Elsevier Editorial System(tm) for Plant Physiology and Biochemistry 

Manuscript Number: PLAPHY‐D‐09‐00335 

Differential and functional interactions emphasize the multiple roles of polyamines

in plants1

Avtar K. Handaa and Autar K. Mattoob

a
Department of Horticulture & Landscape Architecture, Purdue University, W. Lafayette, IN,

USA
b
Sustainable Agricultural Systems Laboratory, United States Department of Agriculture,

Agricultural Research Service, The Henry A. Wallace Beltsville Agricultural Research Center,

Building 001, Beltsville, MD 20705-2350, USA

E-mail addresses: ahanda@purdue.edu (A.K. Handa), autar.mattoo@ars.usda.gov (A.K. Mattoo)

Keywords:

Biogenic amines; Correlation coefficients; Fruit proteins; Metabolome; Plurality model; Protein

profiles; Tomato; Transcriptome; Transgenic

Abbreviations:

ACC, 1-aminocyclopropane-1-carboxylic acid; ADC, arginine decarboxylase; Brix, total insoluble

solids; CrtL, lycopene cyclase; CyP, cyclophilin; dsDNA, double stranded DNA; eIF5A,

initiation/elongation factor eIF5A; Hsp, heat shock proteins; ODC, ornithine decarboxylase; PAO,

polyamine oxidases; PAs, polyamines; PME, pectin methylesterase; PPT, precipitate weight

ratio; Put, putrescine; SAM, S-adenosylmethionine; SAMDC, SAM decarboxylase; Spd,

spermidine; Spm, spermine; SSAT, spermidine/spermine N1-acetyltransferase

1
Dedicated to the memory of our friend and colleague Prof. Nello Bagni

1
*Manuscript

Click here to view linked References

ABSTRACT

Biogenic amines putrescine, spermidine and spermine are ubiquitous in nature and have

interested researchers because they are essential for cell division and viability, and due to a large

body of their pharmacological effects on growth and development in most living cells. The genes

and enzymes involved in their biosynthetic pathways are now established and characterized. In

recent years, molecular aspects of polyamine action have also begun to emerge. Our model is the

ripening tomato fruit in which processes of cell division, cell expansion and cell growth have

ceased, and yet the cells are responsive at biochemical and molecular levels to genetically

manipulated concentrations of putrescine (Put), spermidine (Spd) and spermine (Spm). Thus,

transcriptome, limited protein profiling, and metabolome studies of transgenic tomato fruit have

yielded significant new information on cellular processes impacted by polyamine manipulation.

We have used these datasets to determine the linear correlation coefficients between the

endogenous levels of Put, Spd and Spm with several parameters. Results of our analysis

presented here show that effects of the diamine Put generally contrast those with polyamines Spd

and Spm, emphasizing that individual biogenic amines should be considered to have defined

action in plant biology and that they differentially affect growth and development. A multiple function

model of polyamine action is discussed to explain the role of polyamines in most organisms, in

general, and ripening fruit, in particular.

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1. Introduction

The field of polyamines can be said to have had its origin from the first discovery, in the 17th

century, of the presence of spermine as one of the crystalline inclusions in human seminal fluid

[34]. Since then a number of diamines and polyamines have been shown to be ubiquitous and

essential for cell viability in living organisms [14], [46], [50] and [53]. Abundant reports exist on

profound effects of these biogenic amines on growth, development and senescence in eukaryotic cells

[14], [46] and [50]. In plants, putrescine (Put) is a major diamine and a direct substrate for triamine

spermidine (Spd) and tetraamine spermine (Spm) [53]. Put is synthesized from either arginine (Arg) by

Arg decarboxylase (ADC) or ornithine (Orn) by Orn decarboxylase (ODC). Spd synthase catalyzes

Spd synthesis from Put and decarboxylated S-adenosylmethionine (SAM) (dcSAM). The

formation of dcSAM from SAM by SAM decarboxylase is a committed and rate-limiting step in

polyamine pathway. Spm is synthesized from Spd and dcSAM via a reaction catalyzed by Spm

synthase. SAM is a molecule central to 1C metabolism and, in plants, also for ethylene

biosynthesis [19]. Evidence has been presented for an alternative biosynthetic route for sym-norSpd,

which utilizes aspartate δ-semialdehyde as an aminopropyl group donor for formation and for

diaminopropane production [32] and [56]. Intracellular concentrations of polyamines seem tightly

regulated at several levels, impacting not only their biosynthesis but also their catabolism which are

responsible for their homeostasis [4], [18], [31], [38], [44], [50] and [51]. ADC, ODC, SAM

decarboxylase and spermidine/spermine N1-acetyltransferase (SSAT) are among the well studied

enzymes that regulate cellular levels of polyamines [1], [2], [13], [38], [44] and [50].

