Ascorbate Peroxidase (APX) is a heme-containing, non-glycosylated enzyme that destroys harmful hydrogen peroxide via the ascorbate glutathione pathway in plants. This enzyme is considered to be an indispensable part of the electron-scavenging pathway and is involved in preventing oxidative damage in plants. Using differential display RT-PCR and 5' RACE a full length c-DNA clone was isolated, from citrus, with very high similarity at the nucleotide and amino acid level, to ascorbate peroxidases from several plant species. It is well known that APXs have highly conserved motifs like the Arg-38, Ars-71, Glu-65 and Asp-208 residues around the distal Hist-42 and proximal His-163. These residues are essential for binding the ligand heme. Additionally, Trp-179 is conserved in most APXs and is the third participant in hydrogen bonding network, together with His-163 and Asp-208. All these conserved motifs were present in the putative APX from citrus in addition of the presence of the peroxidase active site motif residues (APITLRLAWHSA) and the peroxidase heme-ligand motif (DIVVLSGGHTL). Expression analysis in E. Coli reviewed a recombinant protein of 27 Kda.
In Texas, the freezes of 1951 and 1962 together killed 125,000 acres of citrus trees and the freeze of 1983 killed 40,000 acres. The low temperature is one of the most important abiotic stresses to be understood and manipulated molecularly. Cold hardiness is found in the deciduous citrus relative, trifoliate orange, which can withstand temperatures as low as -26 °C when it is cold acclimated. Exposure of the cold hardy trifoliate orange plants to temperature from 28 °C to -5 °C enabled us to isolate and characterize one novel citrus low temperature gene (clt) with two transcripts, called clt-a and clt-b from leaves and twigs. Clt-a was produced when plants were subjected to low temperatures (starting at 10 °C), while cltb was constitutively expressed. Both clt-a and clt-b have the same open reading frame of 165 nucleotides and encodes a small protein of 54 amino acid. However, clt-a has an additional 98 bp nucleotides at the 3'-untranslated region (UTR), which is absent in clt-b. Expression analysis using relative quantitative RT-PCR demonstrated that clt-a is expressed exclusively at low temperatures, while clt-b is expressed constitutively (expression verified from 2 °C to -5 °C). In the process of deacclimation from -1 °C to 28 °C, the clt-a transcript degraded dramatically after 2 °C and was completely absent at 28 °C, while the clt-b transcript remain stable. When the acclimated plant was taken from -1 °C to room temperature, the clt-a gene degraded within 2 hours. Moreover, when acclimated plant was continuously exposed at -1 °C for 20 days, both transcripts clt-a and clt-b remained stable. Involvement of alternative splicing in transcript stability will be discussed.
Grapefruit [Citrus ×aurantium (synonym C. ×paradisi)] is an important citrus commodity that originated in Barbados in the 17th century. Grapefruit is the youngest member of the genus Citrus. Most commercially important grapefruit cultivars arose through natural and induced mutations, not traditional breeding, of the white-fleshed and seedy Duncan grapefruit. Now, cultivars with a range of flesh colors exist; the pigmentation is correlated with lycopene content. A bud sport mutant of grapefruit discovered in Texas has a deep golden-colored flesh, significantly different from the typical reddish pigmentation. In this review, we discuss grapefruit’s journey from its origin in Barbados and its global establishment including production, marketing, drug interactions, cultivar development, genetic diversity, and commercially significant cultivars.
The development of improved orange and grapefruit varieties via conventional breeding is not possible due their high degree of apomixis. The currently available varieties originated through natural or induced mutation. The development of a alternative breeding method is urgently needed for these citrus group. Microprotoplast Mediated Chromosome Transfer (MMCT) provides a direct way to transfer a very limited portion of the genome (one or more chromosome) from a donor species to a recipient species. In mammalian cells this procedure has been a powerful tool for gene mapping and to study the regulation of gene expression. Until recently, no chemical treatment was known for an efficient induction of microprotoplat in plants. Recently, amiprophosmethyl (APM) and cremart was found to be very efficient for the mass production of microprotoplasts in the Solanaceae family enabling a single chromosome to be transferred from potato to tomato and tobacco. To establish this technology in citrus, the efficiency of APM for the mass induction of microprotoplast from Swiglea glutinosa, a wild relative of citrus, was studied. APM ranging from 16 to 32 μmol was effective on promoting the scattering of the chromosomes and to create multinucleated cells. The microprotoplasts will be used in chromosome transfer experiments.
Reactive oxygen species (ROS) are continuously produced during the normal aerobic metabolism and also under environmental stress conditions. They are the major damaging factors to the photosynthetic machinery under stress conditions and need to be scavenged for the normal growth of the plant. Ascorbate peroxidase (APX) is the key enzyme in detoxifying H2O2, one of ROS from chloroplast and cytosol. A cDNA encoding a putative APXcit was isolated from mature `Dancy' tangerine (Citrus reticulata Blanco) juice vesicles using differential display reverse transcription-polymerase chain reaction (RT-PCR). Subsequently, full-length APXcit cDNA clone and genomic clone were obtained and sequenced. The full-length APXcit sequence is composed by 1082-bp nucleotides, including an open reading frame (ORF) of 753 bp, encoding a protein of 250 amino acids (27 kDa). The 5' un-translated region (UTR) of the APXcit gene consisted of 91 nucleotides and the 3' UTR consisted of 238 nucleotides. Homology search for APXcit at GenBank database showed high similarity to APX from several plant species.
