The morphometric parameters measured in the roots were area, diameter, number and perimeter of the tracheal element cells, regardless of the stage of ontogenetic development (primary or secondary) and, from the primary xylem, only the metaxylem was measured, because the protoxylem collapsed at the end of its differentiation (Figures 1 and 2).

methodology reported by Phillips and Hayman (1970).

**2.4. Determination of reserve substances**

gram for determination of reserve substances.

substances the method described by Priestley (1965).

**2.5. Histological studies**

were made using a manual microtome.

analyzed). To determine the colonization rate the radicels were stained following

Samples of the aerial part (leaves, stems and stem) and dried roots were ground in the mill, coupled with a sieve of 20 meshes per inch. Each sample was collected approximately one

A similar procedure was carried out with samples of roots. After each sample individually packaged in bags made of special screen for the filtration of food products and brought back to 65C oven to constant weight, recording the weight of each bag, after, were digested in order to extract all components of plant tissue (carbohydrates, fats, fatty acids, etc.) that were not fibers (cellulose, hemicellulose and lignin), as conventionally known as reserve

The samples were placed in one liter Erlenmeyer flask containing an aqueous solution with 5% trichloroacetic acid (99%) and 35% methanol (99.8%) remained on heating gas burner, under a hood with hood, by eight hours. From the third hour to eight hours, distilled water was added to the solution, as it would evaporate in order to always maintain the same

After the samples were rinsed with distilled water again and put in stove to dry at 65C until constant weight. The difference in mass of the samples before and after digestion

Secondary roots with similar diameter were used for the morphological studies, as shown in Tables 1 and 2. The histological studies followed the methods described by Johansen (1940), where 1 cm long samples were dehydrated and blocked in paraffin, and 10-15µm thick slices

The slices were placed on slides, removed the paraffin with xylol, rehydrated for later staining with aqueous Safranin (1%) and Toluidine Blue O (0.05%), and than dehydrated

These sections were observed under a Leica DM microscope with 400X magnification. The images were captured with a Nikon CoolPix 990 digital camera (Photos of José Luis da Silva

The morphometric parameters measured in the roots were area, diameter, number and perimeter of the tracheal element cells, regardless of the stage of ontogenetic development (primary or secondary) and, from the primary xylem, only the metaxylem was measured,

because the protoxylem collapsed at the end of its differentiation (Figures 1 and 2).

volume of liquid sufficient to maintain the samples immersed in the solution.

constisted substance content of the buffer that contained samples.

again and the preparations mounted in Canada balsam with a coverslip.

Nunes ) and analyzed using the "WCIF Image J" software.

Alterations in Root Morphology of Rootstock Peach Trees Caused by Mycorrhizal Fungi 395

**Figure 1.** Cross sections of secondary roots of peach tree rootstock Okinawa without inoculation (1) and inoculated with AMF (2 – *G. clarum*, 3 – *G. etunicatum*, 4 – *Acaulospora sp*.). MX – metaxylem; PX – protoxylem; XS – secondary xylem. Scale 50 µm.

Alterations in Root Morphology of Rootstock Peach Trees Caused by Mycorrhizal Fungi 395

394 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

**Figure 1.** Cross sections of secondary roots of peach tree rootstock Okinawa without inoculation (1) and inoculated with AMF (2 – *G. clarum*, 3 – *G. etunicatum*, 4 – *Acaulospora sp*.). MX – metaxylem; PX

– protoxylem; XS – secondary xylem. Scale 50 µm.

396 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology Alterations in Root Morphology of Rootstock Peach Trees Caused by Mycorrhizal Fungi 397

**Figure 2.** Cross sections of secondary roots of peach tree rootstock Aldrighi without inoculation (1) and inoculated with AMF (2 – *G. clarum*, 3 – *G. etunicatum*, 4 – *Acaulospora sp*., 5 - *Scutellospora heterogama*).

The data were submitted to an analysis of variance by the SAS program and the measurements

The results regarding the effect of the AMF on the conductor tissue of the roots of the Okinawa cultivar showed that the treatments with the *Acaulospora sp.* and *G. etunicatum* species performed similarly for the number and diameter of the metaxylem and secondary xylem cells, superior to the other treatments, while for cell area and perimeter, *Acaulospora sp.* was superior to the other treatments. The treatment with *G. etunicatum* presented cell area, perimeter and diameter similar to *G. clarum*, superior to the controls. The treatment with *G. clarum* presented the number of cells of these xylem classes similar to that of the controls. There was an inverse performance for cortical thickness, where the control plants presented the highest results, followed by the plants inoculated with *G. clarum* while those inoculated with *Acaulospora sp.* and *G. etunicatum* presented similar but lower results,

were compared by the Duncan test (Duncan, 1955) at the level of 5% significance.

MX – metaxylem; PX – protoxylem; XS – secondary xylem. Scale 50 µm.

**2.6. Statistics**

**3. Results**

compared to the control plants (Table 1).

**Figure 2.** Cross sections of secondary roots of peach tree rootstock Aldrighi without inoculation (1) and inoculated with AMF (2 – *G. clarum*, 3 – *G. etunicatum*, 4 – *Acaulospora sp*., 5 - *Scutellospora heterogama*). MX – metaxylem; PX – protoxylem; XS – secondary xylem. Scale 50 µm.

## **2.6. Statistics**

The data were submitted to an analysis of variance by the SAS program and the measurements were compared by the Duncan test (Duncan, 1955) at the level of 5% significance.

## **3. Results**

The results regarding the effect of the AMF on the conductor tissue of the roots of the Okinawa cultivar showed that the treatments with the *Acaulospora sp.* and *G. etunicatum* species performed similarly for the number and diameter of the metaxylem and secondary xylem cells, superior to the other treatments, while for cell area and perimeter, *Acaulospora sp.* was superior to the other treatments. The treatment with *G. etunicatum* presented cell area, perimeter and diameter similar to *G. clarum*, superior to the controls. The treatment with *G. clarum* presented the number of cells of these xylem classes similar to that of the controls. There was an inverse performance for cortical thickness, where the control plants presented the highest results, followed by the plants inoculated with *G. clarum* while those inoculated with *Acaulospora sp.* and *G. etunicatum* presented similar but lower results, compared to the control plants (Table 1).

Alterations in Root Morphology of Rootstock Peach Trees Caused by Mycorrhizal Fungi 399

Nutrients (%) Colonization

Nutrients (%) Colonization

N P K (%)

N P K (%)

The inoculation with AMF species accelerated the growth of the plants of the Okinawa cultivar rootstock, inducing greater height, diameter, leaf area and greater nitrogen, phosphorus and potassium content, compared with the control. All presented root colonization rates were over 90%. *Acaulospora sp.* was the species that was most efficient among the AMF tested giving greatest height, diameter and nutritional state compared to the other species. The plants inoculated with *G. clarum* and *G. etunicatum* presented to

> L.A. (cm²/ plant)

*Acaulospora sp.* 136,46a 8,42a 197,01a 2,35a 0,16a 2,07a 97,00a *G. clarum* 126,65b 7,79b 163,00b 2,23b 0,15b 1,74b 91,76b *G. etunicatum* 129,04b 7,87b 167,00b 2,22b 0,15b 1,82b 92,62b Control 119,23c 7,24c 142,03c 2,05c 0,14c 1,60c 00,00c V. C. (%) 3,88 2,17 2,54 2,56 2,61 4,75 2,42

**Table 3.** Height (H), root-stem junction diameter (D), leaf area (L.A.), percentage of nitrogen (N), phosphorus (P) and potassium (K) in the plant tissue and root colonization (%) of the Okinawa cultivar rootstock inoculated with three AMF species (*Acaulospora sp*., *G. clarum* and *G. etunicatum*), collected 360 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not

For the plants of the Aldrighi cultivar, only the *S. heterogama* and *G. etunicatum* species were

L.A. (cm²/ plant)

*Acaulospora sp.* 130,94c 6,28c 125,00c 2,99c 0,16b 2,40b 30,00c *G. clarum* 129,80c 6,24c 119,01c 2,96c 0,17b 2,44b 28,50c *G. etunicatum* 138,36b 6,82b 137,00b 3,33b 0,20a 2,72a 91,50b *S. heterogama* 143,97a 7,29a 173,10a 3,74a 0,22a 2,76a 97,75a Control 129,70c 5,88d 105,00d 2,65d 0,16b 2,29c 00,00d V. C. (%) 1,55 2,77 2,44 6,64 10,60 3,97 2,91

**Table 4.** Height (H), root-stem junction diameter (D), leaf area (L.A.), percentage of nitrogen (N), phosphorus (P) and potassium (K) in the plant tissue and root colonization (%) of the Aldrighi cultivar rootstock inoculated with four AMF species (*Acaulospora sp*., *G. clarum*, *G. etunicatum* and *S. heterogama*), collected 180 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the

mediate growth and nutritional state, and were similar (Table 3).

D (mm)

H (cm)

differ by the Duncan test at 5% significance.

shown to be efficient for the height parameter (Table 4).

D (mm)

H (cm)

column do not differ by the Duncan test at 5% significance.

Treatment

Treatment

**Table 1.** Root morphology of secondary roots of the Okinawa cultivar rootstock inoculated with three AMF species (*Acaulospora sp*., *G. clarum* and *G. etunicatum*), collected 360 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not differ by the Duncan test at 5% significance. nsNon-significative.

For the Aldrighi cultivar, the treatment with the *S. heterogama* species presented better results than the other treatments for the parameters number of cells, cell area and perimeter of the metaxylem and the secondary xylem, but performed similarly to *G. etunicatum* for cell diameter, and was superior to the other treatments for this parameter (Table 2).

**Table 2.** Root morphology of secondary roots of the Aldrighi cultivar rootstock inoculated with four AMF species (*Acaulospora sp*., *G. clarum*, *G. etunicatum* and *S. heterogama*) collected 180 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not differ by the Duncan test at 5% significance. nsNon-significative.

*G. etunicatum* performed similarly to *Acaulospora sp.* regarding the number of cells that was better than *G. clarum* and the control. Furthermore, *G. etunicatum* performed better than *Acaulospora sp.*, *G. clarum* and the controls for the other parameters. The plants inoculated with *Acaulospora sp.* performed better than *G. clarum* for the number of cells and similarly to the other parameters. The control plants presented lower results for all the parameters in the assessment of the xylem classes. However, regarding cortical thickness, the control plants performed better than the other treatments, followed by *G. clarum* that was better than *Acaulospora sp.* that was better than the treatments with *S. heterogama* and *G. etunicatum* that presented similar results.

The inoculation with AMF species accelerated the growth of the plants of the Okinawa cultivar rootstock, inducing greater height, diameter, leaf area and greater nitrogen, phosphorus and potassium content, compared with the control. All presented root colonization rates were over 90%. *Acaulospora sp.* was the species that was most efficient among the AMF tested giving greatest height, diameter and nutritional state compared to the other species. The plants inoculated with *G. clarum* and *G. etunicatum* presented to mediate growth and nutritional state, and were similar (Table 3).

398 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

Cortex thickness (µm)

Number of Cell

*Acaulospora sp.* 957,14ns 118,12c 38,53a 5,50a 16,86a 58,55a *G. clarum* 958,32ns 129,85b 26,00b 5,11b 15,61b 48,46b *G. etunicatum* 961,35ns 119,31c 34,60a 5,26ab 15,70b 50,45b Testemunha 952,61ns 139,39a 22,00b 4,04c 11,77c 41,14c V. C. (%) 5,17 7,41 5,01 10,09 6,02 13,22 **Table 1.** Root morphology of secondary roots of the Okinawa cultivar rootstock inoculated with three AMF species (*Acaulospora sp*., *G. clarum* and *G. etunicatum*), collected 360 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not differ by the Duncan test at

For the Aldrighi cultivar, the treatment with the *S. heterogama* species presented better results than the other treatments for the parameters number of cells, cell area and perimeter of the metaxylem and the secondary xylem, but performed similarly to *G. etunicatum* for cell

> Number of Cell

*Acaulospora sp.* 945,01ns 118,08c 33,40b 4,87b 12,95c 52,95c *G. clarum* 939,11ns 128,85b 24,93c 4,93b 13,10c 53,10c *G. etunicatum* 955,23ns 107,31d 30,20b 6,17a 16,86b 56,86b *S. heterogama* 960,12ns 105,22d 37,00a 7,06a 22,70a 62,70a Testemunha 940,04ns 138,39a 16,33d 3,87c 11,57d 41,57d V. C. (%) 4,76 6,11 5,54 7,84 6,84 11,32 **Table 2.** Root morphology of secondary roots of the Aldrighi cultivar rootstock inoculated with four AMF species (*Acaulospora sp*., *G. clarum*, *G. etunicatum* and *S. heterogama*) collected 180 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not differ by the Duncan

*G. etunicatum* performed similarly to *Acaulospora sp.* regarding the number of cells that was better than *G. clarum* and the control. Furthermore, *G. etunicatum* performed better than *Acaulospora sp.*, *G. clarum* and the controls for the other parameters. The plants inoculated with *Acaulospora sp.* performed better than *G. clarum* for the number of cells and similarly to the other parameters. The control plants presented lower results for all the parameters in the assessment of the xylem classes. However, regarding cortical thickness, the control plants performed better than the other treatments, followed by *G. clarum* that was better than *Acaulospora sp.* that was better than the treatments with *S. heterogama* and *G. etunicatum* that

diameter, and was superior to the other treatments for this parameter (Table 2).

