The influence of seed morpho-physiological and meristematic cell wall traits on seed desiccation sensitivity in three angiosperm tree species

Flora 261 (2019) 151490

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The influence of seed morpho-physiological and meristematic cell wall traits on seed desiccation sensitivity in three angiosperm tree species

T

Ashley Subbiah, Wynston R. Woodenberg, Boby Varghese, Norman W. Pammenter, Sershen* School of Life Sciences, University of KwaZulu-Natal, Westville Campus, Private Bag X 54001, Durban, 4000, South Africa

A R T I C LE I N FO

A B S T R A C T

Edited by Alessio Papini

The structure and composition of physical barriers to water loss such as the seed coat and embryo meristematic cell walls (CWs) can potentially affect the rate and topographical patterns of water loss and consequently desiccation sensitivity in recalcitrant seeds. This study provides insights into the phenomenon of seed recalcitrance, by investigating the influence of seed morpho-physiological and (root) meristematic CW structure and composition on seed desiccation sensitivity in three recalcitrant-seeded angiosperm tree species, viz. a mangrove tree, Avicennia marina (Forssk.) Vierh. (Acanthaceae); a sub-tropical forest tree, Trichilia dregeana Sond. (Meliaceae); and a tropical fruit tree, Artocarpus heterophyllus Lamk. (Moraceae). Whole seed water content (WC), dry mass (DM), seed coat ratio (SCR), and root meristem CW thickness and composition were related to embryo desiccation sensitivity and drying rate in each of the three species. The embryos of the heavier A. marina and A. heterophyllus seeds were faster drying and more desiccation sensitive than the relatively lighter T. dregeana seeds. The apportioning of more water towards the embryo in A. marina and A. heterophyllus than in T. dregeana, may be an evolutionary consequence of their faster drying rate and greater desiccation sensitivity. In both A. marina and A. heterophyllus, the ratio of highly esterified to unesterified pectins was lower, and the meristem CW thickness was greater than in T. dregeana. The thick, rigid CWs of A. marina and A. heterophyllus probably contributes to their greater desiccation sensitivity, since desiccation tolerance in vegetative tissues has previously been associated with thin flexible CWs. Seed water distribution patterns and CW thickness and rigidity may be useful indicators of seed desiccation sensitivity. The degree and nature to which these morpho-physiological and ultrastructural traits influence seed desiccation sensitivity in individual species may be a consequence of habitatspecific evolutionary pressures.

Keywords: Desiccation tolerance Recalcitrant Ultrastructure Zygotic embryo

1. Introduction Desiccation sensitive or recalcitrant seeds are shed highly hydrated, characteristically display variable degrees of viability loss upon removal of structure-associated water and exhibit various signs of dehydration stress when free water is removed (Pammenter and Berjak, 1999). This can be ascribed to the incomplete expression of protective mechanisms that confer desiccation tolerance in orthodox seeds (Pammenter and Berjak, 1999). Cryopreservation, storage at ultra-low temperatures (generally around −196 °C), is widely recognised as the most feasible strategy for the long-term ex situ conservation of recalcitrant zygotic seed germplasm (Engelmann, 2011). Partial

dehydration is a necessary and common pre-conditioning treatment for the cryopreservation of recalcitrant seed germplasm (Ballesteros et al., 2014). However, the inherent variability in drying rate and desiccation sensitivity of embryonic explants across species (Subbiah et al., 2017), challenges the success of cryopreservation protocols for recalcitrantseeded species (Berjak and Pammenter, 2014). Differences in drying rate and desiccation sensitivity across recalcitrant-seeded species have a number of ecophysiological implications. For instance, desiccation sensitivity of recalcitrant seeds may be a significant disadvantage post-shedding, particularly if seeds are shed in dry spells characteristic of more temperate habitat types or in the dry season in tropical and sub-tropical habitats. This highlights the

Abbreviations: ANOVA, analysis of variance; BSA, bovine serum albumin; CW, cell wall; DM, dry mass; DSI, desiccation stress index; EXP, exponential; FBS, foetal bovine serum; LM2, antibody for arabinogalactan protein; LM5, antibody for galactan protein; LM6, antibody for arabinan side-chains of homogalacturonan; LM7, antibody for epitopes of partially methyl-esterified homogalacturonan; LM14, antibody for epitopes of arabinogalactan protein; LM19, antibody for unesterified epitopes of homogalacturonan; LM20, antibody for highly methyl-esterified epitopes of homogalacturonan; IH, modified inverse hyperbolic; P50, 50% germination; PBS, phosphate buffered saline; PCW, primary cell wall; RWC, relative water content; SCR, seed coat ratio; SOP, second order polynomial; td, point of significant damage accumulation; WC, water content ⁎ Corresponding author; [email protected] https://doi.org/10.1016/j.flora.2019.151490 Received 17 July 2019; Received in revised form 17 October 2019; Accepted 24 October 2019 Available online 31 October 2019 0367-2530/ © 2019 Elsevier GmbH. All rights reserved.

