FUNCTION OF ROOT BORDER CELLS AND THEIR EXUDATES ON PLANT

DEFENSE IN HYDROPONIC SYSTEMS

by

Gilberto Curlango-Rivera

_____________________

A Dissertation Submitted to the Faculty of the

SCHOOL OF PLANT SCIENCES

In Partial Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY
WITH A MAJOR IN PLANT PATHOLOGY
In the Graduate College

THE UNIVERSITY OF ARIZONA

2011
 2

THE UNIVERSITY OF ARIZONA

GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation

prepared by Gilberto Curlango-Rivera

entitled Function of Root Border Cells and their Exudates on Plant Defense in
Hydroponic Systems

and recommend that it be accepted as fulfilling the dissertation requirement for the

Degree of Doctor of Philosophy

_______________________________________________________________________ Date: July 20th, 2011

Martha C. Hawes

_______________________________________________________________________ Date: July 20th, 2011

Hans D. VanEtten

_______________________________________________________________________ Date: July 20th, 2011

Chieri Kubota

_______________________________________________________________________ Date: July 20th, 2011

Mary Olsen

Final approval and acceptance of this dissertation is contingent upon the candidate‟s
submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and
recommend that it be accepted as fulfilling the dissertation requirement.

________________________________________________ Date: July 20th, 2011

Dissertation Director: Martha C. Hawes
 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an

advanced degree at the University of Arizona and is deposited in the University Library
to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided
that accurate acknowledgment of source is made. Requests for permission for extended
quotation from or reproduction of this manuscript in whole or in part may be granted by
the head of the major department or the Dean of the Graduate College when in his or her
judgment the proposed use of the material is in the interests of scholarship. In all other
instances, however, permission must be obtained from the author.

SIGNED: Gilberto Curlango-Rivera

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ACKNOWLEDGEMENTS

I want to honestly express my gratitude to Dr. Martha Hawes for giving me the

opportunity of doing my doctoral studies in her research laboratory. Her advice and

guidance helped me to accomplish this important goal of my academic career and life.

In the same manner, I want to thanks to Dr. Hans VanEtten for his advice and

disposition. His example as a scientist has been a very important part of my formation.

Thanks to Dr. Chieri Kubota, Dr. Sandy Pierson, and Dr. Mary Olsen for being part of

my committee and for their advice and help on my research.

I want to say thanks to all my friends that helped me during my studies in different

ways. Special thanks to Dr. Fushi Wen, Dr. Gerard White, Dr. Yolanda Flores-Lara, and

MS Rhodesia Celoy for their help, advice on research, and friendship.

Thanks to all members of my family for the strength they have given me throughout

my studies. Thanks to my father, to my mother, to my sisters, and to my grandparents for

their support.

Thanks to my wife for her help and the good moments.

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DEDICATION

To all members of my family,

To my parents,

To my sisters,

To my grandparents,

To my wife,

and

Specially to my mother and grand mother

Julieta and Julieta

 6

TABLE OF CONTENTS

LIST OF FIGURES……………………………………………………………….... 8

ABSTRACT……………………………………………………………………….... 9

I INTRODUCTION………………………………………………………………… 11

I.1 Context of Research…………………………………………………………... 11

I.2 Literature Review…………………………………………………………….. 12

I.2.1 Controlled Environment Agriculture…………………………………….. 12

I.2.2 Diseases in Controlled Environment Agriculture…………………........... 14
I.2.3 Root Border Cells……………………………………………………....... 16
I.2.4 Defense Mechanisms by Border Cells…………………………………… 19
I.2.5 Border Cell Exudates: Lectins…………………………………………… 21
I.2.6 Border Cell in Hydroponics…………………………………………........ 28

I.3 Objectives…………………………………………………………………….. 28

I.4 Format of this Dissertation…………………………………………………… 29

II PRESENT STUDY………………………………………………………………. 30

II.1 Summary of appendix A: Dynamics of root border cells in hydroponic

systems………………………………………………………………………… 30

II.2 Summary of appendix B: Proteins among the polysaccharides. A new

perspective on root cap slime………………………………………………….. 31

II.3 Summary of appendix C: Contribution of the root cap to soil fertility:

Extracellular plant lectins……………………………………………………… 33

II.4 Summary of appendix D: Transient exposure of root tips to primary and

secondary metabolites: Impact on root growth and production of border cells.. 34

II.5 Summary of appendix E: Root tips moving through soil. An intrinsic

vulnerability........................................................................................................ 36
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TABLE OF CONTENTS – Continued

II.6 Summary of appendix F: Extracellular DNA: The tip of root defenses?......... 37

II.7 Summary of appendix G: Altered root tip morphology in pea hairy roots
with altered expression of a border cell specific gene………………………… 38

REFERENCES……………………………………………………………...………. 40

APPENDIX A DYNAMICS OF BORDER CELLS IN HYDROPONIC

SYSTEMS: PRODUCTION AND FUNCTION……………….………………... 48

APPENDIX B PROTEINS AMONG THE POLYSACCHARIDES: A NEW

PERSPECTIVE ON ROOT CAP „SLIME‟……………………….……………... 70

APPENDIX C CONTRIBUTION OF THE ROOT CAP TO SOIL FERTILITY:

EXTRACELLULAR PLANT LECTINS ……………………………………….. 75

APPENDIX D TRANSIENT EXPOSURE OF ROOT TIPS TO PRIMARY AND

SECONDARY METABOLITES: IMPACT ON ROOT GROWTH AND
PRODUCTION OF BORDER CELLS……...…………………………………... 92

APPENDIX E ROOT TIPS MOVING THROUGH SOIL: AN INTRINSIC

VULNERABILITY ………………………………………...…………………… 103

APPENDIX F EXTRACELLULAR DNA: THE TIP OF ROOT DEFENSES?…... 107

APPENDIX G ALTERED ROOT TIP MORPHOLOGY IN PEA HAIRY ROOTS

WITH ALTERED EXPRESSION OF A BORDER CELL SPECIFIC GENE….. 114
 8

LIST OF FIGURES

Figure 1. Root tips and border cells………………………………………………… 17

Figure 2. Root cap structure organized in structured tiers………………………….. 18

Figure 3. Fungal infection zone…………………………………………………….. 21

Figure 4. Lectins in the root cap……………………………………………………. 25

 9

ABSTRACT

Controlled environment agriculture offers a solution to challenges including less

available land, water deficits, and consumer demand for pesticide free produce. However,

control of soil-borne diseases is a major limiting factor. The goal of this dissertation was

to examine predictions of the hypothesis that border cells function to protect plant health

by controlling microorganisms associated with plants grown in hydroponic culture.

Border cells separate from root tips upon immersion in water, and appear to have

important roles in the defense mechanisms of plant roots.

The general objectives were (1) to study the delivery of border cells in

hydroponics; (2) to evaluate interactions between border cells and microorganisms in

hydroponics; and (3) to explore approaches to alter border cell production for improved

root disease control.

In this study it was confirmed that border cells can be released continuously into

the solution of hydroponic culture suggesting that plants grown in this system may use

extra energy in the production of new border cells. Free border cells interacted with

microorganisms present in the hydroponic solution by secreting an extracellular capsule.

Previous studies showed that proteins are a key component of this capsule, including

lectins. The interaction of pea lectin and Nectria haematococca spores therefore was

explored. Results demonstrated that pea lectin agglutinates fungal spores in a hapten-

specific manner, and inhibits their germination. Lectin had no negative effect on root

development suggesting that it could be used as a potential control for soil-borne diseases

in hydroponics.
 10

To control the production of border cells, subsequent studies measured the impact

of a transient exposure of root tips to different metabolites secreted by root caps and

border cells. Exposure to specific metabolites altered the production of border cells

without measurable effects on root growth and development. This is in contrast to results

obtained with altered gene expression. For example, gene silencing of a border cell

specific gene resulted in altered root growth.

 11

I. INTRODUCTION

I.1 Context of Research

Traditional agriculture faces emerging challenges to be both more productive and

environmentally friendly (Schnotzler et al., 2003). The development of controlled

environment agriculture techniques offers a viable alternative to obtain a high efficiency

in water use, and higher productivity of safe produce. As a result, the development of

controlled environment agriculture has been increasing worldwide (Enoch and Enoch,

1998). The North American region plays an important role especially in the use of high-

technology approaches such as closed-hydroponics systems, achieving average yields of

378 metric tons of tomato per hectare and obtaining better efficiencies in the use of water

(Cook and Calvin, 2005).

However, although in these systems the crop is protected and the diversity of

plant pathogens is reduced considerably, disease remains a significant problem that may

limit application of the economic and cultural potential of controlled environment

systems (Dasberg, 1998). In most cases, the problem is soil-borne pathogens

(Stanghellini and Rasmussen, 1994). In soil ecosystems, plants have different defense

mechanisms to overcome soil-borne infections, including those carried out by root border

cells and their exudates (reviewed in Hawes et al., 1998, 2000, 2003). Border cells are

plant cells present on root tips with the particular characteristic that they separate from

the root cap upon immersion in water. An important function of root caps is that they are

programmed to replace those cells that have separated. Border cells and their exudates
 12

appear to have important roles in the defense mechanisms of plant roots, such as

protecting the growing root tip from pathogenic organisms and harsh conditions in the

rhizosphere (Miyasaka and Hawes, 2000; Wen et al., 2007a).

The goal of this dissertation work is to examine predictions of the hypothesis that

border cells function to protect plant health by controlling microbial populations

associated with plants grown using hydroponic cultures. If correct, then control of

border cells and their products may prove to be a key target for improved efficiency of

safe, ecologically sound crop production using controlled environmental systems.

I.2 Literature Review

I.2.1 Controlled Environment Agriculture. In the course of the development of

conventional agriculture, there have been modifications in the cultural practices used to

increase productivity. Higher yields have been reached by the management of higher

plant densities, monocultures, and high amounts of synthetic fertilizers (Chrispeels and

Sadava, 1994). However, these practices have caused serious outbreaks of diseases,

which have been partially controlled with toxic chemicals causing serious environmental

and human health problems (Lenteren, 2000). Other limiting factors are the reduced

availability of water (Yeston et al., 2006) and arable land acreage (Liang et al., 2005).

The development of controlled environment agriculture techniques has been

shown to be a viable alternative. The productivity in these protected systems is very high

leading to much higher yields per unit of land, and increased efficiencies in the use of

water (Schnotzler et al., 2003). For instance, water usage in field conditions is at least
 13

two fold higher than in greenhouses (Rouphael et al., 2005). Also, since the crop is

protected against harsh environments and disease agents, the production of free-pesticide

produces is possible resulting in an environment-friendly agriculture system. Other

advantages of controlled environment agriculture is that it needs a higher number of

trained workers (8-10 persons per hectare versus one in open fields) which may help to

reactivate the agricultural economy by the generation of more employees, mainly in

developing countries (Tiwari, 2003).

With these advantages, the development of controlled environment agriculture has

been increasing worldwide. According to Enoch and Enoch (1998), the total area of

greenhouses around the world is about 800,000 hectares. This area is distributed mainly

in Asiatic countries, the European Union and recently North America.

