Lloyd T. Evans

Regulation of Flowering in the Long-Day Grass Lolium temulentum by Gibberellins and the FLOWERING LOCUS T Gene

Seasonal control of flowering often involves leaf sensing of daylength coupled to time measurement and generation and transport of florigenic signals to the shoot apex. We show that transmitted signals in the grass Lolium temulentum may include gibberellins (GAs) and the FLOWERING LOCUS T (FT) gene. Within 2 h of starting a florally inductive long day (LD), expression of a 20-oxidase GA biosynthetic gene increases in the leaf; its product, GA20, then increases 5.7-fold versus short day; its substrate, GA19, decreases equivalently; and a bioactive product, GA5, increases 4-fold. A link between flowering, LD, GAs, and GA biosynthesis is shown in three ways: (1) applied GA19 became florigenic on exposure to LD; (2) expression of LtGA20ox1, an important GA biosynthetic gene, increased in a florally effective LD involving incandescent lamps, but not with noninductive fluorescent lamps; and (3) paclobutrazol, an inhibitor of an early step of GA biosynthesis, blocked flowering, but only if applied before the LD. Expression studies of a 2-oxidase catabolic gene showed no changes favoring a GA increase. Thus, the early LD increase in leaf GA5 biosynthesis, coupled with subsequent doubling in GA5 content at the shoot apex, provides a substantial trail of evidence for GA5 as a LD florigen. LD signaling may also involve transport of FT mRNA or protein because expression of LtFT and LtCONSTANS increased rapidly, substantially (>80-fold for FT), and independently of GA. However, because a LD from fluorescent lamps induced LtFT expression but not flowering, the nature of the light response of FT requires clarification.

Gibberellin structure and florigenic activity in Lolium temulentum, a long day plant.

Structural requirements for florigenic activity among gibberellins (GAs) and GA derivatives, including several new ones, applied once to leaves of Lolium temulentum, were examined. The compounds were applied to plants kept either in non-inductive short days (SD) or exposed to one inductive long day (LD). Inflorescence initiation and stem-elongation responses were assessed three weeks later. Among the GAs used, the range in effective dose for inflorescence initiation was more than 1000-fold, but substantially less for stem elongation. Some GAs promoted both stem elongation and inflorescence initiation, some promoted one without the other, and some affected neither. The structural features enhancing florigenic activity were often different from those enhancing stem elongation. Except in the case of 2,2-dimethyl GA4, a double bond in the A ring at either C-1,2 or C-2,3 was essential for high florigenic activity, though not for stem elongation. A free carboxy group was needed for both. Inflorescence initiation in Lolium was enhanced by hydroxylation at C-12, −13 and −15, whereas hydroxylation at C-3 reduced the effect on inflorescence initiation but increased that on stem elongation. A 12β-hydroxyl was more effective than the α epimer for inflorescence initiation whereas the reverse was true for stem elongation. Although such differential effectiveness of GAs for inflorescence initiation and for stem elongation could reflect differences in uptake, transport or metabolism, we suggest that it is indicative of specific structural requirements for inflorescence initiation.

Floral determination and in-vitro floral differentiation in isolated shoot apices of Lolium temulentum L.

Shoot apices of Lolium temulentum excised after exposure of the plants to one long day (LD) undergo floral development in vitro, whereas those excised from plants in short days (SD) remain vegetative. Floral differentiation in vitro is reasonably normal and responds quantitatively to the preceding LD induction when three conditions are met. First, excision must not precede the arrival at the apex of the LD stimulus from the leaves. This appears to begin about 22 h after the start of the LD and is completed after a further 14 h, by which time all isolated apices have become capable of initiating inflorescence differentiation, i.e. florally determined. Second, for apices excised on the day after the LD (Day II) or early on Day III, the presence of gibberellic acid in the medium is required for floral differentiation to occur in most explants. By contrast, neither kinetin (N6-furfurylaminopurine) nor indole-3-acetic acid is required or beneficial, while abscisic acid in the medium is inhibitory to both survival and floral differentiation in excised apices. The third requirement is for an adequate supply of sugar, particularly after floral differentiation begins. Sucrose is taken up rapidly to reach high levels in the excised apices, but high sugar concentration in the medium, either alone or with gibberellic acid, does not suffice for floral differentiation to begin, and there is an absolute requirement for receipt of the LD stimulus.

