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Identification of HvLRX, a new dehydration and light responsive gene in Tibetan hulless barley (Hordeum vulgare var. nudum)

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Abstract

Background

Tibetan hulless barley (Hordeum vulgare var. nudum), adjusting to the harsh environment on Qinghai–Tibet Plateau, is a good subject for analyzing drought tolerance mechanism. Several unannotated differentially expressed genes (DEGs) were identified through our previous RNA-Seq study using two hulless barley accessions with contrasting drought tolerance. One of these DEGs, HVU010048.2, showed up-regulated pattern under dehydration stress in both drought tolerant (DT) and drought susceptible (DS) accessions, while its function in drought resistance remains unknown. This new gene was named as HvLRX (light responsive X), because its expression was induced under high light intensity while suppressed under dark.

Objective

To provide preliminary bioinformatics prediction, expression pattern, and drought resistance function of this new gene.

Methods

Bioinformatics analysis of HvLRX were conducted by MEGA, PlantCARE, ProtParam, CELLO et al. The expression pattern of HvLRX under different light intensity, dehydration shock, gradual drought stress, NaCl stress, polyethylene glycol (PEG) 6000 stress and abscisic acid (ABA) treatment was investigated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The function of HvLRX was analyzed by virus induced gene silencing (VIGS) in hulless barley and by transgenic method in tobacco.

Results

Full cDNAs of HvLRX were cloned and compared in three hulless barley accessions. Homologues of HvLRX protein in other plants were excavated and their phylogenetic relationship was analyzed. Several light responsive elements (ATC-motif, Box 4, G-box, Sp1, and chs-CMA1a) were identified in its promoter region. Its expression can be promoted under high light intensity, dehydration shock, gradual drought stress, PEG 6000, and NaCl stress, but was almost unchanged in ABA treatment. HvLRX-silenced plants had a higher leaf water loss rate (WLR) and a lower survival rate (SR) compared with controls under dehydration stress. The infected leaves of HvLRX-silenced plants lost their water content quickly and became withered at 10 dpi. The SR of HvLRX overexpressed transgenic tobacco plants was significantly higher than that of wild-type plants. These results indicated HvLRX play a role in drought resistance. Besides, retarded vegetative growth was detected in HvLRX-silenced hulless barley plants, which suggested that this gene is important for plant development.

Conclusions

This study provided data of bioinformatics, expression pattern, and function of HvLRX. To our knowledge, this is the first report of this new dehydration and light responsive gene.

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Abbreviations

ABA:

Abscisic acid

ANOVA:

Analysis of variance

BPs:

Bootstrap probabilities

BSMV:

Barley stripe mosaic virus

DEGs:

Differentially expressed genes

DPI:

Days post inoculation

DT:

Drought tolerant

DS:

Drought susceptible

DW:

Dry weight

EF:

Elongation factor

FW:

Fresh weight

GFP:

Green fluorescent protein

LSD:

Least significant differences

MS:

Murashige and Skoog

NCBI:

National Center for Biotechnology Information

PEG:

Polyethylene glycol

PPDK:

Pyruvate phosphate dikinase

RT-qPCR:

Quantitative reverse transcription-polymerase chain reaction

SD:

Standard deviation

SR:

Survival rate

RT-PCR:

Reverse transcription-polymerase chain reaction

TDW:

Total dry weight

VIGS:

Virus induced gene silencing

WC:

Water content

WLR:

Water loss rate

WT:

Wild type

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Acknowledgements

Prof. Daowen Wang of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, are kindly acknowledged for providing BSMV vectors.

Funding

This work was financially supported by Applied Basic Research Program of Sichuan Province (2019YJ0011); Science and Technology Service Network Initiative (KFJ-STS-QYZD-2021–22-001) by Chinese Academy of Sciences; Major Tibet Science and Technology Projects (XZ2021NA01) by Science and Technology Department of Tibet; Innovation Team of Triticeae Crops of Sichuan Province by Sichuan Provincial Department of Agriculture and Rural Affairs. All the funding bodies did not participate in the design of the study, collection, analysis, interpretation of data, or in writing the manuscript.

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Authors and Affiliations

  1. Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, China

    Junjun Liang, Haili Zhang, Ling Yi, Hai Long, Maoqun Yu & Guangbing Deng

  2. Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa, 850032, China

    Yawei Tang

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  1. Junjun Liang
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  2. Haili Zhang
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  3. Ling Yi
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  4. Yawei Tang
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  5. Hai Long
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  6. Maoqun Yu
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  7. Guangbing Deng
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Contributions

JL and GD conceived and designed the experiments; JL, HZ and LY conducted the experiments; JL and HZ analyzed the data; JL and HL wrote the manuscript; MY and YT revised the manuscript. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Guangbing Deng.

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13258_2021_1147_MOESM1_ESM.xlsx

Fig. S1: Map of vector pJG081. Fig. S2: Nucleotide sequences alignment of HvLRX and AK353794.1. Fig. S3: Amino acid sequences alignment of HvLRX and AK353794.1. Fig. S4: Nucleotide sequences alignment of HvLRX, TaLRX-2A, TaLRX-2B, and TaLRX-2D. Fig. S5: Protein domain of HvLRX analyzed by Pfam. Fig. S6: Details for all protein motifs in Fig. 2. Fig. S7: Sequence analysis of HvLRX promoter by PlantCARE. Fig. S8: Secondary structure prediction of HvLRX and AK353794.1 using GOR 4. Fig. S9: Subcellular localization predication of HvLRX (Z772) using CELLO v.2.5. Fig. S10: PCR amplification analysis for transgenic lines confirmation. Table S1: Primers for gene cloning, vector construction, and RT-qPCR. Table S2: Nucleotide sequences of HvLRX in Z013, Z033, and Z772. Table S3: The deduced amino acid sequence of HvLRX protein in Z013, Z033, and Z772. Table S4: Nucleotide sequences of TaLRX-2A, TaLRX-2B, and TaLRX-2D. Table S5: Motifs found in the promoter of HvLRX (2000 bp before ATG) detected using PlantCare algorithm. Table S6: Protein properties prediction of HvLRX and AK353794.1 using ProtParam. Table S7: Secondary structure prediction of HvLRX and AK353794.1 using GOR 4. (XLSX 16405 KB)

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Liang, J., Zhang, H., Yi, L. et al. Identification of HvLRX, a new dehydration and light responsive gene in Tibetan hulless barley (Hordeum vulgare var. nudum). Genes Genom 43, 1445–1461 (2021). https://doi.org/10.1007/s13258-021-01147-3

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