A complex tissue-specific interplay between the Arabidopsis transcription factors AtMYB68, AtHB23, and AtPHL1 modulates primary and lateral root development and adaptation to salinity.

Adaptation to different soil conditions is a well-regulated process vital for plant life. AtHB23 is a homeodomain-leucine zipper I transcription factor (TF) that was previously revealed as crucial for plant survival under salinity conditions. We wondered whether this TF has partners to perform this essential function. Therefore, TF cDNA library screening, yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays were complemented with expression analyses and phenotypic characterization of silenced, mutant, overexpression, and crossed plants in normal and salinity conditions. We revealed that AtHB23, AtPHL1, and AtMYB68 interact with each other, modulating root development and the salinity response. The encoding genes are coexpressed in specific root tissues and at specific developmental stages. In normal conditions, amiR68 silenced plants have fewer initiated roots, the opposite phenotype to that shown by amiR23 plants. AtMYB68 and AtPHL1 play opposite roles in lateral root elongation. Under salinity conditions, AtHB23 plays a crucial positive role in cooperating with AtMYB68, whereas AtPHL1 acts oppositely by obstructing the function of the former, impacting the plant's survival ability. Such interplay supports the complex interaction between these TF in primary and lateral roots. The root adaptation capability is associated with the amyloplast state. We identified new molecular players that through a complex relationship determine Arabidopsis root architecture and survival in salinity conditions.

AtHB23 participates in the gene regulatory network controlling root branching, and reveals differences between secondary and tertiary roots.

In Arabidopsis, lateral root (LR) development is mainly controlled by several known auxin-regulated transcription factors (TFs). Here, we show that AtHB23 (a homeodomain-leucine zipper I TF) participates in this intricate network. Our study of the expression pattern of AtHB23 revealed that it is transcriptionally activated in the early stages of secondary LR primordium (LRP). We found that AtHB23 directly limits the expression of LBD16, a key factor in LR initiation, and also directly induces the auxin transporter gene LAX3. We propose that this HD-Zip I mediates the regulation of LAX3 by ARF7/19. Furthermore, AtHB23 plays distinct roles during the formation of secondary and tertiary roots, exhibiting differential expression patterns. ATHB23 is expressed throughout the tertiary root primordium, whereas it is restricted to early stages in secondary primordia, likely later repressing LBD16 in tertiary LR development and further inhibiting root emergence. Our results suggest that different genetic programs govern the formation of LRP from the main or secondary roots, thereby shaping the global dynamic architecture of the root system.

Functional characterization of the homeodomain leucine zipper I transcription factor AtHB13 reveals a crucial role in Arabidopsis development.

AtHB13 is a homeodomain leucine zipper I transcription factor whose function in development is largely unknown. AtHB13 and AtHB23 mutant and silenced lines were characterized by expression studies, reciprocal crosses, complementation, molecular analyses, and developmental phenotypes. The athb13-1 and athb13-2 mutants, athb23 silenced, and athb13/athb23 double-silenced plants exhibited faster elongation rates of their inflorescence stems, whereas only athb13-1 and the double-knockdown athb13/athb23 exhibited shorter siliques, fewer seeds, and unfertilized ovules compared with the wild type (WT). The cell sizes of mutant and WT plants were similar, indicating that these transcription factors probably affect cell division. Reciprocal crosses between athb13-1 and the WT genotype indicated that the silique defect was male specific. Pollen hydration assays indicated that the pollen grains of the athb13-1 mutant were unable to germinate on stigmas. AtHB23-silenced plants exhibited normal siliques, whereas double-knockdown athb13/athb23 plants were similar to athb13-1 plants. Both AtHB13 and AtHB23 were able to rescue the abnormal silique phenotype. AtHB23 was upregulated in athb13-2 plants, whereas its transcript levels in athb13-1 mutants were not significantly increased. Transcriptome analysis comparing athb13-1 and WT inflorescences revealed that a large number of genes, including several involved in pollen coat formation, are regulated by AtHB13. Finally, athb13-1 complementation with mutated versions of AtHB13 confirmed that two different tryptophans in its C terminus are essential. We conclude that AtHB13 and AtHB23 play independent, negative developmental roles in stem elongation, whereas only AtHB13 is crucial for pollen germination. Furthermore, AtHB23, which does not normally exert a functional role in pollen, can act as a substitute for AtHB13.

The Arabidopsis transcription factors AtPHL1 and AtHB23 act together promoting carbohydrate transport from pedicel-silique nodes to seeds.

Carbohydrates are produced in green tissues through photosynthesis and then transported to sink tissues. Carbon partitioning is a strategic process, fine regulated, involving specific sucrose transporters in each connecting tissue. Here we report that a screening of an Arabidopsis transcription factor (TF) library using the homeodomain-leucine zipper I member AtHB23 as bait, allowed identifying the TF AtPHL1 interacting with the former. An independent Y2H assay, and in planta by BiFC, confirmed such interaction. AtHB23 and AtPHL1 coexpressed in the pedicel-silique nodes and the funiculus. Mutant plants (phl1, and amiR23) showed a marked reduction of lipid content in seeds, although lipid composition did not change compared to the wild type. While protein and carbohydrate contents were not significantly different between mutants and control mature seeds, we observed a reduced carbohydrate content in mutant plants young siliques (7 days after pollination). Moreover, using a CFDA probe, we revealed an impaired transport to the seeds, and the gene encoding the carbohydrate transporters SWEET10 and SWEET11, usually expressed in connecting tissues, was repressed in the amiR23 and phl1 mutant plants. Altogether, the results indicated that AtHB23 and AtPHL1 act together, promoting sucrose transport, and the lack of any of them provoked a reduction in seeds lipid content.

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

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.