Jinxin WANG, Jingmiao HUANG, Jianming LI, Jie HAO, Xueying LI

Shijiazhuang Pomology Institute, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050061, China

Abstract Flowering is a prerequisite for apple fruiting, and apple flower buds are mixed buds, that is, the vegetative organs and flower structure exist in the same terminal bud simultaneously, which are formed in the year before flowering and fruiting, mainly including spur terminal buds and axillary buds. The infrequent formation of flower buds during its growth and biennial bearing are closely related to flower bud differentiation. Therefore, this paper reviews the research progress of flower bud differentiation of apple from the morphological differentiation, plant hormones and flowering-related genes, in order to provide a theoretical reference for efficient cultivation and stable yield of apple.

Key words Apple, Flower bud differentiation, Hormone, Gene

1 Introduction

Apple (MalusdomesticaBorkh.) is an important fruit tree resource in the world, and the planting area and yield of apple in China rank first in the world[1]. The flower buds of apple are mixed buds, that is, the vegetative organs and flower structure exist in the same terminal bud simultaneously, which are formed in the year before flowering and fruiting, mainly including spur terminal buds and axillary buds[2]. The number of flower bud formation, the quality of flower buds and the time of flowering seriously affect the economic benefits of apple. For apple, there are some problems in the cultivation and production, such as difficulties in flower bud formation, biennial bearing and long juvenile period, which are closely related to flower bud differentiation. Therefore, this paper reviews the research progress of flower bud differentiation of apple from the morphological differentiation, plant hormones and flowering-related genes, in order to provide a theoretical reference for efficient cultivation and stable yield of apple.

2 Flower bud morphological differentiation of apple

Flower bud differentiation is completed through three stages of flower induction (flowering determination), flower bud initiation and flower bud development. Flower induction stage, also known as physiological differentiation stage of flower buds, is a critical period for regulating flower bud differentiation. Leaf picking (or ring stripping plus leaf picking) and fruit picking are the main research methods[3-4]. This period is closely related to the time when branches stop growing, and apple trees enter physiological differentiation at that time. The time required for physiological differentiation of the same variety in the same region is relatively stable in different years. In Shunping area of Baoding, it takes about 40 d for ‘Tianhong 2’ apple from growth cessation of short branches to the end of physiological differentiation[5], and there are significant differences in time from growth cessation of short branches to the end of physiological differentiation of different varieties in the same region. In Yangling area, it takes 56 d for ‘Changfu 2’ and 49 d for ‘Yanfu 6’. Morphological differentiation of axillary flower buds begins in late July[6]. Further studies have shown that the flower bud differentiation of autumn shoots of early maturing apple ‘Liaofu’ is in succession, and the flower bud morphological differentiation in the middle part starts at 3-4 weeks post growth cessation of corresponding leaves[7], while the time required for physiological differentiation of axillary flower buds of different varieties needs to be further studied. The stage of flower bud initiation is an irreversible process[8]. Fosteretal.[9]took the widening of apex growth point as a sign of the beginning of this period. The stage of flower bud development is the process that various primordia of flower organs successively differentiate and grow, and further develop into a complete flower organ. Predecessors dedicated many efforts to the morphological anatomy of flower buds. In recent years, the process of flower bud morphological differentiation has also attracted much attention. The conversion stage (flower initiation stage), flower bud differentiation stage, inflorescence primordial stage, calyx primordial stage, petal primordial stage, stamen primordial stage and pistil primordial stage occur successively among different buds and overlap mutually. There are differences in the start time and end time of terminal bud morphological differentiation between ‘Gala’ and ‘Fuji’ spurs in the same region[4,10]. The occurrence time and duration of flower bud differentiation of ‘Tianhong 2’ in the same region are affected by rootstock and year, but the concentrated differentiation stage is not affected[5]. Axillary flower bud differentiation of autumn shoots occurs successively in apple[7], and the process of morphological differentiation of each flower bud has not been elaborated in detail.

3 Plant hormones and flower bud differentiation of apple

Flower bud differentiation is closely related to various hormone levels in the body, and requires the coordination of various hormones. Therefore, hormone levels and hormone balance at different stages are crucial. Endogenous hormones play an important regulatory role in all stages of flower bud development[11]. Kondoetal.[12]suggested that IAA content might be related to flower bud differentiation of apple. Some scholars held that high IAA content could promote the activation of flower bud initiation of apple[13], while some scholars proposed that high IAA content might have an inhibitory effect on apple flowering[14]. Cytokinin promotes the formation of flower buds[15]. Exogenous application of 6-BA induces the increased expression ofMdTFL1 and promotes apple flowering[16-17]. Sanyaletal.[18]showed that the decrease of ZR content increased the proportion of flower buds. Wangetal.[19]put forward that the decrease of ZR content was conducive to floral induction. GA3 inhibits the flowering of woody plants[20], and low GA3 content is conducive to floral induction[21-22]. Spraying GA3 would break the balance of ZR/GAs, and inhibit the expression ofMdSPLs, thus inhibiting the flower bud formation of apple[23]. Changetal.[24]found that GA3 might promote flowering. High content of ABA promotes floral induction of apple, and spraying uniconazole increases ABA content in terminal bud and accelerates the process of flower bud differentiation[25]. In addition, the balance of endogenous hormones is considered to be a key factor in flower bud differentiation[26-27]. Hence, there are different views about the effect of endogenous hormones on flower formation of apple.

