CONTENTS:

 Introduction

 The competence of an abscission zone to abscise is dependent upon regulatory events in multiple plant tissues

 Model systems for stress ethylene-mediated cell

Regulation of ethylene biosynthesis during environmental abscission

We have cloned multiple ethylene biosyntheticenzymes and receptors from abscising abscission zones.

ACC oxidase gene expression is differentially regulated in abscission zones and flanking tissues during water stress-mediated abscission.

Preliminary results demonstrate a significant role for ACC oxidase-mediated ethylene biosynthesis in the regulation of water stress-associated abscission.

An understanding of how ACC oxidase-mediated ethylene synthesis mediates organ abscission requires methods that will reduce ethylene production in tissues distal to abscission zones.

Acknowledgments

Selected publications

Ethylene regulates numerous plant developmental processes, including vegetative growth, flowering, senescence and abscission.  Ethylene is also an integral component of the mechanisms by which plants sense and respond to their surroundings.  The synthesis of this hormone in response to such diverse stresses as flooding, drought and dim light illustrates the central role played by ethylene in coordinating plant responses to environmental stress.  Abscission and dehiscence (‘shatter’) are two cell separation processes regulated by biotic and abiotic cues. My research integrates molecular, physiological and genomic approaches to the study of plant mechanisms that link water deficit signals with cell separation responses. Although the current emphasis in the lab is on the role of ethylene in coordinating stress responses involving abscission, future studies will address common and unique regulatory mechanisms involved in abscission and dehiscence.

The competence of an abscission zone to abscise is dependent upon regulatory events in multiple plant tissues
Molecular approaches to abscission and its regulation have generally focused on cell separation determinants operating in the abscission zone.  These include cell wall hydrolases and, more recently, ethylene receptors (Lashbrook et al. 1994; 1998a; 1998b and see published work from the groups of Alan Bennett, Elena del Campillo, Jerry Roberts, Roy Sexton, Mark Tucker, and Don Grierson).  Nevertheless, a great body of information is available showing organ detachment to be regulated by processes occurring in tissues outside the abscission zone.  Many of these studies were conducted in cotton, where Elmo Beyer, Page Morgan and others have demonstrated that initial changes in ethylene biosynthesis and perception in response to a abscission stimulus occur in tissues distal to the future site of cell separation, e.g. leaf blades attached to petioles destined to abscise.

Thus, our studies of cell separation and its hormonal control consider molecular mechanisms operating within a whole plant context.  From this broader perspective, abscission is considered to be a process initiated with the receipt of an abscission signal, propagated with the transduction of that signal to the abscission zone and culminated by organ detachment.  What role does ethylene biosynthesis and perception play in each step of this process leading to organ detachment, and in what tissues?

Model systems for stress ethylene-mediated cell separation
Certain crops are prone to premature abscission and dehiscence, partially because of their extreme sensitivity to environmental fluctuations.  These species, which include cotton and soybeans, represent excellent model systems for understanding not only how cell separation is regulated by stress-associated ethylene but also fundamental aspects of how plants respond to their environment. The economic importance of the two crops listed above underscores their value as potential targets for genetic improvement using transgenic technologies.

For the past five years, my work has focused on the regulation of cotton abscission in response to drought stress. A wealth of physiological information on the regulation of cell separation in this species from the Morgan group prompted its use as one of our models for environmental abscission. Cotton exhibits extreme sensitivity to environmental change.  High rates of shedding occur in buds, flowers, bolls and leaves in response to fluctuations in ambient temperature, water availability, nutritional status and insect pressure.  Detachment of cotton organs in response to these diverse cues is attended by significant changes in ethylene synthesis and perception.  Early abscission work in cotton has provided a strong physiological foundation on which we have initiated studies aimed to establish the molecular genetic mechanisms by which ethylene may promote and coordinate organ shedding in response to stress.

The use of cotton as a physiological system complements recent applications of classical genetic strategies to cell separation in Arabidopsis.  Mutants impaired in various aspects of flower petal abscission and silique dehiscence have been identified in Arabidopsis (e.g. see published work of Tony Bleecker, Sara Patterson and Marty Yanofsky).  The identification of the genes harboring these mutations promises to reveal novel determinants of abscission, including some whose regulation lies outside of ethylene and/or auxin control.  Although the rapid, annual life cycle of Arabidopsis obviates its use in some of our physiological and biochemical studies on the role of ethylene in promoting long-term adaptive responses to environmental stress, we are exploiting the genetic strengths of Arabidopsis in other aspects of our work as discussed below.

