Vipen K. Sawhney

Proteome profile and functional classification of proteins in Arabidopsis thaliana (Landsberg erecta) mature pollen

Proteome analysis of mature Arabidopsis thaliana (Landsberg erecta ecotype) pollen was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 960 spots were resolved on pH 4–7 IPG strips and 110 distinct proteins were identified from 150 spots analyzed. The identified proteins were categorized based on their functional role in the pollen, which included proteins involved in energy regulation, defense-related mechanisms, calcium-binding and signaling, cytoskeletal formation, pollen allergens, glycine-rich proteins (GRPs), and late embryogenesis abundant (LEA) proteins. These proteins potentially play important roles in pollen function at maturity and during subsequent germination and tube growth. Some of the proteins identified were related to known pollen-specific transcripts, while some were similar to proteins found in the seed. In this study, 66 new proteins were identified which were not reported in two other recent studies on Arabidopsis pollen, 17 proteins were common in all three studies, and 35 or 26 proteins reported here had an overlap with one or the other two studies. These differences may be attributed to the methods of protein extraction, spot selection for analysis, and the ecotype used. Together, the three studies provide a broad spectrum of the Arabidopsis pollen proteome.

Differential expression of proteins in the wild type and 7B-1 male-sterile mutant anthers of tomato (Solanum lycopersicum): A proteomic analysis

In the 7B-1 male-sterile mutant of tomato, pollen development breaks down prior to meiosis in microspore mother cells (MMCs). We have used the proteomic approach to identify differentially expressed proteins in the wild type (WT) and mutant anthers with the objective of analyzing their roles in normal pollen development and in male sterility. By using 2-DE and DIGE technologies, over 1800 spots were detected and of these 215 spots showed 1.5-fold or higher volume ratio in either WT or 7B-1 anthers. Seventy spots, either up-regulated in WT, or in 7B-1, were subjected to mass spectrometry and 59 spots representing 48 distinct proteins were identified. The proteins up-regulated in WT anthers included proteases, e.g., subtilase, proteasome subunits, and 5B-protein with potential roles in tapetum degeneration, FtsZ protein, leucine-rich repeat proteins, translational and transcription factors. In 7B-1 anthers, aspartic protease, superoxide dismutase, ACP reductase, ribonucleoprotein and diphosphate kinase were up-regulated. Also, cystatin inhibitory activity was high in the mutant and correlated with the expression of male sterility. Other proteins including calreticulin, Heat shock protein 70, glucoside hydrolase, and ATPase, were present in both genotypes. The function of identified proteins in tapetum and normal pollen development, and in male sterility is discussed.

Benzylaminopurine induces phenocopies of floral meristem and organ identity mutants in wild-type Arabidopsis plants

Arabidopsis thaliana (L.) Heynh. has been used as a model system to investigate the regulatory genes that control and coordinate the determination, differentiation and morphogenesis of the floral meristem and floral organs. We show here that benzylaminopurine (BAP), a cytokinin, influences flower development inArabidopsis and induces partial phenocopies of known floral homeotic mutants. Application of BAP to wild-type inflorescences at three developmental stages results in: (i) increase in floral organ number; (ii) formation of abnormal floral organs and (iii) induction of secondary floral buds in the axils of sepals. These abnormalities resemble the phenotypes of mutants,clv1 (increase in organ number),ap1,ap2,ap3 (abnormal floral organs) andap1 (secondary floral buds in the axils of first-whorl organs). In addition, BAP induces secondary floral buds in the axils of perianth members ofapt2-6, ap3-1 andag mutants, and accentuates the phenotype of theapt2-1 mutant to resemble theapt2-6 mutant. These observations suggest that exogenous BAP suppresses the normal functioning of the genes for floral meristem identity and thereby affects flower development and the later stages of floral organ differentiation.

