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Root Hairs (RHs) growth is influenced by endogenous and by external environmental signals that coordinately regulate its final cell size. We have recently determined that RH growth was unexpectedly boosted when Arabidopsis thaliana seedlings are cultivated at low temperatures. It was proposed that RH growth plasticity in response to low temperature was linked to a reduced nutrient availability in the media. Here, we explore the molecular basis of this RH growth response by using a Genome Wide Association Study (GWAS) approach using Arabidopsis thaliana natural accessions. We identify the poorly characterized PEROXIDASE 62 (PRX62) and a related protein PRX69 as key proteins under moderate low temperature stress. Strikingly, a cell wall protein extensin (EXT) reporter reveals the effect of peroxidase activity on EXT cell wall association at 10 °C in the RH apical zone. Collectively, our results indicate that PRX62, and to a lesser extent PRX69, are key apoplastic PRXs that modulate ROS-homeostasis and cell wall EXT-insolubilization linked to RH elongation at low temperature. Arabidopsis root hair growth is enhanced at low temperatures. Here the authors show that the class III peroxidases PRX62 and PRX69 modulate ROS homeostasis and cell wall characteristics, and promote root hair elongation at low temperature.

Bromus auleticus (Trin.) is a grass native to the southern cone with important agronomical potential as fodder. Different breeding programs have been initiated with this grass, but plant tissue culture techniques could not be used because B. auleticus is recalcitrant. The aim of the present study was to develop a micropropagation protocol in the genus Bromus and to investigate if the association between B. auleticus and Epichloë endophytes affected in vitro culture and growth of micropropagated plantlets. In different micropropagation stages, better results were obtained with endophyte-infected (E+) seeds compared to endophyte-free (E−) seeds. The E+ seeds presented higher percentages of in vitro germination (82 ± 5 vs. 57 ± 6%), callus induction (72 ± 6 vs. 37 ± 6%), and plant regeneration from callus (89 ± 5 vs. 13 ± 5%). We also compared the biomass of shoot complexes and regenerated plantlets. After 4 weeks of culture, shoot complexes obtained from E+ seeds reached greater weight than the ones regenerated from E− seeds (173 ± 24 vs. 74 ± 9 mg). More than the 80% of the regenerated shoot complexes were rooted ex vitro and acclimated, regardless of their origin (E+ or E−). Finally, after 4 weeks of acclimatization, the plantlets regenerated from E+ seeds reached a greater weight than the ones from E− seeds, (461 ± 64 vs. 172 ± 25 mg). These results indicate that the use of endophyte-infected (E+) seeds enhances significantly B. auleticus micropropagation and promotes growth of the regenerated plantlets.

The root hair (RH) cells can elongate to several hundred times their initial size, and are an ideal model system for investigating cell size control. Their development is influenced by both endogenous and external signals, which are combined to form an integrative response. Surprisingly, a low temperature condition of 10°C causes an increased RH growth in Arabidopsis and in several monocots, even when the development of the rest of the plant are halted. Previously, we demonstrated a strong correlation between the RH growth response and a significant decrease in nutrient availability in the medium under low temperature conditions. However, the molecular basis responsible for receiving and transmitting signals related to the availability of nutrients in the soil, and their relation to plant development, remain largely unknown. In this study, we have discovered two antagonic gene regulatory networks (GRNs) controlling RH early transcriptome responses to low temperature. One GNR enhances RH growth and it is commanded by the transcription factors (FTs) ROOT HAIR DEFECTIVE 6 (RHD6), HAIR DEFECTIVE 6-LIKE 2 and 4 (RSL2-RSL4) and a member of the homeodomain leucine zipper (HD-Zip I) group I 16 (AtHB16). On the other hand, a second GRN was identified as a negative regulator of RH growth at low temperature and it is composed by the trihelix TF GT2-LIKE1 (GTL1) and the associated DF1, a previously unidentified MYB-like TF (AT2G01060) and several members of HD-Zip I group (AtHB3, AtHB13, AtHB20, AtHB23). Functional analysis of both GRNs highlights a complex regulation of RH growth response to low temperature, and more importantly, these discoveries enhance our comprehension of how plants synchronize the RH growth in response to variations in temperature at the cellular level.Competing Interest StatementThe authors have declared no competing interest.Footnotes* New data was added. Specifically, we have validated several of the AlphaFold Multimer protein-protein predictions by BiFC (Bimolecular Fluorescence Complementation) assays.

