Differential expression of AtXTH17, AtXTH18, AtXTH19 and AtXTH20 genes in Arabidopsis roots. Physiological roles in specification in cell wall construction

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of enzymes that are capable of splitting and reconnecting xyloglucan molecules, and are implicated in the construction and restructuring of the cellulose/xyloglucan framework. Thirty-three members of the XTH gene family are found in the genome of Arabidopsis thaliana, but their roles remain unclear. Here, we describe the tissue-specific and growth stage-dependent expression profiles of promoter::GUS fusion constructs for four Arabidopsis XTH genes, AtXTH17, AtXTH18, AtXTH19 and AtXTH20, which are phylogenetically closely related to one another. AtXTH17 and AtXTH18 were expressed in all cell types in the elongating and differentiating region of the root, while AtXTH19 was expressed in the apical dividing and elongating regions, as well as in the differentiation zone, and was up-regulated by auxin. In contrast, AtXTH20 was expressed specifically in vascular tissues in the basal mature region of the root. This expression analysis also disclosed cis-regulatory sequences that are conserved among the four genes, and are responsible for the root-specific expression profile. These results indicate that the four XTH genes, which were generated by gene duplication, have diversified their expression profile within the root in such a way as to take responsibility for particular physiological roles in the cell wall dynamics.     

A xyloglucan endotransglucosylase/hydrolase involves in growth of primary root and alters the deposition of cellulose in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of enzymes that mediate the construction and restructure of the cellulose/xyloglucan framework by splitting and reconnecting xyloglucan molecule cross-linking among cellulose microfibrils. Remodification of cellulose microfibrils within cell-wall matrices is realized to be one of the most critical steps in the regulation of cells expansion in plants. Thirty-three XTH genes have been found in Arabidopsis thaliana but their roles remain unclear. AtXTH21 (At2g18800), an Arabidopsis XTH gene that mainly expresses in root and flower, exhibits different expression profiles from other XTH members under hormone treatment. We examined loss-of-function mutants using T-DNA insertion lines and overexpression lines and found that the AtXTH21 gene played a principal role in the growth of the primary roots by altering the deposition of cellulose and the elongation of cell wall.     

Effect of hypergravity stimulus on XTH gene expression in Arabidopsis thaliana.

Hypergravity stimulus suppresses plant shoot growth by making the cell wall rigid. Xyloglucan endotransglucosylase/hydrolase (XTH) is involved in determining the rigidity of cell walls. We demonstrated that hypergravity influenced the expression of some XTH genes in shoots of Arabidopsis thaliana L.; in response to hypergravity stimulus of 300 g, the expression of AtXTH22 was up-regulated, while that of AtXTH15 was down-regulated. The effect of hypergravity on the expression of these genes was nullified by lanthanum chloride at 0.1 mM, suggesting that the expression of these XTH genes in Arabidopsis is under the control of the mechanoreceptor.     

A principal role for AtXTH18 in Arabidopsis thaliana root growth: a functional analysis using RNAi plants

Rearrangement of cellulose microfibrils within cell-wall matrices is considered one of the most critical steps in the regulation of both the orientation and extent of cell expansion in plants. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of load-bearing cross links among cellulose microfibrils. The Arabidopsis thaliana XTH genes AtXTH17, 18, 19, and 20 are phylogenetically closely related to one another and are preferentially expressed in the roots. However, they exhibit different expression profiles within the root and respond to hormonal signals differently. To investigate their functions in root growth, we examined phenotypes of loss-of-function mutants for these genes using T-DNA insertion lines and RNAi plants. These functional analyses disclosed a principal role for the AtXTH18 gene in primary root elongation. Of the four XTH genes, AtXTH18 exhibits the highest level of mRNA expression. We also determined auxin-signaling pathways for these genes using a mutant with a defect in the AXR2/IAA7 gene and found that the expression of AtXTH19 in the elongation/maturation region of the root is under the control of the AXR2/IAA7 signaling pathway.     

