Arif Hasan Khan Robin

Identification and expression analysis of glucosinolate biosynthetic genes and estimation of glucosinolate contents in edible organs of Brassica oleracea subspecies.

Glucosinolates are anti-carcinogenic, anti-oxidative biochemical compounds that defend plants from insect and microbial attack. Glucosinolates are abundant in all cruciferous crops, including all vegetable and oilseed Brassica species. Here, we studied the expression of glucosinolate biosynthesis genes and determined glucosinolate contents in the edible organs of a total of 12 genotypes of Brassica oleracea: three genotypes each from cabbage, kale, kohlrabi and cauliflower subspecies. Among the 81 genes analyzed by RT-PCR, 19 are transcription factor-related, two different sets of 25 genes are involved in aliphatic and indolic biosynthesis pathways and the rest are breakdown-related. The expression of glucosinolate-related genes in the stems of kohlrabi was remarkably different compared to leaves of cabbage and kale and florets of cauliflower as only eight genes out of 81 were expressed in the stem tissues of kohlrabi. In the stem tissue of kohlrabi, only one aliphatic transcription factor-related gene, Bol036286 (MYB28) and one indolic transcription factor-related gene, Bol030761 (MYB51), were expressed. The results indicated the expression of all genes is not essential for glucosinolate biosynthesis. Using HPLC analysis, a total of 16 different types of glucosinolates were identified in four subspecies, nine of them were aliphatic, four of them were indolic and one was aromatic. Cauliflower florets measured the highest number of 14 glucosinolates. Among the aliphatic glucosinolates, only gluconapin was found in the florets of cauliflower. Glucoiberverin and glucobrassicanapin contents were the highest in the stems of kohlrabi. The indolic methoxyglucobrassicin and aromatic gluconasturtiin accounted for the highest content in the florets of cauliflower. A further detailed investigation and analyses is required to discern the precise roles of each of the genes for aliphatic and indolic glucosinolate biosynthesis in the edible organs.

Expression Profiling of Glucosinolate Biosynthetic Genes in Brassica oleracea L. var. capitata Inbred Lines Reveals Their Association with Glucosinolate Content

Glucosinolates are the biochemical compounds that provide defense to plants against pathogens and herbivores. In this study, the relative expression level of 48 glucosinolate biosynthesis genes was explored in four morphologically-different cabbage inbred lines by qPCR analysis. The content of aliphatic and indolic glucosinolate molecules present in those cabbage lines was also estimated by HPLC analysis. The possible association between glucosinolate accumulation and related gene expression level was explored by principal component analysis (PCA). The genotype-dependent variation in the relative expression level of different aliphatic and indolic glucosinolate biosynthesis genes is the novel result of this study. A total of eight different types of glucosinolates, including five aliphatic and three indolic glucosinolates, was detected in four cabbage lines. Three inbred lines BN3383, BN4059 and BN4072 had no glucoraphanin, sinigrin and gluconapin detected, but the inbred line BN3273 had these three aliphatic glucosinolate compounds. PCA revealed that a higher expression level of ST5b genes and lower expression of GSL-OH was associated with the accumulation of these three aliphatic glucosinolate compounds. PCA further revealed that comparatively higher accumulation of neoglucobrassicin in the inbred line, BN4072, was associated with a high level of expression of MYB34 (Bol017062) and CYP81F1 genes. The Dof1 and IQD1 genes probably trans-activated the genes related to biosynthesis of glucoerucin and methoxyglucobrassicin for their comparatively higher accumulation in the BN4059 and BN4072 lines compared to the other two lines, BN3273 and BN3383. A comparatively higher progoitrin level in BN3273 was probably associated with the higher expression level of the GSL-OH gene. The cabbage inbred line BN3383 accounted for the significantly higher relative expression level for the 12 genes out of 48, but this line had comparatively lower total glucosinolates detected compared to the other three cabbage lines. The reason for the genotypic variation in gene expression and glucosinolate accumulation is a subject of further investigation.

Exogenous Methyl Jasmonate and Salicylic Acid Induce Subspecies-Specific Patterns of Glucosinolate Accumulation and Gene Expression in Brassica oleracea L.

