Role of Dark-Fixation of CO2 in Plants

In this article we will discuss about the role of dark-fixation of CO₂ in plants.

In several plants a highly specialized cellular adaptation in most of the developmental systems exists in which dark-fixation of CO₂ utilizes phosphoenolpyruvate (PEP) derived from carbohydrates as a substrate plays a vital role (Fig).

Malate produced from dark fixation of CO₂ acted as an osmoregulatory solute during turgor-driven cell expansion of several developmental systems e.g. pollen tube growth; embryo growth; fruit development and also during shoot formation in calli of several species. It may also supply reducing power (NADPH) through its decarboxylation by malic enzyme.

The key differentiating events leading to pollen tube growth, cell division, enlargement of embryo sac, the formation of meristemoids within the heart-embryo and its growth, shoot formation in calli of several species are high energy requiring processes where endogenous carbohydrates (sugars, starch), exogenous free sugars from the culture medium were utilized.

Pollen tubes grow through the styles while embryos grow and differentiate within the ovary/seed. Evidently these tissues grow in darkness and also undergo active respiration. The importance of dark fixation of this respired CO₂ has been demonstrated in several systems. Besides being a source of malate and a reducing power, this process could affect detoxification of respired CO₂.

During embryogenesis, prior to heart-shape stage histological changes leading to organogenesis, enzymes involved in malate metabolism increase sharply. There is an apparent burst in enzyme activities just before the heart-embryo.

There is also a continuous increase in malate content between globular and heart- embryo and the increase is drammatic. Malate decarboxylation through NADP- malic dehydrogenase enzyme is also seemingly taking place at a greater rate.

Clearly the internally generated CO₂ is increasingly recycled. Heart embryo can also metabolize ¹⁴C- glucose at a faster rate than cotyledonary or young embryo. Obviously there is an increased endogenous CO₂ production during late globular-or early heart-embryo.

The embryos of this stage have high actual ability to fix CO₂ in the dark. The depletion of malate in the heart and cotyledonary-embryos strongly suggests its role in supplying NADPH for reductive biosynthesis of cellular constituents.

NADPH possibly originates from transhydrogenation when net hydrogen transfer from NADH to NADP+ occurs and through several reactions generates the internal cycling of CO₂. The decrease in malate is also accompanied by an increase in the pentose phosphate pathway—a fact ascertained through glucose 6-phosphate dehydrogenase or CVC, ratio using labelled glucose (6¹⁴_ C glucose and l¹⁴_ C glucose).

This clearly points towards an additional source of reducing power. The sugars obtained through suspenser undergo glycolysis and produce PEP which is a substrate for dark CO₂ fixation. Triose phosphate could enter glycolysis through PPP and additionally supply PEP.

Supplementing pollen suspension cultures with CO₂ (3-5%) caused marked increase in germination and pollen tube growth in vitro in pollen culture of Brassica campestris var toria. There was weakening of self-incompatibility by increased CO₂, levels.

When pistils are enclosed in a chamber with 5% CO₂, high percentage of pollen germination, pollen tube penetration of stigma hair and also seed set was observed in self-pollinated pistils.

Recently dark fixation of CO₂ by the self-and cross-pollinated pistils has been shown. Cross-pollinated pistils had double the level of fixation. PEP- carboxylase activity increased in the self-pollinated pistils following supply of CO₂. Recent studies have also demonstrated very high level of PPP in the cross- pollinated pistils and also enhanced respiration.

It seems that in the self-compatible state a quick carbon flow through malate may be making available NADPH to meet special metabolic requirements of pollen tube growth. The role of malate in the compatible state seems vital.

Seemingly free sugars were not available in the self- incompatible state and were converted into callose and PPP was reduced or inhibited. There is also a possibility that PEP was not sufficiently available. Thus reactions leading to malate formation were not expressed.

In the developing fruits of pea, bean and okra dark fixation of CO₂ during early periods of fruit growth and differentiation are also seen.

Non-autotrophic carbon fixation has also been described during growth of tobacco callus cultured in dark under shoot-forming and non-shoot forming conditions. Malate derived from dark fixation of CO₂ plays differing roles in the two conditions. In the non- shoot formation state it acts chiefly as an osmotic solute regulating partly cell expansion between successive cell divisions while in shoot forming state malate provides NADPH for the reductive biosynthesis.

The role of dark CO₂ fixation in regulating the fibers development in cotton ovules has also been described recently.

In developing fruits and seeds of Phaseolus similar observations are made. In summary seeds in this bean species also possess a dark fixation system of CO₂. Indeed all the above mentioned systems develop in the perpetual low light intensity or dark. The relatively high activity of PEP- carboxylase found in these systems suggests that recycling of respired CO₂ could occur in them at the site of origin.

In summary PEP-carboxylase, which indicates dark fixation of CO₂ and is usually considered to be associated with CAM and C₄ systems, is also a predominant attribute of the developing systems of many C₃ plants. In the C₃ plants PEP-carboxylase activity is maximal during the developing stages when the rate of respiration is very high. Such a system facilitating dark CO₂ fixation helps in the accumulation and generation of malate and also provides the reducing power.

Calmodulin:

In 1980 Cheung and others reported the discovery of calmodulin—a calcium-binding protein that could modulate the activity of key regulatory enzymes in plants and animals. Subsequently two plant proteins, NAD kinase, and a plasma membrane calcium pump or Ca²⁺-ATPase were shown to be regulated by calmodulin and also phytochrome.

Then a working hypothesis of the Ca²⁺ action became apparent which included the following transduction sequence: first, photoactivation of phytochrome; second, an increase in cytosolic Ca²⁺; and third, activation of calmodulin and/or other calcium-binding modulator proteins, and lastly activation of cAMP-dependent enzymes. Following the type of enzyme(s) activation, several kinds of pleiotropic effects follow.