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

One of the richest sources of CKs in various plants species are developing seeds. In fact, zeatin, a major natural CK, was first discovered in developing seeds of maize (Letham, 1963). Not surprisingly, developing seeds of maize, rice and Lupinus albus have played a major role in CK research (reviewed in Emery & Atkins, 2005). A major focus of these studies has been on the possible role of CKs in sink strength of developing seeds. The role of CKs in controlling sink strength is inferred largely from (a) the well known role of CK in stimulating cell division that may lead to increased organ size of the sink tissue, and (b) the coincidence of CK accumulation with seed cell division profiles in several plant species (Dietrich et al., 1995; Arnau et al., 1999; Yang et al., 2002).


Introduction
Cytokinins (CKs) are plant hormones promoting cell division and differentiation, morphogenesis in tissue culture, leaf expansion, bud formation, delay of leaf senescence and chloroplast development (reviewed in Rijavec & Dermastia, 2010).Natural CKs are adenine derivatives and based on the side chain moiety, they can be divided into two subgroups: isoprenoid and aromatic cytokinins.
One of the richest sources of CKs in various plants species are developing seeds.In fact, zeatin, a major natural CK, was first discovered in developing seeds of maize (Letham, 1963).Not surprisingly, developing seeds of maize, rice and Lupinus albus have played a major role in CK research (reviewed in Emery & Atkins, 2005).A major focus of these studies has been on the possible role of CKs in sink strength of developing seeds.The role of CKs in controlling sink strength is inferred largely from (a) the well known role of CK in stimulating cell division that may lead to increased organ size of the sink tissue, and (b) the coincidence of CK accumulation with seed cell division profiles in several plant species (Dietrich et al., 1995;Arnau et al., 1999;Yang et al., 2002).
Nearly all CK metabolites identified thus far have been reported to be present in developing seeds of different plants.In maize, the zeatin type of isoprenoid cytokinins is most common (Brugière et al. 2003;Veach et al., 2003;Rijavec et al., 2009Rijavec et al., , 2011)).Developmental profiles of various CKs in maize caryopsis, a single seeded fruit of plants from the grass family, have shown high levels of zeatin riboside (Brugière et al., 2008;Rijavec et al., 2009Rijavec et al., , 2011) ) during the early stages of endosperm development (Dietrich et al., 1995;Brugière et al., 2003;Rijavec et al., 2011).In maize caryopsis there are also many reports on biochemical and molecular aspects of genes and proteins involved in CK biosynthesis, conjugation, and degradation.Enzyme activity for isopentenyl transferase (IPT) that marks the first committed step in CK biosynthesis was reported in immature maize caryopsis by Blackwell and Horgan (1994).Recently, Brugière et al., (2008) described a small family of IPT genes from maize based on the similarity with Arabidopsis IPT proteins.The Ipt genes ZmIpt1, ZmIpt2 and ZmIpt10 are expressed in the pedicel, endosperm and embryo (Brugière et al., 2008;Šmehilová et al., 2009;Vyroubalová et al., 2009;Rijavec et al., 2011), indicating local biosynthesis of CKs in these parts of the caryopsis.Degradation and reversible/irreversible inactivation are key regulators of CK levels.Several maize CK dehydrogenase genes (Ckx) are expressed in the caryopsis pedicel region, endosperm and embryo (Massonneau et al., 2004;Šmehilová et al., 2009;Vyroubalová et al., 2009), and their corresponding protein products irreversibly cleave the N 6 -side chain from the main purine ring (Massonneau et al., 2004).Recently, the importance of conjugation has also been described.In maize caryopsis cis-zeatin riboside-O-glucoside was shown to be a major CK metabolite in caryopsis (Veach et al., 2003), and similarly, increased levels of zeatin-O-glucoside have been reported in roots and leaves of maize transformants harboring the Zog1 gene encoding a zeatin-Oglucosyltransferase from Phaseolus lunatus (Rodó et al., 2008).Although the exact role of the conjugates CK-O-glucosides is not clear, they are generally assumed to be the storage products.Finally, information on the molecular biology of CK-N-glucosides, a group of metabolically inactive CKs (Brzobohatý et al., 1994) is scarce (Hou et al., 2004;Rijavec et al., 2011).However, in maize caryopsis there is a notably high concentration of trans-zeatin-9glucoside (Z-9-G) recorded in the developmental phase following intense mitotic activity (Rijavec et al., 2009(Rijavec et al., , 2011)).
