The Effect of High Temperature on the Reproductive Success of Trianthema Portulacastrum

The Effect of High Temperature on the Reproductive Success of Trianthema portulacastrum

Haley Anne Branch

A thesis submitted in conformity with the requirements for the degree of Master of Science Ecology and Evolutionary Biology University of Toronto

The Effect of High Temperature on the Reproductive Success of Trianthema portulacastrum

Haley Anne Branch

Master of Science

Ecology and Evolutionary Biology University of Toronto

2016 Abstract

Plant reproduction is highly sensitive to rising temperatures, which can lead to pollen abortion, and lower yield in many crop species. It remains uncertain whether wild plant species adapted to hot climates are able to reproduce at high temperature. I studied heat sterility thresholds in

Trianthema portulacastrum, a weedy species found throughout the tropics and subtropics, often on barren soils where temperature exceeds 40°C. Plants were grown at seven day/night temperatures: 30/24°C, 33/24°C, 36/24°C, 40/24°C, 44/24°C, 24/40°C, and 40/40°C. Pollen viability significantly declined with increasing temperature, but this did not significantly affect percent pollen germination or seed set. In contrast, seed set was significantly reduced under high night temperature. The results show high night temperatures have a greater impact on reproduction than day temperature, indicating T. portulacastrum is using a night escape strategy to maintain reproductive success in its natural habitat.

Acknowledgments

I would like to thank my supervisor, Prof. Rowan Sage, who inspired me to pursue an academic career in plant ecology and helped formulate the ideas for this thesis. He encouraged me to think outside the box, taught me to question everything, and has shown me the importance of passion in research throughout my MSc and during my undergraduate degree. Secondly, thank you to Prof. John Stinchcombe and Prof. Art Weis. I have greatly appreciated your advice and support throughout my Master’s. Thirdly, to Prof. Asher Cutter, thank you for allowing me access to your lab equipment, without you this thesis would not have been completed.

A special thank you to Dr. Corlett Wood, who generously gave her time to assist me with my statistical methods.

Thank you to my friends and colleagues at the University of Toronto. Stefanie Sultmanis, Colin Bonner, Matt Stata, Vanessa Lundsgaard-Nielsen, and Dr. Roxana Khoshravesh helped me with various aspects of my study and engaged with me in numerous scientific discussions. Michael Foisy, thank you for joining me at libraries and coffee shops while I took on the task of writing this thesis.

To my husband, Graham Hassell, thank you for your unconditional support throughout my degree. You listened patiently as I practiced my talks over and over again, waded through unfamiliar jargon to edit my work, and dedicated much time to assist me in nighttime laboratory excursions. For all that you have done, I am grateful. Thank you to my parents, Donald and Cathy Branch, for providing me with the foundation to pursue science intellectually and creatively. To my big sister, Cara-Lynn, you have rooted for me since the very beginning, thank you.

This research was funded by an NSERC CGS M scholarship.

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Table of Contents

Acknowledgments ...... iii

Table of Contents ...... iv

List of Figures ...... v

List of Tables ...... vi

Abbreviations ...... vii

Introduction ...... 1

1.1 Heat Studies in Wild Species ...... 6

Methods ...... 12

2.1 Growth Conditions ...... 12

2.2 Experiment 1: Variable day temperature, constant night temperature ...... 13

2.3 Experiment 2: Elevated night temperature ...... 14

2.4 Analysis of Anther and Pollen Development ...... 14

2.5 Pollen Viability Characterization ...... 15

2.6 Characterizing Reproductive Success ...... 16

2.7 Statistical Analysis ...... 16

Results ...... 19

Discussion ...... 25

Conclusion and Future Directions ...... 29

6 References ...... 31

List of Figures

Figure 1-1 Heat-induced abortion in eight diverse genera……………………………………4

Figure 1-2 Average temperatures in the Mojave Desert……………………………………..10

Figure 1-3 Trianthema portulacastrum growth and morphology…………………………....11

Figure 2-1 Flower and air temperature during control and 44/24°C treatments………….....14

Figure 3-1 Anther and pollen development in T. portulacastrum…………………………...20

Figure 3-2 Heat stress affects pollen and anther development…………………..…………..21

Figure 3-3 High temperature affects pollen viability but has no effect on yield...... 22

Figure 3-4 T. portulacastrum does not exhibit apomictic reproduction………..……………23

List of Tables

Table 2-1 Control treatment growing conditions…………………………………………...12

Table 2-2 T. portulacastrum growth stage and timing of sampling………………………...18

Table 3-1 Night growth temperature effects on reproduction………………………………24

Abbreviations

AB – Aniline blue

ANOVA – Analysis of variance

ATS – Alexander’s Triple Stain

DAPI – 4’,6-diamidino-2-phenylindole

FDA – Fluorescein diacetate

FITC – Fluorescein isothiocyanate

HNT – High night temperature

HSP – Heat shock protein

ROS – Reactive oxygen species

TT – Treatment temperature

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Introduction

Climate change is one of the greatest threats of this century. Average global temperatures are expected to increase by up to 2°C by 2050, along with increased frequency and intensity of extreme heat events (Collins et al., 2013). This will have profound impacts on the Earth’s biota, as most biological processes have optimal thermal ranges. Plant reproduction is among the most sensitive of processes to increases in temperature, because a relatively small rise in temperature above 30°C can lead to heat-induced sterility (Hedhly, Hormaza, and Herrero, 2008; Schlenker and Roberts, 2009; Harsant et al., 2013; Sage et al., 2015). Because of this, heat stress is of particular concern with climate warming in regions of the world that currently experience maximum growing season temperatures above 30°C (Hedhly, Hormaza, and Herrero, 2008; Jagadish et al., 2010; De Storme and Geelen, 2014). For example, mean daily temperature during the growing season in Sub-Saharan Africa are frequently above 30°C, and maize production in this region can experience >10% yield decline for every additional 1°C increase in average temperature above 25°C (Lobell et al., 2011). Heat inhibition can increase to over 20% yield loss when compounded by the effects of drought (Lobell et al., 2011). Similarly, maize and soybean in the United States could experience a 17% yield decline for each degree increase in temperature (Lobell and Asner, 2003). At the International Rice Research Institute in the Philippines, Peng et al. (2004) compiled annual temperature and rice yield data over an eleven- year period. Temperatures were consistently near 30°C during the day, but night temperatures increased more rapidly over the time period, such that for each 1°C increase in night temperature rice yield declined 10% (Peng et al., 2004). This pattern is observed in many plant genera, raising concerns about yield in a wide range of crops as the Earth warms during a period when population expansion will substantially increase food demands.

Warming temperatures can affect vegetative growth and development in many ways. Early in development, high temperatures disrupt cell elongation and differentiation (Bita and Gerats, 2013), reduce membrane stability and function (Maestri et al., 2002; Bita and Gerats, 2013), and lower the ability of seeds to germinate (Balyan and Bhan, 1986; Wahid et al., 2007). Plants that have germinated can experience lower shoot and root growth because of increased respiration and decreased resource assimilation (Wahid et al., 2007; Bita and Gerats, 2013). Throughout development, growth can be greatly impacted by the effects of heat stress on photosynthesis

1 2

(Sharkey, 2005). For instance, reactive oxygen species may accumulate in cell membranes, resulting in cell damage, negatively affecting photosynthesis (Bita and Gerats, 2013). Heat stress also disrupts the electron transport chain by reducing the photochemical efficiency of photosystem II (Rekika, Monneveux, and Havaux, 1997; Masetri et al., 2002) and also affects plant growth by reducing Rubisco activity (Sharkey et al., 2001), and increasing photorespiration (Schrader et al., 2004).

Although heat stress affects vegetative tissues in many ways, reproductive tissues are considered more stress-prone (Prasad et al., 2008; Schlenker and Roberts, 2009; Harsant et al., 2013; Sage et al., 2013). This is largely due to the lower thermal range often seen in reproductive organs (Hatfield and Prueger, 2015). For example, maize photosynthesis has an optimal temperature of ~35°C (Oberhuber and Edwards, 1993), but its reproduction is significantly affected by temperatures ≥35°C (Hatfield et al., 2011; Lobell et al., 2011; Sage et al., 2015). Both male and female reproductive organs are affected (Hasanuzzaman et al., 2013), with heat stress promoting bud and flower abscission (Aloni et al., 1991; Djanaguiraman et al., 2013), inhibiting pollen development and anther dehiscence (Gross and Kigel, 1994; Ahmed, Hall, and DeMason, 1992; Erickson and Markhart, 2002), disrupting carbon allocation to reproductive tissues (Aloni et al., 1991), impeding ovule development (Iwahori, 1966; Snider and Oosterhuis, 2011) and pollen tube growth (Gross and Kigel, 1994; Young, Wilen, and Bonham-Smith 2004), and reducing yield (Lobell et al., 2012). Additionally, plants may advance their reproductive phenology with warmer and longer growing seasons, resulting in less time for reproductive structures to develop (Barnabás, Jäger, and Fegér, 2008; Mathieu et al., 2014).