China is the world‟s leader in protected agriculture area with seventy-five percent

of the world‟s total, and specifically this area is designated to vegetable production

(Enoch and Enoch, 1998). Japan and South Korea are important regions of Asia with

42,000 and 3,807 hectares respectively (Enoch and Enoch, 1998).

The European community has a representative area of greenhouse designated to

the production of vegetables (Enoch and Enoch, 1998). Spain represents the largest area

of greenhouses with 18,500 hectares, followed by Italy, France, and the Netherlands with

9,000, 6,450, and 4,500 hectares respectively. Countries including Belgium, United

Kingdom, and Germany have an area that ranges between 2,000 and 2,500 hectares,

while Portugal, Denmark, and Ireland have 1250, 140, and 55 hectares respectively. This

European region represents about twenty-five percent of the global greenhouse

 14

distribution excluding China (Enoch and Enoch, 1998). However, recently this number

has been increasing because of the increasing development of controlled environment

agriculture in other regions in North America. The use of techniques as closed-

hydroponics systems, where plants are grown in a nutrient solution that is recovered and

recycled through the root system of plants, allows achievement of higher yields and

higher efficiencies in the use of water (Cook and Calvin, 2005).

In 2003, the tomato industry of Canada, Mexico and United States produced

528,000 metric tons of greenhouse tomatoes, which represent around thirty-seven percent

of all fresh tomatoes sold in United States. This production was accomplished in an area

of 1,726 hectares, with average yields of 378 metric tons/hectare compared with 25

metric tons/hectare from conventional agriculture. The North American fresh tomato

industry represents thirteen percent of the total worldwide greenhouse production (Cook

and Calvin, 2005).

I.2.2 Diseases in Controlled Environment Agriculture. In controlled

environment agriculture the crop is protected from external environmental conditions

such as excessive heat, cold, wind, and rains, and from pests and pathogens organisms

such as bacteria, viruses and fungi. However, organisms can enter the protected system

by a number of ways. Examples include the ventilation system, water source, dust,

humans, insects and contaminated plant material. Also, in spite of the reduction of the

diversity and amount of disease causing agents within these systems, serious outbreaks of

diseases remain a problem (Dasberg, 1998). These are mainly caused by soil-borne

pathogens since the root system is difficult to monitor in order to carry out a disease
 15

preventive program. Causal agents are mostly fungal species such as Fusarium,

Verticillium, Rhizoctonia, and also oomycetes, such as Phytophthora, Olpidium,

Plasmopara and several species of Pythium (Stanghellini and Rasmussen, 1994).

Oomycetes are of considerable importance in hydroponics because they are favored by

aquatic environments that allow rapid spread through the nutrient solution, infecting roots

through the whole system causing serious economical losses or even losing the whole

crop (Stanghellini and Rasmussen, 1994). Fungi such as Fusarium spp. also cause serious

diseases in many plant species worldwide (Fujinaga et al., 2003; Katan et al., 1997; Punja

and Parker, 2000; Stanghellini and Rasmussen, 1994).

An approach to prevent soil-borne diseases in controlled environment systems is

the use of grafted transplants, which also enhance plant productivity (Lee, 1994). Another

approach to solve this problem is the disinfection of the nutrient solution by ultraviolet

radiation, which increases the costs of an already expensive technology. This radiation

penetrates the cell wall of microorganisms causing damage to their DNA, so that they

cannot multiply and eventually are eradicated. Ultraviolet radiation has been effective in

control of Pythium aphanidermatum (Stanghellini, 1984), but unfortunately iron chelates

are broken down by this radiation causing iron chlorosis in plants (Daughtrey, 1980;

Nederhoff, 2001).

Plants have different defense mechanisms in order to prevent infections from soil-

borne pathogens. One of these defense mechanisms is carried out by border cells and

their exudates (Hawes et al, 2000).

 16

I.2.3 Root Border Cells. Border cells (Fig. 1) are living plant cells generated at

root caps (Fig. 2) forming a sheath of cells that surrounds and gives protection to the root

tip, having the characteristic that they separate from the root cap once in contact with

water (reviewed in Hawes et al., 1998, 2000, 2003). This phenomenon of border cell

separation is visualized with the aid of a stereoscope (Fig. 1). For instance, when root tips

from pea (Pisum sativum L.) seedlings are submerged in water, border cells disperse

immediately. This triggers cell cycle activation within the root cap meristem, with

immediate production of border cell production. After 24 h a new set of border cells is

completed, the root cap turns off the border cell cycle and no more border cells are

produced, unless border cells are again removed by immersion in water or other signals

(Brigham et al., 1998). It is hypothesized that once the whole set of border cells is

formed, border cells release an unknown signal which is sensed by the root cap, which

consequently blocks cell cycle (Brigham et al., 1998). Therefore, in order to stop the

border cell cycle, the root cap must sense the unknown signal; otherwise the production

of border cells by the root cap will be continuous requiring a constant use of energy. In

addition, it has been shown that the interruption of the border cell cycle increases plant

susceptibility against pathogenic organisms (Woo et al., 2004).

 17

A B C

Figure 1. Root tips and border cells. Root tip in dry conditions visualized with a
stereoscope (A). Visualization of border cells attached to root tip in dry
conditions with scanning electron microscopy (From Hawes and Brigham,
1992; photo by Perkins S, Calvert HE and Bauer WD) (B). Detached border
cells from root tip in the presence of free water, visualized with a stereoscope
(C). Magnification A,C 10X; B 20X. (From Hawes et al., 2003).
 18

Figure 2. Root cap structure organized in structured tiers (from Barlow,

1975).

Root caps release a variable number of border cells depending on plant species. A

single radicle of tomato, pepper, corn, pea, and cucumber will produce a set of 20, 90,

2700, 3300, and 3800 border cells, respectively (Hawes and Pueppke, 1986). The energy

required to produce such numbers of cells is regulated within roots and the

photosynthetic mechanism of plants (Farrar et al., 2003). For example, it has been shown

that tomato plants translocate forty-three percent of photosynthates to the root system of

tomato plants grown in soil, and from this percentage, seventy percent is released as

rhizodeposition (Lynch and Whipps, 1991), including border cells. Nevertheless, in soil

ecosystems the energy necessary for the extra production of border cells may be limited
 19

by the normal control of border cell production. Thus, at ninety-nine percent humidity

which is typical for soil conditions most of the time, border cell production ceases

(Brigham et al., 1998; Guinel and McCully, 1986). Only upon exposure to continuous

film of water do border cells disperse rapidly, inducing renewed production.

I.2.4 Defense Mechanisms by Border Cells. To date, results obtained under

controlled laboratory conditions, have been consistent with the hypothesis that border

cells function to protect plant health by protecting the root tip, which houses the root

meristem from which all new cells are derived (reviewed in Hawes et al., 1998, 2000,

2003, 2005).

Root tips and border cells are of considerable importance for plants because of

their interaction with the microbial population in the rhizosphere (Jaroszuk-Scisel et al.,

2009; Liljeroth et al., 1991; McCully and Canny, 1988). For example, root border cell

production is positively correlated with root colonization by beneficial organisms such as

arbuscular micorrhizae (Niemira, et al., 1996). Also, Tsai and Phillips (1991) found that

flavonoids from alfalfa (Medicago sativa) root exudates promote spore germination,

hyphal elongation and branching in the vesicular-arbusclar mycorrhizae Glomus

etunicatum. Moreover, Arriola (1997) found that the mycorrhizal fungus Glomus

intraradices was positively correlated with maximum border cell production by four

species from the Amaranthaceae family (Gomphrena globosa, Amaranthus tricolor,

Amaranthus caudatus, and Celosia cristata).

In addition, root border cells may attract pathogenic microorganisms as a 'decoy'

defense mechanism. For instance, nematodes are attracted toward border cells, but after
 20

30 min of interaction, nematodes are immobilized in response to exudates release by

border cells. After a few hours or few days, nematodes regain motility. By that time the

growing root tip has escaped from the danger of infection (Zhao et al., 2000b; Hubbard et

al., 2005). A similar defense mechanism against pathogenic fungi is carried out by root

border cells. Gunawardena et al. (2001, 2002, 2005) observed that fungal spores of N.

haematococca (mating population VI) are localized among pea (P. sativum L.) border

cells. As a result, a “mantle” is formed among border cells, their secretions and fungal

hyphae (Fig. 3) (Gunawardena et al., 2005). The mantle detaches spontaneously allowing

the uninfected root tip to keep growing, thus escaping infection. New border cells start to

regenerate right away forming a new set in 24 h giving renewed protection to the growing

root tip. Similar responses are shown when root tips are in adverse abiotic conditions

such as the presence of aluminum or high carbon dioxide levels (Chen et al., 2008; Liu et

al., 2007; Miyasaka and Hawes, 2001; Zhao et al., 2000a).

Recent discoveries have revealed the mechanism by which border cells may

function in prevention of infection by fungal spores (Wen et al., 2007b). When a set of

ca. 120 proteins secreted by pea root tips (the root cap „secretome‟) were treated with

protein degrading agents, infection by the pea pathogen N. haematococca was increased

from lees than five percent to one hundred percent. Multidimensional protein

identification technology analysis revealed that border cells secrete a number of different

proteins including those involved in plant defense mechanisms (Wen et al., 2007b). Of

particular interest was the discovery that lectins are among the secretome. The possibility

that this protein plays a key role in border cell function, and therefore is a target for
 21

increased crop protection in hydroponics, was explored. Their rationale for this is

summarized in the following paragraphs.

Figure 3. Fungal infection zone (From Gunawardena, 2005).

I.2.5 Border Cell Exudates: Lectins. Lectins are plant, animal and microbial

proteins that bind to certain specific carbohydrate groups on cell surfaces and agglutinate

them (Lehninger, 1982). Lectins are abundant in plants, particularly those of the legume

family. Plant extracts with the ability to agglutinate human cells such as erythrocytes

were first described as ricin from seeds of the castor bean (Ricinus commuinis) and abrin
 22

from Abrus precatorius (reviewed in Lis and Sharon, 1972, 1977, 1981, and 2004;

Reviewed in Sharon, 2007). The first plant lectin was isolated by Sumner and Howell

(1936) in a purified form from seeds of the jack bean (Canavalia ensiformis) and named

concanavalin A. The same authors demonstrated that this lectin was hapten-specific, this

is, it precipitated glycogen and starch from solution and this agglutination activity was

inhibited by sucrose from sugar cane. Cell specificity of lectins was demonstrated by lima

bean (Phaseolus lunatus syn. limensis) lectin agglutination on human erythrocytes type

A, but not those of type B or O; based on these findings, Boyd and Shapleigh (1954)

proposed the term lectin (Latin lego, to choose or to pick out) for these sugar-binding

proteins.

Lectin composition. Most lectins are glycoproteins with carbohydrate content that

can be as high as fifty percent. The sugar components are the same as those found in

other plant glycoproteins, with the exception of L-arabinose. However, concanavalin A,

wheat germ, and peanut lectins are exceptions that do not contain covalently bound

carbohydrates. The amino acid pattern of lectins is characteristic of plant proteins, and

there are no structural features common to all of them. Many are rich in aspartic acid,

serine, and threonine, which comprise about thirty percent of their amino acid content.

