The Role of Cytosolic Potassium and pH in the Growth of Barley Roots1

In an earlier paper we showed that in fully developed barley (Hordeum vulgare L.) root epidermal cells a decrease in cytosolic K+ was associated with an acidification of the cytosol (D.J. Walker, R.A. Leigh, A.J. Miller [1996] Proc Natl Acad Sci USA 93: 10510–10514). To show that these changes in cytosolic ion concentrations contributed to the decreased growth of K+-starved roots, we first measured whether similar changes occurred in cells of the growing zone. Triple-barreled ion-selective microelectrodes were used to measure cytosolic K+ activity and pH in cells 0.5 to 1.0 mm from the root tip. In plants growing from 7 to 21 d after germination under K+-replete conditions, the mean values did not change significantly, with values ranging from 80 to 84 mm for K+ and 7.3 to 7.4 for pH. However, in K+-starved plants (external [K+], 2 μm), the mean cytosolic K+ activity and pH had declined to 44 mm and 7.0, respectively, after 14 d. For whole roots, sap osmolality was always lower in K+-starved than in K+-replete plants, whereas elongation rate and dry matter accumulation were significantly decreased after 14 and 16 d of K+ starvation. The rate of protein synthesis in root tips did not change for K+-replete plants but declined significantly with age in K+-starved plants. Butyrate treatment decreased cytosolic pH and diminished the rate of protein synthesis in K+-replete roots. Procaine treatment of K+-starved roots gave an alkalinization of the cytosol and increased protein synthesis rate. These results show that changes in both cytosolic pH and K+ can be significant factors in inhibiting protein synthesis and root growth during K+ deficiency.

Opposite Effects on Spodoptera littoralis Larvae of High Expression Level of a Trypsin Proteinase Inhibitor in Transgenic Plants1

This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI.

Developmental Regulation of Intercellular Protein Trafficking through Plasmodesmata in Tobacco Leaf Epidermis1

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the 3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.

Metallothioneins 1 and 2 Have Distinct but Overlapping Expression Patterns in Arabidopsis1

The spatial and temporal expression patterns of metallothionein (MT) isoforms MT1a and MT2a were investigated in vegetative and reproductive tissues of untreated and copper-treated Arabidopsis by in situ hybridization and by northern blotting. In control plants, MT1a mRNA was localized in leaf trichomes and in the vascular tissue in leaves, roots, flowers, and germinating embryos. In copper-treated plants, MT1a expression was also observed in the leaf mesophyll and in vascular tissue of developing siliques and seeds. In contrast, MT2a was expressed primarily in the trichomes of both untreated and copper-treated plants. In copper-treated plants, MT2a mRNA was also expressed in siliques. Northern-hybridization studies performed on developing seedlings and leaves showed temporal variations of MT1a gene expression but not of MT2a expression. The possible implications of these findings for the cellular roles of MTs in plants are discussed.

Changes in Growth CO2 Result in Rapid Adjustments of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunit Gene Expression in Expanding and Mature Leaves of Rice1

The accumulation of soluble carbohydrates resulting from growth under elevated CO2 may potentially signal the repression of gene activity for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS). To test this hypothesis we grew rice (Oryza sativa L.) under ambient (350 μL L−1) and high (700 μL L−1) CO2 in outdoor, sunlit, environment-controlled chambers and performed a cross-switching of growth CO2 concentration at the late-vegetative phase. Within 24 h, plants switched to high CO2 showed a 15% and 23% decrease in rbcS mRNA, whereas plants switched to ambient CO2 increased 27% and 11% in expanding and mature leaves, respectively. Ribulose-1,5-bisphosphate carboxylase/oxygenase total activity and protein content 8 d after the switch increased up to 27% and 20%, respectively, in plants switched to ambient CO2, but changed very little in plants switched to high CO2. Plants maintained at high CO2 showed greater carbohydrate pool sizes and lower rbcS transcript levels than plants kept at ambient CO2. However, after switching growth CO2 concentration, there was not a simple correlation between carbohydrate and rbcS transcript levels. We conclude that although carbohydrates may be important in the regulation of rbcS expression, changes in total pool size alone could not predict the rapid changes in expression that we observed.

