Planta (2015) 241:371–385 DOI 10.1007/s00425-014-2187-y

ORIGINAL ARTICLE

Identification of differentially expressed transcripts associated with bast fibre development in Corchorus capsularis by suppression subtractive hybridization Pradipta Samanta • Sanjoy Sadhukhan Asitava Basu

•

Received: 6 May 2014 / Accepted: 30 September 2014 / Published online: 16 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Main conclusion The present study documented the predominant role of WRKY transcription factor in controlling genes of different pathways related to fibre formation in jute and could be a candidate gene for the improvement of jute fiber. Abstract Understanding of molecular mechanism associated with bast fibre development is of immense significance to achieve desired improvement in jute (Corchorus sp.). Therefore, suppression subtractive hybridization was successfully applied to identify genes involved in fibre developmental process in jute. The subtracted library of normal Corchorus capsularis as tester with respect to its fibre-deficient mutant as driver resulted in 2,685 expressed sequence tags which were assumed to represent the differentially expressed genes between two genotypes. The identified expressed sequence tags were assembled and clustered into 225 contigs and 231 singletons. Among these 456 unigenes, 377 were classified into 15 different functional categories while others were of unknown functional category. Reverse Northern analysis of the unigenes showed distinct variation in hybridization intensity of 11 transcripts between two genotypes tested. The findings were also documented by Northern and real-time PCR analysis. Varied expression level of these transcripts

Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2187-y) contains supplementary material, which is available to authorized users. P. Samanta  S. Sadhukhan  A. Basu (&) Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology, Kharagpur 721302, India e-mail: [email protected]; [email protected]

suggested their crucial involvement in fibre development in this species. Among these transcripts, WRKY transcription factor was documented to be a most important transcript which was in agreement with its known role in other plant species in possible regulation related to cell wall biosynthesis, expansion and lignification. This report constitutes first systematic analysis of genes involved in fibre development process in jute. It may be suggested that the information generated in this study would be useful for genetic improvement of fibre traits in this plant species. Keywords Corchorus  Expressed sequence tags  Fibre-deficient mutant  Northern hybridization  Real-time PCR  Suppression subtractive hybridization Abbreviations AGP Arabinogalactan protein CAD Cinnamyl alcohol dehydrogenase CCR Cinnamoyl-CoA reductase C4H Cinnamate-4-hydroxylase CHS Chalcone synthase 4CL 4-Coumarate CoA:ligase COMT Caffeic acid 3-O-methyltransferase Das Days after sowing EST Expressed sequence tags FF Fibre forming F5H Ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydoxylase GRP Glycine rich protein HCT Hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase HD-Zip Homeodomain leucine zipper protein PAL Phenylalanine ammonia lyase PEL Pectate lyase PME Pectin methylesterase

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SSH XET ZFP

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Suppression subtractive hybridization Xyloglucan endotransglycosylase Zinc finger protein

Introduction Jute (Corchorus sp. 2n = 14) is a commercially important crop, cultivated mainly for its bast fibres, and is of great concern today with its cheaper availability and fibres having relatively higher tensile strength than found in other plants. The fibre is mostly used for bio-degradable packaging materials, and the economy of the marginal farming community largely depends on the success of this crop in tropical South-East Asia. Improvement of this crop species in terms of fibre yield and quality is a long-awaited demand in textile, non textile and construction industries. Continuous attempts have been made for the generation of jute varieties with better fibre quality by breeding methods and by chemical and physical mutagenesis, yet without any significant outcome. The biotechnological approaches were severely restricted due to lack of sufficient information about the genetic factors involved in the fibre developmental processes. In general, fibre development in plants has a number of consecutive stages like differentiation, expansion/elongation and deposition of secondary metabolites till maturity. These events are accompanied by multiple changes during transcription, post-transcription, and translation (Yadun 2010). Each of the developmental stages is expected to have its specific and crucial role in certain properties of the jute fibre also. Current elucidation on the molecular processes underlying the mechanism of fibre development in any bast fibre forming plant species is far from adequate. So, any attempt for identification of genetic factors related to its fibre developmental processes in jute is considered worth pursuing. Rational use of a suitable mutant would be a powerful approach for identification of the genetic factor(s) involved in fibre formation in any plant species and has already been demonstrated to explore the genetic basis of a trait in different plant systems (Hsing et al. 2007; Bolton et al. 2009). Among them, elucidation of genetic factors involved in cotton fibre development using a lintless mutant is of great significance (Bolton et al. 2009). Though anatomically fibres of cotton and jute are different, the similar strategy could be applied in jute also. Fortunately, an artificially induced fibre-deficient mutant, known as ‘soft-stem’ mutant, is available in the genetic stock of Corchorus capsularis. The mutant shows significant morphological differences in comparison to normal cultivated genotype (Sengupta and Palit 2004). The most conspicuous

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morphological difference in this mutant is in its architecture of stem. The mutant has undulated stem compared to the normal one. It has been reported that the mutant produce less fibre in spite of its photosynthetic rate comparable to the normal cultivated variety (Sengupta and Palit 2004). Anatomically, the retted fibre cells of the mutant were of less thickness having more pith area in lumen and rough surface compared to those of normal jute plant (Sadhukhan 2007). Moreover, chemical composition analysis of the fibre showed that the mutant fibre contains 54 and 27 % less lignin and hemicellulose, respectively, compared to the normal one (Sadhukhan 2007; Sengupta and Palit 2004). These differences between the two genotypes were expected to be due to alteration in expression of the genetic factor(s) as the development of fibre was suggested to be genetically controlled (Ghosh 1983). Thus, this mutant could be a valuable resource for comparative gene expression study related to bast fibre formation in C. capsularis. Understanding the alteration in expression pattern of the functional genes in either of the genotypes may help to decipher the involvement of genetic factor(s) in the fundamental process of fibre development in this plant species. Suppression subtractive hybridization (SSH) is a powerful and efficient method to identify genes those are differentially expressed in one or the other population. Though large extent of efforts has been employed to identify differentially expressed gene(s) related to a certain trait in several plant species, no such attempt yet has been made in jute to make it more amenable to fulfil agricultural and/or industrial demands. Thus, an attempt has been made to identify differentially expressed gene(s) at fibre forming stages in the stem of normal jute plant with respect to ‘softstem’ mutant using SSH technique for understanding of the fibre development process in this fibre crop.

Materials and methods Plant material Seeds, obtained from the inbred genetic stock of CRIJAF (Barrackpore, West Bengal, India), of cultivated variety of C. capsularis L., JRC 321 and its ‘soft stem’ mutant were allowed to grow in the field and used as the source of plant materials in the present study. The plants were grown in loamy soils at warm humid climate with temperature in between 25 and 40 °C and rainfall of 150–200 cm. Developmental stages were categorized into two: one was ‘fibre forming stage’ (‘FF’ stage) and other was ‘non-fibre forming stage (‘non-FF stage) following histological study. Stage 7–14 days after sowing (das) for normal and 14–21 das for mutant were designated as ‘FF’ stage whereas prior to ‘FF’ stages for both of the plants were considered as

