Discussion

Discussion

It is an interesting fact that in rape seeds，as in other oil-bearing seeds，the carbohydrates store is abruptly and rapidly converted into fats usually at the age of 10—20 days or more days after flowering.This observation reported in a variety of seeds suggests that there are some factors controlling the“turn about”of metabolism and synthesis of fats in the maturation process［14］.Of these factors one may be the activity of the TCA cycle［15］and another the generation of NADPH2，as discussed in this paper.

In the light of the present knowledge it is generally contended that fatty acids were synthesized in the following way：acetyl-CoA is first carboxylated in the presence of CO2，ATP，Mn2+，and biotin，forming malonyl-CoA，then decarboxylated and condensed to form higher fatty acids［16］.It is well known that in the process of fatty acid synthesis a large amount of NADPH2 is needed as a specific electron donor for the reduction of the carbonyl to methylene groups.The chief sources of NADPH2 are two dehydrogenation reactions：first，the two primary reactions of the HMP pathway（G-6-P DH and 6-PG DH）；second，the reaction of the extramitochondrial NADP-linked isocitrate dehydrogenase（the NAD-linked isocitrate dehydrogenase is located in the mitochodria and is coupled with the TCA cycle and oxidative phosphorylation）［17］.Obviously，the HMP pathway plays a more important part in the supply of NADPH2，since 12 molecules of NADPH2 can be generated from a molecule of G-6-P.Evidences on the relationship between fat formation and the HMP pathway have been reported in animal tissues，but this approach has not yet been made with tissues of higher plants.

There are considerable experimental evidences for the existence of a pathway of glucose catabolism other than the EMP or glycolytic route in developing rape seeds.In a preliminary account［6］we have shown that besides all the enzymes of the EMP pathway the rape siliques contain a very active G-6-P DH and 6-PG DH as well as enzymes concerned with the breakdown of R-5-P.The identification of several intermediates of the HMP pathway and the ability of the cell-free extract of the siliques to resynthesize hexosemonophosphate from R-5-P indicate the existence of the complete cycle in this tissue［6］.In later experiments in which the tape seeds were incubated with glucosel-C14 or glucose-6-C14，we found that the aldehyde carbon of the glucose is converted more rapidly into C14O2 than the carbon atom 6.It was further calculated from the relative incorporation of C14 from specifically labelled glucose molecules into lipid that in rape seeds approaching maturation approximately 40%of glucose utilization proceeded via the HMP pathway［18］.

It is shown in the present experiments that the activities of enzymes of the HMP pathway including G-6-P DH，6-PG DH，and enzymes utilizing R-5-P in the rape siliques rise noticeably with the development of rape seeds whereas the activities of enzymes of the HMP pathway，namely phosphofructokinase，aldolase，3-PGA kinase，and enzymes involved in the conversion of 3-PGA into pyruvic acid decline rapidly.The metabolic shift from the predominance of the EMP pathway to that of the HMP pathway in siliques takes place between 10 to 20 days or more days after flowering，and corresponds to the period when soluble sugars in the seeds decrease sharply and fats increase markedly［19］.Taking the activities of the isolated enzymes as a criterion of the intensity of a metabolic pathway in vivo，as suggested by Glock and McLean［20］and Gibbs and Earl［21］，we may conclude that the advance of fat synthesis in rape seeds is correlated with the increase in the activity of the HMP pathway and the decrease in the activity of the EMP pathway.This conclusion is supported by the fact that the fraction of respiration sensitive to iodoacetamide and NaF，powerful inhibitors of two important enzymes of the EMP pathway，3-phosphoglyceride dehydrogenase and enolase［6］，declines rapidly with the age of the siliques.Further confirmation comes from feeding experiment with specifically labelled glucose，in which it was demonstrated that the ratios C1/C6 and C1/Cu of the liberated CO2 rise with the development of seeds and reach maxima on approaching maturation parallel with the course of fat accumulation［18］.The finding that the activity of the TCA cycle which is closely linked to the EMP pathway declines noticeably during the development is also in line with the above conclusion［15］.

Among the enzymes of the EMP pathway only phosphoglucoisomerase and 3-phosphoglyceraldehyde dehydrogenase behave somewhat differently from others.Their activities decrease from 8 to 15 days after flowering，but increase gradually again after 15 days.As phosphoglucoisomerase is involved in the HMP pathway，its enhancement in the later period is to be expected.The similar behaviour of 3-phosphoglyceraldehyde dehydrogenase is，however，difficult to understand.

