Chang Qu·Xiuyan Bian·Rui Han·Jing Jiang·Qibin Yu·Guifeng Liu

Abstract Auxin polar transport genes PIN (PINFORMED)determine the concentration gradient of auxin in plants. To understand the relationship between the development of different tissues in Betula pendula‘Dalecartica',BpPIN gene expression and indole-3-acetic acid(IAA)content were analyzed using qRT-PCR,ELISA,and GUS staining.Gene expression of BpPIN genes and IAA levels in the leaves,buds,stems,xylem,and roots of B.pendula‘Dalecartica'and B.pendula as a control were measured.BpPIN1,BpPIN5 and BpPIN6 were upregulated during development in both species,suggesting a dominant role in the development of B.pendula‘Dalecartica'leaves.Moreover,BpPIN1 gene expression was positively associated with IAA levels during leaf, vein and petiole development in B.pendula‘Dalecartica'only.The correlation coefficient of the first three leaves was 0.69(P=0.04),while that of the first three petioles was 0.85(P=0.001). In addition, GUS staining of the pro-DR5::GUS transgenic line of cultivar was correlated with the results of BpPIN1 expression.Overall,these findings suggest that BpPIN1 is associated with the formation of lobed leaves in B.pendula‘Dalecartica'.

Keywords Betula pendula·Betula pendula‘Dalecartica'·BpPIN genes·Gene expression·IAA

Introduction

Betula pendula‘Dalecartica'is an intraspecies variety of B.pendula,cutleaf weeping birch,was selected breeding of B.pendula and is distinguished by 7-9 lobes on its incised,ovate,triangular leaves.The lobes,3-4 per side,gradually increase in size similar to the leaves of Acer ginnala Maxim (Valobra and James 1990; Mu et al. 2013).Although the leaves of B.pendula are also triangular ovate,the leaf margin does not contain lobes. Furthermore,viewed with a microscope,the leaf veins of the variant are obvious on the raised abaxial surface of the leaf along with thorns and a dense covering of villi.Thus,because of its white trunk and attractive leaves,the variant has significant ornamental value and is widely used in landscaping in Europe and America.

Previous studies suggest that the formation of the lobes at the leaf margin is closely related to the polar transport and concentration gradient of auxin in different parts of the leaf margin,with higher concentrations of indole-3-acetic acid(IAA)near lobed leaves or sinuses(Hay et al.2006;Bilsborough et al.2011;Adamowski and Friml 2015).In Arabidopsis, the genes PIN, CUC2 and miR164 are involved in the IAA concentration gradient(Liu et al.2009,2014;Zhao 2009;Bilsborough et al.2011;Zhou et al.2011;Wang et al.2016).PIN proteins,polar transport carriers of IAA(Hu et al.2015),regulate IAA flow and local accumulation (Li 2015). AtPIN genes consist of multiple genes,while AtPIN proteins represent transmembrane proteins.The AtPIN protein family is further divided into two subfamilies according to the length of the hydrophilic ring.Members of the long hydrophilic-loop subfamily are located on the cell membrane and are responsible for transporting auxin out of the cell,thereby playing a role in auxin transport between cells.In contrast,members of the short hydrophilic domain subfamily are localized on the endoplasmic reticulum and are responsible for transport of auxin between the cytoplasm and endoplasmic reticulum,thereby helping regulate the balance of auxin in the cell(Wang et al.2015).Previous studies have also found that the concentration of hormones at the lobes and leaf margins of the variant are higher than in the middle of the leaves(Qu et al.2017).However,while six BpPIN genes are known to exist in birch,their biological function during the formation of birch lobes remains unknown.In this study,BpPIN gene expression was measured in the leaves of B.pendula and the variant,and the relationship between BpPIN gene expression and IAA content was investigated.The findings provide insight into the formation of the variant leaves.

Materials and methods

Plant materials

To analyze expression characteristics of the BpPIN genes in B.pendula and the variant and the relationship between IAA content and the formation of the variant leaf lobes,we used 1-year-old plants of the variant and B.pendula(Rousi and Pusenius 2005).Plants of both species had more than 100 ramets.From both species,buds(B),1st to 4th leaves without the main vein(L1-L4),petioles(P1-P4),2nd to 4th leaves'main veins(V2-V4),stems(S),xylem(X)and roots(R)were collected.Samples were collected on 3 June 2015 between 07:00 and 08:00 a.m(Fig.1).Tissue samples were divided into 3-4 portions,wrapped in foil then immediately placed in liquid nitrogen and stored in the laboratory at -80°C until analysis of BpPIN gene expression and IAA content.

