Sabrina M. Elias

Enhanced Salt Tolerance Conferred by the Complete 2.3 kb cDNA of the Rice Vacuolar Na+/H+ Antiporter Gene Compared to 1.9 kb Coding Region with 5′ UTR in Transgenic Lines of Rice

Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na+/H+ antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions (UTRs) of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5′ UTR with CDS and the other of 2.3 kb, including 5′ UTR, CDS, and the 3′ UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5′ and 3′ UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3′ UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P < 0.05) and the electrolyte leakage significantly lower (P < 0.05) in the order 2.3 kb > 1.9 kb > and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P < 0.01) compared, respectively, to the best 1.9 kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance.

Comparative genomics of two jute species and insight into fibre biogenesis

Jute (Corchorus sp.) is one of the most important sources of natural fibre, covering ∼80% of global bast fibre production1. Only Corchorus olitorius and Corchorus capsularis are commercially cultivated, though there are more than 100 Corchorus species2 in the Malvaceae family. Here we describe high-quality draft genomes of these two species and their comparisons at the functional genomics level to support tailor-designed breeding. The assemblies cover 91.6% and 82.2% of the estimated genome sizes for C. olitorius and C. capsularis, respectively. In total, 37,031 C. olitorius and 30,096 C. capsularis genes are identified, and most of the genes are validated by cDNA and RNA-seq data. Analyses of clustered gene families and gene collinearity show that jute underwent shared whole-genome duplication ∼18.66 million years (Myr) ago prior to speciation. RNA expression analysis from isolated fibre cells reveals the key regulatory and structural genes involved in fibre formation. This work expands our understanding of the molecular basis of fibre formation laying the foundation for the genetic improvement of jute.

Physiology and gene expression of the rice landrace Horkuch under salt stress

Good donors in breeding for salt tolerance are a prerequisite for food security under changing climatic conditions. Horkuch, a farmer-popular salt tolerant rice (Oryza sativa L.) variety from the south-west coast of Bangladesh was characterised up to maturity under NaCl stress, together with a modern variety (BRRI dhan41), a sensitive control (BRRI dhan29) and Pokkali, the salt-tolerant benchmark for rice. Horkuch had low reduction in shoot biomass, a low Na:K ratio in flag leaves, a low percent reduction in yield and good partitioning of Na in the older leaves, and maintained high levels of Ca and Mg in the flag leaves. In order to understand the physiology at the molecular level, the expression of salt-responsive genes was investigated using microarray analysis. Salt-stressed cDNA of Horkuch seedlings were hybridised with cDNA probes synthesised mainly from database sequences of Arabidopsis thaliana (L.) Heynh. The upregulated genes included transcription factors, signal transducers, metabolic enzymes, reactive oxygen species (ROS) scavengers, osmoprotectants and some specific salt-induced transcripts. An increase in expression of photosynthesis-related genes as well ROS scavengers suggested that this could be the reason for the better yield performance of Horkuch. The data therefore indicate Horkuch as a potential donor alternative to Pokkali in breeding programs for salt tolerance.

Reproductive stage physiological and transcriptional responses to salinity stress in reciprocal populations derived from tolerant (Horkuch) and susceptible (IR29) rice

Global increase in salinity levels has made it imperative to identify novel sources of genetic variation for tolerance traits, especially in rice. The rice landrace Horkuch, endemic to the saline coastal area of Bangladesh, was used in this study as the source of tolerance in reciprocal crosses with the sensitive but high-yielding IR29 variety for discovering transcriptional variation associated with salt tolerance in the resulting populations. The cytoplasmic effect of the Horkuch background in leaves under stress showed functional enrichment for signal transduction, DNA-dependent regulation and transport activities. In roots the enrichment was for cell wall organization and macromolecule biosynthesis. In contrast, the cytoplasmic effect of IR29 showed upregulation of apoptosis and downregulation of phosphorylation across tissues relative to Horkuch. Differential gene expression in leaves of the sensitive population showed downregulation of GO processes like photosynthesis, ATP biosynthesis and ion transport. Roots of the tolerant plants conversely showed upregulation of GO terms like G-protein coupled receptor pathway, membrane potential and cation transport. Furthermore, genes involved in regulating membrane potentials were constitutively expressed only in the roots of tolerant individuals. Overall our work has developed genetic resources and elucidated the likely mechanisms associated with the tolerance response of the Horkuch genotype.

Unique Genotypic Differences Discovered among Indigenous Bangladeshi Rice Landraces

Bangladesh is a reservoir of diverse rice germplasm and is home to many landraces with unique, important traits. Molecular characterization of these landraces is of value for their identification, preservation, and potential use in breeding programs. Thirty-eight rice landraces from different regions of Bangladesh including some high yielding BRRI varieties were analyzed by 34 polymorphic microsatellite markers yielding a total of 258 reproducible alleles. The analysis could locate 34 unique identifiers for 21 genotypes, making the latter potentially amenable to identity verification. An identity map for these genotypes was constructed with all the 12 chromosomes of the rice genome. Polymorphism information content (PIC) scores of the 34 SSR markers were 0.098 to 0.89 where on average 7.5 alleles were observed. A dendogram constructed using UPGMA clustered the varieties into two major groups and five subgroups. In some cases, the clustering matched with properties like aromaticity, stickiness, salt tolerance, and photoperiod insensitivity. The results will help breeders to work towards the proper utilization of these landraces for parental selection and linkage map construction for discovery of useful alleles.

Microsatellite marker diversity and sequence polymorphism in the red gene locus of indigenous rice populations of Bangladesh

Bangladesh is home to diverse rice germplasm, including red rice, many of which are preferred for cultivation by farmers over high-yielding rice varieties due to their special characteristics, color, taste, and nutritional value. Red color of seed pericarp is unusual among modern cultivated varieties, though it is a common characteristic in wild relatives of rice, making the trait an important parameter when studying the domestication and evolution of crop plants. Diversity analysis using microsatellite markers and sequence variation of the red rice loci of the indigenous rice population in Bangladesh was therefore performed. Microsatellite fingerprinting could successfully cluster cultivars according to their specific phenotypic characteristics such as stickiness or aroma, irrespective of their pericarp color, and locate a set of unique identifiers. Sequence analysis of a portion of the bHLH transcription factor gene Rc, which controls the red pigment, confirmed the occurrence of the 14-bp deletion in white rice accessions which has been reported previously. The analysis included a group of rice cultivars which are known by the same name but produce two different colored seeds and are indistinguishable morphologically unless dehusked. Statistical and internal transcribed spacer region sequence diversity analyses established that such cultivars, although genetically different, had very low diversity, suggesting a close evolutionary relationship between them. Red and white seeds from cultivars with the same name were planted individually over two generations and dehusked to check for loss/gain of pigmentation. Red-seeded plants produced some dirty-white seeds in addition to red ones in the first generation, the proportion of which increased when planted in the successive generation. This loss in pigment was probably due to faulty transcription, since no deletion in the respective region of the genome of the dirty-white seeds was noted.

In Planta transformation for conferring salt tolerance to a tissue-culture unresponsive indica rice (Oryza sativa L.) cultivar

Many farmer-popular indica rice (Oryza sativa L.) cultivars are recalcitrant to Agrobacterium-mediated transformation through tissue culture and regeneration. In planta transformation using Agrobacterium could therefore be a useful alternative for indica rice. A simple and reproducible in planta protocol with higher transformation efficiencies than earlier reports was established for a recalcitrant indica rice genotype. Agrobacterium tumefaciens containing the salt tolerance-enhancing Pea DNA Helicase45 (PDH45) gene, with the reporter and selectable marker genes, gus-INT (β-glucuronidase with intron) and hygromycin phosphotransferase (hpt), respectively, were used. Overnight-soaked mature embryos were infected and allowed to germinate, flower, and set T1 seeds. T0 plants were considered positive for the transgene if the spikelets of one or more of their panicles were positive for gus. Thereafter, selection at T1 was done by germination in hygromycin and transgenic status re-confirmation by subjecting plantlet DNA/RNA to gene-specific PCR, Southern and semi-quantitative RT-PCR. Additionally, physiological screening under saline stress was done at the T2 generation. Transformation efficiency was found to be 30–32% at the T0 generation. Two lines of the in planta transformed seedlings of the recalcitrant rice genotype were shown to be saline tolerant having lower electrolyte leakage, lower Na+/K+, minimal leaf damage, and higher chlorophyll content under stress, compared to the WT at the T2 generation.

Helicases and Their Importance in Abiotic Stresses

Helicases are a ubiquitous class of ATP-dependent nucleic acid unwinding enzymes crucial for life processes in all living organisms. There are six classes of helicases based on their conserved amino acid sequences. All eukaryotic RNA helicases belong to the SF1 and SF2 groups. Groups SF3–SF5 are mainly viral and bacterial DNA helicases, while SF6 includes the ubiquitous mini-chromosome maintenance or MCM group of helicases. SF3–SF6 are also characterized as hexameric ring-forming, whereas SF1 and SF2 groups are usually monomeric. The SF2 class is the largest group of helicases, including both DNA and RNA helicases with the widest range of function in replication, transcription, translation, repair, as well as chromatin remodeling. There is no clear sequence-based separation between DNA and RNA helicases. SF2 also includes both RNA and DNA helicases that are involved in biotic and abiotic stresses. While both DNA and RNA helicases play important roles in normal cellular function, the latter are more markedly involved in stress alleviation. This functional divergence was also evident in promoter sequence comparisons of the 113 A. thaliana helicases. Some DNA helicases like those from SF6 (MCM) and SF2 (CHR) are also active under stressed conditions. However, the most prominent stress-activated helicases are those with the conserved amino acid motifs, DEAD/H. Overexpression of DEAD/H helicases in many crops confers a growth advantage in the transgenic plants and has resulted in their protection against major abiotic stresses, such as salinity, drought, and oxidative stresses with minimal loss in yield potential.