Plant Biology ISSN 1435-8603

RESEARCH PAPER

Physiological history may mask the inherent inducible desiccation tolerance strategy of the desert moss Crossidium crassinerve L. R. Stark1, J. L. Greenwood1, J. C. Brinda2 & M. J. Oliver3 1 School of Life Sciences, University of Nevada, Las Vegas, NV, USA 2 Missouri Botanical Garden, St. Louis, MO, USA 3 U.S. Department of Agriculture-ARS-MWA-PGRU, University of Missouri, Columbia, MO, USA

Keywords Acclimation; chlorosis; chlorophyll fluorescence; deacclimation; dehardening; slow-dry; constitutive desiccation tolerance. Correspondence L. R. Stark, School of Life Sciences, University of Nevada, 4505 Maryland Parkway, Las Vegas, NV 89154-4004, USA. E-mail: [email protected] Editor D. Byers Received: 6 June 2013; Accepted: 22 October 2013 doi:10.1111/plb.12140

ABSTRACT Shoots of bryophytes collected in the desiccated state from the field are likely to be hardened to desiccation tolerance (DT) to varying degrees. To account for this, most studies on DT include a relatively short deacclimation period. However, no study has experimentally determined the appropriate deacclimation time for any bryophyte species. Our purposes are to (i) determine if ‘field effects’ are biologically relevant to DT studies and how long a deacclimation period is required to remove them; and (ii) utilise field versus cultured shoot responses within the context of a deacclimation period to elucidate the ecological strategy of DT. Our hypothesis (based on an extensive literature on DT) is that a deacclimation period from 24 to 72 h should be sufficient to eliminate historical stress effects on the physiology of the shoots and allow an accurate determination of the inherent ecological DT strategy (constitutive or inducible). We determined, however, using chlorophyll fluorescence and visual estimates of shoot damage, that field-collected shoots of the desert moss Crossidium crassinerve required an experimental deacclimation period of >7 days before field effects were removed, and revealed an ecological DT strategy of inducible DT. If the deacclimation period was 3 weeks 0h 2–3 weeks (protonema) >10 days ≥7 days 48 h ~7 days 3–7 days >24 h 3 days

Beckett & Hoddinott (1997), Beckett (1999, 2001), Mayaba et al. (2001), Mayaba & Beckett (2003) Beckett et al. (2005) Beckett et al. (2000) Werner et al. (1991), Werner & Bopp (1992) Streusand et al. (1986) Arscott et al. (2000) Streusand et al. (1986) Egunyomi (1979) Frank et al. (2005), Oldenhof et al. (2006), Saavedra et al. (2006), Cuming et al. (2007), Koster et al. (2010), Cui et al. (2011) Mansour & Hallet (1981) Stark et al. (2013) Arscott et al. (2000) Bates & Phoon (2008) Bates & Phoon (2008) Hajek & Beckett (2008) Schipperges & Rydin (1998) Stark et al. (2012), Yang et al. (2012) Barker et al. (2005) Stark et al. (2011) Oliver et al. (1993) Stark et al. (2005), Brinda et al. (2011) Stark et al. (2012) Oliver et al. (1993) Takacs et al. (2000) Tuba et al. (1996, 1998), Csintalan et al. 1998; Okoloko & Bewley (1982), Tucker & Bewley (1974, 1976), Oliver & Bewley (1984a,b,c), Oliver (1991), Platt et al. (1994), Velten & Oliver (2001), Chen et al. (2002), Zeng et al. (2002) Bewley & Thorpe (1974) Bewley et al. (1978) Oliver et al. (1993) Mahan et al. (1998), Scott & Oliver (1994), O’Mahony & Oliver (1999), Wood & Oliver (1999), Wood et al. (1999), Oliver et al. (2009) Schonbeck & Bewley (1981) Badacsonyi et al. (2000) Stark et al. (2007) Alpert & Oechel (1987) Burch (2003) Dilks & Proctor (1976b) Nabe et al. (2007) Pressel & Duckett 2010; Tobiessen et al. (1979) Alpert & Oechel (1987) Cleavitt (2002) Dilks & Proctor (1976a) Streusand & Ikuma (1986) Proctor (2003) Davey (1997)

