Lateral root development differs between main and secondary roots and depends on the ecotype

ABSTRACT

Root architecture depends on the development of the main root and also on the number and density of lateral roots. Most molecular knowledge about the development of lateral roots was acquired studying primary roots, and it was implied that high order roots follow the same pattern. Recently, we informed that AtHB23 is differentially regulated in primary and secondary roots. Here we show that LBD16, a target of AtHB23, also is differentially regulated; it is expressed in the tip of secondary and tertiary roots but not in primary ones. Moreover, the key hormone auxin exhibits a different distribution pattern in secondary and tertiary roots, according to the reporter DR5. Finally, we show that in Col 0 and Ler ecotypes development of secondary and tertiary roots exhibits significant variations. Altogether, we can conclude that different genetic programs govern secondary and tertiary roots development and such processes are dependent on the Arabidopsis genotype.

Introduction

Plant adaptation to soil depends on root architecture and the latter is defined by the length of the main root together with the number and density of lateral roots (LRs). LRs develop from de novo meristems dependent on auxin and this process can be repeated several times in higher-order LRs.¹ Notably, most studies were focused on the formation of secondary roots from primary roots and tacitly accepted that the molecular mechanisms involved are subsequently repeated.

Most knowledge about LR development has been acquired in the model dicot Arabidopsis thaliana. This process involves transcription factors (TFs) as main players, particularly those from the Lateral organ Boundaries Domain (LBD) and Auxin Response Factors (ARF) families, and auxin as the hormone responsible for the integration of many internal and external signals.²

AtHB23 is a homeodomain-leucine zipper (HD-Zip) I TF³ expressed at the base of the secondary LR primordium and we showed by Chromatin Immunoprecipitation assays (ChIP-qPCR) that it directly controls the expression of the auxin carrier LAX3 and the TF LBD16.⁴ LBD16 is a TF associated with the acquisition of LR founder cell polarity and cell cycle activation^5-7 and it is directly modulated by ARF7 SUMOylation in response to water availability.⁸

Using mutant lines, it was revealed that tertiary roots do not develop from secondary roots following the same molecular pathways that the latter from the primary ones because the expression of the HD-Zip I TF AtHB23 differs between secondary and tertiary root development.⁴

Adding complexity to LR developmental process and taking into account that available mutant lines are not always on the same genetic backgrounds, we considered this issue in our studies. Severald evelopmental events may occur via different pathways when comparing two Arabidopsis genotypes. For example, petal development in kin13A mutants significantly differs between Columbia (Col 0) and Landsberg erecta (Ler) genotypes.⁹ A second example is the different response to phosphate starvation¹⁰ and the high difference in the root tip transcriptomes of such genotypes including mRNAs, lncRNAs, and small RNAs.¹¹ Regarding particularly roots, the size of these organs exhibits a natural variation between Col 0 and Ler accessions when plants are subjected to osmotic stress. In such conditions, the total LR number in Ler plants was significantly higher than in Col 0 seedlings.¹²

The above-mentioned observations make necessary to revise several conclusions about root architecture determination derived only from the study of LR development from primary roots and also consider the genotype in which studies are carried out. This is because these processes are certainly more complex than thought so far. In this manuscript, we contribute with experimental evidence supporting that high order LRs exhibit key-genes differential expression patterns than primary roots. Furthermore, we reveal here that root architecture and development follow different programs in Col 0 and Ler ecotypes.

Results and discussion

AtHB23 positively regulates auxin distribution in tertiary roots

To further investigate LR development from main and secondary roots, we analyzed the expression of the auxin carriers AUX1 and LAX1 and the peak of auxin response (shown by the DR5 synthetic reporter) in tertiary roots using prAUX1:GUS, prLAX1:GUS, and prDR5:GUS transgenic plants. These analyses were performed by GUS histochemistry¹³ both in the WT background as well as in AtHB23-silenced plants (Figure 1). The expression of these carriers in secondary roots was previously informed.¹⁴ The assessment of auxin-carrier promoters (prAUX1 and prLAX1) driving GUS expression in tertiary roots was almost not affected when the plants were crossed with amiR23 ones.⁴ In contrast to previous reports of what is observed in secondary roots,⁴ histochemical analysis of tertiary roots in the above-mentioned crosses indicated that the auxin peak (revealed by DR5 activity) disappeared in tertiary lateral roots primordia and tertiary lateral roots, suggesting a key and different role for AtHB23 in secondary and tertiary roots development.

Figure 1.

