Limited growth of the first root
The first root of a seedling develops from the embryonic radicle and forms the initial axis of the underground plant. Its elongation depends on the production of new cells in the root apical meristem and on their subsequent increase in length. The meristem is a region that retains the capacity for cell division. As development proceeds, cells leave this region, expand and acquire the characteristics of particular tissues. In Pachycereus pringlei, gradual differentiation eventually also encompasses the primary root apex itself. [Dubrovsky and Gómez-Lomelí 2003] [Rodriguez-Alonso et al. 2018]
This sequence is termed determinate growth. It describes a limited period of activity at a particular apex, followed by the end of further elongation along that axis. The fate of one root must be considered in relation to the organization of the whole plant. The seedling produces lateral roots that establish new surfaces of contact with the substrate. Development of the root system can therefore proceed while growth of its first axis comes to an end. This distinction in scale is especially important when comparing a young seedling with a large, long lived cactus. [Dubrovsky and Gómez-Lomelí 2003]
Water availability changes developmental timing
In 2003, Dubrovsky and Gómez-Lomelí studied seedlings from seeds collected near La Paz in Mexico. Plants grew on agar medium in vertically positioned Petri dishes. The location of each root tip was marked daily, allowing growth of the same root to be followed through time. Polyethylene glycol was introduced into some media to lower water potential. Under these conditions, water becomes less available to plant tissues. Comparing several treatments made it possible to distinguish the effects of moderate and severe water deficit from the passage of developmental time itself. [Dubrovsky and Gómez-Lomelí 2003]
The primary root ceased growth in every treatment tested. Severe water deficit brought this endpoint forward and reduced the rate of elongation. A moderate reduction in water potential produced a much weaker response, and final root length could remain similar to that of control plants. In one comparison, the average growth period was approximately eight days in the control and under moderate deficit, compared with approximately six days under severe stress. These values refer to the particular media and experimental conditions used. They demonstrate a change in the timing of a developmental programme in response to water availability. [Dubrovsky and Gómez-Lomelí 2003]
Changing proportions of dividing and differentiating cells
Microscopy identified the changes underlying the difference in root length. The meristematic region gradually diminished, while cells with features of mature epidermis appeared progressively closer to the apex. Root hairs provided a visible marker. These slender extensions of epidermal cells increase the surface in contact with the surrounding environment. At the final stage, hairs also covered the most apical portion of the axis. The authors therefore followed tissue transformation through the location and structural characteristics of cells, rather than relying only on measurements of total root length. [Dubrovsky and Gómez-Lomelí 2003]
Meristem reduction was gradual. Cell divisions continued during part of the observation period even as the pool of cells retaining a meristematic state became smaller. Meanwhile, cells produced earlier could continue to elongate. The period of active cell production and the period during which the whole root increased in length therefore had different endpoints. Severe stress restricted both cell production and subsequent elongation. The root response consequently involved two linked processes that the researchers distinguished by making measurements at the levels of individual cells and the entire organ. [Dubrovsky and Gómez-Lomelí 2003]
Denser branching along a shorter axis
Lateral root development began early, while the primary root was still growing. The researchers counted both roots already visible at the surface and primordia concealed within the tissues. Under severe water deficit, more of these structures occurred per millimetre of the main axis. However, the total number of roots and primordia per plant remained similar among treatments. Increased density therefore reflected the distribution of a comparable number of branches along a shorter root. This distinction is essential for describing the change in the architecture of the underground part of the seedling. [Dubrovsky and Gómez-Lomelí 2003]
Many lateral roots were very short and also stopped growing early. Some produced further branches, while certain axes maintained activity for longer. The result was a small, branched system in which individual apices followed different developmental trajectories. The authors associated this organization with economical resource use and the potential to take up water during brief periods of availability. This is an interpretation of the significance of the observed structure. The experiment measured seedling development under controlled conditions, whereas any survival advantage in a natural population requires evaluation through field studies. [Dubrovsky and Gómez-Lomelí 2003]
RNA reveals successive states of the apex
In 2018, Rodriguez-Alonso and colleagues investigated this process at the level of the transcriptome, the collection of RNA molecules reflecting gene activity in sampled material. They distinguished three stages of the Pachycereus pringlei root apex. The first contained an active meristem, the next represented meristem reduction, and the last consisted of an apex with differentiated cells. Seedlings grew on a medium providing access to water. The comparison therefore concerned changes accompanying development, rather than a direct response to an imposed drought episode. Material was collected from one millimetre apical segments. [Rodriguez-Alonso et al. 2018]
