The Arabidopsis SHORTROOT network coordinates shoot apical meristem development with auxin-dependent lateral organ initiation
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eLife assessment
This is a valuable study of Arabidopsis shoot apical meristem maintenance and organ initiation, defining the expression, interactions and functions of four transcription factors (SHR, SCR, JKD, and SCL23) whose roles were initially described in the root apical meristem. The imaging, genetics and FRET-FLIM evidence supporting the claims of the authors is comprehensive, extensive, and solid, although similar mechanisms, protein interactions, and gene regulatory interactions were previously reported in the root. The work will be of interest and importance for plant developmental biologists.
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Abstract
Plants produce new organs post-embryonically throughout their entire life cycle. This is due to stem cells present in the shoot and root apical meristems, the SAM and RAM, respectively. In the SAM, stem cells are located in the central zone where they divide slowly. Stem cell daughters are displaced laterally and enter the peripheral zone, where their mitotic activity increases and lateral organ primordia are formed. How the spatial arrangement of these different domains is initiated and controlled during SAM growth and development, and how sites of lateral organ primordia are determined in the peripheral zone is not yet completely understood. We found that the SHORTROOT (SHR) transcription factor together with its target transcription factors SCARECROW (SCR), SCARECROW-LIKE23 (SCL23) and JACKDAW (JKD), promotes formation of lateral organs and controls shoot meristem size. SHR, SCR, SCL23, and JKD are expressed in distinct, but partially overlapping patterns in the SAM. They can physically interact and activate expression of key cell cycle regulators such as CYCLIND6;1 ( CYCD6;1 ) to promote the formation of new cell layers. In the peripheral zone, auxin accumulates at sites of lateral organ primordia initiation and activates SHR expression via the auxin response factor MONOPTEROS (MP) and auxin response elements in the SHR promoter. In the central zone, the SHR-target SCL23 physically interacts with the key stem cell regulator WUSCHEL (WUS) to promote stem cell fate. Both SCL23 and WUS expression are subject to negative feedback regulation from stem cells through the CLAVATA signaling pathway. Together, our findings illustrate how SHR-dependent transcription factor complexes act in different domains of the shoot meristem to mediate cell division and auxin dependent organ initiation in the peripheral zone, and coordinate this activity with stem cell maintenance in the central zone of the SAM.
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Author Response
Reviewer #1 (Public Review):
In this genetic and imaging based analysis of stem-cell maintenance and organ initiation, two phases important for continued production of shoot organs in plants, the authors tested whether SHR and targets/partners (SCR, SCL23, JKD) provide the circuitry to maintain stem cell pool and contribute to the production of lateral organs. Finding that these factors are indeed expressed in and required for SAM activities, and furthermore, behaviors of SHR and SCR in the root are recapitulated in the meristem, including mobility of SHR (here to epidermis from internal layers), activation of SCR by SHR, and "trapping" of SHR movement by complexing with SCR. Strengths include high quality imaging of reporters and FRET-FLIM measurement to assess in vivo complex formation. The analysis is then extended …
Author Response
Reviewer #1 (Public Review):
In this genetic and imaging based analysis of stem-cell maintenance and organ initiation, two phases important for continued production of shoot organs in plants, the authors tested whether SHR and targets/partners (SCR, SCL23, JKD) provide the circuitry to maintain stem cell pool and contribute to the production of lateral organs. Finding that these factors are indeed expressed in and required for SAM activities, and furthermore, behaviors of SHR and SCR in the root are recapitulated in the meristem, including mobility of SHR (here to epidermis from internal layers), activation of SCR by SHR, and "trapping" of SHR movement by complexing with SCR. Strengths include high quality imaging of reporters and FRET-FLIM measurement to assess in vivo complex formation. The analysis is then extended to link SHR and SCR to shoot-specific factors and auxin, again by testing expression, genetic dependencies and physical interaction. This is repeated for a number of factors and individually, each is well done experiment. Conclusions about causal relationships are somewhat overstated (for example, the idea that SHR-SCR act through CYCD6 to alter cell division is based on expression patterns, not a functional analysis of cycd6).
We concluded that SHR and cofactors drive cell proliferation through CYCD6;1, substantiated by the significant reduction in pCYCD6;1-GFP expression within the lateral organ primordia of the shr-2 mutant. This decrease in expression corresponds with the reduction in the number of cell layers within the L3 of the lateral organ primordia in shr-2 mutants, compared to wild-type. To further support this conclusion, we have added new data by analyzing the meristem of the cycd6;1 mutant. Our findings reveal a small, but significant reduction in both meristem size and the number of cell layers in the L3, relative to the wild type, as depicted in Fig4-FigSuppl2I-N. Collectively, these findings underscore our assertion that the SHR regulatory network plays a role in activating CYCD6;1 expression, thereby promoting cell division within the lateral organ primordia.