Root hairs (RH) as a mixed tip- and non-tip growing protrusions that develop from root epidermal cells are important for nutrient and water uptake, root anchoring, and interaction with soil microorganisms. Although nutrient availability and temperature are critical interlinked factors for sustained plant growth, the molecular mechanisms underlying their sensing and downstream signaling pathways remain unclear. Here, we identified a moderate low temperature (10°C) condition that triggers a strong RH elongation response involving several molecular components of the auxin pathway. Then, we have determined that auxin biosynthesis carried out by YUCCAs/TAA1, the auxin transport conducted by PIN2/PIN4 and AUX1/PGP4, and the auxin signaling controlled by TIR1/AFB2 coupled to four specific ARFs (ARF6/ARF8 and ARF7/ARF19), are all crucial for the RH response at moderate low temperature. These results uncover the auxin pathway as one central hub under moderate low temperature in the roots to trigger RH growth. Our work highlights the importance of moderate low temperature stimulus as a complex nutritional signal from the media soil into the roots that may be fine-tuned for future biotechnological applications to enhance nutrient uptake.

Plant genomes encode a unique group of papain-type Cysteine EndoPeptidases (CysEPs) containing a KDEL endoplasmic reticulum (ER) retention signal (KDEL-CysEPs or CEPs). CEPs process the cell-wall scaffolding EXTENSIN proteins (EXTs), which regulate de novo cell wall formation and cell expansion. Since CEPs are able to cleave EXTs and EXT-related proteins, acting as cell wall-weakening agents, they may play a role in cell elongation. Arabidopsis thaliana genome encodes three CEPs (AtCPE1-AtCEP3). Here we report that the three Arabidopsis CEPs, AtCEP1-AtCEP3, are highly expressed in root-hair cell files. Single mutants have no evident abnormal root-hair phenotype, but atcep1-3 atcep3-2 and atcep1-3 atcep2-2 double mutants have longer root hairs (RHs) than wild type (Wt) plants, suggesting that expression of AtCEPs in root trichoblasts restrains polar elongation of the RH. We provide evidence that the transcription factor NAC1 activates AtCEPs expression in roots to limit RH growth. Chromatin immunoprecipitation indicates that NAC1 binds the promoter of AtCEP1, AtCEP2, and to a lower extent to AtCEP3 and may directly regulate their expression. Indeed, inducible NAC1 overexpression increases AtCEP1 and AtCEP2 transcript levels in roots and leads to reduced RH growth while the loss of function nac1-2 mutation reduces AtCEP1-AtCEP3 gene expression and enhances RH growth. Likewise, expression of a dominant chimeric NAC1-SRDX repressor construct leads to increased RH length. Finally, we show that RH cell walls in the atcep1-1 atcep3-2 double mutant have reduced levels of EXT deposition, suggesting that the defects in RH elongation are linked to alterations in EXT processing and accumulation. Taken together, our results support the involvement of AtCEPs in controlling RH polar growth through EXT-processing and insolubilization at the cell wall.

Root hairs (RH) are excellent model systems for studying cell size and polarity since they elongate several hundred-fold their original size. Their tip growth is determined both by intrinsic and environmental signals. Although nutrients availability and temperature are key factors for a sustained plant growth, the molecular mechanisms underlying their sensing and downstream signaling pathways remain unclear. Here, we identified that low temperature (10C) triggers a strong RH elongation response involving the cell surface receptor kinase FERONIA (FER) and the nutrient sensing TOR Complex 1 (TORC1). In this study, we found that FER is required to perceive limited nutrients availability caused by low temperature. FER interacts with and activates TORC1-downstream components to trigger RH growth. In addition, the small GTPase Rho-related protein from plants 2 (ROP2) (and possibly ROP4) is also involved in this RH growth response linking FER and TORC1. We also found that limited nitrogen nutrient availability can mimic the RH growth response at 10C in a NRT1.1-dependent manner. These results uncover a molecular mechanism by which a central hub composed by FER-ROP2-TORC1 is involved in the control of RH elongation under low temperature and nitrogen deficiency. Competing Interest Statement The authors have declared no competing interest. Footnotes * We added new results on the temperature effect to demonstrate that the diffusion of nitrates is reduced at low temperature 10oC when compared to 22oC (see Fig. S4). We show evidences that higher agar concentrations induces a nitrate lower mobility regulating RH growth independently of the temperature (see Fig. S5). In addition, we performed a 3-way ANNOVA analysis to quantify the effect of genotype, conditions and RH growth. We added RH growth ratios 10oC/22oC or low nitrate/high nitrate on scatter-plots to asses RH growth variations between conditions in all the figures. We have added western blots as duplicates or triplicates in a separate file as Supplementary Material 1. In addition, we have answered most of the comments from the three reviewers and make some changes in the text. For example, the ROP2 part is now before the beginning of the nitrate NRT1.1 section. The Final Figure7 was also modified as suggested by one of the reviewers.