Extensive research into the effects of high temperatures on crops indicates that pollen development is one of the most sensitive stages of the reproductive process (Jain et al., 2007; Ainsworth and Ort, 2010; Bita and Gerats, 2013; Harsant et al., 2013; Sage et al., 2015). Exposure to 32°C during early reproductive development results in inviable pollen in common bean, Phaseolus vulgaris (Gross and Kigel, 1994). Temperatures that exceed a mid-30°C threshold drastically impair pollen germination, resulting in sterility and causing reduced yield in rice (Matsui et al., 1997), soybean (Djanaguiraman et al. 2013), and cotton (Song, Chen, and Tang, 2014). Abortion of pollen during development was observed in the following plants: cotton at 35-39°C daytime temperatures (Min et al., 2014), wheat (Saini and Aspinall, 1982) and

3 barley at 30°C daytime temperature (Oshino et al., 2007), snap bean at 28°C average air temperature (Suzuki et al., 2001), and sorghum at 36°C daytime temperature (Jain et al., 2007). Anther dehiscence can be arrested at daytime growing temperatures of 33-38°C in rice (Prasad et al., 2006b), 33°C in bell pepper (Erickson and Markhart, 2002), and 32°C in common bean (Gross and Kigel, 1994; Porch and Jahn, 2001). Night temperatures of 30°C reduced anther dehiscence by 84% and pollen viability by 78% in cowpea (Ahmed, Hall, and DeMason, 1992). These patterns are so widespread that it has led to suggestions that flowering plants in general experience high rates of heat-induced pollen sterility at temperatures near 36°C, a widely reported thermal sterility threshold (Sage et al., 2015). Indeed, I studied the effect of 36/24°C day/night temperatures in comparison to 28/24°C on eight diverse heat-adapted species: Amaranthus cruentus, Capsicum annuum, Capsicum baccatum, Cucurbita palmata, Eragrostis tef, Phaseolus acutifolius, Phaseolus coccineus, and Zea mays var. Yuman yellow (Figure 1-1) (Sage et al., 2015). At 36°C all species experienced >60% pollen abortion at the uninucleate stage; in contrast at 28°C ≤ 10% of pollen aborted at the uninucleate stage of development (Sage et al., 2015). While this phenomenon is documented in many crops, little research into whether wild plant populations experience similar sterility patterns to crops; representing a critical gap in our understanding of pollen heat sensitivity. Given that there are many mechanisms of heat sterility (Bita and Gerats, 2013; De Storme and Geelen, 2014), crops and wild plants may respond differently under heat stress.

Climate change has the potential to cause an agricultural crisis should pollination fail in our crop plants. In response to this realization, research into understanding how heat stress affects pollen viability in crops has gained priority in recent years (Hedhly, 2011; Sage et al., 2015). Three of the leading hypotheses for pollen injury due to heat include: accumulation of reactive oxygen species, irregularities in tapetum development, and carbohydrate starvation (Bita and Gerats, 2013; De Storme and Geelen, 2014; Sage et al., 2015). However, these three are not mutually exclusive: indeed, one may be a prerequisite for another.

100 ● ● ● ● 100 ● ● ● ●

● ● ●

75 ● 4 ● 75 ●

● Treatment Day/NightTreatment Temperature● 28/24 (°C) 50 ● ● ● 36/24 Abortion 28/24 50 ● 36/24 Abortion

100 ● ● 100 ● ● 25 ● ● ● ● ● 25 ● ● ● ● ●

● ● ● 75 ● ● ● 75● ● ● 0 ● ● ● ● ● 0 ● ● ● Treatment ● Treatment ● ● 28/24 28/24 50 Eragrostis tef 50 ● ● 36/24 Abortion 36/24 Abortion Zea mays Yuman Capsicum annuum Cucurbita palmata Eragrostis tef Amaranthus cruentus Capsicum baccatum Phaselous acutifoliusPhaseolus coccineus

Pollen Abortion (%) Pollen Zea mays Yuman Capsicum annuum Cucurbita palmata Amaranthus cruentus Capsicum baccatum Phaselous acutifoliusPhaseolus coccineus

25 25

● ● ● ●

● ● ● ● ● ●● ● ● 0 0 ● ●

Eragrostis tef Eragrostis tef Zea mays YumanZea mays Yuman Capsicum annuumCapsicum annuumCucurbita palmataCucurbita palmata Capsicum baccatum Amaranthus cruentusAmaranthus cruentusCapsicum baccatum Phaselous acutifoliusPhaseolusPhaselous coccineusacutifoliusPhaseolus coccineus Species

Figure 1-1. Growth temperature effects on pollen abortion in 8 plant taxa at growth regimes of 28/24°C and 36/24°C day/night temperatures. Abortion was determined with Alexander’s Triple Stain (n=3, except n=4 for P. acutifolius and n =5 for P. coccineus) Bars are standard deviation. Adapted from Sage et al. 2015. Field Crops Res. 182: 30-4.

Reactive oxygen species (ROS) accumulate in male reproductive organs during heat events, particularly in microspores/pollen grains (Prasad and Djanaguiraman, 2011; De Storme and Geelen, 2014; Sage et al., 2015) and the tapetum (De Storme and Geelen, 2014; Sage et al., 2015). While ROS is part of the normal heat stress response and can be beneficial by inducing production of heat shock proteins (HSPs) and antioxidants that mediate heat stress effects (Frank et al., 2009), when stress is prolonged or extreme, there can be an overwhelming accumulation of ROS, leading to injury (Karuppanapandian et al., 2011; Mittler, Finka, and Goloubinoff, 2012; Bita and Gerats, 2013). Evidence of oxidative injury includes membrane disruption, protein degradation, DNA mutation and damage, and cell death (Karuppanapandian et al., 2011; Sage et al., 2015). At high levels, oxidative injury caused by ROS can lead to male reproductive abortion (Karuppanapandian et al., 2011; Mittler, Finka, and Goloubinoff, 2012; Sage et al., 2015), as observed in ROS accumulation in uninucleate rice pollen (Bagha, 2014). While undergoing heat stress, plants also produce ROS-scavenging enzymes, such as ascorbate peroxidase and superoxide dismutases, which reduce ROS levels (Frank et al., 2009; Karuppanapandian et al., 2011; Mittler, Finka, and Goloubinoff, 2012; Bita and Gerats, 2013; Siebers et al., 2015). This could be a mechanism that explains higher genotypic variation between plant genotypes/species from warm climates then genotypes/species from cool climates, possibly through exhibiting greater ROS scavenging potential (Karuppanapandian et al., 2011). HSPs bind to membranes and proteins to act as chaperones to prevent denaturation and repair misfolding (Frank et al., 2009; Bita and Gerats, 2013). Similarly, in membranes, heat injury is ameliorated by production of tocopherols and tocotrienols, that scavenge oxidized lipids in the membranes (Munné-Bosch and Alegre, 2002). Heat tolerant plants may have a greater capacity to protect proteins and lipids from damage by increased concentration or efficiency of HSPs and tocopherols/tocotrienols.

The tapetum is an ephemeral cell layer of the anther, surrounding the developing pollen grains (D’Arcy, 1996; Pacini, 1996; Sage et al., 2015). It serves an important function for proper pollen development, transporting essential elements and sugars, thereby ensuring an adequate nutrient supply (Wu and Cheung, 2000; Suzuki et al., 2001; Sage et al., 2015). Additionally, it builds the sporopollenin layer that protects the pollen from UV damage (Bedinger, 1992; Erickson and Markhart, 2002; Albert, et al., 2010). As part of its normal function, the tapetum undergoes programmed cell death and in the process releases components for the pollen outer surface, adhesives, and signaling molecules, which are essential for the completion of pollen maturation

(Wu and Cheung, 2000; Dolferus, Ji, and Richards, 2011). At the uninucleate stage of pollen development, when the microspores are released from the tetrad, the tapetum is highly active (Parish et al., 2012; Sage et al., 2015). At this time, both the microspores and the tapetum are highly vulnerable to heat stress (Ahmed, Hall, and DeMason, 1992; Quilichini, Douglas, and Samuels, 2014; Sage et al., 2015). Exposure to 39/30°C day/night temperature resulted in retention of the tapetum in rice (Endo et al., 2009), restricting the release of necessary pollen wall components. In contrast, in bean and cowpea, premature degradation of the tapetum occurred at average air temperatures of 28°C (Suzuki et al., 2001) and 33°C respectively (Ahmed, Hall, and DeMason, 1992), causing loss of pollen. In bean this was attributed to disruption of endoplasmic reticulum development in the tapetum (Suzuki et al., 2001).

Carbohydrates play a critical role in pollen development. Anthers are large sinks for carbohydrates, particularly as they are elongating (Clement, Burrus, and Audran, 1996). As the anthers mature there is high turnover in the types of carbohydrates and their concentration; this turnover is vulnerable to heat stress (Clement, Burrus, and Audran, 1996; Aloni et al., 2001; Pressman, Peet, and Pharr, 2002; Sato et al., 2006). Flowers of bell peppers grown at 35°C maximum temperature were allocated less sugar than at 25°C maximum growing temperatures (Aloni et al., 1991). However, high temperature did not alter sugar content of vegetative tissues, but rather the amount exported from the leaves (Aloni, et al., 1991). This reduction in sucrose and starch concentrations in anthers at high temperature is associated with a disruption in source- sink relationships (Pressman, Peet, and Pharr, 2002; Aloni et al., 1991; Aloni et al., 2001). For instance, in tomato, high temperature resulted in a reduced starch accumulation and therefore reduced available sugars in the developing pollen (Pressman, Peet, and Pharr, 2002). Photosynthesis, as previously mentioned, can be affected by high temperatures, resulting in reduced carbon availability to flowers, leading to pollen starvation during development (Rekika, Monneveux, and Havaux, 1997; Masetri et al 2002; Boyer and Westgate, 2004) or abscission of floral buds, as seen in peppers grown at 32/26°C day/night temperatures (Aloni et al., 1991).

1.1 Heat Studies in Wild Species

The reproductive sensitivity to heat in wild plants has largely been ignored, such that the vulnerability of the Earth’s natural flora to heat-induced sterility is poorly understood. From the crop literature, it is widely acknowledged that plants begin to experience extensive pollen

7 sterility above 32°C up to a threshold near 36°C, above which complete abortion is typically observed (Sage et al., 2015). If pollen development in natural plant species exhibit similar patterns of heat sensitivity as noted for crops, then many ecosystems could be threatened by widespread loss of reproductive potential in their resident flora.

