Silicon Alleviates Cadmium Toxicity in Two Cypress Varieties by Strengthening the Exodermis Tissues and Stimulating Phenolic Exudation of Roots

The aim of this work was to investigate the effect of silicon (Si) on phenolic exudation of plant roots and cadmium (Cd) bioavailability in rhizospheres. For this purpose, pot experiments with two cypress varieties, Juniperus chinensis and Platycladus orientalis, each subjected to 100 mg kg^?1 Cd and/or 400 mg kg^?1 Si for 220 days, were conducted using a rhizobag technique. The results showed that P. orientalis accumulated a higher amount of Cd, hence caused higher growth inhibition on the leaves compared with J. chinensis. Si alleviated the growth inhibition induced by Cd toxicity on both varieties, but the mechanisms involved were species specific. For J. chinensis, Si did not affect the root exudation but enhanced the Cd retention of the roots by strengthening the exodermis tissues, restraining Cd translocation from the roots to the shoots. For P. orientalis, Si exposure significantly elevated the phenolic exudation (for example, ferulic acid, catechin, and gallic acid) of the roots, which caused greater Cd mobility in the rhizosphere and enhancement of Cd accumulation in the shoots compared with Cd treatment alone. These results suggest that Cd-chelating with the Si-induced phenolics in the rhizosphere is involved in the Cd detoxification in P. orientalis.     

Effect of short-term heat exposure on physiological traits of indica rice at grain-filling stage

Heat stress impacts the quantity and quality of rice grains, particularly during grain-filling stage needed for grain development. In this study, the effect of short heat stress (42 °C, 30 min) on indica rice plants at the grain-filling stage (dough grain stage) was found by determining their physiological and growth traits F_v/F_m, ?F/F_m′, chlorophyll content, leaf water potential (LWP), membrane stability, relative leaf area (RLA), relative plant height (RPH), total grain weight per panicle (TGW) and 1000 GW. Thai economic rice cvs. KDML105 and Pathumthani 1 (PTT1) were compared to the heat-tolerant rice cultivars N22 and Dular and to the heat-sensitive rice cultivar IR64. The results showed that short heat stress exhibited effects on physiology and growth greater than the control (35 °C, 30 min) by reducing of F_v/F_m, ?F/F_m′, chlorophyll content, LWP, membrane stability and RLA. This result impacted the TGW and 1000 GW. A higher reduction of physiological traits was shown in IR64, followed by KDML105. In contrast, N22 and Dular were minimally affected by heat stress and were able to adapt and recover based on their grain weight that exhibited less of an effect. PTT1 was also impacted by heat stress similarly to Dular. Thus, short heat stress affected the physiological parameters of five rice cultivars at the dough grain stage. In addition, the five indica rice cultivars were classified into three groups: (1) the heat-tolerant group (N22, Dular and PTT1), (2) the moderately heat-tolerant group (KDML105), and (3) the heat-sensitive group (IR64) by PC-ORD program at 50% of similarity of the 13 physiological traits.     

The crucial role of roots in increased cadmium-tolerance and Cd-accumulation in the pea mutant <Emphasis Type="Italic">SGECd</Emphasis><Superscript><Emphasis Type="Italic">t</Emphasis></Superscript>

Elucidation of mechanisms underlying plant tolerance to cadmium, a widespread toxic soil pollutant, and accumulation of Cd in plants are urgent tasks. For this purposes, the pea (Pisum sativum L.) mutant SGECd^t (obtained by treatment of the laboratory pea line SGE with ethylmethane sulfonate) was reciprocally grafted with the parental line SGE, and four scion/rootstock combinations were obtained: SGE/SGE, SGECd^t/SGECd^t, SGE/SGECd^t, and SGECd^t/SGE. They were grown in hydroponics in the presence of 1 μM CdCl₂ for 30 d. The SGE and SGECd^t scions on the SGECd^t rootstock had a higher root and shoot biomass and an elevated root and shoot Cd content compared with the grafts having SGE rootstock. Only the grafts with the SGE rootstock showed chlorosis and roots demonstrating symptoms of Cd toxicity. The content of nutrient elements in roots (Fe, K, Mg, Mn, Na, P, and Zn) was higher in the grafts having the SGECd^t rootstock, and three elements, namely Ca, Fe, and Mn, were efficiently transported by the SGECd^t root to the shoot of these grafts. The content of other measured elements (K, Mg, Na, P, and Zn) was similar in the root and shoot in all the grafts. Then, the non-grafted plants were grown in the presence of Cd and subjected to deficit or excess concentrations of Ca, Fe, or Mn. Exclusion of these elements from the nutrient solution retained or increased differences between SGE and SGECd^t in growth response to Cd toxicity, whereas excess of Ca, Fe, or Mn decreased or eliminated such differences. The obtained results assign a principal role of roots to realizing the increased Cd-tolerance and Cdaccumulation in the SGECd^t mutant. Efficient translocation of Ca, Fe, and Mn from roots to shoots appeared to counteract Cd toxicity, although Cd was actively taken up by roots and accumulated in shoots.     

