Morphological analysis of seed shape in Arabidopsis thaliana reveals altered polarity in mutants of the ethylene signaling pathway

The shape of Arabidopsis thaliana dry seed is described here as a prolate spheroid. The accuracy of this approximation is discussed. Considering its limitations, it allows a geometric approximation to the analysis of changes occurring in seed shape during imbibition prior to seed germination as well as the differences in shape between genotypes and their changes during imbibition. The triple mutant ein2-1, ers1-2, etr1-7 presents notable alterations in seed shape. In addition, seeds of this and other mutants in the ethylene signaling pathway (ctr1-1, eto1-1, etr1-1, ein2-1) show different response to imbibition than the wild type. Imbibed seeds of the wild type increase their asymmetry compared with the dry seeds. This is detected by the relative changes in the curvature values in both poles. Thus, during imbibition of the wild-type seeds, the reduction in curvature values observed in the basal pole gives them an ovoid shape. In contrast, in the seeds of the ethylene mutants, reduction in curvature values occurs in both basal and apical poles, and its shape remains as a prolate spheroid. Our data indicate that the ethylene signaling pathway is involved, in general, in the complex regulation of seed shape and, in particular, in the establishment of polarity in seeds, controlling curvature values in the seed poles.     

Ethylene responses in Arabidopsis seedlings include the reduction of curvature values in the root cap

Recently, curvature was described as a new trait useful in the analysis of root apex shape. Treating the root profile as a geometric curve revealed that root apex curvature values are lower in ethylene-insensitive mutants (Cervantes E, Tocino A. Geometric analysis of Arabidopsis root apex reveals a new aspect of the ethylene signal transduction pathway in development. J Plant Physiol 2005;162:1038-45). This fact suggests that curvature is regulated by ethylene. In this work, we have determined the curvature values in embryonic roots of wild-type Columbia as well as in ethylene signal-transduction mutants, and found smaller values in embryos of the mutants. We also report on the evolution of root curvature during early development after seed germination. The line Lt16b that expresses GFP in the cell wall has allowed us to investigate the evolution of curvature values in three successive cell layers of seedling roots by confocal microscopy. Treatment of seedlings with norbornadiene resulted in lower curvature values. Our results show details illustrating the effect of ethylene in root curvature.     

Dynamic analysis of Arabidopsis seed shape reveals differences in cellulose mutants

In a previous work, the shape of Arabidopsis seed was described as a cardioid modified by a factor of Phi. In addition, J index was defined as the similarity of the seed (in an orthogonal, bi-dimensional image) to a cardioid, thus allowing the quantitative comparison of seed shape in seeds of varieties and mutants at different stages of development. Here, J index is used for modeling changes in seed morphology during the dynamic process of seed imbibition before germination. The analysis was carried out by means of a general linear model with two fixed factors (genotype and time) applied to two Arabidopsis varieties: Columbia and Wassilewskija and two mutants in cellulose synthesis: prc1-1 (CESA6 in Columbia) and kor1-1 (in Wassilewskija). Equations representing the changes in seed form during imbibition are given. The analysis of changes in seed shape by this procedure provides (1) a quantitative method to record changes in seed shape and to compare between genotypes or treatments showing the time points with maximum differences, and (2) the observation of remarkable differences between wild-type seeds and mutants in cellulose biosynthesis, indicating new phenotypic characteristics previously unknown in the latter. While wild-type seeds increase their J index values during imbibition, in the cellulose mutants J index values decrease. In addition, shape comparisons were done with other mutants. Seeds of ga1-1 mutants behave like cellulose mutants, whereas different ethylene mutants present varied responses. Quantitative analysis of seed morphology is a new basis for the record of differences between wild-type and mutants as well as for phenotypic characterization.     

Seed shape in model legumes: Approximation by a cardioid reveals differences in ethylene insensitive mutants of Lotus japonicus and Medicago truncatula

Seed shape in the model legumes Lotus japonicus and Medicago truncatula is described. Based in previous work with Arabidopsis, the outline of the longitudinal sections of seeds is compared with a cardioid curve. L. japonicus seeds adjust well to an unmodified cardioid, whereas accurate adjustment in M. truncatula is obtained by the simple transformation of scaling the vertical axis by a factor equal to the Golden Ratio. Adjustments of seed shape measurements with simple geometrical forms are essential tools for the statistical analysis of variations in seed shape under different conditions or in mutants. The efficiency of the adjustment to a cardioid in the model plants suggests that seed morphology may be related to genome complexity. Seeds of ethylene insensitive mutants present differences in size and shape as well as altered responses to imbibition. The biological implication and meaning of these relationships are discussed.     

