Growth and development of Brassica genotypes differing in endogenous gibberellin content. II. Gibberellin content, growth analyses and cell size

Three rapid cycling Brassica rapa genotypes were grown in greenhouse conditions to investigate the possible relationships between endogenous gibberellin (GA) content and shoot growth. Endogenous GA₁ GA₃ and GA₂₀ were extracted from stem samples harvested at 3 weekly intervals and analyzed by gas chromatography-mass spectrometry with selected ion monitoring, using [²H₂]-GA₁ and [²H₂]-GA₂₀ as quantitative internal standards. During the first 2 weeks, GA levels of the dwarf, rosette ( ros ), averaged 36% of levels in normal plants (on a per stem basis). Levels in the tall mutant, elongated internode (ein) , were consistently higher, averaging 305% of levels in normal plants.
Differences in shoot height across the genotypes resulted from varying internode length which resulted from epidermal cell length and number being increased in ein and decreased in ros relative to the normal genotype. The exogenous application of GA₃ to normal plants increased cell length while the application of paclobutrazol (PP333), a triazole plant growth retardant, reduced cell size. Thus, exogenous GA manipulations mimicked the influence of the mutant genes ros and ein. The dwarf, ros , had reduced shoot dry weights and relative growth rates compared to the other genotypes. Total dry weights were similar in ein and the normal genotype but stem weights were increased in ein , compensating for decreased leaf weights. Thus, the gibberellin-deficiency of ros resulted in generally reduced shoot growth. The overproduction of endogenous GA by ein did not result in enhanced shoot growth but rather a specific enhancement of internode elongation and stem growth at the expense of leaf size.     

A Gibberellin-Deficient Brassica Mutant-rosette

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 internode 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 internal standard. Fourteen-day-old rosette and normal seedlings contained 5.3 and 23.2 ng GA₁ 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.     

Floral transition and nitric oxide emission during flower development in Arabidopsis thaliana is affected in nitrate reductase-deficient plants

The nitrate reductase (NR)-defective double mutant of Arabidopsis thaliana (nia1 nia2) has previously been shown to present a low endogenous content of NO in its leaves compared with the wild-type plants. In the present study, we analyzed the effect of NR mutation on floral induction and development of A. thaliana, as NO was recently described as one of the signals involved in the flowering process. The NO fluorescent probes diaminofluorescein-2 diacetate (DAF-2DA) and 1,2-diaminoanthraquinone (1,2-DAA) were used to localize NO production in situ by fluorescence microscopy in the floral structures of A. thaliana during floral development. Data were validated by incubating the intact tissues with DAF-2 and quantifying the DAF-2 triazole by fluorescence spectrometry. The results showed that NO is synthesized in specific cells and tissues in the floral structure and its production increases with floral development until anthesis. In the gynoecium, NO synthesis occurs only in differentiated stigmatic papillae of the floral bud, and, in the stamen, only anthers that are producing pollen grains synthesize NO. Sepals and petals do not show NO production. NR-deficient plants emitted less NO, although they showed the same pattern of NO emission in their floral organs. This mutant blossomed precociously when compared with wild-type plants, as measured by the increased caulinar/rosette leaf number and the decrease in the number of days to bolting and anthesis, and this phenotype seems to result from the markedly reduced NO levels in roots and leaves during vegetative growth. Overall, the results reveal a role for NR in the flowering process.     

