Anthony H. C. Huang

Oleosins and oil bodies in seeds and other organs.

Diverse organisms store lipids in subcellular particles as food reserves, which will be mobilized during a forthcoming period of active metabolism. These lipid particles can be found in the seeds, pollens, flowers, roots, and stems of flowering plants, the spores and vegetative organs of nonflowering plants, and algae. They are also present in some animal cells, fungi, and Euglena. Of a11 these subcellular storage lipid particles, those from seeds have been studied most extensively. Seeds of most plant species store TAGs as food reserves for germination and postgerminative growth. The TAGs are present in small subcellular spherical oil bodies of approximately 1 pm in diameter. Each oil body has a matrix of TAGs surrounded by a layer of PLs. The small entities provide a large surface area per unit TAG, which would facilitate lipase binding and lipolysis during germination. Oil bodies inside the cells of mature seeds or in isolated preparations are remarkably stable and do not aggregate or coalesce. This stability is in contrast to the instability of artificial liposomes made from amphipathic and neutra1 lipids; the liposomes slowly coalesce after formation. Seed oil bodies are stable because their surface is shielded by a layer of unique proteins, termed oleosins. Oleosins, in addition to being present in the storage oil bodies of seeds and pollens, have recently been found in the nonstorage tissue of floral tapetum. The preliminary findings suggest that the tapetum oleosins are similar to seed oleosins in structure and function. They may prevent subcellular lipid droplets from coalescing; these lipid droplets are to be deposited onto the surface of the maturing

Lipids, Proteins, and Structure of Seed Oil Bodies from Diverse Species

Oil bodies isolated from the mature seeds of rape (Brassica napus L.), mustard (Brassica juncea L.), cotton (Gossypium hirsutum L.), flax (Linus usitatis simum), maize (Zea mays L.), peanut (Arachis hypogaea L.), and sesame (Sesamum indicum L.) had average diameters that were different but within a narrow range (0.6–2.0 [mu]m), as measured from electron micrographs of serial sections. Their contents of triacylglycerols (TAG), phospholipids, and proteins (oleosins) were correlated with their sizes. The correlation fits a formula that describes a spherical particle surrounded by a shell of a monolayer of phospholipids embedded with oleosins. Oil bodies from the various species contained substantial amounts of the uncommon negatively charged phosphatidylserine and phosphatidylinositol, as well as small amounts of free fatty acids. These acidic lipids are assumed to interact with the basic amino acid residues of the oleosins on the surface of the phospholipid layer. Isoelectrofocusing revealed that the oil bodies from the various species had an isoelectric point of 5.7 to 6.6 and thus possessed a negatively charged surface at neutral pH. We conclude that seed oil bodies from diverse species are very similar in structure. In rapeseed during maturation, TAG and oleosins accumulated concomitantly. TAG-synthesizing acyltransferase activities appeared at an earlier stage and peaked during the active period of TAG accumulation. The concomitant accumulation of TAG and oleosins is similar to that reported earlier for maize and soybean, and the finding has an implication for the mode of oil body synthesis during seed maturation.

Endoplasmic Reticulum, Oleosins, and Oils in Seeds and Tapetum Cells

Diverse organisms contain neutral lipids in subcellular particles for food reserves and other purposes. These lipid particles are present in seeds, flowers, pollen and fruit of higher plants, the vegetative and reproductive organs of primitive plants, algae, fungi, nematodes, mammalian glands and

Tapetosomes in Brassica Tapetum Accumulate Endoplasmic Reticulum–Derived Flavonoids and Alkanes for Delivery to the Pollen Surface