Molecular studies have provided compelling evidence in favor of important roles polyamines play in

plant biology. Overexpression of heterologous ODC and ADC genes led to increased Put levels with

only slight increase in Spd and Spm levels [17]. Such a genetic intervention in cell cultures has provided

interesting information on Put mediated transcriptional and posttranscriptional changes [38].

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Overexpression of yeast SAMDC gene in tomato resulted in a 2- to 3-fold increase in the intracellular

levels of Spd and Spm at the cost of Put in ripening fruit [40], providing a strong evidence in favor of

the key role SAM decarboxylase plays in regulating triamine and tetraamine levels in plants [17], [20],

[40] and [59].

Spm and its related form thermospermine (tSpm), an abundant polyamine in Thermus thermophilus, are

now recognized to have important functions in living organisms [23], [41] and [47]. Thermospermine is

required for stem elongation [27] while the thicker vein (tkr) (vascular tissue differentiation) phenotype

of a recessive mutant was linked to Spm synthase (ACL5/TKV) [12] and [24], both in Arabidopsis. Spm

has also been linked to salt [30], [31], [57] and [60], and drought [8] and [61]) tolerance. These

molecular genetics inroads into plant polyamine field have produced seminal discoveries on the function

of polyamines in plant development and responses to environmental extremes. Clearly, the field is ripe

for discovery research on additional regulatory mechanisms that regulate the biosynthesis of polyamines

as well as their action.

Catabolism of polyamines is another path that potentially can regulate polyamine levels and

at the same time generate signaling molecules such as hydrogen peroxide [42] and [62]. Thus, Spm can

be back-converted to Spd and Spd to Put [28], [45] and [58]. Polyamine oxidases (PAO) that catalyze

back conversion pathway have been reviewed [15] and [45]. These advances have further intensified

interest in the function and role of polyamines in plants. In terms of polyamine transport and efflux

pathways, not much is well understood as yet in most organisms but significant progress has been made

in mammalian systems [50].

Reverse genetics approaches have been effectively employed in providing direct evidence for

4
the involvement of polyamines in fruit quality, ripening, and processing attributes [37], [38], [39], [40]

and [55]. These biogenic amines seem involved in regulating ion channels, scavenging free radicals and

regulating gene expression [50] and [55]. Inroads into molecular aspects of polyamine action have

brought to light the implications of polyamines in cellular processes such as chromatin

condensation, stability and function, maintenance of DNA structure, RNA processing,

translation, and protein activation [9]. One of the well studied functions involving polyamines

and protein synthesis is the posttranslational hypusine (derived from Spd) modification of the

initiation/elongation factor eIF5A that regulates the transport and processing of specific RNAs

[26] and [49]. Also, the participation of polyamines is well established in a translational frameshift of

at least two known genes via novel RNA-decoding mechanism, influencing protein kinase casein

kinase 2 action [5] and [11].

In spite of significant new knowledge on polyamine biosynthesis and action, the

differential role, if any, of each polyamine (Put, Spd, Spm and tSpm) in regulating cellular metabolism

is largely not known except for a role of Spd in modifying eIF5A initiation factor and Spm (tSpm) in

vascular development. Generally, little attention is paid to discriminate polyamine function in terms of a

diamine, triamine, tetraamine, or hexamine. In recent years, it has become clear that some amino acids

have a signaling function; for example, the signaling role of glutamate, glutamine, asparagine

and -aminobutyric acid in nitrogen and intermediary metabolism [literature cited in [38] and [39]. The

datasets we generated on gene expression, endogenous protein levels, metabolome and plant

phenotype by analyzing transgenic plants engineered for altered levels of Put, Spd and Spm

provided a means to determine significant correlative associations between the endogenous

levels of Put, Spd and Spm and specific cellular and developmental processes. Our analyses

presented here have revealed significant associations that pinpoint opposite and, generally,

contrasting functions of the diamine Put versus those of the polyamines Spd and Spm.