Selection and Characterization of a Gibberellin-Deficient Mutant of Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for reproductive development. Previous work in this laboratory has demonstrated that the bolting aspect (rapid stem growth) of reproductive development is mediated by gibberellins (GA). The present paper describes the selection and characterization of a mutant lacking the capacity for thermoinduced stem growth. Seeds of an inbred field pennycress line (CR₁) were treated with the chemical mutagen ethyl methane sulfonate, germinated, and allowed to produce seed. Plants derived from these seeds were screened for reduced stem growth. A mutant line, EMS-141, in which the lack of stem growth can be fully overcome with exogenous GA₃, was selected for further analysis. Other phenotypic abnormalities exhibited by the mutant line include reduced petiole growth, slightly delayed floral initiation, and failure of flowers to develop fully. These are also reversed with exogenous GA₃. Evidence is presented indicating that all of the alterations in growth and development exhibited by EMS-141 are conferred by a recessive mutation of a single nuclear gene. Through quantitative analysis of endogenous GA and GA precursors and a comparison of the abilities of various compounds to restore normal growth when applied to plants of EMS-141, the physiological basis for the mutant phenotype was determined to be the result of highly reduced endogenous GA levels. Moreover, the affected site in GA biosynthesis appears to be the accumulation of ent-kaurene, probably at the level of ent-kaurene synthase. The relative abilities of exogenous GA and GA precursors to restore normal growth of petioles and stems are compared, and the results are used to make inferences on the functions of the two different pathways of GA metabolism that exist in field pennycress.     

Effects of Inhibitors of Protein Serine/Threonine Phosphatases on Pollination in Brassica

We have examined the effect of the protein phosphatase inhibitors okadaic acid and microcystin on pollen-pistil interactions in Brassica. Inhibitor-treated flowers or floral buds were pollinated with untreated pollen and examined for pollen tube growth by fluorescence microscopy. Our results show that type 1 or type 2A serine/threonine phosphatases play a crucial role in the pollination responses of Brassica. We observed two distinct effects of protein phosphatase inhibitors on pollination: (a) the inhibition of pollen tube growth during cross-pollination in flowers, and (b) the break-down of self-incompatibility or promotion of pollen tube growth during self-pollination in flower buds just prior to anthesis. Thus, treatment of flower pistils with protein phosphatase inhibitors resulted in the inhibition of pollen tube growth at the surface of the papillar cells of the stigma in crosses between different self-incompatible Brassica oleracea strains, in an interspecific cross between B. oleracea and Brassica campestris, and in self-pollinations of a self-fertile Brassica napus cultivar. With four different self-incompatibility genotypes, treatment of mature flowers with protein phosphatase inhibitors had no effect on self-pollination response. In contrast, treatment of flower buds just prior to the anthesis stage allowed self-pollen tube invasion of papillar cells. However, the magnitude of this effect was genotype dependent, being most pronounced in the S22 genotype. The data support the conclusion that pollinations in Brassica are controlled in part by the presence of phosphorylated proteins in the papillar cells of the stigma, and that the quantity of these proteins or their levels of phosphorylation changes during stigma development.     

Characterisation of the procera mutant of tomato and the interaction of gibberellins with end-of-day far-red light treatments

The tomato ( Solanum lycopersicum L.) slender mutant procera ( pro ) was analysed for its relationship with gibberellin (GA) by combining it with GA deficiency due to the gib-1 mutation. The sensitivity to GA biosynthesis inhibitors and the GA content were measured in the pro gib-1 double mutant. In the gib-1 mutant background, the pro mutation strongly reduced the GA requirement for seed germination and stem growth and almost fully restored the morphological leaf defects of the gib-1 mutant. An end-of-day far-red light treatment, when applied to the various genotypes, indicated that GAs are required for a response to this treatment, but that it act independently of the Pro gene product.     

