**3. Cytokinin profiles and transcript levels of CK metabolism related genes**  The total concentration of nine isoprenoid CKs (*trans*-zeatin, *t*Z; dihydrozeatin, DHZ; N6 isopentenyl adenine, iP; *trans*-zeatin riboside, ZR; dihydrozeatin riboside, DHZR; N6 isopentenyl adenosine, iPA; *trans*-zeatin-9-glucoside, Z-9-G; dihydrozeatin-9-glucoside, DHZ-9-G; N6-isopentenyladenine-9-glucoside, iP-9-G) detected in maize caryopses with applied immunoaffinity chromatography followed by HPLC analyses (Rijavec et al., 2009, 2011) was similar in all examined genotypes (Fig. 4a). Depending on the mutant, a CK peak was recorded in the period between 8 and 12 DAP, followed by a drop of cytokinin content at 16 DAP and a slight increase at 20 DAP (Fig. 4a). Similar CK patterns have been reported in maize caryopses before (Dietrrich et al., 1995; Brugière et al., 2008; Rijavec et al., 2009, 2011). Specific CK concentrations at peak stages were 40.87 ± 5.35, 81.7 ±33.3 and 90,68 ±23.21 pmol CKs per caryopsis from *mn1-89/mn1-89/mn1-1*, *Mn1* and *mn1-1/mn1-1/mn1-89*, respectively at 8 DAP; 93,54 ± 13.91 pmol per caryopsis from *mn1-1/mn1-1/mn1-1* caryopsis at 10 DAP; and 88.62 ±17.99 of total CKs per caryopsis from *mn1-89/mn1-89/mn1-1* at 12 DAP (Fig. 4a). The peak of CK concentration before 12 DAP has been previously suggested to be associated with the induction of programmed cell death in the placento-chalazal region of the caryopsis pedicel (Rijavec et al., 2011), as its essential developmental phase (Kladnik et al., 2004). The proper activity of the placento-chalazal region is crucial for the transport of photosynthates from the maternal to the filial tissues of the caryopsis. An interesting observation in the present research was the lowest detected amount of total CKs at 10 DAP in *mn1-89/mn1-89/mn1-1,* in which the total content never exceeded 41 pmol per caryopsis and differed substantially from the CK concentrations in *mn1-89/mn1-89/mn1-89* (Fig. 4a). However, the overall appearance of caryopses remained similar. It has been shown before that the absolute concentration of CKs in plants is not the crucial factor controlling their development, but that local changes in cytokinin level can have global consequences for the plant (Dermastia & Ravnikar, 1996; Dermastia et al., 1996). Indeed, specific distribution of CK metabolites in various caryopsis

The observed CK distribution was associated with a temporal expression of several CK metabolic genes (Fig. 5). The transcript abundance of isopentenyl transferase 2 (*ZmIpt2*), cytokinin dehydrogenase 1 (*ZmCkx1*), *cis*-zeatin-*O*-glucosyl transferase (*ZmCzog*), histidine kinase 1 (*ZmHK1*) and a putative *N-*glucosyl transferase (*ZmCngt*; Rijavec et al., 2011) were

tissues has been demonstrated (Rijavec et al., 2009, 2011).

determined in the period from 6 DAP to 28 DAP using quantitative real-time PCR. The temporal expression profiles of genes *ZmIpt2*, *ZmCkx*, *ZmCzog* and *ZmcHK1* roughly clustered into two groups related to the genotype; the first group consisted of *Mn1*, *mn1- 89/mn1-89/mn1-89* and *mn1-89/mn1-89/mn1-1*, and the second group of *mn1-1/mn1-1/mn1-89*  and *mn1-1/mn1-1/mn1-1* (Fig. 5).

Fig. 4. Temporal profiles of cytokinins in caryopses with genotypes *Mn1*, *mn1-89/mn1-89/mn1- 89, mn1-89/mn1-89/mn1-1, mn1-1/mn1-1/mn1-89* and *mn1-1/mn1-1/mn1-1* between 6 and 28 days after pollination*.* Data represent the average relative amount of cytokinin metabolites ± SE for 3 separate quantifications. a) Total CK concentration per caryopsis of CKs *t*Z, ZR, Z-9-G, DHZ, DHZR, DHZ-9-G, iP, iPA, and iP-9-G; b) percent of CK free bases *t*Z, DHZ and iP from total detected CKs; c) percent of CK ribosides ZR, DHZR and iPA from total detected CKs; d) percent of CK 9-glucosides Z-9-G, DHZ-9-G, iP-9-G from total detected CKs.

The *ZmIpt2* transcript profile of the first group of endosperm genotypes was low at 6 DAP, peaked between 8 DAP and 12 DAP and dropped after 12 DAP. The transcript abundance was similar in the two mutant genotypes and was significantly higher than in *Mn1*. In the second group the transcript profile showed level amount of the *ZmIpt2* transcript between 6 DAP and 12 DAP and, again, steep decrease after 12 DAP (Fig. 5a). The broader expression peak of the CK biosynthetic gene *ZmIpt2* in the wild type, in the homozygous *mn-1* mutant and in *mn1-1/mn1-1/mn1-89* (Fig. 5a) preceded the elevated CK concentration through several DAP (Fig. 4a). On the contrary, a narrow increase and subsequent drop in *ZmIpt2* transcript in *mn1-89/mn1-89/mn1-89* was associated with a

Cytokinins and Their Possible Role in Seed Size and Seed Mass Determination in Maize 301

narrow peak of CK concentration at 12 DAP. However, *ZmIpt2* transcript abundance was not related to the quantity of detected CKs. Interestingly, in a heterozygote with two alleles of *mn1-89* no clear increase in CKs was observed (Fig. 4a), but a high and sharp

The transcript abundance of *ZmCkx1* in the first group of genotypes peaked at 10 DAP. In *mn1-89/mn1-89/mn1-89* stayed approximately at the same level until 20 DAP and then decreased (Fig. 5b). However, in *Mn1* and *mn1-89/mn1-89/mn1-1* the transcript level decreased after 10 DAP, increased again between16 DAP and 20 DAP and declined after that. The absence of a clear CK concentration peak in *mn1-89/mn1-89/mn1-1* (Fig. 4a) might be explained because the highest detected level of the *ZmCkx* transcript peak corresponds with the expression peak of *ZmIpt2* (Fig. 5a), suggesting enhanced regulation of CKs during this time period.. In the group of *mn1-1/mn1-1/mn1-89* and *mn1-1/mn1-1/mn1-1* the transcript abundance was similarly high at 6 DAP, decreased in the period between 6 DAP and 8 DAP, slightly increased after 10 DAP, decrease again between 16 DAP and 20 DAP and at 28 DAP

The expression profile of the *ZmCzog* gene was low between 6 DAP and 16 DAP. It remained low until 28 DAP in the first group of genotypes, but it steeply increased after 20

Regardless of the genotype, the profile of the transcript *ZmCngt*, encoding the corresponding enzyme *N*-glucosyl transferase, was very low at 6 DAP, followed by a clear expression peak at 8 DAP and rapid drop afterward (Fig. 5d). However, its abundance was similar in all mutants except the homozygous *mn1* mutant, where it was two-fold lower (Fig. 5d). Previously reported high concentration of the putative product of the *N*-glucosyl transferase reaction, Z-9-G, in the developmental phase following intense mitotic activity in maize endosperm (Rijavec et al., 2009, 2011) was confirmed in mutant hybrids. The concentration of this metabolically inactive CK-*N-*glucoside (Brzobohatý et al., 1994) in mutants with the *mn1-89/mn1-89/mn1-89* genotype was close to the concentration previously reported for the wild type (Fig. 4d) (Rijavec et al., 2011). On the other hand, its concentrations were lower in the other examined mutant hybrids, especially at 28 DAP (Fig.

In addition, the expression of the *ZmHk1* gene was evaluated in caryopses with different genotypes (Fig. 5e). Histidine kinases (HK) are transmembrane receptors that bind cytokinin on the outer side of the cell's membrane and transducer the signal across the membrane to the inner side (reviewed in Rijavec & Dermastia, 2010). When cytokinin is not bound to the receptor, the latter is not active, suppressing downstream CK signaling. The oscillating expression patterns of *ZmHk1* were similar for all genotypes until 20 DAP, but by 28 DAP a distribution into two groups was evident again (Fig. 5e). Whether this higher *ZmHk1* expression was related to more active CK forms in *mn1-1/mn1-1/mn1-89* and *mn1-1/mn1- 1/mn1-*1 at this time point is currently not known. However, it has been suggested that the cellular cytokinin level modulates signaling and that mutual control mechanisms exists between metabolism and signaling, which may contribute to fine-tuning of the cytokinin response (Riefler et al., 2006). Study of cytokinin receptor mutants has also led to the conclusion that the seed size is a direct consequence of loss of receptor functions and their

increase of *ZmIpt2* transcript was detected at 10 DAP (Fig. 5a).

DAP in *mn1-1/mn1-1/mn1-89* and *mn1-1/mn1-1/mn1-1* (Fig. 5c).

4d), but the peaks corresponded to the gene expression profile of *ZmCngt*.

reached the level of 6 DAP (Fig. 5b).

role in growth control (Riefler et al., 2006).

Fig. 5. Temporal expression profiles of cytokinin metabolism related genes. Gene expression of (a) *ZmIpt2*, (b) *ZmCkx1*, (c) *ZmCzog* d) *ZmCngt*, and (e) *ZmHk1*, genes was examined in developing caryopsis 6 to 28 DAP in four *miniature1* endosperm mutants with different relative numbers of *mn1-89* and *mn1-1* mutant alleles. Data represents average transcript abundance ±SE for 6-9 real-time quantification reaction prepared from two individual RNA isolations and three subsequent reverse transcription reactions. Gene-specific primers were based on the sequences with the following GenBank Accesion numbers: *ZmCkx1*, Y18377; *ZmIpt2*, DV527975; *ZmCngt*, BT016809; *ZmHk1*, AB042270; *ZmCzog*, AF318075.

Fig. 5. Temporal expression profiles of cytokinin metabolism related genes. Gene expression of (a) *ZmIpt2*, (b) *ZmCkx1*, (c) *ZmCzog* d) *ZmCngt*, and (e) *ZmHk1*, genes was examined in developing caryopsis 6 to 28 DAP in four *miniature1* endosperm mutants with different relative numbers of *mn1-89* and *mn1-1* mutant alleles. Data represents average transcript abundance ±SE for 6-9 real-time quantification reaction prepared from two individual RNA isolations and three subsequent reverse transcription reactions. Gene-specific primers were based on the sequences with the following GenBank Accesion numbers: *ZmCkx1*, Y18377;

*ZmIpt2*, DV527975; *ZmCngt*, BT016809; *ZmHk1*, AB042270; *ZmCzog*, AF318075.

narrow peak of CK concentration at 12 DAP. However, *ZmIpt2* transcript abundance was not related to the quantity of detected CKs. Interestingly, in a heterozygote with two alleles of *mn1-89* no clear increase in CKs was observed (Fig. 4a), but a high and sharp increase of *ZmIpt2* transcript was detected at 10 DAP (Fig. 5a).

The transcript abundance of *ZmCkx1* in the first group of genotypes peaked at 10 DAP. In *mn1-89/mn1-89/mn1-89* stayed approximately at the same level until 20 DAP and then decreased (Fig. 5b). However, in *Mn1* and *mn1-89/mn1-89/mn1-1* the transcript level decreased after 10 DAP, increased again between16 DAP and 20 DAP and declined after that. The absence of a clear CK concentration peak in *mn1-89/mn1-89/mn1-1* (Fig. 4a) might be explained because the highest detected level of the *ZmCkx* transcript peak corresponds with the expression peak of *ZmIpt2* (Fig. 5a), suggesting enhanced regulation of CKs during this time period.. In the group of *mn1-1/mn1-1/mn1-89* and *mn1-1/mn1-1/mn1-1* the transcript abundance was similarly high at 6 DAP, decreased in the period between 6 DAP and 8 DAP, slightly increased after 10 DAP, decrease again between 16 DAP and 20 DAP and at 28 DAP reached the level of 6 DAP (Fig. 5b).

The expression profile of the *ZmCzog* gene was low between 6 DAP and 16 DAP. It remained low until 28 DAP in the first group of genotypes, but it steeply increased after 20 DAP in *mn1-1/mn1-1/mn1-89* and *mn1-1/mn1-1/mn1-1* (Fig. 5c).

Regardless of the genotype, the profile of the transcript *ZmCngt*, encoding the corresponding enzyme *N*-glucosyl transferase, was very low at 6 DAP, followed by a clear expression peak at 8 DAP and rapid drop afterward (Fig. 5d). However, its abundance was similar in all mutants except the homozygous *mn1* mutant, where it was two-fold lower (Fig. 5d). Previously reported high concentration of the putative product of the *N*-glucosyl transferase reaction, Z-9-G, in the developmental phase following intense mitotic activity in maize endosperm (Rijavec et al., 2009, 2011) was confirmed in mutant hybrids. The concentration of this metabolically inactive CK-*N-*glucoside (Brzobohatý et al., 1994) in mutants with the *mn1-89/mn1-89/mn1-89* genotype was close to the concentration previously reported for the wild type (Fig. 4d) (Rijavec et al., 2011). On the other hand, its concentrations were lower in the other examined mutant hybrids, especially at 28 DAP (Fig. 4d), but the peaks corresponded to the gene expression profile of *ZmCngt*.

