Khurram Ziaf

Over-expression of microRNA169 confers enhanced drought tolerance to tomato.

Plant miRNA regulates multiple developmental and physiological processes, including drought responses. We found that the accumulation of Sly-miR169 in tomato (Solanum lycopersicum) was induced by drought stress. Consequently, Sly-miR169 targets, namely, three nuclear factor Y subunit genes (SlNF-YA1/2/3) and one multidrug resistance-associated protein gene (SlMRP1), were significantly down-regulated by drought stress. Constitutive over-expression of a miR169 family member, Sly-miR169c, in tomato plant can efficiently down-regulate the transcripts of the target genes. Compared with non-transgenic plants, transgenic plants over-expressing Sly-miR169c displayed reduced stomatal opening, decreased transpiration rate, lowered leaf water loss, and enhanced drought tolerance. Our study is the first to provide evidence that the Sly-miR169c negatively regulates stomatal movement in tomato drought responses.

Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner

Expression of artificial microRNAs (amiRNAs) in plants can target and degrade the invading viral RNA, consequently conferring virus resistance. Two amiRNAs, targeting the coding sequence shared by the 2a and 2b genes and the highly conserved 3′ untranslated region (UTR) of Cucumber mosaic virus (CMV), respectively, were generated and introduced into the susceptible tomato. The transgenic tomato plants expressing amiRNAs displayed effective resistance to CMV infection and CMV mixed with non-targeted viruses, including tobacco mosaic virus and tomato yellow leaf curl virus. A series of grafting assays indicate scions originated from the transgenic tomato plant maintain stable resistance to CMV infection after grafted onto a CMV-infected rootstock. However, the grafting assay also suggests that the amiRNA-mediated resistance acts in a cell-autonomous manner and the amiRNA signal cannot be transmitted over long distances through the vascular system. Moreover, transgenic plants expressing amiRNA targeting the 2a and 2b viral genes displayed slightly more effective to repress CMV RNA accumulation than transgenic plants expressing amiRNA targeting the 3′ UTR of viral genome did. Our work provides new evidence of the use of amiRNAs as an effective approach to engineer viral resistance in the tomato and possibly in other crops.

SpUSP, an annexin-interacting universal stress protein, enhances drought tolerance in tomato

Universal stress protein (USP) appears to play an active role in the abiotic stress response, but their functions remain largely unknown in plants. A USP gene (SpUSP) was cloned from wild tomato (Solanum pennellii) and functionally characterized in cultivated tomato in the present study. The SpUSP transcript is abundantly accumulated in leaf stomata and its expression varied with the circadian rhythm. SpUSP was remarkably induced by dehydration, salt stress, oxidative stress, and the phytohormone abscisic acid (ABA) etc. This protein was predominantly localized in the nucleus and cell membrane. Overexpressing SpUSP increased drought tolerance of tomato in the seedling and adult stages. Under drought stress, the ABA content significantly increased in the SpUSP-overexpressing plants, which induced stomatal closure and reduced water loss, leading to the enhancement of drought tolerance. Based on the microarray data, a large number of chlorophyll a/b-binding proteins and photosystem-related genes were up-regulated in the SpUSP-overexpressing plants under drought conditions, which possibly enhanced the stomatal sensivitity to ABA and maintained the photosynthetic function. SpUSP overexpression also alleviated the oxidative damage accompanied by oxidative stress-responsive gene activation and osmolyte accumulation. Annexin (SGN-U314161) was found to interacte with SpUSP in the yeast two-hybrid method. This interaction was further confirmed by the bimolecular fluorescence complementation assay. The present study demonstrated that the annexin-interacting SpUSP plays important roles in the drought tolerance of tomato by influencing ABA-induced stomatal movement, increasing photosynthesis, and alleviating oxidative stress.

