Here, we combined SSH and microarray techniques to investigate potential mechanism underlying seedlessness in Ponkan mandarin. SSH was proved to be an efficient and popular approach to enrich and identify differentially expressed genes between wild-type and its mutant or treatment
[35, 36]. However, because of high sensitivity of SSH, usually a large number of clones could be obtained but inevitably included some false-positive ones. Screening the SSH libraries to identify some candidate genes using microarray and to validate using qRT-PCR has proved to be a high-throughput and efficient way
[37–39]. However, relatively few clones were isolated in this study. Of the 6,000 clones, only 279 cDNA clones were identified as differentially expressed. Such results may suggest that there were little variations between QS and EG mandarins in gene expression. It was hypothesized that bud sport mutant was likely caused by single gene mutation, DNA methylation or retroelement activity
[40, 41]. In this research, various types of DNA markers including SCAR
, and SSR (172 pairs of primers), MSAP (96 pairs of primers) and AFLP (13 pairs of primers) were employed to analyze the polymorphism between these two mandarins, and no repeatable polymorphic bands were detected (data no shown). These results suggested that very few nuclear genes were altered during the developmental stages.
For the four developmental stages we chose, immense efforts were taken to determine which time-point was pivotal for stamen development, but there has no criteria for citrus gametophyte development. Though criteria for gametophyte development was available in model plant Arabidopsis, it can not be directly applied herein. Semi-thin and paraffin sections were performed in this study to survey the microsporogenesis of QS, and it was found that abnormal tetrads produced at the tetrad stage and subsequently the microsporocyte underwent abnormal meiosis. This process mainly occurred at SF stage (the diameter of floral organs is about 3 mm) (unpublished data). Additionally, large proportion (about 59.7%) of differentially expressed genes was found in BF when the anthers and pollen grains were almost mature, indicating that this time-point might be also important.
Amino acid metabolic process
Of the metabolic pathways with altered expressed genes, 25% were involved in amino acid metabolism. Amino acids were not only primary metabolic products for normal growth and development but also cell signaling molecules and regulators of gene expression and protein phosphorylation cascade
. Interestingly, among these amino acid metabolism pathways, two genes were down-regulated across the developmental stages in QS versus EG, one (JU497356) encoding glutamate-ammonialigase (EC 22.214.171.124), the other (JU497338) encoding beta-glucosidase (EC 126.96.36.199). In higher plants, glutamate-ammonialigase catalyzes ATP-dependent conversion of glutamate and ammonia into glutamine which occupies a central position of amino acid metabolic pathway
, and this metabolic process is critical for coordinating metabolic balance in rice
. And beta-glucosidase could be used for the cellulosic ethanol industry
 and has diversity of functions in plants. In maize, Zm-p60.1 encoding a beta-glucosidase could release active cytokinin, and might function in vivo to supply the developing maize embryo
. Additionally, some beta-glucosidases affect the properties of cell wall
 and are associated with freezing tolerance, such as the SFR2 in Arabidopsis. Some beta-glucosidases are related to the efficiency of microspore embryogenesis
. It is noteworthy that a gene (JU497374) encoding asparagine synthase (EC 188.8.131.52) was down-regulated exclusively at SF (early stage of stamen development). And asparagine is one central intermediate in nitrogen assimilation and transportation in plant
[52, 53]. Recent studies showed that this gene played important role in defense against pathogens and salt stress
[54, 55]. Additionally, genes related to carbohydrate metabolism and energy metabolism also showed down-regulated expression in QS mainly at BF and OV (late stage of stamen development). These results suggested that the vital activities of QS weakened during early development stages of stamen, and the metabolic process of nutrition and energy was also impaired at subsequent stages of stamen development especially when the stamen was mature.
Two genes involved in cysteine/methionine metabolism and participated in the biosynthesis of ethylene were also identified in this study. One (JU497321) encodes 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (EC 184.108.40.206) is likely involved in the biosynthesis of L-methionine. And the methionine can be transformed into S-adenosylmethionine (SAM) (the precursor of ethylene)
. The other one (JU497373) encodes aminocyclopropane carboxylate oxidase (EC 220.127.116.11) and is a pivotal enzyme during the biosynthesis of ethylene. In addition, genes involved in the synthesis of IAA (indole-3-acetic acid) were also identified such as a gene (JU497377) encoding Indole-3-acetatebeta-glucosyltransferase (EC 18.104.22.168). These results implied that the endogenous phytohormones might be involved in the male gametophyte development of citrus.