Cortex thickness (µm)

Metaxylem e secondary xylem

Metaxylem e secondary xylem

Cell perimeter (µm)

Cell area (µm²)

Cell diameter (µm)

Cell perimeter (µm)

Cell area (µm²)

Cell diameter (µm)

Diameter of root (µm)

Diameter of root (µm)

test at 5% significance. nsNon-significative.

presented similar results.

Treatment

Treatment

5% significance. nsNon-significative.

**Table 3.** Height (H), root-stem junction diameter (D), leaf area (L.A.), percentage of nitrogen (N), phosphorus (P) and potassium (K) in the plant tissue and root colonization (%) of the Okinawa cultivar rootstock inoculated with three AMF species (*Acaulospora sp*., *G. clarum* and *G. etunicatum*), collected 360 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not differ by the Duncan test at 5% significance.

For the plants of the Aldrighi cultivar, only the *S. heterogama* and *G. etunicatum* species were shown to be efficient for the height parameter (Table 4).

**Table 4.** Height (H), root-stem junction diameter (D), leaf area (L.A.), percentage of nitrogen (N), phosphorus (P) and potassium (K) in the plant tissue and root colonization (%) of the Aldrighi cultivar rootstock inoculated with four AMF species (*Acaulospora sp*., *G. clarum*, *G. etunicatum* and *S. heterogama*), collected 180 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not differ by the Duncan test at 5% significance.

These species were the only ones to present root colonization rates of over 90%. All of the AMF species were efficacious for the root-stem junction diameter and leaf area parameters and only varied in the response intensity. In all the assessments of plant growth and nutritional states, invariably *S. heterogama* induced greater growth compared to the other AMF species, while *G. etunicatum* induced intermediate performance and *Acaulospora sp.* and *G. clarum* presented similar performance.

Alterations in Root Morphology of Rootstock Peach Trees Caused by Mycorrhizal Fungi 401

For the shoot, plants inoculated with *G. clarum*, *G. etunicatum* and *S. heterogama* showed percentages of reserve substances statistically similar but superior to *Acaulospora sp*., which in turn was higher than the control. In roots, the plants were inoculated with *S. heterogama* presented the greatest results, while those inoculated with *G. etunicatum* showed intermediate levels, statistically similar to *G. clarum* that, in turn, was similar to those

It was observed that inoculation with AMF reduced the cortex thickness of inoculated plants in both cultivars, associated to increase in most of the morphological parameters of the root xylem assessed for the Okinawa cultivar and for all of those of the Aldrighi cultivar (Tables

The main effect of the AMF occurred on the metaxylem, that is, one of the categories of the primary xylem, whose conductor cells differentiate later and are larger in diameter (Costa et al., 2003) and also on the secondary xylem cells. On the other hand, the AMF not did not seem to exercise effect on the protoxylem, that are conductor cells of the primary xylem that differentiate first, that is, they acquire secondary lignin walls early (Apezzato-da-Glória & Hayashi, 2003) that reduce the possibility of the AMF acting on the growth of this category

The decrease in the cortex area seems to be directly linked to the increase in the number of cells in the metaxylem and the secondary xylem of the plants inoculated with AMF. The control plants presented a smaller number of metaxylem and secondary xylem cells that were smaller in diameter compared to the cells of the inoculated plants, especially in the case of the treatments with the species *Acaulospora sp.* (cv. Okinawa) and *S. heterogama* (cv.

The mycelia of endomycorrhizal fungi were extracted from roots of *Ophrys lutea*  (Orchidaceae) and placed in culture medium kept and in the dark for three weeks (Barroso et al., 1986). Then the derivatives released in the culture medium were extracted, whose greatest concentrations consisted of indol-3-acetic acid and indol-3-ethanol acid, showing the ability of these fungi to synthesize hormones. The authors concluded that the identification of these compounds in the mycelia extract suggested the transference of these compounds from the fungi to the host plants in the phase when symbiosis was established.

Roots colonized by AMF presented an increase in auxin and cytokinin production that are involved in the increase or continuity of the growth of the conductor tissue cells, especially in the size and number of the cells of the metaxylem and the secondary xylem (Hirsch et al., 1997). According to the same authors, the establishment of symbiosis would lead to the production of biochemical signals that would activate genes involved in the production of these plant hormones, and thus the same signals would be responsible for the formation of

inoculated with *Acaulospora sp*. and the control.

**4.1. Anatomy and morphology changes in roots**

**4. Discussion**

of cells of the primary xylem.

1 and 2).

Aldrighi).

Inoculation with AMF increased content of reserve substances to plants of cv. Okinawa, especially when inoculated with *Acaulospora sp*. In the shoots of rootstock plants inoculated with *G. etunicatum* and *G. clarum* showed intermediate levels. In roots, the plants inoculated with *G. etunicatum* also showed intermediate values, while those inoculated with *G. clarum* not differ from the controls (Table 5).

**Table 5.** Reserve substances of shoots (leaves and stems) and roots of plants of the Okinawa cultivar rootstock inoculated with three AMF species (*Acaulospora sp*., *G. clarum* and *G. etunicatum*), collected 360 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not differ by the Duncan test at 5% significance.

In reviewing the data on the percentage of reserve substances from plants of cv. Aldrighi, present in the tissue of the shoot, it appears that the plants were inoculated with the AMF species had percentages higher than uninoculated plants (Table 6).

**Table 6.** Reserve substances of shoots (leaves and stems) and roots of plants of the Okinawa cultivar rootstock inoculated with four AMF species (*Acaulospora sp*., *G. clarum*, *G. etunicatum* and *S. heterogama*), collected 180 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not differ by the Duncan test at 5% significance.

For the shoot, plants inoculated with *G. clarum*, *G. etunicatum* and *S. heterogama* showed percentages of reserve substances statistically similar but superior to *Acaulospora sp*., which in turn was higher than the control. In roots, the plants were inoculated with *S. heterogama* presented the greatest results, while those inoculated with *G. etunicatum* showed intermediate levels, statistically similar to *G. clarum* that, in turn, was similar to those inoculated with *Acaulospora sp*. and the control.

## **4. Discussion**

400 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

*G. clarum* presented similar performance.

not differ from the controls (Table 5).

differ by the Duncan test at 5% significance.

These species were the only ones to present root colonization rates of over 90%. All of the AMF species were efficacious for the root-stem junction diameter and leaf area parameters and only varied in the response intensity. In all the assessments of plant growth and nutritional states, invariably *S. heterogama* induced greater growth compared to the other AMF species, while *G. etunicatum* induced intermediate performance and *Acaulospora sp.* and

Inoculation with AMF increased content of reserve substances to plants of cv. Okinawa, especially when inoculated with *Acaulospora sp*. In the shoots of rootstock plants inoculated with *G. etunicatum* and *G. clarum* showed intermediate levels. In roots, the plants inoculated with *G. etunicatum* also showed intermediate values, while those inoculated with *G. clarum*

Treatment Reserve substances (% in the plant)

*Acaulospora sp.* 39,81a 28,38a *G. clarum* 35,05b 21,02c *G. etunicatum* 35,53b 24,28b Control 27,29c 19,41c V. C. (%) 5,24 2,58

**Table 5.** Reserve substances of shoots (leaves and stems) and roots of plants of the Okinawa cultivar rootstock inoculated with three AMF species (*Acaulospora sp*., *G. clarum* and *G. etunicatum*), collected 360 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the column do not

In reviewing the data on the percentage of reserve substances from plants of cv. Aldrighi, present in the tissue of the shoot, it appears that the plants were inoculated with the AMF

Treatment Reserve substances (% in the plant)

*Acaulospora sp.* 34,69b 22,03c *G. clarum* 35,50a 22,99bc *G. etunicatum* 36,61a 24,92b *S. heterogama* 38,25a 28,27a Control 28,57c 19,80c V. C. (%) 2,76 3,26

**Table 6.** Reserve substances of shoots (leaves and stems) and roots of plants of the Okinawa cultivar rootstock inoculated with four AMF species (*Acaulospora sp*., *G. clarum*, *G. etunicatum* and *S. heterogama*), collected 180 days after sowing. Eldorado do Sul, RS, 2005. Means followed by the same letter in the

species had percentages higher than uninoculated plants (Table 6).

column do not differ by the Duncan test at 5% significance.

Shoot Roots

Shoot Roots

## **4.1. Anatomy and morphology changes in roots**

It was observed that inoculation with AMF reduced the cortex thickness of inoculated plants in both cultivars, associated to increase in most of the morphological parameters of the root xylem assessed for the Okinawa cultivar and for all of those of the Aldrighi cultivar (Tables 1 and 2).

The main effect of the AMF occurred on the metaxylem, that is, one of the categories of the primary xylem, whose conductor cells differentiate later and are larger in diameter (Costa et al., 2003) and also on the secondary xylem cells. On the other hand, the AMF not did not seem to exercise effect on the protoxylem, that are conductor cells of the primary xylem that differentiate first, that is, they acquire secondary lignin walls early (Apezzato-da-Glória & Hayashi, 2003) that reduce the possibility of the AMF acting on the growth of this category of cells of the primary xylem.

The decrease in the cortex area seems to be directly linked to the increase in the number of cells in the metaxylem and the secondary xylem of the plants inoculated with AMF. The control plants presented a smaller number of metaxylem and secondary xylem cells that were smaller in diameter compared to the cells of the inoculated plants, especially in the case of the treatments with the species *Acaulospora sp.* (cv. Okinawa) and *S. heterogama* (cv. Aldrighi).

The mycelia of endomycorrhizal fungi were extracted from roots of *Ophrys lutea*  (Orchidaceae) and placed in culture medium kept and in the dark for three weeks (Barroso et al., 1986). Then the derivatives released in the culture medium were extracted, whose greatest concentrations consisted of indol-3-acetic acid and indol-3-ethanol acid, showing the ability of these fungi to synthesize hormones. The authors concluded that the identification of these compounds in the mycelia extract suggested the transference of these compounds from the fungi to the host plants in the phase when symbiosis was established.

Roots colonized by AMF presented an increase in auxin and cytokinin production that are involved in the increase or continuity of the growth of the conductor tissue cells, especially in the size and number of the cells of the metaxylem and the secondary xylem (Hirsch et al., 1997). According to the same authors, the establishment of symbiosis would lead to the production of biochemical signals that would activate genes involved in the production of these plant hormones, and thus the same signals would be responsible for the formation of

the root nodes on legumes colonized by *Rhizobium sp*. These authors showed that in roots of the MN 1008 alfalfa mutant cultivar, that carries out the transcription of these signals in the absence of the symbiants, the responses of the root tissue were identical to those of roots of plants colonized both by AMF and *Rhizobium meliloti*.

Alterations in Root Morphology of Rootstock Peach Trees Caused by Mycorrhizal Fungi 403

2005). The route of transfer of carbohydrates from the plant to the AMF follows the opposite

The benefits given by the AMF on xylem development is associated to many action mechanisms of these fungi, that act directly or indirectly on the plants (Souza et al., 2000). One of the positive effects of the AMF is in function of the presence of the external mycelia, which play an important role in slow diffusion nutrient absorption, such as phosphorus and potassium (Minhoni & Auler, 2003; Souza et al., 2000; Tobar et al., 1994), increasing the nutritional content of the plants (An et al., 1993; Barea, 1991). Associated to this, the modifications caused by the AMF in the xylem structure, such as increase in the number and diameter of the metaxylem and secondary xylem cells, permitted a greater flow of nutrient absorption, such as nitrogen, phosphorus and potassium, translocated to the upper part of

The fact that the AMF species induce major development parameters such as height, diameter and leaf area per plant provides greater photosynthesis and, consequently, a higher level of production of assimilates (Nunes et al., 2006). This report confirms the data obtained in this study with respect to the reserve substances of shoots of both cultivars, for all species used provided an increase in leaf area compared to control (Tables 3 and 4). There is also agreement with other authors, who found higher levels of reserve substances in the tissues of plants inoculated with AMF (Theodore et al., 2003; Sena et al., 2004, Souza et al.,

Another fact to be noted is that only the AMF species that provided the greatest results for height, diameter and leaf area for both cultivars (*Acaulospora sp*. Okinawa cultivar, *S. heterogama* for Aldrighi cultivar and *G. etunicatum* for both cultivars) yielded significant differences in plant reserves, both to the tissues of shoots and roots to the tissues, compared to other treatments. Plants with greater height and leaf area, are more light-gathering capacity and production of assimilates, which allows a higher flow of carbohydrates into the root system where one part would be used by the AMF in its nutrition and accumulation in structures buffer (vesicles, where *Acaulospora sp*. and *G. etunicatum*), and the rest would be accumulated in the storage tissue of the plant in the form of reserve substances (Souza et al., 1999; Scatena & Scremin-Dias, 2003). Moreover, the larger diameter provided by the AMF, would increase the upward flow of water and nutrients, and sap formulated in the

Plants inoculated with AMF have changes in the morphological structure of the roots, such as reduction of the cortex and increased the number and size of cells of the metaxylem, which provides greater volume of water and nutrients translocated to the top of the plant. This benefits plants, accelerating its vegetative growth, improving the content of

macronutrients and allowing the production and accumulation of assimilates.

the plant, culminating in accelerated growth (Souza, 2000; Souza et al., 2000).

direction (Nagy et al., 2005; Ohtomo et al., 2005).

downward (Mazzoni-Viveiros & Trufem, 2004).