New QTLs identified for leaf correlative traits in rice seedlings under cadmium stress

Cadmium (Cd) is a non-essential toxic metal that is primarily released into the environment from artificial sources in recent decades. To investigate the genetics of Cd toxicity tolerance at the seedling stage in rice, a QTL analysis was carried out under cadmium stress conditions with two toxicity-linked traits—leaf rolling (LR) and the green leaf ratio (GLR). Using 127 rice lines of doubled haploid (DH) population derived from a cross between a japonica JX17 and indica ZYQ8, two QTLs for LR (qLR-1 and qLR-9) and one QTL for GLR (qGLR-3) were detected. Among them, the phenotypic variation of qLR-1 and qGLR-3 were 19.27 and 16.09, values which are useful for marker-assistant selection in breeding elite rice cultivars that have the capacity to tolerate Cd. The results further demonstrate that visual measurements of both LR and GLR in seedlings are effective methods for screening tolerant rice germplasm in cadmium stress scenarios.     

Comprehensive identification of major flowering time genes and their combinations,which determined rice distribution in Northeast China

Flowering time of rice (Oryza sativa L.) is among the most important agronomic traits for regional adaptation and grain yield. To date, a number of genes or quantitative trait loci (QTLs) controlling flowering time have been identified in rice, and diverse natural allelic variations for these flowering genes have been revealed, which suggested that the underlying regulation mechanism of flowering time in rice is very complicated. Northeast China is a major cultivation region for temperate japonica rice, where the temperature is cooler and the day length is longer. The regional adaptability of local rice cultivar is substantially different from that of other regions. Recently, some flowering genes have been proved to play roles in regulating flowering time of local cultivars. However, a comprehensive analysis of the effectiveness of these flowering genes has not been performed. In the present study, 395 cultivars collected from Northeast China is re-sequenced, SNP and InDel markers were called for 23 selected flowering-related genes. The heading date of these cultivars was also investigated for three consecutive years. Through association analysis, we found that Hd2, Hd4, and Hd5 are major flowering repressors, whereas Dth2 and Hd18 are major flowering promoters. Furthermore, Hd6 and Hd16 were identified as minor flowering repressors, and Hd17 was minor flowering promoter, in that their effectiveness can exclusively be detected when both Hd2 and Hd4 are functional. Collectively, we comprehensively identified the major and minor flowering genes which determine flowering time of temperate japonica rice grown in Northeast China.     

Growth,ionic response,and gene expression of shoots and roots of perennial ryegrass under salinity stress

Growth, ionic responses, and expression of candidate genes to salinity stress were examined in two perennial ryegrass accessions differing in salinity tolerance. The salinity tolerant (PI265349) and sensitive accessions (PI231595) were subjected to 75-mM NaCl for 14 days in a growth chamber. Across two accessions, salinity stress increased shoot dry weight and concentrations of malondialdehyde (MDA) and Na⁺ in the shoots and roots, but decreased shoot Ca²⁺ and root K⁺ concentrations. Salinity stress also increased root expressions of SOS1, PIP1, and TIP1. Plant height and chlorophyll content were unaffected by salinity stress in the tolerant accession but significantly decreased in the sensitive accession. Shoot MDA content did not change in the tolerant accession but increased in the sensitive accession. A more dramatic increase in Na⁺ was found in the roots of the sensitive accession. Relative to the control, salinity stress reduced expression of SOS1, NHX1, PIP1, and TIP1 in the shoots but increased expression of these genes in the roots of the tolerant accession. Expression levels of SOS1 increased in the roots and expression of NHX1 increased in the shoots but decreased in the roots of the sensitive accession under salinity stress. A decline in PIP1 expression in the shoots and dramatic increases in TIP expression in both shoots and roots were found in the sensitive accession under salinity stress. The results suggested maintenance of plant growth and leaf chlorophyll content, lesser Na⁺ accumulation in the roots, and lower lipid peroxidation in the shoots which could be associated with salinity tolerance. The decreased expressions of SOS1, NHX1, and TIP1 in the shoots, and increased expressions of NHX1 and PIP1 in the roots might also be related to salinity tolerance in perennial ryegrass.     

