The Effect of Drying on Desiccation Tolerance and Late Embryogenesis Abundant Protein Gene Expression in Immature Seeds of Phalaenopsis Amabilis" (2014)

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Masters Theses Graduate Research and Creative Practice

2-2014 The ffecE t of Drying on Desiccation Tolerance and Late Embryogenesis Abundant Protein Gene Expression in Immature Seeds of Phalaenopsis amabilis Huijing Zhu Grand Valley State University

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Recommended Citation Zhu, Huijing, "The Effect of Drying on Desiccation Tolerance and Late Embryogenesis Abundant Protein Gene Expression in Immature Seeds of Phalaenopsis amabilis" (2014). Masters Theses. 714. http://scholarworks.gvsu.edu/theses/714

This Thesis is brought to you for free and open access by the Graduate Research and Creative Practice at ScholarWorks@GVSU. It has been accepted for inclusion in Masters Theses by an authorized administrator of ScholarWorks@GVSU. For more information, please contact [email protected]. The Effect of Drying on Desiccation Tolerance and Late Embryogenesis Abundant

Protein Gene Expression in Immature Seeds of Phalaenopsis amabilis

Huijing Zhu

A Thesis Submitted to the Graduate Faculty of

GRAND VALLEY STATE UNIVERSITY

Partial Fulfillment of the Requirements

For the Degree of

Master of Science

Cell and Molecular Biology

February 02, 2014 Abstract

Orchidaceae as the most diverse family of flowering plants are endangered due to losing habitats or destruction of their unique symbiotic living style. Long-term seed storage banks can be a solution for threatened plant species but the stored seeds must tolerate extreme drying and cold. This ability is acquired during the last stage

(maturation drying) of seed development and is correlated with a decline in water content and expression of the Late Embryogenesis Abundant (LEA) protein genes. Our goal is to investigate if premature orchid seeds can be artificially induced to become desiccation tolerant and the potential role of LEA genes in this process. In this work I monitored changes in water content, germinability, desiccation tolerance and LEA protein gene expression in seeds undergoing artificial drying in Phalaenopsis amabilis. Seeds were processed in 4-day Slow Drying (SD) treatment where they are placed in atmospheres of progressively lower relative humidity and High Relative Humidity (HRH) control where they are placed above water. During SD, seeds maintained their starting moisture content of 70% for 3 days, and then dried to 10-15% moisture on the 4th day. Seeds that had undergone SD become desiccation tolerant but seeds in the HRH control did not.

Transcript level of all three LEA genes we’ve studied (TG1: Group4 LEA; TG2: group3

LEA; Dehydrin: Group2 LEA) increased after excision in both treatments but the transcript level of LEA genes from SD treatment were higher than control group. Our results suggest that premature seeds of Phalaenopsis amabilis can tolerate desiccation by appropriate post-harvest treatments and the accumulation of LEA proteins may contribute to the tolerance of desiccation.

Table of Contents

Abstract ….………………………………………………………………………….. 3

List of Tables ……………………………………………………………………….. 6

List of Figures ………………………………………………………………………. 7

List of Abbreviations ……………………………………………………………...... 9

Chapter 1 - Introduction to Thesis - An Introduction to Phalaenopsis amabilis and the Prospects for Successful Storage of their Immature Seeds ………………… 10

An Introduction to the Orchids………………………………………………….. 10 Taxonomy, Distribution and Habitat………………………………………... 10 Conservation Status ………………………………………………………… 11 Mutualistic Relationships between Orchids and Other Organisms………… 12 Economic Importance ……………………………………………………… 13

Our Research Model: Phalaenopsis amabilis ………………………...... 15

Structure and development of the Angiosperm Seed ………………………….. 19 Fertilization………………………………………………………………….. 22 Histodifferentiation ………………………………………………………… 24 Reserve Deposition ………………………………………………………… 26 Maturation Drying …………………………………………………………. 27

Seed Storage Banks ……………………………………………………………. 30

Late Embryogenesis Abundant Proteins ………………………………………. 33 LEA group 2 ……………………………………………………………….. 34 LEA group 3 ……………………………………………………………….. 37 LEA group 4 ……………………………………………………………….. 39

Literature Cited ………………………………………………………………… 42

Chapter 2 – Manuscript - The Effect of Drying on Desiccation Tolerance and Late Embryogenesis Abundant Protein Gene Expression in Immature Seeds of Phalaenopsis amabilis………………………………………………………….. 56

Abstract ………………………………………………………………………… 57

Introduction……………………………………………………………………… 58

Materials and Methods ………………………………………………………… 63 Growth conditions and seed harvesting…………………………………… 63

Post-harvest treatments…………………………………………………….. 63 Physiological tests ……………………………………………………….... 64 Biochemical tests ………………………………………………………….. 65

Results ………………………………………………………………………….. 72 Slow drying induced desiccation tolerance………………………………. 72 LEA genes were up-regulated by slow drying and high relative humidity treatment…………………………………………………….. 74 Dehydrin protein level was slightly elevated on day 4 of high relative humidity control treatment …………………………………………….. 79

Discussion ……………………………………………………………………….. 81

Literature Cited …………………………………………………………………... 88

Supplementary Data ……………………………………………………………… 91

List of Tables

Table 1. Sequences of primers used in this work...... 68

List of Figures

Chapter 1

Figure 1.1 Contribution of different floricultural crops ...... 14

Figure 1.2 Sketch of Phalaenopsis ...... 15

Figure 1.3 A typical angiosperm seed (Dicot) ...... 19

Figure 1.4 Structure of a typical angiosperm carpel ...... 20

Figure 1.5 Seed structure of Phalaenopsis amabilis ...... 21

Figure 1.6 The mega-gametophyte ...... 22

Figure 1.7 Array of the distinctive motifs in group2 LEA proteins...... 34

Figure 1.8 Sequence analysis of the dehydrin amino acid sequence ...... 35

Figure 1.9 Array of the distinctive motifs in group3 LEA proteins...... 37

Figure 1.10 The predicted protein product of a group3 LEA gene in Phalenopsis amabilis aligns with LEA Group 3A motifs and orthologues ...... 38

Figure 1.11 Array of the distinctive motifs in group4 LEA proteins...... 39

Figure 1.12 The predicted protein product of TG1 aligns with LEA Group 4 motifs and

orthologues ...... 41

Chapter 2

Figure 2.1 Morphological evaluation of germinating orchid seeds ...... 65

Figure 2.2 Nucleotide sequence of dehydrin from OrchidBase ...... 67

Figure 2.3 The translated amplified dehydrin sequence of Phalaenopsis aligns with other dehydrins...... 69

Figure 2.4 Amino acid sequence of dehydrin gene of Phalaenopsis ...... 70

Figure 2.5 The effect of Slow-drying and High-relative humidity treatments on moisture content and germinability ...... 72

Figure 2.6 Effect of desiccation on germination ...... 74

Figure 2.7 Expression of TG2, TG1 and Dehydrin in seeds of 130 DAP...... 75

Figure 2.8 The effect of Slow Drying (SD) and High Relative Humidity (HRH) treatment for 4 days on the expression level of TG1, TG2 and Dehydrin ...... 77

Figure 2.9 Difference in cycles required for actin transcript and target gene transcript to cross threshold fluorescence...... 78

Figure 2.10 The effect of slow drying and high relative humidity treatment on dehydrin protein level as determined by Western blot with anti-dehydrin ...... 79

List of Abbreviations

LEA: Late Embryogenesis Abundant

DAP: Date after Pollination

FW: Fresh Weight

SD: Slow Drying

HRH: High Relative Humidity

RH: Relative Humidity

RER: Relative Expression Ratio

ABA: Abscisic Acid

Chapter 1. An Introduction to Phalaenopsis amabilis and the Prospects for

Successful Storage of their Immature Seeds

An Introduction to the Orchids

Orchids, well known for their varied and unique flower patterns, are loved by amateur collectors and scientists alike. However, many species are threatened with extinction primarily by human activities. The orchid family is one of the largest families of flowering plants with 25000 estimated species (Cribb et al., 2003). Although this family is distributed throughout the world (including desert, semiscrub, rain cloud forest, and even tundra ecosystems) its greatest diversity is achieved in the tropics and semitropics where over half of the species are found (Dressier, 1993). Each orchid species often requires a specific pollinator (usually an insect or a bird). In addition, seed germination of most species depends on specific symbiotic mycorrhizae which are required to transport nutrients from the environment to the seed. These complex interactions are partly due to their long evolutionary history – the family originated 80 million years ago (Bradt, 2007). This fascinating lifestyle as well as the commercial popularity of orchids has inspired some research targeted at conservation. This section summarizes our knowledge about the distribution of orchids, their conservation status, their unique biology, and finishes with the economic importance of orchids.

Taxonomy, distribution and habitat

Orchidaceae is divided into five sub families; Apostasioideae, Cypripedioideae,

Vanilloideae, Orchidoideae and Epidendroideae. The latter three subfamilies make up

99% of the orchid species (Royal Botanic Gardens, 2013). Epidendroideae, Vanilloideae

10 and Apostasioideae are mainly restricted to tropical and subtropical habitats by their living requirements (climate and existence of required pollinators and mycorrhizae). The remaining two subfamilies: Cypripedioideae and Orchidoideae also have representatives in Europe and North America (Royal Botanic Gardens, 2013). Over half of orchid species are epiphytic with the remainder being terrestrial. Most epiphytic species belong to

Epidendroideae and Vanilloideae (Sanford, 1974) and the Cypripedioideae and

Orchidoideae and Apostasioideae are predominately terrestrial (Pridgeon et al, 1999).

Conservation status

Currently (2013), 450 orchid species are included on the IUCN Red list of threatened species. Two hundred and six of these are critically endangered (41 species), endangered (99 species) or vulnerable (66 species). The largest threats to orchid populations are human activities, most notably, habitat alteration, and extraction or collection of wild plants for trade (Hagsater and Dumont, 1996). Whereas habitat destruction may directly cause wholesale extinction of all orchid species living in a given area, collection targets specific orchids and can result in a precipitous decline in their numbers. For example, most species of Paphiopedilum (a terrestrial genus that is a main target of collection in south-east Asia) are rare in the wild and 25 of the 60 species recognized by Cribb are seriously endangered (Cribb, 1987).

Habitat alteration (including destruction or modification, and fragmentation) is universally recognized as a major threat to wild biodiversity, but it is especially acute in the tropical areas where most orchid species are endemic. The annual deforestation rate in tropical areas reached a peak estimated at 0.9% during the 90s (World Conservation

Monitoring Center 1992). Even though the deforestation rate has declined in recent years,

11 it remains high in many tropical countries due to agriculture (which accounts for 50%-65% of deforestation) and logging (10%-15%) (FAO 2005; Butler, 2007).

Habitat alteration or loss not only affects the orchids themselves but also the unique mutualistic symbioses required for their growth and reproduction. These mutualistic relationships include mycorrhizal relationships with specific fungal species during germination to supply nutrients, and relationships with a very specific and limited group of pollinators (Swarts & Dixon, 2009). The obligate nature of these relationships from the orchids’ perspective makes orchids especially vulnerable to habitat loss or degradation. The threats from losing such components of the orchids’ ecosystems are discussed in the following sections.

Mutualistic Relationships between Orchids and Other Organisms

Since orchids have a high level of pollinator specialization, reduction or even extinction of pollinator populations constitutes a serious threat to orchid persistence

(Roberts, 2003). Charles Darwin wrote about the intricate and specialized pollination biology of orchids (Swarts and Dixon, 2009) and the subject remains intriguing to scientists to this day. The pollinators include birds (mostly humming birds) and insects such as ants, butterflies, flies, carrion beetles, wasps, bees, and fungus gnats (Horak, 2004;

Tremblay, 2006; Gaskett, 2011). Each orchid flower’s intricacy poses a barrier to all potential pollinators except for the specialized partner. These ―hybridization barriers‖

(Tremblay, 2006), coupled with the high number of seeds, high dispersal rates and preadaptation for epiphytism may be responsible for the rapid radiation and evolution of the diversity we see today in the orchid family (Hagsater and Dumont, 1996). This same

12 pollinator specialization now is causing a high risk of extinction as pollinator populations themselves teeter.

