. subtilis to obtain mannan [161]. Four anthocyanin synthetic genes, sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY, are introduced into rice endosperm to produce astaxanthin-rich rice with high antioxidant activity [162]. Therefore, it is promising that glucomannan biofortified crops in the future will contribute to reducing hidden hunger and chronic diseases and promoting human health. Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data used to support the findings of this study is available.