Advertisement

The occurrence and accumulation of d-pinitol in fenugreek (Trigonella foenum graecum L.)

  • Lesław Bernard Lahuta
  • Joanna Szablińska
  • Monika Ciak
  • Ryszard Józef Górecki
Open Access
Original Article
  • 205 Downloads

Abstract

This article presents changes in concentrations of d-pinitol (and other cyclitols as well as low molecular weight carbohydrates) in vegetative and reproductive organs of fenugreek (Trigonella foenumgraecum L.) during an entire plant growing period. d-Pinitol was the major cyclitol in all tested organs, representing 43–94% of total cyclitols and 2–77% of total soluble carbohydrates. The highest concentration of d-pinitol was found in pods (14–23 mg g−1 of dry weight, DW), lower in leaves and stems (5–20 and 9–10 mg g−1 DW, respectively), and the lowest in maturing seeds (2–5 mg g−1 DW). Although maturing seeds accumulate α-d-galactosides of d-pinitol (galactosyl pinitols, up to 6.6 mg g−1 DW), the major storage sugars were raffinose family oligosaccharides (RFOs, 65.37 mg g−1 DW). Both RFOs and galactosyl pinitols are hydrolyzed during seed germination, releasing sucrose and d-pinitol, respectively. Accumulation of free galactose was not detected. Owing to the high concentration of d-pinitol (up to 23.70 mg g−1 DW) and low concentration of soluble sugars, developing pods seem to be the best source of d-pinitol.

Keywords

Fenugreek d-pinitol Galactosides Pod Seed Shoot 

Introduction

Fenugreek (Trigonella foenum-graecum L., Family: Fabaceae) is a herb grown widely in India, Egypt and the Middle East. It is one of the oldest medicinal plants, whose seeds, leaves or whole plants are used to prepare powders and extracts for medicinal use, as well as to make vegetable and food additives (Acharya et al. 2006; Zandi et al. 2015). Today, fenugreek is considered as one of the leading functional foods in markets in Asia, Africa and in the Mediterranean countries. Fenugreek seeds contain substantial amounts of several bioactive compounds, indicating therapeutic and medicinal properties (Aher et al. 2016). It contains beneficial metabolites, including galactomannans (mucilaginous fibers), pyridine alkaloids (mostly trigonelline), steroidal sapogenins (spirostanols), amino acid 4-hydroxyisoleucine, antioxidants and flavonoids (Zandi et al. 2015). Thus, fenugreek seeds/leaves (or extracts) possess antioxidant, anti-inflammatory, hypocholesterolemic, anti-diabetic and hypoglycemic properties. Unripe, mature and germinating seeds of fenugreek have the potential to be used in treatment of diabetes (Gaddam et al. 2015), obesity (Basch et al. 2003) and cancer (Lin et al. 2013). Fenugreek seed extracts are tested (Deshpande et al. 2016) and used for treatment of some metabolic disorders (Rao et al. 2016). Possible therapeutic mechanisms and biological effects of some seed compounds were last summarized in reviews concerning the health-promoting properties of fenugreek seeds (Zandi et al. 2015; Aher et al. 2016).

Unexpectedly, data on the presence of myo-inositol and d-pinitol (both cyclitols indicating healthful properties and therapeutic activity similar to properties of fenugreek seeds/extracts) in fenugreek seeds and plants are rare. The potential role and therapeutic effects of myo-inositol (cyclitol ubiquitous in plants) in metabolic diseases were last reviewed by Croze and Soulage (2013) and Muscogiuri et al. (2016). Myo-inositol derivative, d-pinitol (1d-3-O-methyl-chiro-inositol) also reveals anti-diabetic (Do et al. 2008), anti-hyperglycemic (Hernández-Mijares et al. 2013), anti-oxidative (Nascimento et al. 2006), anti-inflammatory (Sivakumar et al. 2010), lipid lowering (Choi et al. 2009), anti-cancer (Sethi et al. 2008; Lin et al. 2013) and immunosuppressive (Chauhan et al. 2011) properties. Thus, it can be supposed that therapeutic effects of fenugreek seeds and plants (Aher et al. 2016) partially arise from the biological properties of d-pinitol. Thus far, d-pinitol in fenugreek has been detected only in seeds (Shang et al. 2002).

Changes in the content of sucrose, reducing sugars and raffinose family of oligosaccharides (RFOs: raffinose, stachyose and verbascose) in developing (Singh et al. 1994), mature and germinating fenugreek seeds were reported earlier (Reid 1971). However, no data are available on the occurrence and accumulation of d-pinitol and its α-d-galactosides (galactosyl pinitols) in fenugreek plants. In our preliminary study, d-pinitol and galactosyl pinitols were identified as an important share of soluble carbohydrates (accounting for 10% of total soluble carbohydrates) in mature fenugreek seeds (unpublished data). The current study is the first one to have demonstrated changes in the concentration of d-pinitol (and other cyclitols as well as low molecular weight carbohydrates) in vegetative and reproductive organs of fenugreek plants during a whole plant growing period. Moreover, our results can support a choice of developing pods, seeds, germinating seeds or seedlings to be used as ingredients in functional foods (Thomas et al. 2011) and/or as a new source for isolation of d-pinitol (Poongothai and Sripathi 2013).

Materials and methods

Plant material

Seeds of fenugreek (Trigonella foenum-graecum L.) were obtained from the W. Legutko Seed Breeding and Production Company (Poland).

Germinating seeds and seedlings

Mature fenugreek seeds (200 seeds in each of 20 replicates) were placed on wet sheet germination paper (Eurochem BGD, Poland), rolled and transferred into 250 ml glass cylinders. After addition of 50 ml of redistilled water, cylinders were incubated in a germination chamber (ILW 115-T STD, Pol-Eko-Aparatura, Poland) at 22 °C in the dark for 5 days. Seedlings were collected at 24 h intervals, weighed, frozen in liquid nitrogen, stored at − 76 °C (for a week) and freeze dried for 48 h (freeze dryer Alpha 1-2LD, Christ, Germany). The water content in tissues was calculated by subtracting the dry weight (DW, after freeze drying) from the fresh weight (FW) of the tissues.

Vegetative organs

For analyses of low molecular weight carbohydrates in early developing plants, fenugreek seeds were sown (in March) in 1 dm3 pots (five per pot), containing a mixture of soil and sand (3:1), and cultivated in a greenhouse (up to 30th day after sowing) without artificial illumination. After emergence, cotyledons grew into leaves and during the subsequent 6 weeks, plants developed six true leaves, reaching 10–15 cm in height. Cotyledons, leaves (blades with the petiole), the apical bud and stem were collected separately from ten plants (in each of the three replicates), weighed and freeze-dried.

Fenugreek was planted in April 2016, in an experimental field near Olsztyn (53.66°N, 20.48°E) in north-eastern Poland. Vegetative organs (leaves with leaf petioles and stems, separately) were separated from the stems of whole shoots, weighed and frozen in liquid nitrogen. Shoots were collected five times: at the onset of the reproductive development (at 56 day after sowing, DAS), then at 76, 86, 116 DAS and finally at 126 DAS (maturation of seeds). At each harvest time, leaves (fully opened trifoliolates with petioles) and stems were separated from shoots (ten plants in each of three replicates), weighed, frozen in liquid nitrogen and freeze-dried.

Pods and maturing seeds

Pods with seeds were collected from July–September, at six stages of seed development, starting from day 30 after flowering (DAF) up to full maturity of seeds (80 DAF). Pods were collected from the first and second node. Pods (20 at each harvest time) and isolated seeds (Online resources, Plate 1) were weighed separately and freeze-dried.