Gibberellins and seed development in maize. I. Evidence that gibberellin/abscisic acid balance governs germination versus maturation pathways

Abscisic acid (ABA) is required for the regulation of seed maturation in maize (Zea mays L.). Mutants blocked in ABA synthesis (such as viviparous-5) do not mature to quiescent, desiccation-tolerant seeds, but germinate on the ear midway through kernel development. Because gibberellins (GA) and ABA act antagonistically in many aspects of plant development, we hypothesized that ABA antagonizes a positive GA signal for precocious germination in maize. In these experiments, we show that a GA deficiency early in seed development, induced genetically or via biosynthesis inhibitors, suppresses vivipary in ABA-deficient developing kernels. The resulting seeds have both desiccation tolerance and storage longevity. Temporal analysis of GA accumulation in wild-type kernels revealed the accumulation of bioactive GA(1) and GA(3) prior to the peak in ABA content. We speculate that these GAs stimulate a developmental program leading to vivipary in the absence of normal amounts of ABA, and that a reduction of GA content re-establishes an ABA/GA ratio appropriate for suppression of germination and induction of maturation. In contrast, the induction of a GA deficiency did not suppress vivipary in viviparous-1 mutant kernels, suggesting that VP1 acts downstream of both GA and ABA in programming seed development.     

Photoperiod and temperature effects on in vitro growth and flowering of P. pusilla, an epiphytic orchid.

Psygmorchis pusilla Dodson and Dressler, an epiphytic orchid, has been shown to be an interesting model to study in vitro flower formation. In the present study, the effects of photoperiod and temperature on vegetative and reproductive development were investigated. Although photoperiod had limited effects on leaf number, an etiolating process was verified in darkness and a higher growth was detected under long days. A positive relationship was observed between long days and floral spike formation. However, plant incubation under 20 h photoperiod or longer days negatively affected floral bud development, inhibiting anthesis and reducing flower longevity. Higher soluble sugar and starch levels were detected in plants cultivated under long days, while chlorophyll and carotenoids contents were negatively affected under these conditions. Plants showed great sensitivity to temperature variations; 27 degrees C being the most adequate for growth, leaf and floral spike formation. Temperatures of 22 and 32 degrees C were not appropriate for in vitro development of P. pusilla.     

High temperature arrest of inflorescence development in broccoli (Brassica oleracea var. italica L.)

High temperature causes unevenly-sized flower buds on broccoli inflorescences. This deformity limits production of broccoli to areas where summer temperatures rarely exceed 30 C. The stage of development sensitive to heat was determined by exposing plants of 'Galaxy' broccoli at varying developmental states to 35 C day temperature for 1 week, and subsequently analysing the head structure. During the high temperature exposure, the development of certain flower buds was arrested. There was no corresponding cessation of bud initiation at the apex. No injury resulted if heat was applied before the reproductive induction, or was their injury to differentiated flower buds. Meristems were affected only if heat was applied during inflorescence production or the floral initiation process. Shorter heat exposures produced little injury, and longer exposures were lethal. The plant's development at this sensitive period still appeared vegetative externally, but the youngest leaves had just begun to reorientate as a consequence of the reduced stem elongation rate. The meristem was less than 1 mm wide, and floral primordia were just forming, still subtended by leaf primordia. The injury was fully expressed by the time the head was first exposed (approximately 5-10 mm wide), though it became more apparent as the head matured. The buds that were delayed in development by the high temperature developed into normal flowers.Key words: Brassica oleracea, broccoli, flowering, heat injury, developmental arrest      

The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo.     

Genetic Regulation of Development in Sorghum bicolar: I. Role of the Maturity Genes