2 months >2 months (cultured) continuous culture continuous culture >3 weeks

Sollows et al. (2001) Hellwege et al. (1994) Pence et al. (2005) Pence (1998) Pressel et al. (2009)

7 days up to 2 weeks 2–3 weeks (protonema) >48 h 12 h >48 h 24 h (leaves) continuous culture (protonema) >2 weeks continuous culture 12 h up to 2 weeks up to 2 weeks ≥6 weeks 3 days 0h 1h 2h 36 h 3 days 27 days 36 h 4h 12 h 24 h

>24 h 30 h 36 h 48 h

Tortula inermis 3 spp. 3 spp. 3 spp. 4 spp. 4 spp. 4 spp. 5 spp. 6 spp. 6 spp. 10 spp. 11 spp. 14 spp. liverworts Bazzania trilobata Exormotheca holstii 3 spp. 4 spp. 6 spp.

reference

Plant Biology 16 (2013) 935–946 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

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Field effects and desiccation strategy

Stark, Greenwood, Brinda & Oliver

strategies directly to mechanistic processes is not tenable, since it would require a more detailed examination of the gene expression and cellular characteristics of the desiccation response. Our goal here is to determine the ecological strategy employed in a desert moss by assessing the shoot response to desiccation following a variety of deacclimation periods, including those shoots used directly from the field and shoots that have been in continuous culture. This study does not attempt to resolve mechanistic aspects of the ecological strategies we have described, but rather investigates the ecological implications of the two possibilities with regard to Crossidium crassinerve. Purpose and hypothesis To our knowledge, no study has explicitly addressed the DT effects of varying the deacclimation period on field-collected shoots and compared the effects to shoots of the same species placed into continuous culture, with the goal of elucidating the inherent ecological DT strategy of a particular species. Our purposes are to (i) determine if ‘field effects’ are biologically relevant to DT studies, and how long a deacclimation period is required to remove them; and (ii) utilise field versus cultured shoot responses within the context of a deacclimation period to elucidate the ecological strategy of DT. We selected one of the most, at least ostensibly, DT species in the world, Crossidium crassinerve. It is a very small perennial plant (Stark & Delgadillo 2003) typically found in low-elevation regions of the Mojave Desert, where annual precipitation is on the order of 100 mm and long dry periods of several months are common (Stark 2005). Our working hypothesis followed the conventions of the DT literature, that a deacclimation period from 24 to 72 h should be sufficient to eliminate historical stress effects on the physiology of the shoots and allow an accurate determination of the ecological DT strategy (CDT or IDT) utilised by the species.

Experimental design For field-collected shoots, the deacclimation period was from 0 (directly from the field) to 30 days. For regenerant shoots, a single shoot (genotype) from the field population was cloned and subcultured for ~6 months prior to testing (culture methods follow Horsley et al. 2011), such that from this culture all shoots tested represented regenerant adult shoots (Table 2). Deacclimation From each of the ten patches sampled in the field, a randomly selected patch section measuring ~4 9 4 mm (consisting of ~40–80 shoots) was removed, hydrated (by placing the patch section directly into several drops of sterile water on a glass microscope slide), cut to a depth (height) of ~2 mm (including substrate and plants), placed on a hydrated folded double chemical wipe inside a lidded glass bottle (7 9 6 cm, height 9 diameter), and allowed to remain hydrated for up to 30 days in a growth chamber set to these conditions simulating the field environment during conditions of growth for the species: 12 h photoperiod, 20 °C light, 8 °C dark, RH ~65%, Photosynthetically active radiation (PAR) ~115–147 lmolm 2s 1. It should be noted that when shoots were deacclimated for 30 days, shoots had begun to regenerate and produce new protonemata and regenerant shoots. Rapid desiccation of deacclimated shoots