In addition, the expression of the *ZmHk1* gene was evaluated in caryopses with different genotypes (Fig. 5e). Histidine kinases (HK) are transmembrane receptors that bind cytokinin on the outer side of the cell's membrane and transducer the signal across the membrane to the inner side (reviewed in Rijavec & Dermastia, 2010). When cytokinin is not bound to the receptor, the latter is not active, suppressing downstream CK signaling. The oscillating expression patterns of *ZmHk1* were similar for all genotypes until 20 DAP, but by 28 DAP a distribution into two groups was evident again (Fig. 5e). Whether this higher *ZmHk1* expression was related to more active CK forms in *mn1-1/mn1-1/mn1-89* and *mn1-1/mn1- 1/mn1-*1 at this time point is currently not known. However, it has been suggested that the cellular cytokinin level modulates signaling and that mutual control mechanisms exists between metabolism and signaling, which may contribute to fine-tuning of the cytokinin response (Riefler et al., 2006). Study of cytokinin receptor mutants has also led to the conclusion that the seed size is a direct consequence of loss of receptor functions and their role in growth control (Riefler et al., 2006).

Cytokinins and Their Possible Role in Seed Size and Seed Mass Determination in Maize 303

The expression level of *ZmCycD3-1*was 2- to 4- fold lower in the mutant caryopsis compared to the wild type at 0 DAP. It increased during 0 - 5 DAP and was higher in comparison to the wild type until 20 DAP, with an additional peak of expression at 16 DAP. However, at 34 DAP the expression of *ZmCycD3-1*in the mature caryopsis was lower than that of the

The expression profile of the gene *CycD3-2* was similar to that of *CycD3-1*, but more differentially expressed (data not shown). Fig. 6c shows its relative expression analysed in detail at three time points. It is evident that its expression at 20 DAP was significantly (2.6-

The expression of *CycD2* and *CycD3* has been previously characterized in *Arabidopsis*. It has been shown to be induced by sucrose and glucose, but the increase in *CycD2* levels is more responsive to glucose (Riou-Khamlichi et al., 2000). It has been recently demonstrated in maize caryopsis that in the period between 8 and 12 DAP sucrose levels remain constant in the basal part and only slightly decrease in the upper part of the wild type caryopsis, but increase in both parts of the mutant maize caryopsis (LeClere et al., 2010). On the other hand, in the same time period glucose decrease in both caryopses (LeClere et al., 2010). In rough accordance with those results was a down-regulation of *ZmCycD2* between 8 and 12 DAP (Fig. 6a). The subsequent up-regulation of *ZmCycD2* between 12 and 16 DAP is consistent with the constant concentration of sucrose in the whole *Mn1* caryopsis, the slight decrease of glucose in its upper part and strong increase of glucose in its basal part. Similar patterns of sugars and *ZmCycD2* expression were observed in the *mn1* caryopsis. The differences in the expression profiles of the *ZmCycD3* genes in the *Mn1* and *mn1* caryopsis in comparison with *ZmCycD2* might be at least partially explained by the levels of cytokinins present, immunolocalized as described (Rijavec et al., 2011) (Fig. 7). The immunosignal was similarly high in the pedicel regions, slightly stronger in the mutant embryo but very abundant throughout *mn1* endosperm development. While the signal was relatively strong in the aleurone layer of both caryopses, it was very faint in the upper part of the *Mn1* endosperm. On the other hand it was very strong in the upper endosperm of *mn1*. It has been shown, that *CycD3* expression is induced by

Another interesting observation from this study is the increased concentration of the *ZmCycD3-2* transcript, which was shown to be significantly higher at 20 DAP (Fig. 6c) in the *mn1* caryopsis in comparison with the wild type. The peak of *ZmCycD3-1* in *mn1* at 16 DAP (Fig.6b) corresponds to the peak of endosperm endoreduplication. In the *mn1* mutant at the endoreduplication developmental stage endosperm comprise 55 % fewer cells with reduced cell size (Vilhar et al., 2002). Despite this, the progress of endoreduplication was not affected in the *mn1* endosperm in comparison with the wild type. In the model of caryopsis growth (Fig. 1) (Rijavec et al., 2009) it has been proposed that growth of *mn1* caryopsis that is highly reduced in size due to the absence of the invertase *Incw2* gene may be partially compensated by *CycD2* and *CycD3* induced cell division regulated by sugars and cytokinins. The results of this and other studies support the model. Specifically, in transgenic tobacco lines expressing *Arath-CycD3* gene the ploidy levels of mature stem tissues were not affected, suggesting no effect on the extent of endoreduplication. Moreover, the *Arath-CycD3* overexpressing lines have an increased cell number together with a reduced cell size (Boucheron et al., 2005). Similar effects were also observed in *Arabidopsis* that expressed very high levels of *CycD3;1* (Dewitte et al., 2003). However, in *Arabidopsis* endoreduplication was

fold) higher in the *mn1* caryopsis in comparison with *Mn1*.

cytokinins when sucrose remains present (Riou-Khamlichi et al., 1999).

also inhibited in plants overexpressing *CycD3;1*.

wild type (Fig. 6b).

Although absolute CK concentrations (Fig. 4a) did not cluster into two groups as did expression levels of CK metabolic genes (Fig. 5), specific groups of CKs, specifically ribosides and 9-glucosides, clearly followed this pattern (Fig. 4c, d). Specifically, in *Mn1* and the hybrids with two or three *mn1-89* alleles the share of CK ribosides continuously decreased after 10 DAP, while in the hybrids with one *mn1-89* allele or in the homozygous hybrid with three alleles of *mn1* their share increased and was 2.7-fold higher at 28 DAP. The share of 9-glucosides, which in maize caryopses were represented solely by Z9G, did not exceed 17% from 6 DAP to 12 DAP. However, in *Mn1*, *mn1-89/mn1-89/mn1-89 and mn1- 89/mn1-89/mn1-1* it continuously rose and represented at 28 DAP about 57% in the hybrids and even 88% in the wild type (Fig. 4d). On the other hand, the share of Z-9-G in *mn1-1/mn1- 1/mn1-89* and *mn1-1/mn1-1/mn1-1* increased to 34 % and 39 %, respectively, at 16 DAP but subsequently decreased, and in the homozygous genotype represented only 8 % of total cytokinin metabolites at 28 DAP (Fig. 4d).

#### **4. Differential expression of type D2 and type D3 cyclin genes in the** *Mn1* **and**  *mn1* **caryopsis supports the alternative development of** *mn1*

The degree of change in transcript abundance of putative maize cycling genes *ZmCycD2*, *ZmCycD3-1* and *ZmCycD3-2* recovered by homology searches against all translated maize sequences (TBLASTN) with known protein sequences from *Arabidopsis* and rice was quantified by a real-time PCR in *Mn1* and *mn1* caryopsis samples at developing stages from 0 to 32 DAP. The *ZmCycD2* expression profile was similar in both, wild type and mutant caryopsis. The gene was highly expressed at pollination, down-regulated in the period between 0 and 12 DAP, upregulated from 12 to 20 DAP, and in the wild type the transcript abundance afterward decreased. However, the the *ZmCycD2* transcript in *mn1* mutant caryopses was slightly more abundant from 0 to 8 DAP than in *Mn1*; the gene was more heavily down-regulated at 12 DAP and was up-regulated in the late stages of caryopsis development (Fig. 6a).

Fig. 6. The expression profiles of a) *ZmCycD2*, b) *ZmCycD3-1* and c) *ZmCycD3-2* genes in caryopsis from 0 DAP to 28 DAP. Three independent RT preparations from three separate RNA isolations were used for transcript quantification each in three replicate real-time PCR reactions. Final data represented the mean ±SE of nine quantification reactions; \*\* P < 0.01. The relative quantification of gene transcripts was done after normalization of all samples to a geometric average of the expression levels of cytochrome oxidase (COX), elongation factor 1α (EF-1α) and cyclophilin (CyP) genes. Gene specific primers based on the sequences with the following GenBank Accesion numbers: *ZmCycD2* (NM\_001111578), *ZmCycD3-1* (NM\_001158642) and *ZmCycD3-2* (NM\_001196691).

Although absolute CK concentrations (Fig. 4a) did not cluster into two groups as did expression levels of CK metabolic genes (Fig. 5), specific groups of CKs, specifically ribosides and 9-glucosides, clearly followed this pattern (Fig. 4c, d). Specifically, in *Mn1* and the hybrids with two or three *mn1-89* alleles the share of CK ribosides continuously decreased after 10 DAP, while in the hybrids with one *mn1-89* allele or in the homozygous hybrid with three alleles of *mn1* their share increased and was 2.7-fold higher at 28 DAP. The share of 9-glucosides, which in maize caryopses were represented solely by Z9G, did not exceed 17% from 6 DAP to 12 DAP. However, in *Mn1*, *mn1-89/mn1-89/mn1-89 and mn1- 89/mn1-89/mn1-1* it continuously rose and represented at 28 DAP about 57% in the hybrids and even 88% in the wild type (Fig. 4d). On the other hand, the share of Z-9-G in *mn1-1/mn1- 1/mn1-89* and *mn1-1/mn1-1/mn1-1* increased to 34 % and 39 %, respectively, at 16 DAP but subsequently decreased, and in the homozygous genotype represented only 8 % of total

**4. Differential expression of type D2 and type D3 cyclin genes in the** *Mn1* **and**

The degree of change in transcript abundance of putative maize cycling genes *ZmCycD2*, *ZmCycD3-1* and *ZmCycD3-2* recovered by homology searches against all translated maize sequences (TBLASTN) with known protein sequences from *Arabidopsis* and rice was quantified by a real-time PCR in *Mn1* and *mn1* caryopsis samples at developing stages from 0 to 32 DAP. The *ZmCycD2* expression profile was similar in both, wild type and mutant caryopsis. The gene was highly expressed at pollination, down-regulated in the period between 0 and 12 DAP, upregulated from 12 to 20 DAP, and in the wild type the transcript abundance afterward decreased. However, the the *ZmCycD2* transcript in *mn1* mutant caryopses was slightly more abundant from 0 to 8 DAP than in *Mn1*; the gene was more heavily down-regulated at 12 DAP

Fig. 6. The expression profiles of a) *ZmCycD2*, b) *ZmCycD3-1* and c) *ZmCycD3-2* genes in caryopsis from 0 DAP to 28 DAP. Three independent RT preparations from three separate RNA isolations were used for transcript quantification each in three replicate real-time PCR reactions. Final data represented the mean ±SE of nine quantification reactions; \*\* P < 0.01. The relative quantification of gene transcripts was done after normalization of all samples to a geometric average of the expression levels of cytochrome oxidase (COX), elongation factor 1α (EF-1α) and cyclophilin (CyP) genes. Gene specific primers based on the sequences with

the following GenBank Accesion numbers: *ZmCycD2* (NM\_001111578), *ZmCycD3-1*

(NM\_001158642) and *ZmCycD3-2* (NM\_001196691).

*mn1* **caryopsis supports the alternative development of** *mn1*

and was up-regulated in the late stages of caryopsis development (Fig. 6a).

cytokinin metabolites at 28 DAP (Fig. 4d).

The expression level of *ZmCycD3-1*was 2- to 4- fold lower in the mutant caryopsis compared to the wild type at 0 DAP. It increased during 0 - 5 DAP and was higher in comparison to the wild type until 20 DAP, with an additional peak of expression at 16 DAP. However, at 34 DAP the expression of *ZmCycD3-1*in the mature caryopsis was lower than that of the wild type (Fig. 6b).

The expression profile of the gene *CycD3-2* was similar to that of *CycD3-1*, but more differentially expressed (data not shown). Fig. 6c shows its relative expression analysed in detail at three time points. It is evident that its expression at 20 DAP was significantly (2.6 fold) higher in the *mn1* caryopsis in comparison with *Mn1*.

The expression of *CycD2* and *CycD3* has been previously characterized in *Arabidopsis*. It has been shown to be induced by sucrose and glucose, but the increase in *CycD2* levels is more responsive to glucose (Riou-Khamlichi et al., 2000). It has been recently demonstrated in maize caryopsis that in the period between 8 and 12 DAP sucrose levels remain constant in the basal part and only slightly decrease in the upper part of the wild type caryopsis, but increase in both parts of the mutant maize caryopsis (LeClere et al., 2010). On the other hand, in the same time period glucose decrease in both caryopses (LeClere et al., 2010). In rough accordance with those results was a down-regulation of *ZmCycD2* between 8 and 12 DAP (Fig. 6a). The subsequent up-regulation of *ZmCycD2* between 12 and 16 DAP is consistent with the constant concentration of sucrose in the whole *Mn1* caryopsis, the slight decrease of glucose in its upper part and strong increase of glucose in its basal part. Similar patterns of sugars and *ZmCycD2* expression were observed in the *mn1* caryopsis. The differences in the expression profiles of the *ZmCycD3* genes in the *Mn1* and *mn1* caryopsis in comparison with *ZmCycD2* might be at least partially explained by the levels of cytokinins present, immunolocalized as described (Rijavec et al., 2011) (Fig. 7). The immunosignal was similarly high in the pedicel regions, slightly stronger in the mutant embryo but very abundant throughout *mn1* endosperm development. While the signal was relatively strong in the aleurone layer of both caryopses, it was very faint in the upper part of the *Mn1* endosperm. On the other hand it was very strong in the upper endosperm of *mn1*. It has been shown, that *CycD3* expression is induced by cytokinins when sucrose remains present (Riou-Khamlichi et al., 1999).