Plant lectins are low or devoid of sulfur-containing amino acids. A few exceptions

include potato, pokeweed and wheat germ lectins which are rich in cysteine with 11.5,

18, and 20 percent of the total amino acid residues, respectively. Most lectins are soluble

cell components and also they are membrane-bound (reviewed in Lis and Sharon, 1981).
 23

Properties of lectins. Lectins are important tools in cell differentiation and cell

surface characterization, as well as detection, isolation, and characterization of

carbohydrate-containing materials such as polysaccharides, glycoproteins, and

glycolipids (reviewed in Lis and Sharon, 1981). For example, lectins were used in the

1950s to show that the determinants of the specificity of the blood types in humans were

sugars: α-N-acetylgalactosamine of type A, α-galactose of type B, and α-L-fucose of type

O specificity (reviewed in Lis and Sharon, 1981). In addition, cell differentiation by

lectins was shown in experiments in the 1960s, when the wheat germ agglutinin from

Triticum vulgare was found to agglutinate tumor cells at concentrations much lower than

those required for the agglutination of normal cells. In the same manner, concanavalin A,

soybean (Glycine max) lectin, and many others agglutinated malignant cells

preferentially, suggesting that tumor cells must have a different surface structure from

normal cells since the specific carbohydrate residues to which lectins bind are apparently

more exposed on tumor cell surfaces. These findings led to the use of lectins for studying

changes on cell surfaces during growth, differentiation, and malignant transformation

(Lehninger, 1982; reviewed in Lis and Sharon, 1981). Moreover, lectins from the red

kidney bean (P. vulgaris) cause lymphocyte cells to enlarge. This cell enlargement

facilitated the study of chromosomes increasing the understanding of relationships

between chromosomes abnormalities and diseases, and the expansion of cytogenetics

(Nowell, 1960). Others functions of lectins include enzymatic properties such as the

mung bean lectin which has a strong enzymatic activity comparable to that of α-

galactosidase (Hankins and Sharon, 1978).

 24

Localization of plant lectins. In plants, lectins occur mostly in seeds representing

three to four percent of the dry mass (Summer and Howell, 1936). Mishkind et al. (1983)

showed the presence of wheat germ agglutinin in different tissues of wheat, barley, rye,

and rice plants. These included the germ portion of the grain, such as the surface layers

of various organs including the coleoptile, scutellum, coleorhiza, and the first

adventitious roots. The functions remain unknown. Of special interest to this dissertation

was the discovery that lectins were restricted to the root cap and root border cells (Fig. 4).

A small population of cells in the embryo contains most of the lectin present in the grain,

which represent one percent or less. In addition, Gatehouse and Boulter (1980) have also

shown the presence of lectins in seeds, and besides that, they reported the detection of

lectin in pea roots. Pea lectins purified from seeds appear to be different from pea lectins

purified from roots, since they have different sugar specificity and agglutination activity,

suggesting that they may have different function and structures. Specifically, root lectins

were detected in the root cortical cells and on the surface of root hairs, showing their

presence at sites of rhizobial infection. At higher magnifications, lectins were detected in

root cap cells and border cells (Mishkind et al. 1983).

 25

A B C D

Figure 4. Lectins in the root cap. Mishkind et al. (1984) used

immunolocalization to demonstrate that in the embryonic root caps of wheat
(A) lectin is localized at the cap periphery (B). As the root emerges (C), lectin
is secreted into the extracellular environment of border cells (D).

The presence of lectins in phloem exudates from cucurbits species such as

pumpkin (Cucurbita maxima Duch.), cucumber (Cucumis sativus), and melon (Cucumis

melo) has been reported by Sabnis and Hart (1978). Lectins were found in five day old

seedlings, and no detection was possible in seeds. The agglutinating activity was shown

at concentrations as low as 0.1 μg/mL, and interestingly, sugars such as raffinose and

stachyose (which are transported in the phloem of these species), sucrose, galactose,

glucose, fucose, mannose, xylose, and arabinose, do not inhibit agglutination. From the

total phloem protein content, fifteen to twenty-five percent was found as lectin, which is a

higher content compared to that of seeds from other plant species. According to these

authors, one possible function of phloem lectin may be the protection of the sugar-rich
 26

phloem from invasions of microorganisms, since inhibition of pathogenic

microorganisms has been reported as described below.

Lectin-microbe interaction. For the majority of plants in the leguminosae family,

emergent root hairs are the sites of infection by Rhizobium spp. Diaz et al (1986), found a

positive correlation on the colonization of the bacterial symbiont Rhizobium

leguminosarum on sites where lectins from pea (P. sativum L.) roots were accumulated.

Lectins were observed on the tips of newly formed, growing root hairs and on epidermal

cells located just below the young hairs. Most nodulation (seventy-three to ninety

percent) occurred on sites where lectin was localized. Contrary, few plants showed

nodulation at sites where the lectin was absent.

Plant lectins also interact with pathogenic organisms, showing antifungal,

antiviral, antibacterial, and anti-insect properties, as well as toxicity toward herbivorous

organisms (reviewed in Peumans and Van Damme, 1995a; 1995b). For example, wheat

germ lectin agglutinates and inhibits growth of Trichoderma viride and Fusarium solani

(Mirelman et al., 1975; Cristinzio et al., 1988). Wheat (Triticum aestivum L.) germ

agglutinin levels in seedling roots are induced two-fold by fungal species such as

Rhizoctonia solani, Fusarium culmorum, Pythium ultimum and Neurospora crassa, as

well as by fungal elicitors. These findings suggest that lectins could be involved in the

defense of wheat against fungal attack. Moreover, lectin from stinging nettle (Urtica

dioica L.) rhizomes bind chitin, inhibiting the growth of phytopathogenic and saprophytic

chitin-containing fungi such as Botrytis cinerea, Collectotrichum lindemuthianum,

Phoma betae, Phycomyces blakesleeanus, Septoria nodorum, Trichoderma hamatum and

 27

T. viride (Broekaert et al., 1974) This lectin inhibition is different from the one caused by

chitinase, but both the nettle lectin and chitinase acts synergistically against fungal

growth.

In the case of pathogenic bacteria, Pueppke et al. (1982), showed that the lectin

concanavalin A binds to Agrobacterium tumefaciens strains, reducing the number of

tumors produced on potato. In contrast, the potato lectin did not bind to A. tumefaciens

and neither reduced the number of tumors produced. Isolated lectins from maize (Zea

mays) were tested for agglutination of virulent and avirulent Erwinia stewartii strains.

Results showed that lectins bind better to the avirulent strains rather than to the virulent

ones, which produce higher amounts of extracellular polysaccharide. When the

extracellular polysaccharide was washed from virulent E. stewartii strains, lectin

agglutination was higher than in unwashed virulent-bacterial cells. According to these

results, the extracellular polysaccharide may be involved in pathogenicity, since it may

prevents bacterial agglutination in the host and consequently increasing its multiplication

(Bradshaw-Rouse et al., 1981).

Lectins found in cell walls of organisms are implicated in host-microbe

interactions (Sharon, 1987). For example, the human pathogen Escherichia coli produces

mannose specific surface lectins, functioning primarily in the initiation of infection by

mediating bacterial adherence to epithelial cells. This mannose specific lectin also acts as

recognition molecules in lectinophagocytosis by mouse, rat, and human peritoneal

macrophages and human polymorphonuclear leukocytes (Sharon, 1987).

 28

I.2.6 Root Border Cells in Hydroponics. Root border cells have been

implicated in protection of the root from injury and infection. Also, it has been shown

that, the phenomenon of border cell separation is influenced by aquatic conditions (Yu et

al., 2006; Endo et al., 2011). Yet their impact in hydroponics remains unexamined.

Border cells were first reported by Knudson (1919), who found free border cells released

into the nutrient solution of pea and maize plants grown in hydroponics. Also, he made

the important observation that these cells remained metabolically active for at least forty-

five days after they were released by root tips. This observation was later supported by

Hawes and Pueppke (1986) who additionally found that border cells are able to divide in

response to the proper supply of nutrients. Griffin et al. (1976) estimated that root

exudates including border cells, released by peanut plants in hydroponics may contribute

ninety-five to ninety-eight percent of the rhizodeposition present in the nutrient solution.

These findings suggest that in hydroponics, there is a frequent release of border cells

which are metabolically active after separation of their root caps. The significance of

these cells in plant development and root disease, and potentially as a target for improved

crop protection, is the focus of this dissertation.

I.3 Objectives

I.3.1 To define the dynamics of border cell production in model hydroponic

systems, using agronomically important species such as cereals and legumes, and with

special emphasis on common greenhouse crops including tomato, cucumber, pepper and

lettuce.
 29

I.3.2 To compare results of the dynamics of border cell production in model

hydroponics with the dynamics of border cell production in semi-commercial

hydroponics.

I.3.3 To explore specifically the ways border cells may interact with microbial

populations to influence plant health.

I.3.4 To examine the functional impact of border cells on root function, with

special emphasis on the role of secreted compounds in root growth and resistance to

infection.

I.4 Format of this Dissertation.

Results of this dissertation will be presented as appendices containing a copy of

either a published article or a draft manuscript in preparation for publication. A summary

of each appendix containing the contribution of the author of this dissertation is presented

in the following section.

 30

II. PRESENT STUDY

The methods, results, and conclusions of this study are presented in the papers

appended to this dissertation. The following is a summary of the most important findings

in this document.

II.1 Summary of appendix A: Dynamics of root border cells in hydroponic systems.

Appendix A is a draft manuscript in progress to be submitted to Journal of

American Society for Horticultural Science. Root border cells have been implicated in

protection of the root from injury and infection (reviewed in Hawes et al., 1998). It has

been shown that border cell separation occurs in the presence of free water (reviewed in

Wen et al., 2007a), but little is known about their impact on the rhizosphere of

hydroponic systems. Border cells of roots grown in hydroponic systems were first

described by Knudson (1919), who observed that border cells from pea (P. sativum L.)

and maize (Zea mays L.) remained alive for at least 45 days after they separated from

root tips. Griffin et al. (1976) estimated that border cells and their associated exudates

from the root cap (ca. 1 mm of the apex) are up to ninety-eight percent of the

rhizodeposition released by peanuts roots grown in hydroponic culture. The observation

that these cells continue metabolically active after separation of their root caps was later

supported by Hawes and Pueppke (1986). Considering these findings it is hypothesized

that in hydroponics there is a constant release of border cells that are metabolically active

after separation of their root caps. In this study, the number of border cells released into
 31

the nutrient solution from different important crops grown in model hydroponic systems

was determined and compared to experimental semi-commercial hydroponic systems.

Results of the current study confirmed results reported by Knudson (1919)

regarding the presence of viable border cells released into the nutrient solution. During

the first days of culture root tips remain free of border cells, as border cells detach

continuously into liquid. Pea and cucumber (Cucumis sativus L.) root tips were shown to

accumulate a sheath of border cells surrounding the root tip. In contrast, root tips remain

free of border cells border cells in semi-commercial hydroponic settings. Samples

analyzed were taken during the day when the crop was being irrigated. In samples

analyzed before the irrigation system started, root tips showed accumulation of border

cells, suggesting that the presence of free water due to frequent irrigation caused border

cell separation. However, border cells were not found in the nutrient solution at any time.