Analysis of amino acid sequences of BpPIN family proteins in B.pendula‘Dalecartica'

To determine the function of the BpPIN genes,we compared PIN family genes in Arabidopsis thaliana and Populus trichocarpa.Conserved domains of A.thaliana AtPIN and P.trichocarpa PrtPIN gene sequences were obtained from the NCBI website (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Two conserved domains were subsequently compared with the annotated full-length sequences of the six BpPIN genes on the birch genome(http://birch.genomics.com).Amino acid sequences of the BpPIN proteins were also analyzed,and their functions were predicted using TMHMM Server,v.2.0(http://www.cbs.dtu.dk/services/TMHMM/).

Fig.1 Sketch map of material location.Buds(B);The first to forth pieces of leaves without the main veins(L1-L4);Petioles(P1-P4);The second to fourth leaf veins(V2-V4);Spears(S);Xylems(X);Roots(R)

Gene expression analysis of the BpPIN genes

Spatiotemporal expression of the BpPIN genes was analysed with qRT-PCR.Primer 5.0 software was used to design the target gene and internal primer sequences.Primer sequences were compared using the BLAST program on the NCBI website to ensure primer specificity(Table 1),then synthesised by Invitrogen Group Trading Co.,Ltd.(Shanghai,China).

Total RNA was extracted using a Plant RNA Extraction Kit(Beijing BioTeke Biotechnology Co.,Ltd.,China)and reverse-transcribed to cDNA using a PrimeScript RT reagent Kit with a gDNA Eraser(Toyobo Co.,Ltd,Osaka,Japan).Reverse-transcribed cDNA was diluted tenfold and used as a template for qRT-PCR amplification of BpPIN1,BpPIN2, BpPIN3, BpPIN4, BpPIN5 and BpPIN6. A quantitative SYBR green PCR Kit(Toyobo Co.,Ltd)with 18S as a housekeeping gene were used as described previously(Huang 2014).

Relative expression of each exogenous gene(XN)was calculated using the formula:XN=-ΔΔCt,where Ct is the cycle threshold representing the number of cycles required for the fluorescence signal in the reaction tube toreach the set threshold value,ΔCt is the average Ct value of the target gene-the mean Ct value of the housekeeping gene,and ΔΔCt is ΔCt(sample 1)-ΔCt(sample 2),and-ΔΔCt represents the original data.Expression levels of BpPIN genes in the buds of B.pendula were used as a control.Data processing and the relative expression of each gene in each organ were determined using Excel 2013(Microsoft,Redmond,WA,USA).

Table 1 Primers used for quantitative real time PCR

Pro-BpPIN::GUS vector construction and genetic transformation

BpPIN1,BpPIN3 and BpPIN5 promoters were cloned to create pro-BpPIN::GUS vectors for genetic transformation.GUS staining was then used to detect BpPIN gene expression in each sample(Qu et al.2017).

Promoter sequence cloning and vector construction

Using birch total DNA as a template,2000-bp upstream sequences of BpPIN1,BpPIN3 and BpPIN5 genes were selected.According to the characteristics of the carriers and pro-BpPIN gene sequences,restriction enzyme sites were added at the 5′end of the upstream and downstream primers before PCR amplification(Table 2)(Yang 2015).The PCR products were detected by 1%agarose gel electrophoresis and stained by gel red,and the target fragments were recovered and purified using a gel recovery kit(Biospin Gel Extraction Kit,the BioFlux company).Purified fragments of the BpPIN1, BpPIN3 and BpPIN5promoters were then connected to the carrier cut by enzymes.The recombinant plasmid(pro-BpPIN::GUS)was transformed into Escherichia coli using the heat shock method,and EHA105(pro-BpPIN1::GUS),EHA105(pro-BpPIN3::GUS) and EHA105 (pro-BpPIN5::GUS) were obtained using the three-parent mating method(Yang et al.2015a,b).