Another interesting observation from this study is the increased concentration of the *ZmCycD3-2* transcript, which was shown to be significantly higher at 20 DAP (Fig. 6c) in the *mn1* caryopsis in comparison with the wild type. The peak of *ZmCycD3-1* in *mn1* at 16 DAP (Fig.6b) corresponds to the peak of endosperm endoreduplication. In the *mn1* mutant at the endoreduplication developmental stage endosperm comprise 55 % fewer cells with reduced cell size (Vilhar et al., 2002). Despite this, the progress of endoreduplication was not affected in the *mn1* endosperm in comparison with the wild type. In the model of caryopsis growth (Fig. 1) (Rijavec et al., 2009) it has been proposed that growth of *mn1* caryopsis that is highly reduced in size due to the absence of the invertase *Incw2* gene may be partially compensated by *CycD2* and *CycD3* induced cell division regulated by sugars and cytokinins. The results of this and other studies support the model. Specifically, in transgenic tobacco lines expressing *Arath-CycD3* gene the ploidy levels of mature stem tissues were not affected, suggesting no effect on the extent of endoreduplication. Moreover, the *Arath-CycD3* overexpressing lines have an increased cell number together with a reduced cell size (Boucheron et al., 2005). Similar effects were also observed in *Arabidopsis* that expressed very high levels of *CycD3;1* (Dewitte et al., 2003). However, in *Arabidopsis* endoreduplication was also inhibited in plants overexpressing *CycD3;1*.

Cytokinins and Their Possible Role in Seed Size and Seed Mass Determination in Maize 305

The authors thank the Slovenian Research Agency (grant 1000-05-310055 to T.R.) and the USA-Slovenia Cooperation in Science and Technology (grant BI-US/06-07-031). The authors also thank Drs. Aleš Kladnik for his help with data visualization, S. Chen and Mukesh Jain for critical review of the manuscript and Elizabeth Covington for proof-reading. M.

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Carlson, S.J., Shanker, S. & Chourey, P.S. (2000.) A point mutation at the *Miniature1* seed

Cheng, W.H., Taliercio, E.W., & Chourey, P. S. (1996). The *Miniature1* seed locus of maize

Dermastia, M., & Ravnikar, M. (1996) Altered cytokinin pattern and enhanced tolerance to

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Endogenous levels during early fruit development. *Plant Physiology and*

Habben, J. E. (2003). Cytokinin oxidase gene expression in maize is localized to the vasculature, and is induced by cytokinins, abscisic acid, and abiotic stress. *Plant*

isopentenyl transferase gene family, *Zea mays isopentenyl transferase 2* (*ZmIPT2*), encodes a cytokinin biosynthetic enzyme expressed during kernel development.

plant growth and development. *Plant Molecular Biology* 26, p.p. 1483-1497, ISSN

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locus reduces levels of the encoded protein, but not its mRNA, in maize. *Molecular*

encodes a cell wall invertase required for normal development of endosperm and maternal cells in the pedicel. *The Plant Cell* 8, no. 6, p.p. 971-983, ISSN 1040-4651 Chourey, P. S., Jain, M., Li, Q.-B., & Carlson, S. J. (2006). Genetic control of cell wall

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Dermastia and P.S. Chourey contributed equally to this work.

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**6. Acknowledgement**

**7. References**

0167-4412

0889

5765

Fig. 7. Immunolocalization of cytokinins on the central longitudinal section of wild type *Mn1* and *mn1-1/ mn1-1/ mn1-1* caryopsis at 12 DAP.

#### **5. Conclusions**

The results here confirm the previously demonstrated linear gene-dose relationship of the *Incw2* transcripts with the number of *mn1-89* copies. Additionally we show that the seed mass was markedly different between the homozygous *Mn1* and the *mn1-89* series beginning at 12 to 16 DAP. Quantitative measurements of nine CK metabolites showed that the genotypes with reduced levels of invertase activity have reduced concentrations of the metabolically inactive CK form zeatin-9-glucoside and increased concentrations of active CK ribosides. Quantitative real-time PCR analyses for the CK genes showed that the *cytokinin-N-glucosyl transferase* gene (*ZmCngt1*) was expressed at the highest level at 10 DAP and was greatly reduced in expression in the *mn1* mutant. Additionally, this study supports our model (Rijavec et al., 2009) which proposes that high amounts of CKs and a higher ratio of their active forms may partially compensate the INCW2-deficiency in regulating CycD3 and CycD2 induced cell divisions by CKs and sucrose in the *mn1* endosperm. Consistent with the model, the sink strength is greater in caryopses with the *Mn1* genotype. In contrast, the levels of glucose, fructose and sucrose were higher in the upper part of the mutant caryopsis, due to as yet unknown mechanisms, and may contribute to the increased expression of the CycD3 gene and to normal, although substantially slower, development of *mn1* caryopses.

#### **6. Acknowledgement**

304 Advances in Selected Plant Physiology Aspects

*mn1-1mn1-1mn1-1*

Fig. 7. Immunolocalization of cytokinins on the central longitudinal section of wild type

The results here confirm the previously demonstrated linear gene-dose relationship of the *Incw2* transcripts with the number of *mn1-89* copies. Additionally we show that the seed mass was markedly different between the homozygous *Mn1* and the *mn1-89* series beginning at 12 to 16 DAP. Quantitative measurements of nine CK metabolites showed that the genotypes with reduced levels of invertase activity have reduced concentrations of the metabolically inactive CK form zeatin-9-glucoside and increased concentrations of active CK ribosides. Quantitative real-time PCR analyses for the CK genes showed that the *cytokinin-N-glucosyl transferase* gene (*ZmCngt1*) was expressed at the highest level at 10 DAP and was greatly reduced in expression in the *mn1* mutant. Additionally, this study supports our model (Rijavec et al., 2009) which proposes that high amounts of CKs and a higher ratio of their active forms may partially compensate the INCW2-deficiency in regulating CycD3 and CycD2 induced cell divisions by CKs and sucrose in the *mn1* endosperm. Consistent with the model, the sink strength is greater in caryopses with the *Mn1* genotype. In contrast, the levels of glucose, fructose and sucrose were higher in the upper part of the mutant caryopsis, due to as yet unknown mechanisms, and may contribute to the increased expression of the CycD3 gene and

to normal, although substantially slower, development of *mn1* caryopses.

*Mn1* and *mn1-1/ mn1-1/ mn1-1* caryopsis at 12 DAP.

**5. Conclusions**

*Mn1*

The authors thank the Slovenian Research Agency (grant 1000-05-310055 to T.R.) and the USA-Slovenia Cooperation in Science and Technology (grant BI-US/06-07-031). The authors also thank Drs. Aleš Kladnik for his help with data visualization, S. Chen and Mukesh Jain for critical review of the manuscript and Elizabeth Covington for proof-reading. M. Dermastia and P.S. Chourey contributed equally to this work.

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**14**

*Greece*

**Nutritional and Proteomic Profiles in**

Christina K. Kitsaki, Nikos Maragos and Dimitris L. Bouranis *Laboratory of Plant Physiology, Department of Agricultural Biotechnology*

Flowering is a crucial phase of the plant life cycle hardly programmed through floral time genes, cadastral genes and floral organ identity genes (Jack, 2004). Four pathways control flowering time, floral meristem identity genes and floral organs genes: light and photoperiod, temperature, energy supplies (mainly through carbohydrates) and hormones

Plant nutrient status and especially nitrogen is determinant for flowering and completion of the plant life cycle. Tree nutrient status, as well as its seasonal nutrient fluctuations have been based almost exclusively on leaf analysis. Flower analysis has been used for the study of the distribution of different nutrients through the various parts of the fruit tree (Drossopoulos et al., 1996, Sanchez et al., 1992), as well as for the detection and handling of

Olive tree (*Olea europaea L.)* is a very important indeterminate long lived, evergreen fruit tree, cultivated mainly in the Mediterranean basin, since experimental evidences have demonstrated both fruits and leaves to be important organs for synthesis of several interesting biological compounds. The tree has been cultivated for approximately 6000 years. Some specimens have been reported to live for thousand, and it may be in the spirit of peace that olive branches appear on the flag of the United Nation Organization (Rugini and

Olive tree has a plentiful bloom but a low percentage of normal fruit set. (Reale et al., 2006). In plants nutrient status affects flowering, and play a crucial role for the completion of the life cycle. Except from nutrients, proteins play a special role in inflorescence development, as they play crucial role in flower organ development (Taiz and Zeiger, 2010). In olive tree, nitrogen status affects flower quality and ovule longevity (Fernandez-Escobar et al., 2008). Proteomic analyses in trees have also gained little interest, as they are difficult as plant materials. Especialy, olive tree has major difficulty, because of the presence of many phenolic compounds. In spite of the importance of olive tree for human diet, no interest has been focused on olive inflorescence development. In this work we studied the profile of nutrients, as well as the proteomic profile in olive inflorescence cv "Konservolia" in order to

deficiencies during this early stage of growth (Sanz and Montanes, 1995).

**1. Introduction**

(Taiz and Zeiger, 2010).

Fedeli, 1990; Martin, 2009).

add data on this interest subject.

**Developing Olive Inflorescence**

*Agricultural University of Athens, Athens*

associated with reduced mitotic activity in the developing endosperm. *Plant Physiology* 129, no. 1 (May 1), p.p. 23-30, ISSN 0032-0889

### **Nutritional and Proteomic Profiles in Developing Olive Inflorescence**

Christina K. Kitsaki, Nikos Maragos and Dimitris L. Bouranis *Laboratory of Plant Physiology, Department of Agricultural Biotechnology Agricultural University of Athens, Athens Greece*

#### **1. Introduction**

308 Advances in Selected Plant Physiology Aspects

Vyroubalova, S., Vaclavikova, K., Tureckova, V., Novak, O., Šmehilová , M., Hluska, T.,

Yang, J., Zhang, J., Huang, Z., Wang, Z., Zhu, Q., & Liu, L. ( 2002). Correlation of cytokinin

*Physiology* 129, no. 1 (May 1), p.p. 23-30, ISSN 0032-0889

p.p. 433-447, ISSN 0032-0889

0305-7364

associated with reduced mitotic activity in the developing endosperm. *Plant*

Ohnoutkova, L., Frebort, I., & Galuszka. P. (2009). Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels. *Plant Physiology*, no. 1 (September 1),

levels in the endosperms and roots with cell number and cell division activity during endosperm development in rice. *Annals of Botany* 90, 3, p.p. 369 -377, ISSN

> Flowering is a crucial phase of the plant life cycle hardly programmed through floral time genes, cadastral genes and floral organ identity genes (Jack, 2004). Four pathways control flowering time, floral meristem identity genes and floral organs genes: light and photoperiod, temperature, energy supplies (mainly through carbohydrates) and hormones (Taiz and Zeiger, 2010).

> Plant nutrient status and especially nitrogen is determinant for flowering and completion of the plant life cycle. Tree nutrient status, as well as its seasonal nutrient fluctuations have been based almost exclusively on leaf analysis. Flower analysis has been used for the study of the distribution of different nutrients through the various parts of the fruit tree (Drossopoulos et al., 1996, Sanchez et al., 1992), as well as for the detection and handling of deficiencies during this early stage of growth (Sanz and Montanes, 1995).

> Olive tree (*Olea europaea L.)* is a very important indeterminate long lived, evergreen fruit tree, cultivated mainly in the Mediterranean basin, since experimental evidences have demonstrated both fruits and leaves to be important organs for synthesis of several interesting biological compounds. The tree has been cultivated for approximately 6000 years. Some specimens have been reported to live for thousand, and it may be in the spirit of peace that olive branches appear on the flag of the United Nation Organization (Rugini and Fedeli, 1990; Martin, 2009).

> Olive tree has a plentiful bloom but a low percentage of normal fruit set. (Reale et al., 2006). In plants nutrient status affects flowering, and play a crucial role for the completion of the life cycle. Except from nutrients, proteins play a special role in inflorescence development, as they play crucial role in flower organ development (Taiz and Zeiger, 2010). In olive tree, nitrogen status affects flower quality and ovule longevity (Fernandez-Escobar et al., 2008).

> Proteomic analyses in trees have also gained little interest, as they are difficult as plant materials. Especialy, olive tree has major difficulty, because of the presence of many phenolic compounds. In spite of the importance of olive tree for human diet, no interest has been focused on olive inflorescence development. In this work we studied the profile of nutrients, as well as the proteomic profile in olive inflorescence cv "Konservolia" in order to add data on this interest subject.

