In flowering plants, pollen grains are produced in the anther and released to the external environment with the primary function of delivering sperm cells to the female gametophyte. This study was conducted to identify proteins in tomato pollen and to analyse their roles in relation to pollen function. Tomato is an important crop which is grown worldwide and is an excellent experimental system. Proteins were extracted from pollen, separated by two-dimensional gel electrophoresis (2-DE), and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDITOF MS) and peptide mass fingerprinting. Of the 960 spots observed on Colloidal Coomassie Blue (CCB)stained 2-DE gels, 190 were selected for analysis. Of these, 158 spots, representing 133 distinct proteins, were identified by searching the NCBInr and Expressed Sequence Tag databases. The identified proteins were classified based on designated functions and the majority included those involved in defence mechanisms, energy conversions, protein synthesis and processing, cytoskeleton formation, Ca 2+ signalling, and as allergens. A number of proteins in tomato pollen were similar to those reported in the pollen of other species; however, several additional proteins with roles in defence mechanisms, metabolic processes, and hormone signalling were identified. The potential roles of the identified proteins in the survival strategy of the small, independent, two-celled pollen grain of tomato, and subsequently in pollen germination and tube growth are discussed.

Proteomic analysis of tomato (Lycopersicon esculentum) pollen

Cryopreservation of tropical plant germplasm

The cyclin-dependent protein kinases (CDKs) have a central role in cell cycle regulation and can be inhibited by the binding of small protein CDK inhibitors. The first plant CDK inhibitor gene ICK1 was previously identified in Arabidopsis thaliana. In comparison to known animal CDK inhibitors, ICK1 protein exhibits unique structural and functional properties. The expression of ICK1 directed by the constitutive CaMV 35S promoter was shown to inhibit cell division and plant growth. The aim of this study was to determine the effects of ICK1 overexpression on particular organs and cells. ICK1 was expressed in specific tissues or cells of Brassicanapus L. plants using two tissue-specific promoters, ArabidopsisAP3 and BrassicaBgp1. Transgenic AP3-ICK1 plants were morphologically normal except for some modified flowers either without petals or with petals of reduced size. Surprisingly, petals of novel shapes such as tubular petals were also observed, indicating a profound effect of cell division inhibition on morphogenesis. The cell size in the smaller modified petals was similar to that in control petals, suggesting that the reduction of petal size is mainly due to the reduction of cell numbers and that the inhibition of cell division does not necessarily lead to an increase in cell size. Transgenic Bgp1-ICK1 plants were normal morphologically; however, dramatic decreases in seed production were observed in some plants. In those plants, the ability of pollen to germinate and pollen nuclear number were affected. These results are discussed in relation to the cell cycle and plant development.

Control of petal and pollen development by the plant cyclin-dependent kinase inhibitor ICK1 in transgenic Brassica plants.

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.

https://link.springer.com/content/pdf/10.1007%2Fs00497-006-0035-3.pdf

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

Winter-grown canola (Brassica napus L.) production is limited mostly by frost and winter kill in the southern canola-growing regions of the United States. Tolerance to cold and heat were assessed by studying percentage of pollen viability (PV), in vitro pollen germination (PG) and pollen tube length (PTL) for 12 field-grown cultivars. Freshly collected pollen from all cultivars were incubated on artificial solid growth media at a constant temperature ranging from 10 to 35 � Ca t 5� C interval for 30 h to determine PG and PTL. A modified bilinear model best described the temperature response functions of PG and PTL. Canola cultivars showed significant variability (P < 0.001) for PV (61.3 % to 89.7 %), PG (29.0 % to 48.2 %) and PTL (463 to 931 lm). The average cardinal temperatures, Tmin ,T opt and Tmax, for PG and PTL were 6.4, 24.3 and 33.7 � C, respectively. Principal component analysis revealed that maximum PG, PTL, Tmin and Topt of both PG and PTL were the most important factors in determining cold tolerance, whereas Tmax of PG and PTL, and maximum PG and PTL were more responsible in separating the cultivars for heat tolerance. The canola cultivar, KS3077, was the most cold tolerant with the lowest Tmin and the widest temperature adaptability range, and the cultivar Kadore was the most heat tolerant with the highest Tmax for the PG. The identified cold- and heat-tolerant cultivars may be useful in canola-breeding programmes to develop cultivars suitable for a niche environment.

Assessment of Cold and Heat Tolerance of Winter‐grown Canola (Brassica napus L.) Cultivars by Pollen‐based Parameters

The culture media for the in vitro pollen assay of Brassica species have so far shown good percentage germination, but limited pollen tube growth. It is reported here that by lowering sucrose concentration from 20% to 5 or 10 % and by adding polyethylene glycol 4000 in the culture medium, high percentage germination and high tube growth (10 times that in the standard media) can be achieved in three species of cultivated Brassica. This improved medium should be useful in fundamental and applied studies on the pollen biology of Brassica species.

K.R. Shivanna

Papers

Polyethylene glycol improves the in vitro growth of Brassica pollen tubes without loss in germination