MATERIAL AND METHODS Species and study site Crossidium crassinerve (De Not.) Jur. (Pottiaceae) is a xeric, perennial, monoecious (bisexual gametophytes) species native to deserts of the world (Delgadillo 2007). It forms tightly packed colonies of ~4 ramets mm 2, with the shoots elongating only 0.10 mmyear 1 (13 lgyear 1, with 90% of the biomass as leaves), the shortest growth interval reported for bryophytes (Stark & Delgadillo 2003). It is distinguished from other species in the genus by its smooth leaf cells and thin-walled, branched costal filaments that are two to 12 cells long and have terminal cells conic, smooth or with two to four papillae (Delgadillo 1975). On 20 May 2012, a ‘walking transect’ was established along the edge of a large population of C. crassinerve spanning ~50 9 6 m along the north-facing embankment of a desert wash (Fig. 1A; USA, Nevada, Clark County, River Mountains, 6.4 km northeast of downtown Henderson, 36°04′ N, 114°55′W, Township 22S, Range 63E, Section 2, 638 m a.s.l.; vouchers deposited at UNLV as Stark NV-660v01, NV660v02). This population consisted of a series of desiccated patches, and was the subject of a previous study determining the length of the field hydration period (Stark 2005). Other bryophyte species conspicuously present in the area included 938

Tortula inermis, Pterygoneurum ovatum, P. lamellatum and Grimmia orbicularis; additional site vegetation is described in Stark (2005). At intervals of 3 m, a section (~3 9 3 cm) of the nearest patch (if present) was collected along the embankment; ten patches were collected in this manner. Specimens were placed into paper bags, transported to the lab, and stored in the lab until use (21.5 °C, 15–25% RH).

All shoot manipulations were carried out in a walk-in environmental control room set to constant temperature, RH, and light (20 °C, 50% RH, 2–4 lmolm 2s 1), with internal variation within the room 95%), indicating water loss from shoots under high RH conditions (Fig. 3). Chlorophyll fluorescence Deacclimated control shoots showed no ill effects of continuous hydration under growth chamber settings through an 11-day period, using Fv/Fm as an indication of PSII health (Figure S1). Following rehydration, deacclimation time had a significant negative effect on Fm (P = 0.007, t = 2.917, df = 28; Fig. 4), Fv/Fm (P = 0.002, t = 3.527, df = 28; Fig. 5) and quantum yield (P < 0.001, t = 4.622, df = 28; Fig. 6). Longer deacclimation times resulted in lower Fm, Fv/Fm and quantum yield values, with the effect particularly marked at the two longest deacclimation times (8 and 11 days). The recovery (rehydration) period of 72 h during which chlorophyll fluorescence measurements were made had no effect on Fm (P = 0.381, t = 0.917, df = 10), a significant positive effect on Fv/Fm (P < 0.002, t = 4.092, df = 10), and a significant positive effect on quantum yield (P = 0.012, t = 3.082, df = 10). There

Plant Biology 16 (2013) 935–946 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Stark, Greenwood, Brinda & Oliver

Fig. 2. Water content and relative humidity experienced by shoots of Crossidium crassinerve during an experimental rapid drying event (mean  SE, N = 4 for each, SE values too small to show on graph for RH; upper dotted line is 50% RH, lower dotted line is 0.0% WC). Trials for RH and WC were independent tests, because the occupation of space within the Petri dish by an iButton is significant and may affect water loss. RH = relative humidity, WC = water content, DW = dry weight; WC = [shoot mass equilibrated at 50% RH DW]/DW, equilibrated at 8.0  0.1%).

