Maarten J. Chrispeels

Lectins, lectin genes, and their role in plant defense.

Lectins are carbohydrate-binding proteins that bind gly? cans of glycoproteins, glycolipids, or polysaccharides with high affinity (Goldstein and Hayes, 1978). Because of their binding specificity, they have the capability to serve as recognition molecules within a cell, between cells, or be? tween organisms. It is assumed that lectins play fundamental biological roles in plants because they are found in many different species and in many different organs and tissues.

Water Transport Activity of the Plasma Membrane Aquaporin PM28A Is Regulated by Phosphorylation

PM28A is a major intrinsic protein of the spinach leaf plasma membrane and the major phosphoprotein. Phosphorylation of PM28A is dependent in vivo on the apoplastic water potential and in vitro on submicromolar concentrations of Ca2+. Here, we demonstrate that PM28A is an aquaporin and that its water channel activity is regulated by phosphorylation. Wild-type and mutant forms of PM28A, in which putative phosphorylation sites had been knocked out, were expressed in Xenopus oocytes, and the resulting increase in osmotic water permeability was measured in the presence or absence of an inhibitor of protein kinases (K252a) or of an inhibitor of protein phosphatases (okadaic acid). The results indicate that the water channel activity of PM28A is regulated by phosphorylation of two serine residues, Ser-115 in the first cytoplasmic loop and Ser-274 in the C-terminal region. Labeling of spinach leaves with 32P-orthophosphate and subsequent sequencing of PM28A-derived peptides demonstrated that Ser-274 is phosphorylated in vivo, whereas phosphorylation of Ser-115, a residue conserved among all plant plasma membrane aquaporins, could not be demonstrated. This identifies Ser-274 of PM28A as the amino acid residue being phosphorylated in vivo in response to increasing apoplastic water potential and dephosphorylated in response to decreasing water potential. Taken together, our results suggest an active role for PM28A in maintaining cellular water balance.

The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes.

The vacuolar membrane (tonoplast) of higher plant cells contains an abundant 27 kDa protein called TIP (tonoplast intrinsic protein) that occurs in different isoforms and belongs to a large family of homologous channel‐like proteins found in bacteria, plants and animals. In the present study, we identified and characterized the function of gamma‐TIP from Arabidopsis thaliana by expression of the protein in Xenopus oocytes. gamma‐TIP increased the osmotic water permeability of oocytes 6‐ to 8‐fold, to values in the range 1–1.5 × 10(−2) cm/s. Similar results were obtained with the homologous human erythrocyte protein CHIP28, recently identified as the erythrocyte water channel. The bacterial homolog GlpF did not affect the osmotic water permeability of oocytes, but facilitated glycerol uptake, in accordance with its known function. By contrast, gamma‐TIP did not promote glycerol permeability. Voltage clamp experiments provided evidence showing that gamma‐TIP induced no electrogenic ion transport in oocytes, especially during osmotic challenge that resulted in massive transport of water. These results allow us to conclude that the various protein members of the MIP family have unique and specific transport functions and that the plant protein gamma‐TIP likely functions as a water specific channel in the vacuolar membrane.

Genomic Analysis of the Unfolded Protein Response in Arabidopsis Shows Its Connection to Important Cellular Processes

We analyzed the breadth of the unfolded protein response (UPR) in Arabidopsis using gene expression analysis with Affymetrix GeneChips. With tunicamycin and DTT as endoplasmic reticulum (ER) stress–inducing agents, we identified sets of UPR genes that were induced or repressed by both stresses. The proteins encoded by most of the upregulated genes function as part of the secretory system and comprise chaperones, vesicle transport proteins, and ER-associated degradation proteins. Most of the downregulated genes encode extracellular proteins. Therefore, the UPR may constitute a triple effort by the cell: to improve protein folding and transport, to degrade unwanted proteins, and to allow fewer secretory proteins to enter the ER. No single consensus response element was found in the promoters of the 53 UPR upregulated genes, but half of the genes contained response elements also found in mammalian UPR regulated genes. These elements are enriched from 4.5- to 15-fold in this upregulated gene set.

cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection.

We isolated a full-length cDNA for apoplastic (extracellular or cell wall-bound) beta-fructosidase (invertase), determined its nucleotide sequence, and used it as a probe to measure changes in mRNA as a result of wounding of carrot storage roots and infection of carrot plants with the bacterial pathogen Erwinia carotovora. The derived amino acid sequence of extracellular beta-fructosidase shows that it is a basic protein (pl 9.9) with a signal sequence for entry into the endoplasmic reticulum and a propeptide at the N terminus that is not present in the mature protein. Amino acid sequence comparison with yeast and bacterial invertases shows that the overall homology is only about 28%, but that there are short conserved motifs, one of which is at the active site. Maturing carrot storage roots contain barely detectable levels of mRNA for extracellular beta-fructosidase and these levels rise slowly but dramatically after wounding with maximal expression after 12 hours. Infection of roots and leaves of carrot plants with E. carotovora results in a very fast increase in the mRNA levels with maximal expression after 1 hour. These results indicate that apoplastic beta-fructosidase is probably a new and hitherto unrecognized pathogenesis-related protein [Van Loon, L.C. (1985). Plant Mol. Biol. 4, 111-116]. Suspension-cultured carrot cells contain high levels of mRNA for extracellular beta-fructosidase and these levels remain the same whether the cells are grown on sucrose, glucose, or fructose.

