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Plant Growth Promoting and Abiotic Stress Tolerant Chickpea (Cicer arietinum L.) Rhizobial Isolates from Some Areas of South Wollo Zone, Ethiopia

Chickpea (Cicer arietinum L) is an important pulse crop prized for its high protein content and is grown as a food source worldwide, including Ethiopia. However, the yield of chickpea is low due to low soil fertility and the ever-changing abiotic stresses. erefore, this study aimed at isolation, characterization, and selection of chickpea rhizobia eective in their nitrogen xation and abiotic stress tolerance potential. As a result, 150 nodule samples were collected from three districts of chickpea-producing areas in South Wollo. e nodules were crushed, and the rhizobia were isolated and characterized by using standard microbiological procedures. Based on the presumptive tests conducted, 103 (68.7%) of the rhizobial isolates were tentatively categorized as Rhizobium species. Regarding phosphate solubilization, only 48 (46.7%) solubilized phosphate with a solubilization index ranging from 2.1 to 2.7mm. Twenty-four (50%) of the isolates were found to be hydrogen cyanide producers. Among the rhizobial isolates tested under greenhouse conditions, 37 (77.1%) of them induced nodulation on their host plant (chickpea). eir symbiotic eectiveness evaluation test conrmed that 16(47.1%), 6(17.6%), 26.47%, and 3(8.8%) were highly eective, eective, low eective, and ineective, respectively. Of the authenticated rhizobial isolates, 12 (35.5%) of them, includingWuCR-15, 16, 17, 18, 19, 20, 23, 30, 31, 32, 36, 38, and 48, accumulated higher shoot dry matter than the positive control. Isolates WuCR11, 17, and 36 showed resistance to low and high extreme abiotic stresses of pH, temperature, and salt. Consequently, rhizobial isolates, WuCR11, 17, and 36, which were eective and competent in all the tested parameters, were recommended as good rhizobial candidates for applications under greenhouse and eld conditions.

Introduction
Chickpea (Cicer arietinum L.), a member of the Leguminosae family, is a popular pulse crop recognized for its high protein content [1]. It is a staple basic food crop in many tropical and subtropical Afro-Asian countries, and it is one of the world's primary pulse crops, typically grown in marginal and semi-arid environments [2]. It is the third most signi cant pulse crop in the world [3]. Chickpeas were initially cultivated about 7,000 years ago in the Middle East. India is the largest producer of chickpeas worldwide, which accounts for 64% of global production [4]. Apart from India, Australia (12.35%), Myanmar (3.25%), and Ethiopia (2.92%)

Study Sites and Nodule Sample Collection.
e study sites of this research comprise three districts of South Wollo, including Kutaber, Kombolcha, and Tehulederie ( Figure 1) that were known as major chickpea growing areas with no previous history of chickpea rhizobial inoculation. Nodules that looked pink or red in color were collected randomly from the selected Chick pea grown farms in the study areas [23]. e collected nodules were placed inside vials containing a desiccant (Silica gel) and brought to Wollo University Biology laboratory at 4°C until isolation was conducted.

Isolation of Chickpea Rhizobium Species.
e dehydrated or desiccated root nodules were surface sterilized using 70% ethanol for 10 seconds, followed by a 3% (v/v) solution of sodium hypochlorite for 4 minutes after being immersed in sterile distilled water overnight using Petri-dishes with labels.
e surface-sterilized nodules were then rinsed in sterile distilled water five times to completely remove the sterilizing chemicals [24]. e nodules were then transferred into sterile Petri-dishes and crushed in a drop of normal saline solution (0.85% NaCl) inside a laminar airflow hood with an alcohol-flamed sterile glass rod [23]. Finally, 0.1 mL (loop-full) of each suspension was streaked onto a Congo red Yeast Extract Mannitol Agar (YEMA-CR) plate and incubated at 28 ± 2°C for 3-4 days. Isolates that did not absorb the congo red were selected for future purification work.

Purification of Isolates.
A single, well-separated rhizobial colony was selected and placed into 6 mL of sterilized yeast extract mannitol broth by using a sterile inoculating loop [23]. e test tubes were then vortexed and shaken for 48 hours at room temperature on a rotary shaker. After two days, a loop of culture suspensions from each test tube was taken and streaked on sterile yeast extract mannitol agar (YEMA) and cultured at 28 ± 2°C for 3-4 days. e colony's purity and consistency were rigorously checked by repeated re-streaking.

Preservation of the Isolates.
After repeated purification, a single well-isolated colony was selected and streaked into a YEMA slant containing 0.3% (w/v) CaCO 3 in a culture test tube, where it was cultured for 3-4 days at 28 ± 2°C [24]. e culture slants were moved and kept inside a refrigerator set at 40°C once sufficient growth was noticed. For future investigation, the pure cultures were kept at −20°C in an Eppendorf tube containing 20% glycerol.

Presumptive
Test of the Isolates. According to Ref. [23], the isolates' growth was measured using Peptone Glucose Agar (PGA), Gram staining, and YEMA-CR media.

Congo Red Absorption.
Congo red stock solution was prepared by dissolving 0.25 g Congo red in 100 mL sterilized distilled water. Ten (10) milliliters of Congo red was added to a liter of YEMA media and sterilized. Finally, a loop full of test isolates was streaked on the medium, covered with aluminum foil, and incubated at 28 ± 2°C for 3-4 days. e absorption of Congo red by the rhizobial isolates, which indicated contaminant growth, was recorded [24].

PEPTONE-GLUCOSE Test.
e growth of the rhizobia on peptone glucose agar was determined by using the protocol described by Ref. [25]. e media composition comprised (g/l): glucose (5), peptone (10), agar (15), bromocresol purple (BCP) (10 mL), and distilled water (1000 mL). e pH was adjusted to pH 7.0-7.1 by using 1N NaOH and HCl. e stock solution was prepared by dissolving 1 g of BCP in 100 mL of ethanol. A loop full of bacterial cells grown in yeast extract mannitol broth (YEMB) was streaked on peptone glucose medium and incubated at 28 ± 2°C for 3-4 days. After incubation, the absence of growth or appearance of poor growth indicated rhizobia.

Gram Reaction Test.
e Gram reaction of the rhizobial isolates was determined by using the KOH technique [26]. A loop full of rhizobial isolates grown for 48 h on YEMA was taken and mixed well with 3% KOH on a clean microscope slide. e presence of stringiness that can be raised to 1 cm from the slide indicated Gram-positive rhizobia, whereas the appearance of viscosity property showed Gram-negative rhizobia.

Phosphate Solubilization.
e ability of the rhizobial isolates to solubilize tri calcium phosphate (TCP) was evaluated by growing them on the Pikovskaya (PKV) agar medium [25] containing the media composition (g/l): glucose (10), yeast extract (0.5), NaCl (0.1), (NH 4 ) 2 SO 4 (0.2), 25), and agar (15). After the pH of the media was adjusted to pH 7.2, it was autoclaved to sterilize the contaminants. A loop of bacteria (about 10 6 cells per mL) was inoculated on PVK agar media and incubated at 28 ± 2°C for 3-4 days. After 3-4 days of incubation, the formation of a clear zone indicated phosphate solubilizing rhizobia. e phosphate solubilizing index (PSI) of the rhizobial isolates was calculated by (27,28,29): Where CD � colony diameter, HD� Halo zone diameter.

Acid/Base Reaction.
e ability of the isolates to acid or alkaline in YEMA media amended with bromothymol blue (BTB) (0.025 w/v) was determined (31). A loop of isolates grown for 48 h in YEMB was streaked onto the YEMA-BTB medium and cultured at 28 ± 2°C for 3-4 days. After four days of incubation, the color change of the medium was recorded.

Authentication of the Isolates and Preliminary Screening of eir Symbiotic Effectiveness in Sand
Experiment. e ability of the rhizobial isolates to infect and nodulate on the host Advances in Agriculture plant was carried out under greenhouse conditions by using the desi chickpea variety brought from Sirinka Agricultural Research Centre (23). Pots that were surface sterilized with 95% ethanol and rinsed with 1N sulfuric acid were used for conducting the experiment. Pots of sand were sterilized, of which 3 Kg was added into sterilized pots. Seeds of uniform color, size, and shape were selected. e seeds were then surface sterilized, washed with sterilized distilled water, and placed on sterilized plates containing 0.75(w/v) water agar. ey were then cultured for 3-4 days at 28 ± 2°C for germination.
ree days after germination, five germinated seeds were dipped into each pot for germination after three days. e seedlings were thinned down to three and inoculated with 1 mL of selected rhizobial grown in YEMB for 72 h. e pots were set up in a randomized design (CRD) with triplicates in a greenhouse with 12 h photoperiods and an average of 25 and 18°C day and night temperature. e sampling includes negative control without treatment and positive control without bacterial inoculation receiving 10 mL of 0.05% KNO 3 once a week. All plants were irrigated with 10 mL of N-free medium nutrients once a week (Table 1) and 100 mL of water every two days [29].

Relative Effectiveness of the Isolates.
After sixty days of cultivation, the plants were uprooted, and vegetative growth data, including nodule number, nodule dry weight, number of branches, shoot length and shoot dry weight, were recorded. e relative symbiotic effectiveness of the isolates in accumulating the shoot dry weight was determined [31] as follows: %SE � Inoculated plant DM × 100 or N − Fertilized plant DM.