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‘non-FF’ stage. The growth of both the plants was same till 14 das. After that, the growth of the normal plants was faster compared to the mutant one throughout the developmental stages. Histological analysis Histological analysis was done to define the ‘fibre forming’ stage (‘FF’ stage) through microscopic visualization of the appearance of fibre and deposition of fibre-related components between C. capsularis L., JRC 321 and ‘soft-stem’ mutant plants using lignin-specific stain phloroglucinol, as lignin is known to be one of the major components of jute fibre. Phloroglucinol staining was performed according to the standard protocol (Jensen 1962) with a little modification in incubation time of the sections in staining solution. Fresh hand cut transverse sections of stem of the normal and mutant plants were made at regular interval from days after sowing till phloem fibres were distinctly visualized on phloroglucinol staining. Sections were dipped in phloroglucinol solution for 10 min. After that excess phloroglucinol was washed with water and sections were treated with 18 % HCl for 5 min. Finally, stained sections were photographed by a colour digital camera under light microscope (Leica DMLS) at 1009. Suppression subtractive hybridization (SSH) library construction Total RNA was isolated from 1 g of stem tissue at different developmental stages of the plants following the protocol of Samanta et al. (2011). For extraction of RNA, ten plants were killed at the same time (14 das) and pooled. Messenger RNA (mRNA) was isolated from total RNA with a mRNA purification kit (GE Healthcare) according to the manufacturer’s instructions. cDNA synthesis, digestion with RsaI, hybridization, and PCR amplification were carried out using the PCR-Select cDNA Subtraction Kit (Clontech). Forward subtraction was performed using normal plant, C. capsularis, JRC 321 as tester and that of the mutant plant as driver to generate a cDNA library enriched in sequences derived from genes that are upregulated in the ‘fibre forming stage’ in normal plants. PCR products were purified using QIAquick PCR Purification Kit (Qiagen), ligated into pTZ57R/T vector (Fermentas) and transformed in E. coli strain to generate subtracted library. Colonies obtained from subtractive hybridization were further screened by colony hybridization to identify and isolate differentially expressed genes. Colonies were blotted on Hybond-N? membranes (GE Healthcare). a-32P labelled probes were prepared from cDNA isolated at the ‘fibre forming’ stage of the mutant plant.

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Sequence analysis and annotation Plasmid DNA from bacterial culture was isolated using QIAprep Spin Miniprep Kit (Qiagen). The DNA samples were subjected to single-pass DNA sequencing by automated capillary sequencer (ABI 3100, Applied Biosystems) using BigDye terminator (BDT) v3.1 cycle sequencing kit (PE Applied Biosystems). Raw sequence data were manipulated to remove vector and terminal sequences of low reliability. The cDNA sequences were edited by VecScreen in NCBI to remove the vector sequences and ambiguous regions. Poly(A) or poly(T) tails, if present, were removed manually. Moreover, sequences with less than 100 nucleotides were rejected. The sequences were used for BLASTX searches followed by BLASTN alignment against the non-redundant database for dicotyledonous species deposited as ESTs in TIGR and EGO sequences (http://combio.dfci.harvard.edu/tgi/Blast/index.cgi). The tentative annotation was cross checked using BLASTX followed by BLASTN against GenBank nucleotide collection in NCBI (http://www.ncbi.nlm.nih.gov/). The BLASTX results were ranked by e value. EST with an e value higher than 10-10 was rejected. The annotated sequences were subjected for assembling using the software Sequencher version 4.7 for generation of unigene sets. Annotated unigenes were further classified and mapped into three Gene Ontology (GO) categories like biological process, molecular function and cellular component via AmiGO (http://amigo.geneontology.org/). Redundancy of EST for a particular transcript was considered as a measure for the expression level of the transcript and was calculated using the formula [(1 - Number of Unigenes/Number of ESTs) 9 100 %] (Sui et al. 2011). Differential screening using reverse northern analysis DNA samples isolated from selected clones were printed on nylon membrane (Hybond N?, Amersham Biosciences) using HYBRI-BOT apparatus (BRL) in duplicate at 3 mm apart. The spotted DNAs were fixed on a nylon filter by UV cross-linking. Membranes were denatured in 0.6 M NaOH for 3 min, neutralized in 0.5 M Tris–HCl (pH 7.5) for 3 min and rinsed in distilled water for 30 s. Uubiquitin gene isolated from jute was printed on each membrane as internal control. Distilled water and vector DNA were used as negative control. Hybridization of the prepared blots was done in the same procedure as described in the case of colony blot hybridization using the [a-32P] dCTP labelled cDNA of the normal and mutant plant as probe separately. The signal was detected by exposing the blot on the super resolution screen (Type SR, Size 12.5 9 25.2 cm) of CycloneÒ Plus Storage Phosphor System (Perkin-Elmer).

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Northern hybridization of total RNA Total RNA was isolated from the stem tissue of normal and mutant plant at two developmental stages ‘fibre forming’ and ‘non-fibre forming’ and subjected to electrophoresis on 1.2 % denaturing agarose gel and transferred onto nylon membrane (Hybond N?, Amersham Biosciences) with 209 SSC using vacuum gel transfer apparatus (Vacuum Blotter, Bio-Rad). The RNA was allowed to be cross linked with the nylon membrane under UV (245 nm) at 0.15 J/cm2. The membrane was hybridized with [a-32P] dCTP labelled fragment of desired gene as probe prepared using Rediprime kit (GE Healthcare) following the manufacture’s instruction. The hybridization was carried out for 16–18 h at 42 °C. The hybridization was followed by washing with 29, 19, 0.59, 0.29 and 0.19 SSC buffer containing 0.1 % SDS at 42 °C for 15–30 min in each step. The signal was detected by exposing the blot on the Super Resolution screen (Type SR, Size 12.5 9 25.2 cm) of PerkinElmer’s CycloneÒ Plus Storage Phosphor System. Semi quantitative RT-PCR of the differentially expressed transcripts To confirm the Northern results, cDNA was prepared for both the plants at two developmental stages. Gene transcripts for 11 of the selected ESTs were quantified by realtime RT-PCR using the Eppendorf Realplex 2 Master Cycler and SYBR GREEN. Gene-specific primers were designed from EST sequences considering the parameters of optimum primer GC content of 50 %, Tm value of the primers as 65 °C [ Tm [ 50 °C primer length 18–30 nucleotides and an expected amplicon size of 80–200 bp (see Supplement Table S1). Ubiquitin was used as internal standard. For relative quantification, the CT value of the transcript of interest was normalized against the CT value of the housekeeping internal control ubquitin gene of jute (Accession No. FK826547). Initial validation experiments showed that ubiquitin gene was expressed at almost similar level in all experimental jute samples. Real-time PCR for gene of interest and housekeeping internal control gene were conducted using 1st strand cDNA, gene and gene specific forward and reverse primers. The PCR was carried out in 10 ll volume having 19 SYBRGREEN PCR master mix (5 Prime from Eppendorf), 29 SYBR GREEN (5 Prime from Eppendorf), 0.5 lM of each primer and 25 ng of cDNA for each of the samples. The thermal profile was as follows: 95 °C for 2 min for 1 cycle, 40 cycles of 95 °C for 15 s, 50–62 °C for 15 s (depending on Tm of the respective primers) and 62 °C for 30 s. All reactions were carried out in triplicate. Specificity of each amplified product was checked by analysis of the melting curve. Melting curves were generated automatically for every

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product by plotting the fluorescence signal as the function of temperature. The CT value calculation was performed by CalQplex algorithm. The 2DDCT method (Livak and Schmittgen 2001) was adapted for relative quantification of expression of the target gene at different developmental stages for both the normal and the mutant plants. Two important parameters were considered for estimation of DDCT; first the efficiency of a given PCR amplification must be as close to as 100 %, the other being that the amplification efficiencies should be comparable in the PCR system for both of the target and the housekeeping genes. Confirmation of the PCR efficiency was carried out by diluting the cDNA samples in 10-fold serial order. The range and the CT values at each dilution steps were estimated and plotted to judge the efficiency of the estimation. To measure relative efficiency, amplifications were done with the same diluted samples, using primers for the ubiquitin gene as reference and the target genes. The CT values were estimated in triplicate for each of the samples. The average CT was calculated for both of the reference and target genes and DCT (CT of target gene–CT of ubiquitin) was determined. Plots of the log DNA dilution versus DCT were made. The efficiencies of the target and reference genes were found to be similar, based on which the DDCT was estimated for each sample. The change in expression levels (R) was calculated as follows: R ¼ 2DDCT ¼ 2ðDCT sampleDCT calibratorÞ ¼ 2½ðCT sampleCT ubiquitin geneÞðCT calibratorCT ubiquitin geneÞ The normal plant at the ‘fibre forming’ stage was considered as the calibrator.