These results suggest that in hydroponic culture the dynamics of border cell separation is

different from soil systems and it may influence the rhizosphere in different ways. For

example, the constant release of border cells may cause extra production of border cells

by plants and a change in the establishment of a microbial community on the rhizosphere.

My contribution to this study was the collection of samples and detailed

microscopic analysis of the number and viability of border cells from plants of different

crops grown in model hydroponic cultures and in a semi-commercial hydroponic system.

II.2 Summary of appendix B: Proteins among the polysaccharides: A new

perspective on root cap ‘slime.’

 32

Appendix B is an article published in Plant Signaling and Behavior (2007), 2(5):

410-412. Root border cells, whose long-term viability in hydroponic culture was

described by Knudson (1919), have been implicated in protection of the root from injury

and infection (Hawes et al., 1998). In the previous appendix Knudson‟s (1919)

observation that metabolically active border cells are released into the nutrient solution of

model hydroponic systems was confirmed. However, the impact of these cells in this type

of culture is unknown. Thus, in this study the ways border cells may interact with

microbial populations present in hydroponic cultures were measured. In previous studies

it has been demonstrated that root caps are covered by border cells contained in a slime-

mucilage material that is mainly composed of polysaccharides and a small portion of

proteins (Bacic, 1986; Chaboud, 1983; Wright 1975). This small part includes ca. 120

proteins that are secreted into the extracellular environment with different functions such

as signaling, structure, and defense (Brigham et al., 1995; Wen et al., 2007b).

In the current study India ink was used to measure dynamics of the slime-

mucilage material in which border cells are contained. Also, a slime-mucilage layer or

capsule surrounding single border cells was visualized. When border cells were treated

with proteinase, this capsule was eliminated and could not be visualized with the India

ink assay, suggesting that proteins are a key component of this slime-mucilage material.

In addition, capsule surrounding single border cells responded differently to the presence

of different microorganisms, which was determined by measuring the increase of the

border cell capsule. For example, the presence of a seed-born epiphyte, Bacillus sp.,

caused a dramatic 50 X increase in size of the capsule of corn border cells grown for 7-10
 33

days in model hydroponic culture. The impact in crop production is unknown. In

conclusion, this study establishes that further research is necessary to examine the cost-

benefit of border cell release into hydroponic crop systems. Metabolically active border

cells released in hydroponic culture may have a positive impact since they interact with

the microbial populations present in these systems. If so, the extra energy used by plants

in hydroponics for the continuous production of border cells might foster a beneficial

microbial population in the hydroponic rhizosphere. An alternative hypothesis is that

uncontrolled border cell production wastes energy and stimulates growth and microbial

populations that are not beneficial.

My contribution to this article was setting up and running a model hydroponic

system in which pea and corn plants were grown, detailed microscopic analysis of border

cells from hydroponic solution samples using India ink assays, and training of graduate

student Mariana Del Olmo-Ruiz during her rotation program.

II.3 Summary of appendix C: Contribution of the root cap to soil fertility:

Extracellular plant lectins.

Appendix C is an invited research article published in Soil Fertility (2009),

Lucero DP and Boggs JE (eds.), Nova Science Publishers, Inc. pp. 65-79. ISBN: 978-1-

60741-466-7. In this study it was determined that pea lectins are involved in recognition

and agglutination of fungal spores. Previous studies have demonstrated that border cells

respond to specific signals released by microorganisms, which triggers defense responses

(reviewed in Hawes et al., 1998, 2000, 2003). These signals are cell wall components,
 34

such as sugars, and it has been shown that these components recognize and bind to lectins

(Halverson and Stacey, 1986). Lectins are part of a set of ca. 120 proteins secreted by

border cells (Wen et al., 2007b), and have been reported to be involved in plant defense

mechanisms (Peumans and Van Damme, 1995a, 1995b). Based on that, it was

hypothesized that lectins function as signals involved in border cell defense mechanisms

such as recognition and agglutination of pathogens. During this study I tested the

prediction that recognition of the pathogen is carried out by the carbohydrate component

of lectins from border cells, implementing agglutination assays and using the pea – N.

haematoccoca model system.

Results revealed that N. haematococca spores are recognized and agglutinated by

pea lectin. This response is correlated with inhibition of spore germination and growth.

The results suggest that aspects of recognition involve lectins from border cells.

My contribution to the content of this article is the implementation of

agglutination assays and detailed microscopic examination of border cell-fungal spore

binding. Training of undergraduate Gabriela Albala (University of Arizona) during her

independent study, winter term students David Olsen (Luther College), Jimp Kemp

(Luther College), and Emily Hildebrand (Centre College), was also a component of this

study.

II.4 Summary of appendix D: Transient exposure of root tips to primary and

secondary metabolites: Impact on root growth and production of border cells.

 35

Appendix D is an article published in Plant and Soil (2010), 332: 267-275. In this

study the impact of pea lectin, and other root secreted metabolites on root development

was evaluated. Specifically, the effect of pea lectin, sugars, amino acids and secondary

metabolites on root cap cell cycle was measured based on border numbers. Border cell

viability, root growth, and morphology of pea seedlings also were evaluated. If lectins are

involved in pathogen recognition and agglutination during plant defense mechanisms

(appendix C), then the accumulation of root border cell-secreted lectins in the nutrient

solution of a hydroponic system may be predicted to have a positive effect in plant health.

If so, then lectins are a potential tool in the control of soil borne pathogens in

hydroponics. However, glucose and mannose as sugar components of lectins may be a

limiting factor in this control strategy, mainly in closed agricultural systems where the

accumulation of different compounds including sugars and different metabolites may

reach a high concentration in the nutrient solution becoming toxic to plants (Jung, 2003;

Knudson, 1917). These hypotheses were explored by developing a transient-exposure

assay to measure effects of metabolites released from roots.

Results demonstrated that pea lectin (1 µg/mL) and the amino acids valine,

alanine, threonine, and asparagine do not have a negative effect on pea at a concentration

of 10 mM. The sugars galactose, glucose, mannose, fucose, arabinose, xylose, rhamnose,

acetylglucosamine and acetylgalactosamine did not have a negative effect as well, at

concentrations up to 50 mM. In contrast, rhamnose reduced the root growth and border

cell production. Metabolites such as ferulic acid and naringenin decreased the root length

and border cell production at 1 mM concentrations, and salicylic acid at 50 mM. Our
 36

results show that pea lectin (1 µg/mL) and its carbohydrate contents glucose and mannose

are not toxic to pea seedlings, which supports the use of lectins as potential tool to control

soil-borne disease.

My contribution to this research was to setting up the transient treatments and

analysis of growth, detailed microscopic analysis of border cell number and viability.

Also, training of undergraduate Gabriela Albala (University of Arizona) during her

independent studies, and helping Dr. Denise Duclos to design and set up experiments

during her post-doctoral research.

II.5 Summary of appendix E: Root tips moving through soil: An intrinsic

vulnerability.

Appendix E is an article published in Plant Signaling and Behavior (2011), 6(5):

726-727. In this article, results show the attraction of Pythium dissotocum to cotton root

tips where border cells are present, and that this attraction occurs in a transient manner,

within seconds. This suggests that a transient presence of metabolites secreted by border

cells into the rhizosphere can play a critical role in plant defense.

My contribution to this study supports the general significance of this

phenomenon by demonstrating the attraction of microorganisms to root tips using

different model systems, specifically, the attraction of Erwinia carotovora carotovora

and Pseudomonas fluoresncens to pea root tips. Studies to determine host specificity of

the attraction of different microorganisms to border cells is in progress.

 37

II.6 Summary of appendix F: Extracellular DNA: The tip of root defenses?

Appendix F is an invited review article published in Plant Science (2011), 180:

741-745. In human pathogenesis, the role of extracellular DNA (exDNA) is not limited to

specific types of pathogens but appears to play a role in bacterial, fungal, and protozoan

infection. Similarly the role of exDNA in plant defense has been documented (Wen et al.,

2009). To evaluate a broader role for exDNA in plant pathogenesis, we selected the causal

agent of bacterial wilt, whose invasion of legume roots has recently been characterized in

detail, using Medicago truncatula as a model (Turner et al. 2009).

Results revealed that susceptibility to infection of pea roots by Ralstonia

solanacearum GMI1000, occurred when the bacteria were co-inoculated with DNase 1.

This increased infection was ameliorated when actin, a specific inhibitor of DNase 1

activity, was added. Control plants treated with DNase 1, alone, showed no change in

growth or development. The results obtained by treating with DNase 1 at the time of

inoculation of plants with N. haematococca or R. solanacearum suggest that, as in

mammalian systems, the presence of exDNA influences host susceptibility to bacterial as

well as fungal plant pathogens. If correct, the capacity to degrade exDNA by the production

of extracellular DNase will be predicted to be a virulence factor in plant pathogens as it is

now known to be in human pathogens. If so, then inhibition of pathogen exDNase is a

promising target for disease prevention in hydroponic systems. Gene silencing of a N.

haematococca exDNase to test its role in disease is in progress.

My contribution to this article is the survey carried out to identify the production

of exDNase activity by plant pathogens using in vitro assays, to measure the effect of
 38

exDNases in bacteria root rot using a model hydroponic system, and to use gene silencing

to study exDNase role in virulence root pathogens.

II.7 Summary of appendix G: Altered root tip morphology in pea hairy roots with

altered expression of a border cell specific gene.

Appendix G is a draft manuscript in progress to be submitted to Annuals of

Botany. The preceding appendixes show that the controlled production and release of

border cells and their exudates may provide a tool to modify root-microbe associations in

hydroponic cropping systems. Altered production of the secreted products by border cells

could be a feasible approach to deliver specific compounds into the rhizosphere in order

to control diseases in a more efficient manner (Hawes et al., 1998, 2000, 2003; Lilley et

al., 2010). In this study, a specific gene expressed only in root border cells was identified.

The coding region of this gene, showed a putative motif that includes a flavin binding

protein site. Flavins have been implicated in plant defense and root growth (Bonner,

1942; Liu et al., 2010, Mishina and Zeier, 2006; Verdrengh and Tarkowski, 2005).

The impact of flavins in the transient exposure assay was measured. Pea and corn

seedlings grown in hydroponic culture and treated with different flavins showed a neutral

impact on root growth, border cell viability and production, and mucilage-slime capsule

response. No protection by riboflavin was observed on pea seedlings inoculated with N.

haematoccoca, a pea pathogen. In addition, transformed hairy roots with altered

expression of this gene caused negative effects on root development such as altered root

growth and abnormal border cell formation. Similar results were obtained in efforts to
 39

modify other root cap genes (Table 1 in Curlango-Rivera et al., 2010). This suggests that

genetic modification as an approach to deliver specific compounds into the rhizosphere

may not be a viable alternative for the control of soil-borne pathogens.

My contribution to this article was to carry out transient assays for root tip

responses to flavins, infection assays in model hydroponics systems to evaluate the

influence of flavins on disease protection, and detailed microscopic examination of

border cell mucilage-slime capsules in response to different flavins.