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and Renzaglia, 2014). The esterification state of homogalacturonan plays a key role in controlling porosity and mechanical properties of the CW (Merced and Renzaglia, 2014; Woodenberg et al., 2018). Highly methyl-esterified homogalacturonan confers a greater flexibility in the CW (Merced and Renzaglia, 2014; Zhang et al., 2017). In contrast, unesterified homogalacturonan provides sites for calcium binding to confer greater rigidity in the CW (Merced and Renzaglia, 2014; Zhang et al., 2017). Interestingly, recent studies have shown the implications of embryo CW composition and structure on desiccation sensitivity in recalcitrant seeds of gymnosperms (Woodenberg et al., 2015, 2018); however, at the time of this study no published data on the composition and structure of meristematic CWs of recalcitrant seeds from angiosperms were available. For this reason, selected embryo root meristem CW traits (thickness, flexibility/ rigidity and porosity) were also compared across the three species in the context of their relative desiccation sensitivity.

ecological importance of mechanisms that may prevent water loss in seeds, which can reduce seed mortality post-shedding (Xia et al., 2012). This may also explain why recalcitrant seeds, which exhibit a global incidence of approximately c. 8%, are produced largely by mature phase forest species that occur in the mesic tropics and sub- tropics (Tweddle et al., 2003; Wyse and Dickie, 2017). The recalcitrant trait may actually be an advantage in these environments, reducing the risk of desiccation and predation by germinating rapidly and allocating more resources towards the seed embryo than seed defensive covering structures (Pritchard et al., 2004; Daws et al., 2005; Xia et al., 2012). Several studies have focused on biochemical, metabolic, physiological and molecular factors that influence desiccation sensitivity in recalcitrant seeds (Varghese et al., 2011; Ballesteros et al., 2014; Costa et al., 2016; Sershen et al., 2016). Comparatively less consideration has been afforded to seed morphological and ultrastructural adaptations that may influence their germination rate, drying rate and most importantly desiccation sensitivity (but see Xia et al., 2012 and Woodenberg et al., 2018). Studies that have investigated the influence of morphological and physiological seed traits on recalcitrant seed survival have shown that they are generally relatively large, sphere-shaped, prone to rapid germination, are shed at relatively high water content (WC) and possess thin seed coats (Hong et al., 1998; Dickie and Pritchard, 2002; Tweddle et al., 2003; Pritchard et al., 2004; Daws et al., 2006). However, the presence and nature of physical barriers to water loss such as the seed coat, embryo cuticle and endosperm/cotyledon(s) (Sobrino Vesperinas and Viviani, 2000; Pammenter et al., 2002; Ntuli and Pammenter, 2009; Xia et al., 2012) and the meristematic cell walls (CWs) (Woodenberg et al., 2015, 2018) also have the potential to influence the rate of water loss and consequently desiccation sensitivity. This formed the focus of the present study on the influence of seed morpho-physiological and meristematic cell wall traits on seed desiccation sensitivity in three angiosperm species: a mangrove tree, Avicennia marina (Forssk.) Vierh. (Acanthaceae); a sub-tropical forest tree, Trichilia dregeana Sond. (Meliaceae); and a tropical fruit tree, Artocarpus heterophyllus Lamk. (Moraceae). Seed dry mass (DM) and WC, seed coat ratio (SCR), and root meristem cell CW thickness and composition were determined in each of the three species and these parameters were then related to drying rate and desiccation sensitivity. Recent studies on the relationship between drying rate and desiccation sensitivity in recalcitrant seeds (Ballesteros et al., 2014; Sershen et al., 2016) have suggested that indices of desiccation sensitivity must integrate the effects of stress intensity and duration. For this reason, this study used a desiccation stress index (DSI) that was recently shown to successfully integrate the effects of stress intensity (WC) and duration (drying rate) (Subbiah et al., 2017) to compare desiccation sensitivity across the three species. Seed DM, WC and SCR, have previously been shown to be relatively easy to determine and offer readily comparable ‘correlates’ of desiccation sensitivity across species (Tweddle et al., 2003; Pritchard et al., 2004; Daws et al., 2005, 2006). The three dimensional structural conformation and chemical composition of CWs are integral influences on their inherent and developmental functional properties (Reiter, 2002; Sarkar et al., 2009). Therefore, elucidating the structure and composition of CWs, can help understand their functional characteristics within specific tissues (Reiter, 2002), and in our case their possible influence on the drying rate and desiccation sensitivity of embryonic axes of the recalcitrant seeds. For example, the proportion of hemicellulose and pectin fractions of cellulose microfibrils, which typically make up 30% of the macromolecules constituting the CW, may change in response to biotic and abiotic (e.g. desiccation) stresses (Sarkar et al., 2009; Woodenberg et al., 2015). The pectic polysaccharides, of which homogalacturonan is the major group, are abundant (as much as 30% in CWs of dicotyledonous angiosperms (Zhang et al., 2017). Homogalacturonan, secreted in the Golgi bodies in a highly methyl-esterified form, is modified in the CW by removal of their methyl groups (Merced