2005).

**5. Conclusion**

Thus it can be inferred that the presence of AMF would favor the constant differentiation of the xylem tracheal elements, that coincides with the results obtained in this study for both the root stock cultivars.

There appear to be possible variable effects on root morphology, according to the AMF species and the plant species involved in the symbiosis that also influences the size and growth of the xylem cells, that was also observed in this study, because some species presented variable performance in increasing the size and number of cells, in function of the cultivar used, and in function of the AMF species used for the same cultivar (Atkinson et al., 1994). The species *G. fasciculatum* and *G. etunicatum* induced modification in the roots of the Elsanta and Cambridge Favorite strawberry cultivars, but did not cause any alteration in the morphology of the roots of the Rhapsody cultivar, shedding the variable effect of these AMF species on different cultivars of the same plant species (Norman et al., 1996).

Moreover, roots of plants were colonized by AMF may or may not show increases in longevity, depending on plant species and the fungi involved in symbiosis (Atkinson et al, 2003; Eissenstat et al., 2000; Hodge et al., 2000). However, the morphological attributes of the roots that may be affected by the AMF, such as roots and branches of the diameter of the conducting tissue, has a direct influence on increasing the longevity of roots (Wells et al., 2002). In addition to increasing longevity, root colonization by AMF provides a quick renewal of the root system, increasing the rate of substitution of roots that have collapsed.

## **4.2. Acquisition of nutrients and benefits**

The increase in the absorption and transport volume of nutrients such as nitrogen, that is a constituent of proteins (Tedesco et al., 1995), phosphorus that is essential for a cell division and photosynthesis metabolism and potassium that acts on the electric equilibrium of the cells and on the stomata opening and closing (Tedesco et al., 1995), is vital for plant growth. This contributed to greater responses of the inoculated plants in terms of plant development that was observed in this study for both the cultivars, especially in the plants where there were the highest percentages of root colonization (Tables 3 and 4).

The AMF obtain carbohydrates from their host plants and provide nutrients, especially phosphate. In the case of phosphate, depending on the combination plant-fungus, the acquisition can be performed wholly or partly by the fungus (Smith et al., 2003). The metabolic pathway of nutrient acquisition starts with the uptake by hyphae-soil interface (Benedetto et al., 2005). In hyphae, the nutrient is transported to structures of the fungus in the roots (Ohtomo et al., 2005), where it is transferred to the plant via arbúsculo (Nagy et al., 2005). The route of transfer of carbohydrates from the plant to the AMF follows the opposite direction (Nagy et al., 2005; Ohtomo et al., 2005).

The benefits given by the AMF on xylem development is associated to many action mechanisms of these fungi, that act directly or indirectly on the plants (Souza et al., 2000). One of the positive effects of the AMF is in function of the presence of the external mycelia, which play an important role in slow diffusion nutrient absorption, such as phosphorus and potassium (Minhoni & Auler, 2003; Souza et al., 2000; Tobar et al., 1994), increasing the nutritional content of the plants (An et al., 1993; Barea, 1991). Associated to this, the modifications caused by the AMF in the xylem structure, such as increase in the number and diameter of the metaxylem and secondary xylem cells, permitted a greater flow of nutrient absorption, such as nitrogen, phosphorus and potassium, translocated to the upper part of the plant, culminating in accelerated growth (Souza, 2000; Souza et al., 2000).

The fact that the AMF species induce major development parameters such as height, diameter and leaf area per plant provides greater photosynthesis and, consequently, a higher level of production of assimilates (Nunes et al., 2006). This report confirms the data obtained in this study with respect to the reserve substances of shoots of both cultivars, for all species used provided an increase in leaf area compared to control (Tables 3 and 4). There is also agreement with other authors, who found higher levels of reserve substances in the tissues of plants inoculated with AMF (Theodore et al., 2003; Sena et al., 2004, Souza et al., 2005).

Another fact to be noted is that only the AMF species that provided the greatest results for height, diameter and leaf area for both cultivars (*Acaulospora sp*. Okinawa cultivar, *S. heterogama* for Aldrighi cultivar and *G. etunicatum* for both cultivars) yielded significant differences in plant reserves, both to the tissues of shoots and roots to the tissues, compared to other treatments. Plants with greater height and leaf area, are more light-gathering capacity and production of assimilates, which allows a higher flow of carbohydrates into the root system where one part would be used by the AMF in its nutrition and accumulation in structures buffer (vesicles, where *Acaulospora sp*. and *G. etunicatum*), and the rest would be accumulated in the storage tissue of the plant in the form of reserve substances (Souza et al., 1999; Scatena & Scremin-Dias, 2003). Moreover, the larger diameter provided by the AMF, would increase the upward flow of water and nutrients, and sap formulated in the downward (Mazzoni-Viveiros & Trufem, 2004).

## **5. Conclusion**

402 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

plants colonized both by AMF and *Rhizobium meliloti*.

**4.2. Acquisition of nutrients and benefits**

were the highest percentages of root colonization (Tables 3 and 4).

the root stock cultivars.

collapsed.

the root nodes on legumes colonized by *Rhizobium sp*. These authors showed that in roots of the MN 1008 alfalfa mutant cultivar, that carries out the transcription of these signals in the absence of the symbiants, the responses of the root tissue were identical to those of roots of

Thus it can be inferred that the presence of AMF would favor the constant differentiation of the xylem tracheal elements, that coincides with the results obtained in this study for both

There appear to be possible variable effects on root morphology, according to the AMF species and the plant species involved in the symbiosis that also influences the size and growth of the xylem cells, that was also observed in this study, because some species presented variable performance in increasing the size and number of cells, in function of the cultivar used, and in function of the AMF species used for the same cultivar (Atkinson et al., 1994). The species *G. fasciculatum* and *G. etunicatum* induced modification in the roots of the Elsanta and Cambridge Favorite strawberry cultivars, but did not cause any alteration in the morphology of the roots of the Rhapsody cultivar, shedding the variable effect of these AMF

Moreover, roots of plants were colonized by AMF may or may not show increases in longevity, depending on plant species and the fungi involved in symbiosis (Atkinson et al, 2003; Eissenstat et al., 2000; Hodge et al., 2000). However, the morphological attributes of the roots that may be affected by the AMF, such as roots and branches of the diameter of the conducting tissue, has a direct influence on increasing the longevity of roots (Wells et al., 2002). In addition to increasing longevity, root colonization by AMF provides a quick renewal of the root system, increasing the rate of substitution of roots that have

The increase in the absorption and transport volume of nutrients such as nitrogen, that is a constituent of proteins (Tedesco et al., 1995), phosphorus that is essential for a cell division and photosynthesis metabolism and potassium that acts on the electric equilibrium of the cells and on the stomata opening and closing (Tedesco et al., 1995), is vital for plant growth. This contributed to greater responses of the inoculated plants in terms of plant development that was observed in this study for both the cultivars, especially in the plants where there

The AMF obtain carbohydrates from their host plants and provide nutrients, especially phosphate. In the case of phosphate, depending on the combination plant-fungus, the acquisition can be performed wholly or partly by the fungus (Smith et al., 2003). The metabolic pathway of nutrient acquisition starts with the uptake by hyphae-soil interface (Benedetto et al., 2005). In hyphae, the nutrient is transported to structures of the fungus in the roots (Ohtomo et al., 2005), where it is transferred to the plant via arbúsculo (Nagy et al.,

species on different cultivars of the same plant species (Norman et al., 1996).

Plants inoculated with AMF have changes in the morphological structure of the roots, such as reduction of the cortex and increased the number and size of cells of the metaxylem, which provides greater volume of water and nutrients translocated to the top of the plant. This benefits plants, accelerating its vegetative growth, improving the content of macronutrients and allowing the production and accumulation of assimilates.

## **Author details**

José Luis da Silva Nunes *BADESUL Desenvolvimento, Brasil*

Paulo Vitor Dutra de Souza, Gilmar Arduino Bettio Marodin and Jorge Ernesto de Araújo Mariath *Universidade Federal do Rio Grande do Sul, Brasil*

José Carlos Fachinello *Universidade Federal de Pelotas, Brasil*

## **Acknowledgement**

To Ministério da Agricultura, Pecuária e Abastecimento (MAPA) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for research support and grants of the authors.

Alterations in Root Morphology of Rootstock Peach Trees Caused by Mycorrhizal Fungi 405

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San Diego, USA

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**Author details**

José Luis da Silva Nunes

José Carlos Fachinello

**Acknowledgement**

authors.

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ISSN 0304-4238

Viçosa, Brasil

Switzerland

New York, USA

*BADESUL Desenvolvimento, Brasil*

and Jorge Ernesto de Araújo Mariath

*Universidade Federal de Pelotas, Brasil*

*Universidade Federal do Rio Grande do Sul, Brasil*

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**Chapter 19**

© 2012 Morkunas et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Morkunas et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Plant Responses to Sugar Starvation**

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51569

**1. Introduction**

Iwona Morkunas, Sławomir Borek, Magda Formela and Lech Ratajczak

The production of carbohydrates via photosynthesis is the most fundamental activity in plant life. Carbohydrate synthesis, transport, utilization, and storage are dynamic processes, strongly dependent on cell physiology, plant organ, environmental conditions, and developmental stage of the plant. The plant's ability to monitor and respond to the level of carbohydrates may act as a controlling mechanism, integrating the influence of environmental conditions (e.g. light, nutrients, biotic and abiotic stress factors) with internal developmental programs, controlled directly by hormones (Koch 2004, Rolland et al. 2006, Hammond and White 2008, Loreti et al. 2008, Ramon et al. 2008, Agulló-Antón et al. 2011). Studies conducted in recent years have provided an extensive body of information on the participation of carbohydrates in metabolic reactions of plant cells. In plants, sugars are essential as respiratory substrates for the generation of energy and metabolic intermediates that are then used for the synthesis of macromolecules. Binding to sugar is required for proper functioning of many proteins and lipids. Moreover, carbohydrates have important hormone-like functions as physiological signals, which cause activation or repression of many plant genes, and this in turn leads to specific metabolic effects. Sugar-signaling networks have the ability to regulate directly the expression of genes and to interact with other signaling pathways. The progress in research on molecular mechanisms of sugar sensing and signaling in plants shows that signal molecules include glucose, fructose, sucrose, and trehalose (Jang and Sheen 1994, Koch 1996, Müller et al. 1999, Rolland et al. 2002, Koch 2004, Gibson 2005, Gonzali et al. 2006, Rolland et al. 2006, Ramon et al. 2008, Rosa et al. 2009, Cho and Yoo 2011). Plants have developed effective mechanisms of perception and transduction of sugar signals. Perception of the sugar signal may take place already in the apoplast during transport across membranes or within the cell, in the cytosol. Sugar signal perception and transduction may involve cell wall invertases (CW-INV), sucrose and glucose transporters (and specific sugar receptors), and hexokinase (HXK) (Sheen et al. 1999, Smeekens 2000, Loreti et al. 2001, Rolland et al. 2002, Harrington and Bush 2003, Moore et al. 2003, Sherson et al. 2003, Koch 2004, Rolland and Sheen 2005,

## **Plant Responses to Sugar Starvation**

Iwona Morkunas, Sławomir Borek, Magda Formela and Lech Ratajczak

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51569

## **1. Introduction**

408 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

Marcel Dekker, ISBN 0-8247-0631-5, New York, USA

Wilcox, H. E. (2002). Mycorrhizae. In: *Plants roots*, Waisel, Y., Eshel, A., Kafkafi, U. (eds),