Intermediates of Salicylic Acid Biosynthesis in Tobacco1

Salicylic acid (SA) is an important component of systemic-acquired resistance in plants. It is synthesized from benzoic acid (BA) as part of the phenylpropanoid pathway. Benzaldehyde (BD), a potential intermediate of this pathway, was found in healthy and tobacco mosaic virus (TMV)-inoculated tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaf tissue at 100 ng/g fresh weight concentrations as measured by gas chromatography-mass spectrometry. BD was also emitted as a volatile organic compound from tobacco tissues. Application of gaseous BD to plants enclosed in jars caused a 13-fold increase in SA concentration, induced the accumulation of the pathogenesis-related transcript PR-1, and increased the resistance of tobacco to TMV inoculation. [13C6]BD and [2H5]benzyl alcohol were converted to BA and SA. Labeling experiments using [13C1]Phe in temperature-shifted plants inoculated with the TMV showed high enrichment of cinnamic acids (72%), BA (34%), and SA (55%). The endogenous BD, however, contained nondetectable enrichment, suggesting that BD was not the intermediate between cinnamic acid and BA. These results show that BD and benzyl alcohol promote SA accumulation and expression of defense responses in tobacco, and provide insight into the early steps of SA biosynthesis.

Prenylcysteine α-Carboxyl Methyltransferase in Suspension-Cultured Tobacco Cells1

Isoprenylation is a posttranslational modification that is believed to be necessary, but not sufficient, for the efficient association of numerous eukaryotic cell proteins with membranes. Additional modifications have been shown to be required for proper intracellular targeting and function of certain isoprenylated proteins in mammalian and yeast cells. Although protein isoprenylation has been demonstrated in plants, postisoprenylation processing of plant proteins has not been described. Here we demonstrate that cultured tobacco (Nicotiana tabacum cv Bright Yellow-2) cells contain farnesylcysteine and geranylgeranylcysteine α-carboxyl methyltransferase activities with apparent Michaelis constants of 73 and 21 μm for N-acetyl-S-trans,trans-farnesyl-l-cysteine and N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, respectively. Furthermore, competition analysis indicates that the same enzyme is responsible for both activities. These results suggest that α-carboxyl methylation is a step in the maturation of isoprenylated proteins in plants.

Control of Cl− Efflux in Chara corallina by Cytosolic pH, Free Ca2+, and Phosphorylation Indicates a Role of Plasma Membrane Anion Channels in Cytosolic pH Regulation1

Enhanced Cl− efflux during acidosis in plants is thought to play a role in cytosolic pH (pHc) homeostasis by short-circuiting the current produced by the electrogenic H+ pump, thereby facilitating enhanced H+ efflux from the cytosol. Using an intracellular perfusion technique, which enables experimental control of medium composition at the cytosolic surface of the plasma membrane of charophyte algae (Chara corallina), we show that lowered pHc activates Cl− efflux via two mechanisms. The first is a direct effect of pHc on Cl− efflux; the second mechanism comprises a pHc-induced increase in affinity for cytosolic free Ca2+ ([Ca2+]c), which also activates Cl− efflux. Cl− efflux was controlled by phosphorylation/dephosphorylation events, which override the responses to both pHc and [Ca2+]c. Whereas phosphorylation (perfusion with the catalytic subunit of protein kinase A in the presence of ATP) resulted in a complete inhibition of Cl− efflux, dephosphorylation (perfusion with alkaline phosphatase) arrested Cl− efflux at 60% of the maximal level in a manner that was both pHc and [Ca2+]c independent. These findings imply that plasma membrane anion channels play a central role in pHc regulation in plants, in addition to their established roles in turgor/volume regulation and signal transduction.

High Expression of the Tonoplast Aquaporin ZmTIP1 in Epidermal and Conducting Tissues of Maize1

Aquaporins are integral membrane proteins of the tonoplast and the plasma membrane that facilitate the passage of water through these membranes. Because of their potentially important role in regulating water flow in plants, studies documenting aquaporin gene expression in specialized tissues involved in water and solute transport are important. We used in situ hybridization to examine the expression pattern of the tonoplast aquaporin ZmTIP1 in different organs of maize (Zea mays L.). This tonoplast water channel is highly expressed in the root epidermis, the root endodermis, the small parenchyma cells surrounding mature xylem vessels in the root and the stem, phloem companion cells and a ring of cells around the phloem strand in the stem and the leaf sheath, and the basal endosperm transfer cells in developing kernels. We postulate that the high level of expression of ZmTIP1 in these tissues facilitates rapid flow of water through the tonoplast to permit osmotic equilibration between the cytosol and the vacuolar content, and to permit rapid transcellular water flow through living cells when required.