Like the obligate relationships between orchids and their pollinators, the orchid- mycorrhizal relationship has been researched since the 19th century and this subject continues to fascinate scientists (see, for example Bonnardeaux et al., 2007; Brundrett et al., 2003 and Ramsay et al., 1986). Epiphytic species are not as dependent on such mutualistic relationships as terrestrial taxa and this may explain why epiphytic species outnumber terrestrial species by 2:1 (Zettler et al., 2003). This mutualistic relationship has been recognized as unilateral, in favor of the plant (Rasmussen and Rasmussen,

2009). Like the pollinator partnership, the orchids’ dependency on mycorrhizae might also contribute to their rarity since any decline in the population of an obligate partner would pose a threat to the orchids themselves (Swarts and Dixon 2009).

Economic importance

Despite the precariousness of wild orchid populations, some species and artificial hybrids are increasingly popular, mainly as flowering potted plants but also as cut flowers.

In the US orchids now constitute the largest contributor to flowering potted plant sales, with over 30 % of the total retail sales (Fig 1.1). Furthermore, orchid sales in dollar amounts continue to increase – the only potted flowering plant to do so.

“Other” Figure 1.1 Contribution of 1 Roses, etc. (26%) different floricultural crops to (14%) Bulbs (8%) wholesale flowering potted Orchids plant sales in the US in 2011. Poinsettias (30%) Data from USDA (2012). (22%) 1Roses, Easter Lilies, Chrysanthemums, Azaleas, African Violets

Our Research Model: Phalaenopsis amabilis

The genus Phalaenopsis (commonly referred to as the ―moth orchids‖) belongs to the sub family Epidendroideae (Dressler, 1993). It is naturally distributed from northern

India, Tibet and China to Southeast Asian island countries, northern Australia and New

Guinea (Sweet, 1980). Members of the genus are mostly epiphytes on trees (Davis, 1949) while some are lithophytes (Comber, 1972). The majority of Phalaenopsis species share a common floral structure: long inflorescences, star-shaped waxy flowers; and oblong or elliptic leaves at the base (Batchelor, 1982). Among 62 species taxonomically identified

(Christenson, 2001), 3 (Phalenopsis hainanensis, Phal lindenii, Phal michollitzii) are on the IUCN Red list.

The flowers of Phalaenopsis are attractive because of their unique structure Anther cap consisting of three sepals, two petals and one labellum (Fig 1.2). Typically, the Labellum Petal Sepal sepals are large, colored, and all about the same size. They are evenly spread, and thus form an attractive triangular ―frame‖ for the rest of the flower. The two unmodified petals are round and large Arial roots (usually bigger than sepals). These two petals arise on each side of the flower and resemble the wings of a moth (hence the

Figure 1.2 Sketch of Phalaenopsis common name). They fill in the space

15 between the sepals. The labellum or lip is a modified petal that hangs at the base of the flower. The labellum in all orchids is the most complex and variable part of the flower and, indeed, the part that attracts pollinators and admirers alike. In Phalaenopsis, the labellum consists of 3 lobes. Two elliptically-shaped side lobes rise at a steep angle from their point of attachment to the petal’s long axis while a third highly variable and distinctively shaped lobe protrudes outward from the point of attachment of the labellum to the other two petals. In addition the labellum has a ―callus‖ – a raised ―landing platform‖ for pollinators which is precisely positioned below the tip of the gynostemium

(a column comprising stamen filaments and style). This arrangement virtually guarantees that pollinators will encounter the adhesive anther cap and the two pollinia (sacks of pollen) to which it is attached at the tip of the gynostemium. The pollinator will thus leave the flower with the anther cap and pollinia firmly attached (Batchelor, 1982).

Phalaenopsis is a monopodial orchid which means the plant produces or reproduces from a single apical growing point (Batchelor, 1982). The leaves of

Phalaenopsis are fleshy and closely clustered when they emerge from the growing point.

They are elliptical and range in length between 20 and 30 cm long. Phalaenopsis commonly has only a couple of leaves with the older (lower) leaves being replaced by new leaves every few years (Batchelor, 1982). The aerial roots of Phalaenopsis also initiate from the growing point and ramble out of the pot seeking open air (Batchelor,

1982).

Phalaenopsis flowers range from pink, purple, white, brown, yellow or red and sometime possess unique patterns such as stripes, dots, or gradients. The color of the labellum is typically different from the rest of the flower and the color of individual lobes

16 can also vary. Some Phalaenopsis have mottled leaves while some display solid, dark green leaves. The color of the roots is often whitish while the growing tips are green or brown (Batchelor, 1982).

Phalaenopsis amabilis is distributed in the Philippines, Borneo, Indonesia, Papua

New Guinea and Australia (Queensland). It was first discovered in 1750 by Georg

Eberhard Rumphius and named Angreacum album majus (Rumphius, 1750; Hagsater and

Dumont, 1996). It was then renamed by Linnaeus as Epidendrum amabile since it was thought to belong to Epidendrum and the word ―amabile‖ means lovely (Christenson,

2001). In 1825 Dr. Karl Ludwig Blume defined a new genus named Phalaenopsis within the subfamily Epidendroideae (Christenson, 2001).

Phalaenopsis amabilis (L.) Blume is epiphytic and typically grows on tree trunks, branches and rocks in lowland tropical rain forest. This species is not evaluated on the

IUCN Red list but one of the sub species rosenstromii is evaluated as endangered on the

CITES (Convention on International Trade in Endangered Species) Appendix II

(Hagsater and Dumont, 1996).

Phalaenopsis amabilis is a major genetic component of many of the Phalaenopsis hybrids since all the large, round, white Phalaenopsis hybrids are originated from

Phalaenopsis amabilis (Moses, 1981). The flower of Phalaenopsis amabilis is entirely white, except for the three-lobed labellum which is yellow.

Phalaenopsis amabilis is pollinated naturally by large carpenter bees (Hagsater and Dumont, 1996). It is also readily selfed by hand pollination which is one of the main reasons that I chose Phalaenopsis amabilis as my model plant. This property, coupled

17 with plentiful commercial supplies guarantees the availability of plant material for our research.

Despite its prevalence, Phalaenopsis amabilis is closely related taxonomically and in seed storage behavior to other orchids, many of which are threatened (Swamy,

1949). Thus, results with Phalaenopsis amabilis may provide a useful reference for related species. To underscore this point, Phalaenopsis was also the first orchid genus to be propagated in vitro (Arditti and Ernst, 1993). The methods devised for propagation of

Phalaenopsis have since been applied with few modifications to other epiphytic taxa, and fueled a revolution in both orchid commercialization and conservation. I hope that my work with Phalaenopsis amabalis will yield similar insights for all orchid taxa.

Structure and Development of the Angiosperm Seed

Seeds are the propagation units of the Spermatophyta (plants that produce seeds) which store the genetic material within the enclosed embryo and guarantee the extending of the plant’s next generation. The focus of my work is the development of orchid seeds, which differ morphologically and anatomically from crop and most other orthodox seeds.

These ―rudimentary‖ seeds form through a much abbreviated course of development in comparison with more commonly studied seeds. In this section the morphophysiological events that typify angiosperm seed development and as they are completed during orchid seed development are discussed.

The seed (Fig. 1.3) develops from a single ovule located in the ovary of the angiosperm carpel (Fig 1.4). In addition to the ovary, the carpel possesses a stigma and a style. The stigma is an extension of the ovary wall where the pollen grain will attach. It is connected to the ovary by the style - a tubular process down which the pollen tube must grow to reach the ovule and embryo sac. An ovule

(Fig 1.4) consists of the nucellus, one or two

Figure 1.3 A typical angiosperm seed (Dicot) integuments and the funiculus. The nucellus is the inner part of the ovule in which the embryo sac develops. The integuments are the outer cell layers covering the nucellus. The funiculus is the stalk that connects the ovule to the ovary. The micropyle is the opening between the integuments where the pollen

19 tube enters. The chalaza is located opposite from the micropyle where the integuments and nucellus join.

Pollen grain Stigma st Style

Pollen tube

Nucellus Embryo sac Chalaza

Ovule Inner integument Ovary Outer integument Funiculus Micropyle

Figure 1.4 Structure of a typical angiosperm carpel which consists of stigma, style and ovary.

Each structure in a typical mature angiosperm seed (Fig 1.3) is a development of part of the ovule. The seed (Fig 1.3) consists of (1) the embryo which is an immature sporophyte that develops after the egg is fertilized by one of the two sperm nuclei; (2) the perisperm, a development of the nucellus and; (3) the testa, a protective structure surrounding the seed, that develops from one or both of the integuments surrounding the ovule. In addition, an endosperm, which develops from the fertilization of the polar nuclei with the second sperm nucleus, may be present. This tissue supplies nutrients to the embryo as it develops within the ovule. In some species, this endosperm remains in the mature seed where it continues to supply nutrients to the embryo when it germinates

(Bewley and Black, 1994). In other species, it is partially or wholly absorbed by the cotyledons of the embryo. The one or two cotyledon(s) is/are attached just above the hypocotyl of the embryonic axis (consisting of the epicotyl, hypocotyl and radicle). The number of cotyledons is used to categorize plants, with the embryo of monocots having one and the embryo of eudicots having two.

The mature ovary of orchids forms a capsule containing numerous seeds most of which contain a globular-shaped embryo and lack defined endosperm (Yam et al., 2002).

The seed of Phalaenopsis amabilis, like all orchids, consists only of a tiny, rudimentary

(globular stage) 120-celled embryo enclosed in a papery thin, translucent seed coat (Fig

1.5). Orchid seeds are ―dust‖ seeds - reliant on wind for dispersal, but - lacking any fruit- derived structures to aid in this dispersal - the seed is extremely light and small (0.18-3.8 mm in length) (Yoder et al., 2010).

Seed coat

0.5mm

Embryo proper

Chalazal end

Figure 1.5 Seed structure of Phalaenopsis amabilis (130 DAP) under white light (left) and dark field (right)

Fertilization

Angiosperm seed development Chalazal end starts when a mature female gametophyte is fertilized. The common Antipodal cells ―Polygonum-type‖ female (―mega-‖) Central cell gametophyte (also commonly called the Polar nuclei (will ―embryo sac‖) (Fig 1.6) develops within fuse before or after fertilization) the ovule after one or more of the four Synergid cells haploid megaspores undergoes mitosis. Egg cell It is a 7-celled, 7 or 8-nucleate organism.

It includes 3 haploid antipodal cells at Micropylar end Figure 1.6 The mega-gametophyte the chalazal end of the ovule, a central cell containing either 2 haploid polar nuclei or 1 diploid central cell nucleus (that results from the fusion of the 2 polar nuclei), and 3 micropylar cells – 2 synergid cells and the one egg cell (Higashiyama et al., 2001). While this seven- or eight-celled megagametophyte is the final form in some species (Huang and Russell, 1992), the antipodals degenerate in

Arabodopsis and proliferate in Zea mays prior to fertilization (Drews et al., 1998), illustrating a wide variation in development. Antipodal cells are believed to play a part as transfer cells based on the fact that the cell wall that is adjacent to the nucellus is papillate.

However, this hypothesis has yet to be experimentally confirmed (Murgia et al., 1993).

Once the pollen grain attaches to the stigma the pollen tube starts to germinate through the style (Fig 1.4). As the pollen tube approaches the micropyle of an ovule, the synergid cells send out an attractive signal which guides the pollen tube to the egg

(Higashiyama et al., 2001). The details of pollen tube guidance and interaction with the synergid cells are fascinating and complex (Beale and Johnson, 2013) and beyond the scope of this review. It is sufficient here to state that the pollen tube ruptures at the synergid cell membrane to release the sperm cells into one of the synergid cells, thus triggering cell death in the penetrated synergid cell. The two sperm cells migrate through the degenerating synergid cytoplasm – one to the plasma membrane of the egg and the other to the plasma membrane of the central cell. Fusion of the plasma membranes and then of the sperm nuclei with the egg nucleus and the central cell nucleus forms the diploid zygote and triploid primary endosperm nucleus respectively (Yadegari and Drews,

2004). The zygote divides mitotically and develops into the young sporophyte while mitosis of the primary endosperm nucleus gives rise to the endosperm during seed development (Bewley and Black, 1994).