 40

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2003. Efficient water use for high quality vegetables through the
environmentally sound hydroponic production „ecoponics.‟ Acta Horticulturae
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Sharon N. 2007. Lectins: Carbohydrate-specific reagents and biological

recognition molecules. The Journal of Biological Chemistry 282: 2753-2764.

Stanghellini ME, Stowell LJ, and Bates ML. 1984. Control of root rot of
spinach caused by Pythium aphanidermatum in a recirculating hydroponic
 46

system by ultraviolet irradiation. Plant Disease 68: 1075-1076.

Stanghellini ME, and Rasmussen SL. 1994. Hydroponics. A solution for

zoosporic pathogens. Plant disease 78: 1129-1138.

Sumner JB, and Howell SF. 1936. The identification of the hemagglutinin of
the jack bean with concanavalin A. Journal of Bacteriology 32: 227-237.

Tiwari GN. 2003. Greenhouse technology for greenhouse environment. Ed.

Alpha Science International. England. Printed in India. 544 p.

Tsai SM, and Phillips DA. 1991. Flavonoids released naturally from alfalfa
promote development of symbiotic Glomus spores in vitro. Applied and
Environmental Microbiology 57: 1485-1488.

Turner M, Jauneau A, Genin S, Tavella M, Vailleau F, Gentzbittel L, and

Jardinaud M. 2009. Dissection of bacterial wilt on Medicago truncatula revealed
two Type III secretion system effectors acting on root infection process and
disease development. Plant Physiology 150: 1713-1722.

Verdrengh M, and Tarkowski A. 2005. Riboflavin in innate and acquired immune

responses. Inflammation Research 54: 390-393.

Wen F, Laskowski M, and Hawes MC. 2007a. Cell Separation on roots. Chapter
5. In: Plant cell separation and adhesion. Gonzalez-Carranza Z and Roberts JA
(eds.). Annual Plant Reviews 25: 91-105.

Wen F, VanEtten HD, Tsaprailis G, and Hawes MC. 2007b. Extracellular

proteins in pea root tip and border cells exudates. Plant Physiology
143: 773-783.

Wen F, White GJ, Xiong X, VanEtten HD, and Hawes MC. 2009. Extracellular
DNA is required for root tip resistance to fungal infection. Plant Physiology 151:
820-829.

Woo HH, Hirsch AM, and Hawes MC. 2004. Altered susceptibility to infection
by Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with
altered cell cycle. Plant Cell Reports 22: 967-973.

Wright K. 1975. Ploysaccharides of root-cap slime from five maize varieties.

Photochemistry 14: 759-63.

Yeston J, Coontz R, Smith J, and Ash C. 2006. Freshwater resources.

Science 313: 1067-1090.
 47

Yu M, Feng YM, and Goldbach HE. 2006. Mist culture for mass harvesting of root
border cells: aluminum effects. J Plant Nutr Soil Sci 169: 670-674.

Zhao X, Misaghi I, and Hawes MC. 2000a. Stimulation of border cell production
in response to increased carbon dioxide levels. Plant Physiology 122: 1-8.

Zhao X, Schmidt M, and Hawes MC. 2000b. Species-dependent effects of

root border cells on nematode chemotaxis and motility. Phytopathology
90: 1239-1245.
 48

APPENDIX A

DYNAMICS OF BORDER CELLS IN HYDROPONIC SYSTEMS:

PRODUCTION AND FUNCTION

Gilberto Curlango-Rivera

Martha C. Hawes

Draft manuscript in progress to be submitted to Journal of American Society for

Horticultural Science.
 49

Dynamics of root border cells in hydroponic systems: Production and function.

Gilberto Curlango-Rivera,1 Martha C. Hawes*

Draft manuscript in progress to be submitted to Journal of American Society for

Horticultural Science.

Author affiliation:
1
Division of Plant Pathology and Microbiology, Controlled Environment Agriculture

Center. School of Plant Sciences, University of Arizona, Tucson, AZ, USA.

*
Corresponding author; Department of Soil, Water, and Environmental Science,

University of Arizona, Tucson, AZ, USA; E-mail mhawes@u.arizona.edu.

 50

ABSTRACT

Root border cells have been implicated in protection of the root from injury and

infection. It has been shown that border cell separation occurs in the presence of water,

and that the cells can remain alive after they separate from root tips. Root tip exudates,

including border cells, can contribute with up to ninety-eight percent of the

rhizodeposition from peanuts and other legumes grown in hydroponics. However, little is

known about their impact on growth, development, and susceptibility to disease. We

hypothesized that in hydroponics there is a constant release of border cells that are

metabolically active after separation of their root caps. In this study, the number of border

cells released into the nutrient solution from different important crops grown in model

hydroponic systems was determined and compared to experimental semi-commercial

hydroponic systems. Results confirmed the presence of active border cells released into

the nutrient solution. During the first days of culture root tips remained free of border

cells, and overtime, pea and cucumber root tips accumulated a sheath of border cells

surrounding the root tip. In contrast, root tips remain free of border cells border cells in

semi-commercial hydroponic settings. However, border cells were not found in the

nutrient solution at any time. These results suggest that in hydroponic culture the

dynamics of border cell separation is different from soil systems and it may influence the

rhizosphere in different ways. For example, the constant release of border cells may

stimulate changes in associated microbial populations on the rhizosphere.

 51

INTRODUCTION

The development of controlled environment agriculture techniques has been

shown to be a viable alternative to conventional agriculture (Cook and Calvin, 2005;

Tiwari 2003). The use of techniques such as closed-hydroponics systems, where plants

are grown in a nutrient solution that is recovered and recycled through the root system of

plants, allows achievement of higher yields and higher efficiencies in the use of water

(Cook and Calvin, 2005; Schnotzler et al., 2003). According to Enoch and Enoch (1998),

the total area of greenhouses around the world is about 800,000 hectares. In spite of the

protection these systems offer, diseases remain a problem (Dasberg, 1998), mainly

caused by soil-borne pathogens (Stanghellini and Rasmussen, 1994). Prevention of soil-

borne diseases in controlled environment systems increases the costs of operation, and

relies on the use of pesticides or are not totally effective (Daughtrey, 1980; Lee, 1994;

Nederhoff, 2001; Stanghellini, 1984).

Plants have different defense mechanisms in order to prevent infections from soil-

borne pathogens. One of these defense mechanisms is carried out by border cells and

their exudates (Hawes et al, 2000). Border cells are living plant cells generated at root

caps forming a sheath of cells that surrounds and protects the root tip (reviewed in Hawes

et al., 1998, 2000, 2003). Border cells separate from the root tip upon immersion in

water (Hawes et al., 1998). After border cell separation the root cap starts to produce new

border cells within minutes, completing a whole set in 24 h. After the new set of border

cells is complete, the root cap cell cycle is blocked and no more border cells are

produced, unless border cells are again separated by immersion in water. It is

 52

hypothesized that once the whole set of border cells is formed, border cells release an

unknown signal which is sensed by the root cap, which consequently blocks the root cap

cell cycle (Brigham et al., 1998). Therefore, in order to stop the cell cycle, the root cap

must sense the unknown signal; otherwise the production of border cells by the root cap

will be continuous requiring a constant use of energy. Conversely, it has been shown that

the interruption of the root cap cell cycle to inhibit border cell production increases plant

susceptibility against pathogenic organisms (Woo et al., 2004).

Root caps release a variable number of border cells depending on plant species. A

single radicle of tomato, pepper, corn, pea, and cucumber will produce a set of 20, 90,

2700, 3300, and 3800 border cells, respectively (Hawes and Pueppke, 1986). The energy

required to produce such numbers of cells is regulated within roots and the

photosynthetic mechanism of plants (Farrar et al., 2003). For example, it has been shown

that tomato plants translocate forty-three percent of photosynthates to the root system of

tomato plants grown in soil, and from this percentage, seventy percent is released as

rhizodeposition (Lynch and Whipps, 1991), including border cells. Nevertheless, in soil

ecosystems the energy necessary for the extra production of border cells may be limited

by the normal control of border cell production. Thus, at ninety-nine percent humidity

which is typical for soil conditions most of the time, border cell production ceases

(Brigham et al. 1998). Only upon exposure to free water do border cells disperse rapidly,

inducing renewed production.

Metabolically active border cells were first reported by Knudson (1919), who

found free border cells released into the nutrient solution of pea and maize plants grown
 53

in hydroponics. Also, he made the important observation that these cells remained one

hundred percent viable for at least forty-five days after they were released by root tips.

This observation was later supported by Hawes and Pueppke (1986) who additionally

found that border cells are able to divide in response to the proper supply of nutrients.

Griffin et al. (1976) estimated that root exudates including border cells, released by

peanut plants in hydroponics may contribute ninety-five to ninety-eight percent of the

rhizodeposition present in the nutrient solution. These findings suggest that in

hydroponics, there is a frequent release of border cells which are metabolically active

after separation of their root caps.

Since border cells are the main product released into the rhizosphere, and

evidence suggests that they function in defense, it is reasonable to propose that their

production in hydroponic culture may influence growth, development, and susceptibility

to disease. The objective of this work will be to study the release of border cells of

important agronomic plant species and greenhouse crops using model hydroponic

cultures and experimental semi-commercial hydroponic systems.

METHODOLOGY

Border cell production in model hydroponic systems.

Plant species used for border cells quantification in laboratory conditions are

described in Table 1. Z. mays and C. sativus were surface sterilized in 95% (v/v) ethanol

for ten minutes and 50% (v/v) commercial bleach (6 % sodium hypochlorite) for ten

minutes. Legume seeds were surface sterilized in 95% ethanol for ten minutes (v/v),
 54

followed by full strenghth commercial bleach (6 % sodium hypochlorite) for thirty

minutes, eliminating floating seeds. Seeds from the other species were surface sterilized

in 95% (v/v) ethanol and 1% (v/v) commercial bleach (6 % sodium hypochlorite) for five

minutes each. All seeds were rinsed five times and imbibed in distilled water for 1 hours,

with the exception of P. sativum which was imbibed for 6 hours. After imbibition, seeds

were plated onto 1 % water-agar plates which were overlaid with Whatman # 1 filter

paper in order to prevent loss of border cells by root penetration of the agar. Seeds were

incubated at 25 °C (Hawes and Pueppke, 1986).

Quantification of border cells.

After seedlings reached a length of 25 mm, root tips were washed from border

cells, as described (Hawes and Pueppke, 1986). Then, one set of three seedlings was put

back onto Petri plates, and another was kept separated in 1.5 ml microfuge tubes with 1

ml of distilled water simulating hydroponic conditions. After 24 hours, border cells from

each seedling on agar plates were harvested in 100 μl of distilled water, and then three

samples of 10 μl each were counted using a light microscope. For seedlings in water, a

sample of 100 μl and a subsample of 10 μl were used, repeating three times for each

microfuge. This experiment was repeated three times for each plant species.

After quantifying border cells in water-agar plates, we proceeded to quantify

border cell number in hydroponics just by setting seedlings in 1.5 ml centrifuge tubes

containing 1 ml of distilled water. Previously, border cells were removed and incubated.

After 24 hours, the new set of border cells formed was counted.
 55

Border cells in semi-commercial hydroponic systems.