Tapetosomes are abundant organelles in tapetum cells during the active stage of pollen maturation in Brassicaceae species. They possess endoplasmic reticulum (ER)–derived vesicles and oleosin-coated lipid droplets, but their overall composition and function have not been established. In situ localization analyses of developing Brassica napus anthers revealed flavonoids present exclusively in tapetum cells, first in an ER network along with flavonoid-3′-hydroxylase and then in ER-derived tapetosomes. Flavonoids were absent in the cytosol, elaioplasts, vacuoles, and nuclei. Subcellular fractionation of developing anthers localized both flavonoids and alkanes in tapetosomes. Subtapetosome fractionation localized flavonoids in ER-derived vesicles, and alkanes and oleosins in lipid droplets. After tapetum cell death, flavonoids, alkanes, and oleosins were located on mature pollen. In the Arabidopsis thaliana mutants tt12 and tt19 devoid of a flavonoid transporter, flavonoids were present in the cytosol in reduced amounts but absent in tapetosomes and were subsequently located on mature pollen. tt4, tt12, and tt19 pollen was more susceptible than wild-type pollen to UV-B irradiation on subsequent germination. Thus, tapetosomes accumulate ER-derived flavonoids, alkanes, and oleosins for discharge to the pollen surface upon cell death. This tapetosome-originated pollen coat protects the haploidic pollen from UV light damage and water loss and aids water uptake.

Ubiquitous and Endoplasmic Reticulum–Located Lysophosphatidyl Acyltransferase, LPAT2, Is Essential for Female but Not Male Gametophyte Development in Arabidopsis

Lysophosphatidyl acyltransferase (LPAT) is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid into different phosphatidic acids in diverse tissues. We examined putative LPAT genes in Arabidopsis thaliana and characterized two related genes that encode the cytoplasmic LPAT. LPAT2 is the lone gene that encodes the ubiquitous and endoplasmic reticulum (ER)–located LPAT. It could functionally complement a bacterial mutant with defective LPAT. LPAT2 and 3 synthesized in recombinant bacteria and yeast possessed in vitro enzyme activity higher on 18:1-CoA than on 16:0-CoA. LPAT2 was expressed ubiquitously in diverse tissues as revealed by RT-PCR, profiling with massively parallel signature sequencing, and promoter-driven β-glucuronidase gene expression. LPAT2 was colocalized with calreticulin in the ER by immunofluorescence microscopy and subcellular fractionation. LPAT3 was expressed predominately but more actively than LPAT2 in pollen. A null allele (lpat2) having a T-DNA inserted into LPAT2 was identified. The heterozygous mutant (LPAT2/lpat2) had minimal altered vegetative phenotype but produced shorter siliques that contained normal seeds and remnants of aborted ovules in a 1:1 ratio. Results from selfing and crossing it with the wild type revealed that lpat2 caused lethality in the female gametophyte but not the male gametophyte, which had the redundant LPAT3. LPAT2-cDNA driven by an LPAT2 promoter functionally complemented lpat2 in transformed heterozygous mutants to produce the lpat2/lpat2 genotype. LPAT3-cDNA driven by the LPAT2 promoter could rescue the lpat2 female gametophytes to allow fertilization to occur but not to full embryo maturation. Two other related genes, putative LPAT4 and 5, were expressed ubiquitously albeit at low levels in diverse organs. When they were expressed in bacteria or yeast, the microbial extract did not contain LPAT activity higher than the endogenous LPAT activity. Whether LPAT4 and 5 encode LPATs remains to be elucidated.

Plastid Lysophosphatidyl Acyltransferase Is Essential for Embryo Development in Arabidopsis