2. Materials and methods 2.1. Plant material Mature fruits of A. marina, T. dregeana and A. heterophyllus were harvested directly from parent trees in Durban, South Africa, and conveyed to the laboratory immediately. Fruits (propagules – seeds) of A. marina were collected from the ground during low tide and care was taken to avoid seeds displaying visible signs of pericarp browning (indicative of deteriorative metabolic processes). Upon arrival in the laboratory the seeds were stored hydrated at 25 °C (after Calistru et al., 2000). All seeds were used within 24 h of being collected. Seeds of T. dregeana were collected once fruit/capsules had split and care was taken to avoid seeds displaying any visible signs of damage and predation. Fruits of A. heterophyllus were collected directly from parent trees and maintained at room temperature until fully mature (indicated by splitting of the rind and softening of the fruit pulp). Once the arils of T. dregeana were removed and the seeds of A. heterophyllus were extracted from mature fruits, the seeds were stored hydrated at 16 °C (after Goviea et al., 2004). Seeds of both species were used within 14 days of being collected as seeds of these species are known to store for months in this condition (Wesley-Smith et al., 2001; Goviea et al., 2004), compared with those of A. marina which can start deteriorating/ germinating after just four days in hydrated storage (Calistru et al., 2000). 2.2. Quantification of drying rate and degree of desiccation sensitivity 2.2.1. Drying kinetics and curve fitting Embryonic axes (whole axes for T. dregeana and A. heterophyllus, and root apices for A. marina) were excised and flash-dried at 24 °C (Pammenter et al., 2002) for a range of drying times, which differed across species. Preliminary studies indicated that upon rapid partial dehydration, water loss only in the root apices of the large elongated embryonic axes of A. marina affected the overall viability of these seeds. At each interval, 10 axes were removed and weighed with a six-place electronic balance (Mettler MT5; Germany), to determine fresh weight. These axes were then dried in an oven at 80 °C for 48 h to determine their DM and absolute WC was subsequently expressed on a dry mass basis (g H2O g−1 DM; referred to as g g-1). This absolute WC data were converted to relative water contents (RWC) (Pammenter et al., 2003), to account for wide differences in the shedding/initial WC and allow for consistency in the application of different functions to fit the drying data of all three species. The modified inverse hyperbolic (MIH) function of the form y = ab/(b + x) (Pammenter et al., 2003), an exponential (EXP) function of the form y = eax (Liang and Sun, 2000; Sun, 2002) and a second order polynomial (SOP) function of the form y = ax2 + bx + c (Subbiah et al., 2017), were used to fit drying data for each species, to evaluate the Quality of fit provided by each function in 2

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software version 1.45 (NIH, Bethesda, MD, USA).

describing the drying kinetics across species. Flash drying was conducted on a single batch of seeds within 7 days of seed collection for T. dregeana and A. heterophyllus and within 24 h of collection for A. marina.

2.4.2. Immunocytochemistry Cell wall composition of root meristem cell CWs was determined immunocytochemically following the methods outlined by Merced and Renzaglia (2014) and Woodenberg et al. (2018). Samples (n = 10 for each species) were excised and fixed as for determination of CW thickness, precluding post-fixation with osmium tetroxide. Samples embedded in LR White resin (London Resin Company, Berkshire, UK) were then sectioned (as described for determination of CW thickness) and ultra-thin sections were collected on 300-mesh nickel grids. Sections were then treated with 0.01 M phosphate-buffered saline (PBS) (pH 7.4) containing 0.2% polyethylene glycol 20 000 (Fluka, Buchs, Switzerland) for five minutes at room temperature. They were then incubated in a blocking solution of 0.01 M PBS (pH 7.4) containing 10% foetal bovine serum (FBS) (Delta Bioproducts, Kempton Park, South Africa), 1% bovine serum albumin (BSA) (Sigma, St Louis, Mo, USA), 0.05% Tween-20 (Sigma) and 0.2% sodium azide (Fluka), for 30 min at room temperature. Following this, they were incubated in 40 μl of a primary antibody preparation, Rat IgG (Plantprobes, Leeds, UK) prepared with a 1:20 dilution in 0.01 M PBS (pH 7.4), overnight at 4 °C in a moist chamber. After washing with 0.01 M PBS (pH 7.4) containing 1% BSA and 0.05% Tween-20, sections were treated with a secondary antibody preparation of goat antiserum to rat IgG (Sigma), conjugated with 10 nm colloidal gold and prepared with a 1:10 dilution in 950 μl of 0.01 M PBS (pH 7.4), containing 1% BSA, 0.05% Tween-20 and 50 μl of FBS, for one hour at room temperature. After washing with 0.01 M PBS (pH 7.4), samples were fixed with 1% glutaraldehyde for two minutes. After washing with sterile distilled water, sections were post-stained with saturated uranyl acetate. Sections were then viewed at an accelerating voltage of 100 kV. Specificity was assessed on control sections incubated in only one of the antibody preparations (i.e. either the primary or secondary preparation only). Primary antibodies used included LM2 for arabinogalactan protein (Smallwood et al., 1996), LM5 for galactan protein (Jones et al., 1997), LM6 for arabinan side-chains of homogalacturonan (Willats et al., 1998), LM7 for partially methyl-esterified homogalacturonan, LM14 for arabinogalactan protein, LM19 for unesterified epitopes of homogalacturonan (Verhertbruggen et al., 2009), and LM20 for highly methyl-esterified epitopes of homogalacturonan (Verhertbruggen et al., 2009).

