Arabidopsis TCH3 encodes a novel Ca2+ binding protein and shows environmentally induced and tissue-specific regulation.

The Arabidopsis touch (TCH) genes are up-regulated in response to various environmental stimuli, including touch, wind, and darkness. Previously, it was determined that TCH1 encodes a calmodulin; TCH2 and TCH3 encode calmodulin-related proteins. Here, we present the sequence and genomic organization of TCH3. TCH3 is composed of three repeats; remarkably, the first two repeats share 94% sequence identity, including introns that are 99% identical. The conceptual TCH3 product is 58 to 60% identical to known Arabidopsis calmodulins; however, unlike calmodulin, which has four Ca2+ binding sites, TCH3 has six potential Ca2+ binding domains. TCH3 is capable of binding Ca2+, as demonstrated by a Ca(2+)-specific shift in electrophoretic mobility. 5' Fragments of the TCH3 locus, when fused to the beta-glucuronidase (GUS) reporter gene, are sufficient to confer inducibility of expression following stimulation of plants with touch or darkness. These TCH3 sequences also direct expression to growing regions of roots, vascular tissue, root/shoot junctions, trichomes, branch points of the shoot, and regions of siliques and flowers. The pattern of expression of the TCH3/GUS reporter genes most likely reflects expression of the native TCH3 gene, because immunostaining of the TCH3 protein shows similar localization. The tissue-specific expression of TCH3 suggests that expression may be regulated not only by externally applied mechanical stimuli but also by mechanical stresses generated during development. Consequently, TCH3 may perform a Ca(2+)-modulated function involved in generating changes in cells and/or tissues that result in greater strength or flexibility.     

Life in a changing world: TCH gene regulation of expression and responses to environmental signals

The Arabidopsis TCH genes were discovered as a consequence of their marked upregulation of expression in response to seemingly innocuous stimuli, such as touch. Further analyses have indicated that these genes are upregulated by a variety of diverse stimuli. Understanding the mechanism(s) and factors that control TCH gene regulation will shed light on the signalling pathways that enable plants to respond to changing environmental conditions. The TCH proteins include calmodulin, calmodulin-related proteins and a xyloglucan endotransglycosylase. Expression analyses and localization of protein accumulation indicate that the potential sites of TCH protein function include expanding cells and tissues under mechanical strain. We hypothesize that the TCH proteins may collaborate in cell wall biogenesis.     

Regulation of expression of calmodulin and calmodulin-related genes by environmental stimuli in plants.

Plants are very sensitive to environmental stimuli and have evolved the ability to adapt to many environmental stresses by altering development. In particular, mechanical stimuli such as touch or wind, result in growth changes that result in plants with greater resistance to such mechanical stimuli. We have initiated a molecular dissection of the pathways that enable perception of and responses to these environmental stimuli in plants. We have discovered five genes--termed the TCH genes--whose expression levels are strongly and rapidly increased in response to stimuli such as touch, wind, rain, wounding and darkness. Three of the TCH genes encode proteins related to calmodulin thereby implicating roles for calcium ions and calmodulin in the transduction of signals from the environment.     

Cellular localization of the Ca2+ binding TCH3 protein of Arabidopsis

TCH3 is an Arabidopsis t ou ch ( TCH ) gene isolated as a result of its strong and rapid upregulation in response to mechanical stimuli, such as touch and wind. TCH3 encodes an unusual calcium ion-binding protein that is closely related to calmodulin but has the potential to bind six calcium ions. Here it is shown that TCH3 shows a restricted pattern of accumulation during Arabidopsis vegetative development. These data provide insight into the endogenous signals that may regulate TCH3 expression and the sites of TCH3 action. TCH3 is abundant in the shoot apical meristem, vascular tissue, the root columella and pericycle cells that give rise to lateral roots. In addition, TCH3 accumulation in cells of developing shoots and roots closely correlates with the process of cellular expansion. Following wind stimulation, TCH3 becomes more abundant in specific regions including the branchpoints of leaf primordia and stipules, pith parenchyma, and the vascular tissue. The consequences of TCH3 upregulation by wind are therefore spatially restricted and TCH3 may function at these sites to modify cell or tissue characteristics following mechanical stimulation. Because TCH3 accumulates specifically in cells and tissues that are thought to be under the influence of auxin, auxin levels may regulate TCH3 expression during development. TCH3 is upregulated in response to low levels of exogenous indole-3-acetic acid (IAA), but not by inactive auxin-related compounds. These results suggest that TCH3 protein may play roles in mediating physiological responses to auxin and mechanical environmental stimuli.     

Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase.

Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.     

