Garlic (Allium sativum L.) fertility: transcriptome and proteome analyses provide insight into flower and pollen development

Abstract

Commercial cultivars of garlic, a popular condiment, are sterile, making genetic studies and breeding of this plant challenging. However, recent fertility restoration has enabled advanced physiological and genetic research and hybridization in this important crop. Morphophysiological studies, combined with transcriptome and proteome analyses and quantitative PCR validation, enabled the identification of genes and specific processes involved in gametogenesis in fertile and male-sterile garlic genotypes. Both genotypes exhibit normal meiosis at early stages of anther development, but in the male-sterile plants, tapetal hypertrophy after microspore release leads to pollen degeneration. Transcriptome analysis and global gene-expression profiling showed that >16,000 genes are differentially expressed in the fertile vs. male-sterile developing flowers. Proteome analysis and quantitative comparison of 2D-gel protein maps revealed 36 significantly different protein spots, 9 of which were present only in the male-sterile genotype. Bioinformatic and quantitative PCR validation of 10 candidate genes exhibited significant expression differences between male-sterile and fertile flowers. A comparison of morphophysiological and molecular traits of fertile and male-sterile garlic flowers suggests that respiratory restrictions and/or non-regulated programmed cell death of the tapetum can lead to energy deficiency and consequent pollen abortion. Potential molecular markers for male fertility and sterility in garlic are proposed.

Keywords: energy deficiency; gene expression; microsporogenesis; mitochondrial dysfunction; protein profiling; tapetum.

Figure 1

Flowchart of experimental design, including morphological and anatomical studies of garlic genotypes F87 and MS96, transcriptome and proteome analyses and validation of candidate genes.

Figure 2

Comparative developmental anatomy of anthers in the F87 (A–D) and MS96 (E–H) garlic genotypes during microgametogenesis. Comparisons were made between early, mid and late stages of flower development. (A) Cross section of F87 pollen sac at the tetrad stage. Bar = 40 μm. (B) Longitudinal section of F87 pollen sac after microspore (arrow) release from the callose. Endothecium (et), and tapetum (t) are visible. Bar = 60 μm. (C) Longitudinal section of an anther with mature microspores (arrow) that contain vegetative and generative cells. Tapetum (t) is degenerated, and only remains are visible. Bar = 30 μm. (D) Mature F87 flower. Long filaments, dehisced anthers (a) and long style are visible. Bar = 1.5 mm. (E) Longitudinal section of MS96 pollen sac at the tetrad (arrow) stage. Typical tapetum (t) cells are visible. Bar = 30 μm. (F) Cross section of an MS96 anther, with microspores released from the callose. Note hypertrophy of the tapetum (t) cells. Bar = 45 μm. (G) Considerable enlargement of the tapetum (t) cells and degenerated microspores (arrow) in MS96. Bar = 45 μm. (H) Mature MS96 flower. Degenerated yellow anthers (a) are visible and the style is elongated. Bar = 1.5 mm.

Figure 3

Differentially expressed genes (DEGs) between garlic genotypes F87 and MS96 at three stages of flower development. A total of 16,271 DEGs were differentially expressed; 1872 DEGs were common for all three stages, while 967, 4839, 3054 were specific for the early, mid and late stages, respectively.

Figure 4

Hierarchical cluster analysis of gene-expression patterns at the three developmental stages of garlic genotypes F87 and MS96 shows the relative expression levels of each gene (column) in each sample (row). Two large (1, 2) and three small gene clusters were differentially expressed in one or more samples. The expression values (FPKM; average of three replications) were log₂-transformed and then median-centered by transcript.

Figure 5

Biological processes in clusters 1 and 2 (see Figure 4), as revealed by analysis of GO term distribution using Blast2GO and REVIGO algorithms. GO terms are represented by circles and are plotted according to semantic similarities to other GO terms (adjoining circles are most closely related). Circle size is proportional to the abundance of the GO term in the cluster, while color indicates semantic similarities. Only GO terms with higher than 1% frequency in the cluster are shown. (A) Cluster 1. Main patterns are related to the general development of reproductive tissues, metabolism, microsporogenesis and cell-division processes and specific related fertility processes. (B) Cluster 2. Main patterns are related to energy-consuming activities and/or response to stress.

Figure 6

Hierarchical cluster analysis of the expression patterns of 23 genes with high similarity to the published sequences of plant mitochondrial genes at three flower-development stages of garlic genotypes F87 and MS96. Three clusters were identified. Note enhanced representation in the early stage of MS96. The relative expression levels of each gene (column) in each sample (row) are shown. Average of three replications' expression values (FPKM) were log₂-transformed and then median-centered by transcript.

Figure 7

Six transcripts mapped to the predicted protein ADP-ribosylation factor 1, accumulated in garlic genotype MS96. The protein was identified by mass spectrometry analysis, followed by matching with NCBI-nr. Expression was calculated using TMM normalization and FPKM calculations. The comparison was made with transcript expression in roots, leaves, cloves, basal plates and inflorescences, using the organ-specific garlic transcriptome catalog (NCBI bioproject PRJNA243415). Transcript-expression levels of (A) 14342_c0_seq1, (B) 14342_c0_seq2, (C) 14638_c0_seq1, (D) 47637_c0_seq4, (E) 47637_c0_seq1, (F) 47637_c0_seq2.

Figure 8

Validation of the expression of eight candidate genes by rt-qPCR and transcriptome analyses. Red columns represent gene expression in MS96, blue columns in F87. (A) Homologs of the genes AP3, MS2, MMD1, and GPAT2, expressed mainly in F87. (B) Homologs of the genes nad7, ccmC, COX2, and 18S rRNA, expressed mainly in MS96.

Figure 9

Accumulation patterns of two predicted proteins and their matched transcripts, as estimated by bioinformatics tools and rt-qPCR. Red columns represent the expression in MS96, blue columns in F87.