As in other eukaryotes the plant cell cycle is governed by cyclin-dependent kinases (CDKs).While the catalytic CDK subunits are responsible for recognizing the target motif, which is present in substrate protein, the regulatory proteins -cyclins play a role in discriminating between distinct protein substrates and thus regulate different cell cycle transitions.Plants contain many cyclins.In Arabidopsis, for example, at least 32 cyclins in seven classes have been described (reviewed in Inzé and De Veylder 2006).In particular, cyclins from the class D were proposed to have a role as external growth factor sensor that integrates the external signals with the cell cycle machinery (Sherr and Roberts 1999;Planchais et al., 2004).In Arabidopsis, the expression of CycD2 and CycD3 during the G1 phase is controlled by the availability of sugars (Riou-Khamlichi et al., 2000).In addition, CycD3, but not CycD2, expression responds to CKs.CycD3 is elevated in an Arabidopsis mutant, exhibiting high CK levels, and is rapidly induced by CK application in both cell cultures and whole plants (Riou-Khamlichi et al., 1999).On the other hand, in maize CKs also stimulated the expression of CycD2 at the late stages of germination (Gutiérrez et al., 2005).In Arabidopsis developing leaves CycD3 function contributes to the control of cell number by regulating the duration of the mitotic phase and timing of the transition to the endoreduplication stage (Dewitte et al., 2007), in which nuclear DNA content is increased by rounds of full genome duplication without intervening mitoses, giving rise to cells with higher ploidy levels (Joubès and Chevalier 2000).It has also been suggested that cellular expansion and its accompanying endoreduplication are inhibited in Arabidopsis plants overexpressing the CycD3;1 gene (Dewitte et al., 2003).
In this study, the role of various CK metabolites in the control of seed mass in maize was investigated using an allelic series of miniature 1 (mn1), a loss-of-function mutation in the www.intechopen.comCytokinins and Their Possible Role in Seed Size and Seed Mass Determination in Maize 295 Mn1 gene that codes for an endosperm-specific cell wall invertase 2 (INCW2) (Miller & Chourey, 1992, Cheng et al., 1996).The mn1 seed mutation is associated with a conspicuous loss of seed mass at maturity and small seed size, related to the decreased cell number and size in developing endosperm (Vilhar et al., 2002).Several lines of correlative evidence from various plant species are available to show that cell wall invertase plays a major role in controlling sink strength through source-to-sink unloading of sucrose (Chourey et al., 2006).As expected from the invertase-deficiency, the mn1 mutation also exhibits much altered sugar metabolism relative to Mn1 (LeClere et al., 2010).The mn1 mutant accumulates higher levels of sucrose and lower levels of hexoses in the basal region of the caryopsis, while its upper regions accumulate either similar or even increased concentrations of sugars compared to the upper regions of the wild type caryopsis.Given the importance of hexose signaling, these changes may also be associated with the levels of CKs and auxin (Rolland et al., 2002).Indeed, CK metabolism in the mn1 mutant differs from that of the wild type (Rijavec et al., 2009(Rijavec et al., , 2011) ) and also has greatly reduced levels of the auxin indole-3-acetic acid (IAA) throughout caryopsis development (LeClere et al., 2008).Given these relationships, it was reasoned that the four lineagerelated genotypes of the mn1 allelic series are an ideal genetic system in which to analyze possible relationship among various CK metabolites, RNA level expression of a few selected genes of CK metabolism and sink strength in developing seeds.In this regard, a previously described mn1-89 mutation in maize is of special interest.It is an EMS-induced point mutation representing a single amino acid change from the conserved proline to leucine at the position that is critical for either efficient translation or for stabilization of the protein in planta (Carlson et al., 2000).The mn1-89 allele encodes normal or higher levels of Incw2 RNA compared to the wild type, but exceedingly low levels, ~ 6% of the Mn1, of the INCW2 protein and enzyme activity (Cheng et al., 1996).More importantly for the studies here, genetic analyses showed that the mn1-89 allele is semi-dominant in seed phenotype (Cheng et al., 1996) when crossed to the standard null allele, mn1-1, which has approximately ~1% of the wild type levels of invertase activity encoded by the non-allelic gene, Inc1, a paralog of the Mn1 locus (Chourey et al., 2006).The reciprocal hybrids from such crosses yield gene-dose dependent invertase activity levels of ~4 and 2% encoded by two and one mn1-89 alleles, respectively, present in the triploid endosperms.Thus, the invertase activity is strictly gene-dose dependent in that the number of allele copies determines the amount of enzyme activity.