Results Histological characterization of the fibre developmental process in C. capsularis and its mutant Analysis of the transverse section of stem tissue at the actively growing stage (42 days old) of the plants showed distinct differences in fibre development process between normal JRC 321 and its mutant. It was observed that despite the well-defined growth and organized structure of fibre in normal plants in active fibre development stage, the mutant was found to lack in proper secondary phloem development (Supplement Fig. S1). Bundles of thick-walled fibre cells occupied most of the phloem tissue in the normal while it was absent in the mutant. Moreover, the deposition of lignin was found to be markedly less in mutant compared to the normal plant. The organizations of the fibre cells were irregular in mutant. Further analysis of progressive fibre development processes in both of the

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Fig. 1 Phloroglucinol stained transverse sections of C. capsularis stems at different developmental stages showing the time lag of fibre initiation between normal and mutant plants. Transverse section of

stem of normal (a, c, e, g, i) and mutant plants (b, d, f, h, j) at 3, 5, 7, 14 and 21 das, respectively. Bar 500 lm

plants at different days after showing, viz, 3, 5, 7, 14, 21 and 28 days revealed clearly that the mutant had a delayed start of fibre formation and/or had poorly developed fibre throughout the developmental stages compared to the normal one. The initiation of fibre formation started in between 7 to 14 das in normal plants while that was delayed by at least 7 days and initiated at 14 to 21 das in mutant (Fig. 1). Hence, the respective time periods were considered as ‘FF’ stage for the normal and the mutant plants. The time periods prior to ‘FF’ stage was considered as ‘non-fibre forming’ (‘non-FF’) stage. The results indicated that the mutant lacks the proper initiation and/or differentiation of cell layers required for fibre formation which could be due to differential expression of genes related to fibre developmental process.

Table 1 Overview of the subtracted library of C. capsularis Library titre (cfu/ml)

3.2 9 104

Total number of unique sequences

3,154

ESTs passed quality check

2,685 (85 %)

Frequency of recombinant clones

60 %

Total number of unique sequences

3,154

Average cDNA length per EST (bp)

483

Average (G ? C) content

44 %

Homology to nr database

98 %

Number of known genes Number of unknown/putative genes

2,176 509

Number of contigs

225

Number of singletons

231

Number of unigenes submitted to dbEST

456

Redundancy of the library

83 %

Differential screening of genes identified by SSH About 5,000 clones were collected by suppression subtractive hybridization of the normal plant as tester and the mutant plant as driver. Differential screening of the subtracted colonies using labelled driver cDNA as probe resulted in easily detectable 3,154 un-hybridized and/or faintly hybridized colonies. These selected recombinant clones were subjected to single-pass sequencing from the 30 end to identify a putative set of genes involved in phloem fibre formation, to characterize their predicted functions and redundancy. The sequences showing low quality and/or having short length, less than 100 bp were rejected and finally, 2,730 sequences were found to be of good quality indicating the success of cloning to approximately 87 %. The majority of the selected clones (83 %) were found to contain poly(A) tails. The absence of the poly(A) tail in rest

of the sequences may be due to presence of RsaI restriction site within the cDNA fragments. Comparison of these sequences against the non-redundant plant database in TIGR (http://compbio.dfci.harvard.edu/tgi/Blast/index.cgi) using BLASTN algorithm showed that a total of 2,685 sequences have significant match with protein sequences available in the database. Among 2,685 ESTs, 2,176 ESTs (81 %) were found to be similar with genes of known function, 54 ESTs (2 %) showed significant homology with the sequences marked at putative and 455 ESTs (17 %) were similar to genes of unknown function (Supplement Fig. S2). These 2,685 EST sequences were assembled to 456 non-redundant sequences or unigenes indicating that the redundancy value was significantly high (83 %), calculated using the following the formula: [(1 - Number of

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Unigenes/Number of ESTs) 9 100 %] (Sui et al. 2011). Out of 2,685 ESTs, 231 ESTs did not form any cluster with other ESTs examined in this study and considered as singleton (containing only one EST). The remaining 2,454 ESTs of this dataset generated 225 contigs. The contigs were formed with variable number of reads from 2 to 20. More than 50 % of the contigs contained 2–4 ESTs, whereas contigs with more than 16 were scarce (Supplement Fig. S3). All the 456 unigenes were submitted to dbEST and assigned with the accession numbers FK826398 to FK826618, GH985150 to GH985272, JK714287 to JK714342 and JK743765 to JK743820 (Supplement Table S1). The analysis of subtracted EST library and sequencing statistics was summarized in Table 1. Functional categorization of annotated unigenes via AmiGO BLAST The initial annotations of 456 unigenes via TIGR BLAST search were verified by AmiGo blast (http://amigo.gen eontology.org/) to obtain additional insights into the putative functions of unigenes (Supplement Table S1). Gene ontology (GO) terms based on the automated annotation of each unigenes were designated using the Arabidopsis database. A significant percentage of unigenes (17 %) was classified as ‘unknown function’ which paralleled the findings of TIGR blast. The rest of the unigenes (83 %) were found to be distributed into the three Gene Ontology (GO) categories including biological process (40 %), molecular function (29 %) and cellular component (14 %) (Fig. 2a). The largest category was found to be the biological process with 182 unigenes. The highly represented groups in this category were ‘energy’, ‘growth and development’, and ‘metabolism’ comprised of 35, 35 and 27 % unigenes, respectively (Fig. 2b). All cellular processes require an input of energy to proceed. Most of the transcripts of ‘Energy’ group in the presently described library include genes related to photosynthesis. The over-representation of this category indicated a possible role in fibre formation. Apart from, the genes belonging to the ‘metabolism’ category are of great interest. Over-expression of the genes of secondary metabolism in normal plants indicated their critical role in fibre formation. Under the molecular function (132 unigenes), the largest proportion of functionally assigned unigenes fell into ‘defence and cell rescue’ consisting of 46 unigenes (35 %) followed by ‘signalling and gene regulation’ having 37 unigenes (28 %) (Fig. 2c). Rest 49 unigenes of this category were grouped into ‘stress response’, ‘transporters’ and ‘transcription factors’. Among them, genes related to ‘signalling’ and ‘transcription factors’ are important as they affect several traits through a cascade of biosynthetic pathway. In cellular component category, a total of 63 unigenes were mostly

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grouped into ‘protein synthesis and processing’ and ‘cell wall components’ (Fig. 2d). Differential expression of ‘cell wall component’ related genes indicated that it may have some critical role in proper formation of fibre in jute. Overall, ‘growth and development’, ‘signalling and gene regulation’, ‘cell wall components’, ‘transcription factors’ and ‘metabolism’ were found to be most important among the categories, as some of the transcripts in these categories were reported previously to be associated with initiation, elongation, maturation in cellular development process and its regulation in other fibre producing plant species. Therefore, ESTs belong to these categories were expected to be involved in the fibre formation in jute also and considered for further characterization. These categories were comprised a total of 123 ESTs. Reverse Northern analysis of fibre-specific cDNAs Reverse Northern analysis of 123 clones showed that most of the clones were hybridized weakly or remain unhybridized with the driver cDNA probe (Fig. 3) while the clones were hybridized strongly with the tester cDNA probe (Fig. 3a) indicating that the genes were up-regulated at ‘FF’ stage in the normal plant in comparison to the mutant one. Surprisingly, few clones were found to be over-expressed in reverse direction, that is, clones were hybridized strongly with the driver (mutant) cDNA probe (Fig. 3b). These are arabinogalactan protein (AGP), xyloglucan endotransglycosylase (XET) and homeodomain leucine zipper protein (HD-Zip) representing their downregulation at ‘FF’ stage in the normal plant compared to the mutant. The result indicated the limitation of the SSH method. The clones hybridized strongly in either of the blots may have significant role in jute fibre formation compared to the other transcripts. Overall, 11 such transcripts were selected based on remarkable differences in their hybridization signals in either of the blots detectable by naked eye (Table 2). Finally, these transcripts were considered for their expression analysis at qualitative and quantitative levels to avoid any ambiguity. Expression analysis of selected differentially expressed transcripts by Northern hybridization Expression level of the selected transcripts was studied using RNA isolated from ‘FF’ and ‘non-FF’ stages for both of the normal and the mutant plants. Among the transcripts, three transcripts caffeic acid 3-O-methyltransferase (COMT) (FK826456), cinnamoyl-CoA reductase (CCR) (FK826506) and 4-coumarate CoA:ligase (4CL) (JK714316) were found to be related to lignin formation in plants. It was revealed by Northern analysis that the expressions of all these three enzymes were