The production of carbohydrates via photosynthesis is the most fundamental activity in plant life. Carbohydrate synthesis, transport, utilization, and storage are dynamic processes, strongly dependent on cell physiology, plant organ, environmental conditions, and developmental stage of the plant. The plant's ability to monitor and respond to the level of carbohydrates may act as a controlling mechanism, integrating the influence of environmental conditions (e.g. light, nutrients, biotic and abiotic stress factors) with internal developmental programs, controlled directly by hormones (Koch 2004, Rolland et al. 2006, Hammond and White 2008, Loreti et al. 2008, Ramon et al. 2008, Agulló-Antón et al. 2011). Studies conducted in recent years have provided an extensive body of information on the participation of carbohydrates in metabolic reactions of plant cells. In plants, sugars are essential as respiratory substrates for the generation of energy and metabolic intermediates that are then used for the synthesis of macromolecules. Binding to sugar is required for proper functioning of many proteins and lipids. Moreover, carbohydrates have important hormone-like functions as physiological signals, which cause activation or repression of many plant genes, and this in turn leads to specific metabolic effects. Sugar-signaling networks have the ability to regulate directly the expression of genes and to interact with other signaling pathways. The progress in research on molecular mechanisms of sugar sensing and signaling in plants shows that signal molecules include glucose, fructose, sucrose, and trehalose (Jang and Sheen 1994, Koch 1996, Müller et al. 1999, Rolland et al. 2002, Koch 2004, Gibson 2005, Gonzali et al. 2006, Rolland et al. 2006, Ramon et al. 2008, Rosa et al. 2009, Cho and Yoo 2011). Plants have developed effective mechanisms of perception and transduction of sugar signals. Perception of the sugar signal may take place already in the apoplast during transport across membranes or within the cell, in the cytosol. Sugar signal perception and transduction may involve cell wall invertases (CW-INV), sucrose and glucose transporters (and specific sugar receptors), and hexokinase (HXK) (Sheen et al. 1999, Smeekens 2000, Loreti et al. 2001, Rolland et al. 2002, Harrington and Bush 2003, Moore et al. 2003, Sherson et al. 2003, Koch 2004, Rolland and Sheen 2005,

© 2012 Morkunas et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Morkunas et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Rolland et al. 2006, Ramon et al. 2008, Cho et al. 2009, Hanson and Smeekens 2009, Smeekens et al. 2010). Additionally, biochemical studies provide evidence for the involvement of a variety of protein kinases, i.e. Snf1-related kinases (SnRKs) (Rolland et al. 2006, Smeekens et al. 2010), calcium-dependent protein kinases (CDPKs), mitogen-activated protein kinases (MAPKs), and protein phosphatases, 14-3-3 proteins, Ca2+ ions as a second messenger, and G-proteins (Rolland et al. 2006) in sugar signal transduction (Rolland et al. 2002, Sinha et al. 2002). However, it must be stressed that HXK plays a significant role as a component of the sugar sensing machinery. Genetic analyses have revealed a central role for HXK as a conserved glucose sensor (Moore et al. 2003, Harrington and Bush 2003, Rolland and Sheen 2005, Rolland et al. 2006, Ramon et al. 2008, Cho et al. 2009, Hanson and Smeekens 2009, Smeekens et al. 2010). HXK sensing and signaling functions are probably dependent on HXK's subcellular localization, translocation, and/or interactions with downstream effectors. The HXK sugar sensor, as a cytosolic protein or associated with mitochondria or other organelles, then could activate a signaling cascade through HXKinteracting proteins (HIPs) or affect transcription directly after nuclear translocation (Rolland et al. 2002, Rolland et al. 2006, Hanson and Smeekens 2009). Various sugar signals activate many HXK-dependent and HXK-independent pathways and use different molecular mechanisms to control transcription, translation, protein stability, and enzymatic activity. It has been shown that in 7-day-old *Arabidopsis* seedlings, glucose (3%) during 6 h increased the expression of 983 genes and decreased the expression of 771 genes (Li et al. 2006). Sucrose (3%) in *Arabidopsis* cell suspension during 48 h modulated the expression of 243 genes in light and 193 genes in darkness (Nicolaï et al. 2006). Glucose-regulated transcription factors (TFs) account for 8.3% (82 factors among 978) of all glucose-regulated genes, where most of TFs showed repression (Price et al. 2004).

Plant Responses to Sugar Starvation 411

generally considered to be autotrophs but sometimes they can be heterotrophs, e.g. at some stages of development (such as seed germination, as long as the seedling grows in darkness, before it emerges above the soil surface) and in non-photosynthetic organs, such as most roots, stems, and flowers. In germinating seeds, in periods of unfavorable environmental conditions or after too deep sowing, mobilization of storage materials in cotyledons may be delayed or there may be some disturbances in distribution of carbohydrates. This may lead to a decrease in available carbohydrates in embryo axes (i.e. sugar starvation) and in a lower seed germination rate. Additionally, high carbohydrate losses can be observed in most plant species under the influence of environmental conditions, such as water or temperature, attack of pathogens or herbivores. These factors may lead to a remarkable decrease in photosynthetic rate in donor tissues (i.e. leaves that synthesize and export carbohydrates), and this reduces the supply of carbohydrates to acceptor tissues (i.e. non-photosynthetic tissues importing carbohydrates for respiration, growth, and development). Besides, in some conditions, e.g. during dormancy or leaf shedding, photosynthesis is switched off or slowed down. In such conditions, the stored carbohydrates must be used, so their reserves may be greatly diminished in non-photosynthetic tissues then. Knowledge of the response to sugar starvation and of adaptive mechanisms in plants is both fundamental and

**2. Materials and methods of research on sugar starvation**

Carbohydrate starvation has been studied in many of plant species, e.g. in common wheat (Wittenbach 1977, Wittenbach et al. 1982), maize (Saglio and Pradet 1980, Pace et al. 1990), barley (Farrar 1981), pearl millet (Baysdorfer et al. 1988), pea (Webster and van't Hof 1973, Sahulka and Lisa 1978, Webster and Henry 1987, Morkunas et al. 1999, 2000), soybean (Kerr et al. 1985, Walsh et al. 1987), sycamore (Journet et al. 1986, Dorne et al. 1987, Roby et al. 1987, Genix et al. 1990), tobacco (Moriyasu and Ohsumi 1996), lupine (Morkunas et al. 1999, Borek and Ratajczak 2002, Morkunas et. al. 2003, Borek et al. 2006, Borek and Nuc 2011, Borek et al. 2011, 2012a, 2012b), *Arabidopsis thaliana* (Rose et al. 2006). Metabolic changes caused by sugar starvation are usually analysed in cell cultures, callus tissue cultures, and in isolated plant organs cultured *in vitro*. In all types of the cultures it is easy to control nutrition of the plant material by manipulating the medium composition. Cell cultures clearly react by metabolic changes when sugar is omitted from the nutrient medium, e.g. in sycamore (*Acer pseudoplatanus*) cells (Journet et al. 1986, Gout et al. 2011), but similar sensitivity is observed also in excised root tips (Brouquisse et al. 1991, 1992). *In vitro* cultured embryo axes (isolated from seeds), cut off from the natural source of nutrients, i.e. cotyledons, are a good model for research on the effect of sugars on seedling development and metabolism (Borek et al. 2001, Borek and Ratajczak 2002, Borek et al. 2003, 2006, Borek and Nuc 2011, Borek et al. 2012a, 2012b), or even for research on the role of sugar in plant resistance to pathogens (Morkunas et al. 2005, Morkunas and Gmerek 2007, Morkunas et al. 2007, Morkunas and Bednarski 2008, Morkunas et al. 2008, 2011). The influence of sugar on leaf senescence was assessed on excised leaves and on leaves of intact plants. The effect of sugar starvation was achieved by keeping the plant material in darkness or by its spraying

agronomically important.

Genes encoding the enzymes of the phenylpropanoid biosynthesis pathway (Hara et al. 2003, Solfanelli et al. 2006, Morkunas et al. 2011) as well as sink-specific enzymes, such as sucrose synthase (Salanoubat and Belliard 1989), granule-bound starch synthase (Visser et al. 1991), and extracellular invertase (Roitsch et al. 1995), are induced by sucrose or glucose. Sugar-induced gene expression has also been detected for enzymes involved in pathogen and stress response, such as proteinase inhibitor II of potato (Johnson and Ryan 1990), chalcone synthase (Tsukaya et al. 1991), and flavonoid biosynthetic enzymes (Morkunas et al. 2011). In contrast, sugar repression of photosynthetic genes was observed. For example, genes encoding photosynthetic proteins, e.g. the small subunit of the Calvin cycle enzyme ribulose bisphosphate carboxylase/oxygenase (Rubisco) and the chlorophyll *a* binding protein, are repressed by carbohydrates (Sheen 1990, Krapp et al. 1993).

Due to their regulatory and signal function, sugars affect all phases of the plant life cycle by controlling the number of essential metabolic processes. In animals, the level of sugar in cells is strictly controlled, but in plants the level and composition of carbohydrates varies widely, depending on tissue type and environmental conditions. An excess or loss of carbohydrates or their derivatives triggers various reactions in plants and significantly affects the metabolism, growth, and development. Moreover, all abiotic and biotic stress responses are regulated, at least in part, by sugars (Koch 1996, Rolland et al. 2002). Plants are generally considered to be autotrophs but sometimes they can be heterotrophs, e.g. at some stages of development (such as seed germination, as long as the seedling grows in darkness, before it emerges above the soil surface) and in non-photosynthetic organs, such as most roots, stems, and flowers. In germinating seeds, in periods of unfavorable environmental conditions or after too deep sowing, mobilization of storage materials in cotyledons may be delayed or there may be some disturbances in distribution of carbohydrates. This may lead to a decrease in available carbohydrates in embryo axes (i.e. sugar starvation) and in a lower seed germination rate. Additionally, high carbohydrate losses can be observed in most plant species under the influence of environmental conditions, such as water or temperature, attack of pathogens or herbivores. These factors may lead to a remarkable decrease in photosynthetic rate in donor tissues (i.e. leaves that synthesize and export carbohydrates), and this reduces the supply of carbohydrates to acceptor tissues (i.e. non-photosynthetic tissues importing carbohydrates for respiration, growth, and development). Besides, in some conditions, e.g. during dormancy or leaf shedding, photosynthesis is switched off or slowed down. In such conditions, the stored carbohydrates must be used, so their reserves may be greatly diminished in non-photosynthetic tissues then. Knowledge of the response to sugar starvation and of adaptive mechanisms in plants is both fundamental and agronomically important.

### **2. Materials and methods of research on sugar starvation**

410 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

genes, where most of TFs showed repression (Price et al. 2004).

protein, are repressed by carbohydrates (Sheen 1990, Krapp et al. 1993).

Rolland et al. 2006, Ramon et al. 2008, Cho et al. 2009, Hanson and Smeekens 2009, Smeekens et al. 2010). Additionally, biochemical studies provide evidence for the involvement of a variety of protein kinases, i.e. Snf1-related kinases (SnRKs) (Rolland et al. 2006, Smeekens et al. 2010), calcium-dependent protein kinases (CDPKs), mitogen-activated protein kinases (MAPKs), and protein phosphatases, 14-3-3 proteins, Ca2+ ions as a second messenger, and G-proteins (Rolland et al. 2006) in sugar signal transduction (Rolland et al. 2002, Sinha et al. 2002). However, it must be stressed that HXK plays a significant role as a component of the sugar sensing machinery. Genetic analyses have revealed a central role for HXK as a conserved glucose sensor (Moore et al. 2003, Harrington and Bush 2003, Rolland and Sheen 2005, Rolland et al. 2006, Ramon et al. 2008, Cho et al. 2009, Hanson and Smeekens 2009, Smeekens et al. 2010). HXK sensing and signaling functions are probably dependent on HXK's subcellular localization, translocation, and/or interactions with downstream effectors. The HXK sugar sensor, as a cytosolic protein or associated with mitochondria or other organelles, then could activate a signaling cascade through HXKinteracting proteins (HIPs) or affect transcription directly after nuclear translocation (Rolland et al. 2002, Rolland et al. 2006, Hanson and Smeekens 2009). Various sugar signals activate many HXK-dependent and HXK-independent pathways and use different molecular mechanisms to control transcription, translation, protein stability, and enzymatic activity. It has been shown that in 7-day-old *Arabidopsis* seedlings, glucose (3%) during 6 h increased the expression of 983 genes and decreased the expression of 771 genes (Li et al. 2006). Sucrose (3%) in *Arabidopsis* cell suspension during 48 h modulated the expression of 243 genes in light and 193 genes in darkness (Nicolaï et al. 2006). Glucose-regulated transcription factors (TFs) account for 8.3% (82 factors among 978) of all glucose-regulated