In most orchid species studied the megagametophyte develops only after pollination (Cocucci and Jensen, 1969). The time required for this development results in an unusually long interval between pollination and fertilization. For example, fertilization of Paphiopedilum delenatii occurs 60 days after pollination (Lee et al., 2006). In addition, the fertilization process itself is relatively prolonged, but other than this time requirement, fertilization appears to be similar to the process that occurs in other angiosperms (Cocucci and Jensen, 1969, Ye et al., 2002). Following fertilization, seed development is initiated. It includes 3 stages: histodifferentiation, reserve deposition and maturation drying (Bewley and Black, 1994).

Histodifferentiation

The earliest stage (histodifferentiation) can be further sub-divided into 3 phases: postfertilization, globular-heart transition, and organ expansion (Goldberg et al., 1994).

During postfertilization (which is universal in flowering plants), the zygote retains the polarity inherent in the egg, which possesses a large, vacuolated basal end and a more cytoplasm-rich apical end. This polarity is retained in the early embryo when it divides asymmetrically to form a smaller cytoplasmic terminal cell and a larger more vacuolated basal cell. The terminal cell proliferates to an 8-cell embryo proper which will become most of the embryo, while the basal cell is more vacuolated and divides to become a stalk-like multinucleate suspensor with the top cell called the hypophysis (Goldberg et al.,

1994). The hypophysis is the only derivative of the basal cell which persists – it becomes the root cap and quiescent center of the root apical meristem when the embryo germinates.

During the second phase (the globular-heart transition), embryonic organs and tissue types differentiate. The spherical 8-cell embryo proper first divides into a 16-cell stage.

At this stage, the protoderm (the progenitor of the epidermis) becomes apparent. As mitosis of the embryo proper continues, cell differentiation proceeds to produce an inner layer of procambium and a ground meristem layer between it and the protoderm. These 3 basic tissue layers form a radial ―globular‖ embryo. In a dicot embryo, two protuberances form on opposite sides of the spherical globular embryo, thus establishing bilateral symmetry. These protuberances will form the cotyledons and this stage is commonly called the heart embryo. Meanwhile, the elongation of the hypocotyl and the hypophysis

(which becomes part of the root meristem) at the top of the suspensor is triggered (Dolan et al., 1993). In the final ―organ expansion phase‖, the further development and division

24 of each cell layer defines the shapes of embryonic cotyledon(s), epicotyl (the apical region distal to the point of attachment of the cotyledon(s) which will become the shoot), radical (the basal, distal region which will grow into the root), and hypocotyl (the area between the radicle and the point of attachment of the cotyledons). This phase of histodifferentiation actually overlaps with reserve deposition.

In monocots (such as orchids) histodifferentiation resembles that of dicots, but the structures are formed differently and have different names. The embryo proper in the globular stage is similar to dicots while the suspensor is less differentiated (Bewley and

Black, 1994). In dicots, differentiation of cells into progenitors of the root and shoot meristem can be detected during the globular stage - in monocots, it is the single cotyledon (called the scutellum) that develops first. The radicle and plumule (shoot) then develop as a protuberance off the side of the scutellum. In addition, specialized tissues - the coleorhiza and the coleoptile - develop to cover the root and shoot, respectively, to aid in emergence during germination. The coleorhiza eventually degenerates and the coleoptile develops into the first leaf (McMahon, 2008). Last, the meristems of each organ (scutellum, plumule (epicotyl+coleoptile), and radicle) are formed at both chalazal end and micropylar end of the embryo and the embryo should have reached full size at this stage.

In orchids, while the terminal cell divides into the embryo proper, the basal cell continues dividing periclinally to form 8 filamentous suspensor cells. Four of them elongate toward the micropylar end while the remaining four elongate toward the chalazal end (Lee et al., 2008). The suspensor cells eventually get through the micropyle opening and into the lumen between inner and outer integument (Lee et al., 2008). As

25 with other flowering plants, the suspensor collapses during development (Lee et al.,

2008). The embryo proper reaches the globular stage rapidly with smaller cells toward the chalazal end and larger cells toward the micropylar end (Lee et al., 2008). Eventually the protoderm layer can be defined within the globular embryo proper (Lee et al., 2008) but the embryos undergo no further differentiation to form defined organs (scutellum, radicle or epicotyl) or a defined tissue pattern (procambium, ground meristem) as do other flowering plants. They remain in the globular stage through maturity (Lee et al.,

2008). Thus their development appears to be arrested, at least anatomically, before the completion of the histodifferentiation phase as we know it in agricultural species.

Reserve deposition

After embryogenesis, the cotyledons begin to synthesize and store large amount of macromolecules such as starch, protein, and lipids which provide a nutrient reserve for germination. Starch is stored in amyloplasts in the cells of cotyledons (dicots) and at least initially, in the cells of the endosperm in monocots (the endosperm is dead at maturity). After starch synthesis ceases, carbohydrates such as hemicelluloses may start to accumulate in cell walls (Bewley and Black, 1994). Lipids accumulate in lipid bodies within the cells of the embryo (as opposed to the endosperm), especially in oil-rich embryos such as sunflower, peanut, and soybean, etc (Bewley and Black, 1994). Storage protein synthesis is precisely coordinated in seed development, with mRNA for storage proteins reaching a peak when maximum protein is deposited (Bewley and Black, 1994).

Reserve proteins are stored in protein bodies in the cells of cotyledons and persistent endosperms of dicots or monocots.

In orchids, neither the endosperm nor an embryo storage organ (scutellum) develops. Starch, accumulates transiently at 120 DAP in Phalaenopsis amabilis within the embryo proper but is absent by 150 DAP (Lee et al., 2008). Between 120 and 150

DAP, as the starch content declines, lipids and protein accumulate and these are the major reserves when the seed finally matures (Lee et al., 2008). These reserves are inadequate to support germination. Hence, in nature, the tiny embryo is very dependent on finding an external source of carbon (through a fungal symbiont) (Yoder et al., 2010). In culture, these embryos must be grown on a medium containing reduced carbon and vitamins as well as minerals (Knudson, 1922).

Maturation Drying

Following reserve deposition, the embryo and endosperm of most species start to lose water. While water declines during reserve accumulation due to the increasing quantity of dry matter, actual water loss per seed begins during maturation drying as the vascular connection through the funiculus to the mother plant is severed. Water content at the end of maturation drying in orthodox seeds reaches as low as 0.04-0.06 g g-1 dry weight. Along with the water loss, morphological changes such as size and weight reduction and color changes can normally be observed. As well as these observable changes, the concentration of the plant hormone, Abscisic Acid (ABA), decreases following the cessation of storage protein synthesis. Maturation drying appears to act as a switch to suppress the expression of development-related genes while activating germination-related genes (Bewley and Black, 1994).

Orthodox seeds must be ―pre-adapted‖ for water loss as they enter the maturation drying phase. They do not react to drought in the same way as vegetative tissue

(essentially, desiccation ―avoidance‖ by adaptations to reduce moisture loss). It is thought that ABA, which peaks during the later stages of reserve accumulation (Guttierez et al., 2007) is important in this ―pre-adaptation‖. As water loss first initiates, and then progresses, the seed responds with a progression of distinct mechanisms targeted to allow it to survive the many distinct stresses imposed by the changing water content. These stresses cause lethal damage to recalcitrant seeds and this is believed to be due to the deficiency or absence of some or all of these protective processes in these types of seeds

(Berjak and Pammenter, 2002). These processes have been suggested to include: changes in intracellular physical characteristics (including reduction in vacuolation (Farrant et al.,

1992); formation of intracellular ―glasses‖ (Koster and Leopold 1988) and; restructuring of the nuclear DNA (Berjak and Pammenter, 2002)); de-differentiation (Vertucci and

Farrant, 1995), reducing metabolism to prevent accumulation of free radicals (Berjak and

Pammenter, 2002; Finch-Savage et al., 1994); engagement of antioxidant systems

(Leprince et al., 1993); deployment and preservation of effective repair mechanisms for use during rehydration (Veltin and Oliver, 2001) and; accumulation of molecules (eg. proteins, sugars) that serve a protective role, such as by contributing to the glassy state, or interacting directly with macromolecules or structures to prevent damage (e.g. denaturation of proteins and coalescence of membranes) (Berjak and Pammenter, 2002).

My work concentrates on the accumulation of the Late Embryogenesis Abundant

(LEA) proteins which are suggested to stabilize subcellular structures under desiccation

(Dure, 1993b). Their genes are switched on as water content starts to decline and embryonic ABA level increases during the late stages of reserve deposition and early stages of maturation drying (Farrant et al., 1996). At least some LEA proteins may act

28 together with the high levels of soluble sugars that also accumulate during the late stage of orthodox seed development. Two hypotheses (not mutually exclusive) could explain the protective roles played by sugars and LEA proteins during desiccation and rehydration: water replacement and glassy state formation (reviewed in Berjak and

Pammenter, 2002). The ―water replacement hypothesis‖ (Clegg, 1986; Crowe et al., 1992) suggests a replacement of water in the hydration shells of critically important macromolecules, during desiccation and/or rehydration. In the glassy state, sugars along with LEA proteins could form a highly viscous, metastable ―glass‖ when they become super-saturated as water content declines during maturation drying (Koster and Leopold

1988, Leopold et al., 1994). The ensuing reduced diffusion may halt intracellular metabolic activity and eliminate the possibility of damaging interactions that result from diffusion (Koster and Leopold 1988).

In orchids, the maturation drying stage hasn’t been well studied yet but the seeds from Phalaenopsis are desiccation tolerant when mature and undergo water loss

(Schwallier et al., 2011). Furthermore, accumulation of two LEA protein transcripts has been documented to occur concomitant with the acquisition of desiccation tolerance

(Godfrey et al., in preparation). So far, the evidence suggests that orchid seeds undergo maturation drying to become desiccation tolerant by mechanisms similar to those reported for other orthodox seeds.

Seed Storage Banks

The idea of storing seeds originated at the dawn of plant domestication. The storage period and conditions vary depending on both the objectives and species concerned (Hong and Ellis, 1996). For the purposes of farming or plant breeding, short-

(from one growing season to the next) or medium- (a few years) term storage is adequate.

However, for maintenance of the breadth of species & genetic diversity long-term seed storage is required. Most seeds naturally lend themselves to long-term storage because they easily dry to 3%-7% water (fresh weight basis) a water content that allows them to be easily stored at -20° or lower without viability loss (FAO/IPGRI, 1994). The ability to tolerate such extreme desiccation is a common adaptive mechanism for organisms or tissues (such as temperate zone seeds (Roberts 1972, Bewley and Black 1994); pollen

(Hoekstra 1986); spores of Bryophyta (Sussman and Halvorson 1966), Pteridophyta

(Keilin 1959) and lichens (Bewley 1972), etc.) that routinely experience periods of abiotic stress since desiccation permits metabolic activity to be suspended. However, seeds differ in their ability to tolerate such desiccation.

On the basis of their response to storage at different water contents and temperatures, seeds have been divided into three categories: orthodox, recalcitrant, and intermediate. Most plant species produce ―orthodox‖ (or ―poikilohydric‖) seeds. By definition these seeds can remain viable through long periods when stored in the cold in the desiccated state. Their longevity increases as seed moisture content and temperature decrease (Roberts 1973). On the other hand, some seeds do not tolerate desiccation or cold and these are termed ―recalcitrant‖ (or ―homeohydric‖). Recalcitrant seeds differ in their ability to tolerate desiccation. Most more-or-less retain viability with decreasing

30 moisture content but hit a ―critical moisture content‖ below which viability decreases drastically (Hong and Ellis, 1996). In addition, a new category of seed storage behavior

(―intermediate‖) has emerged. ―Intermediate‖ seeds are cold- and desiccation- tolerant

(thus are both poikilothermic and poikilohydric), but appear to lose viability rapidly under storage conditions (Ellis et al., 1990, Volk et al., 2006). In the case of intermediate seeds, the geographical distribution of the species correlates with seed longevity. Seeds of species originating from hot, dry areas live longer than those from cooler, humid areas

(Commander et al., 2009).

Of the 82 orchid species listed in the seed database (Royal Botanic Gardens Kew,

2013) whose storage behavior has been studied, 31 are defined as orthodox, 6 as

―probably orthodox‖, 15 as ―likely orthodox‖ , 4 as intermediate, 17 as ―likely intermediate‖ and 9 (including Phalaenopsis amabilis) uncertain. Work on

Phalaenopsis amabalis hybrids from our lab revealed that seeds become tolerant to desiccation to 5-10% moisture (FW basis) between 150 and 165 DAP and dehisce at 180

DAP (Schwallier et al., 2011).