In order to determine the presence of border cells in the root system of hydroponic

systems, we proceeded to take root samples of different greenhouse crops grown under

aggregate-hydroponic conditions and determine the presence of border cells. In these

systems, rock wool is used as an artificial substrate which gives plant support. This

substrate is drip-irrigated during 10 hours per day with irrigation frequencies of 3-4 times

per hour.

Most of the root system was distributed on the bottom and out of the substrate.

Thus, root tips samples were taken from the bottom of the substrate, and the number of

root tips with and without border cells was determined. After that, root tips were plated

overnight on water-agar plates, and next day were analyzed for border cell number and

viability.

To measure the effect of irrigation effect on border cell production, root tip

samples were taken after the last schedule of the irrigation system (6:00 p.m.). To see if

root tips were viable and able to produce more border cells during the overnight period

when the irrigation system was off, samples were taken before the initiation of the

irrigation system (7:00 a.m.). In addition, samples were taken from plants irrigated with

different electrical conductivity.

RESULTS

Border cell production in model hydroponic culture.

 56

Table 2 presents the number of border cells produced by root tips from seedlings

grown for 24 hours on water agar and in hydroponics for 24 h. These results show

differences in border cell production depending in the plant species. Cucumber, tomato

and pepper species produced a high number of border cells in water culture. Alfalfa

produced the same number of border cells either growing on water-agar or in water

culture conditions. Corn, pea and lettuce released fewer border cells in water culture than

on water agar. Fluorescein diacetate used as a vital stain to measure cell viability (Hawes

and Pueppke, 1986) showed that detached border cells were metabolically active. Also,

root tips in water culture conditions were free of border cells or had only a few attached

to the root cap, with the exception of cucumber and pea seedlings. During the first days

of culture root tips from all species remained free of border cells, and overtime, pea and

cucumber root tips accumulated a sheath of border cells surrounding the root tip (Fig. 1).

A similar phenomenon was observed on roots of water hyacinth grown in ponds in

Gainesville, Florida (Hawes, unpublished results).

Border cells in semi-commercial hydroponic systems.

Plants grown in hydroponics had a higher number of root tips without border

cells, and when these roots tips were placed on water-agar conditions root caps restored

the ability to accumulate border cells (Table 3). It is important to mention that root tips

registered as “roots with border cells” were root tips with a few cells attached showing or

not showing border cell separation after immersion in water. Root tips with a normal set

of border cells were not observed. All the root tips analyzed for high and low electric
 57

conductivity conditions were free of border cells after a 10 hour period of irrigation, and

after overnight some root tips showed accumulation of a few border cells. Fluorescein

diacetate assays showed that accumulated border cells were metabolically active.

DISCUSSIONS

Our results support previous studies by Knudson (1919) and Griffin et al. (1976).

This is, in model hydroponics under laboratory conditions, the roots of important

agronomic crops (Table 2) can constantly release border cells. Interestingly, some plant

species release even more than one set of border cells daily. In addition, root caps

remained free of border cells suggesting that plant species tested release border cells

continuously, as they are produced. Moreover, the use of the vital stain fluorescein

diacetate, confirmed Knudson's (1919) observations that metabolically active border cells

are delivered into the nutrient solution of hydroponic systems.

Results from semi-commercial hydroponic systems (Tables 3 and 4), support the

observations seen in model hydroponic cultures. In commercial hydroponic systems,

plants are drip-irrigated frequently for a 10 h period during the day, with irrigation

frequencies of 3-4 times per hour. Root tips analyzed confirmed that this constant

irrigation left root tips largely free of border cells. Border cells remains on root tips

sampled after overnight without irrigation.

These results suggest that fundamental differences may exist in border cell

function in hydroponics, compared with soil ecosystems. One prediction of this

hypothesis is that due to the constant production and release of border cells, defense
 58

mechanisms of border cells are no longer functional in hydroponic systems because of

the absence of border cells surrounding the root tip leave it susceptible to pathogen

infection. Another prediction of this hypothesis is that a larger proportion of fixed carbon

would be translocated to roots, consequently increasing the rhizodeposition rate (Lynch

and Whipps, 1991). If these predictions are true, then production of border cells in

hydroponics may be deleterious to crop production.

CONCLUSION

Results confirmed that border cells are released into nutrient solution in model

hydroponic culture. A cost-benefit analysis to determine the impact of this process on

plant growth, development, and disease response throughout the growing season is

needed. Methods to control border cell production may facilitate crop improvement.
 59

REFERENCES

Arriola LL. 1997. Arbuscular mycorrhizal fungi and Trichoderma harzianum in

relation to border cell production and Fusarium root rot of Asparagus. M.S.

Thesis. Department of Botany and Plant Pathology. Michigan State University.

USA. 68 p.

Brigham LA, Woo HH, Wen F, and Hawes MC. 1998. Meristem specific

suppression of mitosis and a global switch in gene expression in the root cap of

pea by endogenous signals. Plant Physiology 118: 1223-1231.

Cook R, and Calvin L. 2005. Greenhouse tomatoes change the dynamics of the

North American Fresh Tomato Industry. Economic Research Report Number 2.

United States Department of Agriculture. Economic Research Service. 86 p.

Daughtrey ML, and Schippers PA. 1980. Root death and associated

problems. Acta Horticola 98: 283-291.

Enoch HZ, and Enoch Y. 1998. The history and geography of the greenhouse.

In: Ecosystems of the world 20. Greenhouse ecosystems. Edited by Stanhill G,

and Enoch HZ. 1-15 pp.

Farrar J, Hawes MC, Jones D, and Lindow S. 2003. How roots control the flux of carbon

to the rhizosphere. Ecology 84: 827–837.

Griffin GJ, Hale MG, and Shay FJ. 1976. Nature and quantity of sloughed

organic matter produced by roots of axenic peanuts plants. Soil Biology and

Biochemistry 8: 29-32.

Gunawardena U, and Hawes MC. 2002. Tissue specific localization of root

 60

Infection by fungal pathogens: role of border cells. Molecular Plant-Microbe

Interactions 15: 1128-1136.

Gunawardena U, VanEtten HD, and Hawes MC. 2005. Tissue specific

localization of pea (Pisum sativum L.) root infection by Nectria haematococca:

Mechanisms and consequences. Plant Physiology 137: 1363-1374.

Gunawardena U, Zhao X, and Hawes MC. 2006. Roots: Contribution to the

Rhizosphere. Encyclopedia of Life Sciences 1.

Knudson L. 1919. Viability of detached root cap cells. American Journal of

Botany 6: 309-310.

Hawes MC, and Pueppke SG. 1986. Sloughed peripheral root cap cells: Yield

from different species and callus formation from single cells. American Journal of

Botany 73(10): 1466-1473.

Hawes MC, Brigham LA, Wen F, Woo HH, and Zhu Y. 1998. Function of root

Border cells: Pioneers in the rhizosphere. Annual Review of

Phytopathology 36: 311-327

Hawes MC, Gunawardena U, Miyasaka S, and Zhao X. 2000. The role of root

border cells in plant defense. Trends in Plant Science 5: 128-133.

Hawes MC, Bengough GA, and Cassab G. 2003. Root caps and rhizosphere. Journal of

Plant Growth Regulation 21: 352-367.

Hubbard JE, Schmitt N, McClure M, Stock SP, and Hawes MC. 2005.

Characterization of root exudate induced quiescence in parasitic,

entomophathogenic, and free-living nematode species. Nematology 7: 321-331.

 61

Lynch JM, and Whipps JM. 1991. Substrate flow in the rhizosphere. In: The

rhizosphere and plant growth. Eds. D.L. Keister and P.B. Cregan. Kluwer

Academic Publishers. pp. 15-24.

Miyasaka S, and Hawes MC. 2001. Possible role of root border cells in detection

and avoidance of aluminum toxicity. Plant Physiology 125: 1978-87.

Nederhoff E. 2001. Ultraviolet treatment for water disinfection. Vegfed‟s

Greenhouse Vegetables Technical Leaflet No. 4. 4 p.

Niemira BA, Safir GR, and Hawes MC. 1996. Arbuscular mycorrhizal

colonization and border cell production: A possible correlation.

Phytopathology 86: 563-565.

Stanghellini ME, Stowell LJ, and Bates ML. 1984. Control of root rot of

spinach caused by Pythium aphanidermatum in a recirculating hydroponic system

by ultraviolet irradiation. Plant Disease 68: 1075-1076.

Stanghellini ME and Rasmussen SL. 1994. Hydroponics. A solution for

zoosporic pathogens. Plant Disease 78(12): 1129-1138.

Tiwari GN. 2003. Greenhouse technology for greenhouse environment. Ed.

Alpha Science international. Ingland. Printed in India. 544 p.

Tsai SM, and Phillips DA. 1991. Flavonoids released naturally from alfalfa

promote development of symbiotic Glomus spores in vitro. Applied and

Environmental Microbiology 57(5): 1485-1488.

Wen F, Laskowski M, and Hawes MC. 2007. Cell Separation on roots. Chapter 5.
 62

In: Plant cell separation and adhesion. Gonzalez-Carranza Z and Roberts JA

(eds.). Annual Plant Reviews 25: 91-105.

Woo HH, Hirsch AM, and Hawes MC. 2004. Altered susceptibility to infection by

Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with altered cell

cycle. Plant Cell Reports 22: 967-973.

Zhao X, Misaghi I, and Hawes MC. 2000. Stimulation of border cell production in

response to increased carbon dioxide levels. Plant Physiology 122: 1-8.

Zhao X, Schmidt M, and Hawes MC. 2000. Species-dependent effects of root

border cells on nematode chemotaxis and motility. Phytopathology

90: 1239-1245.
 63

Table 1. Plant species tested in model hydroponic systems.

Plant species
Pisum sativum L. “Little Marvel” 1
Medicago truncatula
Zea mays L. “Golden Bantam” 2
Lycopersicon esculentum L. “Mariachi RZ” 3 F1 hybrid
L. esculentum L. “DRO 83” 4 F1 hybrid rootstock
Cucumis sativus L. “Langley Hybrid” 5
Capsicum annuum L. “Crusader Hybrid” 5
Lactuca sativa L. “Black Seeded Simpson” 6
L. sativa L. “Paris White Romain” 7
1
Meyer Quality Seeds, 2 Vegetable Seed Warehouse, 3 Rijk Zwaan, 4 De Ruiter,
5
Rogers, 6 Lawn and Garden, 7 Burpee.
 64

Table 2. Border cell production from plant species in model hydroponic cultures.

Plant species # of border cells per root tip # of border cells per root tip
in Agar in water
Corn, Z. mays 3,500 900
Alfalfa, M. truncatula 2,090 1,970
Pea, P. sativum (25 °C) 4,700 2,560
Pea, P. sativum (15 °C) 5,870 2,100
Cucumber, C. sativus 1,440 4,870
Tomato, L. esculentum 360 1,010
Tomato rootstock, L. 135 250
esculentum
Lettuce, L. sativa 170 40
Pepper, C. annuum 6 165
 65

Table 3. Presence of border cells attached to the root tips of different greenhouse crops
grown in aggregate hydroponics (CEAC‟s greenhouse).