Genes encoding the enzymes of the phenylpropanoid biosynthesis pathway (Hara et al. 2003, Solfanelli et al. 2006, Morkunas et al. 2011) as well as sink-specific enzymes, such as sucrose synthase (Salanoubat and Belliard 1989), granule-bound starch synthase (Visser et al. 1991), and extracellular invertase (Roitsch et al. 1995), are induced by sucrose or glucose. Sugar-induced gene expression has also been detected for enzymes involved in pathogen and stress response, such as proteinase inhibitor II of potato (Johnson and Ryan 1990), chalcone synthase (Tsukaya et al. 1991), and flavonoid biosynthetic enzymes (Morkunas et al. 2011). In contrast, sugar repression of photosynthetic genes was observed. For example, genes encoding photosynthetic proteins, e.g. the small subunit of the Calvin cycle enzyme ribulose bisphosphate carboxylase/oxygenase (Rubisco) and the chlorophyll *a* binding

Due to their regulatory and signal function, sugars affect all phases of the plant life cycle by controlling the number of essential metabolic processes. In animals, the level of sugar in cells is strictly controlled, but in plants the level and composition of carbohydrates varies widely, depending on tissue type and environmental conditions. An excess or loss of carbohydrates or their derivatives triggers various reactions in plants and significantly affects the metabolism, growth, and development. Moreover, all abiotic and biotic stress responses are regulated, at least in part, by sugars (Koch 1996, Rolland et al. 2002). Plants are Carbohydrate starvation has been studied in many of plant species, e.g. in common wheat (Wittenbach 1977, Wittenbach et al. 1982), maize (Saglio and Pradet 1980, Pace et al. 1990), barley (Farrar 1981), pearl millet (Baysdorfer et al. 1988), pea (Webster and van't Hof 1973, Sahulka and Lisa 1978, Webster and Henry 1987, Morkunas et al. 1999, 2000), soybean (Kerr et al. 1985, Walsh et al. 1987), sycamore (Journet et al. 1986, Dorne et al. 1987, Roby et al. 1987, Genix et al. 1990), tobacco (Moriyasu and Ohsumi 1996), lupine (Morkunas et al. 1999, Borek and Ratajczak 2002, Morkunas et. al. 2003, Borek et al. 2006, Borek and Nuc 2011, Borek et al. 2011, 2012a, 2012b), *Arabidopsis thaliana* (Rose et al. 2006). Metabolic changes caused by sugar starvation are usually analysed in cell cultures, callus tissue cultures, and in isolated plant organs cultured *in vitro*. In all types of the cultures it is easy to control nutrition of the plant material by manipulating the medium composition. Cell cultures clearly react by metabolic changes when sugar is omitted from the nutrient medium, e.g. in sycamore (*Acer pseudoplatanus*) cells (Journet et al. 1986, Gout et al. 2011), but similar sensitivity is observed also in excised root tips (Brouquisse et al. 1991, 1992). *In vitro* cultured embryo axes (isolated from seeds), cut off from the natural source of nutrients, i.e. cotyledons, are a good model for research on the effect of sugars on seedling development and metabolism (Borek et al. 2001, Borek and Ratajczak 2002, Borek et al. 2003, 2006, Borek and Nuc 2011, Borek et al. 2012a, 2012b), or even for research on the role of sugar in plant resistance to pathogens (Morkunas et al. 2005, Morkunas and Gmerek 2007, Morkunas et al. 2007, Morkunas and Bednarski 2008, Morkunas et al. 2008, 2011). The influence of sugar on leaf senescence was assessed on excised leaves and on leaves of intact plants. The effect of sugar starvation was achieved by keeping the plant material in darkness or by its spraying

with photosynthetic inhibitors (Mohapatra et al. 2010). To examine the effects of sugar on senescence of various flower parts, cut flowers were treated with various sucrose concentrations in the vase solution (Azad et al. 2008, Arrom and Munné-Bosch 2012). Brouquisse et al. (1998) show that in roots (the main sink organs of maize plants) the consequences of carbon depletion induced by extended darkness are identical to those observed in the excised root tip, but some aspects of sugar starvation (e.g. effect on translocation of assimilates, links with the metabolism of nitrogen and phosphorus, interactions with plant hormones) were investigated in intact plants (Ciereszko et al. 2005). By spraying of sugar-starved plants with various sugars, it can be determined which of them are signal molecules and which of them are sugar sensors (Lothier et al. 2010).

Plant Responses to Sugar Starvation 413

intercellular spaces. Many of the observed cells were dividing or have just divided. The cells lacked a central vacuole; instead, they contained very numerous small vacuoles. The major feature distinguishing cortical cells of starved lupine (as compared to sugar-fed lupine) was

**Figure 1.** Ultrastructure of root meristematic zone cells of yellow lupine embryo axes grown for 96 h *in vitro* on medium without sucrose (A; sugar starvation) and on medium with 60 mM sucrose (B). CW cell wall, M mitochondrion, N nucleus, Nu nucleolus, OB oil body, Ph phytoferritin, S starch, SP storage

Ultrastructural studies of the root meristematic zone of sucrose-starved lupine embryo axes showed that (in contrast to cells of embryo axes fed with sucrose) their cytoplasm, along with endoplasmic reticulum, mitochondria, plastids, ribosomes, and nuclei, were forced to the periphery by growing vacuoles (Fig. 1A, B). In spite of gradual autolysis of cytoplasmic proteins, in starved cells mitochondria were protected as organelles maintaining cellular respiration. Electron micrographs reveal that the inner mitochondrial membrane in starved pea cells is well developed, forming numerous cristae (Fig. 2A), in contrast to sucrose-fed cells, with less developed cristae (Fig. 2B). Oxygraphic studies of mitochondria isolated from starved embryos of *Pisum sativum* and *Lupinus angustifolius* have shown that they are active and exercise respiratory control (Morkunas et al. 2000, 2003). The phenomenon of mitochondria protection against autolysis under sugar starvation was observed also in other

protein, V vacuole.

the presence of a large central vacuole with marginal cytoplasm.

Particularly valuable information is obtained as a result of research on various types of mutants, which sometimes supplies surprising information about the role of sugars as signal molecules (Gibson 2005, Ramon et al. 2008, Usadel et al. 2008, Hanson and Smeekens 2009).

Plant reactions to sugar deficits have been studied with the use of a wide range of research methods: electron microscopy (Borek and Ratajczak 2002, Borek et al. 2006, 2011, 2012a), confocal microscopy (Morkunas and Bednarski 2008, Morkunas et al. 2011, 2012), enzymatic activity assays and metabolite assays, e.g. by using HPLC (Morkunas et al. 2010), nuclear 1HNMR spectroscopy (Brouquisse et al. 2007, Kim et al. 2007), and 13C and 31P-NMR spectroscopy (Vauclare et al. 2010, Gout et al. 2011). Electron paramagnetic resonance (EPR) spectroscopy is applied to measure free radicals (Morkunas et al. 2004, Morkunas and Bednarski 2008, Morkunas et al. 2008, 2012). Novelties include various methods of molecular biology: assays of gene expression (Buchanan-Wollaston et al. 2005) and application of reporter genes (Lee et al. 2007). Moreover, sugar-starvation-induced promoters have been used to obtain useful recombinant proteins (Xu et al. 2011a, 2011b).

## **3. Morphological, anatomical, and ultrastructural changes under sugar starvation**

Sugar starvation causes changes in growth, as shown in organ cultures *in vitro* and in cell suspensions. For example, significant differences are observed in morphological structure of embryo axes of narrowleaf lupine (*Lupinus angustifolius* L.), white lupine (*Lupinus albus* L.), yellow lupine (*Lupinus luteus* L.), Andean lupine (*Lupinus mutabilis* Sweet) and garden pea (*Pisum sativum* L.) cultured *in vitro* under sugar starvation and with 60 mM sucrose. Radicles of *Pisum* and *Lupinus* spp. cultured under sugar starvation were shorter and thicker than sugar-fed radicles. The length and fresh weight of starved isolated embryo axes was about 2-fold lower than those of sugar-fed axes (Morkunas 1997, Borek et al. 2012a). In contrast to lupine isolated embryo axes, lupine seedlings were much bigger when they were grown on medium without sucrose (Borek et al. 2012a).

Light micrographs of radicle cross sections revealed that starved radicles were thicker than sugar-fed radicles. Cortical cells of sugar-fed embryo axes were rounded, with large intercellular spaces. Many of the observed cells were dividing or have just divided. The cells lacked a central vacuole; instead, they contained very numerous small vacuoles. The major feature distinguishing cortical cells of starved lupine (as compared to sugar-fed lupine) was the presence of a large central vacuole with marginal cytoplasm.

412 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

Smeekens 2009).

**starvation**

with photosynthetic inhibitors (Mohapatra et al. 2010). To examine the effects of sugar on senescence of various flower parts, cut flowers were treated with various sucrose concentrations in the vase solution (Azad et al. 2008, Arrom and Munné-Bosch 2012). Brouquisse et al. (1998) show that in roots (the main sink organs of maize plants) the consequences of carbon depletion induced by extended darkness are identical to those observed in the excised root tip, but some aspects of sugar starvation (e.g. effect on translocation of assimilates, links with the metabolism of nitrogen and phosphorus, interactions with plant hormones) were investigated in intact plants (Ciereszko et al. 2005). By spraying of sugar-starved plants with various sugars, it can be determined which of

them are signal molecules and which of them are sugar sensors (Lothier et al. 2010).

used to obtain useful recombinant proteins (Xu et al. 2011a, 2011b).

medium without sucrose (Borek et al. 2012a).

Particularly valuable information is obtained as a result of research on various types of mutants, which sometimes supplies surprising information about the role of sugars as signal molecules (Gibson 2005, Ramon et al. 2008, Usadel et al. 2008, Hanson and

Plant reactions to sugar deficits have been studied with the use of a wide range of research methods: electron microscopy (Borek and Ratajczak 2002, Borek et al. 2006, 2011, 2012a), confocal microscopy (Morkunas and Bednarski 2008, Morkunas et al. 2011, 2012), enzymatic activity assays and metabolite assays, e.g. by using HPLC (Morkunas et al. 2010), nuclear 1HNMR spectroscopy (Brouquisse et al. 2007, Kim et al. 2007), and 13C and 31P-NMR spectroscopy (Vauclare et al. 2010, Gout et al. 2011). Electron paramagnetic resonance (EPR) spectroscopy is applied to measure free radicals (Morkunas et al. 2004, Morkunas and Bednarski 2008, Morkunas et al. 2008, 2012). Novelties include various methods of molecular biology: assays of gene expression (Buchanan-Wollaston et al. 2005) and application of reporter genes (Lee et al. 2007). Moreover, sugar-starvation-induced promoters have been

**3. Morphological, anatomical, and ultrastructural changes under sugar**

Sugar starvation causes changes in growth, as shown in organ cultures *in vitro* and in cell suspensions. For example, significant differences are observed in morphological structure of embryo axes of narrowleaf lupine (*Lupinus angustifolius* L.), white lupine (*Lupinus albus* L.), yellow lupine (*Lupinus luteus* L.), Andean lupine (*Lupinus mutabilis* Sweet) and garden pea (*Pisum sativum* L.) cultured *in vitro* under sugar starvation and with 60 mM sucrose. Radicles of *Pisum* and *Lupinus* spp. cultured under sugar starvation were shorter and thicker than sugar-fed radicles. The length and fresh weight of starved isolated embryo axes was about 2-fold lower than those of sugar-fed axes (Morkunas 1997, Borek et al. 2012a). In contrast to lupine isolated embryo axes, lupine seedlings were much bigger when they were grown on

Light micrographs of radicle cross sections revealed that starved radicles were thicker than sugar-fed radicles. Cortical cells of sugar-fed embryo axes were rounded, with large

**Figure 1.** Ultrastructure of root meristematic zone cells of yellow lupine embryo axes grown for 96 h *in vitro* on medium without sucrose (A; sugar starvation) and on medium with 60 mM sucrose (B). CW cell wall, M mitochondrion, N nucleus, Nu nucleolus, OB oil body, Ph phytoferritin, S starch, SP storage protein, V vacuole.