In general, there are many high-quality studies included in this paper, and the presentation of imaging data (both the images themselves and quantification of data) is excellent. There is also a lot of data, and while each section was presented in a logical way, connections between sections, and the overarching developmental questions were sparse. Because the authors found that many of the relationships defined in the root were recapitulated in the shoot, the present organization leaves one with somewhat of a sense that little new was learned, and yet, the shoot meristem IS different and there are shoot specific inputs into the core regulatory factors. Rewriting to highlight the different activities (and thus expectation about regulation) could make the finding of the same network more interesting and creating a summary figure that highlights the input of shoot specific signals would bring the unique analysis to the forefront.
We greatly appreciate your positive feedback on the imaging data presentation and the quality of the included studies! We tried to address your and the other reviewer´s comments and strengthened the connections between the different sections of the manuscripts. We made substantial revisions to the organization and presentation of the paper. Our focus has been on highlighting the distinct activities and regulatory aspects of the SHR network within the shoot meristem, underscoring the novel insights gained from this analysis. We also created a summary figure that features the input of shoot-specific signals, thereby emphasizing the unique analysis conducted. These changes have allowed us to better convey the significance of our findings and showcase the novel aspects of shoot meristem regulation. We believe these revisions align more closely with the paper's objectives and will make the study's contributions more engaging and apparent.
Reviewer #2 (Public Review):
This study contains a huge amount of data and the images are of high quality. However, the conclusions are not really well supported. The authors may have reached too far from their results. The roles of SHR, SCR and SCL23 in the shoot apex are not really clarified. The manuscript by Bahafid et al., reports a study of the functions of SHORTROOT (SHR), a well-established root development regulator in the shoot apical meristem (SAM) development with focus on lateral organ initiation. A large amount of data is included in this paper. This study highly depends on imaging, and the images are in general of very good quality. The authors show reciprocal interactions between SHR and SCR with auxin/MP. There are also a large amount of genetic interactions among several genes, including WUS and CLV3. Although the study provides a vast amount of data, the conclusions are not so well supported. There seem to be many interactions, at the protein level, and at the transcriptional regulation level, but the conclusion is nevertheless ambiguous.
We have refined our manuscript.
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eLife assessment
This is a valuable study of Arabidopsis shoot apical meristem maintenance and organ initiation, defining the expression, interactions and functions of four transcription factors (SHR, SCR, JKD, and SCL23) whose roles were initially described in the root apical meristem. The imaging, genetics and FRET-FLIM evidence supporting the claims of the authors is comprehensive, extensive, and solid, although similar mechanisms, protein interactions, and gene regulatory interactions were previously reported in the root. The work will be of interest and importance for plant developmental biologists.
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Reviewer #1 (Public Review):
In this genetic and imaging based analysis of stem-cell maintenance and organ initiation, two phases important for continued production of shoot organs in plants, the authors tested whether SHR and targets/partners (SCR, SCL23, JWD) provide the circuitry to maintain stem cell pool and contribute to the production of lateral organs. Finding that these factors are indeed expressed in and required for SAM activities, and furthermore, behaviors of SHR and SCR in the root are recapitulated in the meristem, including mobility of SHR (here to epidermis from internal layers), activation of SCR by SHR, and "trapping" of SHR movement by complexing with SCR. Strengths include high quality imaging of reporters and FRET-FLIM measurement to assess in vivo complex formation. The analysis is then extended to link SHR and …
Reviewer #1 (Public Review):
In this genetic and imaging based analysis of stem-cell maintenance and organ initiation, two phases important for continued production of shoot organs in plants, the authors tested whether SHR and targets/partners (SCR, SCL23, JWD) provide the circuitry to maintain stem cell pool and contribute to the production of lateral organs. Finding that these factors are indeed expressed in and required for SAM activities, and furthermore, behaviors of SHR and SCR in the root are recapitulated in the meristem, including mobility of SHR (here to epidermis from internal layers), activation of SCR by SHR, and "trapping" of SHR movement by complexing with SCR. Strengths include high quality imaging of reporters and FRET-FLIM measurement to assess in vivo complex formation. The analysis is then extended to link SHR and SCR to shoot-specific factors and auxin, again by testing expression, genetic dependencies and physical interaction. This is repeated for a number of factors and individually, each is well done experiment. Conclusions about causal relationships are somewhat overstated (for example, the idea that SHR-SCR act through CYCD6 to alter cell division is based on expression patterns, not a functional analysis of cycd6).