The relative significance for stem elongation and flowering in Lolium temulentum of 3β-hydroxylation of gibberellins

In previous experiments with many gibberellins (GAs) and GA derivatives applied to Lolium temulentum L., quite different structural requirements were evident for stem elongation on the one hand and for the promotion of flowering on the other. Whereas hydroxylation at carbons 12, 13 and 15 enhanced flowering relative to stem growth, the reverse was the case at carbon 3 (L.T. Evans et al. 1990, Planta 182, 97–106). The significance of hydroxylation at carbon 3 is examined in this paper. The application of inhibitors of 3β-hydroxylation, including C/D-ring-rearranged GAs, reduced stem growth but, in the case of the two acylcyclohexanediones, increased the flowering response when applied on the inductive long day. Later applications of the acylcyclohexanediones, made after floral initiation had occurred, were inhibitory to flowering, suggesting that subsequent inflorescence development requires 3β-hydroxylated GAs. Applications of the 3α-hydroxy epimers of GA1, GA3 and GA4 gave slightly less promotion of flowering in comparison with the 3β-hydroxy GAs, but far less promotion of stem elongation, except in the case of 3-epi-GA4, which was comparable to GA4. The 3α-hydroxy epimer of 2,2-dimethyl GA4 gave less promotion of flowering than its 3β-hydroxy epimer but almost no promotion of stem elongation. The 3α-hydroxy epimers of GA3 and 2,2-dimethyl GA4 did not act as competitive inhibitors of the stem elongation elicited by GA3 and 2,2-dimethyl GA4, respectively. These results extend the differences in GA structure which favour flowering as opposed to stem elongation, and indicate that 3-hydroxylation and its epimeric configuration are of much greater importance to stem elongation than to flower initiation in Lolium.

Synthesis of gibberellin GA6 and its role in flowering of Lolium temulentum.

The induction of flowering by one long day (LD) in the grass Lolium temulentum is most closely mimicked by application of the gibberellins (GAs) GA(5) or GA(6), both of which occur naturally. These gibberellins promote floral development but have little effect on stem elongation. Endogenous GA(5) and GA(6) contents in the shoot apex double on the day after the LD and, for GA(5) (and we presume for GA(6) as well) reach a concentration known to be inductive for the excised shoot apex in vitro. They are, therefore, strong candidates as LD floral stimuli in this grass. The synthesis of GA(6) and an examination of its florigenic properties in L. temulentum are described.

The differential effects of C-16,17-dihydro gibberellins and related compounds on stem elongation and flowering in Lolium temulentum

Plants of Lolium temulentum L. cv. Ceres grown under short days (SDs) can be induced to initiate inflorescences either by exposure to one long day (LD) or by single applications of some gibberellins (GAs), which also enhance the flowering response to one LD. Single doses of up to 25 μg per plant of C-16, 17-dihydro-GA5 were about as effective as GA5 for promoting flowering after one LD but inhibited stem elongation by up to 40% over three weeks. The promotion of flowering but not the inhibition of elongation by 16, 17-dihydro-GA5 was reduced in SDs or in LDs low in far-red (FR) radiation. With shoot apices cultured in vitro, 16, 17-dihydro-GA5 was more florigenic than GA3 for apices excised after one LD of 14 h or more, but less florigenic for apices excised from plants in shorter days. 16, 17-Dihydro-GA5 was ineffective compared with GA1, GA3 and GA5 for α-amylase production by half-seeds of Lolium, a response concordant with its effect on stem elongation. As with GA5, 16, 17-dihydro derivatives of GA1, GA3, GA20 and several other GAs were more effective for flowering and less effective for stem elongation than the GAs from which they were derived. Hydroxylation at C-17 and/or C-16 generally reduced the effectiveness of 16, 17-dihydro-GA5 for flowering. These results extend the known features of GA structure which favour flowering relative to stem elongation in L. temulentum. Moreover, C-16, 17-dihydro-GA5 mimics, in its daylength- and wavelength-dependence and lack of stem elongation, characteristics of the LD stimulus in L. temulentum.

