Water status and associated processes mark critical stages in pollen development and functioning

Abstract

Background: The male gametophyte developmental programme can be divided into five phases which differ in relation to the environment and pollen hydration state: (1) pollen develops inside the anther immersed in locular fluid, which conveys substances from the mother plant--the microsporogenesis phase; (2) locular fluid disappears by reabsorption and/or evaporation before the anther opens and the maturing pollen grains undergo dehydration--the dehydration phase; (3) the anther opens and pollen may be dispersed immediately, or be held by, for example, pollenkitt (as occurs in almost all entomophilous species) for later dispersion--the presentation phase; (4) pollen is dispersed by different agents, remaining exposed to the environment for different periods--the dispersal phase; and (5) pollen lands on a stigma and, in the case of a compatible stigma and suitable conditions, undergoes rehydration and starts germination--the pollen-stigma interaction phase.

Scope: This review highlights the issue of pollen water status and indicates the various mechanisms used by pollen grains during their five developmental phases to adjust to changes in water content and maintain internal stability.

Conclusions: Pollen water status is co-ordinated through structural, physiological and molecular mechanisms. The structural components participating in regulation of the pollen water level, during both dehydration and rehydration, include the exine (the outer wall of the pollen grain) and the vacuole. Recent data suggest the involvement of water channels in pollen water transport and the existence of several molecular mechanisms for pollen osmoregulation and to protect cellular components (proteins and membranes) under water stress. It is suggested that pollen grains will use these mechanisms, which have a developmental role, to cope with environmental stress conditions.

Fig. 1.

A semi-diagrammatic scheme of male gametophyte development from the tetrad stage to pollen tube emission, indicating changes in volume (solid line) and water content (dashed line). The scheme does not consider species with tricellular pollen. Vacuoles are in blue, starch is in red and cytoplasmic carbohydrates are in pink. (1) Late tetrad stage. (2) Early microspore stage; small vacuoles are formed. (3) Late vacuolated microspore stage, before the first haploid mitosis; small vacuoles merge into a large one. (4) Early bicellular stage; starch deposition begins, as well as vacuole reduction. (5) Late bicellular stage; vacuoles are absent, starch deposition reaches maximum levels. From stage 5, two possibilities are illustrated (6 and 6¹). (6) Almost mature pollen grain; starch is completely hydrolysed and carbohydrates are present in the cytoplasm. (6¹) Almost mature pollen grain; starch is only partially hydrolysed. (7) Mature pollen, of the partially hydrated type (recalcitrant; Nepi et al., 2001), containing cytoplasmic carbohydrates. (7¹) Mature pollen of the partially dehydrated type (orthodox; Nepi et al., 2001), containing cytoplasmic carbohydrates. (7²) Mature recalcitrant pollen with starchy carbohydrate reserves. (7³) Mature orthodox pollen with starchy carbohydrate reserves. (8) After landing on the stigma, pollen, independently of the hydration state and type of carbohydrate reserves, rehydrates and small vacuoles are formed. (9) Small vacuoles merge into a large one and a pollen tube is emitted. Starch and cytoplasmic carbohydrates interconvert during presentation and dispersal, enabling control of water loss and water influx, and are consumed (or transformed) during rehydration and germination. Pollen water content (dependent on water absorbed from the anther through the tapetal cells and then through the loculus) starts increasing at the microspore stage, in parallel with the initial increase in volume, up to a few days before flower opening (according to the species; phase 1). Upon beginning the dehydration phase (phase 2), pollen water content starts to decrease, reaching a minimum at maturity. During presentation (phase 3) and dispersal (phase 4), water content adjusts to environmental changes (depending on the species) and will increase sharply during rehydration, upon landing on a compatible stigma (phase 5). This precedes the emission of the pollen tube which grows in the style and will reach the ovule where sperm cells will be delivered for fertilization. It should be noted that the illustrated changes in pollen water content during development are speculative since, to the best of our knowledge, no such measurements have ever been reported. The time scale for water content during the different phases varies widely according to the species and is deeply affected by the environment during dispersal. In general: phase 1 is the longest, lasting from several days to several months, whilst the other phases last from seconds to days. Pollen volume starts increasing significantly at the early microspore stage and continues to increase until partial dehydration. Pollen volume decreases during anther and pollen dehydration and, similar to water content, adapts to environmental conditions during dispersal, increasing during rehydration.

Fig. 2.

Cryo-scanning electron micrograph of Petunia hybrida pollen after anther opening. (A) Pollen before flower opening. (B) Pollen after flower opening. Pollen grains lose water after flower opening and change in size and shape, from spheroid to ovoid. The furrow area is distended in the closed flower (asterisks) whereas it is folded in the open flower after partial pollen dehydration (arrows). Pictures were taken at the Department of Plant Cytology and Morphology, University of Wageningen, The Netherlands, in 1999 using a JEOL 6300F field emission cryo-SEM.

Fig. 3.

Cryo-fractures of Cucurbita pepo pollen observed with a cryo-SEM after freeze-drying (10 min at –90 °C in a vacuum of 5 × 10⁻⁸ hPa). The sites containing water appear as vesicle-like structures (dark holes) in the cytoplasm. These vesicles are highly abundant in the cytoplasm of C. pepo pollen from just-opened anthers (water content 43 %). They decrease in pollen dehydrated to 13 % water content and they are no longer evident in pollen dehydrated to 8·5 % water content. Amyloplasts (a) and their imprints (asterisks) are evident in C. pepo pollen. Pollen viability is high at both 43 and 13 % water content, but it decreases drastically upon further dehydration to 8·5 % water content. This degree of dehydration is induced by pollen exposure to 30 % relative humidity for 90 min (see data in Nepi et al., 2010). Pictures were taken at the Department of Plant Cytology and Morphology, University of Wageningen, The Netherlands, in 1999 using a JEOL 6300F field emission cryo-SEM. Scale bar = 2 µm.

Fig. 4.

Changes in sucrose concentration in developing pollen grains of tomato ‘Hazera 3017’. Sucrose concentration was analysed according to Hubbard et al. (1990) as described in Firon et al. (2006), at six stages of development, 9, 7, 5, 3, 1 and 0 d before anthesis (A-9 to A-1 and A, respectively). Data represent the average of at least three biological replicates. Vertical bars represent the s.e.