Analyses of soluble carbohydrates

Dry samples of plant tissues were pulverized in a mixer mill (MM200, Retsch, Verder Group, Netherlands). Soluble carbohydrates were extracted from 40 to 45 mg of flour with 800 µL of ethanol: water (1:1, v/v, at 90 °C for 30 min with continuous shaking at 300 rpm), containing 100 µg of xylitol (internal standard). Homogenates were centrifuged (20,000g at 4 °C for 20 min) and aliquots (400 µL) of clear supernatants were deionized (with a 300 µL of a mixture of Dowex ion-exchanger resins) and brought to dryness in a speed vacuum rotary evaporator. The dry residues were derivatized with a 200 µL of mixture of TMSI/pyridine (1:1, v/v) at 80 °C for 45 min. The TMS derivatives of soluble carbohydrates were analyzed with the high-resolution gas chromatography method on a capillary column (Rtx-1, 15 m length, 0.25 mm diameter, 0.1 µm thickness of 100% dimethyl polysiloxane layer, Restek, USA) in a gas chromatograph (GC2010, Shimadzu, Japan), under conditions described previously (Lahuta and Górecki 2011). The carbohydrate content was calculated using the internal standard method. Standards of carbohydrates (fructose, galactose, glucose, sucrose, maltose, raffinose, stachyose and verbascose), xylitol and myo-inositol were purchased from Sigma (USA). Standards of other cyclitols (d-pinitol, d-chiro-inositol and d-ononitol) were obtained from Fine Chemicals (New Zealand), and galactinol was supplied by Wako Pure Chemicals Industries Ltd. (Japan). Standards of α-d-galactosides of d-pinitol (mono-, di- and tri-galactosyl pinitol A), commercially unavailable, were extracted from seeds of Vicia villosa Roth. (winter vetch) and purified as described previously (Szczeciński et al. 2000). Standards of α-d-galactosides of d-chiro-inositol (fagopyritols) were a gift from Prof. Marcin Horbowicz (Siedlce University of Natural Sciences and Humanities, Poland).

Statistical analysis

The results were presented (in mg g−1 of dry weight, DW) as means from three replications ± SD. The significance of differences in the concentrations of various carbohydrates was determined by one-way ANOVA with the Tukey’s test.

Results

The GC analyses of soluble carbohydrates revealed the presence of sugars (glucose, fructose, galactose and sucrose), cyclitols (myo-inositol, d-pinitol, d-ononitol and d-chiro-inositol), raffinose family oligosaccharides (RFOs: raffinose, stachyose and verbascose) and galactosyl cyclitols in all parts of fenugreek plants (Fig. 1). However, RFOs and galactosyl cyclitols were accumulated mainly in maturing seeds (Fig. 1a), being detected in trace amounts in vegetative tissues (leaves and stems) (Fig. 1b, c). d-Pinitol was the major cyclitol, regardless of the type of tissues and plant developmental stage. The qualitative and quantitative differences in soluble carbohydrates were found between plant organs and developmental stages.

Fig. 1

The GC chromatograms of TMS derivatives of soluble carbohydrates extracted from dry mature seeds (a), leaves and stems at 86 DAS (b, c, respectively) of fenugreek (Trigonella foenum graecum L.) plants. Denotations: 1—xylitol (internal standard), 2—d-pinitol, 3—myo-inositol, 4—sucrose, 5—GPA, 6—GPB, 7—galactinol, 8—raffinose, 9—ciceritol, 10—stachyose, 11—TGPA, 12—verbascose, 13 and 14—fructose, 15 and 16—glucose, 17 and 18—maltose

Changes in carbohydrates during seed germination

The concentration of total soluble carbohydrates (TSCs) in fenugreek seeds (used for germination) was 88.56 mg g−1 DW (Table 1). RFOs, sucrose and d-pinitol were the major soluble carbohydrate (61.40, 6.41 and 4.43 mg g−1 DW, respectively), constituting 81% of TSCs. The prevalent RFO was stachyose (85% of total RFOs). Seeds also contained considerable amounts (11.25 mg g−1 DW) of α-d-galactosides of d-pinitol (galactosyl pinitols, GPs), with galactosyl pinitol A (GPA) being prevalent (5.87 mg g−1 DW, Online Resources, Table 1). Concentrations of the other identified galactosyl cyclitols: α-d-galactosides of myo-inositol (galactinol and di-galactosyl myo-inositol) and α-d-galactosides of d-chiro-inositol (fagopyritols) were much lower (2.28 and 0.40 mg g−1 DW, respectively) than those of GPs (Online Resources, Table 1). Monosaccharides (glucose, fructose) and myo-inositol (Table 1) occurred at much lower concentrations (1.18 and 0.67 mg g−1 DW). Moreover, seeds contained traces of d-chiro-inositol.

Table 1

Changes in the concentration of: total soluble carbohydrates (TSCs), oligosaccharides (RFOs and GPs), major sugars (sucrose, glucose, fructose) and major cyclitols (d-pinitol, myo-inositol,) in fenugreek seeds and seedlings during 5 days of germination (DAI, days after imbibition)

DAI (day)

TSCs*

Oligosaccharides (mg g−1 DW )

Sugars (mg g−1 DW )

Cyclitols

RFOs

GPs

Sucrose

Glucose

Fructose

d-Pinitol

Myo-Inositol

0

88.56 ± 2.21bc

61.40 ± 1.61a

11.29 ± 0.34a

6.41 ± 0.26d

0.12 ± 0.02e

0.81 ± 0.02d

4.43 ± 0.15c

0.67 ± 0.02d

1

70.51 ± 2.21d

42.48 ± 1.71b

9.68 ± 0.18b

9.56 ± 0.69d

0.55 ± 0.21d

0.79 ± 0.23d

4.10 ± 0.09d

0.57 ± 0.02d

2

77.34 ± 4.77 cd

6.30 ± 1.08c

3.38 ± 0.21c

36.65 ± 1.49a

8.67 ± 0.97c

9.69 ± 1.06c

6.32 ± 0.07b

1.40 ± 0.03c

3

105.56 ± 1.23a

0.79 ± 0.09d

0.64 ± 0.01d

28.29 ± 0.67b

36.26 ± 0.53b

25.09 ± 1.41a

7.77 ± 0.10a

2.04 ± 0.06b

4

101.43 ± 6.78ab

0.33 ± 0.01d

0.50 ± 0.01d

16.39 ± 0.37c

44.32 ± 3.28a

23.65 ± 2.75a

8.35 ± 0.09a

2.14 ± 0.04ab

5

78.83 ± 8.20 cd

0.00 ± 0.00d

0.00 ± 0.00e

7.61 ± 0.22d

38.34 ± 4.61ab

18.40 ± 3.04b

8.07 ± 0.15a

2.22 ± 0.07a

Values (in mg g−1 DW) are means (n = 3). Values with different superscripts (a–e) are significantly different (P < 0.05) after a Tukey’s correction for multiple comparisons for each compound separately (valid within columns only)

*TSCs—total soluble carbohydrates include: RFOs (raffinose, stachyose and verbascose), GPs, sugars, cyclitols, galactosides of myo-inositol and galactosides of d-chiro-inositol (presented in online resources, Table 1) and unknown compounds (data not shown)

Dry seeds imbibed very fast—the fresh weight increased three-fold (from 13 up to 40 mg seed−1) during the first day of seed imbibition (Online Resources, Plate 1A). Rapid degradation of RFOs and galactosyl pinitols started at the onset of seed germination (Table 1). Most of oligosaccharides were degraded during the first 2 days of seed germination, and RFOs and GPs were undetected in growing seedlings after the following 3 days (Table 1). The concentrations of sucrose, glucose, fructose and cyclitols (myo-inositol and d-pinitol) significantly increased (P < 0.05) between the first and second day of germination, due to the degradation of oligosaccharides and galactosyl cyclitols (Table 1). The concentration of sucrose was the highest (36.65 mg g−1 DW) after 2 days of seed germination and decreased subsequently (to 7.61 mg g−1 DW, after fifth day of germination, Table 1). Between the second and third day of seed germination, the concentrations of glucose and fructose dramatically increased (from 8.67 to 36.26 and from 9.69 to 25.09 mg g−1 DW, for glucose and fructose, respectively). It is worth noting that the increase in the concentrations of glucose and fructose (Table 1) was much higher than the simultaneous decrease in the concentration of sucrose (from 36.65 to 28.29 mg g−1 DW).

After the first day of seed germination, the concentration of free cyclitols decreased slightly (from 5.13 to 4.75 mg g−1 DW, Table 1), despite the obvious onset of the degradation of all galactosyl cyclitols (by about 10%). The hydrolysis of individual types of galactosyl cyclitols: fagopyritols, galactosides of myo-inositol and galactosyl pinitols, finished after 2, 4 and 5 days of germination (Online Resources, Table 1). Accordingly, the concentrations of free cyclitols increased: d-chiro-inositol—up to 0.10 mg g−1 DW (after second day of germination, not presented data), myo-inositol—up to 2.14 mg g−1 DW (fourth day), and d-pinitol—up to 8.07 mg g−1 DW (fifth day, Table 1).

