Here it comes! The first full-length transcriptome of the shuttle and the white shuttle responding to drought stress!

Background

2022 is known as the Year of Drought, and the continued high temperature weather has caused droughts around the world, which will lead to reduced crop yields
.
The light will affect the growth and development of the plant, and the heavy will lead to the death of
the plant.

The molecular mechanism of drought stress has been extensively studied in broad-leaved tree species, such as black poplar, birch, etc.
, but only a few studies have focused on important drought-tolerant plants in northwest China
, Haloxylon ammodendron and Haloxylon persicum.

Shuttlecock and Nymphalus are plants of the genus Phyllonoliaceae that are drought-tolerant, high-temperature, saline-alkalid, wind- and cold-resistant
.
They usually form large areas of pure forest in desert and semi-desert areas, with the function of fixing sand dunes and maintaining the structure of the ecosystem, and are known as the guardians of the desert
.
Therefore, it is of great significance to reveal the drought tolerance mechanism of
the genus Phylloscopus sinensis.

Recently, the team of Professor Lu Guanghui of the Institute of Arid Ecological Environment of Xinjiang University published a report entitled "Characterization of the gene expression profile response to drought stress in Haloxylon using PacBio single-molecule real-time" in Frontiers in Plant Science and Illumina sequencing", which obtained the first full-length transcriptome of PacBio full-length transcription (Iso-seq) in response to drought stress, laying a foundation
for further study of its regulatory mechanisms.

1 Technical difficulties

Due to the complex genetic background and large genome of the genus Phylloscopus, the existing whole genome sequence remains incomplete, which limits the study of
its molecular mechanisms of drought tolerance.
In particular, there are great limitations in the discovery of new transcripts and the mining of key genes
.

Although it is possible to sequence different transcripts based on the second-generation sequencing technology, due to the limitation of reading length, it is necessary to infer the whole picture of the transcript through the exon splicing site, and it is impossible to obtain a complete and correctly stitched transcript
.
Therefore, the stitching and annotation of full-length transcripts is still a difficult point
for second-generation sequencing.

2 Solutions

To uncover the transcriptional regulatory network of Passerox and Whitefly in response to drought, the researchers selected long-read PacBio Iso-seq sequencing and Illumina RNA sequencing for full-length transcripts
.

3 Analysis of results

3.
1 Experimental materials

Haloxylon ammodendron
growing environment: wet: soil moisture content 9.
70–15.
00%; Drought: soil moisture content 2.
41–4.
00% Haloxylon persicum
growth environment: wet: soil moisture content 3.
38–5.
12%
; Drought: Land moisture content 1.
05–3.
11%

3.
2 Sequencing results

In order to explore the molecular mechanism of the response of the genus Phylloscopus to drought stress, the researchers performed Iso-seq
on the Shuttlecock and the White Shuttlecock in arid and humid environments.
Shuttle obtained 20246576 subreads, 435938 CCS reads and 35,096 single genes, and Bai Suo obtained 908053 subreads, 210334 CCS reads, and 20,621 single genes
.
Among them, there are 9404 and 8426 single genes up to 2000-3000 bp, and 4281 and 6603 single genes with a length of more than 3000 bp
, respectively.

Table 1 SMRT sequencing results

Note: The final number of single genes in this article is mutually corrected according to PacBio and Illumina sequencing, and the corrected transcripts are clustered according to the 95% similarity between the sequences to eliminate redundancy, and the final number of single genes is 15238 and 10135 (Table 1).

SAB and BAB are general terms for
mixed samples of shuttle and white shuttle under dry and wet conditions.

The difficulty of second-generation sequencing—simple sequence repeats (SSRs) and unknown long non-coding RNAs (LncRNAs), requires sequencing techniques that are both long and accurate
.
The PacBio Iso-seq has just these two advantages:

SSR is widely present in eukaryotic genomes, generally composed of 1-6 bp low degree of repeating sequences, mainly with 2-3 nucleotides as duplicates such as (GA)n, (AC)n and (GAA)n, etc.
, the length is generally below 200 bp, has been widely used in the construction of genetic maps, calibration of target genes and fingerprint mapping
.
Since SSR markers have great application value, researchers use MISA to mine SSR information
in the sequencing data of shuttle and white shuttle.
The results showed that 9794 and 7330 SSRs were identified from mixed samples of these two species, respectively, with single repeats having the highest abundance, followed by triple repeats and double repeats (Figures 1A, B).

LncRNAs are noncoding RNAs larger than 200 nt in length that can be used as competitive endogenous RNAs (ceRNAs) to participate in plant responses to drought stress, but they have not been fully identified
.
Based on the sequencing data of PacBio Iso-seq, the researchers used four different software (PLEK, CNCI, CPC, and Pfam) to make predictions, and 838 and 71 LncRNAs were obtained in the shuttle and 71 LncRNAs in the white shuttle (Figure 1C, D).

This will contribute to a better understanding of the mechanisms of response to drought stress at the molecular level in the future
.

Figure 1 SSR analysis (A-B) and LncRNA prediction (C-D)
of shuttle and white shuttle.

SAB and BAB are general terms for
mixed samples of shuttle and white shuttle under dry and wet conditions.

3.
3 Analysis of drought resistance mechanism

Plant drought tolerance is a complex trait involving multiple genes
.
The researchers analyzed differentially expressed genes (DEGs) involved in drought regulation in the shuttle and the white shuttle under drought and wet conditions, and identified 11803 and 15217 DEGs
, respectively.
Among them, there are 319 and 415 DEGs related to drought stress signal perception and transmission pathways, indicating that the Ca2+ signaling pathway, ABA signaling pathway and MAPK cascade signaling pathway all play an important role
in drought conditions.
However, the number and expression patterns of genes involved in regulation are very different
.

Fig.
2 Heat map
of DEGs of shuttle (A) and white shuttle (B).
The x-axis represents the change in the expression of genes in different samples; The y-axis represents the significance of differences in gene expression;
Genes with significant expression differences are represented
by green dots (upregulated) and bluespots (downregulated).
LS and LB represent the shuttle and the white shuttle in arid environment, respectively; HS and HB
represent shuttles and white shuttles in wet environments
, respectively.

Table 2 Number of transcription factors regulated by the^Ca2+ signaling pathway, ABA signaling pathway, and MAPK cascade signaling pathway under drought conditions

ZEP: zeaxanthin epoxidase (zeaxanthin cyclooxygenase); PYL: abscisic acid receptor (ABA receptor); PP2C: protein phosphatase type-2C (protein phosphatase type-2C); SnRK2: SNF1-related protein kinase 2 (SNF1-related protein kinase 2); CaMs: calmodulin (calmodin); CIPK: CBL-interacting protein kinase

Plant drought stress responses are controlled
by a complex network of transcription factor (TF) regulation.
The study found that the expression of 217 and 398 TFs in the shuttle and the white shuttle under drought conditions varied, including the highly expressed FAR1, MYB, bZIP, C2H2 and AP2/ERF
.
Most of the transcription factors involved in the regulation of the shuttle are upregulated, while the opposite
is true of the white shuttle.

Fig.
3 Transcription factors associated with drought stress in the differential expression of the shuttle (A) and the white shuttle (B) (the first 20 differentially expressed transcription factors were selected).


LS and LB represent the shuttle and the white shuttle in arid environment, respectively; HS and HB represent shuttles and white shuttles in wet environments
, respectively.

Physiological index measurement and bioinformatics analysis showed that under drought stress, the expression patterns of reactive oxygen species (ROS) reactive oxygen species (ROS) removal, glucose metabolism, functional protein and lignin biosynthesis related genes were different, indicating that due to the different degrees of drought, the reactive oxygen species (ROS) clearance, glucose metabolism, functional protein and lignin biosynthesis related genes were different, indicating that due to the different degrees of drought in which they lived, the reactive oxygen species and the white sophora had different regulatory mechanisms and drought tolerance for drought stress
.

3.
4 Schematic model of drought stress response of shuttle and white shuttle

Based on the differential expression genes obtained by sequencing and their related pathways, the researchers propose a shuttle drought response model (Figure 4
).
Under drought stress, the expression of a large number of genes in the shuttle and the white shuttle changed, including stress signal-sensitive genes, antioxidant stress genes, functional protein genes, lignin biosynthesis genes, and glucose metabolism genes
.

