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Comparative Transcriptome Analysis of Hypocotyls During the Developmental Transition of C3 Cotyledons to C4 Leaves in Halimocnemis mollissima Bunge

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Abstract

Identification of signaling pathways that control C4 photosynthesis development is essential for introducing the C4 pathway into C3 crops. Species with dual photosynthesis in their life cycle are interesting models to study such regulatory mechanisms. The species used here Halimocnemis mollissima Bunge, belonging to the Caroxyleae tribe, displays C3 photosynthesis in its cotyledons and a NAD-ME subtype of C4 photosynthesis in the First leaves (FLs) onwards. We explored the long-distance signaling pathways that are probably implicated in the shoot–root coordination associated with the manifestation of the C4 traits, including efficient resource usage by comparing the mRNA content of hypocotyls before and after the C4 first leave’s formation. Histological examination showed the presence of C3 anatomy in cotyledons and C4 anatomy in the FLs. Our transcriptome analyses verified the performance of the NAD-ME subtype of C4 in FLs and revealed differential transcript abundance of several potential mobile regulators and their associated receptors or transporters in two developmentally different hypocotyls of H. mollissima Bunge. These differentially expressed genes (DEGs) belong to diverse functional groups, including various transcription factor (TF) families, phytohormones metabolism, and signaling peptides, part of which could be related to hypocotyl development. Our findings support the higher nitrogen and water use efficiency associated with C4 photosynthetic and provide insights into the coordinated above- and under-ground tissue communication during the developmental transition of C3–C4 photosynthesis in this species.

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Abbreviations

TF:

Transcription factor

DEG:

Differentially expressed gene

FLs:

First leaves

PEPC:

Phosphoenolpyruvate carboxylase

BCA:

Beta-carbonic anhydrase

PPdK:

Pyruvate orthophosphate dikinase

Asp-AT:

Aspartate aminotransferas

Ala-AT:

Alanine aminotransferase

NAD-ME:

NAD-dependent malic enzyme

NADP-ME:

NADP-dependent malic enzyme

TPT:

Triosephosphate translocator

PPase6:

Pyrophosphatase

PPdK-RP1:

Pyruvate orthophosphate dikinase-related protein1

PPT2:

PEP/phosphate translocator

BASS4:

Bile acid:sodium symporter family protein

BASS2:

Bile acid:sodium symporter family protein

AMK2:

Adenosine monophosphate kinase

HY5:

Elongated hypocotyl 5

SHR:

Short-root

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Acknowledgements

We thank Stanislav Kopriva (University of Cologne) for the critical reading of the manuscript. We also would like to thank Alexander Rudov and Hossein Akhani (University of Tehran) for their support during seed collection.

Funding

A.M.B-M gratefully acknowledges the financial support from the Iran National Science Foundation (INSF) (Funding Reference No. 95838484).

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Authors and Affiliations

  1. Laboratory of Genomics and Epigenomics (LGE), Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

    Mahdis Zolfaghar & Ali Mohammad Banaei-Moghaddam

  2. Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Stadt Seeland/OT Gatersleben, Germany

    Twan Rutten

  3. Systems Biology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education, and Extension Organization (AREEO), Karaj, Iran

    Mohammad Reza Ghaffari

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  1. Mahdis Zolfaghar
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  2. Twan Rutten
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  4. Ali Mohammad Banaei-Moghaddam
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Contributions

MZ performed the experiments, analyzed data, and wrote the original draft. TR performed the microscopic analysis and critically revised the manuscript. MRG analyzed data. AMB-M conceived and designed the research, supervised the experiment, and critically revised the manuscript. All authors contributed to the article and approved the submitted version.

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Correspondence to Mohammad Reza Ghaffari or Ali Mohammad Banaei-Moghaddam.

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Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file 1

. Primers and their sequences (PDF 62 kb)

Supplementary file 2

. The accession number of raw sequences uploaded to National Center for Biotechnology Information. (PDF 6 kb)

Supplementary file 3

. Summary of A sequencing information. B De novo transcriptome assembly results by Trinity. (PDF 9 kb)

Supplementary file 4

. Active functional classes in the First leaves, hypocotyl A and hypocotyl B. (XLSX 13 kb)

Supplementary file 5

. Abundance (total mean TPM) of C4-related proteins and genes categorized in the CRP and NCRP signaling peptide groups (XLSX 13 kb)

Supplementary file 6

. Transcript abundance, statistics of differentially expressed genes, and their annotations. (XLSX 2683 kb)

Supplementary file 7

. Venn diagram analysis of DEGs of two developmental H. mollissima's hypocotyls and identified genes related to the hypocotyl developments in flax (Roach and Deyholos 2008) and hemp (Behr et al. 2018) based on their assigned Arabidopsis ID (PDF 237 kb)

Supplementary file 8

. List of the unique and shared genes compared to DEGs of two developmental H. mollissima's hypocotyls and identified genes related to the hypocotyl developments in flax (Roach and Deyholos 2008) and hemp (Behr et al. 2008) based on their assigned Arabidopsis ID. (XLSX 65 kb)

Supplementary file 9

. Functional analysis of the Cell signaling genes using Mapman. (XLSX 202 kb)

Supplementary file 10

. Differentially transcription factor genes between hypocotyl A and hypocotyl B using PlantTFDB v5.0. (XLSX 29 kb)

Supplementary file 11

. Cis-elements located upstream of orthologous transcription factor genes with mobile mRNA in Arabidopsis. (XLSX 12 kb)

Supplementary file 12

. Validation of RNA-Seq results using quantitative RT-PCR (qRT-PCR) (PDF 207 kb)

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Zolfaghar, M., Rutten, T., Ghaffari, M.R. et al. Comparative Transcriptome Analysis of Hypocotyls During the Developmental Transition of C3 Cotyledons to C4 Leaves in Halimocnemis mollissima Bunge. J Plant Growth Regul 43, 1076–1092 (2024). https://doi.org/10.1007/s00344-023-11162-1

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