Regulation of ethylene biosynthesis during environmental abscission

We have cloned multiple ethylene biosynthetic enzymes and receptors from abscising abscission zones.  Ethylene biosynthesis is dependent upon the sequential action of ACC synthase and ACC oxidase. The perception of synthesized ethylene by one or more ethylene receptors initiates a cascade of events leading to an appropriate physiological response.   Using heterologous probes derived from Arabidopsis and other species, multiple ACC synthases (ACSs), ACC oxidases (ACOs) and ethylene receptors (ETR1 homologs) were cloned from cotton abscission zones abscising in response to ethylene (Lashbrook and Klee, 2001).

ACC oxidase gene expression is differentially regulated in abscission zones and flanking tissues during water stress-mediated abscission. Our environmental abscission model system is based upon the abscission of water stressed leaves and buds from young cotton plants.  Water is withheld from plants bearing up to a dozen main leaves and multiple closed flower buds until the plants exhibit a “full-wilt” value of ~10-12% plant available water (% PAW).  % PAW is a measure of the water available to a plant for transpiration.   At full wilt, leaf water potential (y), a secondary index of stress magnitude, approaches -4 mPa.  At this stage, plants are watered to run-off and enter the rehydration phase: the stress phase associated with abscission.  Following the attainment of full turgor in wild-type plants (where y values approach 0), the oldest three leaves abscise rapidly, with younger leaves sequentially abscising over the next 2-4 days.

Preliminary data show that all four Gh-ACOs are expressed in leaf abscission zones abscising in response to relief of water stress, but significant up-regulation of ACC oxidase gene expression in abscission zones is restricted to a subset of Gh-ACO genes.  In petiole tissues distal to leaf abscission zones, a more complex expression pattern is observed.   Stimulation of specific Gh-ACO mRNA accumulation in petioles during the wilting phase of imposed stress is followed by significant induction of a different complement of Gh-ACOs during the rehydration phase associated with abscission.  In our studies, we wish to consider that organ detachment due to water stress may be dependent not only upon events occurring in abscission zones but also upon these earlier changes in ethylene synthesis occurring in flanking tissues.

Preliminary results demonstrate a significant role for ACC oxidase-mediated ethylene biosynthesis in the regulation of water stress-associated abscission.  A transgenic approach has been taken to determine the functional significance of Gh-ACO gene expression in and around cotton abscission zones. Gh-ACO4 was placed in its antisense orientation under the control of the constitutive FMV promoter and used to transform cotton via Agrobacterium-mediated transfection (Lashbrook, Rangwala, Perlak and Klee; unpublished).  Transgenic expression inhibited ethylene synthesis by suppressing Gh-ACO4 sense transcript accumulation.  Relative to controls, ethylene production was suppressed by up to ~98% in most water-stressed transgenic tissues including shoots, leaves and petioles.  Technical constraints prevented measurement of ethylene production in the few cell layers comprising leaf abscission zones.  Thus, in order to estimate ethylene “seen” by these zones we measured hormone produced by tissue of the adjacent pulvinus, which was also greatly reduced relative to controls.  Pulvini are swollen pads of specialized tissue that lie at the base of petioles and leaf blades of species including cotton and bean, where they may play a role in regulating leaf movements.

Correlated with the significant reduction in ethylene production by transgenic plants is a dramatic decrease in leaf abscission rates of plants subjected to severe wilt and rehydration. For example, transgenic leaf abscission incidence in fruiting cotton was reduced by as much as two-thirds relative to control plants (Lashbrook, Rangwala, Perlak and Klee; unpublished).  Interestingly, reduction of leaf abscission in anti-Gh-ACO4 plants following relief of water deficit is not accompanied by decreases in rates of floral bud abscission. These data raise the interesting possibility that mechanistically distinct hormone regulation pathways may operate in abscission tissues of leaves and buds.
It should be noted, though, that ethylene production in peduncle tissue bordering reproductive organ abscission zones is incompletely suppressed from especially high initial levels in transgenic plants. Whatever the explanation for maintenance of physiologically active ethylene levels in floral bud peduncles of at least some transgenic Gh-ACO4 plants, it is clear that an analysis of the role of ethylene biosynthesis in reproductive organ abscission will require methods that specifically reduce biosynthesis in tissues that border abscission zones.