Selective Deactivation of Gibberellins below the Shoot Apex is Critical to Flowering but Not to Stem Elongation of Lolium

Gibberellins (GAs) cause dramatic increases in plant height and a genetic block in the synthesis of GA(1) explains the dwarfing of Mendels pea. For flowering, it is GA(5) which is important in the long-day (LD) responsive grass, Lolium. As we show here, GA(1) and GA(4) are restricted in their effectiveness for flowering because they are deactivated by C-2 hydroxylation below the shoot apex. In contrast, GA(5) is effective because of its structural protection at C-2. Excised vegetative shoot tips rapidly degrade [14C]GA(1), [14C]GA(4), and [14C]GA(20) (>80% in 6 h), but not [14C]GA(5). Coincidentally, genes encoding two 2beta-oxidases and a putative 16-17-epoxidase were most expressed just below the shoot apex (<3 mm). Further down the immature stem (>4 mm), expression of these GA deactivation genes is reduced, so allowing GA(1) and GA(4) to promote sub-apical stem elongation. Subsequently, GA degradation declines in florally induced shoot tips and these GAs can become active for floral development. Structural changes which stabilize GA(4) confirm the link between florigenicity and restricted GA 2beta-hydroxylation (e.g. 2alpha-hydroxylation and C-2 di-methylation). Additionally, a 2-oxidase inhibitor (Trinexapac Ethyl) enhanced the activity of applied GA(4), as did limiting C-16,17 epoxidation in 16,17-dihydro GAs or after C-13 hydroxylation. Overall, deactivation of GA(1) and GA(4) just below the shoot apex effectively restricts their florigenicity in Lolium and, conversely, with GA(5), C-2 and C-13 protection against deactivation allows its high florigenicity. Speculatively, such differences in GA access to the shoot apex of grasses may be important for separating floral induction from inflorescence emergence and thus could influence their survival under conditions of herbivore predation.

Effects of D-ring modified gibberellins on flowering and growth in Lolium temulentum

Abstract The modified gibberellins, 16-methyl-16,17-dihydro-GA 5 , 16,17-methano-16,17-dihydro-GA 5 , 16,17-methano-16,17-dihydro-GA 3 and several halogenated derivatives of 16,17-methano-16,17-dihydro-GA 3 and GA 5 were prepared and tested for bioregulating properties in Lolium temulentum . The effects ranged from the promotion of both stem elongation and flowering, the promotion of flowering but not elongation, and vice versa , the inhibition of stem elongation combined with the promotion of flowering, and the inhibition of both.

Shifts in the semidian rhythm of flowering do determine night-break timing and critical night length in Pharbitis nil

Abstract.There is a semidian (≈12 h) rhythm in the flowering response of the short-day plant Pharbitis nil Choisy following 90 min exposure to either far-red light/darkness or a temperature drop (27 °C to 12 °C) given at various times in constant conditions before an inductive dark period. This semidian rhythmic response to the temperature-drop pretreatments in the light is also evident through the inductive dark period without change of phase. Furthermore, those pretreatments which increase flowering also advance the time of maximum sensitivity to red light (R) interruptions of the dark period by up to 1.5 h and shorten the critical night length. Conversely, pretreatments which reduce flowering delay the time of maximum R inhibition by up to 1.5 h and increase the critical night length by the same amount. However the phase of a circadian rhythm of flowering response had no effect on either the time of maximum R inhibition or the critical night length. Thus, the semidian rhythm determines both the time of maximum R inhibition and the critical night length in Pharbitis.