Carbohydrates in stems and leaves during plant growth

Sucrose, glucose and d-pinitol were the main soluble carbohydrates in vegetative organs of fenugreek plants at the stage of shoot growth (six true leaves) (Online Resources, Table 2). The highest concentration of TSCs (76.61 mg g−1 DW) was found in cotyledons (evolved into the first photosynthetic active leaves), whereas the lowest were in the stem (35.16 mg g−1 DW). Sucrose made up 22–24% of TSCs in the stem, cotyledons and leaves, and 11.29% in the apical bud. Pinitol was the main carbohydrate in the stem (61% of TSCs), leaves (43%) and apical bud (58%, Online Resources, Table 2). The concentration of d-pinitol was higher in stems, leaves and the apical, but (20–26 mg g−1 DW) than in cotyledons (12.29 mg g−1 DW). Moreover, the high concentration of glucose in cotyledons (35.50 mg g−1 DW, 43% of TSCs) is presumably a result of the degradation of transitory starch.

Table 2

Changes in the water content (WC), total soluble carbohydrates (TSCs), predominant sugars (sucrose, glucose, fructose) and cyclitols (d-pinitol, myo-inositol) in stems and leaves of fenugreek plants during generative period of plant vegetation (between 56th and 126th day after sowing, DAS, corresponding with 1–70th day after flowering, DAF)

Plant organ

DAS

DAF

WC (% of FW)

TSCs

Sugars

Cyclitols

Sucrose

Glucose

Fructose

d-Pinitol

Myo-inositol

mg g−1 DW

Stem

56

1*

88.56

32.87 ± 2.76d

7.11 ± 0.11d

11.03 ± 2.30b

0.89 ± 0.24b

9.50 ± 0.09b

0.76 ± 0.01b

 

76

20

79.52

88.74 ± 9.07b

32.54 ± 0.56b

35.87 ± 7.21a

4.70 ± 1.39b

10.19 ± 0.33a

0.82 ± 0.03a

 

86

30

76.01

112.91 ± 8.56a

48.53 ± 1.12a

36.57 ± 4.78a

11.26 ± 2.34a

10.57 ± 0.25a

0.44 ± 0.01c

 

116

60

75.83

51.84 ± 6.28c

22.10 ± 5.39c

12.92 ± 7.31b

4.26 ± 3.16b

9.16 ± 0.28b

0.43 ± 0.03c

 

126

70

72.99

39.41 ± 4.56d

15.54 ± 1.25c

5.37 ± 2.23b

1.31 ± 0.78b

9.01 ± 0.07b

0.39 ± 0.00d

Leaves

56

1*

86.39

26.12 ± 0.62d

8.07 ± 0.14b

1.96 ± 0.27b

0.88 ± 0.18b

5.07 ± 0.09d

3.99 ± 0.10a

 

76

20

85.36

63.03 ± 3.29ab

21.26 ± 0.77a

14.21 ± 1.69a

2.03 ± 0.07b

16.15 ± 0.36b

2.85 ± 0.06b

 

86

30

85.33

70.68 ± 5.61a

26.07 ± 2.44a

15.22 ± 0.98a

4.97 ± 0.30a

14.23 ± 1.37c

1.63 ± 0.14 cd

 

116

60

82.17

60.88 ± 2.47b

13.31 ± 5.52b

12.02 ± 4.26a

6.81 ± 2.17a

17.94 ± 0.50b

1.42 ± 0.04d

 

126

70

81.28

45.09 ± 1.23c

11.10 ± 0.05b

4.71 ± 0.71b

1.96 ± 0.44b

19.84 ± 0.16a

1.76 ± 0.01c

Values are means (n = 3) ± SD. Values with different superscripts (a–d) are significantly different (P < 0.05) after a Tukey’s correction for multiple comparisons for each compound separately (comparisons for stem and leaves separately, valid within columns only)

*One day after full opening of the first flower

In plants collected at the initial stage of flowering (56 day after sowing, DAS) and subsequently up to seed maturation (126 DAS), the concentrations of carbohydrates differed between organs and plant developmental stages. The concentration of TSCs significantly (P < 0.05) increased in both the stem and leaves up to 76–86 DAS, and decreased thereafter (Table 2). The major sugars were sucrose, glucose and fructose. The concentrations of these sugars were higher in the stem than in leaves throughout the entire plant growing period. d-Pinitol and myo-inositol dominated among cyclitols, whereas d-ononitol and d-chiro-inositol were present in trace amounts. The concentration of each cyclitol was higher in leaves than in the stem. Moreover, d-pinitol accumulated in leaves (to 19.84 mg g−1 DW, at 126 DAS), remaining on a relatively stable level in the stem (9–10 mg g−1 DW). Opposite to changes in d-pinitol, the concentration of myo-inositol decreased during plant growth in both leaves and stems (Table 2). Changes in the profiles of soluble carbohydrates in fenugreek shoots indicate that at 86 DAS plants were fully developing, whereas 30 days later, the ability of plants to synthesize soluble carbohydrates decreased (Table 2). At 126 DAS, senescence of leaves from the bottom of plants started, and those leaves were not collected for analyses.

Changes in soluble carbohydrate during pod and seed maturation

The first sample of pods with developing seeds was collected at day 30 after flowering (DAF), corresponding to 86 DAS (Online Resources, Plate 1B). At this stage of the development of pod and seeds, the concentration of TSCs was the highest (111.08 and 181.76 mg g−1 DW, in pods and seeds, respectively, Table 3). Sugars and cyclitols dominated among TSCs (Table 3). However, glucose and fructose dominated in pods (42.35 and 10.26 mg g−1 DW, respectively), whereas sucrose was prevalent in seeds (149.75 mg g−1 DW). During pod maturation, the concentration of TSCs gradually decreased (to 20.95 mg g−1 DW, at 70 DAF) due to a decrease in the concentrations of sugars: sucrose and glucose (Table 3 and Online Resources Table 3). Concentrations of cyclitols, RFOs and galactosyl cyclitols fluctuated. The concentrations of d-pinitol (constituting above 94% of total cyclitols), RFOs and galactosyl pinitols increased up to 50 DAF and diminished thereafter (Table 3). However, at 70 DAF, GPs were absent, RFOs were present in trace amounts only, whereas the level of d-pinitol was eight-fold higher (16.23 mg g−1 DW) than that of total sugars (Table 3), including sucrose and glucose (Online Resources, Table 3).

Table 3

Changes in the fresh weight (FW), water content (WC), concentration of: total soluble carbohydrates (TSCs), d-pinitol, total sugars, total RFOs and total galactosyl pinitols (GPs) in maturing pods and seeds of fenugreek

Plant organ

DAF

FW (mg part−1)

WC (% FW)

TSCs (mg g−1 DW )

d-Pinitol (mg g−1 DW )

Total sugars (mg g−1 DW )

Total RFOs (mg g−1 DW )

Total GPs (mg g−1 DW )

Pod

30

533.14

81.21

111.08 ± 11.83a

14.49 ± 0.68c

89.41 ± 10.70a

3.61 ± 0.10c

0.15 ± 0.01c

 

40

529.36

82.48

43.91 ± 2.43b

21.94 ± 0.35b

14.57 ± 2.05b

5.12 ± 0.04b

0.21 ± 0.01b

 

50

490.85

83.82

48.14 ± 0.34b

23.70 ± 0.44a

11.05 ± 1.36b

9.94 ± 0.48a

0.39 ± 0.03a

 

60

343.48

80.87

26.37 ± 0.59c

19.66 ± 1.01a

3.15 ± 0.16b

1.41 ± 0.08d

0.17 ± 0.01bc

 

70

75.60

58.33

20.95 ± 0.51c

16.23 ± 0.66c

2.25 ± 0.13b

0.08 ± 0.01e

0.00 ± 0.00d

Seed

30

31.54

77.52

181.16 ± 7.53a

5.30 ± 0.54a

157.15 ± 7.77a

1.44 ± 0.08e

0.00 ± 0.00d

 

40

33,88

71.99

90.25 ± 8.16d

3.84 ± 0.34b

66.51 ± 5.84b

9.84 ± 0.59d

0.10 ± 0.02d

 

50

35.23

65.37

77.75 ± 2.43d

2.61 ± 0.07c

44.30 ± 1.22c

21.25 ± 0.82c

0.19 ± 0.01d

 

60

36.58

59.85

95.99 ± 0.76 cd

2.22 ± 0.04c

35.65 ± 0.10 cd

50.05 ± 1.40b

1.41 ± 0.07c

 

70

26.17

58.52

112.03 ± 0.83b

2.27 ± 0.04c

28.68 ± 0.37d

68.54 ± 1.16a

3.41 ± 0.06b

 

80a

13.65

18.28

106.15 ± 1.66bc

4.44 ± 0.11b

15.41 ± 0.42e

65.37 ± 2.44a

6.56 ± 0.25a

Values are means (n = 3) ± SD

Values with different superscripts (a–e) are significantly different (P < 0.05) after a Tukey’s correction for multiple comparisons for each compound separately (comparisons for pods and seeds separately valid within columns only)

aMature seeds

In developing fenugreek seeds, the concentration of d-pinitol was the highest at 30 DAF (5.30 mg g−1 DW) and decreased later on, down to 2.22–2.27 mg g−1 DW (at 60–70 DAF, Table 3), most likely as a result of successive accumulation of galactosyl pinitols. Galactosyl pinitol A (GPA) was detected in fenugreek seeds at 40 DAF, di-galactosyl pinitol A (ciceritol)—at 60 DAF, and tri-galactosyl pinitol A TGPA—at 80 DAF (Online Resources, Table 3). However, the biosynthesis of galactosyl pinitols started later than that of RFOs and was less intensive.