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Fig. 2 Distribution and relative frequencies of C. capsularis unigenes to the gene ontology (GO) functional categories (a), biological process (b), molecular function (c) and cellular components (d) according to AmiGo BLAST

Fig. 3 Representative figures of differential screening by reverse Northern analysis of the selected clones (spotted in duplicate) of C. capsularis. Duplicate blots were prepared and hybridized with a32P labelled tester (normal JRC 321) (a) and driver (mutant cDNA)

probes (b). Ubiquitin gene (FK826547) as positive control (spots: 1A and 2A) and vector (spots: 3A and 4A) and water (spots: 5A and 6A) as negative control were used in both the blots indicated by arrows

significantly higher in normal plant than those of the mutant, irrespective of their developmental stages (Fig. 4). Moreover, the enzymes were found to be developmentally regulated as expression level of the enzymes was increased with the advancement of the developmental stages in both the plants tested. The results indicated a positive correlation between the expression of these

transcripts and lignin deposition in the jute fibre as revealed by histological analysis. The abundance of these transcripts may have a critical role in initiation of fibre development in the normal plant in comparison to the mutant one. Analysis of the selected transcripts showed that the expression of GRP (FK826399) and PME (FK826535) was higher in normal plants than of the

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378 Table 2 Selected clones of different functional categories for expression analysis

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Functional categories Secondary metabolism

Name of the transcripts

Accession number

Redundancy

Caffeic acid 3-O-methyltransferase

FK826456

14

Cinnamoyl-CoA reductase

FK826506

19

4-Coumarate: CoA ligase

JK714316

20

Xyloglucan endotransglycosylase

FK826511

14

Pectin methylesterase

FK826535

17

Glycine rich protein

FK826399

17

Homeodomain-leucine zipper

FK826421

8

WRKY transcription factors

FK826433

18

Signalling and gene regulation

Zinc finger protein

JK714288

11

Growth and development

Pectate lyase

FK826563

15

Cell wall components

Transcription factors

transcription factor was almost same irrespective of developmental stages in normal plant while its expression gradually increases with the developmental stages in the mutant. This indicated that it could have a potential role in fibre formation in jute. In case of zinc finger protein (ZFP) (JK714288), the expression of this transcript was conspicuously higher in normal plant than the mutant in both of the developmental stages (Fig. 4). Furthermore, zinc finger protein was expressed in significantly high level irrespective of developmental stages in normal plant but in the mutant its expression gradually increases with the advancement of the developmental stages.

Fig. 4 Northern analyses of the selected genes. Lanes 1 and 2, RNA samples from C. capsularis, JRC 321 at ‘FF’ and ‘non-FF’, respectively; lanes 3 and 4, RNA samples from ‘soft-stem’ mutant at ‘FF’ and ‘non-FF’stages, respectively. The blots were subsequently hybridized with ubiquitin gene (FK826547) to represent the uniform loading of RNA samples

mutant plant. In contrast, the expression of both of the AGP (FK826429) and XET (FK826511) was higher in the mutant than the normal plant which was in contradiction with the SSH analysis (Fig. 4). The results indicated that the former two transcripts have a positive correlation while the latter two transcripts carry a negative correlation with the fibre development process in C. capsularis. Analysis of the transcript pectate lyase (PEL) (FK826563) showed a noticeable difference in its expression between the normal and the mutant plants. Additionally, the difference in its expression was also observed at two developmental stages, ‘FF’ and ‘non- FF’ in both of the plants (Fig. 4). Qualitative analysis by Northern hybridization of homeodomain leucine zipper (HD-Zip) protein showed less expression in normal plant in comparison to the mutant one while the reverse is true for the WRKY transcription factor (Fig. 4). The expression of WRKY

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Validation of relative expression of the transcripts by real-time polymerase chain reaction (Real time RTPCR) The relative expression of COMT was found to be significantly higher (1.6-fold) in ‘FF’ stage of the normal plant than that of the mutant plant. It was observed that its expression level in the normal plant at ‘FF’ stage was much higher than that of the ‘non-FF’ stage, whereas in mutant the expression was found to be almost same at these two development stages (Fig. 5a). On the other hand, the expression of CCR increased with the advancement of the developmental stages for both of the normal and mutant plant, though level of expression was found to be much higher in the normal plant than the mutant one (Fig. 5b). The drastic reduction (3.2-fold) was observed in the expression of 4CL at ‘non-FF’ stage compared to the ‘FF’ stage in normal plant, whereas its expressions in the mutant at both of the stages were negligible compared to the normal plant (Fig. 5c). The results indicated that among the three transcripts 4CL plays a major role during lignin accumulation in jute plant. The AGP and XET were found to be preferentially expressed in the mutant plant irrespective of the developmental stages. The expression of the AGP in the mutant plant was 2.17- and 2.78-fold higher at ‘FF’ stage and ‘non-FF’ stages, respectively, than in the

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Fig. 5 Relative expression of the selected genes at two developmental stages for both of the normal and mutant plants. The relative mRNA levels of individual genes were normalized to house keeping ubiquitin gene as standard. The expressions of the transcripts were

presented as percentage change considering the expression level at fibre forming stage in normal plant as 100 %. Results are expressed as mean ± SD, n = 3. Significant difference (P \ 0.01) was estimated using one-way ANOVA

normal plant (Fig. 5d). In the case of XET, the expression in the mutant plant was found to be 4.53-fold and 6.43-fold higher than in the normal plant at ‘FF’ stage and ‘non-FF’ stages, respectively (Fig. 5e). The transcripts PME and GRP were found to be expressed less in mutant plants compared to the normal ones. It was found by RT-PCR that the expression of PME at the ‘FF’ stage and ‘non-FF’ stages in the normal plant was higher than that in the mutant to the tune of *1.7-fold and *3-fold, respectively (Fig. 5f). Similarly, over-expression of GRP was also observed in the normal plant by *2.1- and *1.25-fold at

the ‘FF’ stage and ‘non-FF’ stages, respectively, when compared with its expression in the mutant plant (Fig. 5g). Overall, the result is in accordance with the reverse Northern as well as Northern analysis and indicated that the transcripts AGP and XET may have a negative influence, whereas PME and GRP have a positive influence during the fibre development in C. capsularis. The RT-PCR assessment of the relative expression of pectate lyase (PEL) showed a significant change in its expression at ‘FF’ stage of the normal plant compared to the mutant one. The expression at ‘FF’ stage was found to be around 1.4-fold

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higher than that of the ‘non-FF’ stage of the normal plant as well as ‘FF’ and ‘non-FF’ stages of the mutant (Fig. 5h). Thus, it could be expected that a threshold level of its expression is required for normal development of the fibre in jute. Relative expressions of three differentially expressed transcripts viz., HD-Zip, WRKY and ZFP are known to have significant roles in regulation of cellular development through interaction with hormone and cellular components. It was revealed that the expressions of both WRKY transcription factor (Fig. 5j) and zinc finger protein (Fig. 5i) were at high level at the ‘non-FF’ stage and maintained steadily during the development of the normal plant, whereas its expression gradually increased in case of the mutant one. This steady-state expression of these factors may have significant contribution in early initiation of fibre formation in the normal plant. The expression level of these two transcripts in the mutant, however, was much lower compared to those of normal plants in either stage of development. On the other hand, the expression of HD-Zip was found to be similar at the ‘non-FF’ stage in both the genotypes, but increased sharply (*1.75-fold) in the mutant plants compared to the normal one at ‘FF’ stage (Fig. 5k) indicating its negative impact in cellular differentiation processes during fibre development in C. capsularis.

