Genetic control of petiole length in Arabidopsis thaliana

Shade-avoidance syndrome is characterized by the formation of elongated petioles and unexpanded leaf blades under low-intensity light, but the genetic basis for these responses is unknown. In this study, two-dimensional mutational analysis revealed that the gene for phytochrome B, PHYB, had opposing effects in the leaf petioles and leaf blades of Arabidopsis, while the ROT3, ACL2, and GAI genes influenced the length of leaf petioles more significantly than the length of leaf blades. Anatomical analysis revealed that the PHYB and ACL2 genes control the length of leaf petioles exclusively via control of the length of individual cells, while the GAI, GA1 and ROT3 genes appeared to control both the elongation and proliferation of petiole cells, in particular, under strong light. By contrast, both the size and the number of cells were affected by the mutations examined in leaf blades. The differential control of leaf petiole length and leaf blade expansion is discussed.     

Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness

Field-emission scanning electron microscopy was used to measure wall thicknesses of different cell types in freeze-fractured hypocotyls of Arabidopsis thaliana. Measurements of uronic acid content, wall mass, and wall volume suggest that cell wall biosynthesis in this organ does not always keep pace with, and is not always tightly coupled to, elongation. In light-grown hypocotyls, walls thicken, maintain a constant thickness, or become thinner during elongation, depending upon the cell type and the stage of growth. In light-grown hypocotyls, exogenous gibberellic acid represses the extent of thickening and promotes cell elongation by both wall thinning and increased anisotropy during the early stages of hypocotyl elongation, and by increased wall deposition in the latter stages. Dark-grown hypocotyls, in the 48 h period between cold imbibition and seedling emergence, deposit very thick walls that subsequently thin in a narrow developmental window as the hypocotyl rapidly elongates. The rate of wall deposition is then maintained and keeps pace with cell elongation. The outer epidermal wall is always the thickest ( approximately 1 mum) whereas the thinnest walls, about 50 nm, are found in inner cell layers. It is concluded that control of wall thickness in different cell types is tightly regulated during hypocotyl development, and that wall deposition and cell elongation are not invariably coupled.     

ERECTA controls low light intensity-induced differential petiole growth independent of phytochrome B and cryptochrome 2 action in Arabidopsis thaliana