Galactinol, raffinose and stachyose were detected in seeds at 30 DAF (Online Resources, Table 3). Raffinose and stachyose were accumulated up to 70 DAF, whereas the accumulation of verbascose, starting between 40 and 50 DAF, was continued to full seed maturity. In mature seeds, stachyose was the predominant RFO. It should be underlined that the accumulation of the highest homologues of GPA and raffinose (TGPA and verbascose, respectively) occurred most intensively during seed natural desiccation (between 70 and 80 DAF).

Discussion

The composition of soluble carbohydrates in mature seeds of fenugreek was similar to that in soybean (Yasui and Ohashi 1990). The prevalent RFO in fenugreek seeds was stachyose (85% of total RFOs), which was consistent with data obtained by Reid (1971). Our data also indicate presence of considerable amounts of galactosyl pinitols (accounting for 13% of TSCs, Table 1), with prevalent GPA (Online Resources, Table 1). Changes in the concentrations of RFOs, sucrose and monosaccharides in germinating fenugreek seeds, found in our study (Table 1), correspond well with the results obtained earlier by Dirk et al. (1999) and are generally similar to the ones occurring in germinating legume seeds (Górecki et al. 2000; Kadlec et al. 2008; Martin-Cabrejas et al. 2008). Moreover, the fast degradation of galactosyl cyclitols confirms their role as reserve materials important for early germination of fenugreek seeds, same as in seeds of winter vetch (Lahuta and Goszczyńska 2009). The hydrolysis of RFOs and galactosyl cyclitols in fenugreek seeds coincided with an increase in the concentrations of sucrose (initially) and then (after the second day of germination) monosaccharides (glucose and fructose), myo-inositol and cyclitols. However, it can be expected that the level of galactose released from α-d-galactosides (both RFOs and galactosyl cyclitols) should be much higher than estimated in our study (up to 2.8 mg g−1 DW, at second day of germination, data not presented). Initially, galactose is released from RFOs and galactosyl cyclitols (Lahuta and Goszczyńska 2009), but later on it originates from galactomannans (deposited in the endosperm tissues), which are hydrolyzed after 3–4 days of a fenugreek seedling’s development (Reid 1971; Buckeridge and Dietrich 1996; Dirk et al. 1999). In starchless seeds of fenugreek, galactomannans are the main carbohydrate reserves, representing up to 50% of dry seed weight (Zandi et al. 2015). However, in our study, mannose and galactose were not accumulated during fenugreek seed germination and seedling development. Similarly, no accumulation of galactose and/or mannose in germinating fenugreek seeds was detected by Dirk et al. (1999). A much lower concentration of galactose (0.16–2.82 mg g−1 DW) than that of glucose and fructose (Table 1) found in our study can be explained by the fast metabolic conversion of both galactose and mannose into substrates (UDP-glucose and fructose-6-phosphate, respectively) used for the synthesis of sucrose and cell wall compounds of growing seedling (Buckeridge et al. 2000).

The high concentration of sucrose after 2 days of seed germination (Table 1) can be a signal for both elongation of the hypocotyl (Penfield et al. 2004) and the synthesis of starch in cotyledons, as it was suggested by Dirk et al. (1999). Synthesis of starch in cotyledons is initiated early, i.e., during the first several hours of seed germination (Srivastava and Kayastha 2014), it increases up to 50–60 h and later on decreases (Dirk et al. 1999). Starch degradation in cotyledons occurs by the action of β-amylase (Srivastava and Kayastha 2014), releasing maltose. Maltose was detected at 1.25 mg g−1 DW after the second day of fenugreek seed germination, after which it gradually increased, up to 2.27 mg g−1 DW.

The significantly innovative aspect of our research lies in the determination of qualitative and quantitative changes in cyclitols and their galactosides during fenugreek seed germination and plant vegetation. After the first day of seed germination, the concentration of free cyclitols decreased slightly (Table 1), despite the obvious onset of the degradation of all galactosyl cyclitols. However, the utilization of cyclitols at initial stages of seed germination remains to be explained. Leakage of free d-pinitol from imbibed seeds is possible, as it has been documented in soybean (Nordin 1984). The hydrolysis of individual types of galactosyl cyclitols: fagopyritols, galactosides of myo-inositol and galactosyl pinitols, finished after 2, 4 and 5 days of germination (Online Resources, Table 1). Accordingly, the concentrations of free cyclitols increased (Table 1). In germinating yellow lupin seeds, the concentration of d-pinitol decreased during the first 2 days of germination, and an increase in d-chiro-inositol (a product of d-pinitol demethylation) was noted (Zalewski et al. 2007). In germinating seeds of soybean (Gu et al. 2017), yellow lupin (Górecki et al. 1997) and winter vetch (Lahuta and Górecki 2011), the concentration of d-pinitol increased due to the degradation of galactosyl pinitols. However, high differences in concentrations of d-pinitol occur between species. A higher concentration of d-pinitol (up to 40–80 mg g−1 DW) was found in the epicotyl and root of 7-day-old seedlings of winter vetch (Lahuta and Górecki 2011).

The average content of d-pinitol, as free and bound in total galactosyl pinitols (calculated on the basis of the molecular formula of individual galactosyl pinitols), in dry fenugreek seeds was 0.05 and 0.06 mg seed−1, respectively. During 4 days of germination, the increase in the content of free d-pinitol in seedlings by 1.35 mg (to 1.40 mg seedling−1) was much higher than the content of d-pinitol released from galactosyl pinitols, which indicates that the de novo synthesis of d-pinitol is initiated at early stages of fenugreek seedling development. Data documenting the beginning of d-pinitol accumulation during seedling development are rare (Górecki et al. 1997; Lahuta and Górecki 2011). The biosynthesis of d-pinitol occurs through four-step conversion of glucose phosphate precursor (Ishitani et al. 1996). At first, myo-inositol is synthesized from glucose-6-phosphate in two reactions catalyzed by myo-inositol 1-phosphate synthase (MIPS) and myo-inositol monophosphatase (IMP). The biosynthesis of d-pinitol occurs through transfer of the methyl group from S-adenosyl-l-methionine (SAM) to myo-inositol (in reaction catalyzed by high substrate specify inositol methyl transferase, IMT), producing d-ononitol (1d-4-O-methyl-chiro-inositol), and then ononitol is converted to d-pinitol through an epimerase reaction (Dittrich and Brandl 1987). However, that epimerase(-s) has yet to be isolated and characterized. The methyl transfer rate from SAM to myo-inositol can be regulated by availability of myo-inositol, presence of functional IMT and the intracellular ratio of SAM to SAH (S-adenosyl homocysteine), a potent competitive inhibitor of SAM-dependent methyl transferase reactions (Sengupta et al. 2008).