The seeds from cultivated orchid plants are generally collected from non-dehisced fruit at approximately 2/3rds of the way from pollination to maturity (1/2 of the time from fertilization to maturity) to facilitate the in vitro culture which requires sterile seeds.

This strategy is termed the ―green pod culture process‖ (American Orchid Society) and, even if the mature seeds are orthodox, it is quite likely that the immature seeds are not.

In fact in work from our lab, the seeds of the hybrids of Phalaenopsis amabalis were not desiccation tolerant at the time recommended for green pod culture (Schwallier et al.,

2011). This is to be expected since, as mentioned in previous sections, orthodox seeds

31 acquire the ability to tolerate desiccation and cold only during the maturation drying stage and evidence suggests this only occurs in Phalaenopsis amabilis between 150 and

165 DAP (Schwallier et al, 2011). In order to successfully store premature orchid seeds that have been harvested during ―green pod‖ period, proper post-harvest treatment is required since immature orthodox seeds may still show damage upon cold or desiccation

(Hong and Ellis, 1996). One strategy to induce desiccation tolerance that has been applied successfully to other immature orthodox seeds like soybean (Blackman et al.,

1992) is a slow-drying treatment. Our preliminary work shows immature seeds of

Phalaenopsis amabilis become desiccation tolerant as the water content drops during slow-drying treatment. Further, it is worth asking if the same chemical change(s) are responsible for this adaption as are responsible for desiccation tolerance in other seeds.

Late Embryogenesis Abundant proteins

Plants must respond to the many biotic and abiotic stresses they encounter in their environment with mechanisms that eliminate damage and allow survival (Bartels and

Sunkar, 2005). The abiotic stresses of cold, freezing, salinity and drought all cause damage through decreasing cellular water activity. These stresses induce the accumulation of a group of highly hydrophilic proteins called Late Embryogenesis

Abundant (LEA) proteins (Battaglia et al., 2008). The name derives from the fact that the first LEA protein transcripts to be discovered were found to accumulate in cotton

(Gossypium hirsutum) cotyledons during the late embryogenesis stage (Galau et al.,

1986). At that time, their function was not known. Since then, LEA proteins have been shown to accumulate in response to any of the abiotic stresses that cause cellular water deficit as well as water loss that occurs naturally during growth and development (for example, seed development) (Vicient et al., 2000; Sheoran et al., 2006). Members of this broad group are ubiquitous in plants from algae and mosses (Saavedra et al., 2006), through gymnosperm and angiosperm species. Other than in seeds and pollen which are desiccation tolerant organs, LEA proteins are also induced in vegetative tissues such as roots and shoots under environmentally-induced water deficit (reviewed in Battaglia et al., 2008). More recently, LEA-like proteins have also been shown to accumulate in certain invertebrates and some bacteria in response to water loss (Hand et al., 2011; Clark et al., 2012).

Based on their possession of distinct sequence motifs, all but group 5 (the hydrophobic ―atypical‖) LEA proteins can be divided into 6 hydrophilic groups that belong to a larger group of proteins called hydrophilins (Battaglia et al., 2008). All of

33 these are characterized by an abundance of charged amino acid residues as well as glycine, alanine, serine, or threonine, and a lack of tryptophans and cysteines (Garay-

Arroyo et al., 2000). Most groups of LEA proteins are predicted to be unstructured in aqueous solutions based on their chemical properties (Dure, 1993a) and the few structural studies that have been done confirm that LEA groups 1, 2, 3 and 4 are over 50% unfolded

(reviewed in Tunnacliffe and Wise, 2007). My work focuses on representatives of LEA groups 2, 3, and 4 and these groups will be discussed in depth below.

LEA group 2

Prevalence

Group2 LEA proteins (also known as ―dehydrins‖) are the most characterized group of LEA proteins. They are also highly hydrophilic, with a high proportion of charged and polar amino acids and absence of Trp and Cys residues (Battaglia et al.,

2008). This group of LEAs was first identified in developing cotton embryos (Battaglia et al., 2008).

Group 2 LEA proteins typically accumulate in seeds during desiccation (Nylander et al., 2001), and in most vegetative tissues in response to abscisic acid (ABA) application, low temperature or water Figure 1.7 Array of the distinctive motifs in deficit. In some species (barley and group2 LEA proteins. (Fig. 3 from Battaglia et al., 2008)

34 some Solanum species), they are expressed constitutively (Rorat et al., 2004).

Accumulation of dehydrins, sometimes along with sugars, was shown to be correlated with desiccation and freezing tolerance in protocorm-like bodies of Dendrobium candidum and protocorms of Spathoglottis plicata (Wang et al., 2002; Bian et al., 2002)

– suggesting these proteins accumulate in some orchids in response to stress.

Sequence

Group2 LEA proteins or dehydrins are distinguished by their conserved Lys-rich

15-residue motif ―EKKGIMDKIKEKLPG‖ (also called the ―K-segment‖ (Close et al.,

1993)) which exists in one to 11 copies per polypeptide (Battaglia et al., 2008). An additional motif present in most group2 LEA proteins is a Y-segment usually found in 1 to 35 tandem copies at the N-terminus (Close, 1996). Many members of this group also have an S-segment which is a tract of Ser residues (Plana et al., 1991). Depending on whether these three motifs are present and in which order, several sub groups of dehydrins can be identified, such as the K sub-group in which only the K-segment is present, and the SK or KS sub-groups in which K-segments follow S-segments or vice versa. In addition there are YSK and YK sub groups (Fig 1.7). Proteins in these subgroups are detected in various organisms within the plant kingdom (Battaglia et al.,

2008).The Y motif is absent in the gymnosperms so far examined and the YK subgroup is likewise thus far absent in the monocots (Supplemental data from Battaglia et al., 2008).

Figure 1.8 Sequence analysis of the dehydrin amino acid sequence of Phalaenopsis aphrodite (UniProt Accession number: AAT08674.1). Sections highlighted in red are S-segments and in green are K-segments.

The dehydrin sequence that I identified using Orchidbase (Fu et al., 2011) from

Phalaenopsis aphrodite (Fig 1.8) consists of one S-segment followed by 2 K-segments

(Battaglia et al., 2008). Thus, this protein belongs to the SK sub group.

Suggested function

Several studies on group2 LEAs confirm that they accumulate during seed desiccation. They also accumulate ubiquitously in vegetative tissue in response to water deficit caused by drought or low temperature (Nylander et al., 2001, Rorat et al., 2004) and they have been shown to accumulate in buds that overwinter at the onset of dormancy in the fall (reviewed by Battaglia et al., 2008). Up regulation of group2 LEA gene expression appears to be mediated by the stress hormone, ABA, which induces many group2 LEA genes (Nylander et al., 2001). However, certain group2 LEAs are also abundant during optimal growth conditions and a few dehydrins show a constitutive expression pattern (Battaglia et al., 2008). Predictably, these members of group2 LEA proteins do not respond to ABA (Battaglia et al., 2008).

Because of their expression pattern, group2 LEAs have been suggested to protect against damage caused by water deficit. Group2 LEA proteins are localized in all cellular compartments (Rorat et al., 2004).One predicted function of group2 LEA proteins is membrane stabilization since the K-segment is predicted to form amphipathic

α-helices, similar in structure to those found in apolipoproteins (Segrest et al., 1992).

This is supported by some in vitro experiments that found that group2 LEA proteins protect the structure of anionic phospholipid vesicles or isolated membranes during freezing (Koag et al 2009; Kosová et al., 2007).

LEA group 3

Prevalence

Group3 LEA proteins are relatively diverse in sequence and widely distributed in the plant kingdom. Related proteins also accumulate in several nonplant organisms in response to dehydration (Battaglia et al., 2008). In plants, group3 LEAs are abundant in mature embryos (Roberts et al., 1993) and localized in the cytoplasm and protein storage vacuoles (Marttila et al., 1996). In Phalaenopsis hybrids, one member of group3 LEA proteins was found to accumulate during the late seed development phase and its accumulation correlated with the desiccation tolerance (Godfrey et al., in preparation).

Sequence

Group 3 LEA proteins are characterized by a repeating 11-mer motif which itself varies among species (Dure 1993b). This group of

LEA proteins is relatively diverse compared to other groups not only because of the variation of the 11-mer motif but also because of the changes in sequence of other motifs. Group3

LEA proteins are further divided into two subgroups according to the 11-mer motif. The subgroup 3A shares a highly conserved 11-mer motif: TAQ [A/S] AK [D/E] KT[S/Q]E (motif

3 and 5 in Fig. 1.9) (Dure 2001; Battaglia et Figure 1.9 Array of the distinctive motifs in group3 LEA proteins. (Fig. 3 from al., 2008).- Other than this conserved motif, in Battaglia et al., 2008)

37 subgroup 3A, motif 4 (SYKAGETKGRKT) is found at the N-terminus while motifs 1 and 2 are found at the C-terminus. In subgroup 3B however, 4 variations of the 11mer motif were found (motifs 1-4) as shown in Fig 1.9. In addition, a conserved motif (5 in

Fig 1.9, ―Group 3B‖) is unique to this subgroup (Battaglia et al., 2008).

In Phalaenopsis, one group3A LEA protein sequence was successfully identified

(Fig 1.10) (Godfrey et al., in preparation). The Phalaenopsis sequence possesses 4 of the

5 motifs characteristic of this group of LEA genes (highlighted in Fig 1.10).

Figure 1.10 The predicted protein product of TG2 aligns with LEA Group 3A motifs and orthologues from Bamboo (Ampelocalamus calcareus; Poales, Poaceae), Wheat (Triticum aestivum; Poales, Poaceae), corn (Zea mays; Poales, Poaceae), Peanut (Arachis hypogaea; Fabales, Fabaceae)), and Arabidopsis thaliana (Brassicales; Brassicaceae) (NCBI GenBank accessions GU433601.1, AB115914.1, DQ244556.1, HM543579.1, and AY093968.1, respectively) . Color-coded shaded blocks represent the areas of conserved or conservative amino acid sequences that correspond to the motifs identified in Battaglia (et al., 2008) as follows: blue, motif 4; orange: motif 3; purple, motif 5; red, motif 1; green, motif 2. Motif 5 is absent from Phalaenopsis, peanut and Arabidopsis. (from Godfrey et al., in preparation).

Suggested function

A variety of studies suggest potential roles for LEA group 3 proteins. Some in vitro experiments suggest that they interact in a 1:1 molar stoichiometry to prevent the inactivation of enzymes such as Lactate or Malate dehydrogenase (Reyes et al., 2005,

2008; Goyal et al., 2005). Similar tests with higher LEA:enzyme ratios also suggest a role in preventing protein aggregation (Chakrabortee et al., 2007). These proteins may also work together with the soluble sugars that also accumulate during late embryogenesis by forming a tight hydrogen-bonding network and stabilizing the glassy state (Wolkers et al., 2001). Some over-expression and knock-down experiments in different organisms also support a protective role for group3 LEA protein during water deficit (reviewed by Battaglia et al., 2013). Group3 LEA proteins are also proposed to associate with membranes based on models of their potentially amphipathic structure

(Woods et al., 2007). Further, LEA group3 proteins were found to interact with liposomes in vitro so as to protect them when desiccated (Tolleter et al., 2007). Similar to dehydrins, group3 LEA proteins are also up-regulated by ABA during stress or certain development stages (reviewed by Battaglia et al., 2008).

LEA group 4

Prevalence

Group 4 LEA proteins are wide-spread in the plant kingdom.

They were first found in dry Figure 1.11 Array of the distinctive motifs in group4 LEA proteins. (Fig3 from Battaglia et embryos of cotton (Roberts et al., al., 2008)

1993) and later in vegetative tissues exposed to drought (reviewed in Battaglia et al.,

2008). In Phalaenopsis hybrids, one group 4 LEA protein (Fig 1.12) was found to accumulate during the late seed development phase and this accumulation correlated with desiccation tolerance (Godfrey et al., in preparation).

