Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes

In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism.     

Heat-inducible expression of FLP gene in maize cells

The soybean heat-shock gene promoter ( Gmhsp 17.5-E ) has been used to direct expression of gusA and FLP genes in maize cells. At inducible temperatures, in transient expression assays, gusA gene expression controlled by the heat-shock promoter is about 10-fold higher than the expression directed by the CaMV 35S promoter. The Gmhsp 17.5-E promoter preserves its regulatory functions in heterologous maize cells after random integration into genomic DNA.
Heat-shock inducible expression of the FLP gene was investigated by co-transformation of the FLP expression vector (pHsFLP) and a recombination test vector (pUFNeo-FmG) into maize protoplasts. Co-transformed protoplasts were incubated at 42°C for 2 h. This treatment induced recombination of 20–25% of the available FRT sites in transient assays. As a result of heat-shock treatment of stably co-transformed maize cells, activation of gusA gene expression and an associated decrease or elimination of NPT-II activity in transgenic maize lines was observed. Molecular evidence was obtained of the expected DNA excision process catalyzed by the FLP protein in maize transgenic cells. Thus, the experiments presented in this paper indicate that the FLP protein can recognize and subsequently recombine the FRT target sites that had integrated into plant genomic DNA, and that regulated expression of the FLP gene is possible in maize cells using the soybean heat-shock promoter.     

Sugar-responsive gene expression, invertase activity, and senescence in aborting maize ovaries at low water potentials

• Background and Aims Ovary abortion can occur in maize(Zea mays) if water deficits lower the water potential (_w) sufficientlyto inhibit photosynthesis around the time of pollination. Theabortion decreases kernel number. The present work exploredthe activity of ovary acid invertases and their genes, togetherwith other genes for sucrose-processing enzymes, when this kindof abortion occurred. Cytological evidence suggested that senescencemay have been initiated after 2 or 3 d of low _w, and the expressionof some likely senescence genes was also determined. • Methods Ovary abortion was assessed at kernel maturity.Acid invertase activities were localized in vivo and in situ.Time courses for mRNA abundance were measured with real timePCR. Sucrose was fed to the stems to vary the sugar flux. • Key Results Many kernels developed in controls but mostaborted when _w became low. Ovary invertase was active in controlsbut severely inhibited at low _w for cell wall-bound forms invivo and soluble forms in situ. All ovary genes for sucroseprocessing enzymes were rapidly down-regulated at low _w exceptfor a gene for invertase inhibitor peptide that appeared tobe constitutively expressed. Some ovary genes for senescencewere subsequently up-regulated (RIP2 and PLD1). In some genes,these regulatory changes were reversed by feeding sucrose tothe stems. Abortion was partially prevented by feeding sucrose. • Conclusions A general response to low _w in maize ovarieswas an early down-regulation of genes for sucrose processingenzymes followed by up-regulation of some genes involved insenescence. Because some of these genes were sucrose responsive,the partial prevention of abortion with sucrose feeding mayhave been caused in part by the differential sugar-responsivenessof these genes. The late up-regulation of senescence genes mayhave caused the irreversibility of abortion.     

Genome-wide analysis of the invertase gene family from maize

Key message

The recent release of the maize genome (AGPv4) contains annotation errors of invertase genes and therefore the enzymes are bestly curated manually at the protein level in a comprehensible fashion

Abstract

The synthesis, transport and degradation of sucrose are determining factors for biomass allocation and yield of crop plants. Invertase (INV) is a key enzyme of carbon metabolism in both source and sink tissues. Current releases of the maize genome correctly annotates only two vacuolar invertases (ivr1 and ivr2) and four cell wall invertases (incw1, incw2 (mn1), incw3, and incw4). Our comprehensive survey identified 21 INV isogenes for which we propose a standard nomenclature grouped phylogenetically by amino acid similarity: three vacuolar (INVVR), eight cell wall (INVCW), and ten alkaline/neutral (INVAN) isogenes which form separate dendogram branches due to distinct molecular features. The acidic enzymes were curated for the presence of the DPN tripeptide which is coded by one of the smallest exons reported in plants. Particular attention was placed on the molecular role of INV in vascular tissues such as the nodes, internodes, leaf sheath, husk leaves and roots. We report the expression profile of most members of the maize INV family in nine tissues in two developmental stages, R1 and R3. INVCW7, INVVR2, INVAN8, INVAN9, INVAN10, and INVAN3 displayed the highest absolute expressions in most tissues. INVVR3, INVCW5, INVCW8, and INVAN1 showed low mRNA levels. Expressions of most INVs were repressed from stage R1 to R3, except for INVCW7 which increased significantly in all tissues after flowering. The mRNA levels of INVCW7 in the vegetative stem correlated with a higher transport rate of assimilates from leaves to the cob which led to starch accumulation and growth of the female reproductive organs.