 
Figure 4 Hypothetical model
of the drought tolerance mechanism of the shuttle and the white shuttle.
The arrows indicate positive regulation, the line ending with a bar indicates negative regulation, and the dashed arrow indicates neither upward nor downward adjustment
.
The specific drought tolerance mechanisms are shown above, when threatened by drought stress: (1) plant cells can sense stress signals and pass them through ABA (abscisic acid 8'-hydroxylase, PYL, NCED, PP2C, ZEP, SnRK2, XERICO), Ca2+ (CaMBP, CBL, CaM, CML, CIPK, CDPK, CABP) AND MAPK (MAPK, MAPKK, MAPKKK) cascaded signal transduction of the associated protein kinase gene regulation, which is transmitted from outside the cell to the intracellular, and then transcribed
by the relevant TF (MYB, FAR1, AP2/ERF) in the nucleus that regulates the expression of the relevant gene.
(2) TFs promote reactive oxygen species clearance system related genes (APX, CAT, SOD; GPX, GST, POD) upregulation and reduce the content of reactive oxygen species in cells to reduce drought damage
to cells.
(3) Osmotic regulation related genes are regulated
by TFs in the nucleus.
Upregulation of β-amylase and ADH1 increases the synthesis of soluble sugars, macromolecular proteins such as (AQP and HSP; ASR and HSP) begin to accumulate in large quantities, which can balance the intracellular and extracellular water potential
to some extent.
In addition, PIP proteins located on cell membranes and on vacuole membranes can also regulate water balance in cells and vacuoles
.
(4) Drought stress to promote lignin biosynthesis genes (4CL, β-glucosidase, CCOMT, CCR, COMT, F5H, laccase; β-glucosidase, CADH, CONT, etc.
) expression increases, thereby enhancing the drought tolerance of plants
.

Sum up

Based on PacBio Iso-seq, the researchers obtained the first full-length transcriptome on the response of shuttle and white shuttle to drought stress without the reference gene being perfect, and resolved the response
of shuttle and white shuttle to drought stress through the synergy of physiological and molecular reactions.
Compared with the shuttle, the growth environment of the white shuttle is more harsh (the soil moisture content is lower) and the drought tolerance is stronger
.
Shuttlecock, on the other hand, grows and changes physiology more dramatically in arid conditions, and responds faster and more intensely
.
These results provide a basis
for further study of the adaptation mechanism and breeding improvement strategy of plants under drought stress.

Iso-seq

Based on HiFi sequencing, it can not only reveal the transcript diversity brought about by variable splicing, but also be used for SSR molecular markers, variable polyadenylate, gene annotation, fusion gene transcripts, phasing of transcript subtype variants, and LncRNA analysis and detection
.

tips

The use of PacBio Iso-seq binding to Illumina RNA-seq is based on the technical route
of published articles before 2019.
Because the data measured by PacBio Iso-seq before 2019 were not accurate enough, the PacBio Iso-seq data needed to be corrected with Illumina RNA-seq data (before the release of HiFi
).
In fact, since the advent of HiFi sequencing technology featuring single-molecule real-time (SMRT) sequencing in 2019, PacBio Iso-seq has been
based on HiFi sequencing.
HiFi sequencing uses Circular consensus sequencing (CCS) sequencing, and the accuracy rate can reach Q30
after the random error is corrected by continuous reading between molecules.
With its advantages of being both long (up to 25 kb) and accurate (accuracy Q30), high-quality full-length transcript information can be obtained directly by HiFi sequencing without reference genome, without interruption and assembly
.
Therefore, HiFi-based sequencing now eliminates the need for second-generation sequencing to be corrected, and HiFi sequencing alone can obtain full-length and accurate Iso-form sequence information
.

Genes Ltd

As the exclusive agent of PacBio in China, Gene Co.
, Ltd.
has introduced PacBio's third-generation single-molecule real-time sequencing technology into China since 2011, and has been providing domestic users with professional third-generation sequencing system installation training, technical support, application training and after-sales maintenance work, winning the praise and trust
of customers.
Genetics Ltd.
will continue to support more and more PacBio users
.