Sequence

Group 4 proteins are conserved in their N-terminus and less conserved in the C- terminal 70 to 80 amino acids (Dure, 1993b) (Fig. 1.11). Motifs 2 and 1 in the N-terminus are characteristic of all group 4 LEA proteins (Battaglia et al., 2008). Four more motifs are also distinguished in some of the group 4 LEA proteins. Sub group 4A consists of motifs 2 and/or 3 flanking motif 1, while the addition of motifs 4 and/or 5 forms the subgroup 4B (Battaglia et al., 2008). Among seeds studied, motif 4 is absent from monocots and gymnosperms (Battaglia et al., 2008).

In Phalaenopsis one group 4 LEA protein sequence was successfully identified

(Fig 1.12). Three of the motifs (1, 2 and 3) characterizing group 4 proteins were identified within the Phalaenopsis sequence, thus suggesting this belongs to the group 4A protein subgroup.

Suggested function

The accumulation of LEA group 4 proteins correlate with water deficit in vegetative tissue (Cohen et al., 1991) and also developing seeds (Ali-Benali et al.,

2005), and, as with other LEA proteins, the genes respond to ABA which presumably up-regulates expression during dehydration in vegetative tissue and the late stage of seed development (Luo et al., 2008). Like the LEA group 3 proteins, proteins from this group prevent the inactivation of Lactate dehyrogenase during water loss in vitro (Reyes

40 et al., 2005). Further, one group 4 LEA protein from soybean was found to interact with sucrose and raffinose and promote the glass transition of the sugar-protein matrix.

Contribution to the formation of glass matrices in mature seeds may be a common role for both group 3 and 4 LEA proteins (Shih et al., 2004).

Figure 1.12 The predicted protein product of TG1 aligns with LEA Group 4 motifs and orthologues from representatives of the Fabales (peanut (Arachis hypogea) and Woolly glycine (Glycine tomentella)); the Brassicales (Rape Seed (Brassica napus) and Arabidopsis thaliana) and; the Poales (corn (Zea mays)) (NCBI GenBank accessions HM543585.1, AY007511.1, AY572958.1 , AY091171.1, NM_001157274.1, respectively). Color-coded shaded blocks represent areas of conserved or conservative amino acid sequences corresponding to motif 1 (blue), motif 2 (yellow), motif 3 (green), motif 4 (purple) (Battaglia et al. , 2008) Motifs 1 through 3 were identified in Phalaenopsis. The one-letter amino acid codes are color-coded to indicate common side- chain chemical properties (from Godfrey et al., in preparation).

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Chapter 2 The Effect of Drying on Desiccation Tolerance and Late

Embryogenesis Abundant Protein Gene Expression in Immature Seeds of

Phalaenopsis amabilis

Huijing Zhu

Abstract

To separate the effects of drying and excision alone on these processes, seeds were subjected to different treatments: a 4-day Slow Drying (SD) treatment where they were placed in atmospheres of progressively lower relative humidity and; a High Relative

Humidity (HRH) treatment where they were placed above water. During SD, seeds maintained their starting moisture content of 70% for 3 days, and then dried to 10-15% moisture on the 4th day whereas during the HRH treatment, moisture content did not decrease. Seeds that had undergone SD became desiccation tolerant but seeds in the

HRH control did not. Transcript level of all three LEA genes we studied (TG1: Group4

LEA; TG2: group3 LEA; Dehydrin: Group2 LEA) increased after excision in both treatments but the transcript level of LEA genes from the SD treatment was higher than in the HRH control group. Our results suggest that immature seeds of Phalaenopsis amabilis can tolerate desiccation by appropriate post-harvest treatments and the accumulation of LEA proteins may contribute to the tolerance of desiccation.

Introduction

The orchid family, with over 25000 estimated species (Cribb et al., 2003), is best known for its various and unique floral patterns which are loved by amateur collectors and scientists. However, many species are threatened to extinction by habitat alteration, and extraction or collection of wild plants for trade (Hagsater and Dumont, 1996).

Habitat alteration (including destruction or modification, and fragmentation) is especially acute in the tropical areas where over half of the orchid species are endemic (Dressier,

1993). Each orchid species often requires a specific pollinator (usually an insect or a bird). In addition, seed germination of most species depends on specific symbiotic mycorrhizae which are required to transport nutrients from the environment to the seed.

This fascinating lifestyle as well as the commercial popularity of orchids has inspired some research targeted at conservation.

Most species in the genus Phalaenopsis are united by their unique oblong or elliptic leaves at the base, long, graceful inflorescences and unique-shaped flower which consist of three sepals, two petals and one labellum (Batchelor, 1982). Among 62 species taxonomically identified (Christenson, 2001), 3 are on the IUCN Red list: Phalaenopsis hainanensis, P. lindenii,and P. michollitzii (IUCN red list). Phalaenopsis amabilis is not evaluated on the IUCN Red list but one of the sub species (rosenstromii) is classified as endangered by the CITES (Convention on International Trade in Endangered Species)

Appendix II (Hagsater and Dumont, 1996). Phalaenopsis amabilis is a major genetic component of many of the Phalaenopsis hybrids (Moses, 1980) and is closely related taxonomically and in seed storage behavior to other orchids, many of which are threatened (Swamy, 1949). Thus, research with Phalaenopsis amabilis may provide a

58 useful reference for studies on related species. Similarly, Phalaenopsis was the first orchid genus that was propagated in vitro, but now most epiphytic orchids can be propagated in a similar manner (Arditti and Ernst, 2009).

The charisma coupled with vulnerability embodied in Phalaenopsis and other orchids ought to inspire ample research on potential long-term preservation strategies.

However the unique biology of orchid seeds poses special technical challenges to progress in this area. Orchid seeds are tiny (1/2 mm) and light, making them difficult to count and weigh. Further, the capsules wherein the millions of seeds develop are large and sufficiently drain the plant’s resources such that only one or two capsules are recommended to be produced simultaneously per plant. They also mature very slowly (in the case of Phalaenopsis amabilis, seed maturation requires 6 months from pollination).

This makes it difficult to generate the numbers of biological replicates necessary for sound conclusions. The germination of the seeds takes several weeks which is longer than common model plants like soybeans or Arabidopsis and the seeds have to be cultured aseptically on nutrient media (Knudson et al., 1922) since the fungal symbiont is often difficult to identify and culture. For the sake of sterility capsules are usually harvested prior to dehiscence (Sauleda, 1976).

One promising and effective strategy for species conservation is long-term seed banking. Common seed-banking methodology dries seeds to 3%-7% water content (fresh weight basis) which allows orthodox seeds (those that can survive desiccation and freezing) to be easily stored at -20°or lower (FAO/IPGRI, 1994). However, some seeds

(termed ―recalcitrant‖) fail to tolerate desiccation or cold and require special preparation and conditions for storage. There is also an ―intermediate‖ category of seeds that tolerate

59 cold and desiccation, but appear to lose viability rapidly under storage conditions (Ellis et al., 1990, Volk et al., 2006). Despite decades of reports, the storage behavior of most orchid seeds is uncertain. Only 82 species have been studied and of these, 31 are defined as orthodox, 6 as probably orthodox, 15 as likely orthodox, 4 as intermediate, 17 as likely intermediate and 9 (including Phalaenopsis amabilis) as uncertain (Royal Botanic

Gardens Kew, 2013).

Seeds acquire the ability to tolerate desiccation and cold during the last of the three stages of seed development (histodifferentiation, reserve accumulation and maturation drying) (Bewley and Black, 1994). Recalcitrant seeds’ failure to tolerate desiccation or cold is believed to be due, in part, to failure to complete this stage

(Pammenter and Berjak, 1999). Developing orchid seeds have much abbreviated histodifferentiation and reserve deposition stages (Lee et al., 2008) and this leads to the question of whether maturation drying is similarly abbreviated. The effect of harvest time on longevity during storage is particularly difficult to study in orchid seeds since they are enclosed within dehiscent fruits that display little outward clue as to the maturity level.

Furthermore, as mentioned previously, even if the developmental history is tracked, the seeds from cultivated orchid plants are generally collected from non-dehisced fruit to facilitate the in vitro culture which requires sterile seeds. This strategy (termed ―green pod culture‖ (American Orchid Society)) yields desiccation intolerant (―recalcitrant‖) seeds even if the mature seeds may be orthodox. Thus, difficulty in categorizing orchid seed storage behavior may arise from variable maturity at seed harvest.

Phalaenopsis seeds are not desiccation tolerant until 160 DAP - far beyond the time (130 DAP) recommended for green pod culture (Schwallier et al., 2011). Other

60 work has shown that immature orthodox seeds can be induced to tolerate desiccation and hence long term storage if they are treated by a ―slow drying‖ protocol (Blackman et al.,

1992). Our preliminary work showed that seeds of a Phalaenopsis hybrid as young as

120 DAP, but not younger, if subjected to slow drying over days, can tolerate water contents as low as those that would kill them if the drying were imposed quickly. This suggests that immature seeds do have the ability to sense imminent desiccation and respond with adaptations that protect them from desiccation-induced damage if given sufficient time. One of the objectives of my work is to confirm and extend this work to determine more closely the parameters under which this occurs.

The knowledge that desiccation tolerance can be induced in immature seeds can only be practically useful when studying seeds gathered from a variety of species with unknown developmental histories if they can be precisely staged at harvest. Therefore, my second objective is to identify biochemical markers for the development of desiccation tolerance which could be useful for precisely identifying an orchid seed’s physiological age and desiccation tolerance.

Substantial changes in enzyme activities and gene expression are commonly detected in plants as they prepare to protect themselves against damage caused by water deficit (Battaglia et al., 2008). In plants, highly hydrophilic Late Embryogenesis

Abundant (LEA) proteins (Dure et al., 1989) are associated with desiccation tolerance

(Cumming, 1999). LEA proteins were first discovered in cotton (Gossypium hirsutum) seeds where they accumulated to high levels during the last stages of seed maturation as the seeds acquired desiccation tolerance. They also accumulate in response to water loss or ABA in vegetative organs (Cohen et al., 1991; Shih et al., 2004; Ali-Benali et al.,

2005). This expression pattern suggests some of these proteins may play a protective role during drought stress (Garay-Arroyo et al., 2000; Hoekstra et al., 2001).

Based on sequence similarity LEA proteins can be divided into seven groups

(Battaglia et al., 2008). Six of these groups (the ―typical‖ LEA proteins) share the property of being highly hydrophilic. Most of the hydrophilic LEA proteins adopt a flexible random coil structure in dilute solution, but many of these intrinsically unstructured proteins (hydrophilic LEA proteins from groups 2, 3, and 4) tend to form ordered conformations under water-deficit conditions in vitro (Goyal et al., 2003;

Tolleter et al., 2007). In this work the expression of 2 previously isolated and sequenced

Phalaenopsis LEA genes (―TG1‖ and ―TG2‖) and one new LEA gene (Dehydrin) are quantified by quantitative PCR (qPCR). These three genes represent three of the most well-characterized groups of LEA genes – groups 2 (dehydrin), 3 (TG2) and 4 (TG1)

(Battaglia et al., 2008). All of these groups of LEA genes are proposed to be involved in plants’ response to dehydration (Battaglia et al., 2008). To fulfill my second objective

(to develop biochemical markers of desiccation tolerance), my hypothesis is that the expression level of these LEA genes will be correlated with desiccation tolerance in orchid seeds during development. In addition, I will quantify the protein level of one of these genes (dehydrin) by Western blotting using a commercially available antibody to the well-conserved but unique lysine-rich block (KIKEKLPG) motif of dehydrin (which the orchid protein possesses)(Orchidbase (http://lab.fhes.tn.edu.tw/est)).

Materials and Methods

Growth conditions and seed harvesting

Phalaenopsis amabilis was grown and the flowers were pollinated as described previously (Schwallier et al., 2011). Two separate experiments were performed using capsules harvested at 125 and 130 DAP and sterilized as previously described (Schwallier et al., 2011). Briefly, capsules were rinsed for 30 seconds in 95% ethanol, followed by a

15 minute wash in 50% bleach and three rinses in sterile water. Capsules were split longitudinally with a scalpel and the sterile seeds within scooped out.