Nutritional and Proteomic Profiles in Developing Olive Inflorescence 311

carried out overnight at -20 oC. A 10 min centrifugation in cold at 10,000 g resulted in a three phase system, an upper aqueous, an intermediate protein phase, and a lower organic phase. The upper aqueous phase was discarded and the remained proteins were homogenized with the lower organic phase by vortexing. Proteins were then rinsed three times by adding a twofold volume of 10% aqueous TCA, 0.07% β-mercaptoethanol, vortexing and centrifugation for 5 min in cold at 10,000 g. Purification of proteins completed by washing once with 5fold volume ddH2O. Finally, the protein pellet was washed twice with ice cold acetone, 0.07% βmercaptoethanol and centrifuged for 5 min in cold at 10,000 g. The pellet was dried under a gentle stream of air. Total water extractable proteins (WSP) were quantified using bovine

The pellets for SDS-PAGE analysis (about 50 μg) were solubilised in sample buffer 62,5 mM Tris-HCl, pH 6.8, 2% SDS, 5% β-mercaptoethanol, 10% glycerol and traces of bromophenol blue (Garcia et al. 2000) enhanced with 8.0 M urea, and denatured by heating in boiling water for 5 min. Then cooled to room temperature and centrifuged for 5 min at 14,000g. Electrophoresis was carried out on a Perfect Blue M 14x16 cm (Peqlab Biotechnologie GmbH) vertical electrophoresis system using 12,5% continuous pH 8,3 SDS running gel (1,5 mm thick), with a 4% stacking gel parallel with Sigma markers (14 to 205 kDa), according to Hoefer (1994) at 30 mA for about 1.5 hours. The gels were stained with Coomassie Brilliant

For 2-DE about 300 μg of purified proteins were diluted with rehydration buffer (9.5 M urea, 2% CHAPS, 2% IPG buffer pH 4-7 and 20 mM DTT (Amersham Biosciences). The samples were then put on Immobiline Drystrip 13 cm with a linear pH range 4.0-7.0, the strips were covered with cover fluid and left to rehydrate for at least 10 h. Isoelectric focusing was carried out on a horizontal Multiphor II Amersham Biosciences system under a four phases program (300 V/1 Vh, 300 V/1800 Vh, 3500 V/9500 Vh and 3500 V/19250 Vh). Prior to running the second dimension the proteins were reduced with 65 mM DTT followed by alkylation with 135 mM iodoacetamide and traces of bromophenol blue, both diluted in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol and 2% SDS). The second dimension was carried out as in SDS-PAGE analysis but with 0.8 mm thick gels, without stacking gel. Sigma markers (14-205 KDa) were run parallel to samples. Proteins were stained with silver nitrate and data were assessed by mean of Photoshop CS3 program.

Flower and inflorescence development is one of the most complicated and well regulated plant process (Rolland-Lagan et al., 2003). Flowering time genes, floral meristem identity genes, and floral organ identity genes have well studied in *Arabidopsis*, *Pisum and Antirrhinum*, as well as in other annual plants. Tetramers of MADS proteins are thought to determine floral organ identity (Jack, 2004; Taiz and Zeiger, 2010). The size and the shape of petals as well as of the rest whorls is genetically determined (Laitinen et al., 2007). However little data are available until now concerning flower proteomic (Dafny-Yelin et al., 2005;

Olive inflorescence is a panicle which arises with a central axis terminated by a flower, with lateral axes branching from the peduncle. The lateral axes may in turn branch, resulting in a

Moccia et al., 2009; Sun et al., 2009; Logacheva et al. 2011; Zhu et al., 2011).

tertiary branched panicle (Weis et al., 1988; Seifi et al., 2008; Ganinoa et al., 2011).

serum albunin as standard (Bradford, 1976; Bearden,1978).

Blue R-250, according to Sigma (1994) protocol.

**3. Results and discussion**

**3.1 Olive inflorescence development**

### **2. Materials and methods**

#### **2.1 Plant material**

Three about 30-year-old fruit bearing olive trees (*Olea europaea* L. cv "Konservolia"), were selected. The experiment started about one week after floral bud burst, nine weeks before full bloom, in early March, and lasted up to one week after full bloom, in late May. Hanging or horizontal reproductive shoots 15-25 cm long with ten or more inflorescence bearing nodes were cut from positions symmetrically distributed around the crown of each tree at weekly intervals. The inflorescences from the central region of each reproductive shoot (3rd to 6th node measuring from the top of the shoot) were used for further analysis. Three groups of samples were collected each sampling date at 09:00 h i.e. (i) for measurements of developmental parameters of inflorescences (length, FW, DW, and morphological observations), (ii) for nutritional profile analyses and (iii) for proteomic analyses.

#### **2.2 Developmental parameters of inflorescence development**

Twenty inflorescences per tree were used for measurements of the inflorescence length and fresh and dry weight of the organ. For detailed morphological and anatomical observations two representative inflorescences with four lateral branches were selected from four reproductive shoots of each of the three trees and fixed in FAA.

Anatomy (median longitudinal section of perfect flowers) were observed on flowers from the first branch (counting from the apex of the inflorescence). Photographs were taken with an Olympus SZX12 stereoscope, equipped with an Olympus DP71 P-30N camera.

#### **2.3 Nutritional analyses**

Three samples per tree were separately analysed. Fresh weight per sample was recorded, samples were oven-dried at 80 °C, dry weight was recorded and the samples were ground to pass a 40-mesh screen using an analytical mill (IKA, model A10) prior to chemical analysis (Mills and Jones 1996). N was analyzed by micro-Kjeldahl digestion followed by distillation (Jones, 1991). P, K, Mg, Ca, Cu, Fe, Zn and Mn were determined following a wet acid digestion procedure based on the combination of HNO3 and 30% H2O2 (Mills and Jones 1996). Phosphorus quantitative analysis in the diluted digests was carried out colorimetrically by determining the absorption of the blue phosphomolybdate complex at 660 nm, using the ammonium molybdate and stannus chloride procedure (Peach and Tracey 1956). The concentrations of all other nutrients were determined in the diluted digests by atomic absorption spectrophotometry using a GBC Avanta spectrophotometer. For the determination of Ca and Mg, 1% (w/v) lanthanum was added in the digests.

#### **2.4 Proteomic analyses**

Three separate replicates were used for each of the proteomic analyses we followed. A modified protocol based on those suggested by Garcia et al. (2000) Süle et al. (2004), and Wang et al. (2003, 2004) for olive was followed for protein extraction and purification. Proteins were extracted using a 10fold volume/weight buffer (50 mM sodium borate, 50 mM ascorbic acid pH 9.0, 1% β-mercaptoethanol, 1% soluble PVP, 1% insoluble PVP, 10 mM PMSF) under continuous vortexing for 60 min at 4 oC. The homogenate was centrifuged at 4 oC for 30 min at 35,000 g and the pellet was discarded. Proteins in the supernatant were precipitated by adding equal volume of cold 10% TCA, 0.07% β-mercaptoethanol in acetone. The precipitation was

Three about 30-year-old fruit bearing olive trees (*Olea europaea* L. cv "Konservolia"), were selected. The experiment started about one week after floral bud burst, nine weeks before full bloom, in early March, and lasted up to one week after full bloom, in late May. Hanging or horizontal reproductive shoots 15-25 cm long with ten or more inflorescence bearing nodes were cut from positions symmetrically distributed around the crown of each tree at weekly intervals. The inflorescences from the central region of each reproductive shoot (3rd to 6th node measuring from the top of the shoot) were used for further analysis. Three groups of samples were collected each sampling date at 09:00 h i.e. (i) for measurements of developmental parameters of inflorescences (length, FW, DW, and morphological observations), (ii) for

Twenty inflorescences per tree were used for measurements of the inflorescence length and fresh and dry weight of the organ. For detailed morphological and anatomical observations two representative inflorescences with four lateral branches were selected from four

Anatomy (median longitudinal section of perfect flowers) were observed on flowers from the first branch (counting from the apex of the inflorescence). Photographs were taken with

Three samples per tree were separately analysed. Fresh weight per sample was recorded, samples were oven-dried at 80 °C, dry weight was recorded and the samples were ground to pass a 40-mesh screen using an analytical mill (IKA, model A10) prior to chemical analysis (Mills and Jones 1996). N was analyzed by micro-Kjeldahl digestion followed by distillation (Jones, 1991). P, K, Mg, Ca, Cu, Fe, Zn and Mn were determined following a wet acid digestion procedure based on the combination of HNO3 and 30% H2O2 (Mills and Jones 1996). Phosphorus quantitative analysis in the diluted digests was carried out colorimetrically by determining the absorption of the blue phosphomolybdate complex at 660 nm, using the ammonium molybdate and stannus chloride procedure (Peach and Tracey 1956). The concentrations of all other nutrients were determined in the diluted digests by atomic absorption spectrophotometry using a GBC Avanta spectrophotometer. For the

Three separate replicates were used for each of the proteomic analyses we followed. A modified protocol based on those suggested by Garcia et al. (2000) Süle et al. (2004), and Wang et al. (2003, 2004) for olive was followed for protein extraction and purification. Proteins were extracted using a 10fold volume/weight buffer (50 mM sodium borate, 50 mM ascorbic acid pH 9.0, 1% β-mercaptoethanol, 1% soluble PVP, 1% insoluble PVP, 10 mM PMSF) under continuous vortexing for 60 min at 4 oC. The homogenate was centrifuged at 4 oC for 30 min at 35,000 g and the pellet was discarded. Proteins in the supernatant were precipitated by adding equal volume of cold 10% TCA, 0.07% β-mercaptoethanol in acetone. The precipitation was

an Olympus SZX12 stereoscope, equipped with an Olympus DP71 P-30N camera.

determination of Ca and Mg, 1% (w/v) lanthanum was added in the digests.

**2. Materials and methods**

**2.3 Nutritional analyses**

**2.4 Proteomic analyses**

nutritional profile analyses and (iii) for proteomic analyses.

**2.2 Developmental parameters of inflorescence development**

reproductive shoots of each of the three trees and fixed in FAA.

**2.1 Plant material**

carried out overnight at -20 oC. A 10 min centrifugation in cold at 10,000 g resulted in a three phase system, an upper aqueous, an intermediate protein phase, and a lower organic phase. The upper aqueous phase was discarded and the remained proteins were homogenized with the lower organic phase by vortexing. Proteins were then rinsed three times by adding a twofold volume of 10% aqueous TCA, 0.07% β-mercaptoethanol, vortexing and centrifugation for 5 min in cold at 10,000 g. Purification of proteins completed by washing once with 5fold volume ddH2O. Finally, the protein pellet was washed twice with ice cold acetone, 0.07% βmercaptoethanol and centrifuged for 5 min in cold at 10,000 g. The pellet was dried under a gentle stream of air. Total water extractable proteins (WSP) were quantified using bovine serum albunin as standard (Bradford, 1976; Bearden,1978).

The pellets for SDS-PAGE analysis (about 50 μg) were solubilised in sample buffer 62,5 mM Tris-HCl, pH 6.8, 2% SDS, 5% β-mercaptoethanol, 10% glycerol and traces of bromophenol blue (Garcia et al. 2000) enhanced with 8.0 M urea, and denatured by heating in boiling water for 5 min. Then cooled to room temperature and centrifuged for 5 min at 14,000g. Electrophoresis was carried out on a Perfect Blue M 14x16 cm (Peqlab Biotechnologie GmbH) vertical electrophoresis system using 12,5% continuous pH 8,3 SDS running gel (1,5 mm thick), with a 4% stacking gel parallel with Sigma markers (14 to 205 kDa), according to Hoefer (1994) at 30 mA for about 1.5 hours. The gels were stained with Coomassie Brilliant Blue R-250, according to Sigma (1994) protocol.

For 2-DE about 300 μg of purified proteins were diluted with rehydration buffer (9.5 M urea, 2% CHAPS, 2% IPG buffer pH 4-7 and 20 mM DTT (Amersham Biosciences). The samples were then put on Immobiline Drystrip 13 cm with a linear pH range 4.0-7.0, the strips were covered with cover fluid and left to rehydrate for at least 10 h. Isoelectric focusing was carried out on a horizontal Multiphor II Amersham Biosciences system under a four phases program (300 V/1 Vh, 300 V/1800 Vh, 3500 V/9500 Vh and 3500 V/19250 Vh). Prior to running the second dimension the proteins were reduced with 65 mM DTT followed by alkylation with 135 mM iodoacetamide and traces of bromophenol blue, both diluted in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol and 2% SDS). The second dimension was carried out as in SDS-PAGE analysis but with 0.8 mm thick gels, without stacking gel. Sigma markers (14-205 KDa) were run parallel to samples. Proteins were stained with silver nitrate and data were assessed by mean of Photoshop CS3 program.