The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis

The plant leaf provides an ideal system to study the mechanisms of organ formation and morphogenesis. The key factors that control leaf morphogenesis include the timing, location and extent of meristematic activity during cell division and differentiation. We identified an Arabidopsis mutant in which the regulation of meristematic activities in leaves was aberrant. The recessive mutant allele blade-on-petiole1-1 (bop1-1) produced ectopic, lobed blades along the adaxial side of petioles of the cotyledon and rosette leaves. The ectopic organ, which has some of the characteristics of rosette leaf blades with formation of trichomes in a dorsoventrally dependent manner, was generated by prolonged and clustered cell division in the mutant petioles. Ectopic, lobed blades were also formed on the proximal part of cauline leaves that lacked a petiole. Thus, BOP1 regulates the meristematic activity of leaf cells in a proximodistally dependent manner. Manifestation of the phenotypes in the mutant leaves was dependent on the leaf position. Thus, BOP1 controls leaf morphogenesis through control of the ectopic meristematic activity but within the context of the leaf proximodistality, dorsoventrality and heteroblasty. BOP1 appears to regulate meristematic activity in organs other than leaves, since the mutation also causes some ectopic outgrowths on stem surfaces and at the base of floral organs. Three class I knox genes, i.e., KNAT1, KNAT2 and KNAT6, were expressed aberrantly in the leaves of the bop1-1 mutant. Furthermore, the bop1-1 mutation showed some synergistic effect in double mutants with as1-1 or as2-2 mutation that is known to be defective in the regulation of meristematic activity and class I knox gene expression in leaves. The bop1-1 mutation also showed a synergistic effect with the stm-1 mutation, a strong mutant allele of a class I knox gene, STM. We, thus, suggest that BOP1 promotes or maintains a developmentally determinate state in leaf cells through the regulation of class I knox genes.     

<Emphasis Type="Italic">ASYMMETRIC LEAVES1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis</Emphasis> gene that is involved in the control of cell differentiation in leaves

During leaf development, the formation of dorsal-ventral and proximal-distal axes is central to leaf morphogenesis. To investigate the genetic basis of dorsoventrality and proximodistality in the leaf, we screened for mutants of Arabidopsis thaliana (L.) Heynh. with defects in leaf morphogenesis. We describe here the phenotypic analysis of three mutant alleles that we have isolated. These mutants show varying degrees of abnormality including dwarfism, broad leaf lamina, and aberrant floral organs and fruits. Genetic analysis revealed that these mutations are alleles of the previously isolated mutant asymmetric leaves1 ( as1). In addition to the leaf phenotypes described previously, these alleles display other phenotypes that were not observed. These include: (i) some rosette leaves with petiole growth underneath the leaf lamina; (ii) leaf vein branching in the petiole; and (iii) a leaf lamina with an epidermis similar to that on the petiole. The mutant phenotypes suggest that the ASYMMETRIC LEAVES1 ( AS1) gene is involved in the control of cell differentiation in leaves. As the first step in determining a molecular function for AS1, we have identified the AS1 gene using map-based cloning. The AS1 gene encodes a MYB-domain protein that is homologous to the Antirrhinum PHANTASTICA ( PHAN) and maize ROUGH SHEATH2 ( RS2) genes. AS1 is expressed nearly ubiquitously, consistent with the pleiotropic mutant phenotypes. High levels of AS1 expression were found in tissues with highly proliferative cells, which further suggests a role in cell division and early cell differentiation.     

A series of novel mutants of Arabidopsis thaliana that are defective in the formation of continuous vascular network: calling the auxin signal flow canalization hypothesis into question

For the genetic analysis of molecular mechanisms underlying temporal and spatial regulation of vascular pattern formation, we isolated mutants of Arabidopsis thaliana that are impaired in vascular patterning. Microscopic examination of the cotyledonary venation of 3,400 M(3) lines led to the identification of 12 mutant lines. Genetic analysis of 8 of these mutant lines indicated that vein pattern formation in these lines resulted from monogenic recessive mutations in 7 different genes, designated VAN1 through VAN7. Mutations in VAN1 through VAN6 genes caused fragmentation (disconnection or partial loss) of lateral veins of the cotyledon and tertiary veins of the rosette leaf whereas they were less injurious to the formation of major veins. Detailed characterization of the van3 mutant using pAthb8::GUS and pTED3::GUS, as molecular markers for the early stage of vascular tissue formation showed that the provascular tissue of the cotyledonary lateral veins was differentiated in fragments during late embryogenesis. These phenotypes of the van mutants are discussed in relation to the auxin signal flow canalization hypothesis and the diffusion-reaction prepattern hypothesis, with the fragility of the continuity in the minor vein formation favoring the latter hypothesis.     