DR5 reporter activity is repressed in the tertiary lateral roots of AtHB23-silenced plants. Left panel: (a-c) Histochemistry of GUS in single transgenic (prDR5:GUS, prAUX1:GUS, and prLAX1:GUS) 15 day-old plants. Right panel: plants described in the left panel crossed with the amiR23-1 plants. Each picture illustrates a lateral root of each genotype of different order and was carried out with N: 15 per genotype and repeated at least three times. Staining reactions were carried out overnight.Black bars represent 50 µm.

LBD16 is expressed in the tip of secondary and tertiary roots in contrast with primary roots

It was recently reported that AtHB23 directly regulates LBD16.⁴ Transgenic plants carrying LBD16 promoter driving the expression of the reporter GUS were previously described,⁵ obtained from the ABRC and analyzed by histochemistry. LBD16 is a deeply characterized gene, including its expression pattern as well as its role in the promotion of LR initiation.^2,6,15–17 Using prLBD16:GUS transgenic plants, we investigated LBD16 expression in secondary and tertiary roots. Surprisingly, we observed GUS staining in the cap as well as in the vascular tissue of lateral root and tertiary lateral roots whereas, and in agreement with the literature, it was not expressed at all in the tip of main roots (Figure 2). This experimental evidence further supports that secondary and tertiary roots follow different molecular programs.

Figure 2.

LBD16 promoter is active in the root tip of secondary and tertiary lateral roots. GUS histochemistry of 15-day-old prLBD16:GUS roots grown in control conditions. (a) Tip of the main root; (b) Tips of two secondary roots representing different developmental emergence stages; and (c) tip of the tertiary lateral root. Each picture represents an illustration of each genotype and root order, and was carried out with N: 15 per genotype and repeated at least three times.

Lateral root development significantly differs between Col 0 and Ler genotypes

We analyzed LR development from main and secondary roots in Col 0 and Ler genotypes and we detected clear differences between them. Main roots are longer in 8-day-old Ler plants than in Col 0 ones whereas secondary roots showed the opposite scenario, i.e. longer in Col 0 than in Ler plants (Figure 3a). Eight-day-old Ler seedlings exhibit more lateral root primordia (LRP) than their Col 0 counterparts whereas no significant differences were detected in this parameter in 14-day-old plants (secondary to tertiary roots; Figure 3b). Similarly, as LRP, the number of LR in the main root was higher in Ler than in Col 0 genotype in 8-day-old seedlings and the opposite was observed in secondary roots in 14-day-old plants (Figure 3c). In agreement with these observations, total more LR were developed from main roots in Ler than in Col 0 individuals whereas those from secondary roots were slightly less in the opposite sense (Figure 3d). These observations can be directly visualized in the illustrative pictures of Col 0 and Ler 8- and 14-day-old seedlings shown in Figure 3e,f.

Figure 3.

Secondary and tertiary root development differs between Col 0 and Ler ecotypes. (a) Root length of the main root of 8-day-old plants and total lateral secondary roots length of 14-day-old plants. (b) Lateral Root Primordium (LRP) in the main root of 8-day-old plants and in secondary roots of 14-day-old plants. (c) Lateral root (LR) in the main root of 8-day-old plants and in the secondary root of 14-day-old plants. (d) Total Lateral Root (LRP + LR) in the main root of 8-day-old plants and in secondary roots of 14-day-old plants. (e) Col 0 8-day-old seedlings. (f) Ler 8-day-old seedlings. (g) Col 14-day-old seedlings. (h) Ler 14-day-old seedlings. The assays were repeated at least three times with N: 15/genotype. Error bars represent SEM. Asterisks indicate statistical significance determined using the Sidak-Bonferroni method, with alpha = 5.0% (*P < 0.0001). Black bars represent 1 cm.

Concluding remarks

It is well known that root architecture is essential for plant adaptation to the soil and environment and there is a vast literature showing that main and lateral roots development is modulated by environmental factors. At the molecular level, architecture plasticity follows complex mechanisms that involve many actors tightly regulated at the transcriptional level. In the present work, we showed another layer of complexity, i.e. LR development, both in main and secondary roots, is differentially modulated in Col 0 and Ler genotypes. Moreover, we added experimental evidence demonstrating that key molecular actors in Col0 behave differently in the development of secondary and tertiary roots. Although tertiary roots development remains poorly studied, it is not so surprising that plant plasticity evolved for soil adaptation with highly regulated programs subjected to natural variation.

Funding Statement

This work was supported by the Fondo para la Investigación Científica y Tecnológica [PICT 0305].

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.