Aims: To isolate and identify endophytic nitrogen-fixing bacteria in sugarcane growing in Cuba without chemical fertilizers. Methods and Results: Two N 2 -fixing isolates, 9C and T2, were obtained from surface-sterilized stems and roots, respectively, of sugarcane variety ML3-18. Both isolates showed acetylene reduction and H 2 production in nitrogenfree media. Nitrogenase activity measured by H2 production was about 15 times higher for isolate 9C than for T2 or for Gluconoacetobacter diazotrophicus (PAL-5 standard strain, ATCC 49037). The nifH gene segment was amplified from both isolates using specific primers. Classification of both T2 and 9C was made on the basis of morphological, biochemical, PCR tests and 16S rDNA sequence analysis. Conclusions: Isolate 9C was identified as a Pantoea species from its 16S rDNA, but showed considerable differences in physiological properties from previously reported species of this genus. For example, 9C can be cultured over a wide range of temperature, pH and salt concentration, and showed high H 2 production (up to 67AE7 nmol H 2 h)1 10 10 cell )1 ). Isolate T2 was a strain of Gluconacetobacter diazotrophicus.Significance and Impact of the Study: A new N 2 -fixing endophyte, i.e. Pantoea, able to produce H 2 and to grow in a wide range of conditions, was isolated from sugarcane stem tissue and characterized. The strain with these attributes may well be valuable for agriculture.

Ferrochelatase (FC) is the final enzyme for haem formation in the tetrapyrrole biosynthesis pathway and encoded by two genes in higher plants. FC2 exists predominantly in green tissue, whereas FC1 is constitutively expressed. We intended to substantiate the specific roles of FC1. The embryo‐lethal fc1–2 mutant was used to express the two genomic FC‐encoding sequences under the FC1 and FC2 promoter and explore the complementation of the FC1 deficiency. Apart from the successful complementation with FC1, expression of FC2 under control of the FC1 promoter (pFC1::FC2) compensates for missing FC1 but not by FC2 promoter expression. The complementing lines pFC1FC2(fc1/fc1) succeeded under standard growth condition but failed under salt stress. The pFC1FC2(fc1/fc1) line exhibited symptoms of leaf senescence, including accelerated loss of haem and chlorophyll and elevated gene expression for chlorophyll catabolism. In contrast, ectopic FC1 expression (p35S::FC1) resulted in increased chlorophyll accumulation. The limited ability of FC2 to complement fc1 is explained by a faster turnover of FC2 mRNA during stress. It is suggested that FC1‐produced haem is essential for embryogenesis and stress response. The pFC1::FC2 expression readily complements the fc1–2 embryo lethality, whereas higher FC1 transcript content contributes essentially to stress tolerance.

In plants, two genes encode ferrochelatase (FC), which catalyzes iron chelation into protoporphyrin IX at the final step of heme biosynthesis. FERROCHELATASE1 (FC1) is continuously, but weakly expressed in roots and leaves, while FC2 is dominantly active in leaves. As a continuation of previous studies on the physiological consequences of FC2 inactivation in tobacco, we aimed to assign FC1 function in plant organs. While reduced FC2 expression leads to protoporphyrin IX accumulation in leaves, FC1 down-regulation and overproduction caused reduced and elevated FC activity in root tissue, respectively, but were not associated with changes in macroscopic phenotype, plant development or leaf pigmentation. In contrast to the lower heme content resulting from a deficiency of the dominant FC2 expression in leaves, a reduction of FC1 in roots and leaves does not significantly disturb heme accumulation. The FC1 overexpression was used for an additional approach to re-examine FC activity in mitochondria. Transgenic FC1 protein was immunologically shown to be present in mitochondria. Although matching only a small portion of total cellular FC activity, the mitochondrial FC activity in a FC1 overexpressor line increased 5-fold in comparison with wild-type mitochondria. Thus, it is suggested that FC1 contributes to mitochondrial heme synthesis.

This study was aimed to isolate and identify the N 2 -fixing bacterium Gluconacetobacter diazotrophicus from 11 sugarcane varieties, grown under field conditions in four Cuban provinces, and from their associated mealybugs Saccharicoccus sacchari. Identification was based on morphological and biochemical tests and PCRamplification of 16S rRNA genes using species-specific primers. From all sugarcane varieties and numerous mealybug colonies sampled, G. diazotrophicus isolates were recovered from inside sugarcane stems of only three varieties, and one from S. sacchari colony. These four isolates showed acetylene reduction activity in nitrogen-free media and contained nifH genes which were PCR-amplified using specific primers. ERIC-PCR fingerprinting was used to compare the Cuban G. diazotrophicus isolates with type and reference strains of N 2 -fixing Gluconacetobacteria. The very low frequency of G. diazotrophicus isolates recovered is probably related with the high doses of nitrogen fertilizers applied to the sugarcane in the Cuban fields for almost 30 years. Some genetic differences, using ERIC-PCR, were detected among G. diazotrophicus strains, which could be related with its source.

ABSTRACT Dry land systems spread all over the world and comprise 41.3% of the terrestrial area, which host 34.7% of the global population, so it is convenient to propose crops able to grow there. Jatropha curcas is a plant adapted to arid and semiarid regions as well as sub-humid conditions, being a potential source of biodiesel. The challenge is to understand the physiology of J. curcas, which enables it to live under saline and drought conditions. The seeds of J. curcas used came from Ciego de Ávila Province, Cuba. Seven-day-old seedlings were cultivated in 1.5 L pots with half strength Hoagland solution for 42 days under semi-controlled conditions. NaCl added to solutions in pots provided 75 or 150 mM treatments for 240 h before measurements. Leaf growth, net photosynthesis and stomatal pore area were affected by 150 mM NaCl. Non-photochemical quenching of leaves was only changed by 150 mM NaCl after 24 h; the electron transport rate had a tendency to decrease in leaves under saline conditions. The gene expression pattern changed for SOS1 and HKT1 according to the NaCl used in the medium, indicating active mechanism to deal with Na+ in the cell. In general, Cuban J. curcas plants were able to grow and perform photosynthesis under 75 mM NaCl, which represents 7 dS m-1, a condition that restricts growth for many plant species.