A Gibberellin-Deficient Brassica Mutant— rosette

Abstract

A single-gene mutant (rosette [ros/ros]) in which shoot growth and development are inhibited was identified from a rapid cycling line of Brassica rapa (syn campestris). Relative to normal plants, the mutant germinated slowly, had delayed or incomplete floral development, and reduced leaf, petiole, and intemode growth. The exogenous application of GA3 by foliar spray or directly to the shoot tip of rosette resulted in rapid flowering, bolting (shoot elongation), and viable seed production. Shoots of rosette contained endogenous levels of total gibberellin (GA)-like substances ('Tan-ginbozu' dwarf rice assay) of about one-tenth of that of the normal rapid-cycling line of B. rapa which consisted almost entirely of a very nonpolar, GA-like substance which yielded GA1 and GA3 upon mild acid hydrolysis. In a normal rapid-cycling B. rapa line, the nonpolar putative GA1 and GA3 conjugates were present, but additionally, free GA1 and GA3 were abundant and identified by gas chromatography-mass spectrometry-selected ion monitoring. The quantities of free GA1 and GA3 in the normal line and in rosette were quantified by GC-MS-SIM using [2H2]GA1 as an intemal standard. Fourteen-day-old rosette and normal seedlings contained 5.3 and 23.2 ng GA, per plant, respectively. At day 21 the rosette plants contained 7.7 and 26.1 nanograms per plant of GA1 and GA3, while normal plants contained 31.1 and 251.5 nanograms per plant, respectively. Thus, normal plants contained from four to ten times higher levels of total GA-like substances, GA1, or GA3, than rosette. The ros allele results in reduced GA level, yielding the rosette phenotype whose delayed germination and flowering, and reduced shoot growth responses indicate a probable role for endogenous GA1 and GA3 in the regulation of these processes in Brassica. There are three general experimental approaches that are used to investigate phytohormone physiology. First, and most simply, the exogenous application of the hormone may influence a given process. Exogenous GAs often have a profound effect on higher plants, generally promoting shoot elongation and often altering reproductive development (14). Second, correlations between endogenous levels of the hormone and specific physiological processes are investigated. Third, the biosynthesis, distribution, or action of the hormone can often be manipulated by applying chemicals which interfere with these processes. However, these agents are seldom entirely specific and, hence, other metabolic pathways may be influ-'Supported through National Sciences and Engineering Research Council of Canada grants U0286 and A-2585 to S. B. R. and R. P. P. respectively. enced. For example, numerous growth retardant type plant growth regulators have been developed which block kaurene cyclization or other steps leading to GA biosynthesis (17). However, these also frequently block cyclization of other terpenoids and hence, alter levels of sterols and possibly other compounds (2). A more specific block of GA biosynthesis results from certain single gene mutations (6, 13, 16, 23). These mutations appear to influence only specific steps in GA biosynthesis. and hence complete recovery of the phenotype can result from the application of GA (15). These specific GA-deficient maize, pea, and rice dwarf mutants have been valuable for elucidating the pivotal role of endogenous GA, in the regulation of shoot elongation (6, 9, 13, 16, 23). Further, other pleiotropic effects of reduced GA level, such as altered influ-orescence sexuality in maize (21), are indicated. A clarification of the role(s) of endogenous GAs and specifically the C-3,13 dihydroxylated GAs, GA, and GA3, in other plants will similarly benefit from the identification and study of GA-deficient mutants. In the present paper, we describe a dwarf mutant of Brassica rapa (25, 26) which responds to exogenous GA3, and quantitatively compare the endogenous GAs of the mutant with those in a normal line using a combination of bioassay and GC-MS-SIM.2 MATERIALS AND METHODS Plant Material The study involved a normal, rapid-cycling line of Brassica rapa (syn campestris) (designation: CrGC 51 [formerly CrGC No.