Identification of endogenous gibberellins, and metabolism of tritiated and deuterated GA4, GA9 and GA20, in Scots pine (Pinus sylvestris) shoots

Abstract

The application of gibberellin A4/7 (GA4/7) to the stem of previous‐year (1‐year‐old) terminal shoots of Scots pine (Pinus sylvestris) seedlings has been observed to stimulate cambial growth locally, as well as at a distance in the distal current‐year terminal shoot, but the distribution and metabolic fate of the applied GA4/7, as well as the pathway of endogenous GA biosynthesis in this species, has not been investigated. As a first step, we analysed for endogenous GAs and monitored the transport and metabolism of labelled GAs 4, 9 and 20. Endogenous GAs from the elongating current‐year terminal shoot of 2‐year‐old seedlings were purified by column chromatography and high‐performance liquid chromatography and analysed by combined gas chromatography‐mass spectrometry (GC‐MS). GAs 1, 3, 4, 9, 12 and 20 were identified in the stem, and GAs 1, 3 and 4 in the needles, by full‐scan mass spectrometry (GAs 1, 3, 4, 9 and 12) or selected‐ion monitoring (GA20) and Kovats retention index. Tritiated and deuterated GA4, GA9 or GA20 were applied around the circumference at the midpoint of the previous‐year terminal shoot, and metabolites were extracted from the elongating current‐year terminal shoot, the application point, and the 1‐year‐old needles and the cambial region above and below the application point. After purification, detection by liquid scintillation spectrometry and analysis by GC‐MS, it was evident that, for each applied GA, unmetabolised [2H2]GA and [3H]radioactivity were present in every seedling part analysed. Most of the radioactivity was retained at the application point when [3H]GA9 and [3H]GA20 were applied, whereas the largest percentage of radioactivity derived from [3H]GA4 was recovered in the current‐year terminal shoot. It was also found that [2H2]GA9 was converted to [2H2]GA20 and to both [2H2]GA4 and [2H2]GA1, [2H2]GA4 was metabolised to [2H2]GA1, and [2H2]GA20 was converted to [2H2]GA29. The data indicate that for Pinus sylvestris shoots (1) GAs applied laterally to the outside of the vascular system of previous‐year shoots not only are absorbed and translocated extensively throughout the previous‐year and current‐year shoots, but also are readily metabolised, (2) the GA metabolic pathways found are closely related to the endogenous GAs identified, and (3) GA9 metabolism follows two distinctly different routes: in one, GA9 is converted to GA1 through GA4, and in the other it is converted to GA20, which is then metabolised to GA29. The results suggest that the late 13‐hydroxylation pathway is an important route for GA biosynthesis in shoots of Pinus sylvestris, and that the stimulation of cambial growth in Scots pine by exogenous GA4/7 may be due to its conversion to GA1, rather than to it being active per se.