Temperature, pH, And Salt Tolerance of the Rhizobial
Isolates.
e abiotic tolerance of the isolates to extreme temperature, pH, and salt concentrations was determined. e potential of the rhizobial isolates to grow and tolerate several temperature ranges such as 5,10,15,20,25,35,40,45, and 50°C was evaluated [1]. A loop full of rhizobial isolates grown on YEMA media were streaked and incubated by adjusting at the specific temperature for 3-4 days. e growth and tolerance of the isolates were recorded. Regarding pH tolerance of the isolates, a loop full of the isolates was streaked on YEMA media adjusted at a different pH of 4.0, 4.5, 5.0, 5.5, 8.0, 8.5, 9.0, 9.5, and 10.0 [32,33]. e pH of the media was adjusted by adding 1N HCl and NaOH to pH 7.0 before autoclaving. Similarly, the salt tolerance of the isolates was determined by inoculating each isolate on YEMA media containing 1-10% NaCl concentrations [23]. In all the physiological tolerance tests, the rhizobial inoculated and streaked plates were incubated at 28 ± 2°C for 3-4 days. After incubation, the growth and tolerance of the isolates to the tested tolerance tests were recorded.

HCN
Production. All isolates were tested for HCN production using a slant YEMA medium inserted with filter paper strips dipped in picric acid and 2% sodium carbonate [34]. e test tubes were sealed by using parafilm to prevent the escape of gases produced. e test tubes were incubated at 28 ± 2°C for 3-5 days. e color change of the filter paper from deep yellow to orange, then to brown, indicated the production of HCN.

Data Analysis.
One-way ANOVA was used to analyze and interpret the recorded data. Duncan's multiple range test (DMRT) was used to evaluate and contrast the experimental treatments with their controls using SPSS ver. 20.
e Pearson correlation coefficient was used to examine the correlation between different characteristics. e dendrogram was constructed by using 28 phenotypic parameters that were recorded as 1 for the presence of growth and 0 for the absence of growth.

Isolation and Purification of Rhizobial Isolates.
In this study, chickpea (Cicer arietinum L.) rhizobia were isolated from the root nodule bacteria collected from the three sampling districts of South Wollo, namely, Kutaber, Tehulederie, and Kombolcha. Consequently, among 150 rhizobial isolates streaked on YEMA-CR, 69% (103) did not absorb Congo red, whereas the rest showed absorbance indicating contamination (S1a and S1b). Furthermore, the Gram reaction test conducted by using KOH and the BCP growth test results revealed that 103 (69%) of the rhizobial isolates were tentatively classified as Rhizobium. In addition to these, the isolates changed BTB supplemented YEMA medium into yellow color, indicating their acid production characteristics (S1c). Among the rhizobial isolates [35] taken to the greenhouse for authentication, 37 (77.1%) of them induced nodulation. Concerning cultural characterization of the rhizobial isolates, 35 (72.9%) of the isolates exhibited a

Symbiotic Effectiveness Test on Sand
Culture. e ability of the isolates to induce nodules, accumulate nodule dry weight, shoot dry weight, increase shoot length, and fix nitrogen was presented ( Table 2). In the current study, since 37 (71%) of the rhizobial isolates induced nodules on the host plant (chickpea), they were identified as root nodulating chickpea Rhizobium species.
In this study, the result of the tested symbiotic parameters showed a significant difference at P < 0.01 (Table 2). Regarding nodule number, plants inoculated with the rhizobial isolates Wu-36 and WuCR 25 induced the maximum and minimum nodule numbers of 85 and 1 per plant, respectively. Chickpea plants inoculated by rhizobial isolates WuCR-36, 17, and 38-accumulated the highest consecutive nodule dry weights of 0.21 g, 0.17 g, and 0.16 g, respectively. Likewise, WuCR-36, 20, and 17-inoculated plants accumulated a shoot dry weight of 1.70 g, 1.12 g, and 1.07 g, respectively. Plants inoculated by rhizobial isolate Wu-36 performed better in all, i.e., nodule number, nodule dry weight, and shoot dry weight parameters consistently. e maximum and minimum nodule number, nodule dry weight, and shoot dry weight of this study were 85, 86, and 15 higher than the negative control, respectively. Chickpea plants inoculated with rhizobial isolates Wu-36, 38, and 17 recorded the highest shoot length in order. e shoot dry matter of all the rhizobia-inoculated plants was higher than the negative control, and most of them were also higher than the positive control (S2a and S2b). Among the rhizobial isolates, 47.1%, 17.6%, 26.5%, and 8.8% of the isolates were  (Table 3) indicated that nodule number, nodule dry weight, shoot dry weight, shoot length, and symbiotic effectiveness showed a strong positive correlation of 0.798, 0.79, 0.747, and 0.692, respectively, at p < 0.01. e correlation result indicates that the parameters taken were important in determining symbiotic effectiveness.

Physiological Characterization of Isolates.
e rhizobial isolates showed variation in their growth and tolerance to different salt concentrations, temperature, and pH ranges (Figure 3-Figure 5).

pH Tolerance.
e potential of the isolates to grow and tolerate various pH ranges was presented ( Figure 5). All the isolates grew at pH ranges of 7-9 showing good rhizobial colony characteristics. At pH 4 and 5, 5 (20.8%) and 6 (29.2%) of the rhizobial isolates showed growth, whereas 19 (79.2%) of the isolates showed growth at 6 and 10 pH. In this study, most of the rhizobial isolates showed better growth to higher pH than to lower pH. Isolates WuCR-11, 17, 29, 34, 36, and 44 grew at all the tested pH ranges.
In general, most rhizobial isolates grew and tolerated both lower and upper abiotic stresses. Isolates WuCR-11, 17, and 36 showed the best potential for abiotic tolerance properties, showing growth in all extreme ranges of pH, temperature, and salt concentrations.

