Integrating membrane transport with male gametophyte development and function through transcriptomics

Abstract

Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::beta-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

Figure 1.

Coexpression of genes encoding transporters revealed many genes are expressed either early or late during microgametogenesis. Shown is the relative expression of each gene at the four stages of pollen development: microspore (MS), bicellular (BC), tricellular (TC), and mature pollen (MP). Protein names are provided when available; all other genes are listed by their Arabidopsis Genome Initiative (AGI) names. Data are taken from Supplemental Table I. A, Coexpression of 23 transporter genes late in pollen development (Cluster 1). B, Coexpression of 32 genes late in development in Cluster 2. AHA8 was omitted as its expression peaked at 6,225. C, Coexpression of 12 genes showing peak levels at the tricellular stage (Cluster 18). D, Early pollen-expressed genes are corepressed as pollen matures (Cluster 29).

Figure 2.

Discrete subsets of genes within selected gene families are expressed in male gametophyte in a developmentally regulated manner. Relative expression of all genes within a gene family was monitored at the microspore (MS), bicellular (BC), tricellular (TC), and mature pollen (MP) stages. Protein names are provided when available; all other genes are listed by their AGI numbers. Gene names in red and blue indicate those that are either specifically or preferentially expressed in pollen, respectively. Genes that do not have unique probes on the ATH1 gene chip are highlighted in pink. Data are taken from Supplemental Table I. A, Channels. A.1, VIC K⁺ channel, TC number 1.A.1, FAM number 114; A.2, VIC CNGC, TC number 1.A.1, FAM number 156; A.3, MIP family, TC number 1.A.8, FAM numbers 349 and 622; A.4, MIP family (continued), TC number 1.A.8, FAM number 622. B, Sugar transporters. B.1, MFS monosaccharide-proton symporter (STP), TC number 2.A.1.1, FAM number 44.1; B.2, GPH Suc-proton symporter/Suc transporter (SUC), TC number 2.A.2, FAM numbers 439 and 686. C, Cation transporters. C.1, Ctr2 copper transporter, TC number 9.A.12, FAM number 535. D, Anion transporters. D.1, SulP sulfate permease, TC number 2.A.53, FAM numbers 33 and 263; D.2, AE boron transporter, TC number 2.A.31, FAM number 78. E, Nitrogen-containing compound and amino acid transporters. E.1, OPT oligopeptide transporter, TC number 2.A.67, FAM number 246; E.2, AAAP amino acid/auxin permease, TC number 2.A.18, FAM number 487. F, Pumps. F.1, P-ATPase PM P3A-type H⁺-ATPase, TC number 3.A.3, FAM number 38; F.2, P-ATPase calmodulin-regulated Ca²⁺-transporting P2B-type ATPase, TC number 3.A.3, FAM number 212; F.3, V-ATPase Vo membrane sector, TC number 3.A.2, FAM numbers 283, 289, and 411; F.4, P-ATPase heavy metal-transporting P1B-type ATPase, TC number 3.A.3, FAM numbers 277, 616, and 620. G, Unclassified proteins. G.1, Putative olfactory receptor, FAM number 359. FAM, AMPL family.

Figure 2.

Discrete subsets of genes within selected gene families are expressed in male gametophyte in a developmentally regulated manner. Relative expression of all genes within a gene family was monitored at the microspore (MS), bicellular (BC), tricellular (TC), and mature pollen (MP) stages. Protein names are provided when available; all other genes are listed by their AGI numbers. Gene names in red and blue indicate those that are either specifically or preferentially expressed in pollen, respectively. Genes that do not have unique probes on the ATH1 gene chip are highlighted in pink. Data are taken from Supplemental Table I. A, Channels. A.1, VIC K⁺ channel, TC number 1.A.1, FAM number 114; A.2, VIC CNGC, TC number 1.A.1, FAM number 156; A.3, MIP family, TC number 1.A.8, FAM numbers 349 and 622; A.4, MIP family (continued), TC number 1.A.8, FAM number 622. B, Sugar transporters. B.1, MFS monosaccharide-proton symporter (STP), TC number 2.A.1.1, FAM number 44.1; B.2, GPH Suc-proton symporter/Suc transporter (SUC), TC number 2.A.2, FAM numbers 439 and 686. C, Cation transporters. C.1, Ctr2 copper transporter, TC number 9.A.12, FAM number 535. D, Anion transporters. D.1, SulP sulfate permease, TC number 2.A.53, FAM numbers 33 and 263; D.2, AE boron transporter, TC number 2.A.31, FAM number 78. E, Nitrogen-containing compound and amino acid transporters. E.1, OPT oligopeptide transporter, TC number 2.A.67, FAM number 246; E.2, AAAP amino acid/auxin permease, TC number 2.A.18, FAM number 487. F, Pumps. F.1, P-ATPase PM P3A-type H⁺-ATPase, TC number 3.A.3, FAM number 38; F.2, P-ATPase calmodulin-regulated Ca²⁺-transporting P2B-type ATPase, TC number 3.A.3, FAM number 212; F.3, V-ATPase Vo membrane sector, TC number 3.A.2, FAM numbers 283, 289, and 411; F.4, P-ATPase heavy metal-transporting P1B-type ATPase, TC number 3.A.3, FAM numbers 277, 616, and 620. G, Unclassified proteins. G.1, Putative olfactory receptor, FAM number 359. FAM, AMPL family.

Figure 3.

Differential expression of CHX genes in developing pollen is confirmed by promoter∷GUS analyses of the inflorescence. A, CHX17 promoter∷GUS. Flowers at different stages (S9–S13) were taken from one inflorescence that was stained for GUS activity. B, CHX24 promoter∷GUS. Flowers at different stages (S10–S14) were taken from one inflorescence stained for GUS activity. C, Relative expression of CHX17 and CHX24 in male gametophyte as revealed by ATH1 gene chip. MS, BC, TC, and MP refer to the microspore, bicellular, tricellular, and mature pollen stages. Data were taken from Supplemental Table I.