ABSTRACT Root Hairs (RHs) growth is highly influenced by endogenous as well as by external environmental signals that coordinately regulate its final cell size. RHs actively expand the root surface responsible for nutrient uptake and water absorption. We have recently determined that RH growth was unexpectedly boosted when Arabidopsis thaliana seedlings are cultivated at low temperatures. It was proposed that RH growth plasticity in response to low temperature was linked to a reduced nutrient availability in the media. Here, we explored the molecular basis of this strong RH growth response by using the Genome Wide Association Studies (GWAS) approach on Arabidopsis thaliana natural accessions. We identified the poorly characterized PEROXIDASE 62 (PRX62) as a key protein triggering this conditional growth under a moderate low-temperature stress. In addition, we identified the related protein PRX69 as an important factor in this developmental process. The prx62 prx69 double mutant and the PRX62 and PRX69 over-expressing lines showed contrasting RH phenotypes, peroxidase activities and cyt/apoReactive Oxygen Species (ROS) levels. Strikingly, a cell wall protein extensin (EXT) reporter revealed the effect of peroxidase activity on the EXT cell wall association at 10°C in the RH apical zone. EXT cell wall insolubilization was enhanced at 10°C, which was completely abolished under the PRX inhibitor salicylhydroxamic acid (SHAM) treatment. Finally, we demonstrated that the Root Hair defective 6-like 4 (RSL4) bHLH family transcription factor directly controls the expression of PRX69. Collectively, our results indicate that both PRX62 and PRX69 are key apoplastic PRXs that modulate ROS-homeostasis and cell wall EXT-insolubilization linked to RH elongation at low-temperature. Competing Interest Statement The authors have declared no competing interest.

Plant genomes encode a unique group of papain-type Cysteine EndoPeptidases (CysEPs) containing a KDEL endoplasmic reticulum (ER) retention signal (KDEL-CysEPs or CEPs). CEPs process the cell-wall scaffolding EXTENSIN proteins (EXTs), which regulate de novo cell wall formation and cell expansion. Since CEPs are able to cleave EXTs and EXT-related proteins, acting as cell wall-weakening agents, they may play a role in cell elongation. Arabidopsis thaliana genome encodes three CEPs (AtCPE1-AtCEP3). Here we report that the three Arabidopsis CEPs, AtCEP1-AtCEP, are highly expressed in root-hair cell files. Single mutants have no evident root-hair phenotype, but atcep1-3 atcep3-2 and atcep1-3 atcep2-2 double mutants have longer root hairs (RHs) than wild type (Wt) plants, suggesting that expression of AtCEPs in root trichoblasts restrains polar elongation of the RH. We provide evidence that the transcription factor NAC1 activates AtCEPs expression in roots to limit RH length. Chromatin immunoprecipitation indicates that NAC1 binds the promoter of AtCEP2 and may directly regulate its expression. Indeed, Inducible NAC1 overexpression increases AtCEP1 and AtCEP2 transcript levels in roots and leads to reduced RH growth while the loss of function nac1-2 mutation reduces AtCEP1-AtCEP3 gene expression and enhances RH growth. Likewise, expression of a dominant chimeric NAC1-SRDX repressor construct leads to increased RH length. Finally, we show that RH cell-walls in the atcep1-1 atcep3-2 double mutant have reduced levels of EXT secretion/insolubilization, suggesting that the defects in RH elongation are linked to alterations in EXT processing and accumulation. Taken together, our results support the involvement of the NAC1-AtCEP regulatory module in controlling RH polar growth through EXT processing and insolubilization at the cell wall.