5
2. Put, Spd and Spm in relation to the transcriptome

The relative abundance of gene transcripts in several functional categories was found to be

different in transgenic tomato fruit engineered to accumulate high levels of Spd and Spm as compared to

the wild type fruits that had several-fold lower levels of these polyamines [55]. These functional

categories represent transcription, translation, signal transduction, chaperones, stress responses,

amino acid biosynthesis, ethylene biosynthesis, ethylene action, polyamine biosynthesis,

isoprenoid pathway and flavonoid biosynthesis. About one-quarter of the 1066 unique fruit genes

evaluated exhibited differential regulation, and the genes up-regulated in the presence of elevated Spd

and Spm were twice as abundant as the down-regulated genes, as compared to wild type fruits. About

44% of the differentially regulated genes were novel with no known function [55]. Northern analysis

validated higher transcripts in the transgenic fruit for chaperone protein genes including cyclophilin

(CyP, encodes a protein that plays role in the mitochondrial permeability transition during cell

death [41], pectin methylesterase (PME, fruit carbohydrate metabolism enzyme), lycopene cyclase

(CrtL), mitochondrial cytochrome oxidase I (CoXI) and ethylene biosynthesis (ACS2) as compared to

wild type tomato fruit [36]. Collectively, these results suggested that Spd and Spm levels are linked to a

differential accumulation of specific gene transcripts that encompass a wide variety of cellular processes

[36] and [55].

To determine which transcripts of the above-specified genes are influenced by a particular

polyamine (Put, Spd and/or Spm), correlation coefficients between them were assessed. Data

from three genotypes (wild type, transgenic 556HO and transgenic 579HO) at two stages of

ripening (pink and red) were pooled and correlation between relative transcript abundance for

each gene with endogenous Put, Spd and Spm levels was determined. As illustrated in Figure 1,

6
the gene transcript abundance that was negatively correlated with endogenous Put levels was

found positively correlated with endogenous levels of Spd and Spm. Among the genes that

showed positive correlation with Put include ACC oxidase (ACO1), tomato SAM decarboxylase

(LeSAMDC), MADS-box transcription factor (MADS-RIN) and three genes whose functions are yet to

be characterized. Transcript levels of actin (internal control), light harvesting chlorophyll a/b binding

protein (LHCII), G-protein β-subunit (ArcA1), heat shock protein genes (hsp17.6, hsp20.0 and hsp20.1),

CyP, PME, ACC synthase, CoxI and CoxII, and an unknown EST were positively correlated with Spd

and Spm and all showed negative correlations with Put.

3. Put, Spd and Spm in relation to protein abundance

Immunoblot analysis was employed to identify and quantify the levels of proteins, ACC

Oxidase (ACO), polygalacturonase (PG), SAM synthetase, heat shock proteins (Hsp17.6, Hsp90), and δ-

and γ-subunits of TIP in ripening fruits from two transgenic genotypes and the parental wild type

[36]. Although the levels of ACC oxidase and Hsp17.6 proteins closely paralleled those of their

transcripts, a significant deviation was seen between the TIP transcript expression and the levels

of its δ- and γ- protein subunits. SAM synthetase protein was similar between the transgenics and

the wild type. PG protein is known to increase during fruit ripening [6]. Its level increased in

all the three genotypes but the effect of altered polyamine levels was not consistent. Lack of

synchronization between transcript levels of some of the genes and their corresponding protein

products is indicative of a role of Spd-Spm in posttranslational regulation of proteins [36].

Like the analysis made in the previous section, we sought to determine which particular

polyamine (Put, Spd and/or Spm) was correlated with a change in the abundance of the specified

proteins. Results are presented in Figure 2. Put positively affected ACC oxidase protein while

Spd and Spm had a negative effect, a result that is consistent with corresponding effects at the
7
level of transcripts. The heat shock proteins were positively correlated with Spd and Spm levels

and negatively with higher levels of Put; Hsp17.6 exhibited much more stronger association with

Spd and Spm than was found for Hsp70. Only a week association was found between Hsp90 and

the three polyamines. SAM synthetase and PG exhibited week negative associations with all

three polyamines. Interestingly, while γ-TIP was positively correlated with Spd and Spm and

negatively with Put, δ-TIP showed a week negative correlation with Put and no significant

correlation with Spd and Spm. These data bring to light effects of Put, Spd and Spm on specific

proteins and further emphasize the differential effects of diamine Put versus higher polyamines

Spd and Spm, Put effects being opposite to those of Spd and Spm.