Tomato SlDREB gene restricts leaf expansion and internode elongation by downregulating key genes for gibberellin biosynthesis

Plants have evolved and adapted to different environments. Dwarfism is an adaptive trait of plants that helps them avoid high-energy costs under unfavourable conditions. The role of gibberellin (GA) in plant development has been well established. Several plant dehydration-responsive element-binding proteins (DREBs) have been identified and reported to be induced under abiotic and biotic stress conditions. A tomato DREB gene named SlDREB, which is a transcription factor and was cloned from cultivated tomato M82, was found to play a negative role in tomato plant architecture and enhances drought tolerance. Tissue expression profiles indicated that SlDREB was expressed mainly in the stem and leaf and could be induced by abscisic acid (ABA) but suppressed by GA and ethylene. SlDREB altered plant morphology by restricting leaf expansion and internode elongation when overexpressed, and the resulting dwarfism of tomato plants could be recovered by application of exogenous gibberellic acid (GA3). Transcriptional analysis of transgenic plants revealed that overexpression of SlDREB caused the dwarf phenotype by downregulating key genes involved in GA biosynthesis such as ent-copalyl diphosphate synthase (SlCPS) and GA 20-oxidases (SlGA20ox1, -2, and -4), thereby decreasing endogenous GA levels in transgenic plants. A yeast activity assay demonstrated that SlDREB specifically bound to dehydration-responsive element/C-repeat (DRE/CRT) elements of the SlCPS promoter region. Taken together, these data demonstrated that SlDREB can downregulate the expression of key genes required for GA biosynthesis and that it acts as a positive regulator in drought stress responses by restricting leaf expansion and internode elongation.

A multiple stress-responsive gene ERD15 from Solanum pennellii confers stress tolerance in tobacco.

Wild species often show more tolerance to environmental stress factors than their cultivated counterparts. An early responsive-to-dehydration gene was cloned from a drought- and salt-tolerant wild tomato Solanum pennellii (SpERD15). SpERD15 transcript accumulated differentially in different organs, and was remarkably induced by dehydration, salinity, cold and treatment with plant growth regulators. The protein encoded by SpERD15 was predominantly localized in the nucleus. Interestingly, we found that the majority of the transgenic tobacco plants were co-suppressed along with the overexpressing line. Overexpressing plants manifested stress tolerance accompanied by the accumulation of more soluble sugars and proline, and limited lipid peroxidation compared with co-suppression lines, which were more sensitive than the wild type. The differential contents of these compatible solutes in different transgenic lines were related to the changes in the expression of the genes involved in the production of some important osmolytes (P5CS and Sucrose synthase). Reduced lipid peroxidation over a broad range of stress factors was in agreement with increased expression of stress-responsive genes (ADH and GAPDH). Overexpression of SpERD15 increased the efficiency of PSII (F(v)/F(m)) in transgenic tobacco plants by maintaining PSII quinone acceptors in a partially oxidized form. The results show that SpERD15 augments stress tolerance by enhancing the efficiency of PSII through the protection of cellular membranes, as conferred by the accumulation of compatible solutes and limited lipid peroxidation.

Identification and expression pattern of one stress-responsive NAC gene from Solanum lycopersicum

NAC (for NAM, ATAF1, 2, and CUC2) family genes have been found to play an important role in diversified developmental processes and environmental responses. A new NAC-type transcription factor SlNAC3 was primarily identified and isolated from the cDNA libraries of tomato cultivar Ailsa Craig. It contains three exons and two introns within genomic DNA sequence and encodes a polypeptide of 329 amino acids. A plant-specific and conserved NAC domain is located in the N-terminus of SlNAC3. The protein SlNAC3 is subcellularly localized in the nucleus of onion epidemical cells and it has a transcriptional activation domain in the C-terminal region which shows extremely divergent among NACs. Phylogenetic analysis showed that SlNAC3 belonged to the OsNAC3 subgroup of the NAC protein family. Tissue expression profile analysis revealed that SlNAC3 was expressed mainly in flower, fruit and root. The transcription expression of SlNAC3 was inhibited by salt, drought stress and ABA treatment. These data demonstrate that SlNAC3 might interact with environmental and endogenous stimuli and probably function when plants response to salt and drought stresses through ABA signaling pathways as a transcriptional activator.