Plasma Membrane Aquaporins Play a Significant Role during Recovery from Water Deficit

The role of plasma membrane aquaporins (PIPs) in water relations of Arabidopsis was studied by examining plants with reduced expression of PIP1 and PIP2 aquaporins, produced by crossing two different antisense lines. Compared with controls, the double antisense (dAS) plants had reduced amounts of PIP1 and PIP2 aquaporins, and the osmotic hydraulic conductivity of isolated root and leaf protoplasts was reduced 5- to 30-fold. The dAS plants had a 3-fold decrease in the root hydraulic conductivity expressed on a root dry mass basis, but a compensating 2.5-fold increase in the root to leaf dry mass ratio. The leaf hydraulic conductance expressed on a leaf area basis was similar for the dAS compared with the control plants. As a result, the hydraulic conductance of the whole plant was unchanged. Under sufficient and under water-deficient conditions, stomatal conductance, transpiration rate, plant hydraulic conductance, leaf water potential, osmotic pressure, and turgor pressure were similar for the dAS compared with the control plants. However, after 4 d of rewatering following 8 d of drying, the control plants recovered their hydraulic conductance and their transpiration rates faster than the dAS plants. Moreover, after rewatering, the leaf water potential was significantly higher for the control than for the dAS plants. From these results, we conclude that the PIPs play an important role in the recovery of Arabidopsis from the water-deficient condition.

Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

The vacuolar membrane protein alpha‐TIP is a seed‐specific protein of the Major Intrinsic Protein family. Expression of alpha‐TIP in Xenopus oocytes conferred a 4‐ to 8‐fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha‐TIP forms water channels and is thus a new aquaporin. alpha‐TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha‐TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha‐TIP in oocytes, suggesting that phosphorylation of alpha‐TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8‐bromoadenosine 3′,5′‐cyclic monophosphate, forskolin and 3‐isobutyl‐1‐methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha‐TIP by 80‐100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha‐TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP‐dependent increase in the Pf of oocytes expressing alpha‐TIP, as well as for in vitro phosphorylation of alpha‐TIP. These findings demonstrate that the alpha‐TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)

The Plasma Membrane of Arabidopsis thaliana Contains a Mercury-Insensitive Aquaporin That Is a Homolog of the Tonoplast Water Channel Protein TIP

Plant cells contain proteins that are members of the major intrinsic protein (MIP) family, an ancient family of membrane channel proteins characterized by six membrane-spanning domains and two asparagine-proline-alanine (NPA) amino acid motifs in the two halves of the protein. We recently demonstrated that [gamma]-TIP, one of the MIP homologs found in the vacuolar membrane of plant cells, is an aquaporin or water channel protein (C. Maurel, J. Reizer, J.I. Schroeder, M.J. Chrispeels [1993] EMBO J 12: 2241–2247). RD28, another MIP homolog in Arabidopsis thaliana, was first identified as being encoded by a turgor-responsive transcript. To find out if RD28 is a water channel protein, rd28 cRNA was injected into Xenopus laevis oocytes. Expression of RD28 caused a 10- to 15-fold increase in the osmotic water permeability of the oocytes, indicating that the protein creates water channels in the plasma membrane of the oocytes and is an aquaporin just like its homolog [gamma]-TIP. Although RD28 has several cysteine residues, its activity is not inhibited by mercury, and in this respect it differs from [gamma]-TIP and all but one of the mammalian water channels that have been described. Introduction of a cysteine residue next to the second conserved NPA motif creates a mercury-sensitive water channel, suggesting that this conserved loop is critical to the activity of the protein. Antibodies directed at the C terminus of RD28 were used in combination with a two-phase partitioning method to demonstrate that RD28 is located in the plasma membrane. The protein is present in leaves and roots of well-watered plants, suggesting that its presence in plants does not require a specific desiccation regime. These results demonstrate that plant cells contain constitutively expressed aquaporins in their plasma membranes (RD28), as well as in their tonoplasts ([gamma]-TIP).

The Major Intrinsic Protein Family of Arabidopsis Has 23 Members That Form Three Distinct Groups with Functional Aquaporins in Each Group

Aquaporins, proteins that enhance the permeability of biological membranes to water, are widely distributed in living organisms. They are 26- to 29-kD proteins that belong to the major intrinsic protein (MIP) family of channels. By searching the Arabidopsis thaliana expressed sequence tag database and by using the polymerase chain reaction with oligonucleotides to conserved plant aquaporin domains, we identified 23 expressed Arabidopsis MIP genes. Eight of these had been previously identified as active aquaporins, and two additional ones are now reported to have water-transport activity in Xenopus laevis oocytes. One of these is highly expressed in suspension-cultured cells. On a dendrogram these 23 MIP sequences cluster into three groups: the first group has 11 members and contains the plasma membrane aquaporins, the second group also has 11 members and contains the tonoplast aquaporins, and the third group has only a single member. This MIP protein, provisionally called At-NLM1, is most closely related to the Gm-NOD26 protein that is found in the bacteroid membranes of soybean (Glycine max L.) nodules; At-NLM1 is an active aquaporin when expressed in oocytes. With a semiquantitative slot-blot analysis technique, we determined the expression levels of 22 MIP genes in the various organs. The quantitative polymerase chain reaction was used to determine the effects of various stress treatments on the expression of NLM1.

Isolation of a Mutant Arabidopsis Plant That Lacks N-Acetyl Glucosaminyl Transferase I and Is Unable to Synthesize Golgi-Modified Complex N-Linked Glycans

The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of [beta]1->2 xylose and [alpha]1->3 fucose residues, are derived from typical mannose9(N-acetylglucosamine)2 (Man9GlcNAc2) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arabidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man5GlcNAc1 glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man9GlcNAc2 and Man8GlcNAc2 glycans, suggesting that the bio-chemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, we obtained a unique strain that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan.