The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α), tubulin beta (β-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types.

The chloroplast HSP100/ClpB is a newly documented member of the ClpB family, but little was known about its role in imparting thermotolerance to cells. A cDNA coding for a HSP100/ClpB homolog has been cloned from Lycopersicon esculentum and termed as Lehsp100/ClpB (the cDNA sequence of Lehsp100/ClpB has been submitted to the GenBank database under accession number: AB219939). The protein encoded by the cDNA was most similar to the putative chloroplast HSP100/ClpBs in higher plants and the ClpB from Cyanobacterium Synechococcus sp. A 97 kDa protein, which matched the predicted size of mature LeHSP100/ClpB, was immunologically detected in chloroplast isolated from heat-treated tomato plants. In addition, the fusion protein, combining the transit sequence of LeHSP100/ClpB and GFP, was found to be located in chloroplast based on the observations of fluorescent microscope images. These results indicated the chloroplast-localization of LeHSP100/ClpB. Both the transcript and the protein of Lehsp100/ClpB were not detected under normal growth conditions, but they were induced by increasingly higher temperatures. An antisense Lehsp100/ClpB cDNA fragment was introduced into the tomato by Agrobacterium-mediated transformation. Antisense lines exhibited an extreme repression of heat-induced expression of Lehsp100/ClpB. The levels of chloroplast HSP60 and small HSP in antisense lines were identical to those of the control plants. After plants preconditioned at 38 degrees C for 2 h were exposed to a lethal heat shock at 46 degrees C for 2 h, the antisense lines were greatly impaired and withered in 21 days of the recovery phase, whereas the untransformed control plants and the vector-transformed plants survived. Furthermore, chlorophyll fluorescence measurements showed that PS II in antisense lines were more susceptible to the thermal irreversible inactivation than the untransformed and vector-transformed control plants. This work provides the first example that induction of chloroplast LeHSP100/ClpB contributes to the acquisition of thermotolerance in higher plants.

Summary Salt stress is an important environmental cue impeding poplar nitrogen nutrition. Here, we characterized the impact of salinity on proton‐driven nitrate fluxes in ectomycorrhizal roots and the importance of a Hartig net for nitrate uptake. We employed two Paxillus involutus strains for root colonization: MAJ , which forms typical ectomycorrhizal structures (mantle and Hartig net), and NAU , colonizing roots with a thin, loose hyphal sheath. Fungus‐colonized and noncolonized Populus × canescens were exposed to sodium chloride and used to measure root surface pH , nitrate ( NO 3 − ) flux and transcription of NO 3 − transporters (NRTs; Pc NRT 1.1 , ‐ 1.2 , ‐ 2.1 ), and plasmalemma proton ATPases (HAs; Pc HA 4 , ‐ 8 , ‐11 ). Paxillus colonization enhanced root NO 3 − uptake, decreased surface pH , and stimulated NRT s and HA 4 of the host regardless the presence or absence of a Hartig net. Under salt stress, noncolonized roots exhibited strong net NO 3 − efflux, whereas beneficial effects of fungal colonization on surface pH and HA s prevented NO 3 − loss. Inhibition of HA s abolished NO 3 − influx under all conditions. We found that stimulation of HA s was crucial for the beneficial influence of ectomycorrhiza on NO 3 − uptake, whereas the presence of a Hartig net was not required for improved NO 3 − translocation. Mycorrhizas may contribute to host adaptation to salt‐affected environments by keeping up NO 3 − nutrition.

A targeted heptasaccharide library was synthesised to prepare a heparan sulphate (HS) microarray. The array was probed with two glycan-binding proteins, HS 3-O-sulphotransferase 1 and antithrombin, demonstrating the binding selectivity between HS and proteins. The HS microarray technique will accelerate the understanding of the structure and function relationships of HS.