MS32-regulated timing of callose degradation during microsporogenesisin Arabidopsis is associated with the accumulation of stacked rough ER in tapetal cells

Abstract  In the male sterile32(ms32)mutant in Arabidopsis thaliana, pollen development is affected during meiosis of pollen mother cells (PMCs). In normal wild-type (WT) anthers, callose is deposited around PMCs before and during meiosis, and after meiosis the tetrads have a complete callose wall. In ms32, PMCs showed initial signs of some callose deposition before meiosis, but it was degraded soon after, as was part of the cellulosic wall around the PMCs. The early dissolution of callose in ms32 was associated with the occurrence of extensive stacks of rough ER (RER) in tapetal cells. The stacks of RER were also observed in the WT tapetum, but at a later stage, i.e., after the tetrads were formed and when callose is normally broken down for release of microspores. Based on these observations it is suggested that: (1) callose degradation around developing microspores is linked to the formation of RER in tapetal cells, which presumably synthesize and/or secrete callase into the anther locule, and (2) mutation in MS32 disrupts the timing of these events.

Dynamics of protein expression during pollen germination in canola (Brassica napus).

The proteome of mature (MP) and in vitro germinating pollen (GP) of canola (Brassica napus) were analyzed using the DIGE technology with the objective of identifying proteins and their function in pollen germination. Of the 2,238 protein spots detected in gel images, 344 were differentially expressed in MP and GP samples of which 165 were subjected to MALDI-TOF/TOF and 130 were successfully identified using the NCBInr and Brassica EST databases. The major proteins up-regulated in GP, relative to MP, have roles in carbohydrate metabolism, protein metabolism, and cell wall remodeling. Others with roles in cytoskeleton dynamics, nucleotide and amino acid metabolism, signal transduction, and stress response also showed higher expression in GP. Proteins concerned with transcriptional regulation and ion transport were similar in MP and GP, and some catalases and LEA proteins were down-regulated in GP. A number of proteins including, oleosin, cruciferin, and enolase, were released into the pollen germination medium indicating their potential role in pollen–stigma interaction. Glycosylated proteins were also identified in MP and GP, but their protein profiles were not different. This study has documented the dynamics of protein expression during pollen germination and early tube growth in B. napus and provides insights into the fundamental mechanisms involved in these processes, and in cell growth, cell–cell communication, and cell signaling.

Seed germination in a tomato male-sterile mutant is resistant to osmotic, salt and low-temperature stresses

Abstract Seed germination in a male-sterile 7B-1 mutant in tomato is reletively more resistant to the inhibitory effects of a high osmoticum induced by mannitol and polyethylene glycol, to various salts, including NaCl, Na2SO4, KCl and K2SO4, and to low-temperature stress, compared to the wild-type (WT) seeds. The inhibitory effects of various stresses could be partly or completely overcome by fluridone (FLU), an inhibitor of abscisic acid (ABA) biosynthesis. However, lower concentration of fluridone was required for the 7B-1 mutant than for WT seeds, and the mutant seeds were more sensitive to the inhibitory effects of exogenous ABA. The data suggest that 7B-1 seed has a pre-existing level of elevated ABA which imparts resistance to the various stresses. The ability to regulate male sterility in the 7B-1 mutant by photoperiod, as previously reported by Sawhney (1997), and its resistance to abiotic stresses, as reported here, makes this a useful system for tomato breeding and in hybrid programs.

The Role of Plant Growth Regulators, Sucrose and pH in the Development of Floral Buds of Tomato (Lycopersicon esculentum Mill.) Cultured in vitro

Summary The relative efficiency of different cytokinins i.e., BAP, kinetin, zeatin and zeatin riboside, and the role of NAA, GA3, sucrose and pH, on the in vitro growth and development of young floral buds of tomato (Lycopersicon esculentum Mill.) was examined. Of the various cytokinins tested, BAP was the most and zeatin the least effective in terms of the number of floral buds attaining maturity. Naphthaleneacetic acid alone was without effect, as well, it antagonized the promotory effect of BAP on flower development. Gibberellic acid alone was also ineffective, but in combination with BAP — and depending upon the GA3 concentration — it was either inhibitory, ineffective, or promotory for the growth of different floral organs. The optimal concentration of sucrose for the maturation of flowers was 3%, but the growth of floral organs was maximal at 4% sucrose level. Low pH (3.8–4.5) enhanced the growth of some floral parts, but the number of flowers reaching maturity was maximum at pH 5.8. This study shows that the in vitro growth and maturation of floral organs of tomato require different levels of various factors.