2.2.2. Viability assessment At each drying interval on the flash drying curve viability (expressed as percentage germination) was assessed by in vitro culture of freshly excised and flash-dried embryonic axes (n = 10 for each time interval, for each of three experiments). Flash-dried embryonic axes were rehydrated in an aqueous solution of magnesium chloride (0.1 μM) and calcium chloride (0.01 μM). The rehydrated embryonic axes were then surface sterilised with 1% sodium hypochlorite for five minutes and rinsed thrice with dH2O before being cultured in Petri dishes containing full strength MS (Murashige and Skoog, 1962) with 3% (w/v) sucrose. Petri dishes were initially maintained in the dark until the first signs of germination, after which they were transferred to a growth room (18 h photoperiod; c. 23 °C). Germination was scored by the production of radicals (c. 2–3 mm). As described by Subbiah et al. (2017), flash drying times that coincided with 50% viability retention (P50) were used as an indicator of significant damage accumulation (td) in subsequent analyses. 2.2.3. Calculation of mean drying rate and desiccation stress index (DSI) Mean drying rate was calculated using the SOP fitted drying data and the following formula: mean drying rate = (RWC at initial – RWC at P50) / tP50 (minute−1) (Subbiah et al., 2017). Desiccation sensitivity was quantified as a DSI and calculated for each species by integration of the SOP function used to fit the drying data, to determine the area above the drying curve, with a horizontal limit at the initial RWC and a vertical limit at td, using the following formula: Area = td - [(a/3).(td3) + (b/2).(td2) + td], where td was represented by drying times at P50 (Subbiah et al., 2017). 2.3. Seed morpho-physiology Immediately after collection, 20 seeds (dispersal unit) of each of the investigated species were dissected and intact embryos (embryonic axis and cotyledonary body) were excised from the respective endocarp and testa. The embryo, endocarp and testa were then weighed on a six-place balance, to determine fresh mass, and subsequently dried in an oven at 80 °C for 48 h to determine the whole seed, embryo and endocarp and testa DM (g) and WC expressed on a dry mass basis (g g−1). To calculate SCR, the ratio of the DM of the covering structures (endocarp and testa) to the DM of the total seed was determined (Grubb and Burslem, 1998; Pritchard et al., 2004). We also describe the gross seed anatomy and in situ germination biology of the three species based on qualitative observations and existing literature in order to aid our interpretation of potential inter-species differences in seed morpho-physiology, meristematic CW traits and desiccation sensitivity.

2.5. Statistical analysis A Shapiro-Wilk test was conducted to test normality of the data. Parameters displaying non-normal distribution were log10 transformed for statistical analysis. Where data did not conform to parametric assumptions, even after transformation, non-parametric tests were applied. Differences in the DM and WC of seed components (whole seed, embryo and endocarp and testa), as well as SCR across species (data for all species were pooled) were tested by Analysis of variance (ANOVA) or a Kruskal-Wallis test (non-parametric data). Means were separated using Tukey’s HSD post-hoc test or were Bonferroni corrected by pairwise comparison (non-parametric data). Similarly, differences in root meristem cell CW thickness and the label density for all immunocytochemical probes, in freshly excised embryonic axes across species (data for all species were pooled), were also evaluated. Relationships between the DM and WC of seed components (whole seed, embryo and endocarp and testa), SCR, root meristem cell CW thickness and label density of all immunocytochemical probes in freshly excised embryonic axes, and drying rate across all species (data for all species were pooled) was assessed by Spearman’s rank correlation(ρ). Relationships between the above-mentioned parameters and DSI across all species (data for all species were pooled) was evaluated in a similar fashion. All statistical tests were performed at the 0.05 level of significance using IBM SPSS Statistics version 25 (IBM, New York, USA).

2.4. Transmission electron microscopy 2.4.1. Determination of CW thickness Cell wall thickness of root meristem cell CWs was determined by transmission electron microscopy and sample (small cubes of 1–2 mm3 excised from root apices of fresh undried embryonic axes; n = 3 for each species) preparation followed the methods outlined in Woodenberg et al. (2014). Resin-embedded samples were sectioned using a Riechert-Jung Ultracut E microtome and ultra-thin sections (100 μm) were collected on 200-mesh copper grids. Sections were then post-stained sequentially with saturated uranyl acetate and Reynolds lead citrate (Reynolds, 1963). Sections were viewed with a Jeol JEM 1010 transmission electron microscope at an accelerating voltage of 80 kV, using iTEM Soft Imaging System GmbH imaging software. Cell wall thickness of ten individual cells, within the root meristem, in each of the three samples, for each species, was measured using ImageJ 3