Table 2 Primers for vector construction

Genetic transformation of B.pendula

Mature seeds from the half-sibling of the same birch tree were used as transgenic material for genetic transformation.Zygotic embryos were infected with Agrobacterium tumefaciens and incubated in co-cultivation medium(Zhang 2009).The above-mentioned transformed bacteria were then used to infect sterile birch embryos.The callus was obtained by co-culture in WPM+2.0 mg/L 6-Benzylaminopurine (6-BA)+0.2 mg/L 1-naphthlcetic acid(NAA)then selective culture(WPM+2.0 mg/L6-BA+0.2 mg/L NAA+0.5 mg/L Gibberellin A3(GA3)+50 mg/L hygromycin (Hyg) or 100 mg/L Kanamucin(Kan)+200 mg/L cefotaxime)was used to form adventitious buds,which gradually differentiated into clumps seedlings after 20-30 days.Clustered plants were subcultured on WPM+1.0 mg/L 6-BA+50 mg/L Hyg or 40 mg/L Kan+200 mg/L cefotaxime,then transferred to rooting medium(WPM+0.2 mg/L NAA).After 30 days of culture,rooted plants were stained with β-glucuronidase(GUS)for tissue expression analysis of the above genes(pro-BpPIN1,pro-BpPIN3 and pro-BpPIN5).

Determination of IAA content and localization analysis

To analyze the relationship between IAA content and BpPIN gene expression, we used an enzyme-linked immunosorbent assay(ELISA)to determine the content of endogenous IAA in the different organs of B.pendula and the variant(Wang et al.2000).Mature seeds were used to determine the distribution of IAA.After transferring pro-DR5::GUS into the two birch species,the distribution of IAA was observed directly through GUS staining.The DR5 sequence was as described previously(Liu 2011;Yang et al.2015a,b;Yang 2015).Primer synthesis,genetic transformation and GUS staining were carried out as described previously(Qu et al.2017).

Results

Phylogenetic tree of BpPIN genes from B.pendula,A.thaliana and P.trichocarpa

Phylogenetic tree analysis revealed high homology between the PIN genes of B.pendula and P.trichocarpa,which made by ClustalW(Fig.2).A single group contained the six BpPIN genes, with BpPIN1, BpPIN4,BpPIN5 and BpPIN6 clustered with the PtrPINs of P.trichocarpa on multiple branches.In contrast,the relationship between AtPIN genes of A.thaliana and BpPIN genes of B.pendula didn't as close as that between B.pendula and P.trichocarpa.The highest similarity(68%)among the six BpPIN genes of B.pendula was observed between BpPIN1 and BpPIN6,suggesting a close relationship between these two genes.In the tree,they were clustered with PtrPIN1a and PtrPIN1b,and PtrPIN1c and PtrPIN1d of P.trichocarpa,respectively.

Structure of the BpPIN proteins

The six BpPIN proteins had a similar structure,each possessing a hydrophobic region on each end and a hydrophilic region in the middle(Fig.3).However,they were further divided into two subfamilies according to the number of amino acid residues in each PIN,and the degree and function of membrane folding in the hydrophobic zone.One subfamily consisted of four proteins;namely,BpPIN1,BpPIN2,BpPIN3,and BpPIN6,all of which had approximately 600 amino acid residues.The hydrophobic regions at both ends of these proteins had 4-5 transmembrane folds,while the middle hydrophilic region was long,categorizing them in the long hydrophilic domain subfamily.According to a previous study of AtPIN proteins in A.thaliana(Li and Zhai 2006),the function of this subfamily is IAA transport between cells.The second subfamily was composed of BpPIN4 and BpPIN5,which had 400 and 350 amino acid residues,respectively,and a shorter middle hydrophilic zone,categorizing them in the short hydrophilic domain subfamily.Similarly,according to the above study of A.thaliana,this subfamily functions in IAA transport within the cell.

Fig.2 Phylogenetic tree of BpPIN amino acid sequence in B.pendula and other plants

BpPIN gene expression in different leaf tissues in B.pendula‘Dalecartica'and B.pendula

Three similar expression patterns were observed in the leaf tissues of the two birch species(Fig.4).The first pattern reflected obvious upregulation of BpPIN1,BpPIN5 and BpPIN6,while the second showed significant downregulation of the BpPIN2 gene only.The third pattern consisted of up- and down-regulation of BpPIN3 and BpPIN4,respectively. Differences in BpPIN gene expression between the two species were significant(P ＜0.05)or highly significant(P ＜0.01).The leaf tissues of the variant had higher expression of BpPIN proteins than those of B.pendula.

Fig.3 The transmembrane region of the protein encoded by BpPIN genes

BpPIN gene expression in different organs in B.pendula‘Dalecartica'and B.pendula

Relative expression levels in the leaves,stems,xylem and roots of the two birch species were subsequently determined.Expression of all six BpPIN genes was consistent with that in the leaves(Fig.5),that is,upregulation of BpPIN1,BpPIN5 and BpPIN6 in most tissues in both species,with downregulation of BpPIN2 in all samples.Expression of BpPIN3 and BpPIN4 was up-and downregulated,respectively.

