CEPCEB Members

Linda L. Walling Professor of Genetics Associate Dean of Biological Sciences, College of Natural and Agricultural Sciences Department of Botany and Plant Sciences 3207 Batchelor Hall University of California Riverside, CA 92521 Phone: (951) 827-4687 FAX: (951) 827-4437 Areas of Expertise Plant/pest Interactions Defense Mechanisms Aminopeptidases and Transferases That Modify the N-terminus of Proteins

Background Role of Aminopeptidases in Defense and Development Selected Publications Related to Leucine Aminopeptidase (Bibliography page) Mechanisms of Herbivore Perception in Plants Selected Publications Related to Phloem-feeding Insects (Bibliography page) Selected Collaborative Publications (Bibliography page) Current Laboratory Personnel NSF 2010: Functional Genomics of N-terminal Processing Enzymes

Background

I was trained as an Escherichia coli bacteriophage geneticist and received by Ph.D. from the Department of Microbiology at the University of Rochester Medical School in Rochester, New York in 1980. My first postdoctoral fellowship was performed under the guidance of Dr. James Darnell (Rockefeller University) where I studied mechanisms of gene expression in mouse liver. My entry into the plant world was initiated with my second postdoctoral fellowship with Dr. Robert Goldberg (UCLA). Under his mentorship, I investigated transcriptional and post-transcriptional control of seed protein gene expression in soybeans.

In 1984, I joined the Department of Botany and Plant Sciences at UC Riverside as an Assistant Professor of Genetics and progressed through the ranks to Full Professor. Initially, my laboratory studied the interactions of developmental and light regulatory signals in the regulation of the chlorophyll a/b binding protein genes of soybean. In 1990, my laboratory's emphasis shifted dramatically to focus on understanding plant responses to wounding, pathogens, and herbivores. Two research projects dominate our current research initiatives. First, we are dissecting the mechanisms used to perceive phloem-feeding whiteflies in squash, tomato and Arabidopsis. Second, we identified a peptidase (leucine aminopeptidase) that responds to bacterial pathogens, wounding and tissue-damaging herbivores. This enzyme has led us into studies to understand the role of N-terminal processing enzymes during development and in response to stress. We utilize multidisciplinary approaches in both projects by incorporating the tools of biochemistry, genetics, cell biology, and genomics.

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Role of Aminopeptidases in Defense and Development

Co- and posttranslational modifications of eukaryotic proteins, which may number as many as two hundred separate types of reactions, can occur throughout the polypeptide chain and are essential for a variety of functions, such as translocation, activation, regulation, and, ultimately, degradation. Indeed, these modifications are sufficiently extensive that it can be predicted that essentially all proteins are modified at least once during their lifetime. The N-terminal region is a particularly active area for such alterations and the earliest known modifications are those that occur at or near the N-terminus. In eukaryotes, three types of reactions predominate: 1) limited proteolysis to remove one or more amino acids; 2) modification of the a-amino group; and 3) side chain-specific changes. My laboratory is particularly interested in the limited proteolysis that is executed by aminopeptidases.
The wide variety of aminopeptidases that are found in eukaryotes act primarily on mature proteins (or peptides derived there from) (Walling and Gu, 1996). Their roles are varied and ill defined and virtually unstudied in plants. Many aminopeptidases are certainly involved in protein turnover acting post-proteasomally to complete the conversion of the peptides released by the 26S proteasome to free amino acids. Other aminopeptidases are likely involved in activation/deactivation processes, particularly of bioactive peptides. Given the rising number of bioactive peptides in plants that control plant developmental and stress responses, the role of aminopeptidases in biogenesis or catabolism of critical regulatory molecules is likely. Finally, aminopeptidases may have important roles in dictating half-lives of proteins within cellular compartments.
We were drawn into this field by the identification of a tomato leucine aminopeptidase gene that is induced by Pseudomonas syringae pv. tomato (Pautot et al., 1993). Our studies have shown that in tomato there are two classes of LAP proteins (Gu et al., 1996). The constitutive LapN expressed ubiquitously being detected in all plants examined to date. LAP-N is present in all organs and is not response to hormone treatments or stress signals. In contrast, LapA RNAs, proteins and activities are limited to a subset of the Solanaceae (Chao et al., 2000) and are expressed in fruit and flowers (Fig. 1).

Figure 1. Panel A: GUS activity in flowers of transgenic tomatoes expressing the LapA:GUS gene. Panel B: GUS activity in LapA:GUS plants after JA treatments. GUS activity in untreated LapA:GUS, 35S:GUS plants and non-transformed control plants. Panel C:Accumulation of the 55-kD LAP-A (Blue arrow), 55-kD LAP-N and LAP-like proteins (66-kD and 77-kD) in response to wound and abiotic stress signals.

LapA genes are expressed in foliage only after being challenged with biotic or abiotic stress stimuli including, P.s. tomato, chewing insects (Manduca sexta or Spodoptera littoralis), mechanical wounding, and water-deficit and salinity stress, but not heat shock (Chao et al., 1999; Gu et al., 1999). Treatments with defense signals indicate that LapA genes are regulated by the wound-induced octadecanoid pathway (Chao et al., 1999). Analysis of transgenic tomato plants expressing an antisense LapA construct indicates that LAP-A may have a role in modulating some aspect of the wound response. 35S:asLapA plants have an impaired wound response because proteinase inhibitor 2 RNA levels are diminished relative to control plants after wounding. Surprisingly, there was no substantial impairment of the growth of P.s. tomato or M. sexta (Pautot et al., 2001) on these plants. However, the presence of trace amounts of this enzyme in the 35S:asLapA plants makes these data difficult to interpret. Analysis of LapA:asLapA and silenced plants, which more tightly down-regulate LapA expression, are being analyzed currently. Transgenic tomatoes that ectopically express LAP-A are also being investigated. These reverse genetic approaches are likely to elucidate the roles of LAP in both abiotic and biotic stress. The role of LAP-A and LAP-N in the N-end rule pathway for protein turnover is being tested using novel transgenic tomato plants expressing chimeric reporter genes.