Lysophosphatidyl acyltransferase (LPAAT) is a pivotal enzyme controlling the metabolic flow of lysophosphatidic acid into different phosphatidic acids in diverse tissues. A search of the Arabidopsis genome database revealed five genes that could encode LPAAT-like proteins. We identified one of them, LPAAT1, to be the lone gene that encodes the plastid LPAAT. LPAAT1 could functionally complement a bacterial mutant that has defective LPAAT. Bacteria transformed with LPAAT1 produced LPAAT that had in vitro enzyme activity much higher on 16:0-coenzyme A than on 18:1-coenzyme A in the presence of 18:1-lysophosphatidic acid. LPAAT1 transcript was present in diverse organs, with the highest level in green leaves. A mutant having a T-DNA inserted into LPAAT1 was identified. The heterozygous mutant has no overt phenotype, and its leaf acyl composition is similar to that of the wild type. Selfing of a heterozygous mutant produced normal-sized and shrunken seeds in the Mendelian ratio of 3:1, and the shrunken seeds could not germinate. The shrunken seeds apparently were homozygous of the T-DNA-inserted LPAAT1, and development of the embryo within them was arrested at the heart-torpedo stage. This embryo lethality could be rescued by transformation of the heterozygous mutant with a 35S:LPAAT1 construct. The current findings of embryo death in the homozygous knockout mutant of the plastid LPAAT contrasts with earlier findings of a normal phenotype in the homozygous mutant deficient of the plastid glycerol-3-phosphate acyltransferase; both mutations block the synthesis of plastid phosphatidic acid. Reasons for the discrepancy between the contrasting phenotypes of the two mutants are discussed.

The Predominant Protein on the Surface of Maize Pollen Is an Endoxylanase Synthesized by a Tapetum mRNA with a Long 5′ Leader

In plants, the pollen coat covers the exine wall of the pollen and is the outermost layer that makes the initial contact with the stigma surface during sexual reproduction. Little is known about the constituents of the pollen coat, especially in wind-pollinated species. The pollen coat was extracted with diethyl ether from the pollen of maize (Zea mays L.), and a predominant protein of 35 kDa was identified. On the basis of the N-terminal sequence of this protein, a cDNA clone of theXyl gene was obtained by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of the 35-kDa protein shared similarities with the sequences of many microbial xylanases and a barley aleurone-layer xylanase. The 35-kDa protein in the pollen-coat extract was purified to homogeneity by fast protein liquid chromatography and determined to be an acidic endoxylanase that was most active on oat spelt xylan. Northern and in situhybridization showed that Xyl was specifically expressed in the tapetum of the anther after the tetrad microspores had become individual microspores. Southern hybridization and gene-copy reconstruction studies showed only one copy of the Xyl gene per haploid genome. Analyses of the genomic DNA sequence ofXyl and RNase protection studies with the transcript revealed many regulatory motifs at the promoter region and an intron at the 5′ leader region of the transcript. The Xyl transcript had a 562-nucleotide (nt) 5′ leader, a 54-nt sequence encoding a putative signal peptide, a 933-nt coding sequence, and a 420-nt 3′-untranslated sequence. The unusually long 5′ leader had an open reading frame encoding a putative 175-residue protein whose sequence was most similar to that of a microbial arabinosidase. The maize xylanase is the first enzyme documented to be present in the pollen coat. Its possible role in the hydrolysis of the maize type II primary cell wall (having xylose, glucose, and arabinose as the major moieties) of the tapetum cells and the stigma surface is discussed.

Oil Bodies and Oleosins in Physcomitrella Possess Characteristics Representative of Early Trends in Evolution

Searches of sequenced genomes of diverse organisms revealed that the moss Physcomitrella patens is the most primitive organism possessing oleosin genes. Microscopy examination of Physcomitrella revealed that oil bodies (OBs) were abundant in the photosynthetic vegetative gametophyte and the reproductive spore. Chromatography illustrated the neutral lipids in OBs isolated from the gametophyte to be largely steryl esters and triacylglycerols, and SDS-PAGE showed the major proteins to be oleosins. Reverse transcription-PCR revealed the expression of all three oleosin genes to be tissue specific. This tissue specificity was greatly altered via alternative splicing, a control mechanism of oleosin gene expression unknown in higher plants. During the production of sex organs at the tips of gametophyte branches, the number of OBs in the top gametophyte tissue decreased concomitant with increases in the number of peroxisomes and level of transcripts encoding the glyoxylate cycle enzymes; thus, the OBs are food reserves for gluconeogenesis. In spores during germination, peroxisomes adjacent to OBs, along with transcripts encoding the glyoxylate cycle enzymes, appeared; thus, the spore OBs are food reserves for gluconeogenesis and equivalent to seed OBs. The one-cell-layer gametophyte could be observed easily with confocal microscopy for the subcellular OBs and other structures. Transient expression of various gene constructs transformed into gametophyte cells revealed that all OBs were linked to the endoplasmic reticulum (ER), that oleosins were synthesized in extended regions of the ER, and that two different oleosins were colocated in all OBs.