Plant Roots Roots with Roots without Roots with Roots

species sampled Bcells Bcells Bcells (ON without
(hydroponics) (hydroponics) on agar Bcells
plates) (ON on
agar
plates)
Cucumber 32 11 21 21 6
Pepper 32 10 22 16 11
Tomato 70 22 48 No tested No tested

ON = overnight; Bcells= border cells

 66

Table 4. Presence of border cells attached to root tips of tomato plants irrigated with high
and low electric conductivity (EC).

Plant Roots Roots Roots Roots Roots Roots

species sampled with without sampled with without
Bcells Bcells Bcells Bcells
(7 am) (7 am) (6 pm) (6 pm)
Tomato 7 2 5 12 0 12
(high EC)
Tomato 11 4 7 6 0 6
(low EC)

Bcells = border cells

 67

Figure Captions:

Figure 1. Normal separation of border cells. Normal pea root tip (A). Pea root tip in free

water showing normal separation of border cells (B).

Figure 2. Cucumber root tip showing a sheath of border cells formed after days of culture

in model hydroponics. Normal microscopic visualization (A). (B) Visualization using

India ink stain (B).

 68

Figure 1.

A B
 69

Figure 2.

A B
 70

APPENDIX B

PROTEINS AMONG THE POLYSACCHARIDES: A NEW PERSPECTIVE ON

ROOT CAP ‘SLIME’

Fushi Wen

Gilberto Curlango-Rivera

Martha C. Hawes

Plant Signaling and Behavior (2007), 2(5): 410-412.

71
72
73
74
 75

APPENDIX C

CONTRIBUTION OF THE ROOT CAP TO SOIL FERTILITY:

EXTRACELLULAR PLANT LECTINS

Gilberto Curlango-Rivera

Gabriela Albala

Jim P. Kemp

Denise V. Duclos

Martha C. Hawes

Soil Fertility (2009), Lucero DP and Boggs JE (eds.), Nova Science Publishers, Inc. pp.

65-79. ISBN: 978-1-60741-466-7.

76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
 92

APPENDIX D

TRANSIENT EXPOSURE OF ROOT TIPS TO PRIMARY AND SECONDARY

METABOLITES: IMPACT ON ROOT GROWTH AND PRODUCTION OF

BORDER CELLS

Gilberto Curlango-Rivera

Denise V. Duclos

Jean J. Ebolo

Martha C. Hawes

Plant and Soil (2010), 332: 267-275.

93
94
95
96
97
98
99
100
101
102
 103

APPENDIX E

ROOT TIPS MOVING THROUGH SOIL: AN INTRINSIC VULNERABILITY

Gilberto Curlango-Rivera

Martha C. Hawes

Plant Signaling and Behavior (2011), 6(5): 726-727.

104
105
106
 107

APPENDIX F

EXTRACELLULAR DNA: THE TIP OF ROOT DEFENSES?

Martha C. Hawes

Gilberto Curlango-Rivera

Fushi Wen

Gerard J. White

Hans D. VanEtten

Zhongguo Xiong

Plant Science (2011), 180: 741-745.

108
109
110
111
112
113
 114

APPENDIX G

ALTERED ROOT TIP MORPHOLOGY IN PEA HAIRY ROOTS WITH

ALTERED EXPRESSION OF A BORDER CELL SPECIFIC GENE

Fushi Wen

Gilberto Curlango-Rivera

Lindy A. Brigham

Martha C. Hawes

Draft manuscript in progress to be submitted to Annuals of Botany.

 115

Altered root tip morphology in pea hairy roots with altered expression of a border

cell specific gene.

Fushi Wen, Gilberto Curlango-Rivera1, Lindy A. Brigham, Martha C. Hawes*

Draft manuscript in progress to be submitted to Annuals of Botany.

Author affiliation:
1
Division of Plant Pathology and Microbiology, Controlled Environment Agriculture

Center. School of Plant Sciences, University of Arizona, Tucson, AZ, USA.

*
Corresponding author; Department of Soil, Water, and Environmental Science,

University of Arizona, Tucson, AZ, USA; E-mail mhawes@u.arizona.edu

 116

Background and Aims. Root tips of higher plants are dynamic sensory organs. Three

distinct tissues within the apex--the root cap, root apical meristem, and elongation zone--

control root development. Surrounding the apex in most species are populations of root

border cells programmed to detach from the root cap periphery into the external

environment. Gene expression in border cells is markedly distinct from that of the root.

Methods. mRNA differential display was used to identify border cell specific sequences.

Expression was confirmed using RNase protection assay, Southern and mRNA northern

blot analysis. Reporter gene and antisense mRNA were expressed in transgenic hairy

roots.

Key results. BRD13 was identified as a low abundance message expressed constitutively

in border cells but not in leaves, stems, or roots without border cells. The predicted

protein shares sequence similarity with flavin binding proteins. Transgenic hairy roots

expressing antisense mRNA exhibited abnormal growth and morphology. Root tip

exposure to riboflavin and other flavins had no effect on border cell production or

viability, or on direction or rate of root growth.

Conclusions. Flavin binding proteins play key roles in development, defense, and local

auxin biosynthesis. Altered expression of a border cell specific gene with a putative

flavin binding motif resulted in altered root morphology and direction of growth.
 117

INTRODUCTION

Root growth occurs by the generation of cells within the apical meristem followed by

rapid growth of the new cells within the region of elongation. A second, independently

regulated meristem generates cells of the root cap. The root cap, at the apex, controls

direction of growth in response to gravity and environmental stimuli. Root border cell

populations are programmed to separate from the root cap periphery into the external

environment. Root border cells, previously termed 'sloughed root cap cells' are

programmed to detach as a population of single cells from the root periphery into the

external environment. These populations constitute a differentiated tissue which

influences microbial colonization in a manner which appears to be parallel to that of

mammalian neutrophils (Hawes and Brigham, 1992; Hawes et al. 2011; Wen et al. 2009).

As such, genes expressed in border cells potentially provide a tool for delivery of

chemicals into the root tip region where water and nutrient uptake as well as infection by

pathogens and symbionts is initiated (Lilley et al. 2010).

The separation of border cells from the root cap is the endpoint of a coordinated set

of processes within the root cap. As cell division occurs in the root cap meristem, older

cells are displaced towards the periphery of the cap. Tiers of cells have distinct

morphological characteristics that reflect distinct functions including cell division,

gravity sensing and mucilage production. Once a cell reaches the periphery of the cap,

pectolytic enzymes degrade the middle lamella such that the cell, while still lodged in its

original position, is physically separated from the root cap and from adjacent border cells.
 118

The entire population of separated border cells is encased within a high molecular weight

mucilage consisting of DNA, proteins, polysaccharides, and diverse soluble metabolites.

The cells disperse immediately into suspension if the cap is placed in water as the

surrounding mucilage absorbs water.

Border cell populations synthesize a set of proteins whose profile is markedly

distinct from that of proteins from progenitor cells in the root cap (Brigham et al., 1995).

The change in function and morphology that occurs when border cells separate from the

cap is controlled at least in part at the level of transcription (Brigham et al., 1995). This

switch was exploited to identify a gene whose expression is specific to root border cells

and shares sequence homology with flavin binding proteins. Altered expression resulted

in morphological changes including altered direction of growth.

MATERIALS AND METHODS

Plant material and handling

Seeds of Pisum sativum L. cv. 'Little Marvel' (Royal Seed Company, Kansas City,

MO, USA) and Zea mays cv. Golden Bantam were surface sterilized and germinated as

described (Brigham et al., 1995). Border cells were isolated from the root tips of

seedlings when the radicle was 2.5 cm long. Each root tip was immersed for 30-60

seconds in 2 mL of sterile distilled water which was agitated to release the cells into

suspension (Hawes and Pueppke, 1989). Border cell preparations were assayed for
 119

microbial contamination by plating samples onto plates with solidified Luria broth (10%

tryptone (w/v), 5% yeast extract (w/v), 5% NaCl (w/v)); any samples that developed

bacterial or fungal colonies were discarded.

mRNA differential display

mRNA differential display was used to identify border cell-specific messages, by

comparing mRNA patterns of root border cells with those of cells in the root tip (Liang

and Pardee 2007). First strand cDNA was synthesized from either 100 ng of poly A+-

mRNA or 200 ng of total RNA by Superscript reverse transcriptase (Gibco-BRL Co.,

Bethesda, MD, USA). Total RNA was treated with RNase-free DNase I (Ambion Inc.,

Austin, TX, USA) to remove chromosomal DNA contamination. Poly A+-mRNA was

isolated using the PolyATtract mRNA isolation system (Promega Co., Madison, WI,

USA). First strand cDNA synthesis was primed by one of the T12MN primers (M stands

for the degenerate primer except T and N is one of the four dNTPs). A portion of this first

strand reaction was used for polymerase chain reaction (PCR) amplification using sets of

the arbitrary 10-mer primer for the 5'-end and same T12MN primer for the 3'-end. PCR

was performed with Taq DNA polymerase (exonuclease-) (Boehringer Mannheim Corp.,

Indianapolis, IN, USA) and [-35S]dATP for 40 cycles at 940C for 30 s, 400C for 2 min,

and 720C for 30 s, and 5 min extension at 720C. The amplified PCR products were size-

fractionated on a 6% denaturing PAGE gel for 4 h. After drying, the gel was exposed to

XAR-5 film. Each set of experiments was repeated three times with independent batches

of RNA samples, as described (Brigham et al. 1995). One mRNA found to be specific to
 120

border cells, designated BRD13, was subjected to detailed sequence and functional

analysis.

mRNase protection assay and Southern blot analysis

Radioactive riboprobe of BRD13 was generated by in vitro transcription of cDNA

with 32P-CTP. For RNase protection, the radioactive riboprobe was hybridized with 20 ug

of total RNA from different tissues. After RNase A/T1 treatment, the resulting

radioactive riboprobe was analyzed by a denaturing polyacrylamide gel. After drying of

the polyacrylamide gel, X-ray film was exposed for 1-24 hour. As a control to document

equal loading of total RNA in each lane, ribonuclease protection of PsUBC4 mRNA (pea

ubiquitin conjugating enzyme) (Brigham et al. 1995; Woo et al., 1994) was also

performed.

Cloning and sequencing of cDNA

Using BRD13 as a probe, a -ZAP (Strategene) cDNA library of whole root tips

(root cap and border cells) was screened. A full length cDNA was designated BRD13

and sequenced using the T7 and T3 promoter sequences of the pBlueScript vector

(Strategene). Internal primers were designed to complete the sequencing. Sequencing

was performed 3 times in each direction to verify accuracy. Sequence analysis was

performed using the Wisconsin GCG software. Southern blot analysis was performed

using the full length cDNA, as described.

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Construction of transformation vectors and transgenes

The promoterA 1244 bp fragment of BRD13 was PCR-amplified with primer 1,

5‟-ATCAGGAGCTCAGACTATGGTGACGCCATTG-3‟ containing a created Sst1 site

and primer 2, 5‟-AGCTCCCGGGTCTATGAGAGAGAATTAATTT-3” containing a

created SmaI site (position 10 and 1254 in the BRD13 sequence, respectively). This

PCR-amplified fragment was digested by SmaI and SstI simultaneously, then inserted in

antisense orientation under the control of the BRD13 promoter in vector pBI121 whose

GUS gene was removed by digestion with SmaI and SstI. The resulting constructs, or

reporter genes using GFP, were mobilized into A. rhizogenes strain R1000 through

triparental mating using pRK2013 as helper strain and kanamycin as selectable markers

(Ditta et al., 1980; Tieman et al., 1992).