Plants can respond quickly and profoundly to changes in their environment. Several species, including Arabidopsis thaliana, are capable of differential petiole growth driven upward leaf movement (hyponastic growth) to escape from detrimental environmental conditions. Recently, we demonstrated that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA, explains a major effect Quantitative Trait Locus (QTL) for ethylene-induced hyponastic growth in Arabidopsis. Here, we demonstrate that ERECTA controls the hyponastic growth response to low light intensity treatment in a genetic background dependent manner. Moreover, we show that ERECTA affects low light-induced hyponastic growth independent of Phytochrome B and Cryptochrome 2 signaling, despite that these photoreceptors are positive regulators of low light-induced hyponastic growth.Key words: hyponastic growth, petiole, Arabidopsis, low light, ERECTA, differential growth, phytochrome B, cryptochrome 2Plants must adjust growth and reproduction to adverse environmental conditions. Among the strategies that plants employ to escape from unfavorable conditions is differential petiole growth-driven upward leaf movement, called hyponastic growth. Arabidopsis thaliana is able to exhibit a marked hyponastic response upon flooding, which is triggered by endogenous accumulation of the gaseous phytohormone ethylene.¹ Moreover, a similar response is triggered upon low light intensity perception and in response to supra-optimal temperatures.^2–5 By tilting the leaves to a more vertical position during submergence and shading, the plants restore contact with the atmosphere and light, respectively. The kinetics of the hyponastic growth response induced by the various stimuli is remarkably similar. This led to the hypothesis that shared functional genetic components may be employed to control hyponastic growth. Yet, at least part of the signaling cascades is parallel, as the hormonal control of the response differs between the stimuli. Low light-induced hyponastic growth for example does not require ethylene action.² Whereas the response to heat is antagonized by this hormone.⁵ The abiotic stress hormone abscisic acid (ABA) antagonizes ethylene-induced hyponastic growth and stimulates heat-induced hyponastic growth.^5,6 Moreover, ethylene-induced hyponasty does not involve auxin action⁷ whereas both heat- and low light-induced hyponasty require functional auxin signaling and transport components.^2,5In our recent paper, published in The Plant Journal,⁸ we employed Quantitative Trait Locus (QTL) analysis to identify loci involved in the control of ethylene-induced hyponastic petiole growth. By analyzing induced mutants and by complementation analysis of naturally occurring mutant accessions, we found that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA (ER) is a positive regulator of ethylene-induced hyponastic growth and most likely is causal to one of the identified QTLs. In addition, we demonstrated that the ER dependency is not via ER mediated control of ethylene production or sensitivity.Since low light-induced hyponasty does not require ethylene action,² ER may be part of the proposed shared signaling cascade leading to hyponastic growth where ethylene and low light signals meet. Therefore, we studied low light intensity-induced hyponasty in various erecta mutants. Moreover, natural occurring er mutant accessions complemented with a functional, Col-0 derived, ER allele were tested. The response of Lan-0 (Lan-0; with functional ER) to low light was indistinguishable from the response of Landsberg erecta (Ler) (Fig. 1A). However, complemented Ler (ER-Ler) showed an enhanced response compared to Ler (Fig. 1B). The response of mutant er105 was slightly attenuated compared to the wild type Columbia-0 (Fig. 1C). Mutant er104, however, showed an indistinguishable hyponastic growth phenotype to low light compared to the wild type Wassilewskija-2 (Ws-2) (Fig. 1D). Complementation of the natural occurring erecta mutant accession Vancouver-0 (Van-0) resulted in an enhanced hyponastic growth response to low light (Fig. 1E), whereas this was not the case for Hiroshima-1 (Hir-1) (Fig. 1F). Together, these data suggest that ER acts as positive regulator of low light-induced hyponastic growth and therefore may be part of the shared signaling cascade towards differential petiole growth. Yet, the effect is strongly dependent on the genetic background since the effects were not observed in every accession tested.Open in a separate windowFigure 1ERECTA involvement in low light-induced hyponasty. Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth in Arabidopsis thaliana. (A) mutant (circles) Ler and wild type (dashed line) Lan-0, (B) Ler and Ler complemented (ER-; squares) with the Col-0 ERECTA allele (ER-Ler), (C) er105 and Col-0 wild type, (D) er104 and Ws-2 wild type, (E) natural mutant Van-0 and Van-0 complemented with the Col-0 ER allele (ER-Van-0), (F) natural mutant Hir-1 and Hir-1 complemented with the Col-0 ER allele (ER-Hir-1). Petiole angles were measured using time-lapse photography and subsequent image analysis. Data is pairwise subtracted, which corrects for diurnal petiole movement in control conditions. For details on this procedure, growth conditions and materials, transformation protocol, treatments, data acquirement and all analyses see.^1,8 Error bars represent standard errors; n ≥ 12.Phytochrome B (PhyB) and Cryptochrome 2 (Cry2) photoreceptor proteins are required for a full induction of low light-induced hyponastic growth.² We transformed the phyb5 cry2 mutant9 (Ler genetic background) with Col-0 derived ER. This complementation did not restore the ability of phyb5 cry2 to induce hyponastic growth to neither ethylene (data not shown) nor low light conditions (Fig. 2A). Mutant phyb5 cry2 plants have a typical constitutive shade avoidance phenotype, reflected by severely elongated organs. This includes enhanced inflorescence and silique length and thin inflorescences (Fig. 2B-D). Complementation with ER resulted in a significant additional effect on these parameters (Fig. 2B-D). Together, this suggests that ER is not an integral part of PhyB nor Cry2 signaling with respect to (hyponastic) growth. Moreover, PhyB and Cry2 control of plant architecture does not require ER action. Rather, ER seems to mediate growth via genetic interaction with light-reliant growth mechanisms, instead of being downstream of photoreceptor action. Studies on the effects of ER on shade avoidance responses and various hormone responses, including cytokinin and auxin, led to the similar conclusion, suggesting a possible role for ER as a molecular hub coordinating light- and hormone-mediated plant growth.^10,11 One could speculate that ER fine-tunes other (than light) environmental clues with light signaling components. A comparable conclusion was drawn previously for gibberellin (GA) reliant growth mechanisms, as er enhanced the negative effect on plant size of the short internode (shi) mutation12 and er represses the positive effect of the spindly mutation in a GA independent manner.¹³Open in a separate windowFigure 2Effects of ERECTA on light signaling. (A) Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth of Ler (dashed lines), the photoreceptor double mutant phyb5 cry2 (circles) and this mutant complemented with the Col-0 ERECTA (ER-phyb cry2; squares). For details see legend Figure 1. (B) Plant height, (C) silique length and (D) inflorescence stem thickness of the above mentioned lines. These parameters were measured when the last flower on the plant developed a silique. Plant height was measured from root/shoot junction to inflorescence top. Stem thickness was measured ∼1 cm above the root/shoot junction with a caliper and silique lengths were measured from representative pedicels in the top ∼10 cm of the main inflorescence stem. Error bars represent standard errors; n ≥ 12. Significance levels; *p < 0.05; **p < 0.01; ***p < 0.001; ns = non significant, by Students t-test.     