The AtXTH28 Gene, a Xyloglucan Endotransglucosylase/Hydrolase, is Involved in Automatic Self-Pollination in Arabidopsis thaliana

Successful automatic self-pollination in flowering plants isdependent on the correct development of reproductive organs.In the stamen, the appropriate growth of the filament, whichlargely depends on the mechanical properties of the cell wall,is required to position the anther correctly close to the stigmaat the pollination stage. Xyloglucan endotransglucosylase/hydrolases(XTHs) are a family of enzymes that mediate the constructionand restructuring of xyloglucan cross-links, thereby controllingthe extensibility or mechanical properties of the cell wallin a wide variety of plant tissues. Our reverse genetic analysishas revealed that a loss-of-function mutation of an ArabidopsisXTH family gene, AtXTH28, led to a decrease in capability forself-pollination, probably due to inhibition of stamen filamentgrowth. Our results also suggest that the role of AtXTH28 inthe development of the stamen is not functionally redundantwith its closest paralog, AtXTH27. Thus, our finding indicatesthat AtXTH28 is specifically involved in the growth of stamenfilaments, and is required for successful automatic self-pollinationin certain flowers in Arabidopsis thaliana.     

拟南芥莲座叶片发育过程中P1基因的表达特性

该实验对CDF1类似蛋白基因(P1)在拟南芥叶片发育不同阶段的定量PCR结果显示,P1基因在拟南芥叶片发育的所有时期均可表达,但在茎生叶和衰老叶中的表达水平明显高于成熟叶和幼叶。GUS报告基因表达的组织化学染色结果显示,P1启动子在拟南芥叶片中有较高的驱动活性;在营养生长阶段的幼苗和植株(4~5周)的所有叶片中均能检测到GUS表达,但在植株转入生殖生长阶段后(6周及以后),GUS表达主要集中在逐渐衰老的叶中,并随着叶片衰老程度加剧GUS染色程度也越深,这一结果与GUS荧光定量检测结果一致。通过分析P1基因启动子上可能存在的顺式调控元件,发现茉莉酸甲酯、热压、干旱和水杨酸等均能够引起叶片衰老调控元件的响应,证实P1的表达受到这些因素的调控。研究表明,P1在拟南芥莲座叶片中很可能参与了对上游衰老信号的响应,该研究结果为进一步探究P1在叶片衰老过程中的分子功能验证奠定了基础。     

Mutations in the RETICULATA gene dramatically alter internal architecture but have little effect on overall organ shape in Arabidopsis leaves

A number of mutants have been described in Arabidopsis, whose leaf vascular network can be clearly distinguished as a green reticulation on a paler lamina. One of these reticulate mutants was named reticulata (re) by Rédei in 1964 and has been used for years as a classical genetic marker for linkage analysis. Seven recessive alleles of the RE gene were studied, at least four of which seem to be null. Contrary to many other leaf mutants studied in Arabidopsis, very little pleiotropy was observed in the external morphology of the re mutants, whose only aberration obvious at first sight is the reticulation exhibited by cotyledons and leaves. The re alleles caused a marked reduction in the density of mesophyll cells in interveinal regions of the leaf, which does not result from perturbed plastid development in specific cells, but rather from a dramatic change in internal leaf architecture. Loss of function of the RE gene seems to specifically perturb mesophyll cell division in the early stages of leaf organogenesis. The leaves of re mutants were nearly normal in shape in spite of their extremely reduced mesophyll cell density, suggesting that the epidermis plays a major role in regulating leaf shape in Arabidopsis. The RE gene was positionally cloned and found to be expressed in all the major organs studied. RE encodes a protein of unknown function and is identical to the LCD1 gene, which was identified based on the increased sensitivity to ozone caused by its mutant allele lcd1-1. Double mutant analyses suggest that RE acts in a developmental pathway that involves CUE1 but does not include DOV1.     