Many reports documented accumulation of free myo-inositol, its isomers and methylated ethers (mainly d-pinitol and d-ononitol) in different plant species under abiotic stresses, like drought (Ahn et al. 2011; Sanchez et al. 2012; Yates et al. 2014), high salinity (Kumari et al. 2015; Slama et al. 2015), elevated (Deslauriers et al. 2014) or lowered temperature (Turner and Pollock 1998; Dauwe et al. 2012). The expression of IMT genes seems to be stress up-regulated (Vernon and Bohnert 1992; Sengupta et al. 2008). Several studies, conducted on transgenic plants expressing MIPS or IMT genes (and accumulating elevated amounts of myo-inositol or d-ononitol/d-pinitol, respectively), confirm the protective role of cyclitols under stress conditions (Sheveleva et al. 1997; Sengupta et al. 2008; Patra et al. 2010; Joshi et al. 2013; Kaur et al. 2013). Cyclitols can function as “compatible solutes” (Valluru and Van den Ende 2011), osmolytes (Matos et al. 2010) and antioxidants (scavenging of hydroxyl radicals, Nishizawa et al. 2008). Moreover, cyclitols can regulate the antioxidant systems activity (Tan et al. 2013; Zhang et al. 2017) and facilitate ion transport under salinity stress (Sengupta et al. 2008). However, under stress conditions affecting osmotic potential in cells, the short-term turnover of cyclitols is very low (Smith and Philips 1982; Merchant and Richter 2011). In leaves and stems of different plant species, both herbaceous (Bieleski 1994; Streeter et al. 2001) and woody plants (Adams et al. 2010; Warren et al. 2011; Dauwe et al. 2012; Lintunen et al. 2016), the concentration of d-pinitol (or other species-specific cyclitols) is as high or higher than that of sucrose, the main photoassimilate transported from sources to sink tissues. Pinitol can be translocated through both phloem (Kordan et al. 2011) and xylem (Krishnan et al. 2011), and diurnal changes in its concentration were detected (Krishnan et al. 2011). Thus, it is most likely that an important role of d-pinitol is to act as a carbon sink, enabling the allocation of carbon from carbohydrates and supporting primary metabolism during changes in resource availability at changing environmental conditions (Merchant and Richter 2011). Indeed, the differences in the concentrations of d-pinitol between organs of juvenile fenugreek plants (higher in stems, leaves and the apical bud − 20–26 mg g−1 DW, than in cotyledons − 12.29 mg g−1 DW, Online Resources, Table 2) seem to provide confirmation of the upward transport of d-pinitol from sources (leaves, cotyledons) to growing tissues (apical bud), as it was suggested by Kawai et al. (1985). During fenugreek plant’s vegetation, d-pinitol and myo-inositol dominated among cyclitols, whereas d-ononitol and d-chiro-inositol were present in trace amounts. The concentration of each cyclitol was higher in leaves than in the stem. The low concentration of d-ononitol, as intermediate in biosynthesis of d-pinitol (Dittrich and Brandl 1987), indicates efficient conversion of d-ononitol into d-pinitol. However, the lowest concentration of d-chiro-inositol (in all parts of fenugreek shoot), a product of d-pinitol demethylation, needs explanation in the future. Moreover, there are no data documenting metabolic route of d-pinitol in sink tissues.

The accumulation of d-pinitol in leaves throughout fenugreek plant growth (up to 19.84 mg g−1 DW, at 126 DAS) and its relatively stable level in the stem (9–10 mg g−1 DW) can be explained by the lowering sink demand of ageing organs, like roots and senescent leaves. Contrary to changes in d-pinitol, the concentration of myo-inositol decreased during plant growth in both leaves and stems (Table 2). The concentration of d-pinitol in soybean (Streeter et al. 2001) and Cicer arietinum (Matos et al. 2010) was similar to the one determined in fenugreek in our study, and ranged from 15 to 30 mg g−1 DW of leaf blades, depending on genotypes and rainfall levels. Moreover, there was a d-pinitol gradient in soybean leaf blades from the bottom to the top of the plant, suggesting translocation of d-pinitol from lower to upper nodes during plant development (Streeter et al. 2001). In leaves of red clover, the concentration of d-pinitol ranged from 14 to 20 mg g−1 DW under optimal water conditions, and up to 110–120 mg g−1 DW under drought stress (Yates et al. 2014). An increase in the d-pinitol concentration under drought stress was also found in soybean (Streeter et al. 2001), Cicer arietinum (Matos et al. 2010), alfalfa (Kang et al. 2011) and Medicago truncatula (Zhang et al. 2017). In our study, fenugreek plants grew under suitable watering conditions. Changes in the profiles of soluble carbohydrates in fenugreek shoots indicate that at 86 DAS plants were fully developing, whereas 30 days later, the ability of plants to synthesize soluble carbohydrates decreased (Table 2), presumably due to lowering photosynthetic activity.

The pods, covering developing seeds, play an important dual function: (i) they participate in the regulation of transport of water, minerals and nutrients from the shoot to seeds, and (ii) as transitory photosynthetic active organs they synthesize additional pool of reduced carbon supplying nutrition of seeds (Patrick and Offler 2001). In maturing pods of fenugreek, the concentration of TSCs gradually decreased from 89.41 (at 30 DAF) to 2.25 mg g−1 DW (at 70 DAF), as result of a decrease in levels of sucrose and glucose (Online Resources Table 3). It can mean that sugars were transferred into developing seeds (Weber et al. 1997). The concentrations of d-pinitol (constituting above 94% of total cyclitols), RFOs and galactosyl pinitols increased up to 50 DAF and diminished thereafter (Table 3). The physiological significance of the transitory biosynthesis of RFOs and galactosyl pinitols in tissues of developing pods remains to be explained. Neither type of α-d-galactosides is transported through the phloem in legume plants. Moreover, RFOs and galactosyl pinitol were degraded during pods desiccation in contrast to their fate in developing seeds. Additionally, a relatively high concentration of d-pinitol in naturally aged (and dehydrated) pods remains intriguing. It can be expected that during seed maturation, most of soluble carbohydrates in pods, not only sugars (Lemoine et al. 2013) but also cyclitols, should be remobilized from pod tissues and transported into developing/maturing seeds. In fact, the concentration of d-pinitol in seeds doubled between 70 and 80 DAF, that is at the later stages of seed maturation.

The increasing concentration of d-pinitol in maturing seeds can lead to an increase in the biosynthesis of galactosyl pinitols in embryos, as it was earlier documented in developing seeds of soybean (Gomes et al. 2004), vetch (Lahuta et al. 2005a, b, 2010), pea (Lahuta and Dzik 2011), buckwheat (Ma et al. 2005) and grains of cereals (Lahuta and Goszczyńska 2010). In developing fenugreek seeds, the concentration of d-pinitol was the highest at 30 DAF (5.30 mg g−1 DW) and decreased later on, down to 2.22–2.27 mg g−1 DW (at 60–70 DAF, Table 3), most likely as a result of successive accumulation of galactosyl pinitols. Galactosyl pinitol A (GPA) was detected in fenugreek seeds at 40 DAF, di-galactosyl pinitol A (ciceritol)—at 60 DAF, and tri-galactosyl pinitol A TGPA—at 80 DAF (Online Resources, Table 3). The biosynthesis of galactosyl pinitols started later than that of RFOs and was less intensive, like in seeds of Vicia spp. (Lahuta et al. 2005), yellow lupine (Górecki et al. 1997), soybean (Obendorf et al. 1998) and chickpea (Gangola et al. 2014). Typically, seeds accumulate more RFOs than galactosyl pinitols (Obendorf and Górecki 2012), except seeds of some Vicia spp. accumulating equal amounts of both RFOs and galactosyl pinitols (Lahuta 2006) or more galactosyl pinitols than RFOs (Lahuta et al. 2010).

Galactinol (a galactosyl donor in the biosynthetic pathway for both RFOs and GPs; Peterbauer and Richter 2001), raffinose and stachyose were detected in seeds at 30 DAF (Online Resources, Table 3). Raffinose and stachyose were accumulated up to 70 DAF, whereas the accumulation of verbascose, starting between 40 and 50 DAF, was continued to full seed maturity. In mature seeds, stachyose was the predominant RFO, as it was noted earlier (Reid 1971). It should be underlined that the accumulation of the highest homologues of GPA and raffinose (TGPA and verbascose, respectively) occurred most intensively during seed natural desiccation (between 70 and 80 DAF). Although the concentration of d-pinitol also increased (almost two-fold) during seed desiccation (Table 3), the possible utilization of d-pinitol for the synthesis of elevated amounts of its galactosides was presumably restricted by the water loss and lowering rate of cellular metabolism.

It can be speculated that the preferential accumulation of RFOs was a result of a much higher concentration of sucrose than d-pinitol in seeds throughout seed development and maturation (Table 3 and Online Resources Table 3). Both sucrose and d-pinitol are the first galactose acceptors in the biosynthetic pathway of RFOs and GPs. Thus, the higher concentration of sucrose enabled preferential biosynthesis of raffinose and, later on, stachyose and verbascose (Peterbauer and Richter 2001; Obendorf and Górecki 2012).

