Apple is an important crop and a focus of research worldwide. ... Key words: Apple, Malus Q domestica Borkh., development, shoot apical meristem, floral ...
Annals of Botany 92: 199±206, 2003 doi:10.1093/aob/mcg120, available online at www.aob.oupjournals.org
A Morphological and Quantitative Characterization of Early Floral Development in Apple (Malus 3 domestica Borkh.) T O S H I FO S T E R * , R O B Y N JO H N S T O N and A L L A S E L E Z N Y O V A The Horticulture and Food Research Institute of New Zealand Ltd, Palmerston North Research Centre, Private Bag 11 030, Palmerston North, New Zealand Received: 23 December 2002 Returned for revision: 13 March 2003 Accepted: 17 April 2003 Published electronically: 12 June 2003
Apple is an important crop and a focus of research worldwide. However, some aspects of ¯oral commitment and morphogenesis remain unclear. A detailed characterization of bourse shoot apex development was undertaken to provide a framework for future genetic, molecular and physiological studies. Eight morphologically distinct stages of shoot apex development, prior to winter dormancy, were de®ned. Based on measurements of meristem diameter, two stages of vegetative development were recognized. Vegetative meristems were ¯at, and either narrow (stage 0) or broad (stage 1). Pronounced doming of the apex marked stage 2. During stage 3, the domed meristem initiated four to six lateral ¯oral meristems and subtending bracts before converting to a terminal ¯oral meristem (stage 4). The terminal ¯oral meristem proceeded directly with bractlet and sepal initiation, while lateral ¯oral meristems initiated bractlets (stage 5). Sepal initiation began on the basal lateral ¯ower (stage 6) and continued in an acropetal direction until all ¯oral meristems had completed sepal initiation (stage 7). In this study, only stage 0 and stage 7 apices were observed in dormant buds, indicating that stages 1±6 are transient. The results suggest that broadening of the apex (stage 1) is the ®rst morphological sign of commitment to ¯owering. ã 2003 Annals of Botany Company Key words: Apple, Malus 3 domestica Borkh., development, shoot apical meristem, ¯oral morphogenesis, in¯orescence, scanning electron microscopy.
INTRODUCTION In the post-genomics era, it is increasingly simple to clone genes from non-model species. However, issues of what constitutes an orthologous gene, and what the exact functions of orthologous genes are, remain dif®cult. Comparing loss/gain of function phenotypes or gene expression patterns with those characterized in model species can provide useful information about the mechanisms of gene action, but only if there is a ®rm understanding of the developmental processes in the species being studied. In a landmark paper, Smyth and co-workers undertook a detailed characterization of ¯oral development in wild-type Arabidopsis thaliana, providing reference observations that de®ne 12 distinct developmental stages (Smyth et al., 1990). This work has provided a developmental context in which gene expression patterns and mutant phenotypes can be compared and interpreted in Arabidopsis. Despite the utility of this approach, many non-model systems lack comparable developmental analyses. One such example is apple (Malus 3 domestica Borkh.), a crop of worldwide horticultural importance, and a useful system to study ¯owering in woody angiosperms. Elucidating the genetic and environmental factors that control the transition to ¯owering and ¯oral development in apple is an active area of research. A number of genes that control the transition to reproductive development have been identi®ed and characterized in model species such as * For correspondence. Fax + 64 06 351 7031, e-mail tfoster@ hortresearch.co.nz