Comparative modeling of the three-dimensional structure of the calmodulin-related TCH2 protein from arabidopsis

Plants adapt to various stresses by developmental alterations that render them less easily damaged. Expression of the TCH2 gene of Arabidopsis is strongly induced by stimuli such as touch and wind. The gene product, TCH2, belongs to the calmodulin (CaM) family of proteins and contains four highly conserved Ca²⁺-binding EF-hands. We describe here the structure of TCH2 in the fully Ca²⁺-saturated form, constructed using comparative molecular modeling, based on the x-ray structure of paramecium CaM. Like known CaMs, the overall structure consists of two globular domains separated by a linker helix. However, the linker region has added flexibility due to the presence of 5 glycines within a span of 6 residues. In addition, TCH2 is enriched in Lys and Arg residues relative to other CaMs, suggesting a preference for targets which are more negatively charged. Finally, a pair of Cys residues in the C-terminal domain, Cys126 and Cys131, are sufficiently close in space to form a disulfide bridge. These predictions serve to direct future biochemical and structural studies with the overall aim of understanding the role of TCH2 in the cellular response of Arabidopsis to environmental stimuli. Proteins 27:144–153 © 1997 Wiley-Liss, Inc.     

Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid

Plants, in common with all organisms, have evolved mechanisms to cope with the problems caused by high temperatures. We examined specifically the involvement of calcium, abscisic acid (ABA), ethylene, and salicylic acid (SA) in the protection against heat-induced oxidative damage in Arabidopsis. Heat caused increased thiobarbituric acid reactive substance levels (an indicator of oxidative damage to membranes) and reduced survival. Both effects required light and were reduced in plants that had acquired thermotolerance through a mild heat pretreatment. Calcium channel blockers and calmodulin inhibitors increased these effects of heating and added calcium reversed them, implying that protection against heat-induced oxidative damage in Arabidopsis requires calcium and calmodulin. Similar to calcium, SA, 1-aminocyclopropane-1-carboxylic acid (a precursor to ethylene), and ABA added to plants protected them from heat-induced oxidative damage. In addition, the ethylene-insensitive mutant etr-1, the ABA-insensitive mutant abi-1, and a transgenic line expressing nahG (consequently inhibited in SA production) showed increased susceptibility to heat. These data suggest that protection against heat-induced oxidative damage in Arabidopsis also involves ethylene, ABA, and SA. Real time measurements of cytosolic calcium levels during heating in Arabidopsis detected no increases in response to heat per se, but showed transient elevations in response to recovery from heating. The magnitude of these calcium peaks was greater in thermotolerant plants, implying that these calcium signals might play a role in mediating the effects of acquired thermotolerance. Calcium channel blockers and calmodulin inhibitors added solely during the recovery phase suggest that this role for calcium is in protecting against oxidative damage specifically during/after recovery.     

Mechanically stimulated TCH3 gene expression in Arabidopsis involves protein phosphorylation and EIN6 downstream of calcium

Mechanical signals are important both as environmental and endogenous developmental cues in plants. Among the quickest measurable responses to mechanical stimulation (MS) in plants is the up-regulation of specific genes, including TCH3, in Arabidopsis. Little is known about the signaling events and components that link perception of mechanical signals to gene expression in plants. Calcium has been identified previously as being potentially involved, and a role for ethylene has also been suggested. Using the protein kinase inhibitor staurosporine, we determined that MS up-regulation of TCH3 expression requires protein kinase activity in young Arabidopsis seedlings. Our data from studies on the Arabidopsis ein6 mutant demonstrate that the EIN6 protein is also required, but that its role in mechanically induced TCH3 expression appears to be independent of ethylene. Challenge of seedlings with protein phosphatase inhibitors calyculin A and okadaic acid stimulated TCH3 expression even in the absence of MS, implying protein phosphatase activity acting to negatively regulate TCH3 gene expression. This phosphatase activity acts either downstream or independently of EIN6. EIN6 and protein kinase activity, on the other hand, operate downstream of calcium to mediate mechanically stimulated TCH3 expression.     

Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.)

Full- and partial-length cDNAs encoding calmodulin mRNA have been cloned and sequenced from barley (Hordeum vulgare L.). Barley leaf mRNA, size-fractionated in sucrose density gradients, was used to synthesize double-stranded cDNA. The cDNA was cloned in λgt10 and screened with a synthetic, 14-nucleotide oligonucleotide probe, which was designed using the predicted coding sequences of the carboxy termini of spinach and wheat calmodulin proteins. The primary structure of barley calmodulin, predicted from DNA sequencing experiments, consists of 148 amino acids and differs from that of wheat calmodulin in only three positions. In two of the three positions, the amino acid changes are conservative, while the third change consists of an apparent deletion/insertion. The overall nucleotide sequence similarity between the amino acid coding regions of barley and vertebrate calmodulin mRNAs is approximately 77%. However, a region encoding 11 amino acids of the second Ca²⁺-binding domain is very highly conserved at the nucleotide level compared with the rest of the coding sequences (94% sequence identity between barley and chicken calmodulin mRNAs). Genomic Southern blots reveal that barley calmodulin is encoded by a single copy gene. This gene is expressed as a single size class of mRNA in all tissues of 7-day-old barley seedlings. In addition, these analyses indicate that a barley calmodulin cDNA coding region subclone is suitable as a probe for isolating calmodulin genes from other plants.     