#### **3. Results and discussion**

Flower and inflorescence development is one of the most complicated and well regulated plant process (Rolland-Lagan et al., 2003). Flowering time genes, floral meristem identity genes, and floral organ identity genes have well studied in *Arabidopsis*, *Pisum and Antirrhinum*, as well as in other annual plants. Tetramers of MADS proteins are thought to determine floral organ identity (Jack, 2004; Taiz and Zeiger, 2010). The size and the shape of petals as well as of the rest whorls is genetically determined (Laitinen et al., 2007). However little data are available until now concerning flower proteomic (Dafny-Yelin et al., 2005; Moccia et al., 2009; Sun et al., 2009; Logacheva et al. 2011; Zhu et al., 2011).

#### **3.1 Olive inflorescence development**

Olive inflorescence is a panicle which arises with a central axis terminated by a flower, with lateral axes branching from the peduncle. The lateral axes may in turn branch, resulting in a tertiary branched panicle (Weis et al., 1988; Seifi et al., 2008; Ganinoa et al., 2011).

Nutritional and Proteomic Profiles in Developing Olive Inflorescence 313

plate c). The DW/FW ratio (plate d) showed a decreasing trend for a period of seven weeks before full bloom, stabilized on the lower level the two weeks before full bloom, increased at full bloom when floral whorls were separated by each other, and increased sharply the week after full bloom. This decreasing trend is obviously due to the enlargement of the floral organ cells and the contemporary hydration of them. The opening of petals and thereafter the higher transpiration rate, as well as the abscission of floral organs even before full bloom (Weis et al., 1988) and mainly after full bloom leads to a recovery of the DW/FW ratio at full

In Fig.2 are shown representative developmental stages of perfect flowers of the first lateral branch of four-branching inflorescence on median longitudinal sections five weeks before full bloom up to full bloom. Bracts which covered individual flowers were already shed five weeks before full bloom (plate -5). The stamen sac development found to be completed about two weeks before full bloom (plate -2). Pistil development followed that of the stamen and continued until full bloom in perfect flowers. Nucellus was already visible in the ovary five weeks before full bloom, while two well developed embryosacs were found on sections two and one week before full bloom (plates –2, –1). At full bloom, the opened petals

revealed the flattened anthers and a global-like ovary (plate 0).

Fig. 2. Median longitudinal sections of olive perfect flowers (*Olea europaea* L. cv

weeks before full bloom (plate -5) to full bloom (plate 0).

"Konservolia") showing the developmental stages of the organ at weekly intervals from 5

bloom and the next week.

Under normal conditions, olive inflorescence development last a prolonged period starting in March and full bloom occurs in May or June (Cirik, 1989; Weis et al., 1991).

The inflorescence development in "Konservolia" (Fig. 1, photos above plate a) seemed to constitute of three stages, as was also reported for "Kalamon" inflorescence (Bouranis et al., 2010) and lasted about 10 weeks until full bloom. Nine to seven weeks before full bloom inflorescence looked as a small berry covered with bracts and the elongation of the main axis prevailed, obviously due to the elongation of its cells. Six to four weeks before full bloom lateral axes having compact floral meristems on them were developed on the inflorescence, which suggest mitotic activity, as well as cell enlargement in the organs. At this stage the development of floral meristems and their whorls on the lateral axes indicates the implication of proteins derived from floral organ identity genes transcription. The next three weeks up to full bloom the development of flower whorls tended to complete. The week after full bloom only fertilized ovaries were remained on inflorescence, while lateral axes, unfertilized flowers and bracts were massively abscised during this week.

Fig. 1. The developmental stages of olive cv "Konservolia" inflorescence from floral bud burst 9 week before full bloom (plate a) up to one week after full bloom as well as the profiles of the changes of the length (plate b), of the fresh weights (plate c), dry weights (plate d) and the DW/FW ratio (plate d) (full bloom week 0). Vertical bars represent SE.

The increase of inflorescence length (Fig. 1, plate a) followed a sigmoid type of development and reached a maximum one week before full bloom. The fresh weight of inflorescence (Fig. 1, plate b) was progressively increased for a period of five weeks, with an intensive increase up to one week before full bloom, decreasing rapidly afterwards. Dry weight presented a similar but milder progress in comparison to that of FW during the same period (Fig. 1,

Under normal conditions, olive inflorescence development last a prolonged period starting

The inflorescence development in "Konservolia" (Fig. 1, photos above plate a) seemed to constitute of three stages, as was also reported for "Kalamon" inflorescence (Bouranis et al., 2010) and lasted about 10 weeks until full bloom. Nine to seven weeks before full bloom inflorescence looked as a small berry covered with bracts and the elongation of the main axis prevailed, obviously due to the elongation of its cells. Six to four weeks before full bloom lateral axes having compact floral meristems on them were developed on the inflorescence, which suggest mitotic activity, as well as cell enlargement in the organs. At this stage the development of floral meristems and their whorls on the lateral axes indicates the implication of proteins derived from floral organ identity genes transcription. The next three weeks up to full bloom the development of flower whorls tended to complete. The week after full bloom only fertilized ovaries were remained on inflorescence, while lateral axes, unfertilized flowers and bracts were massively abscised

Fig. 1. The developmental stages of olive cv "Konservolia" inflorescence from floral bud burst 9 week before full bloom (plate a) up to one week after full bloom as well as the profiles of the changes of the length (plate b), of the fresh weights (plate c), dry weights (plate d) and the DW/FW ratio (plate d) (full bloom week 0). Vertical bars represent SE.

The increase of inflorescence length (Fig. 1, plate a) followed a sigmoid type of development and reached a maximum one week before full bloom. The fresh weight of inflorescence (Fig. 1, plate b) was progressively increased for a period of five weeks, with an intensive increase up to one week before full bloom, decreasing rapidly afterwards. Dry weight presented a similar but milder progress in comparison to that of FW during the same period (Fig. 1,

in March and full bloom occurs in May or June (Cirik, 1989; Weis et al., 1991).

during this week.

plate c). The DW/FW ratio (plate d) showed a decreasing trend for a period of seven weeks before full bloom, stabilized on the lower level the two weeks before full bloom, increased at full bloom when floral whorls were separated by each other, and increased sharply the week after full bloom. This decreasing trend is obviously due to the enlargement of the floral organ cells and the contemporary hydration of them. The opening of petals and thereafter the higher transpiration rate, as well as the abscission of floral organs even before full bloom (Weis et al., 1988) and mainly after full bloom leads to a recovery of the DW/FW ratio at full bloom and the next week.

In Fig.2 are shown representative developmental stages of perfect flowers of the first lateral branch of four-branching inflorescence on median longitudinal sections five weeks before full bloom up to full bloom. Bracts which covered individual flowers were already shed five weeks before full bloom (plate -5). The stamen sac development found to be completed about two weeks before full bloom (plate -2). Pistil development followed that of the stamen and continued until full bloom in perfect flowers. Nucellus was already visible in the ovary five weeks before full bloom, while two well developed embryosacs were found on sections two and one week before full bloom (plates –2, –1). At full bloom, the opened petals revealed the flattened anthers and a global-like ovary (plate 0).

Fig. 2. Median longitudinal sections of olive perfect flowers (*Olea europaea* L. cv "Konservolia") showing the developmental stages of the organ at weekly intervals from 5 weeks before full bloom (plate -5) to full bloom (plate 0).

Nutritional and Proteomic Profiles in Developing Olive Inflorescence 315

Fig. 3b. The profiles of concentration and accumulation of iron, manganese, copper, zink, and manganese in olive inflorescence (*Olea europaea* L. cv "Konservolia") from floral bud

burst (nine weeks before full bloom) to one week after full bloom.

Fig. 3a. The profiles of concentration and accumulation of water soluble proteins, total nitrogen, phosphorus, potassium and calcium in olive inflorescence (*Olea europaea* L. cv "Konservolia") from floral bud burst (nine weeks before full bloom) to one week after full bloom.

Fig. 3a. The profiles of concentration and accumulation of water soluble proteins, total nitrogen, phosphorus, potassium and calcium in olive inflorescence (*Olea europaea* L. cv "Konservolia") from floral bud burst (nine weeks before full bloom) to one week after full

bloom.

Fig. 3b. The profiles of concentration and accumulation of iron, manganese, copper, zink, and manganese in olive inflorescence (*Olea europaea* L. cv "Konservolia") from floral bud burst (nine weeks before full bloom) to one week after full bloom.

Nutritional and Proteomic Profiles in Developing Olive Inflorescence 317

while magnesium and manganese showed not considerable fluctuations during inflorescence development. On the other hand accumulation of the above nutrients showed a slow or medium increasing rate up to full bloom, when a rapid increase was observed in the accumulations (except from that of Cu). All the above nutrients are constituents of enzymes which implicate in the basic metabolic activity and especially Mg and Fe. Iron and copper seemed to have special interest during the massive flower organ abscission one week after full bloom, as their concentrations was increased considerably. On the other hand three to two weeks before full bloom (late stages of ovary and stamens development) zink concentration showed to be of increased interest. The strong positive linear regression among DW and water soluble proteins, total N, as well as macro- and micro-nutrients accumulation during inflorescence development (Table 1) reinforces the concept that olive

 **WSP N P K Mg Ca Cu Fe Zn Mn R2** 0.78\*\* 0.98\*\* 0.97\*\* 0.98\*\* 0.97\*\* 0.64\*\* 0.81\*\* 0.67\*\* 0.76\*\* 0.90\*\* **SE** 14.2 7.0 6.3 18.6 1.8 70.6 0.25 3.1 0.5 0.4 **n** 11 11 11 11 11 11 11 11 11 11 Table 1. The R2 values among dry weight accumulation and of total water soluble proteins (WSP), as well as mineral nutrients (N, P, K, Mg, Ca, Cu, Fe, Zn and Mn) in developing olive

In Fig. 4 is shown the profile of proteins revealed by SDS-PAGE analysis in developing olive inflorescence nine weeks before full bloom up to one week after full bloom. In table 2 are summarized the number and the kDa of the detected protein bands as well as the most abundant and the common protein bands in developing olive inflorescence by SDS-

Fig. 4. The protein profile in developing olive inflorescence (*Olea europaea* L. cv "Konservolia") nine weeks (-9) before full bloom up to one week (1) after full bloom. Proteins (50 μg) were resolved in 12,5% SDS/acrylamide gels (1,5 mm thick) parallel with Sigma markers (14 to 66 and 36 to 205 kDa) and gels were stained with Coomassie Brilliant

inflorescence is a strong sink organ.

PAGE analysis.

inflorescence (*Olea europaea* L. cv "Konservolia").

Blue R-250, according to Sigma protocol.

**3.3 Proteomic changes in developing olive inflorescence**

#### **3.2 Profiles in total water extractable proteins and macro- and micro-nutrients**

A pulse like profile found in total water extractable proteins in olive inflorescence which seemed to be consisted of three phases. The two lower levels at water soluble proteins found six and 2 weeks before full bloom. This profile showed to be similar to that of the progress of inflorescence development as discussed above. Water soluble protein accumulation showed a sigmoid like profile in which the phase of the higher rate of accumulation localized in the period of five to one week before full bloom, which corresponds to the period of flower and floral whorls development on inflorescence. On the other hand, the profile of nitrogen concentration showed to be more uniform, having a decreasing trend. Nitrogen accumulation showed an increasing trend up to one week before full bloom. In spite the similar trend, the rate of increase of nitrogen accumulation was lower to that of DW accumulation, resulting in a progressive dilution of nitrogen level in the developing inflorescence. A pulse like profile on water soluble proteins was found in differentiating olive floral buds during late winter and thought to be due to a need of a relaxing phase of the dividing cells (Kitsaki et al., 2010). Proteins may be enzymes for the different metabolic processes among which mitosis, structural proteins, membrane proteins, or storage proteins. In flowers tetramers of proteins which determine floral organ identity and development, as well as those implicated to meiosis have a special role (Jack, 2004; Luu and Maurel 2005; Ahsan and Komatsu, 2009; Malumbres et al., 2009; Echalier et al., 2010; Taiz and Zeiger, 2010). As different physiological processes take place during floral organ development, among them meiosis, a fluctuated profile of water soluble proteins during this period could be expected. The observed fluctuations in the water soluble proteins could also be attributed to interchanges between soluble and insoluble protein fractions in the organ.

In plant cells except from proteins nitrogen is a constituent of free amino acids, amides, nucleic acids nucleotides, coenzymes, amines and others substances Therefore, nitrogen deficiency rapidly inhibits plant growth and if persists transition of plant from vegetative to reproductive phase may not be completed (Marschner, 1995; Taiz and Zeiger, 2010). Differences in the level of nitrogenous compounds between vegetative and reproductive shoots on the same plant have been reported for the majority of plant, among them for olive (Bouranis et al., 2004). In olive tree, nitrogen status affects flower quality and ovule longevity (Fernandez-Escobar et al., 2008).