4. Differential effects of Put, Spd and Spm on metabolism

Coordination of transcriptome and proteome has to synchronize with downstream signaling

to effect cellular metabolism that generally translates into altering the endogenous levels of

metabolites. Thus, a metabolome reflects this translation, a final determinant of a certain genetic

alteration that has a consequence on cellular metabolism. To obtain an insight into the metabolic

processes that Spd-Spm influence in plants, nuclear magnetic resonance (NMR) spectroscopy

was used to characterize metabolite profiles of transgenic fruit that accumulate the higher

polyamines Spd and Spm and the wild type tomato fruit [39]. Distinct changes in the metabolite

trends between the SAMdc-transgenic and wild-type/azygous fruits ripened off the vine were

revealed. Glutamine, asparagine, choline, citrate, fumarate, malate and an unidentified compound

A were among the metabolites that accumulated in the red transgenic fruit whereas the levels of

valine, aspartic acid, sucrose, and glucose significantly decreased compared to the control (wildtype

and azygous) red fruit. Isoleucine, glucose, γ-aminobutyrate, phenylalanine and fructose

were metabolites that maintained a similar content in the non-transgenic and transgenic fruits.

Along with these metabolite trends, pathways of nitrogen sensing/signaling, carbon metabolism,

8
and respiration were preferentially activated in the high Spd-Spm transgenics. A clear coordination

was apparent between N sensing and up-regulation of the N:C indicator genes,

phosphoenolpyruvate carboxylase and NADP-dependent isocitrate dehydrogenase, in the

transgenic fruit, consistent with our previous findings [39].

To unveil the significant interactions between the various metabolites in relation to the

endogenous levels of Put, Spd and Spm, the datasets on the red-ripe fruit from all the three

genotypes were pooled and statistically analyzed to determine the linear correlation coefficients

(Fig. 3). Positive correlation coefficients of > 0.8 were found between Spd-Spm and the

metabolites glutamine, asparagine, isoleucine, alanine, threonine, choline, fumarate, malate and

citrate. These metabolites were negatively correlated with Put at < -0.8. However, Put was

positively correlated (correlation coefficient > 0.8) with metabolites such as valine, glucose and

sucrose. Glucose showed a strong negative correlation with Spd and Spm whereas sucrose and

valine were weakly associated with Put levels. Thus, the metabolome patterns resonate with

those of transcriptome (and partial protein analysis) and corroborate the theme that Put effects

are opposite to those of Spd and Spm in affecting cellular metabolism in a ripening fruit.

5. Differential effects of Put, Spd and Spm on fruit phenotype

The genetic intervention by expressing yeast SAM decarboxylase gene in a fruit-specific

manner not only resulted in several-fold increase in Spd and Spm levels but also impacted the

transcriptome, proteins and the primary metabolism. These effects led to phenotypic changes

such as an increase in lycopene (a secondary metabolite), prolonged vine life, and enhanced fruit

juice quality [40]. It was thus important to determine in what manner the manifested phenotypes

caused by one-step metabolic engineering were related to Put versus Spd-Spm. W determined the

correlation coefficients between Put, Spd and Spm and parameters such as rate of

9
ethylene production, lycopene, and vine life. From this analysis, it was evident (Fig. 4) that,

except for pH, all other juice parameters including total soluble solids, Brix, acidity, and

viscosity were positively associated with Spd and Spm and negatively with Put. Rate of ethylene

production, lycopene levels and vine life were also positively associated with Spd and Spm and

negatively with Put.

6. A plurality model of polyamine action

Polyamines as ubiquitous molecules have wide-ranging function in a number of

physiological processes. Being an indispensable component of cellular milieu, polyamines

directly or indirectly function in many metabolic processes. A whole body of literature is filled

with data implicating Put, Spd and Spm in the maintenance of DNA structure, chromatin

condensation, transcription, RNA processing, translation, enzymatic activation, and modifying

ion channels. It is likely that all these functions involve diverse modes of action. Polyamines can

interact with different classes of macromolecules, alter structural states and influence metabolic

activity, all of which can manifest in one or more of the several phenotypic changes reported thus

far. The identification of the targets, as done here, is a first step for developing strategies to

elucidate mechanisms involved. We present here a plurality model that can be an avenue to

discern the genetic, biochemical, physiological roles of polyamines (Fig. 5). We propose that due

to the chemical attributes of polyamines (Put, Spd, Spm) and their derivatives/conjugates, they

interact differentially with cellular components including chromatin, transcriptional machinery,

translational machinery, and macromolecules (DNA,RNA and proteins) and cause modified

metabolic profiles responsible for the observed phenotypes.