As previously mentioned, research on heat sterility in heat-adapted plants observed high rates of pollen abortion at 36°C day temperature in all species examined including, Cucurbita palmata, Amaranthus cruentus, Capscium annuum, and Zea mays var. Yuman yellow (Figure 1-1) (Sage et al., 2015). C. palmata is found in the Mojave Desert and has vegetative tissue heat tolerance of 47.5°C (Seeman, Berry, and Downton, 1984), but it exhibited 90% pollen abortion when exposed to 36°C daytime temperature (Sage et al., 2015). A. cruentus, found throughout Mexico (National Research Council, 1984), C. annuum, a pepper from the warm sub-tropics, and Z. mays var. Yuman, a cultivar from the Mojave Desert region (Sage et al., 2015), all experience warm temperatures regularly. These results indicate there is a consistent sterility threshold across higher plant taxa (Matsui et al., 1997; Djanaguiraman et al., 2013; Song, Chen, and Tang, 2014; Sage et al., 2015) and suggests that heat-adapted species may also be vulnerable to climate warming that could raise exposure temperatures above 35°C in their natural habitat during flowering.

In warm climates, many wild species routinely encounter daytime temperatures that exceed 35°C (Berry and Björkman, 1980). In particular, plants from low latitude climates grow and reproduce during seasons when daily temperatures exceed 40°C (Berry and Björkman, 1980); for example, Mojave and Sonoran deserts summer annuals (Berry and Björkman, 1980). Here and elsewhere, for example low latitude arid zones of Australia and Africa, it is not uncommon to observe plants in hot climates flowering close to the ground on substrate of a dark colour, where surface temperatures can exceed 50°C (R Sage, unpublished).

Species from such climates have vegetative heat tolerance up to 50°C (Berry and Björkman, 1980). For example, Downton, Berry, and Seemann (1984) observed photosynthesis in 41 desert species and 2 crops, maize and cotton, to tolerate temperatures from 39-46°C without obvious injury, and could acclimate to temperatures up to 52°C. Additionally they found that cacti, such as Lophocereus schottii, could tolerate temperatures between 50-58°C (Seeman et al., 1984). One Mojave Desert species, Tidestromia suffruticosa var. oblongifolia, exhibits a thermal optimum of

8 photosynthesis at 46°C, and still maintains higher photosynthetic rates near 55°C relative to cooler adapted species (Berry and Björkman, 1980). The capacity of leaves to avoid heat injury at such warm temperatures is evidence that the reproductive system may also be adapted to such high temperatures. However, if pollen development has a particularly heat sensitive step, then even species from the world’s hot deserts may be vulnerable to reproductive failure at temperatures their photosynthetic and other physiological processes would otherwise withstand. A primary goal of this study is to examine the thermal tolerance of pollen maturation in a hot desert species.

To investigate whether hot climate species have greater heat sterility thresholds, I established a series of criteria to identify the ideal species for the study. First, my intention was to identify a plant that clearly flowers in a hot situation, ideally as hot as possible. The range of my collecting possibilities was limited to North America; the low-elevation Mojave Desert represents the ideal location within this range. June to August peak temperatures in this region exceed 40°C, and summer monsoons are limited in strength, though still sufficient to support a robust population of plants (Figure 1-2) (Western Regional Climate Center, 2016). Second, the plant should occur and flower in hot microsites and during the day, so heat escape is not possible. Prostrate species flowering in the surface boundary layers of the soil cannot avoid heat exposure or sunny days (Körner, 2003), which are common in the Mojave during the summer. Third, the species should be a C4 plant, as the high thermal optimum of the C4 pathway will ensure an abundant carbohydrate supply during summer heat (Berry and Björkman, 1980; Prasad et al., 2006a). Additionally, the species should be easily obtained, germinated, and grown in a laboratory setting. Many desert species are difficult to germinate and grow, due to strong dormancy mechanisms and sensitivity to growth in pots. The species I identified that best fits these criteria is Trianthema portulacastrum, a widespread herbaceous weed from hot climates of low latitude (Figure 1-3) (Correll and Johnston, 1996; Western Australian Herbarium, 1998-; Flora of North America, 1993+).

There are 20 described species in the genus Trianthema (family Aizoaceae, order Caryophyllales), many of which occur in some of the hottest places in the world, such as Western Australian and Southwestern North American deserts (Correll and Johnston, 1996; Western Australian Herbarium, 1998-; Flora of North America, 1993+). T. portulacastrum is a self-compatible species that is able to proliferate under a range of warm temperatures, and has an

9 optimal seed germination temperature of 35°C (Baylan and Bhan, 1986), further indicating its suitability for reproductive heat tolerance studies. This species is found globally in tropical and subtropical areas growing in sandy soils and waste grounds (Balyan and Bhan, 1986; Correll and Johnston, 1996; Flora of North America, 1993+). Prostrate growth places its ~4mm flowers at ground level (Flora of North America, 1993+), where the hot soil and boundary layer resistance can cause heat buildup and reduction in convectional cooling for the plant. In the Mojave, it has been observed proliferating and flowering along roadsides when air temperature exceeds 45°C and surface temperature exceeds 50°C (Figure 1-3) (R Sage, unpublished). In prior studies (Muhaidat, Sage, and Dengler, 2007), the Sage lab has developed germination and growth protocols that demonstrate the ease of growing T. portulacastrum in Toronto, in greenhouse and growth chambers, and that it performs well in hot (>35°C) conditions.

It is critical that we understand how rising temperatures will affect plant reproduction, not only in our agriculturally important species, but in natural populations, and particularly species in habitats that currently proliferate near the upper limits of growth season temperatures, such as desert and tropical species. This thesis examines the heat tolerance of pollen and seed set in

Trianthema portulacastrum, also known as desert horse purslane, a C4 herbaceous plant that proliferates in hot climates around the world and is an important agricultural weed (Baylan and Bhan, 1986). The ability of T. portulacastrum to compete against crops in hot climates, as well as grow along marginal areas in the Mojave Desert, suggests that it may have higher heat tolerance than the predicted 36°C threshold. This paper seeks to address the effect that rising temperatures will have on the reproduction of heat-adapted species, using T. portulacastrum as a model. Two hypotheses will specifically be addressed. First, pollen development in T. portulacastrum has a significantly higher sterility threshold than the 36°C value commonly observed in crop species. Alternatively, if heat sterility shows the same thermal threshold as warm season crops, then T. portulacastrum may utilize a night escape mechanism, where pollen development is occurring primarily at night. A third possibility is T. portulacastrum is as heat sensitive as other species, which would suggest low fecundity in its summer environment.

Average Max. Average Min. Las Vegas, NV Needles, CA

● ● Las Vegas, NV Needles, CA ● 40 ● 40 40 ● ● ● ● ●

● ● 3030 30 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 20 ● 20 C) ● 20 ● ° ● ●

( ● ● ● ● ● ● ● ● 1010 ● 10 Temperature (degrees Celsius) Temperature ● (degrees Celsius) Temperature ● ● ● ● ● ● 0 0 Palm Springs, CA 0 Yuma, AZ Jan Feb Mar Ap May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Ap May Jun Jul Aug Sep Oct Nov Dec Temperature

● Palm Springs, CA ● Yuma, AZ ● ● 40 40 ● 40 ● ● ● ● ● ● ● ● 30 30 ● 30 ● ● ● ● ● ● ● ● ●

AverageAir ● ● ● ● ● ● 20 ● 20 ● ● ● ● 20 ● ● ● ● ● ● ● ● ● 10 ● 10 10 ● Temperature (degrees Celsius) Temperature Temperature (degrees Celsius) Temperature ● ● ●

0 0 0 Jan Feb Mar Ap May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Ap May Jun Jul Aug Sep Oct Nov Dec

Month

Figure 1-2. Average monthly maximum and minimum temperatures from four locations in the Mojave Desert: Las Vegas, Nevada (1996-2008), Needles, California (2001-2008), Palm Springs, California (1998-2008), and Yuma, Arizona (1996-2008). Records retrieved from Western Regional Climate Center, 2016 at http://www.wrcc.dri.edu/summary/lcdus08.html.

A B

Figure 1-3. Trianthema portulacastrum growth habit and morphology. A) Image taken along Highway 64, California, USA about 7km west of Nipton, CA B) Small (~3-5mm diameter) pink flowers are found at branching points of the plant. Photos taken by Rowan F. Sage, 2004.

Methods 2.1 Growth Conditions

Seeds of T. portulacastrum (family Aizoaceae) were originally collected in August 2002 by Rowan Sage from a waste ground on a barren lot in St. George, Utah, USA. These seeds were used to grow subsequent plants from which the seeds I used were produced. To promote germination, seeds were heat stratified for 72 hours at 50°C; then soaked in water and placed on filter paper in a petri dish. Seedlings were planted in 3’’ pots containing 2/5 sand, 2/5 loam, and 1/5 Sunshine Mix. After the emergence of 3-5 true leaves they were transplanted into individual

3.8L pots. Plants were watered daily and fertilized twice a week with 1g/L Ca(NO3)2, 0.26g/L

MgSO4, and a 2:1 mixture of two commercial fertilizers: 1.7g/L Miracle Gro (24-8-16), and 0.9g/L Plant-Prod (30-10-10) at the recommended dose. The commercial fertilizers were complete with the exception of Ca and MgSO4.

Plants were initially grown at 30/24°C day/night temperatures, which is the average daytime temperature during the year (Western Regional Climate Center, 2016). Photon flux density was maintained at 350-450 µmolm2s-1 at plant canopy height in Conviron PGR15 growth chambers. Photoperiod was set to 12 hours to stimulate flowering (Table 2-1). The temperatures of the air and flowers were monitored using microthermistors and recorded by HOBO model Pv2 data loggers (Figure 2-1).

Table 2-1. Experimental chamber temperature and lighting schedule, where TT is treatment temperature. Lights are fluorescent and incandescent bulbs. During the first half hour of the lights turning on, the chambers gradually ramped to the daytime temperature.