Some observations in our study: (1) relatively high concentration of d-pinitol in all parts of fenugreek shoot, (2) much lower changes in its concentration in leaves, stem and pods, as compared to the concentrations of sucrose and monosaccharides and (3) occurrence of d-chiro-inositol, a product of d-pinitol demethylation at extremely low concentration, lead us to the conclusion that the metabolic route of d-pinitol utilization in sink tissues needs explanation.

Conclusions

Concentrations of d-pinitol change in different plant organs during fenugreek growth and development. The increase in d-pinitol at early stages of seed germination is a result of the hydrolysis of galactosyl pinitols, but later on it occurs as a result of the de novo synthesis of d-pinitol in photosynthetic active organs, i.e., cotyledons/leaves. d-Pinitol seems to be transported (like sucrose) from the source (leaves) to sink organs (pods/seeds). d-Pinitol reaches the highest concentration in pods at 96–106 DAS (40–50 DAF), but 3 weeks later its content peaks in leaves—even at 116–126 DAS (60–70 DAF). Because of the high concentration of d-pinitol and the simultaneously low level of low molecular carbohydrates, pods seem to be the best source of d-pinitol.

Notes

Acknowledgements

This work was financed from the grant titled: “Cultivated plants and natural products as a source of biologically active substances destined for the production of cosmetic and pharmaceutical products as well as diet supplements” (No. BIOSTRATEG2/298205/9/NCBR/2016) awarded by the National Center for Research and Development (Poland).

Author contribution statement

LBL: determining the purpose of research, developing research methodology, participating in chromatographic analyzes, interpretation and discussion of results. JS: collection and analyses (using gas chromatography method) of plant material. MC: collection and analyses of plant material. RJG: discussion of results.

Supplementary material

11738_2018_2734_MOESM1_ESM.docx (16 kb)
Supplementary material 1 (DOCX 16 KB)
11738_2018_2734_MOESM2_ESM.doc (644 kb)
Supplementary material 2 (DOC 643 KB)

