Riboflavin as Photoreceptor in the Induction of Two-dimensional Growth in Fern Gametophytes

Abstract

Two distinct phases of growth may be recognized in the development of the gametophyte of the polypodiaceous fern, Phymatodes nigrescens. In the first phase, the spore germinates to form a 4to 5-celled filamentous protonema (1-dimensional growth). During subsequent growth, either the apical or subapical cell of the protonema divides in all directions to form a heart-shaped prothallium (2-dimensional growth). The initiation of 2-dimensional growth is inhibited by base analogs of RNA like 8-azaadenine, 8-azaguanine, 2-thiocytosine and 2-thiouracil (Yeoh, unpublished observations). These results which are in accord with previous work from this laboratory (4) indicate that 2-dimensional growth is mediated through the endogenous RNA. Two-dimensional growth in the gametophytes is also dependent upon a blue-l:ght mediated photoreaction. It is now well established that 2-dimensional growth occurs in blue light, while in red light, continued 1-dimensional growth is sustained (2). From these results, it appears unlikely that RNA has a direct role in the induction of 2-dimensional growth in the gametophytes. Rather, it has been suggested that RNA metabolism may be related to the synthesis of a pigment (Raghavan, unpublished observations), probably a flavoprotein (3) which acts as a photoreceptor in blue-light mediated 2dimensional growth. In the present work we have investigated this possibility by analyzing the effect of the blue-absorbing pigment, riboflavin, in reversing the inhibition of 2-dimensional growth in the gametophytes induced by the pyrimidine analogs, 2-thiocytosine and 2-thiouracil. Spores of P. nigrescens (Bl.) J. Sm., were collected from the greenhouse. They were sieved through 2 layers of lens paper to separate the sporangial walls, and were stored at 250. The spores were sown on the surface of 15 ml of a modified mineral salt solution (4) contained in 7 cm Petri dishes. The basal meditum was supplemented with 2thiocytosine and 2-thiouracil at 35.0 mg/l and 2.0 mg/I, respectively. Preliminary experiments using a range of concentrations of the analogs showed that at these concentrations, 2-dimensional growth in the gametophytes was completely inhibited. In attempts to reverse the inhibition, the reversers were added with the specified concentration of analog to give different reverser/analog ratios in the medium. Data for reversals are given from media which induced optimum 2-dimensional growth. The pH of the medium was adjusted to 5.6 with 0.1 N HCl or KOH. The conditions of cultture were the same as in earlier work (4). Although our quantitative experiments were done in white lght, in brief experiments we have found that blue l.ght was as effective as white light. For certain experiments ulsing red light, light was filtered throuigh red cellophane (max transmission at 650 m,u). Growth was evaluated in terms of width, cell number and sturface area of the gametophytes at 28 days, according to previously described procedures (4). Results are presented in table I. From the low values of width, cell ntumber and suirface area, it is clear that addition of 2-thWocytosine and 2-thiouracil inhibited 2-dimensional growth in the gametophytes. In the presence of the inhbbitors, the plants retained their filamentous form throtughout the duration of the experiment (f:g 1C) while those in the basal medium formed normal 2-dimensional prothallia (fig 1A). Addition of riboflavin with the inhibitors completely reversed the inhibition and reinstated 2-dimensional growth in the gametophytes (fig 1 D). With 2-thiocytosine and 2-thiouracil, optimum reversal was obtained on addition of riboflavin at 1X (35.0 mg//l) and 15X (30.0 mg/l), respectively, the concentration of the inhibitor. This evidently shows that the amount of riboflavin needed for reversal of inhibition is independent of the concentration of the inhibitor and is more or less constant. It was also noted that with both analogs riboflavin was twice as effective as their corresponding bases in nullifying the inhibition (fig 1E). Still better reversal was obtained with lumichrome (kindly supplied by Dr. John S. Tzou1 The work described in this paper forms part of a thesis of 0. C. Yeoh for the M.Sc degree, University of Malava.