The higher levels of phosphorus concentration observed during the early stages of flower development (-7 to -5 weeks), while the nutrient was accumulated progressively up to full bloom in the inflorescence. Potassium also accumulated in the inflorescence up to full bloom but changes in the concentration showed a rather mild profile. The accumulation of calcium remained at low levels up to full bloom when a huge increase was observed. On the other hand the fluctuations in calcium concentration were the most dramatic (Fig. 3a). Except from the special role of P in energy storage, of K in osmoregulation and of Ca in the structure of cell wall and as a second messenger, as well as their participation in many enzymes and coenzymes, they occupy a special role in the pollen grain germination as components of phytate salts (Fu et al., 2008; Taiz and Zeiger, 2010). The decreasing trend in Ca up to full bloom is possibly the result of the immobile character of this nutrient (Marschner, 1995), while the rapid increase in its accumulation after full bloom may be due to the dehydration of inflorescence at this stage and the consequent increase of percentage of cell walls in the organ.

Profiles of iron and copper concentration showed an increasing trend during inflorescence development, zink concentration also tended to increase up to two weeks before full bloom,

A pulse like profile found in total water extractable proteins in olive inflorescence which seemed to be consisted of three phases. The two lower levels at water soluble proteins found six and 2 weeks before full bloom. This profile showed to be similar to that of the progress of inflorescence development as discussed above. Water soluble protein accumulation showed a sigmoid like profile in which the phase of the higher rate of accumulation localized in the period of five to one week before full bloom, which corresponds to the period of flower and floral whorls development on inflorescence. On the other hand, the profile of nitrogen concentration showed to be more uniform, having a decreasing trend. Nitrogen accumulation showed an increasing trend up to one week before full bloom. In spite the similar trend, the rate of increase of nitrogen accumulation was lower to that of DW accumulation, resulting in a progressive dilution of nitrogen level in the developing inflorescence. A pulse like profile on water soluble proteins was found in differentiating olive floral buds during late winter and thought to be due to a need of a relaxing phase of the dividing cells (Kitsaki et al., 2010). Proteins may be enzymes for the different metabolic processes among which mitosis, structural proteins, membrane proteins, or storage proteins. In flowers tetramers of proteins which determine floral organ identity and development, as well as those implicated to meiosis have a special role (Jack, 2004; Luu and Maurel 2005; Ahsan and Komatsu, 2009; Malumbres et al., 2009; Echalier et al., 2010; Taiz and Zeiger, 2010). As different physiological processes take place during floral organ development, among them meiosis, a fluctuated profile of water soluble proteins during this period could be expected. The observed fluctuations in the water soluble proteins could also be attributed

**3.2 Profiles in total water extractable proteins and macro- and micro-nutrients**

to interchanges between soluble and insoluble protein fractions in the organ.

longevity (Fernandez-Escobar et al., 2008).

In plant cells except from proteins nitrogen is a constituent of free amino acids, amides, nucleic acids nucleotides, coenzymes, amines and others substances Therefore, nitrogen deficiency rapidly inhibits plant growth and if persists transition of plant from vegetative to reproductive phase may not be completed (Marschner, 1995; Taiz and Zeiger, 2010). Differences in the level of nitrogenous compounds between vegetative and reproductive shoots on the same plant have been reported for the majority of plant, among them for olive (Bouranis et al., 2004). In olive tree, nitrogen status affects flower quality and ovule

The higher levels of phosphorus concentration observed during the early stages of flower development (-7 to -5 weeks), while the nutrient was accumulated progressively up to full bloom in the inflorescence. Potassium also accumulated in the inflorescence up to full bloom but changes in the concentration showed a rather mild profile. The accumulation of calcium remained at low levels up to full bloom when a huge increase was observed. On the other hand the fluctuations in calcium concentration were the most dramatic (Fig. 3a). Except from the special role of P in energy storage, of K in osmoregulation and of Ca in the structure of cell wall and as a second messenger, as well as their participation in many enzymes and coenzymes, they occupy a special role in the pollen grain germination as components of phytate salts (Fu et al., 2008; Taiz and Zeiger, 2010). The decreasing trend in Ca up to full bloom is possibly the result of the immobile character of this nutrient (Marschner, 1995), while the rapid increase in its accumulation after full bloom may be due to the dehydration of inflorescence at this stage and the consequent increase of percentage of cell walls in the organ. Profiles of iron and copper concentration showed an increasing trend during inflorescence development, zink concentration also tended to increase up to two weeks before full bloom, while magnesium and manganese showed not considerable fluctuations during inflorescence development. On the other hand accumulation of the above nutrients showed a slow or medium increasing rate up to full bloom, when a rapid increase was observed in the accumulations (except from that of Cu). All the above nutrients are constituents of enzymes which implicate in the basic metabolic activity and especially Mg and Fe. Iron and copper seemed to have special interest during the massive flower organ abscission one week after full bloom, as their concentrations was increased considerably. On the other hand three to two weeks before full bloom (late stages of ovary and stamens development) zink concentration showed to be of increased interest. The strong positive linear regression among DW and water soluble proteins, total N, as well as macro- and micro-nutrients accumulation during inflorescence development (Table 1) reinforces the concept that olive inflorescence is a strong sink organ.

Table 1. The R2 values among dry weight accumulation and of total water soluble proteins (WSP), as well as mineral nutrients (N, P, K, Mg, Ca, Cu, Fe, Zn and Mn) in developing olive inflorescence (*Olea europaea* L. cv "Konservolia").

#### **3.3 Proteomic changes in developing olive inflorescence**

In Fig. 4 is shown the profile of proteins revealed by SDS-PAGE analysis in developing olive inflorescence nine weeks before full bloom up to one week after full bloom. In table 2 are summarized the number and the kDa of the detected protein bands as well as the most abundant and the common protein bands in developing olive inflorescence by SDS-PAGE analysis.

Fig. 4. The protein profile in developing olive inflorescence (*Olea europaea* L. cv "Konservolia") nine weeks (-9) before full bloom up to one week (1) after full bloom. Proteins (50 μg) were resolved in 12,5% SDS/acrylamide gels (1,5 mm thick) parallel with Sigma markers (14 to 66 and 36 to 205 kDa) and gels were stained with Coomassie Brilliant Blue R-250, according to Sigma protocol.

Nutritional and Proteomic Profiles in Developing Olive Inflorescence 319

flowers and leaves at various developmental stages revealed organ specific functional differentiation of proteins (Ahsan and Komatsu, 2009). Moreover, Dafny-Yelin et al. (2005) through expression analysis of resolved proteins from rose petals at advanced stage development found about 30% of them to be stage specific. Recently Zhu et al. (2011) identified differentially expressed proteins in mature and germinated maize pollen and found 26 proteins (among about 470) to be changed between the two stages, 13 of them (upregulated) were mainly involved in tube wall modification, actin cytoskeleton organization and energy metabolism. However, although genetic control of flowering and floral organ development has been extensively studied (Jack, 2004; Leitanen at al., 2007) much remains to be done concerning proteomic approach. Three groups of proteins (about 20 kDa, 31 kDa and 40 kDa) which were present in a wide range of developmental stages of inflorescence may be related to the basic cell metabolic activity or may be storage proteins. Ahsan and Komatsu (2009) referred an abundance of a glycoprotein of 31 kDa in soybean petals, which may be similar to that we also realized. Except from any relation to pollen germination, pollination and fertilization, the presence of the high molecular weight proteins exclusively at the late stage of inflorescence development, may also be related to genes that regulate synthesis of secondary metabolites, as flavonoids, carotenoids and other aromatic substances, which have been reported that appear at the late stage of floral organ

Further proteome analyses with 2-D electrophoresis carried out in three sampling dates distributed in the three phases of inflorescence development: (a) eight weeks before full bloom (middle of the phase of inflorescence axis elongation), (b) four weeks before full bloom (middle of the phase of floral organ development and (c) at full bloom. In Fig. 5a, 5b, 5c are shown the correspond to the above sampling dates profiles of proteins revealed by 2-DE analysis. In table 3 are summarized the kDa and the pIs of the detected proteins by 2-DE analyses. Proteins were distributed all over the range of 4.0 to 7.0 pH, while molecular weights fluctuated between about 10 to 55 kDa the -8 and the -3 weeks and between 10 to 105 kDa at full bloom (Table 3). Most of the abundant proteins were found at full bloom. Two protein groups (42.5 kDa/4.12-4.60 pIs and 28.2 kDa/4.64-5.83 pIs) were visualized in the three sampling dates. Some of proteins were common in the three stages of development, while other were present in one or two of the three phases. The most abundant protein was that of 44.3 kDa/4.67-5.19 pIs, especially at -8 week and at -4 week. Proteins with high molecular weights (over 60 kDa) were monitored exclusively at

Early tree proteome studies were focused on the process of xylogenesis mainly in *Populus* and *Pinus* (Costa et al., 1999; Mijnsbruge et al., 2000), but some fruit trees have recently gained interest for proteome analysis (Cartu et al., 2008; Paiva et al., 2008). However, little effort have been done on olive proteomic, probably because of the difficulty on sample handling, due to the presence of many phenolic compounds (Garcia et al. 2000, Wang et al.,

The pIs of most of the proteins we visualized by 2-DE in developing olive inflorescence were localized in the low pH region. Similar data have been reported for olive leaf ( Wang et al., 2003), as well as for other plants (Dafny-Yelin et al., 2005; Ahsan and Komatsu, 2009; Chen et al., 2009). The fluctuation in abundance in the three stages of inflorescence development may be related to developmental stages specific functional proteins (Ahsan

development (Ben-Meir et al., 2002).

2003, Wang et al., 2004, Wang et al., 2010,).

full bloom.

Table 2. The kDa of the detected protein bands as well as the most abundant (bold numbers) and the common (shadowed cells) protein groups in developing olive inflorescence by SDS PAGE analysis nine weeks before (-9) full bloom to one week after full bloom (1).

Protein bands molecular weights were fluctuated between 10 and about 100 kDa. Protein bands of 44, 40, 32, 19, 15 and 14 kDa were the most frequently appeared, while the groups of 14 and 12 kDa were the most abundant. The number of protein bands was fluctuated between 5 and 9 during the three developmental stages of inflorescence. Most of protein groups were visualized at full bloom (9 groups), at the middle of the phase of inflorescence elongation (-8 week), as well as at the late stage of floral organ development (-2 week), 7 protein groups. As full bloom was coming, new groups of high molecular weights were appeared (3 weeks before full bloom to full bloom). These groups were disappeared the week after full bloom. In this week the range of molecular weights of protein groups was reduced to 10 to 44 kDa (Table 2). The fluctuation of the number of protein bands may be related with the intensity of meristematic activity, as well as cell enlargement processes, which are correlated with enzyme systems relative to mitosis, as cyclin-dependent-kinases, structural proteins for dauter cells and others, as mentioned above (Dembinsky et al., 2007; Malumbres et al., 2009; Echalier et al., 2010; Nafati et al., 2011). This concept is reinforced by the pulse like data we found in the profile of water extractable proteins. At full bloom new physiological processes take place e.g pollen tube growth, pollination and fertilization which need specific enzymatic activity (Chen et al., 2009). Proteome analysis of soybean

9 42.78 **43.62** 43.34 43.89 43.89 44.20 10 **41.39** 39.02 39.54 40.15 40.59 40.41

12 33.18 **33.60** 32.27

16 20.13 20.45 20.66 **20.78**

19 14.31 **13.40 13.94 13.87 13.58**

22 **10.61** 10.52 Table 2. The kDa of the detected protein bands as well as the most abundant (bold numbers) and the common (shadowed cells) protein groups in developing olive inflorescence by SDS

Protein bands molecular weights were fluctuated between 10 and about 100 kDa. Protein bands of 44, 40, 32, 19, 15 and 14 kDa were the most frequently appeared, while the groups of 14 and 12 kDa were the most abundant. The number of protein bands was fluctuated between 5 and 9 during the three developmental stages of inflorescence. Most of protein groups were visualized at full bloom (9 groups), at the middle of the phase of inflorescence elongation (-8 week), as well as at the late stage of floral organ development (-2 week), 7 protein groups. As full bloom was coming, new groups of high molecular weights were appeared (3 weeks before full bloom to full bloom). These groups were disappeared the week after full bloom. In this week the range of molecular weights of protein groups was reduced to 10 to 44 kDa (Table 2). The fluctuation of the number of protein bands may be related with the intensity of meristematic activity, as well as cell enlargement processes, which are correlated with enzyme systems relative to mitosis, as cyclin-dependent-kinases, structural proteins for dauter cells and others, as mentioned above (Dembinsky et al., 2007; Malumbres et al., 2009; Echalier et al., 2010; Nafati et al., 2011). This concept is reinforced by the pulse like data we found in the profile of water extractable proteins. At full bloom new physiological processes take place e.g pollen tube growth, pollination and fertilization which need specific enzymatic activity (Chen et al., 2009). Proteome analysis of soybean

18 15.28 15.90 15.08 **15.00**

20 **12.53 12.37 12.45**

PAGE analysis nine weeks before (-9) full bloom to one week after full bloom (1).

