Tandem oleosin genes in a cluster acquired in Brassicaceae created tapetosomes and conferred additive benefit of pollen vigor Chien Yu Huanga, Pei-Ying Chenb, Ming-Der Huangb, Chih-Hua Tsoub, Wann-Neng Janeb, and Anthony H. C. Huanga,b,1 aCenter for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, CA 92521; and bInstitute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan Edited by June B. Nasrallah, Cornell University, Ithaca, NY, and approved July 12, 2013 (received for review March 19, 2013) During evolution, genomes expanded via whole-genome, segmental, their presence in diverse organisms and their differential expres- tandem, and individual-gene duplications, and the emerged redun- sion in specific tissues and under various physiological conditions dant paralogs would be eliminated or retained owing to selective and environmental cues. The findings have led to the delineation neutrality or adaptive benefit and further functional divergence. of the mechanism of gene duplication, deletion, and retention fi Here we show that tandem paralogs can contribute adaptive quan- due to adaptive neutrality or bene t, and additional functional fi fi divergence. Studies of paralogs in tandem, unlike those dispersed titative bene t and thus have been retained in a lineage-speci c fi manner. In Brassicaceae, a tandem oleosin gene cluster of five to nine throughout the genome, have inherent technical dif culties be- paralogs encodes ample tapetum-specific oleosins located in abun- cause of the lack of knockout mutants of more than one of the dant organelles called tapetosomes in flower anthers. Tapeto- tightly linked paralogs. As a consequence, studies involving loss- of-function and even gain-of-function approaches are difficult. In somes coordinate the storage of lipids and flavonoids and their addition, a seldom-addressed but important conceptual issue is transport to the adjacent maturing pollen as the coat to serve whether tandem paralogs offer quantitative adaptive benefitin various functions. Transfer-DNA and siRNA mutants of Arabidopsis thaliana boosting gene expression, such that they have been retained and with knockout and knockdown of different tandem oleo- expanded in number. sin paralogs had quantitative and correlated loss of organized A tandem oleosin gene cluster in Arabidopsis thaliana has been PLANT BIOLOGY structures of the tapetosomes, pollen-coat materials, and pollen studied in terms of evolution and function. A. thaliana has 17 tolerance to dehydration. Complementation with the knockout oleosin genes: 5 expressing in seed, 3 in both seed and pollen, paralog restored the losses. Cleomaceae is the family closest to and 9 in the tapetum of flower anthers (9–11). The oleosin Brassicaceae. Cleome species did not contain the tandem oleosin paralogs specifically expressed in the tapetum are termed gene cluster, tapetum oleosin transcripts, tapetosomes, or pollen T-oleosin genes. They were termed genes encoding oleosin-like tolerant to dehydration. Cleome hassleriana transformed with an proteins because the proteins possessed oleosin sequence char- Arabidopsis oleosin gene for tapetum expression possessed primi- acteristics (next paragraph) (11), or glycine-rich-protein genes tive tapetosomes and pollen tolerant to dehydration. We propose because one or two of the paralogs encode oleosins with short that during early evolution of Brassicaceae, a duplicate oleosin gene glycine-rich repeats at the nonconserved C termini (10, 12). mutated from expression in seed to the tapetum. The tapetum oleo- These earlier terms were used before the function of the oleosins sin generated primitive tapetosomes that organized stored lipids in anthers was delineated (13, 14). All of the nine T-oleosin and flavonoids for their effective transfer to the pollen surface for paralogs locate on chromosome 5; eight are tightly linked in a greater pollen vitality. The resulting adaptive benefit led to reten- 30-kb locus, and the remaining one is 23 Mb downstream of tion of tandem-duplicated oleosin genes for production of more the cluster. oleosin and modern tapetosomes. Comparisons of the T-oleosin paralogs in species of Brassi- caceae show that the paralogs evolved more rapidly than did their neighbor genes, as is expected for reproductive genes (10, evolutionary appearance of organelle | tandem gene cluster 11). This rapid evolution applies to the sequences encoding the two less-conserved N- and C-terminal motifs of oleosin but not n evolution, genomes expanded via whole-genome, segmental, to the sequence encoding the essential central hairpin. Each of Itandem, and individual-gene duplications (1–3). The mecha- the T-oleosin paralogs contains two exons, and the location of nisms of these duplications have been explored. The newly the exons/intron differs from those of the seed- and seed/pollen- emerged paralogs would be eliminated over time, unless they expressed oleosin paralogs (9). The T-oleosin gene cluster has conferred adaptive neutrality or benefit to the organism. Further been found in several genera of Brassicaceae (10, 