Discussion
In this study, chickpea (Cicer arietinum L.) root nodule bacteria were collected from three districts of South Wollo, namely, Kutaber, Tehulederie, and Kombolicha. Consequently, 150 bacterial isolates were streaked on YEMA-CR of which 69% (103) did not absorb Congo red. Moreover, the Gram reaction test conducted using KOH, and the PGA result proved that the rhizobial isolates were tentatively categorized as Rhizobium species [25]. Besides, the isolates changed BTB supplemented YEMA medium into yellow color, indicating their acid production characteristics. ese same isolates were yellow in color, indicating that they were quick in growth and acid makers, both of which are typical of Rhizobium species [31]. As similarly reported in Ref. [36], the isolates did not show any growth on peptone glucose agar containing BCP, implying that they were Rhizobium species. Among the rhizobial isolates authenticated under greenhouse, 34 (70.8%) of them induced nodulation which is close to the findings of Refs. [37][38][39], who reported 77% similar cultural characteristics. e remaining isolates failed to form nodules on their host plant which could be due to poor root inoculation or plasmid loss due to long storage in the refrigerator. In general, based on the presumptive tests, cultural characterization, and greenhouse authentication tests conducted, 34 (71%) of the rhizobial isolates were considered as Rhizobium species.
Concerning cultural characterization of the rhizobial isolates, 35 (72.9%) of the isolates exhibited a milky and mucus-like appearance, while the remaining 13 (27.1%) of them showed a watery and transparent appearance. Moreover, 32 (66.66%) of the isolates produced round forms, whereas 16 (33.33%) of the isolates were flat with a complete circular edge. According to Ref. [22], 89% and 11% of isolates had a white-milky mucoid and watery-transparent texture with a full circular form which is nearly similar to the cultural properties of the isolates of the current study.
Out of the culturally characterized and tentatively classed Rhizobium isolates using confirmatory and greenhouse authentication tests, 48 (46.6%) of them solubilized tricalcium phosphate (TCP) adjusted on PVK media. is result coincided with Ref. [22], who reported 42% of TCP solubilizing Chickpea rhizobia. e isolates of this study showed a phosphate solubilization index ranging from 2.1 to 2.7, which is nearly similar to Ref. [39], which reported a maximum solubilization index of 2.85 by chickpea rhizobial isolates indicating that the isolates of the current study can be applied as candidates of phosphate solubilizing rhizobia.
In this study, 34 (71%) of the isolates induced nodules and were authenticated as root-nodulating Rhizobium  species, which is low as compared to the findings of Refs. [21,22,40], who reported 100% authenticated rhizobial chickpea isolates. Regarding nodule number, the maximum and minimum nodule number was recorded from plants inoculated with isolates Wu-36 and Wu-25 with nodule number of 85 and 1 p/plant, respectively, which is close to the result of Ref. [21] who reported maximum and minimum nodule number of 61 and 2 p/plants, respectively. e difference between the maximum and minimum nodule number, nodule dry weight, and shoot dry weight of this study was 85, 86, and 15, respectively, whereas the study conducted by Refs. [21,22,40] reported very less results than the current findings indicating the effectiveness of the study of rhizobial isolates.
In the present study, 47.1%, 17.6%, 26.5%, and 8.8% of the isolates were highly effective, effective, lowly effective, and ineffective, respectively. According to a previous study [41], 14.4%, 59%, and 27% of chickpea rhizobia isolates were very effective, lowly effective, and ineffective, respectively. Furthermore, Ref. [21] found that chickpea rhizobial isolates were 7% and 67% highly effective and effective, respectively, indicating the better performance of isolates of this study. Plants inoculated with rhizobial isolates that scored the highest shoot dry weight were effective in nitrogen fixation and considered as good candidate isolates for developing microbial inoculants for field application. On the other hand, plants inoculated with some rhizobial isolates that induced the least number of nodules were lowly effective and ineffective indicating variation in nodulation potential and symbiotic efficiency, which could be due to differences in their evolutionary link, ecological factors, and genetic diversity of rhizobia and their host plants [42]. All the symbiotic parameters showed a moderately to strongly positive correlation with each other at a significance level of 0.01. However, shoot dry matter is a good indicator of relative effectiveness that describes a sound correlation between the nitrogen-fixing capacity of legumes and their shoot dry matter accumulation [33].
In the present study, chickpea rhizobial isolates showed a wide range of variations in their tolerance to different salt concentrations. e rhizobial isolates of this study grew and tolerated all the tested salt concentrations of 1-8%, which is nearly similar to the findings of Ref. [41], who reported chickpea rhizobial isolates that tolerated up to 8% of NaCl concentrations. Among the isolates, 16.7% of them showed growth at 8% salt concentrations which were better than the findings of Ref. [37], who reported 7.4% of chickpea rhizobia grown at 8% salt concentrations. e investigation of such type of rhizobial isolates that tolerated this high amount of salt concentration is categorized as fast-growing rhizobia and could be used as a remedy in areas having high soil salinity problems [43]. e isolates of this study showed wide variations in growth to different temperature ranges of 5-50°C. Consequently, all 12.5%, 18.75%, 66.67%, 20.85%, and 4.17% of the isolates showed growth to 25-35, 5, 10, 20, 45, and 50°C, which were close to the findings of Ref. [44] who reported chickpea rhizobia that tolerated temperature at a range of 10-42°C. Moreover, Refs. [22,41] reported 17% and 8% of chickpea rhizobial isolates that grew up to 40°C. Among all the isolates, only two strains (Wu17 and Wu36) were grown in all the tested temperature ranges (5-50°C), implying that these rhizobial isolates are endowed with the potential for resisting high-and low-temperature ranges and can be used for developing rhizobial inoculants serving as biofertilizer for tropical and polar areas with low and high soil temperatures.
Regarding pH tolerance, among the isolates, all 87.5%, 18.75%, and 12.5% of the isolates showed growth at pH 5.5-7.5, 8, 9, and 10, respectively, which is nearly similar to Ref. [45], who reported the tolerance of rhizobial isolates to pH 10 and 11. Similarly, Refs. [41,44] reported rhizobial chickpea isolates grown at a pH level of 9 that was nearly similar to the current study's findings. Moreover, 22.9% and 54.2% of them grew at pH 4 and 4.5, respectively, which is close to the findings of Ref. [22], who reported 31% of chickpea rhizobia that tolerated a pH of 4.5. In this study, 12.5% and 22.9% of the isolates showed growth at pH 10 and 4. Likewise, Refs. [21,22] reported nearly similar findings. In general, isolates of this study exhibited tolerance to lower and higher pH levels, indicating that they can be used as important microbial inoculants in alkaline and acidic soils under greenhouse and field conditions. e isolates of this study showed variations in different salt concentrations and different pH and temperature ranges which are important parameters used as phenotypic markers [46,47]. In this study, the investigation of the isolates that tolerated both low and high levels of salinity, pH, and temperature is vital at times of the current climate change and could play an important role in boosting crop productivity by increasing their successful symbiotic association with their host plants [48,49]. Among the tested rhizobial isolates, 13 (27%) produced HCN that coincided with the findings of Ref. [50], who reported the production of HCN by Mesorhizobium species isolated from chickpea. ese isolates can be used as biocontrol agents against fungal pathogens like Fusarium wilt by inhibiting their growth by affecting the respiratory system of the pathogen [51][52].
In conclusion, this study screened rhizobial isolates that were highly effective in nitrogen fixation. Moreover, the study also determined that rhizobial isolates have a high potential for resisting extreme abiotic factors such as salt, pH, and temperature. Isolates WuCR-17 and 36 performed best in all the symbiotic nitrogen fixation and abiotic stress tests. Consequently, these isolates can be recommended as good rhizobial candidates to be applied as biofertilizers as a better option for the unaffordable and eco-friendly chemical fertilizer.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there exists no conflict of interest. 8 Advances in Agriculture


provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

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Repetitive Bathing and Skin Poultice with Hydrogen-Rich Water Improve Wrinkles and Blotches Together with Modulation of Skin Oiliness and Moisture

Hydrogen-rich warm water (HW) has not been verified yet for skin anti-aging effects. Daily 10 min HW (dissolved hydrogen: 338–682 μg/mL, 41 ◦C) bathing and skin poultice with HW-impregnated towels for 11–98 days were demonstrated to improve wrinkle degrees (29 skin-loci) from 3.14 ± 0.52 to 1.52 ± 0.74 (p < 0.001) and blotch degrees (23 loci) from 3.48 ± 0.67 to 1.74 ± 0.92 (p < 0.001) in five healthy subjects (49–66 years old), by densito-/planimetrically evaluating with an Image-J software, and ranking into six hierarchies (0, 1–5). Meanwhile, skin oiliness was evaluated to increase for the oil-poor skins, but inversely decrease for excessively oily skins, suggesting the HW’s function as skin-oiliness modulation, with an appreciably negative correlation in prior oiliness contents versus change after HW application (r = −0.345, 23 loci). Skin moisture increased upon HW application, with a negative correlation (r = −0.090, 23 loci) in prior moisture contents versus post-HW-application moisture-changing rates, meaning that HW application compensated moisture for water-deficient skins (27.5–40% moisture), but not for wet skins (>41% moisture). Thus, the HW bath together with HW poultice exerted beneficial effects on skin appearances such as wrinkles, blotches and moisture/oiliness, some of which might ensue from enhanced antioxidant ability in blood, as was previously demonstrated for the HW bath.

Among many antioxidant-application strategies, hydrogen molecules (H 2 ) are utilized as a novel medical gas with antioxidant properties [2,12,13]. Numerous experimental and clinical studies have demonstrated that hydrogen could protect cells, tissues and organs from oxidative stress-induced injuries by scavenging ROS [14,15]. Hydrogen-enriched electrolyzed water is guaranteed for safety in human body, and hydrogen also has beneficial effects on the human body, including preventive effects on many diseases and radiationprotective effects [16][17][18][19]. Several methods of administering hydrogen are represented by inhaling hydrogen gas/mist, drinking hydrogen water (HW) and bathing in HW [13]. The living cells also constantly produce ROS, even under healthy conditions, whereas excess amounts of ROS are produced by inflammation and stress [19]. It is generally known that skin aging is also caused by stress due to overproduction of ROS [1][2][3][4][5][6]. Therefore, it has been reported that there are many substances that suppress skin aging [2,3,[6][7][8][9][10]14]. Recently, hydrogen water is also one of the methods being used for anti-aging [12].
The newly developed instrument, designated as Lita Life Ver. 2, produces hydrogenrich water by electrolysis, forming colloidal nanobubbles and stabilizing hydrogen nanobubbles dispersed in water for a long time [13]. Hydrogen-rich water has been suggested to be effective for alleviating oxidative stress [16,17,19]. It has improved the heightening of antioxidant activity, and its nanobubbles are markedly important for permeation into the depth of the living body and organs. By bathing with nanobubble hydrogen-rich water that is prepared by the electrolyzer, the oxygen radical absorption capacity (ORAC)-based antioxidant capacity increased and C-reactive protein (CRP, an index of degree of inflammation) level of human serum as well as inflammatory symptoms decreased [13]. Therefore, the hydrogen-rich bath is also considered to bring about skin-quality improvement. It has been reported that inhibitions by hydrogen-occluding silica microcluster to melanogenesis in human pigment cells and to the melanin-generating key enzyme tyrosinase reaction [12].
For this reason, we aim to investigate the effects of a hydrogen-rich water bath based on the improvement of the cosmetological state of the skin.

Preparation of HW Bath
A hydrogen-rich water (HW) generator, Lita Life Ver.2 (WCJ Co., Ltd., Osaka, Japan), equipped with a lattice-shaped three-blade-type electrode surface-plated with platinum, and which produces hydrogen bubbles via electrolysis for Sakai-city-supplied tap water, was used according to the manufacturer's protocol. The dissolved hydrogen concentrations were evaluated to be 338-682 µg/mL, after 30-60 min electrolysis, with a polarographic diaphragm-type device with hydrogen-selective electrode (DH meter, KM2100DH, Kyoei Electronics Research Institute, Saitama, Japan) [13]. The nano-sized bubbles, which contain gaseous hydrogen, were produced via the electrolysis of warm (41 • C) water of 200 L poured into a bathtub and evaluated with a Nano Sight LM10V-HS (Malvern Co., Ltd., Malvern, UK) system, combined with a sCMOS camera (Hamamatsu Photonics, Hamamatsu, Japan).