Discussion The present study constitutes the first effort to understand the molecular basis of fibre developmental process in C. capsularis. Variation in fibre development among two different developmental stages, viz., ‘FF’ and ‘non-FF’ stages of normal plant and its mutant as revealed by anatomical analysis indicated a distinct time lag of at least 7 days for fibre initiation in mutant plant (Fig. 1). This suggested that these developmental stages were crucial to gain understanding about the gene(s) related to fibre initiation and formation through comparative molecular studies between these two inbred genetic stocks, JRC321 and its fibre-deficient ‘soft stem’ mutant. Suppression subtractive hybridization (SSH) of the normal plant with the mutant one at the ‘FF’ stage generated a total of 456 unigenes which were assumed to be up-regulated in the ‘FF’ stage of the normal plant. These genes may have some cognizable roles in the fibre formation and developmental process in C. capsularis. The unigenes generated in the present study were found to fall into 16 different tentative functional categories. High percentage (17 %) of unigenes (Fig. 2) corresponding to unknown or hypothetical proteins as observed in the present study was comparable to other EST programme in different plant species (Zhang et al. 2008; Leida et al. 2012; Long et al. 2012).

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Correlation between the size of the ESTs and ‘unknown function’ could not be established as was demonstrated earlier in EST analysis of Citrus sinensis whole seedling library (Bausher et al. 2003). However, these unigenes could provide an interesting pool of novel proteins that may be of special relevance for fibre development, and further functional analysis may lead to greater insight into the molecular processes of fibre formation. The rest ESTs (83 %) were found to have known function (Table S1) indicating their definite involvement in a variety of physiological and molecular events related to the development process in C. capsularis. Transcripts belong to ‘growth and development’, ‘signalling and gene regulation’, ‘cell wall components’, ‘transcription factors’ and ‘metabolism’ were found to be most important among the categories, as some of the transcripts in these categories were known to be associated with initiation, elongation, maturation in cellular development process and its regulation in other fibre producing plant species. Therefore, ESTs belonging to these categories were expected to be involved in the fibre formation in jute also and considered for further characterization. These categories were comprised of a total of 123 ESTs. Further screening of 123 clones by reverse Northern revealed that some of the clones have significantly higher signal intensity and are easily detectable by naked eye with driver probes. These clones could be the potential candidate genes associated with early stage of fibre development process in jute as distinct anatomical differences were observed between two genotypes. Apart from the influence on phloem fibre development, the identified transcriptomes may also have some role in xylem formation as it was found to be less developed in the mutant line compared to wild type (Fig. 1). However, predominant role of the transcriptomes in relation to fibre bundle formation, the important economic trait in jute, has only been analysed in the present discussion. On the other hand, among the clones tested three clones, viz., AGP, XET and HD-Zip showed bright signal intensity with the tester probe (Fig. 3) indicating partial success of the SSH technique that could be due to presence of sequence-related genes from the same multigene family (Sahu et al. 2009; Abid et al. 2011). These findings indicated that these genes may also have some crucial roles in controlling fibre development in C. capsularis. Finally, 11 such strongly hybridized clones of different category were selected (Table 2) as these genes was reported previously to be associated with initiation, elongation, maturation in cellular development process and its regulation in other fibre producing plant species. The cellular basis of fibre differentiation in plants is a concerted function of fibre cell initiation, expansion, deposition of wall materials and action of cell wall modifying enzymes. The degree of cell expansion and strength

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of fibre cells are closely associated with the relative deposition of cellulose with hemicellulose and structural heteropolysaccharide molecules. Furthermore, cell elongation is mainly caused by regulation of cell-wall loosening factors (Pritchard 1994). In this context, the cDNAs encoding AGP, XET, PME, GRP and PEL may have primary importance in the present study as these are known to be involved in controlling cell expansion, cell to cell adhesion and strength of fibre cells (Yang and Showalter 2007; Li et al. 2010). The resulting differential transcript profile in jute was found to be dissimilar in comparison to other phloem-fibre bearing plant species like flax and hemp. This could be due to differences in fibre composition. The former is lignocellulosic whereas latter are mostly cellulosic in nature. In the present study, relatively higher accumulation of AGP was observed in the mutant compared to normal ones (Fig. 5). Yet, delayed appearance of fibre and its irregular elongation (Fig. 1, Supplement Fig. S1) indicated that the process of cell extension and/or cell to cell adhesion might have been affected adversely in the mutant. On the other hand, relatively less expression of AGP did not show any negative effect on the fibre development in the normal plants. A similar low level of occurrence of AGP was also observed in other fibre crops like flax and hemp (Blake et al. 2008; Gea et al. 2013). Thus, it could be assumed that not the AGP alone, rather a balanced combinatorial effect of other factors, might be involved in required cell elongation and expansion, in turn, for proper fibre formation in the presently studied species, C. capsularis. XET is thought to play a major role in the regulation of cell wall expansion. Studies on XET expression in primary vascular tissues and the internal phloem of tobacco stems (Herbers et al. 2001; Bourquin et al. 2002), flax and hemp (Day et al. 2005; Blake et al. 2008) suggested that XET may be involved in the formation of secondary walls of xylem and/or primary phloem cells and an optimal expression of XET may be required for cell wall expansion. It was proposed that xyloglucan, the most abundant hemicellulose in plant cell, forms crosslink to the cellulose microfibril and breakage of this xyloglucan-cellulose network is necessary for the expansion of cellulose microfibril, in turn, wall expansion (Cosgrove 2000; Nishikubo et al. 2011). The breakage of this crosslink is mediated mainly by intra-molecular cleavage of xyloglucan molecules and re-joining of the reducing ends of newly formed products to the non-reducing ends of another xyloglucan chain which is controlled by XET (Lee et al. 2010). However, it was also proposed that grafting of nascent xyloglucan chain which is much shorter in length than that of original xyloglucan produced in the cell wall cellulose microfibril, made the cell wall extensively compact and less extensible (Cosgrove 2000). It was further suggested that the amount and length of nascent xyloglucan

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is an important determinant of the cell wall extensibility (Nishikubo et al. 2011). Thus, the deficiency of fibre quantity as well as fragmented nature of the fibre in the mutant as observed earlier (SenGupta and Palit 2004) and in the present anatomical study (Supplement Fig. S1) could be explained by the much higher expression of XET that may degrade the xyloglucan molecule at higher rates compared to the normal plant. In plants, cells grow in a symplastic manner. However, few specialized cells grow intrusively by penetrating through pectin-rich middle lamella, resulting in cellular adhesion. The intrusive tip growth of cells is of great significance as it is the major determinant of phloem and xylem fibre elongation and thereby influences a fibre quality of the commercially cultivated bast fibre producing plants, such as sisal, abaca, jute, flax, ramie, hemp and kenaf (Siedlecka et al. 2008). During intrusive growth, the cells invade neighbouring cells by tip growth only and make a firm attachment along its length through methylesterification of pectin (Siedlecka et al. 2008). The enzymes PME and PEL are known to modify the degree of methylesterification of pectin by de-esterification and depolymerization of de-esterified pectin, respectively (Pelloux et al. 2007). It has been demonstrated in transgenic hybrid aspen (Populus tremula 9 P. tremuloides) trees that up-regulation of PtPME1 increased pectin modification and strengthened cellular adhesion due to modification of PME activity (Siedlecka et al. 2008). Similar to aspen, cell adhesion promoting role of PME was also observed in flax and tomato (Orfila et al. 2001; Lacoux et al. 2003). These evidences supported the present findings of higher expression of PME and resulted in organized fibre structure in the normal plant compared to the mutant plants as revealed from anatomical study (Fig. 1, Supplement Fig. S1). PEL is known to have high correlation with fibre properties, particularly in controlling fibre cell elongation (Wang et al. 2010). This justified further the production of fragmented or short fibre strands in the presently studied mutant as was evidenced earlier (SenGupta and Palit 2004; Sadhukhan 2007). Thus, it could be suggested safely that preferential expression of PEL in normal plants is essential for the fibre formation in C. capsularis. GRP are known to contribute to the strengthening of biological structures, i.e., tensile strength of fibres (Ringli et al. 2001). It has been reported that glycine rich proteins help in cell elongation as well as in deposition of lignin in Arabidopsis thaliana (Mangeon et al. 2010). Thus, it could be suggested that the variations in expression of these transcripts between two genotypes, in the present study, could be responsible for less deposition of lignin and altered fibre bundle structure in the mutant of C. capsularis as were observed in anatomical studies (Fig. 1, Suppement Fig. S1).