Hydrogen peroxide treatment results in reduced curvature values in the Arabidopsis root apex

Curvature of a plane curve is a measurement related to its shape. A Mathematica code was developed [Cervantes E, Tocino A. J Plant Physiol 2005;162:1038-1045] to obtain parametric equations from microscopic images of the Arabidopsis thaliana root apex. In addition, curvature values for these curves were given. It was shown that ethylene-insensitive mutants (etr1-1 and ein2-1) have reduced curvature values in the root apex. It has also been shown that blocking ethylene action by norbornadiene, an ethylene inhibitor, results in reduced curvature values in the two outer cell layers of the root apex [Noriega A, Cervantes E, Tocino A. J Plant Physiol 2008, in press]. Because ethylene action has been related with hydrogen peroxide [Desikan R, Hancock JT, Bright J, Harrison J, Weir I, Hooley R, Neill SJ. Plant Physiol 2005;137:831-834], the effect of a treatment with hydrogen peroxide in the curvature values of three successive layers of the root apex in Arabidopsis thaliana was investigated by confocal microscopy. Treatment with 10mM hydrogen peroxide resulted in reduced curvature values in the three layers. The effect was associated with smaller cells having higher circularity indices. The results are discussed in the context of the role of ethylene in development.     

Ethylene modulates flavonoid accumulation and gravitropic responses in roots of Arabidopsis

Plant organs change their growth direction in response to reorientation relative to the gravity vector. We explored the role of ethylene in Arabidopsis (Arabidopsis thaliana) root gravitropism. Treatment of wild-type Columbia seedlings with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC) reduced root elongation and gravitropic curvature. The ethylene-insensitive mutants ein2-5 and etr1-3 had wild-type root gravity responses, but lacked the growth and gravity inhibition by ACC found in the wild type. We examined the effect of ACC on tt4(2YY6) seedlings, which have a null mutation in the gene encoding chalcone synthase, the first enzyme in flavonoid synthesis. The tt4(2YY6) mutant makes no flavonoids, has elevated indole-3-acetic acid transport, and exhibits a delayed gravity response. Roots of tt4(2YY6), the backcrossed line tt4-2, and two other tt4 alleles had wild-type sensitivity to growth inhibition by ACC, whereas the root gravitropic curvature of these tt4 alleles was much less inhibited by ACC than wild-type roots, suggesting that ACC may reduce gravitropic curvature by altering flavonoid synthesis. ACC treatment induced flavonoid accumulation in root tips, as judged by a dye that becomes fluorescent upon binding flavonoids in wild type, but not in ein2-5 and etr1-3. ACC also prevented a transient peak in flavonoid synthesis in response to gravity. Together, these experiments suggest that elevated ethylene levels negatively regulate root gravitropism, using EIN2- and ETR1-dependent pathways, and that ACC inhibition of gravity response occurs through altering flavonoid synthesis.     

A subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats

During Arabidopsis seed development large quantities of mucilage, composed of pectins, are deposited into the apoplast underneath the outer wall of the seed coat. Upon imbibition of mature seeds, the stored mucilage expands through hydration and breaks the outer cell wall that encapsulates the whole seed. Mutant seeds carrying loss-of-function alleles of AtSBT1.7 that encodes one of 56 Arabidopsis thaliana subtilisin-like serine proteases (subtilases) do not release mucilage upon hydration. Microscopic analysis of the mutant seed coat revealed no visible structural differences compared with wild-type seeds. Weakening of the outer primary wall using cation chelators triggered mucilage release from the seed coats of mutants. However, in contrast to mature wild-type seeds, the mutant's outer cell walls did not rupture at the radial walls of the seed coat epidermal cells, but instead opened at the chalazal end of the seed, and were released in one piece. In atsbt1.7, the total rhamnose and galacturonic acid contents, representing the backbone of mucilage, remained unchanged compared with wild-type seeds. Thus, extrusion and solubility, but not the initial deposition of mucilage, are affected in atsbt1.7 mutants. AtSBT1.7 is localized in the developing seed coat, indicating a role in testa development or maturation. The altered mode of rupture of the outer seed coat wall and mucilage release indicate that AtSBT1.7 triggers the accumulation, and/or activation, of cell wall modifying enzymes necessary either for the loosening of the outer primary cell wall, or to facilitate swelling of the mucilage, as indicated by elevated pectin methylesterase activity in developing atsbt1.7 mutant seeds.     

Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy

Freshly harvested seeds of Arabidopsis thaliana, Columbia (Col) accession were dormant when imbibed at 25°C in the dark. Their dormancy was alleviated by continuous light during imbibition or by 5 weeks of storage at 20°C (after-ripening). We investigated the possible role of reactive oxygen species (ROS) in the regulation of Col seed dormancy. After 24 h of imbibition at 25°C, non-dormant seeds produced more ROS than dormant seeds, and their catalase activity was lower. In situ ROS localization revealed that germination was associated with an accumulation of superoxide and hydrogen peroxide in the radicle. ROS production was temporally and spatially regulated: ROS were first localized within the cytoplasm upon imbibition of non-dormant seeds, then in the nucleus and finally in the cell wall, which suggests that ROS play different roles during germination. Imbibition of dormant and non-dormant seeds in the presence of ROS scavengers or donors, which inhibited or stimulated germination, respectively, confirmed the role of ROS in germination. Freshly harvested seeds of the mutants defective in catalase (cat2-1) and vitamin E (vte1-1) did not display dormancy; however, seeds of the NADPH oxidase mutants (rbohD) were deeply dormant. Expression of a set of genes related to dormancy upon imbibition in the cat2-1 and vet1-1 seeds revealed that their non-dormant phenotype was probably not related to ABA or gibberellin metabolism, but suggested that ROS could trigger germination through gibberellin signaling activation.     

拟南芥胼胝质合酶基因GSL8参与细胞壁形成和根端静止中心建立与维持

用T-DNA插入和RNA干扰技术敲除拟南芥(Arabidopsis thaliana)胼胝质合酶基因GSL8, 在光学和透射电子显微镜下观察野生型和突变体种子的细胞壁和胚根结构, 比较未敲除和敲除该基因幼苗细胞壁及根端分生组织结构。结果表明, 敲除该基因可导致细胞壁发育不良, 壁上出现大小不等的缺口, 缺口处没有质膜将相邻细胞分隔。用T-DNA插入法抑制该基因表达, 发现在种子发育阶段胚根不能形成正常的静止中心。用小RNA干扰技术抑制该基因表达, 结果显示根端失去静止中心。综合以上结果, 认为该基因不仅参与细胞壁发育, 也参与根端静止中心的建立与维持。     

Interactions between abscisic acid and ethylene signaling cascades

We screened for mutations that either enhanced or suppressed the abscisic acid (ABA)-resistant seed germination phenotype of the Arabidopsis abi1-1 mutant. Alleles of the constitutive ethylene response mutant ctr1 and ethylene-insensitive mutant ein2 were recovered as enhancer and suppressor mutations, respectively. Using these and other ethylene response mutants, we showed that the ethylene signaling cascade defined by the ETR1, CTR1, and EIN2 genes inhibits ABA signaling in seeds. Furthermore, epistasis analysis between ethylene- and ABA-insensitive mutations indicated that endogenous ethylene promotes seed germination by decreasing sensitivity to endogenous ABA. In marked contrast to the situation in seeds, ein2 and etr1-1 roots were resistant to both ABA and ethylene. Our data indicate that ABA inhibition of root growth requires a functional ethylene signaling cascade, although this inhibition is apparently not mediated by an increase in ethylene biosynthesis. These results are discussed in the context of the other hormonal regulations controlling seed germination and root growth.     

Drought-induced Short Roots in Arabidopsis thaliana: Structural Characteristics

In Arabidopsis thaliana, as in other Brassicaceae species, a progressive drought stress induced changes in root morphogenesis: from a threshold plant water deficit, the new emerging roots remain short, hairless and often take a tuberized shape at their base while drought persists. The organization of these drought-induced roots was examined in light microscopy in Arabidopsis thaliana, Columbia wild-type ecotype, and compared to the normal, well-watered lateral roots. The main structural traits were the absence of elongation zone, the arrest of cell cap expansion, the lack of root hairs (despite epidermal differentiation in trichoblasts and atrichoblasts) and the radial enlargement of epidermal and cortical cells. The early differentiation, close to the short root apex, of large and highly lignified metaxylem elements, the absence of starch accumulation in hypertrophied cortical cells appeared to be characteristic of the species Arabidopsis, as compared to other Brassicaceae. These structural alterations are discussed in terms of drought-induced changes in gene expression with regard to similar modifications described in root morphogenesis and root hair-defective Arabidopsis mutants.     

