Translocation of various ¹⁴C-compounds from maternal tissue into developing maize seeds was examined by using caryopsis cultures (Gengenbach, 1977, Planta 134:191.) in order to test a hypothesis that the absence of auxotrophs in higher plants is due to inadequate translocation of essential compounds from maternal tissue to developing seeds. Fertilized zygotes homozygous for a mutant in a locus coding for a step in the synthesis of an essential compound have to receive the missing metabolite from the maternal tissue to survive to a dormant stage. Therefore, a lack of adequate translocation of the metabolites from the maternal tissue to developing seeds would lead to the death of mutant zygotes.

Ears of inbred W22 (5DAP) were cut into cob blocks containing four caryopses, grown on the standard medium for 10 days, transferred onto ¹⁴C-containing medium, and incubated for 7 days. Five cob blocks were placed on the medium (50 ml) in a 125 ml Erlenmeyer flask, and duplicate flasks were used for each compound. At harvest, the five cob blocks within each flask were bottled for processing. The material was divided into four tissue groups; embryo, endosperm, pericarp and pedicel, and cob were dried, ground to powder, and radioactivities counted by using a liquid scintillation counter.

Table 1 shows total uptake of ¹⁴C-compounds by cultured cob blocks. The tested compounds can be grouped into three distinct classes according to their relative efficiencies of uptake into the cultured cob blocks. The first class of compounds which was most efficiently taken up into the cultured tissue includes the three amino acids tested, adenine, and nicotinic acid. The second group having the intermediate degree of uptake consists of fructose and thiamine HCl. Thymine, characterized by its extremely low efficiency of uptake, can be included in the third class. Because the amount of sucrose in the medium was approximately five orders of magnitude higher than other compounds, this compound was not grouped into the three classes mentioned above.

Table 1. Total ¹⁴C Uptake by Cultured Cob Blocks
 

14C Compound	Total 14C Added	Total Amounts	Specific Activity	Total 14C Taken Up		Total Uptake		Total Uptake		% Total 14C Taken Up/g D.W.
	(cpmx 10-6 /flask)	(nmole/flask)	(cpm/nmole)	(cpmx10-4)		(nmole/flask)		(nmole/g D.W.)
				F1.1	F1.2	F1.1	F1.2	F1.1	F1.2	F1.1	F1.2
Sucrose	27.8	7.3x106	3.8x106	18.0	17.3	4.7x104	4.5x104	1.1x105	1.0x105	1.5	1.4
Fructose	23.1	62	3.7x1011	51.2	ND	1.38	ND	2.7	ND	4.4	ND
Leucine	16.5	152	1.0x1011	163.9	161.7	14.90	14.70	41.0	29.4	27.3	19.3
Phenyl-alanine	28.7	30	9.6x1011	383.4	454.4	4.00	4.73	7.8	9.0	26.0	30.0
Proline	10.8	19	5.7x1011	103.9	126.6	1.82	2.22	4.2	5.7	22.1	30.0
Adenine	11.9	107	1.1x1011	190.2	185.2	17.29	16.83	31.9	31.0	29.8	29.0
Thymine	13.5	128	1.1X1011	3.9	3.9	0.35	0.35	0.7	0.8	0.5	0.6
Thiamine HCl	11.2	289	3.9x1010	29.2	41.6	7.49	10.67	17.5	20.7	6.1	7.2
Nicotinic acid	9.1	75	1.2x1011	105.6	110.5	8.80	9.21	20.1	18.8	26.8	25.1

Table 2 shows the distribution of ¹⁴C in various tissues of cultured cob blocks. Each compound had a characteristic pattern of ¹⁴C distribution between different tissues. For instance, sucrose and fructose had a gradient of increasing ¹⁴C concentration in the order of cob-pericarp and pedicel-endosperm-embryo. However, for leucine and phenylalanine, this order was completely reversed. The rest of the compounds tested had an order, endosperm-embryo-pericarp and pedicel-cob from low to high. Nonetheless, the considerable enrichment of ¹⁴C in the cob and the pericarp and pedicel was also found for these compounds. To obtain an idea of relative concentration of ¹⁴C in various tissues, ratios of ¹⁴C concentrations between different tissues are shown in the right half of Table 2. For example, the endosperm had approximately five times more counts than the cob when ¹⁴C-sucrose or ¹⁴C-fructose was supplied. Conversely, the cob accumulated 10-50 times greater amounts of ¹⁴C than the endosperm did when amino acids, adenine, or vitamins were supplied. When the ratios of ¹⁴C concentrations between the endosperm and the embryo were examined, the unique aspect of thiamine HCl became apparent. This compound was almost ten times higher in the embryo than in the endosperm.