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survive within this environment (Berjak et al., 2011). They possess a velvety, grey-green pericarp that bursts open very shortly after shedding to expose the radicle (and hypocotyl), which is very loosely surrounded by cotyledons. The distal portion of the hypocotyl protrudes out of the cotyledons and is exposed to full sunlight, ambient air (during low tide), and flooding (during high tide) shortly (minutes to hours) after shedding. A thick mass of bristle-like hairs covers the distal end of the hypocotyl, protecting multiple (five or more) root primordia that are covered by a very thin layer of epidermal tissue and can germinate rapidly (within 3–4 days; Calistru et al., 2000). Trichilia dregeana (Fig. 1b) and A. heterophyllus (Fig. 1c) are both mature phase forest species native to the mesic tropics and sub-tropics and their seeds are large and highly hydrated at shedding, which facilitates fast (i.e. weeks as opposed to days as in A. marina) germination in seasonally wet environments. Trichilia dregeana seeds are shed in velvety green three-lobed capsules, most often containing six seeds with a waxy, thick protective aril. The embryonic axis, which is very small relative to the rest of the seed, is completely and tightly enclosed by large fleshy cotyledons. The axis only becomes exposed to ambient air and sunlight after the waxy aril has rotted away and the cotyledons separate, which can take a few weeks to a month (Ramlall et al., 2015). Narrow, elliptical to egg-shaped seeds of A. heterophyllus are shed in very large fleshy fruits (∼300-400 mm diameter) and can contain hundreds of seeds. The small embryonic axis (relative to the rest of the seed) is completely and tightly enclosed by large fleshy cotyledons. The axis is exposed to ambient air and full sunlight after the seed coat sloughs off and the cotyledons separate; this can take between 1–2 weeks to occur (authors’ unpublished observations). Artocarpus heterophyllus seeds possessed the highest whole seed DM and embryo DM (Fig. 2a). Seeds of T. dregeana possessed the lowest whole seed DM but embryo DM was marginally higher than in A. marina (Fig. 2a). Dry mass and initial WC of the whole seed, embryo, endocarp and testa were significantly different across species (Figs. 2a and b). While embryo WC was significantly different across species (Fig. 2b), embryo WC was significantly lower in T. dregeana than in A. marina and A. heterophyllus, with no significant difference between A. marina and A. heterophyllus (Fig. 2b). In A. heterophyllus seeds, which as mentioned above exhibited the lowest whole seed initial WC, 70% of the water was apportioned to the embryo, relative to the endocarp and testa (Fig. 2b). Similarly, whole seeds of A. marina (shed at a relatively higher WC) exhibited a greater apportioning of water to the embryo (54%), relative to the endocarp and testa (Fig. 2b). In contrast, in T. dregeana seeds (which exhibited the highest shedding WC) 70% of the water was allocated to the

Table 1 Initial water content (WC), drying time to P50 and % water loss at P50 for flashdried Artocarpus heterophyllus, Avicennia marina and Trichilia dregeana embryonic axes. Mean drying rate and desiccation stress index (DSI) (n = 10), were calculated for each species using the second order polynomial (SOP) model. Species

A. heterophyllus A. marina T. dregeana

Initial WC

Water loss at P50 %

Mean drying rate (minute−1)

DSI

(g g−1)

Drying time to P50 (minutes)

1.89 ± 0.44 1.94 ± 0.16 2.39 ± 0.38

30 15 180

46 14 69

0.020 0.009 0.004

8 1 102

3. Results 3.1. Drying kinetics Initial WC of excised embryonic axes revealed that seeds of all three species were shed highly hydrated, with explants of T. dregeana displaying the highest initial WC (Table 1). The A. marina and A. heterophyllus embryonic axes displayed similar initial WC but while A. marina reached P50 (15 min) after only 14% water loss, A. heterophyllus experienced 46% water loss and took twice as long (in terms of drying time) in reaching P50. Trichilia dregeana axes reached P50 after 69% water loss and 180 min of drying, six times longer than A. heterophyllus and 12 times longer than A. marina. The SOP function provided the best quality of fit for drying data across the three species. Based on SOP fitted drying data, A. heterophyllus embryonic axes displayed the fastest drying rate and T. dregeana the slowest (Table 1). Since lower DSI values indicate a greater sensitivity to desiccation and vice versa (Subbiah et al., 2017), the species could be ranked from most sensitive to least sensitive as follows: A. marina > A. heterophyllus > T. dregeana (Table 1). Initial WC and WC at P50 were significantly different (p < 0.05) when compared across species (data not shown). There was no significant relationships between DSI and initial WC, WC at P50 and drying rate. 3.2. Seed gross anatomical and morpho-physiological characteristics The fruit of the mangrove species, A. marina (Fig. 1a) occur in coastal habitats exposed to tidal and inter-tidal oscillations. Their large highly hydrated propagules, typically dispersed into water, have evolved specialised morphological and anatomical adaptations to

Fig. 1. (a) Avicennia marina propagules showing thin pericarp which encloses seeds upon shedding (inset) and the radicles that emerge from the multiple meristems when the pericarp is sloughed hours to 1–2 days after shedding; (b) Trichilia dregeana seeds showing the waxy red and black aril that encloses them and the capsule within which the seeds develop; (c) Artocarpus heterophyllus fruit (usually > 200 mm in length) showing pulp (top inset) which encloses seeds (bottom inset). Bar = 10 mm (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article). Source for (C): https://i.ebayimg.com/images/g/51sAAOSwB4NWyBDZ/s-l300.jpg 4

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Table 2 Results of Spearman’s rank correlation analysis (ρ) for Artocarpus heterophyllus, Avicennia marina and Trichilia dregeana seeds, between dry mass (DM) and water content (WC) of each seed component (whole seed, embryo and endocarp and testa), and drying rate and desiccation stress index (DSI). Data for all species were pooled for regression analysis. Values in bold represent significant correlations (WC = water content, DM = dry mass and SCR = seed coat ratio). Drying rate

Whole seed WC Embryo WC Endocarp and testa WC Whole seed DM Embryo DM Endocarp and testa DM SCR

DSI

P

p

ρ

P

−0.63 0.14 −0.68 0.84 0.70 −0.27 −0.47

< 0.01 0.28 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01

0.11 −0.45 0.14 −0.40 0.02 −0.56 −0.47

0.38 < 0.01 0.29 < 0.01 0.87 < 0.01 < 0.01

Fig. 3. Cell wall thickness of Avicennia marina, Trichilia dregeana and Artocarpus heterophyllus root meristem cells. Vertical bars represent mean ± SD (n = 3 for all species). Bars labelled with different letters are significantly different when compared across species (p < 0.05, Kruskal-Wallis test).