Glucosinolates have anti-carcinogenic properties. In the recent decades, the genetics of glucosinolate biosynthesis has been widely studied, however, the expression of specific genes involved in glucosinolate biosynthesis under exogenous phytohormone treatment has not been explored at the subspecies level in Brassica oleracea. Such data are vital for strategies aimed at selective exploitation of glucosinolate profiles. This study quantified the expression of 38 glucosinolate biosynthesis-related genes in three B. oleracea subspecies, namely cabbage, broccoli and kale, and catalogued associations between gene expression and increased contents of individual glucosinolates under methyl jasmonate (MeJA) and salicylic acid (SA) treatments. Glucosinolate accumulation and gene expression in response to phytohormone elicitation was subspecies specific. For instance, cabbage leaves showed enhanced accumulation of the aliphatic glucoiberin, progoitrin, sinigrin and indolic neoglucobrassicin under both MeJA and SA treatment. MeJA treatment induced strikingly higher accumulation of glucobrassicin (GBS) in cabbage and kale and of neoglucobrassicin (NGBS) in broccoli compared to controls. Notably higher expression of ST5a (Bol026200), CYP81F1 (Bol028913, Bol028914) and CYP81F4 genes was associated with significantly higher GBS accumulation under MeJA treatment compared to controls in all three subspecies. CYP81F4 genes, trans-activated by MYB34 genes, were expressed at remarkably high levels in all three subspecies under MeJA treatment, which also induced in higher indolic NGBS accumulation in all three subspecies. Remarkably higher expression of MYB28 (Bol036286), ST5b, ST5c, AOP2, FMOGS-OX5 (Bol031350) and GSL-OH (Bol033373) was associated with much higher contents of aliphatic glucosinolates in kale leaves compared to the other two subspecies. The genes expressed highly could be utilized in strategies to selectively increase glucosinolate compounds in B. oleracea subspecies. These results promote efforts to develop genotypes of B. oleracea and other species with enhanced levels of desired glucosinolates.

Genome-Wide Identification, Characterization and Expression Profiling of ADF Family Genes in Solanum lycopersicum L.

The actin depolymerizing factor (ADF) proteins have growth, development, defense-related and growth regulatory functions in plants. The present study used genome-wide analysis to investigate ADF family genes in tomato. Eleven tomato ADF genes were identified and differential expression patterns were found in different organs. SlADF6 was preferentially expressed in roots, suggesting its function in root development. SlADF1, SlADF3 and SlADF10 were predominately expressed in the flowers compared to the other organs and specifically in the stamen compared to other flower parts, indicating their potential roles in pollen development. The comparatively higher expression of SlADF3 and SlADF11 at early fruit developmental stages might implicate them in determining final fruit size. SlADF5 and SlADF8 had relatively higher levels of expression five days after the breaker stage of fruit development, suggesting their possible role in fruit ripening. Notably, six genes were induced by cold and heat, seven by drought, five by NaCl, and four each by abscisic acid (ABA), jasmonic acid (JA) and wounding treatments. The differential expression patterns of the SlADF genes under different types of stresses suggested their function in stress tolerance in tomato plants. Our results will be helpful for the functional characterization of ADF genes during organ and fruit development of tomato under different stresses.

Diversity and Inheritance of Intergenic Spacer Sequences of 45S Ribosomal DNA among Accessions of Brassica oleracea L. var. capitata

Ribosomal DNA (rDNA) of plants is present in high copy number and shows variation between and within species in the length of the intergenic spacer (IGS). The 45S rDNA of flowering plants includes the 5.8S, 18S and 25S rDNA genes, the internal transcribed spacer (ITS1 and ITS2), and the intergenic spacer 45S-IGS (25S-18S). This study identified six different types of 45S-IGS, A to F, which at 363 bp, 1121 bp, 1717 bp, 1969 bp, 2036 bp and 2111 bp in length, respectively, were much shorter than the reported reference IGS sequences in B. oleracea var. alboglabra. The shortest two IGS types, A and B, lacked the transcription initiation site, non-transcribed spacer, and external transcribed spacer. Functional behavior of those two IGS types in relation to rRNA synthesis is a subject of further investigation. The other four IGSs had subtle variations in the transcription termination site, guanine-cytosine (GC) content, and number of tandem repeats, but the external transcribed spacers of these four IGSs were quite similar in length. The 45S IGSs were found to follow Mendelian inheritance in a population of 15 F1s and their 30 inbred parental lines, which suggests that these sequences could be useful for development of new breeding tools. In addition, this study represents the first report of intra-specific (within subspecies) variation of the 45S IGS in B. oleracea.