In general, there are many high-quality studies included in this paper, and the presentation of imaging data (both the images themselves and quantification of data) is excellent. There is also a lot of data, and while each section was presented in a logical way, connections between sections, and the overarching developmental questions were sparse. Because the authors found that many of the relationships defined in the root were recapitulated in the shoot, the present organization leaves one with somewhat of a sense that little new was learned, and yet, the shoot meristem IS different and there are shoot specific inputs into the core regulatory factors. Rewriting to highlight the different activities (and thus expectation about regulation) could make the finding of the same network more interesting and creating a summary figure that highlights the input of shoot specific signals would bring the unique analysis to the forefront.
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Reviewer #2 (Public Review):
This study contains a huge amount of data and the images are of high quality. However, the conclusions are not really well supported. The authors may have reached too far from their results. The roles of SHR, SCR and SCL23 in the shoot apex are not really clarified.
The manuscript by Bahafid et al., reports a study of the functions of SHORTROOT (SHR), a well-established root development regulator in the shoot apical meristem (SAM) development with focus on lateral organ initiation. A large amount of data is included in this paper. This study highly depends on imaging, and the images are in general of very good quality. The authors show reciprocal interactions between SHR and SCR with auxin/MP. There are also a large amount of genetic interactions among several genes, including WUS and CLV3. Although the …
Reviewer #2 (Public Review):
This study contains a huge amount of data and the images are of high quality. However, the conclusions are not really well supported. The authors may have reached too far from their results. The roles of SHR, SCR and SCL23 in the shoot apex are not really clarified.
The manuscript by Bahafid et al., reports a study of the functions of SHORTROOT (SHR), a well-established root development regulator in the shoot apical meristem (SAM) development with focus on lateral organ initiation. A large amount of data is included in this paper. This study highly depends on imaging, and the images are in general of very good quality. The authors show reciprocal interactions between SHR and SCR with auxin/MP. There are also a large amount of genetic interactions among several genes, including WUS and CLV3. Although the study provides a vast amount of data, the conclusions are not so well supported. There seem to be many interactions, at the protein level, and at the transcriptional regulation level, but the conclusion is nevertheless ambiguous.
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Reviewer #3 (Public Review):
This is a comprehensive and extensive investigation of the auxin-dependent role of four GRAS family proteins (SHR, SCR, JKD, and SCL23) in regulating organ initiation and shoot apical meristem (SAM) maintenance. The authors present a detailed phenotypical analysis of the shr and scr mutants, which have fewer cell layers, reduced auxin maximum, and halted proliferation of cells in the G1 phase, indicating SHR and SCR both influence SAM maintenance and organ initiation. Auxin distribution, mediated through MONOPTEROS, was also found to regulate SHR activity in organ initiation. Furthermore, the authors hypothesize and show evidence for a coordinated regulation of CYCD6;1, a known marker for asymmetric cell divisions in the root, by SHR, SCR, JKD, and SCL23 in the SAM. Finally, the roles of SCL23 and WUSCHEL …
Reviewer #3 (Public Review):
This is a comprehensive and extensive investigation of the auxin-dependent role of four GRAS family proteins (SHR, SCR, JKD, and SCL23) in regulating organ initiation and shoot apical meristem (SAM) maintenance. The authors present a detailed phenotypical analysis of the shr and scr mutants, which have fewer cell layers, reduced auxin maximum, and halted proliferation of cells in the G1 phase, indicating SHR and SCR both influence SAM maintenance and organ initiation. Auxin distribution, mediated through MONOPTEROS, was also found to regulate SHR activity in organ initiation. Furthermore, the authors hypothesize and show evidence for a coordinated regulation of CYCD6;1, a known marker for asymmetric cell divisions in the root, by SHR, SCR, JKD, and SCL23 in the SAM. Finally, the roles of SCL23 and WUSCHEL were investigated with respect to SHR-SCR activity to investigate their roles in stem cell maintenance. Overall, the authors presented a thorough and sound analysis of SAM organ initiation and this reviewer applauds the authors for this extensive systematic and comprehensive study, which has produced as well as leveraged new and established material to demonstrate similar mechanisms for organ initiation found in the shoot and root apical meristems.
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