Body regeneration through formation of new organs is a major question in developmental biology. We investigated de novo root formation using whole leaves of Arabidopsis (Arabidopsis thaliana). Our results show that local cytokinin biosynthesis and auxin biosynthesis in the leaf blade followed by auxin long-distance transport to the petiole leads to proliferation of J0121-marked xylem-associated tissues and others through signaling of INDOLE-3-ACETIC ACID INDUCIBLE28 (IAA28), CRANE (IAA18), WOODEN LEG, and ARABIDOPSIS RESPONSE REGULATORS1 (ARR1), ARR10, and ARR12. Vasculature proliferation also involves the cell cycle regulator KIP-RELATED PROTEIN2 and ABERRANT LATERAL ROOT FORMATION4, resulting in a mass of cells with rooting competence that resembles callus formation. Endogenous callus formation precedes specification of postembryonic root founder cells, from which roots are initiated through the activity of SHORT-ROOT, PLETHORA1 (PLT1), and PLT2. Primordia initiation is blocked in shr plt1 plt2 mutant. Stem cell regulators SCHIZORIZA, JACKDAW, BLUEJAY, and SCARECROW also participate in root initiation and are required to pattern the new organ, as mutants show disorganized and reduced number of layers and tissue initials resulting in reduced rooting. Our work provides an organ regeneration model through de novo root formation, stating key stages and the primary pathways involved.Plants have striking regeneration capacities, and can produce new organs from postembryonic tissues (Hartmann et al., 2010;Chen et al., 2014;Liu et al., 2014) as well as reconstitute damaged organs upon wounding (Xu et al., 2006;Heyman et al., 2013; PerianezRodriguez et al., 2014;Melnyk et al., 2015;Efroni et al., 2016). Intriguingly, root regeneration upon stem cell damage recruits embryonic pathways (Hayashi et al., 2006;Efroni et al., 2016), whereas in contrast, postembryonic formation of whole new organs, such as lateral roots, appears to use specific postembryonic pathways (Lavenus et al., 2013).Cross talk between auxin and cytokinin signaling is required for many aspects of plant development and regeneration (El-Showk et al., 2013), although how their synergistic interaction is implemented at the molecular level has not been clarified (Skoog and Miller, 1957;Chandler and Werr, 2015). Exogenous in vitro supplementation of these two hormones results in continuous cell proliferation, to form a characteristic structure termed "callus". Callus emerges as a common regenerative mechanism for almost all plant organs through in vitro culture (Atta et al., 2009;Sugimoto et al., 2010). There is increasing evidence that callus formation requires hormone-mediated activation of a lateral and meristematic root development program in pericycle-like cells defined by expression of the J0121 marker 1 This work was supported by grants from Ministerio de Economía y Competitividad (MINECO) of Spain, the European Regional Development Fund (ERDF) and FP7 Funds of the European Commission, BFU2013-41160-P, BFU2016-80315-P, and PCIG11-GA-2012-322082 to M.A....

In Arabidopsis, the root clock regulates the spacing of lateral organs along the primary root through oscillating gene expression. The core molecular mechanism that drives the root clock periodicity and how it is modified by exogenous cues such as auxin and gravity remain unknown. We identified the key elements of the oscillator (AUXIN RESPONSE FACTOR 7, its auxin-sensitive inhibitor IAA18/POTENT, and auxin) that form a negative regulatory loop circuit in the oscillation zone. Through multilevel computer modeling fitted to experimental data, we explain how gene expression oscillations coordinate with cell division and growth to create the periodic pattern of organ spacing. Furthermore, gravistimulation experiments based on the model predictions show that external auxin stimuli can lead to entrainment of the root clock. Our work demonstrates the mechanism underlying a robust biological clock and how it can respond to external stimuli.

Stem cells divide and differentiate to form all of the specialized cell types in a multicellular organism. In theArabidopsisroot, stem cells are maintained in an undifferentiated state by a less mitotically active population of cells called the quiescent center (QC). Determining how the QC regulates the surrounding stem cell initials, or what makes the QC fundamentally different from the actively dividing initials, is important for understanding how stem cell divisions are maintained. Here we gained insight into the differences between the QC and the cortex endodermis initials (CEI) by studying the mobile transcription factor SHORTROOT (SHR) and its binding partner SCARECROW (SCR). We constructed an ordinary differential equation model of SHR and SCR in the QC and CEI which incorporated the stoichiometry of the SHR-SCR complex as well as upstream transcriptional regulation of SHR and SCR. Our model prediction, coupled with experimental validation, showed that high levels of the SHR-SCR complex are associated with more CEI division but less QC division. Furthermore, our model prediction allowed us to propose the putative upstream SHR regulators SEUSS and WUSCHEL-RELATED HOMEOBOX 5 and to experimentally validate their roles in QC and CEI division. In addition, our model established the timing of QC and CEI division and suggests that SHR repression of QC division depends on formation of the SHR homodimer. Thus, our results support that SHR-SCR protein complex stoichiometry and regulation of SHR transcription modulate the division timing of two different specialized cell types in the root stem cell niche.