Ultrastructural studies of the root meristematic zone of sucrose-starved lupine embryo axes showed that (in contrast to cells of embryo axes fed with sucrose) their cytoplasm, along with endoplasmic reticulum, mitochondria, plastids, ribosomes, and nuclei, were forced to the periphery by growing vacuoles (Fig. 1A, B). In spite of gradual autolysis of cytoplasmic proteins, in starved cells mitochondria were protected as organelles maintaining cellular respiration. Electron micrographs reveal that the inner mitochondrial membrane in starved pea cells is well developed, forming numerous cristae (Fig. 2A), in contrast to sucrose-fed cells, with less developed cristae (Fig. 2B). Oxygraphic studies of mitochondria isolated from starved embryos of *Pisum sativum* and *Lupinus angustifolius* have shown that they are active and exercise respiratory control (Morkunas et al. 2000, 2003). The phenomenon of mitochondria protection against autolysis under sugar starvation was observed also in other

species and plant tissues (Baysdorfer et al. 1988, Brouquisse et al. 1991). However, Couée et al. (1992) reported that under conditions of carbohydrate starvation, mitochondria could be degraded. Those authors revealed heterogeneity of mitochondria isolated from root tips of maize (*Zea mays*) and fractionated on Percoll density gradients, i.e. higher- and lower-density mitochondria. The higher-density mitochondria from glucose-starved maize root tips retained the ultrastructure and most of the respiratory properties of nonstarved mitochondria. By contrast, lower- and intermediate-density mitochondria were absent in the mitochondrial fractions from glucose-starved maize root tips and were not detected *in situ*. Interestingly, in plastids of starved pea cells, large amounts of phytoferritin were accumulated (Fig. 3A), while only starch grains and tubular structures were found in plastids of cells with a high level of sucrose (Fig. 3B) (Morkunas 1997). Lowering carbohydrate level in storage tissues of germinating seeds enhances the breakdown of reserves. In cotyledons of 4-day-old lupine seedlings and in excised lupine cotyledons grown on medium without sucrose, the deposits of storage protein were clearly smaller than in organs fed with sucrose. Starch granules and oil bodies were also smaller and less numerous (Fig. 4A, B). Similarly, the cell walls, containing hemicelluloses (important storage compounds in lupine seeds) were thinner (Borek et al. 2006, 2011, 2012a).

Plant Responses to Sugar Starvation 415

**Figure 3.** Plastids of pea embryo axes grown for 72 h *in vitro* on medium without sucrose (A; sugar starvation) and on medium with 60 mM sucrose (B). CW cell wall, ER endoplasmic reticulum, M

Research conducted on *Arabidopsis* cell suspension revealed that sugar starvation caused an immediate arrest of cell growth, together with a rapid degradation of cellular proteins. Cell divisions, as indicated by cell number and accumulation of biomass, were stopped immediately after the initiation of sucrose starvation. Observations of the morphology of starved cells using Nomarski interference microscopy and confocal laser-scanning microscopy revealed that in sucrose-free medium, the width of the cytoplasm was markedly decreased, while the vacuole was enlarged (Rose et al. 2006). Carbon starvation induced autophagy in plant cells, as confirmed by experiments on model systems, e.g. on cell suspensions of sycamore (Journet et al. 1986), rice (Chen et al. 1994), *Arabidopsis* (Rose et al. 2006) and in pea and lupine embryo axes grown *in vitro* (Morkunas 1997, Morkunas et al. 2003, Borek et al. 2011, 2012a, 2012b). Vacuolar autophagy starts as early as several hours after sugar starvation. Sequestration of portions of the cytoplasm with organelles (except for the nucleus) by endomembranes results in formation of autophagic vacuoles, followed by their fusion with other vacuoles. During periods of nutrient starvation, the autophagic process can be reinitiated in plant cells that are already vacuolated (Chen et al. 1994, Aubert et al. 1996, Moriyasu and Ohsumi 1996, Moriyasu and Klionsky 2003, Moriyasu et al. 2003). Carbohydrate starvation-induced autophagy is associated with an increase in intracellular proteolysis (James et al. 1996, Moriyasu and Ohsumi 1996, Thompson and Vierstra 2005) and a marked degradation of membrane polar lipids (Aubert et al. 1996, Inoue and

mitochondrion, P plastid, Ph phytoferritin, Pl plastoglobule, S starch, V vacuole.

**Figure 2.** Mitochondria of pea embryo axes grown for 72 h *in vitro* on medium without sucrose (A; sugar starvation) and on medium with 60 mM sucrose (B). CW cell wall, ER endoplasmic reticulum, GA Golgi apparatus, M mitochondrion, V vacuole.

2012a).

species and plant tissues (Baysdorfer et al. 1988, Brouquisse et al. 1991). However, Couée et al. (1992) reported that under conditions of carbohydrate starvation, mitochondria could be degraded. Those authors revealed heterogeneity of mitochondria isolated from root tips of maize (*Zea mays*) and fractionated on Percoll density gradients, i.e. higher- and lower-density mitochondria. The higher-density mitochondria from glucose-starved maize root tips retained the ultrastructure and most of the respiratory properties of nonstarved mitochondria. By contrast, lower- and intermediate-density mitochondria were absent in the mitochondrial fractions from glucose-starved maize root tips and were not detected *in situ*. Interestingly, in plastids of starved pea cells, large amounts of phytoferritin were accumulated (Fig. 3A), while only starch grains and tubular structures were found in plastids of cells with a high level of sucrose (Fig. 3B) (Morkunas 1997). Lowering carbohydrate level in storage tissues of germinating seeds enhances the breakdown of reserves. In cotyledons of 4-day-old lupine seedlings and in excised lupine cotyledons grown on medium without sucrose, the deposits of storage protein were clearly smaller than in organs fed with sucrose. Starch granules and oil bodies were also smaller and less numerous (Fig. 4A, B). Similarly, the cell walls, containing hemicelluloses (important storage compounds in lupine seeds) were thinner (Borek et al. 2006, 2011,

**Figure 2.** Mitochondria of pea embryo axes grown for 72 h *in vitro* on medium without sucrose (A; sugar starvation) and on medium with 60 mM sucrose (B). CW cell wall, ER endoplasmic reticulum, GA

Golgi apparatus, M mitochondrion, V vacuole.

**Figure 3.** Plastids of pea embryo axes grown for 72 h *in vitro* on medium without sucrose (A; sugar starvation) and on medium with 60 mM sucrose (B). CW cell wall, ER endoplasmic reticulum, M mitochondrion, P plastid, Ph phytoferritin, Pl plastoglobule, S starch, V vacuole.

Research conducted on *Arabidopsis* cell suspension revealed that sugar starvation caused an immediate arrest of cell growth, together with a rapid degradation of cellular proteins. Cell divisions, as indicated by cell number and accumulation of biomass, were stopped immediately after the initiation of sucrose starvation. Observations of the morphology of starved cells using Nomarski interference microscopy and confocal laser-scanning microscopy revealed that in sucrose-free medium, the width of the cytoplasm was markedly decreased, while the vacuole was enlarged (Rose et al. 2006). Carbon starvation induced autophagy in plant cells, as confirmed by experiments on model systems, e.g. on cell suspensions of sycamore (Journet et al. 1986), rice (Chen et al. 1994), *Arabidopsis* (Rose et al. 2006) and in pea and lupine embryo axes grown *in vitro* (Morkunas 1997, Morkunas et al. 2003, Borek et al. 2011, 2012a, 2012b). Vacuolar autophagy starts as early as several hours after sugar starvation. Sequestration of portions of the cytoplasm with organelles (except for the nucleus) by endomembranes results in formation of autophagic vacuoles, followed by their fusion with other vacuoles. During periods of nutrient starvation, the autophagic process can be reinitiated in plant cells that are already vacuolated (Chen et al. 1994, Aubert et al. 1996, Moriyasu and Ohsumi 1996, Moriyasu and Klionsky 2003, Moriyasu et al. 2003). Carbohydrate starvation-induced autophagy is associated with an increase in intracellular proteolysis (James et al. 1996, Moriyasu and Ohsumi 1996, Thompson and Vierstra 2005) and a marked degradation of membrane polar lipids (Aubert et al. 1996, Inoue and

Moriyasu 2006). In addition, Rose et al. (2006) revealed that concomitantly, the number of transvacuolar strands is decreased dramatically. Starvation-induced acidic compartments were most frequent in the dense perinuclear cytoplasm, close to the large central vacuole. In vacuoles of starved cells, quinacrine was strongly accumulated. The role of autophagy in adaptation to sugar starvation has been investigated primarily because of its impact on basal metabolism and biomass production. The intracellular degradation of cytoplasmic components by means of autophagy produces amino acids, phospholipids, and other elements that are necessary for basic metabolism and essential biosynthetic pathways. In starved cells the initiation of autophagy is needed to obtain respiratory substrates (Journet et al. 1986, Chen et al. 1994, Aubert et al. 1996).

Plant Responses to Sugar Starvation 417

(1992) report that during sugar starvation of maize root meristems, 3 phases can be

1. acclimation (from 0 to 30-35 h), when cellular carbohydrate levels and respiration rate decrease, while nitrogen is released from storage proteins through their degradation; 2. survival phase (from 30-35 to 90-100 h), involving intensive breakdown of proteins and lipids, release of Pi phosphorylcholine, and free amino acids; this phase can be reversed

3. cell disorganization (more than 100 h), when the level of all metabolites and enzymatic activity are significantly decreased and the changes are irreversible, leading to death.

During sugar starvation at the acclimation and survival phases, the total protein content decreases. This is associated with a temporary increase in free amino acids and an increase in proteolytic activity (Tassi et al. 1992, James et al. 1993, Moriyasu and Ohsumi 1996, Borek and Ratajczak 2002). *In vitro* studies of embryo axes of lupines and garden pea have revealed a dramatic decrease in protein concentration (especially the soluble fraction) in starved cells (Morkunas 1997, Borek et al. 2012a). The decrease in cytosolic proteins in starved cells was correlated with an increase in the activity of proteolytic enzymes, i.e. endo- and exopeptidases (Morkunas et al. 1999, Borek and Ratajczak 2002). The increased activity of proteases could result from activation of enzymatic proteins that were already present in cells but in a bound or inactive form. Their increased activity could also result from *de novo* synthesis of the enzymes, caused by release of their genes from catabolite repression. The increased activity of proteases does not confirm unambiguously that the lack of sugar causes an enhanced expression of genes of these enzymes, because enzymatic activity can be controlled in other ways, e.g. by allosteric regulation or by phosphorylation and dephosphorylation, which is the most common type of reversible covalent modification (Berg et al. 2002). However, a study of starved maize root meristems shows that the increased activity of proteolytic enzymes under sugar starvation is an effect of enhanced transcription and translation (James et al. 1993). It also has been evidenced that proteases are closely associated with some cell compartments. Optimal acidic pH for endo- and carboxypeptidase, which increased under starvation, suggested that proteases were located in vacuoles or in lysosomes (Feller 1986, Mikola and Mikola 1986, Huffaker 1990). Accumulation of autolysosomes was observed in tobacco suspension cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor E-64. Experiments with fluorescent dyes, green fluorescent protein (GFP), and endocytosis markers (FM4-64 and Lucifer Yellow CH) suggested that there is a membrane flow from the plasma membrane to autolysosomes. Using fluorescent dyes and markers of the central vacuole (GFP-AtVam3p, sporamin-GFP and gamma-VM23-GFP), the transport of components of the central vacuole to autolysosomes was displayed (Yano et al. 2004). When cells of tobacco were treated with E-64c cysteine protease inhibitor, then both protein degradation and protease activation were inhibited. Simultaneously, many spherical bodies accumulated in the cytosol, beside the nucleus. The bodies, with acidic pH inside, contained acid phosphatase, which is a marker enzyme of autolysosomes. The bodies accumulated because E-64c inhibitor blocked the degradation of the proteins contained in them.

distinguished:

by sugar feeding;

**Figure 4.** Ultrastructure of parenchyma cells of white lupine excised cotyledons grown for 96 h *in vitro* on medium without sucrose (A) and on medium with 60 mM sucrose (B). CW cell wall, ER endoplasmic reticulum, M mitochondrion, N nucleus, Nu nucleolus, OB oil body, Ph phytoferritin, S starch, SP storage protein, V vacuole.

## **4. Plant metabolism under sugar starvation**

The studies have shown that in most cases, sugar starvation triggers a specific sequence of events in plant cells. The cells subjected to sugar starvation at first adapt to the lack of carbohydrates through gradual replacement of carbohydrate metabolism by protein and lipid metabolism. Such metabolic reorganization may result in autophagy (Saglio and Pradet 1980, Journet et al. 1986, Brouquisse et al. 1991, Rose et al. 2006). Brouquisse et al. (1992) report that during sugar starvation of maize root meristems, 3 phases can be distinguished:

416 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

al. 1986, Chen et al. 1994, Aubert et al. 1996).

storage protein, V vacuole.