A loss‐of‐function allele of OsHMA3 associated with high cadmium accumulation in shoots and grain of Japonica rice cultivars

Excessive cadmium (Cd) accumulation in rice poses a risk to food safety. OsHMA3 plays an important role in restricting Cd translocation from roots to shoots. A non‐functional allele of OsHMA3 has been reported in some Indica rice cultivars with high Cd accumulation, but it is not known if OsHMA3 allelic variation is associated with Cd accumulation in Japonica cultivars. In this study, we identified a Japonica cultivar with consistently high Cd accumulation in shoots and grain in both field and greenhouse experiments. The cultivar possesses an OsHMA3 allele with a predicted amino acid mutation at the 380^th position from Ser to Arg. The haplotype had no Cd transport activity when the gene was expressed in yeast, and the allele did not complement a known nonfunctional allele of OsHMA3 in F₁ test. The allele is present only in temperate Japonica cultivars among diversity panels of 1483 rice cultivars. Different cultivars possessing this allele showed greatly increased root‐to‐shoot Cd translocation and a shift in root Cd speciation from Cd―S to Cd―O bonding determined by synchrotron X‐ray absorption spectroscopy. Our study has identified a new loss‐of‐function allele of OsHMA3 in Japonica rice cultivars leading to high Cd accumulation in shoots and grain.     

Genomic analysis and expression pattern of <Emphasis Type="Italic">OsZIP1, OsZIP3</Emphasis>, and <Emphasis Type="Italic">OsZIP4</Emphasis> in two rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes with different zinc efficiency

The ZRT-and IRT-like proteins (ZIP) comprise a large family of transition metal transporters in plants that have diverse functions to transport zinc, iron, copper, etc. Here, we provided a complete overview of this gene family in rice (Oryza sativa L.). Based on the hidden Markov model and BLAST analysis, a total of 17 ZIP-coding genes were identified and further studied by semi-quantitative RT-PCR analysis. Sequence analysis revealed 17 putative genes distributed randomly on eight chromosomes. Although most of the predicted proteins had typical characteristics of the ZIP protein family, the extent of their sequence similarity varied considerably. The expression patterns of OsZIP1, OsZIP3, and OsZIP4, which encode Zn²⁺ transporters in rice, were studied in the Zn-efficient and Zn-inefficient rice genotypes (IR8192 and Erjiufeng) by semi-quantitative RT-PCR analysis of roots, shoots, and panicle from the plants grown under Zn deficiency and normal conditions. OsZIP1 was expressed only in the roots and very weakly if at all in the panicles, while the other two genes were expressed in all parts of plants under study. The Zn-deficient conditions up-regulated the expression of OsZIP1, OsZIP3, and OsZIP4 in the roots and that of OsZIP4 in the shoots of both genotypes, indicating that all these genes may participate in rice zinc nutrition. Furthermore, the expression of OsZIP3 and OsZIP4 was found to be much stronger in the roots of IR8192 than those of Erjiufeng, which suggests that these genes may contribute to high Zn efficiency in rice. The expression patterns and the roles of other OsZIPs are also discussed on the basis of the phylogenetic tree of ZIP proteins and RT-PCR analysis of the two rice genotypes with different zinc efficiency.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of commercially elite rice restorer line using <Emphasis Type="Italic">npt</Emphasis>II gene as a plant selection marker

Transformation of commercially important indica cultivars remains challenging for the scientific community even though Agrobacterium-mediated transformation protocols for a few indica rice lines have been well established. We report successful transformation of a commercially important restorer line JK1044R of indica rice hybrid JKRH 401. While following existing protocol, we optimized several parameters for callusing, regeneration and genetic transformation of JK1044R. Calli generated from the rice scutellum tissue were used for transformation by Agrobacterium harboring pCAMBIA2201. A novel two tire selection scheme comprising of Geneticin (G418) and Paramomycin were deployed for selection of transgenic calli as well as regenerated plantlets that expressed neomycin phosphotransferase-II gene encoded by the vector. One specific combination of G418 (30 mg l^?1) and Paramomycin (70 mg l^?1) was very effective for calli selection. Transformed and selected calli were detected by monitoring the expression of the reporter gene uidA (GUS). Regenerated plantlets were confirmed through PCR analysis of nptII and gus genes specific primers as well as dot blot using gus gene specific as probe.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis>-mediated salinity tolerance in <Emphasis Type="Italic">Aloe vera</Emphasis> plantlets