Exogenous Application of Polyamines Improves Germination and Early Seedling Growth of Hot Pepper

Las poliaminas son compuestos organicos de bajo peso molecular implicados en numerosos procesos biologicos en todos los organismos vivos. El acondicionamiento de semillas es una tecnica que mejora el comportamiento de las semillas respecto a una germinacion mas rapida y uniforme, con plantulas normales y vigorosas. Se realizo un estudio de laboratorio para explorar los beneficios del acondicionamiento de semillas con poliaminas sobre la germinacion y el crecimiento de plantulas de aji (Capsicum annuum L.) Las semillas de aji fueron colocadas en solucion acuosa aireada de putrescina, espermina o espermidina (25, 50, 75 y 100 mM), durante 48 h a 25 ± 2 °C. Se observaron resultados significativos (P < 0,05) para los diferentes atributos de germinacion de semillas y crecimiento de plantulas. El acondicionamiento con poliaminas adelanto la germinacion y sincronizo el porcentaje final de germinacion, tiempo hasta 50% de germinacion, tiempo medio de germinacion, energia de germinacion, velocidad de germinacion, e indice de germinacion en comparacion con el control. El mejoramiento de longitud de tallo y raiz, peso fresco y seco de plantulas, tasa de crecimiento de plantulas y el indice de vigor de las plantulas fue claramente indicativo de los efectos positivos de la imprimacion de semillas con poliamina. De todas las poliaminas, putrescina demostro ser mejor a bajas concentraciones (es decir 25 y 50 mM) para la mayoria de los rasgos relacionados con emergencia de semillas y crecimiento de plantulas.

Characterization of ERD15 gene from cultivated tomato (Solanum lycopersicum).

Plant physiology, morphology and biochemistry are genetically controlled by a large set of genes. Expression of these genes is influenced by a number of factors, internal as well as external, including stage of plant development, diseases, drought, salinity, cold, and many others (Yoshioka et al., 2003; Zhang et al., 2001). Some genes are activated or suppressed by a specific stress factor while, many genes respond to more than one stimulus (Kariola et al., 2006). These genes can be classified, on the basis of their expression patterns and/or their responsiveness to environmental stressors, in different groups viz., responsive to dehydration (RD), cold regulated (COR), cold inducible (KIN), lowtemperature induced (LTI), early responsive to dehydration (ERD), salt overly sensitive (SOS), dehydration-responsive element-binding proteins (DREBs) and many more (Shinozaki and Yamaguchi-Shinozaki, 2007; Mahajan and Tuteja, 2005). Among these genes, ERDs have emerged as a new class or group of genes, identified first time by Kiyosue et al. (1994) in Arabidopsis plants dehydrated for one hour. Some members of the ERD group have been studied such as ERD1 (that encodes a Clp protease regulatory subunit; Nakashima et al., 1997), ERD5 (proline dehydrogenase; Kiyosue et al., 1996), ERD8 (hsp8-1; Takahashi et al., 1992), ERD10 (Group II LEA protein = lti29/lti45; Welin et al., 1995) and ERD15 (Kariola et al., 2006; Alves et al., 2011; Ziaf et al., 2011). Among these ERD genes, ERD15 has been used as a stress responsive gene in various stress experiments (Dunaeva and Adamska, 2001; Park et al., 2009; Li et al., 2010) in Arabidopsis and wheat. Nevertheless, its induction and function has been contradictory (Kariola et al., 2006; Ziaf et al., 2011), which can be due to difference in sequence among the species and/or due to some mutations in the sequence from specific crop. Transcript of several genes is accumulated at a specific stage of plant growth or in specific organs, varying to some extent with their function. Some are under the control of endogenous circadian system and thus affect the physiology and metabolism (Yakir et al., 2007). About 6% genes in Arabidopsis, involved in various functions such as photosynthesis, responses to stresses, sugar metabolism and flowering in response to day-length, fluctuate daily and are controlled by circadian clock (Harmer et al., 2000). Wang and Grumet (2004) reported diurnal pattern for transcript level of Pak. J. Agri. Sci., Vol. 53(1), 27-33; 2016 ISSN (Print) 0552-9034, ISSN (Online) 2076-0906 DOI: 10.21162/PAKJAS/16.3695 http://www.pakjas.com.pk

