Isolation, sorting, and characterization of uni- and binucleate tapetal protoplasts from anthers of normal and Texas cytoplasmic male-sterileZea mays L.

1 De part ment of Plant Ge net ics and Breed ing, Cen tre of Ag ri cul tural Re search Gent, Caritasstraat 21, 9090 Melle, Bel gium, e-mail: t.eeckhaut@clo.fgov.be 2 De part ment of Bio tech nol ogy, Ag ri cul ture and Land scape Ar chi tec ture, Hogeschool Gent, Voskenslaan 270, 9000 Gent, Bel gium Key words: cell sort ing, hap loid, interspecific hybrid iza tion, ploidy, polyploid Ab stractIn this re view, the dif fer ent ap pli ca tions of flow cytometry in plant breed ing are high lighted. Four main breed ing re lated pur poses can be dis tin guished for flow cytometry: (i) Char acteri sa tion of avail able plant ma te rial, in clud ing screen ing of pos si ble par ent plants for breed ing pro grams as well as eval u ation of pop u la tion biodiversity; (ii) Off spring screen ing af ter inter specific, interploidy or ab er rant crosses; (iii) Ploidy level de ter mi na tion af ter haploidization and polyploidization treatments and (iv) Par ti cle sort ing, that al lows sep a ra tion of plant cells based on mor pho log i cal or flu o res cent char ac ter is tics. An over view and dis cus sion of these var i ous ap pli ca tions in dicates that flow cytometry is a rel a tively quick, cheap and re liable tool for many breed ing re lated ob jec tives. List of ab bre vi a tions:DAPI (4', 6-diamidino-2-phenylindol), EB (ethidium bro mide), FCM (flow cytometry), FDA (fluorescein diacetate), FSC (for ward scatter), ORY (oryzalin), PI (propidium io dide), PMT (photomultiplier), SSC (sideward scat ter), TRI (trifluralin) In tro duc tion Bases and his tory of flow cytometryCytometry re fers to the mea sure ment of phys i cal and chem i cal char ac ter is tics of par ti cles. In short, flow cytometry (FCM) in volves the anal y sis of fluo res cence and light-scat ter ing prop er ties of sin gle par ti cles dur ing their pas sage within a nar row, precisely de fined, liq uid stream (Galbraith et al. 1983, Dolezel et al. 1989, Dolezel, 1991.The most im por tant fea ture of FCM is that it enables mea sure ments to be made on in di vid ual cells at high speed. This al lows the de tec tion of subpopulations and the quan ti fi ca tion of the het er o gene ity of the pop u la tion of in ter est rather than merely the ob tain ing of av er age val ues for a pop ula tion (Shapiro 2003).It took an other de cade be fore Galbraith et al.(1983) came up with a rad i cally prac ti cal so lu tion, in which the sus pen sions of in tact nu clei are prepared by chop ping a small amount of fresh tis sue in a suit able iso la tion buffer. This sim ple, con ve nient and rapid tech nique al lowed the wide spread use of FCM for DNA-con tent re lated re search in dif fer ent plant spe cies (De Laat et al.

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“…All kinds of par ticles can be sorted, e.g. proto plasts (Hor ner et al 1993) and chro mo somes (Li and Arumuganathan 2003).…”

Section: Par Ti Cle Sort Ingmentioning

confidence: 99%

Exploitation of flow cytometry for plant breeding

2005

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“…Among these other species, maize (Zea mays) has been the most studied. Electron microscopy (EM) studies have shown that tapetum cells at a late developmental stage in maize do not possess elaioplasts and tapetosomes (Skvarla and Larson, 1966;Horner et al, 1993;this report), so the pollen-coat materials have to be synthesized, stored, and delivered via other mechanisms.…”

mentioning

confidence: 99%

The Maize Tapetum Employs Diverse Mechanisms to Synthesize and Store Proteins and Flavonoids and Transfer Them to the Pollen Surface

Suen

Huang

et al. 2012

Plant Physiology

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In anthers, the tapetum synthesizes and stores proteins and flavonoids, which will be transferred to the surface of adjacent microspores. The mechanism of synthesis, storage, and transfer of these pollen-coat materials in maize (Zea mays) differs completely from that reported in Arabidopsis (Arabidopsis thaliana), which stores major pollen-coat materials in tapetosomes and elaioplasts. On maize pollen, three proteins, glucanase, xylanase, and a novel protease, Zea mays pollen coat protease (ZmPCP), are predominant. During anther development, glucanase and xylanase transcripts appeared at a mid developmental stage, whereas protease transcript emerged at a late developmental stage. Protease and xylanase transcripts were present only in the anther tapetum of the plant, whereas glucanase transcript was distributed ubiquitously. ZmPCP belongs to the cysteine protease family but has no closely related paralogs. Its nascent polypeptide has a putative amino-terminal endoplasmic reticulum (ER)-targeting peptide and a propeptide. All three proteins were synthesized in the tapetum and were present on mature pollen after tapetum death. Electron microscopy of tapetum cells of mid to late developmental stages revealed small vacuoles distributed throughout the cytoplasm and numerous secretory vesicles concentrated near the locular side. Immunofluorescence microscopy and subcellular fractionation localized glucanase in ER-derived vesicles in the cytoplasm and the wall facing the locule, xylanase in the cytosol, protease in vacuoles, and flavonoids in subdomains of ER rather than in vacuoles. The nonoverlapping subcellular locations of the three proteins and flavonoids indicate distinct modes of their storage in tapetum cells and transfer to the pollen surface, which in turn reflect their respective functions in tapetum cells or the pollen surface.