Treatments with HW Bath and HW Poultice
The bath water of 180-200 L was electrolyzed for 60 min, immediately followed by 10 min bathing concurrently with twice continuously compressing of a fresh HW-soaked towel on the face, and finally further washing face. The subjects were two men and three women, aged from 49 to 66 years with no disease nor medical treatment and no intake of hydrogen-related drinking/supplement for the last 60 days. The research was officially approved in 2016 with an approval number of 15C01, under conditions of noninvasive research such as no hemorrhage, painless treatment, and no trace/sign, using a widespread commercially available apparatus, by the Medical Ethics Committee of the Japanese Center for Anti-Aging Medical Sciences, which was officially authenticated by the Hiroshima Prefecture Government of Japan, and all subjects also provided their signed informed consent forms.

Quantitative Assessments for Degrees of Wrinkles and Blotches
Immediately before and after the HW bath concurrently with the HW poultice compressing the concerned skin parts, the subjects underwent photography of wrinkles (at forehead, eye outer corner, between eyebrows, nasolabial folds, Marionette line and hand) and blotches (at forehead, eye outer corner, cheek, lower head and neck and upper limbs) under the conditions unified for background brightness and illuminating angle. The identification of the concerned area as a blotch was performed where both the subject and the inspector recognized it as a blotch without any pathological symptoms. A digital camera (Compact Digital Camera PowerShot SX730 HS, Canon, Tokyo, Japan) was used to photograph the wrinkles and blotches on the face and hands. The degrees of wrinkle and blotch on the skin were evaluated in quadruplicate for each affected part as a percentage of the area and density distribution that were objectively recognized by application of the software Image J (NIH, Bethesda, MD, USA) as changes on the surface of the skin, and further expressed in the average value ± SD of the results of each datum, calculated using Microsoft Excel 2013 for Windows as six hierarchies (−, ±, +, ++, +++, ++++, +++++). To investigate the statistically significant difference between before and after HW bath and face poultice, data of six hierarchies of wrinkles and blotches were described as graphics.
The HW application with bath and poultice for the prearranged periods were evaluated for effects on skin oiliness and moisture of the concerned face parts (23 loci, respectively: at eye corner, cheek and hand neck) of five subjects in quadruplicate, by a bioelectricity-impedance-/electroconductivity-based skin-oiliness/moisture meter (HKJ-SK03P, HuntKey Japan, Co., Ltd., Tokyo, Japan), after gently wiping the concerned skin part with purified water-soaking cloth and drying up for 5 min.

Statistical Analysis
All data were analyzed statistically by Student's t-examination of Microsoft Excel (Redmond, WA, USA). Statistical significance was considered when p < 0.05.

Effect of Improving Wrinkles on Five Subjects by Bathing and Face Poultice with Hydrogen-Rich Warm Water
Five subjects received bathing in hydrogen-rich warm water (HW) of 338-682 µg/L (another expressed unit: ppb) (41 • C) with 6.86-9.02 × 10 7 nano-bubbles/mL, once a day for 11-98 consecutive days. During the 10 min bathing, the subjects were repeatedly applied with a towel reimpregnated with fresh HW to the relevant skin area every 3 min. For every subject, loci where wrinkles became narrow, shallow, faint or disappeared after HW bathing and face washing were photographed ( Figure 1A-H).
In three subjects-a 49-year-old female, 59-year-old male and 57-year-old male-a reduction in small wrinkles around the right-eye outer corner was observed ( Figure 1A-C). In one subject-the 49-year-old female-a reduction in wrinkles around the forehead mole was observed ( Figure 1D). In one subject-the 59-year-old male-a reduction in wrinkles on the left cheek was observed ( Figure 1E). In one subject-a 66-year-old female-a reduction in wrinkles between the eyebrows was observed ( Figure 1F). In one subject-a 57-year-old male-an improvement of wrinkles of both nasolabial folds was observed ( Figure 1G). In one subject-a 66-year-old female-the second wrinkle from the top among four wrinkles in the total on the outer corners of the right eye almost disappeared, and the other three wrinkles also became shallower and shorter ( Figure 1H). Figure 2A shows changes of degrees of wrinkles for all observed loci of five subjects. Figure 2B shows changes of degrees for each region of five subjects. Figure 2C shows changes for total loci and five subjects. A total of 29 examined wrinkles were improved (H): Before hydrogen application, among 4 wrinkles on the outer corners of the right eye (indicated by circles), the second wrinkle (indicated by an arrow) from the top was shallow and thin. However, the second wrinkle had almost disappeared, and the other three wrinkles had also become shallower and shorter. Figure 2A shows changes of degrees of wrinkles for all observed loci of five subjects. Figure 2B shows changes of degrees for each region of five subjects. Figure 2C shows changes for total loci and five subjects. A total of 28 out of 29 examined wrinkles were improved after the hydrogen-rich warm bath, with a significant decrease in wrinkle degrees of 3.14 ± 0.52 to 1.52 ± 0.74 (p < 0.001) (Figure 2A,B).   Figure 2B,C, mean values ± SD before and after treatments are described and analyzed by the Student's t-test. * p < 0.05.

Effect of Improving Skin Blotches on Five Subjects by Bathing and Face Poultice with Hydrogen-Rich Warm Water
As in Figure 1A-H, five subjects received the bathing and face poultice with hydrogenrich warm water (HW) for 10 min, for 11-98 consecutive days.
For every subject, several loci were found where the blotches became narrow, faint or disappeared after HW bathing and face poultice ( Figure 3A-D).
Hydrogen 2022, 3, FOR PEER REVIEW 9 For every subject, several loci were found where the blotches became narrow, faint or disappeared after HW bathing and face poultice ( Figure 3A-D).
In the 66-year-old female subject, a reduction in blotches on the right cheek was observed ( Figure 3A). In the 58-year-old female subject, a reduction in widespread blotches on the right cheek was observed ( Figure 3B). In the 66-year-old female subject, a reduction in blotches on the left cheek was observed ( Figure 3C). In the, 57-year-old male subject, a reduction in blotches at the back of the left hand was observed ( Figure 3D).   Figure 4A shows changes of degrees of blotches for all observed loci of five subjects. Figure 4B shows changes of degrees for each region of five subjects. Figure 4C shows changes for total loci of five subjects. The HW application was found to bring about improvements in all out of 23 examined blotches, with a significant decrease in blotch degrees of 3.48 ± 0.65 to 1.74 ± 0.90 (p < 0.001, Figure 4C).
(A) Figure 3. Improvements to blotches in five subjects by bathing and poultice with hydrogen-rich warm water. As in Figure 1A-H, five subjects received the bathing and face poultice with hydrogen-rich warm bath (HW) for 10 min, for 11-98 consecutive days. The face-blotch loci were classified to the degree of area and density of blotches into six hierarchies (−, ±, +, ++, +++, ++++, +++++). In the 66-year-old female subject, a reduction in blotches on the right cheek was observed ( Figure 3A). In the 58-year-old female subject, a reduction in widespread blotches on the right cheek was observed ( Figure 3B). In the 66-year-old female subject, a reduction in blotches on the left cheek was observed ( Figure 3C). In the, 57-year-old male subject, a reduction in blotches at the back of the left hand was observed ( Figure 3D). Figure 4A shows changes of degrees of blotches for all observed loci of five subjects. Figure 4B shows changes of degrees for each region of five subjects. Figure 4C shows changes for total loci of five subjects. The HW application was found to bring about improvements in all 23 examined blotches, with a significant decrease in blotch degrees of 3.48 ± 0.65 to 1.74 ± 0.90 (p < 0.001, Figure 4C).   Figure 4A shows changes of degrees of blotches for all observed loci of five subjects. Figure 4B shows changes of degrees for each region of five subjects. Figure 4C shows changes for total loci of five subjects. The HW application was found to bring about improvements in all 23 examined blotches, with a significant decrease in blotch degrees of 3.48 ± 0.67 to 1.74 ± 0.92 (p < 0.001, Figure 4C).   Figure 4B,C, the mean values ± SD before and after treatment are described and analyzed by the Student's t-test. * p < 0.05.

Improvements to Oiliness in Five Subjects by Bathing and Face Poultice with Hydrogen-Rich Warm Water
Meanwhile, skin-oiliness contents were evaluated as a comparison between oiliness values before and after HW bathing/face poultice. Interestingly, the HW application was found to make the oil-poor skin gain oiliness, but inversely, the oily skin was altered to less-oily or moderate skin, suggesting the HW's function as an oiliness modulation, with an appreciably negative correlation (r = −0.345; 23 loci) of prior oiliness values versus the fluctuation for before/after HW application ( Figure 5).

Improvements to Skin-Moisture Content in Five Subjects by Bathing or Face Poultice with Hydrogen-Rich Warm Water
It is suggested that the water content of skin is related to the scavenging action of hydrogen on reactive oxygen species. [7][8][9] Thus, the effect of hydrogen-rich water treatment on the amount of water content of the face was examined. HW application was found to make dry skins (moistures: 27-35%, 35-45.5%) moist (moisture gains: 2.0%, 0.6%,

Improvements to Skin-Moisture Content in Five Subjects by Bathing or Face Poultice with Hydrogen-Rich Warm Water
It is suggested that the water content of skin is related to the scavenging action of hydrogen on reactive oxygen species. [7][8][9] Thus, the effect of hydrogen-rich water treatment on the amount of water content of the face was examined. HW application was found to make dry skins (moistures: 27-35%, 35-45.5%) moist (moisture gains: 2.0%, 0.6%, respectively) concurrently, with scarce changes for the wet skin (moisture: above 40.5%), with a negative correlation (r = −0.090; 23 loci) of prior moisture contents versus the fluctuation in before/after HW application ( Figure 6). respectively) concurrently, with scarce changes for the wet skin (moisture: above 40.5%), with a negative correlation (r = −0.090; 23 loci) of prior moisture contents versus the fluctuation in before/after HW application ( Figure 6).

