Читать книгу Plant Nucleotide Metabolism - Hiroshi Ashihara - Страница 26

Plants contain complex mixtures of free nucleotides. The principal components of such mixtures can be isolated and determined using HPLC combined with absorbance or, preferably, mass spectrometric detection. However, the extraction of nucleotides from cells is a crucial step. With some plant tissues, the extraction procedure has a profound influence on the resultant analytical profile, and special precautions must be taken if the results are to truly represent the nucleotide composition of living cells. Because of the presence of enzymes that hydrolyse nucleotides and nucleosides, such as non-specific acid phosphatases and nucleosidases, extraction of nucleotide-related compounds from plant material is extremely challenging. These hydrolytic enzymes are not denatured during extraction and remain active even in cold methanolic extracts.

Free nucleotides are separated from cellular macromolecular components by cold extraction procedures in which the macromolecules are precipitated; the usual extraction solvents are dilute perchloric acid or trichloroacetic acid. Prior to analysis, perchloric acid extracts are neutralized with KOH, precipitating KClO₄, while those treated with trichloroacetic acid are partitioned against diethyl ether to remove the acid. Usually, the concentration of nucleotides is low, so the aqueous fraction is lyophilized, and redissolved in small amounts of an aqueous solvent prior to HPLC.

Some examples of the levels of purine and pyrimidine nucleotide levels in cultured plant cells and leaves are shown in Table 2.1. The results were obtained with cultured cells of Arabidopsis thaliana (thale cress), Nicotiana tabacum (tobacco), Lycopersicon esculentum (tomato), and Catharanthus roseus (Madagascar periwinkle) at the exponential stage of growth. For comparison, nucleotide levels of cultured C. roseus cells grown in a phosphate (Pi)-deficient medium are also shown. Table 2.1 also lists the levels in young leaves of A. thaliana, N. tabacum, L. esculentum and Ca. sinensis.

Table 2.1 Concentration of nucleotides in plant cells and tissues.

Source: Based on Ashihara and Crozier (1999) and Riondet et al. (2005).

	Arabidopsis thaliana	Nicotiana tabacum	Lycopersicon esculentum	Camellia sinensis	Catharansus roseus
Nucleotide	Cells	Leaves	Cells	Leaves	Cells	Leaves	Leaves	+Pi	−Pi
ATP	145	40	124	48	190	50	177	100	22
ADP	19	77	8	23	38	30	35	18	16
AMP	17	59	9	33	—	55	14	16	12
GTP	26	tr	23	tr	24	tr	26	26	8
GDP	—	—	—	—	—	—	tr	3	3
GMP	tr	9	tr	—	10	—	tr	3	2
IMP	—	8	—	20	—	20	—	—	—
XMP	—	—	—	—	—	—	29	—	—
UTP	18	20	49	7	27	6	88	55	10
UDP	tr	18	—	23	59	16	46	29	22
UMP	178	81	187	74	174	50	55	33	24
CTP	9	tr	4	12	—	—	tr	7	2
CDP	tr	—	—	9	—	—	tr	4	2
CMP	—	tr	—	tr	—	tr	32	6	3
Relative rate
ΣAN (%)	44	56	35	42	44	60	48	45	40
ΣGN (%)	6	3	6	0	7	0	6	11	10
ΣUN (%)	48	38	59	42	50	0	40	39	44
ΣCN (%)	2	0	1	8	0	32	7	6	6
Energy status
EC	0.85	0.45	0.91	0.57	0.92	0.48	0.86	0.81	0.60
ATP/ADP	7.6	0.5	14.9	2.0	5.1	1.7	3.3	5.6	1.4

The values (nmol g⁻¹ f.w.) are obtained from the suspension-cultured cells at the exponential phase of growth and young leaves. For comparison, the values from phosphate-deficient Catharanthus roseus cells are also shown. tr: trace and EC: energy charge.

There are similar profiles of nucleotide pools in almost all plants. The pools of adenine nucleotides and uridine nucleotides (aka uracil nucleotides) always exceed the guanine nucleotide pool. The cytidine nucleotide pool is the smallest of these pools. The nucleoside triphosphate levels are usually much more substantial than those of nucleoside mono- and diphosphates.

The intracellular levels of nucleotides in plant cells are influenced by several environmental factors. For example, Pi starvation of cultured plant cells results in a marked decrease in the levels of nucleotides, especially nucleoside triphosphates (Table 2.1). The proportion of adenylates, i.e. the adenylate energy charge ([ATP] + ½[ADP])/(ATP + ADP + AMP) originally proposed by Atkinson (1977), is maintained at an almost constant ratio of 0.8–0.9 in actively growing plant cells. The energy charge in young tea leaves, 0.86, is within this range, but leaves of A. thaliana, tobacco, and tomato showed lower values, 0.5−0.6, because of the higher concentrations of ADP and AMP. While the energy charge fluctuates with diverse growth conditions, some reported changes might be ascribed to the presence of hydrolytic enzymes in extracts. The ATP/ADP ratio is also a major parameter of interest in the investigation of metabolic aspects of bioenergetics. The ratios are usually high in actively growing cells and tissues.