It was known that floral organ formation and function were influenced by TFs regulation. In our research, twelve unigenes were assigned to the category of transcription factor, and six of them were identified as AP2-ERF family members. AP2-ERF TF containing highly conserved AP2/ERF DNA-binding domain, is a large family unique in plant. In our research, four AP2-ERF members showed similar expression pattern. AP2-EREBP TF1 was closely homologous with atERF107 (AT1G19210). This gene was likely involved in the regulation of gene expression by stress factors and by components of stress signal transduction pathways. However, until now, no experimental evidence was available. AP2-EREBP TF3 showed high similarity with ERF5 (AT5G47230.1). ERF5 might play an important role in plant innate immunity likely through coordinating chitin and other defense pathways
. Other research suggested that ERF5 and ERF6 might potentially overlap in their function and acted as positive regulators of JA/ethylene-mediated defense
. In tomato, this gene was mainly involved in responses to drought and salt stresses
. As for AP2/ERF domain containing TF2, its closest relative was ERF104 (AT5G61600.1). Recent studies showed that ERF104 was in vivo substrate of MPK6, and ethylene could release ERF104 and allow liberated ERF104 to access target genes related to plant defense
. CBF/DREB-like TF was of high similarity with CBF4 (AT5G51990.1) which was critical regulator involved in cold acclimation and drought adaptation
In addition, AP2-EREBP TF2 was highly homologous with RAP2.4 (AT1G78080.1). RAP2.4 acted at or downstream of a converging point of light and ethylene signaling pathways, and it coordinately regulated multiple developmental processes and stress responses
. As for AP2-ERF domain containing TF1, its expression pattern was different from other five members. It showed high similarity with DREB26 (AT1G21910.1). In plant, RAP2.6, RAP2.6 L, DREB26 and DREB19 exhibited tissue specific expression and participated developmental processes as well as biotic and/or abiotic stress signaling
. Though previous researches emphasized the functions of these AP2-ERF TFs on resistance against biotic and abiotic stresses, AP2-ERF TFs were also participated in plant development such as embryo patterning
, and stamen emergence
Additionally, two MYB (R2R3-MYB) transcription factors also showed differential expression between QS and EG. In plant, MYB TF family was categorized into 3 subfamilies according to the number of adjacent repeats of MYB-domain. Of them, R2R3-MYB subfamily contains the largest number of members. Like the AP2-ERF TF family proteins, MYB family proteins also function in various plant-specific processes. In Arabidopsis, MYB TFs were found as key regulators involved in development, metabolism and biotic and abiotic stress responses. Among these MYB TFs of Arabidopsis, AtMYB26 is involved in determining endothecial cell development within the anther and is essential for anther dehiscence
. AtMYB33 and AtMYB65 redundantly facilitate anther and pollen development
. AtMYB80 regulates exine formation and acts downstream of AtMYB35; and AtMYB103 is required for tapetal development and microsporogenesis, especially for callose dissolution and exine formation
[69, 70]. AtMYB125 positively control male germ cell division and commit progenitor germ cells to sperm cell differentiation
[71, 72]. In rice, CSA gene encoding MYB TF functions as a key transcriptional regulator for sugar partitioning during male reproductive development, and the CSA mutant showed reduced levels of sugars and starch in floral organs which lead to MS.
Interestingly, in our results, one MYB TF showed similar expression pattern with AP2-ERF TFs that down-regulated at BF stage when the anther and pollen grains are mature. This MYB TF termed as R2R3-MYB TF was closely related to ATMYBR1/ATMYB44 (AT5G67300.1), and AtMYB44 was likely to enhance drought and salt stress tolerance by suppressing the expression of genes encoding PP2Cs, which was described as negative regulators of ABA signaling
. Previous report showed that AtMYB44 was with changed expression during late embryogenesis and seed maturation
. And notably there was a NAC domain protein (JU497421) highly homologous with ANAC102 (AT5G63790.1). ANAC102 was an important regulator of seed germination and activated a seed-specific subset of genes under low-oxygen stress; it was also necessary for the viability of Arabidopsis seeds following low-oxygen treatment
In summary, these results suggested that these AP2-ERF TFs and the MYB TF functioned redundantly and coordinated with other TFs which involved in the complex network regulating floral organ development. Further research should emphasize on the isolation of proteins interacted with these TFs.