Effects of the gibberellin biosynthetic inhibitor uniconazol on mutants of Arabidopsis

Using the gibberellin (GA) biosynthetic inhibitor Uniconazol, we determined that det1, a mutant that no longer requires light to be germinated, still requires GA synthesis for germination. This result suggests that dark inhibition of germination in Arabidopsis may be due to inhibition of GA synthesis by the DET1 gene product in mature wild-type seeds. Similar experiments with mutants that lack seed dormancy due to a reduced sensitivity to abscisic acid (abi) have shown that abi1 and abi3 no longer require GA for germination. Furthermore, by shifting wild-type seeds to inhibitor at 6-hour intervals during imbibition, we determined that GA synthesis is only required during the first 24 hours of the imbibition process to reverse abscisic acid-induced dormancy in Arabidopsis.     

Genetic differences in seed longevity of various Arabidopsis mutants

Seeds gradually lose their viability during dry storage. The damage that occurs at the biochemical level can alter the seed physiological status and is affected by the storage conditions of the seeds. Although these environmental conditions controlling loss of viability have been investigated frequently, little information is available on the genetics of seed longevity. Using Arabidopsis mutants in defined developmental or biochemical pathways such as those affected in seed coat composition, seed dormancy, hormone function and control of oxidative stress, we tried to gain insight into the genes and mechanisms controlling viability of stored seeds. Mutations like abscisic acid insensitive3 ( abi3 ) as well as abscisic acid deficient1 ( aba1 ) show reduced longevity, which may be partially related to the seed dormancy phenotype of these mutants. Mutants with seed coat alterations, especially aberrant tests shape ( ats ), showed a stronger reduction in germination percentage after storage, indicating the importance of a 'functional' seed coat for seed longevity. A specific emphasis was placed on mutants affected in dealing with Reactive Oxygen Species (ROS). Because several pathways are involved in protection against ROS and because gene redundancy is a common feature in Arabidopsis , 'double' mutants were generated. These 'double' mutants and the corresponding single mutants were subjected to a controlled deterioration test (CDT) and a germination assay on hydrogen peroxide (H₂O₂) after prolonged storage at two relative humidities. CDT and germination on H₂O₂ affected all genotypes, although it appears that other effects like genetic background are more important than the deficiencies in the ROS scavenging pathway. Explanations for this limited effect of mutations affecting ROS scavenging are discussed.     

The isolation and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana

Abscisic acid (ABA) insensitive mutants of Arabidopsis thaliana (L.) Heynh. were isolated by selecting plants which grew well on a medium containing 10 μ M ABA. From the progeny of approximately 3500 mutagen-treated seeds, five mutants of at least three different loci were isolated. Three mutants were characterized, moreover, by a reduced seed dormancy and by symptoms of withering, indicating disturbed water relations and, therefore, resembled phenotypically the ABA-deficient mutants we described earlier in this species. Two mutants showed in addition only a reduction of seed dormancy. Compared to wild type, all mutants showed similar or increased levels of endogenous ABA in developing seeds and fruits (siliquae). The role of the different genes involved is discussed in relation to the mechanism of ABA action.     

Novel software for analysis of root gravitropism: comparative response patterns of Arabidopsis wild-type and axr1 seedlings

In an earlier study (Evans, Ishikawa & Estelle 1994, Planta 194, 215-222) we used a video digitizer system to compare the kinetics of auxin action on root elongation in wild-type seedlings and seedlings of auxin response mutants of Arabidopsis thaliana (L.) Heynh. We have since modified the system software to allow determination of elongation on opposite sides of vertical or gravistimulated roots and to allow continuous measurement of the angle of orientation of sequential subsections of the root during the response. We used this technology to compare the patterns of differential growth that generate curvature in roots of the Columbia ecotype and in the mutants axr1-3, axr1-12 and axr2, which show reduced gravitropic responsiveness and reduced sensitivity to inhibition by auxin. The pattern of differential growth during gravitropism differed in roots of wild-type and axr1 seedlings. In wild-type roots, initial curvature resulted from differential inhibition of elongation in the distal elongation zone (DEZ). This was followed by an acceleration of elongation along the top side of the DEZ. In roots of axr1-3, curvature resulted from differential stimulation of elongation whereas in roots of axr1-12 the response was variable. Roots of axr2 did not exhibit gravitropic curvature. The observation that the pattern of differential growth causing curvature is dramatically altered by a change in sensitivity to auxin is consistent with the classical Cholodny-Went theory of gravitropism which maintains that differential growth patterns induced by gravistimulation are mediated primarily by gravi-induced shifts in auxin distribution. The new technology introduced with this report allows automated determination of stimulus response patterns in the small but experimentally popular roots of Arabidopsis.