Table 2. Distribution of ¹⁴C in Various Tissues
 

			Total 14C Uptake (cpm/mg DW)				Ratio of 14C Concentration Between Tissues
14C compound		Flask	Cob	Per & Ped	Endo	Emb	Per & Ped/Cob	Endo/Cob	Emb/Endo	Emb & Endo/Cob
Sucrose		1	174	276	818	2624	1.59	4.70	2.47	5.05
		2	169	246	845	2854	1.46	5.00	3.38	5.46
	Mean		172	261	832	2439	1.52	4.84	2.92	5.26
Fructose		1	378	497	1804	3423	1.31	4.77	1.90	5.03
Leucine		1	11927	2602	1085	660	0.22	0.09	0.61	0.09
		2	9875	1860	828	631	0.19	0.08	0.76	0.09
	Mean		10901	4462	957	646	0.20	0.09	0.69	0.09
Phenylalanine		1	19526	3964	1476	1061	0.20	0.08	0.72	0.07
		2	19812	5487	2501	1890	0.28	0.13	0.76	0.12
	Mean		19669	4726	1989	1476	0.24	0.10	0.74	0.10
Proline		1	6860	1860	363	437	0.27	0.05	1.20	0.05
		2	7504	2355	342	405	0.31	0.05	1.18	0.05
	Mean		7182	2108	353	421	0.29	0.05	1.19	0.05
Adenine		1	8209	3626	183	210	0.44	0.02	1.15	0.02
		2	7504	2762	161	203	0.37	0.02	1.26	0.02
	Mean		7857	3144	172	207	0.41	0.02	1.21	0.02
Thymine		1	104	83	53	121	0.80	0.51	2.28	0.57
		2	124	70	49	144	0.56	0.40	2.94	0.48
	Mean		114	77	51	133	0.78	0.45	2.61	0.53
Thiamine HCl		1	1310	553	74	683	0.42	0.06	9.23	0.09
		2	1634	952	95	954	0.58	0.06	9.95	0.10
	Mean		1472	753	85	814	0.50	0.06	9.59	0.10
Nicotinic Acid		1	3642	2969	518	876	0.81	0.14	1.69	0.15
		2	3990	2833	506	1061	0.71	0.13	2.07	0.14
	Mean		3817	2901	512	969	0.76	0.13	1.88	0.14

Distribution of ¹⁴C among various compounds in water or 5% TCA extracts from cultured tissues was analyzed by paper chromatography, and the results are shown in Tables 3-5. After the entry into the cob tissue, vitamins appeared to experience little conversion (Table 3). For instance, the counts supplied as nicotinic acid remained in nicotinic acid in the cob tissue. Similarly, approximately 80% of total ¹⁴C in cob extracts was recovered as thiamine HCl. This was also true in the endosperm and embryo extracts. A limited amount of conversion of adenine and thymine in the cob was observed (Table 3). Corresponding phosphorylated nucleosides were the only compounds which had appreciable ¹⁴C other than the original compounds. In contrast, amino acids were often converted to other compounds within the cob tissue (Table 4). Approximately 20% of total ¹⁴C activity in the cob and endosperm extracts was recovered as the amino acids supplied. In addition, the ¹⁴C label was also found in other amino acids and simple sugars. Among the amino acids derived from originally supplied ones, aspartic acid (also possibly glycine and alanine) and glutamic acid (also possibly arginine) were found in greatest amounts. They were tentatively identified based on their color reactions and Rf values. As expected, sucrose and fructose were converted to a variety of compounds in the cob tissue although 16 and 27% of ¹⁴C was still found in simple sugars where ¹⁴C-sucrose and ¹⁴C-fructose, respectively, were fed (Table 5).