Molecular Characterization and Expression Profiling of Tomato GRF Transcription Factor Family Genes in Response to Abiotic Stresses and Phytohormones

Growth regulating factors (GRFs) are plant-specific transcription factors that are involved in diverse biological and physiological processes, such as growth, development and stress and hormone responses. However, the roles of GRFs in vegetative and reproductive growth, development and stress responses in tomato (Solanum lycopersicum) have not been extensively explored. In this study, we characterized the 13 SlGRF genes. In silico analysis of protein motif organization, intron–exon distribution, and phylogenetic classification confirmed the presence of GRF proteins in tomato. The tissue-specific expression analysis revealed that most of the SlGRF genes were preferentially expressed in young and growing tissues such as flower buds and meristems, suggesting that SlGRFs are important during growth and development of these tissues. Some of the SlGRF genes were preferentially expressed in fruits at distinct developmental stages suggesting their involvement in fruit development and the ripening process. The strong and differential expression of different SlGRFs under NaCl, drought, heat, cold, abscisic acid (ABA), and jasmonic acid (JA) treatment, predict possible functions for these genes in stress responses in addition to their growth regulatory functions. Further, differential expression of SlGRF genes upon gibberellic acid (GA3) treatment indicates their probable function in flower development and stress responses through a gibberellic acid (GA)-mediated pathway. The results of this study provide a basis for further functional analysis and characterization of this important gene family in tomato.

Expression Profiling of MLO Family Genes under Podosphaera xanthii Infection and Exogenous Application of Phytohormones in Cucumis melo L.

Powdery mildew disease caused by Podosphaera xanthii is a major concern for Cucumis melo production worldwide. Knowledge on genetic behavior of the related genes and their modulating phytohormones often offer the most efficient approach to develop resistance against different diseases. Mildew Resistance Locus O (MLO) genes encode proteins with seven transmembrane domains that have significant function in plant resistance to powdery mildew fungus. We collected 14 MLO genes from ‘Melonomics’ database. Multiple sequence analysis of MLO proteins revealed the existence of both evolutionary conserved cysteine and proline residues. Moreover, natural genetic variation in conserved amino acids and their replacement by other amino acids are also observed. Real-time quantitative PCR expression analysis was conducted for the leaf samples of P. xanthii infected and phytohormones (methyl jasmonate and salicylic acid) treated plants in melon ‘SCNU1154’ line. Upon P. xanthii infection using 7 different races, the melon line showed variable disease reactions with respect to spread of infection symptoms and disease severity. Three out of 14 CmMLO genes were up-regulated and 7 were down-regulated in leaf samples in response to all races. The up- or down-regulation of the other 4 CmMLO genes was race-specific. The expression of 14 CmMLO genes under methyl jasmonate and salicylic acid application was also variable. Eleven CmMLO genes were up-regulated under salicylic acid treatment, and 7 were up-regulated under methyl jasmonate treatments in C. melo L. Taken together, these stress-responsive CmMLO genes might be useful resources for the development of powdery mildew disease resistant C. melo L.