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Besides cellulose, hemicellulose and structural proteins, the other essential component in plant cell wall is lignin that is known to be impregnated in cellulose-hemicellulose matrix. Such types of cross-linking are known to provide strength and continuity of fibre strand (Roy 1953; Meredith 1956). Lack of these components may reduce cell to cell adhesion and may result in discontinuity of the fibre strand. The biosynthesis of monolignols commences with the deamination of phenylalanine and proceeds through the general phenylpropanoid pathway involving a numbers of genes (Rastogi and Diwedi 2008). Thus, any reduction in the expression of any of these genes involved in monolignol biosynthesis is expected to have significant impact on fibre development process. It was demonstrated that individual and/or simultaneous down-regulation of the transcripts COMT, CCR and 4CL resulted in reduction of lignin content in different plant species including N. tabacum, A. thaliana, M. sativa, Populus sp. (Stephens and Halpin 2007; Rastogi and Diwedi 2008; Hisano et al. 2009). On the other hand, lower expression of lignin biosynthesis-related gene was demonstrated in the bast tissue of flax and hemp (Van den Broeck et al. 2008) which could be explained by low abundance of lignin in these plant species. In some cases, the reduction of lignin was found to be compensated by increment of cellulose production (Li et al. 2008). Detection of differential expression of the above mentioned transcripts between the genotypes studied presently justified the variation in their lignin deposition as was revealed from anatomical analysis of the stem (Fig. 1, Supplement Fig. S1). The present result corroborated the earlier evidence of less lignin content by 50 % and more cellulose by 30 % in the mutant compared to normal plant (Sengupta and Palit 2004). The less lignin content was also observed in ‘non-FF’ stage compared to ‘FF’ stage in the normal plant. Thus, it may be concluded that the biosynthesis of lignin is triggered prior to onset of fibre initiation. Taken together, it could be suggested that variation in expression of these transcripts might be the reason for variation of lignin content in the fibre cells between the normal and mutant plants and between the developmental stages of either genotype as was observed in histological analysis (Fig. 1, Supplement Fig. S1). Among the transcripts, 4CL was found to be suppressed more than COMT and CCR in the presently studied mutant compared to normal plants as was revealed by Northern hybridization and RT-PCR (Fig. 5). The differences in degree of suppression among these three transcripts could be due to variation in number of isoforms of the respective transcripts present in jute. The present analyses have uncovered several genes, including genes encoding cell wall structural proteins and various enzymes associated with the biosynthesis of secondary cell wall components, whose expressions were

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found to be differentially regulated during fibre development in the normal plant and the mutant plant. These findings led to the assumption that concerted action of these genes might regulate fibre differentiation in a coordinated manner in the presently studied species. In fact, differential expressions of such genes have been demonstrated in developing stem of different plant species (Demura and Fukuda 2007). So, it could further be assumed that such co-coordinated expression was regulated by some transcription factors/signal transduction molecules. Three such genes, viz., HD-Zip, ZFP and WRKY transcription factor, were found to be expressed differentially in normal and the mutant plants during fibre formation. HD-Zip family proteins are a class of homeobox containing transcription factor (TF) whose gain-in-function or loss-of-function contributes towards the plant growth and development. It has been suggested that HD-Zip has a predominant role in regulating vascular differentiation in plants (Hofer et al. 2009; Harris et al. 2011; Sanchez et al. 2012). Transgenic studies in A. thaliana (Zhong and Ye 1999) and P. trichocarpa (Coˆte´ et al. 2010; Du et al. 2011) further demonstrated that over-expression of such homeobox genes delayed differentiation of secondary xylem and severe reduction of phloem fibre differentiation with restricted lignification. The past analyses indicated that the function of HD-Zip gene regulated activation and/or deactivation of several downstream genes, though most of them have not yet been elucidated (Elhiti and Stasolla 2009). Surprisingly, the present study elucidated a clear and distinguishable delayed appearance and/or lack of differentiating phloem fibres in the mutant plant in comparison to the normal ones (Fig. 1) having abundant expression of HD-Zip (Fig. 5). Thus, it could be proposed with certainty that the expression of this protein may play a crucial role in fibre differentiation in C. capsularis. Furthermore, the fibres were found to be less lignified in the mutant plant as was observed by anatomical studies (Fig. 1, Supplement Fig. S1) and has been quantified earlier (SenGupta and Palit 2004). However, the variations of the lignin deposition between the genotypes could not be explained definitely at present with respect to the expression of this particular transcript. On the other hand, WRKY transcription factor, an atypical zinc finger protein, was found to act directly or indirectly on both the repression and de-repression of many genes involved in plant biological processes including defence signalling, biosynthetic pathways of secondary metabolites and cell wall components, disease resistance, and metabolite transport (Tuskan et al. 2006; Wang et al. 2010; Wei et al. 2012; Motamayor et al. 2013; Dou et al. 2014). The amino acid sequence identity of the presently identified fragment of this transcript was found to be relatively close to that of cotton, cacao and poplar.

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The highest homology was observed with GhWRKY97 (87 %) which was suggested to participate in leaf senescence and development of tissues (Dou et al. 2014). This transcription factor has also been found to be up-regulated during secondary growth of stem and in xylem tissue in Arabidopsis and tension wood in aspen (Dharmawardhana et al. 2010). In indirect influences, WRKY transcription factor functions as an upstream regulator of NAC master switches and in turn, negatively regulates the expression of the transcription factor MYB. This MYB protein was known to bind AC-element in the promoter region of target genes of lignin biosynthetic pathway and regulated their expression (Wang et al. 2011). There are several reports of MYB-mediated negative regulation of several target genes involved in lignin biosynthesis in different plant species (Wang et al. 2011). It has been demonstrated that the over-expression of AmMYB from Antirrhinum majus decreased transcript levels of 4CL, cinnamate-4hydroxylase (C4H) and cinnamyl alcohol dehydrogenase (CAD) (Tamagnone et al. 1998). Similarly, the overexpression of AtMYB4 was shown to decrease transcript levels of C4H, 4CL, CAD and chalcone synthase (CHS) in Arabidopsis (Jin et al. 2000) and the over-expression of ZmMYB42 down-regulated phenylalanine ammonia lyase (PAL), C4H, 4CL, hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT), ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydoxylase (F5H), COMT and CAD genes in maize (Sonbol et al. 2009). Reduction of the expression level of CCR has also been demonstrated in T. aestivum (Ma et al. 2011). All the previous information suggested that WRKY acts as the key regulating factor for redirecting the metabolic flux of lignin biosynthesis in plants through repression of MYB proteins. The present findings of higher expression of WRKY and the genes related to lignin biosynthetic pathway in normal plants compared to the mutant ones suggested that the WRKY could be the prime regulatory molecule for 4CL, COMT and CCR expressions in lignin biosynthetic pathway in C. capsularis and responsible for variation in lignin deposition between these two genotypes. Although the effect of WRKY is mediated through MYB, none of the genes related to MYB was found differentially expressed in the present study. Such observation could be due to restricted application of SSH in forward direction only. Furthermore variations in the choice of target genes for WRKY transcription factor in C. capsularis were similar to the earlier observation in other plant species stated above. However, the reasons behind the variations in expression level of the target lignin biosynthetic genes need further investigation. Regulation of the cell wall components including AGP, XET, GRP, PME have also been reported to be regulated by the expression of either WRKY directly or through MYB. It has been