Genetic transformation and gene expression of pro-BpPIN::GUS

To further examine expression of BpPIN1,BpPIN3 and BpPIN5,we inserted pro-BpPIN1::GUS,pro-BpPIN3::GUS and pro-BpPIN5::GUS vectors into the birch genome to obtain transgenic resistant plants(Fig.6).GUS staining was then performed in resistant rooted plants.The results are shown in Fig.7.

The wild-type nontransgenic line had no staining,but the degree of GUS staining was similar in the roots of the three transgenic lines.Staining was strongest in young leaves and less so in mature leaves,with obvious variation among the different lines.Of the transgenic lines,pro-BpPIN1::GUS and pro-BpPIN5::GUS had the strongest staining,suggesting that BpPIN1 and BpPIN5 were highly expressed. Meanwhile, the light staining of pro-BpPIN3::GUS suggested low expression of BpPIN3.These results were consistent with the quantitative results of BpPIN gene expression.

Fig.4 Expression characteristics of BpPIN genes in leaves and tissues of two species of birch.Buds(B);The first to forth leaves(L1-L4);The second to fourth leaf veins(V2-V4);The first to forth petioles(P1-P4)

Combined analysis of IAA content and BpPIN gene expression in B.pendula‘Dalecartica'and B.pendula

IAA content in leaf tissues

Comparisons of IAA content between the two species revealed a higher content of IAA in most samples from the variant compared with B.pendula(Table 3).The average IAA content was 69.97 ng/g fw in the variant and 48.31 ng/g fw in B.pendula,indicating a 44.8%higher content in the variant.The IAA content was much higher in the petiole of the first leaf in both birch species(495.25 and 276.89 ng/g fw in the variant and B.pendula,respectively)compared with all other samples by an average of 16.6-and 10.6-fold,respectively.In addition,in the variant,the IAA content of the first four leaves increased with increasing leaf size up to the fourth leaf,followed by a significant reduction. No such phenomenon was observed in B.pendula.

Fig.5 Tissue expression characteristics of BpPINs gene.Buds(B);The first leaves(L);Spears(S);Xylems(X);Roots(R)

BpPIN gene expression and IAA contents

Expression of BpPIN1,BpPIN5 and BpPIN6 in the variant was correlated with leaf development and the change in IAA content(Table 3).Expression of BpPIN1 increased with increasing leaf size and increasing IAA content.The correlation coefficient between BpPIN1 gene expression and the content of IAA in the first three leaves was 0.69(P=0.04),while in the petioles a gradual decrease in expression was observed in both species,with a highly significant correlation coefficient of 0.85 (P=0.001).Expression of BpPIN1 was negatively correlated with BpPIN6 expression in the leaves(r=-0.59,P=0.094)and with BpPIN5 gene expression in the petioles of the variant(r=-0.66,P=0.054)(Table 3).

IAA localization analysis via GUS staining

When IAA levels in different tissues that developed from GUS-transformed embryos were examined(Fig.8),young leaves had stronger staining than in mature leaves.The petiole of first leaves also had stronger staining than in that of second,third and fourth leaves.The intensity of GUS staining represented BpPIN gene expression and was consistent with the IAA levels in different samples.

Fig.6 Genetic transformation of pro-BpPIN::GUS.a Genetic transformation of zygotic embryo;b resistant callus;c resistant subculture;d the resistant rooting seedlings of pro-BpPIN1::GUS;e the resistant rooting seedlings of pro-BpPIN3::GUS; f the resistant rooting seedlings of pro-BpPIN5::GUS

Fig.7 Gus staining of pro-BpPIN::GUS transgenic lines

Discussion

Relationship between the phylogenetic tree and protein structure and function

By comparing DNA sequences between species or protein sequences within a gene family and constructing phylogenetic trees,we can accurately determine the evolutionarystatus between species and genes.In this study,we compared the amino acid sequences of PIN family genes of B.pendula,P.trichocarpa and A.thaliana and confirmed a close phylogenetic relationship between B.pendula and P.trichocarpa.Both species are broadleaf deciduous trees,very different from the annual herbaceous plant A.thaliana.In the traditional classification of plants based on catkin characteristics,both are classified into the same family.This study confirms this reasonable evolutionary origin.