Both in vitro and in vivo studies point to a differential involvement of Put, Spd and Spm in

determining DNA conformation and chromatin condensation [9]. Polyamines preferentially

10
stabilize A-DNA rather than B-DNA with the major groove of A-DNA being the preferred site

for binding. Differential binding of Spd and Spm along the backbone and Put to the sugar phosphate

backbone in both the major and minor grooves in B-DNA has also been reported [7]. Due to its

relatively smaller size, Put can bind to both the major and minor grooves of DNA with

preference for the double-stranded DNA (dsDNA) whereas Spd preferentially localizes to the

minor groove of DNA [52]. Put is restricted to the intrastrand binding while Spd to the

interstrand [9]. In vivo studies indicate that polyamines affect chromatin structure and likely

chromatin remodeling since they target histone acetylation [25], [35] and [54]. Spd has the ability to

protect DNA against damage due to ROS and gamma-rays [22]. Such properties of

polyamines can positively impact stability and functioning of chromatin.

Our results clearly pinpoint the differential effects of diamine Put, and triamine Spd

together with tetraamine Spm on transcriptional profiles of fruit cells. Among the 21 genes

examined, Put was positively correlated with only 3 genes at a correlation coefficient > 0.5 but

negatively with 10 genes that had a correlation coefficient of < -0.5. In contrast, Spm was

positively correlated with 10 genes at a coefficient of > 0.5 and negatively with 3 genes (at a

coefficient of < -0.5). Spd was positively correlated with 6 genes at a coefficient of > 0.5 and

negatively with 5 genes at a correlation coefficient of < -0.5. The genes such as ACC oxidase

and SAMDC, which were upregulated by Put were downregulated by higher levels of Spd-Spm.

The opposite scenario characterized the effects on the chaperone gene such as Hsp17.6, Hsp20.0

and Hsp20.1. These results are consistent with the findings of other investigators who presented

transcriptome analyses showing that polyamines affect transcription profiles of Escherichia coli

and higher organisms [10], [16] and [29]. However, the latter studies did not discriminate or dissect the

effects specific to an individual polyamine, analyses that needs to be carried out in the context of

our theme that diamines regulate cell transcription and metabolome differently than do higher

11
polyamines.

In spite of the terminal stage of fruit development bereft of cell division or cell

expansion, the transgenic tomato was characterized by not only changes at the transcriptome

level but also in the immunorective levels of several proteins, which were differentially

associated with the intracellular levels of Put, Spd and Spm (Figure 2). The proteins that were

positively correlated with Spd and Spm showed a negative correlation with Put. Some of these

observed effects on proteins paralleled their transcript levels; for example, positive association

between ACC oxidase protein and Put (and its negative correlation with Spd and Spm) was

mimicked by its transcripts. Nonetheless, polyamines may affect protein levels by either

increasing or decreasing the rate of degradation of a particular protein [33], a possibility

supported by the reports on polyamine-mediated inhibition of plant proteases [9] and [21].

At the level of the metabolome as well, transgenic interventions have been shown to

result in dramatic changes in metabolite profiles of non-growing tomato fruit cells and actively

multiplying poplar cell culture [36], [39], [43] and [48]. Correlative coefficient analyses of these have

also revealed that Put effects are opposite those of Spd-Spm [38]. The differential behavior of

individual polyamines could involve one or more of the following mechanisms: enhancement or

inhibition of translation (a differential effect at the synthesis of enzymes in a pathway), structural

modification of enzymes, enzyme stability, or posttranslational modification of enzyme(s)

leading to either activation or inhibition of key enzymes in a pathway.

7. Conclusions

Three main themes emerge from the analysis presented here: 1) Functions of Put, Spd and

Spm in cellular and developmental processes are differential and, therefore, cannot be boxed

together; 2) Spd and Spm effects on various physiological and metabolic processes seem similar

12
and more positive in nature than found for Put; and 3) Put impacts cellular metabolism in a manner

generally opposite to that of Spd and Spm. However, “correlation does not imply causation”,

therefore we do not interpret our analysis as a causal relationship between the variables.

Nonetheless, high correlation coefficients reveal the existence of a relationship between the

parameters and provide the initial evidence for a direct or an indirect association between

parameters. Our model is the ripening fruit, which represents a plant tissue at a terminal

developmental state by which time the cells no longer undergo cell division or cell expansion.

Therefore, the observed profound metabolic effects of increased levels of polyamines Spd and

Spm unearth early events uncomplicated by the complexities in a cell undergoing growth, cell

division or cell expansion. Also, these results clearly demonstrate that a senescing plant tissue

maintains intact the sensing and signaling processes for important molecules such as the higher

polyamines late into development.