The photoperiodic behavior and other developmental and morphological differences of 11 maturity genotypes (as identified by JR Quinby 1967, Adv Agron 19: 267-305) of the milo group of Sorghum bicolor (L.) Moench were studied under 8, 10, 12, and 14 hour photoperiods. Sorghum is a quantitative short day plant. The genotypes studied differ in genes which modify photoperiodic behavior and thus maturity; the alleles are designated as Ma(1), ma(1), Ma(2), ma(2), Ma(3), ma(3), and ma(3) (R) (single symbols indicate homozygosity at the indicated gene loci). Based on floral initiation (differentiation) under 10, 12, and 14 hour photoperiods the 11 genotypes were assigned to three clases: (I) flower initiation delayed by 12 hour photoperiods (all genotypes with Ma(1)Ma(2) but not ma(3) (R)), (II) flower initiation delayed by 14 hour photoperiods (all genotypes with Ma(1)ma(2), ma(1)Ma(2), or ma(1)ma(2) but not ma(3) (R)), (III) flower initiation not drastically delayed by 14 hour photoperiods (all genotypes with ma(3) (R)). All of the class III genotypes were taller, had longer leaf sheaths, narrower and longer leaf blades, and less leaf area, than the other genotypes. In addition, the class III genotypes initiated rapid culm and thus internode elongation sooner after floral initiation than any of the class I or II genotypes. Dry weight did not differ between the class III genotypes and the others. The rate of leaf emergence in the class III genotypes and all others was indistinguishable until after floral initiation in the former. The allelic combination unique to class I, Ma(1)Ma(2), makes plants very photoperiod sensitive without causing observable changes in morphology or other development events. The allelic combination unique to class III, ma(3) (R), makes plants relatively photoperiod insensitive and results in several differences in morphology and development.     

The rice FON1 gene controls vegetative and reproductive development by regulating shoot apical meristem size

Most plant organs develop from meristems. Rice FON1, which is an ortholog of Clv1, regulates stem cell proliferation and organ initiation. The point muta-tions, fon1-1 and fon1-2, disrupt meristem balance, resulting in alteration of floral organ numbers and the architecture of primary rachis branches. In this study, we identified two knockout alleles, fon1-3 and fon1-4, generated by T-DNA and Tos17 insertion, respectively. Unlike the previously isolated point mutants, the null mutants have alterations not only of the reproductive organs but also of vegetative tissues, producing fewer tillers and secondary rachis branches. The mutant plants are semi-dwarfs due to delayed leaf emergence, and leaf senescence is delayed. SEM analysis showed that the shoot apical meristems of fon1-3 mutants are enlarged. These results indicate that FON1 controls vegetative as well as reproductive development by regulating meristem size.     

A petunia MADS box gene involved in the transition from vegetative to reproductive development.

We have identified a novel petunia MADS box gene, PETUNIA FLOWERING GENE (PFG), which is involved in the transition from vegetative to reproductive development. PFG is expressed in the entire plant except stamens, roots and seedlings. Highest expression levels of PFG are found in vegetative and inflorescence meristems. Inhibition of PFG expression in transgenic plants, using a cosuppression strategy, resulted in a unique nonflowering phenotype. Homozygous pfg cosuppression plants are blocked in the formation of inflorescences and maintain vegetative growth. In these mutants, the expression of both PFG and the MADS box gene FLORAL BINDING PROTEIN26 (FBP26), the putative petunia homolog of SQUAMOSA from Antirrhinum, are down-regulated. In hemizygous pfg cosuppression plants initially a few flowers are formed, after which the meristem reverts to the vegetative phase. This reverted phenotype suggests that PFG, besides being required for floral transition, is also required to maintain the reproductive identity after this transition. The position of PFG in the hierarchy of genes controlling floral meristem development was investigated using a double mutant of the floral meristem identity mutant aberrant leaf and flower (alf) and the pfg cosuppression mutant. This analysis revealed that the pfg cosuppression phenotype is epistatic to the alf mutant phenotype, indicating that PFG acts early in the transition to flowering. These results suggest that the petunia MADS box gene, PFG, functions as an inflorescence meristem identity gene required for the transition of the vegetative shoot apex to the reproductive phase and the maintenance of reproductive identity.     