11); whether it substitutions in the retained paralogs have resulted in refinement is present outside of Brassicaceae has not been determined. and divergence of functions. The refinement could give rise to Oleosins are structural proteins on lipid droplets in seeds, gene expression in restricted cells or tissues and under particular pollen, and tapeta (15–17). The protein has a hallmark of ∼72 physiological conditions and environmental cues. The divergence uninterrupted nonpolar residues that form a hairpin penetrating could include new structural properties, binding specificity, or into the lipid droplets. This hairpin, together with the adjacent enzymic reactions of the encoded proteins. The retained paral- ogs in modern genomes appear as numerous gene families, each consisting of a few to hundreds of members. Members of each Author contributions: C.Y.H., C.-H.T., and A.H.C.H. designed research; C.Y.H., P.-Y.C., family may be present as individual paralogs dispersed throughout M.-D.H., C.-H.T., W.-N.J., and A.H.C.H. performed research; C.Y.H., P.-Y.C., M.-D.H., C.-H.T., the whole genome, in linkage with neighboring unrelated genes of W.-N.J., and A.H.C.H. analyzed data; and C.Y.H. and A.H.C.H. wrote the paper. the original duplicated genome or segment, and/or as tandem The authors declare no conflict of interest. repeats, depending on the original mechanisms of duplication and This article is a PNAS Direct Submission. the subsequent retention, deletion, and rearrangement. Data deposition: The sequences reported in this paper have been deposited in the Gen- Tandem paralogs are ubiquitous in sequenced genomes. Par- Bank database (accession nos. KC777372, KC777373, KC777374, KF270089,and alogs that have been studied intensively include genes encoding KF270090). major histocompatibility complex (4) and homeobox (5) in ani- 1To whom correspondence should be addressed. E-mail: [email protected]. mals, as well as lectins (6) and other stress-resistant proteins (7, 8) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in plants. The functional study of tandem paralogs has focused on 1073/pnas.1305299110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1305299110 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 amphipathic N- and C-terminal motifs, stabilizes the hydropho- (Fig. S2). Overall, the reported order of levels of transcripts was bic lipid droplet in the cytoplasm. In the tapetum of Brassica- T3 ≥ T5 ∼ T4 ∼ T6 > other paralogs. ceae, T-oleosins are components of the abundant organelles The relative level of oleosin proteins, as visually determined by called the tapetosomes (14, 18, 19). Each tapetosome contains SDS/PAGE and Coomassie blue staining, was T3 oleosin ≥ other numerous oleosin-coated alkane lipid droplets associated ioni- individual oleosins in tapetosomes from wild-type Columbia (9) cally with many flavonoid-containing, endoplasmic reticulum- and on the pollen surface of wild-type Ws2 (12, 20). Similar derived vesicles (14). In the tapetum cells, the tapetosomes tem- findings of T3-ortholog oleosin being more abundant than other porarily store alkanes and flavonoids, which will be discharged individual T-oleosins in tapetosomes and pollen coat were ob- to the adjacent maturing pollen grains, forming the pollen coat. tained in Brassica napus (13, 18) and Brassica rapa (19). The The coat waterproofs the pollen grain and protects it against higher amount of T3 oleosin determined visually by SDS/PAGE UV radiation. Mutational loss of a major T-oleosin in Arabi- could be due to its high molecular mass (53 kDa) compared with dopsis leads to pollen having a delay in hydration on the stigma T5 (10 kDa), T4 (22 kDa), and T6 (15 kDa) oleosins, as well as surface (20). Similarly, mutational loss of pollen-coat flavonoids an 8-kDa portion of these T-oleosins being the conserved non- in Arabidopsis results in pollen being more susceptible to UV polar hairpin motif that was less reactive to Coomassie blue. The radiation (14). overall findings suggest that the relative numbers of T-oleosin The tandem T-oleosin gene cluster produces abundant oleo- molecules are on the order of T3 ≥ T5 ∼ T4 ∼ T6 > others. sins in the tapetum. We investigated the clustered paralogs in conferring functional neutrality, adaptive benefit, and/or quan- Arabidopsis Mutants of Various T-Oleosin Paralogs Had Similar titative benefit. We probed the existence of the T-oleosin gene Phenotypes of Reduced Levels of Transcripts, Tapetosome Structures, cluster outside Brassicaceae and found that the closest family, Pollen Coat, and Pollen Dehydration Tolerance. Our observations did Cleomaceae (21–23), did not possess the T-oleosin gene cluster, not suggest that individual paralogs of the T-oleosin gene cluster tapetosomes, and dehydration-tolerant pollen. We thus exam- contributed to functional divergence. Thus, we tested whether the ined whether transforming a Cleome species with an Arabidopsis individual paralogs conferred quantitative benefits rather than T-oleosin gene could generate tapetosomes, which in turn would functional neutrality.