Piriformospora indica, a root endophytic fungus, has been reported to promote growth of many plants under normal condition and allow the plants to survive under stress conditions. However, its impact on an important medicinal plant Aloe vera L. has not been well studied. Therefore, this study was undertaken to investigate the effect of P. indica on salinity stress tolerance of A. vera plant. P. indica inoculated and non-inoculated A. vera plantlets were subjected to four levels of salinity treatment- 0, 100, 200 and 300 mM NaCl. The salinity stress decreased the ability of the fungus to colonize roots of A. vera but the interaction of A. vera with P. indica resulted in an overall increase in plant biomass and greater shoot and root length as well as number of shoots and roots. The photosynthetic pigment (Chl a, Chl b and total Chl) and gel content were significantly higher for the fungus inoculated A. vera plantlets, at respective salinity concentrations. Furthermore, the inoculated plantlets had higher phenol, flavonoid, flavonol, aloin contents and radical scavenging activity at all salinity concentrations. The higher phenolic and flavonoid content may help the plants ameliorate oxidative stress resulting from high salinity.     

Deciphering the Mechanisms of Endophyte-Mediated Biofortification of Fe and Zn in Wheat

An investigation was carried out to understand the mechanism(s) underlying enhanced Fe or Zn uptake in low Fe–Zn accumulator wheat genotype 4HPYT-414, due to inoculation of siderophore-producing and zinc-solubilizing endophytes—Arthrobacter sulfonivorans DS-68 and Arthrobacter sp. DS-179. Root anatomical features, using transmission electron microscopy (TEM), qualitative and quantitative aspects of production of organic acids and sugars in root exudates, and expression of TaZIP genes were analysed to relate to endophyte-mediated higher concentrations of Fe and Zn in the roots and shoots of wheat plants. TEM studies revealed that the endodermis, cortical region, root hair extension, xylem and xylem vessels, pericycle and vascular bundles were more pronounced and thicker in inoculated treatments, as compared to control. The organic acid profile of root exudates revealed five types of organic acids, with citric acid being predominant. Inoculation of A. sulfonivorans and Arthrobacter sp. brought about 5- and eightfold increases in the amounts of acids, respectively, as compared to control, particularly citric acid, succinic acid and acetic acid. Among the four TaZIP genes targeted, expression was achieved only for TaZIP3 and TaZIP7 genes, which showed 1–2 fold increases in the inoculated treatments. The results clearly indicated that the endophyte-mediated overexpression of TaZIP3 and TaZIP7 genes in roots and shoots, and the observed anatomical and exudate changes were acting synergistically in facilitating higher Fe and Zn translocation in roots and shoots.     

<Emphasis Type="Italic">In Planta</Emphasis> transformation for conferring salt tolerance to a tissue-culture unresponsive indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> 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.     

<Emphasis Type="Italic">Lsi1</Emphasis>-regulated Cd uptake and phytohormones accumulation in rice seedlings in presence of Si

Silicon (Si) is known for its role in regulating the response of plants to imposed abiotic stresses. Since the stresses generally hinder production of a crop, such as rice, the exploration of the biochemistry and plant physiology relating to the function is of interest. Indeed, recently, there were reports on the function of Lsi1 in regulating the tolerance of rice to cadmium (Cd) stress. This study compared the kinetics of the Cd uptakes in Lemont wild type rice and its transgenic lines exposed to Cd with or without exogenous Si supply. At the same time, changes on the endogenous phytohormones and growth of the rice seedlings were monitored. Genetically, Lsi1 overexpression was found to downregulate K_m and V_max of Cd uptake kinetics in the plants under Cd stress, especially in the presence of Si. On the other hand, Lsi1 RNAi upregulated K_m and V_max regardless whether Si was present or not. It implied that Lsi1 could be capable of regulating Si as well as Cd transports. Under Cd stress, addition of Si reduced the Cd uptake of the rice lines in the order of Lsi1-overexpression line?>?Lemont?>?Lsi1-RNAi line. In addition, it also affected the chlorophyll biosynthesis and dry mass accumulation of the rice plants under Cd stress. Analyses on phytohormones including IAA, GA₃, JA, SA and ABA, as well as physiological functions, of the seedlings further verified the active involvement of Lsi1 in the complex defense system of the plants against Cd stress.