References

  1. Acharya SN, Thomas JE, Basu SK (2006) Fenugreek: an “old world” crop for the “new world”. Biodiversity 7(3–4):27–30CrossRefGoogle Scholar
  2. Adams MA, Simon J, Pfautsch S (2010) Woody legumes: a (re)view from the South. Tree Physiol 30:1072–1082CrossRefPubMedGoogle Scholar
  3. Aher RR, Belge SA, Kadam SR, Kharade SS, Misal AV, Yeole PT (2016) Therapeutic importance of fenugreek (Trigonella foenum-graecum L.): a review. J Plant Sci Res 3(1):149Google Scholar
  4. Ahn C, Park U, Park PB (2011) Increased salt and drought tolerance by d-ononitol production in transgenic Arabidopsis thaliana. Biochem Biophys Res Comm 415:669–674CrossRefPubMedGoogle Scholar
  5. Basch E, Ulbricht C, Kuo G, Szapary P, Smith M (2003) Therapeutic applications of fenugreek. Altern Med Rev 8(1):20–27PubMedGoogle Scholar
  6. Bieleski RL (1994) Pinitol is a major carbohydrate in laves of some coastal plants indigenous to New Zealand. N Zeal J Bot 32:73–78CrossRefGoogle Scholar
  7. Buckeridge MS, Dietrich SMC (1996) Mobilisation of the raffinose family oligosaccharides and galactomannan in germinating seeds of Sesbania marginata Benth. (Leguminosae-Faboidae). Plant Sci 17:33–43CrossRefGoogle Scholar
  8. Buckeridge MS, dos Santos HP, Tine MAS (2000) Mobilisation of storage cell wall polysaccharides in seeds. Plant Physiol Biochem 38(1/2):141–156CrossRefGoogle Scholar
  9. Chauhan PS, Gupta KK, Bani S (2011) The immunosuppressive effects of Agyrolobium roseum and pinitol in experimental animals. Int Immunopharmacol 11:286–291CrossRefPubMedGoogle Scholar
  10. Choi MS, Lee MK, Jung UJ, Kim HJ, Do GM, Park YB, Jeon SM (2009) Metabolic response of soy pinitol on lipid-lowering, antioxidant and hepatoprotective action in hamsters fed-high fat and high cholesterol diet. Mol Nutr Food Res 53:751–759CrossRefPubMedGoogle Scholar
  11. Croze ML, Soulage CO (2013) Potential role and therapeutic interests of myo-inositol in metabolic diseases. Biochimie 95:1811–1827CrossRefPubMedGoogle Scholar
  12. Dauwe R, Holliday JA, Aitken SN, Mansfield SD (2012) Metabolic dynamics during autumn cold acclimation within and among populations of Sitka spruce (Picea sitchensis). New Phytol 194:192–205CrossRefPubMedGoogle Scholar
  13. Deshpande P, Mohan V, Thakurdesai P (2016) Preclinical safety evaluation of low molecular weight galactomannans based standardized fenugreek seeds extract. EXCLI J 15:446–459PubMedPubMedCentralGoogle Scholar
  14. Deslauriers A, Beaulieu M, Balducci L, Giovannelli A, Gagnon MJ, Rossi S (2014) Impact of warming and drought on carbon balance related to wood formation in black spruce. Ann Bot 114:335–345CrossRefPubMedPubMedCentralGoogle Scholar
  15. Dirk LMA, van der Krol AR, Vreugdenhil D, Hilhort HWM, Bewley JD (1999) Galactomannan, soluble sugar and starch mobilization following germination of Trigonella foenum-graecum seeds. Plant Physiol Biochem 37(1):41–50CrossRefGoogle Scholar
  16. Dittrich P, Brandl A (1987) A revision of the pathway of d-pinitol formation in Leguminosae. Phytochem 26:1925–1926CrossRefGoogle Scholar
  17. Do GM, Choi MS, Kim HJ, Woo MN, Lee MK, Jeon SM (2008) Soy pinitol acts partly as an insulin sensitizer or insulin mediator in 3T3-L1 preadipocytes. Genes Nutr 2:359–364CrossRefPubMedGoogle Scholar
  18. Gaddam A, Galla C, Thummisetti S, Marikanty RK, Palanisamy UD, Rao PV (2015) Role of fenugreek in the prevention of type 2 diabetes mellitus in prediabetes. J Diabetes Metab Disorders 14:74.  https://doi.org/10.1186/s40200-015-0208-4 CrossRefGoogle Scholar
  19. Gangola MP, Jaiswal S, Khedikar YP, Chibbar RN (2014) A reliable and rapid method for soluble sugars and RFO analysis in chickpea using HPAEC-PAD and its comparison with HPLC-RI. Food Chem 154:127–133CrossRefPubMedGoogle Scholar
  20. Gomes CI, Obendorf RL, Horbowicz M (2004) myo-Inositol, d-chiro-inositol, and d-pinitol synthesis, transport, and galactoside formation in soybean explants. Crop Sci 45(2):1312–1319Google Scholar
  21. Górecki RJ, Piotrowicz-Cieślak AI, Lahuta LB, Obendorf RL (1997) Soluble carbohydrates in desiccation tolerance of yellow lupin seeds during maturation and germination. Seed Sci Res 7:107–115Google Scholar
  22. Górecki RJ, Fordoński G, Halmajan H, Horbowicz M, Jones R, Lahuta LB (2000) Seed physiology and biochemistry. In: Hedley C (ed) Carbohydrates in legume seeds. CAB International, pp 117–144Google Scholar
  23. Gu E-J, Kim DW, Jang G-J, Song SH, Lee J-I, Lee SB, Kim B-M, Cho Y, Lee H-J, Kim H-J (2017) Mass-based metabolomic analysis of soybean sprouts during germination. Food Chem 217:311–319CrossRefPubMedGoogle Scholar
  24. Hernández-Mijares A, Bañuls C, Peris E, Monzó JE, Jover N, Bellod A, Victor LM, Rocha VM M (2013) A single acute dose of pinitol from a naturally-occurring food ingredient decreases hyperglycemia and circulating insulin levels in healthy subjects. Food Chem 141(2):1267–1272CrossRefPubMedGoogle Scholar
  25. Ishitani M, Majumder AL, Bornhouser A, Michalowski CB, Jensen RG, Bohnert HJ (1996) Coordinate transcriptional induction of myo-inositol metabolism during environmental stress. Plant J 9(4):537–548CrossRefPubMedGoogle Scholar
  26. Joshi R, Ramanarao MV, Baisakh N (2013) Arabidopsis plants constitutively overexpressing a myo-inositol 1-phosphate synthase gene (SaINO1) from the halophyte smooth cordgrass exhibits enhanced level of tolerance to salt stress. Plant Physiol Biochem 65:61–66CrossRefPubMedGoogle Scholar
  27. Kadlec P, Dostáálova J, Bernáškova J, Skulinová (2008) Degradation of α-d-galactosides during the germination of grain legume seeds. Czech J Food Sci 26(2):99–108CrossRefGoogle Scholar
  28. Kang Y, Han Y, Torrez-Jerez I, Wang M, Tang Y, Monteros M, Udvardi M (2011) System responses to long-term drought and re-watering of two contrasting alfalfa varieties. The Plant J 68:871–889CrossRefPubMedGoogle Scholar
  29. Kaur H, Verma P, Petla BP, Rao V, Saxena SC, Majee M (2013) Ectopic expression of the ABA-inducible dehydration-responsive chickpea L-myo-inositol 1-phosphate synthase 2 (CaMIPS2) in Arabidopsis enhances tolerance to salinity and dehydration stress. Planta 237:321–335CrossRefPubMedGoogle Scholar
  30. Kawai S, Ohyama T, Kumazawa K (1985) Possibility of upward transport of D-pinitol in soybean plant: Investigation by petiole girdling treatment. Soil Sci Plant Nutr 31(2):287–292.  https://doi.org/10.1080/00380768.1985.10557434 CrossRefGoogle Scholar
  31. Kordan B, Lahuta LB, Dancewicz K, Sądej W, Gabryś B (2011) Effect of lupin cyclitols on pea aphid probing behaviour. J Plant Protect Res 51:171–178CrossRefGoogle Scholar
  32. Krishnan HB, Natarajan SS, Bennett JO, Sicher RC (2011) Protein and metabolite composition of xylem sap from field-grown soybeans (Glycine max). Planta 233:921–931CrossRefPubMedGoogle Scholar
  33. Kumari A, Das P, Parida AK, Agarwal PK (2015) Proteomics, metabolomics, and ionomics perspectives of salinity tolerance in halophytes. Front Plant Sci  https://doi.org/10.3389/fpls.2015.00537
  34. Lahuta LB (2006) Biosynthesis of raffinose family oligosaccharides and galactosyl pinitols in developing and maturing seeds of winter vetch (Vicia villosa Roth.). Acta Soc Bot Pol 75:219–227CrossRefGoogle Scholar
  35. Lahuta LB, Dzik T (2011) d-chiro-Inositol affects accumulation of raffinose family oligosaccharides in developing embryos of Pisum sativum. J Plant Physiol 168:352–358CrossRefPubMedGoogle Scholar
  36. Lahuta LB, Górecki RJ (2011) Raffinose in seedlings of winter vetch (Vicia villosa Roth.) under osmotic stress and followed by recovery. Acta Physiol Plant 33:725–733CrossRefGoogle Scholar
  37. Lahuta LB, Goszczyńska J (2009) Inhibition of raffinose family oligosaccharides and galactosyl pinitols breakdown delays germination of winter vetch (Vicia villosa Roth.) seeds. Acta Soc Bot Pol 78(3):203–208CrossRefGoogle Scholar
  38. Lahuta LB, Goszczyńska J (2010) Cyclitols in maturing grains of wheat, triticale and barley. Acta Soc Bot Pol 79(3):181–187CrossRefGoogle Scholar
  39. Lahuta LB, Górecki RJ, Gojło E, Horbowicz M (2005a) Differences in accumulation of soluble α-galactosides during seed maturation of several Vicia species. Acta Physiol Plant 27(2):163–171CrossRefGoogle Scholar
  40. Lahuta LB, Górecki RJ, Horbowicz M (2005b) High concentrations of d-pinitol or d-chiro-inositol inhibit the biosynthesis of raffinose family oligosaccharides in maturing smooth tare (Vicia tetrasperma [L.] Schreb.) seeds. Acta Physiol Plant 27(4A):505–513CrossRefGoogle Scholar
  41. Lahuta LB, Goszczyńska J, Horbowicz M (2010) Seed α-D-galactosides of selected Vicia species and enzymes involved in their biosynthesis. Acta Biol Crac Ser Bot 52/1:27–35Google Scholar
  42. Lemoine R, LaCamera S, Atanassova R, Dédaldéchamp F, Allario T, Pourtau N, Bonnemain J-L, Laloi M, Coutos-Thévenot P, Maurousset L, Faucher M, Girousse C, Lemonnier P, Parrilla J, Durand M (2013) Source- to-sink transport of sugar and regulation by environmental factors. Front Plant Sci 4:272.  https://doi.org/10.3389/fpls.2013. 00272CrossRefPubMedPubMedCentralGoogle Scholar
  43. Lin TH, Tan TW, Tsai TH, Chen CC, Hsieh TF, Lee SS, Liu HH, Chen WC, Tang CH (2013) D-Pinitol inhibits prostate cancer metastasis through Inhibition of αVβ3 integrin by modulating FAK, c-Src and NF-κB pathways. Int J Mol Sci 14:9790–9802CrossRefPubMedPubMedCentralGoogle Scholar
  44. Lintunen A, Paljakka T, Jyske T, Peltoniemi M, Sterck F et al (2016) Osmolality and non-structural carbohydrate composition in the secondary phloem of trees across a latitudinal gradient in Europe. Front Plant Sci.  https://doi.org/10.3389/fpls.2016.00726
  45. Ma JM, Horbowicz M, Obendorf RL (2005) Cyclitols galactosides in embryos of buckwheat stem-leaf-seed explants fed d-chiro-inositol, myo-inositol or d-pinitol. Seed Sci Res 15:329–338CrossRefGoogle Scholar