Time (24hr) Temperature (°C) Lights 0000 24 off 1000 24 on(half) 1030 TT on(full) 2130 TT on(half) 2200 24 off 2359 24 off

12 13

2.2 Experiment 1: Variable day temperature, constant night temperature

The experimental approach for the main series of treatments consisted of first growing plants at a non-stressful “control temperature” (30/24°C) for four weeks. Plants were then transferred to their designated treatment daytime temperature in plant growth chambers (model GC-20, BioChambers Inc, Winnipeg, MN) and exposed for one hour to induce a heat shock response (Prasad and Djanaguiraman, 2011; Mittler, Finka, and Goloubinoff, 2012). After one hour, plants were returned to the control growth regime (30/24°C). This heat-shock treatment was intended to induce heat acclimation that would reduce acute heat stress upon permanent transfer to the thermal treatments. In field conditions, plants would not normally experience a rapid shift in temperature, but rather a gradual increase. This heat-shock treatment ensured proper transition to the high temperature treatments. After 24 hours, the plants were transferred to their treatment regime for the duration of the experiment (two weeks). Treatment temperatures were 30/24°C (control), 33/24°C, 36/24°C, 40/24°C and 44/24°C day/night temperature. Five plants were randomly chosen for each treatment and each experiment was fully replicated three times at each temperature using a different chamber per replicate treatment. Treatment at 44/24°C required watering twice a day to prevent drought stress. Sampling of floral tissues commenced after twelve consecutive days in treatment conditions to ensure that sampled flowers had initiated during the treatment temperature.

14 d

T30 T44 5050 A 50 B

C) ● Air ● Air ° ● ( ● ● ● ● Flower Flower ● ● ● ● ●●● ●● ●●●●●●●●●●● ●●●●●●● ● ●●●●●● ●●●●●●●●●●●● ●●●● ●●● ●●● ● ●●● ● ● ●●●●●●●●●●●● ● ● ● ● 40 40 ● 40 ●

●

● ● ● ● ● ● ● ●●●●● ●●● ● ● ●●●● ●●●●●● ●●●● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● 3030 ●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● 30 ● ● ● ● Temperature Temperature ● ● ● ● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●● ●● ●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●● ●●●●●●●● 2020 20 Average Temperature 0 :00 0:00 3:00 6:00 9:00 0:00 3:00 6:00 9:00 :00 :00 :00 :00 :00 d d 12:00 15:00 18:00 21:00 24:00 12:00 15:00 18:00 21:00 24:00 0:00 3 0:00 3 6 9 6 9 12:00 15:00 12:00 15:00 18:00 24:00 21:00 18:00 24:00 Time Time 21:00 Time (24hr)

Figure 2-1. Average air (solid circle) and flower (empty circle) temperatures measured with thermistors and recorded by HOBO Pv2 data loggers in the A) control treatment, 30/24°C and B) 44/24°C treatment during a 72-hour period. Error bars are standard deviation.

2.3 Experiment 2: Elevated night temperature

This experiment examined the effect of HNT on reproduction. The experimental approach remained unchanged, except for the use of 24/40°C and 40/40°C thermal treatments commencing 24 hours following a 40°C pre-treatment for 1 hour. Five plants were chosen randomly for each treatment and the same measurements were collected for these treatments as the five previous experimental treatments. Plants at 40/40°C require watering three times a day to prevent drought. These treatments were conducted once (n=5).

2.4 Analysis of Anther and Pollen Development

To assess pollen abortion, Alexander’s Triple Stain (ATS) was used (Alexander, 1969). ATS stains pollen cytoplasm purple and the pollen wall blue; an aborted pollen grain appears blue and the cell wall is generally collapsed (Alexander, 1969; Firon et al., 2006). Thus pollen could be classified as either halting development prematurely (aborting) or not aborting.

For all treatment groups, five buds per plant were excised per replicate. Anthers were removed prior to dehiscence and stained with ATS (Alexander, 1969). The slides were then placed in the dark for at least 72 hours before examining under a compound microscope. Total pollen grains were counted as either aborted (stained only blue) or not aborted (Alexander, 1969; Firon et al., 2006; Harsant et al., 2013). Total number of pollen grains was also recorded to determine whether heat affected pollen production.

Timing of anthesis was documented during all treatments, which remained consistent. To determine timing of anther dehiscence, three plants were reexamined per treatment. Five buds were removed every 20 minutes from 07:00-14:00. Lights in the chamber turned on at 10:00AM. The buds were dissected and examined (Table 2-2).

Anther dehiscence was examined at all temperature treatments. Whole anthers from 3 flowers per plant were excised 24 hours after pollination. Using a light microscope, anthers were characterized as either retaining pollen (≥ 40% of the anther is full) or dehisced (<40% filled with pollen).

2.5 Pollen Viability Characterization

Pollen viability was examined using an enzyme activity assessment and pollen germination (Rao, Jain, and Shivanna, 1992; Young, Wilen, and Bonham-Smith, 2004; Djanaguiraman et al., 2013; Harsant et al., 2013; Choudhary et al., 2014). Pollen enzyme activity was assessed by performing a fluorochromatic reaction with fluorescein diacetate (FDA), which enters the pollen wall and is hydrolyzed by esterase (Choudhary et al., 2014). The FDA stock solution contained 2mg/mL FDA in acetone and was stored at -20°C. The FDA stain contained 15% sucrose with 20µL/mL of stock solution in distilled water. Three buds from each plant (n=15) were removed and whole anthers were abscised prior to dehiscence. Pollen grains were removed, placed in FDA, and observed under a fluorescent microscope with a FITC filter. Total pollen number was recorded and pollen was characterized as either viable (indicated by fluorescence) or inviable (no fluorescence) (Heslop-Harrison and Heslop-Harrison, 1970).

Pollen germination was examined on a stigma rather than on germination medium, which allows for a more natural germination response. Three flowers from each plant (n=15) were pollinated at their respective thermal treatments. These flowers were removed 24 hours later and fixed in

3:1 ethanol-acetic acid for 12 hours, washed five times with distilled water, and placed in Aniline blue (AB). The washes removed unadhered pollen grains only (Samuel et al., 2009); compatible pollen grains have a strong adhesion to the stigma (and therefore would not have been dislodged during the washing process (Samuel et al., 2009). After at least 3 hours, flowers were dissected and observed using a fluorescent microscope with a DAPI (blue/cyan) filter. As pollen tubes lengthen, callose plugs are deposited and stained by aniline blue, enabling visualization of germinated pollen grains and pollen tube growth (Martin, 1959). Total pollen on the stigma and the percent that had germinated was recorded. Germination was identified as pollen tubes that had elongated ³ diameter of the pollen grain (Prasad et al., 2006a; Djanaguiraman et al., 2013).

2.6 Characterizing Reproductive Success

Documenting the seed set is the ultimate viability test, because it shows that healthy pollen grains that deposited on the stigma had germinated and fertilized ovules. Twenty flowers from each plant (n=15) were pollinated and labeled. Plants were moved back into the 30/24°C growing conditions 24 hours after pollination. Fruits were allowed to develop under these control conditions. Fruits were removed after one week, before maturity, to ensure all seeds per fruit were recorded. The average number of seeds per fruit was calculated.

To determine differences between seed set and ovule number, AB was used to count the average number of ovules per flower. Ovule number was recorded from buds (3 per plant) used for the pollen germination characterization. The average number of ovules per flower was recorded for each treatment. Additionally, to assess apomictic reproduction, ten flowers from five plants at 30/24°C and 40/24°C were emasculated and labeled. They were then measured for seed set.

2.7 Statistical Analysis

The five temperature treatments were replicated three times in different chambers. Each treatment contained fifteen plants, unless noted otherwise above.

To analyze changes in a) the number of pollen grains produced, b) pollen abortion rates, c) anther dehiscence rates, d) pollen viability (esterase activity), e) germination, f) ovules produced and g) seed set per fruit between the five daytime temperature treatments I generated linear

17 models and assessed these models using two-way ANOVAs in R (R Core Team, 2016). Models included treatment, replicate, and the treatment by replicate interaction as independent variables and observations made from the same plant were pooled to generate a mean. Treatment was treated as a categorical variable in these analyses. This statistical design accounted for chamber effects, allowing the sampling unit to be individual plants. Significance was tested using type 3 sums of squares in the car package (Fox and Weisberg, 2011). Tests of normality and homoscedasticity, including histograms of the residuals, normal quantile-quantile plots, scale- location plots, and Levene’s test for homogeneity of variance, were performed to ensure the assumptions of ANOVA were met. A Tukey’s Test was performed to make pairwise comparisons to examine the specific differences between treatment groups. For cases in which there were significant differences among treatments, I reanalyzed the data treating daytime temperature as a continuous variable to test for a linear relationship between daytime temperature and each dependent variable. A Kruskal-Wallis rank sum test was used to analyze anther dehiscence. This is because the assumptions of normality and homogeneity of variance required for parametric statistics was violated.

Experiment two contained nighttime temperature treatments, 24/40°C and 40/40°C, which were replicated once, due to time limitations. The measured variables were analyzed along with the 40/24°C daytime temperature treatment. Treatments were all conducted in the same growth chamber to eliminate chamber effects. The same tests of normality and variance as described previously were used, as well as a one-way ANOVA assessment.

Table 2-2. Growth conditions and flowering stages in T. portulacastrum during timing of experimental methods. TT refers to temperature treatment (30, 33, 36, 40, 44).