Table 3 IAA content and expression of BpPIN1,BpPIN5 and BpPIN6 genes in B.pendula and B.pendula Dalecartica

Fig.8 Gus staining of pro-DR5::GUS transgenic lines

The structural characteristics of PIN proteins determine its location in cells and whether hormone transport is intercellular or intracellular(Hu et al.2015).For example,the eight AtPIN genes of A.thaliana(Adamowski and Friml 2015)can be divided into two subfamilies,those possessing a long hydrophilic ring and those with a short hydrophilic ring.Members of the long hydrophilic-loop subfamily are located on the cell membrane and are responsible for auxin transport from the cell,suggesting a role in auxin transport between cells.In contrast,members of the short hydrophilic domain subfamily are localized on the endoplasmic reticulum and are responsible for auxin transport between the cytoplasm and endoplasmic reticulum,thereby helping regulate the balance of auxin in the cell(Wang et al.2015).There are six BpPIN genes in the birch genome,which were divided into two subfamilies.The BpPIN1, BpPIN2, BpPIN3 and BpPIN6 proteins belong to the long hydrophilic subfamily,while BpPIN4 and BpPIN5 belong to the short hydrophilic domain subfamily.According to the PIN gene functions of the A.thaliana subfamilies,these two subgroups of BpPIN proteins in birch are thought to be responsible for intracellular and intracellular hormone transport, respectively. In addition,the BpPIN4 and BpPIN5 proteins had only one transmembrane fold at the C-terminus,differing from the reported 5 or 6 transmembrane folds at both ends of PIN proteins in other plant species(Liu et al.2009).

Tissue-specific expression of BpPIN1 genes was correlated with IAA content

The PIN proteins also had tissue-or cell-specific characteristics,with different PIN members expressed differentially.Temporal expression of PIN genes is also strongly correlated with the polar transport of auxin(Okada et al.1991;Peer et al.2004;Zhu et al.2013).The existence of multiple highly conserved PIN genes therefore indicates that there are many paths of auxin transport in the plants,reflecting the complexity of auxin polar transport.These findings also confirm that the PIN family regulates different growth and developmental processes.The spatiotemporal expression of the BpPIN genes also had tissuespecific characteristics.For example,BpPIN1,BpPIN5 and BpPIN6 were expressed in most samples and were upregulated,suggesting a leading role in birch growth and as the main carrier of IAA(Figs.4,5,7).Although expression of BpPIN2,BpPIN3 and BpPIN4 was relatively low,their expression was higher in the roots than in all other samples,suggesting a role in root development(Figs.4,5,7).During the development of B. pendula ‘Dalecartica',expression of BpPIN1 increased with increasing leaf size,while expression in the veins and petioles gradually decreased with increasing leaf size.Furthermore,expression of BpPIN1 also reflected the change in IAA content.In contrast,no such correlation was observed in B.pendula(Table 3),suggesting that the BpPIN1 gene is associated with the formation of leaf lobes in birch.

Comparison of the IAA transport system and transport volume in the two species of birch

The distribution of auxin in plants depends on two interconnected transport systems, rapid and nondirectional transport,which occur in sieve tubes and are accompanied by photosynthates and slow directional intercellular transport.Transport through sieve tubes is commonly used to deliver auxin from the site of synthesis to the receiving organ,while polar auxin transport is an efficient way of providing auxin locally at the developmental site.In general,the more developed the conducting tissue,the more hormone can be transported.Thus,the variant leaves have well-developed conducting tissues.For example,the crosssectional area of mature leaves is as high as 26.42 dm2,21.46%larger than in B.pendula(Qu et al.2017).Moreover,the average IAA content of the variant leaf veins and petioles was 36.96%higher than in B.pendula.In addition,auxin content in first leaf petioles was very high in both the variant and B.pendula at 495.25 and 276.89 ng/g fw,respectively,and 12-and sixfold higher than in second leaf petioles, respectively. These findings suggest that the majority of hormones required for leaf development are acquired via long-distance transmission through conducting tissues,taking the form of passive nonpolar transport in a combined state(Ansari et al.2004;Kumar et al.2011).When transported to the petioles,auxin is then oxidized by light or enzymatically degraded into its free form.The petioles subsequently store the auxin, while the IAA required for growth and development of young leaves is polar-transported by BpPIN carrier proteins.To summarize,we confirmed that the location of BpPIN genes is similar to that of IAA.Moreover,of the 6 BpPIN genes,BpPIN1,BpPIN5,and BpPIN6 encoded the main carriers of IAA during development of the variant leaves and other organs,suggesting a role in the formation of the lobed leaves.