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demonstrated that over expression of MYB led to high expression of AGP in P. glauca and A. thaliana (Bedon et al. 2007; Jung et al. 2008). On the other hand, expression of PME has been demonstrated to be down-regulated by MYB (Phan et al. 2011). Since WRKY has been demonstrated as the upstream regulatory factor of MYB expression (Dharmawardhana et al. 2010), it could be assumed that expression of AGP and PME might be a consequence of the expression pattern of WRKY which regulates negatively and positively the expression of AGP and PME, respectively. Thus, variation of AGP and PME between genotypes in the present study could be explained by the variation of expression level of WRKY. Similar negative regulation of WRKY on the expression of XET has been demonstrated in V. vinifera (Guillaumie et al. 2010) while positive regulation was observed with GRP, ZFP and PEL (Miao et al. 2004). Taken together, it could be suggested that WRKY transcription factor has the predominant role as regulatory element in controlling sets of genes of different pathways related to components required for fibre formation and could be a candidate gene for the improvement of jute fibre. However, it is not clear whether single or multiple isoforms of this gene are involved in carrying out such regulation. Detail analysis of this WRKY transcription factor is necessary to improve our understanding of the molecular mechanism in fibre formation in C. capsularis. Acknowledgments Financial assistance in the form the grant support to this laboratory from the Department of Biotechnology, Government of India is acknowledged.

References Abid G, Muhoviski Y, Jacquemin J-M, Mingeot D, Sassi K, Toussaint A, Baudoin JP (2011) Changes in DNA-methylation during zygotic embryogenesis in interspecific hybrids of beans (Phaseolus sp.). Plant Cell Tiss Organ Cult 105:383–393 Bausher M, Shatters R, Chaparro J, Dang P, Hunter W, Niedz R (2003) An expressed sequence tag (EST) set from Citrus sinensis L. Osbeck whole seedlings and the implications of further perennial source investigations. Plant Sci 165:415–422 Bedon F, Grima-Pettenati J, Mackay J (2007) Conifer R2R3-MYB transcription factors: sequence analyses and gene expression in wood-forming tissues of white spruce (Picea glauca). BMC Plant Biol 7:17–34 Blake AW, Marcus SE, Copeland JE, Blackburn RS, Knox JP (2008) In situ analysis of cell wall polymers associated with phloem fibre cells in stems of hemp, Cannabis sativa L. Planta 228:1–13 Bolton JJ, Soliman KM, Wilkins TA, Jenkins JN (2009) Aberrant expression of critical genes during secondary cell wall biogenesis in a cotton mutant, Ligon Lintless-1 (Li-1). Comp Funct Genomics 2009:1–8 Bourquin V, Nishikubo N, Abe H, Brumer H, Denman S, Eklund M, Christiernin M, Teeri TT, Sundberg B, Mellerowicz EJ (2002) Xyloglucan endotransglycosylases have a function during the

123

384 formation of secondary cell walls of vascular tissues. Plant Cell 14:3073–3088 Cosgrove DJ (2000) Loosening of plant cell walls by expansins. Nature 407:321–326 Coˆte´ CL, Boileau F, Roy V, Ouellet M, Levasseur C, Morency MJ, Cooke JEK, Se´guin A, Coˆte´ JJM (2010) Gene family structure, expression and functional analysis of HD-Zip III genes in angiosperm and gymnosperm forest trees. BMC Plant Biol 10:273 Day A, Addi M, Kim W, David H, Bert F, Mesnage P, Rolando C, Chabbert B, Neutelings G, Hawkins S (2005) ESTs from the fibre-bearing stem tissues of flax (Linum usitatissimum L.): expression analyses of sequences related to cell wall development. Plant biol 7:23–32 Demura T, Fukuda H (2007) Transcriptional regulation in wood formation. Trends Plant Sci 12:64–70 Dharmawardhana P, Brunner AM, Strauss SH (2010) Genome-wide transcriptome analysis of the transition from primary to secondary stem development in Populus trichocarpa. BMC Genom 11:150–169 Dou L, Zhang X, Pang C, Song M, Wei H, Fan S, Yu S (2014) Genome-wide analysis of the WRKY gene family in cotton. Mol Genet Genomics. doi:10.1007/s00438-014-0872-y Du J, Miura E, Robischon M, Martinez C, Groover A (2011) The Populus class III HD ZIP transcription factor POPCORONA affects cell differentiation during secondary growth of woody stems. PLoS One 6:e17458 Elhiti M, Stasolla C (2009) Structure and function of homodomainleucine zipper (HD-Zip) proteins. Plant Signal Behav 4:86–88 Gea G, Kjell S, Jean-Franc¸ois H (2013) Integrated -Omics: a powerful approach to understanding the heterogeneous lignification of fibre crops. Int J Mol Sci 14:10958–10978 Ghosh T (1983) Handbook on jute. FAO plant production and protection. Paper No. 51. Rome: FAO Guillaumie S, Mzid R, Me´chin V, Le´on C, Hichri I, Destrac-Irvine A, Trossat-Magnin C, Delrot S, Lauvergeat V (2010) The grapevine transcription factor WRKY2 influences the lignin pathway and xylem development in tobacco. Plant Mol Biol 72:215–234 Harris JC, Hrmova M, Lopato S, Langridge P (2011) Modulation of plant growth by HD-Zip class I and II transcription factors in response to environmental stimuli. New Phytol 190:823–837 Herbers K, Lorences EP, Barrachina C, Sonnewald U (2001) Functional characterization of Nicotiana tabacum xyloglucan endotransglycosylase (Nt XET-1): generation of transgenic tobacco plants and changes in cell wall xyloglucan. Planta 212:279–287 Hisano H, Nandakumar R, Wang ZY (2009) Genetic modification of lignin biosynthesis for improved biofuel production. In Vitro Cell Dev Biol-Plant 45:306–313 Hofer J, Turner L, Moreau C, Ambrose M, Isaac P, Butcher S, Weller J, Dupin A, Dalmais M, Le Signor C, Bendahmane A, Ellis N (2009) Tendril-less regulates tendril formation in pea leaves. Plant Cell 21:420–428 Hsing YI, Cher CG, Fan MJ, Lu PC, Chen KT, Lo SF, Sun PK, Ho SL, Lee KW, Wang YC, Huang WL, Ko SS, Chen S, Chen JL, Chung CI, Lin YC, Hour AL, Wang YW, Chang YC, Tsai MW, Lin YS, Chen YC, Yen HM, Li CP, Wey CK, Tseng CS, Lai MH, Huang SC, Chen LJ, Yu SM (2007) A rice gene activation/ knockout mutant resource for high throughput functional genomics. Plant Mol Biol 63:351–364 Jensen WA (1962) Botanical histochemistry—principles and practice. Freeman and Company, University of California, Berkeley Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, Weisshaar B, Martin C (2000) Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J 19:6150–6161