Since the discovery of G-6-P DH in erythrocyte haemolysate and yeast autolysate by Warburg et al.［22，23］nearly 30 years ago，a great amount of evidence has been accumulated on the contribution of the HMP pathway in the breakdown of glucose in many animal and plant tissues.The cycle mechanism，the metabolic intermediates，and the properties of enzyme of the pathway have been studied rather tliorouglily［24-26］.In the last 10 years，the interest of investigators has turned to the physiological role and the regulatory mechanism of the HMP pathway.It has been demonstrated，e.g.，that the age of tissues is related to the HMP pathway，thus，Gibbs et al.［21］observed that while immature tissues respire exclusively or to a large extent via the EMP glycolytic pathway，but as the tissue ages and diffetentiates，the HMP pathway becomes increasingly important.Shaw and Samborski［27］，and Daly et al.［28］stressed the significance of the HMP pathway in the immunity of plant tissues.Other workers reported that aureomycin formation in Streptomyces aurefaciens［29］，the abscission of organs of plants［30］，and the differentiation of flowering buds［31］，may also be correlated to the HMP pathway.The evidence presented in this investigation as well as in our other report［18］for the intimate association of lipogenesis and the HMP pathway further emphasized the significance of the HMP pathway in the metabolism and physiological activities of the higher plants.

The regulation and control of the HMP pathway of carbohydrate metabolism in organisms are of great interest.There is growing evidence showing that the supply of NADP is one of the factors controlling the activity of the HMP pathway in many tissues.The rate of NADPH2 reoxidation and the ratio of NADP/NADPH2 can affect the rate of channelling of glucose into the HMP pathway.As has been shown in our laboratory，the presence of artificial electron acceptors such as phenazine methosulpbate，methylene blue，and triphenyltetrazolium，or the supply of substrates which require NADPH2 for their metabolism（e.g.，the synthesis of fatty acids），can increase the rate of the relative formation of C1402 from glucose-1-C14 by rape seeds［32］.Similar effects have been observed in other tissues such as liver slices［33，34］，erythrocytes［35］，corneal epithelium［36］，aspites tumour cells［37］，mammary gland［38］，and carrot slices［39］.In addition to NADP，the regulatory effects of phosphate［40，41］，glucose concentration［42］，Cu2+content，the Michaelis constants of enzymes of two pathways competiting for G-6-P［43］，and 6-PG［44］on the HMP pathway and EMP pathway have also been frequently discussed.(https://www.daowen.com)

The most interesting result of the present investigation is the finding of a natural inhibitor of G-6-P DH and 6-PG DH in the reproductive organs of rape seed.Its presence was evidenced by the facts that activities of both G-6-P DH and 6-PG DH rose markedly when the extract of acetone powder of the buds was diluted，and that the extract of the buds could strongly inhibit the G-6-P DH derived from different sources.On the basis of its solubility and stability properties，absorption spectra，fluorescence under ultra-violet，etc.，the inhibitor was presumed to be a flavonoid or its derivative.

Experiments showed that the natural inhibitor is present in buds，flowers，and young siliques，but nearly absent in the old siliques.These results，together with the changes of the activities of G-6-P DH and 6-PG DH in rape reproductive organs during seed development，led us to believe that the inhibitor may take part in controlling the activities of G-6-P DH and 6-PG DH and therefore regulate the rate of fat formation.In the early periods of seed development，the content of inhibitor is high enough to block the dehydrogenase of the HMP pathway and no fat is synthesized.Later，when the inhibitor decreases and disappears and the enzymes of the HMP pathway become active，the“valva”of the HMP pathway becomes to be opened，and fat is actively synthesized，as a result of the abundant supply of NADPH2 generated.Thus，it seems that the inhibitor may play an important role in the regulation of carbohydrate breakdown and fat synthesis.Such mechanism of regulation is quite different from that discovered in animal tissues by Tepperman and Tepperman［45］，who suggested that the hepatic G-6-P DH increase seen on refeeding and after fructose feeding probably represents denovo protein synthesis.Here，according to our findings，it seems more likely that，as the seeds develop，the natural inhibitor decreases and eventually disappears，thus releasing the G-6-P DH and 6-PG DH from inhibition，and accelerating the operation of the HMP pathway.This results in an increase of generation of NADPH2，which together with other unknown factors，e.g.，the formation or activation of the enzyme complex required for fatty acid synthesis，as suggested by Stumpf［14］，would stimulate the synthesis of fatty acids.The increased speed of fatty acid synthesis would in turn accelerate the oxidation of NADPH2 to NADP，which further quickens the entrance of glucose into the HMP pathway，resulting thus in a positive“feedback”.Such mechanism，schematically postulated in Figure 6，could explain why the carbohydrates are suddenly and increasingly converted into fats during the development of oil-bearing seeds.It must be emphasized that the mechanism of regulation of fat synthesis in oil-bearing seeds is very complicated.The simple interpretation given here does not claim to be adequate or even correct.It only serves as a starting clue to approach the complex and interlocking problems of seed metabolism and lipogenesis，the theoretical and practical importance of which is obvious.

The authors wish to express their gratitude to Prof.Ying Hung-chang for his advice and encouragement throughout the course of this work，and to Comrade C.F.Chen of the Institute of Organic Chemistry for the measurement of infrared absorption of the natural inhibitor.