Cell wall polysaccharides are involved in P-deficiency-induced Cd exclusion in Arabidopsis thaliana

The physiological and molecular mechanisms leading to the competitive interactions between phosphorus (P) and metal elements are a matter of debate. In this study, we found that P deficiency can alleviate cadmium (Cd) toxicity in Arabidopsis thaliana (Col-0). Under P deficiency (-P), less Cd was accumulated in the plants and the root cell walls, indicating the operation of a P-deficiency-induced Cd exclusion mechanism. However, organic acid efflux was similar under -P+Cd and +Cd treatments, suggesting that organic acid efflux is not responsible for the Cd exclusion. Interestingly, P deficiency significantly decreased cell wall polysaccharides (pectin and hemicellulose) contents and pectin methylesterase activity, and decreased the Cd retained by the extracted root cell wall. Therefore, we conclude that the modification of cell wall composition is responsible for the Cd exclusion of the root under P deficiency.     

The involvement of gibberellin 20-oxidase genes in phytochrome-regulated petiole elongation of Arabidopsis

Long day (LD) exposure of rosette plants causes rapid stem/petiole elongation, a more vertical growth habit, and flowering; all changes are suggestive of a role for the gibberellin (GA) plant growth regulators. For Arabidopsis (Arabidopsis thaliana) L. (Heynh), we show that enhancement of petiole elongation by a far-red (FR)-rich LD is mimicked by a brief (10 min) end-of-day (EOD) FR exposure in short day (SD). The EOD response shows red (R)/FR photoreversibility and is not affected in a phytochrome (PHY) A mutant so it is mediated by PHYB and related PHYs. FR photoconversion of PHYB to an inactive form activates a signaling pathway, leading to increased GA biosynthesis. Of 10 GA biosynthetic genes, expression of the 20-oxidase, AtGA20ox2, responded most to FR (up to a 40-fold increase within 3 h). AtGA20ox1 also responded but to a lesser extent. Stimulation of petiole elongation by EOD FR is reduced in a transgenic AtGA20ox2 hairpin gene silencing line. By contrast, it was only in SD that a T-DNA insertional mutant of AtGA20ox1 (ga5-3) showed reduced response. Circadian entrainment to a daytime pattern provides an explanation for the SD expression of AtGA20ox1. Conversely, the strong EOD/LD FR responses of AtGA20ox2 may reflect its independence of circadian regulation. While FR acting via PHYB increases expression of AtGA20ox2, other GA biosynthetic genes are known to respond to R rather than FR light and/or to other PHYs. Thus, there must be different signal transduction pathways, one at least showing a positive response to active PHYB and another showing a negative response.     

Initiation of dedifferentiation and structural changes in in vitro cultured petiole of Arabidopsis thaliana

Although the method of tissue culturing has been used widely in practice for a long time, and there are numerous hypotheses to explain the dedifferentiation phenomenon in the tissue culturing, many details of mechanism of dedifferentiation remain unclear. In the study, dedifferentiation process is initiated in the residual procambium, followed by the procambium-derived cells and finally xylem parenchyma cells under the culturing of Arabidopsis thaliana petiole explants. The procambium may induce its derivative cells to undergo dedifferentiation, which in turn induce the xylem parenchyma cells to dedifferentiate. This phenomenon is very similar to the activity of interfascicular cambium induced by intrafascicular cambium in secondary growth of plant stems. In the present study, only the paired procambium-derived cells and xylem parenchyma truly underwent dedifferentiation, whereas the initial changes in the procambium simply recovered the inherent meristematic capacity of those cells. In transverse section of petiole of A. thaliana, parenchyma cells outside the vascular bundle did not participate in dedifferentiation and gradually disintegrated under the culture conditions. Obviously, the time for initiation and difficulty underlain for undergoing dedifferentiation are dependent on the differential degree and location of parenchyma cells in the petiole.     

Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling

In plants, nucleoside diphosphate kinases (NDPKs) play a key role in the signaling of both stress and light. However, little is known about the structural elements involved in their function. Of the three NDPKs (NDPK1-NDPK3) expressed in Arabidopsis thaliana, NDPK2 is involved in phytochrome-mediated signal transduction. In this study, we found that the binding of dNDP or NTP to NDPK2 strengthens the interaction significantly between activated phytochrome and NDPK2. To better understand the structural basis of the phytochrome-NDPK2 interaction, we determined the X-ray structures of NDPK1, NDPK2, and dGTP-bound NDPK2 from A.thaliana at 1.8A, 2.6A, and 2.4A, respectively. The structures showed that nucleotide binding caused a slight conformational change in NDPK2 that was confined to helices alphaA and alpha2. This suggests that the presence of nucleotide in the active site and/or the evoked conformational change contributes to the recognition of NDPK2 by activated phytochrome. In vitro binding assays showed that only NDPK2 interacted specifically with the phytochrome and the C-terminal regulatory domain of phytochrome is involved in the interaction. A domain swap experiment between NDPK1 and NDPK2 showed that the variable C-terminal region of NDPK2 is important for the activation by phytochrome. The structure of Arabidopsis NDPK1 and NDPK2 showed that the isoforms share common electrostatic surfaces at the nucleotide-binding site, but the variable C-terminal regions have distinct electrostatic charge distributions. These findings suggest that the binding of nucleotide to NDPK2 plays a regulatory role in phytochrome signaling and that the C-terminal extension of NDPK2 provides a potential binding surface for the specific interaction with phytochromes.     

Control of hypocotyl elongation in Arabidopsis thaliana by photoreceptor interaction

In order to test the interaction of different phytochromes and blue-light receptors, etiolated seedlings of wild-type Arabidopsis thaliana (L.) Heynh., a phytochrome (phy) B-overexpressor line (ABO), and the photoreceptor mutants phyA-201, phyB-5, hy4-2.23n, fha-1, phyA-201/phyB-5, and phyA-201/hy4-2.23n were exposed to red and far-red light pulses after various preirradiations. The responsiveness to the inductive red pulses is primarily mediated by phyB which is rather stable in its far-red-absorbing form as demonstrated by a very slow loss of reversibility. Without preirradiation the red pulses had an impact on hypocotyl elongation only in PHYA mutants but not in the wild type. This indicates a suppression of phyB function by the presence of phyA. Preirradiation with either far-red or blue light resulted in an inhibition of hypocotyl elongation by red pulses in the wild type. Responsiveness amplification by far-red light is mediated by phyA and disappears slowly in the dark. The extent of responsiveness amplification by blue light was identical in the wild type and in the absence of phyA, or the cryptochromes cryl (hy4-2.23n) or cry2 (fha-1). Therefore, we conclude that stimulation of phyB by blue light preirradiation is either mediated by an additional still-unidentified blue-light-absorbing pigment or that phyA, cry1 and cry2 substitute for each other completely. Both blue and red preirradiation established responsiveness to red pulses in phyA-201/phyB-5 double mutants. These results demonstrate that inhibition of hypocotyl elongation by red pulses is not only mediated by phyB but also by a phytochrome(s) other than phyA and phyB. Received: 21 July 1998 / Accepted: 7 December 1998     

Ethylene-induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion

? Hyponastic growth is an upward petiole movement induced by plants in response to various external stimuli. It is caused by unequal growth rates between adaxial and abaxial sides of the petiole, which bring rosette leaves to a more vertical position. The volatile hormone ethylene is a key regulator inducing hyponasty in Arabidopsis thaliana. Here, we studied whether ethylene-mediated hyponasty occurs through local stimulation of cell expansion and whether this involves the reorientation of cortical microtubules (CMTs). ? To study cell size differences between the two sides of a petiole in ethylene and control conditions, we analyzed epidermal imprints. We studied the involvement of CMT orientation in epidermal cells using the tubulin marker line as well as genetic and pharmacological means of CMT manipulation. ? Our results demonstrate that ethylene induces cell expansion at the abaxial side of the- petiole and that this can account for the observed differential growth. At the abaxial side, ethylene induces CMT reorientation from longitudinal to transverse, whereas, at the adaxial side, it has an opposite effect. The inhibition of CMTs disturbed ethylene-induced hyponastic growth. ? This work provides evidence that ethylene stimulates cell expansion in a tissue-specific manner and that it is associated with tissue-specific changes in the arrangement of CMTs along the petiole.     

Distinctive core histone post-translational modification patterns in Arabidopsis thaliana

Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants.     