123

Planta (2015) 241:371–385 Jung C, Seo JS, Han SW, Koo YJ, Kim CH, Song SI, Nahm BH, Choi YD, Cheong JJ (2008) Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant Physiol 146:623–635 Lacoux J, Klein D, Domon JM, Burel C, Lamblin F, Alexandre F, Sihachakr D, Roger D, Balange AP, David A, Morvan C, Laine´ E (2003) Antisense transgenesis of Linum usitatissimum with a pectin methylesterase cDNA. Plant Physiol Biochem 41:241–249 Lee J, Burns TH, Light G, Sun Y, Fokar M, Kasukabe Y, Fujisawa K, Maekawa Y, Allen RD (2010) Xyloglucan endotransglycosylase/ hydrolase genes in cotton and their role in fiber elongation. Planta 232:1191–1205 Leida C, Romeu JF, Garcı´a-Brunton J, Rı´os G, Badenes ML (2012) Gene expression analysis of chilling requirements for flower bud break in peach. Plant Breed 131:329–334 Li X, Weng JK, Chapple C (2008) Improvement of biomass through lignin modification. Plant J 54:569–581 Li Y, Liu D, Tu L, Zhang X, Wang L, Zhu L, Tan J, Deng F (2010) Suppression of GhAGP4 gene expression repressed the initiation and elongation of cotton fiber. Plant Cell Rep 29:193–202 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–DDCT method. Methods 25:402–408 Long SH, Deng X, Wang YF, Li X, Qiao RQ, Qiu CS, Guo Y, Hao DM, Jia WQ, Chen XB (2012) Analysis of 2,297 expressed sequence tags (ESTs) from a cDNA library of flax (Linum ustitatissimum L.) bark tissue. Mol Biol Rep 39:6289–6296 Ma Y, Hong Z, Wu Q, Yao X (2011) Transcriptome subtractive hybridization and its reliability validated by an E. coli model. Biosci Method 2:38–42 Mangeon A, Junqueira RM, Sachetto-Martins G (2010) Functional diversity of the plant glycine-rich proteins superfamily. Plant Signal Behav 5:99–104 Meredith R (1956) Mechanical properties of textile fibres, vol Chapters VI and VII. Interscience Publisher, New York Miao Y, Laun T, Zimmermann P, Zentgraf U (2004) Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol Biol 55:853–867 Motamayor JC, Mockaitis K, Schmutz J, Haiminen N, Livingstone D III, Cornejo O, Findley SD, Zheng P, Utro F, Royaert S, Saski C, Jenkins J, Podicheti R, Zhao M, Scheffler BE, Stack JC, Feltus FA, Mustiga GM, Amores F, Phillips W, Marelli JP, May GD, Shapiro H, Ma J, Bustamante CD, Schnell RJ, Main D, Gilbert D, Parida L, Kuhn DN (2013) The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color. Genome Biol 14:1–24 Nishikubo N, Takahashi J, Roos AA, Derba-Maceluch M, Piens K, Brumer H, Teri TT, Stalbrand H, Mellerowicz EJ (2011) Xyloglucan endo transglycosylase mediated xyloglucan rearrangements in developing wood of hybrid Aspen. Plant Physiol 155:399–413 Orfila C, Huisman MMH, Willats WGT, Van Alebeek GJWM, Schols HA, Seymour GB, Knox JP (2001) Altered middle lamella homogalacturonan and disrupted deposition of (1 ? 5)-a-Larabinan in the pericarp of cnr, a ripening mutant of tomato. Plant Physiol 126:210–221 Pelloux J, Ruste´rucci C, Mellerowicz EJ (2007) New insights into pectin methylesterase structure and function. Trends Plant Sci 12:267–277 Phan HA, Iacuone S, Li SF, Parish RW (2011) The MYB80 transcription factor is required for pollen development and the regulation of tapetal programmed cell death in Arabidopsis thaliana. Plant Cell 23:2209–2224 Pritchard J (1994) The control of cell expansion in roots. New Phytol 127:3–26

Planta (2015) 241:371–385 Rastogi S, Dwivedi UN (2008) Manipulation of lignin in plants with special reference to O-methyltransferase. Plant Sci 174:264–277 Ringlia C, Keller B, Ryse U (2001) Glycine-rich proteins as structural components of plant cell walls. Cell Mol Life Sci 58:1430–1441 Roy MM (1953) Mechanical properties of jute. The study of chemically treated fibres. J Tex Inst Trans 44:44–52 Sadhukhan S (2007) Characterization of jute fibre and generation of developmental stage specific expressed sequence tags in relation to fibre formation. PhD Thesis, Indian Institute of Technology, Kharagpur, West Bengal Sahu BB, Shaw BP (2009) Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization. BMC Plant Biol 9:69 Samanta P, Sadhukhan S, Das S, Joshi A, Sen SK, Basu A (2011) Isolation of RNA from field-grown jute (Corchorus capsularis) plant in different developmental stages for effective downstream molecular analysis. Mol Biotechnol 49:109–115 Sanchez P, Nehlin L, Greb T (2012) From thin to thick: major transitions during stem development. Trends Plant Sci 17:113–121 Sengupta G, Palit P (2004) Characterization of a lignified secondary phloem fibre-deficient mutant of jute (Corchorus capsularis). Ann Bot 93:211–220 Siedlecka A, Wiklund S, Pe´ronne M-A, Micheli F, Les´niewska J, Sethson I, Edlund U, Richard L, Sundberg B, Mellerowicz EJ (2008) Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of populus. Plant Physiol 146:554–565 Sonbol F-M, Fornale´ S, Capellades M, Encina A, Tourin˜o S, Torres J-L, Rovira P, Ruel K, Puigdome`nech P, Rigau J, Caparro´s-Ruiz D (2009) The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana. Plant Mol Biol 70:283–296 Stephens J, Halpin C (2007) Lignin manipulation for fibre improvement. In: Ranalli P (ed) Improvement of crop plants for industrial end uses, chapter 5. Springer, Netherlands, pp 129–153 Sui S, Luo J, Ma J, Zhu Q, Lei X, Li M (2011) Generation and analysis of expressed sequence tags from Chimonanthus praecox (Wintersweet) flowers for discovering stress-responsive and

385 floral development-related genes. Comp Func Genomics. doi:10. 1155/2012/134596 Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C (1998) The AmMYB308 and AmMYB330 transcription factors from Antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell 10:135–154 Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S et al (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596–1604 Van den Broeck HC, Maliepaard C, Ebskamp MJM, Toonen MAJ, Koops AJ (2008) Differential expression of genes involved in C1 metabolism and lignin biosynthesis in wooden core and bast tissues of fibre hemp (Cannabis sativa L.). Plant Sci 174:205–220 Wang HZ, Dixon RA (2011) On–off switches for secondary cell wall biosynthesis. Mol Plant 5:297–303 Wang H, Guo Y, Lv F, Zhu H, Wu S, Jiang Y, Li F, Zhou B, Guo W, Zhang T (2010) The essential role of GhPEL gene, encoding a pectate lyase, in cell wall loosening by depolymerization of the de-esterified pectin during fiber elongation in cotton. Plant Mol Biol 72:397–406 Wang H, Zhao Q, Chen F, Wang M, Dixon RA (2011) NAC domain function and transcriptional control of a secondary cell wall master switch. Plant J 68:1104–1114 Wei KF, Chen J, Chen YF, Wu LJ, Xie DX (2012) Molecular phylogenetic and expression analysis of the complete WRKY transcription factor family in maize. DNA Res 19:153–164 Yadun SL (2010) Plant fibers: initiation, growth, model plants, and open questions. Russ J Plant Physiol 57:305–315 Yang J, Showalter AM (2007) Expression and localization of AtAGP18, a lysine-rich arabinogalactan-protein in Arabidopsis. Planta 226:169–179 Zhang JZ, Li ZM, Liu L, Mei L, Yao JL, Hu CG (2008) Identification of early-flower-related ESTs in an early-flowering mutant of trifoliate orange (Poncirus trifoliata L. Raf.) by suppression subtractive hybridization and macroarray analysis. Tree Physiol 28:1449–1457 Zhong R, Ye ZH (1999) IFL1, a gene regulating interfascicular fiber differentiation in Arabidopsis encodes a homeodomain-leucine zipper protein. Plant Cell 11:2139–2152

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