Time Growth Conditions (24hr) (Temp °C, Light) Flowering Observations Experimental Method 7:00 24, lights off Mature anthers, containing mature pollen grains ATS -9:30 Emasculation FDA 9:30 24, lights off Anthers begin to dehisce -10:00 24, lights on (half) 11:00 TT, lights on (full) Flowers elongated, majority of anthers have dehisced, flowers begin opening 12:00 TT, lights on (full) Majority of flowers open AB preparation -13:00 Anthers dehisced Labelling for seed set 13:00 TT, lights on (full) Flowers begin to close 13:30 TT, lights on (full) Majority of flowers closed

Results

The timing of flowering was consistent across temperature treatments. Pollen was released from the tetrad stage after 22:00 (Figure 3-1). Pollen grains were fully mature by 0:700 (Table 2-2). Anthers began to dehisce at 09:30-10:00, half an hour before the lights in the chamber turned on (Table 2-2). Two hours after the lights in the chamber had turned on, the majority of the flowers were open and could be pollinated between 12:00-13:00 before closing.

The number of pollen grains averaged about 150 per anther at the 30/24°C, 33/24°C, and 36/24°C treatments, and rose to a mean of 198 in the 44/24°C treatment (Figure 3-2A). In all treatments, the number of mature pollen grains not aborted as indicated by Alexander’s Triple Stain was 80 to 95% (Figure 3-2B), however the sample size was small (n=5). The majority of anthers dehisced across all treatments (c2= 6.95, df = 4, p>0.1) (Figure 3-2C).

The viability of the pollen, as determined by FDA, was 60% or above in all treatments

(F4,60=23.2, p<0.001), but showed a gradual decline from near 80% in the 30/24°C treatment to near 60% in the 44/24°C treatment (R2=0.37, p<0.001) (Figure 3-3A). Germination percentages were elevated (~70%) in the 30/24°C treatment but between ~45-60% in the warmer 4 treatments

(F4,40=7.11, p<0.001) (Figure 3-3B). Three to four ovules were present in each fruit at all treatment temperatures (F4,53=5.5962, p<0.001) (Figure 3-3C), while seed set showed no temperature treatment response, averaging just above 3 per fruit in all conditions (F4,60=1.9766, p=0.11) (Figure 3-3D). T. portulacastrum is not apomictic, as emasculated flowers had marginal seed set (<1 per fruit) (F3,16=229.9275, p<0.001) (Figure 3-4).

To evaluate sensitivity of reproduction to HNT, I also examined pollen, ovule and seed set responses to 40°C night temperatures in tandem with either 24°C day or 40°C day temperatures. Anther dehiscence was reduced from ~90% in the 40/24°C treatment to ~50% in the 24/40°C treatment, and ~60% in the 40/40°C treatment (Table 3-1). Pollen viability was also reduced >50% in the two 40°C night treatments compared to the 40/24°C treatment. Pollen germination percentage was similar in the three treatments of Experiment 2. Ovule number per fruit was slightly elevated in the two 40°C night treatments relative to the 40/24°C treatment, while seed number per fruit was reduced in the two 40°C night treatments. At 24/40°C, three seeds per fruit were observed, but only 2.2 at 24/40°C and 1.5 at 40/40°C. By comparison, over three seeds per fruit were measured in the control (30/24°C) treatment of Experiment 1.

A B C D *

50µm 100µm 50µm 50µm E F G H

50µm 10µm 10µm 50µm

Figure 3-1. Anther and pollen development in T. portulacastrum at 30/24°C observed with Alexander’s Triple Stain. A,E) Microspore mother cell stage; B,F) Anther following microspore mother cell meiosis at 22:00. Pollen grains are in a tetrad; G) Tetrad of pollen grains prior to separation; C) Anther following tetrad separation, uninucleate pollen grains; D) Pollen grains after mitosis; H) Mature anther with fully developed pollen grains at 09:30. Double asterisk indicates microspore mother cell. Arrow head indicates tetrad of uninucleate pollen grains. Arrow indicates thick callose walls. Single asterisk indicates tapetum.

250250 A

200200 ●

●

● 150150 ab b ● ● ab a a 100100 Number of pollen grains

5500 Number of pollen grains

00 30/24 33/24 36/24 40/24 44/24 5050 Treatment B

4040

3030 ab ab b

2020 ● ●

Pollen abortion (%) Pollen ● a Pollen abortion (%) 1010 ● a ●

00 30/24 33/24 36/24 40/24 44/24 Treatment C

● 100100 ● ● ●

8080 ●

6060

4040 Anther dehiscence (%)

Anther dehiscence (%) 20

00 30/2430/24 33/2433/24 36/2436/24 40/2440/24 44/2444/24 Treatment Day/Night Temperature (°C)

Figure 3-2. Growth temperature effects on A) Pollen grain number per anther (n=5); B) the fraction of pollen aborting as determined by Alexander’s Triple Stain (n=5); and C) the proportion of anthers dehiscing (n=5 for day temperatures 30°C and 33°C, n=10 for 36°C, 40°C, and 44°C). Bars are standard deviation. Lowercase letters beside symbols indicate statistically distinct groups based on Two-way ANOVA with a Tukey’s post-hoc test.

A B 100100 100 100

a a ab 8080 ab b 80 80 PollenGermination (%) ● bc bc ● ● ● b ● ● b ● 60 60 b 60 ● 60

● ●

4400 40 40 Pollen viability (%) Pollen Pollen Viability (%) Viability Pollen Pollen germination (%) Pollen

2200 20 20

00 0 0 30/24 33/24 36/24 40/24 44/24 30/24 33/24 36/24 40/24 44/24 55 Treatment 5 Treatment 5 CC D

4 4 4 ● 4 ● SeedNumber perFruit

● ● ● ● ● ● 3 ab b 3 ● ● 3 a 3 a a

22 2 2 Number of seeds Number of ovules

Ovule Number per Flower per Number Ovule 11 1 1

0 0 0 30/24 33/24 36/24 40/24 44/24 30/24 33/24 36/24 40/24 44/24 0 Treatment Treatment Day/Night Temperature (°C) Day/Night Temperature (°C)

Figure 3-3. Growth temperature effects on A) pollen viability as determined by fluorescein diacetate (n=15); B) pollen germination percentage determined with aniline blue (n=15, exception 33/24°C n=10); C) Average number of ovules per flower (n=10, exception 36/24°C and 44/24°C n=15); D) Average seed set per fruit (n=15). Bars are standard deviation. Lowercase letters beside symbols indicate statistically distinct groups based on Two-way ANOVA with a Tukey’s post-hoc test.

a 4 ● b

● 33 d 2 Number of seeds

11 c

Seed Number per Fruit c ● ● 0

30/2430/24 40/2440/24 30/24E30/24 E 40/2440/24E E

Treatmentd Day/Night Temperature (°C)

Figure 3-4. Growth temperature effects on seed set, where E indicates emasculated flowers (n=5). Bars are standard deviation. Lowercase letters beside symbols indicate statistically distinct groups based on Two-way ANOVA with a Tukey’s post-hoc test.

Table 3-1. Nighttime growth temperature effects on anther dehiscence, pollen viability, pollen germination, ovule number per flower, and seed set per fruit. LS mean and standard error from Sample size is 5 in one replicate. Letters beside means indicate statistically distinct groups based on One-way ANOVA with a Tukey’s post-hoc test.

F statistic Day/Night Least Squares Standard (df=2, Temperature Mean Deviation residuals =12) P-value Anther Dehiscence 24/40 53b ±10 10.213 <0.01 (%) 40/24 93a ±19 40/40 61b ±15

Pollen Viability 24/40 36b ±2 47.198 <0.001 (%) 40/24 77a ±8 40/40 27b ±11

Pollen 2.9637 0.09 Germination 24/40 26 ±10 (%) 40/24 51 ±16 40/40 42 ±22

Ovule Number 24/40 4b ± <1 43.2 <0.001 per Flower 40/24 3c ± <1 40/40 5a ± <1

Seed Set 24/40 2b ± <1 30.05 <0.001 per Fruit 40/24 3a ± <1 40/40 1c ± <1

Discussion

To date, the vast majority of studies, largely examining crops, indicate that higher plants are vulnerable to heat sterility above 32-36°C (Matsui et al., 1997; Harsant et al., 2013; Sage et al., 2015). Due to the increasing growth season temperatures associated with climate warming, there is a potential risk of widespread loss in fecundity resulting from heat sterility. In crop plants, this could substantially reduce yield, particularly at low latitude where plants already experience temperatures approaching sterility thresholds (Figure 1-2) (Western Regional Climate Center, 2016). In the natural flora of the Earth, particularly the species-rich tropical biomes, widespread loss of fecundity may result if wild species exhibit similar patterns as seen in crops. To evaluate this, I selected a species from one of the hottest landscapes on Earth, the Mojave Desert, where summer air temperatures exceed 40°C (MacMahon, 1986; Sage et al., 2015; Western Regional Climate Center, 2016) and temperatures within the surface boundary layer can rise above 50°C (R Sage, unpublished). This species, T. portulacastrum, is a prostrate herb which produces flowers in the boundary layer, and I thus hypothesized that T. portulacastrum evolved greater heat tolerance and reduced sterility well above 35°C.

My results showed that hot days up to 40°C did not reduce pollen grain number, pollen abortion rates, anther dehiscence, or ovule and seed number per fruit. Pollen viability and germination rates were slightly reduced above 30°C. Growth at 44°C day temperature also did not strongly affect pollen parameters or seed set; only the number of anthers dehiscing was substantially reduced. From these results, I conclude that T. portulacastrum has evolved exceptional heat tolerance and shows little sterility at high daytime temperatures that would otherwise sterilize crop species. While it is probable that other hot climate species have as high a heat tolerance as T. portulacastrum, our results represent the highest example of reproductive heat tolerance so far demonstrated in the literature. Thus, there exists in the natural flora sufficient heat tolerance to ensure fecundity in very hot environments, a feature that may be of value to crop breeders.