Two biological replicates were prepared for RNA sequencing at each stage. Each replicate pooled many root apices to obtain sufficient material for analysis. Reads were assembled into transcript sequences without using an established reference genome for the species. The resulting collection contained 49045 sequences at least 400 nucleotides long. This number describes the outcome of transcriptome assembly. Individual sequences can represent RNA variants and fragments, so their number cannot be equated directly with the number of genes in the plant. [Rodriguez-Alonso et al. 2018]
Differentiation changes the expression profile
The largest expression changes were detected when earlier stages were compared with the terminal stage. An apex composed of differentiated cells had an RNA profile distinct from a region containing dividing and elongating cells. The researchers also compared these profiles with data from developmental zones of the Arabidopsis thaliana root. Similarity between the terminal cactus stage and the Arabidopsis differentiation zone supported the interpretation based on tissue structure. However, the analysis used a mixture of cells from each sampled segment, so changing cellular proportions also contributed to the measured expression profile. [Rodriguez-Alonso et al. 2018]
At the terminal stage, transcripts associated with stress responses and abscisic acid signalling were more strongly represented. Because the plants had access to water, the authors proposed that part of a programme commonly associated with drought is activated during normal root development. This is a hypothesis about the organization of gene regulation. Similarity in the annotated functions of transcripts points towards further experiments, but does not by itself establish which regulator causes meristem activity to end. Resolving that question requires direct manipulation of the functions of selected genes. [Rodriguez-Alonso et al. 2018]
From sequence similarity to a regulatory network
The cactus sequences contained putative counterparts of meristem regulators known from Arabidopsis, including proteins in the PLETHORA, SCARECROW, SHORT-ROOT and WOX groups. A predicted regulatory network was then constructed by transferring information about interactions from the model plant. Such a network organizes candidates for further investigation. Its individual connections represent relationships inferred through comparison. Activity of a similar transcript in two species can coexist with differences in the location, regulation or biological role of the encoded protein. [Rodriguez-Alonso et al. 2018]
Selected expression profiles were additionally examined by RT-qPCR, which measures the relative abundance of particular RNAs after their conversion into DNA. For twelve of the thirteen genes tested, the qualitative direction of change agreed with the sequencing result. Agreement between two methods strengthens confidence in the expression description. It remains a measurement of RNA abundance, distinct from measurements of protein quantity and activity. Combining microscopy with the transcriptome therefore provides a description of root development at several levels, while preserving the distinction between the observed process and the proposed mechanism regulating it. [Rodriguez-Alonso et al. 2018]
Two experiments describe different levels of the process
The two studies used seeds collected at different localities and different growth media. The physiological experiment compared treatments including sucrose supplementation, then selected conditions that reduced variation in root length for subsequent measurements. In the transcriptome study, seeds came from the vicinity of Bahía Kino, and seedlings grew without added sucrose at 28°C under a twelve hour day. Comparing the findings connects cellular observations with RNA profiles. Nevertheless, particular developmental ages retain the meaning assigned to them within each experiment. [Dubrovsky and Gómez-Lomelí 2003] [Rodriguez-Alonso et al. 2018]
The findings describe early root organization, from radicle emergence to differentiation of its apex. During this brief period, axes and branches are established that provide the starting point for subsequent development. The first study shows how water availability changes the course of this stage. The second identifies groups of transcripts and regulators associated with successive cellular states. Together, they provide a basis for further investigation of the relationship between tissue differentiation, branching of the root system and the conditions under which seedlings of a desert cactus become established. [Dubrovsky and Gómez-Lomelí 2003] [Rodriguez-Alonso et al. 2018]
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)
Dubrovsky J. G., Gómez-Lomelí L. F., 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), American Journal of Botany, 2003, 90(6):823-831, doi: 10.3732/ajb.90.6.823
Experiments on Pachycereus pringlei seedlings describe termination of primary root growth and the influence of water deficit on its meristem. Measurements of cells and branching distinguish reduced root elongation from lateral root initiation. Severe stress accelerates growth termination while lateral primordium number is maintained.
Transcriptomics insights into the genetic regulation of root apical meristem exhaustion and determinate primary root growth in Pachycereus pringlei (Cactaceae)
Rodriguez-Alonso G., Matvienko M., López-Valle M. L., Lázaro-Mixteco P. E., Napsucialy-Mendivil S., Dubrovsky J. G., Shishkova S., Transcriptomics insights into the genetic regulation of root apical meristem exhaustion and determinate primary root growth in Pachycereus pringlei (Cactaceae), Scientific Reports, 2018, 8:8529, doi: 10.1038/s41598-018-26897-1
The transcriptome of Pachycereus pringlei root apices was compared at three developmental stages. The analysis identifies expression changes accompanying cell differentiation and putative regulators of meristem maintenance. A regulatory network was inferred by comparison with Arabidopsis, and selected RNA profiles were checked by RT-qPCR.