     

Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin

The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M_r 63,000, pH optimum at 4.7, K_{m sucrose} 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl₂ but is insensitive to H₂O₂, N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink.     

Intron-specific stimulation of anaerobic gene expression and splicing efficiency in maize cells

Most of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes characterized in plants and algae to date have one intron very close to the 5 end of the gene. To study the functional relevance of some of these introns for gene expression we have analysed the influence of three 5 introns on transient gene expression of the anaerobically inducible maizeGapC4 promoter in maize cells. Under aerobic conditions, reporter gene expression is increased in the presence of the first introns of theGapC4 andGapC1 genes, and the first intron of the nuclear encoded chloroplast-specificGapA1 gene. In contrast, theGapC4 intron increases anaerobic gene expression above the level obtained for the intronless construct, while anaerobic expression of constructs harboring theGapA1 andGapC1 introns was similar to the anaerobic expression level of the intronless construct. Splicing analysis revealed that theGapC4 intron is processed more efficiently under anaerobic conditions, while no change in splicing efficiency is observed for theGapC1 and theGapA1 introns when subjected to anaerobic conditions. These results suggest that an increase in splicing efficiency contributes to the anaerobic induction of the maizeGapC4 gene.     

DNA array profiling of gene expression changes during maize embryo development

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)⁺RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)⁺ RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6. Electronic Publication     

Chimeric gene expression using maize intron in cultured cells of breadwheat

High voltage electrical pulses were used to introduce the CAT reporter gene into cultured protoplasts of breadwheat,Triticum aestivum. Four DNA constructs harboring the CAT gene and the 35S or mannipine synthase promoter were tested for levels of CAT activity 40–45 hr after electroporation of protoplasts. One construct, containing a maize intron sequence between 35S and CAT sequences, conferred 30 to 185 fold greater CAT activity over the other three constructs. Data from these experiments suggest that a maize intron or sequences with similar effects may be required in DNA constructs for efficient heterologous gene expression in cultured cells of breadwheat.Abbreviations CAT Chloramphenicol acetyl transferase - NPT II neomycin phosphotransferase - 35S the 35S promoter of Cauliflower Mosaic Virus - PEG Polyethylene glycol - MES 2-[N-morpholino] ethanesulfonic acid     

Constitutive expression of cell wall invertase genes increases grain yield and starch content in maize

Grain size, number and starch content are important determinants of grain yield and quality. One of the most important biological processes that determine these components is the carbon partitioning during the early grain filling, which requires the function of cell wall invertase. Here, we showed the constitutive expression of cell wall invertase–encoding gene from Arabidopsis, rice (Oryza sativa) or maize (Zea mays), driven by the cauliflower mosaic virus (CaMV) 35S promoter, all increased cell wall invertase activities in different tissues and organs, including leaves and developing seeds, and substantially improved grain yield up to 145.3% in transgenic maize plants as compared to the wild‐type plants, an effect that was reproduced in our 2‐year field trials at different locations. The dramatically increased grain yield is due to the enlarged ears with both enhanced grain size and grain number. Constitutive expression of the invertase‐encoding gene also increased total starch content up to 20% in the transgenic kernels. Our results suggest that cell wall invertase gene can be genetically engineered to improve both grain yield and grain quality in crop plants.     

Elevation of cytosolic calcium precedes anoxic gene expression in maize suspension-cultured cells.

Based on pharmacological evidence, we previously proposed that intracellular Ca2+ mediates the perception of O2 deprivation in maize seedlings. Herein, using fluorescence imaging and photometry of Ca2+ in maize suspension-cultured cells, the proposal was further investigated. Two complementary approaches were taken: (1) real time analysis of anoxia-induced changes in cytosolic Ca2+ concentration ([Ca]i) and (2) experimental manipulation of [Ca]i and then assay of the resultant anoxia-specific responses. O2 depletion caused an immediate increase in [Ca2+]i, and this was reversible within a few seconds of reoxygenation. The [Ca]i elevation proceeded independent of extracellular Ca2+. The kinetics of the Ca2+ response showed that it occurred much earlier than any detectable changes in gene expression. Ruthenium red blocked the anoxic [Ca]i elevation and also the induction of adh1 (encoding alcohol dehydrogenase) and sh1 (encoding sucrose synthase) mRNA. Ca2+, when added along with ruthenium red, prevented the effects of the antagonist on the anoxic responses. Verapamil and bepridil failed to block the [Ca]i rise induced by anoxia and were equally ineffective on anoxic gene expression. Caffeine induced an elevation of [Ca]i as well as ADH activity under normoxia. The data provide direct evidence for [Ca]i elevation in maize cells as a result of anoxia-induced mobilization of Ca2+ from intracellular stores. Furthermore, any manipulation that modified the [Ca]i rise brought about a parallel change in the expression of two anoxia-inducible genes. Thus, these results corroborate our proposal that [Ca]i is a physiological transducer of anoxia signals in plants.