Discussion
The examination periods were flexibly set for each subject for as long it was possible to be permitted, in the viewpoint of subjects' individual life styles, for improvements of wrinkles and blotches, although it is difficult to secure sufficiently long-term cooperation among subjects; thus, the limitation of this study is to be terminated in a case study.
As compared to other diverse antioxidants, the ROS-scavenging effects of hydrogen by itself may be much weaker or moderate. However, because certain levels of ROS are crucial as triggering or relaying signals for some biological processes, excessively strong antioxidants could occasionally have some undesired effects by blocking the ROSinvolved cellular-signaling pathway. Hydrogen-dissolved water has been found to be a safe alternative and has few side effects [13]. The effects might be achieved by compression of the HW poultice on concerned skin parts, in addition to hydrogen permeation into skin depth, as previously suggested by elevation of blood antioxidant ability caused by the HW bath, as shown by our previous study [13]. By taking an HW bath and HW skin poultice, hydrogen nanobubbles could rapidly infiltrate into the stratum corneum, reach the epidermis and dermis, and be absorbed into the blood stream. Repeated HW bathing has been clinically demonstrated to repress the inflammatory blood-marker CRP (C-reactive protein) value, which is majorly synthesized in and secreted from the liver, in contrast to no significant CRP value change for normal warm water, suggesting a possibility for the penetration and widespread distribution of hydrogen molecules into the whole liver [13]. In contrast to no effect for a normal tap-water warm bath, the hydrogen-rich warm-water bath was demonstrated previously to promote the ORAC (oxygen radical absorbing capacity)-based antioxidative activity in the blood [13], which might improve skin conditions including wrinkle/blotch prevention and moisture/oiliness modulation, all of which have been numerically and visually verified in the present clinical study. Hydrogen molecules can be distributed in the blood and tissues such as the skin of an organism. Once the hydrogen molecules reach the blood stream, they scavenge ROS and protect the cells and tissues against inflammation-/oxidative-stress-induced damages [13]. Therefore, the hydrogenrich water bath was used to deliver hydrogen molecules to subjects in the present study. Comparatively, in the present study, our data showed that hydrogen-rich water brought out repetitive effects on human skin.
Repression by HW treatments on wrinkles (Figures 1 and 2) might ensue from combinative effects of HW-bath-induced systemic-circulation-based hydrogen-distribution [13] and HW-poultice-induced local permeation-based hydrogen distribution. Wrinkles in human skin are considered to depend on the ability of collagens to moisturize or retain dermal structural robustness. Collagens are essential components that support the strength and flexibility of the skin structure. Studies have shown that wrinkles in human skin depend on either the deteriorated moisturizing ability of collagen or its reduced expression and the resultant intra-/intermolecular cross-linkage [3,7,8,10,21,22]. In human artificial three-dimensional skin, type I collagen is known to be widely distributed inside the dermis [10,19,23,25]; meanwhile, type IV collagen is localized inside the basement membrane that connects the dermis and the epidermis [22,29]. The "basement membrane" forms a "scaffold" for the growth of keratinocytes that occupy most of the epidermis, and type IV collagen creates a lattice-like structure that becomes the basement membrane.
The ROS-scavenging ability of nanobubbles as abundant as 9.02 × 10 7 /mL (as previously evaluated by NanoSight method [13]) in a hydrogen-rich water bath helps in the lightening and size reduction of the dark dermal spots and stains, improving skinwhitening quality [12]. The present study also revealed that the hydrogen-rich water bath confers anti-aging effects on human skin blotches (Figures 3 and 4). The blotch-repressive effects of the HW bath/poultice might be attributed to both the abilities of hydrogen to reductively bleach the existent blotches and to extracellularly expel the melanin-containing old epidermal keratinocytes and old stratum corneum through cell-proliferation promotion. Hydrogen molecules are assumed to penetrate from the surface to the target pigment-cell layer, through the minimum molecular mass combined with neutral electrostatic property, and alleviate ROS in the target skin part, suppressing skin melanin, as presumed by the fact that nanospheres easily penetrate into the skin [30]. It was presumed that, the skin epidermal layers containing melanin and lipofuscin, which cause blotching, were expelled out of the skin surface, such as dirt through hydrogen-activated keratinocyte proliferation, and/or were reductively bleached by the whitening power of hydrogen [12,31].
Hydrogen application inclined to modify oil-deficient skin into oily skin, and, inversely, excessively oily skin into less-oily or intermediate skin, bringing out the appropriate modulation for skin-oiliness degrees ( Figure 5). This finding (oiliness) means that HW application optimizes skin-oiliness degrees, through either hydrogen functions for a possible modulation to the sebum-secreting glands or hydrogen adjusting to adipogenesis by subcutaneous adipocytes, as previously demonstrated using three-dimensional human skin equivalents [32].
Hydrogen-application-induced heightening of skin-moisture contents ( Figure 6) is considered to be caused by improvements of skin-moisture-retaining extracellular matrices such as type I/IV collagens, which are demonstrated to be increased by administration of hydrogen-generating silica microparticles to three-dimensional human skin-tissue equivalents [31,32].
Thus, the HW bath in combination with the HW poultice on concerned skin parts exerts diverse beneficial effects on cosmetological aspects of human skin appearances such as wrinkles, blotches and oiliness/moisture. In the future, the issue is whether to promote the expression of stress-related genes such as COX-2, p53, Nfr2, MAPK signaling by hydrogen water, as well as other antioxidants [15,20,21]. Similar effects of the hydrogenrich water bath could be considered for other tissues and blood/capillaries, helping the systemic improvements of health.

Conclusions
Bathing and skin poultice with hydrogen-rich warm water (HW) for 11-98 days improved both wrinkle degrees and blotch degrees in five healthy subjects (49-66 years old). Meanwhile, HW functioned as skin-oiliness modulation, and compensated moisture for the water-deficient skins. Thus, the HW bath together with HW poultice exerted beneficial effects on skin appearances such as wrinkles, blotches and oiliness/moisture, some of which might ensue from enhanced antioxidant ability in blood. Funding: The present study was supported in part by a Grant-in-Aid No. 1902 to YT for the Scientific Research on Anti-Aging Promotion from the Non-Profitable Organization Corporate Japanese Center for Anti-Aging MedSciences, authenticated by Hiroshima Prefectural Government.

Institutional Review Board Statement:
This research was officially approved in 2016 with an approval number of 15C01, under conditions of noninvasive research such as no hemorrhage, painless treatment, and no trace/sign, using a widespread commercially available apparatus, by the Medical Ethics Committee of the Japanese Center for Anti-Aging Medical Sciences, which was officially authenticated by the Hiroshima Prefecture Government of Japan.
Informed Consent Statement: Informed consent was obtained from all subjects involved in this study.


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Genetic Mechanisms of Cold Signaling in Wheat (Triticum aestivum L.)

Cold stress is a major environmental factor affecting the growth, development, and productivity of various crop species. With the current trajectory of global climate change, low temperatures are becoming more frequent and can significantly decrease crop yield. Wheat (Triticum aestivum L.) is the first domesticated crop and is the most popular cereal crop in the world. Because of a lack of systematic research on cold signaling pathways and gene regulatory networks, the underlying molecular mechanisms of cold signal transduction in wheat are poorly understood. This study reviews recent progress in wheat, including the ICE-CBF-COR signaling pathway under cold stress and the effects of cold stress on hormonal pathways, reactive oxygen species (ROS), and epigenetic processes and elements. This review also highlights possible strategies for improving cold tolerance in wheat.