Citrus Huanglongbing (HLB, also known as “citrus greening”), an important disease worldwide, is associated with three species of phloem-limited Candidatus liberibacter, of which Candidatus L. asiaticus (CLas) is the predominant one that has severely affected citrus production. TaqMan real-time polymerase chain reaction (PCR) (TM) has been the standard and very efficient method to diagnose several strains of Candidatus Liberibacter in citrus; however, it detects total bacteria and is unable to differentiate dead from live Liberibacter. The detection of only live bacteria is essential for testing methods of control for this important citrus disease. It is well known that ethidium monoazide and propidium monoazide (PMA) are compounds that supposedly enter only dead or membrane-damaged bacteria, intercalate the DNA strand, and make the DNA unavailable for amplification by PCR. These compounds are widely used when extracting the plant DNA to detect only live bacteria. In this research, we tested primers amplifying products from 79 to 1160 bp in TM and SYBR Green real-time PCR (SG) and PMA as DNA intercalating compound. Specifically, primers amplifying a 500-bp amplicon in SG provided the most reliable live-only detection, whereas those producing a smaller amplicon were unable to distinguish between live and dead. This is the first report of testing primers amplifying various amplicon sizes for the detection of only live CLas cells in citrus.
Protoplasm culture following polyethylene glycol-induced fusion resulted in the regeneration of tetraploid somatic hybrid plants from the following attempted parental combinations: Cleopatra mandarin (Citrus reticulata Blanco) + Argentine trifoliate orange [Poncirus trifoliata (L.) Raf.]; `Succari' sweet orange [C. sinensis (L.) Osb.] + Argentine trifoliate orange; sour orange (C. aurantium L.) + Flying Dragon trifoliate orange (P. trifolita); sour orange + Rangpur (C. limonia Osb.); and Milam lemon (purported sexual hybrid of C. jambhiri Lush × C. sinensis) + Sun Chu Sha mandarin (C. reticulate Blanco). Protoplasm isolation, fusion, and culture were conducted according to previously published methods. Regenerated plants were classified according to leaf morphology, chromosome number, and peroxidase, phosphoglucomutase, and phosphoglucose isomerase leaf isozyme profiles. All of the somatic hybrid plants were tetraploid, as expected (2n = 4x = 36), and all five selections have been propagated and entered into commercial citrus rootstock trials.
Hispanics lag behind all other U.S. ethnic groups in education, and are especially poorly represented in science careers. Undergraduate research is an efficient method to attract undergraduate students to science, and many universities are taking advantage of this; however, much still needs to be done to fully explore its potential. In 2000, Texas A&M University-Kingsville, in collaboration with the University of Texas at Brownsville and the University of Texas Pan-American, initiated a undergraduate research internship program in citrus biotechnology to channel Hispanic undergraduate students into graduate education. To date, 51 internships have been provided, and 20 students have been channeled into graduate school, including four at the doctoral level. Most were first-generation college students.
The Asian citrus psyllid, Diaphorina citri Kuwayama, one of the known vectors for citrus greening disease or Huanglongbing (HLB) pathogens, has been present in Texas for over a decade, but the detection of the disease is recent. HLB has been confirmed in only two adjacent commercial citrus groves of grapefruit and sweet orange. A study was conducted to compare the population of Candidatus Liberibacter asiaticus (CLas) cells in different plant parts including peduncle, columella, leaves, seeds, young shoots, flower buds, flowers, and bark of 6-year-old known infected grapefruit and sweet orange trees. The bacterial population was estimated using a previously described grand universal regression equation Y = 13.82 – 0.2866X, where Y is the log of the target copy number and X is the Ct (threshold cycle) of the assay. Except for bark tissue, there was no significant difference in the concentration of CLas cells in other plant parts between the two cultivars. Within the cultivar, the bacterial concentration also varied with the plant part, with peduncle, columella, midrib having significantly higher titer of CLas compared with other plant parts. The obtained results here are in agreement with previous studies conducted on Florida samples, but the consistently lowest bacterial titer recorded in young shoots, leaf blade, and especially leaf margins relative to the midrib has never been previously reported.
Seeds from four citrus rootstocks including sour orange, Bitters-C22 citrandarin, Sarawak pummelo Rio Red grapefruit, and Sarawak pummelo Bower mandarin were exposed to high inoculum levels of Phytophthora nicotianae to screen for tolerance. Inoculation of pregerminated seeds (PGIS) and non-PGIS was carried out. The average P. nicotianae propagule counts from the soil samples where these seedlings were raised ranged from 424 to 1361 colony forming units/cm3. The proportion of live to dead plants was recorded at 11 months postinoculation, which showed that Sarawak Bower performed significantly better than other rootstocks. Evaluation of the rootstocks 18 months postinoculation resulted in only one surviving sour orange plant, which suggests potential rootstock resistance.