Cell Wall Reactive Proteins in the Coat and Wall of Maize Pollen POTENTIAL ROLE IN POLLEN TUBE GROWTH ON THE STIGMA AND THROUGH THE STYLE

The surface of a pollen grain consists of an outermost coat and an underlying wall. In maize (Zea mays L.), the pollen coat contains two major proteins derived from the adjacent tapetum cells in the anthers. One of the proteins is a 35-kDa endoxylanase (Wu, S. S. H., Suen, D. F., Chang, H. C., and Huang, A. H. C. (2002) J. Biol. Chem. 277, 49055–49064). The other protein of 70 kDa was purified to homogeneity and shown to be a β-glucanase. Its gene sequence and the developmental pattern of its mRNA differ from those of the known β-glucanases that hydrolyze the callose wall of the microspore tetrad. Mature pollen placed in a liquid medium released about nine major proteins. These proteins were partially sequenced and identified via GenBank™ data bases, and some had not been previously reported to be in pollen. They appear to have wall-loosening, structural, and enzymatic functions. A novel pollen wall-bound protein of 17 kDa has a unique pattern of cysteine distribution in its sequence (six tandem repeats of CX3CX10–15) that could chelate cations and form signal-receiving finger motifs. These pollen-released proteins were synthesized in the pollen interior, and their mRNA increased during pollen maturation and germination. They were localized mainly in the pollen tube wall. The pollen shell was isolated and found to contain no detectable proteins. We suggest that the pollen-coat β-glucanase and xylanase hydrolyze the stigma wall for pollen tube entry and that the pollen secrete proteins to loosen or become new wall constituents of the tube and to break the wall of the transmitting track for tube advance.

Analyses of Advanced Rice Anther Transcriptomes Reveal Global Tapetum Secretory Functions and Potential Proteins for Lipid Exine Formation

The anthers in flowers perform important functions in sexual reproduction. Several recent studies used microarrays to study anther transcriptomes to explore genes controlling anther development. To analyze the secretion and other functions of the tapetum, we produced transcriptomes of anthers of rice (Oryza sativa subsp. japonica) at six progressive developmental stages and pollen with sequencing-by-synthesis technology. The transcriptomes included at least 18,000 unique transcripts, about 25% of which had antisense transcripts. In silico anther-minus-pollen subtraction produced transcripts largely unique to the tapetum; these transcripts include all the reported tapetum-specific transcripts of orthologs in other species. The differential developmental profiles of the transcripts and their antisense transcripts signify extensive regulation of gene expression in the anther, especially the tapetum, during development. The transcriptomes were used to dissect two major cell/biochemical functions of the tapetum. First, we categorized and charted the developmental profiles of all transcripts encoding secretory proteins present in the cellular exterior; these transcripts represent about 12% and 30% of the those transcripts having more than 100 and 1,000 transcripts per million, respectively. Second, we successfully selected from hundreds of transcripts several transcripts encoding potential proteins for lipid exine synthesis during early anther development. These proteins include cytochrome P450, acyltransferases, and lipid transfer proteins in our hypothesized mechanism of exine synthesis in and export from the tapetum. Putative functioning of these proteins in exine formation is consistent with proteins and metabolites detected in the anther locule fluid obtained by micropipetting.