cell wall epitopes (LM2, LM5, LM6, LM7, LM14, LM19, and LM20). However, LM7, which binds to partially methyl-esterified homogalacturonan, and LM14, which labels arabinogalactan protein were present in T. dregeana only. Hence, the presence of the remaining five epitopes, which could be detected in CWs of all three species, were quantified in terms of abundance (i.e. label density; number of spots per μm2) and compared across species (Fig. 4). Sparse labelling of arabinogalactan protein (LM2) was localised predominantly in the CW and cytomatrix within close proximity to the plasmalemma across all species (Figs. 5a, 6 a and 7 a). Dense labelling of galactan protein (LM5) was localised predominantly in the primary cell walls (PCWs) of A. marina (Fig. 5b). Also, there was dense LM5 labelling throughout the CWs of A. heterophyllus (Fig. 6b) and T. dregeana (Fig. 7b). Dense labelling of arabinan side-chains of homogalacturonan (LM6) was localised in the CWs of A. heterophyllus (Fig. 6c). However, sparse LM6 labelling was localised predominantly in the PCWs of A. marina (Fig. 5c) and T. dregeana (Fig. 7c). Dense labelling of unesterified epitopes of homogalacturonan (LM19) was localised predominantly in the middle lamella of CWs of T. dregeana (Fig. 7D). Also, there was dense LM19 labelling throughout the CWs of A. marina (Fig. 5d) and A. heterophyllus (Fig. 6d). Dense labelling of highly methyl-esterified epitopes of homogalacturonan (LM20) was localised predominantly in the PCWs of A. marina (Fig. 5e). Also, dense LM20 labelling was evenly distributed throughout the CWs of A. heterophyllus (Fig. 6e) and T. dregeana (Fig. 7e). Drying rate was significantly positively correlated with CW abundance of both LM19 and LM20, when data for all species were pooled for analysis (Table 3). Desiccation stress index was significantly positively correlated with CW abundance of LM5, LM6, and LM20 and significantly negatively correlated with CW abundance of LM19, when data for all species were pooled for analysis (Table 3).

Fig. 2. Whole seed, embryo and endocarp and testa (a) dry mass (DM), (b) water content (WC) and (c) the seed coat ratio (SCR) of Avicennia marina, Trichilia dregeana and Artocarpus heterophyllus seeds. Bars represent mean ± SD (n = 20 for all species) and when labelled with different letters are significantly different when compared within and across seed components (p < 0.05, Kruskal-Wallis test or *ANOVA).

endocarp and testa (Fig. 2b). Artocarpus heterophyllus possessed the lowest SCR, indicative of the thinnest seed coat, while A. marina seeds possessed the highest SCR (Fig. 2c). Drying rate was significantly positively correlated with whole seed and embryo DM, and significantly negatively correlated with endocarp and testa DM, whole seed and endocarp and testa WC, and SCR (Table 2). The DSI was significantly negatively correlated with whole seed and endocarp and testa DM, embryo WC and SCR (Table 2). 3.3. Cell wall thickness Artocarpus heterophyllus possessed the thickest and T. dregeana the thinnest CWs, and these differences were significant (Fig. 3). Cell wall thickness was significantly positively correlated with drying rate (ρ = 0.55, p < 0.01) and significantly negatively correlated with DSI (ρ = -0.45, p < 0.01). 3.4. Cell wall composition The three species were initially assessed for the presence of seven 5

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label density of LM5 and LM6 (LM5:LM6) were also calculated. Label density of LM5 was considerably higher than LM6 in all species (∼5:1), with no specific trend related to drying rate or DSI across species (data not shown).

4. Discussion Inter- and even intra- species differences in desiccation sensitivity have been studied in a number of species (Ntuli and Pammenter, 2009; Ballesteros et al., 2014; Sershen et al., 2016) but these differences have very rarely been interpreted in the context of seed morpho-physiological and/ or embryo ultrastructural traits (but see Pammenter et al., 2011). A systematic analysis of desiccation sensitivity in the three angiosperm tree species investigated here revealed the embryonic axes of A. marina and A. heterophyllus to be significantly more sensitive than T. dregeana (Table 1). Generally, recalcitrant seeds are shed at WC > 0.4 g g−1 (Pammenter and Berjak, 1999) and are most often larger in seed size and heavier than desiccation tolerant orthodox types (Dickie and Pritchard, 2002; Pritchard et al., 2004; Daws et al., 2005, 2006). The relatively greater sensitivity of A. marina and A. heterophyllus axes may be related to the fact that these species produced much heavier seeds than T. dregeana (Fig. 2A). Daws et al. (2006) in an investigation of 104 species from a semi-deciduous forest in Panama, showed that seeds with greater seed mass may be more likely to be desiccation sensitive. Embryo DM of A. marina and T. dregeana were comparable, while that of A. heterophyllus was considerably higher (Fig. 2a). Previous studies (Pritchard et al., 2004; Daws et al., 2006) have also suggested no correlation between embryo size and desiccation sensitivity. Our findings do, however, suggest that the amount of total seed water apportioned to the embryo relative to other structures may influence drying rate and desiccation sensitivity; for example, the more desiccation sensitive and faster drying A. marina and A. heterophyllus seeds exhibited a greater apportioning of water to the embryo than T. dregeana (Fig. 2b). The SCR of a seed is a quantitative representation of the resource

Fig. 4. Quantified immunocytochemistry gold label density of cell wall epitopes in Avicennia marina, Trichilia dregeana and Artocarpus heterophyllus embryos. Bars represent mean ± SD (n = 3 for all species). Bars labelled with different letters are significantly different (p < 0.05) within and across species (Kruskal-Wallis or *ANOVA); label density is plotted on a semi-logarithmic scale, with the y-axis transformed with log10 (LM2 = antibody for arabinogalactan protein, LM5 = antibody for galactan protein, LM6 = antibody for arabinan side-chains of homogalacturonan, LM19 = antibody for unesterified epitopes of homogalacturonan and LM20 = antibody for highly methyl-esterified epitopes of homogalacturonan).

