Arabidopsis thaliana mitogen-activated protein kinase 6 is involved in seed formation and modulation of primary and lateral root development.

Mitogen-activated protein kinase (MAPKs) cascades are signal transduction modules highly conserved in all eukaryotes regulating various aspects of plant biology, including stress responses and developmental programmes. In this study, we characterized the role of MAPK 6 (MPK6) in Arabidopsis embryo development and in post-embryonic root system architecture. We found that the mpk6 mutation caused altered embryo development giving rise to three seed phenotypes that, post-germination, correlated with alterations in root architecture. In the smaller seed class, mutant seedlings failed to develop the primary root, possibly as a result of an earlier defect in the division of the hypophysis cell during embryo development, but they had the capacity to develop adventitious roots to complete their life cycle. In the larger class, the MPK6 loss of function did not cause any evident alteration in seed morphology, but the embryo and the mature seed were bigger than the wild type. Seedlings developed from these bigger seeds were characterized by a primary root longer than that of the wild type, accompanied by significantly increased lateral root initiation and more and longer root hairs. Apparently, the increment in primary root growth resulted from an enhanced cell production and cell elongation. Our data demonstrated that MPK6 plays an important role during embryo development and acts as a repressor of primary and lateral root development.

The lateral root initiation index: an integrative measure of primordium formation.

BACKGROUND AND AIMS: Lateral root initiation is an essential and continuous process in the formation of root systems; therefore, its quantitative analysis is indispensable. In this study a new measure of lateral root initiation is proposed and analysed, namely the lateral root initiation index (I(LRI)), which defines how many lateral roots and/or primordia are formed along a parent-root portion corresponding to 100 cortical cells in a file. METHODS: For data collection, a commonly used root clearing procedure was employed, and a new simple root clearing procedure is also proposed. The I(LRI) was determined as 100dl, where d is the density of lateral root initiation events (number mm(-1)) and l is the average fully elongated cortical cell length (mm). KEY RESULTS: Analyses of different Arabidopsis thaliana genotypes and of a crop plant, tomato (Solanum lycopersicum), showed that I(LRI) is a more precise parameter than others commonly used as it normalizes root growth for variations in cell length. Lateral root primordium density varied in the A. thaliana accessions Col, Ler, Ws, and C24; however, in all accessions except Ws, I(LRI) was similar under the same growth conditions. The nitrogen/carbon ratio in the growth medium did not change the lateral root primordium density but did affect I(LRI). The I(LRI) was also modified in a number of auxin-related mutants, revealing new root branching phenotypes in some of these mutants. The rate of lateral root initiation increased with Arabidopsis seedling age; however, I(LRI) was not changed in plants between 8 and 14 d post-germination. CONCLUSIONS: The I(LRI) allows for a more precise comparison of lateral root initiation under different growth conditions, treatments, genotypes and plant species than other comparable methods.

Determinate root growth and meristem maintenance in angiosperms.

BACKGROUND: The difference between indeterminate and determinate growth in plants consists of the presence or absence of an active meristem in the fully developed organ. Determinate root growth implies that the root apical meristem (RAM) becomes exhausted. As a consequence, all cells in the root tip differentiate. This type of growth is widely found in roots of many angiosperm taxa and might have evolved as a developmental adaptation to water deficit (in desert Cactaceae), or low mineral content in the soil (proteoid roots in various taxa). SCOPE AND CONCLUSIONS: This review considers the mechanisms of determinate root growth to better understand how the RAM is maintained, how it functions, and the cellular and genetic bases of these processes. The role of the quiescent centre in RAM maintenance and exhaustion will be analysed. During root ageing, the RAM becomes smaller and its organization changes; however, it remains unknown whether every root is truly determinate in the sense that its RAM becomes exhausted before senescence. We define two types of determinate growth: constitutive where determinacy is a natural part of root development; and non-constitutive where determinacy is induced usually by an environmental factor. Determinate root growth is proposed to include two phases: the indeterminate growth phase, when the RAM continuously produces new cells; and the termination growth phase, when cell production gradually decreases and eventually ceases. Finally, new concepts regarding stem cells and a stem cell niche are discussed to help comprehend how the meristem is maintained in a broad taxonomic context.

Lateral root initiation in Arabidopsis: developmental window, spatial patterning, density and predictability.

BACKGROUND AND AIMS: The basic regulatory mechanisms that control lateral root (LR) initiation are still poorly understood. An attempt is made to characterize the pattern and timing of LR initiation, to define a developmental window in which LR initiation takes place and to address the question of whether LR initiation is predictable. METHODS: The spatial patterning of LRs and LR primordia (LRPs) on cleared root preparations were characterized. New measures of LR and LRP densities (number of LRs and/or LRPs divided by the length of the root portions where they are present) were introduced and illustrate the shortcomings of the more customarily used measure through a comparative analysis of the mutant aux1-7. The enhancer trap line J0121 was used to monitor LR initiation in time-lapse experiments and a plasmolysis-based method was developed to determine the number of pericycle cells between successive LRPs. KEY RESULTS: LRP initiation occurred strictly acropetally and no de novo initiation events were found between already developed LRs or LRPs. However, LRPs did not become LRs in a similar pattern. The longitudinal spacing of lateral organs was variable and the distance between lateral organs was proportional to the number of cells and the time between initiations of successive LRPs. There was a strong tendency towards alternation in LR initiation between the two pericycle cell files adjacent to the protoxylem poles. LR density increased with time due to the emergence of slowly developing LRPs and appears to be unique for individual Arabidopsis accessions. CONCLUSIONS: In Arabidopsis there is a narrow developmental window for LR initiation, and no specific cell-count or distance-measuring mechanisms have been found that determine the site of successive initiation events. Nevertheless, the branching density and lateral organ density (density of LRs and LRPs) are accession-specific, and based on the latter density the average distance between successive LRs can be predicted.