Introduction
Higher plants are sessile organisms that suffer from various environmental stresses throughout their life cycle, such as cold, heat, drought, and salinity. Cold stress is vital for limiting plant geographical distribution and influencing plant growth and development, and ultimately determines yield and quality [1,2]. After a long interaction with their environment, plants have evolved complex and sophisticated mechanisms to adapt to cold stress. Cold signals are transduced from the plasma membrane to the nucleus, leading to a series of cold-induced cellular responses and the induction of cold-responsive genes. The main cold-responsive genes in plants are C-REPEAT BINDING FACTORs (CBFs), INDUCER of CBF EXPRESSION (ICE), and COLD-REGULATED (COR) genes [3,4]. It has been established that the ICE-CBF-COR signaling pathway is a universal pathway associated with cold tolerance in plants [2,5,6]. In this pathway, CBFs/DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR 1s (DREB1s) is rapidly induced by cold conditions. Additionally, CBFs/DREB1s proteins can bind to the promoter regions of COR genes to activate their transcription in Arabidopsis [7,8].
Low-temperature conditions seriously affect the growth and development of wheat grown in temperate regions [9]. Exposure to low temperatures changes various biochemical processes and induces membrane damage in wheat [9,10]. The effects of cold stress on wheat growth, development, and yield are determined not only by the degree and duration of low-temperature conditions but also by the growth stage in which the cold stress events occur [11]. Cold stress can significantly reduce the viable leaf area and the soluble carbohydrate accumulation, ultimately negatively affecting the final yield [12,13]. During the reproductive phase, wheat is susceptible to cold stress. The grain number will be decreased by low temperatures if stress occurs before anthesis [14]. Moreover, pollen tube elongation and gametophyte tissue development will be disrupted under cold stress, particularly in pollen tapetal cells, which can lead to pollen sterility [15,16]. Cold Life 2022, 12, 700 2 of 14 stress can alter sink-source distribution by increasing the accumulation of soluble carbohydrates to regulate grain filling in wheat [9]. In addition, low-temperature events could also happen during vegetative stages in wheat and are detrimental to wheat growth and development since they cause leaves to wilt [17]. The leaf mass ratio and relative growth rate are significantly increased under low-temperature conditions. Furthermore, the flag leaf size and wheat biomass both decrease under cold stress [18,19]. Biomass allocation is essential for grain yield formation under cold stress, and the appropriate allocation is responsible for reproductive growth proportion and yield formation [20]. Diploid and tetraploid wheat have large leaf areas and produce no or low yield under cold stress. In contrast, hexaploid wheats have relatively low leaf areas and higher rates of grain yield among these species [13].
Cold stress has been categorized into two primary groups: chilling stress (0-15 • C) and freezing stress (<0 • C), which depends on how the plants are affected [1,21]. The cellular and molecular responses of plants to cold stress have been intensively studied. At present, plants have acquired highly sophisticated systems to cope with cold stress. For instance, plants activate a series of biochemical and physiological changes in their cells, such as altering the transcription of cold-responsive genes, regulating hormone levels and responses, producing ROS to stimulate the accumulation of compatible osmolytes and antioxidants, and remodeling genome-wide epigenetic modifications [8,22]. Significant progress has been made over the past few decades in understanding how signaling pathways control cold stress responses in plants. However, current knowledge of the cold signal transduction pathway in wheat is limited. In this review, we summarize the most recent studies assessing cold stress response in wheat and highlight possible strategies for improving cold tolerance in wheat.

ICE-CBF-COR Signaling Pathway in Cold Stress
Plants in temperate regions, such as winter wheat, have evolved adaptive responses known as cold acclimation, where plants acquire freezing tolerance after prior exposure to low non-freezing temperatures [8]. It is well known that the ICE-CBF-COR signaling pathway is essential for cold acclimation [3]. In addition to Arabidopsis, the ICE-CBF-COR cascade has been identified in rice [23] and wheat [6,24].
ICE genes encode a class of MYC-like bHLH transcriptional factors upstream of the cold signaling pathway [25]. The C-terminal regions of ICE have highly conserved regions for specific interactions with downstream cold regulatory genes [5,[24][25][26][27]. The homologs of ICE have been identified as TaICE41 and TaICE87 in wheat ( Figure 1). Overexpression of TaICE41 or TaICE87 in Arabidopsis enhanced cold tolerance, suggesting the significance of ICE homologs in cold stress response [24]. HOS1 (HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1), an E3 ubiquitin ligase, reduces the stability of ICE1 protein by ubiquitination under cold stress [28]. In addition, the stability of ICE1 protein is enhanced by SUMO E3 ligase SIZ1 (SAP and Miz) through sumoylation in response to cold stress [29]. ICE1 is phosphorylated by the cold-activated protein kinase OPEN STOMATA 1 (OST1), resulting in weakened interaction between ICE1 and HOS1 to increase the stability of ICE1 under cold stress [30]. Furthermore, the stability of OsICE1 is up-regulated by OsMPK3 (MAP KINASE 3) through phosphorylation in rice in response to cold stress [31]. These results indicate that the posttranslational modification of ICE1 is crucial for its role in response to cold stress. However, whether TaICEs have similar regulatory mechanisms in wheat responses to cold stress needs further study.
CBFs (CBF1, CBF2, and CBF3), which belong to the AP2/ERF multi-gene family, can be activated by ICE in the cold signaling pathway of plants [3,25]. CBFs are key components for increasing the cold tolerance of plants [32][33][34]. The overexpression of CBFs in rice, maize, barley, wheat, and other plant species significantly enhances the cold tolerance of transgenic plants [35][36][37][38][39]. However, the cbfs triple mutant in Arabidopsis show reduced cold tolerance and larger biomass than wild type [40]. These results indicate CBFs may act to balance cold tolerance and plant growth. However, whether CBFs are important regulators of growth and cold tolerance to enhance the biomass of wheat requires further study. Several CBF genes have been characterized in Triticeae species, including 37 genes from hexaploid wheat [41], 20 genes from barley [36], 13 genes from Triticum monococcum [42], 11 genes from rye [43], ten genes from durum wheat [44], ten genes from Aegilops biuncialis [5], four genes from Brachypodium distachyon [45,46], and one gene from Aegilops tauschii [41]. TaCBF14 and TaCBF15, two wheat CBF transcription factors, play significant roles in cold stress response (Figure 1) [38]. Overexpression of TaCBF14 or TaCBF15 in barley enhances the expression of HvCOR14b, a cold-regulated gene in barley, increasing cold tolerance [38]. Additionally, T. aestivum ABIOTIC STRESS-INDUCED DNA BINDING FACTOR a (TaAIDFa) is markedly activated by cold stress [47]. Overexpression of TaAIDFa in Arabidopsis increases the transcription of the cold-regulated genes like RD29A and COR15A to enhance the cold tolerance of transgenic lines [47].
pression of TaAIDFa in Arabidopsis increases the transcription of the cold-regulat like RD29A and COR15A to enhance the cold tolerance of transgenic lines [47].
CORs generally refer to the protective substances encoded by cold-regulate The protective substances such as osmolytes and cryoprotective proteins accum facilitate cold acclimation and freezing tolerance [1,6]. CBFs are known to bind t REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) sequence (TACC in the promoters of COR genes for their transcription activation in response to co [48,49]. The expression of ABA-dependent COR genes (Wrab15/17/18/19) and AB pendent COR genes (WCS19, WCS120, Wcor14, and Wcor15) are significantly incr cold stress in wheat ( Figure 1) [50]. The expression of DRE-BINDING PRO (TaDREB1), a wheat homolog of Arabidopsis DREB2, is elevated under cold stress transcription of the WHEAT COLD SPECIFIC 120 (WCS120) gene is activated by T and increases cold tolerance in winter wheat [52]. The expression of wheat (WDREB2), also a wheat homolog of Arabidopsis DREB2, is activated by cold [ WDREB2 transcription factor directly affects the expression of wheat COR genes Wrab19 in response to cold stress [53]. Cold stress alters the fluidity of plasma membrane and activates protein kinases. Furthermore, kinases positively regulate cold tolerance in wheat by phosphorylating TaICE proteins, including TaICE41, TaICE87. TaICE directly binds to the promoters of TaCBFs to regulate its expression. Additionally, TaCBFs bind to the CRT/DRE sequence in the promoters of TaCOR genes, such as Wrab15, Wrab17, Wrab18, Wrab19, WCS19, WCS120, Wcor14, and Wcor15, for their transcription activation in response to cold stress.
CORs generally refer to the protective substances encoded by cold-regulated genes. The protective substances such as osmolytes and cryoprotective proteins accumulate to facilitate cold acclimation and freezing tolerance [1,6]. CBFs are known to bind to the C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) sequence (TACCG-CAT) in the promoters of COR genes for their transcription activation in response to cold stress [48,49]. The expression of ABA-dependent COR genes (Wrab15/17/18/19) and ABAindependent COR genes (WCS19, WCS120, Wcor14, and Wcor15) are significantly increased by cold stress in wheat ( Figure 1) [50]. The expression of DRE-BINDING PROTEIN 1 (TaDREB1), a wheat homolog of Arabidopsis DREB2, is elevated under cold stress [51]. The transcription of the WHEAT COLD SPECIFIC 120 (WCS120) gene is activated by TaDREB1 and increases cold tolerance in winter wheat [52]. The expression of wheat DREB2 (WDREB2), also a wheat homolog of Arabidopsis DREB2, is activated by cold [53]. The WDREB2 transcription factor directly affects the expression of wheat COR genes such as Wrab19 in response to cold stress [53].