How does T. portulacastrum achieve this result? One possibility is that it has inherent tolerance in developing flowers and anthers, preventing heat-induced sterility. There are many potential mechanisms of pollen heat-induced sterility, such as ROS accumulation, abnormal tapetum development, and carbohydrate starvation (Bita and Gerats, 2013; De Storme and Geelen, 2014; Sage et al., 2015). The sensitivity of male reproduction during/following meiosis, when pollen is

26 at the uninucleate stage, has been documented in a number of species (Dolferus, Ji, and Richards, 2011), such as cowpea (Ahmed, Hall, and DeMason, 1992), rice (Bagha, 2014; Sage et al., 2015), and wheat (Saini and Aspinall, 1982). The ability of T. portulacastrum to produce abundant mature pollen grains at 40°C and 44°C demonstrates that the uninucleate stage is not affected by high daytime temperature, as it is in crop species. Although I did not quantify ROS accumulation under heat stress, T. portulacastrum did not exhibit premature abortion and showed marginal reductions in viability/germination at high daytime temperature, suggesting that there may be little ROS accumulation within the anther when T. portulacastrum experiences high day temperatures. Wheat cultivars with higher heat tolerance had greater activity of ROS-scavenging enzymes, such as superoxide dismutase, and increased ascorbic acid concentration, which reduced the amount of oxidative damage (Sairam, Srivastava, and Saxena, 2000; Almeselmani et al., 2006). Additionally, heat stress can cause misfolding of proteins and alterations in membrane structure and fluidity (Sairam, Srivastava, and Saxena, 2000; Barnabás, Jäger, and Fegér, 2008). These otherwise detrimental effects can be ameliorated by chaperones, such as HSPs (Giorno et al., 2013). Thermotolerance could potentially be linked to upregulation of genes encoding ROS- scavenging enzymes and HSPs, but more research is needed in this area. If so, T. portulacastrum may have higher capacities to scavenge ROS, and protect membranes and proteins; thereby protecting pollen from degradation that would otherwise lead to reduced viability. Alternatively, T. portulacastrum may have mechanisms that reduce ROS production in hot conditions. Either way the exceptional thermotolerance exhibited by T. portulacastrum could reveal promising new mechanisms for improving heat tolerance in a wider range of crop species.

The pollen carbohydrate starvation hypothesis relies on an alteration in the photosynthate sink from reproductive organs to other parts of the plant (Boyer and Westgate, 2004; Barnabás, Jäger, and Fegér, 2008). T. portulacastrum is a C4 species, meaning it has a greater capacity to photosynthesize at high temperatures, in comparison to C3 species (Berry and Björkman, 1980;

Prasad et al., 2006a). The majority of our agricultural plants utilize the C3 photosynthetic pathway, with a few exceptions, such as maize and sorghum (Prasad et al., 2006a). Out of the total exported photosynthate in pepper plants, Aloni et al. (1991) found 3.1% of the carbon was allocated to developing flowers under normal temperatures (25/18°C), whereas only 0.3% of the total carbon was exported to the flowers at 35/25°C. This was proposed to result in flower abscission in the heat stressed plants (Aloni et al., 1991). C3 plants exhibit higher rates of

27 photorespiration at high temperature, meaning they not only uptake carbon, but they also expel carbon (Berry and Björkman, 1980). Similarly, plants experiencing drought stress may reduce carbon allocation to reproductive structures, which could result from heat-induced water deficiency in vegetative organs (Saini, 1997). C4 plants have higher water-use efficiency (Prasad et al., 2006a); therefore, they may not experience indirect carbohydrate starvation in the developing flowers to the same extent as C3 plants. Therefore, C4 plants, such as T. portulacastrum, may be able to maintain carbon exports or have less of a reduction of carbohydrates to reproductive structures, because they have a lower photorespiration rate.

However, maize, a C4 crop, lacks reproductive heat tolerance (Lobell et al., 2011; Sage et al., 2015), suggesting it is unable to maintain carbon allocation to flowers.

Anther indehiscence is commonly observed in crop species at elevated temperature (Ahmed, Hall, and DeMason, 1992; Porch and Jahn, 2001; Hedhly, 2011; Bagha, 2014). A heat-sensitive cultivar of common bean (Phaseolus vulgaris) experienced dehiscence inhibition when exposed to 9 or more days at 32/27°C day/night temperatures (Porch and Jahn, 2001). This response was associated with the lack of endothecium wall thickenings, preventing proper tension in the anther wall (Ahmed, Hall, and DeMason, 1992; Porch and Jahn, 2001). T. portulacastrum anther dehiscence was on average 80% under extreme temperatures of 44/24°C. The results indicate greater capacity to withstand heat stress at the level of the anther cell wall, potentially by maintaining endothecium thickness. Matsui, Omasa, and Horie (2001) found rice with greater heat tolerance had a much thicker endothecium and formed cavities within the anther, which were believed to promote dehiscence. In contrast, a heat sensitive cultivar had neither, and exhibited lower dehiscence rates (Matsui, Omasa, and Horie, 2001).

I also hypothesized that T. portulacastrum could escape heat sterility by shifting flower development to nights and early morning. Heat escape would be apparent if critical stages of pollen development occurred at night. As previously mentioned, the uninucleate stage of pollen development, when microspores are released from the tetrad, is highly vulnerable to heat stress (Ahmed, Hall, and DeMason, 1992; Quilichini, Douglas, and Samuels, 2014; Sage et al., 2015). Sampling of T. portulacastrum under control temperatures (30/24°C) during the night found uninucleate pollen grains in tetrads. At these conditions, pollen viability was ~80%. My observations indicate the critical uninucleate stage of pollen development occurred at 22:00 (Figure 3-1). When the nighttime temperature was raised to 40°C in Experiment 2, pollen

28 viability was reduced 40-50%, indicating T. portulacastrum is using a night escape strategy. Such a night escape strategy would have adaptive value in the field environment, where the nighttime temperatures during the summer typically decline to ~26°C (Figure 1-2). Night temperatures are expected to increase disproportionately more than daytime temperatures in the next century (Karl, Kukla, and Razuvayev, 1991; Peng et al., 2004; Jagadish, Murty, and Quick, 2015). Average night temperatures in the Philippines have already risen 1.13°C between 1979- 2003 (Peng et al., 2004). This raises concerns for regions such as Needles, CA where average night temperature during July was 30°C between the years 2001-2008 (Figure 1-2) (Western Regional Climate Center, 2016). If desert plant flowers are developing at night temperatures that already exceed 30°C, increased nighttime warming represents a critical vulnerability for desert plant reproduction.

Conclusion and Future Directions

In this study, I show that T. portulacastrum has the highest reproductive thermal tolerance yet recorded. Daytime temperatures up to 44°C had at most a modest impact on pollen viability and seed set per fruit. Night temperatures of 40°C did reduce seed set and pollen viability significantly, but plants still maintained substantial seed production, averaging 1- 2 seeds per fruit in the two 40°C night treatments. Therefore, I can conclude that while the evidence supports heat escape at night, T. portulacastrum still exhibits greater reproductive heat tolerance than has been observed in all crop and non-domesticated species examined to date. T. portulacastrum can thus be considered an excellent model to examine reproductive heat tolerance, both to identify explanatory mechanisms, as well as to study how its tolerance sustains fitness in field settings, where it would work in tandem with night escape.

Going forward, research on T. portulacastrum should focus on its mechanism of heat tolerance. In particular, how does it manage ROS accumulation at elevated temperatures, and what role do HSPs play in ameliorating heat injury in anthers and developing pollen. Additionally, a more holistic understanding of heat impacts on reproduction is needed. Here I focused on pollen performance and seed set during 12 days of reproduction. T. portulacastrum has indeterminate flowering, producing many more flowers over the growing season than during these 12 days. It will be important to examine the reproductive effort of whole plants over the growing season where timing effects can be coupled with flower production and seed set per flower. Additional efforts should address other aspects of the reproductive process, notably pollen tube growth, fertilization success, and seed viability. In its natural habitat, the diurnal timing of T. portulacastrum development could be examined in the context of actual temperatures, and its interaction with pollinators examined. It would be interesting to determine if T. portulacastrum can shift its floral timing to night only when high temperatures occur, as opposed to showing constitutive night development. Furthermore, its floral timing may reflect pollinator capabilities. It is likely bee or fly pollinated, and these animals may restrict activity to the cooler mornings, which is when I observed opening of T. portulacastrum flowers.

In conclusion, T. portulacastrum demonstrates that heat-adapted species have evolved impressive reproductive thermal tolerance to withstand extreme heat stress. Further research should explore other wild species from hot climates to better understand the natural diversity of heat tolerance in higher plant taxa. By doing so, we can better understand the vulnerability of these biomes to climate warming and therefore predict future outcomes more effectively. Additionally, crop improvement studies seeking to generate heat tolerant lines for the future should look to T. portulacastrum as a model of reproductive heat tolerance. As global temperatures rise along with the looming possibility of widespread crop failure all avenues of possible mitigation should be explored. I believe that reproductive heat tolerance in wild species, specifically T. portulacastrum, comprises one of these avenues.