**W -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1**

2 **101.6** 101.1

4 77.36 77.36 5 65.56 **67.25**  5 53.81 54.85 **54.51**

3 80.26 **79.74**

7 49.04

13 31.93 31.22 31.22 32.33 31.43 **31.85**

14 29.58 15 24.44 24.28

21 **11.83**

17 19.48 19.07 19.86 19.43 19.06

1 121.5

6 51.06 **52.80** 50.73

11

8 45.90

flowers and leaves at various developmental stages revealed organ specific functional differentiation of proteins (Ahsan and Komatsu, 2009). Moreover, Dafny-Yelin et al. (2005) through expression analysis of resolved proteins from rose petals at advanced stage development found about 30% of them to be stage specific. Recently Zhu et al. (2011) identified differentially expressed proteins in mature and germinated maize pollen and found 26 proteins (among about 470) to be changed between the two stages, 13 of them (upregulated) were mainly involved in tube wall modification, actin cytoskeleton organization and energy metabolism. However, although genetic control of flowering and floral organ development has been extensively studied (Jack, 2004; Leitanen at al., 2007) much remains to be done concerning proteomic approach. Three groups of proteins (about 20 kDa, 31 kDa and 40 kDa) which were present in a wide range of developmental stages of inflorescence may be related to the basic cell metabolic activity or may be storage proteins. Ahsan and Komatsu (2009) referred an abundance of a glycoprotein of 31 kDa in soybean petals, which may be similar to that we also realized. Except from any relation to pollen germination, pollination and fertilization, the presence of the high molecular weight proteins exclusively at the late stage of inflorescence development, may also be related to genes that regulate synthesis of secondary metabolites, as flavonoids, carotenoids and other aromatic substances, which have been reported that appear at the late stage of floral organ development (Ben-Meir et al., 2002).

Further proteome analyses with 2-D electrophoresis carried out in three sampling dates distributed in the three phases of inflorescence development: (a) eight weeks before full bloom (middle of the phase of inflorescence axis elongation), (b) four weeks before full bloom (middle of the phase of floral organ development and (c) at full bloom. In Fig. 5a, 5b, 5c are shown the correspond to the above sampling dates profiles of proteins revealed by 2-DE analysis. In table 3 are summarized the kDa and the pIs of the detected proteins by 2-DE analyses. Proteins were distributed all over the range of 4.0 to 7.0 pH, while molecular weights fluctuated between about 10 to 55 kDa the -8 and the -3 weeks and between 10 to 105 kDa at full bloom (Table 3). Most of the abundant proteins were found at full bloom. Two protein groups (42.5 kDa/4.12-4.60 pIs and 28.2 kDa/4.64-5.83 pIs) were visualized in the three sampling dates. Some of proteins were common in the three stages of development, while other were present in one or two of the three phases. The most abundant protein was that of 44.3 kDa/4.67-5.19 pIs, especially at -8 week and at -4 week. Proteins with high molecular weights (over 60 kDa) were monitored exclusively at full bloom.

Early tree proteome studies were focused on the process of xylogenesis mainly in *Populus* and *Pinus* (Costa et al., 1999; Mijnsbruge et al., 2000), but some fruit trees have recently gained interest for proteome analysis (Cartu et al., 2008; Paiva et al., 2008). However, little effort have been done on olive proteomic, probably because of the difficulty on sample handling, due to the presence of many phenolic compounds (Garcia et al. 2000, Wang et al., 2003, Wang et al., 2004, Wang et al., 2010,).

The pIs of most of the proteins we visualized by 2-DE in developing olive inflorescence were localized in the low pH region. Similar data have been reported for olive leaf ( Wang et al., 2003), as well as for other plants (Dafny-Yelin et al., 2005; Ahsan and Komatsu, 2009; Chen et al., 2009). The fluctuation in abundance in the three stages of inflorescence development may be related to developmental stages specific functional proteins (Ahsan

Nutritional and Proteomic Profiles in Developing Olive Inflorescence 321

Fig. 5a. Protein profile in developing olive inflorescence eight weeks before full bloom. Proteins (300 μg) were resolved in 13 cm linear immobilized dry strips pH 4.0-7.0 and in the

Fig. 5b. Protein profile in developing olive inflorescence four weeks before full bloom. Proteins (300 μg) were resolved in 13 cm linear immobilized dry strips pH 4.0-7.0 and in the

second dimension in 12.5 % SDS/acrylamide 0.8 mm thick gels.

second dimension in 12.5 % SDS/acrylamide 0.8 mm thick gels.

and Komatsu, 2009). The majority of the abundance found at full bloom is in accordance with the recent data of Ahsan and Komatsu (2009) and Zhu et al. (2011), concerning stage specific functional proteins.

Table 3. The kDa and the pIs of the detected proteins by 2-DE analyses in developing olive inflorescence (*Olea europaea* L. cv "Konservolia") eight weeks before full bloom (-8 week), three weeks before full bloom (-3 week) and at full bloom (0 week). The most abundant proteins are indicated with bold numbers (SN: spot number).

and Komatsu, 2009). The majority of the abundance found at full bloom is in accordance with the recent data of Ahsan and Komatsu (2009) and Zhu et al. (2011), concerning stage

**-8 week (middle of the phase of inflorescence axis elongation) SN kDa/pI SN kDa/pI SN kDa/pI SN kDa/pI SN kDa/pI**  1 55.6/4.61 10 25.6/4.20 20 13.6/4.92 25 **11.8/6.28** 36 10.5/4.80 2 **46.8/4.96** 11 24.7/6.91 21 13.6/5.20 32 **11.5/5.34** 38 **10.0/4.40**  3 **44.3/4.95** 12 23.8/6.90 22 **12.9/6.03** 33 11.6/5.49 40 10.0/4.29 4 **42.7/4.96** 13 22.7/4.54 26 12.9/4.03 16 11.5/5.88 41 10.0/4.88 5 **40.9/4.96** 15 18.9/4.40 28 12.5/4.41 29 11.5/4.30 43 **10.0/5.81**  6 34.8/4.21 14 18.5/4.23 27 12.5/4.76 31 10.9/5.20 39 10.0/4.05 7 29.7/4.43 17 16.6/4.35 23 **12.3/5.95** 37 10.9/4.30 42 **10.0/5.29**  8 29.4/4.69 18 15.7/4.37 24 **12.3/6.27** 34 **10.5/5.77** 44 **10.0/6.03**  9 28.4/5.11 19 14.8/4.92 30 **12.3/4.84** 35 **10.5/5.93** 45 **10.0/6.10 -4 week (middle of the phase of floral organ development) SN kDa/pI SN kDa/pI SN kDa/pI SN kDa/pI SN kDa/pI**  1 **52.8/5.2** 7 **42.3/4.21** 16 28.0/4.64 26 17.3/4.27 31 **14.4/4.84**  2 **50.5/5.19** 13 **35.4/5.51** 15 27.2/4.20 25 17.5/4.27 29 14.4/4.47 3 **48.7/5.19** 10 **33.5/4.20** 22 23.7/5.46 27 17.5/4.68 28 14.4/4.28 4 **47.5/5.19** 11 33.5/4.83 21 22.6/5.40 35 15.3/5.33 36 14.0/6.43 5 **44.8/5.19** 12 33.5/5.16 20 22.6/4.69 34 **15.3/5.27** 37 14.0/6.48 9 **44.7/4.67** 14 31.7/6.72 19 21.3/4.28 33 15.3/5.20 38 10.7/4.27 8 **43.5/4.38** 17 29.4/4.91 23 19.7/4.27 32 15.3/4.95 39 10.0/4.47 6 **43.5/5.19** 18 29.4/5.15 24 19.7/4.70 30 15.3/4.72 40 10.0/4.48 **0 week (full bloom) SN kDa/pI SN kDa/pI SN kDa/pI SN kDa/pI SN kDa/pI**  8 106.5/6.29 6 93.5/5.34 17 **42.9/4.12** 25 32.3/4.84 32 16.5/4.39 10 105.2/6.47 4 92.0/5.19 19 **42.9/4.47** 26 **28.4/5.83** 34 **16.5/5.39**  9 104.3/6.45 12 68.7/4.57 20 **42.9/4.60** 27 **28.4/5.19** 35 **15.2/5.36**  2 **104.3/4.35** 11 67.3/4.38 21 **41.3/5.20** 28 26.3/4.50 36 **14.3/4.57**  3 **103.2/4.74** 13 **56.6/5.19** 18 **41.3/4.31** 29 **26.3/4.23** 37 14.3/4.36 7 103.2/5.86 14 **49.7/5.47** 22 **36.6/5.41** 30 20.7/4.70 38 13.2/4.16 1 **102.5/4.10** 15 **48.3/5.27** 23 **32.3/5.41** 31 20.7/4.34 39 **10.0/4.45**  5 93.5/5.34 16 **46.6/5.20** 24 **32.3/5.20** 33 17.2/4.59 40 **10.0/4.08**  Table 3. The kDa and the pIs of the detected proteins by 2-DE analyses in developing olive inflorescence (*Olea europaea* L. cv "Konservolia") eight weeks before full bloom (-8 week), three weeks before full bloom (-3 week) and at full bloom (0 week). The most abundant

proteins are indicated with bold numbers (SN: spot number).

specific functional proteins.

Fig. 5a. Protein profile in developing olive inflorescence eight weeks before full bloom. Proteins (300 μg) were resolved in 13 cm linear immobilized dry strips pH 4.0-7.0 and in the second dimension in 12.5 % SDS/acrylamide 0.8 mm thick gels.

Fig. 5b. Protein profile in developing olive inflorescence four weeks before full bloom. Proteins (300 μg) were resolved in 13 cm linear immobilized dry strips pH 4.0-7.0 and in the second dimension in 12.5 % SDS/acrylamide 0.8 mm thick gels.

Nutritional and Proteomic Profiles in Developing Olive Inflorescence 323

This work was supported by the Ministry of National Education Affairs. The authors are grateful to the staff of the Laboratory of Pomology of Agricultural University of Athens,

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**5. Acknowledgments**

**6. References**

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Fig. 5c. Protein profile in developing olive inflorescence at full bloom . Proteins (300 μg) were resolved in 13 cm linear immobilized dry strips pH 4.0-7.0 and in the second dimension in 12.5 % SDS/acrylamide 0.8 mm thick gels.

#### **4. Conclusions**

Olive inflorescence development seems to be separated in three stages: a first one corresponding to the inflorescence axis elongation, a second corresponding to the development of flowers and floral whorls development and the last one which corresponds to the full bloom and flower fertilization. Changes in DW/FW ratio revealed intense hydration of the organ two weeks before full bloom. Strong positive linear regression found among DW and water soluble proteins, as well as macro- and micronutrients. A pulse like profile found in total water extractable proteins in olive inflorescence which seemed to be consisted of three phases. This profile showed to be similar to that of the progress of inflorescence development. Most of protein groups were visualized at full bloom, at the middle of the phase of inflorescence elongation, as well as at the late stage of floral organ development. As full bloom was coming, new groups of high molecular weights were appeared. Proteins with high molecular weights (over 60 kDa) were monitored exclusively at full bloom. The pIs of most of the proteins we visualized by 2-DE in developing olive inflorescence were localized in the low pH region.

#### **5. Acknowledgments**

This work was supported by the Ministry of National Education Affairs. The authors are grateful to the staff of the Laboratory of Pomology of Agricultural University of Athens, which take care of the olive orchard.

#### **6. References**

322 Advances in Selected Plant Physiology Aspects

Fig. 5c. Protein profile in developing olive inflorescence at full bloom . Proteins (300 μg) were resolved in 13 cm linear immobilized dry strips pH 4.0-7.0 and in the second

Olive inflorescence development seems to be separated in three stages: a first one corresponding to the inflorescence axis elongation, a second corresponding to the development of flowers and floral whorls development and the last one which corresponds to the full bloom and flower fertilization. Changes in DW/FW ratio revealed intense hydration of the organ two weeks before full bloom. Strong positive linear regression found among DW and water soluble proteins, as well as macro- and micronutrients. A pulse like profile found in total water extractable proteins in olive inflorescence which seemed to be consisted of three phases. This profile showed to be similar to that of the progress of inflorescence development. Most of protein groups were visualized at full bloom, at the middle of the phase of inflorescence elongation, as well as at the late stage of floral organ development. As full bloom was coming, new groups of high molecular weights were appeared. Proteins with high molecular weights (over 60 kDa) were monitored exclusively at full bloom. The pIs of most of the proteins we visualized by 2-DE in developing olive

dimension in 12.5 % SDS/acrylamide 0.8 mm thick gels.

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**4. Conclusions**

Nutritional and Proteomic Profiles in Developing Olive Inflorescence 325

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**15**

*Japan*

Aiko Amagai

**Regulatory Mechanism in Sexual and Asexual**

Cellular slime molds exhibit dimorphism in development: sorocarp formation as an asexual cycle and macrocyst formation as a sexual cycle. These two developmental forms are regulated by environmental conditions, such as light and water. *Dictyostelium mucoroides 7* (Dm 7), a species of cellular slime molds forms sorocarps in the light, while forming

Fig. 1. Two developmental forms in Dm7. A: Starved Dm7 cells were developed on 1.5 % agar in the light. After 48 hours of incubation, they formed sorocarps. A globular spore mass

In an asexual cycle, amoeboid cells grow and multiply feeding on bacteria. Upon exhaustion of the bacterial food supply (starvation), starving cells stop growing and start the differentiation process. They gather together to form cell aggregates. A tip is formed on the top of each cell aggregate, which then migrates as a slug-shaped mass. After migration, the slug changes its shape dramatically to form a sorocarp consisting of a stalk with an apical mass of spores.