Cold Stress Influences Hormonal Responses
Plant hormones (Phytohormones), which function as small molecules to regulate various cellular processes and work as chemical messengers to communicate cellular activities, are produced in very low concentrations in higher plants [54]. Phytohormones are needed for plants to deal with abiotic stresses, including salinity, drought, and low temperature, by mediating a wide range of adaptive responses [55]. These phytohormones include auxin, abscisic acid (ABA), ethylene, cytokinins (CKs), gibberellins (GAs), jasmonic acid (JA), brassinosteroids (BRs), salicylic acid (SA), and strigolactones (SLs). In recent years, the phytohormone signaling pathway has been investigated by genetic and biochemical approaches, and a growing body of evidence indicates that the elements in hormonal signaling pathways contribute to regulating plant cold tolerance [33].
Auxin, a tryptophan derivative most commonly present in the form of indole-3-acetic acid (IAA), plays an essential role in plant development and cold stress response. The YUCCA genes encode the key rate-limiting enzymes in the auxin biosynthetic pathway and are involved in the regulation of plant growth and development. The transcript levels of OsYUCCAs are strongly induced by low temperatures; however, the expression of IAA catabolism-related genes, Oryza sativa GRETCHEN HAGENs (OsGHs), is down-regulated, resulting in significantly increased IAA content in rice under cold stress (Table 1) [56]. In colder/ambient temperatures, CLAVATA (CLV) peptide signaling promotes flower development by stimulating auxin-dependent growth. In contrast, at higher temperatures, YUCCA genes are activated to maintain flower development bypass CLV signaling [57][58][59]. There are 15 genes among 63 TaYUCCAs that are induced by drought and heat stress in wheat, though it is unclear whether the expression of these genes is regulated by cold stress. Arabidopsis AUXIN RESPONSE FACTOR (ARF) genes, which regulate the expression of auxin-responsive genes by binding to the auxin response element in their promoters, are up-regulated during cold acclimation (Table 1) [60]. In wheat, 46 genes from 69 TaARFs are also up-regulated in response to cold stress (Table 1) [61].
Abscisic acid (ABA) is the most important phytohormone due to its role in plant adaptation to biotic and abiotic stresses [62]. ABA-deficient mutants in Arabidopsis show defects in freezing tolerance, with the induced expression of COR genes, suggesting that ABA is involved in cold signaling [63,64]. Additionally, ABA contents are moderately decreased after cold treatment [30]. SUCROSR NON-FERMENTING 1-RELATED PROTEIN KINASE 2s (SnRK2s) are important protein kinases in ABA signaling, and their role in abiotic and biotic stress signaling has been extensively characterized in Arabidopsis. The SnRK2 homologs in wheat appear to play a critical role in cold signaling. PKABA1, the first SnRK2 protein identified in wheat, is rapidly induced in seedlings when ABA levels increase in response to cold stress [65]. Furthermore, the expression of TaSnRK2.3, TaSnRK2.4, and TaSnRK2.8 can be induced by cold stress, suggesting that they are essential in cold signal transduction (Table 1) [66][67][68]. Overexpression of TaSnRK2.3 or TaSnRK2.8 in Arabidopsis increases cold tolerance, which is due to the increased expression of cold-responsive genes, and the enhanced accumulation of stress-associated metabolites such as proline [67]. Recent studies have identified 10 SnRK2 homologs in wheat, and the expression of these genes is induced by cold stress [69]. Although ABA and cold signaling are closely related, it is unclear what the exact role of ABA in regulating plant cold stress responses is. Further work is needed to elucidate the molecular mechanisms of ABA when regulating cold signaling pathways.
Ethylene, a gaseous plant hormone, is important in various cellular and developmental processes, as well as during abiotic and biotic stress responses [70][71][72][73][74][75]. It is reported that cold stress can alter endogenous ethylene levels in many plant species. Cold stress inhibits ethylene production in Arabidopsis [76]; however, the ethylene levels are increased in winter rye under cold stress [77]. T. aestivum ethylene-responsive factor 1 (TaERF1), the first member of the ERF gene family identified in wheat, is induced by cold stress (Table 1). Additionally, TaERF1 overexpression can activate COR genes and improve freezing tolerance in transgenic Arabidopsis [78]. Pathogen-induced ethylene response factor 1 (TaPIE1) in wheat positively regulates freezing stresses by activating cold-regulated genes downstream of the ethylene signaling pathway and by modulating related physiological traits (Table 1) [79].
Gibberellins (GAs) play vital roles in abiotic stress response and adaptation. DELLA proteins are master regulators of GA-responsive growth and development [80]. Cold stress activates the expression of GA 2-oxidase genes to reduce the content of GA, resulting in the enhanced accumulation of DELLA proteins [81]. It is reported that overexpression of CBFs reduces the bioactive GA levels to suppress plant growth and flowering. CBF1overexpression plants exhibit dwarfism and late-flowering phenotypes due to limited accumulation of bioactive GA [81]. Additionally, the cbfs mutants display impaired cold tolerance and larger architecture than the wild type after cold acclimation [40,82]. These results indicate that both the content and signal components of GA are related to cold signaling and CBFs may be associated with GA signaling to balance low-temperature adaption and growth. DELLAs act early in the cold signaling pathway as regulators of GROWTH REGULATORY FACTORs (GRFs). Cold-induced CBF genes are decreased in GRF5-overexpression lines, indicating that GRFs can repress CBF expression under cold stress (Table 1) [83]. Overexpression of SLENDER RICE 1 (SLR1), a gene that encodes the rice DELLA protein, enhances chilling tolerance. When rice seedlings are subjected to chilling stress, the cold-induced SLR1 (Table 1) releases the repressive effect of OsGRF6 on OsGA2ox1. The increased OsGA2ox1 expression then decreases the active GA levels to enhance rice chilling tolerance [84]. Rht-B1b and Rht-D1b, the most important and common semi-dwarfing genes, encode GA-insensitive forms of DELLA proteins that likely have a reduced affinity for the GA receptor in wheat [85]. It has been reported that the Rht-B1b and Rht-D1b mutant alleles are not responsive to GA at warmer temperatures but are responsive at colder temperatures (Table 1) [86]. This suggests that Rht-B1b and Rht-D1b play vital roles in response to cold stress.
The phytohormone jasmonic acid (JA) and its methyl ester, methyl jasmonate (MJ), act as signaling molecules in response to environmental stimuli. Cold stress rapidly increases endogenous JA levels by up-regulating the expression of JA biosynthesis genes, such as LIPOXYGENASE 1 (LOX1), ALLENE OXIDE SYNTHASE 1 (AOS1), ALLENE OXIDE CYCLASE 1 (AOC1), JASMONATE RESISTANT 1 (JAR1) in Arabidopsis and OsLOX2, OsAOS, OsAOC, Oryza sativa 12-OXOPHYTODIENOATE REDUCTASE 1 (OsOPR1) in rice (Table 1) [56,87]. The accumulation of JA induced by cold stress is due to the repression of ICE1 by JASMONATE ZIM-DOMAIN 1/4 (JAZ1/4), repressors of jasmonate signaling, resulting in the induction of CBFs expression in Arabidopsis [87]. Wheat TaJAZ genes are up-regulated in response to low temperatures (Table 1) [88]. Additionally, endogenous JA levels increase under cold stress in wheat [89]. Exogenous MJ treatment tends to upregulate of the transcription of COR genes, such as WCS19 and WCS120, and increase the activity of superoxide dismutase (SOD) and peroxidase (PO) to promote wheat cold tolerance [90,91]. Rice HAN1 ("han" means "chilling" in Chinese), which functions as an oxidase to reduce the accumulation of the active to inactive, decreases the expression of CBF/DREB1s in rice under cold stress [92]. Arabidopsis OPR3 is one of the major players in the JA biosynthesis pathway. Transgenic wheat plants with AtOPR3-overexpression have increased the accumulation of JA and improved cold tolerance [93]. Brassinosteroids (BRs) play a vital role in plant development and stress tolerance. COR gene expression and cold tolerance in Arabidopsis are increased by exogenous BR treatment [94]. Exogenous BR treatment promotes growth recovery of maize seedlings following chilling treatment [95] and increases cold tolerance in winter rye and winter wheat [96,97]. BRASSINOSTEROID INSENSITIVE 2 (BIN2) negatively regulates the freezing tolerance in Arabidopsis [98]. Knockout mutants of Oryza sativa GLYCOGEN SYNTHASE KINASE 3-LIKE GENE 1 (OsGSK1), an ortholog of Arabidopsis BIN2, show enhanced cold tolerance (Table 1) [99]. The expression of T. aestivum SHAGGY KINASE 5 (TaSK5), an abiotic stress-inducible GSK3/SHAGGY-like kinase in wheat, is induced at the early stages of cold acclimation (Table 1) [100]. The BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a transmembrane receptor kinase as a BR receptor. Its mutation results in defective BR signaling and increases cold stress tolerance in Arabidopsis (Table 1) [101]. The enhanced expression of its wheat homologous TaBRI1 in Arabidopsis leads to better cold tolerance than the wild-type plants by maintaining membrane integrity [102]. Furthermore, overexpression of TaBRI1 in Arabidopsis and the ortholog of BRI1 in rice or barley increases the silique size and seed yield [103,104]. Therefore, TaBRI1 is involved in cold tolerance and is a suitable gene for improving crop yields under conditions of extreme environmental stress.