Enzymatic changes in post-meiotic anther development in Petunia hybrida. I: Anther ontogeny and isozyme analyses

Summary The relationship of the occurrence of isozymes of esterase, peroxidase, alcohol dehydrogenase (ADH) and malate dehydrogenase (MDH) with nine stages of the post-meiotic anther development of Petunia hybrida was examined. The esterases were primarily found in stages i to iii when tapetum degeneration was taking place, and later a single band was observed during pollen maturation. Peroxidases were also observed during the earlier stages and their occurrence was related to the differentiation of the endothecium. The activity of ADH was not observed until stage iv and it increased through to anther maturation. The occurrence of ADH was considered to be due to the anaerobic conditions of the maturing pollen and the dessication of anther tissue at later stages of development. Different forms of MDH were present at all nine stages of development and were suggested to be involved with the differentiation of various tissues.

Pleiotropic effects of the male sterile33 (ms33) mutation in Arabidopsis are associated with modifications in endogenous gibberellins, indole-3-acetic acid and abscisic acid

AbstractEarlier, we reported that mutation in the Male Sterile33 (MS33) locus in Arabidopsis thaliana causes inhibition of stamen filament growth and a defect in the maturation of pollen grains [Fei and Sawhney (1999) Physiol Plant 105:165–170; Fei and Sawhney (2001) Can J Bot 79:118–129]. Here we report that the ms33 mutant has other pleiotropic effects, including aberrant growth of all floral organs and a delay in seed germination and in flowering time. These defects could be partially or completely restored by low temperature or by exogenous gibberellin A4 (GA4), which in all cases was more effective than GA3 Analysis of endogenous GAs showed that in wild type (WT) mature flowers GA4 was the major GA, and that relative to WT the ms33 flowers had low levels of the growth active GAs, GA1 and GA4, and very reduced levels of GA9, GA24 and GA15, precursors of GA4. This suggests that mutation in the MS33 gene may suppress the GA biosynthetic pathway that leads to GA4 via GA9 and the early 13-H C20 GAs. WT flowers also possessed a much higher level of indole-3-acetic acid (IAA), and a lower level of abscisic acid (ABA), relative to ms33 flowers. Low temperature induced partial restoration of male fertility in the ms33 flowers and this was associated with partial increase in GA4. In contrast, in WT flowers GA1 and GA4 were very much reduced by low temperature. Low temperature also had little effect on IAA or ABA levels of ms33 flowers, but did reduce (>2-fold) IAA levels in WT flowers. The double mutants, ms33 aba1-1 (an ABA-deficient mutant), and ms33 spy-3 (a GA signal transduction mutant) had flower phenotypes similar to ms33. Together, the data suggest that the developmental defects in the ms33 mutant are unrelated to ABA levels, but may be causally associated with reduced levels of IAA, GA1 and GA4, compared to WT flowers.

Enzymatic Changes in Post-meiotic Anther Development in Petunia hybrida. II. Histochemical Localization of Esterase, Peroxidase, Malate- and Alcohol dehydrogenase

Summary The histochemical localization of esterase, peroxidase, malate (MDH), and alcohol dehydrogenase (ADH) was examined in nine stages of post-meiotic anther development in Petunia hybrida . Esterase was found mostly in the tapetum at early stages and later only in the pollen grains. Peroxidase was also found in the early stages, but in the cell walls of inner wall layers and in the endothecium. MDH was observed in most tissues of the anther including stomium, inner wall layers, tapetum and endothecium, but not in pollen grains. In contrast, ADH was observed only in the pollen grains. The role of these enzymes in the differentiation of the various tissues of the anther is discussed.