A novel calcium/calmodulin-regulated kinesin-like protein is highly conserved between monocots and dicots

Recently, a novel kinesin-like protein (KCBP) that is regulated by Ca2+/calmodulin was isolated from dicot plants. A homolog of KCBP has not been reported in monocots. To determine if this motor protein is present in phylogenetically divergent flowering plants, Arabidopsis KCBP cDNA was used as a probe to screen a genomic library of maize, an evolutionarily divergent species. This screening resulted in isolation of a KCBP homolog. Comparison of the predicted amino acid sequence of the KCBP from maize (ZmKCBP), a monocot, with the previously reported KCBP sequences from dicot species showed that the amino acid sequence, domain organization, and gene structure are highly conserved between monocots and dicots. The C-terminal region of ZmKCBP, containing the motor domain and the calmodulin-binding domain, and the N-terminal tail, with a myosin tail homology region (MyTH4) and talin-like region, showed strong sequence similarity to the KCBP homolog from dicots. However, the coiled-coil region is less conserved between monocots and dicots. The ZmKCBP gene contained 22 exons and 21 introns. The location of 19 of the 21 introns of ZmKCBP is also conserved. The ZmKCBP protein is encoded by a single gene and expressed in all tissues. Affinity-purified antibody to the calmodulin-binding domain of Arabidopsis KCBP detected a protein in both the soluble and the microsomal fractions. The C-terminal region of ZmKCBP, containing the motor and calmodulin-binding domains, bound calmodulin in the presence of calcium and failed to bind in the presence of EGTA. The ZmKCBP, along with other KCBPs from dicots, was grouped into a distinct group in the C-terminal subfamily of kinesin-like proteins. These data suggest that the KCBP is ubiquitous and highly conserved in all flowering plants and the origin of KCBP predated the divergence of monocots and dicots.     

The bacterial stringent response, conserved in chloroplasts, controls plant fertilization

The chloroplast, an essential organelle for plants, performs a wide variety of metabolic processes for host cells, which include photosynthesis as well as amino acid and fatty acid biosynthesis. The organelle conserves many bacterial systems in its functions, implicating its origin from symbiosis of a photosynthetic bacterium. In bacterial cells, the stringent response acts as a global regulatory system for gene expression mediated by a small nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), that is necessary for cell adaptation to diverse environmental stimuli such as amino acid starvation. Recent studies indicated that proteins similar to the bacterial ppGpp synthase/hydrolyase are conserved in plants, although their precise roles are not known. Here we show that the stringent response in chloroplasts is crucial for normal plant fertilization. Specifically, one of the Arabidopsis ppGpp synthase homologs, CRSH (Ca(2+)-activated RelA/SpoT homolog), exhibits calcium-dependent ppGpp synthesis activity in vitro, and is localized in chloroplasts in vivo. A knockdown mutation of CRSH in Arabidopsis results in a significant reduction in silique size and seed production, indicating that plant reproduction is under the control of chloroplast function through a ppGpp-mediated stringent response.     

Isolation and characterization of calmodulin from the motile green alga Chlamydomonas reinhardtii

Calmodulin, a calcium-binding protein with no known enzymatic activity but multiple, in vitro effector activities, has been purified to apparent homogeneity from the unicellular green alga Chlamydomonas reinhardtii and compared to calmodulin from vertebrates and higher plants. Chlamydomonas calmodulin was characterized in terms of electrophoretic mobility, amino acid composition, limited amino acid sequence analysis, immunoreactivity, and phosphodiesterase activation. Chlamydomonas calmodulin has two histidine residues similar to calmodulin from the protozoan Tetrahymena. However, unlike the protozoan calmodulin, only one of the histidinyl residues of Chlamydomonas calmodulin is found in the COOH-terminal third of the molecule. Chlamydomonas calmodulin lacks trimethyllysine but does have a lysine residue at the amino acid sequence position corresponding to the trimethyllysine residue in bovine brain and spinach calmodulins. The lack of this post-translational modification does not prevent Chlamydomonas calmodulin from quantitatively activating bovine brain phosphodiesterase. These studies also demonstrate that this unique calmodulin from a phylogenetically earlier eukaryote may be as similar to vertebrate calmodulin as it is to higher plant calmodulins, and suggest that Chlamydomonas calmodulin may more closely approximate the characteristics of a putative precursor of the calmodulin family than any calmodulin characterized to date.     