Post-harvest treatments

First, I wished to determine whether P. amabilis seeds could be induced to become desiccation tolerant by slowly drying them and if so, what extent and rate of drying were necessary for achieving this effect. For these experiments, capsules at 130

DAP were utilized. The seeds from individual capsules were designated for each of the following days of the slow drying protocol: 0, 1, 2, 3, and 4. The seeds from each capsule, upon completion of their designated treatment were subjected to the five physiological and gene expression tests described below.

Having established that a 4 day treatment was sufficient to induce tolerance and all of the transcripts of the LEA genes of interest, I wished to further investigate whether this response was repeatable with different capsules and thus whether any of the LEA genes could be used as a marker for desiccation tolerance. For these experiments, 4 capsules at 125 DAP were harvested and the enclosed seeds split into three different groups. The first group was split into four sub-groups, each of which was tested directly

63 for: germination, desiccation tolerance, moisture content and transcript abundance (by RT qPCR). The second group was slowly dried for 4 days after which it was split into four sub-groups to test for the same four parameters. The third group was excised but held at high relative humidity for 4 days after which it, too, was split into four subgroups for the same tests. The results of these experiments were tested for statistical significance by paired two-sample t test for means.

Slow-drying treatment

Isolated seeds were dried during the course of 4 days by transferring them daily to successive sealed jars in which atmospheres of progressively lower relative humidity (75,

51, 45, and 32.5%) were maintained over saturated solutions of NaCl, Mg(NO3)2, K2CO3, and MgCl2, respectively (Blackman et al., 1992).

High RH treatment

Seeds were placed in desiccators over water-saturated filter paper for 4 days

(Blackman et al., 1992).

Physiological tests

Germination

Before and after all treatments, four subsamples of 150-500 seeds were placed on

60 mm Petri dishes containing one-third concentration of the vitamins and macro- and micro- nutrients of Murashige and Skoog (1962), 30 g/l sucrose and 8 g/l agar. All media components were purchased from Sigma chem. co., St. Louis, MO. Plates were incubated under the same conditions as plants, and percent germination was scored after 6 weeks.

Germinants were scored as category ―1‖ (with a recognizable shoot and root apex, green color, and smooth epidermis), category ―2‖ (having undergone cell division to form a

64 visible callus not having the characteristics of category 1); category ―3‖ (enlarged to break through the seed coat but no further cell division), category ―4‖ (embryo enlarged by did not rupture the seed coat) and category ―5‖ (no change in embryo) (Fig. 2.1).

Figure 2.1. Morphological evaluation of germinating orchid seeds. Categories 1, 2 and 3 (a, b and c, respectively) had grown sufficiently to rupture the testa within 6 weeks and were thus deemed viable while categories 4 and 5 (d and e, respectively) had not and were deemed non-viable.

Desiccation tolerance

Isolated seeds were allowed to dry in a sterile petri dish under the sterile flow of air from a laminar flow hood for 1 day. Under these conditions, seeds equilibrate within

-1 one day to 5-15 g H2O g FW. After drying, these seeds were tested for germination as described above.

Moisture content

Moisture content in the seed was determined gravimetrically in harvested seed by measuring samples (0.005 to 0.03 g dry weight) before and after drying at 67˚C for 48 hours.

Biochemical tests

Tissue Processing

Upon harvest from the capsule (for freshly harvested seed) or removal from the slow-drying or high relative humidity treatment, a portion of the seeds were frozen in

65 liquid nitrogen and pulverized. If this powdered seed material was not used immediately, it was stored at -80 °C until use.

RNA Extraction and cDNA Synthesis

Total RNA was extracted from powdered seeds from all samples using a Qiagen

RNeasy Plant Mini Kit (Cat. No. 74904) following the manufacturer’s manual with an additional on-column digestion with 80 μl DNase I (BioLabs Inc. Cat. No. M0303S) solution (10 μl stock (consisting of 1500 units in 550 μl RNase-free water) mixed with 70

μl RDD buffer supplied in the Qiagen RNeasy Plant Mini Kit) for 15 min to remove genomic DNA (RNAeasy mini handbook, 2012). The concentration of RNA was determined by Nanodrop 2000 (Nanodrop products, Wilmington, DE). The quality of the total RNA was tested by electrophoresing 1 μg on a 1.5% Agarose (Sigma, Cat No.

A9539) gel with 0.5-10kb RNA ladder (Ambion, Cat No. 15623-200) in 10mM NaPO4,

1mM EDTA (supplemental data, figure 2.11). RNA samples (brought to 10 μl with

RNase free water) or RNA ladder (1.5 μl) were mixed with 5μl Glyoxal Sample Load buffer (Ambion Cat No. 8551) and heated at 50°C for 30min prior to loading according to the manufacture manual (www.lifetechnologies.com/order/catalog/product/AM8551).

The gel was stained for 10 min in 5μg/ml ethidium bromide in 0.5 M ammonium acetate and then destained in 0.5 M ammonium acetate for 10 min then visualized under UV light to confirm that samples are equalized. The template cDNA was then synthesized from

1μg of the extracted RNA via RT-PCR using a Qiagen Omniscript RT kit (Qiagen, Cat.

No. 205111) kit with Oligo dT primers (Ambion) following manufacturer’s guidelines, and stored at -20°C.

Quantitative polymerase chain reaction

Primer design. Two primer sets for previously isolated and sequenced LEA genes (―TG1‖ and ―TG2‖) (Godfrey et al., in preparation), one newly designed primer set for dehydrin, a LEA 2 gene (Battaglia et al., 2008), and Actin (a reference gene) were used for amplification. The specific primers (Table 1) were designed by DNAStar

(www.dnastar.com/) using previously reported sequences (TG1 and TG2) (Godfrey et al., in preparation), a sequence in NCBI (Actin: GenBank accession No. U18102) or the

OrchidBase EST database (Dehydrin) (http://lab.fhes.tn.edu.tw/est) (Fig. 2.2) to amplify a 100-200 bp region - in some cases, including conserved regions of the 5’ and 3’UTR.

Figure 2.2. Nucleotide sequence of dehydrin from Phalaenopsis aphrodite from OrchidBase (Accession No. AAT08674.1). The dehydrin primers are in red font. Phalaenopsis aphrodite is very closely related to P. amabilis.

All primers were from Integrated DNA Technologies (Coralville, IA). With these three genes I have representatives for three of the most well-characterized groups of LEA genes – groups 2 (dehydrin), 3 (TG2) and 4 (TG1) (Battaglia et al., 2008). All of these groups of LEA genes are proposed to be involved in responses to dehydration (Battaglia et al., 2008).

Table 1. Sequences of primers used in this work. Primer Sequence Expected Amplicon size (bp) G1F 5’-ACCGGAGTCGGCCACGAAACT-3’ 136 TG1R 5’-CCCCACCAGTAGTGTGCCCAGT-3’ TG2F 5’-GCCGCAGAAGCCAAGGACAA-3’ 136 TG2R 5’-CTGTAGTGCCCTGAGCAGCACT-3’ ActinF 5’-TGACCAGGAGCTCGAGACAGC-3’ 122 ActinR 5’-GCTGGAAAAGCACCTCCGCAC-3’ DhF 5’- AGAAAGCGGCGGAGGAGAGC-3’ 190 DhR 5’- TCAGATCAATGGCTGCCAGGAC-3’

Use of all primer sets in PCR yielded only one amplicon when the amplification reaction was run on a 1.5% agarose gel in TAE buffer (Tris 40 mM; Acetate20 mM;

EDTA 1 mM) (Godfrey et al., unpublished and Supplemental data, Fig. 2.12). To verify that our dehydrin primers were, indeed, amplifying a dehydrin transcript, I sequenced the amplicon as follows. Traditional PCR was carried out using 1μl of synthesized cDNA as template in a 25μL reaction using TaKaRa Ex Taq® Hot Start supplied with dNTPs and

PCR buffer (Clontech, Mountainview, CA, Cat No. RR006A) following manufacturer’s suggested conditions with an annealing temperature of 60°C, cycled 25 times. After reaction the entire sample was electrophoresed with 6X EZ-Vision One loading dye

(Amresco®, Sydney, Australia, Cat No. N472) through 1.5% (w/v) low melting temperature agarose (Sigma, Cat No. A9414) in TAE buffer (supplemental data, Fig.

2.12). The target product was recovered with QIAquick Gel Extraction Kit (Qiagen, Cat

No.28704). The purified product was sequenced at the Grand Valley State University

Annis Water Resources Institute Sequencing facility (www.gvsu.edu/dna/).

Sequences similar to the translation product of the amplified dehydrin sequence were identified in the NCBI non‐ redundant protein database

(blast.ncbi.nlm.nih.gov/Blast.cgi) using BLASTx. This amplified section shared high sequence identity with other dehydrins (Fig. 2.3), showing that our primers indeed amplified a dehydrin transcript.

Figure 2.3. The amplified dehydrin sequence of Phalaenopsis amabilis (“Phalaenopsis”) aligns with other dehydrins from Musa, Oryza sativa, Hyacinthus orientalis, Panax ginseng, Solanum chilense, Solanum tuberosum, Capsicum annuum, Salvia miltiorrhiza (NCBI accessions AEI54683, BAA24978, AAT08674, ABF48477, ADQ73955, AAR25792, AFU61110, AAU29458) and Phalaenopsis aphrodite (―P. aphrodite‖) from Orchidbase (Fu et al., 2011) .

Testing the suitability of Actin as a reference gene. In Phalaenopsis amabilis actin gene expression level did not change significantly over the course of these physiological treatments (Supplemental data, Fig. 2.13).

Quantitation of transcripts. Quantitative PCR was performed on three replicates using 1 μl of the synthesized cDNA solution (equivalent to approximately 1 ng of total

RNA) as template. Actin transcript was amplified in parallel with the target genes for normalization. Transcript sequences were amplified using Agilent’s Brilliant II SYBR®

Green QPCR Master mix (Cat. No. 600828) along with the supplied reference dye according to manufacturer’s instructions except that total reaction volumes were decreased to 12.5 μl. At the end of the PCR run, a melting curve was generated

(supplemental data, Fig. 2.14). PCR cycling conditions were: 10 minutes at 95°C; 40 cycles consisting of 30 sec at 95°C, 1 min at 60 °C, and 1 min at 72°C, followed by a

69 final incubation for 1 min at 95°C. The CT value for each sample was the cycle at which the sample ΔRn crossed a threshold calculated by the instrument software by averaging the ΔRn values of all samples at 1/3rd of their maximum values.

Protein extraction and Western blotting

To correspond with the transcript data, protein level of dehydrin was determined with western blot using commercially available antibodies to dehydrin. Antibodies raised to the highly conserved KIKEKLPG motif of dehydrins (Enzo Life Sciences) that should cross-react with the 21.4 kDa orchid protein (since it also possesses this motif (Fig. 2.4)) were used.

Figure 2.4. Amino acid sequence of dehydrin gene of Phalaenopsis with conserved motif to which antibodies were raised highlighted in purple and the amplified sequence highlighted in yellow (Fu et al., 2011).

Treated, pulverized and frozen seed material was homogenized in extraction buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10 mM DTT, 10% Glycerol) and centrifuged at 12,000xg. Supernatant containing 50 µg of protein (quantified by the Bio-

Rad dye-binding assay according to manufacturer’s instructions) was electrophoresed through 0.75mm 18% polyacrylamide gels with pre-stained molecular weight markers as reference. Proteins were then electro-transferred onto nitrocellulose membranes (Towbin et al., 1979) which were then stained with Ponceau red to ensure protein transfer

(Sambrook and Russell, 2001). Membranes were blocked with blocking buffer (5% gelatin in 20 mM Tris-Cl buffer, pH 7.5) for 30 min. Membranes were blotted with

1:500 dehydrin antibodies (Enzo Life Sciences Cat No. ADI-PLA-100-D) in blotting

70 buffer (1% gelatin in Tris-Cl buffer pH 7.5) for 2 h at room temperature and then washed for 10 min in blotting buffer twice. Membranes were incubated with 1:3000 goat anti- rabbit IgG secondary antibody (Biorad Immun-Star™ Goat Anti- Rabbit (GAR)-AP

Detection Kit Cat.No.170-5011) in blotting buffer for 1hour at room temperature, washed for 10min with 20mM Tris-Cl buffer pH 7.5, and then protein bands were visualized by developing the blot in the dark with peroxidase substrates according to manufacturer’s directions (Biorad Immun-Star™ Goat Anti- Rabbit (GAR)-AP Detection Kit cat.no.170-

5011). The immune blot was photographed with LI-COR Odyssey® Fc image system under 600nm wave length (for visible color bands on blotted membrane or protein gel) for 10min.