Arabidopsis and Antirrhinum (Mandel et al., 1992; Weigel et al., 1992; Blazquez et al., 1997; Bradley et al., 1997; Hempel et al., 1997). Apple orthologues of several of these genes have been isolated (Sung and An, 1997; Sung et al., 1999, 2000; Wada et al., 2002). However, the developmental context in which these genes act is still poorly understood because the published descriptions of ¯oral morphogenesis in apple are inadequate. For instance, a number of authors use the terms in¯orescence and ¯ower interchangeably, and most fail to describe the sequence of lateral ¯ower initiation (Pratt et al., 1959; Fulford, 1966b; Buban and Faust, 1982; Hirst and Ferree, 1995). In apple, ¯owers are produced on terminal in¯orescences (Fig. 1). New growth is initiated from one or more axillary meristem(s) proximal to the terminal in¯orescence (Crabbe and Escobedo Alvarez, 1991). During the growing season, these axillary meristems initiate a series of vegetative leaves, bud scales and leaf primordia that comprise the bourse shoot. A bourse shoot may either terminate in an in¯orescence (Fig. 1) or remain vegetative (Fulford, 1966a). Due to the horticultural importance of apple, much of the work on ¯owering has focused on the later stages of ¯oral morphogenesis and fruit development (for a review, see Pratt et al., 1988). There have been a number of studies on the timing of the transition from vegetative to ¯oral development, but much of the early literature focused on the numbers, type, and rate of lateral organ appearance rather than events occurring at the shoot apex itself (Fulford, 1965, 1966a, b). Doming of the apex has been identi®ed as an indication of the transition from vegetative to
Annals of Botany 92/2, ã Annals of Botany Company 2003; all rights reserved
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Foster et al. Ð Characterization of Floral Commitment in Apple
F I G . 1. Apple ¯oral bud before and after bud break. A, Schematic illustrating the organization of a winter dormant ¯oral bud. In the previous season, the terminal meristem initiated a series of vegetative leaves (not represented), then bud scales (curved lines), transition leaves (cross-hatched) and leaf primordia (®lled), before terminating in an in¯orescence. Subsequent vegetative growth is from a `bourse' shoot (circled) that develops from the axil of one of the vegetative primordia. B, Macroscopic image of ¯oral bud after bud break. Flowers and expanded vegetative leaves (VL) were initiated in the previous season; new leaves (L1±L3) are initiated by the bourse shoot (arrowhead) terminal meristem.
reproductive development, but it is unclear if this is truly the ®rst morphological sign of ¯oral commitment (Buban and Faust, 1982; Fulford, 1966b; Luckwill and Silva, 1979). Pratt et al. (1959) noted that a broadening of the apex precedes doming, although this observation was not quanti®ed. To date, there has been no comprehensive developmental analysis of the morphogenetic events between vegetative development and ¯oral organ initiation. In this paper, scanning electron micrographs and images of sectioned shoot apices are presented, illustrating the progression from vegetative to ¯oral development. Based on these, eight morphologically distinct stages of shoot apex development prior to winter dormancy are de®ned. We discuss these stages with reference to meristem identity. This detailed description provides morphological markers that are more indicative of the developmental state of the shoot apex than temporal references, such as dates, degree days, or days relative to blooming. Quantitative analyses of meristem diameter over time and transitions between meristem identity states are presented. These data provide quantitative support for the observations of Pratt et al. (1959) and suggest that broadening of the apex is the ®rst morphological sign of the transition to ¯owering. It is believed that this study will provide a framework for the interpretation of future molecular, physiological and developmental studies. MATERIALS AND METHODS Samples were collected from 15-year-old `Royal Gala'/ MM.106 trees growing at the HortResearch Lawn Road orchard near Havelock North, New Zealand. For each collection, approx. 50 bourse shoots (Fig. 1B) were removed from spurs (short shoots) carrying ¯owers/fruit. One bourse shoot per branch was collected from random positions on well-exposed branches. Two to three branches were sampled per tree, and 16±25 different trees were sampled at each time-point. A previous study of Royal Gala grown in the same geographical location found no correlation between
heat accumulation and the onset of ¯oral bud development (McArtney et al., 2001), thus sampling dates are presented as days after full bloom (DAFB). Full bloom is de®ned as the date when 80 % of the terminal (king) ¯owers on spurs are open. At 21 DAFB, long (>50 mm) and short (