The effect of 2,4-D and kinetin on dedifferentiation of petiole cells in Arabidopsis thaliana

Phytohormones are indispensable factors regulating plant cell dedifferentiation. In this paper, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) were incorporated in the culture medium and the anatomy of dedifferentiated cells prior to callus formation from Arabidopsis thaliana petiole explants was examined. The results indicated that the cytoplasm of parenchyma cells in the vascular bundle gradually became denser with time of culture only if 2,4-D was included in the medium. The WUSCHEL (WUS) gene was expressed in derivative cells of the vascular bundle after culture for 24 h in the presence of 2,4-D and there was no obvious signal in these cells of cultured petioles with KIN alone. These results suggest that 2,4-D plays an important role in the process of dedifferentiation of vascular bundle cells in Arabidopsis petioles and KIN has no obvious effect on it.     

Cell wall proteins in apoplastic fluids of Arabidopsis thaliana rosettes: identification by mass spectrometry and bioinformatics

Weakly bound cell wall proteins of Arabidopsis thaliana were identified using a proteomic and bioinformatic approach. An efficient protocol of extraction based on vacuum-infiltration of the tissues was developed. Several salts and a chelating agent were compared for their ability to extract cell wall proteins without releasing cytoplasmic contaminants. Of the 93 proteins that were identified, a large proportion (60%) was released by calcium chloride. From bioinformatics analysis, it may be predicted that most of them (87 out of 93) had a signal peptide, whereas only six originated from the cytoplasm. Among the putative apoplastic proteins, a high proportion (67 out of 87) had a basic pI. Numerous glycoside hydrolases and proteins with interacting domains were identified, in agreement with the expected role of the extracellular matrix in polysaccharide metabolism and recognition phenomena. Ten proteinases were also found as well as six proteins with unknown functions. Comparison of the cell wall proteome of rosettes with the previously published cell wall proteome of cell suspension cultures showed a high level of cell specificity, especially for the different members of several large multigenic families.     

表油菜素内酯对拟南芥细胞分化的影响

研究了表油菜素内酯（ｅｐｉ－ＢＲ）对拟南芥细胞体外分化的影响．表明ｅｐｉ－ＢＲ不仅能促进愈伤组织的增殖,而且还能有效地诱导愈伤组织转绿,继而分化绿芽和长成小植株,其诱导频率高达７０％以上。电镜观察表明,ｅｐｉ－ＢＲ诱导的转绿细胞中的叶绿体发育正常。     

拟南芥细胞中存在中间纤维的研究

利用整装电镜制样与选择性抽提技术,在拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ （Ｌ．） Ｈｅｙｎｈ） 愈伤组织细胞质中观察到直径１０ ｎｍ 左右的纤维网络结构。免疫印迹分析表明纤维的主要成分是６ 种多肽,它们分别与动物角蛋白单克隆抗体ＡＥ１ 、ＡＥ３ 有免疫交叉反应。利用间接免疫荧光技术,与ＡＥ１ 和ＡＥ３ 反应的抗原呈弥散状定位于整个细胞质中,而且１０ ｎｍ 纤维可以在体外重新组装。以上结果表明,在拟南芥细胞质中存在类角蛋白的中间纤维。以动物中间纤维基因的保守序列为引物,采用ＲＴ＿ＰＣＲ技术,进一步从这一模式植物中克隆到一个ｃＤＮＡ片段,这可能为从分子水平上证明植物中间纤维的存在提供了一个线索     

Cell wall integrity controls root elongation via a general 1-aminocyclopropane-1-carboxylic acid-dependent, ethylene-independent pathway

Cell expansion in plants requires cell wall biosynthesis and rearrangement. During periods of rapid elongation, such as during the growth of etiolated hypocotyls and primary root tips, cells respond dramatically to perturbation of either of these processes. There is growing evidence that this response is initiated by a cell wall integrity-sensing mechanism and dedicated signaling pathway rather than being an inevitable consequence of lost structural integrity. However, the existence of such a pathway in root tissue and its function in a broader developmental context have remained largely unknown. Here, we show that various types of cell wall stress rapidly reduce primary root elongation in Arabidopsis (Arabidopsis thaliana). This response depended on the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC). In agreement with the established ethylene signaling pathway in roots, auxin signaling and superoxide production are required downstream of ACC to reduce elongation. However, this cell wall stress response unexpectedly does not depend on the perception of ethylene. We show that the short-term effect of ACC on roots is partially independent of its conversion to ethylene or ethylene signaling and that this ACC-dependent pathway is also responsible for the rapid reduction of root elongation in response to pathogen-associated molecular patterns. This acute response to internal and external stress thus represents a novel, noncanonical signaling function of ACC.