Acknowledgments

This study was supported in part by a US-Israel BARD grant to AKH and AKM (Grant N0. IS-

3441-03) and a grant from the USDA- IFAFS program (Award No. 741740) to AKH. Mention of trade

names or commercial products in this publication is solely for the purpose of providing specific

information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

13
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Figure Legends

Fig. 1. Differential effects of Put, Spd and Spm on steady state levels of transcripts.

Correlation of gene transcripts, representing diverse metabolic pathways, with the intracellular

Put, Spd, Spm, and total polyamine (Total PA) levels. Most of the genes shown were obtained after

subtraction of wild-type red-ripe fruit transcripts from the transgenic fruit [40] at the same stage

of ripening [36] and [55]. Transcript levels were quantified by determining relative hybridization

intensity for each gene transcript using northerns [36]. Levels of the indicated polyamines, in

nmol/g fresh weight, for each sample represent the average of several determinations from fruits at a

defined stage of ripening for the each genotype [40]. Pearson’s correlation coefficients were calculated

using correlation function of Microsoft Excel program. For theses analyses, all the data obtained from

analysis of pink and red ripe fruits from wild type and two transgenic lines 556HO and 579HO were

pooled. Meaningful calculations for p-values by randomizing were not possible; therefore, correlation

threshold below 0.5 (for positive correlation) and above - 0.5 (for negative correlation) were not

considered consequential. Gene identity [36] and [55]: light harvesting chlorophyll a/b apoprotein

(LHCII), tonoplast intrinsic protein (TIP), ACC oxidase 1 (ACO1), G-protein β-subunit (ArcA1), heat

shock protein genes (Hsp 17.6, Hsp 20.o and Hsp 20.1), tomato SAMDC (Le SAMdc), cyclophilin

(CyP), pectin methylesterase 2 (PME2), eukaryotic translation initiation factor 5A2 (eIF5A2),

unidentified genes (EST2, pSC-B, pSC-C, pSC-G, pSC-K), ACC synthase 2 (ACS), cytochrome

oxidases I (CoxI) and II (CoxII), and MADS-box transcription factor (MADS-RIN).

Fig. 2. Differential effects of Put, Spd and Spm on protein abundance. Correlation of cellular

levels of indicated proteins with the intracellular Put, Spd, Spm, and total polyamine (PA) contents.

Immunoreactive levels of each protein were determined from immunoblotting [36]. Other details

are same as in Figure 1. Proteins quantified are: heat shock proteins (HSP 17.6, HSP 70, and HSP 90),

large subunit of ribulose,1-5-bisphosphate carboxylase/oxygenase (RbcL), SAM synthetase (SAM S),

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ACC oxidase (ACO), δ-subunit of tonoplast intrinsic protein (δ-TIP), γ-subunit of TIP (γ-TIP),

polygalacturonase (PG) and calcium dependent protein kinase (CDPK).

Fig. 3. Differential effects of Put, Spd and Spm on metabolome constituents. Correlation of the indicated

metabolites with the intracellular Put, Spd, Spm, and total polyamine (PA) levels. NMR spectroscopy

was used to identify and characterize metabolite profiles of wild type and two SAMDC-transgenic

fruits [39]. Phe did not show any correlation (data not shown). Other details are the same as in

Figure 1. Thick lines (Red and Blue) represent correlation coefficients of > 0.8; thinner lines

represent correlation coefficients in the range of 0.55 to 0.79. Red lines, positive correlation; blue

broken lines, negative correlation.

Fig. 4. Differential effects of Put, Spd and Spm on fruit phenotype. Correlation of indicated

metabolites with the intracellular Put, Spd, Spm, and total polyamine levels. Levels of ethylene

production, juice precipitate weight ratio (PPT), pH, total insoluble solids (Brix), acidity, viscosity (vis),

vine life, and lycopene levels were taken from [40]. Correlation coefficients were determined as

described in Figure 1 except that data from only ripe fruit were analyzed. Other details are the same as in

Figure 1.

Fig. 5. A multiple function model of polyamine action. Shown are major cellular processes in a

sequential order, from chromatin to phenotype. Due to their chemistry Put, Spd and Spm (and possibly

their derivatives) affect these components differentially. The incomplete circle around each

component indicates that polyamines act to stabilize as well as move the cellular function

forward to the next level. A partial circle of arrows in the middle of the model starting from

chromatin to phenotype signifies the continuous requirement of polyamines throughout the life

cycle of an organism.

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