**4. Plant metabolism under sugar starvation**

Moriyasu 2006). In addition, Rose et al. (2006) revealed that concomitantly, the number of transvacuolar strands is decreased dramatically. Starvation-induced acidic compartments were most frequent in the dense perinuclear cytoplasm, close to the large central vacuole. In vacuoles of starved cells, quinacrine was strongly accumulated. The role of autophagy in adaptation to sugar starvation has been investigated primarily because of its impact on basal metabolism and biomass production. The intracellular degradation of cytoplasmic components by means of autophagy produces amino acids, phospholipids, and other elements that are necessary for basic metabolism and essential biosynthetic pathways. In starved cells the initiation of autophagy is needed to obtain respiratory substrates (Journet et

**Figure 4.** Ultrastructure of parenchyma cells of white lupine excised cotyledons grown for 96 h *in vitro* on medium without sucrose (A) and on medium with 60 mM sucrose (B). CW cell wall, ER endoplasmic reticulum, M mitochondrion, N nucleus, Nu nucleolus, OB oil body, Ph phytoferritin, S starch, SP

The studies have shown that in most cases, sugar starvation triggers a specific sequence of events in plant cells. The cells subjected to sugar starvation at first adapt to the lack of carbohydrates through gradual replacement of carbohydrate metabolism by protein and lipid metabolism. Such metabolic reorganization may result in autophagy (Saglio and Pradet 1980, Journet et al. 1986, Brouquisse et al. 1991, Rose et al. 2006). Brouquisse et al.

During sugar starvation at the acclimation and survival phases, the total protein content decreases. This is associated with a temporary increase in free amino acids and an increase in proteolytic activity (Tassi et al. 1992, James et al. 1993, Moriyasu and Ohsumi 1996, Borek and Ratajczak 2002). *In vitro* studies of embryo axes of lupines and garden pea have revealed a dramatic decrease in protein concentration (especially the soluble fraction) in starved cells (Morkunas 1997, Borek et al. 2012a). The decrease in cytosolic proteins in starved cells was correlated with an increase in the activity of proteolytic enzymes, i.e. endo- and exopeptidases (Morkunas et al. 1999, Borek and Ratajczak 2002). The increased activity of proteases could result from activation of enzymatic proteins that were already present in cells but in a bound or inactive form. Their increased activity could also result from *de novo* synthesis of the enzymes, caused by release of their genes from catabolite repression. The increased activity of proteases does not confirm unambiguously that the lack of sugar causes an enhanced expression of genes of these enzymes, because enzymatic activity can be controlled in other ways, e.g. by allosteric regulation or by phosphorylation and dephosphorylation, which is the most common type of reversible covalent modification (Berg et al. 2002). However, a study of starved maize root meristems shows that the increased activity of proteolytic enzymes under sugar starvation is an effect of enhanced transcription and translation (James et al. 1993). It also has been evidenced that proteases are closely associated with some cell compartments. Optimal acidic pH for endo- and carboxypeptidase, which increased under starvation, suggested that proteases were located in vacuoles or in lysosomes (Feller 1986, Mikola and Mikola 1986, Huffaker 1990). Accumulation of autolysosomes was observed in tobacco suspension cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor E-64. Experiments with fluorescent dyes, green fluorescent protein (GFP), and endocytosis markers (FM4-64 and Lucifer Yellow CH) suggested that there is a membrane flow from the plasma membrane to autolysosomes. Using fluorescent dyes and markers of the central vacuole (GFP-AtVam3p, sporamin-GFP and gamma-VM23-GFP), the transport of components of the central vacuole to autolysosomes was displayed (Yano et al. 2004). When cells of tobacco were treated with E-64c cysteine protease inhibitor, then both protein degradation and protease activation were inhibited. Simultaneously, many spherical bodies accumulated in the cytosol, beside the nucleus. The bodies, with acidic pH inside, contained acid phosphatase, which is a marker enzyme of autolysosomes. The bodies accumulated because E-64c inhibitor blocked the degradation of the proteins contained in them.

Consequently, the normally short life span of these bodies was significantly prolonged, and their observation was possible. In all probability, the observed spherical bodies were autolysosomes participating in protein degradation during autophagy (Moriyasu and Ohsumi 1996). During advanced sugar starvation, autophagy involves cytosolic proteins and even whole organelles can be degraded. Plastids, ribosomes, and endoplasmic reticulum are consumed relatively early, whereas the plasma membrane, mitochondria, and peroxisomes persist longer (Baker and Graham 2002). The increased activity of proteolytic enzymes and autophagic processes observed under sugar starvation are certainly adaptive processes enabling the cells to maintain continuous energy supply under sugar starvation. Amino acids released from degraded cytosolic proteins, storage proteins, and organelles are used as respiratory substrates (Brouquisse et al. 1991, Ratajczak et al. 1996, Yu 1999, Borek et al. 2001, Gonzali et al. 2006).

Plant Responses to Sugar Starvation 419

specific proteins appear, known as carbohydrate-responsive proteins (Baysdorfer et al. 1988). They may be the proteins associated with the phase of tissue acclimation to sugar starvation. Prolonged starvation results in synthesis of many specific proteins known as starvation-related proteins (STP) (Tassi et al. 1992). The genes encoding them (at least some of them) are normally under catabolite repression caused by carbohydrates. Their

During sugar starvation, respiration rate declines rapidly in embryos of garden pea (Morkunas et al. 2000) and yellow lupine (Borek et al. 2011). The lower respiration rate is caused by the deficit of respiratory substrates rather than by mitochondrial degradation. In mitochondria isolated from starved pea embryos, respiratory activity (with glutamate as a substrate) is 60% higher than in those isolated from embryos fed with 60 mM sucrose (Morkunas et al. 2000). A similar relationship was observed in mitochondrial fractions from starved and control (sugar-fed) embryos of narrowleaf lupine (Morkunas et al. 2003). The concentration of mitochondrial proteins in starved cells was slightly lower than in control cells, and the activity of a mitochondrial marker enzyme, NAD+-dependent isocitrate dehydrogenase, was reduced in starved embryos only slightly. Besides, mitochondria isolated from lupine embryo axes cultivated without sucrose, exhibited respiration coupled with oxidative phosphorylation. They exhibited a respiration control ratio of 2 for succinate as a respiratory substrate. Mitochondria isolated from starved embryo axes oxidized glutamate and malate more intensively than mitochondria from embryo axes fed with sucrose. Ultrastructural analysis of cells of starved pea embryo axes shows that the inner mitochondrial membrane is highly convoluted, forming many folds (cristae, Fig. 2A). A similar finding was reported by Journet et al. (1986) who studied sycamore cell suspension. Mitochondria were protected in sycamore cells even as late as after 60 h of starvation, and then an addition of sugar to the medium resulted in intensive respiration. In starved cultures, sugar reserves were used up very quickly. Respiration rate was maintained at a high level for up to 30 h of starvation. In that period, respiratory substrates were provided by degradation of starch, phosphate esters, and proteins. Morkunas et al. (2000) also showed that the transfer of pea embryo axes from sugar starvation conditions onto the medium with sucrose after 48 h recovered their respiration activity. Metabolic adaptations of starved embryos of lupine (Morkunas et al. 2003) and pea (Morkunas et al. 2000) were quite effective physiologically, enabling their survival, and their transfer to a medium with sucrose allowed restoration of normal growth. Similarly, Couée et al. (1992), who studied maize root tips cultured *in vitro*, found that after 48 h of sugar starvation they still had a significant pool of functional mitochondria. Carbohydrate stress caused degeneration of lower-density mitochondria, but not of higher-density mitochondria, which was proved by measurements of respiratory activity of mitochondria and of fumarase activity (mitochondrial marker enzyme). Under sugar starvation, the increased utilization of cytosolic proteins and storage proteins, is accompanied by a more intensive catabolism of lipids, aimed to supply respiratory substrates. In turn, sugar feeding significantly retards the breakdown of storage lipid. In the research on the regulatory function of sugars in lipid metabolism, sucrose and glucose are predominantly taken under consideration. Sucrose is particularly important in seed tissues

expression is initiated when the level of sugar in the cell falls below a critical level.

A perfect example of enzymes induced under sugar starvation is glutamate dehydrogenase (GDH). This is a mitochondrial enzyme that catalyzes the synthesis and degradation of glutamate, which is one of the central amino acids of nitrogen metabolism in plants (Lehmann and Ratajczak 2008, Borek et al. 2011). The reversibility of the reaction catalyzed by GDH *in vivo* is considered as alternative and depends on carbon and ammonium status (Lehmann et al. 2011). In sucrose-starved pea and lupine embryo axes, a remarkable increase in GDH activity was observed (Morkunas et al. 2000, Lehmann et al. 2003, 2010, Borek and Nuc 2011, Borek et al. 2012a). Sugar starvation caused also a significant increase in a number of GDH isoenzymes (Morkunas et al. 2000, Lehmann et al. 2003, 2010). The increased GDH activity caused by sugar deficit was also recorded in callus of *Nicotiana plumbaginifolia* (Maestri et al. 1991) and in carrot cell suspension (Robinson et al. 1992). Under sugar starvation, plant mitochondria may adapt to the stress conditions by increasing their ability to use amino acids as respiratory substrates. Thus the increase in GDH activity under sugar starvation may be the reason for the higher oxidation of glutamate by mitochondria. GDH catalyzing the oxidative deamination of glutamate, in cooperation with the corresponding aminotransferases, is the main pathway of amino acid catabolism in plants (Lea 1993, Lehmann and Ratajczak 2008). During this process, a large quantity of ammonia is produced. Nitrogen in this form is toxic, so its utilization is necessary. One of the possibilities of ammonia detoxification is the synthesis of asparagine, whose content increases during sugar starvation. In sugar-starved suspension cells of sycamore, asparagine content increased steadily, whereas the cell protein content declined progressively (Genix et al. 1990).

The higher activity of the enzymes of protein and amino acid catabolism under sugar starvation probably results from the release of the genes encoding them from catabolite repression (caused by sugar). Catabolite repression is of fundamental importance since it allows cells to adapt their metabolism to carbon sources other than sugar when they are in a habitat where sugar is unavailable. If sugar is present in the medium, then the synthesis of enzymes of the catabolism of carbon sources other than carbohydrates is inhibited. This applies to proteolytic enzymes, amino acid dehydrogenases (mostly GDH), alcohol dehydrogenase, and isocitrate dehydrogenase. Other studies of the influence of sugar deficit on plant cell metabolism show that already in the initial phase of starvation (after 1.5 h), specific proteins appear, known as carbohydrate-responsive proteins (Baysdorfer et al. 1988). They may be the proteins associated with the phase of tissue acclimation to sugar starvation. Prolonged starvation results in synthesis of many specific proteins known as starvation-related proteins (STP) (Tassi et al. 1992). The genes encoding them (at least some of them) are normally under catabolite repression caused by carbohydrates. Their expression is initiated when the level of sugar in the cell falls below a critical level.

418 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

al. 2001, Gonzali et al. 2006).

progressively (Genix et al. 1990).

Consequently, the normally short life span of these bodies was significantly prolonged, and their observation was possible. In all probability, the observed spherical bodies were autolysosomes participating in protein degradation during autophagy (Moriyasu and Ohsumi 1996). During advanced sugar starvation, autophagy involves cytosolic proteins and even whole organelles can be degraded. Plastids, ribosomes, and endoplasmic reticulum are consumed relatively early, whereas the plasma membrane, mitochondria, and peroxisomes persist longer (Baker and Graham 2002). The increased activity of proteolytic enzymes and autophagic processes observed under sugar starvation are certainly adaptive processes enabling the cells to maintain continuous energy supply under sugar starvation. Amino acids released from degraded cytosolic proteins, storage proteins, and organelles are used as respiratory substrates (Brouquisse et al. 1991, Ratajczak et al. 1996, Yu 1999, Borek et

A perfect example of enzymes induced under sugar starvation is glutamate dehydrogenase (GDH). This is a mitochondrial enzyme that catalyzes the synthesis and degradation of glutamate, which is one of the central amino acids of nitrogen metabolism in plants (Lehmann and Ratajczak 2008, Borek et al. 2011). The reversibility of the reaction catalyzed by GDH *in vivo* is considered as alternative and depends on carbon and ammonium status (Lehmann et al. 2011). In sucrose-starved pea and lupine embryo axes, a remarkable increase in GDH activity was observed (Morkunas et al. 2000, Lehmann et al. 2003, 2010, Borek and Nuc 2011, Borek et al. 2012a). Sugar starvation caused also a significant increase in a number of GDH isoenzymes (Morkunas et al. 2000, Lehmann et al. 2003, 2010). The increased GDH activity caused by sugar deficit was also recorded in callus of *Nicotiana plumbaginifolia* (Maestri et al. 1991) and in carrot cell suspension (Robinson et al. 1992). Under sugar starvation, plant mitochondria may adapt to the stress conditions by increasing their ability to use amino acids as respiratory substrates. Thus the increase in GDH activity under sugar starvation may be the reason for the higher oxidation of glutamate by mitochondria. GDH catalyzing the oxidative deamination of glutamate, in cooperation with the corresponding aminotransferases, is the main pathway of amino acid catabolism in plants (Lea 1993, Lehmann and Ratajczak 2008). During this process, a large quantity of ammonia is produced. Nitrogen in this form is toxic, so its utilization is necessary. One of the possibilities of ammonia detoxification is the synthesis of asparagine, whose content increases during sugar starvation. In sugar-starved suspension cells of sycamore, asparagine content increased steadily, whereas the cell protein content declined