Merced and Renzaglia (2014) suggest that it is more significant to consider the interaction between unesterified and methyl-esterified homogalacturonan than the total abundance of each component in the CW in isolation. Therefore, the ratio of label density between LM19 (unesterified homogalacturonan) and LM20 (highly esterified homogalacturonan) was used as an indication of the rigidity/ flexibility of the CW. The LM19:LM20 ratio was equivalent to ∼2:1 for A. marina, ∼1:1 for A. heterophyllus and ∼1:3 for T. dregeana (data not shown). It has also been suggested that galactan and arabinan may interact in a similar fashion, with arabinan involved in conferring greater flexibility and galactan rigidity (Woodenberg et al., 2018). Therefore, ratios between

Fig. 5. Immuno-gold localisation of Avicennia marina embryo cell walls. (a) LM2 labelling was present in the cell wall (CW) and cytomatrix (Cy) adjacent to the plasmalemma. (b) LM5 labelling was present predominantly in the primary cell wall (PCW), while (c) LM6 exhibited sparse labelling (arrows). (d) LM19 exhibited dense labelling throughout the cell wall (CW), while (e) LM20 label appeared limited to the primary cell wall (PCW). Gold particles ∼ 10 nm. Bar =0.5 μm. 6

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Fig. 6. Immuno-gold localization of Artocarpus heterophyllus embryo cell walls. (a) LM2 label was present in the cell wall (CW) and cytomatrix (Cy) adjacent to the plasmalemma. (b) Dense LM5, (c) LM6, (d) LM19 and (e) LM20 labelling was exhibited throughout the cell wall (CW). Gold particles ∼ 10 nm. Bar =0.5 μm.

thin seed coats (Daws et al., 2006). Artocarpus heterophyllus seeds possessed thinner seed coats than the least sensitive species, T. dregeana. However, the most sensitive species (A. marina) possessed the thickest seed coat (Fig. 2c), but were lighter than those of A. heterophyllus (Fig. 2a), which may explain why SCR, which is a function of DM, was

allocation towards physical defence, in the form of endocarp and testa, in any given species (Pritchard et al., 2004; Daws et al., 2006) and recalcitrant seeds have been shown to generally possess a SCR within the range of 0.01 to 0.53 (Daws et al., 2006). All three of the species investigated possessed SCR within this range, indicative of relatively

Fig. 7. Immuno-gold localisation of Trichilia dregeana embryo cell walls. (a) Sparse labelling was exhibited in the cell wall (CW) in close proximity to the plasmalemma. (b) Dense LM5 labelling was exhibited throughout the cell wall (CW); however, (c) LM6 labelling was present mostly in the primary cell wall (PCW). (d) LM19 labelling was present predominantly in the middle lamella (ML), while (e) LM20 displayed a relatively uniform distribution in the cell wall (CW). Gold particles ∼ 10 nm. Bar =0.5 μm. 7

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(Table 1). This suggestion is based on the fact that in A. marina root meristem CWs exhibited a greater proportion of unesterified homogalacturonan (LM19:LM20 of ∼2:1), usually indicative of CW rigidity (Merced and Renzaglia, 2014), than T. dregeana (LM19:LM20 of ∼1:3) and A. heterophyllus (LM19:LM20 of ∼1:1). Cell wall extension and porosity have also been shown to be key features in desiccation tolerant systems (Farrant et al., 1997; Farrant, 2000; Woodenberg et al., 2018), and CW composition may be one of the factors that influences desiccation sensitivity in recalcitrant seeds (Woodenberg et al., 2018). While complex pectic polysaccharides can influence the retention/passage of water and solutes across CWs (Voiniciuc et al., 2018), to effect drying rate, the current contribution, has identified LM19:LM20 as a potential indicator of CW rigidity and hence, desiccation sensitivity. High levels of methyl-esterified homogalacturonan is normally associated with developing CWs immediately following cell division, as developing CWs require greater flexibility for cell growth and increases in cell size (Reiter, 2002; Merced and Renzaglia, 2014; Woodenberg et al., 2018). Once cells have attained their mature size and the CW plate has been formed, there is a switch from high levels of methylesterified homogalacturonan to unesterified homogalacturonan, to confer greater rigidity of the mature CW (Merced and Renzaglia, 2014). Flexible, newly formed CWs rich in methyl-esterified homogalacturonan have been shown to be more porous in comparison with mature, unesterified homogalacturonan-rich, rigid CWs (Merced and Renzaglia, 2014; Woodenberg et al., 2015, 2018). Hence, the high levels of methyl esterified homogalacturonan (LM20) in A. heterophyllus (approximately three-fold greater than T. dregeana and six-fold greater than A. marina) may explain why this species exhibited the fastest drying rate (Table 1). However, these results suggest that while methyl esterified homogalacturonan levels may influence drying rate and hence, desiccation sensitivity, Merced and Renzaglia (2014) suggested that thicker CWs are more rigid, while thinner CW offer a greater flexibility. Our results also indicate that the least sensitive species T. dregeana, possessed the thinnest and therefore possibly the most flexible CWs. The greater flexibility in T. dregeana root meristem CWs, may have allowed embryos to lose water non-lethally to much lower WCs than the other two species, by minimising CW-plasmalemma separation.