Direct Evidence for Rapid Degradation of Bacillus thuringiensis Toxin mRNA as a Cause of Poor Expression in Plants1

It is well established that the expression of Bacillus thuringiensis (B.t.) toxin genes in higher plants is severely limited at the mRNA level, but the cause remains controversial. Elucidating whether mRNA accumulation is limited transcriptionally or posttranscriptionally could contribute to effective gene design as well as provide insights about endogenous plant gene-expression mechanisms. To resolve this controversy, we compared the expression of an A/U-rich wild-type cryIA(c) gene and a G/C-rich synthetic cryIA(c) B.t.-toxin gene under the control of identical 5′ and 3′ flanking sequences. Transcriptional activities of the genes were equal as determined by nuclear run-on transcription assays. In contrast, mRNA half-life measurements demonstrated directly that the wild-type transcript was markedly less stable than that encoded by the synthetic gene. Sequences that limit mRNA accumulation were located at more than one site within the coding region, and some appeared to be recognized in Arabidopsis but not in tobacco (Nicotiana tabacum). These results support previous observations that some A/U-rich sequences can contribute to mRNA instability in plants. Our studies further indicate that some of these sequences may be differentially recognized in tobacco cells and Arabidopsis.

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca2+]cyt response. Cyclic-nucleotide-mediated elevations in [Ca2+]cyt involved both internal and external Ca2+ stores. Both cAMP- and cGMP-mediated [Ca2+]cyt elevations could be inhibited by the Ca2+-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca2+]cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca2+]cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca2+ signaling.

Expression of Tobacco Carbonic Anhydrase in the C4 Dicot Flaveria bidentis Leads to Increased Leakiness of the Bundle Sheath and a Defective CO2-Concentrating Mechanism

Flaveria bidentis (L.) Kuntze, a C4 dicot, was genetically transformed with a construct encoding the mature form of tobacco (Nicotiana tabacum L.) carbonic anhydrase (CA) under the control of a strong constitutive promoter. Expression of the tobacco CA was detected in transformant whole-leaf and bundle-sheath cell (bsc) extracts by immunoblot analysis. Whole-leaf extracts from two CA-transformed lines demonstrated 10% to 50% more CA activity on a ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This increased CA activity resulted in plants with moderately reduced rates of CO2 assimilation (A) and an appreciable increase in C isotope discrimination compared with the controls. With increasing O2 concentrations up to 40% (v/v), a greater inhibition of A was found for transformants than for wild-type plants; however, the quantum yield of photosystem II did not differ appreciably between these two groups over the O2 levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher O2 concentrations, the transformants had increased rates of photorespiration. Thus, the expression of active tobacco CA in the cytosol of F. bidentis bsc and mesophyll cells perturbed the C4 CO2-concentrating mechanism by increasing the permeability of the bsc to inorganic C and, thereby, decreasing the availability of CO2 for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.

Implications of a Developmental-Stage-Dependent Thylakoid-Bound Protease in the Stabilization of the Light-Harvesting Pigment-Protein Complex Serving Photosystem II during Thylakoid Biogenesis in Red Kidney Bean1

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.

An Arabidopsis VPS45p Homolog Implicated in Protein Transport to the Vacuole1

The Sec1p family of proteins is required for vesicle-mediated protein trafficking between various organelles of the endomembrane system. This family includes Vps45p, which is required for transport to the vacuole in yeast (Saccharomyces cerevisiae). We have isolated a cDNA encoding a VPS45 homolog from Arabidopsis thaliana (AtVPS45). The cDNA is able to complement both the temperature-sensitive growth defect and the vacuolar-targeting defect of a yeast vps45 mutant, indicating that the two proteins are functionally related. AtVPS45p is a peripheral membrane protein that associates with microsomal membranes. Sucrose-density gradient fractionation demonstrated that AtVPS45p co-fractionates with AtELP, a potential vacuolar protein sorting receptor, implying that they may reside on the same membrane populations. These results indicate that AtVPS45p is likely to function in the transport of proteins to the vacuole in plants.

Indole-3-Acetic Acid Controls Cambial Growth in Scots Pine by Positional Signaling1

The vascular cambium produces secondary xylem and phloem in plants and is responsible for wood formation in forest trees. In this study we used a microscale mass-spectrometry technique coupled with cryosectioning to visualize the radial concentration gradient of endogenous indole-3-acetic acid (IAA) across the cambial meristem and the differentiating derivatives in Scots pine (Pinus sylvestris L.) trees that had different rates of cambial growth. This approach allowed us to investigate the relationship between growth rate and the concentration of endogenous IAA in the dividing cells. We also tested the hypothesis that IAA is a positional signal in xylem development (C. Uggla, T. Moritz, G. Sandberg, B. Sundberg [1996] Proc Natl Acad Sci USA 93: 9282–9286). This idea postulates that the width of the radial concentration gradient of IAA regulates the radial number of dividing cells in the cambial meristem, which is an important component for determining cambial growth rate. The relationship between IAA concentration in the dividing cells and growth rate was poor, although the highest IAA concentration was observed in the fastest-growing cambia. The radial width of the IAA concentration gradient showed a strong correlation with cambial growth rate. The results indicate that IAA gives positional information in plants.