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“…This website program indicates a possibility that the protein is targeted to the peroxisomes because of the presence the tripeptide AKL in the 60-kDa pre-xylanase and the 35-kDa xylanase. We discount this possibility, because the tripeptide AKL, unlike those in peroxisomal proteins, is not present at or very near the C terminus, and because we (data not shown), as well as others (13,14), did not observe apparent peroxisomes in the tapetum cells. We subfractionated the extract of stage-3 anthers by differential centrifugation after the microspores (and the attached 35-kDa xylanase) in the extract had been removed by a short and low speed centrifugation.…”

Section: There Is No Apparent Sequence Signal In the 60-kda Prexylanamentioning

confidence: 65%

“…The coat also contains minor proteins involved in self-incompatibility that are synthesized in the pollen interior (11,12). In wind-pollinating species, the pollen coat is thin (13,14), and major biochemical studies have been carried out only with maize (15). The coat of maize pollen contains undefined neutral lipids and a predominant protein.…”

mentioning

confidence: 99%

Maize Tapetum Xylanase Is Synthesized as a Precursor, Processed and Activated by a Serine Protease, and Deposited on the Pollen

Suen

Chang

et al. 2002

Journal of Biological Chemistry

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Pollen coat contains ingredients that interact with the stigma surface during sexual reproduction. In maize (Zea mays L.) pollen coat, the predominant protein is a 35-kDa endoxylanase, whose mRNA is located in the tapetum cells enclosing the maturing pollen in the anthers. This 2.0-kb mRNA was found to have an open reading frame of 1,635 nucleotides encoding a 60-kDa pre-xylanase. In developing anthers, the pre-xylanase protein appeared prior to the 35-kDa xylanase protein and enzyme activity and then peaked and declined, whereas the 35-kDa xylanase protein and activity continued to increase until anther maturation. An acid protease in the anther extract converted the inactive prexylanase to the active 35-kDa xylanase in vitro. The protease activity was inhibited by inhibitors of serine proteases but unaffected by inhibitors of cysteine, aspartic, or metallic proteases. Sequence analysis revealed that the 60-kDa pre-xylanase was converted to the 35-kDa xylanase with the removal of 198 and 48 residues from the N and C termini, respectively. During in vitro and in vivo conversions, no intermediates of 60 -35 kDa were observed, and the 35-kDa xylanase was highly stable. The pre-xylanase was localized in the tapetum-containing anther wall, whereas the 35-kDa xylanase was found in the pollen coat. The significance of having a large non-active pre-xylanase and the mode of transfer of the xylanase to the pollen coat are discussed. A gene encoding the barley (Hordeum vulgare L.) tapetum xylanase was cloned; this gene and the gene encoding the seed aleurone-layer xylanase had strict tissuespecific expressions.A major step in sexual reproduction in plants is the interaction between the male gamete-containing pollen and the pollen-receiving stigma in flowers (1-4). This interaction manifests at the contact between the pollen coat and the surface structures of the stigma. The coat of pollen contains special constituents that are essential to the initial sexual contact and thus the success of fertilization. Its chemical compositions vary, depending on the species. In insect or self-pollinating species, of which Brassica and Arabidopsis are the best studied (5-10), the pollen coat is thick and contains steryl esters and very non-polar lipids as the major lipids and oleosins as the predominant proteins. The lipids are for water-proofing, whereas the amphipathic oleosins may act as a "wick" for water uptake to initiate germination. These major coat lipids and proteins are synthesized and accumulated initially in the tapetum cells, which enclose the pollen locule in the anthers.

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Isolation, sorting, and characterization of uni- and binucleate tapetal protoplasts from anthers of normal and Texas cytoplasmic male-sterileZea mays L.

Cited by 12 publications

References 48 publications

Exploitation of flow cytometry for plant breeding

Exploitation of flow cytometry for plant breeding

The Maize Tapetum Employs Diverse Mechanisms to Synthesize and Store Proteins and Flavonoids and Transfer Them to the Pollen Surface

Maize Tapetum Xylanase Is Synthesized as a Precursor, Processed and Activated by a Serine Protease, and Deposited on the Pollen

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