Table 3 Results of Spearman’s rank correlation analysis (ρ) for Artocarpus heterophyllus, Avicennia marina and Trichilia dregeana embryonic axes, between label density of cell wall epitopes and drying rate and desiccation stress index (DSI). Data for each epitope across species were pooled for regression analysis. Values in bold represent significant correlations (LM2 = antibody for arabinogalactan protein, LM5 = antibody for galactan protein, LM6 = antibody for arabinan side-chains of homogalacturonan, LM19 = antibody for unesterified epitopes of homogalacturonan and LM20 = antibody for highly methyl-esterified epitopes of homogalacturonan). Drying rate

LM2 LM5 LM6 LM19 LM20

DSI

ρ

p

ρ

P

−0.15 0.02 0.16 0.68 0.46

0.17 0.84 0.14 < 0.01 < 0.01

0.18 0.47 0.35 −0.34 0.31

0.09 < 0.01 < 0.01 < 0.01 < 0.01

higher in the latter. These inter-species difference may be a consequence of the ecology and dispersal syndromes of these species. Avicennia marina seeds need to be light enough to be buoyant due to their dispersal into water, as in many other water dispersed species (Nilsson et al., 2010), but are also highly susceptible to predation by crabs and insects within estuarine habitats (author’s unpublished observation), which may explain their characteristically thick seed coat. Of the three species investigated, T. dregeana was the smallest, lightest, slowest drying and least sensitive to desiccation (Table 1). Seeds of this species also possess a SCR of ∼0.32, which is commonly associated with the seeds of several ecologically similar recalcitrantseeded climax forest tree species native to the mesic tropics, sub-tropics and some more temperate regions (Pritchard et al., 2004; Daws et al., 2006). A significantly large proportion of water within T. dregeana seeds was located in the endocarp and testa (Fig. 2b). Interestingly, small desiccation tolerant and dormant orthodox seeds generally show a greater apportionment of water towards the seed coat during developmental stages, prior to maturational drying and shedding (Manz et al., 2005; Garnczarska et al., 2007). Orthodox and recalcitrantseeded species do have a common ancestor (Subbiah et al., 2019) and these inter-species differences in water distribution patterns amongst recalcitrant-seeded species may be the product of habitat-specific evolutionary drivers. At a finer scale, while A. marina exhibited the greatest sensitivity to desiccation and A. heterophyllus possessed the fastest drying rate (Table 1), both species possessed similar root meristem CW thickness (Fig. 3). The slower drying rate of A. marina embryos compared with A. heterophyllus, may be attributed to the presence of a distinct mucilaginous layer in the former, which may have evolved as a barrier to the combination of the extremely harsh, saline environment into which A. marina seeds are shed and desiccation during low tide events. In studies on leaf and vegetative tissues of resurrection plants, the relatively thin CWs have been shown to be highly flexible, which allow them to cope with the effects of cellular shrinkage upon dehydration (Farrant et al., 1997; Farrant, 2000). Accompanying this shrinkage is the accumulation of insoluble reserves such as lipid bodies, amyloplasts and starch bodies, to provide stabilisation during this shrinkage (Farrant, 2000). However, Farrant et al. (1997) have shown that meristematic cells of A. marina seeds do not accumulate an abundance of insoluble reserves, but possess large quantities of soluble sugars. Upon dehydration, without the stability offered by insoluble reserves, the plasmalemma of recalcitrant seeds may separate from the CW, causing the CWs to fold inwards, resulting in a loss of ultrastructural integrity (Woodenberg et al., 2018). This can be lethal in recalcitrant seeds (Woodenberg et al., 2018) and is definitely a contributory factor to the loss of viability upon drying in A. marina axes observed here and possibly the relatively higher degree of desiccation sensitivity observed in this species

5. Conclusions While it is difficult to draw conclusive findings from a comparison of only three species, this study provides some key insights into the influence of seed morpho-physiology and meristematic CWs on drying rate and desiccation sensitivity in recalcitrant seeds. The findings suggest that seed water distribution patterns and CW structural and compositional traits may be useful indicators of seed desiccation sensitivity. A higher apportionment of water to the embryo (relative to other seed structures) appears to be indicative of faster drying rates and greater desiccation sensitivity. Thick, rigid meristematic CWs appear to be associated with greater desiccation sensitivity. The results also suggest that the degree to which these morpho-physiological and ultrastructural traits influence seed desiccation sensitivity in individual species may be a consequence of habitat-specific evolutionary pressures. Furthermore, in light of global climate change, these findings may aid in the prioritisation and design of conservation strategies for recalcitrant-seeded species in future.

Funding This work was made possible through the financial support of the National Research Foundation (South Africa) and the Oppenheimer Memorial Trust (South Africa). 8

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Declaration of Competing Interest

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