ROS and Cold Stress
Abiotic stresses typically increase ROS levels, including hydrogen peroxide (H 2 O 2 ), superoxide radical (O 2 •− ), hydroxyl radical (OH•), and singlet oxygen ( 1 O 2 ), all of which are toxic to plant cells [105][106][107]. Several pieces of evidence suggest that plant responses to cold stress are directly linked to ROS signaling [108][109][110][111]. It has been proven that lowtemperature conditions depress the activities of ROS-scavenging enzymes, such as ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), glutathione S-transferase (GST), glutathione reductase (GR), and peroxiredoxin (PRX). These cold-regulated antioxidant enzymes play a key role in enhancing cold tolerance [107,112,113]. The H 2 O 2 contents of 'dongnongdongmai1 ('dn1 ), a winter wheat variety, are significantly increased under cold stress. Additionally, ABA treatment enhances cold tolerance in wheat by increasing the activities of TaSOD, TaAPX, TaCAT, TaGR, TaDHAR, and TaMDHAR [107]. The ABA-stress-ripening (ASR), which functions as a transcription factor, can be induced by low temperatures [114]. The levels of ROS and the activities of antioxidant enzymes under abiotic stress are regulated by ASRs, suggesting that ASR plays an important role in regulating ROS homeostasis [87,115]. Ectopic expression of the cold-induced OsASR1 gene exhibits enhanced cold tolerance in transgenic rice plants [116]. It has been reported that TaASR genes respond strongly to low temperatures [117]. In addition, overexpression of TaASR1-D confers enhanced antioxidant capacity and stress tolerance in transgenic wheat plants [118]. T. aestivum GTP-BINDING PROTEIN β SUBUNIT LIKE GENE (TaGPBL), the first G-protein gene in wheat, contributes to cold stress response. TaGBPL overexpression reduces the activity of cold-responsive genes and reduces the activities of ROS scavengers and producers under cold stress [119].

Cold-Induced Epigenetic Processes and Elements
Epigenetic mechanisms play an important role in response to cold stress. The plant epigenome is highly dynamic, and cold stress can quickly reshape genome-wide epigenetic modifications [120]. Changes in DNA methylation and histone modification and the regulation of epigenetic elements, such as small RNA (sRNA) and long noncoding RNA (lncRNA), are the key modulators of plant stress responses [121,122].
The proteins containing methyl-CpG-binding domain (MBD) can recognize DNA methylation. TaMBD6, including a typical MBD domain at the N-terminal, is induced by prolonged chilling in wheat, indicating that the protein is potentially involved in recognizing DNA methylation during vernalization [123]. Wheat requires various vernalization genes in response to cold stress to adjust floral initiation, such as T. aestivum VERNAL-IZATION 1 (TaVRN1), TaVRN2, TaVRN3/FLOWERING LOCUS T1 (TaFT1), TaVRN-D4, and VERNALIZATION-RELATED 2 (VER2) [124,125]. In wheat and barley (Hordeum vulgare), the TaVRN1, HvVRN1, TaVRN2, and TaVRN3/TaFT1 gene are regulated by epigenetic modification (Figure 2). Two histone modification markers include histone 3 lysine 4 trimethylation (H3K4me3), which is a modification associated with active gene transcription, and H3K27me3, which is a modification associated with gene repression. Vernalization enriches H3K4me3 levels at the TaVRN1 and TaVRN3/TaFT1 promoters (Figure 2), while no significant changes are observed in H3K27me3 levels at the same regions of the TaVRN1 and TaVRN3/TaFT1 promoters in winter wheat. Furthermore, TaVRN1 and TaVRN3/TaFT1 are up-regulated by vernalization to accelerate floral transition in winter wheat [126]. TaVRN2, a dominant repressor of flowering, is down-regulated by vernalization [127]. Increased levels of H3K27me3 at the TaVRN1 promoter explain the repression of TaVRN2 gene expression in winter wheat (Figure 2) [126]. Before cold (vernalization), the increased levels of H3K27me3 at the HvVRN1 chromatin reduce the transcription of HvVRN1 in barley. Vernalization increases levels of H3K4me3, the active histone modification marks, and decreases levels of H3K27me3 at HvVRN1 (Figure 2) [128]. The novel transcript TaVRN1 ALTERNATIVE SPLICING (VAS), induced by vernalization, functions as a lncRNA derivative from the sense strand of the TaVRN1 gene to regulate TaVRN1 transcription during the flowering of winter wheat [129]. Additionally, TaVRN1 is the earliest target of TaVRN-D4 among the TaVRN1, TaVRN2, and TaVRN3 genes [130]. VER2 encodes a jacalin-like lectin and promotes TaVRN1 upregulation by physically interacting with the RNA-binding protein GLYCINE-RICH RNA-BINDING PROTEIN 2 (TaGRP2) after prolonged cold exposure [131]. However, whether the expression of TaVRN-D4 and VER2 is associated with DNA methylation requires further study. as a lncRNA derivative from the sense strand of the TaVRN1 gene to regulate TaVRN1 transcription during the flowering of winter wheat [129]. Additionally, TaVRN1 is the earliest target of TaVRN-D4 among the TaVRN1, TaVRN2, and TaVRN3 genes [130]. VER2 encodes a jacalin-like lectin and promotes TaVRN1 upregulation by physically interacting with the RNA-binding protein GLYCINE-RICH RNA-BINDING PROTEIN 2 (TaGRP2) after prolonged cold exposure [131]. However, whether the expression of TaVRN-D4 and VER2 is associated with DNA methylation requires further study. Histone acetylation is up-regulated in cold-responsive genes like ZmDREB1 in maize under cold stress [132]. Additionally, cold stress induces higher levels of histone acetylation in the OsDREB1b promoter [133]. The level of acetylation is decreased by the up-regulated expression of HISTONE DEACETYLASEs (HDACs) in maize during cold acclimation [132]. MicroRNA (miRNA) is a class of sRNA that plays a critical role in plant growth and development. miRNA398 (miR398) participates in regulating plant responses to low temperatures in winter turnip rape (Brassica rapa L.) [134]. Additionally, the expression of wheat miR398 (tae-miR398) decreases in response to low temperature [135]. It is reported that tae-miR398 regulates cold tolerance by downregulating its target, COPPER-ZINC SU-PEROXIDE DISMUTASE 1 (CSD1). Furthermore, the expression of CSD1 is indirectly regulated by lncRNAs (lncR9A, lncR117, and lncR616). The regulation of miR398 induces a regulatory loop that is critical for cold tolerance in wheat [135]. Genome-wide association studies and annotations should be performed to outline the intricately epigenomic landscape, particularly in cereal crops subject to cold stress.

Conclusions and Perspectives: Improving Cold Tolerance in Wheat
Global food security is a problem of worldwide importance. The rapid increased population and unpredictable climatic events highlight the need to increase crop productivity. Understanding the perception and signaling cascades activated by cold stress response can help develop new technologies that can alleviate yield losses triggered by cold stress. Advances in molecular technologies and a rapidly expanding knowledge of the mechanisms regulating wheat response to cold stress will contribute to improvements in the efficiency of cereal crops. Histone acetylation is up-regulated in cold-responsive genes like ZmDREB1 in maize under cold stress [132]. Additionally, cold stress induces higher levels of histone acetylation in the OsDREB1b promoter [133]. The level of acetylation is decreased by the up-regulated expression of HISTONE DEACETYLASEs (HDACs) in maize during cold acclimation [132]. MicroRNA (miRNA) is a class of sRNA that plays a critical role in plant growth and development. miRNA398 (miR398) participates in regulating plant responses to low temperatures in winter turnip rape (Brassica rapa L.) [134]. Additionally, the expression of wheat miR398 (tae-miR398) decreases in response to low temperature [135]. It is reported that tae-miR398 regulates cold tolerance by downregulating its target, COPPER-ZINC SUPEROXIDE DISMUTASE 1 (CSD1). Furthermore, the expression of CSD1 is indirectly regulated by lncRNAs (lncR9A, lncR117, and lncR616). The regulation of miR398 induces a regulatory loop that is critical for cold tolerance in wheat [135]. Genome-wide association studies and annotations should be performed to outline the intricately epigenomic landscape, particularly in cereal crops subject to cold stress.

Conclusions and Perspectives: Improving Cold Tolerance in Wheat
Global food security is a problem of worldwide importance. The rapid increased population and unpredictable climatic events highlight the need to increase crop productivity. Understanding the perception and signaling cascades activated by cold stress response can help develop new technologies that can alleviate yield losses triggered by cold stress. Advances in molecular technologies and a rapidly expanding knowledge of the mechanisms regulating wheat response to cold stress will contribute to improvements in the efficiency of cereal crops.
Phytohormones are dominating regulatory factors of plant growth, development, and signaling networks involved in various abiotic stress responses. This indicates that Life 2022, 12, 700 9 of 14 phytohormones are associated with the cross-talk between environmental stress signals and plant growth. In addition, a growing body of evidence suggests the vital role of the ROS signaling pathway in plant development and stress response in wheat. However, the regulatory mechanisms of plant hormones and ROS in response to cold stress at the biochemical level are still poorly understood. Building comprehensive regulation networks in phytohormones, ROS signaling, and cold tolerance in wheat requires a combination of transcriptomes, proteomics, and metabolomics methods while analyzing mutants and protein-protein interactions.
Systematic research into epigenetic mechanisms in response to abiotic stress, including cold stress, heat stress, drought stress, and salt stress, must be performed under field conditions where multiple stress factors frequently coexist. Inheritable epigenetic processes and elements such as sRNA and lncRNA regulatory mechanisms, histone modification, and DNA methylation could provide within-generation and trans-generational stress memory. More powerful and versatile tools are needed to study epigenetic mechanisms in cereals like wheat in a trans-generational memory context since these epigenetic variations could improve stress tolerance in the offspring.
To successfully develop varieties equipped for cold stress, it is necessary to identify the extent of genetic variation for these traits in wheat. Therefore, future work must identify core components involved in the wheat cold signaling pathway that improve cold tolerance in wheat and increase its production in cold temperatures.