Field effects and desiccation strategy

Fig. 3. Water content and relative humidity experienced by shoots of Crossidium crassinerve during an experimental slow drying event; dotted line is both 50% RH and 0.0% WC (mean  SE, N = 4 for each, SE values too small to show on graph for RH). Trials for RH and WC were independent tests, because the occupation of space within the Petri dish by an iButton is significant and may affect water loss. RH = relative humidity, WC = water content, DW = dry weight; WC = [shoot mass equilibrated at 50% RH DW]/DW, equilibrated at 8.6  0.2%).

was also a significant interaction effect between deacclimation time and recovery time (P ≤ 0.001, t ≤ 3.293, df = 280), such that the strength of the effect of deacclimation time on all three fluorescence parameters becomes more obvious after the early (first few hours) measurements. Cultured, non-dried controls (C1) and cultured slowly dried controls (C2) behaved similarly to each other and similar to field shoots that were deacclimated up to 4 days (Figs 4–6). However, rapidly dried cultured controls (C3) behaved similarly to field shoots that were deacclimated longer than 6 days (Figs 4–6). Leaf damage For rapidly dried shoots, longer deacclimation times led to increased leaf damage (P < 0.001, z = 9.737, estimated df = 4; Fig. 7) upon rehydration. The statistical threshold (the statistical model prediction for the time duration when, on average, the plants change from one leaf damage category to the next) for the change from green to partially chlorotic leaves was 5.2  0.5 days (mean  1 SE) of deacclimation, and the threshold for the change from partially chlorotic to entirely chlorotic leaves was 9.0  0.7 days of deacclimation. Shoots from the field and non-dried cultured shoots exhibited very little leaf damage among the five distal leaves (98–100% chlorophyllose). Those shoots taken from the field and deacclimated for 1, 2, 3 and 4 days prior to administering a RD and allowing to rehydrate, all exhibited very healthy leaves that were at or above 93% chlorophyllose. However, leaf damage increased from day 5 (86% chlorophyllose) through day 8 (58% chlorophyllose) of deacclimation, and leaves were heavily damaged after 11 (18%) and 30 (6% chlorophyllose) days of deacclimation (followed by a RD and allowed to rehydrate for 7 days). Cultured shoots that were rapidly dried (C3) became heavily damaged (27% chlorophyllose). However, if shoots were allowed to deacclimate for 30 days and then slowly dried (C4), or the non-dried cultured shoots (C1) were slowly dried (C2),

Fig. 4. The relationship between deacclimation period and mean Fm over a 24-h rehydration period for shoots of Crossidium crassinerve exposed to varying periods of deacclimation followed by a rapid-dry event (mean  1 SE, N = 3; similar shoot masses used for each measurement). Results for shoots acclimated for 3, 5 and 30 days have been removed for clarity; C = Control: C1 = non-dried cultured shoots, C2 = slowly dried cultured shoots, C3 = rapidly dried cultured shoots, each at 24-h post-rehydration.

virtually no damage was detected (96–99% chlorophyllose). Leaves from field-collected shoots appeared more pigmented than cultured shoots (Fig. 1). DISCUSSION Ecological strategies of desiccation tolerance and deacclimation time Some bryophytes are capable of hardening to desiccation stress through either partial desiccation (Abel 1956; Beckett 1999; Beckett et al. 2005) or through a slow-dry (SD) event (Cruz de

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Fig. 5. The relationship between deacclimation period and mean Fv/Fm over a 24-h rehydration period for shoots of Crossidium crassinerve exposed to varying periods of deacclimation followed by a rapid-dry event (mean  1 SE, N = 3). Results for shoots acclimated for 3, 5 and 30 days have been removed for clarity; C = Control: C1 = non-dried cultured shoots, C2 = slowly dried cultured shoots, C3 = rapidly dried cultured shoots, each at 24-h post-rehydration.