Flowering in the uniflora mutant of tomato (Lycopersicon esculentum Mill.): description of the reproductive structure and manipulation of flowering time

The uniflora (uf) mutant of tomato (Lycopersicon esculentum Mill.) is known to produce solitary, normal, fertile flowers instead of inflorescences. Histological and SEM studies revealed that this unusual reproductive structure resulted from the inability of the plant to produce an inflorescence and not from post-initiation abortion processes affecting young flower buds. Development prior to floral transition was apparently not affected by the mutation since rates of germination and leaf initiation were identical in both uf and the Ailsa Craig (AC) initial cultivar. However, the time of flowering of the mutant was always delayed as compared to AC. In uf, environmental conditions markedly influenced flowering time which occurred early in all individuals in summer, but was strongly delayed during winter, with less than 20% plants reaching flowering before having initiated 40 leaves. Defoliation treatments stimulated floral transition in uf plants since 100% flowering occurred whatever the season and since the time of floral transition was usually advanced in comparison to the non-defoliated control plants. Similarly, compared to intact uf plants, flowering of terminal meristem of cuttings and upper axillary bud of decapitated plants was promoted. The involvement of correlative influences and assimilate availability in the control of flowering in tomato is suggested by these findings.     

Effects of Ring D-Modified Gibberellins on Gibberellin Levels and Development in Selected Sorghum bicolor Maturity Genotypes

Plants of early flowering mutant and wild type genotypes of Sorghum bicolor were treated with ring D-modified gibberellins (GAs), and the effects on endogenous GA levels were determined. The growth and timing of floral initiation in 58M plants grown under 18-h days (which significantly delays floral initiation in this short day plant) following treatment with these compounds, relative to GA₃ and GA₅ treatments, were also investigated. Application of the endo-isomer of C16,17-dihydro-GA₅ (endo-DiHGA₅), the exo-isomer of C16,17-dihydro-GA₅ (exo-DiHGA₅), and C16α,17-dichloromethanodihydro-GA₅ (DMDGA₅) altered GA levels in both genotypes. Each ring D-modified GA significantly inhibited shoot growth while significantly decreasing levels of GA₁ and increasing levels of its immediate precursor, GA₂₀. Gibberellin A₈ levels also decreased. Tillering was not affected by any treatment. For the early flowering genotype 58M, grown under noninductive long days, both dihydro-GA₅ isomers promoted floral initiation while shoot growth was strongly inhibited, and floral development was strongly advanced beyond floral stage 4. Gibberellin A₃ and GA₅, applied under the same conditions, promoted shoot growth slightly and gave ``floral-like' apical meristems that did not develop past floral stage 1. These results suggest that the reduced shoot growth of sorghum, which follows application of those ring D-modified GAs, is due to their inhibiting the 3β hydroxylation of GA₂₀ to GA₁, thereby reducing the GA₁ content. That floral initiation was hastened and floral development promoted in genotype 58M by application of both isomers of DiHGA₅ are in contrast to the effects of other GA biosynthesis inhibitors, which act earlier in the GA biosynthesis pathway, but are consistent with results seen for long day grasses. This suggests that endo-DiHGA₅ and exo-DiHGA₅ may be acting directly in promoting floral initiation and subsequent floral apex development of this short day plant under long day conditions. Received October 3, 1996; accepted January 22, 1997     

Plant development from microspore-derived embryos in oilseed rape as affected by chilling, desiccation and cotyledon excision

The present study evaluated the effects of chilling, partial desiccation, cotyledon excision and successive subculture of microspore-derived embryos on plant development in oilseed rape (Brassica napus L.). The results showed that out of the five media, all the genotypes showed the best response when the embryos were cultured on the half-strength Murashige and Skoog medium with 2.0 mg dm⁻³ benzylaminopurine. A cold treatment for 3 or 5 d further increased frequencies of embryo germination (90.0 %) and plantlet development (58.46 %). Desiccation for one day also increased the embryo germination and plantlet development in all genotypes tested. Cutting the cotyledons of the embryos at late cotyledonary stage significantly increased the frequency of plantlet development. The highest rate of plantlet development was obtained from cultures of embryos sampled with size of less than 4.0 mm. The successive subculture further improved the germination and development of plantlets from embryos. In the genotype ZJU452, the rate of plantlet development reached 99.78 % after the second subculture of embryos.