华山姜中钙调素基因的克隆及其RNA原位杂交

本文报道了华山姜中钙调素cDNA的核苷酸序列以及由此推导的氨基酸序列。并用原化杂交的方法检测其在花器官中的时间表达模式。用[^32P]d-CTP标记的小草蔻-Alpinia hainanensis AGAMOUS（AG）cDNA的MADS-domain作为探针筛选华山姜的cDNA文库．得到一个钙调素蛋白相关克隆．命名为AoCAM。华山姜钙调素AoCAM的cDNA全长518bp．有一个包含149个氨基酸的开放读码框．编码区起始于第54个核苷酸,终止于第501个核苷酸。AoCAM与拟南芥、小麦、大豆、矮牵牛、玉米的钙调素氨基酸序列比较同源性高达95％。RNA原位杂交表明钙凋素基因和花瓣、雄蕊、雌蕊细胞中大量表达。钙调素基因的表达强度随不同的发育阶段而变化：花发育早期在花的各器官中部表达强烈,以后逐渐减弱并向特定部位集中．如花粉囊、唇瓣、花柱和胚珠等分生能力较强的细胞中表达较强。     

Characterisation of a novel gene family of putative cyclic nucleotide- and calmodulin-regulated ion channels in Arabidopsis thaliana

In plants, cyclic GMP is involved in signal transduction in response to light and gibberellic acid. For cyclic AMP, a potential role during the plant cell cycle was recently reported. However, cellular targets for cyclic nucleotides in plants are largely unknown. Here we report on the identification and characterisation of a new gene family in Arabidopsis, which share features with cyclic nucleotide-gated channels from animals and inward-rectifying K⁺ channels from plants. The identified gene family comprises six members (Arabidopsis thaliana cyclic nucleotide-gated channels, AtCNGC1–6) with significant homology among the deduced proteins. Hydrophobicity analysis predicted six membrane-spanning domains flanked by hydrophilic amino and carboxy termini. A putative cyclic nucleotide binding domain (CNBD) which contains several residues that are invariant in other CNBDs was located in the carboxy terminus. This domain overlaps with a predicted calmodulin (CaM) binding site, suggesting interaction between cyclic nucleotide and CaM regulation. We demonstrated interaction of the carboxy termini of AtCNGC1 and AtCNGC2 with CaM in yeast, indicating that the CaM binding sites are functional. Furthermore, it was shown that both AtCNGC1 and AtCNGC2 can partly complement the K⁺-uptake-deficient yeast mutant CY162. Therefore, we propose that the identified genes constitute a family of plant cyclic nucleotide- and CaM-regulated ion channels.     

gamma-Tubulin in Arabidopsis: gene sequence, immunoblot, and immunofluorescence studies.

gamma-Tubulin is a protein associated with microtubule (Mt)-organizing centers in a variety of eukaryotic cells. Unfortunately, little is known about such centers in plants. Genomic and partial cDNA clones encoding two gamma-tubulins of Arabidopsis were isolated and sequenced. Comparisons of genomic and cDNA sequences showed that both genes, TubG1 and TubG2, contain nine introns at conserved locations. The sequences of the two genes both predict proteins containing 474 amino acids, with molecular masses of 53,250 and 53,280 D, respectively. The predicted gamma 1- and gamma 2-tubulins exhibit 98% amino acid identity with each other and approximately 70% amino acid identity with the gamma-tubulins of animals and fungi. RNA gel blot results demonstrated that both genes are transcribed in suspension culture cells, seedlings, and roots and flowers of mature plants. Immunoblots of Arabidopsis proteins using an antibody specific to a conserved peptide of gamma-tubulin showed a major cross-reacting polypeptide with an M(r) of 58,000. The same antibody stained all Mt arrays in tissue and suspension culture cells of this species. Binding was inhibited by the homologous oligopeptide in the gamma-tubulins predicted by the two Arabidopsis gene sequences. Antibody staining avoided the plus ends of Mts at the kinetochores and cell plate, but unlike the case in animal cells, seemed to be localized over broad stretches of the kinetochore fibers and phragmoplast toward the minus ends. We concluded that at least two gamma-tubulin protein homologs are present in Arabidopsis and that at least one of them is localized along Mt arrays. Its distribution is correlated with and may help explain unique characteristics of Mt organization in plants.