6 References

Ahmed, F.E., Hall, A.E., DeMason, D.A. 1992. Heat injury during floral development in cow pea (Vigna unguiculata, Fabaceae). American Journal of Botany, 79(7), 784-791. Ainsworth, E.A., Ort, D.R. 2010. How do we improve crop production in a warming world? Plant Physiology, 154, 526-530. Albert, B., Nadot, S., Dreyer, L., Ressayre, A. 2010. The influence of tetrad shape and intersporal callose wall formation on pollen aperture pattern ontogeny in two eudicot species. Annals of Botany, 106(4), 557-564. Alexander, M.P. 1969. Differential staining of aborted and nonaborted pollen. Stain Technology, 44, 117-122. Almeselmani, M., Deshmukh, P.S., Sairam, R.K., Kushwaha, S.R., Singh, T.P. 2006. Protective role of antioxidant enzymes under high temperature stress. Plant Science, 171, 382-388. Aloni, B., Pashkar, T., Karni, L. 1991. Partitioning of [14C]sucrose and acid invertase activity in reproductive organs of pepper plants in relation to their abscission under heat stress. Annals of Botany, 67, 371-377. Aloni, B., Peet, M., Pharr, M., Karni, L. 2001. The effect of high temperature and high

atmospheric CO2 on carbohydrate changes in bell pepper (Capsicum annuum) pollen in relation to its germination. Physiologia Plantarum, 112, 505-512. Bagha, S. 2014. The impact of high temperatures on anther and pollen development in cultivated Oryza species. (Ph.D. dissertation). University of Toronto. Balyan, R.S., Bhan, V.M. 1986. Emergence, growth, and reproduction of horse purslane (Trianthema portulacastrum) as influenced by environmental conditions. Weed Science, 34(4), 516-519. Barnabás, B., Jäger, K., Fegér, A. 2008. The effect of drought and heat stress on reproductive processes in cereals. Plant, Cell and Environment, 31, 11-38. Bedinger, P. 1992. The remarkable biology of pollen. The Plant Cell, 4, 879-887. Berry, J., Björkman, O. 1980. Photosynthetic response and adaptation to temperature in high plants. Annual Review of Plant Physiology, 31, 491-543. Bita, C.E., Gerats, T. 2013. Plant tolerance to high temperature in a changing environment: scientific fundamentals and production of heat stress-tolerant crops. Frontiers in Plant Science, http://dx.doi.org/10.3389/fpls.2013.00273 31 32

Boyer, J.S., Westgate, M.E. 2004. Grain yields with limited water. Journal of Experimental Botany, 55(407), doi:10.1093/jxb/erh219 Choudhary, N., Siddiqui, M.B., Bi, S., Khatoon, S. 2014. Effect of seasonality and time after anthesis on the viability and longevity of Cannabis sativa pollen. Palynology, 48(2), 235- 241. Clement, C., Burrus, M., Audran, J.-C. 1996. Floral organ growth and carbohydrate content during pollen development in Lilium. American Journal of Botany, 83(4), 459-469. Collins, M., Knutti, R., Arblaster, J., Dufresne, J.-L., Fichefet, T., Friedlingstein, P., Gao, X. Gutowski, W.J., Johns, T., Krinner, G., Shongwe, M., Tebaldi, C., Weaver, A.J., Wehner, M. 2013. Long-term Climate Change: Projections, Com-mitments and Irreversibility. In: Stocker, T.F., Qin, D., Plattner, G.-K., Tignor, M.., Allen, S.K., Boschung, J., Nauels, A., Xia, Y., Bex, V., Midgley, P.M. (Eds.), Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge /New York. Correll, D.S., Johnston, M.C. 1996. Manual of the Vascular Plants of Texas. Dallas: The University of Texas. Djanaguiraman, M., Prasad, P.V.V., Boyle, D.L., Schapaugh, W.T. 2013. Soybean pollen anatomy, viability and pod set under high temperature stress. Journal of Agronomy and Crop Science, 199, 171-177. D’Arcy, W. 1996. Anthers and stamens and what they do. In: D’Arcy, W.G., Keating, R.C. (Eds.), The Anther: Form, function and phylogeny. Cambridge University Press, Cambridge. De Storme, N. Geelen, D. 2014. The impact of environmental stress on male reproductive development in plants: biological processes and molecular mechanisms. Plant, Cell and Environment, 37, 1-18. Dolferus, R., Ji, X., Richards, R.A. 2011. Abiotic stress and control of grain number in cereals. Plant Science, 181, 331-341. Downton, W.J.S., Berry, JA., Seemann, J.R. 1984. Tolerance of photosynthesis to high temperature in desert plants. Plant Physiology, 74, 786-790.

Endo, M., Tsuchiya, T., Hamada, K., Kawamura, S., Yano, K., Ohshima, M., Higashitani, A., Watanabe, M., Kawagishi-Kobayashi, M. 2009. High temperatures cause male sterility in rice plants with transcriptional alterations during pollen development. Plant and Cell Physiology, 50(11), 1911-1922. Erickson, A.N., Markhart, A.H. 2002. Flower developmental stage and organ sensitivity of bell pepper (Capsicum annuum L.) to elevated temperature. Plant, Cell and Environment, 25, 123-130. Firon, N., Shaked, R., Peet, M.M., Pharr, D.M., Zamski, E., Rosenfeld, K., Althan, L., Pressman, E. 2006. Pollen grains of heat tolerant tomato cultivars retain higher carbohydrate concentration under heat stress conditions. Scientia Horticulturae, 109, 212-217. Flora of North America Editorial Committee, eds. 1993+. Flora of North American North of Mexico. 19+ vols. New York and Oxford. Fox, J., Weisberg, S. 2011. An {R} Companion to Applied Regression, Second Edition. Thousand Oaks CA: Sage. URL: http://socserv.socsci.mcmaster.ca/jfox/Books/Companion Frank, G., Pressman, E., Ophir, R., Althan, L., Shaked, R., Freedman, M., Shen, S., Firon, N. 2009. Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response. Journal of Experimental Botany, 60(13), 3891- 3908. Galen, C. 2000. High and dry: drought stress, sex-allocation trade-offs, and selection on flower size in the alpine wildflower Polemonium viscosum (Polemoniaceae). The American Naturalist, 156(1):72-83. Giorno, F., Wolters-Arts, M., Mariani, C., Rieu, I. 2013. Ensuring reproduction at high temperatures: the heat stress response during anther and pollen development. Plants, 2, 489-506. Gross, Y., Kigel, J. 1994. Differential sensitivity to high temperature of stages in the reproductive development of common bean (Phaseolus vulgaris L.). Field Crops Research, 36, 201-212.

Harsant, J., Pavlovic, L., Chiu, G., Sultmanis, S., Sage, T.L. 2013. High temperature stress and its effect on pollen development and morphological components of harvest index in the C3 model grass Brachypodium distachyon. Journal of Experimental Botany, 64(10), 2971-2983. Hasanuzzaman, M., Nahar, K., Alam, M.M., Roychowdhury, R., Fujita, M. 2013. Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants. International Journal of Molecular Sciences, 14, 9643-9684. Hatfield, J.L., Boote, K.J., Kimball, B.A., Ziska, L.H., Izaurralde, R.C., Ort, D., Thomson, A.M., Wolfe, D. 2011. Climate impacts on agriculture: implications for crop production. Agronomy Journal, 103(2) 351-370. Hatfield, J.L., Prueger, J.H. 2015. Temperature extremes: effect on plant growth and development. Weather and Climate Extremes, 10, 4-10. Hedhly, A. 2011. Sensitivity of flowering plant gametophytes to temperature fluctuations. Environmental and Experimental Botany, 74, 9-16. Hedhly, A., Hormaza, J.L., Herrero, M. 2008. Global warming and sexual plant reproduction. Trends in Plant Science, 14(1), 30-36. Heslop-Harrison, J., Heslop-Harrison, Y. 1970. Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Technology, 45, 115-120. Huang, Z., Footitt, S., Finch-Savage, W.E. 2014. The effect of temperature on reproduction in the summer and winter annual Arabidopsis thaliana ecotypes Bur and Cvi. Annals of Botany, 113, 921-929. Iwahori, S. 1966. High temperature injuries in tomato. V. Fertilization and development of embryo with special reference to the abnormalities caused by high temperature. Journal of the Japanese Society for Horticultural Science, 35(4), 55-62. Jagadish, S.V.K., Murty, M.V.R., Quick, W.P. 2015. Rice responses to rising temperatures – challenges, perspectives and future directions. Plant, Cell and Environment, 38, 1686- 1698. Jagadish, S.V.K., Muthurajan, R., Oane, R., Wheeler, T.R., Heuer, S., Bennett, J., Craufurd, P.Q. 2010. Physiological and proteomic approaches to address heat tolerance during anthesis in rice (Oryza sativa L.). Journal of Experimental Botany, 61(1), 143-156.

Jain, M., Prasad, P.V.V., Boote, K.J., Hartwell Jr, A.L., Chourey, P.S. 2007. Effects of season- long high temperature growth conditions on sugar-to-starch metabolism in developing microspores of grain sorghum (Sorghum bicolor L. Moench). Planta, 227, 67-79. Karl, T.R., Kukla, G., Razuvayev, V.N. 1991. Global warming: evidence for asymmetric diurnal temperature change. Geophysical Research Letters, 18(12), 2253-2256. Karuppanapandian, T., Moon, J.-C., Kim, C., Manoharan, K., Kim, W. 2011. Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms. Australian Journal of Crop Science, 5(6), 709-725. Körner, C. 2003. Alpine plant life: functional plant ecology of high mountain ecosystems (2nd ed.). Springer, Verlag, Berlin, Heidelberg, Germany. Lobell, D.B., Asner, G.P. 2003. Climate and management contributions to recent trends in U.S. agricultural yields. Science, 299, 1032. Lobell, D.B., Bänziger, M., Magorokosho, C., Vivek, B. 2011. Nonlinear heat effects on African maize evidenced by historical yield trials. Nature Climate Change, 1, 42-45. Lobell, D.B., Sibley, A., Ortiz-Monasterio, J.I. 2012. Extreme heat effects on wheat senescence in India. Nature Climate Change, 2, 186-189. MacMahon, J.A. 1986. Deserts. The Audubon Society nature guides. Alfred A. Knopf, Inc., New York. Maestri, E., Klueva, N., Perrotta, C., Gulli, M., Nguyen, H.T., Marmiroli, N. 2002. Molecular genetics of heat tolerance and heat shock proteins in cereals. Plant Molecular Biology, 48, 667-681. Mathieu, A.-S., Lutts, S., Vandoorne, B., Descamps, C., Périlleux, C., Dielen, V., Van Herck, J.- C., Quinet, M. 2014. High temperatures limit plant growth but hasten flowering in root chicory (Cichorium intybus) independently of vernalisation. Journal of Plant Physiology, 171, 109-118. Matsui, T., Omasa, K., Horie, T. 2001. Comparison between anthers of two rice (Oryza sativa L.) cultivars with tolerance to high temperatures at flowering or susceptibility. Plant Production Science, 4, 36-40.