Water deficit accelerates determinate developmental program of the primary root and does not affect lateral root initiation in a Sonoran Desert cactus (Pachycereus pringlei, Cactaceae).

Determinate root growth is an important adaptation feature for seedling establishment in some Cactaceae. We show that seedlings of Pachycereus pringlei have primary roots with a stable determinate developmental program. How water stress affects determinate root growth and lateral root development has not been studied. Here we address this question. Root growth was analyzed in plants growing in vitro under well-watered and water-deficient (created by polyethylene glycol) growth conditions. Under severe water stress roots terminated their growth earlier and the rate of growth was significantly decreased as a result of inhibition of both cell elongation and cell production. Under severe water stress the number of lateral roots and primordia per millimeter of primary root was 1.5-1.7 times greater than under well-watered conditions; however, the total number of lateral roots and primordia was the same under all conditions. Lateral roots resembled root spurs found in some Opuntioideae. Analysis of the dynamics of meristem exhaustion indicated that initial-cell activities are required for the maintenance of proliferation before meristem exhaustion. We conclude that lateral root formation is a stable developmental process resistant to severe water stress and that water stress accelerates the determinate developmental program of the primary root. Both of these features appear to be important for successful seedling establishment in a desert.

Early primordium morphogenesis during lateral root initiation in Arabidopsis thaliana.

The first morphogenetic events of lateral root primordium (LRP) formation in the Arabidopsis thaliana (L.) Heynh. pericycle occur soon after cells of the primary root complete elongation. Pericycle cells in direct contact with underlying protoxylem cells participate in LRP formation. Two types of LRP initiation were found, longitudinal uni- and bi-cellular. These occur when a single or two pericycle cells within a file, respectively, become founder cells for the entire longitudinal extent of the LRP. Histochemical and cytological analysis suggests that three is the minimum number of cells required to initiate an LRP. In young primordia comprising less than 32 cells, the average cell-doubling time was 3.7 h, indicating a drastic acceleration of cell cycle progression after lateral root initiation. Early in LRP development, cell growth is limited and therefore cytokinesis leads to a reduction of cell volume, similar to cleavage division cycles during animal and plant embryogenesis. The striking coordination of proliferation between pericycle cells in adjacent files in direct contact with the underlying protoxylem implies that intercellular signaling mechanisms act in the root apical meristem or later in development.

Pericycle cell proliferation and lateral root initiation in Arabidopsis.

In contrast with other cells generated by the root apical meristem in Arabidopsis, pericycle cells adjacent to the protoxylem poles of the vascular cylinder continue to cycle without interruption during passage through the elongation and differentiation zones. However, only some of the dividing pericycle cells are committed to the asymmetric, formative divisions that give rise to lateral root primordia (LRPs). This was demonstrated by direct observation and mapping of mitotic figures, cell-length measurements, and the histochemical analysis of a cyclin-GUS fusion protein in pericycle cells. The estimated duration of a pericycle cell cycle in the root apical meristem was similar to the interval between cell displacement from the meristem and the initiation of LRP formation. Developmentally controlled LRP initiation occurs early, 3 to 8 mm from the root tip. Thus the first growth control point in lateral root formation is defined by the initiation of primordia in stochastic patterns by cells passing through the elongation and young differentiation zones, up to where lateral roots begin to emerge from the primary root. Therefore, the first growth control point is not restricted to a narrow developmental window. We propose that late LRP initiation is developmentally unrelated to the root apical meristem and is operated by a second growth control point that can be activated by environmental cues. The observation that pericycle cells divide and lateral root primordia form without intervening mitotic quiescence suggests that lateral organ formation in roots and shoots might not be as fundamentally different as previously thought.

A squash preparation method for root meristem field studies.

We present a field method for squash preparation of root apical meristem that does not require dry ice for cell adhesion to a slide. The method is based on the use of a weak gelatin solution as a stabilizing agent to prevent cell loss. Alcian blue 8GX was used as a counterstain for better recognition of cell shapes. Root meristems of Sonoran Desert succulents were used to illustrate the method. Fixed root tips were stained by Feulgen and squashes were prepared in a 1:2 mixture of solution A (45% acetic acid) and solution B (3:1 mixture of 0.5% gelatin in water:0.02% of alcian blue 8GX in 45% acetic acid). After squashing, the material was lightly dried for 5-6 min, then dehydrated and mounted in Canada balsam. Our method permits observation of chromosomes, recognition of meristematic and nonmeristematic cells, and estimation of mitotic indices and thus recognition of growing, dormant, and dead roots. The method can be used for estimation of cell division cycle time by colchicine and other methods where squash preparation is required.