Macrocyst formation as a sexual cycle is characterized by the formation of large aggregates after starvation. Large aggregates are subdivided into smaller masses (precysts), each of

is supported by a stalk (arrow). B: When starved Dm7 cells were developed under submerged conditions, they formed macrocysts. Each macrocyst is surrounded by a thick

**1. Introduction**

macrocysts in the dark or water (Fig.1).

wall (arrow). Bars: 200 μm.

**Cycles of** *Dictyostelium*

*Graduate School of Life Sciences, Tohoku University*

### **Regulatory Mechanism in Sexual and Asexual Cycles of** *Dictyostelium*

Aiko Amagai *Graduate School of Life Sciences, Tohoku University Japan*

#### **1. Introduction**

326 Advances in Selected Plant Physiology Aspects

Weis, K., G., Webster, B., D., Goren, R., Martin, G., C. (1991). Inflorescence abscission in

Zhu, Y., Zhao, P., Wu, X., Wang, W., Scali, M., Cresti, M. (2011). Proteomic identification of

*Gaz*. 152 pp 51-58.

*Physiol Plant* 33 pp 1467–1474.

olive: Anatomy and Histochemistry in response to ethylene and ethephon. . *Bot.*

differentially expressed proteins in mature and germinated maize pollen *Acta*

Cellular slime molds exhibit dimorphism in development: sorocarp formation as an asexual cycle and macrocyst formation as a sexual cycle. These two developmental forms are regulated by environmental conditions, such as light and water. *Dictyostelium mucoroides 7* (Dm 7), a species of cellular slime molds forms sorocarps in the light, while forming macrocysts in the dark or water (Fig.1).

Fig. 1. Two developmental forms in Dm7. A: Starved Dm7 cells were developed on 1.5 % agar in the light. After 48 hours of incubation, they formed sorocarps. A globular spore mass is supported by a stalk (arrow). B: When starved Dm7 cells were developed under submerged conditions, they formed macrocysts. Each macrocyst is surrounded by a thick wall (arrow). Bars: 200 μm.

In an asexual cycle, amoeboid cells grow and multiply feeding on bacteria. Upon exhaustion of the bacterial food supply (starvation), starving cells stop growing and start the differentiation process. They gather together to form cell aggregates. A tip is formed on the top of each cell aggregate, which then migrates as a slug-shaped mass. After migration, the slug changes its shape dramatically to form a sorocarp consisting of a stalk with an apical mass of spores.

Macrocyst formation as a sexual cycle is characterized by the formation of large aggregates after starvation. Large aggregates are subdivided into smaller masses (precysts), each of

Regulatory Mechanism in Sexual and Asexual Cycles of *Dictyostelium* 329

regulator. The signal transduction pathways involved in zygote formation are also noted, specifically paying attention to the function of a novel protein ZYG1, whose expression is

**2. Advantages of using cellular slime molds for cellular and developmental**

**Organisms**

Cellular slime molds are known as model organisms and have a lot of advantages as materials in the fields of cellular and developmental biology. As the differentiation phase of the cellular slime molds starts by removal of nutrients (starvation), it is temporarily separated from the growth phase. The pattern of cell differentiation is also relatively simple: *Dictyostelium* cells eventually differentiate into mainly two cell types, spore and stalk cells in an asexual cycle. These characters are quite suitable for studies of differentiation including the mechanisms of the transition from growth to the differentiation phase. As they are usually haploid, it is easier to manipulate genes, such as clone, knockout genes and so on. In fact, many transformants were cloned and used in this chapter. Transformants described

ACOOE Dm 7 *Dd-aco D. discoideum* Dm-ACOOE Dm 7 *Dm-aco* Dm 7 OH10 Dm 7 *zyg1* Dm 7 GFP/ZYG1OE *D. discoideum* (Ax2) *gfp/zyg1 AequoreaVictoria*/Dm 7 GFPOE *D. discoideum* (Ax2) *gfp Aequorea victoria* HA/ZYG1 myoblasts (C2C12) *ha/mzyg1 Influenza virus*/Dm 7

ACO-RNAi Dm 7 *Dd-aco D. discoideum* Table 1. Transformants used in this study. *Dd-aco*: an acc oxidase homologue gene isolated from *D. discoideum*. *Dm-aco* : an acc oxidase homologue gene isolated from Dm 7. *zyg1*: a novel gene isolated from Dm 7. *gfp:* a green fluorescent protein gene isolated from *Aequorea Victoria. gfp/zyg1*: a fusion gene of *gfp* and *zyg1*. *ha/mzyg1*: a fusion gene of a hemagglutinin gene (*ha*) and a humanized *zyg1* gene (*mzyg1*). Introduction of the vector constructs into *Dictyostelium* cells was performed by electroporation as described by Howard et al. (1988). In case of C2C12 cells (myoblasts), they were placed on coverslips and transfected with a

manufacturer's instruction. After 6 hours of transfection in Opti-MEM (Invitrogen), samples were washed twice with phosphate buffered saline (PBS) and incubated with Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS) and 80 μg/ml of Kanamycin for 24 h at 37°C in 5% carbon dioxide (CO2) at 95% humidity. Transformants underexpressing *Dd-aco* (ACO-RNAi) were obtained by means of the RNAi method. For this purpose, a vector construct of the stem-loop RNA directed against the full

*ha/mzyg1* fusion gene, using LipofectamineTM2000 (Invitrogen) according to the

length of Dd-aco was infected into Dm 7 cells (Amagai et al., 2007).

**transformed Name of genes Origin of genes**

augmented by ethylene.

here are summarized in Table 1.

**Name of transformants**

(Over expression)

(Under expression)

**studies**

which is surrounded by a fibrillar sheath. At the center of each precyst, a cytophagic cell (a giant cell) arises, which in turn engulfs all the other cells in the precyst. The engulfed cells (endocytes) are eventually broken down into granular remnants.  After a resting period, the macrocyst germinates to release several amoeboid cells and initiates a new life cycle (Nickerson & Raper, 1973). Cytophagic cells (giant cells) formed during macrocyst formation have been proved to be zygotes that are produced by cell fusion and subsequent nuclear fusion (Amagai, 1989).

Fig. 2. The developmental process of macrocyst formation. Starved Dm7 cells were developed by shaking. A: 21 hours development after starvation. The first few endocytes have formed at the center of a precyst. A boundary delimits the cluster of endocytes (e) from the surrounding peripheral cells (p) (arrow). B: 24 hours of development. The number of endocytes has increased as more peripheral cells have been engulfed by the cytophagic cell (delimited by arrows). C: 6 day-old cyst filled with granules in Brown motion. After all peripheral cells have been transformed into endocytes, endocytes are broken down into granular remnants. S: fibrillar sheath, W: macrocyst wall. Drawings show the same stages as photographs shown at the top (modified data cited from Filosa & Dengler, 1972). Bars: 35μm.

There are two kinds of mating systems in the macrocyst formation; homothallic and heterothallic (Clark et al., 1973; Erdos et al., 1973). Dm 7 forms macrocysts without mating types as a homothallic strain. On the other hand, *Dictyostelium discoideum* (*D. discoideum*), a heterothallic strain, undergoes mating with an opposite mating type, V12M2 cells.

In this chapter, the regulatory mechanism for determining one of two cycles in Dm 7 and *D. discoideum* is reviewed, particularly focusing on the function of ethylene as a potent

which is surrounded by a fibrillar sheath. At the center of each precyst, a cytophagic cell (a giant cell) arises, which in turn engulfs all the other cells in the precyst. The engulfed cells (endocytes) are eventually broken down into granular remnants.  After a resting period, the macrocyst germinates to release several amoeboid cells and initiates a new life cycle (Nickerson & Raper, 1973). Cytophagic cells (giant cells) formed during macrocyst formation have been proved to be zygotes that are

Fig. 2. The developmental process of macrocyst formation. Starved Dm7 cells were developed by shaking. A: 21 hours development after starvation. The first few endocytes have formed at the center of a precyst. A boundary delimits the cluster of endocytes (e) from the surrounding peripheral cells (p) (arrow). B: 24 hours of development. The number of endocytes has increased as more peripheral cells have been engulfed by the cytophagic cell (delimited by arrows). C: 6 day-old cyst filled with granules in Brown motion. After all peripheral cells have been transformed into endocytes, endocytes are broken down into granular remnants. S: fibrillar sheath, W: macrocyst wall. Drawings show the same stages as photographs shown at

There are two kinds of mating systems in the macrocyst formation; homothallic and heterothallic (Clark et al., 1973; Erdos et al., 1973). Dm 7 forms macrocysts without mating types as a homothallic strain. On the other hand, *Dictyostelium discoideum* (*D. discoideum*), a

In this chapter, the regulatory mechanism for determining one of two cycles in Dm 7 and *D. discoideum* is reviewed, particularly focusing on the function of ethylene as a potent

heterothallic strain, undergoes mating with an opposite mating type, V12M2 cells.

the top (modified data cited from Filosa & Dengler, 1972). Bars: 35μm.

produced by cell fusion and subsequent nuclear fusion (Amagai, 1989).

regulator. The signal transduction pathways involved in zygote formation are also noted, specifically paying attention to the function of a novel protein ZYG1, whose expression is augmented by ethylene.

#### **2. Advantages of using cellular slime molds for cellular and developmental studies**

Cellular slime molds are known as model organisms and have a lot of advantages as materials in the fields of cellular and developmental biology. As the differentiation phase of the cellular slime molds starts by removal of nutrients (starvation), it is temporarily separated from the growth phase. The pattern of cell differentiation is also relatively simple: *Dictyostelium* cells eventually differentiate into mainly two cell types, spore and stalk cells in an asexual cycle. These characters are quite suitable for studies of differentiation including the mechanisms of the transition from growth to the differentiation phase. As they are usually haploid, it is easier to manipulate genes, such as clone, knockout genes and so on. In fact, many transformants were cloned and used in this chapter. Transformants described here are summarized in Table 1.

Table 1. Transformants used in this study. *Dd-aco*: an acc oxidase homologue gene isolated from *D. discoideum*. *Dm-aco* : an acc oxidase homologue gene isolated from Dm 7. *zyg1*: a novel gene isolated from Dm 7. *gfp:* a green fluorescent protein gene isolated from *Aequorea Victoria. gfp/zyg1*: a fusion gene of *gfp* and *zyg1*. *ha/mzyg1*: a fusion gene of a hemagglutinin gene (*ha*) and a humanized *zyg1* gene (*mzyg1*). Introduction of the vector constructs into *Dictyostelium* cells was performed by electroporation as described by Howard et al. (1988). In case of C2C12 cells (myoblasts), they were placed on coverslips and transfected with a *ha/mzyg1* fusion gene, using LipofectamineTM2000 (Invitrogen) according to the manufacturer's instruction. After 6 hours of transfection in Opti-MEM (Invitrogen), samples were washed twice with phosphate buffered saline (PBS) and incubated with Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS) and 80 μg/ml of Kanamycin for 24 h at 37°C in 5% carbon dioxide (CO2) at 95% humidity. Transformants underexpressing *Dd-aco* (ACO-RNAi) were obtained by means of the RNAi method. For this purpose, a vector construct of the stem-loop RNA directed against the full length of Dd-aco was infected into Dm 7 cells (Amagai et al., 2007).

Regulatory Mechanism in Sexual and Asexual Cycles of *Dictyostelium* 331

producing ethylene (Amagai et al., 2007). *Dd-aco,* an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase homologue gene, isolated from *D. discoideum* (DDBJ, EMBL and GenBank databases, accession no. AB105858) was introduced into Dm7 cells to obtain the transformant overproducing ethylene. As ACC oxidase (ACO) catalyzes the last step in the biosynthesis of ethylene, the transformant overexpressing *Dd-aco* (ACOOE) was expected to increase the production of ethylene (Fig. 3). When the production of ethylene was determined by gas chromatography, ACOOE actually produced a larger amount of ethylene as compared with wild type, Dm7. On the other hand, the transformant underexpressing *Dd-aco* (ACO-RNAi) which had been isolated by the method of RNAi, produced a smaller amount of ethylene as compared with Dm7. Depending upon the amount of ethylene produced, ACOOE cells formed macrocysts, while ACO-RNAi cells failed to form them, regardless of culture conditions. The relationship between the amount of ethylene produced and the developmental forms in the transformants is

Fig. 3. Ethylene biosynthesis in the cellular slime molds. Ethylene is synthesized from methionine through S-adenosyl-L-methionine (SAM) and 1-aminocyclopropane-1-

prevents ethylene synthesis by blocking ACC synthase activity.

**3.4 How to induce macrocyt formation by ethylene**

carboxylic acid (ACC) in *Dictyostelium* as the case in higher plants. ACC synthase catalyzes the production of the ethylene precursor ACC from SAM (S-adenosylmethionine). ACC oxidase catalyzes the last step in the ethylene biosynthesis. AOA (amino-oxy acetic acid)