Transgenic analysis of sugar beet xyloglucan endo-transglucosylase/hydrolase Bv-XTH1 and Bv-XTH2 promoters reveals overlapping tissue-specific and wound-inducible expression profiles

The identification and analysis of tissue-specific gene regulatory elements will improve our knowledge of the molecular mechanisms that control the growth and development of different plant tissues and offer potentially useful tools for the genetic engineering of plants. A polymerase chain reaction (PCR)-based 5'-genome walk from sequences of an isolated sugar beet xyloglucan endo-transglucosylase hydrolase (XTH) gene led to the isolation of two independent upstream fragments. They were 1332 and 2163 base pairs upstream of the XTH ATG start site, respectively. In vivo transgenic assays in sugar beet hairy roots and Arabidopsis thaliana revealed that both fragments had promoter function and, in A. thaliana, directed expression in vascular tissues within the root, leaves and petals. Promoter activity was also observed in the leaf trichomes and within rapidly expanding stem internodes. Expression driven by both promoters was found to be wound inducible. Overall, the spatial and temporal expression pattern of these promoters suggested that the corresponding Bv-XTH genes (designated Bv-XTH1 and Bv-XTH2) may be involved in secondary cell wall formation. This work provides new insights on molecular mechanisms that could be exploited for the genetic engineering of sugar beet crops.     

Effect of hypergravity stimulus on XTH gene expression in Arabidopsis thaliana.

There are several reports indicating that hypergravity and microgravity influence the mechanical properties of cell walls in shoots, resulting in changes in the growth rate. The mechanical properties of cell walls in dicots are mainly determined by the physicochemical properties of xyloglucan, a matrix polysaccharide. An increase in the molecular mass of xyloglucan correlated with a decrease in cell wall extensibility. Hypergravity is known to increase the molecular mass of xyloglucan. The cell wall enzyme, xyloglucan endotransglucosylase/hydrolase (XTH) is involved in xyloglucan metabolism. Using Arabidopsis, it was examined whether or not the expression of XTH genes in the floral stem and rosette leaf is influenced by hypergravity. RT-PCR analysis revealed that the expression of XTH genes changes in response to hypergravity of 300 g.     

KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis.

Plant development depends on the activity of apical meristems, which are groups of indeterminate cells whose derivatives elaborate the organs of the mature plant. Studies of knotted1 (kn1) and related gene family members have determined potential roles for homeobox genes in the function of shoot meristems. The Arabidopsis kn1-like gene, KNAT1, is expressed in the shoot apical meristem and not in determinate organs. Here, we show that ectopic expression of KNAT1 in Arabidopsis transforms simple leaves into lobed leaves. The lobes initiate in the position of serrations yet have features of leaves, such as stipules, which form in the sinus, the region at the base of two lobes. Ectopic meristems also arise in the sinus region close to veins. Identity of the meristem, that is, vegetative or floral, depends on whether the meristem develops on a rosette or cauline leaf, respectively. Using in situ hybridization, we analyzed the expression of KNAT1 and another kn1-like homeobox gene, SHOOT MERISTEMLESS, in cauliflower mosaic virus 35S::KNAT1 transformants. KNAT1 expression is strong in vasculature, possibly explaining the proximity of the ectopic meristems to veins. After leaf cells have formed a layered meristem, SHOOT MERISTEMLESS expression begins in only a subset of these cells, demonstrating that KNAT1 is sufficient to induce meristems in the leaf. The shootlike features of the lobed leaves are consistent with the normal domain of KNAT1's expression and further suggest that kn1-related genes may have played a role in the evolution of leaf diversity.     

Modification of cell proliferation patterns alters leaf vein architecture in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Formation of leaf vascular pattern requires regulation of a number of cellular processes, including cell proliferation. To assess the role of cell proliferation during vein order formation, leaf development in genetic lines exhibiting aberrant cell proliferation patterns due to altered expression patterns of ANT and ICK1 genes was analyzed. Modification of cell proliferation patterns alters the number of higher order veins and the number of minor tertiary veins remodeled as intersecondary veins in Arabidopsis rosette leaves. Minor vein complexity, as indicated by branch point and freely ending veinlet number, is highly correlated with a decrease or increase in cell proliferation. Observations of procambial strand formation in modified cell proliferation pattern lines showed that vein pattern is specified early in leaf development and that formation of freely ending veinlets is temporally correlated with the expansion of ground meristem when cell proliferation is terminated prematurely. Taken together, our observations indicate that: (1) genes that modulate cell proliferation play a key role in regulating the meristematic competence of ground meristem cells to form procambium and vein pattern during leaf development, and (2) ANT is a crucial part of this regulation.