Screening of tomato genotypes for salinity tolerance based on early growth attributes and leaf inorganic osmolytes

ABSTRACT The experiment containing three replicates of completely randomized factorial treatments was conducted in a glasshouse under controlled conditions with three simulated soil salinity levels (control, 10 and 15 dS m−1). Morpho-physiological traits (i.e. lengths, fresh weights and dry weights of root and shoot, number of leaves, root/shoot ratio, shoot Na+ accumulation, K+/Na+ ratio, Ca2+/Na+ ratio, membrane stability index, lycopene contents, chlorophyll-a and -b) were recorded to determine mechanism of salt tolerance of tomato at seedling stage. Principal component analysis (PCA) was used to express a three-way interaction of genotype × salinity level × traits that scattered the 25 tomato genotypes based on their morpho-physiological response to different NaCl levels. The negative association of Na+ with all other traits except root/shoot ratio and the morpho-physiological response trend of genotypes exposed that probable mechanism of salt tolerance was initially Na+ exclusion by abscising older leaves to have younger physiologically energetic, and lastly a higher activity of plants for root development to sustain them in saline soil. PCA three-way biplot efficiently recognized ANAHU, LA-2821, LO-2752, LO-2707, PB-017909, LO-2831-23 and 017860 as salt tolerant genotypes. On the other hand, ZARNITZA, GLACIER, LO-2692, LO-2576, BL-1079, 006233, 006232, 017856, NUTYT-701 and NAGINA were found to be salt susceptible.

Stability of Capsaicinoids and Antioxidants in Dry Hot Peppers under Different Packaging and Storage Temperatures

The maintenance of the quality and storage life of perishable fruits and vegetables is a major challenge for the food industry. In this study, the effects of different temperatures, packaging materials and storage time on the stability of capsaicinoids and antioxidants, such as total carotenoids, ascorbic acid and total phenolic compounds, were studied in three commercially cultivated hot pepper hybrids, namely Sky Red, Maha and Wonder King. For this purpose, dry whole pods were packed in jute bags and low-density polyethylene bags (LDPE), stored for five months under controlled conditions at 20, 25 or 30 ○C and analyzed on Day 0 and at 50-day intervals until Day 150. The three hot pepper hybrids differed significantly with respect to their capsaicinoids and antioxidant concentrations, but the results indicated that with the increase in storage temperature and time, a gradual and steady decrease in these levels was equally observed for all hybrids. Overall, mean concentrations after five months were significantly reduced by 22.6% for ascorbic acid, 19.0% for phenolic compounds, 17% for carotenoids and 12.7% for capsaicinoids. The trends of capsaicinoids and antioxidants evolution were decreasing gradually during storage until Day 150, this effect being more pronounced at higher temperature. Furthermore, the disappearance rates of capsaicinoids and antioxidants were higher in peppers packed in jute bags than in those wrapped with LDPE. In conclusion, despite the sensitivity of capsaicinoids and antioxidants to oxygen, light and moisture, the packaging in natural jute or synthetic LDPE plastic bags, as well as the storage at ambient temperature preserved between 77.4% and 87.3% of the initial amounts of these health- and nutrition-promoting compounds during five months’ storage.