It has recently been shown that both Mn1 and mn1 genotypes have extremely high, but similar CK levels during the very early stages of development from 6 to 8 days after pollination (DAP), which are followed by a marked and genotype-specific reduction (Rijavec et al., 2009).While the decrease of CKs in Mn1 was associated with their deactivation by 9-glucosylation, the absolute CK concentrations as well as concentrations of the biologically active CKs remained higher in the mutant until 16 DAP.Based on the correlative results from different studies, a developmental model showing possible crosstalk among CKs, cyclins CycD2 and CycD3, and INCW as causal to increase of cell number and sink strength of the Mn1 developing endosperm has been proposed (Fig. 1) (Rijavec et al., 2009).In the present work the proposed hypotheses that CycD2 and CycD3 genes are temporally differentially expressed in the mutant mn1 caryopsis and that their transcript abundance is associated with the elevated CK level or CK activation were further explored.
The general temporal profiles of the ZmIncw2 transcript levels measured with quantitative real-time PCR were similar for all endosperm genotypes (Fig. 3).Transcript levels were low at 6 DAP and between 16 and 28 DAP, while showing a distinct peak in transcript abundance at 8 or 10 DAP (Fig. 3).The highest abundance of the transcript preceded the peak of INCW2 activity at 12 DAP (Cheng et al., 1996).In spite of the similar profiles, ZmIncw2 transcript levels showed a gene dose dependent trend in the mn1-89/mn1-1 mutant series.Accordingly, genotypes mn1-89/mn1-89/mn1-89 and mn1-89/mn1-89/mn1-1 showed the highest transcript levels and even exceeded those of the wild type, confirming previous observations of high Incw2 transcript levels in the mn1-89 homozygous mutant using Northern blotting (Carlson et al., 2000).On the other hand, Incw2 transcript levels were low in the heterozygous mutant with one mn1-89 allele and almost undetectable in the homozygous mn1-1 mutant genotype (Fig. 3).It is noteworthy that there is a discrepancy between protein and enzyme activity levels and Incw2 transcript levels in Mn1 and mn1 endosperms (Chourey et al., 2006), since high RNA level in the homozygous mn1-89 mutant results in only ~6 % of the Mn1 invertase activity (Cheng et al., 1996).It has been suggested that a large proportion of INCW2 is dispensable without a significant change in seed phenotype (Cheng et al., 1996).
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 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).
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(Rijavec et al., , 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).

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, upr e g u l a t e d f r o m 1 2 t o 2 0 D A P , a n d i n t h e 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).
The expression level of ZmCycD3-1was 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 ZmCycD3-1in 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 (2.6fold) 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.

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.

Fig. 1 .
Fig. 1.Model of two alternative developmental pathways induced by cytokinins that lead to different sizes of filial tissues (endosperm and embryo) in wild-type (Mn1) and mutant miniature1 (homozygous for the mn1 allele) maize caryopsis.Cytokinin pie charts indicate the ratio of metabolically active cytokinins (dark) versus metabolically inactive trans-zeatin-9-glucoside (Z-9-G) (white) in both genotypes.The pathway that utilizes INCW2 for increasing cell number and sink strength is shaded gray.The images of Mn1 and mn1 caryopsis are bubble graph presentations of their actual longitudinal sections at 16 DAP.Steps in the model presented by dotted boxes and arrows with open heads have been proven in experimental systems other than maize.(From Rijavec et al., 2009; with the permission of the Journal of Integrative Plant Biology).