Stem cells divide and differentiate to form all the specialized cell types in a multicellular organism. In the Arabidopsis root, stem cells are maintained in an undifferentiated state by a less mitotically active population of cells called the Quiescent Center (QC). Determining how the QC regulates the surrounding stem cell initials, or what makes the QC fundamentally different from the actively dividing initials, is important for understanding how stem cell divisions are maintained. Here, we gained insight into the differences between the QC and the Cortex Endodermis Initials (CEI) by studying the mobile transcription factor SHORTROOT (SHR) and its binding partner SCARECROW (SCR). We constructed an Ordinary Differential Equation (ODE) model of SHR and SCR in the QC and CEI which incorporated the stoichiometry of the SHR-SCR complex as well as upstream transcriptional regulation of SHR and SCR. Our model prediction coupled with experimental validation showed that high levels of the SHR-SCR complex is associated with more CEI division but less QC division. Further, our model prediction allowed us to establish the timing of QC and CEI division and propose that SHR repression of QC division depends on the formation of SHR homodimer. Thus, our results support that SHR-SCR protein complex stoichiometry and regulation of SHR transcription modulate the division timing of two different specialized cell types in the root stem cell niche.

Fluorescence-activated cell sorting (FACS) is a technique used to isolate specific cell populations based on characteristics detected by flow cytometry. FACS has been broadly used in transcriptomic analyses of individual cell types during development or under different environmental conditions. Different protoplast extraction protocols are available for plant roots; however, they were designed for accessible cell populations, which normally were grown in the presence of light, a non-natural and stressful environment for roots. Here, we report a protocol using FACS to isolate root protoplasts from Arabidopsis green fluorescent protein (GFP)-marked lines using the minimum number of enzymes necessary for an optimal yield, and with the root system grown in darkness in the D-Root device. This device mimics natural conditions as the shoot grows in the presence of light while the roots grow in darkness. In addition, we optimized this protocol for specific patterns of scarce cell types inside more differentiated tissues using the mCherry fluorescent protein. We provide detailed experimental protocols for effective protoplasting, subsequent purification through FACS, and RNA extraction. Using this RNA, we generated cDNA and sequencing libraries, proving that our methods can be used for genome-wide transcriptomic analyses of any cell-type from roots grown in darkness.

The current biodiversity crisis represents a historic challenge for natural communities: the environmental rate of change exceeds the population’s adaptation capability. Integrating both ecological and evolutionary responses is necessary to make reliable predictions regarding the loss of biodiversity. The race against extinction from an eco-evolutionary perspective is gaining importance in ecological risk assessment. Here, we performed a classical study of population dynamics—a fluctuation analysis—and evaluated the results from an adaption perspective. Fluctuation analysis, widely used with microorganisms, is an effective empirical procedure to study adaptation under strong selective pressure because it incorporates the factors that influence demographic, genetic and environmental changes. The adaptation of phytoplankton to beryllium (Be) is of interest because human activities are increasing the concentration of Be in freshwater reserves; therefore, predicting the effects of human-induced pollutants is necessary for proper risk assessment. The fluctuation analysis was performed with phytoplankton, specifically, the freshwater microalgae Chlamydomonas reinhardtii, under acute Be exposure. High doses of Be led to massive microalgae death; however, by conducting a fluctuation analysis experiment, we found that C. reinhardtii was able to adapt to 33 mg/l of Be due to pre-existing genetic variability. The rescuing adapting genotype presented a mutation rate of 9.61 × 10−6 and a frequency of 10.42 resistant cells per million wild-type cells. The genetic adaptation pathway that was experimentally obtained agreed with the theoretical models of evolutionary rescue (ER). Furthermore, the rescuing genotype presented phenotypic and physiologic differences from the wild-type genotype, was 25% smaller than the Be-resistant genotype and presented a lower fitness and quantum yield performance. The abrupt distinctions between the wild-type and the Be-resistant genotype suggest a pleiotropic effect mediated by an advantageous mutation; however, no sequencing confirmation was performed.

The root system is essential for the survival of terrestrial plants, plant development, and adaptation to changing environments. The development of the root system relies on post-embryonic organogenesis and more specifically on the formation and growth of lateral roots (LR). The spacing of LR along the main root is underpinned by a precise prepatterning mechanism called the Root Clock. In Arabidopsis, the primary output of this mechanism involves the generation of periodic gene expression oscillations in a zone close to the root tip called the Oscillation Zone (OZ). Because of these oscillations, pre-branch sites (PBS) are established in the positions from which LR will emerge, although the oscillations can also possibly regulate the root wavy pattern and growth. Furthermore, we show that the Root Clock is present in LR. In this review, we describe the recent advances unraveling the inner machinery of Root Clock as well as the new tools to track the Root Clock activity. Moreover, we discuss the basis of how Arabidopsis can balance the creation of a repetitive pattern while integrating both endogenous and exogenous signals to adapt to changing environmental conditions. These signals can work as entrainment signals, but in occasions they also affect the periodicity and amplitude of the oscillatory dynamics in gene expression. Finally, we identify similarities with the Segmentation Clock of vertebrates and postulate the existence of a determination front delimiting the end of the oscillations in gene expression and initiating LR organogenesis through the activation of PBS in an ARF7 dependent-manner.