  46. Martín-Cabrejas M, Díaz MF, Aguilera Y, Benítez V, Mollá E, Esteban RM (2008) Influence of germination on the soluble carbohydrates and dietary fibre fractions in non-conventional legumes. Food Chem 107:1045–1052CrossRefGoogle Scholar
  47. Matos MC, Campos PS, Passarinho JA, Semedo JN, Marques NM, Ramalho JC, Ricardo CP (2010) Drought effect on photosynthetic activity, osmolyte accumulation and membrane integrity of two Cicer arietinum genotypes. Photosynthetica 48(2):303–312CrossRefGoogle Scholar
  48. Merchant A, Richter AA (2011) Polyols as biomarkers and bioindicators for 21st century plant breeding. Func Plant Biol 38:934–940CrossRefGoogle Scholar
  49. Muscogiuri G, Palomba S, Laganà AS, Orio F (2016) Inositols in the treatment of insulin-mediated diseases. Int J Endocrinol article ID 3058393,  https://doi.org/10.1155/2016/3058393
  50. Nascimento NRF, Lessa LMA, Kerntopf MR, Sousa RS, Queiroz MGR, Price J, Heimark DB, Larner J, Du X, Brownlee M, Gow A, Davis C, Fonteles MC (2006) Inositols prevent and reverse endothelial dysfunction in diabetic rat and rabbit vasculature metabolically and by scavenging superoxide. PNAS 103(1):218–223CrossRefPubMedGoogle Scholar
  51. Nishizawa-Yokoi A, Yabuta Y, Shigeoka S (2008) The contribution of carbohydrates including raffinose family oligosaccharides and sugar alcohols to protection of plant cells from oxidative damage. Plant Signal Behav 3(11):1016–1018CrossRefPubMedPubMedCentralGoogle Scholar
  52. Nordin P (1984) Preferential leaching of pinitol from soybeans during imbibition. Plant Physiol 76:313–315CrossRefPubMedPubMedCentralGoogle Scholar
  53. Obendorf RL, Górecki RJ (2012) Soluble carbohydrates in legume seeds. Seed Sci Res 22:219–242CrossRefGoogle Scholar
  54. Obendorf RL, Horbowicz M, Dickerman AM, Brenac P, Smith ME (1998) Soluble oligosaccharides and galactosyl cyclitols in maturing soybean seeds in planta and in vitro. Crop Sci 38:78–84CrossRefGoogle Scholar
  55. Patra B, Ray S, Richter A, Majumder (2010) Enhanced salt tolerance of transgenic tobacco plants by co-expression of PcINO1 and McIMT1 is accompanied by increased level of myo-inositol and methylated inositol. Protoplasma 245:143–152CrossRefPubMedGoogle Scholar
  56. Patrick JW, Offler C (2001) Compartmentation of transport and transfer events in developing seeds. J Exp Bot 52:551–564CrossRefPubMedGoogle Scholar
  57. Penfield S, Rylott EL, Gilday AD, Graham S, Larson TR, Graham IA (2004) Reserve mobilization in the Arabidopsis endosperm fuels hypocotyl elongation in the dark, is independent of abscisic acid, and requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1. Plant Cell 16:2705–2718CrossRefPubMedPubMedCentralGoogle Scholar
  58. Peterbauer T, Richter A (2001) Biochemistry and physiology of raffinose family oligosaccharides and galactosyl cyclitols in seeds. Seed Sci Res 11:185–197Google Scholar
  59. Poongothai G. Sripathi SK (2013) A review on insulinomimetic pinitol from plants. Int J Pharm Bio Sci 4(2):992–1009Google Scholar
  60. Rao A, Steels E, Inder WJ, Abraham S, Vitetta L (2016) Testofen, a specialised Trigonella foenum-graecum seed extract reduces age-related symptoms of androgen decrease, increases testosterone levels and improves sexual function in healthy aging males in a double-blind randomised clinical study. The Aging Male 19(2):134–142CrossRefPubMedGoogle Scholar
  61. Reid JSG (1971) Reserve carbohydrate metabolism in germinating seeds of trigonella foenum-graecum L. (Leguminosae). Planta 100(2):131–142CrossRefPubMedGoogle Scholar
  62. Sanchez DH, Schwabe F, Erban A, Udvardi MK, Kopka J (2012) Comparative metabolomics of drought acclimation in model and forage legumes. Plant Cell Environ 35:136–149CrossRefPubMedGoogle Scholar
  63. Sengupta S, Patra B, Ray S, Majumder AL (2008) Inositol methyl transferase from a halophytic wild rice Porteresia coarctata Roxb. (Tateoka): regulation of pinitol synthesis under abiotic stress. Plant Cell Environ 31:1442–1459CrossRefPubMedGoogle Scholar
  64. Sethi G, Ahn KS, Sung B, Aggarwal BB (2008) Pinitol targets nuclear factor-κB activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis. Mol Cancer Ther 7(6):1604–1614CrossRefPubMedGoogle Scholar
  65. Shang MY, Cai SQ, Lin WH, Wang MC, Park JH (2002) Studies on chemical constituents from the seed of Trigonella foenum-graecum. Zhongguo Zhong Yao Za Zhi 27(4):277–279 (in Chinese) PubMedGoogle Scholar
  66. Sheveleva E, Chmara W, Bohnert HJ, Jensen RG (1997) Increased salt and drought tolerance by d-ononitol production in transgenic Nicotiana tabacum L. Plant Physiol 115:1211–1219CrossRefPubMedPubMedCentralGoogle Scholar
  67. Singh J, Gupta K, Arora SK (1994) Changes in the anti-nutritional factors of developing seeds and pod walls of fenugreek (Trigonella foenum-graecum. L.) Plant Foods Hum Nutr 46(1):77–84CrossRefPubMedGoogle Scholar
  68. Sivakumar S, Palsamy P, Subramanian SP (2010) Impact of D-pinitol on the attenuation of proinflammatory cytokines, hyperglycemia-mediated oxidative stress and protection of kidney tissue ultrastructure in streptozotocin-induced diabetes rats. Chem Biol Inter 188:237–245CrossRefGoogle Scholar
  69. Slama I, Abdelly C, Bouchereau A, Flowers T, Savouré (2015) Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress. Ann Bot 115:433–447CrossRefPubMedPubMedCentralGoogle Scholar
  70. Smith AE, Philips DV (1982) Influence of sequential prolonged periods of dark and light on pinitol concentration in clover and soybean tissue. Physiol Plant 54:31–33CrossRefGoogle Scholar
  71. Srivastava G, Kayastha AM (2014) B-amylase from strachless seeds of Trigonella foenum-graecum and its localization in germinating seeds. PLoS One 9(2):e88697.  https://doi.org/10.1371/journal.pone.0088697 CrossRefPubMedPubMedCentralGoogle Scholar
  72. Streeter JG, Lohnes DG, Fioritto RJ (2001) Patterns of pinitol accumulation in soybean plants and relationships to drought tolerance. Plant Cell Environ 24:429–438CrossRefGoogle Scholar
  73. Szczeciński P, Gryff-Keller A, Horbowicz M, Lahuta LB (2000) Galactosylpinitols isolated from vetch (Vicia villosa Roth.) seeds. J Agric Food Chem 48:2717–2720CrossRefPubMedGoogle Scholar
  74. Tan J, Wang C, Xiang B, Han R, Guo Z (2013) Hydrogen peroxide and nitric oxide mediated cold- and dehydration-induced myo-inositol phosphate synthase that confers multiple resistances to abiotic stresses. Plant Cell Environ 36:288–299CrossRefPubMedGoogle Scholar
  75. Thomas JE, Bandara M, Lee EL, Driedger D, Acharya S (2011) Biochemical monitoring in fenugreek to develop functional food and medicinal plant variants. New Biotech 28(2):110–117CrossRefGoogle Scholar
  76. Turner LB, Pollock CJ (1998) Changes in stolon carbohydrates during the winter in four varieties of white clover (Trifolium repens L.) with contrasting hardiness. Ann Bot 81:97–107CrossRefGoogle Scholar
  77. Valluru R, Van den Ende W (2011) Myo-inositol and beyond—emerging networks under stress. Plant Sci 181:387–400CrossRefPubMedGoogle Scholar
  78. Vernon DM, Bohnert HJ (1992) Increased expression of a myo-inositol methyl transferase in Mesembryanthemum crystallinum is part of a stress response distinct from Crassulacean Acid Metabolism induction. Plant Physiol 99:1695–1698CrossRefPubMedPubMedCentralGoogle Scholar
  79. Warren CR, Aranda I, Cano FJ (2011) Responses to water stress of gas exchange and metabolites in Eucalyptus and Acacia spp. Plant Cell Environ 34:1609–1629CrossRefPubMedGoogle Scholar
  80. Weber H, Borisjuk L, Wobus U (1997) Sugar import and metabolism during seed development. Trends Plant Sci 2:169–174CrossRefGoogle Scholar
  81. Yasui T, Ohashi H (1990) The low molecular weight carbohydrate composition of seeds in the Leguminosae - a new taxonomic character in the family. Sci Rep Tohoku Univ (Biol) 4th Ser 39:257–393Google Scholar
  82. Yates SA, Swaing MT, Hegarty MJ, Chernukin I, Lowe M, Allison GG, Ruttink T, Abberton MT, Jenkins G, Skøt L (2014) De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response, gene discovery and marker identification. BMC Genom 15:453.  https://doi.org/10.1186/1471-2164-15-453 CrossRefGoogle Scholar
  83. Zalewski K, Lahuta LB (2007) Studies on soluble carbohydrates in yellow lupin (Lupinus luteus L.) seeds of different age. Acta Soc Bot Pol 76(4):309–312CrossRefGoogle Scholar
  84. Zandi P, Basu SK, Khatibani LB, Balogun MO, Aremu MO, Sharma M, Kumar A, Sengupta R, Li X, Li Y, Tashi S, Hedi A, Cetzal-Ix W (2015) Fenugreek (Trigonella foenum-graecum L.) seed: a review of physiological and biochemical properties and their genetic improvement. Acta Physiol Plant 37:1714.  https://doi.org/10.1007/s11738-014-1714-6 CrossRefGoogle Scholar
  85. Zang JY, Cruz de Carvalho MH, Torrez-Jerez I, Kang Y, Allen SN, Huhman DV, Tang Y, Murray J, Sumner LW, Udvardi MK (2014) Global reprogramming of transcription and metabolism in Medicago truncatula during progressive drought and after rewatering. Plant Cell Environ 37:2553–2576CrossRefGoogle Scholar
  86. Zhang RX, Qin LJ, Zhao DG (2017) Overexpression of the OsIMP gene increases the accumulation of inositol and confers enhanced cold tolerance in tobacco through modulation of the antioxidant enzymes’ activities. Genes 8:179.  https://doi.org/10.3390/genes8070179 CrossRefPubMedCentralGoogle Scholar

Copyright information

© The Author(s) 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • Lesław Bernard Lahuta
    • 1
  • Joanna Szablińska
    • 1
  • Monika Ciak
    • 1
  • Ryszard Józef Górecki
    • 1
  1. 1.Department of Plant Physiology, Genetics and BiotechnologyUniversity of Warmia and Mazury in OlsztynOlsztynPoland

Personalised recommendations