Fig. 6. The relationship between deacclimation period and mean quantum yield (ΦPSII) over a 24-h rehydration period for shoots of Crossidium crassinerve exposed to varying periods of deacclimation followed by a rapid-dry event (mean  1 SE, N = 3). Results for shoots acclimated for 3, 5 and 30 days have been removed for clarity; C = Control: C1 = non-dried cultured shoots, C2 = slowly dried cultured shoots, C3 = rapidly dried cultured shoots, each at 24-h post-rehydration.

Carvalho et al. 2011; Stark et al. 2013). Given this phenomenon, and its potential to be widespread, it is a reasonable practice to deharden (deacclimate) material collected from the field prior to conducting experiments on DT. This is recognised in the ‘Austin Protocol’, where the suggested deacclimation time, based on input from a variety of scientists, is 24 h (Wood 2007). At the time of this suggestion, however, there was no experimental determination of the appropriate dehardening time of a species, leaving some doubt to the suitability of a 24-h standard deacclimation time for all species. In pilot experiments on several species of desert mosses grown in 942

Fig. 7. The relationship between deacclimation period and visual leaf damage on shoots of Crossidium crassinerve exposed to varying periods of deacclimation followed by a rapid-dry event (mean  1 SE, N = 16). Leaf damage visually assessed on day 7 post-rehydration (see Material and Methods for details). C = Control: C1 = non-dried cultured shoots, C2 = slowly dried cultured shoots, C3 = rapidly dried cultured shoots; C4 = slowly dried field shoots deacclimated for 30 days.

continuous culture versus those same species freshly collected from the field, it became apparent that there was variability in the results depending upon the length of time a deacclimation period was administered (including C. crassinerve). The potential for pretreatment effects was taken into account in Alpert & Oechel (1987); such variation was also noted as a distinct possibility by Bopp & Werner (1993), and subsequently appreciated by Hajek & Beckett (2008). Specifically, a possibility exists that the ecological DT strategy of a species may be mis-determined as constitutive (CDT) when using a 24-h deacclimation period if 24 h is an insufficient time to allow the plants to become dehardened of field effects. Whereas, if cultured (fully dehardened) plants were used, the inherent DT strategy would give a clear signal of ecological IDT. Alternatively, when an inherently constitutively protected species is so tested, deacclimation effects should be minimal, and we have unpublished (LRS) data indicating this to be true in species of Syntrichia. Experimental derivation of deacclimation time Crossidium crassinerve is capable of tolerating several months of desiccation in the field and can survive a rapid drying event in the field (Stark 2005). Field-collected shoots are expected to have hardened to varying degrees depending on the location, season, species and recent hydration history (Dilks & Proctor 1976a; Beckett & Hoddinott 1997). The C. crassinerve plants in this study were intentionally collected in late spring, when bryophytes of the Mojave Desert are expected to be fully hardened. Most precipitation occurs during the winter months, and as temperatures rise patches of the moss desiccate in preparation to endure the summer dry months. Such a desert terrestrial species should exhibit a CDT ecological strategy of DT, as predicted by a number of workers in the field (Deltoro et al.

Plant Biology 16 (2013) 935–946 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Stark, Greenwood, Brinda & Oliver