An understanding of how ACC oxidase-mediated ethylene synthesis mediates organ abscission requires methods that will reduce ethylene production in tissues distal to abscission zones.  An analysis of the role of ACC oxidase-mediated ethylene biosynthesis in mediating organ abscission will require methods that lead to the reduction of biosynthesis not only in abscission zones but in tissues distal to abscission zones. One strategy to accomplish this goal is to identify tissue-specific promoters capable of driving transgene expression in these domains.  To identify prospective genes for future promoter isolation, we have generated an ordered array of ~4000 cDNAs from an unamplified cDNA library of cotton flowers abscising in response to ethylene treatment (Lashbrook and Klee, unpublished).   A portion of this library was provided to Rod Wing at Clemson University, who has prepared additional arrays.  High throughput sequencing of arrayed clones has identified  ~3300 expressed sequence tags (ESTs) thus far: 2000 from our effort (Lashbrook, Rangwala, Perlak and Klee; unpublished) and ~1300 from the Wing group published on GenBank.

Promoters cloned from two pathogenesis-related (PR) proteins that were highly represented in our cotton abscission zone EST collection were used to construct promoter-GUS fusions for transforming Arabidopsis.   GUS staining of transgenic Arabidopsis expressing one such cotton PR gene promoter-GUS construct was restricted to flower petal abscission zones, confirming that proper targeting of heterologous promoter expression had occurred (Shrode, Lashbrook and Klee, unpublished).  Cloning of potential tissue-specific promoters from cotton followed by testing of expression specificity in Arabidopsis is a potentially expedient way to evaluate means of modulating ethylene biosynthesis or action specifically within abscission zones.  We are currently initiating similar strategies for identifying promoters useful for targeting reductions in ethylene biosynthesis to tissues bordering leaf and bud abscission zones

ACKNOWLEDGEMENTS
The work described herein was developed during postdoctoral work with Harry Klee at the University of Florida and is being continued at Iowa State. Our Monsanto colleagues Fred Perlak and Tasneem Rangwala have been instrumental in the generation of the transgenic plants discussed here.  We acknowledge the financial support of the USDA Plant Responses to the Environment Panel (USDA-NRICGP Postdoctoral Fellowship 97-35100-4192 to CC Lashbrook) and support from the Monsanto Co. to Harry Klee.

SELECTED PUBLICATIONS
Lashbrook, CC and Klee, HJ.  2001.  Differential regulation of the cotton ACC oxidase gene family during drought stress and abscission. (manuscript submitted)

Lashbrook, CC and Klee, HJ.  1999.  Ethylene Regulation of Abscission Competence.  In: Biology and Biotechnology of the Plant Hormone Ethylene II. (Kanellis, AK., Bleecker, AB and Klee, eds.).  pp 227-233.  Dordrecht, The Netherlands: Kluwer Academic Press

Lashbrook, CC, Tieman, DM and Klee, HJ. 1998a.  Differential regulation of the tomato ETR gene family throughout tomato development. Plant Journal 15: 243-252

Lashbrook, CC, Giovannoni, JJ, Hall, BD, Fischer, RL and Bennett, AB. 1998b.  Transgenic analysis of tomato endo-b-1,4-glucanase gene function. Role of cel1 in floral abscission.  Plant Journal 13: 303-310

Brummell, DA, Catala, C, Lashbrook, CC and Bennett, AB. 1997. A membrane-anchored E-type endo-1, 4-b-glucanase is localized on Golgi and plasma membranes of higher plants.  Proc Nat Acad Sci USA 94: 4794-4799

Rose, JKC, Catala, C, Brummell, DA, Lashbrook, CC, Gonzalez-Bosch, C and AB Bennett. 1997.  The tomato endo-b-1,4-glucanase gene family: Regulation by both ethylene and auxin.  In: Biology and Biotechnology of the Plant Hormone Ethylene (AK Kanellis, C Chang, H Kende, D Grierson, eds). pp 197-205.  Mass: Kluwer Academic Publishers

Brummell, DA, CC Lashbrook and AB Bennett. 1994.  Plant endo-1, 4-b-D-glucanases: Structure, properties and physiological function. Am Chem Soc Symp Ser 566: 100-129

Lashbrook, CC, C Gonzalez-Bosch and AB Bennett. 1994.  Two divergent endo-b-1,4-glucanase genes exhibit overlapping expression in ripening fruit and abscising flowers. Plant Cell 6: 1485-1493

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