The higher activity of the enzymes of protein and amino acid catabolism under sugar starvation probably results from the release of the genes encoding them from catabolite repression (caused by sugar). Catabolite repression is of fundamental importance since it allows cells to adapt their metabolism to carbon sources other than sugar when they are in a habitat where sugar is unavailable. If sugar is present in the medium, then the synthesis of enzymes of the catabolism of carbon sources other than carbohydrates is inhibited. This applies to proteolytic enzymes, amino acid dehydrogenases (mostly GDH), alcohol dehydrogenase, and isocitrate dehydrogenase. Other studies of the influence of sugar deficit on plant cell metabolism show that already in the initial phase of starvation (after 1.5 h), During sugar starvation, respiration rate declines rapidly in embryos of garden pea (Morkunas et al. 2000) and yellow lupine (Borek et al. 2011). The lower respiration rate is caused by the deficit of respiratory substrates rather than by mitochondrial degradation. In mitochondria isolated from starved pea embryos, respiratory activity (with glutamate as a substrate) is 60% higher than in those isolated from embryos fed with 60 mM sucrose (Morkunas et al. 2000). A similar relationship was observed in mitochondrial fractions from starved and control (sugar-fed) embryos of narrowleaf lupine (Morkunas et al. 2003). The concentration of mitochondrial proteins in starved cells was slightly lower than in control cells, and the activity of a mitochondrial marker enzyme, NAD+-dependent isocitrate dehydrogenase, was reduced in starved embryos only slightly. Besides, mitochondria isolated from lupine embryo axes cultivated without sucrose, exhibited respiration coupled with oxidative phosphorylation. They exhibited a respiration control ratio of 2 for succinate as a respiratory substrate. Mitochondria isolated from starved embryo axes oxidized glutamate and malate more intensively than mitochondria from embryo axes fed with sucrose. Ultrastructural analysis of cells of starved pea embryo axes shows that the inner mitochondrial membrane is highly convoluted, forming many folds (cristae, Fig. 2A). A similar finding was reported by Journet et al. (1986) who studied sycamore cell suspension. Mitochondria were protected in sycamore cells even as late as after 60 h of starvation, and then an addition of sugar to the medium resulted in intensive respiration. In starved cultures, sugar reserves were used up very quickly. Respiration rate was maintained at a high level for up to 30 h of starvation. In that period, respiratory substrates were provided by degradation of starch, phosphate esters, and proteins. Morkunas et al. (2000) also showed that the transfer of pea embryo axes from sugar starvation conditions onto the medium with sucrose after 48 h recovered their respiration activity. Metabolic adaptations of starved embryos of lupine (Morkunas et al. 2003) and pea (Morkunas et al. 2000) were quite effective physiologically, enabling their survival, and their transfer to a medium with sucrose allowed restoration of normal growth. Similarly, Couée et al. (1992), who studied maize root tips cultured *in vitro*, found that after 48 h of sugar starvation they still had a significant pool of functional mitochondria. Carbohydrate stress caused degeneration of lower-density mitochondria, but not of higher-density mitochondria, which was proved by measurements of respiratory activity of mitochondria and of fumarase activity (mitochondrial marker enzyme).

Under sugar starvation, the increased utilization of cytosolic proteins and storage proteins, is accompanied by a more intensive catabolism of lipids, aimed to supply respiratory substrates. In turn, sugar feeding significantly retards the breakdown of storage lipid. In the research on the regulatory function of sugars in lipid metabolism, sucrose and glucose are predominantly taken under consideration. Sucrose is particularly important in seed tissues

because storage lipid is synthesized from this sugar in developing seeds and sucrose is one of the main end products of storage lipid breakdown during seed germination. In seedlings of *Arabidopsis* (a model oil-seed plant), 1% sucrose significantly retards the breakdown of storage lipid (Eastmond et al. 2000), while 0.3 M glucose nearly eliminates the mobilization of this storage compound (To et al. 2002). Seedlings grown for 22 days on glucose solution contained about 80% of their seed storage lipid. In contrast, 22-day-old seedlings grown in equi-molar sorbitol retained only 4-5% of their seed storage lipid. This result additionally proves that the effect of glucose is not due to the osmotic potential of the media (To et al. 2002).

Plant Responses to Sugar Starvation 421

matter). Restriction in sucrose feeding of developing embryos of yellow lupine (lipid content about 6% of seed dry matter), white lupine (lipid content 7-14%), and Andean lupine (lipid content about 20%) caused a decrease in storage lipid accumulation during seed development (Borek et al. 2009). Ultrastructural investigations of cotyledons of 4-day-old yellow, white, and Andean lupine seedlings showed that oil bodies were smaller and less numerous when sugar level was decreased in tissues (Borek et al. 2006, 2011, 2012a). A peculiar and puzzling feature was observed in 4-day-old sucrose-starved isolated embryo axes of yellow, white, and Andean lupine grown *in vitro*. Their lipid level was by 43, 44, and 70% higher, respectively, than in sucrose-fed organs (Borek et al. 2012b). Lipase and catalase activity increased when sugar reserves were depleted in yellow lupine germinating seed organs (Borek et al. 2006). However, the enzymes involved in further steps of lipid breakdown (cytosolic aconitase, isocitrate lyase, NADP+-dependent cytosolic isocitrate dehydrogenase) were more active in sucrose-fed tissues (Borek and Nuc 2011). Changes in enzymatic activity caused by sucrose starvation were accompanied by modification of gene expression. Lipase mRNA level was higher, while mRNA levels for cytosolic aconitase and NADP+-dependent isocitrate dehydrogenase were lower in yellow lupine cotyledons and embryo axes whose sugar content was decreased (Borek and Nuc 2011). During yellow lupine seed germination, lipid-derived carbon skeletons are used for sugar synthesis but some of them are also used for amino acid formation (mainly asparagine, glutamine, and glutamate). In sugar-deficient conditions in tissues, this carbon flow from storage lipid to

amino acids is significantly inhibited (Borek et al. 2003, Borek and Ratajczak 2010).

**5. The role of sugar starvation in plant growth and development**

and in plant responses to biotic and abiotic stresses.

As mentioned above, sugar in plants is not only an energetic and structural substrate but it also regulates the expression of many genes. Sugar may play a role of hormonal-like signal in a variety of eukaryotic cell types. The best known are the signaling pathways in yeasts (*Saccharomyces cerevisiae*), but many similar pathways have also been discovered in plants (Rolland et al. 2006). The time-course monitoring of gene expression profiles in suspension cells was investigated by Wang et al. (2007). After 12, 24, and 48 h of sucrose-starvation in rice cells, the expression of 867 genes was increased, while the expression of 855 genes was inhibited. Most of these genes encoded enzymes associated with metabolism. It is characteristic that genes associated with catabolism were up-regulated, while genes involved in biosynthetic pathways were down-regulated. Importantly, Wang et al. (2007) succeeded to identify the genes responsible for transcription and translation, which were the second and third major categories of genes in each group significantly affected by sucrose starvation. Response-to-stress genes were the fourth largest group of regulated genes, which suggests that most of the stress-associated genes were also significantly up-regulated by sucrose starvation. Admittedly, the model of cell suspension used by Wang and his research team is a rather "artificial" system, but identification of the genes affected by sugar starvation proved to be very useful for interpretation of results of research on the role of sugar starvation in the regulation of various processes of plant growth and development

Transcriptome analysis of sucrose-starved rice suspension cells has revealed a decrease in the expression of transcripts involved in fatty acid synthesis but induced those involved in fatty acid degradation, such as 3-ketoacyl-CoA thiolase, fatty acid multifunctional proteins, and acyl-CoA oxidase (Wang et al. 2007). An increased expression of genes encoding lipase and enzymes linked to fatty acid β-oxidation was noted in sucrose-starved *Arabidopsis* suspension cells (Contento et al. 2004). Similarly, in dark-induced senescent *Arabidopsis* leaves an enhanced gene expression of lipase and enzymes involved in fatty acid βoxidation was observed (Buchanan-Wollaston et al. 2005). Glucose starvation remarkably enhances the intensity of lipid degradation in isolated maize root tips. After 24 h of glucose starvation, the rate of oxidation of palmitic acid to CO2 was increased 2.5-fold. The overall βoxidation of fatty acids (measured as acetyl-CoA formation) was increased up to 5-fold in glucose-starved roots. An increase in catalase activity was observed as well (Dieuaide et al. 1992). Catalase is not directly involved in fatty acid β-oxidation but is involved in detoxification of H2O2 produced in glyoxysomes by the activity of acyl-CoA oxidase when acyl-CoA is converted into trans-2-enoyl-CoA. Sucrose starvation in *Arabidopsis* suspension cells caused a 5.7-fold increase in the expression of acyl-CoA oxidase-4, which is the first committed step of short-chain fatty acid β-oxidation. A significant increase in acyl-CoA oxidase-4 activity in suspension cells was observed after 12 h of sucrose starvation, whereas a similar effect in one-week-old seedlings was noted after 2 days of sucrose starvation. The significant increase in gene expression of catalase-3 (glyoxysomal isoform) was observed already after 6 h of the removal of sucrose from the medium of *Arabidopsis* suspension cells. An increase in catalase activity was observed 6 h later, i.e. after 12 h of sucrose starvation. In seedlings the increase in catalase activity was observed after 2 days of sucrose starvation. About half of the catalase activity observed during sucrose starvation was due to glyoxysomal isoform 3 (Contento and Bassham 2010). In cucumber suspension cells, the expression of genes encoding 2 marker enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase) was induced during deficits of sucrose, mannose and fructose in the medium (Graham et al. 1994). The above-mentioned results suggest that the enhanced gene expression patterns and enzymatic activities involved in lipolysis, fatty acid β-oxidation, and glyoxylate cycle, facilitate the production of acetyl-CoA from the lipid, which may be directly utilized by respiration to sustain energy production and growth when carbohydrates are exhausted.

Another set of data concerns the regulation of lipid metabolism by sucrose in developing and germinating protein lupine seeds (storage protein content up to 45% of seed dry matter). Restriction in sucrose feeding of developing embryos of yellow lupine (lipid content about 6% of seed dry matter), white lupine (lipid content 7-14%), and Andean lupine (lipid content about 20%) caused a decrease in storage lipid accumulation during seed development (Borek et al. 2009). Ultrastructural investigations of cotyledons of 4-day-old yellow, white, and Andean lupine seedlings showed that oil bodies were smaller and less numerous when sugar level was decreased in tissues (Borek et al. 2006, 2011, 2012a). A peculiar and puzzling feature was observed in 4-day-old sucrose-starved isolated embryo axes of yellow, white, and Andean lupine grown *in vitro*. Their lipid level was by 43, 44, and 70% higher, respectively, than in sucrose-fed organs (Borek et al. 2012b). Lipase and catalase activity increased when sugar reserves were depleted in yellow lupine germinating seed organs (Borek et al. 2006). However, the enzymes involved in further steps of lipid breakdown (cytosolic aconitase, isocitrate lyase, NADP+-dependent cytosolic isocitrate dehydrogenase) were more active in sucrose-fed tissues (Borek and Nuc 2011). Changes in enzymatic activity caused by sucrose starvation were accompanied by modification of gene expression. Lipase mRNA level was higher, while mRNA levels for cytosolic aconitase and NADP+-dependent isocitrate dehydrogenase were lower in yellow lupine cotyledons and embryo axes whose sugar content was decreased (Borek and Nuc 2011). During yellow lupine seed germination, lipid-derived carbon skeletons are used for sugar synthesis but some of them are also used for amino acid formation (mainly asparagine, glutamine, and glutamate). In sugar-deficient conditions in tissues, this carbon flow from storage lipid to amino acids is significantly inhibited (Borek et al. 2003, Borek and Ratajczak 2010).

420 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

carbohydrates are exhausted.

because storage lipid is synthesized from this sugar in developing seeds and sucrose is one of the main end products of storage lipid breakdown during seed germination. In seedlings of *Arabidopsis* (a model oil-seed plant), 1% sucrose significantly retards the breakdown of storage lipid (Eastmond et al. 2000), while 0.3 M glucose nearly eliminates the mobilization of this storage compound (To et al. 2002). Seedlings grown for 22 days on glucose solution contained about 80% of their seed storage lipid. In contrast, 22-day-old seedlings grown in equi-molar sorbitol retained only 4-5% of their seed storage lipid. This result additionally proves that the