Results

Slow drying induced desiccation tolerance.

0.8

content FW

0.6 1

- SD O g O

2 0.4 HRH

Moisture Moisture H g 0.2

0 B 1 2 3 4 100 80

60 SD SD-D 40

Percent Germination 20

0 1 2 3 4 5 100 C 80

60 HRH HRH-D 40 1 2 3 4

Percent Germination 20

DAP 0 10 21 23 34 45 Day after treatment Figure 2.5. The effect of Slow-drying and High-relative humidity treatments on moisture content (A) and germinability before (B & C: closed symbols) and after (B -1 & C; open symbols) rapid (2 d) desiccation to 0.124 g H2O g Fresh weight.

The water content remained high in the seeds (Fig. 2.5A) during the slow drying

-1 process until day 4 when it declined dramatically to 0.156g H2O g FW. This moisture

72 content level is similar to the moisture content of rapidly dried seeds. The Slow Drying treatment did not affect germinability of the seeds (Fig. 2.5B). After removal from the

-1 treatment and rapid desiccation to 0.124 g H2O g FW, only 26.7% of freshly excised seeds could germinate compared to more than 50% of seeds removed from the SD treatment after 3 or 4 days of treatment (Fig. 2.5B). This suggests that seeds of

Phalaenopsis harvested at 130 DAP become desiccation tolerant after the 3rd day of the slow drying treatment. This increase in desiccation tolerance precedes the decline in

-1 -1 water content from approximately 0.60 g H2O g Fresh weight to 0.156g H2O g FW between the 3rd and 4th day of treatment (Fig. 2.5A).

On the other hand, seeds kept at High Relative Humidity (HRH) retain their high moisture content (Fig. 2.5A) and germination rates (over 60%) (Fig. 2.5C, closed circles) until day 4, when the germination rate dropped to 40%. However, the germinability of desiccated HRH treated seeds remained low (under 30%). Since the HRH treatment failed to induce desiccation tolerance in seeds of Phalaenopsis amabilis at 130 DAP, slow-drying (rather than excision alone) was critical for the development of desiccation tolerance in seeds of Phalaenopsis amabilis.

In the fixed-time experiment, the germination rate of freshly harvested seeds, and seeds after four 4 days of HRH or SD treatment did not differ significantly (Fig. 2.6).

However, after desiccation, 97% of freshly harvested seeds were not viable. After 4 days of slow drying the percentage of seeds that were desiccation tolerant (71.99%) was indistinguishable statistically from the percentage of seeds that were germinable

(72.83%). This confirms that seeds of 125 DAP, while not desiccation tolerant when freshly excised, can be induced to become desiccation tolerant by a 4-day SD treatment.

Drying (rather than simple excision) appears to be a critical component of this treatment since a 4-day course of HRH treatment only induces desiccation tolerance to 26.89% – significantly different from the percentage of seeds that are desiccation tolerant after slow drying. In addition, capsules left to mature for an equivalent 4 days on the plant did not develop desiccation tolerance. They had a viability of 70% when freshly excised but 0% when desiccated (data not shown).

100 90 a 80 a a

70 a 60 Not desiccated 50 Desiccated 40

Percent Viable Percent c 30 20 10 b 0 Fresh HRH SD

Figure 2.6. Effect of desiccation on germination of freshly excised (“Fresh”), Slowly dried (“SD”), or High Relative Humidity-treated (“HRH”) seeds of 125 DAP. Values are the means ± SD of the 4 biological replicates. Means with different letters differ significantly when tested by the paired two sample t-test for means (α=0.05).

LEA genes were up-regulated by slow drying and high relative humidity treatment.

Of the three LEA genes tested, the expression of TG2 (LEA group 3) increased the most with SD treatment. Transcript level was approximately 270-fold higher after the day 1 of SD than it was in untreated seed and remained at approximately the same level for the duration of the treatment (Fig. 2.7A). Like TG2, TG1 (LEA group 4) increased dramatically upon SD treatment but only 10-fold. Thereafter, transcript level of TG1

continued to increase to a final value of 38-fold after 4 days of treatment (Fig. 2.7B).

Dehydrin (LEA group 2) also increased after the 1st day of slow-drying treatment by 9.5-

fold, and continued to increase to the end of the second day (18-fold) where it remained

for the duration of the rest of the treatment.

1,000.0 A 100.0

SD 10.0 RER HRH

1.0 B

0 1 2 3 4

10.0 SD RER HRH

1.0 C

0 1 2 3 4

10.0 SD RER HRH

1.0 1,000.0 0 1 1,000.02 3 4 Days after Treatment 100.0 Figure 2.7. Expression of TG2 (LEA100.0 Group 3) (A), TG1 (LEA Group 4) (B) and Dehydrin (LEA Group 2) (C) SDin seeds of 130 DAP as they undergo slow drying ( )SD 10.0 or high relative humidity ( HRH) treatment.10.0 Expression relative to actin is expressed as a HRH ratio (Relative Expression Ratio, RER) of that at Day 0 of treatment (Pfaffl, 2001). 1.0 1.0 0 1 To2 disco3 ver whether4 the onset of the rapid0 rise in1 transcript2 level3 was4 due to

10.0 simple excision of the seeds fromSD the mother plant, I also incubated some seeds at high 10.0 SD HRH HRH 75 1.0 0 1 2 3 4 1.0 0 1 2 3 4

10.0 SD HRH10.0 SD HRH 1.0 0 1 2 3 4 1.0 0 1 2 3 4 relative humidity for an equivalent time. During this treatment, the seeds did not lose water (Fig. 2.5A). This treatment also induced the accumulation of transcripts for all three LEA genes, though not to the same extent as the slow drying protocol. The expression of TG2 increased from day 0 by 80-fold after day 1 and remained between 80 and 100- fold higher than it was on day 0 for the remainder of the treatment. Expression of TG1 and dehydrin increased after the first day of this control treatment (to 4.6-fold and

3- fold higher, respectively, than the level in the freshly harvested seeds). Thereafter,

TG1 transcripts continued to accumulate (to a high of 12.2 fold at day 3) whereas dehydrin transcripts remained at about 3 fold higher than at day 0 for the duration of the treatment. In addition to extracting RNA from fresh, untreated seeds, I also extracted

RNA from fresh seeds that had been rapidly dried. The RER for TG2, TG1, and dehydrin was 1.2, 2.8 and 4.2 respectively (data not shown).

Taken together, our results show all of our LEA transcripts increase dramatically and rapidly after excision, regardless of whether any detectable water loss occurs.

However, the increase is more dramatic during the slow-drying treatment, and occurs prior to a noticeable decline in water content (Fig. 2.5A).

In order to determine whether the increase I saw in my time course experiment was a general response of orchid seeds at this age to these treatments, I replicated this experiment with 4 different capsules of 125 DAP, splitting each capsule and testing the seeds’ responses using a paired sample t-test. These experiments generally corroborated the time-course experiments. TG2 transcript level increased the most after 4 days of slow-drying treatment relative to freshly excised seeds (204-fold), while TG1 increased

55-fold, and dehydrin increased 4.4-fold. As with the time course experiment these genes

76 were also induced during HRH treatment: TG2 reached 82.8-fold increase over day 0,

TG1 reached 32.8-fold, and dehydrin reached 2.4-fold. In all cases, the RER of seeds that were slowly dried for 4 days was significantly different from the RER of seeds that were treated for 4 days at high relative humidity.

1000 P=0.03581 SD

HRH

P=0.04257 100

RER 10 P=0.00077

1 TG2 TG1 Dehydrin

Figure 2.8. The effect of Slow Drying (SD) and High Relative Humidity (HRH) treatment for 4 days on the expression level of TG1, TG2 and Dehydrin in seeds from 4 capsules at 125 DAP expressed relative to their level at 0 days of treatment (freshly excised). Values represent the mean±SD of four replicates. A paired sample t-test was conducted to test for significant differences between the two treatments. P-values are listed above each gene.

To discover whether the expression level of these LEA genes relative to actin could be used as a marker for desiccation tolerance without knowing the expression ratio in freshly excised seeds, I tested whether the Ct value of the target LEA gene relative to

Actin was significantly different in the seeds with different treatments. Slow drying and high relative humidity treatment both significantly (P<0.05) affected the Ct value of the target LEA gene relative to Actin for TG2 (P=7.6*10-5 and 1.8*10-4 for SD and HRH, respectively) and TG1 (P=1.6*10-3 and 3.1*10-4 for SD and HRH, respectively)

77 transcripts when compared with freshly isolated seeds. The Ct value of the target LEA transcript relative to that of actin also significantly differed between the SD and HRH treated seeds (P=0.02 and P=0.03 for TG2 and TG1, respectively). However, neither

HRH treatment nor slow drying significantly affected dehydrin expression relative to actin when compared with freshly excised seeds (P= 0.08 and 0.06 for HRH and SD treatments, respectively), nor was dehydrin expression relative to actin in SD vs. HRH treatment significantly different (P=0.08). Thus, TG2 and TG1, but not dehydrin, have the potential as marker genes to determine the age or desiccation tolerance of

Phalaenopsis amabilis seeds, bearing in mind that all three of these genes are induced to their highest levels before desiccation tolerance is achieved (figs. 2.5 and 2.7).

7.5

6.5 5.5 4.5 3.5

TG2 Ct(Target gene) Ct(Target

2.5 - TG1 1.5 Dhn 0.5

Ct (Actin) Ct -0.5 -1.5 -2.5 Fresh HRH SD Figure 2.9. Difference in cycles required for actin transcript and target gene transcript in 4 capsules at 125 DAP to cross threshold before (“Fresh”) and after seeds underwent Slow Drying (SD) or High Relative Humidity (HRH) treatment for 4 days. Values represent the mean±SD of four replicates. A paired sample t-test was conducted to test for significant differences between the two treatments.

Dehydrin protein level was slightly elevated on day 4 of High Relative Humidity

control treatment

The fact that the presence of high levels of transcripts for the three LEA groups I

tested does not, by itself, confer desiccation tolerance (see, for example Fig. 2.6 - day 1

of the slow drying treatment, when transcript level is high, but desiccation tolerance is

low), prompted me to ask whether some of the LEA gene products might be

accumulating differently than their transcripts.

25kD 20kD

15kD

L SD1 SD2 SD3 SD4 H1 H2 H3 H4 D0

Figure 2.10. The effect of slow drying and high relative humidity treatment on dehydrin protein level as determined by Western blot with anti-dehydrin. The total protein amount in each sample is equalized. SD1 through 4 represent protein samples taken from seeds undergoing slow drying on days 1 through 4; H1 through 4 represent protein samples taken from seeds undergoing the High Relative Humidity treatment on days 1 through 4. D0 represents freshly excised seeds, and L represents the molecular weight ladder.

The commercially available anti-dehydrin antibody detected a protein of

molecular weight 25kD (Fig. 2.10, white arrow), similar to the reported molecular weight

of dehydrin from the closely related species Phalaenopsis aphrodite (21.4kD) (Fu et al.,

2011), but it also detected a lower molecular weight polypeptide of 17 kD (Fig. 2.10,

black arrow). Although the signal from the western blot was extremely weak, it appears

that the both forms of the protein are highest in the freshly harvested seeds and decreased

after excision from the mother plant, whether or not drying occurred. During both SD and

HRH treatment, the 25kD form (after an initial apparent decline from Day 0) remained at a similar level for the duration of the treatment, with the possible exception of the HRH – day 4 seeds, which appeared to have a moderate increase in protein level relative to day

3. The signal from the smaller form of cross-reacting protein (17kD) decreased after the

1st day of both treatments. Whereas the protein level decreased during the HRH treatment, it appeared to increase in level after the 2nd day of SD treatment and then decline again. Finally at the 4th day of the HRH treatment, multiple bands with smaller molecule weights (14kD, 15kD) appeared.