To date, LAP-A is the best biochemically characterized aminopeptidase in plants. This homohexameric enzyme has a high temperature and pH optima (Gu et al., 1999). Based on the analysis of over 30 chromogenic substrates and 68 peptide substrates LAP-A preferentially cleaves substrates with N-terminal (P1) Leu, Met, Arg and Ala (Gu et al., 1999; Gu and Walling, 2000). It will not cleave substrates with acidic residues in the P1 or P1' position. We have recently shown by site directed mutagenesis that the plant LAP-A is likely to utilize a reaction mechanism similar to that used by the bovine LAP and E. coli PepA (Gu and Walling, 2001). A crystal structure for the tomato LAP-A is being determined.

Despite the fact that over 50 aminopeptidases have been biochemically purified from plants (Walling and Gu, 1996), the Lap genes of tomato are the only well characterized aminopeptidases in plants. Given the availability of the complete Arabidopsis genome, we are now able to assess the minimal complement of aminopeptidases present in plants. To data we have identified 12 different classes of aminopeptidases. There is a curious paucity of membrane-associated aminopeptidases relative to animals and yeast. Aminopeptidases with putative locations in the cytosol, mitochondrion, and plastid have been identified. GFP-aminopeptidase fusions will be assessed to determine their subcellular localization. To date we have identified T-DNA knock-outs for 25% of these genes. The analysis of these mutants and others will enable us to assess the role of each aminopeptidase gene in development and stress. Of particular importance is the functional redundancy of gene members within each aminopeptidase class and if divergent aminopeptidase families are functionally redundant. This integrated biochemical, cellular and genomic approach to peptidases in a higher eukaryote is unprecedented.

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Selected Publications Related to Leucine Aminopeptidase (Bibliography page)

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Mechanisms of Herbivore Perception in Plants

Defense signaling is complex and involves the intertwining of numerous signaling pathways. While these signaling networks are being dissected for plant pathogens, relatively little is known about the nature of the signaling networks important for plant defense against phloem-feeding insects (Walling, 2000). We have examined plant responses to phloem-feeding whiteflies in tomato, squash and Arabidopsis. These projects were initiated because the silverleaf whitefly causes several novel developmental disorders in plants including irregular ripening in tomato, stem and leaf blanching in Arabidopsis, and leaf silvering in squash (Fig 2).
Using tomato-whitefly infestations, we were able to determine that plants perceive tissue-damaging and phloem-feeding herbivores distinctly. Two species of phloem-feeding whiteflies activate pathogenesis-related protein gene expression and do not activate the wound octadecanoid pathway. JA-responsive defense signaling pathways were preferentially activated by whiteflies (Puthoff et al, in revision). Defense responses were activated both locally and systemically.

Our studies with silverleaf whitefly (Bemisia argentifolii) and sweetpotato whitefly (B. tabaci Type A) indicate that there are profound changes in local and systemic gene expression in response to phloem-feeding whiteflies. Leaf silvering develops in all leaves that develop after the silverleaf whitefly nymphs initiate feeding. Leaf silvering is not induced by silverleaf whitefly adults or by sweetpotato whitefly adults or nymphs. For this reason, we searched for genes that were preferentially expressed in apical non-infested leaves after silverleaf whitefly infestation and not after sweetpotato whitefly infestation (van de Ven et al., 2000). Two novel genes (SLW1 and SLW3) were identified that were not previously associated with plant defense responses and careful temporal and spatial expression studies do not provide evidence that indicates either is strictly correlated with silvering. SLW1 is a M20b peptidase that is preferentially induced locally and systemically by the silverleaf whitefly, water-deficit stress, and JA. SLW3 encodes a b-glucosidase that is preferentially expressed systemically in response to the silverleaf whitefly. Local expression in infested leaves is similar in response to both whitefly species. SLW3 is regulated by a novel signaling pathway since neither JA, ABA, SA, reactive oxygen species (NO or H2O2) nor ethylene cause SLW3 RNAs to accumulate (van de Ven et al., 2000).
Furthermore, SLW3 RNAs do not accumulate in response to wounding, the cotton aphid, nematodes or pathogens. We are using SLW3:GUS transgenic tomatoes and Arabidopsis to identify the components of the whitefly saliva that activate SLW3 gene expression locally and systemically. Genetic and biochemical approaches to the role of SLW1 and SLW3 proteins in defense are being pursued. The recent success in over-expressing SLW1 and SLW3 in yeast will allow the substrate specificity for each enzyme to be assessed.

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Selected Publications Related to Phloem-feeding Insects (Bibliography page)

Selected Collaborative Publications (Bibliography page)

Current Laboratory Personnel

• Fran Holzer, staff research associate
• Sang-Youl Park, postdoc
• Virginia Alonzo, graduate student
• Mikiko Matsui, graduate student
• Sonia Zarate, graduate student
• Angel Chen, graduate student
• Jon Fowler, graduate student
• Lorem Que, graduate student
  
  

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