1998; Proctor & Pence 2002; Marschall & Beckett 2005; Pressel et al. 2009; Toldi et al. 2009). This hypothesis maintains that if a species inhabits a substrate that is vulnerable to rapid drying (e.g. exposed microhabitats), then the plants should have evolved a mechanism to protect tissues undergoing rapid water loss, and employ a CDT ecological strategy. On the other hand, if a species is native to habitats such as streamside or deeply shaded forest soils, selection should favour the development of an inducible strategy, since in all likelihood such plants will be subject to very gradual or bouts of partial desiccation prior to reaching a fully desiccated state. Pertaining to this correlation of strategy to habitat, the CDT strategy of DT is considered to be metabolically expensive compared to the IDT strategy of DT, with a trade-off predicted between growth rate and degree of DT, i.e. the Productivity Trade-off hypothesis (Alpert 2006). Pilot studies of a variety of chaparral and desert acrocarpous mosses incorporating tests on cultured plants indicate that the CDT and IDT ecological strategies of DT are approximately equally represented (LRS, unpublished data). The recent finding of another desert species incorporating an inherently IDT ecological strategy (Pterygoneurum lamellatum; Stark et al. 2013) suggests that IDT bryophytes can tolerate the rigours of a desert environment. We found that plants of C. crassinerve require at least 7 days of continuous hydration before they become dehardened. If tested for desiccation responses prior to 6 days, the plants give a strong signal of constitutive protection due to the presence of field hardening effects. Plants of Atrichum androgynum also displayed hardening effects persisting at least 7 days after a partial desiccation (hardening) treatment (Beckett 1999). ‘Recovery’ from desiccation can be construed as (i) recovery from desiccation in a dehardened physiological state; and (ii) recovery from desiccation in a hardened state. In the IDT species studied, dehardened shoots largely failed to recover from a RD event, while hardened shoots apparently enlisted little physiological repair time because damage was minimal. Both lines of evidence (chlorophyll fluorescence and visual leaf damage) in this study support the assertion that plants of C. crassinerve incorporate an inherently IDT ecological strategy of DT, i.e. capable of hardening to DT. Visual shoot damage to leaves does not show up until the shoots are rehydrated for a period of at least 4 days; this is the probable time for chlorophyll degradation to occur. Use of chlorophyll fluorescence, however, offers an immediate insight into shoot damage and can indicate the presence of a compromised photosystem within a few hours. Five types of controls were implemented in the experiments, and these can help to understand the deacclimation process. Control 1 (C1) represents non-dried shoots from continuous culture; these behaved very similarly (Fm, shoot damage) or slightly better (Fv/Fm, quantum yield) than field shoots deacclimated for 0–4 days. Control 2 (C2) represents shoots grown in continuous culture that were then slowly dried (SD); these shoots behaved very similar to Control 1 shoots in all respects, indicative of hardening capacity. Control 3 (C3) represents non-dried shoots from continuous culture that were then rapidly dried (RD); these shoots behaved very similar to field shoots deacclimated for 8 or more days, showing widespread chlorosis and breakdown of the photosynthetic apparatus. Control 4 (C4) represents those field shoots that were deacclimated for 30 days, which were then SD. We looked at visual shoot damage in (C4), and shoots behaved nearly

Field effects and desiccation strategy

identically with Controls 1, 2 and field shoots deacclimated for 0–4 days (Fig. 7). The last control was a field-collected series deacclimated for 0–11 days and assayed for Fv/Fm without a desiccation treatment. These shoots showed a healthy PSII signal, indicating that the transfer of shoots from the field to the lab and subsequent deacclimation treatment did not compromise the photosystem or apparent health of the shoots (Fig. S1). These data are understandable by postulating that (i) field shoots are hardened to DT; (ii) dehardening of field shoots requires about 7 days or longer; (iii) hardening to DT can occur during a single SD event; and (iv) field shoots can be experimentally dehardened to behave similarly to shoots from continuous culture. Implications of field conditions The finding that C. crassinerve exhibits an ecological DT strategy that is IDT invites an assessment of field conditions attending patch hydration; i.e. how frequently are field patches actually dehardened to desiccation? Over a 4-year period, mean patch hydroperiod (the time during which a patch was fully hydrated) was 4–5 days. A single patch was followed in a detailed fashion, and found to be hydrated 25 times over this 4-year period. In 20 of the 25 hydroperiods followed, the length of hydration was

Physiological history may mask the inherent inducible desiccation tolerance strategy of the desert moss Crossidium crassinerve.

Shoots of bryophytes collected in the desiccated state from the field are likely to be hardened to desiccation tolerance (DT) to varying degrees. To a...
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