4 Flowering genes and flower bud differentiation of apple

There have been reports on molecular biology related to flower bud differentiation of apple. In apple, in addition to the two homologuesAFL1 andAFL2, there are other homologues ofLFYgene, butAFL1 andAFL2 have low homology with other homologous genes. Taking ‘Jonathan’ apple as the test material, Kotodaetal.[28]and Masatoetal.[29]pointed out thatAFL1 was only expressed during the transformation process from vegetative buds to floral buds, whileAFL2 was expressed at the tip of vegetative shoots, floral buds, floral organs and roots. Caoetal.[30]also studiedLFYhomologous genes in ‘Jonathan’ apple; the results showed thatAFL1 andAFL2 were homologous genes ofLFY, and their base sequences had 90% homology in the coding region, but only about 60% homology in the 3’ non-coding region;AFL2 was expressed in sepals, ovaries, roots, stems, leaves and terminal buds, whileAFL1 was only expressed in terminal buds;AFL1 was clearly expressed in the terminal buds at different developmental stages after the terminal buds stopped vegetative growth and turned to reproductive growth, whileAFL2 was expressed from June to October, consistent with previous studies. Zhang[31]took three main cultivars ‘Starkrimson’, ‘Gala’ and ‘Fuji’ as materials, and found thatAFL1 expression lasted from the beginning to winter dormancy, whileAFL2 also lasted from the beginning of expression, which was somewhat different from previous reports.

Two AP1 homologous genes, namedMdAP1 andMdMADS2, have been isolated from apple.MdAP1 is detected only during the period of sepal differentiation in flower meristem, so it does not directly cause the initiation of flower, but it is associated with sepal and fruit development. Kotodaetal.[28]concluded thatMdAP1 was only expressed in sepals of ‘Jonathan’ apple, nearly 2 months later thanAFL. Kotodaetal.[32]also studiedMdMADS5 from ‘Jonathan’ apple; overexpression ofMdMADS5 gene inArabidopsisthalianaresulted in earlier flowering, shorter inflorescence, and fewer rosette leaves, which was considered to be the same product asMdAP1. There were only a small part of base differences in the 3’ non-coding region, and the expression pattern was consistent.MdMADS2, another homologous gene ofAP1, was expressed at all stages of flower bud differentiation and in four flower organs. Transgenic tobacco expressing theMdMADS2 gene showed early flowering and shorter bolts, but did not show any homeotic changes in the floral organs, indicating thatMdMADS2 plays an important role during early stages of flower development[33].

TFL1 gene is a flowering inhibitory gene related to flowering induction signal, and it is a functional gene maintaining the development of inflorescence meristem. Kotodaetal.[34]studied the expression pattern ofMdTFL1 (homolog ofTFL1) from ‘Jonathan’ apple and the performance of transformingA.thaliana. The study showed that the gene was expressed in vegetative tissues such as vegetative buds, stems and roots of apples, and the expression became weak about two weeks before flower bud differentiation. The transgenicA.thalianablossomed later than the wild type, and its phenotype was similar to that of transgenicA.thalianaoverexpressingTFL1.

Most of the flower organ identity genes belong to the MADS-box gene family (such asAP1,AP3,PI,AG,etc.), whose family members play an important role in the process of flower bud differentiation as transcriptional regulators[35]. At present, a cDNA containing the conserved region of MADS has been isolated from ‘Fuji’, namedMdMADS1. This gene is expressed in all young fruits and flower organs, and the expression level is high in the early stage of flower and fruit development, indicating thatMdMADS1 mainly acts on the initial stage of flower bud differentiation. The expression pattern ofMdMADS1 is similar toAGL2 gene inA.thaliana, except that the former is not expressed in leaves[36].MdMADS3 andMdMADS4 isolated from ‘Fuji’ apple have high homology withAGL2 andAGL4 ofA.thaliana.MdMADS3 is expressed in stamen primordia, petal primordia and carpellary primordia, but not in fruit.MdMADS4 is expressed in inflorescence meristem, flower meristem, four flower organs and fruits.MdMADS4 is highly expressed in style vascular bundle and carpel vascular bundle of fruits in the early stage, and is transcriptionally accumulated in developing seed embryos[37]. TheAP2 homologous gene,MAP2, isolated from apple flower buds exists in the genome in low copy and is expressed in vegetative tissues, flower buds and different flower organs, consistent with theAP2 gene expression pattern ofA.thaliana[38].

5 Prospects

Flower bud differentiation of apple is a complex physiological and biochemical process. Signal changes in internal and external factors are transmitted to stem meristem, to initiate a series of physiological and molecular regulatory mechanisms of flower bud differentiation. The research on apple flower bud differentiation mainly focuses on the observation of morphological structure in buds, the changes in related physiological level, the cloning and expression of related genes and functional verification during differentiation of terminal buds. At present, few efforts have been dedicated to the influencing factors of flower bud differentiation of apple axillary buds and the flowering mechanism, which can be explored and studied in the future.