Discussion

The objectives of this work were two-fold: First, I wanted to confirm and extend preliminary work to characterize the requirements for development of desiccation tolerance in immature orchid seeds. Further, I wanted to test the hypothesis that the expression level of LEA genes will be correlated with desiccation tolerance in orchid seeds during the development of this desiccation tolerance. I found that, consistent with previous work, desiccation tolerance was induced in immature orchid seeds if the drying rate was sufficiently slow. Furthermore, while simple excision of the seeds from the mother plant resulted in the development of tolerance to some extent, full tolerance was not achieved unless the seeds were dried.

During slow drying, the water content of immature seeds (125-130 DAP) of P. amabilis declined gradually between day 1 and day 3 and dropped dramatically at the 4th day of slow-drying treatment to levels comparable to those in desiccated seeds. Seeds kept at High Relative Humidity (HRH) retained their high moisture content. The seeds that were slowly dried, but not those treated at HRH, became desiccation tolerant just prior to the dramatic water loss. In orthodox species, water loss to less than 15 % (FW basis) is one of the significant physiological events that occurs during maturation drying

(reviewed in Pammenter and Berjak, 1999). Orchid seeds have a relatively high water content at maturity (approximately 50% FW in Phalaenopsis amabilis hybrids

(Schwallier et al., 2011), and between 42% and 47% FW from several terrestrial and epiphytic North American species (Yoder et al., 2010)). Such a high moisture content at maturity is more typical of recalcitrant seeds whose moisture content is generally between 30% and 80% (FW) (Berjak and Pammenter, 2002). However the seeds from

Phalaenopsis hybrids are not recalcitrant in that they survive desiccation to less than 15%

(FW) when mature (Schwallier et al., 2011). A water content between 55% and 68% FW appears to be critical for induction of full desiccation tolerance in Phalaenopsis

(Schwallier et al., 2011) and other species such as soybean (Blackman et al., 1992) and wheat (Black et al., 1999). My work supports the hypothesis that two critical factors are necessary for the development of desiccation tolerance during maturation drying in orchid seeds: 1) a decline in water content within this critical window (shown by the fact that seeds that do not experience a decline in water content are not desiccation tolerant) and 2) a rate of drying that is sufficiently slow to permit adaptive changes critical for the development of tolerance (shown by the fact that seeds that experience rapid drying are not desiccation tolerant). I propose that this strategy will facilitate the storage of seeds harvested prior to maturity as in the ―green pod‖ harvest practice in orchids where harvest at 110-120 DAP is recommended (Sauleda, 1976). In this case a slow-drying post- harvest treatment should induce the property of desiccation tolerance if the seeds are harvested at 125 DAP. Whether these seeds can be stored in the long term will be a subsequent research question in the laboratory.

My work does not address whether slow drying treatment induces desiccation tolerance in seeds of P. amabilis that are younger than 125 DAP but preliminary work suggests that these seeds do not in hybrids (unpublished). Morphological studies on P. amabilis reveals that at 120 DAP cell division within the embryo ceases and starch grains are abundant around the nucleus (Lee et al., 2008). It is possible that cessation of cell division and accumulation of starch are necessary for the development of desiccation tolerance. Cessation of cell division may be necessary in order for the necessary nuclear

―remodeling‖ that appears to be a prerequisite for desiccation tolerance (Berjak and

Pammenter, 2002) and the abundance of starch may serve as substrate for synthesis of soluble oligosaccharides or galactosyl cyclitols necessary for the development of desiccation tolerance (reviewed in Berjak and Pammenter, 2002). During slow drying ex situ, abundant starch grains have been shown to disappear as sugars accumulate in soybean (Blackman et al., 1992) and, during normal maturation in situ these starch grains have been shown to disappear by 150 DAP in P. amabilis (Lee et al., 2008) suggesting a similar process occurs. Desiccation tolerance of the freshly harvested seeds differed between the time-course experiment and the fixed-time experiment. This difference may be due to differences in maturation rate between individual capsules (for example, due to small differences in ambient atmospheric conditions, nutrient status, or other uncontrolled differences in developmental rate between capsules).

The percent germination of these immature (non-desiccated) seeds was not very high (65%). Both slow drying and high relative humidity treatment slightly increased germination rate (figure 5). Such promotion of germination in excised, immature seeds or embryos when they are held for some time under water availabilities not permissive to germination has been observed in soybeans, Ricinus, and Sitka (reviewed in Blackman et al., 1992).

Next, I addressed the hypothesis of whether LEA gene expression was correlated with the acquisition of desiccation tolerance. Expression of representatives of

LEA groups 3 (TG2) and 4 (TG1) increased dramatically after 1 day of slow drying and, to a slightly lesser extent, during high relative humidity treatment to values comparable to values described for seed developing in situ between 160 and 172 DAP (Godfrey et al., in

83 preparation). The expression of both of these genes remained high and perhaps increased slightly throughout the remainder of both of the experimental regimes. During SD, the increase in gene expression preceded the increase in desiccation tolerance by two days.

In addition, seeds undergoing rapid drying (during the desiccation tolerance test) did not accumulate transcript. In as much as LEA gene expression is correlated with the maturation drying phase of seed development (Blackman et al., 1992), these results support the hypothesis that seeds that have not yet completed maturation drying can be induced to undergo this phase of seed development.

Similarly to transcripts for TG1 and TG2, transcript level of dehydrin of seeds from both SD-treatment and HRH-control accumulated after the first day following excision, but not to the same extent as TG1 and TG2. Like groups 3 and 4 LEA genes, dehydrin expression has been shown to respond to ABA or water stress in other systems

(Nylander et al., 2001) but in my hands, an involvement of dehydrin in desiccation tolerance, is much harder to support for 3 reasons. First, transcript level was also increased to the same level during the rapid drying of the desiccation tolerance test.

Second, the slight decrease in transcript level of actin with both SD and HRH treatment relative to its level at day 0 (Supplemental data, Fig.2.13) may appear spuriously as an increase in dehydrin expression to this level. Third, the protein level of dehydrin appeared to decrease during the period both under SD- treatment and HRH-control although these results are too weak to allow definitive conclusions to be drawn. This decline, if real, may be due to the competition for resources for translation because TG1 and TG2 transcript level increased so much more. Translation of these proteins would consume the majority of resources available. The presence of smaller bands on the

Western blot may also indicate degradation is occurring. It will be interesting to measure transcript accumulation for TG1 and TG2 in future work.

Since LEA gene expression increases prior to detectable water loss, some event other than drying appears to be responsible. The event that is common between seeds at the onset of maturation drying in situ and those I have excised manually is separation of the fruit and seeds from the mother plant. It is possible that some changes occur in the seeds in response to this event that induce the LEA gene expression. One possible candidate that might change during this time is abscisic acid (ABA) a hormone that increases shortly before the onset of maturation drying (King, 1976) and that has been shown to induce the expression of most LEA genes during specific developmental stages or stress conditions (Luo et al., 2008; reviewed by Battaglia et al., 2008). Perhaps the excision triggers an increase in synthesis or concentration of ABA in the seeds which further induces the expression of certain LEA genes. In addition, other changes that might be common to both manual excision and funicular senescence in situ are reduced water and nutrient supply from the mother plant and changes in respiration rate.

For all three LEA genes quantified in this experiment, the expression from the

High Relative Humidity control group increased, consistent with the suggestion that the inducing trigger is excision rather than water loss. Nevertheless, higher expression level is detected among all three genes from the Slow Drying treatment. Although this higher transcript level could reflect a higher rate of synthesis (caused, for example, by a high

ABA concentration or further desiccation), degradation of transcripts also affects their accumulation and a lower level could reflect an increased rate of degradation. Since the metabolism of seeds kept in HRH would remain highly activate the transcripts or

85 translation products might degrade at a faster rate in HRH control seeds than in those seeds that are slowly dried.

A practical outcome of this study is that a high level of LEA transcripts in seeds collected from capsules of unknown age (such as those collected in the field for the purposes of seed collection and storage) could serve as a marker to determine the age of the seeds. In these cases, there would not be a day 0 reference to determine the Relative

Expression Ratio (Pfaffl et al., 2001). I determined (Fig. 2.9) that the change in the difference between the Ct-value of actin and either TG1 or TG2 upon excision is dramatic and highly significant. This suggests that it would be possible to use TG1 or

TG2 as marker genes when actin is used as a reference to determine the suitability of seeds to survive desiccation. This difference is also detected in naturally matured seeds - in our previous work with Phalaenopsis hybrids, the Ct difference between actin and

TG1 and TG2 was 2.8 and 2.6, respectively when seeds are at 172 DAP and desiccation tolerant compared with -2.7 and -4 at 100 DAP and -0.6 and -1.7 at 120 DAP (data not shown). This confirmed that a critical value of the difference of Ct value between marker gene and reference gene could be developed as a marker to determine the age or desiccation tolerance of seeds of Phalaenopsis.

Nonetheless, the correlation coefficients between desiccation and expression level of TG1 and TG2 and desiccation tolerance are around 0.7 in all seeds, too low to support a direct causative relationship between the accumulation of these transcripts and desiccation tolerance. The breakdown in this correlation is intuitively seen in the fact that both gene transcripts accumulate to very high levels in seeds maintained at High Relative

Humidity (which never develop appreciable desiccation tolerance) and 2 days prior to the

86 development of desiccation tolerance in slow-dried seeds. The fact that the transcript level accumulates prior to desiccation tolerance may be due to the time required for translation since water loss may reduce the rate of protein synthesis (Dhindsa and

Cleland, 1975) and protein is the actual molecule that may function against desiccation.

Thus it might be ideal to detect protein level as an indicator for desiccation tolerance and my work suggests that the TG2 gene product would be a good candidate for this marker.

In addition, additional mechanisms have been proposed to contribute to desiccation tolerance in orthodox seeds (Berjak and Pammenter, 2002), including the accumulation of certain sugars (Leopold et al., 1994) and these may be an additional marker to indicate the maturation level or desiccation tolerance of orchid seeds.

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Supplementary Data

Day 0 0 1 2 3 4

Treatment Desiccated Fresh SD HRH SD HRH SD HRH SD HRH Kb Ladder Ladder 10.0 - 6.0 - 4.0 - 3.0 - 2.0 - 1.5 -

1.0 -

0.5 -

Figure 2.11. Gel of extracted RNA from seeds of Phalaenopsis amabilis. To test the quality of RNA samples, 1 μg RNA of each sample and 1.5 μl RNA ladder were mixed with 5μl Glyoxal Sample Load buffer and heated at 50°C for 30min prior to loading. Gel was stained in 5μg/ml ethidium bromide in 0.5M ammonium acetate and destained in 0.5 ammonium acetate for 10 min then visualized under UV light.

Figure 2.12. DNA gel of PCR products with Dehydrin primers. PCR was carried out using 1μl of synthesized cDNA as template in a 25μL reaction using TaKaRa Ex Taq® Hot Start FD D0 S1 H1 S2 H2 S3 H3 S4 H4 supplied with dNTPs and PCR buffer (Clontech, Mountainview, CA, Cat No. RR006A) with an annealing temperature of 60°C, cycled 25 times. After the reaction the entire sample was electrophoresed with 6X EZ-Vision One loading dye (Amresco®, Sydney, Australia, Cat No. N472) through 1.5% (w/v) low melting temperature agarose (Sigma, Cat No. A9414) in TAE buffer. The dehydrin band was excised, and the DNA was recovered with QIAquick Gel Extraction Kit (Qiagen, Cat No.28704), prior to verifying identify as dehdryin by sequencing at the Grand Valley State University Annis Water Resources Institute Sequencing facility (http://www.gvsu.edu/dna/).

27.00 26.50 26.00

25.50 25.00 24.50

Ct Value Ct 24.00 23.50 23.00 22.50 Untreated SD HRH

Figure 2.13. Expression of actin in seeds of 125 DAP after slow drying (SD) or high relative humidity (HRH) treatment. Values represent the mean±standard deviation of 4

replicates. Values were not statistically different (P=0.20) when tested by ANOVA.

A B

C D

Figure 2.14. Melting curves of qPCR products with primers of TG1 (A), TG2 (B), dehydrin (C) and actin (D).

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Title of thesis: The effect of drying on desiccation tolerance and Late Embryogenesis Abundant protein gene expression in immature seeds of Phalaenopsis amabilis

Signature of author: ______Date: ______

Printed name of author: Huijing Zhu

Keywords: Seed, Phalaenopsis amabilis, LEA, desiccation tolerance, qPCR, slow- drying.