Table 3
 

Distribution of 14C Among Various Compounds in Tissue Extracts: Nucleic Acid Bases and Vitamins
		% Total 14C in Extracts
14C Compound Added in the Medium	Tissue	Original Form	Phosphory Nucleosides	Total
Adenine	Cob	31.0	55.2	86.2
	Endo	43.9	56.1	100.0
Thymine	Cob	52.6	15.5	68.1
	Endo	34.3	27.3	61.6
Thiamine HCl	Cob	70.9
	Endo	85.1
	Emb	81.0
Nicotinic Acid	Cob	100.0

Table 4. Distribution of ¹⁴C Among Various Compounds in Tissue Extracts: Amino Acids
 

		% Total 14C in Extracts
14C Compound Added In the Medium	Tissue	Original Form	Amino* Acids	Amino** Acids	Sub-Total	Sucrose	Glucose	Fructose	Sub- Total	Total
Leucine	Cob	29.5	6.1	9.2	44.8	3.1	2.2	3.9	9.2	54.0
	Endo	31.4	3.8	6.1	41.3	4.9	3.8	6.1	14.8	56.1
Phenylalanine	Cob	23.9	6.6	8.8	39.3	3.9	3.3	13.9	21.1	60.4
	Endo	23.2	6.8	6.8	36.3	5.7	4.0	5.3	15.0	51.8
Proline	Cob	18.7	18.5	11.0	48.2	10.9	10.9	6.5	28.3	76.5
	Endo	31.4	21.5	13.0	65.9	6.6	7.6	5.9	20.1	86.0

*Aspartic acid and some others.
**Glutamic acid and some others.

Table 5. Distribution of ¹⁴C Among Various Compounds in Tissue Extracts: Sugars

*Aspartic acid and some others.
**Glutamic acid and some others.

Based on relative ¹⁴C concentrations in the cob and the endosperm, it is concluded that the translocation of the vitamins, thiamine HCl and nicotinic acid, from the cob to the endosperm is strongly inhibited in in vitro conditions. Nucleic acid bases and their phosphorylated nucleosides appeared also poorly translocated into the endosperm through the cob tissue.

Due to extensive metabolic conversions observed in the cob during the culture period, the observations of the translocation of amino acids should be evaluated with caution. With regard to compounds converted from an amino acid taken up into the cob, they would be grouped into two classes according to their properties of translocation. The first class includes those compounds which have similar or lower relative efficiencies of translocation from the cob to the endosperm. Presumably, amino acids derived from the originally labeled one would be in this class. Also, intermediates in catabolic processes could possibly be included in this class. This class of compounds would be a major class of converted labeled compounds and would not considerably affect the endo/cob ¹⁴C ratio. The second class of compounds derived from a labeled amino acid would be those that are more easily transported into the endosperm through the cob than the originally labeled amino acid. Simple sugars would be included in this category. This class of labeled compounds is a minor group of derivatives but increases the endo/cob ¹⁴C ratio of an originally labeled amino acid. Based on above discussions, the efficiency of translocation for amino acids deduced from the relative enrichment of ¹⁴C labels in the endosperm and the cob could possibly be overestimates. Therefore, it is not unreasonable to conclude that there is also a strong translocation barrier between the cob and the endosperm for amino acids in in vitro conditions.

Supposing that a similar restriction of translocation operates in vivo, the results suggest the following: 1) Most (or all) of nutritionally essential compounds other than simple sugars are subjected to a strong inhibition of translocation from maternal tissue to the seeds. This inhibition may be strong enough to prevent the development of mutant zygotes lacking the capacity to synthesize a vital metabolite to a mature dormant seed. 2) That thiamine auxotrophs are the only obligate auxotrophs known in higher plants may be partly explained by the concentration of thiamine HCl in the embryo. Although the translocation of this compound from the cob to the endosperm is no better than that of other compounds, this vitamin is accumulated in the embryo almost ten times more efficiently than in the endosperm. In addition, the small requirement for vitamins for normal growth compared with amino acids or nucleic acid precursors may possibly be an additional factor explaining the presence of thiamine auxotrophs in higher plants.

Ko Shimamoto and Oliver E. Nelson

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