Identification ofGibberellins inSpinach andEffects of Light andDarkness on their Levels1

Theendogenous gibberellin (GA)content ofspinach (Spinacia oleracea) wasreinvestigated bycombined gaschromatographymassspectrometry analysis. The13-hydroxy GAs:GAw3, GA44, GA19, GA17, GA2o, GA5, GA1,GA29, andGA$; thenon-3,13-hydroxy GAs:GA12, GA15, GA,,andGA51; andthe3#-hydroxy GAs:GA4, GA7,andGA34, wereidentified inspinach extracts bycomparing full-scan massspectra andKovats retention indices withthose ofreference GAs.Inaddition, spinach plants contained GA7isolactone, 16,17-dihydro-17-hydroxy-GAw, GA29-catabolite, 3epi-GA1, and10uncharacterized GAswithmassspectra indicative ofmono-anddihydroxy-GA12, monohydroxy-GA25, dihydroxyGA24, anddihydroxy-GA9. Theeffect oflight-dark conditions on theGA levels ofthe13-hydroxylation pathway wasstudied by using labeled intemal standards inselected ionmonitoring mode. Inshort day,theGA levels werehigher attheendofthelight period thanattheendofthedarkperiod. Levels ofGAsatthe endofeachshort daywererelatively constant. During thefirst supplementary light period oflongdaytreatment, GA,1andGA19 declined dramatically, GA." andGA1decreased slightly, andGA2o increased. During thesubsequent high-intensity light period, the GA2o level decreased andthelevels ofGA53, GA",GA19, andGA1 increased slightly. Within 7daysafter thebeginning oflongday treatment, similar patterns forGAs3andGA19occurred. Furthermore,whentheseplants weretransferred todarkness, anincrease inthelevels ofGA,3 andGA1,9 wasobserved. Theseresults arecompatible withtheideathat inspinach, theflowthrough the GA biosynthetic pathway ismuchenhanced during thehighintensity light period, although GA tumoveroccursalsoduring thesupplementary period oflongday, botheffects being responsible fortheincrease ofGA29 andGA1inlongday. under noninductive photoperiods stimulated stemelongation, whereas inhibitors ofGA biosynthesis arrested growth induced byLDconditions (24). Later, six members oftheearly13-hydroxylation pathway, GA53,GA44, GA19, GA17, GA20, andGA29, wereidentified asendogenous GAsinspinach shoots (16). Itwasalso shownthat, under LD conditions, levels ofGA19decreased, whereas levels ofbothGA20and GA29increased. Thisobservation ledtothesuggestion that thephotoperiod regulates theconversion ofGA19toGA20 (17). Furthermore, invivo feeding experiments with[2H]GA53 (8)and['4C]GA19 (25) indicated that these GAswereonly metabolized toGA20whenplants werekeptincontinuous light. Results obtained withcell-free extracts fromspinach leaves confirmed that theoxidations ofbothGA53toGA44, andofGA19toGA20areunderphotoperiodic control (9). Thus, LDregulates GAmetabolism bymodulating theactivityoftwoenzymes oftheGA biosynthetic pathway, viz. GA53- andGA19-oxidases. These enzymeactivities areonly present inplants growing inlight, which enhances thelevels