Translocation of 14C-compounds from maternal tissue into maize seeds grown in vitro: A test of a hypothesis concerning the absence of auxotrophs in higher plants

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

% Total 14C in Extracts


 
14C Compound Added in the Medium Tissue Sucrose Glucose Fructose Sub-Total Amino* Acids Amino** Acids Sub-Total Sum Total
Sucrose Cob 4.7 2.3 9.4 16.4 9.2 14.9 24.1 40.5
  Endo 10.0 12.7 15.8 38.5 15.1 27.2 42.3 80.8
Fructose Cob 6.0 2.9 17.9 26.8 7.1 20.6 27.7 54.5
  Endo 8,0 11.3 13.2 32.5 13.7 22.3 36.0 68.5

*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


Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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