Antisense mRNA expression in pea hairy roots

Constructs were transformed into pea stems using A. rhizogenes R1000

containing a kanamycin resistance gene as a selectable marker. Pea seeds were sterilized

as described at the beginning of methods. Sterilized seeds were germinated on 1% water

agar in magenta vessels at 24oC in the dark until hypocotyls reached approximately 1 cm

in length. Subsequently, seedlings were incubated at 24oC with a 16 hr light period.

Sterile stem segments (1.5-2 cm long) were transferred aseptically in an inverted position

to TM-1 solid medium (Shahin, 1985) containing 500 mg/L carbenicillin. A drop of

bacterial suspension (A600 is about 1.0) was then placed on the upper surface of the stem

section with a 10 l Pippetman. The plates were incubated at 24oC, with a 16 hr

 122

photoperiod, and 2 E m-2sec-1 light intensity. Ten to 15 days after inoculation, hairy

roots emerge from the upper surface of the inoculated stem (Nicoll et al., 1995).

One to 2 weeks after emergence, primary hairy roots were excised and cultured on

hormone free Gamborg‟s B5 medium (Sigma), pH 5.8, with 1% Difco agar, 100 mg

kanamycin, 500 mg carbenicillin, and 20 g of sucrose per L. Putative positive hairy root

clones (selected on kanamycin) were subcultured once a month on the same medium

without kanamycin. Two to four weeks after subculture, sufficient material was available

for RNA gel blot analysis. For confirmation of transformation, genomic DNA from

independent transformants was digested with BamHI and analyzed by DNA gel blotting
32
using a P-labeled CaMV 35S promoter fragment as a probe. The frequency of

transformed stems that gave rise to hairy roots was approximately 85%. Results reported

here represent ten independent transformations conducted over an 18-month period, with

dozens of replicate plate cultures and hundreds of roots.

Transient assay for root tip responses to flavins

Impact of riboflavin, lumichrome, flavin adenine dinucleotide (FAD) and flavin

adenine mononucleotide (FMN) on root growth, development, border cell production,

viability, and slime production, and susceptibility to infection were assayed as described

(Curlango-Rivera et al. 2010).

RESULTS AND DISCUSSION

 123

Border cell expressed sequences were compared to root tip expressed sequences

using mRNA differential display (Brigham et al. 1995). BRD13, consisting of 250 base

pairs of the 3‟ end of a target cDNA, was one of several sequences identified in border

cells but not the root tip. This sequence was further analyzed to verify specificity. Using

Brd13 as a probe to identify the cDNA, full length BRD13 was cloned and sequenced

(Genbank Accession Number AF139187). The cDNA of BRD13 consists of 1723

nucleotides. Southern blot analysis of pea genomic DNA revealed the presence of a

single major band (Figure 1).

The largest open reading frame (frame 3) consists of 975 nucleotides. There are two

ATG start sequences at the beginning of the sequence consistent with other pea genes

(Zhu, 2000). An unknown protein from Arabidopsis Chromosome III (Accession #

AAF00629.1) shows 70% identity within a 200 amino acid region in the middle of the

longest reading frame for the deduced protein sequence – nucleotides 144 to 743. Motifs

identified within the coding region include 3 casein kinase II (CK-2) phosphorylation

sites, 2 N-myristoylation sites, 5 protein kinase C phosphorylation sites, and a flavin

binding protein site. RNase protection was used to analyze the expression patterns.

BRD13 expression was detected only in border cells and root tips which included border

cells (Figure 2). Northern blot analysis yielded the same outcome (Figure 3). The

BRD13 promoter linked to green fluorescent protein (GFP) confirmed the pattern of

border cell specific expression (Figure 4).

Root tips of some species, including legumes, produce riboflavin which is found in

the extracellular environment (Bonner 1942, Higa et al 2010, Rovira and Campbell 1961,
 124

Susin et al. 1994, Vorweiger et al. 2007, Welkie et al. 1988). Riboflavin and its

derivatives have been implicated in root infection by Rhizobium, and in activation of

defense responses (Liu et al. 2010, Mishina and Zeier 2006, Ramamani et al. 2008, Streit

et al. 1996). Experiments were carried out to examine the possibility that a border cell

specific flavin binding protein might condition altered defense response in response to

dynamic changes in flavins (Table 1, 2). No signficant changes in response of corn or

pea roots to fungal infection were seen.

Soluble chemicals in the mucilage surrounding the root tip and border cells have

been implicated as modulators of root growth (Baluska et al. 1996). Altered riboflavin in

cultured roots has been reported to result in changes in root growth (Drew et al. 1993,

Gorst et al 1983). Moreover, flavin binding proteins increasingly are recognized as key

regulators of local auxin biosynthesis and associated growth responses (Chandler 2009,

Forneris et al. 2009, Roje 2007, White et al. 1988). Transient changes in riboflavin and

derivatives resulted in no significant alterations in corn and pea root growth, border cell

production and viability, or response to gravity (Table 3, 4). However, transformed hairy

roots expressing antisense mRNA exhibited marked phenotypic changes commonly

observed in response to auxin (Figure 5). The root tip appears enlarged with altered

morphology including direction of growth. It will be of interest in future studies to

determine if BRD13 plays a role in local auxin synthesis fostering dynamic changes in tip

growth.
 125

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Table 1. Transient efffect of riboflavin on pea infection by Nectria haematococca. Pea

seedlings inoculated at 24 h post riboflavin treatment.

Treatment Seedling

1 2 3 4 5

H2O 0 0 0 0 0

0.1 mM 0 0 0 0 0

1 mM 0 0 0 0 0

2mM 0 0 0 0 0

H2O + N 3 3 3 3 EZ 2

H2O + 0.1 2 3 3 EZ 2 EZ 2

mM

H2O + 1 3 T 3 EZ 1 EZ 2 3 3

mM

H2O + 2 3 EZ 2 3 3 EZ 2

mM

T = root tip, EZ = elongation zone, 0 = no symptoms, 1 = brownish, 2 = either T or EZ

brownish/blackish, 3 = both T and EZ brownish/blackish.

 133

Table 2. Transient efffect of riboflavin on pea infection by Nectria haematococca. Pea

seedlings inoculated at 2 h post riboflavin treatment.

Treatment Seedling

1 2 3 4 5

H2O 0 0 0 0 0

0.1 mM 0 0 0 0 0

1 mM 0 0 0 0 0

2mM 0 0 0 0 0

H2O + N 3 T 2 EZ 1 3 3

H2O + 0.1 mM EZ 2 T 1 EZ 1 3 EZ 2

H2O + 1 mM EZ 2 3 3 3

H2O + 2 mM T2 T2 T2

T = root tip, EZ = elongation zone, 0 = no symptoms, 1 = brownish, 2 = either T or EZ

brownish/blackish, 3 = both T and EZ brownish/blackish.

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Table 3. Effect of flavins on root length (RL), viability (V) and border cell (BC) number

of pea at 24 h.

Trial Treatment n RL (mm) A V% BC # Gravity

± SD test
1 H2O 3 9.3 ± 0.6 85 5633
0.1 mM 3 11.7 ± 4.0 86 7333
Riboflavin
1 mM 3 12.3 ± 2.9 84 6100
Riboflavin

2 H2O (8 h) 1 1940
0.1 mM 2 2450
Riboflavin
(8 h)
1 mM 3 1870
Riboflavin
(8 h)
H2O (24 h) 6 4690 ± 1117
0.1 mM 7 3890 ± 495
Riboflavin
(24h)
1 mM 8 3530 ± 976
Riboflavin
(24 h)

3 H2O 10 8.1 ± 4.9 90 ± 1.4 5870 ± 1853

0.1 mM 10 9.9 ± 3.4 88 ± 4.2 5440 ± 792
Riboflavin
1 mM 10 13.1 ± 4.2 89 ± 2.1 4640 ± 1075
Riboflavin

4 H2O 10 11.5 ± 2.8 88 ± 1.4 5220 ± 198

5 nM 10 14.1 ± 4.0 88 ± 4.2 4980 ± 85
Lumichrome
1 μM 10 13.5 ± 4.2 82.5 ± 2.1 5800 ± 226
Lumichrome
H2O (48 h) 10 37.6 ± 5.2
5 nM 10 44.6 ± 5.1
Lumichrome
(48 h)
 135

Table 3. Continued.

Trial Treatment n RL (mm) A V% BC # Gravity

± SD test
5 H2O 4 11.5 ± 3.5 64 4325
1 μM FAD 4 14.3 ± 1.5 57 3200
100 μM FAD 4 9.3 ± 1.5 76 4175

6 H2O 4 13.5 ± 1.7 39 5025 +

1 mM FAD 4 13.5 ± 7.4 51 5800 +
10 mM FAD 4 11.0 ± 3.6 64 6050 +

7 H2O 4 11.5 ± 3.5 64 4325

1 μM FMN 4 10.0 ± 2.2 64 3900
100 μM FMN 4 12.0 ± 3.7 67 4750

8 H2O 4 13.5 ± 1.7 39 5025 +

1 mM FMN 4 14.0 ± 3.9 49 5000 +
10 mM FMN 4 13.8 ± 6.8 75 5000 +

Data show the average ± standard deviation (Avg ± SD) from at least two replicates;

other data not showing standar deviation are from a single observation.
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Table 4. Effect of flavins on root length (RL), viability (V) and border cell (BC) number

of corn at 24 h.

Trial Treatment n RL (mm) V% BC # Gravity test

1 H2O 4 13.3 ± 19 97 1325 +

1 μM FAD 4 21.0 ± 6.8 100 1150 +

100 μM FAD 4 16.5 ± 2.4 93 1300 +

2 H2O 4 20.3 ± 4.2 100 850 +

1 μM FMN 4 21.0 ± 4.1 100 1800 +

100 μM FMN 4 19.0 ± 1.6 100 1850 +

3 H2O 4 13.5 ± 1.3 100 1150 +

100 μM Riboflavin 4 15.3 ± 3.2 100 975 +

1 mM Riboflavin 4 12.8 ± 4.0 100 925 +

Data show the average ± standard deviation from four replicates; other data not showing

standard deviation are from a single observation.

 137

Figure captions:

Figure 1. Southern blot analysis.

Figure 2. RNase protection assay.

Figure 3. mRNA northern.

Figure 4. Border cell specific expression of GFP under the control of the BRD13

promoter.

Figure 5. Morphological changes in transgenic hairy roots expressing BRD13 antisense

mRNA under the control of the BRD13 promoter.

 138

Figure 1.
 139

Figure 2.
RNase Protection of BRD13

1 2 3 4 5 6 7
1 = Leaves
2 = Stems
3 = Roots
4 = Roots without root tips
5 = Border cells
6 = Internal control
7 = Undigested Probe ( ) and Internal control
, Indicates the protected probes
, Internal control
 140

Figure 3.
 141

Figure 4.
 142

Figure 5.

A B

C D