Matsui, T., Namuco, O.S., Ziska, L.H., Horie, T. 1997. Effects of high temperature and CO2 concentration on spikelet sterility in indica rice. Field Crops Research, 51, 213-219. Martin, F.W. 1959. Staining and observing pollen tubes in the style by means of fluorescence. Stain Technology, 34(3), 125-128.

Min, L., Li, Y., Hu, Q., Zhu, L., Gao, W., Wu, Y., Ding, Y., Liu, S., Yang, X., Zhang, X. 2014. Sugar and auxin signaling pathways respond to high-temperature stress during anther development as revealed by transcript profiling analysis in cotton. Plant Physiology, 164, 1293-1308. Mittler, R., Finka, A., Goloubinoff, P. 2012. How do plants feel the heat?, Trends in Biochemical Sciences, 37(3), 118-125. Morrison, M.J., Stewart, D.W. 2002. Heat stress during flowering in summer Brassica. Crop Science, 42: 797-803. Muhaidat, R., Sage, R.F., Dengler, N.G. 2007. Diversity of Kranz anatomy and biochemistry in

C4 eudicots. American Journal of Botany, 94(3), 362-381. Munné-Bosch, S., Alegre, L. 2002. The function of tocopherols and tocotrienols in plants. Critical Reviews in Plant Sciences, 21(1), 31-57. National Research Council. 1984. Amaranth: modern prospects for an ancient crop. National Academy Press, Washington, D.C. Oberhuber, W., Edwards, G.E. 1993. Temperature dependence of the linage of quantum yield of

photosystem II to CO2 fixation in C4 and C3 plants. Plant Physiology, 101, 507-512. Oshino, T., Abiko M., Saito, R., Ichiishi, E., Endo, M., Kawagishi-Kobayashi, M., Higashitani, A. 2007. Premature progression of anther early development programs accompanied by comprehensive alterations in transcription during high-temperature injury in barley plants. Molecular Genetics and Genomics, 278, 31-42. Pacini, E. 1996. Types and meaning of pollen carbohydrate reserves. Sexual Plant Reproduction, 9, 362-366. Parish, R.W., Phan, H.A., Iacuone, S., Li, S.F. 2012. Tapetal development and abiotic stress: a centre of vulnerability. Functional Plant Biology, 39, 553-559. Peng, S., Huang, J., Sheehy, J.E., Laza, R.C., Visperas, R.M., Zhong, X., Centeno, G.S., Khush, G.S., Cassman, K.G. 2004. Rice yields decline with higher night temperature from global warming. Proceedings of the National Academy of Science, 101(27), 9971-9975. Porch, T.G., Jahn, M. 2001. Effects of high-temperature stress on microsporogenesis in heat- sensitive and heat-tolerant genotypes of Phaseolus vulgaris. Plant, Cell and Environment, 24, 723-731.

Prasad, P.V.V., Boote, K.J., Allen, L.H. Jr. 2006a. Adverse high temperature effects on pollen viability, seed-set, seed yield and harvest index of grain-sorghum [Sorghum bicolor (L.) Moench] are more severe at elevated carbon dioxide due to higher tissue temperatures. Agricultural and Forest Meteorology, 139, 237-251. Prasad, P.V.V., Boote, K.J., Allen, L.H. Jr., Sheehy, J.E., Thomas, J.M.G. 2006b. Species, ecotype and cultivar differences in spikelet fertility and harvest index of rice in response to high temperature stress. Field Crops Research, 95, 398-411. Prasad, P.V.V., Djanaguiraman, M. 2011. High night temperature decreases leaf photosynthesis and pollen function in grain sorghum. Functional Plant Biology, 38, 993-1003. Prasad, P.V.V., Pisipati, S.R., Mutava, R.N., Tuinstra, M.R. 2008. Sensitivity of grain sorghum to high temperature stress during reproductive development. Crop Science, 48, 1911- 1917. Pressman, E., Peet, M.M., Pharr, M. 2002. The effect of heat stress on tomato pollen characteristics is associated with changes in carbohydrate concentration in the developing anthers. Annals of Botany, 90, 631-636. Quilichini, T.D., Douglas, C.J., Samuels, A.L. 2014. New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana. Annals of Botany, doi:10.1093/aob/mcu042. R Core Team. 2016. R: a language and environment for statistical computer. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/ Rao, G.U., Jain, A., Shivanna, K.R. 1992. Effects of high temperature stress on Brassica pollen: viability, germination and ability to set fruits and seeds. Annals of Botany, 68, 193-198. Rekika, D., Monneveux, P., Havaux, M. 1997. The in vivo tolerance of photosynthetic membranes to high and low temperatures in cultivated and wild wheats of Triticum and Aegilops genera. Journal of Plant Physiology, 150, 734-738. Sage, T.L., Bagha, S., Lundsgaard-Nielsen, V., Branch, H.A., Sultmanis, S., Sage, R.F. 2015. The effect of high temperature stress on male and female reproduction in plants. Field Crops Research, 182, 30-42. Saini, H.S. 1997. Effects of water stress on male gametophyte development in plants. Sexual Plant Reproduction, 10, 67-73. Saini, H.S., Aspinall, D. 1982. Abnormal sporogenesis in wheat (Triticum aestivum L.) induced by short periods of high temperature. Annals of Botany, 49, 835-846.

Sairam, R.K., Srivastava, G.C., Saxena, D.C. 2000. Increased antioxidant activity under elevated temperatures: a mechanism of heat stress tolerance in wheat genotypes. Biologia Plantarum, 43(2), 245-251. Samuel, M.A., Chong, Y.T., Haasen, K.E., Aldea-Brydges, M.G., Stone, S.L., Goring, D.R. 2009. Cellular pathways regulating response to compatible and self-incompatible pollen in Brassica and Arabidopsis stigmas intersect at Exo70A1, a putative component of the Exocyst Complex. The Plant Cell, 21, 2655-2671. Sato, S., Kamiyama, M., Iwata, T., Makita, N., Furukawa, H., Ikeda, H. 2006. Moderate increase of mean daily temperature adversely affects fruit set of Lycopersicon esculentum by disrupting specific physiological processes in male reproductive development. Annals of Botany, 97, 731-738. Schlenker, W., Roberts, M.J. 2009. Nonlinear temperature effects indicate severe damages to U.S. crop yields under climate change. Proceedings of the National Academy of Science, 106(37), 15594-15598. Schrader, S.M., Wise, R.R., Wacholtz, W.F., Ort, D.R., Sharkey, T.D. 2004. Thylakoid membrane responses to moderately high leaf temperature in Pima cotton. Plant, Cell and Environment, 27, 725-735. Seemann, J.R. Berry, J.A., Downton, J.S. 1984. Photosynthetic response and adaptation to high temperature in desert plants. Plant Physiology, 75, 364-368. Seemann, J.R., Downton, W.J.S., Berry, J.A. 1986. Termperature and leaf osmotic potential as factors in the acclimation of photosynthesis to high temperature in desert plants. Plant Physiology, 80, 926-930. Sharkey, T.D. 2005. Effects of moderate heat stress on photosynthesis: importance of thylakoid reactions, rubisco deactivation, reactive oxygen species, and Thermotolerance provided by isoprene. Plant, Cell and Environment, 28, 269-277. Sharkey, T.D., Badger, M.R., von Caemmerer, S., Andrews, T.J. 2001. Increased heat sensitivity of photosynthesis in tobacco plants with reduced Rubisco activase. Photosynthesis Research, 67, 147-156. Siebers, M.H., Yendrek, C.R., Drag, D., Locke, A.M., Acosta, L.R., Leakey, A.D.B., Ainsworth, E.A., Bernacchi, C.J., Ort, D.R. 2015. Heat waves imposed during early pod development in soybean (Glycine max) cause significant yield loss despite a rapid recovery from oxidative stress. Global Change Biology, 21, 3114-3125.

Song, G., Chen, Q., Tang, C. 2014. The effects of high-temperature stress on the germination of pollen grains of upland cotton during square development. Euphytica, 200, 175-186. Snider, J.L., Oosterhuis, D.M. 2011. How does timing, during and severity of heat stress influence pollen-pistil interactions in angiosperms?. Plant Signaling and Behavior, 6(7), 930-933. Suzuki, K., Takeda, H., Tsukaguchi, T., Egawa, Y. 2001. Ultrastructural study on degeneration of tapetum in anther of snap bean (Phaseolus vulgaris L.) under heat stress. Sexual Plant Reproduction, 13, 293-299. Wahid, A., Gelani, S., Ashraf, M., Foolad, M.R. 2007. Heat tolerance in plants: an overview. Environmental and Experimental Botany, 61, 199-223. Western Australian Herbarium (1998-2016). FloraBase-the Western Australian Flora. Department of Parks and Wildlife. https://florabase.dpaw.wa.gov.au/ Western Regional Climate Center, cited 2016: LCD Summaries for the Western U.S. [Available online at http://www.wrcc.dri.edu/summary/lcdus08.html] Wu, H., Cheung, A.Y. 2000. Programmed cell death in plant reproduction. Plant molecular biology, 44, 267-281. Young, L.W., Wilen, R.W., Bonham-Smith, P.C. 2004. High temperature stress of Brassica napus during flowering reduces micro- and megagametophyte fertility, induces fruit abortion, and disrupts seed production. Journal of Experimental Botany, 55(396), 485- 495.