Salinity-induced reduction in root surface area and changes in major root and shoot traits at the phytomer level in wheat

The aim of this study was to investigate the effect of salinity stress on root growth at the phytomer level in wheat to provide novel site-specific understanding of salinity damage in roots. Seedlings of 13 wheat varieties were grown hydroponically. Plants were exposed to three concentrations of NaCl, 0 (control), 50 and 100mM, from 47 days after sowing. In a destructive harvest 12 days later we determined the number of live leaves, adventitious roots, seminal roots and newly formed roots at the youngest phytomer; length and diameter of main axes; and length and diameter of root hairs and their number per millimetre of root axis. Elongation rate of main axes and root hair density were then derived. Root surface area at each root-bearing phytomer (Pr) was mechanistically modelled. New root formation was increased by salt exposure, while number of live leaves per plant decreased. The greatest salinity effect on root axis elongation was observed at the youngest roots at Pr1 and Pr2. Both the 50mM and the 100mM levels of salinity reduced root hair length by approximately 25% and root hair density by 40% compared with the control whereas root hairs alone contributed around 93% of the estimated total root surface area of an individual tiller. Decrease in main axis length of new roots, root hair density and root hair length combined to reduce estimated root surface area by 36-66% at the higher NaCl concentration. The varietal response towards the three salinity levels was found to be trait-specific. The data highlight reduction in root surface area as a major but previously largely unrecognized component of salinity damage. Salinity resistance is trait-specific. Selection for retention of root surface area at a specific phytomer position following salt exposure might be useful in development of salinity-tolerant crop varieties.

Detection of Ribosomal DNA Sequence Polymorphisms in the Protist Plasmodiophora brassicae for the Identification of Geographical Isolates

Clubroot is a soil-borne disease caused by the protist Plasmodiophora brassicae (P. brassicae). It is one of the most economically important diseases of Brassica rapa and other cruciferous crops as it can cause remarkable yield reductions. Understanding P. brassicae genetics, and developing efficient molecular markers, is essential for effective detection of harmful races of this pathogen. Samples from 11 Korean field populations of P. brassicae (geographic isolates), collected from nine different locations in South Korea, were used in this study. Genomic DNA was extracted from the clubroot-infected samples to sequence the ribosomal DNA. Primers and probes for P. brassicae were designed using a ribosomal DNA gene sequence from a Japanese strain available in GenBank (accession number AB526843; isolate NGY). The nuclear ribosomal DNA (rDNA) sequence of P. brassicae, comprising 6932 base pairs (bp), was cloned and sequenced and found to include the small subunits (SSUs) and a large subunit (LSU), internal transcribed spacers (ITS1 and ITS2), and a 5.8s. Sequence variation was observed in both the SSU and LSU. Four markers showed useful differences in high-resolution melting analysis to identify nucleotide polymorphisms including single- nucleotide polymorphisms (SNPs), oligonucleotide polymorphisms, and insertions/deletions (InDels). A combination of three markers was able to distinguish the geographical isolates into two groups.

Genome-wide identification, characterization and expression profiling of LIM family genes in Solanum lycopersicum L.

LIM domain proteins, some of which have been shown to be actin binding proteins, are involved in various developmental activities and cellular processes in plants. To date, the molecular defense-related functions of LIM family genes have not been investigated in any solanaceous vegetable crop species. In this study, we identified 15 LIM family genes in tomato (Solanum lycopersicum L.) through genome-wide analysis and performed expression profiling in different organs of tomato, including fruits at six different developmental stages. We also performed expression profiling of selected tomato LIM genes in plants under ABA, drought, cold, NaCl and heat stress treatment. The encoded proteins of the 15 tomato LIM genes were classified into two main groups, i.e., proteins similar to cysteine-rich proteins and plant-specific DAR proteins, based on differences in functional domains and variability in their C-terminal regions. The DAR proteins contain a so far poorly characterized zinc-finger-like motif that we propose to call DAR-ZF. Six of the 15 LIM genes were expressed only in flowers, indicating that they play flower-specific roles in plants. The other nine genes were expressed in all organs and at various stages of fruit development. SlβLIM1b was expressed relatively highly at the later stage of fruit development, but three other genes, SlWLIM2a, SlDAR2 and SlDAR4, were expressed at the early stage of fruit development. Seven genes were induced by ABA, five by cold, seven by drought, eight by NaCl and seven by heat treatment respectively, indicating their possible roles in abiotic stress tolerance. Our results will be useful for functional analysis of LIM genes during fruit development in tomato plants under different abiotic stresses.